U.S. patent application number 12/301021 was filed with the patent office on 2010-08-05 for modified dendritic cells having enhanced survival and immunogenicity and related compositions and methods.
Invention is credited to Natalia Lapteva, Dongsu Park, David Spencer.
Application Number | 20100196336 12/301021 |
Document ID | / |
Family ID | 38724115 |
Filed Date | 2010-08-05 |
United States Patent
Application |
20100196336 |
Kind Code |
A1 |
Park; Dongsu ; et
al. |
August 5, 2010 |
MODIFIED DENDRITIC CELLS HAVING ENHANCED SURVIVAL AND
IMMUNOGENICITY AND RELATED COMPOSITIONS AND METHODS
Abstract
Modified antigen presenting cells provided herein have improved
lifespan and immunogenicity compared to unmodified antigen
presenting cells, and are useful for immunotherapy. The modified
antigen presenting cells express an altered protein kinase,
referred to herein as "Akt." The altered Akt associates with the
cell membrane with greater frequency than unaltered Akt, and is
referred to herein as "membrane-targeted Akt."
Inventors: |
Park; Dongsu; (Houston,
TX) ; Spencer; David; (Houston, TX) ; Lapteva;
Natalia; (Houston, TX) |
Correspondence
Address: |
GRANT ANDERSON LLP;C/O PORTFOLIOIP
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Family ID: |
38724115 |
Appl. No.: |
12/301021 |
Filed: |
May 23, 2007 |
PCT Filed: |
May 23, 2007 |
PCT NO: |
PCT/US07/69586 |
371 Date: |
June 4, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60803025 |
May 23, 2006 |
|
|
|
Current U.S.
Class: |
424/93.21 ;
435/235.1; 435/29; 435/375; 435/455; 435/456; 530/350;
536/23.5 |
Current CPC
Class: |
A61K 2039/5154 20130101;
A61P 35/00 20180101; C12N 15/85 20130101; C12N 15/861 20130101;
C12N 2710/10343 20130101; C12N 9/1205 20130101; A61K 2039/5156
20130101; C07K 2319/00 20130101; C07K 2319/033 20130101; A61P 37/04
20180101 |
Class at
Publication: |
424/93.21 ;
536/23.5; 530/350; 435/455; 435/456; 435/375; 435/29;
435/235.1 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07H 21/04 20060101 C07H021/04; C07K 14/435 20060101
C07K014/435; A61P 37/04 20060101 A61P037/04; C12N 15/00 20060101
C12N015/00; C12N 15/86 20060101 C12N015/86; C12N 5/0784 20100101
C12N005/0784; C12Q 1/02 20060101 C12Q001/02; C12N 7/00 20060101
C12N007/00; A61P 35/00 20060101 A61P035/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made in part with government support
under Grant No. DAMD17-03-1-0156 awarded by the Department of
Defense. The government has certain rights in this invention.
Claims
1-130. (canceled)
131. A method for preparing a modified dendritic cell, which
comprises: contacting a dendritic cell with a nucleic acid that
encodes a membrane-targeted Akt protein comprising a mammalian Akt
region lacking a functional pleckstrin homology (PH) domain,
whereby the modified dendritic cell survives longer than dendritic
cells that do not include the nucleic acid.
132. The method of claim 131, wherein the modified dendritic cell
presents a greater amount of antigen than cells that do not include
the nucleic acid.
133. The method of claim 131, wherein the modified dendritic cell
is more immunogenic than cells that do not include the nucleic
acid.
134. The method of claim 131, which comprises contacting the
modified dendritic cell with an antigen.
135. The method of claim 134, wherein the antigen is prostate
specific membrane antigen.
136. The method of claim 135, wherein the antigen has a sequence
substantially identical to SEQ ID NO: 10.
137. The method of claim 131, wherein the dendritic cell is a human
cell.
138. The method of claim 131, wherein the membrane-targeted Akt
comprises one or more binding partner components.
139. The method of claim 138, wherein the dendritic cell comprises
an acylation region having one or more binding partner components,
wherein at least one of the binding partner components binds to a
binding partner component of the membrane-targeted Akt.
140. The method of claim 131, wherein the membrane-targeted Akt
comprises at least one acylation region.
141. The method of claim 140, wherein the acylation region is a
dual acylation region.
142. The method of claim 140, wherein one acylation region is a
myristoyl region.
143. The method of claim 140, wherein the acylation region is from
a protein kinase.
144. The method of claim 143, wherein the protein kinase is Fyn or
Lck.
145. The method of claim 143, wherein the protein kinase is
Src.
146. The method of claim 140, wherein the acylation region is
linked to the N-terminus of the Akt region.
147. The method of claim 140, wherein the acylation region is
linked to the C-terminus of the Akt region.
148. The method of claim 140, wherein the acylation region
comprises a cys-ala-ala sequence.
149. The method of claim 148, wherein the cys-ala-ala sequence is
from a G-protein.
150. The method of claim 131, wherein the mammalian Akt region is
substantially identical to mouse Akt lacking a functional
pleckstrin homology (PH) domain.
151. The method of claim 131, wherein the mammalian Akt region is
substantially identical to human Akt lacking a functional
pleckstrin homology (PH) domain.
152. The method of claim 131, wherein the mammalian Akt region is
substantially identical to the amino acid sequence of SEQ ID NO:
6.
153. The method of claim 131, wherein the mammalian Akt region
consists of the amino acid sequence of SEQ ID NO: 6.
154. The method of claim 140, wherein the acylation region is
substantially identical to the amino acid sequence of SEQ ID NO:
8.
155. The method of claim 140, wherein the acylation region consists
of the amino acid sequence of SEQ ID NO: 8.
156. The method of claim 140, wherein the acylation region having
one or more binding partner components is encoded by a nucleotide
sequence in the dendritic cell.
157. The method of claim 131, wherein the membrane-targeted Akt
comprises a membrane protein or membrane protein region linked to
the mammalian Akt region.
158. The method of claim 138, wherein the dendritic cell comprises
a membrane protein or membrane protein region having one or more
binding partner components, wherein at least one of the binding
partner components binds to a binding partner component of the
membrane-targeted Akt.
159. The method of claim 158, wherein the membrane protein or
membrane protein region having one or more binding partner
components is encoded by a nucleotide sequence in the dendritic
cell.
160. The method of claim 131, wherein the nucleic acid is from a
virus.
161. The method of claim 160, wherein the dendritic cell is
contacted with a virus that contains the nucleic acid.
162. The method of claim 131, wherein the nucleic acid comprises a
consitutively active promoter operably linked to the nucleic acid
that encodes the membrane-targeted Akt protein.
163. A method for loading a modified dendritic cell with an
antigen, comprising: contacting a modified dendritic cell with an
antigen or antigen precursor, wherein the modified dendritic cell
expresses a membrane-targeted Akt protein lacking a functional
pleckstrin homology (PH) domain, whereby the modified dendritic
cell is loaded with the antigen.
164. A method for inducing an immune response against an antigen,
which comprises contacting a dendritic cell that expresses a
membrane-targeted Akt protein lacking a functional pleckstrin
homology (PH) domain with an antigen or antigen precursor; and
administering the dendritic cell to a subject; whereby the immune
response against the antigen is induced.
165. A method for inducing an immune response against an antigen,
which comprises contacting a dendritic cell that expresses a
membrane-targeted Akt protein lacking a functional pleckstrin
homology (PH) domain with an antigen or antigen precursor; and
administering the dendritic cell to a subject; whereby the immune
response against the antigen is induced.
166. A method for detecting an immune response against an antigen,
which comprises: contacting a dendritic cell that expresses a
membrane-targeted Akt protein lacking a functional pleckstrin
homology (PH) domain with an antigen; administering the dendritic
cell to a subject; and detecting the immune response.
167. A method for reducing cell proliferation in a subject, which
comprises: contacting a dendritic cell that expresses a
membrane-targeted Akt protein lacking a functional pleckstrin
homology (PH) domain with an antigen produced in proliferating
cells; and administering the dendritic cell to a subject; whereby
cell proliferation is reduced.
168. A method for inhibiting tumor growth in a subject, which
comprises: contacting a dendritic cell that expresses a
membrane-targeted Akt protein lacking a functional pleckstrin
homology (PH) domain with an antigen produced by cells in a tumor;
and administering the dendritic cell to a subject having a tumor;
whereby tumor growth is inhibited.
169. A kit which comprises a nucleic acid comprising a nucleotide
sequence that encodes a membrane-targeted Akt protein lacking a
functional pleckstrin homology (PH) domain.
170. An isolated nucleic acid which comprises a nucleotide sequence
that encodes a protein containing: a first region comprising a
human Akt sequence lacking a functional pleckstrin homology (PH)
domain; and a second region linked to the N-terminus of the Akt
sequence comprising two or more acylation sites.
171. An adenovirus which comprises the nucleotide sequence of claim
170.
172. A method for inducing an immune response, which comprises: a.
contacting a dendritic cell from a human subject with an antigen;
b. contacting the dendritic cell with a nucleic acid that comprises
a nucleotide sequence that encodes a protein containing: a first
region comprising a human Akt sequence lacking a pleckstrin
homology (PH) domain; and a second region linked to the N-terminus
of the Akt sequence comprising two or more acylation sites; and
administering the dendritic cell after steps a and b to the
subject, whereby an immune response is induced.
173. A method for inducing an immune response, which comprises: a.
contacting a dendritic cell from a human subject with an antigen;
b. contacting the dendritic cell with a nucleic acid that comprises
a nucleotide sequence that encodes a protein containing: a first
region comprising a human Akt sequence lacking a functional
pleckstrin homology (PH) domain; and a second region linked to the
N-terminus of the Akt sequence comprising two or more acylation
sites; and proliferating in vitro antigen-specific CTLs against the
dendritic cell after steps a and b, whereby an immune response is
induced.
174. A composition comprising (i) a first polynucleotide sequence
that encodes a first chimeric protein comprising an Akt lacking a
functional pleckstrin homology (PH) domain, and further comprising
a membrane-association region, and (ii) a second polynucleotide
sequence that encodes a second chimeric protein comprising a
membrane-association region, a multimeric ligand binding region and
a CD40 cytoplasmic polypeptide region lacking the CD40
extracellular domain.
175. A protein comprising: a mammalian Akt region lacking a
functional pleckstrin homology (PH) domain; and a dual acylation
region covalently linked to the mammalian Akt region.
176. A protein comprising: a mammalian Akt region lacking a
functional pleckstrin homology (PH) domain; and a membrane protein
or membrane protein region covalently linked to the mammalian Akt
region.
Description
RELATED PATENT APPLICATION
[0001] This patent application claims the benefit of U.S.
provisional patent application No. 60/803,025 filed on May 23,
2006, entitled "Modified Dendritic Cells Having Enhanced Survival
and Immunogenicity and Related Compositions and Methods," naming
Park et al. as inventors, and designated by attorney docket no.
BEL-1001-PV. The entire content of this provisional patent
application is incorporated herein by reference in jurisdictions
permitting such incorporation.
FIELD OF THE INVENTION
[0003] The invention pertains generally to immunotherapy, and more
specifically to antigen-presenting cells.
BACKGROUND
[0004] Immunotherapy treatments generally involve inducing an
immune response against a disease-associated antigen in a subject
by sensitizing the subject's immune system to the antigen. The
immune response often is induced by a vaccine that bears the
antigen. Upon treatment with the vaccine, the immune system attacks
cells bearing the antigen, which leads to a therapeutic effect. The
immunity induced by vaccines depends largely on the efficiency of
the antigen presenting cells (APC) that process and present the
antigen. Dendritic cells (DCs) are APCs that can be responsible for
vaccine efficacy by capturing and processing antigen and
stimulating T cell immunity It is possible to generate ex vivo
functional DCs from a subject's peripheral blood monocytes or CD34
haemopoietic stem cells. In ex vivo approaches, dendritic cells
generated from a patient's peripheral blood monocytes or CD34
haemopoietic stem cells can be loaded with a disease-associated
antigen and reinfused into the patient with the aim of generating
effective anti-disease immunity.
SUMMARY
[0005] Dendritic cells (DCs) are potent antigen-presenting cells
(APCs), justifying their widespread use in vaccination protocols
for the treatment of various malignancies. The lifespan of
activated DCs is limited to only a few days in draining lymph nodes
and phagocytosis or "reprocessing" of antigens from dying DCs to
immature APCs can lead to tolerance. The limited lifespan of DCs
therefore has been a shortcoming of immunotherapy.
[0006] Modified DCs provided herein have improved lifespan and
immunogenicity, and are useful for immunotherapy. The modified DCs
express an altered protein kinase, referred to herein as "protein
kinase B" or "Akt." The altered Akt associates with cell membranes
with greater frequency than unaltered Akt, and is referred to
herein as "membrane-targeted Akt."
[0007] Thus, provided herein is a dendritic cell which comprises a
nucleotide sequence that encodes a membrane-targeted Akt protein.
The dendritic cell (DC) often originates from a mammal, including,
for example, a rodent, (e.g., rat, mouse, rabbit, hamster, guinea
pig), dog, cat, ungulate, fish, avian, monkey, ape or human. DCs
often are isolated from a subject (e.g., a human) and then cultured
ex vivo under conditions suitable for maintaining DCs for one or
more days.
[0008] The membrane-targeted Akt protein comprises a mammalian Akt
protein or a fragment thereof. A fragment often includes the
protein kinase catalytic domain of Akt, and sometimes does not
include the N-terminal pleckstrin homology (PH) domain of native
Akt Amino acid sequences of mammalian Akt proteins or fragments are
known, and sometimes the amino acid sequence is a mouse or a human
Akt protein, a fragment thereof, a substantially identical variant
of the foregoing. A human Akt protein may have the amino acid
sequence encoded by SEQ ID NO: 2, 3, or 4, or a substantially
identical variant thereof. An Akt protein fragment may have a
portion of the amino acid sequence encoded by SEQ ID NO: 2, 3, or 4
or a substantially identical variant thereof, and in some
embodiments, the fragment has the amino acid sequence of SEQ ID NO:
6 (i.e., encoded by SEQ ID NO: 5).
[0009] In certain embodiments, the membrane-targeted Akt protein is
in association with a membrane association region, the latter of
which can increase the frequency with which the Akt protein is in
association with a cell membrane or portion thereof. In some
embodiments, the membrane-targeted Akt interacts with (e.g., binds
with) affinity and selectivity to membrane rafts in cells. The term
"in association" as used herein with respect to an Akt region and a
membrane association region refers to covalent or non-covalent
association of the membrane association region to an Akt protein or
fragment. The N- or C-terminus of an Akt protein or fragment may be
covalently linked to a membrane association region, and a membrane
association region may be linked to a non-terminal portion of an
Akt protein or fragment. A protein having an Akt protein region and
a membrane association region may be referred to as a "fusion
protein" or "chimeric protein." In certain embodiments, an Akt
chimeric protein does not include a heterologous multimerization
region, such as a region that binds to a FK506 molecule or analog
thereof, and in such embodiments, the chimeric protein can be
constitutively active. An Akt protein or fragment component may be
non-covalently associated with a membrane association region
component via a complementary member of a binding pair linked to
each component. The membrane association region and the Akt protein
or fragment in the membrane-targeted Akt often are homologous
(e.g., each are a human amino acid sequence), and sometimes are
heterologous (e.g., one is a human amino acid sequence and the
other is a mouse amino acid sequence).
[0010] A membrane association region often is an amino acid
fragment of a native mammalian protein, or a substantially
identical sequence thereof. Membrane association regions include,
for example, acylation regions, transmembrane proteins, and
transmembrane protein fragments. Acylation regions are capable of
being linked to one or more acyl moieties. For example a dual
acylation region is capable of being linked to two acyl moieties.
In certain embodiments, the acylation region is from a protein
kinase, such as Fyn, Lck or Src, for example, which can be
myristoylated and may be linked to the N-terminus of the Akt
protein or fragment. In some embodiments, the acylation region
comprises a cys-ala-ala sequence, and sometimes the region is from
a G-protein, which can be linked to the C-terminus of the Akt
protein or fragment.
[0011] The nucleotide sequence that encodes the membrane-targeted
Akt can be incorporated in the dendritic cell in a variety of
manners. For example, one or more copies of the nucleotide sequence
may be stably inserted into the dendritic cell genomic DNA by
random insertion or by non-random insertion (e.g., knock-in), or
the nucleotide sequence may be non-stably inserted in the dendritic
cell (e.g., in plasmid DNA). For embodiments in which (a) an Akt
protein or fragment-binding partner component and (b) a membrane
association region-binding partner component, are expressed in a
dendritic cell, each component may be expressed from a nucleotide
sequence in functional association with a common promoter or from
different promoters.
[0012] Modified dendritic cells comprising a nucleotide sequence
that expresses a membrane-targeted Akt generally (a) survive longer
on average than counterpart dendritic cells that do not include the
nucleotide sequence, (b) present a greater amount of antigen on
average than cells that do not include the nucleotide sequence, and
(c) are more immunogenic on average than cells that do not include
the nucleotide sequence. Processes for assessing dendritic cell
survival periods and antigen presentation and immunogenicity are
well known to the person of ordinary skill in the art.
[0013] A modified dendritic cell described herein sometimes is
provided in a composition comprising components suitable for
administering the dendritic cell to a subject. For example,
dendritic cells can be mixed with Toll-like receptor (TLR) ligands
that can increase DC activation locally, such as monophosphoryl
lipid A (TLR4 ligand), imiquimod (TLR7/8 ligand), unmethylated CpG
oligonucleotides (TLR9 ligand), and others. Additional activation
ligands, such as those found in monocyte-conditioned media
"maturation cocktail" also can be included. Thus, included is a
non-human organism, sometimes a mammalian organism, comprising a
modified dendritic cell described herein.
[0014] Also provided are methods for preparing a modified dendritic
cell, which comprises: contacting a dendritic cell with a nucleic
acid having a nucleotide sequence that encodes a membrane-targeted
Akt protein, whereby the modified dendritic cell survives longer on
average than dendritic cells that do not include the nucleotide
sequence. The resulting modified dendritic cell often presents a
greater amount of antigen on average than cells that do not include
the nucleotide sequence, and the resulting modified dendritic cell
is more immunogenic on average than cells that do not include the
nucleic acid. The nucleic acid sometimes is isolated from a source
(e.g., a cell or virus) before contacting the dendritic cell with
the nucleic acid, and sometimes the nucleic acid is within a virus
and is delivered to the dendritic cell by contacting the cell with
the virus, whereby the virus inserts the nucleic acid into the
cell.
[0015] In some embodiments, the modified dendritic cell is
contacted with an antigen or antigen precursor. Any antigen or
precursor suitable for immunotherapy can be utilized, such as an
antigen expressed more frequently in cancer cells than in
non-cancer cells, for example. The antigen may result from
contacting the modified dendritic cell with prostate specific
membrane antigen (PSMA) or a fragment thereof. In certain
embodiments, the modified dendritic cell is contacted with a PSMA
fragment having the amino acid sequence of SEQ ID NO: 10 (e.g.,
encoded by the nucleotide sequence of SEQ ID NO: 9).
[0016] Provided also are methods for loading a modified dendritic
cell with an antigen, comprising: contacting a modified dendritic
cell with an antigen or antigen precursor, where the modified
dendritic cell expresses a membrane-targeted Akt protein, whereby
the modified dendritic cell is loaded with the antigen. The antigen
can be delivered as a purified protein or purified fragment, or by
a viral or non-viral sequence. Also provided are methods for
manufacturing a virus having a nucleotide sequence that encodes a
membrane-targeted Aid protein, which comprise: transfecting a
producer cell with a virus having a nucleotide sequence that
encodes a membrane-targeted Akt; and isolating the virus produced
by the cells.
[0017] Also provided are isolated membrane-targeted Akt proteins.
For example, included is a protein comprising: (a) a mammalian Akt
portion lacking a pleckstrin homology (PH) domain; and (b) a dual
acylation portion, or (c) a membrane protein or membrane protein
fragment. Portion (a) may be covalently linked or non-covalently
linked to portion (b) or portion (c). In certain embodiments,
portion (a) is non-covalently linked to portion (b) or portion (c)
by components of a binding pair linked to each of portion (a) and
portion (b) or (c). In certain embodiments, the dual acylation
portion is from a protein kinase, such as Fyn, Lck or Src, for
example. In certain embodiments, the dual acylation region
comprises two acyl moieties, where one or both of the acyl moieties
may be selected from the group consisting of myristoyl, geranyl,
farnesyl and prenyl. The acylation region may comprise a
cys-ala-ala sequence, and may be from a G-protein. In some
embodiments, the dual acylation portion is identical to or
substantially identical to the amino acid sequence of SEQ ID NO: 8.
The acylation portion, membrane protein or membrane protein
fragment may be linked to the N-terminus or C-terminus of the Akt
portion. The mammalian Akt portion may be identical to or
substantially identical to mouse Aid, or a fragment thereof, or may
be identical to or substantially identical to human Akt, or a
fragment thereof. In certain embodiments, the mammalian Akt portion
is identical to or substantially identical to the amino acid
sequence encoded by the nucleotide sequence of SEQ ID NO: 6, or a
fragment thereof. Also provided is an antibody or antibody fragment
that specifically binds to a membrane-targeted Akt protein.
[0018] In some embodiments, a membrane-targeted Akt protein may
include one or more multimerization regions (e.g., one, two, three,
four or more multimerization regions), and one or more
multimerization regions may be located at the N-terminus of the
chimeric protein, within the protein (e.g., between an Aid region
and a membrane association region), or at the C-terminus of the
protein. A multimeriation region often is heterologous to other
regions of the membrane-targeted Akt protein (i.e., the nucleic
acid encoding the multimerization region is not linked in vivo to a
nucleic acid sequence encoding the Akt protein or the acylation
region). A multimerization region may be covalently linked or
non-covalently linked to a membrane-targeted Akt protein. An
example of a multimerization region is an amino acid sequence that
binds to a FK506 molecule or analog thereof. A membrane-targeted
Akt protein containing a multimerization region can be referred to
as an "inducible membrane-targeted Akt protein." In certain
embodiments, a membrane-targeted does not contain a multimerization
region or a heterologous multimerization region. A
membrane-targeted Akt protein not containing a multimerization
region or heterologous multimerization region can be referred to as
a "constitutively active membrane-targeted Akt protein."
[0019] Provided also is a nucleic acid which comprises a nucleotide
sequence that encodes a membrane-targeted Akt protein described
herein. In certain embodiments, the nucleic acid comprises a
promoter operably linked to the nucleotide sequence. The promoter
sometimes is constitutively active, at times is inducible, and can
be from a virus. The nucleic acid in some embodiments comprises one
or more nucleotide sequences from a virus, and in certain
embodiments, the nucleic acid is derived from a virus. Any suitable
virus capable of expressing a protein in cells, and optionally
replicating in cells, can be utilized, such as adenovirus.
[0020] Also provided is a cell which comprises a nucleic acid
described herein. The cell can be an antigen presenting cell, such
as a dendritic cell for example, and the cell sometimes is a human
cell. The cell containing the nucleic acid often (a) survives
longer on average than a counterpart cell that does not include the
nucleic acid (i.e., the same type of cell that does not contain the
nucleic acid), (b) presents a greater amount of antigen on average
than a counterpart cell that does not include the nucleic acid,
and/or (c) is more immunogenic on average than a cell that does not
include the nucleic acid. In the cell, the membrane-associated Akt
protein encoded by the nucleic acid can be in association with a
raft membrane with higher frequency than other portions of the cell
membrane, in certain embodiments.
[0021] Provided also is a method for inducing an immune response
against an antigen, which comprises contacting a dendritic cell
that expresses a membrane-targeted Akt protein with an antigen or
antigen precursor; and administering the dendritic cell to a
subject; whereby the immune response against the antigen is
induced. Also included is a method for detecting an immune response
against an antigen, which comprises: contacting a dendritic cell
that expresses a membrane-targeted Akt protein with an antigen;
administering the dendritic cell to a subject; and detecting the
immune response. Provided in addition is a method for reducing cell
proliferation in a subject, which comprises: contacting a dendritic
cell that expresses a membrane-targeted Akt protein with an antigen
produced in proliferating cells; and administering the dendritic
cell to a subject; whereby cell proliferation is reduced. In
certain methods, the antigen or a fragment thereof sometimes is
co-administered to the subject (e.g., before, during or after
administration of the dendritic cell). Administration of the
dendritic cell and/or antigen may be pulsed (e.g., administered
multiple times over a period of time (e.g., within a day, once
every five days). Also provided is a method for inhibiting tumor
growth in a subject, which comprises: contacting a dendritic cell
that expresses a membrane-targeted Akt protein with an antigen
produced by cells in a tumor; and administering the dendritic cell
to a subject having a tumor; whereby tumor growth is inhibited.
[0022] Also provided is a kit which comprises a nucleic acid
comprising a nucleotide sequence that encodes a membrane-targeted
Akt protein described herein. In certain embodiments, the nucleic
acid is packaged in a virus, and sometimes the virus is an
adenovirus, such as a replication deficient adenovirus. In some
embodiments, the kit comprises one or more transfection components
for inserting the nucleic acid into a dendritic cell. A kit may
comprise an antigen, and/or a second nucleic acid that encodes an
antigen. Some kits comprise instructions, or directions to obtain
instructions (e.g., obtained from a website), for preparing a
dendritic cell with the nucleic acid comprising a nucleotide
sequence that encodes the membrane-targeted Akt.
[0023] Also provided is an isolated nucleic acid which comprises a
nucleotide sequence that encodes a protein containing: a first
region comprising a human Akt sequence lacking a pleckstrin
homology (PH) domain; and a second region linked to the N-terminus
of the Akt sequence comprising two or more acylation sites. In
certain embodiments, the second region comprises a Gly-Cys-Xaa-Cys-
sequence, and sometimes the second region is about 5 to about 25
amino acids in length and from the N-terminus of the nucleic acid
kinase Fyn, Yes or Lck. The second region sometimes comprises two
acyl moieties, such as a myristoyl and palmitoyl region, for
example. In certain embodiments, the Akt sequence is identical to
or substantially identical to a fragment of an amino acid sequence
encoded by SEQ ID NO: 2, 3, or 4. Sometimes the Akt sequence is
identical to or substantially identical to the amino acid sequence
of SEQ ID NO: 6, and at times the Akt sequence is identical to or
substantially identical to an amino acid sequence encoded by SEQ ID
NO: 5. In certain embodiments, the second region is identical to or
substantially identical to the amino acid sequence encoded by SEQ
ID NO: 7 (from Fyn). Provided also is an adenovirus which comprises
the nucleotide sequence of any one of the preceding aspects. Also
provided is a cell which comprises the nucleotide sequence of any
one of the preceding aspects. In some embodiments the cell is a
human antigen presenting cell, such as a human dendritic cell. In
certain embodiments, the cell has been contacted with an antigen,
such as a prostate specific membrane antigen or fragment thereof
(e.g., SEQ ID NO: 10).
[0024] Also provided is a method for inducing an immune response,
which comprises: (a) contacting a dendritic cell from a human
subject with an antigen; (b) contacting the dendritic cell with a
nucleic acid that comprises a nucleotide sequence that encodes a
protein containing: a first region comprising a human Akt sequence
lacking a pleckstrin homology (PH) domain; and a second region
linked to the N-terminus of the Akt sequence comprising two or more
acylation sites; and administering the dendritic cell after steps
(a) and (b) to the subject, whereby an immune response is induced.
The immune response often is or includes an antigen-specific CTL
response, and sometimes the antigen is a prostate specific membrane
antigen or fragment thereof (e.g., SEQ ID NO: 10). Provided also is
a method for inducing an immune response, which comprises: (a)
contacting a dendritic cell from a human subject with an antigen;
(b) contacting the dendritic cell with a nucleic acid that
comprises a nucleotide sequence that encodes a protein containing:
a first region comprising a human Akt sequence lacking a pleckstrin
homology (PH) domain; and a second region linked to the N-terminus
of the Akt sequence comprising two or more acylation sites; and
proliferating in vitro antigen-specific CTLs against the dendritic
cell after steps (a) and (b), whereby an immune response is
induced. In certain embodiments the CTLs are administered to the
subject, and sometimes the antigen is a prostate specific membrane
antigen or fragment thereof (e.g., SEQ ID NO: 10).
[0025] These and other embodiments are described hereafter in the
Detailed Description and in the Claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A to 1F show LPS and CD40 prevent DC death by
blocking the down-regulation of Akt and Bcl-2.
[0027] FIGS. 2A-2F show Akt1 is critical for LPS and CD40-mediated
DC survival and activation.
[0028] FIGS. 3A-3F show functionally optimized Akt induces DC
activation and protects DCs from PI3K inhibition.
[0029] FIG. 4 shows Akt-mediated DC longevity requires Bcl-2.
[0030] FIGS. 5A-5E show MF-.DELTA.Akt and M-Akt induce BMDC
longevity in vitro and in vivo.
[0031] FIGS. 6A-6C show MF-.DELTA.Akt and M-Akt-expressing BMDCs
reveal improved T cell activation and proliferation.
[0032] FIGS. 7A-7E show MF-.DELTA.Akt expression enhances the
efficacy of DC-based therapeutic tumor vaccines.
[0033] FIGS. 8A-8C show MF-.DELTA.hAkt induces human MoDC longevity
leading to improved antigen-specific CTL activation and
proliferation.
[0034] FIGS. 9A-9C show effects of Akt inhibitors on DC
survival.
[0035] FIG. 10 shows a representative analysis of surface markers
of Akt1.sup.+/+ or Akt1.sup.-/- DCs.
[0036] FIG. 11 shows a comparison of bone marrow and spleens
isolated from Akt1.sup.+/+ or Ak t.sup.-/- mice.
[0037] FIGS. 12A-12E show effects of different acylation
regions.
[0038] FIGS. 13A-13D show effects of modified DCs described herein
on tumor growth and animal survival.
[0039] FIG. 14 shows representative memory recall responses for
particular antigens.
[0040] FIGS. 15A-15B show effects of a combination
immunotherapy.
DETAILED DESCRIPTION
Akt Molecules
[0041] The term "Akt molecule" refers to a molecule, such as a
protein, polypeptide, nucleic acid or expression vector, for
example, comprising a native Akt sequence, or fragment thereof or
substantially identical variant of the foregoing. An Akt molecule
often is capable of enhancing antigen presenting cell longevity and
immunogenicity when expressed in an antigen presenting cell in such
a manner that it is membrane-targeted, and constitutively active in
certain embodiments. An Akt molecule also may include other
portions in addition to the Akt sequence, and in such embodiments,
the Akt sequence in the Akt molecule sometimes is referred to
herein as an "Akt portion" or "Akt region." Additional sequences
that may be included optionally in an Akt molecule are described
herein, such as a membrane association sequence and/or a
multimerization sequence, for example.
[0042] An Akt sequence may be a native Akt sequence, a fragment of
an Akt sequence or a substantially identical variant of the
foregoing. An Akt sequence often is mammalian (e.g., mouse or
human), or a fragment or variant sequence thereof. Examples of
native polynucleotide sequences that encode Akt polypeptides
include, but are not limited to, SEQ ID NO: 1 (mouse Akt1), SEQ ID
NO: 2 (human Akt1), SEQ ID NO: 2 (human Akt2), SEQ ID NO: 4 (human
Akt3), and Akt homologs from other species, often mammals, and
including Akt oncogenic viral sequences.
[0043] As noted above, Akt sequences include Akt fragment
sequences. An AKT fragment sequence may lack one or more
nucleotides, amino acids or regions, the latter of which may be a
functional region or domain. An Akt fragment sequence can include
one or more functional regions, and may lack one or more functional
regions compared to a native Akt sequence. Functional regions
include a pleckstrin homology (PH) domain (e.g., from about
position 6 to about position 107 in human AKT1), a serine/threonine
protein kinase catalytic region (e.g., from about position 149 to
about position 408 in human AKT1) and a catalytic domain extension
region (e.g., from about position 409 to about position 476 in
human AKT1). Where an Akt fragment sequence includes one or more
functional regions, the region may be flanked on each side by a
native amino acid sequence from a native Akt sequence. In certain
embodiments, an Akt amino acid fragment sequence is 10 or more, 15
or more, 20 or more, 25 or more, 50 or more, 100 or more, 200 or
more, 300 or more, 400 or more or 450 or more amino acids from a
native Akt protein. An Akt molecule often includes an Akt protein
kinase catalytic domain, and therefore often is capable of
catalyzing Ser/Thr protein phosphorylation. An Akt fragment can
exclude a PH domain or includes a modified PH domain. A modified PH
domain may be truncated or mutated, generated by using standard
mutagenesis, insertions, deletions, or substitutions, and the
modified form may or may not be functional. Examples of Akt nucleic
acids lacking a functional PH domain sequence have the nucleotide
sequences of SEQ ID NO: 5 (mouse Akt) and SEQ ID NO: 6 (human
Akt).
[0044] Akt sequences include homologs, alternative transcripts,
alleles, functionally equivalent fragments, variants, and analogs
of native Akt sequences (e.g., nucleotide sequences described
herein). The term "substantially identical variant" as used herein
refers to a nucleotide or amino acid sequence sharing sequence
identity to a nucleotide sequence or amino acid sequence of Akt or
another molecule described herein (e.g., membrane association
region). Included are nucleotide sequences or amino acid sequences
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 (each sometimes
within a 1%, 2%, 3% or 4% variability) identical to a nucleotide
sequence or encoded amino acid sequence described herein, or has
one to ten nucleotide or amino acid substitutions. One test for
determining whether two nucleotide sequences or amino acids
sequences are substantially identical is to determine the percent
of identical nucleotide sequences or amino acid sequences
shared.
[0045] Calculations of sequence identity can be performed as
follows. Sequences are aligned for optimal comparison purposes
(e.g., gaps can be introduced in one or both of a first and a
second amino acid or nucleic acid sequence for optimal alignment
and non-homologous sequences can be disregarded for comparison
purposes). The length of a reference sequence aligned for
comparison purposes is sometimes 30% or more, 40% or more, 50% or
more, often 60% or more, and more often 70% or more, 80% or more,
90% or more, or 100% of the length of the reference sequence. The
nucleotides or amino acids at corresponding nucleotide or
polypeptide positions, respectively, are then compared among the
two sequences. When a position in the first sequence is occupied by
the same nucleotide or amino acid as the corresponding position in
the second sequence, the nucleotides or amino acids are deemed to
be identical at that position. The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, introduced for optimal alignment of the two
sequences. Comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. Percent identity between two amino acid or
nucleotide sequences can be determined using the algorithm of
Meyers & Miller, CABIOS 4: 11-17 (1989), which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4. Also, percent identity between two amino acid sequences can
be determined using the Needleman & Wunsch, J. Mol. Biol. 48:
444-453 (1970) algorithm which has been incorporated into the GAP
program in the GCG software package (available at the http address
www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix,
and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight
of 1, 2, 3, 4, 5, or 6. Percent identity between two nucleotide
sequences can be determined using the GAP program in the GCG
software package (available at http address www.gcg.com), using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. A set of parameters often
used is a Blossum 62 scoring matrix with a gap open penalty of 12,
a gap extend penalty of 4, and a frameshift gap penalty of 5.
[0046] Another manner for determining whether two nucleic acids are
substantially identical is to assess whether a polynucleotide
homologous to one nucleic acid will hybridize to the other nucleic
acid under stringent conditions. As use herein, the term "stringent
conditions" refers to conditions for hybridization and washing.
Stringent conditions are known to those skilled in the art and can
be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y., 6.3.1-6.3.6 (1989). Aqueous and non-aqueous
methods are described in that reference and either can be used. An
example of stringent hybridization conditions is hybridization in
6.times. sodium chloride/sodium citrate (SSC) at about 45.degree.
C., followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50.degree. C. Another example of stringent hybridization conditions
are hybridization in 6.times. sodium chloride/sodium citrate (SSC)
at about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 55.degree. C. A further example of
stringent hybridization conditions is hybridization in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
60.degree. C. Often, stringent hybridization conditions are
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 65.degree. C. More often, stringency
conditions are 0.5M sodium phosphate, 7% SDS at 65.degree. C.,
followed by one or more washes at 0.2.times.SSC, 1% SDS at
65.degree. C.
[0047] An example of a substantially identical nucleotide sequence
to a base nucleotide sequence described herein is one that has a
different nucleotide sequence but still encodes the same amino acid
sequence encoded by the base nucleotide sequence. Another example
is a nucleotide sequence that encodes a protein having an amino
acid sequence 70% or more identical to, sometimes 75% or more, 80%
or more, or 85% or more identical to, and often 90% to 99%
identical to an amino acid sequence encoded by the base nucleotide
sequence.
[0048] Nucleotide sequences and encoded amino acid sequences
described herein can be used as "query sequences" to perform a
search against public databases to identify other family members or
related sequences, for example. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul et
al., J. Mol. Biol. 215: 403-10 (1990). BLAST nucleotide searches
can be performed with the NBLAST program, score=100, wordlength=12
to obtain nucleotide sequences homologous to nucleotide sequences
described herein. BLAST polypeptide searches can be performed with
the XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences homologous to those encoded by nucleotide sequences
described herein. To obtain gapped alignments for comparison
purposes, Gapped BLAST can be utilized as described in Altschul et
al., Nucleic Acids Res. 25(17): 3389-3402 (1997). When utilizing
BLAST and Gapped BLAST programs, default parameters of the
respective programs (e.g., XBLAST and NBLAST) can be used (see the
http World Wide Web address ncbi.nlm.nih.gov). Thus, a protein
having a substantially identical amino acid sequence to (i) an
amino acid sequence described herein or (ii) an amino acid sequence
encoded by a nucleotide sequence described herein, identified by a
query sequence search can be considered a substantially identical
sequence.
[0049] Substantially identical nucleotide sequences may include
altered codons for enhancing expression of an amino acid sequence
in a particular expression system. One or more codons may be
altered, and sometimes 10% or more or 20% or more of the codons are
altered for optimized expression in an expression system that may
include bacteria (e.g., E. coli.), yeast (e.g., S. cervesiae),
human (e.g., 293 cells or antigen presenting cells), insect, or
rodent (e.g., hamster) cells (e.g., antigen presenting cells).
[0050] An Akt protein, polypeptide or fragment variant can include
one or more amino acid substitutions, deletions or insertions. Any
amino acid may be substituted by a conservative or non-conservative
substitution. For example, phosphorylatable amino acids (e.g.,
serine, threonine or tyrosine) in an Akt protein or fragment may be
modified (e.g., deleted or substituted with an amino acid that
cannot be phosphorylated).
[0051] An Akt protein, polypeptide or fragment variant may contain
one or more unnatural amino acids. Unnatural amino acids include
but are not limited to D-isomer amino acids, ornithine,
diaminobutyric acid, norleucine, pyrylalanine, thienylalanine,
naphthylalanine and phenylglycine, alpha and alpha-disubstituted
amino acids, N-alkyl amino acids, lactic acid, halide derivatives
of natural amino acids such as trifluorotyrosine,
p-Cl-phenylalanine, p-Br-phenylalanine, p-I-phenylalanine,
L-allyl-glycine, beta-alanine, L-alpha-amino butyric acid,
L-gamma-amino butyric acid, L-alpha-amino isobutyric acid,
L-epsilon-amino caproic acid, 7-amino heptanoic acid, L-methionine
sulfone, L-norleucine, L-norvaline, p-nitro-L-phenylalanine,
L-hydroxyproline, L-thioproline, methyl derivatives of
phenylalanine (Phe) such as 4-methyl-Phe, pentamethyl-Phe, L-Phe
(4-amino), L-Tyr (methyl), L-Phe (4-isopropyl), L-Tic
(1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid),
L-diaminopropionic acid, L-Phe (4-benzyl), 2,4-diaminobutyric acid,
4-aminobutyric acid (gamma-Abu), 2-amino butyric acid (alpha-Abu),
6-amino hexanoic acid (epsilon-Ahx), 2-amino isobutyric acid (Aib),
3-amino propionic acid, ornithine, norleucine, norvaline,
hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic
acid, t-butylglycine, t-butylalanine, an amino acid derivitized
with a heavy atom or heavy isotope (e.g., Au, deuterium, 15N;
useful for synthesizing protein applicable to X-ray
crystallographic structural analysis or nuclear magnetic resonance
analysis), phenylglycine, cyclohexylalanine, fluoroamino acids,
designer amino acids such as beta-methyl amino acids, Ca-methyl
amino acids, Na-methyl amino acids, naphthyl alanine, and the
like.
[0052] Membrane Association Regions
[0053] Molecules in association with cell membranes contain certain
regions that facilitate the membrane association, and such regions
can be incorporated into or associated with a protein containing an
Akt sequence to generate membrane-targeted Akt molecules (e.g., Akt
chimeras). For example, some proteins contain sequences at the
N-terminus or C-terminus that are acylated, and these acyl moieties
facilitate membrane association. Such sequences are recognized by
acyltransferases and often conform to a particular sequence motif.
Certain acylation motifs are capable of being modified with a
single acyl moiety and others are capable of being modified with
multiple acyl moieties. For example, the N-terminal sequence of the
protein kinase Src can comprise a single myristoyl moiety. Dual
acylation regions are located within the N-terminal regions of
certain protein kinases (e.g., Yes, Fyn, Lck) and G-protein alpha
subunits. Such dual acylation regions often are located within the
first eighteen amino acids of such proteins, and conform to the
sequence motif Met-Gly-Cys-Xaa-Cys, where the Met is cleaved, the
Gly is N-acylated and one of the Cys residues is S-acylated. The
Gly often is myristoylated and a Cys can be palmitoylated.
Acylation regions conforming to the sequence motif Cys-Ala-Ala-Xaa
(so called "CAAX boxes"), which can modified with C15 or C10
isoprenyl moieties, from the C-terminus of G-protein gamma subunits
and other proteins (e.g., http World Wide Web address
ebi.ac.uk/interpro/DisplayIproEntry?ac=IPRO01230) also can be
utilized. These and other acylation motifs are known to the person
of ordinary skill in the art (e.g., Gauthier-Campbell et al.,
Molecular Biology of the Cell 15: 2205-2217 (2004); Glabati et al.,
Biochem. J. 303: 697-700 (1994) and Zlakine et al., J. Cell Science
110: 673-679 (1997)), and can be incorporated in Akt molecules to
induce membrane localization. In certain embodiments, a native
sequence from a protein containing an acylation motif can be linked
to an Akt protein. For example, in some embodiments, an N-terminal
portion of Lck, Fyn or Yes or a G-protein alpha subunit, such as
the first twenty-five N-terminal amino acids or fewer from such
proteins (e.g., about 5 to about 20 amino acids, about 10 to about
19 amino acids, or about 15 to about 19 amino acids of the native
sequence with optional mutations), may be linked to the N-terminus
of an Akt protein (e.g., a Fyn fragment-Akt molecule is described
in the Examples section hereafter). In certain embodiments, a
C-terminal sequence of about 25 amino acids or less from a
G-protein gamma subunit containing a CAAX box motif sequence (e.g.,
about 5 to about 20 amino acids, about 10 to about 18 amino acids,
or about 15 to about 18 amino acids of the native sequence with
optional mutations) can be linked to the C-terminus of an Akt
protein.
