Note: Descriptions are shown in the official language in which they were submitted.
1
SELECTIVE ANTI-HLA ANTIBODY REMOVAL DEVICE AND
METHODS OF PRODUCTION AND USE THEREOF
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0001] Not Applicable.
BACKGROUND OF THE INVENTIVE CONCEPT(S)
1. Field of the Invention
[0002] The presently disclosed and claimed inventive concept(s) relates
generally to a
methodology of removing anti-HLA antibodies from a sample, as well as a device
utilized
therefor.
2. Description of the Background Art
[0003] Human cells express on their surface an incredibly large number of
membrane-
bound proteins, all of which display individual properties and physiological
functions. From
this large array of surface cell proteins, a number of clinical procedures
require
characterization of the human major histocompatibility complex (MHC) class I
and II
membrane-bound molecules. The human MHC class I and class II molecules are
known as
human leukocyte antigens, or HLA. The HLA class I and class ll molecules are
responsible for
presenting peptide antigens to receptors located on the surface of T-
Iymphocytes, Natural
Killer Cells (NK), and possibly other immune effector and regulatory cells.
Display of peptide
antigens on the MHC I and MHC II molecules are the basis for the recognition
of "self vs.
non-self" and the onset of important immune responses such as transplant
rejection, graft-
versus-host-disease, autoimmune disease, and healthy anti-viral and anti-
bacterial immune
responses.
[0004] HLA class I and class II molecules differ from person to person.
Each person
expresses a different complement of class I and class II on the surface of
their cells. For
transplant purposes it is important to determine which of the multiple HLA
expressed on a
cell are recognized by the antibodies of another individual. The presence of
anti-HLA
antibodies in a transplant recipient can lead to hyperacute organ rejection.
It is often
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difficult to determine which of many HLA are recognized by antibodies because
sera can
have antibodies to non-HLA proteins and multiple HLA molecules, and sera may
crossreact
among different HLA molecules. With many human proteins, many HLA proteins,
antibodies
to multiple human proteins, and antibodies crossreactive to various HLA
proteins, it can be
difficult when screening patients for organ transplantation to ascertain which
of the many
HLA in the population, and expressed on an organ to be transplanted, are
recognized by
antibodies. Antibodies to HLA proteins may also lead to problems during the
transfusion of
blood products, whereby antibodies in the blood of the blood donor may react
with the HLA
class I and class ll antigens of the recipient of the blood product.
Antibodies in the blood
product that recognize the recipient's HLA may lead to transfusion related
acute lung injury
(TRALI).
[0005] Class I MHC molecules, designated HLA class I in humans, bind and
display
peptide antigen ligands upon the cell surface. The peptide antigen ligands
presented by the
class I MHC molecule are derived from either normal endogenous proteins
("self") or foreign
proteins ("nonser) introduced into the cell. Nonself proteins may be products
of malignant
transformation or intracellular pathogens such as viruses. In this manner,
class I MHC
molecules convey information regarding the internal fitness of a cell to
immune effector cells
including but not limited to, CD8+ cytotoxic T lymphocytes (CTLs), which are
activated upon
interaction with "nonser peptides, thereby lysing or killing the cell
presenting such
"nonser peptides.
[0006] Class ll MHC molecules, designated HLA class ll in humans, also bind
and display
peptide antigen ligands upon the cell surface. Unlike class I MHC molecules
which are
expressed on virtually all nucleated cells, class II MHC molecules are
normally confined to
specialized cells, such as B lymphocytes, macrophages, dendritic cells, and
other antigen
presenting cells which take up foreign antigens from the extracellular fluid
via an endocytic
pathway. The peptide antigens bound and presented by HLA class II are derived
from
extracellular foreign antigens, such as products of bacteria that multiply
outside of cells,
wherein such products include protein toxins secreted by the bacteria or any
other bacterial
protein to which the human immune system might respond in a protective manner.
In this
manner, class ll molecules convey information regarding the existence of
pathogens in
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extracellular spaces that are accessible to the cell displaying the class ll
molecule. HLA class
II expressing cells then present peptide antigens derived from the
extracellular
antigen/bacteria to immune effector cells, including but not limited to, CD4+
helper T cells,
thereby helping to eliminate such pathogens. The elimination of such pathogens
is
accomplished by both helping B cells make antibodies against microbes, as well
as toxins
produced by such microbes, and by activating macrophages to destroy ingested
microbes.
[0007] HLA class I and class II molecules exhibit extensive polymorphism
generated by
systematic recombinatorial and point mutation events; as such, hundreds of
different HLA
types exist throughout the world's population, resulting in substantial
immunologic diversity.
Such extensive HLA diversity throughout the population results in tissue or
organ transplant
rejection between individuals as well as differing susceptibilities and/or
resistances to
infectious diseases. HLA molecules also contribute significantly to
autoimmunity and cancer.
Because HLA molecules mediate most, if not all, adaptive immune responses, and
because of
their tremendous diversity, large quantities of individual HLA proteins are
required in order
to effectively study transplantation, autoimmunity disorders, and for vaccine
development.
[0008] Antibodies that recognize class I and class II human leukocyte
antigens (HLA)
currently represent a contraindication at multiple stages of the organ
transplant process.
Prior to transplantation, patients who have been sensitized to produce HLA-
specific
antibodies typically wait longer to receive a transplant. Post-
transplantation, antibodies that
recognize the HLA of the donor organ contribute to hyperacute, acute, and
chronic rejection
of a transplanted organ. However, it is likely that not all antibodies that
recognize HLA
promote organ failure. A more thorough understanding of anti-HLA antibodies
would
therefore indicate those immunoglobulins that are truly a contraindication for
transplantation.
[0009] It has been difficult to evaluate the phenotypic and functional
traits of antibodies
to any given HLA molecule because anti-HLA humoral responses tend to be
polyclonal and
these antibodies cannot be readily isolated for individual characterization.
Antibody
concentration, isotype, epitope specificity, cross-reactivity, and the ability
to fix complement
have all been implicated as factors that contribute to the pathogenicity of
anti-HLA
antibodies (6). More advanced tools such as bead-based semi-quantitative
assays have
4
recently provided a more definitive indication for these antibodies' HLA
specificity. Nonetheless, the
complex nature of human sera and the inability to study antibodies reactive
against individual HLA
antigens continue to cloud the contribution of antibody isotype,
concentration, and specificity to
transplant rejection.
[0010] The current methods of antibody removal only remove antibodies of
broad specificity.
The PROSORBA (Cypress Bioscience, San Diego, CA) and follow-on IMMUNOSORBA
(Fresenius Medical
Care, Waltham, MA) products (and others like them) use Protein A to bind a
broad range of antibodies.
Plasma is filtered through the IMMUNOSORBA device to rid the majority of IgG
antibodies from the sera.
However, IgG3 and IgM and other subtypes are NOT removed. These current
devices provide no method
of selecting between "wanted" and "not-wanted" antibodies.
[0011] Therefore, there exists a need in the art for improved devices
that selectively remove
anti-MHC/HLA antibodies from a sample, as well as methods of production and
use thereof, that overcome
the disadvantages and defects of the prior art.
SUMMARY OF THE INVENTION
Thus, in one aspect the present invention provides an extracorporeal anti-
major
histocompatibility complex (MHC) antibody removal device, for removing anti-
MHC antibodies from a
plasma sample obtained from a patient, the device comprising: one or more anti-
MHC antibody removal
columns, wherein each removal column comprises a solid support matrix and a
glycosylated, serologically
active, soluble MHC trimolecular complex covalently coupled to the solid
support matrix, wherein the
MHC trimolecular complex comprises a soluble MI-IC alpha chain protein
associated with a soluble MHC
beta chain protein and an endogenously loaded peptide ligand in an antigen
binding groove of the MHC
trimolecular complex, wherein the MHC trimolecular complex lacks cytoplasmic
and transmembrane
domains, and wherein the soluble MHC alpha chain protein and the soluble MHC
beta chain protein are
associated by a super secondary structural motif, wherein the MHC trimolecular
complex comprises an
alpha chain allele or beta chain allele that is known to be an antigenic
target for anti-MHC antibodies,
whereby the anti-MHC antibodies in the plasma sample that are specific for the
MHC trimolecular complex
present in the removal column will bind thereto, resulting in removal of said
antibodies from the plasma
sample, and wherein the MHC trimolecular complex has been recombinantly
produced in a mammalian
cell line.
In another aspect, the present invention provides a kit for removing anti-
major
histocompatibility (MHC) antibodies from a biological sample, the kit
comprising: a first anti-MI-IC antibody
removal column comprising a first solid support matrix and a glycosylated,
serologically active, soluble
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MHC trimolecular complex covalently coupled to the first solid support matrix,
wherein the trimolecular
complex comprises a first MHC alpha chain protein, a first MHC beta chain
protein, and an endogenously
loaded peptide ligand in an antigen binding groove of the first MHC
trimolecular complex; and a second
anti-MHC antibody removal column comprising a second solid support matrix and
a glycosylated,
serologically active, soluble MHC trimolecular complex covalently coupled to
the second solid support
matrix, wherein the trimolecular complex comprises a second WIC alpha chain
protein, a second WIC
beta chain protein, and a second endogenously loaded peptide ligand in an
antigen binding groove of the
second MHC trimolecular complex, wherein the first MHC alpha chain protein is
a different allele than the
second MHC alpha chain protein; wherein the first and second MHC trimolecular
complexes lack
cytoplasmic and transmembrane domains, and wherein the soluble MHC alpha and
beta chain proteins of
the first and second MHC trimolecular complexes are each associated by a super
secondary structural
motif; and wherein the first and second MHC trimolecular complexes have been
recombinantly produced
in a mammalian cell line.
In another aspect, the present invention provides a method of removing anti-
major
histocompatibility (WIC) antibodies from a plasma sample, the method
comprising the steps of: (a)
contacting a plasma sample from a patient with an extracorporeal anti-MHC
antibody removal device as
defined in any one of claims 1 to 18, whereby antibodies specific for the MHC
trimolecular complex are
removed from the plasma sample; and (b) recovering the plasma sample, whereby
the antibodies specific
for the MHC trimolecular complex are substantially reduced in the recovered
plasma sample.
In another aspect, the present invention provides a commercial package
comprising an
extracorporeal anti-major histocompatibility complex (MHC) antibody removal
device as described herein,
together with instructions for the use thereof to remove anti-MI-IC antibodies
from a plasma sample
obtained from a patient.
In a particular aspect, the commercial package of the present invention
comprises a plurality
of removal columns with different alpha chain alleles or beta chain alleles
known to be antigenic targets
for anti-MHC antibodies and includes instructions for selecting which removal
columns are to be used
on the basis of the patient's plasma sample having been screened for the
presence of anti-MHC antibodies
that bind to the alpha chain alleles or beta chain alleles present in the
removal columns.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] This
patent or application file contains at least one drawing executed in color.
Copies of this
patent or patent application publication with color drawing(s) will be
provided by the Office upon request
and payment of the necessary fee.
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[0013] Figure 1 is a schematic representation of a soluble HLA class II
trimolecular complex
produced in accordance with the presently disclosed and claimed inventive
concept(s).
[0014] Figure 2 is a schematic diagram of a method of producing the
soluble 1-ILA (sHLA) class II
trimolecular complex (of Figure 1) in accordance with the presently disclosed
and claimed inventive
concept(s).
[0015] Figure 3 is a schematic diagram of sHLA class II trimolecular
complex production in a
hollow fiber bioreactor unit.
[0016] Figure 4 graphically depicts the production of sHLA class II
DRBI*0103 produced in
transfected cells, demonstrating the ability to scale up production from a
T175 flask to a hollow fiber
bioreactor unit (CELL PHARMn.
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[0017] Figure 5 graphically demonstrates the ability of commercially
available
monoclonal antibodies (mAb) and patient sera to specifically detect the sHLA
DRB1*0103
produced in Figure 4.
[0018] Figure 6 graphically depicts the ability to produce multiple
different sHLA class II
complexes from transfected cells in accordance with the presently disclosed
and claimed
inventive methods.
[0019] Figure 7 graphically depicts production in a bioreactor of milligram
quantities of
sHLA class ll overtime.
[0020] Figure 8 demonstrates quantification of sHLA class ll
DRB*0103/DRA*0101
(produced in Figure 7) using electrospray mass spectroscopy.
[0021] Figure 9 illustrates the molecular weight results and analysis of
the proteins from
Figure 8 and using electrospray ionization TOF mass spectrometry.
[0022] Figure 10 graphically depicts coupling of soluble DRB1*1101 ZP HLA
Class ll
molecule to a solid support and use thereof to facilitate removal of HLA Class
ll specific
antibodies in an ELISA format. Panel A: a diagram of the consecutive
absorption matrix ELISA
performed for specific antibody removal. Panel B: absorbance and retentate
values from 3
different HLA Class II specific mAb antibodies: L243, OL (One Lambda), and
2H11 were
subjected to the consecutive absorbance matrix.
[0023] Figure 11 graphically depicts that DRB1*1101-specific human sera was
recognized
by soluble DRB1*1101 in an ELISA format.
[0024] Figure 12 graphically depicts that soluble DRB1*1101 can be coupled
to
SEPHAROSE and used to absorb HLA Class ll specific antibody, 9.3F10. Panel A:
soluble
DRB1*1101 was coupled to SEPHAROSE Fast Flow and packed into a gravity
column. mAb
9.3F10, which has DR reactivity, was passed over the column and flow thru was
collected as
fractions. Then the mAb was eluted using DEA (diethanolamine) buffer, pH 11.3,
was added
to the column, and fractions were collected. Panel B: two separate ELISAs for
total mouse
IgG and human HLA were also performed on the Flow Thru and Eluate to detect
specific
antibodies versus HLA proteins that might have been eluted off the column.
[0025] Figure 13 graphically depicts that antibodies contained in human
sera specific for
DRB1*1101 can be removed by a DRB1*1101 specific column. Donor #1 sera was
passed
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over the DRB1*1101 SEPHAROSE column, and two 2 ml fractions of flow thru were
collected. To elute, DEA buffer pH 11.3, was added to the column, and two 2 ml
fractions
were collected. Panel A: a direct DRB1*1101 ELISA was performed to detect the
amount of
DRB1*1101 specific antibodies that were left in the flow thru and eluate.
Panel B: a total
human IgG sandwich ELISA was also performed to evaluate passage of total human
IgG.
[0026] Figure 14 graphically depicts that soluble DRB1*1101 coupled
SEPHAROSE is
specific for DRB1*1101 and not other DR alleles. Donor #2 sera was passed over
the same
DRB1*1101 column in the same manner as Figure 13, and two fractions of the
flow thru and
one fraction of the eluate were evaluated for multi-allele DR reactivity.
[0027] Figure 15 depicts the nucleic acid (SEQ ID NO:1) and amino acid (SEQ
ID NO:2)
sequences of a DRA*0101 alpha chain-leucine zipper construct. The highlighted
sequence
encodes a linker that connects DRA1*0101 allele's sequence to the leucine
zipper motif's
sequence. The underlined sequence encodes the leucine zipper motif.
[0028] Figure 16 depicts the nucleic acid (SEQ ID NO:3) and amino acid (SEQ
ID NO:4)
sequences of a DRB1*0401 beta chain-leucine zipper construct. The highlighted
sequence
encodes a linker that connects DRB1*0401 allele's sequence to the leucine
zipper motif's
sequence. The underlined sequence encodes the leucine zipper motif.
