U.S. patent application number 09/802359 was filed with the patent office on 2003-07-10 for biodegradable immunomodulatory formulations and methods for use thereof.
Invention is credited to Tuck, Stephen, Van Nest, Gary.
Application Number | 20030129251 09/802359 |
Document ID | / |
Family ID | 26883938 |
Filed Date | 2003-07-10 |
United States Patent
Application |
20030129251 |
Kind Code |
A1 |
Van Nest, Gary ; et
al. |
July 10, 2003 |
Biodegradable immunomodulatory formulations and methods for use
thereof
Abstract
The invention provides new compositions and methods for
immunomodulation of individuals. Immunomodulation is accomplished
by administration of immunomodulatory polynucleotide/microcarrier
(IMP/MC) complexes. The IMP/MC complexes may be covalently or
non-covalently bound, and feature a polynucleotide comprising at
least one immunostimulatory sequence bound to a biodegradable
microcarrier or nanocarrier.
Inventors: |
Van Nest, Gary; (Martinez,
CA) ; Tuck, Stephen; (Oakland, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
755 PAGE MILL RD
PALO ALTO
CA
94304-1018
US
|
Family ID: |
26883938 |
Appl. No.: |
09/802359 |
Filed: |
March 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60188303 |
Mar 10, 2000 |
|
|
|
Current U.S.
Class: |
424/493 ;
514/44R |
Current CPC
Class: |
A61K 9/1647 20130101;
A61P 27/16 20180101; A61P 31/06 20180101; A61P 31/12 20180101; A61K
9/1075 20130101; A61P 35/00 20180101; Y10T 428/2982 20150115; A61P
1/04 20180101; A61P 13/12 20180101; A61P 37/02 20180101; A61P 27/14
20180101; A61P 31/00 20180101; A61P 43/00 20180101; A61P 17/00
20180101; A61P 31/04 20180101; A61K 47/6925 20170801; A61P 33/02
20180101; A61K 9/1617 20130101; A61P 11/00 20180101; A61P 29/00
20180101; A61K 9/167 20130101; A61P 33/10 20180101; Y02A 50/423
20180101; A61P 37/08 20180101; Y02A 50/30 20180101; A61P 11/06
20180101 |
Class at
Publication: |
424/493 ;
514/44 |
International
Class: |
A61K 048/00; A61K
009/16; A61K 009/50 |
Claims
What is claimed is:
1. An immunomodulatory polynucleotide/microcarrier (IMP/MC)
complex, comprising: a polynucleotide comprising an
immunostimulatory sequence (ISS) linked to a biodegradable
microcarrier (MC), wherein the ISS comprises the sequence 5'-C,
G-3' and wherein said MC is less than 10 .mu.m in size.
2. The IMP/MC complex of claim 1, wherein said polynucleotide is
covalently linked to said microcarrier.
3. The IMP/MC complex of claim 1, wherein said polynucleotide is
non-covalently linked to said microcarrier.
4. The IMP/MC complex of claim 1, wherein said microcarrier is a
liquid phase microcarrier.
5. The IMP/MC complex of claim 1, wherein said microcarrier is a
solid phase microcarrier.
6. The IMP/MC complex of claim 1, wherein said microcarrier is from
25 nm to 5 .mu.m in size.
7. The IMP/MC complex of claim 6, wherein said microcarrier is from
1.0 .mu.m to 2.0 .mu.m in size.
8. The IMP/MC complex of claim 7, wherein said microcarrier is 1.4
.mu.m in size.
9. The IMP/MC complex of claim 7, wherein said microcarrier is
cationic.
10. The IMP/MC complex of claim 1, wherein said complex is
antigen-free.
11. The IMP/MC complex of claim 1, wherein the ISS comprises the
sequence 5'-T, C, G-3'.
12. The IMP/MC complex of claim 1, wherein the ISS comprises the
sequence 5'-C, G, pyrimidine, pyrimidine, C, G-3'.
13. The IMP/MC complex of claim 1, wherein the ISS comprises the
sequence 5'-purine, purine, C, G, pyrimidine, pyrimidine, C,
G-3'.
14. The IMP/MC complex of claim 1, wherein the ISS comprises the
sequence SEQ ID NO:1.
15. A method of modulating an immune response in an individual
comprising administering to an individual an immunomodulatory
polynucleotide/microcarrier (IMP/MC) complex, said complex
comprising a polynucleotide comprising an immunostimulatory
sequence (ISS) linked to a biodegradable microcarrier (MC), wherein
the ISS comprises the sequence 5'-C, G-3' and wherein said MC is
less than 10 .mu.m in size, in an amount sufficient to modulate an
immune response in said individual.
16. The method of claim 15, wherein said microcarrier is a solid
phase microcarrier.
17. The method of claim 15, wherein said microcarrier is a liquid
phase microcarrier.
18. The method of claim 15, wherein the IMP/MC complex is
covalently linked.
19. The method of claim 15, wherein the IMP/MC complex is
non-covalently linked.
20. The method of claim 15, wherein said complex is
antigen-free.
21. The method of claim 15, wherein a Th1-type immune response is
stimulated.
22. The method of claim 15, wherein a Th2-type immune response is
suppressed.
23. The method of claim 15, wherein the ISS comprises the sequence
5'-T, C, G-3'.
24. The method of claim 15, wherein the ISS comprises the sequence
5'-C, G, pyrimidine, pyrimidine, C, G-3'.
25. The IMP/MC complex of claim 15, wherein the ISS comprises the
sequence 5'-purine, purine, C, G, pyrimidine, pyrimidine, C,
G-3'.
26. The IMP/MC complex of claim 15, wherein the ISS comprises the
sequence SEQ ID NO:1.
27. A method of increasing interferon-gamma (IFN-.gamma.) in an
individual, comprising: administering an effective amount of an
immunomodulatory polynucleotide/microcarrier (IMP/MC) complex to
said individual, said complex comprising a polynucleotide
comprising an immunostimulatory sequence (ISS) linked to a
biodegradable microcarrier (MC), wherein the ISS comprises the
sequence 5'-C, G-3', wherein said MC is less than 10 .mu.m in size
and wherein an effective amount is an amount sufficient to increase
IFN-.gamma. in said individual.
28. The method of claim 27, wherein said microcarrier is a solid
phase microcarrier.
29. The method of claim 27, wherein said microcarrier is a liquid
phase microcarrier.
30. The method of claim 27, wherein the IMP/MC complex is
covalently linked.
31. The method of claim 27, wherein the IMP/MC complex is
non-covalently linked.
32. The method of claim 27, wherein said complex is
antigen-free.
33. The method of claim 27, wherein the ISS comprises the sequence
5'-T, C, G-3'.
34. The method of claim 27, wherein the ISS comprises the sequence
5'-C, G, pyrimidine, pyrimidine, C, G-3'.
35. The IMP/MC complex of claim 27, wherein the ISS comprises the
sequence 5'-purine, purine, C, G, pyrimidine, pyrimidine, C,
G-3'.
36. The IMP/MC complex of claim 27, wherein the ISS comprises the
sequence SEQ ID NO:1.
37. A method of increasing interferon-alpha (IFN-.alpha.) in an
individual, comprising: administering an effective amount of an
immunomodulatory polynucleotide/microcarrier (IMP/MC) complex to
said individual, said complex comprising a polynucleotide
comprising an immunostimulatory sequence (ISS) linked to a
biodegradable microcarrier (MC), wherein the ISS comprises the
sequence 5'-C, G-3', wherein said MC is less than 10 .mu.m in size
and wherein an effective amount is an amount sufficient to increase
IFN-.alpha. in said individual.
38. The method of claim 37, wherein said individual has a viral
infection.
39. The method of claim 37, wherein said microcarrier is a solid
phase microcarrier.
40. The method of claim 37, wherein said microcarrier is a liquid
phase microcarrier.
41. The method of claim 37, wherein the IMP/MC complex is
covalently linked.
42. The method of claim 37, wherein the IMP/MC complex is
non-covalently linked.
43. The method of claim 37, wherein said complex is
antigen-free.
44. The method of claim 37, wherein the ISS comprises the sequence
5'-T, C, G-3'.
45. The method of claim 37, wherein the ISS comprises the sequence
5'-C, G, pyrimidine, pyrimidine, C, G-3'.
46. The IMP/MC complex of claim 37, wherein the ISS comprises the
sequence 5'-purine, purine, C, G, pyrimidine, pyrimidine, C,
G-3'.
47. The IMP/MC complex of claim 37, wherein the ISS comprises the
sequence SEQ ID NO:1.
48. A method of reducing levels of IgE in an individual,
comprising: administering an effective amount of an
immunomodulatory polynucleotide/microcarrier (IMP/MC) complex to
said individual, said complex comprising a polynucleotide
comprising an immunostimulatory sequence (ISS) linked to a
biodegradable microcarrier (MC), wherein the ISS comprises the
sequence 5'-C, G-3', wherein said MC is less than 10 .mu.m in size
and wherein an effective amount is an amount sufficient to reducing
levels of IgE in said individual.
49. The method of claim 48, wherein said microcarrier is a solid
phase microcarrier.
50. The method of claim 48, wherein said microcarrier is a liquid
phase microcarrier.
51. The method of claim 48, wherein the IMP/MC complex is
covalently linked.
52. The method of claim 48, wherein the IMP/MC complex is
non-covalently linked.
53. The method of claim 48, wherein said complex is
antigen-free.
54. The method of claim 48, wherein the ISS comprises the sequence
5'-T, C, G-3'.
55. The method of claim 48, wherein the ISS comprises the sequence
5'-C, G, pyrimidine, pyrimidine, C, G-3'.
56. The IMP/MC complex of claim 48, wherein the ISS comprises the
sequence 5'-purine, purine, C, G, pyrimidine, pyrimidine, C,
G-3'.
57. The IMP/MC complex of claim 48, wherein the ISS comprises the
sequence SEQ ID NO:1.
58. A kit, comprising: a container comprising an immunomodulatory
polynucleotide/microcarrier (IMP/MC) complex, wherein the ISS
comprises the sequence 5'-C, G-3', wherein said MC is a
biodegradable MC and wherein said MC is less than 10 .mu.m in size;
and instructions for use of IMP/MC complex in immunodulation of an
individual.
59. The kit of claim 58, wherein said polynucleotide is covalently
linked to said microcarrier.
60. The kit of claim 58, wherein said polynucleotide is
non-covalently linked to said microcarrier.
61. The kit of claim 58, wherein said microcarrier is a liquid
phase microcarrier.
62. The kit of claim 58, wherein said microcarrier is a solid phase
microcarrier.
63. The kit of claim 58, wherein said microcarrier is from 25 nm to
5 .mu.m in size.
64. The kit of claim 63, wherein said microcarrier is from 1.0
.mu.m to 2.0 .mu.m in size.
65. The kit of claim 64, wherein said microcarrier is 1.4 .mu.m in
size.
66. The kit of claim 58, wherein said microcarrier is cationic.
67. The kit of claim 58, wherein said complex is antigen-free.
68. The kit of claim 58, wherein the ISS comprises the sequence
5'-T, C, G-3'.
69. The kit of claim 58, wherein the ISS comprises the sequence
5'-C, G, pyrimidine, pyrimidine, C, G-3'.
70. The kit of claim 58, wherein the ISS comprises the sequence
5'-purine, purine, C, G, pyrimidine, pyrimidine, C, G-3'.
71. The kit of claim 58, wherein the ISS comprises the sequence SEQ
ID NO:1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
Provisional application 60/188,303, filed Mar. 10, 2000, which is
hereby incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The present invention relates to immunomodulatory
compositions comprising an immunostimulatory oligonucleotide
sequence (ISS) and methods of use thereof. In particular, the
invention relates to immunomodulatory compositions comprising an
ISS bound to a biodegradable microparticle. It also relates to the
administration of the polynucleotide/microcarrier complex to
modulate at least one immune response.
BACKGROUND ART
[0003] The type of immune response generated to infection or other
antigenic challenge can generally be distinguished by the subset of
T helper (Th) cells involved in the response. The Th1 subset is
responsible for classical cell-mediated functions such as
delayed-type hypersensitivity and activation of cytotoxic T
lymphocytes (CTLs), whereas the Th2 subset functions more
effectively as a helper for B-cell activation. The type of immune
response to an antigen is generally influenced by the cytokines
produced by the cells responding to the antigen. Differences in the
cytokines secreted by Th1 and Th2 cells are believed to reflect
different biological functions of these two subsets. See, for
example, Romagnani (2000) Ann. Allergy Asthma Immunol. 85:9-18.
[0004] The Th1 subset may be particularly suited to respond to
viral infections, intracellular pathogens, and tumor cells because
it secretes IL-2 and IFN-.gamma., which activate CTLs. The Th2
subset may be more suited to respond to free-living bacteria and
helminthic parasites and may mediate allergic reactions, since IL-4
and IL-5 are known to induce IgE production and eosinophil
activation, respectively. In general, Th1 and Th2 cells secrete
distinct patterns of cytokines and so one type of response can
moderate the activity of the other type of response. A shift in the
Th1/Th2 balance can result in an allergic response, for example,
or, alternatively, in an increased CTL response.
[0005] For many infectious diseases, such as tuberculosis and
malaria, Th2-type responses are of little protective value against
infection. Proposed vaccines using small peptides derived from the
target antigen and other currently used antigenic agents that avoid
use of potentially infective intact viral particles, do not always
elicit the immune response necessary to achieve a therapeutic
effect. The lack of a therapeutically effective human
immunodeficiency virus (HIV) vaccine is an unfortunate example of
this failure. Protein-based vaccines typically induce Th2-type
immune responses, characterized by high titers of neutralizing
antibodies but without significant cell-mediated immunity.
[0006] Moreover, some types of antibody responses are inappropriate
in certain indications, most notably in allergy where an IgE
antibody response can result in anaphylactic shock. Generally,
allergic responses also involve Th2-type immune responses. Allergic
responses, including those of allergic asthma, are characterized by
an early phase response, which occurs within seconds to minutes of
allergen exposure and is characterized by cellular degranulation,
and a late phase response, which occurs 4 to 24 hours later and is
characterized by infiltration of eosinophils into the site of
allergen exposure. Specifically, during the early phase of the
allergic response, allergen cross-links IgE antibodies on basophils
and mast cells, which in turn triggers degranulation and the
subsequent release of histamine and other mediators of inflammation
from mast cells and basophils. During the late phase response,
eosinophils infiltrate into the site of allergen exposure (where
tissue damage and dysfunction result).
[0007] Antigen immunotherapy for allergic disorders involves the
subcutaneous injection of small, but gradually increasing amounts,
of antigen. Such immunization treatments present the risk of
inducing IgE-mediated anaphylaxis and do not efficiently address
the cytokine-mediated events of the allergic late phase response.
Thus far, this approach has yielded only limited success.
[0008] Administration of certain DNA sequences, generally known as
immunostimulatory sequences or "ISS," induces an immune response
with a Th1-type bias as indicated by secretion of Th1-associated
cytokines. Administration of an immunostimulatory polynucleotide
with an antigen results in a Th1-type immune response to the
administered antigen. Roman et al. (1997) Nature Med. 3:849-854.
For example, mice injected intradermally with Escherichia coli (E.
coli) .beta.-galactosidase (.beta.-Gal) in saline or in the
adjuvant alum responded by producing specific IgG1 and IgE
antibodies, and CD4.sup.+ cells that secreted IL-4 and IL-5, but
not IFN-.gamma., demonstrating that the T cells were predominantly
of the Th2 subset. However, mice injected intradermally (or with a
tyne skin scratch applicator) with plasmid DNA (in saline) encoding
.beta.-Gal and containing an ISS responded by producing IgG2a
antibodies and CD4.sup.+ cells that secreted IFN-.gamma., but not
IL-4 and IL-5, demonstrating that the T cells were predominantly of
the Th1 subset. Moreover, specific IgE production by the plasmid
DNA-injected mice was reduced 66-75%. Raz et al. (1996) Proc. Natl.
Acad. Sci. USA 93:5141-5145. In general, the response to naked DNA
immunization is characterized by production of IL-2, TNF.alpha. and
IFN-.gamma. by antigen-stimulated CD4.sup.+ T cells, which is
indicative of a Th1-type response. This is particularly important
in treatment of allergy and asthma as shown by the decreased IgE
production. The ability of immunostimulatory polynucleotides to
stimulate a Th1-type immune response has been demonstrated with
bacterial antigens, viral antigens and with allergens (see, for
example, WO 98/55495).
[0009] ISS-containing oligonucleotides bound to microparticles
(SEPHAROSE.RTM. beads) have previously been shown to have
immunostimulatory activity in vitro (Liang et al., (1996), J. Clin.
Invest. 98:1119-1129). However, recent results show that
ISS-containing oligonucleotides bound to gold, latex and magnetic
particles are not active in stimulating proliferation of 7TD1
cells, which proliferate in response to ISS-containing
oligonucleotides (Manzel et al., (1999), Antisense Nucl. Acid Drug
Dev. 9:459-464).
[0010] Other references describing ISS include: Krieg et al. (1989)
J. Immunol. 143:2448-2451; Tokunaga et al. (1992) Microbiol.
Immunol. 36:55-66; Kataoka et al. (1992) Jpn. J. Cancer Res.
83:244-247; Yamamoto et al. (1992) J. Immunol. 148:4072-4076;
Mojcik et al. (1993) Clin. Immuno. and Immunopathol. 67:130-136;
Branda et al. (1993) Biochem. Pharmacol. 45:2037-2043; Pisetsky et
al. (1994) Life Sci. 54(2):101-107; Yamamoto et al. (1994a)
Antisense Research and Development. 4:119-122; Yamamoto et al.
(1994b) Jpn. J. Cancer Res. 85:775-779; Raz et al. (1994) Proc.
Natl. Acad. Sci. USA 91:9519-9523; Kimura et al. (1994) J. Biochem.
(Tokyo) 116:991-994; Krieg et al. (1995) Nature 374:546-549;
Pisetsky et al. (1995) Ann. N.Y. Acad. Sci. 772:152-163; Pisetsky
(1996a) J. Immunol. 156:421-423; Pisetsky (1996b) Immunity
5:303-310; Zhao et al. (1996) Biochem. Pharmacol. 51:173-182; Yi et
al. (1996) J. Immunol. 156:558-564; Krieg (1996) Trends Microbiol.
