Note: Descriptions are shown in the official language in which they were submitted.
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SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODlNG THE~/i
This application is a continuation-in-part of application Ser. No. 08/667,231, filed July
9, 1996.
FELD 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
Teehnology aimed at the discovery of protein factors ~including e.g., cytokines, such
as Iymphokines, hlte~ uns, 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 DNAlamino acid sequence of the protein in the case of
hybridization cloning; activity of the protein in the case of expression cloning). More recent
2 0 "indirect" cloning techniques such as signa] sequence cloning, which isolates DNA sequences
based on the ~ sencc of a now well-recognized secrelory leader sequf nce motif, a~ well as
various PCR-based or low stringency hybridization cloning techniques, have advanced the state
of the art by making available large nu.-.be.~ of DNA/amino acid sequences for proteins that
are known to have biological activity by vinue of their secreted nature in the case of leader
2 5 s~u~ -~e cloning, or by virtue of the cell or tissue source in the case of PCR-based techniques.
It is to these proteins and the polynucleotides encoding them that the present invention is
directed.
SUMMARY OF THE INVENTION
3 0 In one embo~limPn~, the presen~ 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:I;
(b) a polynucleo~ide cc .,.ising the nucleotide sequ~ nee of SEQ ID NO: I
3 5 from nucleotide 281 to nucleotide 621;
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(c ) a polynucleotide comprising the nucleoti-~e sequence of the full length
protein coding seq-~en~e of clone BM46_10 deposited under accession number ATCC
98152;
(d) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone BM46_10 deposited under accession number ATCC 9815~;
(e) a polynucleotide comprising the nucleotide seql-ence of the mature
protein coding sequence of clone BM46_10 deposited under ~ccession number ATCC
98 1 52;
(f) a polynucleotide enco ling the mature protein encoded by ~he cDNA
insert of clone BM46_10 deposited under accession number ATCC 98152;
(g) a polynucleotide encoding a protein col,~pli~."g the amino acid
sequence of SEQ lD NO:2;
(h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:2 having biologicai activity;
(i) a polynucleotide which is ~n allelic variant of a polynucleotide of (a)-
(d) above;
(j) a polynucleotide which encodec a species homologue of the protein
of (g) or (h) above .
Preferably. such polynucleotide colll~ es the nucleotide sequence of SEQ ID NO: I
from nucleotide 2~1 to nucleotide 621; the nucleotide sequence of the full length protein
coding sequ~n~e of clone BM46_10 deposited under accession number ATCC g8 152; or the
nucleotide sequence of the mature protein coding s~ quence of clone BM46_10 deposited under
accession number ATCC 981~2. In other preferred embodimP,n~c, the polynucleotide encodes
the full length or mature protein encoded by the cDNA insert of clone BM46_10 deposited
under accession number ATCC 98152. In yet other preferred embo-~im~ntc, the present
invention provides a polynucleotide encoding a protein comprising the amino acid s~quence
of SEQ ID NO:2 from amino acid I to amino acid 79.
Other embodiments provide the gene co, l~ ronding to the cDNA sequence of SEQ
ID NO: I or SEQ ID NO:3.
3 0 In other emb~dhn~ ts, 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:2;
(b~ the arnino acid sequen~e of SEQ ID NO:2 from amlno acid I to amino
3 5 acid 79;
.. . .. ..
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(c) fragments of lhe amino acid sequence of SEQ ID NO:2; and
(d) the arnino acid sequence encoded by the cDNA inscrt of clone
BM46_10 deposited under accession number ATCC 98152;
the protein being substantially free from other mammalian proteins. Preferably such protein
5 comprises the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID
NO:2 from amino acid I to amino acid 79.
ln alternate embodiments, isolate BM46_3 deposited under accession number ATCC
98101 may be ~lbslitllled for BM46_10 in any of the foregoing.
In certain prefcrred embodiments, the polynucleotide is operably linked to an
10 expression control 5~ ce The invention also provides a host cell, including bacterial, yeast,
insect and rnz~mm~ n cells, transformed with such polynucleotide cc,.-.posilions.
Processes are also provided for producing a protein, which comprise:
(a) ~rowing a culture of the host cell transfonned 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 i~ a
mature form of the protein.
Protein co,~ ositions of the present invention may further comprise a ph.u ,.,~r~utic~ily
2 0 ~cc~ le 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 co...~ es aflminicr~oring to a m:lmm~ subject a t~ ic~lly effective amount of
a cou.~osilion comprising a protein of the present invention and a pha.... ~e..lir~ily accept~hle
2 5 carrier.
DETALED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequenres are reported below for each clone and protein
3 0 ~liccloced in the present application. In some . ~ .c the seqvencec are preliminary and may
include some incorrect or ambiguous bases or amino acids. The actual nucleotide sequence
of each clone can readily be dcte....illed by sequencing of the deposited clone in accordance
with known methods. The predicted amino acid sequence (both full length and mature) can
then be deterrnined from such nucleotide sequence. The amino acid sequence of the protein
.. . . . .... . . . ....
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encoded by a particular clone can also be deterrnined by expression of the clone in a suitable
host cell, collecting the protein and deterrnining its sequence.
For each disclosed prolein applicants have identified what they have determined to be
the reading frame best identifiable with sequence information av~ilable at the time of filing
5 Because of the partial ~llbi~uity in reported sequence information. reported protein sequences
include "Xaa" de~ign~ors These "Xaa" designators indicate either ( I ) a residue which cannot
be identified because of nucleotide sequence ambiguity or (2) a stop codon in the determined
nucleolide sequence where applicants believe one should not exist (if the nucleotide sequence
were deterrnined more accurately)
1 0 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
s~qUçn~ in its amino acid seguenre "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 limita~ion proteins which arc
1 5 transported across the n,e.,.b,dne of the endoplpasmic reticulum
Clone "BM46 10"
A polynucleotidc of the present invention has been identified as clone "BM46_10"BM46_10 was isolated from a human adult muscle cDNA library using methods which are
2 0 selective for cDNAs encoding secreted proteins. BM46_10 is a full-length clone, including
the entire coding se~uence of a secreted protein (also referred to herein as "BM46_10
protein").
The nucleotide sequence of the 5' portion of BM46_10 as pl~sently determined is
reported in SEQ ID NO: I . What applicants presently believe is the proper reading frame for
2 5 the coding region is inf~i~ 1 in SEQ ID NO:2. The predicted acid ~uenc~ of the BM46_10
protein col.~,ponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2.
Additional nucleotide se~luence from the 3' ponion of BM46_10, including the polyA tail, is
reported in SEQ ID NO:3.
The EcoRIlNotl restriction fragment obtainable from the deposit containing clone30 BM46_10 should be approximately 3600 bp.
The nucleotide sequence disclosed herein for BM46_10 was searched against the
GenBank database using BLASTAtBLASTX and FASTA search protocols BM46_10
d~...ol.s~ at least some identity with ESTs identified as "zb43c09.sl Homo sapiens cDNA
clone 306352 3"' (N79027, BlastN) and "H sapiens EST sequence 00~-X" (F19321, Fasta)
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Based upon identity, BM46_10 proteins and each identical protcin or peptidc may share at
least some activity.
Deposit of Clone
Clone BM46_10 was deposited on August 23, 1996 with the American Type Culture
Collection under accession number 98152.
