NZ742216B2 - Induction of immune tolerance by using methotrexate - Google Patents
Induction of immune tolerance by using methotrexate Download PDFInfo
- Publication number
- NZ742216B2 NZ742216B2 NZ742216A NZ74221612A NZ742216B2 NZ 742216 B2 NZ742216 B2 NZ 742216B2 NZ 742216 A NZ742216 A NZ 742216A NZ 74221612 A NZ74221612 A NZ 74221612A NZ 742216 B2 NZ742216 B2 NZ 742216B2
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- NZ
- New Zealand
- Prior art keywords
- methotrexate
- cells
- treatment
- alemtuzumab
- matg
- Prior art date
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- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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Abstract
Disclosed is the use of methotrexate in the preparation of a medicament for treating a subject in need of protein replacement therapy, wherein methotrexate reduces undesired immune responses, such as anti- drug antibody (ADA) responses and other T- and/or B-cell-mediated immune responses.
Description
INDUCTION OF IMMUNE TOLERANCE USING METHOTREXATE
CROSS REFERENCE TO OTHER ATIONS
This application claims priority from US. Provisional Application No.
61/486,697, filed May 16, 2011, the disclosure of which is incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
This invention relates generally to immunology, and more specifically to the use
of methotrexate to reduce undesired immune responses in patients.
BACKGROUND OF THE INVENTION
[0003] Currently, the US. Food and Drug Administration (FDA) has ed more
than 130 protein therapeutics for clinical use (Leader et al., Nat Rev Drug Discov,
7(l):2l-39 (2008)). These therapeutics include peptides, inant human proteins,
protein vaccines, polyclonal dy preparations derived from a variety of animal
species, and monoclonal antibodies. Depending upon their sequence and mational
homology to endogenous self-antigens, glycosylation status, dose level, route of
administration, localization, and manufacturing process-related characteristics, the
therapeutic proteins may elicit antibody responses in the patient (Schellekens, Nat Rev
Drug Discov, l(6):457-62 (2002); Zinkemagel, Semin Immunol, 12(3): 163-71 ,
discussion 4; Thorland et al., Haemophilia, 5(2): 101-5 (1999); Goodeve, Blood
Coagul Fibrinolysis, 14 Suppl l:Sl7-2l ). These responses, referred to as anti-
drug antibody (ADA) responses, can impact patient safety and drug efficacy sometimes.
In the case of the lysosomal storage disorder, Pompe e, ADA can develop
in the enzyme-replacement therapy (ERT) against recombinant human acid alpha-
glucosidase. In patients who do not express measurable amounts of the endogenous
enzyme, sustained levels of high antibody titer correlate with patient decline (CRIM-
Pompe patients) (Kishnani et al., Mol Genet Metab., 99(1):26-33 (2010); Hunley et al.,
Pediatrics, 114(4):e532-5 (2004); tano et al., Genet Med, 3(2): 132-8 (2001)).
Similar compromise in drug safety and efficacy has been observed in hemophilia patients
who develop ADA to factor IX (Thorland et al., Haemophilia, 5(2):101-5 (1999);
Ewenstein et al., Blood, 89(3): 1 1 15-6 (1997)). In rare instances, ADA can also induce
autoimmune disease as in the case of recombinant human erythropoietin (Schellekens,
Clin Ther, 24(11):1720-40 (2002), discussion 1719; Locatelli et al., Perit Dial Int, 27
Suppl 2:S303-7 (2007)).
ADA responses can also occur with antibody therapeutics less of r
the eutics are non-human derived, humanized or even fillly human. The
genicity of both monoclonal and onal antibody therapeutics can influence
patient safety and drug efficacy. Antibodies that develop t therapeutic monoclonal
antibodies such as infliximab, adalimumab, rituximab and natalizumab have been
associated with decreased serum levels and efficacy of the therapeutic antibodies
(Bendtzen et al., Arthritis Rheum, 54(12):3782-9 (2006); Schmidt et al., Clin Immunol,
l32(3):334-4l (2009); Bartelds et al., Ann Rheum Dis, 66(7):92l-6 (2007); Baert et al., N
Engl J Med, 348(7):601-8 (2003); Tahir et al., tology (Oxford), 44(4):561-2
(2005); Maini et al., Arthritis Rheum, 41(9): 1552-63 (1998)). Allergic reactions have
been ated with anti-infliximab antibodies (Baert et al., N Engl J Med, 348(7):601-8
(2003)). Infusion-related hypersensitivity ons have been observed in a small
percentage of relapsing-remitting multiple sclerosis patients treated with zumab
(Phillips et al., Neurology, 67(9): 1717-8 ).
Alemtuzumab is another antibody therapeutic that can generate ADA in
relapsing-remitting multiple sclerosis patients (Coles et al., N Engl J Med, 2008.
359(17): 1786-801 (2008)). Alemtuzumab is a lymphocyte-depleting monoclonal
antibody that interacts with CD52, a cell surface n expressed on immune cells.
Alemtuzumab is in tage clinical trials for treating relapsing-remitting multiple
sclerosis and has also been evaluated in rheumatoid arthritis. A group of researchers have
shown that anti-alemtuzumab antibodies developed in 63% of rheumatoid arthritis
patients treated in a single dose escalation study (Weinblatt et al., tis Rheum, 1995.
38(11):1589-94 (1995)). In that study, the efficacy of zumab appeared to be
altered by the presence ofADA (Id).
The polyclonal antibody therapeutic Thymoglobulin® is also associated with
deleterious ADA in a small subset of patients. Serum sickness, acute renal failure and
cardiovascular reactions have been observed in Thymoglobulin®-treated transplant
ents (Boothpur et al., Am J Kidney Dis., 55(1): 141-3 ; ist et al., Liver
Transpl, 13(5):647-50 (2007); Busani et al., Minerva Anestesiol, 72(4):243-8 (2006);
Tanriover et al., Transplantation, 80(2):279-81 (2005); Buchler et al., Clin Transplant,
539-45 (2003)).
Researches have tried to find ways to minimize the deleterious effects ofADA.
Tools that have been tested for their ability to induce immunotolerance and reduce ADA
in protein therapies e, for example, non-depleting anti-CD4 antibodies (Cobbold et
al., Semin Immunol, 2(6):377-87 (1990); Winsor—Hines et al., J Immunol, 173(7): 4715-
23 (2004)), non-cell-binding minimal mutants of alemtuzumab (Gilliland et al., J
Immunol, :3663-71 (1999); leld et al., J Immunol., 185(1):763-8 (2010)),
immunosuppressive therapies (Bennett et al., Blood, 106(10):3343-7 ; n et
al., J Clin Invest, :2868-76 (2008)), phosphatidylinositol-containing lipidic
particles binding to Factor VIII (Peng et al., AAPS. J ., 12(3):473-81 (2010)), and liver-
specific administration of recombinant acid alpha-glucosidase via adeno-associated virus
infection (Sun et al., Am J Human Genet, 81(5): 1042-9 (2007); Sun et al., Mol Ther
18(2):353-60 (2009); Ziegler et al., Hum Gene Ther, 19(6):609-21 (2008)). There
s a need, however, to develop improved methods for reducing undesired dy
responses in protein therapies and other settings.
SUMMARY OF THE INVENTION
The invention provides a method of inducing immune tolerance in a subject in
need of treatment with a therapeutic. In this method, one administers to the subject an
effective amount of methotrexate in a single cycle, thereby inducing immune tolerance
toward the therapeutic in the subject.
The invention also provides a method of ting antibody responses to a
therapeutic in a subject in need of treatment with the therapeutic. In this , one
administers to the subject an effective amount of methotrexate in a single cycle, thereby
inhibiting antibody responses to the therapeutic in the subject.
The invention also provides a method of alleviating an infusion reaction to a
therapeutic in a subject in need of treatment with the therapeutic. In this method, one
administers to the subject an effective amount of methotrexate, thereby alleviating an
infilsion reaction to the protein therapeutic in the subject.
The invention also provides a method of reducing secondary autoimmunity in an
autoimmune subject in need of treatment with a therapeutic. In this , one
administers to the subject an effective amount of methotrexate, thereby reducing
secondary autoimmunity in the t.
The invention also provides a method of increasing the efficacy of a therapeutic
in a subject in need of treatment with the eutic. In this method, one administers to
the subject an effective amount of methtrexate, y increasing the efficacy of the
protein therapeutic in the subject.
The invention also es a method of increasing the tage of T
regulatory cells in the T cell population in a subject treated with a lymphocyte-depleting
therapy, e.g., an alemtuzumab y or a lobulin® therapy. In this method, one
sters to the t an effective amount of rexate, thereby increasing said
percentage in said subject.
The invention also es a method of increasing the percentage of B
regulatory cells in the B cell population in a subject in need of treatment with a protein
therapeutic such as an antibody therapeutic. In this method, one administers to the
subject an effective amount of methotrexate, thereby increasing said percentage in said
subject. In some embodiments, the effective amount of methotrexate is administered in a
single cycle. In related embodiments, the invention provides a method of increasing
TGF-beta-, IL-lO-, and/or FoxP3-expressing B cells in a subject in need of treatment with
a protein therapeutic, comprising administering an effective amount of methotrexate in a
single cycle.
The invention also provides a method of prolonging the pharmacokinetics of a
therapeutic agent in a subject. In this method, one administers methotrexate to the
subject, before or during or after administration of the therapeutic agent, in an amount
effective to prolong the pharmacokinetics of the therapeutic agent.
[0017 ] The invention r provides a method of depleting lymphocytes in a human
patient in need thereof. In this method, one treats the patient with a lymphocyte -depleting
agent and administers methotrexate to the patient, before or during or after the tr eatment
with the lymphocyte -depleting agent, in an amount effective to induce immune tolerance
in the patient toward the cyte -depleting agent or to reduce secondary
autoimmunity. In some embodiments, the effective amount of rexate is
ste red in a single cycle. In some embodiments, the lymphocyte -depleting agent
may be a monoclonal antibody therapeutic ( e.g. , alemtuzumab or rituximab). In certain
of these embodiments, the patient may be a multiple sclerosis patient ( e.g. , a remitting -
rela psing le sclerosis patient). In some embodiments, the lymphocyte -depleting
agent may be a polyclonal dy eutic ( e.g. , anti -thymocyte in polyclonal
antibody).
[0018 ] The invention also provides a method of in ducing immune tolerance in a subject
in need of tissue transplantation. In this method, one sters to the subject an
effective amount of methotrexate, thereby inducing immune nce toward the
transplanted tissue in the subject. In some embodimen ts, the ive amount of
methotrexate is administered in a single cycle. In some embodiments, the transplanted
tissue is renal tissue or cardiac tissue. In some embodiments, the subject also receives an
agent for immune -modulation ( e.g. , an immunosupp ressant), such as polyclonal anti -
thymocyte globulin antibody).
[0019 ] The invention also provides a method of inhibiting T cell responses in a subject
in need of a eutic or tissue transplantation. In this method, one administers to the
subject an effective amount of methotrexate prior to, concurrently with, or aft er, treating
the subject with the therapeutic or tissue transplantation, thereby ting T cell
responses in the subject.
[0019a] The invention also provides a method of treatment of a disease or condition of
the immune system in a subject in need of said treatment. The disease or condition of the
immune system may be mult iple sclerosis, or an autoimmun e condition, or tissue or o rgan
transplantation, or aplastic anaemia or graft -verus -host disease.
[0019b] The invention also provides for the use o f methotrexate and a n
therapeutic in the manufacture of a medicament for the treatment of a disease or condition
of the immune system in a subject in need of said treatment, wherein the medicament is
formulated such that in a single -cycle regimen of one or more s ng prior to,
concurrently with or after a first dosage of said protein therapeutic, and ending before a
second dosage of the said protein therapeutic, the medicament is capable of providing an
effective amount of methotrexate the reby inducing immune intolerance to said protein
therapeutic enabling treatment of said disease or condition of the immune system.
[0020 ] In some ments of the methods of the invention, the therapeutic is a
protein therapeutic.
[0021 ] In some embodiments of the methods of the invention, the therapeutic is an
antibody therapeutic. For example, the antibod y therapeutic may be a monoclonal
antibody eutic and/or a cyte -depleting agent ( e.g. , alemtuzumab , rituximab ),
or a onal antibody therapeutic ( e.g. , polyclonal rabbit anti -thymocyte globulin
antibody). In further embodiments wherein the antibody therapeutic is alemtuzumab, the
subject may be a le sclerosis patient. In further embodiments wherein the antibody
therapeutic is rituximab, the subj ect may be a human patient who has an mune
condition. In r embodiments wherein the dy therapeutic is polyclonal rabbit
anti -thymocyte globulin antibody, the subject may be a human patient who is in need of
organ transplantation, has apla stic anemia, and/or has or is at risk of having graft -versus -
host disease. In other embodiments of the methods of the invention, the therapeutic is an
enzyme. For example, the enzyme may be human alpha -galactosidase A or human acid
alpha -glucosidase.
[00 22 ] In some embodiments of the methods of the invention, the subject is a human.
[0023 ] In some embodiments of the methods of the invention, the effective amount of
methotrexate may be 0.1 mg/kg to 5 mg/k g. In some embodiments, the single cycle of
methotrexate may consist of 1 day of methotrexate administration, or 2, 3, 4, 5, 6, 7, 8, 9,
, or 11 consecutive days of methotrexate administration. In some embodiments, the
single cycle of methotrexate may be administered between 48 hours prior to and 48 hours
after the onset of the therapeutic treatment.
BRIEF DESCRIPTION OF THE FIGURES
[0024 ] In each of the Figures bed below, sks or stars indicate measurements
with statistically significant differences (*, p < 0.05; **, p ≤ 0.001; ***, p ≤ 0.0001).
[0025 ] FIGS. 1A -B show anti -rabbit IgG responses to a si ngle and multiple courses of
mATG, a rabbit anti -murine thymocyte globulin polyclonal antibody. Average anti -rabbit
IgG titers were measured, and mice treated with rabbit IgG (rbIgG) only were used as a
control. shows responses to a single course of mATG over an 8 week period.
shows responses to multiple courses of mATG over a 20 week period. Arrows
indicate time points at which mATG or rbIgG were administered.
[0026 ] shows average alemtuzumab -specific IgG titers following five monthly
treatments with alemtuzumab. Arrows te time points at which alemtuzumab was
administered.
[0027 ] shows suppression of anti -mATG IgG respo nses by methotrexate (MTX)
following a single course of mATG. Mice were treated with mATG only, rbIgG only, or
mATG and methotrexate.
[0028 ] FIGS. 4A -C show the effects of methotrexate on anti -rabbit IgG responses
throughout a cou rse of five monthly treatments with mATG. Arrows indicate time points
at which mATG or methotrexate were administered. shows that three cycles of
rexate significantly reduce average anti -rabbit IgG . shows that a
single cours e of rexate significantly reduces average anti -rabbit IgG . compares the average anti -rabbit IgG titers of mice treated with mATG alone, mATG
and a single cycle of methotrexate, or mATG and three cycles of methotrexate. A single
cycle of methotrexate reduces anti -rabbit IgG titers more significantly than three cycles of
rexate.
[0029 ] shows that average anti -rabbit IgG titers are reduced after mATG re -
challenge in methotrexate -treated mice (single and multiple methotrexate cycles), as
compared to mice d with mATG but not methotrexate. Arrows indicate time points
at which mATG or methotrexate were administered.
[0030 ] shows that the average anti -rabbit IgG titer in mice treated with mATG
and a single cycle of methotrexate is 100 fold less than that in mice treated with mATG
alone. Arrows indicate time points at which mATG or methotrexate were administered
[0031 ] FIGS. 7A -C show that methotrexate can reduce average anti -alemtuzumab IgG
titers in huCD52 Tg mice. : Anti uzumab titers are lower in mice treated
with a single cycle of 1 mg/kg or 5 mg/kg of methotrexate than in mice d with 0.5
mg/k g or no rexate. Mice were treated with alemtuzumab alone, or alemtuzumab
and a single cycle of methotrexate at 0.5 mg/kg, 1 mg/kg, or 5 mg/kg. : Study
design to test anti uzumab titers. Titer data was determined at 24 hours following
the fifth dose of alemtuzumab, as indicated by the star. : Methotrexate reduced
anti -alemtuzumab antibody titers in mice receiving alemtuzumab and methotrexate,
compared to mice receiving only alemtuzumab, 24 hours after the fifth dose of
alemtuzuma b.
[0032 ] FIGS. 8A -D show the absolute cell number/µl of whole blood of circulating T
cells in mice treated with five daily doses of 0.5 mg/kg alemtuzumab or phosphate -
buffered saline (PBS). shows that total T cells (CD3 +) were reduced two, three,
and four weeks post -treatment. shows that total B cells (CD19 +) were reduced
three weeks post -treatment. shows that T helper cells (CD4 +) were reduced at
CONTINUES ON PAGE 8
two, three, and four weeks post-treatment, and shows that cytotoxic T cells
(CD8+) were reduced at two, three, and four weeks post-treatment.
FIGS. 9A-B show alemtuzumab-specific IgG ses. shows
responses following three cycles of treatment with alemtuzumab at 0.5 mg/kg, and one
cycle of treatment with methotrexate at 0.5 mg/kg, 1 mg/kg, or 2 mg/kg. The first cycle
of zumab treatment consisted of five consecutive days of dosing, and the second
and third cycles each ted of three utive days of dosing. shows anti-
alemtuzumab IgG titers of each animal per group at week 14.
shows that a single cycle of 5 mg/kg of rexate restores circulating
mATG levels in mice d with five monthly doses ofmATG.
