Title of Invention

METHODS OF DONOR SPECIFIC CROSSMATCHING

Abstract The invention relates to a method for isolation of precursor endothelial cells and the use of these cells for donor specific crossmatching. Today lymphocyte crossmatching is performed before transplantation and is used for detecting antibodies against HLA. Lymphocyte crossmatches does not permit detection of clinically relevant endothelial/monocyte reactive and endothelial specific antibodies. The invention describes a method for isolation of cells that can be used for crossmatching and detection of non-HLA antigens.
Full Text METHOPS OF DONOR SPECIFIC CROSSMATCHING
FIELD OF THE INVENTION
The invention relates to a method for the direct isolation of endothelial cells from
whole blood for routine donor-specific crossmatching to detect anti-endothelial cell
antibodies prior to organ transplantation.
BACKGROUND OF THE INVENTION
The presence of donor lymphocyte-reactive Human Leukocyte Antigen (HLA)-
specific antibodies either before and/or after renal allograft transplantation has been
associated with hyperacute rejections, early acute rejections, and poor graft survival.
However, rejections may occur in the absence of detectable lymphocytotoxic antibodies,
suggesting that non-HLA antigenic systems may also play a role in renal allograft
hyperacute and acute rejections. Antibodies reactive with endothelial cells and monocytes
(also called the EM-antigenic system), or only with endothelial cells, have been described
and reported to have a deleterious effect in several organ transplantations.
Recently, the major histocompatibility class I-rclated chain A antigen (MICA)
expressed on endothelial cells was identified as one of the target antigens of humoral
immunity associated with irreversible rejections of kidney allografts. Studies of HLA-
identical living-related donor allografts showed tliat the presence of endothelial
ccll/monocyte reactive antibodies correlated with rejection, graft loss, and poor allograft
function. It was reported that this reactivity could be responsible for up to 80% of
irreversible rejections in this group of patients However, the routinely used lymphocyte
cross-match (LXM) does not permit detection of the clinically relevant HLA class-I, class-
II, endothclial/monocytc-rcactive and endothelial cell-specific antibodies. Although the
presence of circulating endothelial cells in whole blood has been a subject of debate: for
many years, the existence of circulating precursor endothelial cells in adult humans has
recently been reported by some investigators. However, there is currently no suitable
method available to perform a routine donor-specific endothelial cell-crossrnatch (ECXM).
Therefore, there is a need to efficiently perform routine donor-specific endothelial
cell cross-matching to aid in the identification of better donor-recipient combinations,
which will thereby have a greater impact on transplant survival than the current method of
lymphocyte cross-match.
SUMMARY OF THE INVENTION
The invention pertains to a method of isolating endothelial cells which are useful in
donor specific crossmatching prior to transplantation. The isolated endothelial cells are
also useful in diagnosing various vascular and immune related disorders.
The invention includes a method of isolating an endothelial cell from a biological
sample by providing a sample known to contain, or suspected of containing, an endothelial
cell. The endothelial cell is Tie-2 positive. Alternatively, the isolated endothelial cell is
VEGFR positive, The isolated endothelial cell is a mature cell. Alternatively, the isolated
endothelial cell is an endothelial cell precursor. The sample is contacted with a detection
reagent to form a detection reagent-cndothclial cell complex. The endothelial cell is
isolated by separating the complex form the sample. Separation is accomplished, for
example, by flow cytometry or use of a magnetic field.
The detection reagent is a ligand for an endothelial sell surface receptor such as
ongiopoietin or vascular endothelial growth factor (VEGF). Alternatively the detection
reagent is an antibody specific for an endothelial cell surface receptor such as Tje-2 or the
vascular endothelial growth factor receptor (VEGFR). The antibody is an intact antibody
or an antibody fragment (e.g. Fab, or Fv). The detection reagent is attached to a solid
support, such as a non-magnetic, magnetic, or paramagnetic bead.
The biological sample is whole blood, sera, tissue homogenate, peripheral blood
mononuclear cells (PBMCs) or leucapherisatc.
The invention further provides a method of cross-matching a donor and a recipient
by providing a biological sample from a donor that is known to contain an endothelial cell,
contacting the donor sample with detection reagent to isolate an endothelial cell. A
recipient biological sample is screened for icactivity with the donor isolated endothelial
cell. No reactivity between the recipient sample and the isolated endothelial cell indicates
compatibility between tlie donor and recipient and a higher probability that the organ
transplant will be successful.
In a further aspect, the invention includes a method of diagnosing an immune
related or vascular disorder in a subject by providing a first sample from a subject known to
contain, or suspected of containing, an endothelial cell. The first sample is contacted with
a second sample. The second sample is from a subject known to contain, or suspected of