An additional isolate of BM46, BM46_3, was deposited on luly 9, 199fi with the
American Type Culture Collection as part of a ~ol~,posite deposit (with other clones) under
accession number ATCC 98101, from which each clone comprising a particular polynucleotide
is obtainable. Each clone has been transfected into separate bacterial cells (E. coli) in this
composite deposit. Each clone can be removed from the vector in which it was deposited by
performing an ~coRI/Notl digestion ~5' cite. EcoRI; 3' cite~ Notl) to produce the appropriate
f~ for such clone (approximate clone si~ r,~.~"ne~1t are identified above). Bacterial cells
containing a particular clone can be obtained from the composite deposit as follows:
An oligonucleotide probe or probcs should be designed to the sequencc that is known
for that particular clone. This se~uence can be derivcd from the sequ~nces provided herein,
or from a combination of those sequences. The sequence of the oligonucleotide probe that was
used to isolate each full-lenglh clone is identified below, and should be moct reliable in
2 0 isolating the clone of interest.
Clone Probe Sequence
BM46_10 SEQ ID NO:4
2 5 In the sequences listed above which include an N at position 2, that position is occupied in
preferred probestprimers by a biotinylated phosphoaramidite re.sidue rather than a nucleotide
~such as, for example. that produced by use of biotin phospho-a~"iJ~ dimethoxytrityloxy-
2-(N-biotinyl-4-a 1pn~obulyl)-propyl-3-o-(2-cyanoethyl)-(N~N-diisopropyl)-phosphc~lanlddite)
(Glen Research, cat. no. 10-1953)).
The design of the oligonucleotide probe should preferably follow these parameters:
(a) lt should be designed IO an area of the sequence which has the fewest
ambiguous bases ("N'c"), if any;
(b) lt should be deci~n~d to have a Tm of approx. 80 ~ C (assurning 2~ for each A
3 5 or T and 4 degrees for each G or C).
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Thc oligonucleotide should preferably be labeled with g 32p ATP (specific activity 6000
Ci/mmole) and T4 polynucleotidc 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 othcr cstahlishcd methods. Thc
amount of radioactivity incorporated into the probe should be 4uantitated 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 ~1 of the stock used to inoculate a sterile culture flask containing ~5 ml of
sterile L-broth cu~ h~g ampicillin at 100 ~lg/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 deterrnine the dilution and volume
which will yield approximately 5000 distinct and well-separated colonies on solid
bacteriologicaJ media contail,h~g L-broth containing ampicillin at 100 IJg/ml and agar at 1.5%
in a 150 mm petri dish when grown overnight at 37~C. Other known methods of obtaining
distinct. well-separated colonies can also be employed.
Standard colony hybridization procedures should then be used to transfer the colonies
to nitrocellulose filters and Iyse, denature and bake them.
The filter is then preferably incubated at 65''C for I hour Wilh gentle agitalion in 6X
2 0 SSC (20X stock is 175.3 g NaCUliter, ~8.2 g N~ citrate/liter, adjusted to pH 7.0 with NaOH)
containing 0.5% SDS, 100 ~g/ml of yeast RNA, and 10 mM EDTA (approximately 10 rnL per
150 mm filter). Preferably, the probe is then added to the hybridization mix at a concentration
greater than or equal to le+6 dpmlmL. The filter is then preferably incubated at 65~C with
gentle agitation overnight. The filter is then preferably washed in 500 mL of 2X SSC/0.5~c
2 5 SDS at room tc.~ ul~ without agitation, plG~ldbly followed by 500 mL of 2X SSC10. 1%
SDS at room tell"~.atLIlc; with gentle shaking for 15 minutes. A third wash with 0.1X
SSC/0.5% SDS al 65~C for 30 minutes to I hour is optional. The filter is then preferably dried
and subjected to autoradiography for sufficient time to visualize the positives on lhe X-ray
film. Other known hybridization methods can also be employed.
3 0 The positive colonies are picked, grown in culturc, and plasmid DNA isolated using
standard procedures. The clones can then be verified by restriction analysis, hybridization
analysis, or DNA sequencing.
.. .. . ..... . ...... .
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Fragments of the proteins of the presen~ invention which are capable of exhibiting
biological activity are also encompassed by the prescnt invention. Fragments of the protein
may be in linear form or they may be cyclized using known methods, for example, as descnbed
in H U. Saragovi. et al., BiolTechnology 10. 773-778 ( 1992) and in R.S. McDowell. et c~.. 1.
Amer. Chem. Soc. 1 14, 9~45-9253 ( l 992), both of which are incorporated herein by referencc.
Such fragmen~s may be fused to carrier molecules such as immunoglobulins for many
purposes, including increasing the valency of protein binding sites. ~or cxample, 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
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 lis~ing by
translation of the nucleotide seguence 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 r~ mm~ n cell or other host cell. The sequence of
the mature form of the protein may also be detcrminable from the amino acid se4uence of thc
full-length form.
2 0 The present invention also provides genes corresponding to the cDNA sequences
disclosed herein. The corresponding genes can be isolated in accolddnce 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 and/or
amplification of genes in appropriate genomic libraries or other sources of gcnomic materials.
2 5 Where the protein of the present invention is ~ blane-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 intracellular and l~dns~,.c.~b.dnc domains of the protein are deleted such that the protein
is fully secreted from the cell in which it is expressed. The intracellular and ~ldns,,,~,,b,dne
domains of proteins of the invention can be identified in accordance with known techniques
3 0 for determination of such domains from sequence information.
Species homologs of the disclosed polynucleotides and proteins are also provided 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.
. . .
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The invention also encon.passes allelic vananls of the disclosed polynucleotidcs or
proteins; ~hat is, naturally-occumng alternative forms of the isolated polynucleotide which also
encodc proteins which are identicah homologous or related ~o lhat encoded by th~polynucleotides .
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. 19, 4485-4490 (l991), in order to produce the protein recombinantly.
Many suitable expression control sPquences are known in the art. General methods of
expressing ~~co--lbi-,ant proteins are also known and are exemplified in R. Kaufman, Methods
in Enzymology }85. 537-566 (l990). As defined herein "operably linked" means that the
isolated polynucleotide of the invention and an expression control sequenre are situated within
a vector or cell in such a way that the protein is expressed by a host cell which has been
transforrned (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
(CHO) cells, human kidney 293 cells, human cpiderrnal A43 I cells, human Colo205 cells, 3T3
cells. CV- I 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 lurkat cells.
2 0 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 cerev~si~e, Schizo~accharomyces pombe, Kluyveromyces strains, Candida, or
any yeast strain capable of expressing heteroJogous proteins. Potentially suitable bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonelkl ~yphimuri~lm, or any bacterial
2 5 strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria,
it may be nece ,Sa.y 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 polynucleotide of
3 0 the invention to suitable control sequences in one or more insect expression vectors, and
employing an insect expression system. Materials and methods for baculoviruslinsect 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 E~xperiment Station Bulletin No. 1555
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(1987), incorporated herein by ~cfe~eilce. 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 cells under
culture conditions suitable to express the recombinant protein. The resulting expresscd protein
5 may then be purified from such culture (i.efrom culture medium or cell 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~3 or Cibacrom blue 3GA Sepha use~); one or more steps involving
hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or
propyl ether; or i~ .unoaffinity chromatography.
Altematively, 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 cxpression and purification of such fusion proteins are colllm~.~ially available from New
England BioLab (Beverly, MA), Pharmacia (Piscataway, Nl) 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 cu~"llle,c,ally available from
Kodak (New Haven, Cl ).
2 0 Finally, one or more reverse-phase high perforrnance liquid chromatography (RP-
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 thc
foregoing purification steps, in various combinations, can also be employed to provide a
substantially homogeneous isolated recombinant protein. The protein thus purified is
2 5 substantially free of other m:~mn~ n proteins and is defined in accordace with the present
invention as an "isolated protein."
