U.S. patent application number 10/429314 was filed with the patent office on 2004-01-15 for induction of antigen specific immunologic tolerance.
Invention is credited to Kakkis, Emil D., Lester, Thomas, Passage, Merry, Tanaka, Christopher, Yang, Rebecca.
Application Number | 20040009906 10/429314 |
Document ID | / |
Family ID | 29399718 |
Filed Date | 2004-01-15 |
United States Patent
Application |
20040009906 |
Kind Code |
A1 |
Kakkis, Emil D. ; et
al. |
January 15, 2004 |
Induction of antigen specific immunologic tolerance
Abstract
Antigen specific immune tolerance is induced in a mammalian host
by administration of a toleragen in combination with a regimen of
immunosuppression. The methods optionally include a preceding
conditioning period, where immunosuppressive agents are
administered in the absence of the toleragen. After the tolerizing
regimen, the host is withdrawn from the suppressive agents, but is
able to maintain specific immune tolerance to the immunogenic
epitopes present on the toleragen. Optimally, the toleragen will
have high uptake properties that allow uptake in vivo at low
concentrations in a wide variety of tolerizing cell types.
Inventors: |
Kakkis, Emil D.; (Novato,
CA) ; Lester, Thomas; (Harbor City, CA) ;
Passage, Merry; (Torrance, CA) ; Tanaka,
Christopher; (Gardena, CA) ; Yang, Rebecca;
(Los Angeles, CA) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
6300 SEARS TOWER
233 S. WACKER DRIVE
CHICAGO
IL
60606
US
|
Family ID: |
29399718 |
Appl. No.: |
10/429314 |
Filed: |
May 5, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10429314 |
May 5, 2003 |
|
|
|
10141668 |
May 6, 2002 |
|
|
|
Current U.S.
Class: |
424/185.1 ;
514/14.1; 514/20.5; 514/5.4; 514/5.9; 514/8.5 |
Current CPC
Class: |
A61K 31/522 20130101;
A61P 37/02 20180101; A61K 31/7024 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 38/00 20130101; A61P 11/06 20180101;
A61K 31/7024 20130101; A61P 37/06 20180101; A61P 43/00 20180101;
A61K 38/095 20190101; A61P 37/00 20180101; A61K 38/13 20130101;
A61P 37/08 20180101; A61K 31/522 20130101; A61K 38/095 20190101;
A61K 38/13 20130101; A61K 39/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
514/11 ;
424/185.1 |
International
Class: |
A61K 038/13; A61K
039/00 |
Claims
What is claimed is:
1. A method of inducing antigen specific immune tolerance in a
mammal having an anamnestic response to said antigen, comprising
administering an effective dose of a toleragen comprising a high
uptake moiety during a tolerization regimen wherein said host is
maintained on a T cell immunosuppressive agent; wherein following
said tolerization regimen, upon withdrawal of said T cell
immunosuppressive agent, the immunoglobulin titer against said
antigen in said mammalian host is reduced as compared to the
immunoglobulin titer against said antigen prior to said
tolerization regimen, and wherein said mammal otherwise comprises a
substantially normal immune system.
2. The method according to claim 1, wherein tolerization regimen
further comprises administration of an antiproliferative agent.
3. The method according to claim 2, wherein said tolerization
regimen is preceded by a conditioning period, said conditioning
period comprising: administering a combination of said T cell
immunosuppressive agent and said antiproliferative agent in the
absence of said toleragen for a period of time sufficient to
suppress host T cell responses.
4. The method according to claim 2, wherein an initial dose of said
T cell immunosuppressive agent is administered to maintain
suppressive levels of said agent in the host blood at trough
level.
5. The method according to claim 2, wherein during said
tolerization regimen, said host is maintained on a combination of a
T cell immunosuppressive agent and an antiproliferative agent for a
period of at least 6 weeks.
6. The method according to claim 2, wherein said T cell
immunosuppressive agent is an immunophilin.
7. The method according to claim 6, wherein said immunophilin is
cyclosporin A.
8. The method according to claim 7, wherein an initial dose of said
cyclosporin A is administered such that the trough level is at
least about 200 ng/m.
9. The method according to claim 7, wherein an initial dose of said
cyclosporin A is administered such that the trough level is at
least about 300 ng/mls.
10. The method according to claim 2, wherein said antiproliferative
agent is a nucleotide analog.
11. The method according to claim 10, wherein said nucleotide
analog is a 6-mercaptopurine drug.
12. The method according to claim 10, wherein said 6-mercaptopurine
drug is azathioprine.
13. The method according to claim 2 wherein said antiproliferative
agent is an antimetabolite drug.
14. The method according to claim 13, wherein said antimetabolite
drug is an inhibitor of dihydrofolate reductase or other enzymes
involved in nucleotide metabolism.
15. The method according to claim 13, wherein said nucleotide
metabolic enzyme is dihydrofolate reductase and said inhibitor is
methotrexate, or an analogs thereof.
16. The method according to claim 12, wherein said azathioprine is
maintained at a dose from 1 to 5 mg/ml/day for at least about two
weeks.
17. The method according to claim 2, comprising: administering an
effective dose of said toleragen at least 6 times during a
tolerization regimen of at least about 6 weeks.
18. The method according to claim 17, wherein said effective dose
of said toleragen is less than 50% of the normal therapeutic
dose.
19. The method according to claim 17, wherein said effective dose
of said toleragen is less than 10% of the normal therapeutic
dose.
20. The method according to claim 17, wherein said toleragen is a
polypeptide for which a high uptake receptor is widely expressed in
said mammalian host.
21. The method according to claim 17, wherein said toleragen
comprises mannose 6 phosphate.
22. The method according to claim 21, wherein said toleragen is a
mannose 6 phosphate conjugate of an antigen of interest.
23. The method according to claim 17, wherein said toleragen is a
therapeutic polypeptide.
24. The method according to claim 23, wherein said therapeutic
polypeptide is selected from the group consisting of antibodies,
clotting factors, enzymes and growth factors.
25. The method according to claim 17, wherein said toleragen is an
autoantigen.
26. The method according to claim 25, wherein said toleragen
comprises a plurality of autoantigens.
27. The method according to claim 16, wherein said toleragen
comprises a transplantation antigen.
28. The method according to claim 27, wherein said toleragen
comprises a plurality of transplantation antigens.
29. The method according to claim 23, wherein said toleragen is
iduronidase.
30. The method according to claim 29, wherein said iduronidase
comprises a mannose 6 phosphate moiety.
31. The method according to claim 24, wherein said clotting factor
is Factor VIII.
32. The method according to claim 31, wherein said Factor VIII is
wherein said Factor VIII is conjugated to a high uptake moiety.
33. The method according to claim 32, wherein said high uptake
moiety is iduronidase comprising a high affinity uptake marker on
its N-linked carbohydrates.
34. The method according to claim 1, wherein said toleragen
comprises a .beta.-cell antigen.
35. The method according to claim 34, wherein said .beta.-cell
antigen is selected from the group consisting of glutamic acid
decarboxylase (GAD), IA2 and insulin.
36. The method according to claim 35, wherein said .beta.-cell
antigen is GAD.
37. The method according to claim 36, wherein said GAD is
conjugated to a high uptake moiety.
38. The method according to claim 36, wherein said high uptake
moiety is a peptide that binds to a receptor selected from the
group consisting of transferrin receptor, melanotransferrin
receptor and mannose 6-phosphate receptor.
39. The method according to claim 36, wherein said high uptake
moiety is a peptide that binds to a mannose 6-phosphate
receptor.
40. The method according to claim 39, wherein said peptide is
insulin-like growth factor-2 (IGF2).
Description
[0001] The present application is a continuation-in-part
application of U.S. patent application Ser. No. 10/141,668, filed
May 6, 2002. The entire text of the aforementioned application is
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present application is directed to methods and
compositions for inducing immune tolerance in a mammal. The methods
comprise administration of a high-uptake toleragen in combination
with immunosuppressive agents in a tolerization regimen.
BACKGROUND OF THE INVENTION
[0003] Immune tolerance is highly relevant to a wide range of
clinically important applications. Antigen-specific tolerance
induction is a major goal for the treatment or prevention of
autoimmune disease and graft rejection, which are currently
controlled by nonspecific, immunosuppressive therapies that result
in increased rates of infections, cancers and drug-related
pathology. Other applications of immune tolerance induction include
allergies and asthma, bone marrow replacement and protein based
therapeutics.
[0004] One of the first practical applications of molecular biology
has been the ability to produce large quantities of rare biological
agents, many of which have therapeutic activity. It has been found,
however, that during administration of these agents, a patient can
mount an immune response, leading to the production of antibodies
that bind and interfere with the therapeutic activity as well as
cause acute or chronic immunologic reactions. This problem is most
significant for therapeutics that are proteins because proteins are
complex antigens and in many cases, the patient is immunologically
naive to the antigens.
[0005] This type of immune response has been found in at least some
patients with deficiency disorders such as hemophilia A (Aledort
(1994) Am. J. Hemat. 47:208-217), diabetes mellitus (Gossain et al.
(1985) Ann. Allergy 55:116-118), adenosine deaminase deficiency
(Chaffee et al. (1993) J. Clin. Inv. 89:1643-1651), Gaucher disease
(Richards et al. (1993) Blood 82:1402-1409), and Pompe disease
(Amalfitano (2001) Genet Med 3:132-138. In hemophilia A, antibodies
can inhibit factor VIII function requiring alternative treatment
with activated prothrombin complex concentrates. In adenosine
deaminase deficiency, antibodies to PEG modified adenosine
deaminase enhance the clearance of the enzyme and lower its
efficacy. In Gaucher disease, the induction of IgG1 antibodies has
been associated with anaphylactoid reactions due to complement
activation during infusions. In Pompe disease, replacement therapy
with recombinant alpha-glucosidase resulted in the induction of
antibodies in two of three patients treated, which resulted in
declining efficacy of the therapy.
[0006] Similar immune responses have been found in the delivery of
protein therapeutics by gene therapy. For example, viral proteins
associated with vectors are targets of an immune response that can
cause inflammation, shortened expression and prevented repeat
administration of vector (Wilson and Kay (1995) Nature Med.
1:887-889). Antibody responses to the therapeutic protein have also
been observed in gene therapy experiments and are part of an
overall immune response that prevents long-term expression (Shull
et al. (1996) Blood 88:377-379). Generalized immune suppression,
blockade and immune deviation from humoral to cellular responses
have been utilized to address this problem, but are not likely to
ensure long lasting tolerance to the antigen.
[0007] Tolerance may be defined as the absence of an immune
response to a specific antigen in the setting of an otherwise
normal immune system. This state of specific immunological
tolerance to self-components involves both central and peripheral
mechanisms. Central tolerance (negative selection) is a consequence
of immature T cells receiving strong intracellular signaling while
still resident in the thymus, resulting in clonal deletion of
autoreactive cells. Peripheral tolerance occurs when the immune
system becomes unreactive to an antigen presented in the periphery,
where, in contrast to the thymus, T cells are assumed to be
functionally mature. Peripheral tolerance has been proposed to be
the result of various mechanisms, including the development of
antigen specific suppressor cells or other means of active
tolerance, clonal deletion, and anergy. Autoreactive cells may be
physically deleted by the induction of apoptosis after recognition
of tolerizing antigen, may become anergic without deletion, or may
be functionally inhibited by regulatory cytokines or cells.
Although much has been learned in recent years, it is still
difficult to predict the outcome of antigenic exposure in vivo, and
further elucidation of tolerance mechanisms is needed at the basic
level.
[0008] Numerous strategies have been developed to induce antigen
specific tolerance in animal models, for example with respect to
autoimmune disorders, such as multiple sclerosis (or experimental
allergic encephalitis, EAE) or diabetes, as well as to prevent
rejection of allogeneic tissue transplants. The major methods
developed in mouse and rat models involve administration of high
doses of soluble antigen, oral ingestion of antigens or intrathymic
injection. The efficacy of these methods depends to varying degrees
on clonal deletion, clonal anergy, active suppression by
antigen-specific T cells and immune deviation from cellular to
humoral immune responses.
[0009] Although administration of large quantities of soluble
antigens has long been known to induce non-responsiveness to
subsequent immunological challenge, studies of EAE have also
highlighted the difficulties in this approach. The high doses
required and the inconsistency of tolerance versus immune deviation
make soluble antigen administration alone impractical for most gene
therapy situations.
[0010] Administration of very large doses of antigen orally to mice
has also been demonstrated to induce tolerance to protein antigens.
However, extraordinary doses are required, and the results are
complex. Certain doses are found to result in anergy, while other
doses induce a form of antigen-specific bystander suppression,
characterized by antigen-specific TH2 type responses. In addition,
oral antigen can sensitize the immune system and lead to more
severe disease.
[0011] Intrathymic injection of antigens or cells has been widely
explored as a technique to induce central immune tolerance.
Antigens injected and presented within the thymus can cause
apoptosis or anergy of CD4.sup.+, CD8.sup.+ T cells in a process
that may take up to 10 days. Like oral and soluble antigen
tolerance, the tolerizing effect is dose dependent: low doses of
antigen are sensitizing or provide only partial protection, whereas
larger doses of antigen are tolerizing. The context of antigen
presentation within the thymus is also important. Tolerance is most
efficiently induced when the antigens are presented by host antigen
presenting cells (APC). Once tolerized, the continued presence of
antigen within the animal is needed for maintenance of tolerance,
and depletion of mature T cells may also be required.
[0012] One strategy for tolerance induction is based on the
discovery that optimal T cell activation requires both
antigen-specific signals and non-antigen-specific signals. During
antigen presentation, a variety of important bidirectional cognate
interactions take place, with signaling to both the T cell and the
antigen presenting cell. The best understood costimulatory signal
is provided through the T cell surface molecule CD28. CD28 has two
ligands, the homologous molecules CD80 (B7-1) and CD86 (B7-2); both
are expressed on activated APC and some other cell types. Another
pathway that has received significant attention and is important in
T cell costimulation is that mediated by CD40 and its ligand CD154.
