U.S. patent application number 08/987756 was filed with the patent office on 2002-05-09 for soluble recombinant alpha beta adhesion receptor.
Invention is credited to BROWN, ALEX, CULLEN, EILISH, DIEFENBACH, BEATE, GOODMAN, SIMON L., GUESSOW, DETLEF, MEHTA, RAJ J..
Application Number | 20020055136 08/987756 |
Document ID | / |
Family ID | 8223479 |
Filed Date | 2002-05-09 |
United States Patent
Application |
20020055136 |
Kind Code |
A1 |
GOODMAN, SIMON L. ; et
al. |
May 9, 2002 |
SOLUBLE RECOMBINANT ALPHA BETA ADHESION RECEPTOR
Abstract
The invention relates to a novel purified recombinant
.alpha..sub.v.beta..sub.3 adhesion receptor which shows an
unimpaired ligand binding activity, and a process for preparing
said soluble non-membrane bound receptor in excellent yields by
recombinant techniques using a baculovirus-insect cell expression
system. The so-synthesized soluble receptor may be used very easily
as screening tool for new therapeutic compounds which may inhibit
the natural .alpha..sub.v.beta..sub.3 adhesion receptor. Such
therapeutic compounds which can be discovered very easily, fast and
without health risk by means of the souluble receptors according to
the invention may be, for example, RGD peptides or non-peptidic
compounds mimicking the natural ligand epitopes. The invention
relates, furthermore, to a corresponding process for preparing
recombinant full-length .alpha..sub.v.beta..sub.3 adhesion receptor
in excellent yields, additionally using detergents to dissolve the
membrane bound receptor from the surface of the host cell.
Inventors: |
GOODMAN, SIMON L.;
(DARMSTADT, DE) ; DIEFENBACH, BEATE; (DARMSTADT,
DE) ; GUESSOW, DETLEF; (DARMSTADT, DE) ;
MEHTA, RAJ J.; (DARMSTADT, DE) ; CULLEN, EILISH;
(DARMSTADT, DE) ; BROWN, ALEX; (DARMSTADT,
DE) |
Correspondence
Address: |
MILLEN WHITE ZELANO & BRANIGAN
220 CLARENDON BLVD
SUITE 1400
ARLINGTON
VA
22201
|
Family ID: |
8223479 |
Appl. No.: |
08/987756 |
Filed: |
December 9, 1997 |
Current U.S.
Class: |
435/69.1 ;
435/7.1; 436/63; 530/350 |
Current CPC
Class: |
C07K 14/70557 20130101;
C12N 2799/026 20130101 |
Class at
Publication: |
435/69.1 ;
530/350; 436/63; 435/7.1 |
International
Class: |
G01N 033/53; G01N
033/48; C12P 021/06; C07K 001/00; C07K 014/00; C07K 017/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 9, 1996 |
GB |
EP96119700.1 |
Claims
What is claimed is:
1. A purified soluble recombinant .alpha..sub.v.beta..sub.3
adhesion receptor which retains a ligand binding activity.
2. A soluble human .alpha..sub.v.beta..sub.3 adhesion receptor of
claim 1.
3. A receptor of claim 2, comprising the .alpha..sub.v chain and
the .beta..sub.3 chain of said receptor, wherein each chain is
shortened at its C-terminus by a portion containing the
transmembrane domain or a portion thereof and the complete
cytoplasmic domain.
4. A receptor of claim 3, comprising a truncated .alpha..sub.v
chain containing approximately 957 amino acids and a truncated
.beta..sub.3 chain containing approximately 692 amino acids, each
starting at the N-terminal of the corresponding mature protein
chain.
5. A human .alpha..sub.v.beta..sub.3 adhesion receptor, obtainable
by a process as defined in claim 7.
6. A receptor of claim 12, wherein the truncated .alpha..sub.v
chain has a molecular weight of 120 kD (.+-.12 kD) and the
truncated .beta..sub.3 chain has a molecular weight of
approximately 80 kD (.+-.8 kD), each determined by SDS-PAGE under
non-reducing conditions.
7. A process for preparing a large amount of a highly purified
soluble recombinant human .alpha..sub.v.beta..sub.3 adhesion
receptor which retains a ligand binding activity, comprising (i)
subcloning a first cDNA coding for the .alpha..sub.v chain of said
receptor, shortened by a portion comprising at least 52 amino acids
calculated from the C-terminus, and a second cDNA coding for the
.beta..sub.3 chain of said receptor, shortened by a portion
comprising at least 61 amino acids calculated from the C-terminus,
into a baculovirus transfer vector of a baculovirus expression
system, (ii) transferring said vector comprising said first and/or
second DNA into the genomic DNA of a baculovirus of said expression
system, (iii) infecting an insect cell with said complete
recombinant baculovirus, (iv) cultivating said infected insect
cells in a culture medium whereby said heteromeric truncated
.alpha..sub.v.beta..sub- .3 receptor is expressed into the medium,
and (v) purifying said expressed receptor from the medium by
antibody affinity chromatography, wherein the antibody is specific
to the human .alpha..sub.v.beta..sub.3 adhesion receptor or its
individual component chains.
8. A process of claim 7, wherein the first and second cDNA are
sub-cloned into the same baculovirus vector.
9. A process of claim 7, wherein the baculovirus expression system
is the BacPAK system.
10. A process of claim 7, wherein the insect cells infected are
High Five (BTI-TN-5B1-4) cells.
11. A process of claim 7, wherein the specific antibody used for
the antibody affinity chromatography is mAb 17E6 produced by a
hybridoma cell line having the designation 272-17E6 (DSM
ACC2160).
12. A process of claim 7, wherein the first cDNA codes for the
.alpha..sub.v chain of said receptor, shortened by a portion
encoding approximately 61 amino acids starting at the C-terminus
and which corresponds to a mature protein chain containing
approximately 957 amino acids, and the second cDNA codes for the
.beta..sub.3 chain of said receptor, shortened by a portion
encoding approximately 70 amino acids starting at the C-terminus
and which corresponds to a mature protein chain containing
approximately 692 amino acids.
13. A process of claim 12, wherein the cDNA of the truncated
.alpha..sub.v chain is generated by PCR using the oligonucleotide
primers
7 5'-GAC CAG CAT TTA CAG TGA-3' and 5'-CA CAG GTC TAG ACT ATG GCT
GAA TGC CCC AGG-3',
and the cDNA of the truncated .beta..sub.3 chain is generated by
PCR using the oligonucleotide primers 5'
8 5'-GCG CGC AAG CTT GCC GCC ACC ATG CGA GCG CGG CCG-3' and 5'GAT
CGA TCT AGA CTA GGT CAG GGC CCT TGG GAC ACT-3'.
14. A method of screening a compound for its capability to inhibit
an activity of a natural .alpha..sub.v.beta..sub.3 receptor,
comprising contacting a soluble .alpha..sub.v.beta..sub.3 receptor
of claim 1 with a compound which may inhibit said
.alpha..sub.v.beta..sub.3 receptor, and determining of the receptor
activity is inhibited.
15. A method of claim 14, wherein the compound is an RGD-peptide or
a non-peptidic analogue.
16. A method of claim 15, wherein the RGD-peptide or a non-peptidic
analogue compound is modified by a detectable marker and reacted
with said immobilized purified soluble .alpha..sub.v.beta..sub.3
receptor, and the amount of ligand bound to said receptor is
measured.
17. A process for preparing large amounts of a highly purified
intact full-length recombinant human .alpha..sub.v.beta..sub.3
adhesion receptor, comprising (i) subcloning a first cDNA coding
for the complete a chain of said receptor and a second cDNA coding
for the .beta..sub.3 chain of said receptor into a baculovirus
transfer vector of a baculovirus expression system, (ii)
transferring said vector comprising said first and/or second DNA
into the genomic DNA of a baculovirus of said expression system,
(iii) infecting insect cells with said complete recombinant
baculovirus, (iv) cultivating said infected insect cells in a
culture medium, whereby said receptor is expressed into the cell
memembrane, (v) solubilizing said expressed cell membrane-bound
receptor with a detergent, and (vi) purifying said solubilized
receptor from the cell-free medium by antibody affinity
chromatography, wherein the antibody is specific to the human
.alpha..sub.v.beta..sub.3 adhesion receptor or its individual
component chains.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The invention relates to a novel purified recombinant
.alpha..sub.v.beta..sub.3 adhesion receptor which shows an
unimpaired ligand binding activity, and a process for preparing
said soluble non-membrane bound receptor in excellent yields by
recombinant techniques using a baculovirus-insect cell expression
system.
