IE84574B1 - Fusion proteins with parts of immunoglobulins, their production and use - Google Patents
Fusion proteins with parts of immunoglobulins, their production and useInfo
- Publication number
- IE84574B1 IE84574B1 IE1998/1059A IE981059A IE84574B1 IE 84574 B1 IE84574 B1 IE 84574B1 IE 1998/1059 A IE1998/1059 A IE 1998/1059A IE 981059 A IE981059 A IE 981059A IE 84574 B1 IE84574 B1 IE 84574B1
- Authority
- IE
- Ireland
- Prior art keywords
- fusion protein
- fusion
- human
- receptor
- immunoglobulin
- Prior art date
Links
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- 108020001507 fusion proteins Proteins 0.000 title claims description 40
- 102000018358 Immunoglobulins Human genes 0.000 title claims description 23
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- 238000004519 manufacturing process Methods 0.000 title claims description 4
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Description
PATENTS ACT, 1992
FUSION PROTEINS WITH PARTS OF IMMUNOGLOBULINS, THEIR
PRODUCTION AND USE
HOECHST AKTIENGESELLSCHAFT
THE GENERAL HOSPITAL CORPORATION
Description
The invention relates to the field of
engineered soluble fusion proteins consisting of human
genetically
proteins not belonging to the immunoglobulin family, or
the
The
functional properties of the two fusion partners are,
of parts thereof, and of various portions of
constant region of immunoglobulin molecules.
surprisingly, retained in the fusion protein.
EP—A O 325 262 and EP-A O 314 317 disclose corresponding
fusion proteins consisting of various domains of the CD4
membrane protein. of human T cells and of human IgGl
portions. some of these fusion proteins bind with the
same affinity to the glycoprotein gpl2O of human immuno-
deficiency virus as the cell—bound CD4 molecule. The CD4
the
consequently, has a very similar tertiary structure to
molecule belongs to immunoglobulin family and,
that of immunoglobulin molecules. This also applies to
the a chain of the T-cell antigen receptor, for which
such fusions have also been described (Gascoigne et al.,
Proc. Natl. Acad. Sci. USA, Vol. 84 (1987), 2937-2940).
Hence, on the basis of the very similar domain
structure, in this case retention of the biological
activity of the two fusion partners in the fusion
protein was to be expected.
The humarx proteins coupled. according to the invention
preferably to the amino terminus of the constant region
of immunoglobulixi do not belong to the immunoglobulin
family and are to be assigned to the following classes:
(i) membrane-associated proteins
whose extracellular
domain is wholly or partly introduced into the fusion.
These are in particular thromboplastin and cytokine
receptors and growth factor receptors such as the
cellular receptors for interleukin 4, interleukin 7,
tumor necrosis factor, GM-CSF, G-CSF,
(ii) non—membrane—associated soluble proteins which are
erythropoietin;
wholly or partly introduced into the fusion. These are
w . 845
in particular proteins of therapeutic interest, such as,
for example, erythropoietin and other cytokines and
growth factors.
The fusion proteins can be prepared in known pro- and
eukaryotic expression systems, but preferably in mammal-
ian cells (for example CHO, COS and BHK cells).
EP—A O 269 455 describes
protein in. which the sequence Ala—Pro—Thr—Ser—Ser—Thr~
a highly purified fusion
Lys-Lys-Thr-Gln-Arg-Asn-Ser-Met-Leu is coupled to the N
terminus of a human IgE Fc fragment, and a process for
preparing and pmrifying this fusion protein, which is
beneficial in the treatment of allergies.
The fusion proteins according to the invention are, by
reason of their immunoglobulin portion, easy to purify
by" affinity chromatography and have improved
pharmacokinetic properties in vivo.
In many cases, the Fc part in the fusion protein is
quite advantageous for use in therapy and diagnosis and
thus results, for example, in improved pharmacokinetic
(EP—A. 0 232 262). On the other hand, the
possibility of removing the Fc part would be desirable
properties
for some applications, after the fusion protein has been
expressed, detected and purified in the advantageous
manner described. This is the case when the Fc portion
proves an impediment for use in therapy and diagnosis,
e.g. when the fusion protein is to serve as antigen for
immunizations.
There are in existence various proteases whose use for
this purpose appears conceivable. Papain or pepsin are
employed, for example, to generate F(ab)
immunoglobulins ed. Roitt, I.