containing, an auto antibody. Alternatively the second sample contains reagents (e.g.
antibodies) that recognize endothelial cell surface markers associated with the particular
disorder. Complex formation between the endothelial cell and the second sample is
determined. The presence of the complex indicates the disorder in the subject.
In yet another aspect, the invention includes a method of determining the efficacy of
treatment or prognosis of an immune related or vascular disorder in a subject by providing
a first sample from a subject known to contain, or suspected of containing, an endothelial
cell, contacting the endothelial cell with a second sample from a subject known to contain,
or suspected of contnining. an atonntihody mrnsuring nnv autoantibody-endothelial cell
precursor complex present to yield a subject profile. The subject profile is compared with a
reference profile, wherein a similarity between the subject profile and reference profile
indicates that the treatment is efficacious or a favorable prognosis.
Immune related or vascular disorders include vasculitis, atherosclerosis, bleeding
disorders, angiogenesis, thrombosis, defective wound healing, and transplantation.
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 speciticution, inciuding. defination, will control in addition, the
materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following
detailed description, and from the claims
ACCOMPANYING
BRIEF DESCRIPTION OF TIIE/dRA WINGS
Figure J shows dot plots representing forward and side scatter profile as well as
fluorescence of peripheral blood mononuclear cells (PBMCs) stained with anti-Tie-2+
monoclonal antibodies (Mabs). Tie-2+ cells (grey dots) appeared in the lymphocyte gate.
Histograms show the percentage of Tie-2+ cells (app. 2±3%) in PBMCs stained with
negative control abs and Tie-2 Mabs.
Figures 2a-c are photographs of the morphology of Tic-2+ cells under light
microscope at various time points after isolation from peripheral blood. Initially, these
cells appeared either as single cells or as clusters of round cells (a), which after few days of
culture converted into adherent cells with extended cytoplasm, (the dark objects arc beads)
(b). After 7-12 days in culture the cells gradually developed a spindle shape (c).
Figure 3a is a series of photographs showing Tie-2+ cells stained positive for the
endothelial associated markers, acetylated low density lipoprotein (Ac-LDL), von
Willebrand factor (vWF), vascular endothelial growth factor receptor-1 (VEGFR-1) and the
vascular cell adhesion molecule (VCAMXon activated Tie-2+ cells). Tie-2+ cells also
constitutivcly expressed the human leukocyte antigen class 1 (HLA class 1) and the
monocyte/macrophage marker CD68.
Figure 3b shows that quiescent Tie-2+ cells expressed small amounts of the
clinically important MICA antigens (dark grey), and HLA class II (light grey), control
antibodies (black).
Figure 4a is a dot plot that represents the forward and side scatter of rosettes of Tie-
2+ cells and paramagnetic beads (gate R1) and paramagnetic beads only (gate R2).
Figures 4b-g are histograms showing that prc-transplant sera from two kidney-
transplanted patients with hyperacute rejections reacted strongly with their respective
donor-specific Tie-2+ cells (b & e), while their Tie-2- fractions, which included
lymphocytes did not (d & g). Histograms c & f show that the paramagnetic beads alone
did not react with the sera non-specifically. The grey lines represent control or negative
sera, and the black lines represent reactivity with patient's sera.
DETAILED DESCRIPTION OF THE INVENTION
The invention is based in part on the discovery that targeting a single specific cell
population allows for the detection of clinically relevant donor-specific human leukocyte
antigcns(HLA) class I, class II, endothelial-monocytes, or endothelial cell-specific
antibodies prior to transplantation of organs. Routine use of the endothelial cell cross
matching will aid in identifying better donor-recipient combinations and thus have a greater
impact on transplant survival as compared to traditional lymphocyte cross matching.
The clinical importance of endothelial cell (EC) antibodies in allo-transplantation
has been reported. However, lack of a suitable method for isolation of donor-specific ECs
has prevented routine detection of these antibodies prior to transplantation. The invention
provides a quick and simple method for the direct isolation of ECs from whole blood, for
routine cross-matching to detect anti-EC antibodies. The presence of endothelial cell-
reactive antibodies has previously been detected using human umbilical vein endothelial
cell (HUVEC) lines, keratrnocyte cell lines, or monocytes as targets, or by
immunohistochemistry. However, these methods are cumbersome because endothelial cell
culturing is tedious and a generally large panel of donors of HUVEC lines/keratinocytes is
required to represent all the known polymorphic alleles for screening of endothelial cell-
reactive antibodies. Moreover, these methods do not permit the detection of donor-specific
endothelial cell antibodies. Thus, use of an endothelial cell cross-match is advantageous
over use of lymphocyte cross-match because lymphocyte cross-match docs not allow for
such detection or isolation of donor-specific endothelial cell-reactive antibodies.
ECs were isolated using magnetic beads coated with antibodies against the
