U.S. patent application number 17/453736 was filed with the patent office on 2022-02-17 for lymphangiogenesis for therapeutic immunomodulation.
This patent application is currently assigned to The University of Chicago. The applicant listed for this patent is The University of Chicago. Invention is credited to Priscilla S. Briquez, Maria Broggi, Manuel Fankhauser, Sachiko Hirosue, Jeffrey A. Hubbell, Lambert Potin, Maria Stella Sasso, Melody A. Swartz, Efthymia Vokali, Shann S. Yu.
Application Number | 20220047700 17/453736 |
Document ID | / |
Family ID | |
Filed Date | 2022-02-17 |
United States Patent
Application |
20220047700 |
Kind Code |
A1 |
Hubbell; Jeffrey A. ; et
al. |
February 17, 2022 |
LYMPHANGIOGENESIS FOR THERAPEUTIC IMMUNOMODULATION
Abstract
The present invention concerns methods and compositions for
evoking protective immune responses against pathogen infection or
cancer. In certain embodiments, the methods and compositions
comprise a lymphangiogenesis inducer and an antigen.
Inventors: |
Hubbell; Jeffrey A.;
(Chicago, IL) ; Swartz; Melody A.; (Chicago,
IL) ; Yu; Shann S.; (Chicago, IL) ; Vokali;
Efthymia; (Chicago, IL) ; Fankhauser; Manuel;
(Chicago, IL) ; Hirosue; Sachiko; (Chicago,
IL) ; Briquez; Priscilla S.; (Chicago, IL) ;
Broggi; Maria; (Chicago, IL) ; Potin; Lambert;
(Chicago, IL) ; Sasso; Maria Stella; (Chicago,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of Chicago |
Chicago |
IL |
US |
|
|
Assignee: |
The University of Chicago
Chicago
IL
|
Appl. No.: |
17/453736 |
Filed: |
November 5, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17301099 |
Mar 24, 2021 |
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17453736 |
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16096990 |
Oct 26, 2018 |
10980877 |
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PCT/US2017/030242 |
Apr 28, 2017 |
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17301099 |
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62329133 |
Apr 28, 2016 |
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International
Class: |
A61K 39/39 20060101
A61K039/39; A61K 38/18 20060101 A61K038/18; A61P 35/00 20060101
A61P035/00; A61K 39/00 20060101 A61K039/00; A61K 38/19 20060101
A61K038/19; C07K 14/49 20060101 C07K014/49; C07K 16/28 20060101
C07K016/28; G01N 33/574 20060101 G01N033/574; C12N 9/10 20060101
C12N009/10; A61P 37/04 20060101 A61P037/04; A61P 35/04 20060101
A61P035/04; A61K 9/06 20060101 A61K009/06; A61K 45/06 20060101
A61K045/06; A61K 47/42 20060101 A61K047/42 |
Claims
1. A method of eliciting an immune response to an antigen in a
subject comprising administering to the subject one or more
lymphangiogenesis inducers and an effective amount of the
antigen.
2. The method of claim 1, wherein the one or more lymphangiogenesis
inducers comprise vascular endothelial growth factor C (VEGF-C) or
vascular endothelial growth factor D (VEGF-D).
3. The method of claim 1 or 2, wherein the one or more
lymphangiogenesis inducers comprise CCL21.
4. The method of any of claims 1-3, wherein the antigen is
bacterial antigen, a viral antigen, a fungal antigen, a protozoal
antigen, a helminth antigen or a cancer antigen.
5. The method of any of claims 1-4, wherein the lymphangiogenesis
inducer and the antigen are administered in a single
composition.
6. The method of any of claims 1-5, wherein the lymphangiogenesis
inducer and the antigen are provided over multiple
administrations.
7. The method of any of claims 1-6, wherein the subject is also
administered an adjuvant.
8. The method of any of claims 1-7, wherein the lymphangiogenesis
inducer and the antigen are administered in a single composition
comprising an adjuvant.
9. The method of any of claims 1-8, wherein the lymphangiogenesis
inducer and the antigen are administered in a single composition
comprising a pharmaceutically acceptable excipient.
10. The method of any of claims 5-9, wherein the composition is
administered parenterally, subcutaneously or intramuscularly.
11. The method of any of claims 1-10, wherein the subject is
administered an effective amount of a second antigen.
12. The method of any of claims 1-11, wherein the lymphangiogenesis
inducer is incorporated into a matrix.
13. The method of claim 12, wherein the antigen is bound or
incorporated into the matrix and is cleavable.
14. The method of claims 12-13, wherein the matrix incorporates one
or more cleavable chemoattractants or one or more cleavable
cytokines.
15. The method of any one of claims 12-14, wherein the matrix is a
hydrogel.
16. The method of claim 15, wherein the hydrogel is a fibrin
hydrogel or a fibrin domain modified polyethylene glycol
hydrogel.
17. The method of any of claims 12-16, wherein the
lymphangiogenesis inducer is capable of binding to the matrix or
hydrogel.
18. The method of any of claims 12-17, wherein the
lymphangiogenesis inducer comprises a matrix binding domain.
19. The method of any of claims 12-18, wherein the
lymphangiogenesis inducer comprises a protease cleavage site.
20. The method of claim 18 or 19, wherein the lymphangiogenesis
inducer is a modified vascular endothelial growth factor C (VEGF-C)
protein comprising a fibrin-binding domain.
21. The method of claim 19 or 20, wherein the protease cleavage
site is a matrix metalloprotease cleavage site.
22. The method of claim 20 or 21, wherein the VEGF-C is
recombinant, the antigen is recombinant or both the VEGF-C and the
antigen are recombinant.
23. The method of claim 22, wherein the VEGF-C, the antigen or both
the VEGF-C and the antigen are expressed by a heterologous cell
line.
24. The method of any of claims 12-23, wherein the matrix or
hydrogel is implanted into the subject.
25. The method of any of claims 1-24, wherein the lymphangiogenesis
inducer and antigen are administered separately.
26. The method of claim 25, wherein the lymphangiogenesis inducer
and the antigen are administered up to 1 month apart.
27. The method of any of claims 1-26, wherein the subject is a
mammal.
28. The method of any of claims 1-27, wherein the subject is a
human.
29. The method of any of claims 1-28, wherein the immune response
is a protective immune response.
30. The method of any of claims 1-29, wherein the immune response
produces antibodies that specifically bind to the antigen or the
second antigen.
31. The method of any of claims 1-30, wherein the antigen is
encoded by a recombinant nucleic acid molecule.
32. A method of treating or preventing an infection in a subject,
the method comprising administering to the subject an isolated
lymphangiogenesis inducer and an effective amount of an
antigen.
33. The method of claim 32, wherein the lymphangiogenesis inducer
is vascular endothelial growth factor C (VEGF-C) or vascular
endothelial growth factor D (VEGF-D).
34. The method of claim 32 or 33, wherein the antigen is a
bacterial antigen, a viral antigen, a fungal antigen, a helminth
antigen or a protozoal antigen.
35. The method of any of claims 32-34, wherein the
lymphangiogenesis inducer and the antigen are administered in a
single composition.
36. The method of any of claims 32-35, wherein the
lymphangiogenesis inducer and the antigen are provided over
multiple administrations.
37. The method of any of claims 32-36, wherein the subject is also
administered an adjuvant.
38. The method of any of claims 32-37, wherein the
lymphangiogenesis inducer and the antigen are administered in a
single composition comprising an adjuvant.
39. The method of any of claims 32-38, wherein the
lymphangiogenesis inducer and the antigen are administered in a
single composition comprising a pharmaceutically acceptable
excipient.
40. The method of any of claims 35-39, wherein the composition is
administered parenterally, subcutaneously or intramuscularly.
41. The method of any of claims 32-40, wherein the subject is
administered an effective amount of a second antigen.
42. The method of any of claims 32-41, wherein the
lymphangiogenesis inducer is incorporated into a matrix.
43. The method of claim 42, wherein the antigen is bound or
incorporated into the matrix and is cleavable.
44. The method of claims 42-43, wherein the matrix incorporates one
or more cleavable chemoattractants or one or more cleavable
cytokines.
45. The method of any one of claims 42-44, wherein the matrix is a
hydrogel.
46. The method of claim 45, wherein the hydrogel is a fibrin
hydrogel or a fibrin domain modified polyethylene glycol
hydrogel.
47. The method of any of claims 42-46, wherein the
lymphangiogenesis inducer is capable of binding to the matrix or
hydrogel.
48. The method of any of claims 42-47, wherein the
lymphangiogenesis inducer comprises a matrix binding domain.
49. The method of any of claims 42-47, wherein the
lymphangiogenesis inducer comprises a protease cleavage site.
50. The method of claim 48 or 49, wherein the lymphangiogenesis
inducer is a modified vascular endothelial growth factor C (VEGF-C)
protein comprising a fibrin-binding domain.
51. The method of claim 49 or 50, wherein the protease cleavage
site is a matrix metalloprotease cleavage site.
52. The method of claim 50 or 51, wherein the VEGF-C is
recombinant, the antigen is recombinant or both the VEGF-C and the
antigen are recombinant.
53. The method of claim 52, wherein the VEGF-C, the antigen or both
the VEGF-C and the antigen are expressed by a heterologous cell
line.
54. The method of any of claims 42-53, wherein the matrix or
hydrogel is implanted into the subject.
55. The method of any of claims 32-54, wherein the
lymphangiogenesis inducer and antigen are administered
separately.
56. The method of claim 55, wherein the lymphangiogenesis inducer
and the antigen are administered up to 1 month apart.
57. The method of any of claims 32-56, wherein the subject is a
mammal.
58. The method of any of claims 32-57, wherein the subject is a
human.
59. The method of any of claims 32-58, wherein administration of
the lymphangiogenesis inducer and antigen or second antigen
produces antibodies that specifically bind to the antigen or the
second antigen.
60. The method of any of claims 32-59, wherein the antigen is
encoded by a recombinant nucleic acid molecule.
61. A pharmaceutical composition comprising one or more
lymphangiogenesis inducers, an effective amount of an antigen and a
pharmaceutically acceptable excipient.
62. The composition of claim 61, wherein the one or more
lymphangiogenesis inducer comprise vascular endothelial growth
factor C (VEGF-C) or vascular endothelial growth factor D
(VEGF-D).
63. The composition of claim 61 or 62, wherein the one or more
lymphangiogenesis inducers comprise CCL21.
64. The composition of any of claims 61-63, wherein the antigen is
a bacterial antigen, a viral antigen, a fungal antigen, a protozoal
antigen or a cancer antigen.
65. The composition of any of claims 61-64, wherein the composition
comprises an adjuvant.
66. The composition of any of claims 61-65, wherein the composition
is adapted to be administered parenterally, subcutaneously or
intramuscularly.
67. The composition of any of claims 61-66, wherein the composition
comprises an effective amount of a second antigen.
68. The composition of any of claims 61-67, wherein the
lymphangiogenesis inducer is incorporated into a matrix.
69. The composition of claim 68, wherein the antigen is bound or
incorporated into the matrix and is cleavable.
70. The composition of claims 68-69, wherein the matrix
incorporates one or more cleavable chemoattractants or one or more
cleavable cytokines.
71. The composition of any of claims 68-70, wherein the matrix is a
hydrogel.
72. The composition of claim 71, wherein the hydrogel is a fibrin
hydrogel or a fibrin domain modified polyethylene glycol
hydrogel.
73. The composition of any of claims 68-72, wherein the
lymphangiogenesis inducer is bound or incorporated into the matrix
or hydrogel.
74. The composition of any of claims 68-73, wherein the
lymphangiogenesis inducer comprises a matrix binding domain
75. The composition of claim 74, wherein the lymphangiogenesis
inducer comprises a protease cleavage site.
76. The composition of claim 75, wherein the lymphangiogenesis
inducer is a modified vascular endothelial growth factor C (VEGF-C)
protein comprising a fibrin-binding domain and a matrix
metalloprotease cleavage site.
77. The composition of claim 76, wherein the VEGF-C is recombinant,
the antigen is recombinant or both the VEGF-C and the antigen are
recombinant.
78. The composition of claim 77, wherein the VEGF-C, the antigen or
both the VEGF-C and the antigen are expressed by a heterologous
cell line.
79. A vaccine comprising the composition of any one of claims
61-78.
80. A method of treating neoplasia, dysplasia or cancer, the method
comprising administering to a subject one or more lymphangiogenesis
inducers and an effective amount of one or more neoplasia,
dysplasia or cancer antigens.
81. The method of claim 80, wherein the one or more
lymphangiogenesis inducers comprise vascular endothelial growth
factor C (VEGF-C) or vascular endothelial growth factor D
(VEGF-D).
82. The method of claim 80 or 81, wherein the one or more
lymphangiogenesis inducers comprise CCL21.
83. The method of claim 80 or 81, wherein the one or more antigens
are cancer antigens.
84. The method of any of claims 80-83, wherein the one or more
antigens are in a cell lysate.
85. The method of any of claims 80-84, wherein the
lymphangiogenesis inducer and the one or more neoplasia, dysplasia
or cancer antigens are administered in a single composition.
86. The method of any of claims 80-85, wherein the
lymphangiogenesis inducer and the one or more neoplasia, dysplasia
or cancer antigens are provided over multiple administrations.
87. The method of any of claims 80-86, wherein the subject is also
administered an adjuvant.
88. The method of any of claims 80-87, wherein the
lymphangiogenesis inducer and the one or more neoplasia, dysplasia
or cancer antigens are administered in a single composition
comprising an adjuvant.
89. The method of any of claims 80-88, wherein the
lymphangiogenesis inducer and the one or more neoplasia, dysplasia
or cancer antigens are administered in a single composition
comprising a pharmaceutically acceptable excipient.
90. The method of any of claims 85-89, wherein the composition is
administered parenterally, subcutaneously or intramuscularly.
91. The method of any of claims 80-90, wherein the subject is
administered an effective amount of a second neoplasia, dysplasia
or cancer antigen.
92. The method of any of claims 80-91, wherein the
lymphangiogenesis inducer is incorporated into a matrix.
93. The method of claim 92, wherein the antigen is bound or
incorporated into the matrix and is cleavable.
94. The method of claims 92-93, wherein the matrix incorporates one
or more cleavable chemoattractants or one or more cleavable
cytokines.
95. The method of any one of claims 92-94, wherein the matrix is a
hydrogel.
96. The method of claim 95, wherein the hydrogel is a fibrin
hydrogel or a fibrin domain modified polyethylene glycol
hydrogel.
97. The method of any of claims 92-96, wherein the
lymphangiogenesis inducer is capable of binding to the matrix or
hydrogel.
98. The method of any of claims 92-97, wherein the
lymphangiogenesis inducer comprises a matrix binding domain.
99. The method of any of claims 92-98, wherein the
lymphangiogenesis inducer comprises a protease cleavage site.
100. The method of claim 98, wherein the lymphangiogenesis inducer
is a modified vascular endothelial growth factor C (VEGF-C) protein
comprising a fibrin-binding domain.
101. The method of claim 99 or 100, wherein the lymphangiogenesis
inducer is a modified vascular endothelial growth factor C (VEGF-C)
protein comprising a matrix metalloprotease cleavage site.
102. The method of claim 100 or 101, wherein the VEGF-C is
recombinant, the one or more neoplasia, dysplasia or cancer
antigens are recombinant or both the VEGF-C and the one or more
antigens are recombinant.
103. The method of claim 102, wherein the VEGF-C, the antigen or
both the VEGF-C and the one or more neoplasia, dysplasia or cancer
antigens are expressed by a heterologous cell line.
104. The method of any of claims 80-101, wherein the matrix or
hydrogel is implanted into the subject.
105. The method of any of claims 80-104, wherein the
lymphangiogenesis inducer and one or more neoplasia, dysplasia or
cancer antigens are administered separately.
106. The method of claim 105, wherein the lymphangiogenesis inducer
and one or more neoplasia, dysplasia or cancer antigens are
administered up to 1 month apart.
107. The method of any of claims 80-106, wherein the subject is a
mammal.
108. The method of any of claims 80-107, wherein the subject is a
human.
109. The method of any of claims 80-108, wherein administration of
the lymphangiogenesis inducer and the one or more neoplasia,
dysplasia or cancer antigens or the second neoplasia, dysplasia or
cancer antigen produces antibodies that specifically bind to the
one or more neoplasia, dysplasia or cancer antigens or to the
second neoplasia, dysplasia or cancer antigen.
110. The method of any of claims 80-109, wherein the one or more
neoplasia, dysplasia or cancer antigens are encoded by a
recombinant nucleic acid molecule.
111. The method of any one of claims 80-110, wherein the cancer
comprises breast cancer or melanoma or a neoplasia or dysplasia of
the breast or skin.
112. The method of any one of claims 80-111, wherein the method
further comprises administration of an immunotherapy.
113. The method of claim 112, wherein the immunotherapy comprises a
checkpoint blockade inhibitor, adoptive T cell therapy, a chimeric
antigen receptor, a STING agonist, cytolytic virus therapy, one or
more additional antigens, tumor cell lysate, tolerance-breaking
peptide antigen, a dendritic cell vaccine, or an antibody-antigen
conjugate.
114. A method for treating a cancer patient with cancer
immunotherapy comprising administering cancer immunotherapy to the
patient after detecting in a serum sample from that patient a level
of VEGF-C and/or CCL21 that is indicative of response to cancer
immunotherapy.
115. The method of claim 114, further comprising comparing the
level of VEGF-C and/or CCL21 to a control level.
116. The method of claim 115, wherein the control level is a level
of VEGF-C or CCL21 expression from serum of patients who
affirmatively respond to the cancer immunotherapy.
117. The method of claim 116, wherein patients who affirmatively
respond to the cancer immunotherapy exhibit at least a 25%
reduction in tumor growth following treatment with the cancer
immunotherapy.
118. The method of claim 115, wherein the control level is a level
of VEGF-C or CCL21 expression that is the median level in serum of
cancer patients.
119. The method of claim 118, wherein the patient is administered
immunotherapy after being determined to have a level of expression
of VEGF-C or CCL21 that is increased as compared to the level of
expression in serum from noncancer patients.
120. The method of claim 115, wherein the control level is a level
or range of level of VEGF-C or CCL21 expression from serum of
patients who do not respond affirmatively to the cancer
immunotherapy.
121. The method of claim 115, wherein the control level is a level
or range of level of VEGF-C or CCL21 expression in serum from
patients who do not respond affirmatively to the cancer
immunotherapy.
122. The method of any of claims 114-121, further comprising
obtaining serum from the patient.
123. The method of any of claims 114-122, further comprising
measuring the level of VEGF-C and/or CCL21 expression in serum from
the patient.
124. The method of claim 123, wherein measuring the level of VEGF-C
and/or CCL21 expression comprises using a peptide or polypeptide
that binds VEGF-C and/or CCL21.
125. The method of any of claims 114-124, wherein the immunotherapy
is a checkpoint blockade inhibitor, adoptive T cell therapy, a
chimeric antigen receptor, a STING agonist, cytolytic virus
therapy, one or more additional antigens, tumor cell lysate,
tolerance-breaking peptide antigen, a dendritic cell vaccine, or an
antibody-antigen conjugate.
126. A method for predicting the efficacy of cancer immunotherapy
in a patient comprising: measuring a level of VEGF-C and/or CCL21
protein expression in a serum sample from the patient; and,
comparing the level of VEGF-C and/or CCL21 protein expression to a
level in a control sample.
127. The method of claim 126, further comprising predicting
efficacy of the cancer immunotherapy in the patient if the level of
VEGF-C and/or CCL21 protein expression is increased compared to the
level of VEGF-C and/or CCL21 protein expression in a patient
without cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 17/301,099 filed Mar. 24, 2021, which is a
continuation of U.S. patent application Ser. No. 16/096,990 filed
Oct. 26, 2018, now U.S. Pat. No. 10,980,877 issued Apr. 20, 2021,
which is a national phase application under 35 U.S.C. .sctn. 371 of
International Application No. PCT/US2017/030242 filed Apr. 28,
2017, which claims the benefit of priority of U.S. Provisional
Patent Application No. 62/329,133 filed Apr. 28, 2016. The entire
contents of all of the above-referenced disclosures are
specifically incorporated herein by reference without
disclaimer.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0002] The present invention relates generally to the field of
immunology. More particularly, it concerns methods and compositions
for evoking protective immune responses against pathogen infection
or cancer comprising a lymphangiogenesis inducer and an antigen
against which an immune response is desired.
2. Description of Related Art
[0003] Adaptive immune responses are induced in vivo by stimulation
of T and B lymphocytes by antigen-presenting cells (APCs), most
notably dendritic cells (DCs). Induction of antigen-specific immune
responses is important in medical applications such as vaccination,
where pathogen-derived antigens or recombinant or synthetic
antigens are administered to the body in conjunction with adjuvant
molecules to induce expansion of antigen-specific CD4 and CD8 T
cells, expansion of antigen-specific B cells and differentiation
into plasma cells, and production of antibodies (Bachmann, et al.,
2010; Hubbell, et al., 2009; Moon, et al., 2012; Seder, et al.,
2008). This is commonly accomplished by administering the antigen
admixed with adjuvants such as alum, which is a non-specific immune
activator, or with adjuvants such as biomolecular activators of
Toll like receptors, such as monophosphoryl lipid A, gardiquimod,
resiquimod, or CpG oligodeoxynucleotides, for example (Maisonneuve,
et al., 2014; Scott et al., 2012). APCs such as DCs collect the
antigens and present them to lymphocytes; under the influence of
the adjuvant, the APCs present epitopes from the antigens with
co-simulation, so as to induce an effector immune response
(Banchereau & Steinman, 1998; Chen & Flies, 2013). In the
effector phase, T and B lymphocytes expand, and in a memory phase,
those cells populations contract and transition into memory T and B
lymphocytes, capable of responding again rapidly upon re-exposure
to the antigen (Sallusto, et al., 1999; Gerlach, et al., 2010;
Wherry, et al., 2003; Kaech & Wherry, 2007; Dorner &
Radbruch, 2007). Thus, in the classical understanding of adaptive
immune responses, the DC plays a central role in coordinating
response to antigens and inducing effector responses which then
contract and mature into memory T and B cell responses.
[0004] Standard vaccination strategies target conventional APCs,
such as DCs, and initiate an adaptive immune response through
induction of an effector immune phase. Thus, the T cell populations
that are antigen-specific expand, exhibiting activated and
inflammatory behavior, and after some time, their populations
contract, producing memory cells. Indeed, in prophylactic
vaccination, the ultimate goal is to produce enough of these memory
cells, since these are the cells that eventually respond to an
antigenic challenge. In this sense, the initial effector response
is not beneficial per se, but it is generally accepted that a
strong effector phase response leads to a beneficial memory
response, and the literature on vaccination judges strong effector
phase responses as being harbingers of strong generation of memory
cells (Kaech & Wherry, 2007; Sprent & Surh, 2001).
SUMMARY OF THE INVENTION
[0005] Methods and compositions are provided for treating cancer
using VEGF-C and/or CCL21 in conjunction with an antigen against
which an immune response is desired.
[0006] In certain embodiments a method of eliciting an immune
response to an antigen in a subject comprising administering to the
subject one or more lymphangiogenesis inducers and an effective
amount of the antigen is provided. In some embodiments, one or more
lymphangiogenesis inducers and/or antigens are administered. In
specific embodiments, the lymphangiogenesis inducer is vascular
endothelial growth factor C (VEGF-C) or vascular endothelial growth
factor D (VEGF-D). In specific embodiments, the lymphangiogenesis
inducers are vascular endothelial growth factor C (VEGF-C) and
vascular endothelial growth factor D (VEGF-D). In certain
embodiments VEGF-C is human VEGF-C. A human VEGF-C protein may
comprise a sequence that is at least or at most 70%, 80%, 85%, 90%,
95%, 98%, 99% or 100% similar or identical to SEQ ID NO: 1 (or any
range derivable therein) (UniProtKB #P49767; available on the world
wide web at uniprot.org/uniprot/P49767. In other embodiments,
VEGF-C is mouse VEGF-C A mouse VEGF-C protein may comprise a
sequence that is at least or at most 70%, 80%, 85%, 90%, 95%, 98%,
99% or 100% similar or identical to SEQ ID NO: 2 (or any range
derivable therein) (UniProtKB #P97953; available on the world wide
web at uniprot.org/uniprot/P97953. In certain embodiments VEGF-D is
human VEGF-D. A human VEGF-D protein may comprise a sequence that
is at least or at most 70%, 80%, 85%, 90%, 95%, 98%, 99% or 100%
similar or identical to SEQ ID NO: 3 (or any range derivable
therein) (UniProtKB #043915; available on the world wide web at
uniprot.org/uniprot/043915. In other embodiments, VEGF-D is mouse
VEGF-D A mouse VEGF-D protein may comprise a sequence that is at
least or at most 70%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar
or identical to SEQ ID NO: 4 (or any range derivable therein)
(UniProtKB #P97946; available on the world wide web at
uniprot.org/uniprot/P97946.
[0007] In specific embodiments, the lymphangiogenesis inducer
comprises CCL21. In specific embodiments, the lymphangiogenesis
inducers are vascular endothelial growth factor C (VEGF-C) and
CCL21. In some embodiments, the lymphangiogenesis inducer comprises
VEGF-D and CCL21. In some embodiments, the lymphangiogensis inducer
comprises VEGF-C, VEGF-D, and CCL21. In certain embodiments CCL21
is human CCL21. A human CCL21 protein may comprise a sequence that
is at least or at most 70%, 80%, 85%, 90%, 95%, 98%, 99% or 100%
similar or identical to SEQ ID NO: 5 (or any range derivable
therein) (UniProtKB #000585; available on the world wide web at
uniprot.org/uniprot/000585. In other embodiments, CCL21 is mouse
CCL21. A mouse CCL21 protein may comprise a sequence that is at
least or at most 70%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar
or identical to SEQ ID NO: 6 (or any range derivable therein)
(UniProtKB #P86792; available on the world wide web at
uniprot.org/uniprot/P86792.
[0008] The lymphangiogenesis inducer may be wild-type protein or
truncated protein. In some embodiments, the lymphangiogenesis
inducer is released in a controlled-release device or polymer. In
some embodiments, the lymphangiogenesis induces is fused to or
associated with a mistrix-binding sequence.
[0009] In some embodiments, the antigen comprises secreted exosomes
from the patient's tumor cells. In some embodiments, the method
further comprises obtaining a tumor cell sample from the patient.
In some embodiments, the method further comprises isolating
exosomes from the sample from the patient. In some embodiments, the
antigen comprises irradiated tumor cells, tumor lysate, or antigens
described herein.
[0010] Similar polypeptides, peptides and proteins, in some
embodiments, are limited to those proteinaceous compounds whose
substitutions are only with conservative amino acids. In other
embodiments, only conservative substitutions are contemplated,
while in others, deletions of nonessential amino acids or the
addition of other amino acids in an area that is not involved in
the compound's function are contemplated. In other embodiments, the
antigen is a bacterial antigen, a viral antigen, a fungal antigen,
a protozoal antigen, a helminth antigen or a cancer antigen. In
still other aspects, the antigen is one or more bacterial antigens,
one or more viral antigens, one or more fungal antigens, one or
more protozoal antigens, one or more helminth antigens or one or
more cancer antigens. In some aspects an antigen may be a
biomolecule, a protein, a polypeptide, a carbohydrate, a
polysaccharide, a lipid, a nucleic acid, a fatty acid, a
glycolipid, a sterol, a polyterpene, a glycerolipid or a
combination of these.
[0011] The polypeptides described herein may include 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more variant amino acids (or
any range derivable therein) or be at least 70%, 80%, 85%, 90%,
95%, 98%, 99% or 100% similar or identical within at least, or at
most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,
104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,
117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,
130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,
143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,
156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,
169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,
182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,
195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,
208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,
221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,
234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246,
247, 248, 249, 250, 300, 400, 500, 550, 1000 or more contiguous
amino acids, or any range derivable therein, of SEQ ID NO: 1-6. The
contiguous amino acids may start at, include, or exclude amino
acids starting at the N-terminus at positions 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,
108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,
121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,
134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,
147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,
160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,
173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,
186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,
199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,
212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,
225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,
238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250
(or any length therein) in the polypeptide, such as of SEQ ID
NO:1-6.
[0012] A polypeptide segment as described herein may include 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,
106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,
119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,
132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,
145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,
158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,
171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,
184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,
197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,
210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,
223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,
236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,
249, 250, 300, 400, 500, 550, 1000 or more contiguous amino acids,
or any range derivable therein, of SEQ ID NO: 1-6.
[0013] In some embodiments, the lymphangiogenesis inducer or the
antigen or both the lymphangiogenesis inducer and the antigen may
be isolated. The term "isolated" can refer to a nucleic acid or
polypeptide that is substantially free of cellular material,
bacterial material, viral material, or culture medium (when
produced by recombinant DNA techniques) of their source of origin,
or chemical precursors or other chemicals (when chemically
synthesized). Moreover, an isolated compound refers to one that can
be administered to a subject as an isolated compound; in other
words, the compound may not simply be considered "isolated" if it
is adhered to a column or embedded in an agarose gel. Moreover, an
"isolated nucleic acid fragment" or "isolated peptide" is a nucleic
acid or protein fragment that is not naturally occurring as a
fragment and/or is not typically in the functional state.
[0014] In some embodiments, the subject is administered an
antibacterial, antiviral, antifungal, antibiotic, antineoplastic or
chemotherapeutic agent. In some aspects, administration of the
antibacterial, antiviral, antifungal, antibiotic, antineoplastic or
chemotherapeutic agent is concurrent with the lymphangiogenesis
inducer and the antigen. In other aspects the antibacterial,
antiviral, antifungal, antibiotic, antineoplastic or
chemotherapeutic agent is administered separately. The
antibacterial, antiviral, antifungal, antibiotic, antineoplastic or
chemotherapeutic agent may be administered up to 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, 18, 19, 20, 21, 22, 23 or
24 hours apart or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30 or
31 days apart or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12
months apart (or any range derivable therein).
[0015] In particular aspects, the methods and compositions
described are aimed at treating, preventing, ameliorating,
suppressing, resolving, improving or otherwise addressing the
symptoms of a subject or patient with an infection, disease or
condition related to a bacterial infection, a viral infection, a
fungal infection, a protozoal infection, a helminth infection or a
cancer condition. In certain aspects, the subject exhibits one or
more symptoms of a bacterial infection, a viral infection, a fungal
infection, a protozoal infection, a helminth infection or a cancer
condition. In other embodiments, the subject has been diagnosed
with a bacterial infection, a viral infection, a fungal infection,
a protozoal infection, a helminth infection or a cancer condition.
In still other embodiments, the subject is at risk for a bacterial
infection, a viral infection, a fungal infection, a protozoal
infection, a helminth infection or a cancer condition. In specific
embodiments the bacterial infection, viral infection, fungal
infection, protozoal infection, helminth infection or cancer
condition affects one or more organ systems. For example, a
bacterial infection, a viral infection, a fungal infection, a
protozoal infection, a helminth infection or a cancer condition may
affect the circulatory system, integumentary system, skeletal
system, reproductive system, digestive system, urinary system,
respiratory system, endocrine system, lymphatic system, muscular
system, nervous system or immune system. Specifically, a bacterial
infection, a viral infection, a fungal infection, a protozoal
infection, a helminth infection or a cancer condition may infect or
affect the heart, blood, blood vessels, skin, hair, fat, nails,
bones, cartilage, ligaments, tendons, sex organs, ovaries,
fallopian tubes, uterus, vagina, mammary glands, testes, vas
deferens, seminal vesicles, prostate, salivary glands, esophagus,
stomach, liver, gallbladder, pancreas, intestines, rectum, anus,
kidneys, ureters, bladder, urethra, pharynx, larynx, bronchi,
lungs, diaphragm, hypothalamus, pituitary gland, pineal body,
pineal gland, thyroid, parathyroid and adrenals, adrenal glands,
lymph nodes, lymph vessels, skeletal muscles, smooth muscles,
cardiac muscle, brain, spinal cord or peripheral nervous
system.
[0016] In specific embodiments, the lymphangiogenesis inducer and
the antigen are administered in a single composition. In certain
aspects, the lymphangiogenesis inducer and the antigen are provided
over multiple administrations. For example, the lymphangiogenesis
inducer and the antigen may be provided over at least or at most 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or
20 administrations (or any range derivable therein). In a further
aspect, the subject is also administered an adjuvant. In still
other aspects, the lymphangiogenesis inducer and the antigen are
administered in a single composition comprising an adjuvant. In
further aspects, the lymphangiogenesis inducer and the antigen are
administered in a single composition comprising a pharmaceutically
acceptable excipient. In specific embodiments, the composition is
administered parenterally, subcutaneously or intramuscularly. In
some embodiments, the adjuvant is operapably linked to the tumor
antigen. In some embodiments, the adjuvant is fused through a
peptide bond to the tumor antigen. In some embodiments, the
adjuvant is coupled to the tumor antigen chemically or encapsulated
with the tumor antigen. In some embodiments, the adjuvant is
injected into the site of the tumor and/or site of administration
of the lymphanogenesis inducer and antigen. In some embodiments,
the adjuvant is applied topically at the site of the tumor and or
site of administration of the lymphanogensis inducer and
antigen.
[0017] In certain aspects, administering to a patient or a subject
an effective amount of a composition comprising a lymphangiogenesis
inducer and the antigen comprises more than one administration of
the composition. In certain aspects, the composition is
administered orally, intravenously, subcutaneously, intradermally,
intramuscularly, nasally, by injection, by inhalation, and/or using
a nebulizer.
[0018] In still other embodiments, the subject is administered an
effective amount of a second antigen. For example, the second
antigen may be a bacterial antigen, a viral antigen, a fungal
antigen, a protozoal antigen, a helminth antigen or a cancer
antigen or a second bacterial antigen, a second viral antigen, a
second fungal antigen, a second protozoal antigen, a second
helminth antigen or a second cancer antigen. In some aspects the
second antigen may be a biomolecule, a protein, a polypeptide, a
carbohydrate, a polysaccharide, a lipid, a nucleic acid, a fatty
acid, a glycolipid, a sterol, a polyterpene, a glycerolipid or a
combination of these.
[0019] In further aspects, the lymphangiogenesis inducer is
incorporated into a matrix. In certain aspects, the antigen is
bound or incorporated into the matrix and is cleavable. In other
aspects, the matrix incorporates one or more cleavable chemokines,
chemoattractants, chemorepellants or one or more cleavable
cytokines. For example, the chemokine may be one or any combination
of chemokines selected from the group consisting of CCL1, CCL2,
CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/CCL10, CCL11, CCL12,
CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21,
CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2,
CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11,
CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2 and
CX3CL1. A cytokine for use with the methods or compositions
disclosed herein may include members of the IL 17, IL-10,
Interleukin, Lymphokine, Monokine, Myokine, Tumor necrosis factor
or Proinflammatory cytokine families. Specific examples of
cytokines for use with the methods and compositions described
herein include one or any combination of Erythropoietin, GcMAF,
Granulocyte colony-stimulating factor, Granulocyte macrophage
colony-stimulating factor, Hepatocyte growth factor, ILIA,
Interferon, Interferon beta-1a, Interferon beta-lb, Interferon
gamma, Interferon type I, Interferon type II, Interferon type III,
Interleukin 1 beta, Interleukin 1 receptor antagonist, Interleukin
10, Interleukin 12, Interleukin 13, Interleukin 16, Interleukin 2,
Interleukin 23, Interleukin 23 subunit alpha, Interleukin 34,
Interleukin 35, Interleukin 6, Interleukin 7, Interleukin 8,
Interleukin-1 family, Interleukin-12 subunit beta, Interleukin-36,
Leukemia inhibitory factor, Leukocyte-promoting factor,
Lymphotoxin, Lymphotoxin alpha, Lymphotoxin beta, Macrophage
colony-stimulating factor, Macrophage inflammatory protein,
Macrophage-activating factor, Myonectin, Nicotinamide
phosphoribosyltransferase, Oncostatin M, Oprelvekin, Platelet
factor 4, Promegapoietin, RANKL, Stromal cell-derived factor 1,
Tumor necrosis factor alpha or Vascular endothelial growth
inhibitor.
[0020] In some embodiments the matrix is a gel. In yet other
embodiments the matrix is a hydrogel. In certain instances, a
hydrogel refers to three-dimensional hydrophilic crosslinked
polymer networks that can absorb large volumes of water and
biological fluids without dissolving. Hydrogels may be composed of
polymers that are insoluble due to the presence of physical
crosslinks (e.g., crystalline regions, intermolecular interactions
and entanglements) or chemical crosslinks (e.g., covalent bonding).
In specific embodiments, the hydrogel is a fibrin hydrogel or a
fibrin domain modified polyethylene glycol hydrogel.
[0021] In one embodiment, the hydrogel is a fibrin hydrogel gel
which is cross-linked with a cross-linking agent. In certain
embodiments, the cross-linked fibrin hydrogel has chemical,
physical or mechanical properties that are suitable for their use
in implantation into a subject or patient, in particular
subcutaneous implantation.
