U.S. patent application number 10/394092 was filed with the patent office on 2003-12-18 for immunostimulatory polynucleotide/immunomodulatory molecule conjugates.
Invention is credited to Carson, Dennis A., Raz, Eyal, Roman, Mark.
Application Number | 20030232780 10/394092 |
Document ID | / |
Family ID | 21841670 |
Filed Date | 2003-12-18 |
United States Patent
Application |
20030232780 |
Kind Code |
A1 |
Carson, Dennis A. ; et
al. |
December 18, 2003 |
Immunostimulatory polynucleotide/immunomodulatory molecule
conjugates
Abstract
Immunostimulatory polynucleotide-immunomodulatory molecule
conjugate compositions are disclosed, These compositions include a
polynucleotide that is linked to an immunomodulatory molecule,
which molecule comprises an antigen and may further comprise
immunomodulators such as cytokines and adjuvants. The
polynucleotide portion of the conjugate includes at least one
immunostimulatory oligonucleotide nucleotide sequence (ISS).
Methods of modulating an immune response upon administration of the
polynucleotide-immunomodulatory conjugate preparation to a
vertebrate host are also disclosed.
Inventors: |
Carson, Dennis A.; (Del Mar,
CA) ; Raz, Eyal; (Del Mar, CA) ; Roman,
Mark; (San Diego, CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
200 MIDDLEFIELD RD
SUITE 200
MENLO PARK
CA
94025
US
|
Family ID: |
21841670 |
Appl. No.: |
10/394092 |
Filed: |
March 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10394092 |
Mar 20, 2003 |
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09308036 |
Feb 16, 2000 |
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6610661 |
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09308036 |
Feb 16, 2000 |
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PCT/US97/19004 |
Oct 9, 1997 |
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60028118 |
Oct 11, 1996 |
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Current U.S.
Class: |
514/44R ;
514/20.9; 514/54 |
Current CPC
Class: |
A61K 47/54 20170801;
A61K 2039/55561 20130101; A61K 39/385 20130101; A61K 38/00
20130101; A61K 2039/57 20130101; C12N 15/117 20130101; C12N
2310/315 20130101; A61K 39/36 20130101; A61K 2039/6025 20130101;
Y02A 50/30 20180101; A61K 2039/55516 20130101; C12N 2310/3513
20130101; A61K 2039/55522 20130101; C12N 2310/313 20130101; A61P
37/06 20180101; C12N 2310/17 20130101; Y02A 50/466 20180101; A61P
37/02 20180101 |
Class at
Publication: |
514/44 ; 514/8;
514/54 |
International
Class: |
A61K 048/00 |
Goverment Interests
[0002] Support for the research disclosed herein may have been
provided by the National Institutes of Health under Grant Nos.
AI37305 and/or AR25443.
Claims
The invention claimed is:
1. An immunomodulatory composition comprising an immunomodulatory
molecule, which molecule comprises an antigen, conjugated to an
polynucleotide that contains at least one immunostimulatory
nucleotide sequence (ISS).
2. The composition of claim 1, wherein the antigen is selected from
the group consisting of proteins, glycoproteins, polysaccharides
and gangliosides.
3. The composition of claim 2, wherein the ISS comprises a
nucleotide sequence selected from the group CpG, p(GC) and
p(IC).
4. The composition of claim 2, wherein the ISS comprises a CG
containing oligonucleotide.
5. The composition of claim 4, wherein the ISS further comprises a
pG nucleotide sequence.
6. The composition of claim 4, wherein the CG containing
oligonucleotide has the sequence 5'-Purine, Purine, CG, Pyrimidine,
Pyrimidine-3'.
7. The composition of claim 3, wherein the CpG, p(GC) or p(IC)
containing nucleotide sequence is a palindromic double-stranded or
non-palindromic single-stranded oligonucleotide.
8. The composition of claim 6, wherein the oligonucleotide sequence
is selected from the group consisting of AACGTT, AGCGTT, GACGTT,
GGCGTT, AACGTC, AGCGTC, GACGTC, GGCGTC, AACGCC, AGCGCC, GACGCC,
GGCGCC, AACGCT, AGCGCT, GACGCT, AND GGCGCT:
9. The composition of claim 6, wherein the oligonucleotide sequence
is selected from the group consisting of AACGTT, AGCGTT, GACGTT,
GGCGTT, AACGTC, and AGCGTC.
10. The composition of claim 6, wherein the oligonucleotide
sequence is selected from the group consisting of AACGTT, AGCGTT,
and GACGTT.
11. The composition of claim 2, wherein the polynucleotide further
comprises a linear DNA sequence.
12. The composition of claim 2, wherein the polynucleotide further
comprises a circular DNA sequence.
13. The composition of claim 2, wherein the polynucleotide further
comprises an RNA nucleotide sequence.
14. The composition of claim 13, wherein the RNA nucleotide
sequence comprises a sequence selected from the group consisting of
AACGUU, AACGpI, AACGpC, AGCGUC, AGCGpI, AGCGpC, GACGCU, GACGCpI,
GACGCpC, GACGUU, GACGpI, GACGpC, GACGUC, GACGpI, GACGpC.
15. The composition of claim 13, wherein the RNA nucleotide
sequence comprises a double-stranded poly(I.cndot.C) sequence.
16. The composition of claim 13, wherein the RNA nucleotide
sequence comprises a sequence selected from the group consisting of
AACGUU, AACGpI, AACGpC, AGCGUC, AGCGpI, AGCGpC.
17. The composition of claim 13, wherein the RNA nucleotide
sequence comprises a sequence selected from the group consisting of
AACGUU, AACGpI, AACGpC.
18. The composition of claim 2, wherein the polynucleotide further
comprises at least one modified oligonucleotide.
19. The composition of claim 11, wherein the ISS is contained
within the linear DNA sequence, and further wherein the ISS
comprises a Purine, Purine, CG, Pyrimidine, Pyrimidine nucleotide
sequence.
20. The composition of claim 11, wherein the ISS is contained
within the linear DNA sequence, and further wherein the ISS
comprises a CG containing pG nucleotide sequence.
21. The composition of claim 12, wherein the ISS is contained
within the circular DNA nucleotide sequence, and further wherein
the ISS comprises a Purine, Purine, CG, Pyrimidine, Pyrimidine
nucleotide sequence.
22. The composition of claim 12, wherein the ISS is contained
within the circular DNA nucleotide sequence, and further wherein
the ISS comprises a CG containing pG nucleotide sequence.
23. The composition of claim 13, wherein the ISS is contained
within the RNA nucleotide sequence, and further wherein the ISS
comprises a Purine, Purine, CG, Pyrimidine, Pyrimidine nucleotide
sequence.
24. The composition of claim 13, wherein the ISS is contained with
the RNA nucleotide sequence, and further wherein the ISS comprises
CG containing pG nucleotide sequence.
25. The composition of claim 4, wherein the CG containing
nucleotide sequence further comprises a modified
oligonucleotide.
26. The composition of claim 6, wherein the 5'-Purine, Purine, CG,
Pyrimidine, Pyrimidine-3' nucleotide sequence further comprises a
modified oligonucleotide.
27. An immunomodulatory composition comprising an immunomodulatory
molecule, which molecule comprises an antigen and an
immunostimulatory peptide, conjugated to an polynucleotide that
contains at least one ISS.
28. The composition of claim 27, wherein the polynucleotide is DNA
or RNA.
29. The composition of claim 27, wherein the immunostimulatory
peptide is selected from the group consisting of co-stimulatory
molecules, cytokines, chemokines, targeting protein ligands, and
trans-activating factors.
30. The composition of claim 27, wherein the ISS comprises a DNA or
RNA nucleotide sequence selected from the group CG, p(GC) and
p(IC).
31. The composition of claim 27, wherein the ISS comprises a CG
containing oligonucleotide.
32. The composition of claim 31, wherein the ISS further comprises
a pG nucleotide sequence.
33. The composition of claim 31, wherein the CG containing
nucleotide sequence is the nucleotide sequence 5'-Purine, Purine,
CG, Pyrimidine, Pyrimidine-3'.
34. The composition of claim 31, wherein the CG containing
nucleotide sequence is a palindromic double-stranded or
non-palindromic single-stranded oligonucleotide.
35. The composition of claim 33, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, GACGTT,
GGCGTT, AACGTC, AGCGTC, GACGTC, GGCGTC, AACGCC, AGCGCC, GACGCC,
GGCGCC, AACGCT, AGCGCT, GACGCT, and GGCGCT.
36. The composition of claim 33, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, GACGTT,
GGCGTT, AACGTC, and AGCGTC.
37. The composition of claim 33, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, and
GACGTT.
38. The composition of claim 29, wherein the polynucleotide further
comprises a linear DNA nucleotide sequence.
39. The composition of claim 29, wherein the polynucleotide further
comprises a circular DNA nucleotide sequence.
40. The composition of claim 29, wherein the polynucleotide portion
further comprises an RNA nucleotide sequence.
41. The composition of claim 40, wherein the RNA nucleotide
sequence comprises a nucleotide sequence selected from the group
consisting of AACGUU, AACGpI, AACGpC, AGCGUC, AGCGpI, AGCGpC,
GACGCU, GACGCpI, GACGCpC, GACGUU, GACGpI, GACGpC, GACGUC, GACGpI,
GACGpC.
42. The composition of claim 40, wherein the RNA nucleotide
sequence comprises a double-stranded poly(I-C) nucleotide
sequence.
43. The composition of claim 40, wherein the RNA nucleotide
sequence comprises a nucleotide sequence selected from the group
consisting of AACGUU, AACGpI, AACGpC, AGCGUC, AGCGpI, AGCGpC.
44. The composition of claim 40, wherein the RNA nucleotide
sequence comprises a nucleotide sequence selected from the group
consisting of AACGUU, AACGpI, AACGpC.
45. The composition of claim 29, wherein the polynucleotide portion
further comprises at least one modified oligonucleotide.
46. The composition of claim 38, wherein the ISS is contained
within the linear DNA nucleotide sequence, and further wherein the
ISS comprises a Purine, Purine, CG, Pyrimidine, Pyrimidine
nucleotide sequence.
47. The composition of claim 38, wherein the ISS is contained
within the linear DNA nucleotide sequence, and further wherein the
ISS comprises a CG containing pG nucleotide sequence.
48. The composition of claim 39, wherein the ISS is contained
within the circular DNA nucleotide sequence, and further wherein
the ISS comprises a Purine, Purine, CG, Pyrimidine, Pyrimidine
nucleotide sequence.
49. The composition of claim 39, wherein the ISS is contained
within the circular DNA nucleotide sequence, and further wherein
the ISS comprises a CG containing pG nucleotide sequence.
