U.S. patent application number 11/987956 was filed with the patent office on 2008-05-29 for activation and inhibition of the immune system.
Invention is credited to Marc Feldmann, Brian Foxwell.
Application Number | 20080124322 11/987956 |
Document ID | / |
Family ID | 10867023 |
Filed Date | 2008-05-29 |
United States Patent
Application |
20080124322 |
Kind Code |
A1 |
Foxwell; Brian ; et
al. |
May 29, 2008 |
Activation and inhibition of the immune system
Abstract
Activation of the immune response by NF-kB inducers, induction
of an anergic response by NF-kB inhibitors and the inhibition and
activation of immune response by the administration of an activator
or inhibitor of NF-kB is disclosed. Examples of NF-kB inhibitors
include IkB.alpha., PSI, a nucleotide sequence encoding IkB.alpha.
anti-sense nucleic acid encoding an NF-kB sequence, such as Rel B,
and anti-NF-kB antibodies. Examples of NF-kB inducers include NIK,
MEKK, IKK2, TFRRF2, and Rel B. Also disclosed are vectors encoding
inducers and inhibitors of NF-kB, for example adenoviral
vectors.
Inventors: |
Foxwell; Brian; (London,
GB) ; Feldmann; Marc; (London, GB) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Family ID: |
10867023 |
Appl. No.: |
11/987956 |
Filed: |
December 6, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10168805 |
Jan 31, 2003 |
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PCT/GB00/04925 |
Dec 22, 2000 |
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11987956 |
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Current U.S.
Class: |
424/130.1 ;
424/184.1; 424/277.1; 424/93.2; 435/375; 514/17.8; 514/19.3;
514/2.3; 514/44A; 536/23.1; 536/23.5 |
Current CPC
Class: |
A61P 25/28 20180101;
A61K 38/1709 20130101; A61K 48/00 20130101; A61K 38/177 20130101;
A61P 31/00 20180101; C12N 2799/06 20130101; A61K 2039/53 20130101;
A61P 37/02 20180101; C12N 2799/022 20130101; A61K 38/45 20130101;
A61P 37/00 20180101; A61P 37/08 20180101; A61P 37/06 20180101; A61P
37/04 20180101; A61P 35/00 20180101; A61K 38/07 20130101; A61K
39/0008 20130101; A61K 2039/5154 20130101; A61K 39/0011
20130101 |
Class at
Publication: |
424/130.1 ;
424/184.1; 514/44; 435/375; 424/277.1; 536/23.1; 536/23.5; 514/12;
424/93.2 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; A61P 37/00 20060101
A61P037/00; A61P 37/08 20060101 A61P037/08; C07H 21/04 20060101
C07H021/04; A61K 35/12 20060101 A61K035/12; A61K 38/16 20060101
A61K038/16; A61K 39/00 20060101 A61K039/00; A61K 31/711 20060101
A61K031/711; C12N 5/06 20060101 C12N005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 24, 1999 |
GB |
9930616.9 |
Claims
1. (canceled)
2. A method of inhibiting antigen presentation or inducing an
anergic response in a mammal, such as a human, comprising
administering a pharmaceutically-effective dose of an NF-.kappa.B
inhibitor, wherein the mammal, such as a human, is administered an
antigenic molecule or polynucleotide encoding an antigenic
molecule.
3. (canceled)
4. A method of treating an autoimmune disease comprising
administering a pharmaceutically-effective dose of an NF-.kappa.B
inhibitor to induce an anergic response in a mammal, wherein the
mammal, such as a human, is administered an antigenic molecule or
polynucleotide encoding an antigenic molecule.
5. (canceled)
6. A method of preventing transplant rejection in a mammal, such as
a human, comprising administering a pharmaceutically-effective dose
of an NF-.kappa.B inhibitor to induce an anergic response in a
mammal, wherein the mammal, such as a human, is administered an
antigenic molecule or polynucleotide encoding an antigenic
molecule.
7. A method according to claim 2, comprising administering a vector
comprising a nucleic acid encoding an NF-.kappa.B inhibitor
operatively linked to regulatory elements necessary for expression
of said nucleic acid.
8. A method according to claim 7, wherein the vector is an
adenovirus or a lentivirus.
9-13. (canceled)
14. A method according to claim 2, wherein the inhibitor is
I.kappa.B.alpha. or PSI or an IKK2 dominant negative mutant.
15. A method according to claim 2, wherein the inhibitor is an
anti-sense nucleic acid effective against at least a portion of an
NF-.kappa.B.
16. A method according to claim 15, wherein the anti-sense nucleic
acid is effective against Rel B.
17. A method according to claim 2, wherein the inhibitor is encoded
by a nucleic acid in a vector, the vector additionally comprising
regulatory elements operatively linked to said nucleic acid.
18. A method according to claim 17, wherein the inhibitor is an Fv
fragment of an antibody.
19. A method according to claim 2, wherein the inhibitor is an
antibody or an effective fragment thereof, or a cDNA encoding an
antibody or an effective fragment thereof, capable of binding
NF-.kappa.B or a fragment thereof.
20. A method according to claim 19, wherein the antibody is an
anti-Rel B antibody or an effective fragment thereof or a cDNA
encoding Rel B or an effective fragment thereof.
21. (canceled)
22. A method of stimulating antigen presentation or of stimulating
an immune response in a mammal, such as a human, comprising
administering a pharmaceutically effective amount of an NF-.kappa.B
inducer, wherein the mammal, such as a human, is administered an
antigenic molecule or polynucleotide encoding an antigenic
molecule.
23. A method according to claim 22 for treating an infectious
disease or a cancer.
24-27. (canceled)
28. A method according to claim 22, wherein the inducer is selected
from the group consisting of MEKK1, NIK, IKK2, TRAF2, TRAF5, TRAF6,
TAK, a dominant negative mutant of Myd88, TP L-2, IRAK, IKK5, Toll
receptors, Rel B, and fragments and muteins thereof which are
capable of inducing NF-.kappa.B.
29. A method according to claim 22, wherein the NF-.kappa.B inducer
is encoded by a nucleic acid within a vector, the vector
additionally comprising regulatory elements operatively linked to
said nucleic acid and necessary for expression of said nucleic
acid.
30. A method according to claim 29, wherein the vector is an
adenovirus or a lentivirus.
31. A method according to claim 22, wherein the NF-.kappa.B inducer
is in the form of a DNA vaccine.
32. A method of inhibiting maturation and activation of APCs, such
as dendritic cells in vivo or in vitro, comprising administering an
effective amount of an inhibitor of NF-.kappa.B.
33. A method of stimulating maturation and activation of APCs, such
as dendritic cells in vivo or in vitro, comprising administering an
effective amount of an inducer of NF-.kappa.B.
34. (canceled)
35. The method according to claim 33, wherein the inducer is
targeted to APCs such as dendritic cells or precursors thereof.
36. The method according to claim 33, wherein APCs, such as
dendritic cells or precursors thereof are exposed to the inducer in
vitro and introduced into a mammal.
37-40. (canceled)
41. A kit of parts or composition or a chimaeric molecule,
comprising (1) a modulating portion comprising or encoding an
NF-.kappa.B inhibitor or inducer and (2) an antigenic portion
comprising or encoding an antigenic molecule.
42. The kit of parts to of claim 41, wherein the antigenic molecule
comprises an epitope present on transformed or cancerous cells or
on a pathogenic organism, or on a cell infected by a pathogenic
organism, or a polypeptide expressed in a pathologic condition such
as Alzheimer's disease or a spongiform encephalopathy.
43. A vaccine effective against cancer or tumour cells, or against
a pathogenic organism or cell infected with a pathogenic organism,
or a disease characterised by expression of a polypeptide in a
pathologic condition such as Alzheimer's disease or a spongiform
encephalopathy comprising an effective amount of an NF-.kappa.B
inducer or polynucleotide encoding an NF-.kappa.B inducer and
further comprising an antigen or polynucleotide encoding an
antigen, having an epitope present on the cancer or tumour cells,
or the pathogenic organism or cell infected with a pathogenic
organism or present in a polypeptide associated with a pathologic
condition.
44. (canceled)
45. The vaccine of claim 43, wherein the vaccine is a nucleic acid
vaccine.
46. A pharmaceutical composition comprising an NF-.kappa.B
inhibitor as defined in claim 16 and a pharmaceutically acceptable
carrier.
47-49. (canceled)
50. A method of killing target cells which aberrantly express an
epitope in a patient, the method comprising the steps of (1)
obtaining APCs, such as dendritic cells, from said patient; (2)
contacting said APCs, such as dendritic cells, with an NF-.kappa.B
inducer ex vivo; (3) optionally contacting said cells with the said
epitope or with a polynucleotide or expression vector encoding the
said epitope and (4) reintroducing the so treated APCs, such as
dendritic cells, into the patient.
51. A method of killing target cells in a patient according to
claim 50, wherein the target cells are cancer cells.
52-57. (canceled)
58. A method of treating an allergy in a mammal, such as a human,
comprising administering a pharmaceutically-effective dose of an
NF-.kappa.B inhibitor to induce an anergic response in a mammal
wherein the mammal, such as a human, is administered an antigenic
molecule or polynucleotide encoding an antigenic molecule.
59. The method according to claim 2, wherein the inhibitor is
targeted to APCs such as dendritic cells or precursors thereof.
60. The method according to claim 2, wherein APCs, such as
dendritic cells or precursors thereof are exposed to the inhibitor
in vitro and introduced into the mammal.
61. The method according to claim 2, wherein the antigenic molecule
comprises an epitope present on transformed or cancerous cells or
on a pathogenic organism, or on a cell infected by a pathogenic
organism, or a polypeptide expressed in a pathologic condition such
as Alzheimer's disease or a spongiform encephalopathy.
62. The method according to claim 22, wherein the inducer is
targeted to APCs such as dendritic cells or precursors thereof.
63. The method according to claim 22, wherein APCs, such as
dendritic cells or precursors thereof are exposed to the inducer in
vitro and introduced into the mammal.
64. The method according to claim 22, wherein the antigenic
molecule comprises an epitope present on transformed or cancerous
cells or on a pathogenic organism, or on a cell infected by a
pathogenic organism, or a polypeptide expressed in a pathologic
condition as such as Alzheimer's disease or a spongiform
encephalopathy.
65. The method according to claim 32, wherein the inhibitor is
targeted to APCs such as dendritic cells or precursors thereof.
66. The method according to claim 32, wherein APCs, such as
dendritic cells or precursors thereof are exposed to the inhibitor
in vitro and introduced into the mammal.
67. The method according to claim 32, wherein the antigenic
molecule comprises an epitope present on transformed or cancerous
cells or on a pathogenic organism, or on a cell infected by a
pathogenic organism, or a polypeptide expressed in a pathologic
condition such as Alzheimer's disease or a spongiform
encephalopathy.
68. The method according to claim 33, wherein the antigenic
molecule comprises an epitope present on transformed or cancerous
cells or on a pathogenic organism, or on a cell infected by a
pathogenic organism, or a polypeptide expressed in a pathologic
condition such as Alzheimer's disease or a spongiform
encephalopathy.
69. A polynucleotide encoding an antigenic molecule and an
NF-.kappa.B inhibitor.
70. The polynucleotide of claim 69, wherein the antigenic molecule
comprises an epitope present on transformed or cancerous cells or
on a pathogenic organism, or on a cell infected by a pathogenic
organism, or a polypeptide expressed in a pathologic condition such
as Alzheimer's disease or a spongiform encephalopathy.
71. A polynucleotide encoding an antigenic molecule and an
NF-.kappa.B inducer.
72. The polynucleotide of claim 71, wherein the antigenic molecule
comprises an epitope present on transformed or cancerous cells or
on a pathogenic organism, or on a cell infected by a pathogenic
organism, or a polypeptide expressed in a pathologic condition such
as Alzheimer's disease or a spongiform encephalopathy.
73. The kit of parts, composition or chimaeric molecule of claim
41, wherein the modulating portion comprises or encodes an
NF-.kappa.B inducer.
74. The kit of parts or composition or chimaeric molecule of claim
41, wherein the modulating portion comprises or encodes an
NF-.kappa.B inducer.
75. A pharmaceutical composition comprising an NF-.kappa.B
inhibitor as defined in claim 20 and a pharmaceutically acceptable
carrier.
76. A pharmaceutical composition comprising an NF-.kappa.B
inhibitor as defined in claim 43 and a pharmaceutically acceptable
carrier.
77. A pharmaceutical composition comprising a vaccine of claim 69
and a pharmaceutically acceptable carrier.
78. A pharmaceutical composition comprising a polynucleotide of
claim 71 and a pharmaceutically acceptable carrier.
Description
[0001] This application is a continuation of application Ser. No.
10/168,805, filed Jan. 31, 2003, now pending; which is a U.S.
national phase under 35 U.S.C. 371 of International Application No.
PCT/GB00/04925, filed Dec. 22, 2000; the entire contents of each of
which are incorporated by reference herein.
[0002] The invention relates to the activation and inhibition of
the immune system by using inducers and inhibitors of NF-.kappa.B,
and to the use of such inducers and inhibitors to treat transplant
rejection, autoimmune disease, allergy, infectious disease and
cancer.
[0003] Antigen presentation is a critical step in the initiation of
the immune response, and dendritic cells (DC) are acknowledged to
be the most potent antigen presenting cells for naive T cells. This
is partly due to their high expression of MHC and costimulatory
molecules (Hart (1997) Blood 90, 3245-3287). However, little is
known about the biochemical pathways which regulate antigen
presenting function, partly due to the difficulty in transfecting
DC. Using an inhibitor of I.kappa.B degradation, PSI that produces
an effective inhibition of NF-.kappa.B activation, the inventors
show here abrogation of the capacity of DC to induce a mixed
lymphocyte reaction. The mechanism suppressed DC function and
diminished antigen presentation involved downregulation of multiple
steps, including costimulatory molecules (CD86 and CD80), HLA class
II (DQ>DR) as well as cytokines such as IL-12. Moreover, T cell
exposed to such DC were unable to be stimulated by subsequent
encounters with normal DC. These results point out NF-.kappa.B as
an effective target for blocking antigen presentation.
[0004] Dendritic cells are of major importance in the presentation
of antigen to naive T cells in the primary immune response. They
are bone marrow derived cells which were first described in the
early 1970's by Steinman and Cohn (1973) J. Exp. Med. 179, 1109.
Studies on dendritic cells were initially hampered by the
difficulty in isolating them in sufficient numbers, but this
problem was overcome in part by the realisation that a subset of DC
could be generated in vitro by culture of CD34+ cells or human
monocytes with GM-CSF and IL-4. These cultured DC have the
phenotype of immature DC, and can be matured into high MHC, high
CD80/86 expressing cells through incubation with TNF.alpha. or LPS
(Bender et al (1996) J. Immunol. Methods 196, 121; Romani et al
(1996) J. Immunol. Methods (1996) 196, 137; Reddy et al (1997)
Blood 90, 3640).
[0005] DC can also be derived from a post colony-forming unit
CD14.sup.+ intermediate in the peripheral blood. DC migrate to
peripheral sites in skin, mucosa, spleen and thymus. They have been
implicated in a variety of clinically important processes,
including allograft rejection, atopic disorders, autoimmunity and
anti-tumour immunity.
[0006] DC can be cultured ex vivo from CD34.sup.+ stem cells or
CD14.sup.+ peripheral blood monocytes using cytokines, principally
GM-CSF, IL-4 and TNF.alpha. Scabolsc et al (1995) J. Immunol. 154,
5651-5661. DC from both these sources are immunocompetent and can
take up exogenously presented antigen, process it and then present
it to cytotoxic T-cells (Grabbe et al (1995) Immunology Today 16,
117-121; Girolomoni & Ricciardi-Castagnoli (1997) Immunology
Today 18, 102-104). DC can transfer antigen-specific tumour
immunity generated in vivo (Kwak et al (1995) Lancet 345,
1016-1020) and autologous DC pulsed with tumour antigen ex vivo can
induce a measurable anti-tumour effect (Hsu et al (1996) Nature
Medicine 2, 52-58). DC can be effectively pulsed using a crude
tumour membrane lysate, purified peptides or peptide fragments. The
ex vivo expansion of autologous dendritic cells from patients,
loading with a peptide antigen and reinfusion as adoptive
immunotherapy, is described in, for example, WO/00/26249.