FIG. ll shows that mice treated with five monthly treatments ofmATG and a
single cycle of methotrexate have ed mATG-mediated CD4+ and CD8+ T cell
depletion in blood after the fifth dose ofmATG. These data are pooled from two
experiments.
[0036] FIGS. lZA-B show that mice treated with a single cycle of methotrexate exhibit
increased percentages of T regulatory (CD25IFoxp3I) cells ing a fifth monthly
mATG treatment. Mice were treated with five monthly treatments ofmATG alone, or
five monthly treatment ofmATG and a single cycle of methotrexate, or five monthly
treatments ofmATG and three cycles of methotrexate. A shows T regulatory cell
levels in the spleen. B shows T regulatory cell levels in the blood.
A shows that an anti-rabbit IgG titer greater than 10,000 can interfere
with pharmacokinetics ofmATG. B shows that an anti-rabbit titer greater than
100,000 can interfere with CD4 and CD8 cell depletion. % of pretreatment control refers
to the percent of CD4 and CD8 titers relative to their respective levels prior to mATG
treatment.
shows that mice treated with 5 mg/kg of rexate exhibit enhanced
alemtuzumab depletion of circulating CD3+ T cells and CD19+ B cells after a fifth
monthly dose of alemtuzumab. Mice were treated with alemtuzumab alone, or
alemtuzumab and a single cycle of methotrexate for the first three days of the study.
Asterisks indicate measurements with statistically significant differences (*, p < 0.05;
***, p 5 0.0001).
A shows that a single cycle of methotrexate can reduce recombinant
human acid glucosidase (rhGAA)—specific IgG titers throughout treatment, and
even four weeks after the last rhGAA treatment, in a 16-week study. Arrows indicate
time points at which rhGAA and methotrexate were administered. Mice were treated
with rhGAA alone, or rhGAA and a single cycle of methotrexate, or rhGAA and three
cycles of methotrexate. B shows that, in a six-week study, a single cycle of
rexate reduces rhGAA-specific IgG titers. C shows that a single cycle of
methotrexate reduces rhGAA-specific IgG titers in an 18 week study.
A shows that methotrexate decreases anti-rabbit IgG titers in a murine
allogeneic heart transplant model when administered with mATG, as compared to mATG
alone. B shows that methotrexate increases circulating levels ofmATG in a
murine allogeneic heart transplant model. Mice were treated with saline, mATG alone, or
mATG and a single cycle of methotrexate. mATG was administered at 20 mg/kg at days
0 and 4 of the study, while 2 mg/kg of methotrexate was administered on days 0-6 of the
study.
shows a -Meier plot indicating that a combined treatment of
mATG and methotrexate prolongs the survival of allogeneic hearts transplanted into
recipient mice. Mice were left untreated, or were treated with 20 mg/kg ofmATG alone
at days 0 and 4 of the study, or with 2 mg/kg of methotrexate alone on days 0-6 of the
study, or with both mATG and 2 mg/kg of methotrexate on days 0-6, or with both mATG
and 0.5 mg/kg of methotrexate on days 0-6 or days 0-11.
shows that mice with an neic heart transplanted treated with either
methotrexate alone or methotrexate in combination with mATG experience a reduction in
anti-allograft antibody responses. A shows recipient IgG binding to allogeneic
fibroblasts on day 21. B shows alloantibody levels on day 21 in mice given
syngeneic lants (Syn TX) or an allogeneic transplant (Allo TX) with the indicated
treatments. Shown is the binding of dual recipient mouse serum IgG to allogeneic
asts and is expressed as a ratio of the mean fluorescence intensity (MFI) to
unstained fibroblasts. C shows alloantibody levels on day 21 in mice given no
treatment, mATG, methotrexate, or mATG and methotrexate. tibody levels were
significantly lower in mice d with methotrexate or mATG and methotrexate
(p=0.0008 and p<0.0001, respectively).
shows that BlO regulatory B cells are significantly increased ing
methotrexate treatment. Mice were treated with rhGAA alone or rhGAA and a single
three-day cycle of methotrexate or saline. Cell numbers were counted on day 7 and day 8
of the study.
shows that activated B cell subpopulations are cantly increased on
day 6 following treatment with rhGAA and a single three-day cycle of methotrexate, as
compared to treatment with rhGAA alone. Absolute cell numbers of CD86+ transitional
2 B cells, CD86+ transitional 3 B cells, CD86+ follicular B cells, and CD86+ marginal
zone B cells were counted on day 6 of the study. Asterisks indicate measurements with
statistically significant differences (*, p < 0.05; **, p 5 0.001).
shows that for ted splenic B cell subpopulations, such as activated
al zone B cells, activated follicular B cells, and activated transitional 3 B cells, cell
s remain enhanced even following treatment with rhGAA and methotrexate, as
compared to treatment with rhGAA alone. Arrows represent the treatment with rhGAA
and methotrexate. rhGAA was administered on day l and day 8. Methotrexate was
administered on days 1, 2, and 3, and days 8, 9, and 10. Significant differences are
represented by stars (*, p < 0.05). Data not shown for days 8 and 9 (indicated by al
dotted lines).
shows that activated splenic T cell populations, such as activated T
helper cells, activated T cytotoxic cells, and activated T regulatory cells, remain largely
unchanged following treatment with rhGAA and methotrexate, as compared to treatment
with rhGAA alone. Arrows represent the treatment with rhGAA and methotrexate.
rhGAA was administered on day l and day 8. Methotrexate was administered on days 1,
2, and 3, and days 8, 9, and 10. Significant differences are represented by stars (*, p <
0.05). Data not shown for days 8 and 9 (indicated by vertical dotted .
shows a six month-long study design for examining methotrexate
treatment in combination with mATG. Solid arrows ent 5 mg/kg mATG injections,
dashed arrows represent 5 mg/kg methotrexate injections, and dotted arrows represent
terminal ces.
[0048] FIGS. 24A-B show that a single cycle of methotrexate in connection with
mATG enriches splenic B cells, as compared to mATG alone or three cycles of
methotrexate. A: activated follicular B cells; B: activated transitional 3 B
cells.
FIGS. 25A-B show the effects of methotrexate on alemtuzumab’s
pharmacodynamics in the blood. Mice were treated with 0.5 mg/kg of alemtuzumab for
five months, either with or without three daily doses of 5 mg/kg/day of methotrexate in
connection with the first administration of alemtuzumab. A: Methotrexate
enhances depletion of total T cells (CD3+), T helper cells (CD4+), and T regulatory cells
(CD4+CD25+Foxp3+) by zumab (AZM). Black bars represent measurements in
mice treated with alemtuzumab alone, while white bars ent measurements in mice
treated with alemtuzumab and methotrexate. B: Methotrexate es depletion
of B cells by alemtuzumab. Stars indicate measurements with tically significant
differences (*, p < 0.05).
shows the s of methotrexate on alemtuzumab’s pharmacodynamics
in the spleen. Mice were treated with 0.5 mg/kg of alemtuzumab for five months, either
with or without three daily doses of 5 mg/kg/day of rexate in connection with the
first administration of alemtuzumab. T cells are depleted following the fifth treatment
with alemtuzumab. CD8 CEN: CD8+ central memory T cells; CD8 EFF MEM: CD8+
effector memory T cells; CD4 CEN MEM: CD4+ central memory T cells; CD4 EFF
MEM: CD4+ effector memory T cells. Stars indicate measurements with statistically
significant ences (*, p < 0.05).
FIGS. 27A-B show the effects of rexate on B cell numbers in the spleen.
Mice were treated with 0.5 mg/kg of zumab for five months, either with or without
three daily doses of 5 mg/kg/day of methotrexate in connection with the first
administration of alemtuzumab. Stars indicate measurements with statistically significant
differences (*, p < 0.05).
FIGS. 28A-B show depletion of splenic lymphocytes three days after a single
dose of zumab. A: T cells and B cells are significantly depleted. Small
checks represent PBS-treated control mice, and large checks represent alemtuzumab-
treated mice. B: B cells (CD l9+) are 92% ed at 24 hours after treatment,
and remain 36% depleted three days after treatment. The three graphs represent different
group of animals, each of which was bled at different time points. Stars indicate
ements with statistically significant differences (*, p < 0.05; **, p 5 0.001; ***, p
S ).
FIGS. 29A-B show the effects of methotrexate on cytokine levels. FIG 29A
shows a study design for a six month study to assess cytokine levels in the spleen and
lymph nodes. The star indicates that data were collected 24 hours after the fifth treatment
with alemtuzumab. Arrows indicate time points at which alemtuzumab or rexate
were administered, or terminal sacrifices were performed, as indicated. B shows
levels of B cell activating factor belonging to the TNF family (BAFF) in mice d
with alemtuzumab alone or with alemtuzumab and methotrexate.
[0054] FIGS. 30A-B show the levels of cytokines after treatment with alemtuzumab
and methotrexate. FIG 30A shows a study design for a four month study. The star
indicates that data were collected one week after the second cycle of methotrexate.
Arrows indicate time points at which alemtuzumab or methotrexate were administered, or
terminal sacrifices were performed, as ted. B shows the levels of various
cytokines in mice treated with alemtuzumab (AZM) alone or with 0.5 mg/kg, 1.0 mg/kg,
or 2.0 mg/kg of methotrexate.
shows the effects of methotrexate on anti-rhGAA titers in IL10 -/-
(knockout) and C57BL/6 mice. 20 mg/kg of rhGAA was administered weekly for nine
weeks in IL10 -/- knockout mice and C57BL/6 wild-type mice, with or without 5
mg/kg/day of methotrexate at 0, 24, and 48 hours after the first three weekly treatments of
rhGAA.
shows that some, but not all, c B cell populations are ed at
one and/or two weeks ing treatment with alemtuzumab. Small hatched bars
represent phosphate buffered saline (PBS) treated huCD52 transgenic control mice; large
hatched bars depict huCD52 transgenic mice treated with 0.5 mg/kg of alemtuzumab for
five consecutive days. Asterisks indicate measurements with statistically significant
differences (*, p < 0.05; **, p 5 0.001; ***, p 5 0.0001).
shows a study design for examining the effects of methotrexate on B
cell populations in the context of treatment with alemtuzumab. Thyroid and lymph nodes
(LN) were used for pathological evaluation. Arrows indicate time points at which
alemtuzumab and methotrexate were administered, or terminal ices as indicated.
shows the effects of three daily doses of 5 mg/kg/day methotrexate
alone, 0.5 mg/kg alemtuzumab alone, and 0.5 mg/kg alemtuzumab and 5 day
methotrexate in combination on the ion of B cell populations. Asterisks te
measurements with statistically significant differences (*, p < 0.05; **, p 5 0.001; ***, p
S 0.0001); ns, not significant).
shows the effects of methotrexate on B cell depletion after five cycles of
treatment with 0.5 mg/kg alemtuzumab alone or three daily doses of 5 mg/kg/day
methotrexate alone or 0.5 mg/kg alemtuzumab and 5 mg/kg/day methotrexate in
combination. Asterisks indicate measurements with statistically significant differences
(*, p < 0.05).
shows that levels of the cytokines MCP-l, IL-13, IL-6, and IL-12 are
decreased in mice treated with a single cycle of three days of 5 mg/kg of methotrexate
stered on the first day of alemtuzumab treatment. Data were collected 24 hours
after a fifth dose of alemtuzumab, four months after ent with methotrexate.
[0061] FIGS. 37A-B show rhGAA titers at weeks 2, 6, and 12 in nu/nu nude mice.
A shows rhGAA titers at weeks 2 and 6. From left to right, week 2 measurements
in control mice treated with saline, mice treated with rhGAA, and mice treated with
rhGAA and methotrexate, followed by week 6 measurements in control mice treated with
saline, mice treated with rhGAA, and mice treated with rhGAA and methotrexate. 37B shows week 12 measurements in, from left to right, nu/nu mice treated with rhGAA
alone (Myo), nu/nu mice treated with methotrexate and rhGAA, BL6 mice d with
rhGAA alone, and BL6 treated with methotrexate and rhGAA.
FIGS. 38A-B show that the numbers and percentages of IL-10 expressing B10 B
cells is increased in mice tolerized to rhGAA with methotrexate. B10 B cells isolated
from animals treated with rhGAA or rhGAA and methotrexate were assessed for IL-10
protein expression by flow try.
FIGS. 39A-B show that IL-10 is expressed in both activated (CD86+) and non-
activated ) B10 B cells. A depicts a FACS plot of B10 B cells stained
with CD86, while B s the numbers of CD86+IL10+ B10 B cells and CD86
IL10+ B10 B cells in response to treatment with rhGAA or rhGAA and methotrexate.
FIGS. 40A-B show that methotrexate treatment with rhGAA s B10 B
cells to increase their expression of TGF-beta. The second and third panels of A
are FACS plots showing BlO B cells stained with ta and CD86 from animals
treated with rhGAA or rhGAA and methotrexate. The first panel of A depicts the
number of TGF-beta+ BlO B cells in animals treated with rhGAA or rhGAA and
methotrexate. B s CD86+TGF-beta+ BlO B cell and CD86'TGF-beta+ BlO
B cell counts.
A depicts that BlO B cells appear to express FoxP3 in animals treated
with rhGAA (A). B depicts that the numbers of FoxP3+ B cells increase
with treatment with both methotrexate and rhGAA. C depicts that both activated
(CD86+) and non-activated (CD86-) B10 B cells express FoxP3.
[0066] FIGS. 42A-C show that follicular, transitional 2, and transitional 3 B cells (top
to bottom) express IL-10 and that the cell numbers of the IL-lO-expressing B cell subsets
increase with methotrexate as compared to mice treated with rhGAA alone.
FIGS. 43A-C show that follicular, transitional 2, and tional 3 B cells (top
to ) s ta and that the cell numbers of the TGF-beta-expressing B cell
subsets increase with methotrexate as compared to mice treated with rhGAA alone.
FIGS. 44A-C show that ular, transitional 2, and transitional 3 B cells (top
to bottom) express FoxP3 and that the cell numbers of the FoxP3-expressing B cell
subsets se with methotrexate as compared to mice treated with rhGAA alone.
shows anti-rhGAA titers at week 6 in animals treated with rhGAA or
rhGAA and methotrexate, in the presence or absence of 5 mg/kg of anti-TGF-beta
antibody (lDl l, Genzyme) or the isotype control (l3C4). Antibody titers were assessed
bi-weekly in the four different groups of animals.
FIGS. 46A-C show that lDll treatment interfered with methotrexate-induced
expansion of B10 B cells expressing TGF-beta, IL-10, or FoxP3. Spleens were isolated
from animals treated with rhGAA or rhGAA and methotrexate that also were co-
administered lDll or l3C4 seven days following a single rhGAA treatment or a single
rhGAA and methotrexate treatment. Cells in each group were then pooled and cultured
for two days and then counted using flow cytometry.
FIGS. 47A-C show that lDll ent interfered with methotrexate-induced
ion of follicular B cells expressing TGF-beta or IL-10, although FoxP3+ Follicular
B cells did not appear to experience lDll effects. Spleens were ed from animals
treated with rhGAA or rhGAA and methotrexate that also were co-administered lDll or
l3C4 seven days following a single rhGAA ent or a single rhGAA and
methotrexate ent. Cells in each group were then pooled and cultured for two days
and then counted using flow cytometry.
FIGS. 48A-C show that, in tional 2 B cells, while lDll treatment
interfered with methotrexate-induced expansion of TGF-beta-expressing transitional 2 B
cells, no effects were seen on IL-10+ transitional 2 B cells. Spleens were isolated from
s treated with rhGAA or rhGAA and methotrexate that also were co-administered
lDll or l3C4 seven days following a single rhGAA treatment or a single rhGAA and
methotrexate treatment. Cells in each group were then pooled and cultured for two days
and then counted using flow cytometry.
FIGS. 49A-C show that in transitional 3 B cells there is detectable ta, IL-
and FoxP3, but no apparent effect of lDll treatment on the cells. Spleens were
isolated from animals treated with rhGAA or rhGAA and methotrexate that also were co-
administered lDll or l3C4 seven days following a single rhGAA ent or a single
rhGAA and methotrexate treatment, and cells were counted using flow try.
FIGS. SOA-C show that lDll treatment does not affect basal levels of IL-lO,
TGF-beta, and FoxP3 in follicular B cells, transitional 2 B cells, and transitional 3 B cells
(top to bottom).
A is a schematic showing the transfer of total c B cells from a
mouse tolerized to rhGAA (Myozyme® or “MYO”) into an rhGAA-na'ive recipient
mouse. After transfer, the recipients (along with non-transferred control animals treated
with either rhGAA or rhGAA and methotrexate) were treated weekly with 20 mg/kg of
rhGAA.
B shows titer analysis demonstrating that total splenic B cells isolated
from animals treated with rhGAA and single cycle of rexate can transfer immune
tolerance to rhGAA in naive hosts.
depicts cell counts from the blood or spleen of normal mice treated with
rabbit IgG (rbIgG), mATG alone, rbIgG and methotrexate, or mATG and methotrexate.