Medical Publishing, (1989)),
cleave in a particularly specific manner. Blood coagula-
fragments from
et al.,
but they do not
(Immunology, Gower
London
tion factor Xa by contrast recognizes in a protein the
relatively rare tetrapeptide sequence Ile—Glu—Gly—Arg
and carries out a hydrolytic cleavage of the protein
after the arginine residue. Cleavage sequences which
contain the described tetrapeptide were introduced first
by Nagai and Thogersen into a hybrid protein by genetic
H.C.,
These authors were
engineering means (Nagai, K. and Thogersen,
309 (1984), 8l0~8l2).
able to show that the proteins expressed in E.
Nature, vol.
coli
actually are specifically cleaved by factor Xa. However,
there is as yet no published example of the possibility
of such proteins also being expressed in eukaryotic and,
animal cells after their
especially, in and,
purification, being cleaved by factor Xa. However,
expression of the proteins according to the invention in
animal cells is preferable because only in a cell system
of this type is there expected to be secretion of, for
example, normally membrane—associated. receptors as
fusion partners with retention of their native structure
and thus of their‘ biological activity; Secretion into
the cell culture supernatant facilitates the subsequent
straightforward purification of the fusion protein.
The
soluble fusion proteins consisting of human proteins not
the
invention thus relates to genetically engineered
belonging to immunoglobulin family, or parts
thereof, and various portions of the constant regions of
heavy or light chains of immunoglobulins of various
(IgG, IgM, IgA, IgE). The
immunoglobulin is the constant part of the heavy chain
subclasses preferred
of human IgG, particularly preferably of human IgGl, the
fusion taking place at the hinge region. In a particular
embodiment, the Fc part can be detached in a simple way
by a cleavage sequence which is also incorporated and
can be cleaved by factor Xa.
Furthermore, the invention relates to processes for the
preparation of these fusion proteins by genetic
engineering, and to the use thereof for diagnosis and
therapy.
Finally, the invention is explained in further examples.
Example 1: Thromboplastin fusion proteins
Blood coagulation is a process of central importance in
the human body. There is appropriately delicate regula-
tion of the coagulation cascade, in which a large number
of cellular factors and plasma proteins cooperate. These
proteins (and their cofactors)
are
The final products of the
thrombin, which the
aggregation of blood platelets, and fibrin which stabil-
the platelet thrombus. Thrombin the
formation of fibrin from fibrinogen and itself is formed
in their entirety
called coagulation factors.
induces
coagulation cascade are
izes catalyzes
by limited proteolysis of prothrombin. Activated factor
X (factor Xa) is responsible for this step and, in the
presence of factor Va and calcium ions, binds to
platelet membranes and cleaves prothrombin.
Two ways exist for factor X to be activated, the extrin-
sic and the intrinsic pathway. In the intrinsic pathway
a series of factors is activated by proteolysis in order
for each of them to form active proteases themselves. In
the extrinsic pathway, there is increased synthesis of
thromboplastin (tissue factor) by damaged cells, and it
with VIIa
calcium ions. It was formerly assumed that the activity
activates factor X, together factor and
of thromboplastin is confined to this reaction. However,
the thromboplastin/VIIa
activate the intrinsic pathway at the level of factor
complex also intervenes to
IX. Thus, a thromboplastin/VIIa complex is one of the
most important physiological activators of blood
coagulation.
It is therefore conceivable that thromboplastin, apart
from its use as diagnostic aid (see below), can also be
employed as constituent of therapeutic agents for treat-
ing inborn or acquired blood coagulation deficiencies.
Examples of this are chronic hemophilias caused. by‘ a
deficiency of factors VIII, IX or XI or else acute
disturbances of blood coagulation as a consequence of,
for example,
liver or kidney disease. Use of such a
therapeutic agent after surgicial intervention would
also be conceivable.
Thromboplastin is an integral membrane protein which
the
Thromboplastin CDNA sequences have been published by a
does not belong to immunoglobulin family.
total of four groups (Fisher et al., Thromb. Res., vol.