[0007] The importance of antigen presentation in the generation of
immune response was confirmed by demonstration that blocking
antigen presentation downregulates immune responses and is useful
in treating animal models of disease. Thus antibody to murine MHC
class II has been used to treat experimental allergic
encephalomyelitis (Smith et al (1994) Immunology 83, 1), and
blocking the CD80/86 costimulatory molecules with antibodies or
CTLA4-Ig fusion protein is beneficial in transplants or animal
models of arthritis (Lu et al (1999) Gene Ther. 6, 554-563). This
has led to a search of new ways of downregulating antigen
presentation which may be useful in human diseases or in
transplantation.
[0008] NF-.kappa.B has been speculated as being involved in the
immune system. This is summarised in, for example, the paper by
Baeueurle P. A. and Henkel T. (Annual Reviews in Immunology, 1994,
Vol. 12, pages 141-179). However, no-one has shown that NF-.kappa.B
is indeed crucial in the activation and inhibition of the immune
system, as the effects of activating or inactivating NF-.kappa.B on
antigen representing cells were not amenable to study previously.
The inventors, for the first time, have demonstrated the key role
of NF-.kappa.B in the immune response.
[0009] The activation of the transcription factor NF-.kappa.B like
proteins results from post-translational modification permitting
translocation of the preformed transcription factor from the
cytoplasm to the nucleus. This translocation is controlled by the
phosphorylation and degradation of an inhibitor protein called
I.kappa.B, which forms a complex with NF-.kappa.B, and thereby
holds it in the cytoplasm. Stimulation of the cell by appropriate
signals leads to modification of I.kappa.B which in turn results in
its dissociation and/or degradation from NF-.kappa.B.
[0010] Binding of the I.kappa.B protein to NF-.kappa.B masks the
nuclear localisation signal (NLS) of NF-.kappa.B. Upon stimulation
of the cell with specific agents, which depend on the cell type and
stage of cell development, I.kappa.B is modified in a way that
disables binding to NF-.kappa.B, leading to dissociation of
NF-.kappa.B from I.kappa.B.
[0011] NF-.kappa.B is a heterodimeric protein consisting of a 50 kD
subunit (p50) and a 65 kD subunit (p65). The cDNAs for p50 and p65
have been cloned and have been shown to be homologous over a region
of 300 amino acids.
[0012] Recently an additional member of the NF-.kappa.B family, Rel
B, has been cloned as an immediate early response gene from
serum-stimulated fibroblasts.
[0013] Both p50 and p65 are capable of forming homodimers, although
with different properties: whereas p50 homodimers have strong DNA
binders affinity but cannot transactivate transcription, the p65
homodimers can only weakly bind to DNA but are capable of
transactivation. P50 is synthesised as the amino-terminal part of
the 110 kD precursor (p110), which has no DNA binding and
dimerisation activity. The carboxyl-terminal part contains eight
ankyrin repeats, a motif found in several proteins involved in cell
cycle control and differentiation.
[0014] Five I.kappa.B family members have been identified:
I.kappa.B.alpha., I.kappa.B.beta., p105/I.kappa.B.gamma.,
p110/I.kappa.B.DELTA. and I.kappa.B.epsilon. (Baeuerle and
Baltimore, Cell 1996, Vol. 87, pages 13-20). All I.kappa.B-like
family members contain multiple ankyrin repeats, which are
essential for inhibition of NE-.kappa.B activation.
[0015] The inventors have found that many of the key features of
the inflammatory response in human macrophages and in the
rheumatoid synovium were dependent on the transcription factor
NF-.kappa.B. The inventors have studied the proteosome inhibitory
drug PSI, which was initially described as an inhibitor of the
chymotrypsin-like activity of the proteosome. It was found that the
production of many proinflammatory mediators, such as TNF.alpha.,
IL-6, IL-2 were dependent on NF-.kappa.B (inhibitable by
AdvI.kappa.B.alpha. disclosed in PCT/GB98/02753) whereas the
anti-inflammatory cytokines and mediators, namely IL-10, IL-1
receptor antagonist, IL-11 were not affected. This suggested to us
that NF-.kappa.B segregated accurately between these two classes of
mediators, and so raised the question, in view of the close
relationship of the inflammatory and immune systems of what the
role of NF-.kappa.B might be in the induction of immunity.
[0016] The inventors first investigated the effect of the
proteosome inhibitory drug PSI, which does not require the use of
gene therapy. This is known to inhibit I.kappa.B degradation and
hence NF-.kappa.B activation on the immunostimulatory function of
dendritic cells, which is the key early event on the generation of
a primary immune response.
[0017] They report that PSI treatment of mature, monocyte derived
DC inhibited their capacity to induce T cell proliferation in the
mixed lymphocyte response. To elucidate the mechanism of this
effect, cell surface analysis, cytokine assays and co-cultures were
performed, which suggested that blocking NF-.kappa.B permits
immunosuppressive mechanisms to become operational in the
interaction between the dendritic cells and the T cells.
[0018] Furthermore, the inventors have demonstrated that the
changes result in an anergic response. That is, they result in the
inability to produce an immune response, even after removal of the
original inhibiting compound.
[0019] The inventors have also realised that NF-.kappa.B can also
be used as a target to induce or modulate an immune response. This
is unexpected, as it has been shown Feuillard et al (1996) Eui. J
Immunol 26, 2547-2551; Granelli-Piperno et al (1995) Proc Natl.
Acad. Sci. USA 92, 10944-10948 the NF-.kappa.B is already activated
in DC, and hence further activation would not have been expected to
be beneficial.
[0020] A first aspect of the invention provides a method of
inhibiting antigen presentation or inducing an anergic response in
a mammal, such as a human, comprising administering a
pharmaceutically-effective dose of an intracellular inhibitor of
APC, such as DC, function.
[0021] By "intracellular inhibitor of APC function" we include any
suitable inhibitor of antigen presenting cell function. By "APC
function" we include the ability to present antigen, the ability to
express MHC Class II, the ability to express cell surface molecules
such as costimulatory molecules including CD80 and CD86, the
ability to produce cytokines and the ability to induce anergy
rather than activation. Typically the inhibitor of APC function is
an inhibitor of DC function. Preferably, the inhibitor is an
inhibitor of intracellular signalling within the APC. By
"intracellular signalling within the APC" we include communication
between the membrane and the nucleus, signalling which controls
gene expression (including expression of CD80 and CD86) and control
of cytoskeletal organisation. Inhibition of intracellular
signalling include, for example, an inhibitor of NF-.kappa.B as
described in more detail below.
[0022] For the avoidance of doubt, cytokines and molecules
containing CPG motifs are not intracellular inhibitors of APC
function since they act extracellularly. Clearly, the inhibitors
are ones that do not kill the cell.
[0023] A further aspect of the invention provides a method of
inhibiting antigen presentation or inducing an anergic response in
a mammal comprising administering a pharmaceutically-effective dose
of an inhibitor of NF-.kappa.B.
[0024] By pharmaceutically-effective dose, we mean an amount
sufficient to induce the desired response in a mammal. This amount
can be determined by routine clinical and experimental trials known
in the art.
[0025] By mammal, we mean any mammal but especially a human.
[0026] Anergy is a form of immunological tolerance in which
lymphocytes, in this case T lymphocytes after exposure to antigen
in an inappropriate setting become refractory to subsequent
optional immunogenic stimulus, usually the immunogenic dose of
antigen in the context of activated antigen presenting cells (see
Roitt, I., Broskoff, J. and Male, D. (1998, 5th edition)
Immunology, Mosby, London). Thus, the invention provides methods in
which a tolerogenic response is induced in a mammal.
[0027] Antigen presentation describes the display of antigen as
peptide fragments bound to MHC molecules on the surface of a cell;
T cells recognise antigen only when it is presented in this
way.
[0028] A further aspect of the invention provides a method of
treating an allergy or an autoimmune disease comprising
administering a pharmaceutically-effective dose of an intracellular
inhibitor of APC, such as DC, function to induce an anergic
response in a mammal.
[0029] A further aspect of the invention provides a method of
treating an allergy or an autoimmune disease comprising
administering a pharmaceutically-effective amount of an inhibitor
of NF-.kappa.B to induce an anergic response in a mammal. The
autoimmune disease may be any autoimmune disease such as rheumatoid
arthritis, type I diabetes, multiple sclerosis, Crohn's disease,
Hashimoto's thyroiditis, coeliac disease, myasthenia gravis,
pemphigus vulgaris, systemic lupus erythromatosus and Graves
disease.
[0030] Allergies which may be treatable by the method described
herein include allergies to the following allergens: Fel d 1 (the
feline skin and salivary gland allergen of the domestic cat Felis
domesticus--the amino acid sequence of which is disclosed in WO
91/06571), Der p I, Der p II, Der fI or Der fII (the major protein
allergens from the house dust mite dermatophagoides-amino acid
sequences disclosed in WO 94/24281).
[0031] The invention is applicable substantially to any allergy,
including those caused by allergens present in any of the
following: grass, tree and weed (including ragweed) pollens; fungi
and moulds; foods eg fish, shellfish, crab lobster, peanuts, nuts,
wheat gluten, eggs and milk; stinging insects eg bee, wasp and
hornet and the chirnomidae (non-biting midges); spiders and mites,
including the house dust mite; allergens found in the dander,
urine, saliva, blood or other bodily fluid of mammals such as cat,
dog, cows, pigs, sheep, horse, rabbit, rat, guinea pig, mouse and
gerbil; airborne particulates in general; latex; and protein
detergent additives.
[0032] Allergies to proteins from the following insects may also be
treated: housefly, fruit fly, sheep blow fly, screw worm fly, grain
weevil, silkworm, honeybee, non-biting midge larvae, bee moth
larvae, mealworm, cockroach and larvae of Tenibrio molitor
beetle.
[0033] A further aspect of the invention provides a method of
preventing transplant rejection in a mammal, such as a human,
comprising administering a pharmaceutically-effective dose of an
intracellular inhibitor of APC, such as DC, function to induce an
anergic response in a mammal.
[0034] A further aspect of the invention provides a method of
preventing transplant rejection comprising administering a
pharmaceutically-effective amount of NF-.kappa.B inhibitor to
induce an anergic response.
[0035] The invention further relates to inhibitor of APC function
such as NF-.kappa.B inhibitors for use as a medicament, to induce
an anergic response, to inhibit the rejection of transplanted
tissue, or as anti-autoimmune disease agents or to treat
allergy.
[0036] The invention also provides a NF-.kappa.B inhibitor for use
in the manufacture of a medicament to treat transplant rejection,
allergy or an auto-immune disease.
[0037] Preferably the NF-.kappa.B inhibitor is an inhibitor of
proteolysis, for example a proteosome inhibitor. The inhibitor may
be PSI, available from Calbiochem. This is known as an inhibitor of
proteosomes (Traechner, et al., EMBO J. (1994), Vol. 13, pages
5433-41; Griscavage, et al., PNAS (1996), Vol. 93, pages 3308-12;
Bondeson, et al., J Immunol. (1999), Vol. 162, pages 2939-45). ALLN
(Jobin, et al., Hepatology (1998), Vol. 27, pages 1285-95);
Lactacystin (Delic, et al. (1998), Vol 77, pages 1103-07); MG-132
(Jobin, et al. Supra); C-LFF and Calpain Inhibitors (Neauparfant
and Hiscott, Cytokine & Growth Factor Reviews (1996), Vol. 7
pages 175-190); or CVT-134 (Lum, et al., Biochem. Pharmacol (1998),
Vol. 55, pages 1391-97) may also be used as inhibitors.
[0038] Other inhibitors include: Caffeic acid phenethyl ester
(Natarajan, et al., PNAS (1992), Vol. 93 pages 9090-95);
Pyrrolidine dithiocarbonate (Schreck, et al., J. Exp. Med. (1992),
Vol. 175, pages 1181-94); Lovastatin (Guijarro, et al., Nephrol
Dial Transplant (1996), Vol. 11, pages 990-996); Aselastine HCL
(Yoneda, Japan. J. Pharmacol. (1997), Vol. 73, pages 145-153);
Tepaxalin (Kazmi, et al., J Cell. Biochem. (1995), Vol. 57, pages
299-310); (-)-epi gallocatechin-3-gallate (Lin & Lin, Mol.
Pharmacol. (1997), Vol. 52, pages 465-472); deoxyspergualin
(Tapper, et al., J Immunol. (1995), Vol. 155, pages 2427-36);
Phenyl-N-tert-butylnitrone (Kotake, et al., Biochem. Biophys Acta
(1998), Vol. 1446, pages 77-84; Quercutin (Sato, et al., J
Rheumatol. (1997), Vol 24, pages 1680-84); Cucumin (Chan, Biochem,
Pharmacol. (1998), Vol. 55, pages 965-973); or E3330 (Goto, et al.,
Mol. Pharmacol (1996), Vol. 49, pages 860-873).
[0039] As is clear from the examples of NF-.kappa.B inhibitors and
activators indicated herein, it is preferred that the inhibitor or
activator enters the cell and acts within the cell, ie acts as an
intracellular NF-.kappa.B inhibitor or activator, for example an
intracellular modulator of intracellular signalling events leading
to NF-.kappa.B inhibition or activation.
[0040] In another embodiment of the invention, NF-.kappa.B may be
inhibited by inhibitors of NF-.kappa.B, ie that act directly on the
level (quantity), cellular location or activity of NF-.kappa.B. For
example, the inhibitor may be a naturally occurring regulator of
NF-.kappa.B that interacts directly with NF-.kappa.B, such as an
I.kappa.B.
[0041] Preferably the inhibitor is an I.kappa.B, especially
I.kappa.B.alpha. is described in, for example, paper by Makarov,
Gene Therapy, 1997, Vol. 4, pages 846-852, and in PCT/GB98/02753.
Other inhibitors of NF-.kappa.B include antisense cDNA or
oligonucleotides encoding for any of the known NF-.kappa.B
subunits, e.g. p50, p65, Rel B. Bondeson et al (1999) Proc. Natl.
Acad. Sci. USA 96, 5668 describes an I.kappa.B-encoding
adenovirus.
[0042] The inhibitor may also be a ribozyme which selectively
destroy mRNA encoding NF-.kappa.B, or an antisense molecule which
prevents transcription of NF-.kappa.B or an antibody or
antibody--like molecule which blocks NF-.kappa.B action. These
inhibitors are described in more detail below. It will be
appreciated that inhibitors of APC, such as DC function, may also
comprise ribozymes or antisense molecules or antibodies or
antibody-like molecules which, for example, inhibit intracellular
signalling within the APC.
[0043] It will be appreciated that inhibitors of inhibitors of
NF-.kappa.B may act as inducers of NF-.kappa.B. Thus, antibodies or
antisense molecules or ribozymes that block I.kappa.B.alpha.
function or expression may act as inducers of NF-.kappa.B. The
utility of such inducers is described below.
[0044] Ribozymes which may be encoded in the genomes of the viruses
or virus-like particles herein disclosed are described in Cech and
Herschlag "Site-specific cleavage of single stranded DNA" U.S. Pat.
No. 5,180,818; Altman et al "Cleavage of targeted RNA by RNAse P"
U.S. Pat. No. 5,168,053, Cantin et al "Ribozyme cleavage of HIV-1
RNA" U.S. Pat. No. 5,149,796; Cech et al "RNA ribozyme restriction
endoribonucleases and methods", U.S. Pat. No. 5,116,742; Been et al
"RNA ribozyme polymerases, dephosphorylases, restriction
endonucleases and methods", U.S. Pat. No. 5,093,246; and Been et al
"RNA ribozyme polymerases, dephosphorylases, restriction
endoribonucleases and methods; cleaves single-stranded RNA at
specific site by transesterification", U.S. Pat. No. 4,987,071, all
incorporated herein by reference.
[0045] Preferably the inhibitor is encoded by a nucleic acid
sequence, for example within a vector, such as an adenovirus. The
nucleic acid sequence encoding the inhibitor is preferably
operatively linked to regulatory elements necessary for expression
of said sequence. Such vectors may be used for gene therapy to
enable the nucleic acid sequence encoding the inhibitor to be
inserted into the body of a mammal. Methods of gene therapy, such
as by using an adenovirus, are known in the art. The vector may
also comprise a nucleic acid sequence encoding an antigenic
molecule.