[0054] In some embodiments, an acyl moiety has a log p value of +1
to +6, and sometimes has a log p value of +3 to +4.5. Log p values
are a measure of hydrophobicity and often are derived from
octanol/water partitioning studies, in which molecules with higher
hydrophobicity partition into octanol with higher frequency and are
characterized as having a higher log p value. Log p values are
published for a number of lipophilic molecules and log p values can
be calculated using known partitioning processes (e.g., Chemical
Reviews, Vol. 71, Issue 6, page 599, where entry 4493 shows lauric
acid having a log p value of 4.2). Any acyl moiety can be linked to
a peptide composition described above and tested for antimicrobial
activity using known methods and those described hereafter. The
acyl moiety sometimes is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20
alkynyl, C3-C6 cycloalkyl, C1-C4 haloalkyl, C4-C12 cyclalkylalkyl,
aryl, substituted aryl, or aryl (C1-C4)alkyl, for example. Any
acyl-containing moiety sometimes is a fatty acid, and examples of
fatty acid moieties are propyl (C3), butyl (C4), pentyl (C5), hexyl
(C6), heptyl (C7), octyl (C8), nonyl (C9), decyl (C10), undecyl
(C11), lauryl (C12), myristyl (C14), palmityl (C16), stearyl (C18),
arachidyl (C20), behenyl (C22) and lignoceryl moieties (C24), and
each moiety can contain 0, 1, 2, 3, 4, 5, 6, 7 or 8 unsaturations
(i.e., double bonds). An acyl moiety sometimes is a lipid molecule,
such as a phosphatidyl lipid (e.g., phosphatidyl serine,
phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidyl
choline), sphingolipid (e.g., shingomyelin, sphigosine, ceramide,
ganglioside, cerebroside), or modified versions thereof. In certain
embodiments, one, two, three, four or five or more acyl moieties
are linked to a membrane association region.
[0055] An Aid protein also may be linked to a single-pass or
multiple pass transmembrane sequence (e.g., to the N-terminus or
C-terminus of an Akt protein sequence). Single pass transmembrane
regions are found in certain CD molecules, tyrosine kinase
receptors, serine/threonine kinase receptors, TGFbeta, BMP, activin
and phosphatases. Single pass transmembrane regions can include a
signal peptide region and a transmembrane region of about 20 to
about 25 amino acids, many of which are hydrophobic amino acids and
can form an alpha helix. A short track of positively charged amino
acids can follow the transmembrane span. Multiple pass proteins
include ion pumps, ion channels, and transporters, and include two
or more helices that span the membrane multiple times. All or
substantially all of a multiple pass protein can be attached to an
Akt molecule. Sequences for single pass and multiple pass
transmembrane regions are known and can be selected for
incorporation into an Akt molecule by the person of ordinary skill
in the art.
[0056] Akt and Membrane Association Component Combinations
[0057] A membrane association region may be covalently linked to
the N-terminus or C-terminus of an Akt protein or fragment, and
such fusions sometimes are referred to herein as "chimeric
proteins." Such chimeric proteins can be encoded by a nucleotide
sequence in which the membrane association region-encoding sequence
is adjacent to the Akt protein or fragment-encoding sequence, with
or without an intervening linker sequence. Covalent linkages also
can be generated by a chemically reactive binding pair, whereby a
one member of the binding pair is linked to the Akt molecule and
another member of the binding pair is linked to the membrane
association region. Examples of chemically reactive binding pairs
include without limitation sulfhydryl/maleimide,
sulfhydryl/haloacetyl derivative, amine/isotriocyanate,
amine/succinimidyl ester, and amine/sulfonyl halides. When binding
pairs are utilized to generate a covalent linkage, it is possible
to join the membrane association region to the N-terminus or
C-terminus of the Akt molecule, or to a chemical moiety within the
Akt molecule.
[0058] In certain embodiments, the membrane association portion may
not be covalently attached and may non-covalently associate with
the Akt molecule. Non-covalent linkages can be generated by
interactive binding pairs, wherein one binding pair member is
linked to the membrane association region and the other binding
pair member is linked to the Akt molecule. Any suitable interactive
binding pair can be utilized to effect a non-covalent linkage,
including without limitation biotin/avidin, biotin/streptavidin,
folic acid/folate binding protein, vitamin B12/intrinsic factor,
nucleic acid/complementary nucleic acid (e.g., DNA, RNA, PNA), and
may be antibody/antigen, antibody/antibody, antibody/antibody
fragment, antibody/antibody receptor, antibody/protein A or protein
G, or hapten/anti-hapten pairs.
[0059] Where the membrane association region and the Akt molecule
are provided separately and then joined in a covalent or
non-covalent manner, nucleotide sequences encoding each portion can
be located on separate nucleic acids or the same nucleic acid. The
nucleotide sequence encoding each portion often are under the
control of the same regulatory sequences, and can be under the
control of different regulatory sequences (e.g., promoter, internal
ribosome entry sequence).
[0060] Multimerization regions also are known and can be utilized
to inducibly join an Akt protein, fragment or variant with a
membrane association region in a non-covalent manner. Such
inducible systems are referred to herein as "chemically induced
dimerization (CID)." In addition to being inducible, CID also is
reversible, due to the degradation of the labile dimerizing agent
or administration of a monomeric competitive inhibitor.
[0061] CID systems often involve synthetic bivalent ligands to
rapidly crosslink signaling molecules that are fused to
ligand-binding domains CID. This system has been used to trigger
the oligomerization and activation of cell surface (Spencer et al.,
1993; Spencer et al., 1996; Blau et al., 1997), or cytosolic
proteins (Luo et al., 1996; MacCorkle et al., 1998), the
recruitment of transcription factors to DNA elements to modulate
transcription (Ho et al., 1996; Rivera et al., 1996) or the
recruitment of signaling molecules to the plasma membrane to
simulate signaling (Spencer et al., 1995; Holsinger et al.,
1995).
[0062] CID systems often are based upon the notion that surface
receptor aggregation effectively activates downstream signaling
cascades. In one embodiment, the CID system uses a dimeric analog
of the lipid permeable immunosuppressant drug, FK506, which loses
its normal bioactivity while gaining the ability to crosslink
molecules genetically fused to the FK506-binding protein, FKBP12.
By fusing one or more FKBPs and a myristoylation sequence to the
cytoplasmic signaling domain of a target receptor, one can
stimulate signaling in a dimerizer drug-dependent, but ligand and
ectodomain-independent manner. This approach provides the system
with temporal control, reversibility using monomeric drug analogs,
and enhanced specificity. The high affinity of third-generation
AP20187/AP1903 CIDs for their binding domain (e.g., Pollock and
Rivera, Methods Enzymo1306:263-81 (1999)), FKBP12 permits specific
activation of the recombinant receptor in vivo without the
induction of non-specific side effects through endogenous FKBP12.
In addition, the synthetic ligands are resistant to protease
degradation, making them more efficient at activating receptors in
vivo than most delivered protein agents.
[0063] In specific embodiments, rapamycin analogs crosslink
endogenous FKBP12 with a 89 amino acid domain from FRAP/mTOR,
called FRB (FRAP rapamycin binding domain, residues 2025-2113).
Thus, in certain embodiments, activation of iAkt is based on
ligand-dependent recruitment of chimeric Akt (first chimeric
protein) to a membrane association protin (second chimeric
protein).
[0064] Ligands utilized for CID are capable of binding to two or
more of the ligand-binding domains. One skilled in the art realizes
that the chimeric proteins may be able to bind to more than one
ligand when they contain more than one ligand-binding domain. The
ligand sometimes is a non-protein or a chemical. Exemplary ligands
include, but are not limited to dimeric FK506 (e.g., FK1012),
AP1903, rapamycin or a derivative thereof.
[0065] In specific embodiments, the ligand-binding region linked to
the membrane association region is heterologous to the
ligand-binding region linked to the Akt molecule. In certain
embodiments, the ligand-binding region is a rapamycin-binding
domain, FRB, from FRAP/mTOR. Representative sequences and methods
of incorporating them into expression vectors are set forth in the
Examples section hereafter.
[0066] Nucleic Acid Constructs
[0067] In certain immunotherapy procedures, antigen presenting
cells are transfected or transformed with a nucleic acid having a
polynucleotide sequence that encodes an Akt molecule described
herein. The nucleic acid bearing such a nucleotide sequence can be
transferred into the antigen presenting cell in a variety of
manners, as described hereafter (e.g., delivery of a naked nucleic
acid or encapsulation of the nucleic acid in a liposome or virus).
Based on nucleotide sequences within the nucleic acid, a target
nucleotide sequence encoding an Akt molecule and/or other target
molecules may be stably integrated into the genomic DNA of the
antigen presenting cell, in a random or non-random manner (e.g.,
knock-in), or may be transiently deposited to the antigen
presenting cell.
[0068] Nucleic acids containing an Akt nucleotide sequence
sometimes are referred to herein as "nucleic acid compositions." A
nucleic acid composition can be from any source or composition,
such as DNA, cDNA, RNA or mRNA, for example, and can be in any
suitable form (e.g., linear, circular, supercoiled,
single-stranded, double-stranded, and the like). A nucleic acid
composition sometimes is a plasmid, phage, autonomously replicating
sequence (ARS), centromere, artificial chromosome or other nucleic
acid able to replicate or be replicated in vitro or in a host cell
(e.g., dendritic cell). Such nucleic acid compositions are selected
for their ability to guide production of the desired protein or
nucleic acid molecule. When desired, the nucleic acid composition
can be altered as known in the art such that codons encode for a
different amino acid than is normal, including unconventional or
unnatural amino acids (including detectably labeled amino
acids).
[0069] A nucleic acid composition can comprise certain elements
often selected according to the intended use of the nucleic acid.
Any of the following elements can be included in or excluded from a
nucleic acid composition. A nucleic acid composition, for example,
may include one or more or all of the following nucleotide
elements: one or more promoter elements, one or more 5'
untranslated regions (5'UTRs), one or more regions into which a
target nucleotide sequence may be inserted (an "insertion
element"), one or more target nucleotide sequences, one or more 3'
untranslated regions (3'UTRs), and a selection element. A nucleic
acid composition is provided with one or more of such elements and
other elements may be inserted into the nucleic acid before the
template is contacted with a transcription and/or translation
system. In some embodiments, a provided nucleic acid composition
comprises a promoter, 5'UTR, optional 3'UTR and insertion
element(s) by which a target nucleotide sequence is inserted (i.e.,
cloned) into the template. In certain embodiments, a provided
nucleic acid composition comprises a promoter, insertion element(s)
and optional 3'UTR, and a 5'UTR/target nucleotide sequence is
inserted with an optional 3'UTR. The elements can be arranged in
any order suitable for transcription and/or translation, and in
some embodiments a nucleic acid composition comprises the following
elements in the 5' to 3' direction: (1) promoter element, 5'UTR,
and insertion element(s); (2) promoter element, 5'UTR, and target
nucleotide sequence; (3) promoter element, 5'UTR, insertion
element(s) and 3'UTR; and (4) promoter element, 5'UTR, target
nucleotide sequence and 3'UTR.
[0070] A promoter element typically is required for DNA synthesis
and/or RNA synthesis. A promoter often interacts with a RNA
polymerase to generate message RNA suitable for translation of a
protein, polypeptide or peptide. Promoter sequences are readily
accessed and obtained by the artisan, and are readily adapted to
nucleic acid compositions described herein. The particular promoter
employed to control the expression of a polynucleotide sequence of
interest is not believed to be important, so long as it is capable
of directing the expression of the polynucleotide in the targeted
cell. Thus, where a human cell is targeted, it is preferable to
position the polynucleotide sequence-coding region adjacent to and
under the control of a promoter that is capable of being expressed
in a human cell. Generally speaking, such a promoter might include
either a human or viral promoter. Examples of promoters include
human cytomegalovirus (CMV) immediate early gene promoter, SV40
early promoter, Rous sarcoma virus long terminal repeat,
.beta.-actin, elongation factor 1-alpha (EF-1.alpha.), rat insulin
promoter and glyceraldehyde-3-phosphate dehydrogenase. The use of
other viral or mammalian cellular or bacterial phage promoters
which are well known in the art to achieve expression of a coding
sequence of interest is contemplated as well, provided that the
levels of expression are sufficient for a given purpose. By
employing a promoter with well-known properties, the level and
pattern of expression of the protein of interest following
transfection or transformation can be optimized.
[0071] In some circumstances, it is desirable to regulate
expression of a transgene in an immunotherapy vector. For example,
different viral promoters with varying strengths of activity are
utilized depending on the level of expression desired. In mammalian
cells, the CMV immediate early promoter can be used to provide
strong transcriptional activation. Modified versions of the CMV
promoter that are less potent have also been used when reduced
levels of expression of the transgene are desired. When expression
of a transgene in hematopoetic cells is desired, retroviral
promoters such as the LTRs from MLV or MMTV can be used. Other
viral promoters that are used depending on the desired effect
include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters
such as from the E1A, E2A, or MLP region, AAV LTR, HSV-TK, and
avian sarcoma virus.
[0072] Tissue specific promoters sometimes are used to effect
transcription in specific tissues or cells so as to reduce
potential toxicity or undesirable effects to non-targeted tissues.
For example, promoters such as the alpha myosin heavy chain
(.alpha.MHC) promoter, directing expression to cardiac
myocytes.
[0073] In certain indications, it is desirable to activate
transcription at specific times after administration of an
immunotherapy vector. Promoters that are hormone or cytokine
regulatable often are utilized. Cytokine and inflammatory protein
responsive promoters that can be used include K and T Kininogen
(Kageyama et al., 1987), c-fos, TNF-alpha, C-reactive protein
(Arcone et al., 1988), haptoglobin (Oliviero et al., 1987), serum
amyloid A2, C/EBP alpha, IL-1, IL-6 (Poli and Cortese, 1989),
Complement C3 (Wilson et al., 1990), IL-8, alpha-1 acid
glycoprotein (Prowse and Baumann, 1988), alpha-1 antityrpsin,
lipoprotein lipase (Zechner et al., 1988), angiotensinogen (Ron et
al., 1991), fibrinogen, c-jun (inducible by phorbol esters,
TNF-alpha, UV radiation, retinoic acid, and hydrogen peroxide),
collagenase (induced by phorbol esters and retinoic acid),
metallothionein (heavy metal and gluccocorticoid inducible),
Stromelysin (inducible by phorbol ester, interleukin-1 and EGF),
alpha-2 macroglobulin and alpha-1 antichymotrypsin. CID promoters
also can be utilized (Ho et al., 1996; Rivera et al., 1996). Full
citations of certain documents referenced herein are in U.S.
20030144204, published Jul. 31, 2003.
[0074] Other inducible promoters are known and can be utilized. An
ecdysone system (Invitrogen, Carlsbad, Calif.) is one such system.
This system is designed to allow regulated expression of a gene of
interest in mammalian cells. It consists of a tightly regulated
expression mechanism that allows virtually no basal level
expression of the transgene, but over 200-fold inducibility. The
system is based on the heterodimeric ecdysone receptor of
Drosophila, and when ecdysone or an analog such as muristerone A
binds to the receptor, the receptor activates a promoter to turn on
expression of the downstream transgene high levels of mRNA
transcripts are attained. In this system, both monomers of the
heterodimeric receptor are constitutively expressed from one
vector, whereas the ecdysone-responsive promoter, which drives
expression of the gene of interest is on another plasmid.
Engineering of this type of system into the gene transfer vector of
interest would therefore be useful. Cotransfection of plasmids
containing the gene of interest and the receptor monomers in the
producer cell line would then allow for the production of the gene
transfer vector without expression of a potentially toxic
transgene. At the appropriate time, expression of the transgene
could be activated with ecdysone or muristeron A. Another inducible
system is the Tet-Off.TM. or Tet-On.TM. system (Clontech, Palo
Alto, Calif.) originally developed by Gossen and Bujard (Gossen and
Bujard, 1992; Gossen et al., 1995). This system also allows high
levels of gene expression to be regulated in response to
tetracycline or tetracycline derivatives such as doxycycline. In
the Tet-On.TM. system, gene expression is turned on in the presence
of doxycycline, whereas in the Tet-Off.TM. system, gene expression
is turned on in the absence of doxycycline. These systems are based
on two regulatory elements derived from the tetracycline resistance
operon of E. coli. The tetracycline operator sequence to which the
tetracycline repressor binds, and the tetracycline repressor
protein. The gene of interest is cloned into a plasmid behind a
promoter that has tetracycline-responsive elements present in it. A
second plasmid contains a regulatory element called the
tetracycline-controlled transactivator, which is composed, in the
Tet-Off.TM. system, of the VP16 domain from the herpes simplex
virus and the wild-type tertracycline repressor. Thus in the
absence of doxycycline, transcription is constitutively on. In the
Tet-On.TM. system, the tetracycline repressor is not wild type and
in the presence of doxycycline activates transcription. For gene
therapy vector production, the Tet-Offm system would be preferable
so that the producer cells could be grown in the presence of
tetracycline or doxycycline and prevent expression of a potentially
toxic transgene, but when the vector is introduced to the patient,
the gene expression would be induced constitutively.
[0075] It is envisioned that any of the above promoters alone or in
combination with another can be useful according to the present
invention depending on the action desired. In addition, this list
of promoters should not be construed to be exhaustive or limiting,
those of skill in the art will know of other promoters that are
used in conjunction with the promoters and methods disclosed
herein.
[0076] A 5' UTR may comprise one or more elements endogenous to the
nucleotide sequence from which it originates, and sometimes
includes one or more exogenous elements. A 5' UTR can originate
from any suitable nucleic acid, such as genomic DNA, plasmid DNA,
RNA or mRNA, for example, from any suitable organism (e.g., virus,
bacterium, yeast, fungi, plant, insect or mammal). The artisan may
select appropriate elements for the 5' UTR based upon the
transcription and/or translation system being utilized. A 5' UTR
sometimes comprises one or more of the following elements known to
the artisan enhancer sequence (e.g., Eukaryotic Promoter Data Base
EPDB), translational enhancer sequence, transcription initiation
site, transcription factor binding site, translation regulation
site, translation initiation site, translation factor binding site,
ribosome binding site, replicon, enhancer element, internal
ribosome entry site (IRES), and silencer element.
[0077] A 5'UTR in the nucleic acid composition can comprise a
translational enhancer nucleotide sequence. A translational
enhancer nucleotide sequence often is located between the promoter
and the target nucleotide sequence in a nucleic acid composition. A
translational enhancer sequence often binds to a ribosome,
sometimes is an 18S rRNA-binding ribonucleotide sequence (i.e., a
40S ribosome binding sequence) and sometimes is an internal
ribosome entry sequence (IRES). An IRES generally forms an RNA
scaffold with precisely placed RNA tertiary structures that contact
a 40S ribosomal subunit via a number of specific intermolecular
interactions. Examples of ribosomal enhancer sequences are known
and can be identified by the artisan (e.g., Mignone et al., Nucleic
Acids Research 33: D141-D146 (2005); Paulous et al., Nucleic Acids
Research 31: 722-733 (2003); Akbergenov et al., Nucleic Acids
Research 32: 239-247 (2004); Mignone et al., Genome Biology 3(3):
reviews0004.1-0001.10 (2002); Gallie, Nucleic Acids Research 30:
3401-3411 (2002); Shaloiko et al., http address
www.interscience.wiley.com, DOI: 10.1002/bit.20267; and Gallie et
al., Nucleic Acids Research 15: 3257-3273 (1987)). A translational
enhancer sequence sometimes is a eukaryotic sequence, such as a
Kozak consensus sequence or other sequence (e.g., hydroid polyp
sequence, GenBank accession no. U07128). A translational enhancer
sequence sometimes is a prokaryotic sequence, such as a
Shine-Dalgarno consensus sequence. In certain embodiments, the
translational enhancer sequence is a viral nucleotide sequence. A
translational enhancer sequence sometimes is from a 5'UTR of a
plant virus, such as Tobacco Mosaic Virus (TMV), Alfalfa Mosaic
Virus (AMV); Tobacco Etch Virus (ETV); Potato Virus Y (PVY); Turnip
Mosaic (poty) Virus and Pea Seed Borne Mosaic Virus, for example.
In certain embodiments, an omega sequence about 67 bases in length
from TMV is included in the nucleic acid composition as a
translational enhancer sequence (e.g., devoid of guanosine
nucleotides and includes a 25 nucleotide long poly (CAA) central
region). In some embodiments, a translational enhancer sequence
comprises one or more ARC-1 or ARC-1 like sequence, such as one of
the following nucleotide sequences GCCGGCGGAG, CUCAUAAGGU,
GACUUUGAUU, CGGAACCCAA, AUACUCCCCC and CCUUGCGACC, or a
substantially identical sequence thereof. In certain embodiments, a
translational enhancer sequence comprises an IRES sequence, such as
one or more of EMBL nucleotide sequences J04513, X87949, M95825,
M12783, AF025841, AF013263, AF006822, M17169, M13440, M22427,
D14838 and M17446, or a substantially identical nucleotide sequence
thereof. An IRES sequence may be a type I IRES (e.g., from
enterovirus (e.g., poliovirus), rhinovirus (e.g., human
rhinovirus)), a type II IRES (e.g., from cardiovirus (e.g.,
encephalomyocraditis virus), aphthovirus (e.g., foot-and-mouth
disease virus)), a type III IRES (e.g., from Hepatitis A virus) or
other picornavirus sequence (e.g., Paulos et al. supra, and Jackson
et al., RNA 1: 985-1000 (1995)).
[0078] A 3' UTR may comprise one or more elements endogenous to the
nucleotide sequence from which it originates and sometimes includes
one or more exogenous elements. A 3' UTR may originate from any
suitable nucleic acid, such as genomic DNA, plasmid DNA, RNA or
mRNA, for example, from any suitable organism (e.g., a virus,
bacterium, yeast, fungi, plant, insect or mammal). The artisan can
select appropriate elements for the 3' UTR based upon the
transcription and/or translation system being utilized. A 3' UTR
sometimes comprises one or more of the following elements known to
the artisan: transcription regulation site, transcription
initiation site, transcription termination site, transcription
factor binding site, translation regulation site, translation
termination site, translation initiation site, translation factor
binding site, ribosome binding site, replicon, enhancer element,
silencer element and polyadenosine tail. A 3' UTR can include a
polyadenosine tail, and sometimes may not. If a polyadenosine tail
is present, one or more adenosine moieties may be added or deleted
from the native length (e.g., about 5, about 10, about 15, about
20, about 25, about 30, about 35, about 40, about 45 or about 50
adenosine moieties may be added or subtracted).
[0079] The term a "target nucleotide sequence" as used herein
encodes a nucleic acid, peptide, polypeptide or protein of
interest, and may be a ribonucleotide sequence or a
deoxyribonucleotide sequence. An Akt nucleotide sequence (e.g., a
chimeric sequence encoding an Akt sequence and a
membrane-association sequence) may be incorporated into a nucleic
acid composition as a target nucleotide sequence. The term "nucleic
acid" as used herein is generic to polydeoxyribonucleotides
(containing 2'-deoxy-D-ribose or modified forms thereof), to
polyribonucleotides (containing D-ribose or modified forms
thereof), and to any other type of polynucleotide which is an
N-glycoside of a purine or pyrimidine bases, or modified purine or
pyrimidine bases. A target nucleic acid can include an untranslated
ribonucleic acid and sometimes is a translated ribonucleic acid. An
untranslated ribonucleic acid may include, but is not limited to, a
small interfering ribonucleic acid (siRNA), a short hairpin
ribonucleic acid (shRNA), other ribonucleic acid capable of RNA
interference (RNAi), an antisense ribonucleic acid, or a ribozyme.
A translatable target nucleotide sequence (e.g., a target
ribonucleotide sequence) sometimes encodes a peptide, polypeptide
or protein, which are sometimes referred to herein as "target
peptides," "target polypeptides" or "target proteins." The term
"protein" as used herein refers to a molecule having a sequence of
amino acids linked by peptide bonds. This term includes fusion
proteins, oligopeptides, polypeptides, cyclic peptides,
polypeptides and polypeptide derivatives. A protein or polypeptide
sometimes is of intracellular origin (e.g., located in the nucleus,
cytosol, or interstitial space of host cells in vivo) and sometimes
is a cell membrane protein in vivo.
[0080] A translatable nucleotide sequence generally is located
between a start codon (AUG in ribonucleic acids and ATG in
deoxyribonucleic acids) and a stop codon (e.g., UAA (ochre), UAG
(amber) or UGA (opal) in ribonucleic acids and TAA, TAG or TGA in
deoxyribonucleic acids), and sometimes is referred to herein as an
"open reading frame" (ORF). A nucleic acid composition sometimes
comprises one or more ORFs. An ORF may be from any suitable source,
sometimes from genomic DNA, mRNA, reverse transcribed RNA or
complementary DNA (cDNA) or a nucleic acid library comprising one
or more of the foregoing, and is from any organism species, such as
human, insect, nematode, bovine, equine, canine, feline, rat or
mouse, for example. An Akt nucleotide sequence often is utilized as
an ORF herein, and sometimes a membrane association region-encoding
nucleotide sequence is utilized as an ORF.
[0081] A nucleic acid composition sometimes comprises a nucleotide
sequence adjacent to an ORF that is translated in conjunction with
the ORF and encodes an amino acid tag. The tag-encoding nucleotide
sequence is located 3' and/or 5' of an ORF in the nucleic acid
composition, thereby encoding a tag at the C-terminus or N-terminus
of the protein or peptide encoded by the ORF. Any tag that does not
abrogate or substantially reduce transcription and/or translation
may be utilized and may be appropriately selected by the artisan. A
tag sometimes specifically binds a molecule or moiety of a solid
phase or a detectable label, for example, thereby having utility
for isolating, purifying and/or detecting a protein or peptide
encoded by the ORF. In some embodiments, a tag comprises one or
more of the following elements: FLAG (e.g., DYKDDDDKG), AU1 (e.g.,
DTYRYI), V5 (e.g., GKPIPNPLLGLDST), c-MYC (e.g., EQKLISEEDL), HSV
(e.g., QPELAPEDPED), influenza hemaglutinin, HA (e.g., YPYDVPDYA),
VSV-G (e.g., YTDIEMNRLGK), bacterial glutathione-S-transferase,
maltose binding protein, a streptavidin- or avidin-binding tag
(e.g., pcDNA.TM.6 BioEase.TM. Gateway.RTM. Biotinylation System
(Invitrogen)), thioredoxin, .beta.-galactosidase, VSV-glycoprotein,
a fluorescent protein (e.g., green fluorescent protein or one of
its many color variants (e.g., yellow, red, blue)), a polylysine or
polyarginine sequence, a polyhistidine sequence (e.g., His.sub.6)
or other sequence that chelates a metal (e.g., cobalt, zinc,
copper) and/or a cysteine-rich sequence that binds to an
arsenic-containing molecule. In certain embodiments, a
cysteine-rich tag comprises the amino acid sequence CC-X.sub.n-CC,
wherein X is any amino acid and n is 1 to 3, and the cysteine-rich
sequence sometimes is CCPGCC. In certain embodiments, the tag
comprises a cysteine-rich element and a polyhistidine element
(e.g., CCPGCC and His.sub.6).
[0082] A tag often conveniently binds to a binding partner. For
example, some tags bind to an antibody (e.g., FLAG) and sometimes
specifically bind to a small molecule. For example, a polyhistidine
tag specifically chelates a bivalent metal, such as copper, zinc
and cobalt; a polylysine or polyarginine tag specifically binds to
a zinc finger; a glutathione S-transferase tag binds to
glutathione; and a cysteine-rich tag specifically binds to an
arsenic-containing molecule. Arsenic-containing molecules include
LUMIO.TM. agents (Invitrogen, California), such as FIAsH.TM.
(EDT.sub.2[4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2--
ethanedithiol).sub.2]) and ReAsH reagents (e.g., U.S. Pat. No.
5,932,474 to Tsien et al., entitled "Target Sequences for Synthetic
Molecules;" U.S. Pat. No. 6,054,271 to Tsien et al., entitled
"Methods of Using Synthetic Molecules and Target Sequences;" U.S.
Pat. Nos. 6,451,569 and 6,008,378; published U.S. Patent
Application 2003/0083373, and published PCT Patent Application WO
99/21013, all to Tsien et al. and all entitled "Synthetic Molecules
that Specifically React with Target Sequences"). Such antibodies
and small molecules sometimes are linked to a solid phase for
convenient isolation of the target protein or target peptide, as
described in greater detail hereafter.
[0083] A tag sometimes comprises a sequence that localizes a
translated protein or peptide to a component in a system, which is
referred to as a "signal sequence" or "localization signal
sequence" herein. A signal sequence often is incorporated at the
N-terminus of a target protein or target peptide, and sometimes is
incorporated at the C-terminus. Examples of signal sequences are
known to the artisan, are readily incorporated into a nucleic acid
composition, and often are selected according to the cells from
which a cell-free extract is prepared. A signal sequence in some
embodiments localizes a translated protein or peptide to a cell
membrane. Examples of signal sequences include, but are not limited
to, a nucleus targeting signal (e.g., steroid receptor sequence and
N-terminal sequence of SV40 virus large T antigen); mitochondia
targeting signal (e.g., amino acid sequence that forms an
amphipathic helix); peroxisome targeting signal (e.g., C-terminal
sequence in YFG from S. cerevisiae); and a secretion signal (e.g.,
N-terminal sequences from invertase, mating factor alpha, PHO5 and
SUC2 in S. cerevisiae; multiple N-terminal sequences of B. subtilis
proteins (e.g., Tjalsma et al., Microbiol. Molec. Biol. Rev. 64:
515-547 (2000)); alpha amylase signal sequence (e.g., U.S. Pat. No.
6,288,302); pectate lyase signal sequence (e.g., U.S. Pat. No.
5,846,818); precollagen signal sequence (e.g., U.S. Pat. No.
5,712,114); OmpA signal sequence (e.g., U.S. Pat. No. 5,470,719);
lam beta signal sequence (e.g., U.S. Pat. No. 5,389,529); B. brevis
signal sequence (e.g., U.S. Pat. No. 5,232,841); and P. pastoris
signal sequence (e.g., U.S. Pat. No. 5,268,273)).
[0084] A tag sometimes is directly adjacent to the amino acid
sequence encoded by an ORF (i.e., there is no intervening sequence)
and sometimes a tag is substantially adjacent to a the ORF encoded
amino acid sequence (e.g., an intervening sequence is present). An
intervening sequence sometimes includes a recognition site for a
protease, which is useful for cleaving a tag from a target protein
or peptide. In some embodiments, the intervening sequence is
cleaved by Factor Xa (e.g., recognition site I(E/D)GR), thrombin
(e.g., recognition site LVPRGS), enterokinase (e.g., recognition
site DDDDK), TEV protease (e.g., recognition site ENLYFQG) or
PreScission.TM. protease (e.g., recognition site LEVLFQGP), for
example.
[0085] An intervening sequence sometimes is referred to herein as a
"linker sequence," and may be of any suitable length selected by
the artisan. A linker sequence sometimes is about 1 to about 20
amino acids in length, and sometimes about 5 to about 10 amino
acids in length. The artisan may select the linker length to
substantially preserve target protein or peptide function (e.g., a
tag may reduce target protein or peptide function unless separated
by a linker), to enhance disassociation of a tag from a target
protein or peptide when a protease cleavage site is present (e.g.,
cleavage may be enhanced when a linker is present), and to enhance
interaction of a tag/target protein product with a solid phase. A
linker can be of any suitable amino acid content, and often
comprises a higher proportion of amino acids having relatively
short side chains (e.g., glycine, alanine, serine and
threonine).
[0086] A nucleic acid composition sometimes includes a stop codon
between a tag element and an insertion element or ORF, which can be
useful for translating an ORF with or without the tag. Mutant tRNA
molecules that recognize stop codons (described above) suppress
translation termination and thereby are designated "suppressor
tRNAs." Suppressor tRNAs can result in the insertion of amino acids
and continuation of translation past stop codons (e.g., U.S. Patent
Application No. 60/587,583, filed Jul. 14, 2004, entitled
"Production of Fusion Proteins by Cell-Free Protein Synthesis,";
Eggertsson, et al., (1988) Microbiological Review 52(3):354-374,
and Engleerg-Kukla, et al. (1996) in Escherichia coli and
Salmonella Cellular and Molecular Biology, Chapter 60, pps 909-921,
Neidhardt, et al. eds., ASM Press, Washington, D.C.). A number of
suppressor tRNAs are known, including but not limited to, supE,
supP, supD, supF and supZ suppressors, which suppress the
termination of translation of the amber stop codon; supB, glT,
supL, supN, supC and supM suppressors, which suppress the function
of the ochre stop codon and glyT, trpT and Su-9 suppressors, which
suppress the function of the opal stop codon. In general,
suppressor tRNAs contain one or more mutations in the anti-codon
loop of the tRNA that allows the tRNA to base pair with a codon
that ordinarily functions as a stop codon. The mutant tRNA is
charged with its cognate amino acid residue and the cognate amino
acid residue is inserted into the translating polypeptide when the
stop codon is encountered. Mutations that enhance the efficiency of
termination suppressors (i.e., increase stop codon read-through)
have been identified. These include, but are not limited to,
mutations in the uar gene (also known as the prfA gene), mutations
in the ups gene, mutations in the sueA, sueB and sueC genes,
mutations in the rpsD (ramA) and rpsE (spcA) genes and mutations in
the rpIL gene.
[0087] Thus, a nucleic acid composition comprising a stop codon
located between an ORF and a tag can yield a translated ORF alone
when no suppressor tRNA is present in the translation system, and
can yield a translated ORF-tag fusion when a suppressor tRNA is
present in the system. In some embodiments, the stop codon is
located 3' of an insertion element or ORF and 5' of a tag, and the
stop codon sometimes is an amber codon. Suppressor tRNA sometimes
are within a cell-free extract (e.g., the cell-free extract is
prepared from cells that produce the suppressor tRNA), sometimes
are added to the cell-free extract as isolated molecules, and
sometimes are added to a cell-free extract as part of another
extract. A provided suppressor tRNA sometimes is loaded with one of
the twenty naturally occurring amino acids or an unnatural amino
acid (described herein). Suppressor tRNA can be generated in cells
transfected with a nucleic acid encoding the tRNA (e.g., a
replication incompetent adenovirus containing the human tRNA-Ser
suppressor gene can be transfected into cells). Vectors for
synthesizing suppressor tRNA and for translating ORFs with or
without a tag are available to the artisan (e.g., Tag-On-Demand.TM.
kit (Invitrogen Corporation, California); Tag-On-Demand.TM.
Suppressor Supernatant Instruction Manual, Version B, 6 Jun. 2003,
at http address
www.invitrogen.com/content/sfs/manuals/tagondemand_supernatant_man.pdf;
Tag-On-Demand.TM. Gateway.RTM. Vector Instruction Manual, Version
B, 20 Jun., 2003 at http address
www.invitrogen.com/content/sfs/manuals/tagondemandvectors_man.pdf;
and Capone et al., Amber, ochre and opal suppressor tRNA genes
derived from a human serine tRNA gene. EMBO J. 4:213, 1985).
[0088] Any convenient cloning strategy known to the artisan may be
utilized to incorporate an element, such as an ORF, into a nucleic
acid composition. Known methods can be utilized to insert an
element into the template independent of an insertion element, such
as (1) cleaving the template at one or more existing restriction
enzyme sites and ligating an element of interest and (2) adding
restriction enzyme sites to the template by hybridizing
oligonucleotide primers that include one or more suitable
restriction enzyme sites and amplifying by polymerase chain
reaction (described in greater detail herein). Other cloning
strategies take advantage of one or more insertion sites present or
inserted into the nucleic acid composition, such as an
oligonucleotide primer hybridization site for PCR, for example, and
others described hereafter.
[0089] In some embodiments, the nucleic acid composition includes
one or more recombinase insertion sites. A recombinase insertion
site is a recognition sequence on a nucleic acid molecule that
participates in an integration/recombination reaction by
recombination proteins. For example, the recombination site for Cre
recombinase is loxP, which is a 34 base pair sequence comprised of
two 13 base pair inverted repeats (serving as the recombinase
binding sites) flanking an 8 base pair core sequence (e.g., FIG. 1
of Sauer, B., Curr. Opin. Biotech. 5:521-527 (1994)). Other
examples of recombination sites include attB, attP, attL, and attR
sequences, and mutants, fragments, variants and derivatives
thereof, which are recognized by the recombination protein .lamda.
Int and by the auxiliary proteins integration host factor (IHF),
FIS and excisionase (Xis) (e.g., U.S. Pat. Nos. 5,888,732;
6,143,557; 6,171,861; 6,270,969; 6,277,608; and 6,720,140; U.S.
patent application Ser. No. 09/517,466, filed Mar. 2, 2000, and
09/732,914, filed Aug. 14, 2003, and in U.S. patent publication no.
2002-0007051-A1; Landy, Curr. Opin. Biotech. 3:699-707 (1993)).
Examples of recombinase cloning nucleic acids are in Gateway.RTM.
systems (Invitrogen, California), which include at least one
recombination site for cloning a desired nucleic acid molecules in
vivo or in vitro. In some embodiments, the system utilizes vectors
that contain at least two different site-specific recombination
sites, often based on the bacteriophage lambda system (e.g., att1
and att2), and are mutated from the wild-type (att0) sites. Each
mutated site has a unique specificity for its cognate partner att
site (i.e., its binding partner recombination site) of the same
type (for example attB1 with attP1, or attL1 with attR1) and will
not cross-react with recombination sites of the other mutant type
or with the wild-type att0 site. Different site specificities allow
directional cloning or linkage of desired molecules thus providing
desired orientation of the cloned molecules. Nucleic acid fragments
flanked by recombination sites are cloned and subcloned using the
Gateway.RTM. system by replacing a selectable marker (for example,
ccdB) flanked by att sites on the recipient plasmid molecule,
sometimes termed the Destination Vector. Desired clones are then
selected by transformation of a ccdB sensitive host strain and
positive selection for a marker on the recipient molecule. Similar
strategies for negative selection (e.g., use of toxic genes) can be
used in other organisms such as thymidine kinase (TK) in mammals
and insects.
[0090] In certain embodiments, the nucleic acid composition
includes one or more topoisomerase insertion sites. A topoisomerase
insertion site is a defined nucleotide sequence recognized and
bound by a site-specific topoisomerase. For example, the nucleotide
sequence 5'-(C/T)CCTT-3' is a topoisomerase recognition site bound
specifically by most poxvirus topoisomerases, including vaccinia
virus DNA topoisomerase I. After binding to the recognition
sequence, the topoisomerase cleaves the strand at the 3'-most
thymidine of the recognition site to produce a nucleotide sequence
comprising 5'-(C/T)CCTT-PO.sub.4-TOPO, a complex of the
topoisomerase covalently bound to the 3' phosphate via a tyrosine
in the topoisomerase (e.g., Shuman, J. Biol. Chem. 266:11372-11379,
1991; Sekiguchi and Shuman, Nucl. Acids Res. 22:5360-5365, 1994;
U.S. Pat. No. 5,766,891; PCT/US95/16099; and PCT/US98/12372). In
comparison, the nucleotide sequence 5'-GCAACTT-3' is a
topoisomerase recognition site for type IA E. coli topoisomerase
III. An element to be inserted often is combined with
topoisomerase-reacted template and thereby incorporated into the
nucleic acid composition (e.g., http address
www.invitrogen.com/downloads/F-13512_Topo_Flyer.pdf; http address
at
www.invitrogen.com/content/sfs/brochures/710.sub.--021849%20_B_TOPOClonin-
g_bro.pdf; TOPO TA Cloning.RTM. Kit and Zero Blunt.RTM. TOPO.RTM.
Cloning Kit product information).
[0091] A nucleic acid composition sometimes contains one or more
origin of replication (ORI) elements. In some embodiments, a
template comprises two or more ORIs, where one functions
efficiently in one organism (e.g., a bacterium) and another
functions efficiently in another organism (e.g., a eukaryote). In
some embodiments, an ORI may function efficiently in insect cells
and another ORI may function efficiently in mammalian cells. A
nucleic acid composition also sometimes includes one or more
transcription regulation sites.
[0092] A nucleic acid composition often includes one or more
selection elements. Selection elements often are utilized using
known processes to determine whether a nucleic acid composition is
included in a cell. In some embodiments, a nucleic acid composition
includes two or more selection elements, where one functions
efficiently in one organisms and another functions efficiently in
another organism. Examples of selection elements include, but are
not limited to, (1) nucleic acid segments that encode products that
provide resistance against otherwise toxic compounds (e.g.,
antibiotics); (2) nucleic acid segments that encode products that
are otherwise lacking in the recipient cell (e.g., essential
products, tRNA genes, auxotrophic markers); (3) nucleic acid
segments that encode products that suppress the activity of a gene
product; (4) nucleic acid segments that encode products that can be
readily identified (e.g., phenotypic markers such as antibiotics
(e.g., .beta.-lactamase), .beta.-galactosidase, green fluorescent
protein (GFP), yellow fluorescent protein (YFP), red fluorescent
protein (RFP), cyan fluorescent protein (CFP), and cell surface
proteins); (5) nucleic acid segments that bind products that are
otherwise detrimental to cell survival and/or function; (6) nucleic
acid segments that otherwise inhibit the activity of any of the
nucleic acid segments described in Nos. 1-5 above (e.g., antisense
oligonucleotides); (7) nucleic acid segments that bind products
that modify a substrate (e.g., restriction endonucleases); (8)
nucleic acid segments that can be used to isolate or identify a
desired molecule (e.g., specific protein binding sites); (9)
nucleic acid segments that encode a specific nucleotide sequence
that can be otherwise non-functional (e.g., for PCR amplification
of subpopulations of molecules); (10) nucleic acid segments that,
when absent, directly or indirectly confer resistance or
sensitivity to particular compounds; (11) nucleic acid segments
that encode products that either are toxic (e.g., Diphtheria toxin)
or convert a relatively non-toxic compound to a toxic compound
(e.g., Herpes simplex thymidine kinase, cytosine deaminase) in
recipient cells; (12) nucleic acid segments that inhibit
replication, partition or heritability of nucleic acid molecules
that contain them; and/or (13) nucleic acid segments that encode
conditional replication functions, e.g., replication in certain
hosts or host cell strains or under certain environmental
conditions (e.g., temperature, nutritional conditions, and the
like).
[0093] Certain nucleotide sequences sometimes are added to,
modified or removed from one or more of the nucleic acid
composition elements, such as the promoter, 5'UTR, target sequence,
or 3'UTR elements, to enhance or potentially enhance transcription
and/or translation before or after such elements are incorporated
in a nucleic acid composition. In some embodiments, one or more of
the following sequences may be modified or removed if they are
present in a 5'UTR: a sequence that forms a stable secondary
structure (e.g., quadruplex structure or stem loop stem structure
(e.g., EMBL sequences X12949, AF274954, AF139980, AF152961, S95936,
U194144, AF116649 or substantially identical sequences that form
such stem loop stem structures)); a translation initiation codon
upstream of the target nucleotide sequence start codon; a stop
codon upstream of the target nucleotide sequence translation
initiation codon; an ORF upstream of the target nucleotide sequence
translation initiation codon; an iron responsive element (IRE) or
like sequence; and a 5' terminal oligopyrimidine tract (TOP, e.g.,
consisting of 5-15 pyrimidines adjacent to the cap). A
translational enhancer sequence and/or an internal ribosome entry
site (IRES) sometimes is inserted into a 5'UTR (e.g., EMBL
nucleotide sequences J04513, X87949, M95825, M12783, AF025841,
AF013263, AF006822, M17169, M13440, M22427, D14838 and M17446 and
substantially identical nucleotide sequences). An AU-rich element
(ARE, e.g., AUUUA repeats) and/or splicing junction that follows a
non-sense codon sometimes is removed from or modified in a 3'UTR. A
polyadenosine tail sometimes is inserted into a 3'UTR if none is
present, sometimes is removed if it is present, and adenosine
moieties sometimes are added to or removed from a polyadenosine
tail present in a 3'UTR. Thus, some embodiments are directed to a
process comprising: determining whether any nucleotide sequences
that reduce or potentially reduce translation efficiency are
present in the elements, and removing or modifying one or more of
such sequences if they are identified. Certain embodiments are
directed to a process comprising: determining whether any
nucleotide sequences that increase or potentially increase
translation efficiency are not present in the elements, and
incorporating such sequences into the nucleic acid composition.