CD154 is expressed on activated T cells, primarily CD4.sup.+ T
cells.
[0013] In the past several years, a variety of laboratories have
shown that blockade of T cell costimulatory signals can improve
long-term allograft survival rates and induce transplantation
tolerance. Most of these studies have used either CTLA4Ig, a fusion
protein of CTLA-4 and human Ig that competitively binds CD80 and
CD86, or a blocking monoclonal antibody to CD154. Costimulatory
blockade has been partially successful in mouse and rat models of
cardiac, hepatic, islet, renal, lung, and bone marrow
transplantation. Although a single agent alone such as CTLA4Ig or
anti-CD154 antibody can improve long-term graft survival rates,
these agents by themselves are unlikely to yield indefinite graft
survival; late allograft loss resulting from chronic rejection is
the rule. Most commonly, either a transfusion of donor-specific
lymphocytes or the combination of CTLA4Ig and anti-CD154 is
required for long-term survival, with or without tolerance.
Furthermore, the results in nonhuman primates are not as good as
those in rodent models.
[0014] In murine models in which CTLA4Ig and/or anti-CD40 antibody
has been used to induce tolerance, it has been shown that
concomitant administration of cyclosporine prevents tolerance
induction. It seems that the induction of tolerance in T cells
deprived of costimulatory signals is an active process involving
TCR signaling events that are sensitive to cyclosporine. Therefore,
in the presence of cyclosporine, tolerance cannot be achieved by
this means. These references therefore teach away from the use of
cyclosporine in tolerance induction regimens.
[0015] In a protocol in which the combination of CTLA4Ig given 2 d
after transplantation and donor-specific lymphocytes is used to
induce cardiac allograft tolerance in mice, blockade of CTLA-4 at
the time of transplantation prevents tolerance induction and leads
to early rejection. Therefore, early CTLA-4 signals may be
permissive for some T cell toleragenic/inhibitory strategies;
without these signals, it may prove difficult to turn off the
immune response. Similarly, in murine models of autoimmune disease,
blockade of CTLA-4 exacerbates the duration and severity of the
illness. Because agents such as CTLA4Ig prevent CD80 and CD86 from
binding to both CD28 and CTLA-4, they have both the potential to
block positive signals (through CD28) and the undesired ability to
block negative signals (through CTLA-4).
[0016] In all of these methods, the tolerance is either unreliably
induced, has not been achieved in humans or is not therapeutically
or clinically useful. There is a clinical need for methods of
preventing immune responses to antigens. The present invention
addresses this problem.
SUMMARY OF THE INVENTION
[0017] Methods are provided for inducing antigen specific immune
tolerance in a mammalian host. A toleragen, which comprises
substantially all of the immunogenic epitopes present in the
antigen of interest, is administered to a mammalian host over a
period of time in combination with a T cell immunosuppressant, at a
does sufficient to provide profound immunosuppression. The regimen
may further comprise administration of an anti-proliferative agent.
In one embodiment of the invention, this tolerizing regimen is
preceded by a conditioning period, wherein the T cell
immunosuppressant is administered in the absence of the toleragen.
After the tolerizing regimen, the host is withdrawn from the
immunosuppressive agent, but is able to maintain specific immune
tolerance to the immunogenic epitopes present on the toleragen.
Maintenance doses of the toleragen are optionally administered
after the tolerizing regimen is completed.
[0018] The toleragen can be any antigen, particularly soluble
protein antigens, e.g. therapeutic proteins, cocktails of
transplantation antigens, etc., and may be identical to the antigen
of interest, or may be a modified form of the antigen with enhanced
toleragenic properties, for example to increase uptake by
non-professional antigen presenting cells. Preferably the toleragen
comprises a high uptake moiety, and is taken up efficiently by a
widely present high-affinity receptor, e.g. mannose 6-phosphate
receptor, etc. Other receptors that are widely present and equal in
affinity are also suitable. An antigen without a suitable high
uptake capability may be modified to contain a moiety that provides
this function to allow uptake by tolerizing cell types.
[0019] In one embodiment of the invention the host is
immunologically naive to the antigen of interest, i.e. there is no
pre-existing, or memory immune response to the antigen. In another
embodiment of the invention the host has been exposed to the
antigen. For the latter case it may be necessary to ablate cells of
the immune system responsible for the pre-existing immune
response.
[0020] The invention further contemplates use of a toleragen
comprising an antigen and a high uptake moiety, for the manufacture
of a medicament for use in combination therapy with a T cell
immunosuppressive agent in the prophylactic induction of immune
tolerance to the antigen component of the toleragen.
[0021] In particular aspects of the present invention, it should be
noted that the dose of the immunosuppressive agent is to be
sufficient to substantially suppress T cells. Variations in dosage
of the drugs may be combined to reach the same degree of T cell
suppression in different subjects and under different conditions.
The level of T cell suppression is monitored as that level at which
the T cells do not proliferate in response to antigen stimulation.
Methods for monitoring T cell proliferation are known to those
skill in the art, and may be used in conjunction with the present
invention.
[0022] In preferred embodiments, the range of dose for the
antigen/toleragen may be between 0.001 mg/kg to 5 mg/kg/week. More
preferably, the dose range of the toleragen is between 0.01 mg to I
mg/kg and more preferably 0.03 mg/kg/week to 0.1 mg/kg/week. In
preferred embodiments, the dose for the antigen/toleragen is 0.056
mg/kg body weight once per week.
[0023] In those preferred embodiments in which the
immunosuppressive agent is CsA, CsA is given to reach a plasma
concentration of >400 ng/ml although 300ng/ml or greater may
also be used. A preferred dose range may be between 1 mg/kg to 30
mg/kg, most preferably in humans the dose range may be between 5
and 15 mg/kg/day. Such a daily dose may be administered by dividing
the dose into two, three, four or more fractions of the complete
dose, which would be administered at spaced intervals during the
day. Alternatively, the dose may be administered as one single
dose.
[0024] The medicaments of the invention further may comprise a
nucleotide analog. In those embodiments in which the analog is
azathioprine, the range of dose of this agent may be 1 mg/kg/day to
10 mg/kg/day administered daily or every other day.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1. A diagram of the tolerance regimen is shown. The
common example for the tolerance regimen requires only the CsA+Aza
treatment (with CsA dose at 25 mg/kg/day) along with toleragen
infusions on the schedule shown. Additional treatments indicated
such as intrathymic injection and monoclonal antibodies are shown
as examples of treatments that did not work or matter to tolerance
induction. These non-effective regimens are included in the
examples to provide contrast with the response of those canines
optimally tolerized to the antigen by the invention.
[0026] For the common tolerance regimen, the canines received daily
CsA (25 mg/kg/day) plus every other day Aza from day 0 to day 18 as
shown on the timeline at the bottom of the figure. Weekly
iduronidase enzyme infusions are initiated at day 18 as indicating
by the upward arrows above the timeline. The canines then received
weekly infusions of toleragen at 0.056 mg/kg/wk. At 2 week
intervals thereafter, the CsA+Aza dose was halved, and finally
quartered from the initial dose in the final 2 week segment. Drugs
CsA+Aza were then terminated after a total 60 days and toleragen
infusions continued on a weekly basis.
[0027] For non-tolerance inducing regimens, the canines received
intrathymic injection or monoclonal antibodies and 25 mg/kg dose of
CsA and 5 mg/kg of Aza on alternate days (i.e. each drug on an
every other day ). On day 4, the canines receive intrathymic
injection, if scheduled. In the top protocol diagram for IT+drugs,
the canine receives CsA+Aza for 0-4 days, and then receives an
intrathymic injection (IT Inj. Or ITI). If monoclonal antibodies
are indicated, they are given in the days prior to IT injection.
The exact dosing performed depends on the experiment. The CsA and
Aza dose administered on alternate days was halved on day 18, the
first day that the canine was infused. The canines began receiving
0.56 mg/kg of enzyme on a weekly basis. For the subsequent 2 weeks,
the canines received CsA and Aza at 1/2 the initial doses on
alternate days. Thereafter, the drug dose was halved again to 1/4
the initial and after a further 2 weeks, it was halved again to
1/8.sup.th the initial dose. After 2 weeks at 1/2.sup.th the
initial dose, the drugs were terminated and the canine continued to
receive weekly infusions of enzyme.
[0028] FIG. 2. Induction of tolerance with daily CsA and every
other day Aza during infusions of recombinant human iduronidase
(rhIDU). In the figure, the antibody titer to iduronidase from 6
tolerant (open symbols) and 11 non-tolerant (closed symbols)
canines is shown as OD Units/.mu.L undiluted serum as measured by
ELISA is plotted against the week of enzyme infusion. Beginning 18
days before iduronidase infusion, the canines received a tolerance
regimen as described in example 1. Beginning at week 1, the canine
is infused with weekly intravenous infusions rh-Idu at 0.056
mg/kg/week iduronidase. Low antibody levels (<20 for the
iduronidase ELISA) with continued iduronidase challenge indicate
tolerance in the dogs receiving the optimal tolerance regimen and
titers exceeding 50 and up to 500 indicate an active immune
response. The CsA+Aza drug regimen ends at week 7 of enzyme
challenge and low antibody response beyond that point indicates
that the tolerance is not dependent on continued immune
suppression.
[0029] FIG. 3. Successful induction of tolerance in
iduronidase-deficient MPS I dogs and tolerance to subsequent high
therapeutic levels of enzyme. Three MPS I dogs were tolerized using
the tolerance regimen and one MPS I dog served as a control and did
not receive the tolerance regimen. The antibody titer to
iduronidase in the three tolerant MPS I dogs (open symbols) and 1
non-tolerant (closed symbol) MPS I dog is shown with OD Units/.mu.L
undiluted serum as measured by ELISA plotted versus the week of
enzyme challenge. The tolerant canines received the Tolerance Drug
Regimen as described in example 2; the non-tolerant control
received no drug treatment. Low antibody levels (<20 OD) with
continued antigen challenge indicate tolerance. The tolerizing drug
regimen ends at week 7 of enzyme challenge and low antibody levels
beyond that point is indicative of induced tolerance. The 4 canines
received 0.056 mg/kg/week intravenous iduronidase infusions at
weeks 1-12. Subsequently, the canines received a stepwise increase
in iduronidase dose over 3 weeks to therapeutic doses of 0.500
mg/kg/week at week 15 of enzyme challenge. Antibody levels in
tolerant canines remained <20 OD Units at week 15 compared to
control RU antibody levels of >500 OD Units at week 15 in
response to increasing iduronidase antigen dose. At week 16,
non-tolerant canine RU had a serious clinical anaphylactic reaction
during the infusion and treatment was ended. The tolerant canines
did not exhibit anaphylaxis during the infusions.
[0030] FIG. 4. Successful induction of antigen-specific tolerance
to rhGAA, a second lysosomal enzyme being developed to treat Pompe
disease. The antibody titer to rh-alpha-glucosidase (rhGAA) for 1
tolerant (SC, open symbol) and 1 non tolerant (ST, closed symbol)
canine is shown as OD Units/.mu.L undiluted serum as measured by
ELISA versus the week of enzyme challenge. The tolerant canine
received the tolerance drug regimen as described in the example 3.
At week 1, the canine is tolerized with weekly intravenous
infusions of rhGAA at 0.056 mg/kg/week. The cyclosporine and
azathioprine (CsA(daily)+Aza) is continued and then the dose halved
every 2 weeks until the canine is off drugs at week 7 of enzyme
challenge. The non tolerant control received no drug treatment. The
tolerizing drug regimen ends at week 7 of enzyme challenge and low
antibody levels beyond that point is indicative of induced
tolerance that is maintained in the absence of continued immune
suppression. The 2 canines received 0.056 mg/kg/week intravenous
rhGAA infusions at weeks 1-12. Antibody levels in the tolerant
canine remained <5 OD Units at the highest point compared to the
non tolerant control antibody levels of >150 OD Units at the
highest point. The result confirms that the tolerance regimen can
succeed with another high uptake immunogenic enzyme with
therapeutic implications.
[0031] FIG. 5. Data from 6 non-tolerant canines is shown compared
with one canine (JH) who became tolerant. The non-tolerant canines
have ELISA titers exceeding 50 and in some cases exceeding 200 OD
U/.mu.l/serum. In contrast, JH had an antibody titer of less than
20 OD U/.mu.l/serum throughout 18 weeks of iduronidase infusions.
JH was distinguished by cyclosporine levels of greater than 500
ng/ml in blood, while the other canines were less than 400 ng/ml in
blood and often less than 200 ng/ml in blood.
[0032] FIG. 6. Antigen-specific tolerance is long-lasting. The
figure shows the antibody titer of canines tolerized (JO, RO) and
not tolerized (RU) over time. In the first segment, the titer
during and after tolerization is shown. RU has a high titer,
whereas the other canines have a low titer. After gaps in time of 5
weeks, 4.5 mos, 6 mos or 2.5 years, the canines were rechallenged
with iduronidase. Tolerant dogs (PE, RO) showed no immune response
to the iduronidase even after 5 weeks to 6 months of hiatus from
toleragen infusions. The non-tolerant canine RU, showed a response
to antigen of >20 fold after only 2 doses of enzyme challenges
following a 4.5 months hiatus. The original tolerant dog JH shows a
partial response after 2.5 years of hiatus. The data show a
long-lasting effect of the induced tolerant state.
[0033] FIG. 7. Tolerance regimen prevents the induction of other Ig
subtypes, in addition to IgG. FIGS. 7A and 7B show ELISA titers of
non-tolerant control dogs before and after 12 weekly infusions with
iduronidase. FIGS. 7C and 7D show ELISA titers of tolerant control
dogs before and after 12 weekly infusions with iduronidase.