Thc 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
characterized by somatic or germ cells cc ~It~ ng 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
~o those skilled in the art. The syn~h~tic~lly-constructed protein sequence~, by virtue of sharing
pnmary, secondary or tertiary structural and/or conformational characteristics with proteins
may possess ~iological properties in co therewith. including protein activity. Thus. they
3 5 may be employed as biologically active or immunological substitutes for natural, purified
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proteins in screcning of therapeutic compounds and in immunological processes for the
dcvelopment of antibodies
The proteins provided herein also include proteins characterized hy amino acid
sequences similar to those of purified proteins but into which mo~ification are naturally
5 provided or deliberately engineered. For example, modifications in the peptide or DNA
sequences can be made by those skilled in the art using known lechniques. Modifications of
interest in the protein sequences may include the alteration, substitution, replacement, insertion
or deletion of a selected amino acid residue in the coding sequence. For examplc, one or more
of the cysteine residues may be deleted or replaced with another amino acid to alter the
10 conforrnation of the molecule. Tcchniques for such alteration, substitution, replacement,
insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No.
4.518,584). Preferably, such alteration, substi~ution. repl:~rement insertion or deletion retains
the desired activity of the protein.
Other fid~ ,lts and derivatives of the sequPncPc of proteins which would be expected
15 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 an given the
disclosures herein. Such modifications are believed to be encompassed by the present
invcntion.
20 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 tho~e ~ssociated with assays cited herein)
identified below. Uses or activities described for proteins of the present invention may be
provided by aflministration or use of such proteins or by ~ dtion or use of
25 polynucleoti~l~s encoding such proteins (such as, for éxample. 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
3 0 community for various purposes. The polynucleotides can be used to express recombinant
protein for analysis, characterization or the.dpc.~lic use; as markers for tissues in which the
C~ vn~ling protein is preferentially expressed (either constitutively or at a parlicular stage
of tissue differ~nti ~ion or development or in disease states); as molecular weight markers on
Southem gels; as chromosome markers or tags (when labeled) to identify chromosomes or to
3 5 map related gene positions; to COIIIpdlt: with endogenous DNA sequences in patients to identify
.. . ~ ........... . . ..... .. .. .
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potential genetic disorders; as probes to hybridize and thus discover novel, related DNA
sequences; as a source of information to denve PCR primers for genetic fingerprintin~; as a
probe to "subtract-out" known sequences in thc process of discovering other novel
polynucleotides; for selecting and making oligomers for at~achment to a "genc chip" or o~her
5 support, including for exzl~in~tion of expression patterns; to raise anti-protein antibodies using
DNA jmml-ni7:l~ion techniques; and as an antigen to raise anti-DNA antibodies or elicit
anothcr immune response. Where the polynucleotide encodes a protein which binds or
potcntially binds to another protein (such as, for example, in a receptor-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 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
15 labeled reagent) in assays designed to quantitatively determine levels of the protein (or 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
2 0 receptor-ligand interaction), the protein can be used to identify the other 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 rcagent gradc
2 5 or kit format for commercialization as research products.
Methods for perforrning the uses listed ~bove 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: Guidc to Molecular Cloning
3 0 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
35 supplement, use as a carbon source, use as a nitrogen source and use as a source of
11
. .
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carbohydrale. In such cases the protein or polynucleotide of the invention can be added to the
feed of a particular organism or can bc administercd a~i a ~eparate solid or liquid ~"~pa, ~tion,
such as in the form of powder. pills, solutions, su~pensions or capsules. In the case of
microorganisms, the protein or polynucleotide of the invention can be added to the medium
5 in or on which the mic-uo-ganism 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
10 induce production of other cytokines in certain cell populations. Many protein factors
discovered to date, in~lu~ g all known cytokines, have exhibited activity in one or more factor
depenfl~nt cell proliferation assays, and hence the assays serve as a convenient confinnation
of cytokine activity. The activity of a protcin of the prescnt invention is evidenced by any one
of a number of routine factor dependent cell proliferation assays for cell lines including,
without lirnitation, 32D, DA2, DAIG, T10, B9, B9/11. BaF3, MC9/G, M+ (preB M+), 2E~,
RB5, DAI, 123, T1165, HT2, CTLL2, TF-I, Mo7c and CMK.
The activity of a protein of the invention may, among other means, be mea~ured by the
following methods:
Assays for T-cell or thymocyte proliferation include without limitation those described
2 0 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-l"te.~ience (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, 19~6; Bertagnolli et al.,1. Immunol.
I 45: 1706- 1712, 1990; Bertagnolli et al ., Cellu lar Immunology 133 :327-341, I 991;
2 5 Bertagnolli, et al., l. Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-
1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, Iymph node cells
or thymocytes include, without limitation, those described in: Polyclonal T cell slimul:--ion,
Kruisbeek, A.M. and Shevach, E.M. In C~(rren~ Prolocols in Immunology. l.E.e.a. Coligan
30 eds. Vol I pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of
mouse and human Intelrelon ~, Schreiber, R.~. In Curren~ Pro~ocols in Immunologv. I.E.e.a.
Coligan eds. Vol I pp. 6.X.1-6.8.8, John Wiley and Sons, Toronto. 1994.
Assays for proliferation and differentiation of hematopoietic and Iymphopoietic cells
include, without li---it~ti~ . those dese.ibed in: Measurement of Human and Murine Interleukin
3 5 2 and Interleukin 4, Bottomly, K., Davi~, L.S. and ~ipsky, P.E. In Curren~ Pro~oools in
12
.....
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Immunolog~. J.E.e.a. Coligan eds. Vol I pp. 6.3.1-6.3.12, John Wiley and Sons. Toronto.
I 991; deVries et al., J. Exp. Med. 173: 1205- 1211, I 991; Moreau et al., Nature 336 690-69?,
198~; Greenbergeret al., Proc. Natl. Acad. Sci. U.S.A. 80:293]-293~, 1983; Measurement of
mouse and human interleukin 6 - Nordan, R. In Current Protocol~ in Immunol~. J.E.e.a.
5 Coligan eds. Vol I 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; Measu,~.ne.~t of human Interleukin 11 - Bennelt.
F.. Giannotti, J., Clark, S.C. and Turner, K. J. In Current Pr(7tocols in Immunolog!. J.E.e.a.
Coligan eds. Vol I 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 Current
0 Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.13.1, lohn Wiley and Sons,
Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among others,
proteins that affec~ APC-T cell interactions as well as direct T-cell effects by measuring
proliferation and cytokine production) include. without limitation, those described in: Curren~
15 Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. l~Jlargulies, E.M.
Shcvach, W Strober. Pub. Greene Publishing Associates and Wiley-lnterscience (Chapter 3,
In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular
receptors; Chapter 7, Immunologic studies in Humans~; Weinberger ct al., Proc. Natl. Acad.
Sei. USA 77:6091 -6095, 1980; Weinberger et al., Eur. J. Immun. I I :405-411, 1981; Takai
20 et al.. J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.
lmmune Stimula~ing or Suppressing Activity
A protein of the present invention may also exhibit immune stimulating or immune~u~J~ncssing activity, including without limitation the activities for which assays ate described
2 5 herein~ A protein may be useful in the IIL~ I.,..t of various immune deficienciçs and disorders
(including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down)
growth and prolir~ldtiun of T and/or B Iyrnphocytes, 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
3 0 autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial. fungal
or other infection may be treatable using a protein of the present invention, including
infections by HIV, hepatitis viruses, herpesviruses. my~oba~telia, 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
3 5 be desirable, i.e., in the treatment of cancer.
. .
