U.S. patent application number 13/336523 was filed with the patent office on 2012-06-14 for multivalent entrain-and-amplify immunotherapeutics for carcinoma.
This patent application is currently assigned to MannKind Corporation. Invention is credited to Adrian Ion Bot, Chih-Sheng Chiang, David C. Diamond, Jian Gong, Liping Liu, Xiping Liu, Zhiyong Qiu, Kent Andrew Smith.
Application Number | 20120148613 13/336523 |
Document ID | / |
Family ID | 37309646 |
Filed Date | 2012-06-14 |
United States Patent
Application |
20120148613 |
Kind Code |
A1 |
Bot; Adrian Ion ; et
al. |
June 14, 2012 |
MULTIVALENT ENTRAIN-AND-AMPLIFY IMMUNOTHERAPEUTICS FOR
CARCINOMA
Abstract
The present invention provides a method of treating a cell
proliferative disease such as cancer by providing to a subject in
need thereof an immunogenic composition comprising plasmid and
peptide(s) or analogues thereof. In embodiments of the present
invention there is provided methods and compositions for inducing,
entraining, and/or amplifying the immune response to MHC class-I
restricted epitopes of carcinoma antigens to generate an effective
anti-cancer immune response.
Inventors: |
Bot; Adrian Ion; (Valencia,
CA) ; Chiang; Chih-Sheng; (Chatsworth, CA) ;
Diamond; David C.; (West Hills, CA) ; Gong; Jian;
(Northridge, CA) ; Smith; Kent Andrew; (Ventura,
CA) ; Liu; Liping; (Manassas, VA) ; Liu;
Xiping; (Temple City, CA) ; Qiu; Zhiyong; (Los
Angeles, CA) |
Assignee: |
MannKind Corporation
Valencia
CA
|
Family ID: |
37309646 |
Appl. No.: |
13/336523 |
Filed: |
December 23, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11455279 |
Jun 16, 2006 |
8084592 |
|
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13336523 |
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60691581 |
Jun 17, 2005 |
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Current U.S.
Class: |
424/185.1 |
Current CPC
Class: |
A61K 39/001184 20180801;
A61K 39/00 20130101; A61K 39/001156 20180801; A61K 39/001195
20180801; A01K 2227/105 20130101; A61K 39/001191 20180801; C07K
14/70539 20130101; A61K 2039/53 20130101; A61K 2039/545 20130101;
A61K 39/0011 20130101; A61K 39/001188 20180801; A01K 2207/15
20130101; A61P 35/00 20180101; A01K 67/0275 20130101; A01K 2267/03
20130101; A61K 39/001189 20180801; C07K 14/4748 20130101; A01K
2217/00 20130101; C12N 15/8509 20130101; A01K 2217/05 20130101 |
Class at
Publication: |
424/185.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61P 35/00 20060101 A61P035/00 |
Claims
1-43. (canceled)
44. An immunogenic product comprising a plurality of compositions
comprising one or more nucleic acid compositions and one or more
peptide compositions; wherein the one or more nucleic acid
compositions are capable of expressing two or more class I MHC
restricted epitopes, wherein the two or more class I MHC restricted
epitopes comprise a Melan-A epitope, or a cross reactive analogue
thereof, and a Tyrosinase epitope, or a cross-reactive analogue
thereof, and wherein the one or more peptide compositions comprise
one or more class I MHC restricted epitopes, wherein the one or
more class I MHC restricted epitopes comprise the Melan-A epitope,
or a cross-reactive analogue thereof, or the Tyrosinase epitope, or
a cross-reactive analogue thereof, or both.
45. The immunogenic product of claim 44, wherein the Melan-A
epitope is selected from Melan-A.sub.26-35 (SEQ ID NO. 9) or an
analogue thereof.
46. The immunogenic product of claim 45, wherein the analogue of
said Melan-A epitope is E{Nva}AGIGILTV (SEQ ID NO. 11).
47. The immunogenic product of claim 45, wherein the analogue of
said Melan-A epitope is the A27L analogue of the Melan-A.sub.26-35
(SEQ ID NO. 9).
48. The immunogenic product of claim 44, wherein the Tyrosinase
epitope is selected from Tyrosinase.sub.369-377 (SEQ ID NO. 10), or
an analogue thereof.
49. The immunogenic product of claim 48, wherein the analogue of
said Tyrosinase epitope is YMDGTMSQ{Nva} (SEQ ID NO. 12).
50. The immunogenic product of claim 44, wherein the one or more
nucleic acid compositions is one composition.
51. The immunogenic product of claim 44, wherein one molecule is
capable of expressing the two or more class I MHC restricted
epitopes.
52. The immunogenic product of claim 44, wherein the one or more
nucleic acid compositions comprise a sequence encoding the
liberation sequence of SEQ ID NO. 15.
53. The immunogenic product of claim 44, wherein the one or more
nucleic acid compositions comprise a sequence encoding the
immunogenic polypeptide of SEQ ID NO. 18.
54. The immunogenic product of claim 53, wherein the one or more
nucleic acid compositions comprise pSEM (SEQ ID NO:19).
55. The immunogenic product of claim 44, further comprising at
least one of: i. a nucleic acid molecule capable of expressing an
SSX-2 class I MHC restricted epitope, or analogue thereof; ii. a
nucleic acid molecule capable of expressing an NY-ESO-1 class I MHC
restricted epitope, or analogue thereof; iii. a nucleic acid
molecule capable of expressing a PRAME class I MHC restricted
epitope, or analogue thereof; iv. a nucleic acid molecule capable
of expressing a PSMA class I MHC restricted epitope, or analogue
thereof; v. a peptide consisting essentially of an SSX-2 class I
MHC restricted epitope, or analogue thereof; vi. a peptide
consisting essentially of an NY-ESO-1 class I MHC restricted
epitope, or analogue thereof; vii. a peptide consisting essentially
of a PRAME class I MHC restricted epitope, or analogue thereof or
viii. a peptide consisting essentially of a PSMA class I MHC
restricted epitope, or analogue thereof.
56. The immunogenic product of claim 55, wherein the SSX-2 epitope
is SSX-2.sub.41-49 (SEQ ID NO. 1) or its analogue KVSEKIFYV (SEQ ID
NO. 5); the NY-ESO-1 epitope is NY-ESO-1.sub.157-165 (SEQ ID NO. 2)
or its analogue S{Nva}LMWITQV (SEQ ID NO. 6); the PRAME epitope is
PRAME.sub.425-433 (SEQ ID NO. 3) or its analogue S{Nva}LQHLIG{Nle}
(SEQ ID NO. 7); or the PSMA epitope is PSMA.sub.288-297 (SEQ ID NO.
4) or its analogue GLPSIPVHPV (SEQ ID NO. 8).
57. The immunogenic product of claim 55, wherein said one or more
nucleic acid compositions comprise a plasmid selected from the
group consisting of pSEM, pBPL and pRP12.
58. A method of treating cancer comprising administering the
product of claim 44 to a patient in need thereof.
59. The method of claim 58, wherein the cancer is a skin cancer, a
melanoma, or a glioblastoma.
60. The method of claim 58, further comprising a step of
administering to a patient in need thereof a composition
comprising: i) a nucleic acid molecule capable of expressing an
SSX-2 class I MHC restricted epitope, or analogue thereof; or ii) a
nucleic acid molecule capable of expressing an NY-ESO-1 class I MHC
restricted epitope, or analogue thereof; or iii) a nucleic acid
molecule capable of expressing an PRAME class I MHC restricted
epitope, or analogue thereof; or iv) a nucleic acid molecule
capable of expressing an PSMA class I MHC restricted epitope, or
analogue thereof; or v) a peptide consisting essentially of an
SSX-2 class I MHC restricted epitope, or analogue thereof; vi) a
peptide consisting essentially of an NY-ESO-1 class I MHC
restricted epitope, or analogue thereof; vii) a peptide consisting
essentially of a PRAME class I MHC restricted epitope, or analogue
thereof; or viii) a peptide consisting essentially of a PSMA class
I MHC restricted epitope, or analogue thereof.
61. The method of claim 58, comprising administering the plurality
of compositions for entraining and amplifying a T cell response in
a subject.
62. The method of claim 58, comprising administering the plurality
of compositions for inducing an anti-cancer immune response in a
subject.
Description
[0001] The present application claims the benefit of the tiling
date of U.S. Provisional Patent Application Ser. No. 60/691,581,
filed on Jun. 17, 2005, the entire text of which is incorporated
herein by reference without disclaimer.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention disclosed herein relates to methods and
compositions for inducing an MHC class-I restricted immune
response, controlling the nature and magnitude of the response,
particularly a multivalent response, and promoting effective
immunologic intervention in pathogenic processes. Disclosed herein
are methods and compositions for inducing an immune response
against various combinations of tumor-associated antigens, which
can promote effective immunologic intervention in
pathogenic-processes.
[0004] 2. Description of the Related Art
[0005] The American Cancer Society has estimated that over one
million people get cancer each year, and that approximately one out
of every two American men and one out of every three American women
will have some type of cancer at some point during their
lifetime.
[0006] Normal body cells grow, divide, and die in an orderly
fashion. In cell proliferative diseases such as cancer, cells,
instead of dying, continue to grow out of control and divide.
Although there are many kinds of cancer, they usually start because
of out-of-control growth of abnormal cells.
[0007] Usual treatment options for cancer include surgery,
radiation therapy, and chemotherapy. A fourth branch of treatment,
which is referred to as immunotherapy, has more recently become
established. Immunotherapies are designed to help the immune system
recognize cancer cells, and/or to strengthen a response against
cancer cells in order to destroy the cancer. Immunotherapies
include active and passive immunotherapies. Active immuotherapies
attempt to stimulate the body's own immune system to fight the
disease. Passive immunotherapies generally do not rely on the
patient's immune system to attack the disease; instead, they use
immune system components (such as antibodies) created outside of
the patient's body.
[0008] The immune system can be categorized into two discrete
effector arms, innate and adaptive immunity. Innate immunity
involves numerous cellular components and soluble factors that
respond immediately, but generally to foreign stimuli. Adaptive
immunity is customized to respond specifically to precise epitopes
from foreign agents. The adaptive immune response is further
divided into two effector arms known as the humoral and cellular
immune systems. The humoral arm is centered on the production of
antibodies by B-lymphocytes while the cellular arm involves the
cytolytic activity of cytotoxic T lymphocytes.
[0009] Cytotoxic T lymphocytes (CTL) do not recognize epitopes on
the targeted antigens themselves. Rather, CTL detect fragments of
antigens that are displayed on the surface of cells. As a result
antigens are visible to CTL only after they have been processed by
the cell and displayed on the surface of the cell. The antigen
processing and display system of cells has been well established.
CTL recognize short peptide antigens, which are displayed on the
surface in non-covalent association with class I major
histocompatibility complex molecules (MHC). These class I peptides
are in turn derived from the degradation of cytosolic proteins.
[0010] Despite various types of cancer treatments, a continuing
need exists for additional and more effective treatment
alternatives. One such alternative envisions methodologies of
medical treatment that require or benefit from an ability to
initiate, stimulate, and/or enhance an immune response by
immunization. These methodologies include those depending upon the
creation of an immune response against a desired antigenic
polypeptide and those that depend upon the initiation or modulation
of an innate immune response. Thus one approach in the treatment of
cancer is the manipulation of the immune system by use of a
therapeutic anticancer vaccine.
[0011] To generate a vaccine or other immunogenic composition, an
antigen or epitope against which an immune response can be mounted
is introduced into a subject. Although neoplastic cancer cells are
derived from and therefore are substantially identical to normal
cells on a genetic level, many neoplastic cells are known to
present tumor-associated antigens (TuAAs). These antigens can be
used by a subject's immune system to recognize and attack the
neoplastic cells as foreign. Unfortunately, neoplastic cells
generally appear to be ignored by the host's immune system.
[0012] A number of different strategies have been developed in the
art in an attempt to generate vaccines with activity against
neoplastic cells; however, an effective and marketable product has
not emerged. The present invention therefore serves to overcome the
deficiencies in the art and provides a plurality of immunogenic
compositions, disclosed herein, for targeting cancer or tumor
cells.
SUMMARY OF THE INVENTION
[0013] The present invention relates to methods and compositions
for inducing, entraining, and/or amplifying the immune response to
MHC class-I restricted epitopes of carcinoma antigens to generate
an effective anti-cancer immune response.
[0014] Embodiments of the disclosed invention are directed to the
use of combinations of tumor-associated antigens (TuAAs) for the
immunotherapy of patients with various types of cancer. In
preferred embodiments, the TuAAs are antigens expressed by the
cancer cell itself. Examples of such TuAAs are Melan-A, tyrosinase,
SSX-2, NY-ESO-1, and PRAME. In alternate embodiments, the TuAAs are
antigens associated with non-cancerous components of the tumor,
such as tumor-associated neovasculature or other stroma. An example
of such an antigen is PSMA--though, in prostate cancer PSMA is
expressed by cancerous cells. In particularly preferred embodiments
both types of antigen are targeted. Different aspects of the
invention include the immunogenic compositions, their collection
into defined products, and methods for their use.
[0015] Some specific embodiments relate to an immunogenic product
comprising a plurality of compositions comprising one or more
nucleic, acid compositions and one or more peptide compositions;
wherein the one or more nucleic acid compositions are capable of
expressing one or more class I MHC restricted epitopes, or an
analog thereof, selected from the group consisting of an SSX-1
epitope, an NY-ESO-1 epitope, a PRAME epitope, a PSMA epitope, a
tyrosinase epitope, and a Melan-A epitope; wherein the one or more
peptide compositions consist essentially of said one or more class
I MHC restricted epitopes, or an analog thereof, selected from the
group consisting of an SSX-1 epitope, an NY-ESO-1 epitope, a PRAME
epitope, a PSMA epitope, a tyrosinase epitope, and a Melan-A
epitope; and wherein the one or more peptides correspond to the
epitopes expressed by the selected nucleic acids.
[0016] In some embodiments of the immunogenic product the one or
more nucleic acid compositions comprise a plasmid selected from the
group consisting of pSEM, pBPL and pRP12. In some embodiments the
peptide compositions comprise a peptide selected from the group
consisting of SSX-2.sub.41-49 (SEQ ID NO. 1), its analogue
KVSEKIFYV (SEQ ID NO. 5); NY-ESO-1.sub.157-165 (SEQ ID NO. 2), its
analogue SNvaLMWITQV (SEQ ID NO. 6); PRAME.sub.415-433 (SEQ ID NO.
3), its analogue S(Nva)LQHLIG(Nle) (SEQ ID NO. 7); PSMA.sub.288-297
(SEQ ID NO. 4), its analogue GLPSIPVHPV (SEQ ID NO. 8);
Melan-A.sub.26-35 (SEQ ID NO. 22), the A27L analogue of
Melan-A.sub.26-35 (SEQ ID NO. 9), the Melan-A.sub.26-35 analogue
ENvaAGIGILTV (SEQ ID NO. 11); tyrosinase.sub.369-377 (SEQ ID NO.
10), and its analogue yMdgtmsqNva (SEQ ID NO. 12). In some
embodiments the plurality of compositions comprise: a nucleic acid
molecule capable of expressing an SSX-2 class I MHC restricted
epitope, or analogue thereof; a nucleic acid molecule capable of
expressing an NY-ESO-1 class I MHC restricted epitope, or analogue
thereof; a nucleic acid molecule capable of expressing a PRAME
class I MHC restricted epitope, or analogue thereof; a nucleic acid
molecule capable of expressing a PSMA class I MHC restricted
epitope, or analogue thereof; a peptide consisting essentially of
said SSX-2 epitope, or analogue thereof; a peptide consisting
essentially of said NY-ESO-1 epitope, or analogue thereof; a
peptide consisting essentially of said PRAME epitope, or analogue
thereof; and a peptide consisting essentially of said PSMA epitope,
or analogue thereof.
[0017] In some embodiments of the immunogenic product the included
nucleic acid molecules are part of the same composition. In some
embodiments the nucleic acid molecules are the same. In some
embodiments the nucleic acid molecule comprises a sequence encoding
the liberation sequence of pBPL (SEQ ID NO. 13). In some
embodiments the nucleic acid comprises a sequence encoding the
immunogenic polypeptide of pBPL (SEQ ID NO. 16). In some
embodiments the nucleic acid molecule is pBPL (SEQ ID NO. 20). In
some embodiments the nucleic acid molecule comprises a sequence
encoding the liberation sequence of pRP12 (SEQ ID NO. 14). In some
embodiments the nucleic acid comprises a sequence encoding the
immunogenic polypeptide of pRP12 (SEQ ID NO. 17). In some
embodiments the nucleic acid molecule is pRP12 (SEQ ID. 21). In
some embodiments the SSX-2 epitope is SSX-.sub.241-49 (SEQ ID NO.
1). In some embodiments the NY-ESO-1 epitope is
NY-ESO-1.sub.157-165 (SEQ ID NO. 2). In some embodiments the PRAME
epitope is PRAME.sub.425-433 (SEQ ID NO. 3). In some embodiments
the PSMA epitope is PSMA.sub.288-297 (SEQ ID NO. 4). In some
embodiments the SSX-2 analogue is KVSEKIFYV (SEQ ID NO. 5). In some
embodiments the NY-ESO-1 analogue is SNvaLMWITQV (SEQ ID NO. 6). In
some embodiments the PRAME analogue in is S(Nva)LQHLIG(Nle) (SEQ ID
NO. 7). In some embodiments the PSMA analogue in is GLPSIPVHPV (SEQ
ID NO. 8).
[0018] In some embodiments of the immunogenic product the plurality
of compositions comprise: a nucleic acid molecule capable of
expressing a Melan-A class I MHC restricted epitope, or analogue
thereof; a nucleic acid molecule capable of expressing a Tyrosinase
class I MHC restricted epitope, or analogue thereof; a peptide
consisting essentially of said Melan-A epitope, or analogue
thereof; and a peptide consisting essentially of said Tyrosinase
epitope, or analogue thereof. In some embodiments the nucleic acid
molecules are part of the same composition. In some embodiments the
nucleic acid molecules are the same. In some embodiments the
nucleic acid molecule comprises a sequence encoding the liberation
sequence of pSEM (SEQ ID NO. 15). In some embodiments the nucleic
acid comprises a sequence encoding the immunogenic polypeptide of
pSEM (SEQ ID NO. 18). In some embodiments the nucleic acid molecule
is pSEM (SEQ ID NO. 19). In some embodiments the Melan-A epitope is
the A27L analogue of Melan-A.sub.26-35 (SEQ ID NO. 9). In some
embodiments the Tyrosinase epitope is Tyrosinase.sub.369-377 (SEQ
ID NO. 10). In some embodiments the immunogenic product further
comprises: a nucleic acid molecule capable of expressing an SSX-2
class I MHC restricted epitope, or analogue thereof; and a nucleic
acid molecule capable of expressing an NY-ESO-1 class I MHC
restricted epitope, or analogue thereof. In some embodiments the
immunogenic product also comprises a peptide consisting essentially
of an NY-ESO-1 epitope. In some embodiments the immunogenic product
also comprises a peptide consisting essentially of an SSX-2
epitope.