50. The composition of claim 40, wherein the ISS is contained
within the RNA nucleotide sequence, and further wherein the ISS
comprises a Purine, Purine, CG, Pyrimidine, Pyrimidine nucleotide
sequence.
51. The composition of claim 40, wherein the ISS is contained with
the RNA nucleotide sequence, and further wherein the ISS comprises
CG containing pG nucleotide sequence.
52. The composition of claim 31, wherein the CG containing
nucleotide sequence further comprises a modified
oligonucleotide.
53. The composition of claim 33, wherein the 5'-Purine, Purine, CG,
Pyrimidine, Pyrimidine-3' nucleotide sequence further comprises a
modified oligonucleotide.
54. A method of modulating an immune response comprising the
administration of an immunomodulatory composition, comprising an
immunomodulatory molecule, which molecule comprises an antigen,
conjugated to an polynucleotide that contains at least one ISS.
55. The method of claim 54, wherein the route of administration is
a dermal route.
56. The method of claim 54, wherein the route of administration is
low-frequency ultrasonic delivery.
57. The method of claim 54, wherein the antigen is selected from
the group consisting of proteins, glycoproteins, polysaccharides
and gangliosides.
58. The method of claim 57, wherein the ISS comprises a DNA or RNA
nucleotide sequence selected from the group CG, p(GC) and
p(IC).
59. The method of claim 57, wherein the ISS comprises a CG
containing oligonucleotide.
60. The method of claim 59, wherein the ISS further comprises a pG
nucleotide sequence.
61. The method of claim 59, wherein the CG containing nucleotide
sequence is the nucleotide sequence 5'-Purine, Purine, CG,
Pyrimidine, Pyrimidine-3'.
62. The method of claim 59, wherein the CG containing nucleotide
sequence is a palindromic or non-palindromic oligonucleotide
nucleotide sequence.
63. The method of claim 59, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, GACGTT,
GGCGTT, AACGTC, AGCGTC, GACGTC, GGCGTC, AACGCC, AGCGCC, GACGCC,
GGCGCC, AACGCT, AGCGCT, GACGCT, and GGCGCT.
64. The method of claim 59, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, GACGTT,
GGCGTT, AACGTC, and AGCGTC.
65. The method of claim 59, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, and
GACGTT.
66. The method of claim 54, wherein the immune response modulation
comprises the induction of a Th1 response.
67. The method of claim 66, wherein the antigen molecule is
selected from the group consisting of proteins, glycoproteins and
polysaccharides.
68. The method of claim 67, wherein the ISS comprises a DNA or RNA
nucleotide sequence selected from the group CG, p(GC) and
p(IC).
69. The method of claim 67, wherein the ISS comprises a CG
containing oligonucleotide.
70. The method of claim 69, wherein the ISS further comprises a pG
nucleotide sequence.
71. The method of claim 69, wherein the CG containing nucleotide
sequence is the nucleotide sequence 5'-Purine, Purine, CG,
Pyrimidine, Pyrimidine-3'.
72. The method of claim 69, wherein the CG containing nucleotide
sequence is a double-stranded palindromic or single-stranded
non-palindromic oligonucleotide nucleotide sequence.
73. The method of claim 69, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, GACGTT,
GGCGTT, AACGTC, AGCGTC, GACGTC, GGCGTC, AACGCC, AGCGCC, GACGCC,
GGCGCC, AACGCT, AGCGCT, GACGCT, and GGCGCT.
74. The method of claim 69, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, GACGTT,
GGCGTT, AACGTC, and AGCGTC.
75. The method of claim 69, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, and
GACGTT.
76. A method of modulating an immune response comprising the
administration of an immunomodulatory composition comprising an
immunomodulatory molecule, which molecule is comprised of an
antigen and an immunostimulatory peptide, conjugated to an
polynucleotide that contains at least one ISS.
77. The method of claim 76, wherein the route of administration is
a dermal route.
78. The method of claim 76, wherein the route of administration is
low-frequency ultrasonic delivery.
79. The method of claim 76, wherein the immunostimulatory peptide
is selected from the group consisting of co-stimulatory molecules,
cytokines, chemokines, targeting protein ligands, and
trans-activating factors.
80. The method of claim 79, wherein the ISS comprises a nucleotide
sequence selected from the group CG, p(GC) and p(IC).
81. The method of claim 79, wherein the ISS comprises a CG
containing oligonucleotide.
82. The method of claim 81, wherein the ISS further comprises a pG
nucleotide sequence.
83. The method of claim 81, wherein the CG containing nucleotide
sequence is the nucleotide sequence 5'-Purine, Purine, CG,
Pyrimidine, Pyrimidine-3'.
84. The method of claim 81, wherein the CG containing nucleotide
sequence is a double-stranded palindromic or single-stranded
non-palindromic oligonucleotide nucleotide sequence.
85. The method of claim 81, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, GACGTT,
GGCGTT, AACGTC, AGCGTC, GACGTC, GGCGTC, AACGCC, AGCGCC, GACGCC,
GGCGCC, AACGCT, AGCGCT, GACGCT, and GGCGCT.
86. The method of claim 81, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, GACGTT,
GGCGTT, AACGTC, and AGCGTC.
87. The method of claim 81, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, and
GACGTT.
88. The method of claim 76, wherein the immune response modulation
comprises the induction of a Th1 response.
89. The method of claim 88, wherein the antigen is selected from
the group consisting of proteins, glycoproteins and
polysaccharides.
90. The method of claim 89, wherein the ISS comprises a nucleotide
sequence selected from the group CG, p(GC) and p(IC).
91. The method of claim 89, wherein the ISS comprises a CG
containing oligonucleotide.
92. The method of claim 91, wherein the ISS further comprises a pG
nucleotide sequence.
93. The method of claim 91, wherein the CG containing nucleotide
sequence is the nucleotide sequence 5'-Purine, Purine, CG,
Pyrimidine, Pyrimidine-3'.
94. The method of claim 91, wherein the CG containing nucleotide
sequence is a double-stranded palindromic or single-stranded
non-palindromic oligonucleotide nucleotide sequence.
95. The method of claim 91, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, GACGTT,
GGCGTT, AACGTC, AGCGTC, GACGTC, GGCGTC, AACGCC, AGCGCC, GACGCC,
GGCGCC, AACGCT, AGCGCT, GACGCT, and GGCGCT.
96. The method of claim 91, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, GACGTT,
GGCGTT, AACGTC, and AGCGTC.
97. The method of claim 91, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, and
GACGTT.
98. A method for introducing a soluble antigen into the Class I MHC
processing pathway of the mammalian immune system to elicit a CTL
response to the antigen comprising administering a polynucleotide
conjugated to an immunomodulatory molecule, which molecule
comprises the antigen, to a mammalian host.
99. The method of claim 98 wherein the polynucleotide includes at
least one ISS.
100. The method of claim 98 wherein the polynucleotide is free of
ISS.
101. The method of claim 98, wherein the antigen is selected from
the group consisting of proteins, glycoproteins and
polysaccharides.
102. The method of claim 98, wherein the ISS comprises a nucleotide
sequence selected from the group CG, p(GC) and p(IC).
103. The method of claim 98, wherein the ISS comprises a CG
containing oligonucleotide.
104. The method of claim 103, wherein the ISS further comprises a
pG nucleotide sequence.
105. The method of claim 103, wherein the CG containing nucleotide
sequence is the nucleotide sequence 5'-Purine, Purine, CG,
Pyrimidine, Pyrimidine-3'.
106. The method of claim 103, wherein the CG containing nucleotide
sequence is a double-stranded palindromic or single-stranded
non-palindromic oligonucleotide nucleotide sequence.
107. The method of claim 102, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, GACGTT,
GGCGTT, AACGTC, AGCGTC, GACGTC, GGCGTC, AACGCC, AGCGCC, GACGCC,
GGCGCC, AACGCT, AGCGCT, GACGCT, and GGCGCT.
108. The method of claim 102, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, GACGTT,
GGCGTT, AACGTC, and AGCGTC.
109. The method of claim 102, wherein the nucleotide sequence is
selected from the group consisting of AACGTT, AGCGTT, and
GACGTT.
110. The method of claim 98 wherein the polynucleotide comprises a
GpG oligonucleotide.
111. The method of claim 110, wherein the nucleotide sequence is
selected from the group consisting of AAGGTT, AGGGTT, GAGGTT,
GGGGTT, AAGGTC, AGGGTC, GAGGTC, GGGGTC, AAGGCC, AGGGCC, GAGGCC,
GGGGCC, AAGGCT, AGGGCT, GAGGCT, and GGGGCT.
112. The composition of claim 110, wherein the nucleotide sequence
is selected from the group consisting of AAGGTT, AGGGTT, GAGGTT,
GGGGTT, AAGGTC, and AGGGTC.
113. The composition of claim 110, wherein the nucleotide sequence
is selected from the group consisting of AAGGTT, AGGGTT, and
GAGGTT.
114. A composition for introducing a soluble antigen into the Class
I MHC processing pathway of the mammalian immune system to elicit a
CTL response to the antigen comprising a polynucleotide conjugated
to an immunomodulatory molecule, which molecule comprises the
antigen.
115. The composition of claim 114, wherein the antigen is selected
from the group consisting of proteins, glycoproteins and
polysaccharides.
116. The composition of claim 114 wherein the polynucleotide
comprises a GpG oligonucleotide.
117. The composition of claim 116, wherein the nucleotide sequence
is selected from the group consisting of AAGGTT, AGGGTT, GAGGTT,
GGGGTT, AAGGTC, AGGGTC, GAGGTC, GGGGTC, AAGGCC, AGGGCC, GAGGCC,
GGGGCC, AAGGCT, AGGGCT, GAGGCT, and GGGGCT.
118. The composition of claim 116, wherein the nucleotide sequence
is selected from the group consisting of AAGGTT, AGGGTT, GAGGTT,
GGGGTT, AAGGTC, and AGGGTC.
119. The composition of claim 116, wherein the nucleotide sequence
is selected from the group consisting of AAGGTT, AGGGTT, and
GAGGTT.
Description
RELATED U.S. PATENT APPLICATIONS
[0001] This is a continuation-in-part and utility conversion of
U.S. Provisional Patent Application Serial No. 60/028,118, filed
Oct. 11, 1996.
FIELD OF THE INVENTION
[0003] The invention relates to compositions comprising an
immunomodulatory molecule (IMM) including an antigen, conjugated to
a polynucleotide that contains or consists of at least one
immunostimulatory oligonucleotide (ISS-PN). It also relates to
methods for modulating the immune response of a vertebrate host to
an antigen.
HISTORY OF THE RELATED ART
[0004] Conventionally, immunization of a host against an antigen is
accomplished by repeatedly vaccinating the host with the antigen.
