U.S. patent application number 10/743398 was filed with the patent office on 2004-10-07 for induction of cytotoxic t-lymphocyte responses.
This patent application is currently assigned to Biogen Idec Inc.. Invention is credited to Black, Amelia, Rastetter, William H., Raychaudhuri, Syamal.
Application Number | 20040197331 10/743398 |
Document ID | / |
Family ID | 27502813 |
Filed Date | 2004-10-07 |
United States Patent
Application |
20040197331 |
Kind Code |
A1 |
Raychaudhuri, Syamal ; et
al. |
October 7, 2004 |
Induction of cytotoxic T-lymphocyte responses
Abstract
Methods and compositions useful for inducing a cytotoxic T
lymphocyte response (CTL) in a human or domesticated or
agriculturally important animal. The method includes the steps of
providing the antigen to which the CTL response is desired and
providing a microfluidized antigen formulation which comprises,
consists, or consists essentially of two or more of a stabilizing
detergent, a micelle-forming agent, and an oil. This antigen
formulation is preferably lacking, in an immunostimulating peptide
component, or has sufficiently low levels of such a component that
the desired CTL response is not diminished. This formulation is
provided as a stable oil-in-water emulsion.
Inventors: |
Raychaudhuri, Syamal; (San
Diego, CA) ; Rastetter, William H.; (Rancho Santa Fe,
CA) ; Black, Amelia; (Cardiff, CA) |
Correspondence
Address: |
PILLSBURY WINTHROP, LLP
P.O. BOX 10500
MCLEAN
VA
22102
US
|
Assignee: |
Biogen Idec Inc.
|
Family ID: |
27502813 |
Appl. No.: |
10/743398 |
Filed: |
December 23, 2003 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10743398 |
Dec 23, 2003 |
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09740003 |
Dec 20, 2000 |
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6733763 |
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09740003 |
Dec 20, 2000 |
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09024220 |
Feb 17, 1998 |
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6197311 |
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09024220 |
Feb 17, 1998 |
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08476674 |
Jun 7, 1995 |
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08476674 |
Jun 7, 1995 |
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08351001 |
Dec 7, 1994 |
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5709860 |
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08351001 |
Dec 7, 1994 |
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07919787 |
Jul 24, 1992 |
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5585103 |
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07919787 |
Jul 24, 1992 |
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07735069 |
Jul 25, 1991 |
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Current U.S.
Class: |
424/144.1 |
Current CPC
Class: |
C01P 2006/90 20130101;
Y02A 50/30 20180101; C01P 2002/60 20130101; A61K 9/1075 20130101;
A61K 2039/55555 20130101; A61K 39/015 20130101; A61K 2039/572
20130101; A61K 39/39 20130101; A61K 2039/525 20130101; A61K
2039/55566 20130101; A61K 39/21 20130101; A61K 39/12 20130101; A61K
2039/585 20130101; A61K 39/0011 20130101; C01P 2002/54 20130101;
A61P 35/00 20180101; C12N 2710/20034 20130101; A61K 39/0005
20130101; C12N 2740/16134 20130101; A61K 39/0011 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
424/144.1 |
International
Class: |
A61K 039/395 |
Claims
What is claimed is:
1. A composition comprising an antigen mixed with a microfluidized
antigen formulation comprising: (a) a stabilizing detergent, (b) a
micelle-forming agent, and (c) a biodegradable and biocompatible
oil, said antigen formulation being formulated as a stable
oil-in-water emulsion, said antigen formulation being substantially
free of immunostimulating peptides and wherein said composition
upon administration to an animal selected from the group consisting
of humans, domesticated animals and agricultural animals is capable
of inducing a specific cytotoxic T-lymphocyte response against the
antigen contained in the composition.
2. The composition of claim 1, wherein said antigen is chosen from
antigenic portions of the HIV antigens: gp160, gag, pol, Nef, Tat,
and Rev; the malaria antigens: CS protein and Sporozoite surface
protein 2; the Hepatitis B surface antigens: Pre-S1, Pre-S2, HBc
Ag, and HBe Ag; the influenza antigens: HA, NP and NA; Hepatitis A
surface antigens; Hepatitis C surface antigens, the Herpes virus
antigens: EBV gp340, EBV gp85, HSV gB, HSV gD, HSV gH, HSV early
protein product, cytomegalovirus gB, cytomegalovirus gH, and IE
protein gp72; the respiratory syncytial virus antigens: F protein,
G protein, and N protein; and the tumor antigens: carcinoma CEA,
carcinoma mutated EGF receptor, prostate carcinoma specific antigen
(PSA), prostate specific membrane associated antigen, carcinoma
associated mucin, carcinoma P21, carcinoma P53, melanoma MPG,
melanoma p97, MAGE-1, MAGE-3, gp100, MART-1, melanoma antigen gp75
carcinoma Neu oncogene product, carcinoma p53 gene product, and
mutated p21 ras protein.
3. A composition comprising an antigen mixed with a microfluidized
antigen formulation comprising: (a) a stabilizing detergent, (b) a
micelle-forming agent, and (c) a biodegradable and biocompatible
oil, said antigen formulation being formulated as a stable
oil-in-water emulsion, said antigen formulation lacking
immunostimulating peptides and wherein said composition upon
administration to an animal selected from the group consisting of
humans, domesticated animals and agricultural animals is capable of
inducing a specific cytotoxic T-lymphocyte response against the
antigen contained in the composition.
4. The composition of claim 3, wherein said antigen formulation
consists essentially of said detergent, agent, and oil.
5. The composition of claim 3, wherein said antigen formulation is
non-toxic to said human or domesticated or agricultural animal.
6. The composition of claim 3, wherein said antigen is chosen from
the HIV antigens: gp160, gag, pol, Nef, Tat, and Rev; the malaria
antigens: Cs protein and Sporozoite surface protein 2; the
Hepatitis B surface antigens: Pre-S1, Pre-S2, HBc Ag, and HBe Ag;
the influenza antigens: HA, NP and NA; Hepatitis A surface
antigens; Hepatitis C surface antigens; the Herpes virus antigens.
EBV gp340, EBV gp85, HSV gB, HSV gD, HSV gH, HSV early protein
product, cytomegalovirus gB, cytomegalovirus gH, and IE protein
gp72; the respiratory syncytial virus antigens: F protein, G
protein, and N--protein; and the tumor antigens: carcinoma CEA,
carcinoma mutated EGF receptor, prostate carcinoma specific antigen
(PSA), prostate specific membrane associated antigen, carcinoma
associated mucin, carcinoma P21, carcinoma P53, melanoma MPG,
melanoma p97, MAGE-1, MAGE-3, gp100, MART-1, carcinoma Neu oncogene
product, carcinoma p53 gene product, and mutated p21 ras
protein.
7. A method for inducing a cytotoxic T-lymphocyte response in an
animal selected from the group consisting of humans, domesticated
animals and agricultural animals, comprising: administering to said
animal an admixture comprising an antigen and a microfluidized
antigen formulation, said antigen formulation comprising: (a) a
stabilizing detergent, (b) a micelle-forming agent, and (c) a
biodegradable and biocompatible oil, said antigen formulation
lacking an immunostimulating peptide component, said antigen
formulation being formulated as a stable oil-in-water emulsion;
wherein said admixture is administered to said animal in an amount
sufficient to induce a cytotoxic T-lymphocyte response in said
animal which is specific for the antigen contained in said
admixture.
8. The method of claim 7, wherein said antigen formulation consists
essentially of said detergent, agent, and oil.
9. The method of claim 7, wherein said method consists essentially
of a single administration of said mixture to said human or said
animal.
10. The method of claim 7, wherein said human or said animal is
infected with a virus or suffers one or more symptoms of infection
from said virus.
11. The method of claim 7, wherein said antigen formulation is
non-toxic to said human or said animal.
12. The method of claim 7, wherein said antigen is chose from the
HIV antigens: gp160, gag, pol, Nef, Tat, and Rev; the malaria
antigens: CS protein and Sporozoite surface protein 2; the
Hepatitis B surface antigens: Pre-S1, Pre-S2, HBc Ag, and HBe Ag;
the influenza antigens: HA, NP and HA; Hepatitis A surface
antigens; Hepatitis C surface antigens; the Herpes virus antigens:
EBV gp340, EBV gp85, HSV gB, HSV gD, HSV gH, HSV early protein
product, cytomegalovirus gB, cytomegalovirus gH, and IE protein
gP72; the respiratory syncytial virus antigens: F protein, G
protein, and N protein; and the tumor antigens: carcinoma CEA,
carcinoma associated mucin, carcinoma P21, carcinoma P53, melanoma
MPG, melanoma p97, MAGE-1, MAGE-3, gp100, MART-1, carcinoma mutated
EGF receptor, prostate carcinoma specific antigen (PSA), prostate
specific membrane associated an tigen, and carcinoma Neu oncogene
product, carcinoma *mutated EGF receptor, carcinoma p53 gene
product, and mutated p21 ras protein.
13. A method of treating a patient infected with HIV virus,
comprising administering a composition comprising an HIV antigen
mixed with a microfluidized antigen formulation comprising: (a) a
stabilizing detergent, (b) a micelle-forming agent, and (c) a
biodegradable and biocompatible oil, said antigen formulation
lacking an immuno-stimulating peptide component, and being
formulated as a stable oil-in-water emulsion; wherein said
composition is administered to said patient in an amount sufficient
to induce a cytotoxic T-lymphocyte response in said patient.
14. The method of claim 13, wherein said HIV antigen is selected
from gp160, gag, pol, Nef, Tat, and Rev. 77
15. A method of treating a patient suffering from malaria,
comprising administering a microfluidized composition comprising a
malaria-associated antigen mixed with an antigen formulation
comprising: (a) a stabilizing detergent, (b) a micelle-forming
agent, and c) a biodegradable and biocompatible oil, said antigen
formulation lacking an immunostimulating peptide component, and
being, formulated as a stable oil-in-water emulsion; wherein said
composition is administered to said patient in an amount sufficient
to induce a cytotoxic T-lymphocyte response in said patient.
16. The method of claim 15, wherein said malaria-associated antigen
is selected from CS protein, and Sporozoite surface protein 2.
17. A method of treating a patient suffering from influenza,
comprising administering a composition comprising an
influenza-associated antigen mixed with a microfluidized antigen
formulation comprising: (a) a stabilizing detergent, (b) a
micelle-forming agent, and (c) a biodegradable and biocompatible
oil, said antigen formulation lacking an immunostimulating peptide
component, and being formulated as a stable oil-in-water emulsion;
wherein said composition is administered to said patient in an
amount sufficient to induce a cytotoxic T-lymphocyte response in
said patient.
18. The method of claim 17, wherein said influenza-associated
antigen is selected from HA, NP, and NA.
19. A method of treating a patient suffering from hepatitis,
comprising administering a com position comprising a
hepatitis-associated antigen mixed with a microfluidized antigen
formulation comprising: (a) a stabilizing detergent, (b) a
micelle-forming agent, and (c) a biodegradable and biocompatible
oil, said antigen formulation lacking an immunostimulating peptide
component, and being formulated as a stable oil-in-water emulsion;
wherein said composition is administered to said patient in an
amount sufficient to induce a cytotoxic T-lymphocyte response in
said patient.