[0019] Embodiments of the current invention relate to compositions
and methods for entraining and amplifying a T cell response. Such
methods include an entraining step where a composition comprising a
nucleic acid encoded immunogen is delivered to an animal. The
composition can be delivered to various locations on the animal,
but preferably the composition is delivered to the lymphatic system
(for example to a lymph node). The entrainment step can include one
or more deliveries of that composition, for example, spread out
over a period of time or in a continuous fashion over a period of
time. The methods can further include an amplification step
comprising administering a composition comprising a peptide
immunogen such as SSX-2.sub.41-49 (SEQ ID NO. 1),
NY-ESO-1.sub.157-165 (SEQ ID NO. 2), PRAME.sub.425-433 (SEQ ID NO.
3), PSMA.sub.288-297 (SEQ ID NO. 4), the A27L analogue of
Melan-A.sub.26-35 (SEQ ID NO. 9), Tyrosinase.sub.369-377 (SEQ ID
NO. 10), and analogues thereof (as represented by SEQ. ID NOS. 5,
6, 7, 8, 11, and 12) having substantial similarity to the
corresponding TuAA epitopes encoded by the nucleic acid
composition. The amplification step can be performed one or more,
times, for example, at intervals over a period of time, in one
bolus, or continuously over a period of time. Although not required
in all embodiments, some embodiments can include the use of
compositions that include an immunopotentiator or adjuvant.
[0020] Further embodiments include those in which the disclosed
plasmids are used individually or in any combination. The peptide
Compositions corresponding to these epitopes and part of the
amplification portion of the immunization strategy can be native
sequences or peptide analogues substantially similar to the native
epitope sequence. The peptides can be incorporated into the
amplification protocol individually or in combinations of 2, 3, 4,
or more of the immunogens.
[0021] Still other embodiments can include alternate epitopes (such
as those described in the U.S. patent application Ser. No.
10/117,937, entitled "Epitope Sequences," filed on Apr. 4, 2002
(Publication No. 20030220239 A1), which is hereby expressly
incorporated by reference) substituted in similar combination as
the epitopes expressed in the pSEM (SEQ ID NO. 19), pBPL (SEQ ID
NO. 20), and pRP12 (SEQ ID NO. 21) plasmids and corresponding
peptide immunogens administered as the amplification portion of the
immunization strategy.
[0022] Embodiments of the invention can encompass, for example, two
monovalent plasmids expressing single immunogens in place of one
bivalent plasmid expressing both immunogens; a trivalent plasmid
expressing three immunogens in place of one bivalent and one
monovalent plasmid; a trivalent plasmid and one monovalent plasmid
in place of a tetravalent plasmid; or two bivalent plasmids in
place of a tetravalent plasmid. Embodiments can also encompass the
use of the various plasmid combinations as part of the entrain step
of the entrain-and-amplify immunization strategy.
[0023] Embodiments of the inventions can encompass a polypeptide or
otherwise conjugated peptide that can be cleaved into individual
peptides in the lymph and its use in the amplification step of the
entrain-and-amplify immunization strategy.
[0024] Embodiments of the current invention relate to methods of
immunization that include administering a series of immunogenic
doses directly into the lymphatic system of a mammal wherein the
series can include at least 1 entraining dose and at least 1
amplifying dose, and wherein the entraining dose can include a
nucleic acid encoding an immunogen and wherein the amplifying dose
can be free of any virus, viral vector, or replication-competent
vector. The methods can further include obtaining an
antigen-specific immune response. The methods can include, in a
non-limiting example, 1-2 entraining doses. The method can include
administering a plurality of entraining doses, wherein said doses
are administered over a course of one to about 7 days. The
entraining doses, amplifying doses, or entraining and amplifying
doses can be delivered in multiple pairs of injections, wherein a
first member of a pair can be administered within about 4 days of a
second member of the pair, and wherein an interval between first
members of different pairs can be at least about 14 days. An
interval between a last entraining dose and a first amplifying dose
can be between about 7 and about 100 days, for example, but is not
limited to such.
[0025] Other embodiments relate to a method of treating carcinoma
comprising a step of administering to a patient in need thereof a
plurality of compositions including a nucleic acid molecule capable
of expressing an SSX-2 class I MHC restricted epitope, or analogue
thereof; a nucleic acid molecule capable of expressing an NY-ESO-1
class I MHC restricted epitope, or analogue thereof; a nucleic acid
molecule capable of expressing a PRAME class I MHC restricted
epitope, or analogue thereof; and a nucleic acid molecule capable
of expressing a PSMA class I MHC restricted epitope, or analogue
thereof. Another embodiment relates to the above-method further
comprising a step of administering one or more peptides selected
from the group epitopes or analogues consisting essentially of
SSX-2, NY-ESO-1, PRAME, and PSMA.
[0026] A method of treating cancer comprising administering an
immunogenic product comprising a plurality of compositions
comprising one or more nucleic acid compositions and one or more
peptide compositions; wherein the one or more nucleic acid
compositions are capable of expressing one or more class I MHC
restricted epitopes, or an analog thereof, selected from the group
consisting of an SSX-1 epitope, an NY-ESO-1 epitope, a PRAME
epitope, a PSMA epitope, a tyrosinase epitope, and a Melan-A
epitope; wherein the one or more peptide compositions consist
essentially of said one or more class I MHC restricted epitopes, or
an analog thereof, selected from the group consisting of an SSX-1
epitope, an NY-ESO-1 epitope, a PRAME epitope, a PSMA epitope, a
tyrosinase epitope, and a Melan-A epitope; and wherein the one or
more peptides correspond to the epitopes expressed by the selected
nucleic acids. Some embodiments relate to the use of the above
method wherein the cancer is a breast cancer, an ovarian cancer, a
pancreatic cancer, a prostate cancer, a colon cancer, a bladder
cancer, a lung cancer, a liver cancer, a stomach cancer, a
testicular cancer, an uterine cancer, a brain cancer, a lymphatic
cancer, a skin cancer, a bone cancer, a kidney cancer, a rectal
cancer, a melanoma, a glioblastoma, or a sarcoma.
[0027] Still other embodiments relate to a method of treating
cancer comprising a step of administering to a patient in need
thereof a plurality of compositions comprising: a nucleic acid
molecule capable of expressing a Melan-A class I MHC restricted
epitope, or analogue thereof; a nucleic acid molecule capable of
expressing a Tyrosinase class I MUG restricted epitope, or analogue
thereof; a peptide consisting essentially of said Melan-A epitope,
or analogue thereof; and a peptide consisting essentially of said
Tyrosinase epitope, or analogue thereof. Other embodiments relate
to the use of the method where the cancer to be treated to is
glioblastoma or melanoma. Yet other embodiments include a further
step of administering to a patient in need thereof a composition
comprising: a nucleic acid molecule capable of expressing an SSX-2
class I MHC restricted epitope, or analogue thereof; and a nucleic
acid molecule capable of expressing an NY-ESO-1 class I MHC
restricted epitope, or analogue thereof; a peptide consisting
essentially of said NY-ESO-1 epitope or analogue thereof; and a
peptide consisting essentially of said SSX-2 epitope or analogue
thereof.
[0028] Other embodiments relate to sets of immunogenic compositions
for inducing an immune response in a mammal including, in a
non-limiting manner, 1-6 entraining doses and at least one
amplifying dose. In such embodiments, the entraining doses can
include a nucleic acid encoding an immunogen, and wherein the
amplifying dose can include a peptide epitope, and wherein the
epitope can be presented by a pAPC expressing the nucleic acid. The
one dose further can include an adjuvant, for example, RNA. The
entraining and amplifying doses can be in a carrier suitable for
direct administration to the lymphatic system, (e.g., a lymph node
and the like). The nucleic acid can be a plasmid. The epitope can
be a class I HLA epitope. The immunogen can include an epitope
array, which array can include a liberation sequence. The immunogen
can consist essentially of a target-associated antigen. The
target-associated antigen can be a tumor-associated antigen but is
not limited to such. The immunogen can include a fragment of a
target-associated antigen that can include an epitope cluster.
[0029] Further embodiments relate to the method of use of the
entrain-and-amplify therapeutic compositions, tetravalent,
bivalent, and/or monovalent plasmids and corresponding peptide
immunogens, in the treatment of carcinoma, including melanoma,
comprising administration via lymph node injection (i.e., directly
into the organs where the immune responses are initiated and
amplified) according to an optimized immunization schedule.
[0030] Yet further embodiments related to the manufacture of
medicaments comprising the compositions of the invention. One
embodiment relates to the manufacture of a medicament suitable for
administration to the lymphatic system of a subject. Another
embodiment relates to the manufacture of a medicament suitable for
inducing an anti-cancer immune response in a subject. Another
embodiment relates to the manufacture of a medicament that entrains
and amplifies a T cell response in a subject. Another embodiment
relates to the manufacture of a medicament suitable for treating
carcinoma in a subject. Another embodiment relates to the use of
one or more nucleic acid compositions capable of expressing one or
more class I MHC restricted epitopes, or an analog thereof, and one
or more peptide compositions corresponding to the said class I MHC
restricted epitopes or analogues thereof, in the manufacture of a
medicament suitable for inducing an anti-cancer immune response in
a subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1. Tetramer analysis of pSEM/pBPL primed animals prior
to peptide boost. Group 1, 2, and 3 animals (n=60) were primed with
four injections of the pSEM/pBPL plasmid mixture on days 1, 4, 15,
and 18 (100 .mu.g/day) in bilateral inguinal lymph nodes. Tetramer
analysis was performed on day 25, 10 days following the final
plasmid injection and compared to untreated naive littermate
controls (n=5). Tetramer values (Melan A, Tyrosinase, SSX-2,
NY-ESO-1) represent the average+/-SEM.
[0032] FIG. 2. Melan-A/Tyrosinase, SSX-2/NY-ESO-1 tetramer analysis
was performed on day 39 demonstrating a tetravalent immune response
in individual animals. Animals were primed with a plasmid mixture
of pBPL+pSEM on days 1, 4, 15, and 18 (100 .mu.g/day) in bilateral
inguinal lymph nodes followed by a peptide boost consisting of
SSX2.sub.41-49 A42V (SEQ ID. NO. 5) in the left lymph node and
Tyrosinase.sub.369-377 V377Nva (SEQ ID. NO. 12) on in the right
lymph node on days 28 and 32 (25 .mu.g/day). Representative animals
(n=3) from Group 2 are shown and compared to tetramer values for an
untreated naive littermate control.
[0033] FIG. 3. Tetramer analysis of pSEM/pBPL primed,
SSX-2/Tyrosinase boosted animals. Melan-A/Tyrosinase,
SSX-2/NY-ESO-1 tetramer analysis was performed on day 39, 7 days
following the last peptide injection. Group 1 animals (n=10) were
primed with a plasmid mixture of pBPL+pSEM on days 1, 4, 15, and 18
(100 .mu.g/day) followed by a boost with a plasmid mixture of
pBPL+pSEM on days 28 and 32 (100 .mu.g/day). Group 2 and 3 animals
(n=50) were primed with a plasmid mixture of pBPL+pSEM similar to
Group I followed by a peptide boost consisting of SSX-2.sub.41-49
A42V (SEQ ID. NO. 5) in the left lymph node and
Tyrosinase.sub.369-377 V377Nva (SEQ ID. NO. 12) in the right lymph
node on days 28 and 32 (25 .mu.g/day). Average tetramer values
(Melan A, tyrosinase, SSX-2, and NY-ESO-1) were compared to
untreated naive littermate controls (n=5) and represent the
average+/-SEM.
[0034] FIGS. 4A-4B. IFN-.gamma. ELISpot analysis following a first
peptide boost (FIG. 4A). ELISPOT analysis was performed on day 41.
Group 1 animals (n=3 sacrificed) were primed with a plasmid mixture
of pBPL+pSEM on days 1, 4, 15, and 18 (100 .mu.g/day) followed by a
boost with a plasmid mixture of pBPL+pSEM on days 28 and 32 (100
.mu.g/day). Group 2 and 3 animals (n=6 sacrificed) were primed with
a plasmid mixture of pBPL+pSEM similar to Group I followed by a
peptide boost consisting of SSX-2.sub.41-49 A42V (SEQ ID. NO. 5) in
the left lymph node and Tyrosinase.sub.369-377 V377Nva (SEQ ID. NO.
12) in the right lymph node on days 28 and 32 (25 .mu.g/day).
Antigen specific (Melan A, Tyrosinase, SSX-2, and NY-ESO-1)
interferon-.gamma. spot forming cells per spleen were compared to
untreated naive littermate controls (n=3); FIG. 4A. IFN-.gamma.
ELISpot analysis was performed in triplicate, values represent
average+/-Stdev. Peptide stimulating concentration was at 10
.mu.g/ml and incubated for 42 hrs. FIG. 4B. IFN-.gamma. ELISpot
analysis following the second peptide boost. ELISPOT analysis was
performed by sacrificing representative animals on day 63. Group 1
animals (n=3 sacrificed) received injections of a mixture of
pBPL+pSEM on Days 1, 4, 15, 18, 28, 32, 49, and 53 (100 .mu.g/day).
Group 2 animals (n=4 sacrificed) received injections of a mixture
of pBPL+pSEM on days 1, 4, 15, and 18 (100 .mu.g/day) followed by a
peptide boost consisting of SSX-2.sub.41-49 A42V (SEQ ID. NO. 5) in
the left lymph node and Tyrosinase.sub.369-377 V377Nva (SEQ ID. NO.
12) in the right lymph node on days 28, 32, 49, and 53 (25
.mu.g/day). Group 3 animals (n=7 sacrificed) received injections of
a mixture of pBPL+pSEM on days 1, 4, 15, and 18 (100 .mu.g/day) in
bilateral inguinal lymph nodes followed by a peptide boost
consisting of SSX-2.sub.41-49 A42V (SEQ ID. NO. 5) in the left
lymph node and Tyrosinase.sub.369-377 V377Nva (SEQ ID. NO. 12) in
the right lymph node on days 28 and 32 (25 .mu.g/day) and a second
peptide boost consisting of NY-ESO-1.sub.157-165 L158Nva (SEQ ID.
NO. 6), C165V (12.5 .mu.g on days 49 and 53) in the left lymph node
and Melan A.sub.26-35 A27Nva (SEQ ID. NO. 11) (25 .mu.g on days 49
and 53) in the right lymph node. Antigen specific (Melan A,
Tyrosinase, SSX-2, and NY-ESO-1) interferon-.gamma. spot forming
cells per spleen were compared to a untreated naive littermate
control (FIG. 4B). IFN-.gamma. ELISpot analysis was performed in
triplicate, values represent average+/-SEM. Peptide stimulating
concentration was at 10 .mu.g/ml and incubated for 42 hrs.
[0035] FIG. 5. Depicts tetramer levels, IFN-.gamma. ELISPOT and
carboxy-fluorescein diacetate, succinimidyl ester (CFSE) histograms
from in vivo studies where animals were challenged with human
melanoma tumor cells expressing all four tumor associated antigens.
Naive control (top left panel); two animals with tetravalent
immunity (top right panel and lower left panel); and an animal with
a monovalent response to Melan A (lower right panel).
[0036] FIG. 6. Tetramer analysis of the "original" versus the
"expanded" protocol. Animals were injected based on a "original
protocol" (Groups 1-3) or an "expanded protocol" (Groups 4-6) with
4 injections of D1 (pRP12) plasmid (4 mg/ml) in the right inguinal
lymph node and 4 injections of D2 (pBPL) plasmid (4 mg/ml) in left
inguinal lymph node. Animals were subsequently boosted with PSMA,
SSX-2, PRAME and NY-ESO-1 peptides. Animals were primed with D1
(pRP12) plasmid and D2 (pBPL) plasmid (4 mg/ml) on days 1, 4, 15,
and 18, followed by boosting with PSMA.sub.288-297 (I297V) peptide
(RLN) (SEQ ID. NO. 8) and SSX-2.sub.41-49 (A42V) peptide (LLN) (SEQ
ID. NO. 5) on days 29 and 31 for the original protocol; and
boosting with PRAME.sub.425-433 (L426Nva, L433Nle)) peptide (RLN)
(SEQ ID. NO. 7) and NY-ESO-1.sub.157-165 (L158Nva, C165V) peptide
(LLN) (SEQ ID. NO. 6) on days 42, 45 for the expanded protocol
(Groups 4-6). Values represent average+/-SEM from individual
animals after peptide boost and are compared to untreated naive
littermate controls (n=5).
[0037] FIG. 7. Tetravalent immune response from a representative
animal in Group I (FIG. 6). Following plasmid priming, PSMA peptide
(25 .mu.g) and PRAME peptide (20 .mu.g) were injected into the
right lymph node. Twenty-five micrograms each of SSX-2 and NY-ESO-1
peptides were injected into the left lymph node. Data shown
measures immune response by both tetramer and ELISpot assays.
[0038] FIG. 8. IFN-.gamma. ELISPOT analysis of the "original"
versus the "expanded" protocol. Total antigen specific (SSX-2,
NY-ESO-1, PRAME, and PSMA) interferon-.gamma. spot forming cells
per spleen are shown comparing the "original" and "expanded"
protocols comprised of low, medium and high peptide boosts.
IFN-.gamma. ELISpot analysis was performed in triplicate, values
represent average+/-SEM after peptide boost. Splenocytes
(3.times.10.sup.5 cells per well) were stimulated, ex vivo in 96
well ELISpot plates, with peptide (SSX-2, NY-ESO-1, PRAME, and
PSMA) at a concentration of 10 .mu.g/ml for 72 hrs. Values are
extrapolated from total nucleated splenocytes and normalized per
spleen from each animal.
[0039] FIG. 9. .sup.51Cr cytotoxicity assays. FIG. 9 depicts CTL
response to PRAME.sub.425-433 (SEQ ID. NO. 3), PSMA.sub.288-297
(SEQ ID. NO. 4), NY-ESO-1.sub.157-165 (SEQ ID. NO. 2) and
SSX-2.sub.41-49 (SEQ ID. NO. 1) after DNA prime and peptide boost
and one round of in vitro stimulation in immunized mice. Data are
presented as follows: the x-axis shows the effector to target
ratio; the y-axis shows the corresponding percentage specific
lysis.
[0040] FIG. 10. Immune response elicited by two cycles of
therapeutic regimens of the PP (PRAME and PSMA) regimen and the NS
(NY-ESO-1 and SSX-2) regimen showing peptide dominance of
PRAME.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0041] Embodiments of the present invention are based upon the
induction of active immunity (therapeutic vaccination) preferably
co-targeted against multiple molecules expressed by cancer cells
and by the underlying neovasculature. This approach preferably
involves targeted delivery of both recombinant DNA (plasmid) and
synthetic peptides directly into the lymph nodes, thereby eliciting
a strong cell-mediated immune response with the potential to
ultimately interfere with the survival and/or viability of tumor
cells within primary and metastatic lesions.
[0042] The methodology of the present invention includes the
combined use of recombinant DNA plasmid and synthetic peptides,
preferably administered using a prime (plasmid)/boost (peptide)
approach via lymph node injection according to an optimized
immunization schedule. In preferred embodiments, the lymph node
injection is directly into the organism where the immune responses
are initiated and amplified. Embodiments of the current invention
can be administered to patients with tumor tissue that express
HLA-A2, particularly HLA-A*0201. It has been observed that by using
this immunization protocol that not only can the plasmid initiate
an immune response, it biases the response and its subsequent
amplification toward an effector as opposed to a regulatory
character. Without this prior nucleic acid-based immunization, the
repeated administration of peptide leads to a response ever more
dominated by regulatory T cells. The long-lived bias toward an
effector response is termed entrainment.
