U.S. patent application number 11/266959 was filed with the patent office on 2006-08-24 for method for treating established spontaneous auto-immune diseases in mammals.
Invention is credited to Jean-Francois Bach, Lucienne Chatenoud.
Application Number | 20060188494 11/266959 |
Document ID | / |
Family ID | 36272220 |
Filed Date | 2006-08-24 |
United States Patent
Application |
20060188494 |
Kind Code |
A1 |
Bach; Jean-Francois ; et
al. |
August 24, 2006 |
Method for treating established spontaneous auto-immune diseases in
mammals
Abstract
A method of treating spontaneous and ongoing auto-immune
diseases in mammals, comprising administering to a mammal, in need
of such a treatment, a therapeutically effective amount of one or
more non mitogenic anti-CD3 active principles to achieve permanent
disease remission through the induction of antigen-specific
unresponsiveness, i.e. immune tolerance.
Inventors: |
Bach; Jean-Francois; (Paris,
FR) ; Chatenoud; Lucienne; (Paris, FR) |
Correspondence
Address: |
CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI,;STEWART & OLSTEIN
5 BECKER FARM ROAD
ROSELAND
NJ
07068
US
|
Family ID: |
36272220 |
Appl. No.: |
11/266959 |
Filed: |
November 4, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
08986568 |
Dec 5, 1997 |
7041289 |
|
|
11266959 |
Nov 4, 2005 |
|
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Current U.S.
Class: |
424/133.1 ;
424/144.1 |
Current CPC
Class: |
Y10S 424/81 20130101;
A61K 2039/505 20130101; Y10S 530/868 20130101; C07K 2317/54
20130101; C07K 16/2809 20130101 |
Class at
Publication: |
424/133.1 ;
424/144.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1-15. (canceled)
16. A method of treating an ongoing autoimmune disease in a human
having said disease, wherein said disease is psoriasis, comprising:
treating said human by administering one or more non-mitogenic
anti-CD3 active compounds selected from the group consisting of CD3
antibodies and fragments of CD3 antibodies in an amount effective
to treat said psoriasis.
17. The method of claim 16 wherein said treatment results in a
durable remission of said psoriasis.
18. The method of claim 16 wherein said administration of said one
or more non-mitogenic anti-CD3 active compounds is a non-chronic
administration.
19. The method of claim 16 wherein said non-mitogenic anti-CD3
active compound is a non-mitogenic anti-CD3 monoclonal antibody
(Fab').sub.2 fragment.
20. The method of claim 16 wherein said non-mitogenic anti-CD3
active compound is a non-mitogenic anti-CD3 antibody.
21. The method of claim 20 wherein said non-mitogenic anti-CD3
active compound is a non-mitogenic anti-CD3 monoclonal
antibody.
22. The method of claim 21 wherein said monoclonal antibody is
selected from the group consisting of murine or humanized
antibody.
23. The method of claim 16 wherein said compound is highly purified
and endotoxin-free.
24. The method of claim 16 wherein said active compound is
administered by injection.
25. The method of claim 16 wherein said non-mitogenic anti-CD3
active compound is administered in an injectable form that contains
from 5 to 20 mg of the non-mitogenic anti-CD3 active compound.
Description
[0001] The invention relates to a method for treating established
and ongoing spontaneous auto-immune diseases in mammals.
[0002] In order to suppress T cell function, immunotherapy based on
the use of antibodies directed at T cell surface receptors,
particularly of monoclonal antibodies (mAbs), has been extensively
investigated. Particularly, mAbs directed against the CD3 complex
of the T cell receptor have been shown to cause transient T-cell
depletion and antigenic modulation of the CD3-T cell receptor
complex.
[0003] In PNAS USA, vol. 91, p 123-127, 1994 Immunology, the
inventors, with other co-authors, have reported that a short term
treatment with low doses of an anti-CD3 mAb could restore self
tolerance to .beta.-cell-associated antigens, thus inducing
complete and durable remission of the spontaneous auto-immune
diabetes, in overtly diabetic NOD (non obese diabetic) mice.
