U.S. patent application number 12/563991 was filed with the patent office on 2010-08-12 for methods and compositions for generating an immune response by inducing cd40 and pattern recognition receptor adapters.
This patent application is currently assigned to BAYLOR COLLEGE OF MEDICINE. Invention is credited to Narayanan Priyadharshini, David Spencer.
Application Number | 20100203067 12/563991 |
Document ID | / |
Family ID | 42039913 |
Filed Date | 2010-08-12 |
United States Patent
Application |
20100203067 |
Kind Code |
A1 |
Spencer; David ; et
al. |
August 12, 2010 |
METHODS AND COMPOSITIONS FOR GENERATING AN IMMUNE RESPONSE BY
INDUCING CD40 AND PATTERN RECOGNITION RECEPTOR ADAPTERS
Abstract
Provided are methods for activating an antigen-presenting cell
and eliciting an immune response by inducing an inducible pattern
recognition receptor adapter, or adapter fragment, and CD40
activity. Also provided are nucleic acid compositions comprising
sequences coding for chimeric proteins that include an inducible
CD40 peptide and an inducible pattern recognition receptor adapter
or adapter fragment.
Inventors: |
Spencer; David; (Houston,
TX) ; Priyadharshini; Narayanan; (Houston,
TX) |
Correspondence
Address: |
GRANT ANDERSON LLP;C/O PORTFOLIOIP
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
BAYLOR COLLEGE OF MEDICINE
Houston
TX
|
Family ID: |
42039913 |
Appl. No.: |
12/563991 |
Filed: |
September 21, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61099163 |
Sep 22, 2008 |
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61153562 |
Feb 18, 2009 |
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61181572 |
May 27, 2009 |
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Current U.S.
Class: |
424/184.1 ;
435/320.1; 435/325; 435/375; 536/23.1 |
Current CPC
Class: |
A61P 37/04 20180101;
A61K 2039/5154 20130101; A61K 39/00 20130101; A61P 31/04 20180101;
C12N 5/0639 20130101; C07K 14/4705 20130101; C12N 2710/10343
20130101; C07K 14/4702 20130101; A61P 31/00 20180101; C12N 7/00
20130101; A61P 35/00 20180101; A61P 43/00 20180101; C12N 2501/70
20130101; C12N 2501/998 20130101; A61K 2039/5156 20130101; C12N
2501/52 20130101; C12N 9/90 20130101; C12N 15/86 20130101; C07K
2319/033 20130101; C12N 2510/00 20130101; C07K 14/70578 20130101;
C12Y 502/01008 20130101 |
Class at
Publication: |
424/184.1 ;
435/375; 536/23.1; 435/320.1; 435/325 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C12N 5/02 20060101 C12N005/02; C07H 21/04 20060101
C07H021/04; C12N 15/63 20060101 C12N015/63; C12N 5/10 20060101
C12N005/10 |
Goverment Interests
STATEMENT OF FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government support under NIH
Grant Number R01-CA120411. The Government may have certain rights
in this invention.
Claims
1. A method for activating an antigen-presenting cell, which
comprises: transfecting or transducing an antigen-presenting cell
with a nucleic acid having a nucleotide sequence that encodes a
chimeric protein, wherein the chimeric protein comprises (i) a
membrane targeting region, (ii) a ligand-binding region (iii) a
cytoplasmic CD40 polypeptide region, and (iv) a peptide selected
from the group consisting of a MyD88 peptide, a truncated MyD88
peptide lacking the TIR domain, a NOD2 peptide, a RIG-1 peptide,
and a TRIF peptide; and contacting the antigen-presenting cell with
a non-protein multimeric ligand that binds to the ligand-binding
region; whereby the antigen-presenting cell is activated.
2. A method for activating an antigen-presenting cell, which
comprises: transfecting or transducing an antigen-presenting cell
with a nucleic acid having a nucleotide sequence that encodes a
chimeric protein, wherein the chimeric protein comprises (i) a
membrane targeting region, (ii) a ligand-binding region, and (iii)
a truncated MyD88 peptide lacking the TIR domain; and contacting
the antigen-presenting cell with a non-protein multimeric ligand
that binds to the ligand-binding region; whereby the
antigen-presenting cell is activated.
3. The method of claim 2, wherein the chimeric protein further
comprises a cytoplasmic CD40 polypeptide region.
4. The method of claim 1, wherein the peptide has a peptide
sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID
NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16 or wherein
the peptide is encoded by a nucleotide sequence selected from the
group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and
SEQ ID NO: 15.
5. The method of claim 1, wherein the truncated MyD88 has the
peptide sequence of SEQ ID NO: 6.
6. The method of claim 1, wherein the truncated MyD88 peptide is
encoded by the nucleotide sequence of SEQ ID NO: 5.
7. The method of claim 1, wherein the membrane targeting region is
selected from the group consisting of myristoylation-targeting
region, palmitoylation targeting region, prenylation region, and
receptor transmembrane region.
8. The method of claim 1, wherein the CD40 cytoplasmic polypeptide
region has a peptide sequence of the cytoplasmic region of SEQ ID
NO: 2.
9. The method of claim 1, wherein the ligand-binding region is
selected from the group consisting of FKBP ligand-binding region,
cyclophilin receptor ligand-binding region, steroid receptor
ligand-binding region, cyclophilin receptor ligand-binding region,
and tetracycline receptor ligand-binding region.
10. The method of claim 1, wherein the ligand-binding region
comprises a Fv'Fvls sequence.
11. The method of claim 1, wherein the ligand is a small
molecule.
12. The method of claim 1, wherein the ligand is dimeric.
13. The method of claim 12, wherein the ligand is dimeric FK506 or
a dimeric FK506 analog.
14. The method of claim 1, wherein the nucleic acid is contained
within a viral vector.
15. The method of claim 14, wherein the viral vector is an
adenoviral vector.
16. The method of claim 1, wherein the nucleic acid is contained
within a plasmid vector.
17. The method of claim 14, wherein the antigen-presenting cell is
contacted with the vector ex vivo.
18. The method of claim 1, wherein the antigen-presenting cell is
contacted with an antigen.
19. The method of claim 17, wherein the antigen-presenting cell is
transfected or transduced with the vector ex vivo and administered
to a subject
20. The method of claim 14, wherein the antigen-presenting cell is
transduced or transfected with the nucleic acid in vivo.
21. The method of claim 19, wherein an immune response is generated
against the antigen.
22. The method of claim 21 wherein the immune response is a
cytotoxic T-lymphocyte (CTL) immune response.
23. The method of claim 1, wherein the antigen-presenting cell is a
dendritic cell.
24. The method of claim 1, wherein the nucleic acid comprises a
promoter sequence operably linked to the polynucleotide
sequence.
25. The method of claim 1, wherein the antigen-presenting cell is
activated without the addition of an adjuvant.
26. A composition comprising a nucleic acid having a nucleotide
sequence that encodes a chimeric protein, wherein the chimeric
protein comprises (i) a membrane targeting region, (ii) a
ligand-binding region (iii) a cytoplasmic CD40 polypeptide region,
and (iv) a peptide selected from the group consisting of a MyD88
peptide, a truncated MyD88 peptide lacking the TIR domain, a NOD2
peptide, a RIG-1 peptide, and a TRIF peptide.
27. A composition comprising a nucleic acid having a nucleotide
sequence that encodes a chimeric protein, wherein the chimeric
protein comprises (i) a membrane targeting region, (ii) a
ligand-binding region, and (iii) a truncated MyD88 peptide lacking
the TIR domain.
28. The composition of claim 27, wherein the chimeric protein
further comprises a cytoplasmic CD40 polypeptide region.
29. The composition of claim 26, wherein the peptide has a peptide
sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID
NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16 or wherein
the peptide is encoded by a nucleotide sequence selected from the
group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and
SEQ ID NO: 15.
30. The composition of claim 26, wherein the truncated MyD88 has
the peptide sequence of SEQ ID NO: 6.
31. The composition of claim 26, wherein the truncated MyD88
peptide is encoded by the nucleotide sequence of SEQ ID NO: 5.
32. The composition of claim 26, wherein the membrane targeting
region is selected from the group consisting of
myristoylation-targeting region, palmitoylation targeting region,
prenylation region, and receptor transmembrane region.
33. The composition of claim 26, wherein the CD40 cytoplasmic
polypeptide region has a peptide sequence of the cytoplasmic region
of SEQ ID NO: 2.
34. The composition of claim 26, wherein the ligand-binding region
is selected from the group consisting of FKBP ligand-binding
region, cyclophilin receptor ligand-binding region, steroid
receptor ligand-binding region, cyclophilin receptor ligand-binding
region, and tetracycline receptor ligand-binding region.
35. The composition of claim 26, wherein the nucleic acid is
contained within a viral vector.
36. The composition of claim 35, wherein the viral vector is an
adenoviral vector.
37. The composition of claim 26, wherein the nucleic acid is
contained within a plasmid vector.
38. The composition of claim 26, wherein the nucleic acid comprises
a promoter sequence operably linked to the polynucleotide
sequence.
39. A composition comprising a cell transduced or transfected with
a nucleic acid of claim 26.
40. The composition of claim 26, wherein the cell is an
antigen-presenting cell.
41. The composition of claim 26, wherein the cell is a dendritic
cell.
42. A method for treating a condition in a subject by enhancing an
immune response, comprising administering to said subject a
composition of claim 26.
43. A method for treating a condition in a subject by enhancing an
immune response, comprising administering to said subject a
composition of claim 39.
44. The method of claim 43, wherein the condition is a
hyperproliferative disease.
45. The method of claim 43, wherein the condition is an infectious
disease.
Description
RELATED PATENT APPLICATIONS
[0001] Priority is claimed to U.S. Provisional Application Ser. No.
61/181,572, filed May 27, 2009, and entitled "Methods and
Compositions for Generating an Immune Response by Inducing CD40 and
Pattern Recognition Receptor Adapters;" to U.S. Provisional
Application Ser. No. 61/153,562, filed Feb. 18, 2009, and entitled
"Methods and Compositions for Generating an Immune Response by
Inducing CD40 and Pattern Recognition Receptor Adapters;" and to
U.S. Provisional Application Ser. No. 61/099,163, filed Sep. 22,
2008, and entitled "Methods and Compositions for Generating an
Immune Response by Inducing CD40 and Pattern Recognition Receptor
Adapters;" which are all referred to and all incorporated herein by
reference in their entirety.
FIELD OF THE INVENTION
[0003] The invention relates generally to the field of immunology,
and in particular, methods and compositions for activating
antigen-presenting cells and for inducing immune responses.
BACKGROUND
[0004] Due to their unique method of processing and presenting
antigens and the potential for high-level expression of
costimulatory and cytokine molecules, dendritic cells (DC) are
effective antigen-presenting cells (APCs) for priming and
activating naive T cells (Banchereau J, Paczesny S, Blanco P, et
al. Dendritic cells: controllers of the immune system and a new
promise for immunotherapy. Ann N Y Acad. Sci. 2003; 987:180-187).
This property has led to their widespread use as a cellular
platform for vaccination in a number of clinical trials with
encouraging results (O'Neill D W, Adams S, Bhardwaj N. Manipulating
dendritic cell biology for the active immunotherapy of cancer.
Blood. 2004; 104:2235-2246; Rosenberg S A. A new era for cancer
immunotherapy based on the genes that encode cancer antigens.
Immunity. 1999; 10:281-287). However, the clinical efficacy of DC
vaccines in cancer patients has been unsatisfactory, probably due
to a number of key deficiencies, including suboptimal activation,
limited migration to draining lymph nodes, and an insufficient life
span for optimal T cell activation in the lymph node
environment.
[0005] A parameter in the optimization of DC-based cancer vaccines
is the interaction of DCs with immune effector cells, such as CD4+,
CD8+ T cells and T regulatory (Treg) cells. In these interactions,
the maturation state of the DCs is a key factor in determining the
resulting effector functions (Steinman R M, Hawiger D, Nussenzweig
M C. Tolerogenic dendritic cells. Annu Rev Immunol. 2003;
21:685-711). To maximize CD4+ and CD8+ T cell priming while
minimizing Treg expansion, DCs need to be fully mature, expressing
high levels of co-stimulatory molecules, (like CD40, CD80, and
CD86), and pro-inflammatory cytokines, like IL-12p70 and IL-6.
Equally important, the DCs must be able to migrate efficiently from
the site of vaccination to draining lymph nodes to initiate T cell
interactions (Vieweg J, Jackson A. Modulation of antitumor
responses by dendritic cells. Springer Semin Immunopathol. 2005;
26:329-341).
[0006] For the ex vivo maturation of monocyte-derived immature DCs,
the majority of DC-based trials have used a standard maturation
cytokine cocktail (MC), comprised of TNF-alpha, IL-1beta, IL-6, and
PGE.sub.2. The principal function of prostaglandin E2 (PGE2) in the
standard maturation cocktail is to sensitize the CC chemokine
receptor 7 (CCR7) to its ligands, CC chemokine ligand 19 (CCL19)
and CCL21 and thereby enhance the migratory capacity of DCs to the
draining lymph nodes (Scandella E, Men Y, Gillessen S, Forster R,
Groettrup M. Prostaglandin E2 is a key factor for CCR7 surface
expression and migration of monocyte-derived dendritic cells.
Blood. 2002; 100:1354-1361; Luft T, Jefford M, Luetjens P, et al.
Functionally distinct dendritic cell (DC) populations induced by
physiologic stimuli: prostaglandin E(2) regulates the migratory
capacity of specific DC subsets. Blood. 2002; 100:1362-1372).
However, PGE2 has also been reported to have numerous properties
that are potentially deleterious to the stimulation of an immune
response, including suppression of T-cell proliferation, (Goodwin J
S, Bankhurst A D, Messner R P. Suppression of human T-cell
mitogenesis by prostaglandin. Existence of a
prostaglandin-producing suppressor cell. J Exp Med. 1977;
146:1719-1734; Goodwin J S. Immunomodulation by eicosanoids and
anti-inflammatory drugs. Curr Opin Immunol. 1989; 2:264-268)
inhibition of pro-inflammatory cytokine production (e.g., IL-12p70
and TNF-alpha (Kalinski P, Vieira P L, Schuitemaker J H, de Jong E
C, Kapsenberg M L. Prostaglandin E(2) is a selective inducer of
interleukin-12 p40 (IL-12p40) production and an inhibitor of
bioactive IL-12p70 heterodimer. Blood. 2001; 97:3466-3469; van der
Pouw Kraan T C, Boeije L C, Smeenk R J, Wijdenes J, Aarden L A.
Prostaglandin-E2 is a potent inhibitor of human interleukin 12
production. J Exp Med. 1995; 181:775-779)), and down-regulation of
major histocompatibility complex (MHC) II surface expression
(Snyder D S, Beller D I, Unanue E R. Prostaglandins modulate
macrophage Ia expression. Nature. 1982; 299:163-165). Therefore,
maturation protocols that can avoid PGE2 while promoting migration
are likely to improve the therapeutic efficacy of DC-based
vaccines.
[0007] A DC activation system based on targeted temporal control of
the CD40 signaling pathway has been developed to extend the
pro-stimulatory state of DCs within lymphoid tissues. DC
functionality was improved by increasing both the amplitude and the
duration of CD40 signaling (Hanks B A, Jiang J, Singh R A, et al.
Re-engineered CD40 receptor enables potent pharmacological
activation of dendritic-cell cancer vaccines in vivo. Nat. Med.
2005; 11:130-137). To accomplish this, the CD40 receptor was
re-engineered so that the cytoplasmic domain of CD40 was fused to
synthetic ligand-binding domains along with a membrane-targeting
sequence. Administration of a lipid-permeable, dimerizing drug,
AP20187 (AP), called a chemical inducer of dimerization (CID)
(Spencer D M, Wandless T J, Schreiber S L, Crabtree G R.
Controlling signal transduction with synthetic ligands. Science.
1993; 262:1019-1024), led to the in vivo induction of
CD40-dependent signaling cascades in murine DCs. This induction
strategy significantly enhanced the immunogenicity against both
defined antigens and tumors in vivo beyond that achieved with other
activation modalities (Hanks B A, et al., Nat. Med. 2005;
11:130-137). The robust potency of this chimeric ligand-inducible
CD40 (named iCD40) in mice suggested that this method might enhance
the potency of human DC vaccines, as well.
[0008] Pattern recognition receptor (PRR) signaling, an example of
which is Toll-like receptor (TLR) signaling also plays a critical
role in the induction of DC maturation and activation; human DCs
express, multiple distinct TLRs (Kadowaki N, Ho S, Antonenko S, et
al. Subsets of human dendritic cell precursors express different
toll-like receptors and respond to different microbial antigens. J
Exp Med. 2001; 194:863-869). The eleven mammalian TLRs respond to
various pathogen-derived macromolecules, contributing to the
activation of innate immune responses along with initiation of
adaptive immunity. Lipopolysaccharide (LPS) and a clinically
relevant derivative, monophosphoryl lipid A (MPL), bind to cell
surface TLR-4 complexes(Kadowaki N, Ho S, Antonenko S, et al.
Subsets of human dendritic cell precursors express different
toll-like receptors and respond to different microbial antigens. J
Exp Med. 2001; 194:863-869), leading to various signaling pathways
that culminate in the induction of transcription factors, such as
NF-kappaB and IRF3, along with mitogen-activated protein kinases
(MAPK) p38 and c-Jun kinase (JNK) (Ardeshna K M, Pizzey A R,
Devereux S, Khwaja A. The PI3 kinase, p38 SAP kinase, and NF-kappaB
signal transduction pathways are involved in the survival and
maturation of lipopolysaccharide-stimulated human monocyte-derived
dendritic cells. Blood. 2000; 96:1039-1046; Ismaili J, Rennesson J,
Aksoy E, et al. Monophosphoryl lipid A activates both human
dendritic cells and T cells. J. Immunol. 2002; 168:926-932). During
this process DCs mature, and partially upregulate pro-inflammatory
cytokines, like IL-6, IL-12, and Type I interferons (Rescigno M,
Martino M, Sutherland C L, Gold M R, Ricciardi-Castagnoli P.
Dendritic cell survival and maturation are regulated by different
signaling pathways. J Exp Med. 1998; 188:2175-2180). LPS-induced
maturation has been shown to enhance the ability of DCs to
stimulate antigen-specific T cell responses in vitro and in vivo
(Lapointe R, Toso J F, Butts C, Young H A, Hwu P. Human dendritic
cells require multiple activation signals for the efficient
generation of tumor antigen-specific T lymphocytes. Eur J. Immunol.
2000; 30:3291-3298). Methods for activating an antigen-presenting
cell, comprising transducing the cell with a nucleic acid coding
for a CD40 peptide have been described in U.S. Pat. No. 7,404,950,
and methods for activating an antigen-presenting cell, comprising
transfecting the cell with a nucleic acid coding for a chimeric
protein including an inducible CD40 peptide and a Pattern
Recognition Receptor, or other downstream proteins in the pathway
have been described in International Patent Application No.
PCT/US2007/081963, filed Oct. 19, 2007, published as WO
2008/049113, which are hereby incorporated by reference herein.
SUMMARY
[0009] An inducible CD40 (iCD40) system has been applied to human
dendritic cells (DCs) and it has been demonstrated that combining
iCD40 signaling with Pattern recognition receptor (PRR) adapter
ligation causes persistent and robust activation of human DCs.
These features form the basis of cancer immunotherapies for
treating such cancers as advanced, hormone-refractory prostate
cancer, for example. Accordingly, it has been discovered that the
combination of inducing CD40 and an inducible PRR adapter
synergistically activates antigen-presenting cells and induces an
immune response against an antigen. Inducible PRR adapters include,
for example, MyD88 and TRIF; inducible Pattern Recognition
Receptors, such as, for example, NOD-like receptors, for example,
NOD1 or NOD2, and RIG-like helicases, for example, RIG-I or Mda-5,
may also be used in combination with inducible CD40. Provided
herein are methods for activating antigen-presenting cells,
comprising transducing an antigen-presenting cell with a nucleic
acid having a nucleotide sequence that encodes a chimeric protein,
wherein the chimeric protein comprises (i) a membrane targeting
region, (ii) a ligand-binding region (iii) a cytoplasmic CD40
polypeptide region, and (iv) a peptide selected from the group
consisting of a truncated MyD88 peptide lacking the TIR domain and
a TRIF peptide; and contacting the antigen-presenting cell with a
non-protein multimeric ligand that binds to the ligand-binding
region; whereby the antigen-presenting cell is activated.
[0010] Thus, provided herein are methods for activating an
antigen-presenting cell, which comprise transfecting or transducing
an antigen-presenting cell with a nucleic acid having a nucleotide
sequence that encodes a chimeric protein, wherein the chimeric
protein comprises a membrane targeting region, a ligand-binding
region, a cytoplasmic CD40 polypeptide region, and a peptide
selected from the group consisting of a MyD88 peptide, a truncated
MyD88 peptide lacking the TIR domain, a NOD2 peptide, a RIG-1
peptide, and a TRIF peptide; and contacting the antigen-presenting
cell with a non-protein multimeric ligand that binds to the
ligand-binding region; whereby the antigen-presenting cell is
activated. The cytoplasmic CD40 polypeptide region of the methods
and compositions may, for example, have a peptide sequence of the
cytoplasmic region of SEQ ID NO: 2, and may, for example, be
encoded by a polynucleotide sequence coding for a cytoplasmic
polypeptide region in SEQ ID NO: 1.
[0011] Also provided are methods for method for activating an
antigen-presenting cell, which comprise transfecting or transducing
an antigen-presenting cell with a nucleic acid having a nucleotide
sequence that encodes a chimeric protein, wherein the chimeric
protein comprises a membrane targeting region, a ligand-binding
region, and a truncated MyD88 peptide lacking the TIR domain; and
contacting the antigen-presenting cell with a non-protein
multimeric ligand that binds to the ligand-binding region; whereby
the antigen-presenting cell is activated. The chimeric protein may
further comprise a CD40 polypeptide region.
[0012] Further provided are compositions comprising a nucleic acid
having a nucleotide sequence that encodes a chimeric protein,
wherein the chimeric protein comprises a membrane targeting region,
a ligand-binding region, a cytoplasmic CD40 polypeptide region, and
a peptide selected from the group consisting of a MyD88 peptide, a
truncated MyD88 peptide lacking the TIR domain, a NOD2 peptide, a
RIG-1 peptide, and a TRIF peptide.
[0013] An antigen-presenting cell is "activated," when one or more
activities associated with activated antigen-presenting cells may
be observed and/or measured by one of ordinary skill in the art.
For example, an antigen-presenting cell is activated when following
contact with an expression vector presented herein, an activity
associated with activation may be measured in the expression
vector-contacted cell as compared to an antigen-presenting cell
that has either not been contacted with the expression vector, or
has been contacted with a negative control vector. In one example,
the increased activity may be at a level of two, three, four, five,
six, seven, eight, nine, or ten fold, or more, than that of the
non-contacted cell, or the cell contacted with the negative
control. For example, one of the following activities may be
enhanced in an antigen-presenting cell that has been contacted with
the expression vector: co-stimulatory molecule expression on the
antigen-presenting cell, nuclear translocation of NF-kappaB in
antigen-presenting cells, DC maturation marker expression, such as,
for example, toll-like receptor expression or CCR7 expression,
specific cytotoxic T lymphocyte responses, such as, for example,
specific lytic activity directed against tumor cells, or cytokine
(for example, IL-2) or chemokine expression. Methods of assaying
the activation of antigen-presenting cells are presented herein,
for example, in Examples 11-17.
[0014] An amount of a composition that activates antigen-presenting
cells that "enhances" an immune response refers to an amount in
which an immune response is observed that is greater or intensified
or deviated in any way with the addition of the composition when
compared to the same immune response measured without the addition
of the composition. For example, the lytic activity of cytotoxic T
cells can be measured, for example, using a .sup.51Cr release
assay, with and without the composition. The amount of the
substance at which the CTL lytic activity is enhanced as compared
to the CTL lytic activity without the composition is said to be an
amount sufficient to enhance the immune response of the animal to
the antigen. For example, the immune response may be enhanced by a
factor of at least about 2, or, for example, by a factor of about 3
or more. The amount of cytokines secreted may also be altered.
[0015] The enhanced immune response may be an active or a passive
immune response. Alternatively, the response may be part of an
adaptive immunotherapy approach in which antigen-presenting cells
are obtained with from a subject (e.g., a patient), then transduced
or transfected with a composition comprising the expression vector
or construct presented herein. The antigen-presenting cells may be
obtained from, for example, the blood of the subject or bone marrow
of the subject. The antigen-presenting cells may then be
administered to the same or different animal, or same or different
subject (e.g., same or different donors). In certain embodiments
the subject (for example, a patient) has or is suspected of having
a cancer, such as for example, prostate cancer, or has or is
suspected of having an infectious disease. In other embodiments the
method of enhancing the immune response is practiced in conjunction
with a known cancer therapy or any known therapy to treat the
infectious disease.
[0016] The steps of the methods provided may be performed using any
suitable method known to and selected by the person of ordinary
skill, these methods include, without limitation, methods of
transducing, transforming, or otherwise providing nucleic acid to
the antigen-presenting cell, presented herein. In some embodiments,
the truncated MyD88 peptide is encoded by the nucleotide sequence
of SEQ ID NO: 5 (with or without DNA linkers). In other
embodiments, the peptide is a TRIF peptide. Often, the peptide is
encoded by the nucleotide sequence of SEQ ID NO: 9 (with or without
DNA linkers). In some embodiments, the CD40 cytoplasmic polypeptide
region is encoded by a polynucleotide sequence in SEQ ID NO: 1. In
some embodiments of the methods or compositions, the peptide has a
peptide sequence selected from the group consisting of SEQ ID NO:
6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16
or wherein the peptide is encoded by a nucleotide sequence selected
from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID
NO: 13, and SEQ ID NO: 15. IN Certain embodiments, the truncated
MyD88 has the peptide sequence of SEQ ID NO: 6, and, may, for
example, be encoded by the nucldotide sequence of SEQ ID NO: 5.
Often, the nucleic acid comprises a promoter sequence operably
linked to the polynucleotide sequence. In general, the term
"operably linked" is meant to indicate that the promoter sequence
is functionally linked to a second sequence, wherein the promoter
sequence initiates and mediates transcription of the DNA
corresponding to the second sequence. Those of ordinary skill in
the art may select an appropriate promoter sequence, including,
without limitation, a promoter sequence discussed herein.
[0017] Those of ordinary skill in the art may select any
appropriate known membrane targeting region, including, without
limitation a myristoylation-targeting region, palmitoylation
targeting region, prenylation region, or receptor transmembrane
region. Often, the membrane targeting region is a myristoylation
targeting region.
[0018] In certain embodiments, the ligand-binding region is
selected from the group consisting of FKBP ligand-binding region,
cyclophilin receptor ligand-binding region, steroid receptor
ligand-binding region, cyclophilin receptors ligand-binding region,
and tetracycline receptor ligand-binding region. Often, the
ligand-binding region comprises a Fv'Fvls sequence. Sometimes, the
Fv'Fvls sequence further comprises an additional Fv' sequence.
[0019] In some embodiments, the ligand is a small molecule. Those
of ordinary skill in the art may select the appropriate ligand for
the selected ligand-binding region. Often, the ligand is dimeric,
sometimes, the ligand is a dimeric FK506 or a dimeric FK506 analog.
In certain embodiments, the ligand is AP1903. In certain
embodiments, the ligand is AP20187.
[0020] In some embodiments, the nucleic acid is contained within a
viral vector. Those of ordinary skill in the art may select the
appropriate viral vector. In certain embodiments, the viral vector
is an adenoviral vector. It is understood that in some embodiments,
the antigen-presenting cell is contacted with the viral vector ex
vivo, and in some embodiments, the antigen-presenting cell is
contacted with the viral vector in vivo.
[0021] In some embodiments, the antigen-presenting cell is a
dendritic cell, for example, a mammalian dendritic cell. Often, the
antigen-presenting cell is a human dendritic cell.
[0022] In certain embodiments, the antigen-presenting cell is also
contacted with an antigen. Often, the antigen-presenting cell is
contacted with the antigen ex vivo. Sometimes, the
antigen-presenting cell is contacted with the antigen in vivo. In
some embodiments, the antigen-presenting cell is in a subject and
an immune response is generated against the antigen. Sometimes, the
immune response is a cytotoxic T-lymphocyte (CTL) immune response.
Sometimes, the immune response is generated against a tumor
antigen. In certain embodiments, the antigen-presenting cell is
activated without the addition of an adjuvant.
[0023] In some embodiments, the antigen-presenting cell is
transduced with the nucleic acid ex vivo and administered to the
subject by intradermal administration. In some embodiments, the
antigen-presenting cell is transduced with the nucleic acid ex vivo
and administered to the subject by subcutaneous administration.
Sometimes, the antigen-presenting cell is transduced with the
nucleic acid ex vivo. Sometimes, the antigen-presenting cell is
transduced with the nucleic acid in vivo.
[0024] Provided also is a method for inducing a cytotoxic T
lymphocyte (CTL) immune response against an antigen, which
comprises: contacting a human antigen-presenting cell sensitized
with an antigen with: (a) a multimeric molecule having two or more
regions that bind to and multimerize native CD40, and (b) an
inducible PRR adapter, for example, MyD88, truncated MyD88, or
TRIF; whereby a CTL immune response is induced against the antigen.
By MyD88 is meant the myeloid differentiation primary response gene
88, for example, but not limited to the human version, cited as
ncbi Gene ID 4615. By TRIF is meant the TIR-domain-containing
adapter-inducing interferon-beta. By "truncated," is meant that the
protein is not full length and may lack, for example, a domain. For
example, a truncated MyD88 is not full length and may, for example,
be missing the TIR domain. One example of a truncated MyD88 is
indicated as MyD88L herein, and is also presented as SEQ ID NOS: 5
(nucleic acid sequence) and 6 (peptide sequence). SEQ ID NO: 5
includes the linkers added during subcloning. Those of ordinary
skill in the art recognize that by a nucleic acid sequence coding
for "truncated MyD88" is meant the nucleic acid sequence coding for
the truncated MyD88 peptide, the term may also refer to the nucleic
acid sequence including the portion coding for any amino acids
added as an artifact of cloning, including any amino acids coded
for by the linkers. In such methods, the multimeric molecule can be
an antibody that binds to an epitope in the CD40 extracellular
domain (e.g., humanized anti-CD40 antibody; Tai et al., Cancer
Research 64, 2846-2852 (2004)), can be a CD40 ligand (e.g., U.S.
Pat. No. 6,497,876 (Maraskovsky et al.)) or may be another
co-stimulatory molecule (e.g., B7/CD28). It is understood by those
of ordinary skill in the art that conservative variations in
sequence, that do not affect the function, as assayed herein, are
within the scope of the present claims.
[0025] Also provided herein are compositions comprising a nucleic
acid having a polynucleotide sequence that encodes a chimeric
protein, wherein the chimeric protein comprises (i) a membrane
targeting region, (ii) a ligand-binding region (iii) a cytoplasmic
CD40 polypeptide region, and (iv) a peptide selected from the group
consisting of a truncated MyD88 peptide lacking the TIR domain and
a TRIF peptide. In some embodiments, the truncated MyD88 peptide is
encoded by the nucleotide sequence of SEQ ID NO: 5. In other
embodiments, the peptide is a TRIF peptide. Often, the peptide is
encoded by the nucleotide sequence of SEQ ID NO: 9. IN some
embodiments, the CD40 cytoplasmic polypeptide region is encoded by
a polynucleotide sequence in SEQ ID NO: 1. Often, the nucleic acid
comprises a promoter sequence operably linked to the polynucleotide
sequence. Those of ordinary skill in the art may select an
appropriate promoter sequence, including, without limitation, a
promoter sequence discussed herein.
[0026] Those of ordinary skill in the art may select any
appropriate known membrane targeting region, including, without
limitation a myristoylation-targeting region, palmitoylation
targeting region, prenylation region, or receptor transmembrane
region. Often, the membrane targeting region is a myristoylation
targeting region.
[0027] In certain embodiments, the ligand-binding region is
selected from the group consisting of FKBP ligand-binding region,
cyclophilin receptor ligand-binding region, steroid receptor
ligand-binding region, cyclophilin receptors ligand-binding region,
and tetracycline receptor ligand-binding region. Often, the
ligand-binding region comprises a Fv'Fvls sequence. Sometimes, the
Fv'Fvls sequence further comprises an additional Fv' sequence.
[0028] In some embodiments, the nucleic acid is contained within a
viral vector. Those of ordinary skill in the art may select the
appropriate viral vector. In certain embodiments, the viral vector
is an adenoviral vector. It is understood that in some embodiments,
the antigen-presenting cell is contacted with the viral vector ex
vivo, and in some embodiments, the antigen-presenting cell is
contacted with the viral vector in vivo.
[0029] In some embodiments, methods are provided for activating an
antigen-presenting cell, comprising transducing an
antigen-presenting cell with a nucleic acid having a nucleotide
sequence that encodes a chimeric protein, wherein the chimeric
protein comprises (i) a membrane targeting region, (ii) a
ligand-binding region, and (iii) a MyD88 peptide or a truncated
MyD88 peptide lacking the TIR domain; and contacting the
antigen-presenting cell with a non-protein multimeric ligand that
binds to the ligand-binding region; whereby the antigen-presenting
cell is activated. Often, the MyD88 peptide is truncated,
including, without limitation, a truncated MyD88 peptide that is
encoded by the nucleotide sequence of SEQ ID NO: 5. The steps of
the methods provided may be performed using any suitable method
known to and selected by the person of ordinary skill, these
methods include, without limitation, methods of transducing,
transforming, or otherwise providing nucleic acid to the
antigen-presenting cell, presented herein. Often, the nucleic acid
comprises a promoter sequence operably linked to the polynucleotide
sequence. Those of ordinary skill in the art may select an
appropriate promoter sequence, including, without limitation, a
promoter sequence discussed herein.
[0030] Those of ordinary skill in the art may select any
appropriate known membrane targeting region, including, without
limitation a myristoylation-targeting region, palmitoylation
targeting region, prenylation region, or receptor transmembrane
region. Often, the membrane targeting region is a myristoylation
targeting region.
[0031] In certain embodiments, the ligand-binding region is
selected from the group consisting of FKBP ligand-binding region,
cyclophilin receptor ligand-binding region, steroid receptor
ligand-binding region, cyclophilin receptors ligand-binding region,
and tetracycline receptor ligand-binding region. Often, the
ligand-binding region comprises a Fv'Fvls sequence. Sometimes, the
Fv'Fvls sequence further comprises an additional Fv' sequence.
[0032] In some embodiments, the ligand is a small molecule. Those
of ordinary skill in the art may select the appropriate ligand for
the selected ligand-binding region. Often, the ligand is dimeric,
sometimes, the ligand is a dimeric FK506 or a dimeric FK506 analog.
In certain embodiments, the ligand is AP1903. In certain
embodiments, the ligand is AP20187.
[0033] In some embodiments, the nucleic acid is contained within a
viral vector. Those of ordinary skill in the art may select the
appropriate viral vector. In certain embodiments, the viral vector
is an adenoviral vector. It is understood that in some embodiments,
the antigen-presenting cell is contacted with the viral vector ex
vivo, and in some embodiments, the antigen-presenting cell is
contacted with the viral vector in vivo.
[0034] In some embodiments, the antigen-presenting cell is a
dendritic cell, for example, a mammalian dendritic cell. Often, the
antigen-presenting cell is a human dendritic cell.
[0035] In certain embodiments, the antigen-presenting cell is also
contacted with an antigen. Often, the antigen-presenting cell is
contacted with the antigen ex vivo. Sometimes, the
antigen-presenting cell is contacted with the antigen in vivo. In
some embodiments, the antigen-presenting cell is in a subject and
an immune response is generated against the antigen. Sometimes, the
immune response is a cytotoxic T-lymphocyte (CTL) immune response.
Sometimes, the immune response is generated against a tumor
antigen. In certain embodiments, the antigen-presenting cell is
activated without the addition of an adjuvant.
[0036] In some embodiments, the antigen-presenting cell is
transduced with the nucleic acid ex vivo and administered to the
subject by intradermal administration. In some embodiments, the
antigen-presenting cell is transduced with the nucleic acid ex vivo
and administered to the subject by subcutaneous administration.
Sometimes, the antigen-presenting cell is transduced with the
nucleic acid ex vivo. Sometimes, the antigen-presenting cell is
transduced with the nucleic acid in vivo.
[0037] Also provided herein are compositions that may be used, for
example, in the methods of the present invention. Thus, provided
are compositions comprising a nucleic acid having a polynucleotide
sequence that encodes a chimeric protein, wherein the chimeric
protein comprises (i) a membrane targeting region, (ii) a
ligand-binding region (iii) a cytoplasmic CD40 polypeptide region,
and (iv) a MyD88 peptide or a truncated MyD88 peptide lacking the
TIR domain. In some embodiments, the MyD88 peptide is truncated.
Sometimes, the truncated MyD88 peptide is encoded by the nucleotide
sequence of SEQ ID NO: 5. Often, the nucleic acid comprises a
promoter sequence operably linked to the polynucleotide sequence.
Those of ordinary skill in the art may select an appropriate
promoter sequence, including, without limitation, a promoter
sequence discussed herein.
[0038] Those of ordinary skill in the art may select any
appropriate known membrane targeting region, including, without
limitation a myristoylation-targeting region, palmitoylation
targeting region, prenylation region, or receptor transmembrane
region. Often, the membrane targeting region is a myristoylation
targeting region.
[0039] In certain embodiments, the ligand-binding region is
selected from the group consisting of FKBP ligand-binding region,
cyclophilin receptor ligand-binding region, steroid receptor
ligand-binding region, cyclophilin receptors ligand-binding region,
and tetracycline receptor ligand-binding region. Often, the
ligand-binding region comprises a Fv'Fvls sequence. Sometimes, the
Fv'Fvls sequence further comprises an additional Fv' sequence.
[0040] In some embodiments, the nucleic acid is contained within a
viral vector. Those of ordinary skill in the art may select the
appropriate viral vector. In certain embodiments, the viral vector
is an adenoviral vector. It is understood that in some embodiments,
the antigen-presenting cell is contacted with the viral vector ex
vivo, and in some embodiments, the antigen-presenting cell is
contacted with the viral vector in vivo.
[0041] Also provided are compositions comprising a cell transduced
with a nucleic acid composition of any of the embodiments presented
herein. In some embodiments, the cell is an antigen-presenting
cell. Often, the cell is a dendritic cell, including, without
limitation, a mammalian cell, for example, but without limitation,
a human dendritic cell.
[0042] Also provided is the use of a composition comprising a
nucleic acid having a nucleotide sequence that encodes a chimeric
protein, wherein the chimeric protein comprises (i) a membrane
targeting region, (ii) a ligand-binding region (iii) a cytoplasmic
CD40 polypeptide region, and (iv) a peptide selected from the group
consisting of a MyD88 peptide, a truncated MyD88 peptide lacking
the TIR domain, a NOD2 peptide, a RIG-1 peptide, and a TRIF
peptide, in the manufacture of a medicament for therapy of a
condition by activating an immune response. In another embodiment
is the use of a composition comprising a cell transduced or
transfected with a nucleic acid having a nucleotide sequence that
encodes a chimeric protein, wherein the chimeric protein comprises
(i) a membrane targeting region, (ii) a ligand-binding region (iii)
a cytoplasmic CD40 polypeptide region, and (iv) a peptide selected
from the group consisting of a MyD88 peptide, a truncated MyD88
peptide lacking the TIR domain, a NOD2 peptide, a RIG-1 peptide,
and a TRIF peptide, in the manufacture of a medicament for therapy
of a condition by activating an immune response. The composition
may be used, for example, to transfect or transduce an
antigen-presenting cell. The peptide may, for example, be a
truncated MyD88 peptide. The condition may, for example, be a
hyperproliferative disease, or, for example, an infectious
disease.