While most current vaccines elicit reasonable antibody responses,
cellular responses (in particular, major histocompatibility complex
(MHC) class I-restricted cytotoxic T cells) are generally absent or
weak. For many infectious diseases, such as tuberculosis and
malaria, humoral responses are of little protective value against
infection.
[0005] Given the weak cellular immune response to protein antigens,
modulation of the immune responses to these antigens has clear
importance. The ability to modify immune responses to protein or
peptide antigen has implications for tumor therapy, for the
treatment of allergic disorders and for treatment of other
conditions achievable through induction of a vigorous cellular
immune response.
SUMMARY OF THE INVENTION
[0006] The present invention provides compositions comprising an
ISS-PN which is conjugated to an IMM (which includes an antigen) to
form ISS-PN/IMM conjugates. The ISS-PN/IMM conjugates of the
invention are biological response modifiers in the sense that they
modify the humoral and cellular immune response of a host to an
antigen.
[0007] Specifically, the ISS-PN and IMM components of the
ISS-PN/IMM conjugates synergistically boost the magnitude of the
host immune response against an antigen to a level greater than the
host immune response to either the IMM, antigen or ISS-PN alone.
The ISS-PN/IMM conjugates also shift the host cellular immune
response away from the helper T lymphocyte type 2 (Th2) phenotype
toward a helper T lymphocyte type 1 (Th1) phenotype. These
responses to ISS-PN/IMM conjugates are particularly acute during
the important early phase of the host immune response to an
antigen.
[0008] To these ends, ISS-PN/IMM conjugates are delivered by any
route through which antigen-sensitized host tissues will be
contacted with the ISS-PN/IMM conjugate. ISS-PN/IMM conjugates
administered in this fashion boost both humoral (antibody) and
cellular (Th1 type) immune responses of the host. Thus, use of the
method to boost the immune responsiveness of a host to subsequent
challenge by a sensitizing antigen without immunization avoids the
risk of Th2-mediated, immunization-induced anaphylaxis by
suppressing IgE production in response to the antigen challenge. An
especially advantageous use for this aspect of the invention is
treatment of localized allergic responses in target tissues where
the allergens enter the body, such as the skin and mucosa.
[0009] Suppression of the Th2 phenotype according to the invention
is also a useful in reducing antigen-stimulated IL-4 and IL-5
production. Thus, the invention encompasses delivery of ISS-PN/IMM
conjugates to a host to suppress the Th2 phenotype associated with
conventional antigen immunization (e.g., for vaccination or allergy
immunotherapy).
[0010] The shift to a Th1 phenotype achieved according to the
invention is accompanied by increased secretion of IFN .alpha.,
.beta. and .gamma., as well as IL-12 and IL-18. Each of these
cytokines enhance the host's immune defenses against intracellular
pathogens, such as viruses. Thus, the invention encompasses
delivery of ISS-PN/IMM conjugates to a host to combat pathogenic
infection.
[0011] Angiogenesis is also enhanced in the Th1 phenotype
(ostensibly through stimulation by IL-12). Thus, the invention
encompasses delivery of ISS-PN/IMM conjugates to a host to
stimulate therapeutic angiogenesis to treat conditions in which
localized blood flow plays a significant etiological role; e.g.,
retinopathies.
[0012] The ISS-PN/IMM conjugates of the invention comprise an IMM
conjugated to a polynucleotide that includes, or consists of, at
least one immunostimulatory oligonucleotide (ISS-ODN) moiety. The
ISS-ODN moiety is a single- or double-stranded DNA or RNA
oligonucleotide having at least 6 nucleotide bases which may
include, or consist of, a modified oligonucleoside or a sequence of
modified nucleosides.
[0013] The ISS-ODN moieties comprise, or may be flanked by, a CpG
containing nucleotide sequence or a p(IC) nucleotide sequence,
which may be palindromic. Where the oligonucleotide moiety
comprises a CpG sequence, it may include a hexamer structure
consisting of: 5'-Purine, Purine, CG, Pyrimidine, Pyrimidine-3'.
Examples of such hexamer structures are AACGTT, AGCGTT, GACGTT,
GGCGTT, AACGTC, and AGCGTC.
[0014] In one aspect of the invention, the ISS-PN consists of an
ISS-ODN. Alternatively, the ISS-PN comprises an ISS-ODN.
[0015] Conjugates of the invention also include PN/IMM wherein the
PN serves as a carrier to introduce the IMM antigen into MHC Class
I processing pathways not normally stimulated by soluble antigen,
but lacks ISS activity and therefore does not stimulate a Th1
phenotype immune response. Examples of such PN/IMM are those
wherein the CpG motif is mutated, for example, to a GpG motif.
[0016] In one aspect of the invention, the IMM conjugate partner to
the ISS-PN consists of an antigen. Such antigens are selected from
the group of antigens consisting of proteins, peptides,
glycoproteins, polysaccharides and gangliosides.
[0017] In another aspect of the invention, the IMM conjugate
partner comprises an antigen and further comprises an
immunostimulatory molecule selected from the group of such
molecules consisting of adjuvants, hormones, growth factors,
cytokines, chemokines, targeting protein ligands, and
trans-activating factors.
[0018] In another aspect of the invention, the ISS-PN/IMM conjugate
is modified for targeted delivery by, for example, attachment to a
monoclonal antibody, receptor ligand and/or liposome.
[0019] Pharmaceutically acceptable compositions of ISS-PN/IMM
conjugates are provided for use in practicing the methods of the
invention. Where appropriate to the contemplated course of therapy,
the ISS-PN/IMM conjugates may be administered with
anti-inflammatory or immunotherapeutic agents. Thus, a particularly
useful composition for use in practicing the method of the
invention is one in which an anti-inflammatory agent (e.g., a
glucocorticoid) is mixed with, or further conjugated to, an
ISS-PN/IMM conjugate.
[0020] The ISS-PN/IMM conjugates can also be provided in the form
of a kit comprising ISS-PN/IMM conjugates and any additional
medicaments, as well as a device for delivery of the ISS-PN/IMM
conjugates to a host tissue and reagents for determining the
biological effect of the ISS-PN/IMM conjugates on a treated
host.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a graph of data demonstrating the vigorous
Th1-type immune response (as measured by production of IgG2a
against an IMM antigen) stimulated by ISS-PN/IMM (1:5 ratio) in
comparison to the levels of Th2-like responses stimulated by an ISS
containing, antigen encoding plasmid (PACB-Z); the antigen alone
(P-gal); the antigen mixed with an ISS (1:5 ratio); the antigen
conjugated to a non-stimulatory PN (mISS conj; 1:5 ratio); the
antigen in adjuvant (alum) and, for reference, the IgG2a levels in
naive (unexposed) mice. The horizontal axis represents the levels
(units/ml) of antibody; the vertical axis represents the number of
weeks following primary antigen exposure.
[0022] FIG. 2 is a graph of data demonstrating the levels of
Th2-type immune responses (as measured by production of IgG1
against an IMM antigen) stimulated by an ISS containing, antigen
encoding plasmid (pACB-Z); the antigen alone (.beta.-gal); the
antigen nixed with an ISS (1:5 ratio); the antigen conjugated to a
non-stimulatory PN (MISS conj; 1:5 ratio); the antigen in adjuvant
(alum) and, for reference, the IgG1 levels in naive (unexposed)
mice, all as compared to the vigorous Th1-type immune response
produced in mice immunized with ISS-PN/IMM (1:5 ratio). The
horizontal axis represents the levels (units/ml) of antibody; the
vertical axis represents the number of weeks following primary
antigen exposure.
[0023] FIG. 3 is a graph of data demonstrating the vigorous
Th1-type immune response (as measured by production of IgG2a
against an IMM antigen) stimulated by ISS-PN/IMM in comparison to
the levels of Th2-like responses stimulated by the antigen alone
(AgE) and antigen conjugated to a non-stimulatory PN (mISS conj).
Antigen to PN ratios are all 1:5. The horizontal axis represents
the levels (units/ml) of antibody; the vertical axis shows the
levels at 4 weeks following. primary antigen exposure (shaded bars)
and at 2 weeks following secondary antigen challenge (solid
bars).
[0024] FIG. 4 is a graph of data demonstrating the levels of
Th2-type immune responses (as measured by production of IgG1
against an IMM antigen) stimulated by the antigen alone (AgE) and
antigen conjugated to a non-stimulatory PN (mISS conj) in
comparison to the vigorous Th1-type immune response stimulated in
ISS-PN/IMM immunized mice. Antigen to PN ratios are all 1:5. The
horizontal axis represents the levels (units/ml) of antibody; the
vertical axis shows the levels at 4 weeks following primary antigen
exposure (shaded bars) and at 2 weeks following secondary antigen
challenge (solid bars).
[0025] FIG. 5 is a graph of data demonstrating suppression of Th2
associated anti-antigen (AgE) IgE production by ISS-PN/IMM in
comparison to the levels of IgE production stimulated by the
antigen alone (AgE) and the antigen conjugated to a non-stimulatory
PN (miSS conj). Antigen to PN ratios are all 1:5. The horizontal
axis represents the levels (counts per minute; cpm) of antibody;
the vertical axis shows the levels at 4 weeks following primary
antigen exposure (shaded bars) and at 2 weeks following secondary
antigen challenge (solid bars).
[0026] FIG. 6 is a graph of data demonstrating the high levels of
Th1 associated interferon .gamma. (IFNg) production stimulated by
ISS-PN/IMM in comparison to the relatively low levels of the Th1
cytokine stimulated by an ISS containing, antigen encoding plasmid
(pACB-Z); the antigen alone (.beta.-gal); the antigen mixed with an
ISS; the antigen conjugated to a non-stimulatory PN (MISS conj);
the antigen in adjuvant (alum) and, for reference, the IFNg levels
in naive (unexposed) mice. Antigen to PN ratios are all 1:5. The
horizontal axis represents the levels (ng/ml) of cytokine; the
vertical axis shows the levels of cytokine at 4 weeks following
primary antigen exposure (shaded bars).
[0027] FIG. 7 is a graph of data demonstrating the vigorous
antigen-specific cytotoxic T lymphocyte (CTL) response stimulated
by ISS-PN/IMM in comparison to the levels of CTL production
stimulated by an ISS containing, antigen encoding plasmid (pACB-Z);
the antigen alone (.beta.-gal); the antigen mixed with an ISS; the
antigen conjugated to a non-stimulatory PN (miSS conj); the antigen
in adjuvant (alum) and, for reference, the CTL levels in naive
(unexposed) mice. Antigen to PN ratios are all 1:5. The horizontal
axis represents the levels of antigen-specific cell lysis obtained
(as a percentage of control; no antigen); the vertical axis shows
the levels of CTL detected at different effector (antigen) to
target ratios, from 0:1 to 10:1. The legend identifies how each
cell population was treated.
