U.S. patent application number 11/861835 was filed with the patent office on 2010-03-25 for dna coding for polypeptide fusion.
This patent application is currently assigned to LABORATOIRES SERONO SA. Invention is credited to Robert K. Campbell, Scott C. Chappel, Bradford A. Jameson.
Application Number | 20100075402 11/861835 |
Document ID | / |
Family ID | 21752599 |
Filed Date | 2010-03-25 |
United States Patent
Application |
20100075402 |
Kind Code |
A1 |
Campbell; Robert K. ; et
al. |
March 25, 2010 |
DNA coding for polypeptide fusion
Abstract
A hybrid protein includes two coexpressed amino acid sequences
forming a dimer. Each sequence contains the binding portion of a
receptor, such as TBP1 or TBP2, or a ligand, such as IL-6,
IFN-.beta. and TPO, linked to a subunit of a natural heterodimeric
scaffold. Each coexpressed sequence contains a corresponding
subunit so as to form a heterodimer upon expression. Corresponding
DNA molecules, expression vectors and host cells are also disclosed
as are pharmaceutical compositions and a method of producing such
proteins.
Inventors: |
Campbell; Robert K.;
(Wrentham, MA) ; Jameson; Bradford A.; (Milton,
MA) ; Chappel; Scott C.; (Milton, MA) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.;624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
LABORATOIRES SERONO SA
Coinsins
CH
|
Family ID: |
21752599 |
Appl. No.: |
11/861835 |
Filed: |
September 26, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10724226 |
Dec 1, 2003 |
7291339 |
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11861835 |
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09756186 |
Jan 9, 2001 |
6663867 |
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10724226 |
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08804166 |
Feb 20, 1997 |
6193972 |
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09756186 |
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60011936 |
Feb 20, 1996 |
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Current U.S.
Class: |
435/254.2 ;
435/320.1; 536/23.1 |
Current CPC
Class: |
C07C 209/22 20130101;
A61P 5/06 20180101; A61P 35/00 20180101; A61K 38/00 20130101; C07K
14/715 20130101; A61P 5/16 20180101; C07K 2319/00 20130101; A61P
15/08 20180101; A61P 5/00 20180101; C07K 14/59 20130101; A61P 15/00
20180101 |
Class at
Publication: |
435/254.2 ;
536/23.1; 435/320.1 |
International
Class: |
C12N 1/16 20060101
C12N001/16; C07H 21/04 20060101 C07H021/04; C12N 15/74 20060101
C12N015/74 |
Claims
1. An isolated DNA molecule coding for a sequence (a) fused to a
sequence (b), wherein: sequence (a) is at least one amino acid
sequence selected from the group consisting of a chain of a
homomeric receptor, a chain of a heteromeric receptor, a ligand
other than a gonadotropin, and a fragment of said chain of said
homomeric receptor, said chain of said heteromeric receptor or said
ligand, wherein said ligand or fragment thereof retains
ligand-receptor binding capability and said chain of said homomeric
receptor or fragment thereof and said chain of said heteromeric
receptor or fragment thereof retain ligand-receptor binding
capability either alone or in association with a homologous or
heterologous chain of said receptor; and sequence (b) is a subunit
of a heterodimeric proteinaceous hormone, or a fragment thereof
which retains the ability of the subunit to form a heterodimer with
other subunits thereof.
2. The isolated DNA molecule of claim 1, wherein the subunit of a
heteromeric proteinaceous hormone, or a fragment thereof, in
sequence (b) is from a heteromeric proteinaceous hormone selected
from the group consisting of hCG, FSH, LH, TSH, inhibin or
fragments thereof.
3. The isolated DNA molecule of claim 1, wherein the subunit of a
heteromeric proteinaceous hormone, or a fragment thereof, in
sequence (b) is from a gonadotropin hormone.
4. The isolated DNA molecule of claim 1, wherein the subunit of a
heteromeric proteinaceous hormone, or a fragment thereof, in
sequence (b) is from hCG.
5. The isolated DNA molecule of claim 1, wherein said sequence (a)
is the amino acid sequence of a ligand other than a
gonadotropin.
6. An isolated plasmid containing the DNA molecule of claim 1.
7. An isolated host cell transformed with the plasmid of claim 6.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a division of application Ser. No.
10/724,226, filed Dec. 1, 2003, which will issue as U.S. Pat. No.
7,291,339, which is a division of 09/756,186, filed Jan. 9, 2001,
which issued as U.S. Pat. No. 6,663,867, which is a division of
application Ser. No. 08/804,166, filed Feb. 20, 1997, which issued
as U.S. Pat. No. 6,193,972, and which claims the benefit of U.S.
Provisional Application No. 60/011,936, filed Feb. 20, 1996, the
entire contents of each of the above applications being
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a hybrid protein comprising
two coexpressed amino acid sequences forming a dimer, each
comprising:
[0003] a) at least one amino acid sequence selected from a
homomeric receptor, a chain of a heteromeric receptor, a ligand,
and fragments thereof; and
[0004] b) a subunit of a heterodimeric proteinaceous hormone or
fragments thereof; in which (a) and (b) are bonded directly or
through a peptide linker, and, in each couple, the two subunits (b)
are different and capable of aggregating to form a dimer
complex.
BACKGROUND OF THE INVENTION
[0005] Protein-protein interactions are essential to the normal
physiological functions of cells and multicellular organisms. Many
proteins in nature exhibit novel or optimal functions when
complexed with one or more other protein chains. This is
illustrated by various ligand-receptor combinations that contribute
to regulation of cellular activity. Certain ligands, such as tumor
necrosis factor .alpha. (TNF.alpha.), TNF.beta., or human chorionic
gonadotropin (hCG), occur as multi-subunit complexes. Some of these
complexes contain multiple copies of the same subunit. TNF.alpha.
and TNF.beta. (collectively referred to hereafter as TNF) are
homotrimers formed by three identical subunits (1-4). Other ligands
are composed of non-identical subunits. For example, hCG is a
heterodimer (5-7). Receptors may also occur or function as
multi-chain complexes. For example, receptors for TNF transduce a
signal after being aggregated to form dimers (8,9). Ligands to
these receptors promote aggregation of two or three receptor
chains, thereby affording a mechanism of receptor activation. For
example, TNF-mediated aggregation activates TNF receptors
(10-12).
[0006] The modulation of protein-protein interactions can be a
useful mechanism for therapeutic intervention in various diseases
and pathologies. Soluble binding proteins, that can interact with
ligands, can potentially sequester the ligand away from the
receptor, thereby reducing the activation of that particular
receptor pathway. Alternatively, sequestration of the ligand may
delay its elimination or degradation, thereby increasing its
duration of effect, and perhaps its apparent activity in vivo. In
the case of TNF, soluble TNF receptors have been primarily
associated with inhibition of TNF activity (13-17).
