U.S. patent application number 12/011828 was filed with the patent office on 2008-12-25 for blood group antigen fusion polypeptides and methods of use thereof.
Invention is credited to Jan Holgersson, Jonas Lofling.
Application Number | 20080319173 12/011828 |
Document ID | / |
Family ID | 23187750 |
Filed Date | 2008-12-25 |
United States Patent
Application |
20080319173 |
Kind Code |
A1 |
Lofling; Jonas ; et
al. |
December 25, 2008 |
Blood group antigen fusion polypeptides and methods of use
thereof
Abstract
The present invention provides compositions and methods for
treating or preventing antibody mediated graft rejection.
Inventors: |
Lofling; Jonas; (Alvsjo,
SE) ; Holgersson; Jan; (Huddinge, SE) |
Correspondence
Address: |
Ivor Elrifi, Esq.;Mintz, Levin, Cohn, Ferris,
Glovsky and Popeo, P.C, One Financial Center
Boston
MA
02111
US
|
Family ID: |
23187750 |
Appl. No.: |
12/011828 |
Filed: |
January 30, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10199342 |
Jul 19, 2002 |
7355017 |
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12011828 |
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60306984 |
Jul 20, 2001 |
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Current U.S.
Class: |
530/391.1 |
Current CPC
Class: |
C07K 14/4727 20130101;
C07K 2319/30 20130101; A61K 38/1709 20130101; A61P 39/00 20180101;
Y10S 530/866 20130101; A61P 37/06 20180101; C07K 2319/00
20130101 |
Class at
Publication: |
530/391.1 |
International
Class: |
C07K 16/46 20060101
C07K016/46 |
Claims
1. An absorber comprising a fusion polypeptide, wherein the fusion
polypeptide comprises a first polypeptide operably linked to a
second polypeptide, wherein the first polypeptide: (a) is a mucin
polypeptide and (b) is glycosylated by an .alpha.1,2
fucosyltransferase and the second polypeptide comprises at least a
region of an immunoglobulin polypeptide.
2. A method removing an antibody from a biological sample, the
method comprising (a) contacting a biological sample with the
absorber of claim 1 to form an absorber-antibody complex; and (b)
removing the complex from the biological sample thereby removing
the antibody from the biological sample.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Ser. No.
10/199,342, filed Jul. 19, 2002, which claims priority to U.S. Ser.
No. 60/306,984, filed Jul. 20, 2001, the contents of which are
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to generally to compositions and
methods for treating or preventing antibody-mediated graft
rejection and more particularly to compositions including fusion
polypeptides comprising blood group determinants
BACKGROUND OF THE INVENTION
[0003] The major human blood group system, the histo-blood group
ABO system, is defined by three carbohydrate determinants, the
blood group A, B, and H epitopes. Glycans carrying the ABH
determinants are found on glycoproteins, on glycolipids, or as free
oligosaccharides. ABH antigens can be found on N- or O-linked
glycans. The most common core structures described so far on
O-linked glycans are core 1 (Gal.beta.3GalNAc), core 2
(Gal.beta.3(GlcNAc.beta.6)GalNAc), core 3 (GlcNAc.beta.3GalNAc),
and core 4 (GlcNAc.beta.3(GlcNAc.beta.6)GalNAc). These have been
shown to carry type 1 (Gal.beta.3GlcNAc), type 2
(Gal.beta.4GlcNAc), and type 3 (Gal.beta.3GalNAc.alpha.) structures
Type 1 structures are mainly found as extensions of the core 3 and
4 structures, whereas type 2 chains (polylactosamine) are seen as
extensions on the GlcNAc.beta.1,6 branch of core 2 structures.
[0004] Transplantation (Tx) across the ABO barrier is usually
avoided in organ Tx because the risk of antibody-mediated rejection
(AMR) due to preformed antibodies is often high This may also hold
true in bone marrow transplantation, though it has long been the
belief that blood group ABH incompatibility does not affect the
outcome. However, in some cases it would still be desirable to
transplant across the ABO barrier, one of the reasons being that it
widens the pool of available donors for a particular recipient,
even if it does not increase the total number of donors
[0005] Removal of anti-A or anti-B antibodies by extracorporeal
immunoabsorption (EIA) or plasmapheresis (PP) has been shown to
improve graft survival following ABO-incompatible organ Tx. Another
method used for the prevention of AMR in both ABO-incompatible Tx
and xeno-Tx is infusion of free oligosaccharides, but low affinity
of antibodies for free saccharides and the short half-life of
low-molecular-weight oligosaccharides in the circulation (Ye et
al., 1994; Simon et al., 1998) prevents a wider use.
SUMMARY OF THE INVENTION
[0006] The invention is based in part on the discovery that blood
group epitopes can be specifically expressed at high density and by
different core saccharides chains on mucin-type protein backbones.
The polypeptides, are referred to herein as ABO fusion
polypeptides.
[0007] In one aspect, the invention provides a fusion polypeptide
that includes a first polypeptide, comprising at least a region of
a mucin polypeptide, glycosylated by a .alpha.1,2
fucosyltransferase operably linked to a second polypeptide. The
first polypeptide is sequentially glycosylated by an .alpha.1,3
N-acetylgalactosaminyl transferase and/or a .alpha.1,3
galactosyltransferase.
[0008] The mucin polypeptide is for example PSGL-1. Preferably, the
mucin polypeptide is the extracellular portion of PSGL-1. The
.alpha.1,2, fucosyltransferase is for example a blood group H or
Secretor .alpha.1,2, fucosyltransferase such as FUT1 or FUT2.
[0009] In preferred embodiments, the second polypeptide comprises
at least a region of an immunoglobulin polypeptide. For example,
the second polypeptide comprises a region of a heavy chain
immunoglobulin polypeptide. Alternatively, the second polypeptide
comprises the FC region of an immunoglobulin heavy chain.
[0010] The ABO fusion polypeptide is a multimer. Preferably, the
ABO fusion polypeptide is a dimer.
[0011] Also included in the invention is a nucleic acid encoding an
ABO fusion polypeptide, as well as a vector containing ABO fusion
polypeptide-encoding nucleic acids described herein, and a cell
containing the vectors or nucleic acids described herein.
Alternatively the vector further comprises a nucleic acid encoding
a .alpha.1,2, fucosyltransferase and/or a .alpha.1,3 N
acetylgalactosaminyltransferase or a .alpha.1,3
galactosyltransferase
[0012] In another aspect, the invention provides a method of
treating antibody-mediated rejection in a subject. The method
includes contacting a biological sample, e.g., whole blood or
plasma from a subject with the ABO fusion polypeptide of the
invention to form an fusion polypeptide-antibody complex. The
complex is removed from the biological sample and the biological
sample is refused into the subject.
[0013] Also included in the invention is a method of removing an
antibody from a sample by contacting the sample with the ABO fusion
peptide of the invention to form an antibody-fusion peptide complex
and removing the complex from the biological sample.
[0014] Also included in the invention are pharmaceutical
compositions that include the ABO fusion polypeptides.
[0015] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0016] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1A-D are photographs of SDS-PAGE and western blot
analysis of immunopurified PSGL-1/m1gG.sub.2b, chimeras produced in
293T cells transfected with the H or Se gene alone, or in
combination with the A gene encoded .alpha.1,3 GalNAcT. Following
separation on an 8% SDS-PAGE and blotting onto nitrocellulose
membranes, the PSGL-1/mIgG.sub.2b, chimeras were probed with an
anti-mouse IgG antibody (A), an anti-blood group A antibody
followed by a goat anti-mouse IgM antibody (B), an anti-H type I
chain-specific antibody followed by a HRP-labelled goat anti-mouse
IgG.sub.3 antibody (C), an anti-H type 2 chain-specific antibody
followed by a goat anti-mouse IgM-HRP antibody (D), and an anti-H
type 3 chain-specific antibody followed by a HRP-labeled goat
anti-mouse IgM antibody (E). In panels A-D, samples analyzed were
from cells transfected with plasmids encoding CDM8 (lanes 1 and 5),
PSGL-1/mIgG.sub.2b, (lanes 2 and 6), PSGL-1/mIgG.sub.2b and the H
gene (lane 3), PSGL-1/mIgG.sub.2b, the Hand A gene (lane 4),
PSGL-1/mIgG.sub.2b, and the Se gene (lane 7), or
PSGL-1/mIgG.sub.2b, and the Se and A gene (lane 8). In E the
duplicate samples from CDM8 and PSGL-1/mIgG.sub.2b transfected
cells were omitted. In C, 250 ng of H-type 1 chain-BSA was used as
a positive control.