48 (1987), 89-99; Morrisey et al., Cell, vol. 50 (1987),
129-135; Scarpati et al., Biochemistry, vol. 26 (1987),
5234-5238; Spicer et al., Proc. Natl. Acad. Sci. USA,
vol. 84 (1987), 5148-5152). Thromboplastin CDNA contains
an open reading frame which codes for a polypeptide of
of which the 32 N—terminal
amino acids act as signal peptide. Mature thromboplastin
amino-acid residues,
comprises 263 amino—acid residues and has a three-domain
structure: i) (219
region (23
amino-terminal extracellular domain
amino—acid residues); ii) transmembrane
amino-acid residues); iii) cytoplasmic domain (carboxyl
terminus; 21 amino—acid residues). In the extracellular
domain there are three potential sites for N-
glycosylation (Asn-X—Thr). Thromboplastin is normally
glycosylated but glycosylation does not appear essential
for the activity of the protein (Paborsky et al.,
Biochemistry, vol. 28 (1989), 8072-8077).
Thromboplastin is required as additive to plasma samples
The
status of the tested person can be found by the one-
in diagnostic tests of coagulation. coagulation
stage prothrombin clotting time determination (for
example Quick's test). The thromboplastin required for
human
difficult to
the yield is low and considerable amounts
diagnostic tests is currently obtained from
tissue, and the preparation process is
standardize,
of human starting material (placentae) must be supplied.
On the other hand, it is to be expected that preparation
of native, membrane-bound thromboplastin by genetic
engineering will also be difficult owing to complex
These
avoided by the fusion according to the invention to
purification processes. difficulties can be
immunoglobulin portions.
The thromboplastin fusion proteins according to the
invention are secreted by mammalian cells
CHO, BHK, cos cells)
by affinity chromatography on protein .A-Sepharose and
(for example
into the culture medium, purified
have surprisingly high activity in one~stage
prothrombin clotting time determination.
Cloning of thromboplastin cDNA
The sequence published by Scarpati et al., Biochemistry,
vol. 26 (1987), 5234-5238,
thromboplastin CDNA. Two oligonucleotide probe molecules
was used for cloning the
(see Fig. 1) were derived from this. These twc> probe
molecules were used to screen a CDNA bank from human
placenta Natl. Acad. Sci. USA,
vol. 83
(Grundmann et al.,
(1986), 8024-8028).
Proc.
CDNA clones of various lengths were obtained. One clone,
2b-Apr5, which is used for the subsequent procedure,
for the the CDNA
2 depicts the total
sequence of the clone 2b-Apr5 with the thromboplastin
codes same amino-acid sequence as
described in Scarpati et al. Fig.
amino—acid sequence deduced therefrom.
Construction of a hybrid plasmid pTF1Fc coding for
thromboplastin fusion protein.
The plasmid pCD4E gamma 1 (EP 0 325 262 A2; deposited at
the ATCC under the 67610) is
expression. of a fusion. protein composed of human CD4
number No. used for
receptor and human IgGl. The DNA sequence coding for the
extracellular domain of CD4 is deleted from this plasmid
using the restriction enzymes HindIII and BamHI. Only
partial cleavage must be carried out with. the enzyme
HindIII in this case, in order to cut at only one of the
two HindIII sites contained in pCD4E gamma 1 (position
2198). The result is an opened vector in which a eukary-
otic transcription regulation sequence (promoter) is
followed by the open HindIII site. The open BamHI site
is located. at the start of the coding regions for a
pentapeptide linker, followed by the hinge and the CH2
and CH3 domains of human IgGl. The reading frame in the
BamHI recognition sequence GGATCC is such that GAT is
translated as aspartic acid. DNA amplification with
thermostable DNA polymerase makes it possible to modify
a given sequence in such a way that
attached at both
oligonucleotides able to hybridize with sequences in the
any desired
sequences are one or ends. Two
'-untranslated region (A: 5'
GATCGATTAAGCTTCGGAACCCGCTCGATCTCGCCGCC 3') or coding
region
(B: 5' GCATATCTGGATCCCCGTAGAATATTTCTCTGAATTCCCC 3') of
thromboplastin cDNA were synthesized. Of these,
nucleotide A is partially homologous with the sequence
oligo-
of the coding strand, and oligonucleotide B is partially
homologous with the non-coding strand; cf. Fig. 3.
Thus, amplification results in a DNA fragment (827 bp)
at the 5'
end before the start of the coding sequence a HindIII
the 3'
three amino—acid residues of the transmembrane region a
which contains (based on the coding strand)
site, and at end after the codon for the first
BamHI site. The reading frame in the BamHI cleavage site
is such that ligation with the BamHI site in pCD4E gamma
1 results in a gene fusion with a frame
the the
thromboplastin CDNA to the stop codon of the heavy chain
of IgGl.
treatment with HindIII and BamHI,
reading
continuous from initiation codon of
after
into the
vector pCD4E gamma 1, as described above, which had been
cut with HindIII and BamHI.
plasmid was called pTFlFc (Fig. 4).
