U.S. patent application number 09/294121 was filed with the patent office on 2002-06-06 for new polypeptides and peptides, nucleic acids coding for them, and their use in the field of tumor therapy, inflammation or immunology.
Invention is credited to DEVOS, KATHLEEN, FRANSEN, LUCIA, VAN DE VOORDE, ANDRE, VAN HEUVERSWYN, HUGO.
Application Number | 20020069422 09/294121 |
Document ID | / |
Family ID | 8211656 |
Filed Date | 2002-06-06 |
United States Patent
Application |
20020069422 |
Kind Code |
A1 |
FRANSEN, LUCIA ; et
al. |
June 6, 2002 |
NEW POLYPEPTIDES AND PEPTIDES, NUCLEIC ACIDS CODING FOR THEM, AND
THEIR USE IN THE FIELD OF TUMOR THERAPY, INFLAMMATION OR
IMMUNOLOGY
Abstract
The invention relates: to a polypeptide containing in its
peptidic chain the amino acid sequence of 311 amino acids of FIG.
3, or a fragment of this sequence, with said fragment being such
that it is liable to produce antibodies capable of forming a
complex with the amino acid sequence of FIG. 3, or an amino acid
sequence having a percentage of homology of at least 50%,
preferably 75%, and advantageously 90% with the amino acid sequence
of FIG. 3, and to pharmaceutical compositions containing, as active
substance, at least one of the polypeptides of the invention or of
the antagonists of the polypeptides of the invention as antitumor
compounds, or antiinflammatory compounds or as growth activators of
T-cells and B-cells, as bone repair compounds as inducer of
immunosuppressive cells, as inhibitors of anti-colony stimulating
factor; or as trypoanocidal agents; or part of the polypeptides of
the invention, capable of binding to the above-defined
receptor.
Inventors: |
FRANSEN, LUCIA;
(NAZARETH-EKE, BE) ; DEVOS, KATHLEEN;
(DESTELBERGEN, BE) ; VAN DE VOORDE, ANDRE;
(LOKEREN, BE) ; VAN HEUVERSWYN, HUGO; (KALKEN,
BE) |
Correspondence
Address: |
CHARLES A MUSERLIAN
C/O BIERMAN MUSERLIAN AND LUCAS
600 THIRD AVENUE
NEW YORK
NY
10016
|
Family ID: |
8211656 |
Appl. No.: |
09/294121 |
Filed: |
April 19, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09294121 |
Apr 19, 1999 |
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08318837 |
Oct 13, 1994 |
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5981277 |
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Current U.S.
Class: |
800/8 ;
435/320.1; 435/325; 435/455; 800/14; 800/3 |
Current CPC
Class: |
C07K 2319/75 20130101;
C07K 14/52 20130101; A61P 35/00 20180101; A01K 2217/05 20130101;
A61P 7/00 20180101; C07K 2319/91 20130101; A61K 38/00 20130101;
C07K 2319/00 20130101; A61P 43/00 20180101; A61P 37/00 20180101;
A61P 33/02 20180101; C07K 14/525 20130101; A61P 29/00 20180101 |
Class at
Publication: |
800/8 ; 800/3;
800/14; 435/325; 435/320.1; 435/455 |
International
Class: |
A01K 067/027; A01K
067/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 30, 1992 |
EP |
92-401,231.3 |
Claims
1. Polypeptide containing in its peptidic chain the amino acid
sequence of 311 amino acids of FIG. 3, or a fragment of this
sequence, with said fragment being such that it is liable to
produce antibodies capable of forming a complex with the amino acid
sequence of FIG. 3, or an amino acid sequence having a percentage
of homology of at least 50%, preferably 75%, and advantageously 90%
with the amino acid sequence of FIG. 3, or a sequence liable to
form a complex with antibodies raised against the amino acid
sequence of FIG. 3 or against pep1(m) or against pep2(m) or against
pep3(m).
2. Polypeptide according to claim 1 containing in its peptidic
chain the amino acid sequence of 311 amino acids of FIG. 2, or a
fragment of this sequence, with said fragment being such that it is
liable to produce antibodies capable of forming a complex with the
amino acid sequence of FIG. 2 or an amino acid sequence having a
percentage of homology of at least 50%, preferably 75%, and
advantageously 90% with the amino acid sequence of FIG. 2, or a
sequence liable to form a complex with antibodies raised against
the amino acid sequence of FIG. 2 or against pep1(h) or against
pep2(h) or against pep3(h).
3. Polypeptide according to claim 1, characterized by the fact that
it is constituted by the sequence represented in FIG. 3, extending
from the extremity constituted by amino acid at position (1) to the
extremity constituted by amino acid at position (311), or that it
contains at least one of the following peptides:
Cys-Ser-Trp-Lys-Gly-Ser-Gly-Leu-Thr
Val-Glu-Trp-Met-Tyr-Pro-Thr-Gly-Ala-Leu-Ile-Val-Asn-Leu-Arg-Pro-Asn-Thr-P-
he-Ser-Pro-Ala
Asp-Ser-Ser-Gly-Ala-Asn-Ile-Tyr-Leu-Glu-Lys-Thr-Gly-Glu-Leu-
-Arg-Leu-Leu-Val
Leu-Glu-Glii-Gly-Gly-Leu-Phe-Val-Glu-Ala-Thr-Pro-Gln-Gln-- Asp-Ile
Arg-Arg-Thr-Thr-Gly-Phe-Gln-Tyr-Glu-Leu Leu-Ser-Ala-Pro-Cys-Arg-Pr-
o-Cys-Ser-Asp-Thr-Glu-Val-Leu-Leu-Ala Arg-Gln-Lys-Ser-Arg-Val-Phe
Cys-Gly-Val-Arg-Pro-Gly-His- Gly Phe-Leu-Phe-Thr-Gly-His
Leu-Gly-Cys-Ala-Pro-Arg-Phe Asp-Phe-Gln-Arg-Met-Tyr-Arg
4. Polypeptide according to claim 2, characterized by the fact that
it is constituted by the sequence represented in FIG. 2, extending
from the extremity constituted by amino acid at position (1) to the
extremity constituted by amino acid at position (311).
5. Muteins deriving from anyone of the polypeptides of claims 1 to
4, containing modifications consisting of substitutions and/or
deletions and/or additions of one or several amino acids, insofar
that said modifications do not alter the hydropathicity profile
such as defined by Kyte and Doolitle (1982) and such as represented
in FIG. 6a and 6b.
6. Polypeptide according to any one of claims 1 to 5 presenting at
least one of the following properties: promoting the incorporation
of .sup.3H-thymidine in rat femur pre- and osteoblasts cells, in
3-week-old mice thymocytes, in splenic cells or lymph node cells,
advantageously upon co-stimulation with IFN-.gamma., promoting the
incorporation of .sup.3H-thymidine in thymocytes, advantageously
upon co-stimulation with IL4, promoting the activation or
cytotoxicity or mobility of LAK cells. promoting the recruitment of
suppressive peritoneal exudate cells upon injection in vivo,
promoting the generation of immunocompetent lymph node cells,
preferantially after ConA, PHA or LPS induction, upon in vivo
intrafootpath injection, exerting a trypanocidal or trypanolytical
activity on the pleomorph bloodstream trypanosomes in vitro.
7. Amino acid sequence constituted by a polypeptide according to
anyone of claims 1 to 6, and a protein or a heterologous sequence
with respect to said polypeptide, with said protein or heterologous
sequence comprising, for instance, from about 10 to about 100 amino
acids.
8. Nucleic acid characterized by the fact that it comprises or is
constituted by: a nucleotide sequence which is effectively
homologous with any of the nucleotide sequence coding for the
polypeptides according to any one of claims 1 to 6, a nucleotide
sequence liable to hybridize with anyone of the nucleotide sequence
coding for the polypeptides according to anyone of claims 1 to 6,
or a nucleotide sequence which, further to translation or further
to transcription and to translation, leads to anyone of the
polypetide according to claims 1 to 6, or the complementary
sequences of the above-mentioned nucleotide sequences.
9. Nucleic acid according to claim 8, which comprises or is
constituted by: a nucleotide sequence which is effectively
homologous with any of the nucleotide sequences of FIG. 1, a
nucleotide sequence liable to hybridize with the complementary
strand of the nucleotide sequence of FIG. 1, the nucleotide
sequence of FIG. 1, the complementary sequences of the
above-mentioned sequences.
10. Nucleic acid according to claim 8, which comprises or is
constituted by: a nucleotide sequence which is effectively
homologous with any of the nucleotide sequences of FIG. 2, a
nucleotide sequence liable to hybridize with the complementary
strand of the nucleotide sequence of FIG. 2, the nucleotide
sequence of FIG. 2, the complementary sequences of the
above-mentioned sequences.
11. Nucleic acid according to claim 8, which comprises or is
constituted by: a nucleotide sequence which is effectively
homologous with any of the nucleotide sequences of FIG. 3, a
nucleotide sequence liable to hybridize with the complementary
strand of the nucleotide sequence of FIG. 3, the nucleotide
sequence of FIG. 3, the complementary sequences of the
above-mentioned sequences.
12. Recombinant nucleic acid containing at least one of the
nucleotide sequences of anyone of claims 8 to 11 combined with or
inserted in a heterologous nucleic acid.
13. Recombinant vector particularly for cloning and/or expression,
comprising a vector sequence, notably of the type plasmid, cosmid,
phage, or virus DNA and a recombinant nucleic acid according to
anyone of claims 8 to 12, inserted in one of the nonessential sites
for its replication.
14. Recombinant vector according to claim 13, containing necessary
elements to promote the expression in a cellular host of
polypeptides coded by nucleic acids according to anyone of claims 8
to 12, inserted in said vector and notably a promoter recognized by
the RNA polymerase of the cellular host, particularly an inducible
promoter and possibly a sequence coding for transcription,
termination, and possibly a signal sequence and/or an anchoring
sequence.
15. Recombinant vector according to claim 13, containing the
elements enabling the expression of a nucleotide sequence according
to anyone of claims 8 to 11, as a mature protein or as part of a
fusion protein, the fusion moiety which is used in the fusion
protein being part of a nonhomologous protein (such as mTNF) chosen
to optimize the expression of a fusion protein.
16. Cellular host chosen from among bacteria such as E. coli or
chosen from among eukaryotic organisms, such as COS cells,
baculovirus or vaccinia virus, which is transformed by a
recombinant vector according to anyone of claims 13 to 15 and
containing the regulatory elements enabling the expression of the
nucleotide sequence coding for the polypeptide according to anyone
of claims 1 to 7 in this host.
17. Expression product of a nucleic acid expressed by a transformed
cellular host according to claim 16.
18. Antibody characterized by being specifically directed against a
polypeptide according to anyone of claim 1 to 7.
19. Nucleotidic probe, hybridizing with any of the nucleic
sequences according to anyone of claims 8 to 11.
20. Process for preparing a recombinant polypeptide according to
anyone of claims 1 to 7, comprising the following steps: the
culture in an appropriate medium of a cellular host which has
previously been transformed by an appropriate vector containing a
nucleic acid according to anyone of claims 8 to 11, and the
recovery of the polypeptide produced by the above-said transformed
cellular host from the above-said culture medium or from the
cellular host.
21. Process for detecting the capacity of a molecule to behave as a
ligand or as a receptor with respect to a polypeptide according to
anyone of claims 1 to 6, characterized by: contacting the molecule
with a cellular host which has previously been transformed by a
vector itself modified by an insert coding for said polypeptide,
this host carrying on its surface one or several specific sites of
this polypeptide, possibly after induction of the expresion of this
insert, with said contacting being carried out under conditions
enabling binding between at least one of these specific sites and
said molecule to be formed if it happens to present an affinity for
said polypeptide, detecting the possible formation of a complex of
the type ligand-polypeptide or receptor-polypeptide.
22. Immunogenic composition containing, as active substance, at
least one of the polypetides of FIG. 2, or anyone of the peptides
pep1(h), pep2(h) or pep3(h).
23. Pharmaceutical compositions containing, as active substance, at
least anyone of the polypeptides of claims 1 to 6 or of the
antagonists of the polypeptides above-defined as antitumor
compounds, or antiinflammatory compounds, as growth activators of
T-cells and B-cells, as bone repair compounds, as inducer of
immunosuppressive cells as inhibitors of anti-colony stimulating
factor; or as trypanocidal agents; or part of the polypeptides of
the invention, capable of binding to the receptor defined
above.
24. Antisense oligonucleotides or antisense mRNA derived from the
nucleotide sequences of anyone of claims 8 to 11.
25. Nonhuman mammalian transgenic animal which contains, in its
genome, a nucleic acid sequence according to anyone of claims 8 to
11, and which can be used to study the effects of pharmacological
compositions and to prepare different cell types from transgenic
animals which express the nucleotide sequence according to anyone
of claims 8 to 11 in a constitutive or inducible way.
26. "Knock-out" nonhuman mammalian transgenic animal in which the
natural gene, effectively homologous with any one of the nucleotide
sequences according to anyone of claims 8 to 11, is rendered
nonfunctional, for instance by homologous recombination, with said
animal being suitable for the study of the possible loss of
functions or the possible restoration effects caused by the
reintroduction into the animals of the polypeptides according to
anyone of claims 8 to 11.
Description
[0001] The invention relates to polypeptides and peptides,
particularly recombinant polypeptides, which can be useful in the
field of tumor therapy, inflammation or immunology.
[0002] The invention also relates to a process for preparing the
above-said polypeptides and peptides.
[0003] It also relates to nucleic acids coding for said
polypeptides and peptides.
[0004] Monocytes/macrophages are cells of great complexity
accomplishing a multitude of different functions related to (i)
responses to environmental challenges such as phagocytosis, antigen
processing and presentation, (ii) enzyme production, (iii) to
differentiation, (iv) to regulatory responses by the synthesis of
macrophage-specific cytokines which function as metabolic or
immunological regulatory proteins and (v) by the production of
complement components, coagulation factors, enzymes, enzyme
inhibitors, and oxygen radicals (reviewed by Adams and Hamilton
(1984).
[0005] Several macrophage-derived cytokines have already been
described: interleukin-1 (IL-1), tumor necrosis factor (TNF),
interleukin-6 (IL-6), colony stimulating factor (CSF), interferon
(IFN), macrophage inflammatory protein (MIP), and monocytic-derived
neutrophil chemotactic factor (MDNCF or IL-8) (Old, 1985; Durum et
al., 1986; Quesenberry, 1986; Billiau, 1987; Yoshimura et al.,
1987; Davatelis et al., 1988; Kishimoto and Hirano, 1988;). In most
instances, the production of these cytokines by the macrophages
requires exposure to one or more signals present in the immediate
microenvironment of the cells. These signals may consist of
particulate matter which can be opsonized, invading parasites,
bacterial infectants, or antibody-covered antigens (Unanue,
(1989)). They invariably lead to a state of enhanced competence of
the macrophage (termed activation), ultimately giving rise to the
synthesis of some of the above-mentioned macrophage-specific
cytokines (monokines).
[0006] A particular set of genes (some of which are specifically
expressed by macrophages, hereafter termed monokine genes) may
correspond to each individual activation process. Such genes can
either be up- or down-regulated. Characterization of some of these
genes is possible by measuring their corresponding biological
function with appropriate bioassays (Ruff and Gifford, 1986; Van
Snick et al, 1986; Van Damme et al., 1987), or using
differential
[0007] hybridization with cDNAs derived from either activated or
nonactivated macrophages. This can result in the isolation of cDNAs
that correspond to genes switched on during the process of
differentiation of resting macrophages to activated cells.
[0008] It is an object of the invention to provide new polypeptides
and their corresponding nucleic acids which can be used in
immunology or in the field of tumor therapy.
[0009] It is another object of the invention to provide nucleic
acids coding for the peptide chains of biologically pure, active
recombinant peptides which enable their preparation on a large
scale.
[0010] It is another aspect of the invention to provide nucleic
sequences which can be used as antisense oligonucleotides.
[0011] It is another aspect of the invention to provide a
chromosomal DNA fragment which can be used for producing a
pathological model of a nonhuman animal such as a transgenic animal
which can be used to study the effects of pharmacological
compositions and to prepare different cell types from these
transgenic animals which express the gene of the invention in a
constitutive or inducible way.
[0012] It is another aspect of the invention to provide "knock-out"
transgenic animals (Capecchi, 1989) in which the natural gene
effectively homologous to the nucleotide sequences of the invention
(definition of effectively homologous given hereafter) is rendered
nonfunctional, for instance, by homologous recombination, with said
animal being suitable for the study of the possible loss of
functions or the possible restoration effects caused by the
reintroduction of the polypeptides of the invention into the
animals.
[0013] The polypeptide of the invention is characterized by the
fact that it contains in its peptidic chain:
[0014] the amino acid sequence of 311 amino acids of FIG. 3,
[0015] or a fragment of this sequence, with said fragment being
such that it is liable to produce antibodies capable of forming a
complex with the amino acid sequence of FIG. 3,
[0016] or an amino acid sequence having a percentage of homology of
at least 50%, preferably 75%, and advantageously 90%, with the
amino acid sequence of FIG. 3,
[0017] or a sequence liable to form a complex with antibodies
raised
[0018] against the amino acid sequence of FIG. 3
[0019] or against pep1(m)
[0020] or against pep2(m)
[0021] or against pep3(m)
[0022] Pep1 (m) has at least 8 contiguous amino acids contained in
the following sequence:
[0023]
Cys.sup.52-Ser-Trp-Lys-Gly-Ser-Gly-Leu-Thr-Arg-Glu-Ala-Arg-Ser-Lys--
Glu-Val-Glu-Gln-Val-Tyr-Leu-Arg-Cys,
[0024] and preferably is the following sequence:
[0025] Arg-Glu-Ala-Arg-Ser-Lys-Glu-Val-Glu.
[0026] Pep2(m) has at least 8 contiguous amino acids contained in
the following sequence:
[0027]
Cys.sup.107-Ile-Lys-Pro-Phe-Arg-Asp-Ser-Ser-Gly-Ala-Asn-Ile-Tyr-Leu-
-Glu-Lys-Thr-Gly-Glu-Leu-Arg-Leu-Leu-Val-Arg-Asp-Ile-Arg-Gly-Glu-Pro-Gly-G-
ln-Val-Gln-Cys, and preferably is the following sequence:
[0028] Arg-Asp-Ile-Arg-Gly-Glu.