[0002] The so-synthesized soluble receptor may be used very easily
as screening tool for new therapeutic compounds which may inhibit
the natural .alpha..sub.v.beta..sub.3 adhesion receptor.
[0003] The invention relates, furthermore, to a corresponding
process for preparing recombinant full-length
.alpha..sub.v.beta..sub.3 adhesion receptor in excellent yields,
additionally using detergents to solve the membrane bound receptor
from the surface of the host cell.
BACKGROUND OF THE INVENTION
[0004] Integrins are a super-family of cell surface adhesion
receptors which control the attachment of cells with the solid
extracellular environment both to the extracellular matrix (ECM),
and to other cells. Adhesion is of fundamental importance to a
cell; it provides anchorage and cues for migration and signals for
growth and differentiation. Integrins are directly involved in
numerous normal and pathological events, such as cell attachment,
migration to blood clotting, inflammation, embryogenesis or cancer
growth and metastasis, and as such, they are primary targets for
therapeutic intervention.
[0005] Integrins are heterodimeric integral transmembrane
glycoproteins, consisting of non-covalently linked .alpha. and
.beta. subunits. The integrins are classified in four overlapping
subfamilies, containing the .beta..sub.1, .beta..sub.2,
.beta..sub.3 or .alpha..sub.v chains, and a particular cell may
express several different integrins from each subfamily.
[0006] The last decade has shown that integrins are major receptors
involved in cell adhesion and so may be a suitable target for
therapeutic intervention. Reports concerning integrins are given,
e.g., by E. Ruoslahti (J. Clin. Invest., 1991, 87) and R. O. Hynes
(Cell, 1992, 69).
[0007] The .alpha..sub.v-series integrins are now seen to be a
major subfamily, with both classical, and novel functions. In
melanoma cell lines, for example, enhanced expression of the
.alpha..sub.v.beta..sub.3 integrin correlated with tumorigenicity
and metastatic properties (e.g. Felding-Habermann et al. 1992, J.
Clin. Invest. 89, 2018; Marshall et al. 1991, Int. J. Cancer 49,
924), suggesting the involvement of .alpha..sub.v-containing
integrins in some steps of the tumor cell metastasis process. A
similar role is discussed for carcinoma adhesion and spreading and
colorectal cancer, respectively, which is one of the most common
epithelial malignancies.
[0008] The .alpha..sub.v-series integrins seem to exclusively
recognize ligands bearing the RGD-
(NH.sub.2--arginine-glycine-aspartic acid--COOH) tripeptide
sequences, including those in vitronectin
(.alpha..sub.v.beta..sub.1, .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5), fibronection
(.alpha..sub.v.beta..sub.1, .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5, .alpha..sub.v.beta..sub.6) and von
Willebrand factor, fibrinogen and osteopontin
(.alpha..sub.v.beta..sub.3) (e.g., Busk et al. 1992, J. Biol. Chem.
267, 5790; Smith and Cheresh 1990, J. Biol. Chem. 265, 2168).
Function blocking anti-integrin antibodies, e.g., antibodies
directed against the .alpha..sub.v.beta..sub.3 integrin are also
known (e.g., Cheresh and Spiro 1987, J. Biol. Chem. 262, 17703;
Chuntharapai et al. 1993, Exp. Cell Res. 205, 345; EP 95 119
233).
[0009] The disruption of ligand-integrin interaction by peptides
and antibodies has highlighted the important roles of
.alpha..sub.v.beta..sub- .3 integrin, "the vitronectin receptor,"
processes as diverse as tumor growth and metastasis, viral
infection, osteoporosis and angiogenesis (Felding-Habermann and
Cheresh 1993, Curr. Opin. Cell Biol. 5, 864; Brooks et al. 1994,
Cell 79, 1157; Brooks et al. 1994, Science 264, 569). The emergence
of .alpha..sub.v.beta..sub.3 as a potential target for therapeutic
intervention has thus led to a search for .alpha..sub.v.beta..sub.3
antagonists--a process that requires purified
.alpha..sub.v.beta..sub.3. The source of biochemical amounts of
.alpha..sub.v.beta..sub.3 has usually been human placental tissue.
In common with most integral membrane proteins, integrins can only
be obtained as active solubilized molecules in the presence of
non-ionic detergents and therefore purification of
.alpha..sub.v.beta..sub.3 from placenta for drug screening is both
cumbersome, costly, and a considerable health risk (Mitjans et al.
1995, J. Cell Sci. 108, 2825).
[0010] Although recombinant integrins have been expressed already
in eukaryotic systems, such as in CHO cells cells (O'Toole et al.
1990, Cell Regul. 1, 883) and in embryonic kidney 293 cells (King
et al. 1994, J. Bone Miner Res. 9, 381) the preparation of
biochemically satisfactory amounts has not been achieved by
recombinant technology.
[0011] There have been no reports on the biological activity of
truncated soluble .alpha..sub.v.beta..sub.3 constructs, and
full-length .alpha..sub.v.beta..sub.3 receptor apparently has not
been produced by recombinant technology in biochemically important
amounts.
[0012] Thus, it would be desirable to provide a soluble recombinant
.alpha..sub.v.beta..sub.3 receptor which can be produced in
biochemically useful amounts and in a purified quality by a
comparably easy and riskless process. Moreover, such a process
would be also advantageous for producing full-length
.alpha..sub.v.beta..sub.3 receptor.
SUMMARY OF THE INVENTION
[0013] This invention describes for the first time high-level
expression and purification of biologically competent soluble
.alpha..sub.v.beta..sub.3 using the baculovirus-insect cell
expression system. To achieve high yields of recombinant proteins
processed in a manner similar to mammalian cells, over-expression
of .alpha..sub.v.beta..sub.3 was achieved by utilizing the
baculovirus polyhedrin gene as a strong viral promotor. The soluble
molecule retains the ligand binding activity and specificity of the
native placental and recombinant full length receptors, and can be
purified in the absence of detergents, thus removing many possible
ligation artefacts and producing a molecule suitable for high
throughput screening and high resolution structural analysis.
[0014] Thus it is object of the present invention to provide an
essentially purified soluble recombinant .alpha..sub.v.beta..sub.3
adhesion receptor with unimpaired ligand binding activity. A
corresponding receptor which derives from human origin is a
preferred embodiment of the invention.
[0015] The receptor according to the invention comprises the
.alpha..sub.v chain and the .beta..sub.3 chain of said receptor,
each shortened at its C-terminus by a portion containing the
transmenmbrane domain or a portion thereof and the complete
cytoplasmatic domain of each individual chain. The amino acid and
DNA sequences of the mature human .alpha..sub.v and .beta..sub.3
protein chain as well their transmembrane and cytoplasmatic domains
are known.
[0016] Especially, it is a preferred object of the invention to
make available a soluble human recombinant receptor comprising a
truncated .alpha..sub.v chain containing approximately 957 amino
and a truncated .beta..sub.3 chain containing approximately 692
amino acids, each calculated from the N-terminal of the
corresponding mature protein chain. From this preferred soluble
receptor the essentially complete transmembrane domain as well as
the complete cytoplasmatic domain of the mature protein chains has
been removed. However, the invention includes also soluble
receptors comprising portions of the transmembrane domain (see
below). The invention, however, is not limited to human
.alpha..sub.v.beta..sub.3 receptor. According to the process
described and claimed here it is no problem to generate
(.alpha..sub.v.beta..sub.3 receptors deriving from non-human
origin.
