U.S. patent application number 17/563392 was filed with the patent office on 2022-06-23 for compositions, comprising improved il-12 genetic constructs and vaccines, immunotherapeutics and methods of using the same.
The applicant listed for this patent is The Trustees of the University of Pennsylvania. Invention is credited to Matthew P. Morrow, David Weiner, Jian Yan.
Application Number | 20220193230 17/563392 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-23 |
United States Patent
Application |
20220193230 |
Kind Code |
A1 |
Weiner; David ; et
al. |
June 23, 2022 |
COMPOSITIONS, COMPRISING IMPROVED IL-12 GENETIC CONSTRUCTS AND
VACCINES, IMMUNOTHERAPEUTICS AND METHODS OF USING THE SAME
Abstract
Nucleic acid molecules and compositions comprising: a nucleic
acid sequence that encodes IL-12 p35 subunit or a functional
fragment thereof and/or a nucleic acid sequence that encodes IL12
p40 subunit or a functional fragment thereof, are disclosed. The
nucleic acid molecules and compositions further comprising a
nucleic acid sequence that encodes an immunogen are also disclosed.
Method of modulating immune response and methods of inducing an
immune response against an immunogen are disclosed. Therapeutic and
prophylactic vaccination methods are also disclosed.
Inventors: |
Weiner; David; (Merion,
PA) ; Morrow; Matthew P.; (Bala Cynwyd, PA) ;
Yan; Jian; (Wallingford, PA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
The Trustees of the University of Pennsylvania |
Philadelphia |
PA |
US |
|
|
Appl. No.: |
17/563392 |
Filed: |
December 28, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15974982 |
May 9, 2018 |
11241496 |
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17563392 |
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15055002 |
Feb 26, 2016 |
9981036 |
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15974982 |
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14365086 |
Jun 12, 2014 |
9272024 |
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PCT/US12/69017 |
Dec 11, 2012 |
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15055002 |
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61569600 |
Dec 12, 2011 |
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International
Class: |
A61K 39/39 20060101
A61K039/39; A61K 38/20 20060101 A61K038/20; C07K 14/54 20060101
C07K014/54; A61K 39/00 20060101 A61K039/00; A61K 39/245 20060101
A61K039/245; C12N 7/00 20060101 C12N007/00; A61K 39/29 20060101
A61K039/29; A61K 41/00 20060101 A61K041/00 |
Claims
1.-67. (canceled)
68. A composition that comprises a) a nucleic acid sequence that
encodes a functional fragment of IL-12 p35 subunit and b) a nucleic
acid sequence that encodes a functional fragment of IL-12 p40
subunit, wherein the nucleic acid sequence that encodes a
functional fragment of IL-12 p35 subunit is at least 80% homologous
to SEQ ID NO:1 and encodes a protein at least 80% homologous to SEQ
ID NO:2, and the nucleic acid sequence that encodes a functional
fragment of IL-12 p40 subunit is at least 80% homologous to SEQ ID
NO:3 and encodes a protein at least 80% homologous to SEQ ID
NO:4.
69. The composition of claim 68 comprising the nucleic acid
sequence that encodes a functional fragment of IL-12 p35 subunit is
at least 83% homologous to SEQ ID NO:1 and encodes a protein at
least 83% homologous to SEQ ID NO:2, and the nucleic acid sequence
that encodes a functional fragment of IL-12 p40 subunit is at least
83% homologous to SEQ ID NO:3 and encodes a protein at least 83%
homologous to SEQ ID NO:4.
70. The composition of claim 68 comprising the nucleic acid
sequence that encodes a functional fragment of IL-12 p35 subunit is
at least 85% homologous to SEQ ID NO:1 and encodes a protein at
least 85% homologous to SEQ ID NO:2, and the nucleic acid sequence
that encodes a functional fragment of IL-12 p40 subunit is at least
85% homologous to SEQ ID NO:3 and encodes a protein at least 85%
homologous to SEQ ID NO:4.
71. The composition of claim 68 comprising the nucleic acid
sequence that encodes a functional fragment of IL-12 p35 subunit is
at least 87% homologous to SEQ ID NO:1 and encodes a protein at
least 87% homologous to SEQ ID NO:2, and the nucleic acid sequence
that encodes a functional fragment of IL-12 p40 subunit is at least
87% homologous to SEQ ID NO:3 and encodes a protein at least 87%
homologous to SEQ ID NO:4.
72. The composition of claim 68 formulated for delivery to an
individual using electroporation.
73. The composition of claim 68, wherein the nucleic acid sequence
that encodes IL-12 p35 subunit is on a different nucleic acid
molecule than the nucleic acid sequence that encodes IL-12 p40
subunit.
74. The composition of claim 68, wherein the nucleic acid sequence
that encodes IL-12 p35 subunit is on a plasmid and the nucleic acid
sequence that encodes IL-12 p40 subunit is on a different
plasmid.
75. The composition of claim 68, wherein the nucleic acid sequence
that encodes IL-12 p35 subunit and the nucleic acid sequence that
encodes IL-12 p40 subunit are on the same nucleic acid
molecule.
76. The composition of claim 68, wherein the nucleic acid sequence
that encodes IL-12 p35 subunit and the nucleic acid sequence that
encodes IL-12 p40 subunit are on the same plasmid.
77. The composition of claim 68, wherein the nucleic acid sequence
that encodes IL-12 p35 subunit and the nucleic acid sequence that
encodes IL-12 p40 subunit are on the same nucleic acid molecule and
operably linked to different promoters.
78. The composition of claim 68, wherein the nucleic acid sequence
that encodes IL-12 p35 subunit and the nucleic acid sequence that
encodes IL-12 p40 subunit are on the same plasmid and operably
linked to different promoters.
79. The composition of claim 68 further comprising a nucleic acid
sequence that encodes an immunogen.
80. The composition of claim 68 further comprising a nucleic acid
sequence that encodes an immunogen from a pathogen selected from
the group consisting of: HIV, HPV, HCV, Influenza, Smallpox,
Chikungunya, foot and mouth disease virus, Malaria, human
cytomegalovirus, human respiratory syncytial virus, and MRSA.
81. The composition of claim 68, wherein the nucleic acid sequence
that encodes IL-12 p35 subunit and the nucleic acid sequence that
encodes IL-12 p40 subunit are incorporated into a viral
particle.
82. The composition of claim 68 further comprising a nucleic acid
sequence that encodes one or more proteins selected from the group
consisting of: IL-15 and IL-28.
83. A method of inducing an immune response against an immunogen
comprising administering to an individual, a composition of claim
68 in combination with a nucleic acid sequence that encodes an
immunogen in an amount effective to induce an immune response in
said individual.
84. The method of claim 83, wherein the composition further
comprises a nucleic acid sequence that encodes an immunogen.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation application claiming benefit to U.S.
patent application Ser. No. 15/974,982, filed May 9, 2018, which is
a continuation application claiming benefit to U.S. patent
application Ser. No. 15/055,002, filed Feb. 26, 2016, issued as
U.S. Pat. No. 9,981,036, which is a continuation application
claiming benefit to U.S. patent application Ser. No. 14/365,086,
filed Jun. 12, 2014, issued as U.S. Pat. No. 9,272,024, which is
the U.S. national stage application filed under 35 U.S.C. .sctn.
371 claiming benefit to International Patent Application No.
PCT/US2012/069017, filed Dec. 11, 2012, which is entitled to
priority under 35 U.S.C. .sctn. 119(e) to U.S. Provisional
Application No. 61/569,600, filed Dec. 12, 2011, each of which is
hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to improved genetic constructs
that encode human IL-12 and nucleic acid molecules which comprise
the same. The present invention also relates to improved expression
vectors, vaccines and immunotherapeutics which include nucleotide
sequences that encode human 11-12 and to methods of using the
same.
BACKGROUND OF THE INVENTION
[0003] Immunotherapy refers to modulating a person's immune
responses to impart a desirable therapeutic effect.
Immunotherapeutics refer to those compositions which, when
administered to an individual, modulate the individual's immune
system sufficient to ultimately decrease symptoms which are
associated with undesirable immune responses or to ultimately
alleviate symptoms by increasing desirable immune responses. In
some cases, immunotherapy is part of a vaccination protocol in
which the individual is administered a vaccine that exposes the
individual to an immunogen against which the individual generates
an immune response in such cases, the immunotherapeutic increases
the immune response and/or selectively enhances a portion of the
immune response (such as the cellular arm or the humoral arm) which
is desirable to treat or prevent the particular condition,
infection or disease.
[0004] In designing vaccines, it has been recognized that vaccines
that produce the target antigen in cells of the vaccinated
individual are effective in inducing the cellular arm of the immune
system. Specifically, live attenuated vaccines, recombinant
vaccines which use avirulent vectors and DNA vaccines each lead to
the production of antigens in the cell of the vaccinated individual
which results in induction of the cellular arm of the immune
system, On the other hand, killed or inactivated vaccines, and
sub-unit vaccines which comprise only proteins do not induce good
cellular immune responses although they do induce an effective
humoral response.
[0005] A cellular immune response is often necessary to provide
protection against pathogen infection and to provide effective
immune-mediated therapy for treatment of pathogen infection, cancer
or autoimmune diseases. Accordingly, vaccines that produce the
target antigen in cells of the vaccinated individual such as live
attenuated vaccines, recombinant vaccines that use avirulent
vectors and DNA vaccines are often preferred.
[0006] There is a need for vaccine approaches that can induce
strong T cell and B cell immunity in humans, Recent concerns over
attenuation, vaccine manufacturing complexity, serological
interference, as was observed in the HIV STEP trial, among a host
of other issues serve to underscore this important issue. In
non-human primate models and in human clinical simple plasmid DNA
as a vaccine platform has not induced levels of immunogenicity
satisfactory for commercial development efforts to be supported. In
head to head comparisons sonic naked plasmid-based vaccines did not
induce either cellular or humoral responses comparable to those
induced by their viral vector counterparts, including the commonly
used adenovirus serotype 5 (Ad5) platform.
[0007] The development of DNA vaccine technology as a stand-alone
method of vaccination, as well as its utility in current
prime-boost platforms, would benefit by the development of
strategies to enhance its immune potency. The manipulation of codon
and RNA encoding sequences as well as changes in leader sequences
have been reported to enhance the expression of plasmid-encoded
immunogens. In addition, the creation of consensus immunogens
attempts to address the need for broad immunological coverage to
account in part for viral diversity.
[0008] In addition, other strategies have been employed that focus
on improving the physical delivery of DNA plasmids by improving
formulations and device driven technologies. DNA vaccines delivered
by electroporation (EP) have been reported to enhance
antigen-specific interferon-.gamma. (IFN.gamma.) production
following immunization of plasmid DNA in rhesus macaques.
[0009] The co-delivery of plasmid-encoded molecular adjuvants to
augment vaccine-induced responses is another important area of this
specific investigation, One of the best-characterized molecular
adjuvants in non-human primates is a IL-12, a TH1 polarizing
cytokine that drives CTL responses by providing the "third signal"
needed for efficient activation and antigen-specific expansion of
naive CD8+ T cells. IL-12 is a heterodimer which contains two
subunits, p35 and p40. It has been shown to be the most impressive
immune enhancing cytokine, particularly for driving CD8 T cells
when engineered as a DNA vaccine. In macaques, IL-12 has been shown
to be an adjuvant that is highly potent for expanding the cellular
Immune potency of a DNA vaccine targeting multiple antigens. In
both macaques as well and in humans such a DNA vaccine adjuvant can
significantly improve the immune responses induced by a DNA
vaccine.
[0010] U.S. Pat. No. 5,723,127, which is incorporated herein by
reference, discloses IL-12 as a vaccine adjuvant. PCT application
no. PCT/US1997/019502 and corresponding U.S. application Ser. No.
08/956,865, which is incorporated herein by reference, discloses
DNA, vaccines and DNA constructs comprising IL-12 coding
sequences.
[0011] There remains a need for improved vaccines and
immunotherapeutics. There is a need for compositions and methods
that produce enhanced immune responses. Likewise, while some
immunotherapeutics are useful to modulate immune response in a
patient, there remains a need for improved immunotherapeutic
compositions and methods. There remains a need for improved
constructs which encode IL-12 and can be used as part of DNA
vaccine strategies. There remains a need for improved constructs
which encode IL-12 and can be used as an immunotherapeutic. There
remains a need for improved constructs which encode IL-12 and can
be used to achieve high levels of expression of IL-12.
SUMMARY FO THE INVENTION
[0012] Compositions are provided that comprises a nucleic acid
sequence that encodes IL-12 p35 subunit or a functional fragment
thereof and a nucleic acid sequence that encodes IL-12 p40 subunit
or a functional fragment thereof. Nucleic acid sequences that
encodes IL-12 p35 subunit may be at least 98% homologous to SEQ ID
NO:1 and encode a protein at least 98% homologous to SEQ NO:2.