[0029] Pep3(m) has at least 8 contiguous amino acids contained in
the following sequence:
[0030]
Gly.sup.283-Cys-Ala-Pro-Arg-Phe-Ser-Asp-Phe-Gln-Arg-Met-Tyr-Arg-Lys-
-Ala-Glu-Glu-Met-Gly-Ile-Asn-Pro-Cys-Glu-Ile-Asn-Met-Glu,
[0031] and preferably is the following sequence:
[0032] Arg-Lys-Ala-Glu-Glu.
[0033] According to another advantageous embodiment of the
invention, the peptide contains in its peptidic chain:
[0034] the amino acid sequence of 311 amino acids of FIG. 2,
[0035] or a fragment of this sequence, with said fragment being
such that it is liable to produce antibodies capable of forming a
complex with the amino acid sequence of FIG. 2,
[0036] or an amino acid sequence having a percentage of homology of
at least 50%, preferably 75%, and advantageously 90%, with the
amino acid sequence of FIG. 2,
[0037] or a sequence liable to form a complex with antibodies
raised:
[0038] against the amino acid sequence of FIG. 2
[0039] or against pep1(h)
[0040] or against pep2(h)
[0041] or against pep3(h).
[0042] Pep1 (h) has at least 8 contiguous amino acids contained in
the following sequence:
[0043]
Cys.sup.52-Ser-Trp-Lys-Gly-Ser-Gly-Leu-Thr-His-Glu-Ala-His-Arg-Lys--
Glu-Val-Glu-Gln-Val-Tyr-Leu-Arg-Cys,
[0044] and preferably is the following sequence:
[0045] Arg-Lys-Glu-Val-Glu.
[0046] Pep2(h) has at least 8 contiguous amino acids contained in
the following sequence:
[0047]
Cys.sup.201-Tlir-Ser-Asp-Phe-Ala-Val-Arg-Gly-Ser-Ile-Gln-Gln-Val-Th-
r-His-Glu-Pro-Glu-Arg-Gln-Asp-Ser-Ala-Ile-His-Leu-Arg-Val-Ser-Arg,
[0048] and preferably is the following sequence:
[0049] Glu-Pro-Glu-Arg-Gln-Asp.
[0050] Pep3(h) has at least 8 contiguous amino acids contained in
the following sequence:
[0051]
Gly.sup.283-Cys-Ala-Pro-Arg-Phe-Lys-Asp-Phe-Gln-Arg-Met-Tyr-Arg-Asp-
-Ala-Gln-Glu-Arg-Gly-Leu-Asn-Pro-Cys-Glu-Val-Gly-Thr-Asp,
[0052] and preferably is the following sequence:
[0053] Arg-Asp-Ala-Gln-Glu-Arg.
[0054] An advantageous polypeptide of the invention is
characterized by the fact that it is constituted by the sequence
represented on FIG. 3, extending from the extremity constituted by
amino acid at position (1) to the extremity constituted by amino
acid at position (311) or that it contains at least one of the
following peptides:
[0055] Cys-Ser-Trp-Lys-Gly-Ser-Gly-Leu-Thr
[0056]
Val-Glu-Trp-Met-Tyr-Pro-Thr-Gly-Ala-Leu-Ile-Val-Asn-Leu-Arg-Pro-Asn-
-Thr-Phe-Ser-Pro-Ala
[0057]
Asp-Ser-Ser-Gly-Ala-Asn-Ile-Tyr-Leu-Glu-Lys-Thr-Gly-Glu-Leu-Arg-Leu-
-Leu-Val
[0058]
Leu-Glu-Gln-Gly-Gly-Leu-Phe-Val-Glu-Ala-Thr-Pro-Gln-Gln-Asp-Ile
[0059] Arg-Arg-Thr-Thr-Gly-Phe-Gln-Tyr-Glu-Leu
[0060]
Leu-Ser-Ala-Pro-Cys-Arg-Pro-Cys-Ser-Asp-Thr-Glu-Val-Leu-Leu-Ala
[0061] Arg-Gln-Lys-Ser-Arg-Val-Phe
[0062] Cys-Gly-Val-Arg-Pro-Gly-His-Gly
[0063] Phe-Leu-Phe-Thr-Gly-His
[0064] Leu-Gly-Cys-Ala-Pro-Arg-Phe
[0065] Asp-Phe-Gln-Arg-Met-Tyr-Arg
[0066] An advantageous polypeptide of the invention is constituted
by the sequence represented on FIG. 2, extending from the extremity
constituted by amino acid at position (1) to the extremity
constituted by amino acid at position (311).
[0067] The invention also relates to the muteins deriving from
anyone of the above-defined polypeptides and containing
modifications consisting of substitution, and/or deletion and/or
addition of one or several amino acids, insofar that said
modifications do not alter the hydropathicity profile as defined in
Kyte and Doolittle (1982) and such as is represented in FIGS. 6a
and 6b.
[0068] The above-mentioned substitution is carried out by replacing
one or more amino acids by their synonymous amino acids. Synonymous
amino acids within a group are defined as amino acids which have
sufficient physicochemical properties to allow substitution between
members of a group in order to preserve the biological function of
the molecule. Synonymous amino acids are those preferably listed in
Table I.
1 Amino acids Synonymous groups Ser Ser, Thr, Gly, Asn Arg Arg,
His, Lys, Glu, Gln Leu Leu, Ile, Met, Phe, Val, Tyr Pro Pro, Ala,
Thr, Gly Thr Thr, Pro, Ser, Ala, Gly, His, Gln Ala Ala, Pro, Gly,
Thr Val Val, Met, Ile, Tyr, Phe, Leu, Val Gly Gly, Ala, Thr, Pro,
Ser Ile Ile, Met, Leu, Phe, Val, Ile, Tyr Phe Phe, Met, Tyr, Ile,
Leu, Trp, Val Tyr Tyr, Phe, Trp, Met, Ile, Val, Leu Cys Cys, Ser,
Thr His His, Gln, Arg, Lys, Glu, Thr Gln Gln, Glu, His, Lys, Asn,
Thr, Arg Asn Asn, Asp, Ser, Gln Lys Lys, Arg, Glu, Gln, His Asp
Asp, Asn, Glu Glu Glu, Gln, Asp, Lys, Asn, His, Arg Met Met, Ile,
Leu, Phe, Val
[0069] As to deletions or insertions of amino acids, they may also
be introduced into the defined sequences provided they do not alter
the biological functions of said sequences. Preferentially such
insertions or deletions should be limited to a few amino acids and
should not remove or physically disturb or displace amino acids
which are critical to the functional conformation.
[0070] Muteins of the proteins of the invention are proteins having
a sequence homologous to the sequence disclosed in the invention in
which amino acid substitutions, deletions, or insertions are
present at one or more amino acid positions. Said muteins may have
a biological activity which is at least 10% of the polypeptides of
the invention and which can be higher than the biological activity
of the invention, and thus do not necessarily have to be identical
to the biological function of the proteins of the disclosure.
[0071] In another embodiment of the invention, muteins derived from
the protein sequences of the invention or polypeptides derived from
said proteins may be used to block the biological function of the
proteins of the invention. Preferably, such an embodiment is
composed of polypeptides minimally containing about 7 amino acids
and maximally having about 100 amino acids.
[0072] Another preferred embodiment contains polypeptides or
muteins of the protein of the invention which comprise more than
about 100 amino acids of a sequence contained in any one of the
polypeptide sequences of the invention.
[0073] The proteins of the invention have interesting biological
functions.
[0074] The term biological function means that the proteins, the
muteins, and the polypeptides either provoke the proliferation of
the target cell line, as can be measured by different methods such
as the incorporation of .sup.3H- thymidine, direct cell counting or
3-(4,5-dimethylthiazolyl-2- )-2,5-diphenyltetrazolium bromide
(MTT)-staining (Mossman, 1983), or provoke alterations in the
differentiation state of the target cell line as can be measured by
the changes in cell membrane marker distribution or the modulation
of the biological activity of the target cell, or provoke
mobilization or chemotaxis of the target cell line as can be
measured by counting the cells migrating through microporous
membranes.
[0075] According to another embodiment of the invention, the
above-defined peptides have at least one of the following
properties:
[0076] promoting the incorporation of .sup.3H-thymidine in rat
femur preosteoblast and osteoblast cells, in 3-week-old mice
thymocytes, in splenic cells or lymph node cells advantageously
upon costimulation with IFN-.gamma.,
[0077] promoting the incorporation of .sup.3H-thymidine in
thymocytes, advantageously upon costimulation with IL4,
[0078] promoting the activation, cytotoxicity or mobility of LAK
cells,
[0079] promoting the recruitment of suppressive peritoneal exudate
cells upon injection in vivo,
[0080] promoting the generation of immunocompetent lymph node
cells, preferentially after ConA, PHA or LPS induction, upon in
vivo intrafootpath injection,
[0081] exerting a trypanocidal or trypanolytical activity on the
pleomorph bloodstream trypanosomes in vitro.
[0082] Promoting the incorporation of .sup.3H-thymidine in rat
femur osteoblast cells corresponds to an increased proliferation of
the cells and can be carried out according to the technique as
described in the examples. Promoting the incorporation of
.sup.3H-thymidine in 3-week-old thymocytes, advantageously upon
costimulation with IL4, corresponds to an enhanced cell
proliferation and can be carried out as described in the
examples.
[0083] Promoting the incorporation of .sup.3H-thymidine in splenic
cells or lymph node cells, advantageously upon costimulation with
IFN-.gamma., corresponds to an enhanced cell proliferation and can
be carried out as described in the examples. Promoting the mobility
of LAK cells can be carried out as described in the examples.
[0084] Promoting the recruitment of supressive peritoneal exudate
cells, promoting the generation of immunocompetent lymph node
cells, and exerting a trypanocidal activity can be measured as
described in the examples. As to the activity or cytotoxicty of LAK
cells, the activity of the polypeptides of the invention can be
shown by the use of anti-sense primers derived from the nucleotides
of the invention which are capable of reducing or blocking the
activation or cytotoxicity of LAK cells, or the IL-2-generated
expression of the polypeptides of the invention. It should be clear
that the addition of IL-2 in order to promote the activation or the
cytotoxicity of LAK cells is accompanied by the induction of the
mRNA of the polypeptides of the invention.
[0085] The protein of the invention induces the uptake of
.sup.3H-thymidine upon its addition to thymocytes in the presence
of a lectin (PHA or ConA or others), with such uptake being
enhanced by coincubation with cytokines (IL4, IL-2, IL-1, IL-6 or
combinations of these). In another preferred embodiment, the
composition of the invention induces the uptake of
.sup.3H-thymidine when added to splenic cells and lymph node cell
populations in the presence of a lectin, with such uptake being
enhanced by coincubation with IFN-.gamma.. In another preferred
embodiment, the composition of the invention induces the
differentiation of splenic cells into LAK cells when added together
with IL-2.
[0086] It is to be noted that the above-mentioned polypeptides are
derived from the expression products from the nucleotide sequence
coding for a protein of 30 or 34 kDa present in the culture fluids
of human and mouse macrophages, respectively, in the human
monocytic cell lines U-937 (ATCC 1593), and in the cell line Mono
Mac 6 (Ziegler-Heitbrock) or in the mouse cell line PU5-1.8. (ATCC
TIB61).
[0087] The invention also relates to the purified natural mammalian
proteins, muteins thereof, and polypeptides derived from them.
"Purified" corresponds to the proteins obtained according to the
process as specified in the examples.
[0088] The invention also relates to the amino acid sequences
constituted by the above-mentioned polypeptides and a protein or a
heterologous sequence with respect to said polypeptide, with said
protein or heterologous sequence comprising, for instance, anywhere
from about 10 to about 100 amino acids.
[0089] The invention also relates to the nucleic acid sequences
containing or constituted by:
[0090] a nucleotide sequence which is effectively homologous with
the nucleotide sequences coding for the above-defined
polypeptides,
[0091] a nucleotide sequence liable to hybridize with anyone of the
nucleotide sequence coding for the above-defined polypeptides,
[0092] or a nucleotide sequence which, further to translation or
further to transcription and to translation, leads to anyone of the
above-defined polypeptides,
[0093] or the complementary sequences of the above-mentioned
nucleotide sequences.
[0094] An "effectively homologous" nucleotide sequence derived from
the sequence of the invention is at least fifty percent homologous
to the sequence to be isolated. More preferably, the effectively
homologous nucleotide sequence is at least seventy-five percent
homologous to the sequence to be isolated. Most preferably, the
effectively homologous nucleotide sequence is at least ninety
percent homologous to the sequence to be isolated. Homology, as
used herein, is a measure of similarity between nucleotides or
amino acids and can be expressed as the fraction of nucleotides or
amino acids in the nucleotide sequence that are identical to the
sequence to be isolated.
[0095] An advantageous nucleic acid of the invention comprises or
is constituted by:
[0096] a nucleotide sequence which is effectively homologous with
the nucleotide sequence of FIG. 1,
[0097] a nucleotide sequence liable to hybridize with the
complementary strand of the nucleotide sequence of FIG. 1,
[0098] the nucleotide sequence of FIG. 1,
[0099] the complementary sequences of the above-mentioned
sequences
[0100] the above-mentioned sequences wherein T is replaced by
U.
[0101] Another advantageous nucleic acid of the invention comprises
or is constituted by:
[0102] a nucleotide sequence which is effectively homologous with
the nucleotide sequence of FIG. 2,
[0103] a nucleotide sequence liable to hybridize with the
complementary strand of the nucleotide sequence of FIG. 2,
[0104] the nucleotide sequence of FIG. 2,
[0105] the complementary sequences of the above-mentioned
sequences,
[0106] the above-mentioned sequences wherein T is replaced by
U.
[0107] Another advantageous nucleic acid of the invention comprises
or is constituted by:
[0108] a nucleotide sequence which is effectively homologous with
the nucleotide sequence of FIG. 3,
[0109] a nucleotide sequence liable to hybridize with the
complementary strand of the nucleotide sequences of FIG. 3,
[0110] the nucleotide sequence of FIG. 3,
[0111] the complementary sequences of the above-mentioned
sequences,
[0112] the above-mentioned sequences wherein T is replaced by
U.
[0113] Appropriate hybridization conditions between human cDNA and
mouse cDNA are the following ones:
[0114] hybridization temperature: 42.degree. C.,
[0115] hybridization medium: 47% deionized formamide, 10% dextrane
sulfate, 3 x SSPE (3.6 M NaC1, 0.2 M NaH.sub.2PO.sub.4, 0.02 M
EDTA, pH 7.4), 1% SDS, 0.5% milk powder,
[0116] wash temperature: 50.degree. C.
[0117] wash medium: 1 x SSC, 0.1% SDS.
[0118] The invention also relates to a recombinant nucleic acid
containing at least one of the above-mentioned nucleic acids
combined with or inserted in a heterologous nucleic acid.
[0119] The invention also relates to a recombinant vector
particularly for cloning and/or expression, comprising a vector
sequence, notably of the type plasmid, cosmid, phage, or virus DNA
and a recombinant nucleic acid as mentioned above, inserted in one
of the nonessential sites for its replication.
[0120] The invention also relates to a recombinant vector as
defined above and containing necessary elements to promote the
expression in a cellular host of polypeptides coded by nucleic
acids of the invention, inserted in said vector and notably a
promoter recognized by the RNA polymerase of the cellular host,
particularly an inducible promoter, and possibly a sequence coding
for transcription, termination and possibly a signal sequence
and/or an anchoring sequence.
[0121] The invention also relates to a recombinant vector as
defined above, containing the elements enabling the expression of a
nucleotide sequence coding for the polypeptide of the invention as
a mature protein or as part of a fusion protein; the fusion moiety
which is used in the fusion protein is a part of a nonhomologous
protein (such as mTNF) chosen to optimize the expression of the
fusion protein.
[0122] The sequence of mTNF in pmTNF is described in FIGS. 8a and
8b.
[0123] The invention also relates to a cellular host chosen from
among bacteria such as E. coli or chosen from among eukaryotic
organisms, such as COS1 cells, which is transformed by a
recombinant vector defined above and containing the regulatary
elements enabling the expression of the nucleotide sequence coding
for the polypeptide of the invention in this host.
[0124] The invention also relates to viral vectors such as vaccinia
virus or baculovirus, in which a recombinant nucleic acid is
inserted in a nonessential site for virus replication, with said
viral vectors being capable of infecting various eukaryotic cells
or cell lines, resulting in the production of biologically active
recombinant polypeptides of invention.
[0125] The invention also relates to an expression product of a
nucleic acid expressed by a transformed cellular host as defined
above.
[0126] The invention also relates to antibodies themselves formed
against the polypeptides according to the invention.
[0127] It goes without saying that this production is not limited
to polyclonal antibodies.
[0128] It also relates to any monoclonal antibody produced by any
hybridoma liable to be formed according to classical methods from
animal splenic cells, particularly from a mouse or rat, the cells
of the animal being immunized against the purified polypeptide of
the invention on the one hand, and of cells from a myeloma cell
line on the other, and to be selected by the ability of the cell
line to produce the monoclonal antibodies recognizing the
polypeptide which has been initially used for the immunization of
the animals.
[0129] The invention also relates to any antibody of the invention
labeled by an appropriate label of the enzymatic, fluorescent, or
radioactive type.
[0130] The peptides which are advantageously used to produce
antibodies, particularly monoclonal antibodies, are the
following:
[0131]
Cys.sup.52-Ser-Trp-Lys-Gly-Ser-Gly-Leu-Thr-Arg-Glu-Ala-Arg-Ser-Lys--
Glu-Val-Glu-Gln-Val-Tyr-Leu-Arg-Cys,
[0132] Arg-Glu-Ala-Arg-Ser-Lys-Glu-Val-Glu,
[0133]
Cys.sup.107-Ile-Lys-Pro-Phe-Arg-Asp-Ser-Ser-Gly-Ala-Asn-Ile-Tyr-Leu-
-Glu-Lys-Tlr-Gly-Glu-Leu-Arg-Leu-Leu-Val-Arg-Asp-Ile-Arg-Gly-Glu-Pro-Gly-G-
ln-Val-Gln-Cys,
[0134] Arg-Asp-Ile-Arg-Gly-Glu,
[0135]
Gly.sup.283-Cys-Ala-Pro-Arg-Phe-Ser-Asp-Phe-Gln-Arg-Met-Tyr-Arg-Lys-
-Ala-Glu-Glu-Met-Gly-Ile-Asn-Pro-Cys-Glu-Ile-Asn-Met-Glu,
[0136] Arg-Lys-Ala-Glu-Glu,
[0137]
Cys.sup.52-Ser-Trp-Lys-Gly-Ser-Gly-Leu-Thr-His-Glu-Ala-His-Arg-Lys--
Glu-Val-Glu-Gln-Val-Tyr-Leu-Arg-Cys,
[0138] Arg-Lys-Glu-Val-Glu,
[0139]
Cys.sup.201-Thr-Ser-Asp-Phe-Ala-Val-Arg-Gly-Ser-Ile-Gln-Gln-Val-Thr-
-His-Glu-Pro-Glu-Arg-Gln-Asp-Ser-Ala-Ile-His-Leu-Arg-Val-Ser-Arg,
[0140] Glu-Pro-Glu-Arg-Gln-Asp,
[0141]
Gly.sup.283-Cys-Ala-Pro-Arg-Phe-Lys-Asp-Phe-Gln-Arg-Met-Tyr-Arg-Asp-
-Ala-Gln-Glu-Arg-Gly-Leu-Asn-Pro-Cys-Glu-Val-Gly-Thr-Asp
[0142] Arg-Asp-Ala-Gln-Glu-Arg
[0143] From the nucleic acids of the invention, probes (i.e. cloned
or synthetic oligonucleotides) can be inferred.
