U.S. patent application number 11/153082 was filed with the patent office on 2005-11-17 for antigenic fusion protein carrying galalpha 1,3gal epitopes.
Invention is credited to Holgersson, Jan, Liu, Jining.
Application Number | 20050255099 11/153082 |
Document ID | / |
Family ID | 20406335 |
Filed Date | 2005-11-17 |
United States Patent
Application |
20050255099 |
Kind Code |
A1 |
Holgersson, Jan ; et
al. |
November 17, 2005 |
Antigenic fusion protein carrying Galalpha 1,3Gal epitopes
Abstract
The present invention relates to an antigenic fusionprotein,
which carries multiple Gal.alpha.1,3Gal epitopes. The fusion
protein according to the invention may also be comprised of a
heavily glycosylated mucin part, which mediates binding to
selecting, such as PSGL-1, and a part, which exhibits
immunoglobulin properties, such as the Fc part of IgG. The
fusionprotein according to the invention is preferably used as an
absorber to prevent a hyperacute rejection of a xenotransplant,
such as a pig tissue or organ transplanted into a human patient. In
addition, the invention relates to a method for the prevention of a
hyperacute rejection reaction in a patient who is to receive a
xenotransplant.
Inventors: |
Holgersson, Jan; (Huddinge,
SE) ; Liu, Jining; (Huddinge, SE) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY
AND POPEO, P.C.
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
20406335 |
Appl. No.: |
11/153082 |
Filed: |
June 15, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11153082 |
Jun 15, 2005 |
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09194396 |
Dec 8, 1998 |
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6943239 |
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09194396 |
Dec 8, 1998 |
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PCT/SE98/00555 |
Mar 26, 1998 |
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Current U.S.
Class: |
424/140.1 ;
435/193; 435/320.1; 435/325; 435/69.7; 530/395; 536/23.5 |
Current CPC
Class: |
C07K 14/47 20130101;
C07K 14/4727 20130101; C07K 2319/40 20130101; C07K 2319/30
20130101; A61P 37/00 20180101; A61P 37/06 20180101 |
Class at
Publication: |
424/140.1 ;
530/395; 435/069.7; 435/193; 435/320.1; 435/325; 536/023.5 |
International
Class: |
A61K 039/00; C07H
021/04; C12P 021/06; A61K 039/395; C12N 009/10; C07K 014/47 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 26, 1997 |
SE |
9701127-4 |
Claims
What is claimed is:
1. A dimerized fusion polypeptide comprising a first polypeptide
operably linked to a second polypeptide, wherein the first
polypeptide: (a) comprises the extracellular portion of a
P-selectin glycoprotein ligand-1; and (b) is glycosylated by an
.alpha.1,3 galactosyltransferase and the second polypeptide
comprises an immunoglobulin Fc region.
2. The fusion polypeptide of claim 1, wherein the first polypeptide
comprises multiple Gal.alpha.1,3Gal epitopes.
3. The fusion polypeptide of claim 1, wherein the .alpha.1,3
galactosyltransferase is porcine.
4. A dimerized fusion polypeptide comprising a first polypeptide
operably linked to a second polypeptide, wherein the first
polypeptide: (a) comprises a part of a P-selectin glycoprotein
ligand-1 that mediates binding to selectin; and (b) is glycosylated
by an .alpha.1,3 galactosyltransferase and the second polypeptide
comprises an immunoglobulin Fc region.
5. The fusion polypeptide of claim 4, wherein the first polypeptide
comprises multiple Gal.alpha.1,3Gal epitopes.
6. The fusion polypeptide of claim 4, wherein the .alpha.1,3
galactosyltransferase is porcine.
7. An nucleic acid molecule encoding the fusion protein of claim
1.
8. A vector comprising the nucleic acid molecule of claim 7.
9. A cell transfected with the vector of claim 8.
10. The cell of claim 9, further comprising a nucleic acid molecule
encoding a al,3 galactosyltransferase.
11. A host cell genetically engineered to express the fusion
polypeptide of claim 1.
12. A cell comprising: a) a nucleic acid encoding the extracellular
portion of a P-selectin glycoprotein ligand-1 operably linked to a
nucleic acid encoding am immunoglobulin Fc region polypeptide and
b) a nucleic acid encoding an .alpha.1,3 galactosyltransferase
polypeptide.
13. The cell of claim 12, wherein the cell is a COS cell, a CHO
cell or a 293T cell.
14. An absorber comprising the fusion polypeptide of claim 1.
15. A method for removing an antibody from blood or plasma, the
method comprising (a) contacting said blood or plasma with the
fusion polypeptide of claim I to form a fusion polypeptide complex;
and (b) removing the complex from said blood or plasma thereby
removing the antibody from said blood or plasma.
16. A method for treating or preventing hyperacute rejection in a
patient in need thereof, the method comprising (a) contacting blood
or plasma from the patient with the fusion polypeptide of claim 1
to form a fusion polypeptide complex; and (b) removing the complex
from said blood or plasma thereby treating or preventing hyperacute
rejection in said patient.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
09/194,396, filed Dec. 8, 1998 which claims priority under 35
U.S.C. .sctn. 120 to PCT International Application No.
PCT/IL98/00379, filed Aug. 13, 1998 and SE 9701127-4, filed Mar.
26, 1997. The contents of these applications are incorporated
herein by reference in their entireties.
TECHNICAL FIELD
[0002] The present invention relates to antigenic fusion proteins
carrying multiple Gal.alpha.1,3Gal-epitopes for the removal of
foreign antibodies, such as xenoreactive human anti-pig antibodies,
by absorption.
BACKGROUND
[0003] Many diseases are today curable only by a transplantation of
tissue or an organ, such as a kidney or heart. It is sometimes
possible to locate a living donor with immunological markers
compatible with the transplant recipient, although organ donation
by a living donor involves great risks and possible deleterious
health effects for the donor. Without any available living donor,
the organ must be obtained from a heartbeating human cadaver of
high quality and, again, there must be a good immunological match
between the donor and the recipient. The situation today is a
steadily increasing demand for human organs suitable for
transplantation and the gap between said demand and the
availability of organs is likely to grow even wider in view of the
continuing improvements made in transplantation procedures and
outcome. The most promising possible answer to this problem is
xenotransplantation, i.e. transplantation of tissue or organs
between different species. For human patients, the pig is
considered the most suitable donor species for medical, practical,
ethical and economical reasons.
[0004] The main problem in xenografting between discordant species,
such as pig to human, is the hyperacute rejection (HAR), which
leads to a cessation of the blood flow within minutes following a
transplantation. Even though other mechanisms of rejection will
ensue after HAR, the general belief is that if HAR could be
prevented, the patient's immune system may undergo a process of
accommodation, whereafter a conventional immunosuppressive regimen
could maintain the compatibility of the patient and the
xenograft.