[0043] The disclosed embodiments relating to entrain-and-amplify
therapeutics for carcinoma and melanoma can be used to achieve a
multivalent attack, offering the advantage of increasing the
sensitivity of the tumor to attack. If more than a single antigen
on a tumor cell is targeted, the effective concentration of
antitumor agent is increased accordingly. Attack on stroma
associated with the tumor, such as vasculature, can also increase
the accessibility of the tumor cells to the agent(s) targeting
them. Thus, even an antigen that is also expressed on some normal
tissue can receive greater consideration as a target antigen if the
other antigens to be targeted in a multivalent attack are not also
expressed by that tissue.
[0044] Practice of such immunization protocols involving disparate
forms of immunogen requires use of at least two different
compositions and, especially when there is more than a single
target antigen, can involve several compositions to be administered
together and/or at different times. Thus, embodiments of the
invention include sets and subsets of immunogenic compositions and
individual doses thereof. Multivalency can be achieved using
compositions comprising multivalent immunogens, combinations of
monovalent immunogens, coordinated use of compositions comprising
one or more monovalent immunogens or various combinations thereof.
Multiple compositions, manufactured for use in a particular
treatment regimen or protocol according to such methods, define an
immunotherapeutic product.
[0045] In some embodiments all or a subset of the compositions of
the product are packaged together in a kit. In some instances, the
inducing and amplifying compositions targeting a single epitope, or
set of epitopes, can be packaged together. In other instances,
multiple inducing compositions can be assembled in one kit and the
corresponding amplifying compositions assembled in another kit.
Alternatively, compositions may be packaged and sold individually
along with instructions, in printed form or on machine-readable
media, describing how they can be used in conjunction with each
other to achieve the beneficial results of the indicated
immunization protocol. Further variations will be apparent to one
of skill in the art. The use of various packaging schemes
comprising less than all of the compositions that might be employed
in a particular protocol or regimen facilitates the personalization
of the treatment, for example, based on tumor antigen expression,
or observed response to the immunotherapeutic or its various
components, as described in U.S. Provisional Application Ser. No.
60/580,969, filed on Jun. 17, 2004; U.S. patent application Ser.
No. 11/155,288 (Publication No 20060008468) filed Jun. 17, 2005,
and U.S. patent application Ser. No. 11/323,964 filed Dec. 29,
2005, all entitled "COMBINATIONS OF TUMOR-ASSOCIATED ANTIGENS IN
DIAGNOSTICS FOR VARIOUS TYPES OF CANCERS"; and U.S. Provisional
Patent Application Ser. No. 60/580,964, and U.S. patent application
Ser. No. 11/155,928 (Publication No. 20050287068), both entitled
"IMPROVED EFFICACY OF ACTIVE IMMUNOTHERAPY BY INTEGRATING
DIAGNOSTIC WITH THERAPEUTIC METHODS", each of which is hereby
incorporated by reference in its entirety.
[0046] Embodiments of the current invention encompass peptides
incorporated into the amplification protocol individually or in
combinations of 2, 3, 4, or more of the immunogens. Reasons for
using less than all peptide epitopes include but are not limited to
the following: 1) sub-optimal expression of any of the antigens; 2)
the patient does not express, or no longer expresses, the
corresponding antigen; 3) a less robust response is being generated
to one or another of the epitopes, in which case such peptide(s)
can be given in the absence of the others in order to obtain a more
balanced response; 4) and a peptide can be discontinued if it is
generating some sort of immunotoxicity.
I. THERAPEUTIC PEPTIDES AND PLASMIDS OF THE PRESENT INVENTION
[0047] A. Therapeutic Peptides and Analogues Thereof
[0048] The present invention contemplates the use of multiple
molecules expressed by cancer cells and by the neovasculature as
therapeutics in the treatment of cancer. Such molecules include
tumor-associated antigens (TuAAs) which are antigens expressed by
the cancer cell itself or associated with non-cancerous components
of the tumor, such as tumor-associated neovasculature or other
stroma. TuAAs help to match a patient's cancer condition or type
with an appropriate immunotherapeutic agent or regimen.
Non-limiting examples of TuAAs contemplated in the present
invention include SSX-2, NY-ESO-1, PRAME, PSMA (prostate-specific
membrane antigen), Melan-A, and tyrosinase. Therefore, in
particular embodiments of the present invention, there is provided
peptides, peptide analogues or epitopes of TuAAs (Table 1) as
cancer therapeutics. In alternate embodiments of the present
invention, the peptides can comprise the native sequence or be
analogues of NY-ESO-1, SSX-2, Melan-A, tyrosinase, PRAME and PSMA,
such as those disclosed in U.S. Provisional Application Ser. Nos.
60/581,001, 60/580,962, and 60/691,889 and their corresponding
patent application Ser. Nos. 11/156,253 (Publication No.
20060063913), 11/155,929, (Publication No. 20060094661), 11/156,369
(Publication No. 20060057673), 11/455,278 (Publication No.
20070060524), filed on the same date as the present application
Ser. No. 11/454,633 (Publication No. 20070049533), filed on the
same date as the present application and 11/454,300 (Publication
No. 20070060518), filed on the same date as the present
application); and U.S. patent application Ser. Nos. 11/156,369
(Publication No. 20060057673) and 11/156,253 (Publication No.
20060063913); each of which is hereby incorporated by reference in
its entirety.
[0049] Tyrosinase, a melanin biosynthetic enzyme, is predominantly
expressed in melanocytes with high levels often observed in
melanomas. Therefore, tyrosinase is considered one of the most
specific markers of melanocytic differentiation. It is also
expressed in glial cells, which like melanocytes, develop from the
neuroectoderm. Tyrosinase is thus also a useful TuAA for
glioblastomas, including glioblastoma multiform. Further details of
tyrosinase as a TuAA is disclosed in U.S. Pat. No. 5,747,271,
incorporated herein by reference in its entirety. In particular
embodiments of the present invention there is provided the
tyrosinase.sub.369-377 epitope represented herein by SEQ. ID NO:
(Table 1).
[0050] Another TuAA employed in the present invention is Melan-A,
also known as MART-1 (Melanoma Antigen Recognized by T cells).
Melan-A/MART-1 is a melanin biosynthetic protein also expressed at
high levels in Melanomas. Melan-A/MART-1 is disclosed as a TuAA in
U.S. Pat. Nos. 5,994,523; 5,874,560; and 5,620,886, each of which
is incorporated herein by reference in its entirety. In preferred
embodiments of the present invention there is provided the Melan-A
TuAA, Melan-A.sub.26-35 A27L analogue, represented herein by SEQ.
ID NO: 9 (Table 1).
[0051] SSX-2, also known as Hom-Mel-40, is a member of a family of
highly conserved cancer-testis (CT) antigens (Gum, A. O. et al.,
Inf. J. Cancer 72:965-971, 1997, which is incorporated herein by
reference in its entirety). Cancer-testis antigens are found in a
variety of tumors, but are generally absent from normal adult
tissues except testis. Expression of different members of the SSX
family has been found in various tumor cell lines. SSX-2 as a TuAA
is disclosed in U.S. Pat. No. 6,025,191, which is hereby
incorporated by reference in its entirety. In particular
embodiments of the present invention there is provided
SSX-2.sub.41-49 (SEQ. ID NO: 1) and an analogue thereof, SSX-2
Analogue (SEQ. ID NO: 5), Table 1.
[0052] NY-ESO-1, also known as CTAG-1 (Cancer-Testis Antigen-1) and
CAG-3 (Cancer Antigen-3), is a cancer-testis antigen found in a
wide variety of tumors. NY-ESO-1 as a TuAA is disclosed in U.S.
Pat. No. 5,804,381, which is incorporated herein by reference in
its entirety. In preferred embodiments, the present invention
provides epitopes of NY-ESO-1 and analogues thereof, as represented
by SEQ. ID NO: 2 and SEQ. ID NO: 6 respectively (Table 1).
[0053] Another TuAA contemplated in the present invention is PRAME,
also known as MAPE, DAGE, and OIP4. PRAME is known in the art as a
cancer-testis (CT) antigen. However, unlike many CT antigens, such
as: MAGE, GAGE and RAGE, it is expressed in acute myeloid
leukemias. PRAME as a TuAA is disclosed in U.S. Pat. No. 5,830,753,
incorporated herein by reference in its entirety. In preferred
embodiments, the present invention provides epitopes of PRAME and
analogues thereof, as represented by SEQ. ID NO: 3 and SEQ. ID NO:
7 respectively (Table 1).
[0054] Yet another TuAA employed in the present invention is the
prostate-specific membrane antigen (PSMA). PSMA is found to be
highly expressed in prostate cancer cells. However, PSMA expression
is also noted in normal prostate epithelium and in the
neovasculature of non-prostatic tumors. PSMA as an
anti-neovasculature preparation is disclosed in U.S. Provisional
Patent Application Ser. No. 60/274,063, and U.S. patent application
Ser. Nos. 10/094,699 (Publication No. 20030046714) and 11/073,347
(Publication No. 20050260234); each of which is incorporated herein
by reference in its entirety. PSMA us a TuAA is described in U.S.
Pat. No. 5,538,866 incorporated herein by reference in its
entirety. In preferred embodiments, the present invention provides
epitopes of PSMA and analogues thereof, as represented by SEQ. ID
NO: 4 and SEQ. ID NO: 8 respectively (Table 1).
TABLE-US-00001 TABLE 1 PARTIAL LISTING OF SEQ. ID NOS. SEQ. ID NO.
IDENTITY SEQUENCE 1 SSX-2.sub.41-49 KASEKIFYV 2
NY-ESO-1.sub.157-165 SLLMWITQC 3 PRAME.sub.425-433 SLLQHLIGL 4
PSMA.sub.288-297 GLPSIPVHPI 5 SSX-2 Analogue KVSEKIFYV 6 NY-ESO-1
Analogue SNvaLMWITQV 7 PRAME Analogue SNvaLQHLIGNIe 8 PSMA Analogue
GLPSIPVHPV 9 Melan-A.sub.26-35 (A27L) ELAGIGILTV Analogue 10
Tyrosinase.sub.369-377 YMDGTMSQV 11 Melan-A Analogue ENvaAGIGILTV
12 Tyrosinase Analogue YMDGTMSQNva 13 pBPL plasmid IKASEKIFTVSLLM
liberation sequence WITQCKASEKIFY VK 14 pRP12 plasmid KR-SLLQHLIGL-
liberation sequence GDAAY- SLLQHLIGL- ISPEKEEQYIA- SLLQHLIGL-
KRPSIKR- GLPSIPVHPV 15 pSEM plasmid MLLAVLYCL- liberation sequence
ELAGIGILTV- YMDGTMSQV 16 pBPL encoded immunogenic MSLLMWITQCKA
polypeptide SEKIFYVGLPSIPV HPIGLPSIPVHPIK ASEKIFYVSLLM
WITQCKASEKIFY VKASEKIFYVRCG ARGPESRLLEFYL AMPFATPMEAEL
ARRSLAQDAPPLP VPGVLLKEFTVSG NILTIRLTAADHR QLQLSISSCLQQL
SLLMWITQCFLPV FLAQPPSGQRR 17 pRP12 encoded immunogenic MNLLHETDSAVA
polypeptide TARRPRWLCAGA LVLAGGFFLLGFL FGWFIKSAQLAG AKGVILYSDPAD
YFAPGVKSYPDG WNLPGGGVQRG NILNLNGAGDPLT PGYPANEYAYRR GIAEAVGLPSIPV
HPIALQSLLQHLI GLSNLTHVLYPVP LESYEDIHGTLHL ERLAYLHARLRE LLCELGRPSMVW
LSANPCPHCGDR TFYDPEPILCPCF MPNKRSLLQHLIG LGDAAYSLLQHLI
GLISPEKEEQYIAS LLQHLIGLKRPSI KRGLPSIPVHPV 18 pSEM encoded
immunogenic MLLAVLYCLELA polypeptide GIGILTVYMDGT MSQVGILTVILGV
LLLIGCWYCRRR NGYRALMDKSLH VGTQCALTRRCP QEGFDHRDSKVS LQEKNCEPV 22
Melan-A26-35 EAAGIGILTV
[0055] The antigens of the invention, as discussed above, may be
employed in various therapeutic regimens in treating a disease such
as, but not limited to, cancer.
[0056] B. Immunogenic Compositions Comprising Plasmids in
Combination with Peptides
[0057] As discussed above, the present invention provides
immunogenic compositions for the treatment of cancer comprising
plasmid(s) used in combination with synthetic peptide(s). Such an
immunogenic protocol elicits a strong cell-mediated immune response
to target a particular cancer thereby eliminating, eradicating or
ameliorating the cancer in a subject. Preferred plasmids employed
in the present invention are the pRP12 plasmid (SEQ ID NO. 21)
(U.S. Provisional Patent Application No. 60/691,579 and the
corresponding U.S. patent application Ser. No. 11/454,616
(Publication No. 20070004662), filed on the same date as the
present application) both entitled "METHODS AND COMPOSITIONS TO
ELICIT MULTIVALENT IMMUNE RESPONSES AGAINST DOMINANT AND
SUBDOMINANT EPITOPES EXPRESSED ON CANCER CELLS AND TUMOR STROMA"),
the pBPL plasmid (SEQ ID NO. 20), and the pSEM plasmid (SEQ ID NO.
19) disclosed in U.S. Provisional Patent Application No. 60/691,579
and U.S. patent application Ser. No. 10/292,413 (Publication No.
20030228634) respectively; each of which is incorporated herein by
reference in its entirety (Note that in those documents pSEM is
referred to as pMA2M). Additional plasmids that can be used are
disclosed in these references and in U.S. patent application Ser.
No. 10/225,568 (Publication No 20030138808).
[0058] Thus, in various embodiments immunotherapeutic products
comprise assemblages of immunogenic compositions. Such assemblages
can comprise 1, 2, or 3 plasmids as a set of individual
compositions or a single composition can comprise two or more
plasmids. Such assemblages can also comprise multiple peptides
corresponding to the epitopes expressed by the plasmids. Similarly,
they can be provided as compositions comprising individual or
multiple peptides. In some embodiments, an entraining plasmid or
plasmids will be sold together with the corresponding amplifying
peptides. In other embodiments, the multiple plasmids will be sold
together, but without corresponding peptides. In still other
embodiments sets of corresponding peptides will be sold together
without the plasmid, for example, for subsequent rounds of
amplification of the entrained response.
[0059] Therefore, in one particular embodiment of the present
invention there is provided an assemblage comprising the pBPL
plasmid (described in detail in U.S. application Ser. No.
10/292,413 (Publication No. 20030228634), entitled "EXPRESSION
VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS AND METHODS
FOR THEIR DESIGN," which is hereby expressly incorporated by
reference in its entirety) expressing the NY-ESO-1.sub.157-165 (SEQ
ID NO. 2) and SSX-2.sub.41-49 (SEQ ID NO. 1) epitopes and the pRP12
plasmid (described in U.S. Provisional Application No. 60/691,579
U.S. patent application Ser. No. 11/454,616 (Publication No.
20070004662), filed on the same date as the present application,
both entitled "METHODS AND COMPOSITIONS TO ELICIT MULTIVALENT
IMMUNE RESPONSES AGAINST DOMINANT AND SUBDOMINANT EPITOPES
EXPRESSED ON CANCER CELLS AND TUMOR STROMA," which are hereby
expressly incorporated by reference in their entirety) expressing
the PRAME.sub.425-433 (SEQ ID NO. 3) and PSMA.sub.288-297 (SEQ ID
NO. 4) epitopes. The liberation sequence for the pBPL and pRP12
plasmids are represented herein as SEQ ID NO. 13 and 14
respectively, and are also disclosed in U.S. patent application
Ser. No. 10/212,413 (Publication No. 20030228634), incorporated
herein by reference. The plasmids encode the epitopes in such a
manner that they can be expressed and presented by pAPC.
[0060] In another particular embodiment of the present invention
there is provided an assemblage comprising the pSEM plasmid,
(described in detail and referred to as pMA2M in U.S. patent
application Ser. No. 10/292,413 (Publication No: 20030228634)
incorporated herein by reference) expressing the A27L analogue of
Melan-A.sub.26-35 epitope (SEQ ID NO. 9) and the native
tyrosinase.sub.369-377 (SEQ ID NO. 10) epitope. The peptide
analogues Melan-A.sub.26-35 A27Nva (SEQ ID NO. 11) and
tyrosinase.sub.369-377 V377Nva (SEQ ID NO. 12) are disclosed in
U.S. patent application Ser. No. 11/156,369, and U.S. Provisional
Patent Application Ser. No. 60/691,889, both entitled "EPITOPE
ANALOGS", each of which is hereby incorporated by reference in its
entirety. The liberation sequence of this plasmid is represented
herein as SEQ ID NO. 15 and is also disclosed in U.S. Provisional
Patent Application No. 60/691,579, filed on Jun. 17, 2005; and U.S.
patent application Ser. No. 11/454,616 (Publication No.
20070004662), filed on the same date as the present application,
both entitled "METHODS AND COMPOSITIONS TO ELICIT MULTIVALENT
IMMUNE RESPONSES AGAINST DOMINANT AND SUBDOMINANT EPITOPES,
EXPRESSED ON CANCER CELLS AND TUMOR STROMA." The pSEM plasmid
encodes the Melan-A and tyrosinase epitopes in a manner that allows
for their expression and presentation by pAPCs.
[0061] In a further particular embodiment of the current invention
there is provided an assemblage comprising the pBPL plasmid
(described above) expressing the NY-ESO-1.sub.157-165 (SEQ ID NO.
2) and SSX-2.sub.41-49 (SEQ ID NO. 1) epitopes, and the pSEM
plasmid (described above) (SEQ ID NO. 19) expressing the A27L
analogue of Melan-A.sub.26-35 epitope (SEQ ID NO. 9) and the native
tyrosinase.sub.369-377 (SEQ ID NO. 10) epitope. The peptide
analogue Melan-A.sub.26-35 A27Nva (SEQ ID NO 11),
tyrosinase.sub.369-377 V377Nva (SEQ ID NO 12), SSX-2.sub.41-49 A42V
(SEQ ID NO. 5), and NY-ESO-1.sub.157-165 L158Nva, C165V (SEQ ID NO
6) are described in U.S. Provisional Application Ser. No.
60/580,962; U.S. patent application Ser. No. 11/155,929; U.S.
Provisional Application Ser. No. 60/581,001; U.S. patent
application Ser. No. 11/156,253; U.S. patent application Ser. No.
11/156,369 and U.S. Provisional Patent Application Ser. No.
60/691,889, each of which is hereby incorporated by reference in
its entirety. The plasmids, pSEM (SEQ ID NO. 19) and pBPL (SEQ ID
NO. 20), encode the respective epitopes and epitope analogues
(Melan-A A27L analogue, tyrosinase, NY-ESO-1, and SSX-2) in a
manner that they can be expressed and presented by pAPCs.
