Note: Descriptions are shown in the official language in which they were submitted.
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USE OF A SOLUBLE RECOMBINANT HUMAN CD40L PROTEIN FOR INHIBITING IN VIVO IMMUNE
RESPONSE
BACKGROUND OF THE INVENTION
(a) Field of the Invention
s The invention relates to a method for
inhibiting in vivo immune response and to the use of a
soluble recombinant human CD40L or a sequence within
said soluble recombinant human CD40L containing the
active binding site with CD40 for inhibiting an immune
response. The invention also relates to a mouse model
of human alloimmunization for testing in vivo effects
of an immunotherapy or inhibition of a human antibody
response.
(b) Description of Prior Art
Platelet alloimmunization occurs as a result of
exposure to "foreign" antigens present in pooled
random donor platelet concentrates. A consequence of
platelet alloimmunization is the development of a
state of refractoriness to subsequent random donor
platelet transfusion. Up to 50% of patients with acute
leukemia, almost 100% of those with aplastic anemia
and 10% of patients with solid tumors, develop
platelet alloantibodies. The alloantibodies are most
often directed against HLA Class I antigens, although
in 10-20% of cases they are directed against
platelet-specific antigens such as P1A, Bak, Pen.
Effective platelet support for such patients is
dependent upon provision of compatible platelets
selected by HLA matching and/or platelet
crossmatching, approaches which are expensive and, in
up to one-third of cases, ineffective. A multitude of
clinical and experimental studies have indicated that
alloimmunization depends upon (or is at least
augmented by) the presence of "contaminating" MHC
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class II bearing antigen presenting cells (APC) in the
transfused blood products.
Investigators have attempted to inactivate
donor APC by ultraviolet radiation, or have applied
s leukofiltration to remove the APC from the transfused
product. Most studies, including a large US-based
multi-centre study (TRAP, Trial to Reduce
Alloimmunization to Platelets) have indicated that the
frequency of patients that become alloimmunized is
decreased when leukofiltered products are used.
However, it is important to note that although these
studies reduced the incidence of alloimmunization by
approximately 50%, many patients still become
alloimmunized.
The major co-stimulatory molecule for B cells
is the CD40 molecule. This surface membrane protein
which is found on B cells as well as some other cells
interacts with a molecule on activated Th cells
designated as CD40 ligand (CD40L, gp39 or CD154)
CD40-CD40L interaction is critical to B cell
activation and differentiation. B cells stimulated
with anti-CD40 antibodies undergo transmembrane
signaling, cell enlargement, and LFA-1-dependent
aggregation. When B cells are stimulated via an
appropriate stimuli in combination with anti-CD40,
these B cells can proliferate or be induced to isotype
switch depending upon the first stimulus. Patients
with defective CD40L function have X-linked hyper-IgM
syndrome characterized in part by low levels of serum
IgG, IgA, and IgE. CD40 and CD40L deficient mice have
numerous immune defects including the inability to
class switch from IgM to IgG1 and the inability to
stimulate allogenic T cells in an in vitro mixed
lymphocyte reaction (MLR) . Injection of animals with
anti-CD40L antibody has been shown to inhibit both a
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primary and secondary antibody response, as well as
prevent the occurrence of anti-DNA antibodies and
disease pathology in lupus-prone mice. Further,
administration of a soluble form of CD40L to human B
cell hybridomas can induce apoptosis (U.S. Patent No.
5,540,926). While the CD40 molecule on the B cell
provides co-stimulation to that cell via interaction
with a Th cell expressing the CD40L, it is important
to note that the T cell also becomes activated by this
mutually synergistic interaction.
It would be highly desirable to be provided
with a method to inhibit in vivo alloimmunization.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide
a method for inhibiting human anti-HLA alloimmune
response.
In accordance with the present invention there
is provided a soluble recombinant human CD40L or a
functional fragment thereof containing the active
binding site of CD40 and capable of binding thereto,
for inhibiting an immune response. Preferably, the
soluble recombinant human CD40L has a sequence
comprised in amino acids 108 to 261 of the following
sequence:
Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly
1 5 10 15
Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu
20 25 30
Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg
40 45
35 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val
50 55 60
Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser
65 70 75 80
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Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys
85 90 95
Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu
100 105 110
Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser
115 120 125
Glu Ala Ser Ser Lvs Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
130 135 140
Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln
145 150 155 160
Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr
165 170 175
Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser
180 185 190
Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala
195 200 205
Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His
210 215 220
Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn
225 230 235 240
Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
245 250 255
Gly Leu Leu Lys Leu
260 SEQ ID NO:1
The immune response inhibited is preferably an
alloimmune response, and more preferably a human
anti-HLA alloimmune response.
In accordance with the present invention, there
is also provided a soluble recombinant human CD40L or
a functional fragment thereof containing the active
binding site of CD40 and capable of binding thereto,
as described above, for inhibiting T cell function.
Preferably, the soluble recombinant human CD40L or the
functional fragment thereof can be used for treating
or preventing T cell dependent or T cell mediated
diseases selected from the group consisting of
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autoimmune diseases, including systemic lupus
erythematosus (SLE), sjogren's syndrome, sleroderma
myositis, Raynaud's syndrome, type 1 diabetes,
arthritis and rheumatoid arthritis, inflammatory bowel
s disease, uveitis, myesthenia gravis, multiple
sclerosis, idiopathic thrombocytopenic -purpura and
graft vs host disease as well as allergies which are
dependent on T cells.
In accordance with the present invention, there
is also provided the use of a soluble recombinant
human CD40L or a functional fragment thereof
containing the active binding site of 1-D40 and capable
of binding thereto,for the preparation of a medicament
for immunotherapy or for treating or preventing a
is disease selected from the group consisting of SLE,
type 1 diabetes, multiple sclerosis, idiopathic
thrombocytopenic purpura and graft vs host disease.