[0018] FIG. 2 A-D are photographs of SDS-PAGE and western blot
analysis of PNGaseF-treated immunopurified PSGL-1/mIgG.sub.2b
produced in 293T cells transfected with the H or Se gene alone or
in combination with the A gene encoded .alpha.1,3 GalNAcT.
Following PNGaseF treatment (+), or not (-), of immunopurified
PSGL-1/mIgG.sub.2b, the PSGL-1/mIgG2b was separated on an 8%
SDS-PAGE and blotted onto nitrocellulose membranes. PSGL-1/mIgG2b,
chimeras were probed with an anti-mouse IgG antibody (A), an
anti-blood group A antibody followed by a goat anti-mouse IgM
antibody (B), an anti-H type 2 chain-specific antibody followed by
a goat anti-mouse IgM-HRP antibody (C), and an anti-H type 3
chain-specific antibody followed by an HRP-labeled goat anti-mouse
IgM antibody (D). In panels A-D, samples analyzed were from cells
transfected with plasmids encoding PSGL-1/mIgG.sub.2b and the H
gene (lanes 1 and 2), PSGL-1/mIgG.sub.2b and the Hand A gene (lanes
3 and 4), PSGL-1/mIgG.sub.2b and the Se gene (lanes 5 and 6), or
PSGL-1/mIgG.sub.2b and the Se and A gene (lanes 7 and 8).
[0019] FIG. 3 A-E are photographs SDS-PAGE and western blot
analysis of immunopurified PSGL-1/mIgG.sub.2b chimeras produced in
COS-7m6 cells transfected with the H or Se gene alone or in
combination with the A gene encoded .alpha.1,3 GalNAcT. Following
separation on an 8% SDS-PAGE and blotting onto nitrocellulose
membranes, the PSGL-1/mIgG.sub.2b chimeras were probed with an
anti-mouse IgG antibody (A), an anti-blood group A antibody
followed by a goat anti-mouse IgM antibody (B), an anti-H type 1
chain-specific antibody followed by an HRP-labeled goat anti-mouse
IgG.sub.3 antibody (C), an anti-H type 2 chain-specific antibody
followed by a goat anti-mouse IgM-HRP antibody (D), and an anti-H
type 3 chain-specific antibody followed by a HRP-labeled goat
anti-mouse IgM antibody (E). In panels A-D, samples analyzed were
from cells transfected with plasmids encoding CDM8 (lanes 1 and 5),
PSGL-1/mIgG.sub.2b (lanes 2 and 6), PSGL-1/mIgG.sub.2b, and the H
gene (lane 3), PSGL-1/mIgG.sub.2b and the Hand A gene (lane 4),
PSGL-1/mIgG.sub.2b and the Se gene (lane 7), or PSGL-1/mIgG.sub.2b
and the Se and A gene (lane 8). In E the duplicate samples from
CDM8 and PSGL1/mIgG.sub.2b transfected cells were omitted. In C,
250 ng of H-type 1 chain-BSA was used as a positive control.
[0020] FIG. 4 A-E are photographs of SDS-PAGE and western blot
analysis of immunopurified PSGL-1/mIgG.sub.2b chimeras produced in
CHO-K1 cells transfected with the H or Se gene alone or in
combination with the A gene encoded .A-inverted.1,3 GalNAcT.
Following separation on an 8% SDS-PAGE and blotting onto
nitrocellulose membranes, the PSGL1/mIgG.sub.2b chimeras were
probed with an anti-mouse IgG antibody (A), an anti-blood group A
antibody followed by a goat anti-mouse IgM antibody (B), an anti-H
type 1 chain-specific antibody followed by an HRP-labeled goat
anti-mouse IgG.sub.3 antibody (C), an anti-H type 2 chain-specific
antibody followed by a goat anti-mouse IgM-HRP antibody (D), and an
anti-H type 3 chain-specific antibody followed by an HRP-labeled
goat anti-mouse IgM antibody (E). Samples analyzed were from cells
transfected with plasmids encoding CDM8 (lane 1),
PSGL-1/mIgG.sub.2b (lane 2), PSGL-1/mIgG.sub.2b and the H gene
(lane 3), PSGL-1/mIgG.sub.2b and the H and A gene (lane 4),
PSGL-1/mIgG.sub.2b, and the Se gene (lane 5), or PSGL-1/mIgG.sub.2b
and the Se and A gene (lane 6). In C, 250 ng of H-type 1 chain-BSA
was used as a positive control.
[0021] FIG. 5 is a bar graph showing the relative blood group A
density on recombinant PSGL-1/mIg.sub.2b, produced in COS, CHO, and
2931 cells coexpressing FUT1 or FUT2 with .alpha.1,3 GalNAcT. The
density was calculated as the ratio of the volumes for the blood
group A and the mIgG reactivities and the values were normalized to
the ones obtained for the A substituted PSGL-1/mIgG.sub.2b produced
in COS cells coexpressing FUT1 and the .alpha.1,3 GalNAcT.
[0022] FIG. 6 is a line chart showing Anti-blood group A reactivity
remaining in serum after absorption on different blood group A
substituted absorbents. The remaining reactivity, in percentage of
nonabsorbed blood group 0 serum, as measured by an ELISA coated
with A-PAA-biotin, was plotted against the amount of absorber used.
A trend line was calculated using logarithmic regression. The
circles represent PSGL-1/mIgG.sub.2b produced in CHO cells with
FUT2 and .alpha.1,3 GalNAcT, and the squares represent A
trisaccharides linked via A-PAA-MPG. B trisaccharides coupled via
B-PAA-MPG were used to obtain the same amount of PAA-MPG in all
absorptions.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The invention is based in part in the discovery that blood
group epitopes can be specifically expressed at high density and by
different core saccharides chains on mucin-type protein backbones.
This higher density of blood group epitopes results in an increased
binding or removal (i.e., absorption) of anti-blood group
antibodies as compared to free saccharides, or AB determinants
linked to solid phase.
[0024] The invention provides mucin-immunoglobulin fusion proteins
(referred to herein as "ABO fusion proteins") containing multiple
blood group epitopes that are useful as an absorber for anti-blood
group antibodies. The ABO fusion peptides are also useful as a
model protein for studies on glycosylation. For example, the ABO
fusion protein are useful in eliminating recipient anti-blood group
ABO antibodies from blood or plasma prior to an ABO incompatible
organ or bone marrow transplantation. The ABO fusion protein
absorbs 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100% of anti-blood
group ABO antibodies from recipient blood or plasma.
[0025] The ABO fusion peptide is more efficient on a carbohydrate
molar basis in removing or binding anti-blood group antibodies as
compared free sacchamides of wild type AB determinants. The ABO
fusion peptide binds 2, 4, 10, 20, 50, 80, 100 or more-fold greater
number of anti-blood group antibodies as compared to an equivalent
amount of free saccharides of wild type AB determinants.
[0026] The ABO fusion proteins of the invention carries an epitope
specific for a blood group determinants. For example, the ABO
fusion protein carries either the A epitope, the B epitope or the H
epitope. Alternatively, the ABO fusion carries two epitope for
blood group antigens. For example the ABO fusion protein carries
both the A and B epitope. In some aspects the ABO fusion protein
carries all three epitopes (i.e., A, B and H).
Fusion Polypeptides
[0027] In various aspects the invention provides fusion proteins
that include a first polypeptide containing at least a portion of a
glycoprotein, e.g., a mucin polypeptide operatively linked to a
second polypeptide. As used herein, a "fusion protein" or "chimeric
protein" includes at least a portion of a mucin polypeptide
operatively linked to a non-mucin polypeptide. A "mucin
polypeptide" refers to a polypeptide having a mucin domain. The
mucin polypeptide has one, two, three, five, ten, twenty or more
mucin domains. The mucin polypeptide is any glycoprotein
characterized by a amino acid sequence substituted with O-glycans.
For example a mucin polypeptide has every second or third amino
acid being a serine or threonine. The mucin polypeptide is a
secreted protein. Alternatively, the mucin polypeptide is a cell
surface protein.
[0028] Mucin domains are rich in the amino acids threonine, serine
and proline, where the oligosaccharides are linked via
N-acetylgalactosamine to the hydroxy amino acids (O-glycans). A
mucin domain comprises or alternatively consists of an O-linked
glycosylation site. A mucin domain has 1, 2, 3, 5, 10, 20, 50, 100
or more O-linked glycosylation sites. Alternatively, the mucin
domain comprises or alternatively consists of a N-linked
glycosylation site. A mucin polypeptide has 50%, 60%, 80%, 90%, 95%
or 100% of its mass due to the glycan. A mucin polypeptide is any
polypeptide encode for by a MUC genes (i.e., MUC1, MUC2, MUC3,
etc.) Alternatively, a mucin polypeptide is P-selectin glycoprotein
ligand 1 (PSGL-1), CD34, CD43, CD45, CD96, GlyCAM-1, MAdCAM or red
blood cell glycophorins. Preferably, the mucin is PSGL-1. Whereas a
"non-mucin polypeptide" refers to a polypeptide of which at least
less than 40% of its mass is due to glycans.