[0007] Soluble binding proteins may be useful for treating human
diseases. For example, soluble TNF receptors have been have
efficacy in animal models of arthritis (18,19).
[0008] Since TNF has three binding sites for its receptor (10-12),
and dimerization of the cell surface receptor is sufficient for
bioactivity (8,9), it is likely that binding of a single soluble
receptor to TNF will leave open the possibility that this 1:3
complex of soluble receptor:TNF (trimer) can still bind and
activate a pair of cell surface TNF receptors. To achieve an
inhibitory effect, it would be expected that two of the receptor
binding sites on the TNF trimer must be occupied or blocked by the
soluble binding protein. Alternatively, the binding protein could
block proper orientation of TNF at the cell surface.
[0009] Generally speaking, the need was felt of synthesizing
proteins that contain two receptor (or ligands) chains, as dimeric
hybrid protein. See Wallach et al., U.S. Pat. No. 5,478,925.
[0010] The primary strategy employed for generating dimeric or
multimeric hybrid proteins, containing binding domains from
extracellular receptors, has been to fuse these proteins to the
constant regions of an antibody heavy chain.
[0011] This strategy led, for example, to the construction of CD4
immunoadhesins (20). These are hybrid molecules consisting of the
first two (or all four) immunoglobulin-like domains of CD4 fused to
the constant region of antibody heavy and light chains. This
strategy for creating hybrid molecules was adapted to the receptors
for TNF (10,16,21) and led to the generation of constructs with
higher in vitro activity than the monomeric soluble binding
proteins.
[0012] It is widely held that the higher in vitro potency of the
dimeric fusion proteins should translate into higher in vivo
activity. One study does support this, revealing an at least
50-fold higher activity for a p75(TBP2)-Ig fusion protein in
protecting mice from the consequences of intravenous LPS injection
(16).
[0013] However, despite the widespread utilization of
immunoglobulin fusion proteins, this strategy has several
drawbacks. One is that certain immunoglobulin Fc domains
participate in effector functions of the immune system. These
functions may be undesirable in a particular therapeutic setting
(22).
[0014] A second limitation pertains to the special cases where it
is desirable to produce heteromeric fusion proteins, for example
soluble analogs of the heteromeric IL-6 or type I interferon
receptors. Although there are numerous methods for producing
bifunctional antibodies (e.g., by co-transfection or hybridoma
fusions), the efficiency of synthesis is greatly compromised by the
mixture of homodimers and heterodimers that typically results (23).
Recently there have been several reports describing the use of
leucine zipper motifs to guide assembly of heterodimers (24-26).
This appears to be a promising approach for research purposes, but
the non-native or intracellular sequences employed may not be
suitable for chronic applications in the clinic due to
antigenicity. The efficiency of assembly and stability post
assembly may also be limitations.
[0015] On the other hand, in the particular case of TNF receptors,
certain modifications to the p55 TNF receptor have been found to
facilitate homodimerization and signaling in the absence of ligand
(27,28). It has been found that a cytoplasmic region of the
receptor, termed the "death domain," can act as a homodimerization
motif (28,30). As an alternative to an immunoglobulin hybrid
protein, fusion of the extracellular domain of the TNF receptor to
its cytoplasmic death domain could conceivably result in a secreted
protein which can dimerize in the absence of TNF. Such fusion
proteins have been disclosed and claimed in the International
Patent Application WO 95/31544.
[0016] A third further strategy employed for generating dimers of
soluble TNF receptors has been to chemically crosslink the
monomeric proteins with polyethylene glycol (31).
SUMMARY OF THE INVENTION
[0017] An alternative for obtaining such dimeric proteins, offering
some important advantages, is the one of the present invention and
consists in using a natural heterodimeric scaffold corresponding to
a circulating non-immunoglobulin protein with a long half-life. A
preferred example is hCG, a protein that is secreted well, has good
stability, and has a long half-life (32-33). Given hCG's prominent
role as a marker of pregnancy, many reagents have been developed to
quantitate and study the protein in vitro and in vivo. In addition,
hCG has been extensively studied using mutagenesis, and it is known
that small deletions to the protein, such as removal of five
residues at the extreme carboxyl-terminus of the .alpha. subunit,
can effectively eliminate its biological activity while preserving
its capability to form heterodimer (34,35). Small insertions, of up
to 30 amino acids, have been shown to be tolerated at the amino-
and carboxyl-termini of the .alpha. subunit (36), while fusion of
the .alpha. subunit to the carboxyl terminus of the .beta. subunit
also had little effect on heterodimer formation (37).
[0018] An analog of hCG in which an immunoglobulin Fc domain was
fused to the C-terminus of hCG .beta. subunit has also been
reported; however, this construct was not secreted and no effort
was made to combine it with an .alpha. subunit (38).
[0019] Therefore, the main object of the present invention is a
hybrid protein comprising two coexpressed amino acid sequences
forming a dimer, each comprising:
[0020] a) at least one amino acid sequence selected among a
homomeric receptor, a chain of a heteromeric receptor, a ligand,
and fragments thereof; and
[0021] b) a subunit of a heterodimeric proteinaceous hormone, or
fragments thereof; in which (a) and (b) are bonded directly or
through a peptide linker, and in each couple the two subunits (b)
are different and capable of aggregating forming a dimer
complex.
[0022] According to the present invention, the linker may be
enzymatically cleavable.
[0023] Sequence (a) is preferably selected among: the extracellular
domain of the TNF Receptor 1 (55 kDa, also called TBP1), the
extracellular domain of the TNF Receptor 2 (75 kDa, also called
TBP2), or fragments thereof still containing the ligand binding
domain; the extracellular domains of the IL-6 receptors (also
called gp80 and gp130); the extracellular domain of the IFN
.alpha./.beta. receptor or IFN .gamma. receptor; a gonadotropin
receptor or its extracellular fragments; antibody light chains, or
fragments thereof, optionally associated with the respective heavy
chains; antibody heavy chains, or fragments thereof, optionally
associated with the respective light chains; antibody Fab domains;
or ligand proteins, such as cytokines, growth factors or hormones
other than gonadotropins, specific examples of which include IL-6,
IFN-.beta., TPO, or fragments thereof.
[0024] Sequence (b) is preferably selected among a hCG, FSH, LH,
TSH, inhibin subunit, or fragments thereof.
[0025] Modifications to the proteins, such as chemical or protease
cleavage of the protein backbone, or chemical or enzymatic
modification of certain amino acid side chains, can be used to
render the components of the hybrid protein of the invention
inactive. This restriction of activity may also be accomplished
through the use of recombinant DNA techniques to alter the coding
sequence for the hybrid protein in a way that results directly in
the restriction of activity to one component, or that renders the
protein more amenable to subsequent chemical or enzymatic
modification.