[0043] In the methods for inducing an immune response presented
herein, the antigen-presenting cell can be transduced ex vivo or in
vivo with a nucleic acid that encodes the chimeric protein. The
antigen-presenting cell may be sensitized to the antigen at the
same time the antigen-presenting cell is contacted with the
multimeric ligand, or the antigen-presenting cell can be
pre-sensitized to the antigen before the antigen-presenting cell is
contacted with the multimerization ligand. In some embodiments, the
antigen-presenting cell is contacted with the antigen ex vivo. In
certain embodiments the antigen-presenting cell is transduced with
the nucleic acid ex vivo and administered to the subject by
intradermal administration, and sometimes the antigen-presenting
cell is transduced with the nucleic acid ex vivo and administered
to the subject by subcutaneous administration. The antigen may be a
tumor antigen, and the CTL immune response can induced by migration
of the antigen-presenting cell to a draining lymph node.
[0044] In the methods herein, the inducible CD40 portion of the
peptide may be located either upstream or downstream from the
inducible PRR adapter protein portion. Also, the inducible CD40
portion and the inducible PRR adapter protein portions may be
transfected or transduced into the cells either on the same vector,
in cis, or on separate vectors, in trans.
[0045] Also provided herein is a method for assessing migration of
an antigen-presenting cell to a lymph node, which comprises: (a)
injecting into a subject an antigen-presenting cell that produces a
detectable protein, and (b) determining the amount of the
detectable protein in the lymph node of the animal, whereby
migration of the antigen-presenting cell to the lymph node is
assessed from the amount of the detectable protein in the lymph
node. In such methods the animal can be a rodent, such as a rat or
a mouse (e.g., irradiated mouse). In some embodiments, the
detectable protein is a luciferase protein, such as a chick beetle
(e.g., Pyrophorus plagiophalamus) red-shifted luciferase protein.
In certain embodiments, the antigen-presenting cell has been
transduced with a nucleic acid having a polynucleotide sequence
that encodes the detectable protein. In certain embodiments, the
lymph node is the popliteal lymph node or inguinal lymph node. The
antigen-presenting cell can be a dendritic cell, such as a human
dendritic cell. In certain embodiments, the lymph node is removed
from the animal before the amount of detectable protein is
determined, and sometimes the D-Luciferin is administered to the
removed lymph node. The amount of the detectable protein may be
qualitative (e.g., relative amounts compared across different
samples) and can be quantitative (e.g., a concentration). The
amount of the detectable protein may be determined by directly
detecting the protein. For example, the protein may be fluorescent
(e.g., green fluorescent protein or a red-shifted or blue-shifted
version) or can be bound to a fluorescent label (e.g., an antibody
linked to a fluorophore). Alternatively, the amount of the
detectable protein can determined indirectly by administering a
substrate to the animal that is converted into a detectable product
by the protein and detecting the detectable product. For example,
the amount of a luciferase protein can determined by administering
D-Luciferin to the animal and detecting the D-Luciferin product
generated by the luciferase produced in the antigen-presenting
cell.
[0046] In certain embodiments, the membrane targeting region is a
myristoylation-targeting region, although the membrane-targeting
region can be selected from other types of transmembrane-targeting
regions, such as regions described hereafter. In some embodiments
the ligand is a small molecule, and sometimes the molecule is
dimeric. Examples of dimeric molecules are dimeric FK506 and
dimeric FK506 analogs. In certain embodiments the ligand is AP1903
or AP20187. In some embodiments, the chimeric protein includes one
or more ligand-binding regions, such as two or three ligand-binding
regions, for example. The ligand-binding regions often are
tandem.
[0047] The nucleic acid in certain embodiments is contained within
a viral vector, such as an adenoviral vector for example. The
antigen-presenting cell in some embodiments is contacted with an
antigen, sometimes ex vivo. In certain embodiments the
antigen-presenting cell is in a subject and an immune response is
generated against the antigen, such as a cytotoxic T-lymphocyte
(CTL) immune response. In certain embodiments, an immune response
is generated against a tumor antigen (e.g., PSMA). In some
embodiments, the nucleic acid is prepared ex vivo and administered
to the subject by intradermal administration or by subcutaneous
administration, for example. Sometimes the antigen-presenting cell
is transduced or transfected with the nucleic acid ex vivo or in
vivo. In some embodiments, the nucleic acid comprises a promoter
sequence operably linked to the polynucleotide sequence.
Alternatively, the nucleic acid comprises an ex vivo-transcribed
RNA, containing the protein-coding region of the chimeric
protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1. Schematic diagram of iCD40 and expression in human
DCs. A. The human CD40 cytoplasmic domain can be subcloned
downstream of a myristoylation-targeting domain (M) and two tandem
domains (Fv)(Clackson T, Yang W, Rozamus L W, et al. Redesigning an
FKBP-ligand interface to generate chemical dimerizers with novel
specificity. Proc Natl Acad Sci USA. 1998; 95:10437-10442). The
expression of M-Fv-Fv-CD40 chimeric protein, referred to here as
inducible CD40 (iCD40) can be under cytomegalovirus (CMV) promoter
control. B. The expression of endogenous (eCD40) and recombinant
inducible (iCD40) forms of CD40 assessed by Western blot. Lane 1,
wild type DCs (endogenous CD40 control); lane 2, DCs stimulated
with 1 microgram/ml of LPS; lanes 3 and 4, DCs transduced with
10,000 VP/cell (MOI.about.160) of Ad5/f35-iCD40 (iCD40-DCs) with
and without AP20187 dimerizer drug respectively; lane 5, iCD40-DCs
stimulated with LPS and AP20187; lane 6, DCs stimulated with CD40L
(CD40 ligand, a protein a TNF family member) and LPS; lane 7, DCs
transduced with Ad5/f35-GFP (GFP-DCs) at MOI 160 and stimulated
with AP20187 and LPS; lane 8, GFP-DCs stimulated with AP20187; lane
9, 293 T cells transduced with Ad5/f35-iCD40 (positive control for
inducible form of CD40). The expression levels of alpha-tubulin
served as internal control.
[0049] FIG. 2. Schematic of iCD40. Administration of the
lipid-permeable dimerizing drug, AP20187/AP1903.sup.1, leads to
oligomerization of the cytoplasmic domain of CD40, modified to
contain AP20187-binding domains and a myristoylation-targeting
sequence.
[0050] FIG. 3 is a schematic of CID-inducible TLRs.
[0051] FIG. 4 is a schematic of CID-inducible composite Toll-like
receptors (icTLRs).
[0052] FIG. 5 is a schematic of CID-inducible composite TLR
(icTLRs)/CD40.
[0053] FIG. 6: The principal relationships between the Toll-like
receptors (TLRs), their adapters, protein kinases that are linked
to them, and downstream signaling effects. Nature 430, 257-263 (8
Jul. 2004).
[0054] FIG. 7. iNod2 and iCD40 in 293 cells. 293 cells were
cotransfected transiently at the rate of 1 million cells/well (of a
6-well plate) with 3 microgram expression plasmids for chimeric
iNod-2 and 1 microgram NF-kappaB-dependent SEAP reporter plasmid
(indicated as R in Figure). iCD40 was used as the positive
control.
[0055] FIG. 8. iRIG-1 and iMyD88 in RAW264.7 cells. RAW 264.7 cells
were cotransfected transiently with 3 micrograms expression
plasmids for iRIG-1 and 1 microgram IFNgamma-dependent SEAP
reporter plasmid; and 3 micrograms iMyD88 with 1 microgram
NF-kappaB-dependent SEAP reporter plasmid.
[0056] FIG. 9. Schematic of Pattern recognition receptors
[0057] FIG. 10A. Schematic of an example iPRR plasmid.
[0058] FIG. 10B. Schematic of an example iPRR plasmid.
[0059] FIG. 10C. Schematic of an example iPRR plasmid.
[0060] FIG. 11 is a graph of induction of NF-kappa B SEAP reporter
in iRIG, iNOD2, and iCD40-transfected 293 cells.
[0061] FIG. 12 is a graph of induction of NF-kappa B SEAP reporter
in iRIG-1 and iCD40 transfected 293 cells.
[0062] FIG. 13 is a graph of induction of NF-kappa B SEAP reporter
in iRIG, iCD40), and iRIG+CD40 transfected 293 cells.
[0063] FIG. 14 is a graph of induction of NF-kappa B SEAP reporter
in iRIG-1 and iCD40 transfected Jurkat Tag cells.
[0064] FIGS. 15A and 15B provide plasmid maps for
pSH1-Sn-RIGI-Fv'-Fvls-E and pSH1-Sn-Fv'-Fvls-RIGI-E, respectively.
The term "Sn" represents "S" with a NcoI site, added for cloning
purposes. The term "S" represents the term non-targeted.
[0065] FIG. 16 is a schematic of inducible CD40 and MyD88 receptors
and induction of NF-kappa B activity.
[0066] FIG. 17 is a schematic of inducible chimeric CD40/MyD88
receptors and induction of NF-kappaB activity.
[0067] FIG. 18 is a graph of NF-kappa B activation in 293 cells by
inducible MyD88 and chimeric MyD88-CD40 receptors. CD40T indicates
"turbo" CD40, wherein the receptor includes 3 copies of the
FKBP12.sub.v36 domain (Fv').
[0068] FIG. 19 is a graph of NF-kappa B activity by inducible
truncated MyD88 (MyD88L) and chimeric inducible truncated
MyD88/CD40 after 3 hours of incubation with substrate.
[0069] FIG. 20 is a graph of NF-kappa B activity by inducible
truncated MyD88 (MyD88L) and chimeric inducible truncated
MyD88/CD40 after 22 hours of incubation with substrate. Some assay
saturation is present in this assay.
[0070] FIG. 21 is a Western blot of HA protein, following
adenovirus-MyD88L transduction of 293T cells.
[0071] FIG. 22 is a Western blot of HA protein, following
adenovirus-MyD88L-CD40 transduction of 293T cells.
[0072] FIG. 23 is a graph of an ELISA assay after adenovirus
infection of bone marrow derived DCs with the indicated inducible
CD40 and MyD88 constructs.
[0073] FIG. 24 is a graph of the results of an ELISA assay similar
to that in FIG. 23.
[0074] FIG. 25 is a graph of the results of an ELISA assay similar
to that in FIGS. 23 and 24, after infection with a higher amount of
adenovirus.
[0075] FIG. 26 is a graph of results of a NF-kappaB SEAP reporter
assay in iRIG-1 and iCD40 transfected cells.
[0076] FIG. 27 is a graph of results of an IFN-beta-SEAP reporter
assay in iRIG-1 and iTRIF transfected cells.
[0077] FIG. 28 is a graph comparing iTRIF activation of NF-kappa B
and IFN-beta reporters in transfected cells.
[0078] FIG. 29 is a graph of iNOD2 activation of a NF-kappaB
reporter in transfected cells.
[0079] FIG. 30 is a construct map of pShuttleX-iMyD88.
[0080] FIG. 31 is a construct map of pShuttleX-CD4-TLR4L3-E.
[0081] FIG. 32 is a construct map of pShuttleX-iMyD88E-CD40.
[0082] FIG. 33 is a bar graph depicting the results of a
dose-dependent induction of IL-12p70 expression in human
monocyte-derived dendritic cells (moDCs) transduced with different
multiplicity of infections of adenovirus expressing an inducible
MyD88.CD40 composite construct.
[0083] FIG. 34 is a bar graph depicting of the results of a
drug-dependent induction of IL-12p70 expression in human
monocyte-derived dendritic cells (moDCs) transduced with
adenoviruses expressing different inducible constructs.
[0084] FIG. 35 is a bar graph depicting the IL-12p70 levels in
transduced dendritic cells prior to vaccination.
[0085] FIG. 36(a) is a graph of EG.7-OVA tumor growth inhibition in
mice vaccinated with transduced dendritic cells; FIG. 36(b)
presents photos of representative vaccinated mice; FIG. 36(c) is
the graph of 36(a), including error bars.
[0086] FIG. 37(a) is a scatter plot, and 37(b) is a bar graph,
showing the enhanced frequency of Ag-specific CD8+ T cells induced
by transduced dendritic cells.
[0087] FIG. 38 is a bar graph showing the enhanced frequency of
Ag-Specific IFN gamma+CD8+ T cells and CD4+ T.sub.H1 cells induced
by transduced dendritic cells.
[0088] FIG. 39 presents a schematic and the results of an in vivo
cytotoxic lymphocyte assay.
[0089] FIG. 40 is a bar graph summarizing the data from an enhanced
in vivo CTL activity induced by dendritic cells.
[0090] FIG. 41 presents representative results of a CTL assay in
mice induced by transduced dendritic cells.
[0091] FIG. 42 presents the results of intracellular staining for
IL-4 producing T.sub.H2 cells in mice inoculated by transduced
dendritic cells.
[0092] FIG. 43 presents the results of a tumor growth inhibition
assay in mice treated with Ad5-iCD40.MyD88 transduced cells.
[0093] FIG. 44 presents a tumor specific T cell assay in mice
treated with Ad5-iCD40.MyD88 transduced cells.
[0094] FIG. 45 presents the results of a natural killer cell assay
using splenocytes from the treated mice as effectors.
[0095] FIG. 46 presents the results of a cytotoxic lymphocyte assay
using splenocytes from the treated mice as effectors.
[0096] FIG. 47 presents the results of an IFN-gamma ELISPot assay
using T cells co-cultured with dendritic cells transduced with the
indicated vector.
[0097] FIG. 48 presents the results of a CCR7 upregulation assay
using dendritic cells transformed with the indicated vector, with
or without LPS as an adjuvant.
[0098] FIG. 49 presents the results of the CCR7 upregulation assay
presented in FIG. 48, with the data from multiple animala included
in one graph.
DETAILED DESCRIPTION
[0099] As used herein, the use of the word "a" or "an" when used in
conjunction with the term "comprising" in the claims and/or the
specification may mean "one," but it is also consistent with the
meaning of "one or more," "at least one," and "one or more than
one." Still further, the terms "having", "including", "containing"
and "comprising" are interchangeable and one of skill in the art is
cognizant that these terms are open ended terms.
[0100] The term "allogeneic" as used herein, refers to HLA or MHC
loci that are antigenically distinct. Thus, cells or tissue
transferred from the same species can be antigenically distinct.
Syngeneic mice can differ at one or more loci (congenics) and
allogeneic mice can have the same background.
[0101] The term "antigen" as used herein is defined as a molecule
that provokes an immune response. This immune response may involve
either antibody production, or the activation of specific
immunologically-competent cells, or both. An antigen can be derived
from organisms, subunits of proteins/antigens, killed or
inactivated whole cells or lysates. Exemplary organisms include but
are not limited to, Helicobacters, Campylobacters, Clostridia,
Corynebacterium diphtheriae, Bordetella pertussis, influenza virus,
parainfluenza viruses, respiratory syncytial virus, Borrelia
burgdorfei, Plasmodium, herpes simplex viruses, human
immunodeficiency virus, papillomavirus, Vibrio cholera, E. coli,
measles virus, rotavirus, shigella, Salmonella typhi, Neisseria
gonorrhea. Therefore, a skilled artisan realizes that any
macromolecule, including virtually all proteins or peptides, can
serve as antigens. Furthermore, antigens can be derived from
recombinant or genomic DNA. A skilled artisan realizes that any DNA
that contains nucleotide sequences or partial nucleotide sequences
of a pathogenic genome or a gene or a fragment of a gene for a
protein that elicits an immune response results in synthesis of an
antigen. Furthermore, one skilled in the art realizes that the
present invention is not limited to the use of the entire nucleic
acid sequence of a gene or genome. It is readily inherent that the
present invention includes, but is not limited to, the use of
partial nucleic acid sequences of more than one gene or genome and
that these nucleic acid sequences are arranged in various
combinations to elicit the desired immune response.
[0102] The term "antigen-presenting cell" is any of a variety of
cells capable of displaying, acquiring, or presenting at least one
antigen or antigenic fragment on (or at) its cell surface. In
general, the term "antigen-presenting cell" can be any cell that
accomplishes the goal of the invention by aiding the enhancement of
an immune response (i.e., from the T-cell or B-cell arms of the
immune system) against an antigen or antigenic composition. Such
cells can be defined by those of skill in the art, using methods
disclosed herein and in the art. As is understood by one of
ordinary skill in the art (see for example Ku by, 2000, Immunology,
4.sup.th edition, W.H. Freeman and company, incorporated herein by
reference), and used herein in certain embodiments, a cell that
displays or presents an antigen normally or preferentially with a
class II major histocompatibility molecule or complex to an immune
cell is an "antigen-presenting cell." In certain aspects, a cell
(e.g., an APC cell) may be fused with another cell, such as a
recombinant cell or a tumor cell that expresses the desired
antigen. Methods for preparing a fusion of two or more cells is
well known in the art, such as for example, the methods disclosed
in Goding, J. W., Monoclonal Antibodies: Principles and Practice,
pp. 65-66, 71-74 (Academic Press, 1986); Campbell, in: Monoclonal
Antibody Technology, Laboratory Techniques in Biochemistry and
Molecular Biology, Vol. 13, Burden & Von Knippenberg,
Amsterdam, Elseview, pp. 75-83, 1984; Kohler & Milstein,
Nature, 256:495-497, 1975; Kohler & Milstein, Eur. J. Immunol.,
6:511-519, 1976, Gefter et al., Somatic Cell Genet., 3:231-236,
1977, each incorporated herein by reference. In some cases, the
immune cell to which an antigen-presenting cell displays or
presents an antigen to is a CD4+ TH cell. Additional molecules
expressed on the APC or other immune cells may aid or improve the
enhancement of an immune response. Secreted or soluble molecules,
such as for example, cytokines and adjuvants, may also aid or
enhance the immune response against an antigen. Such molecules are
well known to one of skill in the art, and various examples are
described herein.
[0103] The term "cancer" as used herein is defined as a
hyperproliferation of cells whose unique trait--loss of normal
controls--results in unregulated growth, lack of differentiation,
local tissue invasion, and metastasis. Examples include but are not
limited to, melanoma, non-small cell lung, small-cell lung, lung,
hepatocarcinoma, leukemia, retinoblastoma, astrocytoma,
glioblastoma, gum, tongue, neuroblastoma, head, neck, breast,
pancreatic, prostate, renal, bone, testicular, ovarian,
mesothelioma, cervical, gastrointestinal, lymphoma, brain, colon,
sarcoma or bladder.
[0104] The terms "cell," "cell line," and "cell culture" as used
herein may be used interchangeably. All of these terms also include
their progeny, which are any and all subsequent generations. It is
understood that all progeny may not be identical due to deliberate
or inadvertent mutations.
[0105] As used herein, the term "iCD40 molecule" is defined as an
inducible CD40. This iCD40 can bypass mechanisms that extinguish
endogenous CD40 signaling. The term "iCD40" embraces "iCD40 nucleic
acids," "iCD40 polypeptides" and/or iCD40 expression vectors. Yet
further, it is understood the activity of iCD40 as used herein is
driven by CID.
[0106] As used herein, the term "cDNA" is intended to refer to DNA
prepared using messenger RNA (mRNA) as template. The advantage of
using a cDNA, as opposed to genomic DNA or DNA polymerized from a
genomic, non- or partially-processed RNA template, is that the cDNA
primarily contains coding sequences of the corresponding protein.
There are times when the full or partial genomic sequence is
preferred, such as where the non-coding regions are required for
optimal expression or where non-coding regions such as introns are
to be targeted in an antisense strategy.
[0107] The term "dendritic cell" (DC) is an antigen-presenting cell
existing in vivo, in vitro, ex vivo, or in a host or subject, or
which can be derived from a hematopoietic stem cell or a monocyte.
Dendritic cells and their precursors can be isolated from a variety
of lymphoid organs, e.g., spleen, lymph nodes, as well as from bone
marrow and peripheral blood. The DC has a characteristic morphology
with thin sheets (lamellipodia) extending in multiple directions
away from the dendritic cell body. Typically, dendritic cells
express high levels of MHC and costimulatory (e.g., B7-1 and B7-2)
molecules. Dendritic cells can induce antigen specific
differentiation of T cells in vitro, and are able to initiate
primary T cell responses in vitro and in vivo.
[0108] As used herein, the term "expression construct" or
"transgene" is defined as any type of genetic construct containing
a nucleic acid coding for gene products in which part or all of the
nucleic acid encoding sequence is capable of being transcribed can
be inserted into the vector. The transcript is translated into a
protein, but it need not be. In certain embodiments, expression
includes both transcription of a gene and translation of mRNA into
a gene product. In other embodiments, expression only includes
transcription of the nucleic acid encoding genes of interest. The
term "therapeutic construct" may also be used to refer to the
expression construct or transgene. One skilled in the art realizes
that the expression construct or transgene may be used, for
example, as a therapy to treat hyperproliferative diseases or
disorders, such as cancer, thus the expression construct or
transgene is a therapeutic construct or a prophylactic
construct.
[0109] As used herein, the term "expression vector" refers to a
vector containing a nucleic acid sequence coding for at least part
of a gene product capable of being transcribed. In some cases, RNA
molecules are then translated into a protein, polypeptide, or
peptide. In other cases, these sequences are not translated, for
example, in the production of antisense molecules or ribozymes.
Expression vectors can contain a variety of control sequences,
which refer to nucleic acid sequences necessary for the
transcription and possibly translation of an operatively linked
coding sequence in a particular host organism. In addition to
control sequences that govern transcription and translation,
vectors and expression vectors may contain nucleic acid sequences
that serve other functions as well and are described infra.
[0110] As used herein, the term "ex vivo" refers to "outside" the
body. One of skill in the art is aware that ex vivo and in vitro
can be used interchangeably.
[0111] As used herein, the term "functionally equivalent," as used
herein, as, for example it refers to a CD40 nucleic acid fragment,
variant, or analog, refers to a nucleic acid that codes for a CD40
polypeptide, or a CD40 polypeptide, that stimulates an immune
response to destroy tumors or hyperproliferative disease.
"Functionally equivalent" refers, for example, to a CD40
polypeptide that is lacking the extracellular domain, but is
capable of amplifying the T cell-mediated tumor killing response by
upregulating dendritic cell expression of antigen presentation
molecules.
[0112] The term "hyperproliferative disease" is defined as a
disease that results from a hyperproliferation of cells. Exemplary
hyperproliferative diseases include, but are not limited to cancer
or autoimmune diseases. Other hyperproliferative diseases may
include vascular occlusion, restenosis, atherosclerosis, or
inflammatory bowel disease.
[0113] As used herein, the term "gene" is defined as a functional
protein, polypeptide, or peptide-encoding unit. As will be
understood by those in the art, this functional term includes
genomic sequences, cDNA sequences, and smaller engineered gene
segments that express, or are adapted to express, proteins,
polypeptides, domains, peptides, fusion proteins, and mutants.
[0114] The term "immunogenic composition" or "immunogen" refers to
a substance that is capable of provoking an immune response.
Examples of immunogens include, e.g., antigens, autoantigens that
play a role in induction of autoimmune diseases, and
tumor-associated antigens expressed on cancer cells.
[0115] The term "immunocompromised" as used herein is defined as a
subject that has reduced or weakened immune system. The
immunocompromised condition may be due to a defect or dysfunction
of the immune system or to other factors that heighten
susceptibility to infection and/or disease. Although such a
categorization allows a conceptual basis for evaluation,
immunocompromised individuals often do not fit completely into one
group or the other. More than one defect in the body's defense
mechanisms may be affected. For example, individuals with a
specific T-lymphocyte defect caused by HIV may also have
neutropenia caused by drugs used for antiviral therapy or be
immunocompromised because of a breach of the integrity of the skin
and mucous membranes. An immunocompromised state can result from
indwelling central lines or other types of impairment due to
intravenous drug abuse; or be caused by secondary malignancy,
malnutrition, or having been infected with other infectious agents
such as tuberculosis or sexually transmitted diseases, e.g.,
syphilis or hepatitis.
[0116] As used herein, the term "pharmaceutically or
pharmacologically acceptable" refers to molecular entities and
compositions that do not produce adverse, allergic, or other
untoward reactions when administered to an animal or a human.
[0117] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
vectors or cells presented herein, its use in therapeutic
compositions is contemplated. Supplementary active ingredients also
can be incorporated into the compositions.
[0118] As used herein, the term "polynucleotide" is defined as a
chain of nucleotides. Furthermore, nucleic acids are polymers of
nucleotides. Thus, nucleic acids and polynucleotides as used herein
are interchangeable. One skilled in the art has the general
knowledge that nucleic acids are polynucleotides, which can be
hydrolyzed into the monomeric "nucleotides." The monomeric
nucleotides can be hydrolyzed into nucleosides. As used herein
polynucleotides include, but are not limited to, all nucleic acid
sequences which are obtained by any means available in the art,
including, without limitation, recombinant means, i.e., the cloning
of nucleic acid sequences from a recombinant library or a cell
genome, using ordinary cloning technology and PCR.TM., and the
like, and by synthetic means. Furthermore, one skilled in the art
is cognizant that polynucleotides include mutations of the
polynucleotides, include but are not limited to, mutation of the
nucleotides, or nucleosides by methods well known in the art.
[0119] As used herein, the term "polypeptide" is defined as a chain
of amino acid residues, usually having a defined sequence. As used
herein the term polypeptide is interchangeable with the terms
"peptides" and "proteins".
[0120] As used herein, the term "promoter" is defined as a DNA
sequence recognized by the synthetic machinery of the cell, or
introduced synthetic machinery, required to initiate the specific
transcription of a gene.
[0121] As used herein, the term "regulate an immune response" or
"modulate an immune response" refers to the ability to modify the
immune response. For example, the composition is capable of
enhancing and/or activating the immune response. Still further, the
composition is also capable of inhibiting the immune response. The
form of regulation is determined by the ligand that is used with
the composition. For example, a dimeric analog of the chemical
results in dimerization of the co-stimulatory polypeptide leading
to activation of the DCs, however, a monomeric analog of the
chemical does not result in dimerization of the co-stimulatory
polypeptide, which would not activate the DCs.
[0122] The term "transfection" and "transduction" are
interchangeable and refer to the process by which an exogenous DNA
sequence is introduced into a eukaryotic host cell. Transfection
(or transduction) can be achieved by any one of a number of means
including electroporation, microinjection, gene gun delivery,
retroviral infection, lipofection, superfection and the like.
[0123] As used herein, the term "syngeneic" refers to cells,
tissues or animals that have genotypes that are identical or
closely related enough to allow tissue transplant, or are
immunologically compatible. For example, identical twins or animals
of the same inbred strain. Syngeneic and isogeneic can be used
interchangeable.
[0124] The term "subject" as used herein includes, but is not
limited to, an organism or animal; a mammal, including, e.g., a
human, non-human primate (e.g., monkey), mouse, pig, cow, goat,
rabbit, rat, guinea pig, hamster, horse, monkey, sheep, or other
non-human mammal; a non-mammal, including, e.g., a non-mammalian
vertebrate, such as a bird (e.g., a chicken or duck) or a fish, and
a non-mammalian invertebrate.
[0125] As used herein, the term "under transcriptional control" or
"operatively linked" is defined as the promoter is in the correct
location and orientation in relation to the nucleic acid to control
RNA polymerase initiation and expression of the gene.
[0126] As used herein, the terms "treatment", "treat", "treated",
or "treating" refer to prophylaxis and/or therapy. When used with
respect to an infectious disease, for example, the term refers to a
prophylactic treatment which increases the resistance of a subject
to infection with a pathogen or, in other words, decreases the
likelihood that the subject will become infected with the pathogen
or will show signs of illness attributable to the infection, as
well as a treatment after the subject has become infected in order
to fight the infection, e.g., reduce or eliminate the infection or
prevent it from becoming worse.
[0127] As used herein, the term "vaccine" refers to a formulation
which contains a composition presented herein which is in a form
that is capable of being administered to an animal. Typically, the
vaccine comprises a conventional saline or buffered aqueous
solution medium in which the composition is suspended or dissolved.
In this form, the composition can be used conveniently to prevent,
ameliorate, or otherwise treat a condition. Upon introduction into
a subject, the vaccine is able to provoke an immune response
including, but not limited to, the production of antibodies,
cytokines and/or other cellular responses.
Dendritic Cells
[0128] The innate immune system uses a set of germline-encoded
receptors for the recognition of conserved molecular patterns
present in microorganisms. These molecular patterns occur in
certain constituents of microorganisms including:
lipopolysaccharides, peptidoglycans, lipoteichoic acids,
phosphatidyl cholines, bacteria-specific proteins, including
lipoproteins, bacterial DNAs, viral single and double-stranded
RNAs, unmethylated CpG-DNAs, mannans and a variety of other
bacterial and fungal cell wall components. Such molecular patterns
can also occur in other molecules such as plant alkaloids. These
targets of innate immune recognition are called Pathogen Associated
Molecular Patterns (PAMPs) since they are produced by
microorganisms and not by the infected host organism (Janeway et
al. (1989) Cold Spring Harb. Symp. Quant. Biol., 54: 1-13;
Medzhitov et al., Nature, 388:394-397, 1997).
[0129] The receptors of the innate immune system that recognize
PAMPs are called Pattern Recognition Receptors (PRRs) (Janeway et
al., 1989; Medzhitov et al., 1997). These receptors vary in
structure and belong to several different protein families. Some of
these receptors recognize PAMPs directly (e.g., CD14, DEC205,
collectins), while others (e.g., complement receptors) recognize
the products generated by PAMP recognition. Members of these
receptor families can, generally, be divided into three types: 1)
humoral receptors circulating in the plasma; 2) endocytic receptors
expressed on immune-cell surfaces, and 3) signaling receptors that
can be expressed either on the cell surface or intracellularly
(Medzhitov et al., 1997; Fearon et al. (1996) Science 272:
50-3).
[0130] Cellular PRRs are expressed on effector cells of the innate
immune system, including cells that function as professional
antigen-presenting cells (APC) in adaptive immunity. Such effector
cells include, but are not limited to, macrophages, dendritic
cells, B lymphocytes and surface epithelia. This expression profile
allows PRRs to directly induce innate effector mechanisms, and also
to alert the host organism to the presence of infectious agents by
inducing the expression of a set of endogenous signals, such as
inflammatory cytokines and chemokines, as discussed below. This
latter function allows efficient mobilization of effector forces to
combat the invaders.
[0131] The primary function of dendritic cells (DCs) is to acquire
antigen in the peripheral tissues, travel to secondary lymphoid
tissue, and present antigen to effector T cells of the immune
system (Banchereau, J., et al., Annu Rev Immunol, 2000, 18: p.
767-811; Banchereau, J., & Steinman, R. M. Dendritic cells and
the control of immunity. Nature 392, 245-252 (1998)). As DCs carry
out their crucial role in the immune response, they undergo
maturational changes allowing them to perform the appropriate
function for each environment (Termeer, C. C., et al., J Immunol,
2000, Aug. 15. 165: p. 1863-70). During DC maturation, antigen
uptake potential is lost, the surface density of major
histocompatibility complex (MHC) class I and class II molecules
increases by 10-100 fold, and CD40, costimulatory and adhesion
molecule expression also greatly increases (Lanzavecchia, A. and F.
Sallusto, Science, 2000. 290: p. 92-96). In addition, other genetic
alterations permit the DCs to home to the T cell-rich paracortex of
draining lymph nodes and to express T-cell chemokines that attract
naive and memory T cells and prime antigen-specific naive THO cells
(Adema, G. J., et al., Nature, 1997, Jun. 12. 387: p. 713-7).
During this stage, mature DCs present antigen via their MHC II
molecules to CD4+ T helper cells, inducing the upregulation of T
cell CD40 ligand (CD40L) that, in turn, engages the DC CD40
receptor. This DC:T cell interaction induces rapid expression of
additional DC molecules that are crucial for the initiation of a
potent CD8+ cytotoxic T lymphocyte (CTL) response, including
further upregulation of MHC I and II molecules, adhesion molecules,
costimulatory molecules (e.g., B7.1,B7.2), cytokines (e.g., IL-12)
and anti-apoptotic proteins (e.g., Bcl-2) (Anderson, D. M., et al.,
Nature, 1997, Nov. 13. 390: p. 175-9; Ohshima, Y., et al., J
Immunol, 1997, Oct. 15. 159: p. 3838-48; Sallusto, F., et al., Eur
J Immunol, 1998, Sep. 28: p. 2760-9; Caux, C. Adv Exp Med. Biol.
1997, 417:21-5;). CD8+ T cells exit lymph nodes, reenter
circulation and home to the original site of inflammation to
destroy pathogens or malignant cells.
[0132] One key parameter influencing the function of DCs is the
CD40 receptor, serving as the "on switch" for DCs (Bennett, S. R.,
et al., Nature, 1998, Jun. 4. 393: p. 478-80; Clarke, S. R., J
Leukoc Biol, 2000, May. 67: p. 607-14; Fernandez, N. C., et al.,
Nat Med, 1999, Apr. 5: p. 405-11; Ridge, J. P., D. R. F, and P.
Nature, 1998, Jun. 4. 393: p. 474-8; Schoenberger, S. P., et al.,
Nature, 1998, Jun. 4. 393: p. 480-3). CD40 is a 48-kDa
transmembrane member of the TNF receptor superfamily (McWhirter, S.
M., et al., Proc Natl Acad Sci USA, 1999, Jul. 20. 96: p. 8408-13).
CD40-CD40L interaction induces CD40 trimerization, necessary for
initiating signaling cascades involving TNF receptor associated
factors (TRAFs) (Ni, C., et al., PNAS, 2000, 97(19): 10395-10399;
Pullen, S. S., et al., J Biol Chem, 1999, May 14.274: p. 14246-54).
CD40 uses these signaling molecules to activate several
transcription factors in DCs, including NF-kappa B, AP-1, STAT3,
and p38MAPK (McWhirter, S. M., et al., 1999).
[0133] A novel DC activation system is provided based on recruiting
signaling molecules or co-stimulatory polypeptides to the plasmid
membrane of the DCs resulting in prolonged/increased activation
and/or survival in the DCs. Co-stimulatory polypeptides include any
molecule or polypeptide that activates the NF-kappaB pathway, Akt
pathway, and/or p38 pathway. The DC activation system is based upon
utilizing a recombinant signaling molecule fused to a
ligand-binding domains (i.e., a small molecule binding domain) in
which the co-stimulatory polypeptide is activated and/or regulated
with a ligand resulting in oligomerization (i.e., a
lipid-permeable, organic, dimerizing drug). Other systems that may
be used to crosslink or for oligomerization of co-stimulatory
polypeptides include antibodies, natural ligands, and/or artificial
cross-reacting or synthetic ligands. Yet further, other
dimerization systems contemplated include the coumermycin/DNA
gyrase B system.
[0134] Co-stimulatory polypeptides that can be used include those
that activate NF-kappaB and other variable signaling cascades for
example the p38 pathway and/or Akt pathway. Such co-stimulatory
polypeptides include, but are not limited to Pattern Recognition
Receptors, C-reactive protein receptors (i.e., Nod1, Nod2, PtX3-R),
TNF receptors (i.e., CD40, RANK/TRANCE-R, OX40, 4-1BB), and HSP
receptors (Lox-1 and CD-91). Pattern Recognition Receptors include,
but are not limited to endocytic pattern-recognition receptors
(i.e., mannose receptors, scavenger receptors (i.e., Mac-1, LRP,
peptidoglycan, techoic acids, toxins, CD11c/CR4)); external signal
pattern-recognition receptors (Toll-like receptors (TLR1, TLR2,
TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10), peptidoglycan
recognition protein, (PGRPs bind bacterial peptidoglycan, and
CD14); internal signal pattern-recognition receptors (i.e.,
NOD-receptors 1 & 2), RIG1, and PRRs shown in FIG. 8. Those of
ordinary skill in the art are also aware of other Pattern
Recognition Receptors suitable for the present methods and
composition, including those discussed in, for example, Werts C.,
et al., Cell Death and Differentiation (2006) 13:798-815; Meylan,
E., et al., Nature (2006) 442:39-44; and Strober, W., et al.,
Nature Reviews (2006) 6:9-20.
Engineering Expression Constructs
[0135] Also provided are expression construct encoding a
co-stimulatory polypeptide and a ligand-binding domain, all
operatively linked. More particularly, more than one ligand-binding
domain is used in the expression construct. Yet further, the
expression construct contains a membrane-targeting sequence. One
with skill in the art realizes that appropriate expression
constructs may include a co-stimulatory polypeptide element on
either side of the above FKBP ligand-binding elements. The
expression construct may be inserted into a vector, for example a
viral vector or plasmid.
[0136] A. Co-Stimulatory Polypeptides
[0137] Co-stimulatory polypeptide molecules are capable of
amplifying the T-cell-mediated response by upregulating dendritic
cell expression of antigen presentation molecules. Co-stimulatory
proteins that are contemplated include, for example, but are not
limited, to the members of tumor necrosis factor (TNF) family
(i.e., CD40, RANK/TRANCE-R, OX40, 4-1B), Toll-like receptors,
C-reactive protein receptors, Pattern Recognition Receptors, and
HSP receptors. Typically, the cytoplasmic domains from these
co-stimulatory polypeptides are used in the expression vector. The
cytoplasmic domain from one of the various co-stimulatory
polypeptides, including mutants thereof, where the recognition
sequence involved in initiating transcription associated with the
cytoplasmic domain is known or a gene responsive to such sequence
is known.
[0138] In specific embodiments, the co-stimulatory polypeptide
molecule is CD40. The CD40 molecule comprises a nucleic acid
molecule which: (1) hybridizes under stringent conditions to a
nucleic acid having the sequence of a known CD40 gene and (2) codes
for an CD40 polypeptide. The CD40 polypeptide may, in certain
examples, lack the extracellular domain. Exemplary polynucleotide
sequences that encode CD40 polypeptides include, but are not
limited to SEQ.ID.NO: 1 and CD40 isoforms from other species. It is
contemplated that other normal or mutant variants of CD40 can be
used in the present methods and compositions. Thus, a CD40 region
can have an amino acid sequence that differs from the native
sequence by one or more amino acid substitutions, deletions and/or
insertions. For example, one or more TNF receptor associated factor
(TRAF) binding regions may be eliminated or effectively eliminated
(e.g., a CD40 amino acid sequence is deleted or altered such that a
TRAF protein does not bind or binds with lower affinity than it
binds to the native CD40 sequence). In particular embodiments, a
TRAF 3 binding region is deleted or altered such that it is
eliminated or effectively eliminated (e.g., amino acids 250-254 may
be altered or deleted; Hauer et al., PNAS 102(8): 2874-2879
(2005)).
[0139] In certain embodiments, the present methods involve the
manipulation of genetic material to produce expression constructs
that encode an inducible form of CD40 (iCD40). Such methods involve
the generation of expression constructs containing, for example, a
heterologous nucleic acid sequence encoding CD40 cytoplasmic domain
and a means for its expression. The vector can be replicated in an
appropriate helper cell, viral particles may be produced therefrom,
and cells infected with the recombinant virus particles.
[0140] Thus, the CD40 molecule presented herein may, for example,
lack the extracellular domain. In specific embodiments, the
extracellular domain is truncated or removed. It is also
contemplated that the extracellular domain can be mutated using
standard mutagenesis, insertions, deletions, or substitutions to
produce an CD40 molecule that does not have a functional
extracellular domain. A CD40 nucleic acid may have the nucleic acid
sequence of SEQ.ID.NO: 1. The CD40 nucleic acids also include
homologs and alleles of a nucleic acid having the sequence of
SEQ.ID.NO: 1, as well as, functionally equivalent fragments,
variants, and analogs of the foregoing nucleic acids. Methods of
constructing an inducible CD40 vector are described in, for
example, U.S. Pat. No. 7,404,950, issued Jul. 29, 2008.
[0141] In the context of gene therapy, the gene will be a
heterologous polynucleotide sequence derived from a source other
than the viral genome, which provides the backbone of the vector.