Further in accordance with the present
invention, there is provided an immunodeficient mouse
model of human alloimmunization for testing in vivo
effects of an immunotherapy or inhibition of a human
antibody response, characterized in that the mouse
model is a severe combined immunodeficient (SCID)
mouse, reconstituted with human peripheral blood
lymphocytes (PBL) from donors. Preferably, the donors
are sensitized to HLA antigens.
Preferably, the SCID mouse is y-irradiated and
asialoGM1 treated for enhancing cellular engraftment.
Also in accordance with the present invention,
there is provided a method for inhibiting an immune
response in a patient, comprising the step of
administering a therapeutically effective amount of a
soluble recombinant human CD40L or a functional
fragment thereof containing the active binding site of
CD40 and capable of binding thereto.
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In accordance 'with the present invention, there
is also provided a method for 'inhibiting T cell
function in a patient, comprising the step of
administering a therapeutically effective amount. of a
S soluble recombinant human CD40L or a functional
fragment thereof containing the active binding site of
CD40 and capable of-binding thereto.
Preferably, the method for inhibiting T cell
function is used for treating or preventing T cell
dependent or T cell mediated diseases selected from
the group consisting autoimmune diseases, including
systemic lupus ervthematosus (SLE), sjogren's
syndrome, sleroderma myositis, Raynaud's syndrome,
type ? diabetes, arthritis and rheumatoid arthritis,
inflammatory bowel disease, uveitis, myesthenia
gravis, multiple sclerosis, idiopathic
thrombocytopenic purpura and graft vs host disease as
well as allergies which are dependent on T cells.
According to one aspect of the present invention,
there is provided use of a soluble recombinant human CD40L
or a functional fragment thereof containing the active
binding site of CD40 and capable of binding thereto, for
inhibiting an alloimmune response.
According to another aspect of the present
invention, there is provided use of a soluble recombinant
human CD40L or a functional fragment thereof containing the
active binding site of CD40 and capable of binding thereto,
for inhibiting T cell function in an alloimmune response.
According to still another aspect of the present
invention, there is provided use of a soluble recombinant
CD40L or functional fragment thereof containing the
active binding site of CD40 and capable of binding
thereto for treating or preventing a disease selected
from the group consisting of systemic lupus
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erythematosus (SLE), Sjogren's syndrome, sleroderma
myositis, Raynaud's syndrome, type 1 diabetes,
arthritis and rheumatoid arthritis, inflammatory bowel
disease, uveitis, myesthenia gravis, multiple
sclerosis, idiopathic thrombocytopenic purpura, graft
vs. host disease and allergies which are dependent on T
cells.
According to yet another aspect of the present
invention, there is provided use of a soluble recombinant
human CD40L or a functional fragment thereof containing the
active binding site of CD40 and capable of binding thereto,
for the preparation of a medicament for immunotherapy.
According to a further aspect of the present
invention, there is provided use of a soluble recombinant
human CD40L or a functional fragment thereof containing the
active binding site of CD40 and capable of binding thereto,
for the preparation of a medicament for treating or
preventing a disease selected from the group consisting of
systemic lupus erythematosus (SLE), Sjogren's syndrome,
sleroderma myositis, Raynaud's syndrome, type 1 diabetes,
arthritis and rheumatoid arthritis, inflammatory bowel
disease, uveitis, myesthenia gravis,. multiple sclerosis,
idiopathic thrombo-cytopenic purpura, graft vs. host disease
and allergies which are dependent on T cells.
According to yet a further aspect of the present
invention, there is provided use of a soluble recombinant
human CD40L or a functional fragment thereof containing
the active binding site of CD40 and capable of binding
thereto, for the manufacture of a medicament for
inhibiting an alloimmune response.
According to still a further aspect of the present
invention, there is provided use of a soluble recombinant
human CD40L or a functional fragment thereof containing the
active binding site of CD40 and capable of binding thereto,
for the manufacture of a medicament for inhibiting T cell
function in an alloimmune response.
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BRIEF DESCRIPTION OF THE DRAWINGS
20 Fia. 1A represents an histogram illustrating
the measurements of the total IQG from Hu-PBL-SCID
mice on day 18 post-engraftment by ELISA;
Fia. 1E represents a plot chart _llustrat~ng
the measurements of human allcantibody from
25 Hu-PBL-SCID mice by flow cvtometry;
Figs. 2A to 2F illustrate mean T_ SEM
concentrations of human IaG and IaM in SCID mice
reconstituted with PBL from a donor;
Figs. 3A to 3F illustrate flow cytome_ric
30 analysis of alloantibodv production in representative
SCID mice reconstituted with PBL from a donor "A" who
was previously sensitized to HILA-A2;
Figs. 4A to 4F 'illustrate flow cvtomet_ic
analysis of alloantibodv production in representative
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SCID mice reconstituted with PBL from a donor
previously sensitized to HLA-AlO;
Fig. 5 illustrates a cumulative comparison of
ailoantibody responses by flow cytometry;
Fig. 6 illustrates Tetanus toxoid specific
antibody production in HLA-A2 challenged 18 KDa-CD154
treated Hu-PBL-SCID mice; and
Fig. 7 illustrates mean + SEM stimulation index
of a 4 day 1 way mixed lymphocyte culture.
DETAILED DESCRIPTION OF THE INVENTION
The method of the present invention has been
evaluated using a Hu-PBL-SCID mouse model. This
immunodeficient mouse model, developed for the present
invention, is a valuable model for testing in vivo
effects of novel immunotherapies or inhibition of the
human antibody response.