4(2):73-76; Krieg et al. (1996) Antisense Nucleic Acid Drug Dev.
6:133-139; Klinman et al. (1996) Proc. Natl. Acad. Sci. USA.
93:2879-2883; Raz et al. (1996); Sato et al. (1996) Science
273:352-354; Stacey et al. (1996) J. Immunol. 157:2116-2122; Ballas
et al. (1996) J. Immunol. 157:1840-1845; Branda et al. (1996) J.
Lab. Clin. Med. 128:329-338; Sonehara et al. (1996) J. Interferon
and Cytokine Res. 16:799-803; Klinman et al. (1997) J. Immunol.
158:3635-3639; Sparwasser et al. (1997) Eur. J. Immunol.
27:1671-1679; Roman et al. (1997); Carson et al. (1997) J. Exp.
Med. 186:1621-1622; Chace et al. (1997) Clin. Immunol. and
Immunopathol. 84:185-193; Chu et al. (1997) J. Exp. Med.
186:1623-1631; Lipford et al. (1997a) Eur. J. Immunol.
27:2340-2344; Lipford et al. (1997b) Eur. J. Immunol. 27:3420-3426;
Weiner et al. (1997) Proc. Natl. Acad. Sci. USA 94:10833-10837;
Macfarlane et al. (1997) Immunology 91:586-593; Schwartz et al.
(1997) J. Clin. Invest. 100:68-73; Stein et al. (1997) Antisense
Technology, Ch. 11 pp. 241-264, C. Lichtenstein and W. Nellen,
Eds., IRL Press; Wooldridge et al. (1997) Blood 89:2994-2998;
Leclerc et al. (1997) Cell. Immunol. 179:97-106; Kline et al.
(1997) J. Invest. Med. 45(3):282A; Yi et al. (1998a) J. Immunol.
160:1240-1245; Yi et al. (1998b) J. Immunol. 160:4755-4761; Yi et
al. (1998c) J. Immunol. 160:5898-5906; Yi et al. (1998d) J.
Immunol. 161:4493-4497; Krieg (1998) Applied Antisense
Oligonucleotide Technology Ch. 24, pp. 431-448, C. A. Stein and A.
M. Irieg, Eds., Wiley-Liss, Inc.; Krieg et al. (1998a) Trends
Microbiol. 6:23-27; Krieg et al. (1998b) J. Immunol. 161:2428-2434;
Krieg et al. (1998c) Proc. Natl. Acad. Sci. USA 95:12631-12636;
Spiegelberg et al. (1998) Allergy 53(45S):93-97; Homer et al.
(1998) Cell Immunol. 190:77-82; Jakob et al. (1998) J. Immunol.
161:3042-3049; Redford et al. (1998) J. Immunol. 161:3930-3935;
Weeratna et al. (1998) Antisense & Nucleic Acid Drug
Development 8:351-356; McCluskie et al. (1998) J. Immunol.
161(9):4463-4466; Gramzinski et al. (1998) Mol.Med. 4:109-118; Liu
et al. (1998) Blood 92:3730-3736; Moldoveanu et al. (1998) Vaccine
16: 1216-1224; Brazolot Milan et al. (1998) Proc. Natl. Acad. Sci.
USA 95:15553-15558; Briode et al. (1998) J. Immunol. 161:7054-7062;
Briode et al. (1999) Int. Arch. Allergy Immunol. 118:453-456;
Kovarik et al. (1999) J. Immunol. 162:1611-1617; Spiegelberg et al.
(1999) Pediatr. Pulmonol. Suppl. 18:118-121; Martin-Orozco et al.
(1999) Int. Immunol. 11:1111-1118; EP 468,520; WO 96/02555; WO
97/28259; WO 98/16247; WO 98/18810; WO 98/37919; WO 98/40100; WO
98/52581; WO 98/55495; WO 98/55609 and WO 99/11275. See also Elkins
et al. (1999) J. Immunol. 162:2291-2298, WO 98/52962, WO 99/33488,
WO 99/33868, WO 99/51259 and WO 99/62923. See also Zimmermann et
al. (1998) J. Immunol. 160:3627-3630; Krieg (1999) Trends
Microbiol. 7:64-65; U.S. Pat. Nos. 5,663,153, 5,723,335, 5,849,719
and 6,174,872. See also WO 99/56755, WO 00/06588, WO 00/16804; WO
00/21556; WO 00/67023 and WO 01/12223.
[0011] Additionally, Godard et al. (1995) Eur. J. Biochem.
232:404-410, discloses cholesterol-modified antisense
oligonucleotides bound to poly(isohexylcyanoacrylate)
nanoparticles.
[0012] All patents, patent applications, and publications cited
herein are hereby incorporated by reference in their entirety.
DISCLOSURE OF THE INVENTION
[0013] The invention relates to new compositions and methods for
modulating immune responses in individuals, particularly human
individuals.
[0014] In one aspect, the invention relates to compositions which
comprise immunomodulatory polynucleotide/microcarrier (IMP/MC)
complexes. An IMP/MC complex comprises a polynucleotide comprising
an immunostimulatory sequence (IMP) linked to a filterable,
biodegradable microcarrier (MC). The IMP may be covalently or
non-covalently linked to the microcarrier in the complex, and the
IMP may be modified to facilitate complex formation. Microcarriers
used in IMP/MC complexes are typically solid phase microcarriers,
although biodegradable liquid phase microcarriers (e.g., an oil in
water emulsion comprising a biodegradable polymer or oil) are also
contemplated. Microcarriers are generally less than about 50-60
.mu.m in size, and may be about 10 nm to about 10 .mu.m or about 25
nm to 5 .mu.m in size. In certain embodiments, the compositions of
the invention comprise an IMP/MC complex and a pharamceutically
acceptable excipient. In certain embodiments, the compositions of
the invention comprise an antigen-free IMP/MC complex, i.e., an
IMP/MC complex not linked to an antigen (either directly or
indirectly).
[0015] In another aspect, the invention relates to methods of
modulating an immune response in an individual, comprising
administering to an individual an IMP/MC complex in an amount
sufficient to modulate an immune response in said individual.
Immunomodulation according to the methods of the invention may be
practiced on individuals including those suffering from a disorder
associated with a Th2-type immune response (e.g., allergies or
allergy-induced asthma), individuals receiving vaccines such as
therapeutic vaccines (e.g., vaccines comprising an allergy epitope,
a mycobacterial epitope, or a tumor associated epitope) or
prophylactic vaccines, individuals with cancer, individuals having
an infectious disease and individuals at risk of exposure to an
infectious agent.
[0016] In a further aspect, the invention relates to methods of
increasing interferon-gamma (IFN-.gamma.) in an individual,
comprising administering an effective amount of an IMP/MC complex
to the individual. Administration of an IMP/MC complex in
accordance with the invention increases IFN-.gamma. in the
individual. Suitable subjects for these methods include those
individuals having idiopathic pulmonary fibrosis (IPF),
sclerodermna, cutaneous radiation-induced fibrosis, hepatic
fibrosis including schistosomiasis-induced hepatic fibrosis, renal
fibrosis as well as other conditions which may be improved by
administration of IFN-.gamma..
[0017] In another aspect, the invention relates to methods of
increasing IFN-.alpha. in an individual, comprising administering
an effective amount of an IMP/MC complex to the individual.
Administration of an IMP/MC complex in accordance with the
invention increases IFN-.alpha. levels in the individual. Suitable
subjects for these methods include those individuals having
disorders which respond to the administration of IFN-.alpha.,
including viral infections and cancer.
[0018] In another aspect, the invention relates to methods of
ameliorating one or more symptoms of an infectious disease,
comprising administering an effective amount of an IMP/MC complex
to an individual having an infectious disease. Administration of an
IMP/MC complex in accordance with the invention ameliorates one or
more symptoms of the infectious disease. The infectious diseases
which may be treated in accordance with the invention include
infectious diseases caused by a cellular pathogen (e.g., a
mycobacterial disease, malaria, leishmaniasis, toxoplasmosis,
schistosomiasis or clonorchiasis), and may include or exclude viral
diseases.
[0019] The invention further relates to kits for carrying out the
methods of the invention. The kits of the invention comprise a
container comprising an IMP/MC complex and instructions for use of
IMP/MC complex in immunodulation of an individual, for example when
the individual suffers from a disorder associated with a Th2-type
immune response (e.g., allergies or allergy-induced asthma), is
receiving vaccines such as therapeutic vaccines (e.g., vaccines
comprising an allergy epitope, a mycobacterial epitope, or a tumor
associated epitope) or prophylactic vaccines, suffers from cancer,
suffers from an infectious disease or is at risk of exposure to an
infectious agent.
Modes of Practicing the Invention
[0020] We have discovered new compositions and methods for
modulating immune responses in individuals, particularly humans.
The compositions of the invention comprise an immunomodulatory
polynucleotide (IMP) complexed with a biodegradable microcarrier
(MC). We have found that immunomodulatory polynucleotides combined
with nanometer-scale microcarriers (50 and 200 nm diameter beads)
efficiently modulate immune cells, including human cells. IMPs
combined with small microcarriers (approximately 1 to 4.5 .mu.m,
less than 2.0 .mu.m or about 1.5 .mu.m diameter) also
immunomodulated human cells. Our discovery is of particular
interest because human cells, as is known in the art, can be more
resistant to immunomodulation by IMPs than cells from commonly used
laboratory animals, such as mice.
[0021] We found that IMP/MC complexes were more effective at lower
doses than free IMP alone in immunomodulation. In human cells,
IMP/MC complexes were more active than free IMP in inducing
IFN-.alpha..
[0022] The IMP/MC complexes may include or exclude an antigen. In
some embodiments, the invention provides compositions comprising
antigen-free IMP/MC complexes, i.e., IMP/MC complexes not linked to
an antigen (directly or indirectly). In other embodiments, the
invention provides compositions comprising IMP/MC complexes mixed
with one or more antigens. In other embodiments, the invention
provides compositions comprising IMP/MC complexes linked to
antigen.
[0023] We have further found that covalently linked IMP/MC
complexes comprising nanocarrier particles are highly active
immunomodulators. Prior teaching in the art indicates that
immunostimulatory oligonucleotides tightly bound to microparticles
and nanoparticles are not effective (Manzel et al., supra). In view
of this understanding in the art, we believe that our results would
be surprising and unexpected to one of skill in the art.
[0024] The immunomodulatory polynucleotide/microcarrier (IMP/MC)
complexes of the invention may be covalently or non-covalently
linked, and comprise a microcarrier (e.g., a carrier of less than
about 10 .mu.m size) that is insoluble and/or filterable in water.
Microcarriers are generally solid phase (e.g., polylactic acid
beads), although biodegradable liquid phase microcarriers (e.g., an
oil in water emulsion comprising a biodegradable polymer or oil)
are also useful. The IMP may be modified to allow or augment
binding to the MC (e.g., by incorporation of a free sulfhydryl for
covalent crosslinking or addition of a hydrophobic moiety such as
cholesterol for hydrophobic bonding).
[0025] The invention provides new compositions comprising an IMP
covalently linked to a biodegradable microcarrier to form a
covalent IMP/MC complex. Linkage between the IMP and MC may be
direct (e.g., via disulfide bond between sulfhydryls on the IMP and
MC) or the constituents may be linked by a crosslinking moiety of
one or more atoms separating the bonds to the IMP and MC.
[0026] Also provided are compositions comprising an IMP
non-covalently linked to a microcarrier to provide a non-covalent
IMP/MC complex. Non-covalent IMP/MC complexes generally comprise an
IMP that has been modified to allow binding to the microcarrier
(e.g., by addition of a cholesterol moiety to the IMP to allow
hydrophobic binding to oil or lipid based microcarrier).
[0027] The invention also provides methods for modulating an immune
response in an individual by administering an IMP/MC complex to the
individual.
[0028] Further provided are kits for practicing the methods of the
invention. The kits comprise instructions for administering an
IMP/MC complex for immunomodulation in a subject and a package or
container comprising IMP/MC complex.
[0029] General Techniques
[0030] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as,
Molecular Cloning: A Laboratory Manual, second edition (Sambrook et
al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984);
Animal Cell Culture (R. I. Freshney, ed., 1987); Handbook of
Experimental Immunology (D. M. Weir & C. C. Blackwell, eds.);
Gene Transfer Vectors for Mammalian Cells (J. M. Miller & M. P.
Calos, eds., 1987); Current Protocols in Molecular Biology (F. M.
Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction,
(Mullis et al., eds., 1994); Current Protocols in Immunology (J. E.
Coligan et al., eds., 1991); The Immunoassay Handbook (D. Wild,
ed., Stockton Press NY, 1994); Bioconjugate Techniques (Greg T.
Hermanson, ed., Academic Press, 1996); and Methods of immunological
Analysis (R. Masseyeff, W. H. Albert, and N. A. Staines, eds.,
Weinheim: VCH Verlags gesellschaft mbH, 1993).
[0031] Definitions
[0032] As used herein, the singular form "a", "an", and "the"
includes plural references unless indicated otherwise. For example,
"an" ISS includes one or more ISS.
[0033] As used interchangeably herein, the terms "polynucleotide"
and "oligonucleotide" include single-stranded DNA (ssDNA),
double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) and
double-stranded RNA (dsRNA), modified oligonucleotides and
oligonucleosides or combinations thereof. The oligonucleotide can
be linearly or circularly configured, or the oligonucleotide can
contain both linear and circular segments. Oligonucleotides are
polymers of nucleosides joined, generally, through phosphoester
linkages, although alternate linkages, such as phosphorothioate
esters may also be used in oligonucleotides. A nucleoside consists
of a purine (adenine or guanine or derivative thereof) or
pyrimidine (thymine, cytosine or uracil, or derivative thereof)
base bonded to a sugar. The four nucleoside units (or bases) in DNA
are called deoxyadenosine, deoxyguanosine, deoxythymidine, and
deoxycytidine. A nucleotide is a phosphate ester of a
nucleoside.
[0034] The term "ISS" as used herein refers to polynucleotide
sequences that effect a measurable immune response as measured in
vitro, in vivo and/or ex vivo. Examples of measurable immune
responses include, but are not limited to, antigen-specific
antibody production, secretion of cytokines, activation or
expansion of lymphocyte populations such as NK cells, CD4+T
lymphocytes, CD8+T lymphocytes, B lymphocytes, and the like.
Preferably, the ISS sequences preferentially activate a Th1-type
response. A polynucleotide for use in the invention contains at
least one ISS. As used herein, "ISS" is also a shorthand term for
an ISS-containing polynucleotide.
[0035] The term "immunomodulatory polynucleotide" or "IMP", as used
herein, refers to a polynucleotide comprising at least one ISS. In
certain embodiments, the IMP is an ISS.
[0036] The term "microcarrier" refers to a biodegradable
particulate composition which is insoluble in water and which has a
size of less than about 50-60 .mu.m, preferably less than about 10,
5, 2.5, 2 or 1.5 .mu.m. Microcarriers include "nanocarriers", which
are microcarriers have a size of less than about 1 .mu.m,
preferably less than about 500 nm. Solid phase microcarriers may be
particles formed from biocompatible naturally occurring polymers,
synthetic polymers or synthetic copolymers, which may include or
exclude microcarriers formed from agarose or cross-linked agarose,
as well as other biodegradable materials known in the art.
Biodegradable solid phase microcarriers may be formed from polymers
which are degradable (e.g., poly(lactic acid), poly(glycolic acid)
and copolymers thereof) or erodible (e.g., poly(ortho esters such
as 3,9-diethylidene-2,4,8,10-tetraoxaspiro[5.5]undecane (DETOSU) or
poly(anhydrides), such as poly(anhydrides) of sebacic acid) under
mammalian physiological conditions. Microcarriers may also be
liquid phase (e.g., oil or lipid based), such liposomes, iscoms
(immune-stimulating complexes, which are stable complexes of
cholesterol, and phospholipid, adjuvant-active saponin) without
antigen, or droplets or micelles found oil in water or oil in water
in oil emulsions, provided the liquid phase microcarriers are
biodegradable. Biodegradable liquid phase microcarriers typically
incorporate a biodegradable oil, a number of which are known in the
art, including squalene and vegetable oils. Microcarriers are
typically spherical in shape, but microcarriers which deviate from
spherical shape are also acceptable (e.g., elipsoid, rod-shaped,
etc.). Due to their insoluble nature (with respect to water),
microcarriers are filterable from water and water-based (aqueous)
solutions.
[0037] The "size" of a microcarrier is generally the "design size"
or intended size of the particles stated by the manufacturer. Size
may be a directly measured dimension, such as average or maximum
diameter, or may be determined by an indirect assay such as a
filtration screening assay. Direct measurement of microcarrier size
is typically carried out by microscopy, generally light microscopy
or scanning electron microscopy (SEM), in comparison with particles
of known size or by reference to a micrometer. As minor variations
in size arise during the manufacturing process, microcarriers are
considered to be of a stated size if measurements show the
microcarriers are .+-. about 5-10% of the stated measurement. Size
characteristics may also be determined by dynamic light scattering.
Alternately, microcarrier size may be determined by filtration
screening assays. A microcarrier is less than a stated size if at
least 97% of the particles pass through a "screen-type" filter
(i.e., a filter in which retained particles are on the surface of
the filter, such as polycarbonate or polyethersulfone filters, as
opposed to a "depth filter" in which retained particles lodge
within the filter) of the stated size. A microcarrier is larger
than a stated size if at least about 97% of the microcarrier
particles are retained by a screen-type filter of the stated size.
Thus, at least about 97% microcarriers of about 10 .mu.m to about
10 nm in size pass through a 10 .mu.m pore screen filter and are
retained by a 10 nm screen filter.
[0038] As above discussion indicates, reference to a size or size
range for a microcarrier implicitly includes approximate variations
and approximations of the stated size and/or size range. This is
reflected by use of the term "about" when referring to a size
and/or size range, and reference to a size or size range without
reference to "about" does not mean that the size and/or size range
is exact.