angiopoietin receptor, Tie-2 that is expressed on EC precursors. Tie genes play an
important role in renal vascular development, and based on transplantation experiments
these precursors have been shown to contribute to the generation of glomerular maturation.
A retrospective analysis of 50 previously well characterized crossmatch sera taken
immediately prior to transplantation from patients with end-stage kidney disease were
tested Tie-2+ cells expressed HLA class T. class TT and other endothelial cell markers
Sera known to contain only EC specific or EC and monocyte (EM) reactive antibodies
reacted positively with Tic-2+ cells, but not with Tic-2- cells from the same individual In
addition, the Tic-2+ cells reacted with sera containing only HLA class 1 or class n abs. In
all, 3/25 sera from patients with stable graft outcome and no rejections reacted with Tie-2+
cells. This antigen-antibody interaction is relevant to the pathogenesis of rejection, since in
many studies these antibodies are not detected in the serum of patients with good graft
function or in non-transplantation patients.
Methods of Isolating Endothelial Cells
The invention includes methods of isolating an endothelial cell from a mixture of
cells by contacting the mixture of cells with a detection reagent to form a endothelial cell-
detection reagent complex. The complex is formed via a specific affinity interaction
between the detection reagent and the cell. The complex is separated from the mixture to
isolate the endothelial cell. The complex is separated from the mixture using techniques
known in the art, such as by, e.g., liquid chromatography {e.g., HPLC or FPLC), High
Performance Membrane Chromatography (HPMC), flow cytometry or use of a magnetic
field. Alternatively, the complex is separated from the mixture by attaching the detection
reagent to a solid support. A washing step may be employed by re-suspending the
complex in a biologically compatible solution. The complex can be re-suspended, i.e.,
washed as many times as desired. Typically the particles arc washed three times. A
biologically compatible solution include biological buffers known in the art such as
phasphate buffer salione (PBS).
The detection reagent is an endothelial cell detection reagent. An endothelial cell
detection reagent is any reagent that specifically identifies an endothelial cell. For
example, the detection reagent is a ligand for a endothelial cell surface receptor. The
endothelial cell surface receptors include, for example, the EC-specific tyrosine kinase
receptor Tie-2 or VEGFR. The ligand is for example angiopoietin, VEGF or an antibody
specific for the cell surface receptor. The antibody is a monoclonal antibody or a
polyclonal antibody. The term antibody encompasses not only an intact antibody, but also
an immunologicalry-active antibody fragment, e.g. , a Fab or (Fab)2 fragment; an
engineered single chain Fv molecule; or a chimeric molecule, e.g.. an antibody which
contains the binding specificity of one anliboily. eg., of murine origin, and the remaining
monoclonal antibody Tic-2 or VEGFR-1
The detection reagent is attached to a solid support. The solid support is a particle,
a polymer (e.g., polystyrene, polyethylene), a vessel, a chamber, a dipstick, beads, particles,
membranes (e.g., nylon, nitrocellulose or polyvinylidenedifluoride (PVDF)), or other forms
known in the art
The solid support may carry functional groups such as hydroxyl, carboxyl, aldehyde
or amino groups. The solid support may be positively charged, negatively charged or
hydrophobic. Functionalized coated supports for use in the present invention may be
prepared by modification of the support For example, uncoated support is treated with a
polymer carrying one or such functional groups, such as polyurethane together with a
poly glycol to provide hydroxyl groups, or a cellulose derivative to provide hydroxyl
groups, a polymer or copolymer of acrylic acid or methacrylic acid to provide carboxyl
groups, or an aminoalkylated polymer to provide amino groups. US Pat No. 4,654,267
describes the introduction of many surface coatings.
The particle is made of metal compounds, silica, latex, polymeric material, or a
silica, latex or polymer nuclei coated with a metal or metal compound. Preferably, the
particle is made of a nielal compound, such as iron, gadolinium, zinc, indium, gold, silver,
cobalt, copper, or magnesium. Most preferably, the particle is magnetizable or magnetic.
By 'magnetizable or magnetic is meant that the particle is capable of having a magnetic
moment impaired to it when it is placed in a magnetic field.
The detection reagent is labeled with a detectable marker. For example the
detection reagent to labeled with a radioactive isotopes (e.g., 125I, and 131 I) , enzymes (e.g.,
peroxidase, beta.-galactosidase, alkaline phosphatase) or fluorescent substances (e.g.,
fluorescein isothiocyanate (FITC). The labels arc quantified by the conventional methods
well-known in the art, thereby the formed immune complex is quantified. The mixture of
cells is any sample known to or suspected of containing an endothelial cell. For example
the mixture is a biological sample such as whole blood, sera, leucapherisate, bone marrow,
peripheral blood mononuclear cells or a tissue homogenatc.
An endothelial cell is any cell derived from any part of the vascular tree. For
example the endothelial cell is from large and small veins and arteries capillaries, the