Methotrexate does not deplete CD4+, CD8+, T regulatory (CD4+CD25+FoxP3+) T cells,
or total CDl9+ B cells in normal animals.
depicts cell counts from the blood or spleen of transplant mice treated
with rbIgG, mATG alone, rbIgG and methotrexate, or mATG and methotrexate 14 days
after transplantation. Methotrexate does not deplete CD4+, CD8+, T regulatory
(CD4+CD25+FoxP3+) T cells, and total CDl9+ B cells in transplant animals.
shows that methotrexate treatment induces statistically significant
increases in IL-lO in multiple cell subsets as viewed by a shift in mean fluorescence
intensity (MFI) of these proteins in animals treated with rhGAA or rhGAA and
methotrexate.
shows that methotrexate treatment induces statistically cant
increases in TGF-beta in multiple cell subsets as viewed by a shift in mean fluorescence
intensity (MFI) of these ns in animals treated with rhGAA or rhGAA and
rexate.
shows that methotrexate treatment induces statistically significant
increases in FoxP3 in multiple cell subsets as viewed by a shift in mean fluorescence
intensity (MFI) of these proteins in animals treated with rhGAA or rhGAA and
methotrexate.
DETAILED DESCRIPTION OF THE ION
The present invention is based on our sing ery that a single cycle or
short course of methotrexate administration reduces red immune responses (such as
ADA responses, and other undesired T- and/or B-cell mediated immune responses) in
patients receiving protein therapeutics such as replacement enzymes or therapeutic
antibodies, and anti-graft antibody responses in tissue transplantation. This discovery
leads to new methods for increasing both safety and y of protein therapies and
organ transplantation.
[0083] More specifically, our studies have shown that a single cycle of rexate
s ADA against antibody therapeutics. As detailed below, one set of studies was
done with a murine version of a polyclonal antibody therapeutic, Thymoglobulin®.
Thymoglobulin® is a rabbit anti-human thymocyte globulin polyclonal antibody used for
suppression in the setting of solid organ transplantation, aplastic anemia and in
prevention of graft-versus-host disease. A rabbit anti-murine thymocyte globulin
polyclonal antibody (mATG) has been developed. It maintains similar characteristics to
lobulin® (Ruzek, et al., Transplantation, 88(2): 170-9 (2009)). We have
demonstrated that a single course of methotrexate can reduce anti-mATG IgG titers by
>95%. In fact, we have surprisingly found that a single cycle of methotrexate works
better in reducing ADA than three cycles of methotrexate. In addition, this reduction in
ADA ins the levels of circulating mATG and enhances mATG-mediated cell
depletion and T regulatory cell percentages upon repeat mATG dosing. Another set of
our studies was done with a onal antibody therapeutic, alemtuzumab. We have
found that a single course of methotrexate can similarly control anti-alemtuzumab
responses and e alemtuzumab-mediated lymphocyte ion.
We have discovered that this single cycle regimen of methotrexate also reduces
ADA where the protein therapeutic is an enzyme. In two of our studies, we trated
that a single cycle of methotrexate could effectively control anti-Myozyme®
binant human alglucosidase alpha or “rhGAA”) responses where originally
multiple cycles were thought to be required to reduce ADA.
We have discovered that methotrexate is also useful in organ transplantation. In
our studies, we found that a single cycle of methotrexate could control anti-allograft
antibody ses in heart allograft transplantation. When combined with mATG, the
survival of a heart aft was significantly longer.
Our studies show that a single cycle of methotrexate given within the first week
of a protein therapy can provide a long-lived reduction of greater than 95% in ADA over
many months of dosing. This reduction in antibody titers was long-lived despite the
absence of methotrexate hout the majority of the studies. Furthermore, we have
found that a single cycle of methotrexate can control anti-allograft responses (e.g., in a
murine neic heart lant model), g control over antibody responses
directed towards multiple antigens simultaneously.
[0087] Methotrexate is classically known as a dihydrofolate reductase nist that is
thought to kill proliferating cells by inhibiting purine metabolism and interfering with de
novo DNA synthesis (Kremer, Arthritis Rheum, 50(5): 1370-82 (2004)). It could be easily
assumed that methotrexate may simply kill the reactive cells through this well-described
mechanism, but this seems unlikely with the single cycle regimen that we have
discovered. Methotrexate has a short half-life and is not likely to be in cells or circulation
long enough to ly kill cells three to four months following treatment (Walling,
Invest New Drugs, 24(1):37-77 (2006); Slavikova et al., Neoplasma, 25(2):211-6 (1978)).
Moreover, we find no evidence of lymphocyte depletion following methotrexate
treatment. Rather, we have singly found that a single cycle regimen of
methotrexate reduces undesired antibody responses by inducing an active mechanism of
immune control, not by indiscriminately depleting lymphocytes.
Our studies show that a single cycle of methotrexate ses B10 regulatory B
cells as well as activated marginal zone B cells, activated follicular B cells and activated
transitional 3 B cells. Our studies also show that a single cycle of methotrexate increase
the number of IL-lO-expressing, TGF-beta-expressing, and expressing B10,
follicular, tional 2 and transitional B cells, and that this expansion is mediated by
TGF-beta. These studies also show that methotrexate increases the expression levels of
IL-lO, TGF-beta, and FoxP3. The expansion of splenic B cell populations is surprising
given the current understanding of the mechanism of action of methotrexate, and suggests
that in this dosing paradigm, methotrexate is working in a unique, usly unknown
way. Low, continual doses of rexate in infliximab-treated rheumatoid arthritis
patients have been shown to reduce anti-infliximab antibody responses; yet as exposure is
continuous, this regimen is more likely to e constant immunosuppression, rather
than tolerance induction. Methotrexate treatment alone has been shown to reduce disease
activity in rheumatoid arthritis when given weekly in low doses. A recent publication
suggests that upon continual administration of low dose methotrexate (every other day),
autoantigen-specific T regulatory cells appear, which may help account for the efficacy of
methotrexate ent in rheumatoid arthritis (Xinqiang et al., Biomed Pharmacother,
64(7):463-47l ). In contrast, the dosing paradigm described herein for
methotrexate is truly unique in that it involves a short course of methotrexate treatment
that can provide long-lasting control of undesired immunological responses.
[0089] Altogether, we have identified a unique dosing regimen of rexate that can
yield long-lasting control over several different types ofADA responses and anti-allograft
antibody responses in tissue lantation, as well as T cell and B cell responses. We
have found that the immune tolerance ped by methotrexate can be transferred from
one animal to another, for example, by transplanting B cells from a tolerized animal to a
non-tolerized animal. Our data suggest that rexate acts through a unique
mechanism of action that involves the expansion of activated B cell subsets that may
represent tory B cells active in suppressing immune ses. Furthermore,
methotrexate also may act through a mechanism of T regulatory cell expansion.
Undesired Immune ses in Bio-therapy
The methods of this invention can control undesired immunological responses
(e.g., ADA ses, and other red T- and/or B-cell mediated immune responses)
in a variety of biological therapies (e.g, y using a biologic such as proteins, nucleic
acids, carbohydrates, lipids, and metabolites). Protein therapy refers to therapy in which
the therapeutic agent is a proteinaceous substance, including peptides and proteins.
Protein eutics can, for example, be enzymes, cytokines, growth factors,
immunomodulators, thrombolytics, antibodies (including polyclonal and monoclonal
antibodies), antibody fragments or modified antibodies (e.g., Fab, F(ab’)2, Fv, Fd, scFv,
and dAb). For e, many enzyme replacement therapies have been developed for
patients with certain genetic diseases, including Fabrazyme® (recombinant human alphagalactosidase
) for Fabry disease, Cerezyme® (imiglucerase) for Gaucher disease,
Aldurazyme® (laronidase) for Mucopolysaccharidosis I (MPS I), and Myozyme® and
Lumizyme® (alglucosidase alpha) for Pompe disease. Examples of antibody therapeutics
include Campath® (alemtuzumab), Thymoglobulin®, Avastin® (bevacizumab),
Lucentis® (ranibizumab), Remicade® imab), Humira® (adalimumab), Rituxan®
(rituximab), Tysabri® (natalizumab), Simulect® (basiliximab), Zenapax® (daclizumab),
OKT3® (muromonab-CD3), x® imab), Mylotarg® (gemtuzumab),
Herceptin® (trastuzumab), and Benlysta® (belimumab). Examples of other protein
therapeutics include Enbrel® (etanercep), and other fusion proteins.
In many cases, undesired immune responses can be generated in a patient
against the protein therapeutic, causing variable effects on t outcome. Such
responses occur because biologic therapeutics often contain sequences and conformations
that are foreign to a human patient. For example, ADA interferes with eutic
efficacy and/or increases safety risks. ADA may cause hypersensitivy reactions,
anaphylaxis, serum sickness, immune complex disease, acute renal e. ADA can be
monitored in patients receiving protein therapy by a clinician using well established
methods, including ELISA and histochemistry.
In some instances, a “protein therapy” as used herein refers to a viral therapy
where a viral vector is used to deliver a c acid therapeutic. Exemplary viruses used
in such therapies include, but are not limited to, adenoviruses, adeno-associated viruses,
and retroviruses. Antibodies may develop against the capsid proteins of the virus,
reducing the efficacy and increasing the safety risks of such ies. The methods of
this invention are useful to control undesired immunological responses (e.g., ADA
responses, and other undesired T- and/or B-cell mediated immune responses) in viral
therapies as well.
The s of this invention also may control undesired immunological
responses in non-protein biological therapies. ary non-protein bio-therapies
include, but are not limited to, nucleic acid therapies, (e.g., antisense therapies, siRNA
therapies, and miRNA therapies).
Anti-Graft Response in Transplantation
The methods of this invention can also be used to induce immune tolerance in a
patient receiving tissue transplantation such as renal transplantation, liver transplantation,
cardiac transplantation, and stem cell transplantation. Host versus graft and graft versus
host rejections often occur in tissue transplantation, especially allograft and xenograft
transplantation. A single cycle of rexate can be used alone or together with
another immune-modulating agent, (e.g., an immunosuppressant such as
Thymoglobulin®) to manage anti-graft antibody response. In addition, the combination
of Thymoglobulin® and methotrexate in transplantation may act to prolong graft al.
Finally, in a case where Thymoglobulin® would be investigated in the settings of c
mune disease such as toid arthritis and multiple sclerosis, methotrexate
may allow for safer re-treatment of Thymoglobulin®, protecting the patient from
developing significant anti-rabbit dies (such as IgG and/or IgM) and/or infilsion-
related reactions.
Managing Undesired Immunological Responses with Methotrexate
We have found that that a single, short cycle of methotrexate can significantly
reduce undesired logical responses such as ADA in subjects receiving biologic
therapeutics (e.g. , protein eutics) and llograft responses in patients receiving
tissue transplantation. Reducing undesired ADA may not only improve patient safety, but
also may improve the efficacy of a protein therapeutic h improving the n
therapeutic’s pharmacodynamics and/or pharmacokinetics.
rexate, a small le compound, has been used to treat patients with
severe active rheumatoid arthritis, severe sis, and certain types of cancer including
cancers that begin in the tissues that form around a fertilized egg in the uterus, breast
cancer, lung cancer, certain cancers of the head and neck, certain types of lymphoma, and
leukemia (cancer that begins in the white blood cells). Methotrexate treats cancer by
slowing the growth of cancer cells. Methotrexate treats psoriasis by slowing the growth
of skin cells to stop scales from forming. Methotrexate may treat rheumatoid arthritis by
decreasing the activity of the immune system.
Methotrexate has been studied in the context of controlling ADA responses
elicited against u-galactosidase A and u-glucosidase. r, those studies were done
with multiple cycles of methotrexate treatment (Garman et al. Clin Exp Immunol,
:496-502 (2004); Joseph et al., Clin Exp Immunol, 152(1): 138-46 (2008);
Mendelsohn et al., N Engl J Med, 360(2): 194-5 (2009)), rather than a single cycle of
methotrexate.
[0098] Our studies surprisingly show that a single cycle of methotrexate suffices to
reduce ADA and anti-allograft dies significantly. In fact, in studies involving
antibody therapeutics (e.g., mATG), we show that single-cycled treatment of
methotrexate is more effective in reducing ADA than multi-cycled treatment of
methotrexate. Previous studies using methotrexate gave no indication that a single cycle
of methotrexate would be sufficient to reduce ADA, much less that it would be more
effective than multi-cycle treatment. Methotrexate is known to be cytotoxic. Therefore,
if one had hypothesized that a mechanism for reduction ofADA by methotrexate relied
upon this property, then reducing the number of cycles of treatment actually would have
been predicted to reduce the beneficial effects of methotrexate. Furthermore, those
previously published studies do not demonstrate the benefits of methotrexate treatment on
pharmacokinetics, codynamics, efficacy, or safety, as surprisingly have been
demonstrated herein with a single course of methotrexate. Nor do they disclose that
rexate can reduce ADA in antibody therapy.
As used , a single cycle regimen refers to a treatment n, or a
treatment unit, of utive or non-consecutive days and are started at preferably no
more than five (e.g., no more than three) days following the dosing of the primary protein
therapeutic or transplantation. If the primary n therapeutic is dosed in multiple
periods, a single cycle of treatment of methotrexate preferably does not extend past the
first period of protein therapeutic dosing. By way of example, in a weekly, monthly, or
annual protein therapy, a single cycle of methotrexate consists of three consecutive days
of methotrexate intake (e.g., orally), starting on day 0, the day when the primary protein
therapeutic is given to the t for the first time, or when the patient receives a tissue
transplant. Then the patient receives a single dose of methotrexate on day 1 (24 hours
later) and on day 2 (48 hours later). A single cycle of methotrexate may also consist of,
for example, 2, 3, 4, 5, 6, 7, or 8 consecutive daily doses of methotrexate, starting on day
0. Methotrexate also can be administered at other times as deemed appropriate, e.g.,
when managing secondary autoimmunity in e.g., a lymphocyte-depleting therapy. A
single cycle of methotrexate preferably does not last longer than 8 days. In some
embodiments, a single cycle of rexate begins between 48 hours prior to and 48
hours after the onset of the primary therapeutic treatment (z'.e., the treatment with the
biologic therapeutic). For example, a single cycle of methotrexate may begin 48 hours
prior to, 36 hours prior to, 24 hours prior to, 12 hours prior to, concurrently with, 12 hours
after, 24 hours after, 36 hours after, or 48 hours after, the onset of the primary therapeutic
treatment.
Our studies also surprisingly show that a low dosage of rexate suffices to
manage undesired immunological responses (e.g., ADA responses, and other undesired T-
and/or B-cell mediated immune responses) in protein therapies and transplantation.
Accordingly, in embodiments of the ion, methotrexate may be administered in
more than one cycle, but at a low total dosage. For instance, the rexate can be
stered in two or more (e.g., 3, 4, 5, 6, etc.) cycles, but with a total combined dosage
of no more than 5 mg/kg in a patient.
[0101] The dosage of methotrexate will be an ive amount of rexate in
reducing undesired immunological responses, such as antibody or cellular responses,
when given in a single cycle. An ive amount of methotrexate in human patients
may be in the range of 0.05 mg/kg to 5 mg/kg. In some embodiments, the effective
amount is 0.1 mg/kg to 1.5 mg/kg. In some embodiments, the effective amount is 0.12
mg/kg to 1.28 mg/kg. In n embodiments, the effective amount is 0.12 mg/kg. In
certain embodiments, the effective amount is 1.28 mg/kg. The ended dosage of
methotrexate may pose minimal safety risks because the dosing regimen involves only a
brief course of methotrexate at dose levels that are more r to doses for rheumatoid
arthritis than low neoplastic doses. In our studies, the total amount of methotrexate tested
in each cycle in mice was 14 or 15 mg/kg. 14 mg/kg of methotrexate in mice is
equivalent to approximately 68 mg or 5.92 mg/m2 in an average adult weighing 60 kg.
Rheumatoid arthritis patients can receive up to 25 mg of methotrexate per week without
ing from signif1cant toxicities. The low neoplastic dose of methotrexate is
considered to be 30 mg/m2, significantly higher than 5.92 mg/mz. Thus, the above
recommended doses, combined with the transient nature of this rexate regimen, is
likely to be well-tolerated in adults. The exact dosage and regimen of methotrexate
should of course be established by a clinician, taking into t the patient’s physical
condition, age, weight, gender, other medications he/she is taking and their known side-
effects, and any other relevant factors. The effect of methotrexate on managing undesired
antibody responses in the patient can be monitored by well known s, including
clinical examination of the patient, symptoms, blood tests assaying ADA or anti-allograft
antibody titers, immunohistochemical assays (e.g., C4 tion assays and other hase
antibody detection methods such as the enzyme-linked immnuosorbent assay
(ELISA) and ased flurometric assays). The effect also can be red by
measuring levels of biomarkers such as MCP-l, IL-l3, IL-6, and IL-l2, which we have
shown to be reduced in level by methotrexate treatment, and transitional 2 B cells,
transitional 3 B cells, follicular B cells, marginal zone B cells, BlO B cells, and B1 B
cells, which we have shown to be increased in number by methotrexate treatment.
Additionally, TGF-beta, FoxP3, IL-5, IL-10, IL-15, and GM-CSF may be used as
biomarkers to monitor the effects of methotrexate on red immune responses as
needed. The levels of biomarkers also may be used to monitor the effects of methotrexate
on T cell responsiveness to a therapeutic (e.g., a protein therapeutic). Biomarkers for T
cell activation such as IL-2, interferon-y, and TNF-u, may also be monitored as readouts
for methotrexate’s effect on T cell responses.