[0039] A microcarrier is considered "biodegradable" if it is
degradable or erodable under normal mammalian physiological
conditions. Generally, a microcarrier is considered biodegradable
if it is degraded (i.e., loses at least 5% of its mass and/or
average polymer length) after a 72 hour incubation at 37.degree. C.
in normal human serum. Accordingly, and conversely, a microcarrier
is considered "nonbiodegradable" if it is not degraded or eroded
under normal mammalian physiological conditions. Generally, a
microcarrier is considered nonbiodegradable if it not degraded
(i.e., loses less than 5% of its mass and/or average polymer
length) after at 72 hour incubation at 37.degree. C. in normal
human serum.
[0040] The term "immunomodulatory polynucleotide/microcarrier
complex" or "IMP/MC complex" refers to a complex of an
ISS-containing polynucleotide and a microcarrier of the invention.
The components of the complex may be covalently or non-covalently
linked. Non-covalent linkages may be mediated by any non-covalent
bonding force, including by hydrophobic interaction, ionic
(electrostatic) bonding, hydrogen bonds and/or van der Waals
attractions. In the case of hydrophobic linkages, the linkage is
generally via a hydrophobic moiety (e.g., cholesterol) covalently
linked to the IMP.
[0041] The term "immunomodulatory" or "modulating an immune
response" as used herein includes immunostimulatory as well as
immunosuppressive effects. Immunomodulation is primarily a
qualitative alteration in an overall immune response, although
quantitative changes may also occur in conjunction with
immunomodulation. An immune response that is immunomodulated
according to the present invention is one that is shifted towards a
"Th1-type" immune response, as opposed to a "Th2-type" immune
response. Th1-type responses are typically considered cellular
immune system (e.g., cytotoxic lymphocytes) responses, while
Th2-type responses are generally "humoral", or antibody-based.
Th1-type immune responses are normally characterized by
"delayed-type hypersensitivity" reactions to an antigen, and can be
detected at the biochemical level by increased levels of
Th1-associated cytokines such as IFN-.gamma., IL-2, IL-12, and
TNF-.beta., as well as IFN-.alpha. and IL-6, although IL-6 may also
be associated with Th2-type responses as well. Th1-type immune
responses are generally associated with the production of cytotoxic
lymphocytes (CTLs) and low levels or transient production of
antibody. Th2-type immune responses are generally associated with
higher levels of antibody production, including IgE production, an
absence of or minimal CTL production, as well as expression of
Th2-associated cytokines such as IL-4. Accordingly,
immunomodulation in accordance with the invention may be recognized
by, for example, an increase in IFN-.gamma. and/or a decrease in
IgE production in an individual treated in accordance with the
methods of the invention as compared to the absence of
treatment.
[0042] The term "conjugate" refers to a complex in which an
ISS-containing polynucleotide and an antigen are linked. Such
conjugate linkages include covalent and/or non-covalent
linkages.
[0043] The term "antigen" means a substance that is recognized and
bound specifically by an antibody or by a T cell antigen receptor.
Antigens can include peptides, proteins, glycoproteins,
polysaccharides, complex carbohydrates, sugars, gangliosides,
lipids and phospholipids; portions thereof and combinations
thereof. The antigens can be those found in nature or can be
synthetic. Antigens suitable for administration with ISS include
any molecule capable of eliciting a B cell or T cell
antigen-specific response. Preferably, antigens elicit an antibody
response specific for the antigen. Haptens are included within the
scope of "antigen." A hapten is a low molecular weight compound
that is not immunogenic by itself but is rendered immunogenic when
conjugated with an immunogenic molecule containing antigenic
determinants. Small molecules may need to be haptenized in order to
be rendered antigenic. Preferably, antigens of the present
invention include peptides, lipids (e.g. sterols, fatty acids, and
phospholipids), polysaccharides such as those used in Hemophilus
influenza vaccines, gangliosides and glycoproteins.
[0044] "Adjuvant" refers to a substance which, when added to an
immunogenic agent such as antigen, nonspecifically enhances or
potentiates an immune response to the agent in the recipient host
upon exposure to the mixture.
[0045] The term "peptide" are polypeptides that are of sufficient
length and composition to effect a biological response, e.g.
antibody production or cytokine activity whether or not the peptide
is a hapten. Typically, the peptides are at least six amino acid
residues in length. The term "peptide" further includes modified
amino acids (whether or not naturally or non-naturally occurring),
such modifications including, but not limited to, phosphorylation,
glycosylation, pegylation, lipidization and methylation.
[0046] "Antigenic peptides" can include purified native peptides,
synthetic peptides, recombinant proteins, crude protein extracts,
attenuated or inactivated viruses, cells, micro-organisms, or
fragments of such peptides. An "antigenic peptide" or "antigen
polypeptide" accordingly means all or a portion of a polypeptide
which exhibits one or more antigenic properties. Thus, for example,
an "Amb a 1 antigenic polypeptide" or "Amb a 1 polypeptide antigen"
is an amino acid sequence from Amb a 1, whether the entire
sequence, a portion of the sequence, and/or a modification of the
sequence, which exhibits an antigenic property (i.e., binds
specifically to an antibody or a T cell receptor).
[0047] A "delivery molecule" or "delivery vehicle" is a chemical
moiety which facilitates, permits, and/or enhances delivery of an
IMP/MC complex to a particular site and/or with respect to
particular timing. A delivery vehicle may or may not additionally
stimulate an immune response.
[0048] An "allergic response to antigen" means an immune response
generally characterized by the generation of eosinophils and/or
antigen-specific IgE and their resultant effects. As is well-known
in the art, IgE binds to IgE receptors on mast cells and basophils.
Upon later exposure to the antigen recognized by the IgE, the
antigen cross-links the IgE on the mast cells and basophils causing
degranulation of these cells, including, but not limited, to
histamine release. It is understood and intended that the terms
"allergic response to antigen", "allergy", and "allergic condition"
are equally appropriate for application of some of the methods of
the invention. Further, it is understood and intended that the
methods of the invention include those that are equally appropriate
for prevention of an allergic response as well as treating a
pre-existing allergic condition.
[0049] As used herein, the term "allergen" means an antigen or
antigenic portion of a molecule, usually a protein, which elicits
an allergic response upon exposure to a subject. Typically the
subject is allergic to the allergen as indicated, for instance, by
the wheal and flare test or any method known in the art. A molecule
is said to be an allergen even if only a small subset of subjects
exhibit an allergic (e.g., IgE) immune response upon exposure to
the molecule. A number of isolated allergens are known in the art.
These include, but are not limited to, those provided in Table 1
herein.
[0050] The term "desensitization" refers to the process of the
administration of increasing doses of an allergen to which the
subject has demonstrated sensitivity. Examples of allergen doses
used for desensitization are known in the art, see, for example,
Fornadley (1998) Otolaryngol. Clin. North Am. 31:111-127.
[0051] "Antigen-specific immunotherapy" refers to any form of
immunotherapy which involves antigen and generates an
antigen-specific modulation of the immune response. In the allergy
context, antigen-specific immunotherapy includes, but is not
limited to, desensitization therapy.
[0052] An "individual" is a vertebrate, preferably a mammal, more
preferably a human. Mammals include, but are not limited to,
humans, primates, farm animals, sport animals, rodents and pets.
Vertebrates also include, but are not limited to, birds (i.e.,
avian individuals) and reptiles (i.e., reptilian individuals).
[0053] An "effective amount" or a "sufficient amount" of a
substance is that amount sufficient to effect beneficial or desired
results, including clinical results, and, as such, an "effective
amount" depends upon the context in which it is being applied. In
the context of administering a composition that modulates an immune
response to an antigen, an effective amount of an IMP/MC complex is
an amount sufficient to achieve such a modulation as compared to
the immune response obtained when the antigen is administered
alone. An effective amount can be administered in one or more
administrations.
[0054] The term "co-administration" as used herein refers to the
administration of at least two different substances sufficiently
close in time to modulate an immune response. Preferably,
co-administration refers to simultaneous administration of at least
two different substances.
[0055] "Stimulation" of an immune response, such as Th1 response,
means an increase in the response, which can arise from eliciting
and/or enhancement of a response.
[0056] An "IgE associated disorder" is a physiological condition
which is characterized, in part, by elevated IgE levels, which may
or may not be persistent. IgE associated disorders include, but are
not limited to, allergy and allergic reactions, allergy-related
disorders (described below), asthma, rhinitis, conjunctivitis,
urticaria, shock, Hymenoptera sting allergies, and drug allergies,
and parasite infections. The term also includes related
manifestations of these disorders. Generally, IgE in such disorders
is antigen-specific.
[0057] An "allergy-related disorder" means a disorder resulting
from the effects of an antigen-specific IgE immune response. Such
effects can include, but are not limited to, hypotension and shock.
Anaphylaxis is an example of an allergy-related disorder during
which histamine released into the circulation causes vasodilation
as well as increased permeability of the capillaries with resultant
marked loss of plasma from the circulation. Anaphylaxis can occur
systemically, with the associated effects experienced over the
entire body, and it can occur locally, with the reaction limited to
a specific target tissue or organ.
[0058] The term "viral disease", as used herein, refers to a
disease which has a virus as its etiologic agent. Examples of viral
diseases include hepatitis B, hepatitis C, influenza, acquired
immunodeficiency syndrome (AIDS), and herpes zoster.
[0059] As used herein, and as well-understood in the art,
"treatment" is an approach for obtaining beneficial or desired
results, including clinical results. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, alleviation or amelioration of one or more
symptoms, diminishment of extent of disease, stabilized (i.e., not
worsening) state of disease, preventing spread of disease, delay or
slowing of disease progression, amelioration or palliation of the
disease state, and remission (whether partial or total), whether
detectable or undetectable. "Treatment" can also mean prolonging
survival as compared to expected survival if not receiving
treatment.
[0060] "Palliating" a disease or disorder means that the extent
and/or undesirable clinical manifestations of a disorder or a
disease state are lessened and/or time course of the progression is
slowed or lengthened, as compared to not treating the disorder.
Especially in the allergy context, as is well understood by those
skilled in the art, palliation may occur upon modulation of the
immune response against an allergen(s). Further, palliation does
not necessarily occur by administration of one dose, but often
occurs upon administration of a series of doses. Thus, an amount
sufficient to palliate a response or disorder may be administered
in one or more administrations.
[0061] An "antibody titer", or "amount of antibody", which is
"elicited" by an IMP/MC complex refers to the amount of a given
antibody measured at a time point after administration of IMP/MC
complex.
[0062] A "Th1-associated antibody" is an antibody whose production
and/or increase is associated with a Th1 immune response. For
example, IgG2a is a Th1-associated antibody in mouse. For purposes
of this invention, measurement of a Th1-associated antibody can be
measurement of one or more such antibodies. For example, in human,
measurement of a Th1-associated antibody could entail measurement
of IgG1 and/or IgG3.
[0063] A "Th2-associated antibody" is an antibody whose production
and/or increase is associated with a Th2 immune response. For
example, IgG1 is a Th2-associated antibody in mouse. For purposes
of this invention, measurement of a Th2-associated antibody can be
measurement of one or more such antibodies. For example, in human,
measurement of a Th2-associated antibody could entail measurement
of IgG2 and/or IgG4.
[0064] To "suppress" or "inhibit" a function or activity, such as
cytokine production, antibody production, or histamine release, is
to reduce the function or activity when compared to otherwise same
conditions except for a condition or parameter of interest, or
alternatively, as compared to another condition. For example, an
IMP/MC complex administered with an antigen or including an antigen
which suppresses histamine release reduced histamine release as
compared to, for example, histamine release induced by antigen
alone.
[0065] As used herein, the term "comprising" and its cognates are
used in their inclusive sense; that is, equivalent to the term
"including" and its corresponding cognates.
[0066] Compositions of the Invention
[0067] The invention provides new compositions for modulating
immune response in individuals. The new compositions are
immunomodulatory polynucleotide/microcarrier (IMP/MC) complexes
which comprise an ISS-containing polynucleotide complexed to a
biodegradable microcarrier. IMP/MC complexes may be covalent
complexes, in which the IMP portion of the complex is covalently
bonded to the MC, either directly or via a linker (i.e.,
indirectly), or they may be non-covalent complexes.
[0068] Immunomodulatory Polynucleotides
[0069] In accordance with the present invention, the
immunomodulatory polynucleotide contains at least one ISS, and can
contain multiple ISSs. The ISSs can be adjacent within the
polynucleotide, or they can be separated by additional nucleotide
bases within the polynucleotide. Accordingly, an IMP may contain
combinations of any one or more ISS described herein, including
those with modifications. In certain embodiments, the IMP consists
of an ISS.
[0070] ISS have been described in the art and may be readily
identified using standard assays which indicate various aspects of
the immune response, such as cytokine secretion, antibody
production, NK cell activation and T cell proliferation. See, e.g.,
WO 97/28259; WO 98/16247; WO 99/11275; Krieg et al. (1995) Nature
374:546-549; Yamamoto et al. (1992a); Ballas et al. (1996); Klinman
et al. (1997); Sato et al. (1996); Pisetsky (1996a); Shimada et al.
(1986) Jpn. J. Cancer Res. 77:808-816; Cowdery et al. (1996) J.
Immunol. 156:4570-4575; Roman et al. (1997); and Lipford et al.
(1997a).
[0071] The ISS can be of any length greater than 6 bases or base
pairs and generally comprises the sequence 5'-cytosine, guanine-3',
preferably greater than 15 bases or base pairs, more preferably
greater than 20 bases or base pairs in length. As is well-known in
the art, the cytosine of the 5'-cytosine, guanine-3' sequence is
unmethylated. An ISS may also comprise the sequence 5'-purine,
purine, C, G, pyrimidine, pyrimidine, C, G-3'. An ISS may also
comprise the sequence 5'-purine, purine, C, G, pyrimidine,
pyrimidine, C, C-3'. As indicated in polynucleotide sequences
below, an ISS may comprise (i.e., contain one or more of) the
sequence 5'-T, C, G-3'. In some embodiments, an ISS may comprise
the sequence 5'-C, G, pyrimidine, pyrimidine, C, G-3' (such as
5'-CGTTCG-3'). In some embodiments, an ISS may comprise the
sequence 5'-C, G, pyrimidine, pyrimidine, C, G, purine, purine-3'.
In some embodiments, an ISS comprises the sequence 5'-purine,
purine, C, G, pyrimidine, pyrimidine-3' (such as 5'-AACGTT-3').
[0072] In some embodiments, an ISS may comprise the sequence
5'-purine, T, C, G, pyrimidine, pyrimidine-3'.
[0073] In some embodiments, the ISS comprises any of the following
sequences:
1 GACGCTCC; GACGTCCC; GACGTTCC; GACGCCCC; AGCGTTCC; AGCGCTCC;
AGCGTCCC; AGCGCCCC; AACGTCCC; AACGCCCC; AACGTTCC; AACGCTCC;
GGCGTTCC; GGCGCTCC; GGCGTCCC; GGCGCCCC; GACGCTCG; GACGTCCG;
GACGCCCG; GACGTTCG; AGCGCTCG; AGCGTTCG; AGCGTCCG; AGCGCCCG;
AACGTCCG; AACGCCCG; AACGTTCG; AACGCTCG; GGCGTTCG; GGCGCTCG;
GGCGTCCG; GGCGCCCG.
[0074] In some embodiments, the ISS comprises any of the following
sequences:
2 GACGCT; GACGTC; GACGTT; GACGCC; GACGCU; GACGUC; GACGUU; GACGUT;
GACGTU; AGCGTT; AGCGCT; AGCGTC; AGCGCC; AGCGUU; AGCGCU; AGCGUC;
AGCGUT; AGCGTU; AACGTC; AACGCC; AACGTT; AACGCT; AACGUC; AACGUU;
AACGCU; AACGUT; AACGTU; GGCGTT; GGCGCT; GGCGTC; GGCGCC; GGCGUU;
GGCGCU; GGCGUC; GGCGUT; GGCGTU.
[0075] In some embodiments, the ISS comprises any of the following
sequences:
3 GABGCTCC; GABGTCCC; GABGTTCC; GABGCCCC; AGBGTTCC; AGBGCTCC;
AGBGTCCC; AGBGCCCC; AABGTCCC; AABGCCCC; AABGTTCC; AABGCTCC;
GGBGTTCC; GGBGCTCC; GGBGTCCC; GGBGCCCC; GABGCTCG; GABGTCCG;
GABGCCCG; GABGTTCG; AGBGCTCG; AGBGTTCG; AGBGTCCG; AGBGCCCG;
AABGTCCG; AABGCCCG; AABGTTCG; AABGCTCG; GGBGTTCG; GGBGCTCG;
GGBGTCCG; GGBGCCCG; GABGCTBG; GABGTCGB; GABGCCBG; GABGTTBG;
AGBGCTBG; AGBGTTBG; AGBGTCBG; AGBGCCBG; AABGTCBG; AABGCCBG;
AABGTTBG; AABGCTBG; GGBGTTBG; GGBGCTBG; GGBGTCBG; GGBGCCBG, where B
is 5-bromocytosine.
[0076] In some embodiments, the ISS comprises any of the following
sequences:
4 GABGCUCC; GABGUCCC; GABGUTCC; GABGTUCC; GABGUUCC; AGBGUUCC;
AGBGTUCC; AGBGUTCC; AGBGCUCC; AGBGUCCC; AABGUCCC; AABGUUCC;
AABGUTCC; AABGTUCC; AABGCUCC; GGBGUUCC; GGBGUTCC; GGBGTUCC;
GGBGCUCC; GGBGUCCC; GABGCUCG; GABGUCCG; GABGUUCG; GABGUTCG;
GABGTUCG; AGBGCUCG; AGBGUUCG; AGBGUTCG; AGBGTUCG; AGBGUCCG;
AABGUCCG; AABGUUCG; AABGUTCG; AABGTUCG; AABGCUCG; GGBGUUCG;
GGBGUTCG; GGBGTUCG; GGBGCUCG; GGBGUCCG; GABGCUBG; GABGUCBG;
GABGUUBG; GABGUTBG; GABGTUBG; AGBGCUBG; AGBGUUBG; AGBGUCBG;
AGBGUTBG; AGBGTUBG; AABGUCBG; AABGUUBG; AABGUTBG; AABGTUBG;
AABGCUBG; GGBGUUBG; GGBGUTBG; GGBGTUBG; GGBGCUBG; GGBGUCBG, where B
is 5-bromocytosine.