The gene is derived from a prokaryotic or eukaryotic source such as
a bacterium, a virus, yeast, a parasite, a plant, or even an
animal. The heterologous DNA also is derived from more than one
source, i.e., a multigene construct or a fusion protein. The
heterologous DNA also may include a regulatory sequence, which is
derived from one source and the gene from a different source.
[0142] B. Ligand-Binding Regions
[0143] The ligand-binding ("dimerization") domain of the expression
construct can be any convenient domain that will allow for
induction using a natural or unnatural ligand, for example, an
unnatural synthetic ligand. The ligand-binding domain can be
internal or external to the cellular membrane, depending upon the
nature of the construct and the choice of ligand. A wide variety of
ligand-binding proteins, including receptors, are known, including
ligand-binding proteins associated with the cytoplasmic regions
indicated above. As used herein the term "ligand-binding domain can
be interchangeable with the term "receptor". Of particular interest
are ligand-binding proteins for which ligands (for example, small
organic ligands) are known or may be readily produced. These
ligand-binding domains or receptors include the FKBPs and
cyclophilin receptors, the steroid receptors, the tetracycline
receptor, the other receptors indicated above, and the like, as
well as "unnatural" receptors, which can be obtained from
antibodies, particularly the heavy or light chain subunit, mutated
sequences thereof, random amino acid sequences obtained by
stochastic procedures, combinatorial syntheses, and the like.
Examples include, for example, those described in Kopytek, S. J.,
et al., Chemistry & Biology 7:313-321 (2000) and in Gestwicki,
J. E., et al., Combinatorial Chem. & High Throughput Screening
10:667-675 (2007); Clackson T (2006) Chem Biol Drug Des 67:440-2;
Clackson, T. "Controlling Protein-Protein Interactions Using
Chemical Inducers and Disrupters of Dimerization," in Chemical
Biology: From Small Molecules to Systems Biology and Drug Design
(Schreiber, s., et al., eds., Wiley, 2007)).
[0144] For the most part, the ligand-binding domains or receptor
domains will be at least about 50 amino acids, and fewer than about
350 amino acids, usually fewer than 200 amino acids, either as the
natural domain or truncated active portion thereof. The binding
domain may, for example, be small (<25 kDa, to allow efficient
transfection in viral vectors), monomeric (this rules out the
avidin-biotin system), nonimmunogenic, and should have
synthetically accessible, cell permeable, nontoxic ligands that can
be configured for dimerization.
[0145] The receptor domain can be intracellular or extracellular
depending upon the design of the expression construct and the
availability of an appropriate ligand. For hydrophobic ligands, the
binding domain can be on either side of the membrane, but for
hydrophilic ligands, particularly protein ligands, the binding
domain will usually be external to the cell membrane, unless there
is a transport system for internalizing the ligand in a form in
which it is available for binding. For an intracellular receptor,
the construct can encode a signal peptide and transmembrane domain
5' or 3' of the receptor domain sequence or may have a lipid
attachment signal sequence 5' of the receptor domain sequence.
Where the receptor domain is between the signal peptide and the
transmembrane domain, the receptor domain will be
extracellular.
[0146] The portion of the expression construct encoding the
receptor can be subjected to mutagenesis for a variety of reasons.
The mutagenized protein can provide for higher binding affinity,
allow for discrimination by the ligand of the naturally occurring
receptor and the mutagenized receptor, provide opportunities to
design a receptor-ligand pair, or the like. The change in the
receptor can involve changes in amino acids known to be at the
binding site, random mutagenesis using combinatorial techniques,
where the codons for the amino acids associated with the binding
site or other amino acids associated with conformational changes
can be subject to mutagenesis by changing the codon(s) for the
particular amino acid, either with known changes or randomly,
expressing the resulting proteins in an appropriate prokaryotic
host and then screening the resulting proteins for binding.
[0147] Antibodies and antibody subunits, e.g., heavy or light
chain, particularly fragments, more particularly all or part of the
variable region, or fusions of heavy and light chain to create
high-affinity binding, can be used as the binding domain.
Antibodies that are contemplated include ones that are an
ectopically expressed human product, such as an extracellular
domain that would not trigger an immune response and generally not
expressed in the periphery (i.e., outside the CNS/brain area). Such
examples, include, but are not limited to low affinity nerve growth
factor receptor (LNGFR), and embryonic surface proteins (i.e.,
carcinoembryonic antigen).
[0148] Yet further, antibodies can be prepared against haptenic
molecules, which are physiologically acceptable, and the individual
antibody subunits screened for binding affinity. The cDNA encoding
the subunits can be isolated and modified by deletion of the
constant region, portions of the variable region, mutagenesis of
the variable region, or the like, to obtain a binding protein
domain that has the appropriate affinity for the ligand. In this
way, almost any physiologically acceptable haptenic compound can be
employed as the ligand or to provide an epitope for the ligand.
Instead of antibody units, natural receptors can be employed, where
the binding domain is known and there is a useful ligand for
binding.
[0149] C. Oligomerization
[0150] The transduced signal will normally result from
ligand-mediated oligomerization of the chimeric protein molecules,
i.e., as a result of oligomerization following ligand-binding,
although other binding events, for example allosteric activation,
can be employed to initiate a signal. The construct of the chimeric
protein will vary as to the order of the various domains and the
number of repeats of an individual domain.
[0151] For multimerizing the receptor, the ligand for the
ligand-binding domains/receptor domains of the chimeric surface
membrane proteins will usually be multimeric in the sense that it
will have at least two binding sites, with each of the binding
sites capable of binding to the ligand receptor domain. Desirably,
the subject ligands will be a dimer or higher order oligomer,
usually not greater than about tetrameric, of small synthetic
organic molecules, the individual molecules typically being at
least about 150 Da and less than about 5 kDa, usually less than
about 3 kDa. A variety of pairs of synthetic ligands and receptors
can be employed. For example, in embodiments involving natural
receptors, dimeric FK506 can be used with an FKBP12 receptor,
dimerized cyclosporin A can be used with the cyclophilin receptor,
dimerized estrogen with an estrogen receptor, dimerized
glucocorticoids with a glucocorticoid receptor, dimerized
tetracycline with the tetracycline receptor, dimerized vitamin D
with the vitamin D receptor, and the like. Alternatively higher
orders of the ligands, e.g., trimeric can be used. For embodiments
involving unnatural receptors, e.g., antibody subunits, modified
antibody subunits or modified receptors, and the like, any of a
large variety of compounds can be used. A significant
characteristic of these ligand units is that each binding site is
able to bind the receptor with high affinity and they are able to
be dimerized chemically. Also, those of ordinary skill in the art
are aware of methods to balance the hydrophobicity/hydrophilicity
of the ligands so that they are able to dissolve in serum at
functional levels, yet diffuse across plasma membranes for most
applications.
[0152] In certain embodiments, the present methods utilize the
technique of chemically induced dimerization (CID) to produce a
conditionally controlled protein or polypeptide. In addition to
this technique being inducible, it also is reversible, due to the
degradation of the labile dimerizing agent or administration of a
monomeric competitive inhibitor.
[0153] The CID system uses synthetic bivalent ligands to rapidly
crosslink signaling molecules that are fused to ligand-binding
domains. This system has been used to trigger the oligomerization
and activation of cell surface (Spencer, D. M., et al., Science,
1993. 262: p. 1019-1024; Spencer D. M. et al., Curr Biol 1996,
6:839-847; Blau, C. A. et al., Proc Natl Acad. Sci. USA 1997,
94:3076-3081), or cytosolic proteins (Luo, Z. et al., Nature 1996,
383:181-185; MacCorkle, R. A. et al., Proc Natl Acad Sci USA 1998,
95:3655-3660), the recruitment of transcription factors to DNA
elements to modulate transcription (Ho, S, N. et al., Nature 1996,
382:822-826; Rivera, V. M. et al., Nat. Med. 1996, 2:1028-1032) or
the recruitment of signaling molecules to the plasma membrane to
stimulate signaling (Spencer D. M. et al., Proc. Natl. Acad. Sci.
USA 1995, 92:9805-9809; Holsinger, L. J. et al., Proc. Natl. Acad.
Sci. USA 1995, 95:9810-9814).
[0154] The CID system is based upon the notion that surface
receptor aggregation effectively activates downstream signaling
cascades. In the simplest 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 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, 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.
[0155] The ligands used 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
is typically a non-protein or a chemical. Exemplary ligands
include, but are not limited to dimeric FK506 (e.g., FK1012).
[0156] Since the mechanism of CD40 activation is fundamentally
based on trimerization, this receptor is particularly amenable to
the CID system. CID regulation provides the system with 1) temporal
control, 2) reversibility by addition of a non-active monomer upon
signs of an autoimmune reaction, and 3) limited potential for
non-specific side effects. In addition, inducible in vivo DC CD40
activation would circumvent the requirement of a second "danger"
signal normally required for complete induction of CD40 signaling
and would potentially promote DC survival in situ allowing for
enhanced T cell priming. Thus, engineering DC vaccines to express
iCD40 amplifies the T cell-mediated killing response by
upregulating DC expression of antigen presentation molecules,
adhesion molecules, TH1 promoting cytokines, and pro-survival
factors.
[0157] Other dimerization systems contemplated include the
coumermycin/DNA gyrase B system. Coumermycin-induced dimerization
activates a modified Raf protein and stimulates the MAP kinase
cascade. See Farrar et al., 1996.
[0158] D. Membrane-Targeting
[0159] A membrane-targeting sequence provides for transport of the
chimeric protein to the cell surface membrane, where the same or
other sequences can encode binding of the chimeric protein to the
cell surface membrane. Molecules in association with cell membranes
contain certain regions that facilitate the membrane association,
and such regions can be incorporated into a chimeric protein
molecule to generate membrane-targeted molecules. 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 (often followed
by several positively charged residues (e.g. human c-Src:
M-G-S-N-K-S-K-P-K-D-A-S-Q-R-R-R) to improve association with
anionic lipid head groups) and others are capable of being modified
with multiple acyl moieties. For example the N-terminal sequence of
the protein tyrosine kinase Src can comprise a single myristoyl
moiety. Dual acylation regions are located within the N-terminal
regions of certain protein kinases, such as a subset of Src family
members (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., World Wide Web address
ebi.ac.uk/interpro/DisplaylproEntry?ac=1PRO01230) 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 chimeric molecules
to induce membrane localization. In certain embodiments, a native
sequence from a protein containing an acylation motif is
incorporated into a chimeric 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 incorporated within the N-terminus of a chimeric protein. 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 a chimeric protein.
[0160] 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, sphingosine, 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.
[0161] A chimeric protein herein also may include a single-pass or
multiple pass transmembrane sequence (e.g., at the N-terminus or
C-terminus of the chimeric protein). 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 often 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 often follows the transmembrane span to anchor the protein in
the membrane. 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 sometimes is incorporated in a chimeric
protein. Sequences for single pass and multiple pass transmembrane
regions are known and can be selected for incorporation into a
chimeric protein molecule by the person of ordinary skill in the
art.
[0162] Any membrane-targeting sequence can be employed that is
functional in the host and may, or may not, be associated with one
of the other domains of the chimeric protein. In some embodiments,
such sequences include, but are not limited to
myristoylation-targeting sequence, palmitoylation-targeting
sequence, prenylation sequences (i.e., farnesylation,
geranyl-geranylation, CAAX Box), protein-protein interaction motifs
or transmembrane sequences (utilizing signal peptides) from
receptors. Examples include those described in, for example, ten
Klooster J P et al, Biology of the Cell (2007) 99, 1-12, Vincent,
S., et al., Nature Biotechnology 21:936-40, 1098 (2003).
[0163] Additional protein domains exist that can increase protein
retention at various membranes. For example, an .about.120 amino
acid pleckstrin homology (PH) domain is found in over 200 human
proteins that are typically involved in intracellular signaling. PH
domains can bind various phosphatidylinositol (PI) lipids within
membranes (e.g. PI (3,4,5)--P.sub.3, PI (3,4)--P.sub.2, PI
(4,5)--P.sub.2) and thus play a key role in recruiting proteins to
different membrane or cellular compartments. Often the
phosphorylation state of PI lipids is regulated, such as by PI-3
kinase or PTEN, and thus, interaction of membranes with PH domains
is not as stable as by acyl lipids.
[0164] E. Selectable Markers
[0165] In certain embodiments, the expression constructs contain
nucleic acid constructs whose expression is identified in vitro or
in vivo by including a marker in the expression construct.
[0166] Such markers would confer an identifiable change to the cell
permitting easy identification of cells containing the expression
construct. Usually the inclusion of a drug selection marker aids in
cloning and in the selection of transformants. For example, genes
that confer resistance to neomycin, puromycin, hygromycin, DHFR,
GPT, zeocin and histidinol are useful selectable markers.
Alternatively, enzymes such as herpes simplex virus thymidine
kinase (tk) are employed. Immunologic surface markers containing
the extracellular, non-signaling domains or various proteins (e.g.
CD34, CD19, LNGFR) also can be employed, permitting a
straightforward method for magnetic or fluorescence
antibody-mediated sorting. The selectable marker employed is not
believed to be important, so long as it is capable of being
expressed simultaneously with the nucleic acid encoding a gene
product. Further examples of selectable markers are well known to
one of skill in the art and include reporters such as EGFP,
beta-gal or chloramphenicol acetyltransferase (CAT).
[0167] F. Control Regions
[0168] 1. Promoters
[0169] 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 may be 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.
[0170] In various embodiments, the human cytomegalovirus (CMV)
immediate early gene promoter, the SV40 early promoter, the Rous
sarcoma virus long terminal repeat, .beta.-actin, rat insulin
promoter and glyceraldehyde-3-phosphate dehydrogenase can be used
to obtain high-level expression of the coding sequence of interest.
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.
[0171] Selection of a promoter that is regulated in response to
specific physiologic or synthetic signals can permit inducible
expression of the gene product. For example in the case where
expression of a transgene, or transgenes when a multicistronic
vector is utilized, is toxic to the cells in which the vector is
produced in, it is desirable to prohibit or reduce expression of
one or more of the transgenes. Examples of transgenes that are
toxic to the producer cell line are pro-apoptotic and cytokine
genes. Several inducible promoter systems are available for
production of viral vectors where the transgene products are toxic
(add in more inducible promoters).
[0172] The 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.
[0173] Another inducible system that would be useful is the
Tet-Off.TM. or Tet-On.TM. system (Clontech, Palo Alto, Calif.)
originally developed by Gossen and Bujard (Gossen and Bujard, Proc.
Natl. Acad. Sci. USA, 89:5547-5551, 1992; Gossen et al., Science,
268:1766-1769, 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-Off.TM. system may be used 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 constitutively on.
[0174] In some circumstances, it is desirable to regulate
expression of a transgene in a gene therapy 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 is often used to provide
strong transcriptional activation. The CMV promoter is reviewed in
Donnelly, J. J., et al., 1997. Annu. Rev. Immunol. 15:617-48.
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 hematopoietic cells
is desired, retroviral promoters such as the LTRs from MLV or MMTV
are often 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.
[0175] Similarly tissue specific promoters are used to effect
transcription in specific tissues or cells so as to reduce
potential toxicity or undesirable effects to non-targeted tissues.
These promoters may result in reduced expression compared to a
stronger promoter such as the CMV promoter, but may also result in
more limited expression, and immunogenicity. (Bojak, A., et al.,
2002. Vaccine. 20:1975-79; Cazeaux., N., et al., 2002. Vaccine
20:3322-31). For example, tissue specific promoters such as the PSA
associated promoter or prostate-specific glandular kallikrein, or
the muscle creatine kinase gene may be used where appropriate.
[0176] In certain indications, it is desirable to activate
transcription at specific times after administration of the gene
therapy vector. This is done with such promoters as those that are
hormone or cytokine regulatable. Cytokine and inflammatory protein
responsive promoters that can be used include K and T kininogen
(Kageyama et al., (1987) J. Biol. Chem., 262, 2345-2351), c-fos,
TNF-alpha, C-reactive protein (Arcone, et al., (1988) Nucl. Acids
Res., 16(8), 3195-3207), haptoglobin (Oliviero et al., (1987) EMBO
J., 6, 1905-1912), serum amyloid A2, C/EBP alpha, IL-1, IL-6 (Poli
and Cortese, (1989) Proc. Nat'l Acad. Sci. USA, 86, 8202-8206),
Complement C3 (Wilson et al., (1990) Mol. Cell. Biol., 6181-6191),
IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, (1988) Mol
Cell Biol, 8, 42-51), alpha-1 antitrypsin, lipoprotein lipase
(Zechner et al., Mol. Cell. Biol., 2394-2401, 1988),
angiotensinogen (Ron, et al., (1991) Mol. Cell. Biol., 2887-2895),
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 glucocorticoid inducible), Stromelysin (inducible
by phorbol ester, interleukin-1 and EGF), alpha-2 macroglobulin and
alpha-1 anti-chymotrypsin. Other promoters include, for example,
SV40, MMTV, Human Immunodeficiency Virus (MV), Moloney virus, ALV,
Epstein Barr virus, Rous Sarcoma virus, human actin, myosin,
hemoglobin, and creatine.
[0177] It is envisioned that any of the above promoters alone or in
combination with another can be useful depending on the action
desired. Promoters, and other regulatory elements, are selected by
those of ordinary skill in the art such that they are functional in
the desired cells or tissue. 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.
[0178] 2. Enhancers
[0179] Enhancers are genetic elements that increase transcription
from a promoter located at a distant position on the same molecule
of DNA. Early examples include the enhancers associated with
immunoglobulin and T cell receptors that both flank the coding
sequence and occur within several introns. Many viral promoters,
such as CMV, SV40, and retroviral LTRs are closely associated with
enhancer activity and are often treated like single elements.
Enhancers are organized much like promoters. That is, they are
composed of many individual elements, each of which binds to one or
more transcriptional proteins. The basic distinction between
enhancers and promoters is operational. An enhancer region as a
whole must be able to stimulate transcription at a distance and
often independent of orientation; this need not be true of a
promoter region or its component elements. On the other hand, a
promoter must have one or more elements that direct initiation of
RNA synthesis at a particular site and in a particular orientation,
whereas enhancers lack these specificities. Promoters and enhancers
are often overlapping and contiguous, often seeming to have a very
similar modular organization. A subset of enhancers are
locus-control regions (LCRs) that can not only increase
transcriptional activity, but (along with insulator elements) can
also help to insulate the transcriptional element from adjacent
sequences when integrated into the genome.
[0180] Any promoter/enhancer combination (as per the Eukaryotic
Promoter Data Base EPDB) can be used to drive expression of the
gene, although many will restrict expression to a particular tissue
type or subset of tissues. (reviewed in, for example, Kutzler, M.
A., and Weiner, D. B., 2008. Nature Reviews Genetics 9:776-88).
Examples include, but are not limited to, enhancers from the human
actin, myosin, hemoglobin, muscle creatine kinase, sequences, and
from viruses CMV, RSV, and EBV. Those of ordinary skill in the art
will be able to select appropriate enhancers for particular
applications. Eukaryotic cells can support cytoplasmic
transcription from certain bacterial promoters if the appropriate
bacterial polymerase is provided, either as part of the delivery
complex or as an additional genetic expression construct.
[0181] 3. Polyadenylation Signals
[0182] Where a cDNA insert is employed, one will typically desire
to include a polyadenylation signal to effect proper
polyadenylation of the gene transcript. The nature of the
polyadenylation signal is not believed to be crucial to the
successful practice of the present methods, and any such sequence
is employed such as human or bovine growth hormone and SV40
polyadenylation signals and LTR polyadenylation signals. One
non-limiting example is the SV40 polyadenylation signal present in
the pCEP3 plasmid (Invitrogen, Carlsbad, Calif.). Also contemplated
as an element of the expression cassette is a terminator. These
elements can serve to enhance message levels and to minimize read
through from the cassette into other sequences. Termination or
poly(A) signal sequences may be, for example, positioned about
11-30 nucleotides downstream from a conserved sequence (AAUAAA) at
the 3' end of the mRNA. (Montgomery, D. L., et al., 1993. DNA Cell
Biol. 12:777-83; Kutzler, M. A., and Weiner, D. B., 2008. Nature
Rev. Gen. 9:776-88).
[0183] 4. Initiation Signals and Internal Ribosome Binding
Sites
[0184] A specific initiation signal also may be required for
efficient translation of coding sequences. These signals include
the ATG initiation codon or adjacent sequences. Exogenous
translational control signals, including the ATG initiation codon,
may need to be provided. One of ordinary skill in the art would
readily be capable of determining this and providing the necessary
signals. It is well known that the initiation codon must be
in-frame with the reading frame of the desired coding sequence to
ensure translation of the entire insert. The exogenous
translational control signals and initiation codons can be either
natural or synthetic. The efficiency of expression may be enhanced
by the inclusion of appropriate transcription enhancer
elements.
[0185] In certain embodiments, the use of internal ribosome entry
sites (IRES) elements is used to create multigene, or polycistronic
messages. IRES elements are able to bypass the ribosome-scanning
model of 5' methylated cap-dependent translation and begin
translation at internal sites (Pelletier and Sonenberg, Nature,
334:320-325, 1988). IRES elements from two members of the
picornavirus family (polio and encephalomyocarditis) have been
described (Pelletier and Sonenberg, 1988), as well an IRES from a
mammalian message (Macejak and Sarnow, Nature, 353:90-94, 1991).
IRES elements can be linked to heterologous open reading frames.
Multiple open reading frames can be transcribed together, each
separated by an IRES, creating polycistronic messages. By virtue of
the IRES element, each open reading frame is accessible to
ribosomes for efficient translation. Multiple genes can be
efficiently expressed using a single promoter/enhancer to
transcribe a single message (see U.S. Pat. Nos. 5,925,565 and
5,935,819, each herein incorporated by reference).
[0186] G. Sequence Optimization
[0187] Protein production may also be increased by optimizing the
codons in the transgene. Species specific codon changes, known to
those of ordinary skill in the art may be used to increase protein
production. Also, codons may be optimized to produce an optimized
RNA, which may result in more efficient translation. By optimizing
the codons to be incorporated in the RNA, elements such as those
that result in a secondary structure that causes instability,
secondary mRNA structures that can, for example, inhibit ribosomal
binding, or cryptic sequences that can inhibit nuclear export of
mRNA can be removed. (Kutzler, M. A., and Weiner, D. B., 2008.
Nature Rev. Gen. 9:776-88; Yan., J. et al., 2007. Mol. Ther.
15:411-21; Cheung, Y. K., et al., 2004. Vaccine 23:629-38; Narum.,
D. L., et al., 2001. 69:7250-55; Yadava, A., and Ockenhouse, C.F.,
2003. Infect. Immun. 71:4962-69; Smith., J. M., et al., 2004. AIDS
Res. Hum. Retroviruses 20:1335-47; Zhou, W., et al., 2002. Vet.
Microbiol. 88:127-51; Wu, X., et al., 2004. Biochem. Biophys. Res.
Commun. 313:89-96; Zhang, W., et al., 2006. Biochem. Biophys. Res.
Commun. 349:69-78; Deml, L. A., et al., 2001. J. Virol.
75:1099-11001; Schneider, R. M., et al., 1997. J. Virol.
71:4892-4903; Wang, S. D., et al., 2006. Vaccine 24:4531-40; zur
Megede, J., et al., 2000. J. Virol. 74:2628-2635).
[0188] H. Leader Sequences
[0189] Leader sequences may be added, as known to those of ordinary
skill in the art, to enhance the stability of mRNA and result in
more efficient translation. The leader sequence is usually involved
in targeting the mRNA to the endoplasmic reticulum. Examples
include, the signal sequence for the HIV-1 envelope glycoprotein
(Env), which delays its own cleavage, and the IgE gene leader
sequence (Kutzler, M. A., and Weiner, D. B., 2008. Nature Rev. Gen.
9:776-88; L1, V., et al., 2000. Virology 272:417-28; Xu, Z. L., et
al. 2001. Gene 272:149-56; Malin, A. S., et al., 2000. Microbes
Infect. 2:1677-85; Kutzler, M. A., et al., 2005. J. Immunol.
175:112-125; Yang., J. S., et al., 2002. Emerg. Infect. Dis.
8:1379-84; Kumar., S., et al., 2006. DNA Cell Biol. 25:383-92;
Wang, S., et al., 2006. Vaccine 24:4531-40). The IgE leader may be
used to enhance insertion into the endoplasmic reticulum (Tepler,
I, et al. (1989) J. Biol. Chem. 264:5912).
[0190] Expression of the transgenes may be optimized and/or
controlled by the selection of appropriate methods for optimizing
expression, known to those of ordinary skill in the art. These
methods include, for example, optimizing promoters, delivery
methods, and gene sequences, (for example, as presented in Laddy,
D. J., et al., 2008. PLoS.ONE 3 e2517; Kutzler, M. A., and Weiner,
D. B., 2008. Nature Rev. Gen. 9:776-88).
Methods of Gene Transfer
[0191] In order to mediate the effect of the transgene expression
in a cell, it will be necessary to transfer the expression
constructs into a cell. Such transfer may employ viral or non-viral
methods of gene transfer. This section provides a discussion of
methods and compositions of gene transfer.
[0192] A transformed cell comprising an expression vector is
generated by introducing into the cell the expression vector.
Suitable methods for polynucleotide delivery for transformation of
an organelle, a cell, a tissue or an organism for use with the
current methods include virtually any method by which a
polynucleotide (e.g., DNA) can be introduced into an organelle, a
cell, a tissue or an organism, as described herein or as would be
known to one of ordinary skill in the art.
[0193] A host cell can, and has been, used as a recipient for
vectors. Host cells may be derived from prokaryotes or eukaryotes,
depending upon whether the desired result is replication of the
vector or expression of part or all of the vector-encoded
polynucleotide sequences. Numerous cell lines and cultures are
available for use as a host cell, and they can be obtained through
the American Type Culture Collection (ATCC), which is an
organization that serves as an archive for living cultures and
genetic materials. In specific embodiments, the host cell is a
dendritic cell, which is an antigen-presenting cell.
[0194] It is well within the knowledge and skill of a skilled
artisan to determine an appropriate host. Generally this is based
on the vector backbone and the desired result. A plasmid or cosmid,
for example, can be introduced into a prokaryote host cell for
replication of many vectors. Bacterial cells used as host cells for
vector replication and/or expression include DH5alpha, JM109, and
KC8, as well as a number of commercially available bacterial hosts
such as SURE.RTM. Competent Cells and SOLOPACK Gold Cells
(STRATAGENE.RTM., La Jolla, Calif.). Alternatively, bacterial cells
such as E. coli LE392 could be used as host cells for phage
viruses. Eukaryotic cells that can be used as host cells include,
but are not limited to yeast, insects and mammals. Examples of
mammalian eukaryotic host cells for replication and/or expression
of a vector include, but are not limited to, HeLa, NIH3T3, Jurkat,
293, COS, CHO, Saos, and PC12. Examples of yeast strains include,
but are not limited to, YPH499, YPH500 and YPH501.
[0195] Nucleic acid vaccines are known to those of ordinary skill
in the art, and include, for example, non-viral DNA vectors,
"naked" DNA and RNA, and viral vectors. Methods of transforming
cells with these vaccines, and for optimizing the expression of
genes included in these vaccines are known and are also discussed
herein.
[0196] A. Examples of Methods of Nucleic Acid or Viral Vector
Transfer
[0197] 1. Ex Vivo Transformation
[0198] Methods for transfecting vascular cells and tissues removed
from an organism in an ex vivo setting are known to those of skill
in the art. For example, canine endothelial cells have been
genetically altered by retroviral gene transfer in vitro and
transplanted into a canine (Wilson et al., Science, 244:1344-1346,
1989). In another example, Yucatan minipig endothelial cells were
transfected by retrovirus in vitro and transplanted into an artery
using a double-balloon catheter (Nabel et al., Science,
244(4910):1342-1344, 1989). Thus, it is contemplated that cells or
tissues may be removed and transfected ex vivo using the
polynucleotides presented herein. In particular aspects, the
transplanted cells or tissues may be placed into an organism. Thus,
it is well within the knowledge of one skilled in the art to
isolate dendritic cells from an animal, transfect the cells with
the expression vector and then administer the transfected or
transformed cells back to the animal.
[0199] 2. Injection
[0200] In certain embodiments, a polynucleotide may be delivered to
an organelle, a cell, a tissue or an organism via one or more
injections (i.e., a needle injection), such as, for example,
subcutaneously, intradermally, intramuscularly, intravenously,
intraperitoneally, etc. Methods of injection of vaccines are well
known to those of ordinary skill in the art (e.g., injection of a
composition comprising a saline solution). Further embodiments
include the introduction of a polynucleotide by direct
microinjection. The amount of the expression vector used may vary
upon the nature of the antigen as well as the organelle, cell,
tissue or organism used.
[0201] Intradermal, intranodal, or intralymphatic injections are
some of the more commonly used methods of DC administration.
Intradermal injection is characterized by a low rate of absorption
into the bloodstream but rapid uptake into the lymphatic system.
The presence of large numbers of Langerhans dendritic cells in the
dermis will transport intact as well as processed antigen to
draining lymph nodes. Proper site preparation is necessary to
perform this correctly (i.e., hair must be clipped in order to
observe proper needle placement). Intranodal injection allows for
direct delivery of antigen to lymphoid tissues. Intralymphatic
injection allows direct administration of DCs.
[0202] 3. Electroporation
[0203] In certain embodiments, a polynucleotide is introduced into
an organelle, a cell, a tissue or an organism via electroporation.
Electroporation involves the exposure of a suspension of cells and
DNA to a high-voltage electric discharge. In some variants of this
method, certain cell wall-degrading enzymes, such as
pectin-degrading enzymes, are employed to render the target
recipient cells more susceptible to transformation by
electroporation than untreated cells (U.S. Pat. No. 5,384,253,
incorporated herein by reference).
[0204] Transfection of eukaryotic cells using electroporation has
been quite successful. Mouse pre-B lymphocytes have been
transfected with human kappa-immunoglobulin genes (Potter et al.,
(1984) Proc. Nat'l Acad. Sci. USA, 81, 7161-7165), and rat
hepatocytes have been transfected with the chloramphenicol
acetyltransferase gene (Tur-Kaspa et al., (1986) Mol. Cell. Biol.,
6, 716-718) in this manner.
[0205] 4. Calcium Phosphate
[0206] In other embodiments, a polynucleotide is introduced to the
cells using calcium phosphate precipitation. Human KB cells have
been transfected with adenovirus 5 DNA (Graham and van der Eb,
(1973) Virology, 52, 456-467) using this technique. Also in this
manner, mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa
cells were transfected with a neomycin marker gene (Chen and
Okayama, Mol. Cell. Biol., 7(8):2745-2752, 1987), and rat
hepatocytes were transfected with a variety of marker genes (Rippe
et al., Mol. Cell. Biol., 10:689-695, 1990).
[0207] 5. DEAE-Dextran
[0208] In another embodiment, a polynucleotide is delivered into a
cell using DEAE-dextran followed by polyethylene glycol. In this
manner, reporter plasmids were introduced into mouse myeloma and
erythroleukemia cells (Gopal, T. V., Mol Cell Biol. 1985 May;
5(5):1188-90).
[0209] 6. Sonication Loading
[0210] Additional embodiments include the introduction of a
polynucleotide by direct sonic loading. LTK-fibroblasts have been
transfected with the thymidine kinase gene by sonication loading
(Fechheimer et al., (1987) Proc. Nat'l Acad. Sci. USA, 84,
8463-8467).
[0211] 7. Liposome-Mediated Transfection
[0212] In a further embodiment, a polynucleotide may be entrapped
in a lipid complex such as, for example, 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. The lipid components undergo self-rearrangement
before the formation of closed structures and entrap water and
dissolved solutes between the lipid bilayers (Ghosh and Bachhawat,
(1991) In: Liver Diseases, Targeted Diagnosis and Therapy Using
Specific Receptors and Ligands. pp. 87-104). Also contemplated is a
polynucleotide complexed with Lipofectamine (Gibco BRL) or
Superfect (Qiagen).
[0213] 8. Receptor Mediated Transfection
[0214] Still further, a polynucleotide may be delivered to a target
cell via receptor-mediated delivery vehicles. These take advantage
of the selective uptake of macromolecules by receptor-mediated
endocytosis that will be occurring in a target cell. In view of the
cell type-specific distribution of various receptors, this delivery
method adds another degree of specificity.
[0215] Certain receptor-mediated gene targeting vehicles comprise a
cell receptor-specific ligand and a polynucleotide-binding agent.
Others comprise a cell receptor-specific ligand to which the
polynucleotide to be delivered has been operatively attached.
Several ligands have been used for receptor-mediated gene transfer
(Wu and Wu, (1987) J. Biol. Chem., 262, 4429-4432; Wagner et al.,
Proc. Natl. Acad. Sci. USA, 87(9):3410-3414, 1990; Perales et al.,
Proc. Natl. Acad. Sci. USA, 91:4086-4090, 1994; Myers, EPO
0273085), which establishes the operability of the technique.
Specific delivery in the context of another mammalian cell type has
been described (Wu and Wu, Adv. Drug Delivery Rev., 12:159-167,
1993; incorporated herein by reference). In certain aspects, a
ligand is chosen to correspond to a receptor specifically expressed
on the target cell population.
[0216] In other embodiments, a polynucleotide delivery vehicle
component of a cell-specific polynucleotide-targeting vehicle may
comprise a specific binding ligand in combination with a liposome.
The polynucleotide(s) to be delivered are housed within the
liposome and the specific binding ligand is functionally
incorporated into the liposome membrane. The liposome will thus
specifically bind to the receptor(s) of a target cell and deliver
the contents to a cell. Such systems have been shown to be
functional using systems in which, for example, epidermal growth
factor (EGF) is used in the receptor-mediated delivery of a
polynucleotide to cells that exhibit upregulation of the EGF
receptor.
[0217] In still further embodiments, the polynucleotide delivery
vehicle component of a targeted delivery vehicle may be a liposome
itself, which may, for example, comprise one or more lipids or
glycoproteins that direct cell-specific binding. For example,
lactosyl-ceramide, a galactose-terminal asialoganglioside, have
been incorporated into liposomes and observed an increase in the
uptake of the insulin gene by hepatocytes (Nicolau et al., (1987)
Methods Enzymol., 149, 157-176). It is contemplated that the
tissue-specific transforming constructs may be specifically
delivered into a target cell in a similar manner.
[0218] 9. Microprojectile Bombardment
[0219] Microprojectile bombardment techniques can be used to
introduce a polynucleotide into at least one, organelle, cell,
tissue or organism (U.S. Pat. No. 5,550,318; U.S. Pat. No.
5,538,880; U.S. Pat. No. 5,610,042; and PCT Application WO
94/09699; each of which is incorporated herein by reference). This
method depends on the ability to accelerate DNA-coated
microprojectiles to a high velocity allowing them to pierce cell
membranes and enter cells without killing them (Klein et al.,
(1987) Nature, 327, 70-73). There are a wide variety of
microprojectile bombardment techniques known in the art, many of
which are applicable to the present methods.
[0220] In this microprojectile bombardment, one or more particles
may be coated with at least one polynucleotide and delivered into
cells by a propelling force. 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) Proc. Nat'l Acad.
Sci. USA, 87, 9568-9572). The microprojectiles used have consisted
of biologically inert substances such as tungsten or gold particles
or beads. Exemplary particles include those comprised of tungsten,
platinum, and, in certain examples, gold. It is contemplated that
in some instances DNA precipitation onto metal particles would not
be necessary for DNA delivery to a recipient cell using
microprojectile bombardment. However, it is contemplated that
particles may contain DNA rather than be coated with DNA.
DNA-coated particles may increase the level of DNA delivery via
particle bombardment but are not, in and of themselves,
necessary.
[0221] B. Examples of Methods of Viral Vector-Mediated Transfer
[0222] In certain embodiments, a transgene is incorporated into a
viral particle to mediate gene transfer to a cell. Typically, the
virus simply will be exposed to the appropriate host cell under
physiologic conditions, permitting uptake of the virus. The present
methods are advantageously employed using a variety of viral
vectors, as discussed below.
[0223] 1. Adenovirus
[0224] Adenovirus 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.
[0225] 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, M. J. (1990) Radiother
Oncol., 19, 197-218). 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 useful for translation.
[0226] In order for adenovirus to be optimized for gene therapy, it
is necessary to maximize the carrying capacity so that large
segments of DNA can be included. It also is very desirable to
reduce the toxicity and immunologic reaction associated with
certain adenoviral products. The two goals are, to an extent,
coterminous in that elimination of adenoviral genes serves both
ends. By practice of the present methods, it is possible to achieve
both these goals while retaining the ability to manipulate the
therapeutic constructs with relative ease.
[0227] The large displacement of DNA is possible 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, R. T., et al., J
Mol. Biol. 1984 Jun. 5; 175(4):493-510). Therefore, inclusion of
these elements in an adenoviral vector should permit
replication.
[0228] In addition, the packaging signal for viral encapsulation is
localized between 194-385 by (0.5-1.1 map units) at the left end of
the viral genome (Hearing et al., J. (1987) Virol., 67, 2555-2558).
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., Gene,
101:195-202, 1991).
[0229] 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.
[0230] 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 derives from the packaging function of adenovirus.
[0231] It has been shown that a packaging signal for adenovirus
exists in the left end of the conventional adenovirus map (Tibbetts
et. al. (1977) Cell, 12, 243-249). 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) J. Mol. Biol. 167, 809-822).
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 toward the interior of the Ad5 DNA molecule (Hearing et al.,
J. (1987) Virol., 67, 2555-2558).
[0232] 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 is packaged selectively when
compared to the helpers. If the preference is great enough, stocks
approaching homogeneity should be achieved.
[0233] To improve the tropism of ADV constructs for particular
tissues or species, the receptor-binding fiber sequences can often
be substituted between adenoviral isolates. For example the
Coxsackie-adenovirus receptor (CAR) ligand found in adenovirus 5
can be substituted for the CD46-binding fiber sequence from
adenovirus 35, making a virus with greatly improved binding
affinity for human hematopoietic cells. The resulting "pseudotyped"
virus, Ad5f35, has been the basis for several clinically developed
viral isolates. Moreover, various biochemical methods exist to
modify the fiber to allow re-targeting of the virus to target
cells, such as dendritic cells. Methods include use of bifunctional
antibodies (with one end binding the CAR ligand and one end binding
the target sequence), and metabolic biotinylation of the fiber to
permit association with customized avidin-based chimeric ligands.
Alternatively, one could attach ligands (e.g. anti-CD205 by
heterobifunctional linkers (e.g. PEG-containing), to the adenovirus
particle.
[0234] 2. Retrovirus
[0235] The 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) In: Virology, ed., New York: Raven Press, pp.
1437-1500). 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).
[0236] 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) Cell, 33,
153-159). 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, J. F., and
Rubenstein, J. L. R., (1988) In: Vectors: a Survey of Molecular
Cloning Vectors and Their Uses, Rodriquez and Denhardt, Eds.).
Nicolas and Rubenstein; Temin et al., (1986) In: Gene Transfer,
Kucherlapati (ed.), New York: Plenum Press, pp. 149-188; 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) Virology, 67, 242-248).
[0237] 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.
[0238] 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) Proc. Nat'l Acad. Sci. USA,
86, 9079-9083). 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).
[0239] 3. Adeno-Associated Virus
[0240] 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.
[0241] 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.
[0242] 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.
[0243] 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., J.
Virol., 61:3096-3101 (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.
[0244] 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) Ann. N.Y. Acad. Sci., 770;
79-90; Chatteijee, et al., (1995) Ann. N.Y. Acad. Sci., 770, 79-90;
Ferrari et al., (1996) J. Virol., 70, 3227-3234; Fisher et al.,
(1996) J. Virol., 70, 520-532; Flotte et al., Proc. Nat'l Acad.
Sci. USA, 90, 10613-10617, (1993); Goodman et al. (1994), Blood,
84, 1492-1500; Kaplitt et al., (1994) Nat'l Genet., 8, 148-153;
Kaplitt, M. G., et al., Ann Thorac Surg. 1996 December;
62(6):1669-76; Kessler et al., (1996) Proc. Nat'l Acad. Sci. USA,
93, 14082-14087; Koeberl et al., (1997) Proc. Nat'l Acad. Sci. USA,
94, 1426-1431; Mizukami et al., (1996) Virology, 217, 124-130).