DETAILED DESCRIPTION OF THE INVENTION
[0028] A. Biological Activity of the ISS-PN/IMM Conjugates
[0029] The immune response stimulated by the ISS-PN/IMM conjugates
of the invention differs from the vertebrate immune response to
conventional vaccination in both magnitude and quality. In the
former respect, the host immune response to an antigen is boosted
to a level greater than achieved on exposure to an ISS-PN or
antigen administered alone or together in an unconjugated form.
Thus, one surprising aspect of the invention is that conjugation of
an ISS-PN to an antigen-containing IMM produces a synergism between
the immunostimulatory activity of the ISS-PN and the
immunomodulatory activity of the IMM that immunizes the host to the
antigen more effectively than one would predict.
[0030] Advantageously, the immune response stimulated according to
the invention differs from the immune response of vertebrates to
conventional vaccination in that the latter develops in a Th2
phenotype while the former develops in a Th1 phenotype. In this
regard, it is helpful to recall that CD4+ lymphocytes generally
fall into one of two distinct subsets; i.e., the Th1 and Th2 cells.
Th1 cells principally secrete IL-2, IFN.gamma. and TNF.beta. (the
latter two of which mediate macrophage activation and delayed type
hypersensitivity) while Th2 cells principally secrete IL-4 (which
stimulates production of IgE antibodies), IL-5 (which stimulates
granulocyte infiltration of tissue), IL-6 and IL-10. These CD4+
subsets exert a negative influence on one another; i.e., secretion
of Th1 lymphokines inhibits secretion of Th2 lymphokines and vice
versa.
[0031] Factors believed to favor Th1 activation resemble those
induced by viral infection and include intracellular pathogens,
exposure to IFN-.beta., IFN-.alpha., IFN.gamma., IL-12 and IL-18
and exposure to low doses of antigen. Th1 type immune responses
also predominate in autoimmune disease. Factors believed to favor
Th2 activation include exposure to IL-4 and IL-10, APC activity on
the part of B lymphocytes and high doses of antigen. Active Th1
(IFN.gamma.) cells enhance cellular immunity and are therefore of
particular value in responding to intracellular infections, while
active Th2 cells enhance antibody production and are therefore of
value in responding to extracellular infections (at the risk of
anaphylactic events associated with IL-4 stimulated induction of
IgE antibody production). Thus, the ability to shift host immune
responses from the Th1 to the Th2 repertoire and vice versa has
substantial clinical significance for controlling host immunity
against antigen challenge (e.g., in infectious and allergic
conditions).
[0032] To that end, the methods of the invention shift the host
immune response to a sensitizing antigen toward a Th1 phenotype
(Example I). Consequently, Th2 associated cytokine production and
antigen stimulated production of IgE (Examples II and III) are
suppressed, thereby reducing the host's risk of prolonged allergic
inflammation and minimizing the risk of antigen-induced
anaphylaxis. CTL production is also stimulated to a greater degree
in animals treated according to the invention. Because CTL
production is tied to antigen processing in Class I MHC pathways,
increased CTL production can be produced from non-immunostimulatory
PN/IMM as well as ISS-PN/IMM (Example IV).
[0033] Although the invention is not limited to any particular
mechanism of action, it is conceivable that PN facilitate uptake of
exogenous antigen by antigen presenting cells for presentation
through host MHC Class I processing pathways not normally
stimulated by soluble antigen. Thus, ISS-PN/IMM carry antigen into
MHC Class I processing pathways (which may also be achieved by
PN/IMM without ISS activity) then stimulate a cytokine cascade in a
Th1 phenotype (as a result of ISS activity). Whatever the mechanism
of action, use of ISS-PN/IMM to boost the host's immune
responsiveness to a sensitizing antigen and shift the immune
response toward a Th1 phenotype avoids the risk of
immunization-induced anaphylaxis, suppresses IgE production in
response to a sensitizing antigen and eliminates the need to
identify the sensitizing antigen for use in immunization.
[0034] With reference to the invention, "boosting of immune
responsiveness in a Th1 phenotype" in an ISS-PN/IMM treated host is
evidenced by:
[0035] (1) a reduction in levels of IL-4 measured before and after
antigen-challenge; or detection of lower (or even absent) levels of
IL-4 in a treated host as compared to an antigen-primed, or primed
and challenged, control;
[0036] (2) an increase in levels of IL-12, IL-18 and/or IFN (a, P
or y) before and after antigen challenge; or detection of higher
levels of IL-12, IL-18 and/or IFN (.alpha., .beta. or .gamma.) in
an ISS-PN/IMM treated host as compared to an antigen-primed or,
primed and challenged, control;
[0037] (3) IgG2a antibody production in a treated host; or
[0038] (4) a reduction in levels of antigen-specific IgE as
measured before and after antigen challenge; or detection of lower
(or even absent) levels of antigen-specific IgE in an ISS-PN/IMM
treated host as compared to an antigen-primed, or primed and
challenged, control.
[0039] Exemplary methods for determining such values are described
further in the Examples.
[0040] Thus, the ISS-PN/IMM conjugates of the invention provide
relatively safe, effective means of stimulating a robust immune
response in a vertebrate host against any antigen.
[0041] B. ISS-PN/IMM Conjugates: Structure and Preparation
[0042] 1. ISS-PN root structure
[0043] The ISS-ODN base of the ISS-PN/IMM conjugates of the
invention includes an oligonucleotide, which may be a part of a
larger nucleotide construct such as a plasmid. The term
"polynucleotide" therefore includes oligonucleotides, modified
oligonucleotides and oligonucleosides, alone or as part of a larger
construct. The polynucleotide may be single-stranded DNA (ssDNA),
double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) or
double-stranded RNA (dsRNA).
[0044] The polynucleotide portion can be linearly or circularly
configured, or the oligonucleotide portion can contain both linear
and circular segments. Modifications of oligonucleotides include,
but are not limited to, modifications of the 3'OH or 5'OH group,
modifications of the nucleotide base, modifications of the sugar
component, and modifications of the phosphate group.
[0045] The oligonucleotide base of ISS-PN/IMM conjugates may
comprise ribonucleotides (containing ribose as the only or
principal sugar component), deoxyribonucleotides deoxyribose as the
principal sugar component), or in accordance with established
state-of-the-art modified sugars or sugar analogs may be
incorporated in the oligonucleotide of the present invention. Thus,
in addition to ribose and deoxyribose, the sugar moiety may be
pentose, deoxypentose, hexose, deoxyhexos, glucose, arabinose,
xylose, lyxose, and a sugar "analog" cyclopentyl group. The sugar
may be in pyranosyl or in a furanosyl form. In the modified
oligonucleotides of the present invention the sugar moiety is
preferably the furanoside of ribose, deoxyribose, arabinose or
2'-0-methylribose, and the sugar may be attached to the respective
heterocyclic bases either in I or J anomeric configuration. The
preparation of these sugars or sugar analogs and the respective
"nucleosides" wherein such sugars or analogs are attached to a
heterocyclic base (nucleic acid base) per se is known, and need not
be described here, except to the extent such preparation may
pertain to any specific example.
[0046] The phosphorous derivative (or modified phosphate group)
which may be attached to the sugar or sugar analog moiety in the
modified oligonucleotides of the present invention may be a
monophosphate, diphosphate, triphosphate, alkylphosphate,
alkanephosphate, phosphoronthioate, phosphorodithioate or the like.
The preparation of the above-noted phosphate analogs, and their
incorporation into nucleotides, modified nucleotides and
oligonucleotides, per se, is also known and need not be described
here.
[0047] The heterocyclic bases, or nucleic acid bases which are
incorporated in the oligonucleotide base of the ISS-PN/IMM
conjugates may be the naturally occurring principal purine and
pyrimidine bases, (namely uracil or thymine, cytosine, adenine and
guanine, as mentioned above), as well as naturally occurring and
synthetic modifications of said principal bases. Those skilled in
the art will recognize that a large number of "synthetic"
non-natural nucleosides comprising various heterocyclic bases and
various sugar moieties (and sugar analogs) have become available in
the prior art, such that oligonucleotide base of the ISS-PN/IMM
conjugates may include one or several heterocyclic bases other than
the principal five base components of naturally occurring nucleic
acids. Preferably, however, the heterocyclic base in the
oligonucleotide base of the ISS-PN/IMM conjugates is selected form
uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl,
guanin-8-yl, 4-aminopyrrolo [2.3-d] pyrimidin-5-yl,
2-amino-4-oxopyrolo [2,3-d] pyrimidin-5-yl, 2-amino-4-oxopyrrolo
[2.3-d] pyrimidin-3-yl groups, where the purines are attached to
the sugar moiety of the oligonucleotides via the 9-position, the
pyrimidines via the 1-position, the pyrrolopyrimidines via the
7-position and the pyrazolopyrimidines via the 1-position.
[0048] Structurally, the root oligonucleotide of the ISS-PN
component of ISS-PN/IMM is a non-coding sequence which may include
at least one unmethylated CpG motif. The relative position of any
CpG sequence in ISS-PN with immunostimulatory activity in certain
mammalian species (e.g., rodents) is 5'-CG-3' (i.e., the C is in
the 5' position with respect to the G in the 3' position). PN/IMM
can be conveniently obtained by substituting the cytosine in the
CpG dinucleotide with another nucleotide; a particularly useful
substitution is with a guanine to form GpG dinucleotide containing
PN.
[0049] Some oligonucleotide ISS (ISS-ODN) are known. In such
ISS-ODN, the CpG motif is flanked by at least two purine
nucleotides (e.g., GA or AA) and at least two pyrimidine
nucleotides (5'-Purine-Purine-[C]-[G]-Pyrimid- ine-Pyrimidine-3').
CpG motif-containing ISS-ODN are believed to stimulate B lymphocyte
proliferation (see, e.g., Krieg, et al., Nature, 374:546-549,
1995).
[0050] The core hexamer structure of the foregoing ISS-PN may be
flanked upstream and/or downstream by any number or composition of
nucleotides or nucleosides. However, ISS-PN are at least 6 bases in
length, and preferably are between 6 and 200 bases in length, to
enhance uptake of the ISS-PN/IMM into target tissues. Those of
ordinary skill in the art will be familiar with, or can readily
identify, reported nucleotide sequences of known ISS-ODN for
reference in preparing ISS-PN. For ease of reference in this
regard, the following sources are especially helpful:
[0051] Yamamoto, et al., Microbiol.Immunol., 36:983 (1992)
[0052] Ballas, et al., J.Immunol., 157:1840 (1996)
[0053] Klinman, et al., J.Immunol., 158:3635 (1997)
[0054] Sato, et al., Science, 273:352 (1996)
[0055] Each of these articles are incorporated herein by reference
for the purpose of illustrating the level of knowledge in the art
concerning the nucleotide composition of known ISS-ODN.