[0026] The above hybrid proteins will result in monofunctional,
bifunctional or multifunctional molecules, depending on the amino
acid sequences (a) that are combined with (b). In each couple, (a)
can be linked to the amino termini or to the carboxy termini of
(b), or to both.
[0027] A monoclonal hybrid protein of the present invention can,
for instance, comprise the extracellular domain of a gonadotropin
receptor linked to one of the corresponding receptor-binding
gonadotropin subunits. According to such an embodiment, the hybrid
protein of the invention can be a molecule in which, for example,
the FSH receptor extracellular domain is linked to FSH to increase
plasma half-life and improve biological activity.
[0028] This preparation can be employed to induce follicular
maturation in assisted reproduction methods, such as ovulation
induction or in vitro fertilisation, and to serve as a means to
dramatically amplify the biological activity of the hormone
essential for the success of the process, thus reducing the
requirement for both the hormone itself and the number of
injections to achieve ovulation.
[0029] The FSH receptor and the production of the extracellular
domain of the human FSH receptor have been described respectively
in WO 92/16620 and WO 96/38575.
[0030] According to a particular embodiment, the extracellular
domain of the FSH receptor (ECD) can be fused in frame with a
peptide linker that contains the thrombin recognition/cleavage site
(29) and represents a "tethered" arm. The peptide linker links the
extracellular domain of FSH with a FSH subunit. This will allow for
removal of the extracellular domain of the FSH receptor by cleavage
at the thrombin cleavage site as the molecule comes in contact with
thrombin in the systemic circulation.
[0031] In another embodiment, instead of the thrombin cleavage
site, an enzyme recognition site for an enzyme that is found in
greatest abundance in the ovary is used. In this way, as the
ECD-FSH molecule travels to the ovary, it will be exposed to
enzymes found in the highest concentrations in that tissue and the
ECD will be removed so that the FSH can interact with the membrane
bound receptor.
[0032] In yet another embodiment, instead of an enzyme recognition
site, a flexible hinge region is cloned between ECD and FSH so that
the ECD will not be enzymatically removed from the hormone. In this
way, when the ECD-FSH molecule arrives at the ovary, a competition
will be established between the hinge attached ECD and the ECD of
the FSH receptor found on the ovarian cell membrane.
[0033] In a further preferred embodiment of the invention, the
hybrid protein consists of the aggregation between a couple of aa
sequences, one of which contains TBP1 (or the fragments from aa 20
to aa 161 or to aa 190) as (a) and the .alpha. subunit of hCG as
(b), and the other contains always TBP1 (or the same fragments as
above) as (a) and the .beta. subunit of hCG, or fragments thereof,
as (b). According to this embodiment, depending on the particular
sequence that is chosen as (b) (the entire .beta. subunit of hCG,
or fragments or modifications thereof), the resulting hybrid
protein will have one activity (only that of TBP1) or a combination
of activities (that of TBP1 with that of hCG). In this latter case
the hybrid protein can be used, for example, in the combined
treatment of Kaposi's sarcoma and metabolic wasting in AIDS.
[0034] In a further embodiment of the invention, one or more
covalent bonds between the two subunits (b) are added to enhance
the stability of the resulting hybrid protein. This can be done,
e.g., by adding one or more non-native interchain disulfide bonds.
The sites for these cross-links can be deduced from the known
structures of the heterodimeric hormones. For example, a suitable
site in hCG could be to place cysteine residues at .alpha. subunit
residue Lys45 and .beta. subunit residue Glu21, replacing a salt
bridge (non-covalent bond) with a disulfide bond (covalent bond).
Another object of the present invention are PEGylated or other
chemically modified forms of the hybrid proteins.
[0035] A further object of the present invention is a DNA molecule
comprising the DNA sequence coding for the above hybrid protein, as
well as nucleotide sequences substantially the same. "Nucleotide
sequences substantially the same" includes all other nucleic acid
sequences which, by virtue of the degeneracy of the genetic code,
also code for the given amino acid sequence.
[0036] For the production of the hybrid protein of the invention,
the DNA sequence (a) is obtained from existing clones, as is (b).
The DNA sequence coding for the desired sequence (a) is ligated
with the DNA sequence coding for the desired sequence (b). Two of
these fused products are inserted and ligated into a suitable
plasmid or each into a different plasmid. Once formed, the
expression vector, or the two expression vectors, is introduced
into a suitable host cell, which then expresses the vector(s) to
yield the hybrid protein of the invention as defined above.
[0037] The preferred method for preparing the hybrid of the
invention is by way of PCR technology using oligonucleotides
specific for the desired sequences to be copied from the clones
encoding sequences (a) and (b).
[0038] Expression of any of the recombinant proteins of the
invention as mentioned herein can be effected in eukaryotic cells
(e.g., yeasts, insect or mammalian cells) or prokaryotic cells,
using the appropriate expression vectors. Any method known in the
art can be employed.
[0039] For example the DNA molecules coding for the proteins
obtained by any of the above methods are inserted into
appropriately constructed expression vectors by techniques well
known in the art (see Sambrook et al, 1989). Double stranded cDNA
is linked to plasmid vectors by homopolymeric tailing or by
restriction linking involving the use of synthetic DNA linkers or
blunt-ended ligation techniques: DNA ligases are used to ligate the
DNA molecules and undesirable joining is avoided by treatment with
alkaline phosphatase.
[0040] In order to be capable of expressing the desired protein, an
expression vector should comprise also specific nucleotide
sequences containing transcriptional and translational regulatory
information linked to the DNA coding the desired protein in such a
way as to permit gene expression and production of the protein.
First in order for the gene to be transcribed, it must be preceded
by a promoter recognizable by RNA polymerase, to which the
polymerase binds and thus initiates the transcription process.
There are a variety of such promoters in use, which work with
different efficiencies (strong and weak promoters).
[0041] For eukaryotic hosts, different transcriptional and
translational regulatory sequences may be employed, depending on
the nature of the host. They may be derived form viral sources,
such as adenovirus, bovine papilloma virus, Simian virus or the
like, where the regulatory signals are associated with a particular
gene which has a high level of expression. Examples are the TK
promoter of the Herpes virus, the SV40 early promoter, the yeast
gal4 gene promoter, etc. Transcriptional initiation regulatory
signals may be selected which allow for repression and activation,
so that expression of the genes can be modulated.
[0042] The DNA molecule comprising the nucleotide sequence coding
for the hybrid protein of the invention is inserted into a
vector(s), having the operably linked transcriptional and
translational regulatory signals, which is capable of integrating
the desired gene sequences into the host cell. The cells which have
been stably transformed by the introduced DNA can be selected by
also introducing one or more markers which allow for selection of
host cells which contain the expression vector. The marker may also
provide for phototrophy to a auxotropic host, biocide resistance,
e.g., antibiotics, or heavy metals such as copper, or the like. The
selectable marker gene can either be directly linked to the DNA
gene sequences to be expressed, or introduced into the same cell by
co-transfection. Additional elements may also be needed for optimal
synthesis of proteins of the invention.