[0094] An ORF sometimes is mutated or modified (for example, by
point mutation, deletion mutation, insertion mutation, and the
like) to alter, enhance or increase, reduce, substantially reduce
or eliminate the activity of the encoded protein or peptide. The
protein or peptide encoded by a modified ORF sometimes is produced
in a lower amount or may not be produced at detectable levels, and
in other embodiments, the product or protein encoded by the
modified ORF is produced at a higher level (e.g., codons sometimes
are modified so they are compatible with tRNA in cells used to
prepare a cell-free extract). To determine the relative activity,
the activity from the product of the mutated ORF (or cell
containing it) can be compared to the activity of the product or
protein encoded by the unmodified ORF (or cell containing it).
[0095] A stop codon at the end of an ORF sometimes is modified to
another stop codon, such as an amber stop codon described above. In
some embodiments, a stop codon is introduced within an ORF,
sometimes by insertion or mutation of an existing codon. An ORF
comprising a modified terminal stop codon and/or internal stop
codon often is translated in a system comprising a suppressor tRNA
that recognizes the stop codon. An ORF comprising a stop codon
sometimes is translated in a system comprising a suppressor tRNA
that incorporates an unnatural amino acid during translation of the
target protein or target peptide. Methods for incorporating
unnatural amino acids into a target protein or peptide are known,
which include, for example, processes utilizing a heterologous
tRNA/synthetase pair, where the tRNA recognizes an amber stop codon
and is loaded with an unnatural amino acid (e.g., http address
www.iupac.org/news/prize/2003/wang.pdf). Examples of unnatural
amino acids are described above.
[0096] A nucleic acid composition is of any form useful for in
vitro or in vivo transcription and/or translation. A nucleic acid
sometimes is a plasmid, such as a supercoiled plasmid, sometimes is
a linear nucleic acid (e.g., a linear nucleic acid produced by PCR
or by restriction digest), sometimes is single-stranded and
sometimes is double-stranded. A nucleic acid composition for
transcription and/or translation can be prepared by any suitable
process. A nucleic acid composition sometimes is prepared by an
amplification process, such as a polymerase chain reaction (PCR)
process or transcription-mediated amplification process (TMA). In
TMA, two enzymes are used in an isothermal reaction to produce
amplification products detected by light emission (see, e.g.,
Biochemistry 1996 Jun. 25; 35(25):8429-38 and http address
www.devicelink.com/ivdt/archive/00/11/007.html). Standard PCR
processes are known (e.g., U.S. Pat. Nos. 4,683,202; 4,683,195;
4,965,188; and 5,656,493), and generally are performed in cycles.
Each cycle includes heat denaturation, in which hybrid nucleic
acids dissociate; cooling, in which primer oligonucleotides
hybridize; and extension of the oligonucleotides by a polymerase
(i.e., Taq polymerase). An example of a PCR cyclical process is
treating the sample at 95.degree. C. for 5 minutes; repeating
forty-five cycles of 95.degree. C. for 1 minute, 59.degree. C. for
1 minute, 10 seconds, and 72.degree. C. for 1 minute 30 seconds;
and then treating the sample at 72.degree. C. for 5 minutes.
Multiple cycles frequently are performed using a commercially
available thermal cycler. PCR amplification products sometimes are
stored for a time at a lower temperature (e.g., at 4.degree. C.)
and sometimes are frozen (e.g., at -20.degree. C.) before
analysis.
[0097] In some embodiments, a nucleic acid of other molecule
described herein is isolated or purified. The term "isolated" as
used herein refers to material removed from its original
environment (e.g., the natural environment if it is naturally
occurring, or a host cell if expressed exogenously), and thus is
altered "by the hand of man" from its original environment. The
term "purified" as used herein with reference to molecules does not
refer to absolute purity. Rather, "purified" refers to a substance
in a composition that contains fewer substance species in the same
class (e.g., nucleic acid or protein species) other than the
substance of interest in comparison to the sample from which it
originated. "Purified," if a nucleic acid or protein for example,
refers to a substance in a composition that contains fewer nucleic
acid species or protein species other than the nucleic acid or
protein of interest in comparison to the sample from which it
originated. Sometimes, a protein or nucleic acid is "substantially
pure," indicating that the protein or nucleic acid represents at
least 50% of protein or nucleic acid on a mass basis of the
composition. Often, a substantially pure protein or nucleic acid is
at least 75% on a mass basis of the composition, and sometimes at
least 95% on a mass basis of the composition.
[0098] Other nucleotide sequences not specifically described herein
can be included in nucleic acid compositions, as selected for an
application of the nucleic acid composition by the person of
ordinary skill in the art. Certain sequences described in the
section below entitled "Nucleic Acid Transfer to Antigen Presenting
Cells," which include viral nucleotide sequences, sometimes are
included in nucleic acid compositions.
[0099] Antigen Presenting Cells
[0100] Any antigen presenting cell (APC) can be used with the
methods of the present invention. The term "APC" encompasses any
cell capable of handling and presenting an antigen to lymphocytes.
Typically, APCs include, e.g., macrophages, Langerhans dendritic
cells and Follicular dendritic cells. In addition, B cells have
also been shown to have an antigen presenting function and are thus
contemplated by the present invention. The APCs often are dendritic
cells. A "dendritic cell" (DC) is an APC with a characteristic
morphology including lamellipodia extending from the dendritic cell
body in several directions. Dendritic cells are able to initiate
primary, antigen-specific T cell responses both in vitro and in
vivo, and direct a strong mixed leukocyte reaction (MLR) compared
to peripheral blood leukocytes, splenocytes, B cells and monocytes.
DCs can be derived from a number of different hematopoietic
precursor cells. For a general description of dendritic cells,
including their differentiation and maturation, see, e.g. Steinman,
Annu Rev Immunol. 9:271-96 (1991), and Lotze and Thomson, Dendritic
Cells, 2nd Edition, Academic Press, 2001.
[0101] APCs can be isolated from any of the tissues where they
reside and which are known to those of skill in the art. In
particular, dendritic cells and their progenitors may be obtained
from any tissue source comprising dendritic cell precursors that
are capable of proliferating and maturing in vitro into dendritic
cells, when cultured and induced to mature according to the methods
of the present invention. Such suitable tissue sources include,
e.g., peripheral blood, bone marrow, tumor-infiltrating cells,
peritumoral tissues-infiltrating cells, lymph node biopsies,
thymus, spleen, skin, umbilical cord blood, monocytes harvested
from peripheral blood, CD34 or CD14 positive cells harvested from
peripheral blood, blood marrow or any other suitable tissue or
fluid. Dendritic cells sometimes are isolated from bone marrow or
from peripheral blood mononuclear cells (PBMCs).
[0102] Peripheral blood can be collected using any standard
apheresis procedure known in the art (see, e.g., Bishop et al.,
Blood 83:610:616 (1994)). PBMCs can then be prepared from whole
blood samples by separating mononuclear cells from red blood cells.
There are a number of methods for isolating PBMCs including, e.g.,
velocity sedimentation, isopyknic sedimentation, affinity
purification, and flow cytometry. Typically, PBMCs are separated
from red blood cells by density gradient (isopyknic)
centrifugation, in which the cells sediment to an equilibrium
position in the solution equivalent to their own density. For
density gradient centrifugation, physiological media should be
used, the density of the solution should be high, and the media
should exert little osmotic pressure. Density gradient
centrifugation uses solutions such as sodium
ditrizoate-polysucrose, Ficoll, dextran, and Percoll (see, e.g.,
Freshney, Culture of Animal Cells, 3rd ed. (1994)). Such solutions
are commercially available, e.g., HISTOPAQUE.RTM. (Sigma). Examples
of methods for isolating dendritic cells from PBMCs are disclosed
in, e.g., U.S. Pat. Nos. 6,017,527 and 5,851,756; and in O'Doherty
et al., J. Exp. Med. 178:1067-1078 (1993); Young and Steinman, J.
Exp. Med. 171:1315-1332 (1990); Freudenthal and Steinman, Proc.
Natl. Acad. Sci. USA 57:7698-7702 (1990); Macatonia et al., Immunol
67: 285-289 (1989); and Markowicz and Engleman, J. Clin. Invest.
85:955-961 (1990).
[0103] CD34+ PBMCs or CD14+ PBMCs can further be selected as a
source of dendritic cells using a variety of selection techniques
known to those of skill in the art. For example, monoclonal
antibodies (or any protein-specific binding protein) can be used to
bind to a cell surface antigen found on the surface of the PBMC
sub-population of interest (e.g., CD34 or CD14 on the surface of
CD34+ or CD 14+ PBMCs, respectively). Binding of such specific
monoclonal antibodies allows the identification and isolation of
the sub-group of PBMCs of interest from a total PBMC population by
any of a number of immunoaffinity methods known to those of skill
in the art. Examples of immunoaffinity methods for isolating
sub-populations of PBMCs are described in, e.g., U.S. Pat. No.
6,017,527.
[0104] Alternatively, the dendritic cells of the present invention
can be isolated from bone marrow. For a general description of
methods for isolating dendritic cells from bone marrow see, e.g.,
U.S. Pat. No. 5,994,126; Dexter et al., in Long-Term Bone Marrow
Culture, pages 57-96, Alan R. Liss, (1984); and Lutz et al., J.
Immunol. Methods 223:77-92 (1999). Dendritic cells from bone marrow
can typically be obtained from a number of different sources,
including, for example, from aspirated marrow. Alternatively, bone
marrow can be extracted from a sacrificed animal by dissecting out
the femur, removing soft tissue from the bone and removing the bone
marrow with a needle and syringe. Dendritic cells can be identified
among the different cell types present in the bone marrow based on
their morphological characteristics. For example, cultured immature
dendritic cells in one or more phases of their development are
loosely adherent to plastic, flattening out with a stellate
shape.
[0105] Optionally, prior to culturing the cells, the tissue source
can be pre-treated to remove cells that may compete with the
proliferation and/or the survival of the dendritic cells or of
their precursors. Examples of such pre-treatments are described,
e.g., in U.S. Pat. No. 5,994,126.
[0106] Those of skill in the art will recognize that APCs can be
cultured for any suitable amount of time. Antigen presenting cells
often are cultured from 4 to 15 days, can be cultured for 10-12
days (Lutz et al., supra) and may be cultured for 5-7 days (Inaba
et al., J. Exp. Med. 176:1693 (1992); Inaba et al., J. Exp. Med.
175:1157 (1992); Inaba et al., Current Protocols Immunol., Unit 3.7
(Coico et al., eds. 1998); Schneider et al., J. Immunol. Meth.
154:253 (1992)). Examples of cell culture conditions for dendritic
cells and dendritic cell precursors are known to the person of
ordinary skill in the art and are described in, e.g., U.S. Pat.
Nos. 6,017,527 and 5,851,756; Inaba et al., J. Exp. Med. 176:1693
(1992); Inaba et al., J. Exp. Med. 175:1157 (1992); Inaba et al.,
Current Protocols Immunol., Unit 3.7 (Coico et al., eds. 1998);
Schneider et al., J. Immunol. Meth. 154:253 (1992); and Lutz et
al., supra.
[0107] In general, a cell culture environment includes
consideration of such factors as the substrate for cell growth,
cell density and cell contract, the gas phase, the medium, the
temperature, and the presence of growth factors (see, e.g.,
Freshney et al., Culture of Animal Cells, 3rd ed. (1994)). Cells
can be grown under conditions that provide for cell to cell
contact, and may be grown in suspension as three dimensional
aggregates. Suspension cultures can be achieved by using, e.g., a
flask with a magnetic stirrer or a large surface area paddle, or on
a plate that has been coated to prevent the cells from adhering to
the bottom of the dish. For example, the cells may be grown in
Costar dishes that have been coated with a hydrogel to prevent them
from adhering to the bottom of the dish. For cells that grow in a
monolayer attached to a substrate, plastic dishes, flasks, roller
bottles, or microcarriers are typically used. Other artificial
substrates can be used such as glass and metals. The substrate is
often treated by etching, or by coating with substances such as
collagen, chondronectin, fibronectin, laminin or poly-L-lysine. The
type of culture vessel depends on the culture conditions, e.g.,
multi-well plates, petri dishes, tissue culture tubes, flasks,
roller bottles, microcarriers, and the like. Cells are grown at
optimal densities that are determined empirically based on the cell
type.
[0108] Important constituents of the gas phase are oxygen and
carbon dioxide. Typically, atmospheric oxygen tensions are used for
dendritic cell cultures. Culture vessels are usually vented into
the incubator atmosphere to allow gas exchange by using gas
permeable caps or by preventing sealing of the culture vessels.
Carbon dioxide plays a role in pH stabilization, along with buffer
in the cell media, and is typically present at a concentration of
1-10% in the incubator. The CO.sub.2 concentration for dendritic
cell cultures often is 5%.
[0109] Cultured cells are normally grown in an incubator that
provides a suitable temperature, e.g., the body temperature of the
animal from which is the cells were obtained, accounting for
regional variations in temperature. Generally, 37.degree. C. often
is the temperature for dendritic cell culture. Most incubators are
humidified to approximately atmospheric conditions.
[0110] Defined cell media are available as packaged, premixed
powders or presterilized solutions. Examples of commonly used media
include Iscove's media, RPMI 1640, DMEM, and McCoy's Medium (see,
e.g., GibcoBRL/Life Technologies Catalogue and Reference Guide;
Sigma Catalogue). Defined cell culture media are often supplemented
with 5-20% serum, e.g., human, horse, calf, or fetal bovine serum.
The culture medium is usually buffered to maintain the cells at a
pH often from about 7.2 to about 7.4. Other supplements to the
media include, e.g., antibiotics, amino acids, sugars, and growth
factors (see, e.g., Lutz et al., supra). GM-CSF sometimes is in
concentrations ranging from 5 ng/ml to about 20 ng/ml in a culture
medium. Other factors described herein and known to stimulate
growth of dendritic cells may be included in the culture medium.
Some factors will have different effects that are dependent upon
the stage of differentiation of the cells, which can be monitored
by testing for differentiation markers specific for the cell's
stage in the differentiation pathway. GM-CSF can be included in the
medium throughout culturing. Other factors that may be included in
a culture medium include granulocyte colony-stimulating factor
(G-CSF), M-CSF, TNF-.alpha., IFN-.gamma, IL-1, IL-3, IL-6, SCF,
LPS, and thrombopoietin. IL-4 may be present in the culture medium,
sometimes at a concentration ranging from 1-100 ng/ml or about 5
ng/ml to about 20 ng/ml. Other factors and methods that can be
utilized for DC cell culture are known to the person of ordinary
skill in the art (e.g., U.S. 20040033213, published Feb. 19,
2004).
[0111] Dendritic cells can be recovered and used after cryogenic
storage. Precultured DCs can be cryogenically stored, e.g., in
liquid nitrogen, for several weeks or months or years. Dendritic
cells may be cultured in the presence of GM-CSF, sometimes for
about 10 days, prior to being stored cryogenically. The DCs can be
stored either as immature cells or as matured APCs, following
stimulation by suitable adjuvants, as described herein. Dendritic
cells can be cryogenically stored before or following exposure to
an antigen of interest. A variety of cryopreservation agents can be
used and are described in, e.g., U.S. Pat. No. 5,788,963.
Controlling the cooling rate, adding cryoprotective agents and/or
limiting the heat of fusion phase where water turns to ice help
preserve the function of the activated DCs. The cooling procedure
can be carried out by use of, e.g., a programmable freezing device
or a methanol bath procedure. After thorough freezing, cells can be
rapidly transferred to a long-term cryogenic storage vessel. The
samples can be cryogenically stored, for example, in liquid
nitrogen (-196.degree. C.) or its vapor (-165.degree. C.). Such
storage is facilitated by the availability of highly efficient
liquid nitrogen refrigerators. For a general description of methods
to store DCs cryogenically see, e.g., U.S. Pat. No. 5,788,963.
[0112] Certain factors may be contacted with dendritic cells to
facilitate proliferation and maturation. GM-CSF has been found to
promote the proliferation in vitro of both nonadherent immature
dendritic cells and adherent macrophages (see, e.g., U.S. Pat. No.
5,994,126; and Lutz et al., supra). Precursor dendritic cells
sometimes are cultured in the presence of GM-CSF at a concentration
sufficient to promote survival and proliferation. The dose of
GM-CSF depends, e.g., on the amount of competition from other cells
(especially macrophages and granulocytes) for the GM-CSF, and on
the presence of GM-CSF inactivators in the cell population (see,
e.g., U.S. Pat. No. 5,994,126). GM-CSF concentration sometimes is
of about 1 ng/ml to 100 ng/ml, and at times about 5 ng/ml to about
20 ng/ml. GM-CSF can be obtained from different sources well known
to those of skill in the art (see, e.g., Lutz et al., supra; and
U.S. Pat. No. 5,994,126).
[0113] In addition to GM-SCF, a variety of cytokines have been
shown to induce proliferation and/or maturation of dendritic cells
and other APCs (see, e.g., Caux et al., J. Exp. Med. 180:1263-1272
(1984); Allison, Archivum Immunologiae et Therapiae Experimentalis
45:141-147 (1997)). Cytokines that can be used to enhance
maturation of dendritic cells ex vivo include, but are not limited
to, TNF-alpha, stem cell factor (SCF; also named c-kit ligand,
steel factor (SF), mast cell growth factor (MGF); see, e.g., EP
423,980; and U.S. Pat. No. 6,017,527), granulocyte
colony-stimulating factor (G-CSF), monocyte-macrophage
colony-stimulating factor (M-CSF), as well as a number of
interleukins, such as, e.g., IL-1.alpha. and IL-1.beta., IL-3,
IL-4, IL-6, and IL-13 (see, e.g., U.S. Pat. Nos. 6,017,527 and
5,994,126). In addition to promoting maturation of dendritic cells,
some interleukins (e.g., IL-4) have been shown to suppresses the
overall growth of macrophages and thus favors higher levels of pure
DC growth. Cytokines are used in amounts which are effective in
increasing the proportion of dendritic cells present in the culture
by enhancing either the proliferation or the survival of dendritic
cell precursors. In certain embodiments, dendritic cells and
precursors are cultured in the presence of GM-CSF, and sometimes
are cultured in the presence of GM-CSF and IL-4. When human
dendritic precursor cells are cultured, the GM-CSF is sometimes is
human GM-CSF (huGM-CSF).
[0114] Adjuvants also can be used to stimulate the maturation ex
vivo of immature dendritic cells. Specifically, immature dendritic
cells can be harvested from the induction cultures described supra
and their maturation to end-stage antigen presenting cells can be
induced by treating the cells with a variety of adjuvants.
Adjuvants that promote maturation of dendritic cells include, but
are not limited to, MPL.RTM. immunostimulant and selected synthetic
lipid A analogs such as aminoalkyl glucosamide phosphate (AGP).
Synthetic lipid A analogs include, for example, lipid A
monosaccharide synthetics such as RC-529, RC-544 and RC-527, and
the disaccharide mimetic, RC-511. These adjuvants can be used as
10% ethanol-in-water formulations, although any other formulation
that promotes the maturation of dendritic cells is suitable for use
with the methods of the present invention. Adjuvants can be
synthesized or obtained from a variety of sources (see, e.g., Lutz
et al., supra; Johnson et al., Bioorganic Medicinal Chemistry
Letters 9:2273-2278 (1999)). Maturation of dendritic cells
sometimes is induced using MPL or AGP in certain embodiments.
[0115] Maturation of DCs can be assessed by monitoring a number of
molecular markers and cell surface phenotypic alterations. These
changes can be analyzed, for example, using flow cytometry
techniques. Maturation markers can be labeled using specific
antibodies and DCs expressing a marker or a set of markers of
interest can be separated from the total DC population using, for
example, cell sorting FACS analysis. Markers of DC maturation
include genes that are expressed at higher levels in mature DCs
compared to immature DCs. Such markers include, but are not limited
to, cell surface MHC Class II antigens (in particular HLA-DR),
ICAM-1, B7-2, costimulating molecules such as CD40, CD80, CD86,
CD83, cell trafficking molecules such as CD54, CD11b, CD18, and the
like. Mature dendritic cells also can be identified based on their
ability to stimulate the proliferation of naive allogeneic T cells
in a mixed leukocyte reaction (MLR).
[0116] It has been shown that immature dendritic cells are
efficient at antigen uptake but are poor antigen presenting cells,
and mature dendritic cells are poor at antigen uptake but are very
efficient antigen presenting cells. Accordingly, the antigen
presenting function of dendritic cells can be used to determine the
degree of maturation. The antigen presenting function of a
dendritic cell can be measured using antigen-dependent,
MHC-restricted T cell activation assays as described herein, as
well as other standard assays well known to those of skill in the
art. T cell activation can further be determined, e.g., by
measuring the induction of cytokine production by the stimulated
dendritic cells. Stimulation of cytokine production can be
quantified using a variety of standard techniques, such as ELISA,
known to those of ordinary skill in the art.
[0117] Thus, a DC utilized in a composition or process described
herein can be a mature DC, an immature DC, a DC having one or more
particular cell markers, a DC contacted with one or more cytokines,
a DC contacted with an adjuvant, a DC from a particular source
(e.g., bone marrow), a DC contacted with cell culture conditions,
and/or a DC contacted with cryogenic conditions, for example, in
certain embodiments.
[0118] Transferring Target Nucleotide Sequences Into Antigen
Presenting Cells
[0119] Transgene expression in cells requires transfer of a
nucleotide sequence encoding the transgene into the cell. Viral and
non-viral methods of gene transfer are known to the person of
ordinary skill in the art.
[0120] Several non-viral methods for transferring nucleic acids
into cells are known. These transfer methods include, for example,
calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen
and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal, 1985),
electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),
direct microinjection (Harland and Weintraub, 1985), DNA-loaded
liposomes (Nicolau and Sene, 1982; Fraley et al., 1979), cell
sonication (Fechheimer et al., 1987), gene bombardment using high
velocity microprojectiles (Yang et al., 1990), and
receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,
1988).
[0121] In certain embodiments, the nucleic acid is complexed to a
cationic polymer. Cationic polymers, which are water-soluble
complexes, are known in the art and have been utilized in delivery
systems for DNA plasmids. This strategy employs the use of a
soluble system, which conveys nucleic acid into cells via
receptor-mediated endocytosis (Wu & Wu 1988). Complexing
nucleic acids with a cationic polymer can neutralize negative
charge of the nucleic acid, which facilitates increased endocytic
uptake. Examples of cationic polymers include, but are not limited
to, polylysine, polyethyleneimine, polyhistidine, protamine,
polyvinylamines, polyvinylpyridine, polymethacrylates, and
polyornithine.
[0122] In a some transfer embodiments, the nucleic acid is
entrapped in a liposome. Liposomes are vesicular structures
characterized by a phospholipid bilayer membrane and an inner
aqueous medium. Multilamellar liposomes have multiple lipid layers
separated by aqueous medium. They form spontaneously when
phospholipids are suspended in an excess of aqueous solution. Lipid
components undergo self-rearrangement before the formation of
closed structures and entrap water and dissolved solutes between
the lipid bilayers (Ghosh and Bachhawat, 1991). Addition of nucleic
acids to cationic liposomes causes a topological transition from
liposomes to optically birefringent liquid-crystalline condensed
globules (Radler et al., 1997). These DNA-lipid complexes are
potential non-viral vectors for use in immunotherapy.
[0123] Liposome-mediated nucleic acid delivery and expression of
foreign DNA in vitro has been very successful. Using the
.beta.-lactamase gene, Wong et al., (1980) demonstrated the
feasibility of liposome-mediated delivery and expression of foreign
DNA in cultured chick embryo, HeLa, and hepatoma cells. Nicolau et
al., (1987) accomplished successful liposome-mediated gene transfer
in rats after intravenous injection. Also included are various
commercial approaches involving "lipofection" technology.
[0124] In certain embodiments, the liposome is complexed with a
hemagglutinating virus (HVJ). This has been shown to facilitate
fusion with the cell membrane and promote cell entry of
liposome-encapsulated DNA (Kaneda et al., 1989). In other
embodiments, the liposome is complexed or employed in conjunction
with nuclear nonhistone chromosomal proteins (HMG-1) (Kato et al.,
1991). In yet further embodiments, the liposome is complexed or
employed in conjunction with both HVJ and HMG-1. In that such
expression constructs have been successfully employed in transfer
and expression of nucleic acid in vitro and in vivo, then they are
applicable for the present invention.
[0125] In other embodiments, the delivery vehicle may comprise a
ligand and a liposome. For example, Nicolau et al., (1987) employed
lactosyl-ceramide, a galactose-terminal asialganglioside,
incorporated into liposomes and observed an increase in the uptake
of the insulin gene by hepatocytes. Thus, it is feasible that a
nucleic acid encoding a therapeutic gene also is specifically
delivered into a cell type such as prostate, epithelial or tumor
cells, by any number of receptor-ligand systems with or without
liposomes. For example, the human prostate-specific antigen (Watt
et al., 1986) is used as the receptor for mediated delivery of a
nucleic acid in prostate tissue.
[0126] In another embodiment of the invention, the expression
construct may simply consist of naked recombinant DNA or plasmids.
Transfer of the construct is performed by any of the methods
mentioned above which physically or chemically permeabilize the
cell membrane. This is applicable particularly for transfer in
vitro, however, it is applied for in vivo use as well. Dubensky et
al., (1984) successfully injected polyomavirus DNA in the form of
CaPO.sub.4 precipitates into liver and spleen of adult and newborn
mice demonstrating active viral replication and acute infection.
Benvenisty and Neshif (1986) also demonstrated that direct
intraperitoneal injection of CaPO.sub.4 precipitated plasmids
results in expression of the transfected genes. It is envisioned
that DNA encoding a CAM also is transferred in a similar manner in
vivo and express CAM.
[0127] Another embodiment of the invention for transferring a naked
DNA expression construct into cells may involve particle
bombardment. This method depends on the ability to accelerate DNA
coated microprojectiles to a high velocity by application of a
propulsion force, allowing them to pierce cell membranes and enter
cells without killing them (Klein et al., 1987). Several devices
for accelerating small particles have been developed. One such
device relies on a high voltage discharge to generate an electrical
current, which in turn provides the motive force (Yang et al.,
1990). The microprojectiles used have consisted of biologically
inert substances such as tungsten or gold beads.
[0128] Viral vectors also may be utilized to effect nucleic acid
transfer to cells. In certain embodiments, a transgene is
incorporated into a viral particle to mediate gene transfer to a
cell. The resulting virus often is exposed to the appropriate host
cell under physiologic conditions, permitting uptake of the virus.
Such methods are advantageously employed using a variety of viral
vectors, as discussed hereafter.
[0129] Adenovirus, sometimes referred to herein as "Ad," is
particularly suitable for use as a gene transfer vector because of
its mid-sized DNA genome, ease of manipulation, high titer, wide
target-cell range, and high infectivity. The roughly 36 kB viral
genome is bounded by 100-200 base pair (bp) inverted terminal
repeats (ITR), in which are contained cis-acting elements necessary
for viral DNA replication and packaging. The early (E) and late (L)
regions of the genome that contain different transcription units
are divided by the onset of viral DNA replication.
[0130] The E1 region (E1A and E1B) encodes proteins responsible for
the regulation of transcription of the viral genome and a few
cellular genes. The expression of the E2 region (E2A and E2B)
results in the synthesis of the proteins for viral DNA replication.
These proteins are involved in DNA replication, late gene
expression, and host cell shut off (Renan, 1990). The products of
the late genes (L1, L2, L3, L4 and L5), including the majority of
the viral capsid proteins, are expressed only after significant
processing of a single primary transcript issued by the major late
promoter (MLP). The MLP (located at 16.8 map units) is particularly
efficient during the late phase of infection, and all the mRNAs
issued from this promoter possess a 5' tripartite leader (TL)
sequence, which makes them often utilized mRNAs for
translation.
[0131] Adenovirus often is optimized for immunotherapy by
maximizing its carrying capacity so that large segments of DNA can
be included. Large displacement of DNA is possible in adenovirus
because the cis elements required for viral DNA replication all are
localized in the inverted terminal repeats (ITR) (100-200 bp) at
either end of the linear viral genome. Plasmids containing ITR's
can replicate in the presence of a non-defective adenovirus (Hay et
al., 1984). Therefore, inclusion of these elements in an adenoviral
vector should permit replication.
[0132] Toxicity and immunologic reaction associated with certain
adenoviral products also are minimized for immunotherapy
applications. The two goals are, to an extent, coterminous in that
elimination of adenoviral genes serves both ends. It is possible
achieve both these goals while retaining the ability to manipulate
the therapeutic constructs with relative ease.
[0133] In addition, the packaging signal for viral encapsidation is
localized between 194-385 by (0.5-1.1 map units) at the left end of
the viral genome (Hearing et al., 1987). This signal mimics the
protein recognition site in bacteriophage .lambda. DNA where a
specific sequence close to the left end, but outside the cohesive
end sequence, mediates the binding to proteins that are required
for insertion of the DNA into the head structure. E1 substitution
vectors of Ad have demonstrated that a 450 by (0-1.25 map units)
fragment at the left end of the viral genome could direct packaging
in 293 cells (Levrero et al., 1991).
[0134] Previously, it has been shown that certain regions of the
adenoviral genome can be incorporated into the genome of mammalian
cells and the genes encoded thereby expressed. These cell lines are
capable of supporting the replication of an adenoviral vector that
is deficient in the adenoviral function encoded by the cell line.
There also have been reports of complementation of replication
deficient adenoviral vectors by "helping" vectors, e.g., wild-type
virus or conditionally defective mutants.
[0135] Replication-deficient adenoviral vectors can be
complemented, in trans, by helper virus. This observation alone
does not permit isolation of the replication-deficient vectors,
however, since the presence of helper virus, needed to provide
replicative functions, would contaminate any preparation. Thus, an
additional element was needed that would add specificity to the
replication and/or packaging of the replication-deficient vector.
That element, as provided for in the present invention, derives
from the packaging function of adenovirus.
[0136] It has been shown that a packaging signal for adenovirus
exists in the left end of the conventional adenovirus map
(Tibbetts, 1977). Later studies showed that a mutant with a
deletion in the E1A (194-358 bp) region of the genome grew poorly
even in a cell line that complemented the early (E1A) function
(Hearing and Shenk, 1983). When a compensating adenoviral DNA
(0-353 bp) was recombined into the right end of the mutant, the
virus was packaged normally. Further mutational analysis identified
a short, repeated, position-dependent element in the left end of
the Ad5 genome. One copy of the repeat was found to be sufficient
for efficient packaging if present at either end of the genome, but
not when moved towards the interior of the Ad5 DNA molecule
(Hearing et al., 1987).
[0137] By using mutated versions of the packaging signal, it is
possible to create helper viruses that are packaged with varying
efficiencies. Typically, the mutations are point mutations or
deletions. When helper viruses with low efficiency packaging are
grown in helper cells, the virus is packaged, albeit at reduced
rates compared to wild-type virus, thereby permitting propagation
of the helper. When these helper viruses are grown in cells along
with virus that contains wild-type packaging signals, however, the
wild-type packaging signals are recognized preferentially over the
mutated versions. Given a limiting amount of packaging factor, the
virus containing the wild-type signals are packaged selectively
when compared to the helpers. If the preference is great enough,
stocks approaching homogeneity should be achieved.
[0138] Retrovirus also are useful for transferring Akt-encoding
nucleotide sequences into cells. Retroviruses are a group of
single-stranded RNA viruses characterized by an ability to convert
their RNA to double-stranded DNA in infected cells by a process of
reverse-transcription (Coffin, 1990). The resulting DNA then stably
integrates into cellular chromosomes as a provirus and directs
synthesis of viral proteins. The integration results in the
retention of the viral gene sequences in the recipient cell and its
descendants. The retroviral genome contains three genes--gag, pol
and env--that code for capsid proteins, polymerase enzyme, and
envelope components, respectively. A sequence found upstream from
the gag gene, termed .psi., functions as a signal for packaging of
the genome into virions. Two long terminal repeat (LTR) sequences
are present at the 5' and 3' ends of the viral genome. These
contain strong promoter and enhancer sequences and also are
required for integration in the host cell genome (Coffin,
1990).
[0139] In order to construct a retroviral vector, a nucleic acid
encoding a promoter is inserted into the viral genome in the place
of certain viral sequences to produce a virus that is
replication-defective. In order to produce virions, a packaging
cell line containing the gag, pol and env genes but without the LTR
and .psi. components is constructed (Mann et al., 1983). When a
recombinant plasmid containing a human cDNA, together with the
retroviral LTR and .psi. sequences is introduced into this cell
line (by calcium phosphate precipitation for example), the .psi.
sequence allows the RNA transcript of the recombinant plasmid to be
packaged into viral particles, which are then secreted into the
culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et
al., 1983). The media containing the recombinant retroviruses is
collected, optionally concentrated, and used for gene transfer.
Retroviral vectors are able to infect a broad variety of cell
types. However, integration and stable expression of many types of
retroviruses require the division of host cells (Paskind et al.,
1975).
[0140] An approach designed to allow specific targeting of
retrovirus vectors recently was developed based on the chemical
modification of a retrovirus by the chemical addition of galactose
residues to the viral envelope. This modification could permit the
specific infection of cells such as hepatocytes via
asialoglycoprotein receptors, should this be desired.
[0141] A different approach to targeting of recombinant
retroviruses was designed in which biotinylated antibodies against
a retroviral envelope protein and against a specific cell receptor
were used. The antibodies were coupled via the biotin components by
using streptavidin (Roux et al., 1989). Using antibodies against
major histocompatibility complex class I and class II antigens, the
infection of a variety of human cells that bore those surface
antigens was demonstrated with an ecotropic virus in vitro (Roux et
al., 1989).
[0142] Adeno-associated virus, also referred to herein as "AAV,"
also may be utilized to transfer Akt-encoding nucleotide sequences
into cells. AAV utilizes a linear, single-stranded DNA of about
4700 base pairs. Inverted terminal repeats flank the genome. Two
genes are present within the genome, giving rise to a number of
distinct gene products. The first, the cap gene, produces three
different virion proteins (VP), designated VP-1, VP-2 and VP-3. The
second, the rep gene, encodes four non-structural proteins (NS).
One or more of these rep gene products is responsible for
transactivating AAV transcription.
[0143] The three promoters in AAV are designated by their location,
in map units, in the genome. These are, from left to right, p5, p19
and p40. Transcription gives rise to six transcripts, two initiated
at each of three promoters, with one of each pair being spliced.
The splice site, derived from map units 42-46, is the same for each
transcript. The four non-structural proteins apparently are derived
from the longer of the transcripts, and three virion proteins all
arise from the smallest transcript.
[0144] AAV is not associated with any pathologic state in humans.
Interestingly, for efficient replication, AAV requires "helping"
functions from viruses such as herpes simplex virus I and II,
cytomegalovirus, pseudorabies virus and, of course, adenovirus. The
best characterized of the helpers is adenovirus, and many "early"
functions for this virus have been shown to assist with AAV
replication. Low-level expression of AAV rep proteins is believed
to hold AAV structural expression in check, and helper virus
infection is thought to remove this block.
[0145] The terminal repeats of the AAV vector can be obtained by
restriction endonuclease digestion of AAV or a plasmid such as
p201, which contains a modified AAV genome (Samulski et al., 1987),
or by other methods known to the skilled artisan, including but not
limited to chemical or enzymatic synthesis of the terminal repeats
based upon the published sequence of AAV. The ordinarily skilled
artisan can determine, by well-known methods such as deletion
analysis, the minimum sequence or part of the AAV ITRs which is
required to allow function, i.e., stable and site-specific
integration. The ordinarily skilled artisan also can determine
which minor modifications of the sequence can be tolerated while
maintaining the ability of the terminal repeats to direct stable,
site-specific integration.
[0146] AAV-based vectors have proven to be safe and effective
vehicles for gene delivery in vitro, and these vectors are being
developed and tested in pre-clinical and clinical stages for a wide
range of applications in potential gene therapy, both ex vivo and
in vivo (Carter and Flotte, 1995; Chatterjee et al., 1995; Ferrari
et al., 1996; Fisher et al., 1996; Flotte et al., 1993; Goodman et
al., 1994; Kaplitt et al., 1994; 1996, Kessler et al., 1996;
Koeberl et al., 1997; Mizukami et al., 1996).
[0147] AAV-mediated efficient gene transfer and expression in the
lung has led to clinical trials for the treatment of cystic
fibrosis (Carter and Flotte, 1995; Flotte et al., 1993). Similarly,
the prospects for treatment of muscular dystrophy by AAV-mediated
gene delivery of the dystrophin gene to skeletal muscle, of
Parkinson's disease by tyrosine hydroxylase gene delivery to the
brain, of hemophilia B by Factor IX gene delivery to the liver, and
potentially of myocardial infarction by vascular endothelial growth
factor gene to the heart, appear promising since AAV-mediated
transgene expression in these organs has recently been shown to be
highly efficient (Fisher et al., 1996; Flotte et al., 1993; Kaplitt
et al., 1994; 1996; Koeberl et al., 1997; McCown et al., 1996; Ping
et al., 1996; Xiao et al., 1996).
[0148] Other viral vectors also may be utilized for transfer of
nucleic acids into cells. Vectors derived from viruses such as
vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar
et al., 1988) canary pox virus, and herpes viruses are employed.
These viruses offer several features for use in gene transfer into
various mammalian cells. Several viral vectors are known and can be
selected by the person of ordinary skill in the art (e.g., U.S.
20020123479, published on Sep. 5, 2002).
[0149] Once the construct has been delivered into the cell, the
nucleic acid encoding the transgene are positioned and expressed at
different sites. In certain embodiments, the nucleic acid encoding
the transgene is stably integrated into the genome of the cell.
This integration is in the cognate location and orientation via
homologous recombination (gene replacement) or it is integrated in
a random, non-specific location (gene augmentation). In yet further
embodiments, the nucleic acid is stably maintained in the cell as a
separate, episomal segment of DNA. Such nucleic acid segments or
"episomes" encode sequences sufficient to permit maintenance and
replication independent of or in synchronization with the host cell
cycle. How the expression construct is delivered to a cell and
where in the cell the nucleic acid remains is dependent on the type
of expression construct employed.
[0150] A nucleic acid may be targeted to a dendritic cell by
associating a targeting molecule having affinity for a molecule
present on the surface of a dendritic cell with the nucleic acid. A
wide variety of targeting elements can be utilized to specifically
direct a nucleic acid to a dendritic cell. As utilized herein,
targeting elements are considered to be capable of interacting with
a molecule present on the surface of a dendritic cell when a
biological effect of the coupled targeting element may be seen in
the cell, or, alternatively, when there is greater than at least
about a 10-fold difference, and sometimes greater than at least
about a 25, 50, or 100-fold difference, between the binding of the
targeting element to dendritic cells as compared to non-dendritic
cells. Generally, it is preferable that the targeting element
interact with a molecule present on the surface of the selected
cell type with a K.sub.D of less than about 10.sup.-5M, preferably
less than about 10.sup.-6M, more preferably less than about
10.sup.-7M, and most preferably less than about 10.sup.-8M, as
determined by Scatchard analysis (Scatchard, Ann N.Y. Acad. Sci.
51:660, 1949). Suitable targeting elements are preferably
non-immunogenic, not degraded by proteolysis, and not scavenged by
the immune system. Particularly preferred targeting elements should
have a half-life within an animal of between about 10 minutes and
about 1 week.
[0151] Targeting elements often are proteins or peptides, although
other non-proteinaceous molecules may also function as targeting
elements. For example, antibodies (or the antigen binding domain
thereof) can be utilized to target dendritic cells. Particularly
useful antibodies are monoclonal antibodies which interact with the
extracellular domains of integral membrane proteins found
predominantly, or preferably exclusively, in the cell membranes of
dendritic cells, (see generally, Wilchek and Bayer, Anal. Biochem
171:1-32, 1988). In some embodiments the Fc portion of such an
antibody will serve as the targeting element, targeting the gene
delivery vehicle to dendritic cells that express Fc receptor
molecules. Suitable markers for antibody generation include CD11c,
CD54, CD58, CD25, CD11a, CD23, CD32, CD40, CD1, CD45, MHC Class I,
MHC Class II, Mac-1, Mac-2, and Mac-3. These and other lineage
specific markers can be used to separate or purify dendritic cells
from more diverse cell populations, in addition to those described
above. Both positive and negative selection strategies may be
employed. Techniques for conducting cell selection include FACS and
affinity chromatography.
[0152] Other suitable targeting elements include hormones, immune
accessory molecules (e.g., B7, IL-2, .alpha.-interferon, and
.gamma.-interferon), cell adhesion molecules (e.g., ICAM-1, ICAM-2,
and ICAM-3), integrins that bind to ICAMs and receptors known to be
expressed on the surface of dendritic cells. Dendritic
cell-specific ligands also may be selected from libraries created
utilizing recombinant techniques (Scott and Smith, Science 249:386,
1990; Devlin et al., Science 249:404, 1990; Houghten et al., Nature
354:84 1991; Matthews and Wells, Science 260:1113, 1993; Nissim et
al., EMBO J. 13(3):692-698, 1994), or equivalent techniques
utilizing organic compound libraries.
[0153] Targeting molecules can be associated with a nucleic acid in
a variety of manners. For example, a targeting molecule can be
linked directly to a nucleic acid (e.g., when a nucleic acid is
transferred to dendritic cell as naked DNA), can be incorporated
into the lipid bilayer of a liposome that bears the nucleic acid,
and can be expressed on the surface of a virus bearing the target
nucleotide sequence.
[0154] A nucleic acid delivery vehicle sometimes is purified before
it is contacted with an antigen presenting cell for transfer.
Techniques utilized for purification are dependent on the type of
vehicle used for nucleic acid delivery. If naked nucleic acid is
delivered, there are a variety of techniques known in the art
including, for example, purification by CsCl-ethidium bromide
gradient, ion-exchange chromatography, gel-filtration
chromatography, and differential precipitation with polyethylene
glycol. Further description of nucleic acids purification is
provided in Sambrook et. al., Molecular Cloning: A Laboratory
Manual, 2d ed. (Cold Spring Harbor Laboratory Press, 1989). For
viral delivery vehicles a sulfated oligosaccharide can be added
directly to a virus-containing preparation for purification.
[0155] When the delivery vehicle is a liposome, a variety of
purification methods known to those skilled in the art may be
utilized and are described in more detail in Mannino and
Gould-Fogerite (BioTechniques 6:682, 1988). Briefly, preparation of
liposomes typically involves admixing solutions of one or more
purified phospholipids and cholesterol in organic solvents and
evaporating the solvents to dryness. An aqueous buffer containing
the delivery vehicle then is added to the lipid film and the
mixture is sonicated to create a fairly uniform dispersion of
liposomes. In certain embodiments, dialysis, gel filtration, or
ultracentrifugation is then be used to separate unincorporated
components from the intact liposomes. (Stryer, L., Biochemistry,
pp:236 1975 (W. H. Freeman, San Francisco): Szoka et al., Biochim.