[0029] Figure 17 depicts the nucleic acid (SEQ ID NO:5) and amino acid (SEQ
ID NO:6)
sequences of a DRB1*0103 beta chain-leucine zipper construct. The highlighted
sequence
encodes a linker that connects DRB1*0103 allele's sequence to the leucine
zipper motif's
sequence. The underlined sequence encodes the leucine zipper motif.
[0030] Figure 18 illustrates the construction of sHLA-DR11. A) The
transmembrane
domains of the alpha (DRA1*01:01) and beta (DRB1*11:01) chains were deleted
and
replaced by a 7 amino acid linker followed by leucine zipper ACIDp1(LZA) and
leucine zipper
BASEp1 (LZB), respectively. B) Amino acid sequences for the mature DRA1*01:01
and
DRB1*11:01 constructs. Red letters represent the sequence covered from the MS
analysis.
Underlined letters show the amino acid sequence for the leucine zipper
domains.
[0031] Figure 19 illustrates removal and recovery of L243 with a sHLA-DR11
column. A)
A280 values for the fractions obtained from the flow through and elution of
the sHLA-DR11
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column. B) Class ll reactivity of the eluted L243 antibody. The raw MFI for
each individual
HLA complex tested is shown, and the results are grouped together by loci.
[0032] Figure 20 illustrates the specific removal of anti-HLA-DR11
antibodies using the
sHLA-DR11 column. A, B) Representative class II HLA reactivities in the
starting sera obtained
from two sensitized donors, (A:Donor1, B:Donor2). HLA types are color coded by
locus
(DR11:black, other DR:shades of blue, DO: shades of red, DP:green). Data are
shown as
background corrected MFI (BCMFI). C, D) Anti-HLA reactivity of fractions in
the column flow-
through and eluate from Donor 1 (C) and Donor 2 (D) were analyzed as in A and
B. Each
trace shows the reactivity profile for a different class II HLA type as shown
in the figure
legend. HLA types are color coded as in A and B.
[0033] Figure 21 illustrates removal of complement and non-complement
fixing
antibodies. A) Complement dependant cytolysis of HLA-DR11 positive cells
(C433, C418,
C428, C423) using anti-HLA-DR11 antibodies. Mean percent cell death is
calculated as
described in the materials and methods. Starting serum is shown in blue, flow
through in red,
and eluate in green. Error bars represent the standard deviation from three
independent
experiments. Significant differences in mean values are shown and were
determined by a
one way ANOVA (analysis of variance) with a Turkey post-hoc test (p<0.05). B)
Representative fluorescent microscope images used for the quantitative
analysis in A. Dead
cells are red (ethidium bromide) and viable cells are green (acridine orange).
[0034] Figure 22 illustrates isotype profiles of purified anti-HLA-DR11
antibodies.
Antibody isotypes in the starting sera, flow through, and eluate were
quantified using a
LUMINEX -based ELISA and expressed as a percentage of total antibody.
[0035] Figure 23 illustrates removal of anti-HLA-DR11 antibodies from
sensitized sera.
The starting sera from two sensitized donors were tested for class II
reactivity using a single
antigen bead assay. Once the sera were passed over the sHLA-DR11 column, the
flow
through, and eluate from the column were tested using the same class ll single
antigen bead
assay.
[0036] Figure 24 illustrates the coupling efficiencies of two different
SEPHAROSE
matrices with class I soluble HLA. 1 mg of sHLA-B was added to 1 ml of either
CNBr-activated
or NHS-activated SEPHAROSE 4 Fast Flow matrix. The coupling was allowed to
react for 1
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hour and was terminated. Coupling efficiency is calculated using the following
equation:
(coupling efficiency = mg starting sHLA / mg sHLA in solution after coupling).
[0037] Figure 25 illustrates the binding capacities of two different
SEPHAROSE matrices
for class I soluble HLA. Saturating quantities of pan class I HLA monoclonal
antibody W6/32
was run over 1 ml of coupled matrix (1 mg @ 1 mg/ml). The matrix was either
CNBr-
activated or NHS-activated SEPHAROSE 4 Fast Flow matrix. The sHLA used in
this
experiment was sHLA-B*07:02. Binding capacity was determined by measuring the
quantity
of antibody recovered in the elution. To adjust for variations in coupling
efficiencies, the data
is shown as ug of W6/32 in the elution per mg of sHLA coupled on the matrix.
[0038] Figure 26 illustrates the regeneration capabilities of two different
SEPHAROSE
matrices loaded with class I soluble HLA. Saturating quantities of pan class I
HLA monoclonal
antibody W6/32 was run over 1 ml of coupled matrix (1 mg @ 1 mg/ml). The
matrix was
either CNBr-activated or NHS-activated SEPHAROSE 4 Fast Flow matrix. The
columns were
then serially loaded and eluted 5 times as indicated on the x axis. Percent of
the original
(cycle 1) antibody binding capacity is shown for each cycle.
[0039] Figure 27 illustrates the coupling efficiencies of two different
SEPHAROSE
matrices with class II soluble HLA. 1 mg of sHLA-DR11 was added to 1 ml of
either CNBr
activated or NHS activated SEPHAROSE 4 Fast Flow matrix. The coupling was
allowed to
react for 1 hour and was terminated. Coupling efficiency is calculated using
the following
equation: (coupling efficiency = mg starting sHLA / mg sHLA in solution after
coupling).
[0040] Figure 28 illustrates the binding capacities of two different
SEPHAROSE matrices
for class ll soluble HLA. Saturating quantities of pan HLA-DR monoclonal
antibody L243 was
run over 1 ml of coupled matrix (1 mg @ 1 mg/ml). The matrix was either CNBr-
activated or
NHS-activated SEPHAROSE 4 Fast Flow matrix. The sHLA used in this experiment
was sHLA-
DR11. Binding capacity was determined by measuring the quantity of antibody
recovered in
the elution. To adjust for variations in coupling efficiencies, the data is
shown as ug of L243 in
the elution per mg of sHLA coupled on the matrix.
[0041] Figure 29 illustrates the regeneration capabilities of two different
SEPHAROSE
matrices loaded with class ll soluble HLA. Saturating quantities of pan HLA-DR
monoclonal
antibody L243 was run over 1 ml of coupled matrix (1 mg @ 1 mg/m1). The matrix
was either
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CNBr-activated or NHS-activated SEPHAROSE 4 Fast Flow matrix. The columns
were then
serially loaded and eluted 5 times as indicated on the x axis. Percent of the
original (cycle 1)
antibody binding capacity is shown for each cycle.
[0042] Figure 30 illustrates monoclonal anti-HLA antibody depletion from
PBS using a
class I HLA SHARC (soluble HLA antibody removal column). Saturating quantities
of pan class
I HLA monoclonal antibody W6/32 was run over 65 ml of coupled matrix (24.4 mg
at 97
[temp. The column was then washed with PBS pH 7.4 and eluted with 0.1 M
Glycine pH 11.
During the load and wash phase, 11.7 mg passed through the column. During the
elution
phase, 8 mg of antibody was recovered.
[0043] Figure 31 illustrates polyclonal anti-HLA-A2 antibody depletion from
patient
plasma with class I HLA-A2 SHARC. 2.5 L of Patient plasma containing anti-HLA
antibodies
was run over the 65 ml sHLA-A2 SHARC. Plasma pre- and post-SHARC were analyzed
using a
multiplexed, LUMINEe-based detection method as described by the manufacturer
(LABScreen Single Antigen, OneLambda, Inc., Canoga Park, CA). This individual
had multiple
HLA specificities, as indicated in the legend. As shown in the figure, anti-
HLA-A2 antibodies,
as well as serologically related antibodies (B57, B58), were reduced from the
starting plasma.
Serologically unrelated anti-HLA antibodies (B61, B81, B18, B60) were
unchanged from the
pre-SHARC plasma as they passed through the SHARC. This demonstrates the
specificity of
the HLA-A2 SHARC.
[0044] Figure 32 illustrates polyclonal anti-HLA-A2 antibody depletion from
patient
plasma with HLA-A2 SHARC. 2.5L of Patient plasma containing anti-HLA
antibodies was run
over the 65m1 sHLA-A2 SHARC. Fractions were collected as the plasma was passed
over the
SHARC. The resulting fractions were analyzed using a multiplexed, LUMINEe-
based
detection method as described by the manufacturer. Data is represented by
percent
reduction in BCMFI (%Reduction in BCMFI = 1-( BCMFI of the fraction / BCMFI
starting
plasma).
[0045] Figure 33 illustrates monoclonal anti-HLA antibody depletion from
PBS using a
class ll HLA SHARC (soluble HLA antibody removal column). Saturating
quantities of pan
HLA-DR monoclonal antibody L243 was ran over 65 ml of coupled matrix (30.0 mg
@ 120
pg/m1). The column was then washed with PBS pH 7.4 and eluted with 0.1 M
Glycine pH 11.
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During the load and wash phase, 2 mg passed through the column. During the
elution phase,
23.1 mg of antibody was recovered.
[0046] Figure 34 illustrates polyclonal anti-HLA-DR11 antibody depletion
from patient
plasma with HLA-DR11 SHARC. 2.5 L of Patient plasma containing anti-HLA
antibodies was
run over the 65 ml sHLA-DR11 SHARC. Plasma pre- and post-SHARC were analyzed
using a
multiplexed, LUMINEe-based detection method as described by the manufacturer
(LABScreen' Single Antigen, OneLambda, Inc., Canoga Park, CA). This individual
had multiple
HLA specificities as indicated in the legend. As shown in the figure, anti-HLA-
DR11
antibodies as well as serologically related antibodies (DR13, DR4, DR17) were
reduced from
the starting plasma. Serologically unrelated anti-HLA antibodies (DQ7, D08,
DQ9) were
unchanged from the pre-SHARC plasma as they passed through the SHARC. This
demonstrates the specificity of the HLA-DR11 SHARC.
[0047] Figure 35 illustrates polyclonal anti-HLA-DR11 antibody depletion
from patient
plasma with HLA-DR11 SHARC. 2.5 L of Patient plasma containing anti-HLA
antibodies was
run over the 65 ml sHLA-DR11 SHARC. Fractions were collected as the plasma was
passed
over the SHARC. The resulting fractions were analyzed using a multiplexed,
LUMINDe-based
detection method as described by the manufacturer. Data is represented by
percent
reduction in BCMFI (% Reduction in BCMFI = 1-( BCMFI of the fraction / BCMFI
starting
plasma).
[0048] Figure 36 illustrates the coupling efficiency of soluble class I HLA
A*0201 to an
NHS-activated SEPHAROSC Fast Flow Matrix column.
[0049] Figure 37 illustrates a repeatability study evaluating the column
profile of Figure
36 based on absorption units (mAU) to detect proteinaceous material.
[0050] Figure 38 illustrates a repeatability study evaluating the column
profile of Figure
36 based on pH.
[0051] Figure 39 illustrates a repeatability study evaluating the column
profile of Figure
36 based on conductivity to detect changes in buffer phases.
[0052] Figures 40-42 illustrate a stability evaluation of the column of
Figure 36, wherein
the column was exposed to multiple rounds of load-elute-equilibrate cycles
with W6/32.
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[0053] Figure
43 illustrates a capacity evaluation of the column of Figure 36, utilizing
varying amounts of W6/32.
[0054] Figure
44 illustrates a capacity evaluation of the column of Figure 36, utilizing
varying amounts of Anti-Pm.
[0055] Figure
45 illustrates a capacity evaluation of the column of Figure 36, utilizing
varying amounts of Ant-VLDL (an antibody against an artificial tail introduced
into the A*0201
molecule).
[0056] Figure
46 illustrates a binding efficiency evaluation of the column of Figure 36,
using W6/32.
[0057] Figure
47 illustrates a binding efficiency evaluation of the column of Figure 36,
using Anti-132m.
[0058] Figure
48 illustrates a binding efficiency evaluation of the column of Figure 36,
using Anti-VLDL.
[0059] Figure
49 illustrates a proposed application scenario in accordance with one
embodiment of the presently disclosed and claimed inventive concept(s).
DETAILED DESCRIPTION OF THE INVENTIVE CONCEPT(S)
[0060] Before
explaining at least one embodiment of the inventive concept(s) in
detail by way of exemplary drawings, experimentation, results, and laboratory
procedures, it
is to be understood that the inventive concept(s) is not limited in its
application to the details
of construction and the arrangement of the components set forth in the
following
description or illustrated in the drawings, experimentation and/or results.
The inventive
concept(s) is capable of other embodiments or of being practiced or carried
out in various
ways. As such, the language used herein is intended to be given the broadest
possible scope
and meaning; and the embodiments are meant to be exemplary - not exhaustive.
Also, it is
to be understood that the phraseology and terminology employed herein is for
the purpose
of description and should not be regarded as limiting.
[0061] Unless
otherwise defined herein, scientific and technical terms used in
connection with the presently disclosed and claimed inventive concept(s) shall
have the
meanings that are commonly understood by those of ordinary skill in the art.
Further, unless
12
otherwise required by context, singular terms shall include pluralities and
plural terms shall
include the singular. Generally, nomenclatures utilized in connection with,
and techniques
of, cell and tissue culture, molecular biology, and protein and oligo- or
polynucleotide
chemistry and hybridization described herein are those well known and commonly
used in
the art. Standard techniques are used for recombinant DNA, oligonucleotide
synthesis, and
tissue culture and transformation (e.g., electroporation, lipofection).
Enzymatic reactions
and purification techniques are performed according to manufacturer's
specifications or as
commonly accomplished in the art or as described herein. The foregoing
techniques and
procedures are generally performed according to conventional methods well
known in the
art and as described in various general and more specific references that are
cited and
discussed throughout the present specification. See e.g., Sambrook et al.
Molecular Cloning:
A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
(1989) and Coligan et al. Current Protocols in Immunology (Current Protocols,
Wiley
Interscience (1994)). The nomenclatures utilized in connection with, and the
laboratory
procedures and techniques of, analytical chemistry, synthetic organic
chemistry, and medicinal
and pharmaceutical chemistry described herein are those well known and
commonly used in the
art. Standard techniques are used for chemical syntheses, chemical analyses,
pharmaceutical
preparation, formulation, and delivery, and treatment of patients.
[0062] All patents, published patent applications, and non-patent
publications
mentioned in the specification are indicative of the level of skill of those
skilled in the art to
which this presently disclosed and claimed inventive concept(s) pertains.
[0063] All of the compositions and/or methods disclosed and claimed
herein can be
made and executed without undue experimentation in light of the present
disclosure. While
the compositions and methods of this invention have been described in terms of
preferred
embodiments, it will be apparent to those of skill in the art that variations
may be applied to
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the compositions and/or methods and in the steps or in the sequence of steps
of the method
described herein without departing from the concept, spirit and scope of the
invention. All
such similar substitutes and modifications apparent to those skilled in the
art are deemed to
be within the spirit, scope and concept of the inventive concept(s) as defined
by the
appended claims.
[0064] As utilized in accordance with the present disclosure, the following
terms,
unless otherwise indicated, shall be understood to have the following
meanings:
[0065] The use of the word "a" or "an" when used in conjunction with the
term
"comprising" in the claims and/or the specification may mean "one," but it is
also consistent
with the meaning of "one or more," "at least one," and "one or more than one."