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Autoimmune disorders which may be treated using a protein of the present 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,
5 graft-versus-host disease and autoimmune infl~rnmAt--ry eye disease Such a protein of the
present invention may also to be useful in the treatment of allergic reactions and conditions,
such a~ asthma (particuiarly allergic asthma) or other respiratory problems Other conditions.
in which immune ~ s~ion 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. Immunosul,pression of T cell responses is
15 generally an active, non-antigen-specific, process which requires continuous exposure of the
T cells to the suppressive agent. Tolerance, which involves inducing non-re~ponsivcness or
anergy in T cells, is distinguishable from immunosuppression in that it i~ generally antigen-
spccific and persists after exposure to the toleri~ing agent has ceased Operalionally, tolerance
can be de.llon~LIdted by the lack of a T cell response upon reexposure to specific ;mtigen in the
2 0 absence of the tolerizing agent.
Down regulating or preventing onc or more antigen functions (including without
limitation B Iymphocytc 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
2 5 cell function should result in reduced tissue destruction in tissue transplantation. Typically,
in tissue transplants, re)ection 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 molecule which inhibits or blocks interaction of a B7 Iymphocyte antigen with its natural
ligand(s) on immune cells (such as a soluble. monomeric form of a peptide having B7-2
3 0 activity alone or in conjunction with a monomeric form of a peptide having an activity of
another B Iymphocyte antigen (e.g., B7-l, 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
t- ~ nilli.,g the corresponding costim~ tory signal. Blocking B Iymphocyte antigen function
in this matter prevents cytokine synthesis by immune cclls. such as T cells, and thus acts as an
3 5 immuno~upp~essant. Moreover. the l~ck of costimulation may also be sufficient to anergize
14
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W O 98~015~3 PCT~US97/11854
the T cells, thereby inducing tolerance in a subject. Induc~ion of long-term tolerance by B
Iymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of
these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subjecl
it may also be necessary to block the function of a combination of B Iymphocyte antigens.
- 5 The efficacy of particular blocking reagents in preventing organ transplimt rejection
or GVHD can be ~c~essed using animal models that are prediclive of efficacy in humans
~ Examples of appropriate systems which can be uscd include allogeneic cardiac grahs in rats
and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examinc
the immllnc,~ ve effects of CTLA41g fusion proteins in viv~ as described in Lenschow
0 et al., Sciencc 57:789-792 (1992) and Turka eS al., Proc. Natl. Acad. Sci USA, ~ 1 1 102
I 1 105 ( 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 Iymphocyte antigen function in vivo on the development of that disease.
Blochng antigen function may also be th~. ~pe~ic~lly useful for treating autoimmunc
diseases. Many ~toimmllne disorders are the result of h,a~p,~""iate activation of T cells that
are reactive against self tissue and which promote the produclion of cytokines and
autoantibodies involved in the pathology of the diseases. Preventing the activation of
autoreactive T cells may reduce or elimin~e disease symptoms. Administration of reagents
which block costimulation of T cells by disrupting receptor:ligand interactions of B
Iymphocyte antigens can be used to inhibit T cell activation and prevent production O
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-lerm relief from the disease. The efficacy of blocking reagents in
preventing or alleviating .lutoh~ e disorders can be determined using a number of well-
characterized animal models of human autoi"l",w~e ~lice~es Examples include murine
experimental autoimmune encephalitis, systemic lupus erythmatosis in MRLllpr/lpr micc or
NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOl) mice and
BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology,
Raven Press, New York, 1989, pp. 840-856).
3 0 l~pregulation of an antigen function (preferably a B Iymphocyte 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 eliciting
an initial immune response. For example, enh ~ncirlg an immune ~ unse through stimulating
B Iymphocyte antigen function may be useful in cases of viral infection. In addition. systemic
CA 02260293 1999-01-08
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viral diseases such as influenza, the co.,."~on cold, and encephalitis might be alleviated by the
administration of stimulatory forms of B Iymphocyte antigens systemically.
Alternatively, anti-viMl immune responses may be enhanced in an infected patient by
removing T cells from the patient. costimulating the T cells in vitr7 with viral antigen-pulsed
5 APCs either expressing a peptide of the present invention or together with a stimulatory forrn
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 would be to isoiate
infected cells from a patient, transfect them with a nucleic acid encoding a protein of the
present invention as des~r-bcd herein such that the cells express all or a portion of the protein
10 on their surface, and le; - uduce the transfected cells into the patient. The infected cells would
now be capable of delivering a costimulatory signal to, and thereby activate, T celJs in vivo.
ln another application, up regulation or enhancement of antigen function (preferably
B Iymphocyte antigen function) may be useful in the induetion of tumor immunity. Tumor
cells (e.g., sarcoma, mPl~nom~ Iymphoma, leukemia, neuroblastoma, carcinoma) transfected
15 with a nucleic acid ~nco ling at least one peptide of the present invention can be administered
to a subject to overcome tumor-specific tolerance in the suoject. If desired, the tumor cell can
be transfected to express a con,bi on of peptides . For examplc, 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-l-like
20 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 tr~nsfected cell. Altematively, gene
therapy techni~ues can be used to target a tumor cell for tr;msfection in vivo.
The presence of the peptide of the present invention having the activity of a B
Iymphocyte antigen(s) on the surface of the tumor cell provides the necessa,y costimulatlon
2 5 signal to T cells to induce a T cell n ~ ed immune response against the transfected tumor
cells. In addition, tumor cells which laclc 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 MHC class II
3 0 ~ chain protein and an MHC class n ~B chain protein to thereby express MHC class I or MHC
class n proteins on the cell surface. Expression of the app~op..ate class I or class n MHC in
conjunction with a peptide having the activity of a B Iymphocyte antigen (e.~., B7- l, B7-2, B7-
3) induces a T cell n ~oAi -l immune response against the transfected tumor cell. Optionally,
a gene encoding an ~nticence construct which blocks expression of an MHC class n associated
3 5 protein, such as the invariant chain. can also be cotransfected with a DNA encoding a peptide
16
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W O 98/01553 PCT~US97111854
having the activity of a B Iymphocyte antigen to promote presentation of tumor as~ociated
an~igens 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.
The activity of a protein of the invention may, among other means~ be mcasured 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 Associales and Wiley-
1 0 l~ nce (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., 1. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol.
]35:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai e~ al, J.
Immunol. 140:508-51~, Ig88; Hemnann et al., Proc. Natl. Acad. Sci. USA 78:2488-249~.
1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al J Immunol.
135:1564-1572, 1985; Takai et al., 1. Immunol. 137:3494-3500, 1986; Bowmanet al., J.
Virology 61:1992-19g8; 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 les~ on~cc and isotype switching (which
2 0 will identify, among others, proteins that mod~ ue T-cell dependent antibody responses and
that affect ThllTh2 profiles) include, without limitation, those described in: Maliszewski. J.
Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vi~ro antibody production,
Mond, J.l. and Brunswick, M. In C~rrent Protocols in Immunology. I.~.e.a. Coligan eds. Vol
I pp. 3.8.1-3.8.16, lohn Wiley and Sons, Toronto. 1994.