[0043] Factors of importance in selecting a particular plasmid or
viral vector include: the ease with which recipient cells that
contain the vector may be recognized and selected from those
recipient cells which do not contain the vector; the number of
copies of the vector which are desired in a particular host; and
whether it is desirable to be able to "shuttle" the vector between
host cells of different species.
[0044] Once the vector(s) or DNA sequence containing the
construct(s) has been prepared for expression, the DNA construct(s)
may be introduced into an appropriate host cell by any of a variety
of suitable means: transformation, transfection, conjugation,
protoplast fusion, electroporation, calcium
phosphate-precipitation, direct microinjection, etc.
[0045] Host cells may be either prokaryotic or eukaryotic.
Preferred are eukaryotic hosts, e.g., mammalian cells, such as
human, monkey, mouse, and Chinese hamster ovary (CHO) cells,
because they provide post-translational modifications to protein
molecules, including correct folding or glycosylation at correct
sites. Also, yeast cells can carry out post-translational peptide
modifications including glycosylation. A number of recombinant DNA
strategies exist which utilize strong promoter sequences and high
copy number of plasmids which can be utilized for production of the
desired proteins in yeast. Yeast recognizes leader sequences on
cloned mammalian gene products and secretes peptides bearing leader
sequences (i.e., pre-peptides).
[0046] After the introduction of the vector(s), the host cells are
grown in a selective medium, which selects for the growth of
vector-containing cells. Expression of the cloned gene sequence(s)
results in the production of the desired proteins.
[0047] Purification of the recombinant proteins is carried out by
any one of the methods known for this purpose, i.e., any
conventional procedure involving extraction, precipitation,
chromatography, electrophoresis, or the like. A further
purification procedure that may be used in preference for purifying
the protein of the invention is affinity chromatography using
monoclonal antibodies which bind the target protein and which are
produced and immobilized on a gel matrix contained within a column.
Impure preparations containing the recombinant protein are passed
through the column. The protein will be bound to the column by the
specific antibody while the impurities will pass through. After
washing, the protein is eluted from the gel by a change in pH or
ionic strength.
[0048] The term "hybrid protein", as used herein, generically
refers to a protein which contains two or more different proteins
or fragments thereof.
[0049] As used herein, "fusion protein" refers to a hybrid protein,
which consists of two or more proteins, or fragments thereof,
linked together covalently.
[0050] The term "aggregation", as used herein, means the formation
of strong specific non-covalent interactions between two
polypeptide chains forming a complex, such as those existing
between the .alpha. and .beta. subunit of a heterodimeric hormone
(such as FSH, LH, hCG or TSH).
[0051] The terms "ligand" or "ligand protein", as used herein,
refer to a molecule, other than an antibody or an immunoglobulin,
capable of being bound by the ligand-binding domain of a receptor;
such molecule may occur in nature, or may be chemically modified or
chemically synthesised.
[0052] The term "ligand-binding domain", as used herein, refers to
a portion of the receptor that is involved in binding a ligand and
is generally a portion or essentially all of the extracellular
domain.
[0053] The term "receptor", as used herein, refers to a membrane
protein, whose binding with the respective ligand triggers
secondary cellular responses that result in the activation or
inhibition of intracellular process.
[0054] In a further aspect, the present invention provides the use
of the hybrid protein as a medicament. The medicament is preferably
presented in the form of a pharmaceutical composition comprising
the protein of the invention together with one or more
pharmaceutically acceptable carriers and/or excipients. Such
pharmaceutical compositions represent yet a further aspect of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] The invention will be better understood by reference to the
appended drawings, in which:
[0056] FIGS. 1(a) and 1(b) show the TBP(20-161)-hCG.alpha. and
TBP(20-161)-hCG.beta. constructs, respectively, and the
corresponding sequences (SEQ ID NOS:1-4).
[0057] FIGS. 2 (a) and 2 (b) show the TBP(20-190)-hCG.alpha. and
TBP(20-190)-hCG.beta. constructs, respectively, and the
corresponding sequences (SEQ ID NOS:5-8).
[0058] FIG. 3 is a schematic summary of the constructs of FIGS. 1
and 2 showing p55 TNFR1, TBP1 and TBP1 fusion constructs. The
linker sequences shown on the last two lines are SEQ ID NO:9
(Ala-Gly-Ala-Ala-Pro-Gly) and SEQ ID NO:10 (Ala-Gly-Ala-Gly).
[0059] FIG. 4 is a graph illustrating the dose dependent protective
effect of CHO cell expressed TBP-hCG(20-190) on TNF.alpha.-induced
cytotoxicity on BT-20 cells and various controls.
[0060] FIG. 5 is a graph illustrating the dose dependent protective
effect of COS cell expressed TBP-hCG(20-190) on TNF.alpha.-induced
cytotoxicity on BT-20 cells and various controls.
[0061] FIG. 6 is a graph illustrating the dose dependent protective
effect of affinity purified CHO cell expressed TBP-hCG(20-161) on
TNF.alpha.-induced cytotoxicity on BT-20 cells and various
controls.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0062] The invention will now be described by means of the
following Examples, which should not be construed as in any way
limiting the present invention.
Examples
Materials and Methods
[0063] Cell lines used in this study were obtained from the
American Type Culture Collection (ATCC), 10801 University
Boulevard, Manassas, Va. 20110-2209, unless otherwise specified.
The CHO-DUKX cell line was obtained from L. Chasin at Columbia
University through D). Houseman at MIT (34). The CHO-DUKX cells,
which lack a functional gene for dihydrofolate reductase, were
routinely maintained in complete .alpha.-plus Modified Eagles
Medium (.alpha.(+)MEM) supplemented with 10% fetal bovine serum
(FBS). The COS-7 cells were routinely maintained in Dulbecco's
Modified Eagles Medium (DMEM) supplemented with 10% FBS. Unless
specified otherwise, cells were split to maintain them in log phase
of growth, and culture reagents were obtained from GIBCO (Grand
Island, N.Y.).
[0064] 1. Assembly of the Genetic Constructs Encoding the Hybrid
Proteins
[0065] The numbering assignments for the p55 TNF receptor are based
on the cloning paper from Wallach (40), while the numbering
assignments for the hCG subunits are based on the numbering
assignments from the Fiddes cloning papers (41,42). The designation
TBP, or TNF binding protein, refers to the extracellular domain
portions of the TNF receptors capable of binding TNF. In these
Examples, the DNA constructs will be named as TBP-hybrid proteins,
with the partner and region of TBP indicated in the construct
nomenclature. All of the TBP-hCG constructs contain the human
growth hormone (hGH) signal peptide in place of the native p55
signal sequence. In addition, the hGH signal peptide has been
placed so that it immediately precedes TBP residue Asp20, which is
anticipated to make this the first residue in the mature, secreted
protein.