[0062] Another particular embodiment of the current invention
relates to the assemblage comprising the pRP12 plasmid (described
above) expressing the PSMA.sub.288-297 (SEQ 10 NO. 4) and
PRAME.sub.425-433 (SEQ ID NO. 3) epitopes and the pSEM plasmid
(described above) expressing the A27L analogue of Melan-A.sub.26-35
epitope (SEQ ID NO. 9) and the native tyrosinase.sub.369-377 (SEQ
ID NO. 10) epitope. The peptide analogues Melan-A.sub.26-35 A27Nva
(SEQ ID NO. 11), tyrosinase.sub.369-377 V377NVa (SEQ ID NO. 12),
PRAME.sub.425-433 L426Nva, I433Nle (SEQ ID NO. 7), and
PSMA.sub.288-297 I297V (SEQ ID NO. 8) are described in U.S.
Provisional Application Ser. No. 60/580,962; U.S. patent
application Ser. No. 11/155,929; U.S. Provisional Application Ser.
No. 60/581,001; U.S. patent application Ser. No. 11/156,253; U.S.
patent application Ser. No. 11/156,369 and U.S. Provisional Patent
Application Ser. No. 60/691,889, each of which is hereby
incorporated by reference in its entirety. Both plasmids encode
their respective epitopes, (Melan-A, tyrosinase, PRAME, and PSMA),
in such a manner that they can be expressed and presented by
pAPCs.
[0063] In further embodiments, each of the assemblages above
include the peptides corresponding (that is capable of amplifying
the response to) to the epitopes expressed by those plasmids. Other
particular embodiments comprise an individual plasmid and one or
both corresponding peptides. (Although the specific plasmids
referred to herein are described as bivalent, they can also be
amplified in a monovalent fashion).
[0064] As referred to herein, a PP therapeutic regimen entails
administration of plasmid and peptide to target the PRAME and PSMA
antigens. Similarly, an MT regimen targets Melan-A/tyrosinase
antigens and an NS therapeutic regimen targets NY-ESO-1 and SSX-2
antigens.
II. CELL PROLIFERATIVE DISEASES AND METHODS OF SCREENING
[0065] The immunogenic compositions of the present invention,
comprising a plasmid and one or more peptides or analogues thereof,
can be administered in treating a cell proliferative disease such
as cancer, in a subject. Cancers that may be treated using the
immunogenic composition of the present invention include, for
example, melanoma, lung cancer including: non-small cell lung
cancer (NSCLC) or small cell lung cancer (SCLC), hepatocarcinoma,
retinoblastoma, astrocytoma, glioblastoma, leukemia, neuroblastoma,
head and neck cancer, breast cancer, pancreatic cancer, renal
cancer, bone cancer, testicular cancer, ovarian cancer,
mesothelioma, cervical cancer, gastrointestinal cancer, lymphoma,
colon cancer, bladder cancer and/or cancers of the blood, brain,
skin, eye, tongue, gum. It is also anticipated that the immunogenic
compositions of the present invention may be used to treat cell
proliferative diseases other than cancer. Other cell proliferative
diseases contemplated in the present invention may include, for
example, dysplasias, pre-neoplastic lesions (e.g., adenomatous
hyperplasia, prostatic intraepithelial neoplasia, cervical
dysplasia, colon polyposis), or carcinoma in situ, but is not
limited to such.
[0066] Cells or tissue obtained from patients predisposed to, or
having a cancer, can be screened in order to better determine the
appropriate immunotherapeutic regimen to administer to the patient.
Such screening can include the steps of assaying the patient's
tumor tissue for two or more expressed tumor associated antigens
(TuAAs) in a preselected panel of antigens to develop an antigen
profile for the tumor. An immunotherapeutic regimen can then be
selected based on the antigen profile obtained. The regimen
selected can comprise administering at least one immunotherapeutic
agent targeting two, three, four, or more of the expressed
antigens. The immunotherapeutic agent can comprise or encode an
epitope restricted by the patient's class I MHC type, for each of
two or more antigens expressed by the tumor. The antigen expression
can be detected on neoplastic cells, or tumor-associated stromal
cells, or both.
[0067] Immunotherapeutic regimens provided in the present invention
include: the PP regimen where the target antigens are PRAME and
PSMA; this regimen co-targets the vasculature and a cancer testes
antigen. Another regimen provided by the present invention is the
MT regimen where the target antigens are Melan-A and tyrosinase;
this regimen targets tissue specific antigens associated with
melanoma and glioblastoma. NS regimen of the invention relates to
target antigens NY-ESO-1 and SSX-2 which are cancer testes antigens
found with varying frequency in a wide variety of cancers. In other
particular embodiments, of the invention, the regimens: PPNS
(co-targeting PRAME, PSMA, NYESO-1, SSX2), NSMT (co-targeting
NYESO-1, SSX2, Melan A and Tyrosinase) or PPMT (co-targeting PRAME,
PSMA, Melan A, Tyrosinase) are provided.
[0068] A screening method employed in the present invention may
include the steps of: assaying a patient's tumor tissue to detect
one or more expressed polypeptides in a preselected panel, wherein
the panel comprises two, or three, or four or more TuAAs and at
least one lineage specific marker; and confirming the cancer
diagnosis based on the assay. The panel can comprise of at least 2,
3, 4 or more TuAAs selected from the group consisting of NY-ESO-1,
PRAME, PSMA, tyrosinase, melan-A/MART-1, and SSX protein. In some
instances, the lineage specific marker can be a TuAA;
alternatively, the lineage specific marker is not a TuAA. For
example, in the case of melanoma and/or glioblastoma, the lineage
specific marker can be tyrosinase, melan-A/MART-1, or gp100; in the
case of prostate cancer, the lineage specific marker can be, PSA or
PSMA.
[0069] As tumor antigen expression tends to be heterogeneous, any
particular biopsy sample is likely not to give a complete
indication of all the antigens expressed. Thus, it is not necessary
that a patient's profile contain all of the antigens for treatment.
The screening methods employed in the present invention may include
an assay of a tumor tissue of the corresponding presumptive type
for expression of a preselected panel of antigens. In, some
instances, a panel of TuAAs assembled for one tumor type can be
used to screen other tumor types that can express at least some of
the same antigens and an expression profile developed.
[0070] The immunogenic compositions of the present invention can be
administered to patients with tumor tissue that express HLA-A2,
particularly HLA-A*0201.
[0071] Exemplary methodology for obtaining a profile of antigen
expression of a particular tumor that can be used to determine
which antigen or combination of antigens are useful in treating a
particular cancer can be is found in U.S. Provisional Application
Ser. No. 60/580,969, filed Jun. 17, 2004; U.S. patent application
Ser. No. 11/155,288 (Publication No. 20060008468), filed Jun. 17,
2005; and U.S. patent application Ser. No. 11/323,964, also filed
on Jun. 17, 2005, all entitled "COMBINATIONS OF TUMOR-ASSOCIATED
ANTIGENS IN DIAGNOSTICS FOR VARIOUS TYPES OF CANCERS"; each
incorporated herein by reference in its entirety. Specific
antigenic combinations of particular benefit in directing an immune
response against particular cancers are disclosed in U.S.
Provisional Application Ser. No. 60/479,554, filed on Jun. 17,
2003, U.S. patent application Ser. No. 10/871,708 (Publication No.
20050118186), filed on Jun. 17, 2004, (both entitled "COMBINATIONS
OF TUMOR-ASSOCIATED ANTIGENS IN COMPOSITIONS FOR VARIOUS TYPES OF
CANCERS"), and PCT Patent Application Publication No. WO
2004/112825, filed Jun. 17, 2004; each of which is incorporated
herein by reference in its entirety.
III. ENTRAINING-AND-AMPLIFYING THERAPEUTICS FOR ADMINISTRATION
[0072] In a preferred embodiment, the present invention provides a
composition comprising the combined use of recombinant DNA plasmid
and synthetic peptides administered using a prime (plasmid)/boost
(peptide) approach. Such a composition may be delivered via lymph
node injection according to an optimized immunization schedule.
Embodiments of the current invention can be administered to
patients with tumor tissue that expresses HLA-A2, particularly
HLA-A*0201. Therefore, the immunogenic compositions comprising a
plasmid and one or more peptides or analogues thereof can be
administered in treating a cancer in a subject. The disclosed
embodiments of the present invention relate to entrain-and-amplify
therapeutics for carcinoma, including melanoma, that can be used to
achieve a multivalent attack, offering the advantage of increasing
the sensitivity of the tumor to attack.
[0073] Therefore, in particular embodiments, the present invention
provides multivalent entraining-and-amplifying therapeutics for the
treatment of cancer. Such multivalent therapeutics may target more
than one antigen on a tumor cell. In instances where more than a
single antigen on a tumor cell is targeted, the effective
concentration of antitumor therapeutic is increased accordingly.
Attack on stroma associated with the tumor, such as vasculature,
can increase the accessibility of the tumor cells to the agent(s)
targeting them. Thus, even an antigen that is also expressed on
some normal tissue can receive greater consideration as a target
antigen if the other antigens to be targeted in a multivalent
attack are not also expressed by that tissue.
A. Bivalent Entrain-and-Amplify Therapeutic
[0074] An embodiment of the present invention relates to a bivalent
entrain-and-amplify therapeutic for melanoma. Therefore, in the
current invention there is provided an assemblage comprising the
pSEM plasmid and peptides corresponding to Melan-A.sub.26-35 (SEQ
ID NO. 22) and tyrosinase.sub.369-377 (SEQ ID NO. 10) epitopes
administered as the MT regimen against melanoma. In preferred
embodiments, the peptide analogues Melan-A.sub.26-35 A27Nva (SEQ ID
NO. 11) and/or tyrosinase.sub.369-377 V377Nva (SEQ ID NO. 12) are
utilized in the amplification step. The entrain-and-amplify
protocol employed in the present invention is as disclosed
above.
[0075] The pSEM plasmid assemblage can be delivered in a manner
similar to that discussed above for the tetravalent
entrain-and-amplify therapeutic for melanoma. Melanoma patients can
be screened according to the methods disclosed herein and the MT
regimen utilized with patients whose tumor antigen profile includes
Melan-A and/or tyrosinase. Administration of the peptide boost can
involve one or both of the antigens expressed by the plasmids.
[0076] Similarly the PP and NS regimens can be used for bivalent
therapy using assemblages comprising pRP12 and peptides
corresponding to the PSMA.sub.288-297 (SEQ ID NO. 4) and
PRAME.sub.425-433 (SEQ ID NO. 3) epitopes, and pBPL and peptides
corresponding to the NY-ESO-1.sub.157-165 (SEQ ID NO. 2) and
SSX-2.sub.41-49 (SEQ ID NO. 1) epitopes, respectively. These
bivalent regimens can be combined to create treatments of higher
valency, selected embodiments of which are described below, and
various sets of immunogenic compositions assembled to support
them.
B. Tetravalent Entraining-and-Amplifying Therapeutics
[0077] One embodiment of the current invention relates to a
tetravalent entrain-and-amplify therapeutic for carcinoma.
Therefore, in one particular embodiment of the present invention
there is provided an assemblage comprising the pBPL plasmid
expressing the NY-ESO-1.sub.157-165 (SEQ ID NO. 2) and
SSX-2.sub.41-49 (SEQ ID NO. 1) epitopes and the pRP12 plasmid
expressing the PRAME.sub.425-433 (SEQ ID NO. 3) and
PSMA.sub.288-297 (SEQ ID NO. 4) epitopes (referred to herein as the
PP regimen), each administered as the entraining immunogens of an
immunization strategy. An "entraining" immunogen as contemplated in
the present invention includes in many embodiments an induction
that confers particular Stability on the immune profile of the
induced lineage of T cells.
[0078] Additionally, four peptide compositions corresponding to the
NY-ESO-1, SSX-2, PRAME and PSMA epitopes are administered as the
amplification portion of the same immunization strategy as that of
the entraining immuogen. In a preferred embodiment, the peptide
analogues NY-ESO-1.sub.157-165 L158Nva, C165V (SEQ ID NO. 6);
SSX-2.sub.41-49 A42V (SEQ ID NO. 5); PSMA.sub.288-297 I297V (SEQ ID
NO. 8); and/or PRAME.sub.425-433 L426Nva, L433Nle (SEQ ID NO. 7)
are utilized in the amplification step. As contemplated in the
present invention, the term "amplifying or amplification", as of a
T cell response, includes in many embodiments a process for
increasing the number of cells, the number of activated cells, the
level of activity, rate of proliferation, or similar parameter of T
cells involved in a specific response.
[0079] The entrain-and-amplify protocol employed in the present
invention is described in greater detail in U.S. Provisional
Application No. 60/640,402, U.S. patent application Ser. No.
10/871,707 (Publication No. 20050079152), and U.S. patent
application Ser. No. 11/323,572, each entitled "METHODS TO ELICIT,
ENHANCE AND SUSTAIN IMMUNE RESPONSES AGAINST MHC CLASS I-RESTRICTED
EPITOPES, FOR PROPHYLACTIC OR THERAPEUTIC PURPOSES" each of which
is incorporated herein by reference in their entirety.
[0080] In preferred embodiments of the present invention, the
plasmids are administered intranodally as an entraining immunogen
to the inguinal lymph nodes, one to the left side and one to the
right. Subsequently, the peptides are sequentially administered
intranodally as amplifying immunogens, two on separate days to the
left node and the other two on separate days to the right node. It
is preferred, but not required, that the peptides be administered
to the same lymph node that received the plasmid encoding the
corresponding epitopes.
[0081] Carcinoma patients, especially those with ovarian,
colorectal, pancreatic, or renal cell carcinoma, can be screened
according to the methods disclosed herein and PP and or NS
therapeutic regimens administered to patients whose tumor profile
includes PRAME, PSMA, NY-ESO-1, and/or SSX-2. It is noted that the
NY-ESO-1 epitope is also found in LAGE la/s, so the presence of
this antigen in a profile can also be considered in the tumor
profile. As tumor antigen expression tends to be heterogeneous, any
particular biopsy sample is likely not to give a complete
indication of all the antigens expressed. Thus, it is not necessary
that a patient's profile contain all four of the antigens for that
patient to be a candidate for treatment with therapeutics of the
invention. However, it is preferred that the profile contain 2, 3,
or 4 of the antigens.
C. Tetravalent Entraining-and-Amplifying Therapeutics for
Melanoma
[0082] An embodiment of the present invention relates to a
tetravalent entrain-and-amplify therapeutic for melanoma,
comprising the plasmids pSEM and pBPL and the corresponding
peptides. The pSEM plasmid encodes the A27L analogue of the
Melan-A.sub.26-35 (SEQ ID NO. 9) epitope and the native tyrosinase
(tyrosinase.sub.369-377 (SEQ ID NO. 10)) epitope sequence (referred
to herein as the MT regimen). The pBPL plasmid encodes
NY-ESO-1.sub.157-165 (SEQ ID NO. 2) and SSX-2.sub.41-49 (SEQ ID NO.
1) native sequences. The assemblage comprising the plasmid is
administered as the entraining portion of an immunization strategy
against melanoma. Additionally, four peptide compositions
corresponding to the NY-ESO-1, SSX-2, Melan-A and tyrosinase
epitopes are administered as the amplification portion of the same
immunization strategy. In a preferred embodiment, the peptide
analogues Melan-A.sub.26-35 A27Nva (SEQ ID NO. 11),
tyrosinase.sub.369-377 V377Nva (SEQ ID NO. 12), SSX-2.sub.41-49
A42V (SEQ ID NO. 5), and NY-ESO-1.sub.157-165 L158Nva, C165V (SEQ
ID NO. 6) are utilized in the amplification step.
[0083] For treatment of a cancer such as melanoma, the plasmids are
administered intranodally to the inguinal lymph nodes as entraining
immunogens. Subsequently the peptides are administered
intranodally, preferably one to the left node, the other to the
right on any particular day as amplifying immunogens. Melanoma
patients can be screened according to the methods disclosed herein
and the appropriate regimens administered to patients whose tumor
antigen profile includes Melan-A and/or tyrosinase. Administration
of the peptide boost can involve 2, 3, or 4 of the antigens
expressed by the plasmids.
D. Tetravalent Entraining-and-Amplifying Therapeutics for
Glioblastoma
[0084] In a further particular embodiment of the present invention
there is provided a tetravalent entrain-and-amplify therapeutic
applicable to melanoma that is applied to other cancers such as
glioblastoma. One such embodiment relates to the composite pRP12
plasmid (described above) expressing the PSMA.sub.288-297 (SEQ ID
NO. 4) and PRAME.sub.425-433 (SEQ ID NO. 3) epitopes and the pSEM
plasmid (described above) expressing the A27L analogue of
Melan-A.sub.26-35 epitope (SEQ ID NO. 9) and the native
tyrosinase.sub.369-377 (SEQ ID NO. 10) epitope administered as the
entraining portion of an immunization strategy. Additionally, four
peptide compositions corresponding to the PSMA, PRAME, Melan-A and
tyrosinase epitopes are administered as the amplification portion
of the same immunization strategy. In a preferred embodiment, the
peptide analogues Melan-A.sub.26-35 A27Nva (SEQ ID NO. 11),
tyrosinase.sub.369-377 V377Nva (SEQ ID NO. 12), PRAME.sub.423-433
L426Nva, 1433Nle (SEQ ID NO. 7), and PSMA.sub.288-297 I297V (SEQ ID
NO. 8) are utilized in the amplification step.
[0085] Cancer patients can be screened according to the methods
disclosed herein and the PP and/or MT regimens administered to
patients whose tumor antigen profile includes PRAME, PSMA, Melan-A
and/or tyrosinase. Administration of the peptide boost can involve
2, 3, or 4 of the antigens expressed by the plasmids.
IV. METHODS OF DELIVERING COMPOSITIONS OF THE PRESENT INVENTION
[0086] In the present invention, the preferred administration of
the immunogenic composition comprising recombinant DNA plasmid as a
prime and synthetic peptide(s) as a boost, is via lymph node
injection. The plasmid (prime) may be administered separately from
the peptide (boost). Embodiments of the present invention can
encompass two monovalent plasmids expressing single immunogens in
place of one bivalent plasmid expressing both immunogens. In other
embodiments, a trivalent plasmid expressing three immunogens in
place of one bivalent and one monovalent plasmid may be employed.
In some instances a trivalent plasmid and one monovalent plasmid in
place of a tetravalent plasmid; or two bivalent plasmids in place
of a tetravalent plasmid may be employed. Whichever combination of
the compositions of the invention is employed, lymph node injection
is preferred as it allows for delivery directly into the organs
where the immune responses are initiated and amplified according to
an optimized immunization schedule.
[0087] To introduce the immunogenic composition into the lymphatic
system of the patient the composition is preferably directed to a
lymph vessel, lymph node, the spleen, or other appropriate portion
of the lymphatic system. In some embodiments each component is
administered as a bolus. In other embodiments one or more
components are delivered by infusion, generally over several hours
to several days. Preferably, the composition is directed to a lymph
node such as an inguinal or axillary node by inserting a catheter
or needle to the node and maintaining the catheter or needle
throughout the delivery. Suitable needles or catheters are
available made of metal or plastic (e.g., polyurethane, polyvinyl
chloride (PVC), TEFLON, polyethylene, and the like). In inserting
the catheter or needle into the inguinal node for example, the
inguinal node is punctured under ultrasonographic control using a
Vialon.TM. Insyte W.TM. cannula and catheter of 24G3/4 (Becton
Dickinson, USA) which is fixed using Tegaderm.TM. transparent
dressing (Tegaderm.TM., St. Paul, Minn., USA). This procedure is
generally done by an experienced radiologist. The location of the
catheter tip inside the inguinal lymph node is confirmed by
injection of a minimal volume of saline, which immediately and
visibly increases the size of the lymph node. The latter procedure
allows confirmation that the tip is inside the node. This procedure
can be performed to ensure that the tip does not slip out of the
lymph node and can be repeated on various days after implantation
of the catheter. In the event that the tip does slip out of
location inside the lymph node, a new catheter can be
implanted.