In accordance with the method of the present
invention, it is proposed that anergy induction via
inhibition or inappropriate activation of the CD40-
CD40L co-stimulatory cascade, is effective in
inhibiting an in vivo immune response. it is believed
that the soluble recombinant CD40L active fragment
competes B cell- (or APC-) CD40 interaction with CD40L
on the Th cell which disallows the Th cell to be
activated to secrete cytokines (such as Th2 cytokines)
which thus reduce the transfusion-induced alloimmune
response.
To develop an in vivo experimental model of
human alloimmunization that would be amenable to
experimental manipulation, a model was developed, in
which mice with severe combined immunodeficiency
(SCID) are repopulated with human peripheral blood
lymphocytes (Hu-PBL-SCID) from healthy blood donors
and challenged with HLA-mismatched lymphocytes.
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An in vivo model of human alloimmunization was
evaluated using severe combined immunodeficient (SCID)
mice. SCID mice were irradiated (200 cGy), and
reconstituted with human peripheral blood lymphocytes
(PBL) from donors sensitized to HLA antigens by prior
pregnancy. The reconstituted SCID mice (Hu-PBL-SCID)
were then challenged with HLA-mismatched PBL.
Alloantibodies were evaluated by flow cytometry (42)
and a standard two stage microlymphocytotoxicity (LCT)
assay (40).
The Hu-PBL-SCID mice (N=22) that were
challenged with PBL expressing the HLA antigens to
which the donors had previously been sensitized, made
significantly increased levels of both IgM and IgG
is alloantibodies as compared to unchallenged mice.
Responses were measurable by 1 week post
reconstitution and challenge. Prior treatment of SCID
mice with anti-asialo-GM1, which depletes murine NK
cells and macrophages, further increased the
alloantibody response of challenged mice. The human
alloantibodies generated were specific for the
challenge HLA antigens as assessed by LCT.
Hu-PBL-SCID mice were divided into 4 groups.
Group 1 consists of mice reconstituted with PBL
from donor A (as described herein except that 10' PBL
were used to reconstitute the mice) . These animals
were bled twice weekly (18 KDa CD40L-untreated and
HLA-unchallenged negative control group).
Group 2 consists of mice similar to the ones of
Group 1, except that the mice were injected with 200
g of 18 KDa CD40L via the intraperitoneal route on
the day of reconstitution (18 KDa CD40L-treated and
HLA-unchallenged negative control group).
Group 3 consists of mice similar to the ones of
Group 1, except that the mice also received twice
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weekly challenges with HLA-mismatched lymphocytes
(18 KDa CD40L-untreated and HLA-challenged positive
control group).
Group 4 consists of mice similar to the ones of
Group 1, except that the mice were injected with 200
g of 18 KDa CD40L via the intraperitoneal route on
the day of reconstitution and also received twice
weekly challenges with HLA-mismatched lymphocytes (18
KDa CD40L-treated and HLA-challenged experimental
group).
All mice were examined for total human IgG
levels as a measure of the success of functional
cellular engraftment as well as to determine if the
recombinant soluble 18 KDa CD40L protein product
inhibits or affects B cell IgG production. Fig. 1A
shows that the total level of human IgG as measured
after 18 days of human PBL engraftment is not
statistically different in any of the groups. For the
purposes of this disclosure, this indicates that the
soluble CD40L therapeutic does not adversely affect
either cellular engraftment nor does it inhibit the
ability of human B cells to produce immunoglobulin. It
is therefore unlikely that the CD40L therapeutic
induces B cell apoptosis or non-specific B cell anergy
when administered in vivo. The numbers under each bar
indicate the group number corresponding to those of
Fig. 1B. Next, in Fig. 1B, the ability of each group
of mice to make alloantibody was examined. Groups 1
and 2 did not make significant levels of alloantibody
(i.e. no anti-HLA antibody), as expected. Group 3 made
enhanced levels of alloantibody, as expected. Group 4
which received a single dose of the soluble 18 Kda
CD40L therapeutic made significantly less alloantibody
than did group 3.
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Administration of a single 200 g inoculation
of soluble recombinant 18 KDa CD40L active component
was able to significantly decrease the human
alloimmune antibody response to challenge with
HLA-mismatched blood cells in a humanized SCID model
system. This work therefore shows that in vivo
administration of a single dose of soluble recombinant
CD40L active component can inhibit a specific antibody
response. Moreover, T cell proliferation was also
prevented in vitro in a mixed lymphocyte culture.
Therefore, T cell function was also inhibited by the
soluble recombinant 18 KDa CD40L active component.
PREPARATION OF A HU-PBL-SCID MOUSE MODEL
SCID Mice
C.B.17 SCID virgin female mice (6-8 weeks of
age) were obtained from the Hospital for Sick
Children, Toronto, Ontario and were housed under
gnotobiotic conditions in the St. Michael's Hospital
research vivarium. Blood from the tail vein (300 l)
was collected into untreated microvette tubes
(Sarstedt, Montreal, Quebec) and the serum was
separated after incubation for 2 h at 22 C. Serum
levels of endogenous murine IgG were determined by
ELISA and animals with a serum murine immunoglobulin
concentration exceeding 10 g/ml ("leaky" phenotype)
were excluded from the study. Commencing 1 week post
reconstitution, mice were bled twice weekly for 5
weeks, and weekly thereafter until day 70 post
reconstitution.
Reconstituting PBL Donors
Female blood donors with a history of prior
pregnancy were screened for evidence of circulating
HLA class I alloantibodies. With informed consent,
blood samples were obtained at the time of whole blood
or platelet apheresis donations and were tested using
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a standard NIH microlymphocytotoxicity test against a
panel of 30 HLA-typed lymphocytes (40). Donor "All was
blood group 0, HLA Al, A3, B7 and B37 positive and had
low levels of circulating anti-HLA-A2 and anti-HLA-B5
alloantibodies. Donor "B" was group A, HLA Al, A2, 37
and B8 positive and had low levels of circulating
anti-HLA-A10 and anti-B5, -B12 and -B17
alloantibodies.