[0144] These probes can be from 15 to the maximum number of
nucleotides of the selected nucleic acids. The oligonucleotides can
also be used either as amplification primers in the PCR technique
(Mullis and Faloona, 1987) to generate specific enzymatically
amplified fragments and/or as probes to detect fragments amplified
between bracketing oligonucleotide primers.
[0145] The specificity of a PCR-assisted hybridization assay can be
controlled at different levels.
[0146] The amplification process or the detection process or both
can be specific. The latter case, giving the higher specificity, is
preferred. Examples of primers are the following:
2 For mouse: 1) sense primer: 1 antisense primer: 2 denaturation T:
95.degree. C. annealing T: 56.degree. C. elongation T: 72.degree.
C. 2) sense primer: 3 antisense primer: 4 denaturation T:
95.degree. C. annealing T: 60.degree. C. elongation T: 72.degree.
C. For man: 1) sense primer: 5 antisense primer: 6 denaturation T:
95.degree. C. annealing T: 64.degree. C. elongation T: 72.degree.
C. 2) sense primer: 7 antisense primer: 8 denaturation T:
95.degree. C. annealing T: 50.degree. C. elongation T: 72.degree.
C.
[0147] The invention also relates to a process for preparing a
polypeptide according to the invention comprising the following
steps:
[0148] the culture in an appropriate medium of a cellular host
which has previously been transformed by an appropriate vector
containing a nucleic acid according to the invention,
[0149] the recovery of the polypetide produced by the above-said
transforrned cellular host from the above-said culture, and
[0150] the purification of the polypeptide produced.
[0151] In case of a fusion protein in which the fusion moiety is,
for instance, part of mTNF, purification can be achieved by
immunoaffinity chromatography.
[0152] If the fusion protein contains in addition to the fusion
moiety a stretch of at least 2 histidines, purification can be
achieved by immobilized metal affinity chromatography (IMAC) as
detailed in the examples.
[0153] In a particular case, the fusion protein is composed of a
polypeptide which, in the host (eukaryotic or prokaryotic) used for
the expression of the protein, acts as a natural signal sequence
for the expression of the polypeptides of the invention in the
culture medium. In a particular embodiment, this signal sequence
can be the naturally occurring signal sequence as present in the
cDNA sequences of the invention. Purification can be achieved by
applying a Mg.sup.++/dextrane sulphate precipitation followed by
sequential liquid chromatography steps, including hydrophobic
interaction chromatography such as phenyl sepharose fast flow
chromatography (Pharmacia), ion exchange chromatography such as
Mono-Q Sepharose (Pharmacia), glycoprotein binding matrices such as
Phenylboronate agarose (Amicon) and gelfiltration such as Superdex
75 (Pharmacia) or TSK100 (Merck).
[0154] Purification of the natural protein or muteins thereof can
be achieved by using sequential liquid chromatography steps as
detailed above.
[0155] The polypeptides of the invention can be prepared according
to the classical techniques in the field of peptide synthesis.
[0156] The synthesis can be carried out in homogeneous solution or
in solid phase.
[0157] For instance, the synthesis technique in homogeneous
solution which can be used is the one described by Houbenweyl
(1974).
[0158] The polypeptides of the invention can also be prepared in
solid phase according to the methods described by Atherton and
Sheppard (1989).
[0159] The invention also relates to a process for preparing the
nucleic acids according to the invention.
[0160] A suitable method for chemically preparing the
single-stranded nucleic acids (containing at most 100 nucleotides
of the invention) comprises the following steps:
[0161] DNA synthesis using the automatic .beta.-cyanoethyl
phosphoramidite method described in Bioorganic Chemistry 4; 274-325
(1986).
[0162] In the case of single-stranded DNA, the material which is
obtained at the end of the DNA synthesis can be used as such.
[0163] A suitable method for chemically preparing the
double-stranded nucleic acids (containing at most 100 bp of the
invention) comprises the following steps:
[0164] DNA synthesis of one sense oligonucleotide using the
automatic .beta.-cyanoethyl phosphoramidite method described in
Bioorganic Chemistry 4; 274-325 (1986), and DNA synthesis of one
antisense oligonucleotide using either the above-mentioned
automatic .beta.-cyanoethyl phosphoramidite method, or enzymatic
transcription of the sense-strand using a specific primer
hybridizing to the 3'-end of the sense strand,
[0165] combining the sense and antisense oligonucleotide by
hybridization in order to form a DNA duplex,
[0166] cloning the DNA duplex obtained into a suitable plasmid
vector and recovery of the DNA according to classical methods such
as restriction enzyme digestion and agarose electrophoresis, or by
PCR amplification according to the procedure outlined above.
[0167] A method for the chemical preparation of nucleic acids with
lengths greater than 100 nucleotides--or base pairs, in the case of
double-stranded nucleic acids--comprises the following steps:
[0168] assembling the synthesized oligonucleotides, provided at
their ends with different restriction sites, the sequences of which
are compatible with the succession of amino acids in the natural
peptide, according to the principle described Urdea et al.,
(1983),
[0169] cloning the DNA thereby obtained into a suitable plasmid
vector and recovery of the desired nucleic acid according to
classical methods such as restriction enzyme digestion and agarose
gel electrophoresis.
[0170] The purified natural mammalian proteins are preferentially
extracted from the culture fluid of human and mouse macrophages and
from the culture fluid from human monocytic cells and cell lines
such as U937 (ATCC 1593) and Mono Mac 6 (Ziegler-Heitbrock) and
from mouse monocytic cell lines such as PU5-1.8. (ATCC TIB61).
[0171] As derived from said culture fluids, the human protein has a
mobility on SDS-polyacrylamide gels corresponding to a molecular
weight of 30 kDa under reduced conditions and of 27 kDa under
nonreduced conditions; the mouse protein has a molecular weight of
34 kDa under reduced conditions and of 30 kDa under nonreduced
conditions, and may be present as proteins carrying
post-translational secondary modification such as glycosylation,
phosphorylation (but not limited to these).
[0172] The invention also relates to the process of purification of
the natural proteins, the recombinant proteins, the muteins thereof
and polypeptides derived from them as specified in the
examples.
[0173] Said compositions can be used for the treatment of mammalian
cells in vitro as shown in the examples.
[0174] The MRNA derived from the cDNAs of the invention can be
found in several mammalian cell lines including, but not limited
to:
[0175] PU5-1.8 (ATCC TIB61), L-929 (ATCC CCL1), NIH 3T3 (ATCC
CRL
[0176] 1658), U-937 (ATCC 1593), Mono Mac 6 (Ziegler-Heitbrock, see
above) or CTLL (ATCC TIB 214).
[0177] The said mRNA can also be found in mammalian cell lines
after application of external stimuli including, but not limited
to: lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate
diester (C.sub.36H.sub.56O.sub.8)(PMA), retinoic acid
(C.sub.20H.sub.28O.sub.2)(R- A) and IL-2. Preferentially, said mRNA
can be detected by Northern blotting (Fourney et al., 1988) or by
the polymerase chain reaction (Saiki et al., 1985) in Mono Mac 6
cells either stimulated or nonstimulated by LPS and in mouse
natural killer (NK) cells derived from mouse spleen after treatment
with IL-2 as specified in the examples.
[0178] The invention also relates to an antibody characterized as
being specifically directed against a polypeptide according to the
invention.
[0179] The invention also relates to polypeptides containing
epitopes which can be used to raise monoclonal or polyclonal
antibodies. Said polypeptides are composed of a string of amino
acids having a sequence homologous to or synonymous with the
disclosed sequence. Preferentially, said epitopes contain minimally
8 amino acids. A preferred embodiment of the invention contains the
sequence (hu clone 5 peptide I, peptide II, or peptide III). It
should be understood that said epitope-containing polypeptides can
be used to generate antibodies capable of interfering with the
blocking of the biological function of the proteins, the muteins
thereof, and the polypeptides derived from them. The polypeptides
may be used themselves or in combination with the antibodies in the
diagnosis of the polypeptides or the antibodies. Either or both may
be labelled or unlabelled for use in diagnostic assays. A large
number of such assays are described in the literature and include
the binding, either directly or indirectly, of these polypeptides
or antibodies to a variety of labels including, but not limited to,
enzymes, radionucleides, fluorescers, chemiluminescers, coenzymes,
particles, or the like. The antibodies to these polypeptides (AB1)
may be used themselves as antigens to produce anti-idiotypes (AB2)
which may serve as competitive antigens having epitopic sites
competitive with the epitopic sites of these polypeptides. These
anti-idiotypes AB2 may therefore be used as substitutes for the
polypeptides or as antagonists to these polypeptides. These AB2
anti-idiotypes may themselves be used as antigens to produce
anti-anti-idiotypes (AB3) to these polypeptides which may serve as
substitutes for AB1, having complementarity-determining regions
competitive with the complementarity-deterinining regions of
AB1.
[0180] The invention also relates to the use of the proteins of the
invention, muteins thereof or peptides derived from them for the
selection of recombinant antibodies by the process of repertoire
cloning (Perrson et al., 1991).
[0181] The invention relates to nucleotidic probes, hybridizing
with any of the above-defined nucleic acid sequences.
[0182] Preferred oligonucleotide probes of the cDNAs of the
invention are the following:
[0183] Human probes:
[0184] probe 1: 5'-TTCACGGACTCCTCGGGGGCCAATA-3'
[0185] probe 2: 5'-TGGCCTGGAGCAGGGCGGCCTGTTC-3'
[0186] probe 3: 5'-ACAGGCTTCCAGTACGAGCTGGTTA-3'
[0187] Mouse probes:
[0188] probe 1: 5'-GGGCTCACCCGAGAGGCACGCAGCA-3'
[0189] probe 2: 5'-ATCAAGCCTTTCAGGGACTCCTCTG-3'
[0190] probe 3: 5'-AACAGGCTTCACAGGCAGAAGAGCA-3'
[0191] By way of example and not intended to be limiting, a typical
protocol for the hybridization of these nucleotidic probes with any
of the above-defined nucleic sequences bound to a solid support
(e.g. a nitrocellulose membrane) is described below.
[0192] The membranes were prehybridized in a mixture containing the
following components: 3.times.SSC (1.times.SSC is 0.15 M NaCl plus
0.015 M sodium citrate, pH 7.0), 25 mM sodium phosphate buffer (pH
7.1), 20% (v/v) deionized formamide, 0.02% Ficoll (type 400,
Sigma), 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, 0.1
mg sheared heat-denatured salmon sperm DNA ml.sup.-1, and 0.2% SDS,
usually for 0.5-1 h at the appropriate temperature. The
hybridization mixture had the same composition except that
approximately 106 c.p.m. of .sup.32P-labelled probe ml.sup.-1 was
added. Hybridizations were performed at the same temperature for
1-2 h. The membranes were washed for 30. min in 3.times.SSC, 25 mM
sodium phosphate buffer (pH 7.1), 20% (v/v) deionized formamide,
0.2% SDS at the hybridization temperature.
[0193] The optimal hybridization and wash temperatures are:
[0194] human probe 1: 57.degree. C.
[0195] human probe 2: 62.degree. C.
[0196] human probe 3: 54.degree. C.
[0197] mouse probe 1: 62.degree. C.
[0198] mouse probe 2: 54.degree. C.
[0199] mouse probe 3: 54.degree. C.
[0200] The invention relates to a process for detecting the
capacity of a molecule to behave as a ligand or as a receptor with
respect to a polypeptide of the invention, characterized by:
[0201] contacting the molecule with a cellular host which has
previously been transformed by a vector itself modified by an
insert coding for said polypeptide, this host carrying on its
surface one or several specific sites of this polypeptide, possibly
after induction of the expression of this insert, with said
contacting being carried out under conditions enabling a binding to
occur between at least one of these specific sites and said
molecule if it happens to present an affinity for said
polypeptide,
[0202] detecting the possible formation of a complex of the type
ligand-polypeptide or receptor-polypeptide.
[0203] The invention also relates to immunogenic compositions
containing, as active substance, at least one of the polypeptides
of FIG. 2, or anyone of the peptides pep1(h), pep2(h), or
pep3(h).
[0204] The invention also relates to pharmaceutical compositions
containing, as active substance, at least one of the polypeptides
of the invention or of the antagonists of the polypeptides of the
invention as antitumor compounds, as anti-inflammatory compounds,
as growth activators of T-cells or B-cells, as bone repair
compounds as inducer of immunosupressive cells, as inhibitors of
anti-colony stimulating factor, or as trypanocidal agents; or part
of the polypeptides of the invention, capable of binding to the
above-defined receptor.
[0205] Said compositions can be used for the treatment of mammalian
cells in vitro as detailed in the examples.
[0206] More particularly, the polypeptides of the invention and the
AB1, AB2 and AB3 antibodies will find utility in various ways
either as diagnostic reagents or as therapeutic agents, especially
in the field of tumor therapy, macrophage activation and
deactivation, T-cell ontogenicity, osteoblast proliferation and
proliferation inhibition, LAK cell mobilization, generation and
cytotoxicity, T- and B-cell growth and anti-colony stimulating
activity, immnuno-supressive activity and trypanocidal
activity.
[0207] The invention also relates to the process in which the
proteins of the invention, their muteins, or polypeptides derived
from them are used for the isolation and characterization of
cellular receptors or binding molecules which are capable of
forming a complex with said compound. As stated herein, a receptor
is characterized by its localization on the cell membrane, its
ability to bind to the compounds of the invention, and its ability
to produce signal transduction upon binding, thereby leading to an
altered state of the cell on which said receptor is present.
Binding molecules as referred to herein are those molecules which
are capable of interacting with the compounds of the invention in
such a way that this interaction is stable under physiological
circumstances. Preferentially such molecules are capable of forming
said complexes between temperatures of 0.degree. C. and 45.degree.
C., between pH 2 or 11 or at ionic strengths not higher than those
of a 2 M NaCl solution.
[0208] In case AB1 antibodies are capable of neutralizing the
biological functions of the composition of the invention, the AB3
antibodies may contain the internal image of the naturally
occurring receptors for the composition of the invention. AB3
antibodies may therefore be used in diagnostic assays for the
measurement of receptor amounts and as antagonist to these
receptors. For the same reason AB2 antibodies can be used as an
agonist of these receptors.
[0209] The invention also relates to antisense oligonucleotides or
antisense mRNA derived from the nucleotide sequences of the
invention.
[0210] Such antisense oligonucleotides may be introduced into cells
and cell lines expressing the nucleotide sequence of the invention
by methods known to those skilled in the art. Antisense mRNA of
which the sequence can be deduced from the sequences of the
invention can also be expressed in cells and cell lines by methods
known to those skilled in the art. In doing so, these antisense
oligonucleotides or antisense mRNA may interfere with the
translation of the sequence of the invention thereby effectively
blocking the biological role of these expressed mRNAs. Preferably
these anti-sense oligonucleotides can be introduced into cells or
cell lines according to methods known by the man skilled in the art
such as those found in Wickstrom et al. (1988).
[0211] Said antisense oligonucleotides contain preferentially a
sequence of 8 or more nucleotides having sequences effectively
homologous to the sequence of the disclosure. In a preferred
embodiment, an antisense oligonucleotide of the sequence
(5'-CACCGCACCCCGCAT-3' reverse complement of the 5' to 3' mouse
sequence from position 187 to 201) is used.
[0212] These antisense oligonucleotides can also be introduced into
cells or cell lines by transfection of a plasmid in which the gene
encoding the protein is in the opposite orientation with respect to
the promoter (Izant and Weintraub (1984, 1985).
[0213] The invention also relates to nonhuman mammalian transgenic
animals which contain, in their genomes, a nucleic acid sequence of
the invention, and which can be used to study the effects of
pharmacological compositions and to prepare different cell types
from these transgenic animals which express the gene of the
invention in a constitutive or inducible way.
[0214] More particularly, a transgenic nonhuman animal can be
prepared according to the protocol described by Gordon (1989).
[0215] Transgenic animals can be prepared by transformation of
suitably adapted polynucleotide sequences derived from the
invention in embryonic stem cells. In a preferred embodiment, the
embryonic stem cells belong to the mouse embryonic stem cell line
ES (Wagner et al., 1985).
[0216] Said sequences can also be introduced by direct injection
into fertilized oocytes. The methods for adaptation of said
nucleotides sequences to make them capable of transformation or for
injection are known by those skilled in the art (Gordon, 1989).
[0217] A variant transgenic animal is a "knock-out" animal prepared
according to Capecchi (1989).
[0218] More particularly, "knock-out" nonhuman mammalian transgenic
animals are such that the natural gene (effectively homogenous with
the nucleotide sequences of the invention) is rendered
nonfunctional, for instance by homologous recombination, with said
animal being suitable for the study of the possible loss of
functions or the possible restoration effects caused by the
reintroduction into the animals of the polypeptides of the
invention.
DESCRIPTION OF THE FIGURES
[0219] FIG. 1 represents the human genomic sequence of the cDNA of
the invention.
[0220] Its characteristics are the following:
[0221] SEQUENCE TYPE: nucleotide with corresponding protein
[0222] SEQUENCE LENGTH: 3741 base pairs
[0223] STRANDNESS: single
[0224] TOPOLOGY: linear
[0225] ORIGINAL SOURCE: human
[0226] IMMEDIATE EXPERIMENTAL SOURCE: spleen tissue from healthy
adult
[0227] FEATURES: from 1980 to 2188 bp intron 1 (only partially
sequenced: estimated length.+-.5400 bp)
[0228] from 2575 to 2766 bp intron 2 (only partially sequenced:
estimated length.+-.7900 bp)
[0229] from 2827 to 2875 bp intron 3 (only partially sequenced:
estimated length.+-.1000 bp)
[0230] S: G or C
[0231] M: A or C
[0232] R: A or C
[0233] K: Tor G
[0234] Y: T or C
[0235] (X): either present or absent.