[0005] The HAR is caused by preformed, natural antibodies in the
receiving species reacting with antigens on the endothelium in
donor organs, an interaction which leads to complement and
endothelial cell activation, thrombosis, extravasation of white
blood cells and, eventually, rejection. Pig antigens reacting with
human, natural antibodies have turned out to be carbohydrates
(7-10); the major one being the Gal1.alpha.1,3Gal epitope which is
not expressed in old world monkeys, apes and humans due to an
inactivation of the .alpha.1,3 galactosyltransferase (GT)
(10-12).
[0006] Several methods have been proposed for the removal or
elimination of xenoreactive antibodies from the blood of a
recipient. Bach et al (Xenotransplantation, Eds: Cooper, D. K. C.,
et al, Springer Verlag, 1991, Chapter 6) proposed the perfusion of
the recipients blood through an organ of the proposed donor species
prior to transplantation of another, fresh organ, whereby anti-pig
antibodies were removed.
[0007] Plasmapheresis has also been proposed for a non-specific
removal of naturally occurring antibodies, whereby the graft
survival is prolonged (e.g. Cairns et al, Rydberg et al). However,
conventional plasmapheresis, or plasma exchange, results in loss of
blood volume, which in turn may require a volume replacement with
pooled preparations of fresh frozen plasma, human albumin,
immunoglobulin etc. In addition, coagulation factors, platelets and
antithrombotic factors must also be replaced. Such a treatment
carries not only the risk of virus transfer, such as HIV, but also
the risk of an anaphylactic reaction to foreign substances. Other
negative side effects of plasmapheresis are recipient sensitization
and activation of the complement and clotting system. Accordingly,
plasmapheresis does not appear to be either practical or safe.
[0008] Other methods for the removal of xenoreactive antibodies
involve non-specific antibody removal. Protein A, a major component
of the cell wall of S. aureus, has a high affinity for a portion of
the Fc-region of sub-classes 1, 2 and 4 of immunoglobulin G (IgG1,
IgG2, IgG4) and has been used for the non-specific removal of
anti-HLA antibodies from hypersensitized patients in need of kidney
transplants. The efficacy of the Protein A column treatment after
kidney transplantation have been reported (Dantal J., et al, New
England J. Med. 550: 7-14,1994; Nilsson, I. M. et al, Blood 58:
38-44, 1981; Palmer, A., et al., The Lancet Jan. 7, 1989, pp.
10-12). One essential drawback with the use of a Protein A column
technique in the context of xenotransplantation is, however, the
fact that only IgG will be removed. Lately, it has been shown that
the antibodies involved in HAR during a transplantation from pig to
human may involve several other immunoglobulin classes. In
addition, the non-specific antibody removal will cause a general
deterioration of the patients immune defense, which quite naturally
is not desirable during such a process as a transplantation
procedure, where the patient is immunosuppressed.
[0009] Leventhal et al (WO 95/31209) propose a method of preventing
or ameliorating a hyperacute reaction occurring after
transplantation of a pig organ to a primate recipient, including a
human. The method involves passing the recipients plasma over a
column with a coupled protein, which binds to and thereby removes
immunoglobulin therefrom. The protein is selected from a group
consisting of Staphylococcus aureus protein A, Streptococcus
protein G and anti-human immunoglobulin antibodies. This method
suffers from the same drawbacks as depicted above for the protein A
column.
[0010] It has been shown (Platt et al, Good et al, Holgersson et
al) that pig antigens reacting with human, natural antibodies are
carbohydrates, the major one being the Gal.alpha.1,3Gal epitope,
which is not expressed in old world monkeys, apes and humans due to
an inactivation of the .alpha.1,3 galactosyltransferase.
[0011] Recently, McKenzie et al showed that COS cells transfected
with the .alpha.1,3-galactosyltransferase cDNA expressed the
Gal.alpha.1,3Gal-epitope on their surfaces and could absorb most of
the human anti-pig activity from human serum.
[0012] Further, Gal.alpha.1,3Gal-derivatized columns have been used
to specifically remove anti-pig activity from human serum (22),
free Gal.alpha.1,3Gal disaccharides have been shown to prevent
binding of anti-pig antibodies to porcine cells, including
endothelium (23), and so has porcine stomach mucin (24). However,
organic synthesis of saccharides is a very laborious and expensive
method, which in addition is rather slow, and accordingly, has not
found any wide spread applicability.
[0013] Apart from the HAR, xenografts are still typically rejected
within days in a process that has been termed delayed xenograft
rejection (DXR) (29). DXR is characterized by mononuclear cell
activation and graft infiltration, as well as cytokine production
(29). The importance of the Gal.alpha.1,3Gal epitope for these
cellular events is not known, although human anti-Gal.alpha.1,3Gal
antibodies were recently shown to be involved in antibody-dependent
cellular cytotoxicity (ADCC) of porcine cells (30).
[0014] Thus, there is still a need of cheaper and more efficient
methods for the elimination of foreign antibodies, such as
pig-antibodies, from the blood from a recipient who is to obtain a
xenotransplant. In addition, within the field of
xenotransplantation, of methods for the prevention of DXR.
SUMMARY OF THE INVENTION
[0015] The objective of the present invention is to fulfill the
above defined need. Accordingly, the present invention provides an
antigenic fusion protein, which carries multiple Gal.alpha.1,3Gal
epitopes. In a preferred embodiment, the fusion protein according
to the present invention is comprised of a heavily glycosylated
mucin part, which may mediate binding to the selecting, and a part
conferring immunoglobulin properties. The fusion protein according
to the invention carries a multitude of Gal.alpha.1,3Gal epitopes,
that effectively absorbs foreign antibodies, such as anti-pig
antibodies, involved in antibody-dependent, complement-mediated
killing and ADCC of endothelial cells, such as porcine cells.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention relates to an antigenic fusion
protein, which carries multiple Gal.alpha.1,3Gal epitopes.
[0017] Thus, the antigenic fusion protein according to the present
invention is capable of binding preformed antibodies as well as
antibodies produced as a response to a transplanted tissue or organ
originating from the species in which the Gal.alpha.1,3epitopes are
expressed, said species preferably being a species different from
the antibody producing species. In a preferred embodiment, the
antibody producing species is a human being producing antibodies
against a foreign transplant, e.g. a pig organ, in which case the
Gal.alpha.1,3 epitopes are synthesized by a .alpha.1,3
galactosyltransferase of pig origin. The transplant may be an organ
such as a liver, a kidney, a heart etc., or tissue thereof.
Accordingly, in the most preferred embodiment of the fusion protein
according to the invention, the antigenic fusion protein carries
multiple Gal.alpha.1,3Gal epitopes synthesized by a .alpha.1,3
galactosyltransferase derived from a porcine species.
[0018] Thus, as the fusion protein according to the present
invention may be prepared by the culture of genetically manipulated
cells, such as COS cells, it is both cheaper and easier to produce
than the previously saccharides produced by organic synthesis. Even
though a COS cell expressing the Gal.alpha.1,3Gal epitope on its
surface has been described (12), the present invention is the first
proposal of a recombinant fusion protein, which carries multiple
such epitopes. The fusion protein according to the invention can
easily be designed to include other peptides and parts, which may
be advantageous for a particular application. Examples of other
components of the fusion protein according to the invention will be
described more detailed below.