PBLs from the donor used to reconstitute the
Hu-PBL-SCID mice were cultured in vitro (2x105/well)
with the same y-irradiated cells used to challenge the
mice (4x105/well), with or without 18KDa-CD154 for 72 h
in a final volume of 200 ml in RPMI-1640 containing
10% fetal calf serum (FCS), 100 U/mi penicillin G, 100
is mg/mi streptomycin sulfate, 0.25 mg/ml Amphotericin B
as fungizone (Gibco-BRL, Grand Island, NY), 100 mM
L-glutamine, and 5x10-5 M 2-mercaptoethanol (CRPMI), in
96-well flat bottomed tissue culture grade plates at
37 C. Plates were then pulsed with 1 mCi 3H-thymidine
for 24 h, wells were harvested onto filter paper and
incorporated radioactivity assessed by scintillation
counting. For in vitro IgG production, plates were
cultured with cells as above and were maintained for
18 days by replenishing CRPMI every 3 days. The
plates were centrifuged at 300xg for 5 min on day 18
and the supernatant fluid assessed for human IgG
levels by ELISA.
Reconstitution
All SCID mice were exposed to 200 cGy of
irradiation prior to reconstitution to enhance
cellular engraftment. To deplete NK cells, some SCID
mice were injected with 20 l of anti-asialoGM1
antisera (Wako Pure Chemical Industries LTD, Dallas,
TX) I day prior to reconstitution. One unit of whole
blood was collected into standard collection bags
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containing CP2D (Citrate Phosphate Double Dextrose);
following centrifugation at 4550 x g for 3.2 min and
removal of the supernatant plasma, buffy coats were
transferred into a satellite bag. To obtain human PBL,
the buffy coat was layered onto a 1.077 g/L
Percoll(Pharmacia LKB, Baie d'Urfe, Quebec) gradient
and separated by centrifugation (1200 x g for 30 min
at 22 C). The PBL were washed three times with
phosphate-buffered saline, pH 7.4 (PBS), adjusted to a
concentration of 8x10' /ml in 80% FCS in RPMI-1640, and
0.5 ml injected into the peritoneal cavity of
recipient SCID mice using a 27 gauge needle.
SCID Mice Challenge
Challenge leukocytes were obtained from
heparinized blood and isolated by Percoll density
centrifugation as described above. Mice reconstituted
with donor "A" cells were challenged with human PBL
from HLA-A2 antigen positive blood donors and those
reconstituted with donor "B" cells were challenged
with human PBL from HLA-A10 antigen positive blood
donors. A large number of different donors were used
for each challenge; all expressed the pertinent
challenge antigen, but expressed a variety of other
antigens as well. In a separate experiment,
Hu-PBL-SCID mice reconstituted with cells from donor
"A" were challenged with cells from 4 individuals
expressing only HLA A2, and B5 as antigens foreign to
donor "A".
All challenge cells were y-irradiated with 2500
cGy prior to administration to prevent engraftment in
recipient mice. The first challenge consisted of 2x10
PBL/mouse and subsequent immunizations were with 10'
PBL/mouse. Immunizations with pooled y-irradiated PBL
(in 0.5 ml of 80% fetal calf serum in RPMI-1640) were
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performed twice weekly for 3 weeks starting on the day
of reconstitution.
Detection of Mouse or Human Immunoglobulin
ELISA plates were coated with 1.25 g/ml of
either goat antimouse or antihuman IgG+IgM (50
l/well;Caltag-Cedarlane Laboratories, Hornby, Ont.)
in 50 mM carbonate /bicarbonate buffer, pH 9.6, for 18
h at 4 C. The plates were then washed three times with
washing buffer (0.05% Tween 20/PBS), blocked with 0.2%
Tween 20/PBS (200 41/well) for 2 h at 37 C, and again
washed three times with washing buffer. Sera from the
mice were serially diluted in PBS, added to the plates
(25 l/well), and incubated for 2 h at 22 C. Serially
diluted normal mouse or human serum and purified mouse
or human IgG were used as controls and standards. The
plates were washed three times in washing buffer and
41 of alkaline phosphatase-conjugated F(ab')2 goat
antimouse or antihuman IgG or Igm (Cedarlane
Laboratories) was added. After incubation at 22 C for
20 2 h, the plates were washed four times and 100 41 of
substrate solution (5 mM p-Nitrophenyl phosphate;
BioRad Laboratories, Mississauga, Ontario) was added
and absorbance was measured at 405nm. The
concentration of IgG and IgM was calculated based upon
25 a standard curve.
Alloantibody Detection
Alloantibodies were detected by flow cytometry
as previously described (42) or by a microlympho-
cytotoxicity test (LCT) (40) using HLA typed target
cells. For flow cytometric analysis, SCID sera were
diluted 1:10 and incubated with 2x105 fresh HLA typed
antigen positive lymphocytes in a volume of 20 41 for
1 h at 22 C. The cells were then washed twice and
incubated at 22 C for 1 h in 100 l each, of 1 g/ml
of affinity-purified fluorescein isothiocyanate
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(FITC) -labeled F(ab')2 antihuman IgG, Fc -specific
antibody and 0.5 g/ml of affinity-purified
phycoerythrin (PE)-labeled F(ab')2 antihuman IgM, g
specific antibody (Tago; Biosource, Camarillo, CA).
s The cells were then washed twice and fixed in 1%
paraformaldehyde in PBS. Cells were analyzed by flow
cytometry as described previously (42). Background
staining was assessed by comparison with a serum
obtained from each animal prior to any manipulation.