[0046] Preferably the vector construct used is AdvI.kappa.B.alpha.,
disclosed in PCT/GB98/02753.
[0047] "Operatively linked" refers to juxtaposition such that the
normal function of the components can be performed. Thus, a coding
sequence "operatively linked" to regulatory elements refers to a
configuration wherein the nucleic acid sequence encoding the
inhibitor (or inducer, which is useful as described in more detail
below), of NF-.kappa.B can be expressed under the control of the
regulatory sequences.
[0048] "Regulatory sequences" refers to nucleic acid sequences
necessary for the expression of an operatively linked coding
sequence in a particular host organism. For example, the regulatory
sequences which are suitable for eukaryotic cells are promotors,
polyadenylation signals, and enhancers.
[0049] "Vectors" means a DNA molecule comprising a single strand,
double strand, circular or supercoiled DNA. Suitable vectors
include retroviruses, adenoviruses, adeno-associated viruses, pox
viruses and bacterial plasmids. Retroviral vectors are retroviruses
that replicate by randomly integrating their genome into that of
the host. Suitable retroviral vectors are described in WO
92/07573.
[0050] Adenovirus is a linear double-standard DNA Virus. Suitable
adenoviral vectors are described in Rosenfeld et al, Science, 1991,
Vol. 252, page 432.
[0051] Adeno-associated viruses (AAV) belong to the parvo virus
family and consist of a single strand DNA or about 4-6 KB.
[0052] Pox viral vectors are large viruses and have several sites
in which genes can be inserted. They are thermostable and can be
stored at room temperature. Safety studies indicate that pox viral
vectors are replication-defective and cannot be transmitted from
host to host or to the environment.
[0053] Vectors comprising nucleic acid encoding an NF-.kappa.B
inhibitor (or inducer, as described below) may be introduced into a
mammal in the form of liposomes in a manner known in the art.
Alternatively, liposomes may be used in the form of aerosols in
order to access the body by means of the mucus membrane or lung.
Such techniques are known in the art.
[0054] Immunoliposomes (antibody-directed liposomes) are especially
useful in targeting to cell types which over-express a cell surface
protein for which antibodies are available, as is possible with
dendritic cells or precursors, for example using antibodies to CD1,
CD14 or CD83 (or other dendritic cell or precursor cell surface
molecule, as indicated above). For the preparation of
immuno-liposomes MPB-PE
(N-[4-(p-maleimidophenyl)butyryl]-phosphatidylethanolamine) is
synthesised according to the method of Martin & Papahadjopoulos
(1982) J. Biol. Chem. 257, 286-288. MPB-PE is incorporated into the
liposomal bilayers to allow a covalent coupling of the antibody, or
fragment thereof, to the liposomal surface. The liposome is
conveniently loaded with the DNA or other genetic construct of the
invention for delivery to the target cells, for example, by forming
the said liposomes in a solution of the DNA or other genetic
construct, followed by sequential extrusion through polycarbonate
membrane filters with 0.6 .mu.m and 0.2 .mu.m pore size under
nitrogen pressures up to 0.8 MPa. After extrusion, entrapped DNA
construct is separated from free DNA construct by
ultracentrifugation at 80 000.times.g for 45 min. Freshly prepared
MPB-PE-liposomes in deoxygenated buffer are mixed with freshly
prepared antibody (or fragment thereof) and the coupling reactions
are carried out in a nitrogen atmosphere at 4.degree. C. under
constant end over end rotation overnight. The immunoliposomes are
separated from unconjugated antibodies by ultracentrifugation at 80
000.times.g for 45 min. Immunoliposomes may be injected, for
example intraperitoneally or directly into a site where the target
cells are present, for example subcutaneously. Naked DNA encoding
an inhibitor of NF-.kappa.B, in the form of a DNA vaccine, may also
be used as an inhibitor of NF-.kappa.B.
[0055] As noted above, an alternative inhibitor is the use of
anti-sense nucleic acid to an NF-.kappa.B sequence, for example Rel
B. Such an anti-sense nucleic acid comprises a nucleic acid
sequence which is capable of binding to an NF-.kappa.B nucleic acid
sequence, inhibiting transcription of the NF-.kappa.B sequence.
Methods of producing anti-sense nucleic acid per se are known in
the art.
[0056] Antisense oligonucleotides are single-stranded nucleic
acids, which can specifically bind to a complementary nucleic acid
sequence. By binding to the appropriate target sequence, an
RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed. These nucleic
acids are often termed "antisense" because they are complementary
to the sense or coding strand of the gene. Recently, formation of a
triple helix has proven possible where the oligonucleotide is bound
to a DNA duplex. It was found that oligonucleotides could recognise
sequences in the major groove of the DNA double helix. A triple
helix was formed thereby. This suggests that it is possible to
synthesise a sequence-specific molecules which specifically bind
double-stranded DNA via recognition of major groove hydrogen
binding sites.
[0057] By binding to the target nucleic acid, the above
oligonucleotides can inhibit the function of the target nucleic
acid. This could, for example, be a result of blocking the
transcription, processing, poly(A)addition, replication,
translation, or promoting inhibitory mechanisms of the cells, such
as promoting RNA degradations.
[0058] Antisense oligonucleotides are prepared in the laboratory
and then introduced into cells, for example by microinjection or
uptake from the cell culture medium into the cells, or they are
expressed in cells after transfection with plasmids or retroviruses
or other vectors carrying an antisense gene. Antisense
oligonucleotides were first discovered to inhibit viral replication
or expression in cell culture for Rous sarcoma virus, vesicular
stomatitis virus, herpes simplex virus type 1, simian virus and
influenza virus. Since then, inhibition of mRNA translation by
antisense oligonucleotides has been studied extensively in
cell-free systems including rabbit reticulocyte lysates and wheat
germ extracts. Inhibition of viral function by antisense
oligonucleotides has been demonstrated in vitro using
oligonucleotides which were complementary to the AIDS HIV
retrovirus RNA (Goodchild, J. 1988 "Inhibition of Human
Immunodeficiency Virus Replication by Antisense
Oligodeoxynucleotides", Proc. Natl. Acad. Sci. (USA) 85(15),
5507-11). The Goodchild study showed that oligonucleotides that
were most effective were complementary to the poly(A) signal; also
effective were those targeted at the 5' end of the RNA,
particularly the cap and 5' untranslated region, next to the primer
binding site and at the primer binding site. The cap, 5'
untranslated region, and poly(A) signal lie within the sequence
repeated at the ends of retrovirus RNA (R region) and the
oligonucleotides complementary to these may bind twice to the
RNA.
[0059] Typically, antisense oligonucleotides are 15 to 35 bases in
length. For example, 20-mer oligonucleotides have been shown to
inhibit the expression of the epidermal growth factor receptor mRNA
(Witters et al, Breast Cancer Res Treat 53:41-50 (1999)) and 25-mer
oligonucleotides have been shown to decrease the expression of
adrenocorticotropic hormone by greater than 90% (Frankel et al, J
Neurosurg 91:261-7 (1999)). However, it is appreciated that it may
be desirable to use oligonucleotides with lengths outside this
range, for example 10, 11, 12, 13, or 14 bases, or 36, 37, 38, 39
or 40 bases.
[0060] The anti-sense nucleic acid may be encoded by a suitable
vector, for example of the type discussed above.
[0061] The inhibitor may alternatively be an anti-NF-.kappa.B
vaccine or an antibody against NF-.kappa.B or fragment thereof such
as an Fv. The vaccine or antibody may be against any suitable part
of NF-.kappa.B providing it results in the inhibition of
NF-.kappa.B. Preferably the antibody is a monoclonal antibody.
Methods of producing vaccines and antibodies are known in the art.
Alternatively, the antibody may be a suitable antibody fragment,
such as an Fab or (Fab).sub.2 fragment or Fv, which still retains
its anti-NF-.kappa.B activity. The antibody may be linked to
polypeptide sequences that permit entry into cells, ie, the
antibody (particularly antibody fragment) may be joined to a moiety
that facilitates uptake of the antibody by a cell, for example a
DC. For example, the antibody may be linked to a lipophilic
molecule or polypeptide domain that is capable of promoting
cellular uptake of the molecule or the interacting polypeptide, as
known to those skilled in the art. Thus, the moiety may derivable
from the Antennapedia helix 3 (Derossi et al (1998) Trends Cell
Biol 8, 84-87), or from sequences of HIV, generally tat, that
permit entry into cells. Such sequences are known and include the
penetration peptide and the sequences of HIV generally engineered
tat that permit entry into cells. Alternatively, the cDNA encoding
such antibodies (preferably antibody fragments) could be delivered
as a vector as described above.
[0062] A further aspect of the invention provides a method of
stimulating antigen presentation or of stimulating an immune
response in a mammal, such as a human, comprising administering a
pharmaceutically effective amount of an intracellular inducer of
APC, such as DC, function.
[0063] By "intracellular enhancer of APC function" we include any
suitable enhancer of antigen presenting cell function. Typically,
the enhancer is an enhancer of DC function. Preferably, the
enhancer is an enhancer of intracellularly signalling within the
APC.
[0064] The terms "APC function" and "intracellular signalling
within the APC" are as defined above.
[0065] For the avoidance of doubt, cytokines and molecules
containing a CPG motif are not intracellular inducers or enhancers
of APC function since they act extracellularly.
[0066] A further aspect of the invention provides a method of
stimulating antigen presentation or of stimulating an immune
response in a mammal by administering a pharmaceutically-effective
amount of an inducer of NF-.kappa.B. Preferably the method is used
to treat (including prophylactically) infectious diseases or
cancers by stimulating the immune system of the mammal. Infectious
diseases include prion-related diseases, including spongiform
encephalopathies. The method may be used to treat (including
prophylachically) diseases or conditions characterised by aberrant
types and/or aberrantly high levels of (harmful) molecules, for
example polypeptides, in the body, for example levels of
inflammatory mediators (for example cytokine) associated with
chronic inflammation; breakdown products of cells or connective
tissue matrix, for example fibronectin fragments; .beta.-amyloid
polypeptide (associated with Alzheimer's disease). Stimulating an
immune response against such molecules may aid removal of the
molecules from the body, thereby helping in resolution or
prevention of the condition.
[0067] It is preferred if the cancer antigen is, or has at least
one epitope present in, any of the following: [0068] i) normal
cellular proteins that are expressed at abnormally high levels in
tumours; eg cyclin D1 in a variety of tumours; cyclin E in breast
cancer; mdm 2 in a variety of tumours; EGF-R, erb-B2, erb-B3,
FGF-R, insulin-like growth factor receptor, Met, myc, p53 and BCL-2
are all expressed in various tumours. [0069] ii) normal cellular
proteins that are mutated in tumours; eg Ras mutations in a variety
of tumours; p53 mutations in a variety of tumours; BCR/ABL
translocation in CML and ALL; CSF-1 receptor mutations in AML and
MDS; APC mutations in colon cancer; RET mutations in MEN2A, 2B and
FMTC; EGFR mutations in gliomas; PML/RARA translocation in PML;
E2A-PBX1 translocation in pre B leukaemias and in childhood acute
leukaemias. [0070] iii) virally encoded proteins in tumours
associated with viral infection; eg human papilloma virus proteins
in cervical cancer; Epstein-Barr virus proteins in B cell lymphomas
and Hodgkin's lymphoma; HTLV-1 proteins in adult T cell leukaemia;
hepatitis B and C virus proteins in hepatocellular carcinoma;
herpes-like virus proteins in Kaposi's sarcoma. [0071] iv) HIV
encoded proteins in HIV infected patients.
[0072] Thus, the above cancer-associated antigens can be divided
into three main categories: (i) normal self antigens expressed at
high levels in tumour cells; (ii) mutated self antigens expressed
in tumour cells; (iii) viral antigens expressed in tumours
associated with viral infection. Category (i) is preferred.
[0073] Three subtypes are included in category (i): [0074] a)
normal cellular proteins that are overexpressed; [0075] b) proteins
that are expressed in a tissue-specific fashion in normal cells but
also in tumours; and [0076] c) proteins that are embryonic
antigens, silent in most adult tissues but aberrantly expressed in
tumours.
[0077] Examples of b) and c) are: [0078] b) tissue-specific
differentiation antigens as targets for tumour-reactive CTL such as
GATA-1, IKAROS, SCL (expressed in the haematopoietic lineage and in
leukaemias); and immunoglobulin constant regions (for treatment of
multiple myeloma); and [0079] c) Wilms-tumour antigen 1 (WT1) for
treatment of leukaemias and Wilms tumour and carcinoembryonic
antigens (CEA a foetal protein) for liver and intestinal
tumours.
[0080] In one embodiment, the cancer--associated antigen may be
provided by a crude extract of a tumour sample.
[0081] Overexpression of oncogene-encoded proteins in human tumours
and mutated oncogenes expressed in human tumours are described in
Stauss & Dahl (1995) Tumour Immunology, Dalgleish/Browning,
Chapter 7, incorporated herein by reference.
[0082] Thus, it is preferred if the patient to be treated has
cancer; more preferably any one of breast cancer; bladder cancer;
lung cancer; prostate cancer; thyroid cancer; leukaemias and
lymphomas such as CML, ALL, AML, PML; colon cancer; glioma;
seminoma; liver cancer; pancreatic cancer; bladder cancer; renal
cancer; cervical cancer; testicular cancer; head and neck cancer;
ovarian cancer; neuroblastoma and melanoma.
[0083] CML is chronic myelocytic leukaemia; ALL is acute
lymphoblastic leukaemia; AML is acute myelocytic leukaemia; and PML
is pro-myelocytic leukaemia.
[0084] Alternatively, the patient have or be at risk of any disease
caused by a pathogen, particularly a bacterium, yeast, virus,
trypanosome and the like. It is preferred if the disease is caused
by a chronic infection with a pathogen. It is also preferred if the
pathogen is one which is not readily cleared by the host immune
system.
[0085] It is preferred if the disease is a viral infection; more
preferably a disease caused by any one of HIV, papilloma virus,
Epstein-Barr virus, HTLV-1, hepatitis B virus, hepatitis C virus,
herpes virus or any virus that causes chronic infection. It is
particularly preferred if the virus is HIV.
[0086] Abnormally elevated amounts of a hormone produced by cells
occur in some diseases such as certain types of thyroid disease.
Thus, the method of the invention may be used to promote ablation
of cells producing the elevated amounts of the hormone. The antigen
may be the hormone the biosynthetic enzymes involved in synthesis
of the hormone, which may be overproduced by the cell.
[0087] Patients with a bacterial infection, particularly an
infection that causes chronic infection may also be usefully
treated. The bacterial infection may be an intracellular infection.
Thus, the method may be useful in treating tuberculosis.
[0088] The inventors have realised that the pivotal role of
NF-.kappa.B allows it to be stimulated to produce an increase in
the immune response within the mammal, for example human.
[0089] A further aspect of the invention provides an NF-.kappa.B
inducer for use as a medicament, in particular as an immune
response stimulant. The NF-.kappa.B inducer may be used as an
anti-infectious disease agent or as an anti-cancer agent or in
treating diseases or conditions characterised by aberrant types
and/or aberrantly high levels of (harmful) molecules as discussed
above.
[0090] Increasing the immune response in patients having an
infectious disease or cancer or condition characterised by aberrant
types and/or aberrantly high levels of molecules in the body will
help to treat the patient by increasing the body's own immune
response against the infectious disease or cancer or condition.
[0091] A further aspect of the invention provides an NF-.kappa.B
inducer for use in the manufacture of a medicament to stimulate an
immune response. Preferably the inducer is used to treat an
infectious disease or a cancer or a condition as defined above.
[0092] Preferred NF-.kappa.B inducers include MEKKI, NIK, IKK1,
IKK2, TRAF2, TRAF5, TRAF6, TAK, TPL-2 or Rel B or other NF-.kappa.B
subunits e.g. p65 or cRel. Fragments and muteins of such inducers
capable of inducing an NF-.kappa.B may also be used. The inducers
may be encoded by suitable vectors, as described above for
NF-.kappa.B inhibitors, and introduced into the cells of a patient
to be treated.