The use of
the term "or" in the claims is used to mean "and/or" unless explicitly
indicated to refer to
alternatives only or the alternatives are mutually exclusive, although the
disclosure supports
a definition that refers to only alternatives and "and/or." Throughout this
application, the
term "about' is used to indicate that a value includes the inherent variation
of error for the
device, the method being employed to determine the value, or the variation
that exists
among the study subjects. For example but not by way of limitation, when the
term "about"
is utilized, the designated value may vary by plus or minus twelve percent, or
eleven percent,
or ten percent, or nine percent, or eight percent, or seven percent, or six
percent, or five
percent, or four percent, or three percent, or two percent, or one percent.
The use of the
term "at least one" will be understood to include one as well as any quantity
more than one,
including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc.
The term "at least one"
may extend up to 100 or 1000 or more, depending on the term to which it is
attached; in
addition, the quantities of 100/1000 are not to be considered limiting, as
higher limits may
also produce satisfactory results. In addition, the use of the term "at least
one of X, Y and Z"
will be understood to include X alone, Y alone, and Z alone, as well as any
combination of X, Y
and Z. The use of ordinal number terminology (i.e., "first", "second",
"third", "fourth", etc.)
is solely for the purpose of differentiating between two or more items and is
not meant to
imply any sequence or order or importance to one item over another or any
order of
addition, for example.
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[0066] As used
in this specification and claim(s), the words "comprising" (and any
form of comprising, such as "comprise" and "comprises"), "having" (and any
form of having,
such as "have" and "has"), "including" (and any form of including, such as
"includes" and
"include") or "containing" (and any form of containing, such as "contains" and
"contain") are
inclusive or open-ended and do not exclude additional, unrecited elements or
method steps.
[0067] The term
"or combinations thereof" as used herein refers to all permutations
and combinations of the listed items preceding the term. For example, "A, B,
C, or
combinations thereof" is intended to include at least one of: A, B, C, AB, AC,
BC, or ABC, and
if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB,
BAC, or CAB.
Continuing with this example, expressly included are combinations that contain
repeats of
one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB,
and so
forth. The skilled artisan will understand that typically there is no limit on
the number of
items or terms in any combination, unless otherwise apparent from the context.
[0068] As used
herein, "substantially pure" means an object species is the predominant
species present (i.e., on a molar basis it is more abundant than any other
individual species
in the composition). Generally, a substantially pure composition will comprise
more than
about 50% percent of all macromolecular species present in the composition,
such as more
than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99%. In one
embodiment,
the object species is purified to essential homogeneity (contaminant species
cannot be
detected in the composition by conventional detection methods) wherein the
composition
consists essentially of a single macromolecular species.
[0069] The
terms "isolated polynucleotide" and "isolated nucleic acid segment" as used
herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or
some
combination thereof, which by virtue of its origin the "isolated
polynucleotide" or "isolated
nucleic acid segment" (1) is not associated with all or a portion of a
polynucleotide in which
the "isolated polynucleotide" or "isolated nucleic acid segment" is found in
nature, (2) is
operably linked to a polynucleotide which it is not linked to in nature, or
(3) does not occur in
nature as part of a larger sequence.
[0070] The term
"isolated protein" referred to herein means a protein of genomic, cDNA,
recombinant RNA, or synthetic origin or some combination thereof, which by
virtue of its
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origin, or source of derivation, the "isolated protein" (1) is not associated
with proteins found
in nature, (2) is free of other proteins from the same source, e.g., free of
murine proteins, (3)
is expressed by a cell from a different species, or, (4) does not occur in
nature.
[0071] The term ''polypeptide" as used herein is a generic term to refer to
native protein,
fragments, or analogs of a polypeptide sequence. Hence, native protein,
fragments, and
analogs are species of the polypeptide genus.
[0072] The term "naturally-occurring" as used herein as applied to an
object refers to the
fact that an object can be found in nature. For example, a polypeptide or
polynucleotide
sequence that is present in an organism (including viruses) that can be
isolated from a
source in nature and which has not been intentionally modified by man in the
laboratory or
otherwise is naturally-occurring.
[0073] The term "antibody" is used in the broadest sense, and specifically
covers
monoclonal antibodies (including full length monoclonal antibodies),
polyclonal antibodies,
multispecific antibodies (e.g., bispecific antibodies), and antibody fragments
(e.g., Fab,
F(ab')2 and Fv) so long as they exhibit the desired biological activity.
Antibodies (Abs) and
immunoglobulins (Igs) are glycoproteins having the same structural
characteristics. While
antibodies exhibit binding specificity to a specific antigen, immunoglobulins
include both
antibodies and other antibody-like molecules which lack antigen specificity.
Polypeptides of
the latter kind are, for example, produced at low levels by the lymph system
and at
increased levels by myelomas.
[0074] "Antibody" or "antibody peptide(s)" refer to an intact antibody, or
a binding
fragment thereof that competes with the intact antibody for specific binding.
Binding
fragments are produced by recombinant DNA techniques, or by enzymatic or
chemical
cleavage of intact antibodies. Binding fragments include Fab, Fab', F(abT)2,
Fv, and single-
chain antibodies. An antibody other than a "bispecific" or "bifunctional"
antibody is
understood to have each of its binding sites identical. An antibody
substantially inhibits
adhesion of a receptor to a counterreceptor when an excess of antibody reduces
the
quantity of receptor bound to counterreceptor by at least about 20%, 40%, 60%
or 80%, and
more usually greater than about 85% (as measured in an in vitro competitive
binding assay).
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[0075] The term "MHC" as used herein will be understood to refer to the
Major
Histocompability Complex, which is defined as a set of gene loci specifying
major
histocompatibility antigens. The term "HLA" as used herein will be understood
to refer to
Human Leukocyte Antigens, which is defined as the major histoconnpatibility
antigens found
in humans. As used herein, "HLA" is the human form of "MHC".
[0076] The terms "MHC class I light chain" and "MHC class I heavy chain" as
used herein
will be understood to refer to portions of the MHC class I molecule.
Structurally, class I
molecules are heterodimers comprised of two noncovalently bound polypeptide
chains, a
larger "heavy" chain (a) and a smaller "light'' chain (13-2-microglobulin or
[32m). The
polymorphic, polygenic heavy chain (45 kDa), encoded within the MHC on
chromosome six,
is subdivided into three extracellular domains (designated 1, 2, and 3), one
intracellular
domain, and one transmembrane domain. The two outermost extracellular domains,
1 and
2, together form the groove that binds antigenic peptide. Thus, interaction
with the TCR
occurs at this region of the protein. The 3'd extracellular domain of the
molecule contains
the recognition site for the CD8 protein on the CTL; this interaction serves
to stabilize the
contact between the T cell and the APC. The invariant light chain (12 kDa),
encoded outside
the MHC on chromosome 15, includes a single, extracellular polypeptide. The
terms "MHC
class I light chain", "13-2-microglobulin", and "132m" may be used
interchangeably herein.
Association of the class I heavy and light chains is required for expression
of class I molecules
on cell membranes.
[0077] Like MHC class I molecules, class II molecules are also
heterodimers, but in this
case consist of two nearly homologous a and 13 chains, both of which are
encoded in the
MHC. The class II MHC molecules are membrane-bound glycoproteins, and both the
a and 13
chains contain external domains, a transmembrane anchor segment, and a
cytoplasmic
segment. Each chain in a class II molecule contains two external domains: the
33-kDa a
chain contains al and a2 external domains, while the 28-kDa 13 chain contains
131 and 32
external domains. The membrane-proximal a2 and 13z domains, like the membrane-
proximal
31-d extracellular domain of class I heavy chain molecules, bear sequence
homology to the
immunoglobulin-fold domain structure. The membrane-distal domain of a class II
molecule
is composed of the al and p, domains, which form an antigen-binding cleft for
processed
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peptide antigen. The peptides presented by class ll molecules are derived from
extracellular
proteins (not cytosolic intracellular peptide antigens as in class I); hence,
the MHC class II-
dependent pathway of antigen presentation is called the endocytic or exogenous
pathway.
Loading of class ll molecules must still occur inside the cell; extracellular
proteins are
endocytosed, digested in lysosomes, and bound by the class II MHC molecule
prior to the
molecule's migration to the plasma membrane. Because the peptide-binding
groove of MHC
class II molecules is open at both ends while the corresponding groove on
class I molecules is
closed at each end, the peptides presented by MHC class II molecules are
longer, generally
between 13 and 24 amino acid residues long. Like class I HLA, the peptides
that bind to class
II molecules often have internal conserved "motifs', but unlike class l-
binding peptides, they
lack conserved motifs at the carboxyl-terminal end, since the open ended
binding cleft
allows a bound peptide to extend from both ends.
[0078] The term "trimolecular complex" as used herein will be understood to
refer to the
MHC heterodimer associated with a peptide. An "MHC class I trimolecular
complex" or "HLA
class I trimolecular complex" will be understood to include the class I heavy
and light chains
associated together and having a peptide displayed in an antigen binding
groove thereof.
The terms "MHC class ll trimolecular complex" and "HLA class II trimolecular
complex" will
be understood to include the class ll alpha and beta chains associated
together and having a
peptide displayed in an antigen binding groove thereof.
[0079] The term "MHC moiety" as used herein will be understood to include
MHC class I
trimolecular complexes, MHC class ll trimolecular complexes, and any portion
or subunit of
MHC class liclass ll molecules.
[0080] The term "biological sample" as used herein will be understood to
include, but
not be limited to, serum, tissue, blood, plasma, cerebrospinal fluid, tears,
saliva, lymph,
dialysis fluid, organ or tissue culture derived fluids, and fluids extracted
from physiological
tissues. The term "biological sample" as used herein will also be understood
to include
derivatives and fractions of such fluids, as well as combinations thereof. For
example, the
term "biological sample" will also be understood to include complex mixtures.
[0081] The term "HLA protein" as used herein will be understood to refer to
any HLA
molecule, complex thereof or fragment thereof that is capable of being
expressed on a
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surface of a non-human cell. Examples of HLA proteins that may be utilized in
accordance
with the presently disclosed and claimed inventive concept(s) include, but are
not limited to,
an HLA class 1 trimolecular complex, an HLA class 11 trimolecular complex, an
HLA class 11 a
chain and an HLA class 11 [3 chain. Specific examples of HLA class 11 a and/or
13 proteins that
may be utilized in accordance with the presently disclosed and claimed
inventive concept(s)
include, but are not limited to, those encoded at the following gene loci: HLA-
DRA; HLA-
DRB1; HLA-DRB3,4,5; HLA-DQA; HLA-DQB; HLA-DPA; and HLA-DPB.
[0082] The term "mammalian cell" as used herein will be understood to refer
to any cell
capable of expressing a recombinant HLA protein (as defined herein above).
Therefore, any
"mammalian cell" utilized in accordance with the presently disclosed and
claimed inventive
concept(s) must contain the necessary machinery and transport proteins
required for
expression of MHC/HLA proteins and/or MHC/HLA trimolecular complexes on a
surface of
such cell. "Mammalian cells" utilized in accordance with the presently
disclosed and claimed
inventive concept(s) must have (A) machinery for chaperoning and loading
MHC/HLA
proteins, such as class 1 and class!! proteins; and (B) such machinery must be
able to interact
and work with human HLA proteins, such as class! and class 11 proteins. Not
all cells express
class 11 MHC protein; only professional immune cells such as but not limited
to dendritic cells
(DC), macrophages, B cells, and the like express class 11 proteins. Therefore,
when it is
desired to express HLA class 11 protein in a mammalian, non-human cell, such
non-human cell
must express class 11 MHC for that species and contain the appropriate
machinery for
interacting and working with both that species' class 11 MHC as well as human
HLA class II.
However, the presently disclosed and claimed inventive concept(s) also
includes the use of
cells of other lineages that have been induced to express class 11 MHC, such
as but not
limited to, cytokines, cells that have been subjected to mutagenesis, and the
like.
[0083] The term "mammalian cell" as used herein refers to immortalized
mammalian cell
lines and does not include animals or primary cells. Examples of "mammalian
cells" that may
be utilized in accordance with the presently disclosed and claimed inventive
concept(s)
include, but are not limited to, human and mouse DC lines, macrophage lines,
and B cell
lines.
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[0084] MHC (major histocompatibility complex) or HLA (Human leukocyte
antigen) Class
II molecules are found only on a few specialized cell types, including
macrophages, dendritic
cells and B cells, all of which are professional antigen-presenting cells
(APCs). The peptides
presented by class ll molecules are derived from extracellular proteins (not
cytosolic as in
class I); hence, the MHC class ll-dependent pathway of antigen presentation is
called the
endocytic or exogenous pathway. Loading of class ll molecules must still occur
inside the cell;
extracellular proteins are endocytosed, digested in lysosomes, and bound by
the class II MHC
molecule prior to the molecule's migration to the plasma membrane.
[0085] Like MHC class I molecules, class II molecules are also
heterodimers, but in this
case consist of two homologous peptides, an a and 13 chain, both of which are
encoded in the
MHC. Class II molecules are composed of two polypeptide chains, both encoded
by the D
region. These polypeptides (alpha and beta) are about 230 and 240 amino acids
long,
respectively, and are glycosylated, giving molecular weights of about 33 kDa
and 28 kDa.
These polypeptides fold into two separate domains; alpha-1 and alpha-2 for the
alpha
polypeptide, and beta-1 and beta-2 for the beta polypeptide. Between the alpha-
1 and bete-
1 domains lies a region very similar to that seen on the class I molecule.
This region, bounded
by a beta-pleated sheet on the bottom and two alpha helices on the sides, is
capable of
binding (via non-covalent interactions) a small peptide. Because the antigen-
binding groove
of MHC class II molecules is open at both ends while the corresponding groove
on class I
molecules is closed at each end, the antigens presented by MHC class II
molecules are
longer, generally between 15 and 24 amino acid residues long. This small
peptide is
"presented" to a T-cell and defines the antigen "epitope" that the T-cell
recognizes.
[0086] Turning now to the presently disclosed and claimed inventive
concept(s), anti-
MHC antibody removal devices, as well as kits containing same, and methods of
production
and use thereof, are disclosed and claimed herein. The devices/kits described
herein may be
utilized for various clinical, diagnostic and therapeutic methods, as
described in more detail
herein below. The anti-MHC antibody removal device includes a soluble MHC
moiety
covalently coupled to a solid support. The soluble MHC moiety attached to the
solid support
is serologically active such that the soluble MHC moiety maintains the
physical, functional
and antigenic integrity of a native MHC trimolecular complex. When a
biological sample is
20
brought into contact with the anti-MHC antibody removal device, anti-MHC
antibodies
specific for the MHC moiety attach to the soluble MHC moiety and are detected
and/or
removed from the biological sample.
[00871 The
soluble MHC moiety may be a class I or class ll soluble MHC moiety produced
by any methods known in the art or otherwise contemplated herein. In certain
embodiments, the soluble MHC moiety is a class I or class II soluble HLA
moiety. Non-
limiting examples of class I soluble HLA moieties that may be utilized in
accordance with the
presently disclosed and claimed inventive concept(s) (as well as methods of
production and
purification thereof) are disclosed in US Serial No. 10/022,066, filed
December 18, 2001 (US Publication
No. 2003/0166057, published September 4, 2003); and US Serial No. 10/337,161,
filed January 2, 2003
(US Publication No. 2003/0191286, published October 9, 2003). Non-limiting
examples of class II
soluble MA moieties that may be utilized in accordance with the presently
disclosed and claimed
inventive concept(s) (as well as methods of production and purification
thereof) are disclosed in US
Serial No. 13/859, 811, filed April 10, 2013 (US Publication 2013/0237689
published September 12,
2013) which is a continuation of application US Serial No. 12/859,002, filed
August 18, 2010, and are
disclosed in further detail herein below.