Nucleic acid sequences that encodes functional fragment of IL-12p35
subunit may be fragments of a nucleic acid sequence that is at
least 98% homologous to SEQ ID NO:1 and encodes a protein at least
98% homologous to a functional fragment of SEQ ID NO:2. Nucleic
acid sequences that encodes IL-12 p40 subunit may be at least 98%
homologous to SEQ ID NO:3 and encode a protein at least 98%
homologous to SEQ ID NO:4. Nucleic acid sequences that encodes
functional fragment of IL-12 p40 subunit may be fragments of a
nucleic acid sequence that is at least 98% homologous to SEQ ID
NO:3 and encodes a protein at least 98% homologous to a functional
fragment of SEQ ID NO0:4. Compositions may further comprise a
nucleic acid sequence that encodes an immunogen.
[0013] Method of modulating immune response are also provided. The
methods comprise the step of administering to an individual, a
composition that comprises a nucleic acid sequence that encodes
IL-12. p35 subunit or a functional fragment thereof and a nucleic
acid sequence that encodes IL-12 p40 subunit or a functional
fragment thereof.
[0014] Method of inducing an immune response against an immunogen
are also provided. The methods comprise the step of administering
to an individual, a composition that encodes IL-12 p35 subunit or a
functional fragment thereof and a nucleic acid sequence that
encodes IL-12 p40 subunit or a functional fragment thereof in
combination with a nucleic acid sequence that encodes an immunogen
in an amount. The methods of inducing an immune response against an
immunogen may be part of methods of inducing a therapeutic immune
response or methods of inducing a prophylactic immune response.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A and 1B shows a graph comparing expression levels of
human IL-12 cells transfected with 2 .mu.g HuIL12-opt or
HuIL12-nonopt (FIG. 1A) or 4 .mu.g HuIL12-opt and HuIL12-nonopt
(FIG. 1B).
[0016] FIG. 2 shows the enhanced PSA and PSMA-specific cellular
immune responses in rhesus macaques.
[0017] FIG. 3 shows the enhanced HBV core and surface
antigen-specific cellular immune responses in rhesus macaques.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0018] In one aspect of the invention, it is desired that the
improved IL-12 constructs provides for improved transcription and
translation, including having one or more of the following: low GC
content leader sequence to increase transcription; mRNA stability
and codon optimization; eliminating to the extent possible
cis-acting sequence motifs (i.e., internal TATA-boxes).
[0019] In some aspects of the invention, it is desired to
incorporate the improved IL-12 constructs into a vaccine regimen,
either as part of the vaccine composition or as a separate
composition delivered in a coordinated fashion with the vaccine in
order to generate a broad immune against vaccine immunogens. In
some aspects of the invention, it is desired to provide the
improved IL-12 constructs as an immunotherapeutic which can be used
to modulate immune responses in an individual. In some aspects of
the invention, it is desired to provide the improved IL-12
constructs in order to provide expression vectors which can be used
to obtain high levels of IL-12 expression.
[0020] Higher potency IL-12 gene adjuvants are provided herein.
These new adjuvants have several advantages over older IL-12
molecules. An enhanced leader sequence that facilitates secretion
of the molecules as well as improves ribosome loading is provided,
thus expanding the impact of these adjuvants and increasing
expression. Significant changes to the RNA sequences further
removes homology to native IL-12 sequences thus preventing
interference between the delivered adjuvant and the host system, as
well as lowering possible deleterious interactions between the host
IL-12 sequences and the gene delivered molecules. Furthermore the
higher potency of the new constructs lowers the dose requirement
thus improving manufacturing as well as delivery issues associated
with such adjuvants. Finally as these molecules have more
bioactivity, they improve performance of the vaccine in vivo.
Together these are important new tools for vaccine as well as
immune therapy applications.
I. Definitions,
[0021] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting. As
used in the specification and the appended claims, the singular
forms "a," "an" and "the" include plural referents unless the
context clearly dictates otherwise.
[0022] For recitation of numeric ranges herein, each intervening
number there between with the same degree of precision is
explicitly contemplated. For example, for the range of 6-9, the
numbers 7 and 8 are contemplated in addition to 6 and 9, and for
the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6,3, 6.4, 6.5, 6,6,
6.7, 6.8, 6,9, and 7.0 are explicitly contemplated.
[0023] a. Adjuvant
[0024] "Adjuvant" as used herein may mean a molecule, including a
nucleic acid molecule that encodes a protein having
immunomodulating activity, added to DNA, plasmid vaccines or other
vaccines to enhance antigenicity of the one or more antigens
encoded by the DNA plasmids or vaccines, and nucleic acid sequences
that encode the adjuvant protein described hereinafter,
[0025] b. Antibody
[0026] "Antibody" may mean an antibody of classes IgG, IgM, IgA,
IgD or IgE, or fragments, fragments or derivatives thereof,
including Fab, F(ab')2, Fd, and single chain antibodies, diabodies,
bispecific antibodies, bifunctional antibodies and derivatives
thereof. The antibody may be an antibody isolated from the serum
sample of mammal, a polyclonal antibody, affinity purified
antibody, or mixtures thereof which exhibits sufficient binding
specificity to a desired epitope or a sequence derived
therefrom.
[0027] c. Coding Sequence
[0028] "Coding sequence" or "encoding nucleic acid" as used herein
may mean refers to the nucleic acid (RNA or DNA molecule) that
comprise a nucleotide sequence which encodes a protein. The coding
sequence may further include initiation and termination signals
operably linked to regulatory elements including a promoter and
polyadenylation signal capable of directing expression in the cells
of an individual or mammal to whom the nucleic acid is
administered.
[0029] d. Complement
[0030] "Complement" or "complementary" as used herein may mean a
nucleic acid may mean Watson-Crick (e.g., and C-G) or Hoogsteen
base pairing between nucleotides or nucleotide analogs of nucleic
acid molecules.
[0031] e. Constant Current
[0032] "Constant current" as used herein to define a current that
is received or experienced by a tissue, or cells defining said
tissue, over the duration of an electrical pulse delivered to same
tissue. The electrical pulse is delivered from the electroporation
devices described herein. This current remains at a constant
amperage in said tissue over the life of an electrical pulse
because the electroporation device provided herein has a feedback
element, preferably having instantaneous feedback. The feedback
element can measure the resistance of the tissue (or cells)
throughout the duration of the pulse and cause the electroporation
device to alter its electrical energy output (e.g., increase
voltage) so current in same tissue remains constant throughout the
electrical pulse (on the order of microseconds), and from pulse to
pulse. In some embodiments, the feedback element comprises a
controller.
[0033] f. Current Feedback or Feedback
[0034] "Current feedback" or "feedback" as used herein may be used
interchangeably and may mean the active response of the provided
electroporation devices, which comprises measuring the current in
tissue between electrodes and altering the energy output delivered
by the EP device accordingly in order to maintain the current at a
constant level. This constant level is preset by a user prior to
initiation of a pulse sequence or electrical treatment. The
feedback may be accomplished by the electroporation component,
e.g., controller, of the electroporation device, as the electrical
circuit therein is able to continuously monitor the current in
tissue between electrodes and compare that monitored current (or
current within tissue) to a preset current and continuously make
energy-output adjustments to maintain the monitored current at
preset levels. The feedback loop may be instantaneous as it is an
analog closed-loop feedback.
[0035] g. Decentralized Current
[0036] "Decentralized current" as used herein may mean the pattern
of electrical currents delivered from the various needle electrode
arrays of the electroporation devices described herein, wherein the
patterns minimize, or preferably eliminate, the occurrence of
electroporation related heat stress on any area of tissue being
electroporated.
[0037] h. Electroporation
[0038] "Electroporation." "electro-permeabilization," or
"electro-kinetic enhancement" ("EP") as used interchangeably herein
may refer to the use of a transmembrane electric field pulse to
induce microscopic pathways (pores) in a bio-membrane; their
presence allows biomolecules such as plasmids, oligonucleotides,
siRNA, drugs, ions, and water to pass from one side of the cellular
membrane to the other.
[0039] i. Feedback Mechanism
[0040] "Feedback mechanism" as used herein may refer to a process
performed by either software or hardware (or firmware), which
process receives and compares the impedance of the desired tissue
(before, during, and/or after the delivery of pulse of energy) with
a present value, preferably current, and adjusts the pulse of
energy delivered to achieve the preset value. A feedback mechanism
may be performed by an analog closed loop circuit.
[0041] j. Fragment
[0042] "Fragment" as used herein may mean a portion or a nucleic
acid that encodes a polypeptide capable of eliciting an immune
response in a mammal substantially similar to that of the
non-fragment for The fragments may be DNA fragments selected from
fragments of SEQ ID NO:1, fragments of a nucleic acid sequence that
is at least 98% homologous to SEQ ID NO:1 and encodes a function
fragments of a protein that is at least 98% homologous to SEQ ID
NO:2; fragments of SEQ ID NO:3, and fragments of a nucleic acid
sequence that is at least 98% homologous to SEQ ID NO:3 and encodes
a function fragments of a protein that is at least 98% homologous
to SEQ ID NO:4.
[0043] The DNA fragments of SEQ ID NO:1, fragments of a nucleic
acid sequence that is at least 98% homologous to SEQ ID NO:1 and
encodes a function fragments of a protein that is at least 98%
homologous to SEQ ID NO:2 may encodes 50 or more amino acids in
length, 55 or more, 6(i or more, 65 or more, 70 or more, 75 or
more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more,
105 or more, 110 or more, 115 or more, 120 or more, 125 or more,
130 or more, 135 or more, 140 or more, 145 or more, 150 or more,
155 or more, 160 or more, 165 or more, 170 or more, 175 or more,
180 or more, 185 or more, 190 or more, 195 or more, 200 or more,
205 or more, 210 or more in length or 215 or more of SEQ ID NO:2 or
a protein that is at 98% homologous to SEQ ID NO:2 The DNA
fragments of SEQ ID NO:1, fragments of a nucleic acid sequence that
is at least 98% homologous to SEQ ID NO:1 and encodes a function
fragments of a protein that is fewer than 53, fewer than 58, fewer
than 63, fewer than 68, fewer than 73, fewer than 78, fewer than
83, fewer than 88, fewer than 93. fewer than 98, fewer than 103,
fewer than 108, fewer than 113, fewer than 118, fewer than 123,
fewer than 128, fewer than 133, fewer than 138, fewer than 143,
fewer than 148, fewer than 153, fewer than 158, fewer than 163,
fewer than 168, fewer than 173, fewer than 178 fewer than 183,
fewer than 188, fewer than 193, fewer than 198, fewer than 203,
fewer than 208, fewer than 213 or fewer than 218 amino acids in
length of SEQ ID NO:2 or a protein that is at least 98% homologous
to SEQ Ill NO:2. In some embodiments, the fragments of a nucleic
acid sequence that is at least 98% homologous to SEQ ID NO:1
encodes functional fragments of a protein that is at least 98%
homologous to SEQ ID NO:2. In some embodiments, the fragments of a
nucleic acid sequence that is at least 98% homologous to SEQ ID
NO:1 encodes functional fragments of a protein that is at least 99%
homologous to SEQ ID NO:2, In some embodiments, the fragments of a
nucleic acid sequence that is at least 98% homologous to SEQ ID
NO:1 encodes functional fragments of SEQ ID NO:2. In some
embodiments, the fragments of a nucleic acid sequence that is at
least 99% homologous to SEQ ID NO:1 encodes functional fragments of
a protein that is at Least 98% homologous to SEQ ID NO:2. In some
embodiments, the fragments of a nucleic acid sequence that is at
least 99% homologous to SEQ ID NO:1 encodes functional fragments of
a protein that is at least 99% homologous to SEQ ID NO:2. In some
embodiments, the fragments of a nucleic acid sequence that is at
least 99% homologous to SEQ ID NO:1 encodes functional fragments of
SEQ ID NO:2. In some embodiments, the fragments are fragments of
SEQ ID NO:1 that encode functional fragments of SEQ ID NO:2.
