U.S. patent application number 17/517632 was filed with the patent office on 2022-03-31 for allogeneic tumor cell vaccine.
This patent application is currently assigned to ALLOPLEX BIOTHERAPEUTICS, INC.. The applicant listed for this patent is ALLOPLEX BIOTHERAPEUTICS, INC.. Invention is credited to Frank Borriello.
Application Number | 20220096631 17/517632 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-31 |
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United States Patent
Application |
20220096631 |
Kind Code |
A1 |
Borriello; Frank |
March 31, 2022 |
ALLOGENEIC TUMOR CELL VACCINE
Abstract
The described invention provides allogeneic tumor cell vaccines
comprising tumor cell lines or tumor cell line variants that are
genetically engineered to express a core group of three
immunomodulatory molecules, and optionally additional R
immunomodulatory polypeptides for induction of one or more
subpopulations of PBMCs to proliferate in response to the expressed
immunomodulatory molecules and to then enter an effector phase for
killing of tumor cells. According to some embodiments, the tumor
cell vaccine candidate can induce an immune response in the
recipient cancer patient that cross reacts with the patient's own
(autologous) tumor cells, the effects of which are sufficient to
result in enhanced anti-tumor immunity contributing to the
increased survival of a vaccinated patient cohort compared to a
matched unvaccinated patient cohort.
Inventors: |
Borriello; Frank;
(Winchester, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALLOPLEX BIOTHERAPEUTICS, INC. |
Woburn |
MA |
US |
|
|
Assignee: |
ALLOPLEX BIOTHERAPEUTICS,
INC.
Woburn
MA
|
Appl. No.: |
17/517632 |
Filed: |
November 2, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16899318 |
Jun 11, 2020 |
11185586 |
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17517632 |
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15821105 |
Nov 22, 2017 |
11058752 |
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16899318 |
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62425424 |
Nov 22, 2016 |
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International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/00 20060101 A61K039/00; A61P 35/00 20060101
A61P035/00 |
Claims
1. An allogeneic tumor cell vaccine comprising: (1) a population of
live, proliferation-incompetent genetically engineered tumor cells
expressing one or more tumor specific antigens, the population
comprising at least three stably expressed immunomodulatory
molecules, wherein the at least three immunomodulatory molecules
are OX40 Ligand (OX40L), CD27 Ligand (CD70) and CD28 Ligand (CD28L)
comprising CD80, CD86, or both, for induction of one or more
subpopulations of PBMCs to proliferate in response to the expressed
immunomodulatory molecules and to then enter an effector phase for
killing of tumor cells; wherein the subpopulations of PBMC cells
comprise one or more of T-lymphocytes, natural killer (NK) cells,
dendritic cells (DCs) or B lymphocytes; and (2) a pharmaceutically
acceptable carrier.
2. The allogeneic tumor cell vaccine of claim 1, wherein (a) the
population of live, proliferation-incompetent genetically
engineered tumor cells expressing one or more tumor specific
antigens further comprises one or more additional stably expressed
immunomodulatory molecules selected from R.sup.1-R.sup.44; or (b)
the tumor cells are rendered proliferation-incompetent by
irradiation; or (c) the tumor cell is derived from a cancer
selected from the group consisting of: melanoma, colorectal
carcinoma, leukemia, chronic myeloid leukemia, prostate cancer,
head and neck cancer, Squamous Cell Carcinoma, tongue cancer,
larynx cancer, tonsil cancer, hypopharynx cancer, nasalpharynx
cancer, breast cancer, colon cancer, lung cancer, pancreatic
cancer, glioblastoma and brain cancer; or (d) the population of
live, proliferation-resistant tumor cells is derived from a
biological sample derived from a subject; or (e) the population of
live proliferation resistant tumor cells is derived from a tumor
cell line; or (f) the immunostimulatory molecules are presented at
the exterior surface of the genetically engineered tumor cells; or
(g) induction of the T-lymphocytes comprises activation of the
subpopulation of T lymphocytes, expansion of the T lymphocytes, or
both; or (h) induction of the NK cells comprises activation of the
subpopulation of NK cells, expansion of the subpopulation of NK
cells or both; or (i) induction of the subpopulation of DCs
comprises activation of the subpopulation of DCs, expansion of the
subpopulation of DCs or both; or (j) induction of the subpopulation
of B lymphocytes comprises activation of the subpopulation of B
lymphocytes, expansion of the subpopulation of B lymphocytes or
both.
3. The allogeneic tumor cell vaccine of claim 1, wherein (a) the
subpopulation of T lymphocytes comprises a subpopulation of CD8+
cytotoxic T-lymphocytes (CTL); or (b) the subpopulation of T
lymphocytes comprises a subpopulation of memory T cells; or (c) the
subpopulation of T lymphocytes comprises a subpopulation of
regulatory T cells; or (d) the subpopulation of T lymphocytes
comprises a subpopulation of helper T cells; or (e) the
subpopulation of B lymphocytes comprises a subpopulation of memory
B cells.
4. The allogeneic tumor cell vaccine of claim 1, wherein: (1) the
vaccine enhances immune activation of cells effective to recognize
and act against those tumor cells that comprise the target tumor
antigen in vivo without systemic inflammation; or (2) the vaccine
reduces immunosuppression in a tumor microenvironment for tumor
cells comprising the target tumor antigen; or (3) the vaccine
increases cell death of tumor cells expressing the target tumor
antigen; or (4) the population of live, proliferation-resistant
tumor cells elicits immune activation without systemic
inflammation; or (5) the vaccine elicits an immune response that
improves progression free survival, overall survival, or both
relative to placebo controls.
5. The allogeneic tumor cell vaccine of claim 2, wherein the
melanoma tumor cell is characterized by expression of one or more
of gp100, tyrosinase, Melan-A, tyrosinase-related protein
(TRP-2-INT2), melanoma antigen-1 (MAGE-A1), NY-ESO-1,
preferentially expressed antigen of melanoma (PRAME) CDK4 and
multiple myeloma oncogene 1 (MUM-1).
6. The allogeneic tumor cell vaccine of claim 2, wherein the
colorectal cancer tumor cell is characterized by expression of one
or more of carcinoembryonic antigen (CEA), MAGE, HPV, human
telomerase reverse transcriptase (hTERT), EPCAM, PD-1, PD-L1, p53,
and cell surface-associated mucin 1 (MUC1).
7. The allogeneic tumor cell vaccine of claim 2, wherein the one or
more additional stably expressed immunomodulatory molecules
selected from R.sup.1-R.sup.44 is a cytokine, a TNF-family member,
a secreted receptor, a chaperone, an IgG superfamily member and/or
a chemokine receptor.
8. The allogeneic tumor cell vaccine of claim 1, produced by a
process comprising: providing an allogeneic parental tumor cell
line comprising a population of live tumor cells; introducing into
the population of live tumor cells an exogenous nucleic acid
encoding a stably expressed immunomodulatory molecule, wherein the
immunomodulatory molecule is OX40 Ligand (OX40L); introducing into
the population of live tumor cells an exogenous nucleic acid
encoding a stably expressed immunomodulatory molecule, wherein the
immunomodulatory molecule is CD27 Ligand (CD70); introducing into
the population of live tumor cells an exogenous nucleic acid
encoding a stably expressed immunomodulatory molecule, wherein the
immunomodulatory molecule is CD28 Ligand (CD28L) comprising CD80,
CD86, or both; wherein stable expression of OX40 Ligand (OX40L),
CD27 Ligand (CD70) and CD28 Ligand (CD28L) comprising CD80, CD86,
or both induces one or more subpopulations of PBMCs to proliferate
in response to the expressed immunomodulatory molecules and to then
enter an effector phase for killing of tumor cells; generating
tumor cell line variants by selecting for tumor cell clones that
stably express an immunogenic amount of the exogenous subset of the
immunomodulatory molecules; and selecting in a mixed lymphocyte
tumor cell reaction clonally derived cell line variants by one or
more of the following parameters selected from: cellular
proliferation, cellular subset differentiation, cytokine release
profile, and tumor cell lysis; wherein the selected clonally
derived cell line variant is effective to stimulate activation of
one or more of T-lymphocytes, natural killer (NK) cells, dendritic
cells (DCs) or B lymphocytes.
9. The allogeneic tumor cell vaccine produced by the process of
claim 8, further comprising introducing into the population of live
tumor cells an exogenous nucleic acid encoding one or more stably
expressed immunomodulatory molecules selected from
R.sub.1-R.sup.44.
10. The allogeneic tumor cell vaccine produced by the process of
claim 8, wherein (a) the tumor cells are rendered proliferation
incompetent by irradiation; or (b) the parental tumor cell line is
from a melanoma or from a colorectal carcinoma; or (c) the
exogenous nucleic acid comprises DNA or RNA; or (d) the introducing
step comprises viral transduction; or (e) the introducing step
comprises electroporation; or (f) the introducing step comprises
utilizing one or more of: liposome mediated transfer, adenovirus,
adeno-associated virus, herpes virus, a retroviral based vector,
lipofection, and a lentiviral vector; or (g) the introducing step
comprises introducing the exogenous nucleic acid by transfection of
a lentiviral vector.
11. A method of inducing an immune response to a cancer in a
subject comprising: (a) administering to the subject parenterally
or locally into a tumor an allogeneic tumor cell vaccine comprising
(1) a population of live, proliferation-incompetent genetically
engineered tumor cells expressing one or more tumor specific
antigens, the population comprising at least three stably expressed
immunomodulatory molecules, wherein the at least three
immunomodulatory molecules are OX40 Ligand (OX40L), CD27 Ligand
(CD70) and CD28 Ligand (CD28L) comprising, wherein CD28 Ligand
comprises CD80, CD86, or both, and (2) a pharmaceutically
acceptable carrier, (b) inducing one or more subpopulations of
peripheral blood mononuclear cells (PBMCs) to proliferate in
response to the expressed immunomodulatory molecules and to then
enter an effector phase for killing of tumor cells; wherein the
subpopulations of PBMC cells comprise one or more of T-lymphocytes,
natural killer (NK) cells, dendritic cells (DCs) or B lymphocytes;
and wherein the allogeneic tumor cell vaccine is type-matched to
the subject's cancer.
12. The method of claim 11, wherein the cancer is selected from
melanoma or colorectal cancer.
13. The method of claim 11, wherein the subject has an infectious
viral disease with progression to a cancer.
14. The method of claim 11, further comprising administering a
checkpoint inhibitor to the subject.
15. The method of claim 11, wherein the population of live,
proliferation-incompetent genetically engineered tumor cells
expressing one or more tumor specific antigens further comprises
one or more additional stably expressed immunomodulatory molecules
selected from R.sup.1-R.sup.44.
16. The method according to claim 11, wherein (a) the population of
live, proliferation-resistant tumor cells is derived from a
biological sample derived from a subject; or (b) the population of
live proliferation resistant tumor cells is derived from a tumor
cell line.
17-22. (canceled)
Description
RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. application
Ser. No. 16/899,318 (filed Jun. 11, 2020), which is a continuation
in part of U.S. application Ser. No. 15/821,105 (filed Nov. 22,
2017), which claims the benefit of priority to U.S. provisional
application No. 62/425,424 (filed Nov. 22, 2016).
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Nov. 2, 2021 is named 128663-00105_SL.txt and is 263,710 bytes
in size.
FIELD OF THE INVENTION
[0003] The described invention relates generally to immunological
approaches to the treatment of cancer, and more particularly to
cancer vaccines comprising modified tumor cells.
BACKGROUND OF THE INVENTION
[0004] The human immune system may generally be divided into two
arms, referred to as "innate immunity" and "adaptive immunity." The
innate arm of the immune system is predominantly responsible for an
initial inflammatory response via a number of soluble factors,
including the complement system and the chemokine/cytokine system;
and a number of specialized cell types including mast cells,
macrophages, dendritic cells (DCs), and natural killer cells. The
adaptive immune arm involves a delayed and a longer lasting
antibody response together with CD8+ and CD4+ T cell responses that
play a critical role in immunological memory against an antigen. A
third arm of the immune system may be identified as involving
.gamma..delta. T cells and T cells with limited T cell receptor
repertoires such as natural killer T (NKT) cells and
Mucosal-associated invariant T (MAIT) cells.
Cells of the Immune System
[0005] There are a large number of cellular interactions that
comprise the immune system. These interactions occur through
specific receptor-ligand pairs that signal in both directions so
that each cell receives instructions based on the temporal and
spatial distribution of those signals.
[0006] Murine models have been highly useful in discovering
immunomodulatory pathways, but clinical utility of these pathways
does not always translate from an inbred mouse strain to an outbred
human population, since an outbred human population may have
individuals that rely to varying extents on individual
immunomodulatory pathways.
[0007] Cells of the immune system include lymphocytes,
monocytes/macrophages, dendritic cells, the closely related
Langerhans cells, natural killer (NK) cells, mast cells, basophils,
and other members of the myeloid lineage of cells. In addition, a
series of specialized epithelial and stromal cells provide the
anatomic environment in which immunity occurs, often by secreting
critical factors that regulate growth and/or gene activation in
cells of the immune system, which also play direct roles in the
induction and effector phases of the response. (Paul, W. E.,
"Chapter 1: The immune system: an introduction," Fundamental
Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven
Publishers, Philadelphia, (1999), at p. 102).
[0008] The cells of the immune system are found in peripheral
organized tissues, such as the spleen, lymph nodes, Peyer's patches
of the intestine and tonsils. Lymphocytes also are found in the
central lymphoid organs, the thymus, and bone marrow where they
undergo developmental steps that equip them to mediate the myriad
responses of the mature immune system. A substantial portion of
lymphocytes and macrophages comprise a recirculating pool of cells
found in the blood and lymph, providing the means to deliver
immunocompetent cells to sites where they are needed and to allow
immunity that is generated locally to become generalized. (Paul, W.
E., "Chapter 1: The immune system: an introduction," Fundamental
Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven
Publishers, Philadelphia, (1999), at p. 102).
[0009] The term "lymphocyte" refers to a small white blood cell
formed in lymphatic tissue throughout the body and in normal adults
making up about 22-28% of the total number of leukocytes in the
circulating blood that plays a large role in defending the body
against disease. Individual lymphocytes are specialized in that
they are committed to respond to a limited set of structurally
related antigens through recombination of their genetic material
(e.g. to create a T cell receptor and a B cell receptor). This
commitment, which exists before the first contact of the immune
system with a given antigen, is expressed by the presence of
receptors specific for determinants (epitopes) on the antigen on
the lymphocyte's surface membrane. Each lymphocyte possesses a
unique population of receptors, all of which have identical
combining sites. One set, or clone, of lymphocytes differs from
another clone in the structure of the combining region of its
receptors and thus differs in the epitopes that it can recognize.
Lymphocytes differ from each other not only in the specificity of
their receptors, but also in their functions. (Paul, W. E.,
"Chapter 1: The immune system: an introduction," Fundamental
Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven
Publishers, Philadelphia, (1999), at p. 102).
[0010] Two broad classes of lymphocytes are recognized: the
B-lymphocytes (B-cells), which are precursors of antibody-secreting
cells, and T-lymphocytes (T-cells).
[0011] B-Lymphocytes
[0012] B-lymphocytes are derived from hematopoietic cells of the
bone marrow. A mature B-cell can be activated with an antigen that
expresses epitopes that are recognized by its cell surface. The
activation process may be direct, dependent on cross-linkage of
membrane Ig molecules by the antigen (cross-linkage-dependent
B-cell activation), or indirect, via interaction with a helper
T-cell, in a process referred to as cognate help. In many
physiological situations, receptor cross-linkage stimuli and
cognate help synergize to yield more vigorous B-cell responses
(Paul, W. E., "Chapter 1: The immune system: an introduction,"
Fundamental Immunology, 4th Edition, Ed. Paul, W. E.,
Lippicott-Raven Publishers, Philadelphia, (1999)).
[0013] Cross-linkage dependent B-cell activation requires that the
antigen express multiple copies of the epitope complementary to the
binding site of the cell surface receptors, because each B-cell
expresses Ig molecules with identical variable regions. Such a
requirement is fulfilled by other antigens with repetitive
epitopes, such as capsular polysaccharides of microorganisms or
viral envelope proteins. Cross-linkage-dependent B-cell activation
is a major protective immune response mounted against these
microbes (Paul, W. E., "Chapter 1: The immune system: an
introduction", Fundamental Immunology, 4th Edition, Ed. Paul, W.
E., Lippicott-Raven Publishers, Philadelphia, (1999)).
[0014] Cognate help allows B-cells to mount responses against
antigens that cannot cross-link receptors and, at the same time,
provides costimulatory signals that rescue B cells from
inactivation when they are stimulated by weak cross-linkage events.
Cognate help is dependent on the binding of antigen by the B-cell's
membrane immunoglobulin (Ig), the endocytosis of the antigen, and
its fragmentation into peptides within the endosomal/lysosomal
compartment of the cell. Some of the resultant peptides are loaded
into a groove in a specialized set of cell surface proteins known
as class II major histocompatibility complex (MHC) molecules. The
resultant class II/peptide complexes are expressed on the cell
surface and act as ligands for the antigen-specific receptors of a
set of T-cells designated as CD4.sup.+ T-cells. The CD4.sup.+
T-cells bear receptors on their surface specific for the B-cell's
class II/peptide complex. B-cell activation depends not only on the
binding of the T cell through its T cell receptor (TCR), but this
interaction also allows an activation ligand on the T-cell (CD40
ligand) to bind to its receptor on the B-cell (CD40) signaling
B-cell activation. In addition, T helper cells secrete several
cytokines that regulate the growth and differentiation of the
stimulated B-cell by binding to cytokine receptors on the B cell
(Paul, W. E., "Chapter 1: The immune system: an introduction,
"Fundamental Immunology, 4th Edition, Ed. Paul, W. E.,
Lippicott-Raven Publishers, Philadelphia, (1999)).
[0015] During cognate help for antibody production, the CD40 ligand
is transiently expressed on activated CD4.sup.+ T helper cells, and
it binds to CD40 on the antigen-specific B cells, thereby
transducing a second costimulatory signal. The latter signal is
essential for B cell growth and differentiation and for the
generation of memory B cells by preventing apoptosis of germinal
center B cells that have encountered antigen. Hyperexpression of
the CD40 ligand in both B and T cells is implicated in pathogenic
autoantibody production in human SLE patients (Desai-Mehta, A. et
al., "Hyperexpression of CD40 ligand by B and T cells in human
lupus and its role in pathogenic autoantibody production," J. Clin.
Invest. Vol. 97(9), 2063-2073, (1996)).
[0016] T-Lymphocytes
[0017] T-lymphocytes derived from precursors in hematopoietic
tissue undergo differentiation in the thymus, and are then seeded
to peripheral lymphoid tissue and to the recirculating pool of
lymphocytes. T-lymphocytes or T cells mediate a wide range of
immunologic functions. These include the capacity to help B cells
develop into antibody-producing cells, the capacity to increase the
microbicidal action of monocytes/macrophages, the inhibition of
certain types of immune responses, direct killing of target cells,
and mobilization of the inflammatory response. These effects depend
on T cell expression of specific cell surface molecules and the
secretion of cytokines (Paul, W. E., "Chapter 1: The immune system:
an introduction", Fundamental Immunology, 4th Edition, Ed. Paul, W.
E., Lippicott-Raven Publishers, Philadelphia, (1999)).
[0018] T cells differ from B cells in their mechanism of antigen
recognition. Immunoglobulin, the B cell's receptor, binds to
individual epitopes on soluble molecules or on particulate
surfaces. B-cell receptors see epitopes expressed on the surface of
native molecules. While antibody and B-cell receptors evolved to
bind to and to protect against microorganisms in extracellular
fluids, T cells recognize antigens on the surface of other cells
and mediate their functions by interacting with, and altering, the
behavior of these antigen-presenting cells (APCs). There are three
main types of APCs in peripheral lymphoid organs that can activate
T cells: dendritic cells, macrophages and B cells. The most potent
of these are the dendritic cells, whose only function is to present
foreign antigens to T cells. Immature dendritic cells are located
in tissues throughout the body, including the skin, gut, and
respiratory tract. When they encounter invading microbes at these
sites, they endocytose the pathogens and their products, and carry
them via the lymph to local lymph nodes or gut associated lymphoid
organs. The encounter with a pathogen induces the dendritic cell to
mature from an antigen-capturing cell to an APC that can activate T
cells. APCs display three types of protein molecules on their
surface that have a role in activating a T cell to become an
effector cell: (1) MHC proteins, which present foreign antigen to
the T cell receptor; (2) costimulatory proteins which bind to
complementary receptors on the T cell surface; and (3) cell-cell
adhesion molecules, which enable a T cell to bind to the APC for
long enough to become activated ("Chapter 24: The adaptive immune
system," Molecular Biology of the Cell, Alberts, B. et al., Garland
Science, NY, (2002)).
[0019] T-cells are subdivided into two distinct classes based on
the cell surface receptors they express. The majority of T cells
express T cell receptors (TCR) consisting of a and (3-chains. A
small group of T cells express receptors made of y and 6 chains.
Among the a/(3 T cells are two sub-lineages: those that express the
coreceptor molecule CD4 (CD4.sup.+ T cells); and those that express
CD8 (CD8.sup.+ T cells). These cells differ in how they recognize
antigen and in their effector and regulatory functions.
[0020] CD4.sup.+ T cells are the major regulatory cells of the
immune system. Their regulatory function depends both on the
expression of their cell-surface molecules, such as CD40 ligand
whose expression is induced when the T cells are activated, and the
wide array of cytokines they secrete when activated.
[0021] CD8+ (cytotoxic) T cells, like CD4+ Helper T cells, are
generated in the thymus and express the T-cell receptor. However,
rather than the CD4 molecule, cytotoxic T cells express a dimeric
co-receptor, CD8, usually composed of one CD8.alpha. and one
CD8.beta. chain. CD8+ T cells recognize peptides presented by MHC
Class I molecules, found on all nucleated cells. The CD8
heterodimer binds to a conserved portion (the .alpha.3 region) of
MHC Class I during T cell/antigen presenting cell interactions.
CD8+ T cells (often called cytotoxic T lymphocytes, or CTLs) are
important for immune defense against intracellular pathogens,
including viruses and bacteria, and for tumour surveillance. When a
CD8+ T cell recognizes its antigen and becomes activated, it has
three major mechanisms to kill infected or malignant cells. The
first is secretion of cytokines, primarily TNF-.alpha. and
IFN.gamma., which have anti-tumour and anti-viral microbial
effects. The second major function is the production and release of
cytotoxic granules. These granules, also found in NK cells, contain
two families of proteins, perforin, and granzymes. Perforin forms a
pore in the membrane of the target cell, similar to the membrane
attack complex of complement. This pore allows the granzymes also
contained in the cytotoxic granules to enter the infected or
malignant cell. Granzymes are serine proteases which cleave the
proteins inside the cell, shutting down the production of viral
proteins and ultimately resulting in apoptosis of the target cell.
CD8+ T cells are able to release their granules, kill an infected
cell, then move to a new target and kill again, often referred to
as serial killing. The third major function of CD8+ T cell
destruction of infected cells is via Fas/FasL interactions.
Activated CD8+ T cells express FasL on the cell surface, which
binds to its receptor, Fas, on the surface of the target cell. This
binding causes the Fas molecules on the surface of the target cell
to trimerize, which pulls together signaling molecules. These
signaling molecules result in the activation of the caspase
cascade, which also results in apoptosis of the target cell.
Because CD8+ T cells can express both molecules, Fas/FasL
interactions are a mechanism by which CD8+ T cells can kill each
other, called fratricide, to eliminate immune effector cells during
the contraction phase at the end of an immune response.
[0022] T cells also mediate important effector functions, some of
which are determined by the patterns of cytokines they secrete. The
cytokines can be directly toxic to target cells and can mobilize
potent inflammatory mechanisms.
[0023] In addition, T cells, particularly CD8.sup.+ T cells, can
develop into cytotoxic T-lymphocytes (CTLs) capable of efficiently
lysing target cells that express antigens recognized by the CTLs
(Paul, W. E., "Chapter 1: The immune system: an introduction,"
Fundamental Immunology, 4th Edition, Ed. Paul, W. E.,
Lippicott-Raven Publishers, Philadelphia, (1999)).
[0024] T cell receptors (TCRs) recognize a complex consisting of a
peptide derived by proteolysis of the antigen bound to a
specialized groove of a class II or class I MHC protein. CD4.sup.+
T cells recognize only peptide/class II complexes while CD8.sup.+ T
cells recognize peptide/class I complexes (Paul, W. E., "Chapter 1:
The immune system: an introduction," Fundamental Immunology, 4th
Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia,
(1999)).
[0025] The TCR's ligand (i.e., the peptide/MHC protein complex) is
created within APCs. In general, class II MHC molecules bind
peptides derived from proteins that have been taken up by the APC
through an endocytic process. These peptide-loaded class II
molecules are then expressed on the surface of the cell, where they
are available to be bound by CD4.sup.+ T cells with TCRs capable of
recognizing the expressed cell surface complex. Thus, CD4.sup.+ T
cells are specialized to react with antigens derived from
extracellular sources (Paul, W. E., "Chapter 1: The immune system:
an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W.
E., Lippicott-Raven Publishers, Philadelphia, (1999)).
[0026] In contrast, class I MHC molecules are mainly loaded with
peptides derived from internally synthesized proteins, such as
viral proteins. These peptides are produced from cytosolic proteins
by proteolysis by the proteosome and are translocated into the
rough endoplasmic reticulum. Such peptides, generally composed of
nine amino acids in length, are bound into the class I MHC
molecules and are brought to the cell surface, where they can be
recognized by CD8.sup.+ T cells expressing appropriate receptors.
This gives the T cell system, particularly CD8.sup.+ T cells, the
ability to detect cells expressing proteins that are different
from, or produced in much larger amounts than, those of cells of
the remainder of the organism (e.g., viral antigens) or mutant
antigens (such as active oncogene products), even if these proteins
in their intact form are neither expressed on the cell surface nor
secreted (Paul, W. E., "Chapter 1: The immune system: an
introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W.
E., Lippicott-Raven Publishers, Philadelphia, (1999)).
[0027] T cells can also be classified based on their function as
helper T cells; T cells involved in inducing cellular immunity;
suppressor T cells; and cytotoxic T cells.
[0028] Helper T Cells
[0029] Helper T cells are T cells that stimulate B cells to make
antibody responses to proteins and other T cell-dependent antigens.
T cell-dependent antigens are immunogens in which individual
epitopes appear only once or a limited number of times such that
they are unable to cross-link the membrane immunoglobulin (Ig) of B
cells or do so inefficiently. B cells bind the antigen through
their membrane Ig, and the complex undergoes endocytosis. Within
the endosomal and lysosomal compartments, the antigen is fragmented
into peptides by proteolytic enzymes, and one or more of the
generated peptides are loaded into class II MHC molecules, which
traffic through this vesicular compartment. The resulting
peptide/class II MHC complex is then exported to the B-cell surface
membrane. T cells with receptors specific for the peptide/class II
molecular complex recognize this complex on the B-cell surface.
(Paul, W. E., "Chapter 1: The immune system: an introduction,"
Fundamental Immunology, 4th Edition, Ed. Paul, W. E.,
Lippicott-Raven Publishers, Philadelphia (1999)).
[0030] B-cell activation depends both on the binding of the T cell
through its TCR and on the interaction of the T-cell CD40 ligand
(CD40L) with CD40 on the B cell. T cells do not constitutively
express CD40L. Rather, CD40L expression is induced as a result of
an interaction with an APC that expresses both a cognate antigen
recognized by the TCR of the T cell and CD80 or CD86. CD80/CD86 is
generally expressed by activated, but not resting, B cells so that
the helper interaction involving an activated B cell and a T cell
can lead to efficient antibody production. In many cases, however,
the initial induction of CD40L on T cells is dependent on their
recognition of antigen on the surface of APCs that constitutively
express CD80/86, such as dendritic cells. Such activated helper T
cells can then efficiently interact with and help B cells.
Cross-linkage of membrane Ig on the B cell, even if inefficient,
may synergize with the CD40L/CD40 interaction to yield vigorous
B-cell activation. The subsequent events in the B-cell response,
including proliferation, Ig secretion, and class switching of the
Ig class being expressed, either depend on or are enhanced by the
actions of T cell-derived cytokines (Paul, W. E., "Chapter 1: The
immune system: an introduction," Fundamental Immunology, 4th
Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia,
(1999)).
[0031] CD4.sup.+ T cells tend to differentiate into cells that
principally secrete the cytokines IL-4, IL-5, IL-6, and IL-10
(T.sub.H2 cells) or into cells that mainly produce IL-2,
IFN.gamma., and lymphotoxin (T.sub.H1 cells). The T.sub.H2 cells
are very effective in helping B-cells develop into
antibody-producing cells, whereas the T.sub.H1 cells are effective
inducers of cellular immune responses, involving enhancement of
microbicidal activity of monocytes and macrophages, and consequent
increased efficiency in lysing microorganisms in intracellular
vesicular compartments. Although CD4.sup.+ T cells with the
phenotype of T.sub.H2 cells (i.e., IL-4, IL-5, IL-6 and IL-10) are
efficient helper cells, T.sub.H1 cells also have the capacity to be
helpers (Paul, W. E., "Chapter 1: The immune system: an
introduction, "Fundamental Immunology, 4th Edition, Ed. Paul, W.
E., Lippicott-Raven Publishers, Philadelphia, (1999)).
[0032] Natural Killer (NK) Cells
[0033] Natural Killer (NK) Cells are lymphocytes in the same family
as T and B cells, coming from a common progenitor. However, as
cells of the innate immune system, NK cells are classified as group
I Innate Lymphocytes (ILCs) and respond quickly to a wide variety
of pathological challenges. NK cells protect against disease, for
example killing virally infected cells, and detecting and
controlling early signs of cancer. NK cells were first noticed for
their ability to kill tumour cells without any priming or prior
activation (in contrast to cytotoxic T cells, which need priming by
antigen presenting cells). They are named for this `natural`
killing. Additionally, NK cells secrete cytokines such as
IFN.gamma. and TNF.alpha., which act on other immune cells like
Macrophage and Dendritic cells to enhance the immune response.
[0034] While on patrol, NK cells constantly contact other cells.
Whether or not the NK cell kills these cells depends on a balance
of signals from activating receptors and inhibitory receptors on
the NK cell surface. Activating receptors recognize molecules that
are expressed on the surface of cancer cells and infected cells,
and `switch on` the NK cell. Inhibitory receptors act as a check on
NK cell killing. Most normal healthy cells express MHC I receptors
which mark these cells as `self`. Inhibitory receptors on the
surface of the NK cell recognize cognate MHC I, and this `switches
off` the NK cell, preventing it from killing. Cancer cells and
infected cells often lose their MHC I, leaving them vulnerable to
NK cell killing. Once the decision is made to kill, the NK cell
releases cytotoxic granules containing perforin and granzymes,
which leads to lysis of the target cell.
[0035] T cell Involvement in Cellular Immunity Induction
[0036] T cells also may act to enhance the capacity of monocytes
and macrophages to destroy intracellular microorganisms. In
particular, interferon-gamma (IFN.gamma.) produced by helper T
cells enhances several mechanisms through which mononuclear
phagocytes destroy intracellular bacteria and parasitism including
the generation of nitric oxide and induction of tumor necrosis
factor (TNF) production. T.sub.H1 cells are effective in enhancing
the microbicidal action, because they produce IFN.gamma.. In
contrast, two of the major cytokines produced by T.sub.H2 cells,
IL-4 and IL-10, block these activities (Paul, W. E., "Chapter 1:
The immune system: an introduction," Fundamental Immunology, 4th
Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia,
(1999)).
[0037] Regulatory T (Treg) Cells
[0038] Immune homeostasis is maintained by a controlled balance
between initiation and downregulation of the immune response. The
mechanisms of both apoptosis and T cell anergy (a tolerance
mechanism in which the T cells are intrinsically functionally
inactivated following an antigen encounter (Scwartz, R. H., "T cell
anergy", Annu. Rev. Immunol., Vol. 21: 305-334 (2003)) contribute
to the downregulation of the immune response. A third mechanism is
provided by active suppression of activated T cells by suppressor
or regulatory CD4.sup.+ T (Treg) cells (Reviewed in Kronenberg, M.
et al., "Regulation of immunity by self-reactive T cells", Nature,
Vol. 435: 598-604 (2005)). CD4.sup.+ Tregs that constitutively
express the IL-2 receptor alpha (IL-2R.alpha.) chain
(CD4.sup.+CD25.sup.+) are a naturally occurring T cell subset that
are anergic and suppressive (Taams, L. S. et al., "Human
anergic/suppressive CD4.sup.+CD25.sup.+ T cells: a highly
differentiated and apoptosis-prone population", Eur. J. Immunol.
Vol. 31: 1122-1131 (2001)). Depletion of CD4.sup.+CD25.sup.+ Tregs
results in systemic autoimmune disease in mice. Furthermore,
transfer of these Tregs prevents development of autoimmune disease.
Human CD4.sup.+CD25.sup.+ Tregs, similar to their murine
counterpart, are generated in the thymus and are characterized by
the ability to suppress proliferation of responder T cells through
a cell-cell contact-dependent mechanism, the inability to produce
IL-2, and the anergic phenotype in vitro. Human CD4.sup.+CD25.sup.+
T cells can be split into suppressive (CD25.sup.high) and
nonsuppressive (CD25.sup.low) cells, according to the level of CD25
expression. A member of the forkhead family of transcription
factors, FOXP3, has been shown to be expressed in murine and human
CD4.sup.+CD25.sup.+ Tregs and appears to be a master gene
controlling CD4.sup.+CD25.sup.+ Treg development (Battaglia, M. et
al., "Rapamycin promotes expansion of functional
CD4.sup.+CD25.sup.+Foxp3.sup.+ regulator T cells of both healthy
subjects and type 1 diabetic patients", J. Immunol., Vol. 177:
8338-8347, (2006)).
[0039] Cytotoxic T Lymphocytes
[0040] CD8.sup.+ T cells that recognize peptides from proteins
produced within the target cell have cytotoxic properties in that
they lead to lysis of the target cells. The mechanism of
CTL-induced lysis involves the production by the CTL of perforin, a
molecule that can insert into the membrane of target cells and
promote the lysis of that cell. Perforin-mediated lysis is enhanced
by granzymes, a series of enzymes produced by activated CTLs. Many
active CTLs also express large amounts of fas ligand on their
surface. The interaction of fas ligand on the surface of CTL with
fas on the surface of the target cell initiates apoptosis in the
target cell, leading to the death of these cells. CTL-mediated
lysis appears to be a major mechanism for the destruction of
virally infected cells.
[0041] T-Memory Cells
[0042] Following the recognition and eradication of pathogens
through adaptive immune responses, the vast majority (90-95%) of T
cells undergo apoptosis with the remaining cells forming a pool of
memory T cells, designated central memory T cells (TCM), effector
memory T cells (TEM), and resident memory T cells (TRM) (Clark, R.
A., "Resident memory T cells in human health and disease", Sci.
Transl. Med., 7, 269rv1, (2015)).
[0043] Compared to standard T cells, these memory T cells are
long-lived with distinct phenotypes such as expression of specific
surface markers, rapid production of different cytokine profiles,
capability of direct effector cell function, and unique homing
distribution patterns. Memory T cells exhibit quick reactions upon
re-exposure to their respective antigens in order to eliminate the
reinfection of the offender and thereby restore balance of the
immune system rapidly. Increasing evidence substantiates that
autoimmune memory T cells hinder most attempts to treat or cure
autoimmune diseases (Clark, R. A., "Resident memory T cells in
human health and disease", Sci. Transl. Med., Vol. 7, 269rv1,
(2015)).
[0044] For an effective immune response to an antigen, antigen
presenting cells (APCs) must process and display the antigen in a
proper major histocompatibility complex (MHC) context to a T cell,
which then will result in T cell stimulation of cytotoxic and
helper T cells. Following antigen presentation, successful
interaction of co-stimulatory molecules on both APCs and T cells
must occur or activation will be aborted. GM-CSF and IL-12 serve as
effective pro-inflammatory molecules in many tumor models. For
example, GM-CSF induces myeloid precursor cells to proliferate and
differentiate into dendritic cells (DCs), although additional
signals are necessary to activate their maturation to effective
antigen-presenting cells necessary for activation of T cells.
Barriers to effective immune therapies include tolerance to the
targeted antigen that can limit induction of cytotoxic CD8 T cells
of appropriate magnitude and function, poor trafficking of the
generated T cells to sites of malignant cells, and poor persistence
of the induced T cell response. DCs that phagocytose tumor-cell
debris process the material for MHC presentation, upregulate
expression of costimulatory molecules, and migrate to regional
lymph nodes to stimulate tumor-specific lymphocytes. This pathway
results in the proliferation and activation of CD4+ and CD8+ T
cells that react to tumor-associated antigens. Indeed, such cells
can be detected frequently in the blood, lymphoid tissues, and
malignant lesions of patients.
[0045] Lymphocytes are a type of white blood cell involved in
immune system regulation. Lymphocytes are much more common in the
lymphatic system, and include B cells, T cells, killer T-cells, and
natural killer (NK) cells. There are two broad categories of
lymphocytes, namely T cells and B cells. T-cells are responsible
for cell-mediated immunity whereas B-cells are responsible for
humoral immunity (relating to antibodies). T-cells are so-named
such because these lymphocytes mature in the thymus; B-cells mature
in bone marrow. B cells make antibodies that bind to pathogens to
enable their destruction. CD4+(helper) T cells co-ordinate the
immune response. CD8+(cytotoxic) T cells and Natural Killer (NK)
cells are able to kill cells of the body that are, e.g., infected
by a virus or display an antigenic sequence.
[0046] Immune Response
[0047] Generally speaking, immune responses are initiated by an
encounter between an individual and a foreign substance, e.g., an
infectious microorganism. The infected individual rapidly responds
with both a humoral immune response with the production of antibody
molecules specific for the antigenic determinants/epitopes of the
immunogen, and a cell mediated immune response with the expansion
and differentiation of antigen-specific regulatory and effector
T-lymphocytes, including both cells that produce cytokines and
killer T cells, capable of lysing infected cells. Primary
immunization with a given microorganism evokes antibodies and T
cells that are specific for the antigenic determinants/epitopes
found on that microorganism, but that usually fail to recognize or
recognize only poorly antigenic determinants expressed by unrelated
microbes (Paul, W. E., "Chapter 1: The immune system: an
introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W.
E., Lippicott-Raven Publishers, Philadelphia, (1999), at p.
102).
[0048] As a consequence of this initial response, the immunized
individual develops a state of immunologic memory. If the same or a
closely related microorganism is encountered again, a secondary
response ensues. This secondary response generally consists of an
antibody response that is more rapid, greater in magnitude and
composed of antibodies that bind to the antigen with greater
affinity and are more effective in clearing the microbe from the
body, and a similarly enhanced and often more effective T-cell
response. However, immune responses against infectious agents do
not always lead to elimination of the pathogen. (Paul, W. E.,
"Chapter 1: The immune system: an introduction," Fundamental
Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven
Publishers, Philadelphia, (1999), at p. 102).
[0049] Immune Tolerance of Cancer
[0050] Cancer is characterized by genetic instability of particular
cells but has also been described as a disorder of the immune
system, based on the fact that the immune system fails, at least in
certain segments of the afflicted human population, to respond
optimally to cancerous cells that have taken on a distinctly
non-self phenotype that should be recognized as foreign. Several
reasons have been advanced to explain the basis of this
observation. For example, first, cancer cells consist mainly of
self-antigens, in striking contrast to the situation with
infectious organisms. Some antigens that are classified as cancer
antigens are actually normal antigens that are overexpressed, or
normal antigens that have a mutation in only one or two amino acids
in the polypeptide chain. Second, cancer cells down-regulate MHC,
and thus do not much present tumor cell-derived peptides by way of
MHC. Third, cancer cells, and associated tumor-associated
macrophages, express cytokines that dampen the immune response
(see, e.g., Yu et al (2007) Nature Rev. Immunol. 7:41-51). This
dampening is caused, for example, by the secretion of
interleukin-10 (IL-10) by the cancer cells or by the associated
macrophages. Fourth, unlike the situation with infections, cancer
cells do not provide any immune adjuvant. Pathogens express a
variety of naturally-occurring immune adjuvants, which take the
form of toll-like receptor (TLR) agonists and NOD agonists (see,
e.g., Kleinnijenhuis et al (2011) Clin. Dev. Immunol. 405310 (12
pages)). Generally, optimal activation of dendritic cells requires
contact of an immune adjuvant with one or more toll-like receptors
(TLRs) expressed by the dendritic cell. Without activation of the
dendritic cell, contact between the dendritic cell and T cells
(immune synapse) fails to result in optimal activation of the T
cell.
[0051] Insights into the mechanisms underlying immune-evasion,
together with combination treatment regimens that potentiate the
potency of therapeutic vaccination--either directly or
indirectly--through combination with immune checkpoint inhibitors
or other therapies, have served as a basis for the development of
vaccines that induce effective antitumor immunity.
[0052] Immune Surveillance and Immune Editing
[0053] Tumor immune editing is divided into three phases: an
elimination phase, an equilibrium phase, and an escape phase. The
elimination phase, also known as immune surveillance, is the
process by which the immune system identifies cancerous or
pre-cancerous cells and eliminates them before they grow out of
control. This phase can be complete when all cancerous or
precancerous cells are eliminated. If some tumor cells are not
eliminated, a temporary state of equilibrium may be achieved
between the immune system and tumor cell growth. In this
equilibrium phase, tumors cells can either remain dormant or
continue to evolve by accumulating further changes to genomic DNA
that can modulate the antigens they present. During this process,
the immune system exerts a selective pressure on evolving cells,
whereby the tumor cells that are less able to be recognized have a
survival advantage. Eventually the immune response is unable to
recognize cells of the tumor, resulting in the transition to the
escape phase wherein tumor cells progressively grow out of
control.
[0054] Tumor Microenvironment
[0055] The tumor microenvironment provides a consistently effective
barrier to immune cell function because tumors actively
downregulate all phases of anti-tumor immune responses using a
spectrum of different strategies and mechanisms. Many molecular
mechanisms that cause dysfunction of immune cells in the tumor
microenvironment have been identified, including those directly
mediated by factors produced by tumors, and others resulting from
alterations of normal tissue homeostasis in the presence of cancer.
Most human tumors appear to be able to interfere with one or more
stages of immune cell development, differentiation, migration,
cytotoxicity and other effector functions (T L Whiteside, The tumor
microenvironment and its role in promoting tumor growth, Oncogene
(2008) 27, 5904-5912).
[0056] One such mechanism involves accumulation in tumors of
T.sub.reg (CD4+CD25.sup.bright Foxp3.sup.+ T cells) and
myeloid-derived cells
(CD34.sup.+CD33.sup.+CD13.sup.+CD11b.sup.+CD15.sup.-), which are
common features of human tumors, and have been linked to poor
prognosis in patients with cancer (T L Whiteside, The tumor
microenvironment and its role in promoting tumor growth, Oncogene
(2008) 27, 5904-5912). Under normal conditions, T.sub.reg cells are
involved in the important role of preventing autoimmunity, but in
cancer, they expand, migrate to tumors, downregulate autologous
effector T-cell proliferation and suppress anti-tumor responses of
both CD4.sup.+CD25.sup.- and CD8.sup.+CD25.sup.- T cells using
distinct molecular pathways. The T.sub.reg cells in the tumor are a
heterogeneous population of regulatory CD3.sup.+CD4.sup.+ T cells,
comprising natural T.sub.reg, antigen-specific Tr1 cells, and other
less well defined subsets of suppressor cells. Tr1 cells are
induced in the tumor microenvironment, which is rich in IL-10,
TGF-.beta., and prostaglandin E2 (PGE2), all of which have been
shown to promote Tr1 generation (T L Whiteside, The tumor
microenvironment and its role in promoting tumor growth, Oncogene
(2008) 27, 5904-5912).
[0057] Myeloid suppressor cells (MSCs) also suppress T-cell
responses in the tumor microenvironment, where they secrete
TGF-.beta. or induce TGF-.beta. secretion. Immunosuppressive
CD34.sup.+ cell-derived myeloid cells have been identified in the
peripheral blood of cancer patients. In tumor-bearing mice, MSCs
accumulate in the spleen and peripheral circulation in very high
amounts, exerting potent immunosuppression and favoring tumor
growth. MSCs also control the availability of essential amino acids
such as L-arginine and produce high levels of reactive oxygen
species. The MSCs found in tumors also constitutively express iNOS
and arginase 1, an enzyme involved in metabolism of L-arginine,
which also synergizes with iNOS to increase superoxide and NO
production, which have been found to interfere with lymphocyte
responses. GM-CSF, which is also often secreted by tumor cells,
recruits MSCs and induces dose-dependent in vivo immune suppression
and tumor promotion, while at the same time, GM-CSF has been used
as immune adjuvant in antitumor vaccines. GM-CSF was observed to
increase a subset of TGF-.beta.-producing MSCs in the circulation
of patients with metastatic melanoma. The concurrent stimulatory
and suppressive roles suggest that GM-CSF and MSCs are involved in
maintaining immune homeostasis in normal tissue, but in the tumor
microenvironment promote tumor cell escape (T L Whiteside, The
tumor microenvironment and its role in promoting tumor growth,
Oncogene (2008) 27, 5904-5912).
[0058] Tumor Immunotherapy
[0059] Cancer therapy is evolving rapidly as new molecular targets
are being discovered. Despite the advent of biologics targeting
specific pathways (e.g., HERCEPTIN.RTM., ERBITUX.RTM.) and small
molecules designed against specific targets (tamoxifen,
GLEEVEC.TM.) nonspecific modalities such as chemotherapy and
radiation remain a standard of care.
[0060] Anti-cancer immunotherapy has been a goal for many years
with a variety of approaches being tested. One difficulty of
developing this immunotherapy is that target antigens are often
tissue specific molecules found on both cancer cells and normal
cells, and either do not elicit immunity or show non-specificity
regarding cell killing (Kaufman and Wolchok eds., General
Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)).
Furthermore, tumor cells have features that make immune recognition
difficult, such as loss of expression of antigens that elicit
immune response, lack of MHC class II, and downregulation of MHC
class I expression. These features can lead to non-recognition of
tumor cells by both CD4+ and CD8+ T cells (Kaufman and Wolchok
eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121
(2007)). Tumors may also evade detection through active mechanisms,
such as the production of immunosuppressive cytokines (Kaufman and
Wolchok eds., General Principles of Tumor Immunotherapy, Chpt 5,
67-121 (2007)).
[0061] DCs generated ex vivo by culturing hematopoietic progenitor
cells or monocytes with cytokine combinations have been tested as
therapeutic vaccines in cancer patients for more than a decade
(Ueno H, et al., Immunol. Rev. (2010) 234: 199-212). For example,
treatment of metastatic prostate cancer with sipuleucel-T (also
known as APC 8015), which is a cellular product based on enriched
blood APCs that are briefly cultured with a fusion protein of
prostatic acid phosphatase (PAP) and granulocyte macrophage
colony-stimulating factor (GM-CSF), resulted in an approximately
4-month-prolonged median survival in Phase III trials (Higano C S,
et al., Cancer (2009) 115: 3670-3679; Kantoff P W, et al., N. Engl.
J. Med. (2010) 363: 411-422). This study concluded that DC-based
vaccines are safe and can induce the expansion of circulating CD4+
T-cells and CD8+ T-cells specific for tumor antigens. As a result
of this and similar studies, sipuleucel-T has been approved by the
US Food and Drug Administration (FDA) for the treatment of
metastatic prostate cancer, thereby paving the clinical development
and regulatory path for the next generation of cellular
immunotherapy products (Palucka K and Banchereau J, Nature Reviews
Cancer (April 2012) 12: 265-276).
[0062] Vaccination strategies involving DCs to induce
tumor-specific effector T cells that can reduce the tumor mass
specifically and that can induce immunological memory to control
tumor relapse have been developed. For example, DCs can be provided
with tumor-specific antigens by culturing DCs ex vivo with an
adjuvant and a tumor-specific antigen, and then injecting these
cells back into the patient. Tumor cells obtained from an excised
tumor, needle biopsy, core biopsy, vacuum-assisted biopsy or
peritoneal lavage have been used to generate immunogenic
compositions comprising tumor-specific-antigen presenting dendritic
cells.
[0063] Cancer Treatment Strategies
[0064] Antibody therapies such as HERCEPTIN.TM. and ERBITUX.TM. are
passive immunotherapies, but have yielded considerable improvement
in clinical outcome, as measured by, e.g. the recurrence rate,
progression free survival and overall survival. More recently, PD-1
and CTLA4 inhibitors have been reported to block discrete
checkpoints in an active host immune response allowing an
endogenous anti-cancer immune response to be sustained. The term
"immune checkpoints" refers to the array of inhibitory pathways
that are necessary for maintaining self-tolerance and modulating
the duration and extent of immune responses to minimize damage to
normal tissue. Immune checkpoint molecules such as PD-1, PD-L1, and
CTLA-4 are cell surface signaling receptors that play an important
role in modulating the T-cell response in the tumor
microenvironment. Tumor cells have been shown to utilize these
checkpoints to their benefit by up regulating their expression and
activity. With the tumor cell's ability to commandeer some immune
checkpoint pathways as a mechanism of immune resistance, it has
been hypothesized that checkpoint inhibitors that bind to molecules
of immune cells to activate or inactivate them may relieve the
inhibition of an immune response. Recent discoveries have
identified immune checkpoints or targets, like PD-1, PD-L1, PD-L2,
CTLA4, TIM3, LAG3, CCR4, OX40, OX40L, IDO, and A2AR, as proteins
responsible for immune evasion. Specific immune checkpoint
inhibitors, including antibodies against CTLA-4, PD-1 receptor or
its ligand PD-L1 have produced impressive results in the clinic in
a range of cancers, leading to FDA approvals for YERVOY.TM.
(Ipilimumab; CTLA-4 antagonist), OPDIVO.TM. (Nivolumab; PD-1
antagonist) and KEYTRUDA.TM. (Pembrolizumab; PD-1 antagonist) in
multiple tumor indications and with ongoing registration trials in
many more. This method of therapy, however, can only be successful
if a pre-existing antitumor immune response is present within a
patient (Pardoll, D., The blockade of immune checkpoints in cancer
immunotherapy, Nature Reviews: Cancer, Vol. 12, April 2012, 253).
Recent cellular therapies, such as chimeric antigen receptor T-cell
therapy (CAR-T), attempt to use synthetic biology to redirect
T-cells to specific cell surface tumor antigens. Genetic
modification of T-cells is used to confer tumor antigen recognition
by transgenic expression of chimeric antigen receptor (CAR). CARs
are engineered molecules that can be introduced into T cells to
enable them to target tumor antigens (Frey, N. V., Porter, D. L.,
The Promise of Chimeric Antigen Receptor T-Cell Therapy, Oncology
(2016); 30(1)) pii 219281). CAR T cells have been shown to have
some efficacy against hematologic malignancies and to a lesser
extent solid tumors. CAR T therapy, however, has been shown to
cause several types of toxicities, including cytokine release
syndrome, neurological toxicity, non-tumor recognition, and
anaphylaxis (Bonifant C L, et al., Toxicity and management in CAR
T-cell therapy, Molecular Therapy--Oncolytics (2016) 3, 16011).
[0065] Therapeutic vaccination against cancer is an important
modality complementing current standard therapies, and may lead to
long-term control of cancer. GVAX.TM., a prototypical example, is a
GM-CSF gene transduced tumor vaccine within either an autologous or
allogeneic population of tumor cells. It is believed that GM-CSF
secretion of genetically modified tumor cells stimulates cytokine
release at the vaccine site to activate antigen presenting cells to
induce a tumor specific cellular immune response (Eager, R. &
Nemunaitis, J GM-CSF Gene-Transduced Tumor Vaccines, Molecular
Therapy, Vol. 12, No. 1, 18 (July 2005)). Lethally irradiated tumor
cell vaccines engineered to secrete GM-C SF (GVAX) showed promising
efficacy in various models of melanoma, renal cell, prostate, and
non-small cell lung, pancreatic, as well as head and neck squamous
cell carcinoma, but due to the multiple immunological checkpoint
blockades, GVAX as a monotherapy is unlikely to be clinically
effective in advanced disease. There remains a need for improved
compositions and methods for immunologic strategies to treating
diseases such as cancer that can be refractory to traditional
therapeutic approaches.
[0066] Dendritic cell (DC)-tumor cell fusions have been developed
to generate hybrid cells that express the relevant tumor associated
antigens derived from the parent tumor cells, and also have the
ability to process and present such antigens to appropriate cells
of the immune system. The DC-tumor cell fusions provide a greater
variety of tumor antigens, but have been met with limited success
in human trials, likely due to the autologous components required,
the heterogeneity of the product caused by maturation of DC cells,
and variations in antigen loading (Browning, M., Antigen presenting
cell/tumor cell fusion vaccines for cancer, Human Vaccines &
Immunotherapeutics 9:7, 1545-1548; July 2013; Butterfield, L.,
Dendritic Cells in Cancer Immunotherapy Clinical Trials: Are We
Making Progress?, Frontiers of Immunology, 2013 4: 454).
[0067] Immunogenic Potential of Vaccines
[0068] Vaccines against infectious agents are prime examples of
specific receptor-ligand interactions being used to shape an immune
response for the therapeutic goal of preventing or reducing
infection (e.g. flu vaccine). Generally, an antigen is presented to
the immune system in the context of an adjuvant (e.g., a synthetic
small molecule immunomodulator).
[0069] The allogeneic tumor vaccines of the described invention are
distinct from such vaccines in several key features. First, they
are designed to be capable of treating existing tumors, although
prevention of tumor formation is theoretically also possible.
Second, their efficacy tends to be limited by the fact that while
tumors express neoantigens (i.e. new, non-self elements) that are
foreign and new to the individual, they are also undoubtedly human
tumor cells and thus not always recognized as foreign (i.e.
non-self) by the individual.
[0070] The aforementioned difficulties notwithstanding, evidence
has now emerged 1) that endogenous antitumor responses exist, 2)
that these immune responses can be modulated, and 3) that this
modulation can be measured in terms of overall survival in standard
clinical trials.
[0071] According to some aspects of the described invention, a
series of immunomodulators that can be co-expressed either on a
tumor cell line or tumor cell line variant derived from a cancer
patient, or on a multiply genetically modified allogeneic tumor
cell line or tumor cell line variant has been identified that, when
used as a tumor vaccine, may serve 1) to efficiently load the broad
array of tumor antigens into the endogenous antigen presenting
cells, 2) to efficiently stimulate several cell types by enhancing
the normal signals received during an immune response, 3) to impede
the mechanisms by which T regulatory cells suppress the immune
response, 4) to impede the signals by which immune responses are
generally resolved, and 5) to result in enhanced overall survival
of cancer patients vaccinated with such a formulation. Although in
certain embodiments, the modified tumor cell line or tumor cell
line variant can be derived from the patient who receives the
vaccine, the allogeneic tumor cell line or tumor cell line variant
vaccine approach is distinct from a personalized therapy approach,
because the modified tumor cells are not necessarily derived from
the individual who ultimately receives the vaccine. Instead, an
allogeneic tumor cell vaccine aims to focus an immune response on
the many elements that individual tumors of the same tumor type
have in common.
[0072] One strategy for exploiting the large number of potential
tumor antigens for each individual type of cancer is to vaccinate
with whole tumor cells to avoid accidentally excluding potentially
relevant antigens. The invention described herein provides, among
other things, a vaccine with whole tumor cells possessing an array
of tumor antigens and modified to express three or more immune
modulators.
BRIEF SUMMARY OF THE INVENTION
[0073] According to some aspects, the described invention provides
an allogeneic tumor cell vaccine comprising: (1) a population of
live, proliferation-incompetent genetically engineered tumor cells
expressing one or more tumor specific antigens, the population
comprising: at least three stably expressed immunomodulatory
molecules, wherein the at least three immunomodulatory molecules
are OX40 Ligand (OX40L), CD27 Ligand (CD70) and CD28 Ligand (CD28L)
comprising CD80, CD86, or both, for induction of one or more
subpopulations of PBMCs to proliferate in response to the expressed
immunomodulatory molecules and to then enter an effector phase for
killing of tumor cells; wherein the subpopulations of PBMC cells
comprise one or more of T-lymphocytes, natural killer (NK) cells,
dendritic cells (DCs) or B lymphocytes; and (2) a pharmaceutically
acceptable carrier.
[0074] According to some embodiments, the population of live,
proliferation-incompetent genetically engineered tumor cells
expressing one or more tumor specific antigens further comprises
one or more additional stably expressed immunomodulatory molecules
selected from R1-R44. According to some embodiments, the tumor
cells are rendered proliferation-incompetent by irradiation.
According to some embodiments, induction of the T-lymphocytes
comprises activation of the subpopulation of T lymphocytes,
expansion of the T lymphocytes, or both. According to some
embodiments, induction of the NK cells comprises activation of the
subpopulation of NK cells, expansion of the subpopulation of NK
cells or both. According to some embodiments, induction of the
subpopulation of DCs comprises activation of the subpopulation of
DCs, expansion of the subpopulation of DCs or both. According to
some embodiments, induction of the subpopulation of B lymphocytes
comprises activation of the subpopulation of B lymphocytes,
expansion of the subpopulation of B lymphocytes or both. According
to some embodiments, the subpopulation of NK cells comprises a
subpopulation of memory-like NK cells. According to some
embodiments, the subpopulation of T lymphocytes comprises a
subpopulation of CD8+ cytotoxic T-lymphocytes (CTL). According to
some embodiments, the subpopulation of T lymphocytes comprises a
subpopulation of memory T cells. According to some embodiments, the
subpopulation of T lymphocytes comprises a subpopulation of
regulatory T cells. According to some embodiments, the
subpopulation of T lymphocytes comprises a subpopulation of helper
T cells. According to some embodiments, the subpopulation of B
lymphocytes comprises a subpopulation of memory B cells.
[0075] According to some embodiments, the vaccine (1) enhances
immune activation of cells effective to recognize and act against
those tumor cells that comprise the target tumor antigen in vivo
without systemic inflammation; (2) reduces immunosuppression in a
tumor microenvironment for tumor cells comprising the target tumor
antigen; or (3) increases cell death of tumor cells expressing the
target tumor antigen.
[0076] According to some embodiments, the tumor cell is derived
from a cancer selected from the group consisting of: melanoma,
colorectal carcinoma, leukemia, chronic myeloid leukemia, prostate
cancer, head and neck cancer, Squamous Cell Carcinoma, tongue
cancer, larynx cancer, tonsil cancer, hypopharynx cancer,
nasalpharynx cancer, breast cancer, colon cancer, lung cancer,
pancreatic cancer, glioblastoma and brain cancer. According to some
embodiments, the melanoma tumor cell is characterized by expression
of one or more of gp100, tyrosinase, Melan-A, tyrosinase-related
protein (TRP-2-INT2), melanoma antigen-1 (MAGE-A1), NY-ESO-1,
preferentially expressed antigen of melanoma (PRAIVIE) CDK4 and
multiple myeloma oncogene 1 (MUM-1).According to some embodiments,
the colorectal cancer tumor cell is characterized by expression of
one or more of carcinoembryonic antigen (CEA), MAGE, HPV, human
telomerase reverse transcriptase (hTERT), EPCAM, PD-1, PD-L1, p53,
and cell surface-associated mucin 1 (MUC1).
[0077] According to some embodiments, the population of live,
proliferation-resistant tumor cells is derived from a biological
sample derived from a subject. According to some embodiments, the
population of live proliferation resistant tumor cells is derived
from a tumor cell line. According to some embodiments, the
population of live, proliferation-resistant tumor cells is
effective to elicit immune activation without systemic
inflammation. According to some embodiments, the vaccine elicits an
immune response that improves progression free survival, overall
survival, or both relative to placebo controls. According to some
embodiments, the one or more additional stably expressed
immunomodulatory molecules selected from R1-R44 is a cytokine, a
TNF-family member, a secreted receptor, a chaperone, an IgG
superfamily member and/or a chemokine receptor. According to some
embodiments, wherein the immunostimulatory molecules are presented
at the exterior surface of the genetically engineered tumor
cells.
[0078] According to another aspect, the described invention
provides a method of inducing an immune response to a cancer in a
subject comprising administering the allogeneic tumor cell vaccine
of claim 1 to the subject parenterally or locally into a tumor,
wherein the allogeneic tumor cell vaccine is type-matched to the
subject's cancer. According to some embodiments, the cancer is
selected from melanoma or colorectal cancer. According to some
embodiments, the subject has an infectious viral disease with
progression to a cancer. According to some embodiments, the method
further comprises administering a checkpoint inhibitor to the
subject.
[0079] According to another aspect, the described invention
provides a method of treating cancer in a subject, comprising
administering to the subject an allogeneic tumor cell vaccine
comprising: (1) a population of live, proliferation-incompetent
genetically engineered tumor cells expressing one or more tumor
specific antigens, the population comprising: at least three stably
expressed immunomodulatory molecules, wherein the at least three
immunomodulatory molecules are OX40 Ligand (OX40L), CD27 Ligand
(CD70) and CD28 Ligand (CD28L) comprising CD80, CD86, or both, for
induction of one or more subpopulations of PBMCs to proliferate in
response to the expressed immunomodulatory molecules and to then
enter an effector phase for killing of tumor cells; wherein the
subpopulations of PBMC cells comprise one or more of T-lymphocytes,
natural killer (NK) cells, dendritic cells (DCs) or B lymphocytes;
and (2) a pharmaceutically acceptable carrier in an amount that
reduces tumor burden in the subject. According to some embodiments,
the effective amount improves clinical outcome. According to some
embodiments, the effective amount improves progression free
survival, overall survival, or both, of the subject relative to a
placebo control. According to some embodiments, the cancer is
melanoma or colorectal cancer.
[0080] According to another aspect, the allogeneic tumor cell
vaccine comprising: (1) a population of live,
proliferation-incompetent genetically engineered tumor cells
expressing one or more tumor specific antigens, the population
comprising: at least three stably expressed immunomodulatory
molecules, wherein the at least three immunomodulatory molecules
are OX40 Ligand (OX40L), CD27 Ligand (CD70) and CD28 Ligand (CD28L)
comprising CD80, CD86, or both, for induction of one or more
subpopulations of PBMCs to proliferate in response to the expressed
immunomodulatory molecules and to then enter an effector phase for
killing of tumor cells; wherein the subpopulations of PBMC cells
comprise one or more of T-lymphocytes, natural killer (NK) cells,
dendritic cells (DCs) or B lymphocytes; and (2) a pharmaceutically
acceptable carrier, is produced by a process comprising: providing
an allogeneic parental tumor cell line comprising a population of
live tumor cells; introducing into the population of live tumor
cells an exogenous nucleic acid encoding a stably expressed
immunomodulatory molecule, wherein the immunomodulatory molecule is
OX40 Ligand (OX40L); introducing into the population of live tumor
cells an exogenous nucleic acid encoding a stably expressed
immunomodulatory molecule, wherein the immunomodulatory molecule is
CD27 Ligand (CD70); introducing into the population of live tumor
cells an exogenous nucleic acid encoding a stably expressed
immunomodulatory molecule, wherein the immunomodulatory molecule is
CD28 Ligand (CD28L) comprising CD80, CD86, or both; wherein stable
expression of OX40 Ligand (OX40L), CD27 Ligand (CD70) and CD28
Ligand (CD28L) comprising CD80, CD86, or both induces one or more
subpopulations of PBMCs to proliferate in response to the expressed
immunomodulatory molecules and to then enter an effector phase for
killing of tumor cells; generating tumor cell line variants by
selecting for tumor cell clones that stably express an immunogenic
amount of the exogenous subset of the immunomodulatory molecules;
and selecting in a mixed lymphocyte tumor cell reaction clonally
derived cell line variants by one or more of the following
parameters selected from: cellular proliferation, cellular subset
differentiation, cytokine release profile, and tumor cell lysis;
wherein the selected clonally derived cell line variant is
effective to stimulate activation of one or more of T-lymphocytes,
natural killer (NK) cells, dendritic cells (DCs) or B
lymphocytes.
[0081] According to some embodiments, the process for producing the
allogeneic tumor cell vaccine further comprises introducing into
the population of live tumor cells an exogenous nucleic acid
encoding one or more stably expressed immunomodulatory molecules
selected from R1-R44. According to some embodiments, the tumor
cells are rendered proliferation incompetent by irradiation.
According to some embodiments, the parental tumor cell line is from
a tumor selected from the group consisting of: melanoma, and
colorectal carcinoma. According to some embodiments, the exogenous
nucleic acid comprises DNA or RNA. According to some embodiments,
the introducing step comprises viral transduction. According to
some embodiments, the introducing step comprises electroporation.
According to some embodiments, the introducing step comprises
utilizing one or more of: liposome mediated transfer, adenovirus,
adeno-associated virus, herpes virus, a retroviral based vector,
lipofection, and a lentiviral vector. According to some
embodiments, the introducing step comprises introducing the
exogenous nucleic acid by transfection of a lentiviral vector.
[0082] These and other advantages of the invention will be apparent
to those of ordinary skill in the art by reference to the following
detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0083] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0084] FIG. 1 shows one embodiment of a heteroclitic cross reaction
between a peptide native to a tumor cell line and a peptide native
to a tumor cell of a patient receiving immunotherapy.
[0085] FIG. 2 shows a schematic of the organization of the
scFv-anti-biotin-G3hinge-mIgG1 vector 1.
[0086] FIG. 3 shows a schematic of the organization of the full
anti-biotin-G3hinge-mIgG1 vector 2.
[0087] FIG. 4 shows a schematic of the organization of the
sGM-CSF/ires/mFLT3L vector 3.
[0088] FIG. 5 shows a schematic of the organization of the
sFLT3L/ires/(FLT3 signal-GM-C SF-Tm) vector 4.
[0089] FIG. 6 shows a schematic of the organization of the mCD40L
vector 5.
[0090] FIG. 7 shows a schematic of the organization of the mTNFa
vector 6.
[0091] FIG. 8 shows a schematic of the organization of the
mRANKL/ires/FLT3 signal-V5-scFV anti-biotin-Tm vector 7.
[0092] FIG. 9 shows a schematic of vector 44.
[0093] FIG. 10 shows a schematic of vector 97.
[0094] FIG. 11 shows a schematic of vector 84.
[0095] FIG. 12 shows a schematic of vector 29.
[0096] FIG. 13 shows a schematic of vector 107.
[0097] FIG. 14 shows a schematic of vector 116.
[0098] FIG. 15 shows a schematic of vector 86.
[0099] FIG. 16 shows a schematic of vector 18.
[0100] FIG. 17 shows a schematic of vector 17.
[0101] FIG. 18 shows a schematic of vector 98.
[0102] FIG. 19 shows a schematic of vector 30.
[0103] FIG. 20 shows a schematic of vector 109.
[0104] FIG. 21 shows a schematic of vector 106.
[0105] FIG. 22 shows a schematic of vector 16.
[0106] FIG. 23 shows a schematic of vector 83.
[0107] FIG. 24 shows a schematic of vector 31.
[0108] FIG. 25 shows a schematic of vector 12.
[0109] FIG. 26 shows a schematic of vector 99.
[0110] FIG. 27 shows a schematic of vector 121.
[0111] FIG. 28 shows a schematic of vector 105.
[0112] FIG. 29 shows a schematic of vector 32.
[0113] FIG. 30 shows a schematic of vector 37.
[0114] FIG. 31 shows a schematic of vector 22.
[0115] FIG. 32 shows a schematic of vector 19.
[0116] FIG. 33 shows a schematic of vector 20.
[0117] FIG. 34 shows a schematic of vector 89.
[0118] FIG. 35 shows a schematic of vector 21.
[0119] FIG. 36 shows a schematic of vector 23.
[0120] FIG. 37 shows a schematic of vector 108.
[0121] FIG. 38 shows a schematic of vector 15.
[0122] FIG. 39 shows a schematic of vector 124.
[0123] FIG. 40 shows a schematic of vector 65.
[0124] FIG. 41 shows a schematic of vector 64.
[0125] FIG. 42 shows a schematic of vector 88.
[0126] FIG. 43 shows a schematic of vector 96.
[0127] FIG. 44 shows a schematic of vector 14.
[0128] FIG. 45 shows a schematic of vector 119.
[0129] FIG. 46 shows a schematic of vector 120.
[0130] FIG. 47 shows a schematic of vector 45.
[0131] FIG. 48 shows a schematic of vector 60.
[0132] FIG. 49 shows a schematic of vector 59.
[0133] FIG. 50 shows a schematic of vector 8.
[0134] FIG. 51 shows a schematic of vector 128.
[0135] FIG. 52 shows a schematic of vector 35.
[0136] FIG. 53 is a schematic that shows the general experimental
format.
[0137] FIG. 54 is a panel of graphs that show the results of flow
cytometry experiments. Forward (FSC) and side scatter (SSC) plots
for size and granularity. SK lines are represented by a number
code; SK, unmodified parent line; 3, secreted GM-CSF and membrane
expressed FLT-3L; 4, secreted FLT3L and membrane expressed GM-CSF;
5, a non-cleavable form of CD40L; 6, a non-cleavable form of
TNF-.alpha.; 3-4 is a combination of 3 and 4; 3-4-5 is a
combination of 3,4 and 5; and 3-4-6 is a combination of 3,4 and 6.