[0077] In some embodiments, the immunomodulatory polynucleotide
comprises the sequence 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO:1).
In other embodiments, the ISS comprises any of the sequences:
5 5'-TGACCGTGAACGTTCGAGATGA-3':; (SEQ ID NO:2)
5'-TCATCTCGAACGTTCCACAGTCA-3'; (SEQ ID NO:3)
5'-TGACTGTGAACGTTCCAGATGA-3' (SEQ ID NO:4)
5'-TCCATAACGTTCGCCTAACGTTCGTC-3' (SEQ ID NO:5)
5'-TGACTGTGAABGTTCCAGATGA-3', (SEQ ID NO:6) where B is
5-bromocytosine; 5'-TGACTGTGAABGTTCGAGATGA-3', (SEQ ID NO:7) where
B is 5-bromocytosine and 5'-TGACTGTGAABGTTBGAGATGA-3', (SEQ ID
NO:8) where B is 5-bromocytosine.
[0078] An ISS and/or IMP may contain modifications. Modifications
of ISS include any known in the art, 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. Various such modifications
are described below.
[0079] An ISS and/or IMP may be single stranded or double stranded
DNA, as well as single or double-stranded RNA or other modified
polynucleotides. An ISS may or may not include one or more
palindromic regions, which may be present in the motifs described
above or may extend beyond the motif. An ISS may comprise
additional flanking sequences, some of which are described herein.
An ISS may contain naturally-occurring or modified, non-naturally
occurring bases, and may contain modified sugar, phosphate, and/or
termini. For example, phosphate modifications include, but are not
limited to, methyl phosphonate, phosphorothioate, phosphoramidate
(bridging or non-bridging), phosphotriester and phosphorodithioate
and may be used in any combination. Other non-phosphate linkages
may also be used. Preferably, oligonucleotides of the present
invention comprise phosphorothioate backbones. Sugar modifications
known in the field, such as 2'-alkoxy-RNA analogs, 2'-amino-RNA
analogs and 2'-alkoxy- or amino-RNA/DNA chimeras and others
described herein, may also be made and combined with any phosphate
modification. Examples of base modifications include, but are not
limited to, addition of an electron-withdrawing moiety to C-5
and/or C-6 of a cytosine of the ISS (e.g., 5-bromocytosine,
5-chlorocytosine, 5-fluorocytosine, 5-iodocytosine). See, for
example, International Patent Application No. WO 99/62923.
[0080] The ISS and/or IMP can be synthesized using techniques and
nucleic acid synthesis equipment which are well known in the art
including, but not limited to, enzymatic methods, chemical methods,
and the degradation of larger oligonucleotide sequences. See, for
example, Ausubel et al. (1987); and Sambrook et al. (1989). When
assembled enzymatically, the individual units can be ligated, for
example, with a ligase such as T4 DNA or RNA ligase. U.S. Pat. No.
5,124,246. Oligonucleotide degradation can be accomplished through
the exposure of an oligonucleotide to a nuclease, as exemplified in
U.S. Pat. No. 4,650,675.
[0081] The ISS and/or IMP can also be isolated using conventional
polynucleotide isolation procedures. Such procedures include, but
are not limited to, hybridization of probes to genomic or cDNA
libraries to detect shared nucleotide sequences, antibody screening
of expression libraries to detect shared structural features and
synthesis of particular native sequences by the polymerase chain
reaction.
[0082] Circular IMP can be isolated, synthesized through
recombinant methods, or chemically synthesized. Where the circular
IMP is obtained through isolation or through recombinant methods,
the IMP will preferably be a plasmid. The chemical synthesis of
smaller circular oligonucleotides can be performed using any method
described in the literature. See, for instance, Gao et al. (1995)
Nucleic Acids Res. 23:2025-2029; and Wang et al. (1994) Nucleic
Acids Res. 22:2326-2333.
[0083] The techniques for making oligonucleotides and modified
oligonucleotides are known in the art. Naturally occurring DNA or
RNA, containing phosphodiester linkages, is generally synthesized
by sequentially coupling the appropriate nucleoside phosphoramidite
to the 5'-hydroxy group of the growing oligonucleotide attached to
a solid support at the 3'-end, followed by oxidation of the
intermediate phosphite triester to a phosphate triester. Once the
desired oligonucleotide sequence has been synthesized, the
oligonucleotide is removed from the support, the phosphate triester
groups are deprotected to phosphate diesters and the nucleoside
bases are deprotected using aqueous ammonia or other bases. See,
for example, Beaucage (1993) "Oligodeoxyribonucleotide Synthesis"
in Protocols for Oligonucleotides and Analogs, Synthesis and
Properties (Agrawal, ed.) Humana Press, Totowa, N.J.; Warner et al.
(1984) DNA 3:401 and U.S. Pat. No. 4,458,066.
[0084] The ISS and/or IMP can also contain phosphate-modified
oligonucleotides. Synthesis of polynucleotides containing modified
phosphate linkages or non-phosphate linkages is also know in the
art. For a review, see Matteucci (1997) "Oligonucleotide Analogs:
an Overview" in Oligonucleotides as Therapeutic Agents, (D. J.
Chadwick and G. Cardew, ed.) John Wiley and Sons, New York, N.Y.
The phosphorous derivative (or modified phosphate group) which can
be attached to the sugar or sugar analog moiety in the
oligonucleotides of the present invention can be a monophosphate,
diphosphate, triphosphate, alkylphosphonate, phosphorothioate,
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 in detail. Peyrottes et al. (1996)
Nucleic Acids Res. 24:1841-1848; Chaturvedi et al. (1996) Nucleic
Acids Res. 24:2318-2323; and Schultz et al. (1996) Nucleic Acids
Res. 24:2966-2973. For example, synthesis of phosphorothioate
oligonucleotides is similar to that described above for naturally
occurring oligonucleotides except that the oxidation step is
replaced by a sulfurization step (Zon (1993) "Oligonucleoside
Phosphorothioates" in Protocols for Oligonucleotides and Analogs,
Synthesis and Properties (Agrawal, ed.) Humana Press, pp. 165-190).
Similarly the synthesis of other phosphate analogs, such as
phosphotriester (Miller et al. (1971) JACS 93:6657-6665),
non-bridging phosphoramidates (Jager et al. (1988) Biochem.
27:7247-7246), N3' to P5' phosphoramidiates (Nelson et al. (1997)
JOC 62:7278-7287) 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. (1989) Nucleic
Acids Res. 17:6129-6141). Oligonucleotides with phosphorothioate
backbones can be more immunogenic than those with phosphodiester
backbones and appear to be more resistant to degradation after
injection into the host. Braun et al. (1988) J. Immunol.
141:2084-2089; and Latimer et al. (1995) Mol. Immunol.
32:1057-1064.
[0085] ISS-containing polynucleotides and/or IMPs used in the
invention can comprise ribonucleotides (containing ribose as the
only or principal sugar component), deoxyribonucleotides
(containing deoxyribose as the principal sugar component), or, as
is known in the art, modified sugars or sugar analogs can be
incorporated in the ISS. Thus, in addition to ribose and
deoxyribose, the sugar moiety can be pentose, deoxypentose, hexose,
deoxyhexose, glucose, arabinose, xylose, lyxose, and a sugar
"analog" cyclopentyl group. The sugar can be in pyranosyl or in a
furanosyl form. In the ISS, the sugar moiety is preferably the
faranoside of ribose, deoxyribose, arabinose or 2'-0-alkylribose,
and the sugar can be attached to the respective heterocyclic bases
either in .alpha. or .beta. anomeric configuration. Sugar
modifications include, but are not limited to, 2'-alkoxy-RNA
analogs, 2'-amino-RNA analogs and 2'-alkoxy- or amino-RNA/DNA
chimeras. 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 can pertain to any specific example. Sugar
modifications may also be made and combined with any phosphate
modification in the preparation of an ISS and/or IMP.
[0086] The heterocyclic bases, or nucleic acid bases, which are
incorporated in the ISS and/or IMP can 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.
[0087] 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)
are available in the art, and that as long as other criteria of the
present invention are satisfied, the ISS can 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 ISS includes, but is not limited to,
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 ISS via the 9-position, the
pyrimidines via the 1-position, the pyrrolopyrimidines via the
7-position and the pyrazolopyrimidines via the 1-position.
[0088] The ISS and/or IMP may comprise at least one modified base
as described, for example, in the commonly owned international
application WO 99/62923. As used herein, the term "modified base"
is synonymous with "base analog", for example, "modified cytosine"
is synonymous with "cytosine analog." Similarly, "modified"
nucleosides or nucleotides are herein defined as being synonymous
with nucleoside or nucleotide "analogs." Examples of base
modifications include, but are not limited to, addition of an
electron-withdrawing moiety to C-5 and/or C-6 of a cytosine of the
ISS. Preferably, the electron-withdrawing moiety is a halogen. Such
modified cytosines can include, but are not limited to,
azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine,
chlorinated cytosine, cyclocytosine, cytosine arabinoside,
5-fluorocytosine, fluoropyrimidine, fluorouracil,
5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil,
5-nitrocytosine, uracil, and any other pyrimidine analog or
modified pyrimidine.
[0089] 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 been described (including,
but not limited to, e.g., U.S. Pat. Nos. 4,849,513, 5,015,733,
5,118,800, 5,118,802) and can be used similarly.
[0090] In some embodiments, an IMP is less than about any of the
following lengths (in bases or base pairs): 10,000; 5,000; 2500;
2000; 1500; 1250; 1000; 750; 500; 300; 250; 200; 175; 150; 125;
100; 75; 50; 25; 10. In some embodiments, an IMP is greater than
about any of the following lengths (in bases or base pairs): 8; 10;
15; 20; 25; 30; 40; 50; 60; 75; 100; 125; 150; 175; 200; 250; 300;
350; 400; 500; 750; 1000; 2000; 5000; 7500; 10000; 20000; 50000.
Alternately, the ISS can be any of a range of sizes having an upper
limit of 10,000; 5,000; 2500; 2000; 1500; 1250; 1000; 750; 500;
300; 250; 200; 175; 150; 125; 100; 75; 50; 25; or 10 and an
independently selected lower limit of 8; 10; 15; 20; 25; 30; 40;
50; 60; 75; 100; 125; 150; 175; 200; 250; 300; 350; 400; 500; 750;
1000; 2000; 5000; 7500, wherein the lower limit is less than the
upper limit.
[0091] Microcarriers
[0092] Microcarriers useful in the invention are less than about
50-60 .mu.m in size, preferably less than about 10 .mu.m in size,
and are insoluble in pure water. Microcarriers used in the
invention are biodegradable. Microcarriers are commonly solid
phase, such as "beads" or other particles, although biodegradable
liquid phase microcarriers such as oil in water emulsions
comprising a biodegradable polymers or oils are also contemplated.
A wide variety of biodegradable materials acceptable for use as
microcarriers are known in the art.
[0093] Microcarriers for use in the compositions or methods of the
invention are generally less than about 10 .mu.m in size (e.g.,
have an average diameter of less than about 10 .mu.m, or at least
about 97% of the particles pass through a 10 .mu.m screen filter),
and include nanocarriers (i.e., carriers of less than about 1 .mu.m
size). Preferably, microcarriers are selected having sizes within
an upper limit of about 9, 7, 5, 2, or 1 .mu.m or 900, 800, 700,
600, 500, 400, 300, 250, 200, or 100 nm and an independently
selected lower limit of about 4, 2, or 1 .mu.m or about 800, 600,
500, 400, 300, 250, 200, 150, 100, 50, 25, or 10 nm, where the
lower limit is less than the upper limit. In some embodiments, the
microcarriers have a size of about 1.0-1.5 .mu.m, about 1.0-2.0
.mu.m or about 0.9-1.6 .mu.m. In certain preferred embodiments, the
microcarriers have a size of about 10 nm to about 5 .mu.m or about
25 nm to about 4.5 .mu.m, about 1 .mu.m, about 1.2 .mu.m, about 1.4
.mu.m, about 1.5 .mu.m, about 1.6 .mu.m, about 1.8 .mu.m, about 2.0
.mu.m, about 2.5 .mu.m or about 4.5 .mu.m. When the microcarriers
are nanocarriers, preferred embodiments include nanocarriers of
about 25 to about 300 nm, 50 to about 200 nm, about 50 nm or about
200 nm.
[0094] Solid phase biodegradable microcarriers may be manufactured
from biodegradable polymers including, but not limited to:
biodegradable polyesters, such as poly(lactic acid), poly(glycolic
acid), and copolymers (including block copolymers) thereof, as well
as block copolymers of poly(lactic acid) and poly(ethylene glycol);
polyorthoesters such as polymers based on
3,9-diethylidene-2,4,8,10-tetra- oxaspiro[5.5]undecane (DETOSU);
polyanhydrides such as poly(anhydride) polymers based on relatively
hydrophilic monomers such as sebacic acid; polyanhydride imides,
such as polyanhydride polymers based on sebacic acid-derived
monomers incorporating amino acids (i.e., linked to sebacic acid by
imide bonds through the amino-terminal nitrogen) such as glycine or
alanine; polyanhydride esters; polyphosphazenes, especially
poly(phosphazenes) which contain hydrolysis-sensitive ester groups
which can catalyze degradation of the polymer backbone through
generation of carboxylic acid groups (Schacht et al., (1996)
Biotechnol. Bioeng. 1996:102); and polyamides such as poly(lactic
acid-co-lysine).
[0095] Solid phase microspheres are prepared using techniques known
in the art. For example, they can be prepared by emulsion-solvent
extraction/evaporation technique. Generally, in this technique,
biodegradable polymers such as polyanhydrates,
poly(alkyl-.alpha.-cyanoac- rylates) and poly(.alpha.-hydroxy
esters), for example, poly(lactic acid), poly(glycolic acid),
poly(D,L-lactic-co-glycolic acid) and poly(caprolactone), are
dissolved in a suitable organic solvent, such as methylene
chloride, to constitute the dispersed phase (DP) of emulsion. DP is
emulsified by high-speed homogenization into excess volume of
aqueous continuous phase (CP) that contains a dissolved surfactant,
for example, polyvinylalcohol (PVA) or polyvinylpirrolidone (PVP).
Surfactant in CP is to ensure the formation of discrete and
suitably-sized emulsion droplet. The organic solvent is then
extracted into the CP and subsequently evaporated by raising the
system temperature. The solid microparticles are then separated by
centrifugation or filtration, and dried, for example, by
lyophilization or application of vaccum, before storing at
4.degree. C.
[0096] Physico-chemical characteristics such as mean size, size
distribution and surface charge of dried microspheres may be
determined. Size characteristics are determined, for example, by
dynamic light scattering technique and the surface charge was
determined by measuring the zeta potential.
[0097] Liquid phase microcarriers include liposomes, micelles, oil
droplets and other lipid or oil-based particles which incorporate
biodegradable polymers or oils. In certain embodiments, the
biodegradable polymer is a surfactant. In other embodiments, the
liquid phase microcarriers are biodegradable due to the inclusion
of a biodegradable oil such as squalene or a vegetable oil. One
preferred liquid phase microcarrier is oil droplets within an
oil-in-water emulsion. Preferably, oil-in-water emulsions used as
microcarriers comprise biodegradable substituents such as
squalene.
[0098] Antigen
[0099] IMP/MC complexes may be prepared which comprise antigen or
which are antigen-free, i.e., IMP/MC complexes not linked to an
antigen. Any antigen may be used in the preparation of IMP/MC
complexes comprising antigen.
[0100] In some embodiments, the antigen is an allergen. Examples of
recombinant allergens are provided in Table 1. Preparation of many
allergens is well-known in the art, including, but not limited to,
preparation of ragweed pollen allergen Antigen E (Amb al) (Rafnar
et al. (1991) J. Biol. Chem. 266:1229-1236), major dust mite
allergens Der pI and Der PII (Chua et al. (1988) J. Exp. Med.
167:175-182; Chua et al. (1990) Int. Arch. Allergy Appl. Immunol.
91:124-129), white birch pollen Bet v1 (Breiteneder et al. (1989)
EMBO J. 8:1935-1938), domestic cat allergen Fel d I (Rogers et al.
(1993) Mol. Immunol. 30:559-568), and protein antigens from tree
pollen (Elsayed et al. (1991) Scand. J. Clin. Lab. Invest. Suppl.
204:17-31). As indicated, allergens from trees are known, including
allergens from birch, juniper and Japanese cedar. Preparation of
protein antigens from grass pollen for in vivo administration has
been reported. Malley (1989) J. Reprod. Immunol. 16:173-186. As
Table 1 indicates, in some embodiments, the allergen is a food
allergen such as peanut allergen, for example Ara h I, and in some
embodiments, the allergen is a grass allergen such as a rye
allergen, for example Lol p 1. Table 1 shows a list of allergens
that may be used.
6TABLE 1 RECOMBINANT ALLERGENS Group Allergen Reference ANIMALS:
CRUSTACEA Shrimp/lobster tropomyosin Leung et al. (1996) J. Allergy
Clin. Immunol. 98: 954-961 Pan s I Leung et al. (1998) Mol. Mar.