[0093] A preferred NF-.kappa.B inducer may be a dominant negative
mutant of MyD88 (ie capable of inhibiting signalling by wild-type
MyD88 molecules, for example in a cell in which wild-type and
inhibitory MyD88 molecules are present). The inhibition may arise
from blocking interaction of endogenous wild-type MyD88 with a
binding partner of the endogenous MyD88, for example a Toll-Like
Receptor (TLR). The dominant negative mutant may be MyD88lpr (Burns
et al (1998) J Biol Chem 273(20), 12203-12209) or a fragment of
MyD88 lacking a death domain (see Burns et al (1998) and references
reviewed therein). The MyD88 (myeloid differentiation protein) is
considered to have a modular organisation consisting of an
N-terminal death domain (DD) separated by a short linker from a
C-terminal Toll domain (reviewed in Burns et al (1998)). The
N-terminal DD is related to a motif of approximately 90 amino acids
that is considered to mediate protein-protein interactions with
other DD sequences forming either homo- or heterodimers (Boldin et
al (1995) J Biol Chem 270, 387-391).
[0094] The inhibitory MyD88 molecule may be a MyD88 molecule that
is less able than MyD88, preferably substantially unable, to bind
to a DD, for example the DD of MyD88 or of IRAK. For example, the
inhibitory MyD88 may be less able than MyD88, preferably
substantially unable, to dimerise via the DD. The inhibitory MyD88
molecule may be a truncated version of MyD88, for example a MyD88
molecule in which all or part of the domain termed the Death Domain
is deleted. It may be a mutated MyD88 molecule, for example a MyD88
molecule that is mutated in the DD, for example with a
non-conservative mutation. For example, it may be mutated at the
position equivalent to Phe56 of full length mouse MyD88, for
example to Asn. It may be the mutated MyD88 molecule termed
MyD88lpr, as noted above in which the N terminal 53 amino acids of
My D88 are also absent Burns et al (1998) J. Biol. Chem. 273,
12203-12209. MyD88lpr has a point mutation (F56N; mouse sequence
numbering) when compared with wild-type MyD88, for example mouse
wild-type MyD88. This point mutation is in the DD and prevents
dimerisation of the DD (Burns et al (1998)). The mutation
corresponds to the lpr.sup.cp mutation known to abolish cytotoxic
signalling of Fas, probably by disrupting the conformation of the
DD domain (Nagata (1994) Semin Immunol 6, 3-8; Huang et al (1996)
Nature 384, 638-641).
[0095] The constructs for the wild-type MyD88 and dominant negative
MyD88 (MyD88-lpr) has been published (Burns K. et al J. Biol Chem
1998) but MyD88-lpr is wrongly described as a single amino acid
mutation in its death domain, where Phe.sup.56 is mutated to Asn.
This mutation corresponds to the lpr.sup.cp mutation present in the
death domain of Fas ligand which abolishes its downstream
signalling by disrupting the conformation of the death domain.
Actually, in addition to the point mutation there is a deletion in
its N-terminal domain of 53 amino acids (1-159 base pairs of the
genebank sequence are missing). This deletion results in part of
the death domain missing.
[0096] It is preferred that the inhibitory MyD88 comprises a
functional Toll domain, ie a Toll domain that is capable of
interacting with a Toll domain, for example the Toll domain of a
wild-type MyD88, for example wild-type human or mouse MyD88 or a
TLR It is preferred that the inhibitory MyD88 comprises the
full-length MyD88 Toll domain. A full-length Toll domain may be
necessary for Toll-Toll domain interaction.
[0097] Methods of measuring protein-protein interactions (and their
enhancement or disruption) will be well known to those skilled in
the art. Suitable methods of measuring DD and Toll-Toll
interactions are also described in Burns et al (1998). Suitable
methods may include, for example, yeast two-hybrid interactions,
co-purification, ELISA, co-immunoprecipitation, fluorescence
resonance energy transfer (FRET) techniques and surface plasmon
resonance methods. Thus, a MyD88 molecule may be considered capable
of binding to or interacting with a DD or Toll domain if an
interaction may be detected between the said MyD88 polypeptide and
a polypeptide comprising a DD or Toll domain by ELISA,
co-immunoprecipitation or surface plasmon resonance methods or by a
yeast two-hybrid interaction or copurification method. The
preferred method is surface plasmon resonance.
[0098] Preliminary work indicates that MEKK1 can induce NF-.kappa.B
and enhance APC such as DC function. It is preferred that the
inducer is capable of inducing NF-.kappa.B in DC or precursors
thereof. Thus, inducers or enhancers of APC function may be useful
in vaccine production. Similarly, inhibitors of APC function may be
useful in preventing cells recognising a particular antigen and
inducing an anergic state.
[0099] Thus, the invention provides a molecule comprising (1) a
portion (modulating portion) comprising or encoding an
intracellular modulator, for example an intracellular inhibitor or
an intracellular enhancer of antigen-presenting cell (APC), such as
DC, function and (2) a portion comprising or encoding an antigenic
molecule (antigenic portion). In particular, the invention provides
a polynucleotide encoding an antigen and a modulator, for example
an inhibitor or an enhancer of DC function. Preferably, the
molecule is or comprises a DNA vaccine encoding an antigen and an
enhancer of APC, such as DC, function. The modulator, for example
enhancer of APC, such as DC, function may be an intracellular
signalling molecule or derivative thereof which retains or has
enhanced intracellular signalling activity. It is preferred if the
derivative is one which retains or enhances DC function. It is
preferably an activator/inducer of NF-.kappa.B. It may be
NF-.kappa.B or a component thereof. The DNA vaccine may comprise a
recombinant polynucleotide comprising a portion encoding the
enhancer of APC, such as DC function and a portion encoding an
antigenic molecule. Alternatively, the antigenic molecule may be
encoded on a separate polynucleotide molecule; this is less
preferred.
[0100] It will be appreciated that preferred inhibitors as
described may be used.
[0101] Preferred enhancers are MEKK and a dominant negative mutant
of My D88, for example My D88lpr.
[0102] It will be appreciated that the preferred enhancers/inducers
as described above may be used in the vaccines of the
invention.
[0103] The antigenic molecule may comprise more than one copy of
one or more epitopes. For example, it may comprise a single copy of
a single epitope-forming amino acid sequence, for example a
sequence of between about 8 and 30 amino acids, preferably about 10
to 18 amino acids, still more preferably about 15 amino acids in
length. It may comprise multiple copies of such an epitope-forming
sequence, or single or multiple copies of at least two different
epitope-forming sequences. The antigenic sequences may be
concatenated to form a domain-like structure, or may be disposed at
different points in a carrier polypeptide. The polynucleotide may
encode one or several different antigenic molecules, each of which
may have one or more antigenic portions or epitopes.
[0104] The invention also includes DNA vaccines encoding an inducer
of NF-.kappa.B for use in the invention. Such vaccines could
include DNA sequences incorporating an antigen of interest derived
from a pathogen, e.g. hepatitis A, B, C, etc., HIV, HTLV,
influenza, tuberculoses, malaria or alternatively a cancer specific
antigen or aberrant molecules such as .beta.-amyloid, as discussed
above, or a prion. In addition, such vaccines would also include an
activator of NF.kappa.B, possibly two or more activators of
NF.kappa.B for maximum effect. Both antigen and activator would be
under the control of suitable promoter sequences to regulate
expression of antigen and activators. An alternative method of
stimulating an immune response may be to provide a vector
comprising a nucleic acid sequence encoding an NF-.kappa.B inducer
operatively linked to regulatory elements necessary for expressing
said sequence. The vector may comprise an inducible promoter to
enable an increased immune response to be produced by the increased
activation of NF-.kappa.B.
[0105] The use of recombinant polyepitope vaccines for the delivery
of multiple CD8 CTL epitopes is described in Thomson et al (1996)
J. Immunol. 157, 822-826 and WO 96/03144, both of which are
incorporated herein by reference. In relation to the present
invention, it may be desirable to include in a single vaccine, a
peptide (or a nucleic acid encoding a peptide) wherein the peptide
includes, in any order, one or more antigenic amino acid sequences
(for example each of between about 8 and 18 amino acids in length),
for example derived from a tumour-associated antigen, and a CD4 T
cell-stimulating epitope (such as from tetanus toxoid). Such
"bead-on-a-string" vaccines are typically DNA vaccines.
[0106] The antigenic molecule may comprise an epitope present on
transformed or cancerous cells (ie a tumour-associated antigen or
epitope, for example the MAGE-1 antigen produced by a high
proportion of human melanoma tymours (van der Bruggen et al (1991)
Science 254, 1643)). Alternatively, it may comprise an epitope
present on a pathogenic organism, for example a virus, or on a cell
(preferably a human cell) infected by a pathogenic organism, for
example a virally-infected cell, as noted above.
[0107] The epitope may be a T-cell epitope ie an epitope that is
capable of inducing a T-cell response (TH-1 response), preferably a
CD8+ cytotoxic T-cell response, but alternatively a CD4+ helper
T-cell response (TH-2 response) as well known to those skilled in
the art. A cytotoxic T-cell response may be undesirable in certain
cases, for example when the antigen is a mycobacterial antigen (for
example Mycobacterium tuberculosis or M. leprae antigen).
[0108] The inhibitor or inducer or antigen may be a peptidomimetic
compound, for example a peptidomimetic compound corresponding to a
polypeptide inhibitor or inducer discussed above.
[0109] The term "peptidomimetic" refers to a compound that mimics
the conformation and desirable features of a particular peptide as
a therapeutic agent, but that avoids potentially undesirable
features. For example, morphine is a compound which can be orally
administered, and which is a peptidomimetic of the peptide
endorphin.
[0110] Therapeutic applications involving peptides may be limited,
due to lack of oral bioavailability and to proteolytic degradation.
Typically, for example, peptides are rapidly degraded in vivo by
exo- and endopeptidases, resulting in generally very short
biological half-lives. Another deficiency of peptides as potential
therapeutic agents is their lack of bioavailability via oral
administration. Degradation of the peptides by proteolytic enzymes
in the gastrointestinal tract is likely to be an important
contributing factor. The problem is, however, more complicated
because it has been recognised that even small, cyclic peptides
which are not subject to rapid metabolite inactivation nevertheless
exhibit poor oral bioavailability. This is likely to be due to poor
transport across the intestinal membrane and rapid clearance from
the blood by hepatic extraction and subsequent excretion into the
intestine. These observations suggest that multiple amide bonds may
interfere with oral bioavailability. It is thought that the peptide
bonds linking the amino acid residues in the peptide chain may
break apart when the peptide drug is orally administered.
[0111] There are a number of different approaches to the design and
synthesis of peptidomimetics. In one approach, such as disclosed by
Sherman and Spatola, J. Am. Chem. Soc., 112: 433 (1990), one or
more amide bonds have been replaced in an essentially isoteric
manner by a variety of chemical functional groups. This stepwise
approach has met with some success in that active analogues have
been obtained. In some instances, these analogues have been shown
to possess longer biological half-lives than their
naturally-occurring counterparts. Nevertheless, this approach has
limitations. Successful replacement of more than one amide bond has
been rare. Consequently, the resulting analogues have remained
susceptible to enzymatic inactivation elsewhere in the molecule.
When replacing the peptide bond it is preferred that the new linker
moiety has substantially the same charge distribution and
substantially the same planarity as a peptide bond.
[0112] Retro-inverso peptidomimetics, in which the peptide bonds
are reversed, can be synthesised by methods known in the art, for
example such as those described in Meziere et al (1997) J. Immunol.
159 3230-3237. This approach involves making pseudopeptides
containing changes involving the backbone, and not the orientation
of side chains. Retro-inverse peptides, which contain NH--CO bonds
instead of CO--NH peptide bonds, are much more resistant to
proteolysis.
[0113] In another approach, a variety of uncoded or modified amino
acids such as D-amino acids and N-methyl amino acids have been used
to modify mammalian peptides. Alternatively, a presumed bioactive
conformation has been stabilised by a covalent modification, such
as cyclisation or by incorporation of .gamma.-lactam or other types
of bridges. See, eg. Veber et al, Proc. Natl. Acad. Sci. USA,
75:2636 (1978) and Thursell et al, Biochem. Biophys. Res. Comm.,
111:166 (1983).
[0114] A common theme among many of the synthetic strategies has
been the introduction of some cyclic moiety into a peptide-based
framework. The cyclic moiety restricts the conformational space of
the peptide structure and this frequently results in an increased
affinity of the peptide for a particular biological receptor. An
added advantage of this strategy is that the introduction of a
cyclic moiety into a peptide may also result in the peptide having
a diminished sensitivity to cellular peptidases.
[0115] One approach to the synthesis of cyclic stabilised
peptidomimetics is ring closing metathesis (RCM). This method
involves steps of synthesising a peptide precursor and contacting
it with a RCM catalyst to yield a conformationally restricted
peptide. Suitable peptide precursors may contain two or more
unsaturated C--C bonds. The method may be carried out using
solid-phase-peptide-synthesis techniques. In this embodiment, the
precursor, which is anchored to a solid support, is contacted with
a RCM catalyst and the product is then cleaved from the solid
support to yield a conformationally restricted peptide.
[0116] Polypeptides in which one or more of the amino acid residues
are chemically modified, before or after the polypeptide is
synthesised, may be used as antigen providing that the function of
the polypeptide, namely the production of a specific immune
response in vivo, remains substantially unchanged. Such
modifications include forming salts with acids or bases, especially
physiologically acceptable organic or inorganic acids and bases,
forming an ester or amide of a terminal carboxyl group, and
attaching amino acid protecting groups such as N-t-butoxycarbonyl.
Such modifications may protect the polypeptide from in vivo
metabolism.
[0117] A further aspect of the invention provides a kit of parts or
composition of chimaeric molecule, comprising (1) an intracellular
modulator, for example intracellular inhibitor or intracellular
enhancer, of APC, such as DC, function and (2) and antigenic
portion comprising or encoding an antigenic molecule.
[0118] A further aspect of the invention comprises a kit of parts,
composition or a chimaeric molecule, for example chimaeric
polypeptide, comprising a NF-.kappa.B inhibiting or
activating/inducing portion and an antigenic portion, as defined
above.
[0119] Either or both portions in these aspects of the invention
may further comprise a translocating portion and/or a cell binding
portion. The cell binding portion is preferably capable of binding
to a dendritic cell or precursor thereof. The translocating portion
may aid in internalisation of the molecule or at least the
antigenic portion and preferably the signalling inhibiting or
enhancing portion. Thus, exogenously applied peptides may be linked
to a HIV tat peptide. This may direct them into the MHC Class I
pathway for presentation by CTL (see, for example, Kim et al (1997)
J. Immunol. 159, 1666-1668. Chimaeric molecules which may be
adapted in accordance with the present invention are described in
WO95/31483.
[0120] Dendritic cells may be characterised by expression of the
CD80, CD86, CD40, CD1a, HLA-DR and/or CD83 cell surface molecules.
Immature dendritic cells may be CD34.sup.+ or CD14.sup.+. Thus, the
cell binding portion may be capable of binding to one or more of
these cell surface molecules (for example, an antibody capable of
binding to such a molecule).
[0121] Immature DCs show increased antigen capture and processing.
They show high intracellular MHC Class I and II' increased
endocytosis and phagocytosis; high CCR1, CCR5 and CCR6; low CCR7;
high CD68; low CD40, CD54, CD80, CD83, and CD86; and no
DC-LAMP.
[0122] Mature DCs show increased antigen processing. They show high
surface MHC Class I and II; low endocytosis and phagocytosis; low
CCR1, CCR5 and CCR6; high CCR7; low CD68; high CD40, CD54, CD58,
CD80, CD83 and CD86; high DC-LAMP; and high p55 fascin.
[0123] Such a cell binding portion may be useful in directing any
inhibitor or activator as herein described, for example nucleic
acid, DNA vaccine or antibody, to an APC such as a DC or immature
DC.