Cell lines 6, 3-4-5 and 3-4-6 display a larger and more granular
phenotype likely owing to the presence of receptors for TNF-.alpha.
and CD40L on cells of epithelial origin
[0138] FIG. 55 is a panel of graphs that show representative flow
cytometry stains for CD4 cells in hPBMC in response to the
indicated engineered cell lines with the indicated
immunomodulators. SK cell lines are represented by the following
code; SK, unmodified parent line; 2, membrane expressed IgG1, 3,
secreted GM-CSF and membrane expressed FLT-3L; 4, secreted FLT3L
and membrane expressed GM-CSF; 5, a non-cleavable form of CD40L;
and 6, a non-cleavable form of TNF.
[0139] FIG. 56 is a panel of graphs that show representative flow
cytometry stains for the indicated engineered surface markers;
GM-CSF, FLT3L, TNF-.alpha. and CD40L. SK lines are represented by a
number code; SK, unmodified parent line; 3, secreted GM-CSF and
membrane expressed FLT-3L; 4, secreted FLT3L and membrane expressed
GM-CSF; 5, a non-cleavable form of CD40L; 6, a non-cleavable form
of TNF-.alpha.; 3-4 is a combination of 3 and 4; 3-4-5 is a
combination of 3,4 and 5; and 3-4-6 is a combination of 3,4 and
6.
[0140] FIG. 57A and FIG. 57B show the results of CyTOF mass
cytometry single-cell phenotype analysis of hPBMC response to SK
melanoma cells with modification by expression of immunomodulatory
factors. FIG. 57A shows viSNE density contour plots of CyTOF
staining data showing relative changes in immune cell subset
abundance and phenotype. FIG. 57B shows single-cell phenotype
analysis. SK lines are represented by a number code; SK, unmodified
parent line; 3, secreted GM-CSF and membrane expressed FLT-3L; 4,
secreted FLT3L and membrane expressed GM-CSF; 5, a non-cleavable
form of CD40L; 6, a non-cleavable form of TNF-.alpha.; 3-4 is a
combination of 3 and 4; 3-4-5 is a combination of 3,4 and 5; and
3-4-6 is a combination of 3,4 and 6.
[0141] FIG. 58A-FIG. 58D shows CyTOF monocyte cluster analysis of
hPBMCs indicating changes in the activation markers CD40 (FIG.
58A), CD86 (FIG. 58B), CD69 (FIG. 58C) and CD25 (FIG. 58D)
expression following 1 day stimulation with the indicated
genetically modified SK lines at a 1:5 cell ratio. SK lines are
represented by a number code; SK, unmodified parent line; 3,
secreted GM-CSF and membrane expressed FLT-3L; 4, secreted FLT3L
and membrane expressed GM-CSF; 5, a non-cleavable form of CD40L; 6,
a non-cleavable form of TNF-.alpha.; 3-4 is a combination of 3 and
4; 3-4-5 is a combination of 3,4 and 5; and 3-4-6 is a combination
of 3,4 and 6. FIG. 58E shows CyTOF monocyte cluster analysis of
hPBMCs indicating relative median expression levels (MEI) of
monocyte markers CD40 and CD86. FIG. 58F shows CyTOF monocyte
cluster analysis of hPBMCs indicating relative median expression
index (MEI) of CD4 T cell markers CD69 and CD25.
[0142] FIG. 59 is a graph that shows the results of luminex
multiplex cytokine profiling of human PBMC responses to SK parent
line and genetically modified SK lines. Control cultures included
SK cells alone, hPBMCs alone, and hPBMCs stimulated with a mixture
of anti-CD3 and anti-CD28 antibodies (1 .mu.g/ml final
concentration). Symbols indicate cytokine levels in pg/ml as
estimated from a standard curve using recombinant cytokines.
Absence of symbols indicates the cytokine was not detected. SK
lines are represented by a number code; SK, unmodified parent line;
3, secreted GM-CSF and membrane expressed FLT-3L; 4, secreted FLT3L
and membrane expressed GM-CSF; 5, a non-cleavable form of CD40L; 6,
a non-cleavable form of TNF-.alpha.; 3-4 is a combination of 3 and
4; 3-4-5 is a combination of 3,4 and 5; and 3-4-6 is a combination
of 3,4 and 6.
[0143] FIG. 60 shows the results of flow cytometry experiments
demonstrating that CD8+ T cells can be activated by genetically
modified SK lines expressing immunomodulatory molecules. Flow
cytometry forward (FSC) and side scatter (SSC) plot for size and
granularity after incubating parental cell line SKMEL2 (FIG. 60(i)
and genetically modified 14-18-30 expressing SK-MEL-2 tumor cells
(FIG. 60(ii) with PBMCs in a mixed lymphocyte tumor response assay
are shown. The dotted oval in FIGS. 60(i) and 60(ii) indicates the
lymphocyte gate. FIG. 60(iii) and FIG. 60(iv) show the CD8
population after incubation of the PBMCs in the mixed lymphocyte
tumor response assay with the parent cell line (FIG. 60(iii)) and
the genetically engineered 14-18-30 expressing SKMEL2 tumor cells
(FIG. 60(iv)). The dotted circle in the bottom panel of graphs
shows the CD8 gate. FIG. 61A and FIG. 61B show that in vitro CD8+ T
cell expansion from hPBMC comparing a day 9 culture of the parent
SKMEL2 cell line (FIG. 61A) and genetically modified 14-18-30 cell
line (FIG. 61B) expressing a combination of the immunomodulators
shown in Table 2, results in tumor cell killing.
[0144] FIG. 62 shows the results of flow cytometry demonstrating
stimulation of dendritic cell (DC), natural killer (NK) cell and B
cell subpopulations in genetically modified SK lines (i) APX/15;
(ii) APX/19; (iii) APX/22; (iv) APX/23; (v) APX/29.
[0145] FIG. 63 shows CyTOF data demonstrating differentiation of
various subsets of PBMCs following stimulation with genetically
modified SK lines expressing immunomodulatory molecules ((i)
parental; (ii) APX/3; (iii) APX/3-4; (iv) APX/3-4-5; (v)
APX/3-4-6)). SK lines are represented by a number code; 3, secreted
GM-CSF and membrane expressed FLT-3L; 4, secreted FLT3L and
membrane expressed GM-CSF; 5, a non-cleavable form of CD40L; 6, a
non-cleavable form of TNF-.alpha..
[0146] FIG. 64 shows detailed CyTOF data showing DC activation
following stimulation with genetically modified SK lines expressing
immunomodulatory molecules ((i) parental; (ii) APX/3; (iii)
APX/3-4; (iv) APX/3-4-5; (v) APX/3-4-6)). SK lines are represented
by a number code; 3, secreted GM-CSF and membrane expressed FLT-3L;
4, secreted FLT3L and membrane expressed GM-CSF; 5, a non-cleavable
form of CD40L; 6, a non-cleavable form of TNF-.alpha..
[0147] FIG. 65 shows the results of flow cytometry comparing day 6
and day 8 time points in a CD8 expansion assay using SK-parent line
(left panels) versus a genetically modified 14-18-30 expressing
SK-MEL-2 tumor cell lines expressing a combination of the
immunomodulators shown in Table 2 (right panels).
[0148] FIG. 66 is a plot showing mean and standard deviation
results of a xenograft treatment study using NGS mice. The ends of
each box are the upper and lower quartiles; the median is marked by
a vertical line inside the box, and the whiskers are the two lines
outside the box that extend to the highest and lowest observations.
Human tumor cells were implanted on the flank of NGS (NOD scid
gamma) mice. The tumors were allowed to grow to 150 mm.sup.3. Mice
were divided into two groups, a control and a treated group, with 6
mice per group. On day 30 (t=0) mices in the control group were
inoculated with vehicle only, and mice in the treated group were
inoculated with 3.times.10.sup.6 PBMCs activated by 14-18-30
expressing ENLIST.TM. cells ("SUPLEXA.TM. cells"). Tumor size was
measured at intervals through 36 days after inoculation. Divergence
between the two groups appeared within 5 days. After day 22, the
divergence became statistically significant (*P<0.05;
**P<005).
DETAILED DESCRIPTION
Definitions
[0149] The term "activation" or "lymphocyte activation" refers to
stimulation of lymphocytes by specific antigens, nonspecific
mitogens, or allogeneic cells resulting in synthesis of RNA,
protein and DNA and production of lymphokines; it is followed by
proliferation and differentiation of various effector and memory
cells. For example, a mature B cell can be activated by an
encounter with an antigen that expresses epitopes that are
recognized by its cell surface immunoglobulin Ig). The activation
process may be a direct one, dependent on cross-linkage of membrane
Ig molecules by the antigen (cross-linkage-dependent B cell
activation) or an indirect one, occurring most efficiently in the
context of an intimate interaction with a helper T cell ("cognate
help process"). T-cell activation is dependent on the interaction
of the TCR/CD3 complex with its cognate ligand, a peptide bound in
the groove of a class I or class II MEW molecule. The molecular
events set in motion by receptor engagement are complex. Among the
earliest steps appears to be the activation of tyrosine kinases
leading to the tyrosine phosphorylation of a set of substrates that
control several signaling pathways. These include a set of adapter
proteins that link the TCR to the ras pathway, phospholipase
C.gamma.1, the tyrosine phosphorylation of which increases its
catalytic activity and engages the inositol phospholipid metabolic
pathway, leading to elevation of intracellular free calcium
concentration and activation of protein kinase C, and a series of
other enzymes that control cellular growth and differentiation.
Full responsiveness of a T cell requires, in addition to receptor
engagement, an accessory cell-delivered costimulatory activity,
e.g., engagement of CD28 on the T cell by CD80 and/or CD86 on the
antigen presenting cell (APC). The soluble product of an activated
B lymphocyte is immmunoglobulins (antibodies). The soluble product
of an activated T lymphocyte is lymphokines.
[0150] B cell activation. A mature B cell can be activated by an
encounter with an antigen that expresses epitopes that are
recognized by its cell surface immunoglobulin Ig). The activation
process may be a direct one, dependent on cross-linkage of membrane
Ig molecules by the antigen (cross-linkage-dependent B cell
activation) or an indirect one, occurring most efficiently in the
context of an intimate interaction with a helper T cell ("cognate
help process"). The soluble product of an activated B lymphocyte is
immunoglobulins (antibodies).
[0151] T-cell activation is dependent on the interaction of the
TCR/CD3 complex with its cognate ligand, a peptide bound in the
groove of a class I or class II MHC molecule. The molecular events
set in motion by receptor engagement are complex. Among the
earliest steps appears to be the activation of tyrosine kinases
leading to the tyrosine phosphorylation of a set of substrates that
control several signaling pathways. These include a set of adapter
proteins that link the TCR to the ras pathway, phospholipase
C.gamma.1, the tyrosine phosphorylation of which increases its
catalytic activity and engages the inositol phospholipid metabolic
pathway, leading to elevation of intracellular free calcium
concentration and activation of protein kinase C, and a series of
other enzymes that control cellular growth and differentiation.
Full responsiveness of a T cell requires, in addition to receptor
engagement, an accessory cell-delivered costimulatory activity,
e.g., engagement of CD28 on the T cell by CD80 and/or CD86 on the
antigen presenting cell (APC). The soluble product of an activated
T lymphocyte is lymphokines.
[0152] Dendritic cell activation. Pathogen invasion induces a rapid
inflammatory response initiated through the recognition of
pathogen-derived molecules by pattern recognition receptors (PRRs)
expressed on both immune and non-immune cells. Joffre, O., et al.,
Immunol. Rev. (2009) 277(1): 234-47. GET The initial wave of
pro-inflammatory cytokines and chemokines limits pathogen spread
and recruits and activates immune cells to eradicate the invaders.
Dendritic cells (DCs) are responsible for initiating a subsequent
phase of immunity, dominated by the action of pathogen-specific T
and B cells. As for the early pro-inflammatory response, DC
activation is triggered by PRR signals, which convert resting DCs
into potent antigen-presenting cells capable of promoting the
expansion and effector differentiation of naive pathogen-specific T
cells. While DCs can be activated indirectly by inflammatory
cytokines, these cells are unable to induce a functional T-cell
response, and may function in tolerance induction.
[0153] As used herein, the terms "activating CD8+ T cells" or "CD8+
T cell activation" refer to a process (e.g., a signaling event)
causing or resulting in one or more cellular responses of a CD8+ T
cell (CTL), selected from: proliferation, differentiation, cytokine
secretion, cytotoxic effector molecule release, cytotoxic activity,
and expression of activation markers. As used herein, an "activated
CD8+ T cell" refers to a CD8+ T cell that has received an
activating signal, and thus demonstrates one or more cellular
responses, selected from proliferation, differentiation, cytokine
secretion, cytotoxic effector molecule release, cytotoxic activity,
and expression of activation markers. Suitable assays to measure
CD8+ T cell activation are known in the art and are described
herein.
[0154] As used herein, the terms "expanding a CD8+ T cell" or "CD8+
T cell expansion" refer to a process wherein a population of CD8+ T
cells undergoes a series of cell divisions and thereby increases in
cell number. The term "expanded CD8+ T cells" relates to CD8+ T
cells obtained through CD8+ T cell expansion. Suitable assays to
measure T cell expansion are known in the art and are described
herein.
[0155] As used herein, the term "activating an NK cell" or "NK cell
activation" refers to a process (e.g., a signaling event) causing
or resulting in an NK cell being capable of killing cells with
deficiencies in MEW class I expression. As used herein, an
"activated NK cell" refers to an NK cell that has received an
activating signal, and is thus capable of killing cells with
deficiencies in MEW class I expression. Suitable assays to measure
NK cell activation are known in the art and are described
herein.
[0156] As used herein, the terms "expanding an NK cell" or "NK cell
expansion" refer to a process wherein a population of NK cells
undergoes a series of cell divisions and thereby increases in cell
number. The term "expanded NK cells" relates to NK cells obtained
through NK cell expansion. Suitable assays to measure NK cell
expansion are known in the art and are described herein.
[0157] As used herein, the term "administration" and its various
grammatical forms as it applies to a mammal, cell, tissue, organ,
or biological fluid, refers without limitation to contact of an
exogenous ligand, reagent, placebo, small molecule, pharmaceutical
agent, therapeutic agent, diagnostic agent, or composition to the
subject, cell, tissue, organ, or biological fluid, and the like.
"Administration" can refer, e.g., to therapeutic, pharmacokinetic,
diagnostic, research, placebo, and experimental methods.
"Administration" also encompasses in vitro and ex vivo treatments,
e.g., of a cell, by a reagent, diagnostic, binding composition, or
by another cell.
[0158] The term "allogeneic" as used herein means that the donor
and the recipient (host) are of different genetic makeup, but of
the same species. As used herein, an "allogeneic cell" refers to a
cell that is not derived from the recipient, meaning the individual
to which the cell is to be administered, that is, it has a
different genetic constitution than the recipient individual. An
allogeneic cell is generally obtained from the same species as the
individual to which the cell is to be administered. For example,
the allogeneic cell can be a human cell, as disclosed herein, for
administering to a human patient such as a cancer patient. As used
herein, an "allogeneic tumor cell" refers to a tumor cell that is
not derived from the recipient, meaning the individual to which the
allogeneic cell is to be administered. Generally, the allogeneic
tumor cell expresses one or more tumor antigens that can stimulate
an immune response against a tumor in an individual to which the
cell is to be administered. As used herein, an "allogeneic cancer
cell," for example, a lung cancer cell, refers to a cancer cell
that is not derived from the recipient individual to which the
allogeneic cell is to be administered.
[0159] The terms "amino acid residue" or "amino acid" or "residue"
are used interchangeably to refer to an amino acid that is
incorporated into a protein, a polypeptide, or a peptide,
including, but not limited to, a naturally occurring amino acid and
known analogs of natural amino acids that can function in a similar
manner as naturally occurring amino acids. The amino acids may be
L- or D-amino acids. An amino acid may be replaced by a synthetic
amino acid, which is altered so as to increase the half-life of the
peptide, increase the potency of the peptide, or increase the
bioavailability of the peptide. The single letter designation for
amino acids is used predominately herein. Such single letter
designations are as follows: A is alanine; C is cysteine; D is
aspartic acid; E is glutamic acid; F is phenylalanine; G is
glycine; H is histidine; I is isoleucine; K is lysine; L is
leucine; M is methionine; N is asparagine; P is proline; Q is
glutamine; R is arginine; S is serine; T is threonine; V is valine;
W is tryptophan; and Y is tyrosine. The following represents groups
of amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic Acid (D),
Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine
(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan
(W).
[0160] The terms "apoptosis" or "programmed cell death" refer to a
highly regulated and active process that contributes to biologic
homeostasis comprised of a series of biochemical events that lead
to a variety of morphological changes, including blebbing, changes
to the cell membrane, such as loss of membrane asymmetry and
attachment, cell shrinkage, nuclear fragmentation, chromatin
condensation, and chromosomal DNA fragmentation, without damaging
the organism.
[0161] Apoptotic cell death is induced by many different factors
and involves numerous signaling pathways, some dependent on caspase
proteases (a class of cysteine proteases) and others that are
caspase independent. It can be triggered by many different cellular
stimuli, including cell surface receptors, mitochondrial response
to stress, and cytotoxic T cells, resulting in activation of
apoptotic signaling pathways.
[0162] The caspases involved in apoptosis convey the apoptotic
signal in a proteolytic cascade, with caspases cleaving and
activating other caspases that then degrade other cellular targets
that lead to cell death. The caspases at the upper end of the
cascade include caspase-8 and caspase-9. Caspase-8 is the initial
caspase involved in response to receptors with a death domain (DD)
like Fas.
[0163] Receptors in the TNF receptor family are associated with the
induction of apoptosis, as well as inflammatory signaling. The Fas
receptor (CD95) mediates apoptotic signaling by Fas-ligand
expressed on the surface of other cells. The Fas-FasL interaction
plays an important role in the immune system and lack of this
system leads to autoimmunity, indicating that Fas-mediated
apoptosis removes self-reactive lymphocytes. Fas signaling also is
involved in immune surveillance to remove transformed cells and
virus infected cells. Binding of Fas to oligimerized FasL on
another cell activates apoptotic signaling through a cytoplasmic
domain termed the death domain (DD) that interacts with signaling
adaptors including FAF, FADD and DAX to activate the caspase
proteolytic cascade. Caspase-8 and caspase-10 first are activated
to then cleave and activate downstream caspases and a variety of
cellular substrates that lead to cell death.
[0164] Mitochondria participate in apoptotic signaling pathways
through the release of mitochondrial proteins into the cytoplasm.
Cytochrome c, a key protein in electron transport, is released from
mitochondria in response to apoptotic signals, and activates
Apaf-1, a protease released from mitochondria. Activated Apaf-1
activates caspase-9 and the rest of the caspase pathway.
Smac/DIABLO is released from mitochondria and inhibits IAP proteins
that normally interact with caspase-9 to inhibit apoptosis.
Apoptosis regulation by Bcl-2 family proteins occurs as family
members form complexes that enter the mitochondrial membrane,
regulating the release of cytochrome c and other proteins. TNF
family receptors that cause apoptosis directly activate the caspase
cascade, but can also activate Bid, a Bcl-2 family member, which
activates mitochondria-mediated apoptosis. Bax, another Bcl-2
family member, is activated by this pathway to localize to the
mitochondrial membrane and increase its permeability, releasing
cytochrome c and other mitochondrial proteins. Bcl-2 and Bcl-xL
prevent pore formation, blocking apoptosis. Like cytochrome c, AIF
(apoptosis-inducing factor) is a protein found in mitochondria that
is released from mitochondria by apoptotic stimuli. While
cytochrome C is linked to caspase-dependent apoptotic signaling,
AIF release stimulates caspase-independent apoptosis, moving into
the nucleus where it binds DNA. DNA binding by AIF stimulates
chromatin condensation, and DNA fragmentation, perhaps through
recruitment of nucleases.
[0165] The mitochondrial stress pathway begins with the release of
cytochrome c from mitochondria, which then interacts with Apaf-1,
causing self-cleavage and activation of caspase-9. Caspase-3, -6
and-7 are downstream caspases that are activated by the upstream
proteases and act themselves to cleave cellular targets.
[0166] Granzyme B and perforin proteins released by cytotoxic T
cells induce apoptosis in target cells, forming transmembrane
pores, and triggering apoptosis, perhaps through cleavage of
caspases, although caspase-independent mechanisms of Granzyme B
mediated apoptosis have been suggested.
[0167] Fragmentation of the nuclear genome by multiple nucleases
activated by apoptotic signaling pathways to create a nucleosomal
ladder is a cellular response characteristic of apoptosis. One
nuclease involved in apoptosis is DNA fragmentation factor (DFF), a
caspase-activated DNAse (CAD). DFF/CAD is activated through
cleavage of its associated inhibitor ICAD by caspases proteases
during apoptosis. DFF/CAD interacts with chromatin components such
as topoisomerase II and histone H1 to condense chromatin structure
and perhaps recruit CAD to chromatin. Another apoptosis activated
protease is endonuclease G (EndoG). EndoG is encoded in the nuclear
genome but is localized to mitochondria in normal cells. EndoG may
play a role in the replication of the mitochondrial genome, as well
as in apoptosis. Apoptotic signaling causes the release of EndoG
from mitochondria. The EndoG and DFF/CAD pathways are independent
since the EndoG pathway still occurs in cells lacking DFF.
[0168] Hypoxia, as well as hypoxia followed by reoxygenation can
trigger cytochrome c release and apoptosis. Glycogen synthase
kinase (GSK-3) a serine-threonine kinase ubiquitously expressed in
most cell types, appears to mediate or potentiate apoptosis due to
many stimuli that activate the mitochondrial cell death pathway.
Loberg, R D, et al., J. Biol. Chem. 277 (44): 41667-673 (2002). It
has been demonstrated to induce caspase 3 activation and to
activate the proapoptotic tumor suppressor gene p53. It also has
been suggested that GSK-3 promotes activation and translocation of
the proapoptotic Bcl-2 family member, Bax, which, upon aggregation
and mitochondrial localization, induces cytochrome c release. Akt
is a critical regulator of GSK-3, and phosphorylation and
inactivation of GSK-3 may mediate some of the antiapoptotic effects
of Akt.
[0169] The term "autologous" as used herein means derived from the
same individual.
[0170] The term "cancer" as used herein refers to diseases in which
abnormal cells divide without control and are able to invade other
tissues. There are more than 100 different types of cancer. Most
cancers are named for the organ or type of cell in which they
start--for example, cancer that begins in the colon is called colon
cancer; cancer that begins in melanocytes of the skin is called
melanoma. Cancer types can be grouped into broader categories. The
main categories of cancer include: carcinoma (meaning a cancer that
begins in the skin or in tissues that line or cover internal
organs, and its subtypes, including adenocarcinoma, basal cell
carcinoma, squamous cell carcinoma, and transitional cell
carcinoma); sarcoma (meaning a cancer that begins in bone,
cartilage, fat, muscle, blood vessels, or other connective or
supportive tissue); leukemia (meaning a cancer that starts in
blood-forming tissue (e.g., bone marrow) and causes large numbers
of abnormal blood cells to be produced and enter the blood;
lymphoma and myeloma (meaning cancers that begin in the cells of
the immune system); and Central nervous system cancers (meaning
cancers that begin in the tissues of the brain and spinal cord).
The term "myelodysplastic syndrome" refers to a type of cancer in
which the bone marrow does not make enough healthy blood cells
(white blood cells, red blood cells, and platelets) and there are
abnormal cells in the blood and/or bone marrow. Myelodysplastic
syndrome may become acute myeloid leukemia (AML).
[0171] The term "cell line" as used herein, means a permanently
established cell culture developed from a single cell and therefore
consisting of cells with a uniform genetic makeup that will
proliferate indefinitely.
[0172] The term "chemotherapy" as used herein refers to a treatment
that uses drugs to stop the growth of cancer cells.
[0173] The term "contact" and its various grammatical forms as used
herein refers to a state or condition of touching or of immediate
or local proximity. Contacting a composition to a target
destination may occur by any means of administration known to the
skilled artisan.
[0174] The term "costimulatory molecule" as used herein refers to
one of two or more molecules that are displayed on the cell surface
that have a role in activating a T cell to become an effector cell.
For example MHC proteins, which present foreign antigen to the T
cell receptor, also require costimulatory proteins which bind to
complementary receptors on the T cell's surface to result in
activation of the T cell.
[0175] As used herein, the term "cytokine" refers to small soluble
protein substances secreted by cells which have a variety of
effects on other cells. Cytokines mediate many important
physiological functions including growth, development, wound
healing, and the immune response. They act by binding to their
cell-specific receptors located in the cell membrane, which allows
a distinct signal transduction cascade to start in the cell, which
eventually will lead to biochemical and phenotypic changes in
target cells. Cytokines can act both locally and distantly from a
site of release. They include type I cytokines, which encompass
many of the interleukins, as well as several hematopoietic growth
factors; type II cytokines, including the interferons and
interleukin-10; tumor necrosis factor ("TNF")-related molecules,
including TNF.alpha. and lymphotoxin; immunoglobulin super-family
members, including interleukin 1 ("IL-1"); and the chemokines, a
family of molecules that play a critical role in a wide variety of
immune and inflammatory functions. The same cytokine can have
different effects on a cell depending on the state of the cell.
Cytokines often regulate the expression of, and trigger cascades of
other cytokines. Non limiting examples of cytokines include e.g.,
Granulocyte-macrophage colony-stimulating factor (GM-CSF),
Granulocyte colony-stimulating factor (G-CSF), Fms-related tyrosine
kinase 3 ligand (FLT3LG), interleukin-1 (IL-1), IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12/IL-23 P40,
IL13, IL-15, IL-15/IL15-RA, IL-17, IL-18, IL-21, IL-23, TGF-.beta.,
MCP-1, TNF-.alpha. and interferon alpha (IFN.alpha.),
IFN.gamma.
[0176] The term "cytotoxic T lymphocytes" (CTLs) refers to effector
CD8+ T cells. Cytotoxic T cells kill by inducing their targets to
undergo apoptosis. They induce target cells to undergo programmed
cell death via extrinsic and intrinsic pathways.
[0177] As used herein, the term "dendritic cell" or "DC" describes
a diverse population of morphologically similar cell types found in
a variety of lymphoid and non-lymphoid tissues that present foreign
antigens to T cells, see Steinman, Ann. Rev. Immunol. 9:271-296
(1991).
[0178] The term "derived from" as used herein encompasses any
method for receiving, obtaining, or modifying something from a
source of origin.
[0179] The term "derivative" or "variant" with respect to a peptide
or DNA sequence (e.g. immune modulator peptide sequence) as used
herein refers to a non-identical peptide or DNA sequence that is
modified from its original sequence. The differences in the
sequences may by the result of changes, by design, in sequence or
structure. Designed changes may be specifically designed and
introduced into the sequence for specific purposes. Such specific
changes may be made in vitro using a variety of mutagenesis
techniques. Such sequence variants generated specifically may be
referred to as "mutants" or "derivatives" of the original sequence.
The terms "derivative" or "variant" with respect to cells as used
herein refers to a cell line that has been modified from its cell
line of origin (e.g. modified to express recombinant DNA
sequences).
[0180] The term "detectable marker" encompasses both selectable
markers and assay markers. The term "selectable markers" refers to
a variety of gene products to which cells transformed with an
expression construct can be selected or screened, including
drug-resistance markers, antigenic markers useful in
fluorescence-activated cell sorting, adherence markers such as
receptors for adherence ligands allowing selective adherence, and
the like.
[0181] The term "detectable response" refers to any signal or
response that may be detected in an assay, which may be performed
with or without a detection reagent. Detectable responses include,
but are not limited to, radioactive decay and energy (e.g.,
fluorescent, ultraviolet, infrared, visible) emission, absorption,
polarization, fluorescence, phosphorescence, transmission,
reflection or resonance transfer. Detectable responses also include
chromatographic mobility, turbidity, electrophoretic mobility, mass
spectrum, ultraviolet spectrum, infrared spectrum, nuclear magnetic
resonance spectrum and x-ray diffraction. Alternatively, a
detectable response may be the result of an assay to measure one or
more properties of a biologic material, such as melting point,
density, conductivity, surface acoustic waves, catalytic activity
or elemental composition. A "detection reagent" is any molecule
that generates a detectable response indicative of the presence or
absence of a substance of interest. Detection reagents include any
of a variety of molecules, such as antibodies, nucleic acid
sequences and enzymes. To facilitate detection, a detection reagent
may comprise a marker.
[0182] The term "differentiate" and its various grammatical forms
as used herein refers to the process of development with an
increase in the level of organization or complexity of a cell or
tissue, accompanied with a more specialized function.
[0183] The term "dose" as used herein refers to the quantity of a
therapeutic substance prescribed to be taken at one time.
[0184] The term "effector cell" as used herein refers to a cell
that carries out a final response or function. The main effector
cells of the immune system, for example, are activated lymphocytes
and phagocytes.
[0185] Engineered Leukocyte Stimulator cells ("ENLIST.TM. cells)
signify a population of proliferation incompetent tumor cells
genetically engineered to express a core group of three
immunomodulatory molecules used to stimulate mononuclear cells for
the treatment of cancer.
[0186] The term "enrich" as used herein refers to increasing the
proportion of a desired substance, for example, to increase the
relative frequency of a subtype of cell compared to its natural
frequency in a cell population. Positive selection, negative
selection, or both are generally considered necessary to any
enrichment scheme. Selection methods include, without limitation,
magnetic separation and FACS. Regardless of the specific technology
used for enrichment, the specific markers used in the selection
process are critical, since developmental stages and
activation-specific responses can change a cell's antigenic
profile.
[0187] As used herein, the term "exogenous polypeptide" refers to a
polypeptide that is not produced by a wild-type cell of that type
or is present at a lower level in a wild-type cell than in a cell
containing the exogenous polypeptide. According to some
embodiments, an exogenous polypeptide is a polypeptide encoded by a
nucleic acid that was introduced into the cell, which nucleic acid
is optionally not retained by the cell.
[0188] As used herein, the term "exogenous immunomodulatory
molecule" includes a polypeptide comprising (e.g., intracellularly
or at the cell surface) an allogeneic cell (e.g. an allogeneic cell
line) that specifically binds a cognate polypeptide (e.g.,
receptor) on an immune cell, such as an immune killer cell (e.g. an
NK cell or a CD8+ T cell), thereby providing a signal which
mediates stimulation of the immune cell, such as the proliferation,
activation, expansion and the like of the immune cell. According to
one embodiment, one or more exogenous immunomodulatory polypeptides
are sufficient to stimulate an immune killer cell ex vivo or in
vivo. Exemplary exogenous immunomodulatory polypeptides are
described in more detail below.
[0189] As used herein, the term "express" or "expression"
encompasses the biosynthesis of mRNA, polypeptide biosynthesis,
polypeptide activation, e.g., by post-translational modification,
or an activation of expression by changing the subcellular location
or by recruitment to chromatin. Expression may be, e.g., increased
by a number of approaches, including: increasing the number of
genes encoding the polypeptide, increasing the transcription of the
gene (such as by placing the gene under the control of a
constitutive promoter), increasing the translation of the gene,
knock out of a competitive gene, or a combination of these and/or
other approaches.
[0190] The term "expression vector" refers to a DNA molecule
comprising a gene that is expressed in a host cell. Typically, gene
expression is placed under the control of certain regulatory
elements including, but not limited to, promoters, tissue specific
regulatory elements, and enhancers. Such a gene is said to be
"operably linked to" the regulatory elements.
[0191] As used herein, the terms "first" and "second" with respect
to exogenous immunomodulatory molecules are used for convenience of
distinguishing when there is more than one type of exogenous
stimulatory polypeptide. Use of these terms is not intended to
confer a specific order or orientation of the exogenous stimulatory
polypeptides unless explicitly so stated.
[0192] The term "flow cytometry" as used herein refers to a tool
for interrogating the phenotype and characteristics of cells. It
senses cells or particles as they move in a liquid stream through a
laser (light amplification by stimulated emission of
radiation)/light beam past a sensing area. The relative
light-scattering and color-discriminated fluorescence of the
microscopic particles is measured. Flow Analysis and
differentiation of the cells is based on size, granularity, and
whether the cell is carrying fluorescent molecules in the form of
either antibodies or dyes. As the cell passes through the laser
beam, light is scattered in all directions, and the light scattered
in the forward direction at low angles)(0.5-10.degree. from the
axis is proportional to the square of the radius of a sphere and so
to the size of the cell or particle. Light may enter the cell;
thus, the 90.degree. light (right-angled, side) scatter may be
labeled with fluorochrome-linked antibodies or stained with
fluorescent membrane, cytoplasmic, or nuclear dyes. Thus, the
differentiation of cell types, the presence of membrane receptors
and antigens, membrane potential, pH, enzyme activity, and DNA
content may be facilitated. Flow cytometers are multiparameter,
recording several measurements on each cell; therefore, it is
possible to identify a homogeneous subpopulation within a
heterogeneous population (Marion G. Macey, Flow cytometry:
principles and applications, Humana Press, 2007).
Fluorescence-activated cell sorting (FACS), which allows isolation
of distinct cell populations too similar in physical
characteristics to be separated by size or density, uses
fluorescent tags to detect surface proteins that are differentially
expressed, allowing fine distinctions to be made among physically
homogeneous populations of cells.
[0193] The term "functional equivalent" or "functionally
equivalent" are used interchangeably herein to refer to substances,
molecules, polynucleotides, proteins, peptides, or polypeptides
having similar or identical effects or use.
[0194] As used herein, the term "gene" is used broadly to refer to
any segment of nucleic acid associated with expression of a given
RNA or protein. Thus, genes include regions encoding expressed RNAs
(which typically include polypeptide coding sequences) and, often,
the regulatory sequences required for their expression. Genes can
be obtained from a variety of sources, including cloning from a
source of interest or synthesizing from known or predicted sequence
information, and may include sequences designed to have
specifically desired parameters.
[0195] The term "heteroclitic" is used herein to refer to peptides
of higher biological potency than the original peptide. A
"heteroclitic immunogen" is an immunogen that elicits an immune
response that cross-reacts to an original poorly immunogenic
antigen.
[0196] The terms "immune response" and "immune-mediated" are used
interchangeably herein to refer to any functional expression of a
subject's immune system, against either foreign or self-antigens,
whether the consequences of these reactions are beneficial or
harmful to the subject.
[0197] The term "immunosuppression" as used herein and its other
grammatical forms refer to a decrease of the body's immune response
and ability of the immune system to fight infections and other
diseases. For example, some immunosuppression may be induced with
drugs, or may result from disease.
[0198] The terms "immunomodulatory", "immune modulator" and "immune
modulatory" are used interchangeably herein to refer to a
substance, agent, or cell that is capable of augmenting or
diminishing immune responses directly or indirectly by expressing
chemokines, cytokines and other mediators of immune responses.
[0199] As used herein the term "immunostimulatory amount" of the
disclosed compositions refers to an amount of an immunogenic
composition that is effective to stimulate an immune response, for
example, as measured by ELISPOT assay (cellular immune response),
ICS (intracellular cytokine staining assay) and major
histocompatibility complex (MEW) tetramer assay to detect and
quantify antigen-specific T cells, quantifying the blood population
of antigen-specific CD4+ T cells, or quantifying the blood
population of antigen specific CD8+ T cells by a measurable amount,
or where the increase is by at least 10%, at least 20%, at least
30%, at least 40%, at least 50%, at least 60%, at least 70%, at
least 80%, at least 90%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99%, at least 100%, when compared to a
suitable control (e.g., a control composition where dendritic cells
are not loaded with tumor-specific cells, or not loaded with
peptide derived from tumor-specific cells).
[0200] The term "integrate into the genome" as used herein refers
to a recombinant DNA sequence being concomitantly joined to the
genomic DNA comprising a host cell's genome.
[0201] The term "Kaplan Meier plot" or "Kaplan Meier survival
curve" as used herein refers to the plot of probability of clinical
study subjects surviving in a given length of time while
considering time in many small intervals. The Kaplan Meier plot
assumes that: (i) at any time subjects who are censored (i.e.,
lost) have the same survival prospects as subjects who continue to
be followed; (ii) the survival probabilities are the same for
subjects recruited early and late in the study; and (iii) the event
(e.g., death) happens at the time specified. Probabilities of
occurrence of events are computed at a certain point of time with
successive probabilities multiplied by any earlier computed
probabilities to get a final estimate. The survival probability at
any particular time is calculated as the number of subjects
surviving divided by the number of subjects at risk. Subjects who
have died, dropped out, or have been censored from the study are
not counted as at risk.
[0202] The term "labeling" as used herein refers to a process of
distinguishing a compound, structure, protein, peptide, antibody,
cell or cell component by introducing a traceable constituent.
Common traceable constituents include, but are not limited to, a
fluorescent antibody, a fluorophore, a dye or a fluorescent dye, a
stain or a fluorescent stain, a marker, a fluorescent marker, a
chemical stain, a differential stain, a differential label, and a
radioisotope.
[0203] The terms "marker" or "cell surface marker" are used
interchangeably herein to refer to an antigenic determinant or
epitope found on the surface of a specific type of cell. Cell
surface markers can facilitate the characterization of a cell type,
its identification, and eventually its isolation. Cell sorting
techniques are based on cellular biomarkers where a cell surface
marker(s) may be used for either positive selection or negative
selection, i.e., for inclusion or exclusion, from a cell
population.
[0204] The term "mediate" and its various grammatical forms as used
herein means to bring about a result.
[0205] The term "minimal residual disease" as used herein refers to
a very small number of cancer cells that remain in the body during
or after treatment. Minimal residual disease can be found only by
highly sensitive laboratory methods that are able to find one
cancer cell among one million normal cells.
[0206] The terms "mixed lymphocyte tumor reaction" or "MLTR" are
used interchangeably herein to refer to a reaction similar to a
mixed lymphocyte reaction but rather than using allogeneic
lymphocytes to stimulate a response, allogeneic tumor cells are
used instead. The MLTR method comprises contacting tumor cells
being tested for immunogenic potential with mixed lymphocytes from
peripheral blood mononuclear cells, followed by measuring one or
more of cellular proliferation of the lymphocytes, cellular subset
differentiation of the lymphocytes, cytokine release profile of the
lymphocytes, and tumor cell death.
[0207] The term "modify" and its various grammatical forms as used
herein refers to a change of the form or qualities of
[0208] The term "modulate" and its various grammatical forms as
used herein means to regulate, alter, adapt, or adjust to a certain
measure or proportion. Such modulation may be any change, including
an undetectable change.
[0209] The term "modified" or "modulated" as used herein with
respect to an immune response to tumor cells refers to changing the
form or character of the immune response to the tumor cells via one
or more recombinant DNA techniques such that the immune cells are
able to recognize and kill tumor cells.
[0210] The term "myeloid suppressor cells" or "myeloid-derived
suppressor cells" as used herein refers to a heterogeneous
population of cells characterized by myeloid origin, immature
state, and ability to potently suppress T cell responses. These
cells regulate immune responses and tissue repair in healthy
individuals and the population rapidly expands during
inflammation.
[0211] As used herein the term "natural killer (NK) cells" refers
to lymphocytes in the same family as T and B cells, classified as
group I innate lymphocytes. They have an ability to kill tumor
cells without any priming or prior activation, in contrast to
cytotoxic T cells, which need priming by antigen presenting cells.
NK cells secrete cytokines such as IFN.gamma. and TNF.alpha., which
act on other immune cells, like macrophages and dendritic cells, to
enhance the immune response. Activating receptors on the NK cell
surface recognize molecules expressed on the surface of cancer
cells and infected cells and switch on the NK cell. Inhibitory
receptors act as a check on NK cell killing. Most normal healthy
cells express MHCI receptors, which mark them as "self" Inhibitory
receptors on the surface of the NK cell recognize cognate MHCI,
which switches off the NK cell, preventing it from killing. Once
the decision is made to kill, the NK cell releases cytotoxic
granules containing perforin and granzymes, which leads to lysis of
the target cell. Natural killer reactivity, including cytokine
secretion and cytotoxicity, is controlled by a balance of several
germ-line encoded inhibitory and activating receptors such as
killer immunoglobulin-like receptors (KIRs) and natural
cytotoxicity receptors (NCRs). The presence of the MHC Class I
molecule on target cells serves as one such inhibitory ligand for
MHC Class I-specific receptors, the Killer cell Immunoglobulin-like
Receptor (KIR), on NK cells. Engagement of KIR receptors blocks NK
activation and, paradoxically, preserves their ability to respond
to successive encounters by triggering inactivating signals.
Therefore, if a KIR is able to sufficiently bind to MHC Class I,
this engagement may override the signal for killing and allows the
target cell to live. In contrast, if the NK cell is unable to
sufficiently bind to MHC Class I on the target cell, killing of the
target cell may proceed. Consequently, those tumors which express
low MHC Class I and which are thought to be capable of evading a
T-cell-mediated attack may be susceptible to an NK cell-mediated
immune response instead.
[0212] The term "nucleic acid" is used herein to refer to a
deoxyribonucleotide or ribonucleotide polymer in either single- or
double-stranded form, and, unless otherwise limited, encompasses
known analogues having the essential nature of natural nucleotides
in that they hybridize to single-stranded nucleic acids in a manner
similar to naturally occurring nucleotides (e.g., peptide nucleic
acids). Nucleic acid molecules useful in the methods of the
invention include any nucleic acid molecule that encodes a
polypeptide of the invention or a fragment thereof. Such nucleic
acid molecules need not be 100% identical with an endogenous
nucleic acid sequence, but will typically exhibit substantial
identity. Polynucleotides having "substantial identity" to an
endogenous sequence are typically capable of hybridizing with at
least one strand of a double-stranded nucleic acid molecule.
Nucleic acid molecules useful in the methods of the invention
include any nucleic acid molecule that encodes a polypeptide of the
invention or a fragment thereof. Such nucleic acid molecules need
not be 100% identical with an endogenous nucleic acid sequence, but
will typically exhibit substantial identity. Polynucleotides having
"substantial identity" to an endogenous sequence are typically
capable of hybridizing with at least one strand of a
double-stranded nucleic acid molecule. By "hybridize" is meant pair
to form a double-stranded molecule between complementary
polynucleotide sequences (e.g., a gene described herein), or
portions thereof, under various conditions of stringency. (See,
e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399;
Kimmel, A. R. (1987) Methods Enzymol. 152:507). Measuring the
effects of base incompatibility by quantifying the rate at which
two strands anneal can provide information as to the similarity in
base sequence between the two strands being annealed. A nucleic
acid that selectively hybridizes undergoes hybridization, under
stringent hybridization conditions, of the nucleic acid sequence to
a specified nucleic acid target sequence to a detectably greater
degree (e.g., at least 2-fold over background) than its
hybridization to non-target nucleic acid sequences and to the
substantial exclusion of non-target nucleic acids.
[0213] By "substantially identical" is meant a polypeptide or
nucleic acid molecule exhibiting at least 50% identity to a
reference amino acid sequence (for example, any one of the amino
acid sequences described herein) or nucleic acid sequence (for
example, any one of the nucleic acid sequences described herein).
For example, such a sequence is at least 60%, at least 61%, at
least 62%, at least 63%, at least 64%, at least 65%, at least 66%,
at least 67%, at least 68%, at least 69%, at least 70%, at least
71%, at least 72%, at least 73%, at least 74%, at least 75%, at
least 76%, at least 77%, at least 78%, at least 79%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% identical at the amino acid level or nucleic acid to the
sequence used for comparison.
[0214] Sequence identity is typically measured using sequence
analysis software (for example, Sequence Analysis Software Package
of the Genetics Computer Group, University of Wisconsin
Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705,
BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software
matches identical or similar sequences by assigning degrees of
homology to various substitutions, deletions, and/or other
modifications. Conservative substitutions typically include
substitutions within the following groups: glycine, alanine;
valine, isoleucine, leucine; aspartic acid, glutamic acid,
asparagine, glutamine; serine, threonine; lysine, arginine; and
phenylalanine, tyrosine. In an exemplary approach to determining
the degree of identity, a BLAST program may be used, with a
probability score between e-3 and e-100 indicating a closely
related sequence.
[0215] The term "open reading frame" as used herein refers to a
sequence of nucleotides in a DNA molecule that has the potential to
encode a peptide or protein: it starts with a start triplet (ATG),
is followed by a string of triplets each of which encodes an amino
acid, and ends with a stop triplet (TAA, TAG or TGA).
[0216] The phrase "operably linked" refers (1) to a first
sequence(s) or domain being positioned sufficiently proximal to a
second sequence(s) or domain so that the first sequence(s) or
domain can exert influence over the second sequence(s) or domain or
a region under control of that second sequence or domain; and (2)
to a functional linkage between a promoter and a second sequence,
wherein the promoter sequence initiates and mediates transcription
of the DNA sequence corresponding to the second sequence.
Generally, operably linked means that the nucleic acid sequences
being linked are contiguous and, where necessary to join two
protein coding regions, are in the same reading frame. According to
some embodiments, the phrase "operatively linked" refers to a
linkage in which two or more protein domains or polypeptides are
ligated or combined via recombinant DNA technology or chemical
reaction such that each protein domain or polypeptide of the
resulting fusion protein retains its original function.
[0217] The term "overall survival" (OS) as used herein refers to
the length of time from either the date of diagnosis or the start
of treatment for a disease, such as cancer, that patients diagnosed
with the disease are still alive.
[0218] The term "parenteral" and its other grammatical forms as
used herein refers to administration of a substance occurring in
the body other than by the mouth or alimentary canal. For example,
the term "parenteral" as used herein refers to introduction into
the body by way of an injection (i.e., administration by
injection), including, for example, subcutaneously (i.e., an
injection beneath the skin), intramuscularly (i.e., an injection
into a muscle); intravenously (i.e., an injection into a vein),
intrathecally (i.e., an injection into the space around the spinal
cord or under the arachnoid membrane of the brain), intrasternal
injection, or infusion techniques.
[0219] The terms "peripheral blood mononuclear cells" or "PBMCs"
are used interchangeably herein to refer to blood cells having a
single round nucleus such as, for example, a lymphocyte or a
monocyte.
[0220] The term "pharmaceutical composition" as used herein refers
to a composition that is employed to prevent, reduce in intensity,
cure or otherwise treat a target condition, syndrome, disorder or
disease.
[0221] The term "pharmaceutically acceptable carrier" as used
herein refers to any substantially non-toxic carrier conventionally
useable for administration of pharmaceuticals in which the isolated
polypeptide of the present invention will remain stable and
bioavailable. The pharmaceutically acceptable carrier must be of
sufficiently high purity and of sufficiently low toxicity to render
it suitable for administration to the mammal being treated. It
further should maintain the stability and bioavailability of an
active agent. The pharmaceutically acceptable carrier can be liquid
or solid and is selected, with the planned manner of administration
in mind, to provide for the desired bulk, consistency, etc., when
combined with an active agent and other components of a given
composition.
[0222] The term "pharmaceutically acceptable salt" as used herein
refers to those salts which are, within the scope of sound medical
judgment, suitable for use in contact with the tissues of humans
and lower animals without undue toxicity, irritation, allergic
response and the like and are commensurate with a reasonable
benefit/risk ratio. When used in medicine the salts should be
pharmaceutically acceptable, but non-pharmaceutically acceptable
salts may conveniently be used to prepare pharmaceutically
acceptable salts thereof. Such salts include, but are not limited
to, those prepared from the following acids: hydrochloric,
hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic,
salicylic, p-toluene sulphonic, tartaric, citric, methane
sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and
benzene sulphonic. Also, such salts may be prepared as alkaline
metal or alkaline earth salts, such as sodium, potassium or calcium
salts of the carboxylic acid group. By "pharmaceutically acceptable
salt" is meant those salts which are, within the scope of sound
medical judgment, suitable for use in contact with the tissues of
humans and lower animals without undue toxicity, irritation,
allergic response and the like and are commensurate with a
reasonable benefit/risk ratio. Pharmaceutically acceptable salts
are well-known in the art. For example, P. H. Stahl, et al.
describe pharmaceutically acceptable salts in detail in "Handbook
of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley
VCH, Zurich, Switzerland: 2002). The salts may be prepared in situ
during the final isolation and purification of the compounds
described within the present invention or separately by reacting a
free base function with a suitable organic acid. Representative
acid addition salts include, but are not limited to, acetate,
adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, camphorate, camphorsufonate, digluconate,
glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate,
hydrochloride, hydrobromide, hydroiodide,
2-hydroxyethansulfonate(isethionate), lactate, maleate,
methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate,
pamoate, pectinate, persulfate, 3-phenylpropionate, picrate,
pivalate, propionate, succinate, tartrate, thiocyanate, phosphate,
glutamate, bicarbonate, p-toluenesulfonate and undecanoate. Also,
the basic nitrogen-containing groups may be quaternized with such
agents as lower alkyl halides such as methyl, ethyl, propyl, and
butyl chlorides, bromides and iodides; dialkyl sulfates like
dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides
such as decyl, lauryl, myristyl and stearyl chlorides, bromides and
iodides; arylalkyl halides like benzyl and phenethyl bromides and
others. Water or oil-soluble or dispersible products are thereby
obtained. Examples of acids which may be employed to form
pharmaceutically acceptable acid addition salts include such
inorganic acids as hydrochloric acid, hydrobromic acid, sulphuric
acid and phosphoric acid and such organic acids as oxalic acid,
maleic acid, succinic acid and citric acid. Basic addition salts
may be prepared in situ during the final isolation and purification
of compounds described within the invention by reacting a
carboxylic acid-containing moiety with a suitable base such as the
hydroxide, carbonate or bicarbonate of a pharmaceutically
acceptable metal cation or with ammonia or an organic primary,
secondary or tertiary amine. Pharmaceutically acceptable salts
include, but are not limited to, cations based on alkali metals or
alkaline earth metals such as lithium, sodium, potassium, calcium,
magnesium and aluminum salts and the like and nontoxic quaternary
ammonia and amine cations including ammonium, tetramethylammonium,
tetraethylammonium, methylamine, dimethylamine, trimethylamine,
triethylamine, diethylamine, ethylamine and the like. Other
representative organic amines useful for the formation of base
addition salts include ethylenediamine, ethanolamine,
diethanolamine, piperidine, piperazine and the like.
Pharmaceutically acceptable salts also may be obtained using
standard procedures well known in the art, for example by reacting
a sufficiently basic compound such as an amine with a suitable acid
affording a physiologically acceptable anion. Alkali metal (for
example, sodium, potassium or lithium) or alkaline earth metal (for
example calcium or magnesium) salts of carboxylic acids may also be
made.
[0223] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers. The essential nature of
such analogues of naturally occurring amino acids is that, when
incorporated into a protein, that protein is specifically reactive
to antibodies elicited to the same protein but consisting entirely
of naturally occurring amino acids.
[0224] The terms "polypeptide", "peptide" and "protein" also are
inclusive of modifications including, but not limited to,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation, and ADP-ribosylation. It
will be appreciated, as is well known and as noted above, that
polypeptides may not be entirely linear. For instance, polypeptides
may be branched as a result of ubiquitination, and they may be
circular, with or without branching, generally as a result of
posttranslational events, including natural processing event and
events brought about by human manipulation which do not occur
naturally. Circular, branched and branched circular polypeptides
may be synthesized by non-translation natural process and by
entirely synthetic methods, as well. According to some embodiments,
the peptide is of any length or size.
[0225] The term "proliferate" and its various grammatical forms as
used herein refers to the process that results in an increase of
the number of cells, and is defined by the balance between cell
division and cell loss through cell death or differentiation.
[0226] The terms "protein domain" and "domain" are used
interchangeably to refer to a portion of a protein that has its own
tertiary structure. Large proteins are generally composed of
several domains connected to one another via flexible regions of
polypeptide chain.
[0227] The following terms are used herein to describe the sequence
relationships between two or more nucleic acids or polynucleotides:
(a) "reference sequence", (b) "comparison window", (c) "sequence
identity", (d) "percentage of sequence identity", and (e)
"substantial identity." (a) The term "reference sequence" refers to
a sequence used as a basis for sequence comparison. A reference
sequence may be a subset or the entirety of a specified sequence;
for example, as a segment of a full-length cDNA or gene sequence,
or the complete cDNA or gene sequence. (b) The term "comparison
window" refers to a contiguous and specified segment of a
polynucleotide sequence, wherein the polynucleotide sequence may be
compared to a reference sequence and wherein the portion of the
polynucleotide sequence in the comparison window may comprise
additions or deletions (i.e., gaps) compared to the reference
sequence (which does not comprise additions or deletions) for
optimal alignment of the two sequences. Generally, the comparison
window is at least 20 contiguous nucleotides in length, and
optionally can be at least 30 contiguous nucleotides in length, at
least 40 contiguous nucleotides in length, at least 50 contiguous
nucleotides in length, at least 100 contiguous nucleotides in
length, or longer. Those of skill in the art understand that to
avoid a high similarity to a reference sequence due to inclusion of
gaps in the polynucleotide sequence, a gap penalty typically is
introduced and is subtracted from the number of matches. Methods of
alignment of sequences for comparison are well-known in the art.
Optimal alignment of sequences for comparison may be conducted by
the local homology algorithm of Smith and Waterman, Adv. Appl.
Math. 2:482 (1981); by the homology alignment algorithm of
Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); by the search
for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci.
85:2444 (1988); by computerized implementations of these
algorithms, including, but not limited to: CLUSTAL in the PC/Gene
program by Intelligenetics, Mountain View, Calif.; GAP, BESTFIT,
BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group (GCG), 575 Science Dr., Madison,
Wis., USA; the CLUSTAL program is well described by Higgins and
Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS 5:151-153
(1989); Corpet, et al., Nucleic Acids Research 16:10881-90 (1988);
Huang, et al., Computer Applications in the Biosciences, 8:155-65
(1992), and Pearson, et al., Methods in Molecular Biology,
24:307-331 (1994). The BLAST family of programs, which can be used
for database similarity searches, includes: BLASTN for nucleotide
query sequences against nucleotide database sequences; BLASTX for
nucleotide query sequences against protein database sequences;
BLASTP for protein query sequences against protein database
sequences; TBLASTN for protein query sequences against nucleotide
database sequences; and TBLASTX for nucleotide query sequences
against nucleotide database sequences. See, Current Protocols in
Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene
Publishing and Wiley-Interscience, New York (1995). Unless
otherwise stated, sequence identity/similarity values provided
herein refer to the value obtained using the BLAST 2.0 suite of
programs using default parameters. Altschul et al., Nucleic Acids
Res. 25:3389-3402 (1997). Software for performing BLAST analyses is
publicly available, e.g., through the National Center for
Biotechnology-Information. This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits then
are extended in both directions along each sequence for as far as
the cumulative alignment score can be increased. Cumulative scores
are calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always>0) and N
(penalty score for mismatching residues; always<0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a word length (W) of 11, an
expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison
of both strands. For amino acid sequences, the BLASTP program uses
as defaults a word length (W) of 3, an expectation (E) of 10, and
the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989)
Proc. Natl. Acad. Sci. USA 89:10915). In addition to calculating
percent sequence identity, the BLAST algorithm also performs a
statistical analysis of the similarity between two sequences (see,
e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA
90:5873-5787 (1993)). One measure of similarity provided by the
BLAST algorithm is the smallest sum probability (P(N)), which
provides an indication of the probability by which a match between
two nucleotide or amino acid sequences would occur by chance. BLAST
searches assume that proteins may be modeled as random sequences.
However, many real proteins comprise regions of nonrandom sequences
which may be homopolymeric tracts, short-period repeats, or regions
enriched in one or more amino acids. Such low-complexity regions
may be aligned between unrelated proteins even though other regions
of the protein are entirely dissimilar. A number of low-complexity
filter programs may be employed to reduce such low-complexity
alignments. For example, the SEG (Wooten and Federhen, Comput.
Chem., 17:149-163 (1993)) and XNU (Claverie and States, Comput.
Chem., 17:191-201 (1993)) low-complexity filters may be employed
alone or in combination. (c) The term "sequence identity" or
"identity" in the context of two nucleic acid or polypeptide
sequences is used herein to refer to the residues in the two
sequences that are the same when aligned for maximum correspondence
over a specified comparison window. When percentage of sequence
identity is used in reference to proteins it is recognized that
residue positions that are not identical often differ by
conservative amino acid substitutions, i.e., where amino acid
residues are substituted for other amino acid residues with similar
chemical properties (e.g. charge or hydrophobicity) and therefore
do not change the functional properties of the molecule. Where
sequences differ in conservative substitutions, the percent
sequence identity may be adjusted upwards to correct for the
conservative nature of the substitution. Sequences that differ by
such conservative substitutions are said to have "sequence
similarity" or "similarity." Means for making this adjustment are
well-known to those of skill in the art. Typically this involves
scoring a conservative substitution as a partial rather than a full
mismatch, thereby increasing the percentage sequence identity.
Thus, for example, where an identical amino acid is given a score
of 1 and a non-conservative substitution is given a score of zero,
a conservative substitution is given a score between zero and 1.
The scoring of conservative substitutions is calculated, e.g.,
according to the algorithm of Meyers and Miller, Computer Applic.
Biol. Sci., 4:11-17 (1988) e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif, USA). (d) The term
"percentage of sequence identity" is used herein mean the value
determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity. (e) The term "substantial identity" of polynucleotide
sequences means that a polynucleotide comprises a sequence that has
at least 70% sequence identity, at least 80% sequence identity, at
least 90% sequence identity and at least 95% sequence identity,
compared to a reference sequence using one of the alignment
programs described using standard parameters. One of skill will
recognize that these values may be adjusted appropriately to
determine corresponding identity of proteins encoded by two
nucleotide sequences by taking into account codon degeneracy, amino
acid similarity, reading frame positioning and the like.
Substantial identity of amino acid sequences for these purposes
normally means sequence identity of at least 60%, or at least 70%,
at least 80%, at least 90%, or at least 95%. Another indication
that nucleotide sequences are substantially identical is if two
molecules hybridize to each other under stringent conditions.
However, nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides that they encode are substantially identical. This may
occur, e.g., when a copy of a nucleic acid is created using the
maximum codon degeneracy permitted by the genetic code. One
indication that two nucleic acid sequences are substantially
identical is that the polypeptide that the first nucleic acid
encodes is immunologically cross reactive with the polypeptide
encoded by the second nucleic acid. Mutations may also be made to
the nucleotide sequences of the present proteins by reference to
the genetic code, including taking into account codon
degeneracy.
[0228] The term "prime" (or "priming") as used herein refers to the
process of increasing sensitivity to. When used in an immunological
sense it refers to a process whereby a specific antigen is
presented to naive lymphocytes causing them to differentiate.
Priming involves several steps: antigen uptake, processing, and
cell surface expression bound to MHC molecules by an antigen
presenting cell, recirculation and antigen-specific trapping of
helper T cell precursors in lymphoid tissue, and T cell
proliferation and differentiation. Janeway, C A, Jr., "The priming
of helper T cells, Semin. Immunol. 1(1): 13-20 (1989).
[0229] The term "progression free survival" or "PFS" as used herein
refers to length of time during and after the treatment of a
disease, such as cancer, that a patient lives with the disease but
it does not get worse. In a clinical trial, measuring the
progression free survival is one way to determine how well a new
treatment works.
[0230] The term "recurrence" as used herein with respect to cancer
refers to a cancer that has recurred (come back), usually after a
period of time during which the cancer could not be detected. The
cancer may come back to the same place as the original (primary)
tumor or to another place in the body.
[0231] The term "relapse-free survival (RFS)" as used herein refers
to the length of time after primary treatment for a cancer during
which the patient survives without any signs or symptoms of that
cancer. Also called disease-free survival (DFS) and progression
free survival (PFS).
[0232] The term "release" or "release of cytokine effector
molecules" is meant to refer to the complex and tightly controlled
process by which soluble mediators of the immune response are
delivered from a given immune cell type to the external milieu
after activation of a signaling cascade in response to receptor
stimulation. In classical secretory pathways, cytokines with signal
peptides are cotranslationally inserted into the endoplasmic
reticulum (ER) for synthesis as either soluble or transmembrane
precursors. They then are trafficked in vesicles to the Golgi
complex for further processing, and at the trans-Golgi network
(TGN) they are loaded into vesicles or carriers for constitutive
delivery to the cell surface or other organelles. In specialized
cell types, additional modes of secretion are proffered by loading
cytokines and other cargo into granules for storage and later
release. See Lacy, P. and Stow, J L, "Cytokine release from innate
immune cells: association with diverse membrane trafficking
pathways," Blood (2011) 118: 9-18. Cytokine release can be directly
evoked by immunoglobulin- or complement receptor-mediated signaling
or by pathogens through a diverse array of cellular receptors,
including pattern recognition receptors such as TLRs.
[0233] The term "response rate" as used herein refers to the
percentage of patients whose cancer shrinks or disappears after
treatment.
[0234] The term "resistant cancer" as used herein refers to a
cancer that does not respond to a treatment at the beginning of
such treatment or sometime during such treatment.
[0235] The term "reporter gene" ("reporter") or "assay marker"
refers to a gene and/or peptide that can be detected, or easily
identified and measured. The expression of the reporter may be
measured at either the RNA level, or at the protein level. The gene
product, which may be detected in an experimental assay protocol,
includes, but is not limited to, marker enzymes, antigens, amino
acid sequence markers, cellular phenotypic markers, nucleic acid
sequence markers, and the like. Researchers may attach a reporter
gene to another gene of interest in cell culture, bacteria,
animals, or plants. For example, some reporters are selectable
markers, or confer characteristics upon on organisms expressing
them allowing the organism to be easily identified and assayed. To
introduce a reporter gene into an organism, researchers may place
the reporter gene and the gene of interest in the same DNA
construct to be inserted into the cell or organism. For bacteria or
eukaryotic cells in culture, this may be in the form of a plasmid.
Commonly used reporter genes may include, but are not limited to,
fluorescent proteins, luciferase, beta-galactosidase, and
selectable markers, such as chloramphenicol and kanomycin.
[0236] The term "secrete" as used herein when referring to a cell
means a process whereby molecules manufactured within the cell are
moved to a space outside of the cell.
[0237] The term"serial killer cell" as used herein refers to a
population of cells that exhibit an ability to kill multiple tumor
or pathogen-infected cells, while showing resistance to such
killing action. There are multiple kinds of cells that display this
effector function, e.g., NK cells, NKT cells, LAK cells, CIK cells,
MAIT cells, CD8+ CTLs, CD4+ CTLs. The serial killer effector
function may be direct, through cytolytic or cytotoxic activities,
or indirect, through the immunoregulation of other cells and
proteins that target pathogenic and cancerous cells.
[0238] The term "stably expressed exogenous immunomodulatory
molecules" as used herein refers to exogenous immunomodulatory
molecules that are expressed for a period of time that is
sufficient to stimulate one or more of T-lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes
sufficient to stimulate the T-lymphocytes, natural killer (NK)
cells, dendritic cells (DCs) or B lymphocytes. According to some
embodiments, the period of time is between 1 hour and 72 hours,
e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 45 50, 55, 60, 65, 70, 71 or 72 hours.
According to some embodiments, the period of time is more than 72
hours.
[0239] The term "stimulate" in any of its grammatical forms as used
herein refers to inducing activation or increasing activity.
[0240] As used herein, the term "stimulate an immune cell" or
"stimulating an immune cell" refers to a process (e.g., involving a
signaling event or stimulus) causing or resulting in a cellular
response, such as activation and/or expansion, of an immune cell,
e.g. an NK cell and/or a CD8+ T cell. According to some
embodiments, stimulating an immune cell (e.g., an NK cell and/or a
CD8+ T cell) refers to providing a stimulus or signal (e.g., a
stimulating polypeptide) that results in the activation and/or
expansion of the immune cell.
[0241] As used herein, the term "sufficient to stimulate an immune
cell" refers to an amount or level of a signaling event or
stimulus, e.g. of exogenous immunomodulatory polypeptide, that
promotes a cellular response of an immune cell.
[0242] As used herein, the terms "subject" or "individual" or
"patient" are used interchangeably to refer to a member of an
animal species of mammalian origin, including humans.
[0243] The phrase "subject in need thereof" as used herein refers
to a patient that (i) will be administered an immunogenic
composition according to the described invention, (ii) is receiving
an immunogenic composition according to the described invention; or
(iii) has received an immunogenic composition according to the
described invention, unless the context and usage of the phrase
indicates otherwise.
[0244] The term "SUPLEXA.TM. cells means autologous blood cells
that have been stimulated in vitro by Engineered Leukocyte
Stimulator (ENLIST.TM.) cells.
[0245] The term "therapeutic agent" as used herein refers to a
drug, molecule, nucleic acid, protein, metabolite, composition or
other substance that provides a therapeutic effect. The term
"active" as used herein refers to the ingredient, component or
constituent of the compositions of the described invention
responsible for the intended therapeutic effect. The terms
"therapeutic agent" and "active agent" are used interchangeably
herein. The term "therapeutic component" as used herein refers to a
therapeutically effective dosage (i.e., dose and frequency of
administration) that eliminates, reduces, or prevents the
progression of a particular disease manifestation in a percentage
of a population. An example of a commonly used therapeutic
component is the ED50 which describes the dose in a particular
dosage that is therapeutically effective for a particular disease
manifestation in 50% of a population.
[0246] The terms "therapeutic amount", "therapeutically effective
amount", an "amount effective", or "pharmaceutically effective
amount" of an active agent is used interchangeably to refer to an
amount that is sufficient to provide the intended benefit of
treatment. However, dosage levels are based on a variety of
factors, including the type of injury, the age, weight, sex,
medical condition of the patient, the severity of the condition,
the route of administration, and the particular active agent
employed. Thus the dosage regimen may vary widely, but can be
determined routinely by a physician using standard methods.
Additionally, the terms "therapeutic amount", "therapeutically
effective amounts" and "pharmaceutically effective amounts" include
prophylactic or preventative amounts of the compositions of the
described invention. In prophylactic or preventative applications
of the described invention, pharmaceutical compositions or
medicaments are administered to a patient susceptible to, or
otherwise at risk of, a disease, disorder or condition in an amount
sufficient to eliminate or reduce the risk, lessen the severity, or
delay the onset of the disease, disorder or condition, including
biochemical, histologic and/or behavioral symptoms of the disease,
disorder or condition, its complications, and intermediate
pathological phenotypes presenting during development of the
disease, disorder or condition. It is generally preferred that a
maximum dose be used, that is, the highest safe dose according to
some medical judgment. The terms "dose" and "dosage" are used
interchangeably herein.
[0247] The term "therapeutic effect" as used herein refers to a
consequence of treatment, the results of which are judged to be
desirable and beneficial. A therapeutic effect can include,
directly or indirectly, the arrest, reduction, or elimination of a
disease manifestation. A therapeutic effect can also include,
directly or indirectly, the arrest reduction or elimination of the
progression of a disease manifestation.
[0248] For any therapeutic agent described herein the
therapeutically effective amount may be initially determined from
preliminary in vitro studies and/or animal models. A
therapeutically effective dose may also be determined from human
data. The applied dose may be adjusted based on the relative
bioavailability and potency of the administered compound. Adjusting
the dose to achieve maximal efficacy based on the methods described
above and other well-known methods is within the capabilities of
the ordinarily skilled artisan.
[0249] General principles for determining therapeutic
effectiveness, which may be found in Chapter 1 of Goodman and
Gilman's The Pharmacological Basis of Therapeutics, 10th Edition,
McGraw-Hill (New York) (2001), incorporated herein by reference,
are summarized below.
[0250] Pharmacokinetic principles provide a basis for modifying a
dosage regimen to obtain a desired degree of therapeutic efficacy
with a minimum of unacceptable adverse effects. In situations where
the drug's plasma concentration can be measured and related to the
therapeutic window, additional guidance for dosage modification can
be obtained.
[0251] Drug products are considered to be pharmaceutical
equivalents if they contain the same active ingredients and are
identical in strength or concentration, dosage form, and route of
administration. Two pharmaceutically equivalent drug products are
considered to be bioequivalent when the rates and extents of
bioavailability of the active ingredient in the two products are
not significantly different under suitable test conditions.
[0252] The term "therapeutic window" refers to a concentration
range that provides therapeutic efficacy without unacceptable
toxicity. Following administration of a dose of a drug, its effects
usually show a characteristic temporal pattern. A lag period is
present before the drug concentration exceeds the minimum effective
concentration ("MEC") for the desired effect. Following onset of
the response, the intensity of the effect increases as the drug
continues to be absorbed and distributed. This reaches a peak,
after which drug elimination results in a decline in the effect's
intensity that disappears when the drug concentration falls back
below the MEC. Accordingly, the duration of a drug's action is
determined by the time period over which concentrations exceed the
MEC. The therapeutic goal is to obtain and maintain concentrations
within the therapeutic window for the desired response with a
minimum of toxicity. Drug response below the MEC for the desired
effect will be subtherapeutic, whereas for an adverse effect, the
probability of toxicity will increase above the MEC. Increasing or
decreasing drug dosage shifts the response curve up or down the
intensity scale and is used to modulate the drug's effect.
Increasing the dose also prolongs a drug's duration of action but
at the risk of increasing the likelihood of adverse effects.
Accordingly, unless the drug is nontoxic, increasing the dose is
not a useful strategy for extending a drug's duration of
action.
[0253] Instead, another dose of drug should be given to maintain
concentrations within the therapeutic window. In general, the lower
limit of the therapeutic range of a drug appears to be
approximately equal to the drug concentration that produces about
half of the greatest possible therapeutic effect, and the upper
limit of the therapeutic range is such that no more than about 5%
to about 10% of patients will experience a toxic effect. These
figures can be highly variable, and some patients may benefit
greatly from drug concentrations that exceed the therapeutic range,
while others may suffer significant toxicity at much lower values.