[0245] 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., Proc. Nat'l Acad.
Sci. USA, 90, 10613-10617, (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) J. Virol., 70, 520-532; Flotte et al., 1993; Kaplitt et
al., 1994; 1996; Koeberl et al., 1997; McCown et al., (1996) Brain
Res., 713, 99-107; Ping et al., (1996) Microcirculation, 3,
225-228; Xiao et al., (1996) J. Virol., 70, 8098-8108).
[0246] 4. Other Viral Vectors
[0247] Other viral vectors are employed as expression constructs in
the present methods and compositions. Vectors derived from viruses
such as vaccinia virus (Ridgeway, (1988) In: Vectors: A survey of
molecular cloning vectors and their uses, pp. 467-492; Baichwal and
Sugden, (1986) In, Gene Transfer, pp. 117-148; Coupar et al., Gene,
68:1-10, 1988) canary poxvirus, and herpes viruses are employed.
These viruses offer several features for use in gene transfer into
various mammalian cells.
[0248] 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.
Enhancement of an Immune Response
[0249] In certain embodiments, a novel DC activation strategy is
contemplated, that incorporates the manipulation of signaling
co-stimulatory polypeptides that activate NF-kappaB pathways, Akt
pathways, and/or p38 pathways. This DC activation system can be
used in conjunction with or without standard vaccines to enhance
the immune response since it replaces the requirement for CD4+ T
cell help during APC activation (Bennett, S. R., et al., Nature,
1998, Jun. 4. 393: p. 478-80; Ridge, J. P., D. R. F, and P. Nature,
1998, Jun. 4. 393: p. 474-8; Schoenberger, S. P., et al., Nature,
1998, Jun. 4. 393: p. 480-3). Thus, the DC activation system
presented herein enhances immune responses by circumventing the
need for the generation of MHC class II-specific peptides.
[0250] In specific embodiments, the DC activation is via CD40
activation. Thus, DC activation via endogenous CD40/CD40L
interactions may be subject to downregulation due to negative
feedback, leading rapidly to the "IL-12 burn-out effect". Within 7
to 10 hours after CD40 activation, an alternatively spliced isoform
of CD40 (type II) is produced as a secretable factor (Tone, M., et
al., Proc Natl Acad Sci USA, 2001. 98(4): p. 1751-1756). Type II
CD40 may act as a dominant negative receptor, downregulating
signaling through CD40L and potentially limiting the potency of the
immune response generated. Therefore, the present methods co-opt
the natural regulation of CD40 by creating an inducible form of
CD40 (iCD40), lacking the extracellular domain and activated
instead by synthetic dimerizing ligands (Spencer, D. M., et al.,
Science, 1993. 262: p. 1019-1024) through a technology termed
chemically induced dimerization (CID).
[0251] The present methods comprise methods of enhancing the immune
response in an subject comprising the step of administering either
the expression vector, expression construct or transduced
antigen-presenting cells to the subject. The expression vector
encodes a co-stimulatory polypeptide, such as iCD40.
[0252] In certain embodiments the antigen-presenting cells are
comprised in an animal, such as human, non-human primate, cow,
horse, pig, sheep, goat, dog, cat, or rodent. The subject may be,
for example, human, for example, a patient suffering from an
infectious disease, and/or a subject that is immunocompromised, or
is suffering from a hyperproliferative disease.
[0253] In further embodiments, the expression construct and/or
expression vector can be utilized as a composition or substance
that activates antigen-presenting cells. Such a composition that
"activates antigen-presenting cells" or "enhances the activity
antigen-presenting cells" refers to the ability to stimulate one or
more activities associated with antigen-presenting cells. Such
activities are well known by those of skill in the art. For
example, a composition, such as the expression construct or vector
of the present methods, can stimulate upregulation of
co-stimulatory molecules on antigen-presenting cells, induce
nuclear translocation of NF-kappaB in antigen-presenting cells,
activate toll-like receptors in antigen-presenting cells, or other
activities involving cytokines or chemokines.
[0254] The expression construct, expression vector and/or
transduced antigen-presenting cells can enhance or contribute to
the effectiveness of a vaccine by, for example, enhancing the
immunogenicity of weaker antigens such as highly purified or
recombinant antigens, reducing the amount of antigen required for
an immune response, reducing the frequency of immunization required
to provide protective immunity, improving the efficacy of vaccines
in subjects with reduced or weakened immune responses, such as
newborns, the aged, and immunocompromised individuals, and
enhancing the immunity at a target tissue, such as mucosal
immunity, or promote cell-mediated or humoral immunity by eliciting
a particular cytokine profile.
[0255] Yet further, an immunocompromised individual or subject is a
subject that has a reduced or weakened immune response. Such
individuals may also include a subject that has undergone
chemotherapy or any other therapy resulting in a weakened immune
system, a transplant recipient, a subject currently taking
immunosuppressants, an aging individual, or any individual that has
a reduced and/or impaired CD4 T helper cells. It is contemplated
that the present methods can be utilized to enhance the amount
and/or activity of CD4 T helper cells in an immunocompromised
subject.
[0256] In specific embodiments, prior to administering the
transduced antigen-presenting cell, the cells are challenged with
antigens (also referred herein as "target antigens"). After
challenge, the transduced, loaded antigen-presenting cells are
administered to the subject parenterally, intradermally,
intranodally, or intralymphatically. Additional parenteral routes
include, but are not limited to subcutaneous, intramuscular,
intraperitoneal, intravenous, intraarterial, intramyocardial,
transendocardial, transepicardial, intrathecal, and infusion
techniques.
[0257] The target antigen, as used herein, is an antigen or
immunological epitope on the antigen, which is crucial in immune
recognition and ultimate elimination or control of the
disease-causing agent or disease state in a mammal. The immune
recognition may be cellular and/or humoral. In the case of
intracellular pathogens and cancer, immune recognition may, for
example, be a T lymphocyte response.
[0258] The target antigen may be derived or isolated from, for
example, a pathogenic microorganism such as viruses including HIV,
(Korber et al, eds HIV Molecular Immunology Database, Los Alamos
National Laboratory, Los Alamos, N. Mex. 1977) influenza, Herpes
simplex, human papilloma virus (U.S. Pat. No. 5,719,054), Hepatitis
B (U.S. Pat. No. 5,780,036), Hepatitis C (U.S. Pat. No. 5,709,995),
EBV, Cytomegalovirus (CMV) and the like. Target antigen may be
derived or isolated from pathogenic bacteria such as, for example,
from Chlamydia (U.S. Pat. No. 5,869,608), Mycobacteria, Legionella,
Meningiococcus, Group A Streptococcus, Salmonella, Listeria,
Hemophilus influenzae (U.S. Pat. No. 5,955,596) and the like.
[0259] Target antigen may be derived or isolated from, for example,
pathogenic yeast including Aspergillus, invasive Candida (U.S. Pat.
No. 5,645,992), Nocardia, Histoplasmosis, Cryptosporidia and the
like.
[0260] Target antigen may be derived or isolated from, for example,
a pathogenic protozoan and pathogenic parasites including but not
limited to Pneumocystis carinii, Trypanosoma, Leishmania (U.S. Pat.
No. 5,965,242), Plasmodium (U.S. Pat. No. 5,589,343) and Toxoplasma
gondii.
[0261] Target antigen includes an antigen associated with a
preneoplastic or hyperplastic state. Target antigen may also be
associated with, or causative of cancer. Such target antigen may
be, for example, tumor specific antigen, tumor associated antigen
(TAA) or tissue specific antigen, epitope thereof, and epitope
agonist thereof. Such target antigens include but are not limited
to carcinoembryonic antigen (CEA) and epitopes thereof such as
CAP-1, CAP-1-6D and the like (GenBank Accession No. M29540), MART-1
(Kawakarni et al, J. Exp. Med. 180:347-352, 1994), MAGE-1 (U.S.
Pat. No. 5,750,395), MAGE-3, GAGE (U.S. Pat. No. 5,648,226), GP-100
(Kawakami et al Proc. Nat'l Acad. Sci. USA 91:6458-6462, 1992),
MUC-1, MUC-2, point mutated ras oncogene, normal and point mutated
p53 oncogenes (Hollstein et al Nucleic Acids Res. 22:3551-3555,
1994), PSMA (Israeli et al Cancer Res. 53:227-230, 1993),
tyrosinase (Kwon et al PNAS 84:7473-7477, 1987) TRP-1 (gp75) (Cohen
et al Nucleic Acid Res. 18:2807-2808, 1990; U.S. Pat. No.
5,840,839), NY-ESO-1 (Chen et al PNAS 94: 1914-1918, 1997), TRP-2
(Jackson et al EMBO J, 11:527-535, 1992), TAG72, KSA, CA-125, PSA,
HER-2/neu/c-erb/B2, (U.S. Pat. No. 5,550,214), BRC-I, BRC-II,
bcr-abl, pax3-fkhr, ews-fli-1, modifications of TAAs and tissue
specific antigen, splice variants of TAAs, epitope agonists, and
the like. Other TAAs may be identified, isolated and cloned by
methods known in the art such as those disclosed in U.S. Pat. No.
4,514,506. Target antigen may also include one or more growth
factors and splice variants of each.
[0262] An antigen may be expressed more frequently in cancer cells
than in non-cancer cells. The antigen may result from contacting
the modified dendritic cell with prostate specific membrane antigen
(PSMA) or fragment thereof. In certain embodiments, the modified
dendritic cell is contacted with a PSMA fragment having the amino
acid sequence of SEQ ID NO: 4 (e.g., encoded by the nucleotide
sequence of SEQ ID NO: 3).
[0263] For organisms that contain a DNA genome, a gene encoding a
target antigen or immunological epitope thereof of interest is
isolated from the genomic DNA. For organisms with RNA genomes, the
desired gene may be isolated from cDNA copies of the genome. If
restriction maps of the genome are available, the DNA fragment that
contains the gene of interest is cleaved by restriction
endonuclease digestion by methods routine in the art. In instances
where the desired gene has been previously cloned, the genes may be
readily obtained from the available clones. Alternatively, if the
DNA sequence of the gene is known, the gene can be synthesized by
any of the conventional techniques for synthesis of
deoxyribonucleic acids.
[0264] Genes encoding an antigen of interest can be amplified, for
example, by cloning the gene into a bacterial host. For this
purpose, various prokaryotic cloning vectors can be used. Examples
are plasmids pBR322, pUC and pEMBL.
[0265] The genes encoding at least one target antigen or
immunological epitope thereof can be prepared for insertion into
the plasmid vectors designed for recombination with a virus by
standard techniques. In general, the cloned genes can be excised
from the prokaryotic cloning vector by restriction enzyme
digestion. In most cases, the excised fragment will contain the
entire coding region of the gene. The DNA fragment carrying the
cloned gene can be modified as needed, for example, to make the
ends of the fragment compatible with the insertion sites of the DNA
vectors used for recombination with a virus, then purified prior to
insertion into the vectors at restriction endonuclease cleavage
sites (cloning sites).
[0266] Antigen loading of dendritic cells with antigens may be
achieved, for example, by incubating dendritic cells or progenitor
cells with the polypeptide, 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). Prior
to loading, the polypeptide may be covalently conjugated to an
immunological partner that provides T cell help (e.g., a carrier
molecule). Alternatively, a dendritic cell may be pulsed with a
non-conjugated immunological partner, separately or in the presence
of the polypeptide. Antigens from cells or MHC molecules may be
obtained by acid-elution or other methods known in the art (see
Zitvogel L, et al., J Exp Med 1996. 183:87-97).
[0267] In further embodiments, the transduced antigen-presenting
cell is transfected with tumor cell mRNA. The transduced
transfected antigen-presenting cell is administered to an animal to
effect cytotoxic T lymphocytes and natural killer cell anti-tumor
antigen immune response and regulated using dimeric FK506 and
dimeric FK506 analogs. The tumor cell mRNA may be, for example,
mRNA from a prostate tumor cell.
[0268] Yet further, the transduced antigen-presenting cell is
pulsed with tumor cell lysates. The pulsed transduced
antigen-presenting cells are administered to an animal to effect
cytotoxic T lymphocytes and natural killer cell anti-tumor antigen
immune response and regulated using dimeric FK506 and dimeric FK506
analogs. The tumor cell lysate may be, for example, a prostate
tumor cell lysate.
[0269] One skilled in the art is fully aware that activation of the
co-stimulatory molecule of the present relies upon oligomerization
of ligand-binding domains, for example CID, to induce its activity.
In specific embodiments, the ligand is a non-protein. For example,
the ligand may be a dimeric FK506 or dimeric FK506 analogs, which
result in enhancement or positive regulation of the immune
response. The use of monomeric FK506 or monomeric FK506 analogs
results in inhibition or reduction in the immune response
negatively.
[0270] T-lymphocytes may be activated by contact with the
antigen-presenting cell that comprises the expression vector
discussed herein where the antigen-presenting cell has been
challenged, transfected, pulsed, or electrofused with an
antigen.
[0271] Electrofusing is a method of generating hybrid cells. There
are several advantages in producing cell hybrids by electrofusion.
For example, fusion parameters can be easily and accurately
electronically controlled to conditions depending on the cells to
be fused. Further, electrofusion of cells has shown to the ability
to increase fusion efficiency over that of fusion by chemical means
or via biological fusogens. Electrofusion is performed by applying
electric pulses to cells in suspension. By exposing cells to an
alternating electric field, cells are brought close to each other
in forming pearl chains in a process termed dielectrophoresis
alignment. Subsequent higher voltage pulses cause cells to come
into closer contact, reversible electropores are formed in
reversibly permeabilizing and mechanically breaking down cell
membranes, resulting in fusion.
[0272] T cells express a unique antigen binding receptor on their
membrane (T-cell receptor), which can only recognize antigen in
association with major histocompatibility complex (MHC) molecules
on the surface of other cells. There are several populations of T
cells, such as T helper cells and T cytotoxic cells. T helper cells
and T cytotoxic cells are primarily distinguished by their display
of the membrane bound glycoproteins CD4 and CD8, respectively. T
helper cells secret various lymphokines, that are crucial for the
activation of B cells, T cytotoxic cells, macrophages and other
cells of the immune system. In contrast, a naive CD8 T cell that
recognizes an antigen-MHC complex proliferates and differentiates
into an effector cell called a cytotoxic CD8 T lymphocyte (CTL).
CTLs eliminate cells of the body displaying antigen, such as
virus-infected cells and tumor cells, by producing substances that
result in cell lysis.
[0273] CTL activity can be assessed by methods described herein or
as would be known to one of skill in the art. For example, CTLs may
be assessed in freshly isolated peripheral blood mononuclear cells
(PBMC), in a phytohaemaglutinin-stimulated IL-2 expanded cell line
established from PBMC (Bernard et al., AIDS, 12(16):2125-2139,
1998) or by T cells isolated from a previously immunized subject
and restimulated for 6 days with DC infected with an adenovirus
vector containing antigen using standard 4 hour .sup.51Cr release
microtoxicity assays. One type of assay uses cloned T-cells. Cloned
T-cells have been tested for their ability to mediate both perforin
and Fas ligand-dependent killing in redirected cytotoxicity assays
(Simpson et al., Gastroenterology, 115(4):849-855, 1998). The
cloned cytotoxic T lymphocytes displayed both Fas- and
perforin-dependent killing. Recently, an in vitro dehydrogenase
release assay has been developed that takes advantage of a new
fluorescent amplification system (Page, B., et al., Anticancer Res.
1998 July-August; 18(4A):2313-6). This approach is sensitive,
rapid, and reproducible and may be used advantageously for mixed
lymphocyte reaction (MLR). It may easily be further automated for
large-scale cytotoxicity testing using cell membrane integrity, and
is thus considered. In another fluorometric assay developed for
detecting cell-mediated cytotoxicity, the fluorophore used is the
non-toxic molecule AlamarBlue (Nociari et al., J. Immunol. Methods,
213(2): 157-167, 1998). The AlamarBlue is fluorescently quenched
(i.e., low quantum yield) until mitochondrial reduction occurs,
which then results in a dramatic increase in the AlamarBlue
fluorescence intensity (i.e., increase in the quantum yield). This
assay is reported to be extremely sensitive, specific and requires
a significantly lower number of effector cells than the standard
.sup.51Cr release assay.
[0274] Other immune cells that are induced by the present methods
include natural killer cells (NK). NKs are lymphoid cells that lack
antigen-specific receptors and are part of the innate immune
system. Typically, infected cells are usually destroyed by T cells
alerted by foreign particles bound to the cell surface MHC.
However, virus-infected cells signal infection by expressing viral
proteins that are recognized by antibodies. These cells can be
killed by NKs. In tumor cells, if the tumor cells lose expression
of MHC I molecules, then it may be susceptible to NKs.
[0275] Formulations and Routes for Administration to Patients
[0276] Where clinical applications are contemplated, it will be
necessary to prepare pharmaceutical compositions--expression
constructs, expression vectors, fused proteins, transduced cells,
activated DCs, transduced and loaded DCs--in a form appropriate for
the intended application. Generally, this will entail preparing
compositions that are essentially free of pyrogens, as well as
other impurities that could be harmful to humans or animals.
[0277] One may generally desire to employ appropriate salts and
buffers to render delivery vectors stable and allow for uptake by
target cells. Buffers also may be employed when recombinant cells
are introduced into a patient. Aqueous compositions comprise an
effective amount of the vector to cells, dissolved or dispersed in
a pharmaceutically acceptable carrier or aqueous medium. Such
compositions also are referred to as inocula. The phrase
"pharmaceutically or pharmacologically acceptable" refers to
molecular entities and compositions that do not produce adverse,
allergic, or other untoward reactions when administered to an
animal or a human. A pharmaceutically acceptable carrier includes
any and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents and the
like. The use of such media and agents for pharmaceutically active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the vectors or
cells, its use in therapeutic compositions is contemplated.
Supplementary active ingredients also can be incorporated into the
compositions.
[0278] The active compositions may include classic pharmaceutical
preparations. Administration of these compositions will be via any
common route so long as the target tissue is available via that
route. This includes, for example, oral, nasal, buccal, rectal,
vaginal or topical. Alternatively, administration may be by
orthotopic, intradermal, subcutaneous, intramuscular,
intraperitoneal or intravenous injection. Such compositions would
normally be administered as pharmaceutically acceptable
compositions, discussed herein.
[0279] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating, such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial an antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In certain examples, isotonic agents, for example,
sugars or sodium chloride may be included. Prolonged absorption of
the injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0280] For oral administration, the compositions may be
incorporated with excipients and used in the form of non-ingestible
mouthwashes and dentifrices. A mouthwash may be prepared
incorporating the active ingredient in the required amount in an
appropriate solvent, such as a sodium borate solution (Dobell's
Solution). Alternatively, the active ingredient may be incorporated
into an antiseptic wash containing sodium borate, glycerin and
potassium bicarbonate. The active ingredient also may be dispersed
in dentifrices, including, for example: gels, pastes, powders and
slurries. The active ingredient may be added in a therapeutically
effective amount to a paste dentifrice that may include, for
example, water, binders, abrasives, flavoring agents, foaming
agents, and humectants.
[0281] The compositions may be formulated in a neutral or salt
form. Pharmaceutically-acceptable salts include, for example, the
acid addition salts (formed with the free amino groups of the
protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups can also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like.
[0282] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms such as injectable solutions, drug
release capsules and the like. For parenteral administration in an
aqueous solution, for example, the solution should be suitably
buffered if necessary and the liquid diluent first rendered
isotonic with sufficient saline or glucose. These particular
aqueous solutions are especially suitable for intravenous,
intramuscular, subcutaneous and intraperitoneal administration. In
this connection, sterile aqueous media, which can be employed, will
be known to those of skill in the art in light of the present
disclosure. For example, one dosage could be dissolved in 1 ml of
isotonic NaCl solution and either added to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, "Remington's Pharmaceutical Sciences" 15th
Edition, pages 1035-1038 and 1570-1580). Some variation in dosage
will necessarily occur depending on the condition of the subject
being treated. The person responsible for administration will, in
any event, determine the appropriate dose for the individual
subject. Moreover, for human administration, preparations should
meet sterility, pyrogenicity, and general safety and purity
standards as required by FDA Office of Biologics standards.
Methods for Treating a Disease
[0283] The present methods also encompasses methods of treatment or
prevention of a disease caused by pathogenic microorganisms and/or
a hyperproliferative disease.
[0284] Diseases may be treated or prevented include diseases caused
by viruses, bacteria, yeast, parasites, protozoa, cancer cells and
the like. The pharmaceutical composition (transduced DCs,
expression vector, expression construct, etc.) may be used as a
generalized immune enhancer (DC activating composition or system)
and as such has utility in treating diseases. Exemplary diseases
that can be treated and/or prevented include, but are not limited,
to infections of viral etiology such as HIV, influenza, Herpes,
viral hepatitis, Epstein Bar, polio, viral encephalitis, measles,
chicken pox, Papilloma virus etc.; or infections of bacterial
etiology such as pneumonia, tuberculosis, syphilis, etc.; or
infections of parasitic etiology such as malaria, trypanosomiasis,
leishmaniasis, trichomoniasis, amoebiasis, etc.
[0285] Preneoplastic or hyperplastic states which may be treated or
prevented using the pharmaceutical composition (transduced DCs,
expression vector, expression construct, etc.) include but are not
limited to preneoplastic or hyperplastic states such as colon
polyps, Crohn's disease, ulcerative colitis, breast lesions and the
like.
[0286] Cancers which may be treated using the pharmaceutical
composition include, but are not limited to primary or metastatic
melanoma, adenocarcinoma, squamous cell carcinoma, adenosquamous
cell carcinoma, thymoma, lymphoma, sarcoma, lung cancer, liver
cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemias,
uterine cancer, breast cancer, prostate cancer, ovarian cancer,
pancreatic cancer, colon cancer, multiple myeloma, neuroblastoma,
NPC, bladder cancer, cervical cancer and the like.
[0287] Other hyperproliferative diseases that may be treated using
DC activation system presented herein include, but are not limited
to rheumatoid arthritis, inflammatory bowel disease,
osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas,
fibromas, vascular occlusion, restenosis, atherosclerosis,
pre-neoplastic lesions (such as adenomatous hyperplasia and
prostatic intraepithelial neoplasia), carcinoma in situ, oral hairy
leukoplakia, or psoriasis.
[0288] In the method of treatment, the administration of the
pharmaceutical composition (expression construct, expression
vector, fused protein, transduced cells, activated DCs, transduced
and loaded DCs) may be for either "prophylactic" or "therapeutic"
purpose. When provided prophylactically, the pharmaceutical
composition is provided in advance of any symptom. The prophylactic
administration of pharmaceutical composition serves to prevent or
ameliorate any subsequent infection or disease. When provided
therapeutically, the pharmaceutical composition is provided at or
after the onset of a symptom of infection or disease. Thus the
compositions presented herein may be provided either prior to the
anticipated exposure to a disease-causing agent or disease state or
after the initiation of the infection or disease.
[0289] The term "unit dose" as it pertains to the inoculum refers
to physically discrete units suitable as unitary dosages for
mammals, each unit containing a predetermined quantity of
pharmaceutical composition calculated to produce the desired
immunogenic effect in association with the required diluent. The
specifications for the novel unit dose of an inoculum are dictated
by and are dependent upon the unique characteristics of the
pharmaceutical composition and the particular immunologic effect to
be achieved.
[0290] An effective amount of the pharmaceutical composition would
be the amount that achieves this selected result of enhancing the
immune response, and such an amount could be determined as a matter
of routine by a person skilled in the art. For example, an
effective amount of for treating an immune system deficiency could
be that amount necessary to cause activation of the immune system,
resulting in the development of an antigen specific immune response
upon exposure to antigen. The term is also synonymous with
"sufficient amount."
[0291] The effective amount for any particular application can vary
depending on such factors as the disease or condition being
treated, the particular composition being administered, the size of
the subject, and/or the severity of the disease or condition. One
of ordinary skill in the art can empirically determine the
effective amount of a particular composition presented herein
without necessitating undue experimentation.
[0292] A. Genetic Based Therapies In certain embodiments, a cell is
provided with an expression construct capable of providing a
co-stimulatory polypeptide, such as CD40 to the cell, such as an
antigen-presenting cell and activating CD40. The lengthy discussion
of expression vectors and the genetic elements employed therein is
incorporated into this section by reference. In certain examples,
the expression vectors may be viral vectors, such as adenovirus,
adeno-associated virus, herpes virus, vaccinia virus and
retrovirus. In another example, the vector may be a
lysosomal-encapsulated expression vector.
[0293] Those of skill in the art are well aware of how to apply
gene delivery to in vivo and ex vivo situations. For viral vectors,
one generally will prepare a viral vector stock. Examples of viral
vector-mediated gene delivery ex vivo are presented in the present
application. For in vivo delivery, depending on the kind of virus
and the titer attainable, one will deliver, for example,
1.times.10.sup.4, 1.times.10.sup.5, 1.times.10.sup.6,
1.times.10.sup.7, 1.times.10.sup.8, 1.times.10.sup.9,
1.times.10.sup.19, 1.times.10.sup.11 or 1.times.10.sup.12
infectious particles to the patient. Similar figures may be
extrapolated for liposomal or other non-viral formulations by
comparing relative uptake efficiencies. Formulation as a
pharmaceutically acceptable composition is discussed below.
[0294] B. Cell based Therapy
[0295] Another therapy that is contemplated is the administration
of transduced antigen-presenting cells. The antigen-presenting
cells may be transduced in vitro. Formulation as a pharmaceutically
acceptable composition is discussed herein.
[0296] In cell based therapies, the transduced antigen-presenting
cells may be, for example, transfected with target antigen nucleic
acids, such as mRNA or DNA or proteins; pulsed with cell lysates,
proteins or nucleic acids; or electrofused with cells. The cells,
proteins, cell lysates, or nucleic acid may derive from cells, such
as tumor cells or other pathogenic microorganism, for example,
viruses, bacteria, protozoa, etc.
[0297] C. Combination Therapies
[0298] In order to increase the effectiveness of the expression
vectors presented herein, it may be desirable to combine these
compositions and methods with an agent effective in the treatment
of the disease.
[0299] In certain embodiments, anti-cancer agents may be used in
combination with the present methods. An "anti-cancer" agent is
capable of negatively affecting cancer in a subject, for example,
by killing one or more cancer cells, inducing apoptosis in one or
more cancer cells, reducing the growth rate of one or more cancer
cells, reducing the incidence or number of metastases, reducing a
tumor's size, inhibiting a tumor's growth, reducing the blood
supply to a tumor or one or more cancer cells, promoting an immune
response against one or more cancer cells or a tumor, preventing or
inhibiting the progression of a cancer, or increasing the lifespan
of a subject with a cancer. Anti-cancer agents include, for
example, chemotherapy agents (chemotherapy), radiotherapy agents
(radiotherapy), a surgical procedure (surgery), immune therapy
agents (immunotherapy), genetic therapy agents (gene therapy),
hormonal therapy, other biological agents (biotherapy) and/or
alternative therapies.
[0300] In further embodiments antibiotics can be used in
combination with the pharmaceutical composition to treat and/or
prevent an infectious disease. Such antibiotics include, but are
not limited to, amikacin, aminoglycosides (e.g., gentamycin),
amoxicillin, amphotericin B, ampicillin, antimonials, atovaquone
sodium stibogluconate, azithromycin, capreomycin, cefotaxime,
cefoxitin, ceftriaxone, chloramphenicol, clarithromycin,
clindamycin, clofazimine, cycloserine, dapsone, doxycycline,
ethambutol, ethionamide, fluconazole, fluoroquinolones, isoniazid,
itraconazole, kanamycin, ketoconazole, minocycline, ofloxacin),
para-aminosalicylic acid, pentamidine, polymixin definsins,
prothionamide, pyrazinamide, pyrimethamine sulfadiazine, quinolones
(e.g., ciprofloxacin), rifabutin, rifampin, sparfloxacin,
streptomycin, sulfonamides, tetracyclines, thiacetazone,
trimethaprim-sulfamethoxazole, viomycin or combinations
thereof.
[0301] More generally, such an agent would be provided in a
combined amount with the expression vector effective to kill or
inhibit proliferation of a cancer cell and/or microorganism. This
process may involve contacting the cell(s) with an agent(s) and the
pharmaceutical composition at the same time or within a period of
time wherein separate administration of the pharmaceutical
composition and an agent to a cell, tissue or organism produces a
desired therapeutic benefit. This may be achieved by contacting the
cell, tissue or organism with a single composition or
pharmacological formulation that includes both the pharmaceutical
composition and one or more agents, or by contacting the cell with
two or more distinct compositions or formulations, wherein one
composition includes the pharmaceutical composition and the other
includes one or more agents.
[0302] The terms "contacted" and "exposed," when applied to a cell,
tissue or organism, are used herein to describe the process by
which the pharmaceutical composition and/or another agent, such as
for example a chemotherapeutic or radiotherapeutic agent, are
delivered to a target cell, tissue or organism or are placed in
direct juxtaposition with the target cell, tissue or organism. To
achieve cell killing or stasis, the pharmaceutical composition
and/or additional agent(s) are delivered to one or more cells in a
combined amount effective to kill the cell(s) or prevent them from
dividing.
[0303] The administration of the pharmaceutical composition may
precede, be co-current with and/or follow the other agent(s) by
intervals ranging from minutes to weeks. In embodiments where the
pharmaceutical composition and other agent(s) are applied
separately to a cell, tissue or organism, one would generally
ensure that a significant period of time did not expire between the
times of each delivery, such that the pharmaceutical composition
and agent(s) would still be able to exert an advantageously
combined effect on the cell, tissue or organism. For example, in
such instances, it is contemplated that one may contact the cell,
tissue or organism with two, three, four or more modalities
substantially simultaneously (i.e., within less than about a
minute) with the pharmaceutical composition. In other aspects, one
or more agents may be administered within of from substantially
simultaneously, about 1 minute, to about 24 hours to about 7 days
to about 1 to about 8 weeks or more, and any range derivable
therein, prior to and/or after administering the expression vector.
Yet further, various combination regimens of the pharmaceutical
composition presented herein and one or more agents may be
employed.
EXAMPLES
[0304] The examples set forth below illustrate but do not limit the
invention.
Example 1
Materials and Methods
[0305] Described hereafter are materials and methods utilized in
studies described in subsequent Examples.
[0306] Tumor Cell Lines and Peptides
[0307] NA-6-MeI, T2, SK-MeI-37 and LNCaP cell lines were purchased
from the American Type Culture Collection (ATCC) (Manassas, Va.).
HLA-A2-restricted peptides MAGE-3 p271-279 (FLWGPRALV), influenza
matrix (IM) p58-66 (GILGFVFTL), and HIV-1 gag p77-85 (SLYNTVATL)
were used to analyze CD8+ T cell responses. In T helper cell
polarization experiments, tetanus toxoid peptide TTp30
FNNFTVSFWLRVPKVSASHLE was used. All peptides were synthesized by
Genemed Synthesis Inc (San Francisco, Calif.), with an
HPLC-determined purity of >95%.
[0308] Recombinant Adenovirus Encoding Human Inducible CD40
[0309] The human CD40 cytoplasmic domain was Pfu I polymerase
(Stratagene, La Jolla, Calif.) amplified from human
monocyte-derived DC cDNA using an Xho I-flanked 5' primer
(5hCD40X), 5'-atatactcgagaaaaaggtggccaagaagccaacc-3', and a Sal
I-flanked 3' primer (3hCD40S),
5'-atatagtcgactcactgtctctcctgcactgagatg-3'. The PCR fragment was
subcloned into Sal I-digested pSH1/M-FvFvls-E15 and sequenced to
create pSH1/M-FvFvls-CD40-E. Inducible CD40 was subsequently
subcloned into a non-replicating E1, E3-deleted Ad5/f35-based
vector expressing the transgene under a cytomegalovirus
early/immediate promoter. The iCD40-encoding sequence was confirmed
by restriction digest and sequencing. Amplification, purification,
and titration of all adenoviruses were carried out in the Viral
Vector Core Facility of Baylor College of Medicine.
[0310] Western Blot
[0311] Total cellular extracts were prepared with RIPA buffer
containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis,
Mo.) and quantitated using a detergent-compatible protein
concentration assay (Bio-Rad, Hercules, Calif.). 10-15 micrograms
of total protein were routinely separated on 12% SDS-PAGE gels, and
proteins were transferred to nitrocellulose membranes (Bio-Rad).
Blots were hybridized with goat anti-CD40 (T-20, Santa Cruz
Biotechnology, Santa Cruz, Calif.) and mouse anti-alpha-tubulin
(Santa Cruz Biotechnology) Abs followed by donkey anti-goat and
goat anti-mouse IgG-HRP (Santa Cruz Biotechnology), respectively.
Blots were developed using the SuperSignal West Dura Stable
substrate system (Pierce, Rockford, Ill.).
[0312] Generation and Stimulation of Human DCs
[0313] Peripheral blood mononuclear cells (PBMCs) from healthy
donors were isolated by density centrifugation of heparinized blood
on Lymphoprep (Nycomed, Oslo, Norway). PBMCs were washed with PBS,
resuspended in CellGenix DC medium (Freiburg, Germany) and allowed
to adhere in culture plates for 2 h at 37.degree. C. and 5%
CO.sub.2. Nonadherent cells were removed by extensive washings, and
adherent monocytes were cultured for 5 days in the presence of 500
U/ml hIL-4 and 800 U/ml hGM-CSF (R&D Systems, Minneapolis,
Minn.). As assessed by morphology and FACS analysis, the resulting
immature DCs (imDCs) were MHC-class I, Ilhi, and expressed CD40lo,
CD80lo, CD83lo, CD86lo. The imDCs were CD14neg and contained <3%
of contaminating CD3+ T, CD19+ B, and CD16+ NK cells.
[0314] Approximately 2.times.10.sup.6 cells/ml were cultured in a
24-well dish and transduced with adenoviruses at 10,000 viral
particle (vp)/cell (.about.160 MOI) for 90 min at 37.degree. C. and
5% CO.sub.2. Immediately after transduction DCs were stimulated
with MPL, FSL-1, Pam3CSK4 (InvivoGen, San Diego, Calif.), LPS
(Sigma-Aldrich, St. Louis, Mo.), AP20187 (kind gift from ARIAD
Pharmaceuticals, Cambridge, Mass.) or maturation cocktail (MC),
containing 10 ng/ml TNF-alpha, 10 ng/ml IL-1beta, 150 ng/ml IL-6
(R&D Systems, Minneapolis, Minn.) and 1 microgram/ml of PGE2
(Cayman Chemicals, Ann Arbor, Mich.). In T cell assays DCs were
pulsed with 50 micrograms/ml of PSMA or MAGE 3 peptide 24 hours
before and after adenoviral transduction.
[0315] Surface Markers and Cytokine Production
[0316] Cell surface staining was done with fluorochrome-conjugated
monoclonal antibodies (BD Biosciences, San Diego, Calif.). Cells
were analyzed on a FACSCalibur cytometer (BD Biosciences, San Jose,
Calif.). Cytokines were measured in culture supernatants using
enzyme-linked immunosorbent assay kits for human IL-6 and IL-12p70
(BD Biosciences).
[0317] Real Time Q-PCR Assay for Human SOCS1
[0318] Total RNA was purified and reverse transcribed with random
hexamers using SuperScript 11 RTase (Invitrogen, Carlsbad, Calif.).
mRNA levels were quantified in DCs by subjecting cDNA to TaqMan PCR
analysis using the GeneAmp 5700 Sequence Detection System (Applied
Biosystems, Foster City, Calif.). Pre-developed sequence detection
reagents (Applied Biosystems) specific for human SOCS1 and 18S
rRNA, including forward and reverse primers as well as a
fluorogenic TaqMan FAM-labeled hybridization probe, were supplied
as mixtures and were used at 1 microliter/20 microliter PCR.
Samples were run in duplicates. The level of SOCS1 expression in
each sample was normalized to the level of 18S rRNA from the same
sample using the comparative 2-delta delta CT method (Livak K J,
Schmittgen T D. Analysis of relative gene expression data using
real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.
Methods. 2001; 25:402-408).
DC Migration Assay
[0319] Chemotaxis of DCs was measured by migration through a
polycarbonate filter with 8 micrometer pore size in 96-Multiwell
HTS Fluoroblok plates (BD Biosciences). Assay medium (250
microliters) containing 100 ng/ml CCL19 (R&D Systems) or assay
medium alone (as a control for spontaneous migration) were loaded
into the lower chamber. DCs (50,000) were labeled with Green-CMFDA
cell tracker (Invitrogen), unstimulated or stimulated for 48 h with
the indicated reagents, and were added to the upper chamber in a
total volume of 50 microliters for 1 hour at 37.degree. C. and 5%
CO.sub.2. Fluorescence of cells, which had migrated through the
microporous membrane, was measured using the FLUOstar OPTIMA reader
(BMG Labtech Inc., Durham, N.C.). The mean fluorescence of
spontaneously migrated cells was subtracted from the total number
of migrated cells for each condition.
[0320] IFN-Gamma ELISPOT Assay
[0321] DCs from HLA-A2-positive healthy volunteers were pulsed with
MAGE-3 A2.1 peptide (residues 271-279; FLWGPRALV) on day 4 of
culture, followed by transduction with Ad-iCD40 and stimulation
with various stimuli on day 5. Autologous T cells were purified
from PBMCs by negative selection (Miltenyi Biotec, Auburn, Calif.)
and mixed with DCs at DC:T cell ratio 1:3. Cells were incubated in
complete RPMI with 20 U/ml hIL-2 (R&D Systems) and 25
micrograms/ml of MAGE 3 A2.1 peptide. T cells were restimulated at
day 7 and assayed at day 14 of culture.
[0322] ELISPOT Quantitation
[0323] Flat-bottom, 96-well nitrocellulose plates (MultiScreen-HA;
Millipore, Bedford, Mass.) were coated with IFN-gamma mAb (2
.mu.g/ml, 1-D1K; Mabtech, Stockholm, Sweden) and incubated
overnight at 4.degree. C. After washings with PBS containing 0.05%
TWEEN 20, plates were blocked with complete RPMI for 2 h at
37.degree. C. A total of 1.times.10.sup.5 presensitized CD8+ T
effector cells were added to each well and incubated for 20 h with
25 micrograms/ml peptides. Plates were then washed thoroughly with
PBS containing 0.05% TWEEN 20, and anti-IFN-mAb (0.2 microg/ml,
7-B6-1-biotin; Mabtech) was added to each well. After incubation
for 2 h at 37.degree. C., plates were washed and developed with
streptavidin-alkaline phosphatase (1 microg/ml; Mabtech) for 1 h at
room temperature. After washing, substrate
(3-amino-9-ethyl-carbazole; Sigma-Aldrich) was added and incubated
for 5 min. Plate membranes displayed dark-pink spots that were
scanned and analyzed by ZellNet Consulting Inc. (Fort Lee,
N.J.).
[0324] Chromium Release Assay
[0325] Antigen recognition was assessed using target cells labeled
with Chromium-51 (Amersham) for 1 hour at 37.degree. C. and washed
three times. Labeled target cells (5000 cells in 50 microliters)
were then added to effector cells (100 microliters) at the
indicated effector:target cell ratios in V-bottom microwell plates
at the indicated concentrations. Supernatants were harvested after
6-h incubation at 37.degree. C., and chromium release was measured
using MicroBeta Trilux counter (Perkin-Elmer Inc, Torrance Calif.).
Assays involving LNCaP cells were run for 18 hours. The percentage
of specific lysis was calculated as: 100*[(experimental-spontaneous
release)/(maximum-spontaneous release)].