[0066] These modifications are not essential to the basic concept
of using hCG as a partner of the hybrid protein.
[0067] The DNAs encoding the hybrid proteins were constructed using
PCR methodology (43).
[0068] a. TBP1 (20-161)-hCG
[0069] The initial TBP-hCG construct was engineered to contain the
ligand binding domain from the extracellular region of the p55 TNF
receptor (from Asp20 inclusive of residue Cys161) fused though a
short linker to the hCG .alpha. and .beta. subunits (starting at
residues .alpha.Cys7 or .beta.Pro7, respectively). This construct,
hereafter referred to as TBP1(20-161)-hCG, is a heterodimer of two
modified hCG subunits, TBP1(20-161)-hCG.alpha. and
TBP1(20-161)-hCG.beta..
[0070] The oligodeoxynucleotide primers used for the
TBP1(20-161)-hCG.alpha. construct were:
TABLE-US-00001 (SEQ ID NO: 11) primer 1 (.alpha..beta.) TTT TCT CGA
GAT GGC TAC AGG TAA GCG CCC (SEQ ID NO: 12) primer 2 (.alpha.) ACC
TGG GGC AGC ACC GGC ACA GGA GAC ACA CTC GTT TTC (SEQ ID NO: 13)
primer 3 (.alpha.) TGT GCC GGT GCT GCC CCA GGT TGC CCA GAA TGC ACG
CTA CAG (SEQ ID NO: 14) primer 4 (.alpha.) TTT TGG ATC CTT AAG ATT
TGT GAT AAT AAC AAG TAC
[0071] These and all of the other primers described in these
Examples were synthesized on an Applied Biosystems Model 392 DNA
synthesis machine (ABI, Foster City, Calif.), using phosphoramidite
chemistry.
[0072] Since both of the TBP-hCG subunit constructs have the same
5'-end (i.e., the 5'-end of the hGH/TBP construct), primer
1(.alpha.B) was used for both TBP-hCG subunit constructs. The other
primers used for the TBP1(20-161)-hCG.beta. construct were:
TABLE-US-00002 (SEQ ID NO: 15) primer 2 (.beta.) CCG TGG ACC AGC
ACC AGC ACA GGA GAC ACA CTC GTT TTC (SEQ ID NO: 16) primer 3
(.beta.) TGT GCT GGT GCT GGT CCA CGG TGC CGC CCC ATC AAT (SEQ ID
NO: 17) primer 4 (.beta.) TTT TGG ATC CTT ATT GTG GGA GGA TCG GGG
TG
[0073] Primers 2 (.alpha.) and 3(.beta.) are reverse complements,
and cover both the 3'-end of the coding region for the p55
extracellular domain, and the 5'-end of the hCG .alpha. subunit.
Similarly, primers 2(.beta.) and 3(.beta.) are also reverse
complements, and cover both the 3'-end of the coding region for the
p55 extracellular domain, and the 5'-end of the hCG .beta.
subunit.
[0074] Two PCR reactions were run for each of the two TBP-hCG
subunit constructs. The first used primers 1(.alpha..beta.) and 2
(.alpha. or .beta.), and used as the template a plasmid encoding
soluble p55 residues 20-180 preceded by the hGH signal peptide
(plasmid pCMVhGHspcDNA.pA4). The second used primers 3 (.alpha. or
.beta.) and 4 (.alpha. or .beta.), and used as the template either
plasmid pSVL-hCG.alpha. or pSVL-hCG.beta. (44). The PCR was
performed using Vent.TM. polymerase from New England Biolabs
(Beverly, Mass.) in accordance with the manufacturer's
recommendations, using for each reaction 25 cycles and the
following conditions: [0075] 100 .mu.g of template DNA [0076] 1
.mu.g of each primer [0077] 2 U of Vent.TM. polymerase (New England
Biolabs) denaturation at 99.degree. C. for 30 seconds annealing at:
5 g.degree. C. for 30 seconds for primers 1(.alpha..beta.) and
2(.alpha.) [0078] 5 g.degree. C. for 30 seconds for primers
3(.alpha.) and 4(.alpha.) [0079] 57.degree. C. for 30 seconds for
primers 1(.alpha..beta.) and 2(.alpha.) [0080] 63.degree. C. for 30
seconds for primers 3 (.beta.) and 4(.beta.) extension at
75.degree. C. for 75 seconds.
[0081] The PCR products were confirmed to be the expected size by
electrophoresis in a 2% agarose gel and ethidium bromide staining.
The fragments were then purified by passage over a Wizard column
(Promega) in accordance with the column manufacturer's
recommendations.
[0082] The final coding sequence for TBP1(20-161)-hCG.alpha. was
assembled by fusion PCR using primer 1(.alpha..beta.) and primer
4(.alpha.), and using as template the purified products from the
p55 and hCG .beta. fragments obtained from the first PCR reactions.
First the two templates, which due to the overlap between primers
2(.alpha.) and 3(.alpha.) could be denatured and annealed together,
were passed through 10 cycles of PCR in the absence of any added
primers. The conditions for these cycles were essentially the same
as those used earlier, except that the annealing was done at
67.degree. C. and the extension was performed for 2 minutes. At the
end of these 10 cycles, primers 1(.alpha..beta.) and 4(.alpha.)
were added, and another 10 cycles were performed. The conditions
for this final set of reactions was the same as used earlier,
except that an annealing temperature of 5 g.degree. C. was used,
and the extension was performed for 75 seconds.
[0083] Analysis of the products of this reaction by electrophoresis
in a 1% agarose gel confirmed that the expected fragment of about
1100 bp was obtained. The reaction was passed over a Wizard column
to purify the fragment, which was then digested with XbaI and BamHI
and re-purified in a 0.7% low-melting point agarose gel. The
purified fragment was subcloned into plasmid pSVL (Pharmacia),
which had first been digested with XbaI and BamHI and gel purified
on a 0.8% low-melting point agarose gel. Following ligation with T4
ligase, the mixture was used to transform AG1 E. coli and then
plated onto LB/ampicillin plates for overnight culture at
37.degree. C. Plasmid DNAs from ampicillin-resistant colonies were
analyzed by digestion with XhoI and BamHI to confirm the presence
of the insert (which is excised in this digest). Six clones were
found to contain inserts, and one (clone 7) was selected for
further advancement and designated pSVLTBPhCG.alpha. (containing
TBP1(20-161)-hCG.alpha.). Dideoxy DNA sequencing (using
Sequenase.TM., U.S. Biochemicals, Cleveland, Ohio) of the insert in
this vector confirmed that the construct was correct, and that no
undesired changes had been introduced.