[0044] The DNA fragments of SEQ ID NO:3, fragments of a nucleic
acid sequence that is at least 98% homologous to SEQ ID NO:3 and
encodes a function fragments of a protein that is at least 98%
homologous to SEQ ID NO:4 may encodes 50 or more amino acids in
length, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more,
80 or more, 85 or more, 90 or more, 95 or more, 100 or more, 105 or
more, 110 or more, 115 or more, 120 or more, 125 or more, 130 or
more, 135 or more, 140 or more, 145 or more, 1.50 or more, 155 or
more, 160 or more, 165 or more, 170 or more, 175 or more, 180 or
more, 185 or more, 190 or more, 195 or more, 200 or more, 205 or
more, 210 or more, 215 or more, 220 or more, 225 or more, 230 or
more, 235 or more, 240 or more, 245 or more, 250 or more, 255 or
more, 260 or more, 265 or more, 270 or more, 275 or more, 280 or
more, 285 or more, 290 or more, 295 or more, 300 or more, 305 or
more, 310 or more, 315 or more, 320 or more, or 325 or more amino
acids of SEQ ID NO:4 or of a protein that is at least 98%
homologous to SEQ ID NO:4 The DNA fragments of SEQ ID NO:3 and
fragments of a nucleic acid sequence that is at least 98%
homologous to SEQ ID NO:3 may encode a function fragments that is
fewer than 53, fewer than 58, fewer than 63, fewer than 68, fewer
than 73, fewer than 78, fewer than 83, fewer than 88, fewer than
93, fewer than 98, fewer than 103, fewer than 108, fewer than 113,
fewer than 118, fewer than 123, fewer than 128, fewer than 133,
fewer than 138, fewer than 143, fewer than 148, fewer than 153,
fewer than 158, fewer than 163, fewer than 168, fewer than 173,
fewer than 178, fewer than 183, fewer than 188, fewer than 193,
fewer than 198, fewer than 203, fewer than 208, fewer than 213,
fewer than 218, fewer than 223, fewer than 228, fewer than 233,
fewer than 238, fewer than 243, fewer than 248, fewer than 253,
fewer than 258, fewer than 263, fewer than 268, fewer than 273,
fewer than 278, fewer than 283, fewer than 288, fewer than 293,
fewer than 298, fewer than 303, fewer than 308, fewer than 313,
fewer than 318 or fewer than 328 amino acids SEQ ID NO:4 or a
protein that is at least 98% homologous to SEQ ID NO:4. In some
embodiments, the fragments of a nucleic acid sequence that is at
least 98% homologous to SEQ ID NO:3 encodes functional fragments of
a protein that is at least 98% homologous to SEQ ID NO:4, In some
embodiments, the fragments of a nucleic acid sequence that is at
least 98% homologous to SEQ ID NO:3 encodes functional fragments of
a protein that is at least 99% homologous to SEQ ID NO:4. In some
embodiments, the fragments of a nucleic acid sequence that is at
least 98% homologous to SEQ ID NO:3 encodes functional fragments of
SEQ ID NO:4, In some embodiments, the fragments of a nucleic acid
sequence that is at least 99% homologous to SEQ ID NO:3 encodes
functional fragments of a protein that is at least 98% homologous
to SEQ ID NO:4. In some embodiments, the fragments of a nucleic
acid sequence that is at least 99% homologous to SEQ ID NO:3
encodes functional fragments of a protein that is at least 99%
homologous to SEQ ID NO:4. In some embodiments, the fragments of a
nucleic acid sequence that is at least 99% homologous to SEQ ID
NO:3 encodes functional fragments of SEQ ID NO:4. In some
embodiments, the fragments are fragments of SEQ Ii) NO:3 that
encode functional fragments of SEQ ID NO:4.
[0045] DNA fragments may be free of coding sequences for IL-12
signal peptide. DNA fragments may comprise coding sequences for the
immunoglobulin signal peptide such as IgE or IgG signal peptide
sequences. Thus for example, DNA fragments that encode an IL-12 p35
subunit not encode amino acids 1-22 of SEQ ID NO:2 and, in some
such embodiments, may comprises sequences that encode an
immunoglobulin signal peptide such as IgE signal peptide sequence
(SEQ ID NO:5) or IgG signal peptide sequence.
[0046] "Fragment" may also refer to polypeptide fragments capable
of functioning substantially substantially similar to that of the
full length polypeptide. The fragment of IL-12 p35 may be a
fragment of SEQ ID NO:2 or a fragment of a polypeptide that is at
least 98% homologous to a fragment of SEQ ID NO:2. The fragment of
IL-12 p35 may be a fragment of a polypeptide that is at least 99%
homologous to a fragment of SEQ ID The fragment of IL-12 p35 may be
a fragment as described above. The fragment of IL-12 p40 may be a
fragment of SEQ ID NO:4 or a fragment of a polypeptide that is at
least 98% homologous to a fragment of SEQ ID NO:4. The Fragment of
IL-12 p40 may be a fragment of a polypeptide that is at least 99%
homologous to a fragment of SEQ ID NO:4. The fragment of IL-12 p40
may be a fragment as described above.
[0047] A "functional fragment" is meant to refer to a fragment of
an IL-12 subunit that less than complete p35 and/or less than
complete p40 sequence, that, can function substantially similarly
to full length p35 or p40. Such substantially similar function
includes interaction with other proteins, subunits and receptors in
a substantially same manner as the full length p35 or p40 and when
delivered in a manner that allows for formation of a heterodimer
results in substantially the same effect as the IL-12 p35/p40
heterodimer.
[0048] k. Genetic Construct
[0049] The term "genetic construct" as used herein refers to the
DNA or RNA molecules that comprise a nucleotide sequence which
encodes one or both IL-12 subunits or a target protein or another
(non-IL-12) immunomodulating protein. The coding sequence includes
initiation and termination signals operably linked to regulatory
elements including a promoter and polyadenylation signal capable of
directing expression in the cells of the individual to whom the
nucleic acid molecule is administered.
[0050] I. Hyperproliferative
[0051] As used herein, the term "hyperproliferative diseases" is
meant to refer to those diseases and disorders characterized by
hyperproliferation of cells and the term
"hyperproliferative-associated protein" is meant to refer to
proteins that are associated with a hyperproliferative disease.
[0052] M. Identical
[0053] "Identical" or "identity" as used herein in the context of
two or more nucleic acids or polypeptide sequences, may mean that
the sequences have a specified percentage of residues that are the
same over a specified region. The percentage may be calculated by
optimally aligning the two sequences, comparing the two sequences
over the specified region, determining the number of positions at
which the identical residue occurs in both sequences to yield the
number of matched positions, dividing the number of matched
positions by the total number of positions in the specified region,
and multiplying the result by 100 to yield the percentage of
sequence identity. In cases where the two sequences are of
different lengths or the alignment produces one or more staggered
ends and the specified region of comparison includes only a single
sequence, the residues of single sequence are included in the
denominator but not the numerator of the calculation. When
comparing DNA and RNA, thymine (T) and uracil (Li) may be
considered equivalent. Identity may be performed manually or by
using a computer sequence algorithm such as BLAST or BLAST 2.0.
[0054] n. Impedance
[0055] "Impedance" as used herein may be used when discussing the
feedback mechanism and can be converted to a current value
according to Ohm's law, thus enabling comparisons with the preset
current.
[0056] o. Immune Response
[0057] "Immune response" as used herein may mean the activation of
a hoses immune system, e.g., that of a mammal, in response to the
introduction of one or more RSV consensus antigen via the provided
DNA plasmid vaccines. The immune response can be in the form of a
cellular or humoral response, or both.
[0058] p. Intracellular Pathogen
[0059] "Intracellular pathogen" as used herein, is meant to refer
to a virus or pathogenic organism that, at least part of its
reproductive or life cycle, exists within a host cell and therein
produces or causes to be produced, pathogen proteins.
[0060] q. Nucleic. Add
[0061] "Nucleic acid" or "oligonucleotide" or "polynucleotide" as
used herein may mean at least two nucleotides covalently linked
together. The depiction of a single strand also defines the
sequence of the complementary strand. Thus, a nucleic acid also
encompasses the complementary strand of a depicted single strand.
Many variants of a nucleic acid may be used for the same purpose as
a given nucleic acid. Thus, a nucleic acid also encompasses
substantially identical nucleic acids and complements thereof. A
single strand provides a probe that may hybridize to a target
sequence under stringent hybridization conditions. Thus, a nucleic
acid also encompasses a probe that hybridizes under stringent
hybridization conditions. Nucleic acids may be single stranded or
double stranded, or may contain portions of both double stranded
and single stranded sequence. The nucleic acid may be DNA, both
genomic and cDNA, RNA, or a hybrid, where the nucleic acid may
contain combinations of deoxyribo- and ribo-nucleotides, and
combinations of bases including uracil, adenine, thymine, cytosine,
guanine, inosine, xanthine hypoxanthine, isocytosine and
isoguanine. Nucleic acids may be obtained by chemical synthesis
methods or by recombinant methods.
[0062] r. Operably Linked
[0063] "Operably linked" as used herein when referring to a gene
operably linked to a promoter refers to the linkage of the two
components such that expression of the gene is under the control of
a promoter with which it is spatially connected. A promoter may be
positioned 5' (upstream) or 3' (downstream) of a gene under its
control. The distance between the promoter and a gene may be
approximately the same as the distance between that promoter and
the gene it controls in the gene from which the promoter is
derived, As is known in the art, variation in this distance may be
accommodated without loss of promoter function. When referring to a
signal peptide operable linked to a protein, the term refers to the
protein having the signal peptide incorporated as part of the
protein in a manner that it can function as a signal peptide. When
referring to coding sequence that encodes a signal peptide operable
linked to coding sequence that encodes a protein, the term refers
to the coding sequences arranged such that the translation of the
coding sequence produces a protein having the signal peptide
incorporated as part of the protein in a manner that it can
function as a signal peptide.
[0064] s. Promoter
[0065] "Promoter" as used herein may mean a synthetic or
naturally-derived molecule which is capable of conferring,
activating or enhancing expression of a nucleic acid in a cell. A
promoter may comprise one or more specific transcriptional
regulatory sequences to further enhance expression and/or to alter
the spatial expression and/or temporal expression of same. A
promoter may also comprise distal enhancer or repressor elements,
which can be located as much as several thousand base pairs from
the start site of transcription. A promoter may be derived from
sources including viral, bacterial, fungal, plants, insects, and
animals. A promoter may regulate the expression of a gene component
constitutively, or differentially with respect to cell, the tissue
or organ in which expression occurs or, with respect to the
developmental stage at which expression occurs, or in response to
external stimuli such as physiological stresses, pathogens, metal
ions, or inducing agents. Representative examples of promoters
include the bacteriophage T7 promoter, bacteriophage T3 promoter,
SP6 promoter, lac operator-promoter, tac promoter, SV40 late
promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter,
SV40 early promoter or SV40 late promoter and the CMV IE
promoter,
[0066] t. Stringent Hybridization Conditions
[0067] "Stringent hybridization conditions" as used herein may mean
conditions under which a. first nucleic acid sequence (e.g., probe)
will hybridize to a second nucleic acid sequence (e.g., target),
such as in a complex mixture of nucleic acids. Stringent conditions
are sequence-dependent and will be different in different
circumstances. Stringent conditions may be selected to be about
5-10.degree. C. lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic strength The Tm may be the
temperature (under defined ionic strength, pH, and nucleic
concentration) at which 50% of the probes complementary to the
target hybridize to the target sequence at equilibrium (as the
target sequences are present in excess, at Tm, 50% of the probes
are occupied at equilibrium). Stringent conditions may be those in
which the salt concentration is fess than about 1.0 M sodium ion,
such as about 0.01-1.0 M sodium ion concentration (or other salts)
at pH 7.0 to 8.3 and the temperature is at least about 30.degree.
C. for short probes (e.g., about 10-50 nucleotides) and at least
about 60.degree. C. for long probes (e.g., greater than about 50
nucleotides). Stringent conditions may also be achieved with the
addition of destabilizing agents such as formamide. For selective
or specific hybridization, a positive signal may be at least 2 to
10 times background hybridization. Exemplary stringent
hybridization conditions include the following: 50% formamide, 5x
SSC, and 1% SDS, incubating at 42.degree. C., or,5.times. SSC, 1%
SDS, incubating at 65.degree. C., with wash in 0.2.times. SSC, and
0.1% SDS at 65.degree. C.,
[0068] u. Substantially Complementary
[0069] "Substantially complementary" as used herein may mean that a
first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
97%, 98% or 99% identical to the complement of a second sequence
over a region of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19,20,21,22,23,24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100 or more nucleotides or amino acids, or that the two
sequences hybridize under stringent hybridization conditions.
[0070] v. Substantially Identical
[0071] "Substantially identical" as used herein may mean that a
first and second sequence are at least 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 97%, 98% or 99% identical over a region of 8,9, 10,
1.1, 12, 13, 14, 15, 1.6, 17, 18,
19,20,21,22,23,24,25,30,35,40,45,50,55,60,65, 70, 75, 80, 85, 90,
95, 100 or more nucleotides or amino acids, or with respect to
nucleic acids, if the first sequence is substantially complementary
to the complement of the second sequence.
[0072] w. Target Protein
[0073] "Target protein" as used herein is meant to refer to
peptides and protein which are part of vaccines or which are
encoded by gene constructs of DNA vaccines that act as target
proteins for an immune response. The terms "target protein" and
"immunogen" are used interchangeably and. refer to a protein
against which an immune response can be elicited. The target
protein is an immunogenic protein that shares at least an epitope
with a protein from the pathogen or undesirable cell-type such as a
cancer cell or a cell involved in autoimmune disease against which
an immune response is desired. The immune response directed against
the target protein will protect the individual against and/or treat
the individual for the specific infection or disease with which the
target protein is associated.
[0074] x. Variant
[0075] "Variant" used herein with respect to a nucleic acid may
mean (i) a portion or fragment of a referenced nucleotide sequence;
(ii) the complement of a referenced nucleotide sequence or portion
thereof; (iii) a nucleic acid that is substantially identical to a
referenced nucleic acid or the complement thereof; or (iv) a
nucleic acid that hybridizes under stringent conditions to the
referenced nucleic acid, complement thereof, or a sequences
substantially identical thereto.