umbilical vein of newborns, blood vessels in the brain or from vascularized solid tumors.
I"he endothelial cell is a mature cell. Alternatively, the endothelial cell endothelial cell
precursor. Preferably, the endothelial cell is Tie-2 positive.
Methods of Donor Specific Crossmatching
Cross-matching detects those antigenic differences to which the recipient is already
sensitized. A donor is crossmatched to a recipient by contacting a donor sample with a
detection reagent to isolate an endothelial cell. The recipient sample is contacted with the
isolated endothelial cell and reactivity of the recipient sample with the isolated endothelial
cell is determined. By "reactivity" it is meant that that a complex is formed via a specific
affinity interaction between the recipient sample and the cell. No reactivity of the recipient
sample with the isolated endothelial cell indicates compatibility between the donor and
recipient sample. In contrast, reactivity of the of the recipient sample with the isolated
endothelial cell indicates non-compatibility between the donor and recipient sample.
Compatibility is measured by no or low hyperacute rejection of the donor transplant by the
recipient.
The donor and the recipient are , e.g., any mammal, e.g., a human, a pig, a cow, or a
horse. The donor and the recipient are the same species. Alternatively, the donor and the
recipient are of different species.
The donor and recipient sample is, for example, whole blood, sera, leucapherisate,
bone marrow, peripheral blood mononuclear cells or a tissue homogenate. Optionally, the
samples are subjected to a pre-purification step prior to crossmatching.
Reactivity is determined my methods known in the art For example reactivity is
measured by an ELISA assay, flow cytometry (e.g. flow cytometric cross-match), or
complement-dependent lymphotoxicity cross-match
Vascular and Immune Disorders
The invention also provides methods of diagnosing, accessing the prognosis or
monitoring the course of treatment of vascular and immune disorders.
In these methods a first test sample is provided from a subject The sample is known
to or suspected of containing an endothelial cell. Optionally, the endothelial cell is isolated
from the first sample.
An immune disorder is diagnosed by contacting the first sample to a second sample
from the subject known to contain, or suspected of containing, an auto-antibody and
identifying an autoantibody-endothelial cell complex. The presence of an autoantibody-
endothelial cell complex indicates the subject is suffering from or predisposed to an
immune disorder. In contrast, the absence of an autoantibody- endothelial cell complex
indicates the subject is not suffering from or predisposed to an immune disorder.
A vascular disorder is diagnosed by contacting the first sample to a second sample.
The second sample is derived from the subject. Alternatively, the second sample comprises
antibodies to cell surface markers know to be associated with a particular vascular disorder
The presence of a second sample-endothelial cell complex indicates the subject is suffering
from or predisposed to a vascular disorder. In contrast, the absence of a second sample-
endothelial cell complex indicates the subject is not suffering from or predisposed to a
vascular disorder.
The methods allow the course of treatment of a vascular or immune to be monitored
or the prognosis of the subject to be determined In this method, a test sample is provided
from a subject undergoing treatment for the disorder. If desired, test samples arc obtained
from the subject at various time points before, during, or after treatment. The presence of
an endothelial cell complex is then determined to create a subject profile. The subject
profile is compared to a reference profile whose vascular disorder or immune disorder state
is known. The reference profile has not been exposed to the treatment. The reference
profile is derived from a sample type as similar to test sample. Optionally, the reference
profile is derived from a database of molecular information derived from samples for which
the assayed parameter or condition is known.
If the reference profile contains no autoantibody-endothelial cell complexes, a
similarity in the amount of complexes between the subject profile and the reference profile
indicates that the treatment is efficacious (e.g., that one or more symptoms of the immune
disorder arc alleviated or that the severity of the disorder is reduced), and thus, a favorable
prognosis for the subject. However, a shift in the amount of complexes between the subject
profile and the reference profile indicates that the treatment is not efficacious, and thus, an
unfavorable prognosis for the subject.
when the reference profile includes complexes taken from the subject at the time of
diagnosis but prior to beginning treatment, a similarity in the expression of complexes
pattern between the subject profile and the reference profile indicates the treatment is not
efficacious. In contrast, a shift in expression complexes in the subject profile and this
reference profile indicates the treatment is efficacious
By "efficacious" it is meant that the treatment leads to a decrease in any of the
symptoms of an autoimmune disorder in a subject previously noted. When treatment is
applied prophylacticaJly, "efficacious" means that the treatment retards or prevents an
immune related or vascular disorder.