2 5 Mixed Iymphocyte reaction (MLR) assays (which will identify, among others. proteins
that generate predominantly Thl and CIL responses) include, without limitation, those
described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.~. Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-lnterscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1 -3.19; Chapler 7,
3 0 Immunologic stlldies in Humans); Takai et al., J. lmml1nol 137:3494-350Q, 1986; Takai et al.,
J. Immunol. 140:508-512, 1988; Bertagnolli et al., l. Immunol. 149:3778-3783, 1992.
Dendritic cell~le~ nd~l ,t assays (which will identify, among others, proteins expressed
by dendntic cells that activate naive T-cells) include, without limitation, those described in:
Guery et al., J. lrnmunol. 134:536-544, 1995; lnaba et al., Journal of Experimental Medicine
173:549-559, 1991; Maca~ori - et al., Journal of Immunology I54:5071-5079, 1995; Porgador
17
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W O 98/01553 PCT~US9~/11854
et al.. lournal of Experimental Medicine 182:2~5-260, 1995; Nair et al.. Journal of Virology
67:4062-4069, 1993; Huang et al.. Sciencc 264:961 -965, 1 ~94; Macatonia et al., Joumal of
~xpenmenlal Medicine 169:1255-1264, 1989; Bhardwa; et al., Journal of Clinical
Investigation 94:797-~07, 1994; and Inaba et al., Journal Or Expenmental Medicine 17~ :631 -
5 640, 1990
Assays for Iymphocyte survival/apoptosis (which will identify, among others, proteins
that prevent apoptosis after superantigen inductioo and proteins that regula~e Iymphocyte
homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry
13:79~-X0~, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al.. Cancer
Research 53:1945-19~1, 1993; Itoh et al., Cell 66:~33-243. 1991; Zacharchuk, Joumal of
lmmunology 145:4037-4045, 1990; Zamai et al., Cytomctry 14:891 -897, 1993; Gorczyca et
al., International Journal of Oncology 1 :639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and development
include, without limitation, those described in: Antica et al., Blood 84: 111 - 117, 1994; Fine
15 et al., Cellular Immunology ISS:111-122, 1994; Galy ct al., Blood 85:2770-2778, 1995; Toki
et al., Proc. Nat. Acad Sci. USA 88:7548-7551, Ig91.
Hematopoiesis Regulating Activity
A protein of the present invention may be useful in regulation of hematopoiesis and,
20 consequently, in the ~Ita~ nt of myeloid or Iymphoid cell deficiencies. Even marginal
biological activity in support of colony forming cells or of factor-de~,~nd~..t 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 cytolcines, thcreby indicating
utility, for example, in treating various anemias or for use in conjunction with2 5 irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid
cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and
monocytes/ma-,lophagcs (i.e., traditional CSF activity) useful, for example, in conjunction with
chemotherapy to prevent or treat conC~quent myelo-su~?plessioll; in supporting the growth and
proliferation of megakaryocytes and consequently of pla~elcts thereby allowing prevention or
3 0 treatment of various platelet disold~ such as thrombocytopenia, and generally for use in place
of or complh.l~ ..~y to platelet transfusions; and/or in supporting the growth and proliferation
of h~m~opoietic stem cells which are capablc 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,
3 5 aplastic anemia and paroxysmal nocturnal hemoglobinuna), as well as in repopulating the stem
18
... .
CA 02260293 1999-01-08
W O g8101553 PCT~US97/11854
cell compartmen~ post irradiationlchemotherapy, either in-vivo or ex-vivo (i.c.~ in conjunction
with bone marrow transplantation or with peripheral progenitor cell transplantation
(homolo~ous or heterologous)) as norrnal cells or genetically manipulated ror ~enc therapy.
The activity of a protein of Ihe invention may, among other means. be measured by the
5 following methods:
Suitable assays for proliferation and differentiation of various hematopoietic lines arc
~ citcd above.
Assays for embryonic stem cell differentiation (which will identify, among other~,
proleins that influence embryonic differentiation hematopoiesis) include, without limitation,
10 thosedescribed in: lohanssonetal. CellularBiology 15:141-151,1995; Kelleret al., Molecular
and CellularBiology 13:473-4~6, 1993; McC!~n~ n etal., Blood 81:2903-291S, 1993.Assays for stem cell survival and differentiation (which will identify, among others,
proteins that regulate Iympho-hematopoiesis) include, without limitalion, those described in:
Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I.
15 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; Primitivc hematopoietic colony forrning
cells with high proliferative potential, McNiece, I.K. and Briddell, R.A. In Cultllre 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 2'':353-359, 1994; Cobblestone area
2 0 forrning cell assay, Ploemacher, R.E. In Culture of Hematopoietic Cells. R.I. Freshney, et al.
eds. Vol pp. I -~ I, 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 o~
Hema~opoietic Cells. R.I. Freshney, et al. eds. Vol pp. 163- 179, Wiley-Liss, Inc., New York,
NY.1994; Long terrn culture initi~ nf~ cell assay, Sutherland, H.J. In Culture of Hematopoletic
25 Cells. R.I. Freshney, etal. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.
lg
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Tissue Growth Activit~/
A protein of the present invention also may havc utility in composi~ions used for bone,
cartilage, tendon, ligament andlor nerve tissue growth or regeneration, as well as for wound
healing and tissue repair and repl~ernent. and in the treatmen~ of bums, incisions ;md ulcers.
A protein of the present invention, which induces cartilage and/or bone growth in
ChLIIII~ 'eS where bone is not normally forrned, has application in the healing of bone
fractures and cartilage damage or defects in humans and other animal.s. Such a preparation
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 al~o is useful in cosmetic
plastic surgery.
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, stim~-lat~ growth of bone-forming cells or induce differentiation of progenitor~
of bone-forming cells. A protein of the invention may also be useful in the treatment of
osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by
blocking inflammation or processes of tissue destruction (collagenasc activity, osteoclast
activity, etc.) mediated by inflammatory processes.
2 0 Another category of tissue reg~ne.dlion activity that may be attributable to the protein
of the present invention is tendon/ligament formation. A protein of the present invention,
which induces tendon/ligament-like tissuc or olhcr tissue formation in circumstances where
such tissue is not norrnally 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
2 5 p~ ,a.dlion employing a tendon/ligament-like tissue inducing protein may have prophylactic
use in pl~c.~lil)g damage to tendon or ligament tissuc, as 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/ligam~nt-like tissue forrnation induced by a composition of the present
invention contributes to the repair of congenital, trauma induced, or other tendon or ligament
3 0 defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair
of tendons or ligaments. The compositions of the present invention may provide an
environment to attract tendon- or ligament-forn~ing cells, stimula~e growth of tendon- or
ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forn ing
cells, or induce growth of tendonAigament cells or progenitors ex vi~o for return in vivo to
3 5 effect tissue repair. The compositions of the invention may also be useful in the treatment of
. .
CA 02260293 1999-01-08
W O 98~01S~3 PCTfUS97/118~4
tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions
may also include an appropnate matrix andlor sequestering agent ~s a carrier as is well known
in the art.
The protein of the presenl invention may also be useful for proliferation of neural cells
~ 5 and for regeneration of nerve and brain tissue, ~.e. for the treatment of central and peripheral
ncrvous system diseascs and neuropathies, as well as mechanical and traumatic disordcrs,
which involve degeneration, death or trauma to neural cells or nerve tissue. More spccifically,
a protein may be used in the treatment of diseases of the peripheMI nervous system, such as
pcli~h~.~l nerve injuries, peripheral neuropathy and localized neuropathies, and central
nervous system ~ljce~cec such as Alzhetmcr's, Parkinson's disease, Huntington's diseasc,
amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be
treated in accordance with the present invention include mechanical and traumatic disordcrs,
such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke.
Pe. i~hel~l neuropathies resulting from chemotherapy or other medical therapies may al~o be
treatable using a protein of the invention.
Proteins of the invention may also be useful to promote better or faster closure of non-
hcaling 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
2 0 gencration or l~gene.d~ion of other tissucs. such as organs (including, for example, pancreas,
liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular
(including vascular endothelium) tissue, or for p.u---oth,g 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 e~hibit angiogenic
2 5 activity.
A protein of the present invention may also be useful for gut protection or ~ e.ation
and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and condilions
resulting from systemic cytokine damage.