The therapeutic goal is to maintain steady-state drug levels within
the therapeutic window. For most drugs, the actual concentrations
associated with this desired range are not and need not be known,
and it is sufficient to understand that efficacy and toxicity are
generally concentration-dependent, and how drug dosage and
frequency of administration affect the drug level. For a small
number of drugs where there is a small (two- to three-fold)
difference between concentrations resulting in efficacy and
toxicity, a plasma-concentration range associated with effective
therapy has been defined.
[0254] In this case, a target level strategy is reasonable, wherein
a desired target steady-state concentration of the drug (usually in
plasma) associated with efficacy and minimal toxicity is chosen,
and a dosage is computed that is expected to achieve this value.
Drug concentrations subsequently are measured and dosage is
adjusted if necessary to approximate the target more closely.
[0255] In most clinical situations, drugs are administered in a
series of repetitive doses or as a continuous infusion to maintain
a steady-state concentration of drug associated with the
therapeutic window. To maintain the chosen steady-state or target
concentration ("maintenance dose"), the rate of drug administration
is adjusted such that the rate of input equals the rate of loss. If
the clinician chooses the desired concentration of drug in plasma
and knows the clearance and bioavailability for that drug in a
particular patient, the appropriate dose and dosing interval can be
calculated.
[0256] As used herein the term "treating" includes abrogating,
substantially inhibiting, slowing or reversing the progression of a
condition, substantially ameliorating clinical symptoms of a
condition, or substantially preventing the appearance of clinical
symptoms of a condition. Treating further refers to accomplishing
one or more of the following: (a) reducing the severity of the
disorder; (b) limiting development of symptoms characteristic of
the disorder(s) being treated; (c) limiting worsening of symptoms
characteristic of the disorder(s) being treated; (d) limiting
recurrence of the disorder(s) in patients that have previously had
the disorder(s); and (e) limiting recurrence of symptoms in
patients that were previously asymptomatic for the disorder(s).
[0257] A "tumor," as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
precancerous and cancerous cells and tissues.
[0258] The terms "tumor burden" or "tumor load" as used herein
refer to the number of cancer cells, the size of a tumor, or the
amount of cancer in the body.
[0259] The term "vaccinated" as used herein refers to being treated
with a vaccine.
[0260] The term "vaccination" as used herein refers to treatment
with a vaccine.
[0261] The term "vaccine" as used herein refers a substance or
group of substances meant to cause the immune system to respond to
a tumor or to microorganisms, or help the body recognize and
destroy cancer cells or microorganisms. The term vaccine also
refers to an artificial stimulus used to stimulate a robust immune
response against that exposure (e.g. infectious agent, cancer
cell).
[0262] The term "vaccine therapy" as used herein refers to a type
of treatment that uses a substance or group of substances to
stimulate the immune system to destroy a tumor or infectious
microorganisms.
[0263] As used herein, the term "variant" refers to a polypeptide
which differs from the original protein by one or more amino acid
substitutions, deletions, insertions, or other modifications. These
modifications do not significantly change the biological activity
of the original protein. In many cases, a variant retains at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the
biological activity of original protein. The biological activity of
a variant can also be higher than that of the original protein. A
variant can be naturally-occurring, such as by allelic variation or
polymorphism, or be deliberately engineered.
[0264] The amino acid sequence of a variant is substantially
identical to that of the original protein. In many embodiments, a
variant shares at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or
more global sequence identity or similarity with the original
protein. Sequence identity or similarity can be determined using
various methods known in the art, such as Basic Local Alignment
Tool (BLAST), dot matrix analysis, or the dynamic programming
method. In one example, the sequence identity or similarity is
determined by using the Genetics Computer Group (GCG) programs GAP
(Needleman-Wunsch algorithm) The amino acid sequences of a variant
and the original protein can be substantially identical in one or
more regions, but divergent in other regions
[0265] The term "wild-type" as used herein refers to the typical
form of an organism, strain, gene, protein, nucleic acid, or
characteristic as it occurs in nature. Wild-type refers to the most
common phenotype in the natural population. The terms "wild-type"
and "naturally occurring" are used interchangeably.
II. Allogeneic Vaccine
[0266] The present disclosure features allogeneic tumor cell
vaccines comprising tumor cells expressing an exogenous
immunomodulatory molecule, and methods of using the allogeneic
tumor cell vaccine to stimulate an immune response. Vaccine
proteins can induce immune responses that find use in the described
invention e.g. in the treatment of cancer or infectious diseases.
According to some embodiments, the allogeneic tumor cell vaccines
described herein are effective to enhance immune activation of
cells effective to recognize and act against tumor cells comprising
the target tumor antigen in vivo without systemic inflammation;
reduce immunosuppression in a tumor microenvironment for tumor
cells comprising the target tumor antigen; or increase cell death
of tumor cells expressing the target tumor antigen. According to
some embodiments, the allogeneic tumor cell vaccines described
herein are capable of immune activation without systemic
inflammation.
[0267] According to some aspects, the disclosure features an
allogeneic tumor cell vaccine comprising (1) a population of
proliferation-incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising (a) a plurality of stably expressed exogenous
immunomodulatory molecules sufficient to stimulate the
T-lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes; and (2) a pharmaceutically acceptable carrier.
Tumor Specific Antigens
[0268] According to some embodiments, the disclosure provides a
population of proliferation-incompetent tumor cells expressing one
or more tumor specific antigens. According to some embodiments, the
tumor specific antigens may be encoded by a primary open reading
frame of gene products that are differentially expressed by tumors,
and not by normal tissues. According to some embodiments, the tumor
specific antigens may be encoded by mutated genes, intronic
sequences, or translated alternative open reading frames,
pseudogenes, antisense strands, or may represent the products of
gene translocation events. According to some embodiments, the tumor
cell provides a broad array of tumor specific antigens, many of
which are of unknown nature. According to some embodiments, the
tumor antigen is a neoantigen.
[0269] According to some embodiments, the tumor specific antigen is
selected from one of the following groups: (a) nonmutated shared
antigens (e.g., melanoma-associated antigen (MAGE), B-melanoma
antigen (BAGE), renal tumor antigen (RAGE), and a cancer testis
antigen (e.g. NY-ESO); (b) differentiation antigens (e.g.,
prostate-specific membrane antigen [PSMA] and prostate-specific
antigen (PSA) in prostate carcinoma, Mart1/MelanA and tyrosinase
present in many melanomas, and carcino embryonic antigen (CEA)
present in a large percentage of colon cancers), which are tissue
restricted and present in lineage-specific tumor cell; (c) mutated
oncogenes and tumor suppressor genes (e.g., mutated ras, rearranged
bcr/abl, mutated p53), which provide novel epitopes for immune
recognition; (d) unique idiotypes (e.g., immunoglobulin antigensin
myeloma and B-cell myeloma, T-cell receptor (TCR) expressed in
CTCL), (e) oncovirus-derived epitopes (e.g., the human
papillomavirus-encoded E6 and E7 proteins, Epstein-Barr
virus-associated antigens present in primary brain lymphoma); and
(f) nonmutated oncofetal proteins such as CEA, .alpha.-fetoprotein,
and survivin. According to some embodiments, the tumor specific
antigen is selected from an antigen listed in the publically
available Cancer Antigenic Peptide Database (on the world wide web
at caped.icp.ucl.ac.be/Peptide/list, incorporated by reference in
its entirety herein). According to some embodiments, the tumor
specific antigen is selected from an antigen set forth in Table 1,
shown below.
TABLE-US-00001 TABLE 1 Gene/Protein Tumor PPP1R3B melanoma
alpha-actinin-4 lung carcinoma ARTC1 melanoma CASP-8 head and neck
squamous cell carcinoma beta-catenin melanoma Cdc27 melanoma CDK4
melanoma CDK12 melanoma CDKN2A melanoma CLPP melanoma CSNK1A1
melanoma EFTUD2 melanoma Elongation factor 2 lung squamous CC FN1
melanoma GAS7 melanoma GPNMB melanoma HAUS3 melanoma HSDL1 ovarian
cancer LDLR- melanoma fucosyltransferaseAS fusion protein HLA-A2d
renal cell carcinoma HLA-A11d melanoma hsp70-2 renal cell carcinoma
bladder tumor MART2 melanoma MATN melanoma k-ras non-small cell
lung carcinoma MUM-1f melanoma MUM-2 melanoma MUM-3 melanoma
neo-PAP melanoma NFYC lung squamous cell carcinoma OS-9 melanoma
PTPRK melanoma N-ras melanoma BRAF600 melanoma SIRT2 melanoma
SNRPD1 melanoma Triosephosphate melanoma isomerase Myosin class I
melanoma BCR-ABL fusion protein chronic myeloid (b3a2) leukemia
B-RAF melanoma CASP-5 colorectal, gastric, and endometrial
carcinoma dek-can fusion protein myeloid leukemia ETV6-AML1 fusion
acute lymphoblastic protein leukemia FLT3-ITD acute myelogenous
leukemia FNDC3B chronic lymphocytic leukemia OGT colorectal
carcinoma p53 head and neck squamous cell carcinoma pml-RARalpha
fusion promyelocytic leukemia protein PRDX5 melanoma K-ras
pancreatic adenocarcinoma SYT-SSX1 or -SSX2 sarcoma fusion protein
KIAAO205 mutation ME1 mutation EGFRvIII Mutation TGF-betaRII
colorectal carcinoma gp100/Pmel17 melanoma mammaglobin-A breast
cancer Melan-A/MART-1 melanoma NY-BR-1 breast cancer OA1 melanoma
PAP prostate cancer PSA prostate carcinoma RAB38/NY-MEL-1 melanoma
TRP-1/gp75 melanoma TRP-2 melanoma tyrosinase melanoma DKK1 testis,
prostate, ENAH (hMena) breast, prostate stroma and epithelium of
colon- rectum, pancreas, endometrium G250/MN/CAIX stomach, liver,
pancreas Kallikrein 4 prostate and ovarian cancer D393-CD20n B cell
lymphomas and leukemias Cyclin-A1 AML, testicular, endometrial and
epithelial ovarian cancer GAGE-1,2,8 GAGE-3,4,5,6,7 GnTVf GPC3
Hepatocellular carcinoma, melanoma, lung squamous cell carcinoma,
hepatoblastoma, ovarian clear cell carcinoma, neuroblastoma,
stomach HERV-E kidney HERV-K-MEL melanoma KK-LC-1 KM-HN-1 LAGE-1
Melanoma, non-small cell lung carcinoma, bladder, prostate and head
and neck cancer LY6K Breast cancer MAGE-A1 melanoma MAGE-A2
melanoma MAGE-A3 melanoma MAGE-A4 melanoma MAGE-A5 melanoma MAGE-A6
melanoma MAGE-A7 melanoma MAGE-A8 melanoma MAGE-A9 melanoma
MAGE-A10 melanoma MAGE-A11 melanoma MAGE-A12m melanoma MAGE-C1
melanoma MAGE-C2 melanoma MAGE-n melanoma mucink NA88-A
NY-ESO-1/LAGE-2 melanoma Neutrophil granule proteases OFA-iLR
PTH-rP S2 SAGE Sp17 SSX-2 SSX-4 TAG TAG-1 TAG-2 hTERT colorectal
carcinoma TPBG TRAG-3 TRP2-6b TRP2-INT2g TTK XAGE-1b/GAGED2a ART-4
CDCA1/NUF2 Cep55/c10orf3 CML28 (EXOSC5) DAM-6, -10 (MAGE-B1) IMP-3
OVA66 OY-TES-1 PASD1 RHAMM/CD168 SART-3 SART-1
[0270] According to some embodiments, the tumor cell is from a
cancer selected from the group consisting of melanoma, colorectal
carcinoma, leukemia, chronic myeloid leukemia, prostate cancer,
head and neck cancer, squamous cell carcinoma, tongue cancer,
larynx cancer, tonsil cancer, hypopharynx cancer, nasalpharynx
cancer, breast cancer, colon cancer, lung cancer, pancreatic
cancer, glioblastoma and brain cancer.
[0271] According to some embodiments, the melanoma tumor cell is
characterized by the expression of one or more of gp100,
tyrosinase, Melan-A, tyrosinase-related protein (TRP-2-INT2),
melanoma antigen-1 (MAGE-A1), NY-ESO-1, preferentially expressed
antigen of melanoma (PRAIVIE) CDK4 and multiple myeloma oncogene 1
(MUM-1).
[0272] According to some embodiments, the colorectal cancer tumor
cell is characterized by the expression of one or more of
carcinoembryonic antigen (CEA), MAGE, HPV, human telomerase reverse
transcriptase (hTERT), EPCAM, PD-1, PD-L1, p53, cell
surface-associated mucin 1 (MUC1).
[0273] Immunologic antigenic specificity may arise from one or more
of the amino acid sequence of the antigen, from the degree of
expression of that antigen by the tumor cell, from
post-translational modification of the antigen, and the like.
[0274] Immunologic antigen specificity to a certain type of cancer
cell may also arise from one or more of a particular fingerprint of
a plurality of tumor antigens, from the fact that a particular
antigen, while expressed by a wide variety of tumor cells, has
particular use in immunotherapy against a smaller number of tumor
types, from the fact that a particular collection of MHC class I
presentable and MEW class II presentable epitopes exist on a
particular polypeptide or polypeptide fragment, and by omitting one
or more peptides that may provoke immunotolerance. The skilled
artisan can locate the relevant nucleic acid and polypeptide
sequences, e.g., on the U.S. government's web site, at
ncbi.nlm.nih.
[0275] According to some embodiments, the tumor cells are derived
from a sample from a subject. According to some embodiments, the
tumor cells are derived from a tumor cell line or tumor cell line
variant.
[0276] According to some embodiments, tumor antigen specificity of
the present invention may be determined by the parental tumor cell
line or tumor cell line variant that is selected for modification
with immunomodulators.
Parent Cell Lines
[0277] According to some embodiments, tumor cell line or tumor cell
line variants may be derived from established cell lines from
either public sources (e.g. NIH, DCTD Tumor Repository operated by
Charles River Laboratories Inc.) or commercial sources (e.g. ATCC,
Sigma Alrich, Thermo Fischer Scientific, Genescript, DSM2).
According to some embodiments, new cell lines can be established de
novo from tumor cells derived from the tumor of a cancer
patient.
[0278] According to some embodiments, cancer tissues, cancer cells,
cells infected with a cancer-causing agent, other preneoplastic
cells, and cell lines of human origin can be used as a source.
According to some embodiments, a cancer cell can be from an
established tumor cell line or tumor cell line variant such as,
without limitation, an established non-small cell lung carcinoma
(NSCLC), a bladder cancer, a melanoma, an ovarian cancer, a renal
cell carcinoma, a prostate carcinoma, a sarcoma, a breast
carcinoma, a squamous cell carcinoma, a head and neck carcinoma, a
hepatocellular carcinoma, a pancreatic carcinoma, or a colon
carcinoma cell line.
[0279] According to some embodiments, the established cell lines
comprise the LNCaP clone FGC (ATCC CRL-1740), which itself is
derived from a metastatic prostate cancer that had migrated to a
lymph node. According to some embodiments, the established cell
lines comprise the PC-3 (ATCC CRL-1435) cell line, which itself is
derived from metastatic prostate cancer that migrated to bone.
According to some embodiments, the tumor cell line or tumor cell
line variants are derived from one or more of the following ATCC
cell lines: VCaP (ATCC CRL-2876); MDA PCa 2b (ATCC CRL-2422); or DU
145 (ATCC HTB-81).
[0280] According to some embodiments, the established cell lines
comprise the SK-MEL-2 clone (ATCC HTB-68), which itself is derived
from metastasis on skin of thigh.
[0281] According to some embodiments, the established cell lines
comprise one or more of mammary carcinoma cell lines designated
COO-G, DU4475, ELL-G, HIG-G, MCF/7, MDA-MB-436, MX-1, SW-613, and
VAN-G. According to some embodiments, the established cell lines
comprise one or more of alveolar soft part sarcoma cell lines
designated ASPS, and ASPS-1. According to some embodiments, the
established cell lines comprise one or more lung cell lines
designated LX-1, COS-G, H-MESO-1, H-MESO-1A, NCI-H23, and
NCI-H460.
[0282] According to some embodiments, the established cell lines
comprise one or more colon cancer cell lines designated CX-5,
GOB-G, HCC-2998, HCT-15, KLO-G, KM20L2, MRI-H-194, LOVO I, LOVO II,
and MRI-H-250. According to some embodiments, the established cell
lines comprise one or more melanoma cell lines designated NIS-G,
TRI-G, WIL-G, MRI-H-121B, MRI-H-187, MRI-H-221, and MRI-H-255.
According to some embodiments, the established cell lines comprise
one or more cervical cancer cell lines designated MRI-H-177,
MRI-H-186, MRI-H-196, and MRI-H-215. According to some embodiments,
the established cell lines comprise one or more kidney cancer cell
lines designated MRI-H-121 and MRI-H-166. According to some
embodiments, the established cell lines comprise one or more
endometrium cancer cell lines designated MRI-H-147 and MRI-H-220.
According to some embodiments, the established cell lines comprise
one or more ovarian cancer cell lines designated MRI-H-258,
MRI-H-273, MRI-H-1834, and SWA-G. According to some embodiments,
the established cell lines comprise one or more sarcoma cell lines
designated HS-1, OGL-G, and DEL-G. According to some embodiments,
the established cell lines comprise the epidermoid cell line
designated DEAC-1. According to some embodiments, the established
cell line comprises the glioblastoma cell line designated SF 295.
According to some embodiments, the established cell line comprises
the prostate cancer cell line designated CWR-22. According to some
embodiments, the established cell line comprises the Burkitt's
lymphoma cell line designated DAU. According to some embodiments,
the foregoing established cell lines described herein are
commercially available, e.g. from American Type Culture Collection
(ATCC), European Collection of Cell Cultures (ECACC), or any
depository listed as an International Depositary Authority (IDA)
under Article 7 of the Budapest Treaty.
[0283] According to some embodiments, exemplary established cell
lines comprise one or more of the following cell lines shown
below:
TABLE-US-00002 Designation Tissue of Origin Histologic Type 786-0
Kidney Renal Cell Carcinoma A2780 Ovary Adenocarcinoma A498 Kidney
Renal Cell Carcinoma A549 Lung Non-small Cell A704 Kidney Renal
Cell Carcinoma ACHN Kidney Renal Cell Carcinoma ASPS-1 Lymph Node
Alveolar Soft Part Sarcoma BT-549 Breast Adenocarcinoma CAKI-1
Kidney Renal Cell Carcinoma CCRF-CEM Lymph Leukemia CCRF-SB Lymph
Leukemia CHA-59 Bone Osteosarcoma COLO 205 Colon Adenocarcinoma
DMS-114 Lung Small Cell DU-145 Prostate Carcinoma EKVX Lung
Adenocarcinoma HCC-2998 Colon Adenocarcinoma HCT-15 Colon Carcinoma
HCT-116 Colon Adenocarcinoma HOP-18 Lung Large Cell Carcinoma
HOP-62 Lung Adenocarcinoma HL-60 Ascites Pro-myelocytic Leukemia
H-MESO-1 Mesothelioma HS 578T Breast Adenocarcinoma HS 913T Lung
Mixed Cell HT-29 Colon Adenocarcinoma IGR-OV1 Ovary Adenocarcinoma
KM-12 Colon Adenocarcinoma KM 20L2 Colon Adenocarcinoma K-562 Lymph
Leukemia LOVO Colon Adenocarcinoma LOX IMVI Lymph Node Amelanotic
Melanoma Metastisis LXFL 529 Lung Large Cell Carcinoma NCI-H1299
Lung Adenocarcinoma NCI-H2887 Lung Adenocarcinoma NCI-H3122 Lung
Adenocarcinoma NCI-H322M Lung Adenocarcinoma NCI-H3255 Lung
Adenocarcinoma NCI-H358M Lung Bronchioalveolar Carcinoma NCI-H460
Lung Large Cell NCI-H522 Lung Adenocarcinoma NCI-H69 Lung Small
Cell Carcinoma NCI-H82 Lung Small Cell Carcinoma NCI-H838 Lung
Adenocarcinoma NCI/ADR-RES Ovary Adenocarcinoma OVCAR-3 Ovary
Adenocarcinoma OVCAR-4 Ovary Adenocarcinoma OVCAR-5 Ovary
Adenocarcinoma OVCAR-8 Ovary Adenocarcinoma PC-3 Prostate Carcinoma
PC-3/M Prostate Carcinoma RPMI-7951 Skin Melanoma RPMI-8226 Lymph
Leukemia RXF 393 Kidney Renal Cell Carcinoma RXF 631 Kidney Renal
Cell Carcinoma TK-10 Kidney Renal Cell Carcinoma UACC-62 Skin
Melanoma UACC-257 Skin Melanoma UCSD 242L Skin Melanoma UCSD 354K
Skin Melanoma UO-31 Kidney Renal Cell Carcinoma U-251 CNS
Glioblastoma WIDR Colon Adenocarcinoma XF 498 CNS Glioblastoma
[0284] According to some embodiments, the choice of the parental
cell line from which the tumor cell line or tumor cell line variant
may be derived affects the specificity of the allogeneic vaccine.
For example, the use of a tumor cell line or tumor cell line
variant derived from metastatic prostate cancer that migrated to
the bone of a patient may result in an allogeneic vaccine that
elicits an immune response specific for metastatic prostate cancer
in the bone of a patient.
[0285] According to some embodiments, the tumor cell line or tumor
cell line variants may be derived from a parental cell that
comprises a universal cancer specific antigen. For example, the use
of a parental tumor cell line or tumor cell line variant derived
from metastatic prostate cancer that migrated to the bone of a
patient may result in an allogeneic vaccine that elicits an immune
response against all prostate cancer cells.
[0286] According to some embodiments, the tumor cell line or tumor
cell line variants are derived from patient derived cells derived
from various cancers. According to some embodiments, fresh tissue
surgically removed from a tumor is enzymatically digested by type
IV collagenase, followed by collection of disaggregated cells.
According to some embodiments, disaggregated cells may then be
grown in vitro in growth media with 10% fetal bovine serum on an
extracellular matrix substrate, such as collagen or fibronectin, to
promote attachment. According to some embodiments, adherent cells
may then be passaged until the immortal cancer cells outgrow the
non-cancerous fibroblast cells.
[0287] For example, according to some embodiments, the tumor cell
line or tumor cell line variants may be derived from a solid tumor
comprising tumor cells, including cancer stem cells, a metastatic
cancer comprising metastatic tumor cells, comprising cancer stem
cells, or a non-metastatic cancer. According to some embodiments,
the cancer may originate in the bladder, blood, bone, bone marrow,
brain, breast, colon, esophagus, duodenum, small intestine, large
intestine, colon, rectum, anus, gum, head, kidney, liver, lung,
nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue,
or uterus. According to some embodiments, the cancer may be of a
histological type, e.g., a cancer that begins in the skin or
tissues that line or cover internal organs (carcinoma); a cancer
that begins in bone or in the soft tissue of the body including
cartilage, fat, muscle, blood vessels, and fibrous tissue
(sarcoma); a cancer that starts in blood-forming tissue (leukemia);
a cancer that begins in cells of the immune system (lymphoma); a
cancer that arises in plasma cells (myeloma), or a brain/spinal
cord cancer.
[0288] Examples of carcinomas include, without limitation, giant
and spindle cell carcinoma; small cell carcinoma; papillary
carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma;
basal cell carcinoma; pilomatrix carcinoma; transitional cell
carcinoma; papillary transitional cell carcinoma; an
adenocarcinoma; a gastrinoma, a cholangiocarcinoma; a
hepatocellular carcinoma; a combined hepatocellular carcinoma and
cholangiocarcinoma; a trabecular adenocarcinoma; an adenoid cystic
carcinoma; an adenocarcinoma in adenomatous polyp; an
adenocarcinoma, familial polyposis coli; a solid carcinoma; a
carcinoid tumor; a branchiolo-alveolar adenocarcinoma; a papillary
adenocarcinoma; a chromophobe carcinoma; an acidophil carcinoma; an
oxyphilic adenocarcinoma; a basophil carcinoma; a clear cell
adenocarcinoma; a granular cell carcinoma; a follicular
adenocarcinoma; a non-encapsulating sclerosing carcinoma; adrenal
cortical carcinoma; an endometroid carcinoma; a skin appendage
carcinoma; an apocrine adenocarcinoma; a sebaceous adenocarcinoma;
a ceruminous adenocarcinoma; a mucoepidermoid carcinoma; a
cystadenocarcinoma; a papillary cystadenocarcinoma; a papillary
serous cystadenocarcinoma; a mucinous cystadenocarcinoma; a
mucinous adenocarcinoma; a signet ring cell carcinoma; an
infiltrating duct carcinoma; a medullary carcinoma; a lobular
carcinoma; an inflammatory carcinoma; paget's disease, a mammary
acinar cell carcinoma; an adenosquamous carcinoma; an
adenocarcinoma w/squamous metaplasia; a sertoli cell carcinoma;
embryonal carcinoma; choriocarcinoma.
[0289] Examples of sarcomas include, without limitation,
glomangiosarcoma; sarcoma; fibrosarcoma; myxosarcoma; liposarcoma;
leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma;
alveolar rhabdomyosarcoma; stromal sarcoma; carcinosarcoma;
synovial sarcoma; hemangiosarcoma; kaposi's sarcoma;
lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma;
chondrosarcoma; mesenchymal chondrosarcoma; giant cell tumor of
bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic
odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma;
myeloid sarcoma; mast cell sarcoma.
[0290] Examples of leukemias include, without limitation, leukemia;
lymphoid leukemia; plasma cell leukemia; erythroleukemia;
lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia;
eosinophilic leukemia; monocytic leukemia; mast cell leukemia;
megakaryoblastic leukemia; and hairy cell leukemia.
[0291] Examples of lymphomas and myelomas include, without
limitation, malignant lymphoma; hodgkin's disease; hodgkin's;
paragranuloma; malignant lymphoma, small lymphocytic; malignant
lymphoma, large cell, diffuse; malignant lymphoma, follicular;
mycosis fungoides; other specified non-hodgkin's lymphomas;
malignant melanoma; amelanotic melanoma; superficial spreading
melanoma; malignant melanoma in giant pigmented nevus; epithelioid
cell melanoma; multiple myeloma.
[0292] Examples of brain/spinal cord cancers include, without
limitation, pinealoma, malignant; chordoma; glioma, malignant;
ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary
astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;
oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;
ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory
neurogenic tumor; meningioma, malignant; neurofibrosarcoma;
neurilemmoma, malignant.
[0293] Examples of other cancers include, without limitation, a
thymoma; an ovarian stromal tumor; a thecoma; a granulosa cell
tumor; an androblastoma; a leydig cell tumor; a lipid cell tumor; a
paraganglioma; an extra-mammary paraganglioma; a pheochromocytoma;
blue nevus, malignant; fibrous histiocytoma, malignant; mixed
tumor, malignant; mullerian mixed tumor; nephroblastoma;
hepatoblastoma; mesenchymoma, malignant; brenner tumor, malignant;
phyllodes tumor, malignant; mesothelioma, malignant; dysgerminoma;
teratoma, malignant; struma ovarii, malignant; mesonephroma,
malignant; hemangioendothelioma, malignant; hemangiopericytoma,
malignant; chondroblastoma, malignant; granular cell tumor,
malignant; malignant histiocytosis; immunoproliferative small
intestinal disease.
[0294] For any given tumor type, several tumor cell line or tumor
cell line variants may be commercially available. According to some
embodiments, pooling of several of these cells lines, either as a
mixture of whole cells or by making a membrane preparation out of
the mixture of whole cells, may provide an array of cell surface
tumor antigens for that tumor type.
[0295] According to some embodiments, the tumor cells or tumor cell
line or tumor cell line variants are rendered proliferation
incompetent by irradiation.
Exogenous Immunomodulatory Molecules
[0296] According to some embodiments, an exogenous immunomodulatory
molecule of the disclosed invention is a polypeptide that, alone or
in combination with other exogenous immunomodulatory molecules,
mediates stimulation of an immune cell. According to some
embodiments, an exogenous immunomodulatory molecule of the
disclosed invention is a polypeptide that, alone or in combination
with other exogenous immunomodulatory molecules, mediates
stimulation of T-lymphocytes, natural killer (NK) cells, dendritic
cells (DCs) or B lymphocytes. According to some embodiments, the NK
cell is a memory-like NK cell. According to some embodiments, the T
lymphocyte is a cytotoxic T-lymphocyte (CTL) (CD8+ T cell).
According to some embodiments, the T lymphocyte is a memory T cell.
According to some embodiments, the T lymphocyte is a regulatory T
cell. According to some embodiments, the T lymphocyte is a helper T
cell. According to some embodiments, the B lymphocyte is a memory B
cell. It is a feature of the present invention that, according to
some embodiments, the exogenous immunomodulatory molecules
comprising the population of tumor cells are effective to stimulate
more than one type of immune cell, e.g. the allogeneic tumor cell
vaccine comprising a population of proliferation incompetent tumor
cells of the present disclosure are effective to stimulate one or
more of T-lymphocytes (e.g. CD8+ T cells), natural killer (NK)
cells, dendritic cells (DCs) or B lymphocytes.
[0297] According to some embodiments, stimulating an immune cell
refers to expansion of the immune cell. According to some
embodiments, stimulating an immune cell refers to activation of the
immune cell. According to some embodiments, stimulating an immune
cell refers to an increase in cytoxicity of the immune cell.
According to some embodiments, stimulating the immune cell refers
to a combination of one or more of expansion, activation and/or
increased cytoxicity of the immune cell. According to some
embodiments, the one or more exogenous immunomodulatory molecules
expressed by the population of tumor cells are effective to
activate and/or expand immune cells (e.g. T-lymphocytes (e.g. CD8+
T cells), natural killer (NK) cells, dendritic cells (DCs) or B
lymphocytes) ex vivo. According to some embodiments, the one or
more exogenous immunomodulatory molecules expressed by the
population of tumor cells are effective to activate and/or expand
immune killer cells (e.g. T-lymphocytes (e.g. CD8+ T cells),
natural killer (NK) cells, dendritic cells (DCs) or B lymphocytes)
in vivo. Assays to detect if the exogenous immunostimulatory
molecules are effective to stimulate an immune killer cell are
described herein. According to one aspect, the disclosure thus
provides an allogeneic tumor cell vaccine comprising a population
of proliferation incompetent tumor cells expressing one or more
tumor specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising a plurality of stably expressed exogenous
immunomodulatory molecules sufficient to stimulate the
T-lymphocytes (e.g. CD8+ T cells), natural killer (NK) cells,
dendritic cells (DCs) or B lymphocytes.
According to some embodiments, the allogeneic vaccine comprises a
population of proliferation incompetent tumor cells expressing one
or more tumor specific antigens, wherein the tumor cells are
genetically engineered to stimulate one or more of T lymphocytes,
natural killer (NK) cells, dendritic cells (DCs) or B lymphocytes,
the population comprising at least three stably expressed exogenous
immunomodulatory molecules effective to stimulate the
T-lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes. According to some embodiments, the allogeneic
vaccine comprises a population of proliferation incompetent tumor
cells expressing one or more tumor specific antigens, wherein the
tumor cells are genetically engineered to stimulate one or more of
T lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes, the population comprising at least four stably
expressed exogenous immunomodulatory molecules effective to
stimulate the T-lymphocytes, natural killer (NK) cells, dendritic
cells (DCs) or B lymphocytes. According to some embodiments, the
allogeneic vaccine comprises a population of proliferation
incompetent tumor cells expressing one or more tumor specific
antigens, wherein the tumor cells are genetically engineered to
stimulate one or more of T lymphocytes, natural killer (NK) cells,
dendritic cells (DCs) or B lymphocytes, the population comprising
at least five stably expressed exogenous immunomodulatory molecules
effective to stimulate the T-lymphocytes, natural killer (NK)
cells, dendritic cells (DCs) or B lymphocytes. According to some
embodiments, the allogeneic vaccine comprises a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising at least five stably expressed exogenous
immunomodulatory molecules effective to stimulate the
T-lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes. According to some embodiments, the allogeneic
vaccine comprises a population of proliferation incompetent tumor
cells expressing one or more tumor specific antigens, wherein the
tumor cells are genetically engineered to stimulate one or more of
T lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes, the population comprising at least six stably
expressed exogenous immunomodulatory molecules effective to
stimulate the T-lymphocytes, natural killer (NK) cells, dendritic
cells (DCs) or B lymphocytes. According to some embodiments, the
allogeneic vaccine comprises a population of proliferation
incompetent tumor cells expressing one or more tumor specific
antigens, wherein the tumor cells are genetically engineered to
stimulate one or more of T lymphocytes, natural killer (NK) cells,
dendritic cells (DCs) or B lymphocytes, the population comprising
at least seven stably expressed exogenous immunomodulatory
molecules effective to stimulate the T-lymphocytes, natural killer
(NK) cells, dendritic cells (DCs) or B lymphocytes. According to
some embodiments, the allogeneic vaccine comprises a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising at least eight stably expressed exogenous
immunomodulatory molecules effective to stimulate the
T-lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes. According to some embodiments, the allogeneic
vaccine comprises a population of proliferation incompetent tumor
cells expressing one or more tumor specific antigens, wherein the
tumor cells are genetically engineered to stimulate one or more of
T lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes, the population comprising at least nine stably
expressed exogenous immunomodulatory molecules effective to
stimulate the T-lymphocytes, natural killer (NK) cells, dendritic
cells (DCs) or B lymphocytes. According to some embodiments, the
allogeneic vaccine comprises a population of proliferation
incompetent tumor cells expressing one or more tumor specific
antigens, wherein the tumor cells are genetically engineered to
stimulate one or more of T lymphocytes, natural killer (NK) cells,
dendritic cells (DCs) or B lymphocytes, the population comprising
at least ten stably expressed exogenous immunomodulatory molecules
effective to stimulate the T-lymphocytes, natural killer (NK)
cells, dendritic cells (DCs) or B lymphocytes. According to some
embodiments, the allogeneic vaccine comprises a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising at least eleven stably expressed exogenous
immunomodulatory molecules effective to stimulate the
T-lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes. According to some embodiments, the allogeneic
vaccine comprises a population of proliferation incompetent tumor
cells expressing one or more tumor specific antigens, wherein the
tumor cells are genetically engineered to stimulate one or more of
T lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes, the population comprising at least twelve stably
expressed exogenous immunomodulatory molecules effective to
stimulate the T-lymphocytes, natural killer (NK) cells, dendritic
cells (DCs) or B lymphocytes. According to some embodiments, the
allogeneic vaccine comprises a population of proliferation
incompetent tumor cells expressing one or more tumor specific
antigens, wherein the tumor cells are genetically engineered to
stimulate one or more of T lymphocytes, natural killer (NK) cells,
dendritic cells (DCs) or B lymphocytes, the population comprising
at least thirteen stably expressed exogenous immunomodulatory
molecules effective to stimulate the T-lymphocytes, natural killer
(NK) cells, dendritic cells (DCs) or B lymphocytes. According to
some embodiments, the allogeneic vaccine comprises a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising at least fourteen stably expressed exogenous
immunomodulatory molecules effective to stimulate the
T-lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes. According to some embodiments, the allogeneic
vaccine comprises a population of proliferation incompetent tumor
cells expressing one or more tumor specific antigens, wherein the
tumor cells are genetically engineered to stimulate one or more of
T lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes, the population comprising at least fifteen stably
expressed exogenous immunomodulatory molecules effective to
stimulate the T-lymphocytes, natural killer (NK) cells, dendritic
cells (DCs) or B lymphocytes. According to some embodiments, the
allogeneic vaccine comprises a population of proliferation
incompetent tumor cells expressing one or more tumor specific
antigens, wherein the tumor cells are genetically engineered to
stimulate one or more of T lymphocytes, natural killer (NK) cells,
dendritic cells (DCs) or B lymphocytes, the population comprising
at least sixteen stably expressed exogenous immunomodulatory
molecules effective to stimulate the T-lymphocytes, natural killer
(NK) cells, dendritic cells (DCs) or B lymphocytes. According to
some embodiments, the allogeneic vaccine comprises a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising at least seventeen stably expressed exogenous
immunomodulatory molecules effective to stimulate the
T-lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes. According to some embodiments, the allogeneic
vaccine comprises a population of proliferation incompetent tumor
cells expressing one or more tumor specific antigens, wherein the
tumor cells are genetically engineered to stimulate one or more of
T lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes, the population comprising at least eighteen stably
expressed exogenous immunomodulatory molecules effective to
stimulate the T-lymphocytes, natural killer (NK) cells, dendritic
cells (DCs) or B lymphocytes. According to some embodiments, the
allogeneic vaccine comprises a population of proliferation
incompetent tumor cells expressing one or more tumor specific
antigens, wherein the tumor cells are genetically engineered to
stimulate one or more of T lymphocytes, natural killer (NK) cells,
dendritic cells (DCs) or B lymphocytes, the population comprising
at least nineteen stably expressed exogenous immunomodulatory
molecules effective to stimulate the T-lymphocytes, natural killer
(NK) cells, dendritic cells (DCs) or B lymphocytes. According to
some embodiments, the allogeneic vaccine comprises a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising at least twenty stably expressed exogenous
immunomodulatory molecules effective to stimulate the
T-lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes. According to some embodiments, the allogeneic
vaccine comprises a population of proliferation incompetent tumor
cells expressing one or more tumor specific antigens, wherein the
tumor cells are genetically engineered to stimulate one or more of
T lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes, the population comprising at least twenty-one stably
expressed exogenous immunomodulatory molecules effective to
stimulate the T-lymphocytes, natural killer (NK) cells, dendritic
cells (DCs) or B lymphocytes. According to some embodiments, the
allogeneic vaccine comprises a population of proliferation
incompetent tumor cells expressing one or more tumor specific
antigens, wherein the tumor cells are genetically engineered to
stimulate one or more of T lymphocytes, natural killer (NK) cells,
dendritic cells (DCs) or B lymphocytes, the population comprising
at least twenty-two stably expressed exogenous immunomodulatory
molecules effective to stimulate the T-lymphocytes, natural killer
(NK) cells, dendritic cells (DCs) or B lymphocytes. According to
some embodiments, the allogeneic vaccine comprises a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising at least twenty-three stably expressed
exogenous immunomodulatory molecules effective to stimulate the
T-lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes. According to some embodiments, the allogeneic
vaccine comprises a population of proliferation incompetent tumor
cells expressing one or more tumor specific antigens, wherein the
tumor cells are genetically engineered to stimulate one or more of
T lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes, the population comprising at least twenty-four
stably expressed exogenous immunomodulatory molecules effective to
stimulate the T-lymphocytes, natural killer (NK) cells, dendritic
cells (DCs) or B lymphocytes. According to some embodiments, the
allogeneic vaccine comprises a population of proliferation
incompetent tumor cells expressing one or more tumor specific
antigens, wherein the tumor cells are genetically engineered to
stimulate one or more of T lymphocytes, natural killer (NK) cells,
dendritic cells (DCs) or B lymphocytes, the population comprising
at least twenty-five stably expressed exogenous immunomodulatory
molecules effective to stimulate the T-lymphocytes, natural killer
(NK) cells, dendritic cells (DCs) or B lymphocytes. According to
some embodiments, the allogeneic vaccine comprises a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising at least twenty-six stably expressed
exogenous immunomodulatory molecules effective to stimulate the
T-lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes. According to some embodiments, the allogeneic
vaccine comprises a population of proliferation incompetent tumor
cells expressing one or more tumor specific antigens, wherein the
tumor cells are genetically engineered to stimulate one or more of
T lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes, the population comprising at least twenty-seven
stably expressed exogenous immunomodulatory molecules effective to
stimulate the T-lymphocytes, natural killer (NK) cells, dendritic
cells (DCs) or B lymphocytes. According to some embodiments, the
allogeneic vaccine comprises a population of proliferation
incompetent tumor cells expressing one or more tumor specific
antigens, wherein the tumor cells are genetically engineered to
stimulate one or more of T lymphocytes, natural killer (NK) cells,
dendritic cells (DCs) or B lymphocytes, the population comprising
at least twenty-eight stably expressed exogenous immunomodulatory
molecules effective to stimulate the T-lymphocytes, natural killer
(NK) cells, dendritic cells (DCs) or B lymphocytes. According to
some embodiments, the allogeneic vaccine comprises a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising at least twenty-nine stably expressed
exogenous immunomodulatory molecules effective to stimulate the
T-lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes. According to some embodiments, the allogeneic
vaccine comprises a population of proliferation incompetent tumor
cells expressing one or more tumor specific antigens, wherein the
tumor cells are genetically engineered to stimulate one or more of
T lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes, the population comprising at least thirty stably
expressed exogenous immunomodulatory molecules effective to
stimulate the T-lymphocytes, natural killer (NK) cells, dendritic
cells (DCs) or B lymphocytes.
[0299] According to some embodiments, the allogeneic vaccine
comprises a population of proliferation incompetent tumor cells
expressing one or more tumor specific antigens, wherein the tumor
cells are genetically engineered to stimulate one or more of T
lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or B
lymphocytes, the population characterized by the expression of
three stably expressed exogenous immunomodulatory molecules
effective to stimulate the T-lymphocytes, natural killer (NK)
cells, dendritic cells (DCs) or B lymphocytes.
[0300] According to some embodiments, the population of tumor cells
comprises a first exogenous immunomodulatory molecule and a second
exogenous immunomodulatory molecule. According to some embodiments,
the population of tumor cells comprises a first exogenous
immunomodulatory molecule, a second exogenous immunomodulatory
molecule and a third exogenous stimulatory molecule. According to
some embodiments, the first exogenous immunomodulatory molecule and
the second exogenous immunomodulatory molecule comprise a chimeric
or fusion molecule, for example a molecule created through the
joining of two or more separate genes, each of which encodes at
least one domain of a protein so that the genes are transcribed and
translated as a single unit, producing a single polypeptide.
According to some embodiments, the allogeneic vaccine described
herein comprises tumor cells comprising one or more exogenous
immunomodulatory molecules, wherein a first tumor cell, or
population of tumor cells, comprises a first immunomodulatory
molecule, and a second tumor cell, or population of tumor cells,
comprises a second immunomodulatory molecule. According to some
embodiments, the allogeneic vaccine described herein comprises
tumor cells comprising one or more exogenous immunomodulatory
molecules, wherein a first tumor cell, or population of tumor
cells, comprises a first and a second immunomodulatory molecule,
and a second tumor cell, or population of tumor cells, comprises a
third immunomodulatory molecule. Thus, it is understood that the
exogenous immunomodulatory molecules described herein can be
present in a tumor cell population in cis (all on the same cell) or
in trans (each, or a combination of each, on different cells).
According to some embodiments, the exogenous immunostimulatory
molecules are presented at the surface of the genetically
engineered tumor cells.
[0301] According to some embodiments, the exogenous
immunomodulatory molecules are particularly selected from a group
for their ability to either initiate an anti-tumor immune response,
and/or to sustain an anti-tumor immune response, and/or for their
ability to abrogate pre-existing immunosuppression
characteristically present in cancer patients, or a combination of
all three. According to some embodiments, combinations of
immunomodulatory molecules are evaluated and selected by a human
mixed lymphocyte tumor cell reaction. According to some
embodiments, the exogenous immunomodulatory molecule is selected
from a cytokine, a TNF-family member, a secreted receptor, a
chaperone, an IgG superfamily member and a chemokine receptor.
[0302] According to some embodiments, the allogeneic tumor cell
vaccine of the present disclosure comprises a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising a plurality of stably expressed exogenous
immunomodulatory molecules effective to stimulate the
T-lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes, wherein the exogenous immunomodulatory molecules
comprise one or more cytokine proteins; wherein the exogenous
immunomodulatory molecules comprise one or more TNF family member
proteins; wherein the exogenous immunomodulatory molecules comprise
one or more secreted receptor proteins; wherein the exogenous
immunomodulatory molecules comprise one or more chaperone proteins;
wherein the exogenous immunomodulatory molecules comprise one or
more IgG superfamily member proteins; and/or wherein the exogenous
immunomodulatory molecules comprise one or more chemokine receptor
proteins.
[0303] According to some embodiments, the allogeneic tumor cell
vaccine of the present disclosure comprises a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising a plurality of stably expressed exogenous
immunomodulatory molecules effective to stimulate the
T-lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes, wherein the exogenous immunomodulatory molecules
comprise one or more cytokine family member proteins and one or
more TNF family member proteins; wherein the exogenous
immunomodulatory molecules comprise one or more cytokine family
member proteins and one or more secreted receptor proteins; wherein
the exogenous immunomodulatory molecules comprise one or more
cytokine family member proteins and one or more chaperone proteins;
wherein the exogenous immunomodulatory molecules comprise one or
more cytokine family member proteins and one or more IgG
superfamily member proteins; wherein the exogenous immunomodulatory
molecules comprise one or more cytokine family member proteins and
one or more chemokine receptor proteins.
[0304] According to some embodiments, the allogeneic tumor cell
vaccine of the present disclosure comprises a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising a plurality of stably expressed exogenous
immunomodulatory molecules effective to stimulate the
T-lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes, wherein the exogenous immunomodulatory molecules
comprise one or more TNF family member proteins and one or more
secreted receptor proteins; wherein the exogenous immunomodulatory
molecules comprise one or more TNF family member proteins and one
or more chaperone proteins; wherein the exogenous immunomodulatory
molecules comprise one or more TNF family member proteins and one
or more IgG superfamily member proteins; wherein the exogenous
immunomodulatory molecules comprise one or more TNF family member
proteins and one or more chemokine receptor proteins.
[0305] According to some embodiments, the allogeneic tumor cell
vaccine of the present disclosure comprises a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising a plurality of stably expressed exogenous
immunomodulatory molecules effective to stimulate the
T-lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes, wherein the exogenous immunomodulatory molecules
comprise one or more secreted receptor proteins and one or more
chaperone proteins; wherein the exogenous immunomodulatory
molecules comprise one or more secreted receptor proteins and one
or more IgG superfamily member proteins; wherein the exogenous
immunomodulatory molecules comprise one or more secreted receptor
proteins and one or more chemokine receptor proteins.
[0306] According to some embodiments, the allogeneic tumor cell
vaccine of the present disclosure comprises a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising a plurality of stably expressed exogenous
immunomodulatory molecules effective to stimulate the
T-lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes, wherein the exogenous immunomodulatory molecules
comprise one or more chaperone proteins and one or more IgG
superfamily member proteins; wherein the exogenous immunomodulatory
molecules comprise one or more chaperone proteins and one or more
chemokine receptor proteins.
[0307] According to some embodiments, the allogeneic tumor cell
vaccine of the present disclosure comprises a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising a plurality of stably expressed exogenous
immunomodulatory molecules effective to stimulate the
T-lymphocytes, natural killer (NK) cells, dendritic cells (DCs) or
B lymphocytes, wherein the exogenous immunomodulatory molecules
comprise one or more IgG superfamily member proteins and one or
more chemokine receptor proteins.
[0308] According to some embodiments, the exogenous
immunomodulatory molecule is an immune stimulatory molecule.
[0309] According to some embodiments, the exogenous
immunomodulatory molecule is selected from the group shown in Table
2. According to some embodiments, the exogenous immunomodulatory
molecule is from a mouse. According to some embodiments, the
exogenous immunomodulatory molecule is from a human.
TABLE-US-00003 TABLE 2 Category Examples Cytokines
Granulocyte-macrophage colony-stimulating factor (GM-CSF),
Granulocyte colony- stimulating factor (G-CSF), Fms-related
tyrosine kinase 3 ligand (Flt3L), Flt3, interleukin-1 (IL-1),
IL-1a, IL-1b, Il-1r.alpha., IL- 2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL- 10, IL-10r.alpha., IL-11, IL-12, IL-12p40, IL-
12p70, IL-12/IL-23 P40, IL13, IL-15, IL- 15/IL15-RA, IL-17, IL-17A,
IL-18, IL-21, IL-23, TGF-.beta., MCP-1, TNF-.alpha. and interferon
alpha (IFN.alpha.), IFN.gamma., MIP1b, Rantes, Tweak, TREM-1,
mIFN.alpha., mIN.gamma. TNF-family Tumor necrosis factor alpha
(TNF.alpha.), TNF, 4- members 1BBL, APRIL, BAFF, LIGHT, RANK ligand
(RANKL), CD40 ligand (CD40L), OX40 Ligand (OX40L), FAS ligand
(FASL), CD27 ligand (CD27L), CD30 ligand (CD30L), CD137 ligand
(CD137L), TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFS12, TNFSF13,
TNFSF13B, TNFSF14, TNFSF15, TNFSF18, TNF.beta., TNFSF1B,
TNF.gamma., Ectodysplasin A (EDA) Receptors TGFbeta Receptor III
Chaperone GRP78/BiP, GRP94, GRP170, Calnexin, Proteins
calreticulin, Hsp47, ERp29, Protein disulfide isomerase (PDI),
Peptidyl prolyl cis-trans- isomerase (PPI), Erp57, Hsp60, Hsp70,
Hsp90, Hsp100 IgG Superfamily CD80, CD86, ICOS ligand, PVR/CD155,
Members CD48, Nectin2, NK-T-B antigen, PD-L2 Chemokine CXCR1,
CXCR2, CXCR3, CXCR5, CXCR6, Receptors CXCR8, CCR8, CCR1, CCR2,
CCR3, CCR5, CCR4, CCR6, CCR7, CCR9, CCR10, XCR1, CXCR3 Others
Transforming Growth Factor Beta (TGFb) receptor, PSGL1, HSP70,
HSP-90B1 (GRP94/96), TL1A
[0310] According to some embodiments, the exogenous
immunomodulatory molecule is selected from one of more of a
TNF-family member, a secreted receptor, a chaperone protein, an IgG
superfamily member, a chemokine receptor. According to some
embodiments, the TNF-family member is selected from a TNF-family
member listed in Table 2. According to some embodiments, the
secreted receptor is selected from a secreted receptor listed in
Table 2. According to some embodiments, the chaperone protein is
selected from a chaperone protein listed in Table 2. According to
some embodiments, the IgG superfamily member is selected from an
IgG superfamily member listed in Table 2. According to some
embodiments, the chemokine receptor is selected from a chemokine
receptor listed in Table 2.
[0311] According to some embodiments, the exogenous
immunomodulatory molecule in Table 2 is in a membrane bound form
(i.e. comprises a membrane anchor). According to other embodiments,
the exogenous immunomodulatory molecule is in a secreted form.
According to some embodiments, the membrane bound form of the
immunomodulator is one or more selected from the group consisting
of 4-1BB ligand, BAFF, April, CD40 ligand, CD80, CD86, Flt3 Ligand,
GM-CSF, HSP90, ICOS ligand, IL-12, IL-15, IL-18, IL-2, IL-21,
IL-23, IL7, LIGHT, OX40 ligand, RANK ligand and TNF. According to
some embodiments, the secreted form of the immunomodulator is one
or more selected from the group consisting of Flt3 ligand, GM-CSF,
IL10R, IL7 and TGFbeta Receptor.
[0312] According to some embodiments, the exogenous
immunomodulatory molecule in Table 2 is a molecule with a wild-type
amino acid sequence. According to some embodiments, the exogenous
immunomodulatory molecule in Table 2 is a molecule with a variant
amino acid sequence.
[0313] According to some embodiments, the exogenous
immunomodulatory molecule is one or more selected from the group
consisting of 4-1BB Ligand, APRIL, BAFF, CD27 Ligand, CD30L, CD40
Ligand, CD80, CD86, FLT-3 Ligand, FLT-3 ligand engineered to remove
transmembrane region, GM-CSF, GMCSF engineered with CD8 membrane
anchor and IRES compatible Signal Sequence, HSP-70, HSP-90, ICOS
Ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7
engineered with CD8 membrane anchor, LIGHT, OX-40 Ligand, RANK
Ligand, TGF-b Receptor, and TNF.
[0314] According to some embodiments, the one or more exogenous
immunomodulatory molecules comprise at least three essential
immunomodulatory molecules, wherein the at least three essential
immunomodulatory molecules are OX40 Ligand (OX40L), CD27 Ligand
(CD70), and CD28 Ligand (CD28L) comprising CD80, CD86 or both.
According to some embodiments, additional immunomodulatory
components identified as R may also be present.
[0315] According to some embodiments, the allogeneic vaccine
comprises a population of proliferation incompetent tumor cells
expressing one or more tumor specific antigens and three stably
expressed essential exogenous immunomodulatory molecules, OX40L,
CD70, and CD28L, effective to stimulate the MNC population.
According to some embodiments, the ENLIST.TM. cells population
comprising a population of tumor cells expressing one or more tumor
specific antigens and the three stably expressed essential
exogenous immunomodulatory molecules OX40L, CD70, and CD28L
comprising CD80, CD86 or both is effective to stimulate synergistic
expansion of CTLs. According to some embodiments, the allogeneic
vaccine further comprises one or more subsets of R immunomodulators
comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, or 25 immunomodulators. According to some
embodiments, the allogeneic vaccine comprises a population of tumor
cells expressing one or more tumor specific antigens, wherein the
tumor cells are genetically engineered to stably express at least
the three stably expressed exogenous immunomodulatory molecules
OX40L, CD70, and CD28L comprising CD80, CD86 or both, plus one R
subset comprising 3-25, inclusive immunomodulators. According to
some embodiments, the allogeneic vaccine comprises a population of
tumor cells expressing one or more tumor specific antigens, wherein
the tumor cells are genetically engineered to stably express at
least the three stably expressed exogenous immunomoculatory
molecules OX40L, CD70, and CD28L comprising CD80, CD86 or both,
plus two R subsets comprising 3-25, inclusive immunomodulators.
According to some embodiments, the allogeneic vaccine comprises a
population of tumor cells expressing one or more tumor specific
antigens, wherein the tumor cells are genetically engineered to
stably express at least the three stably expressed exogenous
immunomoculatory molecules OX40L, CD70, and CD28L comprising CD80,
CD86 or both, plus three R subsets comprising 3-25, inclusive
immunomodulators. According to some embodiments, the allogeneic
vaccine comprises a population of tumor cells expressing one or
more tumor specific antigens, wherein the tumor cells are
genetically engineered to stably express at least the three
essential stably expressed exogenous immunomodulatory molecules
OX40L, CD70, and CD28L comprising CD80, CD86 or both, plus four R
subsets comprising 3-25, inclusive immunomodulators. According to
some embodiments, the allogeneic vaccine comprises a population of
tumor cells expressing one or more tumor specific antigens, wherein
the tumor cells are genetically engineered to stably express at
least the three essential stably expressed exogenous
immunomoculatory molecules OX40L, CD70, and CD28L comprising CD80,
CD86 or both, plus five R subsets comprising 3-25, inclusive
immunomodulators. According to some embodiments, the allogeneic
vaccine comprises a population of tumor cells expressing one or
more tumor specific antigens, wherein the tumor cells are
genetically engineered to stably express at least the three
essential stably expressed exogenous immunomoculatory molecules
OX40L, CD70, and CD28L comprising CD80, CD86 or both, plus six R
subsets comprising 3-25, inclusive immunomodulators. According to
some embodiments, the allogeneic vaccine comprises a population of
tumor cells expressing one or more tumor specific antigens, wherein
the tumor cells are genetically engineered to stably express at
least the three essential stably expressed exogenous
immunomoculatory molecules OX40L, CD70, and CD28L comprising CD80,
CD86 or both, plus seven R subsets comprising 3-25, inclusive
immunomodulators. According to some embodiments, the allogeneic
vaccine comprises a population of tumor cells expressing one or
more tumor specific antigens, wherein the tumor cells are
genetically engineered to stably express at least the three
essential stably expressed exogenous immunomoculatory molecules
OX40L, CD70, and CD28L comprising CD80, CD86 or both, plus eight R
subsets comprising 3-25, inclusive immunomodulators. According to
some embodiments, the allogeneic vaccine comprises a population of
tumor cells expressing one or more tumor specific antigens, wherein
the tumor cells are genetically engineered to stably express at
least the three essential stably expressed exogenous
immunomoculatory molecules OX40L, CD70, and CD28L comprising CD80,
CD86 or both, plus nine R subsets comprising 3-25, inclusive
immunomodulators. According to some embodiments, the allogeneic
vaccine comprises a population of tumor cells expressing one or
more tumor specific antigens, wherein the tumor cells are
genetically engineered to stably express at least the three
essential stably expressed exogenous immunomoculatory molecules
OX40L, CD70, and CD28L comprising CD80, CD86 or both, plus ten R
subsets comprising 3-25, inclusive immunomodulators.
[0316] According to some embodiments, the exogenous
immunomodulatory molecule R.sup.1 is APRIL. According to some
embodiments, the exogenous immunomodulatory molecule R.sup.2 is
BAFF. According to some embodiments, the exogenous immunomodulatory
molecule R.sup.3 is 4-IBB Ligand. According to some embodiments,
the exogenous immunomodulatory molecule R.sup.4 is CD30L. According
to some embodiments, the exogenous immunomodulatory molecule
R.sup.5 is CD40 Ligand. According to some embodiments, the
exogenous immunomodulatory molecule R.sup.6 is CD80. According to
some embodiments, the exogenous immunomodulatory molecule R.sup.7
is CD86. According to some embodiments, the exogenous
immunomodulatory molecule R.sup.8 is FLT-3 Ligand. According to
some embodiments, the exogenous immunomodulatory molecule R.sup.9
is HSP-70. According to some embodiments, the exogenous
immunomodulatory molecule R.sup.10 is HSP-90. According to some
embodiments, the exogenous immunomodulatory molecule R.sup.11 is
ICOS Ligand. According to some embodiments, the exogenous
immunomodulatory molecule R.sup.12 is IL-10R. According to some
embodiments, the exogenous immunomodulatory molecule R.sup.13 is
IL-12. According to some embodiments, the exogenous
immunomodulatory molecule R.sup.14 is IL-15. According to some
embodiments, the exogenous immunomodulatory molecule R.sup.15 is
IL-18. According to some embodiments, the exogenous
immunomodulatory molecule R.sup.16 is IL-2. According to some
embodiments, the exogenous immunomodulatory molecule R.sup.17 is
IL-21. According to some embodiments, the exogenous
immunomodulatory molecule R.sup.18 is IL-23. According to some
embodiments, the exogenous immunomodulatory molecule R.sup.19 is
IL-7. According to some embodiments, the exogenous immunomodulatory
molecule R.sup.20 is LIGHT. According to some embodiments, the
exogenous immunomodulatory molecule R.sup.21 is RANK Ligand.
According to some embodiments, the exogenous immunomodulatory
molecule R.sup.22 is TGF-b Receptor. According to some embodiments,
the exogenous immunomodulatory molecule R.sup.23 is TNF. According
to some embodiments, the exogenous immunomodulatory molecule
R.sup.24 is GM-CSF.
[0317] According to some embodiments, the exogenous
immunomodulatory molecule R comprises between 1 and 30
immunomodulators, inclusive, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30, exogenous immunomodulatory molecules selected from
the group consisting of APRIL, BAFF, 4-IBB Ligand, CD30 Ligand,
CD40 Ligand, CD80, CD86, FLT-3 Ligand, FLT-3 ligand engineered to
remove transmembrane region, GM-CSF, GMCSF engineered with CD8
membrane anchor and IRES compatible Signal Sequence, HSP-70,
HSP-90, ICOS Ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21,
IL-23, IL-7, IL-7 engineered with CD8 membrane anchor, LIGHT, OX-40
Ligand, RANK Ligand, TGF-b Receptor, and TNF. According to some
embodiments, the exogenous immunomodulatory molecule comprises
between 1 and 30, inclusive, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30, exogenous immunomodulatory molecules, wherein at
least three immunomodulatory molecules are, OX40 Ligand (OX40L),
CD27 Ligand, and CD28 Ligand comprising CD80, CD86 or both, and
wherein additional immunomodulatory components identified as
R.sup.1-R.sup.24 are selected from the group consisting of APRIL,
BAFF, 4-IBBL Ligand (4-IBBL), CD30L, CD40 Ligand, CD80, CD86, FLT-3
Ligand, FLT-3 ligand engineered to remove transmembrane region,
GMCSF engineered with CD8 membrane anchor and IRES compatible
Signal Sequence, HSP-70, HSP-90, ICOS Ligand, IL-10R, IL-12, IL-15,
IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered with CD8 membrane
anchor, LIGHT, RANK Ligand, TGF-b Receptor, and TNF.
[0318] According to some embodiments, the exogenous
immunomodulatory molecule R comprises between 1 and 20, inclusive,
i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19 or 20, exogenous immunomodulatory molecules selected from
the group consisting of APRIL, BAFF, 4-IBB Ligand, CD30L, CD40
Ligand, CD80, CD86, FLT-3 Ligand, FLT-3 ligand engineered to remove
transmembrane region, GM-CSF, GMCSF engineered with CD8 membrane
anchor and IRES compatible Signal Sequence, HSP-70, HSP-90, ICOS
Ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7
engineered with CD8 membrane anchor, LIGHT, OX-40 Ligand, RANK
Ligand, TGF-b Receptor, and TNF. According to some embodiments, the
exogenous immunomodulatory molecule comprises between 1 and 20,
inclusive, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19 or 20, exogenous immunomodulatory molecules, wherein
at least three immunomodulatory molecules are OX40 Ligand (OX40L),
CD27 Ligand, and CD28 Ligand, and wherein additional
immunomodulatory components identified as R.sup.1-R.sup.24 are
selected from the group consisting of APRIL, BAFF, 4-IBB Ligand,
CD30L, CD40 Ligand, CD80, CD86, FLT-3 Ligand, FLT-3 ligand
engineered to remove transmembrane region, GMCSF engineered with
CD8 membrane anchor and IRES compatible Signal Sequence, HSP-70,
HSP-90, ICOS Ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21,
IL-23, IL-7, IL-7 engineered with CD8 membrane anchor, LIGHT, RANK
Ligand, TGF-b Receptor, and TNF.
[0319] According to some embodiments, the exogenous
immunomodulatory molecule R comprises between 1 and 10, inclusive,
i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, exogenous immunomodulatory
molecules selected from the group consisting of APRIL, BAFF, 4-IBB
Ligand, CD30L, CD40 Ligand, CD80, CD86, FLT-3 Ligand, FLT-3 ligand
engineered to remove transmembrane region, GM-CSF, GMCSF engineered
with CD8 membrane anchor and IRES compatible Signal Sequence,
HSP-70, HSP-90, ICOS Ligand, IL-10R, IL-12, IL-15, IL-18, IL-2,
IL-21, IL-23, IL-7, IL-7 engineered with CD8 membrane anchor,
LIGHT, OX-40 Ligand, RANK Ligand, TGF-b Receptor, and TNF.
According to some embodiments, the exogenous immunomodulatory
molecule comprises between 1 and 10, inclusive, i.e., 1, 2, 3, 4,
5, 6, 7, 8, 9 or 10, exogenous immunomodulatory molecules, wherein
at least three immunomodulatory molecules are OX40 Ligand (OX40L),
CD27 Ligand, and CD28 Ligand, and wherein additional
immunomodulatory components identified as R.sup.1-R.sup.24 are
selected from the group consisting of APRIL, BAFF, 4-IBB Ligand,
CD30L, CD40 Ligand, CD80, CD86, FLT-3 Ligand, FLT-3 ligand
engineered to remove transmembrane region, GMCSF engineered with
CD8 membrane anchor and IRES compatible Signal Sequence, HSP-70,
HSP-90, ICOS Ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21,
IL-23, IL-7, IL-7 engineered with CD8 membrane anchor, LIGHT, RANK
Ligand, TGF-b Receptor, and TNF.
[0320] According to some embodiments, the exogenous
immunomodulatory molecule R comprises between 5 and 20, inclusive,
i.e., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20,
exogenous immunomodulatory molecules selected from the group
consisting of APRIL, BAFF, 4-IBB Ligand, CD30L, CD40 Ligand, CD80,
CD86, FLT-3 Ligand, FLT-3 ligand engineered to remove transmembrane
region, GM-CSF, GMCSF engineered with CD8 membrane anchor and IRES
compatible Signal Sequence, HSP-70, HSP-90, ICOS Ligand, IL-10R,
IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered with
CD8 membrane anchor, LIGHT, OX-40 Ligand, RANK Ligand, TGF-b
Receptor, and TNF. According to some embodiments, the exogenous
immunomodulatory molecule comprises between 5 and 20, inclusive,
i.e., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
exogenous immunomodulatory molecules, wherein at least three
immunomodulatory molecules are OX40 Ligand (OX40L), CD27 Ligand,
and CD28 Ligand, and wherein additional immunomodulatory components
identified as R.sup.1-R.sup.24 are selected from the group
consisting of APRIL, BAFF, CD27 Ligand, CD30L, CD40 Ligand, CD80,
CD86, FLT-3 Ligand, FLT-3 ligand engineered to remove transmembrane
region, GMCSF engineered with CD8 membrane anchor and IRES
compatible Signal Sequence, HSP-70, HSP-90, ICOS Ligand, IL-10R,
IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered with
CD8 membrane anchor, LIGHT, RANK Ligand, TGF-b Receptor, and
TNF.
[0321] According to some embodiments, the exogenous
immunomodulatory molecule R comprises between 10 and 15, inclusive,
i.e., 10, 11, 12, 13, 14 or 15, exogenous immunomodulatory
molecules selected from the group consisting of APRIL, BAFF, 4IBB
Ligand, CD30L, CD40 Ligand, CD80, CD86, FLT-3 Ligand, FLT-3 ligand
engineered to remove transmembrane region, GM-CSF, GMCSF engineered
with CD8 membrane anchor and IRES compatible Signal Sequence,
HSP-70, HSP-90, ICOS Ligand, IL-10R, IL-12, IL-15, IL-18, IL-2,
IL-21, IL-23, IL-7, IL-7 engineered with CD8 membrane anchor,
LIGHT, OX-40 Ligand, RANK Ligand, TGF-b Receptor, and TNF.
According to some embodiments, the exogenous immunomodulatory
molecule comprises between 10 and 15, inclusive, i.e., 10, 11, 12,
13, 14 or 15 exogenous immunomodulatory molecules, wherein at least
three immunomodulatory molecules are OX40 Ligand (OX40L), CD27
Ligand, and CD28 Ligand, and wherein additional immunomodulatory
components identified as R.sup.1-R.sup.24 are selected from the
group consisting of APRIL, BAFF, 4-IBB Ligand, CD30L, CD40 Ligand,
CD80, CD86, FLT-3 Ligand, FLT-3 ligand engineered to remove
transmembrane region, GMCSF engineered with CD8 membrane anchor and
IRES compatible Signal Sequence, HSP-70, HSP-90, ICOS Ligand,
IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7
engineered with CD8 membrane anchor, LIGHT, RANK Ligand, TGF-b
Receptor, and TNF.
[0322] According to some embodiments, the exogenous
immunomodulatory molecule R comprises 14 exogenous immunomodulatory
molecules selected from the group consisting of APRIL, BAFF, 4-IBB
Ligand, CD30L, CD40 Ligand, CD80, CD86, FLT-3 Ligand, FLT-3 ligand
engineered to remove transmembrane region, GM-CSF, GMCSF engineered
with CD8 membrane anchor and IRES compatible Signal Sequence,
HSP-70, HSP-90, ICOS Ligand, IL-10R, IL-12, IL-15, IL-18, IL-2,
IL-21, IL-23, IL-7, IL-7 engineered with CD8 membrane anchor,
LIGHT, OX-40 Ligand, RANK Ligand, TGF-b Receptor, and TNF.
According to some embodiments, the exogenous immunomodulatory
molecule comprises 14 exogenous immunomodulatory molecules, wherein
at least three immunomodulatory molecules are OX40 Ligand (OX40L),
CD27 Ligand, and CD28 Ligand, and wherein additional
immunomodulatory components identified as R.sup.1-R.sup.24 are
selected from the group consisting of APRIL, BAFF, 4-IBB Ligand,
CD30L, CD40 Ligand, CD80, CD86, FLT-3 Ligand, FLT-3 ligand
engineered to remove transmembrane region, GM-CSF, GMCSF engineered
with CD8 membrane anchor and IRES compatible Signal Sequence,
HSP-70, HSP-90, ICOS Ligand, IL-10R, IL-12, IL-15, IL-18, IL-2,
IL-21, IL-23, IL-7, IL-7 engineered with CD8 membrane anchor,
LIGHT, OX-40 Ligand, RANK Ligand, TGF-b Receptor, and TNF.
[0323] According to some embodiments, each of the exogenous
immunomodulatory molecules 4-1BB Ligand, APRIL, BAFF, CD27 Ligand,
CD30L, CD40 Ligand, CD80, CD86, FLT-3 Ligand, GM-CSF, HSP-70,
HSP-90, ICOS Ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21,
IL-23, IL-7, LIGHT, OX-40 Ligand, RANK Ligand, TGF-b Receptor, and
TNF is a wild type molecule. According to some embodiments, each of
the exogenous immunomodulatory molecules 4-1BB Ligand, APRIL, BAFF,
CD27 Ligand, CD30L, CD40 Ligand, CD80, CD86, FLT-3 Ligand, GM-CSF,
HSP-70, HSP-90, ICOS Ligand, IL-10R, IL-12, IL-15, IL-18, IL-2,
IL-21, IL-23, IL-7, LIGHT, OX-40 Ligand, RANK Ligand, TGF-b
Receptor, and TNF is a mutant or variant sequence.