An immune disorder includes disorders mediated by an immune mechanism such as
deposition of immune complexes, inflammation, direct attack by circulating antibodies
(e.g., autoimmune disorders). An autoimmune disorder or an autoimmune related disorder
includes those disorders caused by an immune response against the body's own tissues.
Autoimmune disorders result in destruction of one or more types of body tissues, abnormal
growth of an organ, or changes in organ function. The disorder may affect only one organ
or tissue type or may affect multiple organs and tissues. Organs and tissues commonly
affected by autoimmune disorders include blood components such as red blood ceils, blood
vessels, connective tissues, endocrine glands such as the thyroid or pancreas, muscles,
joints, and skin. Autoimmune disorders include for example, autoimmune hemolytic
anemia, autoimmune hepatitis, Berger's disease, chronic fatigue syndrome, Crohn's disease,
Hashimoto's thyroiditis, fibromyalgia, systemic lupus erthyeraatosus, Graves' disease,
ivliopatfiic thiombocytopenia purpura, multiple sclerosis, psoriasis, rheumatic fever,
rheumatoid arthritis,
Vascular disorders include disease associated with the vascular system. For
example vasculitis, atherosclerosis, bleeding disorders, defective wound healing.
Symptoms of an autoimmune disorder depend on the specific disease and the organ
or tissue that is affected. For example, systemic lupus erythematosus may cause kidney
failure, arthritis, and a skin rash on the face. Autoimmune hemolytic anemia causes anemia,
or low red blood cell counts. Generally symptoms of autoimmune disorders may include:
low-grade fever, malaise, which is a vague feeling of illness, fatigue, or tiring easily.
Autoimmune disorders arc diagnosed based on symptoms, a physical exam, and die results
of blood tests.
Treatments to reduce symptoms may include: nonsteroidal anti-inflammatory drugs
(NSAIDs), including aspirin or ibuprofen, to relieve fever, joint pain, and muscle aches
corticosteroids, or steroids, help reduce inflanunation. These medications are often used on
a short-term basis to get a person through a sudden episode or flare-up, medications to
suppress the immune system, such as methotrexate, azathioprine, and cyclophosphamide,
which help to reduce inflammation and organ damage. In some cases, other treatments
may be needed. For example, surgery may be needed for blockage of the bowels, which
may occur in Crohn's disease. Blood transfusions may be needed in severe cases of
autoimmune hemolytic anemia. Insulin is given to individuals with type 1 diabetes to
control blood glucose levels.
The subject is preferably a mammal. The mammal can be, e.g., a human, non-
human primate, mouse, rat, dog, cat, horse, or cow.
GENERAL METHODS
The data described herein was generated using the following reagents and methods.
EXAMPLE 1: Coupling of anti-Tie-Z Mabs to niagnctic beads
Mouse anti-human Tie-2 monoclonal abs. (Mabs) (BD Pharmingen, Oxford, UK)
were first coupled to pan-mouse Dynabeads with a DNA-linker (cat. no. 115.19)(DYNAL,
Oslo, Norway). For this purpose, 10 µg of Tie-2 Mabs were added to 500 µls of pan-mouse
Dynabeads The bead-Mab suspension was rotated on a rock n' roller for 24 hrs at 4°C.
Excess Mab was removed and the Tie-2 Mab coated beads were blocked with 2 ml
phosphate buffered saline (PBS) containing 0.1 % bovine scrum albumin (BSA) on a rock n'
roller at 4°C for 10 min. The blocking step was repeated six times after which, the beads
were resuspended in original volume (500 µls) of PBS/0.1% BSA.
EXAMPLE 2: isolation of Tie-2+ cells from peripheral blood mononuclear cells
(PBMC)
Due to the large numbers of cells required to establish the specificity of the Tie-2+
cells, in the initial experiments PRMCs were isolated from leucapherisate of healthy blood
isolated from PBMCs using anti-Tie-2 Mabs coated magnetic beads. PBMCs were first
distributed into several tubes each containing 40 x 106 cells. Fifteen pis of prc-coatcd Tie-
2 magnetic beads were added to each tube and incubated in a volume of 500 pis RPMT
medium (GIBCO, Paisley, UK) supplemented with 2 mM L-glutamine and 10% heat
inactivated fetal calf serum. Cells+bcads were incubated on rock 'n roller at 4°C for 30
min. Tie-2+ cells were separated after extensive washing (5-6 times) with PBS using a
magnet. Tie-2+ cells from all tubes were pooled together and rosettes were counted under
a light microscope.
EXAMPLE 3: Isolation of Tie-2+ cells directly from peripheral blood
40 ml-heparinized blood was obtained from normal healthy donors. The blood was
washed once as follows: 10 ml blood was diluted with 40 ml PBS/0.1%BSA. The blood
was centrifuged at 800 g for 10 min. The supernatant was discarded and the blood cells
were resuspendcd in 10 ml of PBS containing 0.6% sodium citrate. Fifty µls of Tie-2 Mabs
coated magnetic beads were added lo each tube and incubated for 30 min at 4 C on a rock
'n roller. Tie-2+ cells were collected by a magnet and washed once with PBS-Na-citrate.
After 4-5 washes with PBS, Tic-2+ cells were collected and the concentration of the cells
adjusted to approximately 3-4 x 106 cells /ml. These cells surrounded by beads were used
either in a microcytotoxicity assay or the flow cytometcr.
EXAMPLE 4: mniupocvtochcmisiry
The Mabs used for immunocytochemistry and FACs analysis arc given in Table 1.
Tic-2+ cells obtained from leucapbcrisatc of normal volunteers gave sufficient numbers of