[0029] Within an ABO fusion protein of the invention the mucin
polypeptide can correspond to all or a portion of a mucin protein.
In one embodiment, an ABO fusion protein comprises at least a
portion of a mucin protein. "At least a portion" is meant that the
mucin polypeptide contains at least one mucin domain (e.g., an
O-linked glycosylation site). In one embodiment, the mucin protein
comprises the extracellular portion of the polypeptide. For
example, the mucin polypeptide comprises the extracellular portion
of PSGL-1.
[0030] The first polypeptide is glycosylated by one or more blood
group transferases. The first polypeptide is glycosylated by 2, 3,
5 or more blood group transferases. Glycosylation is sequential or
consecutive. Alternatively glycosylation is concurrent or random,
i.e., in no particular order. For example the first polypeptide is
glycosylated by an .alpha.1,2 fucosyltransferase, such as the H- or
Se-gene encoded .alpha.1,2 fucosyltransferases. Exemplary
.alpha.1,2 fucosyltransferases are FUT1 (Gen Bank Acc. Nos: Q10984;
O10983; O10981; AT455028 and NM00148) and FUT2. (Gen Bank Acc. No:
P19526; BAA11638; D82933 and A56098) Alternatively, the first
polypeptide is glycosylated by 1,3
N-acetylgalactosaminyltransferase or .alpha.1,3
galactosaminyltransferase. In some aspects, the first polypeptide
is glycosylated by both an 1,2 fucosyltransferase and a 1,3
N-acetylgalactosaminyltransferase or a .alpha.1,3
galactosaminyltransferase.
[0031] Within the fusion protein, the term "operatively linked" is
intended to indicate that the first and second polypeptides are
chemically linked (most typically via a covalent bond such as a
peptide bond) in a manner that allows for O-linked glycosylation of
the first polypeptide. When used to refer to nucleic acids encoding
a fusion polypeptide, the term operatively linked means that a
nucleic acid encoding the mucin polypeptide and the non-mucin
polypeptide are fused in-frame to each other. The non-mucin
polypeptide can be fused to the N-terminus or C-terminus of the
mucin polypeptide.
[0032] In a further embodiment, the ABO fusion protein may be
linked to one or more additional moieties. For example, the ABO
fusion protein may additionally be linked to a GST fusion protein
in which the ABO fusion protein sequences are fused to the
C-terminus of the GST (i.e., glutathione S-transferase) sequences.
Such fusion proteins can facilitate the purification of ABO fusion
protein. Alternatively, the ABO fusion protein may additionally be
linked to a solid support. Various solid support are know to those
skilled in the art. Such compositions can facilitate removal of
anti-blood group antibodies. For example, the ABO fusion protein is
linked to a particle made of, e.g., metal compounds, silica, latex,
polymeric material; a microtiter plate; nitrocellulose, or nylon or
a combination thereof. The ABO fusion proteins linked to a solid
support are used as an absorber to remove anti-blood group
antibodies from a biological sample, such as blood or plasma.
[0033] In another embodiment, the fusion protein is includes a
heterologous signal sequence (i.e., a polypeptide sequence that is
not present in a polypeptide encoded by a mucin nucleic acid) at
its N-terminus. For example, the native mucin signal sequence can
be removed and replaced with a signal sequence from another
protein. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of polypeptide can be increased through
use of a heterologous signal sequence.
[0034] An chimeric or fusion protein of the invention can be
produced by standard recombinant DNA techniques. For example, DNA
fragments coding for the different polypeptide sequences are
ligated together in-frame in accordance with conventional
techniques, e.g., by employing blunt-ended or stagger-ended termini
for ligation, restriction enzyme digestion to provide for
appropriate termini, filling-in of cohesive ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic ligation. In another embodiment, the fusion gene can be
synthesized by conventional techniques including automated DNA
synthesizers. Alternatively, PCR amplification of gene fragments
can be carried out using anchor primers that give rise to
complementary overhangs between two consecutive gene fragments that
can subsequently be annealed and reamplified to generate a chimeric
gene sequence (see, for example, Ausubel et al. (eds.) CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
Moreover, many expression vectors are commercially available that
encode a fusion moiety (e.g., an Fc region of an immunoglobulin
heavy chain). A glycoprotein Ib.alpha. encoding nucleic acid can be
cloned into such an expression vector such that the fusion moiety
is linked in-frame to the immunoglobulin protein.
[0035] ABO fusion polypeptides may exist as oligomers, such as
dimers, trimers or pentamers. Preferably, the ABO fusion
polypeptide is a dimer.
[0036] The first polypeptide, and/or nucleic acids encoding the
first polypeptide, can be constructed using mucin encoding
sequences are known in the art. Suitable sources for mucin
polypeptides and nucleic acids encoding mucin polypeptides include
GenBank Accession Nos. NP663625 and NM145650, CAD10625 and
AJ417815, XP140694 and XM140694, XP006867 and XM006867 and
NP00331777 and NM009151 respectively, and are incorporated herein
by reference in their entirety.
[0037] In some embodiments, the mucin polypeptide moiety is
provided as a variant mucin polypeptide having a mutation in the
naturally-occurring mucin sequence (wild type) that results in
increased carbohydrate content (relative to the non-mutated
sequence). For example, the variant mucin polypeptide comprised
additional O-linked glycosylation sites compared to the wild-type
mucin. Alternatively, the variant mucin polypeptide comprises an
amino acid sequence mutations that results in an increased number
of serine, threonine or proline residues as compared to a wild type
mucin polypeptide. This increased carbohydrate content can be
assessed by determining the protein to carbohydrate ratio of the
mucin by methods know to those skilled in the art.
[0038] In some embodiments, the mucin polypeptide moiety is
provided as a variant mucin polypeptide having mutations in the
naturally-occurring mucin sequence (wild type) that results in a
mucin sequence more resistant to proteolysis (relative to the
non-mutated sequence).
[0039] In some embodiments, the first polypeptide includes
full-length PSGL-1. Alternatively, the first polypeptide comprise
less than full-length PSGL-1 polypeptide such as the extracellular
portion of PSGL-1. For example the first polypeptide less than 400
amino acids in length, e.g., less than or equal to 300, 250, 150,
100, 50, or 25 amino acids in length. Exemplary PSGL-1 polypeptide
and nucleic acid sequences include GenBank Access No: XP006867;
XM006867; XP140694 and XM140694.
[0040] The second polypeptide is preferably soluble. In some
embodiments, the second polypeptide includes a sequence that
facilitates association of the ABO fusion polypeptide with a second
mucin polypeptide. In preferred embodiments, the second polypeptide
includes at least a region of an immunoglobulin polypeptide. "At
least a region" is meant to include any portion of an
immunoglobulin molecule, such as the light chain, heavy chain, FC
region, Fab region, Fv region or any fragment thereof.
Immunoglobulin fusion polypeptide are known in the art and are
described in e.g., U.S. Pat. Nos. 5,516,964; 5,225,538; 5,428,130;
5,514,582; 5,714,147; and 5,455,165.
[0041] In some embodiments, the second polypeptide comprises a
full-length immunoglobulin polypeptide. Alternatively, the second
polypeptide comprise less than full-length immunoglobulin
polypeptide, e.g., a heavy chain, light chain, Fab, Fab.sub.2, Fv,
or Fc. Preferably, the second polypeptide includes the heavy chain
of an immunoglobulin polypeptide. More preferably the second
polypeptide includes the Fc region of an immunoglobulin
polypeptide.
[0042] In another aspect of the invention the second polypeptide
has less effector function that the effector function of a Fc
region of a wild-type immunoglobulin heavy chain. Fc effector
function includes for example, Fc receptor binding, complement
fixation and T cell depleting activity. (see for example, U.S. Pat.
No. 6,136,310) Methods of assaying T cell depleting activity, Fc
effector function, and antibody stability are known in the art. In
one embodiment the second polypeptide has low or no affinity for
the Fc receptor. In an alternative embodiment, the second
polypeptide has low or no affinity for complement protein C1q.