[0017] Furthermore, a corresponding human receptor can be produced
by the present invention which is obtainable by a process as
defined below and in the claims. This process according to the
invention provides highly purified soluble recombinant human
.alpha..sub.v.beta..sub.3 adhesion receptor with unimpaired ligand
binding activity in good yields, and is characterized by the
following steps:
[0018] (i) a first cDNA coding for the .alpha..sub.v chain of said
receptor, shortened by a portion comprising at least 52 amino acids
calculated from the C-terminus, and a second cDNA coding for the
.beta..sub.3 chain of said receptor, shortened by a portion
comprising at least 61 amino acids calculated from the C-terminus,
is sub-cloned into a baculovirus transfer vector of a baculovirus
expression system,
[0019] (ii) said vector comprising said first and/or second DNA is
transfered into the genomic DNA of a baculovirus of said expression
system,
[0020] (iii) insect cells are infected with said complete
recombinant baculovirus,
[0021] (iv) said infected insect cells are cultivated in a culture
medium and said heteromeric truncated .alpha..sub.v.beta..sub.3
receptor is expressed into the medium, and
[0022] (v) said receptor expressed is isolated from the medium and
purified by antibody affinity chromatography, the antibody used
hereby is specific to the human .alpha..sub.v.beta..sub.3 adhesion
receptor or its individual component chains.
[0023] As another aspect of the invention a similar process for the
preparation of recombinant full-length .alpha..sub.v.beta..sub.3
receptor, especially of human origin, is disclosed. This process is
characterized by the following steps:
[0024] (i) a first cDNA coding for the complete .alpha..sub.v chain
of said receptor and a second cDNA coding for the complete
.beta..sub.3 chain of said receptor, is sub-cloned into a
baculovirus transfer vector of a baculovirus expression system,
[0025] (ii) said vector comprising said first and/or second DNA is
transfered into the genomic DNA of a baculovirus of said expression
system,
[0026] (iii) insect cells are infected with said complete
recombinant baculovirus,
[0027] (iv) said infected insect cells are cultivated in a culture
medium, said cell membrane-bound receptor expressed is solubilized
by a detergent, and
[0028] (v) said solubilized receptor is purified from the cell-free
medium by antibody affinity chromatography, the antibody used
hereby is specific to the human .alpha..sub.v.beta..sub.3 adhesion
receptor or its individual component chains.
[0029] The .alpha..sub.v.beta..sub.3 receptors according to the
invention are optimally suitable for the screening of compounds,
e.g., therapeutic compounds, which may inhibit the natural
receptor. Therefore, the invention relates to the use of said
soluble receptors for the discovery of said compounds. As mentioned
above such compounds according to the invention are, above all,
linear or cyclic peptides comprising one or more RGD units or they
are non-peptidic analogues which show the same biological
activities against the .alpha..sub.v.beta..sub.3 adhesion receptor
as the RGD peptides (see below).
[0030] Finally the invention relates to a process of screening
therapeutic compounds for their capability to inhibit the natural
.alpha..sub.v.beta..sub.3 receptor by using said soluble
.alpha..sub.v62 .sub.3 receptor as defined above and below and a
therapeutic compound, especially RGD-peptides or non-peptidic
analogues thereof, which may inhibit said .alpha..sub.v.beta..sub.3
receptor.
1 Abbreviations: .alpha..sub.V.beta..sub.3 integrin 17E6 mAb AP3
mAb LM609 mAb P4C19 mAb P1F6 mAb .alpha..sub.V(FL) full-length
.alpha..sub.V chain .alpha..sub.V(SL) short-length (truncated)
.alpha..sub.V chain .beta..sub.3(FL) full-length .beta..sub.3 chain
.beta..sub.3(SL) short-length (truncated) .beta..sub.3 chain BacPAK
Baculovirus expression system SF9, HighFive Insect cells M.O.I.
Multiplicity of Infection
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Various other features and attendant advantages of the
present invention will be more fully appreciated as the same
becomes better understood when considered in conjunction with the
accompanying drawings, wherein:
[0032] FIG. 1 shows a diagram of the .alpha..sub.v.beta..sub.3
heterodimer showing the sites for truncation of the .alpha..sub.v
and .beta..sub.3 chains. The .alpha..sub.v chain is normally post
translationally cleaved into heavy (.alpha..sub.vHC) and light
(.alpha..sub.vLC) chains that are linked with a disulfide (S--S)
bond.
[0033] EC=Extracellular; PM=Plasma membrane; IC=Intracellular;
IAC=Integrin Associated Cytoskeleton; Lig=Ligand
[0034] FIG. 2 shows
[0035] (A) Mock infected Sf9 cells (lane 1) or Sf9 cells infected
with null recombinant (lane 2), .alpha..sub.v(FL) (lane 3) or
.beta..sub.v(FL) (lane 4) were metabolically labelled with
.sup.35S-labelled amino acids for 2 hours at 40 hours post
infection. The cell extracts were resolved by SDS-PAGE under
reducing conditions. The molecular mass in kDa of standard protein
markers are indicated on the right.
[0036] (B) Sf9 cells infected with .alpha..sub.v(FL) (lanes 1 and
2), .alpha..sub.v(SL) (lane 3 and 4), .beta..sub.3(FL) (lane 5 and
6) and .beta..sub.3(SL) (lane 7 and 8) were either treated with 10
.mu.M Tunicamycin in DMSO (lanes 1, 3, 5 and 7) or with DMSO alone
(lanes 2, 4, 6 and 8). The infected cells were metabolically pulse
labelled as above. Standard protein markers are indicated on the
right.
[0037] FIG. 3 shows cell shape change after co-infection. Sf9 cells
infected with either null recombinant virus (A),
.alpha..sub.v(FL)+.beta.- .sub.3(FL) (B) or
.alpha..sub.v(SL)+.beta..sub.3(SL) (C) at 48 hours post infection
were photographed under phase contrast.
[0038] FIG. 4 shows immunoprecipitation of cell surface and soluble
.alpha..sub.v.beta..sub.3. Cell extracts (lanes 1-5) and cell
conditioned medium (lanes 6-10) prepared from High Five cells
co-infected with .alpha..sub.v(FL)+.beta..sub.3(FL) (lanes 1 and
6), .alpha..sub.v(FL)+.beta..sub.3(SL) (lanes 2 and 7),
.alpha..sub.v(SL)+.beta..sub.3(FL) (lanes 3 and 8),
.alpha..sub.v(SL)+.beta..sub.3(SL) (lanes 4 and 9) or null
recombinant (lanes 5 and 10). .alpha..sub.v.beta..sub.3 complex was
immunoprecipitated using monoclonal antibody LM609 and resolved by
SDS-PAGE under non-reducing conditions, transfered onto PVDF
membrane and detected with Streptavidin-HRP conjugate.
[0039] FIG. 5 shows analysis of purified receptors. Epitope and
purity analysis of purified full length and truncated
.alpha..sub.v.beta..sub.3 by monoclonal antibodies. Increasing
receptor concentrations were immobilized and analyzed with
antibodies LM609 (anti-.alpha..sub.v.beta..- sub.3), 17E6
(anti-.alpha..sub.v), AP3 (anti-.beta..sub.3), P4C.sub.10
(anti-.beta..sub.1) and P1F6 (anti-.alpha..sub.v.beta..sub.5).
Vertical axis: optical density at 450 nm; horizontal axis:
concentration of receptor coating (.mu.g/ml).
[0040] (.largecircle.) .alpha..sub.v.beta..sub.3 placenta,
(.box-solid.) .alpha..sub.v(FL).times..beta..sub.3(FL),
(.diamond-solid.) .alpha..sub.v(SL).times..beta..sub.3(SL)
[0041] FIG. 6 shows analysis of purified receptors. Analysis of
purified receptors by SDS-PAGE: Lane 1 and 5 HMW, lane 2 and 6
.alpha..sub.v.beta..sub.3 placenta, lane 3 and 7
.alpha..sub.v(FL).times.- .beta..sub.3(FL), lane 4 and 8
.alpha..sub.v(SL).times..beta..sub.3(SL). Lane 1, 3, 5, 7 are under
nonreducing, lane 2, 4, 6, 8 are under reducing conditions.