[0326] Tetramer Staining
[0327] HLA-A2 tetramers assembled with MAGE-3.A2 peptide
(FLWGPRALV) were obtained from Baylor College of Medicine Tetramer
Core Facility (Houston, Tex.). Presensitized CD8+ T cells in 50
.mu.l of PBS containing 0.5% FCS were stained with PE-labeled
tetramer for 15 min on ice before addition of FITC-CD8 mAb (BD
Biosciences). After washing, results were analyzed by flow
cytometry.
[0328] Polarization of naive T Helper Cells
[0329] Naive CD4+CD45RA+ T-cells from HLA-DR11.5-positive donors
(genotyped using FASTYPE HLA-DNA SSP typing kit; BioSynthesis,
Lewisville, Tex.) were isolated by negative selection using naive
CD4+ T cell isolation kit (Miltenyi Biotec, Auburn, Calif.). T
cells were stimulated with autologous DCs pulsed with tetanus
toxoid (5 FU/ml) and stimulated with various stimuli at a
stimulator to responder ratio of 1:10. After 7 days, T cells were
restimulated with autologous DCs pulsed with the
HLA-DR11.5-restricted helper peptide TTp30 and transduced with
adenovector Ad-iCD40. Cells were stained with PE-anti-CD4 Ab (BD
Biosciences), fixed and permeabilized using BD Cytofix/Cytoperm kit
(BD Biosciences), then stained with hIFN-gamma mAb (eBioscience,
San Diego, Calif.) and analyzed by flow cytometry. Supernatants
were analyzed using human TH1/TH2 BD Cytometric Bead Array Flex Set
on BD FACSArray Bioanalyzer (BD Biosciences).
[0330] PSMA Protein Purification
[0331] The baculovirus transfer vector, pAcGP67A (BD Biosciences)
containing the cDNA of extracellular portion of PSMA (residues
44-750) was kindly provided by Dr Pamela J. Bjorkman (Howard Hughes
Medical Institute, California Institute of Technology, Pasadena,
Calif.). PSMA was fused with a hydrophobic secretion signal, Factor
Xa cleavage site, and N-terminal 6.times.-His affinity tag. High
titer baculovirus was produced by the Baculovirus/Monoclonal
antibody core facility of Baylor College of Medicine. PSMA protein
was produced in High 5 cells infected with recombinant virus, and
protein was purified from cell supernatants using Ni-NTA affinity
columns (Qiagen, Chatsworth, Calif.) as previously described (Cisco
R M, Abdel-Wahab Z, Dannull J, et al. Induction of human dendritic
cell maturation using transfection with RNA encoding a dominant
positive toll-like receptor 4. J. Immunol. 2004; 172:7162-7168).
After purification the .about.100 kDa solitary band of PSMA protein
was detected by silver staining of acrylamide gels.
[0332] Migration of Human DCs in Mouse Host
[0333] In order to assess the migration of human DCs in vivo,
adenovector Ad5-CBR, which expresses red-shifted (emission peak=613
nM) luciferase from Pyrophorus plagiophalamus click beetles
(Promega, Madison, Wis.) was developed. Human DCs were transduced
with .about.50 MOI of Ad5-CBR, and 160 MOI of Ad5f35-iCD40. DCs
were then matured with MC or 1 microgram/ml LPS (Sigma-Aldrich, St.
Louis, Mo.). Mouse bone marrow derived DCs were obtained as
described before 13 and were matured with 1 micrograms/ml LPS.
Approximately 2.times.10.sup.6 DCs were injected into the left and
right hind footpads of irradiated (250 Rads) Balb/c mice (both hind
legs of three mice per group, n=6). Mice were i.p. injected with
D-Luciferin (.about.1 mg/25 g animal) and imaged over several days
using an IVIS.TM. 100 imaging system (Xenogen Corp., Alameda,
Calif.). Luminescent signal was measured in 3 mice per group, and
popliteal and inguinal lymph nodes (LN) were removed at day 2
post-DC inoculation. The LNs' signal was measured and the
background was subtracted for each group (n=6).
[0334] Data Analysis
[0335] Results are expressed as the mean.+-.standard error. Sample
size was determined with a power of 0.8, with a one-sided
alpha-level of 0.05. Differences between experimental groups were
determined by the Student t test.
Example 2
Expression of iCD40 and Induction of DC Maturation
[0336] To investigate whether iCD40 signaling can enhance the
immunogenic functions of human DCs, adenovirus, Ad5/f35-ihCD40
(simplified to Ad-iCD40) was generated, expressing inducible human
CD40 receptor, based on the previously described mouse iCD40
vector13 (FIG. 1). Those of ordinary skill in the art will
recognize that this is an example of an assay that may be used to
examine DC maturation after transduction of a chimeric iCD40
protein, such as, for example, iCD40-MyD88, or other chimera
examples herein. The human CD40 cytoplasmic signaling domain was
cloned downstream of a myristoylation-targeting domain and two
tandem domains (from human FKBP12(V36), designated as "Fv'"), which
bind dimerizing drug AP20187 (Clackson T, Yang W, Rozamus L W, et
al. Redesigning an FKBP-ligand interface to generate chemical
dimerizers with novel specificity. Proc Natl Acad Sci USA. 1998;
95:10437-10442). Immature DCs expressed endogenous CD40, which was
induced by LPS and CD40L. Transduction of Ad-iCD40 led to
expression of the distinctly sized iCD40, which did not interfere
with endogenous CD40 expression. Interestingly, the expression of
iCD40 was also significantly enhanced by LPS stimulation, likely
due to inducibility of ubiquitous transcription factors binding the
"constitutive" CMV promoter.
[0337] One of the issues for the design of DC-based vaccines is to
obtain fully matured and activated DCs, as maturation status is
linked to the transition from a tolerogenic to an activating,
immunogenic state (Steinman R M, et al., Annu Rev Immunol. 2003;
21:685-711; Hanks B A, et al., Nat. Med. 2005; 11:130-137;
Banchereau J, Steinman R M. Dendritic cells and the control of
immunity. Nature. 1998; 392:245-252). It has been shown that
expression of mouse variant Ad-iCD40 can induce murine bone
marrow-derived DC maturation (Hanks B A, et al., Nat. Med. 2005;
11:130-137). To determine whether humanized iCD40 affects the
expression of maturation markers in DCs, DCs were transduced with
Ad-iCD40 and the expression of maturation markers CD40, CD80, CD83,
and CD86 were evaluated. TLR-4 signaling mediated by LPS or its
derivative MPL is a potent inducer of DC maturation (Ismaili J, et
al., J. Immunol. 2002; 168:926-932; Cisco R M, et al., J. Immunol.
2004; 172:7162-7168; De Becker G, Moulin V, Pajak B, et al. The
adjuvant monophosphoryl lipid A increases the function of
antigen-presenting cells. Int Immunol. 2000; 12:807-815; Granucci
F, Ferrero E, Foti M, Aggujaro D, Vettoretto K,
Ricciardi-Castagnoli P. Early events in dendritic cell maturation
induced by LPS. Microbes Infect. 1999; 1:1079-1084). It was also
previously reported that endogenous CD40 signaling specifically
up-regulates CD83 expression in human DCs (Megiovanni A M, Sanchez
F, Gluckman J C, Rosenzwajg M. Double-stranded RNA stimulation or
CD40 ligation of monocyte-derived dendritic cells as models to
study their activation and maturation process. Eur Cytokine Netw.
2004; 15:126-134). Consistent with these previous reports, the
expression levels of CD83 were upregulated upon Ad-iCD40
transduction, and CD83 expression was further upregulated following
LPS or MPL addition.
Example 3
Assay for Synergy of iCD40 Signaling and Inducible PRR Adapter
Protein Ligation
[0338] Those of ordinary skill in the art are able to modify the
assays presented in this example to observe synergy between iCD40
signaling and inducible PRR adapter protein ligation.
[0339] Interleukin-12 (IL-12) activates T and NK cell responses,
and induces IFN-gamma production. It also favors the
differentiation of TH1 cells and is a vital link between innate and
adaptive immunity (Banchereau J, et al., Ann N Y Acad. Sci. 2003;
987:180-187; Puccetti P, Belladonna M L, Grohmann U. Effects of
IL-12 and IL-23 on antigen-presenting cells at the interface
between innate and adaptive immunity. Crit. Rev Immunol. 2002;
22:373-390). Therefore, induction of biologically active IL-12p70
heterodimer is likely critical for optimum DC-based vaccines.
Nonetheless, current DC vaccination protocols that include PGE2
produce only limited IL-12 (Lee A W, Truong T, Bickham K, et al. A
clinical grade cocktail of cytokines and PGE2 results in uniform
maturation of human monocyte-derived dendritic cells: implications
for immunotherapy. Vaccine. 2002; 20 Suppl 4:A8-A22). IL-12 is a
heterodimeric cytokine consisting of p40 and p35 chains.
Previously, it was reported that inducible CD40 signaling promotes
the expression of the p35 subunit of IL-12p70 in mouse bone
marrow-derived DCs (Hanks B A, et al., Nat. Med. 2005; 11:130-137).
It was also reported that TLR-4 ligation can promote p40 expression
(Liu J, Cao S, Herman L M, Ma X. Differential regulation of
interleukin (IL)-12 p35 and p40 gene expression and interferon
(IFN)-gamma-primed IL-12 production by IFN regulatory factor 1. J
Exp Med. 2003; 198:1265-1276). Therefore, iCD40-DCs were cultured
in the presence of LPS or MPL and assayed supernatants by ELISA for
production of IL-12p70.
[0340] Predictably, similar to DCs treated with standard MC,
iCD40-DCs did not produce detectable IL-12p70 heterodimer. If PGE2
was withheld from the MC, DCs produced detectable but low levels of
IL-12p70, consistent with a potentially deleterious role for PGE2.
Furthermore, DCs cultured for 12 h in the presence of LPS or MPL
alone also failed to produce IL-12 (<30 pg/ml). However, when
Ad-iCD40-transduced DCs were cultured in the presence of either MPL
or LPS they produced very high levels of IL-12p70 (16.4.+-.7.8
ng/ml for MPL). This level of IL-12 was about 25-fold higher than
levels induced by standard MC lacking PGE2. Interestingly, this
synergism of iCD40 and TLR4 was partially independent of AP20187
addition, implying that basal iCD40 signaling can also synergize
with TLR4 ligation. IL-12p70 production in iCD40-DCs was also
dose-dependent as IL-12 levels correlated with viral particles
dose.
[0341] Since CD40 signaling is normally tightly restricted to a
relatively short time period (Contin C, Pitard V, Itai T, Nagata S,
Moreau J F, Dechanet-Merville J. Membrane-anchored CD40 is
processed by the tumor necrosis factor-alpha-converting enzyme.
Implications for CD40 signaling. J Biol. Chem. 2003;
278:32801-32809; Tone M, Tone Y, Fairchild P J, Wykes M, Waldmann
H. Regulation of CD40 function by its isoforms generated through
alternative splicing. Proc Natl Acad Sci USA. 2001; 98:1751-1756),
potentially limiting adaptive immunity, it was determined whether
iCD40 could induce not only enhanced, but also prolonged,
expression of IL-12p70 in TLR-4-stimulated DCs. To evaluate the
kinetics of IL-12 expression, LPS-treated iCD40-DCs with LPS and
CD40L-stimulated DCs were compared. It was observed that iCD40-DCs
were able to produce IL-12p70 for over 72 hours post stimulation
compared to CD40L or control vector-transduced DCs in which
IL-12p70 expression ceased when LPS stimulation was removed. These
results indicate that inducible CD40 signaling allows DCs to
produce increased levels of IL-12p70 continuously in response to
TLR-4 stimulation.
[0342] Finally, the induction of the suppressor of cytokine
signaling (SOCS1) was evaluated. SOCS1 is negative feedback
inhibitor of DC activation, that can attenuate (Wesemann D R, Dong
Y, O'Keefe G M, Nguyen V T, Benveniste E N. Suppressor of cytokine
signaling 1 inhibits cytokine induction of CD40 expression in
macrophages. J. Immunol. 2002; 169:2354-2360) responsiveness to LPS
and cytokine stimulation (Evel-Kabler K, Song X T, Aldrich M, Huang
X F, Chen S Y. SOCS1 restricts dendritic cells' ability to break
self tolerance and induce antitumor immunity by regulating IL-12
production and signaling. J Clin Invest. 2006; 116:90-100). LPS
stimulation up-regulated SOCS1 expression in DCs, as previously
reported (Wesemann D R, et al., J. Immunol. 2002; 169:2354-2360).
Strikingly, however, in the presence of LPS, iCD40-DCs expressed
3-fold lower levels of SOCS1 than CD40L-stimulated DCs. Moreover,
iCD40 did not induce SOCS1 by itself, unlike CD40L. These data
indicate that iCD40 can partially bypass SOCS1 induction in human
DCs and may partly explain the observed sustained elevation of
IL-12 levels and DC maturation markers.
[0343] In addition to IL-12, IL-6 plays an important role in cell
survival and resistance to T regulatory cells (Rescigno M, et al.,
J Exp Med. 1998; 188:2175-2180; Pasare C, Medzhitov R. Toll
pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression
by dendritic cells. Science. 2003; 299:1033-1036). It was observed
that upon transfection with Ad-iCD40, IL-6 expression was
significantly enhanced and further upregulated when iCD40-DCs were
stimulated with dimerizer drug and TLR-4 ligands. Thus, iCD40
signaling is sufficient for production of some pro-inflammatory
cytokines, but requires additional TLR signaling for production of
the key TH1 cytokine, IL-12.
Example 4
Antigen-Specific TH1 Polarization
[0344] Antigen-specific TH1 polarization assays are presented
herein. Those of ordinary skill in the art are able to modify the
examples presented in order to assay polarization in the context of
inducible PRR and PRR adapter proteins.
[0345] To further investigate whether iCD40-DCs matured with TLR-4
ligands can effectively prime CD4+ T helper (TH) cells, it was
determined whether they can augment CD4+epitope-specific T-cell
responses in vitro. Naive CD4+CD45RA+ T cells were stimulated for 7
days in the presence of autologous Ad-iCD40 DCs pulsed with the
model antigen, tetanus toxoid. At day 7, T cells were stimulated
with the MHC class II-restricted tetanus toxoid epitope, TTp30. The
production of IFN-gamma was significantly increased in the CD4+ T
cells co-cultured with iCD40-DCs and iCD40-DCs stimulated with
either MPL or MC. IFN-gamma production was iCD40-specific, as it
was not induced by control virus Ad-GFP-transduced DCs or by MPL or
MC stimulation alone. In addition, T cell polarization was analyzed
by assessing TH1/TH2 cytokine levels in the supernatants of T cells
using a cytometric bead array. The levels of IFN-gamma, TNF-alpha,
IL-4, and IL-5 secreted cytokines were increased in helper T cells
stimulated by iCD40-DCs, indicating the expansion of both TH1 and
TH2-polarized T cells. However, the levels of TH1 cytokines were
significantly higher than TH2-associated cytokines, indicating a
predominant expansion of TH1 cells. In contrast, induction of
TT-specific CD4+ T-helper cells from naive CD4+CD45RA+ cells, using
MC-matured DCs, led to only a modest bias in TT epitope-specific
TH1 differentiation. These results suggest that iCD40 signaling in
DCs enables them to effectively induce antigen-specific TH1
differentiation, possibly due to higher IL-12 production.
Example 5
Tumor Antigen-Specific CTL Response Assay
[0346] Antigen-specific TH1 polarization assays are presented
herein. Those of ordinary skill in the art are able to modify the
examples presented in order to assay polarization in the context of
inducible PRR and PRR adapter proteins.
[0347] It was determined whether iCD40 and MPL could enhance
cytotoxic T lymphocyte (CTL) responses to poorly immunogenic
melanoma self-antigen MAGE-3. iCD40-DCs from HLA-A2-positive donors
were pulsed with class-1HLA-A2.1-restricted MAGE3-derived
immunodominant peptide, FLWGPRALV, and co-cultured with autologous
T cells. After a series of stimulations, the frequency of
antigen-specific T cells was assessed by IFN-gamma-specific ELISPOT
assay. iCD40-DCs stimulated with MPL led to a 50% increase in
MAGE-3-specific T cells relative to iCD40-DCs stimulated with MC
and about a five-fold increase in antigen-specific T cells compared
to control non-transduced (WT) DCs.
[0348] It also was determined whether iCD40-DCs were capable of
enhancing CTL-mediated killing of tumor cells in an
antigen-specific fashion. Immature DCs from HLA-A2-positive
volunteers were transfected with Ad-iCD40, pulsed with MAGE-3
protein, and used as stimulators to generate CTLs in vitro.
SK-MEL-37 cells (HLA-A2+, MAGE-3+) and T2 cells pulsed with MAGE-3
A2.1 peptide (HLA-A2+, MAGE-3+) were utilized as targets. NA-6-MEL
cells (HLA A2-, MAGE-3+) and T2 cells (HLA-A2+) pulsed with an
irrelevant A2.1-restricted influenza matrix peptide served as
negative controls. CTLs induced by iCD40-DCs were capable of
efficiently recognizing and lysing their cognate targets
(SK-MEL-37), and also T2 cells pulsed with MAGE-3 A2.1 peptide,
indicating the presence of MAGE-3-specific CTLs. In contrast,
control targets were lysed at significantly lower levels. Improved
lytic activity was consistently observed when iCD40-DCs treated
with MPL or MC were used as stimulators compared with
non-transduced DCs treated with MPL or MC alone. In addition, a
significant expansion of MAGE-3/HLA-A2-specific tetramer positive
CD8+CTLs by iCD40-DCs that were treated with MPL was observed.
[0349] Similarly, to test whether LPS and iCD40-stimulated DCs
could enhance CTL lytic activity, their ability to break tolerance
to prostate-specific membrane antigen (PSMA) was examined. DCs
generated from healthy HLA-A2+ volunteers were pulsed with PSMA
protein (Davis M I, Bennett M J, Thomas L M, Bjorkman P J. Crystal
structure of prostate-specific membrane antigen, a tumor marker and
peptidase. Proc Natl Acad Sci USA. 2005; 102:5981-5986) or MAGE-3,
transduced with AD-iCD40 or Ad-Luc, and were co-cultured with
autologous T cells. After three rounds of stimulation,
antigen-specific CTL activity was measured by chromium release
assay using LNCaP cells (HLA-A2+PSMA+) as targets and SK-MeI-37
(HLA-A2+PSMA-) as control cells for PSMA-pulsed DCs. SK-MeI-37
cells (MAGE-3+) were used as targets when DCs of the same donor
were pulsed with MAGE-3, and LNCaP cells (MAGE-3-) were used as
negative controls. Collectively, these data indicate that
iCD40-transduced DCs are capable of inducing significantly more
potent antigen-specific CTL responses in vitro than MC-treated DCs.
Those of ordinary skill in the art may modify this assay to examine
tumor antigen specific CTL responses using chimeric iCD40-inducible
PRR adapter proteins.
Example 6
Inducible CD40 Enhances CCR7 Expression and Migratory Abilities of
DCs Without PGE2
[0350] Antigen-specific TH1 polarization assays are presented
herein. Those of ordinary skill in the art are able to modify the
examples presented in order to assay polarization in the context of
inducible PRR and PRR adapter proteins.
[0351] In addition to other maturation markers, CCR7 is
up-regulated on DCs upon maturation and is responsible for
directing their migration to draining lymph nodes (Cyster J G.
Chemokines and cell migration in secondary lymphoid organs.
Science. 1999; 286:2098-2102). Recently, several reports have
indicated that, apart from chemotaxis, CCR7 also affects DC
"cytoarchitecture", the rate of endocytosis, survival, migratory
speed, and maturation (Sanchez-Sanchez N, Riol-Blanco L,
Rodriguez-Fernandez J L. The Multiple Personalities of the
Chemokine Receptor CCR7 in Dendritic Cells. J. Immunol. 2006;
176:5153-5159). Along with costimulatory molecules and TH1
cytokines, iCD40 specifically up-regulates CCR7 expression in human
DCs. Moreover, CCR7 expression correlated with Ad-iCD40 viral
dose-escalation.
[0352] Because CCR7 expression levels correlate with enhanced
migration toward MIP-3 beta CCL19), it was determined whether human
iCD40-DCs could migrate in vitro toward MIP-3 beta in transwell
assays. iCD40-DCs treated with AP20187 dimerizer have migration
levels comparable to those induced by MC. Moreover, iCD40-DC
migration was further increased by MPL or MC stimulation, even when
PGE2 was not present. These data were highly reproducible and
indicate that iCD40 is sufficient to induce CCR7 expression and DC
migration in vitro in contrast to the widely held belief that PGE2
is essential for lymph node homing of human DC.
[0353] Chemokines and chemokine receptors share a high degree of
sequence identity within a species and between species (De Vries I
J, Krooshoop D J, Scharenborg N M, et al. Effective migration of
antigen-pulsed dendritic cells to lymph nodes in melanoma patients
is determined by their maturation state. Cancer Res. 2003;
63:12-17). On the basis of this knowledge, a novel xenograft model
was developed for monitoring the migration of human DCs in vivo.
Human DCs were transduced with iCD40 and matured with LPS or MC,
and mouse DCs were matured with LPS. Since DCs were co-transduced
with Ad5-CBR, bioluminescence was immediately visible. As expected,
immature DCs did not migrate to the draining popliteal lymph nodes.
However, iCD40-DCs matured with LPS or MC were detectable in the
xenogeneic popliteal lymph nodes within 2 days post-inoculation.
The migration of iCD40-DCs stimulated with LPS was significantly
(p=0.036) higher than non-stimulated DCs and was comparable to
mouse DC migration. Moreover, at day 2 the iCD40-DCs were detected
in inguinal LNs while MC-stimulated DCs were undetectable,
suggesting higher migratory abilities of iCD40-DCs than stimulated
with MC. Collectively, these results indicate that iCD40 signaling
in DCs plays a critical role in controlling CCR7 expression and is
sufficient for DC migration to lymph nodes. The migration of
iCD40-DCs is further enhanced when the cells are stimulated with
LPS, correlating with enhanced CCR7 expression. Those of ordinary
skill in the art may modify this assay for assaying chimeric
iCD40-inducible PRR adapter proteins.
Example 7
Summary of Observations from Assays Presented in Example 2 to
Example 6 with iCD40 and PRRs
[0354] Dendritic cell efficacy depends on many variables,
especially maturation status and efficient migration to lymph
nodes. Several clinical trials in cancer patients showed the
potency of DCs to induce adaptive immunity to tumor-specific
antigens (Nestle F O, Banchereau J, Hart D. Dendritic cells: On the
move from bench to bedside. Nat. Med. 2001; 7:761-765; Schuler G,
Schuler-Thurner B, Steinman R M. The use of dendritic cells in
cancer immunotherapy. Curr Opin Immunol. 2003; 15:138-147; Cranmer
L D, Trevor K T, Hersh E M. Clinical applications of dendritic cell
vaccination in the treatment of cancer. Cancer Immunol Immunother.
2004; 53:275-306). However, clinical responses were transient, and
warrant further improvement in DC vaccine design (Ridgway D. The
first 1000 dendritic cell vaccines. Cancer Invest. 2003;
21:873-886; Dallal R M, Lotze M T. The dendritic cell and human
cancer vaccines. Curr Opin Immunol. 2000; 12:583-588). Limitation
of current DC-based vaccines are the transient activation state
within lymphoid tissues, low induction of CD4+ T cell immunity, and
impaired ability to migrate to the draining lymph nodes (Adema G J,
de Vries I J, Punt C J, Figdor CG. Migration of dendritic cell
based cancer vaccines: in vivo veritas? Curr Opin Immunol. 2005;
17:170-174). Less than 24 hours following exposure to LPS, DCs
terminate synthesis of the TH1-polarizing cytokine, IL-12, and
become refractory to further stimuli (Langenkamp A, Messi M,
Lanzavecchia A, Sallusto F. Kinetics of dendritic cell activation:
impact on priming of TH1, TH2 and nonpolarized T cells. Nat.
Immunol. 2000; 1:311-316), limiting their ability to activate T
helper cells and CTLs. Other studies indicate that less than 5% of
intradermally administered mature DCs reach the lymph nodes,
showing inefficient homing.sup.39. These findings underscore the
need for either prolonging the activation state and migratory
capacities of the DCs and/or temporally coordinating the DC
activation "window" with engagement of cognate T cells within lymph
nodes.
[0355] A method for promoting mouse DC function in vivo was
developed by manipulation of a chimeric inducible CD40 receptor
(Hanks B A, et al., Nat. Med. 2005; 11:130-137). It has been
observed that the inducible CD40 approach is also effective in
enhancing the immunostimulatory function of human DCs. Consistent
with previous reports of the synergistic activity of combining TLR
and CD40 signaling for IL-12p70 secretion, iCD40 plus TLR4
signaling induced high level IL-12 secretion, DC maturation, T cell
stimulatory functions, and extensive migratory capacities (Lapointe
R, et al., Eur J. Immunol. 2000; 30:3291-3298; Napolitani G,
Rinaldi A, Bertoni F, Sallusto F, Lanzavecchia A. Selected
Toll-like receptor agonist combinations synergistically trigger a T
helper type 1-polarizing program in dendritic cells. Nat. Immunol.
2005; 6:769-776).
[0356] It was also demonstrated that increased and prolonged
secretion of IL-12p70 in DCs could break self tolerance, which
likely is attributable in part to over-riding the production of
SOCS1, which inhibits IL-12 signaling (Evel-Kabler K, et al., J
Clin Invest. 2006; 116:90-100). It has been determined that
although endogenous CD40 signaling stimulated by soluble CD40L
leads to SOCS1 upregulation, iCD40 activates DCs without
significant SOCS1 induction. Additionally, iCD40 signaling
unleashes high and prolonged expression of IL-12p70 in DCs, which
exhibit enhanced potency in stimulating CD4+ T cells and CTLs.
[0357] IL-6 is implicated in the survival of many different cell
types by activation of anti-apoptotic pathways, such as p38 MAPK,
ERK1, 2 47 and PI3-kinase (Bisping G, Kropff M, Wenning D, et al.
Targeting receptor kinases by a novel indolinone derivative in
multiple myeloma: abrogation of stroma-derived interleukin-6
secretion and induction of apoptosis in cytogenetically defined
subgroups. Blood. 2006; 107:2079-2089). The induction of IL-6
expression by iCD40 and TLR-4 signaling in DCs also was identified.
This finding could partly explain the prolonged survival of DCs
described previously (Hanks B A, et al., Nat. Med. 2005;
11:130-137). Furthermore, IL-6 expression is critical in the
ability of DCs to inhibit the generation of CD4+CD25+ T regulatory
cells (Pasare C, and Medzhitov R., Science. 2003; 299:1033-1036).
In this context, an iCD40-DCs-based vaccine could potentially
suppress negative regulators in vivo, inhibiting peripheral
tolerance to targeting antigens.
[0358] One major focus of cancer immunotherapy has been the design
of vaccines to promote strong tumor antigen-specific CTL responses
in cancer patients (Rosenberg S A., Immunity. 1999; 10:281-287).
However, accumulating evidence suggests that CD4+ T cells also play
a critical role in antitumor immunity, as they contribute to the
induction, persistence and expansion of CD8+ T cells (Kalams S A,
Walker B D. The critical need for CD4 help in maintaining effective
cytotoxic T lymphocyte responses. J Exp Med. 1998; 188:2199-2204).
Our study showed that iCD40-DCs could effectively prime naive T
cells and effectively expand antigen-specific cells representing
both arms of the immune response (i.e. MAGE-3 and PSMA specific
CTLs and TT-specific CD4+ T cells). It was demonstrated that TH1
(IFN-gamma and TNF-alpha) cytokines were produced predominantly in
the milieu of iCD40-DC-stimulated CD4+ T cells, indicating
expansion of TH1 cells. As expected, these cytokines were not
detected when T cells were stimulated with MC-treated DCs, because
PGE2 (a key MC component) is a powerful suppressor of TH1 responses
(Kalinski P, Hilkens C M, Snijders A, Snijdewint F G, Kapsenberg M
L. Dendritic cells, obtained from peripheral blood precursors in
the presence of PGE2, promote Th2 responses. Adv Exp Med. Biol.
1997; 417:363-367; Mcllroy A, Caron G, Blanchard S, et al.
Histamine and prostaglandin E up-regulate the production of
Th2-attracting chemokines (CCL17 and CCL22) and down-regulate
IFN-gamma-induced CXCL10 production by immature human dendritic
cells. Immunology. 2006; 117:507-516; Meyer F, Ramanujam K S,
Gobert A P, James S P, Wilson K T. Cutting edge: cyclooxygenase-2
activation suppresses Th1 polarization in response to Helicobacter
pylori. J. Immunol. 2003; 171:3913-3917).
[0359] Recent mouse studies have shown that DC migration directly
correlates with T cell proliferation (Martln-Fontecha A, Sebastiani
S, Hopken U E, et al. Regulation of dendritic cell migration to the
draining lymph node: impact on T lymphocyte traffic and priming. J
Exp Med. 2003; 198:615-621). Therefore, the increase in migration
should enhance the efficacy of DC-based vaccines.sup.45. Current DC
vaccine protocols include pre-conditioning the vaccine injection
site with inflammatory cytokines or ex vivo stimulation of DCs with
TLR ligands and pro-inflammatory cytokines, consisting primarily of
MC constituents (Martln-Fontecha A, et al., J Exp Med. 2003;
198:615-621; Prins R M, Craft N, Bruhn K W, et al. The TLR-7
agonist, imiquimod, enhances dendritic cell survival and promotes
tumor antigen-specific T cell priming: relation to central nervous
system antitumor immunity. J. Immunol. 2006; 176:157-164). Despite
its numerous immunosuppressive functions.sup.8-12, PGE2 has been
used for the past few years as an indispensible component of the DC
maturation cocktail because it stimulates the migratory capacity of
DCs by up-regulating both CCR7 and sensitization to its ligands.
Alternative approaches enhancing DCs migration without PGE2, should
be beneficial for DC-based vaccine improvement.
[0360] The results of these studies show that iCD40 signaling not
only up-regulates CCR7 expression on DCs but also stimulates their
chemotaxis to CCL19 in vitro. Additionally, immature DCs transduced
with iCD40 were able to migrate as efficiently as MC-stimulated DCs
both in vitro and in vivo. Moreover, migration of iCD40-DCs was
further induced when cells were stimulated with TLR-4 ligands. It
was recently shown that stimulation of CCR7 increases the migratory
rate of DCs, indicating that this receptor can regulate DC
locomotion and motility (Riol-Blanco L, Sanchez-Sanchez N, Torres
A, et al. The chemokine receptor CCR7 activates in dendritic cells
two signaling modules that independently regulate chemotaxis and
migratory speed. J. Immunol. 2005; 174:4070-4080; Palecek S P,
Loftus J C, Ginsberg M H, Lauffenburger D A, Horwitz A F.
Integrin-ligand-binding properties govern cell migration speed
through cell-substratum adhesiveness. Nature. 1997; 385:537-540;
Yanagawa Y, Onoe K. CCL19 induces rapid dendritic extension of
murine dendritic cells. Blood. 2002; 100:1948-1956). It has been
shown that stimulation of CCR7 enhances the mature phenotype of
DCs58. Thus, by transduction of DCs with iCD40, CCR7 expression, DC
migration and maturation status have been enhanced, obviating the
need for PGE2.
[0361] Finally, iCD40 stimulation of DCs was capable of inducing a
potent cytotoxic T cell response to the prostate-specific antigen,
PSMA, which was capable of significantly increased killing of
target LNCaP cells. (Dudley M E, Wunderlich J R, Yang J C, et al.
Adoptive cell transfer therapy following non-myeloablative but
lymphodepleting chemotherapy for the treatment of patients with
refractory metastatic melanoma. J Clin Oncol. 2005; 23:2346-2357;
Morgan R A, Dudley M E, Wunderlich J R, et al. Cancer Regression in
Patients After Transfer of Genetically Engineered Lymphocytes.
Science. 2006). Other documents cited herein are referenced in U.S.
patent application Ser. No. 10/781,384, filed Feb. 18, 2004,
entitled "Induced Activation In Dendritic Cells," and naming
Spencer et al. as inventors, now issued as U.S. Pat. No.
7,404,950.
Example 8
Inducible CD40
[0362] The innate immune system uses several families of pattern
recognition receptors PRRs to sense pathological infection or
injury. One family of PRRs is the Toll-like receptors (TLRs) that
now include about 11 members in mammals. These typically bind to
multi-valent ligands through a leucine-rich motif (LRM). The
ligands can come from bacteria, viruses, fungi, or host cells and
can bind to TLRs either on the cell surface or within endocytic
vesicles (especially TLR 3, 7, 8 and 9). Within their cytoplasmic
signaling domains, they share a conserved TIR (Toll/IL-1R) domain
that binds to downstream TIR-containing adapter molecules, such as
MyD88 and TRIF/TICAM-1, and adapters TIRAM/TICAM-2 and MAL/TIRAP.
Additional PRRs include the NOD-like receptors (e.g. NOD1 and NOD2)
and the RIG-like helicases, RIG-I and Mda-5. Many PRRs bind to
ligands through flexible LRMs and couple to downstream signaling
molecules through protein-protein binding motifs, such as TIR or
CARD (caspase recruitment domain) domains.
[0363] Stimulation through TLR-4 in conjunction with signaling
through the costimulatory molecule CD40 can promote high-level
maturation and migratory properties in human monocyte-derived
dendritic cells (MoDCs). Based on both published and unpublished
data.sup.2-7, this prolonged and enhanced activation state of human
MoDCs in vitro and/or in vivo may both promote the activation and
expansion of autologous tumor-specific T cells for adoptive
immunotherapy and overcome the problems of self-limiting ex
vivo-matured DCs for vaccination.
[0364] Various approaches are available for assessing the use of
different PRRs in combination with inducible CD40. These
approaches, and the experimental methods used to conduct these
assessments, may also be used to study inducible CD40 in
combination with inducible PRR adapter proteins such as, for
example, MyD88 and TRIF.
[0365] To replace complex, poorly understood MoDC maturation
cocktails or combinations of adjuvants and CD40 signaling,
CID-inducible versions of toll-like receptor 4 (called iTLR4) and
other iTLRs (i.e. TLR3, 7, 8, and 9) are developed and iTLRs are
assayed for synergy with iCD40 either in trans or in cis within the
same polypeptide chain. Efficacy is based on induction of
transcription factors NF-kappaB and IRF3/7s, and phosphorylation of
p38 and JNK in the DC line, D2SC/1. The most potent inducible
receptor is subcloned into an adenovector for efficient
transduction of MoDCs.
[0366] Dendritic cells (DCs) play a critical role in initiating and
regulating adaptive immunity.sup.7,8. Upon detection of "danger
signals", DCs physiologically adapt to their microenvironment by
undergoing a genetic maturation program.sup.6. Using a broad
repertoire of antigen presentation and costimulatory molecules, DCs
are capable of potently activating naive antigen-specific T
lymphocytes and regulating their subsequent phenotype and
function.sup.9. In most cases, the development of robust cytotoxic
T lymphocyte (CTL) immunity by DCs requires a "helper" signal from
CD4.sup.+T cells.sup.10. This signal is comprised of both soluble
cytokines, such as IL-2, as well as CD40L-mediated stimulation of
the surface CD40 receptor on the DC.sup.11-13. A member of the
tumor necrosis factor receptor (TNFR) superfamily, CD40 triggers
various pathways within the DC resulting in the upregulation of
several antigen presentation, costimulatory, cytokine, and
pro-survival genes, which collectively enable the DC to induce CTL
activation.sup.14,15.
[0367] Given the pre-eminent role of DCs as antigen-presenting
cells (APCs), they may be exploited as natural adjuvants in
vaccination protocols for the treatment of various
malignancies.sup.16,17. Typical applications include harvesting
peripheral blood monocytes via leukapheresis, differentiation in
culture in GM-CSF and IL-4, and loading immature monocyte (or
CD34.sup.+ precursor cell)-derived DCs (MoDC) with tumor antigens
by one of several methods, such as pulsing immature DCs with
unfractionated tumor lysates, MHC-eluted peptides, tumor-derived
heat shock proteins (HSPs), tumor associated antigens (TAAs
(peptides or proteins)), or transfecting DCs with bulk tumor mRNA,
or mRNA coding for TAAs (reviewed in 18,19). Antigen-loaded DCs are
then typically matured ex vivo with inflammatory cytokines (e.g.
TNFalpha, IL1beta, IL6, and PGE.sub.2) or other adjuvants (e.g.
LPS, CpG oligonucleotides) and injected into patients. In each
case, the immuno-stimulatory properties of the DCs depend on many
variables, especially the ability to migrate to lymph nodes and
full maturation status. However, the limited success in recent
clinical trials with DC immunotherapy has suggested that current
protocols need to be refined if DC-based immunotherapy is to be
included in the treatment arsenal alongside more conventional
modalities of anti-cancer therapy.sup.20,21.
[0368] Two key limitations of DC-based vaccines are the short
lifespan of matured DCs and their transient activation state within
lymphoid tissues. Less than 24 hours following exposure to
lipopolysaccharide (LPS), DCs terminate synthesis of the
T.sub.H1-polarizing cytokine, IL-12, and become refractory to
further stimuli.sup.22, limiting their ability to activate
cytotoxic T lymphocytes (CTLs). Other studies indicate that the
survival of antigen-pulsed DCs within the draining lymph node (LN)
is limited to only 48 hours following their delivery, due primarily
to elimination by antigen-specific CTLs.sup.23. These findings
underscore the need for improved methods of either prolonging the
activation state and life span of the DCs and/or temporally
coordinating the DC activation "window" with engagement of cognate
T cells within LNs. Thus, enhancing the activation and survival of
DCs may be critical to promoting immunity against tumors.
[0369] DC survival is regulated, at least partly, by
pathogen-derived molecules acting through one or more conserved
Toll-like receptors (TLRs) and T cell-expressed costimulatory
molecules (e.g. CD40L and TRANCE), which are partly dependent on
Bcl-2 and Bcl-x.sub.L for anti-apoptotic activity.sup.3,24-27.
Although the importance of TLR-, CD40-, or Bcl-2-mediated DC
longevity has been well documented, homeostatic feedback mechanisms
are also likely to limit the utility of TLR-ligands or Bcl-2 family
members to extend DC longevity in tumor vaccine protocols. These
include receptor desensitization or downregulation.sup.4,28,29,
expression of negative regulators for TLR/IL-1Rs, like
IRAK-M.sup.30 and SOCS-1.sup.5, and induction of pro-apoptotic
molecules, like Bim.sup.31, resulting in the neutralization of
anti-apoptotic molecules by TLR signals.
[0370] An attractive target for manipulation is the TNF family
receptor, CD40. Unlike pro-inflammatory cytokines or
pathogen-associated molecules that DCs encounter throughout the
periphery, the DC-expressed CD40 receptor is engaged by CD4.sup.+ T
helper cells within the LN paracortex via its cognate ligand,
CD40L.sup.12,13,37. Recent studies have further shown that CD40
stimulation enables DCs to "cross-present" antigen.sup.38 and
overcome peripheral T cell tolerance.sup.39, prompting therapeutic
studies based on CD40 stimulation. Strategies included systemic
delivery of CD40-specific monoclonal antibodies (mAbs) or of
trimerized CD40L.sup.49, the utilization of CD40-stimulated,
antigen-loaded DC-based vaccines.sup.41, and administration of
genetically modified CD40 ligand (CD40L)-expressing DCs.sup.42.
Despite great potential, several properties of CD40 limit its
therapeutic development, including ubiquitous expression of CD40 by
a variety of other cell types, including B cells, macrophages, and
endothelial cells.sup.14, increasing the likelihood for side
effects due to systemic administration of CD40 stimuli. Moreover,
several mechanisms regulate the surface expression of CD40 by
targeting its extracellular domain, including CD40L-induced
cleavage by matrix metalloproteinase enzymes.sup.29, negative
feedback degradation by an alternatively spliced CD40
isoform.sup.28, and CD40L-mediated endocytosis of CD40.