[0084] The final coding sequence for TBP1(20-161)-hCG.beta. was
assembled in a manner similar to that described for
TBP1(20-161)-hCG.alpha. using fusion PCR and primers
1(.alpha..beta.) and 4(.beta.), and using as template the purified
products from the p55 and hCG.beta. fragments obtained from the
first PCR reactions. The resulting pSVL plasmid containing the
insert of interest was designated pSVLTBPhCG.beta..
[0085] b. TBP(20-190)-hCG
[0086] A second set of TBP-hCG proteins was prepared by
modification of the TBP(20-161)-hCG constructs to produce an analog
containing TBP spanning from Asp20 to Thr190, in place of the
20-161 region in the initial analog. This was done by replacing the
fragment between the BglII and XbaI sites in plasmid
pSVLTBPhCG.alpha. with a PCR fragment containing the change.
[0087] This PCR fragment was generated using fusion PCR. The
primers were:
TABLE-US-00003 (SEQ ID NO: 18) primer 1 TTT TAG ATC TCT TCT TGC ACA
GTG GAC (SEQ ID NO: 19) primer 2 TGT GGT GCC TGA GTC CTC AGT (SEQ
ID NO: 20) primer 3 ACT GAG GAC TCA GGC ACC ACA GCC GGT GCT GCC CCA
GGT TG (SEQ ID NO: 21) primer 4 TTT TTC TAG AGA AGC AGC AGC AGC CCA
TG
[0088] Primers 1 and 2 were used to generate the sequence coding
the additional p55 residues from 161-190. The PCR reaction was
performed essentially as described earlier, using 1 .mu.g of each
primer and pUC-p55 as template. Similarly, primers 3 and 4 were
used to generate by PCR the linker between the 3'-end of the
TBP-coding region, and the 5'-end of the hCG .alpha. subunit coding
region, using as a template plasmid pSVLTBPhCG.alpha.. Products
from these, PCR reactions were confirmed to be the correct size
(about 296 bp and 121 bp respectively) by polyacrylamide gel
electrophoresis (PAGE) on an 8% gel, and were then purified using a
Wizard column. The design of primers 2 and 3 was such that they
contained a region of overlap, so that the two PCR products (from
primers 1 and 2, and from primers 3 and 4) could be annealed for
fusion PCR with primers 1 and 4. Subsequent to the fusion reaction,
the desired product of about 400 bp was confirmed and purified
using a 1.5% agarose gel and a Wizard column. This DNA was then
digested with BglII and XbaI, and ligated with BglII/XbaI-digested
pSVLTBPhCG.alpha.. The presence of an insert in plasmids isolated
from transformed AG1 E. coli was confirmed by digestion with BglII
and XbaI. The new construct was designated
pSVLTBP(20-190)-hCG.alpha..
[0089] Similarly, plasmid pSVLTBPhCG.beta. was modified by
substitution of the BglII-XcmI fragment. However, this was done by
subcloning of a single PCR product, rather than with a fusion PCR
product. Primers 1 and 2b (see below) were used with pUC-p55 as the
template.
TABLE-US-00004 (SEQ ID NO: 22) primer 2b TTT TCC ACA GCC AGG GTG
GCA TTG ATG GGG CGG CAC CGT GGA CCA GCA CCA GCT GTG GTG CCT GAG TCC
TCA GTG
[0090] The resulting PCR product (about 337 bp) was confirmed and
purified as described above, digested with BglII and XcmI, and then
ligated into BglII/XbaI-digested pSVLTBPhCG.beta.. The presence of
an insert in plasmids isolated from transformed AG1 E. coli was
confirmed by digestion with BglII and XcmI. The new construct was
designated pSVLTBP(20-190)-hCG.beta..
[0091] The new constructs were subsequently confirmed by DNA
sequencing.
[0092] In addition to producing these new pSVL-based plasmids,
these constructs were also subcloned into other expression vectors
likely to be more suitable for stable CHO, particularly vector
D.alpha., described as plasmid CLH3AXSV2DHFR (45). This was
accomplished by converting a BamHI site flanking the inserts in the
pSVL-based vectors to an XhoI site, and then excising the insert
with XhoI and cloning it into XhoI digested D.alpha..
[0093] 2. Transient and Stable Expression of the Hybrid
Proteins
[0094] Transfections of COS-7 cells (ATCC CRL 1651, ref. 46) for
transient expression of the TBP-hCG hybrid proteins were performed
using electroporation (47). Exponentially growing COS-7 cells were
removed by trypsinization, collected by gentle centrifugation (800
rpm, 4 minutes), washed with cold phosphate buffered saline (PBS),
pH 7.3-7.4, and then repelleted by centrifugation. Cells were
resuspended at a concentration of 5.times.10.sup.6 cells per 400
.mu.l cold PBS and mixed with 10 .mu.g of plasmid DNA in a
prechilled 2 mm gap electroporation cuvette. For cotransfections, 5
.mu.g of each plasmid were used. The cuvette and cells were chilled
on ice for a further 10 minutes, and then subjected to
electroporation using a BTX Model 600 instrument and conditions of
125 V, 950 .mu.F and R=8. Afterward the cells were set to cool on
ice for 10 minutes, transferred to a 15 ml conical tube containing
9.5 ml complete medium (Dulbeccols modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine)
at room temperature, and left at room temperature for 5 minutes.
After gentle mixing in the 15 ml tube, the entire contents was
seeded onto two P100 plates and placed into a 37.degree. C., 5%
CO.sub.2 incubator. After 18 hours the media was changed, and in
some cases the new media contained only 1% or 0% FBS. After another
72 hours, the conditioned media was harvested, centrifuged to
remove cells, and then stored frozen at -70.degree. C.
[0095] Transfections of CHO-DUKX (CHO) cells for transient or
stable expression were performed using calcium phosphate
precipitation of DNA. Twenty-four hours prior to the transfection,
exponentially growing CHO cells were plated onto 100 mm culture
plates at a density of 7.5.times.10.sup.5 cells per plate. On the
day of the transfection, 10 .mu.g of plasmid DNA was brought to 0.5
ml in transfection buffer (see below), 31 .mu.g of 2 M CaCl.sub.2
were added, the DNA-CaCl.sub.2, solution was mixed by vortexing,
and left to stand at room temperature for 45 minutes. After this
the media was aspirated from the plates, the DNA was added to the
cells using a sterile plastic pipette, and the cells were left at
room temperature for 20 minutes. At the end of this period, 5 ml of
complete .alpha.(+)MEM containing 10% FBS was added to the plates,
which were incubated at 37.degree. C. for 4-6 hours. The media was
then aspirated off the plates, and the cells were subjected to a
glycerol shock by incubating them with a solution of 15% glycerol
in transfection buffer at 37.degree. C. for 3.5 minutes. After
removal of the glycerol solution, the cells were washed twice with
PBS, refed with 10 ml complete .alpha.(+)MEM, 10% FBS, and returned
to the 37.degree. C. incubator. For stable transfections, after 48
hours the cells were split 1:10 and fed with selection medium
(complete .alpha.-minus MEM (lacking nucleosides), 10% dialyzed
FBS, and 0.02 pM methotrexate). Non-transfected (non-resistant)
cells were typically eliminated in 3-4 weeks, leaving a population
of transfected, methotrexate-resistant cells.