[0124] Preferably, the polynucleotide or DNA vaccine is capable of
expressing the encoded antisense molecule or polypeptide(s) in the
patient, still more preferably in an APC such as a DC or immature
DC of the patient. The antisense molecule or polypeptide(s), for
example NF-.kappa.B inhibitor or inducer/activator, or antigen, as
appropriate, may be expressed from any suitable polynucleotide
(genetic construct) as is described herein and delivered to the
patient. Typically, the genetic construct which expresses the
antisense molecule or polypeptide comprises the said polypeptide
coding sequence operatively linked to a promoter which can express
the transcribed polynucleotide (eg mRNA) molecule in a cell of the
patient, which may be translated to synthesise the said
polypeptide. Suitable promoters will be known to those skilled in
the art, and may include promoters for ubiquitously expressed, for
example housekeeping genes or for tissue-specific genes, depending
upon where it is desired to express the said polypeptide (for
example, in dendritic cells or precursors thereof). Preferably, a
dendritic cell or dendritic precursor cell-selective promoter is
used, but this is not essential, particularly if delivery or uptake
of the polynucleotide is targeted to the selected cells ie
dendritic cells or precursors.
[0125] Promoters that may be selective for dendritic cells may be
promoters from the CD36 or CD83 genes.
[0126] Targeting the vaccine to specific cell populations, for
example antigen presenting cells, may be achieved, for example,
either by the site of injection, use of targeting vectors and
delivery systems, or selective purification of such a cell
population from the patient and ex vivo administration of the
peptide or nucleic acid (for example dendritic cells may be sorted
as described in Zhou et al (1995) Blood 86, 3295-3301; Roth et al
(1996) Scand. J. Immunology 43, 646-651). In addition, targeting
vectors may comprise a tissue- or tumour-specific promoter which
directs expression of the antigen at a suitable place.
[0127] As noted above, it may be desirable to use an inducible
promoter. It will be appreciated that it may be desirable to be
able to regulate temporally expression of the polypeptide(s) (for
example NF-.kappa.B inhibitor or activator/inducer) in the cell.
Thus, it may be desirable that expression of the polypeptide(s) is
directly or indirectly (see below) under the control of a promoter
that may be regulated, for example by the concentration of a small
molecule that may be administered to the patient when it is desired
to activate or repress (depending upon whether the small molecule
effects activation or repression of the said promoter) expression
of the polypeptide. It will be appreciated that this may be of
particular benefit if the expression construct is stable ie capable
of expressing the polypeptide (in the presence of any necessary
regulatory molecules) in the said cell for a period of at least one
week, one, two, three, four, five, six, eight months or more. A
preferred construct of the invention may comprise a regulatable
promoter. Examples of regulatable promoters include those referred
to in the following papers: Rivera et al (1999) Proc Natl Acad Sci
USA 96(15), 8657-62 (control by rapamycin, an orally bioavailable
drug, using two separate adenovirus or adeno-associated virus (AAV)
vectors, one encoding an inducible human growth hormone (hGH)
target gene, and the other a bipartite rapamycin-regulated
transcription factor); Magari et al (1997) J Clin Invest 100(11),
2865-72 (control by rapamycin); Bueler (1999) Biol Chem 380(6),
613-22 (review of adeno-associated viral vectors); Bohl et al
(1998) Blood 92(5), 1512-7 (control by doxycycline in
adeno-associated vector); Abruzzese et al (1996) J Mol Med 74(7),
379-92 (reviews induction factors e.g., hormones, growth factors,
cytokines, cytostatics, irradiation, heat shock and associated
responsive elements). Tetracycline--inducible vectors may also be
used. These are activated by a relatively-non toxic antibiotic that
has been shown to be useful for regulating expression in mammalian
cell cultures. Also, steroid-based inducers may be useful
especially since the steroid receptor complex enters the nucleus
where the DNA vector must be segregated prior to transcription.
[0128] This system may be further improved by regulating the
expression at two levels, for example by using a tissue-specific
promoter and a promoter controlled by an exogenous
inducer/repressor, for example a small molecule inducer, as
discussed above and known to those skilled in the art. Thus, one
level of regulation may involve linking the appropriate
polypeptide-encoding gene to an inducible promoter whilst a further
level of regulation entails using a tissue-specific promoter to
drive the gene encoding the requisite inducible transcription
factor (which controls expression of the polypeptide (for example
NF-.kappa.B inhibitor or inducer/activator-encoding gene) from the
inducible promoter. Control may further be improved by
cell-type-specific targeting of the genetic construct.
[0129] The methods or constructs of the invention may be evaluated
in, for example, APCs such as dendritic cells generated in vitro,
as known to those skilled in the art, before evaluation in whole
animals. Suitable methods are described in Example 1.
[0130] The genetic constructs of the invention can be prepared
using methods well known in the art.
[0131] A further aspect of the invention provides vectors, vaccines
and antibodies for use in the invention.
[0132] The vaccines and vectors of the invention (therapeutic
molecules of the invention) may be formulated with suitable
pharmaceutically-acceptable carriers, fillers or other additives.
They may be administered by any suitable means such as
intra-muscularly, intra-veinally, orally, anally, intra-nasally,
etc. Subcutaneous or intramuscular administration may be preferred.
It will be appreciated that an inducer, for example small molecule
inducer as discussed above may preferably be administered
orally.
[0133] It may be desirable to locally perfuse an area comprising
target cells with the suitable delivery vehicle comprising the
therapeutic molecule, for example genetic construct, for a period
of time; additionally or alternatively the delivery vehicle or
therapeutic molecule can be injected directly into accessible areas
comprising target cells, for example subcutaneously. Methods of
delivering genetic constructs, for example adenoviral vector
constructs to cells of a patient will be well known to those
skilled in the art.
[0134] In particular, an adoptive therapy protocol may be used or a
gene gun may be used to deliver the construct to dendritic cells,
for example in the skin.
[0135] An adoptive therapy approach may include the steps of (1)
obtaining antigen presenting cells or precursors thereof,
preferably dendritic cells or precursors thereof, from the patient;
(2) contacting said antigen presenting cells with a NF-.kappa.B
inhibitor or inducer/activator as described herein, and optionally
antigen to which an immune response is required, or chimaeric
molecule or polynucleotide as discussed above, ex vivo; and (3)
reintroducing the so treated antigen presenting cells into the
patient.
[0136] Suitably, the dendritic cells are autologous dendritic cells
which are pulsed with polypeptide(s), for example a NF-.kappa.B
activator and an antigen. T-cell therapy using autologous dendritic
cells pulsed with peptides from a tumour associated antigen is
disclosed in Murphy et al (1996) The Prostate 29, 371-380 and Tjua
et al (1997) The Prostate 32, 272-278.
[0137] In a further embodiment the antigen presenting cells, such
as dendritic cells, are contacted with a polynucleotide which
encodes the NF-.kappa.B inhibitor or activator/inducer. The
polynucleotide may be any suitable polynucleotide and it is
preferred that it is capable of transducing the dendritic cell thus
resulting in respectively activation or inhibition of antigen
presentation by the antigen presenting cell.
[0138] Conveniently, the polynucleotide may be comprised in a viral
polynucleotide or virus, as noted above. For example,
adenovirus-transduced dendritic cells have been shown to induce
antigen-specific antitumour immunity in relation to MUC1 (see Gong
et al (1997) Gene Ther. 4, 1023-1028). Similarly, adenovirus-based
systems may be used (see, for example, Wan et al (1997) Hum. Gene
Ther. 8, 1355-1363); retroviral systems may be used (Specht et al
(1997) J. Exp. Med. 186, 1213-1221 and Szabolcs et al (1997) Blood
90, 2160-2167); particle-mediated transfer to dendritic cells may
also be used (Tuting et al (1997) Eur. J. Immunol. 27, 2702-2707);
and RNA may also be used (Ashley et al (1997) J. Exp. Med. 186,
1177-1182).
[0139] The APCs, such as dendritic cells, may be derived from the
patient (ie autologous dendritic cells) or from a healthy
individual or individuals (MHC matched or mismatched), treated in
vitro as indicated above, followed by adoptive therapy, ie
introduction of the so-manipulated dendritic cells in vivo, which
may then activate CTL responses. By "healthy individual" we mean
that the individual is generally in good health, preferably has a
competent immune system and, more preferably, is not suffering from
any disease which can be readily tested for, and detected.
[0140] Thus, the methods of the invention include methods of
adoptive immunotherapy.
[0141] It is preferred if between about 10.sup.3 and 10.sup.11 DCs
are administered to the patient; more preferably between 10.sup.6
and 10.sup.7 DCs.
[0142] The APCs such as DCs may be administered by any convenient
route. It is preferred if the DCs are administered intravenously.
It is also preferred if the DCs are administered locally to the
site of the disease (such as a tumour or local viral or bacterial
infection). Local administration is particularly preferred for
cancer. Conveniently, the DCs are administered into an artery that
supplies the site of the disease or the tissue where the disease is
located.
[0143] The cells (or vaccine) may be given to a patient who is
being treated for the disease by some other method. Thus, although
the method of treatment may be used alone it is desirable to use it
as an adjuvant therapy.
[0144] The APCs, such as DCs, or vaccine may be administered
before, during or after the other therapy.
[0145] When the disease to be treated is a cancer it is preferable
if the cancer has been, is being or will be treated with a
conventional therapy or surgery as well as with the method of the
invention. Conveniently, depending on the therapy, the cancer is
treated by radiotherapy or by chemotherapy.
[0146] When the disease to be treated is an infection by a pathogen
it is preferable if the infection has been, is being or will be
treated with a conventional therapy or surgery.
[0147] If the patient to be treated has HIV infection it is
preferable if the method of the invention is used as an adjuvant to
other treatment, for example treatment with a reverse transcriptase
inhibitor such as AZT or 3TC or combination therapy for example
HART (highly active retroviral therapy).
[0148] When the method of the invention is used to treat a solid
tumour it is preferred if the APCs such as DCs or vaccine are
administered as the first post-surgery treatment.
[0149] When the method of the invention is used to treat leukaemia
it is preferred if the APCs such as DCs or vaccine are administered
after radiotherapy or chemotherapy. It is also preferred if
leukaemia patients are also treated with the DCs in combination
with bone marrow transplantation.
[0150] Cancer therapy, for example adoptive immunotherapy may be
most effective in the control or elimination of minimal residual
disease rather than in the reduction of bulk disease. It is
conceivable that immunotherapy may temporarily increase the
dimensions of bulk disease due to influx of cytotoxic T
lymphocytes. Extent and bulk of disease may be monitored following
therapy but not used as a formal endpoint. Patients are followed up
in the routine manner in the long term to ensure that no long term
adverse events are manifest.
[0151] Further delivery or targeting strategies may include the
following. Ballistic compressed air driven DNA/protein coated
nanoparticle penetration (for example using a BioRad device) of
cells in culture or in vivo may be used Constructs for delivery
should preferably have cell-type specific promoters.
[0152] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents. The
formulations may be presented in unit-dose or multi-dose
containers, for example sealed ampoules and vials, and may be
stored in a freeze-dried (lyophilised) condition requiring only the
addition of the sterile liquid carrier, for example water for
injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets of the kind well known to those skilled in the
art. Nasal sprays may be a useful format.
[0153] The dose of the construct, for example, is dependent on the
size of the construct and the purpose for which is it administered.
In general, the range is calculated based on the surface area of
tissue to be treated. The effective dose of construct may be
dependent on the size of the construct and the delivery
vehicle/targeting method used and chemical composition of the
oligonucleotide but a suitable dose may be determined by the
skilled person, for example making use of data from the animal and
in vitro test systems indicated above.
[0154] The construct, for example, may be administered to the
patient systemically for both therapeutic and prophylactic
purposes. The construct, for example may be administered by any
effective method, as described above, for example, parenterally (eg
intravenously, subcutaneously, intramuscularly) or by oral, nasal
or other means which permit the construct, for example, to access
and circulate in the patient's bloodstream. Construct administered
systemically preferably are given in addition to locally
administered construct, but also have utility in the absence of
local administration.
[0155] It is believed that uptake of the nucleic acid and
expression of the encoded polypeptide by dendritic cells may be the
mechanism of priming of the immune response; however, dendritic
cells may not be transfected but are still important since they may
pick up expressed peptide from transfected cells in the tissue.
[0156] It is preferred if the vaccine, such as DNA vaccine, is
administered into the muscle. It is also preferred if the vaccine
is administered onto or into the skin.
[0157] As noted above, the invention provides a kit of parts or
composition or a chimaeric molecule, comprising (1) a modulating
portion comprising or encoding a NF-.kappa.B inhibitor or inducer
and (2) an antigenic portion comprising or encoding an antigenic
molecule.
[0158] In relation to any previous aspect of the invention,
particularly a vaccine as previously described, or such a kit,
chimaeric molecule or composition, the antigenic molecule
preferably comprises an epitope present on transformed or cancerous
cells or on a pathogenic organism, or on a cell infected by a
pathogenic organism.
[0159] Thus, the invention provides a vaccine effective against
cancer, or cancer or tumour cells, or against a pathogenic organism
or cell infected with a pathogenic organism, comprising an
effective amount of an NF-.kappa.B inducer or other inducer of APC
function as described above or polynucleotide encoding an
NF-.kappa.B inducer or other inducer of APC function. The vaccine
preferably further comprising an antigen or polynucleotide encoding
an antigen, having an epitope present on the cancer or tumour
cells, or the pathogenic organism or cell infected with a
pathogenic organism. The vaccine is preferably a nucleic acid
vaccine.
[0160] A further aspect of the invention provides a pharmaceutical
composition comprising a NF-.kappa.B inducer, NF-.kappa.B
inhibitor, vaccine, molecule, polynucleotide, kit of parts,
composition or chimaeric molecule of any of the preceding aspects
of the invention and a pharmaceutically acceptable carrier.
[0161] The invention further provides a vaccine, molecule,
polynucleotide, kit of parts, composition or chimaeric molecule of
any preceding aspect of the invention for use in medicine. The
invention still further provides the use of a vaccine, molecule,
polynucleotide, kit of parts, composition or chimaeric molecule of
any of the preceding aspects of the invention in the manufacture of
a medicament for the treatment of a patient in need of modulation
of antigen presentation.
[0162] It will be clear that the invention provides a method of
killing target cells in a patient which target cells aberrantly
express a first epitope, the method comprising the steps of (1)
obtaining APCs, such as dendritic cells, from said patient; (2)
contacting said APCs, such as dendritic cells, with an enhancer of
APC, such as DC, function ex vivo; (3) optionally contacting said
cells with the said epitope or with a polynucleotide or expression
vector encoding the said epitope and (4) reintroducing the so
treated APCs such as dendritic cells, into the patient.
[0163] It will also be clear that the invention provides a method
of killing target cells in a patient which target cells aberrantly
express a first epitope, the method comprising the steps of (1)
obtaining dendritic cells from said patient; (2) contacting said
dendritic cells with a NF-.kappa.B inducer ex vivo; (3) optionally
contacting said cells with the said epitope or with a
polynucleotide or expression vector encoding the said epitope and
(4) reintroducing the so treated dendritic cells into the
patient.
[0164] The target cells may be cancer cells.
[0165] The invention also provides methods of inhibiting or
stimulating the maturation and activation of dendritic cells in
vivo or in vitro comprising administering an effective amount of an
inhibitor or inducer of NF-.kappa.B.
[0166] The inhibitors or inducers may be as defined above.
[0167] For the avoidance of doubt, wherever the term "dendritic
cell" or "dendritic cells" are used the term includes any suitable
antigen presenting cell unless the context suggests otherwise. It
is preferred that the antigen presenting cell is a dendritic
cell.
[0168] Preferred embodiments of the invention will now be
described.
FIGURES LEGENDS
[0169] FIG. 1. Cell viability of DC treated with PSI. Mature DC was
either left untreated, or treated with various doses of PSI for 4
h. After wash, cells were cultured in RPMI 1640 supplemented as
described without monocyte condition medium. 6 days after
treatment, cells were resuspended in PBS containing 10 .mu.g/ml. Of
propidium iodide PI and analysed on FACS scan.
[0170] FIG. 2. Immunostimulatory capacity of DC treated with the
proteosome inhibitor Cbz-Ile-Glu(O-tert-bytyl)-Ala-leucinal (PSI).