[0324] According to some embodiments, the exogenous
immunomodulatory molecule R.sup.24 is a CD86 variant that has been
engineered with an IRES compatible signal sequence. According to
some embodiments, the exogenous immunomodulatory molecule R.sup.25
is a FLT3L variant that has been engineered to remove the
transmembrane region. According to some embodiments, the exogenous
immunomodulatory molecule R.sup.26 is a GM-CSF variant that has
been engineered with a CD8 membrane anchor and IRES compatible
Signal Sequence. According to some embodiments, the exogenous
immunomodulatory molecule R.sup.27 is an HSP70 variant that has
been engineered with a CD8 membrane anchor. According to some
embodiments, the exogenous immunomodulatory molecule R.sup.28 is an
HSP-90B1 (GRP94/96) variant that has been engineered with a CD8
membrane anchor. According to some embodiments, the exogenous
immunomodulatory molecule R.sup.29 is an HSP90 variant that has
been engineered with a CD8 membrane anchor. According to some
embodiments, the exogenous immunomodulatory molecule R.sup.30 is an
ICOSL variant that has been engineered with an IRES compatible
signal sequence. According to some embodiments, the exogenous
immunomodulatory molecule R.sup.31 is an IL10R variant that has
been engineered to remove the transmembrane region. According to
some embodiments, the exogenous immunomodulatory molecule R.sup.32
is an IL-Ra variant that has been engineered to remove
transmembrane region (VSV-GM-CSF tag). According to some
embodiments, the exogenous immunomodulatory molecule R.sup.33 is an
IL12 variant that has been engineered to be a single chain with a
CD8 membrane anchor. According to some embodiments, the exogenous
immunomodulatory molecule R.sup.34 is an IL15 variant that has been
engineered with CD8 membrane anchor. According to some embodiments,
the exogenous immunomodulatory molecule R.sup.35 is an IL18 variant
that has been engineered with a CD8 membrane anchor. According to
some embodiments, the exogenous immunomodulatory molecule R.sup.36
is an IL2 variant that has been engineered with a CD8 membrane
anchor and IRES compatible sequence. According to some embodiments,
the exogenous immunomodulatory molecule R.sup.37 is an IL21 variant
that has been engineered with a CD8 membrane anchor. According to
some embodiments, the exogenous immunomodulatory molecule R.sup.38
is an IL23 variant that has been engineered to be a single chain
with a CD8 membrane anchor. According to some embodiments, the
exogenous immunomodulatory molecule R.sup.39 is an IL7 variant that
has been engineered with a CD8 membrane anchor. According to some
embodiments, the exogenous immunomodulatory molecule R.sup.40 is an
TGFb-R variant that has been engineered to remove transmembrane
region. According to some embodiments, the exogenous
immunomodulatory R.sup.41 molecule is an TGFb Receptor III variant
engineered to remove transmembrane region. According to some
embodiments, the exogenous immunomodulatory molecule R.sup.42 is an
mIFN.alpha. variant modified to be membrane bound. According to
some embodiments, the exogenous immunomodulatory molecule R.sup.43
is an mIFN.alpha..gamma. variant which is modified to be membrane
bound. According to some embodiments, the exogenous
immunomodulatory molecule R.sup.44 is an CD40L variant which is
cleavage resistant.
[0325] Table 3 below sets forth R groups R.sup.1-R.sup.44
TABLE-US-00004 TABLE 3 R.sup.x Description R.sup.1 APRIL R.sup.2
BAFF R.sup.3 4-IBB Ligand R.sup.4 CD30 Ligand R.sup.5 CD40 Ligand
R.sup.6 CD80 R.sup.7 CD86 R.sup.8 FLT-3 Ligand R.sup.9 HSP-70
R.sup.10 HSP-90 R.sup.11 ICOS Ligand R.sup.12 IL-10R R.sup.13 IL-12
R.sup.14 IL-15 R.sup.15 IL-18 R.sup.16 IL-2 R.sup.17 IL-21 R.sup.18
IL-23 R.sup.19 IL-7 R.sup.20 LIGHT R.sup.21 RANK ligand R.sup.22
TGF-b Receptor R.sup.23 TNF R.sup.24 CD86 variant engineered with
an IRES compatible signal sequence R.sup.25 FLT3L variant
engineered to remove the transmembrane region R.sup.26 GMCSF
variant with a CD8 membrane anchor and IRES compatible Signal
Sequence R.sup.27 HSP70 variant with a CD8 membrane anchor R.sup.28
HSP-90B1 (GRP94/96) variant engineered with a CD8 membrane anchor
R.sup.29 HSP90 variant engineered with a CD8 membrane anchor
R.sup.30 ICOSL variant engineered with an IRES compatible signal
sequence R.sup.31 IL10R variant engineered to remove the
transmembrane region R.sup.32 IL-R.alpha. variant engineered to
remove transmembrane region (VSV-GM-CSF tag) R.sup.33 IL12 variant
engineered to be a single chain with a CD8 membrane anchor R.sup.34
IL15 variant engineered with CD8 membrane anchor R.sup.35 IL18
variant engineered with a CD8 membrane anchor R.sup.36 IL2 variant
engineered with a CD8 membrane anchor and IRES compatible sequence
R.sup.37 IL21 variant engineered with a CD8 membrane anchor
R.sup.38 IL23 variant engineered to be a single chain with a CD8
membrane anchor R.sup.39 IL7 variant engineered with a CD8 membrane
anchor R.sup.40 TGFb-R variant engineered to remove transmembrane
region R.sup.41 TGFb Receptor III variant engineered to remove
transmembrane region R.sup.42 mIFN.alpha. variant modified to be
membrane bound R.sup.43 mIFN.alpha..gamma. variant which is
modified to be membrane bound R.sup.44 CD40L variant which is
cleavage resistant
[0326] According to some embodiments, at least 12 vectors comprise
14 immunomodulators, wherein at least three immunomodulatory
molecules are OX40 Ligand (OX40L), CD27 Ligand (CD70), and CD28
Ligand (CD28L) comprising CD80, CD86 or both, and wherein the
remaining 11 immunomodulators are selected from R.sup.1-R.sup.44 in
Table 3. According to some embodiments, at least 11 vectors
comprise 14 immunomodulators, wherein at least three
immunomodulatory molecules are OX40 Ligand (OX40L), CD27 Ligand
(CD70), and CD28 Ligand (CD28L) comprising CD80, CD86 or both, and
wherein the remaining 11 immunomodulators are selected from
R.sup.1-R.sup.44 in Table 3. According to some embodiments, at
least 10 vectors comprise 14 immunomodulators, wherein at least
three immunomodulatory molecules are OX40 Ligand (OX40L), CD27
Ligand (CD70), and CD28 Ligand (CD28L) comprising CD80, CD86 or
both, and wherein the remaining 11 immunomodulators are selected
from R.sup.1-R.sup.44 in Table 3. According to some embodiments, 14
immunomodulators are selected from Table 2, wherein at least three
immunomodulatory molecules are OX40 Ligand (OX40L), CD27 Ligand
(CD70), and CD28 Ligand (CD28L) comprising CD80, CD86 or both, and
wherein the remaining 11 immunomodulators are selected from
R.sup.1-R.sup.44 in Table 3 and wherein the 14 immunomodulators are
in 12 vectors. According to some embodiments, 14 immunomodulators
are selected from Table 2, wherein at least three immunomodulatory
molecules are OX40 Ligand (OX40L), CD27 Ligand (CD70), and CD28
Ligand (CD28L) comprising CD80, CD86 or both, and wherein the
remaining 11 immunomodulators are selected from R.sup.1-R.sup.44 in
Table 3 and wherein the 14 immunomodulators are in 11 vectors.
According to some embodiments, 14 immunomodulators are selected
from Table 2, wherein at least three immunomodulatory molecules are
OX40 Ligand (OX40L), CD27 Ligand (CD70), and CD28 Ligand (CD28L)
comprising CD80, CD86 or both, and wherein the remaining 11
immunomodulators are selected from R.sup.1-R.sup.44 in Table 3 and
wherein the 14 immunomodulators are in 10 vectors. The vectors may
further comprise tags.
[0327] According to some embodiments, the immunomodulators are
codon optimized. "Codon optimization" means a modification of a
codon of a polynucleotide encoding a protein with a codon that is
used first before others in a specific organism such that the coded
protein can be more efficiently expressed therein. Because most
amino acids are described by several codons that are referred to as
"synonym" or "synonymous codon", genetic codes have degeneracy.
However, codon usage by a specific organism is not random, and it
is rather biased to specific codon triplets. Such codon usage bias
may be even higher in relation with a certain gene, a gene with
common function or ancestor origin, protein expressed at high level
vs. proteins with low copy number, or a group protein coding region
of a genome of an organism.
Cytokines
[0328] According to one embodiment, the disclosure encompasses an
allogeneic tumor cell vaccine comprising a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising one or more cytokines. Thus, the disclosure
encompasses a cytokine, including a full-length, fragment,
homologue, variant or mutant of the cytokine. A cytokine includes a
protein that is capable of affecting the biological function of
another cell. A biological function affected by a cytokine can
include, but is not limited to, cell growth, cell differentiation
or cell death. According to some embodiments, a cytokine of the
present disclosure is capable of binding to a specific receptor on
the surface of a cell, thereby stimulating an immune cell (e.g. T
lymphocytes (e.g., CD8+ T cell), natural killer (NK) cells,
dendritic cells (DCs) or B lymphocytes).
[0329] According to some embodiments, the cytokine is selected from
Granulocyte-macrophage colony-stimulating factor (GM-CSF),
Granulocyte colony-stimulating factor (G-CSF), Fms-related tyrosine
kinase 3 ligand (FLT3LG), interleukin-1 (IL-1), IL-1a, IL-1b,
Il-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12p40, IL-12p70, IL-12/IL-23 P40, IL13, IL-15,
IL-15/IL15-RA, IL-17, IL-17A, IL-18, IL-21, IL-23, TGF-.beta.,
MCP-1, TNF-.alpha. and interferon alpha (IFN.alpha.), IFN.gamma.,
MIP1b, Rantes, Tweak, and TREM-1. According to some embodiments,
the cytokine is granulocyte-macrophage colony-stimulating factor
(GM-CSF). According to some embodiments, the cytokine is
Fms-related tyrosine kinase 3 ligand (FLT3LG).
[0330] According to some embodiments, the cytokine is secreted.
According to some embodiments, the cytokine is membrane bound.
[0331] Granulocyte-Macrophage Colony Stimulating Factor
(GM-CSF)
[0332] Granulocyte-macrophage colony-stimulating factor (GM-CSF;
colony stimulating factor 2; CSF2) is found in
monocytes/macrophages and activated T cells, and can act as a
growth factor to stimulate and recruit dendritic cells. GM-CSF is a
monomeric glycoprotein secreted by cells of the immune system, as
well as endothelial cells and fibroblasts. Human GM-CSF is a 144
amino acid protein comprising a 17 amino acid signal peptide that
can be cleaved to produce a mature 127 amino acid protein.
Biological activity of GM-CSF occurs via binding to heteromeric
cell surface receptors that are expressed on monocytes,
macrophages, granulocytes, lymphocytes, endothelial cells and
alveolar epithelial cells. The GM-CSF receptor (GM-CSFR) typically
has a low expression (e.g. 20-200/cell), but has a high affinity
(Shi Y et al., Granulocyte-macrophage colony-stimulating factor
(GM-CSF) and T-cell responses: what we do and don't know, Cell
Research (2006) 16: 126-133).
[0333] In some mouse models, vaccination with syngeneic mouse
melanoma cells that secrete GM-CSF stimulates a more potent and
long-lasting antitumor immunity than vaccines produced by other
cytokines. Melanoma patients treated with soluble GM-CSF as an
adjuvant therapy displayed an increase in disease free survival
compared to controls. GM-CSF has been used as an immune adjuvant in
various ways, including, without limitation, systemic and topical
application of soluble GM-CSF, GM-CSF fusion proteins, transfection
of tumor cells with GM-CSF and injection of GM-CSF DNA. Recombinant
GM-CSF has been used an adjuvant for various peptide, protein, and
viral vaccines, and has been shown to be an effective adjuvant in
patients with melanoma, breast, and ovarian cancer. A fusion
protein comprising GM-CSF has also been shown to enhance
immunogenicity of an antigen. GM-CSF has been tested for use in a
gene therapy approach where allogeneic or autologous GM-CSF
expressing cells are used as a vaccine (Kaufman and Wolchok eds.,
General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)).
Such vaccines have had varying degrees of effectiveness among
several different cancer types.
[0334] According to some embodiments, a tumor cell line or tumor
cell line variant may express the GM-CSF peptide of SEQ ID NO: 13.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 60% to the protein of SEQ ID NO: 13. According to some
embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
70% to the protein of SEQ ID NO: 13. According to some embodiments,
a tumor cell line or tumor cell line variant may comprise one or
more proteins with a sequence identity of at least 80% to the
protein of SEQ ID NO: 13. According to some embodiments, a tumor
cell line or tumor cell line variant may comprise one or more
proteins with a sequence identity of at least 90% to the protein of
SEQ ID NO: 13. According to some embodiments, a tumor cell line or
tumor cell line variant may comprise one or more proteins with a
sequence identity of at least 95% to the protein of SEQ ID NO: 13.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 96% to the protein of SEQ ID NO: 13. According to some
embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
97% to the protein of SEQ ID NO: 13. According to some embodiments,
a tumor cell line or tumor cell line variant may comprise one or
more proteins with a sequence identity of at least 98% to the
protein of SEQ ID NO: 13. According to some embodiments, a tumor
cell line or tumor cell line variant may comprise one or more
proteins with a sequence identity of at least 99% to the protein of
SEQ ID NO: 13.
[0335] According to some embodiments, a tumor cell line or tumor
cell line variant may comprise one or more proteins comprising a
fusion between GM-CSF and HLA-I to enable membrane expression.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 60% to the protein of SEQ ID NO: 42 or SEQ ID NO: 5.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 70% to the protein of SEQ ID NO: 42 or SEQ ID NO: 5.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 80% to the protein of SEQ ID NO: 42 or SEQ ID NO: 5.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 90% to the protein of SEQ ID NO: 42 or SEQ ID NO: 5.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 95% to the protein of SEQ ID NO: 42 or SEQ ID NO: 5.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 96% to the protein of SEQ ID NO: 42 or SEQ ID NO: 5.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 97% to the protein of SEQ ID NO: 42 or SEQ ID NO: 5.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 98% to the protein of SEQ ID NO: 42 or SEQ ID NO: 5.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 99% to the protein of SEQ ID NO: 42 or SEQ ID NO:
5.
[0336] Fms-Like Tyrosine Kinase-3 Ligand (Flt-3L)
[0337] The human Flt3L protein is a membrane bound hematopoietic
four helical bundle cytokine encoded by the FLT3LG gene. Flt3L acts
as a growth factor that stimulates proliferation and
differentiation of various blood cell progenitors, and is crucial
for production and development of dendritic cells. Mice that lack
Flt3L have low levels of dendritic cells, while Flt3L administered
to mice or humans results in very high levels of dendritic cells
(Shortman et al., Steady-state and inflammatory dendritic-cell
development, Nature Reviews Immunology, Vol. 7. 19-30 (2007)).
[0338] According to some embodiments, a tumor cell line or tumor
cell line variant expresses the Flt3L peptide of SEQ ID NO: 14.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 60% to the protein of SEQ ID NO: 14. According to some
embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
70% to the protein of SEQ ID NO: 14. According to some embodiments,
a tumor cell line or tumor cell line variant may comprise one or
more proteins with a sequence identity of at least 80% to the
protein of SEQ ID NO: 14. According to some embodiments, a tumor
cell line or tumor cell line variant may comprise one or more
proteins with a sequence identity of at least 90% to the protein of
SEQ ID NO: 14. According to some embodiments, a tumor cell line or
tumor cell line variant may comprise one or more proteins with a
sequence identity of at least 95% to the protein of SEQ ID NO: 14.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 96% to the protein of SEQ ID NO: 14. According to some
embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
97% to the protein of SEQ ID NO: 14. According to some embodiments,
a tumor cell line or tumor cell line variant may comprise one or
more proteins with a sequence identity of at least 98% to the
protein of SEQ ID NO: 14. According to some embodiments, a tumor
cell line or tumor cell line variant may comprise one or more
proteins with a sequence identity of at least 99% to the protein of
SEQ ID NO: 14.
[0339] According to some embodiments, a tumor cell line or tumor
cell line variant comprises a soluble form of Flt3L. According to
some embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
60% to the protein of SEQ ID NO: 44. According to some embodiments,
a tumor cell line or tumor cell line variant may comprise one or
more proteins with a sequence identity of at least 70% to the
protein of SEQ ID NO: 44. According to some embodiments, a tumor
cell line or tumor cell line variant may comprise one or more
proteins with a sequence identity of at least 80% to the protein of
SEQ ID NO: 44. According to some embodiments, a tumor cell line or
tumor cell line variant may comprise one or more proteins with a
sequence identity of at least 90% to the protein of SEQ ID NO: 44.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 95% to the protein of SEQ ID NO: 44. According to some
embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
96% to the protein of SEQ ID NO: 44. According to some embodiments,
a tumor cell line or tumor cell line variant may comprise one or
more proteins with a sequence identity of at least 97% to the
protein of SEQ ID NO: 44. According to some embodiments, a tumor
cell line or tumor cell line variant may comprise one or more
proteins with a sequence identity of at least 98% to the protein of
SEQ ID NO: 44. According to some embodiments, a tumor cell line or
tumor cell line variant may comprise one or more proteins with a
sequence identity of at least 99% to the protein of SEQ ID NO:
44.
[0340] One skilled in the art, once armed with the teachings
provided herein, would appreciate that the invention encompasses
any cytokine, whether well-known in the art now, or discovered in
the future.
[0341] According to some embodiments, an allogeneic tumor cell
vaccine comprising a population of proliferation incompetent tumor
cells expressing one or more tumor specific antigens, comprises one
or more (e.g., 2, 3, 4, 5, or more) cytokines, or variants or
fragments thereof.
TNF-Family Members
[0342] According to one embodiment, the disclosure encompasses an
allogeneic tumor cell vaccine, comprising a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising one or more TNF-family members. Thus, the
disclosure encompasses a TNF-family member protein, including a
full-length, fragment, homologue, variant or mutant of the
TNF-family protein. According to some embodiments, the TNF
superfamily member is selected from one or more of tumor necrosis
factor alpha (TNF.alpha.), CD40 ligand (CD40L), OX40 Ligand
(OX40L), FAS ligand (FASL), CD27 ligand (CD27L), CD30 ligand
(CD30L), CD137 ligand (CD137L), TNFSF8, TNFSF9, TNFSF10, TNF SF11,
TNF S12, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TNFSF18, TNF.beta.,
TNFSF1B, TNF.gamma., Ectodysplasin A (EDA). According to some
embodiments, the TNF superfamily member is TNF.alpha.. According to
some embodiments, the TNF superfamily member is CD40L.
[0343] According to some embodiments, the TNF family member is
membrane bound.
[0344] The tumor necrosis factor (TNF) superfamily is a protein
superfamily of type II transmembrane proteins containing TNF
homology domain and forming trimers. Members of this superfamily
can be released from the cell membrane by extracellular proteolytic
cleavage and function as a cytokine. These proteins are expressed
predominantly by immune cells and regulate diverse cell functions,
including regulation of immune response and inflammation, but also
proliferation, differentiation, apoptosis and embryogenesis. The
superfamily contains 19 members that bind to 29 members of the TNF
receptor superfamily.
OX40L (TNFSF4, bTNF Superfamily Member 4)
[0345] The OX40 Ligand (OX40L)(CD252, TNFSF4) which was originally
termed glycoprotein 34 kDa (GP34) belongs to the TNF superfamily;
it is mainly expressed on the surface of antigen-presenting cells
(APC), including activated dendritic cells (DCs), B cells,
macrophages, T cells as well as endothelial cells [Huang, L. et
al., J. Trans. Med. (2018) 16: 74; doi: 10.1186/s12967-018-1436-4,
citing DeSmedt, T et al, J. Immunol (2002) 168: 661-670. doi:
10.4049/jimmunol.168.2.661; Ohshima, Y. et al., Blood (1998) 92:
3338-3345].
[0346] OX40 (ACT35, CD134, TNFRSF4) is constitutively expressed on
the cell surface of activated CD4+ T cells [Id., citing Ogawa R, et
al., Cytokine Growth Factor Rev. (2008) 19:253-262. doi:
10.1016/j.cytogfr.2008.04.003, Paterson D J, et al. Mol Immunol.
(1987) 24:1281-1290. doi: 10.1016/0161-5890(87)90122-2]. It can
specially bind to OX40L and initiate a series of reactions which
contribute to facilitate the proliferation and survival of CD4+ T
cells and cytokine secretion [Id., citing Kaur D, Brightling C.
Chest. (2012) 141:494-499. doi: 10.1378/chest.11-1730]. The OX-40
receptor (OX-40R) is a transmembrane protein found on the surface
of activated CD4(+) T cells. Weinberg, A D, et al., "OX-40: life
beyond the effector T cell stage," Semin. Immunol. (1998) 10(6):
471-80). When engaged by an agonist such as anti-OX-40 antibody or
the OX-40 ligand (OX-40L) during antigen presentation to T cell
lines, the OX-40R generates a costimulatory signal that is as
potent as CD28 costimulation. Id. Engagement of OX-40R enhances
effector and memory-effector T cell function by up-regulating IL-2
production and increasing the life-span of effector T cells.
Id.
[0347] CD25-Foxp3- naive CD4 T cells can acquire Foxp3 driven by
TGF-.beta.R and IL-2R signals leading to differentiation into an
inducible Treg (iTreg). So, T et al, Cytokine Growth Factor Rev.
(2008) 19 (3-4): 253-62. Costimulatory signals from OX40 have been
found to be antagonistic for Foxp3 induction in antigen-responding
naive CD4 T cells and suppress the development of high numbers of
CD25+Foxp3+iTregs (Id, citing Vu M D, et al. Blood. (2007)
110:2501-10; So T, Croft M. J Immunol. (2007) 179:1427-30).
[0348] According to some embodiments of the disclosed invention, a
tumor cell line or tumor cell line variant may be engineered to
express a membrane bound form of OX40L on the membrane of the tumor
cell of SEQ ID NO: 108. According to some embodiments, a tumor cell
line or tumor cell line variant may comprise one or more proteins
with a sequence identity of at least 60% to the protein of SEQ ID
NO: 108. According to some embodiments, a tumor cell line or tumor
cell line variant may comprise one or more proteins with a sequence
identity of at least 70% to the protein of SEQ ID NO: 108.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 80% to the protein of SEQ ID NO: 108. According to some
embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
90% to the protein of SEQ ID NO: 108. According to some
embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
95% to the protein of SEQ ID NO: 108. According to some
embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
96% to the protein of SEQ ID NO: 108. According to some
embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
97% to the protein of SEQ ID NO: 108. According to some
embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
98% to the protein of SEQ ID NO: 108. According to some
embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
99% to the protein of SEQ ID NO: 108.
CD27 Ligand (CD70)
[0349] CD27 ligand (CD70), a type II transmembrane protein, is a
member of the TNF superfamily. It is expressed on activated T and B
lymphocytes, as well as NK cells. CD27 Ligand and its receptor CD27
regulate the immune response by promoting T cell expansion and
differentiation, as well as NK enhancement. CD27 signals, during
the later phase of the primary CD8+ T cell response, prevent
apoptosis of antigen-specific CD8+ T cells. Lack of CD27 signals
decreases the quality of memory CD8+ T cell responses. Memory CD8+
T cells, which express surface CD27 similar to naive cells,
however, do not require CD27 costimulation during a secondary
response. Thus, in vivo, CD27 acts indirectly to regulate primary
antigen-specific CD8+ T cell responses by preventing apoptosis of
CD8+ T cells during the later phase of the primary response, and is
required for optimal quality of memory cells, but is not required
during normally primed secondary CD8+ T cell responses. Dolfi, D V,
et a., J. Immunol. (2008) 180(5): 2912-2921). Full length CD27
Ligand (CD70) is a 193 amino acid protein, consisting of a 17 amino
acid cytoplasmic domain, a 21 amino acid transmembrane domain, and
a 155 amino acid extracellular domain. Human soluble CD70
corresponds to the 155 amino acid extracellular domain of the full
length CD70 protein.
[0350] According to some embodiments of the disclosed invention, a
tumor cell line or tumor cell line variant may be engineered to
express a membrane bound form of CD70 on the membrane of the tumor
cell.
[0351] According to some embodiments of the disclosed invention, a
tumor cell line or tumor cell line variant may be engineered to
express a soluble form of CD70.
[0352] According to some embodiments of the disclosed invention,
the tumor cell line or tumor cell line variant may be engineered to
express a membrane bound form of CD70 on the membrane of the tumor
cells of SEQ ID NO: 109. According to some embodiments, the tumor
cell line or tumor cell line variant may comprise one or more
proteins with a sequence identity of at least 60% to the protein of
SEQ ID NO: 109. According to some embodiments, the tumor cell line
or tumor cell line variant may comprise one or more proteins with a
sequence identity of at least 70% to the protein of SEQ ID NO: 109.
According to some embodiments, the tumor cell line or tumor cell
line variant may comprise one or more proteins with a sequence
identity of at least 80% to the protein of SEQ ID NO: 109.
According to some embodiments, the tumor cell line or tumor cell
line variant may comprise one or more proteins with a sequence
identity of at least 90% to the protein of SEQ ID NO: 109.
According to some embodiments, the tumor cell line or tumor cell
line variant may comprise one or more proteins with a sequence
identity of at least 95% to the protein of SEQ ID NO: 109.
According to some embodiments, the tumor cell line or tumor cell
line variant may comprise one or more proteins with a sequence
identity of at least 96% to the protein of SEQ ID NO: 109.
According to some embodiments, the tumor cell line or tumor cell
line variant may comprise one or more proteins with a sequence
identity of at least 97% to the protein of SEQ ID NO: 109.
According to some embodiments, the tumor cell line or tumor cell
line variant may comprise one or more proteins with a sequence
identity of at least 98% to the protein of SEQ ID NO: 109.
According to some embodiments, the tumor cell line or tumor cell
line variant may comprise one or more proteins with a sequence
identity of at least 99% to the protein of SEQ ID NO: 109.
4-IBBL
[0353] Naive CD8 T cells require more co-stimulatory activity to
drive them to become activated effector cells than do naive CD4 T
cells. This requirement can be met in two ways. The simplest is
priming by activated DCs, which have high intrinsic co-stimulatory
activity. In some viral infections, dendritic cells become
sufficiently activated to directly induce CD8 T cells to produce
the IL-2 required for their differentiation into cytotoxic effector
cells, without help from CD4 T cells. This property of DCs have
been exploited to generate cytotoxic T cell responses against
tumors. In the majority of viral infections, however, CD8 T-cell
activation requires additional help, which is provided by CD4
effector T cells. CD4 T cells that recognize related antigens
presented by the APC can amplify the activation of naive CD T cells
by further activating the APC. B7 expressed by the DC first
activates the CD4 T cells to express IL-2 and CD40L. CD40L binds
CD40 on the DC, delivering an additional signal that increases the
expression of B7 and 4-IBBL by the dendritic cell, which in turn
provides additional co-stimulation to the naive CD8 T cell. The
IL-2 produced by activated CD4 T cells also acts to promote
effector CD8 T-cell differentiation. Murphy, Kenneth. Janeway's
Immunobiology: 8th ed. Chapter 15: Garland Science. (2012), at
372.
[0354] 4-IBB has a pattern of expression that follows the primary
activation of T cells and is restricted to activated CD4+ and CD8+
T cells. Guinn, B, et al., J. Immuno. (1999) 162: 5003-5010.
Engagement of the 4-IBB receptor has been shown to relay strong
costimulatory signals within activated T cells, which lead to their
enhanced proliferation and cytokine secretion. Id. Such signaling
prevents activation-induced cell death following TCR cross-linking
in the absence of other accessory signals. Id. 4-IBBL, a high
affinity ligand for 4-IBB, expressed on the surface of activated
APCs, is a type II membrane protein that shows homology to members
of the TNF receptor family. T cells purified from CD28 -/- mice
have been shown to secrete cytokines and proliferate in response to
lymphomas expressing 4-IBBL; this response can be inhibited by the
soluble 4-IBB receptor fusion protein. Id. In the absence of a CD28
signal, the 4-IBBL:4-IBB interaction has been shown to play a role
in the production of a Th2 response in mixed lymphocyte reactions.
Id.
[0355] According to some embodiments of the disclosed invention, an
R subset of immunomodulators may comprise a membrane bound form of
4-IBBL. According to some embodiments of the disclosed invention,
an R subset of immunomodulators may comprise a soluble form of
4-D3BL.
CD40L
[0356] The ligand of CD40, known as CD154 or CD40L, is a type II
transmembrane protein, with a variable molecular weight between 32
and 39 kDa because of post-translation modifications (Elgueta R et
al., Molecular mechanism and function of CD40/CD40L engagement in
the immune system. Immunological reviews. 2009;
229(1):10.1111/j.1600-065X.2009.00782.x.
doi:10.1111/j.1600-065X.2009.00782.x, citing van Kooten C et al.,
J. Leukoc Biol. 2000 January; 67(1):2-17.). A soluble form of CD40L
has been reported that has activities similar to the transmembrane
form (Id. citing Graf D et al., Eur J Immunol. 1995 June;
25(6):1749-54; Mazzei G J et al., J Biol Chem. 1995 Mar. 31;
270(13):7025-8.).
[0357] In nature, CD40L is a member of the TNF superfamily and is
characterized by a sandwich extracellular structure that is
composed of a .beta.-sheet, .alpha.-helix loop, and a .beta.-sheet,
which allows for the trimerization of CD40L (Id. citing Karpusas M
et al., Structure. 1995 Oct. 15; 3(10):1031-9). CD40L is expressed
primarily by activated T cells, as well as activated B cells and
platelets; under inflammatory conditions it is also induced on
monocytic cells, natural killer cells, mast cells, and basophils
(Id. citing Carbone E et al., J Exp Med. 1997 Jun. 16;
185(12):2053-60). The widespread expression of the costimulatory
pair of CD40L and CD40 indicates the pivotal roles they play in
different cellular immune processes.
[0358] CD40L has three binding partners: CD40, .alpha.5.beta.1
integrin and .alpha.IIb.beta.3 integrin. CD40L acts as a
costimulatory molecule and is particularly important on a subset of
T cells called T follicular helper cells (TFH cells), where it
promotes B cell maturation and function by engaging CD40 on the B
cell surface facilitating cell-cell communication. A defect in the
CD40L gene results in an inability to undergo immunoglobulin class
switching and is associated with hyper-IgM syndrome. Absence of
CD40L also stops the formation of germinal centers thereby
prohibiting antibody affinity maturation, an important process in
the adaptive immune system.
[0359] CD40 has been found to be expressed on APCs, while its
ligand, CD40L, has been found on activated T cells. CD40 has been
found to play a critical role in the humoral immune response, and
has been identified as enabling APCs to activate T cells. Several
pathologies have been associated with the CD40/CD40L pathway
including lupus and atherosclerosis, but anti-CD40L antibodies have
been limited to clinical applications of thrombic complications
from CD40 expression on activated platelets (Kaufman and Wolchok
eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121
(2007)).
[0360] CD40 has also been found on several types of cancer,
including solid tumors and hematologic malignancies. Signaling
through CD40 in hematological cancer may mediate growth or
regression, while CD40 signaling in solid tumors is only
tumoricidal. These characteristics are found even in SCID mouse
models, and therefore are likely due to TNF death domain signaling.
There is also evidence of immune modulation, for example blockade
of the CD40/CD40L pathway mitigates the protective effect of GM-CSF
secreting melanoma vaccines (Kaufman and Wolchok eds., General
Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)).
[0361] Tumor cell vaccines expressing CD40L have proved useful in
cancer models. For example, ligation of CD40 with CD40L or
anti-CD40 antibodies has shown synergy with GM-CSF, IFN-gamma,
IL-2, and CTLA-4 blockade. Furthermore, anti-CD40 antibodies have
been reported to have anti-tumor activity in a pre-clinical mouse
model (Kaufman and Wolchok eds., General Principles of Tumor
Immunotherapy, Chpt 5, 67-121 (2007)).
[0362] According to some embodiments, an R subset of
immunomodulators may comprise CD40 Ligand (CD40L). According to
some embodiments of the disclosed invention, the tumor cell line or
tumor cell line variant may be engineered to express a noncleavable
CD40L peptide of SEQ ID NO: 6. According to some embodiments, a
tumor cell line or tumor cell line variant may comprise one or more
proteins with a sequence identity of at least 60% to the protein of
SEQ ID NO: 6. According to some embodiments, a tumor cell line or
tumor cell line variant may comprise one or more proteins with a
sequence identity of at least 70% to the protein of SEQ ID NO: 6.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 80% to the protein of SEQ ID NO: 6. According to some
embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
90% to the protein of SEQ ID NO: 6. According to some embodiments,
a tumor cell line or tumor cell line variant may comprise one or
more proteins with a sequence identity of at least 95% to the
protein of SEQ ID NO: 6. According to some embodiments, a tumor
cell line or tumor cell line variant may comprise one or more
proteins with a sequence identity of at least 96% to the protein of
SEQ ID NO: 6. According to some embodiments, a tumor cell line or
tumor cell line variant may comprise one or more proteins with a
sequence identity of at least 97% to the protein of SEQ ID NO: 6.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 98% to the protein of SEQ ID NO: 6. According to some
embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
99% to the protein of SEQ ID NO: 6.
[0363] According to some embodiments, the tumor cell line or tumor
cell line variant may be engineered to express the non-cleavable
membrane bound CD40L peptide of SEQ ID NO: 7 on the membrane
surface of the tumor cell. According to some embodiments, a tumor
cell line or tumor cell line variant may comprise one or more
proteins with a sequence identity of at least 60% to the protein of
SEQ ID NO: 7. According to some embodiments, a tumor cell line or
tumor cell line variant may comprise one or more proteins with a
sequence identity of at least 70% to the protein of SEQ ID NO: 7.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 80% to the protein of SEQ ID NO: 7. According to some
embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
90% to the protein of SEQ ID NO: 7. According to some embodiments,
a tumor cell line or tumor cell line variant may comprise one or
more proteins with a sequence identity of at least 95% to the
protein of SEQ ID NO: 7. According to some embodiments, a tumor
cell line or tumor cell line variant may comprise one or more
proteins with a sequence identity of at least 96% to the protein of
SEQ ID NO: 7. According to some embodiments, a tumor cell line or
tumor cell line variant may comprise one or more proteins with a
sequence identity of at least 97% to the protein of SEQ ID NO: 7.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 98% to the protein of SEQ ID NO: 7. According to some
embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
99% to the protein of SEQ ID NO: 7.
Tumor Necrosis Factor Alpha (TNF.alpha.)
[0364] Tumor necrosis factor (TNF; tumor necrosis factor alpha
(TNF.alpha.); cachexin, cachectin) is a cytokine, primarily
produced by activated macrophages and lymphocytes, which is
involved in systemic inflammation. It is also one of the cytokines
involved in the acute phase of an immunogenic response. TNF may be
produced by other cell types such as, for example, CD4+
lymphocytes, NK cells, neutrophils, mast cells, eosinophils, and
neurons.
[0365] In its primary role as a regulator of immune cells, TNF is
capable of inducing fever, apoptotic cell death, cachexia,
inflammation, and inhibition of tumorigenesis; of inhibiting viral
replication; and of initiating a response to sepsis vial IL-1 and
IL-6 producing cells. Dysregulated TNF production has been
associated with a wide array of human diseases, including
Alzheimer's disease, major depression, psoriasis, and inflammatory
bowel disease (IBD). TNF can be produced ectopically in the setting
of malignancy and parallels parathyroid hormone both in causing
secondary hypercalcemia and in the cancers with which excessive
production is associated.
[0366] TNF comprises a 26 kDa membrane bound form and 17 kDa
soluble cytokine form. The soluble form of TNF is derived from
proteolytic cleavage of the membrane bound form by TNF-alpha
converting enzyme (TACE) (Grell M. et al., The Transmembrane Form
of Tumor Necrosis Factor Is the Prime Activating Ligand of the 80
kDa Tumor Necrosis Factor Receptor, Cell, Vol. 83, 793-802). TACE
is a matrix metalloprotease that recognizes a cleavage site in the
extracellular domain of full-length TNF (Rieger, R., Chimeric form
of tumor necrosis factor-alpha has enhanced surface expression and
antitumor activity, Cancer Gene Therapy, 2009, 16, 53-64). Deletion
of the cleavage site on TNF results in enhanced membrane stability
of TNF (Id.).
[0367] TNF has antiproliferative and cytotoxic effects on cells, is
known to reduce tumor blood flow and tumor vascular damage, and is
able to modulate immune response by stimulating macrophage and NK
cell activity. However, the use of TNF as a therapeutic itself has
been limited by dose-dependent hypotension and capillary leak that
can cause a sepsis-like syndrome. For that reason, it must be
delivered in a manner that limits systemic effects. TNF has been
added to standard chemotherapy agents to improve response rates.
Other approaches to administering TNF include injection of
adenovirus altered to express TNF in gastrointestinal malignancies.
A tumor vascular-targeted TNF compound has also been developed
(Kaufman and Wolchok eds., General Principles of Tumor
Immunotherapy, Chpt 5, 67-121 (2007)). Recombinant TNF has been
used as an immunostimulant under the name tasonermin, while
HUMIRA.RTM. is an antibody to TNF, useful for the treatment of
inflammatory diseases (e.g. psoriasis and rheumatoid arthritis). In
recognition of this role, molecules such as antibodies have been
designed to interfere with TNF activity. However, such therapies
pose the risk of initiating a cytokine storm caused by the
inappropriate systemic release of cytokines, resulting in a
positive feedback loop of white blood cell activation/cytokine
release that potentially can be fatal.
[0368] According to some embodiments, a subset of R
immunomodulators may comprise TNF. According to some embodiments, a
tumor cell line or tumor cell line variant may be genetically
engineered to express the membrane bound form of TNF on the
membrane of the tumor cell. For example, according to some
embodiments, the cell line variants comprise the peptide of SEQ ID
NO: 8. According to some embodiments, a tumor cell line or tumor
cell line variant may comprise one or more proteins with a sequence
identity of at least 60% to the protein of SEQ ID NO: 8. According
to some embodiments, a tumor cell line or tumor cell line variant
may comprise one or more proteins with a sequence identity of at
least 70% to the protein of SEQ ID NO: 8. According to some
embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
80% to the protein of SEQ ID NO: 8. According to some embodiments,
a tumor cell line or tumor cell line variant may comprise one or
more proteins with a sequence identity of at least 90% to the
protein of SEQ ID NO: 8. According to some embodiments, a tumor
cell line or tumor cell line variant may comprise one or more
proteins with a sequence identity of at least 95% to the protein of
SEQ ID NO: 8. According to some embodiments, a tumor cell line or
tumor cell line variant may comprise one or more proteins with a
sequence identity of at least 96% to the protein of SEQ ID NO: 8.
According to some embodiments, a tumor cell line or tumor cell line
variant may comprise one or more proteins with a sequence identity
of at least 97% to the protein of SEQ ID NO: 8. According to some
embodiments, a tumor cell line or tumor cell line variant may
comprise one or more proteins with a sequence identity of at least
98% to the protein of SEQ ID NO: 8. According to some embodiments,
a tumor cell line or tumor cell line variant may comprise one or
more proteins with a sequence identity of at least 99% to the
protein of SEQ ID NO: 8.
[0369] According to some embodiments, a tumor cell line or tumor
cell line variant may be genetically engineered to express a
non-cleavable membrane bound form of TNF. For example, according to
some embodiments, the tumor cell line or tumor cell line variant
may comprise the TNF protein of SEQ ID NO: 8 with one or more of
amino acids VRSSSRTPSDKP (SEQ ID NO: 104) deleted (see e.g. SEQ ID
NO: 26).
[0370] According to some embodiments, a tumor cell line or tumor
cell line variant may be genetically engineered to express a
soluble form of TNF. For example, according to some embodiments,
the tumor cell line or tumor cell line variant may express the TNF
protein of SEQ ID NO: 8 with part or the entire transmembrane
region removed. For example, according to some embodiments, the
tumor cell line or tumor cell line variant may comprise a
derivative TNF protein of SEQ ID NO: 8 with one or more of amino
acids F, S, F, L, I, V, A, G, A, T, T, L, F, C, L, L, H, F, G, V, I
deleted (see e.g. SEQ ID NO: 27).
[0371] According to some embodiments, a tumor cell line or tumor
cell line variant may be genetically engineered to express a
non-cleavable membrane bound chimeric form of CD40L and TNF. For
example, according to some embodiments, the ligand binding portion
of a TNF molecule may be fused with the transmembrane and proximal
extracellular domains of CD40L, such that the TNF lacks a defined
TNF alpha cleaving enzyme (TACE) site. According to some
embodiments, the intracellular, transmembrane, and partial
extracellular portions CD40L may be fused with the extracellular
region of TNF distal to the TACE cleavage site. According to some
embodiments, the chimeric form of CD40L/TNF may comprise the CD40L
sequence of SEQ ID NO: 9 and the TNF sequence of SEQ ID NO: 10.
According to some embodiments, the CD40L/TNF sequences are operably
linked via a linking peptide between 1 and 30 amino acids in
length. According to some embodiments, a tumor cell line or tumor
cell line variant may be genetically engineered to comprise a
fusion protein with a sequence identity of at least 60% to the
proteins of SEQ ID NO: 9 and SEQ ID NO: 10. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise a fusion protein with a sequence
identity of at least 70% to the proteins of SEQ ID NO: 9 and SEQ ID
NO: 10. According to some embodiments, a tumor cell line or tumor
cell line variant may be genetically engineered to comprise a
fusion protein with a sequence identity of at least 80% to the
proteins of SEQ ID NO: 9 and SEQ ID NO: 10. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise a fusion protein with a sequence
identity of at least 90% to the proteins of SEQ ID NO: 9 and SEQ ID
NO: 10. According to some embodiments, a tumor cell line or tumor
cell line variant may be genetically engineered to comprise a
fusion protein with a sequence identity of at least 95% to the
proteins of SEQ ID NO: 9 and SEQ ID NO: 10. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise a fusion protein with a sequence
identity of at least 96% to the proteins of SEQ ID NO: 9 and SEQ ID
NO: 10. According to some embodiments, a tumor cell line or tumor
cell line variant may be genetically engineered to comprise a
fusion protein with a sequence identity of at least 97% to the
proteins of SEQ ID NO: 9 and SEQ ID NO: 10. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise a fusion protein with a sequence
identity of at least 98% to the proteins of SEQ ID NO: 9 and SEQ ID
NO: 10. According to some embodiments, a tumor cell line or tumor
cell line variant may be genetically engineered to comprise a
fusion protein with a sequence identity of at least 99% to the
proteins of SEQ ID NO: 9 and SEQ ID NO: 10.
[0372] According to some embodiments, a tumor cell line or tumor
cell line variant may be genetically engineered to express a
non-cleavable membrane bound form of TNF with a sequence identity
of at least 60% to the protein of SEQ ID NO: 11. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to express a non-cleavable membrane bound
form of TNF with a sequence identity of at least 70% to the protein
of SEQ ID NO: 11. According to some embodiments, a tumor cell line
or tumor cell line variant may be genetically engineered to express
a non-cleavable membrane bound form of TNF with a sequence identity
of at least 80% to the protein of SEQ ID NO: 11. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to express a non-cleavable membrane bound
form of TNF with a sequence identity of at least 90% to the protein
of SEQ ID NO: 11. According to some embodiments, a tumor cell line
or tumor cell line variant may be genetically engineered to express
a non-cleavable membrane bound form of TNF with a sequence identity
of at least 95% to the protein of SEQ ID NO: 11. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to express a non-cleavable membrane bound
form of TNF with a sequence identity of at least 96% to the protein
of SEQ ID NO: 11. According to some embodiments, a tumor cell line
or tumor cell line variant may be genetically engineered to express
a non-cleavable membrane bound form of TNF with a sequence identity
of at least 97% to the protein of SEQ ID NO: 11. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to express a non-cleavable membrane bound
form of TNF with a sequence identity of at least 98% to the protein
of SEQ ID NO: 11. According to some embodiments, a tumor cell line
or tumor cell line variant may be genetically engineered to express
a non-cleavable membrane bound form of TNF with a sequence identity
of at least 99% to the protein of SEQ ID NO: 11.
[0373] According to some embodiments, a tumor cell line or tumor
cell line variant may be genetically engineered to express a
non-cleavable membrane bound chimeric form of CD40L and TNF. For
example, according to some embodiments, the ligand portion of a TNF
molecule may be fused with extracellular portions of CD40L, wherein
CD40L comprises an extracellular portion that is non-cleavable and
the TNF lacks a defined TACE site (e.g. cleavage site between amino
acids 76 and 77). According to some embodiments, some or all of a
CD40L peptide sequence is fused with the extracellular region of a
TNF peptide sequence distal to the TACE cleavage site. According to
some embodiments, the chimeric form of CD40L/TNF may comprise the
sequence of SEQ ID NO: 31. According to some embodiments, a tumor
cell line or tumor cell line variant may be genetically engineered
to comprise a fusion protein with a sequence identity of at least
60% to the protein of SEQ ID NO: 31. According to some embodiments,
a tumor cell line or tumor cell line variant may be genetically
engineered to comprise a fusion protein with a sequence identity of
at least 70% to the protein of SEQ ID NO: 31. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise a fusion protein with a sequence
identity of at least 80% to the protein of SEQ ID NO: 31. According
to some embodiments, a tumor cell line or tumor cell line variant
may be genetically engineered to comprise a fusion protein with a
sequence identity of at least 90% to the protein of SEQ ID NO: 31.
According to some embodiments, a tumor cell line or tumor cell line
variant may be genetically engineered to comprise a fusion protein
with a sequence identity of at least 95% to the protein of SEQ ID
NO: 31. According to some embodiments, a tumor cell line or tumor
cell line variant may be genetically engineered to comprise a
fusion protein with a sequence identity of at least 96% to the
protein of SEQ ID NO: 31. According to some embodiments, a tumor
cell line or tumor cell line variant may be genetically engineered
to comprise a fusion protein with a sequence identity of at least
97% to the protein of SEQ ID NO: 31. According to some embodiments,
a tumor cell line or tumor cell line variant may be genetically
engineered to comprise a fusion protein with a sequence identity of
at least 98% to the protein of SEQ ID NO: 31. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise a fusion protein with a sequence
identity of at least 99% to the protein of SEQ ID NO: 31.
[0374] According to some embodiments, an allogeneic tumor cell
vaccine, comprising a population of proliferation incompetent tumor
cells expressing one or more tumor specific antigens, comprises one
or more (e.g., 2, 3, 4, 5, or more) TNF-family member proteins, or
variants or fragments thereof.
Secreted Receptors
[0375] According to one embodiment, the disclosure encompasses an
allogeneic tumor cell vaccine, comprising a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising one or more secreted receptors.
[0376] According to some embodiments, R immunomodulators may
comprise one or more (e.g., 2, 3, 4, 5, or more) secreted receptor
proteins, or variants or fragments thereof. According to some
embodiments, the secreted receptor is IL10R, TGF.beta.R3, or
both.
[0377] Interleukin-10 (IL-10) is a key immunosuppressive cytokine
that is produced by a wide range of leukocytes, as well as
nonhematopoietic cells. Shouval, D S., et al., Immunity (2014) 40:
706-719. IL-10 mediates its anti-inflammatory effects through IL-10
receptor (IL-10R)-dependent signals emanating from the cell
surface. The IL-10R is a heterotetramer that consists of two
subunits of IL-10Ra and two subunits of IL-10R13. Id., citing
Moore, K W, et al., Annu. Rev. Immunol. (2001) 19: 683-765).Whereas
the IL-10Ra subunit is unique to IL-10 signaling, the IL-10R.beta.
subunit is shared by other cytokine receptors, including IL-22,
IL-26, and interferon X, Id. IL-10 downstream signaling through the
IL-10R inhibits the induction of proinflammatory cytokines by
blocking NF-.kappa.B-dependent signals. (Id., citing Saraiva, M.,
and O'Garra, A. Nat. Rev. Immunol. (2010) 10: 180-181).
[0378] Transforming growth factor-beta receptor 3 (TbetaRIII or
TbetaR3) is an 853 amino acid transmembrane proteoglycan, which
contains a short 41 amino acid cytoplasmic domain. It is
ubiquitously expressed on nearly all cell types. The level of
TbetaRIII expression is cell type specific. It is a member of the
TGF-beta superfamily signaling pathways, which have essential roles
in mediating cell proliferation, apoptosis, differentiation, and
migration in most human tissues. TbetaRIII is the most abundantly
expressed TGF-beta superfamily receptor and functions as a TGF-beta
superfamily co-receptor, by binding the TGF-beta superfamily
members, TGF-beta1, TGF-beta2, or TGF-beta3, inhibin, BMP-2, BMP-4,
BMP-7, and GDF-5 and presents these ligand to their respective
signaling receptors to activate or repress (in the case of inhibin)
TGF-beta1, BMP, or activin signaling to the Smad transcription
factors. For example, in the case of TGF-beta1, 2, or 3, TbetaRIII
presents ligand to the TGF-beta type II receptor (TbetaRII). Once
bound to ligand, TbetaRII then recruits and transphosphorylates the
TGF-beta type I receptor (TbetaRI), activating its kinase function
and leading to the phosphorylation of Smad2/3. Phosphorylation of
Smad2 and Smad3 leads to formation of a complex with Smad4, and
accumulation of this complex in the nucleus, where along with
co-activators and co-repressors they regulate the transcription of
genes involved in proliferation, angiogenesis, apoptosis, and
differentiation. In addition to regulating receptor mediated Smad
signaling, TbetaRIII also mediates ligand dependent and independent
p38 pathway signaling. TbetaRIII can also undergo ectodomain
shedding to generate soluble TbetaRIII (sTbetaRIII), which binds
and sequesters TGF-beta superfamily members to inhibit their
signaling. Although sTbetaRIII expression has been demonstrated to
correlate with the cell surface expression of TbetaRIII, little is
known about the regulation of sTbetaRIII production. TbetaRIII
shedding may be mediated in part by the membrane type matrix
metalloproteases (MT-MMP) MT1-MMP and/or MT3-MMP, and plasmin, a
serine proteinase which has been shown to cleave the extracellular
domain of TbetaRIII In addition, TbetaRIII shedding is modulated by
pervanadate, a tyrosine phosphatase inhibitor. Supporting this,
TAPI-2, a MT-MMP and ADAM protease inhibitor, has been shown to
inhibit TbetaRIII shedding. The regulation of TbetaRIII expression
is sufficient to alter TGF-beta signaling. The cytoplasmic domain
of TbetaRIII interacts with GAIP interacting protein, C terminus
(GIPC), a PDZ-domain containing protein, which stabilizes TbetaRIII
cell surface expression and increases TGF-beta signaling. The
interaction between TbetaRIII and GIPC also plays an important role
in TbetaRIII mediated inhibition of TGF-beta signaling, cell
migration, and invasion during breast cancer progression. The
cytoplasmic domain of TbetaRIII is phosphorylated by TbetaRII,
which results in TbetaRIII binding to the scaffolding protein
beta-arrestin2. The TbetaRIII/beta-arrestin2 interaction results in
the co-internalization of beta-arrestin2/TbetaRIII/TbetaRII and the
down-regulation of TGF-beta signaling. The interaction between
TbetaRIII and beta-arrestin2 regulates BMP signaling as well as
TGF-beta signaling. TbetaRIII complexes with ALK6, a BMP type I
receptor, in a beta-arrestin2 dependent manner to mediate the
internalization of ALK6 and stimulation of ALK6 specific BMP
signaling events. Through its interaction with beta-arrestin2,
TbetaRIII negatively regulates NF.kappa.-B signaling in the context
of breast cancer, regulates epithelial cellular adhesion to
fibronectin, fibrillogenesis, and focal adhesion formation via
regulation of alpha5beta1 internalization and trafficking to
nascent focal adhesions, activates Cdc42, to alter the actin
cytoskeleton and suppresses migration in normal and cancerous
ovarian epithelial cells. During development, TbetaRIII has an
important role in the formation of the atrioventricular cushion in
the heart. Consistent with an important role for TbetaRIII during
development, TGFbetaR3 null mice are embryonic lethal due to heart
and liver defects. TGFbetaR3 has been recently identified as a
tumor suppressor in multiple types of human cancers, including
breast, lung, ovarian, pancreatic and prostate cancer. The loss of
TGFbetaR3 in these cancer types correlates with disease
progression, and results in increased motility and invasion in
vitro and increased invasion and metastasis in vivo.
(http://atlasgeneticsoncology.org/Genes/TGFBR3ID42541ch1p33.html,
visited 8/26/2019).
Chaperones
[0379] According to some embodiments, a subset of R
immunomodulators may comprise one or more chaperone proteins.
According to one embodiment, the disclosure encompasses an
allogeneic tumor cell vaccine, comprising a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising one or more chaperone proteins. Thus, the
disclosure encompasses a chaperone protein, including a
full-length, fragment, homologue, variant or mutant of the
chaperone protein. Chaperones are a functionally related group of
proteins assisting protein folding in the cell under physiological
and stress conditions. According to some embodiments, the chaperone
protein is selected from one or more of GRP78/BiP, GRP94, GRP170,
Calnexin, calreticulin, HSP47, ERp29, Protein disulfide isomerase
(PDI), Peptidyl prolyl cis-trans-isomerase (PPI), Erp57, Hsp60,
Hsp70, Hsp90, Hsp100.
[0380] According to some embodiments, the chaperone protein is
membrane bound.
[0381] According to some embodiments, an allogeneic tumor cell
vaccine, comprising a population of proliferation incompetent tumor
cells expressing one or more tumor specific antigens, comprises one
or more (e.g., 2, 3, 4, 5, or more) chaperone proteins, or variants
or fragments thereof.
Immunoglobulin Superfamily (IgSF)
[0382] According to some embodiments, a subset of R
immunomodulators may comprise one or more IgSF proteins. According
to one embodiment, the disclosure encompasses an allogeneic tumor
cell vaccine, comprising a population of proliferation incompetent
tumor cells expressing one or more tumor specific antigens, wherein
the tumor cells are genetically engineered to stimulate one or more
of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs)
or B lymphocytes, the population comprising one or more IgS family
proteins. Thus, the disclosure encompasses a member of the IgSF
superfamily, including a full-length, fragment, homologue, variant
or mutant of the IgSF superfamily member. The immunoglobulin
superfamily (IgSF) is a class of proteins that are associated with
the adhesion, binding and recognition processes of cells. Molecules
are categorized as members of this superfamily based on shared
structural features with immunoglobulins; they all possess a domain
known as an immunoglobulin domain or fold. Members of the IgSF
include cell surface antigen receptors, co-receptors and
co-stimulatory molecules of the immune system, molecules involved
in antigen presentation to lymphocytes, cell adhesion molecules,
certain cytokine receptors and intracellular muscle proteins.
Members of the IgSF can be classified as follows: antigen receptors
(e.g. antibodies or immunoglobulins: IgA, IgD, IgE, IgG, IgM);
antigen presenting molecules (e.g. MEW class I, MEW class II);
co-receptors (e.g. CD4, CD8); co-stimulatory or inhibitory
molecules (e.g. CD28, Cd80, CD86); receptors on Natural killer
cells (e.g. killer-cell immunoglobulin-like receptors (KIR));
receptors on leukocytes (e.g., leukocyte immunoglobulin-like
receptors (LILR)); IGSF CAMs (e.g., NCAMs, ICAM-1); cytokine
receptors; growth factor receptors; receptor tyrosine
kinases/phosphatases; IgG binding receptors.
[0383] According to some embodiments, the IgSF member is membrane
bound.
[0384] Poliovirus Receptor (PVR/CD155) is a transmembrane
glycoprotein belonging to the immunoglobulin superfamily. PVR/CD155
mediates NK cell adhesion and triggers NK cell effector functions.
PVR/CD155 binds two different NK cell receptors: CD96 and CD226.
These interactions accumulate at the cell-cell contact site,
leading to the formation of a mature immunological synapse between
NK cell and target cell. This may trigger adhesion and secretion of
lytic granules and IFN-gamma (IFN.gamma.) and activate cytoxicity
of activated NK cells, and may also promote NK cell-target cell
modular exchange, and PVR transfer to the NK cell.
[0385] Poliovirus receptor-related 2 (PVRL2), also known as
Nectin-2, is a single-pass type I membrane glycoprotein with two
Ig-like C2-type domains and an Ig-like V-type domain. This protein
is one of the plasma membrane components of adherens junctions.
[0386] CD48 antigen (Cluster of Differentiation 48), also known as
B-lymphocyte activation marker (BLAST-1) or signaling lymphocytic
activation molecule 2 (SLAMF2), is a protein that in humans is
encoded by the CD48 gene. CD48 is a member of the CD2 subfamily of
the IgSF, which includes SLAM (signaling lymphocyte activation
molecules) proteins, such as CD84, CD150, CD229 and CD244. CD48 is
found on the surface of lymphocytes and other immune cells,
dendritic cells and endothelial cells, and participates in
activation and differentiation pathways in these cells.
[0387] NK-T-B antigen (NTBA) is a surface molecule expressed on NK,
T, and B cells. In human NK cells, NTBA has been shown to act
primarily as a coreceptor since it could trigger cytolytic activity
only in cells expressing high surface densities of natural
cytotoxicity receptors (NCR). Molecular cloning revealed that NTBA
is a member of the Ig superfamily characterized by structural
features that allowed its assignment to the CD2 family.
[0388] According to one embodiment, the IgSF protein is IgG.
According to one embodiment, the IgSF protein is PVR/CD155.
According to one embodiment, the IgSF protein is CD48. According to
one embodiment, the IgSF protein is Nectin2. According to one
embodiment, the IgSF protein is NK-T-B antigen.
[0389] Immunoglobulins (Ig) are glycoproteins produced by immune
cells. Antibodies are serum proteins, the molecules of which
possess small areas of their surface that are complementary to
small chemical groupings on their targets. These complementary
regions (referred to as complementary determining regions (CDRs),
or antibody combining sites, or antigen binding sites) of which
there are at least two per antibody molecule, and in some types of
antibody molecules ten, eight, or in some species as many as 12,
may react with their corresponding complementary region on the
antigen (the antigenic determinant or epitope) to link several
molecules of multivalent antigen together to form a lattice.
Immunoglobulins play a critical role in an immune response by
binding to particular antigens, such as those exhibited by bacteria
or viruses. According to some embodiments, the binding of
immunoglobulins to antigens may target them for destruction by the
subject's immune cells.
[0390] The basic structural unit of a whole antibody molecule
consists of four polypeptide chains, two identical light (L) chains
(each containing about 220 amino acids) and two identical heavy (H)
chains (each usually containing about 440 amino acids). The two
heavy chains and two light chains are held together by a
combination of noncovalent and covalent (disulfide) bonds. The
molecule is composed of two identical halves, each with an
identical antigen-binding site composed of the N-terminal region of
a light chain and the N-terminal region of a heavy chain. Both
light and heavy chains usually cooperate to form the antigen
binding surface.
[0391] In mammals, there are five classes of antibodies, IgA, IgD,
IgE, IgG, and IgM, each with its own class of heavy chain-.alpha.
(for IgA), .delta. (for IgD), .epsilon. (for IgE), .gamma. (for
IgG) and .mu. (for IgM). In addition, there are four subclasses of
IgG immunoglobulins (IgG1, IgG2, IgG3, IgG4) having .gamma.1,
.gamma.2, .gamma.3, and .gamma.4 heavy chains respectively. In its
secreted form, IgM is a pentamer composed of five four-chain units,
giving it a total of 10 antigen binding sites. Each pentamer
contains one copy of a J chain, which is covalently inserted
between two adjacent tail regions.
[0392] Diverse libraries of immunoglobulin heavy (VH) and light
(V.kappa. and V.lamda.) chain variable genes from peripheral blood
lymphocytes also can be amplified by polymerase chain reaction
(PCR) amplification. Genes encoding single polypeptide chains in
which the heavy and light chain variable domains are linked by a
polypeptide spacer can be made by randomly combining heavy and
light chain V-genes using PCR.
[0393] According to some embodiments, the tumor cell line or tumor
cell line variants may be engineered to express an IgG1 heavy chain
constant region. In nature, the Ig gamma-1 (IgG-1) chain C region
is a protein encoded by the IGHG1 gene in humans. According to some
embodiments, a tumor cell line or tumor cell line variant may
express a membrane bound form IgG-1 chain C protein of SEQ ID NO:
1. According to some embodiments, a tumor cell line or tumor cell
line variant may be genetically engineered to express a secreted
form of IgG-1 chain C of SEQ ID NO: 2. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to express a secreted form of IgG-1 chain C
of SEQ ID NO: 3. According to some embodiments, a tumor cell line
or tumor cell line variant may be genetically engineered to
comprise one or more proteins with a sequence identity of at least
60% to one or more of proteins with an amino acid sequence SEQ ID
NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise one or more proteins with a
sequence identity of at least 70% to one or more of proteins with
an amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:
3. According to some embodiments, a tumor cell line or tumor cell
line variant may be genetically engineered to comprise one or more
proteins with a sequence identity of at least 80% to one or more of
proteins with an amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2,
and SEQ ID NO: 3. According to some embodiments, a tumor cell line
or tumor cell line variant may be genetically engineered to
comprise one or more proteins with a sequence identity of at least
90% to one or more of proteins with an amino acid sequence SEQ ID
NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise one or more proteins with a
sequence identity of at least 95% to one or more of proteins with
an amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:
3. According to some embodiments, a tumor cell line or tumor cell
line variant may be genetically engineered to comprise one or more
proteins with a sequence identity of at least 96% to one or more of
proteins with an amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2,
and SEQ ID NO: 3. According to some embodiments, a tumor cell line
or tumor cell line variant may be genetically engineered to
comprise one or more proteins with a sequence identity of at least
97% to one or more of proteins with an amino acid sequence SEQ ID
NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise one or more proteins with a
sequence identity of at least 98% to one or more of proteins with
an amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:
3. According to some embodiments, a tumor cell line or tumor cell
line variant may be genetically engineered to comprise one or more
proteins with a sequence identity of at least 99% to one or more of
proteins with an amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2,
and SEQ ID NO: 3.
[0394] According to some embodiments, a tumor cell line or tumor
cell line variant may be genetically engineered to comprise one or
more proteins with a sequence identity of at least 60% to one or
more proteins with amino acid sequence SEQ ID NO: 12, SEQ ID NO:
32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ
ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:
45, and SEQ ID NO: 46. According to some embodiments, a tumor cell
line or tumor cell line variant may be genetically engineered to
comprise one or more proteins with a sequence identity of at least
70% to one or more proteins with amino acid sequence SEQ ID NO: 12,
SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID
NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40,
SEQ ID NO: 45, and SEQ ID NO: 46. According to some embodiments, a
tumor cell line or tumor cell line variant may be genetically
engineered to comprise one or more proteins with a sequence
identity of at least 80% to one or more proteins with amino acid
sequence SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:
34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ
ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 45, and SEQ ID NO: 46.
According to some embodiments, a tumor cell line or tumor cell line
variant may be genetically engineered to comprise one or more
proteins with a sequence identity of at least 90% to one or more
proteins with amino acid sequence SEQ ID NO: 12, SEQ ID NO: 32, SEQ
ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:
37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 45, and
SEQ ID NO: 46. According to some embodiments, a tumor cell line or
tumor cell line variant may be genetically engineered to comprise
one or more proteins with a sequence identity of at least 95% to
one or more proteins with amino acid sequence SEQ ID NO: 12, SEQ ID
NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,
SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID
NO: 45, and SEQ ID NO: 46. According to some embodiments, a tumor
cell line or tumor cell line variant may be genetically engineered
to comprise one or more proteins with a sequence identity of at
least 96% to one or more proteins with amino acid sequence SEQ ID
NO: 12, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35,
SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID
NO: 40, SEQ ID NO: 45, and SEQ ID NO: 46. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise one or more proteins with a
sequence identity of at least 97% to one or more proteins with
amino acid sequence SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 33,
SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID
NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 45, and SEQ ID NO:
46. According to some embodiments, a tumor cell line or tumor cell
line variant may be genetically engineered to comprise one or more
proteins with a sequence identity of at least 98% to one or more
proteins with amino acid sequence SEQ ID NO: 12, SEQ ID NO: 32, SEQ
ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:
37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 45, and
SEQ ID NO: 46. According to some embodiments, a tumor cell line or
tumor cell line variant may be genetically engineered to comprise
one or more proteins with a sequence identity of at least 99% to
one or more proteins with amino acid sequence SEQ ID NO: 12, SEQ ID
NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,
SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID
NO: 45, and SEQ ID NO: 46.
[0395] According to some embodiments, the tumor cell line or tumor
cell line variant may be engineered to express an IgG protein that
is capable of binding to tumor cell specific antigens. For example,
the tumor cell line or tumor cell line variant may be engineered to
express an IgG protein capable of binding to a prostate cancer
specific antigen; e.g., the extracellular region of
prostate-specific membrane antigen (PSMA) (See Chang, S., Overview
of Prostate-Specific Membrane Antigen, Reviews in Urology, Vol. 6
Suppl. 10, S13 (2004)). According to some embodiments, the tumor
cell line or tumor cell line variant may be engineered to express
an IgG protein that is capable of binding to immune cell specific
antigens. For example, the tumor cell line or tumor cell line
variant may be engineered to express an IgG protein capable of
binding to T cell markers, e.g., CD3, CD4, or CD8. According to
another example, the tumor cell line or tumor cell line variant may
be engineered to express an IgG protein capable of binding to
dendritic cell markers, e.g. CD11 c or CD123.
[0396] According to some embodiments, the tumor cell line or tumor
cell line variants may be engineered to express an IgG3 heavy chain
constant region. In nature, the IgG3 heavy chain constant region
comprises CH1-hinge-CH2-CH3 domains, and is encoded by the IGHG3
gene in humans; the IGHG3 gene comprises structural polymorphisms
comprising different hinge lengths. According to some embodiments,
a tumor cell line or tumor cell line variant may be genetically
engineered to express an IgG-3 heavy chain constant region of SEQ
ID NO: 4. According to some embodiments, a tumor cell line or tumor
cell line variant may be genetically engineered to express a
derivative of SEQ ID NO: 4 with amino acids 1-76 missing. According
to some embodiments, a tumor cell line or tumor cell line variant
may be genetically engineered to express the derivative of SEQ ID
NO: 4 with amino acids 1-76 missing. According to some embodiments,
a tumor cell line or tumor cell line variant may be genetically
engineered to express the derivative of SEQ ID NO: 4 with amino
acids 77-98 replaced with amino acids QMQGVNCTVSS (SEQ ID NO: 101).
According to some embodiments, a tumor cell line or tumor cell line
variant may be genetically engineered to express the derivative of
SEQ ID NO: 4 comprising an E213Q variant (SEQ ID NO: 16). According
to some embodiments, a tumor cell line or tumor cell line variant
may be genetically engineered to express the derivative of SEQ ID
NO: 4 comprising a P221L variant (SEQ ID NO: 17). According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to express the derivative of SEQ ID NO: 4
comprising an E224Q variant (SEQ ID NO: 18). According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to express the derivative of SEQ ID NO: 4
comprising a Y226F variant (SEQ ID NO: 19). According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to express the derivative of SEQ ID NO: 4
comprising a D242N variant (SEQ ID NO: 20). According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to express the derivative of SEQ ID NO: 4
comprising a N245D variant (SEQ ID NO: 21). According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to express the derivative of SEQ ID NO: 4
comprising a T269A variant (SEQ ID NO: 22). According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to express the derivative of SEQ ID NO: 4
comprising a S314N variant (SEQ ID NO: 23). According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to express the derivative of SEQ ID NO: 4
comprising a deleted 5314 (SEQ ID NO: 24). According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to express the derivative of SEQ ID NO: 4
comprising F366Y variant (SEQ ID NO: 25).
[0397] According to some embodiments, a tumor cell line or tumor
cell line variant may be genetically engineered to comprise one or
more proteins with a sequence identity of at least 60% to the
protein of SEQ ID NO: 4. According to some embodiments, a tumor
cell line or tumor cell line variant may be genetically engineered
to comprise one or more proteins with a sequence identity of at
least 70% to the protein of SEQ ID NO: 4. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise one or more proteins with a
sequence identity of at least 80% to the protein of SEQ ID NO: 4.
According to some embodiments, a tumor cell line or tumor cell line
variant may be genetically engineered to comprise one or more
proteins with a sequence identity of at least 90% to the protein of
SEQ ID NO: 4. According to some embodiments, a tumor cell line or
tumor cell line variant may be genetically engineered to comprise
one or more proteins with a sequence identity of at least 95% to
the protein of SEQ ID NO: 4. According to some embodiments, a tumor
cell line or tumor cell line variant may be genetically engineered
to comprise one or more proteins with a sequence identity of at
least 96% to the protein of SEQ ID NO: 4. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise one or more proteins with a
sequence identity of at least 97% to the protein of SEQ ID NO: 4.
According to some embodiments, a tumor cell line or tumor cell line
variant may be genetically engineered to comprise one or more
proteins with a sequence identity of at least 98% to the protein of
SEQ ID NO: 4. According to some embodiments, a tumor cell line or
tumor cell line variant may be genetically engineered to comprise
one or more proteins with a sequence identity of at least 99% to
the protein of SEQ ID NO: 4.