[0088] In certain
embodiments, the MHC/HLA is purified substantially away from other
proteins such that the individual MHC/HLA trimolecular complex maintains the
physical,
functional and antigenic integrity of a native MHC/HLA trimolecular complex.
The
functionally active, individual MHC/HLA trimolecular complex may be purified
as described
herein or by any other method known in the art. Upon attachment to the solid
support, the
conformation of the functionally active, individual MHC/HLA trimolecular
complex is
maintained.
[00891 Any solid
support capable of covalent attachment to the MHC/HLA moiety and
capable of otherwise functioning in accordance with the presently disclosed
and claimed
inventive concept(s) may be utilized. In certain embodiments, the solid
support may be
selected from the group consisting of a well, a bead (such as but not limited
to, flow
cytometry bead and/or a magnetic bead), a membrane (such as but not limited
to, a
nitrocellulose membrane, a PVDF membrane, a nylon membrane, and acetate
derivative), a
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microtiter plate, a matrix (such as a SEPHAROSE matrix), a pore, plastic,
glass, a polymer, a
polysaccharide, nylon, nitrocellulose, a paramagnetic compound, and
combinations thereof.
A non-limiting example of a solid support capable of functioning in accordance
with the
presently disclosed and claimed inventive concept(s) includes a device (such
as a column)
that possesses an inlet, an outlet, and a chamber disposed therebetween. The
chamber
contains an inner surface on which the serologically active soluble MHC moiety
is disposed,
whereby the inlet is disposed to introduce the biological sample into the
chamber. As the
biological sample flows through the device, anti-MHC antibodies specific for
the serologically
active MHC moiety attach thereto and are removed from the biological sample.
The flow
through collected from the outlet is substantially free of anti-MHC antibodies
specific for the
serologically active MHC moiety. Particular non-limiting examples of devices
of this type
include human use devices (HUDs), such as an extracorporeal plasmapheresis
HUD.
[0090] In certain embodiments, NHS-activated SEPHAROSE matrix is utilized
as the solid
support. This matrix immobilizes proteins by covalent attachment of their
primary amino
groups to the NHS (N-hydroxysuccinimide) activated group to form a very stable
amide
linkage. This is an important feature for therapeutic uses for the devices and
methods
described herein, as it prevents leaching of the immobilized MHC/HLA complexes
from the
substrate/solid support during a therapy (such as but not limited to, the use
of the device as
an extracorporeal device); leaching of these molecules (as well as fragments
and/or subunits
thereof) could cause deleterious effects to a patient. In addition to
increased stability, the
NHS-activated SEPHAROSE matrix also exhibits increased binding capacity
resulting from a
14 atom spacer arm present therein; the spacer arm allows the MHC/HLA to
reposition as
necessary and thus provide better contact with antibodies.
[0091] In certain other embodiments, alternative coupling linkages are
utilized. Non-
limiting examples of other types of linkages include sugar chemistry, carboxy
linkage, sulfur
linkage, or any other type of linkage chemistry known in the art or otherwise
available to a
person having ordinary skill in the art that would allow the coupling of an
MHC moiety to a
solid support.
[0092] In certain embodiments, the presently disclosed and claimed
inventive concept(s)
uses soluble HLA class I trimolecular complexes produced by the methods
described in the
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US patents/patent applications cited herein above. In a non-limiting example,
soluble HLA
class I trimolecular complexes that are purified substantially away from other
proteins such
that the individual soluble class I MHC trimolecular complexes maintain the
physical,
functional and antigenic integrity of the native class I MHC trimolecular
complex are
provided. The trimolecular complex comprises a recombinant, individual soluble
class I MHC
heavy chain molecule, beta-2-microglobulin non-covalently associated with the
individual
soluble class I MHC heavy chain molecule, and a peptide endogenously loaded in
an antigen
binding groove of the individual soluble class I MHC heavy chain molecule.
These molecules
are produced by providing a nucleotide segment encoding a desired individual
class I MHC
heavy chain that has the coding regions encoding the cytoplasmic and
transmembrane
domains of the desired individual class I MHC heavy chain allele removed such
that the
nucleotide segment encodes a truncated, soluble form of the desired individual
class I MHC
heavy chain molecule. This nucleotide segment may be synthetically produced,
or it may be
produced by locus-specific PCR amplification of the truncated allele (either
from cDNA that
has been reverse transcribed from mRNA isolated from a source, or directly
from gDNA).
The nucleotide segment is then cloned into a mammalian expression vector,
thereby forming
a construct that encodes the desired individual soluble class I MHC heavy
chain molecule. A
mammalian cell line is then transfected with the construct to provide a
mammalian cell line
expressing a construct that encodes a recombinant, individual soluble class I
MHC heavy
chain molecule, wherein the mammalian cell line is able to naturally process
proteins into
peptide ligands for loading into antigen binding grooves of MHC molecules, and
wherein the
mammalian cell line expresses beta-2-microglobulin. The mammalian cell line is
then
cultured under conditions which allow for expression of the recombinant
individual soluble
class I MHC heavy chain molecule from the construct, such conditions also
allowing for
endogenous loading of a peptide ligand into the antigen binding groove of each
recombinant, individual soluble class I MHC heavy chain molecule and non-
covalent
association of native, endogenously produced beta-2-microglobulin to form the
individual
soluble class I MHC trimolecular complexes prior to secretion of the
individual soluble class I
MHC trimolecular complexes from the cell. The soluble class I MHC trimolecular
complexes
are then harvested from the culture while retaining the mammalian cell line in
culture for
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production of additional soluble class I MHC trimolecular complexes, and the
individual,
soluble class I MHC trimolecular complexes are purified substantially away
from other
proteins, wherein the individual soluble class I MHC trimolecular complexes
maintain the
physical, functional and antigenic integrity of the native class I MHC
trimolecular complex,
and wherein each trimolecular complex so purified comprises identical
recombinant,
individual soluble class I MHC heavy chain molecules.
[0093] In other embodiments, the presently disclosed and claimed inventive
concept(s)
uses soluble HLA class II trimolecular complexes produced by the methods
described herein
that provide advancements in the areas of purity, quantity, and applications
over existing
methods; these methods use recombinant DNA methods to alter the protein in a
manner
that allows mammalian host cells to secrete the protein. HLA class ll is
naturally produced as
a trimolecular complex that is endogenously loaded with peptide ligands and is
bound to the
membrane. Obtaining such naturally processed and loaded class II presently
primarily
proceeds by gathering membrane bound forms. Production of membrane bound class
ll
requires cell populations to be lysed for capture of the complex. This method
is known as
cell lysate and represents state-of-the-art for natural mammalian HLA
production for anti-
HLA antibody detection assays. Cell lysate class ll products are a mixture of
numerous cell
surface components, including the membrane anchored HLA class ll trimolecular
complex
and other non-HLA proteins that decorate the cell membrane and that co-purify
with HLA.
Isolation of the HLA from other cell debris and membrane proteins reduces the
yield of HLA
class II. When producing HLA class II from detergent lysates, one is faced
with either
contaminating cell surface proteins and/or low class ll protein yield. As an
alternative, HLA
class II can be obtained from Drosophila Schneider S-2 (insect) cell lines
(Novak et al., 1999;
and US Patent No. 7,094,555 issued to Kwok et al. on August 22, 2006) and P.
postoris (yeast)
(Kalandadze et al. 1996), whereby soluble forms of the HLA class ll molecule
have been
produced. However, class II produced in insect cells lack the endogenously
loaded peptides
that are an integral component of the HLA class II native trimolecular
complex. The HLA
molecules produced in insect cells also lack the native glycosylation of
mammalian cells. As
insect cells lack mammalian protein glycosylation mechanisms and lack the
chaperone
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complexes needed for natural peptide ligand loading, there is a reluctance to
utilize class ll
proteins from insects for clinical applications.
[0094] Thus, certain embodiments of the presently disclosed and claimed
inventive
concept(s) use HLA class ll produced by secretion from mammalian cells as a
means to
produce a native trimolecular complex free of contaminating membrane proteins.
Through
HLA class ll secretion from mammalian cells, a pure product in which the
predominant
species is the desired HLA class ll trimolecular complex is produced. A pure,
secreted
molecule simplifies and enables downstream purification. Soluble HLA complexes
are
conducive to hollow fiber bioreactor production systems, such as but not
limited to, the CELL
PHARM system (McMurtrey et al. 2008; Hickman et al., 2003; and Prilliman et
al., 1999), as
well as other systems designed for recombinant native protein secretion from
mammalian
cells. Highly concentrated harvests are much "cleaner" than cell lysates, thus
allowing for
minimal product loss because purification is simplified.
[0095] Other embodiments of the presently disclosed and claimed inventive
concept(s)
may utilize HLA class ll trimolecular complexes in native form that have been
produced and
purified via cell lysate methods; however, the complexes produced by these
prior art
methods have varying amounts of cell membrane secured to the purified HLA
product,
thereby creating several challenges for the yield of a homogeneous HLA product
as well as
problems associated with the use thereof.
[0096] The presently disclosed and claimed inventive concept(s) includes
the use of
soluble HLA class ll trimolecular complexes produced in mammalian cells by a
method that
solves, in a unique and novel manner, the limitations seen when using cell
lysate and insect
cell techniques (Figure 2 illustrates the method of production, while Figure 1
represents the
sHLA trimolecular complexes produced by said method). This production method
overcomes
the disadvantages and defects of the prior art through the use of a
combination of elements;
first, each of the a and 13 chains of the HLA class II complex is truncated
such that the domain
normally anchoring the complex to the cell surface is removed by recombinant
DNA
techniques. In native form, the alpha and beta chains of the HLA class ll
trimolecular
complexes rely on the transmembrane domain to maintain a native conformation.
While
removal of this transmembrane domain facilitates secretion, this removal
prevents
25
formation of a trimolecular complex. The sHLA production method removes the
transmembrane domain and replaces it with a super secondary structural motif,
such as but
not limited to, a leucine zipper protein sequence, which serves as a tethering
moiety for the
class II alpha and beta chains. The super secondary structural motif (such as
but not limited
to, a leucine zipper) thereby creates adhesion or fusion forces between
proteins.
100971 The sHLA production method may further include the recombinant
production of
the soluble alpha and beta chains of the desired HLA class II in a mammalian
cell line. The
use of a recombinant mammalian cell line provides two distinct advantages over
the prior
art: first, production in a mammalian cell line allows the alpha and beta
chains of the HLA
class II molecule to be glycosylated in the same manner as seen for native HLA
class II alpha
and beta chains. Second, the mammalian cell line contains the appropriate
machinery for
natural endocytosis and lysosomal digestion to produce the same peptide
ligands as would
be produced by a native cell (referred to herein as an "endogenously produced
peptide
ligand"), as well as the appropriate chaperone machinery for trafficking and
loading of the
endogenously produced peptide ligands into an antigen binding groove formed
between the
alpha and beta chains of the HLA class II molecule.
[00981 Therefore, the features of (a) glycosylated, soluble HLA class II a
and 13 chains;
(b) production in a non-human mammalian cell line (or a human cell line that
does not
express endogenous class II molecules); and (c) a non-covalently attached,
endogenously
produced peptide ligand, provide distinct advantages that overcome the
disadvantages and
defects of the prior art cell lysate and non-mammalian cell production
methods.
[00991 Endogenously loaded class II is a key element that distinguishes
from the prior
art. The endogenous peptide allows the class ll trimolecular complex to be
used in multiple
applications not previously possible in soluble forms of the prior art (US
Patent No.
7,094,555; Novak et at., 1999; and Kalandadze et al., 1996). Regarding the
currently claimed
application method, only a HLA class II in its native trimolecular complex
form can properly bind
HLA class II specific antibodies. Similarly, the effects of a non-glycosylated
HLA molecule on the
conformation of class II antibody epitopes when used for HLA specific antibody
detection or T-cell
solicitation are known, but there is some evidence that improper glycosylation
disrupts antigen presentation
CA 2872003 2020-03-06
26
(Guerra et al., 1998). Therefore, the most advantageous format for HLA class
II production is
to maintain all components in a native form. It has been shown that HLA
specific antibody
recognition is impacted indirectly by the peptides that are part of the class
I complexes
(Wilson, 1981). The native binding of HLA specific antibodies is a key element
of the
presently disclosed and claimed inventive concept(s) when the sHLA described
and claimed
herein is used as the antigen in an HLA antibody sera screening/removal assay.
(001001 In certain embodiments, the presently disclosed and claimed
inventive concept(s)
utilizes sMHC/sHLA produced by the method described herein below. In the
method, a first
isolated nucleic acid segment is provided, wherein the first isolated nucleic
acid segment
encodes a soluble form of an alpha chain of at least one HLA class II
molecule, and a second
isolated nucleic acid segment is provided, wherein the second isolated nucleic
acid segment
encodes a soluble form of a beta chain of the at least one HLA class II
molecule. The isolated
nucleic acid segments may be present in a single recombinant vector, or the
isolated nucleic
acid segments may be present on two separate recombinant vectors. The coding
regions
encoding the transmembrane domains of the alpha and beta chains have been
removed and
replaced with a super secondary structural motif that enables the alpha and
beta chains
(which previously interacted through their transmembrane domains) to interact.
In one
embodiment, the super secondary structural motif is a leucine zipper protein
sequence that
acts as a tethering moiety for the alpha and beta chains.
[00101] The isolated nucleic acid segments may be provided by any methods
known in
the art, including commercial production of synthetic segments. In one
embodiment, the
nucleic acid segments may be provided by a method that includes the steps of
PCR
amplification of the alpha and beta alleles from genomic DNA or cDNA. Methods
of
obtaining gDNA or cDNA for PCR amplification of MHC are described in detail in
the
inventor's earlier applications US Serial No. 10/022,066, filed December 18,
2001 and
published as US 2003/0166057 Al on September 4, 2003; and US Patent No.
7,521,202,
issued April 21, 2009. Therefore, while the following non-limiting example
begins with gDNA and
utilizes PCR amplification, it is to be understood that the scope of the
presently disclosed and
claimed inventive concept(s) is not to be construed as limited to any
particular starting
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material or method of production, but rather includes any method of providing
an isolated
nucleic acid segment known in the art.
[00102] In one particular embodiment, gDNA is obtained from a sample,
wherein portions
of the gDNA encode a desired individual HLA class II molecule's alpha chain
and beta chain.
Two PCR products are then produced: a first PCR product encoding a soluble
form of the
desired HLA class II alpha chain, and a second PCR product encoding a soluble
form of the
desired HLA class II beta chain. Each of the PCR products is produced by PCR
amplification of
the gDNA, wherein the amplifications utilize at least one locus-specific
primer having a
leucine sequence incorporated into a 3' primer, thereby resulting in PCR
products that do not
encode the cytoplasmic and transmembrane domains of the desired HLA class ll
alpha or
beta chains and thus produce PCR products that encode soluble HLA class II
alpha or beta
chains. The 3' primer utilized for PCR amplification of the HLA class II alpha
chain may
incorporate the leucine sequence consistent with the acid sequence of the
leucine zipper
dimer, while the 3' primer utilized for PCR amplification of the HLA class II
beta chain may
incorporate the leucine sequence consistent with the basic sequence of the
leucine zipper
dimer. However, it is to be understood that the description of the leucine
zipper moiety is
for purposes of example only, and that the presently disclosed and claimed
inventive
concept(s) encompasses the use of any super secondary structural motif that
enables the
alpha and beta chains (which previously interacted through their transmembrane
domains)
to interact.