Biophys. Acta 600:1, 1980; Bayer et al., Biochim. Biophys. Acta.
550:464, 1979; Rivnay et al., Meth. Enzymol. 149: 119, 1987; Wang
et al., PNAS 84: 7851, 1987 and, Plant et al., Anal. Biochem.
176:420, 1989.
[0156] Priming Modified Antigen Presenting Cells with Antigen
[0157] Loading APCs with antigen can be achieved by a variety of
methods, including pulsing cells with antigenic peptides or
infecting the cells with recombinant viral vectors, for example.
Gene therapy techniques can be applied to dendritic cell vaccines.
Such techniques use recombinant viral vectors incapable of
replication to provide efficient and reliable means of gene
transfer. Genetic material can be introduced into dendritic cells
to provide them with a renewable source of antigen for
presentation; this should lead to more sustained expression of
antigen. The expression of viral (and therefore foreign) genes may
boost the immune response, but this antiviral immunity primed by
dendritic cells may cause the immune system to destroy dendritic
cells rapidly in subsequent rounds of immunization. One solution
can be to use viral vectors that do not result in the expression of
viral genes, such as retroviruses or "gutless" adenoviral
vectors.
[0158] Following expansion in culture and maturation, APCs can be
pulsed with an antigen. Pulsing processes are known to and can be
selected by the person of ordinary skill in the art, and cells may
be pulsed with antigen one or more times in appropriate regular or
variable cycles. APCs pulsed with an antigen of interest will
process and present epitopes of the antigen. Antigens can be from
any source, including, e.g., viruses, bacteria, parasites, etc. In
one embodiment, the antigen is derived from Mycobacterium sp,
Chlamydia sp., Leishmania sp., Trypanosoma sp., Plasmodium sp., or
a Candida sp. APCs can be pulsed with either the entire peptide
(antigen) or with a fragment thereof having immunogenic properties,
e.g., an epitope.
[0159] Briefly, the antigen-activated APCs (e.g., antigen-activated
dendritic cells) can be produced by exposing, ex vivo, an antigen
to the APCs (e.g., the dendritic cells) prepared according to the
methods known to the person of ordinary skill in the art. Dendritic
cells, for example, can be plated in culture dishes and exposed to
an antigen of interest in a sufficient amount and for a sufficient
period of time to allow the antigen to bind to the dendritic cells.
The amount and time necessary to achieve binding of the antigen to
the dendritic cells may be determined by using standard
immunoassays or binding assays. Any other method known to those of
skill in the art may also be used to detect the presence of antigen
on the dendritic cells following their exposure to the antigen.
Methods for pulsing dendritic cells with an antigen of interest are
described, e.g., in U.S. Pat. No. 6,017,527.
[0160] In general, antigens and fragments thereof may be prepared
using any of a variety of procedures well known to those of skill
in the art. For example, antigens can be naturally occurring and
purified from a natural source. Alternatively, antigens and
fragments thereof can be produced recombinantly using a DNA
sequence that encodes the antigen, which has been inserted into an
appropriate expression vector, i.e., a vector which contains the
necessary elements for the transcription and translation of the
inserted coding sequence, and expressed in an appropriate host.
Methods which are well known to those skilled in the art may be
used to construct expression vectors containing sequences encoding
a polypeptide of interest and appropriate transcriptional and
translational control elements. These methods include in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. Such techniques are described in Sambrook et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratories, Cold Spring Harbor, N.Y. (1989); and Ausubel et al.,
Current Protocols in Molecular Biology (1995 supplement). In
addition, antigens and portions thereof may also be generated by
synthetic means. Synthetic polypeptides having fewer than about 100
amino acids, and generally fewer than about 50 amino acids, may be
generated using techniques well known in the art. For example, such
polypeptides may be synthesized using any of the commercially
available solid-phase techniques, such as the Merrifield
solid-phase synthesis method, where amino acids are sequentially
added to a growing amino acid chain (see Merrifield, J. Am. Chem.
Soc. 85:2149-2146 (1963)). Equipment for automated synthesis of
polypeptides is commercially available from suppliers such as
Perkin Elmer/Applied BioSystems Division, Inc., Foster City,
Calif., and may be operated according to the manufacturer's
instructions. Variants of a native antigen may generally be
prepared using standard mutagenesis techniques, such as
oligonucleotide-directed site-specific mutagenesis. Sections of the
DNA sequence may also be removed using standard techniques to
permit preparation of truncated polypeptides.
[0161] Within certain embodiments, the antigen of interest may be a
fusion protein that comprises multiple polypeptides. A fusion
protein may, for instance, include an antigen and a fusion partner
which may, e.g., assist in providing T helper epitopes, and/or
assist in expressing the protein at higher yields than the native
recombinant protein. Other fusion partners may be selected so as to
increase the solubility of the protein or to enable the protein to
be targeted to desired intracellular compartments. Still further
fusion partners include affinity tags, which facilitate the
purification of the protein. Fusion proteins may generally be
prepared using standard techniques, including chemical conjugation
and as recombinant proteins.
[0162] An antigen often is a molecule associated with a disease or
condition. For example, pathogen-associated antigens, such as viral
antigens in virus-associated diseases (e.g., HIV-induced AIDS and
HBV-induced liver disease), and portions thereof, can be utilized
to prime modified APCs of the invention. Representative examples of
pathogen associated antigens include antigens from bacteria (e.g.,
E. coli, streptococcus, staphylococcus, mycobacteria, and the
like). fungi, parasites, and viruses (e.g., influenza virus, HIV,
and Hepatitis A, B and C Virus ("HAV", "HBV" and "HCV",
respectively), human papiloma virus ("HPV"), Epstein-Barr Virus
("EBV"), herpes simplex virus ("HSV"), hantavirus, HTLV I, HTLV II,
cytomegalovirus ("CMV"), and feline leukemia virus). Antigens
critical to the survival and proliferation of the cancer cell in
vivo, and portions thereof, can be selected for priming modified
APCs of the invention, and include, for example: (a)
tissue-specific antigens that are elevated in cancer, such as
carcinoembryonic antigen (CEA, colorectal cancer);
.alpha.-fetoprotein (liver cancer); prostate cancer antigen (PSA,
prostate cancer), mitochondrial creatine kinase (MCK, muscle
cancers), myelin basic protein (MBP, oligodendrocyte specific),
glial fibrillary acidic protein (GFAP, glial cell specific),
tyrosinase (melanoma), and neuron cancer enolase (NSE, neuronal
cancers); (b) mutated forms of tumor suppressor genes, such as
K-ras (colorectal carcinomas), and p53 (J. L. Bos, Cancer Res.
49:4682, 1989; Chiba et al., Oncogene 5:1603, 1990; (c) viral
proteins expressed by virally induced cancers, such as human
papillomavirus 16/18 E6 and E7 proteins (cervical cancer) or
Epstein Barr Virus peptides (EBV, B cell malignancies); (d)
tumor-specific antigens such as MART-1 (melanoma), gp100
(melanoma), HER2/neu (breast and epithelial cancers); NY-ESO-1
(testes and various tumors), PSA or PSMA (prostate cancer),
thymus-leukemia antigen (TL), and proteins of the MAGE family
(hepatocellular cancer and other tumors); (e) survivin and other
apoptosis inhibiting proteins expressed preferentially by tumor
cells (M. Zeiss et al., J. Immunol. 170:5391, 2003); and (f)
components involved in angiogenesis, such as vascular endothelia
growth factor (VEGF, expressed in angiogenic stroma and tumor
cells), VEGF receptor 2, Id2, Id3, and Tie-2 (preferentially
expressed during neoangiogenesis) (US 2004/0115174 A1). These and
other cancer-associated antigens can be selected by the person of
ordinary skill in the art. General reviews for tumor related
antigens useful as cancer vaccine targets include the text Tumor
Antigens Recognized by T Cells and Antibodies by H. J. Stauss, Y.
Kawakami, & G. Parmiani, CRC Press, 2003; and articles by
Rosenberg, Immunity 10:281, 1999; Nestle et al., Nat. Med. 4:328,
1998; Dermime et al., Br. Med. Bull. 62:149, 2002; and Berzofsky et
al., J. Clin. Invest. 113:1515, 2004. Methods for identifying
additional cancer target antigens are described in Barnea et al.,
Eur J Immunol 32:213, 2002; Schirle et al., Eur J. Immunol.
30:2216, 2000; Vinals et al., Vaccine 19:2607, 2001; Perez-Diez et
al., Cell. Mol. Life. Sci. 59:230, 2002; Radvanyi et al., Int.
Arch. Allergy Immunol. 133:179, 2004. An antigen may be a protein,
polypeptide, protein or polypeptide fragment, peptide, dominant
epitope peptide that binds to an HLA class I or II molecule, a
monosaccharide, a polysaccharide or nucleic acid.
[0163] Once an antigen is selected, one or more subfragments (i.e.,
portions) of the antigen having a dominant immunogenic epitope may
be synthesized based upon the knowledge of the ordinary artisan for
selecting epitope subsequences. Sequence motifs for dominant
epitopes are known and can be selected based upon whether they are
presented by specific MHC I (e.g., peptides of about 8-9 amino
acids) and MHC II molecules (e.g., U.S. 20060093617, published on
May 4, 2006, and U.S. 20050271676, published on Dec. 8, 2005). Some
epitopes conform to "pan DR" motifs and are presented by multiple
types of class II DR molecules (e.g., U.S. 20050049197 published on
Mar. 3, 2005), and native epitopes may be modified into
heteroclitic analogs having enhanced immunogenicity (e.g., U.S.
20030143672, published on Jul. 31, 2003).
[0164] Prior to loading, a polypeptide antigen may be covalently
conjugated to an immunological partner that provides T cell help
(e.g., a carrier molecule). Alternatively, an APC may be pulsed
with a non-conjugated immunological partner, separately or in the
presence of the antigen. Often, antigen presenting cells are primed
with an antigen (e.g., a protein, polypeptide, protein or
polypeptide fragment, peptide, dominant immunogenic epitope) for an
hour or more before the cells are matured (e.g., contacted with a
nucleic acid that encodes a membrane-targeted Akt molecule). The
antigen presenting cells may be contacted with a full-length
protein or polypeptide antigen, a fragment of the protein or
polypeptide antigen or a dominant immunogenic epitope from the
antigen that binds to a HLA class I or II molecule. An antigen may
be linked to a cell surface receptor that facilitates uptake of the
antigen by cells (e.g., DEC-205 or DC-sign receptor ligand).
[0165] APCs may be transfected with a polynucleotide sequence
encoding an antigen of interest (e.g., a protein, polypeptide,
protein or polypeptide fragment, peptide, dominant immunogenic
epitope) such that the antigen, or an immunogenic portion thereof,
is expressed on the cell surface. Such transfection may take place
ex vivo, and a composition or vaccine comprising such transfected
cells may then be used for therapeutic purposes, as described
herein. Alternatively, a gene delivery vehicle that targets a
dendritic or other antigen presenting cell may be administered to a
patient, resulting in transfection that occurs in vivo. In the
latter embodiments, the antigen-encoding sequence and
membrane-targeted Akt sequence may be expressed in cis (from the
same nucleic acid) or in trans (from different nucleic acids). In
vivo and ex vivo transfection of dendritic cells, for example, may
generally be performed using any methods known in the art, such as
those described in WO 97/24447, or the gene gun approach described
by Mahvi et al., Immunology and cell Biology 75:456-460 (1997).
Antigen loading of dendritic cells may be achieved by incubating
dendritic cells or progenitor cells with the antigen, DNA (naked or
within a plasmid vector) or RNA; or with antigen-expressing
recombinant bacterium or viruses (e.g., vaccinia, fowlpox,
adenovirus or lentivirus vectors). Vectors that express multiple
antigenic epitopes in "minigenes" also may be utilized (e.g., U.S.
20060093617, published on May 4, 2006).
[0166] In the context of the present invention, the antigens,
antigen fragments or fusion proteins used to pulse the dendritic
cells are preferably immunogenic, i.e., they are able to elicit an
immune response (e.g., cellular or humoral) in a patient, such as a
human, and/or in a biological sample (in vitro). In particular,
antigens that are immunogenic (and portions of such antigens that
are immunogenic) comprise an epitope recognized by a B-cell and/or
a T-cell surface antigen receptor. Antigens that are immunogenic
(and immunogenic portions of such antigens) are capable of
stimulating cell proliferation, interleukin-12 production and/or
interferon-.gamma. production in biological samples comprising one
or more cells selected from the group of T cells, NK cells, B cells
and macrophages, where the cells have been previously stimulated
with the antigen.
[0167] Immunomodulatory factors may be active in vivo and/or ex
vivo, can enhance immunogenicity of an antigen, and may be
co-administered (e.g., before, during or after administration of
antigen(s)). Representative examples of immunomodulatory factors
include, for example, cytokines, such as IL-1, IL-2 (Karupiah et
al., J. Immunology 144:290, 1990: Weber et al., J. Exp. Med.
166:1716, 1987; Gansbacher et al., J. Exp. Med. 172:1217, 1990:
U.S. Pat. No. 4,738,927). IL-3, IL-4 (Tepper et al., Cell 57:503,
1989, Golumbek et al., Science 254:713, 1991 and U.S. Pat. No.
5,017,691), IL-5, IL-6 (Brakenhof et al., J. Immunol. 139:4116,
1987, and WO 90/06370), IL-7 (U.S. Pat. No. 4,965,195), IL-8, IL-9,
IL-10, IL-11, IL-12 (Wolf et al., J. Immuno 46:3074, 1991 and
Gubler et al., PNAS 88:4143, 1991), IL-13 (WO 94/04680), IL-14,
IL-15, .alpha.-interferon (Finter et al., Drugs 42(5):749, 1991,
Nagata et al., Nature 284:316, 1980; Familletti et al., Methods in
Enz. 78:387, 1981, Twu et al., PNAS USA 86:2046, 1989, Faktor et
al., Oncogene 5:867, 1990, U.S. Pat. No. 4,892,743, U.S. Pat. No.
4,966,843, and WO 85/02862), .beta.-interferon (Seif et al., J.
Vir. 65:664, 1991), .gamma.-interferons (Radford et al., The
American Society of Hepatology 9:2008, 1991, Watanabe et al. PNAS
86:9456, 1989, Gansbacher et al., Cancer Research 50:7820, 1990,
Maio et al., Can. Immunol. Immunother. 30:34, 1989, U.S. Pat. No.
4,762,791, and U.S. Pat. No. 4,727,138). G-CSF (U.S. Pat. Nos.
4,999,291 and 4,810,643), GM-CSF (WO 85/04188). tumor necrosis
factors (TNFs) (Jayaraman et al., J. Immunology 144:942, 1990), CD3
(Krissanen et al., Immunogenetics 26:258, 1987), CD8, ICAM-1
(Altman et al., Nature 338:512, 1989; Simmons et al., Nature
331:624, 1988), ICAM-2 (Singer Science 255:1671, 1992), LFA-1
(Altmann et al., Nature 338:521, 1989), LFA-3 (Wallner et al., J.
Exp. Med. 166(4):923, 1987), and other proteins such as HLA Class I
molecules. HLA Class II molecules, B7 (Freeman et al., J. Immuno
143:2714, 1989). B7-2, .beta.sub.2-microglubulin (Parnes et al.,
PNAS 78:2253, 1981), chaperones, and MHC linked transporter
proteins or analogs thereof (Powis et al., Nature 354:528, 1991).
The choice of which immunomodulatory factor(s) to employ is based
upon the therapeutic effects of the factor. Immunomodulatory
factors sometimes utilized include .alpha.-interferon,
.gamma.-interferon, and IL-2.
[0168] A variety of standard assays for measuring the immunogenic
properties of a polypeptide of interest or of a portion thereof are
available and known to those of skill in the art (see, e.g., Paul,
Fundamental Immunology, 3d ed., Raven Press, pp. 243-247 (1993),
and references cited therein), and as described hereafter.
[0169] Assessing Modified Dendritic Cell Longevity and Induced
Immune Response
[0170] In certain embodiments, modified antigen presenting cells
described herein have a greater longevity (i.e., lifespan) than
unmodified counterparts. Such longevity can be expressed in terms
of hours, days weeks, number or fraction of surviving cells and
number or fraction of dead cells (e.g., cells that have or are
undergoing apoptosis), for example. Enhanced longevity can be
expressed in terms of a fold-, fraction- or percentage-increase
longevity of modified cells over the longevity of unmodified
counterpart cells. In certain embodiments, Longevity or lifespan
can be assessed in vitro, ex vivo or in vivo by a number of
techniques know to the person of ordinary skill in the art. In
certain embodiments, methods of observing cell staining (e.g., with
Annexin, propidium iodide (PI), CFSE dyes), enzymatic activity
(e.g., caspase activity), cell sorting (e.g., based upon cell
markers associated with apoptosis or cell death) and/or cell
morphology can be utilized. Representative methods for determining
cell longevity and cell death are described in Examples 9 and 10
hereafter.
[0171] In one aspect of the invention, the modified antigen
presenting cells (e.g., the modified dendritic cells) are used to
generate an immune response to an antigen of interest. The modified
antigen presenting cells described herein often induce a more
intense immune response than unmodified counterparts. An enhanced
immune response can be expressed in terms of a fold-, fraction- or
percentage-increase of an immune response elicited by modified
cells over the immune response elicited by unmodified counterpart
cells. An immune response to an antigen of interest can be detected
by a number of methods available to a person of ordinary skill in
the art (e.g., ELISPOT assay described in Example 9). For example,
an immune response can be assessed by examining the presence,
absence, or enhancement of specific activation of CD4+ or CD8+ T
cells or by antibodies. Typically, T cells isolated from an
immunized individual by routine techniques (e.g., by Ficoll/Hypaque
density gradient centrifugation of peripheral blood lymphocytes)
are incubated with an antigen (e.g., such T cells often are
incubated with APCs expressing antigen, since T cells generally are
poor APCs). For example, T cells may be incubated in vitro for 2-9
days (typically 4 days) at 37.degree. C. with the antigen. It may
be desirable to incubate another aliquot of a T cell sample in the
absence of the antigen to serve as a control.
[0172] Specific activation of CD4+ or CD8+ T cells may be detected
in a variety of ways. Methods for detecting specific T cell
activation include detecting the proliferation of T cells, the
production of cytokines, or the generation of cytolytic activity
(i.e., generation of cytotoxic T cells specific for an antigen).
For CD4+ T cells, an often utilized method for detecting specific T
cell activation is the detection of the proliferation of T cells.
For CD8+ T cells, and an often utilized method for detecting
specific T cell activation is the detection of the generation of
cytolytic activity.
[0173] Detection of the proliferation of T cells may be
accomplished by a variety of known techniques. For example, T cell
proliferation can be detected by measuring the rate of DNA
synthesis. T cells which have been stimulated to proliferate
exhibit an increased rate of DNA synthesis. A typical way to
measure the rate of DNA synthesis is, for example, by
pulse-labeling cultures of T cells with tritiated thymidine, a
nucleoside precursor which is incorporated into newly synthesized
DNA. The amount of tritiated thymidine incorporated can be
determined using a liquid scintillation spectrophotometer. Other
ways to detect T cell proliferation include measuring increases in
interleukin-2 (IL-2) production, Ca.sup.2+ flux, or dye uptake,
such as 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium.
Alternatively, synthesis of lymphokines (e.g., interferon-gamma
(IFN-.gamma.)) can be measured or the relative number of T cells
that can respond to the antigen may be quantified.
[0174] Secretion of IL-2 or IFN-.gamma. can be measured by a
variety of known techniques, including, but not limited to, the
double monoclonal antibody sandwich immunoassay technique of David
et al. (U.S. Pat. No. 4,376,110); monoclonal-polyclonal antibody
sandwich assays (Wide et al., in Kirkham and Hunter, eds.,
Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh (1970));
the "western blot" method of Gordon et al. (U.S. Pat. No.
4,452,901); immunoprecipitation of labeled ligand (Brown et al., J.
Biol. Chem. 255:4980-4983 (1980)); radioimmunoassays (RIA);
enzyme-linked immunosorbent assays (ELISA) as described, for
example, by Raines et al., J. Biol. Chem. 257:5154-5160 (1982);
immunocytochemical techniques, including the use of fluorochromes
(Brooks et al., Clin. Exp. Immunol 39:477 (1980)); and
neutralization of activity (Bowen-Pope et al., Proc. Natl. Acad.
Sci. USA 81:2396-2400 (1984)). In addition to the immunoassays
described above, a number of other immunoassays are available,
including those described in U.S. Pat. Nos. 3,817,827; 3,850,752;
3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and
4,098,876.
[0175] CTLs that are produced ex vivo can be expanded by regular
stimulation with APCs, which can be DCs or "non-professional" APCs
that are modified (e.g., with MHC molecules, B7 and the like) to
act as APCs. An advantage of artificial APCs is that they can be
expanded to appropriate numbers as the T cell populations expand.
Mature DCs are terminally differentiated and are utilized for
initial T cell activation in certain embodiments. Expanded CTLs can
be reinfused into patients by adoptive T cell therapy against
virally infected cells or oncogene-transformed cells in certain
embodiments and are especially useful for targeting comparatively
weak antigens. (e.g., Dudley and Rosenberg, Nature Reviews 2:
666-678 (2003) and Savoldo et al., Blood 100(12): 4059-66
(2002)).
[0176] Administration and Immunotherapy
[0177] APCs can be isolated from a patient, modified with a
membrane-associated AKT molecule described herein, cultured and
exposed in vitro or ex vivo to an antigen of interest (e.g.,
isolated peptide epitope or nucleic acid-encoded epitope), and
after expansion and/or cryogenic storage are administered back to
the patient to stimulate an immune response, including T cell
activation (see, e.g., Thurner et al., J. Immunol. Methods 223:1-15
(1999)), in vivo, in some embodiments. In certain embodiments, the
modified APCs are utilized to generate a CTL response in vitro, the
APCs optionally are separated from the CTLs, and the CTLs
(sometimes in combination with the APCs) are administered to the
subject in vivo.
[0178] In certain embodiments, modified DCs (e.g., obtained as
described herein) are exposed ex vivo to an antigen, washed and
administered to elicit an immune response or to augment an
existing, albeit weak, response. As such, the DCs may constitute a
vaccine and/or an immunotherapeutic agent. In addition, antigen
presenting cells (APCs), and in particular dendritic cells, can be
used as delivery vehicles for administering pharmaceutical
compositions and vaccines. In this context, the APCs may, but need
not, be genetically modified, e.g., to increase the capacity for
presenting the antigen, to improve activation and/or maintenance of
the T cell response and/or to be immunologically compatible with
the receiver (i.e., matched HLA haplotype). APCs generally may be
isolated from any of a variety of biological fluids and organs as
described above, and may be autologous, allogeneic, syngeneic or
xenogeneic cells.
[0179] Modified APCs primed with antigen may be administered by
parenteral (e.g., intravenous, subcutaneous, and intramuscular) or
other traditional direct routes, such as buccal/sublingual, rectal,
oral, nasal, topical, (such as transdermal and ophthalmic),
vaginal, pulmonary, intraarterial, intraperitoneal, intraocular, or
intranasal routes or directly into a specific tissue, such as the
liver, bone marrow, or into the tumor in the case of cancer
therapy. Non-parenteral routes are discussed further in U.S.
application Ser. No. 08/366,788, filed Dec. 3, 1994. Dendritic
cells modified ex vivo sometimes are administered in vivo by
intradermal or subcutaneous injection, sometimes with 5-10 million
cells administered per site. Modified DCs sometimes are
administered interdermally or subcutaneously at between one to five
sites in a subject. Methods for administering dendritic cells to a
patient for eliciting an immune response in the patient are
described, e.g., in U.S. Pat. Nos. 5,849,589; 5,851,756; 5,994,126;
and 6,017,527. CTLs expanded by contact with modified APCs of the
invention also may be administered to a subject via similar routes.
Administration can be repeated at desired intervals based upon the
patient's immune response.
[0180] A DC composition can be administered to a subject via a
desired route and the subject may be tested for a desired
biological response. Such testing may include immunological
screening assays e.g., CTL assays, antibody assays. Other molecules
may be detected after APCs or CTLs are administered to a subject,
such as by assessing whether the amount of a disease-associated
biological marker lowers over time. Administration by many of the
routes of administration described herein or otherwise known in the
art and may be accomplished simply by direct administration using a
needle, catheter or related device, at a single time point or at
multiple time points. Other routes and methods for administration
include non-parenteral routes, such as are disclosed in U.S. Ser.
No. 08/366,788, as well as administration via multiple sites, as
disclosed in U.S. Ser. No. 08/366,784.
[0181] Pharmaceutical compositions described above can be utilized
to elicit a "protective immune response" or "therapeutic immune
response," which as used herein refer to a CTL and/or a T helper
(e.g., "CD4+ T helper) response to an antigen derived from an
infectious agent or a tumor antigen, which prevents or at least
partially arrests or ameliorates disease symptoms or progression.
An immune response may also include an antibody response which has
been facilitated by the stimulation of helper T cells.
[0182] Pharmaceutical compositions described above can include
components other than the modified APCs or CTLs produced from them.
For example, pharmaceutical compositions may include the following
types of proteins, fragments, peptides or nucleic acids, or
nucleotide sequences (within or not within the APC) that encode
such molecules: (a) Toll-like receptor (TLR) ligands that can
increase DC activation locally, such as monophosphoryl lipid A
(TLR4 ligand), imiquimod (TLR7/8 ligand), unmethylated CpG
oligonucleotides (TLR9 ligand), and others, and additional
activation ligands, such as those found in monocyte-conditioned
media "maturation cocktail" also can be included; (b) targeting
ligands; (c) one or more apoptosis-inhibiting factors, such as
Bcl-2 or Bcl-xL, for example; (d) antisense, ribozyme or siRNA
molecules that target apoptosis-inducing molecules (e.g., Bax, Bak,
caspases) and/or molecules that block homeostatic feedback (e.g.,
SOCS-1, c-CBL, CBL-b, SHP-1). APCs also can be administered in
conjunction with other therapeutic regimens. In the treatment of a
cancer, for example, modified APCs described herein primed with a
tumor-associated antigen (e.g., PSMA) can be administered in
conjunction (e.g., before, during or after) with an anti-cancer
therapeutic, including, but not limited to, radiation treatment
(e.g., radioactive implant), chemotherapy or surgery. In the
treatment of a virus infection, for example, modified APCs
described herein primed with a virus-associated antigen (e.g., HIV
antigen(s)) can be administered in conjunction with an antiviral
drug (e.g., HIV protease inhibitor). The person of ordinary skill
in the art can select other therapies in combination with
administration of modified APCs described herein based upon the
disease being treated.
[0183] A DC modified with a membrane-targeted Akt molecule also may
be utilized in combination with one or more other agents. Such
agents include, for example, a nucleic acid, a viral particle, an
adjuvant and/or another modified DC, in some embodiments. For
example, a DC may be may be contacted with a first polynucleotide
sequence that encodes a membrane-targeted Akt described herein, and
a second polynucleotide sequence that encodes an inducible CD40
molecule. Inducible CD40 molecule embodiments are described, for
example, in US publication 20040209836 (published Oct. 21, 2004).
The first and second polynucleotide sequences may be in one nucleic
acid, in separate nucleic acids, in one viral particle or in
separate viral particles. Thus, provided herein is a composition
comprising (i) a first polynucleotide sequence that encodes a first
chimeric protein comprising an Akt and a membrane-association
region, and (ii) a second polynucleotide sequence that encodes a
second chimeric protein comprising a membrane-association region, a
multimeric ligand binding region and a CD40 cytoplasmic polypeptide
region lacking the CD40 extracellular domain. The first and second
polynucleotide sequences may be in one nucleic acid, or in separate
nucleic acids, in certain embodiments. Also provided herein is a
composition comprising the first chimeric protein and the second
chimeric protein. The composition may comprise a DC that includes
the first and second chimeric protein, in certain embodiments. Such
a DC optionally may be contacted with an antigen or fragment
thereof or a third polynucleotide sequence encoding the foregoing,
and optionally may include an antigen or fragment thereof or a
third polynucleotide sequence encoding the foregoing. Also provided
are methods for using such polynucleotides, proteins and DCs.
[0184] Kits
[0185] Kits comprise one or more containers, which contain one or
more of the compositions and/or components described herein. A kit
comprises one or more of the components in any number of separate
containers, packets, tubes, vials, microtiter plates and the like,
or the components may be combined in various combinations in such
containers. A kit in some embodiments includes a component
described herein and provides instructions that direct the user to
another component not included in the kit.
[0186] A kit can include components described herein in a variety
of combinations. A kit may comprise one, two, three, four, five or
more components described herein. For example, a kit can include a
nucleic acid having a nucleotide sequence that encodes an Akt
molecule.
[0187] A kit sometimes is utilized in conjunction with a method
described herein, and sometimes includes instructions for
performing one or more methods described herein and/or a
description of one or more compositions described herein.
Instructions and/or descriptions may be in printed form and may be
included in a kit insert. A kit also may include a written
description of an internet location that provides such instructions
or descriptions.
[0188] Akt related components that may be provided in a kit include
one or more adjuvants, one or more antigens or antigen-encoding
nucleic acids, one or more types of cells (e.g., for producing a
nucleic acid), one or more agents for transferring a nucleic acid
to antigen presenting cells or another type of cell, one or more
reagents or devices for isolating dendritic cells from a subject,
one or more devices for transferring modified dendritic cells to a
subject, and one or more agents or devices for assessing the
presence or absence of an immune response, for example. A kit may
include one or more other components described hereafter.
[0189] A component in a kit sometimes is a molecule that
specifically interacts with (e.g., binds to) a nucleic acid,
protein, polypeptide or peptide described above. The latter class
of components sometimes are referred to herein as "specific
interaction reagents" or "specific binding reagents." A specific
binding reagent sometimes is in association with detectable label
described in greater detail hereafter. Examples of specific binding
reagents include antibodies and antibody fragments; binding
partners; chemical compounds; and antisense, ribozyme and siRNA
nucleic acids.
[0190] A variety of antibodies and antibody fragments are available
to the artisan, and can be generated by the artisan, for use as a
specific binding reagent. Antibodies sometimes are IgG, IgM, IgA,
IgE, or an isotype thereof (e.g., IgG1, IgG2a, IgG2b or IgG3),
sometimes are polyclonal or monoclonal, and sometimes are chimeric,
humanized or bispecific versions of such antibodies. Polyclonal and
monoclonal antibodies that bind specific antigens are commercially
available, and methods for generating such antibodies are known. In
general, polyclonal antibodies are produced by injecting an
isolated antigen (e.g., an Akt protein or fragment) into a suitable
animal (e.g., a goat or rabbit); collecting blood and/or other
tissues from the animal containing antibodies specific for the
antigen and purifying the antibody. Methods for generating
monoclonal antibodies, in general, include injecting an animal with
an isolated antigen (e.g., often a mouse or a rat); isolating
splenocytes from the animal; fusing the splenocytes with myeloma
cells to form hybridomas; isolating the hybridomas and selecting
hybridomas that produce monoclonal antibodies which specifically
bind the antigen (e.g., Kohler & Milstein, Nature 256:495 497
(1975) and StGroth & Scheidegger, J Immunol Methods 5:1 21
(1980)).
[0191] Methods for generating chimeric and humanized antibodies
also are known (see, e.g., U.S. Pat. No. 5,530,101 (Queen, et al.),
U.S. Pat. No. 5,707,622 (Fung, et al.) and U.S. Pat. Nos. 5,994,524
and 6,245,894 (Matsushima, et al.)), which generally involve
transplanting an antibody variable region from one species (e.g.,
mouse) into an antibody constant domain of another species (e.g.,
human). Antigen-binding regions of antibodies (e.g., Fab regions)
include a light chain and a heavy chain, and the variable region is
composed of regions from the light chain and the heavy chain. Given
that the variable region of an antibody is formed from six
complementarity-determining regions (CDRs) in the heavy and light
chain variable regions, one or more CDRs from one antibody can be
substituted (i.e., grafted) with a CDR of another antibody to
generate chimeric antibodies. Also, humanized antibodies are
generated by introducing amino acid substitutions that render the
resulting antibody less immunogenic when administered to
humans.
[0192] A specific binding reagent sometimes is an antibody
fragment, such as a Fab, Fab', F(ab)'2, Dab, Fv or single-chain Fv
(ScFv) fragment, and methods for generating antibody fragments are
known (see, e.g., U.S. Pat. Nos. 6,099,842 and 5,990,296 and
PCT/GB00/04317). In some embodiments, a binding partner in one or
more hybrids is a single-chain antibody fragment, which sometimes
are constructed by joining a heavy chain variable region with a
light chain variable region by a polypeptide linker (e.g., the
linker is attached at the C-terminus or N-terminus of each chain)
by recombinant molecular biology processes. Such fragments often
exhibit specificities and affinities for an antigen similar to the
original monoclonal antibodies. Bifunctional antibodies sometimes
are constructed by engineering two different binding specificities
into a single antibody chain and sometimes are constructed by
joining two Fab' regions together, where each Fab' region is from a
different antibody (e.g., U.S. Pat. No. 6,342,221). Antibody
fragments often comprise engineered regions such as CDR-grafted or
humanized fragments. In certain embodiments the binding partner is
an intact immunoglobulin, and in other embodiments the binding
partner is a Fab monomer or a Fab dimer.
[0193] The artisan may select and prepare a binding partner of Akt
as a specific binding reagent. Multiple binding partners of an Akt
protein exist (e.g., CK2, IRAK1, MAP3K8, TNF11, MTTL7 (http address
www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&dopt=full_rep-
ort&list_uids=207)). The artisan may utilize a fragment of a
binding partner that binds to an Akt protein or fragment as a
specific binding reagent in specific embodiments. The artisan also
may optimize a binding reagent for a specific use or identify new
binding reagents using a variety of procedures. For example,
binding partners may be identified by lysing cells and analyzing
cell lysates by electrophoretic techniques. Alternatively, a
two-hybrid assay or three-hybrid assay can be utilized (e.g., U.S.
Pat. No. 5,283,317; Zervos et al., Cell 72:223-232 (1993); Madura
et al., J. Biol. Chem. 268: 12046-12054 (1993); Bartel et al.,
Biotechniques 14: 920-924 (1993); Iwabuchi et al., Oncogene 8:
1693-1696 (1993); and Brent WO94/10300). A two-hybrid system is
based on the modular nature of most transcription factors, which
consist of separable DNA-binding and activation domains. The assay
often utilizes two different DNA constructs. In one construct, an
Akt nucleic acid (sometimes referred to as the "bait") is fused to
a gene encoding the DNA binding domain of a known transcription
factor (e.g., GAL-4). In another construct, a DNA sequence from a
library of DNA sequences that encodes a potential binding partner
(sometimes referred to as the "prey") is fused to a gene that
encodes an activation domain of the known transcription factor.
Sometimes, an Akt nucleic acid is linked to the activation domain.
If the "bait" and the "prey" molecules interact in vivo, the
DNA-binding and activation domains of the transcription factor are
brought into close proximity. This proximity allows transcription
of a reporter gene (e.g., LacZ) which is operably linked to a
transcriptional regulatory site responsive to the transcription
factor. Expression of the reporter gene can be detected and cell
colonies containing the functional transcription factor can be
isolated and used to identify the potential binding partner.
[0194] The artisan of ordinary skill can select a chemical compound
as a specific binding reagent. Compounds can be obtained using any
of the numerous approaches in combinatorial library methods known
in the art, including: biological libraries; peptoid libraries
(libraries of molecules having the functionalities of peptides, but
with a novel, non-peptide backbone which are resistant to enzymatic
degradation but which nevertheless remain bioactive (see, e.g.,
Zuckermann et al., J. Med. Chem.37: 2678-85 (1994)); spatially
addressable parallel solid phase or solution phase libraries;
synthetic library methods requiring deconvolution; "one-bead
one-compound" library methods; and synthetic library methods using
affinity chromatography selection. Biological library and peptoid
library approaches are typically limited to peptide libraries,
while the other approaches are applicable to peptide, non-peptide
oligomer or small molecule libraries of compounds (Lam, Anticancer
Drug Des. 12: 145, (1997)). Examples of methods for synthesizing
molecular libraries are described, for example, in DeWitt et al.,
Proc. Natl. Acad. Sci. U.S.A. 90: 6909 (1993); Erb et al., Proc.
Natl. Acad. Sci. USA 91: 11422 (1994); Zuckermann et al., J. Med.
Chem. 37: 2678 (1994); Cho et al., Science 261: 1303 (1993);
Carrell et al., Angew. Chem. Int. Ed. Engl. 33: 2059 (1994); Carell
et al., Angew. Chem. Int. Ed. Engl. 33: 2061 (1994); and in Gallop
et al., J. Med. Chem. 37: 1233 (1994). Libraries of compounds may
be presented in solution (e.g., Houghten, Biotechniques 13: 412-421
(1992)), or on beads (Lam, Nature 354: 82-84 (1991)), chips (Fodor,
Nature 364: 555-556 (1993)), bacteria or spores (Ladner, U.S. Pat.
No. 5,223,409), plasmids (Cull et al., Proc. Natl. Acad. Sci. USA
89: 1865-1869 (1992)) or on phage (Scott and Smith, Science 249:
386-390 (1990); Devlin, Science 249: 404-406 (1990); Cwirla et al.,
Proc. Natl. Acad. Sci. 87: 6378-6382 (1990); Felici, J. Mol. Biol.
222: 301-310 (1991); Ladner supra.). A compound often is a small
molecule. Small molecules include, but are not limited to,
peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid
analogs, polynucleotides, polynucleotide analogs, nucleotides,
nucleotide analogs, organic or inorganic compounds (i.e., including
heteroorganic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds.
[0195] The artisan of ordinary skill can select and prepare a
nucleic acid specific binding reagent for use. Nucleic acids may
comprise or consist of analog or derivative nucleic acids, such as
polyamide nucleic acids (PNA) and others exemplified in U.S. Pat.
Nos. 4,469,863; 5,536,821; 5,541,306; 5,637,683; 5,637,684;
5,700,922; 5,717,083; 5,719,262; 5,739,308; 5,773,601; 5,886,165;
5,929,226; 5,977,296; 6,140,482; 5,614,622; 5,739,314; 5,955,599;
5,962,674; 6,117,992; WIPO publications WO 00/56746, WO 00/75372
and WO 01/14398, and related publications. An antisense nucleic
acid sometimes is designed, prepared and/or utilized by the artisan
to inhibit an Akt nucleic acid. An "antisense" nucleic acid refers
to a nucleotide sequence complementary to a "sense" nucleic acid
encoding an Akt protein or fragment (e.g., complementary to the
coding strand of a double-stranded cDNA molecule or complementary
to an mRNA sequence). The antisense nucleic acid can be
complementary to an entire coding strand, or to a portion thereof
or a substantially identical sequence thereof. In another
embodiment, the antisense nucleic acid molecule is antisense to a
"noncoding region" of the coding strand of a nucleotide
sequence.
[0196] An antisense nucleic acid can be complementary to the entire
coding region of an mRNA encoded by an Akt nucleotide sequence, and
often the antisense nucleic acid is an oligonucleotide antisense to
only a portion of a coding or noncoding region of the mRNA. For
example, the antisense oligonucleotide can be complementary to the
region surrounding the translation start site of the mRNA, e.g.,
between the -10 and +10 regions of the target gene nucleotide
sequence of interest. An antisense oligonucleotide can be, for
example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, or more nucleotides in length. An antisense nucleic
acid can be constructed using chemical synthesis or enzymic
ligation reactions using standard procedures. For example, an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and sense nucleic acids
(e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be used). Antisense nucleic acid also can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0197] When utilized in subjects, antisense nucleic acids typically
are administered to a subject (e.g., by direct injection at a
tissue site) or generated in situ such that they hybridize with or
bind to cellular mRNA and/or genomic DNA encoding a polypeptide and
thereby inhibit expression of the polypeptide, for example, by
inhibiting transcription and/or translation. Alternatively,
antisense nucleic acid molecules can be modified to target selected
cells and then are administered systemically. For systemic
administration, antisense molecules can be modified such that they
specifically bind to receptors or antigens expressed on a selected
cell surface, for example, by linking antisense nucleic acid
molecules to peptides or antibodies which bind to cell surface
receptors or antigens. Antisense nucleic acid molecules can also be
delivered to cells using the vectors described herein. Sufficient
intracellular concentrations of antisense molecules are achieved by
incorporating a strong promoter, such as a pol II or pol III
promoter, in the vector construct. Antisense nucleic acid molecules
sometimes are alpha-anomeric nucleic acid molecules. An
alpha-anomeric nucleic acid molecule forms specific double-stranded
hybrids with complementary RNA in which, contrary to the usual
beta-units, the strands run parallel to each other (Gaultier et
al., Nucleic Acids. Res. 15: 6625-6641 (1987)). Antisense nucleic
acid molecules also can comprise a 2'-o-methylribonucleotide (Inoue
et al., Nucleic Acids Res. 15: 6131-6148 (1987)) or a chimeric
RNA-DNA analogue (Inoue et al., FEBS Lett. 215: 327-330 (1987)).
Antisense nucleic acids sometimes are composed of DNA or PNA or any
other nucleic acid derivatives described previously.
[0198] An antisense nucleic acid is a ribozyme in some embodiments.
A ribozyme having specificity for an Akt nucleotide sequence can
include one or more sequences complementary to such a nucleotide
sequence, and a sequence having a known catalytic region
responsible for mRNA cleavage (e.g., U.S. Pat. No. 5,093,246 or
Haselhoff and Gerlach, Nature 334: 585-591 (1988)). For example, a
derivative of a Tetrahymena L-19 IVS RNA is sometimes utilized in
which the nucleotide sequence of the active site is complementary
to the nucleotide sequence to be cleaved in a mRNA (e.g., Cech et
al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No.
5,116,742). Akt mRNA sequences also may be utilized to select a
catalytic RNA having a specific ribonuclease activity from a pool
of RNA molecules (e.g., Bartel & Szostak, Science 261:
1411-1418 (1993)).
[0199] Specific binding reagents sometimes are nucleic acids that
can form triple helix structures with an Akt nucleic acid. Akt
expression can be inhibited by targeting nucleotide sequences
complementary to the regulatory region of a nucleotide sequence
referenced herein or a substantially identical sequence (e.g.,
promoter and/or enhancers) to form triple helical structures that
prevent transcription of a gene in target cells (see e.g., Helene,
Anticancer Drug Des. 6(6): 569-84 (1991); Helene et al., Ann. N.Y.
Acad. Sci. 660: 27-36 (1992); and Maher, Bioassays 14(12): 807-15
(1992). Triple helix formation can be enhanced by generating a
"switchback" nucleic acid molecule. Switchback molecules are
synthesized in an alternating 5'-3',3'-5' manner, such that they
base pair with first one strand of a duplex and then the other,
eliminating the necessity for a sizeable stretch of purines or
pyrimidines being present on one strand of a duplex.