[0034] FIG. 8. Tolerance regimen may be used to induce relative
tolerance or reduce Ig titer with the regimen in canines with
pre-existing immune response. Nitro was allowed a 6-month hiatus
from antigen exposure. Upon reexposure to antigen after this
period, the IgG titer initially rose during the administration of
the CsA+Aza regimen to greater than 100 in a typical anamnestic
response and than rapidly fell to below 20. The immune response was
reduced by the. reinduction of tolerance using the combined
tolerization regimen.
[0035] FIG. 9. Confirmation that high uptake moiety of iduronidase
(the mannose 6-phosphate group) is required for immune tolerance
with iduronidase. To show that an M6P high uptake moiety assists in
the induction of tolerance to antigen, recombinant iduronidase was
dephosphorylated by incubation with acid phosphatase bound to
beads. The dephosphorylated iduronidase enzyme was applied with the
tolerance regimen (CsA+Aza) to canines. The study showed that the
canine treated with dephospho iduronidase was not tolerized to
iduronidase, thereby suggesting that high uptake affinity (the
mannose 6-phosphate moiety) is needed for tolerance.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0036] Antigen specific immune tolerance is induced in a mammalian
host by administration of a toleragen in combination with a regimen
of immunosuppression for a period of time sufficient to tolerize
the host. Immunosuppression is accomplished by administration of a
T cell immunosuppressive agent. The methods may further comprise
administration of an anti-proliferative agent. The methods
optionally include a conditioning period preceding the
administration of the toleragen, where the immunosuppressive agent
is administered in the absence of the toleragen. After the
tolerizing regimen, the host is withdrawn from the
immunosuppression, but is able to maintain specific immune
tolerance to the immunogenic epitopes present on the toleragen.
Maintenance doses of the toleragen may be administered after the
tolerizing regimen is completed.
[0037] In one embodiment of the invention the host is
immunologically naive to the antigen of interest, i.e. there is no
pre-existing, or memory immune response to the antigen. In another
embodiment of the invention the host has been exposed to the
antigen. The latter case it may be necessary to ablate cells of the
immune system responsible for the pre-existing immune response. The
inventors have shown that the tolerance regimen of the present
invention may be used induce relative tolerance or reduce
immunoglobulin titer in canines with pre-existing immune response
against a given antigen.
[0038] The methods are useful in the proactive establishment of
tolerance where a protein or other immunogenic agent is to be
administered to a naive host. For example, the administration of
therapeutic antibodies, of growth factors, enzymes, and other
polypeptides not previously present in the host can give rise to a
significant immune response, which diminishes the effectiveness of
the treatment. By proactive establishment of tolerance, the long
term effectiveness of such treatment is enhanced. The methods of
the invention also find use in establishing tolerance prior to
transplantation; and in the treatment of autoimmune diseases.
[0039] In exemplary studies, the inventors demonstrated that a
method to reduce or prevent a clinically significant
antigen-specific immune response to recombinant human
.alpha.-L-iduronidase (rhIDU) used to treat canine
mucopolysaccharidosis I (MPS I). The method employ an initial 30-60
day regimen of a T-cell immunosuppressive agent such as cyclosporin
A (CsA) and an antiproliferative agent, such as, azathioprine
(Aza), combined with weekly intravenous infusions of low doses of
rhIDU. The typical strong IgG response to weekly infusions of rhIDU
in canines was greatly reduced or prevented using a 60 day regimen
of immunosuppressive drugs, cyclosporin A (CsA) and azathioprine
(Aza), combined with weekly intravenous infusions of low doses of
rhIDU. More specifically, using the regimen, eight canines had a
20-fold reduction in antibody titer after 12 weekly infusions of
rhIDU (6 weeks without CsA+Aza) and low titers did not increase
with further rhIDU infusions up to 6 months and full therapeutic
rhIDU doses (the eight normal and MPS I canines had a mean antibody
titer of 7.2 OD units/.mu.l serum by ELISA compared to 149 OD
units/.mu.l in eight control canines). The canines tolerated higher
therapeutic doses of iduronidase for up to 6 months without an
increase in titer (mean of 7.5 OD/.mu.l serum, n=6) whereas immune
responsive canines had further 2-3 fold increases in antibody titer
(mean of 369 OD/.mu.l serum, n-2). Antiserum from immune responsive
canines inhibited cellular uptake of iduronidase in vitro by
>95% whereas antiserum from non-responsive canines did not. The
data suggest that a simple protocol can prevent or reduce the
clinically significant immune response to lysosomal enzyme
replacement therapy, as well as reducing the immune response to
other clinically significant antigens.
[0040] The studies demonstrated that one key factor determining
success of the tolerizing regimen was a high serum trough level of
CsA of preferably >400 ng/ml. In addition, the studies of the
tolerizing antigen demonstrated that high-affinity, mannose
6-phosphorylated (M6P) enzymes (rhIDU, alpha-glucosidase) can act
as toleragens whereas the non-phosphorylated protein ovalbumin
could not. High affinity M6P makers appear essential since
dephosphorylated rhIDU did not allow induction of the tolerant
state. A model is proposed in which tolerance is induced under
conditions in which all T cell activation is suppressed by CsA+Aza,
while immature and non-professional antigen presenting cells (APC)
are efficiently loaded with antigen via the M6P receptor and
inactivate antigen-responsive T cells or activate regulatory T
cells to induce a tolerant state in the canines. Methods and
compositions for exploiting these discoveries are discussed in
further detail herein below.
[0041] Definitions
[0042] Before the present methods are described, it is to be
understood that this invention is not limited to particular methods
described, as such may, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited
only by the appended claims.
[0043] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range is encompassed within the invention. The
upper and lower limits of these smaller ranges may independently be
included in the smaller ranges, subject to any specifically
excluded limit in the stated range.
[0044] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0045] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise.
[0046] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates, which
may need to be independently confirmed.
[0047] Antigen. As used herein, the term antigen is intended to
refer to a molecule capable of eliciting an immune response in a
mammalian host, particularly a humoral immune response, i.e.
characterized by the production of antigen-specific antibodies.
Antigens of interest are therapeutic agents, e.g polypeptides, and
fragments thereof; autoantigens, e.g. self-polypeptides;
transplantation antigens; and the like. In response to antigens,
antibodies are produced in a variety of classes, subclasses and
isotypes.
[0048] The portion of the antigen bound by the antibody is referred
to as an epitope. Antigens, particular complex antigens such as
polypeptides, usually comprise multiple epitopes. Where the antigen
is a protein, linear epitopes range from about 5 to 20 amino acids
in length. Antibodies may also recognize conformational
determinants formed by non-contiguous residues on an antigen, and
can epitope can therefore require a larger fragment of the antigen
to be present for binding, e.g. a protein domain, or substantially
all of a protein sequence. It will therefore be appreciated that a
therapeutic protein, which may be several hundred amino acids in
length, can comprise a number of distinct epitopes.
[0049] The level of affinity of antibody binding that is considered
to be "specific" will be determined in part by the class of
antibody, e.g. antigen specific antibodies of the IgM class may
have a lower affinity than antibodies of, for example, the IgG
classes. As used herein, in order to consider an antibody
interaction to be "specific", the affinity will be at least about
10.sup.-7 M, usually about 10.sup.-8 M to 10.sup.-9 M, and may be
up to 10.sup.-11 M or higher for the epitope of interest. It will
be understood by those of skill in the art that the term
"specificity" refers to such a high affinity binding, and is not
intended to mean that the antibody cannot bind to other molecules
as well, or that some minor cross-reactivity to an epitope may not
be present in the host.
[0050] Antigen specific immune tolerance. For the purposes of the
present invention, tolerance is the absence of an immune response
to a specific antigen in the setting of an otherwise substantially
normal immune system. Tolerance is distinct from generalized
immunosuppression, in which all, or all of a class such as B cell
mediated immune responses, of immune responses are diminished.
[0051] Therapeutic antigen specific immune tolerance provides a
specific state in a host, where a therapeutic molecule of interest,
e.g. a therapeutic protein, can be administered multiple times at
the normal effective dose. When such antigen specific tolerance
absent, successive administration of a normal dose of the
therapeutic agent leads to decreased efficacy, due to antibody
interference with the therapeutic agent, and therefore the amount
of the agent required for an effective dose will increase. When
antigen specific immune tolerance is achieved according to the
methods of the present invention, the amount required for an
effective dose of a therapeutic agent will increase by not more
than about five fold after successive administration, usually by
not more than about 2.5 fold after successive administration, and
may not be increased above the initial dose.
[0052] Alternatively or in combination with the efficacy benefit
described above, the immune tolerance state provides for an
increase in the safety of therapeutic protein or other drug
administration. Immune responses to drugs can cause anaphylaxis or
anaphylactoid or immune complex reactions based on the production
of specific antibodies when tolerance is absent. The decreased or
absent immune response observed in the presence of tolerance
according to the methods of the present invention, will also
decrease and limit the decreased safety of administration of such
drugs due to a decreased antibody mediated adverse events.
[0053] Where the antigen of interest is an autoantigen, the
induction of antigen specific immune tolerance will be sufficient
to decrease the symptoms of the autoimmune disease in the patient,
for example a patient may be sufficiently improved so as to
maintain normal activities in the absence, or in the presence of
reduced amounts, of general immunosuppressants, e.g.
corticosteroids.
[0054] Where the antigen of interest is one or more transplantation
antigens, the induction of antigen specific immune tolerance
permits the transplanted organ to survive and function in the
recipient host in the absence, or in the presence of reduced
amounts, of general immunosuppressants.
[0055] An alternative method for determining antigen specific
immune tolerance is to observe the presence of antigen-specific
antibodies in the serum of the host animal after successive
administration of the therapeutic agent. In a non-tolerant host,
the titer of antibodies specific for an antigen, e.g. a therapeutic
agent, autoantigen, transplantation antigen, etc. will increase by
many orders of magnitude on successive administration, or exposure.
For example, it is shown herein that specific antibody titers can
rise more than about 50 fold after about 8 weeks of successive
administration of a therapeutic protein, often more than 100 fold,
and over time may increase by as much as 1000 fold or more. In a
tolerant host, the rise in specific antibody titer will be not more
than 10% of the increase for a corresponding non-tolerant host, and
may be not more than about 5% of the increase. For example, an
increase of less than 50 fold in the specific antibody titer may be
observed over a period of from about 8 weeks, to several months.
The specific level of increase observed against a particular agent
vary depending on the nature of the agent, prior exposure of the
host to the agent, the mode by which the agent is administered, the
method by which the response is measured and the like. Methods are
well known in the art for determining the presence of a specific
antibody in patient serum, e.g. RIA, ELISA, etc., and do not need
to be elaborated here.
[0056] Another aspect of tolerance is the presence of an otherwise
substantially normal immune system. The methods of the present
invention are not directed to a general immunosuppression, and
after the tolerizing regimen the immune response to antigens other
than the antigen of interest are substantially normal, usually
reduced by not more than about five fold as compared to an
untreated control, more usually reduced by not more than about two
fold as compared to an untreated control, and may be
undistinguishable from a normal response.
[0057] Mammalian species that may benefit from the methods of the
invention include canines; felines; equines; bovines; ovines; etc.
and primates, particularly humans. Animal models, particularly
small mammals, e.g. murine, lagomorpha, etc. may be used for
experimental investigations. Animal models of interest include
those for models of therapeutic protein administration,
autoimmunity, graft rejection, and the like.
[0058] Toleragen comprising a high uptake moiety. Toleragen is the
form of the antigen of interest that is administered to the host
during the tolerizing regimen, and will comprise substantially all
of the epitopes present in the antigen, which epitopes may be
provided as one or a cocktail of agents. In some cases the
toleragen and the antigen will be identical, but they may also
differ in the presence of modifications, formulations, and the
like. Toleragens are administered in soluble form, i.e.
substantially free of aggregates, and in an acellular form, while
the antigen of interest may not be soluble. Toleragens may also
comprise carriers to enhance interaction with the immune system,
may comprise multiple fragments derived from the antigen of
interest; may by conjugated to groups that increase toleragenicity,
may be fusion proteins comprising polypeptide moieties of interest,
and the like.
[0059] The toleragens either comprise or are conjugated covalently
to a high uptake moiety. High uptake moieties are polypeptides that
are widely recognized and internalized by receptors present on
non-professional antigen presenting cells. Preferably a receptor is
chosen that is widely expressed on peripheral and central cells
that are tolerizing when they present antigen, and which is not
exclusively expressed on macrophages, dendritic cells, or other
professional antigen presenting cells. Cells that tolerize when
presenting antigens include, for example, liver sinusoidal
endothelial cells, cortical/medullary thymic epithelium cells, and
similar cell types.
[0060] It is especially preferred that the receptor has a
K.sub.uptake for the ligand of at least about 10.sup.-6 M, usually
at least about 10.sup.-7 M more usually at least about 10.sup.-8 M
and preferably at least about 10.sup.-9 M where K.sub.uptake
represents the concentration of ligand at which half maximal uptake
occurs in a cell. Receptors of interest include the transferrin
receptor, the melanotransferrin receptor, mannose 6-phosphate
receptor, growth hormone receptor, and the like. Cognate ligands
include: insulin-like growth factor II (IGF2), transferrin, growth
hormone, insulin, and binding fragments thereof, particularly
polypeptides having specific and high affinity receptors on diverse
cell types. Single chain antibodies and binding agents from
randomized phage display systems could be used if they have
adequate affinity for the receptor. To determine the K.sub.uptake
of a ligand and receptor combination, methods known in the art may
be used, for example see Kakkis et al. (1994) Protein Expr Purif
5(3):225-32 for assays used to determine the K.sub.uptake of
alpha-L-iduronidase. The disclosed methods are readily adapted to
any ligand and receptor combination. Such assays are also useful in
verifying the uptake of toleragens comprising exogenous high uptake
moieties.