The desired fragment was obtained and,
ligated
(partially) The resulting
Transfection of pTF1Fc into mammalian cells
The fusion protein encoded. by the plasmid. pTF1Fc is
called pTFlFc pTFlFc
expressed in COS cells. For this purpose, COS cells were
hereinafter. was transiently
transfected with pTF1Fc with the aid of DEAE-dextran
(EP A O 325 262). Indirect immunofluorescence investiga-
tions revealed that the proportion of transfected cells
was about 25%. the cells were
This cell
supernatant was harvested after a further three days.
h after transfection,
transferred into serum—free medium.
Purification of pTF1Fc fusion protein from cell culture
supernatants
ml of supernatant from transiently transfected COS
cells were collected overnight in a batch process in a
column containing 0.8 ml of protein A-Sepharose at 4°C,
(50 mM tris
and eluted in 0.5 ml frac-
washed with 10 volumes of washing buffer
buffer pH 8.6, 150 mM NaCl)
tions with eluting buffer (93:7 100 mM citric acid:
100 mM The first 9
immediately neutralized with 0.1 ml of 2M tris buffer
sodium citrate). fractions were
pH 8.6 in each case and then combined, and the contained
protein was transferred by three concentration/dilution
cycles in an Amicon microconcentrator (Centricon 30)
into TNE buffer (50 mM tris buffer pH 7.4, 50 mM Nacl,
1 mM EDTA). The pTFlFc obtained in this way is pure by
SDS—PAGE electrophoresis (U.K. Lammli, Nature 227 (1970)
680-685). In the absence of reducing agents it behaves
in the SDS-PAGE like a dimer (about 165 KDa).
Biological activity of purified TF1Fc in the prothrombin
clotting time determination
TFlFc fusion protein is active in low concentrations
(> 50 ng/ml)
determination
in the one-stage prothrombin clotting time
{Vinazzer, H. Gerinnungsphysiologie und
Methoden im Blutgerinnungslabor (1979), Fisher Verlag
Stuttgart).
with the clotting times obtained with thromboplastin
The clotting times achieved are comparable
isolated from human placenta.
Example 2: Interleukin-4 receptor fusion proteins
Interleukin-4 (IL-4)
originally" called B—cell growth factor because it is
is synthesized by T cells and was
able to stimulate B-cell proliferation. It exerts a
large number of effects on these cells. one in
particular is the stimulation of synthesis of molecules
of immunoglobulin subclasses IgGl and IgE in activated B
102 (1988)
IL-4 also regulates the proliferation
cells (Coffmann et al., Immunol. vol.
). In addition,
ReV.,
and differentiation of T cells and other hemopoietic
cells. It thus contributes to the regulation of allergic
and other immunological reactions. IL-4 binds with high
affinity to a specific receptor. The CDNA which codes
for the human IL-4 receptor has been isolated (Idzerda
et al., J. Exp. Med. vol. 171 (1990) 861-873. It is
evident from analysis of the amino-acid sequence deduced
from the CDNA sequence that the IL-4 receptor consists
with the 25 N—terminal
amino acids acting as signal peptide. Mature human IL-4
800 like
has a three-domain structure: i) amino-
(207
amino acids)
of a total of 825 amino acids,
receptor consists of amino acids and,
thromboplastin,
terminal extracellular domain amino acids);
ii) transmembrane region (24 and iii)
cytoplasmic domain (569 amino acids). In the extra-
cellular domain there are six potential sites for
(Asn-X-Thr/Ser). IL-4
homologies with human Il-6 receptor, with the B—subunit
IL-2 with
N~glycosylation receptor has
of human receptor, mouse erythropoietin
receptor and with rat prolactin receptor (Idzerda et
al., cit.). Thus, like thromboplastin,
member of the immunoglobulin family but is
together with the homologous proteins mentioned to the
Members of this
loc. it is not a
assigned
new family of hematopoietin receptors.
family have 4 cysteine residues and a conserved sequence
(Trp-Ser-X-Trp-Ser) in the extracellular domain located
near the transmembrane region in common.
On the basis of the described function of the IL-4/IL-4
receptor system, there is a possible therapeutic use of
a recombinant form of the IL-4 receptor for suppressing
ILmediated immune reactions (for example transplant
rejection reaction, autoimmune diseases, allergic reac-
tions).