[0076] "Variant" with respect to a peptide or polypeptide that
differs in amino acid sequence by the insertion, deletion, or
conservative substitution of amino acids, but retain at least one
biological activity. Variant may also mean a protein with an amino
acid sequence that is substantially identical to a referenced
protein with an amino acid sequence that retains at least one
biological activity. A conservative substitution of an amino acid,
i.e., replacing an amino acid with a different amino acid of
similar properties (e.g., hydrophilicity, degree and distribution
of charged regions) is recognized in the art as typically involving
a minor change. These minor changes can be identified, in part, by
considering the hydropathic index of amino acids, as understood in
the art. Kyte et al., J. Mol Biol. 157:105-132 (1982). The
hydropathic index of an amino acid is based on a consideration of
its hydrophobicity and charge. It is known in the art that amino
acids of similar hydropathic indexes can be substituted and still
retain protein function. In one aspect, amino acids having
hydropathic indexes of .+-.2 are substituted. The hydrophilicity of
amino acids can also be used to reveal substitutions that would
result in proteins retaining biological function. A consideration
of the hydrophilicity of amino acids in the context of a peptide
permits calculation of the greatest local average hydrophilicity of
that peptide, a useful measure that has been reported to correlate
well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101,
incorporated fully herein by reference. Substitution of amino acids
having similar hydrophilicity values can result in peptides
retaining biological activity, for example immunogenicity, as is
understood in the art. Substitutions may be performed with amino
acids having hydrophilicity values within .+-.2 of each other. Both
the hydrophobicity index and the hydrophilicity value of amino
acids are influenced by the particular side chain of that amino
acid. Consistent with that observation, amino acid substitutions
that are compatible with biological function are understood to
depend on the relative similarity of the amino acids, and
particularly the side chains of those amino acids, as revealed by
the hydrophobicity, hydrophilicity, charge, size, and other
properties.
[0077] y. Vector
[0078] "Vector" used herein may mean a nucleic acid sequence
containing an origin of replication. A vector may be a plasmid,
bacteriophage, bacterial artificial chromosome or yeast artificial
chromosome. A vector may be a DNA or RNA vector. A vector may be
either a self-replicating extrachromosomal vector or a vector which
integrates into a host genome.
[0079] 2. IL-12
[0080] Provided herein is a synthetic, constructs which encode
human IL-12 p35 (the a subunit) and p40 (the subunit). The human
IL-12 p35 subunit (SEQ ID NO:2) is a 219 amino acid protein which
includes a signal peptide at amino acids 1-22 and a mature protein
sequence at positions 23-219. The human IL-12 p40 subunit (SEQ ID
NO:4) is a 328 amino acid protein which includes a signal peptide
at amino acids 1-22 and a mature protein sequence at positions
23-328. Amino acids 40-90 of the human IL-12 p40 subunit are
referred to as the immunoglobulin domain; amino acids 125-21 of the
human IL-12 p40 subunit are referred to as the cytokine
interleukin-12 p40 C-terminus domain.
[0081] In some embodiments, the IL-12 p35 subunit is encoded by a
construct comprising a coding sequence on one plasmid and the IL-12
p40 subunit is encoded by a construct comprising a coding sequence
on a different plasmid. In some embodiments, the construct which
comprises the IL-12 p35 subunit coding sequence and the construct
which comprises the IL-12 p40 subunit coding sequence are on the
same plasmid but each construct has its own promoter. In some
embodiments, the construct which comprises the IL-12 p35 subunit
coding sequence and the construct which comprises the IL-12 p40
sub-unit coding sequence are on the same plasmid and under the
control of a single promoter and separated by an IRES sequence. In
some embodiments, the construct which comprises the IL-12 p35
subunit coding sequence and the construct which comprises the IL-12
p40 subunit coding sequence are on the same plasmid and under the
control of a single promoter and separated by a coding sequence for
a proteolytic cleavage site. In some embodiments, the construct
which comprises the IL-12 p35 subunit coding sequence and the
construct which comprises the IL-12 p40 subunit coding sequence are
on the same plasmid and under the control of a single promoter and
the subunit are separated by a linker which allows them to be
active as a single chain protein
[0082] HuIL12-opt sequences are optimized sequences that encode
human IL-12 subunits. The sequence have lower homology with the
host genome to change the RNA structure and avoid criptic
regulation sequences. The sequences provide improved mRNA stability
and expression.
[0083] The HuIL12-opt sequence that is the coding sequence that
encodes human IL-12 p35 subunit is disclosed in SEQ ID NO:1. The
HuIL12-opt sequence that is the 219 amino acid IL-subunit amino
acid sequence encoded thereby is disclosed as SEQ ID NO.2. Amino
acids 1-22 correspond to the signal peptide. Amino acids 23-219
correspond to the mature protein region.
[0084] The HuIL12-opt sequence that is the coding sequence that
encodes human IL-12 p40 subunit is disclosed as SEQ ID NO:3. The
HuIL12-opt sequence that is the 328 amino acid IL-12 p40 subunit
amino acid sequence encoded thereby is disclosed as SEQ NO.4. Amino
acids 1-22 correspond to the IL-12 signal peptide and amino acids
23-328 make up the mature protein. Analogous sequences for Rhesus
IL-12 are RhIL12-opt sequences which are optimized. sequences that
encode rhesus IL-12 subunits.
[0085] In some embodiments, the IL-12 signal peptide of the IL-12
p35 or p40 subunit or both may be replaced with a different signal
peptide such as another immunoglobulin signal peptide, for example
IgG or IgE (SEQ ID NO:5). Coding sequences that encode the signal
peptide of the IL-12 p35 or p40 subunit or both may be replaced
with coding sequences that encode a different signal peptide such
as another immunoglobulin signal peptide, for example IgG or IgE
(that is coding, sequences that encode SEQ ID NO:5). In some
embodiments, the IL-12 p35 signal peptide may be replaced with a
different signal peptide such as another immunoglobulin signal
peptide, for example IgG or IgE (SEQ ID NO:5). Functional fragments
of SEQ ID NO.? may be free of the IL-12 p35 signal peptide
sequence. In some embodiments, coding sequence that encodes the
IL-12 p35 signal peptide may be replaced with a coding sequence for
different signal peptide such as a coding sequence for another
immunoglobulin signal peptide, for example a coding sequence for
the signal peptide of IgG or IgE (i.e a coding sequence that
encodes SEQ ID NO:5). Nucleic acid sequences that are fragments of
SEQ ID NO:1 may be free of the coding sequence for IL-12 p35 signal
peptide. Functional fragments of SEQ ID NO.4 may be free of the
IL-12 p40 signal peptide sequence. In some embodiments, coding
sequence that encodes the IL-12 p40 signal peptide may be replaced
with a coding sequence for different signal peptide such as a
coding sequence for another immunoglobulin signal peptide, for
example a coding sequence for the signal peptide of IgG or IgE (i.e
a coding sequence that encodes SEQ ID NO:5). Nucleic acid sequences
that are fragments of SEQ ID NO:3 may be free of the coding
sequence for IL-12 p40 signal peptide. In calculating homology to
SEQ ID NO:1 or SEQ ID NO:3 in coding sequences that do not encode
the IL-12 p35 signal peptide or IL-12 p40 signal peptide,
respectively, the calculation is base upon a comparison of SEQ ID
NO:1 or SEQ ID NO:3 excluding the portion of SEQ ID NO:1 that
encode the IL-12 p35 signal peptide or the portion of SEQ ID NO:3
that encodes the IL-12 p40 signal peptide,
[0086] 3. Plasmid
[0087] Provided herein is a vector that is capable of expressing
the IL-12 constructs in the cell of a mammal in a quantity
effective to modulate an immune response in the mammal Each vector
may comprise heterologous nucleic acid encoding the one or both
subunits. The vector may be a plasmid. The plasmid may be useful
for transfecting cells with nucleic acid encoding IL-12, which the
transformed host cell is cultured and maintained under conditions
wherein expression of the IL-12 takes place.
[0088] The plasmid may comprise a nucleic acid encoding one or more
antigens. The plasmid may further comprise an initiation codon,
Which may be upstream of the coding sequence, and a stop codon,
which may be downstream of the coding sequence. The initiation and
termination codon may be in frame with the coding sequence.
[0089] The plasmid may also comprise a promoter that is operably
linked to the coding sequence The promoter operably linked to the
coding sequence may be a promoter from simian virus 40 (SV40), a
mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency
virus (HIV) promoter such as the bovine immunodeficiency virus
(BIV) long terminal repeat (LTR) promoter, a Moloney virus
promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus
(CMV) promoter such as the CMV immediate early promoter, Epstein
Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter.
The promoter may also be a promoter from a human gene such as human
actin, human myosin, human hemoglobin, human muscle creatine, or
human metalothionein. The promoter may also be a tissue specific
promoter, such as a muscle or skin specific promoter, natural or
synthetic. Examples of such promoters are described in US patent
application publication no. US20040175727, the contents of which
are incorporated by reference herein in its entirety.
[0090] The plasmid may also comprise a polyadenylation signal,
which may be downstream of the coding sequence. The polyadenylation
signal may be a SV40 polyadenylation signal, LTR polyadenylation
signal, bovine growth hormone (bGH) polyadenylation signal, human
growth hormone (hGH) polyadenylation signal, or human -globin
polyadenylation The SV40 polyadenylation signal may be a
polyadenylation signal from a pCEP4 plasmid (Invitrogen, San Diego,
Calif.).
[0091] The plasmid may also comprise an enhancer upstream of the
coding sequence. The enhancer may be human actin, human myosin,
human hemoglobin, human muscle creatine or a viral enhancer such as
one from CMV, FMDV, RSV or EBV. Polynucleotide function enhances
are described in U.S. Pat. Nos. 5,593,972, 5,962,428, and
WO94/016737, the contents of each are fully incorporated by
reference in their entireties.
[0092] The plasmid may also comprise a mammalian origin of
replication in order to maintain the plasmid extrachromosomally and
produce multiple copies of the plasmid in a cell. The plasmid may
be pVAXI, pCEP4 or pREP4 from Invitrogen (San Diego, Calif.), which
may comprise the Epstein Barr virus origin of replication and
nuclear antigen EBNA-1 coding region, which may produce high copy
episomal replication without integration. The backbone of the
plasmid may be pAV0242. The plasmid may be a replication defective
adenovirus type 5 (Ad5) plasmid.
[0093] The plasmid may also comprise a regulatory sequence, which
may be well suited for gene expression in a cell into which the
plasmid is administered. The coding sequence may comprise a codon
that may allow more efficient transcription of the coding sequence
in the host cell.
[0094] The coding sequence may also comprise an 1g leader sequence.
The leader sequence may be 5' of the coding sequence. The consensus
antigens encoded by this sequence may comprise an N-terminal lg
leader followed by a consensus antigen protein. The N-terminal 1 g
leader maybe IgE or IgG.
[0095] The plasmid may be pSE42O (Invitrogen, San Diego, Calif.),
which may be used for protein production in Escherichia coli (E.
coli). The plasmid may also be pYES2 (Invitrogen, San Diego,
Calif.), which may be used for protein production in Saccharomyces
cerevisiae strains of yeast. The plasmid may also be of the
MAXBAC.TM. complete baculovirus expression system (Invitrogen, San
Diego, Calif.), which may be used for protein production in insect
cells. The plasmid may also be pcDNA 1 or pcDNA3 (Invitrogen, San
Diego, Calif.), which maybe used for protein production in
mammalian cells such as Chinese hamster ovary (CHO) cells.
[0096] 4. Vaccine
[0097] According to sonic embodiments of the invention, the
delivery of a nucleic acid sequence that encodes IL-12 or
functional fragments thereof, in combination with a nucleic acid
sequence that encodes an immunogen to an individual enhances the
immune response against the immunogen. When the nucleic acid
molecules that encode the immunogens and IL-1.2 are taken up by
cells of the individual, the immunogen and IL-12 are expressed in
cells and the proteins are thereby delivered to the individual.
Aspects of the invention provide methods of delivering the coding
sequences of the immunogen and IL-12 on a single nucleic acid
molecule, methods of delivering the coding sequences of the
immunogen and IL-12 On different nucleic acid molecules and methods
of delivering the coding sequences of the proteins as part of
recombinant vaccines and as part of attenuated vaccines.
[0098] According to some aspects of the present invention,
compositions and methods are provided which prophylactically and/or
therapeutically immunize an individual against a pathogen or
abnormal, disease-related cells. The vaccine may be any type of
vaccine such as, a live attenuated vaccine, a recombinant vaccine
or a nucleic acid or DNA vaccine. By delivering nucleic acid
molecules that encode an immunogen and IL-12 or functional
fragments thereof the immune response induced by the vaccine may be
modulated. The IL-12 constructs are particularly useful when
delivered in combination with a nucleic acid molecule that encodes
an immunogen such as for example as part of a plasmid or the genome
of a recombinant vector or attenuated pathogen or cell. The IL-12
constructs may be used in vaccines prophylactically in order to
induce a protective immune response in an uninfected or disease
free individual. The IL-12 constructs are particularly useful when
delivered to induce a protective immune response in humans. The
IL-12 constructs may be used in vaccines therapeutically in order
to induce a immune response in an infected or diseased individual.
The IL-12 constructs are particularly useful when delivered to
induce a therapeutic immune response in humans. In some
embodiments, nucleic acid molecules comprising the IL-12 constructs
are delivered in a cell freecomposition. In some embodiments,
nucleic acid molecules comprising the IL-12 constructs are
delivered in a composition free of cancer cells. In some
embodiments, comprising the IL-12 constructs are administered free
of any other cytokine.
[0099] Provided herein are vaccine capable of generating in a
mammal an immune response against pathogens, immunogens expressed
on cells associated with disease and other immunogens against which
an immune response is desired. The vaccine may comprise each
plasmid as discussed above. The vaccine may comprise a plurality of
the plasmids, or combinations thereof. The vaccine may be provided
to induce a therapeutic or prophylactic immune response.