[0200] An artisan may select an interfering RNA (RNAi) or siRNA
specific binding reagent for use. The nucleic acid selected
sometimes is the RNAi or siRNA or a nucleic acid that encodes such
products. The term "RNAi" as used herein refers to double-stranded
RNA (dsRNA) which mediates degradation of specific mRNAs, and can
also be used to lower or eliminate gene expression. The term "short
interfering nucleic acid", "siNA", "short interfering RNA",
"siRNA", "short interfering nucleic acid molecule", "short
interfering oligonucleotide molecule", or "chemically-modified
short interfering nucleic acid molecule" as used herein refers to
any nucleic acid molecule directed against a gene. For example, a
siRNA is capable of inhibiting or down regulating gene expression
or viral replication, for example by mediating RNA interference
"RNAi" or gene silencing in a sequence-specific manner; see for
example Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001, Nature,
411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and
Kreutzer et al., International PCT Publication No. WO 00/44895;
Zernicka-Goetz et al., International PCT Publication No. WO
01/36646; Fire, International PCT Publication No. WO 99/32619;
Plaetinck et al., International PCT Publication No. WO 00/01846;
Mello and Fire, International PCT Publication No. WO 01/29058;
Deschamps-Depaillette, International PCT Publication No. WO
99/07409; and Li et al., International PCT Publication No. WO
00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al.,
2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297,
2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;
Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al.,
2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16,
1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831).
There is no particular limitation in the length of siRNA as long as
it does not show toxicity. Examples of modified RNAi and siRNA
include STEALTH.TM. forms (Invitrogen Corp., Carlsbad, Calif.),
forms described in U.S. Patent Publication No. 2004/0014956
(application Ser. No. 10/357,529) and U.S. patent application Ser.
No. 11/049,636, filed Feb. 2, 2005), and other forms described
hereafter.
[0201] A siNA can be a double-stranded polynucleotide molecule
comprising self-complementary sense and antisense regions, wherein
the antisense region comprises nucleotide sequence that is
complementary to nucleotide sequence in a target nucleic acid
molecule or a portion thereof and the sense region having
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. The siNA can be assembled from two
separate oligonucleotides, where one strand is the sense strand and
the other is the antisense strand, wherein the antisense and sense
strands are self-complementary (i.e. each strand comprises
nucleotide sequence that is complementary to nucleotide sequence in
the other strand; such as where the antisense strand and sense
strand form a duplex or double stranded structure, for example
wherein the double stranded region is about 19 base pairs); the
antisense strand comprises nucleotide sequence that is
complementary to nucleotide sequence in a target nucleic acid
molecule or a portion thereof and the sense strand comprises
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. Alternatively, the siNA is assembled
from a single oligonucleotide, where the self-complementary sense
and antisense regions of the siNA are linked by means of a nucleic
acid based or non-nucleic acid-based linker(s). The siNA can be a
polynucleotide with a duplex, asymmetric duplex, hairpin or
asymmetric hairpin secondary structure, having self-complementary
sense and antisense regions, wherein the antisense region comprises
nucleotide sequence that is complementary to nucleotide sequence in
a separate target nucleic acid molecule or a portion thereof and
the sense region having nucleotide sequence corresponding to the
target nucleic acid sequence or a portion thereof. The siNA can be
a circular single-stranded polynucleotide having two or more loop
structures and a stem comprising self-complementary sense and
antisense regions, wherein the antisense region comprises
nucleotide sequence that is complementary to nucleotide sequence in
a target nucleic acid molecule or a portion thereof and the sense
region having nucleotide sequence corresponding to the target
nucleic acid sequence or a portion thereof, and wherein the
circular polynucleotide can be processed either in vivo or in vitro
to generate an active siNA molecule capable of mediating RNAi. The
siNA can also comprise a single stranded polynucleotide having
nucleotide sequence complementary to nucleotide sequence in a
target nucleic acid molecule or a portion thereof (for example,
where such siNA molecule does not require the presence within the
siNA molecule of nucleotide sequence corresponding to the target
nucleic acid sequence or a portion thereof), wherein the single
stranded polynucleotide can further comprise a terminal phosphate
group, such as a 5'-phosphate (see for example Martinez et al.,
2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell,
10, 537-568), or 5',3'-diphosphate. In certain embodiments, the
siNA molecule of the invention comprises separate sense and
antisense sequences or regions, wherein the sense and antisense
regions are covalently linked by nucleotide or non-nucleotide
linkers molecules as is known in the art, or are alternately
non-covalently linked by ionic interactions, hydrogen bonding, van
der waals interactions, hydrophobic interactions, and/or stacking
interactions. In certain embodiments, the siNA molecules of the
invention comprise nucleotide sequence that is complementary to
nucleotide sequence of a target gene. In another embodiment, the
siNA molecule of the invention interacts with nucleotide sequence
of a target gene in a manner that causes inhibition of expression
of the target gene.
[0202] The double-stranded RNA portions of siRNAs in which two RNA
strands pair are not limited to the completely paired forms, and
may contain non-pairing portions due to mismatch (the corresponding
nucleotides are not complementary), bulge (lacking in the
corresponding complementary nucleotide on one strand), and the
like. Non-pairing portions can be contained to the extent that they
do not interfere with siRNA formation. The "bulge" used herein
often comprises 1 to 2 non-pairing nucleotides, and the
double-stranded RNA region of siRNAs in which two RNA strands pair
up sometimes contains 1 to 7, and at times 1 to 5 bulges. In
addition, the "mismatch" used herein is contained in the
double-stranded RNA region of siRNAs in which two RNA strands pair
up, sometimes 1 to 7, and at times 1 to 5, in number. In an often
utilized mismatch, one of the nucleotides is guanine, and the other
is uracil. Such a mismatch is due to a mutation from C to T, G to
A, or mixtures thereof in DNA coding for sense RNA, but not
particularly limited to them. Furthermore, in the present
invention, the double-stranded RNA region of siRNAs in which two
RNA strands pair up may contain both bulge and mismatched, which
sum up to, sometimes 1 to 7, and at times 1 to 5, in number. The
terminal structure of siRNA may be either blunt or cohesive
(overhanging) as long as siRNA enables to silence the target gene
expression due to its RNAi effect.
[0203] As used herein, siRNA molecules need not be limited to those
molecules containing only RNA, but further encompasses
chemically-modified nucleotides and non-nucleotides. In addition,
as used herein, the term RNAi is meant to be equivalent to other
terms used to describe sequence specific RNA interference, such as
post transcriptional gene silencing, translational inhibition, or
epigenetics. For example, siRNA molecules of the invention can be
used to epigenetically silence genes at both the
post-transcriptional level or the pre-transcriptional level. In a
non-limiting example, epigenetic regulation of gene expression by
siRNA molecules of the invention can result from siRNA mediated
modification of chromatin structure to alter gene expression (see,
for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra
et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297,
1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,
2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,
2232-2237).
[0204] RNAi may be designed by those methods known to those of
ordinary skill in the art. In one example, siRNA may be designed by
classifying RNAi sequences, for example 1000 sequences, based on
functionality, with a functional group being classified as having
greater than 85% knockdown activity and a non-functional group with
less than 85% knockdown activity. The distribution of base
composition was calculated for entire the entire RNAi target
sequence for both the functional group and the non-functional
group. The ratio of base distribution of functional and
non-functional group may then be used to build a score matrix for
each position of RNAi sequence. For a given target sequence, the
base for each position is scored, and then the log ratio of the
multiplication of all the positions is taken as a final score.
Using this score system, a very strong correlation may be found of
the functional knockdown activity and the log ratio score. Once the
target sequence is selected, it may be filtered through both fast
NCBI blast and slow Smith Waterman algorithm search against the
Unigene database to identify the gene-specific RNAi or siRNA.
Sequences with at least one mismatch in the last 12 bases may be
selected.
[0205] Nucleic acid reagents include those which are engineered,
for example, to produce dsRNAs. Examples of such nucleic acid
molecules include those with a sequence that, when transcribed,
folds back upon itself to generate a hairpin molecule containing a
double-stranded portion. One strand of the double-stranded portion
may correspond to all or a portion of the sense strand of the mRNA
transcribed from the gene to be silenced while the other strand of
the double-stranded portion may correspond to all or a portion of
the antisense strand. Other methods of producing dsRNAs may be
used, for example, nucleic acid molecules may be engineered to have
a first sequence that, when transcribed, corresponds to all or a
portion of the sense strand of the mRNA transcribed from the gene
to be silenced and a second sequence that, when transcribed,
corresponds to all or portion of an antisense strand (i.e., the
reverse complement) of the mRNA transcribed from the gene to be
silenced.
[0206] Nucleic acid molecules which mediate RNAi may also be
produced ex vivo, for example, by oligonucleotide synthesis.
Oligonucleotide synthesis may be used for example, to design dsRNA
molecules, as well as other nucleic acid molecules (e.g., other
nucleic acid molecules which mediate RNAi) with one or more
chemical modification (e.g., chemical modifications not commonly
found in nucleic acid molecules such as the inclusion of
2'-O-methyl, 2'-O-ethyl, 2'-O-propyl, 2'-fluoro, etc. groups).
[0207] In some embodiments, a dsRNA to be used to silence a gene
may have one or more (e.g., one, two, three, four, five, six, etc.)
regions of sequence homology or identity to a gene to be silenced.
Regions of homology or identity may be from about 20 by (base
pairs) to about 5 kbp (kilo base pairs) in length, 20 by to about 4
kbp in length, 20 by to about 3 kbp in length, 20 by to about 2.5
kbp in length, from about 20 by to about 2 kbp in length, 20 by to
about 1.5 kbp in length, from about 20 by to about 1 kbp in length,
20 by to about 750 by in length, from about 20 by to about 500 by
in length, 20 by to about 400 by in length, 20 by to about 300 by
in length, 20 by to about 250 by in length, from about 20 by to
about 200 by in length, from about 20 by to about 150 by in length,
from about 20 by to about 100 by in length, from about 20 by to
about 90 by in length, from about 20 by to about 80 by in length,
from about 20 by to about 70 by in length, from about 20 by to
about 60 by in length, from about 20 by to about 50 by in length,
from about 20 by to about 40 by in length, from about 20 by to
about 30 by in length, from about 20 by to about 25 by in length,
from about 15 by to about 25 by in length, from about 17 by to
about 25 by in length, from about 19 by to about 25 by in length,
from about 19 by to about 23 by in length, or from about 19 by to
about 21 by in length.
[0208] A hairpin containing molecule having a double-stranded
region may be used as RNAi. The length of the double stranded
region may be from about 20 by (base pairs) to about 2.5 kbp (kilo
base pairs) in length, from about 20 by to about 2 kbp in length,
20 by to about 1.5 kbp in length, from about 20 by to about 1 kbp
in length, 20 by to about 750 by in length, from about 20 by to
about 500 by in length, 20 by to about 400 by in length, 20 by to
about 300 by in length, 20 by to about 250 by in length, from about
20 by to about 200 by in length, from about 20 by to about 150 by
in length, from about 20 by to about 100 by in length, 20 by to
about 90 by in length, 20 by to about 80 by in length, 20 by to
about 70 by in length, 20 by to about 60 by in length, 20 by to
about 50 by in length, 20 by to about 40 by in length, 20 by to
about 30 by in length, or from about 20 by to about 25 by in
length. The non-base-paired portion of the hairpin (i.e., loop) can
be of any length that permits the two regions of homology that make
up the double-stranded portion of the hairpin to fold back upon one
another.
[0209] Any suitable promoter may be used to control the production
of RNA from the nucleic acid reagent, such as a promoter described
above. Promoters may be those recognized by any polymerase enzyme.
For example, promoters may be promoters for RNA polymerase II or
RNA polymerase III (e.g., a U6 promoter, an H1 promoter, etc.).
Other suitable promoters include, but are not limited to, T7
promoter, cytomegalovirus (CMV) promoter, mouse mammary tumor virus
(MMTV) promoter, metalothionine, RSV (Rous sarcoma virus) long
terminal repeat, SV40 promoter, human growth hormone (hGH)
promoter. Other suitable promoters are known to those skilled in
the art and are within the scope of the present invention.
[0210] Double-stranded RNAs used in the practice of the invention
may vary greatly in size. Further the size of the dsRNAs used will
often depend on the cell type contacted with the dsRNA. As an
example, animal cells such as those of C. elegans and Drosophila
melanogaster do not generally undergo apoptosis when contacted with
dsRNAs greater than about 30 nucleotides in length (i.e., 30
nucleotides of double stranded region) while mammalian cells
typically do undergo apoptosis when exposed to such dsRNAs. Thus,
the design of the particular experiment will often determine the
size of dsRNAs employed.
[0211] In many instances, the double stranded region of dsRNAs
contained within or encoded by nucleic acid molecules used in the
practice of the invention will be within the following ranges: from
about 20 to about 30 nucleotides, from about 20 to about 40
nucleotides, from about 20 to about 50 nucleotides, from about 20
to about 100 nucleotides, from about 22 to about 30 nucleotides,
from about 22 to about 40 nucleotides, from about 20 to about 28
nucleotides, from about 22 to about 28 nucleotides, from about 25
to about 30 nucleotides, from about 25 to about 28 nucleotides,
from about 30 to about 100 nucleotides, from about 30 to about 200
nucleotides, from about 30 to about 1,000 nucleotides, from about
30 to about 2,000 nucleotides, from about 50 to about 100
nucleotides, from about 50 to about 1,000 nucleotides, or from
about 50 to about 2,000 nucleotides. The ranges above refer to the
number of nucleotides present in double stranded regions. Thus,
these ranges do not reflect the total length of the dsRNAs
themselves. As an example, a blunt ended dsRNA formed from a single
transcript of 50 nucleotides in total length with a 6 nucleotide
loop, will have a double stranded region of 23 nucleotides.
[0212] As suggested above, dsRNAs used in the practice of the
invention may be blunt ended, may have one blunt end, or may have
overhangs on both ends. Further, when one or more overhang is
present, the overhang(s) may be on the 3' and/or 5' strands at one
or both ends. Additionally, these overhangs may independently be of
any length (e.g., one, two, three, four, five, etc. nucleotides).
As an example, STEALTH.TM. RNAi is blunt at both ends. Also
included are sets of RNAi and those which generate RNAi. Such sets
include those which either (1) are designed to produce or (2)
contain more than one dsRNA directed against the same target gene.
As an example, the invention includes sets of STEALTH.TM. RNAi
wherein more than one STEALTH.TM. RNAi shares sequence homology or
identity to different regions of the same target gene.
[0213] A protein, fragment or nucleic acid described herein
sometimes is in association with detectable label. The detectable
label can be covalently linked to the reagent, and sometimes is in
association with the reagent in a non-covalent linkage. Methods for
attaching such binding pairs to reagents and effecting binding are
known to the artisan. Any detectable label suitable for detection
of an interaction or biological activity in a system can be
appropriately selected and utilized by the artisan. Examples of
detectable labels are fluorescent labels such as fluorescein,
rhodamine, and others (e.g., Anantha, et al., Biochemistry (1998)
37:2709 2714; and Qu & Chaires, Methods Enzymol. (2000) 321:353
369); radioactive isotopes (e.g., .sup.125I, .sup.131I, .sup.35S,
.sup.31P, .sup.3P, .sup.14C, .sup.3H, .sup.7Be, .sup.28Mg,
.sup.57Co, .sup.65Zn, .sup.67Cu, .sup.68Ge, .sup.82Sr, .sup.83Rb,
.sup.95Tc, .sup.96Tc, .sup.103Pd, .sup.109Cd, and .sup.127Xe);
light scattering labels (e.g., U.S. Pat. No. 6,214,560, and
commercially available from Genicon Sciences Corporation, CA);
chemiluminescent labels and enzyme substrates (e.g., dioxetanes and
acridinium esters), enzymic or protein labels (e.g., green
fluorescence protein (GFP) or color variant thereof, luciferase,
peroxidase); other chromogenic labels or dyes (e.g., cyanine), and
labels described previously. Use of reagents in association with a
detectable label are described in greater detail hereafter.
[0214] Cells also may be provided in a kit. A cell may over-express
or under-express protein, fragment or nucleic acid or other
molecule described herein. A cell can be processed in a variety of
manners. For example, an artisan may prepare a lysate from a cell
and optionally isolate or purify components, may transfect the cell
with a nucleic acid reagent, may fix a cell reagent to a slide for
analysis (e.g., microscopic analysis) and can immobilize a cell to
a solid phase.
[0215] A cell that "over-expresses" an protein or fragment or
nucleic acid described herein produces at least two, three, four or
five times or more of the product as compared to a native cell from
an organism that has not been genetically modified and/or exhibits
no apparent symptom of a cell-proliferative disorder.
Over-expressing cells may be stably transfected or transiently
transfected with a nucleic acid a protein or fragment or nucleic
acid described herein. A cell that "under-expresses" a protein or
fragment or nucleic acid described herein produces at least five
times less of the product as compared to a native cell from an
organism that has not been genetically modified and/or exhibits no
apparent symptom of a cell-proliferative disorder. In some
embodiments, a cell that under-expresses a protein, fragment or
nucleic acid contains no nucleic acid that can encode such a
product (e.g., the cell is from a knock-out mouse) and no
detectable amount of the product is produced. Methods for
generating knock-out animals and using associated cells are known
(e.g., Miller et al., J. Cell. Biol. 165: 407-419 (2004)). A cell
that under-expresses a protein, fragment or nucleic acid described
herein, for example, sometimes is in contact with a nucleic acid
inhibitor that blocks or reduces the amount of the product produced
by the cell in the absence of the inhibitor.
[0216] Cells include, but are not limited to, bacterial cells
(e.g., Escherichia spp. cells (e.g., Expressway.TM. HTP Cell-Free
E. coli Expression Kit, Invitrogen, California) such as DH10B,
Stb12, DH5-alpha, DB3, DB3.1 for example), DB4, DB5, JDP682 and
ccdA-over (e.g., U.S. application Ser. No. 09/518,188), Bacillus
spp. cells (e.g., B. subtilis and B. megaterium cells),
Streptomyces spp. cells, Erwinia spp. cells, Klebsiella spp. cells,
Serratia spp. cells (particularly S. marcessans cells), Pseudomonas
spp. cells (particularly P. aeruginosa cells), and Salmonella spp.
cells (particularly S. typhimurium and S. typhi cells);
photosynthetic bacteria (e.g., green non-sulfur bacteria (e.g.,
Choroflexus spp. (e.g., C. aurantiacus), Chloronema spp. (e.g., C.
gigateum)), green sulfur bacteria (e.g., Chlorobium spp. (e.g., C.
limicola), Pelodictyon spp. (e.g., P. luteolum), purple sulfur
bacteria (e.g., Chromatium spp. (e.g., C. okenii)), and purple
non-sulfur bacteria (e.g., Rhodospirillum spp. (e.g., R. rubrum),
Rhodobacter spp. (e.g., R. sphaeroides, R. capsulatus),
Rhodomicrobium spp. (e.g., R. vanellii)); yeast cells (e.g.,
Saccharomyces cerevisiae cells and Pichia pastoris cells); insect
cells (e.g., Drosophila (e.g., Drosophila melanogaster), Spodoptera
(e.g., Spodoptera frugiperda Sf9 and Sf21 cells) and Trichoplusa
(e.g., High-Five cells); nematode cells (e.g., C. elegans cells);
avian cells; amphibian cells (e.g., Xenopus laevis cells);
reptilian cells; and mammalian cells (e.g., NIH3T3, 293, CHO, COS,
VERO, C127, BHK, Per-C6, Bowes melanoma and HeLa cells). These and
other suitable cells are available commercially, for example, from
Invitrogen Corporation, (Carlsbad, Calif.), American Type Culture
Collection (Manassas, Va.), and Agricultural Research Culture
Collection (NRRL; Peoria, Ill.).
EXAMPLES
[0217] The examples set forth below illustrate but do not limit the
invention. The maturation state and lifespan of dendritic cells
(DCs) are critical for the regulation of immunity. However, current
DC preparations based on ex vivo treatment with differentiation and
maturation factors can lead to transiently active DCs that may
curtail T cell responses following re-infusion. Here, it is shown
that Akt1 levels drop rapidly following growth factor withdrawal
and are critical for pro-inflammatory signal-mediated DC survival
and maturation. Conversely, LPS or CD40 signaling stabilizes Akt1
and promotes both DC activation and survival. Also, it is shown
that Akt1-mediated survival depends on Bcl-2, but not Bcl-xL.
Adenoviral-mediated overexpression of a novel, lipid-raft targeted
Akt allele, MF-.DELTA.Akt, is sufficient for murine bone
marrow-derived DC maturation and survival, resulting in enhanced T
cell proliferation and activation, and eradication of large
pre-established thymoma as well as B16 melanoma. In addition,
transduction of human DCs with an adenovirus expressing human Akt1,
MF-.DELTA.hAkt, also significantly improves DC survival,
antigen-specific T cell proliferation and CD8+ T cell responses.
These data demonstrate that Akt1 is a critical regulator of DC
lifespan and can significantly improve the efficacy of DC-based
tumor vaccines. Results presented herein also is presented in Park
et al., Nat. Biotechnol. 2006 Dec. 24(12):1581-90.
Example 1
Rapid Down-Regulation of Akt Following Cytokine Withdrawal is
Prevented by Signals of Innate and Acquired Immune Responses
[0218] To investigate pathways involved in DC survival following
inflammatory stimuli, signaling proteins induced by LPS that have
been previously implicated in cell survival were assessed. In
addition to the NF-kappaB pathway, a variety of signaling
molecules, such as MAPK30, JAK31, PI3K24 and Src family kinases32,
are activated by LPS treatment of DCs and macrophages. Therefore,
LPS-treated bone marrow-derived DCs (BMDCs) were incubated with
effective concentrations of specific inhibitors for 48 hr to
substantially block these proteins. Treatment with PI3K and Src
kinase inhibitors significantly antagonized LPS-mediated survival,
whereas JAK and MAPK inhibitors had almost no effect (FIG. 1A).
[0219] To further study the role of PI3K in DC survival, kinetics
of Akt expression, a key down-stream effector of PI3K signaling
were determined, during GM-CSF deprivation-mediated DC death.
Within 24 hours of GM-CSF deprivation, prior to DC death, total Akt
protein levels were rapidly down regulated. In addition
down-regulation of Akt closely correlated with decreases in the
protein level of Bcl-2, known to be down-regulated during DC
maturation (FIG. 1B). To test whether innate and adaptive immune
response-triggering molecules modulate Akt protein level, leading
to DC survival, it first was confirmed that anti-CD40 mAb and LPS
both protect DCs against GM-CSF deprivation-mediated death (FIG.
1C). While GM-CSF deprivation consistently down-regulated Akt
protein levels, LPS or anti-CD40 treatment prevented this
down-regulation of Akt. Like previous reports, it was determined
that minimal manipulation of DCs, such as re-plating, contributed
to DC activation, reflected by DC maturation markers (data not
shown) and the increase in Akt phosphorylation at day 2 after
GM-CSF withdrawal. By contrast, LPS and CD40 signals induced high
Akt phosphorylation and protein levels on day 4, suggesting that
LPS and CD40 stimulation regulate not only the phosphorylation
state but also the steady-state protein level of Akt, thereby
promoting DC survival (FIG. 1D).
[0220] To further test the hypothesis that PI3K and Akt are common
regulators for immune response-mediated DC survival, we evaluated
effects of various concentrations (0.05 .mu.M-5 .mu.M) of another
PI3K inhibitor, wortmannin, on DCs treated with LPS or anti-CD40.
Even low wortmannin concentrations (0.05-0.5 .mu.M), having little
effect on other cell types (data not shown), led to death of both
LPS- and anti-CD40-treated DCs, implicating an essential role for
PI3K for DC survival (FIG. 1E). Anti-apoptotic molecules Bcl-x and
Bcl-2 are essential in preventing DC death and are purportedly
differentially regulated during innate and acquired immune
responses 12. Therefore, Bcl-2 and Bcl-xL protein levels were
determined in wortmannin-treated DCs pre-exposed to LPS or
anti-CD40. Consistent with previous findings, GM-CSF deprivation
down-regulated Bcl-xL as well as Bcl-2, but LPS and anti-CD40
treatment reversed this effect. In the presence of wortmannin with
LPS or anti-CD40, only Bcl-2 protein was rapidly down regulated,
whereas protein levels of Bcl-xL were stable for at least 3 days
(FIG. 1F). By contrast, LPS and anti-CD40-induced Bcl-xL did not
prevent DC death triggered by PI3K inhibition, suggesting that Akt
and Bcl-2 were critical effectors of PI3K-dependent DC survival,
and Bcl-xL was regulated independently.
[0221] A description of FIGS. 1A to 1F follows. FIG. 1A shows PI3K
and Src kinase are involved in LPS-mediated DC survival. Bone
marrow-derived CD11c+ DCs were incubated for 16 hr with LPS (1
.mu.g/ml) along with the indicated concentrations (.mu.M) of PI3K
inhibitor Ly294002, JAK inhibitor AG490, MAPK inhibitor PD98059 or
Src kinase inhibitor PP2. Cell viability was assessed 2 days later
using propidium iodide (PI) staining FIG. 1B shows down-regulation
of Akt and Bcl-2 following GM-CSF withdrawal. Total Akt and Bcl-2
protein levels of BMDCs were determined after incubation for 1-4
days without GM-CSF. FIG. 1C shows following GM-CSF withdrawal,
BMDCs were treated for 16 hr with 1 .mu.g/ml of LPS( ), 10 .mu.g/ml
of anti-CD40 antibody (.tangle-solidup.) or neither (.box-solid.)
before incubation for indicated times, and cell viability was
assessed by PI staining FIG. 1D shows LPS and anti-CD40 prevent the
down-regulation in BMDCs of phospho-Akt and total Akt, as
determined by Western blotting using anti-pAkt-S473 and anti-Akt
Abs, respectively. FIG. 1E shows PI3K is essential for both LPS and
anti-CD40-mediated DC survival. DC viability was assessed by PI
staining at indicated time points after LPS or alpha-CD40 treatment
along with 0 (.box-solid.), 0.05 ( ), 0.5 (.largecircle.) or 5
(.quadrature.) .mu.M wortmannin. FIG. 1F shows down-regulation of
Akt correlates with Bcl-2, but not Bcl-xL, expression levels.
Protein levels were determined by Western blotting at indicated
time points. The amount of loaded proteins was normalized to actin.
All data represent two (FIGS. 1B, 1D and 1F) or three (FIGS. 1A, 1C
and 1E) independent experiments with similar results. Error bars
represent S.D. of duplicate measurements (FIGS. 1A and 1E).
Example 2
Activation and Maintenance of DC Survival Requires Akt1
[0222] Despite an essential role for PI3K in DC survival and the
commonly pivotal position of Akt in PI3K signaling, it was also
possible that the observed Akt down-regulation following GM-CSF
withdrawal was a secondary outcome of DC death. Therefore, to
directly evaluate the role of Akt, it was determined whether
endogenous Akt activity was essential for LPS or CD40-mediated DC
survival. First, expression pattern of Akt subtypes was assessed in
DCs. Of the two Akt isoforms commonly found in hematopoietic cells,
Akt1 was identified as the predominant isoform in BMDCs (FIG. 2A).
Based on these results, LPS or CD40-mediated survival of DCs was
tested from Akt1+/+ and Akt1-/- mice. Although the total protein
level of Akt is much less in Akt1-deficient BMDCs (FIG. 2B),
Akt1-/- DCs showed comparable numbers and viability relative to
Akt1+/+ DCs after BMDC isolation (data not shown). However, GM-CSF
withdrawal for 48 hr reduced the survival of Akt1-/- DCs relative
to Akt1+/+ DCs. In addition, the protective effect of LPS and CD40
was completely abolished in Akt1-/- DCs, indicating that innate
(LPS) and acquired (CD40) immune response-related DC survival
requires Akt1 (FIG. 2C).
[0223] To further confirm the role of Akt1 in LPS-mediated DC
survival from cytokine deprivation, growth factor-starved BMDCs
were treated with moderate concentrations (5 .mu.M) of Akt specific
inhibitor Akt-I (IC50=5 .mu.M) along with LPS for 16 hr, followed
by incubation for an additional 48 hr in GM-CSF-free DC medium.
Consistently, Akt-I significantly reduced LPS-mediated DC survival
(FIG. 9C). Moreover, transfection of Akt-RNAi (100 nM), a synthetic
Akt1-specific siRNA36, into BMDCs suppressed Akt expression and
significantly (P<0.005) increased DC death even in the presence
of LPS compared with control RNAi (FIG. 9A, 9B). Therefore, it was
determined Akt1 is a critical mediator of DC survival signals, and
down-regulation of Akt1 is a major regulatory mechanism to control
DC longevity.
[0224] In addition to promoting DC survival, Akt can induce
NF-kappaB activity through IKKbeta phosphorylation, critical for DC
activation and maturation. However, the role of Akt in DC
differentiation from bone marrow precursors has not been tested.
Therefore, to investigate the effects of Akt1 on DC differentiation
and activation, surface expression of several MHC and costimulatory
molecules was assessed in Akt1+/+ and Akt1-/- BMDCs, including
CD40, CD80, MHC class I Kb and MHC class II I-Ab. Although there
was no difference in viability, the surface expression of lineage
markers, CD11b and CD11c, and several MHC and costimulatory
molecules on day 0 and 2 after DC differentiation, Akt1-/- BMDCs
showed significantly reduced expression of maturation markers, CD40
(P<0.01), CD80 (P<0.005) and MHC class I Kb (P<0.01),
relative to Akt1+/+ DCs on day 6 (FIG. 2D, FIG. 9). In addition,
Akt1-/- BMDCs revealed a large defect in the further enhancement of
maturation markers, such as CD40 and MHC class II Kb (FIG. 2F), as
well as CD80 and MHC class II I-Ab (data not shown), even after LPS
and CD40 stimulation, suggesting that Akt1 plays a critical role in
DC differentiation and activation.
[0225] To further investigate the physiological effects of Akt1
deficiency in DCs, size and cellularity of lymph nodes and spleen
were examined in Akt1-/- and Akt1+/+ mice. Although the relative
distribution of T cell, B cell and CD11c+ cells was normal with
only a partial decrease of CD8+ T cells (data not shown), the total
cell number of inguinal lymph nodes and spleen was dramatically
reduced (86% and 43%, respectively) in Akt1-/- mice when compared
to Akt1+/+ littermates (FIG. 2E, FIG. 11), supporting the tenet
that reduced survival and activation defects in Akt1-/- DCs results
in reduction of size, cellularity and likely functions of secondary
lymphoid organs.
[0226] A description of FIGS. 2A-2F follows. FIG. 2A shows analysis
of Akt isoforms in BMDCs. BMDC (DC) or control (A431) cell lysates
were immunoblotted with specific antibodies against Akt1 and Akt2.
Protein level of total Akt was determined by reprobing with
anti-Akt Ab. FIG. 2B shows analysis of Akt1 and total Akt protein
level in Akt1-/- DCs. Cell lysates from Akt1+/+ or Akt1-/- BMDCs
were immunoblotted with antibodies against Akt1 or total Akt.
Protein loading was normalized by actin immunoblotting. FIG. 1C
shows comparison of LPS-- or anti-CD40-mediated survival in Akt1+/+
or Akt-/- DCs. BMDCs from Akt1+/+ or Akt1-/- mice were untreated
(open bars) or treated with LPS (1 .mu.g/ml, gray bars) or
anti-CD40 (10 .mu.g/ml, filled bars) and further incubated in the
presence of wortmannin (0.5 .mu.M) for 48 hrs. Background % PI+ at
day 0 was subtracted from each data point. FIG. 2D shows expression
of surface markers in Akt1+/+ or Akt1-/- DCs. On day 6 after DC
differentiation, surface expression of MHC and costimulatory
molecules in Akt1+/+ or Akt1-/- BMDCs was assessed by CD40, CD80,
MHC class II Kb and MHC class II I-Ab staining along with CD11c.
MFI, is mean fluorescence intensity. FIG. 2E shows a comparison of
lymph nodes from Akt1+/+ or Akt-/- mice. Size of inguinal lymph
nodes isolated from Akt1+/+ or Akt1-/- littermates was measured
(bottom scale bar, 1 mm gap) and trypan blue staining assessed
total cell numbers within each lymph node. FIG. 2F shows reduced
maturation of Akt1-/- DCs. After 6 days of BMDC differentiation,
BMDCs in the starting wells were further treated with indicated
stimuli as above in FIG. 2C for 48 hr. DC maturation was assessed
by CD40 and MHC class II Kb staining. Numbers indicate percentage
of cells in that quadrant. Data represent two (FIGS. 2B and 2F),
three (FIGS. 2C and 2D) or six (FIG. 2E) independent experiments.
Error bars indicate mean.+-.S.D. of results derived from three
(FIGS. 2D and 2E) independent littermates. *, P<0.01, **,
P<0.005, .diamond-solid.P<0.0005.
Example 3
[0227] Akt1 is sufficient for activation and maintenance of
LPS-mediated DC survival
[0228] To further study the molecular mechanism of Akt in PI3K- and
Src kinase-mediated DC survival, we initially utilized a previously
described constitutively active Akt (M-Akt) allele, consisting of
full length Akt, targeted to intracellular membranes with a
myristoylation-targeting sequence from c-Src. However, because dual
acylation, such as palmitoylation and myristoylation, is important
for lipid raft localization of Src family kinases, c-Src, which is
only myristoylated, may be excluded from lipid rafts (FIG. 12A).
Therefore, to test the possibility that efficient lipid raft
localization of Akt improves its function, this construct was
compared to several distinct Akt constructs, containing
myristoylation-targeting sequences from Src family kinases Src, Fyn
and Lck fused to a truncated (denoted ".DELTA.") Akt (FIG. 3A). The
pleckstrin homology (PH) domain (residues 1-102) was removed to
improve Akt activity, important when PI3K activity is low. Among
the three membrane-targeting sequences, the Fyn
myristoylation-targeting sequence (MF) showed the most efficient
lipid raft localization, 2-3-fold NF-kappaB induction, about a
6-fold-increased Akt-5473 and GSK3 phosphorylation, and enhanced
viability of transfected Jurkat cells following treatment with PI3K
inhibitors (FIG. 3A and FIG. 12). To further enhance Akt activity,
Fyn myristoylated .DELTA.Akt (MF-.DELTA.Akt) was generated, which
carried point mutations at both key phosphorylation sites (E308 and
D473). However, these mutations failed to further improve reporter
gene activity (data not shown). Therefore, MF-.DELTA.Akt is
referred to as "functionally optimized" Akt and was used in most
subsequent experiments. For improved expression in BMDCs, we
generated replication-defective adenovirus (Ad),
Ad-MF-.DELTA.Akt.
[0229] To evaluate the functional activity of Ad-MF-.DELTA.Akt in
BMDCs, phosphorylation of GSK3alpha/beta was examined in transduced
cells. Consistently, Ad-MF-.DELTA.Akt led to higher GSK3alpha/beta
phosphorylation in BMDCs, compared with Ad-M-Akt39 (FIG. 3B). It
was next tested whether DCs transduced with either Ad-M-Akt or
Ad-MF-.DELTA.Akt escaped death triggered by PI3K inhibition. In
vitro DC survival assays indicated that both constructs, unlike
Ad-GFP, significantly inhibited wortmannin-mediated lethality in
BMDCs (P<0.005). In addition, Ad-MF-.DELTA.Akt protected DCs
better than Ad-M-Akt (FIG. 3c, d, P<0.01). Meanwhile,
over-expression of functionally optimized Akt had no effect on
PP2-mediated DC death (FIG. 3E). These findings indicated that
functionally optimized Akt almost completely suppressed DC death
initiated by PI3K, but not Src, inhibition, suggesting that PI3K
and Src kinase likely control DC survival by distinct pathways.
[0230] To directly investigate the effects of Akt1 on DC phenotype,
BMDCs were treated with either Ad-MF-.DELTA.Akt or LPS and Ad-GFP
as positive and negative controls, respectively. Flow cytometry
analysis after 48 hr of treatment at about 50% transduction
efficiency, revealed that Ad-MF-.DELTA.Akt-transduced DCs had
consistently enhanced expression of DC activation markers,
including I-Ab, CD40, CD80 and Kb compared with Ad-GFP-transduced
DCs, implicating that Akt activity is sufficient to induce DC
activation and maturation (FIG. 3F).
[0231] A description of FIGS. 3A-3F follows. FIG. 3A shows a
schematic of distinct Akt alleles comprised of
myristoylation-targeting sequences (residues 1-16) from Src (MS),
Fyn (MF) or Lck (ML) fused to PH domain-deleted Akt (.DELTA.Akt
103-408). Activation state and expression of Ms-.DELTA.Akt (MS),
MF-.DELTA.Akt (MF), and ML-.DELTA.Akt (ML) were determined using
anti-pAkt-S473 and anti-HA antibodies, respectively. FIG. 3B shows
phosphorylation of substrate GSK3alpha/beta in BMDCs transduced by
adenovectors expressing constitutive Akt. Activity and expression
of Ad-MF-.DELTA.Akt (MF-.DELTA.Akt) or Ad-MS-Akt (M-Akt) were
determined using anti-phospho-GSK3 (pGSK3) and anti-HA antibodies
respectively. Protein loading was normalized by alpha-tubulin
immunoblotting. FIGS. 3C and 3D show MF-.DELTA.Akt protects BMDCs
from wortmannin-mediated death in vitro. BMDCs transduced with
adenoviruses expressing GFP, M-Akt or MF-.DELTA.Akt at 100 m.o.i.
were further incubated with LPS (1 .mu.g/ml) and wortmannin (W, 0.5
nM) for 2 days. In FIG. 3C cell death was assessed by PI staining
under indicated treatment conditions (open diagrams) compared to
LPS alone (filled reference histogram). Numbers represent %-gated
region of open diagrams. FIG. 3D is a graphical representation of
FIG. 3C. FIG. 3E shows Src and PI3K have independent roles in DC
survival. Viability of BMDCs was assessed as above in FIG. 3D
except for replacement of wortmannin with Src kinase inhibitor, PP2
(20 .mu.M). FIG. 4F shows MF-.DELTA.Akt induces BMDC activation.
Two days after indicated treatments of BMDCs, CD11c+ DC activation
was assessed by CD40, CD80, Kb and I-Ab staining. Numbers indicate
percentage of cells in upper right quadrant. Data represent at
least two independent experiments. Error bars represent S.D. of
triplicate (FIGS. 3D and 3E) measurements. *, P<0.005, **,
P<0.01.
Example 4
Bcl-2 is Required for Akt-Mediated DC Survival
[0232] Since the kinetics of Akt protein down-regulation following
cytokine withdrawal correlated with Bch 2 protein levels (FIG. 1B),
it was hypothesized that Bcl-2 might be a critical downstream
effector of PI3K/Akt. To test this hypothesis, Akt-mediated
survival of DCs was compared in Bcl-2+/- and Bcl-2-/- mice. Overall
viability of Bcl-2-/- DCs was similar to that of Bcl-2+/- DCs in
complete DC medium (data not shown). Consistent with previous
results, treatment of DCs with the PI3K inhibitor, wortmannin,
rapidly induced cell death both in Bcl-2+/- and Bcl-2-/- DCs.
Ad-MF-.DELTA.Akt transduction prior to wortmannin treatment,
however, overcame the deleterious effects of PI3K inhibition,
leading to enhanced survival of Bcl-2+/- DCs (FIG. 4, left). By
contrast, the protective effect of Ad-MF-.DELTA.Akt-transduction
was completely abolished in Bcl-2-/- DCs, indicating that Akt
mediated DC survival requires Bcl-2 (FIG. 4, right). In FIG. 4,
BMDCs from Bcl2+/- or Bcl2-/- mice were left untreated
(.tangle-solidup.) or transduced with Ad-GFP(.box-solid.) or
Ad-MF-.DELTA.Akt ( ) and further incubated in the presence of
wortmannin (W, 0.5 .mu.M) for indicated times. Viability was
assayed by flow cytometry based on PI uptake in triplicate. Data
represent three independent experiments with similar results. Error
bars represent the mean.+-.S.D. of triplicate measurements.
Example 5
Akt-Transduced DCs Show Prolonged Longevity In Vitro and In
Vivo
[0233] Previous reports demonstrated a correlation between
prolonging DC lifespan and the adjuvant potency of DC-based
vaccines and T cell dependent immunity. Therefore, whether the
induction of Akt activity promoted the survival of BMDCs under
various conditions was tested. Initially, effects of Ad-M-Akt and
LPS on DC survival were compared following growth factor
deprivation. As shown in FIGS. 5A and 5B, DCs pre-incubated with
LPS (1 .mu.g/ml) or infected with Ad-M-Akt maintained viability for
at least 5 days after GM-CSF withdrawal, whereas untreated or
Ad-GFP-transduced DCs underwent significant cell death by day 4,
suggesting that the induction of Akt inhibits cell death signals
mediated by GM-CSF withdrawal in vitro.
[0234] To further investigate Akt-mediated survival of DCs in vivo,
the viability of Ad-MF-.DELTA.Akt-transduced DCs with LPS-treated
or Ad-GFP-transduced DCs was compared in draining lymph nodes. DCs
were stained with the fluorescent dye CFSE followed by subcutaneous
(s.c.) delivery into the hind legs of syngeneic mice (FIG. 5C). On
day 5 after delivery, the quantity of CFSE+ MF-.DELTA.Akt-DCs
residing in the draining popliteal lymph nodes was .about.1% of
total lymph node cells, which was a 2-3-fold higher percentage than
control Ad-GFP-DC-treated mice. Consistent with previous findings,
the percentage of CFSE+ DCs from control mice injected with
untreated or LPS-treated DCs rapidly decreased at later time
points, whereas Akt-transduced DCs sustained their disproportionate
representation for at least 10 days post-delivery (FIG. 5D).
Prolonged survival of Ad-GFP-transduced DCs at day 10 may be due to
transfer of long-lived EGFP to resident phagocytes. In addition,
the average volume of draining lymph nodes exposed to
MF-.DELTA.Akt-DCs was approximately 4-8-fold more than control
mice, indicating that Ad-MF-.DELTA.Akt transduction enhances the
number of lymph node resident leukocytes compared to all negative
control groups as well as LPS-treated DCs (FIG. 5E). Because the
arrival of CFSE+ DCs does not apparently differ significantly among
the groups in the first 24 hr after injection, these data strongly
suggest that ex vivo transduction of DCs with Ad-MF-.DELTA.Akt
promotes their prolonged lifespan, which results in sustained
immunity by overcoming various DC death signals in lymphoid
tissues.
[0235] A description of FIGS. 5A-5E follows. FIGS. 5A and 5B show
the anti-apoptotic effect of Ad-M-Akt on DC death, in vitro. BMDCs
were left untreated (.quadrature.), or treated with
LPS(.box-solid.), Ad-GFP(.largecircle.) or Ad-M-Akt ( ) at 100
m.o.i. and further incubated for 2 to 5 days without GM-CSF. In
vitro DC apoptosis was examined by Annexin V-PE staining.
Histograms of Day 5 (open histogram) were compared to that of Day 2
(filled histogram). In FIG. 5A, numbers represent %-gated region of
open histograms. In FIG. 5B, error bars indicate mean.+-.S.D. of
results pooled from three independent experiments. *, P<0.05
between Ad-EGFP and Ad-M-Akt. FIGS. 5C, 5D and 5E show effects of
Ad-MF-.DELTA.Akt on BMDC longevity, in vivo. CFSE-stained BMDCs
were left untreated (.quadrature.), or treated with
LPS(.box-solid.), Ad-GFP(.largecircle.) or Ad-MF-.DELTA.Akt ( ) for
2 hr before injection into the hind legs of syngeneic mice (n=2-4
per time point). After indicated times, cells from draining LNs
(popliteal) were stained with PI. Within PI-populations, CFSE+
cells were analyzed by flow cytometry. FIG. 5C shows representative
dot plots of CFSE+ populations at day 5 (P<0.05, between
Ad-M-Akt and all controls). Boxed numbers indicate CFSE+
populations. In FIG. 5D background CFSE+ from PBS control (-) was
subtracted for each value. Error bars indicate mean.+-.S.E.M. Data
represent three independent experiments with similar results. FIG.