Biol. Biotechnol. 7: 12-20 INSECTS Ant Sol i 2 (venom) Schmidt et
al. J Allergy Clin Immunol., 1996, 98: 82-8 Bee Phospholipase A2
Muller et al. J Allergy Clin Immunol, 1995, 96: 395-402 (PLA)
Forster et al. J Allergy Clin Immunol, 1995, 95: 1229-35 Muller et
al. Clin Exp Allergy, 1997, 27: 915-20 Hyaluronidase (Hya)
Soldatova et al. J Allergy Clin Immunol, 1998, 101: 691-8 Cockroach
Bla g Bd9OK Helm et al. J Allergy Clin Immunol, 1996, 98: 172-180
Bla g 4 (a calycin) Vailes et al. J Allergy Clin Immunol, 1998,
101: 274-280 Glutathione S- Arruda et al. J Biol Chem, 1997, 272:
20907-12 transferase Per a 3 Wu et al. Mol Immunol, 1997, 34: 1-8
Dust mite Der p 2 (major allergen) Lynch et al. J Allergy Clin
Immunol, 1998, 101: 562-4 Hakkaart et al. Clin Exp Allergy, 1998,
28: 169-74 Hakkaart et al. Clin Exp Allergy, 1998, 28: 45-52
Hakkaart et al. Int Arch Allergy Immunol, 1998, 115 (2): 150-6
Mueller et al. J Biol Chem, 1997, 272: 26893-8 Der p2 variant Smith
et al. J Allergy Clin Immunol, 1998, 101: 423-5 Der f2 Yasue et al.
Clin Exp Immunol, 1998, 113: 1-9 Yasue et al. Cell Immunol, 1997,
181: 30-7 Der p10 Asturias et al. Biochim Biophys Acta, 1998, 1397:
27-30 Tyr p 2 Eriksson et al. Eur J Biochem, 1998 Hornet Antigen 5
aka Dol m V Tomalski et al. Arch Insect Biochem Physiol, 1993,
(venom) 22: 303-13 Mosquito Aed a I (salivary Xu et al. Int Arch
Allergy Immunol, 1998, 115: 245-51 apyrase) Yellow jacket antigen
5, King et al. J Allergy Clin Immunol, 1996, 98: 588-600
hyaluronidase and phospholipase (venom) MAMMALS Cat Fel d I Slunt
et al. J Allergy Clin Immunol, 1995, 95: 1221-8 Hoffmann et al.
(1997) J Allergy Clin Immunol 99: 227-32 Hedlin Curr Opin Pediatr,
1995, 7: 676-82 Cow Bos d 2 (dander; a Zeiler et al. J Allergy Clin
Immunol, 1997, 100: 721-7 lipocalin) Rautiainen et al. Biochem
Bioph. Res Comm., 1998, 247: 746-50 .beta.-lactoglobulin (BLG,
Chatel et al. Mol Immunol, 1996, 33: 1113-8 major cow milk Lehrer
et al. Crit Rev Food Sci Nutr, 1996, 36: 553-64 allergen) Dog Can f
I and Can f 2, Konieczny et al. Immunology, 1997, 92: 577-86
salivary lipocalins Spitzauer et al. J Allergy Clin Immunol, 1994,
93: 614-27 Vrtala et al. J Immunol, 1998, 160: 6137-44 Horse Equ c1
(major allergen, Gregoire et al. J Biol Chem, 1996, 271: 32951-9 a
lipocalin) Mouse mouse urinary protein Konieczny et al. Immunology,
1997, 92: 577-86 (MUP) OTHER MAMMALIAN ALLERGENS Insulin Ganz et
al. J Allergy Clin Immunol, 1990, 86: 45-51 Grammer et al. J Lab
Clin Med, 1987,109: 141-6 Gonzalo et al. Allergy, 1998, 53: 106-7
Interferons interferon alpha 2c Detmar et al. Contact Dermatis,
1989, 20: 149-50 MOLLUSCS topomyosin Leung et al. J Allergy Clin
Immunol, 1996, 98: 954-61 PLANT ALLERGENS: Barley Hor v 9 Astwood
et al. Adv Exp Med Biol, 1996, 409: 269-77 Birch pollen allergen,
Bet v 4 Twardosz et al. Biochem Bioph. Res Comm., 1997, 23 9: 197
rBet v 1 Bet v 2: Pauli et al. J Allergy Clin Immunol, 1996, 97:
1100-9 (profilin) van Neerven et al. Clin Exp Allergy, 1998, 28:
423-33 Jahn-Schmid et al. Immunotechnology, 1996, 2: 103-13
Breitwieser et al. Biotechniques, 1996, 21: 918-25 Fuchs et al. J
Allergy Clin Immunol, 1997, 100: 3 56-64 Brazil nut globulin
Bartolome et al. Allergol Immunopathol, 1997, 25: 135-44 Cherry Pru
a I (major allergen) Scheurer et al. Mol Immunol, 1997, 34: 619-29
Corn Zml3 (pollen) Heiss et al. FEBS Lett, 1996, 381: 217-21 Lehrer
et al. Int Arch Allergy Immunol, 1997, 113: 122-4 Grass Phl p 1,
Phl p 2, Phl p 5 Bufe et al. Am J Respir Crit Care Med, 1998, 157:
1269-76 (timothy grass pollen) Vrtala et al. J Immunol Jun 15,
1998, 160: 6137-44 Niederberger et al. J Allergy Clin Immun., 1998,
101: 258- 64 Hol 1 5 velvet grass Schramm et al. Eur J Biochem,
1998, 252: 200-6 pollen Bluegrass allergen Zhang et al. J Immunol,
1993, 151: 791-9 Cyn d 7 Bermuda grass Smith et al. Int Arch
Allergy Immunol, 1997, 114: 265-71 Cyn d 12 (a profilin) Asturias
et al. Clin Exp Allergy, 1997, 27: 1307-13 Fuchs et al. J Allergy
Clin Immunol, 1997, 100: 356-64 Juniper Jun o 2 (pollen) Tinghino
et al. J Allergy Clin Immunol, 1998, 101: 772-7 Latex Hev b 7 Sowka
et al. Eur J Biochem, 1998, 255: 213-9 Fuchs et al. J Allergy Clin
Immunol, 1997, 100: 356-64 Mercurialis Mer a I (profilin) Vallverdu
et al. J Allergy Clin Immunol, 1998, 101: 3 63- 70 Mustard Sin a I
(seed) Gonzalez de la Pena et al. Biochem Bioph. Res Comm.,
(Yellow) 1993, 190: 648-53 Oilseed rape Bra r I pollen allergen
Smith et al. Int Arch Allergy Immunol, 1997, 114: 265-71 Peanut Ara
h I Stanley et al. Adv Exp Med Biol, 1996, 409: 213-6 Burks et al.
J Clin Invest, 1995, 96: 1715-21 Burks et al. Int Arch Allergy
Immunol, 1995, 107: 248-50 Poa pratensis Poa p9 Parronchi et al.
Eur J Immunol, 1996, 26: 697-703 Astwood et al. Adv Exp Med Biol,
1996, 409: 269-77 Ragweed Amb a I Sun et al. Biotechnology Aug,
1995, 13: 779-86 Hirschwehr et al. J Allergy Clin Immunol, 1998,
101: 196- 206 Casale et al. J Allergy Clin Immunol, 1997, 100:
110-21 Rye Lol p I Tamborini et al. Eur J Biochem, 1997, 249:
886-94 Walnut Jug r I Teuber et al. J Allergy Clin Immun., 1998,
101: 807-14 Wheat allergen Fuchs et al. J Allergy Clin Immunol,
1997, 100: 356-64 Donovan et al. Electrophoresis, 1993, 14: 917-22
FUNGI: Aspergillus Asp f 1, Asp f 2, Asp Crameri et al. Mycoses,
1998, 41 Suppl 1: 56-60 f3, Asp f 4, rAsp f 6 Hemmann et al. Eur J
Immunol, 1998, 28: 1155-60 Banerjee et al. J Allergy Clin Immunol,
1997, 99: 821-7 Crameri Int Arch Allergy Immunol, 1998, 115: 99-114
Crameri et al. Adv Exp Med Biol, 1996, 409: 111-6 Moser et al. J
Allergy Clin Immunol, 1994, 93: 1-11 Manganese superoxide Mayer et
al. Int Arch Allergy Immunol, 1997, 113: 213-5 dismutase (MNSOD)
Blomia allergen Caraballo et al. Adv Exp Med Biol, 1996, 409: 81-3
Penicillinium allergen Shen et al. Clin Exp Allergy, 1997, 27:
682-90 Psilocybe Psi c 2 Horner et al. Int Arch Allergy Immunol,
1995, 107: 298- 300
[0101] In some embodiments, the antigen is from an infectious
agent, including protozoan, bacterial, fungal (including
unicellular and multicellular), and viral infectious agents.
Examples of suitable viral antigens are described herein and are
known in the art. Bacteria include Hemophilus influenza,
Mycobacterium tuberculosis and Bordetella pertussis. Protozoan
infectious agents include malarial plasmodia, Leishmania species,
Trypanosoma species and Schistosoma species. Fungi include Candida
albicans.
[0102] In some embodiments, the antigen is a viral antigen. Viral
polypeptide antigens include, but are not limited to, HIV proteins
such as HIV gag proteins (including, but not limited to, membrane
anchoring (MA) protein, core capsid (CA) protein and nucleocapsid
(NC) protein), HIV polymerase, influenza virus matrix (M) protein
and influenza virus nucleocapsid (NP) protein, hepatitis B surface
antigen (HBsAg), hepatitis B core protein (HBcAg), hepatitis e
protein (HBeAg), hepatitis B DNA polymerase, hepatitis C antigens,
and the like. References discussing influenza vaccination include
Scherle and Gerhard (1988) Proc. Natl. Acad. Sci. USA 85:4446-4450;
Scherle and Gerhard (1986) J. Exp. Med. 164:1114-1128; Granoff et
al. (1993) Vaccine 11:S46-51; Kodihalli et al. (1997) J. Virol.
71:3391-3396; Ahmeida et al. (1993) Vaccine 11:1302-1309; Chen et
al. (1999) Vaccine 17:653-659; Govorkova and Smirnov (1997) Acta
Virol. (1997) 41:251-257; Koide et al. (1995) Vaccine 13:3-5;
Mbawuike et al. (1994) Vaccine 12:1340-1348; Tamura et al. (1994)
Vaccine 12:310-316; Tamura et al. (1992) Eur. J. Immunol.
22:477-481; Hirabayashi et al. (1990) Vaccine 8:595-599. Other
examples of antigen polypeptides are group- or sub-group specific
antigens, which are known for a number of infectious agents,
including, but not limited to, adenovirus, herpes simplex virus,
papilloma virus, respiratory syncytial virus and poxviruses.
[0103] Many antigenic peptides and proteins are known, and
available in the art; others can be identified using conventional
techniques. For immunization against tumor formation or treatment
of existing tumors, immunomodulatory peptides can include tumor
cells (live or irradiated), tumor cell extracts, or protein
subunits of tumor antigens such as Her-2/neu, Mart1,
carcinoembryonic antigen (CEA), gangliosides, human milk fat
globule (HMFG), mucin (MUC1), MAGE antigens, BAGE antigens, GAGE
antigens, gp100, prostate specific antigen (PSA), and tyrosinase.
Vaccines for immuno-based contraception can be formed by including
sperm proteins administered with ISS. Lea et al. (1996) Biochim.
Biophys. Acta 1307:263.
[0104] Attenuated and inactivated viruses are suitable for use
herein as the antigen. Preparation of these viruses is well-known
in the art and many are commercially available (see, e.g.,
Physicians' Desk Reference (1998) 52nd edition, Medical Economics
Company, Inc.). For example, polio virus is available as IPOL.RTM.
(Pasteur Merieux Connaught) and ORIMUNE.RTM. (Lederle
Laboratories), hepatitis A virus as VAQTA.RTM. (Merck), measles
virus as ATTENUVAX.RTM. (Merck), mumps virus as MUMPSVAX.RTM.
(Merck) and rubella virus as MERUVAX.RTM.II (Merck). Additionally,
attenuated and inactivated viruses such as HIV-1, HIV-2, herpes
simplex virus, hepatitis B virus, rotavirus, human and non-human
papillomavirus and slow brain viruses can provide peptide
antigens.
[0105] In some embodiments, the antigen comprises a viral vector,
such as vaccinia, adenovirus, and canary pox.
[0106] Antigens may be isolated from their source using
purification techniques known in the art or, more conveniently, may
be produced using recombinant methods.
[0107] Antigenic peptides can include purified native peptides,
synthetic peptides, recombinant proteins, crude protein extracts,
attenuated or inactivated viruses, cells, micro-organisms, or
fragments of such peptides. Immunomodulatory peptides can be native
or synthesized chemically or enzymatically. Any method of chemical
synthesis known in the art is suitable. Solution phase peptide
synthesis can be used to construct peptides of moderate size or,
for the chemical construction of peptides, solid phase synthesis
can be employed. Atherton et al. (1981) Hoppe Seylers Z. Physiol.
Chem. 362:833-839. Proteolytic enzymes can also be utilized to
couple amino acids to produce peptides. Kullmann (1987) Enzymatic
Peptide Synthesis, CRC Press, Inc. Alternatively, the peptide can
be obtained by using the biochemical machinery of a cell, or by
isolation from a biological source. Recombinant DNA techniques can
be employed for the production of peptides. Hames et al. (1987)
Transcription and Translation: A Practical Approach, IRL Press.
Peptides can also be isolated using standard techniques such as
affinity chromatography.
[0108] Preferably the antigens are peptides, lipids (e.g., sterols
excluding cholesterol, fatty acids, and phospholipids),
polysaccharides such as those used in H. influenza vaccines,
gangliosides and glycoproteins. 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
of the molecules are commercially available.
[0109] Examples of viral antigens useful in the subject
compositions and methods using the compositions include, but are
not limited to, HIV antigens. Such antigens include, but are not
limited to, those antigens derived from HIV envelope glycoproteins
including, but not limited to, gp160, gp120 and gp41. Numerous
sequences for HIV genes and antigens are known. For example, the
Los Alamos National Laboratory HIV Sequence Database collects,
curates and annotates HIV nucleotide and amino acid sequences. This
database is accessible via the internet, at
http://hiv-web.lanl.gov/, and in a yearly publication, see Human
Retroviruses and AIDS Compendium (for example, 1998 edition).
[0110] Antigens derived from infectious agents may be obtained
using methods known in the art, for example, from native viral or
bacterial extracts, from cells infected with the infectious agent,
from purified polypeptides, from recombinantly produced
polypeptides and/or as synthetic peptides.
[0111] IMP/MC complex formulations may be prepared with other
immunotherapeutic agents including, but not limited to, cytokine,
adjuvants and antibodies. These IMP/MC complex formulations may be
prepared with or without antigen.
[0112] IMP/MC Complexes
[0113] IMP/MC complexes comprise an IMP bound to the surface of a
microcarrier (i.e., the IMP is not encapsulated in the MC), and
preferably comprise multiple molecules of IMP bound to each
microcarrier. In certain embodiments, a mixture of different IMPs
may be complexed with a microcarrier, such that the microcarrier is
bound to more than one IMP species. The bond between the IMP and MC
may be covalent or non-covalent. As will be understood by one of
skill in the art, the IMP may be modified or derivatized and the
composition of the microcarrier may be selected and/or modified to
accommodate the desired type of binding desired for IMP/MC complex
formation.
[0114] Covalently bonded IMP/MC complexes may be linked using any
covalent crosslinking technology known in the art. Typically, the
IMP portion will be modified, either to incorporate an additional
moiety (e.g., a free amine, carboxyl or sulfhydryl group) or
incorporate modified (e.g., phosphorothioate) nucleotide bases to
provide a site at which the IMP portion may be linked to the
microcarrier. The link between the IMP and MC portions of the
complex can be made at the 3' or 5' end of the IMP, or at a
suitably modified base at an internal position in the IMP. The
microcarrier is generally also modified to incorporate moieties
through which a covalent link may be formed, although functional
groups normally present on the microcarrier may also be utilized.
The IMP/MC is formed by incubating the IMP with a microcarrier
under conditions which permit the formation of a covalent complex
(e.g., in the presence of a crosslinking agent or by use of an
activated microcarrier comprising an activated moiety which will
form a covalent bond with the IMP).
[0115] A wide variety of crosslinking technologies are known in the
art, and include crosslinkers reactive with amino, carboxyl and
sulfhydryl groups. As will be apparent to one of skill in the art,
the selection of a crosslinking agent and crosslinking protocol
will depend on the configuration of the IMP and the microcarrier as
well as the desired final configuration of the IMP/MC complex. The
crosslinker may be either homobifunctional or heterobifunctional.
When a homobifunctional crosslinker is used, the crosslinker
exploits the same moiety on the IMP and MC (e.g., an aldehyde
crosslinker may be used to covalently link an IMP and MC where both
the IMP and MC comprise one or more free amines).
[0116] Heterobifunctional crosslinkers utilize different moieties
on the the IMP and MC, (e.g., a maleimido-N-hydroxysuccinimide
ester may be used to covalently link a free sulfhydryl on the IMP
and a free amine on the MC), and are preferred to minimize
formation of inter-microcarrier bonds. In most cases, it is
preferable to crosslink through a first crosslinking moiety on the
microcarrier and a second crosslinking moiety on the IMP, where the
second crosslinking moiety is not present on the microcarrier. One
preferred method of producing the IMP/MC complex is by `activating`
the microcarrier by incubating with a heterobifunctional
crosslinking agent, then forming the IMP/MC complex by incubating
the IMP and activated MC under conditions appropriate for reaction.
The crosslinker may incorporate a "spacer" arm between the reactive
moieties, or the two reactive moieties in the crosslinker may be
directly linked.
[0117] In one preferred embodiment, the IMP portion comprises at
least one free sulfhydryl (e.g., provided by a 5'-thiol modified
base or linker) for crosslinking to the microcarrier, while the
microcarrier comprises free amine groups. A heterobifunctional
crosslinker reactive with these two groups (e.g., a crosslinker
comprising a maleimide group and a NHS-ester), such as succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carbo- xylate is used to
activate the MC, then covalently crosslink the IMP to form the
IMP/MC complex.
[0118] Non-covalent IMP/MC complexes may be linked by any
non-covalent binding or interaction, including ionic
(electrostatic) bonds, hydrophobic interactions, hydrogen bonds,
van der Waals attractions, or a combination of two or more
different interactions, as is normally the case when a binding pair
is to link the IMP and MC.
