Note: Descriptions are shown in the official language in which they were submitted.
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92/0~6 PCT/US91/05749
LYMP~ORINE 154
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to secreted proteins
(lymphokines) produced by activated T lymphocytes. In
particular, the present invention relates to lymphokine
154.
Back~round Information
Quiescent non-dividing T lymphocytes respond to
antigen and mitogen stimulation by the activation of
cellular genes which are thought to control events that
lead to cell proliferation and the expression of a differ-
entiated phenotype. Treatment of human peripheral blood T
cells with the mitogenic combination of phytohemmagglutin
(P~A) and phorbol 12-myxistate 13-acetate (PMA) appears to
mimic the effects of antigen plus antigen presenting cells
in vivo. These two agents are known to induce the expres-
sion of genes which are important for progression through
the cell cycle (Reed et al., Proc. Natl. Acad. Sci. USA.
83:3982, 1986). -
- T lymphocyte activation further induces genes
producing secreted factors that direct the growth and
differentiation of many cell types, predominantly, but not
exclusively, those of the immune system (Dumonde et al.,
Nature. 224:38, 1969; Mosmann, T.R. "Helper T cells and
their lymphokines." T cells. Feldmann, Lamb and Owen ed.
1988 John Wiley and Sons, New York; Rocklin et al. Adv.
Immunol. 29:55, 1980). Culture supernatants from activat-
ed T cells display multiple biological activities includ-
ing T cell growth (Taniguchi et al., Nature. 302:305,
1983), B cell growth and differentiation (Namen et al.,
Nature. 333:571, 1988; Campbell et al., Proc. Natl. Acad.
Sci. USA. 84:6629, 1987; Hirano et al., Nature, 324:73,
1986; Yokota et al., Proc. Natl. Acad. Sci. USA. 83:5894,
1986), anti-viral activity (Gray et al., Nature. 295:503,
1982), activation of cells involved in inflammation
(Yoshimura et al., Proc. Natl. Acad. Sci. USA. 84:9233,
1987), and hematopoietic cell precursor growth and
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W092/0~66 ~ ~ PCT/US91/0574
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maturation tYang et al., Cell. 47:3, 1986). Numerous
lymphokines and cytokines including IL-2 through IL-8
(Campbell et al., Proc. Natl. Acad. Sci. USA. 84:6629,
1987; Hirano et al., Nature. 324:73, 1986; Namen et al.,
Nature. 333:571, 1988; Taniguchi et al., Nature.
302:305, 1983; Yang et al., Cell. 47:3, 1986; Yokota et
al., Proc. Natl. Acad. S-i. USA. 83:5894, 1986; Yoshimura
et al., Proc. Natl. Acad. Sci. USA. 84:9233, 1987), IFN-~
(Gray et al., Nature. 295:503, 1982), GM-CSF (Wong et
al., Science. 228:810, 1985), TNF (Cuturi et al., J. Exp.
Med. 165:1581, 1987), LT (Cuturi et al., J. Exp. Med.
165:1581, 1987) and others have been identified, purified
and the genes encoding them have subsequently been molecu-
larly cloned. The cloned genes have proven to be immense-
ly useful for the generation of pure recombinant proteins
which can be tested for biological functions in the
absence of other secreted products. In this way, it has
been possible to assign functions unambiguously to indi-
vidual proteins.
Of the numerous lymphokines that have been de-
scribed, each has its own unique spectrum of biological
properties. Although many lymphokines are being developed
for clinical utility, most lymphokines act on such complex
biological systems as to effect a part of the desired
result. Thus, new lymphokines not only present the
potential for entirely new spectrums of biological
activities, but also could act synergistically with
previously described products to effect the overall
efficacy of the treatment.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present
invention to provide a newly identified lymphokine.
In one embodiment, the present invention relates
to a DNA segment encoding protein 154.
In another embodiment, the present invention
relates to protein 154 substantially free of proteins with
which it is normally associated.
In a further embodiment, the present invention
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relates to a recombinantly produced or chemically synthe-
sized protein having all, or a unique portion of the amino
acid sequence given in Figure 2.