[0056] In particular, ISS-PN and PN useful in the invention include
those which have the following hexameric nucleotide sequences:
[0057] 1. For ISS-PN, hexamers having "CpG" motifs or, for PN,
hexamers having XpY motifs, where X cannot be C if Y is G and
vice-versa; and,
[0058] 2. Inosine and/or uracil substitutions for nucleotides in
the foregoing hexamer sequences for use as RNA ISS-ODN.
[0059] For example, DNA based ISS-PN useful in the invention
include those which have the following hexameric nucleotide
sequences:
1 (respectively, SEQ.ID.Nos. 1-18) AACGTT, AGCGTC, GACGTT, GGCGTT,
AACGTC, AGCGTC, GACGTC, GGCGTC, AACGCC, AGCGCC, GACGCC, GGCGCC,
AGCGCT, GACGCT, GGCGCT, TTCGAA, GGCGTT and AACGCC.
[0060] RNA based ISS-PN useful in the invention include those which
have the following hexameric nucleotide sequences:
2 (respectively, SEQ.ID.Nos. 19-33) AACGUU, AACGpI, AACGpC, AGCGUC,
AGCGpI, AGCGpC, GACGCU, GACGCpI, GACGCpC, GACGUU, GACGpI, GACGpC,
GACGUC, GACGpI, GACGpC, and poly(I.cndot.C).
[0061] The ISS-PN may or may not include palindromic regions. If
present, a palindrome may extend only to a CpG motif, if present,
in the core hexamer sequence, or may encompass more of the hexamer
sequence as well as flanking nucleotide sequences.
[0062] In addition, backbone phosphate group modifications (e.g.,
methylphosphonate, phosphorothioate, phosphoroamidate and
phosphorodithioate internucleotide linkages) can confer
anti-microbial activity on the ISS-PN and enhance their stability
in vivo, making them particularly useful in therapeutic
applications. A particularly useful phosphate group modification is
the conversion to the phosphorothioate or phosphorodithioate forms
of ISS-PN. In addition to their potentially anti-microbial
properties, phosphorothioates and phosphorodithioates are more
resistant to degradation in vivo than their unmodified
oligonucleotide counterparts, making the ISS-PN/IMM of the
invention more available to the host.
[0063] 2. IMM conjugate partners.
[0064] The oligonucleotide base of the ISS-PN/IMM conjugate is
conjugated to an IMM which includes an antigen and may further
include an immunomodulatory agent. An "antigen" is a substance that
is recognized and bound specifically by an antibody or by a T cell
antigen receptor. Antigens can include peptides, proteins,
glycoproteins and polysaccharides, including portions thereof and
combinations thereof. The antigens can be those found in nature or
can be synthetic.
[0065] The term "immunomodulatory" as used herein includes
immunostimulatory as well as immunosuppressive effects.
Immunostimulatory effects include, but are not limited to, those
that directly or indirectly enhance cellular or humoral immune
responses. Examples of immunostimulatory effects include, but are
not limited to, increased antigen-specific antibody production;
activation or proliferation of a lymphocyte population such as NK
cells, CD4.sup.+ T lymphocytes, CD8+ T lymphocytes, macrophages and
the like; as well as increased synthesis of Th1 associated
immunostimulatory cytokines including, but not limited to, IL-6,
IL-12, IL-18, IFN-.alpha., .beta., and .gamma., TNF-.alpha. and the
like. Immunosuppressive effects include those that directly or
indirectly decrease cellular or humoral immune responses.
[0066] Examples of immunosuppressive effects include, but are not
limited to, a reduction in antigen-specific antibody production
such as reduced IgE production; activation of lymphocyte or other
cell populations that have immunosuppressive activities such as
those that result in immune tolerance; and increased synthesis of
cytokines that have suppressive effects toward certain cellular
functions. One example of this is IFN-.gamma., which can block IL-4
induced class switch to IgE and IgG1, thereby reducing the levels
of these antibody subclasses.
[0067] Thus, an "immunomodulatory agent" suitable for use as
conjugate partners for ISS-PN/IMM can be a peptide, such as an
antigen or cytokine. Where the ISS-PN/IMM conjugate partner is a
peptide, suitable peptides include purified native peptides,
synthetic peptides, recombinant proteins, crude protein extracts,
attenuated or inactivated viruses, cells, micro-organisms, or
fragments of such peptides.
[0068] Protein antigens that can serve as IMM conjugate partners
include antigens from a wide variety of sources, including
allergens such as plant pollens, dust mite proteins, animal dander,
saliva, and fungal spores as well as infectious microorganims.
Examples of the latter include attenuated or inactivated viruses
such as HIV-1, HIV-2, hepatitis, herpes simplex, rotavirus, polio
virus, measles virus, human and bovine papilloma virus, and slow
brain viruses. For immunization against tumor formation, the
conjugate can include tumor cells (live or irradiated), tumor cell
extracts, or protein subunits of tumor antigens. Vaccines for
immuno-based contraception can be formed by including sperm
proteins as the peptide portion of the conjugate.
[0069] Among the suitable cytokines for use as components of IMM
conjugate partners are the interleukins (IL-1, IL-2, IL-3, etc.),
interferons (e.g., IFN-.alpha., IFN-.beta., IFN-.gamma.),
erythropoietin, colony stimulating factors (e.g., G-CSF, M-CSF,
GM-CSF) and TNF-.alpha..
[0070] IMM conjugate partners can also include amino acid sequences
that mediate protein binding to a specific receptor or that mediate
targeting to a specific cell type or tissue. Examples include, but
are not limited to, antibodies or antibody fragments; peptide
hormones such as human growth hormone; and enzymes. Co-stimulatory
molecules such as B7 (CD80), trans-activating proteins such as
transcription factors, chemokines such as macrophage chemotactic
protein (MCP) and other chemoattractant or chemotactic peptides are
also useful peptide-based conjugate partners.
[0071] More specifically, suitable antigens for use as ISS-PN/IMM
conjugate partners include any molecule capable of being conjugated
to an oligonucleotide and eliciting a B cell or T cell
antigen-specific response. Preferably, antigens elicit an antibody
response specific for the antigen. A wide variety of molecules are
antigens. These include, but are not limited to, sugars, lipids,
autacoids and hormones, as well as macromolecules such as complex
carbohydrates, and phospholipids. Small molecules may need to be
haptenized in order to be rendered antigenic.
[0072] Preferably the antigens are peptides, polysaccharides (such
as the capsular polysaccharides used in Haemophilus influenza
vaccines), gangliosides and glycoproteins. The antigen may be an
intact antigen or T cell epitope(s) of an antigen. These can be
obtained through several methods known in the art, including
isolation and synthesis using chemical and enzymatic methods. In
certain cases, such as for many sterols fatty acids and
phospholipids, the antigenic portions are commercially
available.
[0073] Many antigenic peptides and proteins are known in, and
available to the art; others can be identified using conventional
techniques. Examples of known antigens include, but are not limited
to
[0074] a. Allergens such as reactive major dust mite allergens Der
pI and Der pII (see, Chua, et al., J.Exp.Med., 167:175-182, 1988;
and, Chua, et al., Int.Arch.Allergy Appl. Immunol., 91:124-129,
1990), T cell epitope peptides of the Der pII allergen (see, Joost
van Neerven, et al., J.Immunol., 151:2326-2335, 1993), the highly
abundant Antigen E (Amb aI) ragweed pollen allergen (see, Rafnar,
et al., J.Biol.Chem., 266:1229-1236, 1991), phospholipase A.sub.2
(bee venom) allergen and T cell epitopes therein (see, Dhillon, et
al., J.Allergy Clin.Immunol., ______:42-______, 1992), white birch
pollen (Betvl) (see, Breiteneder, et al., EMBO, 8:1935-1938, 1989),
the Fel dI major domestic cat allergen (see, Rogers, et al.,
Mol.Immunol., 30:559-568, 1993), tree pollen (see, Elsayed et al.,
Scand. J. Clin. Lab. Invest. Suppl., 204:17-31, 1991) and grass
pollen (see, Malley, J. Reprod. Immunol., 16:173-86, 1989).
[0075] b. Live, attenuated and inactivated microorganisms such as
inactivated polio virus (Jiang et al., J. Biol. Stand., 14:103-9,
1986), attenuated strains of Hepatitis A virus (Bradley et al., J.
Med. Virol., 14:373-86, 1984), attenuated measles virus (James et
al., N. Engl. J. Med., 332:1262-6, 1995) and epitopes of pertussis
virus (e.g., ACEL-IMUNE.RTM. acellular DTP, Wyeth-Lederle Vaccines
and Pediatrics).
[0076] c. Contraceptive antigens such as human sperm protein (Lea
et al., Biochim. Biophys. Acta, 1307:263, 1996).
[0077] The published sequence data and methods for isolation and
synthesis of the antigens described in these articles are
incorporated herein by this reference to illustrate knowledge in
the art regarding useful antigen sources. Those of ordinary skill
in the art will be familiar with, or can readily ascertain, the
identity of other useful antigens for use as ISS-PN/IMM conjugate
partners.
[0078] Particularly useful immunostimulatory peptides for inclusion
in IMM are those which stimulate Th1 immune responses, such as
IL-12 (Bliss, et al., J.Immunol., 156:887-894, 1996), IL-18,
INF-.alpha.,.beta. and .gamma. or TGF-.alpha.. Conjugation of
adjuvants (such as keyhole limpet hemocyanin, KLH) to the
ISS-PN/IMM conjugate can further enhance the activity of the
ISS-PN/IMM conjugates of the invention.
[0079] Other useful adjuvants include cholera toxin,
procholeragenoid, cholera toxin B subunit and fungal
polysaccharides including, but not limited to, schizophyllan,
muramyl dipeptide, muramyl dipeptide derivatives, phorbol esters,
microspheres, non-Helicobacter pylori bacterial lysates, labile
toxin of Escherichia coli, block polymers, saponins, and ISCOMs.
For additional adjuvants, those of ordinary skill in the art may
also refer to, for example, Azuma, I., "Synthetic Immunoadjuvants:
Application to Non-Specific Host Stimulation and Potentiation of
Vaccine Immunogenicity" Vaccine, vol. 10, 1000 (1992); Pockley, A.
G. & Montgomery, P. C., "In vivo Adjuvant Effect of
Interleukins 5 and 6 on Rat Tear IgA Antibody Responses"
Immunology, vol. 73, 19-23 (1991); Adam, A. & Lederer, E.