[0043] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding
mucin polypeptides, or derivatives, fragments, analogs or homologs
thereof. In various aspects the vector contains a nucleic acid
encoding a mucin polypeptide operably linked to an nucleic acid
encoding an immunoglobulin polypeptide, or derivatives, fragments
analogs or homologs thereof. Additionally, the vector comprises a
nucleic acid encoding a blood group transferase such as a
.alpha.1,2 fucosyltransferase, a .alpha.1,3 N
acetylgalactosamininytransferase, a .alpha.1,3
galactosyltransferase or any combination thereof. The blood group
transferase facilitates the addition of blood group determinants on
the peptide backbone of the mucin portion of the ABO fusion
protein. As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. One type of vector is a "plasmid", which refers to
a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector,
wherein additional DNA segments can be ligated into the viral
genome. Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) are
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively-linked. Such
vectors are referred to herein as "expression vectors". In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and "vector" can be used interchangeably as the plasmid
is the most commonly used form of vector. However, the invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
[0044] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, that is operatively-linked to the nucleic acid sequence
to be expressed. Within a recombinant expression vector,
"operably-linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner
that allows for expression of the nucleotide sequence (e.g., in an
in vitro transcription/translation system or in a host cell when
the vector is introduced into the host cell).
[0045] The term "regulatory sequence" is intended to includes
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Such regulatory sequences are described,
for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
Regulatory sequences include those that direct constitutive
expression of a nucleotide sequence in many types of host cell and
those that direct expression of the nucleotide sequence only in
certain host cells (e.g., tissue-specific regulatory sequences). It
will be appreciated by those skilled in the art that the design of
the expression vector can depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. The expression vectors of the invention can be
introduced into host cells to thereby produce proteins or peptides,
including fusion proteins or peptides, encoded by nucleic acids as
described herein (e.g., ABO fusion polypeptides, mutant forms of
ABO fusion polypeptides, etc.).
[0046] The recombinant expression vectors of the invention can be
designed for expression of ABO fusion polypeptides in prokaryotic
or eukaryotic cells. For example, ABO fusion polypeptides can be
expressed in bacterial cells such as Escherichia coli, insect cells
(using baculovirus expression vectors) yeast cells or mammalian
cells. Suitable host cells are discussed further in Goeddel, GENE
EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,
San Diego, Calif. (1990). Alternatively, the recombinant expression
vector can be transcribed and translated in vitro, for example
using T7 promoter regulatory sequences and T7 polymerase.
[0047] Expression of proteins in prokaryotes is most often carried
out in Escherichia coli with vectors containing constitutive or
inducible promoters directing the expression of either fusion or
non-fusion proteins. Fusion vectors add a number of amino acids to
a protein encoded therein, usually to the amino terminus of the
recombinant protein. Such fusion vectors typically serve three
purposes: (i) to increase expression of recombinant protein; (ii)
to increase the solubility of the recombinant protein; and (iii) to
aid in the purification of the recombinant protein by acting as a
ligand in affinity purification. Often, in fusion expression
vectors, a proteolytic cleavage site is introduced at the junction
of the fusion moiety and the recombinant protein to enable
separation of the recombinant protein from the fusion moiety
subsequent to purification of the fusion protein. Such enzymes, and
their cognate recognition sequences, include Factor Xa, thrombin
and enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) that fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein.
[0048] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and
pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990)
60-89).
[0049] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant
protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS
IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990)
119-128. Another strategy is to alter the nucleic acid sequence of
the nucleic acid to be inserted into an expression vector so that
the individual codons for each amino acid are those preferentially
utilized in E. coli (see, e.g., Wada, et al., 1992. Nucl. Acids
Res. 20: 2111-2118). Such alteration of nucleic acid sequences of
the invention can be carried out by standard DNA synthesis
techniques.
[0050] In another embodiment, the ABO fusion polypeptide expression
vector is a yeast expression vector. Examples of vectors for
expression in yeast Saccharomyces cerivisae include pYepSec1
(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and
Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987.
Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego,
Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
[0051] Alternatively, ABO fusion polypeptide can be expressed in
insect cells using baculovirus expression vectors. Baculovirus
vectors available for expression of proteins in cultured insect
cells (e.g., SF9 cells) include the pAc series (Smith, et al.,
1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow
and Summers, 1989. Virology 170: 31-39).
[0052] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987.
EMBO J. 6: 187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,
MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[0053] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but also to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0054] A host cell can be any prokaryotic or eukaryotic cell. For
example, glycoprotein Ib.alpha. fusion polypeptides can be
expressed in bacterial cells such as E. coli, insect cells, yeast
or mammalian cells (such as human, Chinese hamster ovary cells
(CHO) or COS cells). Other suitable host cells are known to those
skilled in the art.
[0055] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A
LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[0056] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Various selectable markers
include those that confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding glycoprotein Ib.alpha. fusion polypeptides or can be
introduced on a separate vector. Cells stably transfected with the
introduced nucleic acid can be identified by drug selection (e.g.,
cells that have incorporated the selectable marker gene will
survive, while the other cells die).
[0057] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) ABO fusion polypeptides. Accordingly, the invention
further provides methods for producing ABO fusion polypeptides
using the host cells of the invention. In one embodiment, the
method comprises culturing the host cell of invention (into which a
recombinant expression vector encoding ABO fusion polypeptides has
been introduced) in a suitable medium such that ABO fusion
polypeptides is produced. In another embodiment, the method further
comprises isolating ABO polypeptide from the medium or the host
cell.
[0058] The ABO fusion polypeptides may be isolated and purified in
accordance with conventional conditions, such as extraction,
precipitation, chromatography, affinity chromatography,
electrophoresis or the like. For example, the immunoglobulin fusion
proteins may be purified by passing a solution through a column
which contains immobilized protein A or protein G which selectively
binds the Fc portion of the fusion protein. See, for example, Reis,
K. J., et al., J. Immunol. 132:3098-3102 (1984); PCT Application,
Publication No. WO87/00329. The fusion polypeptide may the be
eluted by treatment with a chaotropic salt or by elution with
aqueous acetic acid (1 M).
[0059] Alternatively, an ABO fusion polypeptides according to the
invention can be chemically synthesized using methods known in the
art. Chemical synthesis of polypeptides is described in, e.g., A
variety of protein synthesis methods are common in the art,
including synthesis using a peptide synthesizer. See, e.g., Peptide
Chemistry, A Practical Textbook, Bodasnsky, Ed. Springer-Verlag,
1988; Merrifield, Science 232: 241-247 (1986); Barany, et al, Intl.
J. Peptide Protein Res. 30: 705-739 (1987); Kent, Ann. Rev.
Biochem. 57:957-989 (1988), and Kaiser, et al, Science 243: 187-198
(1989). The polypeptides are purified so that they are
substantially free of chemical precursors or other chemicals using
standard peptide purification techniques. The language
"substantially free of chemical precursors or other chemicals"
includes preparations of peptide in which the peptide is separated
from chemical precursors or other chemicals that are involved in
the synthesis of the peptide. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of peptide having less than about 30% (by dry
weight) of chemical precursors or non-peptide chemicals, more
preferably less than about 20% chemical precursors or non-peptide
chemicals, still more preferably less than about 10% chemical
precursors or non-peptide chemicals, and most preferably less than
about 5% chemical precursors or non-peptide chemicals.
[0060] Chemical synthesis of polypeptides facilitates the
incorporation of modified or unnatural amino acids, including
D-amino acids and other small organic molecules. Replacement of one
or more L-amino acids in a peptide with the corresponding D-amino
acid isoforms can be used to increase the resistance of peptides to
enzymatic hydrolysis, and to enhance one or more properties of
biologically active peptides, i.e., receptor binding, functional
potency or duration of action. See, e.g., Doherty, et al., 1993. J.
Med. Chem. 36: 2585-2594; Kirby, et al., 1993. J. Med. Chem.
36:3802-3808; Morita, et al., 1994. FEBS Lett. 353: 84-88; Wang, et
al., 1993. Int. J. Pept. Protein Res. 42: 392-399; Fauchere and
Thiunieau, 1992. Adv. Drug Res. 23: 127-159.