20. The method of claim 19, wherein said hepatitis-associated
antigen is selected from hepatitis A surface antigen, Pre-S1,
Pre-S2, HBc Ag, and HBe Ag.
21. A method of treating a patient suffering from a cancer,
comprising administering a composition comprising a
cancer-associated antigen mixed with a microfluidized antigen
formulation comprising: (a) a stabilizing detergent, (b) a
micelle-forming agent, and (c) a biodegradable and biocompatible
oil, said antigen formulation lacking an immunostimulating peptide
component, and being formulated as a stable oil-in-water emulsion;
wherein said composition is administered to said patient in an
amount sufficient-to induce a cytotoxic T-lymphocyte response in
said patient.
22. A method of claim 21, wherein said cancer-associated antigen is
selected from Carcinoma CEA, Carcinoma associated mucin, P21,
carcinoma P53, melanoma MPG, melanoma p97, MAGE-1, MAGE-3, gp100,
MART-1, carcinoma mutated EGF receptor, and carcinoma Neu oncogene
product, carcinoma p53 gene product, and mutated p21 ras
protein.
23. A method of treating a patient infected with herpes virus,
comprising administering a composition comprising a herpes antigen
mixed with a microfluidized antigen formulation comprising: (a) a
stabilizing detergent, (b) a micelle-forming agent, and (c) a
biodegradable and biocompatible oil, said antigen formulation
lacking an immunostimulating peptide component, and being
formulated as a stable oil-in-water emulsion; wherein said
composition is administered to said patient in an amount sufficient
to induce a cytotoxic T-lymphocyte response in said patient.
24. The method of claim 23, wherein said herpes virus antigen is
selected from EBV gp340, EBV gp85, HSV gB, HSV gD, HSV gH, HSV
early protein product, cytomegalovirus gB, cytomegalovirus gH and
IE protein gP72.
25. A method of treating a patient infected with respiratory
syncytial virus, comprising administering a composition comprising
a respiratory syncytial antigen mixed with a microfluidized antigen
formulation comprising: (a) a stabilizing detergent, (b) a
micelle-forming agent, and (c) a biodegradable and biocompatible
oil, said antigen formulation lacking an immunostimulating peptide
component, and being formulated as a stable oil-in-water emulsion;
wherein said composition is administered to said patient in an
amount sufficient to induce a cytotoxic T-lymphocyte response in
said patient.
26. The method of claim 25 wherein said Respiratory Syncytial virus
antigen is selected from F protein, G protein, and N protein.
27. A method for inducing a cytotoxic T-lymphocyte response in a
human or domesticated or agricultural animal, comprising the steps
of: administering a mixture of an antigen mixed with a
microfluidized antigen formulation consisting essentially of two
of: (a) a stabilizing detergent, (b) a micelle-forming agent, and
(c) a biodegradable and biocompatible oil, said antigen formulation
being formulated as a stable oil-in-water emulsion; wherein said
mixture is administered to said human or animal in an amount
sufficient to induce a cytotoxic T-lymphocyte response in said
human or animal.
28. The method of claim 27, wherein said human or domesticated or
agricultural animal is infected with a virus and suffers one or
more symptoms of infection from said virus.
29. The method of claim 27, wherein said antigen formulation is
non-toxic to said human or domesticated or agricultural animal.
30. The method of claim 27, wherein said antigen is chosen from
antigenic portions of the HIV antigens: gp160, gag, pol, Nef, Tat,
and Rev; the malaria anti-gens: CS protein and Sporozoite surface
protein 2; the Hepatitis B surface-antigens: Pre-S1, Pre-S2, HBc
Ag, and HBe Ag; the influenza antigens: HA, NP and NA; Hepatitis A
surface antigens; Hepatitis C surface antigens, the Herpes virus
antigens: EBV gp340, FBV gp85, HSV gB, HSV gD, HSV gH, HSV early
protein product, cytomegalovirus gB., cytomegalovirus gH, and IE
protein gp72; the respiratory syncytial virus antigens: F protein,
G protein, and N protein; and the tumor antigens carcinoma CEA,
prostate carcinoma specific antigen (PSA), prostate specific
membrane associated antigen, carcinoma associated mucin, carcinoma
P21, carcinoma P53, melanoma MPG, melanoma p97, MAGE-1, MAGE-3,
gp100, MART-1, carcinoma Neu oncogene product, carcinoma mutated
EGF receptor, carcinoma p53 gene product, and mutated p21 ras
protein.
31. A method of treating a patient infected with HIV virus,
comprising administering a composition comprising an HIV antigen
mixed with a microfluidized antigen formulation consisting
essentially of two of: (a) a stabilizing detergent, (b) a
micelle-forming agent, and (c) a biodegradable and biocompatible
oil, said antigen formulation being formulated as a stable
oil-in-water emulsion; wherein said composition is administered to
said patient in an amount sufficient to induce a cytotoxic
T-lymphocyte response in said patient.
32. The method of claim 31, wherein said HIV antigen is selected
from gp160, gag, pol, Nef, Tat, and Rv.
33. A method of treating a patient suffering from malaria,
comprising administering a composition comprising a
malaria-associated antigen mixed with a microfluidized antigen
formulation consisting essentially of two of: (a) a stabilizing
detergent, (b) a micelle-forming agent, and (c) a biodegradable and
biocompatible oil, said antigen formulation being formulated as a
stable oil-in-water emulsion; wherein said composition is
administered to said patient in an amount sufficient to induce a
cytotoxic T-lymphocyte response in said patient.
34. The method of claim 33, wherein said malaria-associated antigen
is selected from CS protein, and Sporozoite surface protein 2.
35. A method of treating a patient suffering from influenza,
comprising administering a composition comprising an
influenza-associated antigen mixed with a microfluidized antigen
formulation consisting essentially of two of: (a) a stabilizing
detergent, (b) a micelle-forming agent, and (c) a biodegradable and
biocompatible oil, said antigen formulation being formulated as a
stable oil-in-water emulsion; wherein said composition is
administered to said patient in an amount sufficient to induce a
cytotoxic T-lymphocyte response in said patient.
36. The method of claim 35, wherein said influenza-associated
antigen is selected from HA, NP, and NA.
37. A method of treating a patient suffering from hepatitis,
comprising administering a composition comprising a
hepatitis-associated antigen mixed with a microfluidized antigen
formulation consisting essentially of two of: (a) a stabilizing
detergent, (b) a micelle-forming agent, and (c) a biodegradable and
biocompatible oil, said antigen formulation being formulated as a
stable oil-in-water emulsion; wherein said composition is
administered to said patient in an amount sufficient to induce a
cytotoxic T-lymphocyte response in said patient.
38. The method of claim 37, wherein said hepatitis-associated
antigen is selected from hepatitis A surface antigen, Hepatitis C
surface antigen, Pre-S1, Pre-S2, HBc Ag, and HBe Ag.
39. A method of treating a patient suffering from a cancer,
comprising administering a composition comprising a
cancer-associated antigen mixed with a microfluidized antigen
formulation consisting essentially of two of: (a) a stabilizing
detergent, (b) a micelle-forming agent, and (c) a biodegradable and
biocompatible oil, said antigen formulation being formulated as a
stable oil-in-water emulsion; wherein said composition is
administered to said patient in an amount sufficient to induce a
cytotoxic T-lymphocyte response in said patient.
40. The method of claim 39, wherein said cancer-associated antigen
is selected from Carcinoma CEA, prostate carcinoma specific antigen
(PSA) prostate specific membrane associated antigen, Carcinoma
associated mucin, P21, carcinoma P53, melanoma MPG, melanoma p97,
MAGE-1, MAGE-3, gp100, MART-1, carcinoma Neu oncogene product,
carcinoma mutated EGF receptor, carcinoma p53 gene product, and
mutated p21 ras protein.
41. A method of treating a patient infected with herpes virus,
comprising administering a composition comprising a herpes antigen
mixed with a microfluidized antigen formulation consisting
essentially of two of: (a) a stabilizing detergent, (b) a
micelle-forming agent, and (c) a biodegradable and biocompatible
oil, said antigen formulation being formulated as a stable
oil-in-water emulsion; wherein said composition is administered to
said patient in an amount sufficient to induce a cytotoxic
T-lymphocyte response in said patient.
42. The method of claim 41, wherein said herpes virus antigen is
selected from EBV gp340, EBV gp85, HSV gB, HSV gD, HSV gH, HSV
early protein product, cytomegalovirus gB, cytomegalovirus gH and
IE protein gP72.
43. A method of treating a patient infected with respiratory
syncytial virus, comprising administering a respiratory syncytial
antigen mixed with a microfluidized antigen formulation consisting
essentially of two of: (a) a stabilizing detergent, (b) a
micelle-forming agent, and (c) a biodegradable and biocompatible
oil, said antigen formulation being formulated as a stable
oil-in-water emulsion; wherein said composition is administered to
said patient in an amount sufficient to induce a cytotoxic
T-lymphocyte response in said patient.
44. The method of claim 43 wherein said Respiratory Syncytial virus
antigen is selected from F protein, G protein, and N protein.
45. The method of any of claims 25-44 wherein said antigen
formulation consists essentially of said detergent and said
micelle-forming agent.
46. The method of any of claims 25-44 wherein said antigen
formulation consists essentially of said detergent and said
oil.
47. The method of any of claims 25-44 wherein said antigen
formulation consists essentially of said oil and said
micelle-forming agent.
48. The composition of claim 6 wherein said papillomavirus antigen
is selected from the group consisting of the HPV16 E6 antigen,
HPV16 E7 antigen, HPV18 E6 antigen, HPV18 E7 antigen, HPV6 E4
antigen, HPV6 L1 antigen, HPV11 E4 antigen and HPV11 L1
antigen.
49. A method for treating cervical cancer comprising administering
an effective amount of a human papillomavirus antigen formulation
according to claim 48.
50. A method for treating condyloma acuminata comprising
administering an effective amount of a papillomavirus antigen
formulation according to claim 48.
51. A method for treating prostate cancer comprising administering
a composition according to claim 6 wherein the antigen is the
prostate specific antigen.
52. The composition of claim 1 wherein the stabilizing detergent is
selected from the group consisting of polysorbate 80, Tween 20,
Tween 40, Tween 60, Zwittergent 3-12, Teepol HB7 and Span 85.
53. The composition of claim 1 wherein said detergent is provided
in an amount ranging from approximately 0.05 to 0.5%.
54. The composition of claim 53 wherein said amount of detergent is
about 0.2%.
55. The composition of claim 1 wherein said micelle-forming agent
comprises a hydrophile-lipophile balance of between 0 and 2.
56. The composition of claim 1 wherein said micelle-forming agent
is selected from the group consisting of poloxamer 401, Pluronic
L62LF, Pluronic L101, Pluronic L64, PEG1000, Tetronic 1501,
Tetronic 150R1, Tetronic 701, Tetronic 901, Tetronic 1301, and
Tetronic 130R1.
57. The composition of claim 1 wherein the amount of said
micelle-forming agent ranges from between 0.5 to 10%.
58. The composition of claim 57 wherein the amount of said
micelle-forming agent ranges from between 1.25 and 5%.
59. The composition of claim 1 wherein the oil exhibits a melting
temperature less than 60.degree. C.