[0236] FIG. 2 represents the nucleotide sequence of the human cDNA
homologous to the mouse cDNA sequence of the invention.
[0237] Its characteristics are the following:
[0238] SEQUENCE TYPE: nucleotide sequence with corresponding
protein
[0239] SEQUENCE LENGTH: 1487 base pairs
[0240] STRANDNESS: single
[0241] TOPOLOGY: linear
[0242] MOLECULE TYPE: copy DNA
[0243] ORIGINAL SOURCE
[0244] ORGANISM: human
[0245] IMMEDIATE EXPERIMENTAL SOURCE
[0246] NAME OF THE CELL LINE: THP-1
[0247] FEATURES:
[0248] from 1 to 5 bp: 5' non-coding region
[0249] from 6 to 140 bp: signal sequence as predicted by Von Hejine
G. Nucl. Acids Res. (1986) 14:4683.
[0250] from 141 to 938 bp: mature peptide
[0251] from 939 to 1487 bp: 3' non-coding region.
[0252] FIG. 3 represents the nucleotide sequence of the mouse cDNA
of the invention.
[0253] Its characteristics are the following:
[0254] SEQUENCE TYPE: nucleotide with corresponding protein
[0255] SEQUENCE LENGTH: 1362
[0256] STRANDNESS: single
[0257] TOPOLOGY: linear
[0258] MOLECULE TYPE: copy DNA
[0259] ORIGINAL SOURCE
[0260] ORGANISM: mouse
[0261] IMMEDIATE EXPERIMENTAL SOURCE
[0262] NAME OF CELL LINE: PU5-1.8.
[0263] FEATURES:
[0264] from 1 to 186 bp: 5' non-coding region
[0265] from 187 to 321 bp: signal sequence as predicted by Von
Heijne G. NAR (1986) 14:4683.
[0266] from 322 to 1119 bp: mature peptide
[0267] from 1120 to 1362 bp: 3' non-coding region.
[0268] FIG. 4 represents the sucrose gradient fractionation of
LPS-induced PU5-1.8. cells. mRNA of PU5-1.8. cells which is
prepared using the Nonidet-P40 lysis method followed by a
poly-A.sup.+ purification over oligo-dT as described in section
1.2. 400 .mu.g of poly-A.sup.+-RNA were further fractionated on a
5-20% sucrose gradient prepared in 10 mM Tris-HCl, pH 7.5, 1 mM
EDTA. Gradients were run in a SW40 rotor for 19 hours at 40,000 rpm
in a Beckman ultracentrifuge at 4.degree. C. After centrifugation,
0.4 ml fractions were collected and each fraction was assayed for
the presence of mTNF MRNA by injection of 50 nl of each fraction in
15 oocytes of Xenopus laevis in 200 .mu.l of incubation medium.
After 24 hours, the oocyte incubation medium was assayed for the
presence of biologically active mTNF by incubating 100 .mu.l with
4-5.times.10.sup.4 L-929 cells in the presence of 1 .mu.g/ml of
actinomycin D essentially as described by Ruff and Gifford (1983).
The fractions 16, 17 and 18, containing the maximal TNF activity
and containing the 17S mRNA population were pooled and used for the
preparation of the 17S LPS-induced PU5-1.8. cDNA library.
[0269] The x-axis corresponds to the numbers of fractions, the left
y-axis corresponds to the optical density of 260 nm and the right
y-axis corresponds to the TNF toxicity.
[0270] FIG. 5 represents the alignment of the human and mouse amino
acid sequence encoded by the open reading frame from nucleotide
position 6 to 938 on the human and position 187 to 1061 on the
mouse cDNA sequence presented in FIGS. 2 and 3, respectively. Both
sequences share 77.4% homology. The ten CYS residues conserved
between human and mouse are boxed as well as the computer-predicted
antigenic peptides. The synthetic oligopeptides used to raise
antibodies are underlined.
[0271] FIGS. 6a and FIG. 6b represent the respective hydropathicity
profile of the human and mouse amino acid sequences of the
invention as depicted in FIGS. 2 and 3.
[0272] FIG. 7 represents the Northern blot analysis of mRNA of
different uninduced and LPS-induced cell lines to determine the
degree of macrophage specificity and LPS inducibility of the
selected LPS-induced cDNA clone of mouse PU5-1.8 cells.
Poly-A.sup.+ RNA of the different cell lines here analyzed was
prepared using the Nonidet-P40 lysis method followed by
purification by column chromatography over oligo-dT essentially as
described by Fransen et al. (1985). niRNA was separated on a
denaturating formaldehyde gel as described by Gerard and Miller
(1986) (2.5 .mu.g poly-A.sup.+ RNA per lane) and blotted on a nylon
membrane (Hybond-N, Amersham) as described by Fourney et al.
(1988). The blot was screened by hybridization with the 900 bp
EcoRI-restriction fragment of the selected mouse cDNA clone
labelled to specific activity of 0.5-1.times.10.sup.9 cpms/.mu.g
using a multiprime labeling kit (Amersham, RPN 1600Y).
Prehybridization was for 2 hours at 42.degree. C. in
5.times.Denhardts (100.times.Denhardts: 20 g Ficoll, 20 g
polyvinylpyrrolidone and 20 g BSA (fraction V) per liter),
5.times.SSC (20.times.SSC: 3 M NaCl, 0.3 M Na acetate.2H20), 50 mM
Na Phosphate pH 7.0, 0.1% SDS, 250 .mu.g/ml salmon sperm DNA and
50% deionized formamide. Hybridization was performed for at least
two nights in the same buffer containing 1.times.10.sup.6 cpms/ml
of the labelled probe. Thereafter, the filter was washed two times
in 2.times.SSC, 0.1% SDS followed by two washes in 1.times.SSC,
0.1% SDS, each time for 15 minutes and at 50.degree. C.
Autoradiographical exposure was for two hours at room temperature.
Messenger RNA of the following cell lines are analyzed: mouse
monocytic PU5-1.8. either uninduced (3 h and 24 h: lanes 1 and 3)
or LPS-induced (3 h and 24 h: lanes 2 and 4); 24 hours uninduced
and LPS-induced mouse macrophage 2C11-12 cells (lanes 5 and 6); 24
hours uninduced and LPS-induced mouse T-lymphoma EL-4 (lanes 7 and
8); 24 hours uninduced and LPS-induced mouse B-myeloma NSo (lanes 9
and 10); and 24 hours uninduced and LPS-induced mouse fibrosarcoma
L-29 (lanes 11 and 12). LPS-induction was as described in section
1.1.
[0273] FIG. 8a is a schematic representation of the bacterial
expression plasmid pmTNF-MPH.
[0274] FIG. 8b represents the total DNA sequence of the bacterial
expression plasmid pmTNF-MPH.
[0275] FIG. 9 represents the SDS-PAGE gel analysis of E. Coli
strain transformed with the expression plasmid
pmTNF-MPH-PU1280-Eco47III at different times after
temperature-induced expression.
[0276] Lanes 1 to 5: pmTNF-MPH-PU1280-Eco47III in K12AH after 1 h,
2 h, 3 h, 4 h and 5 h induction at 42.degree. C.; lane 6:
pmTNF-MPH-PU1280-Eco47III in K12AH after 5 h induction at
28.degree. C.; lanes 7-8: pmTNF-MPH in K12AH after 5 h induction at
28.degree. C. and 42.degree. C., respectively.
[0277] A culture of K12AH harbouring pmTNF-MPH-PU1280-Eco47III,
grown overnight in Luria broth a 28.degree. C. with rigorous
shaking in the presence of 10 .mu.g/ml tetracycline, was inoculated
into fresh Luria broth containing tetracycline (10 .mu.g/ml) and
grown to an optical density at 600 nm of 0.2 under the same
conditions as for the overnight culture. At this density of
bacterial growth, half of the culture was shifted to 42.degree. C.
to induce expression, while the other half remained at 28.degree.
C. as a control. At several time intervals, aliquots were taken
which were extracted with one volume of phenol equilibrated against
M9 salts (0.1% ammonium chloride, 0.3% potassium dihydrogen
phosphate, 1.5% disodium hydrogen phosphate, 12 molecules of water)
and 1% SDS. At the same time the optical density at 600 nm of the
culture is measured. The proteins are precipitated from the phenol
phase by addition of two volumes of acetone and stored overnight at
-20.degree. C. The precipitate is pelleted (Biofuge A, 5 min, 13000
rpm, room temperature), air dried, dissolved in a volume of Laemmli
sample buffer (+.beta.-mercaptoethanol) according to the optical
density of the culture sample and boiled for 3 minutes. Samples
were then put on a SDS polyacrylamide gel (12.5%) according to
Laemnimli (1970). Afterwards the gel was first treated for at least
1 hour at 4.degree. C. with a 10% trichloroacetic acid solution and
subsequently immersed in a 1/10 diluted CBB-staining solution (0.5
g CBB-R250 (Serva) in 90 ml of methanol: H.sub.2O (1:1 v/v) and 10
ml glacial acetic acid) and left for about one hour on a gently
rotating platform. After destaining in 30% methanol--7% glacial
acetic acid (two to three washes of about 30 min each) the gel was
dried between two sheets of cellophane at room temperature.
[0278] FIG. 10 is a schematic representation of the expression
vector pSVL used for transient expression of the mouse and human
polypeptide of the invention in COS1 cells. Apart from prokaryotic
sequences (ORI of replication and AMP resistance gene), the vector
contains the SV40 origin of replication (SVORI) and part of the
SV40 late region: SV40 late promoter and enhancer (SV40L) sequence,
the 5' untranslated region (5UTR) followed by a multilinker
sequence, donor and acceptor splice sites of the late 16s MRNA
(INTRON) and the late SV40 polyadenylation site (poly A).
[0279] FIG. 11 is a 2-dimensional nonequilibrium pH gel
electrophoresis (2D-NEPHGE) (non-reducing conditions) and
fluorography of 5 ml conditioned medium of COS1 cells transfected
with the expression plasmid pSV-PU1280-HdIII (A) or the control
plasmid pSV (B) radiolabeled with .sup.35S-methionine (24 h) as
described. The .+-.30 kDa triple peptide spot corresponding to the
mouse polypeptide of the invention is indicated by an arrow.
[0280] FIG. 12 represents the separation on an aquapore butyl 7
.mu.column (Brownlee-10 cm.times.2.1 mm) of peptides generated by
partial formic acid hydrolysis (as described) of the mouse
polypeptide of the invention as secreted by
pSV-PU1280-HdIII-transfected COS1 cells. Peptides were eluted with
a linearly increasing gradient of 0.1% trifluoroacid (TFA) in
acetonitrile and detected by UV absorbance at 214 nm. Peptides
20/24/26 and 27 were selected for sequencing (Applied Biosystems
477 A).
[0281] FIG. 13 represents the 2-dimensional non-equilibrium pH gel
electrophoresis (2D-NEPHGE) (non-reducing conditions) and
fluorography of 5 ml CM of COS1 cells transfected with the
expression plasmid pSV-T1200 containing the human analogue of the
invention cDNA (A) or the control plasmid pSV (B), radiolabeled
with .sup.35S-methionine as described (24 h). The .+-.27-kDa
peptide spot corresponding to the human polypeptide of the
invention is indicated by an arrow.
[0282] FIG. 14 represents the SDS-polyacrylamide gel
electrophoresis (non-reducing conditions) and fluorography of
proteins secreted in 1 ml conditioned medium (CM) of Sf9 cells
(.+-.10.sup.6 cells) infected with either wild type baculovirus
(lane 1) or recombinant baculovirus containing the mouse cDNA of
the invention (lanes 2, 3, 4, 5) and radiolabeled with
.sup.35S-methionine for 18 h, 24 h post-infection as described.
[0283] The .+-.28 kDa protein corresponding to the mouse
polypeptide of the invention is indicated by an arrow.
[0284] FIG. 15 represents the 2D-NEPHGE (reducing conditions)
analysis and fluorography of 5 ml CM of HeLa cells infected with
recombinant vaccinia virus containing the mouse cDNA of the
invention (A) or wild type vaccinia virus (B), labeled with
.sup.35S-methionine for 24 h post- infection. The .+-.34 kDa
protein of the invention is indicated by an arrow.
[0285] FIG. 16 is a Western blot analysis with the anti-human
peptide 3 polyclonal antiserum, of 5 ml CM of HeLa cells infected
with recombinant vaccinia virus containing the human cDNA of the
invention (24 h harvest)(A) or wild type vaccinia virus separated
on 2D-NEPHGE (reducing conditions). The .+-.30 kDa protein of the
invention is indicated by an arrow.
[0286] FIG. 17 is the Western blot analysis with the
anti-mTNF-MPH-mouse cDNA fusion protein antiserum of proteins
secreted in 20 ml CM of LPS-induced (10 .mu.g/ml; 24 h) PU5-1.8
cells, separated on 2D-NEPHGE (reduced conditions). The mouse
polypeptide of the invention is indicated by an arrow. TNF which is
also recognized by the antiserum is also indicated.
[0287] FIG. 18 represents the immunoprecipitation of the native
form of the mouse polypeptide of the invention secreted by
transfected COS1 cells, with the anti-mTNF-MPH-mouse cDNA fusion
protein antiserum.
[0288] 850 .mu.l of .sup.35S-methionine-labeled CM of COS1 cells
transfected with pSV control plasmid (lane 1) or pSV-PU1280-HdIII
plasmid (lane 2) was immunoprecipitated as described and analyzed
by SDS-PAG-fluorography (lanes 3 and 4 correspond to pSV and
pSV-PU1280-HdIII, respectively). The immunoprecipitated mouse
polypeptide of the invention is indicated by an arrow.
[0289] FIG. 19 represents the characterization of the
N-glycosylation of the mouse polypeptide of the invention by
N-glycosidase F treatment:
[0290] 500 .mu.l CM of uninduced (A) or LPS (24 h, 10 .mu.g/ml)
induced PU5-1.8 cells (B), of wild type vaccinia virus infected (C)
or recombinant mouse cDNA vaccinia virus infected HeLa cells (D),
of recombinant mouse cDNA baculovirus-infected Sf9 cells (E) or
pSV-PU1280-HdIII transfected COS1 cells (F) was untreated (-) or
treated with N-glycosidase F (+) (as indicated by the manufacturer)
and analyzed by Western blotting with the anti-mTNF-MPH-mouse cDNA
fusion protein antiserum.
[0291] FIG. 20 represents the thymocyte proliferation assay as
described in section 12.1. performed in the presence of 2 .mu.g/ml
of PHA and a two-fold serial dilution of the mouse polypeptide of
the invention (rec prot: start concentration .+-.5-10 ng/ml) or
pSVL control medium. The proliferation was measured by the
incorporation of .sup.3H-thymidine for 24 hours following a 72-hour
incubation of the cells with the samples (see y-axis representing
the amount of CPM.times.1000). As negative control, conditioned
medium of pSVL-transfected COS1 cells, treated in exactly the same
way as the medium obtained from pSV-PU1280-HdIII-transfe- cted
cells or PBS, was tested in the presence of PHA.
[0292] For each group of three contiguous rectangles, the left
rectangle corresponds to the recombinant protein of the invention,
the middle rectangle corresponds to the negative control (PSVL) and
the right rectangle corresponds to the control. The x-axis
corresponds to the two-fold serial dilutions wherein the number 1
represents the start concentration of 5 to 10 ng/ml of the
polypeptide of the invention, 2 represents 2 times less, etc.
[0293] FIG. 21a and FIG. 21b represent the proliferative effect of
the mouse polypeptide of the invention respectively on rat
pre-osteoblast cells (FIG. 21a) and osteoblast cells (FIG. 21b).
The assay was performed as described in section 12.5. both on
preosteoblast and osteoblast cells by adding a two-fold serial
dilution of the mouse polypeptide of the invention (rec prot: start
concentration .+-.5 ng/ml) or pSVL (see FIG. 20) as negative
control. 5% of fetal calf serum (FCS) and 1% of bovine serum
albumin (BSA) were included as positive and negative assay
controls, respectively.
[0294] The y-axis represents the amount of CPM.times.1000 and the
x-axis corresponds to serial dilutions. For each group of two
contiguous histograms, the left one corresponds to the recombinant
protein of the invention, while the right one corresponds to pSVL;
the highest single histogram corresponds to FCS (5%) while the
lowest single one corresponds to BSA.
[0295] FIG. 22 represente the trypanocidal effect of the
polypeptide of the invention on Trypanosma brucei brucei in vitro.
The assay was performed as described in section 12.6 on
2.times.10.sup.6 parasites by adding a two-fold serial dilution of
the mouse polypeptide of the invention (recombinant protein start
concentration: 50 ng/ml) or PBS (negative control). The y-axis
represents the % of trypanocidal activity of living parasites and
the x-axis corresponds to serial dilutions.
EXAMPLES
[0296] 1. Preparation of Libraries
[0297] 1.1. Lipopolysaccharide (LPS)-induction of the Mouse
Macrophage Cell Line PU5-1.8
[0298] The established mouse monocyte/macrophage cell line PU5-1.8
(PU.5-1R) (purchased from the American Type Culture Collection,
Baltimore, Md., USA; ATCC TIB61) was chosen for lipopolysaccharide,
endotoxin (LPS)-induction. However, other mouse cell lines of the
monocyte-macrophage lineage (such as J-774, RAW309, WR19M, Wehi3B,
etc.) or primary macrophages (peritoneal macrophages, alveolar
macrophages) can also be used. Cells of the PU5-1.8 cell line were
cultured as spinner cultures in RPMI-1640 medium enriched with 10%
non-inactivated preselected batches of fetal calf serum (FCS,
Gibco, Paisley, Scotland). When reaching a density of 1 to
1.5.times.10.sup.6 cells/ml, cells were subcultured at a starting
density of 5.times.10.sup.5 cells/ml in roller bottles in the same
growth medium. At confluence (.+-.1.5.times.10.sup.6 cells/ml), the
cells were washed 3 times with RPMI-1640, resuspended at a cell
concentration of 3.5.times.10.sup.6 cells/ml in RPMI-1640, and
stimulated by addition of 10-15 .mu.g/ml of LPS (LPS E. coli 055:B5
Difco Laboratories, Detroit, Mich., USA) for 20-24 hours. After
induction the cells were collected by centrifugation, washed three
times with icecold phosphate-buffered saline (PBS) and stored at
-70.degree. C. until preparation of the mnRNA. Also, mRNA was
prepared from uninduced PU5-1.8 cells. To this end, cells were
treated as indicated for LPS-induced cells but without addition of
LPS during induction. The conditioned medium of untreated and
LPS-induced PU5-1.8 cells was tested for the presence of
TNF-.alpha., IL-1 and IL-6 using appropriate bioassays. TNF
activity was assayed on L-929 cells (Ruff and Gifford, 1980). IL-1
activity was measured using an indirect assay system (Van Damme et
al. 1987). IL-6 was measured in terms of hybridoma growth activity
(Van Snick et al., 1986)
[0299] 1.2. Preparation of LPS-minus and LPS-plus mRNA of PU5-1.8
Cells
[0300] As a source of mRNA, uninduced (LPS-minus) and LPS-induced
(LPS-plus) PU5-1.8 cells were used. Total cytoplasmic RNA was
extracted by lysing the cells in Nonidet P40 followed by phenol
extraction of the lysate as described (Fransen et al., 1985).