[0019] Thus, in a preferred embodiment, the antigenic fusion
protein according to the invention further comprises a part, which
mediates binding to selectin, such as P-selectin. Said part is
preferably a highly glycosylated protein, such as a protein of
mucin type. The mucins are due to their high content of O-linked
carbohydrates especially advantageous together with the
Gal.alpha.1,3Gal epitope in the fusion protein according to the
invention, as the antigenic properties in the present context
thereby are greatly improved. Thus, it has been shown that the
binding of antibodies which are reactive with the Gal.alpha.1,3Gal
epitope is even more efficient if said epitope is presented by a
protein of mucin type, which indicates that the binding in fact may
involve more than said epitope alone.
[0020] In the hitherto most preferred embodiment of the fusion
protein according to the invention, the part that mediates binding
to selectin is the P-selectin glycoprotein ligand-1 (PSGL-1) or an
essential part thereof. However, other cell membrane-anchored
proteins containing mucin-type domains have been characterized and
may be used in the fusion protein according to the invention as
appropriate, such as CD34, CD43, GlyCAM-1, PSGL-1, MAdCAM, CD96,
CD45 and RBC glycophorins. In the experimental part of the present
application, an example wherein said P-selectin glycoprotein
ligand-1 (PSGL-1) is derived from HL-60 cells, is shown.
Theoretically, the anti-pig antibody repertoire may recognize the
Gal.alpha.1,3Gal epitope in various structural contexts determined
by the core saccharide presenting the epitope, neighbouring
branching points, and the proximity to other sugar residues such as
fucose and sialic acid (31-33). If Gal.alpha.1,3Gal disaccharides
or Gal.alpha.1,3Gal.beta.1,4Gl- cNAc trisaccharides are used as
absorbers some specificities of the repertoire might not be
efficiently absorbed.
[0021] The properties of the part of the fusion protein mediating
selectin binding according to the invention as well as further
reasoning concerning the choice thereof is further discussed below,
see the section "Discussion".
[0022] In an especially advantageous embodiment, the antigenic
fusion protein according the present invention further comprises a
part which confers immunoglobulin properties. The immunoglobulin
parts are advantageous for the design of an efficient and simple
method of coupling the fusion protein according to the invention to
a solid support to be used for purifying plasma from a recipient of
a xenotransplant from antibodies against said transplant. An
immunoglobulin part can also be included in the fusion protein
according to the invention in the preferred case, where the fusion
protein is produced in a cell which secretes it, whereafter the
immunoglobulin part is used for the purification of said secreted
fusion protein from the culture.
[0023] Thus, according to an advantageous embodiment of the
antigenic fusion protein according to the present invention, the
part that confers immunoglobulin properties is an immunoglobulin or
a part thereof, such as IgG or a part thereof. Preferably, said
part that confers immunoglobulin properties is the Fc part of an
immunoglobulin, preferably of IgG, or an essential part thereof. In
a particular embodiment of the present invention, said
immunoglobulin property conferring part is IgG.sub.2b, preferably
the Fc part thereof. In an example of a fusion protein according to
the present invention, said part that confers immunoglobulin
properties is of non-human origin, and is preferably derived from
mouse. However, for certain applications said part might more
preferably be of human origin.
[0024] The fusion protein according to the present invention is
preferably a recombinant fusion protein. It may have been produced
in a recombinant cell line, preferably an eukaryotic cell line,
e.g. a COS cell line, following cotransfection of the cDNA for the
mucin/immunoglobulin fusion protein and the cDNA for the porcine
.alpha.1,3 galactosyltransferase.
[0025] The fusion protein according to the present invention may
for example be used as an absorber for elimination of antibodies
from blood plasma. The use is discussed in more detail below, in
the section "Discussion".
[0026] Another aspect of the present invention is cDNA molecule,
which comprises a cDNA sequence coding for a fusion protein as
defined above or a derivative or variant thereof.
[0027] Yet another aspect of the present invention is a vector,
which comprises the cDNA molecule as described above together with
appropriate control sequences, such as primers etc. The skilled in
the art can easily choose appropriate elements for this end.
[0028] Another aspect of the present invention is a cell line
transfected with the above defined vector. The cell line is
preferably eukaryotic, e.g. a COS cell line. In the preferred
embodiment said cells are secreting the fusion protein according to
the invention into the culture medium, which makes the recovery
thereof easier and, accordingly, cheaper than corresponding methods
for synthesis thereof would be.
[0029] Another aspect of the invention is an absorber comprised of
a fusion protein according to the present invention coupled to a
solid support. The absorber according to the invention is used in a
pretransplant extracorporeal immunoabsorption set up to remove
anti-pig antibodies involved in antibody dependent, complement- as
well as cell-mediated cytotoxicity of pig endothelial cells.
[0030] Finally, a last aspect of the invention is a method of
purifying blood plasma from foreign antibodies, e.g. elimination of
anti-pig antibodies from human blood plasma. The method involves
withdrawal of plasma from a patient, such as a patient who is to
receive a transplant of porcine origin, bringing said plasma in
contact with a fusion protein according to the invention to bind
anti-pig antibodies thereto, whereby the anti-pig antibodies are
eliminated from the plasma, and thereafter reinfusing the plasma
into the patient. The method according to the invention has been
shown to be more efficient than the previously known methods in the
prevention of HAR, possibly thanks to the large amount of
carbohydrate in the fusion protein. Indeed, it is feasible that the
present method also contributes in a prevention of delayed
xenograft rejection (DXR), as the spectra of antibodies eliminated
by the fusion protein according to the invention presumably is
broader than in the prior methods.
Experimental
[0031] In the present application, the following abbreviations are
used: ADCC, antibody-dependent cellular cytotoxicity; BSA, bovine
serum albumin; DXR, delayed xenorejection; ELISA, enzyme-linked
immunosorbent assay; FT, fucosyltransferase; Gal, D-galactose; GT,
galactosyltransferase; Glc, D-glucose; GlcNAc,
D-N-acetylglucosamine; GlyCAM-1, glycosylation-dependent cell
adhesion molecule-1; HAR, hyperacute rejection; Ig, immunoglobulin;
MAdCAM, mucosal addressin cell adhesion molecule; PAEC, porcine
aortic endothelial cells; PBMC, peripheral blood mononuclear cells;
PSGL-1, P-selectin glycoprotein ligand-1; RBC, red blood cell;
SDS-PAGE, sodium dodecyl sulphate--polyacrylamide gel
electrophoresis
[0032] Materials and Methods
[0033] Cell culture. COS-7 m6 cells (35) and the SV40 Large T
antigen immortalized porcine aortic endothelial cell line, PEC-A
(36), were passaged in Dulbecco's modified Eagle's medium (DMEM),
with 10% fetal bovine serum (FBS) and 25 .mu.g/ml gentamicin
sulfate. The human erythroleukemic cell line, K562, and the
Burkitt's lymphoma cell line, Raji, were obtained from ATCC and
cultured in HEPES-buffered RPMI 1640 with 10% FBS, 100 IU/ml
penicillin and 100 .mu.g/ml streptomycin.