[0096] 3. Quantitation of Expression
[0097] Secretion of the hybrid proteins by transfected cells was
assessed using a commercial assay kit for soluble p55 (R&D
Systems; Minneapolis, Minn.) in accordance with the manufacturer's
instructions. This assay also provides an estimate of the hybrid
protein levels in conditioned and processed media, which served as
the basis for selecting doses to be used in the bioassay.
[0098] 4. Assessment of Heterodimer Formation
[0099] To assess the ability of the TBP-hCG subunit fusions to
combine and form heterodimers, a sandwich immunoassay using
antibodies to the hCG subunits was performed. In this assay, a
monoclonal antibody to the hCG .beta. subunit is coated onto
microtiter plates and used for analyte capture. The primary
detection antibody is a goat polyclonal raised against the human
TSH .alpha. subunit (#082422G--Biodesign International;
Kennenbunkport, Me.), which is in turn detected using a horse
radish peroxidase conjugated rabbit anti-goat polyclonal antibody
(Cappel; Durham, N.C.).
[0100] Several different anti-hCG .beta. subunit antibodies were
used in this work, all of which show no detectable crossreactivity
with the free .alpha. subunit. One of these antibodies (3/6) is
used in the commercially available MAIAclone hCG assay kit
(Biodata; Rome, Italy).
[0101] High-protein binding microtiter plates (Costar #3590) were
coated with capture antibody by incubation (2 hours at 37.degree.
C.) with 100 .mu.l/well of a 5 .mu.g/ml solution of antibody in
coating buffer (PBS, pH 7.4, 0.1 mM Ca++, 0.1 mM Mg++). After
washing once with wash solution (PBS, pH 7.4+0.1% Tween 20) the
plate is blocked by completely filling the wells (.apprxeq.400
.mu.l/well) with blocking solution (3% bovine serum albumin (BSA;
fraction V-A-4503 Sigma) in PBS, pH 7.4) and incubating for one
hour at 37.degree. C. or overnight at 4.degree. C. The plate is
then washed twice with wash solution, and the reference and
experimental samples, diluted in diluent (5 mg/ml BSA in PBS, pH
7.4) to yield a 100 .mu.l volume, are added. After incubating the
samples and the plate for two hours at 37.degree. C., the plate is
again twice washed with wash solution. The primary detection
antibody, diluted 1:5000 in diluent, is added (100 .mu.l/well) and
incubated for one hour at 37.degree. C. The secondary detection
antibody (HRP conjugated rabbit anti-goat Ig), diluted 1:5000 in
diluent, is added (100 .mu.l/well) and after incubation for one
hour at 37.degree. C., the plate is washed three times with wash
solution. One hundred .mu.l of TMB substrate solution (Kirkegaard
and Perry Laboratories) is added, the plate is incubated 20 minutes
in the dark at room temperature, and then the enzymatic reaction is
stopped by addition of 50, .mu.l/well 0.3M H.sub.2SO.sub.4. The
plate is then analyzed using a microtiter plate reader set for a
wavelength of 450 nm.
[0102] 5. Partial Purification
[0103] To better quantitate the activities of these hybrid
proteins, TBP-hCG hybrid proteins were partially purified by
immunoaffinity chromatography. The antibody used was a monoclonal
commercially available from R&D Systems (MAB #225). The column
was CNBr-activated sepharose, charged with the antibody by
following the manufacturer's (Pharmacia) instructions.
[0104] Conditioned media was collected from confluent T-175 flasks
of each line using daily harvests of 50 ml SFMII media (GIBCO),
five harvests for each line. The collections were subjected to
centrifugation (1000 RPM) to remove cellular debris. The material
was then assayed for TBP content using the commercial immunoassay
and concentrated (Centricon units by Amicon; Beverly, Mass.) so
that the apparent TBP concentration was about 50 ng/ml.
[0105] Ten ml of the concentrated TBP-hCG (sample #18873) was
brought to approximately 1 M NaCl by addition of NaCl and
adjustment of the solution to a conductivity of approximately 85
mS/cm. This was passed through a 0.5 ml anti-TBP immunoaffinity
column, The flow-through was collected and run through the column a
second time. After this the column was washed with 1 M NaCl in PBS.
The bound TBP(20-161)-hCG was collected after elution with 50 mM
citric acid (pH 2.5). The eluate (approximately 7 ml) was
concentrated by filtration using Amicon Centricon-10's in
accordance with the manufacturer's (Amicon) instructions, to a
volume of approximately 200 .mu.l. Approximately 800 .mu.l of PBS
was added to bring the sample volume to 1 ml, which was stored at
4.degree. C. until tested by bioassay.
[0106] 6. Assessment of Anti-TNF Activity
[0107] Numerous in vitro TNF-induced cytotoxicity assays have been
described for evaluating analogs of soluble TNF receptors. We
utilized an assay employing a human breast carcinoma cell line,
BT-20 cells (ATCC HTB 19). The use of these cells as the basis for
a TNF bioassay has been described previously (48). These cells are
cultured at 37.degree. C. in RPMI 1640 media supplemented with 10%
heat-inactivated FBS. The cells were grown to a maximum 80-90%
confluence, which entailed splitting every 3-4 days with a seeding
density of about 3.times.10.sup.6 cells per T175 cm.sup.2
flask.
[0108] The BT-20 assay uses a cellular stain, crystal violet, as a
detection method to assess survival of cells after treatment with
TNF. Dead cells are unable to take up and retain the dye.
[0109] In brief, the protocol used for the assay of anti-TNF
activity is the following. Recombinant human TNF.alpha. (R&D
Systems) and the experimental samples are constituted in media
(RPMI 1640 with 5% heat-inactivated FBS) and added to the wells of
96-well culture plates. The cells are then plated into these wells
at a density of 1.times.10.sup.5 cells/well. The quantity of
TNF.alpha. added was determined earlier in titration studies, and
represents a dose at which about 50% of the cells are killed.
[0110] After addition of the samples, the cells are cultured for 48
hours at 39.degree. C., after which the proportion of live cells is
determined using crystal violet staining and a microtiter plate
reader (570 nm).