[0042] FIG. 7 shows activity test of purified recombinant
.alpha..sub.v.beta..sub.3. Ligand binding to immobilized receptors:
Biotinylated vitronectin, fibrinogen and fibronectin were allowed
to bind to immobilized receptors. Bound ligand was detected using
an anti-biotin antibody. Vertical axis: ligand binding in optical
density units (405 nm), horizontal axis: vitronectin, fibrinogen,
fibronectin concentration (.mu.g/ml).
[0043] (.largecircle.) .alpha..sub.v.beta..sub.3 placenta,
(.box-solid.) .alpha..sub.v(FL).times..beta..sub.3(FL),
(.diamond-solid.)
.alpha..sub.v.DELTA.(SL).times..beta..sub.3.DELTA.(SL).
[0044] FIG. 8 shows effect of different RGD-peptides on vitronectin
binding to different receptor preparations. Biotinylated
vitronectin was incubated in parallel with incerasing
concentrations of several peptides as indicated. Vertical axis: VN
binding % of control, horizontal axis: peptide concentration;
[0045] --.circle-solid.--.alpha..sub.v.beta..sub.3 placenta
[0046] --.box-solid.--.alpha..sub.v.beta..sub.3 recombinant
truncated (1st preparing)
[0047] --.tangle-solidup.--.alpha..sub.v.beta..sub.3 recombinant
truncated (2nd preparing)
DETAILED DESCRIPTION
[0048] Microorganisms, cell lines, plasmids, phagemids, promoters,
resistance markers, replication origins or other fragments of
vectors which are mentioned in this application are normally
commer-cially or otherwise generally available. In some cases the
above-mentioned materials are not directly purchasable. However,
they are used here only as examples in order to demonstrate
properties or effects of the objects according to the invention,
and are not essential to fulfill the requirements of disclosure.
They can be replaced, as a rule, by other suitable generally
obtainable tools and biological materials.
[0049] The invention includes also DNA and amino acid sequences
having slightly varied or altered sequences or mutants and variants
which can be obtained intentionally or randomly by chemical or
physical processes. Generally, all such mutants and variants are
included which show the described properties and functions.
[0050] The techniques and methods which are essential according to
the invention are described in detail in the specification. Other
techniques which are not described in detail correspond to known
standard methods which are well known to a person skilled in the
art, or are described more in detail in the cited references and
patent applications and in the standard literature (e.g., Sambrook
et al. 1989: Molecular Cloning, a laboratory manual, Cold Spring
Harbor: Cold Spring Harbor Laboratory Press; Harlow and Lane 1988:
Antibodies--A Laboratory Manual, Cold Spring Harbor: Cold Spring
Harbor Laboratory Press).
[0051] Integrin .alpha..sub.v and .beta..sub.3 cDNAs are known and
available (e.g., Fitzgerald et al 1987, J. Biol. Chem. 262, 3936
(.beta..sub.3) or Suzuki et al. 1987, J. Biol. Chem. 262, 14080
(.alpha..sub.v)), or can be produced by known methods, for example,
by nucleotide synthesizers.
[0052] The human .alpha..sub.v mature full-length protein chain
according to the invention has 1018 amino acids, the transmembrane
domain has approximately 29 amino acids and the cytoplasmic domain
has approximately 32 amino acids. Cytoplasmic and transmembrane
regions have about 61 amino acids. Cytoplasmic and transmembrane
regions are located at the N-terminus of the chain. Thus, the
preferred truncated .beta..sub.v chain according to the invention
has approximately 957 amino acids.
[0053] The human .beta..sub.3 mature full-length protein chain
according to the invention has 762 amino acids, the transmembrane
domain has, like the .alpha..sub.v chain, approximately 29 amino
acids and the cytoplasmic domain has approximately 41 amino acids.
Cytoplasmic and transmembrane regions have, therefore, 70 amino
acids. Cytoplasmic and transmembrane regions are located at the
N-terminus of the chain. Thus, the preferred truncated .beta..sub.3
chain according to the invention has approximately 692 amino
acids.
[0054] According to the invention chains are preferred from which
the complete cytoplasmic and transmembrane domain is removed.
However, it is also possible to remove the complete cytoplasmic
domain and only a portion of the amino acids of the transmembrane
domain. Preferably, the truncated .alpha..sub.v or .beta..sub.3
chain comprises additionally up 1 to 10 amino acids, especially 1
to 5 amino acids deriving from said transmembrane domain of each
chain.
[0055] According to their nature the truncated
.alpha..sub.v.beta..sub.3 receptor chains are glycosylated. The
degree of glycosylation depends on their origin and the host cell
system used.
[0056] The baculovirus expression system used according to the
invention is also well known and commonly available. As a preferred
baculovirus expression system, the BacPAK system from Clontech
Laboratories, Inc. (supplied by Cambridge Bioscience, UK) is used.
However, in principle, other baculovirus systems can be used.
[0057] According to the invention insect cells are infected with
recombinant baculovirus DNA. In principal, all insect cell systems
are suitable, however, those systems are preferred which guarantee
a high infection by the virus system and a good and stable
expression. Sf9 cells, and preferably High Five cells, which both
are commercially available, are suitable insect cells. According to
the invention, Sf9 cells are used according to the invention
preferably for sub-cloning steps, whereas High Five cells are used,
preferably for final expression steps (see Examples).
[0058] Sf9 cells (purchasable e.g. from Invitrogen Corporation)
are, for example, maintained in TC100 supplemented with 10% insect
cell-qualified Foetal Bovine Serum (FBS, Life Technologies, Inc.)
and High Five cells (e.g. BTI-TN-5B1-4 from Invitrogen Corporation)
are maintained, for example, in Express Five medium (Life
Technologies, Inc.) supplemented with 16.5 mM L-Glutamine (Life
Technologies, Inc.), Penicillin (50 IU/ml) and Streptomycin (50
.mu.g/ml).
[0059] The generation of recombinant human
.alpha..sub.v.beta..sub.3 receptor by means of a baculovirus-insect
cell expression system is carried out, for example as follows:
[0060] The baculovirus system is used analogously to the methods
given in Kidd and Emery (1993, Appl. Biochem. Biotechnol. 42, 137;
O'Reilly et al., 1992: Baculovirus expression vectors: a laboratory
manual. Oxford University Press, Inc., New York).
[0061] .beta..sub.v and .beta..sub.3 cDNAs truncated at positions
coding for the transmembrane and extracellular interface (FIG. 1)
are prepared by PCR. The .alpha..sub.v and .beta..sub.3 truncated
(.alpha..sub.v(SL) and .beta..sub.3(SL)) and the full length
(.alpha..sub.v(FL) and .beta..sub.3(FL) cDNAs are sub-cloned into
baculovirus transfer vector pBacPAK9. It is also possible according
to the invention to clone the cDNA of each chain into seperate
vectors in order to avoid stability problems. However, cloning into
one single vector is preferred, since this guarantees better that
equimolar amounts of each chain are produced. Surprisingly, the
present results show that the vector comprising the DNA sequences
of both chains are, although rather large, stable enough.
[0062] The truncated .alpha..sub.v and .beta..sub.3 cDNAs usually
comprise a signal sequence (thus, in case where the truncation
comprises the complete transmembrane and cytoplasmic domain, coding
for 987 .alpha..sub.v chain amino acids [957 for mature protein+30
for signal peptide] and 718 .beta..sub.3 chain amino acids [692 for
mature protein+26 for signal peptide]). Recombinant baculovirus
expressing either full length or truncated .alpha..sub.v or
.beta..sub.3 chains are prepared by co-transfection with linearized
baculovirus BacPAK6 genomic DNA followed by enrichment by plaque
purification and virus amplification in Sf9 cells or High Five
cells (O'Reilly et al., 1992: Baculovirus expression vectors: a
laboratory manual. Oxford University Press, Inc., New York).