Polyadenylated RNA (poly A.sup.+-RNA) was purified from total RNA
by oligo dT- cellulose chromatography (Type 3; Collaborative
Research, Boston, MA, USA) as described by Chirgwin et al. (1979).
The resulting RNA was further fractionated on a 5-20% sucrose
gradient in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA by centrifugation at
40,000 rpm for 19 hours at 4.degree. C. 1.3. Construction of a cDNA
Library of a 17S-mRNA Fraction of LPS-induced PU5-1.8 Cells
[0301] The mRNAs from the 17S fraction of the gradient (the
fraction numbers 16, 17, 18 of the gradient as shown in FIG. 4) of
LPS-induced mRNA of PU5-1.8 cells were used for the construction of
the cDNA library. These fractions were selected on the basis of
their capacity to induce the synthesis of mTNF upon injection in
Xenopus laevis oocytes. The conditions used for the construction of
a cDNA library in pAT153 plasmids were chosen according to
established state-of-the-art methods. To this end, the RNA from the
17S fraction was precipitated by addition of 0.1 volume of 2 M Na
acetate pH 5.3 and 2 volumes of ethanol; the precipitate was
redissolved in water and the solution was heated for two minutes at
70.degree. C. and then quickly chilled on ice. The conditions for
the first-strand synthesis were as follows:
[0302] .+-.50 .mu.g poly A.sup.+ RNA/ml
[0303] 50 mM Tris HClI, pH 8.3
[0304] 50 mM KCl
[0305] 10 mM MgCl.sub.2
[0306] 10 mM DTT
[0307] 0.5 mM of each dNTP (N=A, T, C, or G) with 1/1000 dCTP
replaced by .alpha.(.sup.32P)-dCTP at 800 Ci/ummole (code PB 10385,
Amersham, Buckingshamshire, England)
[0308] 60 .mu.g/ml poly dTlo (Pharmacia, Uppsala, Sweden)
[0309] 1000 U/ml human placental RNase inhibitor (Amersham,
Buckingshamshire, England)
[0310] 1000 U/ml reverse transcriptase (Biores, Waerden, The
Netherlands)
[0311] The reaction was performed in a total volume of 100 .mu.l at
41.degree. C. for 1 hour.
[0312] The reaction mixture was then extracted once with
phenol/chloroform/isoamylalcohol (25/24/1), twice with diethyl
ether, and the DNA was precipitated by adding 1 volume of 4 M
ammonium acetate and 4 volumes of ethanol. The pellet was
redissolved in water and the precipitation step was repeated.
[0313] The precipitate was redissolved in 60 .mu.l 15 mM potassium
phosphate buffer, pH 6.9, 0.25 mM EDTA and treated with 2 .mu.g
RNAse A (Boehringer Mannheim, FRG) at 37.degree. C. for 30 minutes.
Subsequently, the mixture was boiled for 2 minutes and immediately
quenched on ice. Potassium phosphate buffer, pH 6.9, MgCl.sub.2,
DTT and dNTPs were added to final concentrations of 100 mM, 10 mM,
10 mM, and 1 mM, respectively. The reaction was initiated by
addition of 330 U/ml E. coli polymerase I (Boehringer Mannheim).
Second-strand synthesis was performed at 15.degree. C. for 6 hours
in a total volume of 300 .mu.l. The reaction was stopped by adding
EDTA, pH 8.0 to a final concentration of 25 mM and the mixture was
phenol-extracted and precipitated as described (see above). The
precipitate was redissolved in 125 mM NaCl, 25 mM sodium acetate, 1
mM zinc acetate, pH 4.5 and treated with 20 units of S1-nuclease
(BRL, Neu-Isenburg, FRG) for 20 minutes at 37.degree. C. The
reaction was stopped by the addition of EDTA pH 8.0 to a final
concentration of 20 mM, neutralized by the addition of Tris-HCl, pH
8.0 to a final concentration of 200 mM and again phenol-extracted
as mentioned above. Finally, the dsDNA was precipitated by addition
of 1/10 volume potassium acetate, pH 4.8 and 1 volume of
isopropanol.
[0314] The pellet was redissolved in buffer containing 30 mM NaCl,
10 mM Tris-HCl, 1 mM EDTA, pH 8.0 and size-fractionated on a Biogel
A 50m gel filtration column (0.8.times.12 cm) (Biorad, Calif., USA)
equilibrated against the same buffer. Fractions containing DNA of
>500 base pairs were pooled and precipitated as above.
[0315] The double-stranded cDNA was oligo(dC) tailed using the
following conditions:
[0316] .+-.2 .mu.g double-stranded cDNA/ml
[0317] 100 mM potassium cacodylate, pH 7.2
[0318] 2 mM CoCl.sub.2
[0319] 200 .mu.M DTT
[0320] 40 .mu.M deoxy (5-.sup.3H)cytidine triphosphate (17
Ci/mmole; Amersham)
[0321] 400 Units/ml terminal deoxynucleotidyl transferase
(Pharmacia)
[0322] The reaction was performed at 37.degree. C. until around
20-25 dC residues were incorporated per 3' OH-end and was stopped
by the addition of EDTA pH 8.0 to a final concentration of 25 mM
followed by phenol extraction, ether extraction, and precipitation
as described above.
[0323] Oligo dG-tailing of the PstI-digested plasmid pAT153 was
carried out under similar conditions except that 4 .mu.M deoxy
(8-.sup.3H) guanosine 5' triphosphate (25 Ci/ummole; Amersham) was
used instead of 40 .mu.M d(.sup.3H) cytidine 5' triphosphate, and
that the concentration of the linearized plasmid DNA was 16
pmole/ml. The oligo dC-tailed double-stranded cDNA was annealed
with the oligo dG-tailed vector as described (Maniatis et al.,
1982.).
[0324] The E. coli strain DH1(.lambda.) was transformed (Hanahan,
1983) using 10 ng of vector DNA per 100 .mu.l of competent cells.
Transformation mixtures were plated on Millipore HATF (0.45 .mu.m)
filters (Millipore, Bedford, Mass., USA) and layered on top of
Luria broth (LB) agar plates containing 10 .mu.g/ml of
tetracycline. After propagation, the filters were placed on fresh
LB-agar plates also containing 20% glycerol and stored at
-20.degree. C. In this way, a 17S mouse cDNA library of about
25,000 clones was obtained.
[0325] 2. Isolation of the CDNA Clones Coding for the Selected
Sequence
[0326] 2.1. Isolation of the Selected Mouse cDNA
[0327] 2.1.1. Plus-minus Screening of the 17S Mouse PU5-1.8 cDNA
Library
[0328] The colonies were lysed in situ and fixed on replicas of the
mouse cDNA library (Hanahan and Meselson, 1980). Two sets of
replicas were screened by differential hybridization: the plus
probe being a .sup.32P-labelled cDNA from LPS-induced PU5-1.8 17S
MRNA, the minus probe being .sup.32P-labelled cDNA from uninduced
PU5-1.8 17S mRNA. This CDNA was synthesized essentially as
previously described (see 1.3.) except that only 15 .mu.M of dCTP
was used, to which .alpha.(.sup.32P) dCTP (6000 Ci/mmole, Amersham)
was added to a concentration of 2 .mu.M. Colony hybridization was
carried out at 42.degree. C. for 40 h in 20% deionised formamide,
5.times.SSC, 5.times.Denhardt solution, 25 mM sodium phosphate
buffer pH 6.5, 20 .mu.g/rnl of sonicated and denaturated E. coli
DNA and .sup.32P-labelled cDNA probe (10.sup.6 cpms/ml) after an
overnight prehybridization in the same buffer but without labelled
cDNA.
[0329] Before autoradiography, the filters were washed three times
for 30 minutes in 2.times.SSC, 0. 1% SDS at 42.degree. C.
[0330] The clones that showed preferential hybridization with the
plus probe were picked up, grown individually, and streaked on new
filters for a second round of plus/minus hybridization. Those
clones that were consistently positive in both rounds of
hybridization were retained. They are referred to as "LPS-induced"
clones.
[0331] 2.1.2. Characterization of a Selected LPS-induced CDNA
Fragment from the PU5-1.8 cDNA Library
[0332] Of all the LPS-induced mouse clones isolated by plus-minus
screening, DNA was prepared using the Triton X100-lysozyme lysis
method essentially as described (Kahn et al., 1979). The length of
the cDNA insert was assessed by digestion with the restriction
enzyme PstI and by separating the insert from the pAT153 vector by
agarose gel electrophoresis.
[0333] The selected LPS-induced clone has an insert of 446 bp,
divided into two subfragments of 332 bp and 114 bp by an internal
PstI site.
[0334] This clone was characterized with respect to its degree of
LPS inducibility, its macrophage cell-type specificity, and its
gene expression in other cells of the immune system (T cells and B
cells) by Northern blot analysis. The selected LPS-induced gene
fragment hybridized only with a MRNA of an approximate length of
1475 base pairs present in uninduced or LPS-induced mouse
macrophage cells and not with MRNA of uninduced or LPS-induced
EL-4, and NSo cells, and very weakly with mRNA of uninduced or
LPS-induced L929 cells. Hence, the selected gene fragment behaved
as LPS-induced and as being dominantly expressed in macrophage.
[0335] 2.1.3. Construction of a LPS-induced PU5-1.8 cDNA Library in
.lambda.ZAP II
[0336] In order to obtain the full-size cDNA information of the
selected LPS-induced PU5-1.8 cDNA fragment, a cDNA library was
constructed in the .lambda.ZAP II vector system (Stratagene, La
Jolla, Calif, USA). To this end, mRNA was prepared from PU5-1.8
cells induced for 3 hours with LPS (see section 1.1.). The
synthesis of cDNA was performed as described (section 1.3.) except
that it was not tailed with dGTP or dCTP but rather was methylated
by dissolving the cDNA pellet in a solution of 100 mM Tris-HCI, 10
mM EDTA, pH 8.0, 80 .mu.M S adenosyl-methionine (Sigma, St. Louis,
Mo., USA) and 1.5 U/mil of RI methylase (Promega, Madison, Wis.
USA) for 60 minutes at 37.degree. C. The enzyme was inactivated by
heat treatment (10 minutes at 70.degree. C.) and, after cooling to
room temperature, MgCl.sub.2, DTT, dXTPs and T4 DNA polymerase
(Boehringer Mannheim, FRG) were added up to a final concentration
of 7 mM, 5 mM, 0.2 mM and 125 units/mil, respectively. The reaction
was performed at 18.degree. C. for 1.5 hours. The enzyme was
heat-inactivated and the reaction mixture was phenol-extracted and
precipitated as above.
[0337] Phosphorylated EcoRI linkers (Pharmacia, Uppsala, Sweden)
were ligated to the blunt-ended dsDNA at 13.degree. C. for 48 hours
at a ratio of 40:1 for the 3 hours LPS-induced PU5-1.8 cDNA
libraries constructed in .lambda.ZAP II in a ligation buffer
containing 1 mM ATP, 50 mM Tris-HCl pH 7.4, 10 mM DTT, 8 mM
MgCl.sub.2 and, 0.5 U/mil T4 ligase (Boehringer-Mannheim, FRG). The
mixture was subsequently heat-treated (10 minutes at 70.degree. C.)
and, after cooling to room temperature, Tris-HCl, pH 7.4, NaCl,
MgCl.sub.2, DTT, and EcoRI enzyme were added to final
concentrations of 50 mM, 100 mM, 10 mM, 10 mM, and 3000 U/ml,
respectively. Digestion was performed for at least 2 hours at
37.degree. C. The material was then phenol-extracted,
ethanol-precipitated, and fractionated by gel filtration over
Biogel A 50-m (Biorad, Richmond Califormia, USA). All DNA fragments
larger than .+-.400 bp were pooled, freeze dried, and redissolved
in 50 mM Tris-HCI, pH 7.4, 12 mM MgCl.sub.2, 12 mM MgCl.sub.2, 10
mM ATP, 1 mM DTT. To this end, a solution of T4-ligase
(Boehringer-Mannheim, FRG) was added to a concentration of 0.5
U/.mu.l and ligation was performed for at least 3 hours at
16.degree. C. at a molar ratio of 1:2 of insert versus vector for
the 3-hour LPS-induced PU5-1.8 cDNA libraries constructed in
.lambda.ZAP II. Packaging of the ligation mixture into phage
particles was performed using a packaging mix from Promega
(Madison, Wis., USA) according to the protocol recommended by the
supplier, except that the chloroform treatment was omitted. The
cDNA library constructed in the KZAP II cloning vector was
amplified on XL-1 blue cells (Stratagene) and contained
1.8.times.106 independent plaques.
[0338] 2.1.4. Screening for the Full-size Mouse cDNA Corresponding
to the LPS-induced Gene Fragment from the PU5-1.8. cDNA Library
[0339] The corresponding full-size mouse sequence was picked up by
screening the LPS-induced PU5-1.8. .lambda.ZAP II cDNA library
(section 2.1.3.). Therefore, the library was plated out and
plaque-lifted in duplo, using 5- and 8-minute adsorption times,
respectively, on Hybond-N membranes (Amersham). The DNA was
denatured by alkaline treatment (0.2 N NaOH, 1.5 M NaCl)
neutralized in a Tris HCl buffer (1 M Tris-HCI, pH 7.5; 1.5 M NaCi)
followed by a fmal wash in 2.times.SSC and fixed on the membranes
by incubation for 2 hours at 80.degree. C. under vacuum. The
filters were screened by hybridization using both PstI cDNA
fragments of the selected LPS-induced mouse clone CDNA fragment as
radioactive probe. The cDNA fragment was labelled to high specific
activity (.+-.8.times.10.sup.8 cpm/.mu.g) with
.alpha.(.sup.32P)dCTP (3000 Ci/ummol; 10 mCi/ml; Amersham) using a
multiprime DNA labelling procedure as provided by Amersham. The
filters were prehybridized for 20-24 hours at 50.degree. C. in a
solution containing 50% deionized formamide, 4.times.SSPE, 1% SDS,
0.5% milk powder and 500 .mu.g/ml denaturated salmon sperm DNA.
Hybridization was allowed to proceed for at least 48 hours at
50.degree. C. in 47% deionized formamide, 10% dextrane sulfate,
3.times.SSPE, 1% SDS, 0.5% milk powder using 0.5-1.times.10.sup.6
cpms of probe/mi.
[0340] A first wash was performed in 2.times.SSC, 0.1% SDS at
30.degree. C. followed by different washes in 1.times.SSC, 0.1% SDS
at 50.degree. C. or at a higher temperature until the background
was acceptable. After autoradiography, plaques showing positive
hybridization on both filters were further plaque-purified.
[0341] Purified plaques were excised in vivo and recircularized by
infecting with fl-helper phage to generate the pBluescript
phagernids as described by the supplier (Stratagene, La Jolla,
Calif., USA). Using these phagemids, DNA was prepared, the cDNA
inserts were characterized by partial restriction mapping, and
inserts were sequenced.
[0342] The combined data allow depiction of the mouse nucleotide
sequence coding for the protein corresponding to the selected
LPS-induced gene (FIG. 3).
[0343] The sequence, numbered from nucleotide 1 to 1362, contains
an ATG initiation signal at nucleotide position 187, opening a
reading frame of 933 bp that codes for a protein of 311 amino acids
(TGA stop codon on nucleotide position 1120). The 3'-end sequence
is 243 nucleotides long and contains the 3'-TTATTAT (position
1329), resembling the cytokine consensus sequence 3'-TTATTTAT
(Caput et al., 1986), and a short poly A stretch of 11 A residus.
However, this part of the cDNA will most propably not be complete
as no AATTAAA polyadenylation signal is present at the end of the
sequence. The derived amino acid sequence encodes a protein with a
calculated molecular weight of 34.5 kDa that contains a
computer-predicted N-terminal signal peptide of around 40 amino
acids with a hydrophobic core of Pro and Leu residues, preceeded by
a rather basic N-terminal region. Algorithms to detect
membrane-spanning or membrane-associated amino acid sequences show
negative results. Furthermore, the sequence contains a putative
N-glycosylation signal (Asn-Leu-Thr; amino acid position 103) and
10 Cys residues.
[0344] 2.2. Isolation of the cDNA Clone Encoding the Human
Homologue of the Selected Mouse Polypeptide of the Invention
[0345] 2.2.1. Induction of the Human THP-1 Cell Line for the
Selected Gene Product
[0346] The human monocytic THP-1 cell line (ATCC TIB202) was chosen
for the screening of a human cDNA library to pick up the human
sequence corresponding to the mouse sequence of the selected
LPS-induced gene. However, other human pre-monocytic cell lines
(e.g. J111 or HL60), macrophage cell lines (U937 or Mono Mac6), or
human macrophage cells isolated from placenta or alveolar fluid can
be used although it should be understood that for each of these
human cells or cell lines a specific induction scheme for the
optimal production of the product may be required. For the
production of our polypeptide, the THP-1 cells were seeded at
2.times.10.sup.5 cells/ml in roller bottles in RPMI-1640 enriched
with 10% fetal calf serum. Three days later, at a cell density of
8.times.10.sup.5 cells/ml, the cells were centrifuged and
concentrated to a cell density of 10.sup.6 cells/ml in
RPMI-1640-10% FCS enriched with 400 IU/ml of human recombinant
interferon-gamma (h-rIFN-.gamma.).
[0347] Twenty-four hours later, the cells were washed twice with
serum-free medium and induced for 6 hours at a cell density of
10.sup.6 cells/ml in serum free-medium with 10-15 .mu.g/ml of LPS.
Thereafter, the cells were collected by centrifugation, washed
twice with icecold PBS and stored as a dry cell pellet at
-70.degree. C. until preparation of the MRNA.