In another embodiment, the present invention
relates to a recombinant DNA construct and to host cells
transformed therewith. The DNA construct comprises a DNA
segment of the present invention encoding protein 154 and
a vector.
In a further embodiment, th~ present invention
relates to a method of producing recombinant protein 154
which comprises culturing host cells of the present
invention, in a manner allowing expression of protein 154
encoded therein and isolating the protein.
In yet another embodiment, the present invention
relates to an antibody specific for protein 154.
BRIEF DESCRIPTION OF THE DRAWINGS
Various other objects and advantages of the
- present invention will become apparent from the following
description of the invention and the figures wherein:
Figures lA, lB and lC show the nucleotide sequence
of the pAT154 cDNA.
Figure 2 shows the putative amino acid sequence of
J the longest open reading frame of the pAT154 cDNA. The
classical hydrophobic leader region contained in the
protein is underlined.
Figure 3 shows the hydrophobicity plot of the
protein 154.
Figure 4 demonstrates in vitro transcription of a
full-length cDNA clone followed by in vitro translation
and SDS polyacrylamide gel electrophoresis (SDS PAGE).
The first two lanes are low molecular weight and high
molecular weiyht standards, respectively. The arrow
points to the 154 translated protein with a molecular
weight of approximately 15 kd.
DETAILED DESCRIPTION OF THE INVENTION
One approach to characterizing additional biologi-
cal activities in supernatants of activated T cells, which
activities may be mediated by unidentified proteins, is to
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WO92/O~K6 PCT/US91/05749-
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directly identify genes that are expressed in the acti-
vated T cells, the protein products of which have struc-
tural features suggesting they are secretory peptides.
With this approach, the present inventors have isolated
more than 60 genes that are induced in human peripheral
blood T cells by mitogens (Zipfel et al., Mol. Cell. Biol.
9:1034-1040, 1989). Of these clones, the inventors have
fully characterized the 154 gene encoding a lymphokine.
Accordingly, the present invention relates to a
DNA segment encoding all, or a unique portion, of protein
154 (lymphokine 154) and to the protein (or polypeptides)
encoded therein, or allelic variations thereof. The
present invention relates, in particular, to the human
protein 154 and to the equivalent protein in other spe-
cies. For example, the mouse protein 154 which has been
cloned and partially sequenced by the present inventors.
A "unique portion" as used herein is defined as consisting
of at least five (or six) amino acids, or correspondingly,
at least 15 (or 18) nucleotides).
The present invention relates to a DNA segment
that encodes the entire amino acid sequence given in
Figure 2 (such as, for example, the DNA segment pAT154
given in Figures lA, lB and lC), or any unique portion
thereof. DNA segments to which the invention relates also
include those encoding substantially the same protein as
that of Figure 2, which includes, for example, allelic
forms thereof or the mouse lymphokine 154. DNA segments
of the present invention can be used as probes for detect-
ing the presence of DNA or RNA encoding lymphokine 154 in
a sample.
The present invention also relates to nucleotide
sequences complementary to the segments referenced above.
The present invention further relates to protein
154 substantially free of proteins with which it is
normally associated. The lymphokine 154 can be purified
by one skilled in the art using protocols known in the art
for protein purification. The present invention also
relates to the lymphokine 154 and to peptide fragments
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~v()s2/o~66 2 0 ~ 9 2 5 ~ PCT/US91/0~749
thereof which are chemically synthesized or recombinantly
produced.
The protein and polypeptides the present invention
can be used as antigens, in protocols known in the art, to
produce antibodies thereto, both monoclonal and poly-
clonal. For example, a polypeptide having an amino acid
sequence corresponding to an epitope of the sequence given
in Figure 2 (or an allelic variation thereo~), can be used
to generate antibodies specific for lymphokine 154. Such
antibodies can be used in bioassays to detect the presence
of protein 154 in samples, such as biological samples.