Accurate integration of the transfer plasmids is confirmed by PCR
and Southern blotting of the recombinant viral genomic DNA.
[0063] Metabolic labelling of Sf9/High Five cells expressing full
length .alpha..sub.v chain shows two major recombinant proteins of
approximately 110 kDa and 150 kDa (FIG. 2A, lane 3). Similarly, Sf9
cells expressing full length .beta..sub.3 show two bands of
approximately 90 kDa and 125 kDa (FIG. 2A, lane 4). Comparison of
mock infected (lane 1) and null recombinant virus infected (lane 2)
also shows the degree of suppression of the host cell genome during
the very late phase of the viral life cycle. Treatment of cells
expressing either individual full length or truncated forms of
.alpha..sub.v or .beta..sub.3 with tunicamycin (FIG. 2B, lanes 1,
3, 5 and 7) inhibits the production of the slower migrating band,
indicating that the 150/110 kDa bands (FIG. 2B, lanes 2 and 4) and
the 125/90 kDa (FIG. 2B, lanes 6 and 8) correspond to glycosylated
and unglycosylated forms of the recombinant proteins. This result
confirms several reports that have suggested that the proportion of
recombinant protein in baculovirus expression systems that undergo
post-translational modifications, such as N-glycosylation,
decreases during later stages of the virus life cycle (Jarvis and
Finn 1995, Virology 212, 500). The relatively large difference in
size between the glycosylated and unglycosylated forms of
.alpha..sub.v and .beta..sub.3 indicated that the proteins undergo
substantial glycosylation, which is also in keeping with the
presence in .alpha..sub.v of thirteen and in .beta..sub.3 of ten
potential N-glycosylation sites.
[0064] Integrins determine and modulate the cell shape by acting as
structural links between the extracellular matrix and the
cytoskeleton (Schwartz et al. 1995, Ann. Rev. Cell Dev. Biol. 11,
549; Montgomery et al. 1994, Proc. Natl. Acad. Sci. USA 91, 8856).
In later phases of baculovirus infection, Sf9/High Five cells are
usually rounded and loosely attached (FIG. 3A), as were the cells
infected with either of the .alpha..sub.v or .beta..sub.3
expressing recombinant baculovirus alone or with the null
recombinant virus (FIG. 3A). However, insect cells co-infected with
.alpha..sub.v(FL)+.beta..sub.3(FL) or
.alpha..sub.v(SL)+.beta..sub.3(FL) are firmly attached and exhibit
a more spread cell shape (FIG. 3B and 3C). No shape change is
observed in cells co-infected with
.alpha..sub.v(FL)+.beta..sub.3(SL) or
.alpha..sub.v(SL)+.beta..sub.3(SL). These results support the
notion that the cytoplasmic domain of the .alpha.-chain is
responsible for negative regulation of the affinity for the
cytoskeleton of the .beta.-chain cytoplasmic domain (Filardo and
Cheresh 1994, J. Biol. Chem. 269, 4641; Akiyama et al. 1994, J.
Biol. Chem. 269, 15961; LaFlamme et al. 1994, J. Cell Biol. 126,
1287). .alpha..sub.v.beta..sub.3 binds many ligands that have the
peptide sequence Arg-Gly-Asp (RGD). Correspondingly, cell shape
change of .alpha..sub.v and .beta..sub.3 virus infected Sf9 cells
on vitronectin coated plates are inhibited when challenged with RGD
peptides (Table 1).
2 TABLE 1 - +RGD +nRGD .alpha..sub.V(FL) + .beta..sub.3(FL) ++ - ++
.alpha..sub.V(FL) + .beta..sub.3(SL) +/- - +/- .alpha..sub.V(SL) +
.beta..sub.3(FL) +++ - +++ .alpha..sub.V(SL) + .beta..sub.3(SL) - -
- Null-Recombinant - - -
[0065] Control peptides, cyc(RGEfV) and cyc(R.beta.ADfV) where
glutamate was substituted for aspartate, or .beta.-alanine for
glycine have no effect on cell shape.
[0066] High Five cells, known to support higher level of production
of baculovirus of expressed secreted proteins than Sf9 cells (Davis
et al. 1993, In Vitro Cell Dev. Bio. Anim. 29A, 388), are
co-infected with various combinations of recombinant baculovirus
expressing the full length and truncated .alpha..sub.v and
.beta..sub.3. Immunoprecipitation of the .alpha..sub.v.beta..sub.3
complex with the MAb LM609 from cell extracts or from cell
conditioned medium (FIG. 4) shows that the recombinant
.alpha..sub.v.beta..sub.3 heterodimer is on the cell surface when
either one or both the individual chains are full length (FIG. 4,
lanes 1-3). More importantly, co-expression of truncated
.alpha..sub.v with truncated .beta..sub.3 results in secretion of a
soluble heterodimer (FIG. 4, lane 9). Production of soluble
.alpha..sub.v.beta..sub.3 in this serum free environment greatly
simplified the downstream processing and also introduced a degree
of flexibility in establishing large scale screening procedures for
anti-.alpha..sub.v.beta..sub.3 compounds.
[0067] The novel truncated .alpha..sub.v.beta..sub.3 receptors
according to the invention as well as their full-length variants
are purified after recombinant expression by antibody affinity
chromatography. Since the expression system according to the
invention is very effective in producing large amounts of said
receptor and only very small amounts of foreign proteins, and
especially free from other human proteins, a single purification
step is usually sufficient to obtain a highly purified product.
[0068] Antibody affinity chromatography is a well known technique:
an antibody with a desired specificity to a certain antigen epitope
is coupled enzymatically or chemically to a suitable activated
support matrix, eg. modified agarose, sepharose, etc. Suitable
antibodies according to the invention must have a good specificity
to the .alpha..sub.v.beta..sub.3 receptor or to the single
.alpha..sub.v chain or the single .beta..sub.3 chain.
[0069] Many such antibodies are known, some of them are public
domain antibodies and, therefore, without problems available. AP3
(anti-.beta..sub.3), LM609 (anti-.alpha..sub.v.beta..sub.3), P4C10
(anti-.beta..sub.1), P1F6 (anti-.alpha..sub.v.beta..sub.5) and 17E6
(anti-.alpha..sub.v) have been characterized in detail (e.g.,
Mitjans et al. 1995, J. Cell Sci. 108, 2825). Monoclonal antibody
17E6 is an antibody which is produced by a hybridoma cell line
having the designation 272-17E6. The cell line was deposited under
accession number DSM ACC2160 at the Deutsche Sammlung fur
Mikroorganismen, Braunschweig, FRG. MAb 17E6 is, in addition,
object of the European patent application 0 719 859.
Anti-.beta..sub.3 antibody A.beta..sub.3 is deposited at the ATCC
under accession number HB-242. Antibodies 17E6 and AP.sub.3 are
especially suitable according to the invention, because they are
easily available. LM609 (Cheresh and Spiro 1987, J. Biol. Chem.
262, 17703) is also of special interest since it is directed to the
alpha as well as to the beta chain.
[0070] After having coupled said antibody to the support, the clear
cell-free medium of the infected and lysed insect cells, containing
the truncated or the solubilized full-length recombinant receptor
according to the invention, is applied to a column filled with said
support, and is recirculated several times over the column to bind
the expressed receptor to the immobilized antibody. The purified
.alpha..sub.v.beta..sub.3 receptor is washed thereafter by means of
an ionic buffer from the column and can be used directly for ligand
binding and other experiments.
[0071] Human vitronectin, fibronectin and fibrinogen can be
purified from human plasma (e.g., Yatohgo et al. 1988, Cell Struct.
Funct. 13, 281; Ruoslahti et al. 1982, Methods Enzymol 82 Pt A,
803; Ruoslahti 1988, Ann. Rev. Biochem. 57, 375; Kazal et al. 1963,
Proc. Soc. Exp. Biol. Med. 113, 989).