[0061] Introduction of covalent cross-links into a peptide sequence
can conformationally and topographically constrain the polypeptide
backbone. This strategy can be used to develop peptide analogs of
the fusion polypeptides with increased potency, selectivity and
stability. Because the conformational entropy of a cyclic peptide
is lower than its linear counterpart, adoption of a specific
conformation may occur with a smaller decrease in entropy for a
cyclic analog than for an acyclic analog, thereby making the free
energy for binding more favorable. Macrocyclization is often
accomplished by forming an amide bond between the peptide N- and
C-termini, between a side chain and the N- or C-terminus [e.g.,
with K.sub.3Fe(CN).sub.6 at pH 8.5] (Samson et al., Endocrinology,
137: 5182-5185 (1996)), or between two amino acid side chains. See,
e.g., DeGrado, Adv Protein Chem, 39: 51-124 (1988). Disulfide
bridges are also introduced into linear sequences to reduce their
flexibility. See, e.g., Rose, et al., Adv Protein Chem, 37: 1-109
(1985); Mosberg et al., Biochem Biophys Res Commun, 106: 505-512
(1982). Furthermore, the replacement of cysteine residues with
penicillamine (Pen, 3-mercapto-(D) valine) has been used to
increase the selectivity of some opioid-receptor interactions.
Lipkowski and Carr, Peptides: Synthesis, Structures, and
Applications, Gutte, ed., Academic Press pp. 287-320 (1995).
Methods of Treating or Preventing Antibody-Mediated Graft
Rejection
[0062] Also included in the invention are methods of treating or
preventing antibody mediated graft rejection (AMR), e.g., organ
transplant rejection. Such transplants include but are not limited
to kidney, liver, skin, pancreas, cornea, or heart. AMR is meant to
include any antibody mediated graft rejection by the recipient. The
method includes contacting a biological sample from a subject with
the ABO fusion peptide of the invention. The biological sample is
for example, blood, i.e., whole blood or plasma. The sample is know
to or suspected of comprising an antibody, e.g., an anti-blood
group antibody. In some aspects, the biological sample is withdrawn
from the subject prior to contacting the sample with the ABO fusion
polypeptide. The biological sample is contacted with the ABO fusion
peptide under conditions to allow formation of an ABO fusion
peptide-anti-blood group antibody complex. The ABO fusion
peptide-complex, if present is separated from the biological sample
to eliminate the anti-blood group antibodies and the biological
sample is reinfused into the subject. AMR is also treated or
prevented by administering to a subject an ABO fusion polypeptide
of the invention.
[0063] The subject can be e.g., any mammal, e.g., a human, a
primate, mouse, rat, dog, cat, cow, horse, pig. The treatment is
administered prior to the subject receiving an ABO-incompatible
transplant. Alternatively, treatment is administered after a
subject receives an ABO incompatible transplant.
[0064] The biological sample is contacted with the ABO fusion
protein by methods know to those skilled in the art. For example,
plasmapheresis or extracorporeal immunoabsorption.
[0065] Essentially, any disorder, which is etiologically linked to
an antibody mediated reaction is considered amenable to prevention
or to treatment. AMR is treated or prevent when the survival rate
of the organ transplant is greater than an organ transplant not
treated by the method of the invention. By survival rate of the
transplant is meant the time before the transplant is rejected by
the recipient For example, AMR is treated or prevent when the
transplant survives at least 1, 2, 4 or 8 weeks after transplant.
Preferably, the transplant survives 3, 6, 13 months. More
preferably, the transplant survives 2, 3, 5 or more years.
Methods of Removing Anti-Blood Group Antibodies from a Sample
[0066] Also included in the invention are methods of removing or
depleting anti-blood group antibodies from a sample. The sample is
a biological fluid such as blood or plasma. Alternatively, the
sample is a biological tissue, such as heart tissue, liver tissue,
skin, or kidney tissue. The method includes contacting a sample
with the ABO fusion peptide of the invention. The sample is
contacted with the ABO fusion peptide under conditions to allow
formation of an ABO fusion peptide-anti-blood group antibody
complex. The ABO fusion peptide-antibody complex, if present is
separated from the biological sample to remove or deplete the
anti-blood group antibodies.
Pharmaceutical Compositions Including ABO Fusion Polypeptides or
Nucleic Acids Encoding Same
[0067] The ABO fusion proteins, or nucleic acid molecules encoding
these fusion proteins, (also referred to herein as "Therapeutics"
or "active compounds") of the invention, and derivatives,
fragments, analogs and homologs thereof, can be incorporated into
pharmaceutical compositions suitable for administration. Such
compositions typically comprise the nucleic acid molecule, protein,
or antibody and a pharmaceutically acceptable carrier. As used
herein, "pharmaceutically acceptable carrier" is intended to
include any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like, compatible with pharmaceutical
administration. Suitable carriers are described in the most recent
edition of Remington's Pharmaceutical Sciences, a standard
reference text in the field, which is incorporated herein by
reference. Preferred examples of such carriers or diluents include,
but are not limited to, water, saline, finger's solutions, dextrose
solution, and 5% human serum albumin. Liposomes and non-aqueous
vehicles such as fixed oils may also be used. The use of such media
and agents for pharmaceutically active substances is well known in
the art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0068] The active agents disclosed herein can also be formulated as
liposomes. Liposomes are prepared by methods known in the art, such
as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:
3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030
(1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with
enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556.
[0069] Particularly useful liposomes can be generated by the
reverse-phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter.
[0070] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (i.e., topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid (EDTA); buffers such as acetates,
citrates or phosphates, and agents for the adjustment of tonicity
such as sodium chloride or dextrose. The pH can be adjusted with
acids or bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0071] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0072] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., an ABO fusion protein) in
the required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions,
methods of preparation are vacuum drying and freeze-drying that
yields a powder of the active ingredient plus any additional
desired ingredient from a previously sterile-filtered solution
thereof.
[0073] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0074] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0075] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0076] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0077] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0078] In some embodiments, oral or parenteral compositions are
formulated in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on the unique characteristics of
the active compound and the particular therapeutic effect to be
achieved, and the limitations inherent in the art of compounding
such an active compound for the treatment of individuals.
[0079] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see, e.g., U.S. Pat. No.
5,328,470) or by stereotactic injection (see, e.g., Chen, et al.,
1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical
preparation of the gene therapy vector can include the gene therapy
vector in an acceptable diluent, or can comprise a slow release
matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells that
produce the gene delivery system.
[0080] Sustained-release preparations can be prepared, if desired.
Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
antibody, which matrices are in the form of shaped articles, e.g.,
films, or microcapsules. Examples of sustained-release matrices
include polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods.
[0081] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0082] The invention will be further illustrated in the following
non-limiting examples.
EXAMPLE 1
General Methods
[0083] The data described herein was generated using the following
reagents and methods.
Cell Culture
[0084] COS-7 m6 cells (Seed, 1987), CHO-K1 (ATCC CCL-61), and the
SV4O Large T antigen expressing 293 human embryonic kidney cell
line (293T; kindly provided by B. Seed), were cultured in
Dulbecco's modified Eagle's medium (GibcoBrl, Life Technologies,
Paisley, Scotland), supplemented with 10% fetal bovine serum
(GibcoBrl, Life Technologies), 25 .mu.g/ml gentamycin sulfate
(Sigma, St. Louis, Mo.) and 2 mM glutamine (GibcoBrl, Life
Technologies). The cells were passaged every 2-4 days. The HH14
hybndoma (ATCC HB-9299; U.S. Pat. No. 4,857,639) were cultured in
RPMI 1640 (GibcoBrl, Life Technologies), supplemented with 10%
fetal bovine serum, 100 U/ml of penicillin, 100 .mu.g/.mu.l of
streptomycin, and 2 mM glutamine.
Purification of HH14 Antibodies
[0085] Supernatant was collected from cultured HH14 cells. Two
liters of supernatant was affinity purified on a goat anti-mouse
IgM (Sigma) column, using a Bio-Rad LP chromatograph (Bio-Rad,
Hercules, Calif.). The bound proteins were eluted by 0.1 M
glycine-Cl, pH 2.5, and the eluate was immediately neutralized
using 1 M Tris-Cl, pH 7.5. The eluate was dialyzed against 1%
phosphate-buffered saline (PBS) and lyophilized. Lyophilized
proteins were dissolved in distilled H.sub.2O to a final
concentration of 3 .mu.g/.mu.l, as measured by the BCA assay.