Antibody specificity for HLA antigens was confirmed
using neat sera in the standard two-stage complement
-dependent microlymphocytotoxicity assay using a typed
panel of lymphocytes from donors (40); a positive
result was defined as > 20% lysis of target cells,
is unless otherwise stated.
IgG Depletion
Sera from NK-depleted challenged Hu-PBL-SCID
mice were pooled and depleted of IgG by affinity
chromatography. A saturating quantity of purified goat
anti-human IgG, Fc-specific antibody (Atlantic
Antibodies, Scarborough ME) was coupled to CNBr-
activated senharoseT" 4B media according to the
manufacturers directions (Pharmacia Biotech, Baie
d'Urfe, PQ). The beads were blocked with excess
glycine and extensively washed. Fifty (50) l of mouse
sera was added to 100 l of packed anti-IgG coated
beads with constant mixing for 1 hour at 25 C followed
by removal of the supernatant fluid. This IgG-
depletion was verified to reduce the IgG content of
the pooled sera from 3.2 mg/ml IgG to 20 g/ml by
ELISA. This IgG-depleted sera (referred to as "pooled
IgM" in Table 1 below) was then added to another 100
41 of packed fresh anti-IgG coated beads with constant
mixing for 1 hour and used in the LCT.
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Ten (10) SCID mice were reconstituted by ip
injection with 4x10' PBL and were either not further
manipulated or received twice weekly ip challenge with
HLA-mismatched ^I-irradiated lymphocytes for 3 weeks
commencing on the day of reconstitution. Challenge
lymphocytes were derived from between 8-12 different
donors for each challenge. These mice are hereafter
referred to as "challenged mice". Both challenged and
unchallenged mice made human IgG and IgM
immunoglobulin (Figs. 2A and 2B), indicating that the
mice were successfully engrafted with human cells.
Human IgG levels in unchallenged and in challenged
mice reached plateau levels by 14 days post
reconstitution and showed little variation until
approximately 50 days post reconstitution (Figs. 2A
and 2C). Human IgM levels in unchallenged and
challenged mice were similar until day 32 post
reconstitution (Figs. 2B and 2D).
Total serum human IgG (Figs. 2A, 2C and 2E) and
IgM (Figs. 2B, 2D and 2F) were quantitated by ELISA.
SCID mice were either reconstituted and not challenged
(Figs. 2A and 23, n=4 mice), reconstituted and
challenged with HLA-A2 antigen positive lymphocytes
(Figs. 2C and 2D, n=7 mice) or pretreated with
anti-asialoGM1, reconstituted and challenged with
HLA-A2 antigen positive lymphocytes (Figs. 2E and 2F,
n=14 mice).
SCID mice reconstituted with lymphocytes from
donor "A" who had anti-HLA-A2 and -B5 alloantibody
were either left unchallenged or challenged with
HLA-A2 antigen positive cells. Sera from these mice
were tested for allo-reactive IgG and IgM antibody by
flow cytometry at each bleed.
Figs. 3A to 3F show reactivity of sera from a
typical mouse from each experimental group against
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HLA-A2 target lymphocytes. The histograms represent
consecutive bleeds over the time period shown by the
number of weeks post reconstitution on the right Y-
axis. Shorter periods shown indicate that the mice
died prior to the next bleed date. Hu-PBL-SCID mice
that remained unchallenged made a low level of IgG
allo-reactive antibody measurable by day 7 post-
reconstitution that did not further increase with time
over the study period (Fig. 3A). The unchallenged
Hu-PBL-SCID mice did not have allo-reactive IgM
antibody detectable by flow cytometry (Fig. 3B). In
contrast, as shown by the shift to the right of the
histograms, representing increased alloantibody
binding, challenged mice made increasing levels of
i5 both allo-reactive IgG (Fig. 2, panel C) as well as
IgM (Fig. 3D) . Both classes of allo-reactive antibody
increased until week 3 (Figs. 3C and 3D) , after which
time the challenge protocol was stopped.
The front histogram in all Figs. 3A to 3F
represents reactivity of serum taken from these mice
prior to reconstitution (prebleed), and each
successive histogram peak is reactivity of serum taken
at the indicated time (weeks post reconstitution) as
indicated on the right y-axis. Sera (1:10 dilution)
were incubated with HLA-A2 antigen positive
lymphocytes followed by antihuman IgG (Figs. 3A, 3C
and 3E) or antihuman IgM (Figs. 3B, 3D and 3F)
fluorochrome-labelled secondary antibody. A shift of
the histogram to the right represents increased
alloantibody binding. Figs. 3A and 3B show findings in
a SCID mouse reconstituted but not challenged, Figs.
3C and 3D in a SCID mouse reconstituted and challenged
with HLA-A2 antigen positive lymphocytes, and Figs. 3E
and 3F in a SCID mouse pretreated with anti-asialoGM,
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to deplete NK cells, reconstituted and challenged with
HLA-A2 antigen positive lymphocytes.
Previous reports have suggested that Nk cells
are present in SCID mice. To determine if NK-depleted
mice could undergo a better alloantibody response, 14
mice were treated with anti-asialoGM, (depletes NK
cells) one day prior to reconstitution and challenge.
NK-depleted challenged mice made significantly more
total human IgG and IgM, indicating better overall
to engraftment (Figs. 2E and 2F). These NK-depleted
challenged mice also produced higher igG and IgM class
alloantibody reactivity (Figs. 3E and 3F). in
addition, these mice attained high steady-state levels
of IgG and IgM more rapidly than non-NK-depleted
is challenged mice (compare Figs. 3E and 3F to 3C and
3D).