Due to its ability to l undesired immune responses, the single cycle
methotrexate regimen of this invention can expand the use ofmany n therapeutics
whose repeated uses in a given t have been limited in the past due to safety and
efficacy concerns. For example, the concomitant use of methotrexate with
Thymoglobulin® may expand the utility of Thymoglobulin® to other disease settings
where re-dosing is desired, such as T cell-mediated autoimmune diseases including, but
not limited to, diabetes, lupus, scleroderma, rheumatoid arthritis, psoriasis and multiple
sclerosis. In addition, methotrexate may expand upon the efficacy and safety of
alemtuzumab, for example, in autoimmune diseases such as le sclerosis, wherein
alemtuzumab is usually administered in repeated annual cycles, or in chronic B-cell
lymphocytic ia, wherein alemtuzumab is administered in a 12-week cycle, dosing
starting at 3 mg/day (until the on reactions are equal to or less than grade 2), then
scaling up to 10 mg/day (until the infusion reactions are equal to or less than grade 2), and
finally moving up to 30 mg/day (on alternate days, 3 times weekly). These types of
dosing regimens may potentiate inhibitory ADA responses. Methotrexate may thus be
used to l ADA and any other undesired immune responses.
Improving Lymphocyte-Depleting Therapy with Methotrexate
An exemplary application of this invention is to use methotrexate to e
lymphocyte-depletion therapy such as alemtuzumab therapy in treating multiple sclerosis
(MS), such as relapsing-remitting MS. “Lymphocyte depletion” is a type of
immunosuppression by reduction of ating lymphocytes, e.g., T cells and/or B cells,
ing in lymphopenia. eutically, lymphocyte depletion can be achieved by a
protein therapeutic such as Thymoglobulin®, humanized anti-CD52 monoclonal antibody
H-lH® (alemtuzumab), and rituximab. Lymphocyte depletion is desired in
treatment of a number of autoimmune conditions, including multiple sis (Coles et
al., Ann. Neurol. 46, 296- 304 (1999); Coles et al., 2008), rheumatoid arthritis, vasculitis,
and lupus.
cyte depletion therapy may cause secondary autoimmunity.
Autoimmunity is referred to herein as “secondary munity” when it arises
subsequent to the onset of a first (“primary”) disease, for example, a ry”
autoimmune disease. Secondary autoimmunity sometimes arises in MS patients having,
or having had, lymphopenia following, e. g., lymphocyte depleting therapy. In some
individuals, ary autoimmunity arises soon after cyte depleting therapy (e.g.,
treatment with alemtuzumab). In other individuals, secondary autoimmunity may not
arise until months or years after lymphocyte depleting therapy; in some of those
individuals, by the time they develop secondary immunity, substantial lymphocyte
recovery (total lymphocyte count) may have occurred so that they may no longer be
penic. cyte depletion may occur in the context of treatment with antibody
therapeutics or small molecule therapeutics.
Secondary autoimmunity arising in lymphopenic MS patients can be any type of
autoimmune condition other than MS, including but not limited to thyroid autoimmunity
(e. g., Graves’ e), thrombocytopenic purpura, immune thrombocytopenia (ITP),
Goodpasture’s disease, autoimmune neutropenia, autoimmune tic anemia, and
autoimmune lymphopenia. In some embodiments, the secondary autoimmunity is B cell
mediated, z'.e., B cell responses and auto-antibodies are directly linked with disease
development and pathology.
[0106] Techniques for diagnosing and monitoring these autoimmune diseases are well
known to those skilled in the art, including assessment of symptoms and medical
ation such as blood analysis. The invention contemplates the use of any known
methods. For e, autoantibody levels in a patient's body fluid (e. g., blood) can be
determined as a means of detecting signs of autoimmunity. cally, anti-nuclear
antibodies, mooth muscle antibodies, and anti-mitochrondrial antibodies can be
measured. In the event anti-nuclear dies are detected, additional assays can be
performed to measure anti-double-stranded DNA antibodies, anti-ribonucleoprotein
antibodies, and anti-La antibodies. Anti-thyroid peroxidase (TPO) and anti-thyroid
stimulating hormone (TSH) receptor antibodies can be measured to detect autoimmune
thyroid diseases; if anti-TPO or anti-TSH receptor antibodies are detected, one can
measure r d function is affected by measuring free T3, free T4 and TSH
levels. Anti-platelet antibodies can be measured to detect autoimmune thrombocytopenia,
and a measurement of blood platelet levels may serve to determine if the presence of anti-
platelet antibodies is causing a reduction in platelet number. See also .
[0107] The single cycle methotrexate regimen of this invention can be used to improve
the safety and efficacy of lymphocyte-depleting therapy by reducing ADA as well as
minimizing secondary autoimmunity. Without g to be bound by theory, we believe
that rexate may reduce secondary autoimmunity by tolerizing multiple
autoantigens simultaneously.
Unless ise defined, all technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the art to which this
invention belongs. Exemplary methods and materials are described below, although
methods and materials similar or equivalent to those described herein can also be used in
the practice or testing of the present invention. All publications and other references
mentioned herein are incorporated by reference in their entirety. In case of conflict, the
present specification, including definitions, will l. Although a number of
documents are cited herein, this citation does not tute an admission that any of these
documents forms part of the common general knowledge in the art. Throughout this
specification and claims, the word “comprise,” or variations such as “comprises” or
ising” will be understood to imply the ion of a stated integer or group of
integers but not the exclusion of any other integer or group of integers. The materials,
methods, and examples are illustrative only and not ed to be limiting.
EXAMPLES
Further details of the invention will be described in the following non-limiting
examples. It should be understood that these examples, while indicating preferred
embodiments of the invention, are given by way of illustration only, and should not be
construed as limiting the appended embodiments. From the present disclosure and these
examples, one skilled in the art can ascertain certain characteristics of this invention, and
without departing from the spirit and scope thereof, can make s changes and
modifications of the invention to adapt it to various usages and conditions. Materials and
methods used in these working examples are described as follows.
Mice
Normal female C57BL/6 mice between 6 and 12 weeks of age were used for the
in vivo studies of rabbit anti-murine thymocyte globulin onal antibody (mATG) and
were obtained from n Laboratories (Bar , ME) or Taconic Laboratories
n, NY). Alemtuzumab-related studies employed human CD52 (huCD52)
transgenic (Tg) mice between 6-12 weeks of age that were obtained from Charles River
Laboratories/Genzyme Corp. Mice were housed and maintained in accordance with the
Guide for Care and Use of Laboratory Animals and under American Association for
Accreditation of Laboratory Animals Care I accreditation and all animal protocols used in
these studies were approved by the Institutional Animals Care and Use Committee.
The huCD52 Tg mouse used for nonclinical pharmacology studies were
generated by Xenogen (Cranbury, NJ, USA). To generate the mouse, a bacmid construct
containing approximately 145 kilobases of genomic DNA from human chromosome 1
was randomly integrated into the mouse genome of CD-l embryonic stem cells. By
virtue of the span of human genomic DNA that this bacmid contained, the construct
included a total of 5 partial or full genes of unknown function in on to human
CD52. The 5 partial or full gene ts contained in the bacmid were as follows: the
human CD52 gene, the 3’ end of a novel gene (DKFZP434L01 17), the SH3BGRL3 gene
(SH3 domain binding glutamic ich protein like 3), the gene for socius (SOC), the
AIMlL (absent in melanoma l-like) gene, and the zinc finger protein 683 gene. Three
founder lines were generated and line 107 was established at Genzyme.
Antibody administration
Polyclonal dies mATG and rbIgG were prepared as described in Ruzek et
al., lantation, 88(2): 170-9 (2009), and administered by intraperitoneal injection in
various regimens depending upon the experiment. Monoclonal antibody alemtuzumab
was administered intravenously as either a single injection of 0.5mg/kg or in either a three
or five day cycle of 0.5mg/kg/day.
Myozyme® treatment
Recombinant human alglucosidase alfa (rhGAA, marketed by Genzyme Corp. as
Myozyme®) was used as a formulated drug product. Mice were treated weekly with 20
mg/kg ofrhGAA by bolus tail vein injection unless stated differently. All mice were
treated prophylactically with 5 to 30 mg/kg diphenhydramine (Baxter Healthcare
Corporation, Deerfield, IL) intraperitoneally prior to rhGAA administration. Control
animals were treated intravenously with either sterile 0.9% saline or formulation
buffer.
Methotrexate treatment
Methotrexate (Calbiochem catalog #454125) was administered at 0.5, 1.0, 2.0 or
mg/kg by intraperitoneal ion for l-3 cycles, where each cycle equals either three,
six or seven consecutive days of injection depending upon the experiment. In studies that
involved monthly mATG treatment, methotrexate was stered intraperitoneally at 5
mg/kg at 0, 24, and 48 hours following either the initial mATG treatment or the first three
mATG treatments. In lant studies where mATG was dosed at days 0 and 4,
methotrexate was given daily at 2 mg/kg from days 0 to 6, daily at 0.5 mg/kg from days 0
to 6, or daily at 0.5mg/kg from days 0 to 11.
mATG treatment
Polyclonal dy mATG was administered as an intraperitoneal injection of 5
mg/kg every four weeks or as two 20 mg/kg doses given four days apart when in the
transplant setting with the first dose given on the day of lant (day 0).
Cell preparations from various tissues
For splenocyte and lymph node cell preparations -cell suspensions were
generated from harvested mouse spleens or al and mesenteric lymph nodes by
homogenization between frosted glass slides into PBS containing 2% FCS. For
splenocyte preparations, red blood cells were lysed by 1-2 minute tion with a red
blood cell lysis solution (BD Biosciences, San Diego, CA). Blood was isolated by retro-
orbital bleeding and cell preparations were performed by lysing red blood cells with red
blood cell lysing solution (BD Biosciences) for inutes. For all tissue preparations,
live cells were enumerated using the ViCell automated counter (Beckman Coulter,
Fullerton, CA). Following isolation, all cell preparations were washed with PBS/2% FCS
prior to use in the assays described below.
Flow try
For evaluation of cell populations within different tissues, single cell
suspensions of the s were incubated with fluorochrome-conjugated dies that
included anti-mouse CD4, CD8, CD25, CD44, CD62L (all antibodies from BD
Biosciences or eBioscience, San Diego, CA). Intracellular Foxp3 expression analysis was
performed according to the anti-Foxp3 manufacturer’s protocol (eBiosciences, San
Diego, CA). Following incubation with the antibodies, cells were washed and analyzed
by flow cytometry (FACSCanto, BD Biosciences and FCS Express software, De Novo
Software, Los Angeles, CA).
Cell populations evaluated were defined as follows:
total CD4 T cells: CD4WCD8',
total CD8 T cells: CD877CD4',
CD4 na'ive cells: CD4+CD25'CD62L+CD44',
CD4 memory cells: CD4+CD25'CD62L'CD44+,
naive CD8 T cells: CD8+CD44'CD62L+,
CD8 memory cells: CD8+CD44+CD62L',
tory T cells: CD4+CD25+Foxp3+,
total B cells: CD191
B2/follicular B cells: CDl9+CD21iH‘CD23hi,
Bl B cells: CD19+CD43+CD11b+,
transitional l B cells: CD19 CD93 CD23'Ithi,
transitional 2 B cells: CD19 CD93 CD23 Ithi,
transitional 3 B cells: CD19 CD93 CD23 IngO,
marginal zone B cells: CDl9+CD2lhiCD231°, and
B10 B cells: CD19+CD5+CD1d+.
In vitro blocking studies determined that up to 100 ug/ml mATG did not prevent
ion of these populations.
Anti-mATG IgG ELISA
The levels of anti-mATG IgG in mouse serum were analyzed by enzyme linked
immunosorbent assay (ELISA). , l plates (Corning Inc., Corning, NY,
USA) were coated overnight with l ug/ml of rabbit IgG in phosphate buffered saline
(PBS). Following blocking with Super Block Blocking Buffer (Thermo Scientific,
Rockford, IL, USA) serial dilutions of serum were added in duplicate to rabbit IgG-
coated plates and incubated at 37°C for l h. The plates were washed and horseradish
peroxidase-conjugated goat anti-mouse IgG secondary antibody (Southern Biotechnology
Associates, Birmingham, AL, USA) was added and allowed to te for l h at 37°C.
Following a final wash, 3,3’,5,5’-tetramethylbenzidine substrate (BioFx, Owings Mills,
MD, USA) was added and allowed to develop for 15 min at room temperature. The
on was stopped by the addition of l N HCl and absorbance values were read at
0 nm on an ELISA plate reader (Molecular Devices, Sunnyvale, CA, USA). End-
point titers were defined as the lowest dilution that averages above an absorbance of
0.100 using Softmax software ular Devices, Sunnyvale, CA, USA).
mATG-specific IgG ELISA
Mouse serum was determined by ELISA. Briefly, 96-well plates (Corning Inc.,
Corning, NY, USA) were coated overnight with l ug/ml of goat anti-rabbit IgG-Fc
fragment dy (Bethyl Laboratories, TX, USA). Following blocking with 0.5% BSA
(high purity), standard controls and serum samples were diluted as necessary and added in
duplicate to the wells of the coated plates and incubated at 36-3 8°C for 1 hour with gentle
shaking. The plates were washed and horseradish peroxidase-conjugated goat anti-rabbit
IgG-Fc fragment antibody (Bethyl Laboratories, TX, USA) was added as appropriate and
incubated for 1 hour at 36-3 8°C with gentle shaking. Following a final wash, 3,3’,5,5’-
tetramethylbenzidine ate (BioFx, Owings Mills, MD, USA) was added and allowed
to develop for 15 min at 23-25°C. The reaction was stopped by adding 1 N HCL and
absorbance values were read at 450/650 nm on an ELISA plate reader (Molecular
Devices, Sunnyvale, CA, USA). Final concentrations were interpolated off the standard
curve. It was predetermined that the measurement of mATG-specific IgG by this method
were only modestly affected by titers of anti-mATG IgG that were greater than 218,000.
lemtuzumab IgG ELISA
Mice were bled 4—6 days ing alemtuzumab treatment and ic anti-
alemtuzumab IgG was measured by ELISA. Briefly, 96-well plates (Corning Inc.,
Corning, NY, USA) were coated overnight with 3 ug/ml of alemtuzumab in PBS (pH
7.2). Following blocking with 0.1% BSA in PBS, serial dilutions of serum were added in
ate to alemtuzumab-coated plates and incubated at 37°C for 1 h. The plates were
washed, and horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody
(Southern Biotechnology Associates, Birmingham, AL, USA) was added and allowed to
incubate for 1 h at 37°C. Following a final wash, TMB substrate (BioFx, Owings Mills,
MD, USA) was added and allowed to develop for 15 min at room temperature. The
on was stopped by the addition of 1 N HCl and absorbance values were read at
450/650 nm on an ELISA plate reader ular Devices, Sunnyvale, CA, USA). End-
point titers were defined as the antilog of the logarithmically transformed sample dilution
interpolated at an absorbance value of 0.2 using Excel software (Microsoft, Redmond,
WA, USA).
Anti-rhGAA IgG ELISA
Mice were bled 4-6 days following rhGAA treatment and specific IgG was
measured by ELISA. Briefly, 96 well plates (Corning Inc., Corning, NY, USA) were
coated overnight with 5 ug/ml ofrhGAA in sodium acetate buffer (pH 5.0). Following
blocking with 0.1% BSA in PBS, serial dilutions of serum were added in duplicate to
rhGAA-coated plates and incubated at 37°C for 1 hour. The plates were , and
njugated goat anti-mouse IgG secondary dy (Southern Biotechnology
Associates, Birmingham, AL) was added and allowed to incubate for 1 hour at 37°C.
Following a final wash, 3,3’,5,5’-tetramethylbenzidine ate (TMB, KPL,
Gaithersburg, MD) was added and allowed to develop for 15 minutes at room
temperature. The reaction was stopped by the addition of 1N HCl and absorbance values
were read at 450/650 nm on an ELISA plate reader ular Devices, Sunnyvale, CA).
Endpoint titers were defined as the reciprocal of the sample dilution resulting in an
absorbance value of 0.2 using SoftmaX software ular Devices, ale, CA).
Ex vivo studies
C57BL/6 (Jackson Laboratories) or E4GAAKO (Charles River) mice 8-12
weeks old were given 5 mg/kg of methotrexate (Calbiochem catalog #454125) by
intraperitoneal injection for 1-3 cycles, where 1 cycle equals 3 consecutive injection days.
mg/kg of Myozyme® (Genzyme Corporation) was given by tail vein ion once or
for 2-6 weekly doses, commencing with the first methotrexate dose. Animals were
sacrificed weekly or daily after initiation of treatment. Spleen, eric, and inguinal
lymph nodes were collected for flow cytometric analysis of T and B cell subsets and sera
was collected for ELISA assays. Spleens were sed between glass slides and red
blood cells (RBC) was lysed with lysing buffer purchased from BD Biosciences
(catalog#555899) according to the manufacturer’s instructions. Lymph nodes were
processed between glass slides and washed with phosphate ed saline (PBS)
containing 2% fetal calf serum (FCS). Cells were resuspended in 200 uL of PBS
containing 4% fetal bovine serum and 25 ug/mL of total mouse IgG and blocked for 30
s at 4 CC. Approximately 3 million spleens cells and 1 million lymph node cells
were stained with different antibody cocktails and analyzed with a high h put
sampler (HTS) on a Becton Dickinson CANTOII flow cytometer. At least 100,000 cell
events were acquired within the lymphocyte gate. Anti-mouse dy cocktails
consisted of PE-CD21/35 catalog #552957, FITC- g #553138, PE- CD138 catalog
#553714, PE- CD127 catalog #552543, APC-Cy7-CD19catalog #557655, FITC-CD43
catalog #553270, PE-Cy7-CD4 catalog #552775, FITC-CD3e catalog #553062, APC-
CD11b catalog #553312, PE-Cy7-IgM catalog 7, APC-Cy7-CD8 catalog
#557654, PE-CD273 (PD-L2) catalog #557796, APC-CD138 catalog #558626, PE-Cy7-
CD11b catalog #552850, PE-CD93 (early B lineage) catalog #558039, APC-CD69
catalog #560689, Pe-Cy7-CD24 catalog #560536, FITC-CDld catalog #553845, APC-
CD5 catalog #550035, and Cy5-7AAD catalog #559925, all purchased from BD
Pharmingen. FITC-FoxP3 intracellular staining kit was purchased from eBioscience.