Mature DC was treated with PSI (0.1 .mu.M:.tangle-solidup.), (0.5
.mu.M:.box-solid.), (1, .mu.M: ) or without PSI (.largecircle.) for
4 h. After wash, different numbers of each category of DC and
10.sub.5 cells of allo-lymphocytes were plated on a 96 well plate.
Proliferation was determined at day 6 using .sup.3H-TdR uptake
assay. (Each point represents mean +/-SEM from five separate
experiments).
[0171] FIG. 3. Effect of PSI on the expression of surface antigens
was studied using monoclonal antibodies, HLA-DR, CD86 (PE
conjugated); Pharmingen, San Diego, Calif.). on day 6 after PSI
treatment, populations of untreated (green line), PSI (0.1 .mu.M)
treated (blue dotted line), PSI (1 .mu.M) treated (red dotted line)
were phenotyped with the MoAbs listed above and analysed on FACScan
(Becton Dickinson).
[0172] FIG. 4. Lack of recovery of proliferation response in
allo-MLR. Mature DC was treated with vehicle (A) or PSI (1 .mu.M)
(B) for 4 h. After wash graded number of each treated DC and
10.sup.5 cells of allo-lymphocytes were plated on a 96 well plate
with vehicle (.largecircle., ), 2 ng/ml. of IL-2 (.quadrature.,
.box-solid.) or 10 g/ml. of anti CD28 Ab (.DELTA.,
.tangle-solidup.). Proliferation was determined at day 6 using
.sup.3H-TdR uptake assay. (Each point represents mean +/-SEM from
three separate experiments).
[0173] FIG. 5. PSI pre-treated DC inhibit the growing ability of
lymphocytes in allo-MLR. Mature DC was treated with PSI as
described above. After washing, each DC was mixed to make different
compositions: vehicle treated DC only (.largecircle.), 1/4 number
of PSI treated DC mixed with 3/4 vehicle treated DC ( ), 1/8 number
of PSI treated DC mixed with % vehicle treated DC (.box-solid.), or
PSI treated DC only (.tangle-solidup.). Graded number of each
mixture and 10.sup.5 cells of allo-lymphocytes were plated on a 96
well plate. Proliferation was determined at day 6 by .sup.3H-TDR
uptake assay.
[0174] FIG. 6. Impaired response of allo-lymphocytes pre-cultured
with PSI treated or non-treated DC. Lymphocytes were co-cultured
with 1/10 number of PSI treated ( , .tangle-solidup.) or
non-treated DC (.largecircle., .DELTA.). After 2 days, each DC were
removed by CD83, CD86 coated plate. Lymphocytes were cultured for
another 4 days using vehicle treated DC (.largecircle., ), or PSI
(1 .mu.M) treated DC (.DELTA., .tangle-solidup.). Proliferation was
determined by .sup.3H-TDR uptake assay. (Each point represents mean
+/-SEM from three separate experiments).
[0175] FIG. 7. Reduced expression of surface antigens on
allo-lymphocytes by PSI pre-treated DC. Lymphocytes were cultured
for 6 days as the same conditions with allo-MLR experiment using
vehicle treated DC (green line), or PSI (1 .mu.M) treated DC (red
line). Each lymphocytes were phenotyped with MoAbs, CD3, CD25 (PE
conjugated; Pharmingen), (CD54) (PE conjugated; Serotec), CD50
(FITC conjugated; Serotec) and analysed by FACScan.
[0176] FIG. 8. Cytokine production of allo-lymphocytes co-cultured
with vehicle treated or PSI treated DC. Mature DC was treated with
vehicle or PSI (1 .mu.M) as described above. Allo-lymphocytes and
each DC were plated at 2.times.10.sup.5 cells per well on a 96 well
plate and stimulated with PMA (10 nM) or left unstimulated for 49
hr. Supernatants were analysed for IL-2, IL-4, and
interferon-.gamma. by ELISA.
[0177] FIG. 9. Cytokine productions of DC treated with vehicle or
PSI. Mature DC was treated with vehicle or PSI (1 .mu.M) as
described above. Each DC and allo-lymphocytes were plated at
2.times.10.sup.5 cells per well on a 96 well plate and stimulated
with PMA (10 nM) or left unstimulated for 48 hr. Supernatants were
analysed for IL-6, IL-8, IL-12 and TNF.alpha. by ELISA.
[0178] FIG. 10. Expression of MEKK-1 greatly enhances
monocyte-derived dendritic cells antigen-presentation in the
allogeneic MLR. Briefly, dendritic cells were generated by
culturing peripheral blood monocytes with 50 ng/ml. GM-CSF and 10
mg/ml. IL-4. At day 5, they were infected for 2 hours with an
adenovirus without insert or encoding MEKK-1 (m.o.i. 100:1), or
were left uninfected. After 2 days, 10.sup.4 dendritic cells were
cultured with 10.sup.5 purified allogeneic T cells for 5 days in
96-well flat-bottomed plates, plused overnight with 0.5 .mu.Ci/well
and harvested the following day. Dendritic cells encoding MEKK-1
were 4-fold more powerful in inducing T cell proliferation than
uninfected DC, whereas this was not true for dendritic cells
infected with an adenovirus without insert.
[0179] FIG. 11: Expression of dominant negative inhibitor of MyD88
(MyD88lpr) in dendritic cells induces NF-.kappa.B activation
[0180] Immature DC were generated from peripheral blood monocytes
after 5 days of culture with 50 ng/ml GM-CSF and 10 ng/ml IL-4.
Then, they were left uninfected, or infected in serum-free medium
with a control adenovirus without insert (Ad0) and an adenovirus
encoding dominant-negative MyD88 (AdMyD88-lpr). A multiplicity of
infection of 100 was used. After 6 h and 24 h expression, cells
were lysed and their nuclear extracts examined for NF-.kappa.B
DNA-binding activity by EMSA. Surprisingly, expression of MyD88lpr
could induce on its own NF-.kappa.B activation which is totally in
constrast with what has been previously found.
[0181] FIG. 12: Expression of dominant negative (inhibitor) but not
wild-type MyD88 induces TNF.alpha. production on its own and does
not inhibit LPS-induced TNF.alpha. production in dendritic
cells
[0182] Immature DC were generated from peripheral blood monocytes
after 5 days of culture with 50 ng/ml GM-CSF and 10 ng/ml IL-4.
Then, they were left uninfected, or infected in serum-free medium
with a control adenovirus encoding .beta.-gal (Ad.beta.-gal), an
adenovirus encoding dominant-negative MyD88 (Adlpr) and an
adenovirus encoding wild-type MyD88 (AdMyD88 wt). Adtoll has been
shown to be non-functional and should be ignored. A multiplicity of
infection of 100 was used. After 24 h, cells were stimulated with
100 ng/ml LPS. Surprisingly, expression of dominant negative MyD88
could induce dendritic cell TNF.alpha. production on its own, in
the absence of additional stimulation. Moreover, it could not
inhibit LPS-induced TNF.alpha. production, a finding that is in
contrast to previous findings.
[0183] FIG. 13: Expression of wild-type MyD88 abrogates the
MyD88-lpr(dominant-negative)-induced TNF.alpha. production in
dendritic cells
[0184] Immature DC were generated from peripheral blood monocytes
after 5 days of culture with 50 ng/ml GM-CSF and 10 ng/ml IL-4.
Then, they were left uninfected, or infected in serum-free medium
with a control adenovirus encoding .beta.-gal (Ad.beta.-gal), an
adenovirus encoding wild-type MyD88 (AdMyD88 wt) and an adenovirus
encoding dominant-negative MyD88 (AdMyD88lpr). In some cases,
double infection by Adlpr and Ad.beta.-gal or AdMyD88 wt at ratios
1:1 and 1:5 was used. A multiplicity of infection of 100 was used
for single infections. After 24 h, supernatants were removed and
analysed. Surprisingly, expression of dominant negative MyD88 could
induce dendritic cell TNF.alpha. production on its own, in the
absence of additional stimulation. Wild-type Myd88 was able to
inhibit TNF production induced by Myd88lpr.
[0185] FIG. 14: Expression of dominant-negative MyD88 in dendritic
cells enhances antigen-specific T cell proliferation
[0186] Immature DC were generated from peripheral blood monocytes
after 5 days of culture with 50 ng/ml GM-CSF and 10 ng/ml IL-4.
Then, they were left uninfected, or infected in serum-free medium
with a control adenovirus without insert (Ad0), an adenovirus
encoding green fluorescent protein as a prototype antigen (AdGFP),
and an adenovirus encoding GFP linked together with the dominant
negative MyD88 (AdGFP-lpr). After 48 h, graded doses of dendritic
cells were cultured with 2.times.10.sup.4 antigen-specific T cells
and proliferation was measured at day 3. Delivery of the antigen
GFP to dendritic cells induced antigen-specific T cell
proliferation that was enhanced by expression of dominant negative
MyD88. This is in agreement with our unexpected result that
inhibition of MyD88 activity in dendritic cells, induces dendritic
cell activation.
[0187] FIG. 15: Expression of dominant-negative MyD88 in dendritic
cells enhances the allogeneic mixed lymphocyte reacfion (MLR)
[0188] Immature DC were generated from peripheral blood monocytes
after 5 days of culture with 50 ng/ml GM-CSF and 10 ng/ml IL-4.
Then, they were left uninfected, or infected in serum-free medium
with a control adenovirus without insert (Ad0), and an adenovirus
encoding the dominant negative form of MyD88 (Adlpr). After 48 h,
graded doses of dendritic cells were cultured with 1.times.10.sup.5
allogeneic T cells and proliferation was measured at day 6.
expression of dominant negative MyD88 enhances the allogeneic T
cell proliferation, a finding that is indicative of increased DC
antigen presentation.
[0189] FIG. 16: Expression of dominant-negative MyD88 in dendritic
cells enhances the expression of costimulatory molecules (CD80,
CD86)
[0190] Immature DC were generated from peripheral blood monocytes
after 5 days of culture with 50 ng/ml GM-CSF and 10 ng/ml IL-4.
Then, they were left uninfected, or infected in serum-free medium
with a control adenovirus encoding GFP, and with an adenovirus
encoding dominant negative MyD88 (Adlpr). After 48 h, dendritic
cells were collected and stained for CD80 and CD86, two very
important costimulatory molecules required for efficient
antigen-presenting function. expression of the dominant negative
form of MyD88 enhanced CD80 and CD86 cell surface expression, which
is indicative of enhanced dendrific cell antigen-presenting
function.
[0191] FIG. 17: Expression of dominant-negative MyD88 in
macrophages induces p38 MAPK phosphorylation
[0192] Human macrophages were differentiated from peripheral blood
monocytes by addition of 100 ng/ml M-CSF for 2-3 days in 5% FCS
RPMI. Then, they were left uninfected, infected in seruim-free
medium with a control adenovirus encoding .beta.-gal, or infected
with an adenovirus encoding dominant negative MyD88 (Adlpr). An moi
of 100 (or 50 in one case) was used as shown. After 6, 24 and 48 h,
cells were lysed and extracts assayed for p38 MAPK activity using
western blotting and phospho-p38 MAPK-specific antibodies.
Unexpectedly, expression of dominant-negative MyD88 induces p38
MAPK activity in human macrophages.
[0193] FIG. 18: Expression of dominant-negative MyD88 in
macrophages induces IRAK phosphorylation
[0194] Human macrophages were differentiated from peripheral blood
monocytes by addition of 100 ng/ml M-CSF for 2-3 days in 5% FCS
RPMI. HELA cells cultured in 5% FCS DMEM were also used Both cell
types were left uninfected, infected in serum-free medium with a
control adenovirus encoding .beta.-gal, an adenovirus encoding
wild-type MyD88 or an adenovirus encoding dominant negative MyD88
(AdMyD88lpr). An moi of 100 was used. After 5 min or 12 h, cells
were lysed and extracts assayed for IRAK and phospho-IRAK using
western blotting. In HELA cells, expression of dominant negative
MyD88 (MyD88-lpr) was found to inhibit IL-1-induced activation of
IRAK. Unexpectedly, however, expression of dominant-negative MyD88
induces IRAK activity in human macrophages that is not increased by
the addition of LPS. This finding suggests that MyD88 activity is
also required for an inhibitory signal in macrophages (but not HELA
cells) that inhibits IRAK phosphorylation, and its blockade results
in the activation of IRAK.
[0195] FIG. 19: Expression of dominant-negative MyD88 in
macrophages induces I.kappa.B.alpha. phosphorylation
[0196] Human macrophages were differentiated from peripheral blood
monocytes by addition of 100 ng/ml M-CSF for 2-3 days in 5% FCS
RPMI. Cells infected in serum-free medium with a control adenovirus
encoding .beta.-gal or an adenovirus encoding dominant negative
MyD88 (Adlpr). An moi of 100 was used. After 24 h, cells were lysed
and extracts assayed for phospho-I.kappa.B.alpha. using western
blotting. Unexpectedly, expression of dominant-negative MyD88
induces I.kappa.B.alpha. phosphorylation in human macrophages.
[0197] FIG. 20: Expression of dominant-negative but not wild-type
MyD88 in macrophages induces TNF.alpha., IL-6 and IL-8 production
in the absence of any stimulus.
[0198] Human macrophages were differentiated from peripheral blood
monocytes by addition of 100 ng/ml M-CSF for 2-3 days in 5% FCS
RPMI. Then, they were left uninfected, infected in serum-free
medium with a control adenovirus encoding .beta.-gal, infected with
an adenovirus encoding wild-type MyD88 or infected with an
adenovirus encoding dominant negative MyD88 (Adlpr). An moi of 100
was used. (a) After 48 h, supernatants were collected and assayed
for TNF.alpha., IL-6 and IL-8 cytokine production in the absence of
any further stimulation. Cytokine production could be detected in
cells encoding dominant negative MyD88 but not cells encoding
wild-type MyD88 or control cells. This suggested that blocking
MyD88 activity in macrophages, as in dendritic cells but not HSF or
HUVEC, results in the activation of cells and the release of
inflammatory cytokines. (b) At 0 h, 4 h, 24 h and 48 h of
expression, supurnatants were collected and assayed by ELISA for
TNF, IL-6 and IL-8. Only the results from cells encoding dominant
negative MyD88 (MyD88-lpr) are shown as control cells or cells
encoding wild-type MyD88 had background levels of cytokine
production.
MATERIALS AND METHODS
1. Reagents
[0199] Human recombinant GM-CSF and TNF.alpha. were kind gifts of
Dr Glenn Larsen (GI) and Dr D Tracey (BASF), respectively. Human
recombinant IL-4 was purchased from R&D Systems (Minneapolis,
USA). PMA, LPS and lonomycin were obtained from Sigma Chemical Co.
(St Louis, USA). The proteasome inhibitor
Cbz-Ile-Glu(O-terr-butyl-Ala-ceremal (PSI) was The proteasome
inhibitor Cbz-Ile-Glu(O-terr-butyl-Ala-ceremal (PSI) was obtained
from Calbiochem (Nottingham, UK). M-CSF was obtained from the
Genetics Institute (Boston, USA).
2. Preparation of Peripheral Blood Mononuclear Cells
[0200] Peripheral blood mononuclear cells (PBMC) were obtained by
density centrifugation of leukopheresis residues from healthy
volunteers (North London Blood Transfusion Service, Colindale, UK).
Heparinised residues were diluted 2.times. with HBSS and 25 ml were
carefully layered over equal volumes of Ficoll-Hypaque lymphoprep
(Nycomed, Oslo, Norway) in 50 ml sterile tubes prior to
centrifugation for 30 minutes at 2000 rpm at room temperature.
After centrifugation, the interface layer was collected and washed
twice with HBSS (centrifuged for 10 minutes at 2000 rpm). PBMC were
then collected and resuspended in 30 ml of RPMI containing 5%
FCS.
3. Isolation of Peripheral Blood T Cells and Monocytes and Culture
of HeLa Cells
[0201] Peripheral blood T cells and monocytes were obtained from
PBMC after cell cell separation in a Beckman JE6 elutriator.