The amount of substance required for therapy‘ make it
necessary to prepare such molecules by genetic
engineering. Because of the straightforward purification
by affinity chromatography and improved pharmacokinetic
properties, according to the invention the synthesis of
soluble forms of the IL-4 receptor as immunoglobulin
fusion protein is particularly advantageous.
The IL-4 receptor fusion proteins are secreted by
(for example CHO, BHK, COS cells)
the culture medium, purified by affinity chromatography
mammalian cells into
on protein A~Sepharose and have, surprisingly, identical
functional properties to the extracellular domain of the
intact membrane-bound IL-4 receptor molecule.
Construction of a hybrid plasmid pIL-4RFc coding for
IL-4 receptor fusion protein.
Cutting of the plasmid pCD4EGamma1 with XhoI and BamHI
results in an opened vector in which the open Xhol site
The
open BamHI site is located at the start of the coding
is located downstream from the promoter sequence.
regions for a pentapeptide linker, followed by the hinge
and the CH2 and CH3 domains of human IgG1. The reading
frame in the BamHI recognition sequence GGATCC is such
that GAT is translated as aspartic acid. DNA amplifica-
tion with thermostable DNA polymerase makes it possible
to modify a given sequence in such a way that any
desired sequences are attached at one or both ends. Two
oligonucleotides able to hybridize with sequences in the
'-untranslated region
(A: 5' GATCCAGTACTCGAGAGAGAAGCCGGGCGTGGTGGCTCATGC 3') or
coding region
(B: 5‘ CTATGACATGGATCCTGCTCGAAGGGCTCCCTGTAGGAGTTGTG 3')
of the IL-4 receptor CDNA which is cloned in the vector
pDC302/T22-8 (Idzerda et al. cit.) were
, loc.
synthesized. Of these, oligonucleotide A is partially
homologous with the sequence of the coding strand, and
oligonucleotide B is partially homologous with the non-
coding strand; cf. Fig. 5. Amplification using thermo-
stable DNA polymerase results in a DNA fragment (836 bp)
which, based on the coding strand, contains at the 5‘
end before the start of the coding sequence an Xhol
site, and at the 3' end before the last codon of the
extracellular domain a BamHI site. The reading frame in
the BamHI cleavage site is such that ligation with the
BamHI site :u1 pCD4E gamma 1. results lJ1 a gene fusion
with a reading frame continuous from the initiation
codon of the IL-4 receptor cDNA to the stop codon of the
heavy chain of IgG1. The desired fragment was obtained
and, after treatment with XhoI and BamHI, ligated into
the vector pCD4E gamma 1, described above, which. had
been cut with XhoI/BamHI. The resulting plasmid was
called pIL4RFc (Fig. 6).
Transfection of pIL4RFc into mammalian cells
The fusion protein encoded by the plasmid pIL4RFc is
called pIL4RFc pIL4RFc was
expressed in COS cells. For this purpose, COS cells were
transfected with pIL4RFc with the aid of DEAE-dextran
(EP A O 325 262). Indirect immunofluorescence investiga-
hereinafter. transiently
tions revealed that the proportion of transfected cells
was about 25%. 24 h after transfection, the cells were
medium. This cell
transferred into serum—free
supernatant was harvested after a further three days.
Purification of IL4RFc fusion protein from cell culture
supernatants
ml of supernatant from transiently transfected COS
cells were collected overnight in a batch process in a
column containing 1.6 ml of protein A-Sepharose at 4°C,
buffer (50 mM tris
and eluted in 0.5 ml frac-
(93:7 100 mM citric acid:
The first 9
immediately neutralized with 0.1 ml of 2M tris buffer
washed with 10 volumes of washing
buffer pH 8.6, 150 mM Nacl)
tions with eluting buffer
100 mM sodium citrate). fractions were
pH 8.6 in each case and then combined, and the contained
protein was transferred by three concentration/dilution
cycles in an Amicon microconcentrator
(Centricon 30)
into TNE buffer (50 mM tris buffer pH 7.4, 50 mM Nacl,
1 mM EDTA). The IL4RFc obtained in this way is pure by
SDS—PAGE electrophoresis (U.K. Lammli, Nature 227 (1970)
680-685). In the absence of reducing agents it behaves
in the SDS-PAGE like a dimer (about 150 KDa).