"Muramyl peptides as Immunomodulators" ISI ATLAS OF SCIENCE 205
(1988); Clements, J. D., et al. "Adjuvant Activity of Escherichia
coli Heat-labile Enterotoxin and Effect on the Induction of Oral
Tolerance in Mice to Unrelated Protein Antigens" Vaccine, vol. 6,
269 (1988); Ben Ahmeida, E. T. S., et al. "Immunopotentiation of
Local and Systemic Humoral Immune Responses by ISCOMs, Liposomes
and FCA: Role in Protection Against Influenza A in Mice" Vaccine,
vol. 11, 1302 (1993); and Gupta, R. K. et al. "Adjuvants--A Balance
Between Toxicity and Adjuvanticity" Vaccine, vol. 11, 290-308
(1993). Those of ordinary skill in the art will appreciate that
non-antigen components of IMM described above can also be
administered in unconjugated form with an ISS-PN/IMM (antigen only)
conjugate. Thus, the co-administration of such components is
encompassed by the invention.
[0080] C. Synthesis of Polynucleotide Conjugates
[0081] 1. Polynucleotide Portion
[0082] ISS-PN can be synthesized using techniques and nucleic acid
synthesis equipment which are well-known in the art For reference
in this regard, see, e.g., Ausubel, et al., Current Protocols in
Molecular Biology, Chs. 2 and 4 (Wiley Interscience, 1989);
Maniatis, et al., Molecular Cloning: A laboratory Manual (Cold
Spring Harbor Lab., New York, 1982); U.S. Pat. No. 4,458,066 and
U.S. Pat. No. 4,650,675. When assembled enzymatically, the
individual units can be ligated with a ligase such as T4 DNA or RNA
ligase as described in, for example, U.S. Pat. No. 5,124,246.
Oligonucleotide degradation could be accomplished through the
exposure of an oligonucleotide to a nuclease, as exemplified in
U.S. Pat. No. 4,650,675. These references are incorporated herein
by reference for the sole purpose of demonstrating knowledge in the
art concerning production of synthetic polynucleotides. Because the
ISS-PN is non-coding, there is no concern about maintaining an open
reading frame during synthesis.
[0083] Alternatively, ISS-PN may be isolated from microbial species
(especially mycobacteria) using techniques well-known in the art,
such as nucleic acid hybridization. Preferably, such isolated
ISS-PN will be purified to a substantially pure state; i.e., to be
free of endogenous contaminants, such as lipopolysaccharides.
ISS-PN isolated as part of a larger polynucleotide can be reduced
to the desired length by techniques well known in the art, such as
by endonuclease digestion. Those of ordinary skill in the art will
be familiar with, or can readily ascertain, techniques suitable for
isolation, purification and digestion of polynucleotides to obtain
ISS-PN of potential use in the invention.
[0084] Circular ISS-PN can be isolated, synthesized through
recombinant methods, or chemically synthesized. Where the circular
ISS-PN is obtained through isolation or through recombinant
methods, the ISS-PN will preferably be a plasmid. The chemical
synthesis of smaller circular oligonucleotides can be performed
using literature methods (Gao et al., Nucleic Acids Res. (1995)
23:2025-9; Wang et al., Nucleic Acids Res. (1994) 22:2326-33).
[0085] The ISS-PN can also contain modified oligonucleotides. These
modified oligonucleotides can be synthesized using standard
chemical transformations. The efficient solid-support based
construction of methylphosphonates has been described. Agrawal et
al. (19) Tet. Lett. 28:3539-3542. The synthesis of other
phosphorous based modified oligonucleotides, such as
phosphotriesters (Miller et al. JACS 93, 6657-6665),
phosphoramidates (Jager et al, Biochemistry 27, 7247-7246), and
phosphorodithioates (U.S. Pat. No. 5,453,496) has also been
described. Other non-phosphorous based modified oligonucleotides
can also be used (Stirchak et al., Nucleic Acids Res. 17,
6129-6141).
[0086] The preparation of base-modified nucleosides, and the
synthesis of modified oligonucleotides using said base-modified
nucleosides as precursors, has been described, for example, in U.S.
Pat. Nos. 4,910,300, 4,948,882, and 5,093,232. These base-modified
nucleosides have been designed so that they can be incorporated by
chemical synthesis into either terminal or internal positions of an
oligonucleotide. Such base-modified nucleosides, present at either
terminal or internal positions of an oligonucleotide, can serve as
sites for attachment of a peptide or other antigen. Nucleosides
modified in their sugar moiety have also bee described (e.g., U.S.
Pat. Nos. 4,849,513, 5,015,733, 5,118,800, 5,118,802) and can be
used similarly.
[0087] The techniques for making phosphate group modifications to
oligonucleotides are known in the art and do not require detailed
explanation. For review of one such useful technique, the an
intermediate phosphate triester for the target oligonucleotide
product is prepared and oxidized to the naturally occurring
phosphate triester with aqueous iodine or with other agents, such
as anhydrous amines. The resulting oligonucleotide phosphoramidates
can be treated with sulfur to yield phophorothioates. The same
general technique (excepting the sulfur treatment step) can be
applied to yield methylphosphoamidites from methylphosphonates. For
more details concerning phosphate group modification techniques,
those of ordinary skill in the art may wish to consult U.S. Pat.
Nos. 4,425,732; 4,458,066; 5,218,103 and 5,453,496, as well as
Tetrahedron Lett. at 21:4149 (1995), 7:5575 (1986), 25:1437 (1984)
and Journal Am. ChemSoc., 93:6657 (1987), the disclosures of which
are incorporated herein for the sole purpose of illustrating the
standard level of knowledge in the art concerning preparation of
these compounds.
[0088] 2. Linking the PN Component to the IMM Component
[0089] The ISS-PN component can be linked to the IMM portion of the
conjugate in a variety of ways. The link can be made at the 3' or
5' end of the ISS-PN, or to a suitably modified base at an internal
position in the PN. If the peptide contains a suitable reactive
group (e.g., an N-hydroxysuccinimide ester) it can be reacted
directly with the N.sup.4 amino group of cytosine residues.
Depending on the number and location of cytosine residues in the
ISS-PN, specific labeling at one or more residues can be
achieved.
[0090] Alternatively, modified oligonucleosides, such as are known
in the art, can be incorporated at either terminus, or at internal
positions in the ISS-PN. These can contain blocked functional
groups which, when deblocked, are reactive with a variety of
functional groups which can be present on, or attached to, a
peptide of interest.
[0091] The IMM portion of the conjugate can be attached to the
3'-end of the ISS-PN through solid support chemistry. For example,
the ISS-PN portion can be added to a polypeptide portion that has
been pre-synthesized on a support (Haralambidis et al., Nucleic
Acids Res. (1990) 18:493-99; Haralambidis et al., Nucleic Acids
Res. (1990) 18:501-505). Alternatively, the PN can be synthesized
such that it is connected to a solid support through a cleavable
linker extending from the 3'-end. Upon chemical cleavage of the
ISS-PN from the support, a terminal thiol group is left at the
3'-end of the ISS-PN (Zuckermann et al., Nucleic Acids Res. (1987)
15:5305-5321; Corey et al., (1987) Science 238:1401-1403), or a
terminal amine group is left at the 3'-end of the PN (Nelson et
al., Nucleic Acids Res. (1989) 17:1781-94). Conjugation of the
amino-modified PN to amino groups of the peptide can be performed
as described in Benoit et al., Neuromethods (1987) 6:43-72.
Conjugation of the thiol-modified ISS-PN to carboxyl groups of the
peptide can be performed as described in Sinah et al.,
Oligonucleotide Analogues: A Practical Approach (1991) IRL
Press.
[0092] The IMM portion of the conjugate can be attached to the
5'-end of the ISS-PN through an amine, thiol, or carboxyl group
that has been incorporated into the ISS-PN during its synthesis.
Preferably, while the ISS-PN is fixed to the solid support, a
linking group comprising a protected amine, thiol, or carboxyl at
one end, and a phosphoramidite at the other, is covalently attached
to the 5'-hydroxyl (Agrawal et al., Nucleic Acids Res. (1986)
14:6227-6245; Connolly, Nucleic Acids Res. (1985) 13:4485-4502;
Coull et al., Tetrahedron Lett. (1986) 27:3991-3994; Kremsky et
al., Nucleic Acids Res. (1987) 15:2891-2909; Connolly, Nucleic
Acids Res. (1987) 15:3131-3139; Bischoff et al., Anal. Biochem.
(1987) 164:336-344; Blanks et al., Nucleic Acids Res. (1988)
16:10283-10299; U.S. Pat. Nos. 4,849,513, 5,015,733, 5,118,800, and
5,118,802). Subsequent to deprotection, the latent amine, thiol,
and carboxyl functionalities can be used to covalently attach the
PN to a peptide (Benoit et al., Neuromethods (1987) 6:43-72; Sinah
et al., Oligonucleotide Analogues: A Practical Approach (1991) IRL
Press).
[0093] A peptide portion can be attached to a modified cytosine or
uracil at any position in the ISS-PN. The incorporation of a
"linker arm" possessing a latent reactive functionality, such as an
amine or carboxyl group, at C-5 of the modified base provides a
handle for the peptide linkage (Ruth, 4th Annual Congress for
Recombinant DNA Research, p. 123).
[0094] The linkage of the ISS-PN to a peptide can also be formed
through a high-affinity, non-covalent interaction such as a
biotin-streptavidin complex. A biotinyl group can be attached, for
example, to a modified base of an oligonucleotide (Roget et al.,
Nucleic Acids Res. (1989) 17:7643-7651). Incorporation of a
streptavidin moiety into the peptide portion allows formation of a
non-covalently bound complex of the streptavidin conjugated peptide
and the biotinylated PN.
[0095] The linkage of the ISS-PN to a lipid can be formed using
standard methods. These methods include, but are not limited to,
the synthesis of oligonucleotide-phospholipid conjugates (Yanagawa
et al., Nucleic Acids Symp. Ser. (1988) 19:189-92),
oligonucleotide-fatty acid conjugates (Grabarek et al., Anal.
Biochem. (1990) 185:131-35; Staros et al., Anal. Biochem. (1986)
156:220-22), and oligonucleotide-sterol conjugates (Boujrad et al.,
Proc. Natl. Acad. Sci. USA (1993) 90:5728-31).
[0096] The linkage of the ISS-PN to a oligosaccharide can be formed
using standard known methods. These methods include, but are not
limited to, the synthesis of oligonucleotide-oligosaccharide
conjugates, wherein the oligosaccharide is a moiety of an
immunoglobulin (O'Shannessy et al., J. Applied Biochem. (1985)
7:347-55).
[0097] Adjuvants and cytokines may also be genetically or
chemically linked to the ISS-ODN conjugates. Examples of this type
of fusion peptide are known to those skilled in the art and can
also be found in Czerkinsky et al., Infect. Immun., 57: 1072-77
(1989); Nashar et al., Vaccine, 11: 235-40 (1993); and Dertzbaugh
and Elson, Infect. Immun., 61: 48-55 (1993).