[0119] Preferred non-covalent IMP/MC complexes are typically
complexed by hydrophobic or electrostatic (ionic) interactions, or
a combination thereof, (e.g., through base pairing between an IMP
and a polynucleotide bound to an MC use of a binding pair). Due to
the hydrophilic nature of the backbone of polynucleotides, IMP/MC
complexes which rely on hydrophobic interactions to form the
complex generally require modification of the IMP portion of the
complex to incorporate a highly hydrophobic moiety. Preferably, the
hydrophobic moiety is biocompatible, nonimmunogenic, and is
naturally occurring in the individual for whom the composition is
intended (e.g., is found in mammals, particularly humans). Examples
of preferred hydrophobic moieties include lipids, steroids, sterols
such as cholesterol, and terpenes. The method of linking the
hydrophobic moiety to the IMP will, of course, depend on the
configuration of the IMP and the identity of the hydrophobic
moiety. The hydrophobic moiety may be added at any convenient site
in the IMP, preferably at either the 5' or 3' end; in the case of
addition of a cholesterol moiety to an IMP, the cholesterol moiety
is preferably added to the 5' end of the IMP, using conventional
chemical reactions (see, for example, Godard et al. (1995) Eur. J.
Biochem. 232:404-410). Preferably, microcarriers for use in IMP/MC
complexes linked by hydrophobic bonding are made from hydrophobic
materials, such as oil droplets or hydrophobic polymers, although
hydrophilic materials modified to incorporate hydrophobic moieties
may be utilized as well. When the microcarrier is a liposome or
other liquid phase microcarrier comprising a lumen, the IMP/MC
complex is formed by mixing the IMP and the MC after preparation of
the MC, in order to avoid encapsulation of the IMP during the MC
preparation process.
[0120] Non-covalent IMP/MC complexes bound by electrostatic binding
typically exploit the highly negative charge of the polynucleotide
backbone. Accordingly, microcarriers for use in non-covalently
bound IMP/MC complexes are generally positively charged (e.g.,
cationic) at physiological pH (e.g., about pH 6.8-7.4). The
microcarrier may intrinsically possess a positive charge, but
microcarriers made from compounds not normally possessing a
positive charge may be derivatized or otherwise modified to become
positively charged (e.g., cationic). For example, the polymer used
to make the microcarrier may be derivatized to add positively
charged groups, such as primary amines. Alternately, positively
charged compounds may be incorporated in the formulation of the
microcarrier during manufacture (e.g., positively charged
surfactants may be used during the manufacture of poly(lactic
acid)/poly(glycolic acid) copolymers to confer a positive charge on
the resulting microcarrier particles, as described, for example, in
Example 5). Thus, microcarriers may comprise a positively charged
moiety.
[0121] Generally, to prepare cationic microspheres, cationic lipids
or polymers, for example,
1,2-dioleoyl-1,2,3-trimethylammoniopropane (DOTAP),
cetyltrimethylammonium bromide (CTAB) or polylysine, are added
either to DP or CP, as per their solubility in these phases.
[0122] Generally, ISS-containing polynucleotides can be adsorbed
onto the cationic microspheres by overnight aqueous incubation of
ISS and the particles at 4.degree. C. Microspheres are
characterized for size and surface charge before and after ISS
association. Selected batches may then evaluated for activity
against suitable controls in, for example, established human
peripheral blood mononuclear cell (PBMC) and mouse splenocyte
assays, as described herein. The formulations may also evaluated in
suitable animal models.
[0123] Non-covalent IMP/MC complexes linked by nucleotide base
pairing may be produced using conventional methodologies.
Generally, base-paired IMP/MC complexes are produced using a
microcarrier comprising a bound, preferably a covalently bound,
polynucleotide (the "capture polynucleotide") that is at least
partially complementary to the IMP. The segment of complementarity
between the IMP and the capture nucleotide is preferably at least
6, 8, 10 or 15 contiguous base pairs, more preferably at least 20
contiguous base pairs. The capture nucleotide may be be bound to
the MC by any method known in the art, and is preferably covalently
bound to the IMP at the 5' or 3' end.
[0124] In other embodiments, a binding pair may be used to link the
IMP and MC in an IMP/MC complex. The binding pair may be a receptor
and ligand, an antibody and antigen (or epitope), or any other
binding pair which binds at high affinity (e.g., K.sub.d less than
about 10.sup.-8). One type of preferred binding pair is biotin and
streptavidin or biotin and avidin, which form very tight complexes.
When using a binding pair to mediate IMP/MC complex binding, the
IMP is derivatized, typically by a covalent linkage, with one
member of the binding pair, and the MC is derivatized with the
other member of the binding pair. Mixture of the two derivatized
compounds results in IMP/MC complex formation.
[0125] Many IMP/MC complex embodiments do not include an antigen,
and certain embodiments exclude antigen(s) associated with the
disease or disorder which is the object of the IMP/MC complex
therapy. In further embodiments, the IMP is also bound to one or
more antigen molecules. Antigen may be coupled with the IMP portion
of an IMP/MC complex in a variety of ways, including covalent
and/or non-covalent interactions, as described, for example, in WO
98/16247. Alternately, the antigen may be linked to the
microcarrier (either directly or indirectly).
[0126] The link between the antigen and the IMP in IMP/MC complexes
comprising an antigen bound to the IMP can be made at the 3' or 5'
end of the IMP, or at a suitably modified base at an internal
position in the IMP. If the antigen is a peptide and 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 IMP, specific coupling at one or more residues can be
achieved.
[0127] Alternatively, modified nucleosides or nucleotides, such as
are known in the art, can be incorporated at either terminus, or at
internal positions in the IMP. 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, the
antigen of interest.
[0128] Where the antigen is a peptide, this portion of the
conjugate can be attached to the 3'-end of the IMP through solid
support chemistry. For example, the IMP portion can be added to a
polypeptide portion that has been pre-synthesized on a support.
Haralambidis et al. (1990a) Nucleic Acids Res. 18:493-499; and
Haralambidis et al. (1990b) Nucleic Acids Res. 18:501-505.
Alternatively, the IMP 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 IMP from the support, a
terminal thiol group is left at the 3'-end of the oligonucleotide
(Zuckermann et al. (1987) Nucleic Acids Res. 15:5305-5321; and
Corey et al. (1987) Science 238:1401-1403) or a terminal amino
group is left at the 3'-end of the oligonucleotide (Nelson et al.
(1989) Nucleic Acids Res. 17:1781-1794). Conjugation of the
amino-modified IMP to amino groups of the peptide can be performed
as described in Benoit et al. (1987) Neuromethods 6:43-72.
Conjugation of the thiol-modified IMP to carboxyl groups of the
peptide can be performed as described in Sinha et al. (1991), pp.
185-210, Oligonucleotide Analogues: A Practical Approach, IRL
Press. Coupling of an oligonucleotide carrying an appended
maleimide to the thiol side chain of a cysteine residue of a
peptide has also been described. Tung et al. (1991) Bioconjug.
Chem. 2:464-465.
[0129] The peptide portion of the conjugate can be attached to the
5'-end of the IMP through an amine, thiol, or carboxyl group that
has been incorporated into the oligonucleotide during its
synthesis. Preferably, while the oligonucleotide 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. (1986)
Nucleic Acids Res. 14:6227-6245; Connolly (1985) Nucleic Acids Res.
13:4485-4502; Kremsky et al. (1987) Nucleic Acids Res.
15:2891-2909; Connolly (1987) Nucleic Acids Res. 15:3131-3139;
Bischoff et al. (1987) Anal. Biochem. 164:336-344; Blanks et al.
(1988) Nucleic Acids Res. 16:10283-10299; and U.S. Pat. Nos.
4,849,513, 5,015,733, 5,118,800, and 5,118,802. Subsequent to
deprotection, the amine, thiol, and carboxyl functionalities can be
used to covalently attach the oligonucleotide to a peptide. Benoit
et al. (1987); and Sinha et al. (1991).
[0130] An IMP-antigen conjugate can also be formed through
non-covalent interactions, such as ionic bonds, hydrophobic
interactions, hydrogen bonds and/or van der Waals attractions.
[0131] Non-covalently linked conjugates can include a non-covalent
interaction such as a biotin-streptavidin complex. A biotinyl group
can be attached, for example, to a modified base of an ISS. Roget
et al. (1989) Nucleic Acids Res. 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 oligonucleotide.
[0132] Non-covalent associations can also occur through ionic
interactions involving an IMP and residues within the antigen, such
as charged amino acids, or through the use of a linker portion
comprising charged residues that can interact with both the
oligonucleotide and the antigen. For example, non-covalent
conjugation can occur between a generally negatively-charged ISS
and positively-charged amino acid residues of a peptide, e.g.,
polylysine, polyarginine and polyhistidine residues.
[0133] Non-covalent conjugation between IMP and antigens can occur
through DNA binding motifs of molecules that interact with DNA as
their natural ligands. For example, such DNA binding motifs can be
found in transcription factors and anti-DNA antibodies.
[0134] The linkage of the IMP 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. (1988) Nucleic Acids Symp. Ser. 19:189-192),
oligonucleotide-fatty acid conjugates (Grabarek et al. (1990) Anal.
Biochem. 185:131-135; and Staros et al. (1986) Anal. Biochem.
156:220-222), and oligonucleotide-sterol conjugates. Boujrad et al.
(1993) Proc. Natl. Acad. Sci. USA 90:5728-5731.
[0135] The linkage of the IMP to an 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. (1985) J. Applied Biochem.
7:347-355.
[0136] The linkage of a circular IMP to a peptide or antigen can be
formed in several ways. Where the circular IMP is synthesized using
recombinant or chemical methods, a modified nucleoside is suitable.
Ruth (1991), pp. 255-282, in Oligonucleotides and Analogues: A
Practical Approach, IRL Press. Standard linking technology can then
be used to connect the circular IMP to the antigen or other
peptide. Goodchild (1990) Bioconjug. Chem. 1:165. Where the
circular IMP 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 other
peptide.
[0137] Additional methods for the attachment of peptides and other
molecules to oligonucleotides can be found in U.S. Pat. No.
5,391,723; Kessler (1992) "Nonradioactive labeling methods for
nucleic acids" in Kricka (ed.) Nonisotopic DNA Probe Techniques,
Academic Press; and Geoghegan et al. (1992) Bioconjug. Chem.
3:138-146.
[0138] Methods of the Invention
[0139] The invention provides methods of modulating an immune
response in an individual, preferably a mammal, more preferably a
human, comprising administering to the individual an IMP/MC complex
(typically in a composition comprising the complex and a
pharmaceutically acceptable excipient) such that the desired
modulation of the immune response is achieved. Immunomodulation may
include stimulating a Th1-type immune response and/or inhibiting or
reducing a Th2-type immune response.
[0140] In some embodiments, the immune modulation comprises
stimulating a (i.e., one or more) Th1-associated cytokine, such as
IFN-.gamma., IL-12 and/or IFN-.alpha.. In some embodiments, the
immune modulation comprises suppressing production of a (i.e., one
or more) Th2-associated cytokine, such as IL-4 and/or IL-5.
Measuring these parameters uses methods standard in the art and has
been discussed herein.
[0141] As described herein, administration of IMP/MC may further
comprise administration of one or more additional immunotherapeutic
agents (i.e., an agent which acts via the immune system and/or is
derived from the immune system) including, but not limited to,
cytokine, adjuvants and antibodies. Examples of therapeutic
antibodies include those used in the cancer context (e.g.,
anti-tumor antibodies). Administration of such additional
immunotherapeutic agents applies to all the methods described
herein.
[0142] In certain embodiments, the individual suffers from a
disorder associated with a Th2-type immune response, such as
allergies or allergy-induced asthma. Administration of an IMP/MC
complex results in immunomodulation, increasing levels of one or
more Th1-type response associated cytokines, which may result in a
reduction of the Th2-type response features associated with the
individual's response to the allergen. Immunomodulation of
individuals with Th2-type response associated disorders results in
a reduction or improvement in one or more of the symptoms of the
disorder. Where the disorder is allergy or allergy-induced asthma,
improvement in one or more of the symptoms includes a reduction one
or more of the following: rhinitis, allergic conjunctivitis,
circulating levels of IgE, circulating levels of histamine and/or
requirement for `rescue` inhaler therapy (e.g., inhaled albuterol
administered by metered dose inhaler or nebulizer).
[0143] In further embodiments, the individual subject to the
immunomodulatory therapy of the invention is an individual
receiving a vaccine. The vaccine may be a prophylactic vaccine or a
therapeutic vaccine. A prophylactic vaccine comprises one or more
epitopes associated with a disorder for which the individual may be
at risk (e.g., M. tuberculosis antigens as a vaccine for prevention
of tuberculosis). Therapeutic vaccines comprise one or more
epitopes associated with a particular disorder affecting the
individual, such as M. tuberculosis or M. bovis surface antigens in
tuberculosis patients, antigens to which the individual is allergic
(i.e., allergy desensitization therapy) in individuals subject to
allergies, tumor cells from an individual with cancer (e.g., as
described in U.S. Pat. No. 5,484,596), or tumor associated antigens
in cancer patients. The IMP/MC complex may be given in conjunction
with the vaccine (e.g., in the same injection or a contemporaneous,
but separate, injection) or the IMP/MC complex may be administered
separately (e.g., at least 12 hours before or after administration
of the vaccine). In certain embodiments, the antigen(s) of the
vaccine is part of the IMP/MC complex, by either covalent or
non-covalent linkage to the IMP/MC complex. Administration of
IMP/MC complex therapy to an individual receiving a vaccine results
in an immune response to the vaccine that is shifted towards a
Th1-type response as compared to individuals which receive vaccine
without IMP/MC complex. Shifting towards a Th1-type response may be
recognized by a delayed-type hypersensitivity (DTH) response to the
antigen(s) in the vaccine, increased IFN-.gamma. and other Th1-type
response associated cytokines, increased IFN-.alpha., production of
CTLs specific for the antigen(s) of the vaccine, low or reduced
levels of IgE specific for the antigen(s) of the vaccine, a
reduction in Th2-associated antibodies specific for the antigen(s)
of the vaccine, and/or an increase in Th1-associated antibodies
specific for the antigen(s) of the vaccine. In the case of
therapeutic vaccines, administration of IMP/MC complex and vaccine
also results in amelioration of the symptoms of the disorder which
the vaccine is intended to treat. As will be apparent to one of
skill in the art, the exact symptoms and manner of their
improvement will depend on the disorder sought to be treated. For
example, where the therapeutic vaccine is for tuberculosis, IMP/MC
complex treatment with vaccine results in reduced coughing, pleural
or chest wall pain, fever, and/or other symptoms known in the art.
Where the vaccine is an allergen used in allergy desensitization
therapy, the treatment results in a reduction in one or more
symptoms of allergy (e.g., reduction in rhinitis, allergic
conjunctivitis, circulating levels of IgE, and/or circulating
levels of histamine).
[0144] Other embodiments of the invention relate to
immunomodulatory therapy of individuals having a pre-existing
disease or disorder, such as cancer or an infectious disease.
Cancer is an attractive target for immunomodulation because most
cancers express tumor-associated and/or tumor specific antigens
which are not found on other cells in the body. Stimulation of a
Th1-type response against tumor cells results in direct and/or
bystander killing of tumor cells by the immune system, leading to a
reduction in cancer cells and a reduction in symptoms.
Administration of an IMP/MC complex to an individual having cancer
results in stimulation of a Th1-type immune response against the
tumor cells. Such an immune response can kill tumor cells, either
by direct action of cellular immune system cells (e.g., CTLs) or
components of the humoral immune system, or by bystander effects on
cells proximal to cells targeted by the immune system.
[0145] Immunomodulatory therapy in accordance with the invention is
also useful for individuals with infectious diseases, particularly
infectious diseases which are resistant to humoral immune responses
(e.g., diseases caused by mycobacterial infections and
intracellular pathogens). Immunomodulatory therapy may be used for
the treatment of infectious diseases caused by cellular pathogens
(e.g., bacteria or protozoans) or by subcellular pathogens (e.g.,
viruses). IMP/MC complex therapy may be administered to individuals
suffering from mycobacterial diseases such as tuberculosis (e.g.,
M. tuberculosis and/or M. bovis infections), leprosy (i.e., M.
leprae infections), or M. marinum or M. ulcerans infections. IMP/MC
complex therapy is also useful for the treatment of viral
infections, including infections by influenza virus, respiratory
syncytial virus (RSV), hepatitis virus B, hepatitis virus C, herpes
viruses, particularly herpes simplex viruses, and papilloma
viruses. Diseases caused by intracellular parasites such as malaria
(e.g., infection by Plasmodium vivax, P. ovale, P. falciparum
and/or P. malariae), leishmaniasis (e.g., infection by Leishmania
donovani, L. tropica, L. mexicana, L. braziliensis, L. peruviana,
L. infantum, L. chagasi, and/or L. aethiopica), and toxoplasmosis
(i.e., infection by Toxoplasmosis gondii) also benefit from IMP/MC
complex therapy. IMP/MC therapy is also useful for treatment of
parasitic diseases such as schistosomiasis (i.e., infection by
blood flukes of the genus Schistosoma such as S. haematobium, S.
mansoni, S. japonicum, and S. mekongi) and clonorchiasis (i.e.,
infection by Clonorchis sinensis). Administration of an IMP/MC
complex to an individual suffering from an infectious disease
results in an amelioration of one or more symptoms of the
infectious disease.
[0146] The invention further provides methods of increasing at
least one Th1-associated cytokine in an individual, including IL-2,
IL-12, TNF-.beta., and IFN-.gamma.. In certain embodiments, the
invention provides methods of increasing IFN-.gamma. in an
individual, particularly in an individual in need of increased
IFN-.gamma. levels, by administering an effective amount of an
IMP/MC complex to the individual. Individuals in need of increased
IFN-.gamma. are those having disorders which respond to the
administration of IFN-.gamma.. Such disorders include a number of
inflammatory disorders including, but not limited to, ulcerative
colitis. Such disorders also include a number of fibrotic
disorders, including, but not limited to, idiopathic pulmonary
fibrosis (IPF), scleroderma, cutaneous radiation-induced fibrosis,
hepatic fibrosis including schistosomiasis-induced hepatic
fibrosis, renal fibrosis as well as other conditions which may be
improved by administration of IFN-.gamma.. Administration of IMP/MC
complex in accordance with the invention results in an increase in
IFN-.gamma. levels, and results in amelioration of one or more
symptoms, stabilization of one or more symptoms, or prevention of
progression (e.g., reduction or elimination of additional lesions
or symptoms) of the disorder which responds to IFN-.gamma.. The
methods of the invention may be practiced in combination with other
therapies which make up the standard of care for the disorder, such
as administration of anti-inflammatory agents such as systemic
corticosteroid therapy (e.g., cortisone) in IPF.