[0022] Without limitations, the gel can comprise any ratio of
cross-linking agent to fibrin. Accordingly, the gel can comprise a
cross-linking agent to fibrin ratio in the range from about 0.1:1
to about 10:1. In some embodiments of the aspects described herein,
the gel comprises a cross-linking agent:fibrin ratio from 0.1:1 to
5:1, from 0.1:1 to 4:1, from 0.1:1 to 2:1, from 0.1:1 to 1.5:1,
from 0.1:1 to 1:1, from 0.1:1 to 0.9:1, from 0.2 to 0.8:1, and/or
from 0.25 to 0.75:1. In some embodiments, the gel comprises a
cross-linking agent:fibrin ratio of 0.20:1 to 0.5:1. In some
embodiments, the gel comprises a cross-linking agent:fibrin ratio
of 0.25:1 or 0.5:1.
[0023] The hydrogels can be made from fibrin solutions comprising a
wide concentration range of fibrin. Accordingly, the gel can be
made from a fibrin solution comprising from about 50 mg/ml to about
500 mg/ml, from about 100 mg/ml to about 400 mg/ml, 150 mg/ml to
about 300 mg/ml, 20 mg/ml to about 250 mg/ml of fibrin, or any
range derivable therein. In some embodiments of the aspects
described herein, the gel is made from a fibrin solution comprising
about 200 mg/ml of fibrin. In some embodiments of the aspects
described herein, the hydrogel is made from a fibrin solution
comprising about 250 mg/ml of fibrin. In still some other
embodiments of the aspects described herein, the hydrogel is made
from a fibrin solution comprising about 300 mg/ml of fibrin.
[0024] In other embodiments, a hydrogel refers to a polymer that is
formed by the free radical polymerization of a hydrophilic monomer
solution gelled and crosslinked to form a three dimensional
polymeric meshwork anchoring macromolecules. The macromolecules may
comprise a constituent of a ground substance of tissue, such as a
native collagen. Collagen may be interspersed within a polymeric
meshwork forming a collagen-hydrogel. In some embodiments the
collagen hydrogel is capable of promoting epithelial cell
growth.
[0025] Soluble collagen for cross-linking can be prepared by
art-recognized techniques. In addition, other proteins are that
support cell attachment and growth may be used to form a
cross-linked hydrogel. One example of an additional protein known
to support cell growth is fibronectin.
[0026] Polysaccharides and mucopolysaccharides can also be added to
hydrogels of the present invention.
[0027] Hydrogel polymers formed by free radical polymerization of
monomer solutions require crosslinking to form the three
dimensional polymeric structure of meshwork to gel the aqueous
solution. The addition of crosslinking agents such as ethylene
glycol dimethacrylate to the polymerization process can change the
resultant hydrogel. Generally, the addition of crosslinking agents
tend to increase the rigidity and mechanical strength of the
hydrogel. Addition of crosslinking agents, such as ethylene glycol
dimethacrylate and methymethacrylate, to the polymerization mixture
in the presence of native collagen, still changes the physical
properties of the hydrogel, and such additions to the
polymerization mixture are compatible with the native collagen, and
result in the collagen-hydrogel. Other known crosslinking agents
that can be used satisfactorily in producing the collagen-hydrogel
include diacrylates and dimethacrylates or other divalent
molecules.
[0028] Without limitations, the gel can comprise any ratio of
cross-linking agent to collagen. Accordingly, the gel can comprise
a cross-linking agent to collagen ratio in the range from about
0.1:1 to about 10:1. In some embodiments of the aspects described
herein, the gel comprises a cross-linking agent:collagen ratio from
0.1:1 to 5:1, from 0.1:1 to 4:1, from 0.1:1 to 2:1, from 0.1:1 to
1.5:1, from 0.1:1 to 1:1, from 0.1:1 to 0.9:1, from 0.2 to 0.8:1,
and/or from 0.25 to 0.75:1. In some embodiments, the gel comprises
a cross-linking agent:collagen ratio of 0.20:1 to 0.5:1. In some
embodiments, the gel comprises a cross-linking agent:collagen ratio
of 0.25:1 or 0.5:1.
[0029] The hydrogels can be made from collagen solutions comprising
a wide concentration range of collagen. Accordingly, the gel can be
made from a collagen solution comprising from about 50 mg/ml to
about 500 mg/ml, from about 100 mg/ml to about 400 mg/ml, 150 mg/ml
to about 300 mg/ml, 20 mg/ml to about 250 mg/ml of collagen, or any
range derivable therein. In some embodiments of the aspects
described herein, the gel is made from a collagen solution
comprising about 200 mg/ml of collagen. In some embodiments of the
aspects described herein, the hydrogel is made from a collagen
solution comprising about 250 mg/ml of collagen. In still some
other embodiments of the aspects described herein, the hydrogel is
made from a collagen solution comprising about 300 mg/ml of
collagen.
[0030] Hydrogels, which can be used as synthetic
"stimuli-responsive" polymers may be based on synthetic polymers,
such as poly (ethylene glycol) (PEG), poly(vinyl alcohol) (PVA),
poly(N-isopropylacrylamide) (poly(NiPAAm)). Such hydrogels have
been used in numerous regenerative medicine applications (see e.g.
N. A. Peppas, P. Bures, W. Leobandung, and H. Ichikawa. Hydrogels
in pharmaceutical formulations. Eur. J. Pharm. Biopharm. 50:27-46
(2000)), incorporated herein by reference.
[0031] In certain aspects hydrogels are prepared with various
polymers such as polyvinyl alcohol (PVA). polyvinyl pyrrolidone
(PVP), or polyacrylamides. Exemplary PVA-based hydrogels are
disclosed in, e.g., U.S. Pat. Nos. 6,231,605; 5,346,935; 5,981,826;
4,663,358; and 4,988,761, the contents of which are herein
incorporated by reference. In certain embodiments polyethylene
glycol (PEG) based hydrogels provide a large degree of swelling in
aqueous solutions. Various PEG based hydrogels are disclosed in
U.S. Pat. Nos. 5,514,379; 6,362,276 and 6,541,015, the contents of
which are herein incorporated by reference. PCT application
WO2006125082, incorporated herein by reference, provides hydrogel
formulation containing pre-solidified hydrogel particles in a
precursor hydrogel solution.
[0032] Without limitations, the gel can comprise any ratio of
cross-linking agent to poly (ethylene glycol) (PEG). Accordingly,
the gel can comprise a cross-linking agent to PEG ratio in the
range from about 0.1:1 to about 10:1. In some embodiments of the
aspects described herein, the gel comprises a cross-linking
agent:PEG ratio from 0.1:1 to 5:1, from 0.1:1 to 4:1, from 0.1:1 to
2:1, from 0.1:1 to 1.5:1, from 0.1:1 to 1:1, from 0.1:1 to 0.9:1,
from 0.2 to 0.8:1, and/or from 0.25 to 0.75:1. In some embodiments,
the gel comprises a cross-linking agent:PEG ratio of 0.20:1 to
0.5:1. In some embodiments, the gel comprises a cross-linking
agent:PEG ratio of 0.25:1 or 0.5:1.
[0033] The hydrogels can be made from PEG solutions comprising a
wide concentration range of PEG. Accordingly, the gel can be made
from a PEG solution comprising from about 50 mg/ml to about 500
mg/ml, from about 100 mg/ml to about 400 mg/ml, 150 mg/ml to about
300 mg/ml, 20 mg/ml to about 250 mg/ml of PEG, or any range
derivable therein. In some embodiments of the aspects described
herein, the gel is made from a PEG solution comprising about 200
mg/ml of PEG. In some embodiments of the aspects described herein,
the hydrogel is made from a PEG solution comprising about 250 mg/ml
of PEG. In still some other embodiments of the aspects described
herein, the hydrogel is made from a PEG solution comprising about
300 mg/ml of PEG.
[0034] In certain aspects the lymphangiogenesis inducer is capable
of binding to the matrix or hydrogel. Hydrogels can further be
prepared according to any of U.S. Pat. No. 4,684,558, 5,346,935,
6,534,083, 6,576,679 or 8,329,763, all of which are herein
incorporated by reference. In specific embodiments, the
lymphangiogenesis inducer comprises a matrix binding domain. In
certain aspects the lymphangiogenesis inducer is a modified
vascular endothelial growth factor C (VEGF-C) or VEGF-D protein
comprising a fibrin-binding domain.
[0035] In some aspects the lymphangiogenesis inducer the antigen or
both the lymphangiogenesis inducer and the antigen are formulated
in or as part of a liposome, micelle or nanoparticle.
[0036] In further aspects, the lymphangiogenesis inducer comprises
a protease cleavage site. Examples of proteases classes that may
act upon or cleave the protease cleavage site include serine
proteases, cysteine proteases, threonine proteases, aspartic
proteases, glutamic proteases, metalloproteases or asparagine
peptide lyases. In specific embodiments, the protease cleavage site
is a matrix metalloprotease cleavage site.
[0037] In yet further aspects, the VEGF-C is recombinant, the
antigen is recombinant or both the VEGF-C and the antigen are
recombinant. In certain aspects, the antigen is encoded by a
recombinant nucleic acid molecule. In certain aspects the VEGF-C or
VEGF-D, the antigen or both the VEGF-C or VEGF-D and the antigen
are expressed by a heterologous cell line.
[0038] In some embodiments, the matrix or hydrogel is implanted
into a subject. In specific embodiments, the lymphangiogenesis
inducer and antigen can be administered separately. In still other
aspects the lymphangiogenesis inducer and the antigen are
administered up to 1 month apart. In other embodiments, the
lymphangiogenesis inducer and the antigen are administered up to 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, 18, 19, 20,
21, 22, 23 or 24 hours apart or up 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29 30 or 31 days apart or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11 or 12 months apart. In specific embodiments, the subject is a
mammal. In further aspects, the subject is a human.
[0039] In certain aspects the immune response induced by the
methods disclosed herein is a protective immune response. In yet
further aspects, the immune response produces antibodies that
specifically bind to the antigen or the second antigen. In certain
aspects the immune response produces antibodies that specifically
bind to antigens present in a cell lysate. In specific aspects the
cell lysate is a tumor cell lysate.
[0040] Embodiments are also directed to a method of treating or
preventing an infection in a subject, the method comprising
administering to the subject an isolated lymphangiogenesis inducer
and an effective amount of an antigen. In some embodiments, the
lymphangiogenesis inducer is vascular endothelial growth factor C
(VEGF-C) or vascular endothelial growth factor D (VEGF-D). In
certain embodiments, the antigen is bacterial antigen, a viral
antigen, a fungal antigen, a helminth antigen or a protozoal
antigen. In yet further embodiments, the method of treating or
preventing an infection in a subject comprises administration of an
effective amount of a second antigen to the subject.
[0041] In certain aspects, the method of treating or preventing an
infection in a subject comprises a lymphangiogenesis inducer and an
antigen administered in a single composition. In some embodiments,
the lymphangiogenesis inducer and the antigen are provided over
multiple administrations. In yet further embodiments, the subject
is also administered an adjuvant. In certain embodiments, the
lymphangiogenesis inducer and the antigen are administered in a
single composition comprising an adjuvant. In some embodiments the
lymphangiogenesis inducer and the antigen are administered in a
single composition comprising a pharmaceutically acceptable
excipient. In certain aspects, the composition is administered
parenterally, subcutaneously or intramuscularly.
[0042] In other aspects, the method of treating or preventing an
infection in a subject comprises a lymphangiogenesis inducer
incorporated into a matrix. Embodiments of the method of treating
or preventing an infection in a subject also include an antigen
that is bound or incorporated into the matrix and is cleavable. In
some embodiments, the matrix incorporates one or more cleavable
chemokines, chemoattractants, chemorepellants or one or more
cleavable cytokines. In certain embodiments, the matrix is a
hydrogel. In certain aspects, the hydrogel is a fibrin hydrogel or
a fibrin domain modified polyethylene glycol hydrogel. In still
further aspects, the lymphangiogenesis inducer is capable of
binding to the matrix or hydrogel. In yet further embodiments, the
lymphangiogenesis inducer comprises a matrix binding domain. In
certain embodiments, the lymphangiogenesis inducer is a modified
vascular endothelial growth factor C (VEGF-C) or vascular
endothelial growth factor D (VEGF-D) protein comprising a
fibrin-binding domain. In some embodiments, the matrix or hydrogel
is implanted into the subject.
[0043] In some embodiments, the method of treating or preventing an
infection in a subject comprises a lymphangiogenesis inducer that
comprises a protease cleavage site. In other aspects, the protease
cleavage site is a matrix metalloprotease cleavage site.
[0044] In yet further embodiments, the VEGF-C or VEGF-D is
recombinant, the antigen is recombinant or both the VEGF-C or
VEGF-D and the antigen are recombinant. In certain embodiments, the
VEGF-C or VEGF-D, the antigen or both the VEGF-C or VEGF-D and the
antigen are expressed by a heterologous cell line. In some
embodiments, the antigen is encoded by a recombinant nucleic acid
molecule.
[0045] In certain aspects, the lymphangiogenesis inducer and
antigen are administered separately. In other aspects, the
lymphangiogenesis inducer and the antigen are administered up to 1
month apart. In certain embodiments, the subject is a mammal.
Embodiments are also included wherein the subject is a human.
[0046] In yet further embodiments, administration of the
lymphangiogenesis inducer and antigen or second antigen produces
antibodies that specifically bind to the antigen or the second
antigen.
[0047] Other embodiments of the present invention include a
pharmaceutical composition comprising a lymphangiogenesis inducer,
an effective amount of an antigen and a pharmaceutically acceptable
excipient. In certain embodiments, the pharmaceutical composition
comprises vascular endothelial growth factor C (VEGF-C) or vascular
endothelial growth factor D (VEGF-D). In some aspects, the
pharmaceutical composition comprises a bacterial antigen, a viral
antigen, a fungal antigen, a protozoal antigen or a cancer antigen.
In still other aspects, the pharmaceutical composition comprises an
adjuvant. In certain embodiments, the composition is adapted to be
administered parenterally, subcutaneously or intramuscularly. In
some aspects, the pharmaceutical composition comprises an effective
amount of a second antigen.
[0048] In certain embodiments, the pharmaceutical composition
comprises a lymphangiogenesis inducer that is incorporated into a
matrix. In still other aspects, the pharmaceutical composition
comprises an antigen which is bound or incorporated into the matrix
and is cleavable. In yet further aspects, the pharmaceutical
composition comprises a matrix that incorporates one or more
cleavable chemokines, chemoattractants, chemorepellants or one or
more cleavable cytokines. In still other aspects, the matrix of the
pharmaceutical composition is a hydrogel. In certain embodiments,
the hydrogel is a fibrin hydrogel or a fibrin domain modified
polyethylene glycol hydrogel. In still other embodiments, the
lymphangiogenesis inducer is bound or incorporated into the matrix
or hydrogel. In some aspects, the lymphangiogenesis inducer
comprises a matrix binding domain. In certain embodiments, the
lymphangiogenesis inducer comprises a protease cleavage site. In
still other aspects, the lymphangiogenesis inducer is a modified
vascular endothelial growth factor C (VEGF-C) protein comprising a
fibrin-binding domain and a matrix metalloprotease cleavage site.
In some aspects, the VEGF-C is recombinant, the antigen is
recombinant or both the VEGF-C and the antigen are recombinant. In
certain embodiments, the VEGF-C, the antigen or both the VEGF-C and
the antigen are expressed by a heterologous cell line. Embodiments
also include a vaccine that comprising any composition as described
herein.
[0049] In some aspects, a method for treating or preventing a
bacterial infection, a viral infection, a fungal infection, a
protozoal infection, a helminth infection or a cancer condition in
a subject or patient comprising administering to the subject or
patient a pharmaceutically acceptable vaccine composition
comprising at least a first lymphangiogenesis inducer and one or
more antigens. In other embodiments, the subject is administered
the vaccine composition multiple times. In still other embodiments,
the composition is administered orally, intravenously,
subcutaneously, intradermally, intramuscularly, nasally, by
injection, by inhalation, and/or using a nebulizer. In certain
aspects, the subject exhibits one or more symptoms of a a bacterial
infection, a viral infection, a fungal infection, a protozoal
infection, a helminth infection or a cancer condition.
[0050] Furthermore, in certain embodiments of the current
compositions or methods, the compositions may contain about, at
least about, or at most about 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5,
2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0,
8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5,
14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0,
19.5, 20.0, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,
110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,
240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,
370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480,
490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610,
620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740,
750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870,
880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000
.mu.g or mg of a biomolecule, a protein, a polypeptide, a
carbohydrate, a polysaccharide, a lipid, a nucleic acid, a fatty
acid, a glycolipid, a sterol, a polyterpene, a glycerolipid or a
combination of these (or any range derivable therein). The
biomolecule, a protein, a polypeptide, a carbohydrate, a
polysaccharide, a lipid, a nucleic acid, a fatty acid, a
glycolipid, a sterol, a polyterpene, a glycerolipid or a
combination of these may be in about, at least about, or at most
about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,
1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,
2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,
3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,
5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,
7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0,
10, 11, 12, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,
310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,
440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,
560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680,
690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810,
820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940,
950, 960, 970, 980, 990, or 1000 .mu.l or ml (or any range
derivable therein). In certain aspects, one or more
lymphangiogenesis inducers or one or more antigens can be
administered as a dose of 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0,
2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,
9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0,
14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5,
20.0, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110,
120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,
250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,
380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490,
500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620,
630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750,
760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880,
890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg
per kg of body weight.
[0051] Certain aspects are directed to a method of treating
neoplasia, dysplasia or cancer, the method comprising administering
to a subject an isolated lymphangiogenesis inducer and an effective
amount of one or more neoplasia, dysplasia or cancer antigens. The
one or more neoplasia, dysplasia or cancer antigens may be one or
more antigens from leukemias, lymphomas, neurological tumors such
as astrocytomas or glioblastomas, melanoma, breast cancer, lung
cancer, head and neck cancer, gastrointestinal tumors, gastric
cancer, colon cancer, liver cancer, pancreatic cancer,
genitourinary tumors such as cervical or uterine tumors, ovarian
cancer, vaginal cancer, testicular cancer, prostate cancer or
penile cancer, bone tumors, vascular tumors, cancer of the lip,
nasopharynx, pharynx, oral cavity, esophagus, rectum, gall bladder,
biliary tree, larynx, lung or bronchus, bladder cancer, kidney
cancer, brain cancer or cancer from other parts of the nervous
system, thyroid cancer, Hodgkin's disease, non-Hodgkin's lymphoma
or multiple myeloma.
[0052] In some embodiments, the method of treating neoplasia,
dysplasia or cancer comprises a method where the lymphangiogenesis
inducer is vascular endothelial growth factor C (VEGF-C) or
vascular endothelial growth factor D (VEGF-D). In certain
embodiments, the one or more antigens are cancer antigens. In yet
other embodiments, the one or more antigens are in or are part of a
cell lysate. The cell lysate may be a cancer, dysplasia, neoplasia
or tumor lysate. After lysis or disruption of cells to generate the
cell lysate, the lysate may be purified or enriched. Enrichment may
be for a specific biomolecule, a protein, a polypeptide, a
carbohydrate, a polysaccharide, a lipid, a nucleic acid, a fatty
acid, a glycolipid, a sterol, a polyterpene, a glycerolipid or a
combination of these.
[0053] In further aspects, the method of treating neoplasia,
dysplasia or cancer comprises a method where the lymphangiogenesis
inducer and the one or more neoplasia, dysplasia or cancer antigens
are administered in a single composition. In some embodiments, the
lymphangiogenesis inducer and the one or more neoplasia, dysplasia
or cancer antigens are provided over multiple administrations. In
still other embodiments, the subject is also administered an
adjuvant. In certain embodiments, the lymphangiogenesis inducer and
the one or more neoplasia, dysplasia or cancer antigens are
administered in a single composition comprising an adjuvant. In yet
other embodiments, the lymphangiogenesis inducer and the one or
more neoplasia, dysplasia or cancer antigens are administered in a
single composition comprising a pharmaceutically acceptable
excipient. In some embodiments, the composition is administered
parenterally, subcutaneously or intramuscularly.
[0054] In certain embodiments, the subject is administered an
effective amount of a second neoplasia, dysplasia or cancer
antigen.
[0055] In further aspects, the lymphangiogenesis inducer is
incorporated into a matrix. In still other embodiments, the antigen
is bound or incorporated into the matrix and is cleavable. In yet
other embodiments, the matrix incorporates one or more cleavable
chemoattractants or one or more cleavable cytokines.
[0056] In some embodiments, the matrix is a hydrogel. In still
other embodiments, the hydrogel is a fibrin hydrogel or a fibrin
domain modified polyethylene glycol hydrogel. In certain
embodiments, the lymphangiogenesis inducer is capable of binding to
the matrix or hydrogel. Embodiments are also included wherein the
lymphangiogenesis inducer comprises a matrix binding domain. In
some embodiments, the lymphangiogenesis inducer comprises a
protease cleavage site. In further aspects, the lymphangiogenesis
inducer is a modified vascular endothelial growth factor C (VEGF-C)
protein comprising a fibrin-binding domain. In certain embodiments,
the lymphangiogenesis inducer is a modified vascular endothelial
growth factor C (VEGF-C) protein comprising a matrix
metalloprotease cleavage site. Specific embodiments are also
included wherein the matrix or hydrogel is implanted into the
subject.
[0057] In yet other embodiments, the VEGF-C is recombinant, the one
or more neoplasia, dysplasia or cancer antigens are recombinant or
both the VEGF-C and the one or more antigens are recombinant. In
still other embodiments, the VEGF-C, the antigen or both the VEGF-C
and the one or more neoplasia, dysplasia or cancer antigens are
expressed by a heterologous cell line.
[0058] In yet other embodiments, the lymphangiogenesis inducer and
one or more neoplasia, dysplasia or cancer antigens are
administered separately. In some embodiments, the lymphangiogenesis
inducer and one or more neoplasia, dysplasia or cancer antigens are
administered up to 1 month apart. In other embodiments, the
lymphangiogenesis inducer and the antigen are administered up to 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, 18, 19, 20,
21, 22, 23 or 24 hours apart or up 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29 30 or 31 days apart or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11 or 12 months apart. In further aspects, the subject is a mammal.
In certain embodiments, the subject is a human. In still other
embodiments, administration of the lymphangiogenesis inducer and
the one or more neoplasia, dysplasia or cancer antigens or the
second neoplasia, dysplasia or cancer antigen produces antibodies
that specifically bind to the one or more neoplasia, dysplasia or
cancer antigens or to the second neoplasia, dysplasia or cancer
antigen.
[0059] In certain embodiments, the one or more neoplasia, dysplasia
or cancer antigens are encoded by a recombinant nucleic acid
molecule.
[0060] In some embodiments, the cancer comprises breast cancer or
melanoma. In some embodiments, the cancer comprises a cancer
disclosed herein. In some embodiments, the neoplasia or dysplasia
is in the breast or skin. In some embodiments, the neoplasia or
dysplasia are pre-cancerous cells that are a precursor to a cancer
described herein. In some embodiments, the cancer, dysplasia,
and/or neoplasia comprises a solid tumor. In some embodiments, the
cancer is non-lymphatic.
[0061] In some embodiments, the method further comprises
administration of an immunotherapy. In some embodiments, the
immunotherapy comprises a checkpoint blockade inhibitor, adoptive T
cell therapy, a chimeric antigen receptor, a STING agonist,
cytolytic virus therapy, one or more additional antigens, tumor
cell lysate, tolerance-breaking peptide antigen, a dendritic cell
vaccine (e.g. Provenge), and/or an antibody-antigen conjugate. It
is contemplated that combinations of immunotherapy may be
administered or that one or more immunotherapies may be
excluded.
[0062] Certain embodiments are directed to a method of inducing
immune tolerance, the method comprising administering to a subject
an isolated lymphangiogenesis inducer and an effective amount of an
antigen. In further embodiments, the method of inducing immune
tolerance comprises administering one or more tolerogenic
adjuvants.
[0063] In particular embodiments, the immune tolerance response
elicited by the lymphangiogenesis inducer and an effective amount
of an antigen may be complemented, supplemented, increased or
augmented. In some embodiments the antigen may be myelin sheath
protein is myelin basic protein (MBP), myelin oligodendrocyte
glycoprotein (MOG), proteolipid protein (PLP), or myelin associated
glycoprotein (MAG). In certain aspects the immune tolerance
response elicited by the lymphangiogenesis inducer and an effective
amount of an antigen may be complemented, supplemented, increased
or augmented by an adjuvant. In certain aspects the adjuvant is a
tolerogenic adjuvant. In certain embodiments the lymphangiogenesis
inducer and an effective amount of an antigen composition further
comprises at least one tolerogenic adjuvant. In certain aspects the
tolerogenic adjuvant is attached or bound to a hydrogel, a
lymphangiogenesis inducer or an antigen. In other aspects, the
tolerogenic adjuvant is conjugated to a hydrogel, a
lymphangiogenesis inducer or an antigen. In still other aspects,
the tolerogenic adjuvant is fused to a hydrogel, a
lymphangiogenesis inducer or an antigen. In specific embodiments,
the tolerogenic adjuvant is selected from IL-10, dexamethasone,
FK506 (Tacrolimus), cholera toxin B subunit, Escherichia coli
heat-labile enterotoxin B subunit, IFN-beta, glucocorticoids,
vitamin D3, and vitamin D3 analogues.
[0064] In some aspects, a method for treating an autoimmune disease
or a demyelinating disease in a subject comprising administering to
the subject a pharmaceutically acceptable composition comprising at
least a first an isolated lymphangiogenesis inducer and an
effective amount of at least one antigen is contemplated. In some
embodiments the antigen may be myelin sheath protein is myelin
basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG),
proteolipid protein (PLP), or myelin associated glycoprotein (MAG).
In some embodiments, the isolated lymphangiogenesis inducer is
VEGF-C or VEGF-D. In other embodiments, the subject is administered
the composition multiple times. In still other embodiments, the
composition is administered orally, intravenously, subcutaneously,
intradermally, intramuscularly, nasally, by injection, by
inhalation, and/or using a nebulizer. In certain aspects, the
subject exhibits one or more symptoms of a demyelinating disease.
In additional aspects, the subject has been diagnosed with a
demyelinating disease. In some embodiments, the subject is at risk
for a demyelinating disease. In other embodiments, the
demyelinating disease affects the central nervous system. In
additional embodiments, the demyelinating disease is an idiopathic
inflammatory demyelinating disease. In certain aspects, the
demyelinating disease is multiple sclerosis, neuropathy, central
pontine myelinolysis, tabes dorsalis, transverse myelitis, Devic's
disease, progressive multifocal leukoencephalopathy, optic
neuritis, or leukodystrophy. In specific embodiments, the
demyelinating disease is multiple sclerosis. In some embodiments,
the demyelinating disease is one of the borderline forms of
multiple sclerosis. In some aspects, the borderline form of
multiple sclerosis is standard multiple sclerosis,
Remitent-Recidivant multiple sclerosis (RRMS), Secondary
Progressive multiple sclerosis (SPMS), Primary progressive multiple
sclerosis (PPMS), KIR4.1 multiple sclerosis, Optic-spinal multiple
sclerosis, Opticospinal multiple sclerosis, Devic's disease, acute
disseminated encephalomyelitis (ADEM), acute hemorrhagic
leukoencephalitis, Balo concentric sclerosis, Schilder disease,
diffuse myelinoclastic sclerosis, Marburg multiple sclerosis,
malignant multiple sclerosis, fulminant multiple sclerosis, acute
multiple sclerosis, Tumefactive multiple sclerosis, or Solitary
sclerosis. In yet other embodiments the demyelinating disease is
Susac's syndrome, myalgic encephalomyelitis or leukoaraiosis. In
other aspects, the demyelinating disease affects the peripheral
nervous system. In specific embodiments, the demyelinating disease
is Guillain-Barre syndrome, chronic inflammatory demyelinating
polyneuropathy, anti-MAG peripheral neuropathy, Charcot-Marie-Tooth
Disease or progressive inflammatory neuropathy. In certain
embodiments, the methods further comprise preparing the
composition. In further embodiments still, the methods further
comprise measuring antibodies against the at least one myelin
sheath protein in the subject after administering the composition.
In some aspects a patient or subject has been diagnosed with or is
at risk for an autoimmune disease or disorder. In some embodiments
a patient or subject has been diagnosed with asthma or an allergic
disorder. In yet other aspects, a method for treating asthma or an
allergic reaction in a subject comprising administering to the
subject a pharmaceutically acceptable composition comprising at
least a first an isolated lymphangiogenesis inducer and an
effective amount of at least one antigen is contemplated.
[0065] In some embodiments, there are methods and compositions for
treating a cancer patient with cancer immunotherapy, for
administering cancer immunotherapy, for predicting efficacy of
cancer immunotherapy in a cancer patient, for prognosing a cancer
patient, for evaluating treatment for a cancer patient, for
evaluating cancer immunotherapy for a cancer patient; for
evaluating efficacy of immunotherapy in a cancer patient, and/or
for determining a cancer treatment for a cancer patient.
[0066] In certain embodiments, there are methods for treating a
cancer patient with cancer immunotherapy comprising administering
cancer immunotherapy to the patient after detecting in a serum
sample from that patient a level of VEGF-C and/or CCL21 that is
indicative of response to cancer immunotherapy. The expression
level or activity level from a control sample or test biological
sample from the patient may be an average value, a median value, a
normalized value, a cut-off value, or an average normalized value.
The expression level or activity level may be an average or mean
obtained from a significant proportion of patient samples. The
expression or activity level may also be an average or mean from
one or more samples from the patient.
[0067] Further steps of methods may include measuring or detecting
the level of expression in a sample from the patient; comparing a
measured or detected level of expression to the level or range of
levels in a control sample; and/or determining the patient is a
good candidate for a particular immunotherapy or predicting that a
particular immunotherapy will have therapeutic efficacy or
predicting that a particular immunotherapy is unlikely to be
efficacious on the patient. A patient is predicted to respond
favorably or positively to a particular immunotherapy if the
expression level of VEGF-C and/or CCL21 in a sample from the
patient is indicative of the level observed in patient who respond
favorably or positively to that particular immunotherapy.
[0068] In certain embodiments, there are methods for treating a
cancer patient with cancer immunotherapy comprising administering
cancer immunotherapy to the patient after detecting in a serum
sample from that patient a level of VEGF-C and/or CCL21 that is
indicative of response to cancer immunotherapy. In additional
embodiments, there are methods for predicting the efficacy of
cancer immunotherapy in a patient comprising: measuring a level of
VEGF-C and/or CCL21 protein expression in a serum sample from the
patient; and, comparing the level of VEGF-C and/or CCL21 protein
expression to a level in a control sample. In certain embodiments,
the level is increased when compared to the level in a control
sample.
[0069] In some embodiments, methods involve further comprising
comparing the level of VEGF-C and/or CCL21 to a control level. In
other embodiments, the control level is a level of VEGF-C or CCL21
expression from serum of patients who affirmatively respond to the
cancer immunotherapy. In additional embodiments, such patients are
those who affirmatively respond to the cancer immunotherapy exhibit
at least a 25% reduction in tumor growth following treatment with
the cancer immunotherapy. In further embodiments, the control level
is a level of VEGF-C or CCL21 expression that is the median level
in serum of cancer patients. In other cases, the patient is
administered immunotherapy after being determined to have a level
of expression of VEGF-C or CCL21 that is increased as compared to
the level of expression in serum from noncancer patients. In other
cases, the control level is a level or range of level of VEGF-C or
CCL21 expression from serum of patients who do not respond
affirmatively to the cancer immunotherapy. In further embodiments,
methods involve a control level that is a level or range of level
of VEGF-C or CCL21 expression in serum from patients who do not
respond affirmatively to the cancer immunotherapy.
[0070] In some methods, there is the additional step of obtaining
serum from the patient. While in other cases, the sample has
already been obtained or a level of expression has already been
measured, though in some embodiments, methods involve measuring the
level of VEGF-C and/or CCL21 expression in serum from the patient.
In some embodiments, there are methods involving measuring the
level of VEGF-C and/or CCL21 expression comprises using a peptide
or polypeptide that binds VEGF-C and/or CCL21. In further
embodiments, an immunotherapy is a checkpoint blockade inhibitor,
adoptive T cell therapy, a chimeric antigen receptor, a STING
agonist, cytolytic virus therapy, one or more additional antigens,
tumor cell lysate, tolerance-breaking peptide antigen, a dendritic
cell vaccine, or an antibody-antigen conjugate.
[0071] In some embodiments, the biological sample from the patient
is a sample from a primary tumor. In some embodiments, the
biological sample is from a tissue or organ as described herein. In
still further embodiments, the method may comprise obtaining a
sample of the subject or patient. Non-limiting examples of the
sample include a tissue sample, a whole blood sample, a urine
sample, a saliva sample, a serum sample, a plasma sample, or a
fecal sample. In particular embodiments, the sample is a serum
sample.
[0072] In some embodiments the subject or patient is one that has
previously been treated for cancer. In some embodiments, the cancer
is recurrent.
[0073] The term subject or patient may refer to an animal (for
example a mammal), including but not limited to humans, non-human
primates, rodents, dogs, or pigs. The methods of obtaining provided
herein include methods of biopsy such as fine needle aspiration,
core needle biopsy, vacuum assisted biopsy, incisional biopsy,
excisional biopsy, punch biopsy, shave biopsy or skin biopsy.
[0074] In certain embodiments the sample is obtained from a biopsy
from gall bladder, skin, heart, lung, breast, pancreas, liver,
muscle, kidney, smooth muscle, bladder, intestine, brain, prostate,
esophagus, or thyroid tissue.
[0075] Alternatively, the sample may include but not be limited to
blood, serum, sweat, hair follicle, buccal tissue, tears, menses,
urine, feces, or saliva. In particular embodiments, the sample may
be a tissue sample, a whole blood sample, a urine sample, a saliva
sample, a serum sample, a plasma sample or a fecal sample.
[0076] In certain aspects the sample is obtained from cystic fluid
or fluid derived from a tumor or neoplasm. In certain aspects of
the current methods, any medical professional such as a doctor,
nurse or medical technician may obtain a biological sample for
testing. In further aspects of the current methods, the patient or
subject may obtain a biological sample for testing without the
assistance of a medical professional, such as obtaining a whole
blood sample, a urine sample, a fecal sample, a buccal sample, or a
saliva sample.
[0077] In further embodiments, the sample may be a fresh, frozen or
preserved sample or a fine needle aspirate. In particular
embodiments, the sample is a formalin-fixed, paraffin-embedded
(FFPE) sample. An acquired sample may be placed in short term or
long term storage by placing in a suitable medium, excipient,
solution, or container. In certain cases storage may require
keeping the sample in a refrigerated, or frozen environment. The
sample may be quickly frozen prior to storage in a frozen
environment. In certain instances the frozen sample may be
contacted with a suitable cryopreservation medium or compound.
Examples of cryopreservation mediums or compounds include but are
not limited to: glycerol, ethylene glycol, sucrose, or glucose.
[0078] Some embodiments further involve isolating nucleic acids
such as ribonucleic or RNA from a biological sample or in a sample
of the patient. Other steps may or may not include amplifying a
nucleic acid in a sample and/or hybridizing one or more probes to
an amplified or non-amplified nucleic acid. The methods may further
comprise assaying nucleic acids in a sample. Further embodiments
include isolating or analyzing protein expression in a biological
sample for the expression of the biomarker.
[0079] In certain embodiments, a microarray or ELISA may be used to
measure or assay the level of CCL21 or VEGF-C in a sample. The
methods may further comprise recording the expression or activity
level in a tangible medium or reporting the expression or activity
level to the patient, a health care payer, a physician, an
insurance agent, or an electronic system.
[0080] In some embodiments, methods will involve determining or
calculating a prognosis score based on data concerning the
expression or activity level of one or more of VEGF-C and CCL21,
meaning that the expression or activity level of one or more of
VEGF-C and CCL21 is at least one of the factors on which the score
is based. A prognosis score will provide information about the
patient, such as the general probability whether the patient is
sensitive to a particular therapy or has poor survival or high
chances of recurrence. In certain embodiments, a prognosis value is
expressed as a numerical integer or number that represents a
probability of 0% likelihood to 100% likelihood that a patient has
a chance of poor survival or cancer recurrence or poor response to
a particular treatment.
[0081] In some embodiments, the subject or patient is determined to
be a candidate for or is further administered an immunotherapy when
the VEGF-C or CCL21 level in the serum sample is greater than 2, 3,
4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, or 40 ng/ml
(or any derivable range therein).
[0082] In some embodiments, the subject or patient is determined to
be a candidate for or is further administered an immunotherapy when
the VEGF-C or CCL21 level is at least 1/3, 2/3, 1, 1.5, 2, 2.5, 3,
3.5, 4, 4.5, or 5 standard deviations from a control.