Pacific Blue (PB)- CD25 catalog #102022, PB-CD23 catalog #101616 and PB-CD86
catalog #105022 were purchased from BioLegend. is of lymphocyte subsets was
performed with FCS express version 3 software provided by De Novo Software.
Percentages were generated with the batch sing option and absolute numbers were
calculated according to the cell counts obtained. Spleen and lymph node cell counts were
obtained with a Beckman Coulter Vi-cell XR cell viability analyzer ing to the
manufacturer’s instructions.
In vitro and cytokine analysis
C57BL/6 (Jackson Laboratories) or E4GAAKO (Charles River) mice 8-12
weeks old were given 5 mg/kg of methotrexate (Calbiochem catalog #454125) by
intraperitoneal injection for 1 cycle (3 consecutive daily doses) commencing with
treatment with 20 mg/kg of rhGAA. For the 1D11 studies, animals were treated with
intraperitoneal injections of 5 mg/kg of either 1D11 or 13C4 (Genzyme ation) 3
time per week, every other day, cing with rhGAA and methotrexate treatment.
Animals were sacrificed on day 6 or 7 post rhGAA initiation depending on the mouse
strain. Spleens were prepared in single cell suspension and loaded onto the RoboSep
(STEMCELL technologies) instrument ing to the manufacturer’s instructions and
subjected to B cell negative selection. Purifled B cells were seeded at 500,000 cells per
well in 96 well round bottom plates (Costar catalog #3799) and either incubated with no
stimulation or with 10 ug/mL of LPS (Sigma g #L5014) for 48 hours at 37 CC. All
wells received Monensin (BD Bioscience catalog #554724) according to the
manufacturer’s instructions. Cells were allowed to incubate for at least 4 hours at 37 oC.
Samples were transferred to V bottom wells (USA Scientific catalog #651201) and spun
at 1200 rpm for 5 minutes at 4 c’C. Cells were ended in 200 uL of PBS containing
4% fetal bovine serum and 25 ug/ml of total mouse IgG and blocked for 30 s at 4
oC. Plates were spun again and ended in 90 uL of PBS/2%FCS with the addition
of 10 ul of antibody cocktail as bed above, and incubated for 20 minutes at 4 0C
with the addition of 5 uL 7AAD for the last 10 minutes of the staining ure.
Addition of 100 uL of buffer to the samples with subsequent spin was used as a wash.
Samples could be resuspended in buffer for surface analysis of protein and immediate
acquisition or resuspended in Fix/Perm (eBioscience catalog #11-5773) for intracellular
ng of IL-10 (BioLegend catalog #505008), TGF-beta (BioLegend catalog #141404)
and FoxP3 (eBioscience catalog #1182) according to the manufacturer’s
instructions. Additional e staining included TGF-beta and Tim-1 (BioLegend
catalog #119506) antibodies. All samples were acquired and analyzed as described
above.
Animals and cardiac allograft model
C57BL/6 and BALB/c mice were obtained from Charles River (Kingston, NY or
Raleigh, NC) and used in these experiments between 8 and 13 weeks of age. The donor
allogenic C57BL/6 mice were first anesthetized with an intraperitoneal injection of
Ketamine (Fort Dodge Animal /Pfizer, Fort Dodge, IA) and Xylazine (Lloyd,
Shenandoah, IA) and a median stemotomy was performed. The donor heart was slowly
perfused in situ with 1 ml of cold heparinized Ringer's e solution (Baxter
Healthcare, Deerfield, IL) through the inferior vena cava and aorta before the or
vena cava and pulmonary veins were ligated and divided. The ascending aorta and
ary artery were then transected, the graft removed from the donor and the heart
was stored in ice-cold saline until engraftment. A recipient mouse (Balb/c) was similarly
anesthetized and prepped as described above for donor mice, except that the abdominal
cavity was opened. Using a surgical microscope to view the opened abdominal cavity,
the abdominal aorta (AA) and the or vena cava (IVC) was isolated. The donor heart
was placed into the ent abdomen (upside down) and the grafts revascularized with
end-to-side anastomoses between the donor pulmonary artery and the recipient inferior
vena cava, as well as the donor's aorta and the ent abdominal aorta. After
hemostasis was med, the abdominal muscle was closed with a running 5-O Vicryl
suture (Ethicon, Johnson & Johnson, Somerville, NJ), and the skin closed with running 5-
0 Ethilon suture (Ethicon). rd post-op pain assessment and ment was
performed. Grafts were assessed by palpation 5-7 times per week for the first 30 day, and
then 3-4 times per week until the end of the study.
C57Bl/6 mice were treated with one 20 mg/kg intravenous dose ofrhGAA
(Genzyme Corporation), 3 consecutive intraperitoneal doses of 5 mg/kg methotrexate
(APP Pharmaceuticals, LLC), and either 1D11 or 13C4 at 5 mg/kg (Genzyme
Corporation) for 3 doses every other day. Methotrexate, 1D11, and 13C4 treatment
commenced with rhGAA injections. Spleens were collected 7 days after treatment
initiation and were processed as described above for B cells, culture and flow analysis.
Additionally, rhGAA titer data was ed by treating animals as described above, with
rhGAA dosed weekly over 12 weeks, methotrexate dosed for either 1 or 3 cycles, and
1D11 or 13C4 dosed 3 times a week every other day for 12 weeks. Serum samples were
ted every 2 weeks for ELISA analysis.
Histopathology and Immunohistochemistry
Cardiac grafts were fixed in 10% neutral buffered formalin, bisected along the
udinal axis to expose the right and left ventricles and the outflow tract, and
routinely processed for paraffin embedding. Sections were cut at 5 microns and were
stained with hematoxylin and eosin (H&E) or ’s trichrome. Serial sections were
also immunostained as described below. Each H&E-stained section was evaluated
qualitatively for various features of allograft rejection ogy (e.g., vasculitis,
myocardial degeneration and necrosis, myocarditis) using a histologic grading scheme.
[0129] Immunohistochemistry was performed using a Bond-Max automated
immunostaining system (Leica Microsystems Inc., Buffalo Grove, IL). To detect CD3
and Foxp3 dual immunopositive cells, graft tissue sections were ted to double
immunostaining with anti-CD3 and anti-Foxp3 antibodies using Bond Polymer Refine
Detection kit and Bond Polymer AP Red kit (Leica, Buffalo Grove, IL) following
cturer’s guidelines. Briefly, deparaffinized sections of paraffin-embedded grafts
were subjected to heat-induced epitope retrieval (25 min at 990C), incubated with serum
free protein block (Dako, Carpentaria, CA), rabbit monoclonal anti-CD3 antibody (Lab
/Neo ), peroxidase-conjugated polymer, dase block, and diamino
benizidene detection reagent followed by rat anti-mouse Foxp3 antibody (eBioscience
Inc., San Diego, CA) and then a rabbit at antibody (Vector Laboratories, Inc.,
Burlingame, CA). Slides were then incubated with Bond r AP and mixed red
detection reagent and y counterstained with hematoxylin. In negative control slides,
primary anti-CD3 and anti-Foxp3 antibodies were replaced with Chromepure whole
rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and rat IgG2a
(AbD Serotec, Raleigh, NC), respectively.
Serum tibody levels
Serum alloantibody levels were determined by incubating serum from cardiac
transplanted or normal mice at 1:50 dilutions with an SV40 transformed 6
fibroblast line (SVB6) followed by detection of st bound antibodies
(alloantibodies) using FITC rabbit anti-mouse IgG (Dako, Carpinteria, CA) and by flow
cytometric analysis. Geometric mean fluorescent intensities of serum stained fibroblasts
were d by isotype control stained fibroblasts to normalize alloantibody levels
between experiments. The g of serum antibodies to allogenic fibroblasts were
specifically alloantibodies because the same serum samples did not bind to a SV40
transformed BALB/c fibroblast line (SVBalb) (data not shown).
Adoptive transfer mouse model
C57BL/6 mice were obtained from Jackson tories and were housed under
specific pathogen free conditions. Control mice were given a single intravenous injection
ofrhGAA at 20 mg/kg. Tolerized mice were given a single intravenous injection of
rhGAA at 20 mg/kg, in addition to three daily consecutive methotrexate intraperitoneal
injections of 0.5 mg. Spleens were harvested on day six after the initial rhGAA injection,
from both donor , and processed into pooled single cell suspensions. Cells were
washed and filtered h a 0.22 uM filter. Cells were then enriched for B cells using a
StemCell logies RoboSep cell separation system, and resuspended to allow for a
200 ul intravenous injection. Tolerized and non-tolerized recipient groups received either
a high cell 10 x 106 or a low cell 5 x 106 concentration via intravenous injections. Control
groups were given rhGAA only, or rhGAA and rexate (single cycle of three
consecutive daily MTX ions). All groups received weekly intravenous rhGAA 20
mg/kg injections, and were retro-orbitally bled biweekly for 16 weeks, into BD
Vacutainer serum separator tubes. Serum was removed and stored below -20 0C until
used for ELISA. Anti-rhGAA antibody titers were determined using ELISA, read on a
SpectraMax M2, and calculated using Softmax to extrapolate titer value. Raw data
Softmax files and EXCEL spreadsheets containing control and titer information were
stored on k servers. All graphs and statistics were generated using GraphPad Prism
software.
Example 1: Anti-drug antibodies are produced in se to antibody therapeutics
Polycloncal dy mATG binds to a variety of immune cell types, including
antigen-presenting cells. Our data show that a single course ofmATG (two doses of 25
mg/kg given three days apart, administered intraperitoneally) could generate anti-mATG
IgG titers as high as 0 () in mice. When given as successive monthly
injections (5 mg/kg, every 4 weeks), anti-mATG titers were r increased with titers
up to 5X106 after five monthly injections (). For both the single course and the
monthly injections, rabbit IgG (rbIgG) was used as a control.
As alemtuzumab does not cross-react with murine CD52, preclnical studies with
zumab must be done in huCD52 Tg mice where the transgene expression pattern is
r to CD52 sion in humans. Similar to mATG, intravenous administration of
alemtuzumab (0.5 mg/kg) elicited significant ADA responses in huCD52 Tg mice. These
responses increased h the first four treatments and then declined such that
following the fifth dose of alemtuzumab, huCD52 Tg mice no longer generated anti-
alemtuzumab antibodies (. This non-responsiveness suggests that natural
tolerance had occurred (Rosenberg et al., Blood, 93(6):2081-8 (1999)).
The data show that the ADA titers against mATG in the C57BL/6 mice and the
ADA titers against alemtuzumab in the huCD52 Tg CD1 mice were high (>100,000).
This high level of immunogenicity may be attributed to the ability ofmATG and
alemtuzumab to bind antigen-presenting cells, thereby enhancing n processing and
presentation for inducing immune responses against them.
Example 2: Methotrexate controls anti-mATG IgG responses with a single cycle of
administration
Elevated antibody titers have been reported following Thymoglobulin®
treatment, and case reports of serum sickness, acute renal failure and cardiovascular
reactions have been described in patients treated with Thymoglobulin® (Boothpur et al.,
supra; Lundquist et al., Liver Transpl, 13(5):647-50 (2007); Busani et al., a
Anestesiol, 243-8 (2006); Tanriover et al., Transplantation, 80(2):279-81 (2005);
Buchler et al., Clin Transplant, 17(6):539-45 (2003)). To determine if methotrexate could
reduce anti-ATG ses, and thus, mitigate these safety concerns, a three-day regimen
of rexate, given only as a single cycle, was ted as a means of controlling
anti-mATG IgG responses in mice. This is distinct from the regimen that was previously
published in that only a single cycle of methotrexate was administered with mATG as
opposed to at least three cycles that were given in the t of ERTs. Methotrexate
(Calbiochem catalog #454125) administered intraperitoneally at 5mg/kg for six
consecutive days ng on the first of two mATG administrations (25mg/kg, 3 days
apart) could suppress anti-mATG IgG responses through at least eight weeks following
treatment by 95% when comparing area under the effect curves (.
Next, the effect of methotrexate on anti-mATG titers following five monthly
injections ofmATG at 5 mg/kg/injection was evaluated. Monthly injections ofmATG
treatment were performed because cyte repopulation is near complete one month
following mATG ent (Ruzek, supra). Anti-drug antibody responses were then
quantified weekly through 20 weeks of monthly treatments. During this period, despite
CD4+ T cell depletion by mATG, antibody titers reached as high as 5 n ().
Interestingly, animals that received non-specific rabbit IgG at the same dose level and
schedule as mATG showed low anti-rabbit IgG responses (). One possibility for
the ed immunogenicity ofmATG may be the specific binding ofmATG to antigen
presenting cells (APCs) such as follicular dendritic cells, which when in the presence of
complement may significantly enhance B cell responses. Two treatment regimens of
methotrexate also were evaluated. In previous work with enzyme-replacement therapy
(ERT), three cycles of methotrexate given during the first three doses of acid alphaglucosidase
provided a sustained reduction in antibody titer through at least eight months
ofweekly ERT dosing (Joseph et al., Clin Exp l, 152(1):138-46 (2008)). A
similar course of methotrexate was evaluated in the context ofmATG where 5 mg/kg of
rexate was given within 15 minutes ofmATG administration as well as 24 and 48
hours following each of the first three monthly mATG treatments. This regimen
successfully sed anti-mATG antibody responses from titers of approximately 4
million to titers of 816,000, yielding a reduction of 79% comparing area under the effect
curves ().
Additionally, the effect of a single course of methotrexate given around only the
first of five monthly mATG treatments on ATG IgG responses was evaluated and
compared directly with a three-cycle regimen. singly, this single cycle regimen
reduced anti-mATG IgG titers even r than the three-cycle regimen, to a titer of
approximately 50,000 (). Comparing area under the effect curves, the single
cycle regimen reduced anti-mATG IgG titers by 98%, while the three cycle regimen
reduced titers by 69% (). The increased effects of the single-cycle versus three-
cycle regimen suggests that increasing rexate exposure may actually antagonize its
tolerizing s, perhaps by killing the cells that are mediating the control over antibody
titer.
Example 3: Methotrexate induces an active mechanism of immune tolerance
As shown above, a very brief course of rexate can significantly control
antibody responses through multiple rounds of antigen challenge. In this context, the
brief cycle was a single cycle of methotrexate that produced lasting effects (on both
antibody titers and cytokine levels) over the course of months of testing. This long-lived
control of the antibody se suggests that methotrexate can successfully induce
immune tolerance. Methotrexate had thus far been evaluated in the context of five
consecutive monthly doses ofmATG. To r evaluate r an immune tolerance
mechanism had been activated, animals that originally received five monthly mATG
injections were withheld from treatment for eight weeks. Following this rest period, the
animals were given a final injection ofmATG. If a ism of immune tolerance was
employed, anti-mATG IgG titers should not increase significantly following the sixth
mATG treatment.
Our data show that animals that were not dosed with methotrexate experienced
an se in anti-mATG IgG titers, as expected (. By contrast, animals that
received just one cycle of methotrexate with the first injection ofmATG did not generate
significantly greater anti-mATG IgG titers (. A similar trend was observed in
animals treated with three cycles of methotrexate, though the effect was not as dramatic
(. When comparing area under the effect curves, the single course of methotrexate
reduced titers by 99% while the three cycle n reduced titers by 85%. These data
indicate that methotrexate can maintain control over recall responses, suggesting that the
mice have developed tolerance against this antigen.
Although rexate-treated animals generated measurable titers that increase
with successive mATG treatments, overall, titer levels in methotrexate-treated s
were consistently lOO-fold lower than those ed in animals treated with mATG
alone (. The lower level of antibody titers should significantly reduce the potential
for safety risks and efficacy effects. Although not wishing to be bound by theory, these
data suggest that an active mechanism of control has been induced that can cantly
reduce anti-mATG IgG responses, and is maintained long after methotrexate treatment.
Example 4: A single cycle of methotrexate can significantly control anti-
alemtuzumab responses
In ing-remitting multiple sclerosis, alemtuzumab is dosed in annual cycles
and patients can generate ADA (Coles et al., N Engl J Med, ): 1786-801 (2008)).
As immunogenicity and pharmacokinetic testing is ongoing in multiple phase III studies,
it is unclear whether lemtuzumab antibodies will impact exposure, efficacy, and/or
safety in a subset of ts. Thus, we evaluated whether a single cycle of methotrexate
could control ADA titers following five y single injection cycles of alemtuzumab.