[00103] Once the isolated nucleic acid segments are provided, they are then
inserted into
at least one mammalian expression vector to form at least one plasmid
containing the PCR
products encoding the soluble HLA class ll alpha chain and the soluble HLA
class II beta chain.
It is to be understood that the two nucleic acid segments may be inserted into
the same
vector or separate vectors.
[00104] The plasmid(s) containing the two PCR products are then inserted
into at least
one suitable immortalized, mammalian host cell line, wherein the cell line
contains the
necessary machinery and transport proteins required for expression of HLA
proteins and/or
are able to naturally process proteins into peptide ligands capable of being
loaded into
antigen binding grooves of HLA class ll molecules.
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[00105] The cell line is then cultured under conditions which allow for
expression of the
individual soluble HLA class II alpha and beta chains and production of
functionally active,
individual soluble HLA class ll trimolecular complexes, wherein the soluble
HLA class ll
trimolecular complexes comprise a soluble alpha chain, a soluble beta chain
and an
endogenously loaded peptide displayed in an antigen binding groove formed by
the alpha
and beta chains. The functionally active, soluble individual HLA class II
trimolecular complex
maintains the physical, functional and antigenic integrity of a native HLA
trimolecular
complex.
[00106] A primary application of the secreted class II product described
herein is the
screening of patients awaiting a transplant for anti-HLA antibodies. The
requirement for an
anti-HLA antibody screening assay is based on the observation that particular
events (such as
but not limited to, blood transfusion, bacterial infection, and pregnancy)
cause one individual
to produce antibodies directed against the HLA of other people (Bohmig et al.,
2000; Emonds
et al., 2000; and Howden et al., 2000). Such anti-HLA antibodies must be
detected before a
patient receives a transplant, or the transplanted organ will be immediately
rejected. Thus,
screening for anti-HLA class II antibodies is a prerequisite for organ
transplantation.
[00107] All transplant patients (approximately 20,000 a year in the U.S.)
and all those
waiting for a transplant (more than 60,000 a year in the U.S.) must regularly
(monthly is
preferred) be screened for antibodies that target the HLA of other people.
Therefore, these
secreted or soluble HLA (sHLA) class II products provide native proteins for
quickly and
accurately identifying anti-HLA antibodies in those awaiting a transplant.
This pre-transplant
diagnostic test will prevent rapid organ failure.
[00108] The presently disclosed and claimed inventive concept(s) is further
directed to a
method of producing any of the anti-MHC removal devices described herein above
or
otherwise contemplated herein. In the method, a serologically active, soluble
MHC moiety
(as described herein above) is covalently coupled to a solid support (as
described herein
above). The soluble MHC moiety is attached to the solid support in such a
manner that the
soluble MHC moiety maintains the physical, functional and antigenic integrity
of a native
MHC trimolecular complex. In addition, the anti-MHC removal device is
constructed so that
a biological sample may be brought into contact with the device in a manner
that allows the
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biological sample to interact with the soluble MHC moiety thereof, whereby
anti-MHC
antibodies specific for the MHC moiety attach to the soluble MHC moiety and
are detected
and/or removed from the biological sample. The method of producing the anti-
MHC
removal device may include any steps contemplated or otherwise described
herein or
otherwise known in the art.
[00109] The presently disclosed and claimed inventive concept(s) is further
directed to a
method for removing anti-MHC antibodies from a biological sample. Such
antibody removal
is useful, for example, when a patient attacks their transplanted organ with
anti-HLA
antibodies. Anti-HLA antibodies can also be removed prior to transplantation
to enable
better outcomes. The removal of antibodies specific for a particular HLA
lessens the need
for immune suppressing drugs. In the method for removing anti-MHC antibodies
from a
biological sample, an anti-MHC removal device as described herein above is
provided. A
biological sample is then brought into contact with the anti-MHC removal
device, whereby at
least a portion of the antibodies present in the biological sample that are
specific for the
serologically active, soluble MHC moiety (that is disposed on the surface of
the anti-MHC
removal device) are removed from the biological sample. The method may further
include
the step of recovering the biological sample following contact with the anti-
MHC removal
device, whereby the antibodies specific for the MHC moiety are substantially
reduced in the
recovered biological sample. The method may further include repeating of the
steps of
contacting the biological sample to the anti-MHC removal device and recovering
the
biological sample following said contact. The use of multiple rounds of
treatment provides
an adequate reduction in antibody titers. In certain embodiments, the
recovered biological
sample may be substantially free of anti-MHC antibodies specific for the
serologically active,
soluble MHC moiety of the anti-MHC removal device.
[00110] When the anti-MHC removal device includes a device (such as a
column) that
possesses an inlet, an outlet, and a chamber disposed therebetween (with an
inner surface
on which the serologically active soluble MHC moiety is disposed), the
biological sample is
introduced into the chamber via the inlet. The biological sample is then
allowed to flow
through the device, and at least a portion of the anti-MHC antibodies specific
for the
serologically active MHC moiety attach thereto and are removed from the
biological sample.
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The flow through is then collected from the outlet, whereby the presence of
anti-MHC
antibodies specific for the serologically active MHC moiety is substantially
reduced.
[00111] When the anti-MHC removal device includes a human use device, the
method
may further include the step of placing the recovered biological sample back
into a patient
from which it was originally taken.
[00112] In certain additional embodiments, the method may further include
the step of
eluting the anti-MHC antibodies from the anti-MHC removal device. This step
may be
performed to allow for regeneration and reuse of the anti-MHC removal device.
Alternatively, the eluted anti-MHC antibodies may be recovered and used as
clinical agents.
For example but not by way of limitation, the eluted, recovered anti-MHC
antibodies may be
utilized for quality control reagents for diagnostics and/or clinical
proficiency testing. Thus,
compositions that include the eluted, recovered anti-MHC antibodies are also
encompassed
by the scope of the presently disclosed and claimed inventive concept(s).
[00113] The presently disclosed and claimed inventive concept(s) further
includes kits
useful for removing anti-MHC antibodies from a biological sample. The kit may
contain any
of the devices described herein, and the kit may further contain other
reagent(s) for
conducting any of the particular methods described or otherwise contemplated
herein. The
nature of these additional reagent(s) will depend upon the particular assay
format, and
identification thereof is well within the skill of one of ordinary skill in
the art. In addition,
positive and/or negative controls may be included with the kit, and the kit
may further
include a set of written instructions explaining how to use the kit. The kit
may further
include a reagent (such as a competitive binding reagent) for elution of the
anti-MHC
antibodies from the device, thus allowing for regeneration and reuse thereof.
Kits of this
nature can be used in any of the methods described or otherwise contemplated
herein.
[00114] Examples are provided hereinbelow. However, the presently disclosed
and
claimed inventive concept(s) is to be understood to not be limited in its
application to the
specific experimentation, results and laboratory procedures. Rather, the
Examples are
simply provided as one of various embodiments and are meant to be exemplary,
not
exhaustive.
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EXAMPLE 1: Production of Class II sHLA Trimolecular Complexes
For Use in Anti-MHC Removal Devices
[00115] This
Example is directed to the expression of soluble individual human HLA class II
trimolecular complexes in mammalian immortal cell lines. The method includes
the use of
modifications that alter the endogenous membrane bound complexes in such a way
that the
membrane bound anchor is disrupted, thereby allowing the cell to secrete the
HLA class ll
trimolecular complexes. In this Example, the Alpha and Beta chain genes
encoding HLA class
II-DR. HLA-DQ, and HLA-DP were truncated such that the transmembrane and
cytoplasmic
domains were deleted. At the site of the truncation, a leucine zipper (a
tethering moiety)
replaced the transmembrane and cytoplasmic that endogenously anchors HLA to
the
membrane. The leucine zipper allows the HLA to be secreted from the cell while
maintaining
the class II trimolecular complex native confirmation (Figures 1 and 2). The
leucine zipper is
comprised of an acid segment tailing the class ll alpha chain with
complementary basic
domain tailing the class ll beta chain. The acid and basic segments fuse by
means of the
amino acid leucine being placed every 7 amino acids in the d position of the
heptad repeat.
The strategy was used by Chang in 1994 to bind the alpha and beta chains of
soluble T cell
Receptors together in the same fashion.
[00116] HLA
class ll complexes are comprised of two different polypeptide chains,
designated a and p. In one method, the alpha and beta constructs were
commercially
purchased and directly ligated into a mammalian expression vector. In
another, the
constructs were produced by PCT amplification as described in the paragraph
below,
followed by purification and ligation into a mammalian expression vector.
[00117]
Amplification of specific HLA class II genes from genomic DNA or cDNA was
accomplished using PCR oligonucleotide primers for alleles at the HLA-DRa HLA-
DRA), DRB
(HLA-DRB); DQa (DQA), DQB (DUB); or DPa (DPA) and DPB (DPB) gene loci. The
beta chain 3'
PCR primer incorporates the leucine sequence consistent with the basic
sequence of the
leucine zipper dimer. The Alpha chain 3' primer incorporates the leucine
sequence
consistent with the acid sequence of the leucine zipper dimer. The truncation
of the class II
genes through placement of the PCR primers eliminates the cytoplasmic and
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32
transmembrane regions, thus resulting in a soluble form of HLA class ll
trimolecular complex
with a leucine zipper moiety.
[00118] Figures 15-17 represent constructs used in the methods of sHLA
production of
the presently disclosed and claimed inventive concept(s). Figure 15
illustrates the nucleic
acid and amino acid sequences for a DRA1*0101 alpha chain-leucine zipper
construct (SEQ ID
NOS:1 and 2, respectively). Figure 16 illustrates the nucleic acid and amino
acid sequences
for a DRB1*0401 beta chain-leucine zipper construct (SEQ ID NOS:3 and 4,
respectively).
Figure 17 illustrates the nucleic acid and amino acid sequences for a
DRB1*0103 beta chain-
leucine zipper construct (SEQ ID NOS:5 and 6, respectively).
[00119] The constructs were then inserted into a mammalian expression
vector. In one
instance, the alpha chain was cut with one set of restriction enzymes, while
the beta chain
was cut with another set of restriction enzymes. The purified and cut alpha
chain
amplification products were ligated into the mammalian expression vector
pcDNA3.1. Next,
this ligated vector containing the sHLA class II alpha gene was transformed
into E. coli strain
JM109. The bacteria were grown on a solid medium containing an antibiotic to
select for
positive clones. Colonies from this plate were picked, grown and checked to
contain insert.
Plasmid DNA was isolated from the identified positive clones and subsequently
DNA
sequenced to insure the fidelity of the cloned alpha gene.
[00120] The alpha vector was re-cut using a second set of restriction
enzymes which
facilitate directional cloning of the purified beta PCR product. The final
ligation product
consisted of both alpha and beta clones. Plasmid DNA was then isolated from
positive
clones, and the beta genes were DNA sequenced from these clones.
[00121] Plasmid DNA for the alpha and beta class ll alleles was prepared
and DNA
sequenced to confirm fidelity of the amplified class II genes. Log phase
mammalian cells and
the plasmid DNA were mixed in a plastic electrocuvette. This mixture was
electroporated,
placed on ice and resuspended in media. Special optimization was required for
the
electroporation step to enable successful enablement of the presently
disclosed and claimed
inventive concept(s). Standard electroporation procedures were unsuccessful in
extensive
trials by the inventors and as reported by other labs in publications.
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[00122] After incubation for 2 days at 37 C in a CO2 incubator, the cells
were subjected to
selection with the antibiotic. First cells were counted and viability was
determined. The cells
were then resuspended in conditioned complete media. Next, cells were placed
into each
well of a 24-well plate and left to undergo selection. Supernatant from each
well was taken,
and an ELISA assay was performed to determine sHLA class II production. High
producers
were expanded and cryopreserved for large-scale production.
[00123] Prior to culture in CELL PHARM bioreactors, the cellular growth
parameters (pH,
glucose, and serum supplementation) for each line was optimized for growth in
bioreactors.
Approximately 8 liters of naive or pathogen infected sHLA-secreting class II
transfectants
were cultured in roller bottles in culture media supplemented with
penicillin/streptomycin
and serum or ITS (insulin-transferrin-selenium) supplement. The total volume
of cells
cultured was adjusted such that approximately 5 x 109 cells were obtained.
Cells were
pelleted by centrifugation and resuspended in 300 ml of conditioned medium in
a CELL
PHARM feed bottle. Cells and conditioned medium were inoculated through the
ECS feed
pump of a Unisyn CP2500 CELL PHARM into 30 kDa molecular-weight cut-off
hollow-fiber
bioreactors previously primed with media supplemented with
penicillin/streptomycin and
serum or ITS. The culture of cells inside the CELL PHARM was continued with
constant
monitoring of glucose, pH and infection. Medium feed rates were monitored and
adjusted
to maintain a glucose level of 70-110 mg/dL. Figure 3 provides an overview of
the CELL
PHARM bioreactor system; the sHLA secreting cells and their sHLA product were
contained
within the extra capillary space (ECS) of the hollow fiber bioreactor.
Nutrients and gases for
the cells were provided by recirculated medium.
[00124] Figure 4A illustrates the increased production of sHLA class II
DRB1*0103
produced from transfected cells when scaled up to the bioreactor production.
The sHLA was
purified from the cell supernatant with the specific anti-HLA class II
antibody L243 coupled to
CNBr-activated SEPHAROSE 4B, and the protein concentration determined by a
micro-BCA
protein assay, UV absorbance and ELISA. The sHLA class ll titer of a typical
production run
was found to be approximately 4 - 5 mg/liter of growth media. Figure 4B
illustrates that
these trimolecular complexes were very stable in a wide variety of buffers and
at a wide
range of pH concentrations using monoclonal antibody L243, which reacts with
virtually all
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DR HLA proteins. L243 is a murine IgG2a anti-HLA-DR monoclonal antibody
previously
described by Lampson & Levy (1980); said monoclonal antibody has been
deposited at the
American Type Culture Collection, Rockville, Md., under Accession number ATCC
HB55.
[00125] In Figure 5, the serologic integrity of the purified sHLA class ll
trimolecular
complexes was confirmed by directly coating the complexes on a plate and
exposing the
coated complexes to defined commercially available mAbs and patient sera. In
addition,
comparison of the sHLA with full-length molecules showed no differences in
antigenicity.
[00126] Figure 6 illustrates the ability to produce multiple different sHLA
class ll
trinnolecular complexes by the methods of the presently disclosed and claimed
inventive
concept(s). While DRB1*0101, DRB1*0103, DRB1*1101, DRB1*1301 and DRB1*1501 are
shown for the purposes of example, multiple other sHLA class ll trimolecular
complexes have
also been produced in milligram quantities in accordance with the presently
disclosed and
claimed inventive concept(s). Trimolecular complexes from each sHLA DR protein
have
been detected and quantitated using the L243 ELISA-based assay.