cells to perform various immunocytochernical analysis. Tie-2+ cells were grown on
fibroncctin-coated tissue culture plates. Cells were allowed to attach (24 hrs) prior to
staining for various EC-specific markers. Cells were left either untreated or stimulated
with TNF-a and IFN-y for 14 hrs. For immunocytochemistry, cells were fixed using 30%
acetone in methanol for I min. After two wastes with PBS, cells were blocked using 1%
bovine serum albumin (BSA) in PBS for I hr at room temperature. The cells were washed
twice with PBS and incubated with the above mentioned primary antibodies diluted 1:100
(in PBS) at 4°C for 2 hrs. The secondary antibody was a poat anti-mouse IpG contueated
with fluorescein isotbiocyanate (FITC) diluted 1:500 (in PBS). After incubation for 1 hr at
4°C, the cells were washed twice and analysed under a fluorescence microscope.
EXAMPLE 5: Flow cytometric assay for detection of apti-endothclial cell
antibodies
A total of 50 sera from kidney-transplanted patients were studied. Over the past
years (1988-2001) the prc-transplant sera from kidney patients has been meticulously
characterized, using various methods, for the presence of endothelial cell specific,
endothelial-monocyte reactive, and HI-A antibodies found to be associated with rejections.
During these years 15 pre-trarrplantation sera, characterized as having either anti-EM or EC
specific antibodies from non-alloimmunized patients with rejections, have been collected.
The general methods described herein are based on these 15 sera. Previous
characterization showed that ten sera gave positive reactions with human umbilical vein
endothelial ceJJ (HUVECs) lines and monocytes (Table 2, pt. nos. 1-10), and five sera with
only ECs (Table 2, pt. nos. 11-15). Since none of the fifteen patients with EM or EC-
specific antibodies had been alloimmunized, no detectable HLA alloantibodics were found
in these sera. In addition, ten wen-characterized sera from alloimmunized patients known
to contain only HLA class I or only class II alloanribodies were selected (Table 2). As
controls, 25 sera from patients with no graft rejections were also tested.
Using these sera, it was determined whether the isolated Tic-2+ cells could be
suitable targets for detection of clinically relevant antibodies in kidney organ
transplantation. A pool of sera from patients who had formed alloantibodics as a result of
multiple blood transfusions or organ transplantations was used as a positive control. Sera
from healthy non-transfused blood group AB males served as negative controls. The
following antibodies, FITC-conjugated F(ab')? fragments of goat anti-human IgG (Fc
specific), or IgM (lmmunotech, USA) were used. For the flow cytometric ECXM 100,000
Tie-2+ cells coupled to beads were used and the procedure carried out as described earlier.
The cells were analyzed on a Becton Dickinson flow cytometer (FACSorter). A shift in the
mean fluorescence of 10 channels in the test sample as compared to negative control was
considered as positive, determined as described before. This value is arbitrary and should
be determined by each transplant laboratory. The Tie-2+ cells were also used in the
addition, Tie-2+ cells immediately after isolation were tested in the flow cytometer for all
the endothelial cell surface markers (Table 1)
EXAMPLE 6: Immunocvtochemistrv and FACS analysis of endothelial cell
markers on isolated Tie-2+ cells
Tic-2+ ECs were isolated from human peripheral blood by magnetic bead selection.
FACS analysis showed that approximately 3± 4% of PBMCs were Tic-2+ (Fig. 1). After
24 hours in culture, the majority of Tie-2+ cells attached to fibronectin-coated 24 well
plates and became adherent. Initially, these cells appeared cither as single cells or as
clusters of round cells, which after few days of culture converted into adherent cells with
extended cytoplasm. After 4 days in culture the cells gradually developed spindle shape
(Fig. 2a-c).
Figure 3 and Tabic 1 summarize the results of the immunocytochernical analyses
using antibodies to known antigens of endothelial cells. Already at 0 hours, FACS analysis
indicated that Tie-2+ cells expressed the acLDL-receptor, vWF, VEGFR-1, and strong
expression of HLA class I and HLA class II antigens. Importantly, the cells expressed the
clinically important MICA antigen, though weakly. Upon cytokine activation, the cells
expressed both the endothelial cell specific adhesion molecules CD62 E (E-selectin) and
CD 106 (VCAM), as well as increased expression of HLA class II. Thus, both the
imuiuuucytochemical and FACS analysis inducted that Tie-2+ cells expressed majority of
the endothelial cell markers.
EXAMPLE 7: Detection of aoti-cndothelial cell reactive antibodies
On an average, approximately, 3± 4 x 104 Tic-2+ cells /106 PBMCs can be obtained
from single donors. Since a large number of sera were analyzed herein Tie-2+ cells from
lcucaphcrisate of healthy blood donors were isolated. The results are shown in Table 2. All
the 10 sera known to have endothelial-monocyte antibodies and 5 sera with endothelial
cell-specific antibodies showed varying patterns of reactivity with the panel of Tie-2+ cells
from six different donors. However, the Tie-2- fractions, which included the lymphocytes
from the same donors, did not react with any of the sera. The reactivity partem with the
Tie-2+ cell panel of both the endothelial-monocyte and endothelial cell-specific antibodies