HuCD52 Tg mice were given alemtuzumab (0.5 mg/kg) intravenously monthly for five
consecutive months. Methotrexate was given at 0.5, 1 or 5 mg/kg 15 minutes prior to the
first monthly umab dose as well as 24 and 48 hours after the dose. 1mg/kg of
methotrexate provided some benefit, as it reduced anti-alemtuzumab responses by 88%
(). 5 mg/kg of methotrexate successfully reduced anti-alemtuzumab IgG
responses by 99% (). rexate appeared to have no effect on natural
tolerance.
A second study confirmed the above findings. As above, huCD52 transgenic
mice were treated with five monthly doses of 0.5 mg/kg alemtuzumab. The mice also
were treated with or without three daily doses of 5 mg/kg/day of methotrexate in
connection with the first administration of alemtuzumab (). Serum samples were
collected throughout the study to assess anti-alemtuzumab titers and confirm tolerance.
Titer data were obtained at 24 hours after the fifth monthly dose. The data demonstrated
that methotrexate reduced anti-alemtuzumab antibody titers ().
Example 5: Methotrexate can l anti-alemtuzumab antibody responses in the
context of a clinically nt alemtuzmab dosing regimen
A series of experiments was ted to evaluate if methotrexate could
successfully control ADA in the context of a clinically-relevant dosing scheme of
alemtuzumab in huCD52 Tg mice. In the clinic, alemtuzumab is dosed as an initial cycle
of five daily treatments of 12 mg/day. Twelve months following the initial treatment
cycle in patients, an additional cycle of three daily 12 mg doses of zumab is
administered. At the time of the second treatment cycle, the levels of ating CD19+
B cells have recovered to baseline values; however, the levels of circulating CD4+ T
helper cells and CD8+ T xic cells have not fully repopulated (Coles et al., N Engl J
Med, 359(17):l786-801 (2008)).
Initially, the ion and repopulation kinetics of circulating T and B cell
subsets following five daily treatments of alemtuzumab was investigated in huCD52 Tg
mice. In the peripheral blood of huCD52 Tg mice treated with alemtuzumab for five
consecutive days with 0.5 mg/kg (equivalent to the 12 mg/kg human dosage) via
intravenous injection, total CD3+ T cells, CD4+ T helper cells, and CD8+ T cytotoxic cells
did not recover to pre-treatment levels four weeks following treatment, while the numbers
of CD19+ B cells returned to control levels (FIGS. 8A-D).
[0145] In order to simulate the cellular environment experienced by patients at the time
of retreatment, alemtuzumab was re-administered in huCD52 Tg mice between 4 and 5
weeks following the first cycle. Since the initial course of alemtuzumab was a five-day
cycle, rexate was administered 15 minutes prior to each daily alemtuzumab
treatment and for two days ards. The maximal cumulative cycle dose of
methotrexate given in this regimen is 14 mg/kg (2 mg/kg/day), which is very similar to
the cumulative dose of 15 mg/kg when methotrexate is given as a three-day course of 5
mg/kg/day. We evaluated the effects of 2, l and 0.5 mg/kg/day administration of
methotrexate on anti-alemtuzumab dies over three alemtuzumab treatment cycles.
At 1 mg/kg, methotrexate appeared to control titers in 7 of the 8 mice tested and l
reduced titers by 79% (). At 2 mg/kg, methotrexate successfully reduced ADA
by 98% when comparing area under the effect curves ().
e 6: Methotrexate can improve the pharmacokinetics and
pharmacodynamics of mATG
ADA can interfere with the pharmacokinetics and pharmacodynamics of protein
therapeutics. We evaluated whether anti-mATG IgG ADA responses ered with
mATG pharmacokinetics. Mice were treated for five months with monthly injections of
either mATG alone or mATG with a single cycle of methotrexate. Methotrexate was
administered at 5 mg/kg daily for three doses. Blood was sampled at various times after
month 1, month 3, and month 5 to assay the level of circulating mATG ().
[0147] At month 1, the levels of circulating mATG were similar among both treatments
, but at months 3 and 5 only the methotrexate-treated group had measurable levels
of circulating mATG. Without methotrexate administration, the levels ofmATG
measured ing the third and fifth mATG doses were significantly lower than those
measured after the first dose (). Previous studies have shown that methotrexate
significantly reduces anti-mATG IgG responses. Thus, it appears that antibodies against
mATG interfere with mATG exposure and pharmacokinetics.
As dies t mATG appear to significantly reduce the levels of
circulating mATG following repeat dosing, it may be expected that the
codynamics ofmATG when d is negatively affected as well. Lymphocyte
depletion in the blood, spleen and lymph nodes was evaluated after the fifth monthly
mATG treatment, when titers were at their highest. As described above, animals treated
with methotrexate were only given a single cycle of treatment.
[0149] Levels of circulating CD4+ and CD8+ T cells were unchanged after the fifth
mATG treatment in s d with mATG but not methotrexate. However, in
animals treated with mATG and one cycle of methotrexate, circulating CD4+ and CD8+ T
cells were significantly depleted (). This effect was similarly observed in spleen
and lymph nodes as well. Methotrexate treatment enhanced the ability ofmATG to
increase the percentage of T regulatory cells in the spleen and blood (FIGS. l2A-B). A
similar effect was observed in blood and lymph nodes. The enhanced presence of T
regulatory cells following mATG and Thymoglobulin® treatment has been postulated to
contribute to the efficacy of this therapeutic (Ruzek et al., Blood, 111(3): 1726-34 (2008)).
The ability of methotrexate to help maintain this effect following successive courses of
mATG is a potential added benefit of significantly reducing antibody anti-rabbit IgG
titers.
Thus far, pharmacokinetic and efficacy studies suggest that anti-mATG
antibodies interfere with the re and efficacy of mATG. A direct comparison
between anti-mATG IgG titer and mATG exposure reveals that when end-point titers are
greater than 10,000, the level of circulating mATG is significantly reduced (a).
er, when anti-mATG IgG titers are greater than 0, ediated cell
ion is inhibited (FIGS. 13b and 13c). The R2 correlation between titer and cell
depletion is > 0.7.
Example 7: Methotrexate can improve the pharmacodynamics of alemtuzumab
[0151] rexate not only enhances the pharmacodynamics ofmATG, but also
es alemtuzumab-mediated depletion of circulating T and B cells when anti-
alemtuzumab responses appear to neutralize some of the depleting activity. In the studies
described in this e, five monthly intravenous injections of alemtuzumab were
given to huCD52 Tg mice with and t rexate. 5 mg/kg of methotrexate was
administered daily for the first three days of the 6 month study. Blood was harvested
from animals of both treatment groups two days prior to the fifth dose and one day after
the fifth dose of alemtuzumab. Cell populations were ed by flow cytometry as
described in Example 6.
Our data show that the fifth monthly dose of zumab ed to no longer
deplete T cells, as the absolute number of circulating T cells seemed similar before and
after treatment ( (each time point represents a different set of animals)). By
contrast, methotrexate appeared to have ed the ability of alemtuzumab to deplete T
cells (P=0.012). When ing the absolute numbers of circulating T cells in animals
treated with zumab alone and animals treated with both alemtuzumab and
methotrexate at either two days prior to or one day following alemtuzumab dosing, the
numbers of circulating T cells were significantly lower in methotrexate-treated animals
(P=0.034 and 0.02; ). Similarly, methotrexate treatment appeared to enhance the
depletion of B cells by alemtuzumab (P: l.2x10'5, P= 0.02 respectively), and the number
of circulating B cells was decreased further in methotrexate-treated animals one day
ing treatment ().
Example 8: A single cycle of methotrexate can significantly control anti-rhGAA
antibody responses
[0153] We also studied the effect of methotrexate in rhGAA enzyme replacement
therapy. In this study, the animals were injected weekly with rhGAA for twelve
consecutive weeks, then rested for four weeks and then re-challenged with rhGAA at
week 16. The animals were also given a single cycle of three consecutive daily doses of 5
mg/kg methotrexate at week 1, or given one cycle at each of weeks 1, 2, and 3 (with a
total of three cycles). The specific IgG titers were measured in the animals at
weeks 0 (prior to any treatment), 6, 8, l2, 16, 18, and 20. Our data show that a single
cycle of methotrexate controlled anti-rhGAA responses through at least 20 weeks as well
as the three cycle regimen ().
Studies also have been conducted in T cell deficient Nu/Nu mice to evaluate the
role of T cells in generating anti-rhGAA titers. In these experiments, we have repeatedly
observed that little to no ADA to rhGAA develop in these T cell nt mice (FIGS.
37A-B). These data support the notion that T cells contribute to anti-rhGAA titers. Thus,
as methotrexate can control ADA to rhGAA, it is also likely affecting T cell responses to
rhGAA.
Example 9: Methotrexate enhances mATG-mediated survival of heart allogeneic
transplants
In addition to evaluating whether methotrexate could enhance the efficacy of
mATG in normal mice, we investigated whether mATG function could be augmented by
methotrexate in a transplantation setting. Since Thymoglobulin® is used clinically as an
induction therapy to prolong transplant survival, we evaluated whether the addition of
methotrexate could augment the efficacy ofmATG in a murine allogeneic heart transplant
model. 20 mg/kg ofmATG was administered on days 0 and 4, while 2 mg/kg
methotrexate was administered as a single cycle of treatment on days 0-6.
In addition, we investigated four fold lower doses of methotrexate (0.5 mg/kg)
given under the same regimen or with an extended regimen of 12 consecutive days.
Groups of mice either received no treatment (saline control), mATG alone, or a
combination of the mATG and methotrexate regimens. Similar to studies in normal mice,
methotrexate coadministered with mATG d anti-drug antibody titers to mATG
less of regimen used (A and Table 1). Moreover, coincidental to the
reduction in antibody titers was an ed increase in mATG exposure in this transplant
g (). Given the likely adjuvant effect under the ions of a potent,
coincident immune response against the transplanted tissue, the anti-drug antibody titers
increased even faster than in a normal mouse setting and resulted in mATG levels being
near undetectable within 7 days of the first mATG administration (B). By
contrast, by seven days following transplant, anti-rabbit IgG antibody titers were
significantly lower in mice treated with methotrexate, and circulating mATG levels were
significantly higher in those mice. This izes that under conditions of an ongoing
inflammatory se, anti-drug antibody responses can be accelerated and perhaps have
an even greater impact on pharmacodynamics and efficacy. Importantly, even under
these conditions, rexate had a profound inhibitory effect on mATG anti-drug
antibodies and enhanced mATG exposure. r, because circulating mATG levels
were still low to undetectable by 21 days with combination mATG and methotrexate
treatment, onal nce mechanisms are likely at play given the >100 day graft
survival. These results trate a remarkable synergy between mATG and
methotrexate treatment on the survival of the allogenic grafts and show a similar level of
reduction in mATG anti-drug antibodies and enhancement ofmATG exposure as
observed in normal mice.
Table 1: Methotrexate regimens reduce ATG antibody titers in cardiac allograft
transplant mice
.....................................\...........................................................................................................................................................................................\
\ \
Average anti -mATG Titers
>““““““““““““““““““‘S““““““““““““““““““E“““““““““““““““““““““““““““““““““““““““““““““““““““““““““““““““““““‘
Days mATG + MTX
post-transplant 3
‘ \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
E Saline mATG g g E
2 m/kg MTX § 0.5 mg/kg MTX § 0.5 mg/kg MTX
Days 0-6 § Days 0-6 § Days 0-11
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Day 14 0 1,880,820 13,500 43,740 5,400
1,202,850 § 79,380 n/a titttttttzgrtttttttttz n/a
92,340
*n/a = Not available
Additional data confirmed that the combination treatment ofmATG and
methotrexate cantly extended the survival of heart afts in addition to reducing
anti-allograft responses (FIGS. 17 and 18). , while both mATG or methotrexate
treatment alone provided a modest benefit of an average extended survival to 15 and 20
days, respectively, the co-administration ofmATG and any of the methotrexate regimens
evaluated demonstrated a dramatic benefit in cardiac graft survival with the majority of
mice retaining their grafts for up to over 100 days (). Since cardiac graft survival
continues long after the early, brief induction ents ofmATG and methotrexate, this
regimen s to be tolerogenic rather than immunosuppressive. The effect was unique
to the ation ofmATG and methotrexate, as other immunosuppressive agents (e.g.,
mycophenolate mofetil, dexamethasone, rapamycin and cyclophosphamide) failed to
cantly prolong graft al when co-administered with mATG. Remarkably,
methotrexate treatment alone was able to significantly reduce anti-allograft dies,
further substantiating the effects of methotrexate on controlling antibody responses in
general (). In this scenario, methotrexate appears to be able to control antibody
responses against multiple antigens of the heart allograft simultaneously. A further
reduction was induced by combining mATG with methotrexate treatment (C).
Example 10: The mechanism of methotrexate-induced nce is unique from the
currently known and accepted function of methotrexate
The dosing regimen of rexate bed in the Examples above appears to
invoke a ism that is unique from that previously bed. Methotrexate is a
folate antagonist that is thought to mediate its suppressive effects by inducing the death of
proliferating cells. mATG data presented above trates that antibody responses are
induced but remain significantly decreased with each successive mATG treatment in
methotrexate-treated s. These data suggest that B cell responses are ly
managed. Without wishing to be bound by theory, we hypothesize that methotrexate may
induce a regulatory cell population(s) that controls these responses as they occur. We
investigated the effect of methotrexate treatment on a variety of splenic B and T cell
subsets in animals d with Myozyme® and methotrexate ed with animals
treated with Myozyme® alone. We observed significant increases in the B10 regulatory
B cell population seven and eight days following Myozyme® treatment (four and five
days after methotrexate treatment; ).
In addition, a number of activated B cell subsets were significantly increased
following methotrexate treatment (). These populations included activated
marginal zone B cells, activated follicular B cells and activated transitional 2 and 3 B
cells. Cell populations were defined as s: B2/follicular B cells:
CD 1 9+CD2 l intCD23hi; transitional 2 B cells: CD 1 9+CD93+CD23WIthi; and transitional 3
B cells: CD19+CD93+CD23+IgM1°; al zone B cells: CD19"CD21hiCD231°. A daily
assessment of c cell populations in animals given two cycles of Myozyme® and
methotrexate, where Myozyme® was administered days 1 and 8 and 5 mg/kg of
methotrexate was given days 1-3 and 8-10, demonstrated that these activated B cell
populations remained increased (). This result is surprising because the expected
response after methotrexate treatment would be death of activated, proliferating cells. In
contrast, activated T helper, T cytotoxic and T regulatory cell populations remained
largely unchanged (). T helper cells were defined as CD4+, T cytotoxic were
defined as CD8+, and T regulatory cells were defined as CD4+CD25+ and FoxP3+.
These findings suggest that the increased B cell tions may help mediate
methotrexate-induced immune tolerance.
Example 11: Methotrexate increases selected B cell populations in combination with
mATG
In the above examples, after each mATG treatment, ADA titers in both the mice
treated with mATG alone and the mice treated with mATG and methotrexate increased,
suggesting that both sets of animals contain B cells that are capable of contributing to an
dy response (. If that is the case, at least two hypotheses can be drawn. A
first hypothesis is that methotrexate may have killed the vast majority of B cells capable
of ding to Myozyme®, and the few that remain are responsible for this slight
se. Although this is possible, flow cytometry data from multiple experiments have
not indicated a se in B cell tions following Myozyme® and methotrexate
treatment. A second hypothesis is that B cell populations that can respond to Myozyme®
are not killed by this brief course of methotrexate, but remain and are lled by
regulatory cells following each exposure to e®. Thus far, phenotypic data
describe an enhancement of B cell subsets following methotrexate and Myozyme®
treatment. The phenotype of these B cells seems similar to regulatory B cell subsets
which have been described in animal studies and in tolerant transplant patients. We
therefore sought to test this second esis in the context of different treatments.
Animals were treated with mATG monthly for five months and received either
a single cycle of 5 mg/kg of methotrexate on the first three days of the study, three cycles
of 5 mg/kg of methotrexate, or no methotrexate (). Differences in cell populations
among the three treatment groups were then assessed by comparing the populations in
s one day prior to the fifth mATG dose and two days after the fifth mATG dose.
singly, five months after treatment with a single cycle of methotrexate,
differences were observed between mice that received a single cycle of methotrexate and
mATG and mice that received either mATG alone or mATG with three cycles of
methotrexate. Two cell tions that unexpectedly demonstrated effects were
activated follicular B cells and activated transitional 3 B cells (FIGS. 24A-B,
tively). In mice treated with either mATG alone or in combination with three
cycles of methotrexate, decreases were observed in the absolute cell number of these cell
populations. However, in mice that received only a single cycle of methotrexate, no
statistically significant decreases in these populations observed, suggesting that this
dosing n of methotrexate induced some enrichment of these populations.
Interestingly, both of these cell populations were also shown to be enriched directly
ing methotrexate ent in combination with in Myozyme (). Similar
subsets also have been identified in tolerant transplant patients. It is possible that a single
cycle of methotrexate treatment can induce these B cell subsets which, upon antigen
re, become ted and suppressive.
Example 12: Methotrexate ses selected B cell populations in combination with
alemtuzumab
As described in Example 4, huCD52 transgenic mice were treated with single
monthly doses of 0.5 mg/kg alemtuzumab for five months, either with or without three
daily doses of 5 mg/kg/day of methotrexate in connection with the first administration of
alemtuzumab. Cell populations were evaluated in the blood and spleen of the mice 2 days
prior to, and l, 7 and/or 28 days following, the fifth dose of alemtuzumab by flow
cytometry.