[0088] The therapeutic composition(s) of the present invention may
be administered to a patient in a manner consistent with standard
vaccine delivery protocols that are well known to one of ordinary
skill in the art. Methods of administering immunogenic compositions
of the present invention comprising plasmids and peptides or
peptide analogues of TuAAs include, without limitation,
transdermal, intranodal, perinodal, oral, intravenous, intradermal,
intramuscular, intraperitoneal, and mucosal administration,
delivery by injection or instillation or inhalation. A particularly
useful method of vaccine delivery to elicit a CTL response is
disclosed in Australian Patent No. 739189; U.S. Pat. Nos. 6,994,851
and 6,977,074 both entitled "A METHOD OF INDUCING A CTL
RESPONSE".
[0089] Various parameters need to be taken into account in
delivering or administering an immunogenic composition to a
subject. In addition, a dosage regimen and immunization schedule
may be employed. Generally the amount of the components in the
therapeutic composition will vary from patient to patient and from
antigen to antigen, depending on such factors as: the activity of
the antigen in inducing a response; the flow rate of the lymph
through the patient's system; the weight and age of the subject;
the type of disease and/or condition being treated; the severity of
the disease or condition; previous or concurrent therapeutic
interventions; the capacity of the individual's immune system to
synthesize antibodies; the degree of protection desired; the manner
of administration and the like, all of which can be readily
determined by the practitioner.
[0090] In general the therapeutic composition may be delivered at a
rate of from about 1 to about 500 microliters/hour or about 24 to
about 12000 microliters/day. The concentration of the antigen is
such that about 0.1 micrograms to about 10,000 micrograms of the
antigen will be delivered during 24 hours. The flow rate is based
on the knowledge that each minute approximately about 100 to about
1000 microliters of lymph fluid flows through an adult inguinal
lymph node. The objective is to maximize local concentration of
vaccine formulation in the lymph system. Some empirical
investigation on patients may be necessary to determine the most
efficacious level of infusion for a given vaccine preparation in
humans.
[0091] In particular embodiments, the immunogenic composition of
the present invention may be administered as a plurality of
sequential doses. Such a plurality of doses may be 2, 3, 4, 5, 6 or
more doses as is needed. In further embodiments of the present
invention, it is contemplated that the doses of the immunogenic
composition would be administered within about seconds or minutes
of each other into the right or left inguinal lymph nodes. For
example, the plasmid (prime) may first be injected into the right
lymph node followed within seconds or minutes by a second plasmid
into the left inguinal lymph node. In other instances the
combination of one or more plasmids expressing one or more
immunogens may be administered. It is preferred that the subsequent
injection following the first injection into the lymph node be
within at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more minutes but
not greater than about 30, 40, 50, or 60 minutes of the first
injection. Similar considerations apply to the administration of
two peptides individually to the right and left lymph nodes. It may
be desirable to administer the plurality of doses of the
immunogenic composition of the invention at an interval of days,
where several days (1, 2, 3, 4, 5, 6, or 7, or more days) lapse
between subsequent administrations. In other instances it may be
desirable for subsequent administration(s) of the compositions of
the invention to be administered via bilateral inguinal lymph node
injection within about 1, 2, 3, or more weeks or within about 1, 2,
3, or more months following the initial dose administration.
[0092] Administration may be in any manner compatible with the
dosage formulation and in such amount as will be therapeutically
effective. An effective amount or dose of an immunogenic
composition of the present invention is that amount needed to
provide a desired response in the subject to be treated.
V. EXAMPLES
[0093] 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 methodology disclosed in the examples
which follow represent methodologies discovered by the inventors 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 one can make many changes to the
specific disclosed embodiments and still obtain a like or similar
result within the spirit and scope of the invention.
Example 1
Experimental Procedure
Animals
[0094] Since the immune response against human T cell epitopes
cannot be studied in original nonclinical models due to the
inherent MHC-restriction of immunity, a genetically manipulated
mouse model was chosen that expresses the human A*0201 gene
(Pascolo et al., 1997), which is frequently expressed in the human
population. In contrast to immune deficient mice (the basis for
xenograft models), the A*0201 transgenic model (HHD) is immune
competent, thus allowing the evaluation of active immunotherapeutic
strategies.
[0095] Therefore, female H-2 class I-negative (knockout)
HLA-A2.1-transgenic HHD mice, 8-12 weeks of age, were used in these
studies. The animals were housed under pathogen-free
conditions.
Methodology
[0096] The bivalent pSEM plasmid (non-replicating recombinant DNA)
encoding for the tumor-associated antigens Melan-A.sub.26-35 A27L
analogue (SEQ ID NO. 9) and Tyrosinase.sub.369-377 (SEQ ID NO. 10)
and pBPL bivalent plasmid encoding for tumor-associated antigens
SSX-2.sub.41-49 (SEQ ID NO. 1) and NY-ESO-1.sub.157-165 (SEQ ID NO.
2) were evaluated regarding the ability to prime a Tcl (gamma
interferon-producing) immune response in Examples 2-6. The
Melan-A.sub.26-35 (A27Nva; ENvaAGIGILTV (SEQ ID NO. 11)) peptide
analogue, Tyrosinase.sub.369-377 (V377Nva; YMDGTMSQNva (SEQ ID NO.
12)) peptide analogue, NY-ESO-1.sub.157-165 (L158Nva, C165V;
S(Nva)LMWITQV (SEQ ID NO. 6)) peptide analogue, and SSX-2.sub.41-49
(A42V; KVSEKIFY (SEQ ID. NO. 5)) peptide analogue were used for
subsequent boosting.
[0097] Plasmids were formulated in clinical buffer (127 mM NaCl,
2.5 mM Na.sub.2HPO.sub.4, 0.88 mM KH.sub.2PO.sub.4, 0.25 mM
Na.sub.2EDTA, 0.5% ETOH, in H.sub.2O). The Melan A.sub.26-35
(A27Nva) (SEQ ID NO. 11) analogue was formulated in PBS at 1.0
mg/ml concentration. Similarly, the Tyrosinase.sub.369-377
(V377Nva) (SEQ ID NO. 12) analogue was formulated in PBS at 1.0
mg/ml concentration. The SSX-2.sub.41-99 (A42V) (SEQ ID NO. 5)
analogue was formulated in PBS at 1.0 mg/ml concentration while the
NY-ESO-1.sub.157-165 (L158Nva, C165V) (SEQ ID NO. 6) peptide
analogue was prepared for immunization in PBS containing 5% DMSO at
a concentration of 0.5 mg/ml. Cytometry data were collected using a
BD FACS Calibur flow cytometer and analyzed using CellQuest
software by gating on the lymphocyte population.
Intranodal Delivery of Plasmids and Peptides
[0098] The dose preparations were administered via bilateral
inguinal intranodal injection on Days 1, 4, 15, 18, 29, 32, 49, and
53 of the study. Mice were anesthetized by inhalation of isoflurane
and surgeries were conducted under aseptic conditions. Following
preparation for surgery, an incision 0.5-1 cm in length was made in
the inguinal fold exposing the inguinal lymph node. A maximum
volume of 25 .mu.L (25 .mu.g in a 1 mg/mL solution) of plasmid or
peptide was injected directly into the right and left inguinal
lymph node using a 0.5 mL insulin syringe. The incision was closed
with sterile 6.0 nylon skin sutures.
Plasmid (Prime)/Peptide (Boost) Immunization Schedule
[0099] Three groups of female HHD animals were immunized, as
described above with a mixture of pSEM/pBPL (100 .mu.g/day) to the
bilateral inguinal lymph nodes. Group 1 (n=10 mice) received
plasmid injections on Days 1, 4, 15, 18, 28, 32, 49, and 53; Group
2 and Group 3 (n=25 mice per group) received plasmid injections on
Days 1, 4, 15, and 18 respectively as shown in Table 2 (below).
[0100] Animals from Group 2 were boosted in the right lymph node
with Tyrosinase V377Nva (SEQ ID NO. 12) (25 .mu.g/day) and in the
left lymph node with SSX-2 A42V (SEQ ID NO. 5) (25 .mu.g/day)
peptides on days 28, 32, 49, and 53. Group 3 animals were boosted
in the right lymph node with Tyrosinase V377Nva (SEQ. ID NO. 12)
(25 .mu.g/day) and in the left lymph node with SSX-2 A42V (SEQ ID
NO. 5) (25 .mu.g/day) peptides on days 28 and 32 then were boosted
in the right lymph node with NY-ESO-1 L158Nva, C165V (SEQ ID NO. 6)
(12.5 .mu.g/day) and in the left lymph node with Melan A A27Nva
(SEQ ID NO. 11) (25 .mu.g/day) peptides on days 49 and 53 as also
shown in Table 2.
TABLE-US-00002 TABLE 2 Immunization Schedule Peptide (boost)
Peptide and Plasmids (prime) Lymph Node Group N* Plasmids Days Each
Dose (R/L) Days Each Dose 1 10 pSEM + 1, 4, 15, 18, 100 .mu.g -- --
-- pBPL 28, 32, 49, 53 2 25 pSEM + 1, 4, 15, 18 100 .mu.g
Tyrosinase (R) 28, 32, 49, 53 25 .mu.g pBPL SSX-2 (L) 28, 32, 49,
53 25 .mu.g 3 25 pSEM + 1, 4, 15, 18 100 .mu.g Tyrosinase (R) 28,
32 25 .mu.g pBPL SSX-2 (L) 28, 32 25 .mu.g NY-ESO-1 (R) 49, 53 12.5
.mu.g.sup. Melan A (L) 49, 53 25 .mu.g
Tetramer Analysis
[0101] Enumeration of CD8.sup.+ antigen-specific T cells requires
cognate recognition of the T cell receptor (TCR) by a Class I
MHC/peptide complex. This can be accomplished using Class I MHC
tetramers which are composed of a complex of four HLA MHC Class I
molecules each bound to the specific peptide and conjugated with a
fluorescent protein. Thus tetramer assays allow quantitation of the
total T cell population specific for a given peptide complexed in a
particular MHC molecule. Flow cytometry is employed in quantifying
binding of cells with the appropriate T cell receptor to the
labeled tetramers. Furthermore, since binding does not depend on
functional pathways, this population includes all specific
CD8.sup.+ T-cells regardless of functional status.
[0102] The CTL response was measured in animals immunized as
described in the above plasmid/peptide immunization schedule, 7
days following the last plasmid (Day 25) and peptide immunizations
(Days 39 and 60). Mononuclear cells were isolated from peripheral
blood after density centrifugation (Lympholyte Mammal, Cedarlane
Labs), and stained with HLA-A*0201 SSX-2 (KASEKIFYV (SEQ ID NO.
1))-PE MHC tetramer (Beckman Coulter, T02001), HLA-A*0201 NY-ESO
(SLLMWITQC (SEQ ID NO. 2))-APC MHC tetramer (Beckman Coulter,
T02001), HLA-A*0201 Melan A A27L (ELAGIGILTV (SEQ ID NO. 9))-PE MHC
tetramer (Beckman Coulter, T02001), HLA-A*0201 Tyrosinase
(YMDGTMSQV (SEQ ID NO. 10))-APC MHC tetramer (Beckman Coulter,
T02001). These cells were then co-stained using FITC conjugated rat
anti-mouse CD8a (Ly-2) monoclonal antibody (BD Biosciences,
553031). Data were collected using a BD FACS Calibur flow cytometer
and analyzed using Cellquest software by gating on the lymphocyte
population and calculating the percent of tetramer positive cells
within the CD8.sup.+ CTL population.
Interferon-.gamma. (IFN-.gamma.) ELISpot Assay
[0103] Instead of measuring cytotoxicity, the CD8.sup.+ CTL
response can be assessed by measuring IFN-.gamma. production by
specific effector cells in an ELISPOT assay. In this assay,
antigen-presenting cells (APC) are immobilized on the plastic
surface of a microtiter well and effector cells are added at
various effector:target ratios. The binding of APCs by
antigen-specific effector cells triggers the production of
cytokines including IFN-.gamma. by the effector cells. The cells
can be stained to detect the presence of intracellular IFN-.gamma.
and the number of positively staining foci (spots) counted under a
microscope.
[0104] Spleens were isolated on days 27 and 62 from euthanized
animals, subjected to the plasmid/peptide immunization schedule as
described above. The mononuclear cells, after density
centrifugation (Lympholyte Mammal, Cedarlane Labs, Burlington,
N.C.), were resuspended in HL-1 medium. Splenocytes
(5.times.10.sup.5, or 3.times.10.sup.5 cells per well) were
incubated with 10 .mu.g of Melan-A.sub.26-35 (A27L) (SEQ ID NO. 9),
Tyrosinase.sub.369-377 (SEQ ID NO. 10), SSX-2.sub.41-49 (SEQ ID NO.
1), or NY-ESO-1.sub.157-165 (SEQ ID NO. 2) natural peptide in
triplicate wells of a 96 well filter membrane plates (Multiscreen
IP membrane 96-well plate, Millipore, Boston, Mass.). Samples were
incubated for 42 hours at 37.degree. C. with 5% CO.sub.2 and 100%
humidity prior to development. Mouse. IFN-.gamma. coating antibody
(IFN-.gamma. antibody pair, U-CyTech Biosciences, The Netherlands)
was used as a coating reagent prior to incubation with splenocytes,
followed by the accompanied biotinylated detection antibody. GABA
conjugate and proprietary substrates from U-CyTech were used for
IFN-.gamma. spot development. The CTL response in immunized animals
was measured 24 hours after development on the AID International
plate reader using ELISpot Reader software version 3.2.3 calibrated
for IFN-.gamma. spot analysis.
.sup.51Chromium-Release Assay
[0105] The chromium release assay, is a well known assay for
evaluating CTL activity. Briefly, target cells expressing antigen
on their surface are labeled with a radioactive isotope of chromium
(.sup.51Cr). Patient cells are then mixed with the target cell and
incubated for several hours. Lysis of antigen-expressing cells
release .sup.51Cr into the medium. Cell-specific lysis is
calculated by comparing lysis of target cells expressing the
antigen(s) of interest or control antigen(s) in the presence or
absence of patient effector cells, and is usually expressed as the
% specific lysis.
Example 2
Immunization with Plasmids pSEM and pBPL Prior to Peptide Boost
[0106] The purpose of this study was to determine whether
immunization with the plasmids pSEM and pBPL could induce a
tetravalent response against the four tumor associated antigens
SSX-2.sub.41-49 (SEQ ID NO. 1), NY-ESO-1.sub.157-165 (SEQ ID NO.
2), Melan-A.sub.26-35(A27L) (SEQ ID NO. 9) and
Tyrosinase.sub.369-377 (SEQ ID NO. 10).
[0107] Three groups of female HHD animals (H-2 class I-negative
(knockout) HLA-A2.1-transgenic HHD Mice, 8-12 weeks of age) were
immunized with a mixture of pSEM/pBPL (100 .mu.g/day) to the
bilateral inguinal lymph nodes. Group I (n=10 mice) received
plasmid injections on Days 1, 4, 15, 18, 28, 32, 49, and 53; Group
2 and Group 3 (n=25 mice per group) received plasmid injections on
Days 1, 4, 15, and 18 respectively (Table 2; above). On day 25,
blood was collected from the immunized animals, and CD8.sup.+ T
cell analysis was performed using a tetramer assay as discussed
elsewhere herein. Responses were compared to naive littermate
control mice (n=5).
[0108] FIG. 1 shows tetramer data from animals that were primed
with four injections of a mixture of the pSEM and pBPL bivalent
plasmids (n=60), which are designed to encode for Melan-A.sub.26-35
(A27L) (SEQ ID NO. 9)/tyrosinase.sub.369-377 (SEQ ID NO. 10), and
NY-ESO-1.sub.157-165 (SEQ ID NO. 2)/SSX-2.sub.41-49 (SEQ ID NO. 1)
respectively, prior to peptide boost. Animals primed with four
injections of the pSEM/pBPL plasmid mixture at a daily dose of 100
.mu.g exhibited a trivalent SSX-2, NY-ESO-1, and Melan-A response
(Groups 1-3, n=60 total) but failed to generate any tyrosinase
specific CTLs as measured by tetramer assay. In addition, Melan-A
and NY-ESO-1 were revealed to be the dominant epitopes expressed by
the bivalent plasmids pSEM and pBPL respectively, as shown in FIG.
1.
Example 3
Individual Immunization with Plasmid Primed SSX-2/Tyrosinase
[0109] It was assessed whether boosting with the subdominant
epitope peptides alone following plasmid priming was sufficient to
achieve a tetravalent immune response. Therefore, animals from
Group 2 above, were boosted with the sub-dominant epitopes,
tyrosinase V377Nva (SEQ ID NO. 12) and SSX-2 A42V (SEQ ID NO. 5)
peptide analogues and immune responses were compared to a naive
control.
[0110] Animals were primed with a plasmid mixture of pBPL+pSEM on
days 1, 4, 15, and 18 (100 .mu.g/day) in bilateral inguinal lymph
nodes followed by a peptide boost consisting of SSX-2.sub.41-49
A42V (SEQ ID NO. 1) in the left lymph node and
Tyrosinase.sub.369-377 V377Nva (SEQ ID NO. 12) on in the right
lymph node on days 28 and 32 (25 .mu.g/day). On day 39, seven days
following the last peptide injection, blood was collected from the
immunized animals, and CD8.sup.+ T cell analysis was performed
using a tetramer assay as discussed elsewhere herein.
[0111] FIG. 2 shows the tetravalent responses from peripheral blood
on day 39 following the Tyrosinase and SSX-2 peptide boost,
generated in three representative immunized animals as compared to
a selected naive control animal using a tetramer flow cytometry
assay. For example, animal 2 demonstrated tetramer responses
specific to SSX-2 (5.8%), NY-ESO-1 (4.1%), tyrosinase (8.7%) and
Melan A (10.8%). These data taken together, represent specific CTL
responses comprised of 29.4% of the total CD8.sup.+ T cell
repertoire. Furthermore, the results show that boosting with the
subdominant epitope peptides alone, following plasmid priming, was
sufficient to achieve a tetravalent immune response.
Example 4
Immunization with Plasmid Primed SSX-2/Tyrosinase
[0112] In order to generate a more balanced tetravalent immune
response, animals were boosted with the sub-dominant-peptide
epitopes Tyrosinase.sub.369-377 (V377Nva) (SEQ ID NO. 12) and
SSX-2.sub.41-49 (A42V) (SEQ ID NO. 5) (Groups 2 and 3, n=50) and
immune responses were compared CO animals boosted with a mixture Of
pSEM/pBPL plasmid (Group 1, n=10) or naive controls (n=10).