as seen in Table 2, indicates the existence of polymorphism in these antigenic systems or
alternatively presence of antibodies with varied specificities.
Sera known to contain only HLA class 1 broadly reactive alloantibodies also gave
positive reactions in every instance with Tie-2 + cells from single donors. Reactivity of
HLA class II alloantibodies (limited specificities) was also observed with unstimulated Tie-
2+ cells. 3/25 (12%) sera from control patients with no rejections and with stable graft
functions gave positive reactions with four of the Tic-2 panel donors (Table 2). Results
obtained using the immuno-magnetic microcytotoxicity assay were in agreement with the
flow cytometric analysis (Table 3).
EXAMPLE 8: Donor-specific endothelial cell cross-match
Donor-specific endothelial cell crossmatches were retrospectively performed in two
cases where frozen donor peripheral blood mononuclear cells were available. The first
kidney grafts of both patients were lost in hyperacute rejections in the absence of
demonstrable donor-specific HLA antibodies. Cross-match sera from one of these patients
had been previously well characterized as having anti-endothelial cell-specific antibodies
using HUVECs.
In this instance, Tic-2+ cells from the peripheral blood mononuclear cells of this
patient's (point, no.l 1, Table 2) first donor (father) were isolated and kept frozen in liquid
N2. A cross-match with the scrum of the patient taken immediately prior to the first
transplant was retrospectively performed. The results are shown in Figure 4b-d. The
results from a second case (point, no. 12, Table 2) are very similar to point no. 11, and are
shown in Figure 4e-g. As seen, in Table 2, the sera from both these points gave similar
pattern of reactivity with Tie-2+ cell panel. No reactivity with the Tie-2- fractions were
observed.
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OTHER EMBODIMENTS
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.
WE CLAIM:
1. An in vitro method of isolating a CD-34 negative precursor endothelial cell from a
biological sample comprising:
(a) collecting a sample containing, or suspected of containing, a precursor
endothelial cell;
(b) contacting the sample with a detection reagent to form a detection reagent-
precursor endothelial cell complex; and
separating the detection reagent-precursor endothelial cell complex from the
biological sample thereby isolating the precursor endothelial cell.
2. The method as claimed in claim 1, wherein the detection reagent is a ligand for a
precursor endothelial cell surface receptor.
3. The method as claimed in claim 2, wherein said ligand is angiopoietin or VEGF.
4. The method as claimed in claim I, wherein the detection reagent is an antibody, or
fragment thereof.
5. The method as claimed in claim 4, wherein the antibody is specific against a
precursor endothelial cell surface receptor.
6. The method as claimed in claim 5, wherein the precursor endothelial cell receptor
is Tie-2.
7. The method as claimed in claim 1, wherein the isolated precursor endothelial cell
is Tie-2 positive.
8. The method as claimed in claim 1, wherein the sample is selected from the group
consisting of whole blood, sera, tissue, peripheral blood mononuclear cells, and
leucapherisate.
9. The method as claimed in claim I, wherein the separation is by flow cytometry or
a magnetic field.
10. The method as claimed in claim 4, wherein the antibody, or fragment thereof, is
attached to a solid support.
11. The method as claimed in claim 10, wherein the solid support is a non-magnetic,
magnetic or a paramagnetic bead.
12. The method as claimed in claim 4, wherein the antibody is a Fab fragment.
13. An in vitro method of cross-matching a donor and a recipient comprising:
a) collecting a donor sample containing or suspected of containing a precursor
endothelial cell;
b) isolating said precursor endothelial cell by contacting the donor sample with a
detection reagent according to the method as claimed in claim 1; and
c) screening a recipient sample for a reactivity with the isolated precursor
endothelial cell;
wherein no reactivity of the recipient sample with the isolated precursor
endothelial cell indicates compatibility between the donor and recipient.
14. The method as claimed in claim 13, wherein the detection reagent is a ligand for a
precursor endothelial cell surface receptor.
15. The method as claimed in claim 14, wherein said ligand is angiopoietin or VEGF.
16. The method as claimed in claim 13, wherein the detection reagent is an antibody,
or fragment thereof.
17. The method as claimed in claim 16, wherein the antibody is specific against a
precursor endothelial cell surface receptor.
18. The method as claimed in claim 17, wherein the precursor endothelial cell
receptor is Tie-2 or VEGFR-1.
19. The method as claimed in claim 13, wherein the isolated precursor endothelial cell
is Tie-2 positive or VEGR-1 positive.
20. The method as claimed in claim 13, wherein the donor sample or recipient sample
is selected from the group consisting of whole blood, sera, tissue, peripheral blood
mononuclear cells, and leucapherisate.
21. The method as claimed in claim 16, wherein the antibody, or fragment thereof, is
attached to a solid support.
22. The method as claimed in claim 21, wherein the solid support is a non-magnetic,
magnetic or a paramagnetic bead.
23. The method as claimed in claim 16, wherein the antibody is a Fab fragment.