[0398] According to some embodiments, a tumor cell line or tumor
cell line variant may be engineered to express one or more IgG
heavy chain variable regions. According to some embodiments, a
tumor cell line or tumor cell line variant may be engineered to
express a lambda/kappa light chain constant and/or light chain
variable region. According to some embodiments, the hinge region of
IgG binds to the Fc.gamma.R receptors on immune cells. According to
some embodiments, the IgG is effective to activate the Fc.gamma.R
and enhance presentation of antigens (e.g. PSA associated with
prostate cancer cells).
[0399] According to some embodiments, a tumor cell line or tumor
cell line variant may be engineered to express an intact monoclonal
or polyclonal antibody on the cell surface of the tumor cell.
According to some embodiments, the intact monoclonal or polyclonal
antibody may be designed to deliver a molecule that elicits an
immunogenic response. For example, according to some embodiments,
the intact monoclonal antibody may be designed to bind to DNA to
deliver CpG motifs to immune cells.
[0400] According to some embodiments, the immunostimulatory
activity of bacterial DNA may be mimicked by engineering an
immunomodulator to deliver unmethylated CpG motifs to immune cells.
For example, according to some embodiments, the IgG may be
engineered to bind to biotin, which is then capable of delivering
biotinylated CpG to cells of the immune system. According to some
embodiments, CpG motifs may be bound directly or indirectly to the
surface of the tumor cells of the tumor cell vaccine to prevent
systemic effects. According to some embodiments, CpG motifs may be
conjugated to one or more antigens presented on the surface of
tumor cells from the tumor cell line or tumor cell line variant.
According to some embodiments, the CpG is a class A CpG. According
to some embodiments, the CpG is a class B CpG. According to some
embodiments, the CpG is a class C CpG. According to some
embodiments, the CpG is a CpG 30-mer of the sequence 5'
EEAACCGTATCGGCGATATCGGTTEEEEEG 3' (SEQ ID NO: 102). As used herein
with respect to CpG motifs, "E" is a G-phosphorothioate and this
linkage refers to the 3' end of the nucleotide (i.e. the
phosphorothioate bond substitutes a sulfur atom for a non-bridging
oxygen in the nucleotide backbone). According to some embodiments,
the CpG is a biotinylated 30-mer of the sequence
5'-biotin-EEAACCGTATCGGCGATATCGGTTEEEEEG-3' (SEQ ID NO: 102).
According to some embodiments, the CpG is a CpG 30-mer of the
sequence 5' EEAACCGTATGCGGCATATCGGTTEEEEEG 3' (SEQ ID NO: 103).
According to some embodiments, the CpG is a biotinylated CpG 30-mer
of the sequence 5'-biotin-EEAACCGTATGCGGCATATCGGTTEEEEEG-3' (SEQ ID
NO: 103).
[0401] According to some embodiments, the IgG may be engineered as
a hybrid of one or more IgG subclasses. For example, according to
some embodiments, the IgG comprises sequences from IgG1 and IgG3.
According to some embodiments, the IgG may be engineered to have an
affinity for biotin. According to some embodiments, a tumor cell
line or tumor cell line variant may be genetically engineered to
comprise one or more proteins with a sequence identity of at least
60% to the protein of SEQ ID NO: 45. According to some embodiments,
a tumor cell line or tumor cell line variant may be genetically
engineered to comprise one or more proteins with a sequence
identity of at least 70% to the protein of SEQ ID NO: 45. According
to some embodiments, a tumor cell line or tumor cell line variant
may be genetically engineered to comprise one or more proteins with
a sequence identity of at least 80% to the protein of SEQ ID NO:
45. According to some embodiments, a tumor cell line or tumor cell
line variant may be genetically engineered to comprise one or more
proteins with a sequence identity of at least 90% to the protein of
SEQ ID NO: 45. According to some embodiments, a tumor cell line or
tumor cell line variant may be genetically engineered to comprise
one or more proteins with a sequence identity of at least 95% to
the protein of SEQ ID NO: 45. According to some embodiments, a
tumor cell line or tumor cell line variant may be genetically
engineered to comprise one or more proteins with a sequence
identity of at least 96% to the protein of SEQ ID NO: 45. According
to some embodiments, a tumor cell line or tumor cell line variant
may be genetically engineered to comprise one or more proteins with
a sequence identity of at least 97% to the protein of SEQ ID NO:
45. According to some embodiments, a tumor cell line or tumor cell
line variant may be genetically engineered to comprise one or more
proteins with a sequence identity of at least 98% to the protein of
SEQ ID NO: 45. According to some embodiments, a tumor cell line or
tumor cell line variant may be genetically engineered to comprise
one or more proteins with a sequence identity of at least 99% to
the protein of SEQ ID NO: 45.
[0402] According to some embodiments, the IgG comprises one or more
mutations relative to wild type IgG that enhance affinity for Fc
receptors for IgG (Fc.gamma.R). According to some embodiments, a
tumor cell line or tumor cell line variant may be genetically
engineered to comprise one or more proteins of SEQ ID NO: 45 with
one or more of mutations T323A and E325A. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise one or more proteins with a
sequence identity of at least 60% to the proteins of one or more of
SEQ ID NO: 41, SEQ ID NO: 30, and SEQ ID NO: 43. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise one or more proteins with a
sequence identity of at least 70% to the proteins of one or more of
SEQ ID NO: 41, SEQ ID NO: 30, and SEQ ID NO: 43. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise one or more proteins with a
sequence identity of at least 80% to the proteins of one or more of
SEQ ID NO: 41, SEQ ID NO: 30, and SEQ ID NO: 43. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise one or more proteins with a
sequence identity of at least 90% to the proteins of one or more of
SEQ ID NO: 41, SEQ ID NO: 30, and SEQ ID NO: 43. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise one or more proteins with a
sequence identity of at least 95% to the proteins of one or more of
SEQ ID NO: 41, SEQ ID NO: 30, and SEQ ID NO: 43. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise one or more proteins with a
sequence identity of at least 96% to the proteins of one or more of
SEQ ID NO: 41, SEQ ID NO: 30, and SEQ ID NO: 43. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise one or more proteins with a
sequence identity of at least 97% to the proteins of one or more of
SEQ ID NO: 41, SEQ ID NO: 30, and SEQ ID NO: 43. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise one or more proteins with a
sequence identity of at least 98% to the proteins of one or more of
SEQ ID NO: 41, SEQ ID NO: 30, and SEQ ID NO: 43. According to some
embodiments, a tumor cell line or tumor cell line variant may be
genetically engineered to comprise one or more proteins with a
sequence identity of at least 99% to the proteins of one or more of
SEQ ID NO: 41, SEQ ID NO: 30, and SEQ ID NO: 43.
[0403] According to some embodiments, an allogeneic tumor cell
vaccine comprises a population of proliferation incompetent tumor
cells expressing one or more tumor specific antigens, the tumor
cells comprising one or more (e.g., 2, 3, 4, 5, or more) IgSF
proteins, or variants or fragments thereof.
Chemokine Receptors
[0404] According to some embodiments, a subset of R
immunomodulators may comprise one or more chemokine receptors.
Chemokine receptors are defined as mediators that activate cellular
responses upon binding of chemokines. Twenty-three subtypes of
human chemokine receptors have been identified, all of which are
members of the seven-transmembrane (7TM) domain superfamily of
receptors. They can be divided into two main groups: the G
protein--coupled chemotactic chemokine receptors (n=19) and the
atypical chemokine receptors (n=4). Chemokine binding, membrane
anchoring, and signaling domains for receptors from both groups
come from a single polypeptide chain. Structural and biochemical
evidence exists that these receptors form homo- and
heterodimers.
[0405] According to some embodiments, the chemokine receptor is
membrane bound.
[0406] According to one embodiment, the disclosure encompasses an
allogeneic tumor cell vaccine, comprising a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more of T lymphocytes, natural
killer (NK) cells, dendritic cells (DCs) or B lymphocytes, the
population comprising one or more chemokine receptors. Thus, the
disclosure encompasses a chemokine receptor, including a
full-length, fragment, homologue, variant or mutant of the
chemokine receptor. A cytokine includes a protein that is effective
to affect the biological function of another cell. A biological
function affected by a cytokine can include, but is not limited to,
cell growth, cell differentiation or cell death. For example, a
chemokine receptor of the present disclosure is capable of
stimulating an immune cell (e.g. T lymphocytes (e.g., CD8+ T cell),
natural killer (NK) cells, dendritic cells (DCs) or B
lymphocytes).
[0407] According to some embodiments, the chemokine receptor is
selected from one or more of CXCR1, CXCR2, CXCR3, CXCR5, CXCR6,
CXCR8, CCR8, CCR1, CCR2, CCR3, CCR5, CCR4, CCR6, CCR7, CCR9, CCR10,
CXCR1, and CXCR3.
CD28 Ligand (CD28L)
[0408] Ligation of the CD28 receptor on T cells provides a critical
second signal alongside T cell receptor (TCR) ligation for naive T
cell activation. Esenstein, J H et al, Immunity (2016) 44(5):
973-988). CD28 drives critical intracellular biochemical events
including unique phosphorylation and transcriptional signaling,
metabolism, and the production of key cytokines, chemokines, and
survival signals that are essential for long-term expansion and
differentiation of T cells (Id., citing Bluestone, J A et al.,
Immunity. (2006)24: 233-238; Bour-Jordan, H. et al., Immunol Rev.
(2011) 241:180-205; Martin, P J et al., J Immunol. (1986) 136:
3282-3287; Weiss, A. et al., J Immunol. (1986) 137:819-825).
[0409] CD28 is the founding member of a subfamily of costimulatory
molecules characterized by an extracellular variable
immunoglobulin-like domain. Other members of the subfamily include
ICOS, CTLA4, PD1, PD1H, and BTLA (Id., citing Chen, L. and Flies,
D. B., Nat Rev Immunol. 2013; 13:227-242). CD28 is expressed
constitutively on mouse T cells, whereas the expression of other
family members ICOS and CTLA4 is induced by T cell receptor
stimulation and in response to cytokines such as interleukin 2
(IL-2). CD28 is expressed on roughly 80% of human CD4+ T cells and
50% CD8+ T cells. The proportion of CD28 positive T cells in humans
declines with age. Although CD28 expression has been identified on
other cell lineages, including bone marrow stromal cells, plasma
cells, neutrophils, and eosinophils, the functional importance of
CD28 on these cells is not completely understood (Id., citing Gray
Parkin, K., et al., J Immunol. (2002) 169:2292-2302; Rozanski, C H
et al., J Exp Med. (2011) 208:1435-1446; Venuprasad, K., et al.,
Eur J Immunol. (2001) 31:1536-1543; Woerly, G. et al., Clin Exp
Allergy. (2004) 34:1379-1387).
[0410] The CD28 ligands CD80 and CD86 diverge in their expression
patterns, multimeric states, and functionality, adding another
layer of complexity to the regulation of CD28 signaling. CD80 is
present in predominantly dimeric form on the cell surface whereas
CD86 is monomeric (Id., citing Bhatia, S. et al., Proc Natl Acad
Sci USA. (2005) 102:15569-155742005). CD86 is expressed
constitutively on antigen presenting cells (APCs) and is rapidly
upregulated by innate stimuli of APCs (Id., citing Lenschow, D J et
al., J Immunol. (1994) 153:1990-1997), whereas the other CD28
ligand, CD80, is upregulated at later time points (Id., citing
Sharpe, A J and Freeman, G J, Nat Rev Immunol. (2002) 2:116-126).
CD86 may therefore be more important in the initiation of immune
responses. CD80 and CD86 are induced by different stimuli in
different cell types and they are not interchangeable in
function.
[0411] CD28 and CTLA4 have opposing effects on T cell stimulation.
CD28 provides an activating signal and CTLA4 provides an inhibitory
signal, which is now considered a prototypical immune checkpoint
(Id., citing Krummel, M F and Allison, JP, J Exp Med. 1995;
182:459-465; Walunas, T L et al., Immunity. (1994) 1:405-413).
ICOS, which also contributes to activation, binds to its ligand
B7H2 (ICOSL), which also serves as a ligand for human CD28 and
CTLA4 (Id., citing Chen, L. and Flies, DB, Nat Rev Immunol. (2013)
13:227-242; Yao, S. et al., Immunity (2011) 34:729-740). Thus, this
family of receptors and ligands has considerable complexity in both
binding pattern and biological effect. Overall, the opposing roles
of CD28 and ICOS compared with CTLA4 allow this family of receptors
and ligands to serve as a rheostat for the immune response through
competing pro- and anti-inflammatory effects. Id.
[0412] It has been suggested that CD80 and CD86 may also act as
signal transducing receptors themselves, since ligation with
CTLA4Ig has been shown to regulate tryptophan metabolism in APCs
(Id., citing Grohmann, U et al., Nat Immunol. (2002) 3:1097-1101).
In addition to T cells, plasma cells also express CD28. CD28
signals may regulate antibody production by plasma cells or plasma
cell survival although the precise role that CD28 plays in plasma
cell biology is still unclear (Id., citing Njau, N M and Jacob, J.,
Adv Exp Med Biol. (2013) 785:67-75).
[0413] The CD28 gene is composed of four exons encoding a protein
of 220 amino acids that is expressed on the cell surface as a
glycosylated, disulfide-linked homodimer of 44 kDa. Members of the
CD28 family share a number of common features. These receptors
consist of paired V-set immunoglobulin superfamily (IgSF) domains
attached to single transmembrane domains and cytoplasmic domains
that contain critical signaling motifs (Id., citing Carreno, B M
and Collins, M, Annu Rev Immunol. (2002) 20: 29-53). The CD28 and
CTLA4 ligands, CD80 and CD86, consist of single V-set and Cl-set
IgSF domains. The interaction of these costimulatory receptors with
ligand is mediated through the MYPPPY motif (SEQ ID NO: 105) within
the receptor V-set domains (Id., citing Evans, E J et al., Nat
Immunol. (2005) 6:271-279; Metzler, W J et al., Nat Struct Biol.
(1997) 4: 527-531).
[0414] CD28 engagement by its ligand initiates signal transduction
events that are dependent on specific associations of proteins with
the cytoplasmic tail of CD28. Despite having no intrinsic enzymatic
activity, the 41 amino acid cytoplasmic tail of human CD28 contains
highly conserved tyrosine-based signaling motifs that are
phosphorylated in response to TCR or CD28 stimulation, and bind
targets with SH2 domains in a phosphotyrosine-dependent manner.
Proline rich sequences within the cytoplasmic tail also bind
SH3-domain containing proteins. In particular, the membrane
proximal YMNM motif (SEQ ID NO: 106), and the distal PYAP motif
(SEQ ID NO: 107) have been shown to complex with several kinases
and adaptor proteins, with some proteins being able to bind to
either or both motifs via SH2 and/or SH3 domain interactions (Id.,
citing Boomer, J S and Green, J M, Cold Spring Harb Perspect Biol.
(2010) 2: a002436). These motifs are important for IL-2 gene
expression, which is mediated by the CD28-dependent activation of
NFAT, AP-1, and NF-.kappa.B family transcription factors (Id.,
citing Fraser, J D et al., Science. (1991) 251:313-316; June, C H
et al., Mol Cell Biol. (1987) 7: 4472-4481; Thompson, C B et al.,
Proc Natl Acad Sci USA. (1989) 86:1333-1337).
[0415] The membrane-proximal YXXM motif is shared between CD28,
CTLA4, and ICOS, and is a consensus site for the p85 subunit of the
lipid kinase phosphatidylinositol 3-kinase (PI3K) (Id., citing
August, A. and Dupont, B. Int Immunol. (1994) 6:769-774; Pages, F.,
et al., Nature. (1994) 369: 327-329; Prasad, K V et al., Proc Natl
Acad Sci USA. (1994) 91: 2834-2838; Rudd, C E and Schneider, H.,
Nat Rev Immunol. (2003) 3: 544-556). In addition to the +3
methionine of the CD28 sequence, YMNM (SEQ ID NO: 106), which
confers PI3K specificity, the +2 asparagine confers specificity for
the adaptor proteins GRB2 and GADS on CD28 (Id., citing Cai, Y C et
al., Immunity. (1995) 3: 417-426; Kim, H H et al., J Biol Chem.
(1998) 273: 296-301; Okkenhaug, K. and Rottapel, R., 1998;
Okkenhaug et al., J Biol Chem. (1998) 273: 21194-21202; Raab,
Metal., Proc Natl Acad Sci USA. (1995) 92: 8891-8895; Stein, P H et
al., Mol Cell Biol. (1994) 14: 3392-3402). Both ICOS and CTLA4 can
bind to PI3K but lack the ability to bind GRB2, which may account
for some of the functional and signaling differences between these
costimulatory receptors (Id., citing Rudd, C E and Schneider,H Nat
Rev Immunol. (2003) 3: 544-556). The importance of the YMNM motif
(SEQ ID NO: 106) in mediating proliferation and IL-2 secretion has
been controversial, Signaling events downstream of the C-terminal
PYAP motif (SEQ ID NO: 107) are thought to include the
phosphorylation and activation of the kinases PDK1 and PKCO, and
the subsequent inactivation of GSK3.beta., ultimately leading to
enhanced transcription of NFAT-dependent genes, including IL-2.
SH3-mediated binding and activation of the Src kinase Lck (Id.,
citing Holdorf, A D et al., J Exp Med. (1999) 190: 375-384; King, P
D et al., J Immunol. (1997) 158: 580-590) is proposed as a
potential regulator of this pathway. The adaptor proteins, GRB2 and
GADS can bind to CD28 either through their SH3 domains at the
distal PYAP motif (SEQ ID NO: 107) or via their SH2 domains to the
membrane proximal YMNM motif (SEQ ID NO: 106). However, it is the
C-terminal PYAP motif (SEQ ID NO: 107) that is thought to play the
greater role in NF-.kappa.B activation, suggesting that other
signaling molecules important for NF-.kappa.B activation bind to
the C-terminal PYAP motif (SEQ ID NO: 107), such as Lck, as
discussed above ((Id., citing Holdorf, A D et al., J Exp Med.
(1999) 190: 375-384; Watanabe, R. et al., J Immunol. (2006)
177:1085-1091).
[0416] Although CD28 ligation is critical in promoting
proliferation and effector function of conventional T cells, it
also promotes the anti-inflammatory function of regulatory T (Treg)
cells. Thus, CD28 serves both pro- and anti-inflammatory roles
depending on the cell type and context in which it is expressed.
CD28 signals are critical for allowing effector T cells to overcome
Treg cell-mediated suppression to immunization (Id., citing
Lyddane, C et al., J Immunol. (2006) 176: 3306-3310), but CD28 in
another context prevents spontaneous autoimmunity by promoting Treg
function (Id., citing Salomon B. et al., Immunity. 2000;
12:431-440).
[0417] CD28 supports T cell homeostasis and function in a variety
of ways. CD28 signals support the expression of miR17-92 family
members, which are critical for maximal IL-10 production by Treg
cells (de Kouchkovsky, D et al., J Immunol. (2013) 191: 1594-1605).
Thymocytes require simultaneous TCR and CD28 signals to upregulate
Foxp3 and differentiate into Treg cells. CD28 is also necessary for
the production of peripheral induced Treg cells. CD4+CD25- T cells
required CD28 ligation to differentiate into functional Foxp3+Treg
cells when activated with TGF-.beta..
[0418] According to some embodiments of the disclosed invention,
the population of proliferation incompetent tumor cells may be
engineered to express a membrane bound form of CD80 on the membrane
of the ENLIST.TM. of SEQ ID NO: 110. According to some embodiments,
the population of proliferation incompetent tumor cells may be
engineered to comprise one or more proteins with a sequence
identity of at least 60% to the protein of SEQ ID NO: 110.
According to some embodiments, the population of proliferation
incompetent tumor cells may be engineered to comprise one or more
proteins with a sequence identity of at least 70% to the protein of
SEQ ID NO: 110. According to some embodiments, the population of
proliferation incompetent tumor cells may be engineered to comprise
one or more proteins with a sequence identity of at least 80% to
the protein of SEQ ID NO: 110. According to some embodiments, the
population of proliferation incompetent tumor cells may be
engineered to comprise one or more proteins with a sequence
identity of at least 90% to the protein of SEQ ID NO: 110.
According to some embodiments, the population of proliferation
incompetent tumor cells may be engineered to comprise one or more
proteins with a sequence identity of at least 95% to the protein of
SEQ ID NO: 110. According to some embodiments, the population of
proliferation incompetent tumor cells may be engineered to comprise
one or more proteins with a sequence identity of at least 96% to
the protein of SEQ ID NO: 110. According to some embodiments, the
population of proliferation incompetent tumor cells may be
engineered to comprise one or more proteins with a sequence
identity of at least 97% to the protein of SEQ ID NO: 110.
According to some embodiments, the population of proliferation
incompetent tumor cells may be engineered to comprise one or more
proteins with a sequence identity of at least 98% to the protein of
SEQ ID NO:110. According to some embodiments, the population of
proliferation incompetent tumor cells may be engineered to comprise
one or more proteins with a sequence identity of at least 99% to
the protein of SEQ ID NO: 110.
[0419] According to some embodiments of the disclosed invention,
the population of proliferation incompetent tumor cells may be
engineered to express a membrane bound form of CD86 on the membrane
of the population of proliferation incompetent tumor cells of SEQ
ID NO: 111. According to some embodiments, the population of
proliferation incompetent tumor cells may be engineered to comprise
one or more proteins with a sequence identity of at least 60% to
the protein of SEQ ID NO: 111. According to some embodiments, the
population of proliferation incompetent tumor cells may be
engineered to comprise one or more proteins with a sequence
identity of at least 70% to the protein of SEQ ID NO: 111.
According to some embodiments, the population of proliferation
incompetent tumor cells may be engineered to comprise one or more
proteins with a sequence identity of at least 80% to the protein of
SEQ ID NO: 111. According to some embodiments, the population of
proliferation incompetent tumor cells may be engineered to comprise
one or more proteins with a sequence identity of at least 90% to
the protein of SEQ ID NO: 111. According to some embodiments, the
population of proliferation incompetent tumor cells may be
engineered to comprise one or more proteins with a sequence
identity of at least 95% to the protein of SEQ ID NO: 111.
According to some embodiments, the population of proliferation
incompetent tumor cells may be engineered to comprise one or more
proteins with a sequence identity of at least 96% to the protein of
SEQ ID NO: 111. According to some embodiments, the population of
proliferation incompetent tumor cells may be engineered to comprise
one or more proteins with a sequence identity of at least 97% to
the protein of SEQ ID NO: 111. According to some embodiments, the
population of proliferation incompetent tumor cells may be
engineered to comprise one or more proteins with a sequence
identity of at least 98% to the protein of SEQ ID NO:111. According
to some embodiments, the population of proliferation incompetent
tumor cells may be engineered to comprise one or more proteins with
a sequence identity of at least 99% to the protein of SEQ ID NO:
111.
[0420] According to some embodiments, the allogeneic vaccine is
adapted for rapid in vitro evaluation using human peripheral blood
mononuclear cells from healthy subjects and cancer patients to
examine inter-individual variability as well as normal to patient
differences, thus avoiding animal experimentation.
[0421] According to some embodiments, the allogeneic vaccine is
adapted to provide clinical benefit in the short term by the
induction of strong anti-allogeneic vaccine responses, and, in the
long term, to provide a long lived and cross reactive response to
the endogenous unmodified host tumor. According to some
embodiments, the immune response against the allogeneic tumor cell
vaccine comprises a heteroclitic cross reaction between a peptide
native to the tumor cell line or tumor cell line variant and a
peptide native to the tumor cells of a patient receiving the
vaccine (See, e.g., FIG. 1). According to some embodiments, the
heteroclitic cross-reaction enhances immunogenicity via enhanced
binding of a T cell receptor with a tumor cell peptide-WIC complex
that normally provides a non-immunogenic surface. According to some
embodiments, the allogeneic tumor cell vaccine comprises peptides
altered relative to tumor cells of a subject with cancer, where the
altered peptides provide an immunogenic surface that results in a
heteroclitic cross-reaction to the non-immunogenic peptide of tumor
cells from the subject with cancer. According to some embodiments,
the heteroclitic recognition and alloreactive antigen recognition
of the tumor cell vaccine provides a broad array of antigens useful
to elicit an immune response against the tumor cells of a patient
receiving the vaccine. According to some embodiments, the
allogeneic vaccine is adapted to provide a clinical benefit, e.g.,
in the form of progression free survival, relapse-free survival, or
overall survival. According to some embodiments, the allogeneic
vaccine is effective to provide heteroclitic immunization induced
tumor immunity (Dyall R., et al., Heteroclitic Immunization Induces
Tumor Immunity, J. Exp. Med., Vol. 188, No. 9, Nov. 2, 1998,
incorporated by reference herein in its entirety).
[0422] According to some embodiments, the allogeneic vaccine is
derived from tumor cell line or tumor cell line variants
genetically modified to comprise recombinant immunomodulatory
signals that are expressed in therapeutic amounts. According to
some embodiments, the allogeneic vaccine is derived from a uniform
starting material, such as a tumor cell line or tumor cell line
variant, wherein multiple discrete biologics are expressed in the
starting material in either soluble or membrane bound form.
According to some embodiments, expression and activity of the
soluble and membrane bound forms are confirmed, in vitro, by flow
cytometry and mixed lymphocyte tumor assays using peripheral blood
mononuclear cells, respectively. According to some embodiments,
expression and activity of the soluble and membrane bound forms are
confirmed, in vitro, by flow cytometry and mixed lymphocyte tumor
assays using peripheral blood mononuclear cells of the vaccinated
cancer patient against the allogeneic tumor cells used to
immunize.
[0423] According to some embodiments, the allogeneic vaccine
comprises exogenous immunomodulatory molecules, each encoding a
membrane bound or secreted signaling molecule. According to some
embodiments, each membrane bound immunomodulatory molecule is
adapted to deliver a therapeutic amount in sub-pharmacologic doses
that is active in a spatially and temporally restricted manner to
provide signaling predominantly at the time and place of antigen
presentation. According to some embodiments, the membrane bound
immunomodulatory molecules are adapted to decrease the probability
of systemic side effects. According to some embodiments, the
secreted immunomodulatory molecules are adapted to deliver local,
not systemic, signals.
[0424] According to some aspects, the allogeneic vaccine comprises
genetic material that is effective to genetically introduce one or
more immunomodulatory molecules into a tumor cell line or tumor
cell line variant. According to some embodiments, the genetic
material can be introduced by viral transduction techniques and
isolated by positive selection for the genetically introduced
immune modulator. For example, according to some embodiments, the
positive selection of the genetically introduced immune modulator
molecule comprises selection using antibodies. According to some
embodiments, the immunomodulatory molecules are diverse and
complementary with respect to impact on key immune cell subsets
such as dendritic cells, and lymphocyte sub-populations (e.g. T
cells, Natural Killer cells, and T-regulatory cells). According to
some embodiments, the allogeneic vaccine comprises a variety of
immunomodulatory molecules directed to a variety of
immunomodulatory pathways on various immune cell subsets, wherein
not all pathways will equally contribute to an immunogenic response
in individual cancer patients. According to some embodiments, the
immunomodulatory molecules genetically introduced into a tumor cell
line or tumor cell line variant are stably expressed.
[0425] According to some embodiments, the allogeneic tumor cell
vaccine of the present disclosure comprises a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, the allogeneic tumor cell comprising a core of
three stably expressed essential exogenous immunomodulatory
molecules, OX40 Ligand (OX40L), CD27 Ligand (CD70), and CD28 Ligand
(CD28L) comprising CD80, CD86 or both. According to some
embodiments, the allogeneic tumor cell vaccine further comprises
one or more additional subsets of stably expressed exogenous
immunomodulator molecules, designated as R groups (by analogy to
those in a core chemical structure), with each subset comprising
3-25 immunomodulators. According to some embodiments, a subset R
can comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, or 25 immunomodulators. According to
some embodiments, the allogeneic tumor cell vaccine of the present
disclosure comprises a population of proliferation incompetent
tumor cells expressing one or more tumor specific antigens, wherein
the tumor cells are genetically engineered to stably express at
least the three stably expressed exogenous immunomoculatory
molecules OX40 Ligand (OX40L), CD27 Ligand (CD70), and CD28 Ligand
(CD28L) comprising CD80, CD86 or both, plus one R subset comprising
3-25 immunomodulators. According to some embodiments, the
allogeneic tumor cell vaccine of the present disclosure comprises a
population of proliferation incompetent tumor cells expressing one
or more tumor specific antigens, wherein the tumor cells are
genetically engineered to stably express at least the three stably
expressed exogenous immunomoculatory molecules 4-IBBL, GM-CSF,
OX40L, plus two R subsets comprising 3-25 immunomodulators.
According to some embodiments, the allogeneic tumor cell vaccine of
the present disclosure comprises a population of proliferation
incompetent tumor cells expressing one or more tumor specific
antigens wherein the tumor cells are genetically engineered to
stably express at least the three stably expressed exogenous
immunomoculatory molecules OX40 Ligand (OX40L), CD27 Ligand (CD70),
and CD28 Ligand (CD28L) comprising CD80, CD86 or both, plus three R
subsets comprising 3-25 immunomodulators. According to some
embodiments, the allogeneic tumor cell vaccine of the present
disclosure comprises a population of proliferation incompetent
tumor cells expressing one or more tumor specific antigens, wherein
the tumor cells are genetically engineered to stably express at
least the three essential stably expressed exogenous
immunomodulatory molecules OX40 Ligand (OX40L), CD27 Ligand (CD70),
and CD28 Ligand (CD28L) comprising CD80, CD86 or both, plus four R
subsets comprising 3-25 immunomodulators. According to some
embodiments, the allogeneic tumor cell vaccine of the present
disclosure comprises a population of proliferation incompetent
tumor cells expressing one or more tumor specific antigens, wherein
the tumor cells are genetically engineered to stably express at
least the three essential stably expressed exogenous
immunomoculatory molecules OX40 Ligand (OX40L), CD27 Ligand (CD70),
and CD28 Ligand (CD28L) comprising CD80, CD86 or both, plus five R
subsets comprising 3-25 immunomodulators. According to some
embodiments, the allogeneic tumor cell vaccine of the present
disclosure comprises a population of proliferation incompetent
tumor cells expressing one or more tumor specific antigens, wherein
the tumor cells are genetically engineered to stably express at
least the three essential stably expressed exogenous
immunomoculatory molecules OX40 Ligand (OX40L), CD27 Ligand (CD70),
and CD28 Ligand (CD28L) comprising CD80, CD86 or both, plus six R
subsets comprising 3-25 immunomodulators. According to some
embodiments, the allogeneic tumor cell vaccine of the present
disclosure comprises a population of proliferation incompetent
tumor cells expressing one or more tumor specific antigens, wherein
the tumor cells are genetically engineered to stably express at
least the three essential stably expressed exogenous
immunomoculatory molecules OX40 Ligand (OX40L), CD27 Ligand (CD70),
and CD28 Ligand (CD28L) comprising CD80, CD86 or both, plus seven R
subsets comprising 3-25 immunomodulators. According to some
embodiments, the allogeneic tumor cell vaccine of the present
disclosure comprises a population of proliferation incompetent
tumor cells expressing one or more tumor specific antigens, wherein
the tumor cells are genetically engineered to stably express at
least the three essential stably expressed exogenous
immunomoculatory molecules OX40 Ligand (OX40L), CD27 Ligand (CD70),
and CD28 Ligand (CD28L) comprising CD80, CD86 or both, plus eight R
subsets comprising 3-25 immunomodulators. According to some
embodiments, the allogeneic tumor cell vaccine of the present
disclosure comprises a population of proliferation incompetent
tumor cells expressing one or more tumor specific antigens, wherein
the tumor cells are genetically engineered to stably express at
least the three essential stably expressed exogenous
immunomoculatory molecules OX40 Ligand (OX40L), CD27 Ligand (CD70),
and CD28 Ligand (CD28L) comprising CD80, CD86 or both, plus nine R
subsets comprising 3-25 immunomodulators. According to some
embodiments, the allogeneic tumor cell vaccine of the present
disclosure comprises a population of proliferation incompetent
tumor cells expressing one or more tumor specific antigens, wherein
the tumor cells are genetically engineered to stably express at
least the three essential stably expressed exogenous
immunomoculatory molecules OX40 Ligand (OX40L), CD27 Ligand (CD70),
and CD28 Ligand (CD28L) comprising CD80, CD86 or both, plus ten R
subsets comprising 3-25 immunomodulators.
Assessing Immunogenic Potential
Mixed Lymphocyte Tumor Cell Reactivity
[0426] According to some embodiments, the genetically introduced
immunomodulators may be assessed for their immunogenic potential by
a mixed lymphocyte tumor cell reaction (MLTR). The MLTR assay
comprises incubating mixed lymphocytes with tumor cell line or
tumor cell line variants (or controls) for several days to allow
the tumor cells of the tumor cell line or tumor cell line variant
to elicit an immune response from the mixed lymphocytes in vitro.
This method can provide a rapid in vitro method to assess mixed
lymphocyte responses (such as cellular proliferation of
lymphocytes, cellular subset differentiation of lymphocytes,
cytokine release profile of lymphocytes, and tumor cell death) to
tumor cells or lysates. This approach can enable comprehensive
monitoring of cellular, humoral, or both, immunity responses to
phenotypically modified transfected tumor cells using human
peripheral blood mononuclear cells. The MLTR also can provide an
alternative to murine tumor survival studies, and can result in
selection of optimal tumor cell line or tumor cell line variants
for anti-tumor response. A similar assay has been described by
Hunter T B et al., (2007) Scandanavian J. Immunology 65, 479-486,
which is incorporated herein by reference in its entirety.
[0427] The MLTR was first described by Stjernsward J, et al. in
1970 (Clin Exp Immunol 6: 963-668, incorporated by reference in its
entirety herein) and was based on the mixed lymphocyte response
(MLR) method. Uchida A, et al. describe generation of specific and
non-specific killer T cells using a MLTR (Int J. Canc (1988) 41:
651-656, incorporated by reference in its entirety herein). The
MLTR is an accepted model of an immune response, as shown for
example by Eini et al. (Biochemical Pharmacology Volume 98, Issue
1, 1 Nov. 2015, Pages 110-118, incorporated by reference in its
entirety herein), which uses the MLTR assay to evaluate immune
response modulation (e.g., elevation in proinflammatory cytokine
release); Wang et al., (Eur J Immunol. 1995 May; 25(5):1163-7,
incorporated by reference in its entirety herein) which assess the
effect of heat stable antigen on T-cell proliferation using a MLTR
assay; and Xiu et al. (J Mol Med (Berl). 2007 May; 85(5):511-21.
Epub 2007 Jan. 12, incorporated by reference in its entirety
herein), which use an MLTR assay to determine the effect of
superantigen staphylococcal enterotoxin A (SEA)-coated tumor cells
on tumor-specific T cell response.
[0428] According to some embodiments, tumor cell line or tumor cell
line variants may be tested for immunogenic potential by contacting
transfected tumor cells with mixed lymphocytes from peripheral
blood mononuclear cells, followed by measuring at least one of
cellular proliferation, cellular subset differentiation, cytokine
release profile, and tumor cell lysate.
[0429] According to some embodiments, mixed lymphocytes may be
obtained from peripheral blood mononuclear cells isolated by a
Ficoll-Paque gradient. Briefly, anticoagulant-treated blood may be
diluted in the range of 1:2 to 1:4 with PBS/EDTA to reduce
aggregation of erythrocytes. The diluted blood may then be layered
above a Ficoll-Paque solution in a centrifuge tube, without mixing.
The layered blood/Ficoll-Paque may be centrifuged for 40 minutes at
400.times.g between 18.degree. and 20.degree. C., without the use
of the centrifuge brake, resulting in the formation of blood
fractions comprising, from top to bottom, a first fraction
comprising blood plasma; a second fraction comprising mononuclear
cells; a third fraction comprising Ficoll-Paque media; and a fourth
fraction comprising granulocytes and erythrocytes.
[0430] The fractions may be further processed to isolate specific
fraction components. For example, to further process mononuclear
cells, the second fraction comprising mononuclear cells may be
carefully removed from the Ficoll-Paque gradient using a Pasteur
pipet. Alternatively, the second fraction may be removed directly
by puncturing the tube with a needle and directly withdrawing the
second fraction. The second fraction may then be washed and
centrifuged at 300.times.g, 18.degree. and 20.degree. C., three
times with PBS/EDTA, discarding the supernatant after each
round.
[0431] According to some embodiments, tumor cell line or tumor cell
line variants may be co-cultured with the PBMCs comprising
lymphocytes for seven days to allow for direct evaluation of
activation of anti-tumor response in the presence of
immunomodulators from the tumor cell line or tumor cell line
variants.
[0432] According to some embodiments, one parameter used for
measuring activation of lymphocytes may be cellular proliferation.
According to some embodiments, proliferation may be detected by
.sup.3H-thymidine incorporation. Briefly, approximately
5.times.10.sup.3 tumor cell line or tumor cell line variant cells
may be co-cultured with approximately 1.times.10.sup.6 mixed
lymphocytes in round bottomed 96-well plates. After three days of
culture, cells may be pulsed with 1 .mu.Ci of .sup.3H-thymidine for
18 hours. The cells may then be harvested onto filter mats, and
.sup.3H-thymidine incorporation may be measured using a
scintillation counter. Proliferation of tumor cell line or tumor
cell line variants compared to non-transfected tumor cell controls
may be measured. An increase, a decrease, or no change in
proliferation relative to controls, are possible outcomes.
[0433] According to some embodiments, another parameter for
measuring activation of lymphocytes may be the cytokine release
profile. For example, the number of responsive T cells in the mixed
lymphocyte population may be quantified by enzyme linked immunospot
(ELISpot) analysis of IFN-gamma and/or IL-2 production by PBMCs.
Briefly, PBMCs comprising mixed lymphocytes and a tumor cell line
or tumor cell line variant may be co-cultured between 3 and 7 days.
Co-cultured cells may then be harvested and incubated on ELISpot
plates pre-coated with anti-IFN-gamma and/or anti-IL-2 antibodies.
After 20 hours, cells may be removed by washing 2 times in
distilled water and two times in washing buffer. ELISpot plates may
then be contacted with biotinylated anti-IFN-gamma and/or anti-IL-2
antibodies and streptavidin alkaline phosphatase in blocking buffer
for 1-2 hours. After washing, plates may be contacted with alkaline
phosphatase substrate until dark spot emerge. Plates may then be
washed in tap water and air dried. Spots are then quantified
manually or by plate reader and compared to non-transfected tumor
cell line or tumor cell line variant control group.
[0434] According to some embodiments, another parameter for
measuring activation of lymphocytes may be by quantifying cellular
subset differentiation. For example, the differentiation of
CD45+/CD3+ T-lymphocytes to CD45+/CD3+/CD4+ helper T-lymphocytes,
CD45+/CD3+/CD8+ cytotoxic T-lymphocytes, and CD45+/CD3+/CD25+
activated T-lymphocytes may be quantified by flow cytometry
analysis.
[0435] According to some embodiments, another parameter for
measuring activation of lymphocytes may be by quantifying tumor
cell cytotoxicity. Cytotoxicity of tumor cells may be measured by
any number of established methods. For example, according to some
embodiments, an LDH-Cytotoxicity colorimetric assay kit (BioVision
Cat. # K311-400) may be used to measure cytotoxicity of tumor cells
by testing for lactate dehydrogenase (LDH) released from damaged
cells into the growth media. Briefly, 100 .mu.l of media from each
of the control group (comprising untransfected tumor cells), the
experimental group (comprising immune modulator transfected tumor
cells), and media alone may be pipetted into the wells of a 96 well
plate. 100 .mu.l of the LDH reaction mixture, comprising dye
solution and catalyst solution, may then be added to the wells of
the 96 well plate and incubated for 30 minutes at room temperature.
Then the samples may be measured for light absorbance at 490-500 nm
using a microliter plate reader.
Stimulation of Immune Cells
[0436] As described herein, the present disclosure provides an
allogeneic tumor cell vaccine comprising a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens, wherein the tumor cells are genetically
engineered to stimulate one or more immune cells (e.g., one or more
of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs)
or B lymphocytes). The stimulation of the immune cells can enhance
normal cellular functions, or initiate normal cell functions in an
abnormal cell.
[0437] According to some embodiments, stimulating the T-lymphocytes
comprises activating and/or expanding the T lymphocytes. According
to some embodiments, stimulating the NK cells comprises activating
and/or expanding the NK cells. According to some embodiments,
stimulating the DCs comprises activating and/or expanding the DCs.
According to some embodiments, stimulating the B lymphocytes
comprises activating and/or expanding the B lymphocytes.
[0438] According to some embodiments, the disclosure provides an
allogeneic tumor cell vaccine comprising a population of
proliferation incompetent tumor cells expressing one or more tumor
specific antigens that are effective to stimulate immune killer
cells. Immune killer cells that may be stimulated include, for
example, cytolytic T cells (CD8+ cells), memory CD8+ T cells, and
NK cells. According to one embodiment, the killer immune cells are
Natural Killer (NK) cells. According to one embodiment, the NK
cells are memory-like NK cells. According to one embodiment, the
killer immune cells are CD8+ T-cells. According to one embodiment,
the CD8+ T-cells are memory T cells. Accordingly, the present
invention also provides populations of cells resulting from
stimulation with the allogeneic tumor cell vaccines described
herein.
[0439] It is a feature of the present invention that, according to
some embodiments, the allogeneic tumor cell vaccines are effective
to stimulate more than one type of immune killer cell at the same
time, for example, more than one of cytolytic T cells (CD8+ cells),
memory CD8+ T cells and NK cells. According to some embodiments,
the allogeneic tumor cell vaccines are effective to stimulate both
CD8+ T cells and NK cells.
[0440] According to some embodiments, the allogeneic tumor cell
vaccine stimulates CD8+ T cells, where the expression of certain
exogenous immunomodulatory molecules, compared to other exogenous
stimulatory molecules, stimulate CD8+ T cells that kill more
effectively than others.
[0441] According to some embodiments, the allogeneic tumor cell
vaccine stimulates NK cells, where the expression of certain
exogenous immunomodulatory molecules, compared to other exogenous
stimulatory molecules, stimulate NK cells that kill more
effectively than others.
[0442] According to some embodiments, "stimulating the immune
killer cells" refers to expansion of the immune killer cell.
According to some embodiments, "stimulating the immune killer
cells" refers to activation of the immune killer cell. According to
some embodiments, "stimulating the immune killer cells" refers to
an increase in cytoxicity of the immune killer cell.
[0443] According to some embodiments, the allogeneic tumor cell
vaccines as described herein are sufficient to stimulate a
population of immune killer cells ex vivo. In other embodiments,
the allogeneic tumor cell vaccines as described herein are
sufficient to stimulate a population of immune killer cells in
vivo.
[0444] NK Cell Activation and Expansion
[0445] According to one embodiment, the allogeneic tumor cell
vaccines described herein are effective to activate NK cells.
[0446] According to one embodiment, the allogeneic tumor cell
vaccines described herein are effective to expand NK cells.
[0447] Degranulation/Cytotoxicity
[0448] The defining functional feature of NK cells remains their
intrinsic ability to conduct "natural killing" of cellular targets
without prior sensitization.
[0449] According to one embodiment, the allogeneic tumor cell
vaccines described herein are effective to activate and expand NK
cells, such that the NK cells that are activated and expanded by
the allogeneic tumor cell vaccines described herein exhibit higher
degranulation activity compared to control NK cells. For example,
degranulation activity can be estimated through the determination
of CD107a expression, for example by flow cytometry. CD107a surface
expression correlates closely with degranulation and release of
cytotoxic granules. Degranulation as measured by CD107a expression
correlates to cytotoxic activity of an effector cell, such as an NK
cell. The method of determining degranulation activity through the
determination of CD107a expression is well known to a person
skilled in the art. See, for example, Alter G, Malenfant J M,
Altfeld M. CD107a as a functional marker for the identification of
natural killer cell activity. J Immunol Methods. 2004; 294: 15-22,
the entire contents of which are incorporated herein by
reference.
[0450] According to one embodiment, the expanded and activated NK
cells, obtained following ex vivo or in vivo stimulation with the
allogeneic tumor cell vaccines of the invention, comprise at least
about 50%, about 60%, about 70%, about 80% or about 90% increased
cytotoxicity, e.g. as measured by degranulation activity, compared
to non expanded NK cells. According to one embodiment, the expanded
and activated NK cells comprise at least about 100% increased
cytotoxicity compared to non expanded NK cells. According to one
embodiment, the expanded and activated NK cells comprise at least
about 200% increased cytotoxicity compared to non expanded NK
cells. According to one embodiment, the expanded and activated NK
cells comprise at least about 300% increased cytotoxicity compared
to non-ex vivo expanded NK cells. According to one embodiment the
expanded and activated NK cells comprise at least about 400%
increased cytotoxicity compared to non-ex vivo expanded NK
cells.
[0451] According to one embodiment the expanded and activated NK
cells, obtained following ex vivo or in vivo stimulation with the
allogeneic tumor cell vaccines of the invention, comprise at least
about 50%, about 60%, about 70%, about 80% or about 90% increased
degranulation activity compared to non expanded NK cells. According
to one embodiment, the expanded and activated NK cells comprise at
least about 100% increased degranulation activity compared to non
expanded NK cells. According to one embodiment, the expanded and
activated NK cells comprise at least about 200% increased
degranulation activity compared to non expanded NK cells. According
to one embodiment, the expanded and activated NK cells comprise at
least about 300% increased degranulation activity compared to
non-ex vivo expanded NK cells. According to one embodiment, the
expanded and activated NK cells comprise at least about 400%
increased degranulation activity compared to non-ex vivo expanded
NK cells.
[0452] Markers of NK Cell Maturation and Activation
[0453] Human NK cells are phenotypically characterized by the
expression of CD56 and the absence of CD3 and can be further
subdivided into a CD56.sup.bright population and a CD56.sup.dim
population. The CD56.sup.bright population produces
immunoregulatory cytokines, including interferon-.gamma.
(IFN.gamma.), tumor necrosis factor-beta (TNF-.beta.), tumor
necrosis factor-.alpha. (TNF-.alpha.), granulocyte
macrophage-colony stimulating factor (GMCSF), IL-10, and IL-13 (4).
The CD56.sup.dim subset is the terminally differentiated successor
of the CD56.sup.bright population and is primarily responsible for
exerting cytolytic functions. However, CD56.sup.dim NK cells can
produce cytokines, specifically IFN.gamma., after cell triggering
via NKp46 of NKp30 activating receptors or after stimulation with
combinations of IL-2, IL-12, and IL-15.
[0454] According to one embodiment, various markers of NK cell
maturation and/or activation can be detected using, e.g. flow
cytometric methods. For example, a classical marker of NK cells is
the activating receptor Fc.gamma.RIII, also called CD16.
[0455] The activation of NK cells leads to the release of cytotoxic
granules containing perforin and various granzymes and to cytokine
production, most prominently interferon-.gamma. (IFN.gamma.). In
addition, the expression at the cell surface of death-inducing
ligands belonging to the tumor necrosis factor (TNF) family, such
as Fas ligand (FasL) and TNF-related apoptosis-inducing ligand
(TRAIL), also drives the activation of the caspase enzymatic
cascade through the binding to the death receptors (DRs), namely,
Fas, DR4 (TRAIL-RI), and DR5 (TRAIL-RII), on target cells.
[0456] According to one embodiment, the allogeneic tumor cell
vaccines described herein upregulate at least one NK cell
activating receptor (e.g., an activating receptor listed in Table
3) by at least about 20%, about 30%, about 40%, about 50%, about
60%, about 70%, about 80%, about 90%, about 100%, about 150%, about
200%, about 300% or more. According to one embodiment, the
allogeneic tumor cell vaccines described herein upregulate at least
one NK cell activating receptor by at least about 75%, i.e., at
least about 76%, at least about 77%, at least about 78%, at least
about 79%, at least about 80%, at least about 81%, at least about
82%, at least about 83%, at least about 84%, at least about 85%, at
least about 86%, at least about 87%, at least about 88%, at least
about 89%, at least about 90%, at least about 91%, at least about
92%, at least about 93%, at least about 94%, at least about 95%, at
least about 96%, at least about 97%, at least about 98%, at least
about 99%, at least about 100%, at least about 110%, at least about
120%, at least about 130%, at least about 140%, at least about
150%, at least about 160%, at least about 170%, at least about
180%, at least about 190%, at least about 200%. According to one
embodiment, the allogeneic tumor cell vaccines described herein
upregulate at least one NK cell activating receptor by at least
about 100%. According to one embodiment, the allogeneic tumor cell
vaccines described herein upregulate at least one NK cell
activating receptor by at least about 200%.
[0457] According to another embodiment, the allogeneic tumor cell
vaccines described herein downregulate expression of at least one
NK cell receptor, such as an inhibitory receptor or a chemokine
receptor (e.g. CCR7). For example, certain NK cell inhibitory
receptors are called KIRs (Killing Inhibitory Receptors or CD158).
Non-limiting examples of inhibitory receptors are inhibitory killer
immunoglobulin-like receptors (KIRs), GL183, KIR2DL 1, Lir-1, NKB1,
and NKG2A.
[0458] According to one embodiment, the allogeneic tumor cell
vaccines described herein downregulate at least one NK cell
inhibitory receptor (e.g., an inhibitory receptor listed in Table 4
below) by at least about 20%, at least about 30%, at least about
40%, at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90%, at least about 100%, at least
about 110%, 120%, at least about 130%, about 140%, at least about
150%, at least about 160%, at least about 170%, at least about
180%, at least about 190%, at least about 200%, at least about
220%, at least about 230%, at least about 240%, at least about
250%, at least about 260%, at least about 270%, at least about
280%, at least about 290%, at least about 300% or more. According
to one embodiment, the allogeneic tumor cell vaccines described
herein downregulate at least one NK cell inhibitory receptor by at
least about 75%. According to one embodiment, the allogeneic tumor
cell vaccines described herein downregulate at least one NK cell
inhibitory receptor by at least about 100%. According to one
embodiment, the allogeneic tumor cell vaccines described herein
downregulate at least one NK cell inhibitory receptor by at least
about 200%.
[0459] The change in receptor expression can be calculated by mean
fluorescence intensity (MFI) ratios:
MFI.sub.dayX/MFI.sub.day0
where x is the number of days of expansion of the NK cell.
[0460] When the MFI for day X samples is higher than for day 0, the
MFI ratio will be higher than 1, which indicates the relative
extent of upregulation in that receptor. Thus, a MFI ratio of e.g.
1.5 would mean a 50% upregulation of a specific receptor. The
calculation of MFI ratios is well known to persons skilled in the
art.
[0461] Various NK cell activating or inhibitory receptors are shown
below in Table 4. Bold type indicates family.
TABLE-US-00005 TABLE 4 Receptor Family Species
Activating/Inhibitory CD16 H Act KIR H Act/Inhib KIR2DL1 Inhib
KIR2DL2/3 Inhib KIR2DL4 Act KIR2DL5 Inhib KIR3DL1 Inhib KIR3DL2
Inhib KIR2DS1 Act KIR2DS2 Act KIR2DS3 Act KIR2DS4 Act KIR2DS5 Act
KIR3DS1 Act CD94-NKG2 H/M Act/Inhib NKG2A Inhib NKG2C Act NKG2E Act
NKG2D H/M Act NCRs H/M Act NKp30 Act NKp44 Act NKp46 Act NKp80 Act
LILR H/M Inhib 2B4 H/M Act/Inhib KLRG1 H/M Inhib DNAM-1 H/M Act
Abbreviations in Table 4: ACT, activation; BAT-3, HLA-B-associated
transcript 3; H, human; HA, hemagglutinin; HLA, human leukocyte
antigen; INHIB, inhibitory; KIR, killer immunoglobulin-like
receptor; KLRG1, killer cell lectin-like receptor G1; LILR,
leukocyte immunoglobulin-like receptor; M, mouse; MHC, major
histocompatibility complex; MULT-1, mouse UL16-binding-like
transcript-1; NCR, natural cytotoxicity receptor; NK, natural
killer; PVR, polio virus receptor; RAE-1, retinoic acid early
transcript-1.
[0462] CD8+ T Cell Activation and Expansion
[0463] According to one embodiment, the allogeneic tumor cell
vaccines described herein are effective to activate CD8+ T-cells.
According to one embodiment, the allogeneic tumor cell vaccines
described herein are effective to expand CD8+ T-cells. According to
one embodiment, the allogeneic tumor cell vaccines described herein
are effective to activate and expand CD8+ T-cells.
[0464] T cell activation and expansion can be measured by various
assays as described herein. For example, T cell activities that may
be measured include the induction of proliferation of T cells, the
induction of signaling in T cells, the induction of expression of
activation markers in T cells, the induction of cytokine secretion
by T cells, and the cytotoxic activity of T cells. For example, in
certain embodiments, CD8+ T cell activation is measured by a
proliferation assay.
[0465] Cytokine Secretion
[0466] The activation of CD8+ T-cells by an allogeneic tumor cell
vaccines of the invention may be assessed or measured by
determining secretion of cytokines, such as gamma interferon
(IFN.gamma.), tumor necrosis factor alpha (TNFa), interleukin-12
(IL-12) or interleukin 2 (IL-2). according to some embodiments,
ELISA is used to determine cytokine secretion, for example
secretion of gamma interferon (IFN.gamma.), tumor necrosis factor
alpha (TNFa), interleukin-12 (IL-12) or interleukin 2 (IL-2). The
ELISPOT (enzyme-linked immunospot) technique may be used to detect
T cells that secrete a given cytokine (e.g., gamma interferon
(IFN.gamma.)) in response to stimulation with the engineered tumor
cells described herein. T cells are cultured with engineered tumor
cells in wells which have been coated with anti-IFN.gamma.
antibodies. The secreted IFN.gamma. is captured by the coated
antibody and then revealed with a second antibody coupled to a
chromogenic substrate. Thus, locally secreted cytokine molecules
form spots, with each spot corresponding to one
IFN.gamma.-secreting cell. The number of spots allows one to
determine the frequency of IFN.gamma.-secreting cells in the
analyzed sample. The ELISPOT assay has also been described for the
detection of tumor necrosis factor alpha, interleukin-4 (IL-4),
IL-5, IL-6, IL-10, IL-12, granulocyte-macrophage colony-stimulating
factor, and granzyme B-secreting lymphocytes (Klinman D, Nutman T.
Current protocols in immunology. New York, N.Y: John Wiley &
Sons, Inc.; 1994. pp. 6.19.1-6.19.8, incorporated by reference in
its entirety herein).
[0467] Flow cytometric analyses of intracellular cytokines may be
used to measure the cytokine content in culture supernatants, but
provides no information on the number of T cells that actually
secrete the cytokine. When T cells are treated with inhibitors of
secretion such as monensin or brefeldin A, they accumulate
cytokines within their cytoplasm upon activation (e.g. with an
engineered erythroid cell of the present invention). After fixation
and permeabilization of the lymphocytes, intracellular cytokines
can be quantified by cytometry. This technique allows the
determination of the cytokines produced, the type of cells that
produce these cytokines, and the quantity of cytokine produced per
cell.
[0468] Cytotoxicity
[0469] The activation of CD8+ T-cells by an allogeneic tumor cell
vaccines of the invention may be assessed by assaying the cytotoxic
activity of the CD8+ T-cells.
[0470] The cytotoxic activity of T cells may be assessed by any
suitable technique known to those of skill in the art. For example,
a sample comprising T cells that have been exposed to the
engineered erythroid cells according to the invention can be
assayed for cytotoxic activity after an appropriate period of time,
in a standard cytotoxic assay. Such assays may include, but are not
limited to, the chromium release CTL assay and the Alamar Blue.TM.
fluorescence assay known in the art.
[0471] Proliferation/Expansion
[0472] The ability of the allogeneic tumor cell vaccines of the
invention to expand T cells can be evaluated using CFSE staining.
To compare the initial rate of cell expansion, the cells are
subject to CFSE staining to determine how well allogeneic tumor
cell vaccines induced the proliferation of T cells. CFSE staining
provides a much more quantitative endpoint and allows simultaneous
phenotyping of the expanded cells. Every day after stimulation, an
aliquot of cells is removed from each culture and analyzed by flow
cytometry. CFSE staining makes cells highly fluorescent. Upon cell
division, the fluorescence is halved and thus the more times a cell
divides the less fluorescent it becomes. The ability of allogeneic
tumor cell vaccines to induce T cell proliferation is quantitated
by measuring the number of cells that divided once, twice, three
times and so on. The allogeneic tumor cell vaccines that induce the
greatest number of cell divisions at a particular time point is
deemed as the most potent expander.
[0473] To determine how well these allogeneic tumor cell vaccines
promote long-term growth of T cells, cell growth curves can be
generated. These experiments are set up as the foregoing CFSE
experiments, but no CFSE is used. Every 2-3 days of culture, T
cells are removed from the respective cultures and counted using a
Coulter counter which measures how many cells are present and the
mean volume of the cells. The mean cell volume is the best
predicator of when to restimulate the cells. In general, when T
cells are properly stimulated they triple their cell volume. When
this volume is reduced to more than about half of the initial
blast, it may be necessary to restimulate the T cells to maintain a
log linear expansion (Levine et al., 1996, Science 272:1939-1943;
Levine et al., 1997, J. Immunol. 159:5921-5930). The time it takes
each engineered erythroid cell to induce 20 population doublings is
calculated. The relative differences of each allogeneic tumor cell
vaccine to induce this level of T cell expansion is one criteria on
which a particular allogeneic tumor cell vaccines is assessed.
[0474] In addition, the phenotypes of the cells expanded by each
allogeneic tumor cell vaccine can be characterized to determine
whether a particular subset is preferentially expanded. Prior to
each restimulation, a phenotype analysis of the expanding T cell
populations is performed to define the differentiation state of the
expanded T cells using the CD27 and CD28 definitions proposed by
Appay et al. (2002, Nature Med. 8, 379-385, incorporated by
reference in its entirety herein) and CCR7 definitions proposed by
Sallusto et al. (1999, Nature 401:708-712, incorporated by
reference in its entirety herein). Perforin and Granzyme B
intracellular staining can be used to perform a gross measure to
estimate cytolytic potential.
[0475] Apoptosis Markers
[0476] According to certain embodiments of the present invention,
stimulation, activation, and expansion of T cells using the
allogeneic tumor cell vaccines as described herein enhances
expression of certain key molecules in T cells that protect against
apoptosis or otherwise prolong survival in vivo or in vitro.
Apoptosis usually results from induction of a specific signal in
the T cell. Thus, the engineered tumor cells of the invention may
provide for protecting a T cell from cell death resulting from
stimulation of the T cell. Therefore, also included in the present
invention is enhanced T cell growth by protection from premature
death or from absence or depletion of recognized T cell growth
markers, such as Bcl-xL, growth factors, cytokines, or lymphokines
normally necessary for T cell survival, as well as from Fas or
Tumor Necrosis Factor Receptor (TNFR) cross-linking or by exposure
to certain hormones or stress.
III. Methods of Making
[0477] Various methods of making allogeneic tumor cell vaccines are
contemplated by the present disclosure.
[0478] According to some embodiments, the disclosure features an
allogeneic tumor cell vaccine comprising a population of
proliferation incompetent tumor cells, comprising at least three
stably expressed exogenous immunomodulatory molecule, produced by a
process comprising providing an allogeneic parental tumor cell
line; introducing an exogenous nucleic acid encoding the at least
one exogenous immunomodulatory molecule into a tumor cell;
generating tumor cell line variants by selecting for tumor cell
clones that stably express an immunogenic amount of the exogenous
immunomodulatory molecule; and selecting in a mixed lymphocyte
tumor cell reaction clonally derived cell line variants by one or
more of the following parameters selected from cellular
proliferation, cellular subset differentiation, cytokine release
profile, and tumor cell lysis; wherein the selected clonally
derived cell line variant is effective to stimulate activation of
one or more of T-lymphocytes, natural killer (NK) cells, dendritic
cells (DCs) or B lymphocytes. According to one embodiment, the
tumor cells are rendered proliferation incompetent by irradiation.
According to one embodiment, the exogenous nucleic acid comprises
DNA or RNA. According to one embodiment, the introducing step
comprises viral transduction. According to one embodiment, the
introducing step comprises electroporation. According to one
embodiment, the introducing step comprises utilizing one or more of
liposome mediated transfer, adenovirus, adeno-associated virus,
herpes virus, a retroviral based vector, lipofection, and a
lentiviral vector. According to one embodiment, the introducing
step comprises introducing the exogenous nucleic acid by
transfection of a lentiviral vector.
Lentiviral Vectors
[0479] The described invention provides nucleic acid constructs
that encode two or more immunomodulators that can be expressed in
prokaryotic and eukaryotic cells. For example, the described
invention provides expression vectors (e.g., DNA- or RNA-based
vectors) containing nucleotide sequences that encode two or more
immunomodulators. In addition, the described invention provides
methods for making the vectors described herein, as well as methods
for introducing the vectors into appropriate host cells for
expression of the encoded polypeptides. In general, the methods
provided herein include constructing nucleic acid sequences
encoding two or more immunomodulators, and cloning the sequences
into an expression vector. The expression vector can be introduced
into host cells or incorporated into virus particles, either of
which can be administered to a subject to, for example, treat
cancer.
[0480] cDNA or DNA sequences encoding two or more immunomodulators
can be obtained (and, if desired, modified) using conventional DNA
cloning and mutagenesis methods, DNA amplification methods, and/or
synthetic methods. In general, a sequence encoding two or more
immunomodulators can be inserted into a cloning vector for genetic
modification and replication purposes prior to expression. Each
coding sequence can be operably linked to a regulatory element,
such as a promoter, for purposes of expressing the encoded protein
in suitable host cells in vitro and in vivo.
[0481] Expression vectors can be introduced into host cells for
producing secreted immunomodulators. There are a variety of
techniques available for introducing nucleic acids into viable
cells. Techniques suitable for the transfer of nucleic acid into
mammalian cells in vitro include the use of liposomes,
electroporation, microinjection, cell fusion, polymer-based
systems, DEAE-dextran, viral transduction, the calcium phosphate
precipitation method, etc. For in vivo gene transfer, a number of
techniques and reagents may also be used, including liposomes; and
natural polymer-based delivery vehicles, such as chitosan and
gelatin; viral vectors are also suitable for in vivo transduction.
In some situations it is desirable to provide a targeting agent,
such as an antibody or ligand specific for a cell surface membrane
protein. Where liposomes are employed, proteins which bind to a
cell surface membrane protein associated with endocytosis may be
used for targeting and/or to facilitate uptake, e.g., capsid
proteins or fragments thereof tropic for a particular cell type,
antibodies for proteins which undergo internalization in cycling,
proteins that target intracellular localization and enhance
intracellular half-life. The technique of receptor-mediated
endocytosis is described, for example, by Wu et al., J. Biol. Chem.
262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci.
USA 87, 3410-3414 (1990).
[0482] Where appropriate, gene delivery agents such as, e.g.,
integration sequences can also be employed. Numerous integration
sequences are known in the art (see, e.g., Nunes-Duby etal.,
Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacteriol.,
165:341-357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et
al., TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm.
Toxicol., 43:413-439, 2003). These include recombinases and
transposases. Examples include Cre (Sternberg and Hamilton, J. Mol.
Biol., 150:467-486, 1981), lambda (Nash, Nature, 247, 543-545,
1974), FIp (Broach, et al., Cell, 29:227-234, 1982), R (Matsuzaki,
et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see, e.g.,
Groth etal., J. Mol. Biol. 335:667-678, 2004), sleeping beauty,
transposases of the mariner family (Plasterk et al., supra), and
components for integrating viruses such as AAV, retroviruses, and
antiviruses having components that provide for virus integration
such as the LTR sequences of retroviruses or lentivirus and the ITR
sequences of AAV (Kootstra et al., Ann. Rev. Pharm. Toxicol.,
43:413-439, 2003).
[0483] Cells may be cultured in vitro or genetically engineered,
for example. Host cells can be obtained from normal or affected
subjects, including healthy humans, cancer patients, private
laboratory deposits, public culture collections such as the
American Type Culture Collection, or from commercial suppliers.
[0484] Cells that can be used for production and secretion of two
or more immunomodulators in vivo include, without limitation,
epithelial cells, endothelial cells, keratinocytes, fibroblasts,
muscle cells, hepatocytes; blood cells such as T lymphocytes, B
lymphocytes, monocytes, macrophages, neutrophils, eosinophils,
megakaryocytes, or granulocytes, various stem or progenitor cells,
such as hematopoietic stem or progenitor cells (e.g., as obtained
from bone marrow), umbilical cord blood, peripheral blood, fetal
liver, etc., and tumor cells (e.g., human tumor cells). The choice
of cell type depends on the type of tumor or infectious disease
being treated or prevented, and can be determined by one of skill
in the art.
[0485] Different host cells have characteristic and specific
mechanisms for post-translational processing and modification of
proteins. A host cell may be chosen which modifies and processes
the expressed gene products in a specific fashion similar to the
way the recipient processes its heat shock proteins (hsps).
[0486] According to some embodiments, an expression construct as
provided herein can be introduced into an antigenic cell. As used
herein, antigenic cells can include preneoplastic cells that are
infected with a cancer-causing infectious agent, such as a virus,
but that are not yet neoplastic, or antigenic cells that have been
exposed to a mutagen or cancer-causing agent, such as a
DNA-damaging agent or radiation, for example. Other cells that can
be used are preneoplastic cells that are in transition from a
normal to a neoplastic form as characterized by morphology or
physiological or biochemical function.
[0487] Typically, the cancer cells and preneoplastic cells used in
the methods provided herein are of mammalian origin. According to
some embodiments, cancer cells (e.g., human tumor cells) can be
used in the methods described herein. Cell lines derived from a
preneoplastic lesion, cancer tissue, or cancer cells also can be
used. Cancer tissues, cancer cells, cells infected with a
cancer-causing agent, other preneoplastic cells, and cell lines of
human origin can be used. According to some embodiments, a cancer
cell can be from an established tumor cell line or tumor cell line
variant such as, without limitation, an established non-small cell
lung carcinoma (NSCLC), bladder cancer, melanoma, ovarian cancer,
renal cell carcinoma, prostate carcinoma, sarcoma, breast
carcinoma, squamous cell carcinoma, head and neck carcinoma,
hepatocellular carcinoma, pancreatic carcinoma, or colon carcinoma
cell line.
[0488] Parent cell lines are described supra.
[0489] Further, according to some embodiments, the allogeneic tumor
cell vaccines provide for an adjuvant effect that further allows
the immune system of a patient, when used in the various methods
described herein, to be activated against a disease of
interest.
[0490] Both prokaryotic and eukaryotic vectors can be used for
expression of the two or more immunomodulators in the methods
provided herein. Prokaryotic vectors include constructs based on E.
coli sequences (see, e.g., Makrides, Microbiol Rev 1996,
60:512-538). Non-limiting examples of regulatory regions that can
be used for expression in E. coli include lac, trp, 1pp, phoA,
recA, tac, T3, T7 and lamda P.sub.L. Non-limiting examples of
prokaryotic expression vectors may include the Agt vector series
such as .lamda.gt11 (Huynh et al., in "DNA Cloning Techniques, Vol.
I: A Practical Approach," 1984, (D. Glover, ed.), pp. 49-78, IRL
Press, Oxford), and the pET vector series (Studier et al., Methods
Enzymol 1990, 185:60-89).
[0491] A variety of regulatory regions can be used for expression
of the allogeneic tumor vaccines in mammalian host cells. For
example, the SV40 early and late promoters, the cytomegalovirus
(CMV) immediate early promoter, and the Rous sarcoma virus long
terminal repeat (RSV-LTR) promoter can be used. Inducible promoters
that may be useful in mammalian cells include, without limitation,
promoters associated with the metallothionein II gene, mouse
mammary tumor virus glucocorticoid responsive long terminal repeats
(MMTV-LTR), the n-interferon gene, and the hsp70 gene (see,
Williams et al., Cancer Res 1989, 49:2735-42; and Taylor et al.,
Mol Cell Biol 1990, 10:165-75). Heat shock promoters or stress
promoters also may be advantageous for driving expression of the
fusion proteins in recombinant host cells.
[0492] Animal regulatory regions that exhibit tissue specificity
and have been utilized in transgenic animals also can be used in
tumor cells of a particular tissue type: the elastase I gene
control region that is active in pancreatic acinar cells (Swift et
al., Cell 1984, 38:639-646; Ornitz et al., Cold Spring Harbor Symp
Quant Biol 1986, 50:399-409; and MacDonald, Hepatology 1987,
7:425-515); the insulin gene control region that is active in
pancreatic beta cells (Hanahan, Nature 1985, 315:115-122), the
immunoglobulin gene control region that is active in lymphoid cells
(Grosschedl etal., Cell 1984, 38:647-658; Adames etal., Nature
1985, 318:533-538; and Alexander et al., Mol Cell Biol 1987,
7:1436-1444), the mouse mammary tumor virus control region that is
active in testicular, breast, lymphoid and mast cells (Leder et
al., Cell 1986, 45:485-495), the albumin gene control region that
is active in liver (Pinkert et al., Genes Devel, 1987, 1:268-276),
the alpha-fetoprotein gene control region that is active in liver
(Krumlauf et al., Mol Cell Biol 1985, 5:1639-1648; and Hammer et
al., Science 1987, 235:53-58); the alpha 1-antitrypsin gene control
region that is active in liver (Kelsey et al., Genes Devel 1987,
1:161-171), the beta-globin gene control region that is active in
myeloid cells (Mogram et al., Nature 1985, 315:338-340; and Kollias
et al., Cell 1986, 46:89-94); the myelin basic protein gene control
region that is active in oligodendrocyte cells in the brain
(Readhead et al., Cell 1987, 48:703-712); the myosin light chain-2
gene control region that is active in skeletal muscle (Sani, Nature
1985, 314:283-286), and the gonadotropic releasing hormone gene
control region that is active in the hypothalamus (Mason et al.,
Science 1986, 234:1372-1378).