5E shows representative LNs isolated from indicated mice on day 7
and 10.
Example 6
[0236] Akt Improves DC Ability to Stimulate T Cell Functions
[0237] In addition to promoting DC survival, optimal maturation and
DC activation, accompanied by IL-12 production, are important for
naive T cell priming, leading to T cell proliferation and IFN-gamma
production. However, it has been suggested that PI3K negatively
regulates IL-12 production in DCs. Therefore, to test whether
enhanced survival and activation of MF-.DELTA.Akt-DCs promotes T
cell function, the proliferative response of allogeneic (BALB/c)
and syngeneic OT-1 T cells (expressing transgenic TCRs specific for
Kb-restricted OVA257-264 peptide (SIINFEKL)) to peptide-pulsed,
Akt-transduced DCs was examined. After 24-hr incubation of DCs with
syngeneic splenocytes from OT-1 mice, Ad-MF-.DELTA.Akt-transduced
DCs induced more T cell proliferation than Ad-GFP-transduced DCs,
which was similar to LPS-treated DCs. Moreover, after .about.72 hr
incubation, MF-.DELTA.Akt-DCs induced about two-fold higher T cell
proliferation than DCs activated with LPS (FIG. 6A).
Ad-M-Akt-transduced DCs also consistently showed 5-7-fold higher
allogeneic T cell proliferation than DCs pulsed with LPS or Ad-GFP
at low DC:effector ratios after 72 hours (FIG. 6B). These data
support a model in which Akt induces DC maturation and survival,
leading to robust T cell proliferation.
[0238] To further determine whether upregulated Akt improves
DC-mediated activation of T cells, secretion of IFN-gamma by OT-1
splenocytes incubated with Ad-MF-.DELTA.Akt- or control
virus-pulsed DCs was examined Peptide-pulsed and Akt or GFP
virus-transduced DCs were cultured with pooled spleen cells from
OT-1 mice for 24 hr or 72 hr. Then, the OVA peptide-specific
splenocyte response was measured ex vivo by IFN-gamma ELISPOT
assay. LPS-- or Ad-GFP-transduced DCs showed approximately the same
numbers of IFN-gamma-producing cells, relative to untreated DCs. In
contrast, MF-.DELTA.Akt-DCs induced at least 7-fold higher
IFN-gamma production by splenocytes (on both day 1 and day 3 (not
shown)) than DCs treated with LPS or Ad-GFP (FIG. 6C). Therefore,
in this experimental model, MF-.DELTA.Akt-DCs elicit relatively
persistent immunity by stimulating proliferation and activation of
antigen specific CD8+ T cells.
[0239] A description of FIGS. 6A-6C follows. FIGS. 6A and 6B show
MF-.DELTA.Akt induces DC function to stimulate antigen-specific and
allogeneic T cell proliferation. BMDCs alone (.quadrature.) or
pulsed with SIINFEKL peptide for 2 hr (FIG. 6A) were left untreated
(.box-solid.) or treated with LPS (A), Ad-EGFP(.tangle-solidup.) or
Ad-M.sub.F-.DELTA.Akt (FIG. 6A, ) and Ad-M-Akt (FIG. 6B, ) for 16
hr. DCs were mixed in various dilutions with 1.times.10.sup.5
splenocytes from syngeneic OT-1 (FIG. 6A) or allogeneic Balb/c
(FIG. 6B) mice and incubated for an additional 1 to 3 days. T cell
proliferative responses were measured by 3H-thymidine uptake (1
mCi/well) for 16 hr. FIG. 6C shows the effect of Ad-MF-.DELTA.Akt
transduction of BMDCs on CD8+ T cell activation. DCs as treated in
FIG. 6A were incubated with splenocytes from syngeneic OT-1 mice
for 3 days. IFN-gamma+ spots were assessed. All data represent two
to three independent experiments.
Example 7
MF-.DELTA.Akt Enhances the Efficacy of DC Vaccines to Eradicate
Pre-Established Tumors
[0240] To better evaluate the clinical relevance of
MF-.DELTA.Akt-DCs, anti-tumor efficacy was tested against two
distinct models. Initially, the induction of antitumor immunity by
Ad-MF-.DELTA.Akt-transduced DC vaccines was monitored after
immunization of C57BL/6 mice bearing large (about 0.4 cm.sup.3)
subcutaneous EG.7-OVA thymomas. While control or LPS-treated and
SIINFEKL-pulsed DC vaccines failed to inhibit tumor growth or
increase survival in most animals, immunization with a single
intraperitoneal dose of peptide-pulsed MF-.DELTA.Akt-DCs led to
significant tumor growth inhibition (P<0.05) (FIG. 7A, FIG. 13).
At early time points, MF-.DELTA.Akt-DCs successfully suppressed all
pre-established EG.7-OVA tumors, although 2 of 5 tumors eventually
relapsed (FIG. 13B). To measure sustained antigen-specific T cell
responses in tumor-bearing mice, H-2 Kb OVA257-264 tetramer
analysis was performed on peripheral blood CD8+ T cells harvested
14 days after vaccination. Consistent with tumor suppression, this
analysis showed that vaccination with peptide-pulsed
MF-.DELTA.Akt-DCs led to an expanded population of OVA257-264
antigen-specific CD8+ T cells (FIG. 7B). Anti-tumor effects of
MF-.DELTA.Akt-DCs against a "natural" tumor associated antigen,
tyrosinase-related protein (TRP)2, that is expressed in poorly
immunogenic B16 melanoma, next was tested. Once again,
peptide-pulsed LPS-treated mature DCs were not significantly better
than control DCs. However, immunization with a single or triple
intraperitoneal dose of TRP-2 peptide-pulsed MF-.DELTA.Akt-DCs
significantly reduced the growth of pre-established B16 melanoma,
leading to increased survival of tumor-bearing mice relative to
vaccination with control DCs (p=0.003) or LPS-DCs (p=0.006). (FIG.
7C, FIG. 7D). In addition, it was observed that only mice
vaccinated with MF-.DELTA.Akt-DCs (5 out of 9) showed clinical
responses, such as extended reduction of tumor growth associated
with tumor necrosis and frequently severe central ulceration. Among
those 5 mice, one mouse exhibited stable tumor size and one mouse
bearing an .about.2 cm.sup.3 peak tumor size completely rejected
its tumor without relapse up to 55 days (FIG. 13C and FIG.
13D).
[0241] To further assess the induction of recall memory responses
in MF-.DELTA.Akt-BMDC-treated mice, long-lived CD8+ memory
responses to TRP-2 were tested in tumor naive mice 1 week after a
booster DC vaccination that followed two months after three initial
biweekly vaccinations. Up to 30% of all peripheral blood CD8+ T
cells were TRP-2-specific from MF-.DELTA.Akt-DC-vaccinated mice,
whereas only 1-3% of all peripheral blood CD8+ T cells from control
DC-treated mice were TRP-2-specific (P<0.0005) (FIG. 7E, FIG.
14). Concomitantly, up to 3 months after the initiation of DC
treatment, no evidence of autoimmunity-induced depigmentation (i.e.
vitiligo) or toxicity was observed, including measurements of body
weight, complete blood cell counts and serum biochemistry (e.g.
AST, ALT, glucose, blood urea nitrogen, creatine kinase, etc) (data
not shown). These findings support the hypothesis that upregulation
of Akt activity in DCs improves DC function, producing enhanced
long-term anti-tumor effects even in the aggressive, poorly
immunogenic B16 tumor model without apparent autoimmunity and
toxicity.
[0242] A description of FIGS. 7A-7E follows. FIG. 7A shows growth
of pre-established EG.7-OVA tumors following single vaccinations
with Akt-transduced DCs. Syngeneic C57BL/6 mice (n=5 per group)
challenged with 2.times.10.sup.6 EG.7-OVA thymoma cells at day 0
were treated at day 7 with PBS (A) or vaccinated with
2.times.10.sup.6 BMDCs pulsed SIINFEKL peptide (20 .mu.g/ml) alone
(.largecircle.) or treated with LPS (1 .mu.g/ml) ( ), 100 m.o.i.
Ad-GFP(.quadrature.) or Ad-M.sub.F-.DELTA.Akt (.box-solid.), and
tumor sizes were estimated as w.sup.2*1*0.5236 and recorded
biweekly. Numbers indicate fraction of mice bearing palpable tumors
(>0.1 cm.sup.3). .diamond-solid., P<0.05; MF-.DELTA.Akt-DCs
versus GFP-DCs. FIG. 7B shows Ad-MF-.DELTA.Akt-transduced BMDCs
significantly increased numbers of tumor antigen-specific CD8+ T
cells. For the left portion of FIG. 7B, PBMCs from indicated group
at day 21 were isolated and stained with PE-conjugated KbSIINFEKL
tetramer and FITC-conjugated CD8. The right portion of FIG. 7B
shows the mean percentage of CD8+ 30 and KbSIINFEKL tetramer
positive population in PBMCs from two to three mice per group.
.diamond-solid..diamond-solid., P<0.005. FIG. 7C shows tumor
growth and FIG. 7D shows animal survival regarding pre-established
B16 melanoma tumors following vaccinations with Akt-transduced DCs.
B16 melanoma cells were injected subcutaneously into syngeneic
C57BL/6 mice (1.times.10.sup.5 cells/mouse, n=9 or 10 per group) at
day 0. Treatment with indicated DC vaccines (2.times.10.sup.6
DCs/mouse) pulsed with TRP2 peptide (10 .mu.g/ml) was done 4 (or
5), 15 and 25 days after tumor injection (arrow). Two tumor
measurements were recorded every 3-4 days. Mice which had about 3
cm.sup.3 tumors were euthanized and plotted with the last recorded
volume. .diamond-solid., P<0.05 based on one-way ANOVA test. In
FIG. 7D, P<0.005, MF-.DELTA.Akt-DCs versus control DCs and
GFP-DCs; P<0.01, MF-.DELTA.Akt-DCs versus LPS-DCs based on log
rank test. Similar trends were observed for single dosing and
triple dosing of DC vaccine and tumors established with
0.5.times.10.sup.5 B16 melanoma cells, and for single dosing of DC
vaccine and tumors established with 1.times.10.sup.5 B16 melanoma
cells. Controls with (1) LPS and CD40 ligand, and (2) MF-.DELTA.Akt
with LPS and CD40 ligand also were tested. The trend for the latter
control followed the trend for the MF-.DELTA.Akt alone, indicating
that LPS and CD40 ligand did not further enhance DC effectiveness.
FIG. 7E shows a memory CD8+ T Cell response by peptide-pulsed
Akt-transduced DCs. Long-lived CD8+ memory responses were monitored
two months after the third vaccination and 1-week after a booster
DC vaccine following the initial three biweekly vaccinations of
tumor naive animals with TRP-2-pulsed, PBS, Ad-GFP-- or
Ad-MF-.DELTA.Akt-transduced BMDCs (2.times.10.sup.6 DCs/mouse, 6
mice/group). Recall response of antigen-specific CD8+ T cell was
measured by the percentage of the CD8+ and H-2 Kb/TRP2 tetramer
positive population in PBMCs using flow cytometry. Data shown is
gated on CD8+ T cells. *, P<0.0005. Data represent two (FIGS. 7A
and 7B) or three (FIGS. 7C and 7D) independent experiments with
comparable results. Error bars represent mean.+-.S.E.M.
Example 8
MF-.DELTA.hAkt is a Potent Adjuvant to Induce Human MoDC Longevity
and CTL Function
[0243] To test whether upregulation of Akt can also enhance human
monocyte-derived DC (MoDC) function, a "humanized" MF-.DELTA.hAkt
was developed, and subcloned it into an Ad5f35 vector to improve
transduction of human DCs. First, viability of
Ad-MF-.DELTA.hAkt-transduced MoDCs was compared with
Ad-GFP-transduced MoDCs and DCs treated with standard maturation
cocktail (MC) (i.e. IL-1, IL-6, TNF .quadrature., and PGE2). As
shown in FIG. 8A, MoDCs pre-incubated with Ad-MF-.DELTA.hAkt
maintained viability up to 4 days after GM-CSF withdrawal, whereas
Ad-GFP and MC-treated MoDCs showed extensive cell death by day 3,
suggesting that targeted upregulation of Akt also inhibits death
signals in human MoDCs and is more anti-apoptotic than standard MC.
In addition, we observed that Ad-MF-.DELTA.hAkt-transduced MoDCs
consistently enhanced expression of DC activation markers,
including CD80, CD86 and CCR7 compared with Ad-GFP-transduced
MoDCs.
[0244] To further determine whether upregulated Akt also improves
MoDC-mediated activation of antigen-specific human T cells, we
performed HLA-A2/MAGE-3271-279 tetramer analysis on peripheral
blood CD8+ T cells from healthy volunteers. Consistent with the
proliferative responses observed for allogeneic (BALB/c) and
syngeneic OT-1 T cells to Ad-MF-.DELTA.Akt-transduced DCs,
vaccination with MF-.DELTA.hAkt-transduced and peptide-pulsed DCs
led to an expanded population of MAGE-3271-279 antigen-specific
CD8+ T cells with higher IFN-gamma production (FIG. 8B). Moreover,
MF-.DELTA.hAkt-DC-mediated proliferative responses were closely
correlated with robust MAGE-3-specific CTL responses against T2
cells pulsed with MAGE-3271-279 peptide and SK-Mel-37
(HLA-A2+/MAGE3+) target cells but not against control target cells,
lacking HLA-A2 or MAGE3 (FIG. 8C). Taken together, these data
clearly showed that administration of activated Akt into human
MoDCs is a more potent adjuvant than conventional cytokine
maturation cocktail and is attractive for clinical
applications.
[0245] MoDCs also can be primed with other antigens, such as a
prostate cancer specific antigen, PSMA (e.g., SEQ ID NO: 10).
Isolated MoDCs are incubated with the antigen for a period of time
(e.g., approximately 1-2 hours) and infected with MF-.DELTA.hAkt
adenovirus described above. After administration of the resulting
MoDCs into human subjects, the proliferation response of
antigen-specific CD8.sup.+ T cells is determined. The immune
response against this antigen sometimes is compared against the
immune response of control cells (e.g., cells lacking the antigen
or a particular HLA molecule).
[0246] A description of FIGS. 8A-8C follows. FIG. 8A shows an
anti-apoptotic effect of Ad-MF-.DELTA.hAkt on human MoDC death.
MoDCs from two donors were left untreated (.largecircle.), or
treated with maturation cocktail (MC) ( ), Ad5f35-GFP(.quadrature.)
or Ad5f35-M.sub.F-.DELTA.hAkt (.box-solid.) at 100 m.o.i. and
further incubated for 1 to 4 days without hGM-CSF. In vitro DC
apoptosis was examined by PI staining FIG. 8B shows enhanced
MAGE3-specific CD8+ T cells after stimulation with
MF-.DELTA.hAkt-transduced MoDCs. Blood CD8+ T cells from a
HLA-A2-positive healthy volunteer were stimulated with DCs
untreated (-), or treated with maturation cocktail (+MC),
Ad5f35-GFP (+GFP) or Ad5f35-MF-.DELTA.hAkt (+MF-hAkt) and stained
with PE-conjugated HLA-A2/MAGE-3271-279 tetramer and
FITC-conjugated CD8. The percentages indicate the fraction of
tetramer-positive cells within the entire populations of T cells.
FIG. 8C shows MF-.DELTA.hAkt transduced DCs enhance antigen
specific CTL killing activity. Human MoDCs were pulsed with MAGE3
peptide and adenoviruses (Ad-GFP, .quadrature., and
Ad-M.sub.F-.DELTA.hAkt, .box-solid.) or MC( ) as indicated. After
three serial stimulations of autologous T cells with DCs at day 7
and 8, antigen-specific CTL activity was evaluated with .sup.51Cr
release assay. Labeled target cells included melanoma cell lines
SK- MeI-37 (MAGE3+, A2+), NA-6-MeI (MAGE3+, A2-), as well as T2
cells (MAGE3-, A2+) loaded with either MAGE3 (positive control) or
influenza M1 (IM A2.1) peptides (as negative control). Data
represent average of two donors (FIG. 8A) or three independent
experiments with similar results (FIGS. 8B and 8C). Error bars
represent mean.+-.S.D.
[0247] A description of FIGS. 9 to 14 are described hereafter. FIG.
9A shows specificity of small interfering RNA for Akt-1 (Akt-RNAi).
BMDCs were transfected with 100 nM of control RNAi (c-RNAi) or
Akt-RNAi and further incubated for indicated times. Akt protein
level was assessed by Akt immunobloting. FIG. 9B shows an effect of
Akt-RNAi on LPS-mediated DC survival. BMDCs (-) and BMDCs
transfected with c-RNAi or Akt-RNAi were incubated with LPS (1
.mu.g/ml) for 3 days. Loaded proteins were normalized by actin
immunobloting (FIG. 9A). (c) Effect of Akt inhibitor on
LPS-mediated DC survival. BMDCs were left untreated (-) or treated
with LPS with or without Akt inhibitor (Akt-i) and further
incubated for 3 days. Cell viability was assayed by PI staining.
Data represent three independent experiments with triplicate
measurements (FIGS. 9B and 9C). Error bars represent mean.+-.S.D.
*, P<0.005.
[0248] FIG. 10 shows a representative analysis of surface markers
of Akt1.sup.+/+ or Akt1.sup.-/- DCs. After 6 days of DC
differentiation, surface expression of MHC and costimulatory
molecules in Akt1.sup.+/+ or Akt1.sup.-/- BMDCs was assessed by
CD40, CD80, MHC class I K.sup.b and MHC class II I-Abstaining along
with CD11c. Numbers indicate percentage of cells in that area.
[0249] FIG. 11 shows a comparison of bone marrow and spleens
isolated from Akt1.sup.+/+ or Ak t.sup.-/- mice. Size of spleens
isolated form Akt1.sup.+/+ or Akt.sup.-/- littermates was measured
(bottom scale bar, 1 mm gap) and total cell numbers of bone marrow
(pooled from two femurs and tibias) and spleens were assessed by
trypan blue staining.
[0250] FIG. 12A shows a comparison of lipid raft localization of
myristoylation-targeting sequences (Myr 1-16) from Src (MS), Fyn
(MF) or Lck (ML) fused to GFP in 293 cells. Membrane lipid rafts
were stained with cholera toxin B (CTxB-TRITC). FIG. 12B shows
inducible lipid raft localization of Akt. 293 cells were
transfected with membrane docking protein with Fyn myristoylation
sequence (M F-FRB12) and three tandem FKBP domains fused with
.DELTA.Akt (F3-GFP-.DELTA.Akt), followed by chemically induced
dimerization (CID, AP22783, L1, B. et al. Gene Therapy (2001) 9: 2
33-244). FIG. 12C shows induced phosphorylation of substrate GSK3
by MS-.DELTA.Akt, MF-.DELTA.Akt, and ML-.DELTA.Akt in 293 cells.
FIG. 12D shows activation of NF-kappaB in Jurkat TAg cells
transiently transfected with indicated amounts of control plasmid
(.box-solid.) or plasmids encoding MS-.DELTA.Akt ( ), MF-.DELTA.Akt
(O), or ML-.DELTA.Akt (.largecircle.) along with NF-kappaB SEAP
reporter. FIG. 12E shows protection of wortmannin-mediated cell
death by MS-.DELTA.Akt (.quadrature.) or MF-.DELTA.Akt ( ) in
Jurkat TAg cells. Viability was assayed by flow cytometry of PI
uptake in triplicate (MFI; mean fluorescent intensity).
[0251] FIG. 13A shows representative examples of EG.7-OVA
tumor-bearing mice vaccinated with Ad-GFP or Ad-MF-.DELTA.Akt
BMDCs. Tumors were compared on day 7 and 14 after vaccination (day
14 and day 21 respectively). FIGS. 13B and 13D show growth of
individual pre-established EG.7-OVA tumors following single
vaccinations with Akt-transduced DCs (FIG. 13B) or individual
pre-established B16 tumors following triple vaccination with GFP-
or Akt-transduced DCs (FIG. 13D). Numbers indicate individual
tumor-bearing mice vaccinated with Akt-transduced DCs (FIG. 13B).
FIG. 13C shows representative examples of B16 melanoma
tumor-bearing mice vaccinated with Ad-GFP (GFP), LPS or
Ad-MF-.DELTA.Akt (MF-.DELTA.Akt) BMDCs. Tumors were compared on day
35 and 42 after tumor cell challenge.
[0252] FIG. 14 shows three representative animals of memory recall
response of antigen-specific CD8+ T cells by PBS (+PBS), Ad-GFP
(+GFP) or Ad-MF-Akt (+MF-.DELTA.Akt)-treated BMDCs. Long-lived CD8+
memory responses were quantified by the percentage of the CD8+ and
H-2 Kb/TRP2 tetramer positive population in PBMCs one week after a
booster vaccination that followed two months after the initial
three biweekly vaccinations. Numbers indicate percentage of cells
in that area.
[0253] Provided hereafter is a discussion of results presented in
this Example and previous Examples. DCs are the most potent
antigen-presenting cells capable of initiating an immune response
by activating rare, naive antigen-specific T cells. The outcome of
immune responses depends on DC longevity and abundance at the site
of T cell priming. Although previous studies suggested that
pro-inflammatory signals increase life span of DCs through
NF-kappaB and Bcl-2 family members, the molecular mechanism of DC
survival is still not clearly defined. Here, we show that the
PI3K/Akt pathway is an essential mediator of LPS- and
CD40-triggered survival signals that occur at the initiation of
innate and adaptive immunity, Bcl-2 is a critical down-stream
effector of Akt-mediated DC survival.
[0254] A recent report showed that pathogen-derived
pro-inflammatory signals induce in bone marrow-derived DCs both a
conserved, Bcl-xL-dependent DC survival pathway and a Bim-dependent
DC apoptotic pathway, which is transiently suppressed by Bcl-2,
serving as a "molecular timer". It was determined by the studies
reported in these Examples that the protein levels of Bcl-2 and
Bcl-xL were independently regulated by pro-inflammatory signals. In
addition, it was determined that Akt acts as a "life switch" to
control Bcl-2 levels and regulates both DC lifespan and their
ability to stimulate an immune response. Furthermore, it was
demonstrated that DC death correlated with a rapid down-regulation
of Akt following GM-CSF withdrawal. Withdrawal of other growth
factors, like IL-3, insulin, and VEGF, has also been shown to
trigger rapid down-regulation of Akt, mediated, in part, by its
degradation. For example, caspase and proteasome inhibitors have a
protective effect on Akt protein level following VEGF removal and
promote endothelial cell viability. Therefore, it is possible that
proteasome and/or caspase-targeted degradation of Akt regulate the
PI3K/Akt signaling pathway to control DC lifespan.
[0255] Although the activity of Akt is dependent on PI3K function,
the precise role of PI3K in DC function is controversial. One group
observed that the reduction of class IA PI3K activity by disruption
of p85-alpha along with wortmannin treatment enhanced IL-12
production in DCs, suggesting that PI3K may have a negative role in
IL-12 production by DCs43. However, an independent report showed
that defective DC migration and subsequent down-regulation of
adaptive immunity occurs in PI3 Kgamma-deficient mice, indicating
complex, non-redundant roles of PI3K isoforms in DC function. In
addition, other reports have shown that CD40L-induced human DC
survival is mediated by PI3K signaling, and inhibition of PI3K
significantly reduces CD40L and CpG-mediated IL-12 production by
BMDCs. A more recent study showed that tyrosine phosphorylation of
TLR3 initiated by double-strand RNA recruits and activates PI3K and
Akt, leading to full phosphorylation and activation of IRF-3,
suggesting also an essential role of PI3K/Akt in TLR3-mediated gene
expression.
[0256] Consistent with a largely stimulatory role for PI3K, it was
observed that expression of PI3K substrate Akt, via
Ad-MF-.DELTA.Akt, induces DC maturation and activation, leading to
enhanced T cell proliferation and IFN-gamma production by activated
T cells. Moreover, it was observed that Akt deficiency reduces BMDC
differentiation following GM-CSF treatment. Although expression of
lineage markers CD11b and CD11c are similar in immature BMDCs from
both Akt1+/+ and Akt1-/- mice, expression of some DC markers, such
as MHC class I molecules, was clearly reduced. In addition, Akt1-/-
BMDCs showed a large defect in the enhancement of DC activation and
maturation markers even after LPS and CD40 stimulation (FIG. 2).
Consistently, overexpression of Akt1 itself was sufficient to
enhance the expression of DC activation markers (FIG. 3F). In
addition, induced surface expression of CCR7 in
MF-.DELTA.hAkt-transduced DCs supports a model in which Akt1 plays
a positive role in DC migration. Together, these observations
demonstrate that Akt is involved in both DC activation and
maturation.
[0257] While Akt activation plays a critical role in preventing
cell death in various cell types and contexts in vitro, only a few
studies have reported that constitutively active Akt promotes
survival of target cells in vivo. Because the Src myristoylation
sequence, used in other Akt constructs, does not target proteins
efficiently to lipid rafts, where Akt is most active, their
efficacy may have been reduced. The optimization studies described
herein led to development of MF-.DELTA.Akt, comprised of the Fyn
myristoylation sequence ("MF") fused to truncated Akt, .DELTA.Akt,
lacking a partially inhibitory pleckstrin homology domain.
MF-.DELTA.Akt functions better than M-Akt in vitro and sustains DC
lifespan for at least 10 days post-delivery in vivo, indicating
that the described Akt improvements translate to improved DC
function. In an independent study, MF-.DELTA.Akt also appeared to
regulate neurite morphology as well as the survival of cultured
sensory neurons. Therefore, in addition to potentiating DC vaccine
efficacy, functionally optimized Akt may have numerous
applications. Targeting other signaling molecules directly to lipid
rafts with the myristoylation-targeting sequence from Fyn, rather
than Src, would likely improve many so-called "constitutive"
alleles.
[0258] Studies provided herein also showed that overexpressing Akt1
in BMDCs led to improved vaccination against both immunogenic
tumors and poorly immunogenic B16 tumors (FIG. 7). In addition,
adenoviral administration of MF-.DELTA.Akt provides a more robust
and extended activation of DCs relative to the potent adjuvants
such as LPS in mouse and maturation cocktails in human. Although
LPS and cytokine cocktails appeared more effective than Akt in
maturing a higher percentage of DCs in vitro, most likely due to
incomplete transduction of target cells by adenovirus,
MF-.DELTA.Akt transduced DCs achieved higher T cell proliferation,
IFN-gamma production and cytolytic activity than DCs treated with
LPS (mouse BMDCs) or maturation cocktails (human MoDCs).
MF-.DELTA.Akt-DCs also efficiently induced long-lived CD8+ memory
responses without detectable signs of autoimmunity (FIG. 7E, FIG.
14). Furthermore, in vivo, Ad-MF-.DELTA.Akt led to a significantly
improved DC-based tumor vaccine compared to antigen-loaded and
LPS-treated DCs, which have been reported to efficiently retard
tumor growth via supra-physiological T cell expansion in C57BL/6
mice bearing poorly immunogenic B16-F10 melanoma cells55. It is
proposed that the anti-tumor effects are due to the ability of
functionally optimized Akt to evade negative feedback mechanisms of
DC activation and survival, such as pro-inflammatory receptor
desensitization or downregulation13-15 and TLR-mediated
upregulation of pro-apoptotic molecules 12 which can limit the
survival signaling capacity of pro-inflammatory signals to maintain
homeostasis. In addition, in vitro transduction of DCs with
Ad-MF-.DELTA.Akt provides an alternative way to overcome some of
the toxicity associated with in vivo administration of microbial
adjuvant or systemic administration of soluble CD40L or CD40
specific monoclonal antibody.
[0259] In summary, the role of Akt1 was characterized in dendritic
cell survival and therapeutic applications were evaluated.
Previously, it has been shown that a synthetic dimerizing
drug-inducible allele of CD40 (called iCD40) leads to increased
lifespan and prolonged activation of DCs, resulting in more potent
T cell-mediated immune responses. The studies presented herein
suggest that at least a subset of the signals provided by iCD40 in
vivo may be attainable independently of pharmacological reagents.
Use of enhanced DCs should complement previous efforts to identify
tumor-associated antigens and apply in vitro expanded and matured
DCs to tumor immunotherapy.
Example 9
Materials and Methods Utilized in Examples 1-8
[0260] Mice and cell lines. Six to eight-week-old female C57BL/6
mice were purchased from the Center for Comparative Medicine
(Baylor College of Medicine, Houston, Tex.). BALB/c mice, Akt1+/-,
Bcl-2+/- and OT-1 TCR transgenic mice, responsive to OVA257-264
peptide (Kb-restricted SIINFEKL), were purchased from Jackson
laboratory. Akt1-/- and Bcl-2-/- mice were obtained by inbreeding.
OVA-expressing EG7 thymoma cells were purchased from American
Tissue Culture Collection and maintained in RPMI 1640 medium
containing 10% FBS, 0.4 mg/ml G418, 50 .mu.M beta-mercaptoethanol,
1 mM sodium pyruvate, and antibiotics. All mice were housed in the
pathogen-free units of Texas Mouse Facility at Baylor College of
Medicine.
[0261] Reagents. Recombinant murine GM-CSF and IL-4 were purchased
from RDI Research Diagnostics. E. Coli LPS and anti-mouse CD40 mAb
(3/23) were purchased from Sigma and BD Pharmingen respectively.
The PI3-Kinase inhibitors LY294002 and wortmannin, ERK inhibitor
PD98059, Src kinase inhibitor PP2, JNK inhibitor AG490 and Akt
inhibitor were purchased from Calbiochem. The following antibodies
were used for Western blotting: anti-Akt, anti-phospho-Akt (Ser473)
and anti-phospho-GSK3alpha/beta (Ser21/9) (Cell Signaling
Technology), anti-Akt1 and anti-Akt2 (Upstate cell signaling
solutions), anti-Bcl-2 and anti-Bcl-XL (BD Biosciences), anti-actin
(1-19) and anti-alpha-tubulin (Santa Cruz Biotechnology), and
anti-mouse hemagglutinin (HA) epitope (HA-11, Covance). CFSE was
purchased from Molecular Probes (Eugene). DC death was assessed
using Propidium Iodide (PI; Sigma) and PE-conjugated Annexin V (BD
Pharmingen). OVA257-264 peptide (SIINFEKL) and MAGE-3271-279
(FLWGPRALV) were purchased from (Tetramer Core Facility, Baylor
College of Medicine). TRP2 peptide (VYDFFVWL) was utilized in the
studies.
[0262] Preparation of Plasmids and Recombinant Adenoviruses. The
recombinant adenovirus encoding constitutively active Akt
(Ad-M-Akt) is described in Guha, M. et al., Lipopolysaccharide
activation of the MEK-ERK1/2 pathway in human monocytic cells
mediates tissue factor and tumor necrosis factor alpha expression
by inducing Elk-1 phosphorylation and Egr-1 expression. Blood 98,
1429-39 (2001). Preparation of plasmids and generation of
recombinant adenovirus encoding myristoylation-targeting sequences
from Fyn kinase fused to a PH domain truncated murine Akt1
(Ad-MF-.DELTA.Akt) or human Akt1 (Ad5f35-MF-.DELTA.hAkt) are
described hereafter. Large-scale expansion of adenoviruses
expressing M-Akt (Ad-M-Akt), MF-.DELTA.Akt (Ad-MF-.DELTA.Akt),
MF-.DELTA.hAkt (Ad5f35-MF-.DELTA.hAkt) and control adenovirus
encoding EGFP (Ad-GFP for mouse DCs and Ad5f35-GFP for human DCs)
was carried out in the Viral Vector Core Facility (Baylor College
of Medicine).
[0263] For the construction of Src-, Fyn- or Lck-myristoylated
.DELTA.Akt or EGFP, annealed oligonucleotide duplexes encoding Src,
Fyn or Lck myristoylation targeting sequences (Src; 5MyrS17:
5'-ggccaccatgggtagcaacaagagcaagcccaaggatgccagccag cggcgccgcagcc-3',
3MyrS17:
5'-tcgaggctgeggcgccgctggctggcatccttgggcttgctcttgttgctacccatggtgg-
ccgc-3', Fyn; 5MyrL17:
5'-ggccaccatgggctgtgtctgcagctcaaaccctgaagatgactggatggagaacattc-3',
3MyrL17:
5'-tcgagaatgttctccatccagtcatcttcagggtttgagctgcagacacagcccatggtgg-
ccgc-3', Lck; 5MyrF17:
5'-ggccaccatgggctgtgtgcaatgtaaggataaagaagcaacaaaactgacggaggagc-3',
3MyrF17:
5'-tcgagctcctccgtcagttttgttgcttctttatccttacattgcacacagcccatggtgg-
ccgc-3') were subcloned into expression vector, pBJ5(Spencer'93) at
Sst II and Xho I. Fragments encoding Sal I-linkered EGFP or Sal
linkered .DELTA.Akt (lacking its PH domain), which were removed
from plasmids pSH1/M-.DELTA.Akt-E (L1, B. et al. Gene Therapy
(2001) 9: 233-244), were subcloned in-frame into the Xho I and Sal
I sites downstream of each myristoylation-targeting sequence. To
generate adenovirus, Ad-MF-.DELTA.Akt, expressing doubly acylated
.DELTA.Akt, MF-.DELTA.Akt fragments were isolated from
pBJ5-MF-.DELTA.Akt by Not I/Klenow and EcoR I digestion and
subcloned into SmaI/EcoRI-digested shuttle vector pDNR-CMV to
create pDNR-MF-.DELTA.Akt. Finally, recombinant adenoviral vector,
pLP-Adeno-X-MF-.DELTA.Akt, encoding MF-.DELTA.Akt, was generated by
Cre-loxP-mediated recombination according to the Adeno-X-Expression
System 2 (BD Biosciences) protocol. To generate adenovirus
expressing MF-.DELTA.hAkt (Ad5f35-MF-human .DELTA.Akt1), human
.DELTA.Akt1 (lacking its PH domain) fused with myristoylation
targeting sequence (MF) was subcloned into shuttle vector,
pShuttle-X, sequenced, I-CeuI/PI-SceI-digested and transferred into
similarly digested Ad5f35 (BCM Gene Vector Lab). Generation,
purification and titration of recombinant adenoviruses expressing
MF-.DELTA.Akt (Ad-MF-.DELTA.Akt) and MF-h.DELTA.Akt
(Ad5f35-MF-h.DELTA.Akt) were performed as described in the
manufacturer's protocol (BD Biosciences).
[0264] Mouse and human DC preparation. Bone marrow derived
dendritic cells (BMDCs) from six to eight-week-old wild type
C57BL/6 mice and Akt1-/- (C57BL/6) mice or two to three-week-old
Bcl-2+/- and Bcl-2-/- mice were generated as described previously
with minor modifications (Inaba, K. et al. Generation of large
numbers of dendritic cells from mouse bone marrow cultures
supplemented with granulocyte/macrophage colony-stimulating factor.
J Exp Med 176, 1693-702 (1992)). Briefly, bone marrow cells were
isolated from mouse femurs and tibias and cultured in DC medium
(RPMI 1640 medium containing 10% FBS, 50 .mu.M
beta-mercaptoethanol, and antibiotics in the presence of 10 ng/ml
each of mouse GM-CSF and IL-4). Half the culture medium was
replaced every other day. On day 6, BMDCs were collected and
enriched with CD11c microbeads (Miltenyi Biotec). For the
generation of human monocyte-derived DCs (MoDCs) from healthy
donors, peripheral blood mononuclear cells (PBMCs) were isolated by
Lymphoprep (Nycomed Pharma AS, Oslo, Norway) and allowed to adhere
in culture plates for 2 hr. Nonadherent cells were removed by
extensive washes and adherent cells were cultured for 5 days in
serum free DC medium (CellGenix) with 500 U/m1 of hIL-4 and 800
U/ml hGM-CSF (R&D Systems). For cell survival experiments,
MoDCs precursors were purified by positive sorting using CD14
microbeads (Miltenyi Biotec) from PBMCs and cultured as described
above.
[0265] In vitro mouse and human DC survival assay. Purified mouse
BMDCs (2.times.10.sup.6 cells/ml) were incubated with combinations
of LPS (1 .mu.g/ml), anti-CD40 (10 .mu.g/ml) and chemical
inhibitors as indicated in the Results section or transduced with
Ad-GFP, Ad-M-Akt or Ad-MF-.quadrature.Akt vectors at 100 m.o.i.
under GM-CSF-free conditions. For human DC survival assays,
purified human MoDCs (2.times.106 cells/ml) were transduced with
adenoviruses (Ad5f35-GFP or Ad5f35-MF-.DELTA.hAkt) at 100 m.o.i. or
stimulated with maturation cocktail (MC) containing 10 ng/ml
TNF-alpha, 10 ng/ml IL-1beta, 150 ng/ml IL-6 (R&D Systems) and
1 .mu.g/ml of PGE2 (Cayman Chemicals). After 16-20 hr, DCs were
washed and further incubated in 96-well plates (2.times.105
cells/well) in complete DC medium without GM-CSF and IL-4. DC death
was assessed by loss of propidium iodide exclusion (PI+) and by
binding of PE-conjugated Annexin-V (Annexin-V+) using flow
cytometry (Epics.RTM. XL-MCL).
[0266] In vivo DC survival assay. To analyze DC survival in vivo,
BMDCs were treated with LPS (1 .mu.g/ml), Ad-GFP or
Ad-MF-.DELTA.Akt for 2 hr, labeled with CFSE dye and injected
subcutaneously into hind legs below knees of syngeneic C57BL/6 mice
(1.times.10.sup.6 cells per mouse) as described previously (Nopora,
A. & Brocker, T. Bcl-2 controls dendritic cell longevity in
vivo. J Immunol 169, 3006-14 (2002)). The draining popliteal lymph
nodes were harvested at various time points after injection, and
percentage CFSE-positive cells among live LN cells (based on
propidium iodide staining) were analyzed by flow cytometry.
[0267] Flow cytometry analysis. Prior to staining with labeled
antibodies, BMDCs were pretreated with Fc blocking antibody
(anti-CD16/CD32 mAb; BD Pharmingen) to avoid nonspecific binding.
DCs were then stained with FITC-conjugated anti-mouse CD11c and
PE-conjugated anti-mouse CD40, CD80, MHC class II Kb, or MHC class
II I-Ab (BD Pharmingen). For counting ovalbumin-specific or
TRP2-specific CD8+ T cells, peripheral blood lymphocytes were
stained with FITC-conjugated anti-mouse CD8alpha (BD Pharmingen)
and PE-conjugated H-2 Kb/OVA257-264 tetramer or H-2 Kb/TRP2181-188
(VYDFFVWL) respectively (Tetramer Core Facility, Baylor College of
Medicine). To detect MAGE-3-specific CD8+ T cells, presensitized
CD8+ T cells were stained with PE-labeled HLA-A2/MAGE-3271-279
(FLWGPRALV) tetramer (Tetramer Core Facility, Baylor College of
Medicine) for 15 min at 37.degree. C. before addition of
FITC-conjugated anti-human CD8 mAb (BD Biosciences). Stained cells
were analyzed by flow cytometry and FlowJo (TreeStar, Inc.,
Ashland, Oreg.) software.
[0268] Allogeneic and autologous T cell proliferation assays.
Purified BMDCs (C57BL/6) were incubated with LPS (1 .mu.g/ml),
Ad-GFP or Ad-MF-.DELTA.Akt virus at 100 m.o.i. For autologous T
cell proliferation assays, BMDCs were pulsed with SIINFEKL peptide
(OVA257-264, Protein Chemistry Core Facility, Baylor College of
Medicine) prior to incubation with LPS or recombinant adenoviruses.
After 16-hr incubation, BMDCs were co-cultured with syngeneic
(C57BL/6) or allogeneic (BALB/c) splenocytes at different ratios
(1:50-1:6400) in 96-well round-bottom plates for 1 to 3 days.
Thereafter, cells were pulsed with 1 .mu.Ci of [.sup.3H]thymidine
(New England Nuclear) per well for 12 hr, and thymidine
incorporation was measured by liquid scintillation (1205 BS
Betaplate). All experiments were conducted in triplicate and
expressed as mean.+-.S.D.
[0269] IFN-gamma ELISPOT assay. BMDC-mediated T cell activation was
assessed by IFN-gamma ELISPOT assay as described previously with
minor modifications (Nikitina, E. Y. et al. Versatile prostate
cancer treatment with inducible caspase and interleukin-12. Cancer
Res 65, 4309-19 (2005)). Briefly, 2.times.105 OT-1 splenocytes and
syngeneic BMDCs treated with SIINFEKL peptide and LPS or
adenoviruses were cultured for 24 or 72 hours in 96-well
Multi-Screen-HA plates (Millipore) coated with 10 .mu.g/mL of
purified rat anti-mouse IFN-gamma monoclonal antibody (clone
R4-6A2; PharMingen). Plates were then incubated overnight at
4.degree. C. with 5 .mu.g/mL biotinylated rat anti-mouse IFN-gamma
monoclonal antibody (clone XMG1.2; PharMingen). Spots were
visualized with 5-bromo-4-chloro-3-indolylphosphate/nitro blue
tetrazolium alkaline phosphatase substrate (Sigma) reaction and
counted per triplicate well using a stereomicroscope.
[0270] In vivo tumor vaccination studies. EG.7-OVA thymoma or B16
melanoma cells (ATCC) were expanded in culture and injected
subcutaneously into C57BL/6 mice (2.times.10.sup.6 EG.7-OVA
cells/mouse or 0.5.times.10.sup.5 or 1.times.10.sup.5 B16 melanoma
cells/mouse). For therapeutic DC vaccines, BMDCs were pulsed with
SIINFEKL peptide (20 .mu.g/ml) for EG.7-OVA cells or
tyrosinase-related protein (TRP)-2 peptide (10 .mu.g/ml, sequence
VYDFFVWL) for B16 cells, transduced with or without adenoviruses or
LPS and injected intraperitoneally (2.times.10.sup.6 DCs/mouse).
Single DC vaccine treatment was done 7 days after EG.7-OVA tumor
injection (tumor sizes reached approximately 0.4 cm.sup.3) or
triple DC vaccination was done 4 (or 5), 15 and 25 days after B16
tumor injection. Two tumor measurements were recorded every 3-4
days. Tumor volumes were estimated as (m12.times.m2.times.0.5236)
w.sup.2*1*0.5236.
[0271] MAGE3-specific CTL activity assay. For T cell assays, DCs
were purified from HLA-A2-positive healthy volunteers, after HLA
typing by flow cytometry with FITC-conjugated anti-HLA-A2 specific
antibody (BD Biosciences, San Diego, Calif.). DCs were pulsed with
MAGE3271-279 (FLWGPRALV) peptide (Genemed Synthesis Inc, San
Francisco, Calif.) and transduced with adenoviruses (Ad5f35-GFP or
Ad5f35-MF-.DELTA.hAkt, 10,000 vp/cell). Cytolytic assays were
performed by coculturing DCs with autologous T cells (isolated
using a Pan T cell isolation kit (Miltenyi Biotec)) at 1:10 ratio.
T cells were restimulated twice in the presence of IL-2 (10 U/m1).