[0113] Melan-A/Tyrosinase, SSX2/NY-ESO-1 tetramer analysis (as
described above), was performed on day 39, seven days following the
last peptide injection. Group 1 animals (n=10) were primed with a
plasmid mixture of pBPL+pSEM on days 1, 4, 15, and 18 (100
.mu.g/day) followed by a boost with a plasmid mixture of pBPL+pSEM
on days 28 and 32 (100 .mu.g/day) in bilateral inguinal lymph
nodes. Group 2 and 3 animals (n=50) were primed with a plasmid
mixture of pBPL+pSEM on days 1, 4, 15, and 18 (100 .mu.g/day) in
bilateral inguinal lymph nodes followed by a peptide boost
consisting of SSX2.sub.41-49 A42V (SEQ ID NO. 5) in the left lymph
node and Tyrosinase.sub.369-377 V377Nva (SEQ ID NO. 12) in the
right lymph node on days 28 and 32 (25 .mu.g/day).
[0114] Average tetramer values for Melan A, Tyrosinase, SSX2, and
NY-ESO-1 were compared to untreated naive littermate controls (n=5)
and represent the average+/-SEM. FIG. 3 shows the immune responses
prior to and following the tyrosinase and SSX-2 boost for Groups 2
and 3 (n=50) compared to Group 1 (n=10; plasmid alone).
[0115] Following the plasmid boost, the predominant immune response
was Melan-A and NY-ESO-1 specific (Group 1), as observed in FIG. 1.
On the other hand, animals primed with the plasmid mixture and
boosted with the subdominant peptides boosted their tyrosinase
response >2 fold and SSX-2 response >2.5 fold, thereby
establishing a balanced tetravalent immune response FIG. 3.
[0116] Thus, the data shows that a balanced tetravalent immune
response was achieved by boosting with the sub-dominant epitope
peptides, Tyrosinase.sub.369-377 (V377Nva) (SEQ ID NO. 12), and
SSX-2.sub.41-49 (A42V) (SEQ ID NO. 5).
Example 5
IFN-.gamma. Elispot Analysis of First and Second Peptide Boost
[0117] The tetramer data obtained in the above Examples was
confirmed by measuring the frequency of interferon gamma producing
(IFN.gamma.) cells following the peptide boost in select animals
from Groups 2. IFN-.gamma. ELISpot analysis was conducted following
the first peptide boost (FIG. 4A) and a second peptide boost (FIG.
4B)
[0118] ELISPOT analysis, as described elsewhere herein, was
performed by sacrificing representative animals on day 41, nine
days following the last peptide boost. Group I animals (n=3
sacrificed) were primed with a plasmid Mixture, of pBPL+pSEM on
days 1, 4, 15, and 18 (100 .mu.g/day) followed by a boost with a
plasmid mixture of pBPL-pSEM on days 28 and 32 (100 .mu.g/day) in
bilateral inguinal lymph nodes. Group 2 animals (n=6 sacrificed)
were primed with a plasmid mixture of pBPL+pSEM on days 1, 4, 15,
and 18 (100 .mu.g/day) in bilateral inguinal lymph nodes followed
by a peptide boost consisting of SSX2.sub.41-49 A42V (SEQ ID NO. 5)
in the left lymph node and Tyrosinase.sub.369-377 V377Nva (SEQ ID
NO. 12) in the right lymph node on days 28 and 32 (25 .mu.g/day).
Antigen specific (Melan A, Tyrosinase, SSX2, and NY-ESO-1)
interferon-.gamma. spot forming cells per spleen were compared to
untreated naive littermate controls (n=3), FIG. 4A.
[0119] Following the second peptide boost, ELISPOT analysis was
performed by sacrificing representative animals on day 63, ten days
following the second peptide boost. Group 1 animals (n=3
sacrificed) received injections of a mixture of pBPL-t-pSEM on days
1, 4, 15, 18, 28, 32, 49, and 53 (100 .mu.g/day) in bilateral
inguinal lymph nodes. Group 2 animals (n=4 sacrificed) received
injections of a mixture of pBPL+pSEM on days 1, 4, 15, and 18 (100
.mu.g/day) in bilateral inguinal lymph nodes followed by a peptide
boost consisting of SSX2.sub.41-49 A42V in the left lymph node and
Tyrosinase.sub.369-377 V377Nva in the right lymph node on days 28,
32, 49, and 53 (25 .mu.g/day). Group 3 animals (n=4 sacrificed)
received, injections of a mixture of pBPL+pSEM on days 1, 4, 15,
and 18 (100 .mu.g/day) in bilateral inguinal lymph nodes followed
by a peptide boost consisting of SSX2.sub.41-49 A42V (SEQ ID NO. 5)
in the left lymph node and Tyrosinase.sub.369-377 V377Nva (SEQ ID
NO. 12) in the right lymph node on days 28 and 32 (25 .mu.g/day)
and a second peptide boost consisting of NY-ESO-1.sub.157-165
L158Nva, C165V (SEQ ID NO. 6) (12.5 .mu.g on Days 49 and 53) in the
left lymph node and Melan A.sub.26-35 A27Nva (SEQ ID NO. 11) (25
.mu.g on Days 49 and 53) in the right lymph node. Antigen specific
(Melan A, Tyrosinase, SSX2, and NY-ESO-1) interferon-.gamma. spot
forming cells per spleen were compared to a untreated naive
littermate control FIG. 4B.
[0120] FIG. 4A shows that animals primed with the plasmid mixture
and boosted with tyrosinase and SSX-2 peptides ((Group 2, n=6)
demonstrated a robust tetravalent response of 2 to 8 fold higher
more IFN.gamma. producing cells than plasmid alone treated animals
(Group 1, n=3). In addition, when the animals received a second
boost of either the subdominant epitope peptides, SSX-2 and
Tyrosinase (Group 2), or the dominant epitope peptides, NY-ESO-1
and Melan A (Group 3), a tetravalent response was maintained as
compared to animals that were primed and boosted with the pSEM and
pBPL plasmid combination alone (Group 1) (FIG. 4B). A more balanced
immune response against all four antigens was achieved simply by
boosting with the subdominant epitope analogues SSX-2 and
Tyrosinase.
[0121] Overall, the data obtained from the above Examples (2-5),
depict the successful generation of a tetravalent immune response
in animals immunized with the NS and/or MT regimens of the present
invention. A comparison of the immune responses (tetramer and
IFN-.gamma. ELISPOT analysis) in naive animals or animals boosted
with a mixture of pSEM/pBPL plasmid alone (Group 1) to animals
boosted with the sub-dominant peptide epitopes tyrosinase and SSX-2
(Groups 2 and 3) on days 28 and 32 confirmed the successful
generation of a tetravalent immune response in animals immunized
with this regimen. Similar results were obtained following the
second peptide boost on days 49 and 53 in where Group 3 (n=25) was
boosted with the dominant epitope peptides, Melan A.sub.26-35
(A27Nva) (SEQ ID NO. 11) and NY-ESO-1.sub.157-165 (L158Nva, C165V)
(SEQ ID NO. 6) and Group 2 (n=25) was boosted again with the
sub-dominant epitope peptides Tyrosinase.sub.369-477 (V377Nva) (SEQ
ID NO. 12) and SSX-2.sub.41-49 (A42V) (SEQ ID NO. 5).
Example 6
Generation of an Immune Response to Human Melanoma
[0122] The carboxy-fluorescein diacetate, succinimidyl ester (CFSE)
assay provides a simple and sensitive means for fluorescently
labeling cells. This method allows for the analysis of antigen
specific and non-specific T cell proliferation.
[0123] The CFSE methodology was employed to evaluate the efficacy
of the immunization protocols. Animals, selected based on their
tetramer levels, were analyzed for their ability to clear human
CFSE labeled melanoma tumor cells in the lung.
[0124] On day 61, two animals from each group (Group 1, 2, and 3)
were selected based on high tetramer levels, and injected
intravenously with CFSE labeled tumor cells. More precisely, human
624.38 cultured melanoma tumor cells (10.times.10.sup.6),
expressing all four tumor associated antigens for SSX-2, NY-ESO-1,
Tyrosinase, and Melan A, were stained with CFSEhi (Vybrant CFDA SE
cell tracer kit, Molecular Probes) fluorescence (1.0 .mu.M for 15
minutes) and co-injected intravenously into Group 1, 2, or 3
immunized mice (N=2/group) or into naive HHD mice (N=2) with an
equal ratio of 624.28 HLA-A2 negative control cells stained with
CFSElo fluorescence (0.1 .mu.M). Animals received a second
injection of target cells two hours later.
[0125] The specific elimination of human target cells was measured
on day 62, approximately 14 hours after the injection of target
cells, by sacrificing the mice, removing lung tissue, and measuring
CFSEhi relative to CFSElo fluorescence (FL1 channel) by flow
cytometry. The formula used to calculate the percent specific lysis
is shown below.
[(1-% CFSEhi/% CFSElo) in immunized-(1-% CFSEhi/% CFSElo) in
naive].times.100
[0126] FIG. 5 shows tetramer levels, IFN.gamma. ELISPOT results,
and two peak CFSE histograms from a naive control (top left panel),
two animals with tetravalent immunity (top right and lower left
panel), and an animal with a monovalent response to Melan A (lower
right panel). As expected, the naive control animal was unable to
clear the target cells as demonstrated by the maintenance of an
equal ratio of both histogram peaks as was the case in the animal
demonstrating the monovalent immune response. On the other hand,
animals displaying an immune response to all four antigens were
much more capable of clearing the human melanoma tumor target cells
with 71% and 95% specific lysis.
Example 7
Generation of an Immune Response by a Original Vs. Expanded
Protocol
[0127] It was assessed whether immunization with the plasmids D1
(pRP12) and D2 (pBPL) could induce a tetravalent response in HHD-1
mice against four tumor-associated antigens: PSMA.sub.288-297 (SEQ
ID NO. 4), PRAME.sub.425-433 (SEQ ID NO. 3), SSX-2.sub.41-49 (SEQ
ID NO. 1), and NY-ESO-1.sub.157-165 (SEQ ID NO. 2).
[0128] Two different boosting strategies were tested with regard to
their ability to enhance the desired immune responses. The first
approach (the "original" protocol) utilized a single injection of
each peptide during the boosting procedure. The second approach
(the "expanded" protocol) tested two injections of each peptide.
Three dosage levels of each peptide (low, mid, and high) were
tested in an effort to determine a dose-response relationship and
to help define the optimum peptide concentration.
[0129] Six groups of 10 female HHD-1 animals/group were immunized
with plasmids D1 and D2 injected directly into the bilateral
inguinal lymph nodes. Animals from Groups 1-3 were boosted using
the "original" protocol, and Groups 4-6 animals were boosted using
the "expanded" protocol.
[0130] Animals on the "original protocol" (Groups 1-3, n=10 per
group) received 4 injections of D1 (pRP12 (SEQ ID NO. 21)) plasmid
(100 .mu.g per dose) in the right inguinal lymph node and 4
injections of D2 (pBPL (SEQ ID NO. 20)) plasmid (100 .mu.g/dose) in
left inguinal lymph node on days 1, 4, 15 and 18. This was followed
by a boost with PSMA.sub.288-297 (I297V) (SEQ ID NO. 8) in the
right lymph node and SSX-2.sub.41-49 (A42V) (SEQ ID NO. 5) in the
left lymph node on day 29, and with PRAME.sub.425-433 (L426Nva,
L433Nle) (SEQ ID NO. 7) in the right lymph node and
NY-ESO-1.sub.157-165 (L158Nva, C165V) (SEQ ID NO. 6) in the left
lymph node on day 32.
[0131] Animals on the "expanded protocol" (Groups 4-6, n=10 per
group) received 4 injections of D1 (pRP12 (SEQ ID NO. 21)) plasmid
(100 .mu.g/dose) in right inguinal lymph node and D2 (pBPL (SEQ ID
NO. 20)) plasmid (100 .mu.g/dose) in left inguinal lymph node on
days 1, 4, 15, and 18. The animals were subsequently boosted with
PSMA.sub.288-297 (I297V) (SEQ ID NO. 8) in the right lymph node and
SSX-2.sub.41-49 (A42V) (SEQ ID NO. 5) in the left lymph node on
days 29 and 32 and with PRAME.sub.425-433 (L426Nva, L433Nle) (SEQ
ID NO. 7) in the right lymph node and NY-ESO-1.sub.157-165
(L158Nva, C165V) (SEQ ID NO. 6) in the left lymph node on days 43
and 46.
[0132] Blood was collected from each group in both protocols, 7
days following the last peptide boost, and CD8.sup.+ T cell
analysis was performed using a tetramer assay (FIG. 6). Responses
were compared to naive littermate control mice (n=5). SSX-2,
NY-ESO-1, PRAME, and PSMA tetramer values are shown comparing the
original and expanded protocols comprised of low, medium and high
peptide boosts in FIG. 6.
[0133] Animals primed with four injections of D1 and D2 plasmid and
subsequently boosted with the peptide analogues PSMA, PRAME,
NY-ESO-1, and SSX-2 demonstrated immune responses to all four
antigens, as assessed by tetramer analysis (FIG. 6), that was
dominated by immune responses to PRAME and PSMA. In addition,
tetravalent immune responses elicited by this immunization strategy
was demonstrated in individual animals (FIG. 7). The responses were
observed to be independent of boosting regimen (original vs.
expanded). In addition, no apparent dose-response was observed,
although the high dose group (25 .mu.g peptide) in each therapeutic
protocol yielded the highest response rate. Furthermore, the
tetramer data indicated that PRAME and PSMA were the dominant
epitopes following immunization of the animals.
Example 8
IFN-.GAMMA. Elispot of an Immune Response by a Original Vs.
Expanded Protocol
[0134] To confirm the results observed with the tetramer assay, an
interferon-.gamma. (IFN-.gamma.) ELiSpot assay was conducted.
Animals from each group in Example 7, were sacrificed 22 days
following the last peptide boost and spleens were removed for
IFN-.gamma. ELISPOT analysis.
[0135] Spleens were isolated on day 68 from euthanized animals and
the mononuclear cells, after density centrifugation (Lympholyte
Mammal, Cedarlane Labs, Burlington, N.C.), were resuspended in HL-1
medium. Splenocytes (3.times.10.sup.5 or 1.5.times.10.sup.5 cells
per well) were incubated with 10 .mu.g of PSMA.sub.288-297 (SEQ ID
NO. 4), PRAME.sub.425-433 (SEQ ID NO. 3), SSX-2.sub.41-49 (SEQ ID
NO. 1), or NY-ESO-1.sub.157-165 (SEQ ID NO. 2), natural peptide in
triplicate wells of a 96 well filter membrane plates (Multi-screen
IP membrane 96-well plate, Millipore, Mass.). Samples were
incubated for 72 hours at 37.degree. C. with 5% CO.sub.2 and 100%
humidity prior to development. Mouse IFN-.gamma. coating antibody
(IFN-.gamma. antibody pair, U-CyTech Biosciences, The Netherlands)
was used as coating reagent prior to incubation with splenocytes,
followed by the accompanied biotinylated detection antibody. GABA
conjugate and proprietary substrates, from U-CyTech Biosciences
were used for IFN-.gamma. spot development. The CTL response in
immunized animals was measured 24 hours after development on the
AID International plate reader using ELISpot Reader software
version 3.2.3 calibrated for IFN-.gamma. spot analysis.
[0136] The IFN.gamma. ELISPOT results shown in FIG. 7 correlate
well with the tetramer data (FIG. 6) and confirm a robust immune
response to PRAME.sub.425-433 (SEQ ID NO. 3), PSMA.sub.288-433 (SEQ
ID NO. 4), SSX-2.sub.41-49 (SEQ ID NO. 1), and NY-ESO-1.sub.157-165
(SEQ ID NO. 2) elicited by the "original" therapeutic protocol. The
"expanded" protocol did not appear to offer any apparent advantage
over the "original" protocol as measured by IFN-.gamma. ELISPOT
analysis.
Example 9
Tetravalent Immune Response Generated by the PP/NS Therapeutic
Regimen
[0137] It was assessed whether a tetravalent immune response can be
elicited by first boosting with the subdominant epitopes PSMA and
SSX-2 followed by boosting with the dominant epitopes PRAME and
NY-ESO-1. A representative animal from Group I ("original
protocol"; high dose) received 4 injections of D1 (pRP12 (SEQ ID
NO. 21)) plasmid (100 .mu.g/dose) in the right inguinal lymph node
and 4 injections of D2 (pBPL (SEQ ID NO. 20)) plasmid (100
.mu.g/dose) in left inguinal lymph node on days 1, 4, 15 and 18.
This was followed by a boost with peptides, PSMA.sub.288-297
(I297V) (SEQ ID NO. 8) in the right lymph node (25 .mu.g) and
SSX-2.sub.41-49 (A42V) (SEQ ID NO. 5) in the left lymph node (25
.mu.g) on day 29 and with PRAME.sub.425-433 (L426Nva, L433Nle) (SEQ
ID NO. 7) in the right lymph node (20 .mu.g) and
NY-ESO-1.sub.157-165 (L158Nva, C165V) (SEQ ID NO. 6) in the left
lymph node (25 .mu.g) on day 32. The data (FIG. 8) shows a
tetravalent immune response as measured by two separate assays,
tetramer and ELISpot analyses.
Example 10
.sup.51Chromium-Release Assay Measuring CTL Activity to PRAME,
PSMA, NY-ESO and SSX-2
[0138] CTL response to PRAME.sub.425-433 (SEQ ID NO. 3),
PSMA.sub.288-297 (SEQ ID NO. 4), NY-ESO-1.sub.157-165 (SEQ ID NO.
2) and SSX-2.sub.41-49 (SEQ ID NO. 1), using .sup.51Cr cytotoxicity
assays, after DNA prime and peptide boost and one round of in vitro
stimulation in immunized mice was assessed. CTLs were generated by
ex vivo stimulation of splenocytes harvested from immunized mice
(N=6) 22 days after the completion of the peptide immunization
regimens.
[0139] Briefly, mice were sacrificed and the spleens were removed.
The spleens were homogenized and the cell suspension was strained
to yield a single-cell suspension. Quantities of 5.times.10.sup.6
cells/well were plated in 24 well tissue culture plates and
1.5.times.10.sup.6 peptide-pulsed, .gamma.-irradiated and LPS
(lipopolysaccharide) blasted B cells were added to each well. Mouse
recombinant IL-2 was also added at a concentration of 1 ng/ml. The
cells were incubated for 4 days for the PRAME group and 6 clays for
each of the PSMA, SSX-2 and NY-ESO-1 groups.
[0140] After the ex vivo stimulation, CTLs were collected from the
plates, washed, and plated into 96 well U-bottom micro-titer assay
plates at concentrations of 10.sup.6, 3.3.times.10.sup.5, and
1.1.times.10.sup.5 cells/well in a total of 100 .mu.L per well. To
assess peptide specific lysis, T2 cells were labeled with .sup.51Cr
and pulsed with 20 .mu.g/mL of each peptide (SSX-2, NY-ESO-1, PSMA,
or PRAME) at 37.degree. C. for 1.5 hours. After the incubation, the
cells were washed and resuspended. Ten thousand .sup.51Cr-labeled
and peptide-pulsed T2 cells were added to each well. The cells were
then incubated at 37.degree. C. for 4 hours.