Results
[0111] 1. Constructs Under Study
[0112] The designs of the hybrid proteins studied are briefly
summarized below; two control proteins, a monomeric soluble p55
(r-hTBP-1) and a dimeric TBP-immunoglobulin fusion protein
(TBP-1gG3) (prepared essentially as described in (10)), were
studied for comparative purposes.
TABLE-US-00005 Fusion Construct TBP N-term TBP C-term partner
r-hTBP-1 mix of 9 and 20 180 none TBP-IgG3 mix of 9 and 20 190 IgG3
heavy chain constant region TBP(20-161)-hcG 20 161 hCG.alpha. and
hCG.beta. (heterodimer) TBP(20-190)-hcG 20 190 hCG.alpha. and
hCG.beta. (heterodimer)
[0113] The sequences of the DNAs encoding, TBP(20-190)-hCG and
TBP(20-161)-hCG are provided in FIGS. 1 and 2, respectively. A
schematic summary of the constructs is provided in FIG. 3.
[0114] 2. Secretion of TBP-hCG Proteins
[0115] All of the constructs tested were found to be produced and
secreted into culture media by transfected mammalian cells. Data
illustrating this are shown in Tables 1 and 2.
TABLE-US-00006 TABLE 1 COS-7 transient expression (TBP ELISA)
Concentration Hybrid Protein (pg/ml) TBP1 66 TBP-hCG.alpha.(20-161)
5.1 TBP-hCG.beta.(20-161) 0.5 TBP-hCG(20-161) 2.7 Control <0.25
Constructs were expressed using pSVL (Pharmacia)
TABLE-US-00007 TABLE 2 COS-7 transient expression (TBP ELISA)
Concentration Hybrid Protein (ng/ml) TBP1 131
TBP-hCG.alpha.(20-190) 81 TBP-hCG.beta.(20-190) 9 TBP-hCG(20-190)
62 Control <1 Constructs were expressed using a mouse
Metallothionein promoter-containing vector = pD.alpha.
[0116] 3. TBP-hCG (.alpha./.beta.) Fusion Proteins Assemble into
Heterodimers
[0117] The combination of TBP-hCG.alpha. and TBP-hCG.beta. was
confirmed using the sandwich assay for the hCG heterodimer.
[0118] Only the combined transfection of .alpha. and .beta. subunit
fusions resulted in heterodimer detection (Table 3).
TABLE-US-00008 TABLE 3 COS-7 transient expression (hCG heterodimer
assay) Concentration Hybrid Protein (ng/ml) TBP1 <0.2
TBP-hCG.alpha.(20-190) <0.2 TBP-hCG.beta.(20-190) <0.2
TBP-hCG(20-190) 38 Control <0.2 Constructs were expressed using
a mouse metallothionein promoter-containing vector - pD.alpha.
[0119] 4. TBP-hCG Hybrid Proteins Exhibit Increased Activity Over
TBP Monomer
[0120] Hybrid proteins produced in either COS-7 or CHO cells were
found to be potent inhibitors of TNF.alpha. in the BT-20 bioassay.
Some of the samples tested are summarized in Table 4.
TABLE-US-00009 TABLE 4 Samples tested for anti-TNF activity Cell
Construct source Nature of sample r-hTBP-1 CHO Purified TBP-lgG3
CHO 1x conditioned media TBP(20-161)hCG CHO Immunopurified
(anti-TBP) TBP(20-190)-hCG CHO 1x conditioned media TBP(20-190)-hCG
COS 1x conditioned media
[0121] Negative controls (conditioned media from mock
transfections) were included for the 1.times. media samples.
[0122] As illustrated in FIGS. 4-6 (points on y-axis), addition of
TNF (2.5 ng/ml) results in a clear reduction in live cell number
(as assessed by OD 570). In every case, active samples have as a
maximal protective effect the restoration of cell viability to the
level seen in the absence of added TNF (i.e., the control labeled
"cells alone").
[0123] The positive controls, r-hTBP-1 and TBP-IgG3, are both
protective, showing a clear dose-dependence and ED50s of
approximately 100 ng/ml for the r-hTBP-1 (FIGS. 4-6) and about 1.5
ng/ml for TBP-IgG3 (FIG. 4) respectively.
[0124] The TBP-hCG constructs from 1.times. media (CHO or COS) or
from the immunopurification show dose-dependent protection, with
approximate EDSOs ranging from 2-11 ng/ml (FIGS. 4-6).
[0125] The results from the in vitro bioassay are reported in Table
5. The data indicate that the hybrid proteins inhibit TNF
cytotoxicity, and that they are substantially more potent than the
TBF monomer. The negative controls were devoid of protective
activity.
TABLE-US-00010 TABLE 5 Preliminary Assessment of the hybrid
proteins in TNF Cytotoxicity Assay Anti-TNF activity (ED50)
Construct Fusion partner in BT-20 bioassay** r-hTBP-1 None 100
ng/ml TBP-lgG3 IgG3 heavy chain constant 1.5 ng/ml region
TBP(20-161)-hCG HCG.alpha. and hCG.beta. 2 ng/ml (heterodimer)
TBP(20-190)-hCG HCG.alpha. and hCG.beta. 8-11 ng/ml (heterodimer)
**The quantitation of material for dosing and estimation of ED50
was made using the TBP ELISA.
[0126] In addition to the possibility that dimerization of TBP may
increase potency, it is also possible that the activity of the
hybrid proteins are not related to dimeric interaction with TBP,
but rather to steric inhibition due to the partner of the hybrid
interfering with soluble TBP/TNF binding to cell-surface TNF
receptors.
[0127] All references cited herein, including journal articles or
abstracts, published or corresponding U.S. or foreign patent
applications, issued U.S. or foreign patents, or any other
references, are entirely incorporated by reference herein,
including all data, tables, figures, and text presented in the
cited references. Additionally, the entire contents of the
references cited within the references cited herein are also
entirely incorporated by reference.
[0128] Reference to known method steps, conventional method steps,
known methods or conventional methods is not in any way an
admission that any aspect, description or embodiment of the present
invention is disclosed, taught or suggested in the relevant
art.
[0129] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying knowledge within the skill of the art (including
the contents of the references cited herein), readily modify and/or
adapt for various applications such specific embodiments, without
undue experimentation, without departing from the general concept
of the present invention. Therefore, such adaptations and
modifications are intended to be within the meaning and range of
equivalents of the disclosed embodiments, based on the teaching and
guidance presented herein. It is to be understood that the
phraseology or terminology herein is for the purpose of description
and not of limitation, such that the terminology or phraseology of
the present specification is to be interpreted by the skilled
artisan in light of the teachings and guidance presented herein, in
combination with the knowledge of one of ordinary skill in the
art.
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Sequence CWU 1
1
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