After 20 days of culture, effector cells were harvested and
analyzed by cytolytic assays. Target cells including melanoma cell
lines SK-Mel-37 (MAGE3 positive, A2 positive), NA-6-MeI (MAGE3
positive, A2 negative) and T2 cells (MAGE3 negative, A2 positive)
loaded with MAGE3271-279 peptide were labeled with 100 .mu.Ci
51Cr-sodium chromate (Amersham Pharmacia Biotech) and cocultured
for 6 hours with effector cells at the indicated cell ratios. The
percentage of specific lysis was calculated as:
100*[(experimental-spontaneous release)/(maximum-spontaneous
release)]. For control purposes, T2 cells were pulsed with flu
matrix peptide (IMP) p58-66 GILGFVFT (Genemed Synthesis Inc).
[0272] Statistical analysis. Statistical significance was
determined based on the student t-test between indicated groups or
one-way ANOVA with a multiple comparison test Animal survival was
analyzed using Kaplan-Meier survival curves with log rank test
(Prism v4, GraphPad Software, Inc.).
[0273] Preparation of siRNAs. The previously identified small
interfering RNA (siRNA) for Akt-1, matching closely all isoforms
(siAKTc; Katome, T., et al. J. Biol. Chem. (2003) 278: 28312), was
synthesized from Integrated DNA Technologies (Coralville, Iowa),
and negative control siRNA was purchased from Ambion (Austin,
Tex.). Transfection of siRNAs into BMDCs was done by
GeneSilencer.TM. siRNA Transfection (GTS, San Diego, Calif.) or
nucleofection using the Human Dendritic Cell Nucleofector Kit
(Amaxa Biosystems, Gaithersburg, Md.).
[0274] NF-kappaB secreted alkaline phosphatase (SEAP) reporter
assay. Jurkat TAg cells were transfected with indicated amounts of
plasmids along with NF-kappaB SEAP reporter plasmid by
electroporation.
[0275] The following day, cells were plated with suboptimal
concentrations of phorbol ester PMA (0.5 nM) and further incubated
for about 24 hr. Thereafter, supernatants were collected and
analyzed for SEAP activity as described previously (L1, B. et al.
Gene Therapy (2001) 9: 233-244).
[0276] Fluorescent confocal microscopy. 293 cells cultured on
poly-L-lysine-coated coverslips were transfected with indicated
plasmids in 6-well plates and further incubated for 24 hr. For
drug-dependent lipid-raft localization, 100 nM of Rapalog (Rapa-B)
was added for an additional 4 hr. After fixation with 4%
paraformaldehyde for 30 min, cells were labeled with cholera toxin
B (CTx-B-TRITC) to detect glycosphingolipid in membrane lipid-rafts
(List Biological Laboratories, Campbell, Calif.), and analyzed by
LSM 430 confocal microscopy.
Example 10
Inducible Akt (iAkt) Membrane Targeting
[0277] Inducible Akt (iAkt) membrane targeting was effected by (a)
linking a modified Akt with a polypeptide that binds to a
bifunctional compound, and (b) linking a membrane association
component to a another polypeptide that binds to the same
bifunctional compound. Adding the bifunctional compound to a system
in which component (a) and (b) are present can induce the two
components to join with one another and the complex can be
recruited to the cell membrane in an inducible manner. In the
example below, the bifunctional compound, a rapamycin derivative,
binds to (a) a derivative of FK binding protein linked to an Akt
protein lacking a PH domain, and (b) a rapamycin binding domain
from mTOR/FRAP/RAFT linked to a N-terminal myristoylation region
from the protein kinase Src. Constructs and methods for generating
and using these components are described in U.S. patent application
publication US2003/0144204 published on Jul. 31, 2003 and filed on
Dec. 19, 2002, entitled "Akt-Based Inducible Survival Switch,"
which can be consulted by the person of ordinary skill in the art
for any details not presented below. Alternative embodiments
include substituting the Src N-terminal myristoylation region with
a dual acylation region from a different protein (e.g., from Fyn or
Lck) or with another membrane protein or membrane protein fragment,
as can be accomplished by the person of ordinary skill in the art
using standard techniques.
[0278] Plasmid Construction. To generate F3-Akt, F3-DPH.Akt,
F3-AktKM and variants, Akt and .DELTA.PH. Akt were Pfu
(Stratagene)-amplified from pCMV6-HA-Akt or pCMV6-HA-AktK179M using
SalI-linkered 5' primers, mAkt5SPH (full-length):
5'-agagcgacaacgacgtagccattgtgaaggag-3' or mAkt5S (truncated
.DELTA.PH): 5'-agagtcgacaccgccattcagactgtggcc-3; and 3' primer,
mAkt3S: 5'-agagtcgacggctgtgccactggctgagtag-3'. PCR products were
subcloned into pCR-Blunt (Invitrogen) or pKSII+(Stratagene) and
sequence verified, to createpSH5/mAkt, pSH5/m.DELTA.PH.Akt, and
pKS/mAkt.KM. The 1440-bp full-length Akt and 1130-bp .DELTA.PH.Akt
fragments were removed with SalI and subcloned into
XhoI/SalI-digested M-Fpk 3-E, or XhoI or SalI-digested S-F.sub.pk
3-E (MacCorkle et al., 1998), to create M-Akt (and Akt variants),
Akt-F3 (and variants) and F3-Akt (and variants). All chimeric
proteins contain the HA epitope (E), but the "E" is left off (along
with "pk" subscripts) for simplicity. The heterodimeric
rapalog/CID.sub.HED can effect the crosslinking of FRB1 and FKPB12
(called F). In these experiments, the non-toxic variant of FKBP12,
Fpk (FKBP12(G89P, I90K)), was used to eliminate background
toxicity.
[0279] To generate myristoylated rapalogue-binding domains, the
rapamycin binding domain (FRB) from human FRAP (res. 2025-2113;
T2098L) was Pfu-amplified from FRAP*-AD using primers SEQ ID NO: 9
(5'-cgatctcgaggagatgtggcatgaaggcctgg-3') and 3FRBS: SEQ ID NO: 10
(5'-cgatgtcgacctttgagattcgtcggaacacatg-3') and subcloned into
pCR-Blunt to produce pSH5/FRB.sub.1. One or two copies of the
XhoI/SalI FRB1 domain were subcloned into XhoI/SalI-digested
M-F.sub.pk 3-E to create M-FRB.sub.1 and M-FRB.sub.1 2. Also, the
NF-.kappa.B-SEAP reporter plasmid was produced. Thus, a
constitutively active, myristoylated Akt (M-Akt) or M-.DELTA.PH.Akt
and kinase-dead mutant versions (i.e. Akt.K179M, named AktKM) of
chimeric Akt constructs were developed.
[0280] To make the bicistronic iAkt constructs, two different
internal ribosome entry sequence (IRES) elements from EMCV or
poliovirus were used to link M-FRB.sub.12 and F3-.DELTA.PH.Akt on
the same transcript. The poliovirus IRES sequence (IRESp) was
PfuI-amplified from pTPOV-3816 with primers, 5pIRES/Mun:
5'-atacaattgccgcggttcgaattctgttttatactcccttcccgtaac-3' and
3pIRES/Mun; 5'-tatcaattggfttaaacagcaaacagatagataatgagtctcac-3'. The
resulting PCR products were subcloned into pCR-Blunt to create
pSH5/IRESp-Mun. The 615-bp IRESp MunI fragment was ligated into
EcoRI-digested pSH1/M-FRB.sub.12-E to create
pSH1/M-FRB.sub.12-E-IRESp. Finally, the NotI/EcoRI F3-.DELTA.PH.Akt
fragment from pSH1/F3-.DELTA.PH.Akt was blunt-ligated into the PmeI
site to create pSH1/M-FRB.sub.1 2-E-IRESp-F3.DELTA.PH.Akt, renamed
as iAkt.sub.b. The bicistronic vector iAkt.sub.a utilizes the EMCV
IRES and was made by a comparable strategy.
[0281] For establishing Jurkat.iAkt cell lines, the bicistronic
NotI/MunI fragment from iAkt.sub.b was subcloned into
NotI/EcoRI-digested pBJ5-neo to create pBJ5-neo/iAkt.sub.b.
[0282] To generate inducible Akt (iAkt) molecules, three tandem
FKBP domains (F3) were fused to the N- or C-termini of wild-type
Akt or a variant.DELTA.PH.Akt), lacking the pleckstrin homology
(PH) domain to reduce natural membrane association.
[0283] Cell Culture. 293T human embryonic kidney cells (ATCC) and
Jurkat (ATCC), Jurkat-TAg and Jurkat.iAkt were maintained in DMEM
or RPMI-1640, respectively, containing 10% fetal bovine serum (FBS)
and antibiotics. The Jurkat.iAkt line was derived by transfecting
Jurkat cells with NdeI-linearized pBJ5-neo/iAkt.sub.b plasmid
followed by G418 (1 mg/ml) selection. Clones were screened by
anti-HA immunoblotting.
[0284] Electroporation and SEAP Assay. Jurkat-TAg cells in
logarithmic-phase growth were electroporated (950 mF, 250 V; Gene
Pulser II (BIO-RAD)) with expression plasmids and the
NF-.kappa.B-SEAP reporter plasmid. After 24 hours, transfected
cells were stimulated with sub-optimal levels of the phorbol ester
PMA (5 ng/ml) along with log dilutions of the heterodimerizing CID,
AP22783, and additional treatments. After an additional 24 hours,
supernatants were assayed for SEAP activity.
[0285] Western Blots. 293T cells seeded in 6-well plates were
transiently transfected with 2.mu.g of different expression
constructs in 6.mu.1 FuGENE6 (Boehringer-Mannheim, Indianapolis,
Ind.) in Opti-MEM-I medium for 24 hours followed by serum
starvation for an additional 24 hours. Cells were then treated with
different agents and harvested at different time points. After
washing (2.times.) in ice-cold PBS, cell pellets were lysed in RIPA
buffer containing protease inhibitors (CytoSignal, Irvine, Calif.).
Equal amounts of protein from each sample were separated on 10%
SDS-PAGE gels and transferred to PVDF membrane (Amersham Pharmacia
Biotech, Piscataway, N.J.). Phospho-specific antibodies against Akt
(T308 or S473 site) (Cell Signaling, Beverly, Mass.) were used for
measuring Akt phosphorylation, and the signal was detected by
AP-conjugated secondary antibodies (NEB, Beverly, Mass.) and
CDP-Star chemiluminescence reagent (NEN life science, Boston,
Mass.).
[0286] Immunoprecipitation and in vitro Akt Kinase Assay.
Jurkat.iAkt.sub.b were serum starved for 24 hours followed by
treatment with AP22783 or serum for 30 min. Cells were then lysed
in a lysis buffer provided with the Akt Kinase Assay kit (Cell
Signaling, Beverly, Mass.), and F.sub.pk3-.DELTA.PH.Akt-E was
immunoprecipitated with polyclonal anti-HA antibody.
Antibody-antigen complexes were washed three times in lysis buffer
and once in kinase buffer. In vitro kinase assays for Akt were
performed using a GSK3.alpha./.beta. "crosstide". The extent of
crosstide phosphorylation was determined by anti-GSK.alpha./.beta.
immunoblotting according to the manufacturer's protocol.
[0287] Apoptosis and Flow Cytometry. Jurkat.iAkt were serum starved
for 24 hours followed by pre-treatment with AP22783 in 0, 2 or 10%
FBS for 40 min. After incubation with apoptosis-inducing stimuli
for the periods indicated, cells were harvested and washed twice in
ice-cold PBS and fixed in 70% ethanol. Cells were stained in
50.mu.g/ml propidium iodide and 100.mu.g/mlRNase A for 30 min at
37.degree. C., and hypodiploid cells were quantitated by flow
cytometry using a Beckman-Coulter EPICS XL-MCL.
[0288] For determination of caspase-3 activation and PARP cleavage
after staurosporine treatment, cell pellets were lysed in Laemmli
sample buffer containing 5% (v/v).beta.-mercaptoethanol (Bio-Rad,
Hercules, Calif.), and equal amounts of protein were separated on 6
(for PARP) or 12% (for caspase3) SDS-PAGE followed by
immunoblotting with anti-caspase-3 and anti-PARP antibodies.
[0289] Inducible membrane recruitment of iAkt. Two key requirements
for efficient synthetic regulation of a biological event are highly
specific conditional dependency and low background. NF-kappaB
induction is a major target of Akt following growth factor
signaling, and multiple reports show that a constitutively active
myristoylated Akt (M-Akt) can enhance protein kinase
C(PKC)-mediated NF-.kappa.B induction by either phosphorylation of
IKK.alpha., the activation domain of p65/RelA, or both. Therefore,
in order to optimize iAkt, an NF-.kappa.B-responsive secreted
alkaline phosphatase (SEAP) reporter plasmid was used as an assay
for Akt activation.
[0290] Briefly, the human T cell line, Jurkat-TAg, was
cotransfected with reporter plasmid, NF-.kappa.B/SEAP, along with
constitutively active M-Akt expression vector or empty control
vector. Twenty-four hours after transfection, cells were divided
into aliquots that were stimulated with sub-optimal levels (5
ng/ml) of the phorbol ester, PMA, or were untreated. After an
additional 24 hours, SEAP activity was measured. Although Akt
activity alone was insufficient to induce measurable NF-.kappa.B
activity, M-Akt expression potentiated (by 3-4 fold) PKC-induced
NF-.kappa.B activity, consistent with multiple reports.
Furthermore, inhibition of PI3K by LY294002 (5.mu.M) or wortmannin
(1.mu.M) did not prevent NF-.kappa.B activation by M-Akt plus PKC,
although inhibition of "typical" PKC isoforms with R0318220
(1.mu.M) led to complete inhibition of NF-.kappa.B as expected
(FIG. 2).
[0291] Since the constitutively active Akt (T308) kinase, PDK1, is
primarily membrane-associated following growth factor stimulation,
membrane recruitment of Akt via its PH domain is necessary for its
activation. Furthermore, although the PH domain has been shown to
suppress basal phosphorylation of T308 and Akt activation when not
bound by its lipid ligand, PIP2, this initial phosphorylation
should still require interaction with membrane-localized PDK1.
[0292] Basal Aid activity and activation following membrane
recruitment of full length and truncated Akt, lacking the PH
domain, was compared using a NF-.kappa.B reporter assay. Both full
length and .DELTA.PH.Akt were fused to a tandem trimer of the
CID-binding domain, F3, at both the amino and carboxyl termini. As
before Jurkat-TAg cells were cotransfected with reporter plasmid
NF-.kappa.B/SEAP along with the membrane docking molecule,
M-FRB.sub.12, alone, various F3-Akt chimeras, alone, or both
together. Twenty-four hours after transfection, cells were
stimulated with 5 ng/ml PMA along with log dilutions of
heterodimerizing CID, AP22783. After additional 24 hr incubation,
SEAP activity was assayed. Wild type Akt showed significant
CID-independent NF-.kappa.B induction that was only slightly
increased by crosslinking to the membrane, via M-FRB 12. This was
true regardless of whether F3 was fused to the N- or C-terminus of
Akt. As expected, membrane recruitment or overexpression of
kinase-deficient Akt.KM (K179M) had no detectable effect on
NF-.kappa.B induction over PMA alone. Thus, membrane recruitment of
full-length Akt only slightly increases its activity due to the
high basal activity from its overexpression. In contrast, membrane
recruitment of F3-.DELTA.PH.Akt showed a very clear CID-dependent
induction of NF-.kappa.B with undetectable CID-independent
activity. Moreover, myristoylated M.DELTA.PH.Akt was more active
than M-Akt in augmenting NF-.kappa.B activation, consistent with an
inhibitory function for the PH domain. Again, M-FRB.sub.1 2 alone
or recruitment of kinase dead F3-.DELTA.PH.AktKM did not influence
NF-.kappa.B induction.
[0293] These results indicated that the chimeric F3-.DELTA.PH.Ald
allele is strongly CID-inducible with very low basal activity.
Also, these results are consistent with previous reports that the
PH domain of Akt kinase is responsible for its translocation to the
plasma membrane and also has an inhibitory function. Since most
applications of CID technology have been based partly, at least, on
empirically designed inducible chimeric proteins, CID-mediated
targeting or crosslinking might not always faithfully reflect
physiological signaling. Further, CID-binding domains, like FKBP
12, could potentially sterically hinder an essential target protein
domain(s). Therefore, .DELTA.PH.Akt with F3 fused to both termini
of .DELTA.PH.Akt was tested. The N-terminal fusion chimera,
F3-.DELTA.PH.Akt, potentiated NF-.kappa.B transactivation somewhat
better than the C-terminal chimera, .DELTA.PH.Akt-F3. Since both
molecules were expressed at similar levels, membrane recruitment of
F3-.DELTA.PH.Akt may place Akt in a more favorable orientation for
interacting with PDK1 or other interacting proteins. In either
orientation, however, both iAkt versions were devoid of detectable
basal NF-.kappa.B signaling.
[0294] Since M-FRB.sub.1 2 could potentially recruit two chimeric
Akt molecules simultaneously, it was determine if membrane
recruitment of one Akt molecule was sufficient for optimal
activation or whether oligomerization of multiple Akt molecules
might enhance activation. Therefore, CID-mediated iAkt activity was
compared when the membrane docking molecule, contained one or two
FRB.sub.1 domains (FRB.sub.1 vs FRB.sub.1 2, respectively). There
was no significant difference in NF-.kappa.B induction by iAkt
whether one or two tandem FRB.sub.1 domains were used for the
docking site, indicating that forced Akt oligomerization is not a
prerequisite for its activation. It was also shown that CID
dependent membrane targeting of Akt resulted in rapid
phosphorylation and activation of endogenous Akt.
Example 12
Combination Immunotherapeutics
[0295] Tumor challenges similar to those described in Example 7 and
reported in FIGS. 7C and 7D were performed to determine effects of
combination immunotherapies. The combined effects of
M.sub.F-.DELTA.Akt and an inducible CD40 (iCD40, described in US
publication 20040209836 (published Oct. 21, 2004)) were assessed.
Briefly, adenoviral particles having a polynucleotide sequence
encoding the M.sub.F-.DELTA.Akt molecule and adenoviral particles
having a polynucleotide sequence encoding the iCD40 construct were
utilized to transduce dendritic cells (DCs). DCs were pulsed with
TRP-2.sub.181-188 peptide (10 micrograms/ml; VYDFFVWL) for about 3
hours and treated with the adenoviruses (about 10,000 viral
particles/DC for iCD40 particles and control adenoviral particles
(Ad-emp) and about 500 viral particles/DC for M.sub.F-.DELTA.Akt
particles) plus or minus LPS (1 ug/ml) as indicated for 2 hours.
The transduced DCs were utilized in a triple intraperitoneal
vaccination (2.times.10.sup.6 CD11c.sup.+ DCs/100 ul PBS) on days
3, 10 and 17. Host animals were C57BL/6 (6-8 wks) female mice (8
mice/group), and tumors were established by subcutaneous injection
of B16 melanoma cells (100,000/100 ul) on the back of each mouse
(day 0). Inducer was administered to the mice intraperitoneally at
a dose of 2 mg/kg AP20187/100 ul/mouse on days 4, 11, 18. Results
are shown in FIGS. 15A and 15B. The combination yielded effects
that were at least additive and may be synergistic. Effects of the
combination are under additional investigation.
Example 13
Immunotherapeutic Effect on Human Tumors
[0296] Six to eight week-old athymic male Balb/c nu/nu mice (NCI at
Frederick, Md.) are used as hosts for human xenografts (e.g., Xie
et al. Cancer Research 61: 6795-6804 (2001)). Mice are maintained
in a specific pathogen-free environment, in compliance with
standard guidelines. Cells in logarithmic-phase growth are lightly
trypsinized and washed twice with OPTI-medium (GIBCO). Cell
suspensions (1.times.10.sup.6-cells/200 ul) of modified DCs primed
with antigen, as described herein (e.g., in Example 8), are
subcutaneously injected into the right flank of the mice. To
establish LNCaP tumors, a 1:4 mix of 100-ul cell suspension
(1.times.10.sup.6 cells) with Matrigel.TM. (Becton Dickinson,
Bedford, Mass.) is injected before, contemporaneously or after,
injection of the modified DCs. The effect of the modified DCs
described herein on tumor growth is determined according to
standard measurements and procedures.
Example 13
Representative Sequences
[0297] Provided hereafter are examples of representative
sequences.
TABLE-US-00001 SEQ ID NO: 1 (Accession no. NM_009652: mouse Akt1) 1
ccgggaccag cggacggacc gagcagcgtc ctgcggccgg caccgcggcg gcccagatcc
61 ggccagcagc gcgcgcccgg acgccgctgc cttcagccgg ccccgcccag
cgcccgcccg 121 cgggatgcgg agcggcgggc gcccgaggcc gcggcccggc
taggcccagt cgcccgcacg 181 cggcggcccg acgctgcggc caggccggct
gggctcagcc taccgagaag agactctgat 241 catcatccct gggttacccc
tgtctctggg ggccacggat accatgaacg acgtagccat 301 tgtgaaggag
ggctggctgc acaaacgagg ggaatatatt aaaacctggc ggccacgcta 361
cttcctcctc aagaacgatg gcacctttat tggctacaag gaacggcctc aggatgtgga
421 tcagcgagag tccccactca acaacttctc agtggcacaa tgccagctga
tgaagacaga 481 gcggccaagg cccaacacct ttatcatccg ctgcctgcag
tggaccacag tcattgagcg 541 caccttccat gtggaaacgc ctgaggagcg
ggaagaatgg gccaccgcca ttcagactgt 601 ggccgatgga ctcaagaggc
aggaagaaga gacgatggac ttccgatcag gctcacccag 661 tgacaactca
ggggctgaag agatggaggt gtccctggcc aagcccaagc accgtgtgac 721
catgaacgag tttgagtacc tgaaactact gggcaagggc acctttggga aagtgattct
781 ggtgaaagag aaggccacag gccgctacta tgccatgaag atcctcaaga
aggaggtcat 841 cgtcgccaag gatgaggttg cccacacgct tactgagaac
cgtgtcctgc agaactctag 901 gcatcccttc cttacggccc tcaagtactc
attccagacc cacgaccgcc tctgctttgt 961 catggagtat gccaacgggg
gcgagctctt cttccacctg tctcgagagc gcgtgttctc 1021 cgaggaccgg
gcccgcttct atggtgcgga gattgtgtct gccctggact acttgcactc 1081
cgagaagaac gtggtgtacc gggacctgaa gctggagaac ctcatgctgg acaaggacgg
1141 gcacatcaag ataacggact tcgggctgtg caaggagggg atcaaggatg
gtgccactat 1201 gaagacattc tgcggaacgc cggagtacct ggcccctgag
gtgctggagg acaacgacta 1261 cggccgtgca gtggactggt gggggctggg
cgtggtcatg tatgagatga tgtgtggccg 1321 cctgcccttc tacaaccagg
accacgagaa gctgttcgag ctgatcctca tggaggagat 1381 ccgcttcccg
cgcacactcg gccctgaggc caagtccctg ctctccgggc tgctcaagaa 1441
ggaccctaca cagaggctcg gtgggggctc tgaggatgcc aaggagatca tgcagcaccg
1501 gttctttgcc aacatcgtgt ggcaggatgt gtatgagaag aagctgagcc
cacctttcaa 1561 gccccaggtc acctctgaga ctgacaccag gtatttcgat
gaggagttca cagctcagat 1621 gatcaccatc acgccgcctg atcaagatga
cagcatggag tgtgtggaca gtgagcggag 1681 gccgcacttc ccccagttct
cctactcagc cagtggcaca gcctgaggcc tggggcagcg 1741 gctggcagct
ccacgctcct ctgcattgcc gagtccagaa gccccgcatg gatcatctga 1801
acctgatgtt ttgtttctcg gatgcgctgg ggaggaacct tgccagcctc caggaccagg
1861 ggaggatgtt tctactgtgg gcagcagcct acctcccagc caggtcagga
ggaaaactat 1921 cctggggttt ttcttaattt atttcatcca gtttgagacc
acacatgtgg cctcagtgcc 1981 cagaacaatt agattcatgt agaaaactat
taaggactga cgcgaccatg tgcaatgtgg 2041 gctcatgggt ctgggtgggt
cccgtcactg cccccattgg cctgtccacc ctggccgcca 2101 cctgtctcta
gggtccaggg ccaaagtcca gcaagaaggc accagaagca cctccctgtg 2161
gtatgctaac tggccctctc cctctgggcg gggagaggtc acagctgctt cagccctagg
2221 gctggatggg atggccaggg ctcaagtgag gttgacagag gaacaagaat
ccagtttgtt 2281 gctgtgtccc atgctgttca gagacattta ggggatttta
atcttggtga caggagagcc 2341 cctgccctcc cgctcctgcg tggtggctct
tagcgggtac cctgggagcg cctgcctcac 2401 gtgagccctc tcctagcact
tgtcctttta gatgctttcc ctctcccgct gtccgtcacc 2461 ctggcctgtc
ccctcccgcc agacgctggc cattgctgca ccatgtcgtt ttttacaaca 2521
ttcagcttca gcatttttac tattataata agaaactgtc cctccaaatt caataaaaat
2581 tgcttttcaa gcttgaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa SEQ ID NO:
2 (Accession no. NM_001014431: human Akt1 mRNA) 1 cggcaggacc
gagcgcggca ggcggctggc ccagcgcagc cagcgcggcc cgaaggacgg 61
gagcaggcgg ccgagcaccg agcgctgggc accgggcacc gagcggcggc ggcacgcgag
121 gcccggcccc gagcagcgcc cccgcccgcc gcggcctcca gcccggcccc
gcccagcgcc 181 ggcccgcggg gatgcggagc ggcgggcgcc ggaggccgcg
gcccggctag gcccgcgctc 241 gcgcccggac gcggcggccc gaggctgtgg
ccaggccagc tgggctcggg gagcgccagc 301 ctgagaggag cgcgtgagcg
tcgcgggagc ctcgggcacc atgagcgacg tggctattgt 361 gaaggagggt
tggctgcaca aacgagggga gtacatcaag acctggcggc cacgctactt 421
cctcctcaag aatgatggca ccttcattgg ctacaaggag cggccgcagg atgtggacca
481 acgtgaggct cccctcaaca acttctctgt ggcgcagtgc cagctgatga
agacggagcg 541 gccccggccc aacaccttca tcatccgctg cctgcagtgg
accactgtca tcgaacgcac 601 cttccatgtg gagactcctg aggagcggga
ggagtggaca accgccatcc agactgtggc 661 tgacggcctc aagaagcagg
aggaggagga gatggacttc cggtcgggct cacccagtga 721 caactcaggg
gctgaagaga tggaggtgtc cctggccaag cccaagcacc gcgtgaccat 781
gaacgagttt gagtacctga agctgctggg caagggcact ttcggcaagg tgatcctggt
841 gaaggagaag gccacaggcc gctactacgc catgaagatc ctcaagaagg
aagtcatcgt 901 ggccaaggac gaggtggccc acacactcac cgagaaccgc
gtcctgcaga actccaggca 961 ccccttcctc acagccctga agtactcttt
ccagacccac gaccgcctct gctttgtcat 1021 ggagtacgcc aacgggggcg
agctgttctt ccacctgtcc cgggagcgtg tgttctccga 1081 ggaccgggcc
cgcttctatg gcgctgagat tgtgtcagcc ctggactacc tgcactcgga 1141
gaagaacgtg gtgtaccggg acctcaagct ggagaacctc atgctggaca aggacgggca
1201 cattaagatc acagacttcg ggctgtgcaa ggaggggatc aaggacggtg
ccaccatgaa 1261 gaccttttgc ggcacacctg agtacctggc ccccgaggtg
ctggaggaca atgactacgg 1321 ccgtgcagtg gactggtggg ggctgggcgt
ggtcatgtac gagatgatgt gcggtcgcct 1381 gcccttctac aaccaggacc
atgagaagct ttttgagctc atcctcatgg aggagatccg 1441 cttcccgcgc
acgcttggtc ccgaggccaa gtccttgctt tcagggctgc tcaagaagga 1501
ccccaagcag aggcttggcg ggggctccga ggacgccaag gagatcatgc agcatcgctt
1561 ctttgccggt atcgtgtggc agcacgtgta cgagaagaag ctcagcccac
ccttcaagcc 1621 ccaggtcacg tcggagactg acaccaggta ttttgatgag
gagttcacgg cccagatgat 1681 caccatcaca ccacctgacc aagatgacag
catggagtgt gtggacagcg agcgcaggcc 1741 ccacttcccc cagttctcct
actcggccag cggcacggcc tgaggcggcg gtggactgcg 1801 ctggacgata
gcttggaggg atggagaggc ggcctcgtgc catgatctgt atttaatggt 1861
ttttatttct cgggtgcatt tgagagaagc cacgctgtcc tctcgagccc agatggaaag
1921 acgtttttgt gctgtgggca gcaccctccc ccgcagcggg gtagggaaga
aaactatcct 1981 gcgggtttta atttatttca tccagtttgt tctccgggtg
tggcctcagc cctcagaaca 2041 atccgattca cgtagggaaa tgttaaggac
ttctgcagct atgcgcaatg tggcattggg 2101 gggccgggca ggtcctgccc
atgtgtcccc tcactctgtc agccagccgc cctgggctgt 2161 ctgtcaccag
ctatctgtca tctctctggg gccctgggcc tcagttcaac ctggtggcac 2221
cagatgcaac ctcactatgg tatgctggcc agcaccctct cctgggggtg gcaggcacac
2281 agcagccccc cagcactaag gccgtgtctc tgaggacgtc atcggaggct
gggcccctgg 2341 gatgggacca gggatggggg atgggccagg gtttacccag
tgggacagag gagcaaggtt 2401 taaatttgtt attgtgtatt atgttgttca
aatgcatttt gggggttttt aatctttgtg 2461 acaggaaagc cctccccctt
ccccttctgt gtcacagttc ttggtgactg tcccaccggg 2521 agcctccccc
tcagatgatc tctccacggt agcacttgac cttttcgacg cttaaccttt 2581
ccgctgtcgc cccaggccct ccctgactcc ctgtgggggt ggccatccct gggcccctcc
2641 acgcctcctg gccagacgct gccgctgccg ctgcaccacg gcgttttttt
acaacattca 2701 actttagtat ttttactatt ataatataat atggaacctt
ccctccaaat tcttcaataa 2761 aagttgcttt tcaaaaaaaa aaaaaaaaaa aaaa
SEQ ID NO: 3 (Accession no. NM_001626: human Akt2 mRNA) 1
gaattccagc ggcggcgccg ttgccgctgc cgggaaacac aaggaaaggg aaccagcgca
61 gcgtggcgat gggcgggggt agagccccgc cggagaggct gggcggctgc
cggtgacaga 121 ctgtgccctg tccacggtgc ctcctgcatg tcctgctgcc
ctgagctgtc ccgagctagg 181 tgacagcgta ccacgctgcc accatgaatg
aggtgtctgt catcaaagaa ggctggctcc 241 acaagcgtgg tgaatacatc
aagacctgga ggccacggta cttcctgctg aagagcgacg 301 gctccttcat
tgggtacaag gagaggcccg aggcccctga tcagactcta ccccccttaa 361
acaacttctc cgtagcagaa tgccagctga tgaagaccga gaggccgcga cccaacacct
421 ttgtcatacg ctgcctgcag tggaccacag tcatcgagag gaccttccac
gtggattctc 481 cagacgagag ggaggagtgg atgcgggcca tccagatggt
cgccaacagc ctcaagcagc 541 gggccccagg cgaggacccc atggactaca
agtgtggctc ccccagtgac tcctccacga 601 ctgaggagat ggaagtggcg
gtcagcaagg cacgggctaa agtgaccatg aatgacttcg 661 actatctcaa
actccttggc aagggaacct ttggcaaagt catcctggtg cgggagaagg 721
ccactggccg ctactacgcc atgaagatcc tgcgaaagga agtcatcatt gccaaggatg
781 aagtcgctca cacagtcacc gagagccggg tcctccagaa caccaggcac
ccgttcctca 841 ctgcgctgaa gtatgccttc cagacccacg accgcctgtg
ctttgtgatg gagtatgcca 901 acgggggtga gctgttcttc cacctgtccc
gggagcgtgt cttcacagag gagcgggccc 961 ggttttatgg tgcagagatt
gtctcggctc ttgagtactt gcactcgcgg gacgtggtat 1021 accgcgacat
caagctggaa aacctcatgc tggacaaaga tggccacatc aagatcactg 1081
actttggcct ctgcaaagag ggcatcagtg acggggccac catgaaaacc ttctgtggga
1141 ccccggagta cctggcgcct gaggtgctgg aggacaatga ctatggccgg
gccgtggact 1201 ggtgggggct gggtgtggtc atgtacgaga tgatgtgcgg
ccgcctgccc ttctacaacc 1261 aggaccacga gcgcctcttc gagctcatcc
tcatggaaga gatccgcttc ccgcgcacgc 1321 tcagccccga ggccaagtcc
ctgcttgctg ggctgcttaa gaaggacccc aagcagaggc 1381 ttggtggggg
gcccagcgat gccaaggagg tcatggagca caggttcttc ctcagcatca 1441
actggcagga cgtggtccag aagaagctcc tgccaccctt caaacctcag gtcacgtccg
1501 aggtcgacac aaggtacttc gatgatgaat ttaccgccca gtccatcaca
atcacacccc 1561 ctgaccgcta tgacagcctg ggcttactgg agctggacca
gcggacccac ttcccccagt 1621 tctcctactc ggccagcatc cgcgagtgag
cagtctgccc acgcagagga cgcacgctcg 1681 ctgccatcac cgctgggtgg
ttttttaccc ctgcc SEQ ID NO: 4 (Accession no. NM_005465: human Akt3
mRNA) 1 gcagcagcag agaatccaaa ccctaaagct gatatcacaa agtaccattt
ctccaagttg
61 ggggctcaga ggggagtcat catgagcgat gttaccattg tgaaagaagg
ttgggttcag 121 aagaggggag aatatataaa aaactggagg ccaagatact
tccttttgaa gacagatggc 181 tcattcatag gatataaaga gaaacctcaa
gatgtggatt taccttatcc cctcaacaac 241 ttttcagtgg caaaatgcca
gttaatgaaa acagaacgac caaagccaaa cacatttata 301 atcagatgtc
tccagtggac tactgttata gagagaacat ttcatgtaga tactccagag 361
gaaagggaag aatggacaga agctatccag gctgtagcag acagactgca gaggcaagaa
421 gaggagagaa tgaattgtag tccaacttca caaattgata atataggaga
ggaagagatg 481 gatgcctcta caacccatca taaaagaaag acaatgaatg
attttgacta tttgaaacta 541 ctaggtaaag gcacttttgg gaaagttatt
ttggttcgag agaaggcaag tggaaaatac 601 tatgctatga agattctgaa
gaaagaagtc attattgcaa aggatgaagt ggcacacact 661 ctaactgaaa
gcagagtatt aaagaacact agacatccct ttttaacatc cttgaaatat 721
tccttccaga caaaagaccg tttgtgtttt gtgatggaat atgttaatgg gggcgagctg
781 tttttccatt tgtcgagaga gcgggtgttc tctgaggacc gcacacgttt
ctatggtgca 841 gaaattgtct ctgccttgga ctatctacat tccggaaaga
ttgtgtaccg tgatctcaag 901 ttggagaatc taatgctgga caaagatggc
cacataaaaa ttacagattt tggactttgc 961 aaagaaggga tcacagatgc
agccaccatg aagacattct gtggcactcc agaatatctg 1021 gcaccagagg
tgttagaaga taatgactat ggccgagcag tagactggtg gggcctaggg 1081
gttgtcatgt atgaaatgat gtgtgggagg ttacctttct acaaccagga ccatgagaaa
1141 ctttttgaat taatattaat ggaagacatt aaatttcctc gaacactctc
ttcagatgca 1201 aaatcattgc tttcagggct cttgataaag gatccaaata
aacgccttgg tggaggacca 1261 gatgatgcaa aagaaattat gagacacagt
ttcttctctg gagtaaactg gcaagatgta 1321 tatgataaaa agcttgtacc
tccttttaaa cctcaagtaa catctgagac agatactaga 1381 tattttgatg
aagaatttac agctcagact attacaataa caccacctga aaaatatgat 1441
gaggatggta tggactgcat ggacaatgag aggcggccgc atttccctca attttcctac
1501 tctgcaagtg gacgagaata agtctctttc attctgctac ttcactgtca
tcttcaattt 1561 attactgaaa atgattcctg gacatcacca gtcctagctc
ttacacatag caggggcacc 1621 ttccgacatc ccagaccagc caagggtcct
cacccctcgc cacctttcac cctcatgaaa 1681 acacacatac acgcaaatac
actccagttt ttgtttttgc atgaaattgt atctcagtct 1741 aaggtctcat
gctgttgctg ctactgtctt actattatag caactttaag aagtaatttt 1801
ccaacctttg gaagtcatga gcccaccatt gttcatttgt gcaccaatta tcatcttttg
1861 atcttttagt ttttccctca gtgaaggcta aatgagatac actgattcta
ggtacatttt 1921 ttaactttct agaagagaaa aactaactag actaagaaga
tttagtttat aaattcagaa 1981 caagcaattg tggaagggtg gtggcgtgca
tatgtaaagc acatcagatc cgtgcgtgaa 2041 gtaggcatat atcactaagc
tgtggctgga attgattagg aagcatttgg tagaaggact 2101 gaacaactgt
tgggatatat atatatatat ataatttttt ttttttaaat tcctggtgga 2161
tactgtagaa gaagcccata tcacatgtgg atgtcgagac ttcacgggca atcatgagca
2221 agtgaacact gttctaccaa gaactgaagg catatgcaca gtcaaggtca
cttaaagggt 2281 cttatgaaac aatttgagcc agagagcatc tttcccctgt
gcttggaaac cttttttcct 2341 tcttgacatt tatcacctct gatggctgaa
gaatgtagac aggtataatg atactgcttt 2401 tcaccaaaat ttctacacca
aggtaaacag gtgtttgcct tatttaattt tttactttca 2461 gttctacgtg
aattagcttt ttctcagatg ttgaaacttt gaatgtcctt ttatgatttt 2521
gtttatattg cagtagtatt tattttttag tgatgagaat tgtatgtcat gttagcaaac
2581 gcagctccaa cttatataaa atagacttac tgcagttact tttgacccat
gtgcaaggat 2641 tgtacacgct gatgagaatc atgcactttt tctcctctgt
taaaaaaaat gataaggctc 2701 tgaaatggaa tatattggtt agaatttggc
tttgggagaa gagatgctgc catttaaccc 2761 cttggtactg aaaatgagaa
aatccccaac tatgcatgcc aaggggttaa tgaaacaaat 2821 agctgttgac
gtttgctcat ttaagaattt gaaacgttat gatgacctgg caacaaaaag 2881
taatgaagaa aattgagacc tgagtgaaga taagaaatga tctttacgtg gcaaaatgaa
2941 cacatcttga gtatttagga aatgggcagt gaaggctaag aacctggtgt
gtttcttggg 3001 atcatggtac atttatcact gaattaagcc atcagggaaa
aaacaacaaa aaaagagaac 3061 acctccagct tttctttttc tgtatatact
catgtccccc agattccaac atttctcact 3121 gaaagggggc atgtatgcaa
acctcatctt tctccttcat taatgatgat cttcagatta 3181 aaccctttgg
tgctaggagc tgacaatttc caaagcagcc tgtgaagtcc taggggctgg 3241
gggccactct tgcggcaagc agaaggccat cctactccgc ggagtgatca tggaaatgta
3301 ttttagttaa actctgacag ctcccaaacg gaagactaca gcatgacgta
gtattatgat 3361 tgcattgtat gaaagagcaa gtgactttct aagtaggatg
aatcatattc atatgcagat 3421 gtcttagcct cttgacgctg gaagtgtgga
tttatagcta tgaaaccact gctggcagtg 3481 ggtgggccac tgggactgac
gggggttaaa gggcatttta ctaaggcagc taagacatat 3541 tcagacatca
acgttatcct tctttttcat atttctacct gagtgaag
Amino acid sequences of the forgoing mRNA sequences can be obtained
by a person of ordinary skill in the art by consulting standard
internes resources. Amino acid sequences encoded by SEQ ID NO: 1-4
are designated by Accession nos. NP.sub.--033782,
NP.sub.--001014432, NP.sub.--001617 and NP.sub.--005456,
respectively. Other cloned sequences for each of these Akt forms
also are available and accessed by the person of ordinary skill in
the art.