A protein of the prescnt invention may also be useful for promoting or inhibiting
3 0 differentiation of tissues describeù above from precursor tissues or cells; or for inhibiting the
growth of tissues described above.
The aclivity of a protein of the invention may, among other means, be measured by the
following methods:
Assays for tissue generation activity include, without limitation, those described in:
International Patent Publication No. W09~116035 (bone, cartilage, tendon); International
CA 02260293 1999-01-08
W O 98/01553 PCTAUS97111854
~atent Publicatlon No. W095/05846 (nerve~ neuronal); In~ernational Patent Publication No.
W091 /07491 (skin, endothelium ).
Assays for wound healing activity include, without limitation. those described in:
Winter, Epidermal Wound Healing, pps. 71~ (Maibach, Hl and Rovee. DT, cds.), Year
Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, ~. Invest.
Dermatol 71:382-84 (1978).
Activinllnhibin Activity
A protein of the present invention may also e~hibit activin- or inhibin-related
1 0 activities. Inhibins are ch~ ized by their ability to inhibit the release of follicle stimulating
hormone (FSH), while activins and are characterized by their ability to slimul~t~ 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 tamily. may be useful as a contraceptive based
on the ability of inhibins to decrease fertility in female ~,r~"",.,als and decrease sperrnatogenesis
1 5 in male mammals. Ad~-lin.~ ion of sufficient amounts of other inhibins can induce infertility
in these m:lrnm~lS. 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, ba~ed upon the ability of activin molecules in slim.ll~ring FSH release
from cells of the anterior pituitary. See, for examplc, United States Patent 4,79g,885. A
2 0 protein of the invention may also be useful for advancement of the onset of fertility in sexually
immature IllA~ c, SO as to increase the lifetime reproductivc 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 m~thocic
2 5 Assays for activin/inhibin activity include, without limi~ inn, those described in: Vale
et al., Endocrinology 91 :562-572, 197~; 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.
3 0 ChemotacticlChemokinetic Activity
A protein of the present invention may have chc"~ota~Lic or chc."oldlletic activity (e.g.,
act as a chemokine) for m~mnl~ljan cells, including, for example, monocytes, fibroblasts,
neutrophils, T-cells, mast cells, eosinophils, epithelial and/or ~.ndothe!i~l cells. Chemotactic
and chemokinetic proteins can be used to mobilize or attract a desired cell population to a
3 5 desired site of action. (-h~ ,ot~ ~ic or ~h~-o~ in~tic proteins provide particular advantages in
CA 02260293 1999-01-08
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treatmcnt of wounds and other trauma to tissues, as well as in treatment of localized infections.
For cxample, attraction of Iymphocytes, monocytes or neutrophils to tumors or sitcs of
infection may resull in improved immune responses against the ~umor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population if it can
5 stimulate~ directly or indircctly, the directed orientation or movemcnt of such cell population.
Preferably, the protein or peptide has the ability to directly stimulatc directed movement of
~ cells. Whether a particular protein has chemotactic activity for a population of cells can be
readily dete~ ed by employing such protein or pcptide in any known assay for cell
chemotaxis.
1 0 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 abilily 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
~5 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. Coligam
A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing
Associates and Wiley-lnterscience ~Chapter 6.1 , 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
103:14()-146, 199S;MullerctalEur.J.Immunol.'~5: 1744-1748;Gruberetal.J.oflmmunol.
5860-5867, l9g4; 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
2 5 a result, such a protein is expected to be useful in ll~atlllcnt of various coagulation disorders
(including hereditary disorders, such as hemophilias) or to enhance coagulation and other
hcl..u~lic 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 ~ nent and prevenlion of conditions resulting the~f.u~ (such ~s, for example~ infarction
3 0 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 the
following methods:
Assay for hemost~tic and thrombolytic activity include. without limitation, those
described in: Linet et al., J. Clin. Pharmacol. 26:131-140~ 1986; Burdick et al., Thrombosis
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Res. 45:413~19,1987; 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 l~ce~llol~, receptor
ligands or inhibitors or agonists of receptor/ligand interactions. Examples of such receptors
and ligands include, without limitation, cytokine receptors and their ligands, receplor kinases
and their ligands, receptor phosphatases and their ligands, receptors involved in cell-ce]l
interactions and their ligands (including without limitation, cellular adhesion molecules ~such
as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen
plc~l,nt~ n, antigen recognition and development of cellular and humora] immune responses).
Receptors and ligands are also useful for screening of potential peptide or small molecule
inhibitors of the relevant receptorAigand interaction. A protein of the present invention
(including, without limitation, fragments of receptors and ligands) may themselves be useful
1 5 as inhibitors of receptorlligand interactions.
The activity of a pro~ein of thc invention may, among other means, be measured by 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.~l. Margulie~,
E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-lnterscience
(Chapter 7.28, Mea~ul~..lent of Cellular Adhesion under static conditions 7.28.1 -7.28.22)~
Takai et al.. Proc. Natl. Acad. Sci. USA 84:6864-686~, 19X7; Bierer et al., J. Exp. Med.
168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenhor~ et
al., ~. lmmunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
Anti-lnfl~mm~rry Activity
Proteins of the present invention may also exhibit anti-infl ~mmz~c ry activity. The anti-
infl:-mm. ~tory activity may be achieved by providing a stimulus to cells involved in the
infl~mm~ ry re~o/)se, by inhibiling or promoting cell-cell inter.lctions (such as. for example,
3 0 cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the infl~mm, tory
process, inhibiting or promoting cell extravasation. or by slimulatin,g or ~u~ e ,sil~g production
of other factors which more directly inhibit or promote an inflammatory response. Proteins
exhibiting such activities can be used to treat inflammalory conditions including chronic or
acute conditions), including without limitation inflammation associated with infection (such
35 as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-
24
.
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rcperfusion injury, endotoxin lethality, arthritis, complement-medi~ d hyperacute rejection,
nephri~is, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn'~;
disease or resulting from over production of cytokines such as TN~ or IL- 1. Proteins of th~
invention may also be us~ful to treat anaphylaxis and hypersensitivity to an antigenic substance
5 or material.
Tumor Inhibition Activity
In addition to the activities d~ ,fibed above for immunological ll~allJlellt or prevention
of tumors, a protein of the invention may exhibit othcr anti-tumor activities. A protein may
10 inhibit tumor growth directly or indirectly (such as, for cxample, via ADCC). A protein may
e~hibit 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 or cell types which
inhibit tumor growth, or by suppl~ssing, eliminating or inhibiting factors, agents or cell types
15 which promotc tumor growth.
Other Activities
A protein of the invention may also exhibit one or more of the following additional
2 0 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, 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 au~;,n~ a~ion or diminution, change in bone form or shape);
2 5 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, vitamins, minerals, cofactors or other
nutritional factors or component(s); effecting behavioral characteristics, including, without
limitation, appetite, Iibido, stress, cognition (including cognitive disorders), depression
3 0 (including depressive disorders) and violent behaviors; providing analgesic effects or other
pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages
other than hematopoietic 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
3 5 (such as, for example, the ability to bind antigens or complement); and the ability to act as an
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antigen in a vaccine cu~l~posilion to raise an immune response agalnst such protein or another
matenal or entity which is cross-rcactive with such protein.