60. The composition of claim 59 wherein the oil is selected from
the group consisting of squalene, squalane, eicosane,
tetratetracontane, pristane, glycerol, and vegetable oils.
61. The composition of claim 1 wherein the amount of the oil ranges
from between 1 and 10%.
62. The composition of claim 61 wherein the amount of the oil
ranges from between 2.5 and 5%.
63. The composition of claim 1 which comprises less than 20
micrograms of muramyl dipeptide.
64. The composition of claim 63 does not comprise any muramyl
dipeptide.
65. The composition of claim 1 wherein the detergent is polysorbate
80, and the micelle-forming agent is poloxamer 401.
66. The composition of claim 65 wherein the oil is squalane.
67. The composition of claim 1 wherein the detergent is selected
from the group consisting of Tween 20, Tween 40 and Tween 80; the
oil is selected from the group consisting of squalane, eicosane,
and pristane and the micelle-forming agent is selected from the
group consisting of Pluronic L62LF and polyoxamer 401.
68. The method of claim 7 wherein the detergent is selected from
the group consisting of polysorbate 80, Tween 20, Tween 40, Tween
60, Zwittergent 3-12, Teepol HB7 and Span 85.
69. The method of claim 7 wherein said detergent is provided in an
amount ranging from approximately 0.05 to 0.5%.
70. The method of claim 69 wherein the amount of detergent is about
0.2%.
71. The method of claim 7 wherein said micelle-forming agent
comprise a hydrophile-lipophile balance of between 0 and-2.
72. The method of claim 7 wherein said micelle-forming agent is
selected from the group consisting polyoxamer 401, Pluronic-L62LF,
Pluronic L101, Pluronic L64, PEG1000, Tetronic 1501, Tetronic
150R1, Tetronic 701, Tetronic 901, Tetronic 1301 and Tetronic
130R1.
73. The method of claim 7 wherein the amount of said
micelle-forming agent ranges from between 0.5 to 10%.
74. The method of claim 71 wherein the amount of said
micelle-forming agent ranges from between 1.25 and 5%.
75. The method of claim 7 wherein the oil exhibits a melting
temperature of less than 60.degree. C.
76. The method of claim 7 wherein the oil is selected from the
group consisting of squalene, eicosane, tetratetracontane,
glycerol, pristane, and vegetable oils.
77. The method of claim 7 wherein the amount of oil ranges from
between land 10%.
78. The method of claim 77 wherein the amount of oil ranges from
between 2.5 and 5%.
79. The method of claim 7 wherein the admixture comprises less than
20 micrograms of muramyl dipeptide.
80. The method of clam 7 wherein the admixture does not contain any
muramyl dipeptides.
81. The method of claim 7 wherein the detergent is polysorbate 80
and the micelle-forming agent is poloxamer 401.
82. The method of claim 81 wherein the oil is squalane.
83. The method of claim 7 wherein the detergent is selected from
the group consisting of Tween 20, Tween 40 and Tween 80, the oil is
selected from the group consisting of squalane, eicosane, olive oil
and pristane and the micelle-forming agent is selected from the
group consisting of polyoxamer 401, and Pluronic L62LF.
84. The method of claim 7 wherein the particle sizes in the
admixture range from 100 to 300 nm.
85. The composition of claim 1 wherein the particle sizes in the
composition range from 100 to 300 nm.
Description
[0001] This application is a continuation-in-part of pending U.S.
application Ser. No. 08/351,001, filed Dec. 7, 1994, which is a
continuation-in-part of pending U.S. Ser. No. 08/919,787 filed Jul.
24, 1992, which is a continuation-in-part of U.S. Ser. No.
07/735,069, filed Jul. 25, 1991, entitled "Induction of Cytotoxic
T-Lymphocyte Responses," by Syamal Raychaudhuri and William H.
Rastetter (now abandoned). All of these applications are
incorporated by reference in their entirety. This invention relates
to methods and compositions useful for inducing cytotoxic T-cell
mediated responses in humans, and domesticated or agricultural
animals.
BACKGROUND OF THE INVENTION
[0002] Cytotoxic T-lymphocytes (CTLs) are believed to be the major
host defense mechanism in response to a variety of viral infections
and neoplastic or cancerous growth. These cells eliminate infected
or transformed cells by recognizing antigen fragments in
association with various molecules (termed class I MHC molecules)
on the infected or transformed cells. CTLs may be induced
experimentally by cytoplasmic loading of certain soluble antigens
within specific cells. Immunization with the soluble antigen alone
is generally insufficient for specific cytotoxic T-lymphocyte
induction.
[0003] One method by which CTL response may be induced involves the
use of recombinant engineering techniques to incorporate critical
components of an antigen in question into the genome of a benign
infectious agent. The aim of such a strategy is to generate
antigen-specific cytotoxic T-lymphocyte responses to the desired
epitope by subjecting the host to a mild, self-limiting infection.
Chimeric vectors have been described using vaccinia, polio, adeno-
and retro-viruses, as well as bacteria such as Listeria and BCG.
For example, Takahashi et al. 85 Proc. Natl. Acad. Sci., USA 3105,
1988 describe use of recombinant vaccinia virus expressing the HIV
gp160 envelope gene as a potential tool for induction of cytotoxic
T-lymphocytes.
[0004] A second method by which a cell mediated response may be
induced involves the use of adjuvants. While the art appears
replete with discussion of the use of adjuvants it is unclear in
such art whether cell mediated immunity was induced and whether
such cell mediated immunity included a cytotoxic T-lymphocyte
response. The following, however, are representative of, various
publications in this area.
[0005] Stover et al., 351 Nature 456, 1991 (not admitted to be
prior art to the present application) describes a CTL response to
.beta.-galactosidase using recombinant BCG containing a
.beta.-galactosidase gene. No such response was detected using
incomplete Freund's adjuvant and .beta.-galactosidase.
[0006] Mitchell et al., 8 J. Clinical Oncology 856, 1990 (which is
not admitted to be prior art to the present invention) describe
treatment of metatastic melanoma patients with an adjuvant termed
"DETOX" and allogeneic melanoma lysates administered five times
over a period of six weeks. In a small portion of the patients an
increase in cytolytic T-cells was observed. The authors describe a
need to enhance the level of cytotoxic T-lymphocyte production, and
suggest a combined therapy of adjuvant with Interleukin-2, as well
as a pretreatment with cyclophosphamide to diminish the level of
tumor specific T-suppressor cells that might exist. DETOX includes
detoxified endotoxin (monophosphoryl lipid A) from Salmonella
minnesota, cell wall skeletons of Mycobacterium phlei, squalene oil
and emulsifier.
[0007] Allison and Gregoriadis, 11 Immunology Today 427, 1990
(which is not admitted to be prior art to the present invention)
note that the only adjuvant "autho-present invention) note that the
only adjuvant "authorized for use" in human vaccines is aluminum
salts (alum) which does not consistently elicit cell mediated
immunity. Allison and Gregoriadis state "[t]here is, therefore, a
need to develop adjuvants with the efficacy of Freund's complete
adjuvant but without its various side effects such as granulomas."
They go on to state that three possible strategies exist, for
example, the use of liposomes; the use of adjuvants, termed
immunostimulating complexes (ISCOMs, which include saponin or Quil
A (a triterpenoid with two carbohydrate chains), cholesterol, and
phosphatidyl choline) which are authorized for use in an influenza
vaccine for horses (Morein et al., Immunological Adjuvants and
Vaccines, Plenum Press, 153); and the use of an emulsion (SAF) of
squalene or Squalane (with or without a pluronic agent) and muramyl
dipeptide (MDP). SAF is said to elicit a cell mediated immunity in
mice, although it "has long been thought that subunit antigens
cannot elicit cytotoxic T-cell (CTL) responses."
[0008] Takahashi et al., 344 Nature 873, 1990, describe class II
restricted helper and cytotoxic T-lymphocyte induction by use of
ISCOMs with a single subcutaneous immunization in mice. They state
that Freund's adjuvant, incomplete Freund's adjuvant, and phosphate
buffered saline did not induce cytotoxic T-lymphocyte activity
against the targets in which they were interested. They state that,
in contrast to results with other forms of exogenous soluble
protein antigen, they have shown that it is possible to prime
antigen specific MHC class I restricted CD8.sup.+ CD4.sup.- CTL by
immunization with exogenous intact protein using ISCOMs. They also
state that the experiments described suggest that it may be
possible to elicit human CTL by using ISCOMs containing HIV
proteins, and that ISCOM-based vaccines may achieve the long sought
goal of induction of both CTL and antibodies by a purified
protein.
[0009] Byars and Allison, 5 Vaccines 223, 1987 describe use of
SAF-1 which includes TWEEN 80, PLURONIC L121, and squalene or
Squalane, with or without muramyl dipeptide, and suggest that their
data indicate that the formulation with muramyl dipeptide will be
useful for human and veterinary vaccines. Booster shots of the
adjuvant were provided without the muramyl dipeptide. The muramyl
dipeptide is said to increase antibody production significantly
over use of the adjuvant without muramyl dipeptide. Cell mediated
immunity was measured as delayed type hypersensitivity by skin
tests to determine T-helper cell induction. Such hypersensitivity
was stronger and more sustained when muramyl dipeptide was provided
in the adjuvant. Similar adjuvants are described by Allison et al.,
U.S. Pat. No. 4,770,874 (where it is stated that the combination of
muramyl dipeptide and pluronic polyol is essential to elicit a
powerful cell mediated and humoral response against egg albumin);
Allison et al., U.S. Pat. No. 4,772,466; Murphy-Corb et al., 246
Science 1293, 1989 (where it is stated that the use of combined
adjuvants with muramyl dipeptide might enhance induction of both
humoral and cellular arms of the immune response); Allison and
Byars, 87 Vaccines 56, 1987 (where it is stated that cell mediated
immunity is elicited by SAF (with muramyl dipeptide) as shown by
delayed type hypersensitivity, by proliferative responses of
T-cells to antigen, by production of Interleukin-2, and by specific
genetically restricted lysis of target cells bearing the immunizing
antigen); Allison and Byars, Immunopharmacology of Infectious
Diseases: vaccine Adjuvants and Modulators of Non-Specific
Resistance 191-201, 1987; Morgan et al., 29 J. Medical Virology 74,
1989; Kenney et al., 121 J. Immunological Methods 157, 1989;
Allison and Byars, 95 J. Immunological Methods 157, 1986 (where
aluminum salts and mineral oil emulsions were shown to increase
antibody formation, but not cell mediated immunity; and muramyl
dipeptide formulations were shown to elicit cell mediated
immunity); Byars et al., 8 Vaccine 49, 1990 (not admitted to be
prior art to the present application, where it is stated that their
adjuvant formulation markedly increases humoral responses, and to a
lesser degree enhances cell mediated reactions to influenzae
haemagglutinin antigen); Allison and Byars, 28 Molecular Immunology
279, 1991 (not admitted to be prior art to the present application;
which states that the function of the muramyl dipeptide is to
induce expression of cytokines and increase expression of major
histocompatibility (MHC) genes; and that better antibody and
cellular responses were obtained than with other adjuvants, and
that it is hoped to ascertain whether similar strategies are
efficacious in humans); Allison and Byars, Technology Advances in
Vaccine Development 401, 1988 (which describes cell mediated
immunity using SAF); Epstein et al., 4 Advance Drug Delivery
Reviews 223, 1990 (which provides an overview of various adjuvants
used in preparation of vaccines); Allison and Byars, 95 J.