[0141] After incubation, supernatants were harvested and the
cytolytic activity was measured in triplicate samples using a gamma
counter. The corrected percent lysis was calculated for each
concentration of effector cells, using the mean cpm for each
replicate of wells (FIG. 8). Percent specific lysis was calculated
using the following formula: Percent
release=100.times.(Experimental release-spontaneous
release)/(Maximum release-spontaneous release). Data are presented
as follows: the x-axis shows the effector to target ratio; the
y-axis shows the corresponding percentage specific lysis.
[0142] The results (FIG. 8) show .sup.51Chromium release assay
(CRA) data for CTL from each group against T2 cells pulsed with
PRAME.sub.425-433 (SEQ ID NO. 3) (panel 1), PSMA.sub.288-297 (SEQ
ID NO. 4) (panel 2), NY-ESO-1.sub.157-165 (SEQ ID NO. 2) (panel 3),
or SSX-2.sub.41-49 (SEQ ID NO. 1) (panel 4) peptides as targets.
Specific lysis values were compared to un-pulsed T2 control cells.
Given that the ELISA analysis (data not shown) indicated that
immunogenicity of the PRAME group is very strong and to avoid
antigen-induced cell deaths, the CRA for the PRAME group was
pursued following a 4-day 1VS protocol. The CRA was done following
6 days 1VS for the other peptide groups. It was found that after in
vitro re-stimulation, T cells isolated from all immunized groups
specifically killed T2 cells pulsed with peptide in contrast with
those from naive animals. CTL responses to PRAME.sub.425-433 (SEQ
ID NO. 3), PSMA.sub.288-797 (SEQ ID NO. 4), SSX-2.sub.41-49 (SEQ ID
NO. 1) and NY-ESO-1.sub.157-165 (SEQ ID NO. 2) were induced in all
groups, as assessed by .sup.51Cr cytotoxicity assays. These CTLs
had no effect on T2 control cells without peptide. The results
demonstrated that T2 target cell lysis by the CTLs isolated from
immunized mice is peptide specific. Compared to the "original"
protocol, the "expanded" protocol offered no significant
enhancement of the lysis percentage, further suggesting that the
"original" protocol is sufficient for eliciting a substantial
immune response against multiple antigens. Furthermore, due to the
increased sensitivity of the CRA assay, the specific NY-ESO-1
responses from each group were more prevalent as compared to the
tetramer and ELISPOT assays.
Example 11
Employing Multiple Therapeutic Cycles
[0143] It was assessed whether immunization with the plasmids D1
(pRP12 (SEQ ID NO. 21)) and D2 (pBPL (SEQ ID NO. 20)) could
maintain robust immune responses in HHD-1 mice against four
tumor-associated antigens: PSMA.sub.288-297 (SEQ. ID NO. 4),
PRAME.sub.423-433 (SEQ ID NO. 3), SSX-2.sub.41-49 (SEQ ID NO. 1),
and NY-ESO-1.sub.157-165 (SEQ ID NO. 2) after more than one cycle
of a therapeutic regimen of the present invention.
[0144] Male and female HHD-1 mice were immunized with plasmids D1
and D2 injected directly into the bilateral inguinal lymph nodes
followed by peptide boost with PSMA.sub.288-297 (SEQ ID NO. 4),
PRAME.sub.425-433 (SEQ ID NO. 3), SSX-2.sub.41-49 (SEQ ID NO. 1),
and NY-ESO-1.sub.157-165 (SEQ ID NO. 2). Animals received 4
injections of D1 (pRP12 (SEQ ID NO. 21)) plasmid in the right
inguinal lymph node and 4 injections of D2 (pBPL (SEQ ID NO. 20))
plasmid in left inguinal lymph node on days 1, 4, 15 and 18. This
was followed by a boost with PSMA.sub.288-297 (I297V) (SEQ ID NO.
8) in the right lymph node and SSX-2.sub.41-49 (A42V) (SEQ ID NO.
5) in the left lymph node on day 29, and with PRAME.sub.425-433
(L426Nva, L433Nle) (SEQ ID NO. 7) in the right lymph node and
NY-ESO-1.sub.157-165 (L158Nva, C165V) (SEQ ID NO. 6) in the left
lymph node on day 32. The second prime (plasmid)/boost (peptide)
therapeutic cycle was repeated following a rest period of 14
days.
[0145] Animals were injected with plasmid vehicle (N=16
animals/group); peptide vehicle (N=16 animals/group); plasmid (400
.mu.g total dose) at high dose (N=16 animals/group); peptide (25
.mu.g total dose) at high dose (N=16 animals/group); plasmid (400
.mu.g total dose) at high dose+peptide (5 .mu.g total dose) at low
dose (N=16 animals/group); plasmid at low dose (100 .mu.g total
dose)+peptide (25 .mu.g total dose) at high dose (N=14
animals/group); or plasmid (400 .mu.g total dose) at high
dose+peptide (25 .mu.g total dose) at high dose (N=16
animals/group) and compared to the naive control group N=7
animals/group.
[0146] Animals from each group, were sacrificed 14 days following
the last peptide boost and spleens were removed for IFN-.gamma.
ELISPOT analysis (FIG. 9).
[0147] The data show that animals can generate robust immune
responses following two cycles of therapeutic regimens of the PP
(PRAME and PSMA) regimen and the NS (NY-ESO-1 and SSX-2)
regimen.
[0148] Overall, the data obtained in Examples 7-11 shows
significant T cell, but no significant antibody responses,
following the PP/NS therapeutic immunization protocol. No
peptide-specific antibodies were detected in the serum of immunized
mice using an ELISA assay following one complete therapeutic cycle
(data not shown). Furthermore, antigen-specific T cell responses
encompassed effector and memory T cells (IFN.gamma. cytokine
producing, cytolytic and tetramer binding) with PRAME and PSMA
leading and SSX-2 and NY-ESO-1 trailing in magnitude. In addition
the results indicate, that while expanding the therapeutic protocol
may not achieve higher T cell immunity, reordering of subdominant
relative to the dominant peptides within a therapeutic cycle may be
needed to improve on immunity against NY-ESO-1 or any other
subdominant epitope.
[0149] In addition to those already disclosed in this application,
the following applications are hereby expressly incorporated by
reference in their entireties. Useful methods for using the
disclosed analogs in inducing, entraining, maintaining, modulating
and amplifying class I MHC-restricted T cell responses, and
particularly effector and memory CTL responses to antigen, are
described in U.S. Pat. Nos. 6,994,851 (Feb. 7, 2006) and 6,977,074
(Dec. 20, 2005) both entitled "A Method of Inducing a CTL
Response"; U.S. Provisional Application No. 60/479,393, filed on
Jun. 17, 2003, entitled "METHODS TO CONTROL MHC CLASS I-RESTRICTED
IMMUNE RESPONSE"; and U.S. patent application Ser. No. 10/871,707
(Pub. No. 2005 0079152) and Provisional U.S. Patent Application No.
60/640,402 filed on Dec. 29, 2004, both entitled "Methods to
elicit, enhance and sustain immune responses against MHC class
I-restricted epitopes, for prophylactic or therapeutic purpose".
The analogs can also be used in research to obtain further
optimized analogs. Numerous housekeeping epitopes are provided in
U.S. application Ser. Nos. 10/117,937, filed on Apr. 4, 2002 (Pub.
No. 20030220239 A 1), and 10/657,022 (20040180354), and in PCT
Application No. PCT/US2003/027706 (Pub. No. WO04022709A2), filed on
Sep. 5, 2003; and U.S. Provisional Application Nos. 60/282,211,
filed on Apr. 6, 2001; 60/337,017, filed on Nov. 7, 2001;
60/363,210 filed on Mar. 7, 2002; and 60/409,123, filed on Sep. 5,
2002; each of which applications is entitled "Epitope Sequences".
The analogs can further be used in any of the various modes
described in those applications. Epitope clusters, which may
comprise or include the instant analogs, are disclosed and more
fully defined in U.S. patent application Ser. No. 09/561,571, filed
on Apr. 28, 2000, entitled EPITOPE CLUSTERS. Methodology for using
and delivering the instant analogs is described in U.S. patent
application Ser. Nos. 09/380,534 and 6977074 (Issued Dec. 20, 2005)
and in PCT Application No. PCTUS98/14289 (Pub. No. WO9902183A2),
each entitled A "METHOD OF INDUCING A CTL RESPONSE". Beneficial
epitope selection principles for such immunotherapeutics are
disclosed in U.S. patent application Ser. Nos. 09/560,465, filed on
Apr. 28, 2000, 10/026,066 (Pub. No. 20030215425 A1), filed on Dec.
7, 2001, and 10/005,905 filed on Nov. 7, 2001, all entitled
"Epitope Synchronization in Antigen Presenting Cells"; 6, 861, 234
(issued 1 Mar. 2005; application Ser. No. 09/561,074), entitled
"Method of Epitope Discovery"; 09/561,571, filed Apr. 28, 2000;
entitled EPITOPE CLUSTERS; 10/094,699 (Pub. No. 20030046714 A1),
filed Mar. 7, 2002, entitled "Anti-Neovasculature Preparations for
Cancer"; application Ser. Nos. 10/117,937 (Pub. No. 20030220239 A1)
and PCTUS02/11101 (Pub. No. WO02081646A2), both filed on Apr. 4,
2002, and both entitled "EPITOPE SEQUENCES"; and application Ser.
Nos. 10/657,022 and PCT Application No. PCT/US2003/027706 (Pub. No.
WO04022709A2), both filed on Sep. 5, 2003, and both entitled
"EPITOPE SEQUENCES". Aspects of the overall design of vaccine
plasmids are disclosed in U.S. patent application Ser. No.
09/561,572, filed on Apr. 28, 2000, entitled "Expression Vectors
Encoding Epitopes of Target-Associated Antigens" and 10/292,413
(Pub. No. 20030228634 A1), filed on Nov. 7, 2002, entitled
"Expression Vectors Encoding Epitopes of Target-Associated Antigens
and Methods for their Design"; 10/225,568 (Pub No. 2003-0138808),
filed on Aug. 20, 2002, PCT Application No. PCT/US2003/026231 (Pub.
No. WO 2004/018666), filed on Aug. 19, 2003, both entitled
"EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED
ANTIGENS"; and U.S. Pat. No. 6,709,844, entitled "AVOIDANCE OF
UNDESIRABLE REPLICATION INTERMEDIATES IN PLASMID PROPAGATION".
Specific antigenic combinations of particular benefit in directing
an immune response against particular cancers are disclosed in
Provisional U.S. patent Application No. 60/479,554, filed on Jun.
17, 2003 and U.S. patent application Ser. No. 10/871,708, filed on
Jun. 17, 2004 and PCT Patent Application No. PCT/US2004/019571
(Pub. No. WO 2004/112825), all entitled "Combinations of
tumor-associated antigens in vaccines for various types of
cancers". Antigens associated with tumor neovasculature (e.g.,
PSMA, VEGFR2, Tie-2) are also useful in connection with cancerous
diseases, as is disclosed in U.S. patent application Ser. No.
10/094,699 (Pub. No. 20030046714 A1), filed Mar. 7, 2002, entitled
"Anti-Neovasculature Preparations for Cancer". Methods to trigger,
maintain, and manipulate immune responses by targeted
administration of biological response modifiers are disclosed U.S.
Provisional Application No. 60/640,727, filed on Dec. 29, 2004.
Methods to bypass CD4+ cells in the induction of an immune response
are disclosed in U.S. Provisional Application No. 60/640,821, filed
on Dec. 29, 2004. Exemplary diseases, organisms and antigens and
epitopes associated with target organisms, cells and diseases are
described in U.S. Application No. 6977074 (issued Dec. 20, 2005)
filed Feb. 2, 2001 and entitled "METHOD OF INDUCING A CTL
RESPONSE". Exemplary methodology is found in U.S. Provisional
Application No. 60/580,969, filed on Jun. 17, 2004, and U.S. Patent
Application No. 2006-0008468-A1, published on Jan. 12, 2006, both
entitled "COMBINATIONS OF TUMOR-ASSOCIATED ANTIGENS IN
DIAGNOTISTICS FOR VARIOUS TYPES. OF CANCERS". Methodology and
compositions are also disclosed in U.S. Provisional Application No.
60/640,598, filed on Dec. 29, 2004, entitled "COMBINATIONS OF
TUMOR-ASSOCIATED ANTIGENS IN COMPOSITIONS FOR VARIOUS TYPES OF
CANCER". The integration of diagnostic techniques to assess and
monitor immune responsiveness with methods of immunization
including utilizing the instant analogs is discussed more fully in
Provisional U.S. Patent Application No. 60/580,964 filed on Jun.
17, 2004 and U.S. Patent Application No. US-2005-0287068-A1,
published on Dec. 29, 2005) both entitled "Improved efficacy of
active immunotherapy by integrating diagnostic with therapeutic
methods". The immunogenic polypeptide encoding vectors are
disclosed in U.S. patent application Ser. No. 10/292,413 (Pub. No.
20030228634 A 1), filed on Nov. 7, 2002, entitled Expression
Vectors Encoding Epitopes of Target-Associated Antigens and Methods
for their Design, and in U.S. Provisional Application No.
60/691,579, filed on Jun. 17, 2005, entitled "Methods and
compositions to elicit multivalent immune responses against
dominant and subdominant epitopes, expressed on cancer cells and
tumor stroma". Additional useful disclosure, including methods and
compositions of matter, is found in U.S. Provisional Application
No. 60/691,581, filed on Jun. 17, 2005, entitled "Multivalent
Entrain-and-Amplify Immunotherapeutics for Carcinoma". Further
methodology, compositions, peptides, and peptide analogs are
disclosed in U.S. Provisional Application Nos. 60/581,001 and
60/580,962, both filed on Jun. 17, 2004, and respectively entitled
"SSX-2 PEPTIDE ANALOGS" and "NY-ESO PEPTIDE ANALOGS." Each of the
applications and patents mentioned in the above paragraphs is
hereby incorporated by reference in its entirety for all that it
teaches. Additional analogs, peptides and methods are disclosed in
U.S. Patent Application Publication No 20060063913, entitled "SSX-2
PEPTIDE ANALOGS"; and U.S. Patent Publication No. 2006-0057673 A1,
published on Mar. 16, 2006, entitled "EPITOPE ANALOGS"; and PCT
Application Publication No. WO/2006/009920, entitled "EPITOPE
ANALOGS"; all filed on Jun. 17, 2005. Further methodology and
compositions are disclosed in U.S. Provisional Application No.
60/581,001, filed on Jun. 17, 2004, entitled "SSX-2 PEPTIDE
ANALOGS", and to U.S. Provisional Application No. 60/580,962, filed
on Jun. 17, 2004, entitled "NY-ESO PEPTIDE ANALOGS"; each of which
is incorporated herein by reference in its entirety. As an example,
without being limited thereto each reference is incorporated by
reference for what it teaches about class I MHC-restricted
epitopes, analogs, the design of analogs, uses of epitopes and
analogs, methods of using and making epitopes, and the design and
use of nucleic acid vectors for their expression. Other
applications that are expressly incorporated herein by reference
are: U.S. patent application Ser. No. 11/156,253 (Publication No.
20060063913), filed on Jun. 17, 2005, entitled."SSX-2 PEPTIDE
ANALOGS"; U.S. patent application Ser. No. 11/155,929, filed on
Jun. 17, 2005, entitled "NY-ESO-1 PEPTIDE ANALOGS" (Publication No.
20060094661); U.S. patent application Ser. No. 11/321,967, filed on
Dec. 29, 2005, entitled "METHODS TO TRIGGER, MAINTAIN AND
MANIPULATE IMMUNE RESPONSES BY TARGETED ADMINISTRATION OF
BIOLOGICAL RESPONSE MODIFIERS INTO LYMPHOID ORGANS"; U.S. patent
application Ser. No. 11/323,572, filed on Dec. 29, 2005, entitled
"METHODS TO ELICIT ENHANCE AND SUSTAIN IMMUNE REPONSES AGAINST MCH
CLASS I RESTRICTED EPITOPES, FOR PROPHYLACTIC OR THERAPEUTIC
PURPOSES"; U.S. patent application Ser. No. 11/323,520, filed Dec.
29, 2005, entitled "METHODS TO BYPASS CD4+ CELLS IN THE INDUCTION
OF AN IMMUNE RESPONSE"; U.S. patent application Ser. No.
11/323,049, filed Dec. 29, 2005, entitled "COMBINATION OF
TUMOR-ASSOCIATED ANTIGENS IN COMPOSITIONS FOR VARIOUS TYPES OF
CANCERS"; U.S. patent application Ser. No. 11,323,964, filed Dec.
29, 2005, entitled "COMBINATIONS. OF TUMOR-ASSOCIATED ANTIGENS IN
DIAGNOSTICS FOR VARIOUS TYPES OF CANCERS"; U.S. Provisional
Application Ser. No. 60/691,889, filed on Jun. 17, 2005 entitled
"EPITOPE ANALOGS."