[0034] Construction of expression vectors. The porcine .alpha.1,3
GT (37-39) was PCR amplified off pig spleen cDNA using a forward
primer having six codons of complementarity to the 5' end of the
coding sequence, a Kozak translational initiation consensus
sequence and a Hind3 restriction site, and a reverse primer with
six codons of complementarity to the 3' end of the coding sequence,
a translational stop and a Not1 restriction site. The amplified
.alpha.1,3GT cDNA was cloned into the polylinker of CDM8 using
Hind3 and Not1 (35). The P-selectin glycoprotein ligand-1
(PSGL-1)--a highly glycosylated mucin-type protein mediating
binding to P-selectin (40)--coding sequence was obtained by PCR off
an HL-60 cDNA library, cloned into CDM8 with Hind3 and Not1, and
confirmed by DNA sequencing. The mucin/immunoglobulin expression
plasmid was constructed by fusing the PCR-amplified cDNA of the
extracellular part of PSGL-1 in frame via a BamH1 site, to the Fc
part (hinge, CH2 and CH3) of mouse IgG.sub.2b carried as an
expression casette in CDM7 (Seed, B. et al, unpublished).
[0035] Production and purification of secreted mucin/immunoglobulin
chimeras. COS m6 cell were transfected using the DEAE-dextran
protocol and 1 .mu.g of CsCl-gradient purified plasmid DNA per ml
transfection cocktail. COS cells were transfected at approximately
70% confluency with empty vector (CDM8), the PSGL1/mIgG.sub.2b
plasmid alone or in combination with the .alpha.1,3GT encoding
plasmid. Transfected cells were trypsinized and transferred to new
flasks the day after transfection. Following adherence for
approximately 12 hrs, the medium was discarded, the cells washed
with phosphate buffered saline (PBS), and subsequently incubated
another 7 days in serum-free, AIM-V medium (cat.nr. 12030, Life
technologies Inc.). After incubation, supernatants were collected,
debris spun down (1400.times.g, 20 minutes), and NaN.sub.3 added to
0.02%. PSGL1/mIgG.sub.2b fusion protein was purified on goat
anti-mouse IgG agarose beads (A-6531, Sigma) by rolling head over
tail, over night at 4.degree. C. The beads were washed in PBS and
subsequently used for SDS-PAGE and Western blot analysis, or for
absorption of human AB serum and purified human
immunoglobulins.
[0036] Purification of human IgG, IgM and IgA. Human IgG, IgM and
IgA were purified from human AB serum--pooled from more than 20
healthy blood donors--using goat anti-human IgG (Fc specific;
A-3316, Sigma), IgM (.mu.-chain specific; A-9935, Sigma), and IgA
(.alpha.-chain specific; A-2691, Sigma) agarose beads. Briefly, 5
ml of slurry (2.5 ml packed beads) were poured into a column of 10
mm diameter and washed with PBS. Ten milliter of human pooled AB
serum was applied at 1 ml/minute using a peristaltic pump, washed
with several column volumns of PBS, and eluted with 0.1M glycine,
0.15M NaCl, pH 2.4 using a flow rate of 1 ml/minute. One milliliter
fractions were collected in tubes containing 0.7 ml of neutralizing
buffer (0.2M Tris/HCl, pH 9). The absorption at 280 nm was read
spectrophotometrically and tubes containing protein were pooled.
dialyzed against 1% PBS, and lyophilized. Lyophilized
immunoglobulins were resuspended in distilled water and the
concentrations adjusted to 16 mg/ml for IgG, 4 mg/ml for IgA and 2
mg/ml for IgM.
[0037] SDS-PAGE and Western blotting. SDS-PAGE was run by the
method of Leammli with a 5% stacking gel and a 6 or 10% resolving
gel using a vertical Mini-PROTEAN II electrophoresis system
(Bio-Rad, Herculus, Calif.) (41). Separated proteins were
electrophoretically blotted onto Hybond.TM.-C extra membranes
(Amersham) using a Mini Trans-Blot electrophoretic transfer cell
(Bio-Rad, Herculus, Calif.) (42). Protein gels were stained using a
silver staining kit according to the manufacturer's instructions
(Bio-Rad, Herculus, Calif.). Following blocking for at least 2 hrs
in 3% BSA in PBS, the membranes were probed for 2 hrs in room
temperature with peroxidase-conjugated Bandereia simplicifolia
isolectin B.sub.4 (L-5391, Sigma) diluted to a concentration of 1
.mu.g/ml in PBS, pH 6.8 containing 0.2 mM CaCl.sub.2. The membranes
were washed 5 times with PBS, pH 6.8, and bound lectin was
visualized by chemiluminescens using the ECL.TM. kit according to
the instructions of the manufacturer (Amersham).
[0038] Quantification of PSGLb1/mIgG.sub.2b by anti-mouse IgG Fc
ELISA. The concentration of fusion protein in cell culture
supernatants before and after absorption was determined by a
96-well ELISA assay, in which fusion proteins were captured with an
affinity purified, polyclonal goat anti-mouse IgG Fc antibody
(cat.nr. 55482, Cappel/Organon Teknika, Durham, N.C.). Following
blocking with 3% BSA in PBS, the fusion proteins were captured and
detected with a peroxidase-conjugated, affinity purified,
polyclonal anti-mouse IgG Fc antibody (cat.nr. 55566, Organon
Teknika, Durham, N.C.) using O-phenylenediamine dihydrochloride as
substrate (Sigma). The plate was read at 492 nm and the ELISA
calibrated using a dilution series of purified mouse IgG Fc
fragments (cat.nr. 015-000-008, Jackson ImmunoResearch Labs., Inc.,
West Grove, Pa.) resuspended in AIM V serum-free medium.
[0039] Porcine aortic endothelial cell ELISA. PEC-A cells were
seeded at a density of 15000 cells/well in gelatin-coated 96-well
plates (Nunclon, Denmark) and cultured for 48 hrs in AIM V
serum-free medium. The plate was washed 5 times in 0.15M NaCl
containing 0.02% Tween 20 and incubated for 1 hr in room
temperature with 50 .mu.l/well of purified human IgG, IgM, and IgA
in PBS, starting at a concentration of 8, 1, and 2 mg/ml,
respectively. The plate was washed again as above, and 50 .mu.l of
alkaline phosphatase-conjugated goat anti-human IgG (.gamma.-chain
specific; A3312, Sigma), IgM (.mu.-chain specific; A1067, Sigma)
and IgA (.alpha.-chain specific; A3062, Sigma) F(ab)'.sub.2
fragments diluted, 1:200 in PBS were added and incubated for 1 hr
at room temperature. The plate was washed as above, incubated with
the substrate p-nitrophenyl phosphate (Sigma 104-105), and read at
405 nm.