[0004] By further investigating the mode of action of anti-CD3 mAb
in this model, the inventors have found that the long term effect
was obtained only when treating animal(s)at a very advanced disease
stage, i.e. overt auto-immunity. They also demonstrate that non
mitogenic, F(ab').sub.2 fragments of the entire CD3 mAb, that are
much better tolerated than the whole entire CD3-mAb, also afford a
long term in vivo effect in overtly diabetic NOD mice as did the
whole anti-CD3 mAb.
[0005] This finding is an unexpected extension of the published
data which until now, in both transplantation and antigen and/or
pharmacologically induced auto-immunity, has proposed (Fab').sub.2
fragments of anti-CD3 mAb as effective tools to only achieve
immunosuppression (an overall depression of immune responses that
is only maintained through the chronic administration of the
drugs), but not to promote permanent antigen-specific
unresponsiveness namely, a state of immune tolerance (an
antigen-specific immune unresponsiveness that is maintained in the
absence of chronic generalized immunosuppression.
[0006] Such results are useful for application to other auto-immune
situations where similar immunoregulatory mechanisms, such as those
present in auto-immune diabetes, have been observed.
[0007] Accordingly, an object of the invention is to provide a
method of treatment of spontaneous and ongoing of auto-immune
diseases in mammals to achieve permanent Ag-specific
unresponsiveness, without the morbidity and with a minimal humoral
response as that encountered when administering the whole mitogenic
antibody.
[0008] Another object of the invention is to provide effective
tools useful for such a method of treatment.
[0009] According to the invention, the method of treating
auto-immune diseases in mammals comprises administering to a
mammal, in need of such a treatment, a therapeutically effective
amount of one or more non mitogenic anti-CD3 active principles to
achieve permanent disease remission through the induction of
antigen-specific unresponsiveness, i.e. immune tolerance.
[0010] Such a treatment was shown to be able to promote durable
remission of the established disease without the clinical side
effects involved when administering mitogenic whole anti-CD3
antibodies.
[0011] Particularly preferred non mitogenic anti-CD3 antibodies are
monoclonal antibodies or fragments thereof, especially F(ab').sub.2
fragments.
[0012] Said fragments are advantageously such as obtained by pepsin
digestion of the whole antibody.
[0013] In view of their therapeutical use, said active principle(s)
are highly purified and particularly endotoxin-free.
[0014] Said non mitogenic anti-CD3 monoclonal antibody, or fragment
thereof is of murine origin or is an humanized antibody.
[0015] The permanent antigen-specific unresponsiveness obtained
with said anti-CD3 active principles make them particularly useful
as therapeutic tools for treating auto-immune pathologies. In
particular, they are suitable for treating diabetes, rheumatoid
arthritis, multiple sclerosis or psoriasis.
[0016] In said applications, they will be administered, if desired,
in combination with other active ingredients and/or compounds which
facilitate their assimilation.
[0017] Said therapeutical tools are advantageously administered in
combination with pharmaceutical carriers under the form of
pharmaceutical preparations.
[0018] Different forms of administration may be used, especially
for injectable route.
[0019] The injectable forms contain 5 to 20 mg of active principle
per unit dose, preferably from 5 to 10 mg.
[0020] For information only, the dose which can be used in the
treatment of auto-immune diseases in humans, for example diabetes,
is 5 to 10 mg/day for 10 to 14 consecutive doses.
[0021] The invention now will be described with respect to the
drawings, wherein:
[0022] FIG. 1A shows the kinetics of expression of Interleukin-2
(IL-2), Interleukin-4 (IL-4), and .beta.-2 microglobulin
(.beta.-2m) RNA in NOD female mice that were given intact CD3
monoclonal antibody, F(ab').sub.2 fragments of CD3 monoclonal
antibody, L2 antibody, or saline; and
[0023] FIG. 1B shows the results of semiquantification of
amplification products using doubling dilutions of cDNA for PCR
reactions for IL-2, IL4, and IL-10.