[0061] Where the antigen is a polypeptide taken up by such
receptors, the antigen and the toleragen may be identical. Where
the antigen of interest is a polypeptide that is not widely taken
up by non-professional antigen presenting cells, the toleragen may
be a modified form of the antigen, e.g. may be a conjugate of the
antigen and a high uptake moiety (ligand), such as mannose
6-phosphate, transferrin, etc.
[0062] Alternatively the toleragen may be a modified form of the
antigenic polypeptide, comprising amino acid changes, e.g.
substitutions, deletions, additions, and the like, which provide
the polypeptide with sequences for high uptake. For example,
glycosylation motifs may be added or altered, in order to provide
suitable post-translational modifications, e.g. a motif for
addition of mannose 6 phosphate (see Cantor et al. (1992) J Biol
Chem 267(32):23349-56). In one embodiment of the invention the
toleragen is a fusion protein comprising (a) all or a part of the
antigen of interest and (b) a fragment of a protein having a high
uptake moiety, where the fragment is sufficient to confer the high
uptake properties. For example, a fragment of .alpha.-L-iduronidase
comprising the necessary motifs for post-translational
glycosylation and addition of mannose 6 phosphate may be fused to
an antigen of interest.
[0063] Methods of conjugating chemical groups to a polypeptide are
well known in the art. Chemical groups that find use in linkage
include carbamate; amide (amine plus carboxylic acid); ester
(alcohol plus carboxylic acid), thioether (haloalkane plus
sulfhydryl; maleimide plus sulfhydryl), Schiff's base (amine plus
aldehyde), urea (amine plus isocyanate), thiourea (amine plus
isothiocyanate), sulfonamide (amine plus sulfonyl chloride),
disulfide; hydrazone, lipids, and the like, as known in the art.
Ester and disulfide linkages are preferred if the linkage is to be
readily degraded in the cytosol after transport of the substance.
Illustrative entities include: azidobenzoyl hydrazide,
N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide),
bis-sulfosuccinimidyl suberate, dimethyladipimidate,.
disuccinimidyltartrate, N-.gamma.-maleimidobutyryloxysuccinimide
ester, N-hydroxy sulfosuccinimidyl-4-azidobenzoate, N-succinimidyl
[4-azidophenyl]-1,3'-dithiopropionate, N-succinimidyl
[4-iodoacetyl]aminobenzoate, glutaraldehyde, NHS-PEG-MAL;
succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate;
3-(2-pyridyldithio)propio- nic acid N-hydroxysuccinimide ester
(SPDP); N, N'-(1,3-phenylene)bismaleim- ide; N,
N'-ethylene-bis-(iodoacetamide); or 4-(N-maleimidomethyl)-cyclohex-
ane-1-carboxylic acid N-hydroxysuccinimide ester (SMCC);
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and
succinimide 4-(p-maleimidophenyl)butyrate (SMPB), an extended chain
analog of MBS. The succinimidyl group of these cross-linkers reacts
with a primary amine, and the thiol-reactive maleimide forms a
covalent bond with the thiol of a cysteine residue.
[0064] Suppressive Agents
[0065] T cell immunosuppressive agent. T cell immunosuppressive
agents are compounds that inhibit the activity of T cells,
particularly T helper cells, usually without general suppression of
the proliferation and activity of other cells, such as B cells,
monocytes, bone marrow hematopoietic progenitors cells, etc.
Methods of assaying for T cell immunosuppression are well known in
the art, include in vitro assays such as release of IL-2 by T
helper cells in the presence of antigen, incorporation of .sup.3H
thymidine into DNA in the presence of antigen or a stimulant such
as Con A, release of .sup.51Cr in the presence of allogeneic
stimulatory cells, etc. In vivo assays may rely upon measuring the
proliferation of T cells, the release of cytokines, inability to
reject a graft while actively suppressed, and the like.
[0066] A group of compounds of particular interest for these
purposes are the immunophilins, also referred to as calcineurin
inhibitors, which inhibit T helper cells. Calcineurin is a
Ca.sup.2+/calmodulin-dependent S/T protein phosphatase 2B, which
has been reported to be important in the calcium signaling pathway.
This enzyme is a heterodimer of a 61 kDa calmodulin-binding
catalytic subunit (calcineurin A) and a small (19 kDa) regulatory
subunit (calcineurin B). The immunosuppressive drugs, cyclosporin
A, rapamycin, FK506, etc. inhibit calcineurin, which is necessary
for the nuclear import of NF-AT (nuclear factor of activated T
cells). The dose of T cell immunosuppressive agent for the purpose
of tolerization may be higher than that normally used for general
immunosuppression, as will be discussed in detail below.
[0067] Immunophilins may be administered in a manner as is
conventionally practiced. See, e.g., Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 7th Ed, 1985, p. 1299. For
example, CSA may be provided as an oral solution of 100 mg/ml with
12.5% alcohol, and for intravenous administration as a solution of
50 mg/ml with 33% alcohol and 650 mg of polyoxyethlated castor oil.
When administered intravenously, CSA may be given as a dilute
solution of 50 mg to 20-100 mg of normal saline or 5% dextrose in
water, by slow infusion over a period of several hours. The
intravenous dose is typically one third of the oral dose. Most
preferably, administration of CSA is orally, either in capsule or
tablet form. Such formulations may be prepared by any suitable
method of pharmacy which includes the step of bringing into
association the active compound and a suitable carrier (which may
contain one or more accessory ingredients). In general, the
formulations can be prepared by uniformly and intimately admixing
the active compound with a liquid or finely divided solid carrier,
or both, and then, if necessary, shaping the resulting mixture. The
preparation of CSA is disclosed in U.S. Pat. No. 4,117,118. CSA
which may be used in the practice of the invention is commercially
available under the name SANDIMMUNE.RTM. from Sandoz
Pharmaceuticals Corporation. FK506, also known as tacrolimus, is
commercially available under the trade name PROGRAF.RTM. from
Fujisawa Healthcare. Rapamycin, also known as sirolimus is
commercially available under the trade name RAPAMUNE.RTM. from
Wyeth-Ayerst Pharmaceuticals Inc.
[0068] Antiproliferative agent. Antiproliferative agents, for the
purposes of the methods of the present invention, are
pharmaceutically active compounds that depress cellular
proliferation. As the cells of the immune system are often actively
dividing, even general anti-proliferative agents frequently have an
immunosuppressive effect. Many such anti-proliferative drugs are
known in the art, for example as used in chemotherapy.
[0069] Anti-proliferative drugs of interest include
antimetabolites, e.g. nucleotide analogs such as azathioprine,
6-mercaptopurine, thioguanine, cytarabine, etc.; other analogs,
such as methotrexate, mycophenolic acid, or
6-(1,3-Dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxy-5-isobenzofuranyl)-4-
-methyl-4-hexanoic acid, and the like. Although less preferred,
alkylating agents such as cyclophosphamide, chlorambucil, etc. may
also find use as immunosuppressive antiproliferatives, for example
where there is a pre-existing immune response to the antigen of
interest.
[0070] In one embodiment of the invention, the antiproliferative
agent is azathioprine (AZA) or 6-mercaptopurine (6-MP). As used
herein, the term "6-mercaptopurine drug" or "6-MP drug" refers to
any drug that can be metabolized to an active 6-mercaptopurine
metabolite that has therapeutic efficacy. Exemplary
6-mercaptopurine drugs as defined herein include 6-mercaptopurine
(6-MP) and azathioprine (AZA). Other 6-MP drugs include, for
example, 6-methylmercaptopurine riboside and 6-TG. 6-TG is a
particularly useful 6-MP drug in patients having high TPMT
activity. Patients exhibiting high TPMT activity are expected to
more easily convert 6-MP drugs such as 6-MP and AZA to 6-MMP. As
used herein, the term "6-thioguanine" or "6-TG" refers to
6-thioguanine or analogues thereof, including molecules having the
same base structure, for example, 6-thioguanine ribonucleoside,
6-thioguanine ribonucleotide mono-, di- and tri-phosphate,
6-thioguanine deoxyribonucleoside and 6-thioguanine
deoxyribonucleotide mono-, di, and triphosphate. The term "6-TG"
also includes derivatives of 6-thioguanine, including chemical
modifications of 6-TG, so long as the structure of the 6-TG base is
preserved. As used herein, the term "6-methyl-mercaptopurine" or
"6-MMP" refers to 6-methyl-mercaptopurine or analogues thereof,
including analogues having the same base structure, for example,
6-methyl-mercaptopurine ribonucleoside, 6-methyl-mercaptopurine
ribonucleotide mono-, di-, and tri-phosphate,
6-methyl-mercaptopurine deoxyribonucleoside, and
6-methyl-mercaptopurine deoxyribonucleotide mono-, di- and
tri-phosphate. The term "6-MMP" also includes derivatives of
6-methyl-mercaptopurine, including chemical modifications of 6-MMP,
so long as the structure of the 6-MMP base is preserved.
[0071] 6-MP drugs may be delivered as a suspension, solution, or
emulsion in oily or aqueous vehicles, and may contain such
formulary agents such as suspending, stabilizing and/or dispersing
agents. Suitable aqueous vehicles include physiological saline,
phosphate-buffered saline, and other vehicles for parenteral drug
delivery, generically referred to as "intravenous solutions".
Alternatively, the active ingredient may be in powder form,
obtained by aseptic isolation of sterile solid or lyophilized from
solution, with a suitable vehicle, e.g. sterile, pyrogen-free
water, before use.
[0072] Methods of the Invention
[0073] Antigen specific immune tolerance is induced in a mammalian
host by administering concurrently a toleragen and a T cell
immunosuppressive agent, which may further be combined with
administration of an antiproliferative agent, as described above.
These agents may be formulated separately or together, usually
separately. The agents are said to be administered concurrently
when introduced into the host simultaneously in time or at
different times during the course of a common treatment schedule.
In the latter case, the compounds are administered sufficiently
close in time to achieve the desired effect. Typically, if one
agent is administered approximately within the in vivo half life of
the other agent, the two agents are considered to be concurrently
administered. Any active agent should be present in the patient at
sufficient combined levels to be therapeutically effective.
[0074] The tolerization regimen is optionally preceded by a
conditioning period, where during the conditioning period the
suppressive agent or agents are administered in the absence of the
toleragen for a period of from about 1 to 3 weeks. The period of
time for conditioning will be sufficient to suppress T cell
responses in the host prior to administration of the toleragen. The
time period for conditioning may vary depending on the host, the
suppressive agents, etc. The period of time may be empirically
determined, or obtained from published sources.
[0075] The tolerization regimen requires that the T cell
immunosuppressive agent be maintained for a period of time
sufficient to induce immune tolerance. Usually the T cell
immunosuppressive agent will be administered to the host for at
least about 3 weeks, usually at least about 5 weeks, and may be at
least about 8 weeks or more. The antiproliferative drug is also
administered during this period of time, in a schedule appropriate
to the drug, usually at least every other day, daily, twice daily
or more. While the tolerization regimen can be continued for longer
periods of time, it is usually desirable to minimize the period of
time in which the patient is treated with the suppressive
agents.
[0076] An important aspect of the invention is maintaining a high
dose of the T cell immunosuppressive agent during the initial
stages of the tolerization procedure. In pharmacokinetics, it is
observed that the concentration of a drug in the bloodstream
reaches a "peak" after administration, and then the blood levels
drop to the lowest point, or "trough", before administration of the
next dose. In the methods of the present invention, it is important
to maintain therapeutic levels of the T cell immunosuppressive
agent during this period of time, such that the "trough" levels do
not drop to non-suppressive levels. This can be empirically
determined by periodically measuring the concentration of the drug
in the bloodstream to determine the trough values for a given
administration protocol. The level of immunosuppression for a given
concentration of drug can be determined empirically, as discussed
previously, or obtained from published values. The concentration of
the drug will be sufficient to prevent the in vivo initiation or
maintenance of a T cell mediated immune response.
[0077] Increasing the trough levels to maintain suppressive levels
of the drug can be accomplished by administering higher doses of
the drug, or by administering a standard dose more frequently. For
example, in humans, with cyclosporine A, it is desirable to have a
trough blood level of not less than about 200 ng/ml, usually not
less than about 300 ng/ml, and may be about 500 ng/ml or higher.
Levels are known to induce tolerance in the canine species are
about 400 ng/ml at the trough, however the appropriate level in
other species will take into account the sensitivity of the host to
the immunosuppressant being administered. In particular, humans may
more sensitive to the effects of cyclosporine than canines. The
dose ranges for humans may be lower than for canines due to the
decreased rate of metabolism in general in humans relative to
canines. It is expected that the human dose may be approximately
one half of the canine dose (about 10 mg/kg/day to about 15
mg/kg/day) based on the differences in metabolic rates in order to
achieve the same plasma levels or the same degree of physiologic
effect. The levels required may be comparable to those used in de
novo renal or other transplants (about 8.+-.3 mg/kg/day) at which
the trough level of about 350.+-.150 ng/ml was achieved, Physicians
Desk Reference, ed. 56 published by Medical Economics Co.,
Montvale, N.J., p 2381).
[0078] For example, the initial dose of the T cell
immunosuppressive agent may be at a dose equivalent to at least
about 125% of the standard dose for immunosuppression, at least
about 150% of the standard dose, or 200% of the standard dose, or
more. In other embodiments, the dose may be the conventional dose,
administered more frequently, e.g. twice daily instead of daily,
etc. A conventional dose for oral tacrolimus is 0.2 mg/kg/day. A
conventional dose for sirolimus is 2 mg/kg/day. It will be
appreciated by one of skill in the art that the standard dose will
vary depending on the specific drug, on the method of
administration, i.e. oral, intravenous, etc., and on the host.