[0040] Porcine aortic endothelial cell cytotoxicity assay. PEC-A
cells were seeded and cultured in 96-well plates as described for
the PEC-A ELISA. Following 48 hrs of culture, the cells were loaded
for 1 hr at 37.degree. C. with Na.sub.2.sup.51CrO.sub.4 (cat.nr.
CJS4, Amersham), 1 .mu.Ci/well, and washed 3 times with AIM V
medium. Fifty microliter of serially diluted, absorbed or
non-absorbed human AB serum or purified human IgG, IgA, or IgM
antibodies were added together with 50 .mu.l of rabbit serum (Cat.
no. 439665, Biotest AG, Dreieich, Germany) as a source of
complement. Following 4 hrs incubation at 37.degree. C. in a 5%
Co.sub.2 atmosphere, the supernatants were harvested using a
Skatron supernatant collection system (Skatro Instruments, Norway)
and analyzed in a .gamma.-counter (1282 Compugamma, LKB Wallac).
Each serum and Ig sample were analyzed in triplicate. The percent
killing was calculated as the measured minus the spontaneous
release divided by the difference between the maximum and the
spontaneous release.
[0041] Antibody-dependent cellular cytotoxicity (ADCC). Human PBMC
were isolated from fresh buffy coats prepared from healthy donors
at the Blood bank of the South hospital, Stockholm. Six milliliter
of buffy coat was mixed in a 50 ml polypropylene tube with 15 ml of
PBS containing 1 mg/ml BSA and 3.35 mg/ml EDTA. Following
centrifugation at 500.times.g for 10 minutes, the platelet rich
supernatant was discarded, and 6 ml of the lower phase was mixed
with 6 ml of Hank's balanced salt solution (HBSS), and underlayered
with 6 ml of Lymphoprep.TM. (Nycomed Pharma AS). Following
centrifugation (800.times.g, 20 min.), the interface was
transferred to a new tube, washed three times in HBSS, and
resuspended in serum-free AIM V medium. The final step in the
effector cell preparation was to transfer the PBMC to tissue
culture flasks that were incubated for 1 hr at 37.degree. C. and 5%
CO.sub.2 to remove plastic-adherent cells. Target cells were K562
and Raji cells kept as described above, or PEC-A cells that had
been trypsinized the day before the assay and subsequently cultured
in AIM V serum-free medium to prevent readhesion to the plastic
surface. At the time for the assay the PEC-A cells were washed off
the bottom of the flask. Target cells were loaded with
Na.sub.2.sup.5ICrO.sub.4, 100 .mu.Ci/1.times.10.sup.6 cells, for 1
hr at 37.degree. C., washed 3 times in HBSS and resuspended in AIM
V to a final concentration of 5.times.10.sup.4/ml. Five thousand
target cells were added to each well with effector cells in 200
.mu.l AIM V medium with and without 10% heat-inactivated, human AB
serum at an effector (E):target (T) ratio ranging from 50:1 in
two-fold dilutions to 6.25:1.
[0042] Spontaneous release was read in wells with 5 000 target
cells incubated in 200 .mu.l AIM V medium without effector cells
and maximum release was read in wells where 5 000 target cells in
100 .mu.l AIM V were incubated with 100 .mu.l 5% Triton X-100. Each
E:T ratio was analyzed in triplicate. Following incubation at
37.degree. C. for 4 hrs, the supernatants were harvested using a
Skatron supernatant collection system (Skatro Instruments, Norway)
and analyzed in a .gamma.-counter (LKB Wallac). The percent killing
was calculated as the measured minus the spontaneous release
divided by the difference between the maximum and the spontaneous
release.
[0043] Results
[0044] Expression and characterization of the PSGL1/mIgG.sub.2b
fusion protein. Supernatants from COS-7 m6 cells transfected with
the vector plasmid CDM8, the PSGL1/mIgG.sub.2b plasmid, or the,
PSGL1/mIgG.sub.2b together with the porcine .alpha.1,3 GT plasmid,
were collected approximately 7 days after transfection. Secreted
mucin/Ig fusion proteins were purified by absorption on anti-mouse
IgG agarose beads and subjected to SDS-PAGE and Western blotting
using the Bandereia simplicifolia isolectin B.sub.4 (BSA IB.sub.4)
for detection. As seen in FIG. 1, the fusion protein migrated under
reducing conditions as a broad band with an apparent molecular
weight of 145 kDa that stained relatively poorly with silver. The
heterogeneity in size, approximately 125 to 165 kDa, and poor
stainability is in concordance with previous observations with
respect to the behavior of highly glycosylated, mucin-type proteins
(43, 44). The fusion protein is most likely produced as a homodimer
because SDS-PAGE under non-reducing conditions revealed a
double-band of an apparent molecular weight of more than 250 kDa.
The amounts of fusion protein affinity-purified from the two
supernatants derived from the same number of COS cells transfected
with the PSGL1/mIgG.sub.2b plasmid alone or together with the
.alpha.1,3GT plasmid, respectively, were similar. Probing the
electroblotted membranes with BSA IB.sub.4 revealed strong staining
of the fusion protein obtained following cotransfection with the
porcine .alpha.1,3 GT (FIG. 1). It is clear, though, that the
PSGL1/mIgG.sub.2b fusion protein produced in COS-7 m6 cells without
cotransfection of the .alpha.1,3 GT CDNA also exhibited weak
staining with the BSA IB.sub.4 lectin, in spite of the fact that
COS cells are derived from the Simian monkey--an old world monkey
lacking .alpha.1,3 GT activity. This indicates that the BSA
IB.sub.4 lectin has a slightly broader specificity than just
Gal.dbd.1,3Gal epitopes (45). Nevertheless, cotransfection of the
porcine .alpha.1,3GT cDNA supported the expression of a highly
Gal.alpha.1,3Gal-substituted PSGL1/mIgG.sub.2b fusion protein.
[0045] Quantification of PSGL1/mIgG.sub.2b chimeras in supernatants
of transfected COS cells, and on goat anti-mouse IgG agarose beads
following absorption. A sandwich ELISA was employed to quantify the
amount of PSGL1/mIgG.sub.2b in the supernatants of transfected COS
cells. Typically, 5 culture flasks (260 ml flasks, Nunclon.TM.)
with COS cells at 70% confluence were transfected and incubated as
described in materials and methods. Following an incubation period
of 7 days in 10 ml of AIM V medium per flask, the medium was
collected. The concentration of fusion protein in the supernatant
from such a transfection, as well as in different volumes of
supernatant following absorption on 100 .mu.l gel slurry of
anti-mouse IgG agarose beads (corresponding to 50 .mu.l packed
beads) was determined by an ELISA calibrated with purified mouse
IgG Fc fragments (FIG. 2). The concentration of PSGL1/mIgG.sub.2b
in the supernatants ranged from 150 to 200 ng/.mu.l, and in this
particular experiment it was approximately 160 ng/.mu.l (FIG. 2A,
the non-absorbed column). The concentration of PSGL1/mIgG.sub.2b
remaining in 2, 4 and 8 ml of supernatant following absorption on
50 .mu.I packed anti-mouse IgG agarose beads was 32, 89 and 117
ng/.mu.l, respectively. This corresponds to 260, 290 and 360 ng of
PSGL1/mIgG.sub.2b being absorbed onto 50 .mu.l packed anti-mouse
IgG agarose beads from 2, 4 and 8 ml of supernatant, respectively.