[0083] In some embodiments, the subject or patient is determined to
be a candidate for or is further administered an immunotherapy when
the VEGF-C or CCL21 level is at least 2, 3, 4, 5, 6, 7, 8, 9, 10,
12, 14, 16, 18, 20, or 30 fold (or any derivable range therein)
higher in a biological sample from the patient compared to the
level in a control.
[0084] In some embodiments, the method further comprises surgical
incision of the primary tumor. In some embodiments, the elevated
level/increased expression or reduced level/decreased expression is
at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10,
12, 14, 16, 18, 20, 50, 100, 150, 200, 250, 500, or 1000 fold (or
any derivable range therein) or at least 10, 20, 30, 40, 50, 60,
70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900%
different than the control, or any derivable range therein.
[0085] In some embodiments, the prognosis score is expressed as a
number that represents a probability of 0, 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, or 100% likelihood (or any range derivable
therein) that a patient will have a poor response to a particular
treatment. Alternatively, the probability may be expressed
generally in percentiles, quartiles, or deciles.
[0086] A control may be serum from healthy or noncancerous
patients; a control level may be the level or range of levels that
represent the level of expression from healthy or noncancerous
patients. In some embodiments, a control may be serum from cancer
patients known to respond positively to the relevant immunotherapy,
such as by demonstrating at least a 25% decrease in tumor volume
after being given a particular immunotherapy. In other embodiments,
a control may be serum from cancer patients known to be
unresponsive to the relevant immunotherapy or to be poor responders
to the relevant immunotherapy, such as by not demonstrating at
least a 25% decrease in tumor volume after being given that
immunotherapy.
[0087] A difference between or among weighted coefficients or
expression or activity levels or between or among the weighted
comparisons may be, be at least or be at most about 0.1, 0.2, 0.3,
0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0,
7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0,
13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5,
19.0, 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,
165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225,
230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290,
295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355,
360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430,
440, 441, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530,
540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640,
650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750,
760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860,
870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970,
975, 980, 990, 1000 times or -fold (or any range derivable
therein).
[0088] In some embodiments, determination of calculation of a
diagnostic, prognostic, or risk score is performed by applying
classification algorithms based on the expression values of VEGF-C
or CCL21 with differential expression p values of about, between
about, or at most about 0.005, 0.006, 0.007, 0.008, 0.009, 0.01,
0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019,
0.020, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028,
0.029, 0.03, 0.031, 0.032, 0.033, 0.034, 0.035, 0.036, 0.037,
0.038, 0.039, 0.040, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046,
0.047, 0.048, 0.049, 0.050, 0.051, 0.052, 0.053, 0.054, 0.055,
0.056, 0.057, 0.058, 0.059, 0.060, 0.061, 0.062, 0.063, 0.064,
0.065, 0.066, 0.067, 0.068, 0.069, 0.070, 0.071, 0.072, 0.073,
0.074, 0.075, 0.076, 0.077, 0.078, 0.079, 0.080, 0.081, 0.082,
0.083, 0.084, 0.085, 0.086, 0.087, 0.088, 0.089, 0.090, 0.091,
0.092, 0.093, 0.094, 0.095, 0.096, 0.097, 0.098, 0.099, 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or higher (or any range derivable
therein).
[0089] Further aspects relate to a kit comprising nucleic acid
probes and/or polypeptides (e.g. antibodies) for detecting the
expression level of VEGF-C or CCL21 in a biological sample. In some
embodiments, the probes or polypeptides are labeled. In some
embodiments, the kit further comprises nucleic acid probes or
polypeptides for detecting a control. In some embodiments, the
probe comprises nucleic acid primers that are capable of amplifying
VEGF-C or CCL21 genes by PCR. In some embodiments, the kit further
comprises reagents for performing one or more of reverse
transcriptase PCR, ELISA, DNA amplification by PCR, and real-time
PCR. In some embodiments, the kit further comprises instructions
for use.
[0090] Any of the methods described herein may be implemented on
tangible computer-readable medium comprising computer-readable code
that, when executed by a computer, causes the computer to perform
one or more operations. In some embodiments, there is a tangible
computer-readable medium comprising computer-readable code that,
when executed by a computer, causes the computer to perform
operations comprising: a) receiving information corresponding to an
expression or activity level of a gene, biomarker or protein in a
sample from a patient; and b) determining a difference value in the
expression or activity levels using the information corresponding
to the expression or activity levels in the sample compared to a
control or reference expression or activity level for the gene.
[0091] In other aspects, tangible computer-readable medium further
comprise computer-readable code that, when executed by a computer,
causes the computer to perform one or more additional operations
comprising making recommendations comprising: wherein the patient
in the step a) is under or after a first treatment for cancer,
administering the same treatment as the first treatment to the
patient if the patient does not have increased expression or
activity level; administering a different treatment from the first
treatment to the patient if the patient has increased expression or
activity level.
[0092] In some embodiments, receiving information comprises
receiving from a tangible data storage device information
corresponding to the expression or activity levels from a tangible
storage device. In additional embodiments the medium further
comprises computer-readable code that, when executed by a computer,
causes the computer to perform one or more additional operations
comprising: sending information corresponding to the difference
value to a tangible data storage device, calculating a prognosis
score for the patient, treating the patient with a traditional
cancer therapy if the patient does not have expression or activity
levels, and/or or treating the patient with an alternative cancer
therapy if the patient has increased expression or activity
levels.
[0093] The tangible, computer-readable medium further comprise
computer-readable code that, when executed by a computer, causes
the computer to perform one or more additional operations
comprising calculating a prognosis score for the patient. The
operations may further comprise making recommendations comprising:
administering a treatment comprising a thymidylate synthase
inhibitor to a patient that is determined to have a decreased
expression or activity level.
[0094] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising", the words "a" or "an" may mean one or
more than one.
[0095] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." As used herein "another" may mean at least a second or
more.
[0096] Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0097] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0098] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0099] FIG. 1A-C--LECs express MHC-II under resting conditions, and
upregulate it under inflammatory conditions, promoting CD4.sup.+ T
cell education. (A) Primary LN-LECs were stimulated with IFN.gamma.
or TNF.alpha. for 6 h, or left in culture medium, prior to
measurement of MHC-II levels by flow cytometry. Representative
histograms of MHC-II expression are shown. (B) BMDCs or primary
LN-LECs or LN-FRCs were stimulated with IFN.gamma. over up to 20 h,
and MHC-II surface expression was detected via flow cytometry. Mean
fluorescence intensities (MFIs) were normalized to negative
controls (dotted line). (C) OVA-reactive CD4.sup.+ T (OT-II) cells
that recognize the OVA.sub.323-339 peptide were co-cultured in the
presence of the peptide with mDCs or LN-LECs, and proliferation
(CFSE dilution) and the expression of memory and effector
phenotypic markers were assessed after 4 d. Numbers indicate
representative percentage of OT-II cells that express CD44 or CD62L
under each education condition.
[0100] FIG. 2A-F--DCs and LECs activate cognate T cells and promote
different T cell phenotypes. DCs, LN-LECs, or LN-FRCs from healthy
wild-type mice, or immortalized LECs (iLECs) were co-cultured with
OT-I cells in the presence of 10 nM SIINFEKL for 3 d. (A)
Representative histograms depicting CFSE dilution of OT-I cells and
(B) quantification of OT-I cell proliferation in the form of
proliferation index, versus the APC used to `educate` the OT-I
cells. (C-F) Quantification of typical T cell activation and
functional markers by flow cytometry. (C) Representative dot plots
of CFSE versus several functional markers to reflect upregulation
or downregulation of certain markers as the T cells proliferated.
(D-F) Percentage of all OT-I cells, regardless of number of cell
divisions undertaken, that express (D) the memory-like phenotype
characterized by CD44.sup.+CD62L.sup.+ staining, (E) ability to
migrate to LNs via the CCR7 receptor, and (F) tissue-resident
phenotype by CD103.sup.+ staining.
[0101] FIG. 3A-C--LEC-educated OT-I cells can be activated
following CD28 stimulation, and can produce effector cytokines as
well as DC-educated OT-I cells. (A) Flow cytometry was used to
analyze the expression of various functional markers on OT-I cells
co-cultured with LN-LECs in the presence of 10 nM SIINFEKL without
(light gray, solid line) or with 2 .mu.g/mL .alpha.CD28 (dotted
lines). Isotype control-stained OT-I cells have been shown as a
negative control for staining (dark gray, filled). (B) Supernatants
were analyzed for IFN.gamma. production by ELISAs, showing that in
the presence of CD28 stimulation, LEC-educated OT-I cells can
produce effector cytokines as effectively as BMDC-educated OT-I
cells. (C) In spite of similar effector cytokine production,
LEC-educated OT-I cells tend to favor a memory-like phenotype
characterized by CD44.sup.+CD62L.sup.+ staining, even in the
presence of CD28 stimulation (Isotype--dotted, gray circles;
+CD28--solid, black squares).
[0102] FIG. 4A-F--Despite a weak effector response, LEC-educated
CD8.sup.+ T cells can be activated for anti-infection immunity. (A)
Schematic of the experiment to characterize early memory phase
response. To determine the functional potential of the transferred
cells, the cells were assessed for cytokine expression (IFN.gamma.,
TNFa, IL-2) and CD107 upon ex vivo restimulation followed by
intracellular staining and flow cytometric analysis. (B)
LEC-educated CD8+ T cells can induce cytokines and cytolytic
molecules at levels similar to DC-educated CD8+ T cells. The
percentage of IFN.gamma., CD107, TNF.alpha., IL-2 positive cells
(gated in transferred cells) is shown for LEC/DC-educated cells in
the spleen. (C) The graph displays the percentage of single
(IFN.gamma.+ only, TNF.alpha.+ only, IL-2+ only), double
(IFN.gamma..sup.+TNF.alpha..sup.+ or TNF.alpha..sup.+IL-2.sup.+,
IL-2.sup.+IFN.gamma..sup.+) or triple
(IFN.gamma..sup.+TNF.alpha..sup.+IL-2.sup.+) positive cells in
LEC/DC-educated CD8.sup.+ T cells in the spleen. The data are from
two independent experiments (n=5 each). Two-way ANOVA followed by
Bonferroni posttest. (D-E) LEC-educated CD8.sup.+ T cells not only
contributed to the generation of effector CTLs against infectious
pathogens but also controlled the bacterial load during infection
with L.m.-OVA. (D) Schematic of the experiment. (E) Representative
examples of bacterial culture plates for the differentially
educated cells and without prior transfer. (F) The graph shows the
bacterial burden in spleen at day 3 after infection with L.m.-OVA.
The data are from one experiment (n=6), *p.ltoreq.0.05 using
two-tailed unpaired Student's t test.
[0103] FIG. 5A-G--Hydrogel-mediated delivery of VEGFC drives local
lymphangiogenesis. (A) Hydrogel precursors and proteins containing
the fibrin-binding, transglutaminase substrate peptide (Tg; yellow)
are mixed and crosslinked in the presence of thrombin and the
clotting factor-XIII to create hydrogels. Inset photo showcases a
hydrogel (arrowhead). In the presence of MMPs, the hydrogel matrix
is degraded, releasing the free protein. (B) Intravital
fluorescence imaging of the release of AF750-labeled VEGFC, in
soluble (sVEGFC) or gel-crosslinkable (TgVEGFC) formats, from
hydrogels following intradermal implantation in the hinds of
healthy wild-type BALB/c over 20 days. Mice injected with buffer
only (naive) or empty hydrogels (empty) have been shown as negative
controls. (C) Release profiles of the VEGFC over the study period.
(D-G) Characterization of lymphangiogenesis at the implant site and
at the draining LNs 22 days after hydrogel implantation. (D-E)
Fibrin-binding VEGFC (TgVEGFC) increases local skin-draining
lymphatic vessels (LYVE-1.sup.+F4/80.sup.-) as observed using
confocal microscopy (D; scale bars=80 .mu.m) and flow cytometry (E)
relative to empty hydrogel controls. (F-G) Soluble VEGFC (sVEGFC)
appears to exert more effects at the draining lymph node. (F) Tiled
fluorescence micrographs (scale=300 .mu.m) showing cross-section of
the draining, brachial LNs with LYVE-1 (red) and DAPI staining
(gray). (G) gp38.sup.+CD31.sup.+ LECs in the brachial LNs were
counted by flow cytometry and normalized against brachial LN mass.
(Data points and error bars represent mean.+-.SD of n=3-4
measurements. *p<0.05 by Student's t-test vs. all other groups;
**p<0.05 relative to all other treatment groups by one-way
ANOVA).
[0104] FIG. 6A-G--Local lymphangiogenesis drives local recruitment
of T cells. Hydrogels containing sVEGFC or TgVEGFC were implanted
intradermally into healthy wild-type C57Bl/6 mice and after 9 or 22
days, implant sites were excised for analysis by flow cytometry and
immunohistochemistry. (A) At day 9 post-implantation, there has not
yet been significant increase in lymphatic vessel density (not
shown), but local recruitment of CD45.sup.+ cells is already
increased. (A, right) Ratio of `area-under-curve` (AUC) of
CD45.sup.+ pixels normalized to AUC of LYVE-1.sup.+ pixels (Each
data point represents ratio quantified from one field-of-view). (B)
At day 22, number of live cells per mg of tissue collected, and (C)
number of live CD45.sup.+ cells per mg of tissue collected. (D) At
day 22, a large number of infiltrating immune cells at the implant
site are CD3.sup.+. (E-H) Flow cytometry analysis of cell types at
the implant site at (E) day 9 and (E-H) day 22 post-implantation.
(E) Pie charts represent composition of all live cells detected at
the implant sites, and bar graphs represent numbers of live cells
within named various CD45.sup.+ subsets normalized per mg of
tissue. Hydrogels containing VEGFC increase the proportion of local
CD45.sup.+ cells over the 22 d study period, and this can be
accounted for by the T cells, DCs, and other CD45+ compartments.
(F-G) increased recruitment of T cells quantified as number of live
CD4.sup.+CD8.sup.- or CD4.sup.-CD8.sup.+ per mg, or as percentage
of total live cells. (H) Conventional DCs (defined as
CD3e.sup.-B220.sup.-CD11b.sup.+CD11c.sup.+) enumerated per mg of
tissue isolated, or as percentage of total live cells. (Data points
and error bars represent mean.+-.SD of n=3-10 measurements pooled
from up to 3 independent experiments. *p<0.05 by Student's
t-test. **p<0.05 by one-way ANOVA with Tukey's post-test).
[0105] FIG. 7A-C--Lymphangiogenic sites promote generation of
effector-memory cells expressing
CD44.sup.+CD62L.sup.-KLRG1.sup.-CD127.sup.+, and can be activated
to produce IFN.gamma.. (A) OT-II cells were adoptively transferred
into healthy wild-type mice 1 d before injection of hydrogels with
VEGFC.+-.OVA. 8 d later, mice were sacrificed and the implant sites
were isolated and analyzed by (B) IFN.gamma. ELISPOTs post-ex vivo
re-stimulation with OVA or (C) flow cytometry for markers of memory
or effector cells. Short-lived effector cells (SLECs) were defined
by CD44.sup.+CD62L.sup.- KLRG1.sup.+CD127.sup.- and effector-memory
Tem cells were defined by
CD44.sup.+CD62L.sup.-KLRG1.sup.-CD127.sup.+. (Each data point=one
individual mouse. *p<0.05 by Student's t-test; **p<0.05
relative to all other treatment groups by one-way ANOVA).
[0106] FIG. 8A-B--Generation of CD4 and CD8 effector-memory like
cells by lymphangiogenic hydrogels. (A) Experimental timeline
showing OT-I and OT-II cells were adoptively transferred into
healthy wild-type mice 1 d before injection of hydrogels with VEGFC
and/or OVA. Mice were bled multiple times to collect blood samples
for longitudinal analysis, and at 22 d post-implantation, mice were
sacrificed and the implant sites and draining brachial LNs (bLNs)
were isolated and analyzed by flow cytometry for memory and
effector phenotype of OT-I and OT-II cells. (B) CD4 and CD8 T cells
infiltrating the implant site or bLNs, or circulating in blood were
gated for CD45.1.sup.+ expression to define OT-I/OT-II cells versus
the endogenously-derived T cells, and then stained for expression
of CD44 and CD62L to define memory and effector compartments. (n=5
per group, **p<0.05 relative to all other treatment groups by
one-way ANOVA).
[0107] FIG. 9A-C--Lymphangiogenic hydrogels provoke less systemic
inflammation than conventional vaccines. (A) Experimental timeline.
Hydrogels containing TgVEGFC and/or TgOVA were implanted
intradermally into mice. As a positive control, a standard vaccine
containing 50 .mu.g soluble OVA and 50 .mu.g CpG were injected into
mice intradermally through the footpads. This group was also
boosted at day 15 with the same formulation, matching published
mouse vaccination schedules. (B) Serum levels of various
inflammatory markers were assayed via Luminex assay at d3
post-implantation or at d35+3 d post-challenge with OVA+LPS,
mimicking an infection. (C) Levels of inflammatory cytokines were
assayed by Luminex assay or by ELISA (IL-15) at the hydrogel
implant site at d35+3 d post-challenge with OVA+LPS. **p<0.05
via one-way ANOVA with Tukey's post-test, n=5 per group.
[0108] FIG. 10A-G. Blocking VEGFR-3 signaling in lymphangiogenic
melanomas decreases T.sub.Reg cell infiltration and delays primary
tumor growth. (A) Representative images of immunostained whole
tumor sections (scale bar=500 .quadrature.m, 200 .quadrature.m in
zoomed images) showing overall LEC density (Lyve-1, green; DAPI,
cyan). (B) Quantification of LECs (CD45.sup.-gp38.sup.+CD31.sup.+)
assessed by flow cytometry. (C) Growth curves of B16-OVA or
B16-OVA/VC tumor-bearing C57BL6 mice treated with control (Iso) or
VEGFR-3 (ER3) blocking antibodies. Quantification of indicated cell
types within the tumor at day 9 post-inoculation: (D) total
leukocytes (CD45.sup.+), (E) regulatory CD4.sup.+ T cells
(T.sub.reg, CD4.sup.+FoxP3.sup.+) and effector CD8.sup.+ T cells
(CD62L.sup.-CD44.sup.+). (F) Representative flow cytometry plots
and gating strategy of tumor cells and (G) quantification of mature
(CD11c.sup.-CD11b.sup.+MHCII.sup.+) and immature
(CD11c.sup.-CD11b.sup.+MHCII.sup.-) myeloid subsets, granulocytic
(CD11c.sup.-CD11b.sup.+MHCII.sup.-Ly6G.sup.+Ly6C.sup.low) and
monocytic (CD11c.sup.-CD11b.sup.+MHCII.sup.-Ly6G.sup.+Ly6C.sup.low)
MDSCs. Results represent 2 independent experiments, n=5 each.
*p.ltoreq.0.05, **p.ltoreq.0.01 by two-tailed Student's t-test. Bar
graphs shown as mean.+-.SEM.
[0109] FIG. 11A-G. VEGF-C/VEGFR-3 signaling increases
responsiveness of melanoma to immunotherapy. B16-OVA/VC
tumor-bearing C57BL/6 mice, treated with control (Iso) or
.quadrature.VEGFR3 (.quadrature.R3) blocking antibodies, received
different immunotherapies after tumors became established (arrows
on growth curves indicate when the immunotherapies were
administered). Tumor growth curves and survival waterfall plots for
(A) Antigen-specific adoptive T cell therapy (ATT) performed by
intravenously (i.v.) injection of 1.times.10.sup.6 ex vivo
activated effector OT-I cells on day 9 after tumor cell inoculation
(data pooled from 3 independent experiments, n.gtoreq.5 each). (B)
ATT performed in tumor-bearing mice lacking dermal lymphatics
(K14-VEGFR-3-Ig mice, n=4). (C) Dendritic cell (DC) vaccination
performed by intraperitoneally (i.p.) injection of 10.sup.6 ex vivo
peptide-pulsed DCs on days 4 and 10 after tumor cell inoculation
(n=6). (D to F) Vaccination on days 4, 7, and 10 with i.d.
injection into the hind legs of CpG (50 .quadrature.g) with (D) no
antigen, (E) 10 .quadrature.g OVA (data from 2 independent
experiments, n.gtoreq.4 each), or (F) 2 .quadrature.g
Trp-2-peptide-conjugated nanoparticles (NP-Trp2, n.gtoreq.6). (G)
Tumor growth curves and survival plot for BRAF GEM treated with
control (Iso) or .quadrature.VEGFR3 (.quadrature.R3) blocking
antibodies, followed by a combined immunotherapy of CpG+gp100
peptide (day 8 and day 12) and anti-PD-1 antibody (day 12 and every
4 days thereafter). *p.ltoreq.0.05, **p.ltoreq.0.01,
***p.ltoreq.0.001 by two-tailed Student's t-test for growth curves
and Log-rank (Mantel-Cox) test for survival curves. Growth curves
shown as mean.+-.SEM over time.
[0110] FIG. 12A-J. VEGFR-3 signaling increases infiltration of
naive T cells in a CCR7-dependent manner. (A to D) B16-OVA and
B16-OVA/VC tumor-bearing mice treated with i.p injection of control
IgG (Iso) or .quadrature.VEGFR3 (.quadrature.R3) blocking
antibodies were euthanized on day 9 after inoculation and tumor
single cell suspensions analyzed by flow cytometry (representative
of 2 independent experiments, n=5 each). (A) Quantification of
conventional (cony) CD4.sup.+ T cells
(CD45.sup.+CD4.sup.+FoxP3.sup.-) and CD8.sup.+ T cells
(CD45.sup.+CD8.sup.+) after live/dead exclusion. (B) Phenotype of
TILs (CD4 (upper graph) or CD8 (lower graph) according to CD44 and
CD62L expression status. Naive (T.sub.naive):
CD44.sup.-CD62L.sup.+, effector/effector memory (T.sub.EM):
CD44.sup.+CD62L.sup.-, central memory (T.sub.CM):
CD44.sup.+CD62L.sup.+T. (C) Ratio of naive versus effector
CD4.sup.+ or CD8.sup.+ T cells. (D) CCL21 concentration as assessed
by ELISA in the tumor, dLNs, and ndLNs. (E) Representative images
of an immunostained section of a lymphangiogenic B16-VC tumor
(scale bar=100 .quadrature.m) showing CCL21 expression and CD4
infiltration within the tumor microenvironment (Lyve-1, white;
CCL21, green; CD4, red; DAPI, blue). (F) CCR7 expression of TILs.
B16-OVA/VC tumor bearing mice treated with control IgG (Iso) or
anti-CCR7 (.quadrature.CCR7) blocking antibodies were euthanized 9
days after inoculation, and tumor single cell suspensions analyzed
by flow cytometry (n=6). CCR7 expression was quantified on
conventional (cony) CD4.sup.+ T cells
(CD45.sup.+CD4.sup.+FoxP3.sup.-CCR7.sup.+), regulatory CD4.sup.+ T
cells (CD4.sup.+FoxP3.sup.+CCR7.sup.+) and CD8.sup.+ T cells
(CD45.sup.+CD8.sup.+CCR7.sup.+). (G) Representative flow cytometry
plots of TIL phenotype based on CD62L and CD44 expression, and (H)
quantification of naive and central memory (CM) phenotype (n=6).
(I) Ratio of naive versus effector T cells. (J) B16-OVA/VC
tumor-bearing mice (CD45.1) were treated with control IgG (Iso) or
anti-CCR7 (.quadrature.CCR7) blocking antibodies on day 0, 3 and 6
after inoculation and adoptive transfer of 1.times.10.sup.6 naive
CD45.2.sup.+ OT-I T cells was performed on day 9. Quantification of
intratumoral OT-I (CD45.sup.+CD3.sup.+CD8.sup.+CD45.2.sup.+) cells
analyzed on day 10 after inoculation (n.gtoreq.6). *p.ltoreq.0.05,
**p.ltoreq.0.01, ***p.ltoreq.0.001 by two-tailed Student's t-test
or one-way ANOVA. Bar graphs shown as mean.+-.SEM.
[0111] FIG. 13A-F. Primary human metastatic melanomas contain
CCL21-expressing LECs, and expression of VEGFC positively
correlates with hallmarks of tumor inflammation. (A) Representative
immunofluorescence images of human primary melanoma tumor sections
(10.times. tiles, scale bar=500 .quadrature.m, 200 .quadrature.m in
zoom-in images) showing nuclei (DAPI, cyan) and LECs (podoplanin,
green). (B) Quantification of lymphatic vessel density in healthy
(when available) and tumor region of skin of melanoma patients.
Paired t-test. Scatter dot plots shown as mean.+-.SEM (n=7 for
normal skin, n=14 for tumors). (C) Representative
immunohistochemistry image of a human primary melanoma tumor
section (scale bar=100 .quadrature.m) showing intratumoral VEGF-C
expression (brown). (D) Representative immunofluorescence image of
an intratumoral lymphatic vessel (podoplanin, green) expressing
CCL21 (red) in a section of human primary melanoma (63.times.,
scale bar=10 .quadrature.m, DAPI, blue). (E and F) Correlations of
gene expression data of human primary melanoma samples from the
cancer genome atlas (TCGA). (E) Heat map showing correlation
between the expression of 30 genes indicative of T cell
inflammation versus VEGFC, -D (FIGF), and -A. Colors indicate min
and max r values using non-parametric Spearman's test. (F) Dot
plots of genes of interest with linear regression curve (n=103,
correlation was tested using non-parametric Spearman's test).
[0112] FIG. 14A-D. Serum VEGF-C levels in human metastatic melanoma
patients predict the magnitude and functionality of a systemic
tumor-specific CD8.sup.+ response after peptide vaccination, and
act as a biomarker for the response to combined anti-PD-1 and
anti-CTLA-4 immunotherapy. (A to C) Correlations of serum VEGF-C
with T cell responses in human melanoma patients (n=20) enrolled in
a Phase I clinical study (NCT00112229) evaluating an anti-tumor
Melan-A/MART-1 peptide vaccine. (A) magnitude and (B) functionality
in terms of IFN.quadrature. expression or (C) polyfunctionality in
terms of IFN.quadrature., TNF.quadrature., IL-2 and CD107
expression. (D) Progression-free survival (PFS) of human melanoma
patients (n=76) enrolled in a Phase II clinical study (NCT01927419)
receiving combined anti-PD-1 and anti-CTLA-4 checkpoint blockade.
Patients were stratified into three groups (high, mid, low)
according to serum VEGF-C concentrations. *p.ltoreq.0.05 by
non-parametric Spearman's test for correlations, two-tailed
Student's t-test for dot plots (shown as mean.+-.SEM) and Log-rank
(Mantel-Cox) test for survival curves.
[0113] FIG. 15A-F. Increased efficacy of immunotherapy in
lymphangiogenic B16 melanomas depends on CCR7 signaling prior to
therapy, and local activation and expansion of TILs post therapy.
(A and B) B16-OVA/VC tumor-bearing mice treated with control IgG
(Iso) or anti-VEGFR3 (.quadrature.R3) blocking antibodies were
euthanized 3 days after adoptive T cell transfer (ATT), and tumor
single cell suspensions analyzed by flow cytometry (n=5, one
experiment). Quantification of overall naive CD8+
(CD45.sup.+CD8.sup.+CD44.sup.-CD62L.sup.+), effector CD8+
(CD45.sup.+CD8.sup.+CD44.sup.+CD62L.sup.-), and OT-I
(CD45.sup.+CD8.sup.+CD45.1.sup.+) T cells (A) in the tumor and (B)
in the dLNs. (C) Tumor growth (left) and survival curves (right) of
B16-OVA/VC tumor-bearing mice treated with anti-CCR7
(.quadrature.CCR7), control IgG (Iso), or .quadrature.R3 antibodies
combined with ATT on day 9. CCR7 blockade was performed only prior
to ATT (day 0, 3, and 6) (data pooled from .gtoreq.2 independent
experiments, n.gtoreq.15 total). (D) Tumor growth curves of
B16-OVA/VC tumor-bearing mice treated with control IgG or
.quadrature.R3 antibodies received daily injections of the small
molecular S1P inhibitor FTY720 starting on the same day as ATT was
performed (day 9) (n.gtoreq.5, one experiment). (E) Representative
flow cytometry plots and (F) quantification of circulating
CD4.sup.+ and CD8.sup.+ T cells (after B220 exclusion) in blood 26
days after tumor inoculation. *p.ltoreq.0.05, **p.ltoreq.0.01,
***p.ltoreq.0.001 by two-tailed Student's t-test or One-way ANOVA.
Bar graphs shown as mean.+-.SEM.
[0114] FIG. 16A-D. Mice rejecting primary lymphangiogenic B16
melanomas in response to immunotherapy show epitope spreading and
long-term protection. B16-OVA/VC tumor bearing mice that rejected
the primary tumor following therapeutic vaccination were
re-challenged with intravenous injections of 2.times.10.sup.5 B16
wildtype (WT) or B16-OVA/VC cells (2.degree. i.v. challenge) at
least 10 days after complete regression. Mice that either received
no treatment (naive) or vaccination only (Vax only) served as
control for the first challenge (1.degree. i.d. challenge). (A)
Representative images of lung metastasis and (B) quantification of
metastatic nodules per lung of mice euthanized 9 days after the
2.degree. i.v. challenge. Flow cytometry analysis of (C) effector
CD4.sup.+ (CD45.sup.+B220.sup.-CD4.sup.+CD44.sup.+CD62L.sup.-),
effector CD8.sup.+
(CD45.sup.+B220.sup.-CD8.sup.+CD44.sup.+CD62L.sup.-), and
OVA-specific CD8.sup.+
(CD45.sup.+B220.sup.-CD8.sup.+SIINFEKL-pentamer.sup.+) 23 days
after inoculation and (D) circulating tumor antigen-specific
CD8.sup.+ T cell responses 9 days after metastatic challenge. Data
pooled from 2 independent experiments, n.gtoreq.3 each.
*p.ltoreq.0.05, **p.ltoreq.0.01, ***p.ltoreq.0.001 by one-way
ANOVA. Bar graphs shown as mean.+-.SEM.
[0115] FIG. 17A-F. VEGFR-3 blocking specifically prevents
lymphangiogenesis in VEGF-C overexpressing B16 melanoma. (A)
Intratumoral VEGFC concentration as assessed by ELISA (n=5) 9 days
after tumor inoculation. (B) Representative images of an
immunostained whole tumor section of a non-lymphangiogenic B16-OVA
tumor (scale bar=500 .quadrature.m) showing overall LEC density
(Lyve-1, green; DAPI, cyan). (C and E) Representative flow
cytometry plots and gating strategy of tumor single cell
suspensions and (D) quantification of BECs
(CD45.sup.-gp38.sup.-CD31.sup.+), macrophages
(CD45.sup.+F4/80.sup.+), and (F) dendritic cell subsets:
conventional DCs (CD11c.sup.+CD11b.sup.-), cross-presenting DCs
(CD11c.sup.+CD11b.sup.-CD8.sup.+), and myeloid DCs
(CD11c.sup.+CD11b.sup.+). Results represent 2 independent
experiments, n=5 each. *p.ltoreq.0.05, **p.ltoreq.0.01 by
two-tailed Student's t-test. Bar graphs shown as mean.+-.SEM.
[0116] FIG. 18A-C. VEGF-C/VEGFR-3 signaling increases melanoma
responsiveness to immunotherapy against endogenous B16 melanoma
antigens. (A) Growth curves of B16-WT (left) or B16-VC (right)
tumor-bearing mice treated with i.p injections of control (Iso) or
.quadrature.VEGFR3 (.quadrature.R3) blocking antibodies (n=5). (B
and C) Growth curves of B16-VC tumor-bearing mice treated with CpG
(50 .quadrature.g) and (B) no antigen or (C) in combination with 2
.quadrature.g Trp-2-peptide-conjugated nanoparticles (NP-Trp2) i.d.
into hind legs on day 4, 7 and 10 after tumor cell inoculation
(n.gtoreq.6). *p.ltoreq.0.05, **p.ltoreq.0.01, ***p.ltoreq.0.001 by
two-tailed Student's t-test for growth curves and Log-rank
(Mantel-Cox) test for survival curves. Growth curves shown as
mean.+-.SEM over time and bar graphs as mean.+-.SEM.
[0117] FIG. 19A-E. VEGFR3 blockade does not broadly affect cytokine
expression in lymphangiogenic B16 tumors, and decreases
intratumoral CCL21 levels and tumor-associated lymphangiogenesis in
BRAF GEMs. (A) Intratumoral concentrations of cytokines as assessed
by protein array (n=5) from B16 tumors 9 days after inoculation. (B
to E) Characterization of BRAF GEM treated with i.p injections of
control (Iso) or .alpha.VEGFR3 (.alpha.R3) blocking antibodies.
Intratumoral concentrations of VEGF-C (B) and CCL21 (D) as assessed
by ELISA (n.gtoreq.6). (C) Representative images of immunostained
sections (scale bar=100 .quadrature.m) showing lymphatic vessels
(Lyve-1, red; DAPI, blue) and (E) zoom at the lymphatic vessel with
combined CCL21 staining (green). *p.ltoreq.0.05 by two-tailed
Student's t-test. Bar graphs shown as mean.+-.SEM.
[0118] FIG. 20. Expression of VEGFC positively correlates with
hallmarks of inflammation within metastatic sites of human
melanoma. Correlations of gene expression data of samples from
human metastatic melanoma sites from the cancer genome atlas
(TCGA). Dot plots shown with linear regression curve (n=369,
correlation was tested using non-parametric Spearman's test).
[0119] FIG. 21A-D. Serum VEGF-C levels in human metastatic melanoma
patients mainly predicts polyfunctionality of systemic
tumor-specific CD8+ response after immunotherapy. (A to D)
Correlations of serum VEGF-C with T cell responses in human
melanoma patients (n=20) enrolled in a Phase I clinical study
(NCT00112229) evaluating an anti-tumor Melan-A/MART-1 peptide
vaccine. Correlation of serum VEGF-C levels with T cell
functionality in terms of (A) TNF.quadrature., (B) IL-2, (C) CD107,
and (D) combined IFN.quadrature. and TNF.quadrature. expression.
*p.ltoreq.0.05 by non-parametric Spearman's test for correlations
and two-tailed Student's t-test for dot plots (shown as
mean.+-.SEM).
[0120] FIG. 22A-F. VEGFC recruits T cells to populate `cold tumors`
and blockade of VEGFC signaling reduces efficacy of checkpoint
blockade immunotherapy. (A) Using a genetically-modified mouse
model (GEMM) of melanoma that mimics the mutations most commonly
found in human melanoma, hydroxytamoxifen (OHT)-treated mice
develop skin tumors within 3 weeks of induction, following which
they were injected with VEGFC weekly for two weeks. Mice were
sacrificed at 5 weeks post-induction of tumors and primary tumors
were collected and analyzed by flow cytometry for immune cell
infiltration. (B) Braf.sup.V600EPTEN.sup.-/CAT-STA mice typically
develop `cold tumors` which were very poorly infiltrated by immune
cells--particularly T cells, which have the ability to kill tumor
cells. However, VEGFC-treated individuals exhibited significantly
increased levels of recruitment of conventional CD4 T cells and
cytotoxic T cells. An insignificant increase in accumulation of
immunosuppressive regulatory T cells was also seen in these mice.
(C) To test the relevance of this data to immunotherapy, a related
mouse model lacking expression of artificial antigens was used.
After tumor induction, a typical therapeutic vaccine (gp100
melanoma antigen with CpG adjuvant) with checkpoint blockade
immunotherapy (anti-PD-1; .alpha.PD-1) schedule was then followed.
Blockade of VEGFC signaling commenced a week before the
vaccine-checkpoint blockade schedule, through administration of an
antibody against its receptor (VEGFR3), and was continued with
.alpha.PD-1 for the duration of the study in order to ensure
complete silencing of this signaling pathway. (D) ELISA
measurements confirm the reduction in local CCL21 levels in mice
that received .alpha.VEGFR3, suggesting a weakened ability to
recruit T cells. (E) Tumors in .alpha.VEGFR3-treated mice grow more
rapidly than in control mice treated with an isotype control
antibody. (F) Survival of tumor-bearing mice was tracked
longitudinally, showing that mice treated with .alpha.VEGFR3
succumb earlier to their disease. *p<0.05 for n=3-7 biological
replicates via Mann-Whitney U-test (A-E) or via Mantel-Cox log rank
test (F).