Construction of Expression Vectors
[0086] The human blood group A gene was polymerase chain reaction
(PCR) amplified off cDNA made from total RNA isolated from the
MKN-45 cell line, using 5'-cgc ggg aag ctt gcc gag acc aga cgc
gga-3' (SEQ ID NO:1) as forward primer and 5'-cgc ggg cgg ccg ctc
acg ggt tcc gga ccg c-3' (SEQ ID NO:2) as reverse primer. The
amplified cDNA (A gene) was subcloned into the polylinker of CDM8
(Seed, 1987) using Hind III and Not I. The blood group H gene was
PCR amplified in two pieces using a human tonsil stroma library as
the template. An internal Sse I site was created by PCR using
internal overlapping primers changing nucleotide 775 (GenBank
accession no. M35531) to a C, creating a Sse I site of the Pst I
site. The cDNA encoding the carboxy terminal of FUT1 was amplified
using 5'-ggg gac tac ctg cag gtt atg cct cag cgc-3' (SEQ ID NO:3)
as forward primer and 5'-cgc ggg gcg gcc gct tca agg ctt agc caa
tgt-3' (SEQ ID NO:4) as reverse primer, cleaved by Sse I and Not I,
and subcloned into the CDM8 expression vector digested with Pst I
and Not I. The eDNA encoding the amino terminal of FUT1 was
amplified using 5'-cgc ggg aag ctt acc atg tgg ctc cgg agc cat-3'
(SEQ ID NO:5) as forward primer and 5'-cca gcg ctg agg cat aac ctg
cag gta gtc-3' (SEQ ID NO:6) as reverse primer, cleaved by Hind III
and Sse I, and subcloned into CDM8 carrying the carboxy terminal
following Hind III and Sse I cleavage.
[0087] The Se gene was similarly PCR amplified from eDNA reversely
transcribed from total RNA isolated from peripheral blood
mononuclear cells donated by a blood group A.sub.2Le(a-b.sup.+)Se
individual, using 5'-cgc ggg aag ctt acc atg ctg gtc gtt cag atg-3'
(SEQ ID NO:7) as forward primer and 5'-cgc ggg cgg ccg ctt agt gct
tga gta agg g-3' (SEQ ID NO:8) as reverse primer. The Se gene cDNA
was subcloned into CDM8 using Hind III and Not I.
Glycosyltransferase cDNAs were sequenced and the enzymatic activity
they encoded checked by flow cytometric analysis of transiently
transfected cells using blood group H and A-specific monoclonal
antibodies. The PSGL-1/mIgG.sub.2b chimera was constructed as
described before (Liu et al., 1997).
Transfections and Production of Secreted PSGL-1/mIgG.sub.2b
Chimeras
[0088] The transfection cocktail was prepared by mixing 39 .mu.l of
20% glucose, 39 .mu.g of plasmid DNA, 127 .mu.l dH.sub.2O, and 15.2
.mu.l 0.1M polyethylenimine (25 kDa; Aldrich, Milwaukee, Wis.) in
5-ml polystyrene tubes. In all transfection mixtures, 13 .mu.g of
the PSGL-1/mIgG.sub.2b plasmid was used. Thirteen micrograms of the
plasmid for the different glycosyltransferases were added, and,
when necessary, the CDM8 plasmid was added to reach a total of 39
.mu.g of plasmid DNA. The mixtures were left in room temperature
for 10 min before being added in 10 ml of culture medium to the
cells, at approximately 70% confluency. After 7 days, cell
supernatants were collected, debris spun down (1400.times.g, 15 mm)
and NaN.sub.3 was added to a final concentration of 0.02%
(w/v).
Purification of Secreted PSGL-1/mIgG.sub.2b, for SDS-PAGE and
Western Blot Analysis
[0089] PSGL-1/mIgG.sub.2b fusion proteins were purified from
collected supernatants on 50 .mu.l goat anti-mIgG agarose beads
(100:1 slurry; Sigma) by rolling head over tail overnight at
4.degree. C. The beads with fusion proteins were washed three times
in PBS and used for subsequent analysis. Typically, the sample was
dissolved in 50 .mu.l of 2.times. reducing sample buffer and 10:1
of sample was loaded in each well.
PNGaseF Treatment of Affinity-Purified PSGL-1/mIgG.sub.2b
[0090] A PNGaseF kit (Roche Diagnostics, Indianapolis, Ind.) was
used for N-glycan deglycosylation, A slight modification of the
protocol provided by the manufacturer was used. In 1.5-ml Eppendorf
tubes, 20 .mu.l of reaction buffer was mixed with purified
PSGL-1/mIgG.sub.2b on agarose beads and boiled for 3 min. The
mixture was spun down, and 10 .mu.l of the supernatant was
transferred to a new Eppendorf tube. Ten microliters of PNGaseF or,
as a negative control, 10 .mu.l of reaction buffer were added. The
tubes were incubated for 1.5 h at 37.degree. C. After incubation,
20 .mu.l of 2.times. reducing sample buffer and 10 .mu.l of
H.sub.2O was added, and the samples were boiled for 3 min.
ELISA for Determination of PSGL-1/mJgG.sub.2b Concentration in
Supernatants
[0091] Ninety-six-well ELISA plates (Costar 3590, Corning, N.Y.)
were coated with 0.5 .mu.g/well of affinity-purified goat anti-mIgG
specific antibodies (Sigma) in 50 .mu.l of 50 mM carbonate buffer,
pH 9.6, for two h in room temperature. After blocking o/n at
4.degree. C. with 300 .mu.l 3% bovine serum albumin (BSA) in PBS
with 0.05% Tween (PBS-T) and subsequent washing, 50 .mu.l sample
supernatant was added, serially diluted in culture medium.
Following washing, the plates were incubated for 2 h with 50 .mu.l
of goat anti-mIgM-HRP (Sigma), diluted 1:10,000 in blocking buffer.
For the development solution, one tablet of
3,3',5,5'-tetramethylbenzidine (Sigma) was dissolved in 11 ml of
0.05 M citrate/phosphate buffer with 3 .mu.l 30% (w/v)
H.sub.2O.sub.2. One hundred microliters of development solution was
added. The reaction was stopped with 25 .mu.l 2 M H.sub.2SO.sub.4.
The plates were read at 450 and 540 nm in an automated microplate
reader (Bio-Tek Instruments, Winooski, Vt.). As a standard, a
dilution series of purified mIgG Fe fragments (Sigma) in culture
medium was used in triplicate.
SDS-PAGE and Western Blotting
[0092] SDS-PAGE was run by the method of Laemmli (1970) with a 5%
stacking gel and an 8% resolving gel, and separated proteins were
electrophoretically blotted onto Hybond.TM.-C extra membranes as
described before (Liu et al., 1997). Following blocking overnight
in Tris-buffered saline with 0.05% Tween-20 (TBS-T) with 3% BSA,
the membranes were washed three times with TBS-T. They were then
incubated for 1 h in room temperature with mouse anti-human blood
group A all types (mIgM, Dako, Carpinteria, Calif.) or anti-human H
type I (mIgG.sub.3, Signet; Dedham, Mass.), H type 2 (mIgM, Dako)
or H type 3 (mIgM, hybridoma HH14, ATCC HB9299). All antibodies
were diluted 1:200 in 3% BSA in TBS-T, except for the H type 3
antibody, which was diluted to a concentration of 1 .mu.g/ml in 3%
BSA in TBS-T. The membranes were washed three times with TBS-T
before incubation for 1 h at room temperature with secondary
horseradish peroxidase (HRP)-conjugated antibodies, goat anti-mIgM
(Cappel, Durham, N.C.) or goat anti-mIgG.sub.3 (Serotec, Oxford,
England) diluted 1:2000 in 3% BSA in TBS-T. Bound secondary
antibodies were visualized by chemiluminescence using the ECL kit
(Amersham Pharmacia Biotech, Uppsala, Sweden) according to the
instructions of the manufacturer. For detection of the
PSGI-1/mIgG2b itself, HRP-labeled goat anti-mIgG (Sigma) was used
at a dilution of 1:10,000 in 3% BSA in TBS-T as described, but
without incubation with a secondary antibody.
Determination of the Relative Blood Group A Epitope Density on
PSGL-1/mIgG.sub.2b
[0093] Western blots were run as described above. The membranes
were visualized in a Fluor-S Max MultImager carrying a CCD camera
operating at -35.degree. C. (BioRad). Using the volume tool in the
analysis window of the Quantity One software (BioRad), the volume
(sum of the intensities of the pixels within the volume
boundary.times.pixel area) for the blood group A reactivity was
divided by the volume for the mIgG reactivity for the
PSGL-1/mIgG.sub.2b made in COS, CHO, and 293T cells. To compare the
A epitope/mouse IgG ratios between PSGL-1/mIgG.sub.2b made in the
different host cells, the ratios were normalized to the ratio
obtained from the A substituted PSGL-1/mIgG.sub.2b made in COS
cells transfected with the Hand A genes.