Biological activity of purified IL4RFc
I—radiolabeled IL-4 with the
(KD=O.5 nM)
IL4RFc proteins binds
same affinity as membrane-bound intact IL-4
receptor. It inhibits the proliferation. of the IL
cell line CTLLHuIL-4RI clone D (Idzerda et
cit.) in concentrations of 10-1000 ng/ml. In
dependent
al.,
addition, it
loc.
is outstandingly suitable for developing
IL-4 binding assays because it can be bound via its Fc
part to uucrotiter plates previously coated with, for
rabbit IgG, this
likewise binds its ligand with high affinity.
example, anti—human and in form
Example 3: Erythropoietin fusion proteins
(EPO) is
consists of 166 amino acids and is
Mature erythropoietin a glycoprotein which
essential for the
stimulates the
development of erythrocytes. It
maturation and the terminal differentiation of erythroid
_ 13 _
precursor cells. The CDNA for human EPO has been cloned
(EP—A-O 267 678)
mature EPO and a signal peptide of 22 amino acids which
The cDNA can be used to
EPO in
modified. mammalian cells and the EPO can be
and codes for the 166 amino acids of
is essential for secretion.
prepare recombinant functional genetically
employed
clinically for the therapy of anemic manifestations of
various etiologies (for example associated. with. acute
renal failure).
of the
improved pharmacokinetic properties,
Because straightforward purification and the
according to the
invention the synthesis of EPO as immunoglobulin fusion
protein is particularly advantageous.
Construction of a hybrid plasmid pEPOFc
erythropoietin fusion protein.
coding for
This construction. was carried. out in analogy to that
described in Example 2 (section: "Construction of a
hybrid plasmid pIL—4RFc coding for IL-4 receptor fusion
protein"). Two oligonucleotides able to hybridize with
sequences in the vicinity of the initiation codon
(A: 5'GATCGATCTCGAGATGGGGGTGCACGAATGTCCTGCCTGGCTGTGG 3')
and of the stop codon
(B: 5' CTGGAATCGGATCCCCTGTCCTGCAGGCCTCCCCTGTGTACAGC 3')
of the EPO CDNA (EP—A
O 267 678) were synthesized. Of these, oligonucleotide A
cloned in the vector pCES
is partially homologous with the sequence of the coding
strand, and oligonucleotide B is partially homologous
Fig. 7.
results with thermostable DNA polymerase in a DNA frag-
(598 bp) which, the
contains at the 5' end in front of the initiation codon
an XhoI site and in which at the 3'
with the non-coding strand, cf. Amplification
ment based on coding strand,
end the codon for
the penultimate C-terminal amino—acid residue of EPO
(Asp) The
reading frame in the BamHI cleavage site is such that
is present iii a BamHI recognition sequence.
ligation with the BamHI site in pCD4E gamma 1 results in
a gene fusion with a reading frame continuous from the
initiation codon of EPO CDNA to the stop codon of the
heavy chain of IgG1. The desired fragment was obtained
and, after treatment with XhoI and BamHI, ligated into
the vector pCD4E gamma 1, described above, which had
been cut with XhoI/BamHI. The resulting plasmid was
called pEPOFC (Fig. 8).
Claims (7)
1. A soluble fusion protein consisting of the extracellular portion of human IL-4 receptor or a functional part thereof and of_ an Fc part of an immunoglobulin molecule selected from one of the immunoglobulin classes IgG, IgM, IgA and IgE.
2. A fusion protein as claimed in claim 1, wherein the portion of the immunoglobulin molecule is connected via its hinge region to the extracellular part of the IL-4 receptor.
3. A fusion protein as claimed in claim 1, wherein the portion of the immunoglobulin molecule consists of the constant region of the heavy chain of human IgG.
4. A fusion protein as claimed in claim 3, wherein the portion of the immunoglobulin molecule consists of the constant region of the heavy chain of human IgGl or a protein A-binding fragment thereof.
5. A process for preparing fusion proteins as claimed in any of claims 1 to 4, which comprises intro- ducing the DNA coding for these constructs into a mam- malian cell expression system and, after expression, purifying the fusion protein which has been formed by affinity chromatography via the immunoglobulin portion.
6. The use of the fusion proteins as claimed in any of claims 1 to 4 for in vitro diagnosis.
7. A fusion protein as claimed in any of claims 1 to 4 as pharmaceutical. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DEGERMANY28/06/1990P4020607.6 |
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Publication Number | Publication Date |
---|---|
IE84574B1 true IE84574B1 (en) | 2007-05-02 |
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