[0121] FIG. 23A-E. Lymphatic expansion in VEGFC-overexpressing
cancer vaccines promotes systemic activation of antigen-specific T
cell immunity. (A) Healthy mice were vaccinated with either
x-irradiated B16OVA/VEGFC plus CFA/OVA (VEGFC vax) or with control
irradiated B16OVA and CFA/OVA (ctrl vax). (B) Following the
vaccination schedule described in A, antigen-specific CD8+ T cells
in vaccinated dLNs and spleen were re-stimulated with a
MHCI-restricted OVA peptide (SIINFEKL) and IFN.gamma. production
was measured by ELISA and intracellular staining. (C) Healthy mice
were vaccinated with either x-irradiated B16OVA/VEGFC plus
Imiquimod (VEGFC vax) or with control irradiated B16OVA plus
Imiquimod (ctrl vax). Imiquimod vas applied on the cell injection
site every other day starting at day 2 from cell injection. (D)
Following the vaccination protocol described in C, antigen-specific
CD8+ T cells in vaccine-dLNs and spleen were re-stimulated with
either a MHCI-restricted OVA peptide (SIINFEKL, left panels) or
MHCI-restricted mixed Trp2 and gp100 peptides (right panels) and
IFN.gamma. secretion was measured by ELISA. (E) Correlation between
IFN.gamma. secretion in dLNs or spleen and LEC proportion in the
vaccine injection site following the vaccination schedule described
in C. IFN.gamma. production was measured post ex vivo T cell
re-stimulation with the SIIKFEKL peptide. *p<0.05, **<0.01.
dLNs: draining-lymph nodes. CFA: Complete Freund's Adjuvant.
B16OVA/VEGFC: VEGFC-overexpressing B16OVA.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0122] The inventors have observed that cells lining the lymphatic
vessels, notably lymphatic endothelial cells (LECs), can serve as
APCs, and that LECs may play a pivotal role in induction of
immunological memory. LECs can collect exogenous antigens and
cross-present them on major histocompatibility complex I (MHC I), a
process more associated with specific classes of DCs (Hirosue et
al., 2014; Lund et al., 2012; Card et al., 2014; Fletcher et al.,
2010; Lukacs-Kornek et al., 2011; Malhotra et al., 2012).
Classically, antigens from cytoplasmic location (such as viral
proteins) are processed and presented on MHC I; as such, this MHC
serves as a sensor for antigens present endogenously within the
cells. Some APCs, however, notably CD8a.sup.+ DCs, are capable of
processing exogenous antigen and loading it on MHC I, a process
referred to as cross-presentation (den Hann et al., 2000; Schulz et
al., 2002). The inventors have observed that LECs can cross-present
exogenous antigens as well. It is thought that antigenic epitopes
presented on MHC I can stimulate responses in the CD8 T cell
compartment. In addition to this mode of antigen presentation, APCs
are also able to present exogenous antigens on major
histocompatibility complex II (MHC II), which stimulates responses
in the CD4 T cell compartment. The inventors have observed that
LECs can do this as well (Dubrot et al., 2014) However, prior art
suggests that LEC-educated T cells tend to exhibit suboptimal
activation profiles, in similar fashion to suppressed T cells
(Hirosue et al., 2014; Lund et al., 2012; Card et al., 2014;
Fletcher et al., 2010; Lukacs-Kornek et al., 2011; Hirosue et al.,
2015; Tewalt et al., 2012).
II. Methods and Compositions
[0123] The inventors describe here the use of lymphangiogenesis
inducers and antigens that serve to evoke protective immune
responses. the inventors find that LECs can function as powerful
APCs in vaccination from the perspective of immunological memory
and memory recall responses. Embodiments focus the way in which
LECs and lymphangiogenesis may be manipulated to induce
immunological responses.
III. Immunotherapies
[0124] In some embodiments, the methods include the administration
of an immunotherapy. Exemplary immunotherapies are described
below.
[0125] A. Checkpoint Inhibitors
[0126] An "immune checkpoint inhibitor" is any molecule that
directly or indirectly inhibits, partially or completely, an immune
checkpoint pathway. Without wishing to be bound by any particular
theory, it is generally thought that immune checkpoint pathways
function to turn on or off aspects of the immune system,
particularly T cells. Following activation of a T cell, a number of
inhibitory receptors can be upregulated and present on the surface
of the T cell in order to suppress the immune response at the
appropriate time. In the case of persistent immune stimulation,
such as with chronic viral infection, for example, immune
checkpoint pathways can suppress the immune response and lead to
immune exhaustion. Examples of immune checkpoint pathways include,
without limitation, PD-1/PD-L1, CTLA4/B7-1, TIM-3, LAG3, By-He, H4,
HAVCR2, ID01, CD276 and VTCN1. In the instance of the PD-1/PD-L1
immune checkpoint pathway, an inhibitor may bind to PD-1 or to
PD-L1 and prevent interaction between the receptor and ligand.
Therefore, the inhibitor may be an anti-PD-1 antibody or anti-PD-L1
antibody. Similarly, in the instance of the CTLA4/B7-1 immune
checkpoint pathway, an inhibitor may bind to CTLA4 or to B7-1 and
prevent interaction between the receptor and ligand. Further
examples of immune checkpoint inhibitors can be found, for example,
in WO2014/144885. Such immune checkpoint inhibitors are
incorporated by reference herein. In some embodiments of any one of
the methods, compositions or kits provided, the immune checkpoint
inhibitor is a small molecule inhibitor of an immune checkpoint
pathway. In some embodiments of any one of the methods,
compositions or kits provided, the immune checkpoint inhibitor is a
polypeptide that inhibits an immune checkpoint pathway. In some
embodiments of any one of the methods, compositions or kits
provided, the inhibitor is a fusion protein. In some embodiments of
any one of the methods, compositions or kits provided, the immune
checkpoint inhibitor is an antibody. In some embodiments of any one
of the methods, compositions or kits provided, the antibody is a
monoclonal antibody.
[0127] Non-limiting examples of immune checkpoint inhibitors
include fully human monoclonal antibodies, such as RG7446,
BMS-936558/MDX-1106, BMS-936559 (anti-PDL1 antibody),
Yervoy/ipilimumab (anti-CTLA-4 checkpoint inhibitor), and
Tremelimumab (CTLA-4 blocking antibody); humanized antibodies, such
as pidilizumab (CT-011, CureTech Ltd.) and lambrolizumab (MK-3475,
Merck, PD-1 blocker); and fusion proteins, such as AMP-224 (Merck).
Other examples of checkpoint inhibitors include anti-OX40, PD-L1
monoclonal Antibody (Anti-B7-H1; MEDI4736), Nivolumab (BMS-936558,
Bristol-Myers Squibb, anti-PD1 antibody), CT-011 (anti-PD1
antibody), BY55 monoclonal antibody, MPLDL3280A (anti-PDL1
antibody), and MSB0010718C (anti-PDL1 antibody), MDX-1105
(Medarex), MPDL3280A (Genentech), Anti-KIR antibodies such as
lirlumab (Innate Pharma) and IPH2101 (Innate Pharma) may perform
similar functions in NK cells. Further examples of checkpoint
inhibitors include agonistic anti-4-1bb antibody; agonistic
anti-CD27 antibody; agonistic anti-GTIR antibody; agonistic
anti-OX40 antibody; and antagonistic anti-TIM3 antibody.
[0128] B. Additional Immunotherapies and Agents
[0129] In some embodiments, the method further comprises
administration of an immunotherapy or an additional agent described
herein. In some embodiments, the additional agent is an
immunostimulator. The term "immunostimulator" as used herein refers
to a compound that can stimulate an immune response in a subject,
and may include an adjuvant. In some embodiments, an
immunostimulator is an agent that does not constitute a specific
antigen, but can boost the strength and longevity of an immune
response to an antigen. Such immunostimulators may include, but are
not limited to stimulators of pattern recognition receptors, such
as Toll-like receptors, RIG-1 and NOD-like receptors (NLR), mineral
salts, such as alum, alum combined with monphosphoryl lipid (MPL) A
of Enterobacteria, such as Escherichia coli, Salmonella minnesota,
Salmonella typhimurium, or Shigella flexneri or specifically with
MPL.RTM. (A504), MPL A of above-mentioned bacteria separately,
saponins, such as QS-21, Quil-A, ISCOMs, ISCOMATRIX, emulsions such
as MF59, Montanide, ISA 51 and ISA 720, AS02 (QS21+squalene+MPL.),
liposomes and liposomal formulations such as AS01, synthesized or
specifically prepared microparticles and microcarriers such as
bacteria-derived outer membrane vesicles (OMV) of N. gonorrheae,
Chlamydia trachomatis and others, or chitosan particles,
depot-forming agents, such as Pluronic block co-polymers,
specifically modified or prepared peptides, such as muramyl
dipeptide, aminoalkyl glucosaminide 4-phosphates, such as RC529, or
proteins, such as bacterial toxoids or toxin fragments.
[0130] In some embodiments, the additional agent comprises an
agonist for pattern recognition receptors (PRR), including, but not
limited to Toll-Like Receptors (TLRs), specifically TLRs 2, 3, 4,
5, 7, 8, 9 and/or combinations thereof. In some embodiments,
additional agents comprise agonists for Toll-Like Receptors 3,
agonists for Toll-Like Receptors 7 and 8, or agonists for Toll-Like
Receptor 9; preferably the recited immunostimulators comprise
imidazoquinolines; such as R848; adenine derivatives, such as those
disclosed in U.S. Pat. No. 6,329,381, U.S. Published Patent
Application 2010/0075995, or WO 2010/018132; immunostimulatory DNA;
or immunostimulatory RNA. In some embodiments, the additional
agents also may comprise immunostimulatory RNA molecules, such as
but not limited to dsRNA, poly I:C or poly I:poly C12U (available
as Ampligen.RTM., both poly I:C and poly I:polyC12U being known as
TLR3 stimulants), and/or those disclosed in F. Heil et al.,
"Species-Specific Recognition of Single-Stranded RNA via Toll-like
Receptor 7 and 8" Science 303(5663), 1526-1529 (2004); J. Vollmer
et al., "Immune modulation by chemically modified ribonucleosides
and oligoribonucleotides" WO 2008033432 A2; A. Forsbach et al.,
"Immunostimulatory oligoribonucleotides containing specific
sequence motif(s) and targeting the Toll-like receptor 8 pathway"
WO 2007062107 A2; E. Uhlmann et al., "Modified oligoribonucleotide
analogs with enhanced immunostimulatory activity" U.S. Pat. Appl.
Publ. US 2006241076; G. Lipford et al., "Immunostimulatory viral
RNA oligonucleotides and use for treating cancer and infections" WO
2005097993 A2; G. Lipford et al., "Immunostimulatory G,U-containing
oligoribonucleotides, compositions, and screening methods" WO
2003086280 A2. In some embodiments, an additional agent may be a
TLR-4 agonist, such as bacterial lipopolysaccharide (LPS), VSV-G,
and/or HMGB-1. In some embodiments, additional agents may comprise
TLR-5 agonists, such as flagellin, or portions or derivatives
thereof, including but not limited to those disclosed in U.S. Pat.
Nos. 6,130,082, 6,585,980, and 7,192,725.
[0131] In some embodiments, additional agents may be
proinflammatory stimuli released from necrotic cells (e.g., urate
crystals). In some embodiments, additional agents may be activated
components of the complement cascade (e.g., CD21, CD35, etc.). In
some embodiments, additional agents may be activated components of
immune complexes. Additional agents also include complement
receptor agonists, such as a molecule that binds to CD21 or CD35.
In some embodiments, the complement receptor agonist induces
endogenous complement opsonization of the synthetic nanocarrier. In
some embodiments, immunostimulators are cytokines, which are small
proteins or biological factors (in the range of 5 kD-20 kD) that
are released by cells and have specific effects on cell-cell
interaction, communication and behavior of other cells. In some
embodiments, the cytokine receptor agonist is a small molecule,
antibody, fusion protein, or aptamer.
[0132] In some embodiments, the additional agent is an
antibody-drug conjugate. In some embodiments, the antibody-drug
conjugate is selected from gemtuzumab ozogamicin, brentuximab
vedotin, and trastuzumab emtansine.
[0133] In some embodiments, the additional agent is a chimeric
antigen receptor (CAR). CARs are artificial T cell receptors which
graft a specificity onto an immune effector cell. The most common
form of these molecules are fusions of single-chain variable
fragments (scFv) derived from monoclonal antibodies, fused to
CD3-zeta transmembrane and endodomain. Such molecules result in the
transmission of a zeta signal in response to recognition by the
scFv of its target. An example of such a construct is 14g2a-Zeta,
which is a fusion of a scFv derived from hybridoma 14g2a (which
recognizes disialoganglioside GD2). When T cells express this
molecule (usually achieved by oncoretroviral vector transduction),
they recognize and kill target cells that express GD2 (e.g.
neuroblastoma cells). The variable portions of an immunoglobulin
heavy and light chain are fused by a flexible linker to form a
scFv. This scFv is preceded by a signal peptide to direct the
nascent protein to the endoplasmic reticulum and subsequent surface
expression (this is cleaved). A flexible spacer allows the scFv to
orient in different directions to enable antigen binding. The
transmembrane domain is a typical hydrophobic alpha helix usually
derived from the original molecule of the signalling endodomain
which protrudes into the cell and transmits the desired signal.
[0134] Additional agents that can act as immunostimulators include
STING agonists. The STING pathway is a pathway that is involved in
the detection of cytosolic DNA. Stimulator of interferon genes
(STING), also known as transmembrane protein 173 (TMEM173) and
MPYS/MITA/ERIS, is a protein that in humans is encoded by the
TMEM173 gene. STING plays an important role in innate immunity.
STING induces type I interferon production when cells are infected
with intracellular pathogens, such as viruses, mycobacteria and
intracellular parasites. Type I interferon, mediated by STING,
protects infected cells and nearby cells from local infection in an
autocrine and paracrine manner.
[0135] STING is encoded by the TMEM173 gene. It works as both a
direct cytosolic DNA sensor (CDS) and an adaptor protein in Type I
interferon signaling through different molecular mechanisms. It has
been shown to activate downstream transcription factors STAT6 and
IRF3 through TBK1, which are responsible for antiviral response and
innate immune response against intracellular pathogen.
[0136] STING resides in the endoplasmic reticulum, but in the
presence of cytosolic DNA, the sensor cGAS binds to the DNA and
forms cyclic dinucleotides. This di-nucleotide binds to STING and
promotes its aggregation and translocation from the ER through the
Golgi to perinuclear sites. There, STING complexes with TBK1 and
promotes its phosphorylation. Once TBK1 is phosphorylated, it
phosphorylates the transcription factor IRF3 that dimerices and
traslocates to the nucleus, where it activates the transcription of
type I IFN and other innate immune genes.
[0137] STING agonsists can include 3'3'-cGAMP fluorinated,
fluorinated cyclic diadenylate monophosphate, ZDHHC1,
2'3'-c-di-AM(PS)2 (Rp,Rp), 2'2'-cGAMP, c-di-IMP, 2'3'-cGAM(PS)2
(Rp/Sp), 3'3'-cGAMP, DMXAA, 2'3'-cGAMP, c-di-GMP, c-di-GMP,
2'3'-c-di-GMP, 2'3'-c-di-AMP, c-di-GMP Fluorinated, and
c-di-AMP.
[0138] In some embodiments, the immunotherapy includes cytolytic
viral therapy, such administration of an onocolytic virus or
modified version thereof. Oncolytic viruses include oncolytic
herpes simplex virus, adenovirus, reovirus, measles, Newcastle
disease virus, and vaccinia virus.
[0139] C. Vaccine Immunotherapies
[0140] The methods of the disclosure may also include the
administration of vaccines. As used herein, the term in vitro
administration refers to manipulations performed on cells removed
from or outside of a subject, including, but not limited to cells
in culture. The term ex vivo administration refers to cells which
have been manipulated in vitro, and are subsequently administered
to a subject. The term in vivo administration includes all
manipulations performed within a subject, including
administrations.
[0141] In certain aspects of the present disclosure, the
compositions may be administered either in vitro, ex vivo, or in
vivo. In certain in vitro embodiments, autologous T cells are
incubated with compositions of this disclosure. The cells can then
be used for in vitro analysis, or alternatively for ex vivo
administration.
[0142] Method aspects of the disclosure include vaccinating a
subject with a variety of different immunotherapeutic compositions.
In some embodiments, the methods further comprise administration of
immune cells to the subject. In some embodiments, the immune cells
are autologous. In some embodiments, the immune cells has been
contacted with an antigen. In some embodiments, the antigen is an
antigen expressed by the subject's cancer cells. In some
embodiments, the antigen is cell free. The term "cell free" refers
to a composition that does not have any cellular components. In
some embodiments, the antigen is an extract from the patient's
tumor. In some embodiments, the antigen is a polypeptide. In some
embodiments, the antigen comprises one or more of tumor cell
lysate, apoptotic tumor cell, tumor-associated antigen, and
tumor-derived mRNA. In some embodiments, the immune cell has been
contacted with a maturation agent. In some embodiments, the
maturation agent is one or more of GM-CSF, IL-1.beta., TNF-.alpha.,
and PGE2. In some embodiments, the immune cell comprises a chimeric
antigen receptor.
[0143] In some embodiments, the immune cell is an antigen
presenting cells. Antigen-presenting cells can be used as a cancer
vaccine. Examples of the antigen-presenting cells include dendritic
cells, macrophages, B cells, and tumor cells (false
antigen-presenting cells) in which a T cell stimulation factor
(e.g., B7 or 4-1 BBL) and the like is forcibly expressed by, for
example, gene transfer. In some embodiments, the antigen presenting
cell is a dendritic cell.
[0144] The route of administration of the immune cell may be, for
example, intratumoral, intracutaneous, subcutaneous, intravenous,
intralymphatic, and intraperitoneal administrations. In some
embodiments, the administration is intratumoral or intrapymphatic.
In some embodiments, the immune cells are administered directly
into a cancer tissue or a lymph node.
[0145] In some embodiments, the immune cell is a T cell. T cells
can also be used as a cancer vaccine. The T cells may be ones that
have been contacted with an antigen or with antigen-presenting
cells. For example, APCs may be cultured with tumor antigen
specific to the patient's cancer to differentiate them, into, for
example, CD8-positive cytotoxic T lymphocytes (CTLs) or
CD4-positive helper T cells. The T cells thus established may be
administered to an individual with cancer.
[0146] The origin of the naive T cells is not specifically limited
and it may be derived from, for example, peripheral blood of a
vertebrate animal. The naive T cell used may be CD8-positive cells
or CD4-positive cells isolated from a PBMC fraction. In some
embodiments, the naive T cells are CD8-positive cells or
CD4-positive cells mixed with other cells and components without
being isolated from the PBMC fraction in terms of the efficiency of
inducing CTLs. For example, when cells of a PBMC fraction are
cultured in a medium supplemented with serum and tumor antigen, the
PBMCs differentiate into dendritic cell precursors. The dendritic
cell precursors then bind to the peptide and differentiate into
dendritic cells as the antigen-presenting cells presenting this
peptide/tumor antigen. The antigen-presenting cells stimulate the
CD8-positive T cells in the PBMCs to differentiate them into CTLs.
Thus, the CTLs capable of recognizing the added peptide can be
obtained. The CTLs thus obtained may be isolated and used as the
cancer vaccine as they are. Alternatively, they may be cultured
further in the presence of interleukin such as IL-2, the
antigen-presenting cell, and tumor antigen before used as the
cancer vaccine. The route of their administration is not
specifically limited and examples include intracutaneous,
subcutaneous, intravenous, and intratumoral administrations.
IV. Antigen
[0147] The methods of the disclosure may include the administration
of an antigen. In one embodiment, the antigen is a cancer antigen.
In a further embodiment, the antigen is specific to the patient's
cancer. In some embodiments, the antigen is chemically or
recombinantly synthesized. In some embodiments, the antigen
comprises one or more of tumor cell lysate, apoptotic tumor cell,
tumor-associated antigen, and tumor-derived mRNA. In some
embodiments, the antigen is cell-free. The antigen may be one known
in the art or described herein. Non-limiting examples of cancer
antigens include antigenic fragments and polypeptides from VEGFR-2,
MMPs, Survivin, TEM8, PMSA, CA125, folate binding protein (FBP),
HER2/neu, MUC1, NYESO 1, PSA, Carcinoembryonic antigen (CEA),
.alpha.-fetoprotein (AFP), heat shock proteins (e.g., hsp70 or
hsp90 proteins) from a particular type of tumor, MICA/B ligands of
NKG2D, epithelial cell adhesion molecule (Ep-CAM/TACSTD1),
mesothelin, tumor-associated glycoprotein 72 (TAG-72), gp100,
Melan-A, MART-1, KDR, RCAS1, MDA7, cancer-associated viral vaccines
(e.g., human papillomavirus antigens), prostate specific antigen
(PSA, PSMA), RAGE (renal antigen), CAMEL (CTL-recognized antigen on
melanoma), CT antigens (such as MAGE-B5, -B6, -C2, -C3, and D;
Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin
antigens (e.g., MUC1, mucin-CA125, etc.), cancer-associated
ganglioside antigens, tyrosinase, gp75, C-myc, Mart1, MelanA,
MUM-1, MUM-2, MUM-3, HLA-B7, Ep-CAM, tumor-derived heat shock
proteins, and the like (see also, e.g., Acres et al., Curr Opin Mol
Ther 2004 February, 6:40-7; Taylor-Papadimitriou et al., Biochim
Biophys Acta. 1999 Oct. 8; 1455(2-3):301-13; Emens et al., Cancer
Biol Ther. 2003 July-August; 2(4 Suppl 1):S161-8; and Ohshima et
al., Int J Cancer. 2001 Jul. 1; 93(1):91-6). Other exemplary cancer
antigen targets include CA 195 tumor-associated antigen-like
antigen (see, e.g., U.S. Pat. No. 5,324,822) and female urine
squamous cell carcinoma-like antigens (see, e.g., U.S. Pat. No.
5,306,811), and the breast cell cancer antigens described in U.S.
Pat. No. 4,960,716.
[0148] The cancer antigen may be an epithelial cancer antigen,
(e.g., breast, gastrointestinal, lung), a prostate specific cancer
antigen (PSA) or prostate specific membrane antigen (PSMA), a
bladder cancer antigen, a skin (melanoma) cancer antigen, a lung
(e.g., small cell lung) cancer antigen, a colon cancer antigen, an
ovarian cancer antigen, a brain cancer antigen, a gastric cancer
antigen, a renal cell carcinoma antigen, a pancreatic cancer
antigen, a liver cancer antigen, an esophageal cancer antigen, a
head and neck cancer antigen, or a colorectal cancer antigen. A
cancer antigen can also be an antigen specifically expressed by the
patient's cancer or an antigen known to be specifically expressed
by the patient's cancer.
V. Nucleic Acids
[0149] In certain embodiments, there are recombinant nucleic acids
encoding the proteins, polypeptides, or peptides described herein.
Polynucleotides contemplated for use in methods and compositions
include those encoding lymphangiogenesis inducers and antigens such
as a bacterial antigen, a viral antigen, a fungal antigen, a
protozoal antigen, a helminth antigen or a cancer antigen.
[0150] As used in this application, the term "polynucleotide"
refers to a nucleic acid molecule that either is recombinant or has
been isolated free of total genomic nucleic acid. Included within
the term "polynucleotide" are oligonucleotides (nucleic acids 100
residues or fewer in length), recombinant vectors, including, for
example, plasmids, cosmids, phage, viruses, and the like.
Polynucleotides include, in certain aspects, regulatory sequences,
isolated substantially away from their naturally occurring genes or
protein encoding sequences. Polynucleotides may be single-stranded
(coding or antisense) or double-stranded, and may be RNA, DNA
(genomic, cDNA or synthetic), analogs thereof, or a combination
thereof. Additional coding or non-coding sequences may, but need
not, be present within a polynucleotide.
[0151] In this respect, the term "gene," "polynucleotide," or
"nucleic acid" is used to refer to a nucleic acid that encodes a
protein, polypeptide, or peptide (including any sequences required
for proper transcription, post-translational modification, or
localization). As will be understood by those in the art, this term
encompasses genomic sequences, expression cassettes, cDNA
sequences, and smaller engineered nucleic acid segments that
express, or may be adapted to express, proteins, polypeptides,
domains, peptides, fusion proteins, and mutants. A nucleic acid
encoding all or part of a polypeptide may contain a contiguous
nucleic acid sequence encoding all or a portion of such a
polypeptide. It also is contemplated that a particular polypeptide
may be encoded by nucleic acids containing variations having
slightly different nucleic acid sequences but, nonetheless, encode
the same or substantially similar protein (see above).
[0152] In particular embodiments, there are isolated nucleic acid
segments and recombinant vectors incorporating nucleic acid
sequences that encode a polypeptide (e.g., a lymphangiogenesis
inducer) that induces formation of lymphatic vessels from
pre-existing lymphatic vessels. The term "recombinant" may be used
in conjunction with a polypeptide or the name of a specific
polypeptide, and this generally refers to a polypeptide produced
from a nucleic acid molecule that has been manipulated in vitro or
that is a replication product of such a molecule.
[0153] The nucleic acid segments, regardless of the length of the
coding sequence itself, may be combined with other nucleic acid
sequences, such as promoters, polyadenylation signals, additional
restriction enzyme sites, multiple cloning sites, other coding
segments, and the like, such that their overall length may vary
considerably. It is therefore contemplated that a nucleic acid
fragment of almost any length may be employed, with the total
length preferably being limited by the ease of preparation and use
in the intended recombinant nucleic acid protocol. In some cases, a
nucleic acid sequence may encode a polypeptide sequence with
additional heterologous coding sequences, for example to allow for
purification of the polypeptide, transport, secretion,
post-translational modification, or for therapeutic benefits such
as targeting or efficacy. As discussed above, a tag or other
heterologous polypeptide may be added to the modified
polypeptide-encoding sequence, wherein "heterologous" refers to a
polypeptide that is not the same as the modified polypeptide.
[0154] In certain embodiments, there are polynucleotide variants
having substantial identity to the sequences disclosed herein;
those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% or higher sequence identity, including all values and
ranges there between, compared to a polynucleotide sequence
provided herein using the methods described herein (e.g., BLAST
analysis using standard parameters). In certain aspects, the
isolated polynucleotide will comprise a nucleotide sequence
encoding a polypeptide that has at least 90%, preferably 95% and
above, identity to an amino acid sequence described herein, over
the entire length of the sequence; or a nucleotide sequence
complementary to said isolated polynucleotide.
[0155] A. Vectors
[0156] Polypeptides may be encoded by a nucleic acid molecule. The
nucleic acid molecule can be in the form of a nucleic acid vector.
The term "vector" is used to refer to a carrier nucleic acid
molecule into which a heterologous nucleic acid sequence can be
inserted for introduction into a cell where it can be replicated
and expressed. A nucleic acid sequence can be "heterologous," which
means that it is in a context foreign to the cell in which the
vector is being introduced or to the nucleic acid in which is
incorporated, which includes a sequence homologous to a sequence in
the cell or nucleic acid but in a position within the host cell or
nucleic acid where it is ordinarily not found. Vectors include
DNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal
viruses, and plant viruses), and artificial chromosomes (e.g.,
YACs). One of skill in the art would be well equipped to construct
a vector through standard recombinant techniques (for example
Sambrook et al., 2001; Ausubel et al., 1996, both incorporated
herein by reference). Vectors may be used in a host cell to produce
lymphangiogenesis inducers and antigens such as a bacterial
antigen, a viral antigen, a fungal antigen, a protozoal antigen, a
helminth antigen or a cancer antigen.
[0157] The term "expression vector" refers to a vector containing a
nucleic acid sequence coding for at least part of a gene product
capable of being transcribed. In some cases, RNA molecules are then
translated into a protein, polypeptide, or peptide. Expression
vectors can contain a variety of "control sequences," which refer
to nucleic acid sequences necessary for the transcription and
possibly translation of an operably linked coding sequence in a
particular host organism. In addition to control sequences that
govern transcription and translation, vectors and expression
vectors may contain nucleic acid sequences that serve other
functions as well and are described herein.
[0158] B. Host Cells
[0159] As used herein, the terms "cell," "cell line," and "cell
culture" may be used interchangeably. All of these terms also
include their progeny, which is any and all subsequent generations.
It is understood that all progeny may not be identical due to
deliberate or inadvertent mutations. In the context of expressing a
heterologous nucleic acid sequence, "host cell" refers to a
prokaryotic or eukaryotic cell, and it includes any transformable
organism that is capable of replicating a vector or expressing a
heterologous gene encoded by a vector. A host cell can, and has
been, used as a recipient for vectors or viruses. A host cell may
be "transfected" or "transformed," which refers to a process by
which exogenous nucleic acid, such as a recombinant
protein-encoding sequence, is transferred or introduced into the
host cell. A transformed cell includes the primary subject cell and
its progeny.
[0160] Some vectors may employ control sequences that allow it to
be replicated and/or expressed in both prokaryotic and eukaryotic
cells. One of skill in the art would further understand the
conditions under which to incubate all of the above described host
cells to maintain them and to permit replication of a vector. Also
understood and known are techniques and conditions that would allow
large-scale production of vectors, as well as production of the
nucleic acids encoded by vectors and their cognate polypeptides,
proteins, or peptides.
[0161] C. Expression Systems
[0162] Numerous expression systems exist that comprise at least a
part or all of the compositions discussed above. Prokaryote- and/or
eukaryote-based systems can be employed for use with an embodiment
to produce nucleic acid sequences, or their cognate polypeptides,
proteins and peptides. Many such systems are commercially and
widely available.
[0163] The insect cell/baculovirus system can produce a high level
of protein expression of a heterologous nucleic acid segment, such
as described in U.S. Pat. Nos. 5,871,986, 4,879,236, both herein
incorporated by reference, and which can be bought, for example,
under the name MAXBAC.RTM. 2.0 from INVITROGEN.RTM. and BACPACK.TM.
BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH.RTM..
[0164] In addition to the disclosed expression systems, other
examples of expression systems include STRATAGENE.RTM.'s COMPLETE
CONTROL Inducible Mammalian Expression System, which involves a
synthetic ecdysone-inducible receptor, or its pET Expression
System, an E. coli expression system. Another example of an
inducible expression system is available from INVITROGEN.RTM.,
which carries the T-REX.TM. (tetracycline-regulated expression)
System, an inducible mammalian expression system that uses the
full-length CMV promoter. INVITROGEN.RTM. also provides a yeast
expression system called the Pichia methanolica Expression System,
which is designed for high-level production of recombinant proteins
in the methylotrophic yeast Pichia methanolica. One of skill in the
art would know how to express a vector, such as an expression
construct, to produce a nucleic acid sequence or its cognate
polypeptide, protein, or peptide.
VI. Proteinaceous Compositions
[0165] Substitutional variants typically contain the exchange of
one amino acid for another at one or more sites within the protein,
and may be designed to modulate one or more properties of the
polypeptide, with or without the loss of other functions or
properties. Substitutions may be conservative, that is, one amino
acid is replaced with one of similar shape and charge. Conservative
substitutions are well known in the art and include, for example,
the changes of: alanine to serine; arginine to lysine; asparagine
to glutamine or histidine; aspartate to glutamate; cysteine to
serine; glutamine to asparagine; glutamate to aspartate; glycine to
proline; histidine to asparagine or glutamine; isoleucine to
leucine or valine; leucine to valine or isoleucine; lysine to
arginine; methionine to leucine or isoleucine; phenylalanine to
tyrosine, leucine or methionine; serine to threonine; threonine to
serine; tryptophan to tyrosine; tyrosine to tryptophan or
phenylalanine; and valine to isoleucine or leucine. Alternatively,
substitutions may be non-conservative such that a function or
activity of the polypeptide is affected. Non-conservative changes
typically involve substituting a residue with one that is
chemically dissimilar, such as a polar or charged amino acid for a
nonpolar or uncharged amino acid, and vice versa.
[0166] Proteins may be recombinant, or synthesized in vitro.
Alternatively, a non-recombinant or recombinant protein may be
isolated from bacteria. It is also contemplated that bacteria
containing such a variant may be implemented in compositions and
methods. Consequently, a protein need not be isolated.
[0167] The term "functionally equivalent codon" is used herein to
refer to codons that encode the same amino acid, such as the six
codons for arginine or serine, and also refers to codons that
encode biologically equivalent amino acids (see Table, below).
TABLE-US-00001 Codon Table Amino Acids Codons Alanine Ala A GCA GCC
GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic
acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA
GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU
Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC
CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG
CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC
ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine
Tyr Y UAC UAU
[0168] It also will be understood that amino acid and nucleic acid
sequences may include additional residues, such as additional N- or
C-terminal amino acids, or 5' or 3' sequences, respectively, and
yet still be essentially as set forth in one of the sequences
disclosed herein, so long as the sequence meets the criteria set
forth above, including the maintenance of biological protein
activity where protein expression is concerned. The addition of
terminal sequences particularly applies to nucleic acid sequences
that may, for example, include various non-coding sequences
flanking either of the 5' or 3' portions of the coding region.
[0169] The following is a discussion based upon changing of the
amino acids of a protein to create an equivalent, or even an
improved, second-generation molecule. For example, certain amino
acids may be substituted for other amino acids in a protein
structure without appreciable loss of interactive binding capacity,
ability to induce lymphangiogenesis or antigenicity with structures
such as, for example, lymphangiogenesis inducers and antigens such
as a bacterial antigen, a viral antigen, a fungal antigen, a
protozoal antigen, a helminth antigen or a cancer antigen. Since it
is the interactive capacity and nature of a protein that defines
that protein's biological functional activity, certain amino acid
substitutions can be made in a protein sequence, and in its
underlying DNA coding sequence, and nevertheless produce a protein
with like properties. It is thus contemplated by the inventors that
various changes may be made in the DNA sequences of genes without
appreciable loss of their biological utility or activity.
[0170] In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring interactive biologic function on a protein is
generally understood in the art (Kyte and Doolittle, 1982). It is
accepted that the relative hydropathic character of the amino acid
contributes to the secondary structure of the resultant protein,
which in turn defines the interaction of the protein with other
molecules, for example, enzymes, substrates, receptors, DNA,
antibodies, antigens, and the like.
[0171] It also is understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by
reference, states that the greatest local average hydrophilicity of
a protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with a biological property of the protein. It is
understood that an amino acid can be substituted for another having
a similar hydrophilicity value and still produce a biologically
equivalent and immunologically equivalent protein.
[0172] As outlined above, amino acid substitutions generally are
based on the relative similarity of the amino acid side-chain
substituents, for example, their hydrophobicity, hydrophilicity,
charge, size, and the like. Exemplary substitutions that take into
consideration the various foregoing characteristics are well known
and include: arginine and lysine; glutamate and aspartate; serine
and threonine; glutamine and asparagine; and valine, leucine and
isoleucine.
[0173] It is contemplated that in compositions there is between
about 0.001 mg and about 10 mg of total biomolecule, protein,
polypeptide, carbohydrate, polysaccharide, lipid, nucleic acid,
fatty acid, glycolipid, sterol, polyterpene, glycerolipid or a
combination of these per ml. Thus, the concentration of a
biomolecule, a protein, a polypeptide, a carbohydrate, a
polysaccharide, a lipid, a nucleic acid, a fatty acid, a
glycolipid, a sterol, a polyterpene, a glycerolipid or a
combination of these in a composition can be about, at least about
or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,
6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or any
range derivable therein). Of this, about, at least about, or at
most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%
may be a lymphangiogenesis inducer and/or antigens such as a
bacterial antigen, a viral antigen, a fungal antigen, a protozoal
antigen, a helminth antigen or a cancer antigen, and may be used in
combination with lymphangiogenesis inducers and antigens such as a
bacterial antigen, a viral antigen, a fungal antigen, a protozoal
antigen, a helminth antigen or a cancer antigen described
herein.
[0174] Included are polypeptides with 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99, or 100% identity (or any derivable range therein) to a peptide
of the disclosure. The peptide or polypeptide may have one or more
conservative or non-conservative substitutions. Substitutional
variants typically contain the exchange of one amino acid for
another at one or more sites within the protein, and may be
designed to modulate one or more properties of the polypeptide,
with or without the loss of other functions or properties.
Substitutions may be conservative, that is, one amino acid is
replaced with one of similar shape and charge. Conservative
substitutions are well known in the art and include, for example,
the changes of: alanine to serine; arginine to lysine; asparagine
to glutamine or histidine; aspartate to glutamate; cysteine to
serine; glutamine to asparagine; glutamate to aspartate; glycine to
proline; histidine to asparagine or glutamine; isoleucine to
leucine or valine; leucine to valine or isoleucine; lysine to
arginine; methionine to leucine or isoleucine; phenylalanine to
tyrosine, leucine or methionine; serine to threonine; threonine to
serine; tryptophan to tyrosine; tyrosine to tryptophan or
phenylalanine; and valine to isoleucine or leucine. Alternatively,
substitutions may be non-conservative such that a function or
activity of the polypeptide is affected. Non-conservative changes
typically involve substituting a residue with one that is
chemically dissimilar, such as a polar or charged amino acid for a
nonpolar or uncharged amino acid, and vice versa.
[0175] The polypeptides described herein may include 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more (or any derivable range
therein) variant amino acids within at least, or at most 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,
107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,
120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,
133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,
146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,
159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,
172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,
185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,
198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,
211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,
224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,
237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249,
250, 300, 400, 500, 550, 1000 or more contiguous amino acids, or
any range derivable therein, of a peptide or polypeptide of the
disclosure.