Immunological Methods 157, 1986 (which states that the addition of
the muramyl dipeptide to the adjuvant markedly augments cell
mediated responses to a variety of antigens, including monoclonal
immunoglobulins and virus antigens); and Morgan et al., 29 J.
Medical Virology 74, 1989 (which describes use of SAF-1 for
preparation of a vaccine for Epstein-Barr virus).
[0010] Kwak et al., Idiotype Networks in Biology and Medicine,
Elsevier Science Publishers, p. 163, 1990 (not admitted to be prior
art to the present application) describe use of SAF without muramyl
dipeptide as an adjuvant for a B-cell lymphoma idiotype in a human.
Specifically, an emulsion of Pluronic L121, Squalane, and 0.4%
TWEEN-80 in phosphate buffered saline was administered with the
idiotype. They state that "[a]ddition of an adjuvant should further
augment . . . humoral responses, and may facilitate induction of
cellular responses as well.
[0011] Other immunological preparations include liposomes (Allison
et al., U.S. Pat. Nos. 4,053,585, and 4,117,113); cyclic peptides
(Dreesman et al., U.S. Pat. No. 4,778,784); Freunds Complete
Adjuvant (Asherson et al., 22 Immunology 465, 1972; Berman et al.,
2 International J. Cancer 539, 1967; Allison, 18 Immunopotentiation
73, 1973; and Allison, Non-Specific Factors Influencing Host
Resistance 247, 1973); ISCOMs (Letvin et al., 87 Vaccines 209,
1987); adjuvants containing non-ionic block polymer agents formed
with mineral oil, a surface active agent and TWEEN 80 (Hunter and
Bennett, 133 J. Immunology 3167, 1984; and Hunter et al., 127 J.
Immunology 1244, 1981); adjuvants composed of mineral oil and
emulsifying agent with or without killed mycobacteria
(Sanchez-Pescador et al., 141 J. Immunology 1720, 1988); and other
adjuvants such as a lipophilic derivative of muramyl tripeptide,
and a muramyl dipeptide covalently conjugated to recombinant
protein (id.).
SUMMARY OF THE INVENTION
[0012] Applicant has discovered a safe and advantageous method and
compositions by which CTL responses may be induced in humans and
domesticated or agriculturally important animals. The method
involves the use of an antigen formulation which has little or no
toxicity to animals, and lacks an immunostimulating peptide, (e.g.,
muramyl dipeptide) the presence of which would decrease the desired
cellular response. In addition, the methodology is simple to use
and does not require extensive in vivo work to alter existing cells
by recombinant DNA techniques to make them more antigenic. This
discovery is surprising since it was unexpected that such a CTL
response could be induced by use of such an antigen formulation
lacking immunostimulating peptides or their equivalent. Applicant's
findings allow the use of such antigen formulations in a broad
spectrum of disease states, or as a prophylactic agent. For
example, such antigen formulation administration can be used for
the treatment of viral diseases in which a CTL response is
important, for example, in the treatment of HIV infection or
influenza; it can also be extended to use in treatment of bacterial
infections, cancer, parasitic infections, and the like. As a
prophylactic agent, the antigen formulation combined with a
suitable antigen is useful in prevention of infection by viruses
responsible for the aforementioned viral diseases, particularly the
prophylaxis of HIV infection, and also for prophylaxis of patients
at risk of cancer, for example, after resection of a primary
tumor.
[0013] Thus, in a first aspect the invention features a method for
inducing a CTL response in a human or domesticated (e.g., a cat or
dog) or agriculturally important animal (e.g., a horse, cow or pig)
to an antigen other than B-cell lymphoma antigen or egg albumin.
The method includes the steps of providing the antigen to which the
CTL response is desired, and providing a non-toxic antigen
formulation which comprises, consists, or consists essentially of,
a stabilizing detergent, a micelle-forming agent, and a
biodegradable and biocompatible oil. This antigen formulation
preferably lacks any immunostimulating peptide component, or has
sufficiently low levels of such a component that the desired
cellular response is not diminished. This formulation is preferably
provided as a stable oil-in-water emulsion. That is, each of the
various components are chosen such that the emulsion will remain in
an emulsion state for a period of at least one month, and
preferably for more than one year, without phase separation. In the
method the antigen and antigen formulation are mixed together to
form a mixture (preferably by microfluidization), and that mixture
administered to the animal in an amount sufficient to induce CTL
response in the animal. Such administration is required only
once.
[0014] By "stabilizing detergent" is meant a detergent that allows
the components of the emulsion to remain as a stable emulsion. Such
detergents include polysorbate, 80 (TWEEN),
(Sorbitan-mono-9-octadecenoat- e-poly(oxy-1,2-ethanediyl;
manufactured by ICI Americas, Wilmington, Del.), TWEEN 40, TWEEN
20, TWEEN 60, Zwittergent 3-12, TEEPOL HB7, and SPAN 85. These
detergents are usually provided in an amount of approximately 0.05
to 0.5%, preferably at about 0.2%.
[0015] By "micelle-forming agent" is meant an agent which is able
to stabilize the emulsion formed with the other components such
that a micelle-like structure is formed. Such agents preferably
cause some irritation at the site of injection in order to recruit
macrophages to enhance the cellular response. Examples of such
agents include polymer surfactants described by BASF Wyandotte
publications, e.g., Schmolka, 54 J. Am. Oil. Chem. Soc. 110, 1977',
and Hunter et al., 129 J. Immunol 1244, 1981, both hereby
incorporated by reference, PLURONIC L62LF, L101, and L64, PEG1000,
and TETRONIC 1501, 150R1, 701, 901, 1301, and 130R1. The chemical
structures of such agents are well known in the art. Preferably,
the agent is chosen to have a hydrophile-lipophile balance (HLB) of
between 0 and 2, as defined by Hunter and Bennett, 133 Journal of
Immunology 3167, 1984. The agent is preferably provided in an
amount between 0.5 and 10%, most preferably in an amount between
1.25 and 5%.
[0016] The oil is chosen to promote the retention of the antigen in
oil-in-water emulsion, i.e., to provide a vehicle for the desired
antigen, and preferably has a melting temperature of less than
65.degree. C. such that emulsion is formed either at room
temperature (about 20.degree. C. to 25.degree. C.), or once the
temperature of the emulsion is brought down to room temperature.
Examples of such oils include squalene, Squalane, EICOSANE,
tetratetracontane, glycerol, and peanut oil or other vegetable
oils. The oil is preferably provided in an amount between 1 and
10%, most preferably between 2.5 and 5%. It is important that the
oil is biodegradable and biocompatible so that the body can break
down the oil over time, and so that no adverse affects, such as
granulomas, are evident upon use of the oil.
[0017] It is important in the above formulation that a peptide
component, especially a muramyl dipeptide (MDP) be lacking. Such a
peptide will interfere with induction of a CTL response if it
provided in an amount greater than about 20 micrograms per normal
human formulation administration. It is preferred that such
peptides be completely absent from the antigen formulation, despite
their apparent stimulation of the humoral compartment of the immune
system. That is, applicant has found that, although such peptides
may enhance the humoral response, they are disadvantageous when a
cytotoxic T-lymphocyte response is desired.
[0018] In other related aspects, the antigen formulation is formed
from only two of the above three components and used with any
desired antigen (which term includes proteins, polypeptides, and
fragments thereof which are immunogenic) except egg albumin (or
other albumins, e.g., HSA, BSA and ovalbumin), to induce a CTL
response in the above animals or humans.
[0019] Applicant believes that the above formulations are
significantly advantageous over prior formulations (including
ISCOMs, DETOX, and SAF) for use in humans. Unlike such
formulations, the present formulation both includes a
micelle-forming agent, and has no peptides, cell wall skeletons, or
bacterial cell components. The present formulation also induces a
CTL response which either does not occur with the prior
formulations, or is significantly enhanced compared to those
formulations.
[0020] By "non-toxic" is meant that little or no side effect of the
antigen formulation is observed in the treated animal or human.
Those of ordinary skill in the medical or veterinary arts will
recognize that this term has a broad meaning. For example, in a
substantially healthy animal or human only slight toxicity may be
tolerated, whereas in a human suffering from an imminently disease
substantially more toxicity may be tolerated.
[0021] In preferred embodiments, the antigen formulation consists
essentially of two or three of the detergent, agent, and oil; the
method consists essentially of a single administration of the
mixture (antigen plus antigen formulation) to the human or the
animal; the human or animal is infected with a virus and suffers
one or more symptoms (as generally defined by medical doctors in
the relevant field), of infection from the virus; and the antigen
formulation is non-toxic to the human or animal.
[0022] In other preferred embodiments, the antigen is chosen from
antigenic portions of the HIV antigens: gp160, gag, pol, Nef, Tat,
and Rev; the malaria antigens: CS protein and Sporozoite surface
protein 2; the Hepatitis B surface antigens: Pre-S1, Pre-S2, HBc
Ag, and HBe Ag; the influenza antigens: HA, NP and NA; Hepatitis A
surface antigens; Hepatitis C surface antigens; the Herpes virus
antigens: EBV gp340, EBV gp85, HSV gB, HSV gD, HSV gH, HSV early
protein product, human papillomavirus antigens (e.g., HPV antigens,
such as L1, E4, E6, E7 antigens, in particular the E6 and E7
antigens from HPV16 and 18, the two most common HPV types
associated with cervical carcinoma, E4 and L1 specific antigen
(PSA), prostate specific membrane associated antigen,
cytomegalovirus gB, cytomegalovirus gH, and IE protein gP72; the
respiratory syncytial virus antigens: F protein, G protein, and N
protein; and the tumor antigens carcinoma CEA, carcinoma associated
mucin, carcinoma mutated EGF receptor, carcinoma P21, carcinoma
P53, melanoma MPG, melanoma p97, MAGE-1 and MAGE-3, gp100, MART-1,
carcinoma Neu oncogene product, carcinoma p53 gene product, called
gp75, melanoma antigen gp75, and mutated p21 ras protein presented
in a variety of malignant tumors.
[0023] In related aspect, the invention features a composition
comprising, consisting, or consisting essentially of an antigen
mixed with an antigen formulation described above, and the antigen
is chosen from those antigenic portions listed above.
[0024] In other related aspects, the invention features methods of
treating a patient infected with HIV virus, suffering from malaria,
suffering from influenza, suffering from hepatitis, suffering from
a cancer, infected with herpes virus, suffering from cervical
cancer, suffering from condyloma acuminata (genital warts), or
infected with respiratory syncytial virus, by administering a
composition including an appropriate antigen (e.g., selected from
those listed above) mixed with one of the above antigen
formulations. These antigens and treatments are only exemplary of
antigens which may be used in the subject antigen formulations.
[0025] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0026] The drawings will first briefly be described.
DRAWINGS
[0027] FIGS. 1A-1C and 4A-4C are graphical presentations of data
comparing CTL induction by various ovalbumin formulations; E:T
represents effector to target ratio in all Figures.