Subsequent evaluation of reducing the numbers
of reconstituting PBL of donor "A" in an otherwise
identical independent experiment, showed that
20 virtually identical results were obtained with 107
reconstituting cells (i.e. 4 times less reconstituting
cells gave rise to a significant and specific
alloantibody response to challenge and the magnitude
of alloantibody produced was again significantly
25 increased by prior NK cell depletion).
The specificities of the alloantibodies
produced in the anti-asialoGM, -treated challenged mice
were determined by LCT and the results are shown in
Table 1 (columns labelled mouse 1, mouse 2, and mouse
30 3). The LCT demonstrated complement-fixing
alloantibodies to HLA-A2, as well as to the A9, and
A28 antigens which share and define the 2C public
epitope. Alloantibodies to HLA-B5 were also detected
as well as to the B17 and B21 antigens which are known
35 to crossreact strongly with B5. The only other
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consistent alloantibodies detected were against A10
and B8 which were not detectable in the donors sera
nor crossreact with A2 or B5.
To determine the specificity of the IgM
alloantibody, the sera from all 3 NK-depleted-
challenged mice were pooled and depleted of IgG by two
rounds of IgG-specific affinity chromatography. Table
1 shows that the "pooled IgM" class alloantibody
reactive with all pertinent HLA-A antigens were
io observed but B5 was the only HLA-B antigen that
reacted with the IgG-depleted sera.
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Table 1
Summary of anti-HLA specificity by lymphocytotoxicity
testing against a 30 cell panel of sera from NK-
depleted challenged mice reconstituted with PBL from
donor A*
pan-HLA challenged HLA-A2 and B5 challenged
HLA Mouse Mouse Mouse Pooled Mouse Mouse Mouse Mouse
antigen 1 2 3 I gm 4 5 6 7
Al -? - - - - - - -
A2 + + + + + + + +
A3 - - - - - - - -
A9 + + + + + + - +
A10 + + + w - - - -
Al1 ? + ? - - -
A28 + + + + + + + +
A30 - - - - - - -
A31 I - - - - - - - -
A34 - - - - - - -
B5 + + + + + + + +
B7 - - - - - - - -
B8 ? + + - - - - -
B13 - - - - - - -
B14 - - - - - - -
B16 - - - - - - - -
B17 + + + -
B21 + + + - I
B22 - ? -
B27 - - - - - - -
B37 ! - - - - - - - I
B40 - - -
B42 - - -
B44 - - - - - -
B75 - - - - - - - -
* The HLA type of donor "A" was HLA-Al, A3, B7, B37; serum from this donor was
also
assessed by LCT at the time of engraftment and alloantibody reactive with the
A2 and B5
antigens only could be detected.
1 A negative (-) sign denotes no reactivity with that HLA antigen; a positive
(+) sign
denotes that sera from that mouse reacted with panel cells expressing the
corresponding
HLA antigen; ? indicates that reactivity with that HLA antigen could not be
verified, w
designates a weak response.
Hu-PBL-SCID mice were repopulated with donor
"A" lymphocytes in a separate experiment and were
challenged with cells expressing HLA-A2 and B5 as the
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only HLA antigens foreign to donor "A". These
challenged mice made alloantibody against HLA typed
panel cells expressing A2, A9, A28, and B5 but not
against those expressing A10 or B8 antigens (Table 1,
see columns labelled mouse 4 through mouse 7).
PBL from a blood donor with low levels of serum
alloantibody to HLA-AlO, BE, B12 and B17 (donor "B")
were also used to reconstitute mice. Mice were divided
into unchallenged, HLA-Al0 challenged, and NK-depleted
and HLA-A10 challenged using essentially the same
protocol as described for donor "A". Sera (1:10
dilution) from unchallenged, challenged, and NK-
depleted challenged Hu-PBL-SCID mice were tested for
allo-reactive antibody by flow cytometry against HLA-
A10 antigen positive target lymphocytes. Unchallenged
donor "B" mice made marginal levels of human IgG and
no IgM class alloantibody as assessed by reactivity
with HLA-AlO target cells (Figs. 4A and 4B). However,
antigenic challenge of these mice did provoke an allo-
response involving IgG but not IgM class antibody
(Figs. 4C and 4D) . NK-depleted challenged mice again
made a stronger alloantibody response (Figs. 4E and
4F) than non-NK-depleted challenged mice and this
"optimization" of the SCID milieu permitted measurable
IgM class alloantibody. As with mice reconstituted
with PBL from donor "A", the time to alloantibody
detection in donor "B" was more rapid in the NK-
depleted challenged mice (compare Figs. 4C and 4D to
Figs. 4E and 4F). The sera from these mice were
subjected to analysis by LCT testing. Alloantibodies
were not detected in unchallenged mice but
alloantibodies reactive with HLA-Al0 were observed in
all challenged mice and all anti-asialoGM1-treated-
challenged mice.
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In Figs. 4A to 4F, sera (1:10 dilution) in this
case were incubated with HLA-AlO antigen positive
lymphocytes followed by antihuman IgG (Figs. 4A, 4C
and 4E) or antihuman IgM (Figs. 4B, 4D and 4F) . Figs.
4A and 4B show findings in a SCID mouse reconstituted
but not challenged, Figs. 4C and 4D in a SCID mouse
reconstituted and challenged with HLA-AlO antigen
positive lymphocytes, and Figs. 4E and 4F in a SCID
mouse pretreated with anti-asialoGM, to deplete NK
io cells, reconstituted and challenged with HLA-AlO
antigen positive lymphocytes. The mice represented in
Figs. 4A/4B and 4E/4F died after 2% and 334 weeks
postreconstitution, respectively.