[0176] A polypeptide segment as described herein may include 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,
106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,
119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,
132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,
145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,
158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,
171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,
184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,
197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,
210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,
223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,
236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,
249, 250, 300, 400, 500, 550, 1000 or more contiguous amino acids,
or any range derivable therein of a peptide or polypeptide of the
disclosure.
[0177] The polypeptides described herein may be of a fixed length
of at least, at most, or exactly 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,
113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,
126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,
139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,
152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,
165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,
178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,
243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or
more amino acids (or any derivable range therein).
[0178] A linker sequence may be included in the compositions of the
disclosure. For example, a linker having at least, at most, or
exactly 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more
amino acids (or any derivable range therein) may separate two
polypeptide components in the compositions of the disclosure.
[0179] A. Polypeptides and Polypeptide Production
[0180] Embodiments involve lymphangiogenesis inducers and antigens
such as a bacterial antigen, a viral antigen, a fungal antigen, a
protozoal antigen, a helminth antigen or a cancer antigen for use
in various aspects described herein. For example, lymphangiogenesis
inducers and antigens such as a bacterial antigen, a viral antigen,
a fungal antigen, a protozoal antigen, a helminth antigen or a
cancer antigen are assayed for or used in methods of eliciting
immune responses, protective immune responses and inducing immune
tolerance. In specific embodiments, all or part of proteins
described herein can also be synthesized in solution or on a solid
support in accordance with conventional techniques. Various
automatic synthesizers are commercially available and can be used
in accordance with known protocols. See, for example, Stewart and
Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany
and Merrifield (1979), each incorporated herein by reference.
Alternatively, recombinant DNA technology may be employed wherein a
nucleotide sequence that encodes a peptide or polypeptide is
inserted into an expression vector, transformed or transfected into
an appropriate host cell and cultivated under conditions suitable
for expression.
[0181] One embodiment includes the use of gene transfer to cells,
including microorganisms, for the production and/or presentation of
proteins. The gene for the protein of interest may be transferred
into appropriate host cells followed by culture of cells under the
appropriate conditions. A nucleic acid encoding virtually any
polypeptide may be employed. The generation of recombinant
expression vectors, and the elements included therein, are
discussed herein. Alternatively, the protein to be produced may be
an endogenous protein normally synthesized by the cell used for
protein production.
[0182] In a certain aspects a lymphangiogenesis inducer comprises
substantially some or all of a lymphangiogenesis inducing protein
which has at least 85% identity, at least 90% identity, at least
95% identity, or at least 97-99% identity, including all values and
ranges there between, to a sequence selected over the length of the
fragment sequence. In yet other aspects, antigens such as a
bacterial antigen, a viral antigen, a fungal antigen, a protozoal
antigen, a helminth antigen or a cancer antigen comprises
substantially some or all of the antigenic portion a protein which
has at least 85% identity, at least 90% identity, at least 95%
identity, or at least 97-99% identity, including all values and
ranges there between, to a sequence selected over the length of the
fragment sequence.
[0183] B. Adjuvants
[0184] In other embodiments an immune adjuvant is included in a
composition comprising a lymphangiogenesis inducer and an antigen
such as a bacterial antigen, a viral antigen, a fungal antigen, a
protozoal antigen, a helminth antigen or a cancer antigen or is
directly fused or otherwise linked to a lymphangiogenesis inducer
or an antigen in order to enhance the efficacy of the
immunotherapeutic. In certain aspects the immune adjuvant may be a
toll-like receptor (TLR) agonist. TLR agonists comprise flagellins
from Salmonella enterica or Vibrio cholerae. In certain aspects the
adjuvant in Flagellin-1 or Flagellin-2. TLR agonists may be
specific for certain TLR classes (i.e., TLRS, TLR7 or TLR9
agonists) and may be presented in any combination or as any
modification. Examples of such immune adjuvants are described in WO
2012/021834, the contents of which are incorporated herein by
reference. Poly ICLC, a TLR3 ligand is also contemplated for use
with a lymphangiogenesis inducer and an antigen comprising
composition. In some embodiments the lymphangiogenesis inducer and
the antigen and Poly ICLC is delivered separately from the antibody
antigen fusion polypeptide. In one embodiment, the Poly ICLC is as
described in U.S. Pat. No. 7,439,349, the contents of which are
incorporated herein by reference. In one embodiment, the Poly ICLC
is Hiltonol.RTM.. Interleukins are also contemplated as adjuvants
that may be administered alongside, separately or fused to a
lymphangiogenesis inducer or an antigen. Non-limiting examples of
such interleukins are IL-21, IL-2, IL-9 and IL-10. In some
embodiments the interleukin proteins are human interleukins. In
certain aspects the adjuvant is an HLA-DR antigen-associated
invariant chain that augments antigen processing. In certain
aspects the adjuvant is interferon alpha. In yet other embodiments
the adjuvant is a toxin that will deliver a death signal to cells
also receiving an myelin sheath protein or component, thereby
augmenting immunotherapeutic efficiency. One example of such a
toxin is PE38. Any adjuvant may be delivered in fused or conjugated
form with a lymphangiogenesis inducer and an antigen or may be
delivered concomitantly as part of the same composition or
preparation without fusion or direct conjugation.
[0185] C. Tolerogenic Adjuvants
[0186] In certain embodiments the immune adjuvant may be a
tolerogenic adjuvant. In certain instances a tolerogenic adjuvant
may refer to an adjuvant that is utilized for tolerogenic
immunization, where the aim of immunization with an antigen is to
generate an immune response such that the antigen is tolerated by
an immunizaed subject. In certain aspects, the goal of a
tolerogenic adjuvant is to enhance tolerogenic immunization such
that tolerance to an antigen is further enhanced. In certain
embodiments a tolerogenic adjuvant is used to tolerize
autoimmunity. In yet other aspects, a tolerogenic adjuvant is used
to tolerize harmful autoimmunity. In some embodiments the
tolerogenic adjuvant is an immunosuppressant. In yet other
embodiments the tolerogenic adjuvant is dexamethasone, FK506
(Tacrolimus), cholera toxin B subunit, Escherichia coli heat-labile
enterotoxin B subunit, IFN-beta, glucocorticoids, vitamin D3, or
vitamin D3 analogues. In certain aspects the tolerogenic adjuvant
is administered concurrently with a lymphangiogenesis inducer and
an antigen as an immunotherapeutic. In other aspects a tolerogenic
adjuvant is administered before or after administration of a
lymphangiogenesis inducer and an antigen. In yet other embodiments
two or more tolerogenic adjuvants are administered concurrently,
before or after administration of a lymphangiogenesis inducer and
an antigen. In certain aspects, the tolerogenic adjuvant may be
fused, conjugated or otherwise linked to the lymphangiogenesis
inducer and antigen. In one embodiment, the tolerogenic adjuvant is
interleukin-10 (IL-10). In another embodiment IL-10 is
co-administered with the lymphangiogenesis inducer and antigen. In
certain aspects, IL-10 is fused by recombinant methods. In other
aspects IL-10 is conjugated. In other embodiments IL-10 is linked
by coupling or other modular domains.
[0187] D. VEGF Proteins
[0188] Human VEGF-C includes the following sequence:
TABLE-US-00002 (SEQ ID NO: 1)
MHLLGFFSVACSLLAAALLPGPREAPAAAAAFESGLDLSDAEPDAGEATA
YASKDLEEQLRSVSSVDELMTVLYPEYWKMYKCQLRKGGWQHNREQANLN
SRTEETIKFAAAHYNTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNT
FFKPPCVSVYRCGGCCNSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVT
ISFANHTSCRCMSKLDVYRQVHSIIRRSLPATLPQCQAANKTCPTNYMWN
NHICRCLAQEDFMFSSDAGDDSTDGFHDICGPNKELDEETCQCVCRAGLR
PASCGPHKELDRNSCQCVCKNKLFPSQCGANREFDENTCQCVCKRTCPRN
QPLNPGKCACECTESPQKCLLKGKKFHHQTCSCYRRPCTNRQKACEPGFS
YSEEVCRCVPSYWKRPQMS.
[0189] MHLLGFFSVACSLLAAALLPGPREAPAAAAA (SEQ ID NO:7) is the signal
peptide for protein secretion. It is usually removed and exchanged
to another leading sequence for protein expression. The N-terminal
propeptide:
FESGLDLSDAEPDAGEATAYASKDLEEQLRSVSSVDELMTVLYPEYWKMYKCQLRKG
GWQHNREQANLNSRTEETIKFAA (SEQ ID NO:8) has been reported to be
involved in the binding of the co-receptor Nrp-1 and enhanced the
binding to Nrp-2, which can act to modulate lymphangiogenic effect
(or kinetics) triggered by VEGF-C.
[0190] The C-terminal propeptides:
SLPATLPQCQAANKTCPTNYMWNNHICRCLAQEDFMFSSDAGDDSTDGFHDICGPNKE
LDEETCQCVCRAGLRPASCGPHKELDRNSCQCVCKNKLFPSQCGANREFDENTCQCVC
KRTCPRNQPLNPGKCACECTESPQKCLLKGKKFHHQTCSCYRRPCTNRQKACEPGFSYS
EEVCRCVPSYWKRPQMS (SEQ ID NO: 9),
CGPNKELDEETCQCVCRAGLRPASCGPHKELDRNSCQCVCKNKLFPSQCGANREFDEN
TCQCVCKRTCPRNQPLNPGKCACECTESPQKCLLKGKKFHHQTCSCYRRPCTNRQKAC
EPGFSYSEEVCRCVPSYWKRPQMS (SEQ ID NO:10), and
CGPNKELDEETCQCVCRAGLRPASCGPHKELDRNSCQCVCKNKLFPSQCGANREFDEN
TCQCVCKRTCPRNQPLNPGKCACEC (SEQ ID NO:11) contain repeats of 16
amino-acids repeats of C-X(10)-C-X-C-X(1-3)-C. These alternate
patterns of CXCXC were also found in other angiogenic proteins. It
is contemplated that variants of VEGF-C may comprise only
conservative mutations in the C-X(10)-C-X-C-X(1-3)-C region. It is
also contemplated that non-conservative mutations may be tolerated
outside of this region and retain the functional activities of this
protein.
[0191] The mature VEGF-C human sequence comprises:
AHYNTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNSEG
LQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLDVYRQVHSIIRR (SEQ ID
NO:12). In some embodiments, the VEGF-C sequence comprises:
TABLE-US-00003 (SEQ ID NO: 13)
AHYNTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYR
CGGCCNSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRC MSKLDVYRQ.
[0192] Human VEGF-D sequence is included as SEQ ID NO:3. This
protein comprises a conserved region of repeats of 16 amino-acids
repeats of C-X(10)-C-X-C-X(1-3)-C that are found in other
angiogenic proteins:
CPIDMLWDSNKCKCVLQEENPLAGTEDHSHLQEPALCGPHMMFDEDRCECVCKTPCPK
DLIQHPKNCSCFECKESLETCCQKHKLFHPDTCSCEDRC (SEQ ID NO:14). It is
contemplated that variants of VEGF-C may comprise only conservative
mutations in the C-X(10)-C-X-C-X(1-3)-C region. It is also
contemplated that non-conservative mutations may be tolerated
outside of this region and retain the functional activities of this
protein.
[0193] The mature VEGF-C human sequence comprises:
TABLE-US-00004 (SEQ ID NO: 15)
FAATFYDIETLKVIDEEWQRTQCSPRETCVEVASELGKSTNTFFKPPCVN
VFRCGGCCNEESLICMNTSTSYISKQLFEISVPLTSVPELVPVKVANHTG
CKCLPTAPRHPYSIIRR.
[0194] E. CCL21
[0195] Mature human CCL21 has the sequence of SEQ ID NO:5. The
signal peptide: MAQSLALSLLILVLAFGIPRTQG (SEQ ID NO:16) may be
removed and still have an active protein. Therefore, in some
embodiments, the CCL21 polypeptide comprises:
SDGGAQDCCLKYSQRKIPAKVVRSYRKQEPSLGCSIPAILFLPRKRSQAELCADPKELWV
QQLMQHLDKTPSPQKPAQGCRKDRGASKTGKKGKGSKGCKRTERSQTPKGP (SEQ ID NO:17).
It is also contemplated that the active form includes the
ECM-binding domain of CCL21, which is underlined in SEQ ID NO:5:
MAQSLALSLLILVLAFGIPRTQGSDGGAQDCCLKYSQRKIPAKVVRSYRKQEPSLGCSIP
AILFLPRKRSQAELCADPKELWVQQLMQHLDKTPSPQKPAQGCrkdrgasktgkkgkgsKGCK
RTERSQTPKGP. However, the lower case amino acid residues may be
unimportant for CCL21 active and can be replaced with additional
sequences such as a spacer, an additional ECM-binding domain, or
any other functional or non-functional domain, or removed
altogether.
[0196] Examples of CCL21 polypeptides are provided as SEQ ID NO: 18
and 19.
TABLE-US-00005 (SEQ ID NO: 18)
MAQSLALSLLILVLAFGIPRTQGSDGGAQDCCLKYSQRKIPAKVVRSYRK
QEPSLGCSIPAILFLPRKRSQAELCADPKELWVQQLMQHLDKTPSPQKPA
QGCRRRPKGRGKRRREKQRKGCKRTERSQTPKGP. (SEQ ID NO: 19)
MAQSLALSLLILVLAFGIPRTQGSDGGAQDCCLKYSQRKIPAKVVRSYRK
QEPSLGCSIPAILFLPRKRSQAELCADPKELWVQQLMQHLDKTPSPQKPA
QGCRKDRGASKTGKKGKGSKGCKRTERSQTPKGPRRRPKGRGKRRREKQR PTDAHL.
[0197] The mature mouse CCL21 sequence comprises SEQ ID NO:6 or SEQ
ID NO: 20:
TABLE-US-00006 (SEQ ID NO: 20)
MAQMMTLSLLSLVLALCIPWTQGSDGGGQDCCLKYSQKKIPYSIVRGYRK
QEPSLGCPIPAILFLPRKHSKPELCANPEEGWVQNLMRRLDQPPAPGKQS
PGCRKNRGTSKSGKKGKGSKGCKRTEQTQPSRG.
VII. Lipids
[0198] In certain aspects, there may be provided methods and
compositions for associating or encapsulating a lymphangiogenesis
inducer, an antigen or both with a lipid and/or liposome. The
lymphangiogenesis inducer, an antigen or both may be encapsulated
in the aqueous interior of a liposome, interspersed within the
lipid bilayer of a liposome, attached to a liposome via a linking
molecule that is associated with both the liposome and the
polynucleotide, entrapped in a liposome, complexed with a liposome,
dispersed in a solution containing a lipid, mixed with a lipid,
combined with a lipid, contained as a suspension in a lipid,
contained or complexed with a micelle, or otherwise associated with
a lipid. The liposome or liposome/lymphangiogenesis inducer, an
antigen or both lymphangiogenesis inducer and antigen-associated
compositions are not limited to any particular structure in
solution. For example, they may be present in a bilayer structure,
as micelles, or with a "collapsed" structure. They may also simply
be interspersed in a solution, possibly forming aggregates which
are not uniform in either size or shape.
[0199] Lipids are fatty substances which may be naturally occurring
or synthetic lipids. For example, lipids include the fatty droplets
that naturally occur in the cytoplasm as well as the class of
compounds which are well known to those of skill in the art which
contain long-chain aliphatic hydrocarbons and their derivatives,
such as fatty acids, alcohols, amines, amino alcohols, and
aldehydes. An example is the lipid dioleoylphosphatidylcholine
(DOPC).
[0200] "Liposome" is a generic term encompassing a variety of
unilamellar, multilamellar, and multivesicular lipid vehicles
formed by the generation of enclosed lipid bilayers or aggregates.
Liposomes may be characterized as having vesicular structures with
a phospholipid bilayer membrane and an inner aqueous medium.
Multilamellar liposomes have multiple lipid layers separated by
aqueous medium. They form spontaneously when phospholipids are
suspended in an excess of aqueous solution. The lipid components
undergo self-rearrangement before the formation of closed
structures and entrap water and dissolved solutes between the lipid
bilayers (Ghosh and Bachhawat, 1991). However, certain embodiments
also encompass compositions that have different structures in
solution than the normal vesicular structure. For example, the
lipids may assume a micellar structure or merely exist as
non-uniform aggregates of lipid molecules. Also contemplated are
lipofectamine-nucleic acid complexes.
[0201] In certain embodiments, the lipid may be associated with a
hemaglutinating virus (HVJ). This has been shown to facilitate
fusion with the cell membrane and promote cell entry of
liposome-encapsulated DNA (Kaneda et al., 1989). In other
embodiments, the lipid may be complexed or employed in conjunction
with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al.,
1991). In yet further embodiments, the lipid may be complexed or
employed in conjunction with both HVJ and HMG-1. In that such
expression vectors have been successfully employed in transfer of a
polynucleotide in vitro and in vivo, then they are applicable.
[0202] Exemplary lipids include, but are not limited to,
dioleoylphosphatidylycholine ("DOPC"), egg phosphatidylcholine
("EPC"), dilauryloylphosphatidylcholine ("DLPC"),
dimyristoylphosphatidylcholine ("DMPC"),
dipalmitoylphosphatidylcholine ("DPPC"),
distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoyl
phosphatidylcholine ("MPPC"), 1-palmitoyl-2-myristoyl
phosphatidylcholine ("PMPC"), 1-palmitoyl-2-stearoyl
phosphatidylcholine ("PSPC"), 1-stearoyl-2-palmitoyl
phosphatidylcholine ("SPPC"), dilauryloylphosphatidylglycerol
("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"),
dipalmitoylphosphatidylglycerol ("DPPG"),
distearoylphosphatidylglycerol ("DSPG"), distearoyl sphingomyelin
("DSSP"), distearoylphophatidylethanolamine ("DSPE"),
dioleoylphosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic
acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), dimyristoyl
phosphatidylethanolamine ("DMPE"), dipalmitoyl
phosphatidylethanolamine ("DPPE"), dimyristoyl phosphatidylserine
("DMPS"), dipalmitoyl phosphatidylserine ("DPPS"), brain
phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"),
dipalmitoyl sphingomyelin ("DPSP"), dimyristyl phosphatidylcholine
("DMPC"), 1,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"),
1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"),
1,2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"),
dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloeoyl
phosphatidylcholine ("POPC"), palmitoyloeoyl
phosphatidylethanolamine ("POPE"), lysophosphatidylcholine,
lysophosphatidylethanolamine, dilinoleoylphosphatidylcholine,
phosphatidylcholines, phosphatidylglycerols,
phosphatidylethanolamines, cholesterol.
[0203] Liposomes and lipid compositions can be made by different
methods. For example, a nucleotide (e.g., siRNA) may be
encapsulated in a neutral liposome using a method involving ethanol
and calcium (Bailey and Sullivan, 2000). The size of the liposomes
varies depending on the method of synthesis. A liposome suspended
in an aqueous solution is generally in the shape of a spherical
vesicle, and may have one or more concentric layers of lipid
bilayer molecules. Each layer consists of a parallel array of
molecules represented by the formula XY, wherein X is a hydrophilic
moiety and Y is a hydrophobic moiety. In aqueous suspension, the
concentric layers are arranged such that the hydrophilic moieties
tend to remain in contact with an aqueous phase and the hydrophobic
regions tend to self-associate. For example, when aqueous phases
are present both within and without the liposome, the lipid
molecules may form a bilayer, known as a lamella, of the
arrangement XY-YX. Aggregates of lipids may form when the
hydrophilic and hydrophobic parts of more than one lipid molecule
become associated with each other. The size and shape of these
aggregates will depend upon many different variables, such as the
nature of the solvent and the presence of other compounds in the
solution.
[0204] Lipids suitable for use can be obtained from commercial
sources. For example, dimyristyl phosphatidylcholine ("DMPC") can
be obtained from Sigma Chemical Co., dicetyl phosphate ("DCP") can
be obtained from K & K Laboratories (Plainview, N.Y.);
cholesterol ("Chol") can be obtained from Calbiochem-Behring;
dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be
obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock
solutions of lipids in chloroform or chloroform/methanol can be
stored at about -20.degree. C. Chloroform may be used as the only
solvent since it is more readily evaporated than methanol.
[0205] Liposomes can be prepared in accordance with known
laboratory techniques. In certain embodiments, liposomes are
prepared by mixing liposomal lipids, in a solvent in a container
(e.g., a glass, pear-shaped flask). The container may have a volume
ten-times greater than the volume of the expected suspension of
liposomes. Using a rotary evaporator, the solvent may be removed at
approximately 40.degree. C. under negative pressure. The solvent
may be removed within about 5 minutes to 2 hours, depending on the
desired volume of the liposomes. The composition can be dried
further in a desiccator under vacuum. Dried lipids can be hydrated
at approximately 25-50 mM phospholipid in sterile, pyrogen-free
water by shaking until all the lipid film is resuspended. The
aqueous liposomes can be separated into aliquots, each placed in a
vial, lyophilized and sealed under vacuum.
[0206] Liposomes can also be prepared in accordance with other
known laboratory procedures: the method of Bangham et al. (1965),
the contents of which are incorporated herein by reference; the
method of Gregoriadis (1979), the contents of which are
incorporated herein by reference; the method of Deamer and Uster
(1983), the contents of which are incorporated by reference; and
the reverse-phase evaporation method as described by Szoka and
Papahadjopoulos (1978). The aforementioned methods differ in their
respective abilities to entrap aqueous material and their
respective aqueous space-to-lipid ratios.
VIII. Nucleic Acid Assays
[0207] Aspects of the methods include assaying nucleic acids to
determine expression or activity levels. Arrays can be used to
detect differences between two samples. Specifically contemplated
applications include identifying and/or quantifying differences
between RNA from a sample that is normal and from a sample that is
not normal, between a cancerous condition and a non-cancerous
condition, or between two differently treated samples. Also, RNA
may be compared between a sample believed to be susceptible to a
particular disease or condition and one believed to be not
susceptible or resistant to that disease or condition. A sample
that is not normal is one exhibiting phenotypic trait(s) of a
disease or condition or one believed to be not normal with respect
to that disease or condition. It may be compared to a cell that is
normal with respect to that disease or condition. Phenotypic traits
include symptoms of, or susceptibility to, a disease or condition
of which a component is or may or may not be genetic or caused by a
hyperproliferative or neoplastic cell or cells.
[0208] An array comprises a solid support with nucleic acid probes
attached to the support. Arrays typically comprise a plurality of
different nucleic acid probes that are coupled to a surface of a
substrate in different, known locations. These arrays, also
described as "microarrays" or colloquially "chips" have been
generally described in the art, for example, U.S. Pat. Nos.
5,143,854, 5,445,934, 5,744,305, 5,677,195, 6,040,193, 5,424,186
and Fodor et al., 1991), each of which is incorporated by reference
in its entirety for all purposes. Techniques for the synthesis of
these arrays using mechanical synthesis methods are described in,
e.g., U.S. Pat. No. 5,384,261, incorporated herein by reference in
its entirety for all purposes. Although a planar array surface is
used in certain aspects, the array may be fabricated on a surface
of virtually any shape or even a multiplicity of surfaces. Arrays
may be nucleic acids on beads, gels, polymeric surfaces, fibers
such as fiber optics, glass or any other appropriate substrate, see
U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and
5,800,992, which are hereby incorporated in their entirety for all
purposes.
[0209] In addition to the use of arrays and microarrays, it is
contemplated that a number of difference assays could be employed
to analyze nucleic acids, their activities, and their effects. Such
assays include, but are not limited to, nucleic amplification,
polymerase chain reaction, quantitative PCR, RT-PCR, in situ
hybridization, Northern hybridization, hybridization protection
assay (HPA) (GenProbe), branched DNA (bDNA) assay (Chiron), rolling
circle amplification (RCA), single molecule hybridization detection
(US Genomics), Invader assay (ThirdWave Technologies), and/or
Bridge Litigation Assay (Genaco).
[0210] A further assay useful for quantifying and/or identifying
nucleic acids is RNAseq. RNA-seq (RNA sequencing), also called
whole transcriptome shotgun sequencing, uses next-generation
sequencing (NGS) to reveal the presence and quantity of RNA in a
biological sample at a given moment in time. RNA-Seq is used to
analyze the continually changing cellular transcriptome.
Specifically, RNA-Seq facilitates the ability to look at
alternative gene spliced transcripts, post-transcriptional
modifications, gene fusion, mutations/SNPs and changes in gene
expression. In addition to mRNA transcripts, RNA-Seq can look at
different populations of RNA to include total RNA, small RNA, such
as miRNA, tRNA, and ribosomal profiling. RNA-Seq can also be used
to determine exon/intron boundaries and verify or amend previously
annotated 5' and 3' gene boundaries.
IX. Protein Assays
[0211] A variety of techniques can be employed to measure
expression levels of polypeptides and proteins in a biological
sample. Examples of such formats include, but are not limited to,
enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot
analysis and enzyme linked immunoabsorbant assay (ELISA). A skilled
artisan can readily adapt known protein/antibody detection methods
for use in determining protein expression levels of biomarkers.
[0212] In one embodiment, antibodies, or antibody fragments or
derivatives, can be used in methods such as Western blots or
immunofluorescence techniques to detect biomarker expression. In
some embodiments, either the antibodies or proteins are immobilized
on a solid support. Suitable solid phase supports or carriers
include any support capable of binding an antigen or an antibody.
Well-known supports or carriers include glass, polystyrene,
polypropylene, polyethylene, dextran, nylon, amylases, natural and
modified celluloses, polyacrylamides, gabbros, and magnetite.
[0213] One skilled in the art will know many other suitable
carriers for binding antibody or antigen, and will be able to adapt
such support for use with the present disclosure. The support can
then be washed with suitable buffers followed by treatment with the
detectably labeled antibody. The solid phase support can then be
washed with the buffer a second time to remove unbound antibody.
The amount of bound label on the solid support can then be detected
by conventional means.
[0214] Immunohistochemistry methods are also suitable for detecting
the expression levels of biomarkers. In some embodiments,
antibodies or antisera, including polyclonal antisera, and
monoclonal antibodies specific for each marker may be used to
detect expression. The antibodies can be detected by direct
labeling of the antibodies themselves, for example, with
radioactive labels, fluorescent labels, hapten labels such as,
biotin, or an enzyme such as horse radish peroxidase or alkaline
phosphatase. Alternatively, unlabeled primary antibody is used in
conjunction with a labeled secondary antibody, comprising antisera,
polyclonal antisera or a monoclonal antibody specific for the
primary antibody. Immunohistochemistry protocols and kits are well
known in the art and are commercially available.
[0215] Immunological methods for detecting and measuring complex
formation as a measure of protein expression using either specific
polyclonal or monoclonal antibodies are known in the art. Examples
of such techniques include enzyme-linked immunosorbent assays
(ELISAs), radioimmunoassays (RIAs), fluorescence-activated cell
sorting (FACS) and antibody arrays. Such immunoassays typically
involve the measurement of complex formation between the protein
and its specific antibody. These assays and their quantitation
against purified, labeled standards are well known in the art. A
two-site, monoclonal-based immunoassay utilizing antibodies
reactive to two non-interfering epitopes or a competitive binding
assay may be employed.
[0216] Numerous labels are available and commonly known in the art.
Radioisotope labels include, for example, .sup.36S, .sup.14C,
.sup.125I, .sup.3H, and .sup.131I. The antibody can be labeled with
the radioisotope using the techniques known in the art. Fluorescent
labels include, for example, labels such as rare earth chelates
(europium chelates) or fluorescein and its derivatives, rhodamine
and its derivatives, dansyl, Lissamine, phycoerythrin and Texas Red
are available. The fluorescent labels can be conjugated to the
antibody variant using the techniques known in the art.
Fluorescence can be quantified using a fluorimeter. Various
enzyme-substrate labels are available and U.S. Pat. Nos. 4,275,149,
4,318,980 provides a review of some of these. The enzyme generally
catalyzes a chemical alteration of the chromogenic substrate which
can be measured using various techniques. For example, the enzyme
may catalyze a color change in a substrate, which can be measured
spectrophotometrically. Alternatively, the enzyme may alter the
fluorescence or chemiluminescence of the substrate. Techniques for
quantifying a change in fluorescence are described above. The
chemiluminescent substrate becomes electronically excited by a
chemical reaction and may then emit light which can be measured
(using a chemiluminometer, for example) or donates energy to a
fluorescent acceptor. Examples of enzymatic labels include
luciferases (e.g., firefly luciferase and bacterial luciferase;
U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,
malate dehydrogenase, urease, peroxidase such as horseradish
peroxidase (HRPO), alkaline phosphatase, .beta.-galactosidase,
glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase,
galactose oxidase, and glucose-6-phosphate dehydrogenase),
heterocyclic oxidases (such as uricase and xanthine oxidase),
lactoperoxidase, microperoxidase, and the like. Techniques for
conjugating enzymes to antibodies are described in O'Sullivan et
al., Methods for the Preparation of Enzyme-Antibody Conjugates for
Use in Enzyme Immunoassay, in Methods in Enzymology (Ed. J. Langone
& H. Van Vunakis), Academic press, New York, 73: 147-166
(1981).
[0217] In some embodiments, a detection label is indirectly
conjugated with an antibody. The skilled artisan will be aware of
various techniques for achieving this. For example, the antibody
can be conjugated with biotin and any of the three broad categories
of labels mentioned above can be conjugated with avidin, or vice
versa. Biotin binds selectively to avidin and thus, the label can
be conjugated with the antibody in this indirect manner.
Alternatively, to achieve indirect conjugation of the label with
the antibody, the antibody is conjugated with a small hapten (e.g.,
digoxin) and one of the different types of labels mentioned above
is conjugated with an anti-hapten antibody (e.g., anti-digoxin
antibody). In some embodiments, the antibody need not be labeled,
and the presence thereof can be detected using a labeled antibody,
which binds to the antibody.
X. Sample Preparation
[0218] In certain aspects, methods involve obtaining a sample from
a subject. The methods of obtaining provided herein may include
methods of biopsy such as fine needle aspiration, core needle
biopsy, vacuum assisted biopsy, incisional biopsy, excisional
biopsy, punch biopsy, shave biopsy or skin biopsy. In certain
embodiments the sample is obtained from a biopsy from colorectal
tissue by any of the biopsy methods previously mentioned. In other
embodiments the sample may be obtained from any of the tissues
provided herein that include but are not limited to non-cancerous
or cancerous tissue and non-cancerous or cancerous tissue from the
serum, gall bladder, mucosal, skin, heart, lung, breast, pancreas,
blood, liver, muscle, kidney, smooth muscle, bladder, colon,
intestine, brain, prostate, esophagus, or thyroid tissue.
Alternatively, the sample may be obtained from any other source
including but not limited to blood, sweat, hair follicle, buccal
tissue, tears, menses, feces, or saliva. In certain aspects the
sample is obtained from cystic fluid or fluid derived from a tumor
or neoplasm. In yet other embodiments the cyst, tumor or neoplasm
is colorectal. In certain aspects of the current methods, any
medical professional such as a doctor, nurse or medical technician
may obtain a biological sample for testing. Yet further, the
biological sample can be obtained without the assistance of a
medical professional.
[0219] A sample may include but is not limited to, tissue, cells,
or biological material from cells or derived from cells of a
subject. The biological sample may be a heterogeneous or
homogeneous population of cells or tissues. The biological sample
may be obtained using any method known to the art that can provide
a sample suitable for the analytical methods described herein. The
sample may be obtained by non-invasive methods including but not
limited to: scraping of the skin or cervix, swabbing of the cheek,
saliva collection, urine collection, feces collection, collection
of menses, tears, or semen.
[0220] The sample may be obtained by methods known in the art. In
certain embodiments the samples are obtained by biopsy. In other
embodiments the sample is obtained by swabbing, scraping,
phlebotomy, or any other methods known in the art. In some cases,
the sample may be obtained, stored, or transported using components
of a kit of the present methods. In some cases, multiple samples,
such as multiple colorectal samples may be obtained for diagnosis
by the methods described herein. In other cases, multiple samples,
such as one or more samples from one tissue type (for example
colon) and one or more samples from another tissue (for example
buccal) may be obtained for diagnosis by the methods. In some
cases, multiple samples such as one or more samples from one tissue
type (e.g. rectal) and one or more samples from another tissue
(e.g. cecum) may be obtained at the same or different times.
Samples may be obtained at different times are stored and/or
analyzed by different methods. For example, a sample may be
obtained and analyzed by routine staining methods or any other
cytological analysis methods.
[0221] In some embodiments the biological sample may be obtained by
a physician, nurse, or other medical professional such as a medical
technician, endocrinologist, cytologist, phlebotomist, radiologist,
or a pulmonologist. The medical professional may indicate the
appropriate test or assay to perform on the sample. In certain
aspects a molecular profiling business may consult on which assays
or tests are most appropriately indicated. In further aspects of
the current methods, the patient or subject may obtain a biological
sample for testing without the assistance of a medical
professional, such as obtaining a whole blood sample, a urine
sample, a fecal sample, a buccal sample, or a saliva sample.
[0222] In other cases, the sample is obtained by an invasive
procedure including but not limited to: biopsy, needle aspiration,
or phlebotomy. The method of needle aspiration may further include
fine needle aspiration, core needle biopsy, vacuum assisted biopsy,
or large core biopsy. In some embodiments, multiple samples may be
obtained by the methods herein to ensure a sufficient amount of
biological material.
[0223] General methods for obtaining biological samples are also
known in the art. Publications such as Ramzy, Ibrahim Clinical
Cytopathology and Aspiration Biopsy 2001, which is herein
incorporated by reference in its entirety, describes general
methods for biopsy and cytological methods. In one embodiment, the
sample is a fine needle aspirate of a colorectal or a suspected
colorectal tumor or neoplasm. In some cases, the fine needle
aspirate sampling procedure may be guided by the use of an
ultrasound, X-ray, or other imaging device.
[0224] In some embodiments of the present methods, the molecular
profiling business may obtain the biological sample from a subject
directly, from a medical professional, from a third party, or from
a kit provided by a molecular profiling business or a third party.
In some cases, the biological sample may be obtained by the
molecular profiling business after the subject, a medical
professional, or a third party acquires and sends the biological
sample to the molecular profiling business. In some cases, the
molecular profiling business may provide suitable containers, and
excipients for storage and transport of the biological sample to
the molecular profiling business.
[0225] In some embodiments of the methods described herein, a
medical professional need not be involved in the initial diagnosis
or sample acquisition. An individual may alternatively obtain a
sample through the use of an over the counter (OTC) kit. An OTC kit
may contain a means for obtaining said sample as described herein,
a means for storing said sample for inspection, and instructions
for proper use of the kit. In some cases, molecular profiling
services are included in the price for purchase of the kit. In
other cases, the molecular profiling services are billed
separately. A sample suitable for use by the molecular profiling
business may be any material containing tissues, cells, nucleic
acids, proteins, polypeptides, genes, gene fragments, expression
products, gene expression products, protein expression products or
fragments, or gene expression product fragments of an individual to
be tested. Methods for determining sample suitability and/or
adequacy are provided.
[0226] In some embodiments, the subject may be referred to a
specialist such as an oncologist, surgeon, or endocrinologist. The
specialist may likewise obtain a biological sample for testing or
refer the individual to a testing center or laboratory for
submission of the biological sample. In some cases the medical
professional may refer the subject to a testing center or
laboratory for submission of the biological sample. In other cases,
the subject may provide the sample. In some cases, a molecular
profiling business may obtain the sample.
XI. Methods of Treatment
[0227] As discussed above, the compositions and methods of using
these compositions can treat a subject (e.g., prevent or treat a
bacterial infection, a viral infection, a fungal infection, a
protozoal infection, a helminth infection or a cancer condition or
evoke a robust immune tolerance to an autoimmune disease) having,
suspected of having, or at risk of developing an infection, cancer
or an autoimmune disorder or related disease.
[0228] As used herein the phrase "immune response" or its
equivalent "immunological response" refers to a humoral (antibody
mediated), cellular (mediated by antigen-specific T cells or their
secretion products) or both humoral and cellular response directed
against a protein, peptide, or polypeptide of the embodiments in a
recipient patient. Treatment or therapy can be an active immune
response induced by administration of immunogen or a passive
therapy effected by administration of antibody, antibody containing
material, or primed T-cells.