5 ADMINISTRATION AND DOSING
A protein of lhe present invention (from whatever source derived, including without
limitation from recombinant and non-recornbinant sources) may be used in a pharmaceutical
composition when combined with a pharTn~ eutic ~lly acceptable carrier. Such a composition
may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers,
10 solubilizers, and other materials well known in the art. The terrn "pharmaccutically
acceptable" means a non-toxic material that does not interfere with the effectiveness of the
biological activity of the active ingredient(s). The ch~a~ lics of the carrier will depend on
the route of ~ ic~alion. The phann~eutic~l composilion of the invention may also contain
cytokines, Iymrho~ines. or other hematopoietic factors such as M-CSF, GM-CSF, TNF. IL- I,
IL-2,IL-3,IL-4,L-5,1L-6,L-7.IL-8,1L-g,lL-lO.lL-ll,lL-12,1L-13,1L-14,1L-15,1FN,
TNF0, TNFI, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor. 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
andlor agents may be inclu(led in the pharmaceutical composition to produce a synergistic
2 0 effect with protein of the invention, or to minimize side effects. Conversely, protein of thc
present invention may be included in formulations of the particular cytokine, Iymphokine, other
hematopoietic factor. thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to
minirnize side effects of the cytokine. Iymphokine, other hematopoietic factor, thrombolytic
or anti-thrombotic factor, or anti-inflammatory agent.
2 5 A protein of the present invention may be active in mullim~rs (e.g., heterodimers or
homodimers) or complexes with itself or other proteins. As a result, pharm~ceutic~l
col.lposilions of the invention may wnl~ise a protein of the invention in such multimeric or
complexed form.
The ph~,l,a~ ic:~l col.lposilion of the invention may be in the form of a complex of
3 0 the protein(s) of present invention along with protein or peptide antigens. The protein and/or
peptide antigen will deliver a stim~ tory signal to both B and T Iymphocytes. B Iymphocytes
will respond to antigen through their surface immunoglobulin receptor. T Iymphocytes will
respond to antigen through the T cell receptor ~TCR) following presentation of the antigen by
MHC proteins. MHC and structurally related proteins inclu-ling those encoded by class I and
3 5 class n MHC genes on host cells will serve to present the peptide antigen(s) to T Iymphocytes.
26
....~ ..
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The antigen components could also be supplied as purified MHC-peptide complexes alone or
with co-stimulatory molecules lhat can directly signal T cells. Alternatively antibodie~ 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 cells can be combined with the pharmaceutical
5 composition of the invention.
Thé pharm~celltic~l composition of the invention may be in the form of a lipocome in
which pro~ein of the present invcntion is combined, in addilion to other pharmaceutically
acceptable carriers, with a~ )hi~JdtlliC agents such as lipids which exist in aggregated forrn as
ellec~ insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable
10 lipids for liposomal forrnulation include, without limitation. monoglycerides, diglycerides.
sulfatides, Iysolecithin. phospholipids, saponin. biie 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,'~35,871; U.S. Patent No 4,S01,728; U.S. Patent No. 4.837,028; and U.S. I'atent
No. 4,737,3~3, all of which are incorporated herein by rcferencc.
As used herein, the terrn "the~ ic llly effective amount" means the total amount of
each active component of the pharm~ceutic~l composition or method that is sufficient lo show
a rnP~nin~ful patient benefit, i.e., treatment. healing~ prevention or amelioration of the relevant
medical condition, or an increase in rate of treatment, healing, prevention or amelioration of
such conditions. When applied to an individual active ingredient, administered alone, the term
2 0 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, whethcr administered in
combination, serially or simultaneously.
In practicing the method of llca~ ,nt or use of the present invention, a Ih~.,.rc.Jlic~lly
effective amount of protein of the present invention is ~-1ministered to a ma~mli having a
2 5 condition to be treated. Protein of the present invention may be a~minictçred in accordance
with the method of the invention either alone or in combination with other therapies such as
treatments employing cytokines, Iympho~inçs or other hematopoietic factors. When co-
~rlminiclçred with one or more cytokines, Iymphokines or other hematopoietic factors, protein
of the present invention may be uiminic~ered either simultaneously with the cytokine(s),
30 Iymphohne(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 administçring protein of the present invention in combination with
cytokine(s), Iymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic
factors.
....
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Administration of protein of the present invention used in the pharrnaceulical
composition or to practice the method of the present invention can be carrieLI out in a vanety
of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous,
subcutaneous, intrapcliloneal, parenteral or intravenous injection. Intravenous administration
to the patient is preferred.
When a thcla~Jeutically effectivé 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 pharmaceutical composition
of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The
tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and
preferably from about 2~ 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
pharrnaceutical composition may further contain physiological saline solution, dextrose or
other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene
glycol. When a~ministered in liquid form, the pha""aceutical composition contains from
about O.5 to 90% by weight of protein of the present invention, and preferably from about I
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, pal~ntGIdlly acceptable aqueous solution. The
preparation of such parenterally acceptable protein solutions, having due regard to pH~
isotonicity, stability, and the like, is within the skill in the art. A preferred pharrnaceutical
composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition
2 5 to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection,
Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, I ~rtsl~.d
Ringer's Iniection, or other vehiclc as known in lhe an. The pha~ acG.Itical composition of
the present invenlion may also contain stabilizers, preservatives, buffers, antioxidants, or other
additives known to those of skill in the art.
3 0 The amount of protein of the present invention in the pharmaceutical composition of
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 which to
treat each individual patient. Initially, the attending physician will ~iminicter low doses Or
3 5 protein of the present invention and observe the patient's response. Larger doses of protein of
28
.
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WO 98101553 PCT/US97/1 1854
the present invention may be adminis~ered until the optimal therapeutic effect is obtained for
the patient~ and at that point the dosage is not increased further. It h; contemplatcd that the
vanous pharrn~ceutic~l compositions used to practicc the method of thc present invention
should contain about 0.01 ~lg to about 100 mg (preferably about O. l,ug to about 10 mg. more
5 prcferably about 0.1 ~Ig to about I mg) of protein of the present invention per kg body wcight
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
condition and potential idiosyncratic response of each individual patient. It is contemplaled
that the duration of each application of the protein of the present invention will be in the range
10 of 12 to 24 hours of continuous intravenous a-h~,i"i~t~dtion. Ultimately the attending physician
will decide on the appropriate duration of intravenous thcrapy using the pharrn l~cutic~l
composition of the present invention.
Protein of the invention may also be used to immunize animals lo obtain polyclonal
and monoclonal antibodies which spccifically react with the protein. Such antibodies may be
15 obtained using either the entire protein or fragments thereof as an immunogen. The peptidc
immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are
conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Methods for synthcsizing
such peptides are known in the an, for example. as in R.P. Merrifield, J. Amer.Chem.Soc. 85,
_149-2154 tl963); JL. Krstenansky, e~ al., FEBS Lett. 211, 10 (1987). Monoclonal2 0 antibodies binding to the protein of thc invention may be useful diagnostic agents for the
immunodetection of the protein. Neutralizing monoclonal antibodies binding to the protein
may also be useful therapeutics for both conditions associated with the protein and also in the
~1eat,.,cnt of some forms of cancer where abnormal expression of the protein is involved. In
the casc of cancerous cells or leukemic cetls, neutralizing monoclonal antibodies against the
2 5 protein may be useful in ~lel~ cting and preventing the ~"r~ l.c spread of thc cancerous cells,
which may be mPtli~-od by the protein.
For co",positions of the present invention which are useful for bone, canilage, tendon
or ligament .ege,)cl~tion, the therapeutic method includes administering the composi~ion
topically, systematically, or locally as an implant or device. When administered, lhe
3 0 therapeutic composition for use in this invention is, of course, in a pyrogen-free,
physiologically acceptable form. Further, the composition may desirably be encapsulated or
iniected in a viscous form for delivery to the site of bonc. canilage 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
3 5 composition as described abovc. may alternatively or additionally, be administered
2g
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~iimult;meously or sequentially with the composltion in the method~ of the invention.