Western blot analysis with the B. simplicifolia IB.sub.4 lectin
revealed that 50 .mu.l of packed beads could absorb out the
PSGL1/mIgG.sub.2b fusion protein from 1 ml supernatant to below
detectability and from 2 ml to barely detectable levels (not
shown).
[0046] The absorption capacity of immobilized,
Gal.alpha.1,3Gal-substitute- d PSGL1/mIgG.sub.2b. Twenty ml of
supernatant from COS cells transfected with the PSGL1/mIgG.sub.2b
plasmid alone or together with the porcine .alpha.1,3GT cDNA, were
mixed with 500 .mu.l gel slurry of anti-mouse IgG agarose beads
each. Following extensive washing the beads were aliquoted such
that 100 .mu.l of gel slurry (50 .mu.l packed beads) were mixed
with 0.25, 0.5, 1.0, 2.0 and 4.0 ml of pooled, complement-depleted,
human AB serum, and rolled head over tail at 4.degree. C. for 4
hrs. Following absorption on PSGL1/mIgG.sub.2b and
Gal.alpha.1,3Gal-substituted PSGL1/mIgG.sub.2b, the serum was
assayed for porcine endothelial cell cytotoxicity in the presence
of rabbit complement using a 4 hr .sup.51Cr release assay (FIG. 3).
As shown in FIG. 3, 100 .mu.l of beads carrying approximately 300
ng of PSGL1/mIgG.sub.2b (see above) can reduce the cytotoxicity of
4 and 2 ml AB serum in each dilution step, and completely absorb
the cytotoxicity present in 1 ml and less of human AB serum. Note
that the same amount of non-Gal.alpha.1,3Gal-substituted
PSGL1/mIgG.sub.2b reduces the cytotoxicity of 0.25 ml absorbed
human AB serum only slightly (FIG. 3).
[0047] The effect of Gal.alpha.1,3Gal-substituted PSGL1/mIgG.sub.2b
on complement-dependent porcine endothelial cell cytotoxicity and
binding. To investigate the efficiency with which PSGL1/mIgG.sub.2b
could absorb individual human immunoglobulin classes, human IgG,
IgM and IgA were purified from human AB serum by immuno-affinity
chromatography on anti-human IgG, IgM and IgA agarose beads.
Following its isolation IgA was passed through anti-IgG and IgM
columns to remove traces of IgG and IgM. This procedure was
performed for the other Ig classes as well. Ig fraction purity was
checked by SDS-PAGE (FIG. 4). In concentrations found in normal
serum, human IgG and IgM, but not IgA, were cytotoxic for PEC-A in
the presence of rabbit complement (FIG. 5). The cytotoxicity
residing in the IgG and IgM fractions was completely removed by
absorption on Gal.alpha.1,3Gal-substituted PSGL1/mIgG.sub.2b. To
investigate whether the lack of cytotoxicity exhibited by the IgA
fraction was due to a lack of binding of human IgA antibodies to
PEC-A, a cell ELISA was run with the same Ig fractions that was
used in the cytotoxicity assay in order to detect bound IgG, IgM
and IgA. Alkaline phosphatase-conjugated, class specific
F(ab)'.sub.2 fragments were used as secondary antibodies.
Eventhough the cytotoxicity of IgG and IgM was completely removed
by absorption on Gal.alpha.1,3Gal-substituted PSGL1/mIgG.sub.2b,
the binding was never reduced with more than 70% (ranging from 30
to 70%) for IgG, and never with more than 55% (ranging from 10 to
55%) for IgM (FIG. 5). Human IgA clearly bound to PEC-A, and the
binding was only slightly reduced (not more than 29%) following
absorption on Gal.alpha.1,3Gal-substituted PSGL1/mIgG.sub.2b. The
lack of cytototoxicity of the IgA fraction could therefore not be
explained by an inability of the IgA fraction to bind PEC-A, but
may be due to an inability to activate complement.
[0048] The effect of Gal.alpha.1,3Gal-substituted PSGL1/mIgG.sub.2b
on ADCC of porcine endothelial cells. Several assays have been
performed under serum-free conditions where PEC-A have had an
intermediate sensitivity to direkt killing by freshly isolated PBMC
when compared to K562 and Raji cells; K562 being sensitive and Raji
non-sensitive to killing by human NK cells (FIG. 6A). In the
presence of 10% human, complement-inactivated AB serum, the killing
rate was almost doubled supporting an ADCC effect in vitro (FIG.
6B) in agreement with previously published data (30). However, if
the serum is absorbed with the Gal.alpha.1,3Gal-substituted
PSGL1/mIgG.sub.2b under conditions known to remove all PEC-A
cytotoxic antibodies (se above), the killing rate decreases to
levels slightly lower than those seen under serum-free conditions.
On the other hand, the PSGL1/mIgG.sub.2b fusion protein itself
without Gal.alpha.1,3Gal epitopes could not absorb out what caused
the increased killing rate in the presence of human AB serum (FIG.
6B). These data support the notion that anti-pig antibodies with
Gal.alpha.1,3Gal specificity can support an antibody-dependent
cell-mediated cytotoxicity in vitro, and that the
Gal.alpha.1,3Gal-substi- tuted PSGL1/mIgG.sub.2b fusion protein can
effectively remove these antibodies just as it effectively removes
the complement-fixing cytotoxic anti-pig antibodies.
DESCRIPTION OF THE DRAWINGS
[0049] FIG. 1. Six percent SDS-PAGE of proteins isolated from
supernatants of COS cells transfected with vector alone (CDM8),
PSGL1/mIgG.sub.2b, or PSGL1/mIgG.sub.2b and porcine .alpha.1,3GT
expression plasmids. Anti-mouse IgG agarose beads were used for
immunoaffinity purification of fusion proteins. Following extensive
washing, the beads were boiled in sample buffer under reducing and
non-reducing conditions to release absorbed proteins. Gels run in
parallel were either silver stained, or used for electrophoretic
transfer of separated proteins onto nitrocellulose membranes. These
were subsequently probed with peroxidase-conjugated Bandeireia
simplicifolia isolectin B.sub.4 lectin and visualized by
chemiluminescens to detect Gal.alpha.1,3Gal epitopes on
immunopurified proteins. The gel migration length of molecular
weight proteins of 220, 97 and 66 kDa is indicated on the left
handside.