[00127] Figures 7-9 illustrate another example of sHLA class ll production
in accordance
with the presently disclosed and claimed inventive concept(s). In this
example, immortalized
cells tranfected with a soluble HLA-DRB*0103/DRA*0101 construct (DRB1*0101
soluble
alpha chain with leucine zipper and DRB1*0103 soluble beta chain with leucine
zipper) were
grown in a roller bottle format until a total 110 cells were obtained. The
cells were then
seeded into the ECS portion of 2 hollow fiber bioreactor units. Cells were
grown in DM EM +
10% FBS in the ECS and no FBS in the ICS. ECS harvest was collected every day
until cells
were dead and no longer producing soluble HLA. Protein was quantified using a
capture
ELISA. For this ELISA an antibody specific for the leucine zipper (2H11) was
used as the
capture antibody, and an antibody specific for class II HLA (L243) as the
detector antibody.
Approximately 8mg of soluble HLA was loaded on an affinity antibody (L243)
column and
eluted in an alkaline buffer (0.1M Glycine, pH 11). Fractions containing
soluble HLA were
pooled and lyophilized. The lyophilate was resuspended in water/20%
acetonitrile and
loaded onto a C18 RP-HPLC column. The soluble HLA was then eluted using a 20%
to 80%
acetonitrile gradient and detected using electrospray ionization TOF mass
spectrometry.
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[00128] As can be seen in Figure 7, milligram quantities of a soluble form
of a single class
II HLA heterodimer were produced in the bioreactor format. Additionally, the
intact
heterodimer was purified with no other contaminating proteins, as determined
by LCMS
(Figure 9). This soluble class ll contains a monoglycosylated beta chain and
diglycosylated
alpha consistent with native class II HLA (Figure 8). Furthermore, the various
glycoforms
were consistent with the natural variation in sugars that occurs as a protein
transits to the
cell surface. For a subpopulation of the class ll molecules, intracellular
proteolytic events
removed all but two amino acids of the leucine zipper domain from both the
alpha and the
beta chains. However, like the full length construct, class ll without the
leucine zipper
domain remain as a heterodimer as both the alpha and beta chains co-elute.
These soluble
class I and class II HLA proteins are amenable to analysis by mass
spectrometry, whereby the
purity and identity of these proteins can be confirmed by TOF analysis of
molecular weights
(Figure 9).
EXAMPLE 2: Use of Class ll sHLA for Antibody Removal
[00129] The soluble HLA class ll trimolecular complexes of the presently
disclosed and
claimed inventive concept(s) have also been demonstrated herein to be
successfully used in
antibody removal techniques, as illustrated in Figures 10-14.
[00130] Figure 10 graphically depicts coupling of soluble DRB1*1101 ZP HLA
Class ll
trimolecular complex to a solid support and use thereof to facilitate removal
of HLA Class II
specific antibodies in an ELISA format. Panel A contains a diagram of the
consecutive
absorption matrix ELISA performed for specific antibody removal. Briefly,
soluble HLA Class II
DRB1*1101/DRA1*0101 ZP (labeled as DRB1*1101) was coated to a standard ELISA
plate
and blocked with BSA. Biotinylated labeled HLAII specific antibodies were then
prepared and
diluted according to a pre-determined titration for optimal binding, and added
to 10 wells as
Si. A small portion of this original dilution (200111) was saved as 5(0). The
antibody was
allowed to bind for 30 minutes at room temperature, after which the entire
contents of each
well (<2000) was moved to a corresponding new well (S2), and BSA buffer was
added to the
Si wells. This entire process was repeated for a total of 9 sample rounds (S1-
S9). For each
round, one well was saved in an eppendorf tube for evaluation of the amount of
antibody
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remaining in the retentate solution. These were marked as S(n). After the
absorption
process was completed, the plate was developed using HRP/OPD peroxidase
substrate and
plotted as "absorbance." The retentate samples were also read on a separate
ELISA plate in
the same manner. These were plotted as "retentate." Panel B depicts absorbance
and
retentate values from 3 different HLA Class II specific mAb antibodies: L243,
OL (One
Lambda), and 2H11 were subjected to the consecutive absorbance matrix. The
L243 and OL
mAbs, specific for the HLA Class ll molecules, and the 2H11 mAb, specific for
the zipper tail
piece recombinantly added to the soluble HLA Class ll molecules, showed a
reduction of HLA
class ll antibodies in the absorption and retentate through each round of the
ELISA. One
control mAb antibody was included, W6/32, which is specific for HLA Class I
molecules, which
was not absorbed to the plate and only found in the retentate.
[00131] Figure
11 graphically depicts that DRB1*1101-specific human sera was recognized
by soluble DRB1*1101 in an ELISA format. Using soluble HLA Class II
DRB1*1101/DRA1*0101
ZP (labeled as DRB1*1101), ELISA plates were directly coated with the HLA
Class II soluble
allele. Serum samples from two human donors known previously to have DRB1*1101
reactivity were added to the plates in a dilution range from lx (no dilution)
to 5000x. Plates
were washed, and a secondary biotinylated goat anti-human IgG antibody was
added.
Plates were developed using HRP/OPD peroxidase substrate and read at
absorbance of 490
nm. Dilution
curves for the sera antibody reactivity can be seen for both donors,
corresponding to specific avidity for DRB1*1101.
[00132] Figure
12 graphically depicts that soluble DRB1*1101 can be coupled to
SEPHAROSE and used to absorb the HLA Class II specific antibody, 9.3F10. In
Panel A, 4 mg
of soluble DRB1*1101 was coupled to 1 ml of SEPHAROSE Fast Flow and packed
into a
gravity column. A known mixture of 100 p.g/m1 of mAb 9.3F10 (in 1xPBS), which
has DR
reactivity, was passed over the column and washed with 1xPBS. A total of 23
200 I
fractions of flow thru were collected, weighed, and measured for OD 280 nm.
Values were
converted to total amount of protein. To elute the column, roughly 4 ml of DEA
(diethanolamine) buffer, pH 11.3, was added to the column, and fractions were
collected in
200 p.I quantities. The eluate was also weighed, measured at an optical
density of 280 nm,
and converted to total amount of protein.
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37
[00133] In Panel B of Figure 12, two separate ELISAs for total mouse IgG
and human HLA
were also performed on the Flow Thru and Eluate to detect specific antibodies
(versus HLA
proteins) that might have been eluted off the column. Due to the increase in
ELISA
sensitivity, the minuscule amount of protein seen in the flow thru gave a
small peak in the
antibody ELISA. Importantly, however, no HLA was seen in the flow thru, but
HLA did elute
off the column when DEA was added.
[00134] Figure 13 graphically depicts that antibodies contained in human
sera specific for
DRB1*1101 can be removed by a DRB1*1101 specific column. Donor tn. sera was
passed
over the DRB1*1101 SEPHAROSE column, and two 2 ml fractions of flow thru were
collected. To elute, DEA buffer, pH 11.3 was added to the column, and two 2 ml
fractions
were collected. In Panel A, a direct DRB1*1101 ELISA was performed to detect
the amount of
DRB1*1101 specific antibodies that were left in the flow thru and eluate. Flow
thru and
eluate fractions were diluted lx (no dilution) to 5000x and developed with a
biotinylated
goat anti-human secondary antibody, followed by HRP/OPD peroxidase substrate.
Plates
were read at an optical density of 490 nm. In Panel B, a total human IgG
sandwich ELISA was
also performed to evaluate passage of total human IgG. Total human IgG was
seen to pass
thru; however only DRB1*1101 antibodies were retained by the column, and only
seen once
the column was eluted.
[00135] Figure 14 graphically depicts that soluble DRB1*1101 coupled
SEPHAROSE is
specific for DRB1*1101 and not other DR alleles. Donor #2 sera was passed over
the same
DR1*1101 column in the same manner as Figure 13, and two fractions of the flow
thru and
one fraction of the eluate were evaluated for multi-allele DR reactivity.
Briefly, multiple
alleles of DR from membrane detergent purifications and two DR alleles
produced solubly
were coated to a 96 well ELISA plate in previously determined optimal amounts
for
reactivity. Two flow thru fractions and one of the eluate fractions were
compared to the
original sera sample for reactivity. The second eluate fraction was not
evaluated given that
most of the specific reactivity was contained in Eluate #1 (Figure 14). Low
reactivity was
seen across the board except for the soluble DRB1*1101 (DRB1*1101 ZP) allele,
which gave
high reactivity to only the sera sample and the eluate but not the flow thrus
(first boxed
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38
area). The sera also contained strongly reactive antibodies to a second
allele, DRB1*1601
(second boxed area), which passed through the flow thru but not the eluate.
[00136] Therefore, this Example demonstrates that sHLA class ll
trimolecular complexes
immobilized in a column format can selectively and efficiently remove the vast
majority of
anti-HLA specific antibodies based on affinity to the bound HLA class II
protein in a single
pass through, while not removing antibodies that bind to antigenically
dissimilar HLA
molecules. These data show that a highly specific and efficient antibody
removal device can
be constructed using the sHLA class ll proteins produced in accordance with
the presently
disclosed and claimed inventive concept(s).
EXAMPLE 3: Isolation of HLA-DR11 Antibodies From Sensitized Human Sera
[00137] To test the hypothesis that antigen-based isolation of naturally
occurring,
polyclonal, anti-HLA antibodies would facilitate the characterization of
allogeneic anti-HLA
antibody responses, appreciable quantities of soluble class II HLA molecules
were produced
in a native conformation. Next, this unique HLA reagent was used to construct
the first
reported HLA immunoaffinity column. Donor sera containing a complex mixture of
anti-HLA
antibodies were then passed over the column. Antibodies specific for a
particular class ll
HLA were retained on the column, and these immunoglobulins were subsequently
recovered
by elution and characterized. The phenotypic and functional profiling of
antigen-specific
antibodies represents a substantial advance in the ability to understand how
anti-HLA
antibodies contribute to organ rejection. A robust application of this
technology would
distinguish complement-fixing antibodies that represent a contraindication for
transplantation from refractory humoral responses that are less of a concern.
These
immunoaffinity columns constructed with native soluble HLA might also provide
a new
generation of therapeutic tools for patients with strong antibody reactivity
directed towards
allogeneic HLA.
[00138] Materials and Methods of Example 3
[00139] Patient serum samples: Donor 1 serum was purchased as HLA-DR11
antiserum
(Gen-Probe, Inc., San Diego, CA). Donor 2 serum was collected from a DR11
sensitized kidney
recipient using informed consent according to a protocol approved by the
University of Texas
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39
Southwestern institutional review board. Donor 2, a 50 year old male, received
a kidney graft
with a 6/6 mismatch (graft HLA: A2, AS, 562, 551, DR4, DR11). After
transplantation donor 2
rejected the graft and developed anti-HLA antibodies. Approximately 5 ml of
whole blood
was collected and allowed to coagulate. The blood was then centrifuged and the
serum was
removed from the pellet. Sera were stored at 4 C until testing.
[00140] sHLA-DR11 Protein Production. To produce secreted HLA-DRB11 (sHLA)
molecules, a-chain cDNAs of HLA-DRA1*01:01 and HLA-DRB1*11:01 were modified by
PCR
mutagenesis to delete codons encoding the transmembrane and cytoplasmic
domains and
add the leucine zipper domains. For DRA*01:01, a 7 amino acid linker (DVGGGGG;
SEQ ID
NO:7) followed by leucine zipper ACIDp1 was added. For DRB*11:01 the same
linker was
used, followed by leucine zipper BASEp1 sequence (Busch et al., 2002). sHLA-
DRA1*01:01
and sHLA-DRB1*11:01 were cloned into the mammalian expression vector pcDNA3.1(-
)
geneticin and zeocin respectively (Invitrogen, Life Technologies, Grand
Island, NY). The HLA
class 11 deficient B-LCL cell line NS1 (ATCC # TIB-18) was transfected by
electroporation
simultaneously with sHLA-DRB1*11:01 and DRA1*01:01. Two days post-
electroporation
cells were transferred into selective growth media containing G418 (0.8 mg /
ml) and zeocin
(1 mg/ml). Drug resistant stable transfectants were tested for production of
sHLA class II
molecules by sandwich ELISA using L243 (Leinco Technologies Inc., St. Louis,
MO) as a
capture and class II specific commercial antibody for detection (One Lambda
Class II, One
Lambda Inc., Canoga Park, CA). Individual wells with clonal cell populations
were tested for
the production of sHLA class!! by ELISA and the highest producing clone was
expanded in an
ACUSYST-MAXIMIZER hollow fiber bioreactor (Biovest International, Inc.,
Minneapolis, MN).
Approximately 25 mg of sHLA-DR11 was harvested from each bioreactor. sHLA-DR11
containing supernatant was loaded on a L243 immunoafffinity column and washed
with 40
column volumes of 20 mM phosphate buffer, pH 7.4. sHLA-DR11 molecules were
eluted
from the affinity column with 50 mM DEA at pH 11.3, neutralized with 1M TRIS
pH 7.0, and
buffer exchanged and stored at 1 mg/ml in sterile PBS.
[00141] Mass spectrometry: 10 pg of purified sHLA-DR11 was reduced and
denatured
with dithiothreitol (Sigma-Aldrich D0632) and incubated at 95 C for 5 minutes.
The sample
was then alkylated with iodoacetamide (Thermo Scientific 89671F), for 1 hour
at room
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temperature. Denatured protein was digested with trypsin using a standard two
step
digestion protocol (Thermo Scientific 90055). Tryptic peptides were
reconstituted in 30%
acetic acid / 70% ultra-pure water, and loaded onto the ULTIMATE 3000 HPLC
system
(Dionex, Thermo Fisher Scientific, Inc., Sunnyvale, CA) with a PEPMAPT"100 C18
751im x
15cm, 311m 100A reverse phase column. Peptides were eluted and analyzed on a
QTOF
QSTAR Elite mass spectrometer (ABI, Thermo Fisher Scientific, MDS Sciex) with
Mascot
software.
[00142] Antibody Removal with DRB1*11:01-Coupled SEPHAROSE Affinity
Columns. For
a 1 ml sHLA-DR11 affinity column, SEPHAROSE 4 Fast Flow (GE Healthcare) was
swollen and
washed 4 times with ice-cold 1 mM HCI pH 3Ø The swollen matrix was mixed
with sHLA-
DR11 (4 mg) in bicarbonate coupling solution at a final reaction concentration
of 1.6 mg/ml.
After the reaction, the matrix was washed three times in coupling buffer and
blocked with
0.1M TRIS, pH 8Ø The coupled matrix was then packed into a small 2m1 column.
[00143] Either 1 ml of a 200 pg/m1 L243 antibody solution or 1 ml of total
human sera was
applied to the matrix and allowed to be absorbed by gravity. After sample
application, 4 ml
of PBS pH 7.4 was added. During this loading step, 25 fractions were collected
manually,
each containing ¨200 I. Finally, the column was eluted by applying 5 ml of 50
mM DEA pH
11.3. 20 fractions were collected in the elution process and immediately
neutralized with
35111 of 1 M TRIS. For L243, collected fractions were measured by 0D280 for
antibody content.
After each procedure, column was mock eluted with DEA, pH11.3 followed by 50
ml of wash
buffer (PBS pH 7.4).
[00144] Class II HLA Single Antigen Bead Assay and Ig Isotyping.
Specificities of anti-HLA
antibodies in the pre-column serum, flow through, and eluate were determined
using a
LUMINEX -based class 11 HLA single antigen assay (Gen-Probe GTI Diagnostics),
according to
manufacturer protocols. Briefly, 40 pl of the bead suspension was incubated
with 10 p.I of
the test sample at room temperature for 30 minutes. Beads were washed and
incubated
with the detecting antibody at room temperature for 30 minutes, then washed
and analyzed
on a LUMINEX 100 analyzer. Data were analyzed using MATCHIT (Gen-Probe GTI
Diagnostics, San Diego, CA) and EXCEL (Microsoft) software. Data for the
starting sera and
flow through are shown as background corrected median florescence intensity
(BCMFI)
41
values based on company defined background levels, which are lot specific and
determined
by a standard negative sera. With the eluate, there was substantially less
background so the
background was defined as the minimum bead MFI. For the flow through, the
BCMFI values
were normalized to the average DQ BCMFI in the starting sera (Tables 1 and 2).