[0493] An expression vector also can include transcription enhancer
elements, such as those found in SV40 virus, Hepatitis B virus,
cytomegalovirus, immunoglobulin genes, metallothionein, and
.beta.-actin (see, Bittner et al., Meth Enzymol 1987, 153:516-544;
and Gorman, Curr Op Biotechnol 1990, 1:36-47). In addition, an
expression vector can contain sequences that permit maintenance and
replication of the vector in more than one type of host cell, or
integration of the vector into the host chromosome. Such sequences
include, without limitation, to replication origins, autonomously
replicating sequences (ARS), centromere DNA, and telomere DNA.
[0494] In addition, an expression vector can contain one or more
selectable or screenable marker genes for initially isolating,
identifying, or tracking host cells that contain DNA encoding the
immunogenic proteins as described herein. For long term, high yield
production of gp96-Ig and T cell costimulatory fusion proteins,
stable expression in mammalian cells can be useful. A number of
selection systems can be used for mammalian cells. For example, the
Herpes simplex virus thymidine kinase (Wigler et al., Cell 1977,
11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalski
and Szybalski, Proc Natl Acad Sci USA 1962, 48:2026), and adenine
phosphoribosyltransferase (Lowy et al., Cell 1980, 22:817) genes
can be employed in tk.sup.-, hgprf.sup.-, or aprf.sup.- cells,
respectively. In addition, antimetabolite resistance can be used as
the basis of selection for dihydrofolate reductase (dhfr), which
confers resistance to methotrexate (Wigler et al., Proc Natl Acad
Sci USA 1980, 77:3567; O'Hare et al., Proc Natl Acad Sci USA 1981,
78:1527); gpt, which confers resistance to mycophenolic acid
(Mulligan and Berg, Proc Natl Acad Sci USA 1981, 78:2072); neomycin
phosphotransferase (neo), which confers resistance to the
aminoglycoside G-418 (Colberre-Garapin et al., J Mol Biol 1981,
150:1); and hygromycin phosphotransferase (hyg), which confers
resistance to hygromycin (Santerre et al., Gene 1984, 30:147).
Other selectable markers such as histidinol and Zeocin.TM. also can
be used.
[0495] A number of viral-based expression systems also can be used
with mammalian cells to produce the allogeneic tumor cell vaccines.
Vectors using DNA virus backbones have been derived from simian
virus 40 (SV40) (Hamer et al., Cell 1979, 17:725), adenovirus (Van
Doren et al., Mol Cell Biol 1984, 4:1653), adeno-associated virus
(McLaughlin et al., J Virol 1988, 62:1963), and bovine papillomas
virus (Zinn et al., Proc Natl Acad Sci USA 1982, 79:4897). When an
adenovirus is used as an expression vector, the donor DNA sequence
may be ligated to an adenovirus transcription/translation control
complex, e.g., the late promoter and tripartite leader sequence.
This fusion gene may then be inserted in the adenovirus genome by
in vitro or in vivo recombination. Insertion in a non-essential
region of the viral genome (e.g., region E1 or E3) can result in a
recombinant virus that is viable and capable of expressing
heterologous products in infected hosts. (See, e.g., Logan and
Shenk, Proc Natl Acad Sci USA 1984, 81:3655-3659).
[0496] Bovine papillomavirus (BPV) can infect many higher
vertebrates, including man, and its DNA replicates as an episome. A
number of shuttle vectors have been developed for recombinant gene
expression, which exist as stable, multicopy (20-300 copies/cell)
extrachromosomal elements in mammalian cells. Typically, these
vectors contain a segment of BPV DNA (the entire genome or a 69%
transforming fragment), a promoter with a broad host range, a
polyadenylation signal, splice signals, a selectable marker, and
"poisonless" plasmid sequences that allow the vector to be
propagated in E. coli. Following construction and amplification in
bacteria, the expression gene constructs are transfected into
cultured mammalian cells by, for example, calcium phosphate
coprecipitation. For those host cells that do not manifest a
transformed phenotype, selection of transformants is achieved by
use of a dominant selectable marker, such as histidinol and G418
resistance.
[0497] Alternatively, the vaccinia 7.5K promoter can be used. (See,
e.g., Mackett et al., Proc Natl Acad Sci USA 1982, 79:7415-7419;
Mackett et al., J Virol 1984, 49:857-864; and Panicali et al., Proc
Natl Acad Sci USA 1982, 79:4927-4931.) In cases where a human host
cell is used, vectors based on the Epstein-Barr virus (EBV) origin
(OriP) and EBV nuclear antigen 1 (EBNA-1; a trans-acting
replication factor) can be used. Such vectors can be used with a
broad range of human host cells, e.g., EBO-pCD (Spickofsky et al.,
DNA Prot Eng Tech 1990, 2:14-18); pDR2 and .lamda.DR2 (available
from Clontech Laboratories).
[0498] Allogeneic tumor cell vaccines also can be made with
retrovirus-based expression systems. Retroviruses, such as Moloney
murine leukemia virus, can be used since most of the viral gene
sequence can be removed and replaced with exogenous coding sequence
while the missing viral functions can be supplied in trans. In
contrast to transfection, retroviruses can efficiently infect and
transfer genes to a wide range of cell types including, for
example, primary hematopoietic cells. Moreover, the host range for
infection by a retroviral vector can be manipulated by the choice
of envelope used for vector packaging.
[0499] For example, a retroviral vector can comprise a 5' long
terminal repeat (LTR), a 3' LTR, a packaging signal, a bacterial
origin of replication, and a selectable marker. The gp96-Ig fusion
protein coding sequence, for example, can be inserted into a
position between the 5' LTR and 3' LTR, such that transcription
from the 5' LTR promoter transcribes the cloned DNA. The 5' LTR
contains a promoter (e.g., an LTR promoter), an R region, a U5
region, and a primer binding site, in that order. Nucleotide
sequences of these LTR elements are well known in the art. A
heterologous promoter as well as multiple drug selection markers
also can be included in the expression vector to facilitate
selection of infected cells. See, McLauchlin et al., Prog Nucleic
Acid Res Mol Biol 1990, 38:91-135; Morgenstern et al., Nucleic Acid
Res 1990, 18:3587-3596; Choulika et al., J Virol 1996,
70:1792-1798; Boesen et al., Biotherapy 1994, 6:291-302; Salmons
and Gunzberg, Human Gene Ther 1993, 4:129-141; and Grossman and
Wilson, Curr Opin Genet Devel 1993, 3:110-114.
[0500] Any of the cloning and expression vectors described herein
may be synthesized and assembled from known DNA sequences using
techniques that are known in the art. The regulatory regions and
enhancer elements can be of a variety of origins, both natural and
synthetic. Some vectors and host cells may be obtained
commercially. Non-limiting examples of useful vectors are described
in Appendix 5 of Current Protocols in Molecular Biology, 1988, ed.
Ausubel et al., Greene Publish. Assoc. & Wiley Interscience,
which is incorporated herein by reference; and the catalogs of
commercial suppliers such as Clontech Laboratories, Stratagene
Inc., and Invitrogen, Inc.
Recombinant Immunomodulators
[0501] According to some embodiments, two or more immunomodulators
may be cloned into two or more plasmid constructs for transfection
(via, e.g., lipids, calcium phosphate, cationic polymers,
DEAE-dextran, activated dendrimers, magnetic beads,
electroporation, biolistic technology, microinjection,
laserfection/optoinjection) or transduction (via, e.g., retrovirus,
lentivirus, adenovirus, adeno-associated virus) into cells of tumor
cell line or tumor cell line variants. According to some
embodiments, recombinant DNA encoding each immune modulator protein
may be cloned into a lentiviral vector plasmid for integration into
the genome of cells of tumor cell line or tumor cell line variants.
According to some embodiments, recombinant DNA encoding the immune
modulator protein may be cloned into a plasmid DNA construct
encoding a selectable trait, such as an antibiotic resistance gene.
According to some embodiments, recombinant DNA encoding the immune
modulator protein may be cloned into a plasmid construct that is
adapted to stably express each recombinant protein in the cells of
the tumor cell line or tumor cell line variant. According to some
embodiments, the transfected or transduced tumor cells may be
clonally expanded to achieve a cell line variant with a homogenous
site of integration of the recombinant DNA encoding each immune
modulator protein into the genome of the cells of the tumor cell
line or tumor cell line variant.
Lentiviral Constructs
[0502] According to some embodiments, the DNA sequences coding for
exogenous immunomodulatory molecules may be cloned into a
lentiviral vector for transduction into mammalian cells. According
to some embodiments, the lentiviral system may comprise a
lentiviral transfer plasmid encoding the two or more immune
modulator sequences, packaging plasmids encoding the GAG, POL, TAT,
and REV sequences, and an envelope plasmid encoding the ENV
sequences. According to some embodiments, the lentiviral transfer
plasmid uses a viral LTR promoter for gene expression. According to
some embodiments, the lentiviral transfer plasmid uses a hybrid
promoter, or other specialized promoter. According to some
embodiments, the promoter of the lentiviral transfer plasmid is
selected to express the two or more immune modulator sequences at a
desired level relative to other immunomodulatory sequences.
According to some embodiments, the relative level is measured on
the level of transcription as mRNA transcripts. According to some
embodiments, the relative level is measured on the level of
translation as protein expression.
Multicistronic Plasmid Constructs
[0503] According to some embodiments, one or more immune modulator
sequence may be cloned in a multicistronic vector for co-expression
of one immune modulator with a second immune modulator or other
recombinant sequence. According to some embodiments, an immune
modulator sequence may be cloned into a plasmid comprising an IRES
element to promote translation of two or more proteins from a
single transcript. According to some embodiments, one or more
immune modulator sequences is cloned into a multicistronic vector
comprising sequences for a self cleaving 2A peptide to produce two
or more exogenous immunomodulatory molecules from a single
transcript.
Genetic Introduction of Exogenous Immunomodulatory Molecules
[0504] According to some embodiments, plasmid constructs comprising
the recombinant immune modulator sequences may be transfected or
transduced into tumor cell line or tumor cell line variants.
[0505] According to some embodiments, up to 25 immunomodulators
(i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 2, 22, 23, 24 or 25) may be cloned into 10 separate
vectors for transduction into mammalian cells. According to some
embodiments, up to 25 immunomodulators (i.e., 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
or 25) may be cloned into 11 separate vectors for transduction into
mammalian cells. According to some embodiments, up to 25
immunomodulators (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) may be cloned
into 12 separate vectors for transduction into mammalian cells.
According to some embodiments, 14 or more immunomodulators (i.e.,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more) may be
cloned into 10 separate vectors for transduction into mammalian
cells. According to some embodiments, 14 or more immunomodulators
(i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more) may
be cloned into 11 separate vectors for transduction into mammalian
cells. According to some embodiments, 14 or more immunomodulators
(i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more) may
be cloned into 12 separate vectors for transduction into mammalian
cells. According to some embodiments, 14 or more immunomodulators
(i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more) may
be cloned into 13 separate vectors for transduction into mammalian
cells. According to some embodiments, 14 or more immunomodulators
(i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more) may
be cloned into 14 separate vectors for transduction into mammalian
cells. According to some embodiments, 14 or more immunomodulators
(i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more) may
be cloned into 14 or more separate vectors for transduction into
mammalian cells.
[0506] According to some embodiments, the vector constructs further
comprise one or more tags, as described herein.
Lentiviral System
[0507] According to some embodiments, the lentiviral system may be
employed where the transfer vector with immune modulator sequences,
an envelope vector, and a packaging vector are each transfected
into host cells for virus production. According to some
embodiments, the lentiviral vectors may be transfected into 293T
cells by any of calcium phosphate precipitation transfection, lipid
based transfection, or electroporation, and incubated overnight.
For embodiments where the immune modulator sequence may be
accompanied by a fluorescence reporter, inspection of the 293T
cells for florescence may be checked after overnight incubation.
The culture medium of the 293T cells comprising virus particles may
be harvested 2 or 3 times every 8-12 hours and centrifuged to
sediment detached cells and debris. The culture medium may then be
used directly, frozen or concentrated as needed.
[0508] Tumor cell line or tumor cell line variants may be grown to
a confluency of about 70% under standard tissue culture conditions.
The cells may then be treated with hexadimethrine bromide (to
enhance transduction of cells) and lentiviral particles comprising
recombinant constructs in fresh media, and incubated for 18-20
hours followed by a media change.
Lipid Based Transfection
[0509] According to some embodiments, cells of tumor cell line or
tumor cell line variants may be transfected with immune modulator
sequences using a lipid based transfection method. According to
some embodiments, established lipid based transfection reagents,
such as LIPOFECTAMINE, may be used. Tumor cell line or tumor cell
line variants may be grown to about 70-90% confluence in a tissue
culture vessel. Appropriate amounts of Lipofectamine.RTM. and
plasmid construct comprising the immune modulator sequences may be
separately diluted in tissue culture media and briefly incubated at
room temperature. The diluted Lipofectamine.RTM. and plasmid
constructs in media may be mixed together and incubated briefly at
room temperature. The plasmid LIPOFECTAMINE mixture may then be
added to the cells of the tumor cell line or tumor cell line
variants in the tissue culture vessel and incubated for 1-3 days
under standard tissue culture conditions.
Selection of Expressing Clones
[0510] According to some embodiments, tumor cells of the tumor cell
line or tumor cell line variant that have been transfected with
immunmodulator sequences may be selected for various levels of
expression.
[0511] According to some embodiments, the immunomodulator sequences
may be accompanied by antibiotic resistance genes, which may be
used to select for clones with stable integration of the
recombinant DNA encoding the immunomodulator sequences. According
to some embodiments, the immunomodulator sequences may be cloned
into a plasmid construct comprising antibiotic resistance, such as
the Neomycin/Kanamycin resistance gene. Transfected cells are
treated with antibiotics according to the manufacturer's protocol
for 1-2 weeks or more with daily media changes. At some point
during antibiotic treatment, there is massive tumor cell death of
all cells that have not stably integrated the antibiotic resistance
gene, leaving behind small colonies of stably expressing clones.
Each of the stably expressing clones may be picked, cultured in a
separate tissue culture container, and tested for levels of immuno
modulator expression by any established method, such as western
blot, flow cytometry, and fluorescence microscopy.
[0512] According to some embodiments, transfected tumor cells may
be selected for high expression of the immunomodulators by
fluorescence activated cell sorting (FACS). According to some
embodiments, immunomodulator sequences may be accompanied by one or
more fluorescent proteins (e.g. GFP), which can be used to quantify
expression of immunomodulator. For example, a bicistronic plasmid
comprising an immunomodulator sequence connected to a GFP sequence
via IRES sequence would result in both an immunomodulator and GFP
protein translated from the same transcript. Thus, the GFP
expression level would act as a proxy for the expression level of
immunomodulator. Single cell suspensions of immunomodulator/GFP
transfected tumor cells could be selected for the desired level of
expression by FACS based on the fluorescence intensity. Any
fluorescent protein may be used in this regard. For example, any of
the following recombinant fluorescent proteins may be used: EBFP,
ECFP, EGFP, YFP, mHoneydew, mBanana, mOrange, tdTomato, mTangerine,
mStrawberry, mCherry, mGrape, mRasberry, mGrape2, mPlum.
[0513] Alternatively, the expression of the recombinant
immunomodulator may be directly observed by fluorescent antibodies
specific to each immunomodulator or specific to a tag engineered
onto each immunomodulator. For example, according to some
embodiments the extracellular region of an immunomodulator sequence
may be fused with a FLAG tag or HA tag. Anti-FLAG or anti-HA
antibodies may be used, along with a fluorophore attached to the
primary antibody or a secondary antibody) to detect the expression
of the immunomodulator on the surface of the transfected tumor
cells. Tumor cells expressing the desired level of immunomodulator
may be selected by FACS sorting and cultured separately.
Sequentially Add New Plasmid Constructs to the Clones
[0514] According to some embodiments, tumor cell line or tumor cell
line variants that express one or more immune modulator sequence(s)
are transfected with additional immunomodulators for stable
expression in a sequential manner. By sequentially adding
recombinant immunomodulators in successive fashion, cells of a
tumor cell line or tumor cell line variant may be created that
express several immunomodulators simultaneously. According to some
embodiments, a tumor cell line or tumor cell line variant may be
created that expresses two immunomodulators simultaneously.
According to some embodiments, a tumor cell line or tumor cell line
variant may be created that expresses three immunomodulators
simultaneously. According to some embodiments, a tumor cell line or
tumor cell line variant may be created that expresses four
immunomodulators simultaneously. According to some embodiments, a
tumor cell line or tumor cell line variant may be created that
expresses five immunomodulators simultaneously. According to some
embodiments, a tumor cell line or tumor cell line variant may be
created that expresses six immunomodulators simultaneously.
According to some embodiments, a tumor cell line or tumor cell line
variant may be created that expresses seven immunomodulators
simultaneously. According to some embodiments, a tumor cell line or
tumor cell line variant may be created that expresses eight
immunomodulators simultaneously. According to some embodiments, a
tumor cell line or tumor cell line variant may be created that
expresses nine immunomodulators simultaneously. According to some
embodiments, a tumor cell line or tumor cell line variant may be
created that expresses ten immunomodulators simultaneously.
According to some embodiments, a tumor cell line or tumor cell line
variant may be created that expresses eleven immunomodulators
simultaneously. According to some embodiments, a tumor cell line or
tumor cell line variant may be created that expresses twelve
immunomodulators simultaneously. According to some embodiments, a
tumor cell line or tumor cell line variant may be created that
expresses thirteen immunomodulators simultaneously. According to
some embodiments, a tumor cell line or tumor cell line variant may
be created that expresses fourteen immunomodulators simultaneously.
According to some embodiments, a tumor cell line or tumor cell line
variant may be created that expresses fifteen immunomodulators
simultaneously. According to some embodiments, a tumor cell line or
tumor cell line variant may be created that expresses sixteen
immunomodulators simultaneously. According to some embodiments, a
tumor cell line or tumor cell line variant may be created that
expresses seventeen immunomodulators simultaneously. According to
some embodiments, a tumor cell line or tumor cell line variant may
be created that expresses eighteen immunomodulators simultaneously.
According to some embodiments, a tumor cell line or tumor cell line
variant may be created that expresses nineteen immunomodulators
simultaneously. According to some embodiments, a tumor cell line or
tumor cell line variant may be created that expresses twenty
immunomodulators simultaneously. According to some embodiments, a
tumor cell line or tumor cell line variant may be created that
expresses twenty-one immunomodulators simultaneously. According to
some embodiments, a tumor cell line or tumor cell line variant may
be created that expresses twenty-two immunomodulators
simultaneously. According to some embodiments, a tumor cell line or
tumor cell line variant may be created that expresses twenty-three
immunomodulators simultaneously. According to some embodiments, a
tumor cell line or tumor cell line variant may be created that
expresses twenty-four immunomodulators simultaneously. According to
some embodiments, a tumor cell line or tumor cell line variant may
be created that expresses twenty-five immunomodulators
simultaneously. According to some embodiments, a tumor cell line or
tumor cell line variant may be created that expresses twenty-six
immunomodulators simultaneously. According to some embodiments, a
tumor cell line or tumor cell line variant may be created that
expresses twenty-seven immunomodulators simultaneously. According
to some embodiments, a tumor cell line or tumor cell line variant
may be created that expresses twenty-eight immunomodulators
simultaneously. According to some embodiments, a tumor cell line or
tumor cell line variant may be created that expresses twenty-nine
immunomodulators simultaneously. According to some embodiments, a
tumor cell line or tumor cell line variant may be created that
expresses thirty immunomodulators simultaneously.
Variably Expressing Clones
[0515] According to one aspect of the disclosed invention, multiple
recombinant immune modulator peptides may be expressed in a single
clonally derived tumor cell line or tumor cell line variant.
According to some embodiments, the amount (or level) of each
individual immune modulator expressed in each cell is the same as
the level of expression of all other immune modulator peptides.
According to some embodiments, however, the level of each
individual immune modulator expressed in each cell is different
from the level of expression of the other immunomodulators
expressed in the cell. According to some embodiments, clonally
derived tumor cell line or tumor cell line variants that express
the same complement of immunomodulators stably express those
immunomodulators in varying amounts relative to each other.
[0516] The relative amount of recombinant immune modulator
expressed within each clonally derived tumor cell line or tumor
cell line variant, and between tumor cell line or tumor cell line
variants, can be measured on the level of transcription or
translation. For example, the relative amount of recombinant immune
modulator can be quantified by western blot, RT-PCR, flow
cytometry, immunofluorescence, and northern blot, among others.
[0517] According to some embodiments, the differences in the amount
of expressed immunomodulators relative to one another may be a
result of random integration into more or less transcriptionally
active regions of the genome of the tumor cell line or tumor cell
line variant. According to some embodiments, the relative
differences in the amount of expressed immune modulator may be
achieved by elements engineered into the transfected or transduced
DNA used to create the tumor cell line or tumor cell line
variant.
[0518] For example, according to some embodiments, the level of
expression of the exogenous immunomodulatory molecules may be
achieved on the transcriptional level by engineering stronger or
weaker gene promoter sequences to control expression of the immune
modulator gene. According to some embodiments, one or more of the
following promoters may be used to control expression of
immunomodulators: simian virus 40 early promoter (SV40),
cytomegalovirus immediate-early promoter (CMV), human Ubiquitin C
promoter (UBC), human elongation factor 1 a promoter (EF1A), mouse
phosphoglycerate kinase 1 promoter (PGK), and chicken .beta.-Actin
promoter coupled with CMV early enhancer (CAGG).
[0519] According to some embodiments, the level of expression of
the exogenous immunomodulatory molecules may be achieved on the
translational level by engineering stronger or weaker Kozak
consensus sequences around the start codon of the immune modulator
transcript. According to some embodiments, the following nucleotide
sequences may be provided to control immune modulator translation:
GCCGCC(A/G)CCAUGG (SEQ ID NO: 15). According to some embodiments, a
sequence that is at least 60% identical to SEQ ID NO: 15 may be
provided to control immune modulator translation. According to some
embodiments, a sequence that is at least 70% identical to SEQ ID
NO: 15 may be provided to control immune modulator translation.
According to some embodiments, a sequence that is at least 80%
identical to SEQ ID NO: 15 may be provided to control immune
modulator translation. According to some embodiments, a sequence
that is at least 90% identical to SEQ ID NO: 15 may be provided to
control immune modulator translation. According to some
embodiments, a sequence that is at least 95% identical to SEQ ID
NO: 15 may be provided to control immune modulator translation.
According to some embodiments, a sequence that is at least 96%
identical to SEQ ID NO: 15 may be provided to control immune
modulator translation. According to some embodiments, a sequence
that is at least 97% identical to SEQ ID NO: 15 may be provided to
control immune modulator translation. According to some
embodiments, a sequence that is at least 98% identical to SEQ ID
NO: 15 may be provided to control immune modulator translation.
According to some embodiments, a sequence that is at least 99%
identical to SEQ ID NO: 15 may be provided to control immune
modulator translation.
[0520] Non-viral approaches can also be employed for the
introduction of a vector encoding one or more immunomodulatory
molecules to a cell derived from a patient having a tumor or a
tumor cell line or variant. For example, a nucleic acid molecule
encoding an immunomodulatory molecule can be introduced into a cell
by administering the nucleic acid molecule in the presence of
lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413,
1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et
al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in
Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation
(Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et
al., Journal of Biological Chemistry 264:16985, 1989), or by
micro-injection under surgical conditions (Wolff et al., Science
247:1465, 1990). Preferably the nucleic acids are administered in
combination with a liposome and protamine.
[0521] Methods for accomplishing transfection in vitro include the
use of calcium phosphate, DEAE dextran, electroporation, and
protoplast fusion. Liposomes can also be potentially beneficial for
delivery of DNA into a cell.
IV. Therapeutic Compositions
[0522] Immunogenic compositions of the invention, including
allogeneic tumor cell vaccines, are useful as therapeutics and
prophylactics for the treatment of specific types of cancers.
Advantageously, these vaccines may be tailored to treat the cancers
of particular individuals, by generating vaccines that target
specific tumor antigens expressed on a tumor in a subject.
Allogeneic vaccines of the invention typically contain inactivated
tumor cells or cells expressing tumor antigens that have been
genetically modified to express exogenous immunomodulatory
molecules, as described herein. According to some embodiments, an
allogeneic tumor cell vaccine may comprise an amount of a tumor
cell line or tumor cell line variant comprising two or more genes
encoding immunomodulatory molecules. According to some embodiments,
clones of tumor cell line or tumor cell line variants that
maximally express the immunomodulatory molecules are identified and
selected. According to some embodiments, expression of the
immunomodulatory molecules by populations of the tumor cell line or
tumor cell line variants is determined by flow cytometry. According
to some embodiments, flow cytometry is used to gate on the
maximally expressing population(s) of tumor cell line or tumor cell
line variants.
[0523] According to some embodiments, the immunogenic amount is
effective to stimulate an anti-tumor immune response to one or more
tumor specific antigens. According to some embodiments, the
immunogenic amount may be titrated to provide both safety and
efficacy.
[0524] According to some embodiments, the immunogenic composition
comprises a pharmaceutically acceptable carrier.
[0525] According to some embodiments, the immunogenic composition
further comprises an adjuvant.
[0526] According to some embodiments, the tumor cell line or tumor
cell line variant may comprise tumor cells derived from an
established cell line. According to some embodiments, the tumor
cell line or tumor cell line variant comprises tumor cells derived
from a patient with cancer, wherein the tumor cells are derived
from a solid tumor.
[0527] According to some embodiments, the tumor cell line or tumor
cell line variant comprises an immunogenic amount of a disrupted
tumor cell line or tumor cell line variant. Examples of methods for
physical disruption include, without limitation, sonication,
cavitation, dehydration, ion depletion, or by toxicity from
exposure to one or more salts.
[0528] According to some embodiments, the immunogenic amount of the
immunogenic composition can comprise at least 1.times.10.sup.3
whole or disrupted tumor cell line or tumor cell line variant
cells. According to some embodiments, the amount of the immunogenic
composition can comprises at least 1.times.10.sup.4 whole or
disrupted tumor cell line or tumor cell line variant cells.
According to some embodiments, the amount of the immunogenic
composition can comprise at least 1.times.10.sup.5 whole or
disrupted tumor cell line or tumor cell line variant cells.
According to some embodiments, the amount of the immunogenic
composition can comprise at least 1.times.10.sup.6 whole or
disrupted tumor cell line or tumor cell line variant cells.
According to some embodiments, the amount of the immunogenic
composition can comprise at least 1.times.10.sup.7 whole or
disrupted tumor cell line or tumor cell line variant cells.
According to some embodiments, the amount of the immunogenic
composition can comprise at least 1.times.10.sup.8 whole or
disrupted tumor cell line or tumor cell line variant cells.
According to some embodiments, the amount of the immunogenic
composition can comprise at least 1.times.10.sup.9 whole or
disrupted tumor cell line or tumor cell line variant cells.
According to some embodiments, the immunogenic amount can be a
therapeutic amount.
[0529] According to some embodiments, the immunogenic amount is
effective (1) to stimulate an immune response that reduces tumor
burden, comprising one or more of a cytotoxic T cell population, a
natural killer cell population, antibodies, APCs, a T cell
population, a B cell population, and a dendritic cell population;
and (2) to improve a clinical outcome parameter selected from one
or more of progression-free survival, disease-free survival, time
to progression, time to distant metastasis, and overall survival of
the subject, when compared to a suitable control.
[0530] According to some embodiments, the immunogenic composition
may be administered once per week, twice per week, once every two
weeks, once every three weeks, once every four weeks, once per
month, once every two months, once every three months, once every
four months, once every five months, once every six months, once
every seven months, once every eight months, once every nine
months, once every ten months, once every eleven months, or once a
year. According to some embodiments, administration occurs in one
day or over 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, 8,
days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days,
16 days, 17 days, 18 days, 19 days, 20 days, or more. According to
some embodiments, administration may involve two or more
administrations on the same day.
Combination Therapies
[0531] According to some embodiments, the disclosure provides
methods that further comprise administering an additional agent to
a subject. According to some embodiments, the invention pertains to
co-administration and/or co-formulation.
[0532] According to some embodiments, administration of the
immunogenic composition acts synergistically when co-administered
with another agent and is administered at doses that are lower than
the doses commonly employed when such agents are used as
monotherapy.
[0533] According to some embodiments, inclusive of, without
limitation, cancer applications, the present invention pertains to
chemotherapeutic agents as additional agents. Examples of
chemotherapeutic agents include, but are not limited to, alkylating
agents such as thiotepa and CYTOXAN cyclosphosphamide; alkyl
sulfonates such as busulfan, improsulfan and piposulfan; aziridines
such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine,
triethylenemelamine, trietylenephosphoramide,
triethiylenethiophosphoramide and trimethylolomelamine; acetogenins
(e.g., bullatacin and bullatacinone); a camptothecin (including the
synthetic analogue topotecan); bryostatin; cally statin; CC-1065
(including its adozelesin, carzelesin and bizelesin synthetic
analogues); cryptophycins (e.g., cryptophycin 1 and cryptophycin
8); dolastatin; duocarmycin (including the synthetic analogues,
KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; a
sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics (e.g., calicheamicin, especially
calicheamicin gammall and calicheamicin omegall; dynemicin,
including dynemicin A; bisphosphonates, such as clodronate; an
esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antibiotic chromophores), aclacinomy sins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN
doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy
doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,
mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,
olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,
rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,
zinostatin, zorubicin; anti-metabolites such as methotrexate and
5-fluorouracil (5-FU); folic acid analogues such as denopterin,
methotrexate, pteropterin, trimetrexate; purine analogs such as
fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine
analogs such as ancitabine, azacitidine, 6-azauridine, carmofur,
cytarabine, dideoxyuridine, doxifluridine, enocitabine,
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals
such as minoglutethimide, mitotane, trilostane; folic acid
replenisher such as frolinic acid; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; demecolcine; diaziquone; elformithine;
elliptinium acetate; an epothilone; etoglucid; gallium nitrate;
hydroxyurea; lentinan; lonidainine; maytansinoids such as
maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;
nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;
podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK
polysaccharide complex; razoxane; rhizoxin; sizofuran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin,
verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., TAXOL paclitaxel (Bristol-Myers Squibb Oncology,
Princeton, N.J.), ABRAXANE Cremophor-free, albumin-engineered
nanoparticle formulation of paclitaxel, and TAXOTERE doxetaxel;
chloranbucil; GEMZAR gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin, oxaliplatin and
carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;
mitoxantrone; vincristine; NAVELBINE. vinorelbine; novantrone;
teniposide; edatrexate; daunomycin; aminopterin; xeloda;
ibandronate; irinotecan (Camptosar, CPT-11) (including the
treatment regimen of irinotecan with 5-FU and leucovorin);
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);
retinoids such as retinoic acid; capecitabine; combretastatin;
leucovorin (LV); oxaliplatin, including the oxaliplatin treatment
regimen (FOLFOX); lapatinib (TYKERB); inhibitors of PKC-.alpha.,
Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce
cell proliferation and pharmaceutically acceptable salts, acids or
derivatives of any of the above. In addition, the methods of
treatment can further include the use of radiation.
Checkpoint Blockade/Blockage of Tumor Immunosuppression
[0534] Some human tumors can be eliminated by a patient's immune
system. For example, administration of a monoclonal antibody
targeted to an immune "checkpoint" molecule can lead to complete
response and tumor remission. A mode of action of such antibodies
is through inhibition of an immune regulatory molecule that the
tumors have co-opted as protection from an anti-tumor immune
response. By inhibiting these "checkpoint" molecules (e.g., with an
antagonistic antibody), a patient's CD8+ T cells may be allowed to
proliferate and destroy tumor cells.
[0535] According to some embodiments, the allogeneic vaccine
composition further comprises one or more checkpoint inhibitors
that may be effective to prevent premature termination of an
effective immune response once such an immune response is
initiated.
[0536] For example, administration of a monoclonal antibody
targeted to by way of example, without limitation, CTLA-4 or PD-1
can lead to a complete response and tumor remission. The mode of
action of such antibodies is through inhibition of CTLA-4 or PD-1
that the tumors have co-opted as protection from an anti-tumor
immune response. By inhibiting these "checkpoint" molecules (e.g.,
with an antagonistic antibody), a patient's CD8+ T cells may be
allowed to proliferate and destroy tumor cells.
[0537] Thus, the allogeneic vaccine compositions provided herein
can be used in combination with one or more blocking antibodies
targeted to an immune "checkpoint" molecule. For instance,
according to some embodiments, the allogeneic vaccine compositions
provided herein can be used in combination with one or more
blocking antibodies targeted to a molecule such as CTLA-4 or PD-1.
For example, the allogeneic vaccine compositions provided herein
may be used in combination with an agent that blocks, reduces
and/or inhibits PD-1 and PD-L1 or PD-L2 and/or the binding of PD-1
with PD-L1 or PD-L2 (by way of non-limiting example, one or more of
nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS
SQUIBB), pembrolizumab (KEYTRUDA, Merck), pidilizumab (CT-011, CURE
TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB),
MPDL328OA (ROCHE)). According to some embodiments, the allogeneic
vaccine compositions provided herein may be used in combination
with an agent that blocks, reduces and/or inhibits the activity of
CTLA-4 and/or the binding of CTLA-4 with one or more receptors
(e.g. CD80, CD86, AP2M1, SHP-2, and PPP2R5A). For instance,
according to some embodiments, the immune-modulating agent is an
antibody such as, by way of non-limitation, ipilimumab (MDX-010,
MDX-101, Yervoy, BMS) and/or tremelimumab (Pfizer). Blocking
antibodies against these molecules can be obtained from, for
example, Bristol Myers Squibb (New York, N.Y.), Merck (Kenilworth,
N.J.), Medlmmune (Gaithersburg, Md.), and Pfizer (New York,
N.Y.).
[0538] Further, the allogeneic immune compositions provided herein
can be used in combination with one or more blocking antibodies
targeted to an immune "checkpoint" molecule such as for example,
BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also referred
to as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, CEACAM
(e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL,
galectin-9, CD244, CD160, TIGIT, SIRP.alpha., ICOS, CD172a, and
TMIGD2 and various B-7 family ligands (including, but are not
limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5,
B7-H6 and B7-H7).
[0539] Adjuvants
[0540] According to some embodiments, the compositions of the
present invention may further comprise one or more additional
substances which, because of their adjuvant nature, can act to
stimulate the immune system to respond to the cancer antigens
present on the inactivated tumor cell(s). Such adjuvants include,
but are not limited to, lipids, liposomes, inactivated bacteria
which induce innate immunity (e.g., inactivated or attenuated
Listeria monocytogenes), compositions which mediate innate immune
activation via Toll-like Receptors (TLRs), (NOD)-like receptors
(NLRs), Retinoic acid inducible gene-based (RIG)-I-like receptors
(RLRs), and/or C-type lectin receptors (CLRs). Examples of PAMPs
include lipoproteins, lipopolypeptides, peptidoglycans, zymosan,
lipopolysaccharide, neisserial porins, flagellin, profillin,
galactoceramide, muramyl dipeptide. Peptidoglycans, lipoproteins,
and lipoteichoic acids are cell wall components of Gram-positive.
Lipopoly saccharides are expressed by most bacteria, with MPL being
one example. Flagellin refers to the structural component of
bacterial flagella that is secreted by pathogenic and commensal
bacterial. a-Galactosylceramide (a-GalCer) is an activator of
natural killer T (NKT) cells. Muramyl dipeptide is a bioactive
peptidoglycan motif common to all bacteria. This list is not meant
to be limiting.
[0541] According to some embodiments, the treatment regimen may
comprise a standard anti-tumor therapy (such as surgery, radiation
therapy, a targeted therapy that precisely identifies and attacks
cancer cells, a hormone therapy, or a combination thereof).
According to some embodiments, the standard anti-tumor therapy is
effective to treat the tumor while preserving any existing
anti-tumor immune response. According to some embodiments, the
immunogenic composition is not applied after chemotherapy.
According to some embodiments, the immunogenic composition is
applied after low-dose chemotherapy.
[0542] According to some embodiments, the immunogenic composition
comprises two or more clonally derived tumor cell line or tumor
cell line variants. According to some embodiments, the two or more
tumor cell line or tumor cell line variants comprise the same
complement of recombinant immunomodulators. According to some
embodiments, the two or more tumor cell line or tumor cell line
variants comprise different panels of recombinant
immunomodulators.
[0543] According to some embodiments, the tumor cell line or tumor
cell line variants are treated with an agent that prevents cell
division prior to administration to a subject. According to some
embodiments, the tumor cell line or tumor cell line variants are
irradiated. According to some embodiments, the tumor cell line or
tumor cell line variants are treated with a chemical agent that
prevents proliferation.
[0544] According to some embodiments, the tumor cell line or tumor
cell line variants may be administered parenterally. According to
some embodiments, the tumor cell line or tumor cell line variants
may be administered locally into a surgical excision cavity.
According to some embodiments, the tumor cell variants may be
administered by intradermal injection. According to some
embodiments, the tumor cell line or tumor cell line variants may be
administered by subcutaneous injection. According to some
embodiments, the tumor cell line or tumor cell line variants may be
administered by intramuscular injection.
V. Methods of Treatment
[0545] According to some embodiments, the allogeneic tumor cell
vaccines described herein are effective to enhance immune
activation of cells, to recognize and act against tumor cells
comprising the target tumor antigen in vivo without systemic
inflammation; to reduce immunosuppression in a tumor
microenvironment for tumor cells comprising the target tumor
antigen; to reduce tumor burden, or to increase cell death of tumor
cells expressing the target tumor antigen. According to some
embodiments, the allogeneic tumor cell vaccines described herein
are effective to induce immune activation without systemic
inflammation. According to some embodiments, the allogeneic tumor
cell vaccine is effective to elicit an immune response that
improves progression free survival, overall survival, or both
relative to placebo controls.
[0546] According to some embodiments, the allogeneic vaccine
composition is administered to a subject diagnosed with cancer in
combination with an agent that inhibits immunosuppressive molecules
produced by tumor cells.
[0547] According to some embodiments, the described invention
comprises an allogeneic tumor cell vaccine for an active
immunotherapy that can be universally administered to all patients
with a particular type of cancer. According to some embodiments,
the allogeneic vaccine comprises a genetically modified allogeneic
tumor-type specific cell, or a membrane lysate derived from
modified allogeneic tumor-type specific cells, formulated in a
pharmaceutically acceptable carrier. According to some embodiments,
the modified allogeneic tumor-type specific cells are derived from
previously established cell lines.
[0548] According to some embodiments, the allogeneic vaccine is
adapted to treat patients with minimal residual disease and a
functional immune system, which includes recognition of self and
response to non-self by innate immunity (resistance) and adaptive
(specific) immunity comprising humoral immunity and cellular
immunity). For example, according to some embodiments, the
allogeneic vaccine is adapted to treat a patient with minimal
residual disease obtained shortly after a primary lesion is
surgically removed. According to some embodiments, the allogeneic
vaccine is adapted for subcutaneous administration of the vaccine.
According to some embodiments, the dose and schedule for
administering the allogeneic vaccine are determined by using
immunologic responses to the vaccine as a guide for eventual
enhancement of overall survival.
[0549] According to some embodiments, the disclosure features a
method of inducing an immune response to a cancer in a subject
comprising administering the allogeneic tumor cell vaccine
described herein, wherein the allogeneic tumor cell vaccine is
type-matched to the subject's cancer.
[0550] Tumor cell line or tumor cell line variants as provided
herein can be incorporated into a composition for administration to
a subject (e.g., a research animal or a mammal, such as a human,
having a clinical condition such as cancer or an infection). For
example, an allogeneic tumor cell vaccine comprising a tumor cell
line or tumor cell line variant genetically engineered to stably
express a core group of three immunomodulatory molecules wherein
the core group of immunomodulator molecules is OX40 Ligand (OX40L),
CD27 Ligand (CD70) and CD28 Ligand (CD28L); and a pharmaceutically
acceptable carrier; can be administered to a subject for the
treatment of cancer. In another example, an allogeneic tumor cell
vaccine comprising a tumor-type specific cell line variant is used
to deliver a broad array of tumor antigens in the context of
immunomodulatory signals sufficient to elicit an effective
anti-tumor response as reflected in improved progression free
survival, overall survival, or both relative to placebo controls,
wherein the immunomodulatory signals comprised at least a core
group of three immunomodulatory molecules wherein the core group of
immunomodulator molecules is OX40 Ligand (OX40L), CD27 Ligand
(CD70) and CD28 Ligand (CD28L); and optionally an additional number
of immunomodulatory molecules comprising 3-25 immunomodulators ("R
groups") selected from those set forth in Table 2.
[0551] Thus, the described invention provides methods for treating
clinical conditions such as cancer with the allogeneic tumor
vaccines provided herein.
[0552] According to various embodiments, the described invention
pertains to cancers and/or tumors; for example, the treatment or
prevention of cancers and/or tumors. The phrase "cancers or tumors"
refers to an uncontrolled growth of cells and/or abnormal increased
cell survival and/or inhibition of apoptosis which interferes with
the normal functioning of the bodily organs and systems. Included
are benign and malignant cancers, polyps, hyperplasia, as well as
dormant tumors or micrometastases. Also included are cells having
abnormal proliferation that is not impeded by the immune system
(e.g. virus infected cells). The cancer may be a primary cancer or
a metastatic cancer. The primary cancer may be an area of cancer
cells at an originating site that becomes clinically detectable,
and may be a primary tumor. In contrast, the metastatic cancer may
be the spread of a disease from one organ or part to another
non-adjacent organ or part. The metastatic cancer may be caused by
a cancer cell that acquires the ability to penetrate and infiltrate
surrounding normal tissues in a local area, forming a new tumor,
which may be a local metastasis. The cancer may also be caused by a
cancer cell that acquires the ability to penetrate the walls of
lymphatic and/or blood vessels, after which the cancer cell is able
to circulate through the bloodstream (thereby being a circulating
tumor cell) to other sites and tissues in the body. The cancer may
be due to a process such as lymphatic or hematogeneous spread. The
cancer may also be caused by a tumor cell that comes to rest at
another site, re-penetrates through the vessel or walls, continues
to multiply, and eventually forms another clinically detectable
tumor. The cancer may be this new tumor, which may be a metastatic
(or secondary) tumor.
[0553] The cancer may be caused by tumor cells that have
metastasized, which may be a secondary or metastatic tumor. The
cells of the tumor may be like those in the original tumor. As an
example, if a breast cancer or colon cancer metastasizes to the
liver, the secondary tumor, while present in the liver, is made up
of abnormal breast or colon cells, not of abnormal liver cells. The
tumor in the liver may thus be a metastatic breast cancer or a
metastatic colon cancer, not liver cancer.
[0554] Illustrative cancers that may be treated include, but are
not limited to, carcinomas, e.g. various subtypes, including, for
example, adenocarcinoma, basal cell carcinoma, squamous cell
carcinoma, and transitional cell carcinoma), sarcomas (including,
for example, bone and soft tissue), leukemias (including, for
example, acute myeloid, acute lymphoblastic, chronic myeloid,
chronic lymphocytic, and hairy cell), lymphomas and myelomas
(including, for example, Hodgkin and non-Hodgkin lymphomas, light
chain, non-secretory, MGUS, and plasmacytomas), and central nervous
system cancers (including, for example, brain (e.g. gliomas (e.g.
astrocytoma, oligodendroglioma, and ependymoma), meningioma,
pituitary adenoma, and neuromas, and spinal cord tumors (e.g.
meningiomas and neurofibroma).
[0555] According to certain embodiments, cancers/tumors that may be
treated are those where the standard of care is no longer
chemotherapy, since chemotherapy is known to interfere with immune
responses, which are expected to occur during a successful
vaccination protocol. Exemplary tumor types include tumor types
treated with hormonal therapies such as prostate and breast cancers
(e.g. Abiraterone.RTM. for prostate cancer and Tamoxifen.RTM. for
breast cancer), tumor types treated with targeted therapies like
antibodies (e.g. Rituxan.RTM. for B cell malignancies,
Herceptin.RTM. for breast cancer), tumor types treated with kinase
inhibitors such as GLEEVEC.TM. for chronic myelogenous leukemia and
tumor types treated with other immune system sparing or enhancing
modalities, such as checkpoint inhibitors, oncolytic viruses and
CAR-T cells
[0556] Representative cancers and/or tumors of the present
invention are described herein.
[0557] The present disclosure also provides compositions containing
an allogeneic tumor cell vaccine comprising a tumor cell line or
tumor cell line variant comprising two or more stably expressed
recombinant exogenous immunomodulatory molecules selected from a
cytokine, a TNF-family member, a secreted receptor, a chaperone, an
IgG superfamily member and a chemokine receptor. The present
disclosure also provides compositions containing an allogeneic
tumor cell vaccine comprising two, three, four or more tumor cell
lines or tumor cell line variants each comprising two or more
stably expressed recombinant exogenous immunomodulatory molecules
selected from a cytokine, a TNF-family member, a secreted receptor,
a chaperone, an IgG superfamily member and a chemokine
receptor.
[0558] The present disclosure also provides compositions containing
an allogeneic tumor cell vaccine comprising a tumor cell line or
tumor cell line variant comprising two or more stably expressed
recombinant membrane bound immunomodulatory molecules selected from
those set forth in table 2, and a pharmaceutically acceptable
carrier, as described herein, in combination with a physiologically
and pharmaceutically acceptable carrier. The physiologically and
pharmaceutically acceptable carrier can include any of the
well-known components useful for immunization. The carrier can
facilitate or enhance an immune response to an antigen administered
in a vaccine. The cell formulations can contain buffers to maintain
a preferred pH range, salts or other components that present an
antigen to an individual in a composition that stimulates an immune
response to the antigen. The physiologically acceptable carrier
also can contain one or more adjuvants that enhance the immune
response to an antigen. Pharmaceutically acceptable carriers
include, for example, pharmaceutically acceptable solvents,
suspending agents, or any other pharmacologically inert vehicles
for delivering compounds to a subject. Pharmaceutically acceptable
carriers can be liquid or solid, and can be selected with the
planned manner of administration in mind so as to provide for the
desired bulk, consistency, and other pertinent transport and
chemical properties, when combined with one or more therapeutic
compounds and any other components of a given pharmaceutical
composition. Exemplary pharmaceutically acceptable carriers
include, without limitation: water, saline solution, binding agents
(e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose);
fillers (e.g., lactose or dextrose and other sugars, gelatin, or
calcium sulfate), lubricants (e.g., starch, polyethylene glycol, or
sodium acetate), disintegrates (e.g., starch or sodium starch
glycolate), and wetting agents (e.g., sodium lauryl sulfate).
Compositions can be formulated for subcutaneous, intramuscular, or
intradermal administration, or in any manner acceptable for
immunization.
[0559] An "adjuvant" refers to a substance which, when added to an
immunogenic agent such as a tumor cell expressing secreted vaccine
protein, nonspecifically enhances or potentiates an immune response
to the agent in the recipient host upon exposure to the mixture.
Adjuvants can include, for example, oil-in-water emulsions,
water-in oil emulsions, alum (aluminum salts), liposomes and
microparticles, such as, polysytrene, starch, polyphosphazene and
polylactide/polyglycosides.
[0560] Adjuvants can also include, for example, squalene mixtures
(SAF-I), muramyl peptide, saponin derivatives, mycobacterium cell
wall preparations, monophosphoryl lipid A, mycolic acid
derivatives, nonionic block copolymer surfactants, Quil A, cholera
toxin B subunit, polyphosphazene and derivatives, and
immunostimulating complexes (ISCOMs) such as those described by
Takahashi et al., Nature 1990, 344:873-875. For veterinary use and
for production of antibodies in animals, mitogenic components of
Freund's adjuvant (both complete and incomplete) can be used. In
humans, Incomplete Freund's Adjuvant (IFA) is a useful adjuvant.
Various appropriate adjuvants are well known in the art (see, for
example, Warren and Chedid, CRC Critical Reviews in Immunology
1988, 8:83; and Allison and Byars, in Vaccines: New Approaches to
Immunological Problems, 1992, Ellis, ed., Butterworth-Heinemann,
Boston). Additional adjuvants include, for example, bacille
Calmett-Guerin (BCG), DETOX (containing cell wall skeleton of
Mycobacterium phlei (CWS) and monophosphoryl lipid A from
Salmonella minnesota (MPL)), and the like (see, for example, Hoover
et al., J Clin Oncol 1993, 11:390; and Woodlock et al., J
Immunother 1999, 22:251-259).
[0561] According to some embodiments, an allogeneic tumor cell
vaccine can be administered to a subject one or more times (e.g.,
once, twice, two to four times, three to five times, five to eight
times, six to ten times, eight to 12 times, or more than 12 times).
An allogeneic tumor cell vaccine as provided herein can be
administered one or more times per day, one or more times per week,
every other week, one or more times per month, once every two to
three months, once every three to six months, or once every six to
12 months. An allogeneic tumor cell vaccine can be administered
over any suitable period of time, such as a period from about 1 day
to about 12 months. According to some embodiments, for example, the
period of administration can be from about 1 day to 90 days; from
about 1 day to 60 days; from about 1 day to 30 days; from about 1
day to 20 days; from about 1 day to 10 days; from about 1 day to 7
days. According to some embodiments, the period of administration
can be from about 1 week to 50 weeks; from about 1 week to 50
weeks; from about 1 week to 40 weeks; from about 1 week to 30
weeks; from about 1 week to 24 weeks; from about 1 week to 20
weeks; from about 1 week to 16 weeks; from about 1 week to 12
weeks; from about 1 week to 8 weeks; from about 1 week to 4 weeks;
from about 1 week to 3 weeks; from about 1 week to 2 weeks; from
about 2 weeks to 3 weeks; from about 2 weeks to 4 weeks; from about
2 weeks to 6 weeks; from about 2 weeks to 8 weeks; from about 3
weeks to 8 weeks; from about 3 weeks to 12 weeks; or from about 4
weeks to 20 weeks.
[0562] According to some embodiments, after an initial dose
(sometimes referred to as a "priming" dose) of an allogeneic tumor
cell vaccine has been administered and a maximal antigen-specific
immune response has been achieved, one or more boosting doses can
be administered. For example, a boosting dose can be administered
about 10 to 30 days, about 15 to 35 days, about 20 to 40 days,
about 25 to 45 days, or about 30 to 50 days after a priming
dose.
[0563] According to some embodiments, the methods provided herein
can be used for controlling solid tumor growth and/or metastasis.
The methods can include administering an effective amount of an
allogeneic tumor cell vaccine as described herein to a subject in
need thereof.
[0564] The vectors and methods provided herein can be useful for
stimulating an immune response against a tumor. Such immune
response is useful in treating or alleviating a sign or symptom
associated with the tumor. A practitioner will appreciate that the
methods described herein are to be used in concomitance with
continuous clinical evaluations by a skilled practitioner
(physician or veterinarian) to determine subsequent therapy. Such
evaluations will aid and inform in evaluating whether to increase,
reduce, or continue a particular treatment dose, mode of
administration, etc.
[0565] The methods provided herein can thus be used to treat a
tumor, including, for example, a cancer. The methods can be used,
for example, to inhibit the growth of a tumor by preventing further
tumor growth, by slowing tumor growth, or by causing tumor
regression. Thus, the methods can be used, for example, to treat a
cancer. It will be understood that the subject to which a compound
is administered need not suffer from a specific traumatic state.
Indeed, the allogeneic tumor cell vaccine described herein may be
administered prophylactically, prior to development of symptoms
(e.g., a patient in remission from cancer).
[0566] Anti-tumor and anti-cancer effects include, without
limitation, modulation of tumor growth (e.g., tumor growth delay),
tumor size, or metastasis, the reduction of toxicity and side
effects associated with a particular anti-cancer agent, the
amelioration or minimization of the clinical impairment or symptoms
of cancer, extending the survival of the subject beyond that which
would otherwise be expected in the absence of such treatment, and
the prevention of tumor growth in an animal lacking tumor formation
prior to administration, i.e., prophylactic administration.
[0567] Therapeutically effective amounts can be determined by, for
example, starting at relatively low amounts and using step-wise
increments with concurrent evaluation of beneficial effects. The
methods provided herein thus can be used alone or in combination
with other well-known tumor therapies, to treat a patient having a
tumor. One skilled in the art will readily understand advantageous
uses of the allogeneic tumor cell vaccines and methods provided
herein, for example, in prolonging the life expectancy of a cancer
patient and/or improving the quality of life of a cancer patient
(e.g., a lung cancer patient).
[0568] According to some embodiments, a subject (i.e. a subject
diagnosed with cancer) is treated by checkpoint inhibitor therapy
prior to or concurrently with administration of the allogeneic
vaccine composition. In certain embodiments, the cancer is a
melanoma.
Subjects
[0569] The methods described herein are intended for use with any
subject that may experience the benefits of these methods. Thus,
"subjects," "patients," and "individuals" (used interchangeably)
include humans as well as non-human subjects, particularly
domesticated animals.
[0570] According to some embodiments, the subject and/or animal is
a mammal, e g., a human, mouse, rat, guinea pig, dog, cat, horse,
cow, pig, rabbit, sheep, or non-human primate, such as a monkey,
chimpanzee, or baboon. In other embodiments, the subject and/or
animal is a non-mammal, such, for example, a zebrafish. According
to some embodiments, the subject and/or animal may comprise
fluorescently-tagged cells (with e.g. GFP). According to some
embodiments, the subject and/or animal is a transgenic animal
comprising a fluorescent cell.
[0571] According to some embodiments, the subject and/or animal is
a human According to some embodiments, the human is a pediatric
human In other embodiments, the human is an adult human. In other
embodiments, the human is a geriatric human In other embodiments,
the human may be referred to as a patient.
[0572] According to certain embodiments, the human has an age in a
range of from about 0 months to about 6 months old, from about 6 to
about 12 months old, from about 6 to about 18 months old, from
about 18 to about 36 months old, from about 1 to about 5 years old,
from about 5 to about 10 years old, from about 10 to about 15 years
old, from about 15 to about 20 years old, from about 20 to about 25
years old, from about 25 to about 30 years old, from about 30 to
about 35 years old, from about 35 to about 40 years old, from about
40 to about 45 years old, from about 45 to about 50 years old, from
about 50 to about 55 years old, from about 55 to about 60 years
old, from about 60 to about 65 years old, from about 65 to about 70
years old, from about 70 to about 75 years old, from about 75 to
about 80 years old, from about 80 to about 85 years old, from about
85 to about 90 years old, from about 90 to about 95 years old or
from about 95 to about 100 years old.
[0573] According to other embodiments, the subject is a non-human
animal, and therefore the invention pertains to veterinary use.
According to a specific embodiment, the non-human animal is a
household pet. According to another specific embodiment, the
non-human animal is a livestock animal According to certain
embodiments, the subject is a human cancer patient that cannot
receive chemotherapy, e.g. the patient is unresponsive to
chemotherapy or too ill to have a suitable therapeutic window for
chemotherapy (e.g. experiencing too many dose- or regimen-limiting
side effects). In certain embodiments, the subject is a human
cancer patient having advanced and/or metastatic disease.
[0574] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range is encompassed within the invention. The
upper and lower limits of these smaller ranges which may
independently be included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either both of those included limits
are also included in the invention.
[0575] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and described the methods and/or materials in
connection with which the publications are cited.
[0576] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
references unless the context clearly dictates otherwise. All
technical and scientific terms used herein have the same
meaning.
[0577] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application and each is incorporated by reference in its entirety.
Nothing herein is to be construed as an admission that the present
invention is not entitled to antedate such publication by virtue of
prior invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed.
EXAMPLES
[0578] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Example 1
[0579] The Examples make use of, but are not limited to, the
methods described hereinbelow.
Western Blotting
[0580] Briefly, cells are lysed with cold lysis buffer and
centrifuged to pellet cellular debris. Protein concentration of the
supernatant is determined by a protein quantification assay (e.g.,
Bradford Protein Assay, Bio-Rad Laboratories). The lysate
supernatant is then combined with an equal volume of 2.times.SDS
sample buffer and boiled at 100.degree. C. for 5 minutes. Equal
amounts of protein in sample buffer are loaded into the wells of an
SDS-PAGE gel along with molecular weight marker and electrophoresed
for 1-2 hours at 100 V. Proteins are then transferred to a
nitrocellulose or PVDF membrane. The membrane is then blocked for 1
hour at room temperature using 5% non-fat dry milk in TBST blocking
buffer. The membrane is then incubated with a 1:500 dilution of
primary antibody in 5% non-fat dry milk in TBST blocking buffer,
followed by three washes in 20 Mn Tris, Ph 7.5; 150 mM NaCl, 0.1%
Tween 20 (TBST) for 5 minutes. The membrane is then incubated with
conjugated secondary antibody at a 1:2000 dilution in 5% non-fat
dry milk in TBST blocking buffer for 1 hour at room temperature,
followed by three washes in TBST for 5 minutes each. Images of the
blot are obtained using dark room development techniques for
chemiluminesence detection, or using image scanning techniques for
colorimetric or fluorescent detection.
Real Time PCR
[0581] Real-time PCR techniques may be performed as described to
analyze expression level of mRNAs (Zhao Y. et al., Biochemical and
Biophysical Research Communications 360 (2007) 205-211). Briefly,
total RNA is extracted from cells using the Quiagen kit (Valencia
Calif.), followed by first strand cDNA synthesis using random
hexamer primers (Fermentas, Hanover Md.). Real-time PCR is
performed on each sample using the Mx3000p Quantitative PCR system
(Stratagene, La Jolla, Calif.), for 40 cycles using validated gene
specific RT-PCR primer sets for each gene of interest. Relative
expression level of each transcript is corrected for that of the
house keeping gene beta-actin as an internal control.
Immunofluorescence
[0582] Briefly, adherent tumor cell line variant cells are fixed
with 4% formaldehyde diluted in warm PBS for 15 minutes at room
temperature. The fixative is aspirated and the cells washed three
times with PBS for 5 minutes each. Cells are blocked in a 5% BSA
blocking buffer for 60 minutes at room temperature. Blocking buffer
is then aspirated and a solution of primary antibody (e.g. 1:100
dilution) is incubated with the cells overnight at 4.degree. C.
Cells are then rinsed three times with PBS for 5 minutes each, and
subsequently incubated with a solution of fluorochrome conjugated
secondary antibody (e.g. 1:1000 dilution) for 1-2 hours at room
temperature. Cells are then washed three times with PBS for 5
minutes each and visualized by fluorescence microscopy.
Flow Cytometry
[0583] Flow Cytomtery analysis may be performed as described (Zhao
Y. et al., Exp. Cell Res., 312, 2454 (2006)). Briefly, tumor cell
line variant cells that are either treated with trypsin/EDTA or
left untreated are collected by centrifugation and re-suspended in
PBS. The cells are fixed in 4% formaldehyde for 10 minutes at
37.degree. C. For extracellular staining with antibodies, cells are
not permeabilized. For intracellular staining, cells are
permeabilized by adding ice-cold 100% methanol to pre-chilled cells
to a final concentration of 90% methanol and incubated on ice for
30 minutes. Cells are immunostained by first resuspending cells in
incubation buffer and adding dilutions of primary antibody. Cells
are incubated with primary antibody for 1 hour at room temperature,
followed by three washes with incubation buffer. Cells are then
resuspended in incubation buffer with dilutions of conjugated
secondary antibody for 30 minutes at room temperature, followed by
three washes in incubation buffer. Stained cells are then analyzed
by flow cytometry.
Enzyme-Linked Immunosorbent Assay (ELISA)
[0584] Briefly, a capture antibody, specific for a protein of
interest, is coated onto the wells of a microplate. Samples,
including a standard containing protein of interest, control
specimens, and unknowns, are pipetted into wells of the microplate,
where the protein antigen binds to the capture antibody. After
washing 4 times, a detection antibody is added to the wells for one
hour, binding to the immobilized protein captured during the first
incubation. After removal of excess detection antibody and washing
4 times, a horse radish peroxidase (HRP) conjugate (secondary
antibody or streptavidin) is added for 30 minutes to bind to the
detection antibody. After washing 4 more times to remove the excess
HRP conjugate, a substrate solution is added for 30 minutes in the
dark to be converted by the enzyme to a detectable form (color
signal). A stop solution is added to each well of the microplate
and evaluated within 30 minutes of stopping the reaction. Intensity
of the colored product may be directly proportional to the
concentration of antigen present in the original specimen.
Human Mixed Lymphocyte Tumor Reaction (MLTR) Testing
[0585] The mixed lymphocyte tumor reaction (MLTR) is an all human,
in vitro assay, designed to optimize lead candidates. In the MLTR,
optimization is achieved through the qualitative and quantitative
assessment of human peripheral blood mononuclear cell (PBMC)
responses to engineered allogeneic tumor cells. The MLTR measures
proliferation and differentiation by flow cytometry and mass
cytometry (CyTOF), by cytotoxicity, measured by lactate
dehydrogenase (LDH) release assay, and by cytokine profile.
According to some embodiments, allogeneic cell pools expressing a
single immunomodulatory protein are used in the MLTR. According to
some embodiments, allogenic cell pools expressing one or more, two
or more, three or more, four or more or five or more
immunomodulatory proteins are used in the MLTR.
[0586] The basic MLTR one day procedure is carried out as
follows:
[0587] A vial of PBMC (20 MN cells) is thawed. Cells are then
washed in dPBS. PMBC cells are resuspended at 2.5.times.10.sup.6
cell per ml in X-VIVO (-8 ml). The cells are characterized by flow
cytometry to document the nature of the cell population.
[0588] Use in the MLTR is carried out as follows:
2.5.times.10.sup.5 cell PBMC (100 .mu.l of stock)
0.5.times.10.sup.5 allogeneic cells (100 .mu.l of stock), when used
0.5.times.10.sup.5 allogeneic cell (100 .mu.l of stock). These
cells will be inactivated with Mitomycin C. Positive control 50
.mu.l of a 6.times. stock (anti-CD28/CD3) Total volume 300 .mu.l in
a 96-well flat bottom-total volume of a 96-well is 360 .mu.l.
Incubate for 4 days 100 .mu.l is removed for cytokine analysis with
Luminex CyTOF is conducted on the remaining 200 .mu.l. Supernatants
for Cytokine Profiling are removed after 1 day.
[0589] CyTOF has been previously described, for example in Bendall
et al. (Science, Vol. 332, 6 May 2011) and Bendall and Nolan
(Nature Biotechnology, Vol. 30 No. 7, July 2012), both of which are
incorporated by reference in their entireties herein. Human markers
employed in CyTOF staining are shown below in Table 6.
TABLE-US-00006 TABLE 6 Human Markers for CyTOF Staining Marker
Clone Metal HLA-DR L243 89Y CD3 UCHT1 115In CD27 O323 141Pr CD19
HIB19 142Nd CD134/OX40 Ber-ACT35 143Nd * Granzyme B GB11 144Nd
CD258/LIGHT 115520 145Nd CD8A RPA t8 146Nd CD45RO UCHL1 147Sm
CD226/DNAM-1 11A8 149Sm CD194/CCR4 L291H4 150Nd PD1 (CD279)
EH12.2H7 151Eu CD170 1A5 152Sm CD69 FN50 153Eu CD70 113-16 154Sm
CD4 RPA T4 155Gd CD8b SIDI8BEE 156Gd IL-17R W15177A 158Gd * CTLA-4
CD152 L3D10 159Tb CD278/ICOS C398.4A 160Gd * AHR FF3399 161Dy CD56
NCAM16.2 162Dy CD195/CCR5 J418F1 163Dy * Ki67 8D5 164Dy * FoxP3 Use
Ebio 165Ho CD40 5C3 166Er * Helios 22F6 168Er * PU.1 puph13 169Tm *
RORgt 1181A 170Er CD127/IL-7R 40131 171Yb CD38 HIT2 172Yb CD25
M-A251 173Yb CD86 IT2.2 174Yb * T-bet 4B10 175Lu * Perforin dG9
176Yb * denotes intracellular target while all other surface
targets
Luminex Multiplex Assay
[0590] The Luminex xMAP technology (formerly LabMAP, FlowMetrix)
uses digital signal processing capable of classifying polystyrene
beads (microspheres) dyed with distinct proportions of red and
near-infrared fluorophores. These proportions define `spectral
addresses` for each bead population. As a result, up to one hundred
different detection reactions can be carried out simultaneously on
the various bead populations in very small sample volumes (Earley
et al. Report from a Workshop on Multianalyte Microsphere Arrays.
Cytometry 2002; 50:239-242; Oliver et al. Clin Chem 1998;
44(9):2057-2060; Eishal and McCoy, Methods 38(4): 317-323, April
2006, all of which are incorporated by reference in their
entireties herein).
[0591] The Luminex Multiplex Assay is commercially available and is
described on the world wide web at
thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-a-
nalysis/luminex-multiplex-assays.html, incorporated by reference in
its entirety herein.
Mitomycin C Preparation of Cells
[0592] Mitomycin C is prepared from dry powder (2 mg per vial)
using 400.mu.1 of DMSO (500.times. stock=5 mg/ml), dissolved
completely and aliquoted into 25 ul volumes, and stored at -80C. 20
.mu.l of 1 aliquot is used in 10 ml warmed C5 to yield 10 .mu.g/ml
final working solution. The solution is filter sterilized.
[0593] The solution can be used on resuspended cells or adherent
cells in flasks.
[0594] Cells are incubated at 37 C for 30 minutes in the dark, then
washed in warm C5 3 times. Cells are resuspended in 1 ml X-VIVO. 40
ul are counted into 200 ul on plate. The cells are resuspended at a
final concentration of 1.times.10.sup.6/ml in X-VIVO (serum free
media, Lonza).
Example 2
[0595] The described invention provides an approach for restoring
immunologic balance in, for example, treating cancer, by targeting
multiple immunomodulators with a single cellular platform. This
approach enables the simultaneous modulation of multiple signals,
and affords a spatially restricted site of action, important
features that have limited traditional approaches for restoring
immunologic balance.
[0596] According to one aspect of the disclosed invention, a tumor
cell line variant expressing five or more recombinant peptides may
be generated for use as a tumor cell vaccine to treat a cancer. For
example, a tumor cell line may be selected for modification, and
lentiviral transfection of recombinant immune modulator sequences
may be used to stably integrate immunomodulators into the cell
genome. Example 3 below describes 7 lentiviral vectors (vector 1,
vector 2, vector 3, vector 4, vector 5, vector 6 and vector 7) that
may be used to stably integrate immunomodulators into the cell
genome.
[0597] According to some embodiments, two recombinant
immunomodulator proteins may be transfected simultaneously,
followed by transfections of two more recombinant immunomodulator
proteins simultaneously, followed by transfection of a single
recombinant immunomodulator protein to achieve the total of five
recombinant peptides for use as a tumor cell vaccine. According to
some embodiments, two recombinant peptides may be transfected
simultaneously, followed by transfection of a single recombinant
peptide, followed by transfection of a single recombinant peptide,
followed by transfection of a single recombinant peptide to achieve
the total of five recombinant peptides for use as a tumor cell
vaccine. According to some embodiments, a single recombinant
peptide is transfected, followed by transfection of two recombinant
peptides simultaneously, followed by transfection of two
recombinant peptides simultaneously to achieve a total of five
recombinant peptide for use as a tumor cell vaccine.
[0598] Example 3 below describes lentiviral vectors (vector 44,
vector 97, vector 84, vector 29, vector 107, vector 116, vector 86,
vector 18, vector 17, vector 98, vector 5, vector 30, vector 109,
vector 3, vector 4, vector 106, vector 16, vector 83, vector 31,
vector 12, vector 99, vector 121, vector 105, vector 32, vector 37,
vector 22, vector 19, vector 20, vector 89, vector 21, vector 23,
vector 108, vector 15, vector 124, vector 65, vector 64, vector 88,
vector 96, vector 14, vector 119, vector 120, vector 45, vector 60,
vector 59, vector 8, vector 128, vector 35, and vector 6) that may
be used to stably integrate immunomodulators into the cell
genome.
[0599] According to one embodiment, vector 44 comprises one or more
TNF family member immunomodulators. According to one embodiment,
vector 29 comprises one or more TNF family member immunomodulators.
According to one embodiment, vector 18 comprises one or more TNF
family member immunomodulators. According to one embodiment, vector
17 comprises one or more TNF family member immunomodulators.
According to one embodiment, vector 5 comprises one or more TNF
family member immunomodulators. According to one embodiment, vector
16 comprises one or more TNF family member immunomodulators.
According to one embodiment, vector 99 comprises one or more TNF
family member immunomodulators. According to one embodiment, vector
15 comprises one or more TNF family member immunomodulators.
According to one embodiment, vector 14 comprises one or more TNF
family member immunomodulators. According to one embodiment, vector
45 comprises one or more TNF family member immunomodulators.
According to one embodiment, vector 6 comprises one or more TNF
family member immunomodulators. According to one embodiment, the
one or more TNF family immunomodulators are selected from those
listed in Table 2 or Table 3.