[0348] 2.2.2. Preparation of THP-1 mRNA and Construction of a Human
LPS-induced, h-rIFN-.gamma.-activated THP-1 cDNA Library and
Screening of the Library for the Full-size Human Sequence
Homologous to the Selected Mouse cDNA Fragment
[0349] The in vitro induced THP-1 cells prepared as described above
were used as a source of human monocytic MRNA. The polyadenylated
RNA was extracted from the cells as described in section 1.2. for
the PU5-1.8. cells. After quality control by sucrose gradient
centrifugation, this mRNA was used for the construction of a human
macrophage cDNA library in the .lambda.ZAP II phagemid.
[0350] The human THP-1 cDNA library was constructed essentially as
described in section 2.1.3. for the PU5-1.8. .lambda.ZAPII cDNA
library. Starting from 0.5 .mu.g THP-1 mRNA, a human macrophage
cDNA library was constructed in .lambda.ZAPII of 1.5.times.10.sup.6
independent plaques. After amplification, the library had a titer
of 10.sup.9 pfu/ml and was stored at -70.degree. C. in the presence
of 7% DMSO.
[0351] Next, 5.times.10.sup.5 pfu of the LPS-induced
h-IFN-.gamma.-activated THP-1 cDNA .lambda.ZAP II library was
screened using the .sup.32P-labelled 990 bp EcoRI restriction
fragment of the previously isolated selected mouse cDNA as
radioactive probe. The cDNA was labelled to high specific activity
(.+-.8.times.108 cpm/.mu.g) with .alpha.(.sup.32P)dCTP (300
Ci/mmol; 10 mCi/mil; Amersham) using a multiprime DNA labelling
procedure as provided by Amersham. The plaque lifts were prepared
as described for the screening of the mouse cDNA libraries (see
section 2.1.4.). The filters were prehybridized for 20-24 hours at
42.degree. C. in a solution containing 50% deionized formamide,
4.times.SSPE, 1% SDS, 0.5% milk powder and 0.5 mg/ml denaturated
salmon sperm DNA. Hybridization was allowed to occur for at least
48 hours at 42.degree. C. in 47% deionised formamide, 10% dextran
sulfate, 3.times.SSPE, 1% SDS, 0.5% milk powder using
0.5-1.times.10.sup.6 cpms of radiolabelled probe/ml. After
hybridization, a first wash was performed in 2.times.SSC, 0.1% SDS
at room temperature for 15 minutes followed by a second wash in
1.times.SSC, 0.1 SDS for 20 minutes at 50.degree. C. and a final
wash in 2.times.SSC, 0.1% SDS for 20 minutes at 55.degree. C. After
autoradiography, phages showing positive hybridization on both
plaque lifts were further plaque-purified. The longest clone we
isolated contains an insert of 1487 bp (FIG. 2) and predicts an
open reading frame starting from the first ATG at position 6 to
position 938, specifying a polypeptide of 311 amino acids. Unlike
the analogously selected LPS-induced mouse clone, the human
sequence does not contain an internal EcoRI site. The 5'-end is 5
nucleotides long and will most propably be incomplete. The
3'-untranslated region is 548 nucleotides long and may be complete
since an AATAAA polyadenylation signal (position 1466) is present
at the end of the sequence. The 3' end region also contains the
5'-TATTAT sequence resembling the cytokine consensus sequence
(Caput et al., 1987), conserved between the selected human and
mouse clone. The nucleotide sequence of both human and mouse share
73.8% homology (data not shown). The sequence predicted by the
human clone encodes for a polypeptide with a calculated MW of 34
kDa and shows 77.4% homology with the amino acid sequence of the
selected mouse clone (FIG. 5). The ten Cys residues are conserved
in both sequences indicating that they may be important in the
folding of the polypeptide. The absence of the putative
N-glycosylation signal in the human sequence in contrast to the
Asn-Leu-Thr code in the mouse sequence suggests that the human cDNA
product is not glycosylated. Furthermore, the hydrophilicity plots
of the human and mouse clone (FIG. 6) are very similar and, in both
sequences, an eukaryotic secretory signal sequence is predicted
with the most probable cleavage site between amino acid position 45
and 46. For both human and mouse, a mature product of .+-.30 kDa
should then be found upon translocation.
[0352] 3. Determination of the Macrophage-specificity and
LPS-inducibility of the PU5-1.8. mRNA Hybridizing with the Selected
LPS-induced cDNA
[0353] To define the degree of the LPS-inducibility of the selected
gene, mRNA of uninduced and LPS-induced PU5-1.8 cells was prepared
after 3 hours or 24 hours of induction. The macrophage-cell type
specificity of the selected LPS-induced mouse cDNA was assessed by
preparing mRNA of uninduced cells and cells treated with LPS
according to the protocol followed for PU5-1.8. induction. As cell
lines were selected (mouse macrophage hybridoma cells; Patent
Application Innogenetics N.V. Analytical Utilisation of Phagocyte
Cell Lines. 19.09.90. EP 0 159 653 B1.), EL-4 cells (mouse T cell
lymphoma; ATCC TIB39), mouse NSo (non-secreting mouse B cell
myeloma, Kearney et al., 1979), and L929 cells (mouse fibrosarcoma,
ATCC CCL1).
[0354] To this end, the cells were grown batch-wise (10.sup.9
cells/batch) for 40 hours in RPMI-1640 medium enriched with 10%
fetal calf serum, washed twice with serum-free medium and incubated
for another 3 hours (for the LPS-induction of PU5-1.8 cells) or 24
hours in serum-free RPMI 1640, in the absence (-LPS) or in the
presence of 10-15 .mu.g/ml of LPS (+LPS). Thereafter, the cells
were washed twice with icecold PBS and stored at -70.degree. C.
until preparation of the mRNA. mRNA was prepared using the NP-40
method as described in section 1.2.
[0355] All MRNA preparations were run on a denaturating
formaldehyde/formamide--1.5% agarose gel (2.5 .mu.g poly A.sup.+
RNA/lane) as described by Maniatis et al., (1982.) and blotted on a
Nylon membrane (Hybond-N, Amersham) in 10.times.SSC by Northern
blotting (Fourney et al. 1988). These blots were subsequently
screened by hybridization using restriction fragments of the
selected clone as radioactive probe (FIG. 7).
[0356] The degree of the LPS-induction of the selected gene in
mouse cells was evaluated by comparison of the strength of the
hybridization signals obtained with the different mRNA preparations
using .beta.-actin as internal standard. Hybridization to a mRNA of
1475 bp was detected in uninduced mouse macrophage cells, but mRNA
levels were slightly increased upon in vitro treatment of the cells
with LPS for three hours.
[0357] 4. Isolation of the Human Gene Containing the Human cDNA of
the Invention
[0358] Starting from high quality genomic DNA, isolated from human
spleen tissue (Maniatis et al., 1982), a human genomic library
(6-8.times.10.sup.6 independent plaques (pfu)) was constructed in
the GEM11 vector (Promega) essentially as described by the
supplier. From this library which has a titer of .+-.10.sup.10
pfu/mi after amplification, 1.2.times.10.sup.6 pfus were plated on
MB406 (Promega) and screened for the human gene of the invention by
hybridization using the full-size human cDNA insert as radioactive
probe. The preparation of the filters and the pre- and
hybridization conditions were as described for the homologous
screening of LPS-induced PU5-1.8. .lambda.ZAP II cDNA library for
the isolation of the full-size mouse cDNA of the invention (section
2.1.4.). Ten positively hybridizing plaques were picked,
plaque-purified, and grown for the preparation of the recombinant
phage DNA. Upon further restriction mapping and Southern blotting
analysis using either the full-size insert or cDNA restriction
fragments located near the 5' or 3' end of the human cDNA of the
invention as radioactive probe, three genomic clones (clones a, b
and c) were retained for subcloning of the different SacI-fragments
in the pBluescript SK(+) (Stratagene) and partially nucleotide
sequencing. The 5000 bp SacI subclone of the genomic clone b
contains the 5' exon fragment of the human cDNA sequence of the
invention, including the ATG initiation site and extends this
information until position 175 of the human cDNA were it transits
in the first intron sequence by the use of a classical splice donor
acceptor site. The 1500 bp SacI-subclone, present in all three
isolated genomic clones, contains the second exon of the human gene
of the invention from position 176 to position 561 of the human
cDNA. Finally, the 3500 bp SacI-subclone contains the third and
fourth exon of the gene, respectively ranging from position 562 to
621 and from position 622 to the end of the human cDNA including
the 3'-end-located AATAAA polyadenylation site.
[0359] 5. Expression of the Mouse Polypeptide of the Invention in
E. coli Cells
[0360] The DNA sequence coding for a polypeptide, or part of it,
can be linked to a ribosome binding site which is part of the
expression vector, or can be fused to the information of another
protein or peptide already present in the expression vector. In the
former case, the information is expressed as such and hence devoid
of any foreign sequences (except possibly for the amino terminal
methionine which is not always removed by E. coli). In the latter
case the expressed protein is a hybrid or a fusion protein.
[0361] Various methods and materials for preparing recombinant
vectors, either of plasmid, bacteriophage or cosmid nature, the
procedures for transformation or infection in different host cells
and expressing polypeptides and proteins are described by Panayatos
(1981) and by Old and Primrose (1981) and are well known to those
skilled in the art.
[0362] A suitable vector is plasmid pmTNF-MPH (Innogenetics). It
contains the tetracycline resistance gene and the origin of
replication of pAT153 (Twigg and Sherratt (1980) (obtainable from
Biores B. V., Woerden, The Netherlands), the PL promoter up to the
MboII site in the N gene 5' untranslated region, followed by a
synthetic ribosome binding site (see sequence data) and the
information encoding the first 25 amino acids of mTNF (except for
the initial Leu which is converted to Val). This sequence is, in
turn, followed by a polylinker sequence encoding six consecutive
His residues downstream of which several proteolytic sites (formic
acid, CNBr, kallicrein and E. coli protease VII sensitive sites)
are incorporated. Each of these proteolytic sites is at the DNA
level accessible by a unique restriction site. The presence of the
[His].sub.6 sequence in the fusion peptide allows
Ni.sup.2+-immobilized metal affinity chromatography (IMAC) based
purification of the recombinant protein of interest. Downstream
from the polylinker, translational stop codons are present in the
three possible reading frames which in turn are followed by the E.
coli trp terminator of transcription (synthetic) and the rrnBT1T2
terminator of transcription (originating from pKK223-3; Pharmacia).
The restriction and genetic map of this plasmid is represented in
FIG. 8a. The total nucleic acid sequence of this plasmid is
represented in FIG. 8b.
[0363] DNA of the pmTNF-MPH-PU1280-Eco47111 containing the mouse
nucleotide sequence of the invention from the Eco47III restriction
site at position 318 to the EcoRI cloning site (position 1364),
cloned in the proper orientation into the StuI site of the
pmTNF-MPH, a technique well known to those skilled in the art, was
transformed into E. coli strain K12.LAMBDA. H (ATCC 33767) using
standard transformation procedures. However, the growth temperature
of the cultures is reduced to 28.degree. C. and the heat shock
temperature is raised to 42.degree. C. A culture of K12D H
harbouring pmTNF-MPH-PU1280-Eco47III, grown overnight in Luria
broth a 28.degree. C. with rigorous shaking in the presence of 10
.mu.g/ml tetracycline, was inoculated into fresh Luria broth
containing tetracycline (10 .mu.g/ml) and grown to an optical
density of 0.2 measured at 600 nm under the same conditions as for
the overnight culture. At this density of bacterial growth, half of
the culture was shifted to 42.degree. C. to induce expression while
the other half remained at 28.degree. C. as a control. At several
time intervals aliquots were taken which were extracted with one
volume of phenol equilibrated against M9 salts (0,1% ammonium
chloride, 0.3% potassium dihydrogen phosphate, 1.5% disodium
hydrogen phosphate, 12 molecules of water) and 1% SDS. At the same
time the optical density at 600 nm of the culture is measured. The
proteins are precipitated from the phenol phase by addition of two
volumes of acetone and storage overnight at -20.degree. C. The
precipitate is pelleted (Biofuge A, 5 min, 13000 rpm, room
temperature), air dried, and dissolved in a volume of Laemmli
(1970) sample buffer (+.beta.-mercaptoethanol) according to the
optical density of the culture sample and boiled for 3 minutes.
Samples were then run on a SDS polyacrylamide gel (12.5%) according
to Laemmli (1970). Temperature induction of
pmTNF-MPH-PU1280-Eco47III was monitored by both Coomassie Brilliant
Blue (CBB) staining and immunoblotting.
[0364] For CBB staining, the gel was first treated for at least 1
hour at 4.degree. C. with a 10% trichloroacetic acid (TCA)
solution. and subsequently immersed in a 1/10 diluted CBB staining
solution (0.5 g CBB-R250 (Serva) in 90 ml of methanol: H.sub.2O
(1:1 v/v) and 10 ml glacial acetic acid) and left for about one
hour on a gently rotating platform. After destaining in 30%
methanol--7% glacial acetic acid (two to three washes of about 30
min each), protein bands were visualized and scanned with a
densitometer (for instance Ultroscan XL Enhanced Laser
sensitometer, Pharmacia LKB).
[0365] For immunoblotting the proteins were transferred onto Hybond
C membranes as described by Towbin et al. (1979). After blotting,
proteins on the membrane were temporarily visualized with Ponceau S
(Serva) and the position of the molecular weight markers was
indicated. The stain was then removed by washing in H.sub.2O.
[0366] Aspecific protein binding sites were blocked by incubating
the blots in 10% non-fat dried milk for about 1 hour on a gently
rotating platform. After washing twice with NT buffer (25 mM Tris
Cl pH 8.0; 150 mM NaCl) blots were incubated with monoclonal
anti-hTNF antibody (1/10) which cross-reacts with mTNF
(Innogenetics No. 17F5D10) for at least 2 hours on a rotating
platform. After washing twice with NT buffer +0.02% Triton X100,
blots were incubated for at least 1 hour with the secondary
antiserum which was alkaline phosphatase-conjugated rabbit
anti-mouse irnmunoglobulins (1/500; Sigma). Blots were washed again
twice with NT buffer +0.02% Triton-X100 and then visualized with
nitro blue tetrazolium (NBT) and
5-bromo-4-chloro-3-indolyl-phosphate (BCIP) from Promega under
conditions recommended by the supplier.
[0367] After induction of K12 H cells transformed with
pmTNF-MPH-PU1280-Eco47III, a band of about 34 kDa appeared on CBB
stained gels, which represents about 15% of total synthesis (FIG.
9). The fusion product between TNF and the selected gene product
reacts clearly with anti-hTNF-monoclonal antibody (No. 17F5D10) on
immunoblot.
[0368] 6. Transient Expression of the Mouse and the Human Sequences
in COS1 cells
[0369] The mouse cDNA of the invention was introduced into an
expression vector comprising the SV40 origin of replication and
part of the SV40 late region containing the strong SV40 late
promoter and enhancer sequence followed by a multilinker sequence
which is flanked by donor and acceptor splice sites of the late 16S
MRNA and the polyadenylation signal of the SV40 region (pSVL)(FIG.
10). This type of expression vector was originally described by
Gheysen et al. (1982).
[0370] The mouse cDNA sequence of the invention was introduced
according to methods known to those skilled in the art into the
multicloning site of this expression plasmid as a HindIII-EcoRV
(multicloning site of pSP73) DNA fragment isolated from the
so-called plasniid pSP73-PU1280 which contains the mouse cDNA
sequence as a SphI-RcoRI fragment. Transfection of the COS1 cells
(Gluzman et al., 1981)(ATCC CRL 1650) with the resulting plasmid
pSV-PU1280-HdIII was done according to an optimized
DEAE-transfection protocol (McCutchan and Pagano, 1968). In case of
in vivo labelling of the cells with 35S-methionine, the
transfection of the cells was followed by two washes with
methionine-free medium (DMEM or RPMI-1640) without serum, a
starvation period of one hour in the same medium, and subsequent
incubation in 1 ml medium per 10.sup.6 cells supplemented with
.sup.35S-methionine for 24 hours (100 .mu.Ci/ml
.sup.35S-methionine; 1150 Ci/mM/ml; 10 m Ci/ml).
[0371] Proteins secreted in conditioned medium of transfected cells
were TCA-precipitated and analyzed on 12.5%
Tricine-SDS-polyacrylamide gels (Schagger and von Jagow, 1987) or
by 2-dimensional nonequilibrium pH gel electrophoresis (NEPHGE) as
described by Van Fleteren et al. (1992). Production of the protein
of the invention was demonstrated by either CBB staining (as
recommended by the supplier, Serva), immunoblotting, or
fluorography (Enhance-Dupont). For immunoblotting, proteins were
electroblotted onto nitrocellulose membranes (Sartorius) and, after
blocking of aspecific protein binding sites with Tween-20 (Sigma)
and 3% BSA (Sigma), the membranes were incubated with polyclonal
antiserum raised against the mTNF-MPH-cDNA fusion protein (1/500)
(section 9.). As a second antibody, alkaline phosphatase-conjugated
mouse anti-rabbit immunoglobulin (1/1000, Sigma) was used and
subsequent visualization was performed with nitro blue tetrazolium
(NBT) and 5-bromo-4-chloro-3-indoly- l-phosphate
(BCIP)(Promega).
[0372] Transfection of COS1 cells with the pSV-PU1280-HdIII
expression plasmid results in production and secretion of an extra
protein with a molecular weight (MW) of approximately .+-.30 kDa
(non-reduced) or .+-.34 kDa (reduced). The reduced form of the
protein (.+-.34 kDa) was only detectable by immunoblotting with
mTNF-MPH-mcDNA antiserum, because a COS1 cell-specific protein
masks the position of the 34 kDa protein. The calculated MW of the
mature polypeptide coded for by the cDNA of the invention is 29.9
kDa. The higher MW of the protein produced in COS1 cells is due to
glycosylation of the mouse protein as indicated by in vitro
transcription-translation experiments (results not shown) and from
results of the Vaccinia expression (see below).
[0373] On 2D-NEPHGE (non-reduced) the protein appears as a triple
peptide spot (due to secondary modifications) with a MW of .+-.30
kDa and a pI of approximately 5.5 to 6.0 (FIG. 11).
[0374] To allow amino acid sequence confirmation of the 30 kDa
protein secreted by pSV-PU1280-HdIII transfected COS1 cells,
preparative amounts (.+-.2 liters) of the COSI conditioned medium
were prepared and the .+-.30 kDa triple protein spot was excised
from preparative Coomassie R stained non-reducing 2D-NEPHGE gels.