Elutriation was performed in RPMI containing 1% FCS (elutriation
medium). Lymphocyte and monocyte purity was assessed by flow
cytometry using fluorochrome-conjugated anti-human monoclonal
antibodies against CD45, CD3, CD14 and CD19 (Becton Dickinson,
Oxford, UK). T lymphocyte fractions typically contained .about.80%
CD3-expressing cells, .about.6% CD19-expressing cells and <1%
CD14-expressing cells. Monocyte fractions routinely consisted of
>85% CD14-expressing cells, <0.5% CD19 cells and <3% of
CD3-expressing cells. HeLa cells were maintained in 5% FCS, 1%
penicillin/streptomycin DMEM.
4. Differentiation of Monocytes with M-CSF for Adenoviral
Infection
[0202] To optimize adenoviral infection, freshly elutriated
monocytes were cultured at 1.times.10.sup.6 cells/ml in 10 cm petri
dishes (Falcon, UK) with 100 ng/ml of M-CSF (Genetics Institute,
Boston, USA). After 2-3 days they were washed with PBS to remove
non-adherent cells and the remaining adherent monocytes were
incubated with 10 ml of cell dissociation solution (Sigma, UK) for
1 h at 37.degree. C. The cell suspension was washed twice in RPMI
containing 5% FCS and cell viability (90%) was assessed by trypan
blue exclusion. Cells at this stage were 99% CD14 positive by FACS
staining and were cultured at 1.times.10.sup.6/ml, in 24-well or
48-well flat-bottomed tissue culture plates (Falcon, UK) for
further experiments.
5. Differentiation of Monocytes to Dendritic Cells
[0203] Freshly elutriated monocytes were cultured at
1.times.10.sup.6 cells/ml in 10 cm petri dishes (Falcon, UK) in 5%
FCS RPMI supplemented with 50 ng/ml GM-CSF and 10 ng/ml IL-4 for
5-6 days. At day 3, cytokines were replenished. This method
presents several advantages as compared to differentiation of
dendritic cells directly from blood or bone marrow precursors.
Besides being easy and giving high numbers of cells, it generates a
homogenous population of cells with a stable "immature DC"
phenotype. This phenotype can be pushed to maturation by addition
of TNF.alpha. (10 ng/ml), LPS (100 ng/ml) or monocyte-conditioned
medium (50% v/v) for a further 2-3 days to the DC.
6. Monocyte Conditioned Medium
[0204] Ig-coated plates (100 mm, Falcon) were prepared immediately
before use by the addition of 5 ml of human gamma-globulin (10
mg/ml, Sigma Chemical Co.) for 10 min. The plates were washed three
times with RPMI 1640 medium (serum free) before use. Elutriated
monocytes (3.times.10.sup.7) were layered on the Ig-coated plates
for 1 hr in 7 ml volumes. Non-adherent cells were washed off, and
gamma-globulin adhered cells were incubated in fresh complete RPMI
1640 medium at 37.degree. C. for 24 hr.
7. Adenoviral Vectors and their Propagation
[0205] Recombinant, replication-deficient adenoviral vectors
encoding E. coli .beta.-galactosidase (Ad.beta.-gal) or having no
insert (Ad0) were provided by Drs A. Byrnes and M. Wood (Oxford
University, UK). The GFP-expressing adenovirus (AdGFP) was
generated by double recombination of AdTrack with AdEasy-1
adenoviral plasmid provided by Prof. B. Vogelstein (The Howard
Huges Medical Institute, Baltimore, Md.). AdMyD88 wt and AdMyD88lpr
were generated from plasmids provided by Dr Xu (University of
Texas, Southwestern) and Dr K. Burns (Lausanne, Switzerland). In
particular, pAdTrackCMV was used for AdMEKK-1wt, whereas for
AdMyD88 wt and AdMyD88lpr, a pAdTrack.CMV vector derivative, termed
AdTrack.CMVKS17, was used. pAdTrack.CMVKS17 was constructed by
removing the EcoRI site of AdTrack.CMV as well as its multiple
cloning site (MCS), and by inserting the larger multiple cloning
site of the vector pBCSK(+) (Stratagene). Recombinant viruses were
generated in BJ5183 bacterial cells transformed by the heat-shock
method with 1 .mu.g of linearised pAdTrack.CMV-MEKK1wt,
AdTrack.CMVKS17-MyD88 wt or AdTrack.CMVKS17-My88lpr constructs and
100 ng of replication-deficient adenoviral vector pAdEasy-1.
Positive recombinant clones were selected through their resistance
to kanamycin. Following selection, DNA extracted was used for virus
propagation in the 293 human embryonic kidney cells. Viruses were
purified by ultracentrifugation through two caesium chloride
gradients, as described in He et al. (He T. C. et al. (1998).
Science 281: 1509-12). Titres of viral stocks were determined by
plaque assay, in HEK 293 cells, after exposure for 1 hour in serum
free DMEM medium (Gibco BRL) and subsequently overlayed with an
(1.5%) agarose/(2.times.DMEM with 4% FCS) mixture (v/v1:1) and
incubated for 10-14 days.(He T. C. et al. (1998). Science 281:
1509-12).
9. Adenoviral Infection of Cells
[0206] M-CSF-differentiated macrophages and immature or mature
dendritic cells were collected, counted and replated. Then, they
were infected in serum-free RPMI with replication-deficient
adenoviruses overexpressing the gene of interest. A multiplicity of
infection of 100 for macrophages and immature DC, and 300, for
mature DC, was used. After 2 h, the virus was removed and cells
were cultured in complete medium for an additional 1-2 days to
allow overexpression of the protein of interest. Then, they were
used in further experiments.
10. Establishment of Antigen-Specific T Cell Lines
[0207] Green fluorescent protein was purchased from Clontech. To
establish antigen-specific polyclonal T cell lines,
1.times.10.sup.6 PBMC/ml were cultured with antigen (1 .mu.g/ml for
green fluorescent protein or 5 .mu.g/ml for tetanus toxoid) for 7
days and then with IL-2 at 20 ng/ml for another 10-14 days. Every 4
days IL-2 was replenished. This resulted in the expansion of
antigen-specific T cells and cell death of most other cell
populations present in PBMC. After 17-21 days of culture, a
restimulation step was included by culturing the T cells with
autologous irradiated PBMC at a 1:1 ratio and fresh antigen in the
absence of IL-2. After 4 days IL-2 was added for another 10-14
days. This restimulation cycle was repeated at least 4 times before
use of antigen-specific T cells in further experiments.
11. Proliferation Assays of Antigen-Specific T Cells
[0208] To assess the specificity of antigen-specific T cell lines,
1.times.10.sup.5 of antigen-specific T cells were cultured with
1.times.10.sup.5 autologous irradiated PBMC and various
concentrations of antigen in 96-well flat-bottomed microtiter
plates. After 3 days, cells were pulsed with [.sup.3H]-Thymidine
overnight and harvested the following day.
[0209] To measure dendritic cell-induced antigen-specific T cell
proliferation, 1.times.10.sup.5 antigen-specific T cells were
cultured with graded doses of irradiated or mitomycin-treated
dendritic cells that were unplulsed, pulsed with antigen,
uninfected or adenovirus-infected. [.sup.3H]-Thymidine
incorporation was measured after 2 days. All antigen-specific
proliferation assays were done in triplicates.
12. Mixed Lymphocyte Reaction (MLR)
[0210] To assay the immunostimulatory capacity of non-irradiated or
irradiated (3000 rad from a .sup.137Cs source) DC, uninfected,
adenovirus-infected, LPS-treated or monocyte-conditioned
medium-treated DC were cultured in graded doses with
1.times.10.sup.5 of allogeneic elutriated T cells in quadruplicate
in a 96-well flat-bottom microtiter plate (Falcon). Proliferation
was measured on day 5 by thymidine incorporation after a 16 h pulse
with [.sup.3H] thymidine (0.5 .mu.Ci/well; Amersham Life Science,
UK).
13. Cytokine Analysis
[0211] Cells were pre-treated with vehicle or 1 .mu.M of PSI for 4
h, or infected with adenoviral vectors for 2 h. PSI-treated DC were
then directly used in experiments whereas infected cells were
further cultured for 2 days to allow overexpression of the relevant
protein to occur. 24 h after stimulation of the cells, culture
supernatants were collected and kept frozen. Cytokine levels in
cell culture supernatants were measured by standard 2 or 3 layer
sandwich ELISA techniques using specific monoclonal and polyclonal
antibodies for TNF.alpha., IL-4, IL-6, IL-8, IL-12 and IFN.gamma..
Antibody pairs and standards for these assays have been purchased
from Pharmingen, with the exception of IL-12 reagents that were
gifted from the Genetics Institute (Boston, USA).
14. Immunofluorescence Staining and Flow Cytometry
[0212] For FACS staining, cells were first harvested. For adherent
cells where surface receptors need to be intact, a warm 2% EDTA in
PBS solution was used for 20 min at 37.degree. C. After cells were
in solution, they were washed once and then resuspended in ice-cold
FACS washing buffer. All subsequent incubations were performed at
4.degree. C. For each analysis, 5.times.10.sup.5 cells were
incubated with the relevant antigen-specific antibody or isotype
control for 30 min and then washed twice with FACS washing buffer.
Cells were then examined by flow cytometry. cells were ready for
analysis on a FACScan flow cytometer (Becton and Dickinson) by
using the CellQuest (Becton Dickinson). Directly conjugated
monoclonal antibodies to HLA-DR, HLA-A,B,C, CD80, CD86, CD3, CD14
and CD25 were purchased by Pharmingen, San Diego, USA).
15. Preparation of Cytosolic Protein Extracts
[0213] Cytosolic extracts were prepared to investigate biochemical
events involved in signal transduction by western blotting.
Adherent cells were scraped from the tissue culture plate/flask
into fresh PBS and harvested by centrifugation (13000 g for 10
seconds at 4.degree. C.). Non-adherent cells were similarly
pelleted by centrifugation and washed once with fresh PBS. After
discarding the supernatants, an appropriate quantity of ice-cold
hypotonic lysis buffer (Whiteside S. T. et al (1992). Nucleic Acids
Res 20:1531-8) was added, depending on the number of cells to be
lysed (50-100 .mu.l per 1.times.10.sup.6 cells). After incubation
on ice for 10 minutes, lysates were centrifuged (13000 g, 5
minutes, 4.degree. C.) in order to remove nuclei and cell debris.
The cleared lysates were then removed to fresh tubes, frozen and
stored at -20.degree. C. for subsequent estimation of protein
concentration and use in western blotting.
16. Preparation of Nuclear Protein Extracts
[0214] Nuclear protein extracts were prepared to study the
NF-.kappa.B activation and translocation from the cytosol to the
nucleus and its DNA-binding ability. After lysis of cells in
hypotonic lysis buffer (see section), nuclei were pelleted by
centrifugation (13000 g for 5 minutes at 4.degree. C.), washed once
in hypotonic lysis buffer to remove contaminating cytosolic
proteins, and then resuspended in hypertonic extraction buffer for
1-2 hours at 4.degree. C. under agitation. Hypotonic lysis buffer
prevents leaching of proteins out of the nucleus during lysis,
whereas hypertonic extraction buffer makes the nuclear membrane
porous, allowing nuclear proteins to escape into solution. After
centrifugation (13000 g for 10 minutes at 4.degree. C.)
supernatants containing the nuclear protein were removed to fresh
tubes and stored at -70.degree. C. This method of nuclear extracts
preparation is based on that of Whiteside S. T. et al (1992).
Nucleic Acids Res 20:1531-8.
17. Immunoprecipitation
[0215] To immunoprecipitate IRAK, cytosolic extracts were incubated
with 3 .mu.g of anti-IRAK antibody for 1 h at 4.degree. C. under
gentle shaking. Then, 50 .mu.l of 50% slurry protein G sepharose
(Amersham) were added and left for another 2 h shaking.
Subsequently, IRAK bound to protein G sepharose was collected,
washed four times, resuspended in Western Blot loading buffer,
boiled for 5 min and then immediately used for Western
blotting.
18. Protein Concentration Assay
[0216] Before using cytosolic or nuclear extracts in any further
experimental procedure (e.g. western blotting, electrophoretic
mobility shift assays), it was necessary to determine their protein
concentration in order to ensure that equivalent amounts of protein
were present in each sample. Protein concentrations were assessed
by the Bradford assay. Briefly, 20 .mu.l of appropriately diluted
extracts were added in triplicates in a 96-well tissue culture
plate along with 20 .mu.l of a series of BSA concentrations (Sigma,
UK) ranging from 10-1000 .mu.g/ml to be used as a standard. 200
.mu.l of Bradford reagent were then added to each well, and
absorbance was measured at 595 nm in a spectrophotometer (Multiscan
Bichromatic, Labsystems). From the linear standard curve formed by
the range of BSA concentrations, protein amounts in the cytosolic
and nuclear extracts were determined.
19. Western Blotting And Electrophoretic Mobility Shift Assay
[0217] Cytosolic proteins were separated by SDS-PAGE on a 10% (w/v)
polyacrylamide gel, followed by electrotransfer onto nitrocellulose
membranes. I.kappa.B.alpha. and IRAK were detected by using
antibodies purchased from Santa Cruz Biotechnology (Santa Cruz,
USA) and Upstate Biotechnology (USA), respectively, whereas the
phosphorylated forms of I.kappa.B.alpha., p38 and p42/44 MAPK were
detected by antibodies from New England Biolabs.
RESULTS
1. PSI Inhibits the Immunostimulatory Function of DC.
[0218] PSI is an inhibitor of proteosome activity and as such is
capable of inhibiting induced I.kappa.B degradation and subsequent
NF-.kappa.B activation. We used PSI to examine the function of
NF.kappa.B in dendritic cell (DC) activity particularly in relation
to antigen presentation and activation of T cells. We have
previously shown that PSI is capable of inhibiting NF.kappa.B
activation at concentrations up to 1 .mu.M. To test the effect of
PSI on DC viability mature DC were incubated with concentrations of
PSI, ranging from 0.1 .mu.M to 5 .mu.M. Concentrations of >5
.mu.M showed some toxicity (FIG. 1), as judged by propidium iodide
(PI) staining method and so functional studies were only performed
over the 0.1 to 2 .mu.M range, using 1 .mu.M in most experiments.
To assess the role of NF.kappa.B in DC function, mature DC were
incubated with 0.3 .mu.M, 0.5 .mu.M or 1 .mu.M PSI for 3 hours and
the cells washed. As PSI is an activated peptide that binds
irreversibly to the proteosomes, the function of the DC can be
assessed over the 6 day period of the MLR despite the short period
of drug exposure.
[0219] Exposure of T cells to PSI treated DC (PSI-DC) in an
allogeneic MLR assay had profound effect on the response of the
lymphocytes that was titratable with PSI concentration. It was
found that while pretreatment of DCs with PSI at 0.1 .mu.M had no
detectable effect, 0.5 .mu.M and 1 .mu.M resulted in a failure of
the T cells to proliferate in response to activation (FIG. 2).
2. PSI Reduces the Surface Expression of Molecules Involved in
Antigen Presentation.
[0220] T cells in the MLR recognise HLA class II antigens, of which
HLA-DR is the most abundant and so its expression was assessed, 6
days after PSI treatment. A minimal effect (by FACS) on DR was
noted at 0.1.mu. PSI, but a major reduction was found at 1 .mu.M
(FIG. 3). CD86 is the major costimulatory molecule expressed on DC,
and its expression was also reduced, to virtually background levels
at 1 .mu.M PSI (FIG. 3).
3. Inhibited MLR with PSI Treated Dendritic Cells is not Rescued by
IL-2 or anti CD28 Antibody.
[0221] There are two broad possibilities as to the mechanism of the
low proliferative response of T cells to PSI-DC. One is that these
DC are not stimulatory, and the T cells are not responding for that
reason. The second is that the T cells have been actively `switched
off`, by processes of tolerance or active immune regulation. To
begin to discriminate between these possibilities, we attempted to
stimulate the T cells with IL-2, or anti CD28 at concentrations
(2-ng/ml. IL-2 or 10 .mu.g/ml. anti CD28).
[0222] The IL-2 or anti CD28 was added at the same time when T
cells and DC were plated. Proliferation was assessed at day 6, and
revealed that IL-2 had a very slight effect at restoring
proliferation, however this was independent of the number of DC and
regardless of PSI treatment (FIG. 4a).