[0024] Other characteristics and advantages of the invention will
appear from the examples given hereinbelow.
EXAMPLE 1
[0025] Treatment of overtly diabetic NOD female mice with purified
F(ab').sub.2 fragments of CD3 anti-mAb.
[0026] NOD mice (K.sup.d, I-A.sup.NOD, D.sup.b) were bred under
specific pathogen-free conditions; in females, spontaneous IDDM
appears by 14 weeks of age (90% incidence at 30 weeks of age) and
is preceded by insulitis at 4 to 6 weeks.
[0027] In a preferred embodiment of the invention said non
mitogenic anti-CD3 active principle is a non mitogenic anti-CD3
antibody or a fragment thereof. Such fragments are advantageously
F(ab').sub.2 fragments.
[0028] The cell line producing the hamster 1452C11 mAb (IgG,
anti-mouse CD3 .epsilon.-chain) was used in those experiments (O.
M. Les et al, 1987, PNAS USA 84:1274)
[0029] The anti-CD3 mAb F(ab').sub.2 fragments were prepared by
pepsin digestion.
[0030] Pepsin (Sigma Chemical Co., St. Louis, Mo.) was used at a
final concentration of 2% (20 .mu.g/mg of purified antibody) in 1M
acetate buffer, pH 3. Digestion was conducted for 2 h at 37.degree.
C. Following dialysis at 4.degree. C. using 0.1 M PBS pH 8,
digested F(ab').sub.2 fragments were purified in two steps : a
protein A-Sepharose CL-4B affinity chromatography column to
eliminate digested Fc fragments, then gel filtration of the
nonretained fraction on an Ultragel AcA54 column (Pharmacia,
Uppsala, Sweden).
[0031] The physico-chemical properties of the fragment preparations
were analyzed by SDS-PAGE.
[0032] The binding capacity was tested by immunofluorescence in a
classical competition assay using purified FITC-labeled whole CD3
mAb.
[0033] The digestion and purification of F(ab').sub.2 fragments was
performed with special caution to avoid endotoxin contamination.
The material used for in vivo treatment was negative in the Limulus
assay.
[0034] NOD females presenting with overt diabetes were included in
the treatment protocol when a fasting glycemia ranging 3.5 to 4 g/L
was scored on two consecutive occasions. Mice were then randomized
to receive a treatment with CD3 mAb F(ab').sub.2 fragments (50
.mu.g/day for 5 consecutive days), whole CD3 mAb (5-20 .mu.g/day
for 5 consecutive days), or as a control normal hamster Igs.
[0035] Complete remission was defined as a return to normal
glycemia and the disappearance of glycosuria in the absence of any
exogenous insulin supply. Histopathology on paraffin sections of
Bovin-fixed or frozen pancreatic tissue were performed as
previously described (1). Scoring of mononuclear cell infiltration
was as follows: grade 0=normal islets; grade 1=focal or peripheral
insulitis (lymphocytes around the islet, but no destruction of
endocrine cells as assessed by labeling with anti-insulin Abs); and
grade 2 invasive destructive insulitis.
[0036] The results regarding the remission of overt diabetes in the
mice following the short treatment with purified F(ab').sub.2
fragements are given in Table 1. TABLE-US-00001 TABLE I % Remission
of IDDM Weeks After Anti-CD3 F(ab').sub.3 Hamster 1 g Treatment n =
42 n - 18 0 0 0 2 55 22 4 62 16 6 64 0 10 67 0 20 67 0
[0037] The difference in percent remission between CD3 mAB
F(ab').sub.2 fragements-treated and control animals is
statistically significant (p<0.0l) using .chi..sup.2 test.