In blood, the pharmacodynamic effect of alemtuzumab was enhanced in
methotrexate-treated animals 24 hours following the fifth dose of alemtuzumab.
Statistically cant cell depletion was observed in both T cell and B cell subsets one
day after the fifth dose, consistent with previous data indicating that anti-alemtuzumab
titers are low in these animals and would not be likely to interfere with zumab-
mediated depletion. By contrast, mice treated with alemtuzumab alone may have more
alemtuzumab-neutralizing antibodies that interfere with alemtuzumab
pharmacodynamics. As shown in A, there was no significant depletion of T cell
subsets in alemtuzumab-treated mice, but mice treated with alemtuzumab and
methotrexate exhibit significant alemtuzumab-mediated depletion in total T cells, T
helper cells and T regulatory cells one day following alemtuzumab dosing. Similar
findings were observed in circulating B cell subsets (B), although trends s
sing cell numbers were also ed in animals treated with alemtuzumab alone
As with circulating T cell populations, splenic T cell populations were significantly
depleted in methotrexate-treated animals one day following the fifth alemtuzumab
treatment (FIGS. .
In contrast to T cell depletion in mice treated with alemtuzumab and
methotrexate, each of the splenic B cell tions that were analyzed, except for
follicular B cells, were not significantly depleted (). This assessment includes the
regulatory B cell population, B10 B cells. Without wishing to be bound by , we
hypothesize that methotrexate may enrich some or all of these B cell populations, which
counters their alemtuzumab-mediated depletion. This ment is not expected to occur
in the fast, fluid environment of the blood because immune cells do not differentiate in
blood. Instead, immune responses occur in the spleen and other peripheral lymphoid
tissue where cell/cell interactions and cytokine/chemokine-initiated responses can occur
in the diverse niches of the tissue. This may explain the differential effects of the
alemtuzumab-mediated depletion of B cells observed in blood and spleen. These data are
similar to data generated with mATG, wherein splenic B cells appeared to be enriched in
mice treated with a single cycle of methotrexate in combination with mATG (A-
B). , in all of the studies described herein, enrichment following methotrexate
treatment has been observed when ing specific populations of ted cells.
However, when assessing total s of a cell population that es both activated
and non-activated cells, such as total follicular B cells, significant enrichment in
methotrexate-treated animals was not observed.
In contrast to splenic cell populations 24 hours after alemtuzumab treatment,
three days after treatment with a single dose of alemtuzumab (and not methotrexate),
splenic cell subsets were significantly depleted (A). It is possible that at 24 hours,
B cell depletion may be greater in alemtuzumab-treated s than at three days
following alemtuzumab treatment, as B cell repopulation may have begun by the threeday
mark. This has been demonstrated in l studies assessing these populations in
peripheral blood. Depletion was observed as early as three hours post-dosing in the
peripheral blood. By three days following treatment, circulating B cell pools were still
significantly lower in alemtuzumab-treated mice than in control mice treated with
phosphate buffered saline (PBS), although the percent depletion was not as great as that at
24 hours. The percent depletion at 24 hours was 92%, whereas the depletion at three days
was 36% (B). B cell reconstitution seems to occur rapidly following a single
dose.
In conclusion, five months after animals ed methotrexate and directly
after antigen exposure, it s that rexate may have enriched B cell populations
that potentially help mediate tolerance induction. The populations that appear enriched
are similar to those that are increased directly after methotrexate treatment ().
Taken together, this suggests that methotrexate may induce an environment that allows
immune ses to be actively controlled at the time of n exposure, even long
after the treatment with methotrexate.
Example 13: The effects of methotrexate on ne levels
Cytokines play a dual role in B cell responses. For instance, B-cell activating
cytokines IL-6 and BAFF are also required for B cell differentiation. Since methotrexate
seems to increase B cell populations, one may expect these cytokines to be increased.
However, IL-6 is also flammatory, and ore, elevated levels may interfere with
methotrexate-induced effects. Like IL-6, IL-10 is involved in B cell differentiation into
plasma cells and immunosuppression as well.
[0169] BAFF data were generated from serum s taken 24 hours after the 5th dose
of alemtuzumab (A). This is a continuation of cellular data from this study
presented above. At this time point no difference in BAFF levels were observed (B).
Table 2: Stud desin in mice treated with alemtuzumab and/or methotrexate
0.5 mg/kg alemtuzumab (x 5 days) 4M/4F
0.5 mg/kg alemtuzumab + methotrexate (0.5 mg/kg 4M/4F
for 8 days)
0.5 mg/kg alemtuzumab -- methotrexate (1 mg/kg 4M/4F
for 8 days)
0.5 mg/kg alemtuzumab -- methotrexate (2 mg/kg 4M/4F
for 8 days)
Cytokine levels were assessed one week after the second cycle of alemtuzumab
(B). Generally, one week after the second cycle of alemtuzumab, cytokine levels
appeared low. At this time point, a statistically significant increase was observed in TNF-
alpha levels in animals treated with 2 mg/kg of methotrexate. This increase may reflect a
change that is related to methotrexate-induced tolerance, or that is a sign of an
inflammatory response. Trends observed in other cytokines, such as apparent increases in
IL-6 and potentially slight ses in IL-7, also were noted ().
Regulatory B cells have been associated with IL-10 ion. One way to
assess whether IL-lO-secreting regulatory B cells play a role in methotrexate-induced
nce is to evaluate whether methotrexate can control antibody responses in IL-lO
deficient animals. This type of assessment may be challenging in that, as mentioned
above, IL-10 is necessary for plasma cell differentiation and therefore antibody responses
may be lower in these animals. With this caveat in mind, we observed sting trends
suggesting that IL-10 may play a role in methotrexate-induced tolerance.
In this study, animals received 20 mg/kg of intravenous Myozyme® weekly for
nine weeks. Three cycles of methotrexate were stered at 5 mg/kg/day 0, 24, and
48 hours after the first three weekly treatments of Myozyme®. Anti-Myozyme® titers
were assessed at weeks 4, 6, and 9 (). ing the average titer values in
rhGAA- and rhGAA/methotrexate-treated IL-lO knockout mice, a significant decrease in
titer was not observed, gh a slight trend was observed at week 9. As expected,
antibody titers were not as high in the IL-lO knockout animals as in the 6 wild-
type animals. Anti-rhGAA responses in C57BL/6 ype animals treated with rhGAA
and methotrexate were decreased at 4, 6 and 9 weeks. By contrast, anti-rhGAA titers at
weeks 4 and 6 were not decreased in IL-lO knockout animals that were treated with
rhGAA and methotrexate. At week 9, there was a slight decrease, which may indicate a
delayed induction of tolerance in IL-lO deficient mice. This would be consistent with
reports that IL-10 is not the only suppressive cytokine secreted by regulatory B cells
(Sagoo et al., J. Clin. Investigation; l20(6):l848-186l (2010)). TGF-beta also has been
ated with the regulatory B cell se. If there is such a delay, other cytokines
such as TGF-beta may be able to help mediate the methotrexate-tolerizing effect. These
data thus far suggest that IL-10 may play a role in methotrexate-induced tolerance.
Example 14: A role for methotrexate in the treatment of alemtumzab—associated
secondary munity
[0173] Alemtuzumab-treated multiple sclerosis patients can p secondary
autoimmunity. The most common autoimmune disorders that develop following
alemtuzumb treatment are those related to thyroid autoimmunity. In addition, immune
thrombocytopenic purpura and Good e’s syndrome also have been observed in
multiple sclerosis patients treated with alemtuzumab. All three types of autoimmunity are
B cell mediated in that B cell responses and auto-antibodies are directly linked with
disease development and pathology. The association of alemtuzumab treatment with the
development of these ary diseases is not well understood.
Following alemtuzumab treatment, T cells and B cells are ed. A large
percentage of these depleted T and B cells are likely to be auto-reactive cells that interact
with antigens expressed in the central nervous system. As a result, their uent
depletion by alemtuzumab is thought to contribute to the therapeutic benefit of this
monoclonal dy therapy. Patients suffering from autoimmune disease have been
described to contain autoreactivities (z'.e., autoreactive B cells and autoreactive
antibodies) against multiple antigens that are associated with a variety of autoimmune
diseases. Environmental, physiological, and c factors all contribute to the
determination of whether autoimmune disease will likely ensue and influence which
autoimmune disease will present in the patient. It is not uncommon for patients who
suffer from one type of autoimmune disease to also develop another.
In the context of alemtuzumab, one hypothesis is that inherent autoreactivities
that were not as prominent as those related to multiple sclerosis are allowed to expand in
the lymphocyte-depleted environment following alemtuzumab treatment. People that
develop an autoimmune disease typically have autoreactivites to a number of different
antigens and therefore a predisposition to develop other autoimmunities (z'.e., ent
activity”). In support of this idea are data in huCD52 Tg mice which show that
alemtuzumab does not lently e all B cell populations (). In fact, there
appears to be an nce in the B cell repertoire that favors a constituency of low
affinity autoreactive B cells, particularly marginal zone B cells. Importantly, marginal
zone B cells have been associated with thyroid autoimmunity (Segundo et al., Thyroid,
11(6):525-530 (2001). Additionally, the regulatory B cell subset, B10 B cells, which
have been shown to help quell autoimmunity in the murine model of multiple sclerosis,
EAE (Matsushita et al., J. Clin. Investigation, 118:3420-3430 (2008)), and have been
shown to exist in humans (Iwata et al., Blood, 117:530-541 (2011)) is depleted for longer
periods of time than marginal zone B cells. A short course of methotrexate treatment
during the first cycle of alemtuzumab may help increase the representation of regulatory
B10 B cells, and to restore the B cell balance such that marginal zone B cells are more
equally ented in the B cell repertoire ing alemtuzumab treatment and/or
entiate the marginal zone B cells into tory marginal zone B cells
(CD 1 d+marginal zone cells).
Splenic B cell populations were studied to determine the effects of
alemtuzumab on B cell depletion (). 0.5 mg/kg of alemtuzumab was administered
intravenously for five consecutive days in huCD52 Tg mice. Specifically, populations of
follicular B cells (which are lly not autoreactive), Bl B cells and marginal zone B
cells (both of which are autoreactive), BlO B cells (which are regulatory), and transitional
B cells and marginal zone B cells (which are thought to be able to differentiate into B
regulatory cells) were examined.
While follicular, B1, and regulatory B cells were ed at one and/or two
weeks following treatment with alemtuzumab, al zone B cells and transitional l
(Tl) and transitional 2 (T2) B cells were not depleted at any of the time-points ().
Transitional 3 B cells (T3) were only ed after one week of treatment, and the
numbers of regulatory B cells generally appear lower in zumab-treated mice than
in control-treated animals, although statistical icance was not observed at weeks 2
and 4.
[0178] To examine the effects of rexate on B cell populations in the context of
treatment with alemtuzumab, a second study was performed as indicated in Table 3 and
. Surprisingly, co-treatment of methotrexate with alemtuzumab may allow for a
stronger depletion of marginal zone B cells shortly following alemtuzumab treatment than
depletion observed with alemtuzumab alone (), thereby promoting a balanced
immune environment with the appropriate level of naturally occurring low-affinity self-
reactive B cells necessary for proper early se to infection.
A group of mice treated with alemtuzumab alone was included in this study,
and revealed that mice treated only with methotrexate in this short cycle regimen
exhibited cellular effects in the absence of antigen stimulation, which can be different
from the effects observed in control mice treated with methotrexate alone (). Data
generated in this study, wherein splenic B cell populations were assessed two days
following the last day of methotrexate treatment, suggest that rexate may enhance
the depletion of al zone B cells. This supports the hypothesis that methotrexate,
when delivered with alemtuzumab, may result in a ar environment wherein naturally
occurring B cell subsets are appropriately in e with non-autoreactive B cell subsets.
Table 3: Study design in mice treated with alemtuzumab and/or methotrexate
Animals Sacrifice
Grou points
Blood,
0.5 mg/kg l.- Lymphnode (LN)
alemtuzumab spleen
0.5 mg/kg Blood,
alemtuzumab + Day 4 LN
methotrexate (5 spleen
mg/kg)
0.5 mg/kg Day 64 LN, thyroid
3 5
alemtuzumab
0.5 mg/kg
zumab + Day 64 LN, thyroid
methotrexate (5
mg/kg)
0.5 mg/kg LN, thyroid
0.5 mg/kg
alemtuzumab + LN, d
methotrexate (5 Day 64
mg/kg)
Blood,
Day 4 LN
7 Methotrexate 5
spleen
Based on the daily assessment of cell populations that followed directly after
methotrexate treatment with e®, we hypothesize that in the context of
alemtuzumab, B10 B cell populations and other potentially regulatory B cell populations
will be enriched by methotrexate no earlier than 5-6 days following methotrexate
treatment. We have not observed such populations to be enriched as early as two days
after rexate ent, which is tent with this hypothesis. By contrast, the
effects seem to be different at longer time periods after methotrexate treatment, as seen in
the context ofmATG and alemtuzumab treatment. In both of those scenarios, five
months after methotrexate treatment, B cell subsets appeared enriched in methotrexate-
treated mice one and two days following mATG and alemtuzumab dosing (see
for alemtuzumab data). Interestingly, marginal zone B cells at this time point also
appeared increased (although this is not statistically significant).
rexate induces tolerance to protein therapies and transplanted c
tissue antigens, thereby abrogating B cell immune responses that relate to the production
and ion ofADA and anti-allograft antibodies. We therefore hypothesize that
methotrexate may not only help control the cellular environment following alemtuzumab
treatment, but that it also may induce tolerance to roteins and mitigate the B cell
immune responses that relate to the generation of auto-antibodies that contribute to the
development and pathology of B cell-mediated autoimmune diseases.
[0182] To test this hypothesis, animals were dosed monthly for five months with
alemtuzumab. Methotrexate was given to a group of animals at 5 mg/kg for three
consecutive days following only the first alemtuzumab treatment. Two days prior to and
one day following the fifth dose of alemtuzumab treatment, s were sacrificed for
is. Serum cytokine levels were assessed before and after alemtuzumab treatment.
Surprisingly, these results suggest that the levels of the pro-inflammatory cytokines MCP-
l, IL-l3, IL-6, and IL-12 were decreased in animals treated with methotrexate 24 hours
after the fifth dose of zumab, five months after being dosed with any methotrexate
(). These nes not only promote B cell responses and immune cell
recruitment, but they also can play a role in hypersensitivity reactions. These data
suggest that methotrexate may help deter on ated reactions.
Example 15: Methotrexate induces immune tolerance through specific induction of
regulatory B cell populations
Our data surprisingly show that methotrexate induces immune tolerance not by
the expected means of killing proliferating cells as suggested by others (Messinger et al.,
Genetics in Medicine 14:135-142 (2012) and Lacana et al., Am J Med Genet Part C
Semin Med Genet 160030-39 (2012)), but through the c induction of regulatory B
cells tions that express TGF-beta, IL-10 and FoxP3. B cells from mice tolerized to
Myozyme® by a single cycle of methotrexate appeared to transfer immune nce to
na'1've animals. Moreover, both IL-10 and ta appeared to be necessary for
methotrexate-induced immune tolerance. In some cell subsets it also appeared that
methotrexate induced TGF-beta, which in turn induced IL-10 and FoxP3. This
mechanism is novel and unexpected, and questions the value of a current clinical immune
tolerance protocol that involves co-treatment with three cycles of methotrexate,
Rituximab® (a B cell-depleting agent), and ally, intravenous immunoglobulin
(IVIG) nger et al., supra). Although this combination treatment appears to be
successful, our data t 1) that a single cycle of methotrexate may potentially
te even lower ADA titers than those currently observed, and 2) that if too much
methotrexate and rituximab are administered, immune tolerance may not be maintained.
The initial doses of rituximab may not be too harmful as rituximab-mediated B cell
depletion is not thought to comprehensively deplete all B cells in the blood and tissues.
As seen with the studies described herein, although alemtuzumab actively depletes B10 B
cells, treatment with rexate still is able to access those cells that seem to help
maintain alemtuzumab tolerance for many months following the initial cycle of
rexate. Moreover, rituximab treatment is rapidly followed by increased
representation of transitional B cells, which, as shown herein, seem influenced by
methotrexate to induce and mediate immune tolerance. As these mechanistic data are
counterintuitive and unexpected, a single cycle of low dose methotrexate is a surprising
and effective method of inducing immune nce to, inter alia, lymphocyte-depleting
protein therapies.
As described above, several B cell subsets are significantly increased in cell
percentage and/or cell numbers soon after co-administration of methotrexate with a
protein therapeutic. Moreover, these subsets appear increased in rexate-tolerized
mice long after a single cycle treatment of methotrexate (see, e. g., FIGS. 24, 27, and 35).
Together, these data suggest that these cell tions may actively mediate both the
induction and nance of immune tolerance induction. To fiarther substantiate this
hypothesis, we investigated whether these cell populations expressed cytokines and other
proteins often associated with immune regulation.