Absorption of Serum
[0094] Six hundred microliters slurry of goat anti-mIgG agarose
beads (Sigma) was transferred into 1.5-ml Eppendorf microcentrifuge
tubes. The beads were spun down by a quick spin at 400.times.g. The
supernatant was then removed, and the beads were washed once with 1
ml PBS, spun down again, and transferred to 180 ml of supernatant
from CHO cells transfected with cDNAs encoding PSGL-1/mIgG.sub.2b
the Se and the A gene. Supernatants containing agarose beads were
incubated head over tail o/n at 4.degree. C. For collection, the
beads were spun down at 400.times.g, 15 min, at room temperature,
and transferred to 1.5-ml Eppendorf microcentrifuge tubes. Washing
was done three times with PBS. These beads are referred to as A
mucin-beads. Six hundred microliters of anti-mIgG agarose beads
were also prepared in the same manner, but they were used for
dilution of the A mucin-beads to obtain a dilution series of the A
mucin-beads as absorbents. These beads are referred to as goat
anti-mIgG beads. The beads were aliquoted into 4-ml Ellerman tubes
according to Table I. A-PAA-MPG (Syntesome, Munich, Germany) and
B-PAA-MPG (Syntesome) were weighed and aliquoted into Ellerman
tubes according to Table 1, and thereafter washed once with
PBS.
[0095] Pooled serum from five patients typed blood group O was
obtained from the Blood Bank at Huddinge University Hospital
(Ethical permission, Dnr. 392/99, approval date Aug. 15, 2000).
Cell debris was removed from the serum by centrifugation at 14,000
rpm for 5 min in a Jouan A-14 microcentrifuge. The cleared serum
was transferred to another tube and incubated in a waterbath at
56.degree. C. for 1 h to inactivate complement. The serum was
stored in aliquots at -20.degree. C. until being used.
TABLE-US-00001 TABLE I Serial dilutions of absorbents used for
absorption of anti-blood group A antibodies from pooled serum of
blood group O individuals Sample Amount of absorbent 1 100 .mu.l of
A mucin-beads 2 80 .mu.l of A mucin-beads, 20 .mu.l of goat
anti-mIgG beads 3 60 .mu.l of A mucin-beads, 40 .mu.l of goat
anti-mIgG beads 4 40 .mu.l of A mucin-beads, 60 .mu.l of goat
anti-mIgG beads 5 20 .mu.l of A mucin-beads, 80 .mu.l of goat
anti-mIgG beads 6 100 .mu.l of goat anti-mIgG beads 7 150 mg of
A~PAA~MPG.sup.a 8 120 mg of A-PAA-MPG, 30 mg of B.PAA.MPG.sup.b 9
90 mg of A-PAA-MPG, 60 mg of B-PAA-MPG 10 60 mg of A-PAA-MPG, 90 mg
of B-PAA-MPG 11 30 mg of A-PAA-MPG, 120 mg of B-PAA-MPG 12 150 mg
of B-PAA-MPG 13 Only serum .sup.aA-PAA-MPG, A trisaccharides linked
via poly[N-(2-hydmxyethyl)acrylamide] to macroporous glass beads.
.sup.bB-PAA-MPG B trisacchaiides linked via
poly[N-(2-hydroxyethyl)acrylamide] to macroporous glass beads.
[0096] Five hundred microliters of serum were added to each tube
and mixed with the beads for 4 h on a rolling table at 4.degree. C.
Following absorption, the beads were spun down and the absorbed
serum transferred to new Ellerman tubes. The absorbed serum was
stored at -20.degree. C. until further analysis.
[0097] To determine the amount of PSGL-1/mIgG.sub.2b on the A
mucin-beads, a goat anti-mIgG Fc ELISA (see previous procedure) was
run on the supernatant before and after incubation with agarose
beads.
ELISA for Quantification of Anti-A Antibodies
[0098] Ninety-six-well ELISA plates (Costar 3590, Corning) were
coated for 2 h at room temperature with 0.05 .mu.l of A-PAA-biotin
(Syntesome) in 50 .mu.l per well of 50 mM carbonate buffer, pH 9.6.
Following blocking o/n at 4.degree. C. with 300 .mu.l 3% BSA in
PBS-T and subsequent washing, 50 .mu.l of serum serially diluted in
PBS were added and the plate was incubated at room temperature for
2 h. After washing, the incubation was done with 50 .mu.l of
mouse-anti-human IgA, G and M-HRP (Jackson, Pa.) diluted 1:10,000
in blocking buffer. Development and reading of the plates was done
as described previously.
Determination of Total Protein Concentration in Serum
[0099] The total protein concentration in serum was determined
before and after absorption of anti-A antibodies. The microtiter
plate protocol of the BCA protein Assay Reagent (Pierce, Rockford,
Ill.) was used according to the manufacturer's instructions, and
samples were run in duplicates or triplicates.
EXAMPLE 2
Blood Group H and A Determinants on Recombinant PSGL-1/mIgG20 Made
in Various Host Cells
[0100] 293T cells. Immunoaffinity-purified PSGL-1/mIgG.sub.2b,
produced by 293T cells transiently transfected with the
PSGL-1/mIgG.sub.2b cDNA with and without the plasmids encoding the
H, Se, or A genes was analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western
blot using anti-mIgG, anti-blood group A, H type 1, H type 2, and H
type 3 specific antibodies for detection (FIG. 1A-E). The fusion
protein migrated under reducing conditions as a doublet with an
apparent molecular weight of approximately 100 and 140 to 160 kDa
(A). The PSGL-1/mIgG.sub.2b stained poorly with silver (not shown)
in concordance with previous observations with respect to the
behavior of highly glycosylated, mucin-type proteins (Carraway and
Hull, 1991; Shimizu and Shaw, 1993). As shown before (Liu et al.,
1997), the fusion protein was produced as a homodimer (data not
shown). Double bands around 65 kDa and 35 kDa were detected with
the anti-mIgG antibody. These bands were not seen in the
supernatants of 293T cells transfected with empty vector alone and
are thus likely to be proteolytic fragments derived from the fusion
protein (Carraway and Hull, 1991). The 47-kDa and 20-kDa bands seen
in cell fractions are derived from the heavy and light Ig chains of
the goat anti-mouse antibody, which is coming off the agarose beads
on boiling in sample buffer.
[0101] Both FUT1 and FUT2 could with the .alpha.1,3 GalNAcT support
the biosynthesis of blood group A chains on the PSGL-1/mIgG.sub.2b
(B). The combination of FUT2 and the .alpha.1,3 GalNAcT seemed to
create more A epitopes on PSGL-1/mIgG.sub.2b, than the combination
of FUT1 and the .alpha.1,3 GalNAcT (cf. these lanes in B). On the
other hand, FUT1 alone supported expression of abundant H type 2
structures (D), whereas FUT2 gave rise to few H type 2 epitopes on
the mucin/Ig (D). There were no detectable H type 1 structures on
PSGL-1/mIgG.sub.2b (C). The H epitopes created by FUT2 were based
almost exclusively on type 3, that is,
Fuc.alpha.2Gal.beta.3GalNAc.alpha.-R, because no H type 1
structures and few H type 2 structures were made on
PSGL-1/mIgG.sub.2b by this enzyme (E). Interestingly,
cotransfection of both the H or Se gene with the A gene gave rise
to abundant epitopes on the PSGL-1/mIgG.sub.2b reactive with the
anti-H type 3 antibody (E). Blood group A, H type 2 and 3 epitopes
were mainly O-linked, because peptide N-glycosidase F (PNGaseF)
treatment of PSGL-1/mIgG.sub.2b, did not decrease antibody staining
using antibodies specific for these epitopes (FIG. 2B-D). Efficient
N-glycan deglycosylation was indicated by a complete mobility shift
of the PSGL-1/mIgG.sub.2b (FIG. 2A-D).
[0102] COS cells. The western blot analysis of PSGL-1/mIgG.sub.2b
made in COS cells is shown in FIG. 3A-E. Although weaker, the
pattern of PSGL-1/mIgG.sub.2b staining using anti-A and H specific
antibodies was somewhat similar to that found for the
PSGL-1/mIgG.sub.2b made in 293T cells. Both FUT1 and FUT2 could
together with the .alpha.1,3 GalNAcT support A epitope expression,
with more epitopes created by FUT2 and the .alpha.1,3 GalNAcT (B).
Only FUT1 could make H type 2 structures (D), and neither FUT1 nor
FUT2 supported expression of H type I structures in COS cells (C).
In contrast to the PSGL-1/mIgG.sub.2b made in 293T-cells, very low
levels of H type 3 epitopes were seen on the PSGL-1/mIgG.sub.2b,
produced in COS cells coexpressing FUT1 and the .alpha.1,3 GalNAcT,
or the FUT2 enzyme alone (E).