[0072] In detail the purification and characterization of
recombinant human .alpha..sub.v.beta..sub.3 is carried out for
example, as follows:
[0073] Antibody affinity chromatography is carried out using 17E6,
A.beta..sub.3 or LM609 antibodies. .alpha..sub.v.beta..sub.3
receptor from human placenta is, for example, purified by affinity
chromatography according to the method of Smith and Cheresh (1988,
J. Biol. Chem. 263, 18726).
[0074] Full length recombinant .alpha..sub.v.beta..sub.3 can be
purified from non-ionic detergent solublilized extracts of High
Five cells co-infected with .alpha..sub.v(FL)+.beta..sub.3(FL).
[0075] Soluble truncated .alpha..sub.v.beta..sub.3 can be purified
from High Five cells co-infected with
.alpha..sub.v(SL)+.beta..sub.3(SL) without using any detergent.
[0076] In ELISA, truncated soluble and full length solubilized
recombinant .alpha..sub.v.beta..sub.3 are both recognized by the
same antibodies (LM609, 17E6 and AP.sub.3) and with similar
affinity, as judged by ELISA titres, as solubilized placental
.alpha..sub.v.beta..sub.3. Each fails to react with
anti-.beta..sub.1 (P4C10) or with anti-.alpha..sub.v.beta..sub-
.5-specific (P1F6) antibodies indicating conservation of epitopes
and low contamination with other integrins (FIG. 5).
[0077] Purified placenta .alpha..sub.v.beta..sub.3 dissociates into
two bands of the a-chain at 160 kDa and of the .beta.-chain at 95
kDa when resolved by SDS-PAGE under non-reducing conditions (FIG.
6, lane 2). Purified full length recombinant
.alpha..sub.v.beta..sub.3 (lane 3) dissociates into .alpha..sub.v
chain of approximately 140 kDa and a .beta..sub.3 chain at 90 kDa.
The lower molecular weights may be due to incomplete or variant
glycosylation of recombinant proteins expressed in baculovirus
infected insect cells. Purified soluble recombinant
.alpha..sub.v.beta..sub.3 dissociates into a truncated
.alpha..sub.v chain of approximately 120 kDa and a truncated
.beta..sub.3 chain of 80 kDa.
[0078] The .alpha..sub.v chain normally undergoes post
translational cleavage into heavy and light chains, linked by a
disulfide bridge (FIG. 1) (Hynes 1992, Cell 69, 11). Therefore,
under reducing conditions the .alpha. chain of placental
.alpha..sub.v.beta..sub.3 further dissociates into the heavy chain
of 140 kDa and the light chain of 25 kDa (FIG. 6, lane 6) while the
.beta..sub.3 chain migrates at 110 kDa. As the transmembrane
truncation in the et chain lies within the light chain, both full
length and truncated recombinant .alpha. chains should migrate
under reducing conditions at .sup..about.140 kDa. However, the
.alpha..sub.v chains of full length and soluble receptors exhibit
the same molecular weights under both reducing and non-reducing
conditions (lane 7 and 8), suggesting that there had only been
partial post-translational cleavage of the .alpha..sub.v chain. The
faint band of approximately 120 kDa in lanes 7 and 8 may represent
the .alpha..sub.v heavy chain. This discrepancy between the
placental and recombinant .alpha..sub.v.beta..sub.3 may be due to
inefficient proteolytic processing during the very late phase of
the baculovirus infection (O'Reilly et al. 1992, Baculuvirus
Expression Vectors: A Laboratory Manual. Oxford University Press,
Inc., New York).
3TABLE 2 depicts an overview of the SDS-PAGE results: kD (.+-.10%)
kD (.+-.10%) human .alpha..sub.V.beta..sub.3 chains non-reduced
reduced Placenta .alpha..sub.V(FL) 160 140 (hc), 25 (lc)
.beta..sub.3(FL) 95 110 recombinant .alpha..sub.V(FL) 140 140
.alpha..sub.V(SL) 120 140 .beta..sub.3(FL) 90 105 .beta..sub.3(SL)
80 95
[0079] According to the process of the invention recombinant
shortened .alpha..sub.v.beta..sub.3 receptor and full-length
receptor can be prepared. In order to demonstrate that so-prepared
receptors, above all the truncated form, show the ligand
specifities of the natural receptor, which was isolated, e.g., from
human placenta and used as comparison embodiment, a binding assay
has to be established. Ligands known to be competitive inhibitors
of the .alpha..sub.v.beta..sub.3 receptor are used therefor. As
mentioned above, linear and cyclic peptides containing the RGD
amino acid sequence (arginine-glycine-asparagine) are excellent
inhibitors of said receptor.
[0080] Suitable peptides are, for example: GRGDSPK, Echistatin,
cyclic-RGDfV, cyclic-RGDfNMeV, cyclic-R.beta.ADfV,
KTADC(Trt)PRNPHKGPAT, GRGESPK, cyclic-KGDfV, and cyclic-RGEfV.
Additionally, also non-peptidic compounds which may mimick
RGD-peptides and bind to the .alpha..sub.v.beta..sub.3 receptor can
be used. Such RGD-peptides and non-peptidic analogues are known
(e.g. from European Appln. No. 96 113 972, European Publication
Nos. 0578 083, 0632 053, 0478 363, 0710 657, 0741 133 and PCT
Publication No. WO 94/12181) and/or can be synthesized by known
standard methods. The ligands are modified by marker molecules or
atoms according to methods known in the prior art. Preferably,
biotinylated ligands are used.
[0081] The recombinant truncated .alpha..sub.v.beta..sub.3 receptor
or its full-length version are immobilized, for example by
adsorption at microtitre plates, and reacted with said marked
ligands. The .alpha..sub.v.beta..sub.3 receptors according to the
invention bind said ligands strongly, very similar to the natural
receptor isolated from human placenta, whereas non-RGD peptides are
not bound. This result demonstrates that the receptors, obtainable
by the claimed process, are functionally intact with respect to
their specific ligand binding capacity.
[0082] In detail, measurement of the biological activity of
recombinant truncated .alpha..sub.v.beta..sub.3 receptor is carried
out, for example, as follows:
[0083] The truncated and full length recombinant
.alpha..sub.v.beta..sub.3 and placenta .alpha..sub.v.beta..sub.3
are immobilized and binding of biotinylated ligands is examined.
Three purified RGD-containing ligands--vitronectin, fibrinogen and
fibronectin--that are known to bind to .alpha..sub.v.sub.3 are
used. The ligands bind both to the full length and soluble receptor
preparations in a concentration dependent manner, and with
overlapping concentration dependencies and saturation
concentrations similar to those of placental
.alpha..sub.v.beta..sub.3 (FIG. 7). Vitronectin is used for testing
specific interaction with the recombinant
.alpha..sub.v.beta..sub.3. Biotinylated vitronectin and increasing
concentrations of above-specified peptides or non-peptidic
analogues are incubated in parallel with immobilized receptor. The
compounds are able to inhibit vitronectin binding to placenta
.alpha..sub.v.beta..sub.3 to full length and to soluble recombinant
.alpha..sub.v.beta..sub.3 (FIG. 8) and are about 4-5 orders of
magnitude more active than the .beta.-alanine-to-glycine control
peptide. Each receptor shows an IC.sub.50-value for cyc(RGDfV) in
the low nanomolar (1-5 nM) range.
[0084] In the same way as described here for known
.alpha..sub.v.beta..sub- .3 receptor specific ligands it is
possible to screen for new drugs and therapeutic compounds which
are regarded to be potent .alpha..sub.v.beta..sub.3 inhibitors.
[0085] This invention shows that the baculovirus insect cell
expression system can be used to express high amounts of fully
functional human recombinant .alpha..sub.v.beta..sub.3 integrins as
soluble receptors secreted into the medium or, as alternative, on
the cell surface. The double (SL)-truncated forms of the receptor
are secreted as a complex into the medium as shown by precipitation
and purification on an .alpha..sub.v.beta..sub.3-complex specific
antibody (LM609, 17E6, AP3).