[0038] As shown by said results, F(ab').sub.2 fragments of the mAb
appeared potent in promoting permanent remission of overt diabetes
in the conditions of the experiments.
EXAMPLE 2
[0039] Study of the triggering effect of F(ab').sub.2 fragements of
CD3 mAb on cytokine gene transcription.
[0040] NOD females received a single i.v. injection of either
intact 145 2C11 CD3 mAb (20 .mu.g) or purified F(ab').sub.2
fragments of 145 2C11 CD3 mAb (50 .mu.g). Mice injected with saline
or with 5 to 50 pg of L2, a hamster mAb specific for recombinant
but not natural mouse IL-2, were used as controls. Three individual
animals were analyzed in each group. Spleen cells were collected
before any treatment and at various times following injection of
the different preparations, and RNA was extracted for RT-PCR.
[0041] Crude RNA was extracted using TRIzol (Life Technologies)
followed by isopropanol precipitation. For reverse transcription
(RT), total RNA (6 .mu.l in a final volume of 12 .mu.l) was added
to 18 .mu.l of cDNA synthesis reaction mixture. Two microliters of
RT product was amplified using PCR for 30 cycles, final volume of
50 .mu.l, with standard buffer conditions and a final Mg.sup.2+
concentration of 1.5 mM (2.5 U Taq UNA polymerase, Life
Technologies) Each PCR cycle consisted of 1 min. at 94.degree. C.,
1 min. at 55.degree. C., and 1 min. at 72.degree. C. on a Techne
thermal cycler (Osi, Paris, France); 100 ng of cDNA was used for
PCR unless stated otherwise. When needed to semi-quantitate the
amplification products obtained, PCR with doubling dilutions of
cDNA was performed. The following primers (Bioprobe Systems,
France) were used IFN-.gamma. 5' primer, CCA GCA GAG AAT GGA AAG
TC; IFN-.gamma. 3' primer. GAT GCT GCT TAC ATG TCT CG; IL.2 5'
primer CCA GCA GAG AAT GGA AAG TC; IL-2 3' primer, GAT GCT GCT TAC
ATG TCT CG; IL-4 5' primer. TCG GCA TTT TGA ACG AGG TC, IL-4 3'
primer. GAA AAG CCC GAA AGA GTC TC; IL-10 5' primer, GGG ATG ACA
GTA GGG GAA CC; IL-10 3' primer, AGA GCA AGG CAG TGG AGC AG:
.beta..sub.2-microglobulin 5' primer, CCA GCA GAG AAT GGA AAG TC
.beta..sub.2-microglobulin 3' primer, GAT GCT GCT TAC ATG TCT CG.
Ten microliters of RT-PCR products were separated by 1.2% agarose
gel electrophoresis in 1.times.TBE (Tris-borate-EDTA) containing
0.2 .mu.g/ml of ethidium bromide and visualized under UV light.
Where products were semiquantified, RT-PCR, .beta..sub.2
microglobulin mRNA was used as a housekeeping reporter gene.
[0042] The kinetics of mRNA expression is shown in FIG. 1, part A.
Semiquantification of amplification products was performed using
doubling dilutions of cDNA for PCR reactions; data are shown for
IL-2, IL-4, and IL-10 in FIG. 1, part B.
[0043] As shown in FIG. 1A, using PCR on splenocytes from anti-CD3
F(ab').sub.2-treated animals, enables identification of the
transcription of mRNAs specific for IL-2, IL-4, IL-10, and
IFN-.gamma.. Semiquantification using serial dilution of cDNA
samples suggested that, as compared with what was observed in NOD
mice treated with intact CD3 mAb, F(ab').sub.2 fragments promote a
less effective transcription of IL-2 mRNA, whereas similar levels
of IL-4, IL-10, and IFN-.gamma. message were detected (FIG.
1B).
EXAMPLE 3
[0044] Cytokine production by stimulated spleen cells from CD3 mAb-
and F(ab').sub.2-treated NOD mice.