[0229] For purposes of this specification and the accompanying
claims the terms "epitope" and "antigenic determinant" are used
interchangeably to refer to a site on an antigen to which B and/or
T cells respond or recognize. B-cell epitopes can be formed both
from contiguous amino acids or noncontiguous amino acids juxtaposed
by tertiary folding of a protein. Epitopes formed from contiguous
amino acids are typically retained on exposure to denaturing
solvents whereas epitopes formed by tertiary folding are typically
lost on treatment with denaturing solvents. An epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino
acids in a unique spatial conformation. Methods of determining
spatial conformation of epitopes include those methods described in
Epitope Mapping Protocols (1996). T cells recognize continuous
epitopes of about nine amino acids for CD8 cells or about 13-15
amino acids for CD4 cells. T cells that recognize the epitope can
be identified by in vitro assays that measure antigen-dependent
proliferation, as determined by 3H-thymidine incorporation by
primed T cells in response to an epitope (Burke et al., 1994), by
antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et
al., 1996) or by cytokine secretion.
[0230] The presence of a cell-mediated immunological response can
be determined by proliferation assays (CD4 (+) T cells) or CTL
(cytotoxic T lymphocyte) assays. The relative contributions of
humoral and cellular responses to the protective or therapeutic
effect of an immunogen can be distinguished by separately isolating
IgG and T-cells from an immunized syngeneic animal and measuring
protective or therapeutic effect in a second subject. As used
herein and in the claims, the terms "antibody" or "immunoglobulin"
are used interchangeably.
[0231] Optionally, an antibody or preferably an immunological
portion of an antibody, can be chemically conjugated to, or
expressed as, a fusion protein with other proteins. For purposes of
this specification and the accompanying claims, all such fused
proteins are included in the definition of antibodies or an
immunological portion of an antibody.
[0232] In some embodiments, a method includes treatment for a
disease or condition caused by a bacterial infection, a viral
infection, a fungal infection, a protozoal infection, a helminth
infection or a cancer condition. Furthermore, in some examples,
treatment comprises administration of other agents commonly used
against a bacterial infection, a viral infection, a fungal
infection, a protozoal infection, a helminth infection or a cancer
condition.
[0233] In one embodiment a method includes treatment for a disease
or condition caused by an autoimmune disorder. Furthermore, in some
examples, treatment comprises administration of other agents
commonly used against autoimmune disorders, such as one or more
immunosuppressant compounds.
[0234] The therapeutic compositions are administered in a manner
compatible with the dosage formulation, and in such amount as will
be therapeutically effective. The quantity to be administered
depends on the subject to be treated. Precise amounts of active
ingredient required to be administered depend on the judgment of
the practitioner. Suitable regimes for initial administration and
boosters are also variable, but are typified by an initial
administration followed by subsequent administrations.
[0235] Compositions of the current methods may be administered to
patients via any route used to introduce vaccines or antibodies to
patients. Such routes include, but are not limited to, mucosal or
intramuscular delivery. In particular embodiments, a composition is
administered to a patient intranasally or by inhalation. In other
embodiments, a composition is administered intravenously or by
intravenous injection. In additional embodiments, the
administration of compositions includes, but is not limited to
oral, parenteral, subcutaneous, intramuscular, intravenous
administration, or various combinations thereof.
[0236] The manner of application may be varied widely. Any of the
conventional methods for administration of a polypeptide
therapeutic are applicable. These are believed to include oral
application on a solid physiologically acceptable base or in a
physiologically acceptable dispersion, parenterally, by injection
and the like. The dosage of the composition will depend on the
route of administration and will vary according to the size and
health of the subject. In one treatment scheme, the patient
receives a subcutaneous dose of the lymphangiogenesis inducer or
inducers and antigens such as a bacterial antigen, a viral antigen,
a fungal antigen, a protozoal antigen, a helminth antigen or a
cancer antigen, together in a single formulation or composition or
separately in multiple formulations or compositions, every week for
three weeks and then every first week for an additional 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, or 12 months. In other aspects the patient
or subject receives two injections spaced a minimum or 7 days apart
within 1-2 months and then every week for an additional 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, or 12 months or every first week of the
month for an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12
months.
[0237] In certain instances, it will be desirable to have multiple
administrations of the composition, e.g., 2, 3, 4, 5, 6 or more
administrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7,
8, to 5, 6, 7, 8, 9, 10, 11, 12 twelve week intervals, including
all ranges there between.
[0238] "Tumor," as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues. The terms "cancer,"
"cancerous," "cell proliferative disorder," "proliferative
disorder," and "tumor" are not mutually exclusive as referred to
herein.
[0239] The cancers amenable for treatment include, but are not
limited to, tumors of all types, locations, sizes, and
characteristics. The methods and compositions of the disclosure are
suitable for treating, for example, pancreatic cancer, colon
cancer, acute myeloid leukemia, adrenocortical carcinoma,
AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix
cancer, astrocytoma, childhood cerebellar or cerebral basal cell
carcinoma, bile duct cancer, extrahepatic bladder cancer, bone
cancer, osteosarcoma/malignant fibrous histiocytoma, brainstem
glioma, brain tumor, cerebellar astrocytoma brain tumor, cerebral
astrocytoma/malignant glioma brain tumor, ependymoma brain tumor,
medulloblastoma brain tumor, supratentorial primitive
neuroectodermal tumors brain tumor, visual pathway and hypothalamic
glioma, breast cancer, lymphoid cancer, bronchial
adenomas/carcinoids, tracheal cancer, Burkitt lymphoma, carcinoid
tumor, childhood carcinoid tumor, gastrointestinal carcinoma of
unknown primary, central nervous system lymphoma, primary
cerebellar astrocytoma, childhood cerebral astrocytoma/malignant
glioma, childhood cervical cancer, childhood cancers, chronic
lymphocytic leukemia, chronic myelogenous leukemia, chronic
myeloproliferative disorders, cutaneous T-cell lymphoma,
desmoplastic small round cell tumor, endometrial cancer,
ependymoma, esophageal cancer, Ewing's, childhood extragonadal Germ
cell tumor, extrahepatic bile duct cancer, eye Cancer, intraocular
melanoma eye Cancer, retinoblastoma, gallbladder cancer, gastric
(stomach) cancer, gastrointestinal carcinoid tumor,
gastrointestinal stromal tumor (GIST), germ cell tumor:
extracranial, extragonadal, or ovarian, gestational trophoblastic
tumor, glioma of the brain stem, glioma, childhood cerebral
astrocytoma, childhood visual pathway and hypothalamic glioma,
gastric carcinoid, hairy cell leukemia, head and neck cancer, heart
cancer, hepatocellular (liver) cancer, Hodgkin lymphoma,
hypopharyngeal cancer, hypothalamic and visual pathway glioma,
childhood intraocular melanoma, islet cell carcinoma (endocrine
pancreas), kaposi sarcoma, kidney cancer (renal cell cancer),
laryngeal cancer, leukemia, acute lymphoblastic (also called acute
lymphocytic leukemia) leukemia, acute myeloid (also called acute
myelogenous leukemia) leukemia, chronic lymphocytic (also called
chronic lymphocytic leukemia) leukemia, chronic myelogenous (also
called chronic myeloid leukemia) leukemia, hairy cell lip and oral
cavity cancer, liposarcoma, liver cancer (primary), non-small cell
lung cancer, small cell lung cancer, lymphomas, AIDS-related
lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin
lymphoma, Non-Hodgkin (an old classification of all lymphomas
except Hodgkin's) lymphoma, primary central nervous system
lymphoma, Waldenstrom macroglobulinemia, malignant fibrous
histiocytoma of bone/osteosarcoma, childhood medulloblastoma,
melanoma, intraocular (eye) melanoma, merkel cell carcinoma, adult
malignant mesothelioma, childhood mesothelioma, metastatic squamous
neck cancer, mouth cancer, multiple endocrine neoplasia syndrome,
multiple myeloma/plasma cell neoplasm, mycosis fungoides,
myelodysplastic syndromes, myelodysplastic/myeloproliferative
diseases, chronic myelogenous leukemia, adult acute myeloid
leukemia, childhood acute myeloid leukemia, multiple myeloma,
chronic myeloproliferative disorders, nasal cavity and paranasal
sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer,
oropharyngeal cancer, osteosarcoma/malignant, fibrous histiocytoma
of bone, ovarian cancer, ovarian epithelial cancer (surface
epithelial-stromal tumor), ovarian germ cell tumor, ovarian low
malignant potential tumor, pancreatic cancer, islet cell paranasal
sinus and nasal cavity cancer, parathyroid cancer, penile cancer,
pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal
germinoma, pineoblastoma and supratentorial primitive
neuroectodermal tumors, childhood pituitary adenoma, plasma cell
neoplasia/multiple myeloma, pleuropulmonary blastoma, primary
central nervous system lymphoma, prostate cancer, rectal cancer,
renal cell carcinoma (kidney cancer), renal pelvis and ureter
transitional cell cancer, retinoblastoma, rhabdomyosarcoma,
childhood Salivary gland cancer Sarcoma, Ewing family of tumors,
Kaposi sarcoma, soft tissue sarcoma, uterine sezary syndrome
sarcoma, skin cancer (nonmelanoma), skin cancer (melanoma), skin
carcinoma, Merkel cell small cell lung cancer, small intestine
cancer, soft tissue sarcoma, squamous cell carcinoma. squamous neck
cancer with occult primary, metastatic stomach cancer,
supratentorial primitive neuroectodermal tumor, childhood T-cell
lymphoma, testicular cancer, throat cancer, thymoma, childhood
thymoma, thymic carcinoma, thyroid cancer, urethral cancer, uterine
cancer, endometrial uterine sarcoma, vaginal cancer, visual pathway
and hypothalamic glioma, childhood vulvar cancer, and wilms tumor
(kidney cancer).
[0240] A. Combination Therapy
[0241] The compositions and related methods, particularly
administration of lymphangiogenesis inducer or inducers and
antigens such as a bacterial antigen, a viral antigen, a fungal
antigen, a protozoal antigen, a helminth antigen or a cancer
antigen, may also be used in combination with the administration of
antibacterial, antiviral, antifungal, antibiotic, antineoplastic or
chemotherapeutic agent effective strategies or traditional
immunomodulatory therapies. In specific aspects, administration of
lymphangiogenesis inducer or inducers and antigens is provided in
combination with programmed cell death protein-1 (PD-1) pathway
inhibitors. In certain aspects, combination therapy may target PD-1
or the PD-L1 or PD-L2 ligands. Such strategies or therapies may be
directed, among other aims, to modify the disease course, treat
exacerbations, manage symptoms or improve a compromised
function.
[0242] In one aspect, it is contemplated that a therapy is used in
conjunction with immunosuppressants. In other aspects, a therapy is
used in conjunction with disease-modifying agents, symptom
controlling agents, or agents to improve compromised function.
Alternatively, the therapy may precede or follow the other agent
treatment by intervals ranging from minutes to weeks. In
embodiments where the other agents and/or a proteins or
polynucleotides are administered separately, one would generally
ensure that a significant period of time did not expire between the
time of each delivery, such that the therapeutic composition would
still be able to exert an advantageously combined effect on the
subject. In such instances, it is contemplated that one may
administer both modalities within about 12-24 h of each other and,
more preferably, within about 6-12 h of each other. In some
situations, it may be desirable to extend the time period for
administration significantly, however, where several days (2, 3, 4,
5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse
between the respective administrations.
[0243] Various combinations of therapy may be employed, for example
immunosuppressant therapy, disease-modifying agents, symptom
controlling agents, or agents to improve compromised function is
"A" and an antibody immunotherapeutic that comprises an antibody
that binds a DC receptor and delivers an myelin sheath protein or
component or a peptide or consensus peptide thereof is "B":
TABLE-US-00007 A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B
A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[0244] Administration of the antibody compositions to a
patient/subject will follow general protocols for the
administration of such compounds, taking into account the toxicity,
if any, of the composition. It is expected that the treatment
cycles would be repeated as necessary. It is also contemplated that
various standard therapies, such as hydration, may be applied in
combination with the described therapy.
[0245] B. General Pharmaceutical Compositions
[0246] In some embodiments, pharmaceutical compositions are
administered to a subject. Different aspects may involve
administering an effective amount of a composition to a subject. In
some embodiments, lymphangiogenesis inducers and antigens such as a
bacterial antigen, a viral antigen, a fungal antigen, a protozoal
antigen, a helminth antigen or a cancer antigen may be administered
to the patient to protect against or treat a bacterial infection, a
viral infection, a fungal infection, a protozoal infection, a
helminth infection or a cancer condition. Alternatively, an
expression vector encoding one or more such antibodies or
polypeptides or peptides may be given to a patient as a
preventative treatment. Additionally, such compositions can be
administered in combination with an adjuvant or if immune tolerance
is desired, an immunosuppressant. Such compositions will generally
be dissolved or dispersed in a pharmaceutically acceptable carrier
or aqueous medium.
[0247] The phrases "pharmaceutically acceptable" or
"pharmacologically acceptable" refer to molecular entities and
compositions that do not produce an adverse, allergic, or other
untoward reaction when administered to an animal or human. As used
herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like. The
use of such media and agents for pharmaceutical active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active ingredients, its use in
immunogenic and therapeutic compositions is contemplated.
Supplementary active ingredients, such as other anti-infective
agents, immunosuppressants and immunotherapeutics, can also be
incorporated into the compositions.
[0248] The active compounds can be formulated for parenteral
administration, e.g., formulated for injection via the intravenous,
intramuscular, sub-cutaneous, or even intraperitoneal routes.
Typically, such compositions can be prepared as either liquid
solutions or suspensions; solid forms suitable for use to prepare
solutions or suspensions upon the addition of a liquid prior to
injection can also be prepared; and, the preparations can also be
emulsified.
[0249] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil, or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form must be sterile and
must be fluid to the extent that it may be easily injected. It also
should be stable under the conditions of manufacture and storage
and must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi.
[0250] The proteinaceous compositions may be formulated into a
neutral or salt form. Pharmaceutically acceptable salts, include
the acid addition salts (formed with the free amino groups of the
protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups can also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like.
[0251] A pharmaceutical composition can include a solvent or
dispersion medium containing, for example, water, ethanol, polyol
(for example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating, such as lecithin, by the maintenance of the
required particle size in the case of dispersion, and by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption, for
example, aluminum monostearate and gelatin.
[0252] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization or an equivalent
procedure. Generally, dispersions are prepared by incorporating the
various sterilized active ingredients into a sterile vehicle which
contains the basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum-drying and
freeze-drying techniques, which yield a powder of the active
ingredient, plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0253] Administration of the compositions will typically be via any
common route. This includes, but is not limited to oral, nasal, or
buccal administration. Alternatively, administration may be by
orthotopic, intradermal, subcutaneous, intramuscular,
intraperitoneal, intranasal, or intravenous injection. In certain
embodiments, a immunotherapeutic composition may be inhaled (e.g.,
U.S. Pat. No. 6,651,655, which is specifically incorporated by
reference). Such compositions would normally be administered as
pharmaceutically acceptable compositions that include
physiologically acceptable carriers, buffers or other
excipients.
[0254] An effective amount of therapeutic or prophylactic
composition is determined based on the intended goal. The term
"unit dose" or "dosage" refers to physically discrete units
suitable for use in a subject, each unit containing a predetermined
quantity of the composition calculated to produce the desired
responses discussed above in association with its administration,
i.e., the appropriate route and regimen. The quantity to be
administered, both according to number of treatments and unit dose,
depends on the protection desired.
[0255] Precise amounts of the composition also depend on the
judgment of the practitioner and are peculiar to each individual.
Factors affecting dose include physical and clinical state of the
subject, route of administration, intended goal of treatment
(alleviation of symptoms versus cure), and potency, stability, and
toxicity of the particular composition.
[0256] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically or prophylactically effective. The formulations are
easily administered in a variety of dosage forms, such as the type
of injectable solutions described above.
XII. Kits
[0257] Certain aspects of the present disclosure also concern kits
containing compositions of the disclosure or compositions to
implement methods of the disclosure. In some embodiments, kits can
be used to evaluate one or more nucleic acid and/or polypeptide
molecules. In certain embodiments, a kit contains, contains at
least or contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 100, 500, 1,000 or more nucleic acid probes,
polypeptide detection agents (e.g. antibodies), synthetic RNA
molecules or inhibitors, or any value or range and combination
derivable therein. In some embodiments, there are kits for
evaluating biomarker levels or activity in a cell.
[0258] Kits may comprise components, which may be individually
packaged or placed in a container, such as a tube, bottle, vial,
syringe, or other suitable container means.
[0259] Individual components may also be provided in a kit in
concentrated amounts; in some embodiments, a component is provided
individually in the same concentration as it would be in a solution
with other components. Concentrations of components may be provided
as 1.times., 2.times., 5.times., 10.times., or 20.times. or
more.
[0260] Kits for using probes, polypeptide detecting agents, and/or
inhibitors or antagonists of the disclosure for prognostic or
diagnostic applications are included. Specifically contemplated are
any such molecules corresponding to any biomarker (e.g. CCL21 or
VEGF-C) nucleic acid or polypeptide.
[0261] In certain aspects, negative and/or positive control agents
are included in some kit embodiments. The control molecules can be
used to verify transfection efficiency and/or control for
transfection-induced changes in cells.
[0262] It is contemplated that any method or composition described
herein can be implemented with respect to any other method or
composition described herein and that different embodiments may be
combined.
[0263] Any embodiment of the disclosure relating to a polypeptide
or nucleic acid is contemplated also to cover embodiments whose
sequences are at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99% identical to the polypeptide or
nucleic acid.
[0264] Embodiments of the disclosure include kits for analysis of a
pathological sample by assessing a nucleic acid or polypeptide
profile for a sample comprising, in suitable container means, two
or more RNA probes, or a biomarker polypeptide detecting agent,
wherein the RNA probes or polypeptide detecting agent detects
biomarker nucleic acids or polypeptides. In some embodiments, the
reagents (i.e. RNA probe and/or polypeptide detecting agent) are
labeled with a detectable label. Labels are known in the art and
also described herein. The kit can further comprise reagents for
labeling probes, nucleic acids, and/or detecting agents. The kit
may also include labeling reagents, including at least one of
amine-modified nucleotide, poly(A) polymerase, and poly(A)
polymerase buffer. Labeling reagents can include an amine-reactive
dye.
XIII. Examples
[0265] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1: Lymphatic Endothelial Cells Present Exogenous Antigens
on MHC II
[0266] The inventors have observed that LECs can present exogenous
antigens on MHC I and MHC II. This is surprising, in that antigen
presentation on MHC II is usually associated with only so-called
professional APCs, including DCs.
[0267] Experimental design: OT-II T cells are CD4 T cells that are
transgenic for the T cell receptor (TCR) that recognizes an
sequence in the model antigen ovalbumin, with the sequence
ISQAVHAAHAEINEAGR (OVA.sub.323-339), on an I-A.sup.d immunological
background. Primary DCs, LECs, and FRCs were compared for their
baseline expression of MHC-II I-A, and for how this expression
changes under inflammatory environments, by stimulating the cells
in inflammatory cytokines such as TNF.alpha. and IFN.gamma.. Then,
OT-II cells were co-cultured with these cells in the presence of
their antigen peptide OVA.sub.323-339 and after 4 d of co-culture,
the surface expression of activation markers was quantified on the
OT-II cells by flow cytometry.
[0268] Methods: Bone marrow-derived dendritic cells (BMDCs) were
differentiated from bone marrow of healthy wild-type mice following
an established method published by other groups (Lutz, et al.,
1999). Mature DCs (mDCs) were obtained by incubating immature BMDCs
with co-culture media supplemented with 10 nM OVA.sub.323-339
peptide and 1 nM CpG-B for 6 h at 37.degree. C., followed by three
washes in PBS to remove all remnants of the CpG-B and antigen,
followed by resuspension of the mDCs in co-culture media. To obtain
primary LECs and FRCs, LNs from healthy wild-type mice were
obtained and digested as above, then plated on tissue-culture
polystyrene for 4 d with multiple washes to remove unbound cells.
This process generally depletes all of the immune cell and blood
cell compartment, and the remaining cells, which consist typically
>99% of LECs and FRCs, were separated from each other using
CD31.sup.+ magnetic bead-based selection (Miltenyi Biotec). Cells
were then stimulated in co-culture media supplemented with 100
ng/mL of IFN.gamma. or TNF.alpha. for various timepoints, harvested
from plates using Accutase, stained with antibodies against MHC-II
I-A/I-E (clone AF6-88.5), CD44 (clone IM7), or CD62L (clone MEL14),
and analyzed via flow cytometry on a BD LSR-II machine.
[0269] Results: The inventors previously observed that LECs are
able to acquire peptide-loaded MHC-II receptors from DCs through
extracellular vesicles such as exosomes..sup.22 Here, the
inventorsdemonstrate that the LECs express low levels of MHC-II
under resting conditions (FIG. 1A), and can upregulate MHC-II
within 24 h of stimulation with IFN.gamma. (FIG. 1B). When OT-II
cells are cultured in the presence of their cognate antigen and
LECs, they are stimulated and acquire an antigen-experienced
CD44.sup.+ phenotype (FIG. 1C), but do not lose CD62L expression
and proliferate much less than OT-II cells co-cultured with mature
DCs.
[0270] The observation that LECs express MHC-II, albeit at much
lower levels than DCs, is particularly relevant in light of the
evidence that free peptides can directly load into the receptors
and further, stimulate cognate CD4 T cells to express markers of
antigen experience (FIG. 1). Similarly to what was observed with
LEC-educated OT-I T cells (FIG. 2), LEC-educated OT-II cells do not
lose CD62L expression, and therefore, appear to have a memory-like
phenotype characterized by CD44.sup.+CD62L.sup.+ expression.
However, OT-II cells as a whole were not as responsive to LEC
education as OT-I cells, in the sense that a majority of
LEC-educated OT-I cells proliferated and expressed activation
markers as described earlier, while a majority of OT-II cells
retained the naive phenotype characterized by a
CD44.sup.-CD62L.sup.+ profile. While this observation may explained
by the low levels of MHC-II expression on LECs, it is nevertheless
clear that LECs possess some ability to prime and activate CD4 T
cell responses, albeit to much weaker extents than mDC-initiated
CD4 responses.
Example 2: Lymphatic Endothelial Cells Educate T Cells to Induce
Memory Responses that are Greater than Those Induced by Dendritic
Cells
[0271] Immunological priming refers to stimulation of lymphocytes
by an APC so as to induce their activation and expansion. The
inventors have observed that LECs can prime T cells so as to induce
their differentiation in to memory T cells, even more so than can
DCs, even though they induce a weaker effector expansion.
[0272] Experimental design: The inventors compared priming in vitro
of OT I T cells on LECs exposed to ovalbumin, versus priming in
vitro of OT-I T cells under the same conditions but on DCs exposed
to ovalbumin. The behavior and biomarker profiles of the OT-I cells
were assessed by flow cytometry after 3 days of co-culture. The
OT-I cells were primed in vitro and then adoptively transferred
into recipient C57BL/6 (which are also of the H2k.sup.b background)
mice to follow immune responses. The memory phase response was
measured at two time points in order to reflect early versus late
memory responses. This was done using a Listeria monocytogenes
challenge model, where the mice were challenged with
ovalbumin-expressing Listeria strain, and the recall response to
this challenge was measured. From the perspective of prophylactic
vaccination, a stronger recall response to DC priming would
indicate that DCs were more valuable than LECs in vaccination, and
a stronger recall response to LEC priming would indicate that LECs
were rather more valuable.
[0273] Methods: Isolation of OT-I cells, LN-LECs, and LN-FRCs, and
following co-cultures were performed as described under Example 1
above. OT-I cell proliferation was quantified by flow cytometry
based on dilution of the CFSE dye as described above. In addition,
the inventors introduced the proliferation index as a method of
quantification of OT-I proliferation, which is calculated
.SIGMA..sub.i=0.sup.gn.sub.i/2.sup.i, where g is the total number
of cell divisions observed (# of peaks in the CFSE histogram+1) and
n.sub.i is the number of OT-I cells within a certain peak. This is
essentially a measure of the number of observed OT-I cells
normalized to the number of progenitor cells that led to that
number of OT-I cells.
[0274] In order to evaluate the functional potential of
LEC-educated CD8.sup.+ T cells, the inventors sought to assess
their functionality when they encounter a bacterial pathogen in
vivo. Ex vivo generated (CD45.1.2) LEC-educated together with
(CD45.1) DC-educated OT-I CD8.sup.+ T cells were mixed at a ratio
of 1:1 and adoptively transferred into C57/B16 mice
(5.times.10.sup.4 total cells/mouse). Mice that did not receive any
T cells served as a positive control. The same mice were then
challenged intravenously five weeks later with L.m.-OVA (10.sup.3
cfu/mouse) to characterize early memory phase responses, or nine
weeks later with 10.sup.4 cfu/mouse to characterize long-term
memory phase responses. L.m.-OVA for challenge was acquired from
log phase of growth in BHI medium. Spleens were collected,
homogenized and resuspended in sterile PBS. Different dilutions of
the cell suspensions were generated, plated on BHI plates and
incubated overnight at 37.degree. C. The following day, bacterial
load was determined by counting colony-forming units (cfu) present
in the plates.
[0275] Results: Following co-culture of OT-I cells with various APC
subtypes in the presence of the cognate antigen, OT-I cells are
activated and proliferate (FIGS. 2A-B). However, the resulting OT-I
cells possess vastly different surface phenotypes, which suggest
different functions (FIG. 2C-F). OT-I cells co-cultured with LECs
and mDCs express high levels of CD25, some OX40, and similar levels
of PD-1 and CTLA-4, which collectively suggest activation. However,
they differ in the context of functional markers related to memory
phenotype (FIG. 2D), LN homing (FIG. 2E), and tissue localization
(FIG. 2F). Nevertheless, the LEC-educated OT-I cells are able to
upregulate various activation markers (FIG. 3A) and produce
effector cytokines (FIG. 3B) in the presence of additional
co-stimulatory signals, even in spite of possessing a central
memory-like phenotype (FIG. 3C).
[0276] Having demonstrated the capacity of LEC-educated CD8.sup.+ T
cells to give rise to functional effectors upon antigen
re-encounter in vivo, the inventors sought to assess their
contribution to immune responses against a real pathogen as well as
evaluate their protective ability in direct competition to
DC-educated T cells (FIG. 4). To this end, the inventors
co-transferred ex vivo generated LEC-educated OT-I CD8.sup.+ T
cells together with DC-educated cells in mice at a 1:1 ratio. The
mice were allowed to rest for five weeks and then challenged with
Listeria monocytogenes (L.m.)-expressing OVA. LEC-educated cells
exhibited effector function with cytotoxic potential, since they
expressed cytokines (FIG. 4B, C) and underwent cytolytic granule
release (FIG. 4B). More specifically, upon ex vivo restimulation,
LEC-educated CD8.sup.+ T cells displayed similar levels of
IFN.gamma. (FIG. 4B), as well as TNF-.alpha. and IL-2, with their
DC-educated counterparts and they were on par in the expression of
CD107, a marker of cytolytic granule exocytosis in the spleen. By
assessing the percentage of single, double or triple positive cells
for IFN.gamma., TNF-.alpha. and IL-2 (FIG. 4C) in order to evaluate
the polyfunctionality of the cells, the inventors observed a
similar distribution between the two populations. In LEC-educated
cells, the inventors detected a dominant subset of cells positive
for one of the three cytokines (42.18.+-.5.35), a subset of cells
positive for two of the three cytokines (30.37.+-.6.25), a smaller
subset of triple positive cells (2.85.+-.0.60), while 25.11%
(.+-.6.75) of the cells did not produce any cytokine.
Interestingly, there was a trend for a greater subset of double
positive and triple positive (p=0.07) in LEC-educated cells
compared to DC-educated ones.
[0277] The inventors observed that OT-I cells primed in vitro on
DCs generally yielded a stronger effector immune response than did
OT-I cells primed in vitro on LECs. By measures accepted in the art
of vaccination, this would indicate that DCs are a more valuable
APC than LECs. However, OT-I cells primed in vitro on LECs yielded
an equivalent memory recall response after adoptive transfer than
did OT-I cells primed on DCs in early memory phase induction, and
further, appeared to provide better long term protection from
bacterial challenge than did the OT-I cells primed on DCs (FIG.
4C,F). Given that the overall goal of prophylactic vaccination is
to induce memory capable of strong memory recall, this indicates
that LECs are a very valuable APC, thereby showing proof-of-concept
of the value of targeting of antigen to LECs in vaccination. This
is a departure from the state-of-the-art in vaccination, where the
DC has been the most important target for antigen delivery.
Example 3: In Vivo Lymphangiogenesis May be Stimulated to Trigger
Induction of Adaptive Immunity with Memory
[0278] Experimental design: As an approach to target antigen to
LECs in vaccination, the inventors have developed a hydrogel
vaccine in which antigen is delivered in a gel along with bioactive
factors that induce lymphangiogenesis. The rationale for this
design is that antigen could be delivered to LECs that were induced
to grow into the gel upon its injection. A powerful inducer of
lymphangiogenesis is vascular endothelial growth factor-C (VEGF-C).
A useful matrix as a gel is fibrin. Thus, a fibrin-binding variant
of VEGF-C was incorporated into gel implants that also comprised
the antigen. In addition, the inventors had also developed an
alternative matrix based on poly(ethylene glycol), where the
polymers were modified to contain the fibrin domains compatible
with the fibrin-binding motifs present on the VEGF-C variant
described above.
[0279] Hydrogels containing VEGF-C with or without the OVA model
antigen were implanted into healthy wild-type mice, which had
previously received CD4.sup.+ T cells recognizing OVA (OT-II cells)
with or without OT-I cells. Activity and biomarker expression on
both cell types were evaluated early post-implantation of the
vaccine (<10 d) or later during the early memory phase (22 d).
Surface marker profiles on T cells can be typically used to
characterize memory and effector phenotypes.
[0280] Methods: Hydro gel synthesis, preparation, and implantation.
8-arm, 40 kDa poly(ethylene glycol) (PEG)-maleimide was reacted
with 9.6.times. molar excess of either Ac-FKGGVPMSMRGGERCG-Amide or
NQEQVSPLERCG-Amide peptides in dimethylformamide, in the presence
of 10.times. molar excess of triethylamine. The reaction was
allowed to proceed for at least 4 h at 37.degree. C. with
agitation, after which the completed polymer-peptide conjugates
(hereinafter referred to as 8APEG-FKGG & 8APEG-NQEQVSPL) were
isolated using a size-exclusion chromatography and characterized by
NMR and BCA assay to confirm peptide conjugation. 5% wt hydrogels
were prepared by mixing 1 mg of 8APEG-FKGG & 1 mg of
8APEG-NQEQVSPL with desired bioactive factors (VEGF-C and/or OVA),
0.2 U Factor XIII, and 0.075 U thrombin, adjusted to a final volume
of 40 .mu.L in TBS with 50 mM CaCl.sub.2). This mixture was
pipetted to mix and injected into the interscapular region of
isoflurane-anesthesized, healthy wild-type mice intradermally,
where it polymerizes within 10-15 min of injection. Each mice
received a single hydrogel.
[0281] Fibrin-binding protein variants. The fibrin-binding domain
use to modify the proteins allows the binding into the hydrogels
described in this example, since they are crosslinked with the
fibrin-crosslinking transglutaminase Factor XIII Cloning of
TgVEGFC: a matrix metalloproteinase (MMP)-cleavage site followed by
the fibrin-binding domain (Tg) was added at the C-terminus of the
mouse VEGFC sequence (accession NM_009506.2, mature peptide, amino
acid 108-223) by sequential polymerase chain reactions. Similarly,
a poly-His tag followed by a thrombin-cleavage site was added at
the N-terminus of VEGFC (TgVEGFC final design:
"polyHis-thrombin-VEGFC-MMP-Tg"). The resulting sequence of TgVEGFC
was then inserted in pSeqTagA plasmid backbone. Cloning of TgOVA: a
MMP-cleavage site followed by the fibrin-binding domain (Tg) was
added at the C-terminus of the chicken ovalbumin (OVA) sequence
(accession P01012, full length), and a poly-His tag followed by a
thrombin-cleavage site was added at the N-terminus of OVA, by
sequential polymerase chain reactions (TgOVA final design:
"polyHis-thrombin-OVA-MMP-Tg"). The resulting sequence of TgOVA was
inserted into pSeqTagA plasmid backbone. Production of proteins:
both TgVEGFC and TgOVA have been produced as follows. The plasmid
was transfected into HEK-293E using 25 kDa polyethyleinimine as a
transfection agent and cells and cultured in FreeStyle medium
(Invitrogen, Carlsbad, USA) supplemented with glutamine (4 mM) and
valproic acid (3.75 mM). At 7 days post-transfection, the expressed
protein was isolated from the cell culture supernatants using
Ni.sup.2+-affinity chromatography (for the poly-His tag). The
poly-His tag was then cleaved with a commercial agarose bead-bound
thrombin cleavage kit (Sigma-Aldrich, St. Louis, USA), and purified
using size-exclusion chromatography. The purified proteins were
further dialyzed against Tris-buffer saline, sterilized through
0.22 .mu.m filtration and stored at -80.degree. C.
[0282] Intravital Imaging and Analysis. All mice were anesthesized
via 4.0% isoflurane and imaged via a Xenogen IVIS Spectrum (Caliper
LifeSciences/Perkin-Elmer, Waltham, Mass., United States).
AlexaFluor750-tagged VEGF-C was detected via excitation and
emission filters set at 745/800 nm and exposure time at 0.5
seconds. To quantify protein release from the hydrogels, ROIs were
defined for each gel injection site using images obtained at the
time of gel implantation. For any given gel injection site, the
same ROIs were kept for the duration of the experiment.
Background-corrected total photon counts within ROIs were
quantified using Perkin-Elmer Living Image software (version 4.0)
and normalized against total photon counts within the ROI at the
time of injection in order to obtain release profiles of the
fluorescently-labeled proteins over time.
[0283] Mice and adoptive transfers. OT-I and OT-II cells were
isolated from spleens of healthy, adult OT-I and OT-II mice using
commercial kits for CD8 and CD4 negative selection, respectively,
according to manufacturer's instructions (Stemcell Technologies,
Vancouver, Canada). Purity was confirmed to be 90-98% by flow
cytometry, at which point cells were resuspended at
5.times.10.sup.6 cells/mL in serum-free RPMI-1640 media. 200 .mu.L
of this suspension (corresponding to 1.times.10.sup.6 cells) was
injected into recipient wild-type C57Bl/6 mice (gender-matched,
8-12 weeks old) via the tail-vein one day prior to hydrogel
implantation.
[0284] Vaccinations and infection-mimicking challenge. As a
positive control vaccine, healthy adult wild-type mice were
injected with 50 .mu.g OVA and 50 .mu.g CpG, in 50 .mu.L PBS,
intradermally via the front footpads (25 .mu.L per footpad). This
was repeated 15 days after the first vaccination in order to boost
the T cell responses in these mice. For the challenge model,
previously-vaccinated mice, or mice receiving hydrogels (with no
vaccination boost) were injected in the front footpads with 50
.mu.g OVA and 10 .mu.g LPS in 50 .mu.L PBS (25 .mu.L per footpad).
All mice were sacrificed 3 d post-challenge.
[0285] Results: Fibrin-mimic PEG hydrogels are prepared liquid,
injected into a mouse intradermally, and polymerize within 10-15
min of injection in vivo (FIG. 5A). Since there is the possibility
of including fibrin-binding variants of proteins of interest into
the hydrogel system, which leads to its slow release over time,
this can be leveraged to create a flexible vaccine delivery
platform which encompasses antigens of interest, chemoattractants
and cytokines for recruitment and modulation of immune responses,
as well as growth factors for encouraging ingrowth of local cell
types, all in a system that slowly releases these proteins over
time (FIG. 5B-C).
[0286] As an example, the inventors showed here the delivery of
soluble VEGFC (sVEGFC) versus a fibrin-binding variant (TgVEGFC)
from the hydrogel system (FIG. 5B-C), where the TgVEGFC is retained
at the implant sites for about 50% longer than the sVEGFC. sVEGFC
levels dropped to 10% of administered amounts within 8 d
post-implantation, while TgVEGFC levels took 4 more days to reach
this level. This led to different effects, as the TgVEGFC, owing to
its longer retention locally, enhanced lymphangiogenesis at the
implant site (FIG. 5D-E), while the sVEGFC enhanced
lymphangiogenesis mainly at the dLNs (FIG. 5F-G). These were
determined based on increased LYVE-1+ staining in the respective
treatment groups detected by microscopy (FIG. 5D,F) and
quantification of local LEC populations by flow cytometry based on
gp38.sup.+CD31.sup.+ staining (FIG. 5E,G).
[0287] Focusing on the TgVEGFC-delivering formulation, the
inventors observed increased recruitment of immune cells, defined
by CD45.sup.+ staining, within 9 d post-implantation into the mice
(FIG. 6A). However, at 22 d post-implantation, TgVEGFC gels further
enhance this difference (FIG. 6B), and this appears to be in large
part due to increased recruitment of T cells (FIG. 6C-D) and
myeloid cells that are CD11c.sup.-CD11b.sup.+ (FIG. 6D).