[0086] Under optimized conditions yields (soluble as well as
full-length receptor) of 1-10 mg, preferably 2-5 mg/L culture
medium (supernatant) can be obtained. This routine production in
biochemical amounts of soluble .alpha..sub.v.beta..sub.3 opens the
way for high throughput screening using .alpha..sub.v.beta..sub.3
and for structural analysis of this important receptor. The yield
of natural receptor isolated from plancenta tissue is about 1-2
mg/kg tissue. Truncated and full-length recombinant
.alpha..sub.v.beta..sub.3 proteins obtained by the process
according to this invention have a purity of 95-99%, preferably
97-99%, as assessed by SDS-PAGE and ELISA. The soluble recombinant
receptor is approximately 4-fold less sensitive to inhibitor than
the full length recombinant molecules. A further advantage of the
truncated soluble receptor is that it can be prepared without using
detergents. The necessary non-ionic detergents are usually very
expensive (e.g., ca. $500/10 g octyl-.beta.-D-glyco-pyranoside).
Approximately 25 g of detergent are necessary for solubilizing 3-4
mg receptor from placenta tissue.
[0087] Cells co-infected with recombinant virus with one full
length and one truncated .alpha.- and .beta.-chain
(.alpha..sub.v(FL).times..beta..s- ub.3(SL) or
.alpha..sub.v(SL).times..beta..sub.3(FL)) express hetero-dimeric
receptor on their surface. But only cells containing either both
full length chains, or full length .beta.-chain and truncated
.alpha.-chain show shape change when adhering on vitronectin. That
this adhesion and spreading was .alpha..sub.v.beta..sub.3-specific
was shown by adding RGD-containing peptides which specifically
block the interaction between ligand and the native receptor while
RGE- or RAD-peptides do not. Cells with fill length .alpha.-chain
and truncated .beta.-chain do not spread.
[0088] ELISA shows that the .beta..sub.3-specific antibody AP3
(Newman et al. 1985, Blood 65, 227) and .alpha..sub.v-specific
antibody 17E6 (Mitjans et al. 1995, J. Cell Sci. 108, 2825) and the
complex-specific antibody LM609 (Cheresh and Spiro 1987, J. Biol.
Chem. 262, 1770323) recognized both full length and truncated
receptors, indicating that the recombinant integrins had the same
epitopes as placenta .alpha..sub.v.beta..sub.3. SDS-PAGE shows
under non-reducing conditions the bands typical for .alpha..sub.v-
and .beta..sub.3-chains. The recombinant full length a chain has a
lower molecular weight compared to the placenta full length a chain
(approx. 140 kDa compared to 160 kDa) which could be explained by a
decreased or different glycosylation in insect cells. Both full
length and soluble recombinant .alpha..sub.v.beta..sub.3 receptors
are able to specifically bind the ligands vitronectin, fibrinogen
and fibronectin, the vitronectin binding was specifically inhibited
by RGD-containing peptides with the same concentration dependency
as the placental receptor.
[0089] With this demonstration of the specificity and similar
saturation concentrations for ligand binding for full length and
truncated human (.alpha..sub.v.beta..sub.3 receptor it is now
possible to embark on large scale preparation of functional
recombinant soluble .alpha..sub.v.beta..sub.3 in the baculovirus
insect cell system without the labor and the health risks
associated using human placental tissue. Furthermore, the system
described by this invention also allows expression of modified or
chimeric receptors that could prove very useful for investigating
at molecular level the ligand binding and the subsequent signal
transduction processes.
[0090] In the foregoing and in the following examples, all
temperatures are set forth uncorrected in degrees Celsius; and,
unless otherwise indicated, all parts and percentages are by
weight.
[0091] The entire disclosure of all applications, patents and
publications, cited above and below, and of corresponding European
application No. 96 119 700. 1, filed Dec. 9, 1996 is hereby
incorporated by reference.
EXAMPLES
Example 1
[0092] Construction of Recombinant Transfer Vectors
[0093] .alpha..sub.v cDNA was excised as an EcoRI fragment from
.alpha..sub.vpcDNA-1Neo(Felding-Habermann 1992, J. Clin. Invest.
89, 2018) and cloned into the transfer plasmid pBacPAK8 (Clontech).
The resulting recombinant was termed .alpha..sub.v(FL)(pBac8). The
integrin .beta..sub.3 cDNA was excised as a XbaI fragment from
.beta..sub.3pcDNA-1Neo and cloned into the transfer plasmid
pBacPAK9 (Clontech). The transmembrane truncated .alpha..sub.v and
.beta..sub.3 cDNAs were constructed using Polymerase Chain Reaction
(PCR) using Pfu Thermostable DNA polymerase (Stratagene). Truncated
.alpha..sub.v cDNA coding for amino acids 1-987 of mature
.alpha..sub.v (signal esquence included) was generated using
oligonucleotide primers:
4 5'-GAC CAG CAT TTA CAG TGA-3' [forward] and 5' CA CAG GTC TAG ACT
ATG GCT GAA TGC CCC AGG-3'
[0094] [reverse primer, containing translation stop codon (bold)
after that coding for aa 987 followed by XbaI restriction site
(underlined)].
[0095] The PCR product was digested with SalI and XbaI restriction
enzymes and the fragment was cloned into SalI/XbaI digested
movpcDNA-1Neo. The truncated .alpha..sub.v cDNA was sub-cloned into
EcoRI/XbaI sites of pBacPAC9 and the resulting clone was termed
.alpha..sub.v(SL) (pBac9). Truncated .beta..sub.3 cDNA coding for
aa 1-718 of mature .beta..sub.3 (signal sequence includes) was
generated using oligonucleotide primers:
5 5'-GCG CGC AAG CTT GCC GCC ACC ATG CGA GCG CGG CCG-3'
[0096] [forward primer containing the translation start codon
(bold) and HindIII restriction side (underlined)] and
6 5'-GAT CGA TCT AGA CTA GGT CAG GGC CCT TGG GAC ACT-3'
[0097] [reverse primer also containing translation stop codon
(bold) after that coding for aa 718 and XbaI restriction site
(underlined)]. The PCR product was digested with HindIII and XbaI
and cloned into HindIII/XbaI sites of pSK+(Stratagene). The
resulting clone, termed .beta..sub.3(SL)(pSK+), was digested with
HindIII and the ends were subsequently polished by filling in with
Klenow fragment of DNA polymerase I (Boehringer Mannheim). The
blunt-end linearized plasmid was then digested with XbaI
restriction enzyme and the fragment was sub-cloned into SmaI/XbaI
sites of pBacPAK9. The resulting clone was termed
.beta..sub.3(SL)(pBac9). The PCR amplified regions of both
.alpha..sub.v(SL)(pBac9) and .beta..sub.3(SL)(pBac9) were verified
by DNA sequencing and all the constructs were tested for expression
of correct sized protein by in vitro transcription and translation
from bacteriophage T7 promoter using TNT Coupled Reticulocyte
Lysate System (Promega).
Example 2
[0098] Generation of Recombinant Baculovirus
[0099] BacPAK6 (Clontech) baculovirus genomic DNA was prepared from
high titre virus stock (Page and Murphy 1990: Methods in Molecular
Biology. Humana Press, Clifton, N.J., USA) and linearized with
Bsu36I (Kitts and Possee 1993, Biotechniques 14, 810).
[0100] Recombinant baculovirus clones expressing full length and
truncated .alpha..sub.v and .beta..sub.3 integrins were prepared
according to the Clontech BacPAK product protocol. A null
recombinant baculovirus clone was also prepared using pBacPAK9 as
transfer plasmid for use as negative control. The correct
recombinant viral clones were identified by PCR and the virus was
propagated and titered in Sf9 cells (O'Reilly et al. 1992,
Baculuvirus expression vectors: a laboratory manual. Oxford
University Press, Inc., New York).
Example 3
[0101] Metabolic Pulse Labelling of Proteins Expressed in Sf9
Cells
[0102] Metabolic labelling of Sf9 cells was performed as described
(Summer and Smith 1987: A manual of methods for baculovirus vectors
and insect cell procedures. Texas Agricultural Experiment Station
Bulletin B-1555). Where indicated, tunicamycin (Sigma) was present
at 10 .mu.g/ml for 16 hours before and during pulse labelling.