[0371] Therefore, a DC activation system based on the CD40
signaling pathway to extend the pro-stimulatory state of DCs within
lymphoid tissues by providing DC-targeted functionality, temporal
control, and resistance to CD40 regulatory mechanisms has been
developed. This engineered recombinant receptor was comprised of
the cytoplasmic domain of CD40 fused to ligand-binding domains and
a membrane-targeting sequence (FIG. 2). Administration of a
lipid-permeable, dimerizing drug intraperitoneally led to the
potent induction of CD40-dependent signaling cascades and greatly
improved immunogenicity against both defined antigens and tumors in
vivo relative to other activation modalities.sup.4. Hence the
chimeric CD40 was named inducible CD40 (iCD40). The high utility of
iCD40-activated DCs in mice, suggested that methods to stabilize
endogenous CD40 signaling might also enhance the potency of DC
vaccines.
[0372] TLRs bind to a variety of viral and bacterial-derived
molecules, which trigger activation of target cells, such as T
cells, macrophages and dendritic cells. Although the majority of
the 10 or so mammalian TLRs utilize a signaling pathway initiated
by the inducible PRR adapter protein, MyD88, leading to NF-kappaB
activation, TLR3 relies instead on the inducible PRR adapter TRIF,
leading to IRF3 and Type I interferon induction. Together, these
signaling pathways can synergize to produce high levels of the Th1
cytokine, IL-12.sup.43. Interestingly, TLR-4 can utilize both
pathways following binding of the potent mitogen, LPS, or
derivatives. Stimulation through TLR-4 in conjunction with
signaling through the costimulatory molecule CD40 can promote
high-level maturation and migratory properties in human MoDCs.
[0373] Like many cell-surface receptors that make a single pass
through the plasma membrane, TLRs are likely to all be activated by
homo or heterodimerization or oligomerization. There have been
reports of homodimerization-mediated activation of TLR-4 and
heterodimerization-mediated activation of TLR2 with TLR1 and
TLR6.sup.44-48. Moreover, in a recent article, Ian Wilson and
colleagues crystallized TLR-3 and identified dimerization regions
within the extracellular domain, suggesting that it signals as a
homodimer followed dsRNA binding.sup.49. Therefore, it is likely
that chemically induced dimerization of TLRs, especially TLR-4,
will lead to their induction.
[0374] Considerations for the development of ex vivo-matured,
monocyte-derived "enhanced" human DCs. Published.sup.4 and
unpublished studies have suggested two potent methods to enhance DC
function in vivo, ectopic expression of an optimized, constitutive
Akt (Myr.sub.F-.DELTA.Akt) and manipulation of a chimeric inducible
CD40 in vivo. Complementing this work, Si-Yi Chen (Baylor College
of Medicine, Houston, Tex.) has shown that lowering SOCS-1 levels
in DCs can also enhance efficacy.sup.5. While significant
supporting data in mice for iCD40, Myr.sub.F-.DELTA.Akt, and SOCS-1
approaches has been accumulated, human MoDCs are not identical to
murine bone marrow-derived DCs. In particular, the most commonly
used human DC vaccine protocol involves differentiation of MoDCs
from monocytes, prior to treatment with the "gold standard"
pro-inflammatory maturation cocktail, containing TNF-alpha,
IL-1-beta, IL-6, and PGE.sub.2. Although PGE.sub.2 is considered
necessary to upregulate CCR7 and gain chemotactic responsiveness to
lymph node-derived chemokines, CCL19 and CCL21.sup.50,51, PGE.sub.2
can also impair DC signaling by suppressing bioactive IL12p70
production.sup.52. While it is unlikely that IL12 suppression is
permanent in vivo, given the slowly building success rate of DC
vaccines', it will be important to determine prior to clinical
applications which of the methods outlined above can best overcome
PGE.sub.2-mediated IL12 suppression in human MoDCs without
interfering with migratory capacity.
[0375] Although clinical success in DC-based vaccines has been
modest.sup.7,20, extremely low side effects and potentially
exquisite specificity and sensitivity make this modality
attractive. Because interaction with antigen-specific T cells is
likely to be prolonged, these enhanced DCs are likely to improve
the clinical outcome of DC vaccines. The development of enhanced
antigen-expressing DCs not only has potential applicability to
treating malignancy, but also should be applicable to the treatment
of numerous pathogens, as well. Moreover, this high impact approach
should complement prior efforts by numerous labs, which have
identified tumor antigens.
[0376] Characterization of iCD40 functionality in primary DCs and
development of an iCD40-expressing DC-based prostate cancer
vaccine. After demonstrating functionality of iCD40 in murine
D2SC/1 cells (4 and not shown), which possess many characteristics
of freshly isolated DCs, iCD40 functionality in primary bone
marrow-derived DCs (BMDCs) by utilizing an iCD40-expressing
adenovirus was examined. A helper-dependent, .DELTA.E1,
.DELTA.E3-type 5 adenoviral vector, named Ad-iCD40-GFP, was
engineered to express both iCD40 and EGFP under the control of the
CMV early/immediate promoter/enhancer. Ad-iCD40-GFP successfully
transduced and expressed the iCD40 transgene, as well as the EGFP
marker, in purified BMDCs. Titrating Ad-iCD40-GFP while measuring
iCD40-induced upregulation of B7.2 (CD86) showed that maximum
drug-mediated iCD40 activation occurred at around 100 moi and
proceeded asymptotically to plateau at higher viral titers (data
not shown). Although the effects were modest, AP20187 induced the
surface expression of MHC class I K.sup.b, B7.2, as well as
endogenous CD40 on iCD40-expressing BMDCs at 100 moi but not on
non-transduced DCs. The effects of Ad-iCD40-GFP on BMDCs using
intracellular cytokine staining to evaluate DC expression of the
T.sub.H1-polarizing cytokine, IL-12 was then investigated. These
findings confirmed numerous previous reports that an empty
adenoviral vector can contribute to background fluorescence
readings by stimulating the production of low levels of this
cytokine.sup.53. These experiments also revealed that the iCD40
transgene could generate a significant level of basal signaling at
these titers even in the absence of CID. However, AP20187 exposure
of these iCD40-expressing DCs managed to reproducibly overcome
these cumulative effects to further increase the percentage of
IL-12.sup.+DCs. Interestingly, the stimulation of IL-12p70/p40
synthesis with LPS and CD40L peaked at 8 hrs and decreased
thereafter, while the percentage of IL-12' DCs continued to
increase until at least 24 hrs following Ad-iCD40-GFP transduction.
Previous work by Langenkamp et al. has demonstrated that prolonged
treatment of DCs with LPS exhausts their capacity for cytokine
production.sup.54. These results imply that the Ad-iCD40-GFP
vector, as opposed to the LPS danger signal, is capable of
promoting and maintaining a more durable IL-12 response by
BMDCs.
[0377] In addition to DC activation state, DC longevity is another
critical variable that influences the generation of T
cell-dependent immunity. In fact, CTL-mediated killing of DCs is
considered to be a significant mechanism for modulating immune
responses while protecting the host from autoimmune
pathologies.sup.55,56. Other work has established that CD40
stimulation of DCs prolongs their survival by a variety of
mechanisms, including upregulation of the anti-apoptotic protein
bcl-X.sub.L and the granzyme B inhibitor, Spi-6.sup.57,58. The
effects of iCD40 relative to CD40L on DC survival were compared in
an in vitro serum-starvation culture assay. By analyzing the vital
dye (propidium iodide (PM-positive cell population by flow
cytometry, iCD40 expressing-BMDCs were found to exhibit greater
longevity under these conditions relative to non-transduced DCs
treated with CD40L. This effect was iCD40-dependent since
Ad-GFP-transduced DCs failed to reflect improved survival under
these conditions. This work also showed that exposure of iCD40
BMDCs to the AP20187 dimerizer drug even further enhanced this
survival effect relative to untreated BMDCs. Moreover, when
Ad-iCD40 transduced DCs were CFSE-stained and injected into
footpads, significantly increased numbers of DCs were found in
popliteal lymph nodes following i.p. injections of AP20187 versus
in vitro stimulated iCD40 DCs or LPS/CD40L-treated DCs.
[0378] Despite well-known Ad-dependent maturation signals and basal
signaling effects of iCD40 in primary BMDCs, enhanced DC activation
was detected in the presence of AP20187. Overall, this data
suggests that an inducible CD40 receptor designed to respond to a
pharmacological agent is capable of maintaining primary DCs in a
sustained state of activation compared to the more transient
effects of CD40L stimulation and the potentially more complex
effects of anti-CD40 antibodies. This data is consistent with
earlier findings describing only short-term DC modulation for
stimuli that target endogenous CD40.
[0379] The iCD40 Activation Switch Functions as a Potent Adjuvant
for Anti-Tumor DNA Vaccines.
[0380] Previous studies have demonstrated that DCs play a critical
role in the processing and presentation of DNA vaccines to
responding T cells.sup.59. The in vivo anti-tumor efficacy of iCD40
DC-based vaccines as well as the in situ role of iCD40-expressing
DCs in tumor immuno-surveillance was then studied. To establish a
therapeutic tumor model, C57BL/6 mice were inoculated
subcutaneously. with the EG.7-OVA thymoma tumor line and allowed to
progress until tumor volumes reached approximately 0.5 cm.sup.3.
These tumor-bearing mice were vaccinated with either
SIINFEKL-pulsed wt or iCD40 BMDCs. Vaccination with wt BMDCs,
either untreated or stimulated in culture with LPS and CD40L or in
vivo with anti-CD40 mAb, failed to slow the overall tumor growth
rate. However, in vivo drug-mediated iCD40 activation of BMDC
vaccines resulted in sustained decreases in tumor size. In
addition, the response rate to in vivo activated iCD40-expressing
BMDC vaccines was significantly higher than the response rates to
wild type BMDCs under all other vaccination conditions (70% vs.
30%). To confirm the elicitation of tumor antigen-specific T cell
responses in tumor-bearing mice, we performed H-2K.sup.b
OVA.sub.257-264 tetramer analysis was performed on peripheral blood
CD8.sup.+ T cells. This analysis verified the presence of a
expanded population of K.sup.bOVA.sub.257-264-specific CD8.sup.+ T
cells exclusively in mice vaccinated with in vivo activated iCD40
BMDCs.
[0381] Although subcutaneous tumor models provide a convenient tool
for approximating tumor size, their utility is typically limited to
non-orthotopic tumors that are reasonably symmetrical. Also
quantitation of metastasis necessitates euthanasia and is limited
to a single measurement. As an improvement on this mainstay
approach, tumor cells were developed that stably express a
red-shifted luciferase from Caribbean click beetles (Pyrophorus
plagiophthalamus). Imaging in mice following administration of
substrate D-Luciferin, confirms easy detection by either a cooled
CCD camera (IVIS.TM. Imaging System, Xenogen Corp.) or standard
calipers. Furthermore, the red-shifted (.about.613 nM emission)
luciferase reporter should permit more linear quantitation of
surface distant metastasis.
[0382] Development of CID-inducible TLRs (iTLRs): There are several
subgroups of TLRs based on sublocalization and signaling pathways
utilized. Regardless of the normal subcellular localization of the
ligand-binding extracellular domains, the signaling domains are
cytoplasmic and should signal properly in all cases if
homodimerization is the normal signaling mechanism. Analogous to
iCD40, the TLR cytoplasmic signaling domains were PCR-amplified
with flanking XhoI and SalI restriction sites for subcloning on the
5' or 3' side of two chemical inducers of dimerization (CID)
binding domains (CBD), FKBP12.sub.V36.sup.1. The chimeric CBD-TLRs
were localized to the plasma membrane using
myristoylation-targeting motifs (FIG. 3).
[0383] Initial testing of TLRs involved co-transfection of
expression vectors into Jurkat-TAg or 293 cells along with an
NF-kappaB-responsive SEAP (secreted alkaline phosphatase) reporter
plasmid.sup.66. Interestingly, the preliminary data suggested that
only iTLR7 and iTLR8 functioned in Jurkat-TAg cells, but not iTLR3,
4, and 9, regardless of the relative position of the CBDs and TLRs.
Additional transfections in a panel of cells will be required to
determine whether this reflects physiological tissue-specific
signaling differences or other idiosyncrasies of these chimeric
constructs.
[0384] Those of ordinary skill in the art will recognize the
modifications that may be made for the use of adapter proteins
rather than TLRs in the following methods.
[0385] Developing inducible TLRs: Inducible chimeric TLRs may be
developed to circumvent the requirement for pathogen-derived (or
synthetic) adjuvant in DC activation. Initially, chimeric iTLR 3,
4, 7, 8, and 9, were developed by cloning the cytoplasmic signaling
domains of TLRs 5' (upstream) or 3' (downstream) of CID-binding
domains (FIG. 3). Initial screening in Jurkat-TAG cells revealed
that iTLR8 (and to a lesser extent iTLR7) triggered the largest
induction of NF-kappaB. However, the relative strength of various
TLRs may be a tissue-specific parameter. To address this. these
constructs may be tested in the DC cell line, D2SC/1 initially with
regard to NF-kappaB activation using an NF-kappaB SEAP reporter
system based on transient transfection of multiple expression
plasmids into target cells. 2DSC/1 cells represent a rare subset of
immortalized DC lines that retain both the immature DC phenotype
and the ability to mature following activation signals.sup.67.
Since NF-kappaB induction is not the only function of TLRs, IRF3/7
induction may also be screened using an interferon (IFN)-stimulated
response element (ISRE)-SEAP reporter plasmid that binds IRFs and
induces reporter activity. To develop ISRE-SEAP, the
ISRE-containing promoter from ISRE-luc (Stratagene) may replace the
SRalpha promoter in the constitutive reporter plasmid pSH1/kSEAP.
As a secondary induction of TLR signaling, JNK and p38
phosphorylation are monitored by Western blotting using
phosphorylation-specific antibodies.
[0386] Since various distinct TLRs can differentially induce IRF
and NF-kappaB and may synergize in DC activation and IL-12
production.sup.43, initial testing of inducible TLRs, may be
followed by combinatorial testing by cotransfection of iTLRs,
two-at-a-time. Although both normal homodimerization and more
unpredictable heterodimerization may occur, this approach should
reveal synergism between different classes of TLRs. Activation of
synergistic TLR pairs should confer enhanced immunostimulatory
capacities to DCs. If synergism can be detected, a new series of
constructs that are comprised of two tandem distinct (or identical)
TLRs, called inducible composite TLRs (icTLRs) are tested (FIG. 4).
In this case cytoplasmic XhoI-SalI-flanked TLR signaling domains
from above are combined in various arrangements upstream and
downstream of CBDs. Finally, the two most potent constructs are
modified to contain the cytoplasmic domain of CD40, previously
demonstrated to be activated by CID (FIG. 5).
[0387] Although transfection of DCs can be problematic, an improved
method of electroporation was recently described by Vieweg and
colleagues.sup.68. In their approach, survival following
electroporation (300 V, 150 mF (Gene Pulser II: Bio-Rad)) is
enhanced by resuspending DCs (4.times.10.sup.7/ml) in high
potassium ion ViaSpan buffer (Barr Laboratories). Additionally, if
the transfection efficiency is still too low, expression vector
pRSV-TAg, containing SV40 large T antigen for amplifying our pSH1
series expression vectors, which all contain the SV40 origin of
replication will be cotransfected.
[0388] Developing an adenovector expressing unified activation gene
icTLR/CD40: Although D2SC/1 is a useful cell model for preclinical
studies, the immunoregulatory genes will next be assayed in primary
mouse and human DCs prior to clinical applications. To facilitate
efficient gene transfer to primary cells, the most potent
construct(s) is subcloned into adenovirus shuttle vector,
pShuttle-X or pDNR-CMV and further transferred into Ad5 vector,
pAdeno-X (BD) or AdXLP (BD), respectively. Preparation of
high-titer virus is carried out. As has been achieved with
previously developed Ad5/f35-iCD40, this vector is tested in both
human and mouse DCs. Although Ad5/f35 pseudotyped adenovectors
improve transduction efficiency a bit in human DCs, "pure" Ad5
enveloped adenovectors may be used to permit additional
transduction of murine DCs.
[0389] For human studies, MoDCs are prepared by standard incubation
of adherent peripheral blood DC precursors in GM-CSF and IL-4.
Immature DCs are transduced with the developed icTLR/CD40 vector
and control vectors (e.g. Ad5/f35-iCD40 and Ad5/f35-EGFP). Standard
MoDC assays for maturation and activity are described herein and
also include, for example, flow cytometry analysis of maturation
markers (e.g. CD40, CD80, CD86, HLA class I and II, CCR7), IL-12
production, migration, and activation of antigen-specific T
cells.
[0390] In the event that placing CD40 and TLR signaling domains in
tandem may interfere with the signaling pathways activated by
isolated domains, the constructs may be coexpressed in viral
vectors using alternative strategies, such as use of bicistronic
expression cassettes or cloning into the E3 region of
deltaE1deltaE3 adenovectors. Also, chimeric receptors may not
signal identical to the endogenous proteins. Although certain PRRs
or PRR adapters may be thought to be the most potent TLR for
activation of myeloid DCs, alternatives may function better when
converted to a CID-activated receptor. Moreover, synergism between
various inducible PRRs and PRR adapters and constitutive Akt,
M.sub.F-deltaAkt, or siRNA SOCS-1 may be found to be more potent.
In these cases, various combinations of immune regulatory genes may
be combined in multicistronic adenovectors.
[0391] Additional Methods of Assaying CID-Inducible Adapters
[0392] Those of ordinary skill in the art will recognize the
modifications that may be made for the use of adapter proteins
rather than TLRs in the following methods.
[0393] Due to the pivotal role that DCs play in regulating adaptive
immunity, there are many homeostatic mechanisms that downregulate
DC activity. Nevertheless, heightened activation may be required
for overcoming tumor- or viral-derived tolerogenic mechanisms.
Several methods to circumvent these homeostatic mechanisms are
discussed herein. Inducible CD40 can be activated in vivo within
the context of an immunological synapse and lacks its extracellular
domain, bypassing several negative feedback mechanisms that target
this domain. "Optimized", constitutively active Akt,
M.sub.F-deltaAkt, is based on lipid-raft targeting of a truncated
Akt1 allele. Reducing the inhibitor SOCS-1 with siRNA technology
increases toll-receptor signaling and Type I interferon production.
Thus, all three methods have the capacity to enhance MoDCs.
[0394] Preparation of MoDCs: for Most Experiments Based on
Optimization of Enhanced Dcs (eDCs), monocyte-derived DCs are
differentiated and enriched from peripheral blood mononuclear cells
obtained from the Blood Bank or healthy volunteers. Briefly, DC
precursors are isolated by buoyant density techniques (Histopaque:
Sigma-Aldrich) and then adherent (and semi-adherent) cells are
cultured for 5 days in serum free X-VIVO 15 DC medium (Cambrex Bio
Science) in the presence of cytokines GM-CSF (800 U/ml) and IL-4
(500 U/ml) (R&D Systems, Minneapolis, Minn.). Following 5 days
in culture, immature DCs are incubated for an additional 24 hours
in the presence of adenovectors expressing iCD40 (i.e.
Ad5/f35-iCD40), constitutive Akt (Ad5/f35-MF-deltaAkt), shRNA SOCS1
(Ad5-shSOCS1), or Ad5-iTLR/CD40 at 10,000 viral particles (vp) per
cell. (Note: Ad5 vectors may be added at 20,000 vp to compensate
partly for somewhat reduced transduction efficiency). In a subset
of samples, additional TLR4 ligand monophosphoryl lipid A (MPL; 1
mg/ml) or dimerizer AP20817 (100 nM; iCD40-DCs only) will be added
for complete maturation.
[0395] Determination of maturation state of MoDCs: A number of
surface proteins ("markers") are induced during MoDC activation,
including CD25, CD40, CD80, CD83, CD86, HLA class I and class II,
CCR7 and others. Preliminary studies demonstrated that iCD40
signaling alone is sufficient to upregulate CD83 and CCR7 on MoDCs
(not shown). Additional TLR4 signaling (via MPL) leads to additive
(or synergistic) activation of all maturation markers. Therefore,
at a fixed vp number, induction of maturation markers (determined
by flow cytometry) by all four viral vectors either alone or in
combination with MPL is evaluated. Maturation by the previous "gold
standard" maturation cocktail (MC), comprised of 1L-1a, IL-6,
TNFalpha, and PGE.sub.2, acts as positive control and non-treated
(mock) immature DCs serve as negative controls in these and the
following experiments. In addition to phenotypic analysis of cell
surface markers, production of IL-12 and other T.sub.H1-polarizing
cytokines (e.g. IL-23, TNFalpha), are also important for optimal
anti-tumor immunity. While iCD40 is not sufficient for IL-12
production, combinations of MPL and iCD40 lead to potent
synergistic production of IL-12. Therefore, DC culture
supernatants, stimulated as above, are harvested 24 and 48 hours
after transduction and maturation. IL-12 p70 levels, IL-12/IL-23
p40 dimers and TNFalpha concentrations are determined by
colorimetric sandwich ELISA assays (BD Biosciences). Alternatively,
multiplex beads developed by BD to simultaneously assay multiple
additional cytokines (e.g. IL-1, IL-6, IFNalpha, etc.) may be
used.
[0396] Determination of migration capacity: Unlike murine bone
marrow-derived DCs (BMDCs) that are competent for LN migration,
immature MoDCs are deficient in this crucial function. While
PGE.sub.2 is typically used to upregulate CCR7 and migratory
capacity, the utility of PGE.sub.2 is tempered by potential
deleterious effects, which include down regulation of CD40
signaling and IL-12 production and upregulation of
IL-10.sup.50,52,69. Moreover, even in the presence of PGE.sub.2,
migration to LNs is modest and around 1-2% of injected
cells.sup.70. Although CCR7 expression is likely a prerequisite for
migration to lymph nodes, chemotactic responsiveness to the
LN-derived CCR7 chemokines, CCL19 and CCL21, is a more direct
measure of likely migration to lymph nodes. Therefore, migration to
CCL19/MIP3b may be compared in a modified 2-chamber assay.
[0397] Preliminary experiments demonstrate the surprising result
that iCD40 signaling is sufficient for migratory capacity even in
the absence of PGE.sub.2. In this assay MoDCs were transduced with
Ad5/f35-ihCD40 and labeled with fluorescent dye, Green-CMFDA
(Molecular Probes). Cells were placed in the top chamber of a
2-chamber 8-mm assay plate and total fluorescence in the bottom
chamber was quantitated and compared with PGE.sub.2-mediated
stimulation. Similarly, the migratory capacity in vitro of
iCD40-TLR-, iCD40-, and Akt-MoDCs, and SOCS1-deficient MoDCs
individually and in combination with and without TLR4 ligands may
be compared.
[0398] As a second more direct assay for migration capacity,
migration in vivo may be compared by injecting eDCs into the lower
leg of non-myeloablatively irradiated immunodeficient SCID mice.
Minimal radiation (.about.250 Rad) is needed to suppress natural
killer (NK) cell activity against xenogeneic cells. Despite species
differences, human MoDCs can respond to murine chemokines and
migrate to draining LNs.sup.71. To visualize successfully migrated
MoDCs, cells are labeled with the fluorescent dye, Green-CMFDA cell
tracker, which is quantitated by flow cytometry. Second, in
addition to adenovector-mediated "enhancement", MoDCs are
transduced with adenovector, Ad5/f35-CBR, expressing red-shifted
(510 nm excitation peak) click beetle (Pyrophorus plagiophthalamus)
luciferase (Promega). Use of the CBR luciferase allele should more
easily allow detection of bioluminescent DCs (using our IVIS.RTM.
Imaging System (Xenogen Corp, Alameda, Calif.)) both within the
draining popliteal LN and at more distant and membrane-distal
sites.
[0399] Activation and polarization of autologous T cells: In
addition to maturation and migration, ability to activate a
T.sub.H1-biased antigen-specific immune response in vivo is the
sine qua non of DC vaccination against solid tumors. Therefore, the
ability of eDCs to stimulate both T helper and cytotoxic function
may be evaluated. Initially, stimulation of proliferation of
allogeneic CD4.sup.+ T cells may be assayed. Enhanced DCs are
matured and activated using the conditions described above,
irradiated (3000 rad) and cultured 1:10 with allogeneic magnetic
bead-purified (Miltenyi Biotec, Auburn, Calif.) CD4.sup.+ T cells.
Proliferation is assessed 4 days later after 16-hour incubation
with [.sup.3H]-thymidine. To complement these studies, the T.sub.H1
polarization (determined by ELISpot assays to IL-4 and IFNgamma)
ability by various standard or eDCs may be determined To more
specifically assay DC maturation state, ability to stimulate naive
CTL function is determined using HLA-A2-restricted tetramer
analysis and CTL assays. (Note: several HLA-A2 carriers have
recently been genotyped). Activation of autologous T cells in
healthy donors by various eDCs presenting 2 distinct cocktails of
HLA-A2-restricted antigens, one strong and one weak, is compared.
CTL assays will be based on antigen-specific lytic activity of T
cells stimulated with standard or eDCs as above. These 4 T cell
assays should provide a balanced preclinical analysis of enhanced
DCs along with a functional analysis of the various approaches.
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Ronchese, F. CD8 T cell-dependent elimination of dendritic cells in
vivo limits the induction of antitumor immunity. Journal of
Immunology 164, 3095-3101 (2000). [0456] 56. Wong, P. a. P., E.
Feedback Regulation of Pathogen-Specific T Cell Priming. Immunity
188, 499-511 (2003). [0457] 57. Miga, A., Masters, S., Durell, B.,
Gonzalez, M., Jenkins, M., Maliszewski, C., Kikutani, H., Wade, W.,
and Noelle, R. Dendritic cell longevity and T cell persistence is
controlled by CD154-CD40 interactions. European Journal of
Immunology 31, 959-965 (2001). [0458] 58. Medema, J., Schuurhuis,
D., Rea, D., van Tongeren, J., de Jong, J., Bres, S., Laban, S.,
Toes, R., Toebes, M., Schumacher, T., Bladergroen, B., Ossendorp,
F., Kummer, J., Melief, C., and Offring a, R. Expression of the
serpin serine protease inhibitor 6 protects dendritic cells from
cytotoxic T lymphocyte-induced apoptosis: differential modulation
by T helper type 1 and type 2 cells. Journal of Experimental
Medicine 194, 657-667 (2001). [0459] 59. Steinman, R. a. P., M.
Exploiting dendritic cells to improve vaccine efficacy. Journal of
Clinical Investigation 109, 1519-1526 (2002). [0460] 60. Woltman,
A. M. et al. Rapamycin specifically interferes with GM-CSF
signaling in human dendritic cells, leading to apoptosis via
increased p27KIP1 expression. Blood 101, 1439-45 (2003). [0461] 61.
Granucci, F. et al. Inducible IL-2 production by dendritic cells
revealed by global gene expression analysis. Nat Immunol 2, 882-8
(2001). [0462] 62. Fujio, Y. & Walsh, K. Akt mediates
cytoprotection of endothelial cells by vascular endothelial growth
factor in an anchorage-dependent manner. J Biol Chem 274, 16349-54
(1999). [0463] 63. Mukherjee, A., Arnaud, L. & Cooper, J. A.
Lipid-dependent recruitment of neuronal Src to lipid rafts in the
brain. J Biol Chem 278, 40806-14 (2003). [0464] 64. Li, B., Desai,
S. A., MacCorkle-Chosnek, R. A., Fan, L. & Spencer, D. M. A
novel conditional Akt `survival switch` reversibly protects cells
from apoptosis. Gene Ther 9, 233-44. (2002). [0465] 65. Sporri, R.
& Reis e Sousa, C. Inflammatory mediators are insufficient for
full dendritic cell activation and promote expansion of CD4+ T cell
populations lacking helper function. Nat Immunol 6, 163-70 (2005).
[0466] 66. Spencer, D. M., Wandless, T. J., Schreiber, S. L. &
Crabtree, G. R. Controlling signal transduction with synthetic
ligands. Science 262, 1019-1024 (1993). [0467] 67. Granucci, F. et
al. Modulation of cytokine expression in mouse dendritic cell
clones. Eur J Immunol 24, 2522-6 (1994). [0468] 68. Su, Z. et al.
Telomerase mRNA-transfected dendritic cells stimulate
antigen-specific CD8+ and CD4+ T cell responses in patients with
metastatic prostate cancer. J Immunol 174, 3798-807 (2005). [0469]
69. Scandella, E., Men, Y., Gillessen, S., Forster, R. &
Groettrup, M. Prostaglandin E2 is a key factor for CCR7 surface
expression and migration of monocyte-derived dendritic cells. Blood
100, 1354-61 (2002). [0470] 70. Morse, M. A. et al. Migration of
human dendritic cells after injection in patients with metastatic
malignancies. Cancer Res 59, 56-8 (1999). [0471] 71. Hammad, H. et
al. Monocyte-derived dendritic cells induce a house dust
mite-specific Th2 allergic inflammation in the lung of humanized
SCID mice: involvement of CCR7. J Immunol 169, 1524-34 (2002).
Example 9
Expression Constructs and Testing
[0472] TLRs 3, 4, 7, 8 and 9 were initially selected to construct
inducible chimeric proteins as they represent TLRs from the
different subfamilies that are know to trigger the Th1 cytokine,
IL-12, in monocyte-derived DCs. Further, TLR4 has been shown to
trigger signaling following cross linking of chimeric TLR4 alleles
via heterologous extracellular domains. The cytoplasmic domains of
each (including TIRs) were PCR-amplified and placed adjacent (5'
and 3') to two (2) FKBP12(V36) (F.sub.v and F.sub.v' (wobbled))
genes, which were attached to the plasma membrane using a
myristoylation-targeting sequence from c-Src. Chimeric proteins
having a third FKBP gene have been developed to improve
oligomerization.
[0473] Additionally, chimeric versions of inducible PRR adapters
MyD88 and TRIF have been generated by fusing these cytoplasmic
proteins to two (2) FKBPs. Finally, the tandem CARD domains from
cytoplasmic PRRs, NOD2 and RIG-I, have been fused to tandem FKBPs.
These constructs and reporter assays are described below.
[0474] Constructs:
[0475] (i) Inducible iTLRs: TLR3, 4, 7, 8 and 9 were PCR-amplified
from cDNA derived from MoDCs. PCR primers were flanked by XhoI and
SalI restriction sites to permit cloning 5' and 3' of tandem FKBPs
in the XhoI and SalI sites, respectively, of
pSH1/M-F.sub.v'-F.sub.vls-E.sup.1, 2. The primers used were (a)
5TLR3cX (5'-cgatcactcgagggctggaggatatctttttattgg-3') and 3TLR3cS
(5'-tgatcggtcgacatgtacagagtttttggatccaagtg-3') to give
pSH1/M-TLR3-F.sub.v'-F.sub.vls-E and
pSH1/M-F.sub.v'-F.sub.vls-TLR3-E; (b) 5TLR4cX
(5'-cgatcactcgagtataagttctattttcacctgatgcttc-3') and 3TLR4cS
(5'-tgatcggtcgacgatagatgttgcttcctgccaattg-3') to give
pSH1/M-TLR4-F.sub.v-F.sub.vls-E and
pSH1/M-F.sub.v-F.sub.vls-TLR4-E; (c) 5TLR7cS
(5'-cgatcagtcgacgatgtgtggtatatttaccatttctg-3') and 3TLR7cS
(5'-tgatcggtcgacgaccgtttccttgaacacctgac-3') to give
pSH1/M-TLR7-F.sub.v'-F.sub.vls-E and
pSH1/M-F.sub.v-F.sub.xls-TLR7-E; (d) 5TLR8cX
(5'-cgatcactcgaggatgtttggtttatatataatgtgtg-3') and 3TLR8cS
(5'-tcggtcgacgtattgcttaatggaatcgacatac-3') to give
pSH1/M-TLR8-F.sub.v-F.sub.vls-E and
pSH1/M-F.sub.v-F.sub.vls-TLR8-E; (e) 5TLR9cX
(5'-cgatcactcgaggacctctggtactgcttccacc-3') and 3TLR9cS
(5'-tgatctgtcgacttcggccgtgggtccctggc-3') to give
pSH1/M-TLR9-F.sub.v-F.sub.vls-E and
pSH1/M-F.sub.v-F.sub.vls-TLR9-E. All inserts were confirmed by
sequencing and for appropriate size by Westernblot to the 3'
hemagluttinin (HA) epitope (E). M, myristoylation-targeting
sequence from c-Src (residues 1-14). pSH1, expression vector.
Additionally, a third XhoI/SalI-linkered F.sub.v' domain was added
to the XhoI sites of pSH1/M-F.sub.v'-F.sub.vls-TLR4-E and
pSH1/M-F.sub.v'-F.sub.vls-TLR8-E to get
pSH1/M-F.sub.v'2-F.sub.vls-TLR4-E and
pSH1/M-F.sub.v'2-F.sub.vls-TLR8-E, respectively, to improve
oligomerization.
[0476] To faithfully reflect physiological TLR4 signaling,
full-length 2.5-kb TLR4 was PCR-amplified from TLR4 cDNA (from the
Medzhitov lab) using SacII and XhoI-linkered primers 5hTLR4
(5'-aatctaccgcggccaccatgatgtctgcctcgcgcctg-3') and 3hTLR4
(5'-tcagttctcgaggatagatgttgcttcctgccaattg-3'), respectively. The
2546-bp PCR product was subcloned into pCR-Blunt-TOPO and
sequenced. The sequence-verified insert was SacII/XhoI-digested and
subcloned into SacII/XhoI digested (and "CIPped")
pSH1/M-F.sub.v'-F.sub.vls-E to give
pSH1/hTLR4-F.sub.v'-F.sub.vls-E. An additional F.sub.v' was added
to XhoI site to give pSH1/hTLR4-F.sub.v'2-F.sub.vls-E.
[0477] (ii) Inducible composite iTLR4-CD40: The 191-bp
XhoI-SalI-linkered human CD40 cytoplasmic domain was PCR-amplified
with primers hCD405X (5'-atatactcgagaaaaaggtggccaagaagccaacc-3')
and hCD403Sns (5'-acatagtcgacctgtctctcctgcactgagatg-3') and
subcloned into the SalI site of pSH1/hTLR4-F.sub.v'-F.sub.vls-E and
pSH1/hTLR4-F.sub.v'2-F.sub.vls-E to get
pSH1/hTLR4-F.sub.v'-F.sub.vls-CD40-E and
pSH1/hTLR4-F.sub.v'2-F.sub.vls-CD40-E.
[0478] (iii) Inducible iNOD2: The .about.800-bp amino terminus of
the PRR NOD2 (containing tandem CARD domains) was PCR-amplified
with XhoI/Sa/1-linkered primers 5NOD2X
(5'-atagcactcgagatgggggaagagggtggttcag-3') and 3N0D2Sb
(5'-cttcatgtcgacgacctccaggacattctctgtg-3') and subcloned into the
XhoI and SalI sites of pSH1/M-F.sub.v'-F.sub.vls-E to give
pSH1/M-NOD2-F.sub.v'-F.sub.vls-E and
pSH1/M-F.sub.v-F.sub.vls-NOD2-E=Fv' NOD2.
[0479] (iv) Inducible iRIG-I: The .about.650 by amino terminus of
the RNA helicase RIG-I (containing tandem CARD domains) was
PCR-amplified with XhoI/SalI-linkered primers 5RIGX
(5'-atagcactcgagaccaccgagcagcgacgcag-3') and 3RIGS
(5'-cttcatgtcgacaatctgtatgtcagaagtttccatc-3') and subcloned into
the XhoI and SalI sites of pSH1/M-F.sub.v'-F.sub.vls-E to give
pSH1/M-RIGI-F.sub.v'-F.sub.vls-E and
pSH1/M-F.sub.v'-F.sub.vls-RIGI-E=Fv'RIG-I.
[0480] (v) Inducible iMyD88: Human TIR-containing inducible PRR
adapter MyD88 (-900-bp) was PCR-amplified from 293 cDNA using
XhoI/Sa/1-linkered primers 5MyD88S
(5'-acatcaactcgagatggctgcaggaggtcccgg-3') and 3MyD88S
(5'-actcatagtcgaccagggacaaggccttggcaag-3') and subcloned into the
XhoI and SalI sites of pSH1/M-F.sub.v'-F.sub.vls-E to give
pSH1/M-MyD88-F.sub.v'-F.sub.vls-E and
pSH1/M-F.sub.v'-F.sub.vls-MyD88-E, respectively.
[0481] (vi) Inducible iTRIF: Human TIR-containing inducible PRR
adapter TRIF2 (.about.2150-bp) was PCR-amplified from 293 cDNA
using XhoI/Sa/1-linkered primers 5TRIFX
(5'-acatcaactcgagatggcctgcacaggcccatcac-3') and 3TRIFS
(5'-actcatagtcgacttctgcctcctgcgtcttgtcc-3') and subcloned into
SalI-digested pSH1/M-F.sub.v'-F.sub.vls-E to give
pSH1/M-F.sub.v-F.sub.vls-TRIF-E.
[0482] (vii) IFNb-SEAP: The minimal IFNbi promoter was
PCR-amplified from human genomic DNA using primers 51FNbMI
(5'-aactagacgcgtactactaaaatgtaaatgacataggaaaac-3') and 3IFNbH
(5'-gacttgaagcttaacacgaacagtgtcgcctactac-3'). The
MluI-HindIII-digested fragment was subcloned into a promoter-less
SEAP reporter plasmid.
[0483] Certain constructs were specifically targeted to plasma
membrane lipid rafts using myristoylation sequences from Fyn as
well as the PIP2 membrane targeting domain of TIRAP.(5)
[0484] Secreted alkaline phosphatase (SEAP) assays: Reporters
assays were conducted in human Jurkat-TAg (T cells) or 293 (kidney
embryonic epithelial) cells or murine RAW264.7 (macrophage) cells.
Jurkat-TAg cells (10.sup.7) in log-phase growth were electroporated
(950 mF, 250 V) with 2 mg expression plasmid and 2 mg of reporter
plasmid NF-kB-SEAP.sup.3 or IFNb-TA-SEAP (see above). 293 or
RAW264.7 cells (.about.2.times.10.sup.5 cells per 35-mm dish) in
log phase were transfected with 6 ml of FuGENE-6 in growth media.
After 24 hr, transformed cells were stimulated with CID. After an
additional 20 h, supernatants were assayed for SEAP activity as
described previously.sup.3.
[0485] Tissue culture: Jurkat-TAg and RAW264.7 cells were grown in
RPMI 1640 medium, 10% fetal bovine serum (FBS), 10 mM HEPES (pH
7.14), penicillin (100 U/ml) and streptomycin (100 mg/ml). 293
cells were grown in Dulbecco's modified Eagle's medium, 10% FBS,
and pen-strep.
[0486] Western blot analysis: Protein expression was determined by
Westernblot using antibodies to the common hemagluttinin (HA)
epitope (E) tag.
[0487] Results
[0488] Chimeric iTLR4 with the PIP2 membrane targeting motif is
activated 2 fold. The construct encoded two ligand-binding domains.
However, the rest of the iTLRs are not induced at robust levels by
CID in 293, RAW or D2SC1 cells, as observed in reporter assays.
This might be attributed to the varied membrane targeting
requirements of the iTLRs. Therefore, we developed inducible Nod2
and RIG-1, which are cytoplasmic PRRs that do not need targeting to
the plasma membrane. While iNod2 was activated 2 fold by the
dimerizer drug in 293 cells, no such effect is observed in RAW
264.7 cells. With the addition of increasing concentrations of CID,
iNod2 activity decreases in RAW cells. Also the effect of iNod2 and
iCD40 together, on NF-kappaB activation, is additive in 293 cells
(FIG. 7). iRIG-1 is activated by 2.5 fold (FIG. 8). Inducible
versions of the full length inducible PRR adapter molecules MyD88
and TRIF that are the primary mediators of signaling downstream of
TLRs are in the screening process.
[0489] Citations referred to in this Example and Providing
Additional Technical Support [0490] 1. Xie, X. et al.