[0079] At the end of the high dose period, the dose of the T cell
immunosuppressive agent will be tapered off until the end of the
tolerizing regimen, at which point it will be discontinued. Any
convenient protocol may be used, for example by halving the dose
every week or two weeks.
[0080] The high dose will be maintained for a period of time
sufficient to allow the toleragen to be taken up by tolerizing
cells, processed and presented on the cell surface, and for T cells
to interact with such tolerizing cells, usually for at least about
2 weeks, more usually at least about 3 weeks, and may be for 4
weeks or more. The dose level and time interval can be determined
to be sufficient by measuring the antibody titer using a method
such as ELISA to assay the serum or plasma of the host at weeks 6
to 8 in the regimen. Hosts that are not tolerant will have mounted
an immune response by near the end of the taper of the immune
suppressive drugs. The period of time for which the high dose is
necessary may include the conditioning period, such that if there
is a two week conditioning period, then the high dose may be
tapered off shortly after initiation of the tolerization
regimen.
[0081] When an antiproliferative agent is included in the regimen,
it will be administered at a conventional dose while the T cell
immunosuppressive agent is administered, usually for at least about
two weeks, more usually at least about 3 weeks, and may be for 4
weeks or more. For example, the standard dose of azathioprine is
from about 1 to 5 mg/kg/day, where the upper end, from about 3 to 5
mg/kg/day is used initially, and the lower range, from about 1 to 3
mg/kg/day is given after establishment of the regimen. The
anti-proliferative agent may also be given every other week. As
described above for the T cell immunosuppressive agent, the period
of time for which the initial dose is necessary may include the
conditioning period, such that if there is a two week conditioning
period, then the high dose may be tapered off shortly after
initiation of the tolerization regimen. Usually it is preferable to
taper off the dose, over the tolerizing regimen, by any convenient
protocol.
[0082] The toleragen is administered at least 2 times, usually at
least about 4 times, and may be administered 6 times or more,
during the tolerization period. The toleragen will not be
administered during the conditioning period, if there is one. In
contrast to the suppressive agents, the toleragen will be
administered less frequently, for example after about 4 days, about
7 days, about 10 days, and the like. Weekly administration is
convenient.
[0083] The dose of toleragen will generally be lower than the
therapeutic dose of the corresponding antigen, and may range from
as much as the normal therapeutic dose of the antigen to as little
as about 5% of the therapeutic dose, about 10% of the therapeutic
dose, 50% of the therapeutic dose, and the like. Where the
toleragen is a polypeptide it will be administered at a dose of at
least about 0.005 mg/kg/week, usually at least about 0.01
mg/kg/week, more usually at least about 0.05 mg/kg/week; and
usually not more than about 1 mg/kg/week. However, it will be
appreciated that the specific dose will depend on the route of
administration, activity of the agent, etc.
[0084] The dose of the toleragen is gradually raised to reach
normal therapeutic doses starting after about 3 weeks, usually
after about 4 weeks, and may be after about 6 weeks or 8 weeks.
Where the toleragen and antigen are not identical, the patient may
be switched to the antigen after the tolerization regimen, which
change may involve administering a mixture of the two for a period
of time.
[0085] The toleragen will be administered by any convenient route,
usually intravenously, in a soluble form. More particularly, the
toleragen can be formulated by combination with appropriate
pharmaceutically acceptable carriers or diluents, and may be
formulated into preparations in solid, semi-solid, liquid or
gaseous forms, such as tablets, capsules, powders, granules,
ointments, solutions, suppositories, injections, inhalants, gels,
microspheres, and aerosols. As such, administration of the
compounds can be achieved in various ways, including oral, buccal,
rectal, parenteral, intraperitoneal, intradermal, transdermal,
intracheal, etc., administration.
[0086] In pharmaceutical dosage forms, the toleragen may be
administered in the form of a pharmaceutically acceptable salts.
They may also be used in appropriate association with other
pharmaceutically active compounds. The following methods and
excipients are merely exemplary and are in no way limiting. The
toleragen can be formulated into preparations for injections by
dissolving, suspending or emulsifying them in an aqueous or
nonaqueous solvent, such as vegetable or other similar oils,
synthetic aliphatic acid glycerides, esters of higher aliphatic
acids or propylene glycol; and if desired, with conventional
additives such as solubilizers, isotonic agents, suspending agents,
emulsifying agents, stabilizers and preservatives.
[0087] The term "unit dosage form", as used herein, refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
compounds of the present invention calculated in an amount
sufficient to produce the desired effect in association with a
pharmaceutically acceptable diluent, carrier or vehicle. The
specifications for the unit dosage forms of the present invention
depend on the particular compound employed and the effect to be
achieved, and the pharmacodynamics associated with each compound in
the host.
[0088] The pharmaceutically acceptable excipients, such as
vehicles, adjuvants, carriers or diluents, are readily available to
the public. Moreover, pharmaceutically acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity
adjusting agents, stabilizers, wetting agents and the like, are
readily available to the public.
[0089] Antigens suitable for the methods of the invention include a
number of therapeutically active agents, particularly polypeptides,
which may be immunologically active, growth factors, hormones,
clotting factors, metabolic enzymes, etc. Examples of therapeutic
proteins of interest include blood clotting factors, such as Factor
VIII; alpha-glucosidase; iduronidase, therapeutic antibodies such
as herceptin; DNAse; human growth factor; insulin; and the
like.
[0090] Where the antigen of interest is an autoantigen, or where
the host has been previously exposed to a therapeutic agent, it may
be necessary to ablate some or all of the mature immune cells
present in the patients. Such methods are known in the art, for
example on may utilize a strong immunosuppressive agent, for
example cyclophosphamide, antithymocyte globulin, total body
irradiation (TBI), etc.
[0091] Autoantigens of interest for tolerization include the
following. The toleragen may comprise one or a cocktail of
autoantigens. For multiple sclerosis, proteolipid protein (PLP);
myelin basic protein (MBP); myelin oligodendrocyte protein (MOG);
cyclic nucleotide phosphodiesterase (CNPase); myelin-associated
glycoprotein (MAG), and myelin-associated oligodendrocytic basic
protein (MBOP); alpha-B-crystalin (a heat shock protein); OSP
(oligodendrocyte specific-protein); citrulline-modified MBP (the C8
isoform of MBP in which 6 arginines have been de-imminated to
citrulline), etc.
[0092] Autoantigens in rheumatoid arthritis include type II
collagen; hnRNP; A2/RA33; Sa; filaggrin; keratin; citrulline;
cartilage proteins including gp39; collagens type I, III, IV, V,
IX, XI; HSP-65/60; IgM (rheumatoid factor); RNA polymerase;
hnRNPB1; hnRNP-D; cardiolipin; aldolase A; citrulline-modified
filaggrin and fibrin.
[0093] Autoantigens in human insulin dependent diabetes mellitus
include tyrosine phosphatase IA-2; IA-2.beta.; glutamic acid
decarboxylase (GAD) both the 65 kDa and 67 kDa forms;
carboxypeptidase H; insulin; proinsulin; heat shock proteins (HSP);
glima 38; islet cell antigen 69 KDa (ICA69); p52; two ganglioside
antigens (GT3 and GM2-1); and an islet cell glucose transporter
(GLUT 2).
[0094] Autoantigens for myasthenia gravis may include epitopes
within the acetylcholine receptor. Autoantigens targeted in
pemphigus vulgaris may include desmoglein-3. Sjogren's syndrome
antigens may include SSA (Ro); SSB (La); and fodrin. The dominant
autoantigen for pemphigus vulgaris may include desmoglein-3.
[0095] Immune rejection of tissue transplants, including lung,
heart; liver, kidney, pancreas, and other organs and tissues, is
mediated by immune responses in the transplant recipient directed
against the transplanted organ. Allogeneic transplanted organs
contain proteins with variations in their amino acid sequences when
compared to the amino acid sequences of the transplant recipient.
Because the amino acid sequences of the transplanted organ differ
from those of the transplant recipient they frequently elicit an
immune response in the recipient against the transplanted organ.
Rejection of transplanted organs is a major complication and
limitation of tissue transplant, and can cause failure of the
transplanted organ in the recipient. The chronic inflammation that
results from rejection frequently leads to dysfunction in the
transplanted organ.
[0096] The toleragen for an intended transplant recipient may
include one or more major histocompatibility antigens, e.g. HLA-A,
HLA-B, HLA-C, HLA-DR, HLA-DQ, HLA-DP, etc., and may comprise a
cocktail of such antigens, where the antigens will include those
not matched between the recipient and the donor. As these are cell
surface proteins, the administered form may be a soluble form, i.e.
one that is truncated at the transmembrane domain.
[0097] While not required, in order to enhance tolerance after
cessation of the suppressive agents, a maintenance dose of the
toleragen may be provided, where the maintenance dose is provided
at a dose equivalent to 0.056 mg/kg per week or lower in dose, or
of {fraction (1/10)} that dose or lower, or less frequent as in
once per month or once every few months or less, or both dose and
frequency. In cases where the toleragen is different from the
antigen, the toleragen will be used for the maintenance phase, if a
maintenance phase is required.
[0098] The agents utilized in the methods of the invention may be
provided in a kit, which kit may further include instructions for
use. Such a kit will comprise a toleragen, usually in a dose and
form suitable for administration to the host. The kit may further
comprise a T cell immunosuppressive agent, in a form suitable for
administration, and may further include assay reagents for
monitoring blood levels of the agent, and/or for determination of
suppression of T cell activity. An anti-proliferative agent may
also be included, in a form suitable for administration.
[0099] A kit may also provided for the conjugation of an antigen,
particularly a polypeptide antigen, to a high uptake moiety, in
order to generate a toleragenic composition. For example, a moiety
such as a mannose 6 phosphate group, either conjugated to a linker
suitable for linking sugars and polypeptides, as described above,
may be provided. The high uptake moiety may also be provided in an
unconjugated form, in combination with a suitable linker, and
instructions for use.
EXAMPLES
[0100] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
[0101] Experimental
Example 1
[0102] Induction of Tolerance to Human .alpha.-L-iduronidase in
Normal and MPS I Dogs
[0103] Mucopolysaccharidosis I is a genetic condition caused by
mutations in the alpha-L-iduronidase gene-leading to a deficiency
in the enzyme iduronidase. This deficiency leads to a progressive
multi-system lysosomal storage disorder that includes coarsened
facial features, large tongue, large liver and spleen, respiratory
problems, heart problems, joint stiffness and bone disease. The
disease leads to death in patients usually in their first or second
decade of life.
[0104] The deficient enzyme iduronidase is a lysosomal hydrolase
that cleaves the terminal iduronide residue of heparan and dermatan
sulfate. The enzyme can be made in recombinant cells and is
produced with a mannose 6-phosphate marker on post-translationally
attached carbohydrates, which is important for its uptake into
cells. Enzyme replacement therapy has been proposed as a method of
treatment, in which a recombinant form of the enzyme is
administered intravenously, distributes to tissues and is taken up
into cells via the mannose 6-phosphate receptor. The therapy has
been studied in dogs and most recently in humans (Kakkis et al.
(2001) NEJM 344:182).
[0105] The administration of recombinant human
.alpha.-L-iduronidase to normal or MPS I dogs induces a strong
immune response to the enzyme (Shull et al. (1994) P.N.A.S.
91:12937-12941; Kakkis et al. (1996) Biochem Mol. Med. 58:156-167)
which mimics that observed in human MPS I patients. These responses
may interfere with the efficacy of enzyme therapy. To study methods
to induce tolerance, normal dogs were administered the heterologous
human .alpha.-L-iduronidase protein under various conditions
designed to prevent an active immune response and to allow the
induction of tolerance. The studies demonstrate that the
appropriate dose of cyclosporine and azathioprine, followed by the
weekly administration of the recombinant human
.alpha.-L-iduronidase and the gradual removal of the
immunosuppressive drugs can induce tolerance to the enzyme that
lasted at least 6 months of weekly enzyme administration. A less
frequent dose of cyclosporine, with or without the addition of the
addition of intrathymic injection or anti-T cell monoclonal
antibodies have no impact on tolerance induction. CsA alone at the
right dose without Aza, did not induce tolerance.
[0106] The tolerance regimen utilized CsA+Aza treatment (with CsA
dose at 25 mg/kg/day), along with toleragen infusions on the
schedule shown in FIG. 1. Additional treatments indicated, such as
intrathymic injection and monoclonal antibodies, are shown as
examples of treatments that did not work or did not enhance
tolerance induction. These non-effective regimens are included in
the examples to provide contrast with the response of those canines
optimally tolerized to the antigen by the invention.
[0107] For non-tolerance inducing regimens using intrathymic
injection or monoclonal antibodies but not the 25 mg/kg/day dose of
CsA, day 4 is the day the canines receive intrathymic injection. In
the bottom regimen diagram (FIG. 1) for IT+drugs, the canine
receives CsA+Aza for 0-4 days, and then receives an intrathymic
injection (IT Inj. or ITI). If monoclonal antibodies are indicated,
they are given in the days prior to IT injection. The exact dosing
performed depends on the experiment. In non-tolerant experiments,
the canines received every other day CsA at 25 mg/kg on alternate
days with the Aza. In tolerizing experiments, the canines received
daily CsA with alternate day Aza. In either case, the dose
administered was halved in the sequence described. For canines
receiving the tolerizing regimen, the top regimen diagram describes
the course of treatment. The canine PA received CsA only, beginning
4 days before infusions begin and tapering following the upper
tolerance inducing regimen.
[0108] Materials and Methods
[0109] Animals. Normal and MPS I canines were obtained from the MPS
I canine colony at Harbor-UCLA. The dogs are a cross between
beagles and Plott hounds and average 12-20 kg in weight. The
canines were under 2 years of age and at least 4 months of age for
these experiments.