[0028] FIGS. 2A and 2B are graphical presentations of data
comparing CTL induction by various .beta.-galactosidase
formulations;
[0029] FIG. 3 is a graphical presentation of data comparing CTL
induction by ovalbumin in a liposome and in an antigen
formulation;
[0030] FIGS. 5 and 6 are graphical presentations of data showing
the effect of CD4 and CD8 cell depletion on CTL induction;
[0031] FIG. 7 is a graphical representation of data showing CTL
induction by gp120;
[0032] FIG. 8 is a graphical representation of data showing CTL
induction by a mixture of pluronic and TWEEN and an antigen;
[0033] FIG. 9 is a graphical representation of data showing CTL
induction with a mixture of squalane and pluronic and an
antigen;
[0034] FIG. 10 is a graphical representation of data showing CTL
induction by a mixture of squalane and pluronic and an antigen;
[0035] FIG. 11 is a graphical representation of the effect of OVA
with various antigen formulations on CTL response;
[0036] FIG. 12 is a graphical representation of the induction of
anti-gp120IIIb antibodies in monkeys with various antigen
formulations;
[0037] FIG. 13 depicts antitumor activity of HOPE2 cells ten days
after a single immunization of soluble E7 protein in adjuvant;
and
[0038] FIG. 14 depicts antitumor activity of HOPE2 cells at days
10, 19 after two immunizations with soluble E7 protein in
adjuvant.
ANTIGEN FORMULATION
[0039] Antigen formulations useful in this invention are generally
described above. Those of ordinary skill in this art will recognize
that equivalent Formulations are readily prepared and can be
expected to have equivalent properties in induction of a CTL
response. Such Formulations are readily tested for their properties
using techniques equivalent to those described in the examples
below.
[0040] There follow examples of the invention with the use of an
antigen formulation (AF) composed of about 2.5% squalane, 5%
pluronic acid, and TWEEN 80 in a phosphate buffered saline.
Specifically, an emulsion of the AF included:15 mg squalane, 37.5
mg Poloxamer 401 (PLURONIC L121), 6 mg polysorbate 80 (TWEEN 80),
0.184 mg potassium chloride, 0.552 mg potassium phosphate
monobasic, 7.36 mg. sodium chloride, 3.3 mg sodium phosphate
dibasic (anhydrous), per 1 ml water, pH 7.4. This emulsion was
microfluidized using standard technique (Microfluidics Model M110F)
with a back-pressure module at 11-14,000 psi *with gradual return
to atmosphere pressure, cooling and packing in wet ice.
[0041] Example 11 which relates to papillomavirus antigen
formulations uses an antigen formulation referred to as AF3.75.
This composition comprises squalane, Tween 80, and pluronic L121
dissolved in phosphate buffered saline at pH 7.4 to form a crude
oil-in-water emulsion containing final concentrations of 15%
(wt/vol) squalane, 0.6% Tween 80, and 3.75% pluronic L121. The
emulsion is then cycled through a microfluidizer multiple times at
a reduced temperature to obtain a stable homogeneous emulsion with
a mean particle size ranging from 100 to 300 nm. The AF is diluted
1:3 with antigen (1 part AF and 2 parts antigen) prior to use.
[0042] Another preferred antigen formulation is identical to AF3.75
except that it contains 1.5% pluronic L121.
[0043] In other examples, antigen was mixed with the microfluidized
squalane (S), pluronic (P) and TWEEN 80 (T) mixture to achieve a
final concentration of 0.2% TWEEN 80, 1.25% pluronic and 5%
squalane respectively. To determine the sub-components necessary
for an antigen specific immune response induction, Squalane-TWEEN
80, pluronic-TWEEN 80 or Squalane-pluronic were prepared at the
same concentration as for the three components mixture. Pluronic,
Squalane or TWEEN 80 was also prepared individually to determine
the effect of individual component on the CTL induction.
Substitutions of TWEEN 20, TWEEN 40 or Zwittergent for TWEEN 80
were also made to determine the effect of various TWEEN derivative
on the, CTL induction in the ova system. Substitutions of Squalane
in the three component formulation were made with Eicosone or
Triacontone and substitution for the co-polymer pluronic in the
same three components formulation were made by PEG 1000, Pleuronic
L62LF, and the Tetronics 1501 and 150R1. As two component
formulations, various analogs in various combinations were mixed
and tested for ova specific CTL induction. They are a mixture of
cholesterol--TWEEN 80, Squalane--TWEEN 20, Pristane--TWEEN 80 or
olive oil--TWEEN 80. For a stabilization study, the micro-fluidized
mixture of Squalane-TWEEN 80 was mixed with dextrose to a final
concentration of 5%. In all cases the combinations of excipients
were mixed in a micro-fluidizer to made a stable emulsion. For
immunization purposes, it is preferable that soluble antigen be
mixed with microfluidized excipients to obtain a stable homogeneous
emulsion with particle sizes ranging from about 100 to 300 nm. In
some experiments, two components formulations were mixed with
various concentration of MDP for CTL and humoral response
inductions. Table 1 describes a comprehensive list of various
formulations used in this study.
1TABLE 1 Effect of various substitution in three or two component
systems percent kill at E:T 100:1 Substitution in three component
formulations STP 84 Tween 40(T) 66 Tween 20(T) 48 T1501(P) 0
T150R1(P) 0 Pluronic L62LF(P) 47 Eicosane(S) * PEG1000(P) *
Triacontane(S) * Zwittergent(T) * Substitution in two component
formulations ST 76 PT 45 SP 26 Cholesterol(S) + Tween 80 0 Squalane
+ Tween 29(T) 65 Pristane(S) + Tween 80 42 Olive Oil(S) + Tween 80
69 1 component formulation Pluronic L121 0 Squalane 0 Tween 80 0
Squalane + Tween 80 + 5% dextrose 86 *CTL assay is being
repeated
[0044] Syntex adjuvant formulation (microfluidized; SAFM) was used
as an adjuvant control and consists of two parts. Part I consists
of phosphate buffered saline containing a final concentration of 5%
Squalane, 1.25% pluronic and 0.2% TWEEN 80 (vehicle or I-SAF). Part
II consists of N-Acetylmuramyl-L-Threonyl-D-Isoglutamine (Thr-MDP),
a derivative of mycobacterium cell wall component. For immunization
purposes, antigen is mixed with microfluidized vehicle (part I) to
obtain a homogeneous emulsion. MDP is added to made SAFm, and
vortexed briefly. The MDP concentration in the mixture was varied
to determine if there was an optimum concentration for CTL
induction. As an adjuvant control, mice were also immunized with
soluble antigens mixed with alum according to the manufacturer's,
manual (Pierce Chemical, Rockford, Ill.) or with Complete Freund's
Adjuvant (CFA).
[0045] This antigen formulation is used for induction of cytotoxic
T-lymphocyte responses in mice. Those of ordinary skill in the art
will recognize that such a mouse model is indicative that
equivalent experiments or treatments will similarly induce
cytotoxic T-lymphocyte responses in humans, domesticated, or
agricultural animals. The amount of antigen formulation and antigen
useful to produce the desired cellular response may be determined
empirically by standard procedures, well known to those of ordinary
skill in the art, without undue experimentation. Thus, if desired
to minimize the side effects of treatment with such a mixture those
of ordinary skill in the art may determine a minimum level of such
a mixture for administration to a human, domesticated, or
agricultural animal in order to elicit a CTL response, and thereby
induce immunity to a desired antigen. In normal use, such a mixture
will be injected by any one of a number of standard procedures, but
particularly preferred is an intramuscular injection at a location
which will allow the emulsion to remain in a stable form for a
period of several days or several weeks.
[0046] Methods
[0047] The following materials and methods were used in the
examples provided below unless otherwise noted:
[0048] Mice
[0049] Female C57BL/6 (H-2.sup.b) and BALB/c (H-2.sup.d) mice were
purchased from Harlen Sprague (San Diego, Calif.).
[0050] Antigens
[0051] Ovalbumin (ova, Grade VII; Sigma Chemical Co., St. Louis,
Mo.) was used in the native form. .beta.-galactosidase,
(.beta.-gal, Grade VIII; BRL) was used in the native form and after
boiling in 1 M NaOH for 2 min to give an alkali digest. Recombinant
gp120 was purchased from American Biotechnology.
[0052] Tumor Cells and Transfectants
[0053] The tumor cells used were the Ia lines EL4 (C57BL/6,
H-2.sup.b thymoma) and P815 (DBA/2, H-2.sup.d mastocytoma).
Derivation of the ova-producing EL4 transfectant, EG7-ova, is
described previously by Moore et al., 54 Cell 777, 1988. The
.beta.-gal-producing transfectant, P13.1, was derived by
electroporation of 10.sup.7 P815 cells in 1 ml of phosphate
buffered saline (PBS) with 10 mg of PstI linearized pCH110
(Pharmacia LKB Biotechnology Inc., Piscataway, N.J.) and 1 mg of
PvuI linearized pSV2 neo (Southern et al., 1 J. Mol. Appl. Genet.
327, 1982) followed by selection in 400 .mu.g/ml of the antibiotic
G418. The C3-4 transfectant was derived from the BALB/c hybridoma
Igm 662 by transfecting with a plasmid encoding the .beta.-gal gene
fused to the third and fourth exon of IgM heavy chain (Rammensee et
al., 30 Immunogenetics 296, 1989). The gp160IIIb expressing 3T3
fibroblast, 15-12, was provided by Dr. Germain of NIH (Bethesda,
Md.). The K.sup.b transfected L cell line was provided by Dr.
Carbone, Monash University, Australia. The D.sup.d and L.sup.d
transfected L cell lines were provided by Dr. Ted Hensen,
Washington University, St. Louis.
[0054] Immunization
[0055] Mice were immunized intravenously with a 200 .mu.l
suspension of 25.times.10.sup.6 splenocytes, after a cytoplasmic
loading as described by Moore et. al. supra, and Carbone et al., J.
Exp. Med. 169:603, 1989). For ova-antigen formulation or
.beta.-gal-antigen formulation immunization, 30 .mu.g of each
protein antigen was injected per mouse in the footpad and the
tailbase subcutaneously. Each injection consists of 67 .mu.l of
microfluidized antigen formulation (made following standard
procedures) and 30 .mu.g of protein antigen in a final volume of
200 .mu.l. The final volume was made up with HBSS, see, Whittaker
manual (Welkersville, Md.). MDP was provided in concentrations
between 0 and 300 .mu.g. Where stated, mice were immunized with
soluble antigens in CFA, or in alum in a total volume of 200
.mu.l.
[0056] In Vitro Stimulation of Effector Populations
[0057] Spleen cells (30.times.10.sup.6) from normal or immunized
mice which had been primed at least 14 days earlier were incubated
with 1.5.times.10.sup.6 EG7-ova (irradiated with 20,000 rad) for
ova responses or 1.5.times.10.sup.6 C3-4 cells (irradiated with
20,000 rad) for .beta.-gal response in 24 well plates at 37.degree.
C. in 7% CO.sub.2/air. All the tissue cultures were performed in a
complete medium consisting of IMDM medium, see, Whittaker Manual
(Welkersville, Md.) supplemented with 10% fetal calf serum (FCS), 2
mM glutamine, gentamycin and 2.times.10.sup.-5 M 2-mercaptoethanol.