[0600] According to one embodiment, vector 44 comprises between
3-14 TNF family member immunomodulators. According to one
embodiment, vector 29 comprises between 3-14 TNF family member
immunomodulators. According to one embodiment, vector 18 comprises
between 3-14 TNF family member immunomodulators. According to one
embodiment, vector 17 comprises between 3-14 TNF family member
immunomodulators. According to one embodiment, vector 5 comprises
between 3-14 TNF family member immunomodulators. According to one
embodiment, vector 16 comprises between 3-14 TNF family member
immunomodulators. According to one embodiment, vector 99 comprises
between 3-14 TNF family member immunomodulators. According to one
embodiment, vector 15 comprises between 3-14 TNF family member
immunomodulators. According to one embodiment, vector 14 comprises
between 3-14 TNF family member immunomodulators. According to one
embodiment, vector 45 comprises between 3-14 TNF family member
immunomodulators. According to one embodiment, vector 6 comprises
between 3-14 TNF family member immunomodulators. According to one
embodiment, the between 3-14 TNF family immunomodulators are
selected from those listed in Table 2 or Table 3.
[0601] According to one embodiment, vector 97 comprises one or more
Ig family member immunomodulators. According to one embodiment,
vector 84 comprises one or more Ig family member immunomodulators.
According to one embodiment, vector 107 comprises one or more Ig
family member immunomodulators. According to one embodiment, vector
98 comprises one or more Ig family member immunomodulators.
According to one embodiment, vector 30 comprises one or more Ig
family member immunomodulators. According to one embodiment, vector
83 comprises one or more Ig family member immunomodulators.
According to one embodiment, vector 121 comprises one or more Ig
family member immunomodulators. According to one embodiment, vector
119 comprises one or more Ig family member immunomodulators.
According to one embodiment, the one or more Ig family member
immunomodulators are selected from those listed in Table 2 or Table
3.
[0602] According to one embodiment, vector 97 comprises between
3-14 Ig family member immunomodulators. According to one
embodiment, vector 84 comprises between 3-14 Ig family member
immunomodulators. According to one embodiment, vector 107 comprises
between 3-14 Ig family member immunomodulators. According to one
embodiment, vector 98 comprises between 3-14 Ig family member
immunomodulators. According to one embodiment, vector 30 comprises
between 3-14 Ig family member immunomodulators. According to one
embodiment, vector 83 comprises between 3-14 Ig family member
immunomodulators. According to one embodiment, vector 121 comprises
between 3-14 Ig family member immunomodulators. According to one
embodiment, vector 119 comprises between 3-14 Ig family member
immunomodulators. According to one embodiment, the between 3-14 Ig
family member immunomodulators are selected from those listed in
Table 2 or Table 3.
[0603] According to one embodiment, vector 116 comprises one or
more chemokine immunomodulators. According to one embodiment, the
one or more chemokine immunomodulators are selected from those
listed in Table 2 or Table 3.
[0604] According to one embodiment, vector 116 comprises between
3-14 chemokine immunomodulators. According to one embodiment, the
between 3-14 chemokine immunomodulators are selected from those
listed in Table 2 or Table 3.
[0605] According to one embodiment, vector 109 comprises one or
more growth factor immunomodulators.
[0606] According to one embodiment, vector 109 comprises between
3-14 growth factor immunomodulators.
[0607] According to one embodiment, vector 3 comprises one or more
cytokine immunomodulators. According to one embodiment, vector 4
comprises one or more cytokine immunomodulators. According to one
embodiment, vector 32 comprises one or more cytokine
immunomodulators. According to one embodiment, vector 22 comprises
one or more cytokine immunomodulators. According to one embodiment,
vector 19 comprises one or more cytokine immunomodulators.
According to one embodiment, vector 20 comprises one or more
cytokine immunomodulators. According to one embodiment, vector 89
comprises one or more cytokine immunomodulators. According to one
embodiment, vector 21 comprises one or more cytokine
immunomodulators. According to one embodiment, vector 23 comprises
one or more cytokine immunomodulators. According to one embodiment,
vector 121 comprises one or more cytokine immunomodulators.
According to one embodiment, vector 65 comprises one or more
cytokine immunomodulators. According to one embodiment, vector 64
comprises one or more cytokine immunomodulators. According to one
embodiment, vector 88 comprises one or more cytokine
immunomodulators. According to one embodiment, vector 96 comprises
one or more cytokine immunomodulators. According to one embodiment,
vector 60 comprises one or more cytokine immunomodulators.
According to one embodiment, vector 59 comprises one or more
cytokine immunomodulators. According to one embodiment, vector 128
comprises one or more cytokine immunomodulators. According to one
embodiment, the one or more cytokine immunomodulators are selected
from those listed in Table 2 or Table 3.
[0608] According to one embodiment, vector 3 comprises between 3-14
cytokine immunomodulators. According to one embodiment, vector 4
comprises between 3-14 cytokine immunomodulators. According to one
embodiment, vector 32 comprises between 3-14 cytokine
immunomodulators. According to one embodiment, vector 22 comprises
between 3-14 cytokine immunomodulators. According to one
embodiment, vector 19 comprises between 3-14 cytokine
immunomodulators. According to one embodiment, vector 20 comprises
between 3-14 cytokine immunomodulators. According to one
embodiment, vector 89 comprises between 3-14 cytokine
immunomodulators. According to one embodiment, vector 21 comprises
between 3-14 cytokine immunomodulators. According to one
embodiment, vector 23 comprises between 3-14 cytokine
immunomodulators. According to one embodiment, vector 121 comprises
between 3-14 cytokine immunomodulators. According to one
embodiment, vector 65 comprises between 3-14 cytokine
immunomodulators. According to one embodiment, vector 64 comprises
between 3-14 cytokine immunomodulators. According to one
embodiment, vector 88 comprises between 3-14 cytokine
immunomodulators. According to one embodiment, vector 96 comprises
between 3-14 cytokine immunomodulators. According to one
embodiment, vector 60 comprises between 3-14 cytokine
immunomodulators. According to one embodiment, vector 59 comprises
between 3-14 cytokine immunomodulators. According to one
embodiment, vector 128 comprises between 3-14cytokine
immunomodulators. According to one embodiment, the between 3-14
cytokine immunomodulators are selected from those listed in Table 2
or Table 3.
[0609] According to one embodiment, vector 37 comprises one or more
receptor immunomodulators. According to one embodiment, vector 124
comprises one or more receptor immunomodulators. According to one
embodiment, vector 88 comprises one or more receptor
immunomodulators. According to one embodiment, vector 8 comprises
one or more receptor immunomodulators. According to one embodiment,
the one or more receptor immunomodulators are selected from those
listed in Table 2 or Table 3.
[0610] According to one embodiment, vector 37 comprises between
3-14 receptor immunomodulators. According to one embodiment, vector
124 comprises between 3-14 receptor immunomodulators. According to
one embodiment, vector 88 comprises between 3-14 receptor
immunomodulators. According to one embodiment, vector 8 comprises
between 3-14 receptor immunomodulators. According to one
embodiment, the between 3-14 receptor immunomodulators are selected
from those listed in Table 2 or Table 3.
[0611] According to one embodiment, vector 86 comprises one or more
other immunomodulators. According to one embodiment, vector 106
comprises one or more other immunomodulators. According to one
embodiment, vector 107 comprises one or more other
immunomodulators. According to one embodiment, vector 31 comprises
one or more other immunomodulators. According to one embodiment,
vector 12 comprises one or more other immunomodulators. According
to one embodiment, vector 105 comprises one or more other
immunomodulators. According to one embodiment, vector 108 comprises
one or more other immunomodulators. According to one embodiment,
vector 120 comprises one or more other immunomodulators. According
to one embodiment, vector 35 comprises one or more other
immunomodulators. According to one embodiment, the one or more
other immunomodulators are selected from those listed in Table 2 or
Table 3.
[0612] According to one embodiment, vector 86 comprises between
3-25 other immunomodulators. According to one embodiment, vector
106 comprises between 3-25 other immunomodulators. According to one
embodiment, vector 107 comprises between 3-25 other
immunomodulators. According to one embodiment, vector 31 comprises
between 3-25 other immunomodulators. According to one embodiment,
vector 12 comprises between 3-25 other immunomodulators. According
to one embodiment, vector 105 comprises between 3-25 other
immunomodulators. According to one embodiment, vector 108 comprises
between 3-25 other immunomodulators. According to one embodiment,
vector 120 comprises between 3-25 other immunomodulators. According
to one embodiment, vector 35 comprises between 3-25 other
immunomodulators. According to one embodiment, the between 3-25
other immunomodulators are selected from those listed in Table 2 or
Table 3.
[0613] According to one embodiment of the disclosed invention,
combinations of allogeneic cell pools, each expressing a single
immunomodulatory protein, are used to model what a single cell
expressing multiple immunomodulatory proteins might do (e.g.
additivity, synergy, interference).
[0614] According to one aspect of the disclosed invention, a tumor
cell line variant expressing one, two three, four or five or more
recombinant peptides may be generated for use as a tumor cell
vaccine to treat skin cancer. For example, the SK-MEL2 human
melanoma cell line (ATCC HTB-68) may be selected for modification,
and lentiviral transfection of recombinant immune modulator
sequences may be used to stably integrate immunomodulators into the
cell genome.
[0615] According to one aspect of the disclosed invention, a tumor
cell line variant expressing one, two three, four or five or more
recombinant peptides may be generated for use as a tumor cell
vaccine to treat a prostate cancer. For example, the DU-145 human
prostate carcinoma cell line may be selected for modification, and
lentiviral transfection of recombinant immune modulator sequences
may be used to stably integrate immunomodulators into the cell
genome. According to some embodiments, two recombinant
immunomodulator proteins may be transfected simultaneously,
followed by transfections of two more recombinant immunomodulator
proteins simultaneously, followed by transfection of a single
recombinant immunomodulator protein to achieve the total of five
recombinant peptides for use as a tumor cell vaccine. According to
some embodiments, two recombinant peptides may be transfected
simultaneously, followed by transfection of a single recombinant
peptide, followed by transfection of a single recombinant peptide,
followed by transfection of a single recombinant peptide to achieve
the total of five recombinant peptides for use as a tumor cell
vaccine. According to some embodiments, a single recombinant
peptide is transfected, followed by transfection of two recombinant
peptides simultaneously, followed by transfection of two
recombinant peptides simultaneously to achieve a total of five
recombinant peptide for use as a tumor cell vaccine.
[0616] According to another aspect of the present invention, two or
more tumor cell line variants expressing one or more recombinant
peptides may be generated for use as a tumor cell vaccine to treat
a prostate cancer. For example, the DU-145 and PC-3 human prostate
carcinoma cell line may be selected for modification, and
lentiviral transfection of recombinant immune modulator sequences
may be used to stably integrate immunomodulators into the cell
genome.
CD40L Immunomodulator
[0617] The CD40L immune modulator cDNA sequence may be cloned into
the lentiviral transfer plasmid construct pLenti-puro (Addgene Cat.
No. 39481) driven by a CMV promoter with puromycin selectable
marker. The CD40L immune modulator cDNA sequence may be engineered
to be non-cleavable, which ultimately keeps the translated CD40L
protein in a membrane bound state (e.g. SEQ ID NO: 7). A human
influenza hemagglutinin tag (HA tag) may be also cloned onto the
extracellular portion of the CD40L sequences. The translated HA tag
has the peptide sequence YPYDVPDYA (SEQ ID NO: 28). Packaging
plasmid psPAX2 (AddGene Cat. No. 12260) and envelope plasmid
pLTR-RD114A (AddGene Cat. No. 17576) may also be selected for the
lentiviral system.
[0618] Each of the lentiviral transfer plasmid, packaging plasmid,
and envelope plasmid may be transfected into log phase growth 293T
cells using Lipofectamine 2000 (ThermoFisher Cat. No. 11668027).
Briefly, cells are seeded at 70% to 90% confluence. On the day of
transfection, 12 .mu.l of Lipofectamine reagent is diluted in 150
.mu.l of serum free cell media. 5 .mu.g of DNA for transfection is
also diluted in 150 .mu.l of serum free media. The diluted DNA is
then added to the diluted Lipofectamine and incubated for 5 minutes
at room temperature. The total volume of the mixture is then added
dropwise to the media of the seeded 293T cells while swirling.
Cells are then incubated for one to three days at 37 degrees.
[0619] The 293T cell culture medium comprising virus particle is
harvested 3 times every 8-12 hours and centrifuged to pellet
detached cells and debris. The culture medium containing virus
particles is used directly to infect the DU-145 cell line.
[0620] The DU-145 cell line is cultured in Eagle's Minimum
Essential Medium (EMEM) with 10% fetal bovine serum to a confluency
of about 70%. Hexadimethrine bromide (Sigma-Aldrich Cat No. H9268)
is then combined with media containing virus particles to make a
final concentration of 8 .mu.g/mL Hexadimethrine bromide. Culture
media of the DU-145 cells is aspirated and replaced with media
containing virus particles and 8 .mu.g/mL Hexadimethrine bromide.
DU-145 cells are cultured for 18-20 hours followed by media
change.
[0621] Infected DU-145 cells are then grown in media containing 1
.mu.g/mL Puromycin (ThermoFisher Cat. No. A1113802) until cell die
off begins after about a week. Multiple surviving colonies of
transfected cells are picked for expansion and tested for CD40L
expression by Western blot. The Western blot is probed with mouse
monoclonal anti-HA primary antibodies (Abcam Cat. No. ab18181) and
goat anti-mouse HRP (Abcam Cat. No. ab205719) secondary antibodies
to quantify the relative amounts of recombinant CD40L expressed in
each clonal line. The highest stably expressing DU-145 line is
labeled DU145-Genl and selected for further manipulation.
TNF-alpha/GM-CSF
[0622] The DU145-Genl cells transfected to express CD40L are
further transfected with a bi-cistronic lentiviral vector
comprising TNF-alpha and GM-CSF sequences. Each of TNF-alpha cDNA
and GM-CSF cDNA is first cloned into the pEF1.alpha.-IRES
bicistronic mammalian expression vector (Clontech Cat. No. 631970)
under the control of the human elongation factor 1 alpha
(EF1.alpha.) promoter. A variant of TNF-alpha that cannot be
cleaved by TACE is used so that the translated protein remains in
membrane bound form. The TNF-alpha sequence is provided with a FLAG
tag sequence on the extracellular region of TNF-alpha for easy
detection of translated protein. The FLAG tag peptide sequence is
DYKDDDDK (SEQ ID NO: 29). GM-CSF sequences capable of forming
soluble GM-CSF are used. The entirety of the pEFl promoter,
TNF-alpha sequences, IRES sequences, and GM-CSF sequences is then
cloned into the pLenti-puro (Addgene Cat. No. 39481) lentiviral
vector (the original CMV promoter from the vector is removed during
this process). Packaging plasmid psPAX2 (AddGene Cat. No. 12260)
and envelope plasmid pLTR-RD114A (AddGene Cat. No. 17576) are also
selected.
[0623] Each of the lentiviral transfer plasmid, packaging plasmid,
and envelope plasmid is transfected into log phase growth 293T
cells using Lipofectamine 2000 (ThermoFisher Cat. No. 11668027).
Briefly, cells are seeded at 70% to 90% confluence. On the day of
transfection, 12 .mu.l of Lipofectamine reagent is diluted in 150
.mu.l of serum free cell media. 5 .mu.g of DNA for transfection is
also diluted in 150 .mu.l of serum free media. The diluted DNA is
then added to the diluted Lipofectamine and incubated for 5 minutes
at room temperature. The total volume of the mixture is then added
dropwise to the media of the seeded 293T cells while swirling.
Cells are then incubated for one to three days at 37 degrees.
[0624] The 293T cell culture medium comprising virus particle is
harvested 3 times every 8-12 hours and centrifuged to pellet
detached cells and debris. The culture medium containing virus
particles is used directly to infect the DU145-Gen1 cell line.
[0625] The DU145-Gen1 cell line is cultured to a confluency of
about 70%. Hexadimethrine bromide (Sigma-Aldrich Cat No. H9268) is
then combined with media containing virus particles to make a final
concentration of 8 .mu.g/mL Hexadimethrine bromide. Culture media
of the DU145-Gen1 cells is aspirated and replaced with media
containing virus particles and 8 .mu.g/mL Hexadimethrine bromide.
DU145-Genl cells are cultured for 18-20 hours followed by media
change.
[0626] The transduced DU145-Gen1 cells are then selected for clones
that stably express the recombinant immunomodulators. The selection
process is performed by fluorescence activated cell sorting using
the FLAG tag on the TNF-alpha to identify cells that have
integrated the immunomodulators. Live cells are probed with mouse
monoclonal anti-FLAG antibody (Sigma Aldrich F3040) and rabbit
anti-mouse FITC conjugated secondary antibody (Sigma Aldrich
ASB3701170) in PBS with blocking buffer. The highest expressing
cells are sorted, isolated, and cultured for further processing.
After sorting based on the presence of the FLAG tag, expression of
soluble GM-CSF is confirmed by Western blot. Concentrated media of
sorted cultured cells is resolved by SDS-PAGE and probed by Western
blot with mouse anti-GM-CSF antibody (ThermoFisher Cat. No. 3092)
and goat anti-mouse HRP conjugated secondary antibody. Cell lysate
may also be resolved by SDS-PAGE and probed for FLAG tag to verify
the presence of TNF. Cell cultures that express high levels of
recombinant GM-CSF and TNF-alpha are designated DU145-Gen2 and
selected for further processing.
Flt-3L
[0627] The DU145-Gen2 cells transfected to express CD40L, GM-CSF,
and TNF are further transfected with a lentiviral vector comprising
Flt-3L immune modulator sequences. The Flt-3L cDNA is cloned into a
pEF1.alpha.-IRES bicistronic mammalian expression vector (Clontech
Cat. No. 631970), along with GFP protein sequences to be used as a
marker for integration and expression. The sequence of Flt-3L is
translated into a membrane bound peptide, while the GFP remains
cytoplasmic. The entirety of the pEF1 promoter, Flt-3L sequences,
IRES sequences, and GFP sequences is then cloned into the
pLenti-puro (Addgene Cat. No. 39481) lentiviral vector (the
original CMV promoter from the vector is removed during this
process). Packaging plasmid psPAX2 (AddGene Cat. No. 12260) and
envelope plasmid pLTR-RD114A (AddGene Cat. No. 17576) are also
selected.
[0628] Each of the lentiviral transfer plasmid, packaging plasmid,
and envelope plasmid is transfected into log phase growth 293T
cells using Lipofectamine 2000 (ThermoFisher Cat. No. 11668027).
Briefly, cells are seeded at 70% to 90% confluence. On the day of
transfection, 12 .mu.l of Lipofectamine reagent is diluted in 150
.mu.l of serum free cell media. 5 .mu.g of DNA for transfection is
also diluted in 150 .mu.l of serum free media. The diluted DNA is
then added to the diluted Lipofectamine and incubated for 5 minutes
at room temperature. The total volume of the mixture is then added
dropwise to the media of the seeded 293T cells while swirling.
Cells are then incubated for one to three days at 37 degrees.
[0629] The 293T cell culture medium comprising virus particle is
harvested 3 times every 8-12 hours and centrifuged to pellet
detached cells and debris. The culture medium containing virus
particles is used directly to infect the DU145-Gen2 cell line.
[0630] The DU145-Gen2 cell line is cultured to a confluency of
about 70%. Hexadimethrine bromide (Sigma-Aldrich Cat No. H9268) is
then combined with media containing virus particles to make a final
concentration of 8 .mu.g/mL Hexadimethrine bromide. Culture media
of the DU145-Gen2 cells is aspirated and replaced with media
containing virus particles and 8 .mu.g/mL Hexadimethrine bromide.
DU145-Gen2 cells are cultured for 18-20 hours followed by media
change.
[0631] The DU145-Gen2 cells are then selected for cells stably
expressing the Flt-3L sequences using the GFP marker. The selection
process is performed by fluorescence activated cell sorting (FACS)
using the GFP marker to identify cells that have integrated the
immune modulator. The highest expressing cells are sorted,
isolated, and cultured for further processing. After sorting based
on the presence of the GFP marker, the expression of Flt-3L is
confirmed by Western blot. Cultured cell lysates are resolved by
SDS-PAGE and probed by Western blot with rabbit polyclonal
anti-Flt-3L antibody (AbCam Cat. No. ab9688) and goat anti-rabbit
HRP conjugated secondary antibody (AbCam Cat. No. ab205718). Cell
cultures that express high levels of recombinant Flt-3L are
designated DU145-Gen3 and are selected for further processing.
IgG Heavy Chain
[0632] The DU145-Gen3 cells transfected to express CD40L, GM-CSF,
TNF-alpha, and Flt-3L are further transfected with a lentiviral
vector comprising IgG 1 (SEQ ID NO: 1), a membrane bound IgG1 heavy
chain fragment. The IgG1 heavy chain cDNA is cloned into
pEF1.alpha.-IRES bicistronic mammalian expression vector (Clontech
Cat. No. 631970), along with RFP protein sequences to be used as a
marker for integration and expression. The sequence of IgG1 heavy
chain is translated into a membrane bound peptide, while the RFP
remains cytoplasmic. The entirety of the pEF1 promoter, IgG1 heavy
chain sequence, IRES sequence, and RFP sequence is then cloned into
the pLenti-puro (Addgene Cat. No. 39481) lentiviral vector (the
original CMV promoter from the vector is removed during this
process). Packaging plasmid psPAX2 (AddGene Cat. No. 12260) and
envelope plasmid pLTR-RD114A (AddGene Cat. No. 17576) are also
selected.
[0633] Each of the lentiviral transfer plasmid, packaging plasmid,
and envelope plasmid is transfected into log phase growth 293T
cells using Lipofectamine 2000 (ThermoFisher Cat. No. 11668027).
Briefly, cells are seeded at 70% to 90% confluence. On the day of
transfection, 12 .mu.l of Lipofectamine reagent is diluted in 150
.mu.l of serum free cell media. 5 .mu.g of DNA for transfection is
also diluted in 150 .mu.l of serum free media. The diluted DNA is
then added to the diluted Lipofectamine and incubated for 5 minutes
at room temperature. The total volume of the mixture is then added
dropwise to the media of the seeded 293T cells while swirling.
Cells are then incubated for one to three days at 37 degrees.
[0634] The 293T cell culture medium comprising virus particle is
harvested 3 times every 8-12 hours and centrifuged to pellet
detached cells and debris. The culture medium containing virus
particles is used directly to infect the DU145-Gen3 cell line.
[0635] The DU145-Gen3 cell line is cultured to a confluency of
about 70%. Hexadimethrine bromide (Sigma-Aldrich Cat No. H9268) is
then combined with media containing virus particles to make a final
concentration of 8 .mu.g/mL Hexadimethrine bromide. Culture media
of the DU145-Gen2 cells is aspirated and replaced with media
containing virus particles and 8 .mu.g/mL Hexadimethrine bromide.
DU145-Gen3 cells are cultured for 18-20 hours followed by media
change.
[0636] The DU145-Gen3 cells are then selected for cells stably
expressing the IgG1 heavy chain sequences using the RFP marker. The
selection process is performed by fluorescence activated cell
sorting (FACS) using the RFP marker to identify cells that have
integrated the immune modulator. The highest expressing cells are
sorted, isolated, and cultured for further processing. After
sorting based on the presence of the RFP marker, the expression of
IgG1 heavy chain is confirmed by Western blot. Cell cultures that
express high levels of recombinant IgG1 heavy chain are designated
DU145-Gen4 and are selected for further processing.
[0637] The DU145-Gen4 tumor cell line transfected to express CD40L,
GM-CSF, TNF, Flt-3L, and IgG1 heavy chain is characterized by
RT-PCR, immunofluorescence, and Western blotting to confirm all
recombinant immunomodulators are expressed by the cells and are in
the right location (e.g. on the membrane of the cell).
Human Mixed Lymphocyte Tumor Reaction (MLTR) Testing
[0638] The DU145-Gen4 cells are tested for their immunomodulatory
potential by primary and secondary MLTR assay against each of the
other generations (i.e. DU145-Gen2 and DU145-Gen3) of modified
cells and unmodified DU145 cells.
[0639] Peripheral blood mononuclear cells (PBMCs) are obtained from
the peripheral blood of healthy individuals and from prostate
cancer patients, and the blood cells separated using a Ficoll-Paque
gradient. Anticoagulant-treated blood is diluted in the range of
1:2 to 1:4 with PBS/EDTA to reduce aggregation of erythrocytes. The
diluted blood is then layered above a Ficoll-Paque solution in a
centrifuge tube, without mixing. The layered blood/Ficoll-Paque is
centrifuged for 40 minutes at 400.times.g between 18.degree. and
20.degree. C., without the use of the centrifuge brake, resulting
in the formation of blood fractions. The fraction comprising
mononuclear cells is selected for further processing.
[0640] Each of the cells from the transfected tumor cell line
variants and from parental tumor cell line DU-145 (control) is
co-cultured with PBMCs for seven days under standard tissue culture
conditions, followed by evaluation for immune cell proliferation,
immune cell differentiation, measured by flow cytometry and CyTOF,
cytokine release profile, and cytoxicity, measured by LDH release
assay.
Example 3
[0641] Vectors as employed herein are described in detail as
follows:
Vector 1. Immunomodulator: scFv-Anti-Biotin-G3hinge-mIgG1 (to
Generate Surface IgG)
[0642] A schematic of the organization of vector 1, used for the
immunomodulator scFv-anti-biotin-G3hinge-mIgG1 is shown in FIG. 2.
Table 7, below, shows the vector component name, the corresponding
nucleotide position in SEQ ID NO. 47, the full name of the
component and a description.
TABLE-US-00007 TABLE 7 Component Nucleotide Name Position Full Name
Description RSV 1-229 Rous sarcoma virus Allows Tat-independent
production promoter (RSV) enhancer/ of viral mRNA. promoter
.DELTA.5' LTR 230-410 HIV-1 truncated 5' Permits viral packaging
and reverse LTRT transcription of the viral mRNA. .PSI. 521-565
HIV-1 psi packaging Allows viral packaging. signal RRET 1075-1308
HIV-1 Rev response Permits Rev-dependent nuclear element export of
unspliced viral mRNA. cPPT 1803-1920 Central polypurine Facilitates
the nuclear import of tract HIV-1 cDNA through a central DNA flap.
EF1A 1959-3137 EF1A Component entered by user Kozak 3162-3167 Kozak
Component entered by user {ORF1} 3168-5005 {ORF1} Component entered
by user WPRE 5044-5641 Woodchuck hepatitis Facilitates effective
transcription virus termination at the 3'TLTR.T posttranscriptional
regulatory element .DELTA.U3/3' LTR 5723-5957 HIV-1 truncated 3'
Allows viral packaging but self- LTR inactivates the 5'LTR for
biosafety purposes. The element also contains apolyadenylation
signal for transcription termination and polyadenylation of mRNA in
transduced cells. SV40 early 6030-6164 SV40 early Allows
transcription termination pA polyadenation signal and
polyadenylation of mRNA. Ampicillin 7118-7978 Ampicillin resistance
Allows selection of the plasmid in gene E. coli. pUC ori 8149-8737
pUC origin of Permits high-copy replication and replication
maintenance in E. coli.
[0643] When vector 1 is employed, anti-IgG is used for flow
detection. A biotin+ fluorescent labelled oligodeoxynucleotides
(ODN) is used as a secondary detection method.
[0644] The following is a description of the immunomodulator
scFv-anti-biotin-G3hinge-IgG1-Tm.
[0645] Type:
Immunoglobulin
[0646] Annotation: [0647] H7 heavy chain leader [0648] Anti-biotin
Variable Heavy chain (VH) allows for loading biotin labeled CpG
[0649] Inter-domain disulfide linkage VH44 (G->C) and VL100
(G->C) [0650] IgG3 hinge to enhance Fc.gamma.R interaction
[0651] Linkage is standard [0652] IgG1 (CH2-CH3-Tm-Cyt) used for
interaction with Fc.gamma.R/FcRn and membrane anchoring [0653]
T233A mutation to enhance FcRn and Fc.gamma.R interaction
[0654] The sequences are shown as follows:
TABLE-US-00008 H7 heavy chain leader (SEQ ID NO. 54)
MEFGLSWVFLVALFRGVQC anti-biotin murine vH with inserted Cys for
inter-domain linkage (SEQ ID NO. 55) QVKLQESGPG LVAPSQSLSI
TCTVSGFSLT AYGVDWVRQP PGKCLEWLGV IWGGGRTNYN SGLMSRLSIR KDNSKSQVFL
TMNSLQTDDT AKYYCVKHTN WDGGFAYWGQ GTTVTVSS linker (SEQ ID NO. 56)
GGGGSGGGGS GGGGS Light Chain Variable (human lambda variable) (SEQ
ID NO. 57) GSPGQSVSIS CSGSSSNIGN NYVYWYQHLP GTAPKLLIYS DTKRPSGVPD
RISGSKSGTS ASLAISGLQS EDEADYYCAS WDDSLDGPVF GCGTKLTVL IgG3 hinge
for greater accessibility to FcyR (SEQ ID NO. 58) LKTPLGDTTHTCPR
CPEPKSCDTP PPCPRCPEPK SCDTPPPCPR CPEPKSCDTP PPCPRCP IgG1 CH2, CH3
Tm and cytoplasmic tail (T256A) (SEQ ID NO. 59) LLGGPSVFLF
PPKPKDTLMI SRAPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV
SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV
SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF
SCSVMHEALH NHYTQKSLSL SPELQLEESC AEAQDGELDG LWTTITIFIT LFLLSVCYSA
TVTFFKVKWI FSSVVDLKQT IIPDYRNMIG QGA*
scFv-anti-biotin-G3hinge-IgG1-Tm (598 ORF1) (SEQ ID NO. 60)
MEFGLSWVFLVALFRGVQCQVKLQESGPGLVAPSQSLSITCTVSGFSLTA
YGVDWVRQPPGKCLEWLGVIWGGGRTNYNSGLMSRLSIRKDNSKSQVFLT
MNSLQTDDTAKYYCVKHTNWDGGFAYWGQGTTVTVSSGGGGSGGGGSGGG
GSGSPGQSVSISCSGSSSNIGNNYVYWYQHLPGTAPKWYSDTKRPSGV
PDRISGSKSGTSASLAISGLQSEDEADYYCASWDDSLDGPVFGCGTKLTV
LLKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPK
SCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRAPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPELQLEESCAEAQDGELDGLWTTI
TIFITLFLLSVCYSATVTFFKVKWIFSSVVDLKQTIIPDYRNIVIIGQGA*
Vector 2. Immunomodulator: Full Anti-Biotin-G3hinge-mIgG1 (Using
Heavy Chain/Ires/Light Chain)
[0655] A schematic of the organization of vector 2, used for the
immunomodulator full anti-biotin-G3hinge-mIgG1 is shown in FIG. 3.
Vector 2 is bicistronic. Table 8, below, shows the vector component
name, the corresponding nucleotide position in SEQ ID NO. 48, the
full name of the component and a description.
TABLE-US-00009 TABLE 8 Component Nucleotide Name Position Full Name
Description RSV 1-229 Rous sarcoma virus Allows Tat-independent
promoter (RSV) enhancer/ production of viral mRNA. promoter
.DELTA.5' LTR 230-410 HIV-1 truncated 5' Permits viral packaging
and LTRT reverse transcription of the viral mRNA. .PSI. 521-56g
HIV-1 psi packaging Allows viral packaging. signal RRET 1075-1308
HIV-1 Rev response Permits Rev-dependent nuclear element export of
unspliced viral mRNA. cPPT 1803-1920 Central polypurine Facilitates
the nuclear import tract of HIV-1 cDNA through a central DNA flap.
EF1A 1950-3128 EF1A Component entered by user Kozak 3153-3158 Kozak
Component entered by user {ORF1} 3159-5342 {ORF2A} Component
entered by user WPRE 6703-7300 Woodchuck hepatitis Facilitates
effective virus transcription termination at posttranscriptional
the 3'TLTR.T regulatory element .DELTA.U3/3' LTR 7382-7616 HIV-1
truncated 3' Allows viral packaging but LTR self-inactivates the
5'LTR for biosafety purposes. The element also contains
apolyadenylation signal for transcription termination and
polyadenylation of mRNA in transduced cells. SV40 early 7689-7823
SV40 early Allows transcription termination pA polyadenation signal
and polyadenylation of mRNA. Ampicillin 8777-9637 Ampicillin
resistance Allows selection of the plasmid gene in E. coli. pUC ori
9808-10396 pUC origin of Permits high-copy replication replication
and maintenance in E. coli.
[0656] When vector 2 is employed, anti-IgG is used for flow
detection. Biotin+ fluorescent labelled ODN is used as a secondary
detection method.
[0657] The following is a description of the immunomodulator full
anti-biotin-G3hinge-mIgG1 (using heavy chain/ires/light chain).
[0658] Type:
Membrane anchored Immunoglobulin
[0659] Annotation: [0660] H7 heavy chain leader [0661] IgG3 hinge
to enhance Fc.gamma.R interaction [0662] T233A mutation to enhance
FcRn and Fc.gamma.R interaction [0663] Anti-biotin Variable H
allows for loading biotin labeled CpG [0664] CH1 (generic) [0665]
LC Variable (human lambda variable) [0666] LC Constant Region 1
from Lambda (http://www.uniprot.org/uniprot/P0CG04) [0667]
Interdomain disulfide linkage VH44 (G->C) and VL100 (G->C)
(ref) [0668] Linkage is standard [0669] IgG1 (CH2-CH3-Tm-Cyt) for
interaction with Fc.gamma.R/FcRn and membrane anchoring [0670] L1
light chain leader (modified for IRES) MATDMRVPAQLLGLLLLWLSGARC
(SEQ ID NO. 61)
[0671] The sequences are shown as follows:
TABLE-US-00010 H7 heavy chain leader (SEQ ID NO. 54)
MEFGLSWVFLVALFRGVQC anti-biotin vH (murine) (SEQ ID NO. 62)
QVKLQESGPG LVAPSQSLSI TCTVSGFSLT AYGVDWVRQP PGKGLEWLGV IWGGGRTNYN
SGLMSRLSIR KDNSKSQVFL TMNSLQTDDT AKYYCVKHTN WDGGFAYWGQ GTTVTVSS CH1
(generic) (SEQ ID NO. 63) PSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS
WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVE
IgG3 hinge for greater accessibility to FcyR (SEQ ID NO. 64) LKTP
LGDTTHTCPR CPEPKSCDTP PPCPRCPEPK SCDTPPPCPR CPEPKSCDTP PPCPRCP IgG1
CH2, CH3 Tm and cytoplasmic tail (T256A) (SEQ ID NO. 65)
APELLGGPSVFLF PPKPKDTLMI SRAPEVTCVV VDVSHEDPEV KFNWYVDGVE
VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP
REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS
FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPELQLEESC AEAQDGELDG
LWTTITIFIT LFLLSVCYSA TVTFFKVKWI FSSVVDLKQT IIPDYRNMIG QGA* Summary
(578 ORF2a) (SEQ ID NO. 66)
MEFGLSWVFLVALFRGVQCQVKLQESGPGLVAPSQSLSITCTVSGFSLTA
YGVDWVRQPPGKGLEWLGVIWGGGRTNYNSGLMSRLSIRKDNSKSQVFLT
MNSLQTDDTAKYYCVKHTNWDGGFAYWGQGTTVTVSSPSVFPLAPSSKST
SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVELKTPLGDTTHTCPRCPEPK
SCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGP
SVFLFPPKPKDTLMISRAPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
KSLSLSPELQLEESCAEAQDGELDGLWTTITIFITLFLLSVCYSATVTFF
KVKWIFSSVVDLKQTIIPDYRNMIGQGA* (SEQ ID NO. 67) IRES L1 Signal
(modified to be IRES compatible) (SEQ ID NO. 61)
MATDMRVPAQLLGLLLLWLSGARC LC Variable (human lambda variable) (SEQ
ID NO. 69) GSPGQSVSIS CSGSSSNIGN NYVYWYQHLP GTAPKLLIYS DTKRPSGVPD
RISGSKSGTS ASLAISGLQS EDEADYYCAS WDDSLDGPVF GGGTKLTVL LC Constant
Region 1 from Lambda (http://www. uniprot.org/uniprot/P0CG04)
(irrelevant) (SEQ ID NO. 70) GQPKANPTVT LFPPSSEELQ ANKATLVCLI
SDFYPGAVTV AWKADGSPVK AGVETTKPSK QSNNKYAASS YLSLTPEQWK SHRSYSCQVT
HEGSTVEKTV APTECS* Summary (229 ORF2b) (SEQ ID NO. 71)
MATDMRVPAQLLGLLLLWLSGARCGSPGQSVSISCSGSSSNIGNNYVYWY
QHLPGTAPKWYSDTKRPSGVPDRISGSKSGTSASLAISGLQSEDEADY
YCASWDDSLDGPVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLV
CLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPE
QWKSHRSYSCQVTHEGSTVEKTVAPTECS*
Vector 3. Immunomodulator: sGM-CSF/Ires/mFLT3L
[0672] A schematic of the organization of vector 3, used for the
immunomodulator sGM-CSF/ires/mFLT3L is shown in FIG. 4. Vector 3 is
bicistronic. Table 9, below, shows the vector component name, the
corresponding nucleotide position in SEQ ID NO. 49, the full name
of the component and a description.
TABLE-US-00011 TABLE 9 Component Nucleotide Name Position Full Name
Description RSV promoter 1-229 Rous sarcoma virus (RSV) Allows
Tat-independent production enhancer/promoter of viral mRNA.
.DELTA.5' LTR 230-410 HIV-1 truncated 5' LTRT Permits viral
packaging and reverse transcription of the viral mRNA. .PSI.
521-565 HIV-1 psi packaging Allows viral packaging. signal RRE
1075-1308 HIV-1 Rev response Permits Rev-dependent nuclear element
export of unspliced viral mRNA. cPPT 1803-1920 Central polypurine
tract Facilitates the nuclear import of HIV-1 cDNA through a
central DNA flap. EF1A 1950-3128 EF1A Component entered by user
Kozak 3153-3158 Kozak Component entered by user {ORF3A_wSPACER}
3159-4040 {ORF3A_wSPACER} Component entered by user IRES 4065-4652
IRES Component entered by user {ORF3B} 4653-5392 {ORF3B} Component
entered by user WPRE 5422-6019 Woodchuck hepatitis virus
Facilitates effective transcription posttranscriptional termination
at the 3' LTR. regulatory element AU3/3' LTR 6101-6335 HIV-1
truncated 3' LTR Allows viral packaging but self- inactivates the
5'LTR for biosafety purposes. The element also contains
apolyadenylation signal for transcription termination and
polyadenylation of mRNA in transduced cells. SV40 early pA
6408-6542 SV40 early polyadenation Allows transcription termination
signal and polyadenylation of mRNA. Ampicillin 7496-8356 Ampicillin
resistance gene Allows selection of the plasmid in E. coli. pUC ori
8527-9115 pUC origin of replication Permits high-copy replication
and maintenance in E. coli.
[0673] When vector 3 is employed, anti-FLT3L is used for flow
detection. The highest surface FLT3L expressor will have the
highest secreted GM-CSF expression.
[0674] The following is a description of the immunomodulator
sGM-CSF/ires/mFLT3L.
[0675] Type:
cytokine, growth and differentiation factor
[0676] Annotation:
wild-type sequence The sequences are shown as follows:
TABLE-US-00012 GM-CSF signal sequence (SEQ ID NO. 72)
MWLQSLLLLGTVACSIS wild type GM-CSF sequence (SEQ ID NO. 73) APA
RSPSPSTQPW EHVNAIQEAR RLLNLSRDTA AEMNETVEVI SEMFDLQEPT CLQTRLELYK
QGLRGSLTKL KGPLTMMASH YKQHCPPTPE TSCATQIITF ESFKENLKDF LLVIPFDCWE
PVQE* (SEQ ID NO. 74) IRES FLT3L signal (modified to be IRES
friendly) (SEQ ID NO. 75) MATVLAPAWSPTTYLLLLLLLSSGLS FLT3L (SEQ ID
NO. 76) GTQDC SFQHSPISSD FAVKIRELSD YLLQDYPVTV ASNLQDEELC
GGLWRLVLAQ RWMERLKTVA GSKMQGLLER VNTEIHFVTK CAFQPPPSCL RFVQTNISRL
LQETSEQLVA LKPWITRQNF SRCLELQCQP DSSTLPPPWS PRPLEATAPT APQPPLLLLL
LLPVGLLLLA AAWCLHWQRT RRRTPRPGEQ VPPVPSPQDL LLVEH* Summary (144
ORF3a) (SEQ ID NO. 77)
MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTA
AEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTP
ETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE* Summary (236 ORF3b) (SEQ ID
NO. 78) MATVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELS
DYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIH
FVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQN
FSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPPLLLLLLLPVGLLLL
AAAWCLHWQRTRRRTPRPGEQVPPVPSPQDLLLVEH*
Vector 4. Immunomodulator: sFLT3L/Ires/(FLT3 Signal-GM-CSF-Tm)
[0677] A schematic of the organization of vector 4, used for the
immunomodulator sFLT3L/ires/(FLT3 signal-GM-CSF-Tm) is shown in
FIG. 5. Vector 4 is bicistronic. Table 10, below, shows the vector
component name, the corresponding nucleotide position in SEQ ID NO.
50, the full name of the component and a description.
TABLE-US-00013 TABLE 10 Component Nucleotide Name Position Full
Name Description RSV promoter 1-229 Rous sarcoma virus (RSV) Allows
Tat-independent production enhancer/promoter of viral mRNA.
.DELTA.5' LTR 230-410 HIV-1 truncated 5' LTRT Permits viral
packaging and reverse transcription of the viral mRNA. .PSI.
521-565 HIV-1 psi packaging Allows viral packaging. signal RRE
1075-1308 HIV-1 Rev response Permits Rev-dependent nuclear element
export of unspliced viral mRNA. cPPT 1803-1920 Central polypurine
tract Facilitates the nuclear import of HIV-1 cDNA through a
central DNA flap. EF1A 1950-3128 EF1A Component entered by user
Kozak 3153-3158 Kozak Component entered by user {ORF4A_wSPACER}
3159-4157 {ORF4A_wSPACER} Component entered by user IRES 4182-4769
IRES Component entered by user {ORF4B} 4770-5557 {ORF4B} Component
entered by user WPRE 5587-6184 Woodchuck hepatitis virus
Facilitates effective transcription posttranscriptional termination
at the 3' LTR. regulatory element AU3/3' LTR 6266-6500 HIV-1
truncated 3' LTR Allows viral packaging but self- inactivates the
5'LTR for biosafety purposes. The element also contains
apolyadenylation signal for transcription termination and
polyadenylation of mRNA in transduced cells. SV40 early pA
6573-6707 SV40 early polyadenation Allows transcription termination
signal and polyadenylation of mRNA. Ampicillin 7661-8521 Ampicillin
resistance gene Allows selection of the plasmid in E. coli. pUC ori
8692-9280 pUC origin of replication Permits high-copy replication
and maintenance in E. coli.
[0678] When vector 4 is employed, anti-GM-CSF is used for flow
detection. The highest surface GMCSF expressor will have highest
secreted FLT3L expression.
[0679] The following is a description of the immunomodulator
sFLT3L/ires/(FLT3 signal-GM-CSF-Tm)
[0680] Type:
cytokine, growth and differentiation factor
[0681] Annotation:
wild-type sequence
[0682] The sequences are shown as follows:
TABLE-US-00014 wild type FLT3L sequence with transmembrane deleted
(SEQ ID NO. 79) MTVLAPAWSP TTYLLLLLLL SSGLSGTQDC SFQHSPISSD
FAVKIRELSD YLLQDYPVTV ASNLQDEELC GGLWRLVLAQ RWMERLKTVA
GSKMQGLLERVNTEIHFVTK CAFQPPPSCL RFVQTNISRL LQETSEQLVA
LKPWITRQNFSRCLELQCQP DSSTLPPPWS PRPLEATAPT APQ* (SEQ ID NO. 80)
IRES FLT3L signal (modified to be IRES friendly) (SEQ ID NO. 81)
MATVLAPAWSP TTYLLLLLLL SSGLS wild type GM-CSF sequence (minus
native signal) (SEQ ID NO. 82) APA RSPSPSTQPW EHVNAIQEAR RLLNLSRDTA
AEMNETVEVI SEMFDLQEPT CLQTRLELYK QGLRGSLTKL KGPLTMMASHYKQHCPPTPE
TSCATQIITF ESFKENLKDF LLVIPFDCWE PVQE CD8alpha transmembrane and
cytoplasmic domain (SEQ ID NO. 83) PTTTP APRPPTPAPTIASQPLSLRP
EACRPAAGGA VHTRGLDFAC DIYIWAPLAG TCGVLLLSLVITLYCNHRNR RRVCKCPRPV
VKSGDKPSLS ARYV* Summary (183 ORF4a) (SEQ ID NO. 84)
MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSD
YLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHF
VTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNF
SRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQ* Summary for CYAGEN (253 ORF4b)
(SEQ ID NO. 85) MATVLAPAWSPTTYLLLLLLLSSGLSAPARSPSPSTQPWEHVNAIQEAR
RLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMAS
HYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWE
PVQEPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
IYIWAPLAGTCGVLLLSLVITLYCNEIRNRRRVCKCPRPVVKSGDKPSLSARYV*
Vector 5. Immunomodulator: mCD40L
[0683] A schematic of the organization of vector 5, used for the
immunomodulator mCD40L is shown in FIG. 6. Vector 5 is
monocistronic. Table 11, below, shows the vector component name,
the corresponding nucleotide position in SEQ ID NO. 51, the full
name of the component and a description.
TABLE-US-00015 TABLE 11 Component Nucleotide Name Position Full
Name Description RSV promoter 1-229 Rous sarcoma virus (RSV) Allows
Tat-independent production enhancer/promoter of viral mRNA.
.DELTA.5' LTR 230-410 HIV-1 truncated 5' LTRT Permits viral
packaging and reverse transcription of the viral mRNA. .PSI.
521-565 HIV-1 psi packaging Allows viral packaging. signal RRE
1075-1308 HIV-1 Rev response Permits Rev-dependent nuclear element
export of unspliced viral mRNA. cPPT 1803-1920 Central polypurine
tract Facilitates the nuclear import of HIV-1 cDNA through a
central DNA flap. EF1A 1959-3137 EF1A Component entered by user
Kozak 3162-3167 Kozak Component entered by user {ORF5} 3168-3991
{ORF5} Component entered by user WPRE 4030-4627 Woodchuck hepatitis
virus Facilitates effective transcription posttranscriptional
termination at the 3' LTR. regulatory element AU3/3' LTR 4709-4943
HIV-1 truncated 3' LTR Allows viral packaging but self- inactivates
the 5'LTR for biosafety purposes. The element also contains
apolyadenylation signal for transcription termination and
polyadenylation of mRNA in transduced cells. SV40 early pA
5016-5150 SV40 early polyadenation Allows transcription termination
signal and polyadenylation of mRNA. Ampicillin 6104-6964 Ampicillin
resistance gene Allows selection of the plasmid in E. coli. pUC ori
7135-7723 pUC origin of replication Permits high-copy replication
and maintenance in E. coli.
[0684] When Vector 5 is employed, anti-CD40L is used for flow
detection.
[0685] The following is a description of the immunomodulator
mCD40L.
[0686] Type:
TNF type II transmembrane protein
[0687] Annotation:
Mutations (UNDERLINED) introduced to make a non-cleavable
version
[0688] The sequences are shown as follows:
TABLE-US-00016 Modified sequence to stop cleavage (SEQ ID NO. 86)
MIETYNQTSP RSAATGLPIS MKIFMYLLTV FLITQMIGSA LFAVYLHRRL DKIEDERNLH
EDFVFMKTIQ RCNTGERSLS LLNCEEIKSQ FEGFVKDIMLNKEETKKENS FEMPRGEEDS
QIAAHVISEA SSKTTSVLQW AEKGYYTMSN NLVTLENGKQ LTVKRQGLYY IYAQVTFCSN
REASSQAPFI ASLCLKSPGR FERILLRAAN THSSAKPCG QSIHLGGVFE LQPGASVFVN
VTDPSQVSHG TGFTSFGLLK L* Summary (261 ORF5) (SEQ ID NO. 87)
MIETYNQTSPRSAATGLPISMKIEMYLLTVFLITQMIGSALFAVYLHRRL
DKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIML
NKEETKKENSFEMPRGEEDSQIAAHVISEASSKTTSVLQWAEKGYYTMSN
NLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGR
FERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVEVNVTDPSQVSHG TGFTSFGLLKL*
Vector 6. Immunomodulator: mTNFalpha (TNF.alpha.) A schematic of
the organization of vector 6, used for the immunomodulator
mTNF.alpha. is shown in FIG. 7. Vector 6 is monocistronic. Table
12, below, shows the vector component name, the corresponding
nucleotide position in SEQ ID NO. 52, the full name of the
component and a description.
TABLE-US-00017 TABLE 12 Component Nucleotide Name Position Full
Name Description RSV promoter 1-229 Rous sarcoma virus (RSV) Allows
Tat-independent production enhancer/promoter of viral mRNA.
.DELTA.5' LTR 230-410 HIV-1 truncated 5' LTRT Permits viral
packaging and reverse transcription of the viral mRNA. .PSI.
521-565 HIV-1 psi packaging signal Allows viral packaging. RRE
1075-1308 HIV-1 Rev response Permits Rev-dependent nuclear element
export of unspliced viral mRNA. cPPT 1803-1920 Central polypurine
tract Facilitates the nuclear import of HIV-1 cDNA through a
central DNA flap. EF1A 1959-3137 EF1A Component entered by user
Kozak 3162-3167 Kozak Component entered by user {ORF6} 3168-3871
{ORF6} Component entered by user WPRE 3910-4507 Woodchuck hepatitis
virus Facilitates effective transcription posttranscriptional
termination at the 3' LTR. regulatory element AU3/3' LTR 4859-4823
HIV-1 truncated 3' LTR Allows viral packaging but self- inactivates
the 5'LTR for biosafety purposes. The element also contains
apolyadenylation signal for transcription termination and
polyadenylation of mRNA in transduced cells. SV40 early pA
4896-5030 SV40 early polyadenation Allows transcription termination
signal and polyadenylation of mRNA. Ampicillin 5984-6844 Ampicillin
resistance gene Allows selection of the plasmid in E. coli. pUC ori
7015-7603 pUC origin of replication Permits high-copy replication
and maintenance in E. coli.
When vector 6 is employed, anti-TNF.alpha. is used for flow
detection. The following is a description of the immunomodulator
mTNF.alpha.. Type: TNF type II transmembrane protein Annotation:
Mutations were introduced to make a non-cleavable version. Vector
7. Immunomodulator: mRANKL/Ires/FLT3 Signal-V5-scFV Anti-Biotin-Tm
A schematic of the organization of vector 7, used for the
immunomodulator mRANKL/ires/FLT3 signal-V5-scFV anti-biotin-Tm is
shown in FIG. 8. Table 13, below, shows the vector component name,
the corresponding nucleotide position, the full name of the
component and a description.
TABLE-US-00018 TABLE 13 Component Nucleotide Name Position Full
Name Description RSV promoter 1-229 Rous sarcoma virus (RSV) Allows
Tat-independent production enhancer/promoter of viral mRNA.
.DELTA.5' LTR 230-410 HIV-1 truncated 5' LTR Permits viral
packaging and reverse transcription of the viral mRNA. .PSI.
521-565 HIV-1 psi packaging Allows viral packaging. signal RRE
1075-1308 HIV-1 Rev response Permits Rev-dependent nuclear element
export of unspliced viral mRNA. cPPT 1803-1920 Central polypurine
tract Facilitates the nuclear import of HIV-1 cDNA through a
central DNA flap. EF1A 1950-3128 EF1A Component entered by user
Kozak 3153-3158 Kozak Component entered by user {ORF7_wSPACER}
3159-4091 {ORF7_wSPACER} Component entered by user IRES 4116-4703
IRES Component entered by user {ORF7B} 4704-5878 {ORF7B} Component
entered by user WPRE 5908-6505 Woodchuck hepatitis virus
Facilitates effective transcription posttranscriptional termination
at the 3' LTR. regulatory element AU3/3' LTR 6587-3821 HIV-1
truncated 3' LTR Allows viral packaging but self- inactivates the
5'LTR for biosafety purposes. The element also contains
apolyadenylation signal for transcription termination and
polyadenylation of mRNA in transduced cells. SV40 early pA
6894-7028 SV40 early polyadenation Allows transcription termination
signal and polyadenylation of mRNA. Ampicillin 7982-8842 Ampicillin
resistance gene Allows selection of the plasmid in E. coli. pUC ori
9013-9601 pUC origin of replication Permits high-copy replication
and maintenance inE. coli.
When vector 7 is employed, anti-RANKL is used for flow detection.
Anti-VS mAb is used as a secondary detection method. The following
is a description of the immunomodulator mRANKL/ires/FLT3
signal-VS-scFV anti-biotin-Tm. Type: TNF type II transmembrane
protein Annotation: wild-type sequence
Vector 44
[0689] FIG. 9 shows a schematic of vector 44. Table 14 below, shows
the vector component name, the corresponding nucleotide position,
the full name of the component and a description.
TABLE-US-00019 TABLE 14 Component Nucleotide Size Name Position
(bp) Description RSV 1-229 229 Rous sarcoma virus enhancer/
promoter promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF44A} 3168-3932 765 None WPRE
3971-4568 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 4650-4884 235 Truncated HIV-1
3' long terminal repeat SV40 early 4957-5091 135 Simian virus 40
early polyadenylation pA signal Ampicillin 6045-6905 861 Ampicillin
resistance gene pUC ori 7076-7664 589 pUC origin of replication
Vector 97
[0690] FIG. 10 shows a schematic of vector 97. Table 15 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00020 TABLE 15 Component Nucleotide Size Name Position
(bp) Description RSV 1-229 229 Rous sarcoma virus enhancer/
promoter promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF97} 3168-4772 1605 None WPRE
4811-5408 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 5490-5724 235 Truncated HIV-1
3' long terminal repeat SV40 early 5797-5931 135 Simian virus 40
early polyadenylation pA signal Ampicillin 6885-7745 861 Ampicillin
resistance gene pUC ori 7916-8504 589 pUC origin of replication
Vector 84.
[0691] FIG. 11 shows a schematic of vector 84. Table 16 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00021 TABLE 16 Component Nucleotide Size Name Position
(bp) Description RSV 1-229 229 Rous sarcoma virus enhancer/
promoter promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF84A} 3168-4709 1542 None IRES +
4710-5501 792 Linker SPACER {ORF84B) 5502-6350 849 None WPRE
6389-6986 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 7068-7302 235 Truncated HIV-1
3' long terminal repeat SV40 early 7375-7509 135 Simian virus 40
early pA polyadenylation signal Ampicillin 8463-9323 861 Ampicillin
resistance gene pUC ori 9494-10082 589 pUC origin of
replication
Vector 29.
[0692] FIG. 12 shows a schematic of vector 29. Table 17 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00022 TABLE 17 Component Nucleotide Size Name Position
(bp) Description RSV 1-229 229 Rous sarcoma virus enhancer/
promoter promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF29a(285aa) + 3159-4242 1084
None SPACER} IRES 4267-4854 588 Encephalomyocarditis virus internal
ribosome entry site {ORF29b(250aa)vi 4855-5604 750 None WPRE
5634-6231 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 6313-6547 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 6620-6754 135 Simian virus 40
early polyadenylation signal Ampicillin 7708-8568 861 Ampicillin
resistance gene pUC ori 8739-9327 589 pUC origin of replication
Vector 107
[0693] FIG. 13 shows a schematic of vector 107. Table 18 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00023 TABLE 18 Component Nucleotide Size Name Position
(bp) Description RSV 1-229 229 Rous sarcoma virus promoter
enhancer/promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF3107A} 3159-5843 2685 None IRES
5868-6455 588 Linker {ORF107B) 6456-7313 858 None WPRE 7343-7940
598 Woodchuck hepatitis virus posttranscriptional regulatory
element .DELTA.U3/3' LTR 8022-8256 235 Truncated HIV-1 3' long
terminal repeat SV40 early 8329-8463 135 Simian virus 40 early pA
polyadenylation signal Ampicillin 9417-10277 861 Ampicillin
resistance gene pUC ori 10448-11036 589 pUC origin of
replication
Vector 116
[0694] FIG. 14 shows a schematic of vector 116. Table 19 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00024 TABLE 19 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF116A} 3159-4421 1263 None IRES
446-5033 588 Linker {ORF116B) 5034-6122 1089 None WPRE 6152-6749
598 Woodchuck hepatitis virus posttranscriptional regulatory
element .DELTA.U3/3' LTR 6831-7065 235 Truncated HIV-1 3' long
terminal repeat SV40 early pA 7138-7272 135 Simian virus 40 early
polyadenylation signal Ampicillin 8226-9086 861 Ampicillin
resistance gene pUC ori 9257-9845 589 pUC origin of replication
Vector 86
[0695] FIG. 15 shows a schematic of vector 86. Table 20 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00025 TABLE 20 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF86A} 3168-4421 1254 None IRES +
spacer 4422-5213 792 Linker {ORF86B) 5214-6788 1575 None WPRE
6827-7424 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 7506-7740 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 7813-7947 135 Simian virus 40
early polyadenylation signal Ampicillin 8901-9761 861 Ampicillin
resistance gene pUC ori 9932-10520 589 pUC origin of
replication
Vector 18
[0696] FIG. 16 shows a schematic of vector 18. Table 21, below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00026 TABLE 21 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF18(193) 3168-3749 582 None WPRE
3788-4385 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 4467-4701 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 4774-4908 135 Simian virus 40
early polyadenylation signal Ampicillin 5862-6722 861 Ampicillin
resistance gene pUC ori 6893-7481 589 pUC origin of replication
Vector 17
[0697] FIG. 17 shows a schematic of vector 17. Table 22 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00027 TABLE 22 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF17} 3168-3872 705 None WPRE
3911-4508 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 4590-4824 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 4897-5031 135 Simian virus 40
early polyadenylation signal Ampicillin 5985-6845 861 Ampicillin
resistance gene pUC ori 7016-7604 589 pUC origin of replication
Vector 98
[0698] FIG. 18 shows a schematic of vector 98. Table 23 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00028 TABLE 23 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF98} 3168-4001 834 None WPRE
4040-4637 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 4719-4953 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 5026-5160 135 Simian virus 40
early polyadenylation signal Ampicillin 6114-6974 861 Ampicillin
resistance gene pUC ori 7145-7733 589 pUC origin of replication
Vector 30
[0699] FIG. 19 shows a schematic of vector 30. Table 24 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00029 TABLE 24 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF30a(288aa) + 3159-4251 1093
None SPACER} IRES 4276-4863 588 Linker {ORF30B(332aa) 4864-5862 999
None WPRE 5892-6489 598 Woodchuck hepatitis virus
posttranscriptional regulatory element .DELTA.U3/3' LTR 6571-6805
235 Truncated HIV-1 3' long terminal repeat SV40 early pA 6878-7012
135 Simian virus 40 early polyadenylation signal Ampicillin
7966-8826 861 Ampicillin resistance gene pUC ori 8997-9585 589 pUC
origin of replication
Vector 109
[0700] FIG. 20 shows a schematic of vector 109. Table 25 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00030 TABLE 25 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF109A} 3159-3947 789 None IRES
3972-4559 588 Linker {ORF109B) 4560-7043 2484 None WPRE 7073-7670
598 Woodchuck hepatitis virus posttranscriptional regulatory
element .DELTA.U3/3' LTR 7752-7986 235 Truncated HIV-1 3' long
terminal repeat SV40 early pA 8059-8193 135 Simian virus 40 early
polyadenylation signal Ampicillin 9147-10007 861 Ampicillin
resistance gene pUC ori 10178-10766 589 pUC origin of
replication
Vector 106
[0701] FIG. 21 shows a schematic of vector 106. Table 26 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00031 TABLE 26 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF106A} 3159-5147 1989 None IRES
5172-5759 588 Linker {ORF106B) 5760-6617 858 None WPRE 6477-7244
598 Woodchuck hepatitis virus posttranscriptional regulatory
element .DELTA.U3/3' LTR 7326-7560 235 Truncated HIV-1 3' long
terminal repeat SV40 early pA 7633-7767 135 Simian virus 40 early
polyadenylation signal Ampicillin 8721-9581 861 Ampicillin
resistance gene pUC ori 9752-10340 589 pUC origin of
replication
Vector 16
[0702] FIG. 22 shows a schematic of vector 16. Table 27 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00032 TABLE 27 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF16A}_w/ 3159-3984 826 None
SPACER IRES 4009-4596 588 Linker {ORF16B) 4597-5742 1146 None WPRE
5772-6369 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 6451-6685 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 6758-6892 135 Simian virus 40
early polyadenylation signal Ampicillin 7846-8706 861 Ampicillin
resistance gene pUC ori 8877-945 589 pUC origin of replication
Vector 83
[0703] FIG. 23 shows a schematic of vector 83. Table 28 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00033 TABLE 28 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF83A} 3168-3704 537 None IRES +
spacer 3705-4496 792 Linker {ORF83B) 4497-5522 1026 None WPRE
5561-6158 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 6240-6474 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 6547-6681 135 Simian virus 40
early polyadenylation signal Ampicillin 7635-8495 861 Ampicillin
resistance gene pUC ori 8666-9254 589 pUC origin of replication
Vector 31
[0704] FIG. 24 shows a schematic of vector 31. Table 29 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00034 TABLE 29 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF31} 3168-5480 2313 None WPRE
5519-6116 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 6198-6432 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 6505-6639 135 Simian virus 40
early polyadenylation signal Ampicillin 7593-8453 861 Ampicillin
resistance gene pUC ori 8624-9212 589 pUC origin of replication
Vector 12
[0705] FIG. 25 shows a schematic of vector 12. Table 30 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00035 TABLE 30 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF12} 3168-5876 None WPRE
5915-6512 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 6594-6828 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 6901-7035 135 Simian virus 40
early polyadenylation signal Ampicillin 7989-8849 861 Ampicillin
resistance gene pUC ori 9020-9608 589 pUC origin of replication
Vector 99
[0706] FIG. 26 shows a schematic of vector 99. Table 31 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00036 TABLE 31 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF99} 3168-4019 852 None WPRE
4058-4655 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 4737-4971 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 5044-5178 135 Simian virus 40
early polyadenylation signal Ampicillin 6132-6992 861 Ampicillin
resistance gene pUC ori 7163-7751 589 pUC origin of replication
Vector 121
[0707] FIG. 27 shows a schematic of vector 121. Table 32 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00037 TABLE 32 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF121A} 3159-3896 738 None IRES
3921-4508 588 Linker {ORF121B) 4509-5444 936 None WPRE 5474-6071
598 Woodchuck hepatitis virus posttranscriptional regulatory
element .DELTA.U3/3' LTR 6153-6387 235 Truncated HIV-1 3' long
terminal repeat SV40 early pA 6460-6594 135 Simian virus 40 early
polyadenylation signal Ampicillin 7548-8408 861 Ampicillin
resistance gene pUC ori 8579-9167 589 pUC origin of replication
Vector 105
[0708] FIG. 28 shows a schematic of vector 105. Table 33 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00038 TABLE 33 Component Nucleotide Size Name Position
(bp) Description RSV 1-229 229 Rous sarcoma virus enhancer/
promoter promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF105A} 3159-4574 1416 None IRES
4599-5186 588 Linker {ORF105B) 5187-6788 1602 None WPRE 6818-7415
598 Woodchuck hepatitis virus posttranscriptional regulatory
element .DELTA.U3/3' LTR 7497-7731 235 Truncated HIV-1 3' long
terminal repeat SV40 early 7804-7938 135 Simian virus 40 early
polyadenylation pA signal Ampicillin 8892-9752 861 Ampicillin
resistance gene pUC ori 9923-14511 589 pUC origin of
replication
Vector 32
[0709] FIG. 29 shows a schematic of vector 32. Table 34 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00039 TABLE 34 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF32A} + 3159-4359 1201 None
SPACER IRES 4384-4971 588 Linker {ORF32B) 4972-6294 1323 None WPRE
6324-6921 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 7003-7237 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 7310-7444 135 Simian virus 40
early polyadenylation signal Ampicillin 8398-9258 861 Ampicillin
resistance gene pUC ori 9429-10017 589 pUC origin of
replication
Vector 37
[0710] FIG. 30 shows a schematic of vector 37. Table 35 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00040 TABLE 35 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF37A} + 3159-4093 935 None
SPACER IRES 4118-4705 588 Linker {ORF37B) 4706-5527 822 None WPRE
5557-6154 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 6236-6470 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 6543-6677 135 Simian virus 40
early polyadenylation signal Ampicillin 7631-8491 861 Ampicillin
resistance gene pUC ori 8662-9250 589 pUC origin of replication
Vector 22
[0711] FIG. 31 shows a schematic of vector 22. Table 36 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00041 TABLE 36 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF22} 3168-5087 1920 None WPRE
5126-5723 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 5805-6039 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 6112-6246 135 Simian virus 40
early polyadenylation signal Ampicillin 7200-8060 861 Ampicillin
resistance gene pUC ori 8231-8819 589 pUC origin of replication
Vector 19
[0712] FIG. 32 shows a schematic of vector 19. Table 37 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00042 TABLE 37 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF19A} 3159-40892 931 None
w_SPACER IRES 4114-4701 588 Linker {ORF19B) 4702-5847 1146 None
WPRE 5877-6474 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 6556-6790 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 6863-6997 135 Simian virus 40
early polyadenylation signal Ampicillin 7951-8811 861 Ampicillin
resistance gene pUC ori 8982-9570 589 pUC origin of replication
Vector 20
[0713] FIG. 33 shows a schematic of vector 20. Table 38 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00043 TABLE 38 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A- 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF20}w_spacer 3159-4218 1060 None
IRES 4243-4830 588 Linker {ORF20B) 4831-5976 1146 None WPRE
6066-6603 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 6685-6919 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 6992-7126 135 Simian virus 40
early polyadenylation signal Ampicillin 8080-8940 861 Ampicillin
resistance gene pUC ori 9111-9699 589 pUC origin of replication
Vector 89
[0714] FIG. 34 shows a schematic of vector 89. Table 39 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00044 TABLE 39 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF89A} + 3159-4194 1036 None
Spacer IRES 4219-4806 588 Linker {ORF89B) 4807-5583 777 None WPRE
5613-6210 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 6292-6526 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 6599-6733 135 Simian virus 40
early polyadenylation signal Ampicillin 7687-8547 861 Ampicillin
resistance gene pUC ori 8718-9306 589 pUC origin of replication
Vector 21
[0715] FIG. 35 shows a schematic of vector 21. Table 40 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00045 TABLE 40 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF21} 3168-3929 762 None WPRE
3968-4565 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 4647-4881 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 4954-5088 135 Simian virus 40
early polyadenylation signal Ampicillin 6042-6902 861 Ampicillin
resistance gene pUC ori 7073-7661 589 pUC origin of replication
Vector 23
[0716] FIG. 36 shows a schematic of vector 23. Table 41 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00046 TABLE 41 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 11959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF23} 3168-5006 1839 None WPRE
5045-5642 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 5724-958 235 Truncated HIV-1 3'
long terminal repeat SV40 early pA 6031-6165 135 Simian virus 40
early polyadenylation signal Ampicillin 7119-7979 861 Ampicillin
resistance gene pUC ori 8150-8738 589 pUC origin of replication
Vector 108
[0717] FIG. 37 shows a schematic of vector 108. Table 42 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00047 TABLE 42 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF108A} 3159-6824 3666 None IRES
6849-7436 588 Linker {ORF108B) 7437-8336 900 None WPRE 8366-8963
598 Woodchuck hepatitis virus posttranscriptional regulatory
element .DELTA.U3/3' LTR 9045-9279 235 Truncated HIV-1 3' long
terminal repeat SV40 early pA 9352-9486 135 Simian virus 40 early
polyadenylation signal Ampicillin 10440-11300 861 Ampicillin
resistance gene pUC ori 11471-12059 589 pUC origin of
replication
Vector 15
[0718] FIG. 38 shows a schematic of vector 15. Table 43 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00048 TABLE 43 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF15} 3168-3890 723 None WPRE
3929-4526 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 4608-4842 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 4915-5049 135 Simian virus 40
early polyadenylation signal Ampicillin 6003-6863 861 Ampicillin
resistance gene pUC ori 7034-7622 589 pUC origin of replication
Vector 124
[0719] FIG. 39 shows a schematic of vector 124. Table 44 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00049 TABLE 44 Component Nucleotide Size Name Position
(bp) Description RSV 1-229 229 Rous sarcoma virus enhancer/
promoter promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF124A} 3159-4112 954 None IRES
4137-4724 588 Linker {ORF124B) 4725-5750 1026 None WPRE 5780-6377
598 Woodchuck hepatitis virus posttranscriptional regulatory
element .DELTA.U3/3' LTR 6459-6693 235 Truncated HIV-1 3' long
terminal repeat SV40 early 6766-6900 135 Simian virus 40 early
polyadenylation pA signal Ampicillin 7854-8714 861 Ampicillin
resistance gene pUC ori 8885-9473 589 pUC origin of replication
Vector 65
[0720] FIG. 40 shows a schematic of vector 65. Table 45 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00050 TABLE 45 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF65} 3168-4121 954 None WPRE
4160-4757 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 4839-5073 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 5146-5280 135 Simian virus 40
early polyadenylation signal Ampicillin 6234-7094 861 Ampicillin
resistance gene pUC ori 7265-7853 589 pUC origin of replication
Vector 64
[0721] FIG. 41 shows a schematic of vector 64. Table 46 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00051 TABLE 46 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HW-1 5' long
terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF64} 3168-4448 1281 None WPRE
4487-5084 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 5166-5400 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 5473-5607 135 Simian virus 40
early polyadenylation signal Ampicillin 6561-7421 861 Ampicillin
resistance gene pUC ori 7592-8180 589 pUC origin of replication
Vector 88
[0722] FIG. 42 shows a schematic of vector 88. Table 47 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00052 TABLE 47 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF88A} + 3159-3900 742 None
spacer IRES 3901-4488 588 Linker {ORF88B) + 4489-5485 997 None
spacer IRES 5510-6097 588 Linker {ORF99C} 6098-6952 855 None WPRE
6982-7579 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 7661-7895 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 7968-8102 135 Simian virus 40
early polyadenylation signal Ampicillin 9056-9916 861 Ampicillin
resistance gene pUC ori 10087-10675 589 pUC origin of
replication
Vector 96
[0723] FIG. 43 shows a schematic of vector 96. Table 48 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00053 TABLE 48 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF96} 3168-4064 897 None WPRE
4103-4700 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 4782-5016 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 5089-5223 135 Simian virus 40
early polyadenylation signal Ampicillin 6177-7037 861 Ampicillin
resistance gene pUC ori 7208-7796 589 pUC origin of replication
Vector 14
[0724] FIG. 44 shows a schematic of vector 14. Table 49 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00054 TABLE 49 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF14(183) 3168-3719 552 None WPRE
3758-4355 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 4647-4671 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 4744-4878 135 Simian virus 40
early polyadenylation signal Ampicillin 5832-6692 861 Ampicillin
resistance gene pUC ori 6863-7451 589 pUC origin of replication
Vector 119
[0725] FIG. 45 shows a schematic of vector 119. Table 50 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00055 TABLE 50 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF119A} 3159-4049 891 None IRES
4074-4661 588 Linker {ORF119B) 4662-5480 819 None WPRE 5510-6107
598 Woodchuck hepatitis virus posttranscriptional regulatory
element .DELTA.U3/3' LTR 6189-6423 235 Truncated HIV-1 3' long
terminal repeat SV40 early pA 6496-6630 135 Simian virus 40 early
polyadenylation signal Ampicillin 7584-8444 861 Ampicillin
resistance gene pUC ori 8615-9203 589 pUC origin of replication
Vector 120
[0726] FIG. 46 shows a schematic of vector 120. Table 51 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00056 TABLE 51 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF120A} 3159-4391 1233 None IRES
4416-5003 588 Linker {ORF120B) 5004-6200 1197 None WPRE 6230-6827
598 Woodchuck hepatitis virus posttranscriptional regulatory
element .DELTA.U3/3' LTR 6909-7143 235 Truncated HIV-1 3' long
terminal repeat SV40 early pA 7216-7350 135 Simian virus 40 early
polyadenylation signal Ampicillin 8304-9164 861 Ampicillin
resistance gene pUC ori 9335-9923 589 pUC origin of replication
Vector 45
[0727] FIG. 47 shows a schematic of vector 45. Table 52 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00057 TABLE 52 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1959-3137 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3162-3167 6 Kozak
translation initiation sequence {ORF45} 3168-4121 954 None WPRE
4160-4757 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 4839-5073 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 5146-5280 135 Simian virus 40
early polyadenylation signal Ampicillin 6234-7094 861 Ampicillin
resistance gene pUC ori 7265-7853 589 pUC origin of replication
Vector 60
[0728] FIG. 48 shows a schematic of vector 60. Table 53 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00058 TABLE 53 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF60A} + 3159-3900 742 None
Spacer IRES 3925-4512 588 Linker {ORF60B) 4513-5367 855 None WPRE
5397-5994 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 6076-6310 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 6383-6517 135 Simian virus 40
early polyadenylation signal Ampicillin 7471-8331 861 Ampicillin
resistance gene pUC ori 8502-9090 589 pUC origin of replication
Vector 59
[0729] FIG. 49 shows a schematic of vector 59. Table 54 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00059 TABLE 54 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF59A} + 3159-4548 1390 None
spacer IRES 4573-5160 588 Linker {ORF59B) 5161-6036 876 None WPRE
6066-6663 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 6745-6979 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 7052-7186 135 Simian virus 40
early polyadenylation signal Ampicillin 8140-9000 861 Ampicillin
resistance gene pUC ori 9171-9759 589 pUC origin of replication
Vector 8
[0730] FIG. 50 shows a schematic of vector 8. Table 55 below, shows
the vector component name, the corresponding nucleotide position,
the full name of the component and a description.