[0100] Genetic constructs may comprise a nucleotide sequence that
encodes a target protein or an immunomodulating protein operably
linked to regulatory elements needed for gene expression. According
to the invention, combinations of gene constructs that include one
construct that comprises an expressible form of the nucleotide
sequence that encodes a target protein and one construct that
includes an expressible form of the nucleotide sequence that
encodes an immunomodulating protein are provided. Delivery into a
living cell of the DNA or RNA molecule(s) that include the
combination of gene constructs results in the expression of the DNA
or RNA and production of the target protein and one or more
immunomodulating proteins. An enhanced immune response against the
target protein results.
[0101] The present invention may be used to immunize an individual
against pathogens such as viruses, prokaryote and pathogenic
eukaryotic organisms such as unicellular pathogenic organisms and
multicellular parasites. The present invention is particularly
useful to immunize an individual against those pathogens which
infect cells and which are not encapsulated such as viruses, and
prokaryote such as gonorrhea, Listeria and Shigella. In addition,
the present invention is also useful to immunize an individual
against protozoan pathogens that include a stage in the life cycle
where they are intracellular pathogens. Table 1 provides a listing
of some of the viral families and genera for which vaccines
according to the present invention can be made. DNA constructs that
comprise DNA sequences that encode the peptides that comprise at
least an epitope identical or substantially similar to an epitope
displayed on a pathogen antigen such as those antigens listed on
the tables are useful in vaccines. Moreover, the present invention
is also useful to immunize an individual against other pathogens
including prokaryotic and eukaryotic protozoan pathogens as well as
multicellular parasites such as those listed on Table 2.
TABLE-US-00001 TABLE 1 Viruses Picornavirus Family Genera:
Rhinoviruses: (Medical) responsible for -50% cases of the common
cold. Etheroviruses: (Medical) includes polioviruses,
coxsackieviruses, echoviruses, and human enteroviruses such as
hepatitis A virus. Apthoviruses: (Veterinary) these are the foot
and mouth disease viruses. Target antigens: VP1, VP2, VP3, VP4, VPG
Calcivirus Family Genera: Norwalk Group of Viruses: (Medical) these
viruses are an important causative agent of epidemic
gastroenteritis. Togavirus Family Genera: Alphaviruses: (Medical
and Veterinary) examples include Sindbis virus, RossRiver virus and
Venezuelan Eastern & Western Equine encephalitis viruses.
Reovirus: (Medical) Rubella virus. Flariviridae Family Examples
include: (Medical) dengue, yellow fever, Japanese encephalitis, St.
Louis encephalitis and tick borne encephalitis viruses. West Nile
virus (Genbank NC001563, AF533540, AF404757, AF404756, AF404755,
AF404754, AF404753, AF481864, M12294, AF317203, AF196835, AF260969,
AF260968, AF260967, AF206518 and AF202541) Representative Target
antigens: E NS5 C Hepatitis C Virus: (Medical) these viruses are
not placed in a family yet but are believed to be either a
togavirus or a flavivirus. Most similarity is with togavirus
family. Coronavirus Family: (Medical and Veterinary) Infectious
bronchitis virus (poultry) Porcine transmissible gastroenteric
virus (pig) Porcine hemagglutinating encephalomyelitis virus (pig)
Feline infectious peritonitis virus (cats) Feline enteric
coronavirus (cat) Canine coronavirus (dog) SARS associated
coronavirus The human respiratory coronaviruses cause about 40% of
cases of common cold. EX, 224E, OC43 Note - coronaviruses may cause
non-A, B or C hepatitis Target antigens: E1 - also called Mor
matrix protein E2 - also called Sor Spike protein E3 - also called
BE or hemagglutin-elterose glycoprotein (not present in all
coronaviruses) N - nucleocapsid Rhabdovirus Family Genera:
Vesiculovirus, Lyssavirus: (medical and veterinary) rabies Target
antigen: G protein, N protein Filoviridae Family: (Medical)
Hemorrhagic fever viruses such as Marburg and Ebola virus
Paramyxovirus Family: Genera: Paramyxovirus: (Medical and
Veterinary) Mumps virus, New Castle disease virus (important
pathogen in chickens) Morbillivirus: (Medical and Veterinary)
Measles, canine distemper Pneumovirus: (Medical and Veterinary)
Respiratory syncytial virus Orthomyxovirus Family (Medical) The
Influenza virus Bunyavirus Family Genera: Bunyavirus: (Medical)
California encephalitis, La Crosse Phlebovirus: (Medical) Rift
Valley Fever Hantavirus: Puremala is a hemahagin fever virus
Nairvirus (Veterinary) Nairobi sheep disease Also many unassigned
bungaviruses Arenavirus Family (Medical) LCM, Lassa fever virus
Reovirus Family Genera: Reovirus: a possible human pathogen
Rotavirus: acute gastroenteritis in children Orbiviruses: (Medical
and Veterinary) Colorado Tick fever, Lebombo (humans) equine
encephalosis, blue tongue Retroyirus Family Sub-Family:
Oncorivirinal: (Veterinary) (Medical) feline leukemia virus, HTLVI
and HTLVII Lentivirinal: (Medical and Veterinary) HIV, feline
immunodeficiency virus, equine infections, anemia virus
Spumavirinal Papovavirus Family Sub-Family: Polyomaviruses:
(Medical) BKU and JCU viruses Sub-Family: Papillomavirus: (Medical)
many viral types associated with cancers or malignant progression
of papilloma. Adenovirus (Medical) EX AD7, ARD., O.B. - cause
respiratory disease - some adenoviruses such as 275 cause enteritis
Parvovirus Family (Veterinary) Feline parvovirus: causes feline
enteritis Feline panleucopeniavirus Canine parvovirus Porcine
parvovirus Herpesvirus Family Sub-Family: alphaherpesviridue
Genera: Simplexvirus (Medical) HSVI (Genbank X14112, NC001806),
HSVII (NC001798) Varicella zoster: (Medical Veterinary)
Pseudorabies varicella zoster Sub-Family betaherpesviridae Genera:
Cytomegalovirus (Medical) HCMV Muromegalovirus Sub-Family.
Gammaherpesviridae Genera: Lymphocryptovirus (Medical) EBV -
(Burkitt's lymphoma) Poxvirus Family Sub-Family: Chordopoxviridae
(Medical - Veterinary) Genera: Variola (Smallpox) Vaccinia (Cowpox)
Parapoxivirus - Veterinary Auipoxvirus - Veterinary Capripoxvirus
Leporipoxvirus Suipoxviru's Sub-Family: Entemopoxviridue
Hepadnavirus Family Hepatitis B virus Unclassified Hepatitis delta
virus
TABLE-US-00002 TABLE 2 Bacterial pathogens Pathogenic gram-positive
cocci include: pneumococcal; staphylococcal; and streptococcal.
Pathogenic gram-negative cocci include: meningococcal; and
gonococcal. Pathogenic enteric gram-negative bacilli include:
enterobacteriaceae; pseudomonas, acinetobacteria and eikenella,
melioidosis; salmonella; shigellosis; haemophilus; chancroid;
brucellosis; tularemia; yersinia (pasteurella); streptobacillus
mortitiformis and spirillum; listeria monocytogenes; erysipelothrix
rhusiopathiae; diphtheria, cholera, anthrax; donovanosis (granuloma
inguinale); and bartonellosis. Pathogenic anaerobic bacteria
include: tetanus; botulism; other clostridia; tuberculosis;
leprosy; and other mycobacteria. Pathogenic spirochetal diseases
include: syphilis; - treponematoses: yaws, pinta and endemic
syphilis; and leptospirosis. Other infections caused by higher
pathogen bacteria and pathogenic fungi include: actinomycosis;
nocardiosis; cryptococcosis, blastomycosis, histoplasmosis and
coccidioidomycosis; candidiasis, aspergillosis, and mucormycosis;
sporotrichosis; paracoccidiodomycosis, petriellidiosis,
torulopsosis, mycetoma, and chromomycosis; and dermatophytosis.
Rickettsial infections include rickettsial and rickettsioses.
Examples of mycoplasma and chlamydial infections include:
mycoplasma pneumoniae; Ilymphogranuloma venereum; psittacosis; and
perinatal chlamydial infections. Pathogenic eukaryotes Pathogenic
protozoans and helminths and infections thereby include: amebiasis;
malaria; leishmaniasis; trypanosomiasis; toxoplasmosis;
pneumocystis carinii; babesiosis; giardiasis; trichinosis;
tilariasis; schistosomiasis; nematodes; trematodes or flukes; and
cestode (tapeworm) infections.
[0102] In order to produce a genetic vaccine to protect against
pathogen infection, genetic material that encodes immunogenic
proteins against which a protective immune response can be mounted
must be included in a genetic construct as the coding sequence for
the target. Because DNA and RNA are both relatively small and can
be produced relatively easily, the present invention provides the
additional advantage of allowing for vaccination with multiple
pathogen antigens. The genetic construct used in the genetic
vaccine can include genetic material that encodes many pathogen
antigens. For example, several viral genes may be included in a
single construct thereby providing multiple targets.
[0103] Tables 1 and 2 include lists of some of the pathogenic
agents and organisms for which genetic vaccines can be prepared to
protect an individual from infection by them.
[0104] In some embodiments, vaccines comprise the optimized IL-12
in combination with one or more DNA vaccine constructs set forth in
the following patent documents which are each incorporated herein
by reference. In some embodiments, vaccines comprise the optimized
IL-12 in combination with (human immunodeficiency virus) an MV
vaccine, an (hepatitis C virus) HCV vaccine, a human papilloma
virus (HPV) vaccine, an influenza vaccine or an hTERT-targeted
cancer vaccines as disclosed in PCT application PCT/US07/74769 and
corresponding U.S. patent application Ser. No.12/375,518, issued as
U.S. Pat. No. 8,168,769. In some embodiments, vaccines comprise the
optimized IL-12 in combination with an Influenza vaccines disclosed
in PCT application PCT/US08/83281 and corresponding U.S. patent
application Ser. No. 12/269,824, issued as U.S. Pat. No. 9,592,285,
or PCT application PCT/US11/22642 and corresponding U.S. patent
application Ser. No. 12/694,238, issued as U.S. Pat. No. 8,298,820.
In some embodiments, vaccines comprise the optimized IL-12 in
combination with an HCV vaccines disclosed in PCT application
PCT/US08/081627 and corresponding U.S. patent application Ser. No.
13/127,008, issued as U.S. Pat. No. 8,829,174, In some embodiments,
vaccines comprise the optimized IL-12 in combination with an HPV
vaccines disclosed in PCT application PCT/US10/21869 and
corresponding U.S. patent application Ser. No. 12/691,588, issued
as U.S. Pat. No. 8,389,706, or U.S. provisional application Ser.
No. 61/442,162, which provided priority to issued U.S. Pat. No.
9,238,679 and U.S. Pat. No. 9,403,879. In some embodiments,
vaccines comprise the optimized IL-12 in combination with an
Smallpox vaccines disclosed in PCT application PCT/US09/045420 and
corresponding U.S. patent application Ser. No. 12/473634, issued as
U.S. Pat. No. 8,535,687. In some embodiments, vaccines comprise the
optimized IL-12 in combination with an Chikungunya vaccines
disclosed in PCT application PCT/US09/039656 and corresponding U.S.
patent application Ser. No. 12/936,186, issued as U.S. Pat. No.
8,852,609. In some embodiments, vaccines comprise the optimized
IL-12 in combination with an foot and mouth disease virus (FMDV)
vaccines disclosed in PCT application PCT/US10/55187 and
corresponding U.S. patent application Ser. No. 13/503,828, issued
as U.S. Pat. No. 9,109,014, corresponding U.S. patent application
Ser. No. 14/816,120, issued as U.S. Pat. No. 10,294,278, and
corresponding U.S. patent application Ser. No. 16/4116,663. In some
embodiments, vaccines comprise the optimized IL-12 in combination
with an Malaria vaccines disclosed in U.S. provisional application
Ser. No. 61/386,973, which provided priority to published PCT
Application No. PCT/US11/53541 and corresponding U.S. patent
application Ser. No. 13/876,148. In some embodiments, vaccines
comprise the optimized IL-12 in combination with an prostate cancer
vaccines disclosed in U.S. provisional application Ser. No.
61/413,176, which provided priority to U.S. Pat. Nos. 8,927,692 and
9,399,056, or U.S. provisional application Ser. No. 61/417,817,
which provided priority to U.S. Pat. Nos. 8,927,692 and 9,399,056.