TABLE-US-00002 SEQ ID NO: 5 (mRNA of human Akt1 lacking a PH
domain; .DELTA.PHAkt-1 (starting at position 101 of the encoded
amino acid sequence)):
accgccatccagactgtggctgacggcctcaagaagcaggaggaggagga
gatggacttccggtcgggctcacccagtgacaactcaggggctgaagaga
tggaggtgtccctggccaagcccaagcaccgcgtgaccatgaacgagttt
gagtacctgaagctgctgggcaagggcactttcggcaaggtgatcctggt
gaaggagaaggccacaggccgctactacgccatgaagatcctcaagaagg
aagtcatcgtggccaaggacgaggtggcccacacactcaccgagaaccgc
gtcctgcagaactccaggcaccccttcctcacagccctgaagtactcttt
ccagacccacgaccgcctctgctttgtcatggagtacgccaacgggggcg
agctgttcttccacctgtcccgggagcgtgtgttctccgaggaccgggcc
cgcttctatggcgctgagattgtgtcagccctggactacctgcactcgga
gaagaacgtggtgtaccgggacctcaagctggagaacctcatgctggaca
aggacgggcacattaagatcacagacttcgggctgtgcaaggaggggatc
aaggacggtgccaccatgaagaccttttgcggcacacctgagtacctggc
ccccgaggtgctggaggacaatgactacggccgtgcagtggactggtggg
ggctgggcgtggtcatgtacgagatgatgtgcggtcgcctgcccttctac
aaccaggaccatgagaagctttttgagctcatcctcatggaggagatccg
cttcccgcgcacgcttggtcccgaggccaagtccttgctttcagggctgc
tcaagaaggaccccaagcagaggcttggcgggggctccgaggacgccaag
gagatcatgcagcatcgcttctttgccggtatcgtgtggcagcacgtgta
cgagaagaagctcagcccacccttcaagccccaggtcacgtcggagactg
acaccaggtattttgatgaggagttcacggcccagatgatcaccatcaca
ccacctgaccaagatgacagcatggagtgtgtggacagcgagcgcaggcc
ccacttcccccagttctcctactcggccagcgcgacggcctga SEQ ID NO: 6 (amino
acid sequence encoded by SEQ ID NO: 5)
ThrAlaIleGlnThrValAlaAspGlyLeuLysLysGlnGluGluGlu
GluMetAspPheArgSerGlySerProSerAspAsnSerGlyAlaGlu
GluMetGluValSerLeuAlaLysProLysHisArgValThrMetAsn
GluPheGluTyrLeuLysLeuLeuGlyLysGlyThrPheGlyLysVal
IleLeuValLysGluLysAlaThrGlyArgTyrTyrAlaMetLysIle
LeuLysLysGluValIleValAlaLysAspGluValAlaHisThrLeu
ThrGluAsnArgValLeuGlnAsnSerArgHisProPheLeuThrAla
LeuLysTyrSerPheGlnThrHisAspArgLeuCysPheValMetGlu
TyrAlaAsnGlyGlyGluLeuPhePheHisLeuSerArgGluArgVal
PheSerGluAspArgAlaArgPheTyrGlyAlaGluIleValSerAla
LeuAspTyrLeuHisSerGluLysAsnValValTyrArgAspLeuLys
LeuGluAsnLeuMetLeuAspLysAspGlyHisIleLysIleThrAsp
PheGlyLeuCysLysGluGlyIleLysAspGlyAlaThrMetLysThr
PheCysGlyThrProGluTyrLeuAlaProGluValLeuGluAspAsn
AspTyrGlyArgAlaValAspTrpTrpGlyLeuGlyValValMetTyr
GluMetMetCysGlyArgLeuProPheTyrAsnGlnAspHisGluLys
LeuPheGluLeuIleLeuMetGluGluIleArgPheProArgThrLeu
GlyProGluAlaLysSerLeuLeuSerGlyLeuLeuLysLysAspPro
LysGlnArgLeuGlyGlyGlySerGluAspAlaLysGluIleMetGln
HisArgPhePheAlaGlyIleValTrpGlnHisValTyrGluLysLys
LeuSerProProPheLysProGlnValThrSerGluThrAspThrArg
TyrPheAspGluGluPheThrAlaGlnMetIleThrIleThrProPro
AspGlnAspAspSerMetGluCysValAspSerGluArgArgProHis
PheProGlnPheSerTyrSerAlaSerAlaThrAla SEQ ID NO: 7 (membrane
association region from human c-Fyn)
atgggctgtgtgcaatgtaaggataaagaagcaacaaaactgacggagga gctcgag SEQ ID
NO: 8 (amino acid sequence encoded by SEQ ID NO: 7)
MetGlyCysValGlnCysLysAspLysGluAlaThrLysLeuThrGlu GluLeuGlu SEQ ID
NO: 9 (nucleotide sequence encoding PSMA)
gcggatccgcatcatcatcatcatcacagctccggaatcgagggacgtgg
taaatcctccaatgaagctactaacattactccaaagcataatatgaaag
catttttggatgaattgaaagctgagaacatcaagaagttcttatataat
tttacacagataccacatttagcaggaacagaacaaaactttcagcttgc
aaagcaaattcaatcccagtggaaagaatttggcctggattctgttgagc
tagcacattatgatgtcctgttgtcctacccaaataagactcatcccaac
tacatctcaataattaatgaagatggaaatgagattttcaacacatcatt
atttgaaccacctcctccaggatatgaaaatgtttcggatattgtaccac
ctttcagtgctttctctcctcaaggaatgccagagggcgatctagtgtat
gttaactatgcacgaactgaagacttctttaaattggaacgggacatgaa
aatcaattgctctgggaaaattgtaattgccagatatgggaaagttttca
gaggaaataaggttaaaaatgcccagctggcaggggccaaaggagtcatt
ctctactccgaccctgctgactactttgctcctggggtgaagtcctatcc
agatggttggaatcttcctggaggtggtgtccagcgtggaaatatcctaa
atctgaatggtgcaggagaccctctcacaccaggttacccagcaaatgaa
tatgcttataggcgtggaattgcagaggctgttggtcttccaagtattcc
tgttcatccaattggatactatgatgcacagaagctcctagaaaaaatgg
gtggctcagcaccaccagatagcagctggagaggaagtctcaaagtgccc
tacaatgttggacctggctttactggaaacttttctacacaaaaagtcaa
gatgcacatccactctaccaatgaagtgacaagaatttacaatgtgatag
gtactctcagaggagcagtggaaccagacagatatgtcattctgggaggt
caccgggactcatgggtgtttggtggtattgaccctcagagtggagcagc
tgttgttcatgaaattgtgaggagctttggaacactgaaaaaggaagggt
ggagacctagaagaacaattttgtttgcaagctgggatgcagaagaattt
ggtcttcttggttctactgagtgggcagaggagaattcaagactccttca
agagcgtggcgtggcttatattaatgctgactcatctatagaaggaaact
acactctgagagttgattgtacaccgctgatgtacagcttggtacacaac
ctaacaaaagagctgaaaagccctgatgaaggctttgaaggcaaatctct
ttatgaaagttggactaaaaaaagtccttccccagagttcagtggcatgc
ccaggataagcaaattgggatctggaaatgattttgaggtgttcttccaa
cgacttggaattgcttcaggcagagcacggtatactaaaaattgggaaac
aaacaaattcagcggctatccactgtatcacagtgtctatgaaacatatg
agttggtggaaaagttttatgatccaatgtttaaatatcacctcactgtg
gcccaggttcgaggagggatggtgtttgagctagccaattccatagtgct
cccttttgattgtcgagattatgctgtagttttaagaaagtatgctgaca
aaatctacagtatttctatgaaacatccacaggaaatgaagacatacagt
gtatcatttgattcacttttttctgcagtaaagaattttacagaaattgc
ttccaagttcagtgagagactccaggactttgacaaaagcaagcatgtca
tctatgctccaagcagccacaacaagtatgcaggggagtcattcccagga
atttatgatgctctgtttgatattgaaagcaaagtggacccttccaaggc
ctggggagaagtgaagagacagatttatgttgcagccttcacagtgcagg
cagctgcagagactttgagtgaagtagcctaagcggccgcatagca SEQ ID NO: 10 (PSMA
amino acid sequence encoded by SEQ ID NO: 9)
MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEAT
NITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQW
KEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPG
YENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKI
VIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPG
GGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYY
DAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTN
EVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVR
SFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYI
NADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKK
SPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYP
LYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDY
AVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERL
QDFDKSKHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQ
IYVAAFTVQAAAETLSEVA
[0298] The entirety of each patent, patent application, publication
and document referenced herein hereby is incorporated by reference.
Citation of the above patents, patent applications, publications
and documents is not an admission that any of the foregoing is
pertinent prior art, nor does it constitute any admission as to the
contents or date of these publications or documents.
[0299] Modifications may be made to the foregoing without departing
from the basic aspects of the invention. Although the invention has
been described in substantial detail with reference to one or more
specific embodiments, those of ordinary skill in the art will
recognize that changes may be made to the embodiments specifically
disclosed in this application, and yet these modifications and
improvements are within the scope and spirit of the invention. The
invention illustratively described herein suitably may be practiced
in the absence of any element(s) not specifically disclosed herein.
Thus, for example, in each instance herein any of the terms
"comprising", "consisting essentially of", and "consisting of" may
be replaced with either of the other two terms. Thus, the terms and
expressions which have been employed are used as terms of
description and not of limitation, equivalents of the features
shown and described, or portions thereof, are not excluded, and it
is recognized that various modifications are possible within the
scope of the invention. Embodiments of the invention are set forth
in the following claims.
Sequence CWU 1
1
4812626DNAMus musculus 1ccgggaccag cggacggacc gagcagcgtc ctgcggccgg
caccgcggcg gcccagatcc 60ggccagcagc gcgcgcccgg acgccgctgc cttcagccgg
ccccgcccag cgcccgcccg 120cgggatgcgg agcggcgggc gcccgaggcc
gcggcccggc taggcccagt cgcccgcacg 180cggcggcccg acgctgcggc
caggccggct gggctcagcc taccgagaag agactctgat 240catcatccct
gggttacccc tgtctctggg ggccacggat accatgaacg acgtagccat
300tgtgaaggag ggctggctgc acaaacgagg ggaatatatt aaaacctggc
ggccacgcta 360cttcctcctc aagaacgatg gcacctttat tggctacaag
gaacggcctc aggatgtgga 420tcagcgagag tccccactca acaacttctc
agtggcacaa tgccagctga tgaagacaga 480gcggccaagg cccaacacct
ttatcatccg ctgcctgcag tggaccacag tcattgagcg 540caccttccat
gtggaaacgc ctgaggagcg ggaagaatgg gccaccgcca ttcagactgt
600ggccgatgga ctcaagaggc aggaagaaga gacgatggac ttccgatcag
gctcacccag 660tgacaactca ggggctgaag agatggaggt gtccctggcc
aagcccaagc accgtgtgac 720catgaacgag tttgagtacc tgaaactact
gggcaagggc acctttggga aagtgattct 780ggtgaaagag aaggccacag
gccgctacta tgccatgaag atcctcaaga aggaggtcat 840cgtcgccaag
gatgaggttg cccacacgct tactgagaac cgtgtcctgc agaactctag
900gcatcccttc cttacggccc tcaagtactc attccagacc cacgaccgcc
tctgctttgt 960catggagtat gccaacgggg gcgagctctt cttccacctg
tctcgagagc gcgtgttctc 1020cgaggaccgg gcccgcttct atggtgcgga
gattgtgtct gccctggact acttgcactc 1080cgagaagaac gtggtgtacc
gggacctgaa gctggagaac ctcatgctgg acaaggacgg 1140gcacatcaag
ataacggact tcgggctgtg caaggagggg atcaaggatg gtgccactat
1200gaagacattc tgcggaacgc cggagtacct ggcccctgag gtgctggagg
acaacgacta 1260cggccgtgca gtggactggt gggggctggg cgtggtcatg
tatgagatga tgtgtggccg 1320cctgcccttc tacaaccagg accacgagaa
gctgttcgag ctgatcctca tggaggagat 1380ccgcttcccg cgcacactcg
gccctgaggc caagtccctg ctctccgggc tgctcaagaa 1440ggaccctaca
cagaggctcg gtgggggctc tgaggatgcc aaggagatca tgcagcaccg
1500gttctttgcc aacatcgtgt ggcaggatgt gtatgagaag aagctgagcc
cacctttcaa 1560gccccaggtc acctctgaga ctgacaccag gtatttcgat
gaggagttca cagctcagat 1620gatcaccatc acgccgcctg atcaagatga
cagcatggag tgtgtggaca gtgagcggag 1680gccgcacttc ccccagttct
cctactcagc cagtggcaca gcctgaggcc tggggcagcg 1740gctggcagct
ccacgctcct ctgcattgcc gagtccagaa gccccgcatg gatcatctga
1800acctgatgtt ttgtttctcg gatgcgctgg ggaggaacct tgccagcctc
caggaccagg 1860ggaggatgtt tctactgtgg gcagcagcct acctcccagc
caggtcagga ggaaaactat 1920cctggggttt ttcttaattt atttcatcca
gtttgagacc acacatgtgg cctcagtgcc 1980cagaacaatt agattcatgt
agaaaactat taaggactga cgcgaccatg tgcaatgtgg 2040gctcatgggt
ctgggtgggt cccgtcactg cccccattgg cctgtccacc ctggccgcca
2100cctgtctcta gggtccaggg ccaaagtcca gcaagaaggc accagaagca
cctccctgtg 2160gtatgctaac tggccctctc cctctgggcg gggagaggtc
acagctgctt cagccctagg 2220gctggatggg atggccaggg ctcaagtgag
gttgacagag gaacaagaat ccagtttgtt 2280gctgtgtccc atgctgttca
gagacattta ggggatttta atcttggtga caggagagcc 2340cctgccctcc
cgctcctgcg tggtggctct tagcgggtac cctgggagcg cctgcctcac
2400gtgagccctc tcctagcact tgtcctttta gatgctttcc ctctcccgct
gtccgtcacc 2460ctggcctgtc ccctcccgcc agacgctggc cattgctgca
ccatgtcgtt ttttacaaca 2520ttcagcttca gcatttttac tattataata
agaaactgtc cctccaaatt caataaaaat 2580tgcttttcaa gcttgaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaa 262622794DNAHomo sapiens 2cggcaggacc
gagcgcggca ggcggctggc ccagcgcagc cagcgcggcc cgaaggacgg 60gagcaggcgg
ccgagcaccg agcgctgggc accgggcacc gagcggcggc ggcacgcgag
120gcccggcccc gagcagcgcc cccgcccgcc gcggcctcca gcccggcccc
gcccagcgcc 180ggcccgcggg gatgcggagc ggcgggcgcc ggaggccgcg
gcccggctag gcccgcgctc 240gcgcccggac gcggcggccc gaggctgtgg
ccaggccagc tgggctcggg gagcgccagc 300ctgagaggag cgcgtgagcg
tcgcgggagc ctcgggcacc atgagcgacg tggctattgt 360gaaggagggt
tggctgcaca aacgagggga gtacatcaag acctggcggc cacgctactt
420cctcctcaag aatgatggca ccttcattgg ctacaaggag cggccgcagg
atgtggacca 480acgtgaggct cccctcaaca acttctctgt ggcgcagtgc
cagctgatga agacggagcg 540gccccggccc aacaccttca tcatccgctg
cctgcagtgg accactgtca tcgaacgcac 600cttccatgtg gagactcctg
aggagcggga ggagtggaca accgccatcc agactgtggc 660tgacggcctc
aagaagcagg aggaggagga gatggacttc cggtcgggct cacccagtga
720caactcaggg gctgaagaga tggaggtgtc cctggccaag cccaagcacc
gcgtgaccat 780gaacgagttt gagtacctga agctgctggg caagggcact
ttcggcaagg tgatcctggt 840gaaggagaag gccacaggcc gctactacgc
catgaagatc ctcaagaagg aagtcatcgt 900ggccaaggac gaggtggccc
acacactcac cgagaaccgc gtcctgcaga actccaggca 960ccccttcctc
acagccctga agtactcttt ccagacccac gaccgcctct gctttgtcat
1020ggagtacgcc aacgggggcg agctgttctt ccacctgtcc cgggagcgtg
tgttctccga 1080ggaccgggcc cgcttctatg gcgctgagat tgtgtcagcc
ctggactacc tgcactcgga 1140gaagaacgtg gtgtaccggg acctcaagct
ggagaacctc atgctggaca aggacgggca 1200cattaagatc acagacttcg
ggctgtgcaa ggaggggatc aaggacggtg ccaccatgaa 1260gaccttttgc
ggcacacctg agtacctggc ccccgaggtg ctggaggaca atgactacgg
1320ccgtgcagtg gactggtggg ggctgggcgt ggtcatgtac gagatgatgt
gcggtcgcct 1380gcccttctac aaccaggacc atgagaagct ttttgagctc
atcctcatgg aggagatccg 1440cttcccgcgc acgcttggtc ccgaggccaa
gtccttgctt tcagggctgc tcaagaagga 1500ccccaagcag aggcttggcg
ggggctccga ggacgccaag gagatcatgc agcatcgctt 1560ctttgccggt
atcgtgtggc agcacgtgta cgagaagaag ctcagcccac ccttcaagcc
1620ccaggtcacg tcggagactg acaccaggta ttttgatgag gagttcacgg
cccagatgat 1680caccatcaca ccacctgacc aagatgacag catggagtgt
gtggacagcg agcgcaggcc 1740ccacttcccc cagttctcct actcggccag
cggcacggcc tgaggcggcg gtggactgcg 1800ctggacgata gcttggaggg
atggagaggc ggcctcgtgc catgatctgt atttaatggt 1860ttttatttct
cgggtgcatt tgagagaagc cacgctgtcc tctcgagccc agatggaaag
1920acgtttttgt gctgtgggca gcaccctccc ccgcagcggg gtagggaaga
aaactatcct 1980gcgggtttta atttatttca tccagtttgt tctccgggtg
tggcctcagc cctcagaaca 2040atccgattca cgtagggaaa tgttaaggac
ttctgcagct atgcgcaatg tggcattggg 2100gggccgggca ggtcctgccc
atgtgtcccc tcactctgtc agccagccgc cctgggctgt 2160ctgtcaccag
ctatctgtca tctctctggg gccctgggcc tcagttcaac ctggtggcac
2220cagatgcaac ctcactatgg tatgctggcc agcaccctct cctgggggtg
gcaggcacac 2280agcagccccc cagcactaag gccgtgtctc tgaggacgtc
atcggaggct gggcccctgg 2340gatgggacca gggatggggg atgggccagg
gtttacccag tgggacagag gagcaaggtt 2400taaatttgtt attgtgtatt
atgttgttca aatgcatttt gggggttttt aatctttgtg 2460acaggaaagc
cctccccctt ccccttctgt gtcacagttc ttggtgactg tcccaccggg
2520agcctccccc tcagatgatc tctccacggt agcacttgac cttttcgacg
cttaaccttt 2580ccgctgtcgc cccaggccct ccctgactcc ctgtgggggt
ggccatccct gggcccctcc 2640acgcctcctg gccagacgct gccgctgccg
ctgcaccacg gcgttttttt acaacattca 2700actttagtat ttttactatt
ataatataat atggaacctt ccctccaaat tcttcaataa 2760aagttgcttt
tcaaaaaaaa aaaaaaaaaa aaaa 279431715DNAHomo sapiens 3gaattccagc
ggcggcgccg ttgccgctgc cgggaaacac aaggaaaggg aaccagcgca 60gcgtggcgat
gggcgggggt agagccccgc cggagaggct gggcggctgc cggtgacaga
120ctgtgccctg tccacggtgc ctcctgcatg tcctgctgcc ctgagctgtc
ccgagctagg 180tgacagcgta ccacgctgcc accatgaatg aggtgtctgt
catcaaagaa ggctggctcc 240acaagcgtgg tgaatacatc aagacctgga
ggccacggta cttcctgctg aagagcgacg 300gctccttcat tgggtacaag
gagaggcccg aggcccctga tcagactcta ccccccttaa 360acaacttctc
cgtagcagaa tgccagctga tgaagaccga gaggccgcga cccaacacct
420ttgtcatacg ctgcctgcag tggaccacag tcatcgagag gaccttccac
gtggattctc 480cagacgagag ggaggagtgg atgcgggcca tccagatggt
cgccaacagc ctcaagcagc 540gggccccagg cgaggacccc atggactaca
agtgtggctc ccccagtgac tcctccacga 600ctgaggagat ggaagtggcg
gtcagcaagg cacgggctaa agtgaccatg aatgacttcg 660actatctcaa
actccttggc aagggaacct ttggcaaagt catcctggtg cgggagaagg
720ccactggccg ctactacgcc atgaagatcc tgcgaaagga agtcatcatt
gccaaggatg 780aagtcgctca cacagtcacc gagagccggg tcctccagaa
caccaggcac ccgttcctca 840ctgcgctgaa gtatgccttc cagacccacg
accgcctgtg ctttgtgatg gagtatgcca 900acgggggtga gctgttcttc
cacctgtccc gggagcgtgt cttcacagag gagcgggccc 960ggttttatgg
tgcagagatt gtctcggctc ttgagtactt gcactcgcgg gacgtggtat
1020accgcgacat caagctggaa aacctcatgc tggacaaaga tggccacatc
aagatcactg 1080actttggcct ctgcaaagag ggcatcagtg acggggccac
catgaaaacc ttctgtggga 1140ccccggagta cctggcgcct gaggtgctgg
aggacaatga ctatggccgg gccgtggact 1200ggtgggggct gggtgtggtc
atgtacgaga tgatgtgcgg ccgcctgccc ttctacaacc 1260aggaccacga
gcgcctcttc gagctcatcc tcatggaaga gatccgcttc ccgcgcacgc
1320tcagccccga ggccaagtcc ctgcttgctg ggctgcttaa gaaggacccc
aagcagaggc 1380ttggtggggg gcccagcgat gccaaggagg tcatggagca
caggttcttc ctcagcatca 1440actggcagga cgtggtccag aagaagctcc
tgccaccctt caaacctcag gtcacgtccg 1500aggtcgacac aaggtacttc
gatgatgaat ttaccgccca gtccatcaca atcacacccc 1560ctgaccgcta
tgacagcctg ggcttactgg agctggacca gcggacccac ttcccccagt
1620tctcctactc ggccagcatc cgcgagtgag cagtctgccc acgcagagga
cgcacgctcg 1680ctgccatcac cgctgggtgg ttttttaccc ctgcc
171543588DNAHomo sapiens 4gcagcagcag agaatccaaa ccctaaagct
gatatcacaa agtaccattt ctccaagttg 60ggggctcaga ggggagtcat catgagcgat
gttaccattg tgaaagaagg ttgggttcag 120aagaggggag aatatataaa
aaactggagg ccaagatact tccttttgaa gacagatggc 180tcattcatag
gatataaaga gaaacctcaa gatgtggatt taccttatcc cctcaacaac
240ttttcagtgg caaaatgcca gttaatgaaa acagaacgac caaagccaaa
cacatttata 300atcagatgtc tccagtggac tactgttata gagagaacat
ttcatgtaga tactccagag 360gaaagggaag aatggacaga agctatccag
gctgtagcag acagactgca gaggcaagaa 420gaggagagaa tgaattgtag
tccaacttca caaattgata atataggaga ggaagagatg 480gatgcctcta
caacccatca taaaagaaag acaatgaatg attttgacta tttgaaacta
540ctaggtaaag gcacttttgg gaaagttatt ttggttcgag agaaggcaag
tggaaaatac 600tatgctatga agattctgaa gaaagaagtc attattgcaa
aggatgaagt ggcacacact 660ctaactgaaa gcagagtatt aaagaacact
agacatccct ttttaacatc cttgaaatat 720tccttccaga caaaagaccg
tttgtgtttt gtgatggaat atgttaatgg gggcgagctg 780tttttccatt
tgtcgagaga gcgggtgttc tctgaggacc gcacacgttt ctatggtgca
840gaaattgtct ctgccttgga ctatctacat tccggaaaga ttgtgtaccg
tgatctcaag 900ttggagaatc taatgctgga caaagatggc cacataaaaa
ttacagattt tggactttgc 960aaagaaggga tcacagatgc agccaccatg
aagacattct gtggcactcc agaatatctg 1020gcaccagagg tgttagaaga
taatgactat ggccgagcag tagactggtg gggcctaggg 1080gttgtcatgt
atgaaatgat gtgtgggagg ttacctttct acaaccagga ccatgagaaa
1140ctttttgaat taatattaat ggaagacatt aaatttcctc gaacactctc
ttcagatgca 1200aaatcattgc tttcagggct cttgataaag gatccaaata
aacgccttgg tggaggacca 1260gatgatgcaa aagaaattat gagacacagt
ttcttctctg gagtaaactg gcaagatgta 1320tatgataaaa agcttgtacc
tccttttaaa cctcaagtaa catctgagac agatactaga 1380tattttgatg
aagaatttac agctcagact attacaataa caccacctga aaaatatgat
1440gaggatggta tggactgcat ggacaatgag aggcggccgc atttccctca
attttcctac 1500tctgcaagtg gacgagaata agtctctttc attctgctac
ttcactgtca tcttcaattt 1560attactgaaa atgattcctg gacatcacca
gtcctagctc ttacacatag caggggcacc 1620ttccgacatc ccagaccagc
caagggtcct cacccctcgc cacctttcac cctcatgaaa 1680acacacatac
acgcaaatac actccagttt ttgtttttgc atgaaattgt atctcagtct
1740aaggtctcat gctgttgctg ctactgtctt actattatag caactttaag
aagtaatttt 1800ccaacctttg gaagtcatga gcccaccatt gttcatttgt
gcaccaatta tcatcttttg 1860atcttttagt ttttccctca gtgaaggcta
aatgagatac actgattcta ggtacatttt 1920ttaactttct agaagagaaa
aactaactag actaagaaga tttagtttat aaattcagaa 1980caagcaattg
tggaagggtg gtggcgtgca tatgtaaagc acatcagatc cgtgcgtgaa
2040gtaggcatat atcactaagc tgtggctgga attgattagg aagcatttgg
tagaaggact 2100gaacaactgt tgggatatat atatatatat ataatttttt
ttttttaaat tcctggtgga 2160tactgtagaa gaagcccata tcacatgtgg
atgtcgagac ttcacgggca atcatgagca 2220agtgaacact gttctaccaa
gaactgaagg catatgcaca gtcaaggtca cttaaagggt 2280cttatgaaac
aatttgagcc agagagcatc tttcccctgt gcttggaaac cttttttcct
2340tcttgacatt tatcacctct gatggctgaa gaatgtagac aggtataatg
atactgcttt 2400tcaccaaaat ttctacacca aggtaaacag gtgtttgcct
tatttaattt tttactttca 2460gttctacgtg aattagcttt ttctcagatg
ttgaaacttt gaatgtcctt ttatgatttt 2520gtttatattg cagtagtatt
tattttttag tgatgagaat tgtatgtcat gttagcaaac 2580gcagctccaa
cttatataaa atagacttac tgcagttact tttgacccat gtgcaaggat
2640tgtacacgct gatgagaatc atgcactttt tctcctctgt taaaaaaaat
gataaggctc 2700tgaaatggaa tatattggtt agaatttggc tttgggagaa
gagatgctgc catttaaccc 2760cttggtactg aaaatgagaa aatccccaac
tatgcatgcc aaggggttaa tgaaacaaat 2820agctgttgac gtttgctcat
ttaagaattt gaaacgttat gatgacctgg caacaaaaag 2880taatgaagaa
aattgagacc tgagtgaaga taagaaatga tctttacgtg gcaaaatgaa
2940cacatcttga gtatttagga aatgggcagt gaaggctaag aacctggtgt
gtttcttggg 3000atcatggtac atttatcact gaattaagcc atcagggaaa
aaacaacaaa aaaagagaac 3060acctccagct tttctttttc tgtatatact
catgtccccc agattccaac atttctcact 3120gaaagggggc atgtatgcaa
acctcatctt tctccttcat taatgatgat cttcagatta 3180aaccctttgg
tgctaggagc tgacaatttc caaagcagcc tgtgaagtcc taggggctgg
3240gggccactct tgcggcaagc agaaggccat cctactccgc ggagtgatca
tggaaatgta 3300ttttagttaa actctgacag ctcccaaacg gaagactaca
gcatgacgta gtattatgat 3360tgcattgtat gaaagagcaa gtgactttct
aagtaggatg aatcatattc atatgcagat 3420gtcttagcct cttgacgctg
gaagtgtgga tttatagcta tgaaaccact gctggcagtg 3480ggtgggccac
tgggactgac gggggttaaa gggcatttta ctaaggcagc taagacatat
3540tcagacatca acgttatcct tctttttcat atttctacct gagtgaag
358851143DNAHomo sapiens 5accgccatcc agactgtggc tgacggcctc
aagaagcagg aggaggagga gatggacttc 60cggtcgggct cacccagtga caactcaggg
gctgaagaga tggaggtgtc cctggccaag 120cccaagcacc gcgtgaccat
gaacgagttt gagtacctga agctgctggg caagggcact 180ttcggcaagg
tgatcctggt gaaggagaag gccacaggcc gctactacgc catgaagatc
240ctcaagaagg aagtcatcgt ggccaaggac gaggtggccc acacactcac
cgagaaccgc 300gtcctgcaga actccaggca ccccttcctc acagccctga
agtactcttt ccagacccac 360gaccgcctct gctttgtcat ggagtacgcc
aacgggggcg agctgttctt ccacctgtcc 420cgggagcgtg tgttctccga
ggaccgggcc cgcttctatg gcgctgagat tgtgtcagcc 480ctggactacc
tgcactcgga gaagaacgtg gtgtaccggg acctcaagct ggagaacctc
540atgctggaca aggacgggca cattaagatc acagacttcg ggctgtgcaa
ggaggggatc 600aaggacggtg ccaccatgaa gaccttttgc ggcacacctg
agtacctggc ccccgaggtg 660ctggaggaca atgactacgg ccgtgcagtg
gactggtggg ggctgggcgt ggtcatgtac 720gagatgatgt gcggtcgcct
gcccttctac aaccaggacc atgagaagct ttttgagctc 780atcctcatgg
aggagatccg cttcccgcgc acgcttggtc ccgaggccaa gtccttgctt
840tcagggctgc tcaagaagga ccccaagcag aggcttggcg ggggctccga
ggacgccaag 900gagatcatgc agcatcgctt ctttgccggt atcgtgtggc
agcacgtgta cgagaagaag 960ctcagcccac ccttcaagcc ccaggtcacg
tcggagactg acaccaggta ttttgatgag 1020gagttcacgg cccagatgat
caccatcaca ccacctgacc aagatgacag catggagtgt 1080gtggacagcg
agcgcaggcc ccacttcccc cagttctcct actcggccag cgcgacggcc 1140tga
11436380PRTHomo sapiens 6Thr Ala Ile Gln Thr Val Ala Asp Gly Leu
Lys Lys Gln Glu Glu Glu1 5 10 15Glu Met Asp Phe Arg Ser Gly Ser Pro
Ser Asp Asn Ser Gly Ala Glu 20 25 30Glu Met Glu Val Ser Leu Ala Lys
Pro Lys His Arg Val Thr Met Asn 35 40 45Glu Phe Glu Tyr Leu Lys Leu
Leu Gly Lys Gly Thr Phe Gly Lys Val 50 55 60Ile Leu Val Lys Glu Lys
Ala Thr Gly Arg Tyr Tyr Ala Met Lys Ile65 70 75 80Leu Lys Lys Glu
Val Ile Val Ala Lys Asp Glu Val Ala His Thr Leu 85 90 95Thr Glu Asn
Arg Val Leu Gln Asn Ser Arg His Pro Phe Leu Thr Ala 100 105 110Leu
Lys Tyr Ser Phe Gln Thr His Asp Arg Leu Cys Phe Val Met Glu 115 120
125Tyr Ala Asn Gly Gly Glu Leu Phe Phe His Leu Ser Arg Glu Arg Val
130 135 140Phe Ser Glu Asp Arg Ala Arg Phe Tyr Gly Ala Glu Ile Val
Ser Ala145 150 155 160Leu Asp Tyr Leu His Ser Glu Lys Asn Val Val
Tyr Arg Asp Leu Lys 165 170 175Leu Glu Asn Leu Met Leu Asp Lys Asp
Gly His Ile Lys Ile Thr Asp 180 185 190Phe Gly Leu Cys Lys Glu Gly
Ile Lys Asp Gly Ala Thr Met Lys Thr 195 200 205Phe Cys Gly Thr Pro
Glu Tyr Leu Ala Pro Glu Val Leu Glu Asp Asn 210 215 220Asp Tyr Gly
Arg Ala Val Asp Trp Trp Gly Leu Gly Val Val Met Tyr225 230 235
240Glu Met Met Cys Gly Arg Leu Pro Phe Tyr Asn Gln Asp His Glu Lys
245 250 255Leu Phe Glu Leu Ile Leu Met Glu Glu Ile Arg Phe Pro Arg
Thr Leu 260 265 270Gly Pro Glu Ala Lys Ser Leu Leu Ser Gly Leu Leu
Lys Lys Asp Pro 275 280 285Lys Gln Arg Leu Gly Gly Gly Ser Glu Asp
Ala Lys Glu Ile Met Gln 290 295 300His Arg Phe Phe Ala Gly Ile Val
Trp Gln His Val Tyr Glu Lys Lys305 310 315 320Leu Ser Pro Pro Phe
Lys Pro Gln Val Thr Ser Glu Thr Asp Thr Arg 325 330 335Tyr Phe Asp
Glu Glu Phe Thr Ala Gln Met Ile Thr Ile Thr Pro Pro 340 345 350Asp
Gln Asp Asp Ser Met Glu Cys Val Asp Ser Glu Arg Arg Pro His 355 360
365Phe Pro Gln Phe Ser Tyr Ser Ala Ser Ala Thr Ala 370 375
380757DNAHomo sapiens 7atgggctgtg tgcaatgtaa ggataaagaa gcaacaaaac
tgacggagga gctcgag 57819PRTHomo sapiens 8Met Gly Cys Val Gln Cys
Lys Asp Lys Glu Ala Thr Lys Leu Thr Glu1 5 10 15Glu Leu
Glu92096DNAHomo sapiens 9gcggatccgc atcatcatca tcatcacagc
tccggaatcg agggacgtgg taaatcctcc 60aatgaagcta ctaacattac tccaaagcat
aatatgaaag catttttgga tgaattgaaa 120gctgagaaca tcaagaagtt
cttatataat tttacacaga taccacattt agcaggaaca 180gaacaaaact
ttcagcttgc aaagcaaatt caatcccagt ggaaagaatt tggcctggat
240tctgttgagc tagcacatta tgatgtcctg ttgtcctacc caaataagac
tcatcccaac 300tacatctcaa taattaatga agatggaaat gagattttca
acacatcatt atttgaacca 360cctcctccag gatatgaaaa tgtttcggat
attgtaccac
ctttcagtgc tttctctcct 420caaggaatgc cagagggcga tctagtgtat
gttaactatg cacgaactga agacttcttt 480aaattggaac gggacatgaa
aatcaattgc tctgggaaaa ttgtaattgc cagatatggg 540aaagttttca
gaggaaataa ggttaaaaat gcccagctgg caggggccaa aggagtcatt
600ctctactccg accctgctga ctactttgct cctggggtga agtcctatcc
agatggttgg 660aatcttcctg gaggtggtgt ccagcgtgga aatatcctaa
atctgaatgg tgcaggagac 720cctctcacac caggttaccc agcaaatgaa
tatgcttata ggcgtggaat tgcagaggct 780gttggtcttc caagtattcc
tgttcatcca attggatact atgatgcaca gaagctccta 840gaaaaaatgg
gtggctcagc accaccagat agcagctgga gaggaagtct caaagtgccc
900tacaatgttg gacctggctt tactggaaac ttttctacac aaaaagtcaa
gatgcacatc 960cactctacca atgaagtgac aagaatttac aatgtgatag
gtactctcag aggagcagtg 1020gaaccagaca gatatgtcat tctgggaggt
caccgggact catgggtgtt tggtggtatt 1080gaccctcaga gtggagcagc
tgttgttcat gaaattgtga ggagctttgg aacactgaaa 1140aaggaagggt
ggagacctag aagaacaatt ttgtttgcaa gctgggatgc agaagaattt
1200ggtcttcttg gttctactga gtgggcagag gagaattcaa gactccttca
agagcgtggc 1260gtggcttata ttaatgctga ctcatctata gaaggaaact
acactctgag agttgattgt 1320acaccgctga tgtacagctt ggtacacaac
ctaacaaaag agctgaaaag ccctgatgaa 1380ggctttgaag gcaaatctct
ttatgaaagt tggactaaaa aaagtccttc cccagagttc 1440agtggcatgc
ccaggataag caaattggga tctggaaatg attttgaggt gttcttccaa
1500cgacttggaa ttgcttcagg cagagcacgg tatactaaaa attgggaaac
aaacaaattc 1560agcggctatc cactgtatca cagtgtctat gaaacatatg
agttggtgga aaagttttat 1620gatccaatgt ttaaatatca cctcactgtg
gcccaggttc gaggagggat ggtgtttgag 1680ctagccaatt ccatagtgct
cccttttgat tgtcgagatt atgctgtagt tttaagaaag 1740tatgctgaca
aaatctacag tatttctatg aaacatccac aggaaatgaa gacatacagt
1800gtatcatttg attcactttt ttctgcagta aagaatttta cagaaattgc
ttccaagttc 1860agtgagagac tccaggactt tgacaaaagc aagcatgtca
tctatgctcc aagcagccac 1920aacaagtatg caggggagtc attcccagga
atttatgatg ctctgtttga tattgaaagc 1980aaagtggacc cttccaaggc
ctggggagaa gtgaagagac agatttatgt tgcagccttc 2040acagtgcagg
cagctgcaga gactttgagt gaagtagcct aagcggccgc atagca 209610719PRTHomo
sapiens 10Met Trp Asn Leu Leu His Glu Thr Asp Ser Ala Val Ala Thr
Ala Arg1 5 10 15Arg Pro Arg Trp Leu Cys Ala Gly Ala Leu Val Leu Ala
Gly Gly Phe 20 25 30Phe Leu Leu Gly Phe Leu Phe Gly Trp Phe Ile Lys
Ser Ser Asn Glu 35 40 45Ala Thr Asn Ile Thr Pro Lys His Asn Met Lys
Ala Phe Leu Asp Glu 50 55 60Leu Lys Ala Glu Asn Ile Lys Lys Phe Leu
Tyr Asn Phe Thr Gln Ile65 70 75 80Pro His Leu Ala Gly Thr Glu Gln
Asn Phe Gln Leu Ala Lys Gln Ile 85 90 95Gln Ser Gln Trp Lys Glu Phe
Gly Leu Asp Ser Val Glu Leu Ala His 100 105 110Tyr Asp Val Leu Leu
Ser Tyr Pro Asn Lys Thr His Pro Asn Tyr Ile 115 120 125Ser Ile Ile
Asn Glu Asp Gly Asn Glu Ile Phe Asn Thr Ser Leu Phe 130 135 140Glu
Pro Pro Pro Pro Gly Tyr Glu Asn Val Ser Asp Ile Val Pro Pro145 150
155 160Phe Ser Ala Phe Ser Pro Gln Gly Met Pro Glu Gly Asp Leu Val
Tyr 165 170 175Val Asn Tyr Ala Arg Thr Glu Asp Phe Phe Lys Leu Glu
Arg Asp Met 180 185 190Lys Ile Asn Cys Ser Gly Lys Ile Val Ile Ala
Arg Tyr Gly Lys Val 195 200 205Phe Arg Gly Asn Lys Val Lys Asn Ala
Gln Leu Ala Gly Ala Lys Gly 210 215 220Val Ile Leu Tyr Ser Asp Pro
Ala Asp Tyr Phe Ala Pro Gly Val Lys225 230 235 240Ser Tyr Pro Asp
Gly Trp Asn Leu Pro Gly Gly Gly Val Gln Arg Gly 245 250 255Asn Ile
Leu Asn Leu Asn Gly Ala Gly Asp Pro Leu Thr Pro Gly Tyr 260 265
270Pro Ala Asn Glu Tyr Ala Tyr Arg Arg Gly Ile Ala Glu Ala Val Gly
275 280 285Leu Pro Ser Ile Pro Val His Pro Ile Gly Tyr Tyr Asp Ala
Gln Lys 290 295 300Leu Leu Glu Lys Met Gly Gly Ser Ala Pro Pro Asp
Ser Ser Trp Arg305 310 315 320Gly Ser Leu Lys Val Pro Tyr Asn Val
Gly Pro Gly Phe Thr Gly Asn 325 330 335Phe Ser Thr Gln Lys Val Lys
Met His Ile His Ser Thr Asn Glu Val 340 345 350Thr Arg Ile Tyr Asn
Val Ile Gly Thr Leu Arg Gly Ala Val Glu Pro 355 360 365Asp Arg Tyr
Val Ile Leu Gly Gly His Arg Asp Ser Trp Val Phe Gly 370 375 380Gly
Ile Asp Pro Gln Ser Gly Ala Ala Val Val His Glu Ile Val Arg385 390
395 400Ser Phe Gly Thr Leu Lys Lys Glu Gly Trp Arg Pro Arg Arg Thr
Ile 405 410 415Leu Phe Ala Ser Trp Asp Ala Glu Glu Phe Gly Leu Leu
Gly Ser Thr 420 425 430Glu Trp Ala Glu Glu Asn Ser Arg Leu Leu Gln
Glu Arg Gly Val Ala 435 440 445Tyr Ile Asn Ala Asp Ser Ser Ile Glu
Gly Asn Tyr Thr Leu Arg Val 450 455 460Asp Cys Thr Pro Leu Met Tyr
Ser Leu Val His Asn Leu Thr Lys Glu465 470 475 480Leu Lys Ser Pro
Asp Glu Gly Phe Glu Gly Lys Ser Leu Tyr Glu Ser 485 490 495Trp Thr
Lys Lys Ser Pro Ser Pro Glu Phe Ser Gly Met Pro Arg Ile 500 505
510Ser Lys Leu Gly Ser Gly Asn Asp Phe Glu Val Phe Phe Gln Arg Leu
515 520 525Gly Ile Ala Ser Gly Arg Ala Arg Tyr Thr Lys Asn Trp Glu
Thr Asn 530 535 540Lys Phe Ser Gly Tyr Pro Leu Tyr His Ser Val Tyr
Glu Thr Tyr Glu545 550 555 560Leu Val Glu Lys Phe Tyr Asp Pro Met
Phe Lys Tyr His Leu Thr Val 565 570 575Ala Gln Val Arg Gly Gly Met
Val Phe Glu Leu Ala Asn Ser Ile Val 580 585 590Leu Pro Phe Asp Cys
Arg Asp Tyr Ala Val Val Leu Arg Lys Tyr Ala 595 600 605Asp Lys Ile
Tyr Ser Ile Ser Met Lys His Pro Gln Glu Met Lys Thr 610 615 620Tyr
Ser Val Ser Phe Asp Ser Leu Phe Ser Ala Val Lys Asn Phe Thr625 630
635 640Glu Ile Ala Ser Lys Phe Ser Glu Arg Leu Gln Asp Phe Asp Lys
Ser 645 650 655Lys His Val Ile Tyr Ala Pro Ser Ser His Asn Lys Tyr
Ala Gly Glu 660 665 670Ser Phe Pro Gly Ile Tyr Asp Ala Leu Phe Asp
Ile Glu Ser Lys Val 675 680 685Asp Pro Ser Lys Ala Trp Gly Glu Val
Lys Arg Gln Ile Tyr Val Ala 690 695 700Ala Phe Thr Val Gln Ala Ala
Ala Glu Thr Leu Ser Glu Val Ala705 710 715115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide motif
11Met Gly Cys Xaa Cys1 51210RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 12gccggcggag
101310RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 13cucauaaggu 101410RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 14gacuuugauu 101510RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 15cggaacccaa 101610RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 16auacuccccc 101710RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 17ccuugcgacc 10189PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 18Asp Tyr Lys Asp Asp Asp
Asp Lys Gly1 5196PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 19Asp Thr Tyr Arg Tyr Ile1
52014PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp
Ser Thr1 5 102110PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 21Glu Gln Lys Leu Ile Ser Glu Glu Asp
Leu1 5 102211PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 22Gln Pro Glu Leu Ala Pro Glu Asp Pro
Glu Asp1 5 10239PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 23Tyr Pro Tyr Asp Val Pro Asp Tyr Ala1
52411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 24Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys1 5
10256PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 6xHis tag 25His His His His His His1 5267PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 26Cys
Cys Xaa Xaa Xaa Cys Cys1 5276PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 27Cys Cys Pro Gly Cys Cys1
5286PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Leu Val Pro Arg Gly Ser1 5295PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 29Asp
Asp Asp Asp Lys1 5307PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 30Glu Asn Leu Tyr Phe Gln
Gly1 5318PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31Leu Glu Val Leu Phe Gln Gly Pro1
5328PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 32Ser Ile Ile Asn Phe Glu Lys Leu1
5339PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 33Phe Leu Trp Gly Pro Arg Ala Leu Val1
5348PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 34Val Tyr Asp Phe Phe Val Trp Leu1
53559DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 35ggccaccatg ggtagcaaca agagcaagcc
caaggatgcc agccagcggc gccgcagcc 593665DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 36tcgaggctgc ggcgccgctg gctggcatcc ttgggcttgc
tcttgttgct acccatggtg 60gccgc 653759DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 37ggccaccatg ggctgtgtct gcagctcaaa ccctgaagat
gactggatgg agaacattc 593865DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 38tcgagaatgt
tctccatcca gtcatcttca gggtttgagc tgcagacaca gcccatggtg 60gccgc
653959DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 39ggccaccatg ggctgtgtgc aatgtaagga
taaagaagca acaaaactga cggaggagc 594065DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 40tcgagctcct ccgtcagttt tgttgcttct ttatccttac
attgcacaca gcccatggtg 60gccgc 65418PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 41Gly
Ile Leu Gly Phe Val Phe Thr1 54232DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 42agagcgacaa cgacgtagcc
attgtgaagg ag 324330DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 43agagtcgaca ccgccattca gactgtggcc
304431DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 44agagtcgacg gctgtgccac tggctgagta g
314532DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 45cgatctcgag gagatgtggc atgaaggcct gg
324634DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 46cgatgtcgac ctttgagatt cgtcggaaca catg
344748DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 47atacaattgc cgcggttcga attctgtttt atactccctt
cccgtaac 484844DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 48tatcaattgg tttaaacagc aaacagatag
ataatgagtc tcac 44
* * * * *
References