[0110] Canines BI, BE, BC, BO, JA, JO, ME, MA, MO, and PA were
normal or carrier canines from the MPS I canine colony and
therefore are unaffected with MPS I, and received a series of
different regimens that did not induce tolerance. When CsA+Aza were
part of the regimen, they received the CsA every other day. BI
received phosphate buffered saline intrathymic injection (ITTI); BE
and BC received iduronidase ITI; BO received CsA(qod)+Aza+ITI; JA,
JO, ME, MA, and MO received CsA+Aza, ITI and various monoclonal
antibodies that deplete mature Tcells. PA received daily CsA dose
but unlike dogs that became tolerant, she did not receive Aza.
[0111] Canines RH, RI, and RO were normal or carrier canines from
the MPS I colony that received at minimum the CsA+Aza regimen that
induces tolerance and in the case of RH and RI, they also received
intrathymic injection. The CsA was administered daily.
[0112] Canine RU is an MPS I affected dog that received no
tolerance regimen and served as a control. Canines PE, SA, and NI
are MPS I affected dogs that received the tolerance inducing
regimen of CsA+Aza.
[0113] Monoclonal antibodies. A series of monoclonal antibodies
were obtained from Peter Moore (UC Davis Veterinary School) and
were specific to canine T cell receptor (anti-TCR), canine CD3
antigen (anti-CD3; IgG2b)) and the canine equivalent of Thy-1
(anti-Thy1; IgG1). The anti-TCR antibody was prepared by growing
the hybridoma in low serum containing medium and purification of
the antibody by protein A chromatography. The anti-CD3 and
anti-thy1 antibodies were prepared by production of ascites in mice
using the hybridomas, CA17.6B3 and CA1.4G8 and protein A
purification, at a contract laboratory (Strategic Biosolutions).
When utilized, the monoclonal antibodies were administered in 2 or
3 doses just prior to ITI.
[0114] Immunosuppressive drugs. Cyclosporine (Neoral or Sandimmune)
and Azathioprine (Imuran) were obtained from a local pharmacy. Both
drugs were dosed orally at the dose and frequency noted in the
experiments. The two regimens tested are shown immediately
below.
[0115] Immunosuppressive Drug Regimen with Daily CsA Dosing that
induces tolerance:
[0116] CsA Neoral.RTM. 25 mg/kg/day divided bid po
[0117] Aza Imuran.RTM. 5 mg/kg qod po
[0118] CsA+Aza given at full dose from day 0-32, 1/2 dose from day
33-46, and 1/4 the dose from 47-60, and then terminated.
[0119] Animals monitored for adverse reactions
[0120] CsA peak and trough levels are monitored and dose adjusted
to maintain a target trough level of 400-500 ng/ml in the
circulation.
[0121] Immunosuppressive Drug Regimen with Every Other Day CsA
dosing that does not induce tolerance:
[0122] CsA Neoral.RTM. 25 mg/kg/every other day divided bid po
[0123] Aza Imuran.RTM. 5 mg/kg every other day on alternate days
from CsA
[0124] CsA+Aza given at full dose from day 0-18, 1/2 dose from day
19-32, and 1/4 dose from 33-46 and 1/8.sup.th from day 47-60, and
then terminated
[0125] Animals monitored for adverse reactions
[0126] CsA peak and trough levels monitored and a trough level of
100-200 ng/ml in the circulation.
[0127] Intrathymic injection. During general anesthesia a 3-inch
incision was made in the left-side at the 3.sup.rd intercostal
space. The incision was continued through all muscle layers until
the chest cavity is reached. A rib separator was employed and the
thymus observed directly as a vascularized, lobed organ beneath the
lungs. The enzyme solution was injected into the thymus with a
syringe and 25 G needle. A "zig-zag" pattern was made when
inserting the needle into the thymus in an attempt to prevent
enzyme leakage upon withdrawal of the needle. The thymus was
visually inspected for any leakage of enzyme solution made visible
by Evan's Blue dye. The surgery site was closed and respiratory
function restored using vacuum drawn through a Foley catheter
inserted in the chest cavity. Antibiotics were applied to all
incision sites and surgery was followed by a short course of
antibiotics and pain management medication as necessary.
[0128] Injection solution, 1.5 mL total: 1 mg/kg of 12.1 mg/mL
iduronidase (3.times.10.sup.6 U/mL). Bring to 1.5 mL with 40% PEG+2
mg/mL Evan's Blue dye in acidic PBS (Final solution approximately
6% PEG, 0.3 mg/mL Evan's).
[0129] Enzyme Infusions. Recombinant iduronidase is prepared in a
highly purified form and has been demonstrated to have high uptake
potential with half maximal uptake into Hurler fibroblasts at an
enzyme concentration of about 1 nanomolar (K.sub.uptake) and a
uptake specification less than 3.3 nanomolar. For toleragenic
infusions, 14,000 U/kg/week .alpha.-L-iduronidase in 50 mL infusion
in saline with 1 mg/mL canine albumin, and 10 mM NaPO4 was
administered intravenously over 2 hours (1.sup.st hour 3,000
U/kg/hr; 2.sup.nd hour 11,000 U/kg/hr). Animals monitored for an
anaphylactic reaction. 250,000 U is equal to 1 mg of protein.
[0130] Results The basic outline of the experimental plan is shown
in FIG. 1. The entire cohort of dogs were pretreated with
immunosuppressive drugs, followed by an IT injection if scheduled,
and then beginning 2 weeks later, a series of weekly challenges of
iduronidase. The canines received either the non-tolerance inducing
regimen with every other day CsA (see regimens above) with or
without other treatments (intrathymic injection, monoclonal
antibodies), or the tolerance inducing regimen with daily CsA
dosing with or without the other treatments. One dog, PA received
the daily CsA dose but no Aza.
[0131] Canines were initiated on immunosuppressive drugs at day 0,
equivalent to 18 days prior to the first enzyme challenge. If
scheduled, the canines received monoclonal antibodies by
intravenous infusion (2-5 mg) just prior to ITI. On day 4, the dogs
received, if scheduled, an intrathymic injection of iduronidase at
1 mg/kg. Two weeks later, the dogs were begun on a weekly schedule
of iduronidase enzyme infusions consisting of a weekly intravenous
infusion of 14,000 U/kg (0.056 mg/kg/wk) of human recombinant
iduronidase. The dose of CsA and Aza were halved every two weeks as
noted in the materials and methods as indicated.
[0132] After 6-8 weeks of enzyme challenges, canines receiving the
optimum protocol of CsA (daily)+Aza with or without IT injection
(FIG. 2 open symbols), were tolerant to iduronidase whereas dogs
receiving other regimens had strong immune responses as assessed by
ELISA (FIG. 2 closed symbols). The iduronidase tolerant canine RO
was continued on weekly enzyme challenges for 6 months without
induction of a significant ELISA titer to iduronidase.
[0133] The antibody titer to iduronidase in the non-tolerant and
tolerant canines is shown in Table 1. Only canines that completed
at least 12 weeks are included in the table; non-tolerant canines
BE, BI, BC and BO were stopped at 7 weeks with titers already of
21-74 OD/.mu.l serum. The non-tolerant canines had induction of 181
fold from a baseline level of 0.8 to a mean induction level of
144.6 ELISA OD units per microliter of serum. The tolerant dogs had
a titer increase of 13 fold, from an initial 0.4 at baseline to 5.2
after treatment with iduronidase. The non-tolerant dogs had a
.about.28 fold higher induction of antibodies to iduronidase than
the tolerant dogs. One dog NI had the highest titer in the tolerant
group of 13.8. This dog vomited the CsA dose on multiple occasions
which may account for the less complete induction of tolerance, and
further supports the critical nature of the CsA dosing. PA received
the preferred daily dosing of CsA but did not receive Aza and did
not tolerize. Though two of the tolerant normal dogs, RH and RI,
also received intrathymic injection, the injection was not
necessary as tolerance in canine RO showed.
1TABLE 1 Immune response in tolerant and non-tolerant canines
Antibody Titer to Iduronidase (OD Units/.mu.L undiluted serum) Pre-
Post- Canine Treatment treatment Mean treatment Mean Non- JA (CsA +
Aza + ITI + TCR mAb) 0 230.7 Tolerant JO (CsA + Aza + ITI + TCR
mAb) 2.6 101.2 (every ME (CsA + Aza + ITI + TCR mAb) 0.3 60.2 other
day MA (CsA + Aza + ITI + CD3/Thyl/TCR mAb) 1.5 0.8 120.8 144.6 CsA
plus MO (CsA + Aza + ITI + CD3/Thyl/TCR mAb) 0 377.9 other PA CSA)
0.3 64.4 treat- RU (No Drug Contol MPS I Affected) 1.2 56.7 ments)
RH (CSA(daily) + Aza + ITI) 0.3 8.7 RI (CSA(daily) + Aza + ITI) 0.3
0.5 RO (CSA(daily) + Aza) 0.2 0.7 PE (CSA(daily) + Aza + MPS I
Affected) 0.6 0.4 0.8 5.2 SA (CSA(daily) + Aza + MPS I Affected)
0.4 6.8 NI (CSA(daily) + Aza + MPS I Affected) 0.8 13.8
[0134] In table 1, the antibody titer to iduronidase for 6 tolerant
canines and 7 non-tolerant canines is shown as OD Units/.mu.L
undiluted serum as measured by ELISA. The canines received drug or
other treatments as described in example 1, and were administered
iduronidase. All canines received 0.056 mg/kg/week rh iduronidase
intravenous challenges. Pre treatment ELISA values represent serum
antibody levels prior to first antigen challenge with iduronidase.
Post treatment points represent serum antibody levels at week 12 of
enzyme challenge, or at latest point measured (ME, MA, MO at week
11; PA at week 9). The mean post treatment antibody titers for
tolerant canines (5.2 OD/microliter) is {fraction (1/25)}.sup.th
the titer of non-tolerant canines (144.6 OD/microliter).
[0135] The greatly reduced response to iduronidase in the tolerant
dogs occurs despite the fact that the dogs were off all
immunosuppressive drugs by the end of week 6. The titer in the
tolerance dogs stayed low for several months and was studied in RO
and SA for 6 months of weekly infusions (FIG. 2). Attempts to
induce tolerance to another antigen ovalbumin with mannose
terminated N-linked oligosaccharides, did not succeed. This data
suggests that the higher uptake and broadly present mannose
6-phosphate receptor may be required or that mannose receptor
mediated uptake is not sufficient for the toleragen to successfully
induce tolerance.
[0136] Induction of tolerance to a therapeutic protein has been
achieved in canines. Canines treated with an optimal regimen of CsA
and Aza, showed a dramatically reduced immune response to weekly
infusions of the iduronidase enzyme that lasted at least 4-6
months. The tolerance does not depend on other reagents or
procedures such as monoclonal antibodies or intrathymic injection.
The tolerant state is maintained in the absence of
immunosuppressive drugs for at least 6 months.
[0137] The key difference between the tolerant dogs and the other
dogs was the use of CsA daily. CsA alone, however, was not
sufficient in dogs, as canine PA did not tolerize using daily CsA
alone. The immunosuppressive drugs alone cannot induce tolerance in
dogs, and the use of antigen infusions under the protective cover
of immunosuppressive drugs is a key part of the tolerance protocol.
The enzyme is likely being presented as a tolerizing antigen while
the T cells that might be activated are held back by the drugs.
[0138] Other parts of the regimen were intended to improve the
chances of inducing tolerance but had no effect. Intrathymic
injection was designed to present antigen in a site known to be
tolerogenic, but this had no effect and if anything, led to earlier
immune response. It appears that such peripheral presentation and
tolerization in this model is more effective.
[0139] Monoclonal antibodies to T cell markers also did not
contribute to tolerance. These monoclonals were intended to deplete
as much as 90% of mature T cells from the circulation as has been
shown in pilot experiments, but their use did not add, and was not
required for, tolerance.
[0140] While the host canine might have been an important factor,
in fact, tolerance to human iduronidase was induced in normal
canines with endogenous iduronidase as well as in MPS I affected
canines with no endogenous iduronidase.
Example 2
[0141] Induction of Tolerance to Therapeutic Iduronidase in MPS I
Affected Dogs and Maintenance of Tolerance During High Therapeutic
Dose Infusions.
[0142] Induction of tolerance must prevent a clinically significant
immune response to the therapeutic protein to be useful in the
clinic. MPS I canines on enzyme replacement therapy with
iduronidase respond with high-titer antibodies that delay clearance
or alter the stability of the enzyme, prevent uptake of the enzyme
and likely limit the efficacy of the enzyme therapy. The same
phenomenon has been reported in other animal models.
[0143] To study whether nave MPS I canines can be tolerized to
iduronidase and subsequently receive high dose therapeutic levels
of enzyme on a weekly basis, a series of four MPS I affected dogs
were tolerized (3 dogs) or kept as control (1 dog). After 12 weeks,
the tolerant canines received an increasing weekly dose of
iduronidase and finally received at least 6 weeks of therapeutic
doses of enzyme, without a significant immune response. The
non-tolerant control dog had a rapidly rising titer to the enzyme
as has been observed previously and infusions were terminated at
week 16 due to an anaphylactic reaction.
[0144] Methods and Materials
[0145] Animals. MPS I dogs were obtained from the MPS I canine
colony at Harbor-UCLA. The dogs are a cross between beagles and
Plott hounds and average 12-20 kg in weight. The canines were under
2 years of age and at least 4 months of age for these
experiments.
[0146] Immunosuppressive drugs. Cyclosporine (Neoral or Sandimmune)
and Azathioprine (Imuran) were obtained from a local pharmacy. Both
drugs were dosed orally at the dose and frequency noted in the
experiments. The regimen tested is shown immediately below.