[0223] The same degree of immunostimulation was seen with untreated
DC (FIG. 4b). Anti CD28 had no restorative effect, which had been
expected in view of the downregulation of the major CD28 ligand
CD86 by PSI (FIG. 3). These results are consistent with multiple
mechanisms and so led to further experiments.
4. Inhibition of the DC Induced MLR by DC Pretreated with PSI.
[0224] A means of detecting whether PSI-DC were generating
inhibitory effects is to culture T cells with a mixture of PSI
treated and untreated DC. If the PSI-DC were just non-stimulatory,
PSI treated DC should have minimal effects on the proliferative
response. However, it was found that the presence of as little as
1/8 or 1/4 PSI-DC, resulted in a 3-5 fold reduction response, as
judged from the slope of the proliferative response curves (FIG.
5). Next we investigated whether exposure to PSI-DC would induce
long term unresponsiveness in the T cells. T cells were exposed to
normal or PSI-DC as previously described after which the T cells
were collected and were treated with a DC for a second time and the
proliferative response measured (FIG. 6). The T cells exposed to
normal DC on both occasions responded and as expected T cells did
not respond to two rounds of PSI-DC. However, T cells that had been
stimulated with PSI-DC prior to normal DC also were unable to
respond (FIG. 6). This would indicate that the effects of exposure
to PSI-DC are prolonged and the T cells acquire a non-responsive or
anergic state. As expected T cells exposed to PSI-DC on the second
round after normal DC also failed to proliferate.
5. Cell Surface Antigen Analysis of T Lymphocytes Exposed to PSI
Treated Dendritic Cells.
[0225] To probe into the mechanism of the effect of PSI treated DC,
we analysed T lymphocyte after 6 days cultures. As anticipated the
yields of T cells were lower. Cell surface expression of CD3 was
reduced. Most dramatic, however, was the marked diminution of CD25
expression, which was virtually abolished compared to controls
(FIG. 7).
[0226] Expression of molecules involved in cell surface adhesion,
such as CD11a (LFA-1) and one of its receptors ICAM-1 CD54 was
analysed. Both were reduced on PSI treated lymphocytes with CD54
reduced to a degree similar to CD3 whereas CD11a expression was
only marginally reduced (FIG. 7).
6. Exposure to T Cells to PSI-DC Results in the Production of IL-4
But not IL-2 or IFN.gamma.
[0227] The production of cytokines is one of the key responses by T
cells to stimulation by DC with IL-2, IFN.gamma. and IL-4 being
expressed (FIG. 8). We therefore investigated what type of response
occurs with exposure to PSI-DC. IL-2 and the key Th1 cytokine,
IFN.gamma. were not produced in response to PSI-DC. However, the
production of IL-4 was unaffected. This data would suggest that
PSI-DC are capable of delivery some signals to T cells. This would
support our prior observation that PSI-DC induce a type of
unresponsiveness state in the T cells which is thought to require
an active signal.
7. DC Production of TNF but not IL-8 is Inhibited by PSI.
[0228] We also investigated the effect of PSI on cytokine produced
by the DC following on MLR. TNF and, to a lesser extent, IL-6
expression was inhibited from PSI-DC during the MLR. In contrast
IL-8 production was unaffected (FIG. 9). Like results on the T cell
this shows the discriminatory nature of PSI treatment, and
indicates that NF.kappa.B has a role in TNF and IL-6 expression but
not that of IL-8.
8. Activating DC with MEKK1 Results in Enhanced MLR Response.
[0229] Since inhibition of NF.kappa.B inhibits the function of DC,
the effect of potentially activating DC using NF.kappa.B
stimulating MEKK was investigated. Activating encoding MEKK1 was
used to infect DC at m.o.i. of 150:1. When used in an MLR such
MEKK1 DC was 4-fold more active than normal DC (FIG. 10).
9. Myd88lpr Activates NF-.kappa.B
[0230] We have found that a potent inducer of NF-.kappa.B is a
mutein of Myd88 that contains a deletion of the first 53 amino
acids and a point mutation PheS6Asn termed Myd88lpr. Myd88lpr is
normally inhibiting to Toll related receptor signalling, for
example, IL-1 receptors (1). However, we have observed that
introduction of Myd88lpr, by adenoviral vectors, into immature DC
induces a potent activation of nuclear NF-.kappa.B activity that is
detectable six hours after infection with Admyd88 and more
strongly, at 24 hours post-infection (FIG. 11).
10. Myd88lpr Induces TNF Production by Immature DC
[0231] As with MEKK1, we were interested to see if Myd88lpr would
also activate DC function. Infection of immature DC with
AdMyd88lpr, resulted in the spontaneous production of TNF by the
cells 24 hours post infection. Moreover, Myd88lpr had no inhibiting
effect on LPS-induced TNF production. Myd88 wt did not induce TNF
production in DC (FIG. 12)
11. Myd88lpr Enhances Antigen Presentation by DC
[0232] Since inhibition of NF-.kappa.B inhibits the function of DC,
the effect of potently activating DC using the NF-.kappa.B
stimulating Myd88lpr was investigated.
[0233] Immature DC were infected with AdMyd88lpr (that also
expresses GFP), Ad0 or AdGFP (Green Fluorescent Protein). After 24
hours, various concentrations of infected DC were cultured with
2.times.10.sup.4 GFP antigen-specific T cells and response measured
as proliferation of the T cells at three days. Delivery of GFP to
dendritic cells induced an antigen specific T cell proliferation
that was enhanced by Myd88lpr (FIG. 14). This result is in
agreement with activation of NF-.kappa.B and TNF production by
Myd88lpr shown in the previous figures (FIGS. 11 and 12).
12. Myd88lpr also Enhances DC Induced Allogenic Mixed Lymphocyte
Reaction (MLR)
[0234] Another test of the effect of activity NF-.kappa.B function
on DC was to investigate the MLR response. Immature DC were
infected with Ad0 and Admyd88lpr and, after 48 hours, graded doses
of the infected DC were cultured with 105 allogenic T cells and
responses measured as proliferation after six days of culture.
Infection with AdMyd88lpr enhanced the MLR response over that of
Ad0 or uninfected cells. This was most notable at lower DC numbers
where the response of the controls was only equal to T cells only,
where the Myd88lpr expressing DC response was higher (FIG. 15).
[0235] 13. Myd88lpr Expression Enhances the Expression of
Costimulating Molecules CD80 and CD86.
[0236] The expression of the cell surface molecules CD80 and CD86
are important to the antigen presenting function of DC. In FIG. 16,
we show that expression of Myd88lpr in DC (by adenoviral infection)
enhanced the expression of both CD80 and CD86, when compared with
uninfected DC's, or cells infected with control virus.
14. Myd88lpr Also Activates Other Signalling Molecules in Addition
to NF-.kappa.B.
[0237] We have shown that Myd88lpr is a potent inducer of
NF-.kappa.B. We also observed that this molecule could activate
other signalling pathways, this time in macrophages, such as p38
MAPK, a kinase known to be involved in cell mechanisms such as the
control of cytokine expression (FIG. 17). Moreover, in macrophages,
Myd88lpr also activated IRAK1, a key element of TLR and IL-IR
signalling mechanisms (FIG. 18).
REFERENCES
[0238] 1. Burns, K., F. Martinon, C. Esslinger, H. Pahl, P.
Schneider, J. L. Bodmer, F. Di Marco, L. French, and J. Tschopp.
1998. MyD88, an adapter protein involved in interleukin-1
signaling. J Biol Chem 273, no. 20:12203.
DISCUSSION
[0239] Treatment of DC with PSI, a proteasome inhibitor, at
concentrations capable of blocking the induction of NF.kappa.B, was
shown to reduce the proliferative response in the MLR. This
suggests that NF.kappa.B function is involved in the stimulation of
unprimed T cells in the MLR. As the specificity of PSI is for the
proteasome, it hence could potentially affect the function of other
transcription factors, although this was not reported in the
original publications describing the action of this drug
(Traenckner et al (1994) EMBO J. 13, 5433-5441; Traenckner &
Baeuerle (1995) J. Cell Sci. Suppl. 19, 79-84; and Haas et al
(1998) J. Leukoc. Biol. 63, 395-404). Other, independent
experiments to verify the role of NF.kappa.B using other approaches
will be needed, and we have initiated studies using infection of DC
with an adenovirus overexpressing I.kappa.B.alpha. under the
control of the CMV promoter. This I.kappa.B.alpha. overexpression
inhibits NF.kappa.B, and also inhibits the antigen presenting
function of dendritic cells (Yoshimura et al., unpublished data),
compatible with the data presented here.
[0240] The mechanism of PSI treated DC reduced immunogenicity was
evaluated at different levels, and it turned out that multiple
aspects of antigen presenting cell function in DC were
downregulated. First, the effects on the expression of cell surface
molecules by which DC stimulate T cells has been studied, and a
marked reduction in the expression of HLA-DR and CD86, both
molecules important for both antigen recognition and CD28
activation, was found. Reductions in DC derived cytokines such as
IL-12 and TNF.alpha., both known to be important in the early
activation of T cells, were also found. In contrast the production
of other cytokines such as IL-6 or IL-8 was not changed in T
cell/DC cocultures. These results indicate that PSI treated DC have
reduced expression of all three major classes of molecules involved
in antigen presentation--the target of T cell recognition (HLA-DR)
the costimulatory molecules (CD86), as well as immunostimulatory
cytokines (IL-12 and TNF.alpha.).
[0241] Further questions as to the mechanism of the lack of the T
cell proliferative response were addressed. It could simply be due
to lack of immunogenicity of the DC, or alternatively it could be
due to the induction of a form of immunological tolerance or of
immune regulation. The mechanism was analysed further by coculture
experiments, adding PSI treated DC to untreated DC, and analysing
the effect of the mixture on unprimed T cells in the MLR. It was
found that if as few as 1/8 (12.5%) PSI treated DC are added to
untreated DC there is a significant reduction in the proliferation
dose response curve, equivalent to 3-5 fold less active DC. This
result, as well as the fact that IL-2 and anti CD28 failed to
stimulate these T cells (data not shown), indicates that there is
an inhibitory (immunomodulatory) effect of PSI treated DC on T cell
function. Furthermore, exposure of T cells to PSI pretreated DC
induced profound changes in the expression of T cell surface
markers, analyzed on day 6 of coculture: there was reduced
expression of CD3, virtually abolished CD25, and some reduction in
ICAM-1 and LFA-1. Similar effects have been reported in a variety
of tolerance models Zanders et al (1983) Nature 303, 625-627;
Zanders et al (1985) J. Immunol. 15, 302-305; Park et al (1997)
Eur. J. Immunol. 15, 302-305; and Waldmann & Cubbold (1998)
Ann. Rev. Immunol. 16, 619-644 and so we asked whether PSI treated
DC induced immunological tolerance. This was assessed by first
exposing allogeneic T cells to PSI treated DC for 2 days, and then
removing them, by panning with anti CD83 and anti CD86. The T cells
were then exposed for 4 days to normal DC/or PSI treated DC from
the same donor, and their lack of response is evidence that at
least `long lasting` (4-6 day) immunological tolerance has been
induced. The formal definition of tolerance includes `antigen
specificity`, that could not be formally tested in this kind of
experiment, but it has later been verified in an independent series
of experiments using T cell lines responsive to a soluble antigen,
tetanus toxoid (Calder et al., unpublished data). However, the term
`immunological tolerance` in this context is an appropriate one,
since rechallenge was performed with DC from the same donor.
[0242] In another set of experiments, the function of the T cells
exposed to PSI treated DC was evaluated further. The T cell
cytokine production was markedly altered. Thus IL-2 production was
inhibited by more than 90%, as was IFN.gamma. production, findings
that are also compatible with the induction of tolerance. IL-2 and
IFN.gamma. are `Th1 ` cytokines, as it was of interest that the
expression of the `Th2` cytokine, IL-4 was not changed. This
suggests that the immunosuppressive effect/tolerance induction
induced by PSI treated DC may be restricted to the Th1 subset.
Further studies with purified Th1 and Th2 T cells are needed to
address this point.
[0243] The studies reported here, and other experiments using an
adenovirus overexpressing the endogenous NF.kappa.B inhibitor
I.kappa.B.alpha. (Yoshimura et al., unpublished data), indicate
that NF.kappa.B has an important role in the regulation of antigen
presentation. This agrees well with the earlier finding that
NF.kappa.B is essential for DC maturation Rescizno et al (1998) J.
Exp. Med 188, 2175-2180. It is also consistent with the concept
that NF.kappa.B is the major mechanism by which innate immunity is
translated into adaptive immunity. The so called `danger signal`,
which activates the immune system Matzinger (1994) Ann. Rev.
Immunol 12, 991-1045; Janeway et al (1196) Curr. Biol. 6, 519-522,
activated via a wide variety of microbial or other noxious agents
thus appears to involve the activation of NF.kappa.B. This has
already been established for LPS that requires TLR4 to induce
NF.kappa.B activation Chow et al (1999) J. Biol. Chem. 274,
10689-10692.
[0244] In the context of regulation of antigen presentation it is
noteworthy that all 3 aspects, expression of target for T cells
(MHC), costimulatory molecules (CD86) as well as inducing cytokines
(IL-12, TNF.alpha.) are all regulated coordinately by NF.kappa.B.
This leads to an obvious prediction, that drugs that block
NF.kappa.B (such as PSI) may be useful immunosuppressive agents in
vivo, depending clearly on their toxicity profile. PSI is likely to
be too toxic for systemic use might nevertheless be useful for
perfusing target organs, such as kidneys, to block APC function
prior to transplantation. An interesting corollary of this work is
that the deliberate activation of NF.kappa.B might provide a good
strategy for a useful adjuvant effect for vaccines. Experiments to
test that hypothesis are under way.
[0245] Further studies are underway to further analyse the exact
mechanism by which PSI treated DC influence T cell function:
1. Blocking NF-.kappa.B in antigen presenting cells is inhibitory
to T cell function, using PSI or other proteosome inhibitors, e.g.
or other inhibitors of NF-.kappa.B, e.g. cDNA inhibitors such as
I.kappa.B, antisense to NF-.kappa.B constitutes drug inhibitors
such as
2. Blocking NF-.kappa.B in DC induces tolerance in T cells
3. Use of NF-.kappa.B inhibitors in) autoimmunity
[0246] ) transplantation [0247] ) allergy 4. Corollary--if
NF-.kappa.B inhibition blocks antigen presentation and promotes
tolerance, then NF-.kappa.B stimulation will upregulate antigen
presentation. This can be achieved in a number of possible ways,
activating pathways which stimulate NF-.kappa.B, in order to
augment vaccination. These include using adenoviruses or other gene
transfer of MEKK1, NIK, IKK2, dominant negative mutants of MyD88,
TRAF2, TRAF6. These sequences could be incorporated into the same
genetic construct as that encoding the antigen to which
immunisation is desired. NF-.kappa.B stimulation may also be useful
in modulating the immune response in an allergic patient to alter
the TH1:TH2 response balance towards a TH1 response.
[0248] We believe that the results show that PSI produces an
anergic state in DCs. That is the drug induces a long term state of
non-responsiveness of the DCs to antigens.
[0249] The corollary to the above observations is that by
activating an intracellular signalling pathway, such as
NF-.kappa.B, in DC, one would activate the cell and enhance antigen
presenting function. We tested this using NF-.kappa.B activating
intracellular signalling molecules MEKK1 and Myd88lpr. MEKK1 has
been previously described as an activator of NF-.kappa.B amongst
other pathways and the introduction of MEKK1 into DC using
adenoviral vectors induced activation of DC function as measured by
MLR.
[0250] We also observed that a mutein of Myd88, Myd88lpr, normally
an inhibitor of signalling by TLR and IL-1 receptors, also
activated NF-.kappa.B in DC. When expressed in DC, Myd88lpr
enhanced DC antigen present function as measured by MLR, or using
antigen specific T cells as well as inducing cytokine production.
These results open the possibility that when associated with
antigens, intracellular signalling molecules, such as NF-.kappa.B,
can act as powerful adjuvants. This could provide a new approach to
vaccine design. The fact that Myd88lpr also activates p38 MAPK
would imply that other signalling molecules that activate DC could
also be used for this purpose.
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