[0103] However, joint expression of FUT2 and the .alpha.1,3 GalNAcT
gave rise to increased reactivity with the H type 3 antibody (E).
Furthermore, weak bands were seen with the anti-A antibody when
only the .alpha.1,2 FTs were expressed, indicating a weak
endogenous activity of an .alpha.1,3 GalNAcT in COS cells, as
previously reported (Clarke and Watkins, 1999). PNGaseF treatment
of PSGL-1/mIgG.sub.2b did not show any detectable reduction in
anti-blood group A, H type 2 or 3 antibody staining (data not
shown), indicating that these epitopes are mainly carried on
PSGL-1/mIgG.sub.2b O-glycans.
[0104] Chinese hamster ovary (CHO) cells. FIG. 4A-E, shows the
staining of PSGL-1/mIgG.sub.2b made in CHO cells,
PSGL-1/mIgG.sub.2b carried blood group A epitopes following
coexpression of FUT1 or FUT2 cDNAs with the .alpha.1,3 GalNAcT (B).
Neither H type 1 (C) nor H type 2 (D) structures could be detected
on the PSGL-1/mIgG.sub.2b chimera made in CHO cells following
cotransfection with either FUT1 or FUT2, suggesting that H type 3
structures are the sole precursors available for the .alpha.1,3
GalNAcT. Staining with the anti-H-type 3 antibody was seen with
both FUT1 and FUT2 (B), supporting the theory that the A epitopes
are based solely on core 1 structures in CHO cells, Further support
to this was obtained after PNGaseF treatment, which did not affect
the antibody staining intensity, indicating an O-glycan restricted
A epitope expression (data not shown). The number of A epitopes and
H type 3 epitopes on the PSGL-1/mIgG.sub.2b chimera were clearly
higher with FUT2 as compared to FUT1 suggesting that the Se gene
product is superior to the H gene product in terms of
.alpha.1,2-fucosylation of core 1 (Gal.beta.3GalNAc.alpha.-Ser/Thr)
structures.
EXAMPLE 3
Relative Blood Group A Epitope Density on PSGL-1/mIgG.sub.2b
Produced in Different Host Cells
[0105] To semi-quantify the relative number of A epitopes on the
PSGL-1/mIgG.sub.2b chimera made in various host cells expressing
the A gene with either of the .alpha.1,2 FTs, western blotting with
anti-A antibodies and anti-mIgG antibodies followed by
chemiluminescence detection in a Fluor-S.RTM.Max MultImager was
used. The ratios of the blood group A and the mIgG reactivities for
each PSGL-1/mIgG.sub.2b are shown in FIG. 5 (one of three
representative experiments shown). As seen, the A epitope density
was highest on the PSGL-1/mIgG.sub.2b made in CHO cells expressing
the A gene together with the Se gene encoded .alpha.1,2 FT. This
PSGL-1/mIgG.sub.2b, carried approximately three times more A
epitopes than the PSGL-1/mIgG.sub.2b, made in 293T cells
transfected with FUT2 and the A gene; the PSGL-1/mJgG.sub.2b,
having the second highest A epitope density (FIG. 5). In each cell
line, the FUT2 gave higher A epitope density together with the A
gene than did FUT1 together with the A gene.
EXAMPLE 4
Absorption of Anti-A Antibodies on PSGL-1/mIgG.sub.2b Carrying
Blood Group A Epitopes
[0106] The efficacy of anti-A antibody absorption on recombinant
PSGL-1/mIgG.sub.2b with or without blood group A epitopes was
compared to that of absorption on A trisaccharides linked via
poly[N-(2-hydroxyethyl)acrylamide] to macroporous glass beads
(A-PAA-MPG). Pre- and postabsorption anti-A-antibody levels were
assessed in an enzyme-linked immunosorbent assay (ELISA) in which
the plate was coated with A trisaccharides linked to
poly[N-(2-hydroxyethyl)acrylamide also substituted with biotin
(A-PAA-biotin). The results are shown in FIG. 6. Twenty pmoles of
recombinant A epitope-substituted PSGL-1/mIgG.sub.2b, were needed
to absorb 60% of the anti-A antibodies as detected in the
A-PAA-biotin ELISA, whereas 164,000 pmoles of A determinants as
A-PAA-MPG were needed to absorb the same amount of anti-A
antibodies. The PSGL-1/mIgG.sub.2b dimer has 106 potential O-linked
glycosylation sites and eight potential N-linked glycosylation
sites (Wilkins et al., 1996; Aeed et al., 1998, 2001), of which the
latter eight may carry branched structures. If one assumes that
each PSGL-1/mIgG.sub.2b carries approximately 100 A epitopes, which
most likely is an over-estimation, the mucin made in CHO cells with
FUT2 and the .alpha.1,3GalNacT is approximately 80 times more
efficient on a carbohydrate molar basis than is A-PAAF-MPG.
EXAMPLE 5
Treating or Preventing Antibody Mediated Graft Rejection
[0107] Organ transplantation across the ABO barrier is
characterized by AMR mediated by preformed or induced antibodies
against the donor organ blood group (Porter, 1963; Sanchez-Urdazpal
et al., 1993; Farges et al., 1995; Tanabe et al, 1998; Alkhunaizi
et al., 1999). Anti-ABO antibodies are removed using a
PSGL-1/mIgG.sub.2b fusion protein carrying A determinants as the
absorber in an EIA setting. Results show that approximately 20
pmoles of recombinant PSGL-1/mIgG.sub.2b (calculated on a molecular
weight of 300 kDa), corresponding to 2 nmoles of A determinants
(based on 100 A determinants per mole of PSGL-1/mIgG.sub.2b)
(Wilkins et al., 1996; Aeed et al., 1998, 2001) made in CHO-K1 with
FUT2 and .alpha.1,3 GalNAcT could absorb 60% of the A-PAA-reactive
antibodies. A-PAA-MPG corresponding to approximately 164,000 pmoles
of A trisaccharides (calculated on 2 .mu.mol/.mu.g) was needed to
absorb the same amount of anti-A antibodies. The amount of
nonspecific protein absorption for both compounds was also
assessed, and the nonspecific absorption was almost fourfold higher
with the PAA-MPG-based compounds. The relatively higher absorption
efficacy of mucin-based absorbers may depend on (1) multivalent
carbohydrate substitution, (2) close spacing of carbohydrate
epitopes, and (3) a structural versatility of the core saccharide
chains that carry the immunodominant determinant. Furthermore,
changing the protein backbone, for example, by making a synthetic
mucin-type protein constructed by optimized mucin tandem repeats,
may improve the absorber (Silverman et al., 2001). Also, cells
engineered to express different combinations of GalNAc:polypeptide
transferases may improve absorption efficacy by optimising the
O-glycan substitution density.
Abbreviations
[0108] AMR, antibody-mediated rejection; BSA, bovine serum albumin;
CHO, Chinese hamster ovary; COS; ETA, extracorporeal
immunoabsorption; ELISA, enzyme-linked immunosorbent assay; FT,
fucosyltransferase; GalNAcT, N-acetylgalactosaminyltransferase;
HRP, horseradish peroxidase; mIgG, mouse IgG; mIgM, mouse IgM; MPG,
macroporous glass beads; PAA, poly[N-(2-hydroxyethyl)acrylamide];
PBS, phosphate buffered saline; PBS-T, PBS with 0.05% Tween; PCR,
polymerase chain reaction; PNGaseF, peptide:N-glycosidase F; PP,
plasmapheresis; PSGL-1, P-selectin glycoprotein ligand-1; SDS-PAGE,
sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBS-T,
Tris-buffered saline with 0.05% Tween-20; Tx, transplantation.
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OTHER EMBODIMENTS
[0167] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
Sequence CWU 1
1
8130DNAartificial sequenceforward primer 1cgcgggaagc ttgccgagac
cagacgcgga 30231DNAartificial sequencereverse primer 2cgcgggcggc
cgctcacggg ttccggaccg c 31330DNAartificial sequenceforward primer
3ggggactacc tgcaggttat gcctcagcgc 30433DNAartificial
sequencereverse primer 4cgcggggcgg ccgcttcaag gcttagccaa tgt
33533DNAartificial sequenceforward primer 5cgcgggaagc ttaccatgtg
gctccggagc cat 33630DNAartificial sequencereverse primer
6ccagcgctga ggcataacct gcaggtagtc 30733DNAartificial
sequenceforward primer 7cgcgggaagc ttaccatgct ggtcgttcag atg
33831DNAartificial sequencereverse primer 8cgcgggcggc cgcttagtgc
ttgagtaagg g 31
* * * * *