The present invention further relates to a recom-
binant DNA construct and to a host cell transformed
therewith. Using standard methodology known in the art, a
recombinant DNA construct comprising the above-described
DNA segment encoding all, or a unique portion of
lymphokine 154 and a vector can be constructed without
undue experimentation. Suitahle vectors for use in the
present invention include, but are not limited to, the
bacterial vector pET-3 (Studier et al., Methods in
Enzymology 185: in press, 1990), the yeast vector YRpRl
(Rymond et al., Gene 25: 249, 1983~, and the eukaryotic
vector pBC12-CMV (Cullen, Cell 46: 973, 1986). The DNA
segment can be isolated for activated T-cells or take the
form of a cDNA clone. The DNA segment can be present in
the vector operably linked to regulatory elements, includ-
ing, for example a promoter. The host cell can be pro-
karyotic (such as bacterial), lower eukaryotic (such as
fungal, including yeast) or higher eukaryotic (such as
mammalian).
Host cells of the present invention can be used to
generate -:ecombinantly produced lymphokine 154. The host
cells can be cultured under conditions such that protein
154 (or a unique polypeptide thereof) encoded in the
construct is expressed. The expressed protein can be
isolated using standard protein isolation protocols.
The present invention also relates to a membrane
bound form of the lymphokine 154 and host cells expressing
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WO9~10~h6 2 ~ ~ 9 ~ 6 - PCT/US91/05740
such on their surfaces. As one skilled in the art will
appreciate, the membrane form of protein 154 can be
generated with the addition of a transmembrane anchor
encoding sequence on the DNA segment encoding the protein.
Furthermore, host cells transformed with a recombinant DNA
construct encoding such a DNA segment will express protein
154 on their surface.
Lymphokines provide a wide array of activities
relating to the immune system. Lymphokine activities
lo range from recruiting cells, such as macrophages, to
specific sites to mount an effective immune response, to
fighting viral infections. Lymphokine 154 of the present
invention may provide such activities and therefore could
be used in various treatment regimens.
For example, the protein or polypeptide of the
present invention may exert cytotoxic effects on certain
cells such as tumor cells and could therefore be used in
the treatment of cancer. Further, the protein or polypep-
tide of the present invention may have anti-viral and/or
anti-parasitic activity (such as the activity of the
lymphokine ~-interferon), in which case the present
invention could be used in the treatment of viral or
parasitic infections. Lymphokine 154 may also effect the
growth and/or differentiation of cells, particularly,
hematopoietic cells (an activity displayed by several of
the interleukin lymphokines). Such activity would be
useful in the treatment of diseases which adversely-affect
and/or deplete cells of the immune system. In addition,
lymphokine 154 may have the ability to augment the immune
system which would allow the protein or polypeptides of
the present invention to be used, for example, to detect
rejection of a transplanted organ by monitoring the
presence of the protein or polypeptide in the blood or
urine of the transplant patient. An increase in the
amount of protein or polypeptide in the biological fluid
of the patient could be an early indication that the
immune system is rejecting the foreign organ.
The following non-limiting examples are provided
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to aid in the understanding of the present invention and
do not restrict the scope of the invention.
EXAMPLES
CLONING.
CDNA clones of pAT154 were originally isolated
from a previously described subtractive hybridization
library (Zipfel et al., Mol. Cell. Biol. 9:1041-1048,
1989). Additional clones were isolated from a AZap
library (Stratagene~ (Short et al., Nuc. Acids. Res.
16:7583-7600, 1988), or from an oligo dT primed )~gtlO
library made following the Guebler-Hoffman method (Gubler
et al., Gene. 25:263, 1983).
Briefly, human peripheral blood T cells were
isolated from whole blood and stimulated for 4.5 hours in
vitro using the mitogenic agents phytohemmagglutin (PHA)
(1 ~g/ml) and phorbol 12-myristate 13-acetate (PMA) (20
ng/ml) in the presence of cycloheximide (CX) (10 ,ug/ml).
cDNA synthesized fr3m the mRNA was cloned into ~gtl0 or
)~Zap and screened using a 453 bp probe of pATl54 original-
ly isolated from the subtractive hybridization library. A
total of seven clones covering the length of the mRNA were
isolated, mapped and sequenced.