In some embodiments, vaccines comprise the optimized IL-12 in
combination with an human cytomegalovirus (CMV) vaccines disclosed
in U.S. provisional application Ser. No. 61/438,089, which provided
priority to U.S. Pat. No. 9,243,041. In some embodiments, vaccines
comprise the optimized IL-12 in combination with
Methicillin-Resistant Staphylococcus aureus (MRSA) vaccines
disclosed in U.S. Provisional Application Ser. No. 61/569,727,
filed on Dec. 12, 2011, entitled "PROTEINS COMPRISING MRSA PBP2A
AND FRAGMENTS THEREOF, NUCLEIC ACIDS ENCODING THE SAME, AND
COMPOSITIONS AND THEIR USE TO PREVENT AND TREAT MRSA INFECTIONS"
and designated attorney docket number 133172.04000 (X5709) and its
corresponding PCT Application (PCT/US12/69014) and the
corresponding U.S. patent application Ser. No. 14/365,071, issued
as U.S. Pat. No. 9,750,795, corresponding U.S. patent application
Ser. No. 15/675,823, issued as U.S. Pat. No. 10,064,931, and
corresponding U.S. patent application Ser. No. 16/111,267, claiming
priority to U.S. Provisional Application Ser. No. 61/569,727, each
of which are incorporated by reference in their entireties. All
patents and patent applications disclosed herein are incorporated
by reference in their entireties.
[0105] Another aspect of the present invention provides a method of
conferring a protective immune response against hyperproliferating
cells that are characteristic in hyperproliferative diseases and to
a method of treating individuals suffering from hyperproliferative
diseases. Examples of hyperproliferative diseases include all forms
of cancer and psoriasis.
[0106] It has been discovered that introduction of a genetic
construct that includes a nucleotide sequence which encodes an
immunogenic "hyperproliferating cell"-associated protein into the
cells of an individual results in the production of those proteins
in the vaccinated cells of an individual. To immunize against
hyperproliferative diseases, a genetic construct that includes a
nucleotide sequence that encodes a protein that is associated with
a hyperproliferative disease is administered to an individual.
[0107] In order for the hyperproliferative-associated protein to be
an effective immunogenic target, it must be a protein that is
produced exclusively or at higher levels in hyperproliferative
cells as compared to normal cells. Target antigens include such
proteins, fragments thereof and peptides; which comprise at least
an epitope found on such proteins. In some cases, a
hyperproliferative-associated protein is the product of a mutation
of a gene that encodes a protein. The mutated gene encodes a
protein that is nearly identical to the normal protein except it
has a slightly different amino acid sequence which results in a
different epitope not found on the normal protein. Such target
proteins include those which are proteins encoded by oncogenes such
as myb, myc, fyn, and the translocation gene bcr/abl, ras, src,
P53, neu, trk and EGRF. In addition to oncogene products as target
antigens, target proteins for anti-cancer treatments and protective
regimens include variable regions of antibodies made by B cell
lymphomas and variable regions of T cell receptors of T cell
lymphomas which, in some embodiments, are also used target antigens
for autoimmune disease. Other tumor-associated proteins can be used
as target proteins such as proteins that are found at higher levels
in tumor cells including the protein recognized by monoclonal
antibody 17-IA and folate binding proteins or PSA.
[0108] While the present invention may be used to immunize an
individual against one or more of several forms of cancer, the
present invention is particularly useful to prophylactically
immunize an individual who is predisposed to develop a particular
cancer or who has had cancer and is therefore susceptible to a
relapse. Developments in genetics and technology as well as
epidemiology allow for the determination of probability and risk
assessment for the development of cancer in individual. Using
genetic screening and/or family health histories, it is possible to
predict the probability a particular individual has for developing
any one of several types of cancer.
[0109] Similarly, those individuals who have already developed
cancer and who have been treated to remove the cancer or are
otherwise in remission are particularly susceptible to relapse and
reoccurrence. As part of a treatment regimen, such individuals can
be immunized against the cancer that they have been diagnosed as
having had in order to combat a recurrence. Thus, once it is known
that an individual has had a type of cancer and is at risk of a
relapse, they can be immunized in order to prepare their immune
system to combat any future appearance of the cancer.
[0110] The present invention provides a method of treating
individuals suffering from hyperproliferative diseases. In such
methods, the introduction of genetic constructs serves as an
immunotherapeutic, directing and promoting the immune system of the
individual to combat hyperproliferative cells that produce the
target protein. In treating or preventing cancer, embodiments which
are free of cells are particularly useful,
[0111] The present invention provides a method of treating
individuals suffering from autoimmune diseases and disorders by
conferring a broad based protective immune response against targets
that are associated with autoimmunity including cell receptors and
cells which produce "self"-directed antibodies.
[0112] T cell mediated autoimmune diseases include Rheumatoid
arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome,
sarcoidosis, insulin dependent diabetes mellitus (IDDM), autoimmune
thyroiditis, reactive arthritis, ankylosing spondylitis,
scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis,
Wegener's granulomatosis, Crohn's disease and ulcerativecolitis.
Each of these diseases is characterized by T cell receptors that
bind to endogenous antigens and initiate the inflammatory cascade
associated with autoimmune diseases, Vaccination against the
variable region of the T cells would elicit an immune response
including CTLs to eliminate those T cells.
[0113] In RA, several specific variable regions of T cell receptors
(TCRs) that are involved in the disease have been characterized.
These TCRs include V.beta.-3, V.beta.-14, 20 V.beta.-17 and
V.alpha.-17. Thus, vaccination with a DNA construct that encodes at
least one of these proteins will elicit an immune response that
will target I cells involved in RA. See: Howell, M. D., et al.,
1991 Proc. Nat. Acad. Sci. USA 88:10921-10925; Piliard, X., et al,
1991 Science 253:325-329; Williams, W. V., et al., 1992 J Clin.
Invest. 90:326-333; each of which is incorporated herein by
reference. In MS, several specific variable regions of TCRs that
are involved in the disease have been characterized. These TCRs
include V.beta.-7, and V.alpha.-10. Thus, vaccination with a DNA
construct that encodes at least one of these proteins will elicit
an immune response that will target T cells involved in MS. See:
Wucherpfennig, K. W., et al., 1990 Science 248:1016-1019;
Oksenberg, J. R., et al, 1990 Nature 345:344-346; each of which is
incorporated herein by reference.
[0114] In scleroderma, several specific variable regions of TCRs
that are involved in the disease have been characterized. These
TCRs include V.beta.-6, V.beta.-8, V.beta.-i4 and V.alpha.-16,
V.alpha.-3C, V.alpha.-7, V.alpha.-14, V.alpha.-15, V.alpha.-16,
V.alpha.-28 and V.alpha.-12. Thus, vaccination with a DNA construct
that encodes at least one of these proteins will elicit an immune
response that will target T cells involved in scleroderma.
[0115] In order to treat patients suffering from a T cell mediated
autoimmune disease, particularly those for which the variable
region of the TCR has yet to be characterized, a synovial biopsy
can be performed. Samples of the T cells present can be taken and
the variable region of those TCRs identified using standard
techniques. Genetic vaccines can be prepared using this
information.
[0116] B cell mediated autoimmune diseases include Lupus (SLE),
Grave's disease, myasthenia gravis, autoimmune hemolytic anemia,
autoimmune thrombocytopenia, asthma, cryoglobulinemia, primary
biliary sclerosis and pernicious anemia. Each of these diseases is
characterized by antibodies that bind to endogenous antigens and
initiate the inflammatory cascade associated with autoimmune
diseases. Vaccination against the variable region of antibodies
would elicit an immune response including CTLs to eliminate those B
cells that produce the antibody.
[0117] In order to treat patients suffering from a B cell mediated
autoimmune disease, the variable region of the antibodies involved
in the autoimmune activity must be identified. A biopsy can be
performed and samples of the antibodies present at a site of
inflammation can be taken. The variable region of those antibodies
can be identified using standard techniques. Genetic vaccines can
be prepared using this information.
[0118] In the case of SLE, one antigen is believed to be DNA. Thus,
in patients to be immunized against SLE, their sera can be screened
for anti-DNA antibodies and a vaccine can be prepared which
includes DNA constructs that encode the variable region of such
anti-DNA antibodies found in the sera.
[0119] Common structural features among the variable regions of
both TCRs and antibodies are well known. The DNA sequence encoding
a particular TCR or antibody can generally be found following well
known methods such as those described in Kabat, et al 1987 Sequence
of Proteins of Immunological Interest U.S. Department of Health and
Human Services, Bethesda Md., which is incorporated herein by
reference. In addition, a general method for cloning functional
variable regions from antibodies can be found in Chaudhary, V. K.,
et al., 1990 Proc. Natl. Acad Sci. USA 87:1066, which is
incorporated herein by reference.
[0120] In addition to using expressible forms of immunomodulating
protein coding sequences to improve genetic vaccines, the present
invention relates to improved attenuated live vaccines and improved
vaccines that use recombinant vectors to deliver foreign genes that
encode antigens. Examples of attenuated live vaccines and those
using recombinant vectors to deliver foreign antigens are described
in U.S. Pat. Nos. 4,722,848; 5,017,487; 5,077,044; 5,110,587;
5,112,749; 5,174,993; 5,223,424; 5,225,336; 5,240,703; 5,242,829;
5,294,441; 5,294,548; 5,310,668; 5,387,744; 5,389,368; 5,424,065;
5,451,499; 5,453,364; 5,462,734; 5,470,734; and 5,482,713, which
are each incorporated herein by reference. Gene constructs are
provided which include the nucleotide sequence of the IL-12
constructs or functional fragments thereof, wherein the nucleotide
sequence is operably linked to regulatory sequences that can
function in the vaccine to effect expression. The gene constructs
are incorporated in the attenuated live vaccines and recombinant
vaccines to produce improved vaccines according to the
invention.
[0121] The vaccine may further comprise a pharmaceutically
acceptable excipient. The pharmaceutically acceptable excipient may
be functional molecules as vehicles, adjuvants, carriers, or
diluents. The pharmaceutically acceptable excipient may be a
transfection facilitating agent, which may include surface active
agents, such as immune-stimulating complexes (ISCOMS), Freunds
incomplete adjuvant, LPS analog including monophosphoryl lipid A,
muramyl peptides, quinone analogs, vesicles such as squalene and
squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral
proteins, polyanions, polycations, or nanoparticles, or other known
transfection facilitating agents.
[0122] The transfection facilitating agent is a polyanion,
polycation, including poly-L-glutamate (LGS), or lipid. The
transfection facilitating agent is poly-L-glutamate, and more
preferably, the poly-L-glutamate is present in the vaccine at a
concentration less than 6 mg/ml. The transfection facilitating
agent may also include surface active agents such as
immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant,
LPS analog including monophosphoryl lipid A, muramyl peptides,
quinone analogs and vesicles such as squalene and squalene, and
hyaluronic acid may also be used administered in conjunction with
the genetic construct. In some embodiments, the DNA plasmid
vaccines may also include a transfection facilitating agent such as
lipids, liposomes, including lecithin liposomes or other liposomes
known in the art, as a DNA-liposome mixture (see for example
WO9324640), calcium ions, viral proteins, polyanions, polycations,
or nanoparticles, or other known transfection facilitating agents.
Preferably, the transfection facilitating agent is a polyanion,
polycation, including poly-L-glutamate (LGS), or lipid.
Concentration of the transfection agent in the vaccine is less than
4 mg/ml, less than 2 mg/ml, less than 1 mg/ml, less than 0.750
mg/ml, less than 0.500 mg/ml, less than 0.250 mg/ml, less than
0.100 mg/ml, less than 0.050 mg/ml, or less than 0.010 mg/ml.
[0123] The pharmaceutically acceptable excipient may be one or more
additional adjuvants. An adjuvant may be other genes that are
expressed from the same or from an alternative plasmid or are
delivered as proteins in combination with the plasmid above in the
vaccine. The one or more adjuvants may be proteins and/or nucleic
acid molecules that encode proteins selected from the group
consisting of: .alpha.-interferon (IFN-.alpha.), .beta.-interferon
(IFN-.beta.), .gamma.-interferon, platelet derived growth factor
(PDGF), TNF.alpha., TNF.beta., GM-CSF, epidermal growth factor
(EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial
thymus-expressed chemokine (TECK), mucosae-associated epithelial
chemokine (MEC), IL-15 including IL-15 having the signal sequence
or coding sequence that encodes the signal sequence deleted and
optionally including a different signal peptide such as that from
IgE or coding sequence that encodes a difference signal peptide
such as that from IgE, IL-28, MHC, CD80, CD86, IL-1, IL-2, IL-4,
IL-6, IL-10, IL-18, MCP-1, MIP-1.alpha., MIP-1.beta., IL-8,
L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1,
LFA-1, VLA-1, p150.95, PECAN, ICAM-1, ICAM-2, ICAM-3, CD2, LFa-3,
M-CSF, G-CSF, mutant forms of IL-18, CD40, CD40L, vascular growth
factor, fibroblast growth factor, IL-7, nerve growth factor,
vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1,
p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER,
TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2,
p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K,
SAP-1,JNK, interferon response genes, NFkB, I3ax, TRAIL, TRAL.rec,
TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40
LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1,
TAP2 and functional fragments thereof, or a combination thereof. In
some embodiments additional adjuvant may be one or more proteins
and/or nucleic acid molecules that encode proteins selected from
the group consisting of: IL-15, IL-28, CTACK, TECK, MEC or RANTES.