The different spots were concentrated according to Rasmussen et al.
(1991) and digested with 2% formic acid at 110.degree. C. for 4
hours (Van Fleteren et al., 1992). Peptides were separated on an
aquapore butyl 7 .mu. (Brownlee-10 cm.times.2.1 mm) column and
peptides AH20 and AH27 (FIG. 12) were sequenced using an Applied
Biosystem 477A protein sequencer. The resulting sequences were 100%
homologous to the polypeptide sequence predicted from the mouse
cDNA sequence of the invention.
[0375] In an analogous manner to the construction of the vector
containing the mouse sequence, the human homologue was inserted
into the vector pSVL as an EcoRI (cDNA cloning sites) DNA fragment.
Transfection of COSI cells with the resulting pSV-T1200 expression
plasmid results in production and secretion of a protein with a MW
of .+-.27 kDa (non-reduced) or .+-.30 kDa (reduced). On
non-reducing 2D-NEPGHE the protein appears as a protein spot with a
MW of .+-.27 kDa and a pI of .+-.6.0 to 7.0, without apparent
indication of secondary modifications, consistent with the lack of
a N-glycosylation site in the human amino acid sequence (FIG.
13).
[0376] 7. Expression of the Selected Mouse cDNA of the Invention in
a Baculovirus Expression System
[0377] The baculovirus expression vector system is a highly
efficient eukaryotic expression vector for producing large amounts
of selected polypeptides in a suitable environment for
posttranslational modifications. This helper-independent
recombinant virus vector has produced recombinant protein at levels
ranging from 1 to 500 mg/liter (Smith et al., 1983; Smith et al.,
1985).
[0378] The mouse cDNA of the invention was inserted into the
intermediate transplacement vector pACYM1 (Matsuura et al., 1987)
according to methods known to those skilled in the art.
[0379] The mouse cDNA of the invention was introduced as a BamHI
fragment, derived from pSV-PU1280-HdIII, into the BamHI insertion
site of the pACYM1 vector downstream from the strong baculoviral
polyhedrin promoter. The resulting transfer vector was
cotransfected with wild type baculovirus DNA (Autographa
californica (mono) nuclear polyhedrosis virus AcMNPV) into
Spodoptera frugiperda cells (Sf9 ATCC 1711) using a modification of
the calcium phosphate precipitation technique (Graham, 1973)
adapted for insect cells. Recombinant virus was selected
visually.
[0380] Infection of Sf9 cells with recombinant baculovirus
containing the mouse cDNA of the invention results in production
and secretion of an extra protein with a MW of .+-.28 kDa
(non-reduced) or .+-.32 kDa (reduced) as determined by SDS-PAGE
(fluorography and/or immunoblotting) (FIG. 14). Upon analysis of
condition medium on 2D-NEPGHE, the protein appears as a spot with
MW .+-.28 kDa (non-reduced), .+-.32 kDa (reduced) and pI .+-.5.5 to
6.0. The difference in MW between the polypeptide of the invention
produced and secreted by either transient expression in COS1 cells
or by recombinant baculovirus-infected Sf9 cells is due to
differences in secondary modifications (glycosylation) of the
polypeptide in the two expression systems, as could be shown by
glycosylation pattern analysis using different glycosidases
(results not shown).
[0381] 8. Expression of the Mouse and Human cDNA of the Invention
in a Vaccinia Expression System
[0382] The vaccinia virus expression system is considered one of
the more promising ways of producing significant amounts of
correctly processed and modified eukaryotic proteins in mammalian
cells. The wide host range of the virus and its ability to infect a
variety of vertebrate cell lines including macrophages makes it an
especially interesting tool to express macrophage-specific proteins
(Moss and Flexner, 1987).
[0383] Both the mouse and human cDNA of the invention were
introduced into the vaccinia genome using the intermediate
transplacement vector pATA18 (Stunnenberg et al., 1988). The cDNA
of the invention was introduced as a BamHI fragment, derived from
pSV-PU1280-HdIII or pSV-T1200, into the BamHI insertion site of the
pATA18 vector (Stunnenberg et al., 1988) downstream from the
vaccinia 11 K late promoter according to methods well-known to
those skilled in the art. The resulting transfer vectors
(pATA-PU1280-HdIII for the mouse cDNA, pATA-T1200 for the human
cDNA) were transfected by the calcium phosphate method into RK13
cells (ATCC CCL 37.) defective for the TK gene (TK-) after prior
infection with wild-type vaccinia virus. Recombinant progeny
virions containing the cDNA of the invention were chosen using
deoxyuridine selection (BrDU). The technology used is well-known to
those skilled in the art.
[0384] In comparison with wild-type virus infected HeLa cells (ATCC
CCL 2.), infection with recombinant vaccinia virus containing the
mouse cDNA of the invention results in production and secretion of
an extra protein with a MW of .+-.kDa (non-reduced) and .+-.34 kDa
(reduced) as determined by SDS-PAGE. Upon analysis of the
conditioned medium produced by these cells on reducing 2D-NEPHGE
the protein appears as a triple peptide spot with MW of .+-.34 kDa
and a pI of 5.5 to 6.0 (FIG. 15).
[0385] Infection of cells (HeLa) with recombinant vaccinia virus
containing the human cDNA of the invention results in the secretion
of a protein with a MW of .+-.27 kDa (non-reduced) and .+-.30 kDa
(reduced). On 2D-NEPHGE, the protein migrates as a peptide spot
with a MW of .+-.30 kDa and a pI of 6.0-7.0 (FIG. 16).
[0386] 9. Production of Polyclonal Antibodies against the Mouse and
Human Polypeptides of the Invention
[0387] A bacterially produced fusion protein between mTNF and the
mouse polypeptide of the invention containing 6 histidine residues
at the fusion position (see section 5.) was prepared by starting
from one liter culture of transformed bacteria
(pmTNF-MPH-PU1280-Eco47III). Bacterial expression products were
sulfonated prior to separation by incubating the bacterial pellet
in 8 M urea, 20 mM Tris-HCl pH 7.0, 1 mM CuSO.sub.4, 130 mM
Na.sub.2S.sub.2O.sub.8 and 610 mM Na.sub.2SO.sub.3. Twenty-four
hours later the mixture was dialyzed against 7 M urea, 30 mM
Tris-HCl pH 7.2 and loaded on a MONO Q-Sepharose column (Pharmacia)
in the same buffer. The material was eluted from the matrix using a
linear salt gradient up to 1 M NaCl and resulting fractions were
tested by SDS-PAGE and immunoblotting using anti-TNF antiserum. The
fractions containing the fusion protein of interest (fractions 48
to 53) eluting from the column between 250 and 350 mM NaCl were
pooled and further purified by Ni.sup.2+-immobilized metal affinity
chromatography (IMAC), according to the manufacturer's
instructions. After elution with 100 mM imidazol the anti-TNF
immunoreactive fusion protein was dialyzed against 7 M urea, 30 mM
Tris-HCl pH 7.2 and subsequently used for immunization of rabbits.
Technology used is well known to those skilled in the art.
[0388] The resulting antiserum (anti-mTNF-MPH-mcDNA) was tested for
specific (cross-) reaction with the mouse and human polypeptide of
the invention both by immunoblotting (detection of the denatured
form of the polypeptide) and immunoprecipitation (detection of the
native soluble form of the polypeptide). Several examples of the
detection by immunoblotting have already been demonstrated (see
above). For immunoprecipitation, 850 11 of
.sup.35S-methionine-labelled conditioned medium of COSI cells
transfected with pSV, pSV-PU1280-HdIII, or pSV-T1200 was incubated
with 30 .mu.of antiserum. After reaction, the inimunoglobulins were
selectively precipitated with 30 .mu.ml of a 50% suspension of
proteina glass beads using the procedure recommended by the
supplier (Porton). After binding, the supernatant was discarded and
the beads were washed with PBS/0.05% Tween 20/0.01% BSA to remove
the aspecific binding proteins. Subsequently, specific antibody
binding proteins were eluted by boiling for 5 min in 30 .mu.l
1.5.times.Laemmli sample buffer and analyzed by
SDS-PAG-fluorography.
[0389] By immunoblotting the anti-mTNF-MPH-mcDNA antiserum
recognizes the polypeptide of the invention produced in COS cells
(30 kDa/34 kDa), recombinant baculovirus- infected Sf9 cells (28
kDa/32 kDa) as well as a 30 kDa/ 34 kDa protein secreted in the
conditioned medium of the macrophage cell line PU5-1.8 (FIG. 17).
The antiserum also cross-reacts with the human analogue of the
polypeptide of the invention produced and secreted by COS1 cells.
In addition the antiserum is also able to immunoprecipitate the
native form of the mouse polypeptide of the invention as produced
in COS1 cells upon transfection with pSV-PU1280-HdIII (FIG.
18).
[0390] Based upon computer prediction of the antigenicity of
different peptides coded for by the human cDNA of the invention, a
C-terminal peptide of 29 amino acids (position 283 to 311:
GCAPRFKDFQRMYRDAQERGLNPCE- VGTD) was chemically synthesized
according to methods known to those skilled in the art (Atherton et
al., 1989), coupled to hemocyanin essentially as described (Harlow,
1988), and injected into rabbits using classical immunization
schemes. The resulting antiserum was tested in immunoprecipitation
and immunoblotting experirments mainly as described earlier. It
recognizes the human polypeptide of the invention secreted by
pSV-T1200-transfected COS1 cells (see above) as well as the product
of the human macrophage cell line Mono Mac 6 either secreted
constitutively or after activation. Furthermore, the antiserum was
also able to immunoprecipitate the native form of the protein as it
recognizes the polypeptide of the invention secreted by
pSV-T1200-transfected COS1 cells.
[0391] 10. N-glycosylation Pattern of the Recombinant and Native
Mouse Polypeptide of the Invention
[0392] In order to study the N-glycosylation patterns of the
recombinant mouse polypeptide of the invention produced in
different expression systems with the native polypeptide secreted
by PU5-1.8 cells, studies using the enzymatic removal of glycosylic
residues by N-glycosidase F (E.C. 3.2.1.96) were performed.
Therefore, conditioned medium (CM) of cells secreting the mouse
polypeptide of the invention or their respective negative controls
were treated with N-glycosidase F under the optimal conditions as
described by the manufacturer. Condition medium was then analyzed
by Western blotting using the anti-mTNF-MPH-mcDNA fusion protein
polyclonal antiserum.
[0393] As was described, the glycosylated form of the protein of
the invention has the same MW (.+-.34 kDa-reduced) when secreted by
PU5-1.8 cells, COS1 cells, or recombinant vaccinia virus-infected
cells. The recombinant baculoviral produced protein has a slightly
lower MW (32 kDa-reduced).
[0394] After N-deglycosylation, both the native protein and the
three eukaryotic expression systems described, shift to an apparent
MW of .+-.29 kDa (reduced), corresponding to the theoretical MW
calculated for the mature unglycosylated polypeptide of the
invention (FIG. 19). These data demonstrate that the lower MW of
the protein produced by insect cells results from a less complex
N-glycosylation pattern of the protein. According to the
literature, insect cells can carry out N-glycosylation, but the
addition of high mannose to complex N-linked oligosaccharides does
not appear to take place (Luckow and Summers, 1988).
[0395] Analogous studies were performed on the human analogue of
the protein of the invention. No evidence for any N-glycosylation
event was demonstrated in accordance with the lack of a
N-glycosylation consensus sequence in the human protein
sequence.
[0396] 11. Purification of the Mouse Recombinant Protein of the
Invention
[0397] Conditioned medium (600 ml) of COS1 cells transfected with
the cDNA of the invention as described in section 6. was collected
after 48 h and filtered over a 0.22 .mu.m filter to remove cell
debris. A typical purification started from 600 to 1000 ml of COSI
transfection medium. To this mgCl.sub.2 and dextranesulphate
500.000 (Pharmacia, Uppsala, Sweden) was added to a final
concentration of 60 mM and 0.02%, respectively. After 1 h
incubation at 4.degree. C. the precipitate was pelleted by
centrifugation (12.000g, 30 min., 4.degree. C.). The supernatant
fraction, containing the mouse polypeptide of the invention was
dialysed against 50 mM Hepes pH 7.0, 4 mM EDTA, adjusted to pH8.0
and located at a flowrate of 0.5 ml/minute on a 4 ml Phenylboronate
agarose (PBA 30, Amicon, MA, USA) column equilibrated in 50 mM
Hepes pH 8.5. The mouse polypeptide of the invention was eluted
from the matrix by 100 mM Sorbitol.
[0398] The Sorbitol eluated peak (.+-.25 ml) is then passed at a
flowrate of 0.5 ml/minute over a 1 ml FPLC Mono Q anion exchange
column (Pharmacia) equilibrated in Hepes pH 8.5 and eluted with a
linear salt gradient of 0 to 1 M NaCl at a flowrate of 1 ml/minute.
The mouse polypeptide of the invention eluted at 125 mM NaCl in a
total volume of .+-.10 ml.
[0399] The eluate was concentrated about 40 fold by Centricon
10.000 (Amicon) and loaded batchwise (3 times 0.25 ml) on a SMART
Superdex 75 gelfiltration column (Pharmacia) equilibrated against
PBS. The highly purified mouse polypeptide of the invention
(>98% pure) eluted at a molecular weight of 34 kDa, well
resolved from a higher molecular weight peak containing aggregated
mouse polypeptide of the invention and higher molecular weight
contaminants.
3 TABLE III sample protein volume conc. purification steps (ml)
(.mu.g) COS1 conditioned medium 600 23.000
Mg.sup.++/dextranesulphate precipitate 600 N.D. PBA Sorbitol eluate
25 750 Mono Q eluate 10--> 0.25 45 Superdex 75 gelfiltration 3
.times. 0.25 or 15 0.75
[0400] 12. Biological Activities of the Mouse Recombinant Protein
of the Invention
[0401] 12.1. Proliferative Effect of the Mouse Polypeptide of the
Invention on Mouse Thymocyte Populations
[0402] Thymocytes were isolated from 3-week-old C3H mice (IFFA
CREDO, France), and seeded in microtiter wells at 5.times.10.sup.5
cells per well in RPMI-1640 5% FCS, 2 .mu.g/ml PHA, and two-fold
serial dilutions of the polypeptide of the invention alone or in
combination with two-fold serial dilutions of the mouse cytokines
IL-1, IL-2, IL4 or IL-6, or combinations of these (start
concentration in the first well being 400 U/ml). Seventy-two hours
later the cells were labelled with .sup.3H-thymidine (1
.mu..mu.Ci/well) and harvested after 24 hours for counting in a
liquid scintillation counter. The mouse polypeptide of the
invention when added alone elicits a thymocyte growth-proliferative
effect in the presence of the lectin PHA. When tested in
combination with the different cytokines, especially in the
presence of IL4, a growth-enhancing effect towards control values
(counts incorporated in the presence of IL4 alone) could be
observed (FIG. 20). When testing combinations of IL4 with other
T-cell interacting cytokines (IL-1, IL-2 or IL-6) and serial
dilutions and/or constant concentrations of the mouse polypeptide
of the invention, a similar invention- specific
growth-proliferative effect could be observed in varying degrees
for the combinations of IL4 and IL-1 or IL-2, but not with
IL-6.
[0403] 12.2. In vitro Activity of the Polypeptide of the Invention
on the Mitogenic and Allogeneic Responses of Lymph Node and Splenic
Cell Populations
[0404] When properly activated, macrophages secrete monokines such
as TNF-.alpha., IL-1-.alpha., IL-1-.beta. and IL-6 that provide
accessory signals for the stimulation of peripheral T cells.
Similarily, certain T-cell derived lymphokines such as IFN-.gamma.
also play an accessory role in the activation of T cells. The mouse
polypeptide of the invention was tested for its function as an
accessory signal in T-cell activation either alone or in
combination with other cytokines (IL-1, IL-6 or IFN-.gamma.) always
in the presence of the lectin PHA or ConA.
[0405] When tested on ConA activation of lymph node cells (LNC)
with and without IL-1, IL-6 or IFN-.gamma. (each time 100 U/ml),
the polypeptide of the invention exhibits an additive
growth-stimulating effect and this effect is significantly
increased when tested in combination with IFN-.gamma.. This effect
is not observed with the accessory molecules IL-1 and IL-6.
[0406] When the growth proliferative effect of the polypeptide of
the invention was tested on the same cell populations (LNC and SPC)
but now combined with the lectin PHA instead of with ConA, a
similar growth- proliferative activity was recorded upon adding the
polypeptide of the invention. This effect also synergized with
IFN-.gamma..
[0407] T cells were depleted of accessory cells by fractionation on
a nylon-wool column, well known to those skilled in the art. The
nonadherent cell fraction represents the T-cell population while
the accessory are retained by the matrix. CD4+ and CD8+ T cell
subpopulations are isolated from the nylon-wool nonadherent
fraction by magnetic cell sorting (MACS) separation using either
anti-CD4+ or anti-CD8+ antibodies, a technique well known to those
skilled in the art. The total LNC T-cell population, the nylon-wool
nonadherent T cells, CD4+ T cells and CD8+ T cells were stimulated
with ConA either as such or supplemented with 5% accessory cells
(mice peritoneal exudate cells). The combined effect of the mouse
polypeptide of the invention with IFN-.gamma. was tested on the
ConA-induced proliferations of the total unfractionated lymph node
cell population or the nylon-wool nonadherent fraction or MACS
purified CD4+ or CD8+ T-cell subpopulations (Table II). On the
total LNC population, a significant enhancement of the
proliferative response by the combined action of the mouse
polypeptide of the invention and IFN-.gamma. could be measured,
while no effect on growth stimulation could be observed on the
nylon-wool nonadherent T-cell population, or on the MACS-purified
CD4+ or CD8+ T cells. However, upon addition of an accessory cell
population (5% of peritoneal exudate cells (PEC)) to the nylon wool
nonadherent subpopulation, the proliferative effect of the mouse
polypeptide of the invention was partially restored. The
restoration of the enhancing effect was not observed when
supplementing the purified CD4+ or CD8+ T cells with 5% PEC.
[0408] Similar experiments were performed with spleen (SPC) cell
populations except that in these experiments the MACS-purified CD4+
and CD8+ T-cell populations were not included. The mouse
polypeptide of the invention again demonstrated a co-stimulatory
effect on the T-cell growth proliferation that requires the
presence of accessory cells.
[0409] On both cell populations (LNC and SPC), similar results
could be recorded when using the lectin PHA instead of ConA.