SEQUENCE ANALYSIS
The nucleic acid sequence was determined on both
strands of pAT154 cDNA's by the Sanger dideoxy chain
termination method (Sanger et al., Pro. Natl. Acad. Sci.
USA. 77:5463-5467, 1977) (See Figures lA, lB and lC).
Two micrograms of pBlueScript (Stratagene) containing 154
cDNA inserts were alkaline denatured in a final concentra-
tion of 0.2N NaOH for 5 min. at room temperature. The DNA
was neutralized with 5M NH40Ac and ethanol precipitated in
3 volumes of -70C ethanol and placed on dry ice for 10
min. The DNA was pelleted and resuspended. Sequencing
reactions were performed according to manufacturer's
instructions using the USB Sequenase II Kit. Elongation
and termination reactions were carried out in the presence
of 10% dimethyl sulfoxide ~DMSO) (Winship, P.R., Nuc.
Acid. Res. 17:1266, 1989). Sequencing reactions were
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WO92/0~W66 2 0 ~ 9 ~ ~ ~ PCT/US91/0574~-
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heated above 80C for 4 min and electrophoresed on 8%
denaturing polyacrylamide gels containing 8M urea at a
constant 65 W. The nucleic acid sequence was determined
after exposure against autoradiographic film. Storage and
analysis of the nucleic acid sequence was performed using
the computer package NicroGenie (Beckman) (Queen et al.,
Nuc. Acids. Res. 12:581-599, 1984). The putative amino
acid sequence was determined (see Figure 2) and shown to
contain a classical hydrophobic leader region but no
transmembrane anchor indicating that the pAT154 protein is
secreted (see Figure 3).
COMPUTER AIDED SEARCH ANALYSIS~
The computer package GenBank (V.63 + updates
through 5/90) was utilized to search the nucleic acid and
protein databanks for statistically relevant homologies in
the published literature to the pA~154 cDNA and amino acid
sequences. No sequences having ~ignificant homology with
pAT154 were found.
IN VITRO TRANSCRIPTION OF 154 OPEN READING FRAME.
A total of 10 ~g pAT154 cDNA containing the 154
open reading frame was linearized by a Cla I digestion of
the pBlueScript's Multicloning site. The final digest was
adjusted to 0.5 M NaCl and Proteinase K (1 mg/ml) treated
for 30 min. at 37C. The cDNA was treated twice with 1:1
phenol/chloroform and ethanol precipitated and dried. The
resulting pellet was resuspended at a final concentration
of 0.5 ~g/~l using diethyl pyrocarbonate treated H2O. In
vitro transcription of the 154 cDNA utilizing T3RNA
polymerase (Stratagene) was performed according to manu-
facturer's instructions. A 1 ~1 aliquot of the mRNA was
qualitatively tested by electrophoresis of a lxTBE 1%
agarose gel for 10 min.
IN VITRO TRANSLATION OF 154 PROTEIN.
The 154 mRNA was translated utilizing a rabbit
reticulolysate system (Promega). Five microliters of 154
mRNA (representing 20% of the total mRNA transcribed) was
mixed with rabbit reticulolysate, amino acids (minus
cysteine) and cysteine, L-[3sS] (NEN) according to the
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~9Z/0~66 PCT/US91/05749
g
Promega protocol. This mixture was incubated for 60 min.
at 30C. Free label was removed from the final translate
by dialysis (EmproTech Prodialyzer) against 0.1 M NH4HC03
overnight at 4C. Samples were lyophilized and resus-
pended in a 50 ~l volume of H20 containing 20% 5x sample
buffer and were boiled for 5 min. Cysteine, L-[35S]
incorporation was analyzed by electrophoresing translates
on a 12.5~ polyacrylamide gel at llOV constant and expos-
ing the gel against autoradiographic film (see Figure 4).
All publications mentioned hereinabove are hereby
incorporated by reference.
The foregoing invention has been described in
detail for purposes of clarity and understanding. Howev-
er, it will be appreciated that those skilled in the art,
upon consideration of this disclosure, may make modifica-
tions and improvements without departing from the true
scope of this invention.
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