[0185] One cell type associated with immune regulation is B10 B cells. B10 B cells in
both human and mouse are terized by their expression of IL-10 (Matsushita et al.,
J. Clin. Invest. 118:3420-3430 (2008), Iwata et al., Blood 117:530-541 (2011)), and can
only suppress immune responses in IL-10 competent mice. B10 B cells were increased in
methotrexate-tolerized mice (), and IL-10 ut animals were unresponsive to
methotrexate-induced immune tolerance (). B10 B cells isolated from animals
treated with Myozyme® or Myozyme® and methotrexate were assessed for IL-lO n
sion by flow cytometry. Although IL-10 was expressed in B10 cells from both
treatment groups, the number of B10 B cells expressing IL-10 was increased in animals
treated with Myozyme® and rexate (). IL-10 was expressed in both
activated, CD86+ and non-activated, CD86- B10 B cells following two days of culture
(). Previous studies appear to show that IL-10 expression is measured only
following in vitro stimulation of cells by stimulants such as PMA/Ionomycin or LPS
(Carter et al., J Immunol 186:5569-5579 (2011); Yanaba et al., J Immunol 182:7459-
7472 (2009)). Surprisingly, in our s, the cultured c B cells did not need any
stimulation or manipulation to allow for the measurement of IL-10, more directly
suggesting that these B10 B cells s IL-10 in vivo. The data ed herein were
generated in non-stimulated cultures. In addition, we believe that we have demonstrated
for the first time that methotrexate can specifically expand cell populations that express
IL-10. er, these cell populations appear to express more IL-10 when isolated
from animals treated with Myozyme® and methotrexate than from animals treated with
Myozyme® alone ().
[0186] TGF-beta expression is associated with immune regulation in T regulatory cells
and is often linked with IL-10 expression in T regulatory cells. Moreover, some reports
seem to indicate that regulatory B cells can express TGF-beta. Although B10 B cells
have never been reported to express TGF-beta, we decided to assess TGF-beta sion
in B10 B cells of mice treated with Myozyme® alone or with Myozyme® and
methotrexate using flow cytometry. Unexpectedly, we found that B10 B cells express
TGF-beta, and that the s of TGF-beta-expressing cells is increased in
methotrexate-tolerized animals (A). Moreover, in these cultured cells, TGF-beta
is expressed in both activated (CD86+) and tivated (CD86-) B10 B cells (B). This is an additional novel observation that methotrexate treatment increases the
s of cells that express TGF-beta. Additionally, rexate increases the
expression level of TGF-beta ().
FoxP3 is another protein associated with immune regulation. FoxP3 is a marker
for T regulatory cells. FoxP3 has not been reported to be expressed in B10 B cells in
mice. We investigated FoxP3 expression in B10 B cells in the ce and absence of
methotrexate-induced immune tolerance by using flow cytometry. B10 B cells appear to
express FoxP3, as seen in animals treated with Myozyme® alone (A). The
numbers of FoxP3+ B cells appear to increase with treatment with both methotrexate and
Myozyme® (B). Additionally, both cultured activated (CD86+) and non-
activated (CD86-) B10 B cells appear to express FoxP3 (). This was the first
report that B10 B cells express FoxP3. We found that FoxP3 was expressed in both
activated ) and unactivated (CD86-) B10 B cells and the sion of FoxP3 is
increased with methotrexate treatment ().
e additional B cell-types are significantly increased with single cycle
methotrexate-induced immune tolerance, we assessed whether some of these cell types
expressed IL-10, TGF-beta and FoxP3. Transitional 2, transitional 3 and follicular B cells
were found to express IL-10 (), TGF-beta () and FoxP3 (), which
was novel and unexpected for each of those B cell subsets. As observed with the B 10
cells, the te cell numbers of the IL-10, TGF-beta, and FoxP3 B cell subsets were
increased with methotrexate (FIGS. 42-44 B and C) as compared to mice treated with
Myozyme® alone (FIGS. 42-44 A and C). Methotrexate treatment also induces
statistically significant increases in IL-10, TGFbeta and FoxP3 in multiple cell subsets as
viewed by the shift in mean fluorescence ity of these proteins in animals treated
with e® alone or Myozyme® and methotrexate (FIGS. 54-56).
To filrther investigate the multiple TGF-beta expressing B cell populations
enriched by treatment with methotrexate and Myozyme®, we next looked to determine
whether TGF-beta is required for methotrexate-induced immune tolerance. Animals were
treated with Myozyme® or Myozyme® and rexate with or without the presence of
mg/kg of anti-TGF-beta antibody (1D11, Genzyme) or the isotype control (13C4) given
three times per week via intraperitoneal ion throughout the study. Antibody titers
were assessed bi-weekly in four different groups of animals. If TGF-beta was required
for methotrexate-induced immune tolerance, then we would expect that animals treated
with the anti-TGF-beta antibody should not exhibit d anti-Myozyme . Week 6
titers are depicted in , and suggest that TGF-beta may be necessary for
rexate-induced immune tolerance. Because this is an early time point, only some of
the animals have had time to generate anti-Myozyme responses. Importantly, at this time
point titers appear similar to those shown in C at week 6, where two animals in
the rhGAA alone and the rhGAA- and methotrexate- and 1D11-treated animals exhibited
high titers. In comparison, none of the animals d with rhGAA and methotrexate or
rhGAA and methotrexate and 13C4 exhibited high titers.
Additionally, spleens were ed from animals treated with Myozyme® or
e® and methotrexate that also were co-administered lDll or l3C4 seven days
following a single e® treatment or a single e® and methotrexate
treatment. At this time point, transitional 2, transitional 3, B10 and follicular B cells that
expressed IL-lO, TGF-beta, and FoxP3 were increased in animals treated with
Myozyme® and methotrexate. Cells in each group were then pooled and cultured for two
days and then assessed by flow cytometry to determine whether the anti-TGF-beta
treatment with lDll interfered with the expansion of cells that expressed TGF-beta, IL-
, and FoxP3 in comparison to the e control antibody (l3C4).
[0191] Quite unexpectedly, lDll treatment interfered with not only the rexate-
induced expansion of cells expressing TGF-beta, but also with the expansion of some
subsets expressing IL-10 and FoxP3. This was true specifically for B10 B cells ()
and ular B cells (), though FoxP3+ Follicular B cells did not appear to
experience lDll effects. In transitional 2 B cells where lDll treatment interfered with
TGF-beta-expressing transitional 2 B cells, no effects were seen with IL-10+ tional
2 B cells (including activated transitional 2 B cells; ). Moreover, effects in
FoxP3+ transitional 2 B cells by lDll did not seem apparent in this small treatment
group unless one looks at activated, CD86+ transitional 2 B cells. Importantly, these data
suggest that methotrexate-induced TGF-beta is associated with IL-10 and FoxP3 in only
some cell types. For transitional 3 B cells, even though there is detectable TGF-beta in the
transitional 3 subset (), there is no apparent effect of lDll treatment on these
cells (P<0.05; ). Notably, in activated CD86+ transitional 3 B cells, there appear
to be higher numbers of IL-lO+ transitional 3 B cells in lDl ted mice. This may
suggest that this population might be expanding to try to help sate for losses in the
numbers of other cell- types due to lDll treatment. Also of note is that lDll treatment
did not affect basal levels of IL-10, TGF-beta, and FoxP3 in these cells (FIG. SOA-C,
respectively), but it appeared to influence methotrexate s on cells that express those
cytokines.
In summary, interfering with TGF-beta by injecting a TGF-beta antibody during
methotrexate-induced immune tolerance reduces the numbers of cells that are expressing
ta in comparison to animals d with the isotype control. Moreover, the
typical increases observed with methotrexate-induced tolerance in IL-10 and FoxP3-
expressing BlO B cells are inhibited by lDll treatment. lDll treatment appears to
influence methotrexate s on TGF-beta, IL-lO, and/or FoxP3 in certain B cell types,
but not all B cell types. These ations are surprising, and suggest that methotrexate
induces TGF-beta, which in turn induces IL-10 and potentially FoxP3 in certain cells,
such as BlO B cells. Although the association of TGF-beta with IL-10 and FoxP3
s to have been reported before, it has not been shown in these cell types.
er, methotrexate has not been simultaneously associated with this complex
signaling cascade.
Thus far, we have demonstrated that certain B cells are significantly increased
with methotrexate-induced tolerance, and that those B cells express proteins associated
with -regulation (suppression). To more directly evaluate if the B cells
themselves are mediating methotrexate-induced tolerance, we performed an adoptive
transfer experiment assessing whether total splenic B cells from methotrexate-tolerized
mice could transfer immune tolerance to na'ive hosts. We performed this experiment
using Myozyme® and a single cycle of methotrexate treatment. We isolated spleens from
animals treated with Myozyme® alone or with Myozyme® and methotrexate, 7 days after
a single treatment of Myozyme® or Myozyme® and methotrexate, (when the above-
mentioned B cell subsets were increased by methotrexate), and then ed all of the
splenic B cells. Those cells were then erred into e®-naive recipient mice
(A). After transfer, the recipients (along with non-transferred control animals
treated with either Myozyme® or Myozyme® and methotrexate) were treated weekly
with 20 mg/kg of Myozyme®. Blood was collected every other week to assess anti-
Myozyme antibody . At the time of harvesting spleens, a subset of B cells from each
donor group was assessed by flow cytometry to confirm the ed increases in TGF-
beta+, IL-lO+ and/or FoxP3+ transitional 2, transitional 3, B10 and follicular B cells.
Donor groups were confirmed to have the expected phenotype. Titer analysis suggested
that total splenic B cells isolated from animals treated with Myozyme® and a single cycle
of methotrexate could transfer immune tolerance to Myozyme® in naive hosts (B). This ts that B cells can mediate methotrexate-induced immune tolerance,
and was the first time that B cells have been described to be enriched (and not killed) by
methotrexate to help e anti-inflammatory effects. These data also ly question
the concomitant use of a B cell depleting agent with methotrexate to induce immune
nce, which is currently being performed in patients (Messinger et al., supra and
Lacana et al., supra).
Example 16: Methotrexate improves graft pathology
We also have generated additional data in the context of murine anti-thymocyte
globulin that demonstrates that methotrexate does not deplete CD4+, CD8+, T regulatory
D25+FoxP3+) T cells, and total CDl9+ B cells in normal animals () and
in transplant animals (). When comparing normal animals d with non-
specific rabbit IgG alone or in ation with methotrexate, there were no significant
changes in the cell tions in the blood and spleen (). Moreover, when
comparing those populations isolated from animals treated with mATG alone or with
mATG and methotrexate, there was no ed depletion. , we observed only an
extension of mATG-mediated CD4+, CD8+, and T regulatory cell effects, which was
most likely due to increased mATG exposure by methotrexate (). In addition,
methotrexate treatment alone in animals receiving an neic transplant did not deplete
those particular cell subsets (). The lower numbers of CD4+ and CD8+ T cells in
animals treated with mATG and methotrexate ed to animals treated with mATG
alone can be explained by the ged effect ofmATG when in the context of
methotrexate treatment. Importantly, methotrexate did not induce ses in B cells as
would have been expected if methotrexate was killing activated B cells to reduce
antibody responses in either normal or transplanted animals (FIGS. 52 and 53). These
data suggest that methotrexate effects on anti-drug antibodies may not be mediated by
anti-folate induced depletion of activated B cells. As expected, mATG treatment also did
not impact total B cell numbers in this heterotopic cardiac allograft model model.
Overall, the combined treatment ofmATG and methotrexate attenuated the severity of
graft rejection ogy and was associated with decreased T cell infiltrate in the graft,
but not with increases in cells with a regulatory T cell phenotype. This is consistent with
the results generated in the context of Myozyme® and alemtuzumab.
To both assess the histological changes in urviving grafts as well as to
understand the mechanism ofmATG and rexate combination on graft survival,
cardiac grafts were collected and evaluated for pathology as well as cellular composition.
In particular, because regulatory T cells have been associated with long-term graft
survival in transplantation, are induced by Thymoglobulin® and mATG, and have been
trated to be responsible for delayed graft rejection following mATG treatment,
CD3+Foxp3+ cells, which bear a phenotype consistent with T regulatory cells, were
evaluated. Specifically, c grafts were collected from the mATG and methotrexate
combination-treated group and the untreated syngeneic group at least 100 days after
transplantation. Grafts from untreated mice and mice treated with mATG alone or
methotrexate alone were taken after graft rejection for comparison. Tissue ns
stained with H&E or Masson’s trichrome or immunostained anti-CD3 and anti-Foxp3
antibodies were microscopically evaluated for histologic changes indicative of transplant
rejection, e.g., mild to moderate myocarditis, myocardial degeneration and necrosis,
cardiac allograft vasculopathy (CAV), and T cell infiltration. At day 100, allografts from
animals co-treated with mATG and methotrexate revealed minimal to mild CAV lesions
and no to minimal myocardial degeneration and myocarditis. Histological changes
suggestive of graft rejection were not apparent in eic grafts at this late time point
(). afts from the combination treated group exhibited mild T cell
infiltration in the myocardium with a few cells infiltrating the myocardial blood vessels.
eic grafts contained rare T cells within the myocardium. Clusters of T cells with
occasional dual CD3 and Foxp3 immunopositive cells were present in the dium of
both syngeneic grafts and combination-treated allografts. Thus, long-surviving grafts
showed minimal signs of graft rejection, which correlates with reduced inflammation.
[0196] Because the effects ofmATG and methotrexate combination treatment were
likely to more actively occur closer to the time of transplantation, we also evaluated
ogy and terized the cellular infiltrate of transplanted heart allografts at 7 (for
untreated mice) or 14 (for all treatment ) days after transplantation. Untreated
allografts displayed graft rejection pathology including myocarditis, myocardial
degeneration and is in both epicardial and intramyocardial branches of coronary
arteries. By contrast, allografts isolated from animals d with both mATG and
methotrexate ed less severe CAV. This was in comparison to allografts from
untreated animals as well as from animals treated with either mATG or methotrexate
(). As ed, syngeneic grafts from untreated mice revealed little or no
pathology at these time points. CD3+ T cell infiltration was observed in the myocardium
and epicardium in allografts from untreated mice and those treated with mATG or
rexate alone. A few CD3+ T cells also were present in the inflammatory cell
infiltrate associated with the CAV lesions in these grafts. By contrast, afts from
animals treated with the combination ofmATG and methotrexate exhibited substantially
lower CD3+ T cell infiltration in the myocardium and only minimal CD3+ T cell
ation in the epicardium. Syngeneic cardiac grafts showed minimal CD3+ T cell
infiltration only in the epicardium. A small proportion of T cells within the inflammatory
cell infiltrates in the epicardium appeared to have a T regulatory cell phenotype as
indicated by dual CD3 and Foxp3 immunoreactiVity, but this frequency appeared no
greater within inflammatory infiltrates than in other groups. Therefore, reduced
ogy also was observed early after treatment with mATG and methotrexate and was
ated with both reduced and epicardium-restricted T cell infiltration.
Claims (15)
1. Use of methotrexate and a protein therapeutic in the manufacture of a medicament for the treatment of a disease or condition of the immune system in a subject in need of said treatment, n the medicament is formulated such that in a single -cycle regim en of one or more dosages starting prior to, concurrently with or after a first dosage of said n therapeutic, and ending before a second dosage of the said protein therapeutic, the medicament is capable of ing an effective amount of methotrexat e thereby inducing immune rance to said protein therapeutic enabling treatment of said disease or condition of the immune system.
2. Use of claim 1 wherein the protein therapeutic is an antibody.
3. Use of claim 1 or 2 wherein the therapeutic is alemtuz umab and the disease or condition of the immune system is multiple sclerosis.
4. Use of claim 1 or 2 wherein the therapeutic is rituximab and the disease or condition of the immune system is an autoimmune condition.
5. Use of claim 1 or 2, wherein the therapeuti c is rabbit anti -thymocyte globulin antibody and the disease or condition of the immune system is tissue or organ transplantation, aplastic anemia, or graft -versus -host disease.
6. The use of any one of claims 1 -5, wherein the single cycle regimen consists of one dose of methotrexate 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 doses of methotrexate over 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 consecutive days.
7. The use of any one of claims 1 -6, wherein the methotrexate effect s: a. inhibition of dy ses to the n therapeutic in the subject; b. ation of an infusion reaction to the protein therapeutic in the subject; c. reduction of secondary autoimmunity in the subject; d. increase in the cy of the n t herapeutic in the subject; e. increase in the percentage of T regulatory cells in the T cell population in the subject; f. increase in the percentage of B regulatory cells in the B cell population in the subject; g. prolongation of the cokinetics of a therapeutic in the t; or h. inhibition of T cell response in the subject.
8. The use of any one of claims 1 -7 n the subject is a human.
9. The use of any one of claims 1 -8, wherein the m edicament comprises an amount of methotrexate of 0.1 mg/kg to 5 mg/kg.
10. The use of any one of claims 1 -9, wherein the medicament is formulated for release of methotrexate at between 48 hours prior to and 48 hours after the onset of the treatment.
11. The use of any one of claims 1 -10, wherein the medicament is formulated to deliver a further agent for immune modulation.
12. The use or the composition for use of claim 11, wherein the agent for immune modulation is an immunosuppressant.
13. The use of claim 3, or of claims 6 -11, wherein the subject has remitting -relapsing multiple sclerosis.
14. The use of claim 13, wherein the rabbit anti -thymocyte globulin antibody is rabbit anti -murine yte globulin antibody.
15. The use of any one of claims 5 -14, wherein the rabbit anti -thymocyte globulin antibody is polyclonal. GENZYME ATION WATERMARK INTELLECTUAL PROPERTY PTY LTD P38189NZ02 —
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US201161486697P | 2011-05-16 | 2011-05-16 | |
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NZ716716A NZ716716B2 (en) | 2011-05-16 | 2012-05-03 | Induction of immune tolerance by using methotrexate |
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