Quantification of the numbers and composition of the T cells and
the dendritic cell compartments (FIG. 6E-G) reveal a 6-fold
increase in accumulation of CD4 T cells at the implant site, while
CD8 T cells and DCs experienced modest levels of accumulation, if
any at all.
[0288] To investigate if the increased recruitment of CD4 T cells
can be leveraged towards the development of a vaccine, OVA and
VEGFC were co-delivered via the hydrogels, and within 8 d
post-implantation, implant sites were isolated for analysis of the
phenotype of the CD4 T cells (FIG. 7). At this early timepoint,
there are typically no significant differences in CD4 T cell
accumulation at the implant site (FIG. 6E), so any differences in
accumulation of any specific CD4 T cell subtypes may better reflect
differences in T cell education. Hydrogels delivering both TgVEGFC
and TgOVA created a local environment such that more cells isolated
from the implant site spontaneously secreted IFN.gamma., and these
differences were preserved when the same cells were stimulated with
OVA (FIG. 7B). Furthermore, these cells contained a larger portion
of effector-memory cells expressing a
CD44.sup.+CD62L.sup.-KLRG1.sup.-CD127.sup.+ marker profile
regardless of whether they were transferred OT-II cells or the
endogenous CD4 T cells. In contrast, there was no detected increase
in short-lived effector cells (SLECs).
[0289] Since these results suggest that hydrogels delivering
TgVEGFC+TgOVA promoted faster generation of effector-memory CD4 T
cells locally, the inventors hypothesized this may lead to higher
numbers of circulating effector-memory CD4 T cells, reflecting the
possibility that this may be used as a platform for vaccination.
The inventors administered OT-I and OT-II cells into recipient mice
1 d prior to administration of the hydrogels (FIG. 8A), and at
various time points, blood samples were obtained for flow cytometry
analysis. Mice were sacrificed at 22 d post-implantation, and
implant sites and the draining LNs--brachial LNs (bLNs) were also
collected for flow cytometry analysis (FIG. 8B). As expected,
relative to negative control hydrogels which lacked TgOVA,
hydrogels delivering TgVEGFC+TgOVA enhanced the proportion of OT-I
and OT-II cells expressing the CD44.sup.+CD62L.sup.- profile
(within which the effector-memory T cells are found) in all organs
analyzed. Notably, this was also observed for the endogenous CD4 T
cells found at the implant site for both hydrogels delivering
TgVEGFC. With regards to the CD8 T cells, both TgOVA-containing
hydrogel conditions produced similar effects in terms of the
distribution of OT-I and endogenous T cells into the various
subpopulations defined by CD44 and CD62L.
[0290] Finally, the performance of the hydrogels delivering
TgVEGFC+TgOVA as a vaccine were compared head-to-head against a
standard vaccine, consisting of soluble antigen and an adjuvant,
injected intradermally in the front limbs of mice (FIG. 9A). To
analyze for the immediate response to vaccination, a cohort of mice
was sacrificed 3 days following hydrogel implantation or the
initial vaccine. Another cohort was incubated for 35 days (mice in
the standard vaccine group were boosted at d15), then challenged
with the antigen and LPS to mimic an infection, incubated for
another 3 days, then sacrificed. Hydrogel implantation sites,
draining LNs, and blood were collected for proteomics analysis via
ELISAs and Luminex. As expected, the standard vaccine elicited a
moderate level of systemic inflammation (FIG. 9B), as characterized
by elevated serum levels of the inflammatory mediators CXCL1,
CXCL10, IL-10, M-CSF, BAFF, and IL-5 relative to all other groups
tested. Following antigenic challenge, there was no difference in
the circulating levels of all of these signals. Notably, the
hydrogels delivering TgVEGFC+TgOVA produced highly localized
responses (FIG. 9C), and the antigen challenge resulted in much
higher local levels of the cytokines IL-15, CXCL1, and CCL20 within
this group relative to all other groups. These are particularly
important in that they stimulate T cell proliferation and memory T
cells (IL-15), while recruiting more activated T cells and APCs
(CCL20) and other immune cells (CXCL1).
[0291] Inducing local lymphangiogenesis increases the local
accumulation of various immune cell subtypes, particularly CD4 T
cells and myeloid cells (the inventors have not further
investigated which subtypes were recruited). Co-delivery of an
antigen with the lymphangiogenic mediators promotes local education
of the T cells, promoting the generation of effector-memory CD4 T
cells and as they disseminate into the bloodstream, they are able
to circulate and potentially provide protection from antigenic
challenges elsewhere in the body. It is also notable that
endogenously-derived CD4 T cells also acquired activated phenotypes
at the implant site. Since all hydrogel materials were confirmed
endotoxin-free prior to injection, this activation may be
potentially due to recognition of other OVA-derived antigens,
resulting in the generation of CD4-directed adaptive responses
against multiple epitopes derived from the same antigen. With CD4 T
cells being capable of aiding in both B cell-mediated as well as
CD8 T cell-mediated immune responses, this generation of CD4
memory, through a combination of lymphangiogenesis and antigen
delivery, represents a vaccination strategy applicable to a large
array of antigens. Moreover, the hydrogel vaccines were able to
induce these memory responses without producing systemic
inflammation that is seen with a standard vaccination strategy. At
the site of the hydrogel implantation, the hydrogel `vaccines` were
even superior to the control vaccine at inducing cytokines and
chemokines related to T cell memory and effector formation as well
as recruitment of accessory immune cells.
Example 4: Lymphangiogenic Growth Factors May be Engineered to
Induce In Vivo Lymphangiogenesis
[0292] Experimental design: For the induction of in vivo
lymphangiogenesis, it is important to provide the means to through
which lymphangiogenic factors such as VEGFC may be retained in a
tissue site. This can be done by incorporating the VEGFC into an
injectable matrix (either co-delivered with the VEGFC, or in a
separate formulation), or by providing the means for the VEGFC to
directly bind the tissues in the injection site (which in this
case, may also function as a matrix). The former is illustrated in
Example 4 in the form of a polymeric hydrogel, which is crosslinked
under the action of the naturally-occurring coagulation
transglutaminase Factor XIIIa. Fibrin serves as another such
material, which may first be crosslinked physically after exposure
to thrombin, and then further crosslinked covalently under the
action of Factor XIIIa. Thus, one means through which VEGFC may be
rendered compatible for retention into such matrices is achieved by
engineering them with a domain that serves as a Factor XIIIa
substrate. The inventors have shown that the N-terminal domain of
the protein alpha2-plasmin inhibitor (the amino acids NQEQVSPL)
serves as such a substrate and can be fused to a terminus of
proteins.sup.27 such as VEGFC, to form TgVEGFC fusion protein.
Factor XIIIa may therefore act on this fusion protein, and use the
engineered peptide tag to directly graft the bioactive factor onto
the polymeric or fibrin matrix. As an alternative, the inventors
have shown that matrix-binding domains from proteins may be
identified and fused to the bioactive factor, which allows binding
to both matrices such as fibrin but also naturally-occurring,
endogenous matrices such as the extracellular matrix (Martino, et
al., 2014). Specifically, the inventors have shown that a domain in
placental growth factor-2 (PlGF-2.sub.123-144) is such a binding
domain, and thus this domain can be used to engineer a
matrix-binding variant of VEGFC, PlGF-2.sub.123-144-VEGFC.
[0293] Methods: TgVEGFC is prepared as described in Example 4. The
sequence of PlGF-2.sub.123-144-VEGFC was made by adding the
matrix-binding domain PlGF-2.sub.123-144 to the C-terminus of the
mouse VEGFC sequence (accession NM_009506.2, mature peptide, amino
acid 108-223). In addition, a polyHis tag followed by a Factor
Xa-cleavage site was added at the N-terminus of the VEGFC sequence.
The resulting sequence was inserted in pXLG plasmid backbone and
expressed in HEK293-E cells under the same conditions as for
TgVEGFC (described in Example 4). The expressed protein was
purified from the cell supernatant after 7 days in culture using
Ni.sup.2+-affinity chromatography and size exclusion
chromatography. The purified proteins were sterile-filtered and
stored in Tris-buffered saline at -80.degree. C.
[0294] Results: TgVEGFC was covalently incorporated into fibrin and
fibrin-mimetic matrices during polymerization by
FactorXIIIa-mediated crosslinking (FIG. 5A-C).
PlGF-2.sub.123-144-VEGFC may be non-covalently sequestered into
fibrin or endogenous matrices, owing to the high affinity of the
PlGF-2.sub.123-144 for endogenous extracellular matrix components.
The spatial sequestration and slow controlled release of bioactive
TgVEGFC and PlGF-2.sub.123-144-VEGFC variants into the delivery
site are likely to sequester the lymphangiogenic effects of VEGFC
(eg. LECs signaling, proliferation, migration and lymphatic vessels
formation) to the injection site, minimizing the systemic side
effects (FIG. 5D-E).
[0295] By using protein engineering approaches, lymphangiogenic
factors can be generated that are more potent in inducing local
lymphangiogenesis in vivo than their wild-type counterparts. The
inventors illustrate that matrix binding is one such approach by
which to achieve this, for example by binding covalently to matrix
under the influence of a transglutaminase (with Factor XIIIa,
although other transglutaminases such as tissue transglutaminase
would be useful) and non-covalently, for example by binding with
high affinity to the extracellular matrix or extracellular matrix
analogs (Martino, et al., 2013).
Example 5: Tumor Lymphangiogenesis Promotes T Cell Infiltration and
Potentiates Immunotherapy in Melanoma
[0296] In melanoma, VEGF-C expression and consequent
lymphangiogenesis correlate with metastasis and poor prognosis.
VEGF-C also promotes tumor immune suppression, suggesting that
lymphangiogenesis inhibitors may be clinically useful in
combination with immunotherapy. The inventors addressed this
hypothesis in mouse melanomas, with VEGFR-3 blocking antibodies and
found, unexpectedly, that VEGF-C signaling enhanced therapeutic
response to various immunotherapies. This was mediated by
VEGF-C-induced CCL21 and infiltration of naive T cells into the
tumor before immunotherapy, since CCR7 blockade reversed the
potentiating effects of VEGF-C. In human metastatic melanoma, gene
expression of VEGF-C strongly correlated with CCL21 and T cell
inflammation, while serum VEGF-C levels associated with T cell
activation and expansion following peptide vaccination. It is
proposed that tumor VEGF-C potentiates immunotherapy by attracting
naive T cells, which are locally activated upon
immunotherapy-induced tumor cell killing to augment the antitumor
immune response. In this way, VEGF-C may serve as a predictive
biomarker for immunotherapy response.
[0297] It these experiments, it was sought to determine whether
inhibiting tumor-associated lymphangiogenesis, and thus reducing
its suppressive effects, would enhance the efficacy of
immunotherapy, but find, surprisingly, that it increases resistance
to immunotherapy. Instead, the inventors have identified a new
mechanism whereby CCL21-dependent recruitment of naive T cells into
lymphangiogenic melanomas renders the tumor microenvironment more
responsive to systemic immunotherapy. It is hypothesized that, once
in the tumor, naive T cells are locally primed and activated
following immunotherapy-induced tumor cell death, leading to
epitope spreading and long lasting anti-tumor immunity. These
results reveal a new role to tumor-associated lymphangiogenesis in
shaping the tumor immune microenvironment. While VEGF-C inhibits
immunotherapy approaches that rely on activation within the
immunosuppressed tumor draining lymph node, it induces a
characteristic immune signature in the primary tumor that
potentiates systemic immunotherapy.
[0298] A. VEGFR-3 Inhibition Decreases Suppressive Features of
VEGFC-Expressing B16 Melanoma
[0299] We first assessed whether anti-VEGFR-3 (.alpha.R3) antibody
treatment specifically altered peri- and intratumoral LEC density
in B16-F10 tumors modified to express ovalbumin (B16-OVA) or OVA
and VEGF-C (B16-OVA/VC). Intratumoral VEGF-C expression was
confirmed in B16-OVA/VC tumors (FIG. 17A). Interestingly, blocking
VEGFR-3 signaling in B16-OVA/VC tumors lead to increased VEGF-C
levels, possibly due to accumulation consequential to decreased
receptor-ligand internalization. As expected, VEGF-C expression
increased the density of intratumoral Lyve-1.sup.+ lymphatic
vessels, while .alpha.R3-treated tumors and non-VEGF-C expressing
B16-OVA tumors were devoid of them (FIG. 17A and FIG. 17B). This
was confirmed by flow cytometry, revealing that LECs
(gp38.sup.+CD31+), but not blood endothelial cells (BECs,
gp38.sup.-CD31.sup.+) or macrophages (F4/80+), were enriched in
lymphangiogenic tumors (FIGS. 10B and 17C-D). Primary tumor growth
of .alpha.R3-treated B16-OVA tumors was unaffected, while
B16-OVA/VC tumors reacted with slightly delayed tumor growth to
.alpha.R3 treatment, which however did not significantly affect
overall survival (FIG. 10C).
[0300] In line with previous reports, we found a VEGFR-3-dependent
trend towards increased CD45.sup.+ immune cell density in
B16-OVA/VC tumors (FIG. 10D) including a significant increase of
CD4.sup.+FoxP3.sup.+ regulatory T cells (FIG. 10E).
Antigen-presenting cells (APCs) including conventional dendritic
cells (DCs), cross-presenting CD8.sup.+ DCs, myeloid DCs, and also
potentially immunosuppressive myeloid-derived suppressor cells
(MDSCs) were generally less abundant in the .alpha.R3--as compared
to control IgG-treated B16-OVA/VC tumors (FIG. 10F-G and FIG.
17E-F). We did not detect any significant effect of anti-VEGFR-3
therapy on the immune cell microenvironment in non-VEGF-C
over-expressing B16-OVA tumors (FIG. 10D-G and FIG. 17D-F). Taken
together, these data demonstrate that VEGF-C expression promotes an
immune suppressive tumor microenvironment, while inhibiting VEGFR-3
prevents VEGF-C-driven tumor lymphangiogenesis and decreases
suppression in B16 melanomas.
[0301] B. Lymphangiogenic Melanomas are Highly Sensitive to
Immunotherapy
[0302] Having confirmed that VEGFR-3 blockade decreases cellular
hallmarks of immuno suppression in B16-OVA/VC tumors, we
hypothesized that a less suppressed environment in
.alpha.R3-treated tumors would enhance the efficacy of anti-tumor
immunotherapy. To test this, we adoptively transferred ex vivo
activated OVA-specific CD8.sup.+ OT-I cells into tumor-bearing mice
and assessed tumor growth over time. In line with our previous
findings, non-lymphangiogenic B16 tumors were more sensitive to
adoptive T cell therapy (ATT) in the short-term, leading to a
significantly decreased tumor volume on day 12 (FIG. 11A). Against
our expectation, .alpha.R3-treated B16-OVA/VC tumors started
progressing shortly after peak regression on day 16, while control
IgG-treated B16-OVA/VC tumors showed a profound and long lasting
response to ATT. This translated into significantly decreased tumor
volume in the progression phase and into increased survival of
control IgG-treated B16-OVA/VC tumor-bearing mice. When we
performed the same experiment in mice that lack dermal lymphatic
vessels (K14-VEGFR-3-IgG mice) there was no difference between
B16-OVA and B16-OVA/VC tumor growth or host survival (FIG. 11B),
confirming that the therapeutic benefit of ATT in B16-OVA/VC tumors
was dependent on host lymphangiogenesis.
[0303] We next asked whether the lymphangiogenic status of B16
melanomas modulates the efficacy towards an immunotherapy approach
that relies on raising an endogenous anti-tumor response. We thus
treated tumor-bearing mice with a therapeutic DC vaccination (DC
vax). DCs were activated ex vivo with LPS and then pulsed with the
immunodominant MHC-I peptide SIINFEKL ex vivo, before being
injected intraperitoneally into mice on days 4 and 10 after tumor
inoculation. As with ATT, lymphangiogenic tumors grew significantly
larger immediately after the DC vax, but then underwent profound
regression (FIG. 11C). Accordingly, the median survival increased
from 21 days for .alpha.R3-treated to over 2 months for control
IgG-treated B16-OVA/VC tumor-bearing mice.
[0304] We next asked whether the lymphangiogenic status of B16
tumors also modulates non-antigen-specific immunotherapy using an
adjuvant-only treatment with the Toll-like receptor 9 (TLR9) ligand
CpG. Indeed, CpG treatment was more effective at controlling tumor
growth and enhancing survival in lymphangiogenic (control
IgG-treated) tumors compared to those treated with VEGFR-3 blocking
antibodies (FIG. 11D). When CpG was combined with OVA protein,
vaccine efficacy was almost complete (FIG. 11E)--with the exception
of one mouse, tumors regressed completely, even after several
months.
[0305] Importantly, the vaccine-potentiating effects of VEGF-C were
not limited to the more immunogenic protein OVA, since an effective
vaccine composed of nanoparticle-bound endogenous melanoma peptide
Trp2 (NP-Trp2) and CpG adjuvant showed similar trends (FIG. 11F).
We performed similar experiments in mice inoculated with wildtype
B16 (B16 WT) and VEGF-C overexpressing (B16-VC) tumors lacking OVA
expression to rule out the possibility that the observed effects
were dependent on the expression of a foreign antigen. VEGFR-3
blocking had no profound effect on B16 WT or B16-VC tumors (FIG.
18A). However, as for the OVA expressing tumors, CpG adjuvant
therapy alone induced delayed tumor growth and enhanced survival
(FIG. 18B), and the NP-Trp2+CpG vaccine induced tumor regression
and long-lasting responses in lymphangiogenic mice (FIG. 18C).
[0306] To ensure that our observations were not specific to the B16
melanoma model, we performed an immunotherapy trial in a more
clinically relevant, genetically engineered mouse (GEM) model of
melanoma driven by mutated BRAF.sup.V600E (further referred to as
BRAF GEM). In order to raise a potent anti-tumor immune response,
BRAF GEM mice received a combinatorial immunotherapy consisting of
a peptide vaccine (CpG+gp100-peptide) combined with anti-PD-1
blockade starting 8 days after enrollment into the trial. Indeed,
as observed in the B16 model, .alpha.R3-treated BRAF GEM mice
responded less well to immunotherapy intervention, while
IgG-treated mice showed delayed tumor outgrowth and increased
survival (FIG. 11G).
[0307] Altogether, these data demonstrate that VEGF-C signaling in
melanoma potentiates the effects of immunotherapy, particularly
with protein or peptide vaccines, despite promoting a more immune
suppressive microenvironment.
[0308] C. CCL21 is Increased in Lymphangiogenic Melanomas and
Drives Recruitment of Naive T Cells into VEGF-C Overexpressing B16
Tumors
[0309] We next asked why the more immunosuppressed, invasive tumors
would be more responsive to immunotherapy. When examining the
immune cell infiltrates, we found a significant increase
(.about.2.5 fold) in CD4.sup.+FoxP3.sup.- T cell density as well as
increased CD8.sup.+ T cell density in control IgG-treated
1B16-OVC/VC tumors as compared to those with VEGFR-3 blocking (FIG.
12A). Interestingly, a large fraction of infiltrating CD4.sup.+
TILs had a naive (CD62L.sup.+CD44.sup.-) phenotype (FIG. 12B),
shifting the balance between naive and effector
(CD62L.sup.-CD44.sup.+) CD4.sup.+ T cells in favor of naive ones in
control IgG-treated B16-OVC/VC tumors (FIG. 12C).
[0310] Since naive, but not effector, T cells express the chemokine
receptor CCR7, we assessed tumor expression of the CCR7 ligand
CCL21, which is normally expressed by LN stromal cells to guide
naive and memory T cells as well as mature DCs into the LN
parenchyma. CCL21, when expressed at physiological levels (i.e., as
in the LN), has been shown to promote local immune suppression by
changing the tumor stroma. Because CCL21 is expressed by LECs and
upregulated in response to VEGF-C/VEGFR-3 signaling, we were not
surprised to find that CCL21 protein was substantially increased in
control IgG-treated B16-OVA/VC as compared to either control
IgG-treated B16-OVA or .alpha.R3-treated B16-OVA/VC tumors (FIG.
12D). Intratumoral LECs could indeed be a major source of CCL21, as
the chemokine could be mainly detected in close proximity to
lymphatic endothelium but not elsewhere in the tumor
microenvironment (FIG. 12E). This effect was both restricted to
CCL21 within the local tumor microenvironment, as no change in
CCL21 levels could be detected in the tumor draining or
non-draining LNs (FIG. 12D) and as no change in other cytokines
levels could be detected within the tumor (FIG. 19A). Accordingly,
and increased number of CCR7 expressing conventional CD4.sup.+ and
CD8.sup.+ T cells was present within B16-OVA/VC as compared to
B16-OVA tumors (FIG. 12F).
[0311] Even though BRAF GEM tumors expressed much lower, possibly
more physiologic, levels of VEGF-C (FIG. 19B) as compared to VEGF-C
overexpressing B16 tumors (FIG. 17A), we observed VEGFR-3 dependent
tumor-associated lymphangiogenesis (FIG. 19C) and CCL21 expression
(FIG. 19D). As in lymphangiogenic tumors, CCL21 accumulations
within BRAF GEM tumors were mainly localized around the lymphatic
endothelium (FIG. 17E).
[0312] To test whether CCL21/CCR7 signaling was indeed a mechanism
underlying increased recruitment of naive T cells into
lymphangiogenic tumors, B16-OVA/VC tumor-bearing mice were treated
with CCR7 blocking antibodies (FIG. 12G-E). Anti-CCR7 (.alpha.CCR7)
treatment mainly reduced the infiltration of naive T cells (FIG.
12G-H), with a very large reduction in the ratio of naive versus
effector phenotype for both tumor-infiltrating CD4.sup.+ and
CD8.sup.+ T cells (FIG. 121). We could also show this more directly
by adoptive transfer of allogeneic (CD45.2) naive OT-I cells into
mice bearing B16-OVA/VC tumors and examining their TILs; after 24
hours, nearly 10-fold fewer OT-I cells were found in
.alpha.CCR7-treated tumors compared with controls (FIG. 12J).
Altogether, these data demonstrate that the CCL21/CCR7 axis not
only drives regulatory CD4.sup.+ but also naive T cells into
lymphangiogenic B16 melanomas.
[0313] D. VEGF-C Correlates with the Expression of CCL21, CCR7, and
a T Cell Signature in Human Melanoma Samples
[0314] We next asked whether the VEGF-C/CCL21 axis was relevant for
shaping the immune microenvironment in human melanoma. We first
performed immunofluorescence analysis of the lymphatic marker
podoplanin in tissue sections of 14 primary human melanomas (FIG.
13A-B). In roughly half of the tumors, we found substantially
higher lymphatic density in the tumor than in the adjacent skin,
implying that these were lymphangiogenic (FIG. 13B). Furthermore,
we stained sections of human primary melanoma for VEGF-C, LECs
(podoplanin+) and CCL21, and found ubiquitous VEGF-C expression
(FIG. 13C) as well as more restricted CCL21 expression by
intratumoral LECs (FIG. 13D).
[0315] Several recent reports have described melanoma gene
signatures that stratify patients that respond to immunotherapy and
whose tumors contain high levels of T cells, from non-responders
with poorly infiltrated tumors. We thus analyzed 469 metastatic
melanoma patients from the Cancer Genome Atlas (TCGA) and found
strong and highly significant correlations between VEGFC, but not
VEGFA or FIGF (VEGF-D), and genes correlating with immunotherapy
response (FIG. 13E, left). In addition, several genes correlated
with immunotherapy resistance, including CTNNB1, MYC, and CXCL1
were inversely correlated with VEGFC (FIG. 13E, right). In line
with our findings in mice, gene expression of VEGFC correlated with
that of CCL21 and CCR7 in human primary melanoma, while expression
of the other main VEGFR-3 ligand, FIGF (VEGF-D), showed no
correlation (FIG. 13F, top 2 rows). Interestingly, VEGFA expression
was inversely correlated with CCR7 expression and showed a similar
trend (though not statistically significant) in CCL21 expression.
According to our findings in mouse melanoma, expression of CD8,
CD4, CD11c, FoxP3 and CD127 (expressed by naive T cells), but not
CD44 (expressed by activated T cells), correlated with that of
VEGFC (FIG. 13F, bottom 3 rows). Interestingly, we observed the
same trends in metastatic tumors from the same database (FIG. 20).
Taken together, these data show that VEGFC and CCL21 expression are
strongly correlated in human melanoma, and are consistent with the
notion that VEGF-C/CCL21 upregulation shifts the immune
microenvironment to be more highly T cell infiltrated.
[0316] E. VEGF-C Correlates with Response to Immunotherapy in Human
Metastatic Melanoma Patients
[0317] Given that we found VEGF-C to be a driver of tumor
inflammation in human metastatic melanoma, we wanted to determine
whether serum VEGF-C represents a biomarker to predict response to
immunotherapy. Using sera stored from an earlier clinical study of
20 patients who underwent Melan-A analog vaccination, we measured
circulating VEGF-C levels to compare with the previously described
tumor-specific CD8+ T cell responses. Interestingly, we found that
all patients with low VEGF-C showed weak responses, measured both
in terms of numbers of circulating Melan-A-specific CD8+ T cells
(FIG. 14A) as well as their expression of the effector cytokine
IFN.gamma. (FIG. 14B), while all responding patients had higher
levels of VEGF-C. While the differences in expression of
TNF.alpha., IL-2 and CD107 did not reach statistical significance
(FIG. 21A-D), Melan-A-specific T cells in patients with higher
VEGF-C levels showed higher percentages of polyfunctionality (FIG.
14C).
[0318] We next wanted to assess whether serum VEGF-C levels could
stratify responders and non-responders in a setting where the
therapeutic interventions leads to actual clinical benefit. We thus
measured serum VEGF-C levels of 76 patients who underwent combined
anti-CTLA-4 (Ipilimumab) and anti-PD-1 (Nivolumab) therapy and
correlated it with progression-free survival (PFS). Indeed, PFS of
metastatic melanoma patients receiving combinatorial checkpoint
blockade could be stratified according to high, medium and low
serum VEGF-C, but not FlGF (VEGF-D, the other ligand for VEGFR-3)
or VEGF-A levels (FIG. 14D). Taken together, these data demonstrate
that serum VEGF-C levels prior to immunotherapy not only predicts
the magnitude and quality of immune responses raised by a cancer
vaccine, but also stratifies patient responses to combined
checkpoint blockade.
[0319] F. Lymphangiogenic Potentiation of Immunotherapy is
Dependent on CCR7 Signaling During Tumor Development and
Independent of T Cells Activated in the Lymph Node.
[0320] It has been previously shown that naive T cells can be
recruited and primed within primary tumors, and that adoptive
transfer of naive OT-I cells into B16-OVA tumors can delay primary
tumor growth. As such, we asked whether there was a mechanistic
link between the increased susceptibility to immunotherapy and the
increased accumulation of naive TILs within lymphangiogenic B16
tumors. We hypothesized that naive CCR7.sup.+ T cells within
lymphangiogenic tumors might be locally activated following
immunotherapy-induced release of tumor antigen and innate immune
activation ("danger signals"). In line with this, we found that
three days after ATT, lymphangiogenic B16-OVA/VC tumors but not the
tumor-draining lymph nodes (tdLNs) contained significantly higher
numbers of endogenous naive and effector CD8.sup.+ T cells as well
as transferred OT-I cells (FIG. 15A-B). To test whether the
efficacy of ATT in B16 melanoma indeed relied on VEGF-C-induced
recruitment of naive immune cells, B16-OVA/VC tumor-bearing mice
were treated with CCR7 blocking antibodies during tumor development
prior to therapy. We found that after the initial response to ATT,
B16-OVA/VC tumors of .alpha.CCR7-treated mice progressed similarly
to .alpha.R3-treated mice, leading to accelerated tumor growth and
significantly decreased survival as compared to isotype treated
tumors (FIG. 15C). To assess whether naive T cells might be
activated within the primary tumor microenvironment, we next
performed ATT while blocking lymphocyte egress from secondary
lymphoid organs using the small molecular inhibitor FTY720. Indeed,
tumor growth in the progression phase after ATT was only dependent
on VEGFR-3 signaling, but independent of lymphocyte egress (FIG.
15D). Accordingly, the blood of FTY720 treated mice was essentially
devoid of lymphocytes (FIG. 15E-F), further indicating that for the
long-term response, anti-tumor immunity does not rely on
circulating lymphocytes, but on intratumoral activation and
expansion of TILs. Together, these data demonstrate that
lymphangiogenic potentiation of immunotherapy in B16 melanomas
depends on CCR7-mediated attraction of naive T cells to and their
subsequent activation and expansion within the primary tumor
microenvironment.
[0321] G. Mice that Reject Lymphangiogenic B16 Melanomas after
Immunotherapy Show Epitope Spreading and Protection to
Re-Challenge
[0322] Immunotherapy can broaden the endogenous anti-tumor immune
response to tumor epitopes that were previously not or not
sufficiently visible, a process called epitope spreading. Because
we found that naive endogenous T cells are activated and expanded
within lymphangiogenic B16 melanomas in response to
antigen-specific immunotherapy, we hypothesized that spreading of
the anti-tumor immune response to antigens other than OVA could
occur in these settings. To determine whether this was the case, we
re-challenged mice that completely rejected the primary tumor (as
shown in FIG. 11E) with an intravenous injection of B16 WT or
B16-OVA/VC cells. Intriguingly, we found that mice that rejected
B16-OVA/VC tumors had increased numbers of overall effector
CD4.sup.+ and CD8.sup.+ T cells as well as of OVA-specific
CD8.sup.+ T cells in blood as compared to mice that had progressing
B16-OVA tumors (FIG. 16A). To assess whether the increased numbers
of effector T cells were the result of epitope spreading within the
tumor microenvironment, we re-challenged mice that were tumor free
for at least 10 days with pulmonary metastasis. While control mice
that only received OVA+CpG vaccination were only partially
protected against B16-OVA but not B16 WT metastasis, mice that
previously rejected B16-OVA/VC tumors were completely protected
against both B16-OVA and B16 WT metastasis (FIG. 16B-C). Moreover,
increased levels of circulating OVA-specific CD8.sup.+ T cells were
detected in mice resistant to pulmonary metastasis (FIG. 16D).
Taken together, these data suggest that antigen-specific
immunotherapy of lymphangiogenic B16 tumors induces secondary
immune responses against a variety of endogenous tumor antigens,
conferring long-term memory and protection against pulmonary
metastasis.
[0323] This work introduces a new and unexpected role of
tumor-associated lymphangiogenesis in enhancing the efficacy of
systemic immunotherapy. Although lymphangiogenic tumors are more
immunosuppressive before immunotherapy, it was surprisingly found
that they turned out to be more sensitive to systemic immunotherapy
as compared to those where VEGFR-3 signaling was blocked (FIGS. 11
and 18). It is hypothesized that lymphangiogenic potentiation of
immunotherapy depends on CCL21-mediated recruitment of CCR7.sup.+
immune cells, particularly naive T cells and DCs, into primary
melanoma tumors (FIGS. 12 and 17). When immunotherapy-induced
cytotoxicity occurs, the release of antigens and danger signals
induce local T cell activation, thereby leading to a
diversification of the endogenous anti-tumor response (`epitope
spreading`) and long-lasting memory (FIGS. 15 and 16). Importantly,
systemic immunotherapy and intratumoral activation of endogenous T
cells do not rely on the tumor draining lymph nodes, which have
acquired robust mechanisms of immuno suppression. These findings
thus point to tumor lymphangiogenesis (inferred from serum VEGF-C
levels) as a biomarker for tumor T cell infiltration, which others
have shown to correlate with responsiveness to immunotherapy. It is
shown here, in two independent clinical immunotherapy settings,
peptide vaccination and checkpoint blockade, that VEGF-C levels in
the serum positively correlated with the numbers and functionality
of circulating tumor-specific CD8.sup.+ T cells (FIG. 14A-C) and
progression-free survival, respectively (FIG. 14D).
[0324] These findings are surprising because VEGF-C expression in
human tumors is strongly correlated with LN metastases and poor
prognosis. Here the inventors find that lymphangiogenic melanomas
express high levels of CCL21 (FIGS. 12D and 19D), a lymphoid homing
chemokine that attracts naive and memory T cells as well as
dendritic cells and show that lymphangiogenic tumors contain
increased numbers of naive TILs that can contribute to increased
efficacy of different immunotherapy approaches (FIGS. 11 and 18).
It is shown here, for the first time, that CCL21 expression and
subsequent naive T cell infiltration within lymphangiogenic mouse
melanomas depends on VEGFR-3 signaling, that intratumoral LECs
express CCL21 and that VEGFC but not VEGFA or -D positively
correlated with CCL21, CCR7, and with a gene signature of T cell
inflammation in a human melanoma TCGA data set (FIG. 13E, F, and
FIG. 20). Taken together, this data show that LECs can actively
influence primary tumor inflammation by secreting CCL21, assigning
a new biological role of intra- and peritumoral lymphatics during
tumor progression.
[0325] We conclude that tumor expression of VEGF-C is a novel
mechanism by which tumor associated lymphatics actively increase
naive immune cell infiltration. Activation of naive T cells can be
induced by immunogenic cell death following immunotherapy,
resulting in a broad and long lasting anti-tumor immune response.
Considering our pre-clinical results, the immune microenvironment
specific to lymphangiogenic melanoma might offer a window of
opportunity to jumpstart an immunosuppressed Cancer-Immunity Cycle
in the clinic. We therefore propose tumor-associated
lymphangiogenesis to be a major determinant of a patients `cancer
immunogram` and speculate that engineering lymphangiogenesis within
primary melanoma might improve response rates of patients with
non-T cell-inflamed primary melanomas.
[0326] H. Materials and Methods
[0327] 1. Mice
[0328] Female wild-type mice, OT-I RAG-1.sup.-/- mice, and CD45.1
mice, all on the C57BL/6 background, were purchased from Harlan
Laboratories (Gannat, France) and used between 8-12 weeks of age.
Tyr:Cre-ER.sup.+/LSL-Braf.sup.V600E/Pten.sup.fl/fl genetically
engineered mice (BRAF GEM) were a kind gift from Dr. T. Gajewski
(University of Chicago). All experiments were performed with
approval from the Veterinary Authority of the Canton de Vaud,
Switzerland, and IACUC of University of Chicago, #72414.
[0329] 2. Tumor Cell Lines
[0330] B16-F10 melanoma cells (ATCC) were maintained in high
glucose DMEM (11995) supplemented with 10% heat-inactivated fetal
bovine serum (FBS) (both from Invitrogen, Zug, Switzerland).
B16-F10 ovalbumin (OVA) expressing cells (gift of Bertrand Huard,
University of Geneva, Geneva, Switzerland) were transfected with
control or VEGF-C lentivirus as described previously (B16-OVA and
B16-OVA/VC, respectively), and expression of VEGF-C and OVA were
confirmed before each experiment by qPCR and ELISA. Cells were
split 1:20 or 1:40 every 3-4 days.
[0331] 3. B16 Tumor Inoculation and Measurements
[0332] Before tumor inoculation, mice were anesthetized with
isoflurane and their backs were shaved. 2.5.times.10.sup.5 B16-OVA
or B16-OVA/VEGF-C cells in 30 .mu.l PBS were injected i.d. on the
front dorsolateral side above the shoulders. Tumor size was
monitored with a caliper, and volume (V) was calculated assuming an
ellipsoid shape (V=1/6.pi..times.length.times.width.times.height).
Mice were sacrificed when tumor volume exceeded 500 mm.sup.3.
[0333] 4. BRAF GEM Induction, Treatment and Measurements
[0334] 8-12-week-old mice were shaved on the back, while under
anesthesia. The next day, 5 .mu.l of hydroxytamoxifen
(4-OH-tamoxifen, Sigma-Aldrich) at 10 mg/mL in pure ethanol was
applied topically on the shaved area. Treatment started two weeks
after hydroxytamoxifen application, when tumor was first palpable.
Tumor volume was calculated as Volume=Surface*Z, where Surface is
generated through ImageJ analysis and Z measured using a digital
caliper, as previously described (Ishihara et al., 2017). Mice were
euthanized when tumor volume reached 1000 mm.sup.3 or when necrosis
occurred.
[0335] 5. Antibody Injections
[0336] To inhibit tumor lymphangiogenesis, mice received i.p.
injections of 500 .mu.g anti-VEGFR-3 antibody (mF4-31C1;
ImClone/Eli Lilly, New York, USA) or isotype rat IgG (I4131; Sigma,
Buchs, Switzerland) every 3-4 days starting the day of B16
inoculation. For CCR7 blocking studies, mice received i.p.
injections of 25 .mu.g anti-CCR7 antibody (16-1971; eBioscience,
Vienna, Austria) or isotype rat IgG on day 0, 3, and 6 after tumor
inoculation. For the GEM study, mice first received i.p. injections
of 500 .mu.g anti-VEGFR-3 antibody or isotype rat IgG every 4 days,
starting when the tumor was palpable. Mice then received i.p.
injections of 250 .mu.g anti-PD-1 antibody (RMP1-14; BioXCell, West
Lebanon, N.H., USA) every 4 days, starting 12 days after start of
VEGFR3 blocking treatment.