Adenovirus-mediated tissue-targeted expression of a caspase-9-based
artificial death switch for the treatment of prostate cancer.
Cancer Res 61, 6795-804. (2001). [0491] 2. Fan, L., Freeman, K. W.,
Khan, T., Pham, E. & Spencer, D. M. in Human Gene Therapy
2273-2285 (1999). [0492] 3. Spencer, D. M., Wandless, T. J.,
Schreiber, S. L. & Crabtree, G. R. Controlling signal
transduction with synthetic ligands. Science 262, 1019-1024 (1993).
[0493] 4. Thompson, B. S., P. M. Chilton, J. R. Ward, J. T. Evans,
And T. C. Mitchell. 2005. The Low-Toxicity Versions Of Lps, Mpl
Adjuvant And Rc529, Are Efficient Adjuvants For Cd4+ T Cells. J
Leukoc Biol 78:1273-1280. [0494] 5. Salkowski, C. A., G. R. Detore,
And S, N. Vogel. 1997. Lipopolysaccharide And Monophosphoryl Lipid
A Differentially Regulate Interleukin-12, Gamma Interferon, And
Interleukin-10 Mrna Production In Murine Macrophages. Infect Immun
65:3239-3247. [0495] 6. Beutler, B. 2004. Inferences, Questions And
Possibilities In Toll-Like Receptor Signalling. Nature 430:257-263.
[0496] 7. Werts, C., S. E. Girardin, And D. J. Philpott. 2006. Tir,
Card And Pyrin: Three Domains For An Antimicrobial Triad. Cell
Death Differ 13:798-815. [0497] 8. Kagan, J. C., And R. Medzhitov.
2006. Phosphoinositide-Mediated Adapter Recruitment Controls
Toll-Like Receptor Signaling. Cell 125:943-955.
Example 10
Drug-Dependent Induction of NF-Kappa B Activity in Cells
Transfected with iRIG-1, iCD40, and iNOD2
[0498] 293 cells were transfected with 1 microgram NF-KappaB-SEAP
reporter construct+1 microgram inducible PRR construct using Fugene
6 transfection reagent. The transfections were performed in a
6-well plate at 1*10.sup.6 cells/well or transfection.
[0499] Jurkat TAg cells were transfected with 2 micrograms NF-kappa
B-SEAP reporter construct and 3 micrograms inducible PRR construct
using electroporation at 950 microF and 0.25 kV. The cells were
transfected at 10*10.sup.6 cells/transfection.
[0500] 24 hours later, the cells were plated in a 96-well plate
with 2 different concentrations of AP20187 (100 nM and 1000 nM).
After a further 24 hour incubation at 37.degree. C., 5% CO.sub.2,
supernatants were collected and analyzed for SEAP activity by
incubation with SEAP substrate, 4-methylumbilliferyl phosphate
(MUP). Fluorescence was determined at excitation 355 nm and
emission 460 nm using a FLUOstar Optima plate reader (BMG
Labtech).
[0501] For iNOD2 and combination experiments, transfections were
normalized for total DNA using an "empty" expression vector,
pSH1/S--Fv'-Fvls-E.
[0502] FIGS. 11-14 are graphs that show drug-dependent induction of
NF-kappaB activity and SEAP reporter counts. Each graph is
representative of a separate individual experiment.
[0503] For purposes of clarity in the graphs, some of the vectors
were renamed for the figures.
Fv'RIG-I=pSH1-Fv'Fvls-RIG-I=pSH1/M-Fv'-Fvls-RIG-I
Fv'NOD2=pSH1-Fv'Fvls-NOD2=pSH1/M-Fv'-Fvls-NOD2-E
Fv'2NOD2=pSH1-Fv'2Fvls-NOD2 Fv'NOD2+=pSH1-Fv'Fvls-NOD2 (same as
Nunez NOD2 sequence) Fv'CD40=pSH1-Fv'Fvls-CD40
Example 11
Inducible MyD88 and Composite MyD88-CD40 Activate NF-KappaB in 293
Cells
[0504] A set of constructs was designed to express inducible
receptors, including a truncated version of MyD88, lacking the TIR
domain. 293 cells were cotransfected with a NF-kappaB reporter and
the SEAP reporter assay was performed essentially as described in
Spencer, D. M., Wandless, T. J., Schreiber, S. L. & Crabtree,
G. R. Controlling signal transduction with synthetic ligands.
Science 262, 1019-1024 (1993). The vector originally designed was
pBJ5-M-MyD88L-Fv'Fvls-E. pShuttleX-M-MyD88L-Fv'Fvls was used to
make the adenovirus. Both of these vectors were tested in SEAP
assays. After 24 hours, AP20187 was added, and after 20 additional
hours, the cell supernatant was tested for SEAP activity. Graphics
relating to these chimeric constructs and activation are provided
in FIGS. 16 and 17. The results are shown in FIG. 18.
Constructs:
[0505] Control: Transfected with NF-kappaB reporter only.
[0506] TLR4 on: pShuttleX-CD4/TLR4-L3-E: CD4/TLR4L3-E is a
constitutive version of TLR4 that contains the extracellular domain
of mouse CD4 in tandem with the transmembrane and cytoplasmic
domains of human TLR4 (as described in Medzhitov R,
Preston-Hurlburt P, Janeway C A Jr, A human homologue of the
Drosophila Toll protein signals activation of adaptive immunity.
Nature. 1997 Jul. 24; 388(6640):394-7.) followed by three 6-amino
acid linkers and an HA epitope.
iMyD88: contains M-MyD88L-Fv'Fvls-E iCD40: contains
M-Fv'-Fvls-CD40-E iCD40T: contains M-Fv'-Fv'-Fvls-CD40-E-iCD40T
contains an extra Fv' (FKBP with wobble at the valine) iMyD88:CD40:
contains M-MyD88L-CD40-Fv'Fvls-E iMyD88:CD40T: contains
M-MyD88LCD40-Fv'Fv'Fvls-E- contains an extra Fv' compared to
iMyD88:CD40.
Example 12
Inducible CD40-MyD88, CD40-RIG-1, and CD40:NOD2
[0507] The following constructs were designed and assayed in the
NF-kappaB reporter system. 293 cells were cotransfected with a
NFkappaB reporter and one of the constructs. After 24 hours,
AP20187 was added, and after an additional 3 hours (FIG. 19) or 22
hours (FIG. 20), the cell supernatant was tested for SEAP activity.
About 20-24 hours after transfection, the cells were treated with
dimer drug AP20187. About 20-24 hours following treatment with
dimer drug, cells were treated with SEAP substrate
4-methylumbelliferyl phosphate (MUP). Following an overnight
incubation (anywhere from 16-22 hrs), the SEAP counts were recorded
on a FLUOStar OPTIMA machine.
MyD88LFv'FvlsCD40: was made in pBJ5 backbone with the
myristoylation sequence upstream from MyD88L Fv'FvlsCD40MyD88L: was
made in pBJ5 backbone with the myristoylation sequence upstream
from Fv'. MyD88LCD40Fv'Fvls: was made in 2 vector backbone (pBJ5)
with the myristoylation sequence upstream from the MyD88L.
CD40Fv'Fv'MyD88L: was made in pBJ5 backbone with the myristoylation
sequence upstream from CD40. Fv'2FvlsCD40stMyD88L: is a construct
wherein a stop sequence after CD40 prevented MyD88L from being
translated. Also named iCD40T'. Fv'2Fvls includes 2 copies of Fv',
separated by a gtcgag sequence.
MyD88LFv'Fvls
[0508] Fv'FvlsMyD88L: was made in pBJ5 backbone with the
myristoylation sequence upstream from the Fv'. Fv'FvlsCD40: is
available in pBJ5 and pShuttleX CD40Fv'Fvls: is available in pBJ5
backbone with the myristoylation sequence upstream from the CD40.
MFv''Fvls: is available in pBJ5 backbone with the myristoylation
sequence indicated by the M. Fv''FvlsNOD2: pBJ5-Sn-Fv'Fvls-NOD2-E
in pBJ5 backbone with no myristoylation sequence, contains 2 FKBPs
followed by 2 CARD domains of NOD2 and the HA epitope.
Fv'FvlsRIG-1: pBJ5-Sn-Fv'Fvls-RIG-1-E in pBJ5 backbone with no
myristoylation sequence, contains 2 FKBPs followed by 2 CARD
domains of RIG-I and the HA epitope.
[0509] Examples of construct maps for pShuttleX versions used for
Adenovirus production are presented in FIGS. 30, 31, and 32. Those
of ordinary skill in the art are aware of methods of modifying
these constructs to produce other constructs used in the methods
and compositions described herein.
Example 13
MyD88L Adenoviral Transfection of 293T Cells Results in Protein
Expression
[0510] The following pShuttleX constructs were constructed for
adenovirus production:
pShuttleX-MyD88L-Fv'Fvls-E pShuttleX-MyD88LCD40-Fv'Fvls-E
pShuttleX-CD4/TLR4-L3-E L3 indicates three 6 amino acid linkers,
having the DNA sequence:
TABLE-US-00001 GGAGGCGGAGGCAGCGGAGGTGGCGGTTCCGGAGGCGGAGGTTCT
Protein sequence: GlyGlyGlyGlySerGlyGlyGlyGlySer
GlyGlyGlyGlySer
E is an HA epitope.
[0511] Recombinant adenovirus was obtained using methods known to
those of ordinary skill in the art, and essentially as described in
He, T. C., S. Zhou, et al. (1998) Proc. Natl. Acad. Sci. USA
95(5):2509-14.
[0512] For each of the adenovirus assays, crude lysates from
several virus plaques were assayed for protein expression by
Western blotting. Viral particles were released from cell pellets
supplied by the Vector Core at Baylor College of Medicine (world
wide web address of http://vector.bcm.tmc.edu/) by freeze thawing
pellets three times. 293T cells were plated at 1.times.10.sup.6
cells per well of a 6 well plate. 24 hours following culture, cells
were washed twice with serum-free DMEM media with antibiotic,
followed by the addition of 25 microliters or 100 microliters virus
lysate to the cell monolayer in 500 microliters serum-free media. 2
hours later, 2.5 ml of serum-supplemented DMEM was added to each
well of the 6-well plate.
[0513] 24-48 hours later, cells were harvested, washed twice with
1.times.PBS and resuspended in RIPA lysis buffer (containing 100
micromolar PMSF) (available from, for example, Millipore, or Thermo
Scientific). Cells were incubated on ice for 30 minutes with mixing
every 10 minutes, followed by a spin at 10,000 g for 15 minutes at
4.degree. C. The supernatants were mixed with SDS Laemmli buffer
plus beta-mercaptoethanol at a ratio of 1:2, incubated at
100.degree. C. for 10 minutes, loaded on a SDS gel, and probed on a
nitrocellulose membrane using an antibody to the HA epitope.
Results are shown in FIGS. 21 and 22. Remaining cell lysates were
stored at -80.degree. C. for future use. The cells were transduced
separately with each of the viruses, viz., Ad5-iMyD88 and Ad5-TLRon
separately.
Example 14
IL-12p70 Expression in MyD88L-Adenoviral Transduced Cells
[0514] Bone marrow-derived dendritic cells (BMDCs) were plated at
0.25.times.10.sup.6 cells per well of a 48-well plate after washing
twice with serum-free RPMI media with antibiotic. Cells were
transduced with 6 microliters crude virus lysate in 125 microliters
serum-free media. 2 hours later, 375 microliters of
serum-supplemented RPMI was added to each well of the 48-well
plate. 48 hours later, supernatants were harvested and analyzed
using a mouse IL-12p70 ELISA kit (BD OptEIA (BD BioSciences, New
Jersey). Duplicate assays were conducted for each sample, either
with or without the addition of 100 nM AP21087. CD40-L is CD40
ligand, a TNF family member that binds to the CD40 receptor. LPS is
lipopolysaccharide. The results are shown in FIG. 23. Results of a
repeat of the assay are shown in FIG. 24, crude adenoviral lysate
was added at 6.2 microliters per 0.25 million cells. FIG. 25 shows
the results of an additional assay, where more viral lysate, 12.5
microliters per 0.25 million cells was used to infect the
BMDCs.
Example 15
Inducible iRIG-1, iNOD2 and iTRIF Activities
[0515] One microgram each of pBJ5-Fv'Fvls-RIG-1 and
pBJ5-Fv'Fvls-CD40 were transfected into 293 cells with one
microgram of NF-kappaB-SEAP reporter and cells were analyzed for
reporter activity on treatment with an increasing dose of dimer
drug AP20187. A pBJ5-RIG-1-Fv'Fvls construct was also tested. The
results are shown in FIG. 26. The results show that iRIG-I could
potentially work well with iCD40 as demonstrated by the additive
effects seen in the NF-kappaB reporter assay when both the iRIG-I
and iCD40 constructs were transfected into 293 cells.
[0516] One microgram of pBJ5-Fv'Fvls-RIG-1 was transfected into 293
cells with 1 micrograms of IFNbeta-SEAP reporter and cells were
analyzed for reporter activity on treatment with half-log dilutions
of dimer drug AP20187. Simultaneously the iTRIF (pBJ5-Fv'Fvls-TRIF)
construct was also tested by transfecting increasing amounts into
293 cells. pBJ5-M-Fv'Fvls-TRIF-E- was made in pBJ5 backbone with a
myristoylation sequence, 2 FKBPs, full length TRIF and a HA
epitope. The results are shown in FIGS. 27 and 28. The results
demonstrate that iTRIF constitutively activates NF-kappaB, and, to
a higher extent, IFNbeta reporters in 293 cells.
[0517] A similar assay was conducted using iNOD2, as shown in FIG.
29. The results show that iNOD2 activates NF-kappaB in a drug
dependent manner. This activation of NF-kappa B increases on the
addition of a third FKBP domain to iNOD2 (iNOD2Turbo).
pBJ5-Sn-Fv'Fv'Fvls-NOD2-E- was made in pBJ5 backbone, and contains
a myristoylation sequence, 3 FKBPs, 2 CARD domains of NOD2 and a HA
epitope.
Example 16
IL-12p70 Expression in MyD88L-Adenoviral Transduced Human
Monocyte-Derived Dendritic Cells
[0518] Immature human monocyte-derived dendritic cells (moDCs) were
plated at 0.25.times.10.sup.6 cells per well of a 48-well plate
after washing twice with serum-free RPMI media with antibiotic.
Cells were transduced with different multiplicity of infections
(M01) of adenovirus AD5-iMyD88.CD40 and stimulated with 100 nM
dimer drug AP20187. The virus used was an optimized version of the
viral lysate used in Examples 13 and 14. 48 hours later,
supernatants were harvested and assayed in an IL12p70 ELISA assay.
FIG. 33 depicts the results of this titration.
[0519] Immature human moDCs were plated at 0.25.times.10.sup.6
cells per well of a 48-well plate after washing twice with
serum-free RPMI media with antibiotic. Cells were then transduced
with either Ad5f35-iCD40 (10,000 VP/cell); Ad5-iMyD88.CD40 (100
MOD; Ad5.1MyD88 (100 MOD or Ad5-TLR4 on (100 MOD and stimulated
with 1 microgram/milliliter LPS where indicated and 100 nM dimer
drug AP20187 where indicated in FIG. 34. 48 hours later,
supernatants were harvested and assayed in an IL12p70 ELISA
assay.
[0520] Ad5f35-iCD40 was produced using pShuttleX-ihCD40 (also known
as M-Fv'-Fvls-hCD40; pShuttleX-M-Fv'-Fvls-hCD40). MyD88, as
indicated in FIGS. 33 and 34, is the same truncated version of
MyD88 as the version indicated as MyD88L herein. The adenovirus
indicated as Ad5.1MyD88 was produced using
pShuttleX-MyD88L-Fv'Fvls-E. The adenovirus indicated as
Ad5-iMyD88.Cd40 was produced using pShuttleX-MyD88LCD40-Fv'Fvls-E.
The adenovirus indicated as Ad5-TLR4On was produced using
pShuttleX-CD4/TLR4-L3-E.
Example 17
Non-viral Transformation of Dendritic Cells
[0521] A plasmid vector is constructed comprising the iMyD88-CD40
sequence operably linked to the Fv'Fvls sequence, such as, for
example, the pShuttleX-MyD88LCD40-Fv'Fvls-E Insert. The plasmid
construct also includes the following regulatory elements operably
linked to the MyD88ICD40-Fv'Fvls-E sequence: promoter, initiation
codon, stop codon, polyadenylation signal. The vector may also
comprise an enhancer sequence. The MyD88L, CD40, and FvFvls
sequences may also be modified using synthetic techniques known in
the art to include optimized codons.
[0522] Immature human monocyte-derived dendritic cells (MoDCs) are
plated at 0.25.times.10.sup.6 cells per well of a 48-well plate
after washing twice with serum-free RPMI media with antibiotic.
Cells are transduced with the plasmid vector using any appropriate
method known to those of ordinary skill in the art such as, for
example, nucleofection using AMAXA kits, electroporation, calcium
phosphate, DEAE-dextran, sonication loading, liposome-mediated
transfection, receptor mediated transfection, or microprojectile
bombardment.
[0523] DNA vaccines are discussed in, for example, U.S. Patent
Publication 20080274140, published Nov. 6, 2008. The iMyD88-CD40
sequence operably linked to the Fv'Fvls sequence is inserted into a
DNA vaccine vector, which also comprises, for example, regulatory
elements necessary for expression of the iMyD88-Cd40 Fv'Fvls
chimeric protein in the host tissue. These regulatory elements
include, but are not limited to, promoter, initiation codon, stop
codon, polyadenylation signal, and enhancer, and the codons coding
for the chimeric protein may be optimized.
Example 18
Evaluation of MyD88CD40 Transformed Dendritic Cells In Vivo Using a
Mouse Tumor Model
[0524] Bone marrow dendritic cells were transduced using adenoviral
vectors as presented in the examples herein. These transduced BMDCs
were tested for their ability to inhibit tumor growth in a EG.7-OVA
model. EG.7-OVA cells (5.times.10.sup.5 cells/100 ml) were
inoculated into the right flank of C57BL/6 female mice. BMDCs of
all groups were pulsed with 50 microgram/ml of ovalbumin protein
and activated as described above. Approximately 7 days after tumor
cell inoculation, BMDCs were thawed and injected subcutaneously
into the hind foot-pads of mice.
[0525] Tumor growth was monitored twice weekly in mice of all
groups. Peripheral blood from random mice of all groups was
analyzed by tetramer staining and by in vivo CTL assays. Table 1
presents the experimental design, which includes non-transduced
dendritic cells (groups 1 and 2), dendritic cells transduced with a
control adenovirus vector (group 3), dendritic cells transduced
with a CD40 cytoplasmic region encoding vector (group 4), dendritic
cells transduced with a truncated MyD88 vector (groups 5 and 6),
and dendritic cells transduced with the chimeric CD40-truncated
MyD88 vector (groups 7 and 8). The cells were stimulated with
AP-1903, LPS, or CD40 ligand as indicated.
TABLE-US-00002 TABLE 1 Other Route of Route of ADV [AP1903]
reagents Administration Administration Group Treatment Dose Level
vp/cell [LPS] (in vitro) (in vitro) (Vaccine) (AP1903) N 1 PBS NA
N/A SC N/A 6 2 DCs + CD40L + LPS 1.5e6 cells 200 ng/ml N/A CD40L SC
N/A 6 2 .mu.g/ml 3 DCs + Ad-Luc + 1.5e6 cells 20K wGJ 200 ng/ml 100
nM SC IP 6 LPS + AP1903 5 mg/kg (AP1903) 4 DCs + Ad-iCD40 + 1.5e6
cells 20K wGJ 200 ng/ml 100 nM SC IP 6 LPS + AP1903 5 mg/kg
(AP1903) 5 DCs + Ad-iMyD88 + 1.5e6 cells 20K wGJ 100 nM SC IP 6
AP1903 5 mg/kg (AP1903) 6 DCs + Ad-iMyD88 1.5e6 cells 20K wGJ N/A
SC N/A 6 7 DCs + Ad-iMyD88.CD40 + 1.5e6 cells 20K wGJ 100 nM SC IP
6 AP1903 5 mg/kg (AP1903) 8 DCs + Ad-iMyD88.CD40 1.5e6 cells 20K
wGJ N/A SC N/A 6
[0526] Prior to vaccination of the tumor-inoculated mice, the
IL-12p70 levels of the transduced dendritic cells were measured in
vitro. The IL-12p70 levels are presented in FIG. 35. FIG. 36 shows
a chart of tumor growth inhibition observed in the transduced mice.
Inoculation of the MyD88 transduced and AP1903 treated dendritic
cells resulted in a cure rate of 1/6, while inoculation of the
MyD88-CD40 transduced dendritic cells without AP1903 resulted in a
cure rate of 4/6, indicating a potential dimerizer-independent
effect. The asterix indicates a comparison of Luc+LPS+AP and
iCD40MyD88+LPS+/-AP1903. FIG. 36 also provides photographs of
representative vaccinated mice.
[0527] FIG. 37 presents an analysis of the enhanced frequency of
Ag-Specific CD8+ T cell induction in mice treated with iMyD88-CD40
transduced dendritic cells. Peripheral bone marrow cells from
treated mice were harvested ten days after vaccination on day 7.
The PBMCs were stained with anti-mCD8-FITC and
H2-K.sup.b-SIINFEKL-tetramer-PE and analyzed by flow cytometry.
[0528] FIG. 38 presents the enhanced frequency of Ag-specific CD8+
T cell and CD4+ T.sub.H1 cells induced in mice after treatment
iMyD88-CD40-transduced dendritic cells. Three mice of all
experimental groups were sacrificed 18 days after the vaccination.
Splenocytes of three mice per group were "pooled" together and
analyzed by IFN-gamma ELISPOT assay. Millipore MultiScreen-HA
plates were coated with 10 micrograms/ml anti-mouse IFN-gamma AN18
antibody (Mabtech AB, Inc., Nacka, Sweden). Splenocytes were added
and cultured for 20 hours at 37 degrees C. in 5% CO.sub.2 in
complete ELISpot medium (RPMI, 10% FBS, penicillin, streptomycin).
Splenocytes were incubated with 2 micrograms/ml OT-1 (SIINFEKL),
OT-2 (ISQAVHAAHAEINEAGR) or TRP-2 peptide (control non-targeted
peptide). After washes, a second biotinylated monoclonal antibody
to mouse IFN-gamma (R4-6A2, Mabtech AB) was applied to the wells at
a concentration of 1 microgram/ml, followed by incubation with
streptavidin-alkaline phosphatase complexes (Vector Laboratories,
Ltd., Burlingame, Calif.). Plates were then developed with the
alkaline phosphatase substrate, 3-amino-9 ethylcarbazole
(Sigma-Aldrich, Inc., St. Louis, Mo.). The numbers of spots in the
wells were scored by ZelINet Consulting, Inc. with an automated
ELISPOT reader system (Carl Zeiss, Inc, Thornwood N Y).
[0529] FIG. 39 presents a schematic and the results of an in vivo
cytotoxic lymphocyte assay. Eighteen days after DC vaccinations an
in vivo CTL assay was performed. Syngeneic naive splenocytes were
used as in vivo target cells. They were labeled by incubation for
10 minutes at 37 degrees C. with either 6 micromolar CFSE
(CFSE.sup.hi cells) or 0.6 micromolar CFSE in CTL medium
(CFSE.sup.lo cells). CFSE.sup.hi cells were pulsed with OT-1
SIINFEKL peptide, and CFSE.sup.lo cells were incubated with control
TRP2 peptide. A mixture of 4.times.10.sup.6 CFSE.sup.hi plus
4.times.10.sup.6 CFSE.sup.lo cells was injected intravenously
through the tail vein. After 16 hours of in vivo incubation,
splenocytes were collected and single-cell suspensions are analyzed
for detection and quantification of CFSE-labeled cells. FIG. 40 is
a chart presenting the enhanced CTL activity induced by
iMyD88-CD40-transduced dendritic cells in the inoculated mice. FIG.
41 shows the raw CTL histograms for select samples, indicating the
enhanced in vivo CTL activity induced by the iMyD88-CD40 transduced
dendritic cells.
[0530] FIG. 42 presents the results of intracellular staining for
IL-4 producing T.sub.H2 cells in the mice vaccinated with the
transduced cells. Splenocytes of mice (pooled cells from three
mice) were reconstituted with 2 micrograms/ml of OT-2 peptide.
Cells were incubated for 6 hours with 10 micrograms/ml of brefeldin
A to suppress secretion. Then cells were fixed and permealized and
analyzed by intracellular staining with anti-mIL-4-APC and
anti-mCD4-FITC.
[0531] The adenoviral vector comprising the iCD40-MyD88 sequence
was again evaluated for its ability to inhibit tumor growth in a
mouse model. In the first experiment, drug-dependent tumor growth
inhibition was measured after inoculation with dendritic cells
modified with the inducible CD40-truncated MyD88 vector
(Ad-iCD40.MyD88). Bone marrow-derived dendritic cells from C57BL/6
mice were pulsed with 10 micrograms/ml of ovalbumin and transduced
with 20,000 viral particles/cell (VP/c) of the adenovirus
constructs Ad5-iCD40.MyD88, Ad5-iMyD88 or Ad5-Luc (control). Cells
were activated with either 2 micrograms/ml CD40L, 200 ng/ml LPS, or
50 nM AP1903 dimerizer drug. 5.times.10.sup.5 E.G7-OVA thymoma
cells were inoculated into the backs of C57BL/6 mice (N=6/group).
When tumors reached .about.5 mm in diameter (day 8 after
inoculation), mice were treated with subcutaneous injections of
2.times.10.sup.6 BMDCs. The next day, after cellular vaccinations,
mice were treated with intraperitoneal injections of 5 mg/kg
AP1903. Tumor growth was monitored twice weekly. The results are
shown in FIG. 43A. In another set of experiments, E.G7-OVA tumors
were established as described above. Mice (N=6/group) were treated
with 2.times.10.sup.6 BMDCs (ovalbumin pulsed) and transduced with
either 20,000 or 1,250 VP/c of Ad5-iCD40.MyD88. BMDCs of AP1903
groups were treated in vitro with 50 nM AP1903. The next day, after
cellular vaccinations, mice of AP1903 groups were treated by
intraperitoneal injection with 5 mg/kg AP1903. The results are
shown in FIG. 43B. FIG. 43C depicts relative IL-12p70 levels
produced following overnight culture of the various vaccine cells
prior to cryopreservation. IL-12p70 was assayed by ELISA assay.
[0532] Blood from mice immunized with the modified bone marrow
dendritic cells was analyzed for the frequency and function of
tumor specific T cells using tetramer staining. FIG. 44A shows the
results of an experiment in which mice (N=3-5) were immunized
subcutaneously with BMDCs pulsed with ovalbumin and activated as
described in FIG. 43. One week after the vaccination, peripheral
blood mononuclear cells (PBMCs) were stained with anti-mCD8-FITC
and SIINFEKL-H2-K.sup.b-PE and analyzed by flow cytometry. FIG. 44B
shows the results of an in vivo CTL assay that was performed in
mice vaccinated with BMDCs as described above. Two weeks after the
BMDC immunization, splenocytes from syngeneic C57BL/6 mice were
pulsed with either TRP-2 control peptide, SVYDFFVWL, or target
peptide, SINFEKL target, and were used as in vivo targets. Half of
the splenocytes were labeled with 6 micromolar CFSE (CFSE.sup.hi
cells) or 0.6 micromolar CFSE (CFSE.sup.lo cells). CFSE.sup.hi
cells were pulsed with OT-1 (SIINFEKL) peptide and CFSE.sup.lo
cells were incubated with control TRP-2 (SVYDFFVWL) peptide. A
mixture of 4.times.10.sup.6 CFSE.sup.hi plus 4.times.10.sup.6
CFSE.sup.lo cells was injected intravenously through the tail vein.
The next day, splenocytes were collected and single-cell
suspensions were analyzed for detection and quantification of
CFSE-labeled cells. FIGS. 44C and 44D show the results of an
IFN-gamma assay. Peripheral blood mononuclear cells (PBMCs) from
E.G7-OVA-bearing mice treated as described in FIG. 43, were
analyzed in IFN-gamma ELISpot assays with 1 microgram/ml of
SIINFEKL peptide (OT-1), ISQAVHAAHAEINEAGR (OT-2) and TRP-2
(irrelevant H2-K.sup.b-restricted) peptides. The number of
IFN-gamma-producing lymphocytes was evaluated in triplicate wells.
Cells from three mice per group were pooled and analyzed by
IFN-gamma ELISpot in triplicate wells. The assays were performed
twice.
[0533] FIG. 45 presents the results of a natural killer cell assay
performed using the splenocytes from mice treated as indicated in
this example. Splenocytes obtained from mice (3 per group) were
used as effectors (E). Yac-1 cells were labeled with .sup.51Cr and
used as targets (T). The EL-4 cell line was used as an irrelevant
control.
[0534] FIG. 46 presents the results of an assay for detection of
antigen-specific cytotoxic lymphocytes. Splenocytes obtained from
mice (3 per group) were used as effectors. EG.7-Ova cells were
labeled with .sup.51Cr and used as targets (T). The EL-4 cell line
was used as an irrelevant control.
[0535] FIG. 47 presents the results of the activation of human
cells transduced with the inducible CD40-truncated MyD88
(iCD40.MyDD) adenovirus vector. Dendritic cells (day 5 of culture)
from three different HLA-A2+ donors were purified by the
plastic-adhesion method and transduced with 10,000 VP/cell of
Ad5-iCD40.MyD88, Ad5-iMyD88 or Ad5-Luc. Cells were activated with
100 nM AP1903 or 0.5 micrograms/ml of CD40L and 250 ng/ml of LPS or
standard maturation cocktail (MC), containing TNF-alpha, IL-1 beta,
IL-6, and prostaglandin E2 (PGE.sub.2). Autologous CD8+ T cells
were purified by negative selection using microbeads and
co-cultured with DCs pulsed with 10 micrograms/ml of
HLA-A2-restricted FLWGPRALV MAGE-3 peptide at 1:5 (DC:T) ratio for
7 days. Five days after the second of round of stimulation with DCs
(on day7) T cells were assayed in standard IFN-gamma ELISpot assay.
Cells were pulsed with 1 micrograms/ml of MAGE-3 or irrelevant
HLA-A2-restricted PSMA peptide (PSMA-P2). Experiments were
performed in triplicate.
[0536] FIGS. 48 and 49 present the results of a cell migration
assay. mBMDCs were transduced with 10,000 VP/cell of Ad5.Luciferase
or Ad5.1MyD88.CD40 in the presence of Gene Jammer (Stratagene, San
Diego, Calif.) and stimulated with 100 nM AP1903 (AP) or LPS (1
microgram/ml) for 48 hours. CCR7 expression was analyzed on the
surface of CD11c+dendritic cells by intracellular staining using a
PerCP.Cy5.5 conjugated antibody. FIG. 48 shows the results of the
experiment, with each assay presented separately; FIG. 49 provides
the results in the same graph.