[0410] The polypeptide of the invention together with IFN-.gamma.
enhances the mitogenic response of T cells. This enhancing effect
requires the presence of accessory cells such as macrophages.
[0411] Table II hereunder relates to the effect of the mouse
polypeptide of the invention and IFN-.gamma. on the ConA-induced
proliferation of LNC populations. T cells were depleted of accesory
cells by fractionation on nylon wool column (nonadherent fraction
represents the T-cell population), essentially as described by
Julius et al. (1973). CD4+ and CD8+ T cell subpopulations were
isolated by magnetic cell sorting (Miltenyl et al., 1990). T cells,
CD4+ T cells and CD8+ T cells (at a cell concentration of
2.times.10.sup.5 cellslml) were stimulated for 24 hours with 2.5
.mu.g/ml of ConA either as such or supplemented with 5% accessory
cells (peritoneal exudate cells). The latter were prepared by
injecting mice intraperitoneally with 5 ml of DMEM-5% glucose
followed by recuperation of the injected material together with the
peritoneal cell population. Twenty-four hours later the cells were
pulsed with .sup.3H-thymidine (1 .mu.Ci/ml) for another 18 hours
and harvested. The combined effect of the conditioned medium of the
COS1-expressed mouse polypeptide of the invention (rec prot) or of
the CM of pSVL-transfected COS1 cells (pSVL) with 100 IU of
IFN-.gamma. was tested on the ConA-induced proliferations of
unfractionated LNC populations or nylon wool nonadherent CD4+ or
CD8+ subpopulations. The ratio 1/10 or 1/2 represent a one-to-ten
or one-to-two dilution of the gel filtration purified material (see
section 6) in the bioassay test medium.
4 TABLE II ConA-induced proliferation .DELTA. cpms .DELTA. cpms
Cell population cpms (-control) (-control-IFN-.gamma.) LNC(total)
20 -- -- +IFN-.gamma. 46 26 -- +IFN-.gamma. + pSVL (1/10) 57 37 11
+IFN-.gamma. + rec prot (1/10) 71 51 25 +IFN-.gamma. + pSVL (1/2)
84 64 38 +IFN-.gamma. + rec prot (1/2) 110 90 64 LNC(nylon
nonadherent) 2 -- -- +IFN-.gamma. 17 15 -- +IFN-.gamma. + pSVL
(1/10) 17 15 0 +IFN-.gamma. + rec prot (1/10) 18 16 1 LNC(nylon
nonadherent) + PEC 11 -- -- +IFN-.gamma. 11 0 -- +IFN-.gamma. +
pSVL (1/10) 18 7 7 +IFN-.gamma. + rec prot (1/10) 31 20 20
LNC(CD4+) 4 -- -- +IFN-.gamma. 7 3 -- +IFN-.gamma. + pSVL (1/10) 8
4 1 +IFN-.gamma. + rec prot (1/10) 10 6 3
[0412]
5TABLE II (continued): Effect of the mouse polypeptide of the
invention and IFN-.gamma. on the ConA-induced proliferation of LNC
subpopulations ConA-induced proliferation .DELTA. cpms .DELTA. cpms
Cell population cpms (-control) (-control-IFN-.gamma.) LNC(CD4+) +
PEC 27 -- -- +IFN-.gamma. 24 0 -- +IFN-.gamma. + pSVL (1/10) 24 0 0
+IFN-.gamma. + rec prot (1/10) 26 0 0 LNC(CD8+) 0.3 -- --
+IFN-.gamma. 1.2 0.9 -- +IFN-.gamma. + pSVL (1/10) 2.2 1.9 1
+IFN-.gamma. + rec prot (1/10) 2.6 2.3 1.4 LNC(CD8+) + PEC 16 -- --
+IFN-.gamma. 12 0 -- +IFN-.gamma. + pSVL (1/10) 14 0 0 +IFN-.gamma.
+ rec prot (1/10) 11 0 0
[0413] 12.3. Effect of the Polypeptide of the Invention on the
Acetyl-LDL Uptake of Mouse Foam Cells
[0414] J774 (ATCC TIB 76) are mouse monocytic cells that can be
differentiated in vitro to mouse foam cells by treatment with
acetylated low density lipoproteins (acetyl-LDL), a process that
can be followed by measuring the intracellular acetyl-LDL content
and the cholesterol esterification. Preincubation or co-incubation
of the mouse monocytic J774 cell line with the mouse polypeptide of
the invention before of during treatment with acetyl-LDL had an
effect on the uptake of acetyl-LDL by the cells. Treatment of the
J774 cells for 24 hours with the mouse polypeptide of the invention
either before or during the treatment with acetyl-LDL increases the
amount of total cholesterol in the cells. This can no longer be
observed after 4 hours treatment indicating that the mouse
polypeptide of the invention modulates the speed of cell uptake of
acetyl-LDL. This effect could be due to an up-regulation of the
expression of the acetyl-LDL scavenger receptor on the J774 cells
or by another interaction of the mouse polypeptide of the invention
with acetyl-LDL or with the J774 cell membrane. Other cytokines
such as IL-1, GM-CSF and M-CSF have been described to have an
effect on the cholesterol metabolism of the macrophages (Ishibashi
et al., 1990). For M-CSF it has been demonstrated that the
preincubation of the macrophage cells with this cytokine enhanced
the uptake and degradation of acetyl-LDL in a dose-dependent
manner. The polypeptide of the invention could act either directly
on the macrophage cell or indirectly by inducing M-CSF or another
of the above-mentioned cytokines or another yet unknown cytokine
having a direct effect on the acetyl-LDL uptake of the macrophage
cell.
[0415] 12.4. Growth Inhibitory Effect of the Mouse Polypeptide of
the Invention on the Colony Stimulating Activity of Wehi-3
Conditioned Medium
[0416] Colony-stimulating activity of bone marrow cells can be
tested in vitro using a standard CFU-GEMM agarose assay (Metcalf
and Johnson, 1978) well known to those skilled in the art. When
enriched with the suitable growth factor (IL-3, GM-CSF, G-CSF,
M-CSF, erythropoietin), this assay system allows the proliferation
of a large variety of clonal cell populations from a total bone
marrow cell mixture including colonies of granulocytes,
macrophages, megakaryocytes, eosinophils, basophils, mast cells
and, if the assay is conducted in the presence of erythropoietin,
even normoblasts and red cells (Bazil et al., 1983). When the mouse
polypeptide of the invention is added to the bone marrow cell
mixture, no cell colonies are formed using the CFU-GEMM assay
either in the absence or presence of erythropoietin indicating that
the protein itself does not have any intrinsic colony-stimulating
activity on this cell population. When combined with conditionned
medium of Wehi-3 (ATCC TIB68) cells, described as containing an
active concentration of mouse IL-3 (Lee et al., 1982) (among other
cytokines such as GM-CSF, G-CSF, IL-1, etc.) allowing the growth of
all the different cell colonies mentioned above, the CSF activity
of the conditioned medium was significantly reduced. This
demonstrates that the mouse polypeptide of the invention exerts an
inhibitory or antagonistic effect on the CSF potential of the
conditioned medium of Wehi-3 cells by interacting either indirectly
or directly with a single cytokine or a mixture of synergizing
cytokines present in the protein mixture.
[0417] 12.5. Osteoblast Growth-promoting Effect of the Mouse
Polypeptide of the Invention using Rat Femur Pre- and Osteoblast
Enriched Cell Populations
[0418] The continuous remodelling of bone occurs as a coordinated
succession of cell-mediated events involving an initial period of
osteoclastic resorption followed by osteoblast-mediated bone
formation. This process is highly regulated by different systemic
factors and by cytokines.
[0419] Osteoblast proliferative activity of the mouse polypeptide
of the invention was tested using femurs of 3-week-old male WISTAR
rats. Osteoblastic cells were isolated from the cleaned and flushed
femur bones by 5 sequential digestions of 30 minutes each with an
enzyme mixture containing collagenase (Sigma), hyaluronidase
(Sigma) and DNase (Boehringer Mannheim) at final concentrations of
0.5, 0.5 and 0.1 mg/ml, respectively. Subsequently, the cell pools
of the first two and the last three enzymatic digestions were
treated separately and are referred to as pre-osteoblasts and
osteoblasts, respectively. Cell pools were washed in Ham F12-DMEM-
Hepes (1:1, v/v) (HD) and plated in HD-10% FCS for 6 days.
Thereafter, the osteoblastic cells were collected by trypsinization
and plated in 48-well plates at 40,000 cells per well in HD -1%
FCS. Twenty-four hours later, the cells were washed and the medium
was replaced by HD -1% BSA for another 24 hours after which serial
dilutions of the polypeptide of the invention were added. Nineteen
hours later the cells were labelled with .sup.3H-thymidine (1
.mu.Ci/well) for 6 hours and harvested for counting. The results
(FIG. 21) point to a specific osteoblast proliferative-inducing
activity of the composition containing the polypeptide of the
invention.
[0420] 12.6. Trypanocidal Activity of the Mouse Polypeptide of the
Invention
[0421] The trypanocidal activity of the mouse polypeptide of the
invention was measured using the following in vitro assay system:
2.10.sup.6 /ml purified bloodstream forms of Trypanosoma brucei
brucei or T. brucei rhodesiense, isolated 1 day before the first in
vivo peak of parasitaemia, were incubated for 5 h in phosphate
buffered saline (PBS), 1% glucose, 1% normal mouse serum
(incubation medium) at 37.degree. C. with various concentrations of
the recombinant mouse polypeptide of the invention. After 5 h of
incubation, the number of living (=moving) parasites was assessed
(counting chamber) and compared to the control wells in which only
incubation medium was added to the trypanosomes. The spontaneous
mortality in the control wells after 5 h was always lower than 10%.
The T. brucei brucei AnTat 1.1 (EATRO 1125) pleomorphic bloodstream
form was provided by Dr. N. Van Meirvenne of the Institute for
Tropical Medicine, Antwerp, Belgium, and the T. brucei rhodesiense
Trp11 pleomorphic bloodstream form was provided by Dr. E. Bajyana
Songa (Dept. Molecular Biology, Free University of Brussels,
Belgium).
[0422] Incubation of purified bloodstream forms of T. brucei brucei
and T. brucei rhodesiense for 5 h with the recombinant mouse
polypeptide of the invention results in the mortality of a part of
the parasites: 50% of the animals are killed with 50 ng/ml of
recombinant mouse polypeptide. In the same assay, 500 pg/ml of
recombinant human or mouse TNF-.alpha. cause a 50% mortality of T.
brucei brucei or T. brucei rhodesiense (data not shown).
[0423] 12.7. The Mouse Polypeptide of the Invention Enhances the
Mobility of LAK Cells.
[0424] Lymphocytes activated killer (LAK) cells can be generated
from murine spleen cells by stimulation with IL2. The mobility of
the cells can then be tested by the use of a Transwell system (3
.mu.m membrane, Costar) wherein the migration of the cells trhough
the membrane is measured after a fixed incubation method.
[0425] To test the effect of the mouse polypeptide of the invention
on the mobility of LAK cells, Fl mice (Balb/c.times.C57b1) were
sacrificed by cervical dislocation, and the spleen was removed
aseptically, crushed with a syringe plunger and pressed through a
syringe with 18G and a 23G needle into a petri-dish containing
DMEM. Cell debris was removed by sedimentation, and the resulting
cell suspension was depleted of B cells and macrophages by passing
through a nylon wool column. Thereto, 0.4 g of nylon wool (Wako,
Japan) was put in a 10 ml syringe and autoclaved. Just before
applying the cells, the column was washed with DMEM -5 % FCS and
incubated for 1 h at 37.degree. C. The spleen cells were
resuspended in DMEM -5% FCS at a cell concentration of 108
cells/ml, loaded on the column (2 ml/run) and incubated for 45 min
at 37.degree. C. Subsequently, the nonadherent cell fraction (these
are all the non-B and non-macrophages cells) was recuperated from
the column by washing the matrix with 10 ml DMEM -5% FCS. The cells
were collected by centrifugation, wasged and resuspended at 106
cells/rnl in RPMI1640 -10% FCS -50 .mu.M .beta.-mercaptoethanol in
the presence of 1000 U/ml of IL2 and 25 ng/ml of the mouse
polypeptide of the invention (LAK+IL2+30 kDa) for 7 days. Northern
blotting analysis as well as cPCR studies demonstrated that the
mRNA of the mouse polypeptide of the invention was induced around
day 5 and that the expression was linked with the generation of the
cytotoxic LAK cell (data not shown).
[0426] After 7 days, the non-adherent cell population was washed
away and the adherent LAK cells were removed from the plates by a
short incubation with 0.01 % EDTA in PBS, washed and resuspended in
RPMI1640 at a cell concentration of 106 cells/ml. The LAK mobility
assay was performed using a 24 well size Transwell cell culture
chamber (Costar Europe, Badhoevedorp, The Netherlands) essentially
as described by the supplier. In the cluster well, 600 .mu.l of
RPMI1640 alone (negative control) or enriched with 50, 25, 12.5,
6.25, or 3.12 ng of the mouse polypeptide of the invention was
added. In the Transwell, 100 .mu.l of the LAK cell suspension
(=10.sup.5 cells) either generated with IL2 alone (LAK) or with IL2
and 50 ng/ml of the mouse polypeptide of the invention (LAK+30kDa)
was added. After 4 h incubation at 37.degree. C., those cells which
migrated through the membrane to the cluster well, were counted.
The results of such an experiment are given in Table IV and clearly
demonstrate that the LAK cells generated in the presence of the
mouse polypeptide of the invention in the cluster well also
enhances the mobility of the LAK cells.
6 TABLE IV LAK + IL2 LAK + IL2 + 30 kDa concentration.sup.a 30 kDa
-- 30 kDa -- 50 ng/ml 86 66 143 104 25 ng/ml 105 96 167 139 12.5
ng/ml 111 58 162 183 6.25 ng/ml 118 48 187 138 3.12 ng/ml 79 79 201
136 0 ng/ml 58 137 .sup.aconcentration of the mouse polypeptide of
the invention (30 kDa) in the cluster well during the mobility
assay.
[0427] 12.8. Intrafootpath (I. fp.) Injection of the Mouse
Polypeptide of the Invention Enhances the Immunoresponsiveness of
Lymph Node Cells in Mice.
[0428] To test the in vivo effect of the mouse polypeptide of the
invention, F1 (Balb/c.times.C57b1) mice were injected intrafootpath
(i.fp.) with 50 ng of the mouse polypeptide of the invention in 50
.mu.l of RPMI1640, or the equivalent volume of PBS in 50 Al
RPMI1640/mouse. Twenty four hours later, the popliteal lymph node
cells were isolated and resuspended in RPMI1640-50.mu.M
.beta.-mercaptoethanol -1% normal mouse serum (NMS) at a cell
concentration of 2.106 cells/ml.
[0429] Subsequently, the cells were plated in a 96-well microtiter
plate at 4.10.sup.5 cells/200 .mu.l/well in the same medium and
stimulated for 2448 h with 3 .mu.g/ml ConA (T-Cell stimulation), or
10 .mu.g/LPS (B-Cell stimulation). .sup.3H-Thymidine was added (1
.mu.Ci/well) for another 18 h and the cells were harvested and
counted. The results are expressed in counts per minute (cpm)
incorporated and are given in Table V.
7TABLE V ConA LPS stimulated LNC proliferation is enhanced after
the in vivo ifp injection of the mouse polypeptide of the
invention. LNC proliferation (cpm .times. 10.sup.-3) ConA LPS
experiment 1 PBS treated 63 2 rec prot treated 141 5 experiment 2
PBS treated 9 N.D. rec prot treated 153 N.D.
[0430] The results clearly demonstrate that the LNC from mice
treated with the mouse polypeptide of the invention are sensitized
to respond more efficiently towards T- and B-cell mitogens. The
mouse polypeptide of the invention might therefore function
directly as a costimulatory factor for T- and B-cells or more
indirectly induce a T- and B-cell stimulatory factor.
[0431] 12.9 Intraperitoneal Injection of the Mouse Polypeptide of
the Invention Augments the Generation of Suppressive
Macrophages.
[0432] To test the effect of the mouse polypeptide of the invention
on peritoneal macrophages, F1 mice (Balb/c.times.C57b1) were
injected intraperitoneal (i.p.) with either 50 ng of the mouse
plypeptide of the invention in 200 .mu.l RPMI1640 or the equivalent
volume of PBS in RPMI1640. Twenty four hours later, the peritoneal
exudate cells (PEC) were isolated by an i.p. wash with 10 ml of
RPMI1640 per mouse, collected by centrifugation and resuspended at
a concentration of 2.106 cells/ml in RPMI1640-50 .mu.M
.beta.-mercaptoethanol-1% NMS. PEC batches contaminated with
erythrocytes were discarded.
[0433] 10.sup.4, 2.10.sup.4, or 3.10.sup.4 PEC were cocultured for
24 hours with normal lymph node cells from normal F1 mice that were
seeded in 96 microtiter plates at a cell concentration of
4.10.sup.9 cells/200 .mu.l/well in RPM11640-1% NMS -3 .mu.g/ml ConA
in the absence (experiment 1) or in the presence of 10 .mu.g/ml
Indomethacin. After an additional 18 h pulse with .sup.3H-thymidine
(1 .mu.Ci/well), the cells were harvested and counted.
[0434] The results summarised in table VI are expressed as %
suppression relative to the proliferation of LNC without PEC.
8TABLE VI The ConA induced proliferation of LNC is reduced by
coculture with PEC isolated from mice injected interperitoneal with
the mouse polypeptide of the invention. % suppression of LNC ConA
proliferation 2.5% 5% 7.5% PEC PEC PEC experiment 1 PBS treated 0
38 97 rec prot treated 60 99 99 experiment 2 (+ indomethacin) PBS
treated 0 0 0 rec prot treated 0 0 84
[0435] From these results the following conclusions can be
drawn:
[0436] (i) PECs from PBS and the mouse recombinant protein of the
invention treated animals are suppressive on LNC. The suppressive
activity of the PECs of the animals treated with the mouse
polypeptide in the invention is, however, more pronounced when
compared with the control animals.
[0437] (ii) The suppressive activity of PECs may be mediated via
Prostaglandin (PG) release of macrophages presence in the cell
mixture. To test this possibility, the PECs were cocultured with
ConA stimulated LNC in the presence of PG synthesis inhibitor
Indomethacin (experiment 3). From these results it is clear that
the PECs from the control animals as well as from the animals
treated with the mouse polypeptide of the invention mediate their
suppressive activity via PG release. However, the mouse polypeptide
of the invention specifically modulates PECs that mediate a part of
their suppressive activity via a PG-independent mechanism.
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