Examples of IL-15 constructs and sequences are disclosed in PCT
application no. PCT/US04/18962 and corresponding U.S. application
Ser. No. 10/560,650, and in PCT application no. PCT/US07/00886 and
corresponding U.S. application Ser. No. 12/160,766, and in PCT
application no. PCT/US10/048827. Examples of IL-28 constructs and
sequences are disclosed in PCT application no. PCT/US09/039648 and
corresponding U.S. application Ser. No. 12/936,192. Examples of
RANTES and other constructs and sequences are disclosed in PCT
application no. PCT/US1999/004332 and corresponding U.S.
application Ser. No. and 09/622452. Other examples of RANTES
constructs and sequences are disclosed in PCT application no.
PCT/US1 1/024098. Examples of RANTES and other constructs and
sequences are disclosed in PCT application no. PCT/US1999/004332
and corresponding U.S. application Ser. No. 09/622452. Other
examples of RANTES constructs and sequences are disclosed in PCT
application no. PCT/US1 1/024098. Examples of chemokines CTACK,
TECK and MEC constructs and sequences are disclosed in PCT
application no. PCT/US2005/042231 and corresponding U.S.
application Ser. No. 11/719,646. Examples of OX40 and other
immunomodulators are disclosed in U.S. application Ser. No.
10/560,653. Examples of DR5 and other immunomodulators are
disclosed in U.S. application Ser. No. 09/622452.
[0124] The vaccine may further comprise a genetic vaccine
facilitator agent as described in U.S. Ser. No. 021,579 filed Apr.
1, 1994, which is fully incorporated by reference.
[0125] The vaccine may comprise the consensus antigens and plasmids
at quantities of from about 1 nanogram to 100 milligrams; about 1
microgram to about 10 milligrams; or preferably about 0.1 microgram
to about 10 milligrams; or more preferably about 1 milligram to
about 2 milligram. In some preferred embodiments, pharmaceutical
compositions according to the present invention comprise about 5
nanogram to about 1000 micrograms of DNA. In some preferred
embodiments, the pharmaceutical compositions contain about 10
nanograms to about 800 micrograms of DNA. In some preferred
embodiments, the pharmaceutical compositions contain about 0.1 to
about 500 micrograms of DNA. In some preferred embodiments, the
pharmaceutical compositions contain about 1 to about 350 micrograms
of DNA. In some preferred embodiments, the pharmaceutical
compositions contain about 25 to about 250 micrograms, from about
100 to about 200 microgram, from about 1 nanogram to 100
milligrams; from about 1 microgram to about 10 milligrams; from
about 0.1 microgram to about 10 milligrams; from about 1 milligram
to about 2 milligram, from about 5 nanogram to about 1000
micrograms, from about 10 nanograms to about $00 micrograms, from
about 0.1 to about 500 micrograms, from about 1 to about 350
micrograms, from about 25 to about 250 micrograms, from about 100
to about 200 microgram of the consensus antigen or plasmid
thereof.
[0126] The vaccine may be formulated according to the mode of
administration to be used. An injectable vaccine pharmaceutical
composition may be sterile, pyrogen free and particulate free. An
isotonic formulation or solution may be used. Additives for
isotonicity may include sodium chloride, dextrose, mannitol,
sorbitol, and lactose. The vaccine may comprise a vasoconstriction
agent. The isotonic solutions may include phosphate buffered
saline. Vaccine may further comprise stabilizers including gelatin
and albumin. The stabilizing may allow the formulation to be stable
at room or ambient temperature for extended periods of time such as
LGS or polycations or polyanions to the vaccine formulation.
5. Methods of Delivery the Vaccine
[0127] Provided herein is a method for delivering a vaccine
including the IL-12 constructs to produce immune responses
effective against the vaccine immunogens. The method of delivering
the vaccine or vaccination may be provided to induce a therapeutic
and prophylactic immune response. The vaccination process may
generate in the mammal an immune response against immunogens. The
vaccine may be delivered to an individual to modulate the activity
of the mammal's immune system and enhance the immune response. The
delivery of the vaccine may be the transfection of sequences
encoding the immunogen and the IL-12 constructs on one or more
nucleic acid molecules. The coding sequences are expressed in cells
and delivered to the surface of the cell upon which the immune
system recognized and induces a cellular, humoral, or cellular and
humoral response. The delivery of the vaccine may be use to induce
or elicit and immune response in mammals against the immunogen by
administering to the mammals the vaccine as discussed above. The
inclusion of the IL-12 constructs results in a more effective
immune response.
[0128] Upon delivery of the vaccine and plasmid into the cells of
the mammal, the transfected cells will express and secrete
immunogens and IL-12 encoded by the plasmids injected from the
vaccine. These immunogens will be recognized as foreign by the
immune system and antibodies will be made against them. These
antibodies will be maintained by the immune system and allow for an
effective response to subsequent infections. The presence of the
IL-12 encoded by the IL-12 constructs results in a greater immune
response.
[0129] The vaccine may be administered to a mammal to elicit an
immune response in a mammal. The mammal may be human, primate,
non-human primate, cow, cattle, sheep, goat, antelope, bison, water
buffalo, bison, bovids, deer, hedgehogs, elephants, llama, alpaca,
mice, rats, and chicken.
[0130] a. Combination Treatments
[0131] The IL-12 construct may be administered in combination with
other proteins or genes encoding one or more of .alpha.-interferon,
.beta.-interferon, platelet derived growth factor (PDGF),
TNF.alpha., TNF.beta., GM-CSF, epidermal growth factor (EGF),
cutaneous T cell-attracting chemokine (CTACK), epithelial
thymus-expressed chemokine (TECK), mucosae-associated epithelial
chemokine (MEC), IL-15 (including IL-15 having the signal sequence
deleted and optionally including the signal peptide from IgE), MHC,
CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-28,
MCP-1, MIP-1.alpha., IL-1.beta., IL-8, RANTES, L-selectin,
P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1,
Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF,
G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth
factor, fibroblast growth factor, IL-7, nerve growth factor,
vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1,
p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER,
TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1,Ap-2,
p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1,
JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec,
TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, O40, Ox40
LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1,
TAP2 and functional fragments thereof or combinations thereof.
[0132] The vaccine may be administered by different routes
including orally, parenterally, sublingually, transdermally,
rectally, transmucosally, topically, via inhalation, via buccal
administration, intrapleurally, intravenous, intraarterial,
intraperitoneal, subcutaneous, intramuscular, intranasal
intrathecal, and intraarticular or combinations thereof. For
veterinary use, the composition may be administered as a suitably
acceptable formulation in accordance with normal veterinary
practice. The veterinarian can readily determine the dosing regimen
and route of administration that is most appropriate for a
particular animal. The vaccine may he administered by traditional
syringes, needleless injection devices, "microprojectile
bombardment gone guns", or other physical methods such as
electroporation ("EP"), "hydrodynamic method", or ultrasound.
[0133] The plasmid of the vaccine may be delivered to the mammal by
several well known technologies including DNA injection (also
referred to as DNA vaccination) with and without in vivo
electroporation, liposome mediated, nanoparticle facilitated,
recombinant vectors such as recombinant adenovirus, recombinant
adenovirus associated virus and recombinant vaccinia. The consensus
antigen may be delivered via DNA injection and along with in vivo
electroporation.
[0134] b. Electroporation
[0135] Administration of the vaccine via electroporation of the
plasmids of the vaccine may be accomplished using electroporation
devices that can be configured to deliver to a desired tissue of a
mammal a pulse of energy effective to cause reversible pores to
form in cell membranes, and preferable the pulse of energy is a
constant current similar to a preset current input by a user. The
electroporation device may comprise an electroporation component
and an electrode assembly or handle assembly. The electroporation
component may include and incorporate one or more of the various
elements of the electroporation devices, including: controller,
current waveform generator, impedance tester, waveform logger,
input element, status reporting element, communication port, memory
component, power source, and power switch. The electroporation may
be accomplished using an in vivo electroporation device, for
example CELLECTRA EP system (inovio Pharmaceuticals, Blue Bell,
Pa.) or Elgen electroporator (Genetronics, San Diego, Calif.) to
facilitate transfection of cells by the plasmid.
[0136] The electroporation component may function as one element of
the electroporation devices, and the other elements are separate
elements (or components) in communication with the electroporation
component. The electroporation component may function as more than
one element of the electroporation devices, which may be in
communication with still other elements of the electroporation
devices separate from the electroporation component. The elements
of the electroporation devices existing as parts of one
electromechanical or mechanical device may not limited as the
elements can function as one device or as separate elements in
communication with one another. The electroporation component may
be capable of delivering the pulse of energy that produces the
constant current in the desired tissue, and includes a feedback
mechanism. The electrode assembly may include an electrode array
having a plurality of electrodes in a spatial arrangement, wherein
the electrode assembly receives the pulse of energy from the
electroporation component and delivers same to the desired tissue
through the electrodes. At least one of the plurality of electrodes
is neutral during delivery of the pulse of energy and measures
impedance in the desired tissue and communicates the impedance to
the electroporation component. The feedback mechanism may receive
the measured impedance and can adjust the pulse of energy delivered
by the electroporation component to maintain the constant
current.
[0137] A plurality of electrodes may deliver the pulse of energy in
a decentralized pattern. The plurality of electrodes may deliver
the pulse of energy in the decentralized pattern through the
control of the electrodes under a programmed sequence, and the
programmed sequence is input by a user to the electroporation
component. The programmed sequence may comprise a plurality of
pulses delivered in sequence, wherein each pulse of the plurality
of pulses is delivered by at least two active electrodes with one
neutral electrode that measures impedance, and wherein a subsequent
pulse of the plurality of pulses is delivered by a different one of
at least two active electrodes with one neutral electrode that
measures impedance.
[0138] The feedback mechanism may be performed by either hardware
or software. The feedback mechanism may be performed by an analog
closed-loop circuit. The feedback occurs every 50 .mu.s, 20 .mu.s,
10 .mu.s or 1 .mu.s, but is preferably a real-time feedback or
instantaneous (i.e., substantially instantaneous as determined by
available techniques for determining response time). The neutral
electrode may measure the impedance in the desired tissue and
communicates the impedance to the feedback mechanism, and the
feedback mechanism responds to the impedance and adjusts the pulse
of energy to maintain the constant current at a value similar to
the preset current. The feedback mechanism may maintain the
constant current continuously and instantaneously during the
delivery of the pulse of energy.
[0139] Examples of electroporation devices and electroporation
methods that may facilitate delivery of the DNA vaccines of the
present invention, include those described in U.S. Pat. No.
7,245,963 by Draghia-Akli, et al., U.S. Patent Pub. 2005/0052630
submitted by Smith, et al., issued as U.S. Pat. No. 8,209,006, the
contents of which are hereby incorporated by reference in their
entirety. Other electroporation devices and electroporation methods
that may be used for facilitating delivery of the DNA vaccines
include those provided in co-pending and co-owned U.S. patent
application Ser. No. 11/874072, filed Oct. 17, 2007, issued as U.S.
Pat. No. 9,452,285, which claims the benefit under 35 USC 119(e) to
U.S. Provisional Applications Ser. Nos. 60/852,149, filed Oct. 17,
2006, and 60/978,982,filed Oct. 10, 2007, all of which are hereby
incorporated in their entirety.
[0140] U.S. Pat. No. 7,245,963 by Draghia-Akli, et al, describes
modular electrode systems and their use for facilitating the
introduction of a biomolecule into cells of a selected tissue in a
body or plant. The modular electrode systems may comprise a
plurality of needle electrodes; a hypodermic needle; an electrical
connector that provides a conductive link from a programmable
constant-current pulse controller to the plurality of needle
electrodes; and a power source. An operator can grasp the plurality
of needle electrodes that are mounted on a support structure and
firmly insert them into the selected tissue in a body or plant. The
biomolecules are then delivered via the hypodermic needle into the
selected tissue. The programmable constant-current pulse controller
is activated and constant-current electrical pulse is applied to
the plurality of needle electrodes. The applied constant-current
electrical pulse facilitates the introduction of the biomolecule
into the cell between the plurality of electrodes. The entire
content of U.S. Pat. No. 7,245,963 is hereby incorporated by
reference.
[0141] U.S. Patent Pub. 2005/0052630 submitted by Smith, et al.
describes an electroporation device which may be used to
effectively facilitate the introduction of a biomolecule into cells
of a selected tissue in a body or plant. The electroporation device
comprises an electro-kinetic device ("EKD device") whose operation
is specified by software or firmware. The EKD device produces a
series of programmable constant-current pulse patterns between
electrodes in an array based on user control and input of the pulse
parameters, and allows the storage and acquisition of current
waveform data. The electroporation device also comprises a
replaceable electrode disk having an array of needle electrodes, a
central injection channel fir an injection needle, and a removable
guide disk. The entire content of U.S. Patent Pub. 2005/0052630 is
hereby incorporated by reference.
[0142] The electrode arrays and methods described in U.S. Pat. No.
7,245,963 and U.S. Patent Pub. 2005/0052630 may be adapted for deep
penetration into not only tissues such as muscle, but also other
tissues or organs. Because of the configuration of the electrode
array, the injection needle (to deliver the biomolecule of choice)
is also inserted completely into the target organ, and the
injection is administered perpendicular to the target issue, in the
area that is pre-delineated by the electrodes. The electrodes
described in U.S. Pat. No. 7,245,963 and U.S. Patent Pub.
2005/005263 are preferably 20 mm long and 21 gauge.