The eluate
BCMFI values were normalized to the DRB1*11:01 BCMFI in the starting sera
(Tables 1 and
2).
[00145] For antibody isotyping and quantification the BIO-PLEX PRO'
immunoglobulin
isotyping kit (Bio-Rad Laboratories, Inc., Hercules, CA) was used according to
manufacturer
protocols. Briefly, 10-fold serial dilutions of the sample were made and 50111
of the sample
was incubated with 50 I of the bead suspension for 30 minutes at room
temperature. Beads
were washed and incubated with the detecting antibody at room temperature for
30
minutes. Last, beads were washed and analyzed on a LUMINEX 100 (One Lambda,
Inc.).
Sample MFI values were translated into Ig concentration using the Ig specific
standard
curves.
[00146] Complement Dependant Cytolysis. Complement dependant cytolysis
(CDC) was
determined using the LambdaTM Cell Tray: 30 B cell panel (One Lambda, Inc.)
Cell lines analyzed
were DR11 positive. Cell line class II HLA haplotypes are as follows. C433:
DR4, DR11, DR52,
DR53, D07. C418: DR4, DR11, DR52, DR53, DQ7. C423: DR11, DR13, DR52, DQ6, DQ7.
C428:
DR11, DR17, DR52, DQ2, DQ7 (One Lambda, Inc.). Lysis was performed on
indicated samples
according to manufacturer protocols. Rabbit complement was used as a source of
complement. After lysis, FLOROQUENCH" dye (One Lambda, Inc.) was used to
differentiate
live cells from lysed cells. Live cells and lysed cells were then analyzed
using a Nikon"TE200-E
florescent microscope. Whole well images were generated for each well using
the 4x
objective lens for both the green filter (excitation: 490nm bp 20, emission:
520nm bp 38) and
the red filter (excitation: 555nm bp 28, emission: 617nm bp 73). Total
florescence in both
channels was determined using MetaMorph v 7.5.5.0 and percent cell death was
calculated
as red florescence / red florescence + green florescence.
[00147] Results for Example 3
[00148] Production and Purification of Soluble Class II HLA. The specific
isolation of anti-
class II HLA antibodies requires a source of plentiful, native class II HLA.
While there are
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several techniques for obtaining HLA proteins, in this Example, soluble
molecules were
produced in mammalian cells because these HLA are glycosylated, naturally
loaded with
ligands, and fully reactive with antibodies. One challenge is that HLA class
II exists as an
alpha / beta heterodimer and these proteins must be specifically paired to be
functional.
Previous studies have stabilized the class II soluble HLA heterodimer by
replacing the
transmembrane and cytoplasmic domains on both the alpha and beta chains with
complementary leucine zipper domains (Busch et al., 2002; and Kalandadze et
at., 1996), but
these studies have only succeeded using non-mammalian cells. Here this
approach was used
to generate constructs for HLA-DRA1*01:01 and HLA-DRB1*11:01, in which the
transmembrane domain is replaced with a 7 amino acid linker followed by an
ACIDp1 or
BASEp1 leucine zipper domain respectively (Figure 18A).
[00149] A murine cell line was chosen for sHLA-DR11 production, because the
inventors
hypothesized that the endogenous mouse class II MHC alpha and beta proteins
(H2-Ad, H2-
Ed) would not pair with the soluble human class II HLA alpha and beta proteins
nor interfere
with the intended pairing of the soluble alpha/beta HLA proteins. To confirm
that the
purified sHLA-DR11 was free from mouse alpha and beta chains, the purified
protein was
digested with trypsin, and the resulting peptides were subjected to liquid
chromatography
mass spectrometry (LCMS) analysis. In a BLAST analysis, the peptide sequences
showed no
matches with the endogenous mouse class II MHC (H2-Ad, H2-Ed), while peptide
sequences
were detected from both the alpha and beta chains of the sHLA-DR11 construct
transfected
into the cells (Figure 18B). Thus, it was concluded that the desired alpha and
beta chain of
sDR11 was produced and purified without contamination from other class II MHC
subunits.
[00150] Column Removal of Anti-HLA Class ll Antibodies. In order to test
sHLA class ll in
an immmunoaffinity column format, sHLA-DR11 was purified and coupled to CNBr
activated
Sepharose-4 Fast Flow solid support matrix. The anti-HLA-DR monoclonal
antibody L243 was
passed over the affinity column to test whether the sHLA-DR11 complexes
remained intact
during the coupling process and to measure the binding capacity of the column.
Fractions of
200 Ld were collected during the loading process (flow through), and bound
L243 was eluted
intact. Between the flow through and the eluate, 78% (170.6 p.g) of the
antibody loaded onto
the column was recovered, of which 28% (47.8 iug) was in the flow through and
72% (122.9
43
mg) in the eluate (Figure 19A). Furthermore the captured and eluted L243
antibody
maintained its HLA-DR binding activity and specificity (Figure 1913). These
results
demonstrated that HLA-DR11 retained its native conformation when coupled to
the affinity
column matrix and that a sHLA-DR11 column could be used to remove and recover
intact
anti-HLA antibodies.
[00151] Depletion
and Recovery of Anti-HLA-DR11 Antibodies from Patient Sera. Nest, it
was tested whether the column could be used to deplete anti-HLA-DR11
antibodies from
patient sera. Sera from two DR11 sensitized patients were analyzed for
reactivity to multiple
class II HLA types in the starting serum (prior to column loading), flow-
through, and eluate.
Both starting sera contained complex mixtures of polyclonal anti-HLA
antibodies reactive
with multiple DR and DQ specificities (Figures 20A and B). Following passage
through the
DR11 column, the flow through and eluate from each donor were quite distinct
in their
patterns of HLA reactivity (Figures 20C and D). In the donor 1 serum, HLA-DQ
(red) and -DP
(green) specific antibodies flowed through the column, while the majority of
antibodies to
DR11, 13, 8, and 4 were depleted from the serum and subsequently recovered in
the eluate.
Likewise, in the donor 2 serum, HLA-DQ and -DP specific antibodies passed
through the
column. However, unlike the donor 1 serum, the majority of DR9 and DR7
specific
antibodies from the donor 2 serum flowed through the column, while antibodies
to DR11
and DR13 were retained and subsequently eluted. Only small amounts of DR9 and
DR7
specific antibodies were recovered in the eluate. All class II HLA reactivity
in the starting
sera, pooled flow-through (fractions 2-11), and pooled eluate (fractions 2-6)
is summarized in
Figure 23.
CA 2872003 2018-09-13
44
[00152] Prior to
column passage, these sera recognized a substantial number of DR
specificities (11 HLA-DR in donor 1 and 17 HLA-DR in donor 2). Strikingly, the
DR11 column
depleted 100% (11/11) of the DR reactive antibodies in donor 1 and 88% (15/17)
in donor 2
(Figure 23, Tables 1 and 2), while no NIA-DO or DP reactive antibodies were
recovered. Thus,
the DR11 column removed antibodies to multiple serologically related HLA-DR
specificities
while antibodies reactive to HLA-DQ and -DP did not bind. These results show
that DR11
specific antibodies can be depleted and recovered from patient sera while
antibodies
reactive with other antigens are not retained.
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TABLE 1
Pre
Bead Antigens Sera Flow Through Eluate
BCMFI BCMFI Normalized* BCMFI MFI Normalizedt MFI
DRB1*11:01 13136 517 498 12063
12619
DRB1*13:03 9245 890 857 7530 7877
DRB1*08:01 5945 49 47 4563 4773
DRB1*01:03 5140 612 589 3964 4147
DRB1*04:02 4890 290 279 3857 4035
DRB1*13:01 4447 280 270 2999 3137
DRB1*16:01 3477 456 439 1705 1784
DRB1*04:01 1767 0 0 1694 1772
DRB1*04:05 1243 0 0 1257 1315
DRB1*12:01 2632 0 0 1172 1225
DRB5*01:01 1496 0 0 574 600
DQA1*05:01, DQB1*02:02 1813 1728 1663 97 101
DQA1*06:01, DQB1*03:03 2743 3084 2969 88 92
DPA1*01:03, DPB1*04:02 1729 1201 1156 82 85
DQA1*03:02, DQB1*02:02 1245 1472 1417 75 78
DPA1*01:03, DPB1*04:01 907 713 686 75 78
DQA1*03:02, DQB1*03:02 2422 2436 2344 65 68
DQA1*03:02, DQB1*03:01 3273 3109 2992 51 53
DQA1*02:01, DQB1*03:02 2674 2780 2676 43 45
DQA1*01:04, DQB1*05:03 1113 1224 1178 36 38
DQA1*05:01, DQB1*03:01 2745 2891 2783 25 26
DQA1*04:01, DQB1*03:03 2229 2322 2235 24 25
Normalization Ratio 0.96 1.05
Background corrected MFI values for Donor 1 used to generate Figure 21A and
Figure 23. * BCMFI values was
normalized to the average DO BCMFI in the starting sera. t BCMFI values was
normalized to the DRB1*11:01
BCMFI in the starting sera.
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TABLE 2
Pre
Bead Antigens Sera Flow Through Eluate
BCMFI BCMFI Normalized* BCMFI MFI Normalizedt
MFI
DRB1*11:01 14320 1094 1386 10721 12934
DRB1*03:03 13703 1618 2050 10567 12748
DRB1*13:03 14101 1980 2509 10146 12241
DRB1*14:01 13267 973 1232 9667 11663
DRB1*13:01 13268 1233 1563 9622 11608
DRB1*03:01 11249 1285 1628 9232 11138
DRB1*08:01 12247 1130 1431 8150 9832
DRB3*03:01 12014 2434 3085 8065 9730
DRB3*02:02 11172 1216 1541 7399 8926
DRB1*12:01 9453 390 494 6599 7961
DRB3*01:01 9915 1073 1360 6078 7333
DRB1*07:01 11299 6741 8543 5247 6330
DRB1*09:01 12218 9504 12044 4370 5272
DRB1*15:01 5333 0 0 3092 3730
DRB1*16:01 3729 0 0 2530 3052
DRB1*15:02 3374 0 0 2076 2505
DRB1*01:01 1569 362 459 180 217
DQA1*02:01, DQB1*06:01 1391 787 997 130 156
D0A1*06:01, DQB1*04:02 4460 3376 4278 93 112
DQA1*05:01, DQB1*02:02 7845 6681 8467 90 108
DQA1*04:01, DQB1*04:02 6815 5123 6492 76 92
DQA1*04:01, DQB1*04:01 6534 4833 6125 71 86
DPA1*02:02, DPB1*01:01 1566 1049 1329 69 83
DQA1*04:01, DQB1*03:03 7082 5599.5 7096 67 81
DQA1*06:01, DQB1*03:03 7024 5252 6656 63 75
DQA1*05:01, DQB1*06:01 7463 5899.5 7476 59 71
DPA1*04:01, DPB1*13:01 2607 2319 2939 30 36
DQA1*05:01, DQB1*03:01 10486 8673 10991 28 34
DQA1*02:01, DQB1*03:02 2645 2499 3167 26 31
DPA1*02:01, DPB1*13:01 3463 3402 4311 21 25
Normalization Ratio 1.27 1.21
Background corrected MFI values for Donor 2 used to generate Figure 21B and
Figure 23. * BCMFI values was
normalized to the average DQ BCMFI in the starting sera. t BCMFI values was
normalized to the DRB1*11:01
BCMFI in the starting sera.
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[00153] Purified HLA-DR11 Antibodies Fix Complement. To evaluate the
functional traits
of antibodies for HLA-DR11, the complement fixing activity of the donor 1 and
donor 2
starting sera, flow through, and eluate were tested. HLA-DR11 positive cells
were incubated
with starting sera, flow through, or eluate in the presence of complement.
Complement
dependent cytolysis (CDC) was measured with florescent microscopy. In donor 1
serum, the
DR11 column depleted CDC activity to all 4 DR11 target cell types (Figure 21;
C433, C418,
C428, C423), and this DR11 specific CDC activity was recovered in the eluate.
Thus, anti-DR11
antibodies were necessary and sufficient for CDC activity in patient 1. The
donor 2 serum
showed heterogeneous reactivity to the different target cell lines in the
assay. On some cell
lines (C433, C418, C428), the removal of DR11 antibodies did not significantly
reduce the CDC
activity in the flow through, likely due to complement fixing antibodies
directed towards the
other HLA present on the target cells. Interestingly, CDC activity on cell
line C423 was
depleted in both the donor 2 flow through and eluate, indicating that anti-
DR11 antibodies
were necessary but not sufficient for CDC activity. These data demonstrate
that antibodies
to individual HLA can be isolated and functionally characterize, and that anti-
HLA CDC
activity can vary between individuals.
[00154] Quantity and Quality of Polyclonal HLA-DR11 Antibodies. The HLA
immunoaffinity column provided a unique opportunity to study patient-derived
populations
of DR11 reactive antibodies. Antibody function is largely dictated by Ig
constant region, or
antibody isotype. Therefore, the isotype of DR11 reactive antibodies was
characterized in
patient sera. Several different isotypes were observed in the starting sera,
the pooled flow
through, and the pooled eluate for both donors (Figure 22). IgG1 predominated
in both the
starting sera and in the flow through, with appreciable IgG2, IgA, IgG3, and
some IgM
present. The isotype profile of antibodies eluted from the DR11 column was
diverse in both
individuals, with 5 of the 7 Ig isotypes represented in the eluate. In the
donor 1 eluate, IgG2
was the most common isotype, with considerable levels of IgG1, IgM, and IgA.
In contrast,
IgG1 predominated in the donor 2 eluate, with appreciable IgG2 and detectable
IgA, IgM,
and IgG3. The antibodies in the donor 1 eluate were 56.1% IgG2, 22.3% IgG1 and
11.6% IgM,
whereas the donor 2 eluate contained 70.5% IgG1, 15.5% IgG2, and 3.3% IgM
(Figure 22).
Both eluate samples showed similar low levels of IgA and IgG3, with IgA
comprising 6.6% and
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48
6.3% and IgG3 comprising 3.3% and 4.2% of the eluate for donor 1 and donor 2,
respectively. This preliminary dataset suggests substantial heterogeneity can
exist in anti-
HLA antibody isotype.
[00155] The column depleted all detectable anti-HLA-DR activity from the
donor 1 serum,
allowing the total concentration of anti-HLA-DR antibodies in this patient to
be estimated.
The pooled eluate of donor 1 contained 17.7 i.teml of antibody. Assuming the
efficiency of
antibody recovery from serum was similar to that of mAb L243, and factoring
for volume
variation, the serum concentration of the anti-HLA-DR antibody in donor 1 was
approximately 23.7 penril, or 0.05% of the total lg. While this may not be
representative of
concentrations in other donor sera, it demonstrates that these immunoaffinity
columns
enable, for the first time, the direct quantification of anti-HLA antibodies
in patient sera.