The cumulative flow cytometric data for all
donor "A" and "B" reconstituted SCID mice over 5
experiments against antigen positive cells is shown in
Fig. 5. The data from the challenged group is the mean
reactivity of sera from 22 mice and the unchallenged
group is the mean of 13 mice. All NK-depleted
challenged mice (22/22) made alloantibody that reacted
with antigen positive challenge cells.
Hu-PBL-SCID mice were unchallenged (p, n=13) or
challenged with leukocytes (^, n=22) and assessed for
IgG class alloantibody reactivity using typed antigen
positive cells. The data on the Y-axis is reported as
the mean log fluorescence intensity ( SEM) for all
mice.
An in vivo model of the secondary immune
response was established in a Hu-PBL-SCID mouse model
system using lymphocytes from previously sensitized
individuals. Intraperitoneai inoculation of SCID mice
with these previously sensitized human PBL resulted in
engraftment of the mice and challenge with HLA-
mismatched lymphocytes resulted in specific allo-
antibody formation in all mice. This model was used on
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12 donors who were not sensitized to HLA antigens. It
was found that HLA-specific antibody was never
observed. Therefore this model can also be used for
defining persons functionally immunized to an antigen
as will be described later.
It is known that SCID mice do not contain
functional B cells or T cells. The mice do however
possess essentially normal NK cells which can inhibit
human lymphocyte engraftment and antibody production,
likely by destroying the engrafting cells. It has been
shown in the present application that pretreatment
with anti-asialoGM, to deplete NK cells resulted in
greater IgG and IgM production and in specific
alloantibody production over that seen in
non-NK-depleted challenged mice. Thus, pretreatment of
SCID mice with anti-asialoGM, did allow maximal
alloantibody production.
The lymphocytes used for antigenic challenge of
mice reconstituted with PBL from donor "A" were from
individuals that were all HLA-A2 antigen positive.
Each challenge consisted of lymphocytes derived from
between 8-12 different individuals and a different
series of individuals was used for each challenge;
this was necessary to obtain sufficient PBL for
immunization of all mice with the same PBL. Thus,
these mice were exposed to lymphocytes from a large
number of different donors possessing a wide spectrum
of other HLA Class I and Class II antigens in addition
to HLA-A2. The response of the mice consisted of
alloantibodies to several HLA antigens in addition to
A2. The phenomenon of "responders" is well recognized
i.e. individuals who make antibody to one challenge
are likely to make antibodies to challenge with new
immunogenic antigens. These other antibodies could
represent primary responses to HLA antigens or
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secondary responses of previously undetected
antibodies, or alloantibodies that are cross reactive
with either A2 or 35 which preexisted in donor "A".
The anti-HLA-A alloantibodies produced included
anti-A2, -A9, -AlO, -All and -A28. Since the A9 and
A28 antigens are well known to cross react with A2,
reactivity with these HLA-A alloantigens is not
unexpected. It is however more difficult to account
for anti-A10 and All reactivity. The anti-AlO and -All
io represent either primary responses, or a secondary
response to a putative paternal HLA antigen where the
antibody was not initially detectable in the serum of
donor "A". Since the paternal HLA typing is unknown,
it was not possible to differentiate between these
possibilities, however, IgM alloantibody was not
produced to the A10 and All antigens and therefore the
alloantibodies reacting against A10 and All are likely
an amnestic response. in the case of responses to HLA-
B antigens; since donor "A" had pre-existing anti-B5,
alloantibody reactive with this antigen is expected.
IgM alloantibody reactive with HLA-B antigens could
only be detected to B5. Donor A's HLA type was HLA Al,
A3, 37 and B37 and none of the mice generated
antibodies that reacted with these "self antigens"
despite the strong likelihood that the pooled
challenge PBL would express these antigens (Al found
in 26% of the Caucasian population, A3 in 25%, and B7
in 22%). This indicates that the mice maintained
specificity for foreign antigens without generating
auto-reactive antibody. In addition, the fact that
challenge with A2- and B5-only expressing cells did
not induce formation of A10 and B8 indicates that a
generalized immune stimulation was not simply induced
but that the allospecificity was maintained for the
stimulating cells.
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Mice reconstituted with PBL from either donor
"A" or "B" made IgM as well as IgG alloantibodies.
This is of interest because although IgG is well known
to be produced in a secondary response from activation
of memory B cell clones, IgM production is not
generally considered to be produced by memory B cell
clones. Antibodies of the secondary response have
however been observed without class switching and
experiments with adoptive primary and secondary
to responses have shown that memory B cells producing IgM
can be observed. An alternative explanation may be
that these previously sensitized donors possess memory
T helper cells to HLA antigens that are able to
efficiently activate "naive B cells" to secrete IgM.
Coupling of secondary T cell carrier epitopes to
primary haptens has been reported to generate primary
immunization in Hu-PBL-SCID mice. The T cells most
often found in Hu-PBL-SCID mice show the CD45RO memory
phenotype and it has been reported that transfer of
CD45RO, but not CD45RA (naive phenotype), T helper cells
could induce purified human B cells in SCID mice to
produce immunoglobulin. It is therefore possible that
primary IgM-secreting B cells were activated via the
memory T cell pool present in the two donors employed
for reconstitution of the SCID mice.
To determine if secondary IgG production from
memory B cells was a non-specific inhibition by 18
KDa-CD154, the IgG anti-tetanus antibody levels from
treated and untreated Hu-PBL-SCID mice were examined.