Example 19
Examples of Particular Nucleic Acid and Amino Acid Sequences
TABLE-US-00003 [0537] SEQ ID NO: 1 (nucleic acid sequence encoding
human CD40; Genbank accession no. NM_001250; cytoplasmic region
indicated in bold) 1 gccaaggctg gggcagggga gtcagcagag gcctcgctcg
ggcgcccagt ggtcctgccg 61 cctggtctca cctcgctatg gttcgtctgc
ctctgcagtg cgtcctctgg ggctgcttgc 121 tgaccgctgt ccatccagaa
ccacccactg catgcagaga aaaacagtac ctaataaaca 181 gtcagtgctg
ttctttgtgc cagccaggac agaaactggt gagtgactgc acagagttca 241
ctgaaacgga atgccttcct tgcggtgaaa gcgaattcct agacacctgg aacagagaga
301 cacactgcca ccagcacaaa tactgcgacc ccaacctagg gcttcgggtc
cagcagaagg 361 gcacctcaga aacagacacc atctgcacct gtgaagaagg
ctggcactgt acgagtgagg 421 cctgtgagag ctgtgtcctg caccgctcat
gctcgcccgg ctttggggtc aagcagattg 481 ctacaggggt ttctgatacc
atctgcgagc cctgcccagt cggcttcttc tccaatgtgt 541 catctgcttt
cgaaaaatgt cacccttgga caagctgtga gaccaaagac ctggttgtgc 601
aacaggcagg cacaaacaag actgatgttg tctgtggtcc ccaggatcgg ctgagagccc
661 tggtggtgat ccccatcatc ttcgggatcc tgtttgccat cctcttggtg
ctggtcttta 721 tcaaaaaggt ggccaagaag ccaaccaata aggcccccca
ccccaagcag gaaccccagg 781 agatcaattt tcccgacgat cttcctggct
ccaacactgc tgctccagtg caggagactt 841 tacatggatg ccaaccggtc
acccaggagg atggcaaaga gagtcgcatc tcagtgcagg 901 agagacagtg
aggctgcacc cacccaggag tgtggccacg tgggcaaaca ggcagttggc 961
cagagagcct ggtgctgctg ctgctgtggc gtgagggtga ggggctggca ctgactgggc
1021 atagctcccc gcttctgcct gcacccctgc agtttgagac aggagacctg
gcactggatg 1081 cagaaacagt tcaccttgaa gaacctctca cttcaccctg
gagcccatcc agtctcccaa 1141 cttgtattaa agacagaggc agaagtttgg
tggtggtggt gttggggtat ggtttagtaa 1201 tatccaccag accttccgat
ccagcagttt ggtgcccaga gaggcatcat ggtggcttcc 1261 ctgcgcccag
gaagccatat acacagatgc ccattgcagc attgtttgtg atagtgaaca 1321
actggaagct gcttaactgt ccatcagcag gagactggct aaataaaatt agaatatatt
1381 tatacaacag aatctcaaaa acactgttga gtaaggaaaa aaaggcatgc
tgctgaatga 1441 tgggtatgga actttttaaa aaagtacatg cttttatgta
tgtatattgc ctatggatat 1501 atgtataaat acaatatgca tcatatattg
atataacaag ggttctggaa gggtacacag 1561 aaaacccaca gctcgaagag
tggtgacgtc tggggtgggg aagaagggtc tggggg SEQ ID NO: 2 (amino acid
sequence encoding human CD40; cytoplasmic region indicated in bold)
MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDT
WNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSD
TICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILL
VLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ
SEQ ID NO: 3 (nucleotide sequence encoding PSMA)
gcggatccgcatcatcatcatcatcacagctccggaatcgagggacgtggtaaatcctccaatgaagcta
ctaacattactccaaagcataatatgaaagcatttttggatgaattgaaagctgagaacatcaagaagtt
cttatataattttacacagataccacatttagcaggaacagaacaaaactttcagcttgcaaagcaaatt
caatcccagtggaaagaatttggcctggattctgttgagctagcacattatgatgtcctgttgtcctacc
caaataagactcatcccaactacatctcaataattaatgaagatggaaatgagattttcaacacatcatt
atttgaaccacctcctccaggatatgaaaatgtttcggatattgtaccacctttcagtgctttctctcct
caaggaatgccagagggcgatctagtgtatgttaactatgcacgaactgaagacttctttaaattggaac
gggacatgaaaatcaattgctctgggaaaattgtaattgccagatatgggaaagttttcagaggaaataa
ggttaaaaatgcccagctggcaggggccaaaggagtcattctctactccgaccctgctgactactttgct
cctggggtgaagtcctatccagatggttggaatcttcctggaggtggtgtccagcgtggaaatatcctaa
atctgaatggtgcaggagaccctctcacaccaggttacccagcaaatgaatatgcttataggcgtggaat
tgcagaggctgttggtcttccaagtattcctgttcatccaattggatactatgatgcacagaagctccta
gaaaaaatgggtggctcagcaccaccagatagcagctggagaggaagtctcaaagtgccctacaatgttg
gacctggctttactggaaacttttctacacaaaaagtcaagatgcacatccactctaccaatgaagtgac
aagaatttacaatgtgataggtactctcagaggagcagtggaaccagacagatatgtcattctgggaggt
caccgggactcatgggtgtttggtggtattgaccctcagagtggagcagctgttgttcatgaaattgtga
ggagctttggaacactgaaaaaggaagggtggagacctagaagaacaattttgtttgcaagctgggatgc
agaagaatttggtcttcttggttctactgagtgggcagaggagaattcaagactccttcaagagcgtggc
gtggcttatattaatgctgactcatctatagaaggaaactacactctgagagttgattgtacaccgctga
tgtacagcttggtacacaacctaacaaaagagctgaaaagccctgatgaaggctttgaaggcaaatctct
ttatgaaagttggactaaaaaaagtccttccccagagttcagtggcatgcccaggataagcaaattggga
tctggaaatgattttgaggtgttcttccaacgacttggaattgcttcaggcagagcacggtatactaaaa
attgggaaacaaacaaattcagcggctatccactgtatcacagtgtctatgaaacatatgagttggtgga
aaagttttatgatccaatgtttaaatatcacctcactgtggcccaggttcgaggagggatggtgtttgag
ctagccaattccatagtgctcccttttgattgtcgagattatgctgtagttttaagaaagtatgctgaca
aaatctacagtatttctatgaaacatccacaggaaatgaagacatacagtgtatcatttgattcactttt
ttctgcagtaaagaattttacagaaattgcttccaagttcagtgagagactccaggactttgacaaaagc
aagcatgtcatctatgctccaagcagccacaacaagtatgcaggggagtcattcccaggaatttatgatg
ctctgtttgatattgaaagcaaagtggacccttccaaggcctggggagaagtgaagagacagatttatgt
tgcagccttcacagtgcaggcagctgcagagactttgagtgaagtagcctaagcggccgcatagca
SEQ ID NO: 4 (PSMA amino acid sequence encoded by SEQ ID NO: 3)
MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENI
KKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFN
TSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFR
GNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYR
RGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTN
EVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFAS
WDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEG
KSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYE
LVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFD
SLFSAVKNFTEIASKFSERLQDFDKSKHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQ
IYVAAFTVQAAAETLSEVA SEQ ID NO: 5 (nucleotide sequence of MyD88L
with SaII linkers)
gtcgacatggctgcaggaggtcccggcgcggggtctgcggccccggtctcctccacatcctcccttcccc
tggctgctctcaacatgcgagtgcggcgccgcctgtctctgttcttgaacgtgcggacacaggtggcggc
cgactggaccgcgctggcggaggagatggactttgagtacttggagatccggcaactggagacacaagcg
gaccccactggcaggctgctggacgcctggcagggacgccctggcgcctctgtaggccgactgctcgagc
tgcttaccaagctgggccgcgacgacgtgctgctggagctgggacccagcattgaggaggattgccaaaa
gtatatcttgaagcagcagcaggaggaggctgagaagcctttacaggtggccgctgtagacagcagtgtc
ccacggacagcagagctggcgggcatcaccacacttgatgaccccctggggcatatgcctgagcgtttcg
atgccttcatctgctattgccccagcgacatcgtcgac SEQ ID NO: 6 (amino acid
sequence of MYD88L)
MAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADP
TGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPR
TAELAGITTLDDPLGHMPERFDAFICYCPSDI SEQ ID NO: 7 (sequence of Fv`FvIs
with XhoI/SaII linkers, (wobbled codons lowercase in Fv`))
ctcgagGGcGTcCAaGTcGAaACcATtagtCCcGGcGAtGGcaGaACaTTtCCtAAaaGgGGaCAaACaT
GtGTcGTcCAtTAtACaGGcATGtTgGAgGAcGGcAAaAAgGTgGAcagtagtaGaGAtcGcAAtAAaCC
tTTcAAaTTcATGtTgGGaAAaCAaGAaGTcATtaGgGGaTGGGAgGAgGGcGTgGCtCAaATGtccGTc
GGcCAacGcGCtAAgCTcACcATcagcCCcGAcTAcGCaTAcGGcGCtACcGGaCAtCCcGGaATtATtC
CcCCtCAcGCtACctTgGTgTTtGAcGTcGAaCTgtTgAAgCTcGAagtcgagggagtgcaggtggaaac
catctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgcactacaccgggatg
cttgaagatggaaagaaagttgattcctcccgggacagaaacaagccctttaagtttatgctaggcaagc
aggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactat
atctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttc
gatgtggagcttctaaaactggaatctggcggtggatccggagtcgag SEQ ID NO: 8
(FV`FVLS peptide sequence)
GlyValGlnValGluThrIleSerProGlyAspGlyArgThrPheProLysArgGlyGlnThrCysVal
ValHisTyrThrGlyMetLeuGluAspGlyLysLysValAspSerSerArgAspArgAsnLysProPhe
LysPheMetLeuGlyLysGlnGluValIleArgGlyTrpGluGluGlyValAlaGlnMetSerValGly
GlnArgAlaLysLeuThrIleSerProAspTyrAlaTyrGlyAlaThrGlyHisProGlyIleIlePro
ProHisAlaThrLeuValPheAspValGluLeuLeuLysLeuGlu (ValGlu)
GlyValGlnValGluThrIleSerProGlyAspGlyArgThrPheProLysArgGlyGlnThrCysVal
ValHisTyrThrGlyMetLeuGluAspGlyLysLysValAspSerSerArgAspArgAsnLysProPhe
LysPheMetLeuGlyLysGlnGluValIleArgGlyTrpGluGluGlyValAlaGlnMetSerValGly
GlnArgAlaLysLeuThrIleSerProAspTyrAlaTyrGlyAlaThrGlyHisProGlyIleIlePro
ProHisAlaThrLeuValPheAspValGluLeuLeuLysLeuGluSerGlyGlyGlySerGly SEQ
ID NO: 9 (TRIF nucleotide sequence with XhoI/SaII linkers)
ctcgagatggcctgcacaggcccatcacttcctagcgccttcgacattctaggtgcagcaggccaggaca
agctcttgtatctgaagcacaaactgaagaccccacgcccaggctgccaggggcaggacctcctgcatgc
catggttctcctgaagctgggccaggaaactgaggccaggatctctctagaggcattgaaggccgatgcg
gtggcccggctggtggcccgccagtgggctggcgtggacagcaccgaggacccagaggagcccccagatg
tgtcctgggctgtggcccgcttgtaccacctgctggctgaggagaagctgtgccccgcctcgctgcggga
cgtggcctaccaggaagccgtccgcaccctcagctccagggacgaccaccggctgggggaaccttcagga
tgaggcccgaaaccggtgtgggtgggacattgctggggatccagggagcatccggacgctccagtccaat
ctgggctgcctcccaccatcctcggctttgccctctgggaccaggagcctcccacgccccattgacggtg
tttcggactggagccaagggtgctccctgcgatccactggcagccctgcctccctggccagcaacttgga
aatcagccagtcccctaccatgcccttcctcagcctgcaccgcagcccacatgggcccagcaagctctgt
gacgacccccaggccagcttggtgcccgagcctgtccccggtggctgccaggagcctgaggagatgagct
ggccgccatcgggggagattgccagcccaccagagctgccaagcagcccacctcctgggcttcccgaagt
ggccccagatgcaacctccactggcctccctgatacccccgcagctccagaaaccagcaccaactaccca
gtggagtgcaccgaggggtctgcaggcccccagtctctccccttgcctattctggagccggtcaaaaacc
cctgctctgtcaaagaccagacgccactccaactttctgtagaagataccacctctccaaataccaagcc
gtgcccacctactcccaccaccccagaaacatcccctcctcctcctcctcctcctccttcatctactcct
tgttcagctcacctgaccccctcctccctgttcccttcctccctggaatcatcatcggaacagaaattct
ataactttgtgatcctccacgccagggcagacgaacacatcgccctgcgggttcgggagaagctggaggc
ccttggcgtgcccgacggggccaccttctgcgaggatttccaggtgccggggcgcggggagctgagctgc
ctgcaggacgccatagaccactcagctttcatcatcctacttctcacctccaacttcgactgtcgcctga
gcctgcaccaggtgaaccaagccatgatgagcaacctcacgcgacaggggtcgccagactgtgtcatccc
cttcctgcccctggagagctccccggcccagctcagctccgacacggccagcctgctctccgggctggtg
cggctggacgaacactcccagatcttcgccaggaaggtggccaacaccttcaagccccacaggcttcagg
cccgaaaggccatgtggaggaaggaacaggacacccgagccctgcgggaacagagccaacacctggacgg
tgagcggatgcaggcggcggcactgaacgcagcctactcagcctacctccagagctacttgtcctaccag
gcacagatggagcagctccaggtggcttttgggagccacatgtcatttgggactggggcgccctatgggg
ctcgaatgccctttgggggccaggtgcccctgggagccccgccaccctttcccacttggccggggtgccc
gcagccgccacccctgcacgcatggcaggctggcacccccccaccgccctccccacagccagcagccttt
ccacagtcactgcccttcccgcagtccccagccttccctacggcctcacccgcaccccctcagagcccag
ggctgcaacccctcattatccaccacgcacagatggtacagctggggctgaacaaccacatgtggaacca
gagagggtcccaggcgcccgaggacaagacgcaggaggcagaagtcgac SEQ ID NO: 10
(TRIF peptide sequence)
MetAlaCysThrGlyProSerLeuProSerAlaPheAspIleLeuGlyAlaAlaGlyGlnAspLysLeu
LeuTyrLeuLysHisLysLeuLysThrProArgProGlyCysGlnGlyGlnAspLeuLeuHisAlaMet
ValLeuLeuLysLeuGlyGlnGluThrGluAlaArgIleSerLeuGluAlaLeuLysAlaAspAlaVal
AlaArgLeuValAlaArgGlnTrpAlaGlyValAspSerThrGluAspProGluGluProProAspVal
SerTrpAlaValAlaArgLeuTyrHisLeuLeuAlaGluGluLysLeuCysProAlaSerLeuArgAsp
ValAlaTyrGlnGluAlaValArgThrLeuSerSerArgAspAspHisArgLeuGlyGluLeuGlnAsp
GluAlaArgAsnArgCysGlyTrpAspIleAlaGlyAspProGlySerIleArgThrLeuGlnSerAsn
LeuGlyC SEQ ID NO: 11 (RIG-I nucleotide sequence (CARD domains
underlined) with XhoI-SaII linkers:
Ctcgagaccaccgagcagcgacgcagcctgcaagccttccaggattatatccggaagaccctggacccta
cctacatcctgagctacatggccccctggtttagggaggaagaggtgcagtatattcaggctgagaaaaa
caacaagggcccaatggaggctgccacactttttctcaagttcctgttggagctccaggaggaaggctgg
ttccgtggctttttggatgccctagaccatgcaggttattctggactttatgaagccattgaaagttggg
atttcaaaaaaattgaaaagttggaggagtatagattacttttaaaacgtttacaaccagaatttaaaac
cagaattatcccaaccgatatcatttctgatctgtctgaatgtttaattaatcaggaatgtgaagaaatt
ctacagatttgctctactaaggggatgatggcaggtgcagagaaattggtggaatgccttctcagatcag
acaaggaaaactggcccaaaactttgaaacttgctttggagaaagaaaggaacaagttcagtgaactgtg
gattgtagagaaaggtataaaagatgttgaaacagaagatcttgaggataagatggaaacttctgacata
cagattgtcgac SEQ ID NO: 12 (RIG-1 peptide sequence (CARD domains
underlined))
TTEQRRSLQAFQDYIRKTLDPTYILSYMAPWFREEEVQYIQAEKNNKGPMEAATLFLKFLLELQEEGWFR
GFLDALDHAGYSGLYEAIESWDFKKIEKLEEYRLLLKRLQPEFKTRIIPTDIISDLSECLINQECEEILQ
ICSTKGMMAGAEKLVECLLRSDKENWPKTLKLALEKERNKFSELWIVEKGIKDVETEDLEDKMETSDIQI
SEQ ID NO: 13 (NOD2 nucleotide sequence (CARD domains underlined)
with XhoI-SaII linkers
Ctcgagatgggggaagagggtggttcagcctctcacgatgaggaggaaagagcaagtgtcctcctcggac
attctccgggttgtgaatgtgctcgcaggaggcttttcaggcacagaggagccagctggtcgagctgctg
gtctcagggtccctggaaggcttcgagagtgtcctggactggctgctgtcctgggaggtcctctcctggg
aggactacgagggcttccacctcctgggccagcctctctcccacttggccaggcgccttctggacaccgt
ctggaataagggtacttgggcctgtcagaagctcatcgcggctgcccaagaagcccaggccgacagccag
tcccccaagctgcatggctgctgggacccccactcgctccacccagcccgagacctgcagagtcaccggc
cagccattgtcaggaggctccacagccatgtggagaacatgctggacctggcatgggagcggggtttcgt
cagccagtatgaatgtgatgaaatcaggttgccgatcttcacaccgtcccagagggcaagaaggctgctt
gatcttgccacggtgaaagcgaatggattggctgccttccttctacaacatgttcaggaattaccagtcc
cattggccctgcctttggaagctgccacatgcaagaagtatatggccaagctgaggaccacggtgtctgc
tcagtctcgcttcctcagtacctatgatggagcagagacgctctgcctggaggacatatacacagagaat
gtcctggaggtcgtcgac SEQ ID NO: 14 (NOD2 peptide sequence (CARD
domains underlined))
MetGlyGluGluGlyGlySerAlaSerHisAspGluGluGluArgAlaSerValLeuLeuGlyHisSer
ProGlyCysGluMetCysSerGlnGluAlaPheGlnAlaGlnArgSerGlnLeuValGluLeuLeuVal
SerGlySerLeuGluGlyPheGluSerValLeuAspTrpLeuLeuSerTrpGluValLeuSerTrpGlu
AspTyrGluGlyPheHisLeuLeuGlyGlnProLeuSerHisLeuAlaArgArgLeuLeuAspThrVal
TrpAsnLysGlyThrTrpAlaCysGlnLysLeuIleAlaAlaAlaGlnGluAlaGlnAlaAspSerGln
SerProLysLeuHisGlyCysTrpAspProHisSerLeuHisProAlaArgAspLeuGlnSerHisArg
ProAlaIleValArgArgLeuHisSerHisValGluAsnMetLeuAspLeuAlaTrpGluArgGlyPhe
ValSerGlnTyrGluCysAspGluIleArgLeuProIlePheThrProSerGlnArgAlaArgArgLeu
LeuAspLeuAlaThrValLysAlaAsnGlyLeuAlaAlaPheLeuLeuGlnHisValGlnGluLeuPro
ValProLeuAlaLeuProLeuGluAlaAlaThrCysLysLysTyrMetAlaLysLeuArgThrThrVal
SerAlaGlnSerArgPheLeuSerThrTyrAspGlyAlaGluThrLeuCysLeuGluAspIleTyrThr
GluAsnValLeuGluVal SEQ ID NO: 15 (MyD88 nucleotide sequence)
atggctgcaggaggtcccggcgcggggtctgcggccccggtctcctccacatcctcccttcccctggctg
ctctcaacatgcgagtgcggcgccgcctgtctctgttcttgaacgtgcggacacaggtggcggccgactg
gaccgcgctggcggaggagatggactttgagtacttggagatccggcaactggagacacaagcggacccc
actggcaggctgctggacgcctggcagggacgccctggcgcctctgtaggccgactgctcgagctgctta
ccaagctgggccgcgacgacgtgctgctggagctgggacccagcattgaggaggattgccaaaagtatat
cttgaagcagcagcaggaggaggctgagaagcctttacaggtggccgctgtagacagcagtgtcccacgg
acagcagagctggcgggcatcaccacacttgatgaccccctggggcatatgcctgagcgtttcgatgcct
tcatctgctattgccccagcgacatccagtttgtgcaggagatgatccggcaactggaacagacaaacta
tcgactgaagttgtgtgtgtctgaccgcgatgtcctgcctggcacctgtgtctggtctattgctagtgag
ctcatcgaaaagaggtgccgccggatggtggtggttgtctctgatgattacctgcagagcaaggaatgtg
acttccagaccaaatttgcactcagcctctctccaggtgcccatcagaagcgactgatccccatcaagta
caaggcaatgaagaaagagttccccagcatcctgaggttcatcactgtctgcgactacaccaacccctgc
accaaatcttggttctggactcgccttgccaaggccttgtccctgccc SEQ ID NO: 16
(MyD88 peptide sequence)
MAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADP
TGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPR
TAELAGITTLDDPLGHMPERFDAFICYCPSDIQFVQEMIRQLEQTNYRLKLCVSDRDVLPGTCVWSIASE
LIEKRCRRMVVVVSDDYLQSKECDFQTKFALSLSPGAHQKRLIPIKYKAMKKEFPSILRFITVCDYTNPC
TKSWFWTRLAKALSLP
[0538] 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.
[0539] 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, yet these modifications and
improvements are within the scope and spirit of the invention.
[0540] 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.
The terms and expressions which have been employed are used as
terms of description and not of limitation, and use of such terms
and expressions do not exclude any equivalents of the features
shown and described or portions thereof, and various modifications
are possible within the scope of the invention claimed. The term
"a" or "an" can refer to one of or a plurality of the elements it
modifies (e.g., "a reagent" can mean one or more reagents) unless
it is contextually clear either one of the elements or more than
one of the elements is described. The term "about" as used herein
refers to a value within 10% of the underlying parameter (i.e.,
plus or minus 10%), and use of the term "about" at the beginning of
a string of values modifies each of the values (i.e., "about 1, 2
and 3" is about 1, about 2 and about 3). For example, a weight of
"about 100 grams" can include weights between 90 grams and 110
grams. Thus, it should be understood that although the present
invention has been specifically disclosed by representative
embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled
in the art, and such modifications and variations are considered
within the scope of this invention.
[0541] Embodiments of the invention are set forth in the claim(s)
that follow(s).
Sequence CWU 1
1
5311616DNAHomo sapiens 1gccaaggctg gggcagggga gtcagcagag gcctcgctcg
ggcgcccagt ggtcctgccg 60cctggtctca cctcgctatg gttcgtctgc ctctgcagtg
cgtcctctgg ggctgcttgc 120tgaccgctgt ccatccagaa ccacccactg
catgcagaga aaaacagtac ctaataaaca 180gtcagtgctg ttctttgtgc
cagccaggac agaaactggt gagtgactgc acagagttca 240ctgaaacgga
atgccttcct tgcggtgaaa gcgaattcct agacacctgg aacagagaga
300cacactgcca ccagcacaaa tactgcgacc ccaacctagg gcttcgggtc
cagcagaagg 360gcacctcaga aacagacacc atctgcacct gtgaagaagg
ctggcactgt acgagtgagg 420cctgtgagag ctgtgtcctg caccgctcat
gctcgcccgg ctttggggtc aagcagattg 480ctacaggggt ttctgatacc
atctgcgagc cctgcccagt cggcttcttc tccaatgtgt 540catctgcttt
cgaaaaatgt cacccttgga caagctgtga gaccaaagac ctggttgtgc
600aacaggcagg cacaaacaag actgatgttg tctgtggtcc ccaggatcgg
ctgagagccc 660tggtggtgat ccccatcatc ttcgggatcc tgtttgccat
cctcttggtg ctggtcttta 720tcaaaaaggt ggccaagaag ccaaccaata
aggcccccca ccccaagcag gaaccccagg 780agatcaattt tcccgacgat
cttcctggct ccaacactgc tgctccagtg caggagactt 840tacatggatg
ccaaccggtc acccaggagg atggcaaaga gagtcgcatc tcagtgcagg
900agagacagtg aggctgcacc cacccaggag tgtggccacg tgggcaaaca
ggcagttggc 960cagagagcct ggtgctgctg ctgctgtggc gtgagggtga
ggggctggca ctgactgggc 1020atagctcccc gcttctgcct gcacccctgc
agtttgagac aggagacctg gcactggatg 1080cagaaacagt tcaccttgaa
gaacctctca cttcaccctg gagcccatcc agtctcccaa 1140cttgtattaa
agacagaggc agaagtttgg tggtggtggt gttggggtat ggtttagtaa
1200tatccaccag accttccgat ccagcagttt ggtgcccaga gaggcatcat
ggtggcttcc 1260ctgcgcccag gaagccatat acacagatgc ccattgcagc
attgtttgtg atagtgaaca 1320actggaagct gcttaactgt ccatcagcag
gagactggct aaataaaatt agaatatatt 1380tatacaacag aatctcaaaa
acactgttga gtaaggaaaa aaaggcatgc tgctgaatga 1440tgggtatgga
actttttaaa aaagtacatg cttttatgta tgtatattgc ctatggatat
1500atgtataaat acaatatgca tcatatattg atataacaag ggttctggaa
gggtacacag 1560aaaacccaca gctcgaagag tggtgacgtc tggggtgggg
aagaagggtc tggggg 16162277PRTHomo sapiens 2Met Val Arg Leu Pro Leu
Gln Cys Val Leu Trp Gly Cys Leu Leu Thr1 5 10 15Ala Val His Pro Glu
Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu 20 25 30Ile Asn Ser Gln
Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val 35 40 45Ser Asp Cys
Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu 50 55 60Ser Glu
Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His65 70 75
80Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr
85 90 95Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys
Thr 100 105 110Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys
Ser Pro Gly 115 120 125Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser
Asp Thr Ile Cys Glu 130 135 140Pro Cys Pro Val Gly Phe Phe Ser Asn
Val Ser Ser Ala Phe Glu Lys145 150 155 160Cys His Pro Trp Thr Ser
Cys Glu Thr Lys Asp Leu Val Val Gln Gln 165 170 175Ala Gly Thr Asn
Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu 180 185 190Arg Ala
Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile 195 200
205Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn
210 215 220Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe
Pro Asp225 230 235 240Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val
Gln Glu Thr Leu His 245 250 255Gly Cys Gln Pro Val Thr Gln Glu Asp
Gly Lys Glu Ser Arg Ile Ser 260 265 270Val Gln Glu Arg Gln
27532096DNAHomo sapiens 3gcggatccgc 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
20964719PRTHomo sapiens 4Met 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
7155528DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 5gtcgacatgg ctgcaggagg tcccggcgcg
gggtctgcgg ccccggtctc ctccacatcc 60tcccttcccc tggctgctct caacatgcga
gtgcggcgcc gcctgtctct gttcttgaac 120gtgcggacac aggtggcggc
cgactggacc gcgctggcgg aggagatgga ctttgagtac 180ttggagatcc
ggcaactgga gacacaagcg gaccccactg gcaggctgct ggacgcctgg
240cagggacgcc ctggcgcctc tgtaggccga ctgctcgagc tgcttaccaa
gctgggccgc 300gacgacgtgc tgctggagct gggacccagc attgaggagg
attgccaaaa gtatatcttg 360aagcagcagc aggaggaggc tgagaagcct
ttacaggtgg ccgctgtaga cagcagtgtc 420ccacggacag cagagctggc
gggcatcacc acacttgatg accccctggg gcatatgcct 480gagcgtttcg
atgccttcat ctgctattgc cccagcgaca tcgtcgac 5286172PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
6Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser1 5
10 15Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg
Arg 20 25 30Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp
Trp Thr 35 40 45Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile
Arg Gln Leu 50 55 60Glu Thr Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp
Ala Trp Gln Gly65 70 75 80Arg Pro Gly Ala Ser Val Gly Arg Leu Leu
Glu Leu Leu Thr Lys Leu 85 90 95Gly Arg Asp Asp Val Leu Leu Glu Leu
Gly Pro Ser Ile Glu Glu Asp 100 105 110Cys Gln Lys Tyr Ile Leu Lys
Gln Gln Gln Glu Glu Ala Glu Lys Pro 115 120 125Leu Gln Val Ala Ala
Val Asp Ser Ser Val Pro Arg Thr Ala Glu Leu 130 135 140Ala Gly Ile
Thr Thr Leu Asp Asp Pro Leu Gly His Met Pro Glu Arg145 150 155
160Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp Ile 165
1707678DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 7ctcgagggcg tccaagtcga aaccattagt
cccggcgatg gcagaacatt tcctaaaagg 60ggacaaacat gtgtcgtcca ttatacaggc
atgttggagg acggcaaaaa ggtggacagt 120agtagagatc gcaataaacc
tttcaaattc atgttgggaa aacaagaagt cattagggga 180tgggaggagg
gcgtggctca aatgtccgtc ggccaacgcg ctaagctcac catcagcccc
240gactacgcat acggcgctac cggacatccc ggaattattc cccctcacgc
taccttggtg 300tttgacgtcg aactgttgaa gctcgaagtc gagggagtgc
aggtggaaac catctcccca 360ggagacgggc gcaccttccc caagcgcggc
cagacctgcg tggtgcacta caccgggatg 420cttgaagatg gaaagaaagt
tgattcctcc cgggacagaa acaagccctt taagtttatg 480ctaggcaagc
aggaggtgat ccgaggctgg gaagaagggg ttgcccagat gagtgtgggt
540cagagagcca aactgactat atctccagat tatgcctatg gtgccactgg
gcacccaggc 600atcatcccac cacatgccac tctcgtcttc gatgtggagc
ttctaaaact ggaatctggc 660ggtggatccg gagtcgag 6788222PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
8Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro1 5
10 15Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu
Asp 20 25 30Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe
Lys Phe 35 40 45Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu
Gly Val Ala 50 55 60Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile
Ser Pro Asp Tyr65 70 75 80Ala Tyr Gly Ala Thr Gly His Pro Gly Ile
Ile Pro Pro His Ala Thr 85 90 95Leu Val Phe Asp Val Glu Leu Leu Lys
Leu Glu Val Glu Gly Val Gln 100 105 110Val Glu Thr Ile Ser Pro Gly
Asp Gly Arg Thr Phe Pro Lys Arg Gly 115 120 125Gln Thr Cys Val Val
His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys 130 135 140Val Asp Ser
Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly145 150 155
160Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser
165 170 175Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala
Tyr Gly 180 185 190Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala
Thr Leu Val Phe 195 200 205Asp Val Glu Leu Leu Lys Leu Glu Ser Gly
Gly Gly Ser Gly 210 215 22092148DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 9ctcgagatgg
cctgcacagg cccatcactt cctagcgcct tcgacattct aggtgcagca 60ggccaggaca
agctcttgta tctgaagcac aaactgaaga ccccacgccc aggctgccag
120gggcaggacc tcctgcatgc catggttctc ctgaagctgg gccaggaaac
tgaggccagg 180atctctctag aggcattgaa ggccgatgcg gtggcccggc
tggtggcccg ccagtgggct 240ggcgtggaca gcaccgagga cccagaggag
cccccagatg tgtcctgggc tgtggcccgc 300ttgtaccacc tgctggctga
ggagaagctg tgccccgcct cgctgcggga cgtggcctac 360caggaagccg
tccgcaccct cagctccagg gacgaccacc ggctggggga acttcaggat
420gaggcccgaa accggtgtgg gtgggacatt gctggggatc cagggagcat
ccggacgctc 480cagtccaatc tgggctgcct cccaccatcc tcggctttgc
cctctgggac caggagcctc 540ccacgcccca ttgacggtgt ttcggactgg
agccaagggt gctccctgcg atccactggc 600agccctgcct ccctggccag
caacttggaa atcagccagt cccctaccat gcccttcctc 660agcctgcacc
gcagcccaca tgggcccagc aagctctgtg acgaccccca ggccagcttg
720gtgcccgagc ctgtccccgg tggctgccag gagcctgagg agatgagctg
gccgccatcg 780ggggagattg ccagcccacc agagctgcca agcagcccac
ctcctgggct tcccgaagtg 840gccccagatg caacctccac tggcctccct
gatacccccg cagctccaga aaccagcacc 900aactacccag tggagtgcac
cgaggggtct gcaggccccc agtctctccc cttgcctatt 960ctggagccgg
tcaaaaaccc ctgctctgtc aaagaccaga cgccactcca actttctgta
1020gaagatacca cctctccaaa taccaagccg tgcccaccta ctcccaccac
cccagaaaca 1080tcccctcctc ctcctcctcc tcctccttca tctactcctt
gttcagctca cctgaccccc 1140tcctccctgt tcccttcctc
cctggaatca tcatcggaac agaaattcta taactttgtg 1200atcctccacg
ccagggcaga cgaacacatc gccctgcggg ttcgggagaa gctggaggcc
1260cttggcgtgc ccgacggggc caccttctgc gaggatttcc aggtgccggg
gcgcggggag 1320ctgagctgcc tgcaggacgc catagaccac tcagctttca
tcatcctact tctcacctcc 1380aacttcgact gtcgcctgag cctgcaccag
gtgaaccaag ccatgatgag caacctcacg 1440cgacaggggt cgccagactg
tgtcatcccc ttcctgcccc tggagagctc cccggcccag 1500ctcagctccg
acacggccag cctgctctcc gggctggtgc ggctggacga acactcccag
1560atcttcgcca ggaaggtggc caacaccttc aagccccaca ggcttcaggc
ccgaaaggcc 1620atgtggagga aggaacagga cacccgagcc ctgcgggaac
agagccaaca cctggacggt 1680gagcggatgc aggcggcggc actgaacgca
gcctactcag cctacctcca gagctacttg 1740tcctaccagg cacagatgga
gcagctccag gtggcttttg ggagccacat gtcatttggg 1800actggggcgc
cctatggggc tcgaatgccc tttgggggcc aggtgcccct gggagccccg
1860ccaccctttc ccacttggcc ggggtgcccg cagccgccac ccctgcacgc
atggcaggct 1920ggcacccccc caccgccctc cccacagcca gcagcctttc
cacagtcact gcccttcccg 1980cagtccccag ccttccctac ggcctcaccc
gcaccccctc agagcccagg gctgcaaccc 2040ctcattatcc accacgcaca
gatggtacag ctggggctga acaaccacat gtggaaccag 2100agagggtccc
aggcgcccga ggacaagacg caggaggcag aagtcgac 214810164PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
10Met Ala Cys Thr Gly Pro Ser Leu Pro Ser Ala Phe Asp Ile Leu Gly1
5 10 15Ala Ala Gly Gln Asp Lys Leu Leu Tyr Leu Lys His Lys Leu Lys
Thr 20 25 30Pro Arg Pro Gly Cys Gln Gly Gln Asp Leu Leu His Ala Met
Val Leu 35 40 45Leu Lys Leu Gly Gln Glu Thr Glu Ala Arg Ile Ser Leu
Glu Ala Leu 50 55 60Lys Ala Asp Ala Val Ala Arg Leu Val Ala Arg Gln
Trp Ala Gly Val65 70 75 80Asp Ser Thr Glu Asp Pro Glu Glu Pro Pro
Asp Val Ser Trp Ala Val 85 90 95Ala Arg Leu Tyr His Leu Leu Ala Glu
Glu Lys Leu Cys Pro Ala Ser 100 105 110Leu Arg Asp Val Ala Tyr Gln
Glu Ala Val Arg Thr Leu Ser Ser Arg 115 120 125Asp Asp His Arg Leu
Gly Glu Leu Gln Asp Glu Ala Arg Asn Arg Cys 130 135 140Gly Trp Asp
Ile Ala Gly Asp Pro Gly Ser Ile Arg Thr Leu Gln Ser145 150 155
160Asn Leu Gly Cys11642DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 11ctcgagacca
ccgagcagcg acgcagcctg caagccttcc aggattatat ccggaagacc 60ctggacccta
cctacatcct gagctacatg gccccctggt ttagggagga agaggtgcag
120tatattcagg ctgagaaaaa caacaagggc ccaatggagg ctgccacact
ttttctcaag 180ttcctgttgg agctccagga ggaaggctgg ttccgtggct
ttttggatgc cctagaccat 240gcaggttatt ctggacttta tgaagccatt
gaaagttggg atttcaaaaa aattgaaaag 300ttggaggagt atagattact
tttaaaacgt ttacaaccag aatttaaaac cagaattatc 360ccaaccgata
tcatttctga tctgtctgaa tgtttaatta atcaggaatg tgaagaaatt
420ctacagattt gctctactaa ggggatgatg gcaggtgcag agaaattggt
ggaatgcctt 480ctcagatcag acaaggaaaa ctggcccaaa actttgaaac
ttgctttgga gaaagaaagg 540aacaagttca gtgaactgtg gattgtagag
aaaggtataa aagatgttga aacagaagat 600cttgaggata agatggaaac
ttctgacata cagattgtcg ac 64212210PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 12Thr Thr Glu Gln Arg
Arg Ser Leu Gln Ala Phe Gln Asp Tyr Ile Arg1 5 10 15Lys Thr Leu Asp
Pro Thr Tyr Ile Leu Ser Tyr Met Ala Pro Trp Phe 20 25 30Arg Glu Glu
Glu Val Gln Tyr Ile Gln Ala Glu Lys Asn Asn Lys Gly 35 40 45Pro Met
Glu Ala Ala Thr Leu Phe Leu Lys Phe Leu Leu Glu Leu Gln 50 55 60Glu
Glu Gly Trp Phe Arg Gly Phe Leu Asp Ala Leu Asp His Ala Gly65 70 75
80Tyr Ser Gly Leu Tyr Glu Ala Ile Glu Ser Trp Asp Phe Lys Lys Ile
85 90 95Glu Lys Leu Glu Glu Tyr Arg Leu Leu Leu Lys Arg Leu Gln Pro
Glu 100 105 110Phe Lys Thr Arg Ile Ile Pro Thr Asp Ile Ile Ser Asp
Leu Ser Glu 115 120 125Cys Leu Ile Asn Gln Glu Cys Glu Glu Ile Leu
Gln Ile Cys Ser Thr 130 135 140Lys Gly Met Met Ala Gly Ala Glu Lys
Leu Val Glu Cys Leu Leu Arg145 150 155 160Ser Asp Lys Glu Asn Trp
Pro Lys Thr Leu Lys Leu Ala Leu Glu Lys 165 170 175Glu Arg Asn Lys
Phe Ser Glu Leu Trp Ile Val Glu Lys Gly Ile Lys 180 185 190Asp Val
Glu Thr Glu Asp Leu Glu Asp Lys Met Glu Thr Ser Asp Ile 195 200
205Gln Ile 21013789DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 13ctcgagatgg gggaagaggg
tggttcagcc tctcacgatg aggaggaaag agcaagtgtc 60ctcctcggac attctccggg
ttgtgaaatg tgctcgcagg aggcttttca ggcacagagg 120agccagctgg
tcgagctgct ggtctcaggg tccctggaag gcttcgagag tgtcctggac
180tggctgctgt cctgggaggt cctctcctgg gaggactacg agggcttcca
cctcctgggc 240cagcctctct cccacttggc caggcgcctt ctggacaccg
tctggaataa gggtacttgg 300gcctgtcaga agctcatcgc ggctgcccaa
gaagcccagg ccgacagcca gtcccccaag 360ctgcatggct gctgggaccc
ccactcgctc cacccagccc gagacctgca gagtcaccgg 420ccagccattg
tcaggaggct ccacagccat gtggagaaca tgctggacct ggcatgggag
480cggggtttcg tcagccagta tgaatgtgat gaaatcaggt tgccgatctt
cacaccgtcc 540cagagggcaa gaaggctgct tgatcttgcc acggtgaaag
cgaatggatt ggctgccttc 600cttctacaac atgttcagga attaccagtc
ccattggccc tgcctttgga agctgccaca 660tgcaagaagt atatggccaa
gctgaggacc acggtgtctg ctcagtctcg cttcctcagt 720acctatgatg
gagcagagac gctctgcctg gaggacatat acacagagaa tgtcctggag 780gtcgtcgac
78914259PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 14Met Gly Glu Glu Gly Gly Ser Ala Ser His Asp
Glu Glu Glu Arg Ala1 5 10 15Ser Val Leu Leu Gly His Ser Pro Gly Cys
Glu Met Cys Ser Gln Glu 20 25 30Ala Phe Gln Ala Gln Arg Ser Gln Leu
Val Glu Leu Leu Val Ser Gly 35 40 45Ser Leu Glu Gly Phe Glu Ser Val
Leu Asp Trp Leu Leu Ser Trp Glu 50 55 60Val Leu Ser Trp Glu Asp Tyr
Glu Gly Phe His Leu Leu Gly Gln Pro65 70 75 80Leu Ser His Leu Ala
Arg Arg Leu Leu Asp Thr Val Trp Asn Lys Gly 85 90 95Thr Trp Ala Cys
Gln Lys Leu Ile Ala Ala Ala Gln Glu Ala Gln Ala 100 105 110Asp Ser
Gln Ser Pro Lys Leu His Gly Cys Trp Asp Pro His Ser Leu 115 120
125His Pro Ala Arg Asp Leu Gln Ser His Arg Pro Ala Ile Val Arg Arg
130 135 140Leu His Ser His Val Glu Asn Met Leu Asp Leu Ala Trp Glu
Arg Gly145 150 155 160Phe Val Ser Gln Tyr Glu Cys Asp Glu Ile Arg
Leu Pro Ile Phe Thr 165 170 175Pro Ser Gln Arg Ala Arg Arg Leu Leu
Asp Leu Ala Thr Val Lys Ala 180 185 190Asn Gly Leu Ala Ala Phe Leu
Leu Gln His Val Gln Glu Leu Pro Val 195 200 205Pro Leu Ala Leu Pro
Leu Glu Ala Ala Thr Cys Lys Lys Tyr Met Ala 210 215 220Lys Leu Arg
Thr Thr Val Ser Ala Gln Ser Arg Phe Leu Ser Thr Tyr225 230 235
240Asp Gly Ala Glu Thr Leu Cys Leu Glu Asp Ile Tyr Thr Glu Asn Val
245 250 255Leu Glu Val15888DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 15atggctgcag
gaggtcccgg cgcggggtct gcggccccgg tctcctccac atcctccctt 60cccctggctg
ctctcaacat gcgagtgcgg cgccgcctgt ctctgttctt gaacgtgcgg
120acacaggtgg cggccgactg gaccgcgctg gcggaggaga tggactttga
gtacttggag 180atccggcaac tggagacaca agcggacccc actggcaggc
tgctggacgc ctggcaggga 240cgccctggcg cctctgtagg ccgactgctc
gagctgctta ccaagctggg ccgcgacgac 300gtgctgctgg agctgggacc
cagcattgag gaggattgcc aaaagtatat cttgaagcag 360cagcaggagg
aggctgagaa gcctttacag gtggccgctg tagacagcag tgtcccacgg
420acagcagagc tggcgggcat caccacactt gatgaccccc tggggcatat
gcctgagcgt 480ttcgatgcct tcatctgcta ttgccccagc gacatccagt
ttgtgcagga gatgatccgg 540caactggaac agacaaacta tcgactgaag
ttgtgtgtgt ctgaccgcga tgtcctgcct 600ggcacctgtg tctggtctat
tgctagtgag ctcatcgaaa agaggtgccg ccggatggtg 660gtggttgtct
ctgatgatta cctgcagagc aaggaatgtg acttccagac caaatttgca
720ctcagcctct ctccaggtgc ccatcagaag cgactgatcc ccatcaagta
caaggcaatg 780aagaaagagt tccccagcat cctgaggttc atcactgtct
gcgactacac caacccctgc 840accaaatctt ggttctggac tcgccttgcc
aaggccttgt ccctgccc 88816296PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 16Met Ala Ala Gly Gly Pro
Gly Ala Gly Ser Ala Ala Pro Val Ser Ser1 5 10 15Thr Ser Ser Leu Pro
Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg 20 25 30Leu Ser Leu Phe
Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr 35 40 45Ala Leu Ala
Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu 50 55 60Glu Thr
Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gln Gly65 70 75
80Arg Pro Gly Ala Ser Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu
85 90 95Gly Arg Asp Asp Val Leu Leu Glu Leu Gly Pro Ser Ile Glu Glu
Asp 100 105 110Cys Gln Lys Tyr Ile Leu Lys Gln Gln Gln Glu Glu Ala
Glu Lys Pro 115 120 125Leu Gln Val Ala Ala Val Asp Ser Ser Val Pro
Arg Thr Ala Glu Leu 130 135 140Ala Gly Ile Thr Thr Leu Asp Asp Pro
Leu Gly His Met Pro Glu Arg145 150 155 160Phe Asp Ala Phe Ile Cys
Tyr Cys Pro Ser Asp Ile Gln Phe Val Gln 165 170 175Glu Met Ile Arg
Gln Leu Glu Gln Thr Asn Tyr Arg Leu Lys Leu Cys 180 185 190Val Ser
Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp Ser Ile Ala 195 200
205Ser Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val Val Val Val Ser
210 215 220Asp Asp Tyr Leu Gln Ser Lys Glu Cys Asp Phe Gln Thr Lys
Phe Ala225 230 235 240Leu Ser Leu Ser Pro Gly Ala His Gln Lys Arg
Leu Ile Pro Ile Lys 245 250 255Tyr Lys Ala Met Lys Lys Glu Phe Pro
Ser Ile Leu Arg Phe Ile Thr 260 265 270Val Cys Asp Tyr Thr Asn Pro
Cys Thr Lys Ser Trp Phe Trp Thr Arg 275 280 285Leu Ala Lys Ala Leu
Ser Leu Pro 290 2951716PRTHomo sapiens 17Met Gly Ser Asn Lys Ser
Lys Pro Lys Asp Ala Ser Gln Arg Arg Arg1 5 10 15185PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Met
Gly Cys Xaa Cys1 5199PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 19Phe Leu Trp Gly Pro Arg Ala
Leu Val1 5209PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 20Gly Ile Leu Gly Phe Val Phe Thr Leu1
5219PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21Ser Leu Tyr Asn Thr Val Ala Thr Leu1
52221PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 22Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val
Pro Lys Val Ser1 5 10 15Ala Ser His Leu Glu202335DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
23atatactcga gaaaaaggtg gccaagaagc caacc 352436DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
24atatagtcga ctcactgtct ctcctgcact gagatg 36256PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag
25His His His His His His1 5268PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 26Ser Ile Ile Asn Phe Glu Lys
Leu1 52736DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 27cgatcactcg agggctggag gatatctttt tattgg
362838DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 28tgatcggtcg acatgtacag agtttttgga tccaagtg
382940DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 29cgatcactcg agtataagtt ctattttcac ctgatgcttc
403037DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 30tgatcggtcg acgatagatg ttgcttcctg ccaattg
373138DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 31cgatcagtcg acgatgtgtg gtatatttac catttctg
383235DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 32tgatcggtcg acgaccgttt ccttgaacac ctgac
353338DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 33cgatcactcg aggatgtttg gtttatatat aatgtgtg
383434DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 34tcggtcgacg tattgcttaa tggaatcgac atac
343534DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 35cgatcactcg aggacctctg gtactgcttc cacc
343632DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 36tgatctgtcg acttcggccg tgggtccctg gc
323738DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 37aatctaccgc ggccaccatg atgtctgcct cgcgcctg
383837DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 38tcagttctcg aggatagatg ttgcttcctg ccaattg
373933DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 39acatagtcga cctgtctctc ctgcactgag atg
334034DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 40atagcactcg agatggggga agagggtggt tcag
344134DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 41cttcatgtcg acgacctcca ggacattctc tgtg
344232DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 42atagcactcg agaccaccga gcagcgacgc ag
324337DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 43cttcatgtcg acaatctgta tgtcagaagt ttccatc
374433DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 44acatcaactc gagatggctg caggaggtcc cgg
334534DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 45actcatagtc gaccagggac aaggccttgg caag
344635DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 46acatcaactc gagatggcct gcacaggccc atcac
354735DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 47actcatagtc gacttctgcc tcctgcgtct tgtcc
354842DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 48aactagacgc gtactactaa aatgtaaatg acataggaaa ac
424936DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 49gacttgaagc ttaacacgaa cagtgtcgcc tactac
365045DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 50ggaggcggag gcagcggagg tggcggttcc
ggaggcggag gttct 455115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 51Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 155217PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 52Ile
Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly1 5 10
15Arg539PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 53Ser Val Tyr Asp Phe Phe Val Trp Leu1 5
* * * * *
References