[0147] In certain embodiments, the invention provides methods of
increasing IFN-.alpha. in an individual, particularly in an
individual in need of increased IFN-.alpha. levels, by
administering an effective amount of an IMP/MC complex to the
individual such that IFN-.alpha. levels are increased. Individuals
in need of increased IFN-.alpha. are those having disorders which
respond to the administration of IFN-.alpha., including recombinant
IFN-.alpha., including, but not limited to, viral infections and
cancer.
[0148] Also provided are methods of reducing levels, particularly
serum levels, of IgE in an individual having an IgE-related
disorder by administering an effective amount of an IMP/MC complex
to the individual such that levels of IgE are reduced. Reduction in
IgE results in an amelioration of symptoms of the IgE-related
disorder. Such symptoms include allergy symptoms such as rhinitis,
conjunctivitis, in decreased sensitivity to allergens, a reduction
in the symptoms of allergy in an individual with allergies, or a
reduction in severity of a allergic response.
[0149] As will be apparent to one of skill in the art, the methods
of the invention may be practiced in combination with other
therapies for the particular indication for which the IMP/MC
complex is administered. For example, IMP/MC complex therapy may be
administered in conjunction with anti-malarial drugs such as
chloroquine for malaria patients, in conjunction with
leishmanicidal drugs such as pentamidine and/or allopurinol for
leishmaniasis patients, in conjunction with anti-mycobacterial
drugs such as isoniazid, rifampin and/or ethambutol in tuberculosis
patients, or in conjunction with allergen desensitization therapy
for atopic (allergy) patients.
[0150] Administration and Assessment of the Immune Response
[0151] The IMP/MC complex can be administered in combination with
other pharmaceutical and/or immunogenic and/or immunostimulatory
agents and can be combined with a physiologically acceptable
carrier thereof.
[0152] Accordingly, the IMP/MC complex can be administered in
conjunction with other immunotherapeutic agents including, but not
limited to, cytokine, adjuvants and antibodies.
[0153] The ISS-containing polynucleotide may be any of those
described above. As indicated in SEQ ID NO:1, preferably, the
ISS-containing polynucleotide administered comprises the sequence
5'-T, C, G-3'. Preferably, the ISS-containing polynucleotide
administered comprises the formula 5' purine, purine, C, G,
pyrimidine, pyrimidine, C, G-3'; more preferably, 5'-A, A, C, G, T,
T, C, G-3'. Another preferred embodiment uses SEQ ID NO:1.
[0154] As with all immunogenic compositions, the immunologically
effective amounts and method of administration of the particular
IMP/MC complex formulation can vary based on the individual, what
condition is to be treated and other factors evident to one skilled
in the art. Factors to be considered include the antigenicity,
whether or not the IMP/MC complex will be administered with or
covalently attached to an adjuvant or delivery molecule, route of
administration and the number of immunizing doses to be
administered. Such factors are known in the art and it is well
within the skill of those in the art to make such determinations
without undue experimentation. A suitable dosage range is one that
provides the desired modulation of immune response to the antigen.
Generally, dosage is determined by the amount of IMP administered
to the patient, rather than the overall quantity of IMP/MC complex.
Useful dosage ranges of the IMP/MC complex, given in amounts of IMP
delivered, may be, for example, from about any of the following:
0.1 to 100 .mu.g/kg, 0.1 to 50 .mu.g/kg, 0.1 to 25 .mu.g/kg, 0.1 to
10 .mu.g/kg, 1 to 500 .mu.g/kg, 100 to 400 .mu.g/kg, 200 to 300
.mu.g/kg, 1 to 100 .mu.g/kg, 100 to 200 .mu.g/kg, 300 to 400
.mu.g/kg, 400 to 500 .mu.g/kg. Alternatively, the doses can be
about any of the following: 0.1 .mu.g, 0.25 .mu.g, 0.5 .mu.g, 1.0
.mu.g, 2.0 .mu.g, 5.0 .mu.g, 10 .mu.g, 25 .mu.g, 50 .mu.g, 75
.mu.g, 100 .mu.g. Accordingly, dose ranges can be those with a
lower limit about any of the following: 0.1 .mu.g, 0.25 .mu.g, 0.5
.mu.g and 1.0 .mu.g; and with an upper limit of about any of the
following: 25 .mu.g, 50 .mu.g and 100 .mu.g. The absolute amount
given to each patient depends on pharmacological properties such as
bioavailability, clearance rate and route of administration.
[0155] The effective amount and method of administration of the
particular IMP/MC complex formulation can vary based on the
individual patient and the stage of the disease and other factors
evident to one skilled in the art. The route(s) of administration
useful in a particular application are apparent to one of skill in
the art. Routes of administration include but are not limited to
topical, dermal, transdermal, transmucosal, epidermal, parenteral,
gastrointestinal, and naso-pharyngeal and pulmonary, including
transbronchial and transalveolar. A suitable dosage range is one
that provides sufficient ISS-containing composition to attain a
tissue concentration of about 1-10 .mu.M as measured by blood
levels. The absolute amount given to each patient depends on
pharmacological properties such as bioavailability, clearance rate
and route of administration.
[0156] As described herein, APCs and tissues with high
concentration of APCs are preferred targets for the IMP/MC
complexes. Thus, administration of IMP/MC complex to mammalian skin
and/or mucosa, where APCs are present in relatively high
concentration, is preferred.
[0157] The present invention provides IMP/MC complex formulations
suitable for topical application including, but not limited to,
physiologically acceptable implants, ointments, creams, rinses and
gels. Topical administration is, for instance, by a dressing or
bandage having dispersed therein a delivery system, by direct
administration of a delivery system into incisions or open wounds,
or by transdermal administration device directed at a site of
interest. Creams, rinses, gels or ointments having dispersed
therein an IMP/MC complex are suitable for use as topical ointments
or wound filling agents.
[0158] Preferred routes of dermal administration are those which
are least invasive. Preferred among these means are transdermal
transmission, epidermal administration and subcutaneous injection.
Of these means, epidermal administration is preferred for the
greater concentrations of APCs expected to be in intradermal
tissue.
[0159] Transdermal administration is accomplished by application of
a cream, rinse, gel, etc. capable of allowing the IMP/MC complex to
penetrate the skin and enter the blood stream. Compositions
suitable for transdermal administration include, but are not
limited to, pharmaceutically acceptable suspensions, oils, creams
and ointments applied directly to the skin or incorporated into a
protective carrier such as a transdermal device (so-called
"patch"). Examples of suitable creams, ointments etc. can be found,
for instance, in the Physician's Desk Reference.
[0160] For transdermal transmission, iontophoresis is a suitable
method. Iontophoretic transmission can be accomplished using
commercially available patches which deliver their product
continuously 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.
[0161] 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 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. Other occlusive patch systems are also suitable.
[0162] For transdermal transmission, low-frequency ultrasonic
delivery is also a suitable method. Mitragotri et al. (1995)
Science 269:850-853. Application of low-frequency ultrasonic
frequencies (about 1 MHz) allows the general controlled delivery of
therapeutic compositions, including those of high molecular
weight.
[0163] Epidermal administration essentially involves mechanically
or chemically irritating the outermost layer of the epidermis
sufficiently to provoke an immune response to the irritant.
Specifically, the irritation should be sufficient to attract APCs
to the site of irritation.
[0164] An exemplary mechanical irritant means employs a
multiplicity of very narrow diameter, short tines which can be used
to irritate the skin and attract APCs to the site of irritation, to
take up IMP/MC complex transferred from the end of the tines. For
example, the MONO-VACC old tuberculin test manufactured by Pasteur
Merieux of Lyon, France contains a device suitable for introduction
of IMP/MC complex-containing compositions.
[0165] The device (which is distributed in the U.S. by Connaught
Laboratories, Inc. of Swiftwater, Pa.) consists of a plastic
container having a syringe plunger at one end and a tine disk at
the other. The tine disk supports a multiplicity of narrow diameter
tines of a length which will just scratch the outermost layer of
epidermal cells. Each of the tines in the MONO-VACC kit is coated
with old tuberculin; in the present invention, each needle is
coated with a pharmaceutical composition of IMP/MC complex
formulation. Use of the device is preferably according to the
manufacturer's written instructions included with the device
product. Similar devices which can also be used in this embodiment
are those which are currently used to perform allergy tests.
[0166] Another suitable approach to epidermal administration of
IMP/MC complex is by use of a chemical which irritates the
outermost cells of the epidermis, thus provoking a sufficient
immune response to attract APCs to the area. An example is a
keratinolytic agent, such as the salicylic acid used in the
commercially available topical depilatory creme sold by Noxema
Corporation under the trademark NAIR. This approach can also be
used to achieve epithelial administration in the mucosa. The
chemical irritant can also be applied in conjunction with the
mechanical irritant (as, for example, would occur if the MONO-VACC
type tine were also coated with the chemical irritant). The IMP/MC
complex can be suspended in a carrier which also contains the
chemical irritant or coadministered therewith.
[0167] Parenteral routes of administration include but are not
limited to electrical (iontophoresis) or direct injection such as
direct injection into a central venous line, intravenous,
intramuscular, intraperitoneal, intradermal, or subcutaneous
injection. IMP/MC formulations suitable for parenteral
administration are generally formulated in USP water or water for
injection and may further comprise pH buffers, salts bulking
agents, preservatives, and other pharmaceutically acceptable
excipients. IMP/MC complexes for parenteral injection may be
formulated in pharmaceutically acceptable sterile isotonic
solutions such as saline and phosphate buffered saline for
injection.
[0168] Gastrointestinal routes of administration include, but are
not limited to, ingestion and rectal. The invention includes IMP/MC
complex formulations suitable for gastrointestinal administration
including, but not limited to, pharmaceutically acceptable powders,
pills or liquids for ingestion and suppositories for rectal
administration. As will be apparent to one of skill in the art,
pills or suppositories will further comprise pharmaceutically
acceptable solids, such as starch, to provide bulk for the
composition.
[0169] Naso-pharyngeal and pulmonary administration include are
accomplished by inhalation, and include delivery routes such as
intranasal, transbronchial and transalveolar routes. The invention
includes IMP/MC complex formulations suitable for administration by
inhalation including, but not limited to, liquid suspensions for
forming aerosols as well as powder forms for dry powder inhalation
delivery systems. Devices suitable for administration by inhalation
of IMP/MC complex formulations include, but are not limited to,
atomizers, vaporizers, nebulizers, and dry powder inhalation
delivery devices.
[0170] The choice of delivery routes can be used to modulate the
immune response elicited. For example, IgG titers and CTL
activities were identical when an influenza virus vector was
administered via intramuscular or epidermal (gene gun) routes;
however, the muscular inoculation yielded primarily IgG2a, while
the epidermal route yielded mostly IgG1. Pertmer et al. (1996) J.
Virol. 70:6119-6125. Thus, one skilled in the art can take
advantage of slight differences in immunogenicity elicited by
different routes of administering the immunomodulatory
oligonucleotides of the present invention.
[0171] The above-mentioned compositions and methods of
administration are meant to describe but not limit the methods of
administering the IMP/MC complex formulations of the invention. The
methods of producing the various compositions and devices are
within the ability of one skilled in the art and are not described
in detail here.
[0172] Analysis (both qualitative and quantitative) of the immune
response to IMP/MC complex formulations can be by any method known
in the art, including, but not limited to, measuring
antigen-specific antibody production (including measuring specific
antibody subclasses), activation of specific populations of
lymphocytes such as CD4+T cells or NK cells, production of
cytokines such as IFN-.gamma., IFN-.alpha., IL-2, IL-4, IL-5, IL-10
or IL-12 and/or release of histamine. Methods for measuring
specific antibody responses include enzyme-linked immunosorbent
assay (ELISA) and are well known in the art. Measurement of numbers
of specific types of lymphocytes such as CD4+T cells can be
achieved, for example, with fluorescence-activated cell sorting
(FACS). Cytotoxicity assays can be performed for instance as
described in Raz et al. (1994) Proc. Natl. Acad. Sci. USA
91:9519-9523. Cytokine concentrations can be measured, for example,
by ELISA. These and other assays to evaluate the immune response to
an immunogen are well known in the art. See, for example, Selected
Methods in Cellular Immunology (1980) Mishell and Shiigi, eds., W.
H. Freeman and Co.
[0173] Preferably, a Th1-type response is stimulated, i.e.,
elicited and/or enhanced. With reference to the invention,
stimulating a Th1-type immune response can be determined in vitro
or ex vivo by measuring cytokine production from cells treated with
ISS as compared to those treated without ISS. Methods to determine
the cytokine production of cells include those methods described
herein and any known in the art. The type of cytokines produced in
response to ISS treatment indicate a Th1-type or a Th2-type biased
immune response by the cells. As used herein, the term "Th1-type
biased" cytokine production refers to the measurable increased
production of cytokines associated with a Th1-type immune response
in the presence of a stimulator as compared to production of such
cytokines in the absence of stimulation. Examples of such Th1-type
biased cytokines include, but are not limited to, IL-2, IL-12, and
IFN-.gamma.. In contrast, "Th2-type biased cytokines" refers to
those associated with a Th2-type immune response, and include, but
are not limited to, IL-4, IL-5, and IL-13. Cells useful for the
determination of ISS activity include cells of the immune system,
primary cells isolated from a host and/or cell lines, preferably
APCs and lymphocytes, even more preferably macrophages and T
cells.
[0174] Stimulating a Th1-type immune response can also be measured
in a host treated with an IMP/MC complex formulation can be
determined by any method known in the art including, but not
limited to: (1) a reduction in levels of IL-4 or IL-5 measured
before and after antigen-challenge; or detection of lower (or even
absent) levels of IL-4 or IL-5 in an IMP/MC complex treated host as
compared to an antigen-primed, or primed and challenged, control
treated without ISS; (2) an increase in levels of IL-12, IL-18
and/or IFN (.alpha., .beta. or .gamma.) before and after antigen
challenge; or detection of higher levels of IL-12, IL-18 and/or IFN
(.alpha., .beta. or .gamma.) in an IMP/MC complex treated host as
compared to an antigen-primed or, primed and challenged, control
treated without ISS; (3) "Th1-type biased" antibody production in
an IMP/MC complex treated host as compared to a control treated
without ISS; and/or (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 IMP/MC
complex treated host as compared to an antigen-primed, or primed
and challenged, control treated without ISS. A variety of these
determinations can be made by measuring cytokines made by APCs
and/or lymphocytes, preferably macrophages and/or T cells, in vitro
or ex vivo using methods described herein or any known in the art.
Some of these determinations can be made by measuring the class
and/or subclass of antigen-specific antibodies using methods
described herein or any known in the art.
[0175] The class and/or subclass of antigen-specific antibodies
produced in response to IMP/MC complex treatment indicate a
Th1-type or a Th2-type biased immune response by the cells. As used
herein, the term "Th1-type biased" antibody production refers to
the measurable increased production of antibodies associated with a
Th1-type immune response (i.e., Th1-associated antibodies). One or
more Th1 associated antibodies may be measured. Examples of such
Th1-type biased antibodies include, but are not limited to, human
IgG1 and/or IgG3 (see, e.g., Widhe et al. (1998) Scand. J. Immunol.
47:575-581 and de Martino et al. (1999) Ann. Allergy Asthma
Immunol. 83:160-164) and murine IgG2a. In contrast, "Th2-type
biased antibodies" refers to those associated with a Th2-type
immune response, and include, but are not limited to, human IgG2,
IgG4 and/or IgE (see, e.g., Widhe et al. (1998) and de Martino et
al. (1999)) and murine IgG1 and/or IgE.
[0176] The Th1-type biased cytokine induction which occurs as a
result of IMP/MC complex administration produces enhanced cellular
immune responses, such as those performed by NK cells, cytotoxic
killer cells, Th1 helper and memory cells. These responses are
particularly beneficial for use in protective or therapeutic
vaccination against viruses, fungi, protozoan parasites, bacteria,
allergic diseases and asthma, as well as tumors.
[0177] In some embodiments, a Th2 response is suppressed.
Suppression of a Th2 response may be determined by, for example,
reduction in levels of Th2-associated cytokines, such as IL-4 and
IL-5, as well as IgE reduction and reduction in histamine release
in response to allergen.
[0178] Kits of the Invention
[0179] The invention provides kits for use in the methods of the
invention. In certain embodiments, the kits of the invention
comprise one or more containers comprising an IMP/MC complex and a
set of instructions, generally written instructions, relating to
the use of the IMP/MC complex for the intended treatment (e.g.,
immunomodulation, ameliorating one or more symptoms of an
infectious disease, increasing IFN-.gamma. levels, increasing
IFN-.alpha. levels, or ameliorating an IgE-related disorder). In
further embodiments, the kits of the invention comprise containers
of materials for producing IMP/MC, instructions for producing
IMP/MC complex, and instructions relating to the use of the IMP/MC
complex for the intended treatment.
[0180] Kits which comprise preformed IMP/MC complex comprise IMP/MC
complex packaged in any convenient, appropriate packaging. For
example, if the IMP/MC complex is a dry formulation (e.g., freeze
dried or a dry powder), a vial with a resilient stopper is normally
used, so that the IMP/MC complex may be easily resuspended by
injecting fluid through the resilient stopper. Ampoules with
non-resilient, removable closures (e.g., sealed glass) or resilient
stoppers are most conveniently used for liquid formulations of
IMP/MC complex. Also contemplated are packages for use in
combination with a specific device, such as an inhaler, nasal
administration device (e.g., an atomizer) or an infusion device
such as a minipump.