[0098] The linkage of a circular ISS-PN to an IMM can be formed in
several ways. Where the circular PN is synthesized using
recombinant or chemical methods, a modified nucleoside (Ruth, in
Oligonucleotides and Analogues: A Practical Approach (1991) IRL
Press). Standard linking technology can then be used to connect the
circular ISS-PN to the antigen or immunostimulatory peptide
(Goodchild, Bioconjugate Chem. (1990) 1: 165). Where the circular
ISS-PN is isolated, or synthesized using recombinant or chemical
methods, the linkage can be formed by chemically activating, or
photoactivating, a reactive group (e.g. carbene, radical) that has
been incorporated into the antigen or immunostimulatory peptide.
Additional methods for the attachment of peptides and other
molecules to ISS-PNs can be found in C. Kessler: Nonradioactive
labeling methods for nucleic acids in L. J. Kricka (ed.)
"Nonisotopic DNA Probe Techniques," Academic Press 1992 and in
Geoghegan and Stroh, Bioconjug. Chem., 3:138-146, 1992.
[0099] D. Methods and Routes for Administration of ISS-PN/IMM to a
Host
[0100] 1. Drug Delivery
[0101] The ISS-PN/IMM of the invention are administered to a host
using any available method and route suitable for drug delivery,
including ex vivo methods (e.g., delivery of cells incubated or
transfected with an ISS-PN/IMM) as well as systemic or localized
routes. However, those of ordinary skill in the art will appreciate
that methods and localized routes which direct the ISS-PN/IMM into
antigen-sensitized tissue will be preferred in most circumstances
to systemic routes of administration, both for immediacy of
therapeutic effect and avoidance of in vivo degradation.
[0102] The entrance point for many exogenous antigens into a host
is through the skin or mucosa. Thus, delivery methods and routes
which target the skin (e.g., for cutaneous and subcutaneous
conditions) or mucosa (e.g., for respiratory, ocular, lingual or
genital conditions) will be especially useful. Those of ordinary
skill in the clinical arts will be familiar with, or can readily
ascertain, means for drug delivery into skin and mucosa. For
review, however, exemplary methods and routes of drug delivery
useful in the invention are briefly discussed below.
[0103] Intranasal administration means are particularly useful in
addressing respiratory inflammation, particularly inflammation
mediated by antigens transmitted from the nasal passages into the
trachea or broncheoli. Such means include inhalation of aerosol
suspensions or insufflation of the polynucleotide compositions of
the invention. Nebulizer devices suitable for delivery of
polynucleotide compositions to the nasal mucosa, trachea and
bronchioli are well-known in the art and will therefore not be
described in detail here. For general review in regard to
intranasal drug delivery, those of ordinary skill in the art may
wish to consult Chien, Novel Drug Delivery Systems, Ch. 5 (Marcel
Dekker, 1992).
[0104] Dermal routes of administration, as well as subcutaneous
injections, are useful in addressing allergic reactions and
inflammation in the skin. Examples of means for delivering drugs to
the skin are topical application of a suitable pharmaceutical
preparation, transdermal transmission, injection and epidermal
administration.
[0105] For transdermal transmission, absorption promoters or
iontophoresis are suitable methods. For review regarding such
methods, those of ordinary skill in the art may wish to consult
Chien, supra at Ch. 7. Iontophoretic transmission may be
accomplished using commercially available "patches" which deliver
their product continuously via electric pulses through unbroken
skin for periods of several days or more. Use of this method allows
for controlled transmission of pharmaceutical compositions in
relatively great concentrations, permits infusion of combination
drugs and allows for contemporaneous use of an absorption
promoter.
[0106] An exemplary patch product for use in this method is the
LECTRO PATCH trademarked product of General Medical Company of Los
Angeles, Calif. This product electronically maintains reservoir
electrodes at neutral pH and can be adapted to provide dosages of
differing concentrations, to dose continuously and/or to dose
periodically. Preparation and use of the patch should be performed
according to the manufacturer's printed instructions which
accompany the LECTRO PATCH product; those instructions are
incorporated herein by this reference.
[0107] Epidermal administration essentially involves mechanically
or chemically irritating the outermost layer of the epidermis
sufficiently to provoke an immune response to the irritant. An
exemplary device for use in epidermal administration employs a
multiplicity of very narrow diameter, short tynes which can be used
to scratch ISS-PN/IMM coated onto the tynes into the skin. The
device included in the MONO-VACC old tuberculin test manufactured
by Pasteur Merieux of Lyon, France is suitable for use in epidermal
administration of ISS-PN/IMM. Use of the device is according to the
manufacturer's written instructions included with the device
product; these instructions regarding use and administration are
incorporated herein by this reference to illustrate conventional
use of the device. Similar devices which may also be used in this
embodiment are those which are currently used to perform allergy
tests.
[0108] Opthalmic administration (e.g., for treatment of allergic
conjunctivitis) involves invasive or topical application of a
pharmaceutical preparation to the eye. Eye drops, topical cremes
and injectable liquids are all examples of suitable mileaus for
delivering drugs to the eye.
[0109] Systemic administration involves invasive or systemically
absorbed topical administration of pharamaceutical preparations.
Topical applications as well as intravenous and intramuscular
injections are examples of common means for systemic administration
of drugs.
[0110] 2. Dosing parameters
[0111] A particular advantage of the ISS-PN/IMM of the invention is
their capacity to exert immunomodulatory activity even at
relatively minute dosages. Although the dosage used will vary
depending on the clinical goals to be achieved, a suitable dosage
range is one which provides up to about 1-1000 .mu.g of
ISS-PN/IMM/ml of carrier in a single dosage. Alternatively, a
target dosage of ISS-PN/IMM can be considered to be about 1-10
.mu.M in a sample of host blood drawn within the first 24-48 hours
after administration of ISS-PN/IMM. Based on current studies,
ISS-PN/IMM are believed to have little or no toxicity at these
dosage levels.
[0112] In this respect, it should be noted that the
anti-inflammatory and immunotherapeutic activity of ISS-PN/IMM in
the invention is essentially dose-dependent. Therefore, to increase
ISS-PN/IMM potency by a magnitude of two, each single dose is
doubled in concentration. Clinically, it may be advisable to
administer the ISS-PN/IMM in a low dosage (e.g., about 1 .mu.g/ml
to about 50 .mu.g/ml), then increase the dosage as needed to
achieve the desired therapeutic goal.
[0113] In view of the teaching provided by this disclosure, those
of ordinary skill in the clinical arts will be familiar with, or
can readily ascertain, suitable parameters for administration of
ISS-PN/IMM according to the invention.
[0114] 3. ISS-PN/IMM compositions
[0115] ISS-PN/IMM will be prepared in a pharmaceutically acceptable
composition for delivery to a host. Pharmaceutically acceptable
carriers preferred for use with the ISS-PN/IMM of the invention may
include sterile aqueous of non-aqueous solutions, suspensions, and
emulsions. Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers
include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers (such as those based on Ringer's
dextrose), and the like. Preservatives and other additives may also
be present such as, for example, antimicrobials, antioxidants,
chelating agents, and inert gases and the like. A composition of
ISS-PN/IMM may also be lyophilized using means well known in the
art, for subsequent reconstitution and use according to the
invention.
[0116] Absorption promoters, detergents and chemical irritants
(e.g., keritinolytic agents) can enhance transmission of an
ISS-PN/IMM composition into a target tissue. For reference
concerning general principles regarding absorption promoters and
detergents which have been used with success in mucosal delivery of
organic and peptide-based drugs, see Chien, Novel Drug Delivery
Systems, Ch. 4 (Marcel Dekker, 1992).
[0117] Examples of suitable nasal absorption promoters in
particular are set forth at Chien, supra at Ch. 5, Tables 2 and 3;
milder agents are preferred. Suitable agents for use in the method
of this invention for mucosal/nasal delivery are also described in
Chang, et al., Nasal Drug Delivery, "Treatise on Controlled Drug
Delivery", Ch. 9 and Table 3-4B thereof, (Marcel Dekker, 1992).
Suitable agents which are known to enhance absorption of drugs
through skin are described in Sloan, Use of Solubility Parameters
from Regular Solution Theory to Describe Partitioning-Driven
Processes, Ch. 5, "Prodrugs: Topical and Ocular Drug Delivery"
(Marcel Dekker, 1992), and at places elsewhere in the text. All of
these references are incorporated herein for the sole purpose of
illustrating the level of knowledge and skill in the art concerning
drug delivery techniques.
[0118] A colloidal dispersion system may be used for targeted
delivery of the ISS-PN/IMM to specific tissue. Colloidal dispersion
systems include macromolecule complexes, nanocapsules,
microspheres, beads, and lipid-based systems including oil-in-water
emulsions, micelles, mixed micelles, and liposomes. The preferred
colloidal system of this invention is a liposome.
[0119] Liposomes are artificial membrane vesicles which are useful
as delivery vehicles in vitro and in vivo. It has been shown that
large unilamellar vesicles (LUV), which range in size from 0.2-4.0
.mu.m can encapsulate a substantial percentage of an aqueous buffer
containing large macromolecules. RNA, DNA and intact virions can be
encapsulated within the aqueous interior and be delivered to cells
in a biologically active form (Fraley, et al., Trends Biochem.
Sci., 6:77, 1981). In addition to mammalian cells, liposomes have
been used for delivery of polynucleotides in plant, yeast and
bacterial cells. In order for a liposome to be an efficient gene
transfer vehicle, the following characteristics should be present:
(1) encapsulation of the genes encoding the antisense
polynucleotides at high efficiency while not compromising their
biological activity; (2) preferential and substantial binding to a
target cell in comparison to non-target cells; (3) delivery of the
aqueous contents of the vesicle to the target cell cytoplasm at
high efficiency; and (4) accurate and effective expression of
genetic information (Mannino, et al., Biotechniques, 6:682,
1988).
[0120] The composition of the liposome is usually a combination of
phospholipids, particularly high-phase-transition-temperature
phospholipids, usually in combination with steroids, especially
cholesterol. Other phospholipids or other lipids may also be used.
The physical characteristics of liposomes depend on pH, ionic
strength, and the presence of divalent cations.
[0121] Examples of lipids useful in liposome production include
phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides, and gangliosides. Particularly useful
are diacylphosphatidylglycerols, where the lipid moiety contains
from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and
is saturated. Illustrative phospholipids include egg
phosphatidylcholine, dipalmitoylphosphatidylcholine and
distearoylphosphatidylcholine.