[00156] Discussion of Example 3
[00157] Donor specific anti-HLA antibodies represent a pre-transplant
contraindication
and a post-transplant risk for graft loss. While it is clear that antibodies
to HLA mediate graft
failure and loss, studies also suggest that not all anti-HLA antibodies are
detrimental
(Wasowska, 2010; and Amico et al., 2009). These observations have sparked
great interest in
discerning what differentiates pathogenic anti-HLA antibodies from those that
are not a
threat to transplanted organs. To date, the tools available for studying
antibodies to HLA
have not been sufficient for characterizing or detecting antibodies that
warrant clinical
intervention. In this Example, HLA-DR11 immunoaffinity columns were used to
characterize
patterns of HLA-DR serologic cross-reactivity, to phenotype DR11 reactive
antibodies, and to
assess the function of antibodies in patient sera. This ability to isolate
anti-HLA antibodies is
positioned to augment both clinical and basic scientific endeavors by
unraveling the complex
nature of humoral responses to HLA.
[00158] Anti-HLA antibody responses are recognized as polyclonal and
heterogeneous. In
particular, allogeneic antibody responses to class ll HLA are highly cross-
reactive, with any
given serum reacting to multiple class ll HLA (El-Awar et at., 2007). Indeed,
antibodies
reactive to the HLA-DR11 column recognized a striking diversity of HLA-DR
specificities. The
HLA-DR11 column depleted 11 different HLA-DR specificities from the donor 1
serum while
15 HLA-DR specificities were removed from donor 2 (Figure 23). The HLA-DR11
reactive
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49
antibodies purified from donor 1 then reacted with HLA-DR103, 4, 8, 12, 13,
16, and 51 with
no reactivity to the remaining 26 DR complexes tested. The pattern of
serologic cross
reactivity observed for donor 1 was consistent with the recognition of a
solvent accessible
Asp residue present at position 70 in the beta chain of all recognized HLA-DR
complexes but
in none of the other HLA-DR complex except HLA-DR7 (El-Awar et al., 2007). The
serologic
reactivity pattern for antibodies recovered from donor 2 was more complex; the
anti- HLA-
DR11 antibodies cross reacted with every HLA-DR tested except for HLA-DR1,
103, 4, 10, 51,
and 53. Interestingly, antibodies directed towards HLA-DR7 and HLA-DR9 were
split into two
groups; those that bound HLA-DR11 and those that did not (Figure 23). This
demonstrates
the availability of two (or more) distinct epitopes in the HLA-DR7 and HLA-DR9
reactive
antibody pool, only one of which is shared with DR11. These data illustrate
the use of HLA
immunoaffinity columns to characterize the target epitopes and cross-
reactivity of anti-HLA
antibodies, and the variability of anti-HLA reactivity profiles from patient
to patient.
[00159] In addition to deciphering patterns of serologic recognition, HLA-
DR11 reactive
sera were analyzed for their isotype profile and ability to fix complement.
The
straightforward relationship between isotype profile and CDC activity in Donor
1 indicated
that complement-fixing anti-HLA-DR11 antibodies (i.e., IgG1 and IgM) were
responsible for
anti-HLA-DR11 CDC activity in the Donor 1 starting serum and that the HLA-DR11
column
removed complement fixing activity from the flow through by depleting HLA-DR11-
reactive
antibodies. The relationship between isotype profile and CDC activity was more
complex for
Donor 2. The Donor 2 eluate was dominated by non-complement-fixing IgG1, and
CDC
activity was lost in both the flow-through and eluate. This finding is
consistent with antibody
synergy, which has been previously described in complement fixation. Murine
models of
MHC class I mismatch during cardiac transplantation demonstrated that modest
amounts of
complement-fixing (IgG2a) antibodies to MHC fix complement much more
effectively when
combined with non-complement-fixing (IgG1) antibodies to MHC (Wasowska, 2010;
and
Murata et al. 2007). Thus, the CDC activity in the Donor 2 starting serum
could have resulted
from a combination of anti-HLA-DR11 IgG1 and complement-fixing antibodies
without
specificity for HLA-DR11, while the HLA-DR11 column eliminated HLA-DR11-
elicited CDC
activity from both the flow-through and eluate by separating these
syngergistic antibodies.
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These results show that HLA immunoaffinity columns absorb complement-fixing
activity in a
sera-specific manner.
[00160] A long-term objective in the development of an HLA immunoaffinity
matrix is
antibody absorption. Antibody reduction therapies such as plasma exchange are
now used
for bulk antibody depletion to facilitate transplants for recipients who are
otherwise
serologically incompatible. One drawback to these existing reduction therapies
is their lack
of specificity, which results in the removal of beneficial as well as
deleterious anti-HLA
antibodies (Schmaldienst et al., 2001). The ability to specifically deplete
anti-HLA antibodies
could significantly improve existing immune reduction therapies. Antigen-
specific antibody
depletion columns are currently in use to remove antibodies specific for blood
group A and B
antigens (Crew et al., 2010; and Takahashi, 2007). While the column and serum
volumes
tested here were on a small scale, this column could be scaled up, similar to
the columns for
blood group antigens, in order to reduce anti-HLA-antibodies from patient
plasma before or
after transplantation.
[00161] In summary, an approach for producing milligram quantities of
native class II HLA
proteins in mammalian cells has been developed, and in this Example, these
proteins have
been successfully coupled to a column support used to purify anti-HLA
antibodies. The DR11
reactive antibodies recovered were functionally intact and highly
crossreactive. Antibodies
that recognized DR11 fixed complement in one of the two donors, and isotype
profiles were
consistent with CDC activity. These observations demonstrate that HLA
immunoaffinity
columns, or perhaps other platforms such as HLA coated magnetic beads, will
provide
transplant physicians and their supporting clinical HLA laboratories with the
means to parse
anti-HLA reactivity into acceptable or unacceptable categories on the basis of
CDC activity,
isotype profile, and serologic cross-reactivity with other HLA. HLA
technologies like this
antibody separation device will help elucidate which antibodies promote
rejection. Lastly,
these results establish the feasibility of using HLA immunoaffinity columns to
study anti-HLA
immunity and to achieve specific immune reduction for organ transplantation.
EXAMPLE 4: SHARC (sHLA Antibody Removal Column) Analysis
[00162] Coupling CNBr (Cyanogen Bromide) vs NHS (N-Hydroxysuccinimide)
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[00163] There are three primary properties of a matrix that indicate the
effectiveness of
the SHARC. These properties are: (1) coupling efficiency ¨ the ability of an
activated matrix to
covalently link sHLA to the solid support; (2) binding capacity ¨ the maximum
quantity of
antibody depleted by the sHLA linked matrix; and (3) regeneration efficiency ¨
the number of
times the matrix can be loaded and eluted (regenerated).
[00164] sHLA can be covalently linked to a solid support such as SEPHAROSE
using a
number of different chemistries. In this Example, the aforementioned
parameters were
tested with either a CNBr- or NHS- SEPHAROSE based chemistry to link sHLA to
a
SEPHAROSE 4 fast flow matrix. Both CNBr and NHS chemistries were tested using
class I
and class ll sHLA. In the case of class I sHLA, the NHS-based chemistry
outperformed in both
coupling efficiency (Figure 24) and regeneration efficiency (Figure 25);
however, it exhibited
a lower binding capacity (Figure 25). For class II sHLA, coupling efficiencies
were similar
between NHS and CNBr (Figure 27), but the binding capacity was higher with the
CNBr
matrix (Figure 28); in addition, the regeneration efficiency was higher with
the NHS matrix.
[00165] Full scale class I and class ll HLA SHARC
[00166] In order to demonstrate that the full scale SHARC was able to
deplete anti-HLA
antibodies, the ability to deplete monoclonal anti-HLA antibodies from PBS was
first
investigated. In these experiments, sHLA-A2 was used as the class I molecule,
and sHLA-
DR11 was used as the class II molecule. The pan-class I antibody W6/32 was
used for
analysis of class I, while the pan-HLA-DR antibody L243 was used for class II.
As shown in
Figures 30 and 33, both the sHLA-A2 (class I) and sHLA-DR11 (class II) SHARC
devices
depleted anti-HLA antibodies from PBS, although the sHLA-DR11 SHARC was more
effective
than the HLA-A2 SHARC.
[00167] Next, the ability of the class I and II SHARC to deplete antibody
from patient
plasma containing anti-HLA antibodies was tested. When patient plasma
containing anti-
HLA-A2 antibodies was passed over the sHLA-A2 SHARC, anti-HLA-A2 antibodies
were
depleted (Figures 31 and 32). In addition to anti-HLA-A2 antibodies,
serologically related
antibodies (B57, B58) were reduced from the starting plasma. The presence of
serologically
unrelated anti-HLA antibodies (B61, B81, B18, B60) was unchanged between pre-
SHARC and
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52
post-SHARC plasma, demonstrating that these antibodies passed through the
SHARC
without binding thereto (Figure 31).
[00168] Like the sHLA-A2 SHARC, the sHLA-DR11 SHARC depleted anti-HLA-DR11
antibodies from patient plasma (Figures 34 and 35). As shown in Figure 34,
anti-HLA-DR11
antibodies as well as serologically related antibodies (DR13, DR4, DR17) were
reduced from
the starting plasma. Serologically unrelated anti-HLA antibodies (DQ7, DQ8,
D09) were
unchanged between pre-SHARC and post-SHARC plasma, demonstrating that these
antibodies passed through the SHARC without binding thereto. Together these
data
demonstrate the ability and specificity of both of the class I and II SHARC
devices.
EXAMPLE 5
[00169] In this Example, several specific HLA-A*0201 columns were generated
to
demonstrate the feasibility of removing defined anti-HLA antibodies (anti-HLA-
mAbs) from a
buffered solution. Soluble class I HLA molecules were produced in a native
conformation in
mammalian cells, purified by affinity chromatography, coupled to a SEPHAROSE
matrix, and
loaded into a column enclosure. The HLA on these columns were shown to
maintain their
structural integrity and function. Multiple passes of the antibody W6/32,
which recognizes
only intact HLA molecules, resulted in consistent and repeatable binding
patterns. During the
entire evaluation process, several parameters were identified determining
capacity and
efficiency. In conclusion, the anti-HLA antibody removal devices have been
demonstrated
herein to be highly efficient in selectively depleting a nti-HLA-mAbs.
[00170] Materials and Methods for Example 5
[00171] Recombinant techniques were used to create cell lines which express
single HLA
class I molecules (as described herein above). Eliminating the cytoplasmic and
transmembrane regions of the molecule resulted in a soluble form of HLA (sHLA)
which is
secreted during production and easily purified by affinity chromatography.
Large-scale
production of sHLA proteins was performed using the CP-2500 CELL PHARM
system. Hollow
fiber bioreactors are designed to produce up to 50 to 100 times more protein
than a
traditional static culture will yield. Affinity chromatography purification
was applied to purify
crude sHLA harvests, resulting in samples of >95% purity. All samples produced
were
53
individually controlled by a QC system. Mass spectroscopy demonstrated that
soluble HLA
proteins were purified so that contaminants are essentially undetectable.
[00172] After purification of sHLA, fractions of the protein stock were
used to couple to
NHS-activated SEPHAROSE 4 Fast Flow and packed into a chromatography column.
Elution
profiling was conducted using an Aktarm Purifier System by applying a specific
run protocol
consisting of a loading cycle, elution cycle, and equilibration cycle. All
parameters were kept
consistent throughout the study, assigning a volume of 12 ml of PBS, pH 7.4 to
the loading
cycle, 8 ml of 0.1 M Glycine pH 11.0 to the elution cycle and 25 ml of PBS, pH
7.4 to
equilibrate the system. Depending on the injection amount and volume,
different loading
loops were used. Data showed that injection conditions are concentration
independent (not
shown).
[00173] Figure 36 shows a typical coupling timeline for binding of the sHLA
to the
SEPHAROSE 4 Fast Flow matrix. A rapid decline of sHLA is visualized within
the first 10
minutes and faded out after 30 minutes, where no additional sHLA is bound to
the matrix.
For this Example, three columns of 0.5, 1.0 and 2.0 mg per ml matrix were
created with
coupling efficiencies above 95%.
[00174] To assure consistency in the measurements, a repeatability study
was started to
record and superimpose elution profiles. For quality purposes, three
parameters were
observed: (1) Absorption Units (mAU) to detect proteinacious material (Figure
37); (2) pH
(Figure 38); and (3) conductivity to follow changes in buffer phases (Figure
39). The graphics
prove great consistency between multiple experiments, validating the
suitability of the
method.
[00175] Using the anti-HLA-mAb W6/32, which recognizes only structurally
intact HLA
molecules, multiple rounds of load-elute-equilibrate cycles were performed to
measure the
stability of sHLA attached to the solid support (Figures 40-42). Overall it
was observed that
freshly coupled HLA-columns lose HLA molecules within the first 3 rounds of
glycine
exposure, but then stabilize with no further loss of functionality. This
effect is most likely
caused by incompletely coupled HLA proteins being trapped within the matrix
and knocked
loose after a drastic pH change. The effect seems to be more profound in
higher coupling
ratios. A similar study was performed manually (data not shown), measuring the
"shedding"
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54
of sHLA after an elution event with the result that no sHLA was detectable
after 5 elutions
(15 cycles).
[00176] Determination of the column's binding capacity is one of the most
important
parameters in establishing feasibility of the technology. The more antibody
that can be
removed, the less sHLA is needed, and smaller/cheaper devices can be created.
Figures 43-
45 show three different anti-HLA mAbs applied to a 2.0 mg column at variable
amounts. The
column's capacity was shown to not be unlimited, but was able to bind a
certain amount of
antibodies before saturation occurred. Anti-VLDL (Figure 45) appeared to be
able to bind the
largest amount of antibody before the column becomes saturated, while Anti-B2m
(Figure
44) bound the lowest amount of antibody before saturation. These differences
were
expected, as each antibody has a different affinity towards its target
epitope. Depending on
the anti-HLA mAb used, capacities ranged from 300 ¨1200 vg of antibody per 1
ml matrix.
[00177] The maximum binding efficiency for the A*02:01 appeared to be at
around 1 mg
of HLA per 1 ml of matrix. This was confirmed by 3 independent tests using
anti-HLA mAbs
W6/32 (Figure 46), anti-B2m (Figure 47) and anti-VLDL (an antibody directed
against an
artificial tail introduced into the A*02:01 molecule; Figure 48). Clear
evidence of sterical
hindrance was detectable, where the 1 mg column reached much higher binding
capacity
than its 2 mg counterpart.
[00178] This Example demonstrates that soluble HLA class I molecules
coupled to an
affinity matrix were capable of binding specific anti-HLA Abs. Elution
profiles become stable
and the column performed without a visual decrease in functionality. All
parameters
measured were highly acceptable to move forward in creating a large-scale
device.
[00179] A proposed application scenario using such a system is shown in
Figure 49. The
large amount of antibody required to be removed necessitates a two column
system where
one column is actively filtering plasma while the second is being regenerated.
[00180] Thus, in accordance with the presently disclosed and claimed
inventive
concept(s), there have been provided anti-MHC removal devices, as well as
methods of
production and use thereof, that fully satisfy the objectives and advantages
set forth
hereinabove. Although the presently disclosed and claimed inventive concept(s)
has been
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described in conjunction with the specific drawings, experimentation, results
and language
set forth hereinabove, it is evident that many alternatives, modifications,
and variations will
be apparent to those skilled in the art. Accordingly, it is intended to
embrace all such
alternatives, modifications and variations that fall within the spirit and
broad scope of the
invention.
56
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