[0143] Additionally, contemplated in some embodiments that
incorporate electroporation devices and uses thereof, there are
electroporation devices that are those described in the following
patents: U.S. Pat. No. 5,273,525 issued Dec. 28, 1993, U.S. Pat.
No. 6,110,161 issued Aug. 29, 2000, U.S. Pat. No. 6,261,281 issued
Jul. 17, 2001, and U.S. Pat. No. 6,958,060 issued Oct. 25, 2005,
and U.S. Pat. No. 6,939,862 issued Sep. 6, 2005. Furthermore,
patents covering subject matter provided in U.S. Pat. No. 6,697,669
issued Feb. 24, 2004, which concerns delivery of DNA using any of a
variety of devices, and U.S. Pat. No. 7,328,064 issued Feb. 5,
2008, drawn to method of injecting DNA are contemplated herein. The
above-patents are incorporated by reference in their entirety.
[0144] c. Method of Preparing DNA Plasmids
[0145] Provided herein is methods for preparing the DNA plasmids
that comprise the DNA constructs and vaccines discussed herein. The
DNA plasmids, after the final subcloning step into the mammalian
expression plasmid, can be used to inoculate a cell culture in a
large scale fermentation tank, using known methods in the art.
[0146] The DNA plasmids for use with the EP devices of the present
invention can be formulated or manufactured using a combination of
known devices and techniques, but preferably they are manufactured
using an optimized plasmid manufacturing technique that is
described in a licensed, co-pending U.S. provisional application
U.S. Ser. No. 60/939,792, which was filed on May 23, 2007. In some
examples, the DNA plasmids used in these studies can be formulated
at concentrations greater than or equal to 10 mg/mL. The
manufacturing techniques also include or incorporate various
devices and protocols that are commonly known to those of ordinary
skill in the art, in addition to those described in U.S. Ser. No.
60/939792, including those described in a licensed patent, U.S.
Pat. No. 7,238,522., which issued on Jul. 3, 2007. The
above-referenced application and patent, U.S. Ser. No. 60/939,792
and U.S. Pat. No. 7,238,522, respectively, are hereby incorporated
in their entirety,
6. Immunomodulating Compositions and Methods
[0147] In some embodiments, the nucleic acid sequences that encode
the IL-12 subunits are delivered without the addition of nucleic
acid sequences that encode an immunogen. In such methods, the
nucleic acid sequences that encode the IL-12 subunits are used as
immunotherapeutics which, when expressed to produce functional
IL-12, impart a desired immunomodulatory effect on the individual.
The nucleic acid sequences that encode the IL-12 subunits are
provided and delivered as described above except for the exclusion
of nucleic acid sequences that encode an immunogen. In such
methods, the nucleic acid sequences that encode the IL-12 subunits
may used as immunotherapeutics alone or in combination with other
immunomodulatory proteins such as those described above in the
section entitled combination treatments.
EXAMPLES
[0148] The present invention is further illustrated in the
following Examples. It should be understood that these Examples,
while indicating preferred embodiments of the invention, are given
by way of illustration only. From the above discussion and these
Examples, one skilled in the art can ascertain the essential
characteristics of this invention, and without departing from the
spirit and scope thereof, can make various changes and
modifications of the invention to adapt it to various usages and
conditions. Thus, various modifications of the invention in
addition to those shown and described herein will be apparent to
those skilled in the art from the foregoing description. Such
modifications are also intended to fall within the scope of the
appended claims
Example 1
[0149] Comparing Expression Levels of phuIL12-opt with
phuIL12-nonopt.
[0150] Comparison of the expression levels of phuIL12-opt with
phuIL12-nonopt was performed to show the important codon/RNA
optimization strategies could boost the expression levels/adjuvant
effects of a designed synthetic IL-12.
[0151] 293T cells (7.5.times.10.sup.5) were transfected ire 6-well
plates with 2 or 4 .mu.g of huIL12-opt or huIL12-nonopt,
respectively, using FuGene6 Transfection Reagent (Roche Applied
Science, Indianapolis, Ind.) per manufacturer's instructions. DNA
and FuGene6 Transfection Reagent were added in sequence to
serum-free media at a DNA:FuGene6 ratio of 1 .mu.g DNA:3 .mu.l
FuGene6 reagent. The volume of serum-free media was determined by
the amount needed to make the entire mixture's total volume equal
200 .mu.l. The mixture was added to each well of cells and
incubated for 48 hours at 37.degree. C. in a 5% CO2 environment. At
the end of the incubation, the supernatant samples were collected
for the ELISA assay.
[0152] High protein binding plates (Nunc, Rochester, N.Y.) were
coated with 100 .mu.I/well of monoclonal antibody MT86/221 from the
human IL-12 ELISA kit (Mabtech, Mariemont, Ohio) and incubated
overnight at 4.degree. C. After the incubation, the plates were
washed twice with PBST (DPBS with 0.1% Tween 20) and blocked for 1
hour with 200 .mu.l/well of a DPBS solution supplemented with 0.05%
Tween 20 and 0.1% BSA. Plates were subsequently washed with PBST.
Using manufacturer's instructions, a positive standard was prepared
using hIL12 p70 (Mabtech, Mariemont, Ohio). The positive standard
and supernatant samples were added to duplicate wells in volumes of
100 .mu.l/well at dilutions of 1:50, 1:150, 1:450, 1:1350, and
1:4050. The samples and positive standard were diluted using the
above blocking solution. The plates were subsequently incubated at
4.degree. C. overnight. Afterwards, the plates were washed with
PBST and incubated with 100 .mu.l/well of mAB MT618-biotin
(Mabtech, Mariemont, Ohio) for 1 hour. After incubation, the plates
were washed again and incubated for 1 hour with 100 .mu.l/well of
Streptavidin-HRP diluted at 1:1000 in blocking buffer. The plates
were then washed again with PBST and developed using TMB and 2N
H.sub.2SO.sub.4. Plates were read at 450 nm using a
photospectrometer.
[0153] As shown in FIGS. 1A and 1B, the huIL12-opt plasmid exhibits
higher levels of expression of IL-12 compared to the huIL12-nonopt.
Clearly, the codon/RNA optimization strategies improve the
expression of IL-12.
Example:2
[0154] Enhanced PSA and PSMA-Specific Cellular Immune Responses
Elicited by Vaccination with pMacIL12-opt.
[0155] Rhesus macaques were immunized with 1 mg of PSA and PSMA in
combination with 0.04 mg of pMacIL-12-opt intramuscularly followed
by electroporation with the Cellectra device from Inovio
Pharmaceuticals. Two weeks after each immunization rhesus macaques
were bled and PBMCs were isolated for the PSA and PSMA-specific
IFN-.gamma. ELISpot assay. The group of animals receiving the
pMacIL12-opt showed about 3-fold increase in peak response compared
to the group of animals not receiving pMacIL12-opt (FIG. 2).
Example3
[0156] Enhanced HBV Core and Surface Antigen-Specific Cellular
Immune Responses Elicted by Vaccination with pMa.cIL12-opt.
[0157] Rhesus macaques were immunized with 1 mg of core and surface
antigens in combination with 0.04 mg of pMacIL-12-opt
intramuscularly followed by electroporation with the Cellectra
device from Inovio Pharmaceuticals. Two weeks after each
immunization rhesus macaques were bled and PBMCs were isolated for
the core and surface antigen-specific IFN-.gamma. ELISpot assay.
The group of animals receiving the pMacIL12-opt showed increased
magnitude and breadth of cellular responses compared to the group
of animals not receiving pMacIL12-opt (FIG. 3).
Sequence CWU 1
1
51660DNAArtificial SequenceIL-12 Optimized p35 subunit nucleic acid
1atgtgccccg ctcggtccct gctgctggtc gctaccctgg tcctgctgga tcacctgtca
60ctggctcgaa atctgcctgt cgctaccccc gatcctggca tgttcccctg cctgcaccat
120agccagaacc tgctgcgggc cgtgtccaat atgctgcaga aagctagaca
gacactggag 180ttttaccctt gtacttctga ggaaatcgac cacgaggata
ttactaagga caaaacctcc 240acagtcgaag cctgcctgcc actggagctg
accaagaacg aatcatgtct gaatagcagg 300gagacttcct tcatcaccaa
cgggtcttgc ctggctagtc gcaagaccag cttcatgatg 360gcactgtgcc
tgagctccat ctacgaggat ctgaagatgt atcaggtgga attcaaaacc
420atgaacgcta agctgctgat ggaccctaaa cgacagatct ttctggatca
gaatatgctg 480gcagtgattg acgagctgat gcaggccctg aacttcaata
gcgaaaccgt cccacagaag 540tctagtctgg aggaacccga cttttataag
acaaaaatca agctgtgcat tctgctgcat 600gcctttcgga ttcgggctgt
cactattgat cgggtcatgt catacctgaa cgcttcctaa 6602219PRTArtificial
SequenceIL-12 Opt p35 subunit amino acid 2Met Cys Pro Ala Arg Ser
Leu Leu Leu Val Ala Thr Leu Val Leu Leu1 5 10 15Asp His Leu Ser Leu
Ala Arg Asn Leu Pro Val Ala Thr Pro Asp Pro 20 25 30Gly Met Phe Pro
Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val 35 40 45Ser Asn Met
Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys 50 55 60Thr Ser
Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser65 70 75
80Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys
85 90 95Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu
Ala 100 105 110Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser
Ser Ile Tyr 115 120 125Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys
Thr Met Asn Ala Lys 130 135 140Leu Leu Met Asp Pro Lys Arg Gln Ile
Phe Leu Asp Gln Asn Met Leu145 150 155 160Ala Val Ile Asp Glu Leu
Met Gln Ala Leu Asn Phe Asn Ser Glu Thr 165 170 175Val Pro Gln Lys
Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys 180 185 190Ile Lys
Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr 195 200
205Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser 210
2153987DNAArtificial SequenceIL-12 Opt p40 subunit nucleic acid
3atgtgccatc agcagctggt catctcttgg tttagtctgg tgtttctggc ttctccactg
60gtcgctatct gggaactgaa aaaggatgtg tacgtggtcg agctggactg gtatccagat
120gcacccggag aaatggtggt cctgacctgc gacacacccg aggaagatgg
catcacttgg 180accctggacc agagctccga ggtgctggga tctggcaaga
cactgactat tcaggtcaaa 240gaattcgggg atgccggaca gtacacatgt
cacaagggcg gggaggtgct gagtcactca 300ctgctgctgc tgcataagaa
agaagacggc atctggtcta ctgacattct gaaggatcag 360aaagagccta
agaacaaaac cttcctgaga tgcgaagcta agaattatag tgggaggttt
420acctgttggt ggctgaccac aatctcaact gacctgacct ttagcgtgaa
atctagtagg 480gggtcaagcg atccacaggg agtgacctgc ggagcagcta
cactgagcgc cgagcgggtg 540agaggagaca acaaggagta cgaatatagt
gtcgagtgcc aggaagattc agcctgtccc 600gcagccgagg aatccctgcc
tatcgaagtg atggtggacg ctgtgcacaa gctgaaatac 660gaaaactaca
catcctcttt ctttattcgc gacatcatta agccagatcc ccctaaaaac
720ctgcagctga agcccctgaa aaattcccga caggtggagg tctcttggga
ataccctgat 780acatggagca ctccacattc ttatttcagt ctgacttttt
gcgtgcaggt ccagggcaag 840agcaaaaggg agaagaaaga ccgcgtgttc
accgataaga catccgctac tgtcatctgt 900cgaaaaaacg caagcatttc
cgtgcgggca caggataggt attattccag cagttggtct 960gagtgggctt
ccgtcccttg tagttga 9874328PRTArtificial SequenceIL-12 Opt p40
subunit amino acid 4Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser
Leu Val Phe Leu1 5 10 15Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys
Lys Asp Val Tyr Val 20 25 30Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro
Gly Glu Met Val Val Leu 35 40 45Thr Cys Asp Thr Pro Glu Glu Asp Gly
Ile Thr Trp Thr Leu Asp Gln 50 55 60Ser Ser Glu Val Leu Gly Ser Gly
Lys Thr Leu Thr Ile Gln Val Lys65 70 75 80Glu Phe Gly Asp Ala Gly
Gln Tyr Thr Cys His Lys Gly Gly Glu Val 85 90 95Leu Ser His Ser Leu
Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp 100 105 110Ser Thr Asp
Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe 115 120 125Leu
Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp 130 135
140Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser
Arg145 150 155 160Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala
Ala Thr Leu Ser 165 170 175Ala Glu Arg Val Arg Gly Asp Asn Lys Glu
Tyr Glu Tyr Ser Val Glu 180 185 190Cys Gln Glu Asp Ser Ala Cys Pro
Ala Ala Glu Glu Ser Leu Pro Ile 195 200 205Glu Val Met Val Asp Ala
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr 210 215 220Ser Ser Phe Phe
Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn225 230 235 240Leu
Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp 245 250
255Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr
260 265 270Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys
Asp Arg 275 280 285Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys
Arg Lys Asn Ala 290 295 300Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr
Tyr Ser Ser Ser Trp Ser305 310 315 320Glu Trp Ala Ser Val Pro Cys
Ser 325518PRTArtificial SequenceIgE leader 5Met Asp Trp Thr Trp Ile
Leu Phe Leu Val Ala Ala Ala Thr Arg Val1 5 10 15His Ser
* * * * *