[0122] The targeting of liposomes can be classified based on
anatomical and mechanistic factors. Anatomical classification is
based on the level of selectivity, for example, organ-specific,
cell-specific, and organelle-specific. Mechanistic targeting can be
distinguished based upon whether it is passive or active. Passive
targeting utilizes the natural tendency of liposomes to distribute
to cells of the reticulo-endothelial system (RES) in organs which
contain sinusoidal capillaries. Active targeting, on the other
hand, involves alteration of the liposome by coupling the liposome
to a specific ligand such as a monoclonal antibody, sugar,
glycolipid, or protein, or by changing the composition or size of
the liposome in order to achieve targeting to organs and cell types
other than the naturally occurring sites of localization.
[0123] The surface of the targeted delivery system may be modified
in a variety of ways. In the case of a liposomal targeted delivery
system, lipid groups can be incorporated into the lipid bilayer of
the liposome in order to maintain the targeting ligand in stable
association with the liposomal bilayer. Various well known linking
groups can be used for joining the lipid chains to the targeting
ligand (see, e.g., Yanagawa, et al., Nuc.Acids Symp.Ser., 19:189
(1988); Grabarek, et al., Anal.Biochem., 185:131 (1990); Staros, et
al., Anal.Biochem., 156:220 (1986) and Boujrad, et al.,
Proc.Natl.Acad.Sci. USA, 90:5728 (1993), the disclosures of which
are incorporated herein by reference solely to illustrate the
standard level of knowledge in the art concerning conjugation of
PNs to lipids). Targeted delivery of ISS-PN/IMM can also be
achieved by conjugation of the ISS-PN/IMM to a the surface of viral
and non-viral recombinant expression vectors, to an antigen or
other ligand, to a monoclonal antibody or to any molecule which has
the desired binding specificity.
[0124] Co-administration of a peptide drug with an ISS-PN/IMM
according to the invention may also be achieved by incorporating
the ISS-PN/IMM in cis or in trans into a recombinant expression
vector (plasmid, cosmid, virus or retrovirus) which codes for any
therapeutically beneficial protein deliverable by a recombinant
expression vector. If incorporation of an ISS-PN/IMM into an
expression vector for use in practicing the invention is desired,
such incorporation may be accomplished using conventional
techniques which do not require detailed explanation to one of
ordinary skill in the art. For review, however, those of ordinary
skill may wish to consult Ausubel, Current Protocols in Molecular
Biology, supra.
[0125] D. Screening for Active ISS-PN/IMM
[0126] Confirmation that a particular compound has the properties
of an ISS-PN/IMM useful in the invention can be obtained by
evaluating whether the ISS-PN/IMM affects cytokine secretion and
IgG antibody isotype production as described in Section A.I, above.
Details of in vitro techniques useful in making such an evaluation
are given in the Examples; those of ordinary skill in the art will
also know of, or can readily ascertain, other methods for measuring
cytokine secretion and antibody production along the parameters
taught herein.
[0127] E. Kits for Use in Practicing the Methods of the
Invention
[0128] For use in the methods described above, kits are also
provided by the invention. Such kits may include any or all of the
following: ISS-PN/IMM (conjugated or unconjugated); a
pharmaceutically acceptable carrier (may be pre-mixed with the
ISS-PN/IMM) or suspension base for reconstituting lyophilized
ISS-PN/IMM; additional medicaments; a sterile vial for each
ISS-PN/IMM and additional medicament, or a single vial for mixtures
thereof; device(s) for use in delivering ISS-PN/IMM to a host;
assay reagents for detecting indicia that the anti-inflammatory
and/or immunostimulatory effects sought have been achieved in
treated animals and a suitable assay device.
[0129] Examples illustrating the practice of the invention are set
forth below. The examples are for purposes of reference only and
should not be construed to limit the invention, which is defined by
the appended claims. All abbreviations and terms used in the
examples have their expected and ordinary meaning unless otherwise
specified.
EXAMPLE I
[0130] selective Induction of a Th1 Response in a Host After
Administration of an ISS-PN/IMM
[0131] In mice, IgG 2A antibodies are serological markers for a Th1
type immune response, whereas IgG 1 antibodies are indicative of a
Th2 type immune response. Th2 responses include the
allergy-associated IgE antibody class; soluble protein antigens
tend to stimulate relatively strong Th2 responses. In contrast, Th1
responses are induced by antigen binding to macrophages and
dendritic cells.
[0132] To determine which response, if any, would be produced by
mice who received ISS-PN/IMM according to the invention, eight
groups of Balb/c mice were immunized with 10 .mu.g
.beta.-galactosidase protein (conjugated to avidin; Sigma, St.
Louis, Mo.) to produce a model allergic phenotype. As set forth in
the Table below, some of the mice received antigen alone, some
received an antigen-ISS-PN conjugate or a conjugate using a mutant,
non-stimulatory PN as a conjugate for the antigen, and others
received the antigen in an unconjugated mixture with an ISS-PN.
Naive mice are shown for reference:
3 Mouse Group ISS-PN/IMM Treatment 1 None (.beta.-gal antigen
vaccinated) 2 DY1018-.beta.gal conjugate (ISS-PN/IMM) 3
DY1019-.beta.gal conjugate (PN/IMM) 4 DY1018 mixed with .beta.gal
(unconjugated) 5 .beta.gal in adjuvant (alum) 6 plasmid DNA
(ISS-ODN present but not expressible with antigen) 7 naive mice (no
antigen priming)
[0133] DY1018 has the nucleotide sequence:
[0134] 5'-TGACTGTGAACGTTCGAGATGA-3' with a phosphothioate
backbone
[0135] and DY1019 has the nucleotide sequence:
[0136] 5'-TGACTGTGAAGGTTGGAGATGA-3' with a phosphothioate
backbone.
[0137] At 2 week intervals, any IgG 2a and IgG 1 to
.beta.-galactosidase present in the serum of each mouse were
measured by enzyme-linked immunoabsorbent assay (using antibodies
specific for the IgG 1 and IgG 2A subclasses) on microtiter plates
coated with the enzyme.
[0138] As shown in FIG. 1, only the mice who received the
ISS-PN/IMM produced high titers of IgG 2A antibodies, which
increased in number over a period of 8 weeks. As shown in FIG. 2,
immunization of the mice with the antigen itself or with the PN/IMM
induced production of relatively high titers of IgG 1 antibodies.
The data shown in the FIGURES comprise averages of the values
obtained from each group of mice.
[0139] To evaluate the effect of treatment of a host before and
after a secondary antigen challenge, 3 groups of Balb/c mice were
immunized with 10 g of antigen E (AgE) in alum to produce a model
allergic phenotype and challenged again with the antigen,
ISS-PN/IMM or mutant (nonstimulatory) PN/IMM at 5 weeks
post-priming. An ELISA for IgG1 and IgG2a antibodies was performed
as described 4 weeks after priming (one week before secondary
antigen challenge) and again at 7 weeks (2 weeks after secondary
challenge).
[0140] Again, the mice who received the ISS-PN/IMM mounted a strong
Th1 type response to the antigen (IMM) as compared to the
antigen-immunized and mutant PN/IMM immunized mice (FIG. 3), while
the reverse was true of a Th2 type response in the same mice (FIG.
4).
[0141] These data indicate that a selective Th1 response is induced
by administration of an ISS-PN/IMM according to the invention to
both an antigen-primed (pre-antigen challenge) and an
antigen-challenged host.
EXAMPLE II
[0142] Suppression of IgE Antibody Response to Antigen by
Immunization with ISS-PN/IMM
[0143] To demonstrate the IgE suppression achieved through
stimulation of a Th1 type cellular immune response in preference to
a Th2 type cellular immune response, five to eight week old Balb/c
mice were immunized with AgE as described in the previous
Example.
[0144] IgE anti-Age were detected using a solid phase
radioimmunoassay (RAST) in a 96 well polyvinyl plate (a
radioisotopic modification of the ELISA procedure described in
Coligan, "Current Protocols In Immunology", Unit 7.12.4, Vol. 1,
Wiley & Sons, 1994), except that purified polyclonal goat
antibodies specific for mouse e chains were used in lieu of
antibodies specific for human Fab. To detect anti-AgE IgE, the
plates were coated with AgE (10 .mu.g/ml). The lowest IgE
concentration measurable by the assay employed was 0.4 ng of
IgE/ml.
[0145] Measuring specifically the anti-antigen response by each
group of mice, as shown in FIG. 5, anti-AgE IgE levels in the
ISS-PN/IMM immunized mice were consistently low both before and
after boosting, while the protein and mutant ISS-PN/IMM injected
mice developed high levels of anti-AgE after antigen challenge.
[0146] These data show that the ISS-PN/IMM immunized mice developed
an antigen specific Th1 response (suppressing the Th2 IgE response)
to the antigen.
EXAMPLE III
INF.gamma. Levels in Mice After Delivery of ISS-PN/IMM
[0147] BALB/c mice were immunized with .beta.gal as described in
Example I then sacrificed 24 hrs later. Splenocytes were harvested
from each mouse.
[0148] 96 well microtiter plates were coated with anti-CD3 antibody
(Pharmingen, La Jolla, Calif.) at a concentration of 1 .mu.g/ml of
saline. The anti-CD3 antibody stimulates T cells by delivering a
chemical signal which mimicks the effects of binding to the T cell
receptor (TCR) complex. The plates were washed and splenocytes
added to each well (4.times.10.sup.5/well) in a medium of RPMI 1640
with 10% fetal calf serum. Supernatants were obtained at days 1, 2
and 3.
[0149] Th1 cytokine (INF.gamma.) levels were assayed with an
anti-INF.gamma. murine antibody assay (see, e.g., Coligan, "Current
Protocols in Immunology", Unit 6.9.5., Vol. 1, Wiley & Sons,
1994). Relatively low levels of INF-.gamma. would be expected in
mice with a Th2 phenotype, while relatively high levels of
INF-.gamma. would be expected in mice with a Th1 phenotype.
[0150] As shown in FIG. 5, levels of Th1 stimulated IFN-.gamma.
secretion were greatly increased in the ISS-PN/IMM treated mice,
but substantially reduced in each other set of mice (as compared to
the control), indicating development of a Th2-type phenotype in the
latter mice and a Th1 phenotype in the ISS-PN/IMM treated mice.
EXAMPLE IV Boosting of CTL Responses by ISS-PN/IMM
[0151] A mixture of lymphoytes was obtained and contacted with
.beta.gal antigen alone or as part of the constructs and mixtures
described in Example I. As shown in FIG. 6, CTL production in
response to ISS-PN/IMM was consistently higher than the response to
antigen delivered in other forms; even twice as high than in
animals treated with an unconjugated mixture of ISS-PN and IMM
antigen.
[0152] In the experiment, the higher values for the mice treated
with M-ISS-PN/IMM after antigen challenge as compared to the
conventionally immunized mice is most likely owing to the antigen
carrier properties of DY1019.
[0153] Thus, longer-term immunity mediated by cellular immune
responses is benefitted by treatment according to the
invention.
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