[0050] FIG. 2. Quantification by anti-mouse IgG Fc ELISA of the
PSGL1/mIgG.sub.2b fusion protein concentration in increasing
volumes of transfected COS cell supernatants before and after
absorption on 50 .mu.l of anti-mouse IgG agarose beads. Triplicate
samples were analyzed.
[0051] FIG. 3. Antibody-dependent, complement-mediated PEC-A cell
cytotoxicity by different volumes of human AB serum following
absorption on 50 .mu.l of anti-mouse IgG agarose beads carrying
approximately 300 ng of Gal.alpha.1,3Gal- or non-substituted
PSGL1/mIgG.sub.2b as estimated in a .sup.51Cr-release assay.
[0052] FIG. 4. Ten percent SDS-PAGE of immunoaffinity purified
human IgG, IgM, and IgA. Four micrograms of each sample were run
under reducing and non-reducing conditions, and proteins were
visualized by silver staining. The gel migration length of
molecular weight proteins of 220, 97, 66, 46 and 30 kDa is
indicated on the left handside.
[0053] FIG. 5. The antibody-dependent, complement-mediated PEC-A
cell cytotoxicity of immunoaffinity purified human IgG, IgM and IgA
before and after absorption on Gal.alpha.1,3Gal-substituted
PSGL1/mIgG.sub.2b was investigated by .sup.51Cr-release assays
(right handside Y-axis; % killing). The PEC-A cell binding of
immunoaffinity purified IgG, IgM and IgA before and after
absorption on Gal.alpha.1,3Gal-substituted PSGL1/mIgG.sub.2b was
estimated in a cell ELISA (left handside Y-axis; O.D. at 405 nm).
The two assays were run in parallel on absorbed and non-absorbed Ig
fractions. The concentration of the different Ig classes were
chosen to be approximately half maximum of what is normally found
in human serum.
[0054] FIG. 6. The direct cytotoxic effect (serum-free conditions)
of human PBMC on K562, Raji and PEC-A cells, and the potentiating
effect on killing by the addition of heat-inactivated, human AB
serum, was investigated in a 4 hr .sup.51Cr-release assay (graph
A). The effect of heat-inactivated, human AB serum on
antibody-dependent cellular cytotoxicity of PEC-A cells was studied
in a 4 hr .sup.51Cr-release assay before and after absorption on
Gal.alpha.1,3Gal- and non-substituted PSGL1/mIgG.sub.2b,
respectively (graph B).
DISCUSSION
[0055] Three major avenues of research have been followed in order
to develop strategies to prevent HAR. Methods (i) to remove or
neutralize anti-pig antibodies, (ii) to interfere with complement
activation, and (iii) to remove or modify Gal.alpha.1,3Gal
determinants, have been tested in assays in vitro, in ex vivo organ
perfusions and in pig to primate transplantations.
[0056] Pretransplant pig organ perfusion (46), plasmapheresis (13,
14, 20), immunoabsorption (13, 14, 17), and absorption on
synthetically made Gal.alpha.1,3Gal-containing oligosaccharides
coupled to a solid phase (22) have been used to remove anti-pig
antibodies. Recently, oligosaccharides derived from pig gastric
mucin were shown to efficiently remove
anti-Gal.alpha.1,3Gal-specific antibodies and thereby anti-pig
cytotoxicity (24). Injection of free saccharides to block anti-pig
antibodies in the circulation is an alternative route used to
prevent HAR. Intravenous carbohydrate therapy was used successfully
in an ABO incompatible, heterotopic cardiac allograft model in the
baboon (47) and in neonates with ABO hemolytic disease (48). No
deleterious effects were noticed in recipient baboons upon
injection of free blood group trisaccharides at rates corresponding
to 500 mg/hr following a bolus injection of 4 g of the
trisaccharide (47). Free Gal.alpha.1,3Gal-contain- ing saccharides
have been used in vitro to neutralize anti-pig antibodies (23), but
synthetic Gal.alpha.1,3Gal-containing oligosaccharides in
sufficient quantities for use in pig to primate xenotransplantation
models are not yet available. However, high concentrations of
melibiose (Gal.alpha.1,6Glc) or arabinogalactan, a naturally
occurring plant polysaccharide containing non-reducing
.alpha.-D-galactose, have been used to prevent the toxic effect of
baboon serum on porcine PK15 cells in vitro and to prolong pig
cardiac xenograft survival in transplanted baboons (49). Lethal
toxic effects of the carbohydrate were observed in this study (49).
Peptides mimicking the Gal.alpha.1,3Gal structure (50, 51) and
murine monoclonal anti-idiotypic antibodies directed against common
idiotopes on naturally occurring human anti-pig antibodies (52),
are new reagents that may be used to absorb or block anti-pig
antibodies. In addition, anti-.mu. chain antibodies have been used
in a guinea pig to rat xenotransplantation model to deplete
xenoreactive IgM antibodies in vivo (15, 16)--a strategy that
warrants further investigation in the pig to primate
combination.
[0057] Cobra venom factor (20) and soluble complement receptor type
1 (21) have been used to perturb complement activation and thereby
to prolong xenograft survival in pig to non-human primate
transplantations. Transgenic pigs have been made in which cDNAs
encoding human sequences of the species-restricted
complement-regulatory proteins, CD55 and CD59, are expressed in
porcine endothelium (53-55). Organs from transgenic pigs have been
shown to be less prone to HAR following organ perfusion with human
blood or transplantation into non-human primates (56-59).
[0058] The most recent approach to prevent HAR involves modulating
the expression of Gal.alpha.1,3Gal epitopes on porcine endothelium.
Sandrin and McKenzie suggested and proved that the expression of a
glycosyltransferase, such as an .alpha.1,2 FT, competing with the
endogenous .alpha.1,3 galactosyltransferase for the same precursor
carbohydrate prevented expression of Gal.alpha.1,3Gal epitopes upon
transfection into porcine LLC-PK1 cells (26). As a result these
cells became less sensitive to antibody-mediated,
complement-dependent lysis (26). Recently, two independent groups
have reported the making of transgenic pigs expressing this blood
group H .alpha.1,2 FT, but no data have been reported on the
sensitivity of the endothelium derived from these pigs to
antibody-mediated cytotoxicity (27, 28).
[0059] We describe the construction and production of a novel,
efficient absorber that can be produced cheaply, in large
quantities, and which can be easily coupled to a solid phase
facilitating extracorporeal immunoabsorption of human blood to
remove anti-pig antibodies before pig organ xenografting. To make
possible the production of a highly glycosylated, recombinant
protein carrying large amounts of Gal.alpha.1,3Gal epitopes (FIG.