Sequence CWU 1
1
2219PRTHOMO SAPIENS 1Lys Ala Ser Glu Lys Ile Phe Tyr Val1
529PRTHOMO SAPIENS 2Ser Leu Leu Met Trp Ile Thr Gln Cys1 539PRTHomo
sapien 3Ser Leu Leu Gln His Leu Ile Gly Leu1 5410PRThomo sapien
4Gly Leu Pro Ser Ile Pro Val His Pro Ile1 5 1059PRTArtificial
SequenceMan made peptide 5Lys Val Ser Glu Lys Ile Phe Tyr Val1
569PRTArtificial SequenceAnalog of NY-ESO-1, amino acids 157-165
6Ser Xaa Leu Met Trp Ile Thr Gln Val1 579PRTArtificial
SequenceAnalog of PRAME, amino acids 425-433 7Ser Xaa Leu Gln His
Leu Ile Gly Xaa1 5810PRTArtificial SequenceMan made peptide 8Gly
Leu Pro Ser Ile Pro Val His Pro Val1 5 10910PRThomo sapien 9Glu Leu
Ala Gly Ile Gly Ile Leu Thr Val1 5 10109PRThomo sapien 10Tyr Met
Asp Gly Thr Met Ser Gln Val1 51110PRTArtificial SequenceAnalog of
Melan-A, amino acids 26-35 11Glu Xaa Ala Gly Ile Gly Ile Leu Thr
Val1 5 10129PRTArtificial SequenceAnalog of tyrosinase, amino acids
369-377 12Tyr Met Asp Gly Thr Met Ser Gln Xaa1 51329PRTArtificial
SequenceMan made peptide 13Ile Lys Ala Ser Glu Lys Ile Phe Tyr Val
Ser Leu Leu Met Trp Ile1 5 10 15Thr Gln Cys Lys Ala Ser Glu Lys Ile
Phe Tyr Val Lys 20 251462PRTArtificial SequenceMan made peptide
14Lys Arg Ser Leu Leu Gln His Leu Ile Gly Leu Gly Asp Ala Ala Tyr1
5 10 15Ser Leu Leu Gln His Leu Ile Gly Leu Ile Ser Pro Glu Lys Glu
Glu 20 25 30Gln Tyr Ile Ala Ser Leu Leu Gln His Leu Ile Gly Leu Lys
Arg Pro 35 40 45Ser Ile Lys Arg Gly Leu Pro Ser Ile Pro Val His Pro
Val 50 55 601528PRTArtificial SequenceMan made peptide 15Met Leu
Leu Ala Val Leu Tyr Cys Leu Glu Leu Ala Gly Ile Gly Ile1 5 10 15Leu
Thr Val Tyr Met Asp Gly Thr Met Ser Gln Val 20 2516179PRTArtificial
SequenceMan made peptide 16Met Ser Leu Leu Met Trp Ile Thr Gln Cys
Lys Ala Ser Glu Lys Ile1 5 10 15Phe Tyr Val Gly Leu Pro Ser Ile Pro
Val His Pro Ile Gly Leu Pro 20 25 30Ser Ile Pro Val His Pro Ile Lys
Ala Ser Glu Lys Ile Phe Tyr Val 35 40 45Ser Leu Leu Met Trp Ile Thr
Gln Cys Lys Ala Ser Glu Lys Ile Phe 50 55 60Tyr Val Lys Ala Ser Glu
Lys Ile Phe Tyr Val Arg Cys Gly Ala Arg65 70 75 80Gly Pro Glu Ser
Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe Ala 85 90 95Thr Pro Met
Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp Ala 100 105 110Pro
Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val Ser 115 120
125Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln Leu
130 135 140Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu
Met Trp145 150 155 160Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala
Gln Pro Pro Ser Gly 165 170 175Gln Arg Arg17275PRTArtificial
SequenceMan made peptide 17Met Asn Leu Leu His Glu Thr Asp Ser Ala
Val Ala Thr Ala Arg Arg1 5 10 15Pro Arg Trp Leu Cys Ala Gly Ala Leu
Val Leu Ala Gly Gly Phe Phe 20 25 30Leu Leu Gly Phe Leu Phe Gly Trp
Phe Ile Lys Ser Ala Gln Leu Ala 35 40 45Gly Ala Lys Gly Val Ile Leu
Tyr Ser Asp Pro Ala Asp Tyr Phe Ala 50 55 60Pro Gly Val Lys Ser Tyr
Pro Asp Gly Trp Asn Leu Pro Gly Gly Gly65 70 75 80Val Gln Arg Gly
Asn Ile Leu Asn Leu Asn Gly Ala Gly Asp Pro Leu 85 90 95Thr Pro Gly
Tyr Pro Ala Asn Glu Tyr Ala Tyr Arg Arg Gly Ile Ala 100 105 110Glu
Ala Val Gly Leu Pro Ser Ile Pro Val His Pro Ile Ala Leu Gln 115 120
125Ser Leu Leu Gln His Leu Ile Gly Leu Ser Asn Leu Thr His Val Leu
130 135 140Tyr Pro Val Pro Leu Glu Ser Tyr Glu Asp Ile His Gly Thr
Leu His145 150 155 160Leu Glu Arg Leu Ala Tyr Leu His Ala Arg Leu
Arg Glu Leu Leu Cys 165 170 175Glu Leu Gly Arg Pro Ser Met Val Trp
Leu Ser Ala Asn Pro Cys Pro 180 185 190His Cys Gly Asp Arg Thr Phe
Tyr Asp Pro Glu Pro Ile Leu Cys Pro 195 200 205Cys Phe Met Pro Asn
Lys Arg Ser Leu Leu Gln His Leu Ile Gly Leu 210 215 220Gly Asp Ala
Ala Tyr Ser Leu Leu Gln His Leu Ile Gly Leu Ile Ser225 230 235
240Pro Glu Lys Glu Glu Gln Tyr Ile Ala Ser Leu Leu Gln His Leu Ile
245 250 255Gly Leu Lys Arg Pro Ser Ile Lys Arg Gly Leu Pro Ser Ile
Pro Val 260 265 270His Pro Val 2751894PRTArtificial SequenceMan
made peptide 18Met Leu Leu Ala Val Leu Tyr Cys Leu Glu Leu Ala Gly
Ile Gly Ile1 5 10 15Leu Thr Val Tyr Met Asp Gly Thr Met Ser Gln Val
Gly Ile Leu Thr 20 25 30Val Ile Leu Gly Val Leu Leu Leu Ile Gly Cys
Trp Tyr Cys Arg Arg 35 40 45Arg Asn Gly Tyr Arg Ala Leu Met Asp Lys
Ser Leu His Val Gly Thr 50 55 60Gln Cys Ala Leu Thr Arg Arg Cys Pro
Gln Glu Gly Phe Asp His Arg65 70 75 80Asp Ser Lys Val Ser Leu Gln
Glu Lys Asn Cys Glu Pro Val 85 90193315DNAArtificial
SequencePlasmid 19atatacgcgt tgacattgat tattgactag ttattaatag
taatcaatta cggggtcatt 60agttcatagc ccatatatgg agttccgcgt tacataactt
acggtaaatg gcccgcctgg 120ctgaccgccc aacgaccccc gcccattgac
gtcaataatg acgtatgttc ccatagtaac 180gccaataggg actttccatt
gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt 240ggcagtacat
caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
300atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
cttggcagta 360catctacgta ttagtcatcg ctattaccat ggtgatgcgg
ttttggcagt acatcaatgg 420gcgtggatag cggtttgact cacggggatt
tccaagtctc caccccattg acgtcaatgg 480gagtttgttt tggcaccaaa
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc 540attgacgcaa
atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg
600gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
actataggga 660gacccaagct ggctagcgtt taaacttaag ccaccatgtt
actagctgtt ttgtactgcc 720tggaactagc agggatcggc atattgacag
tgtatatgga tggaacaatg tcccaggtag 780gaattctgac agtgatcctg
ggagtcttac tgctcatcgg ctgttggtat tgtagaagac 840gaaatggata
cagagccttg atggataaaa gtcttcatgt tggcactcaa tgtgccttaa
900caagaagatg cccacaagaa gggtttgatc atcgggacag caaagtgtct
cttcaagaga 960aaaactgtga acctgtgtag tgagcggccg ctcgagtcta
gagggcccgt ttaaacccgc 1020tgatcagcct cgactgtgcc ttctagttgc
cagccatctg ttgtttgccc ctcccccgtg 1080ccttccttga ccctggaagg
tgccactccc actgtccttt cctaataaaa tgaggaaatt 1140gcatcgcatt
gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
1200aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
ctctatggct 1260tctactgggc ggttttatgg acagcaagcg aaccggaatt
gccagctggg gcgccctctg 1320gtaaggttgg gaagccctgc aaagtaaact
ggatggcttt cttgccgcca aggatctgat 1380ggcgcagggg atcaagctct
gatcaagaga caggatgagg atcgtttcgc atgattgaac 1440aagatggatt
gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
1500gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
gcgcaggggc 1560gcccggttct ttttgtcaag accgacctgt ccggtgccct
gaatgaactg caagacgagg 1620cagcgcggct atcgtggctg gccacgacgg
gcgttccttg cgcagctgtg ctcgacgttg 1680tcactgaagc gggaagggac
tggctgctat tgggcgaagt gccggggcag gatctcctgt 1740catctcacct
tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
1800atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
atcgagcgag 1860cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
tctggacgaa gagcatcagg 1920ggctcgcgcc agccgaactg ttcgccaggc
tcaaggcgag catgcccgac ggcgaggatc 1980tcgtcgtgac ccatggcgat
gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt 2040ctggattcat
cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
2100ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
ctcgtgcttt 2160acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
tcgccttctt gacgagttct 2220tctgaattat taacgcttac aatttcctga
tgcggtattt tctccttacg catctgtgcg 2280gtatttcaca ccgcatcagg
tggcactttt cggggaaatg tgcgcggaac ccctatttgt 2340ttatttttct
aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
2400cttcaataat agcacgtgct aaaacttcat ttttaattta aaaggatcta
ggtgaagatc 2460ctttttgata atctccggaa gagtcaagaa catgtgagca
aaaggccagc aaaaggccag 2520gaaccgtaaa aaggccgcgt tgctggcgtt
tttccatagg ctccgccccc ctgacgagca 2580tcacaaaaat cgacgctcaa
gtcagaggtg gcgaaacccg acaggactat aaagatacca 2640ggcgtttccc
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
2700atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
cacgctgtag 2760gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
tgtgtgcacg aaccccccgt 2820tcagcccgac cgctgcgcct tatccggtaa
ctatcgtctt gagtccaacc cggtaagaca 2880cgacttatcg ccactggcag
cagccactgg taacaggatt agcagagcga ggtatgtagg 2940cggtgctaca
gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
3000tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
gctcttgatc 3060cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
tgcaagcagc agattacgcg 3120cagaaaaaaa ggatctcaag aagatccttt
gatcttttct acggggtctg acgctcagtg 3180gaacgaaaac tcacgttaag
ggattttggt ccggccggaa acgtttggtt gctgactaat 3240tgagatgcat
gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctcg
3300cgatgtacgg gccag 3315203596DNAArtificial SequencePlasmid
20gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
60gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
120ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
acgccaatag 180ggactttcca ttgacgtcaa tgggtggagt atttacggta
aactgcccac ttggcagtac 240atcaagtgta tcatatgcca agtacgcccc
ctattgacgt caatgacggt aaatggcccg 300cctggcatta tgcccagtac
atgaccttat gggactttcc tacttggcag tacatctacg 360tattagtcat
cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
420agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
gggagtttgt 480tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
caactccgcc ccattgacgc 540aaatgggcgg taggcgtgta cggtgggagg
tctatataag cagagctctc tggctaacta 600gagaacccac tgcttactgg
cttatcgaaa ttaatacgac tcactatagg gagacccaag 660ctggctagcg
tttaaactta agccaccatg tccctgttga tgtggatcac gcagtgcaaa
720gcttcggaga aaatcttcta tgtgggtctt ccaagtattc ctgttcatcc
aattggtctt 780ccaagtattc ctgttcatcc aattaaagct tcggagaaaa
tcttctatgt gtccctgttg 840atgtggatca cgcagtgcaa agcttcggag
aaaatcttct atgtgaaagc ttcggagaaa 900atcttctacg tacggtgcgg
tgccaggggg ccggagagcc gcctgcttga gttctacctc 960gccatgcctt
tcgcgacacc catggaagca gagctggccc gcaggagcct ggcccaggat
1020gccccaccgc ttcccgtgcc aggggtgctt ctgaaggagt tcactgtgtc
cggcaacata 1080ctgactatcc gactgactgc tgcagaccac cgccaactgc
agctctccat cagctcctgt 1140ctccagcagc tttccctgtt gatgtggatc
acgcagtgct ttctgcccgt gtttttggct 1200cagcctccct cagggcagag
gcgctagtga gaattctgca gatatccatc acactggcgg 1260ccgctcgagt
ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
1320tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
aggtgccact 1380cccactgtcc tttcctaata aaatgaggaa attgcatcgc
attgtctgag taggtgtcat 1440tctattctgg ggggtggggt ggggcaggac
agcaaggggg aggattggga agacaatagc 1500aggcatgctg gggatgcggt
gggctctatg gcttctactg ggcggtttta tggacagcaa 1560gcgaaccgga
attgccagct ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa
1620actggatggc tttcttgccg ccaaggatct gatggcgcag gggatcaagc
tctgatcaag 1680agacaggatg aggatcgttt cgcatgattg aacaagatgg
attgcacgca ggttctccgg 1740ccgcttgggt ggagaggcta ttcggctatg
actgggcaca acagacaatc ggctgctctg 1800atgccgccgt gttccggctg
tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc 1860tgtccggtgc
cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga
1920cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
gactggctgc 1980tattgggcga agtgccgggg caggatctcc tgtcatctca
ccttgctcct gccgagaaag 2040tatccatcat ggctgatgca atgcggcggc
tgcatacgct tgatccggct acctgcccat 2100tcgaccacca agcgaaacat
cgcatcgagc gagcacgtac tcggatggaa gccggtcttg 2160tcgatcagga
tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
2220ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc
gatgcctgct 2280tgccgaatat catggtggaa aatggccgct tttctggatt
catcgactgt ggccggctgg 2340gtgtggcgga ccgctatcag gacatagcgt
tggctacccg tgatattgct gaagagcttg 2400gcggcgaatg ggctgaccgc
ttcctcgtgc tttacggtat cgccgctccc gattcgcagc 2460gcatcgcctt
ctatcgcctt cttgacgagt tcttctgaat tattaacgct tacaatttcc
2520tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcatc
aggtggcact 2580tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
tctaaataca ttcaaatatg 2640tatccgctca tgagacaata accctgataa
atgcttcaat aatagcacgt gctaaaactt 2700catttttaat ttaaaaggat
ctaggtgaag atcctttttg ataatctccg gaagagtcaa 2760gaacatgtga
gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
2820gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
caagtcagag 2880gtggcgaaac ccgacaggac tataaagata ccaggcgttt
ccccctggaa gctccctcgt 2940gcgctctcct gttccgaccc tgccgcttac
cggatacctg tccgcctttc tcccttcggg 3000aagcgtggcg ctttctcata
gctcacgctg taggtatctc agttcggtgt aggtcgttcg 3060ctccaagctg
ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
3120taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
cagcagccac 3180tggtaacagg attagcagag cgaggtatgt aggcggtgct
acagagttct tgaagtggtg 3240gcctaactac ggctacacta gaagaacagt
atttggtatc tgcgctctgc tgaagccagt 3300taccttcgga aaaagagttg
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 3360tggttttttt
gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
3420tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
aagggatttt 3480ggtccggccg gaaacgtttg gttgctgact aattgagatg
catgctttgc atacttctgc 3540ctgctgggga gcctggggac tttccacacc
tcgcgatgta cgggccagat atacgc 3596213884DNAArtificial
SequencePlasmid 21cgttgacatt gattattgac tagttattaa tagtaatcaa
ttacggggtc attagttcat 60agcccatata tggagttccg cgttacataa cttacggtaa
atggcccgcc tggctgaccg 120cccaacgacc cccgcccatt gacgtcaata
atgacgtatg ttcccatagt aacgccaata 180gggactttcc attgacgtca
atgggtggag tatttacggt aaactgccca cttggcagta 240catcaagtgt
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
300gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
gtacatctac 360gtattagtca tcgctattac catggtgatg cggttttggc
agtacatcaa tgggcgtgga 420tagcggtttg actcacgggg atttccaagt
ctccacccca ttgacgtcaa tgggagtttg 480ttttggcacc aaaatcaacg
ggactttcca aaatgtcgta acaactccgc cccattgacg 540caaatgggcg
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
600agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag
ggagacccaa 660gctggctagc gtttaaactt aagccaccat gaatctcctt
cacgaaaccg actcggctgt 720ggccaccgcg cgccgcccgc gctggctgtg
cgctggggcg ctggtgctgg cgggtggctt 780ctttctcctc ggcttcctct
tcgggtggtt tataaaaagc gctcagctgg caggggccaa 840aggagtcatt
ctctactccg accctgctga ctactttgct cctggggtga agtcctatcc
900agatggttgg aatcttcctg gaggtggtgt ccagcgtgga aatatcctaa
atctgaatgg 960tgcaggagac cctctcacac caggttaccc agcaaatgaa
tatgcttata ggcgtggaat 1020tgcagaggct gttggtcttc caagtattcc
tgttcatcct attgccctgc agagtctctt 1080gcagcacctc atcgggctga
gcaatctgac ccacgtgctg tatcctgtcc ccctggagag 1140ttatgaggac
atccatggta ccctccacct ggagaggctt gcctatctgc atgccaggct
1200cagggagttg ctgtgtgagt tggggcggcc cagcatggtc tggcttagtg
ccaacccctg 1260tcctcactgt ggggacagaa ccttctatga cccggagccc
atcctgtgcc cctgtttcat 1320gcctaacaag cgatcgctcc tgcaacacct
catcgggctg ggggacgccg cctacagtct 1380cctgcaacac ctcatcgggc
tgatttcccc ggagaaggaa gagcagtata tcgccagtct 1440cctgcaacac
ctcatcgggc tgaagaggcc aagtattaag aggggtcttc caagtattcc
1500tgttcatcca gtttagtgag aattctgcag atatccatca cactggcggc
cgctcgagtc 1560tagagggccc gtttaaaccc gctgatcagc ctcgactgtg
ccttctagtt gccagccatc 1620tgttgtttgc ccctcccccg tgccttcctt
gaccctggaa ggtgccactc ccactgtcct 1680ttcctaataa aatgaggaaa
ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 1740gggtggggtg
gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
1800ggatgcggtg ggctctatgg cttctactgg gcggttttat ggacagcaag
cgaaccggaa 1860ttgccagctg gggcgccctc tggtaaggtt gggaagccct
gcaaagtaaa ctggatggct 1920ttcttgccgc caaggatctg atggcgcagg
ggatcaagct ctgatcaaga gacaggatga 1980ggatcgtttc gcatgattga
acaagatgga ttgcacgcag gttctccggc cgcttgggtg 2040gagaggctat
tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
2100ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
gtccggtgcc 2160ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc
tggccacgac gggcgttcct 2220tgcgcagctg tgctcgacgt tgtcactgaa
gcgggaaggg actggctgct attgggcgaa 2280gtgccggggc aggatctcct
gtcatctcac cttgctcctg ccgagaaagt atccatcatg 2340gctgatgcaa
tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
2400gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
cgatcaggat 2460gatctggacg aagagcatca ggggctcgcg ccagccgaac
tgttcgccag gctcaaggcg 2520agcatgcccg acggcgagga tctcgtcgtg
acccatggcg atgcctgctt gccgaatatc 2580atggtggaaa atggccgctt
ttctggattc atcgactgtg gccggctggg tgtggcggac 2640cgctatcagg
acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
2700gctgaccgct tcctcgtgct
ttacggtatc gccgctcccg attcgcagcg catcgccttc 2760tatcgccttc
ttgacgagtt cttctgaatt attaacgctt acaatttcct gatgcggtat
2820tttctcctta cgcatctgtg cggtatttca caccgcatca ggtggcactt
ttcggggaaa 2880tgtgcgcgga acccctattt gtttattttt ctaaatacat
tcaaatatgt atccgctcat 2940gagacaataa ccctgataaa tgcttcaata
atagcacgtg ctaaaacttc atttttaatt 3000taaaaggatc taggtgaaga
tcctttttga taatctccgg aagagtcaag aacatgtgag 3060caaaaggcca
gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
3120ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
tggcgaaacc 3180cgacaggact ataaagatac caggcgtttc cccctggaag
ctccctcgtg cgctctcctg 3240ttccgaccct gccgcttacc ggatacctgt
ccgcctttct cccttcggga agcgtggcgc 3300tttctcatag ctcacgctgt
aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 3360gctgtgtgca
cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
3420ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
ggtaacagga 3480ttagcagagc gaggtatgta ggcggtgcta cagagttctt
gaagtggtgg cctaactacg 3540gctacactag aagaacagta tttggtatct
gcgctctgct gaagccagtt accttcggaa 3600aaagagttgg tagctcttga
tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 3660tttgcaagca
gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
3720ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
gtccggccgg 3780aaacgtttgg ttgctgacta attgagatgc atgctttgca
tacttctgcc tgctggggag 3840cctggggact ttccacacct cgcgatgtac
gggccagata tacg 3884229PRThomo sapien 22Glu Ala Ala Gly Ile Gly Leu
Thr Val1 5
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