Preferably for bone and/or cartilage formation, the composilion 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
resorbecl into the body. Such matrices may be formed of materiahi 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 thc co"~positions will definc the .ll~p~u~iate formulation. Potential matrices for
the compositions may be biodegradable and chemically defmed calcium ~ulfate,
tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.
Other potential materials are biodegradable and biologically wcll-defined, such as bone or
dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix
co,.,p~ ,.,ts. Other potential matrices are nonbiodegradable and chemically defincd, such as
sintered hydroxapatite, bioglass, al~lmin~ec, or other ceramics. Matrices may be comprised
of colllbhlations 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 sizc. particle
size, particle shape. and biodegradability.
2 0 Presently prefemd 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.
2 5 A p~cfel~;d family of s~questf~ring agents is celh-locic materials such as alkylcelluloses
(including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose.
hydroxyethylcellulose, hydroxypropylcellulose~ hydroxypropyl-methylcellulose, and
carboxymethylcellulose, the most preterred being cationic salts of carboxymethylcellulose
(CMC). Other preferred se~ f.ing agents include hyaluronic acid, sodium alginate,
3 0 poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).
The amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1-10 wt% based on
total forrnulation weight, which represents the amount necessary to prevent desorbtion of the
protein from the polymer matrix and to provide ap~ Jp~iate handling of the composition, yet
not so much that the progenitor cells are prevented from infiltrating the matrix, thereby
3 5 providing the protein the opportunity to assist the osteogenic activily of the progenitor cell~.
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In further c~""po~itions, proteins of the invention may be combined with other agents
bencficial to ~he treatment of the bone andlor cartilagc dcfcct, wound, or tis.sue in question.
Thcse agents include various growth factors such as epidermal growth fac~or ~EG~'), platele~
deri~cd growth factor (PDGF), transforming growth factors (TGF-o~ and TGF-~), 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
The dosage regimen of a protein-containing pharmaceutical composition to be usedin tissue regeneration will be deterrnined by the attending physician considering various factors
which modify the action of the proteins, e g., amount of tissue weight desired to be forrned, the
sile of damage, the condition of the damaged tissue, the size of a wound~ type of damaged
lissue (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 matri~ used
in the reconstitution and with inclusion of other proteins in the pharrn~ceutic~l composition.
For example, the addition of other known growth factors, such as IGI~ I (insulin like ~rowth
factor T), to the final composition, may also effect the dosage. Progress can be monitored by
periodic assessment of tissue/bone growth and/or repair, for examplc, X-rays,
histomorphometric determinations and tetracycline labeling.
2 0 Polynucleotides of the present invention can also be used for gene therapy. Such
polynucleotides can be introduced either in vivo or ex vivo into cells for cxpression in a
mammalian subject. Polynucleotides of thc invention may also be administered by other
known methods for introduction of nucleic acid into a cell or olgani~,l, (including, without
limitation, in the form of viral vectors or naked DNA).
2 5 Cells may also be cultured ex vivo in the p,~sence of protcins 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
3 0 set forth.
.. . .
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SEQUENCE LISTING
(l) GENERAL INFORMATION:
(i) APPLICANT: Jacobs, Kenneth
McCoy, ~ohn
LaVallie, Edward
Racie, Lisa
Merber~, David
Treacy, Maurice
Spaulding, Vikki
~ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES
ENCODING THEM
~iii) NUMBER OF SEQUENCES: 4
~iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
~B) STREET: 87 CambridgePark Drive
~C) CITY: Cambridge
~D) STATE: Massachusetts
(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) SOFTWARE: PatentIn Release #l.0, Version ~l.30
(vi) CURRENT APPLICATION DATA:
~A) APPLICATION NUMBER:
~B) FILING DATE:
~C) CLASSIFICATION:
~viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Brown, Scott A.
(B) REGISTRATION NUMBER: 32,724
(ix) TELECOMMUNICATION INFORMATION:
~A) TELEPHONE: ~617) 498-8224
~B) TELEFAX: ~617) 876-5851
~2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 621 base pairs
~B) TYPE: nucleic acid
~C) STRANDEDNESS: double
~D) TOPOLOGY: linear
~ii) MOLECULE TYPE: cDNA
32
.
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-
W O98/01553 PCTAUS97111854
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
ATAACATCTC CCCAGACCCA GAAAAACAGA AAGCTCCACA GAAATTAAAT GTTGAAGAGA 60
AACTCTCAAA GGAAGTTACA GAAGAAAACT ATCTCTTTCC CAGTAAGTTC AGTGGAAAGT l20
GCACTAGAAC ATGAATATGA CTTGGTGAAT TAGATGAAAG TTTTTATGGA CCAGAAAAGG l80
CCACAACATA TTATCTCATC CAGAGACCCA AAGCCAAAAC TCAGCTGACA GGAATGTTTC 240
AAAGGACACA AAGAGAGATG TGGACTCAAA GTCACCGGGG ATGCCTTTAT TTGAAGCAGA 300
GGAAGGAGTT CTATCACGAA CCCAGATATT TCCTACCACT ATTAAAGTCA TTGATCCAGA 360
ATTTCTGGAG GAGCCACCTG CACTTGCATT TTTATATAAG GATCTGTATG AAGAAGCAGT 420
TGGAGAGAAA AAGAAGGAAG AGGAGACAGC TTCTGAAGGT GACAGTGTGA ATTCTGAGGC 480
ATCATTTCCC AGCAGAAATT CTGACACTGA TGATGGAACA GGAATATATT TTGAGAAGTN 540
CATACTCAAA GATGACATTC TCCATGACAC ATCTCTAACT CAAAAGGACC ANGGCCAAGG 600
TCTGGAAAAA AAACAANTTG G 62l
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 112 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Pro Leu Phe Glu Ala Glu Glu Gly Val Leu Ser Arg Thr Gln Ile
l 5 l0 15
Phe Pro Thr Thr Ile Lys Val Ile Asp Pro Glu Phe Leu Glu Glu Pro
Pro Ala Leu Ala Phe Leu Tyr Lys Asp Leu Tyr Glu Glu Ala Val Gly
Glu Lys Lys Lys Glu Glu Glu Thr Ala Ser Glu Gly Asp Ser Val Asn
Ser Glu Ala Ser Phe Pro Ser Arg Asn Ser Asp Thr Asp Asp Gly Thr
.. .. .
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Gly Ile Tyr Phe Glu Lys Xaa Ile Leu Lys Asp Asp Ile Leu His Asp
Thr Ser Leu Thr Gln Lys Asp Xaa Gly Gln Gly Leu Glu Lys Lys Gln
l00 105 ll0
(2) INFORMATION FOR SEQ ID NO:3:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 315 base pairs
~B) TYPE: nucleic acid
~C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
T~ AC TGGTAAAGGA AATCCTCTGA TGGACAGGTC AGAGTGAAGG AAGGTTGTGC 60
TGGTAAGACA TNTCTGANGA AGAGCCATGG ATGCTTTCCA CAAAATGTCA CCTCGCTGCA l20
CTAAAGGATG ATGAATCCTA ATCATTAAAG GAATTGTTTC AGCTGATTTA AATTTATAAT l80
GAACTCTTTT GTAATAATGT ATACTGTAGA ACATGAGTCT CTCCTCCCTA AAATTTTAAA 240
TGTAGAAAAG TGCTATATAT TAGAAATTTC CA~ A AATAAATGGT TAGAGTCTAT 300
AAUUUU~AAA AAAAA 315
(2) INFORMATION FOR SEQ ID NO:4:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: ~inear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide'~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GNCTCCTCCA GAAATTCTGG ATCAATGAC 29
34