[0181] Kits which comprise materials for production of IMP/MC
complex generally include separate containers of IMP and and MC,
although in certain embodiments materials for producing the MC are
supplied rather than preformed MC. The IMP and MC are preferably
supplied in a form which allows formation of IMP/MC complex upon
mixing of the supplied IMP and MC. This configuration is preferred
when the IMP/MC complex is linked by non-covalent bonding. This
configuration is also preferred when the IMP and MC are to be
crosslinked via a heterobifunctional crosslinker; either IMP or the
MC is supplied in an "activated" form (e.g. linked to the
heterobifunctional crosslinker such that a moiety reactive with the
IMP is available).
[0182] Kits for IMP/MC complexes comprising a liquid phase MC
preferably comprise one or more containers including materials for
producing liquid phase MC. For example, an IMP/MC kit for
oil-in-water emulsion MC may comprise one or more containers
containing an oil phase and an aqueous phase. The contents of the
container are emulsified to produce the MC, which may be then mixed
with the IMP, preferably an IMP which has been modified to
incorporate a hydrophobic moiety. Such materials include oil and
water, for production of oil-in-water emulsions, or containers of
lyophilized liposome components (e.g., a mixture of phospholipid,
cholesterol and a surfactant) plus one or more containers of an
aqueous phase (e.g., a pharmaceutically-acceptable aqueous
buffer).
[0183] The instructions relating to the use of IMP/MC complex for
the intended treatment generally include information as to dosage,
dosing schedule, and route of administration for the intended
treatment. The containers of ISS may be unit doses, bulk packages
(e.g., multi-dose packages) or sub-unit doses. Instructions
supplied in the kits of the invention are typically written
instructions on a label or package insert (e.g., a paper sheet
included in the kit), but machine-readable instructions (e.g.,
instructions carried on a magnetic or optical storage disk) are
also acceptable.
[0184] The following Examples are provided to illustrate, but not
limit, the invention.
Examples
Example 1
[0185] Production of Non-Covalent, Liquid Phase IMP/MC
Complexes
[0186] IMP/MC complex comprising a modified IMP and a liquid phase
MC were produced and tested for complex formation.
[0187] An IMP (phosphorothioate oligodeoxynucleotide
5'-TGACTGTGAACGTTCGAGATGA-3') (SEQ ID NO 1) was modified by
addition of a cholesterol molecule to the 5' end of the IMP using
phosphoramidite chemistry. An oil-in-water emulsion was produced by
homogenization of a mixture of 4.5% (w/v) squalene, 0.5% (w/v)
sorbitan trioleate, 0.5% (w/v) TWEEN.RTM. 80 and 10 mM sodium
citrate, pH 6.5, using a microfluidizer. Examination of the
emulsion found that the oil droplets in the emulsion had an average
diameter of approximately 160 nm.
[0188] The emulsion was mixed with the cholesterol-modified IMP or
an unmodified version of the same IMP, then centrifuged to separate
the oil and water phases. RP-HPLC was performed on samples from
each phase to determine nucleotide content. Approximately 75% of
the cholesterol-modified IMP was found in the oil phase, while 100%
of the unmodified IMP was found in the aqueous phase.
Example 2
[0189] Immunomodulation with IMP/MC Mixtures
[0190] Mixtures of an IMP (phosphorothioate oligodeoxynucleotide
5'-TGACTGTGAACGTTCGAGATGA-3') (SEQ ID NO:1) or a control
oligonucleotide (phosphorothioate oligodeoxynucleotide
5'-TGACTGTGAAGGTTAGAGATGA-3') (SEQ ID NO:9) were mixed with
sulphate-derivatized polycarbonate microparticles or nanoparticles
(Polysciences, Inc.) and assayed for immunomodulatory activity on
mouse splenocytes.
[0191] Fragments of BALB/c mouse spleen were digested with
collagenase/dispase (0.1 U/mL/0.8 U/mL) dissolved in phosphate
buffered saline (PBS) for 45 minutes at 37.degree. C., then
mechanically dispersed by forcing the digested fragments through
metal screens. The dispersed splenocytes were pelleted by
centrifugation, then resuspended in fresh medium (RPMI 1640 with
10% fetal calf serum, plus 50 units/mL penicillin, 50 .mu.g/mL
streptomycin, 2 mM glutamine, and 0.05 mM
.beta.-mercaptoethanol).
[0192] 4.times.10.sup.5 mouse splenocytes were dispensed into wells
of 96 well plates and incubated for one hour at 37.degree. C. 100
.mu.L of 2.times.concentration test sample or control was added and
the cells were incubated a further 24 hours. Medium was harvested
from each well and tested for cytokine concentrations by ELISA.
[0193] IFN-.gamma. was assayed using a sandwich-format ELISA.
Medium from the mouse splenocyte assay was incubated in microtiter
plates coated with anti-IFN-.gamma. monoclonal antibody (Nunc).
Bound IFN-.gamma. was detected using a biotinylated
anti-IFN-.gamma. antibody and streptavidin-horseradish peroxidase
conjugated secondary antibody, developed with the chromogenic
peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) in the
presence of peroxidase, and quantitated by measuring absorbance at
450 nm using a Emax precision microplate reader (Molecular
Devices).
[0194] 200 nm beads mixed with IMP substantially increased IL-12,
IL-6 and IFN-.gamma. secretion by mouse splenocytes, and 50 mn
beads mixed with IMP increased IL-12 and IL-6 production. Some
nonspecific activity was associated with 200 nm beads mixed with
the control oligonucleotide, although this was insufficient to
account for the increase in stimulation as compared to IMP alone.
Additionally, microcarriers of 1 .mu.m and 4.5 .mu.m also increased
cytokine secretion. Tables 2-4 summarize assay results for IL-12,
IL-6 and IFN-.gamma., respectively.
7 TABLE 2 DNA dose Test Material 5 .mu.g/ml 1 .mu.g/ml 0.1 .mu.g/ml
IMP IL-12 (pg/mL) alone 6046 4737 915 IMP + 50 nm 8582 4934 364 IMP
+ 200 nm 7377 8393 984 IMP + 500 nm 3680 4260 833 IMP + 1 .mu.m
5082 4652 613 IMP + 4.5 .mu.m 2253 2306 838 Control alone 79 91 65
control + 50 100 100 91 control + 200 661 108 127 control + 500 48
82 82 control + 1000 72 101 147 control + 4500 101 104 141
[0195]
8 TABLE 3 DNA dose Test Material 5 .mu.g/ml 0.1 .mu.g/ml IMP IL-6
(pg/mL) alone 5290 1872 IMP + 50 nm >18000 2127 IMP + 200 nm
6946 3574 IMP + 500 nm 345 2133 IMP + 1 .mu.m 3812 2107 IMP + 4.5
.mu.m 3277 1846 control alone 24 24 control + 50 24 24 control +
200 1842 232 control + 500 24 24 control + 1000 24 24 control +
4500 30 24
[0196]
9 TABLE 4 DNA dose Test Material 5 .mu.g/ml 0.1 .mu.g/ml IMP
IFN.sub.-.gamma.(pg/mL) alone 575 244 IMP + 50 nm 411 232 IMP + 200
nm 3548 3150 IMP + 500 nm 48 426 IMP + 1 .mu.m 252 685 IMP + 4.5
.mu.m 1072 2739 control alone 48 48 control + 50 48 48 control +
200 1907 101 control + 500 48 48 control + 1000 48 48 control +
4500 50 48
Example 3
[0197] Immunomodulation of Mouse Cells by IMP/NC Conjugates
[0198] IMPs covalently linked to non-biodegradable polystyrene
beads (200 nm design size) were tested for immunomodulatory
activity on mouse splenocytes.
[0199] Amine-derivatized polystyrene beads were obtained from
Molecular Probes, Inc., and Polysciences, Inc. Three types of beads
were utilized: amine-derivatized beads (Polysciences, Inc., Catalog
No. 15699), amine-derivatized beads linked to a fluorophore with
excitation/emission maxima of 580 and 605 nm ("Red Beads",
Molecular Probes, Inc., Catalog No. F8763), and amine-derivatized
beads linked to a fluorophore with excitation/emission maxima of
505 and 515 nm ("Yellow Beads", Molecular Probes, Inc., Catalog No.
F8764) were activated with sulfo-SMCC (sulfosuccinimidyl
4-(N-maleimidomethyl)cyclopentane-1-carboxylate, Pierce Chemical
Co.) according to the manufacturer's instructions. The beads were
then linked to IMP (see Examples 1 and 2), a control
phosphorothioate oligonucleotide (5'-TGACTGTGAAGGTTAGAGATGA-3'
(control A) (SEQ ID NO:9), 5'-TGACTGTGAACCTTAGAGATGA-3' (control B)
(SEQ ID NO:10), or 5'-TCACTCTCTTCCTTACTCTTCT-3' (control C) (SEQ ID
NO:11), or treated to quench the free maleimide group for use as a
NC only control.
[0200] Immunomodulatory effects of the IMP/NC complexes were
assayed using mouse splenocytes as described above in Example 2.
IMP/NC complexes demonstrated immunomodulation on the mouse
splenocytes, as shown by increased secretion of IL-12, IL-6 and/or
IFN-.gamma., while control oligonucleotides conjugated to NC did
not stimulate cytokine secretion. Data for IFN-.gamma. secretion is
summarized in Table 5.
10 TABLE 5 IFN-.gamma. (pg/mL) Sample 5 .mu.g/ml dose 1 .mu.g/ml
dose White Beads NC alone 48 48 IMP/NC 903 77 Red Beads NC alone 48
48 IMP/NC 319 63 control A/NC 48 48 control B/NC 48 48 sulfo-SMCC
activated NC 2869 2147 Yellow Beads NC alone-lot 7781-2 1224 147
145-57A: IMP/NC-lot 145-57A 3437 2335 145-57B: IMP/NC-lot 145-57B
4556 5497 145-146: IMP/NC-lot 145-146C 11444 7091 135-171A:
IMP/NC-135-171A 4493 2359 135-171B: control A/NC 147 147 145-148:
control B/NC 147 147 sulfo-SMCC activated NC, BME-inactivated 3163
3723 sulfo-SMCC activated NC, cysteine 3392 3090 inactivated
sulfo-SMCC activated NC 3583 4108 IMP Controls IMP 558 577 control
C 48 48
Example 4
[0201] Immunomodulation of Human Cells by IMP/NC Conjugates
[0202] IMPs covalently linked to non-biodegradable polystyrene
beads (200 nm design size) were tested for immunomodulatory
activity on human peripheral blood mononuclear cells (PBMCs).
[0203] Peripheral blood was collected from volunteers by
venipuncture using heparinized syringes. Blood was layered onto
FICOLL.RTM. (Amersham Pharmacia Biotech) cushion and centrifuged.
PBMCs, located at the FICOLL.RTM. interface, were collected, then
washed twice with cold phosphate buffered saline (PBS). The cells
were resuspended and cultured in 24 or 48 well plates at
2.times.10.sup.6 cells/mL in RPMI 1640 with 10% heat-inactivated
human AB serum plus 50 units/mL penicillin, 50 .mu.g/mL
streptomycin, 300 .mu.g/mL glutamine, 1 mM sodium pyruvate, and
1.times.MEM non-essential amino acids (NEAA).
[0204] The cells were cultured in the presence of test samples
(IMP/NC formulations or controls) for 24 hours, then cell-free
medium was collected from each well and assayed for IFN-.gamma.
concentration. IFN-.gamma. was assayed using a CYTOSCREEN.TM. ELISA
kit from BioSource International, Inc., according to the
manufacturer's instructions.
[0205] IMP/NC complexes stimulated IFN-.gamma. secretion by human
PBMCs. The results are summarized in Table 6.
11 TABLE 6 IFN-.gamma. (pg/mL) Experiment 1 Experiment 2 Sample 5
.mu.g/ml 20 .mu.g/ml 5 .mu.g/ml 10 .mu.g/ml White Beads NC alone
n/a n/a IMP/NC 5 3 Red Beads NC alone 2 8 IMP/NC 39 431 control
A/NC 2 5 control B/NC 3 14 sulfo-SMCC activated NC 15 n/a Yellow
Beads - Old NC alone-lot 7781-2 12 56 8 19 NC alone-lot 6991-1 7
n/a IMP/NC-lot 145-57A 187 n/a IMP/NC-lot 145-57B 777 n/a
IMP/NC-lot 145-57C 536 7752 6356 6413 IMP/NC-lot 145-57D 156 1861
IMP/NC-lot 145-57E 283 1385 IMP/NC-lot 145-57F 140 n/a IMP/NC-lot
145-146 934 6519 IMP/NC-lot 135-171A 123 2400 control A/NC 12 446
control B/NC 24 165 sulfo-SMCC activated NC, BME-inactivated 4 8
sulfo-SMCC activated NC, cysteine inactivated 7 15 sulfo-SMCC
activated NC 7 14 IMP Controls IMP <10 <10
Example 5
[0206] Preparation of Biodegradable, Cationic Microspheres
[0207] Cationic microspheres were prepared as follows. 0.875 g of
poly (D,L-lactide-co-glycolide) 50:50 polymer with an intrinsic
viscosity of 0.41 dl/g (0.1%, chloroform, 25.degree. C.) was
dissolved in 7.875 g of methylene chloride at 10% w/w
concentration, along with 0.3 g of DOTAP. The clear orgainc phase
was then emulsified into 500 ml of PVA aqueous solution (0.35% w/v)
by homogenization at 4000 rpm for 30 minutes at room temperature
using a laboratory mixer (Silverson L4R, Silverson Instruments).
System temperature was then raised to 40.degree. C. by circulating
hot water through the jacket of the mixing vessel. Simultaneously,
the stirring rate was reduced to 1500 rpm, and these conditions
were maintained for 2 hours to extract and evaporate methylene
chloride. The microsphere suspension was allowed to cool down to
room temperature with the help of circulating cold water.
[0208] Microparticles were separated by centrifugation at 8000 rpm
for 10 minutes at room temperature (Beckman Instruments) and
resuspended in deioized water by gentle bath sonication.
Centrifugal wash was repeated two additional times to remove excess
PVA from particle surface. Final centrifugal pellets of particles
are suspended in approximately 10 ml of water, and lyophilized
overnight. Dried microsphere powder was characterized for size and
surface charge: mean size (number weighted, .mu.)=1.4; zeta
potential (mV) 32.4.
[0209] 200 mg of microspheres from the above batch was dispersed in
1.875 ml of 0.1% w/v Tween solution by bath sonication for 5
minutes. 0.75 ml of aqueous ISS solution (SEQ ID NO:1) was added to
the microsphere suspension to yield an approximate and theoreical
drug loading of 2% w/w (ISS to microsphere). After a brief mixing,
the ISS--microsphere suspension was incubated at 4.degree. C.
overnight. Microspheres were separated by centrifugation at 14,000
rpm for 30 minutes at room temperature in an Eppendorf centrifuge.
The supernatant and the microspheres were assayed for free and
bound ISS, respectively, by the standard laboratory analytical
techniques to determine ISS association efficiency or loading.
After ISS association, the preparation was also characterized for
size and surface charge: mean size (number weighted, .mu.)=1.6;
zeta potential (mV)=33.3; % ISS association (drug loading)=88
(1.78% w/w).
Example 6
[0210] Immunomodulatory Effects of IMP/MC Formulations
[0211] Preparations of cationic microspheres with and without
adsorbed ISS were evaluated for immunomodulatory effects in a
standard mouse splenocyte assay. Cationic microspheres were
prepared as described in Example 5, with the exception that 0.2 g
of DOTAP was used. Three doses of adsorbed ISS SEQ ID NO:1, 0.1,
1.0 and 5 .mu.g/ml, were evaluted against the same doses of free
ISS solution and blank (no drug) microspheres. The splenocyte
assays were performed as described in Example 2 and results of
these experiments are presented in Table 7.
[0212] At lower doses tested, IMP/MC formulations were more
effective than free IMP at inducing IL-6, IL-12 and IFN-.gamma. in
the mouse splenocyte assay.
12TABLE 7 Sample IL-6 (pg/mL) IL-12 (pg/mL) IFN-.gamma. (pg/mL) IMP
0.1 .mu.g/ml 27 368 9 1.0 .mu.g/ml 909 1694 273 5.0 .mu.g/ml 3340
1807 435 IMP/MC 0.1 .mu.g/ml 114 1390 161 1.0 .mu.g/ml 226 1086 200
5.0 .mu.g/ml 850 540 200 MC alone (0.1 .mu.g/ml) 27 27 9 (1.0
.mu.g/ml) 31 27 9 (5.0 .mu.g/ml) 27 84 9
[0213] Preparations of cationic microspheres with and without
adsorbed IMP SEQ ID NO:1 were evaluated for immunomodulatory
effects in a human PBMC assay. Cationic microspheres that were made
as described in Example 5. Included as controls were (i) free IMP
and (ii) a mixture of IMP and cationic microspheres. With IMP/MC
formulations, IMP doses of 2 and 10 .mu.g/ml were used. The PBMC
assays were performed as described in Example 4. IFN-.gamma. and
IFN-.alpha. were assayed using CYTOSCREENTM ELISA kits from
BioSource International, Inc., according to the manufacturer's
instructions. Results of such an experiment are presented in Table
8. The IMP/MC formulations were more active than free ISS in
inducing IFN-.alpha. and were comparable to free ISS in inducing
IFN-.gamma. in the human PBMC assay.
13 TABLE 8 Sample IFN-.gamma.(pg/mL)* IFN-.alpha. (pg/mL)* IMP (10
.mu.g/ml) 63 (0, 127) 0 (0, 0) IMP + Microspheres 197 (82, 311) 433
(175, 690) mixture (IMP at 10 .mu.g/ml) IMP/MC 2.0 .mu.g/ml 81 (61,
102) 2584 (2247, 2921) 10 .mu.g/ml 45 (10, 79) 1866 (971, 2762)
*mean of two readings that are in parentheses
[0214] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
and understanding, it will be apparent to those skilled in the art
that certain changes and modifications may be practiced. Therefore,
descriptions and examples should not be construed as limiting the
scope of the invention, which is delineated by the appended
claims.
* * * * *
References