[0147] Immunosuppressive Drug Regimen with Daily CsA Dosing that
induces tolerance: This regimen was as described in Example 1.
[0148] Enzyme Infusions. Recombinant iduronidase is prepared in a
highly purified form and has been demonstrated to have high uptake
potential with half maximal uptake into Hurler fibroblasts at an
enzyme concentration of about 1 nanomolar (K.sub.uptake) and a
uptake specification less than 3.3 nanomolar. Canines were
administered 14,000 U/kg/week .alpha.-L-iduronidase or higher dose
as dose was ramped up, in a 50 mL infusion in saline containing 1
mg/mL canine albumin, 10 mM NaPO.sub.4, pH 5.8. Administered
intravenously over 2 hours (1.sup.sthour 3,000 U/kg/hr; 2.sup.nd
hour 11,000 U/kg/hr, or a higher rate in the 2.sup.nd hour
depending on the dose). Animals were monitored for anaphylactic
reaction.
[0149] Results
[0150] Tolerance induction in three MPS I dogs but not a control
MPS I dog Three MPS I dogs were tolerized using the tolerance
regimen including 18 days of CsA+Aza prior to initiating weekly
enzyme infusions. One MPS I dog served as a control and did not
receive the tolerance regimen. The 4 canines received 0.056
mg/kg/week intravenous iduronidase infusions at weeks 1-12. The
antibody titer to iduronidase in the three tolerant MPS I dogs
(FIG. 3; open symbols) and one non-tolerant MPS I dog (FIG. 3;
closed symbol) are shown as measured by ELISA. Low antibody levels
(<20 OD) with continued antigen challenge indicate tolerance.
The tolerizing drug regimen (CsA+Aza) ends at week 7 of enzyme
challenge and low antibody levels beyond that point are indicative
of induced tolerance.
[0151] Tolerant MPS I dogs are tolerant to full therapeutic dose
enzyme therapy The four MPS I canines received a stepwise increase
in iduronidase dose over 3 weeks to therapeutic doses of 0.500
mg/kg/week at week 15 of enzyme challenge. This is the same dose
used in prior therapeutic trials in the MPS I dogs and is used in
human MPS I enzyme therapy (Kakkis et al 2001, supra.) Antibody
levels in tolerant canines remained <20 OD Units at week 15
compared to control RU antibody levels of >500 OD Units at week
15 in response to increasing antigen dose. Two MPS I canines
previously treated with this dose of enzyme (0.5 mg/kg/wk) had
titers of 1800 and 2000 by week 14 of enzyme therapy for
comparison.
[0152] At week 16, non-tolerant canine RU had a serious clinical
-anaphalactoid reaction during the infusion and treatment was
ended. The tolerant canines did not exhibit anaphalactoid reactions
during the infusions.
[0153] MPS I dogs completely deficient in the antigen iduronidase,
canine or human, can be tolerized to the human iduronidase protein
reproducibly. These tolerant dogs can also receive full therapeutic
doses of enzyme without a significant immune response. This result
indicates that the tolerance induced is robust and can protect a
dog from higher levels of exposure to an antigen including levels
that might be expected during therapeutic protein
administration.
Example 3
[0154] Induction of Tolerance to Alpha Glucosidase
[0155] Induction of tolerance to iduronidase infusions has been
demonstrated in normal and MPS I dogs using a regimen of daily
CsA+Aza, followed by weekly infusions of tolerizing antigen while
tapering the immunosuppressive drugs. To demonstrate that tolerance
can be induced to another enzyme with high affinity uptake
characteristics, recombinant human alpha glucosidase was prepared
and studied with the tolerance regimen. Two normal canines were
studied, one with the tolerance regimen and one control. Weekly
infusions with glucosidase began and by week 3, the control dog had
a rising immune titer. By week 5, the control dog a 100 fold higher
titer, and the treated dog had no significant titer. The data shows
that the tolerance regimen can be successfully used with other
antigens.
[0156] Materials and Methods
[0157] Animals. MPS I dogs were obtained from the MPS I canine
colony at Harbor-UCLA. The dogs are a cross between beagles and
Plott hounds and average 12-20 kg in weight. The canines were under
1 year of age and at least 4 months of age for these
experiments.
[0158] Immunosuppressive drugs. Cyclosporine (Neoral or Sandimmune)
and Azathioprine (Imuran) were obtained from a local pharmacy. Both
drugs were dosed orally at the dose and frequency noted in the
experiments. The regimen tested is shown immediately below.
[0159] Immunosuppressive Drug Regimen with Daily CsA Dosing that
induces tolerance:
[0160] CsA Neoral.RTM. 25 mg/kg/day divided bid po
[0161] Aza Imuran.RTM. 5 mg/kg qod po
[0162] Doses halved for all drugs each 2 weeks after first enzyme
infusion
[0163] Animals monitored for adverse reactions
[0164] CsA peak and trough levels were monitored to maintain an
optimal target trough level of 400-500 ng/ml in the
circulation.
[0165] Enzyme Infusions. Recombinant alpha-glucosidase is prepared
in a highly purified form and has been demonstrated to have high
uptake potential with half maximal uptake into fibroblasts at an
enzyme concentration of about 1 nanomolar (K.sub.uptake) and a
uptake specification less than 3.3 nanomolar. Infusions were
performed with 0.056 mg/kg/week recombinant human alpha glucosidase
in a 50 mL infusion of saline and 10 mM NaPO4, pH 5.8. The
infusions were administered intravenously over 2 hours (1 st hour
21% of the total dose; 2.sup.nd hour the balance of the enzyme).
Animals were monitored for anaphylactic reaction.
[0166] Results
[0167] Control canine ST received no drug treatment and began
receiving alpha-glucosidase by weekly intravenous infusions. By 3
weeks, a significant titer was detected and the titer increased to
more than 100 OD units per microliter of serum by week 4.
Subsequently, the titer ranged between almost 100-200 units per
microliter of serum. In contrast, canine SC received the CsA+Aza
regimen and had minimal if any immune response during 12 weeks of
infusions. This includes weeks 7-12 in which no immunosuppressive
drugs were administered.
[0168] The regimen of CsA+Aza with infusions of tolerizing antigen
on a weekly basis can induce tolerance to other antigens. The
result demonstrates the broader utility of the regimen and
tolerizing antigen protocol in inducing a profound state of immune
tolerance. In particular, immune response to alpha-glucosidase has
been reported to inhibit or limit the utility of enzyme replacement
therapy in Pompe patients. This result demonstrates the utility of
the tolerance regimen in preventing an immune response to this
therapeutic protein.
Example 4
[0169] Induction of Tolerance is Dependent on CsA Dose
[0170] Early work on developing a tolerance protocol focused on a
combination of intrathymic injection, immunosuppressive drugs and
mature T cell-depleting monoclonal antibodies. It was thought that
a complete combination of tolerizing antigen expression
(intrathymic injection), suppression of mature antigen-specific T
cell responses (CsA and Aza) as well as depletion of mature T cells
capable of responding to antigen, was necessary to block an
activating immune response and prepare the immune system to accept
the antigen exposure. During the development of this work, a canine
JH, became tolerant though the canine received only ITI and
CsA+Aza. Further analysis of this canine demonstrated that his
metabolism of CsA led to substantially higher serum levels of CsA.
This finding became the core result that allowed further
experiments into the induction of tolerance with the
immunosuppressive drugs and toleragen only.
[0171] Materials and Methods
[0172] Animals. Normal and MPS I canines were obtained from the MPS
I canine colony at Harbor-UCLA. The dogs are a cross between
beagles and Plott hounds and average 12-20 kg in weight. The
canines were under 2 years of age and at least 4 months of age for
these experiments.
[0173] Monoclonal antibodies. A series of monoclonal antibodies
were obtained from Peter Moore (UC Davis Veterinary School) and
were specific to canine T cell receptor (anti-TCR), canine CD3
antigen (anti-CD3; IgG2b)) and the canine equivalent of Thy-1
(anti-Thy1; IgG1). The anti-TCR antibody was prepared by growing
the hybridoma in low serum containing medium and purification of
the antibody by protein A chromatography. The anti-CD3 and
anti-thy1 antibodies were prepared by production of ascites in mice
using the hybridomas, CA17.6B3 and CA1.4G8 and protein A
purification, at a contract laboratory (Strategic Biosolutions).
When utilized, the monoclonal antibodies were administered in 2 or
3 doses just prior to ITI.
[0174] Immunosuppressive drugs. Cyclosporine (Neoral or Sandimmune)
and Azathioprine (Imuran) were obtained from a local pharmacy. Both
drugs were dosed orally at the dose and frequency noted in the
experiments. The use of Neoral or Sandimmune was evaluated as a
factor in whether tolerance was induced and was not found to be
factor.
[0175] Immunosuppressive Drug Regimen with Every Other Day CsA
dosing that does not induce tolerance:
[0176] CsA Neoral.RTM. 25 mg/kg/every other day divided bid po
[0177] Aza Imuran.RTM. 5 mg/kg every other day on alternate days
from CsA
[0178] CsA+Aza given at full dose from day 0-18, 1/2 dose from day
19-32, and 1/4 th dose from 33-46 and 1/8.sup.th from day 47-60,
and then terminated
[0179] Animals monitored for adverse reactions
[0180] CsA peak and trough levels monitored and a trough level of
100-200 ng/ml in the circulation.
[0181] Intrathymic injection. During general anesthesia a 3-inch
incision was made in the left-side at the 3.sup.rd intercostal
space. The incision was continued through all muscle layers until
the chest cavity is reached. A rib separator was employed and the
thymus observed directly as a vascularized, lobed organ beneath the
lungs. The enzyme solution was injected into the thymus with a
syringe and 25 G needle. A "zig-zag" pattern was made when
inserting the needle into the thymus in an attempt to prevent
enzyme leakage upon withdrawal of the needle. The thymus was
visually inspected for any leakage of enzyme solution made visible
by Evan's Blue dye. The surgery site was closed and respiratory
function restored using vacuum drawn through a Foley catheter
inserted in the chest cavity. Antibiotics were applied to all
incision sites and surgery was followed by a short course of
antibiotics and pain management medication as necessary.
[0182] Injection solution: 1.5 mL total; I mg/kg of 12.1 mg/mL
iduronidase (3.times.10.sup.6 mL). Bring to 1.5 mL with 40% PEG+2
mg/mL Evan's Blue dye in acidic PBS (Final solution approximately
6% PEG, 0.3 mg/mL Evan's). Enzyme injection: 14,000 U/kg/week
.alpha.-L-iduronidase in 50 mL infusion in saline with 1 mg/mL
canine albumin, and 10 mM NaPO.sub.4 administered intravenously
over 2 hours (1st hour 3,000 U/kg/hr; .sub.2nd hour 11,000
U/kg/hr). Animals were monitored for anaphylactic reaction.
[0183] Results
[0184] Early tolerance experiments included CsA dosing at an every
other day interval. These early experiments sought to establish
whether ITI, immunosuppressive drugs and/or monoclonal antibodies
that deplete T cells are required for tolerance induction. FIG. 5
shows the results for some early canines studied using ITI and
drugs.
[0185] BI is a control dog that received an ITI of PBS only, and BE
and BC were two dogs treated with ITI of iduronidase. None of the
three were tolerant as titers exceeded 20 by 7 weeks. BO received
every other day CsA+Aza in addition to ITI and he also was not
tolerant with a titer of 42 by 7 weeks. JA and JO had the same
regimen but in addition, an anti-TCR monoclonal antibody was
infused prior to the ITI to determine if depleting T cells might
allow the induction of tolerance. Both canines mounted immune
responses and reached titers of 148 and 135 by 12 weeks. Curiously,
the control dog JH did not mount an immune response even though he
had the CsA+Aza and ITI protocol that had previously not worked
with BO. The tolerance included a period of modest titer to
iduronidase reaching a peak of 15.1 at week 1 and later declining
to 7.1 at week 18.
[0186] Evaluation of the routine CsA levels showed a pattern that
explains this result. Most dogs with every other day CsA had trough
levels of 60-190 ng/ml though one dog (JO) reached 342 (Table 2).
None of these dogs were tolerant. JH had a level of 570 which
exceeded the other dogs by at least 200 ng/ml. The dog received the
correct regimen based on a review of cage and drug administration
records and yet the level was much higher.
[0187] JH was tolerized to iduronidase infusions using a regimen of
CsA+Aza and ITI. Curiously, he had a much higher level of Csa in
his serum which suggested that the level of CsA might be a critical
factor. Following this experiment, further work on CsA dosing
demonstrated that daily CsA dosing could maintain the trough levels
above 400 ng/ml and that this, with every other day Aza dosing, was
sufficient to induce tolerance.
2TABLE 2 Comparison of Cyclosporine trough blood levels in
iduronidase non-tolerant and iduronidase tolerant canines Trough
CsA Level Canine Treatment (ng/mL) Mean Non- BO (CsA + Aza + ITI)
190 148 Tolerant (every JA (CsA + Aza + ITI + 63 other day CsA TCR
mAb) plus other ME (CsA + Aza + ITI + 60 treatments) TCR mAb) MA
(CsA + Aza + ITI + 120 CD3/Thyl/TCR mAb) MO (CsA + Aza + ITI + 110
CD3/Thyl/TCR mAb) Tolerant JH (CsA + Aza + ITI) 570 (every other
day CsA plus other treatments) Tolerant RH (CsA(daily) + Aza + ITI
520 (Tolerance RI (CsA(daily) + Aza + ITI) 450 drug regimen: RO
(CsA(daily) + Aza) 680 545 daily CsA PE (CsA(daily) + Aza, 440 MPS
I Affected) plus Aza) SA (CsA(daily) + Aza, 520 MPS I Affected) NI
(CsA(daily) + Aza, 660 MPS I Affected) Non-Tolerant PA (CsA(daily)
only) 490 daily CsA, no Aza)
[0188] In table 2, the cyclosporine trough blood levels for
tolerant and non-tolerant canines are shown as ng/mL blood.