Cells were lysed with 100 .mu.l of buffer [1% Nonidet.RTM. P-40, 1
mM CaCl.sub.2, 150 mM NaCl, 0.4 mM Pefabloc.RTM. (Boehringer
Mann-heim), 10 .mu.g/ml Leupeptin and E64 (Sigma), 10 mM Tris/HCl;
pH 7.4; Nonidet P-40 is a detergent and Pefabloc is a protease
inhibitor].The lysate was centrifuged at 14000.times.g in an
Eppendorf microfuge at 4.degree. C. for 10 minutes and resolved by
electrophoresis in 8% SDS-poly-acrylamide gel under reducing
conditions, fixed, and dried before autoradiography (Sambrook et
al. 1989: Molecular Cloning: A Laboratory Manual. Cold Spring
Harbor Laboratory Press, Cold Sring Harbor, New York).
Example 4
[0103] Protein Expression in Insect Cells
[0104] Recombinant proteins were expressed in High Five cells,
either as monolayer or in suspension. Cells were co-infected with
.alpha..sub.v- and .beta..sub.3-expressing recombinant baculovirus,
each at M.O.I. of 5-20 (that means: one cell is infected with 5-20
virus DNA), in a minimum amount of medium with gentle agitation.
After two hours the viral inoculum was removed and replaced with
fresh Express Five medium, the infected cells were incubated at
27.degree. C. for 48-64 hours. For isolation of membrane bound
recombinant integrin, the cells were harvested by centrifugation
(1000.times.g, 10 minutes) and the cell pellet was used. For
isolation of the soluble recombinant integrin the cell-free
supernatant was used.
Example 5
[0105] Immunoprecipitation
[0106] Immunoprecipitation of surface-biotinylated cell extract and
cell conditioned medium was carried out with monoclonal antibody
LM609 which recognises the extracellular domains of intact
.alpha..sub.v.beta..sub.3-- complex, linked to Affigel.RTM. beads
(BIO-RAD) (Mitjans et al. 1995, J. Cell Sci. 108, 2825). After
SDS-PAGE and Western blotting onto Hybond-PVDF membrane (Towbin et
al. 1979, Proc. Natl. Acad. Sci. USA 76, 4350), the biotinylated
proteins were detected by Enhanced-Chemiluminesce- nce using ECL
Western Blotting Detection Reagents (Amersham) as per manufacturers
instructions.
Example 6
[0107] Cell Shape Change Studies
[0108] Sf9 cells (1.times.10.sup.6 cells/well) were allowed to
adhere to six-well plates either virgin or coated with purified
human plasma vitronectin (5 .mu.g/ml in PBS; 2 hours) followed by
blocking of non-specific sites (3% BSA (w/v) in PBS), then infected
with various virus combinations. The cells were observed 48 hours
post-infection for cell shape change. Where indicated, the medium
was supplemented with 10 .mu.M cyclic peptides as competitive
inhibitors of .alpha..sub.v-integrin-ligand interaction
[cyc(RGDfV), cyc(R.beta.ADfV) or cyc(RGEfv), where small letters
are D-amino acids].
Example 7
[0109] Purification of Recombinant Integrins
[0110] High Five cells expressing full length
.alpha..sub.v.beta..sub.3 were harvested, washed with PBS, and then
lysed in ice cold lysis buffer [100 mM n-Octyl-.beta.-D-glucoside,
1 mM CaCl.sub.2, 2 mM Pefabloc in PBS, pH 7.4] for 1 h at 4.degree.
C. The lysate was centrifuged (10,000.times.g; 45 min at 4.degree.
C.) and the supernatant recirculated overnight at 4.degree. C. over
a 17E6-antibody affinity column pre-equilibrated with lysis buffer.
After washing [20 ml bed volumes, 2.times.10 cm column, 50 mM
n-Octyl-.beta.-D-glucoside, 2 mM Pefabloc, 2 mM CaCl.sub.2 in PBS,
pH 7.4]bound protein was eluted [50 mM OG, 2 mM Pefabloc.RTM., 2 mM
CaCl.sub.2, 50 mM Na-Acetate, pH 3.1], the eluant monitored at 280
nm, and the fractions were immediately neutralized (1:50 volume 3M
Tris-HCl pH 8.8). Peak fractions were pooled, dialyzed [10 mM OG, 1
mM CaCl.sub.2 in PBS, pH 7.4] and concentrated, then analyzed by
SDS-PAGE. Aliquots at approx. 1 mg/ml protein were stored at
-80.degree. C. Protein concentration was determined against BSA
standards with the BCA protein assay (Pierce).
[0111] Soluble truncated recombinant .alpha..sub.v.beta..sub.3 was
purified by recirculating the cell free medium of infected High
Five cells over an 17E6 affinity column at 4.degree. C. The
washing, elution and analysis were as described for the full length
.alpha..sub.v.beta..sub.3 except that no detergent was present in
the buffers. ELISA analysis of the purified receptors used
goat-anti-mouse IgG (H+L) HRP conjugate (BIO-RAD) and
3,3',5,5'-Tetramethylbenzidine-dihy- drochloride (Sigma) as
substrate.
Example 8
[0112] The purification of soluble and transmembrane full length
.alpha..sub.v.beta..sub.3 receptor was achieved by using a LM609
antibody column.
Example 9
[0113] Preparation of an Antibody Affinity Chromatography
Column
[0114] The support matrix (Affigel.RTM. BIORAD) was washed with 500
ml cold PBS. Purified antibody was incubated together with the
support matrix (5 mg/ml gel) under circular rotation during ca. 12
hours. The supernatant was removed and still active groups on the
matrix were blocked with 0.1 M ethanolamine. The support matrix was
washed alternately several times with 0.01 M Tris-HCl pH 8.0 and
0.01 M sodium acetate pH 4.5 and was then directly used.
Example 10
[0115] Integrin Ligand Binding and Competition Assays:
Biotinylation of Ligands or Antibodies
[0116] Proteins in PBS were diluted with 5-fold concentrated
ligation buffer [500 mM NaCl, 500 mM NaHCO.sub.3] to 1 mg/ml
protein. Freshly prepared N-hydroxysuccinimidobiotin (Sigma) [1
mg/ml in DMSO] was added to 0.1 mg/ml and incubated for 2 h at
20.degree. C. After dialysis [PBS, 0.025% NaN.sub.3], protein
concentration was determined. The biotinylated proteins were stored
at 4.degree. C.
[0117] Ligand Binding assay Integrins were diluted to 1 .mu.g/ml in
coating buffer [150 mM NaCl, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2, 10
.mu.M MnCl.sub.2, 20 mM Tris-HCl; pH 7.4) and 100 .mu.l were
adsorbed overnight at 4.degree. C. to 96-well microtitre plates.
The plates were washed once with binding buffer [0.1 % (w/v) BSA in
coating buffer] and blocked with blocking buffer (coating buffer
containing 3% (w/v) BSA) 2 h at 37.degree. C. After rinsing with
binding buffer, serially diluted biotinylated ligands were added.
After incubation (3 h, 37.degree. C.), unbound ligand was washed
from the plate with binding buffer, and bound biotin was detected
by incubation with anti-biotin-alkaline-phosphatase conjugated
antibody and detection of bound antibody with
p-Nitro-phenyl-phosphate (BIO-RAD) substrate.
Example 11
[0118] Integrin Ligand Binding and Competition Assays: Competition
Assays
[0119] Integrins were immobilized as described above. Serially
diluted cyclic peptides were added in parallel with biotinylated
vitronectin (1 .mu.g/ml). After 3 h incubation at 37.degree. C.,
bound ligand was detected as described above. Assays were performed
in triplicate and repeated several times.
[0120] The preceding examples can be repeated with similar success
by substituting the generically or specifically described reactants
and/or operating conditions of this invention for those used in the
preceding examples.
* * * * *