For the in vitro depletion experiments, in vivo primed or in vitro
stimulated spleen cells were treated with monoclonal antibodies
(mAbs) RL.172 (anti-CD4) or mAbs 3.168 (anti-CD8) for removal of
CD4.sup.+ or CD8.sup.+T cells (Sarmiento et al., 125 J. Immunol.
2665, 1980, and Ceredig et al., 314 Nature 98, 1985). The mAb
RL.172 and mAb 3.168 were obtained from Dr. Jonathan Sprent at
Scripps Clinic and Research Foundation, La Jolla, Calif.
[0058] Spleen cells (30.times.10.sup.6) from normal or immunized
mice which had been primed at least 21 days earlier were incubated
with 1.5.times.10.sup.6 15-12 cells (treated with 200 ug of
mitomycin C for 45 minutes per 10.sup.8 cells), or with 500 .mu.g
of 18IIIb peptide containing the dominant CTL epitope in Balb/c
mice in complete IMDM media (Irvine Scientific, Santa Ana, Calif.)
containing 10% pre-screened FCS (ICN Flow; ICN Biochemicals, Inc.,
Costa. Mesa, Calif.), 2 mM glutamine, gentamycin and
2.times.10.sup.-5 M 2-mercaptoethanol. For in vitro stimulation
with peptides, spleen cells were cultured in complete IMDM
containing 5% ConA supernatant.
[0059] For depletion experiments, in vivo primed or in vitro
stimulated spleen cells were treated with mAbs RL.172 (anti-CD4) or
mAbs 3.168 (anti-CD8) in presence of low tox. Rabbit complement
(Cederlane Laboratories, Ltd., Hornby Ontario, Canada) for removal
of CD4.sup.+ or CD8.sup.+ T cells (22, 23). The Ab RL.172 and mAb
3.168 were a gift from Dr. Jonathan Sprent at Scripps Clinic and
Research Foundation, La Jolla, Calif.
[0060] Cytoxicity Assay
[0061] Target cells (1.times.10.sup.6) were labeled with 100 .mu.Ci
[.sup.51Cr] sodium chromate for 60 min. For peptide pulsed targets,
50 .mu.l of a 1 mg/ml peptide solution in HBSS was added during the
targets labeling with .sup.51Cr. After washing, 10.sup.4 labeled
targets and serial dilutions of effector cells were incubated in
200 .mu.l of RP10 for 4 h at 37 C. 100 .mu.l of supernatant was
collected and the specific lysis was determined as: Percent
specific lysis=100.times.{(release by CTL-spontaneous
release)/(maximal release-spontaneous release)}. Spontaneous
release in the absence of cytotoxic T-lymphocyte (CTL) was <25%
of maximal release by detergent in all experiments.
[0062] Determination of Antibody Responses in Mice and Monkeys
[0063] Each well of 96-well, U bottomed plates (Costar, Cambridge,
Mass.) were coated with 150 ng of ova or gp120 in 50 ul of HBSS and
incubated overnight at 4.degree. C. For the determination of
anti-gp120 and anti-ova antibody responses in mice, plates were
blocked with 1% BSA for 1 hr. Serially diluted sera were added in
25 .mu.l volume per well and incubated for 2 hrs. Plates were
washed and 50 .mu.l of 1:1000 dilution of goat anti-mouse IgG
conjugated to HRPO (SBT, Alabama) in 1% BSA were added per well.
After 1 hr of incubation, plates were washed and 100 .mu.l of
substrate was added per well. The OD.sub.405 was taken after 10 to
15 minutes. For the determination of monkey anti-gp120 antibody
response, all the steps were the same except both the blocking of
plates and the dilution of sera were done in 5% normal goat serum
in Hank's balanced salt solution.
[0064] Peptide Synthesis
[0065] Synthetic peptides corresponding to amino acid sequences
253-276 (Sequence Listing No. 1: EQLESIINFEKLTEWTSSNVMEER; where
the standard one letter code is used to represent each amino acid)
of ovalbumin (ova 253-276), amino acid sequences 84-102 of myelin
basic protein (MBP 84-102) (Sequence Listing No. 2:
DENPVVHFFKNIVTPRTPP), and synthetic peptides corresponding to amino
acid sequences 308-322 (18IIIb sequence) of gp120IIIb, were
assembled by solid phase peptide synthesis using an Applied
Biosystems 430A synthesizer. Amino acids were coupled via
pre-formed symmetric anhydrides with the exception of asparagine,
glutamine and arginine which were coupled as hydroxybenzotriazole
esters. Coupling efficiency was monitored by ninhydrin reaction
following the method of Kaiser et al. 34 Anal. Biochem. 595, 1970.
The peptides were released from the support with HF following the
"low-high" procedure described by Tam, et al. 21 J. Am. Chem. Soc.
6442, 1983, and the peptides extracted from the resin with 10%
acetic acid. After lyophilization, peptides were desalted on a
Sephadex G-25 column, and samples of the peptides then HPLC
purified by reverse phase chromatography on a Vydac preparative
C-18 column. Purified peptides (98%) were solubilized in HBSS at a
final concentration of 10 mg/ml and diluted to the desired
concentration in the complete media.
[0066] CNBr Digest
[0067] Samples of protein (e.g., .beta.-galactosidase) were treated
with 100 fold molar excess of cyanogen bromide in a solution of 100
mM trifluoroacetic acid. The reaction was allowed to proceed for 18
hours at room temperature (about 20.degree. C.) with rotation.
Following the prescribed reaction time, the peptide fragments were
separated from the reactant using a SEP-PAK C-18 apparatus
(Waters), eluted with 95% acetonitrile, and lyophilized.
[0068] Alkaline Digest
[0069] Protein samples (e.g., .beta.-galactosidase) were treated
with 1 N NaOH and boiled for 2 minutes, and the resulting peptide
fragments were separated from the reactants using a C-18. SEP-PAK
apparatus (Waters), and eluted with 95% acetonitrile and
lyophilized.
EXAMPLE 1
Class I Restricted CTL Priming
[0070] Moore et al., 113 UCLA Symp. Mol. Cell. Biol. 1989 and
Carbone and Bevan, 171 J. Exp. Medicine 377, 1990, demonstrate that
mice immunized with spleen cells loaded cytoplasmically with
soluble ova, were primed for ova specific, class I restricted CTL
response. The ova-expressing EL4 transfectant EG7-ova was employed
for in vitro stimulation of in vivo primed splenic lymphocytes and
also used as target for ova specific CTL mediated killing. This
study also demonstrated that CD8.sup.+ effectors induced by EG7-ova
transfectant or by spleen cells cytoplasmically loaded with ova,
recognize a determinant mapped by the peptide ova 258-276 in the
context of H-2 K.sup.b, lyse EG7-ova, and also kill EL4 cells
coated with ova 258-276. Thus, in order to assess whether an
endogenous class I restricted CD8.sup.+ T cell pathway can be
induced by a soluble antigen, the above system was used to
determine whether certain antigen formulations can be used to drive
soluble antigen into a class I restricted pathway.
[0071] a) ova
[0072] C57BL/6 mice were immunized once with various amounts of ova
(30 .mu.g-1 mg per mouse) with or without an antigen formulation.
Mice were injected subcutaneously and in the tailbase. Spleen cells
were taken from the immunized mice at least two weeks after the
immunizations and in vitro stimulated with the EG7-ova
transfectants. An ova concentration as low as 30 .mu.g was as
effective as a 1 mg dose. Therefore, the CTL studies were routinely
performed with spleen cells from 30 .mu.g ova-primed mice. After
five days of in vitro culture with EG7-ova, priming was assessed by
the presence of ova specific effectors capable of lysing
EG7-ova.
[0073] Mice injected with soluble ova in HBSS as high as 1 mg,
showed no evidence of CTL priming (FIG. 1A). However mice immunized
with 30 .mu.g ova in the antigen formulation described above (shown
as AF in the figures) showed a significant transfectant specific
CTL response (FIG. 1C). Furthermore, the extent of EG7-ova killing
by the ova-AF immunized spleen cells was comparable to that of
ova-loaded spleen cells immunized mice (FIG. 1B).
[0074] That the specificity of CTL priming in vivo was antigen
specific was shown by the lack of spleen cells from
.beta.-galactosidase immunized mice to manifest secondary CTL
response in vitro when stimulated with EG7-ova. No ova specific CTL
induction was observed.
[0075] b) .beta.-galactosidase
[0076] Similar results were obtained using another soluble protein
antigen, .beta.-gal. For assaying .beta.-gal-specific CTL response,
the target used was BALB/c derived .beta.-gal-expressing C3-4
transfectant. Immunization of BALB/c mice with soluble .beta.-gal
gave background CTL response. Therefore, for the determination of
specific CTL response, harvesting was postponed for at least eight
weeks before spleen lymphocytes were harvested, and cultured for
five days in the presence of irradiated C3-4 transfectants.
[0077] FIG. 2B demonstrates that 30 .mu.g of .beta.-galactosidase
in AF induced strong specific CTL response against transfectant. At
an effector-to-target (E:T).ratio of 3:1, .beta.-gal-AF immunized
mice showed about 80% of specific C3-4 killing. However, only 20%
killing of the same target was achieved with effectors isolated
from .beta.-gal in HBSS immunized mice at the same E:T ratio (FIG.
2A). Since neither EL4 nor P815 expresses class II MHC gene
products and the lysis shows syngeneic restriction, these ova and
.beta.-gal specific effectors are class I MHC restricted.
[0078] To demonstrate the usefulness of the antigen formulation
mice were immunized with soluble ova encapsuled in two types of
liposomes, one of which was a pH sensitive liposome. One week
later, spleen cells were stimulated in vitro, as described above,
and tested against .sup.51Cr-labeled EG7-ova or EL4. FIG. 3 shows a
representative result demonstrating that ova in liposome could not
prime mice for substantial CTL induction. Similar results were
observed when ova was immunized in alum.
EXAMPLE 2
Recognition of Epitope by CTL
[0079] Carbone and Bevan, supra, demonstrated that CTL induced in
C57BL/6 mice by EG7-ova transfectant, and by cytoplasmically
ova-loaded splenocytes recognize EL4 cells coated with the peptide
ova 258-276. To determine whether soluble ovalbumin in AF induces
similar CTL responses, spleen cells were prepared from immunized
mice and stimulated in vitro with EG7-ova. The effectors were
tested against EL4 cells coated with the peptide ova 253-276, or
with a control peptide derived from myelin basic protein (MBP
84-102). The results demonstrate that ova-AF primed CTL with a
similar, specificity to those primed by transfectants, or by
cytoplasmically loaded ova (FIGS. 1A, 1B and 1C). ova-AF primed
effector cells effectively lysed EG7-ova, and an untransfected EL4
cells coated with 50 .mu.g/10.sup.8 cells of ova peptide, but did
not lyse EL4 cells coated with 50 .mu.g/10.sup.8 cells of MBP
peptide.