[0045] Spleen cells from CD3 mAb- and F(ab').sub.2-treated NOD mice
were collected at different times after treatment and tested for
their capacity to secrete IFN-.gamma. and IL-4 upon mitogenic
stimulation using Con A.
[0046] Spleen cells from CD3 or F(ab').sub.2 fragments-treated
animals were collected and cultured in vitro
(1.times.10.degree./ml) in 24-well plates, for 24 to 48 h in a
humidified atmosphere containing 5% CO.sub.2, in DMEM-Glu-tamax
(Life Technologies, Paisley, Scotland) supplemented with 100 IU/ml
penicillin, 100 .mu.g/ml streptomycin, sodium pyruvate,
nonessential aminoacids, 0.05 mM .beta.-mercaptoethanol, and 10%
FCS. In stimulated cultures, Con A was added at a final
concentration of 10 .mu.g/ml. Supernatants were collected and
stored frozen at -80.degree. C. until tested. IFN-.gamma. and IL-4
were quantitated using specific ELISA as already described (17).
The Abs used for detection were AN18 (kindly provided by Dr. A.
O'Garra, DNAX, Palo Alto, Calif.) and biotinylated R46A2 for
IFN-.gamma. and 11B11 and biotinylated BVD6 (kindly provided by A.
O'Garra) for IL-4, Mouse rIL-4 (R&D Systems. Minneapolis,
Minn.) and IFN-.gamma. were used as internal standards. Detection
limits were 0.2 ng/ml for IL-4 and 0.1 ng/ml for IFN-.gamma..
[0047] The results are given in Table 2 TABLE-US-00002 TABLE 2 Time
from Treatment Treatment (week) IFN-.gamma. (ng/ml) Anti-CD3 2
68.36 .+-. 11.51* Anti-CD3 F(ab').sub.2 2 16.37 .+-. 3.40* Hamster
Ig 2 101.90 .+-. 13.34* Anti-CD3 7 .sup. 31.74 .+-. 4.14.sup.5
Anti-CD3 F(ab').sub.2 7 .sup. 23.21 .+-. 5.69.sup.1 Hamster Ig 7
35.76 .+-. 4.20 Untreated controls 29.42 .+-. 7.11
[0048] As compared with age-matched controls injected with
irrelevant hamster Ig, polyclonally activated spleen cells from CD3
mAb- and F(ab').sub.2 fragment-related NOD mice showed, for about 5
wk from the end of treatment, a significantly decreased
IFN-.gamma.-producing ability (Table II).
EXAMPLE 4
[0049] Pharmaceutical formulation
[0050] The active principles are formulated under a desaggregated
form and either lyophilyzed or suspended into an appropriate
liquid, each dose containing, as above mentioned, 5 to 20 mg of non
mitogenic antibody or a fragment thereof.
Sequence CWU 1
1
10 1 20 DNA Artificial PCR primer 1 ccagcagaga atggaaagtc 20 2 20
DNA Artificial PCR primer 2 gatgctgctt acatgtctcg 20 3 20 DNA
Artificial PCR primer 3 ccagcagaga atggaaagtc 20 4 20 DNA
Artificial PCR primer 4 gatgctgctt acatgtctcg 20 5 20 DNA
Artificial PCR primer 5 tcggcatttt gaacgaggtc 20 6 20 DNA
Artificial PCR primer 6 gaaaagcccg aaagagtctc 20 7 20 DNA
Artificial PCR primer 7 gggatgacag taggggaacc 20 8 20 DNA
Artificial PCR primer 8 agagcaaggc agtggagcag 20 9 20 DNA
Artificial PCR primer 9 ccagcagaga atggaaagtc 20 10 20 DNA
Artificial PCR primer 10 gatgctgctt acatgtctcg 20 #288216 v1 -
Sequence Listing (Bach, Jean-Francois)
* * * * *