Fig. 6 shows that while stimulation of Hu-PBL-SCID
mice with HLA-mismatched lymphocytes caused a slight
increase in production of anti-tetanus starting at day
10, the administration of 18 KDa-CD154 did not
significantly interfere with the anti-tetanus IgG
levels in these four groups of mice (p>.05). In all
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cases, the reactivity of anti-tetanus IgG in the mice
remained well below the levels of anti-tetanus from
the donor's serum taken at the time of reconstitution
(Fig. 6, dotted line) . Hu-PBL-SLID mice as in Fig. 1B
were assessed for anti-tetanus toxoid specific
antibody. The data on the Y-axis is reported as the
mean absorbance ( SEM, n=4 mice per group).
To determine if administration of 18KDa-CD154
was also able to directly affect cell proliferation,
responder cells (from the individual providing the PBL
for reconstitution) and stimulator cells (challenge
cells) in the presence or absence of 18KDa-CD154 were
set up as mixed lymphocyte cultures. Addition of
y-irradiated stimulator cells to these cultures induced
a 4-fold increase in cell proliferation (Fig. 7).
Addition of 18KDa-CD154 to the stimulator + responder
mixed lymphocyte cultures prevented the increase in
cell proliferation as compared to the absence of
18KDa-CD154 (Fig. 7). The stimulation index was
calculated by dividing the cpm of cultured responder
cells (R) alone by the cpm of responder cells in the
presence of A; unstimulated lymphocytes (R), B;
lymphocytes reacted with 18 KDa-CD154 (R + CD154), C:
lymphocytes stimulated with y-irradiated stimulator
lymphocytes (R + S), D; lymphocytes stimulated with y-
irradiated stimulator lymphocytes and 18 KDa-CD154
(R + S + CD154).
Accordingly, 18 KDa-CD154 could inhibit
alloantibody production by inhibiting T cell
activation or T. cell function. To test this
allegation, mixed lymphocyte cultures were performed
and T cell proliferation was shown to be reduced in
the presence of 18 KDa-CD154. Since n cells do not
play a major role in the mixed lymphocyte culture, T
cell activation and proliferation could be directly
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inhibited by 18 KDa-CD154 treatment. The alloimmune
response to blood transfusion has been suggested to be
a Th2-dominant immune response. Since CD154 is linked
to T cell activation along the Th2 cytokine pathway and
T cell proliferation can be defective when IL-4 is
absent, the 18KDa-CD154 fragment have decreased T cell
proliferation due to decreased Th2 cytokine production.
It is thus demonstrated in the present
application that lymphocytes from individuals
previously sensitized to HLA antigens can reconstitute
SCID mice and can generate reproducible IgG and IgM
allo-immune responses following repeated challenge
with selected "foreign" HLA antigens. The development
of this model will allow detailed study of the
i5 mechanisms of alloimmunization and should facilitate
the in vivo assessment of new strategies for the
modulation of human alloimmunization to blood cell
antigens.
It is also demonstrated in the present
application that administration of a recombinant 18
KDa-CD154 molecule can inhibit an alloimmune response.
This 18 KDa-CD154 molecule may have good therapeutic
potential to inhibit human transfusion-induced
alloimmunization.
The present invention will be more readily un-
derstood by referring to the following examples, which
are given to illustrate the invention rather than to
limit its scope.
EXAMPLE I
Immunotherapy drug screening design for alloantibody
SCID mice (or other immunodeficient mice such
as NOD-SCID, NUDE, etc...) as prepared in accordance
with the present invention are engrafted with human
PBL from pregnancy sensitized blood donors and
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challenged with HLA-mismatched lymphocytes in the
presence or absence of any drug or therapeutic (i.e.
such as an immunotherapeutic to include IVIG,
anti-idiotype antibodies, CTLA4Ig, anti-CD40 ligand
antibody, anti-CD40 antibody, anti-CD-4, anti-IL-2
receptor, anti-CD-28, anti-CD-80, anti-CD-86, anti-CD-
11A, cyclosporine A, FK506, biologically active
peptides such as altered peptide ligands, etc.). The
HLA-specific human IgG and IgM were measured as
described above.
EXAMPLE II
Evaluation of functional immunization
of an individual with an antigen
Immunodificient mice such as SCID mice or NOD-
SCID mice as prepared in accordance with the present
invention are engrafted with PBL from an individual
who has been vaccinated (to polio, tetanus, hepatitis
B surface antigen, HIV, cancer cells or cancer
antigens, etc.) or not. The Hu-PBL-SCID mice are
challenged with the same antigen. The antigen-specific
human IgG and IgM produced in the mice are measured.
The persons exposed to a functional or protective
vaccine make ancigen-specific or neutralizing IgG and
IgM.
EXAMPLE III
Immunotherapy drug screening design for alloantibody
Immunodificient mice such as SCID mice or NOD-
SCID mice as prepared in accordance with the present
invention are engrafted with PBL from an individual
who has been vaccinated as described in Example II
above. The Hu-PBL-SCID mice are challenged with the
same antigen in the presence or absence of any drug or
therapeutic, such as described in Example I above. The
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antigen-specific human IgG and IgM are measured as
described above.
While the invention has been described in con-
nection with specific embodiments thereof, it will be
understood that it is capable of further modifications
and this application is intended to cover any varia-
tions, uses, or adaptations of the invention follow-
ing, in general, the principles of the invention and
including such departures from the present disclosure
io as come within known or customary practice within the
art to which the invention pertains and as may be
applied to the essential features hereinbefore set
forth, and as follows in the scope of the appended
claims.
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SEQUENCE LISTING
<110> Canadian Blood Services
LAZARUS, Alan H.
CROW, Andrew R.
FREEDMAN, John
<120> Method for inhibiting in vivo immune
response
<130> 13453-2"CA" FC/CC
<150> ca 2,223,225
<151> 1997-11-28
<160> 1
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 261
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<213> unknown
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