Cyclosporine levels were measured after at least 4 consecutive
doses of cyclosporine. PA received daily cyclosporine treatment
alone beginning 4 days before initial iduronidase challenge and was
not tolerized to iduronidase. All other canines received
immunosuppressive drugs beginning 18 days before first intravenous
iduronidase challenge (4 days before ITI). All canines received
0.056 mg/kg/week rh iduronidase intravenous challenges. Successful
tolerance to iduronidase was consistently associated with
cyclosporine trough levels of 400-700 ng/mL. Canines with
cyclosporine trough levels less than 400 ng/mL were not tolerized
to iduronidase. (CsA=Cyclosporin A; Aza=Azathioprine;
ITI=Intrathymic injection of iduronidase; mAb=Monoclonal
antibody).
[0189] Table 2 shows additional data from other tolerized dogs
corroborating the role of CsA dose in the tolerizing regimen. The
non-tolerant dogs on the top section of the table received every
other day CsA at 25 mg/kg and the 6 dogs treated with drugs with or
without other treatments had trough CsA levels of 60-190 ng/ml,
with one case JO at 342 ng/ml. These levels are taken after at
least 4 doses of the drugs had been given during the regimen. The
mean CsA level of the non-tolerant dogs is 148 ng/ml. In the third
section, the tolerant dogs had trough CsA levels of 440-680 and a
mean of 545 ng/ml, more than 3 fold higher. JH had level shown in
the second section of the table of 570 ng/ml and though his dosing
was only every other day, the level was high enough and he became
tolerant. In contrast, PA shown in the fourth section of the table
had an adequate CsA level of 490, but did not have Aza as well, and
did not become tolerant. These data demonstrate the importance of
an adequate CsA effect in the tolerance protocol.
Example 5
[0190] Induction of Tolerance is Long-Lasting Without Chronic
Toleragen Stimulation
[0191] The induction of tolerance by a toleragen is particularly
useful if the tolerant state is long-lasting, even in the absence
of toleragen administration. To address the stability of the
tolerant state, canines that were tolerant and non-tolerant were
retained for varying intervals of time and then rechallenged with
iduronidase infusions. The data show that for at least 6 months, a
tolerant animal remains tolerant to iduronidase infusion even
without continuous toleragen or antigen exposure.
[0192] Methods and Materials
[0193] Tolerance was induced as described in the above examples for
each canine noted (the same initials are used). JO was the canine
tolerized with CsA dosing at every other day, unlike the other
canines. This canine did not have complete tolerance initially but
a muted antibody response. Other tolerance canines were fully
tolerant based on studies described in other examples. Iduronidase
was the same high uptake iduronidase as described above. The
canines were challenged with 0.58 mg/kg of enzyme administered
intravenously on a weekly basis for 3-6 doses.
[0194] Results and Discussion
[0195] In FIG. 6, the titers of canines that were tolerant or
non-tolerant are shown with a gap indicated for the period in which
no toleragen or therapeutic infusions occurred. As little as 5
weeks and as much as 6 months of toleragen hiatus did not change
the response of the tolerant dogs, who remained tolerant and did
not mount a significant immune response. The non-tolerant dog RU
showed a >20 fold induction in antibody titer after receiving 2
doses of enzyme after a 4.5 month hiatus. The dog JH showed some
response at 3 weeks of reinduction that exceeded that of most
tolerant dogs but did not reach that of a sensitized non-tolerant
dog RU. JH also had a more significant response when originally
challenged and was not as tolerant as the dogs tolerized with the
optimal regimen. His original titer pattern also plateaued and
declined with continued infusions, which will be studied in this
case.
[0196] These data demonstrate that tolerance induced by the methods
and compositions of the present invention are long lasting and
clinically useful.
Example 6
[0197] Induction of Tolerance in Humans
[0198] Materials and Methods
[0199] Patients. Patients with mucopolysaccharidosis I are selected
for treatment. The patients are evaluated at base line and at 6,
12, 26, and 52 weeks by detailed clinical examinations, magnetic
resonance imaging of the abdomen and brain, echocardiography,
range-of-motion measurements, polysomnography, clinical laboratory
evaluations, measurements of leukocyte .alpha.-L-iduronidase
activity, and urinary glycosaminoglycan excretion.
[0200] Immunosuppressive drugs. Cyclosporine (Neoral or Sandimmune)
and Azathioprine (Imuran) are obtained from commercial sources.
Both drugs are dosed orally at the dose and frequency as follows:
CsA Neoral.RTM. 12.5 mg/kg/every day divided bid po; Aza
Imuran.RTM. 5 mg/kg qod po for two weeks conditioning period. The
drugs are then administered at that dose for an additional two
weeks in the presence of toleragen. Doses are halved for all drugs
each 2 weeks after first toleragen infusion. Patients are monitored
for adverse reactions, and for CsA peak and trough levels.
[0201] Toleragen. Recombinant .alpha.-L-iduronidase is produced in
Chinese-hamster-ovary cells with the use of bioreactors and
standard column chromatography, and extensively analyzed for safety
and purity. The activity of .alpha.-L-iduronidase is measured
according to the method of Shull et al. supra., or with an assay
whose results are reported in SI units (Kakkis et al., 2001,
supra). When the latter assay is used, a dose of 125,000 U of
.alpha.-L-iduronidase per kilogram is equivalent to 100 SI units
per kilogram. Urinary glycosaminoglycan excretion is measured
according to an adaptation of the method of Bjornsson.
Enzyme-linked immunosorbent assays for antibodies to
.alpha.-L-iduronidase uses a variation of the method of Shull et
al., and Western blotting is performed according to a standard
method.
[0202] The toleragen is administered by intravenous infusion
(diluted in normal saline with 0.1 percent human serum albumin) at
a dose of 14,000 U (0.056 mg)/kg, delivered weekly. The first dose
is given after completion of the two week conditioning period, and
weekly thereafter. Patients are premedicated with diphenhydramine
(0.5 to 1.25 mg per kilogram of body weight).
[0203] After induction of tolerance, usually 6 to 8 weeks after
initiation of the conditioning period, the dose is increased to
once weekly, 125,000 U (0.58 mg) per kilogram; the rate is 3000 U
per kilogram during the first hour and 61,000 U per kilogram during
each of the following two hours.
Example 7
[0204] Immune Tolerance Regimen Prevents the Induction of Other
Immunoglobulins in Addition to IgG
[0205] The tolerance regimen in canine prevents the induction of
IgG as manifested by a decrease in total IgG response against
iduronidase as compared to the IgG response observed in
non-tolerant canines. The present Example provides additional
evidence that shows that in addition to preventing the induction of
IgG, the tolerizing regimen prevents the induction of other
immunoglobulins subtypes also. Analyses of the immune sera of
canines that were either tolerant (dogs PE and SA) or canine
controls (dogs RU and UM) that had not received the tolerizing
regimen were studied for the presence of IgG, IgA and IgE
antibodies to iduronidase.
[0206] The data from these determinations are shown in FIGS. 7A to
7D. These data show that tolerant dogs did not have a significant
increase (>3 fold) in titer in these other subtypes and
non-tolerant dogs did have a significant IgA or IgE response
.about.10 fold in some cases. Although the IgA and IgE titers were
not high relative to IgG, these titers were significant and
positive in the non-tolerant canines. The data show that the
tolerance regimen also prevents other Ig subtypes from being
induced consistent with a broader effect on the humoral
response.
Example 8
[0207] Induction of Tolerance in Animals with Pre-existing Immune
Response
[0208] In another aspect of the present invention, the inventors
demonstrated that it is possible to reduce or eliminate an existing
immune response. The present Example discusses data generated from
the canine Nitro. Nitro was originally tolerized, but after a
period of nearly 6 months of hiatus from antigen exposure, a
reexposure induced a profound anamnestic response with a titer
reaching 400+. After several weeks without exposure to antigen, the
canine was placed on the tolerance regimen and given low dose
(0.056 mg) weekly infusions of iduronidase. The titer initially
rose during the administration of the CsA+Aza regimen to greater
than 100 in a typical anamnestic response and than rapidly fell to
below 20 (see FIG. 8). The response was reduced by the regimen and
the immune response to enzyme is relatively muted compared with the
>400 level previously on 0.5 mg/kg/wk infusions. These data
demonstrate that it is possible to reinduce the tolerance to the
toleragen even if the animal already possesses an immune response
against the antigen in the toleragen composition.
Example 9
[0209] Presence of High Affinity Uptake Residue on the Antigen
Renders Antigen More Effective as a Toleragen
[0210] The effects of a high affinity moiety on the toleragen were
discussed in Example 1 above, which showed that the use of a moiety
such as mannose-6-phosphate may be useful in augmenting the
tolerizing capability in an antigen. This aspect of the toleragen
compositions was investigated further and the results are reported
in the present Example and in FIG. 9. Iduronidase normally
comprises a mannose-6-phoshate and this high uptake moiety
facilitates the uptake of the iduronidase by the target cells via a
mannose-6 phosphate receptor.
[0211] In the present example, recombinant iduronidase was
dephosphorylated by incubation with acid phosphatase bound to
beads. The enzyme was successfully dephosphorylated as demonstrated
by the lack of efficient uptake in Hurler fibroblasts and a
K.sub.uptake that was too low to be calculated. The
dephospho-idurondase was used in a tolerization regimen similar to
the tolerization experiments conducted with the iduronidase
discussed in the above examples. The data are shown in FIG. 9. This
study showed that the canine tolerized with dephospho-iduronidase
was not able to tolerize, again suggesting that high uptake
affinity is needed for tolerance.
[0212] Example 10
[0213] Methods of Inducing Tolerance to Factor VIII
[0214] Treatment of hemophilia A relies on the ability to deliver
an effective amount of Factor VIII to the hemophiliac. However,
antibodies or inhibitors to Factor VIII therapy can be a
significant problem in patients with hemophilia A disease.
Hemophiliac patient that have developed antibody inhibitors to
Factor VIII may experience uncontrolled bleeding rendering the
Factor VIII therapy ineffective. Examples 1-9 describe methods and
compositions for inducing an antigen specific immune tolerance
administration of .alpha.-L-iduronidase or alpha-glucosidase as a
toleragen in combination with a regimen of immunosuppression for a
period of time sufficient to tolerize the host to the toleragen.
The present example is directed to inducing immune tolerance to
Factor VIII. The present example provides a protocol. to be used to
prevent and reverse the formation of inhibitor against Factor
VIII.
[0215] Preparation of a toleragenic factor VIII. The toleragenic
Factor VIII is made by conjugating the protein to a high uptake
protein. In the simplest approach Factor VIII protein is conjugated
using a lysosomal enzyme, iduronidase that contains a high affinity
uptake marker on its N-linked carbohydrates. The proteins are
conjugated using a heterobifunctional cross-linking agent (SBDB for
example). Optimally, the cross-linking would occur at a ratio of
1:1 and the cross-linked proteins purified.
[0216] Tolerization of a hemophilia patients: The patient would
receive cyclosporin A at a dose expected to be 12.5 mg/kg/d divided
to achieve a blood level of greater than 400 ng/ml. The patient
would also receive Azathioprine at 2-5 mg/kg every other day as
appropriate for a human patient. After 18 days of the drugs, the
patient would receive 0.6 mg/kg of toleragen in an infusion on a
weekly basis. After day 32, the CsA and Aza are cut in half in dose
size and after day 46 the drugs are cut to 1/4. On day 60, the
CsA+Aza is terminated and the weekly infusions continued.
Monitoring of the immune response would show only a modest if any
response that was less than 20% fold the prior response without
tolerance. The patient receives normal factor VIII infusions and
the toleragen is terminated. If needed, additional toleragen may be
administered to maintain the tolerant state at intervals.
Example 11
[0217] Tolerance to .beta.-cell Antigens in Diabetes
[0218] Antibodies and cellular immune responses to .beta.-cell
antigens, such as, glutamic acid decarboxylase (GAD) are
responsible for the destruction of the pancreatic .beta.-cell and
the onset of diabetes. The induction of tolerance to these antigens
would provide an important method to prevent the progression and
onset of diabetes in patients at risk or having onset of Type 1
diabetes symptoms. GAD is one important antigen but other antigens
such as IA2 and insulin could also be studied this way.
[0219] Preparation of a toleragenic GAD. The toleragenic from of
GAD may be made by fusion with a high-uptake peptide moiety. The
gene for human GAD is connected in frame with the gene for a high
uptake peptide such as IGF2, that is known to bind the mannose
6-phosphate receptor. Such a fusion would allow both proteins to be
made in the right structure and with the right antigenicity and
form.
[0220] Tolerization of a type 1 diabetes patients. A new onset
diabetes patient who was early in the course of the disease and
retained some .beta.-cell function, would preferably be used. The
patient would receive cyclosporin A at a dose expected to be 12.5
mg/kg/d divided bid to achieve a blood level of greater than 400
ng/ml. The patient would also receive azathioprine at 2-5 mg/kg
every other day as appropriate for a human patient. After 18 days
of the drugs, the patient would receive 0.06 mg/kg of toleragen in
an infusion on a weekly basis. After day 32, the CsA and Aza may be
cut in half in dose size and after day 46 the drugs may be cut to
1/4. On day 60, the CsA+Aza is terminated and the weekly infusions
continued. The toleragen dose may be then increased to 0.6 mg/kg
each week. Monitoring of the immune response would likely show only
a modest if any response that was less than 20% fold the prior
response without tolerance. The patient would have normalized
glucose tolerance and the toleragen would be terminated. If needed,
additional toleragen could be administered to maintain the tolerant
state at intervals to be determined by the clinical condition of
the patient.
* * * * *