[0337] 6. Protein and Peptide Vaccinations
[0338] For protein vaccination, mice received 50 .mu.g CpG-B
(5'-TCCATGACGTTCCTGACGTT-3'; Microsynth, Balgach, Switzerland)
combined with 10 OVA grade V (A5503; Sigma) in two i.d. doses 25
.mu.l per hind footpad (targeting non-tumor-draining lymph nodes)
on day 4, 7, and 10 after tumor inoculation.
Trp-2-peptide-conjugated nanoparticles (NP-Trp2) were produced as
described previously, and 2 .mu.g NP-Trp2 combined with 10 .mu.g
OVA grade V administered as described for the protein vaccination.
For the GEM study, mice received two doses of 20 .mu.g of CpG-B and
2 .mu.g of gp100 peptide (KVPRNQDWL; GenScript, Piscataway, N.J.,
USA), at day 8 and 12 after start of VEGFR3 blocking treatment.
[0339] 7. OT-I Ex Vivo Activation and Therapeutic Adoptive
Transfer
[0340] Spleens and LNs were harvested from OT-I mice. Spleens were
placed in a petri dish containing 10 ml IMDM medium (31980-022;
Invitrogen) and disrupted with a scalpel before being transferred
through a 70 .mu.m filter into a 50 ml conical tube. Cells were
then spun down at 2000 rpm for 5 min and resuspended in 1 ml ACK
red blood cells lysis buffer (150 mM NH.sub.4Cl, 10 mM KHCO.sub.3,
and 0.1 mM Na.sub.2EDTA, pH 7.2). After 5 min incubation, 10 ml
IMDM was added, and the cells spun down at 2000 rpm for 5 min. LNs
were placed in a well of a twelve well plate containing 2 ml
digestion medium (IMDM with 1 mg/ml Collagenase D (1108886600;
Roche, Basel, Switzerland)). The lymph node capsules were gently
opened with two syringe needles (26 G) to allow better digestion,
and then incubated for 15 min at 37.degree. C. LN cells were then
spun down at 2000 rpm for 5 min and pooled in 10 ml IMDM with the
splenocytes for further processing. Dendritic cells (DCs) were
first isolated by CD11c.sup.+ magnetic cell sorting (130-052-001;
Milteny Biotec, Bergisch Gladbach, Germany) before CD8.sup.+ T
cells were isolated from the remaining cells by negative magnetic
cell sorting (130-095-236; Milteny Biotech) according to the
manufacturer's protocol. Next, isolated CD11c.sup.+ DCs and
CD8.sup.+ T cells were plated in 96-well plates (10,000 CD11c.sup.+
DCs and 100,000 CD8+ T cells per well) in stimulation medium (IMDM
with 10% FBS, 1% Pen/Strep, 1 nM SIINFEKL, and 10 U/ml mIL-2
(212-12; PreproTech, London, UK)). Cells were collected after 4
days and injected i.v. into tumor-bearing mice (10.sup.6 cells in
200 .mu.l in IMDM).
[0341] 8. Therapeutic Dendritic Cell Vaccination
[0342] BMDCs were generated from the bone marrow of C57BL/6 mice as
previously described. At day 8 of BMDC culture, 10 ng/ml LPS was
added for 12 h. The activated BMDCs were then washed with 20 ml
IMDM with 10% FBS and plated at 3.times.10.sup.6 cells/ml in petri
dishes. The cells were then pulsed with 1 .mu.M of SIINFEKL for 1
h. Cells were then collected and washed 3 times with 20 ml PBS.
Finally, cells were resuspended in PBS at 10.sup.7 cells/ml, and
100 .mu.l was injected i.p. per mouse.
[0343] 9. Lymph Node and Tumor Cell Isolation
[0344] Stromal cells (CD45.sup.-) and immune cells (CD45+) were
recovered from tumors and lymph nodes (LNs) as described. Briefly,
LN capsules were opened with syringe needles and digested in 1
mg/ml Collagenase IV (LS004188; Worthington-Biochem, New Jersey,
USA) and 40 .mu.g/ml DNAse I (11284932001; Roche) for 30 min at
37.degree. C. with magnetic stirring. Supernatant was then
carefully collected and remaining fragments were further digested
with 3.3 mg/ml Collagenase D (11088866001; Roche) and 40 .mu.g/ml
DNAse I for 15 min (for lymph nodes) or 45 min (for tumors)
followed by 500 mM EDTA.
[0345] 10. Immunohistochemistry
[0346] Mouse tumor samples were fixed in Zinc fixation buffer (4.5
mM CaCl.sub.2, 51.5 mM ZnCl.sub.2, 32 mM
Zn(CF.sub.3CO.sub.2).sub.2, and 38.5 mM glycine, pH 6.5),
paraffin-embedded and cut into 8 .mu.m sections. Formalin-fixed
human tumors were paraffin-embedded and cut into 4-5 .mu.m
sections. For immunostaining, paraffin-embedded tissue sections
were deparaffinized and rehydrated. Antigen retrieval was done on
PFA-fixed samples at 98.degree. C. for 15 min in TRIS/EDTA buffer
(pH 9.0). Sections were then stained according to standard
immunohistochemistry protocols. Primary antibodies used were:
rabbit anti-mouse LYVE-1 (103-PASO; RELIATech, Wolfenbuttel,
Germany, 1:200), mouse anti-human D2-40 (SIG-3739; BioLegend,
Lucerne, Switzerland), rabbit anti-human CCL21 (HPA051210; Sigma),
rabbit anti-human VEGFC (PAS-29772, Invitrogen). Quantification of
lymphatic vessels on entire tumor tile images was performed using
Fiji. LYVE-1.sup.+ cells with Prox 1.sup.- nuclei were excluded as
macrophages.
[0347] 11. Flow Cytometry
[0348] Antibody staining for surface targets were performed in PBS
with 2% FCS, and intracellular staining was performed after
fixation and permeabilization according to the manufacturers'
protocols. The following anti-mouse antibodies were used for flow
cytometry: CD45-APC (17-0451-82; eBioscience) or biotinylated CD45
(13-0451-85; eBioscience), CD4-PacBlue (100531; Biolegend) or
CD4-PE-Cy7 (100528; Biolegend), CD8.alpha.-PacOrange (MCD0830;
Invitrogen), F4/80-PerCPCy5.5 (123128; Biolegend), CD25-FITC
(101908; Biolegend), FoxP3-PerCPCy5.5 (45-5773-82; eBioscience),
CD62L-PE (12-0621-82; eBioscience), CD44-APCeF780 (47-0441-82;
eBioscience), CCR7-PE-Cy7 (25-1971-82; eBioscience) or
CCR7-PerCPCy5.5 (45-1971-82; eBioscience), biotinylated CD11b
(13-0112-82; eBioscience). Pentamer staining for H-2 kb-Trp2-PE
(SVYDFFVWL; TC Metrix, Lausanne, Switzerland), H-2Db-gp100-PE
(KVPRNQDWL; TC Metrix) and H-2 kb-SIINFEKL-PE (F093-2B; Prolmmune,
Oxford, UK) was performed according to manufacturers' guidelines.
Cell viability was determined using live/dead aqua (L34957;
Invitrogen) or red (L23102; Invitrogen) dyes. Flow cytometry
acquisition was performed on a Cyan flow cytometer (Beckman
Coulter) and data analysis was performed with FlowJo (Version
9.7.7.).
[0349] 12. ELISAs and Protein Array
[0350] Protein lysates were generated from excised tumors and lymph
node by homogenizing the tissues in 700 .mu.l (tumors) or 400 .mu.l
(lymph nodes) T-Per protein extraction buffer (78510; ThermoFisher)
containing cOmplete protease inhibitor cocktail (1 tablet per 10
ml, 11836145001; Roche). Tissue lysates were spun down at 2000 g
for 2 min, and transferred into new 1.5 ml microcentrifuge tubes.
Tissue lysates were then spun at 12'000 g for 10 min to get rid of
cell debris, and the supernatant was transferred into new 1.5 ml
eppendorf tubes which were stored at -80.degree. C. Lymph node
lysates were normalized to 0.025 mg/ml total protein, tumor lysates
to 1 mg/ml total protein, and ELISAs (CCL21: DY457, RnD Systems,
Abingdon, UK; VEGFC: DVECOO, RnD Systems) were performed according
to standard protocols. Cytokine protein arrays (AAM-CYT-2-2;
RayBiotech, Norcross Ga., USA) were performed according to
manufacturer's specifications.
[0351] 13. Analysis of TCGA Data Set Containing 469 Skin Cutaneous
Melanoma Patients
[0352] TCGA level 3 gene expression data were downloaded for skin
cutaneous melanoma (SKCM) from the Broad GDAC Firehose database
(http://gdac.broadinstitute.org/). The RNAseq data set called
"illuminahiseq_rnaseqv2-RSEM genes normalized (MD5)" with release
date 20151101 contained upper quartile normalized RSEM values
summarized at the gene level. The data was log 2 transformed after
the addition of one pseudoread. The total of 469 SKCM samples were
split into two groups according to whether the tissue biopsy was
retrieved from the primary melanoma site (103 samples) or a
metastatic site (369 samples) including sentinel lymph nodes and
distant organ metastasis. Heat maps were generated using RStudio
(RStudio Team (2015). RStudio: Integrated Development for R.
RStudio, Inc., Boston, Mass., version 0.99.893).
[0353] 14. Vaccination Trial in Melanoma Patients
[0354] For immunotherapy response correlations, we analyzed serum
from Stage III/IV melanoma patients that had been enrolled in a
prospective Phase I clinical study evaluating an anti-tumor peptide
vaccine (ClinicalTrials.gov Identifier NCT00112229) performed by
the Ludwig Institute for Cancer Research (University Hospital,
Lausanne, Switzerland). The trial was conducted according to the
relevant regulatory standards, upon approval by Swissmedic and the
"Commission d'Ethique de la Recherche Clinique de la Faculte de
Biologie et de Medecine, Universite de Lausanne", which also
approved the use of specimens from healthy volunteers. Patients
were enrolled upon written informed consent. Briefly, patients had
received monthly s.c. injections of a vaccine composed of CpG 7909
(PF-3512676) oligonucleotides and Melan-A/MART-1 peptide,
emulsified in Montanide ISA-51. Melan-A-specific CD8.sup.+ T cells
frequency and function was measured in blood by flow cytometry at
the time point of peak response (after a mean of 8 injections).
Serum VEGF-C was assessed using a commercial ELISA kit (DVECOO; RnD
Systems).
[0355] 15. Serum Study of Metastatic Melanoma Patients
[0356] Serum was collected from treatment-naive patients with
unresectable, stage III or stage IV metastatic melanoma in the
randomized, placebo controlled, multi-center, two arm, phase II
trial, BMS Checkmate-069 (CA209-069, NCT01927419). All patients
provided written informed consent within the study for the use of
biological material including serum analyzes. The study was
approved by all IRB s. The study compared ipilimumab (n=47) at 3
mg/kg, every 3 weeks for four doses to combined (n=95) ipilimumab,
3 mg/kg, every 3 weeks and nivolumab 1 mg/kg, every 3 weeks for 4
cycles, followed by nivolumab alone at 3 mg/kg, every 2 weeks until
disease progression or unacceptable toxicity (For details of the
study please also refer to NEJM and www.clinicaltrials.gov). 135
pre-treatment serum samples (46 in the ipilimumab arm and 89 in the
ipilimumab plus nivolumab arm) were analyzed, one sample per
patient. Depending on the volume of available serum, serum was
undiluted (n=103) or diluted (n=33) in median 1.16 times (range of
dilution factor: 1.02-2). 700 .mu.l per sample was analyzed using
the 440 Human Biomarker testing service (RayBiotech, Inc. Parkway
Lane, Suite 100, Norcross Ga., 30092). Only the ipilimumab plus
nivolumab arm was included for our analysis, from which 13 patients
were excluded because they had very short follow-up (<10 days)
and/or had no clear evaluation of response/progression at the first
data cutoff. Progression free survival was defined according RECIST
1.1 criteria. Patients were grouped according to the serum levels
of VEGF-C, -D, and -A into high (concentration>mean+SD/2.5), low
(concentration<mean-SD/2.5), and mid (in between high and
low).
[0357] 16. Statistical Analysis
[0358] Statistical analysis was done using Prism (v5.0d, GraphPad).
Except mentioned differently, statistically significant differences
between two experimental groups were determined by an unpaired
student's t-test, and by One-way ANOVA followed by Tukey's
post-test when more than two groups were compared. Statistical
significance between survival curves assessed with Log-rank
(Mantel-Cox) test. TCGA gene data correlations were tested using
non-parametric Spearman's test. *p<0.05, **p<0.01,
***p<0.001, ns: not significant.
[0359] All of the methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the methods and in the steps or in the sequence of steps
of the method described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
REFERENCES
[0360] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by reference.
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Sequence CWU 1
1
201419PRTHomo sapiens 1Met His Leu Leu Gly Phe Phe Ser Val Ala Cys
Ser Leu Leu Ala Ala1 5 10 15Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro
Ala Ala Ala Ala Ala Phe 20 25 30Glu Ser Gly Leu Asp Leu Ser Asp Ala
Glu Pro Asp Ala Gly Glu Ala 35 40 45Thr Ala Tyr Ala Ser Lys Asp Leu
Glu Glu Gln Leu Arg Ser Val Ser 50 55 60Ser Val Asp Glu Leu Met Thr
Val Leu Tyr Pro Glu Tyr Trp Lys Met65 70 75 80Tyr Lys Cys Gln Leu
Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln 85 90 95Ala Asn Leu Asn
Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala 100 105 110His Tyr
Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys 115 120
125Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe
130 135 140Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser
Val Tyr145 150 155 160Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu
Gln Cys Met Asn Thr 165 170 175Ser Thr Ser Tyr Leu Ser Lys Thr Leu
Phe Glu Ile Thr Val Pro Leu 180 185 190Ser Gln Gly Pro Lys Pro Val
Thr Ile Ser Phe Ala Asn His Thr Ser 195 200 205Cys Arg Cys Met Ser
Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile 210 215 220Ile Arg Arg
Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn225 230 235
240Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys
245 250 255Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp
Asp Ser 260 265 270Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys
Glu Leu Asp Glu 275 280 285Glu Thr Cys Gln Cys Val Cys Arg Ala Gly
Leu Arg Pro Ala Ser Cys 290 295 300Gly Pro His Lys Glu Leu Asp Arg
Asn Ser Cys Gln Cys Val Cys Lys305 310 315 320Asn Lys Leu Phe Pro
Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu 325 330 335Asn Thr Cys
Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro 340 345 350Leu
Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys 355 360
365Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr
370 375 380Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly
Phe Ser385 390 395 400Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser
Tyr Trp Lys Arg Pro 405 410 415Gln Met Ser2415PRTMus musculus 2Met
His Leu Leu Cys Phe Leu Ser Leu Ala Cys Ser Leu Leu Ala Ala1 5 10
15Ala Leu Ile Pro Ser Pro Arg Glu Ala Pro Ala Thr Val Ala Ala Phe
20 25 30Glu Ser Gly Leu Gly Phe Ser Glu Ala Glu Pro Asp Gly Gly Glu
Val 35 40 45Lys Ala Phe Glu Gly Lys Asp Leu Glu Glu Gln Leu Arg Ser
Val Ser 50 55 60Ser Val Asp Glu Leu Met Ser Val Leu Tyr Pro Asp Tyr
Trp Lys Met65 70 75 80Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln
Gln Pro Thr Leu Asn 85 90 95Thr Arg Thr Gly Asp Ser Val Lys Phe Ala
Ala Ala His Tyr Asn Thr 100 105 110Glu Ile Leu Lys Ser Ile Asp Asn
Glu Trp Arg Lys Thr Gln Cys Met 115 120 125Pro Arg Glu Val Cys Ile
Asp Val Gly Lys Glu Phe Gly Ala Ala Thr 130 135 140Asn Thr Phe Phe
Lys Pro Pro Cys Val Ser Val Tyr Arg Cys Gly Gly145 150 155 160Cys
Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr Ser Thr Gly Tyr 165 170
175Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu Ser Gln Gly Pro
180 185 190Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser Cys Arg
Cys Met 195 200 205Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile
Ile Arg Arg Ser 210 215 220Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala
Ala Asn Lys Thr Cys Pro225 230 235 240Thr Asn Tyr Val Trp Asn Asn
Tyr Met Cys Arg Cys Leu Ala Gln Gln 245 250 255Asp Phe Ile Phe Tyr
Ser Asn Val Glu Asp Asp Ser Thr Asn Gly Phe 260 265 270His Asp Val
Cys Gly Pro Asn Lys Glu Leu Asp Glu Asp Thr Cys Gln 275 280 285Cys
Val Cys Lys Gly Gly Leu Arg Pro Ser Ser Cys Gly Pro His Lys 290 295
300Glu Leu Asp Arg Asp Ser Cys Gln Cys Val Cys Lys Asn Lys Leu
Phe305 310 315 320Pro Asn Ser Cys Gly Ala Asn Arg Glu Phe Asp Glu
Asn Thr Cys Gln 325 330 335Cys Val Cys Lys Arg Thr Cys Pro Arg Asn
Gln Pro Leu Asn Pro Gly 340 345 350Lys Cys Ala Cys Glu Cys Thr Glu
Asn Thr Gln Lys Cys Phe Leu Lys 355 360 365Gly Lys Lys Phe His His
Gln Thr Cys Ser Cys Tyr Arg Arg Pro Cys 370 375 380Ala Asn Arg Leu
Lys His Cys Asp Pro Gly Leu Ser Phe Ser Glu Glu385 390 395 400Val
Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro His Leu Asn 405 410
4153354PRTHomo sapiens 3Met Tyr Arg Glu Trp Val Val Val Asn Val Phe
Met Met Leu Tyr Val1 5 10 15Gln Leu Val Gln Gly Ser Ser Asn Glu His
Gly Pro Val Lys Arg Ser 20 25 30Ser Gln Ser Thr Leu Glu Arg Ser Glu
Gln Gln Ile Arg Ala Ala Ser 35 40 45Ser Leu Glu Glu Leu Leu Arg Ile
Thr His Ser Glu Asp Trp Lys Leu 50 55 60Trp Arg Cys Arg Leu Arg Leu
Lys Ser Phe Thr Ser Met Asp Ser Arg65 70 75 80Ser Ala Ser His Arg
Ser Thr Arg Phe Ala Ala Thr Phe Tyr Asp Ile 85 90 95Glu Thr Leu Lys
Val Ile Asp Glu Glu Trp Gln Arg Thr Gln Cys Ser 100 105 110Pro Arg
Glu Thr Cys Val Glu Val Ala Ser Glu Leu Gly Lys Ser Thr 115 120
125Asn Thr Phe Phe Lys Pro Pro Cys Val Asn Val Phe Arg Cys Gly Gly
130 135 140Cys Cys Asn Glu Glu Ser Leu Ile Cys Met Asn Thr Ser Thr
Ser Tyr145 150 155 160Ile Ser Lys Gln Leu Phe Glu Ile Ser Val Pro
Leu Thr Ser Val Pro 165 170 175Glu Leu Val Pro Val Lys Val Ala Asn
His Thr Gly Cys Lys Cys Leu 180 185 190Pro Thr Ala Pro Arg His Pro
Tyr Ser Ile Ile Arg Arg Ser Ile Gln 195 200 205Ile Pro Glu Glu Asp
Arg Cys Ser His Ser Lys Lys Leu Cys Pro Ile 210 215 220Asp Met Leu
Trp Asp Ser Asn Lys Cys Lys Cys Val Leu Gln Glu Glu225 230 235
240Asn Pro Leu Ala Gly Thr Glu Asp His Ser His Leu Gln Glu Pro Ala
245 250 255Leu Cys Gly Pro His Met Met Phe Asp Glu Asp Arg Cys Glu
Cys Val 260 265 270Cys Lys Thr Pro Cys Pro Lys Asp Leu Ile Gln His
Pro Lys Asn Cys 275 280 285Ser Cys Phe Glu Cys Lys Glu Ser Leu Glu
Thr Cys Cys Gln Lys His 290 295 300Lys Leu Phe His Pro Asp Thr Cys
Ser Cys Glu Asp Arg Cys Pro Phe305 310 315 320His Thr Arg Pro Cys
Ala Ser Gly Lys Thr Ala Cys Ala Lys His Cys 325 330 335Arg Phe Pro
Lys Glu Lys Arg Ala Ala Gln Gly Pro His Ser Arg Lys 340 345 350Asn
Pro4358PRTMus musculus 4Met Tyr Gly Glu Trp Gly Met Gly Asn Ile Leu
Met Met Phe His Val1 5 10 15Tyr Leu Val Gln Gly Phe Arg Ser Glu His
Gly Pro Val Lys Asp Phe 20 25 30Ser Phe Glu Arg Ser Ser Arg Ser Met
Leu Glu Arg Ser Glu Gln Gln 35 40 45Ile Arg Ala Ala Ser Ser Leu Glu
Glu Leu Leu Gln Ile Ala His Ser 50 55 60Glu Asp Trp Lys Leu Trp Arg
Cys Arg Leu Lys Leu Lys Ser Leu Ala65 70 75 80Ser Met Asp Ser Arg
Ser Ala Ser His Arg Ser Thr Arg Phe Ala Ala 85 90 95Thr Phe Tyr Asp
Thr Glu Thr Leu Lys Val Ile Asp Glu Glu Trp Gln 100 105 110Arg Thr
Gln Cys Ser Pro Arg Glu Thr Cys Val Glu Val Ala Ser Glu 115 120
125Leu Gly Lys Thr Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Asn Val
130 135 140Phe Arg Cys Gly Gly Cys Cys Asn Glu Glu Gly Val Met Cys
Met Asn145 150 155 160Thr Ser Thr Ser Tyr Ile Ser Lys Gln Leu Phe
Glu Ile Ser Val Pro 165 170 175Leu Thr Ser Val Pro Glu Leu Val Pro
Val Lys Ile Ala Asn His Thr 180 185 190Gly Cys Lys Cys Leu Pro Thr
Gly Pro Arg His Pro Tyr Ser Ile Ile 195 200 205Arg Arg Ser Ile Gln
Thr Pro Glu Glu Asp Glu Cys Pro His Ser Lys 210 215 220Lys Leu Cys
Pro Ile Asp Met Leu Trp Asp Asn Thr Lys Cys Lys Cys225 230 235
240Val Leu Gln Asp Glu Thr Pro Leu Pro Gly Thr Glu Asp His Ser Tyr
245 250 255Leu Gln Glu Pro Thr Leu Cys Gly Pro His Met Thr Phe Asp
Glu Asp 260 265 270Arg Cys Glu Cys Val Cys Lys Ala Pro Cys Pro Gly
Asp Leu Ile Gln 275 280 285His Pro Glu Asn Cys Ser Cys Phe Glu Cys
Lys Glu Ser Leu Glu Ser 290 295 300Cys Cys Gln Lys His Lys Ile Phe
His Pro Asp Thr Cys Ser Cys Glu305 310 315 320Asp Arg Cys Pro Phe
His Thr Arg Thr Cys Ala Ser Arg Lys Pro Ala 325 330 335Cys Gly Lys
His Trp Arg Phe Pro Lys Glu Thr Arg Ala Gln Gly Leu 340 345 350Tyr
Ser Gln Glu Asn Pro 3555134PRTArtificial SequenceSynthetic Peptide
5Met Ala Gln Ser Leu Ala Leu Ser Leu Leu Ile Leu Val Leu Ala Phe1 5
10 15Gly Ile Pro Arg Thr Gln Gly Ser Asp Gly Gly Ala Gln Asp Cys
Cys 20 25 30Leu Lys Tyr Ser Gln Arg Lys Ile Pro Ala Lys Val Val Arg
Ser Tyr 35 40 45Arg Lys Gln Glu Pro Ser Leu Gly Cys Ser Ile Pro Ala
Ile Leu Phe 50 55 60Leu Pro Arg Lys Arg Ser Gln Ala Glu Leu Cys Ala
Asp Pro Lys Glu65 70 75 80Leu Trp Val Gln Gln Leu Met Gln His Leu
Asp Lys Thr Pro Ser Pro 85 90 95Gln Lys Pro Ala Gln Gly Cys Arg Lys
Asp Arg Gly Ala Ser Lys Thr 100 105 110Gly Lys Lys Gly Lys Gly Ser
Lys Gly Cys Lys Arg Thr Glu Arg Ser 115 120 125Gln Thr Pro Lys Gly
Pro 1306133PRTArtificial SequenceSynthetic Peptide 6Met Ala Gln Met
Met Thr Leu Ser Leu Leu Ser Leu Val Leu Ala Leu1 5 10 15Cys Ile Pro
Trp Thr Gln Gly Ser Asp Gly Gly Gly Gln Asp Cys Cys 20 25 30Leu Lys
Tyr Ser Gln Lys Lys Ile Pro Tyr Ser Ile Val Arg Gly Tyr 35 40 45Arg
Lys Gln Glu Pro Ser Leu Gly Cys Pro Ile Pro Ala Ile Leu Phe 50 55
60Leu Pro Arg Lys His Ser Lys Pro Glu Leu Cys Ala Asn Pro Glu Glu65
70 75 80Gly Trp Val Gln Asn Leu Met Arg Arg Leu Asp Gln Pro Pro Ala
Pro 85 90 95Gly Lys Gln Ser Pro Gly Cys Arg Lys Asn Arg Gly Thr Ser
Lys Ser 100 105 110Gly Lys Lys Gly Lys Gly Ser Lys Gly Cys Lys Arg
Thr Glu Gln Thr 115 120 125Gln Pro Ser Arg Gly 130731PRTArtificial
SequenceSynthetic Peptide 7Met His Leu Leu Gly Phe Phe Ser Val Ala
Cys Ser Leu Leu Ala Ala1 5 10 15Ala Leu Leu Pro Gly Pro Arg Glu Ala
Pro Ala Ala Ala Ala Ala 20 25 30880PRTArtificial SequenceSynthetic
Peptide 8Phe Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala
Gly Glu1 5 10 15Ala Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu
Arg Ser Val 20 25 30Ser Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro
Glu Tyr Trp Lys 35 40 45Met Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp
Gln His Asn Arg Glu 50 55 60Gln Ala Asn Leu Asn Ser Arg Thr Glu Glu
Thr Ile Lys Phe Ala Ala65 70 75 809192PRTArtificial
SequenceSynthetic Peptide 9Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln
Ala Ala Asn Lys Thr Cys1 5 10 15Pro Thr Asn Tyr Met Trp Asn Asn His
Ile Cys Arg Cys Leu Ala Gln 20 25 30Glu Asp Phe Met Phe Ser Ser Asp
Ala Gly Asp Asp Ser Thr Asp Gly 35 40 45Phe His Asp Ile Cys Gly Pro
Asn Lys Glu Leu Asp Glu Glu Thr Cys 50 55 60Gln Cys Val Cys Arg Ala
Gly Leu Arg Pro Ala Ser Cys Gly Pro His65 70 75 80Lys Glu Leu Asp
Arg Asn Ser Cys Gln Cys Val Cys Lys Asn Lys Leu 85 90 95Phe Pro Ser
Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr Cys 100 105 110Gln
Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu Asn Pro 115 120
125Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys Cys Leu Leu
130 135 140Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr Arg
Arg Pro145 150 155 160Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly
Phe Ser Tyr Ser Glu 165 170 175Glu Val Cys Arg Cys Val Pro Ser Tyr
Trp Lys Arg Pro Gln Met Ser 180 185 19010140PRTArtificial
SequenceSynthetic Peptide 10Cys Gly Pro Asn Lys Glu Leu Asp Glu Glu
Thr Cys Gln Cys Val Cys1 5 10 15Arg Ala Gly Leu Arg Pro Ala Ser Cys
Gly Pro His Lys Glu Leu Asp 20 25 30Arg Asn Ser Cys Gln Cys Val Cys
Lys Asn Lys Leu Phe Pro Ser Gln 35 40 45Cys Gly Ala Asn Arg Glu Phe
Asp Glu Asn Thr Cys Gln Cys Val Cys 50 55 60Lys Arg Thr Cys Pro Arg
Asn Gln Pro Leu Asn Pro Gly Lys Cys Ala65 70 75 80Cys Glu Cys Thr
Glu Ser Pro Gln Lys Cys Leu Leu Lys Gly Lys Lys 85 90 95Phe His His
Gln Thr Cys Ser Cys Tyr Arg Arg Pro Cys Thr Asn Arg 100 105 110Gln
Lys Ala Cys Glu Pro Gly Phe Ser Tyr Ser Glu Glu Val Cys Arg 115 120
125Cys Val Pro Ser Tyr Trp Lys Arg Pro Gln Met Ser 130 135
1401183PRTArtificial SequenceSynthetic Peptide 11Cys Gly Pro Asn
Lys Glu Leu Asp Glu Glu Thr Cys Gln Cys Val Cys1 5 10 15Arg Ala Gly
Leu Arg Pro Ala Ser Cys Gly Pro His Lys Glu Leu Asp 20 25 30Arg Asn
Ser Cys Gln Cys Val Cys Lys Asn Lys Leu Phe Pro Ser Gln 35 40 45Cys
Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr Cys Gln Cys Val Cys 50 55
60Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu Asn Pro Gly Lys Cys Ala65
70 75 80Cys Glu Cys12116PRTHomo sapiens 12Ala His Tyr Asn Thr Glu
Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg1 5 10 15Lys Thr Gln Cys Met
Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu 20 25 30Phe Gly Val Ala
Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val 35 40 45Tyr Arg Cys
Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn 50 55 60Thr Ser
Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro65 70 75
80Leu Ser Gln Gly Pro Lys Pro Val Thr Ile Ser
Phe Ala Asn His Thr 85 90 95Ser Cys Arg Cys Met Ser Lys Leu Asp Val
Tyr Arg Gln Val His Ser 100 105 110Ile Ile Arg Arg
11513109PRTArtificial SequenceSynthetic Peptide 13Ala His Tyr Asn
Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg1 5 10 15Lys Thr Gln
Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu 20 25 30Phe Gly
Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val 35 40 45Tyr
Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn 50 55
60Thr Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro65
70 75 80Leu Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His
Thr 85 90 95Ser Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln 100
1051497PRTArtificial SequenceSynthetic Peptide 14Cys Pro Ile Asp
Met Leu Trp Asp Ser Asn Lys Cys Lys Cys Val Leu1 5 10 15Gln Glu Glu
Asn Pro Leu Ala Gly Thr Glu Asp His Ser His Leu Gln 20 25 30Glu Pro
Ala Leu Cys Gly Pro His Met Met Phe Asp Glu Asp Arg Cys 35 40 45Glu
Cys Val Cys Lys Thr Pro Cys Pro Lys Asp Leu Ile Gln His Pro 50 55
60Lys Asn Cys Ser Cys Phe Glu Cys Lys Glu Ser Leu Glu Thr Cys Cys65
70 75 80Gln Lys His Lys Leu Phe His Pro Asp Thr Cys Ser Cys Glu Asp
Arg 85 90 95Cys15117PRTHomo sapiens 15Phe Ala Ala Thr Phe Tyr Asp
Ile Glu Thr Leu Lys Val Ile Asp Glu1 5 10 15Glu Trp Gln Arg Thr Gln
Cys Ser Pro Arg Glu Thr Cys Val Glu Val 20 25 30Ala Ser Glu Leu Gly
Lys Ser Thr Asn Thr Phe Phe Lys Pro Pro Cys 35 40 45Val Asn Val Phe
Arg Cys Gly Gly Cys Cys Asn Glu Glu Ser Leu Ile 50 55 60Cys Met Asn
Thr Ser Thr Ser Tyr Ile Ser Lys Gln Leu Phe Glu Ile65 70 75 80Ser
Val Pro Leu Thr Ser Val Pro Glu Leu Val Pro Val Lys Val Ala 85 90
95Asn His Thr Gly Cys Lys Cys Leu Pro Thr Ala Pro Arg His Pro Tyr
100 105 110Ser Ile Ile Arg Arg 1151623PRTArtificial
SequenceSynthetic Peptide 16Met Ala Gln Ser Leu Ala Leu Ser Leu Leu
Ile Leu Val Leu Ala Phe1 5 10 15Gly Ile Pro Arg Thr Gln Gly
2017111PRTArtificial SequenceSynthetic Peptide 17Ser Asp Gly Gly
Ala Gln Asp Cys Cys Leu Lys Tyr Ser Gln Arg Lys1 5 10 15Ile Pro Ala
Lys Val Val Arg Ser Tyr Arg Lys Gln Glu Pro Ser Leu 20 25 30Gly Cys
Ser Ile Pro Ala Ile Leu Phe Leu Pro Arg Lys Arg Ser Gln 35 40 45Ala
Glu Leu Cys Ala Asp Pro Lys Glu Leu Trp Val Gln Gln Leu Met 50 55
60Gln His Leu Asp Lys Thr Pro Ser Pro Gln Lys Pro Ala Gln Gly Cys65
70 75 80Arg Lys Asp Arg Gly Ala Ser Lys Thr Gly Lys Lys Gly Lys Gly
Ser 85 90 95Lys Gly Cys Lys Arg Thr Glu Arg Ser Gln Thr Pro Lys Gly
Pro 100 105 11018134PRTArtificial SequenceSynthetic Peptide 18Met
Ala Gln Ser Leu Ala Leu Ser Leu Leu Ile Leu Val Leu Ala Phe1 5 10
15Gly Ile Pro Arg Thr Gln Gly Ser Asp Gly Gly Ala Gln Asp Cys Cys
20 25 30Leu Lys Tyr Ser Gln Arg Lys Ile Pro Ala Lys Val Val Arg Ser
Tyr 35 40 45Arg Lys Gln Glu Pro Ser Leu Gly Cys Ser Ile Pro Ala Ile
Leu Phe 50 55 60Leu Pro Arg Lys Arg Ser Gln Ala Glu Leu Cys Ala Asp
Pro Lys Glu65 70 75 80Leu Trp Val Gln Gln Leu Met Gln His Leu Asp
Lys Thr Pro Ser Pro 85 90 95Gln Lys Pro Ala Gln Gly Cys Arg Arg Arg
Pro Lys Gly Arg Gly Lys 100 105 110Arg Arg Arg Glu Lys Gln Arg Lys
Gly Cys Lys Arg Thr Glu Arg Ser 115 120 125Gln Thr Pro Lys Gly Pro
13019156PRTArtificial SequenceSynthetic Peptide 19Met Ala Gln Ser
Leu Ala Leu Ser Leu Leu Ile Leu Val Leu Ala Phe1 5 10 15Gly Ile Pro
Arg Thr Gln Gly Ser Asp Gly Gly Ala Gln Asp Cys Cys 20 25 30Leu Lys
Tyr Ser Gln Arg Lys Ile Pro Ala Lys Val Val Arg Ser Tyr 35 40 45Arg
Lys Gln Glu Pro Ser Leu Gly Cys Ser Ile Pro Ala Ile Leu Phe 50 55
60Leu Pro Arg Lys Arg Ser Gln Ala Glu Leu Cys Ala Asp Pro Lys Glu65
70 75 80Leu Trp Val Gln Gln Leu Met Gln His Leu Asp Lys Thr Pro Ser
Pro 85 90 95Gln Lys Pro Ala Gln Gly Cys Arg Lys Asp Arg Gly Ala Ser
Lys Thr 100 105 110Gly Lys Lys Gly Lys Gly Ser Lys Gly Cys Lys Arg
Thr Glu Arg Ser 115 120 125Gln Thr Pro Lys Gly Pro Arg Arg Arg Pro
Lys Gly Arg Gly Lys Arg 130 135 140Arg Arg Glu Lys Gln Arg Pro Thr
Asp Ala His Leu145 150 15520133PRTArtificial SequenceSynthetic
Peptide 20Met Ala Gln Met Met Thr Leu Ser Leu Leu Ser Leu Val Leu
Ala Leu1 5 10 15Cys Ile Pro Trp Thr Gln Gly Ser Asp Gly Gly Gly Gln
Asp Cys Cys 20 25 30Leu Lys Tyr Ser Gln Lys Lys Ile Pro Tyr Ser Ile
Val Arg Gly Tyr 35 40 45Arg Lys Gln Glu Pro Ser Leu Gly Cys Pro Ile
Pro Ala Ile Leu Phe 50 55 60Leu Pro Arg Lys His Ser Lys Pro Glu Leu
Cys Ala Asn Pro Glu Glu65 70 75 80Gly Trp Val Gln Asn Leu Met Arg
Arg Leu Asp Gln Pro Pro Ala Pro 85 90 95Gly Lys Gln Ser Pro Gly Cys
Arg Lys Asn Arg Gly Thr Ser Lys Ser 100 105 110Gly Lys Lys Gly Lys
Gly Ser Lys Gly Cys Lys Arg Thr Glu Gln Thr 115 120 125Gln Pro Ser
Arg Gly 130
* * * * *
References