The invention relates to a method for isolation of precursor endothelial cells and
the use of these cells for donor specific crossmatching. Today lymphocyte
crossmatching is performed before transplantation and is used for detecting
antibodies against HLA. Lymphocyte crossmatches does not permit detection of
clinically relevant endothelial/monocyte reactive and endothelial specific
antibodies. The invention describes a method for isolation of cells that can be
used for crossmatching and detection of non-HLA antigens.

Documents:

1749-kolnp-2004-abstract.pdf

1749-kolnp-2004-assignment.pdf

1749-kolnp-2004-claims.pdf

1749-KOLNP-2004-CORRESPONDENCE-1.1.pdf

1749-kolnp-2004-correspondence.pdf

1749-kolnp-2004-description (complete).pdf

1749-kolnp-2004-drawings.pdf

1749-kolnp-2004-examination report.pdf

1749-kolnp-2004-form 1.pdf

1749-kolnp-2004-form 13.pdf

1749-kolnp-2004-form 18.pdf

1749-KOLNP-2004-FORM 27.pdf

1749-kolnp-2004-form 3.pdf

1749-kolnp-2004-form 5.pdf

1749-KOLNP-2004-FORM-27.pdf

1749-kolnp-2004-gpa.pdf

1749-kolnp-2004-granted-abstract.pdf

1749-kolnp-2004-granted-assignment.pdf

1749-kolnp-2004-granted-claims.pdf

1749-kolnp-2004-granted-correspondence.pdf

1749-kolnp-2004-granted-description (complete).pdf

1749-kolnp-2004-granted-drawings.pdf

1749-kolnp-2004-granted-examination report.pdf

1749-kolnp-2004-granted-form 1.pdf

1749-kolnp-2004-granted-form 13.pdf

1749-kolnp-2004-granted-form 18.pdf

1749-kolnp-2004-granted-form 3.pdf

1749-kolnp-2004-granted-form 5.pdf

1749-kolnp-2004-granted-gpa.pdf

1749-kolnp-2004-granted-reply to examination report.pdf

1749-kolnp-2004-granted-specification.pdf

1749-kolnp-2004-reply to examination report.pdf

1749-kolnp-2004-specification.pdf


Patent Number 236055
Indian Patent Application Number 1749/KOLNP/2004
PG Journal Number 38/2009
Publication Date 18-Sep-2009
Grant Date 17-Sep-2009
Date of Filing 18-Nov-2004
Name of Patentee ABSORBER AB
Applicant Address BOX 7710, DROTTNINGGATAN 33, S-10395 STOCKHOLM
Inventors:
# Inventor's Name Inventor's Address
1 SUMITRAN-HOLGERSSON SUCHITRA VENUSVAGEN 6A, S-14133 HUDDINGS SWEDEN
PCT International Classification Number G01N 33/50
PCT International Application Number PCT/IB2003/02502
PCT International Filing date 2003-05-16
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/381,033 2002-05-16 U.S.A.