1), we made a chimeric protein by fusing the cDNA encoding the
extracellular part of a membrane-anchored mucin, PSGL-1 (40), with
the Fc part of mouse IgG, and coexpressed this with porcine
.alpha.1,3 GT. Mucins are the main constituents of mucus, which is
the gel covering all mucous membranes. The physical characteristics
of mucins are due to their high content of O-linked carbohydrates
(usually more than 60% of the MW). Several cell membrane-anchored
proteins containing mucin-type domains have been characterized:
CD34, CD43, GlyCAM-1, PSGL-1, MAdCAM, CD96, CD45, and RBC
glycophorins among others (43, 44). This group of proteins has
received a lot of attention since many of them have been shown to
carry the carbohydrate ligands of the selectin family of adhesion
molecules; CD34, MAdCAM and GlyCAM-1 carry the carbohydrate epitope
recognized by L-selectin and PSGL-1 carries epitopes recognized by
both E- and P-selectin (60). Two reasons made us choose the PSGL-1
extracellular part as a fusion partner. First, it has one of the
longest mucin-domains known in this family of proteins and may
therefore be expected to carry most O-linked carbohydrates (44,
60). Second, PSGL-1 is not only a functional ligand for E-selectin,
but is the hitherto only identified protein scaffold known to
present siatyl-Le.sup.x to P-selectin (61). So, if one were to make
a fusion protein expressing sialyl-Le.sup.x in order to inhibit P-
and E-selectin-mediated adhesion, this should contain the PSGL-1
extracellular part. We are currently investigating the possibility
of making a fusion protein expressing both Gal.alpha.1,3Gal and
sialyl-Le.sup.x thereby exhibiting the characteristics of a dual
inhibitor; one part neutralizing anti-pig antibodies and the other
preventing E- and P-select-independent rolling which may be a
prerequisite for leukocyte extravasation in xenograft
rejection.
[0060] In a xenotransplantation situation, where pig organs for
transplantation would be readily available, the recipient could be
prepared by extracorporeal immunoabsorption to remove anti-pig
antibodies thereby preventing HAR. Alternative strategies could be
chosen (se above), but a specific removal of anti-pig antibodies
would leave the patient in a more favourable state with regard to
the humoral immunity as compared to plasmapheresis and protein A
absorption. Therefore, immunoabsorption media containing the
epitopes recognized by the anti-pig antibodies themselves would be
ideal. The production of Gal.alpha.1,31Gal-containing di- and
trisaccharides is laborious and costly, although a combination of
organic and enzymatic synthesis have simplified the procedure and
made it more cheap (34). Furthermore, it is not unlikely to expect
the specificity of the human anti-pig antibody repertoire to be
broader than just the Gal.alpha.3Gal.beta.4GlcNAc epitope; i.e.
recognition may be modulated by the core saccharide chain,
neighbouring branching points and monosaccharides, as well as
modifications such as sulfation. In this context it would be better
to use a glycosylated, recombinant glycoprotein modified through
the action of the porcine .alpha.1,3 GT itself resulting in a more
natural array of Gal.alpha.1,3Gal-containing structures. Three
hundred ng of our fusion protein could completely remove the pig
endothelial cell cytotoxic antibodies from 1 ml of human AB serum
(FIG. 3), which should be compared to other studies where 1 g of
oligosaccharide linked to a solid-phase was used to absorb 3 ml of
human AB serum (23). However, direct comparisons are needed to
state a difference in absorption capability. The fusion protein is
most likely efficient due to a polyvalent expression of
Gal.alpha.1,3Gal-containing determinants and a possible expression
of the epitope in a variety of structural contexts facilitating the
absorption of a broader spectrum of the human anti-pig repertoire.
Recent, studies with pig gastric mucin-derived oligosaccharides
indicate that polyvalency is important for absorption effectiveness
(24).
[0061] Human IgG, IgM and IgA were purified from pooled human AB
serum (FIG. 4) in order to investigate their binding to, and
cytotoxicity for, porcine aortic endothelial cells, and to evaluate
the Ig class-specific absorption efficacy of the fusion protein
(FIG. 5). The PAEC cytotoxicity of human IgG at 8 mg/ml and IgM at
1 mg/ml was completely removed by Gal.alpha.1,3Gal-substituted
PSGL1/mIgG.sub.2b. However, the binding was not completely
abolished as estimated in a PAEC ELISA using aliquots of the same
antibody preparations as in the cytotoxicity assay. Whether this
represents residual IgG and IgM binding to epitopes other than
Gal.alpha.1,3Gal on PAEC--epitopes which are not able to induce an
antibody-mediated cytotoxic effect, Fc receptor binding, or just a
non-specific, non-functional binding, is not known at present. In
our hands, purified IgA was not cytotoxic to PAEC although it
clearly bound these cells (FIG. 5). This is in contrast with a
previous study by Schaapherder et al, who demonstrated cytotoxicity
by dimeric human IgA via the alternative pathway of complement
activation (62). However, they used serum from human
agammaglobulinemic donors as complement source, whereas we used
rabbit serum (62). Furthermore, we saw no clear evidence of dimeric
IgA in our IgA preparations (FIG. 4). In any case, IgA clearly
binds PAEC and may be involved in pig xenograft rejection through
other effector mechanisms mediated by, for instance, Fc receptor
binding on macrophages and neutrophils (63, 64). This emphasizes
the importance of using absorbers that remove all anti-pig Ig
classes, something which protein A and anti-A chain antibody
absorptions do not.
[0062] Although anti-pig antibodies to a large extent rely on
complement activation for full cytotoxic effect, there are
additional effector mechanisms involving antibodies that may cause
pig endothelial cell cytotoxicity. Antibody-dependent cellular
cytotoxicity is such a mechanism where anti-pig antibodies with
Gal.alpha.1,3Gal specificity are important for cytotoxicity even in
the absence of complement (30). Therefore, we examined how absorbed
and non-absorbed human AB serum contributed to PAEC cytotoxicity by
human PBMC (FIG. 6). As shown in FIG. 6 human complement-depleted
AB serum increased PEC-A cytotoxicity close to more than 10% in 3
out of 4 effector to target ratios. This increased cytotoxicity
contributed by human AB serum was completely removed by absorption
on Gal.alpha.1,3Gal-substituted PSGL1/mIgG.sub.2b whereas
absorbtion on PSGL1/mIgG.sub.2b without Gal.alpha.1,3Gal had no
effect. This clearly demonstrates that Gal.alpha.1,3Gal-specific
antibodies not only contribute to ADCC against porcine endothelial
cells, but are responsible for all of the enhanced cytotoxic effect
seen upon addition of human AB serum to a human PBMC/PAEC mixed
culture. If this ADCC effect is present also in vivo, strategies
aiming at inhibiting complement activation may not be sufficient to
prevent acute pig xenograft rejection.
[0063] We have presented the construction and production of a new
and effective Gal.alpha.1,3Gal-substituted, mucin domain-containing
absorber that can be used in a pretransplant extracorporeal
immunoabsorption setting to remove anti-pig antibodies involved in
antibody-dependent, complement- as well as cell-mediated
cytotoxicity of pig endothelial cells. By stably transfecting cells
to express the PSGL1/mIgG.sub.2b and porcine .alpha.1,3GT cDNAs,
large amounts of fusion protein can be produced at low costs for
testing in pig to primate xenotransplantation models.
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