[0080] In the .beta.-galactosidase system, Carbone and Bevan,
supra, indicated that .beta.-gal expressing transfectant and
splenocytes cytoplasmically loaded with soluble
.beta.-galactosidase, induced CTL which lysed .beta.-gal expressing
transfectant and nontransfectant P815 cells coated with alkali
digested .beta.-galactosidase. Soluble .beta.-galactosidase induces
CTL having, similar specificity when immunized in AF (FIG. 2).
EXAMPLE 3
CTL Effectors are CD8.sup.+ T Cells
[0081] That soluble protein antigens in AF induce CD8.sup.+
effector T cells was shown as follows. Splenocytes from immunized
mice were cultured for five days with irradiated transfectants in
vitro. Thereafter, cells were harvested and depleted of CD4.sup.+
or CD8.sup.+ T cells by using monoclonal anti-CD4 or anti-CD8
antibodies plus complement. Depleted populations were then tested
against .sup.51Cr-EG7-ova in the ova system or .sup.51Cr-P13.1 in
the .beta.-gal system. The data shown in FIG. 4 indicates that, in
the ova system, depletion of CD8.sup.+ T cells abrogated cytolytic
activity conferred by the whole effector cell population. However,
depletion of CD4.sup.+ T cell population did not have any effect on
the lysis of EG7-ova.
[0082] Similarly, in the .beta.-gal system, depletion of CD8.sup.+
T cells abrogated the cytolytic activity of .beta.-galantigen
formulation immunized spleen cells.
EXAMPLE 4
Soluble ova in AF Prime CD8.sup.+ T Cells
[0083] To demonstrate that ova-AF primes CD8.sup.+ T cell
populations in vivo, and is critical for in vitro secondary
response, CD4.sup.+ or CD8.sup.+ populations were depleted from
spleens of ova-AF immunized mice and from naive mice. These treated
populations were then stimulated in vitro with EG7-ova alone, or in
a combination of CD4.sup.+ and CD8.sup.+ T cells from ova-AF
immunized mice, or in various combination of CD4.sup.+ or CD8.sup.+
T cells from ova-AF immunized mice with the CD4.sup.+ or CD8.sup.+
cells from naive mice. FIG. 5 shows that primed CD8.sup.+ cells are
essential for the manifestation of a secondary CTL response in
vitro. These data also indicate that for the effective secondary
CTL response in vitro, CD4.sup.+ T cells are required. CD4.sup.+
cells are not needed for priming. Similarly, CD8.sup.+ T cells were
required for the manifestation of B-gal specific secondary CTL
response in vitro.
[0084] The above examples demonstrate the effect of the antigen
formulation on the induction of class I restricted CTL responses
against soluble protein antigens. The antigen formulation mediated
soluble antigen induced CTL priming, and is similar in activity to
that induced by transfectants and by splenocytes cytoplasmically
loaded with soluble ova or .beta.-gal. In the ovalbumin system,
EG7-ova, cytoplasmically loaded ova splenocytes, and ova-AF
induced: (a) class I restricted CD8.sup.+ CTL; (b) CTL that
recognize target sensitized with ova 253-276 synthetic peptide; and
(c) long lived CTL after only one immunization. In the
.beta.-galactosidase system, the .beta.-gal-AF induced CTL that
recognize .beta.-gal expressing transfectant C3-4, and also the
untransfected P815 cells sensitized with alkali digested
.beta.-gal. This is analogous to what was observed with CTL induced
by immunization with spleen cells cytoplasmically loaded with
.beta.-galactosidase. The induction of ova-specific CTL by antigen
formulation is unique because neither ova encapsulated in a pH
sensitive liposome, nor in alum, could induce CTL priming in
vivo.
[0085] These examples indicate that the antigen formulation used
above, and its equivalents, are useful in human therapy and in
vaccine development for the induction of CTL in various cancers and
viral diseases.
EXAMPLE 5
[0086] This is a specific example to show the use of the above AF
on producing class I restricted CTL priming by soluble gp120 from
HIV.
[0087] The gp160 IIIB expressing cell line (15-12) was produced in
the Balb/c fibroblast-derived 3T3 cell line. It was obtained from
Drs. Ron Germain and Jay Berzofsky, National Institute of Health,
Bethesda, M.D. The gp160 expressing cell line was employed for in
vitro stimulation of in vivo primed splenic lymphocytes, and also
used as target for gp160 specific CTL induction. Balb/c mice were
immunized once with 10 .mu.g of gp160 per mouse with or without AF.
Mice were injected at footpads and tailbase subcutaneously. Spleen
cells were taken from the immunized mice after two weeks of
immunizations and in vitro stimulated with irradiated gp160
transfectants. After five days of culture in vitro, priming was
assessed by the presence of specific effectors capable of lysing
gp160 transfectants, and not the untransfected cell lines. The
results are shown in FIG. 7, where CTL response is potentiated with
AF and gp120.
[0088] The following example demonstrates the use of antigen
formulations of this invention with use of only one or two
components. These examples demonstrate that CTL-responses can be
induced with only two of the above three components.
EXAMPLE 6
Determination of Critical Components Necessary for CTL
Induction
[0089] To determine whether all the above-noted components are
necessary for antigen specific CTL induction, mice were immunized
with ovalbumin in a microfluidized formulation of various
combinations of two of the three components presented in the AF
above. Two component combinations used were as follows;
Squalane/TWEEN in PBS, Squalane/Pluronic in PBS or Pluronic/TWEEN
in PBS. Another set of groups were included where mice were
immunized with ova formulated in a one component system i.e.,
Squalane in PBS, Pluronic in PBS or TWEEN in PBS only. The above
three component antigen formulation was modified to exclude one
component at a time, constituting PBS in its place.
[0090] The above antigen formulations consist of: 0.300 g TWEEN 80
(Aldrich, Wis.) 1.875 g Pluronic L121 (BASF, NJ), and 7.5 g
Squalane (Aldrich, Wis.), brought to 50 ml with PBS.
[0091] The two-component formulations were:
[0092] Squalane/TWEEN: 0.300 g TWEEN 80, and 7.5 g Squalane,
brought to 50 ml with PBS.
[0093] Pluronic/TWEEN: 1.875 g Pluronic L121, and 0.300 g TWEEN 80,
brought to 50 ml with PBS.
[0094] Pluronic/Squalane: 1.875 g Pluronic L121, and 7.5 g
Squalane, brought to 50 ml with PBS.
[0095] The samples were then processed through a micro-fluidizer,
model 110T, Microfluidics corp, and bottled and stored at 4.degree.
C. until use.
[0096] Ovalbumin (Sigma, MO) was weighted and brought to a 0.3
mg/ml solution in HBSS (Whittaker, Supra). The stock 0.3 mg/ml
solution was combined with the two component formulation in the
following amounts: 5 parts Ovalbumin 0.3 mg/ml solution, 3.3 parts
2 component formulation, and 1.7 parts HBSS.
[0097] The formulation was vortexed and kept on ice until injected.
All solutions were combined just prior to injection.
[0098] Each mouse received 200 .mu.l of one formulation containing
30 .mu.l of OVA by injection in both hind footpads and any
remaining solution was injected subcutaneously at the tail base.
Mice were allowed to rest for two to four weeks prior to spleen
harvest.
[0099] Two weeks after immunizations, spleen cells were prepared
and in vitro stimulated with irradiated EG7-OVA. After five days of
culture, the presence of OVA specific CTL was measured by testing
against .sup.51Cr-EG7-OVA or .sup.51Cr-EL4 in a 4 hour .sup.51Cr
release assay. The data shown in FIGS. 8-10 demonstrate that
Ovalbumin formulated in microfluidized two component system can
prime OVA specific CTLs in vivo.
[0100] We further evaluated the relative contribution of the
individual components for their ability to induce CTL when combined
with protein antigens. For immunization purposes soluble antigen
was mixed with microfluidized excipients to obtain a stable
homogeneous emulsion with particle sizes ranging from 250-300 nm.
To further define the components of squalane-Tween 80-pluronic
(STP) formulation responsible for CTL induction, we immunized mice
with ova in squalane-Tween 80 (ST) mixture, pluronic-Tween 80 (PT)
mixture or squalane-pluronic (SP) mixture and as a control, in
squalane (S), Tween 80. (T) or pluronic (P). Mice were also
immunized with ova-SAFm (containing 70 .mu.g of MDP) or ova-alum as
adjuvant controls. For a positive control, mice were immunized with
spleen cells cytoplasmically loaded with soluble ova. Other
combinations and substitutes were also used, and the results are
presented in Table 1.
[0101] For the detection of CTL priming studies, mice were
immunized once. Two weeks after the immunization, spleen cells were
mixed with irradiated EG7-ova (the ova expressing EL4 cells) for
five days and tested against .sup.51Cr-EG7-ova or .sup.51Cr-EL4
cells. The results (FIG. 11) demonstrate that 30 .mu.g of ova in
combination with STP or ST primes class I restricted CTL response
in mice. The priming-of-ova specific CTL by ova in STP or by ova in
ST appears to be better than that induced by spleen cells
cytoplasmically loaded with soluble ova. Ova in PT or in SP could
induce ova specific CTL responses in mice but inconsistently and
poorly. Unlike SAFm, the addition of MDP to ST formulation did not
compromise the ova specific CTL induction in mice (Table 2). No ova
specific CTL induction occurred when mice were immunized with ova
mixed with the individual components, S, P or T nor when mice were
immunized with ova-SAFm or ova-alum. Mice immunized with as much as
1 mg ova in (a) HBSS, in (b) SAFm or (c) absorbed to alum did not
prime ova specific CTL.
2TABLE 2 Induction of ova specific CTL response is not blocked by
ST + MDP % cytotoxicity in mice immunized with* ova-ST- ova-ST- MDP
MDP ova- 300 .mu.g 72 .mu.g Stimulator Target** ET HBSS ova-ST
mouse mouse EG7-ova EG7-ova 100:1 0 100 82 76 33:1 0 86 67 62 11:1
0 33 39 25 3:1 0 6 13 3 1:1 0 0 0 0 3:1 0 0 0 0 *mice were
immunized with 30 .mu.g ova in various formulations **%
cytotoxicity was calculated by subtracting the percent kill against
antigen non-expressing cell lines
EXAMPLE 7
Components Necessary for ova Specific Antibody Production
[0102] Mice were immunized three times at 2 week intervals with 30
.mu.g of ova in HBSS, STP, ST, PT or SP. As a positive control,
mice were also immunized with ova-SAFm, as SAFm is known to induce
a strong antibody response. Seven days after the second and third
immunizations, mice were bled and the sera tested for ova specific
antibody response. The results are shown in Table 3. They indicate
that mice immunized with ova in STP, ST or in SAFm display similar
anti-ova responses after two immunizations.
3TABLE 3 Induction of anti-ova antibody response 30 .mu.g ova/
animal # mice responded/ 1/dilution formulation # mice injected
sera titer HBSS 0/3 <1/20, <1/20, <1/20 STP 3/3
<1/4860, >1/4860, <1/4860 ST 3/3 >1/4860, >1/4860,
>14860 PT NA NA, NA, NA SP NA NA, NA, NA SAF-M 3/3 1/4860,
1/4860, 1/4860
EXAMPLE 8
HIV gp120 Specific CTL Induction
[0103] HIV gp120 IIIB was used as a second antigen system to
determine CTL induction in STP, ST or in MP-T. Mice were immunized
with 1 .mu.g of gp120 IIIb in HBSS, STP, PT or in ST. As a control,
mice were immunized with 1 .mu.g of gp120IIIb in SAFM or CFA
(Complete Freund's Adjuvant) or in RIBI adjuvant system containing
MPL (monophoshoryl lipid A) and TDM (trehalose dimycolite). Three
weeks after the immunization, spleen cells were prepared and
stimulated in vitro with mitomycin treated transfectant of culture,
the resultant effector cells were tested against vaccinia:gp160
IIIB, or parental vaccinia infected P815 cells as targets. The
results demonstrate that the gp120-Squalane-TWEEN 80 formulation
and not gp120-Squalane-TWEEN 80 pluronic formulation or gp120-HBSS
induced gp120 specific CTL response in mice (Table 4).
4TABLE 4 Induction of gp120 specific CTL response in mice %
cytotoxicity in mice immunized with* gp120- gp120- gp120-
Stimulator Target** E-T HBSS ST STP 18IIIb/IL2 vac:gp120 100:1 23
42 NA*** 33:1 23 38 NA 11:1 0 0 NA 3:1 0 35 NA 18IIIb/IL2 15-12
100:1 0 50 0 33:1 0 35 11:1 0 27 0 3:1 0 18 18IIIb/IL2 3T3 + 18IIIb
100:1 0 59 13 33:1 0 59 2 11:1 0 57 0 3:1 0 29 0 15-12 vac:gp120
100:1 35 84 NA 33:1 19 65 NA 11:1 12 37 NA 3:1 0 22 NA 1:1 0 0 NA
*mice were immunized with 1 .mu.g of gp120III in various
formulations **% cytotoxicity was calculated by subtracting the
percent kill against antigen non-expressing cell lines ***NA; not
available
EXAMPLE 9
Induction of gp120 Specific Humoral Response in Mice
[0104] For the induction of gp120 specific humoral responses, mice
were immunized with 1 .mu.g of gp120IIIb three times at two-week
intervals. The animals were bled and tested for the presence of IgG
antibodies detecting gp120IIIb in a solid phase ELISA assay. The
results demonstrate that gp120-ST is a better immunogen than
gp120-HBSS, gp120SAFm (Table 5), or gp120-STP.
5TABLE 5 Induction of anti-gp120 antibody response 1 .mu.g gp120/
animal # mice responded/ 1/dilution formulation # mice injected
sera titer HBSS 0/3 <1/20, <1/20, <1/20 STP 1/3 <1/20,
>1/4860, <1/20 ST 3/3 >1/4860, >1/4860, >1/4860 PT
3/3 >1/4860, >1/4860, >1/4860 SP 2/3 <1/20, 1/540,
1/540 Saf-M 2/3 1/180, >1/4860, 1/540
EXAMPLE 10
gp120 Specific Antibody Responses in Monkeys
[0105] Monkeys (two per group) were immunized with gp120-SAFm,
gp120-SPT, gp120-ST, or gp120-HBSS. As a control, a group of
monkeys were immunized with recombinant vaccinia containing gp160
IIIb. Monkeys were immunized at two week intervals and bled two
weeks and three weeks after the second immunization. Pre- and
immune sera from each monkey was serially diluted and assayed for
anti-gp120 activity in an ELISA as described in the materials and
methods. The data (FIG. 12) indicate that monkeys immunized with
gp120-STP or gp120-SAFm induced similar responses in monkeys. One
monkey immunized with gp120-ST, induced anti-gp120 response similar
to the gp120-SAFm or gp120'-SPT immunized group. One monkey
immunized with gp120-ST did not induce a strong anti-gp120 response
after two immunizations.
EXAMPLE 11
In Vivo Activity of AF in Combination with HPV 16 E7
[0106] 1. Generation of Recombinant HPV 16 E7 Protein for
Immunization
[0107] a) PCR and Cloning of the E7 Gene
[0108] The HPV 16 E7 gene was cloned from a plasmid obtained from
Dr. Karen Vousden (Ludwig Institute) encoding the E7 gene derived
from the carcinoma cell line CaSki. The coding regions were
amplified by PCR using primers that encode the 5' and 3' ends of
the genes flanked by Bam HI and Sal I cloning sites. The E7 PCR
product was ligated into the pGEX--4T-1 expression vector
(Pharmacia Biotech) resulting in the pGEX.E7 expression plasmid. E.
coli strain XL1--blue (strata-gene) was transfected with the
pGEX.E7 expression plasmid. The sequence of the E7 was obtained
from the plasmids of the, resulting colonies and was identical to
the E7 sequence obtained from CaSki cells.
[0109] b) Production of Purification of Bacterially-Expressed
E7
[0110] The pGEX.E7 bacterial expression plasmid encodes a
glutathione-S-transferase (GST) fusion protein consisting of the
GST at the amino-terminus, a thrombin protease cleavage site and
the E7 protein at the carboxy-terminus. E7 protein was produced and
purified as described in the product information literature from
the manufacturer of the pGEX-4T-1 vector (Pharmacia Biotech).
Briefly, bacteria containing the pGEX.E7 expression plasmid was
induced to express the fusion protein by the addition of isopropyl
b-D-thiogalactosidase to the culture medium. The cells were
harvested and lysed by mild sonication. The lysate was applied to
Glutathione Sepharose 4B (Pharmacia Biotech). After the fusion
protein bound to the matrix, the resin was washed to remove
non-specifically bound proteins. The bound fusion protein was
digested with thrombin to release the E7 protein from the GST
fusion partner.
[0111] The E7 protein preparation was analyzed by SDS-PAGE and the
E7 protein concentration was determined by Bradford analysis
(BioRad). 9 mg soluble E7 protein was obtained per liter of
bacterial culture.
[0112] 2. Generation of the X21 E7 Transfectant
[0113] Coding sequences for the HPV16 E7 protein (see above) have
been inserted into the IDEC proprietary eukaryotic expression
plasmid INPEP4. Within this vector, E7 expression is controlled by
the Cytomegalovirus promoter/enhancer transcriptional elements. In
addition, the first three nucleotides of the E7 coding sequence
have been removed and replaced with an immunoglobulin light chain
leader sequence placed immediately upstream and in frame with the
E7 coding region. Following transfection into the mouse cell line
X21 individual G418 resistant clones were examined by northern blot
analyses for E7 message production. Every clone displayed
detectable E7 message. Western blot analysis of cell lysates from
the two of those clones, 4E7 and 1C7, (HOPE1 and HOPE2
respectively) were then performed and demonstrated E7 protein
production.
[0114] 3. In Vivo Activity of E7/AF Soluble Antigen
Immunization
[0115] Female mice of C3H background (H2.sup.k/k, Harlan Sprague
Dawley) were used in these studies. Animals were maintained
according to "Guide for the Care and Use of Laboratory Animals"
(DHHS Publication No. NIH 86-23, Bethesda, Md.:NIH, 1985), and
received food and water ad libitum. The E7 transfectant cell line
HOPE2H2.sup.k/k) was used in these studies. The tumor cell line was
maintained by serial passage in vitro.
[0116] This cell line has been shown to maintain E7 cytoplasmic
antigen expression, as detected by western blot analysis, following
repeated in vitro passages. Tumors were initiated in syngeneic C3H
mice by subcutaneous injection of 150,000 in vitro passaged
cells.
[0117] Tumors were measured in 2 perpendicular directions at
biweekly intervals. Tumor volume (V) was calculated according to
the following formula:
V(mm.sup.3)=(L.times.W.sup.2) divided by 2
[0118] where:
[0119] L=longest axis measurement in mm
[0120] w=perpendicular axis (mm)
[0121] Data in Table 6 are presented as tumor Mice (number of tumor
bearing animals over the total number of animals injected). Data in
FIGS. 13 and 14 are presented as median tumor size (mm.sup.3) of
each treatment or control group. Each treatment group was compared
to a control group that did not receive therapy. Therapy began 10
days after incoculation of HOPE2 cells, when a majority of the
tumors were palpable (approx. 50-75 mm.sup.3). Therapy was
initiated by immunization of mice with soluble E7 protein in AF3.75
(defined previously) or Alum adjuvants (subcutaneously in a total
volume of 0.2 ml). Directly before immunization, AF3.75 was mixed
for 60 seconds with E7 protein in Hanks Balanced Salt Solution
(HBSS) such that each mouse received either 30 ug or 90 ug E7
protein 0.2 ml. Alum (Pierce Chemical Co.) was mixed with E7
protein, according to instructions by the manufacture, such that
each animal received 90 ug E7 protein in 0.2 ml per mouse. Animals
in a second treatment group received a second immunization 9 days
later (19 days after tumor cell inoculation). Booster Immunization
were prepared immediately before inoculation, as described
above.
[0122] In this example (Table 6: Xp 1233), 41 days after tumor cell
inoculation only 4/8 and 5/8 of mice receiving a single injection
of soluble E7 in AF3.75 (30 ug or 90 ug respectively) had
measurable tumors. In contrast, all of the mice immunized with E7
protein in Alum (8/8) had actively growing tumors. Additionally, as
shown in FIG. 13, significant inhibition of tumor growth was
observed only in treatment groups immunized with E7 protein in
AF3.75 as compared to control (untreated) or Alum treatment groups.
Inhibition of tumor growth (FIG. 13) or increased tumor regression
rates (Table 6 was not observed in mice that received a single
injection of E7 in Alum.
[0123] Similar results were also observed using treatment groups
that received two immunizations at days 10 and 19 after tumor
challenge (Table 6 and FIG. 14), although some tumor growth
retardation was observed with mice receiving two injections of E7
in Alum.
[0124] The results indicate that significant antitumor activity as
measured by a decreased number of tumor bearing mice and inhibition
of tumor growth was observed following immunization of soluble E7
in AF3.75. In contrast, all animals immunized with either a single
or double injection of soluble E7 protein in Alum had growing
tumors. In summary, immunization with soluble E7 protein in AF3.75
resulted in a significant inhibition of tumor cell growth that was
not observed using soluble E7 immunization in Alum.
6TABLE 6 Antitumor activity of soluble E7 immunization in adjuvant
Tumor Animals.sup.a Exp. # Treatment Dose (ug/mouse) Day 41 223
Control -- 7/8 223 E7 in AF3.75 30 ug .times. 1.sup.b 4/8 223 E7 in
AF3.75 90 ug .times. 1 5/8 223 E7 in Alum 90 ug .times. 1 8/8 223
E7 in AF3.75 30 ug .times. 2.sup.c 3/8 223 E7 in AF3.75 90 ug
.times. 2 1/4 223 E7 in Alum 90 ug .times. 2 8/8 .sup.aNumber of
tumor bearing mice/total number inoculated .sup.bAll immunizations
started on Day 10 post implant .sup.cSecond immunication (.times.2)
on Day 19 post implant
[0125] Other embodiments are within the following claims.
Sequence CWU 1
1
2 1 24 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 1 Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu
Thr Glu Trp Thr 1 5 10 15 Ser Ser Asn Val Met Glu Glu Arg 20 2 19
PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 2 Asp Glu Asn Pro Val Val His Phe Phe Lys Asn Ile
Val Thr Pro Arg 1 5 10 15 Thr Pro Pro
* * * * *