TABLE-US-00060 TABLE 55 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF8A} 3159-5960 None IRES
5985-6572 588 Linker {ORF8B) 6573-7456 None WPRE 7486-8083 598
Woodchuck hepatitis virus posttranscriptional regulatory element
.DELTA.U3/3' LTR 8165-8399 235 Truncated HIV-1 3' long terminal
repeat SV40 early pA 8472-8606 135 Simian virus 40 early
polyadenylation signal Ampicillin 9560-10420 861 Ampicillin
resistance gene pUC ori 10591-11179 589 pUC origin of
replication
Vector 128
[0731] FIG. 51 shows a schematic of vector 128. Table 56 below,
shows the vector component name, the corresponding nucleotide
position, the full name of the component and a description.
TABLE-US-00061 TABLE 56 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF128A} 3159-4535 1377 None IRES
4560-5147 588 Linker {ORF128B) 5148-5966 819 None WPRE 5996-6593
598 Woodchuck hepatitis virus posttranscriptional regulatory
element .DELTA.U3/3' LTR 6675-6909 235 Truncated HIV-1 3' long
terminal repeat SV40 early pA 6982-7116 135 Simian virus 40 early
polyadenylation signal Ampicillin 8070-8930 861 Ampicillin
resistance gene pUC ori 9101-9689 589 pUC origin of replication
Vector 35
[0732] FIG. 52 shows a schematic of vector 35 Table 57 below, shows
the vector component name, the corresponding nucleotide position,
the full name of the component and a description.
TABLE-US-00062 TABLE 57 Component Nucleotide Size Name Position
(bp) Description RSV promoter 1-229 229 Rous sarcoma virus
enhancer/ promoter .DELTA.5' LTR 230-410 181 Truncated HIV-1 5'
long terminal repeat .PSI. 521-565 45 HIV-1 packaging signal RRE
1075-1308 234 HIV-1 Rev response element cPPT 1803-1920 118 Central
polypurine tract EF1A 1950-3128 1179 Human eukaryotic translation
elongation factor 1 .alpha.1 promoter Kozak 3153-3158 6 Kozak
translation initiation sequence {ORF35A} + 3159-4140 982 None
SPACER IRES 4165-4752 588 Linker {ORF35B) 4753-5628 876 None WPRE
5658-6255 598 Woodchuck hepatitis virus posttranscriptional
regulatory element .DELTA.U3/3' LTR 6337-6571 235 Truncated HIV-1
3' long terminal repeat SV40 early pA 6644-6778 135 Simian virus 40
early polyadenylation signal Ampicillin 7732-8592 861 Ampicillin
resistance gene pUC ori 8763-9351 589 pUC origin of replication
[0733] According to one embodiment, a tumor cell line is selected
for modification, and vector 2 is used to stably integrate between
3-30 immunomodulators into the cell genome, wherein at least three
immunomodulators are OX40L, CD27L and CD28L, optionally wherein
additional immunomodulators are selected from R.sup.1-R.sup.44 in
Table 3.
[0734] According to one embodiment, a tumor cell line is selected
for modification, and vector 3 is used to stably integrate between
3-30 immunomodulators into the cell genome, wherein at least three
immunomodulators are OX40L, CD27L and CD28L, optionally wherein
additional immunomodulators are selected from R.sup.1-R.sup.44 in
Table 3.
[0735] According to one embodiment, a tumor cell line is selected
for modification, and vector 4 is used to stably integrate between
3-30 immunomodulators into the cell genome, wherein at least three
immunomodulators are OX40L, CD27L and CD28L, optionally wherein
additional immunomodulators are selected from R.sup.1-R.sup.44 in
Table 3.
[0736] According to one embodiment, a tumor cell line is selected
for modification, and vector 5 is used to stably integrate between
3-30 immunomodulators into the cell genome, wherein at least three
immunomodulators are OX40L, CD27L and CD28L, optionally wherein
additional immunomodulators are selected from R.sup.1-R.sup.44 in
Table 3.
[0737] According to one embodiment, a tumor cell line is selected
for modification, and vector 6 is used to stably integrate between
3-30 immunomodulators into the cell genome, wherein at least three
immunomodulators are OX40L, CD27L and CD28L, optionally wherein
additional immunomodulators are selected from R.sup.1-R.sup.44 in
Table 3.
[0738] According to one embodiment, a tumor cell line is selected
for modification, and vector 14 is used to stably integrate between
3-30 immunomodulators into the cell genome, wherein at least three
immunomodulators are OX40L, CD27L and CD28L, optionally wherein
additional immunomodulators are selected from R.sup.1-R.sup.44 in
Table 3.
[0739] According to one embodiment, a tumor cell line is selected
for modification, and vector 18 is used to stably integrate between
3-30 immunomodulators into the cell genome, wherein at least three
immunomodulators are OX40L, CD27L and CD28L, optionally wherein
additional immunomodulators are selected from R.sup.1-R.sup.44 in
Table 3.
[0740] According to one embodiment, a tumor cell line is selected
for modification, and vector 30 is used to stably integrate between
3-30 immunomodulators into the cell genome, wherein at least three
immunomodulators are OX40L, CD27L and CD28L, optionally wherein
additional immunomodulators are selected from R.sup.1-R.sup.44 in
Table 3.
[0741] According to one embodiment, a tumor cell line is selected
for modification, and vector 15 is used to stably integrate between
3-30 immunomodulators into the cell genome, wherein at least three
immunomodulators are OX40L, CD27L and CD28L, optionally wherein
additional immunomodulators are selected from R.sup.1-R.sup.44 in
Table 3.
[0742] According to one embodiment, a tumor cell line is selected
for modification, and vector 19 is used to stably integrate between
3-30 immunomodulators into the cell genome, wherein at least three
immunomodulators are OX40L, CD27L and CD28L, optionally wherein
additional immunomodulators are selected from R.sup.1-R.sup.44 in
Table 3.
[0743] According to one embodiment, a tumor cell line is selected
for modification, and vector 22 is used to stably integrate between
3-30 immunomodulators into the cell genome, wherein at least three
immunomodulators are OX40L, CD27L and CD28L, optionally wherein
additional immunomodulators are selected from R.sup.1-R.sup.44 in
Table 3.
[0744] According to one embodiment, a tumor cell line is selected
for modification, and vector 23 is used to stably integrate between
3-30 immunomodulators into the cell genome, wherein at least three
immunomodulators are OX40L, CD27L and CD28L, optionally wherein
additional immunomodulators are selected from R.sup.1-R.sup.44 in
Table 3.
[0745] According to one embodiment, a tumor cell line is selected
for modification, and vector 29 is used to stably integrate between
3-30 immunomodulators into the cell genome, wherein at least three
immunomodulators are OX40L, CD27L and CD28L, optionally wherein
additional immunomodulators are selected from R.sup.1-R.sup.44 in
Table 3.
[0746] According to one embodiment, a tumor cell line is selected
for modification, and vector 2 and vector 3 are used to stably
integrate between 3-30 immunomodulators into the cell genome,
wherein at least three immunomodulators are OX40L, CD27L and CD28L,
optionally wherein additional immunomodulators are selected from
R.sup.1-R.sup.44 in Table 3.
[0747] According to one embodiment, a tumor cell line is selected
for modification, and vector 2 and vector 4 are used to stably
integrate between 3-30 immunomodulators into the cell genome,
wherein at least three immunomodulators are OX40L, CD27L and CD28L,
optionally wherein additional immunomodulators are selected from
R.sup.1-R.sup.44 in Table 3.
[0748] According to one embodiment, a tumor cell line is selected
for modification, and vector 2 and vector 5 are used to stably
integrate between 3-30 immunomodulators into the cell genome,
wherein at least three immunomodulators are OX40L, CD27L and CD28L,
optionally wherein additional immunomodulators are selected from
R.sup.1-R.sup.44 in Table 3.
[0749] According to one embodiment, a tumor cell line is selected
for modification, and vector 2 and vector 6 are used to stably
integrate between 3-30 immunomodulators into the cell genome,
wherein at least three immunomodulators are OX40L, CD27L and CD28L,
optionally wherein additional immunomodulators are selected from
R.sup.1-R.sup.44 in Table 3.
[0750] According to one embodiment, a tumor cell line is selected
for modification, and vector 3 and vector 4 are used to stably
integrate between 3-30 immunomodulators into the cell genome,
wherein at least three immunomodulators are OX40L, CD27L and CD28L,
optionally wherein additional immunomodulators are selected from
R.sup.1-R.sup.44 in Table 3.
[0751] According to one embodiment, a tumor cell line is selected
for modification, and vector 3 and vector 5 are used to stably
integrate between 3-30 immunomodulators into the cell genome,
wherein at least three immunomodulators are OX40L, CD27L and CD28L,
optionally wherein additional immunomodulators are selected from
R.sup.1-R.sup.44 in Table 3.
[0752] According to one embodiment, a tumor cell line is selected
for modification, and vector 3 and vector 6 are used to stably
integrate between 3-30 immunomodulators into the cell genome,
wherein at least three immunomodulators are OX40L, CD27L and CD28L,
optionally wherein additional immunomodulators are selected from
R.sup.1-R.sup.44 in Table 3.
[0753] According to one embodiment, a tumor cell line is selected
for modification, and vector 2, vector 3 and vector 4 are used to
stably integrate between 3-14 immunomodulators into the cell
genome, wherein at least three immunomodulators are OX40L, CD27L
and CD28L, optionally wherein additional immunomodulators are
selected from R.sup.1-R.sup.44 in Table 3.
[0754] According to one embodiment, a tumor cell line is selected
for modification, and vector 2, vector 3 and vector 5 are used to
stably integrate between 3-30 immunomodulators into the cell
genome, wherein at least three immunomodulators are OX40L, CD27L
and CD28L, optionally wherein additional immunomodulators are
selected from R.sup.1-R.sup.44 in Table 3.
[0755] According to one embodiment, a tumor cell line is selected
for modification, and vector 2, vector 3 and vector 6 are used to
stably integrate between 3-30 immunomodulators into the cell
genome, wherein at least three immunomodulators are OX40L, CD27L
and CD28L, optionally wherein additional immunomodulators are
selected from R.sup.1-R.sup.44 in Table 3.
[0756] According to one embodiment, a tumor cell line is selected
for modification, and vector 2, vector 3 and vector 6 are used to
stably integrate between 3-30 immunomodulators into the cell
genome, wherein at least three immunomodulators are OX40L, CD27L
and CD28L, optionally wherein additional immunomodulators are
selected from R.sup.1-R.sup.44 in Table 3.
[0757] According to one embodiment, a tumor cell line is selected
for modification, and vector 3, vector 4 and vector 5 are used to
stably integrate between 3-30 immunomodulators into the cell
genome, wherein at least three immunomodulators are OX40L, CD27L
and CD28L, optionally wherein additional immunomodulators are
selected from R.sup.1-R.sup.44 in Table 3.
[0758] According to one embodiment, a tumor cell line is selected
for modification, and vector 3, vector 4 and vector 6 are used to
stably integrate between 3-30 immunomodulators into the cell
genome, wherein at least three immunomodulators are OX40L, CD27L
and CD28L, optionally wherein additional immunomodulators are
selected from R.sup.1-R.sup.44 in Table 3.
[0759] According to one embodiment, a tumor cell line is selected
for modification, and vector 2, vector 3, vector 4 and vector 5 are
used to stably integrate between 3-30 immunomodulators into the
cell genome, wherein at least three immunomodulators are OX40L,
CD27L and CD28L, optionally wherein additional immunomodulators are
selected from R.sup.1-R.sup.44 in Table 3.
[0760] According to one embodiment, a tumor cell line is selected
for modification, and vector 2, vector 3, vector 4 and vector 6 are
used to stably integrate between 3-30 immunomodulators into the
cell genome, wherein at least three immunomodulators are OX40L,
CD27L and CD28L, optionally wherein additional immunomodulators are
selected from R.sup.1-R.sup.44 in Table 3.
[0761] According to one embodiment, a tumor cell line is selected
for modification, and vector 2, vector 3, vector 5 and vector 6 are
used to stably integrate between 3-30 immunomodulators into the
cell genome, wherein at least three immunomodulators are OX40L,
CD27L and CD28L, optionally wherein additional immunomodulators are
selected from R.sup.1-R.sup.44 in Table 3.
[0762] According to one embodiment, a tumor cell line is selected
for modification, and vector 3, vector 4, vector 5 and vector 6 are
used to stably integrate between 3-30 immunomodulators into the
cell genome, wherein at least three immunomodulators are OX40L,
CD27L and CD28L, optionally wherein additional immunomodulators are
selected from R.sup.1-R.sup.44 in Table 3.
[0763] According to one embodiment, a tumor cell line is selected
for modification, and vector 2, vector 3, vector 4, vector 5 and
vector 6 are used to stably integrate between 3-30 immunomodulators
into the cell genome, wherein at least three immunomodulators are
OX40L, CD27L and CD28L, optionally wherein additional
immunomodulators are selected from R.sup.1-R.sup.44 in Table 3.
[0764] According to one embodiment, a tumor cell line is selected
for modification, and vector 14 and vector 18 are used to stably
integrate between 3-30 immunomodulators into the cell genome,
wherein at least three immunomodulators are OX40L, CD27L and CD28L,
optionally wherein additional immunomodulators are selected from
R.sup.1-R.sup.44 in Table 3.
[0765] According to one embodiment, a tumor cell line is selected
for modification, and vector 14 and vector 30 are used to stably
integrate between 3-30 immunomodulators into the cell genome,
wherein at least three immunomodulators are OX40L, CD27L and CD28L,
optionally wherein additional immunomodulators are selected from
R.sup.1-R.sup.44 in Table 3.
[0766] According to one embodiment, a tumor cell line is selected
for modification, and vector 18 and vector 30 are used to stably
integrate between 3-30 immunomodulators into the cell genome,
wherein at least three immunomodulators are OX40L, CD27L and CD28L,
optionally wherein additional immunomodulators are selected from
R.sup.1-R.sup.44 in Table 3.
[0767] According to one embodiment, a tumor cell line is selected
for modification, and vector 14, vector 18 and vector 30 are used
to stably integrate between 3-30 immunomodulators into the cell
genome, wherein at least three immunomodulators are OX40L, CD27L
and CD28L, optionally wherein additional immunomodulators are
selected from R.sup.1-R.sup.44 in Table 3.
[0768] According to one embodiment, a tumor cell line is selected
for modification, and one or more of vector 15, vector 19, vector
22, vector 23 and vector 29 are used to stably integrate between
3-30 immunomodulators into the cell genome, wherein at least three
immunomodulators are OX40L, CD27L and CD28L, optionally wherein
additional immunomodulators are selected from R.sup.1-R.sup.44 in
Table 3.
[0769] According to one embodiment, a tumor cell line is selected
for modification, and vector 44 is used to stably integrate between
3-25 immunomodulators into the cell genome. According to one
embodiment, a tumor cell line is selected for modification, and
vector 97 is used to stably integrate between 3-25 immunomodulators
into the cell genome. According to one embodiment, a tumor cell
line is selected for modification, and vector 84 is used to stably
integrate between 3-25 immunomodulators into the cell genome.
According to one embodiment, a tumor cell line is selected for
modification, and vector 29 is used to stably integrate between
3-25 immunomodulators into the cell genome. According to one
embodiment, a tumor cell line is selected for modification, and
vector 107 is used to stably integrate between 3-25
immunomodulators into the cell genome. According to one embodiment,
a tumor cell line is selected for modification, and vector 116 is
used to stably integrate between 3-25 immunomodulators into the
cell genome. According to one embodiment, a tumor cell line is
selected for modification, and vector 86 is used to stably
integrate between 3-25 immunomodulators into the cell genome.
According to one embodiment, a tumor cell line is selected for
modification, and vector 18 is used to stably integrate between
3-25 immunomodulators into the cell genome. According to one
embodiment, a tumor cell line is selected for modification, and
vector 17 is used to stably integrate between 3-25 immunomodulators
into the cell genome. According to one embodiment, a tumor cell
line is selected for modification, and vector 98 is used to stably
integrate between 3-25 immunomodulators into the cell genome.
According to one embodiment, a tumor cell line is selected for
modification, and vector 5 is used to stably integrate between 3-25
immunomodulators into the cell genome. According to one embodiment,
a tumor cell line is selected for modification, and vector 30 is
used to stably integrate between 3-25 immunomodulators into the
cell genome. According to one embodiment, a tumor cell line is
selected for modification, and vector 109 is used to stably
integrate between 3-25 immunomodulators into the cell genome.
According to one embodiment, a tumor cell line is selected for
modification, and vector 3 is used to stably integrate between 3-25
immunomodulators into the cell genome. According to one embodiment,
a tumor cell line is selected for modification, and vector 4 is
used to stably integrate between 3-25 immunomodulators into the
cell genome. According to one embodiment, a tumor cell line is
selected for modification, and vector 106 is used to stably
integrate between 3-25 immunomodulators into the cell genome.
According to one embodiment, a tumor cell line is selected for
modification, and vector 16 is used to stably integrate between
3-25 immunomodulators into the cell genome. According to one
embodiment, a tumor cell line is selected for modification, and
vector 83 is used to stably integrate between 3-25 immunomodulators
into the cell genome. According to one embodiment, a tumor cell
line is selected for modification, and vector 31 is used to stably
integrate between 3-25 immunomodulators into the cell genome.
According to one embodiment, a tumor cell line is selected for
modification, and vector 12 is used to stably integrate between
3-25 immunomodulators into the cell genome. According to one
embodiment, a tumor cell line is selected for modification, and
vector 99 is used to stably integrate between 3-25 immunomodulators
into the cell genome. According to one embodiment, a tumor cell
line is selected for modification, and vector 121 is used to stably
integrate between 3-25 immunomodulators into the cell genome.
According to one embodiment, a tumor cell line is selected for
modification, and vector 105 is used to stably integrate between
3-25 immunomodulators into the cell genome. According to one
embodiment, a tumor cell line is selected for modification, and
vector 32 is used to stably integrate between 3-25 immunomodulators
into the cell genome. According to one embodiment, a tumor cell
line is selected for modification, and vector 37 is used to stably
integrate between 3-25immunomodulators into the cell genome.
According to one embodiment, a tumor cell line is selected for
modification, and vector 22 is used to stably integrate between
3-25 immunomodulators into the cell genome. According to one
embodiment, a tumor cell line is selected for modification, and
vector 19 is used to stably integrate between 3-25 immunomodulators
into the cell genome. According to one embodiment, a tumor cell
line is selected for modification, and vector 20 is used to stably
integrate between 3-25 immunomodulators into the cell genome.
According to one embodiment, a tumor cell line is selected for
modification, and vector 89 is used to stably integrate between
3-25 immunomodulators into the cell genome. According to one
embodiment, a tumor cell line is selected for modification, and
vector 21 is used to stably integrate between 3-25 immunomodulators
into the cell genome. According to one embodiment, a tumor cell
line is selected for modification, and vector 23 is used to stably
integrate between 3-25 immunomodulators into the cell genome.
According to one embodiment, a tumor cell line is selected for
modification, and vector 108 is used to stably integrate between
3-25 immunomodulators into the cell genome. According to one
embodiment, a tumor cell line is selected for modification, and
vector 15 is used to stably integrate between 3-25 immunomodulators
into the cell genome. According to one embodiment, a tumor cell
line is selected for modification, and vector 124 is used to stably
integrate between 3-25 immunomodulators into the cell genome.
According to one embodiment, a tumor cell line is selected for
modification, and vector 65 is used to stably integrate between
3-25 immunomodulators into the cell genome. According to one
embodiment, a tumor cell line is selected for modification, and
vector 64 is used to stably integrate between 3-25 immunomodulators
into the cell genome. According to one embodiment, a tumor cell
line is selected for modification, and vector 88 is used to stably
integrate between 3-25 immunomodulators into the cell genome.
According to one embodiment, a tumor cell line is selected for
modification, and vector 96 is used to stably integrate between
3-25 immunomodulators into the cell genome. According to one
embodiment, a tumor cell line is selected for modification, and
vector 14 is used to stably integrate between 3-25 immunomodulators
into the cell genome. According to one embodiment, a tumor cell
line is selected for modification, and vector 119 is used to stably
integrate between 3-25 immunomodulators into the cell genome.
According to one embodiment, a tumor cell line is selected for
modification, and vector 120 is used to stably integrate between
3-25 immunomodulators into the cell genome. According to one
embodiment, a tumor cell line is selected for modification, and
vector 45 is used to stably integrate between 3-25 immunomodulators
into the cell genome. According to one embodiment, a tumor cell
line is selected for modification, and vector 60 is used to stably
integrate between 3-25 immunomodulators into the cell genome.
According to one embodiment, a tumor cell line is selected for
modification, and vector 59 is used to stably integrate between
3-25 immunomodulators into the cell genome. According to one
embodiment, a tumor cell line is selected for modification, and
vector 8 is used to stably integrate between 3-25 immunomodulators
into the cell genome. According to one embodiment, a tumor cell
line is selected for modification, and vector 128 is used to stably
integrate between 3-25 immunomodulators into the cell genome.
According to one embodiment, a tumor cell line is selected for
modification, and vector 35 is used to stably integrate between
3-25 immunomodulators into the cell genome. According to one
embodiment, a tumor cell line is selected for modification, and
vector 6 is used to stably integrate between 3-25 immunomodulators
into the cell genome.
[0770] According to one embodiment, a tumor cell line is selected
for modification, and one or more of vector 44, vector 29, vector
18, vector 17, vector 5, vector 16, vector 99, vector 15, vector
14, vector 45, and vector 6 are used to stably integrate between
3-14 TNF family immunomodulators into the cell genome. According to
one embodiment, the 3-14 TNF family member immunomodulators are
selected from those listed in Table 2.
[0771] According to one embodiment, a tumor cell line is selected
for modification, and one or more of vector 97, vector 84, vector
107, vector 98, vector 30, vector 83, vector 121, and vector 119
are used to stably integrate between 3-25 Ig family
immunomodulators into the cell genome. According to one embodiment,
the 3-25 Ig family member immunomodulators are selected from those
listed in Table 2.
[0772] According to one embodiment, a tumor cell line is selected
for modification, and vector 109 is used to stably integrate
between 3-25 growth factor immunomodulators into the cell
genome.
[0773] According to one embodiment, a tumor cell line is selected
for modification, and one or more of vector 3, vector 4, vector 32,
vector 22, vector 19, vector 20, vector 89, vector 21, vector 23,
vector 121, vector 65, vector 64, vector 88, vector 96, vector 60,
vector 59, and vector 128 are used to stably integrate between 3-25
cytokine immunomodulators into the cell genome. According to one
embodiment, the 3-25 cytokine immunomodulators are selected from
those listed in Table 2.
[0774] According to one embodiment, a tumor cell line is selected
for modification, and one or more of vector 37, vector 124, vector
88, and vector 8 are used to stably integrate between 3-25 receptor
immunomodulators into the cell genome. According to one embodiment,
the 3-25 receptor immunomodulators are selected from those listed
in Table 2.
[0775] According to one embodiment, a tumor cell line is selected
for modification, and one or more of vector 86, vector 106, vector
107, vector 31, vector 12, vector 105, vector 108, vector 120, and
vector 35 are used to stably integrate between 3-25 other
immunomodulators into the cell genome. According to one embodiment,
the 3-25 other immunomodulators are selected from those listed in
Table 2.
Example 4
[0776] Experiments were carried out to demonstrate that the
immunomodulators described herein, expressed on the melanoma tumor
cell line SK-MEL2 differentially impact the proliferation and
differentiation of human PBMCs. FIG. 53 is a schematic that shows
the general experimental design. The following allogenic cell lines
were tested: [0777] SK-MEL (Parental line) ("SK") [0778] SK
modified with Vector 2 only ("2")) [0779] SK modified with Vector 3
only ("3") [0780] SK modified with Vector 4 only (4'') [0781] SK
modified with Vector 5 only (5'') [0782] SK modified with Vector 6
only (6'' [0783] SK modified with Vector 2 and Vector 3 ("2-3")
[0784] SK modified with Vector 3, Vector 4 and Vector 5 ("3-4-5")
[0785] SK modified with Vector 3, Vector 5 and Vector 6 ("3-5-6")
[0786] SK modified with Vector 3, Vector 4, Vector 5 and Vector 6
("3-4-5-6") [0787] SK modified with Vector 2, Vector 3, Vector 4,
Vector 5 and Vector 6
[0788] Functional characterization of the allogeneic cell lines was
performed using a primary MLTR assay, as described herein. The MLTR
assay was set up with 250,000 freshly thawed PBMC and 50,000 of
select engineered allogeneic cell lines. The following outputs were
measured: 1) Proliferation is measured by flow on CFSE labeled
PMBC; 2) Differentiation is measured by CyTOF on unlabeled PMBC; 3)
Cytokine profiling is performed by Luminex.
Flow Cytometry Data
[0789] "Allorecognition" is a term used to define immunological
recognition of histoincompatible antigens between genetically
disparate individuals within the same species. "Direct
allorecognition" is a mechanism by which recipient T cells
recognize determinants on MHC-molecule-peptide complexes displayed
on the surface of transplanted cells without the requirement for
antigen processing by recipient APCs. The direct response can most
readily be demonstrated in vitro by the mixed lymphocyte reaction
in which only direct allopresentation can occur
[0790] "Indirect allorecognition" refers to recognition of
processed peptides of allogeneic histocompatibility antigens
presented by self-MHC in a self-restricted manner. Indirect
alloantigen presentation invariably results in alloresponses that
are dominated by CD4+ T cells.
[0791] Approximately 10% of peripheral blood T cells bear a TCR
capable of allorecognition of the allogeneic tumor type specific
cells used for vaccination. This is called "direct allorecognition"
and occurs early in the course of events post vaccination. Direct
allorecognition targets a T cell mediated immune response against
the allogeneic cells resulting in their death and release of tumor
type specific neoantigens and shared normal antigens. These tumor
neoantigens (and normal antigens) are taken up by host antigen
presenting cells, processed and presented in the context of host
HLA. This "indirect allorecognition" occurs late in the course of
events post vaccination. The TCRs activated during indirect
allorecognition are different from those involved earlier during
direct allorecognition, but both processes occur in a local
environment repeatedly exposed to allogeneic cells bearing high
density immunomodulators (e.g. monthly vaccinations).
[0792] Epitope spreading is a process of expanding an immune
response to include distinct but closely related T cell epitopes.
This is generally described as a maturation of the immune response.
The differential maturation of the immune response against tumor
neoantigens versus self-antigens is driven by the fact that
tolerance mechanisms are in place to differentially protect against
immune responses against self-antigens. While self-tolerance can be
broken, it is more difficult than the response against a tumor
neoantigen.
[0793] Without being limited by theory, since all tumors of a given
type share many antigens, the T-cell mediated response initially
driven by indirect allorecognition of the immune response will
cross react against the host tumor of the same type. According to
some embodiments, since the tumor microenvironment may provide an
insurmountable negative immunomodulatory hurdle, this approach may
be used in combination with checkpoint inhibitors in the setting of
minimal residual disease after a debulking therapy (e.g. surgery,
radiation or oncolytic viruses).
[0794] The experiments described herein use hPBMC Activation via
Direct Allorecognition versus Pan-Stimulation. Primary human pan-T
cells include CD4 and CD8 T cells as well as some gamma/delta T
cell subsets. In Pan-Stimulation, non-target cells, i.e.,
monocytes, neutrophils, eosinophils, B cells, stem cells, dendritic
cells, NK cells, granulocytes, or erythroid cells are labeled by
using a cocktail of biotin-conjugated antibodies. T cells are
isolated from peripheral blood (PB) mononuclear cells (MNCs) using
the negative immunomagnetic separation technique without the use of
columns. Cells are untouched by the separation process and ready
for downstream usage. It was found that hPBMC activation via direct
allorecognition display a fundamentally different response to tumor
cells. Three key observations were made with regard to hPBMC
activation by this method: 1) that .about.10% of hPBMC proliferate
compared to -50% with anti-CD3/CD28 treatment; 2) it induces more
cell divisions than anti-CD3/CD28 treatment; 3) it induces a more
varied morphology.
[0795] FIG. 54 is a panel of graphs that show the results of flow
cytometry experiments. Forward (FSC) and side scatter (SSC) plots
for size and granularity are shown. SK tumor cell lines are
represented by a number code; SK, unmodified parent line; vector 3,
secreted GM-CSF and membrane expressed FLT-3L; vector 4, secreted
FLT3L and membrane expressed GM-CSF; vector 5, a non-cleavable form
of CD40L; vector 6, a non-cleavable form of TNF-.alpha.; 3-4 is a
combination of vectors 3 and 4; 3-4-5 is a combination of vectors
3,4 and 5; and 3-4-6 is a combination of vectors 3,4 and 6. Cell
lines 6, 3-4-5 and 3-4-6 display a larger and more granular
phenotype likely owing to the presence of receptors for TNF-.alpha.
and CD40L on cells of epithelial origin.
[0796] FIG. 55 is a panel of graphs that show representative flow
cytometry stains for the indicated engineered surface markers;
GM-CSF, FLT3L, TNF-.alpha. and CD40L. SK tumor cell lines are
represented by a number code; SK, unmodified parent line; vector 3,
secreted GM-CSF and membrane expressed FLT-3L; vector 4, secreted
FLT3L and membrane expressed GM-CSF; 5, a non-cleavable form of
CD40L; vector 6, a non-cleavable form of TNF-.alpha.; 3-4 is a
combination of vectors 3 and 4; 3-4-5 is a combination of vectors
3,4 and 5; and 3-4-6 is a combination of vectors 3, 4 and 6.
[0797] FIG. 56 is a panel of graphs that show representative flow
cytometry stains for the indicated engineered surface markers;
GM-CSF, FLT3L, TNF-.alpha. and CD40L. SK tumor cell lines are
represented by a number code; SK, unmodified parent line; vector 3,
secreted GM-CSF and membrane expressed FLT-3L; vector 4, secreted
FLT3L and membrane expressed GM-CSF; vector 5, a non-cleavable form
of CD40L; vector 6, a non-cleavable form of TNF-.alpha.; 3-4 is a
combination of vectors 3 and 4; 3-4-5 is a combination of vectors
3,4 and 5; and 3-4-6 is a combination of vectors 3,4 and 6.
CyTOF Data
[0798] CyTOF mass cytometry single-cell phenotype analysis of hPBMC
response to SK melanoma cells with modification by expression of
immunomodulatory factors is shown in FIG. 14A and FIG. 14B. The SK
melanoma cell line and hPBMCs were cultured for 24 hours. Cells
were harvested from cultures and stained with a 32-marker CyTOF
antibody panel to detect multiple immune cell subsets as well as
cell-surface and intracellular phenotyping markers. CyTOF mass
cytometry data was generated on a Helios instrument. The data were
normalized for signal using equilibration beads. Cell staining data
were analyzed using Cytobank--a cloud computing suite for CyTOF
data analysis that includes cell gating functions and an array of
data visualization methods.
[0799] The data shown in FIG. 57A and FIG. 57B were plotted using
viSNE, which is a dimensional reduction method that converts
multidimensional staining signals from single cells into plots for
visualization. FIG. 57A shows viSNE density contour plots of CyTOF
staining data showing relative changes in immune cell subset
abundance and phenotype. FIG. 57B shows single-cell phenotype
analysis. viSNE density contour plots were generated by viSNE from
ungated total PBMCs that were cultured with SK melanoma cells or
modified SK melanoma cells. The plots illustrate relative changes
in cell density for hPBMC immune cell subsets. The inserted viSNE
plot identifies the immune cell subsets that are found within the
clusters of the viSNE density plots. The arrows in the density
contour plots point to the visible changes in immune cell subsets
between hPBMCs, SK cells, and the modified SK cells. SK tumor cell
lines are represented by a number code; SK, unmodified parent line;
vector 3, secreted GM-CSF and membrane expressed FLT-3L; vector 4,
secreted FLT3L and membrane expressed GM-CSF; vector 5, a
non-cleavable form of CD40L; vector 6, a non-cleavable form of
TNF-.alpha.; 3-4 is a combination of vectors 3 and 4; 3-4-5 is a
combination of vectors 3,4 and 5; and 3-4-6 is a combination of
vectors 3,4 and 6.
[0800] FIG. 58A-FIG. 58D show CyTOF monocyte cluster analysis of
hPBMC indicating changes in the activation markers CD40 (FIG. 58A),
CD86 (FIG. 58B), CD69 (FIG. 58C) and CD25 (FIG. 58D) expression
following 1 day stimulation with the indicated genetically modified
SK lines at a 1:5 cell ratio. FIG. 58E shows CyTOF monocyte cluster
analysis of hPBMC indicating relative median expression levels of
monocyte markers CD40 and CD86. FIG. 58F shows CyTOF monocyte
cluster analysis of hPBMC indicating relative median expression
index (MEI) of CD4 T cell markers CD69 and CD25. SK tumor cell
lines are represented by a number code; SK, unmodified parent line;
vector 3, secreted GM-CSF and membrane expressed FLT-3L; vector 4,
secreted FLT3L and membrane expressed GM-CSF; vector 5, a
non-cleavable form of CD40L; vector 6, a non-cleavable form of
TNF-.alpha.; 3-4 is a combination of vectors 3 and 4; 3-4-5 is a
combination of vectors 3,4 and 5; and 3-4-6 is a combination of
vectors 3,4 and 6.
Cytokine Data
[0801] Luminex multiplex cytokine profiling of human PBMC responses
to SK parent line and genetically modified SK lines is shown in
FIG. 59. SK cells or the indicated modified cell lines were
cultured for 24 hours with human PBMCs at a 1:5 cell ratio. Control
cultures included SK cells alone, hPBMCs alone, and hPBMCs
stimulated with a mixture of anti-CD3 and anti-CD28 antibodies (1
.mu.g/ml final concentration). Supernatants were screened for
cytokine levels using a multiplexed Luminex bead array assay to
detect IL-1a, IL-1b, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10,
IL-12p40, IL-12p70, IL-13, IL-17A, IL-23, TNFa, IFNg, G-CSF,
GM-CSF, MIP1b, MCP-1, Rantes, Tweak, and TREM-1. Those cytokines
found to be specifically induced by the SK parent line and modified
SK lines are shown in the plots. Symbols indicate cytokine levels
in pg/ml as estimated from a standard curve using recombinant
cytokines. Absence of symbols indicates the cytokine was not
detected. SK lines are represented by a number code; SK, unmodified
parent line; 3, secreted GM-CSF and membrane expressed FLT-3L; 3,
secreted FLT3L and membrane expressed GM-CSF; 5, a non-cleavable
form of CD40L; 6, a non-cleavable form of TNF-.alpha.; 3-4 is a
combination of 3 and 4; 3-4-5 is a combination of 3,4 and 5; and
3-4-6 is a combination of 3,4 and 6.
[0802] The described study provides a proof of concept that the
complex combinatorial space of immunomodulators can be rapidly and
efficiently assessed using an all human in vitro MLTR assay.
Example 5
[0803] Experiments were carried out to determine the effect of the
immunomodulators described herein, expressed on the tumor cell line
SK-MEL2, on CD8+ T cell and NK cell activation and expansion.
[0804] The following allogenic cell lines are tested: [0805] SK-MEL
(Parental line, used as control) [0806] SK modified with Vector 14,
Vector 18 and Vector 30 ("14-18-30") [0807] SK modified with Vector
15 only ("15") [0808] SK modified with Vector 19 only ("19") [0809]
SK modified with Vector 22 only ("22") [0810] SK modified with
Vector 23 only ("23") [0811] SK modified with Vector 29 ("29")
[0812] Functional characterization of the allogeneic cell lines was
performed using a primary MLTR assay, as described in Example 1.
The following outputs were measured: 1) CD8+ T-cell proliferation
was measured by flow cytometry (FIG. 60); 2) tumor cell killing
using live/dead staining on day 9 (FIG. 61); 3) Natural Killer (NK)
cell, dendritic cell (DC) and B cell expansion is measured by flow
cytometry (FIG. 62).
[0813] In a first set of experiments, it was found that CD8+ T cell
stimulation can be specifically enhanced by "14-18-30." FIG. 60
compares the effect of the parental line with tumor cells modified
with Vector 14, Vector 18 and Vector 30 ("14-18-30"). The dotted
oval in the top panel of graphs indicates the lymphocyte gate. In
the parent line (i), the lymphocytes are quiescent, whereas in the
"14-18-30" line (ii) there is a considerable expansion of
lymphocytes. The dotted circle in the bottom panel of graphs shows
the CD8 gate. In the parent line (iii), there is a small number of
CD8+ T cells, whereas in the "14-18-30" line (iv) there is a large
increase in the number of CD8+ T cells. It was found that the CD8+
T cell count enhancement was .about.300-fold over baseline, and
that the CD8/CD4 ratio, indicative of immune activation, was found
to increase .about.15-fold over baseline. Further, the CCR7+/CCR7-
ratio (a surrogate for CTL memory phenotype) increased
.about.15-fold over baseline.
[0814] Further, when comparing the parental line and tumor cells
modified with Vector 14, Vector 18 and Vector 30 ("14-18-30") at
culture day 9, in the parental line ("unmodified parental tumor
line+hPBMC) healthy tumor cells and resting lymphocytes could be
visualized, whereas in the tumor cells modified with Vector 14,
Vector 18 and Vector 30 ("APX-14-18-30 vaccine candidate+hPBMC"),
no living tumor cells were visualized, while activated CTL were
seen.
[0815] In a further set of experiments, the effect of tumor cells
modified with Vector 15, Vector 19, Vector 22, Vector 23 and Vector
29 on immune cell stimulation was determined. In particular, as
shown in FIG. 62, it was possible to determine, using flow
cytometry, which subset of immune cell each particular
immunomodulator was stimulating. Killing capacity was determined
visually. As shown in FIG. 62, Vector 15 ("APX/15'') stimulated
DCs, Vector 19 ("APX/19") stimulated NK cells, Vector 22 ("APX/22")
stimulated DCs, Vector 23 ("APX/23") stimulated DCs cells and
Vector 29 ("APX/29") stimulated B cells.
[0816] FIG. 65 shows the results from another set of experiments,
where flow cytometry was used to assess CD8+ T cell expansion and
cell killing. Day 6 and day 8 time points were compared in a CD8
expansion assay using SK-parent line versus modified SK lines
expressing immunomodulatory molecules 14, 18 and 30 ("14-18-30").
The lower left panel (iii) represents CD8+ T cells. Comparing panel
(iii) to panel (iv), it can be seen that the CD8+ T cells increased
in number, with the flow cytometry readout extended to the right,
indicating an activation morphology. The upper right-hand panel
(ii) shows allogeneic cells. Comparing the allogeneic cells (panels
(i) and (ii)), it was observed that the flow cytometry results
shifted to the left in panel (ii), with the modified SK lines
expressing immunomodulatory molecules 14, 18 and 30. These results
represent a transition to dead and dying cells. Thus, not only did
the CD8+ T cells expand, but they also killed the allogeneic cells
in their midst. These results demonstrated that the allogeneic
tumor cells interacted with blood cells to kill the injected cells.
Thus, in a clinical scenario, the allogenic tumor cell vaccine can
be used to activate and expand the patient's lymphocytes, and in
particular, subsets of immune killer cells (CD8+ T cells and NK
cells), which in turn kill the patient's tumor cells. While the
tumor cell vaccine is allogeneic to the subject, the blood cells
and tumor cells have the same HLA.
Dendritic Cell (DC) Expansion
[0817] CyTOF was carried out as described in Example 1. CyTOF mass
cytometry single-cell phenotype analysis of hPBMC response to SK
melanoma cells with modification by expression of immunomodulatory
molecules (Vector 3 ("APX/3"), Vector 3 and Vector 4 ("APX/3-4);
Vector 3, Vector 4, Vector 5 ("APX/3-4-5'') and Vector 3, Vector 4,
Vector 6 ("APX/3-4-6") is shown in FIG. 20 and FIG. 21. CyTOF
provides extreme multiplexing with atomic mass spec resolution, as
compared to flow cytometry, and therefore was used here to define
the various PBMC subpopulations following stimulation with the
various immunomodulatory molecules. The SK melanoma cell line and
hPBMCs were cultured for 24 hours. Cells were harvested from
cultures and stained with a 32-marker CyTOF antibody panel to
detect multiple immune cell subsets as well as cell-surface and
intracellular phenotyping markers. CyTOF mass cytometry data was
generated on a Helios instrument. The data were normalized for
signal using equilibration beads. Cell staining data were analyzed
using Cytobank--a cloud computing suite for CyTOF data analysis
that includes cell gating functions and an array of data
visualization methods.
[0818] The data shown in FIG. 63 and FIG. 64 were plotted using
viSNE, which is a dimensional reduction method that converts
multidimensional staining signals from single cells into plots for
visualization. FIG. 63 shows viSNE density contour plots of CyTOF
staining data showing relative changes in immune cell subset
abundance and phenotype. In FIG. 63, the dotted circles follow
sub-population(s) of cells that were not present in the parent line
("parental"). As can be seen from FIG. 63, populations of NK cells
and myeloid cells that were not present in the parental cell line
are present in FIG. 64, which shows CyTOF monocyte cluster analysis
of hPBMC indicating changes in the markers PD-L1, CD86, CD25, CD16,
CD14, CD141, CD64, CD123, and T-bet expression following
nine-stimulation with the indicated SK lines modified with the
immunomodulatory molecules. As shown in FIG. 64, expression of
CD123 and CD141, known DC markers, was increased in the cell lines
modified with the immunomodulatory molecules. Further, expression
of CD14, a myeloid marker, was increased in the cell lines modified
with the immunomodulatory molecules.
Example 6. In Vivo Xenograft Mouse Experiments
[0819] Six week old female in-bred SCID mice are obtained from
Charles River Laboratories (Hartford, Conn., USA). Animals are
handled according to a protocol approved by the Institutional
Animal Care and Use Committee of the facility. Mice are allowed to
acclimate to animal housing.
[0820] A human tumor xenograft was established in NSG (NOD scid
gamma mice (Jackson Laboratory). Human tumors were implanted on the
flank of the NSG mice Human tumor cells were implanted on the flank
of NGS (NOD scid gamma) mice and allowed to grow to 150 mm.sup.3.
Mice were randomly divided into two groups, a control and a treated
group, with 6 mice per group. The treated group was treated with
expanded activated PBMCs comprising expanded activated serial
killer cells activated by 14-18-30 expressing ENLIST.TM.cells. On
day 30 (t=0) mice in the control group were inoculated with vehicle
only, and mice in the treated group were inoculated with
3.times.10.sup.6 expanded activated PBMCs comprising expanded
activated serial killer cells. Tumor size was measured by caliper
over time after inoculation in both groups. FIG. 66 is a plot
showing mean and standard deviation results of a xenograft
treatment study using NGS mice. The ends of each box are the upper
and lower quartiles; the median is marked by a vertical line inside
the box, and the whiskers are the two lines outside the box that
extend to the highest and lowest observations. Human tumor cells
were implanted on the flank of NGS (NOD scid gamma) mice. The
tumors were allowed to grow to 150 mm.sup.3. Mice were randomly
divided into two groups, a control and a treated group, with 6 mice
per group. On day 30 (t=0) mice in the control group were
inoculated with vehicle only, and mice in the treated group were
inoculated with 3.times.10.sup.6 PBMCs activated by 14-18-30
expressing ENLIST.TM. cells ("SUPLEXA.TM. cells"). Tumor size was
measured at intervals through 36 days after inoculation. Divergence
between the two groups appeared within 5 days. After day 22, the
divergence became statistically significant (*P<0.05;
**P<005).
[0821] While the present invention has been described with
reference to the specific embodiments thereof it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adopt a particular situation,
material, composition of matter, process, process step or steps, to
the objective spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 111 <210> SEQ ID NO 1 <211> LENGTH: 399
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: membrane IgG-1 heavy chain
<400> SEQUENCE: 1 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys
Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys 325 330 335
Ala Glu Ala Gln Asp Gly Glu Leu Asp Gly Leu Trp Thr Thr Ile Thr 340
345 350 Ile Phe Ile Thr Leu Phe Leu Leu Ser Val Cys Tyr Ser Ala Thr
Val 355 360 365 Thr Phe Phe Lys Val Lys Trp Ile Phe Ser Ser Val Val
Asp Leu Lys 370 375 380 Gln Thr Ile Ile Pro Asp Tyr Arg Asn Met Ile
Gly Gln Gly Ala 385 390 395 <210> SEQ ID NO 2 <211>
LENGTH: 330 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<220> FEATURE: <223> OTHER INFORMATION: secreted IgG-1
heavy chain <400> SEQUENCE: 2 Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65
70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 3 <211> LENGTH: 295 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: fragment secreted IgG-1 heavy chain <400>
SEQUENCE: 3 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115
120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu 165 170 175 Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg 180 185 190 Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 195 200 205 Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 210 215 220 Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 225 230 235
240 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
245 250 255 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser 260 265 270 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser 275 280 285 Leu Ser Leu Ser Pro Gly Lys 290 295
<210> SEQ ID NO 4 <211> LENGTH: 377 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: IgG-3 heavy chain constant region <400>
SEQUENCE: 4 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val
Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110
Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115
120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg
Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro
Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His
Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235
240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu
Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360
365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 <210> SEQ ID
NO 5 <211> LENGTH: 184 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
mGM-CSF HLA derived TM and SHORTENED cytoplasmic domain <400>
SEQUENCE: 5 Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys
Ser Ile 1 5 10 15 Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln
Pro Trp Glu His 20 25 30 Val Asn Ala Ile Gln Glu Ala Arg Arg Leu
Leu Asn Leu Ser Arg Asp 35 40 45 Thr Ala Ala Glu Met Asn Glu Thr
Val Glu Val Ile Ser Glu Met Phe 50 55 60 Asp Leu Gln Glu Pro Thr
Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 65 70 75 80 Gln Gly Leu Arg
Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met 85 90 95 Met Ala
Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 100 105 110
Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys 115
120 125 Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln
Glu 130 135 140 Glu Leu Ser Ser Gln Pro Thr Ile Pro Ile Val Gly Ile
Ile Ala Gly 145 150 155 160 Leu Val Leu Leu Gly Ala Val Ile Thr Gly
Ala Val Val Ala Ala Val 165 170 175 Met Trp Arg Arg Lys Ser Ser Asp
180 <210> SEQ ID NO 6 <211> LENGTH: 261 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <223>
OTHER INFORMATION: CD40L <400> SEQUENCE: 6 Met Ile Glu Thr
Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15 Leu Pro
Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30
Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35
40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe
Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg
Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe
Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn Lys Glu Glu Thr
Lys Lys Glu Asn Ser Phe Glu 100 105 110 Met Gln Lys Gly Asp Gln Asn
Pro Gln Ile Ala Ala His Val Ile Ser 115 120 125 Glu Ala Ser Ser Lys
Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130 135 140 Tyr Tyr Thr
Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 145 150 155 160
Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 165
170 175 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala
Ser 180 185 190 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu
Leu Arg Ala 195 200 205 Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly
Gln Gln Ser Ile His 210 215 220 Leu Gly Gly Val Phe Glu Leu Gln Pro
Gly Ala Ser Val Phe Val Asn 225 230 235 240 Val Thr Asp Pro Ser Gln
Val Ser His Gly Thr Gly Phe Thr Ser Phe 245 250 255 Gly Leu Leu Lys
Leu 260 <210> SEQ ID NO 7 <211> LENGTH: 261 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <223>
OTHER INFORMATION: non-cleavable CD40L <400> SEQUENCE: 7 Met
Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10
15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu
20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu
His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His
Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr
Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys
Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn Lys
Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110 Met Pro Arg Gly
Glu Glu Asp Ser Gln Ile Ala Ala His Val Ile Ser 115 120 125 Glu Ala
Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130 135 140
Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 145
150 155 160 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln
Val Thr 165 170 175 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro
Phe Ile Ala Ser 180 185 190 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu
Arg Ile Leu Leu Arg Ala 195 200 205 Ala Asn Thr His Ser Ser Ala Lys
Pro Cys Gly Gln Gln Ser Ile His 210 215 220 Leu Gly Gly Val Phe Glu
Leu Gln Pro Gly Ala Ser Val Phe Val Asn 225 230 235 240 Val Thr Asp
Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 245 250 255 Gly
Leu Leu Lys Leu 260 <210> SEQ ID NO 8 <211> LENGTH: 233
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: TNF <400> SEQUENCE: 8 Met Ser
Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala 1 5 10 15
Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe 20
25 30 Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu
Phe 35 40 45 Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu
Glu Phe Pro 50 55 60 Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln
Ala Val Arg Ser Ser 65 70 75 80 Ser Arg Thr Pro Ser Asp Lys Pro Val
Ala His Val Val Ala Asn Pro 85 90 95 Gln Ala Glu Gly Gln Leu Gln
Trp Leu Asn Arg Arg Ala Asn Ala Leu 100 105 110 Leu Ala Asn Gly Val
Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser 115 120 125 Glu Gly Leu
Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly 130 135 140 Cys
Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala 145 150
155 160 Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser
Pro 165 170 175 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro
Trp Tyr Glu 180 185 190 Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu
Lys Gly Asp Arg Leu 195 200 205 Ser Ala Glu Ile Asn Arg Pro Asp Tyr
Leu Asp Phe Ala Glu Ser Gly 210 215 220 Gln Val Tyr Phe Gly Ile Ile
Ala Leu 225 230 <210> SEQ ID NO 9 <211> LENGTH: 108
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: CD40L piece <400> SEQUENCE: 9
Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5
10 15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe
Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr
Leu His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu
His Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn
Thr Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile
Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn
Lys Glu Glu Thr Lys Lys Glu 100 105 <210> SEQ ID NO 10
<211> LENGTH: 141 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION: TNF
piece <400> SEQUENCE: 10 Val Ala Asn Pro Gln Ala Glu Gly Gln
Leu Gln Trp Leu Asn Arg Arg 1 5 10 15 Ala Asn Ala Leu Leu Ala Asn
Gly Val Glu Leu Arg Asp Asn Gln Leu 20 25 30 Val Val Pro Ser Glu
Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 35 40 45 Lys Gly Gln
Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 50 55 60 Ser
Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 65 70
75 80 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala
Lys 85 90 95 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln
Leu Glu Lys 100 105 110 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro
Asp Tyr Leu Asp Phe 115 120 125 Ala Glu Ser Gly Gln Val Tyr Phe Gly
Ile Ile Ala Leu 130 135 140 <210> SEQ ID NO 11 <211>
LENGTH: 233 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<220> FEATURE: <223> OTHER INFORMATION: mTNF-a
<400> SEQUENCE: 11 Met Ser Thr Glu Ser Met Ile Arg Asp Val
Glu Leu Ala Glu Glu Ala 1 5 10 15 Leu Pro Lys Lys Thr Gly Gly Pro
Gln Gly Ser Arg Arg Cys Leu Phe 20 25 30 Leu Ser Leu Phe Ser Phe
Leu Ile Val Ala Gly Ala Thr Thr Leu Phe 35 40 45 Cys Leu Leu His
Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro 50 55 60 Arg Asp
Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Ser Gly Ser Gly 65 70 75 80
Ser Gly Ser Gly Ser Gly Glu Pro Val Ala His Val Val Ala Asn Pro 85
90 95 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala
Leu 100 105 110 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val
Val Pro Ser 115 120 125 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu
Phe Lys Gly Gln Gly 130 135 140 Cys Pro Ser Thr His Val Leu Leu Thr
His Thr Ile Ser Arg Ile Ala 145 150 155 160 Val Ser Tyr Gln Thr Lys
Val Asn Leu Leu Ser Ala Ile Lys Ser Pro 165 170 175 Cys Gln Arg Glu
Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu 180 185 190 Pro Ile
Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 195 200 205
Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 210
215 220 Gln Val Tyr Phe Gly Ile Ile Ala Leu 225 230 <210> SEQ
ID NO 12 <211> LENGTH: 113 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION: HC
Constant Region 3 from IgG1 <400> SEQUENCE: 12 Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25
30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser
Pro Glu Leu Gln Leu Glu Glu Ser 100 105 110 Cys <210> SEQ ID
NO 13 <211> LENGTH: 144 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
native GM-CSF <400> SEQUENCE: 13 Met Trp Leu Gln Ser Leu Leu
Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 Ser Ala Pro Ala Arg
Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His 20 25 30 Val Asn Ala
Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp 35 40 45 Thr
Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe 50 55
60 Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys
65 70 75 80 Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu
Thr Met 85 90 95 Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr
Pro Glu Thr Ser 100 105 110 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser
Phe Lys Glu Asn Leu Lys 115 120 125 Asp Phe Leu Leu Val Ile Pro Phe
Asp Cys Trp Glu Pro Val Gln Glu 130 135 140 <210> SEQ ID NO
14 <211> LENGTH: 235 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
Flt3L <400> SEQUENCE: 14 Met Thr Val Leu Ala Pro Ala Trp Ser
Pro Thr Thr Tyr Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Ser Ser Gly
Leu Ser Gly Thr Gln Asp Cys Ser Phe 20 25 30 Gln His Ser Pro Ile
Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu 35 40 45 Ser Asp Tyr
Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu 50 55 60 Gln
Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln 65 70
75 80 Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln
Gly 85 90 95 Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr
Lys Cys Ala 100 105 110 Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val
Gln Thr Asn Ile Ser 115 120 125 Arg Leu Leu Gln Glu Thr Ser Glu Gln
Leu Val Ala Leu Lys Pro Trp 130 135 140 Ile Thr Arg Gln Asn Phe Ser
Arg Cys Leu Glu Leu Gln Cys Gln Pro 145 150 155 160 Asp Ser Ser Thr
Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala 165 170 175 Thr Ala
Pro Thr Ala Pro Gln Pro Pro Leu Leu Leu Leu Leu Leu Leu 180 185 190
Pro Val Gly Leu Leu Leu Leu Ala Ala Ala Trp Cys Leu His Trp Gln 195
200 205 Arg Thr Arg Arg Arg Thr Pro Arg Pro Gly Glu Gln Val Pro Pro
Val 210 215 220 Pro Ser Pro Gln Asp Leu Leu Leu Val Glu His 225 230
235 <210> SEQ ID NO 15 <211> LENGTH: 13 <212>
TYPE: RNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <220> FEATURE:
<223> OTHER INFORMATION: Kozak sequence <400> SEQUENCE:
15 gccgccrcca ugg 13 <210> SEQ ID NO 16 <211> LENGTH:
377 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: IgG-3 heavy chain constant region
E213Q <400> SEQUENCE: 16 Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70
75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His
Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro
Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr
Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp
Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190
Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195
200 205 Val Asp Gly Val Gln Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr
Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp
Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315
320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu
325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
Arg Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370
375 <210> SEQ ID NO 17 <211> LENGTH: 377 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <223>
OTHER INFORMATION: IgG-3 heavy chain constant region P221L
<400> SEQUENCE: 17 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys
Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro
Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro
Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro
Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Leu Arg Glu Glu 210
215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu
His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala
Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr
Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330
335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile
340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe
Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375
<210> SEQ ID NO 18 <211> LENGTH: 377 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: IgG-3 heavy chain constant region E224Q <400>
SEQUENCE: 18 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105
110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg
115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro
Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys
Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser
His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Gln 210 215 220 Gln
Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230
235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp
Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355
360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 <210> SEQ
ID NO 19 <211> LENGTH: 377 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
IgG-3 heavy chain constant region Y226F <400> SEQUENCE: 19
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys
Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys
Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135
140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Phe Asn Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260
265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly
Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser
Leu Ser Leu Ser Pro Gly Lys 370 375 <210> SEQ ID NO 20
<211> LENGTH: 377 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
IgG-3 heavy chain constant region D242N <400> SEQUENCE: 20
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys
Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys
Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135
140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asn
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260
265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly
Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser
Leu Ser Leu Ser Pro Gly Lys 370 375 <210> SEQ ID NO 21
<211> LENGTH: 377 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
IgG-3 heavy chain constant region N245D <400> SEQUENCE: 21
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys
Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys
Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135
140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp
Trp Leu Asp Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260
265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly
Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser
Leu Ser Leu Ser Pro Gly Lys 370 375 <210> SEQ ID NO 22
<211> LENGTH: 377 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
IgG-3 heavy chain constant region T269A <400> SEQUENCE: 22
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys
Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys
Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135
140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 260
265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly
Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser
Leu Ser Leu Ser Pro Gly Lys 370 375 <210> SEQ ID NO 23
<211> LENGTH: 377 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
IgG-3 heavy chain constant region S314N <400> SEQUENCE: 23
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys
Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys
Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135
140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260
265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser
Leu Ser Leu Ser Pro Gly Lys 370 375 <210> SEQ ID NO 24
<211> LENGTH: 376 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
IgG-3 heavy chain constant region S314 del <400> SEQUENCE: 24
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys
Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys
Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135
140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260
265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Gly Gln
Pro Glu Asn Asn Tyr 305 310 315 320 Asn Thr Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr 325 330 335 Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe 340 345 350 Ser Cys Ser Val
Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys 355 360 365 Ser Leu
Ser Leu Ser Pro Gly Lys 370 375 <210> SEQ ID NO 25
<211> LENGTH: 377 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
IgG-3 heavy chain constant region F366Y <400> SEQUENCE: 25
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys
Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys
Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135
140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260
265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly
Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn Arg Tyr Thr Gln 355 360 365 Lys Ser
Leu Ser Leu Ser Pro Gly Lys 370 375 <210> SEQ ID NO 26
<211> LENGTH: 221 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION: TNF
- VRSSSRTPSDKP del <400> SEQUENCE: 26 Met Ser Thr Glu Ser Met
Ile Arg Asp Val Glu Leu Ala Glu Glu Ala 1 5 10 15 Leu Pro Lys Lys
Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe 20 25 30 Leu Ser
Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe 35 40 45
Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro 50
55 60 Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Ala His
Val 65 70 75 80 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu
Asn Arg Arg 85 90 95 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu
Arg Asp Asn Gln Leu 100 105 110 Val Val Pro Ser Glu Gly Leu Tyr Leu
Ile Tyr Ser Gln Val Leu Phe 115 120 125 Lys Gly Gln Gly Cys Pro Ser
Thr His Val Leu Leu Thr His Thr Ile 130 135 140 Ser Arg Ile Ala Val
Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 145 150 155 160 Ile Lys
Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 165 170 175
Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 180
185 190 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp
Phe 195 200 205 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu
210 215 220 <210> SEQ ID NO 27 <211> LENGTH: 212
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: TNF - FSFLIVAGATTLFCLLHFGVI del
<400> SEQUENCE: 27 Met Ser Thr Glu Ser Met Ile Arg Asp Val
Glu Leu Ala Glu Glu Ala 1 5 10 15 Leu Pro Lys Lys Thr Gly Gly Pro
Gln Gly Ser Arg Arg Cys Leu Phe 20 25 30 Leu Ser Leu Gly Pro Gln
Arg Glu Glu Phe Pro Arg Asp Leu Ser Leu 35 40 45 Ile Ser Pro Leu
Ala Gln Ala Val Arg Ser Ser Ser Arg Thr Pro Ser 50 55 60 Asp Lys
Pro Val Ala His Val Val Ala Asn Pro Gln Ala Glu Gly Gln 65 70 75 80
Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn Gly Val 85
90 95 Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu Gly Leu Tyr
Leu 100 105 110 Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys Pro
Ser Thr His 115 120 125 Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala
Val Ser Tyr Gln Thr 130 135 140 Lys Val Asn Leu Leu Ser Ala Ile Lys
Ser Pro Cys Gln Arg Glu Thr 145 150 155 160 Pro Glu Gly Ala Glu Ala
Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly 165 170 175 Gly Val Phe Gln
Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu Ile Asn 180 185 190 Arg Pro
Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gln Val Tyr Phe Gly 195 200 205
Ile Ile Ala Leu 210 <210> SEQ ID NO 28 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<223> OTHER INFORMATION: HA tag seq <400> SEQUENCE: 28
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 <210> SEQ ID NO 29
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <223> OTHER INFORMATION: FLAG TAG
<400> SEQUENCE: 29 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5
<210> SEQ ID NO 30 <211> LENGTH: 583 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: mIgG Heavy Chain IgG 1/3 hybrid anti-biotin heavy
chain - E325A mutant <400> SEQUENCE: 30 Met Glu Phe Gly Leu
Ser Trp Val Phe Leu Val Ala Leu Phe Arg Gly 1 5 10 15 Val Gln Cys
Gln Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala 20 25 30 Pro
Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu 35 40
45 Thr Ala Tyr Gly Val Asp Trp Val Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60 Glu Trp Leu Gly Val Ile Trp Gly Gly Gly Arg Thr Asn Tyr
Asn Ser 65 70 75 80 Gly Leu Met Ser Arg Leu Ser Ile Arg Lys Asp Asn
Ser Lys Ser Gln 85 90 95 Val Phe Leu Thr Met Asn Ser Leu Gln Thr
Asp Asp Thr Ala Lys Tyr 100 105 110 Tyr Cys Val Lys His Thr Asn Trp
Asp Gly Gly Phe Ala Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170
175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Leu Lys Thr Pro 225 230 235 240 Leu Gly Asp Thr Thr His Thr
Cys Pro Arg Cys Pro Glu Pro Lys Ser 245 250 255 Cys Asp Thr Pro Pro
Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys 260 265 270 Asp Thr Pro
Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp 275 280 285 Thr
Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu Gly Gly 290 295
300 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
305 310 315 320 Ser Arg Thr Pro Ala Val Thr Cys Val Val Val Asp Val
Ser His Glu 325 330 335 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His 340 345 350 Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg 355 360 365 Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys 370 375 380 Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 385 390 395 400 Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 405 410 415
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 420
425 430 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp 435 440 445 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val 450 455 460 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp 465 470 475 480 Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His 485 490 495 Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 500 505 510 Glu Leu Gln Leu
Glu Glu Ser Cys Ala Glu Ala Gln Asp Gly Glu Leu 515 520 525 Asp Gly
Leu Trp Thr Thr Ile Thr Ile Phe Ile Thr Leu Phe Leu Leu 530 535 540
Ser Val Cys Tyr Ser Ala Thr Val Thr Phe Phe Lys Val Lys Trp Ile 545
550 555 560 Phe Ser Ser Val Val Asp Leu Lys Gln Thr Ile Ile Pro Asp
Tyr Arg 565 570 575 Asn Met Ile Gly Gln Gly Ala 580 <210> SEQ
ID NO 31 <211> LENGTH: 223 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
CD40L-TNFa fusion protein <400> SEQUENCE: 31 Met Ile Glu Thr
Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15 Leu Pro
Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30
Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35
40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe
Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg
Pro Val Ala 65 70 75 80 His Val Val Ala Asn Pro Gln Ala Glu Gly Gln
Leu Gln Trp Leu Asn 85 90 95 Arg Arg Ala Asn Ala Leu Leu Ala Asn
Gly Val Glu Leu Arg Asp Asn 100 105 110 Gln Leu Val Val Pro Ser Glu
Gly Leu Tyr Leu Ile Tyr Ser Gln Val 115 120 125 Leu Phe Lys Gly Gln
Gly Cys Pro Ser Thr His Val Leu Leu Thr His 130 135 140 Thr Ile Ser
Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu 145 150 155 160
Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu 165
170 175 Ala Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln
Leu 180 185 190 Glu Lys Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro
Asp Tyr Leu 195 200 205 Asp Phe Ala Glu Ser Gly Gln Val Tyr Phe Gly
Ile Ile Ala Leu 210 215 220 <210> SEQ ID NO 32 <211>
LENGTH: 19 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic peptide <220>
FEATURE: <223> OTHER INFORMATION: HC Signal <400>
SEQUENCE: 32 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu
Phe Arg Gly 1 5 10 15 Val Gln Cys <210> SEQ ID NO 33
<211> LENGTH: 118 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION: HC
Variable anti-biotin <400> SEQUENCE: 33 Gln Val Lys Leu Gln
Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser
Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ala Tyr 20 25 30 Gly
Val Asp Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40
45 Gly Val Ile Trp Gly Gly Gly Arg Thr Asn Tyr Asn Ser Gly Leu Met
50 55 60 Ser Arg Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln Val
Phe Leu 65 70 75 80 Thr Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Lys
Tyr Tyr Cys Val 85 90 95 Lys His Thr Asn Trp Asp Gly Gly Phe Ala
Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
<210> SEQ ID NO 34 <211> LENGTH: 99 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: HC Constant Region 1 from IgG1 <400> SEQUENCE:
34 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu
<210> SEQ ID NO 35 <211> LENGTH: 61 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: HC Hinge Region from IgG3 <400> SEQUENCE: 35 Leu
Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys Pro 1 5 10
15 Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu
20 25 30 Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
Glu Pro 35 40 45 Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys
Pro 50 55 60 <210> SEQ ID NO 36 <211> LENGTH: 110
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: HC Constant Region 2 from IgG1
<400> SEQUENCE: 36 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100
105 110 <210> SEQ ID NO 37 <211> LENGTH: 63 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <223>
OTHER INFORMATION: HC Transmembrane and Cytoplasmic region from
IgG1 <400> SEQUENCE: 37 Ala Glu Ala Gln Asp Gly Glu Leu Asp
Gly Leu Trp Thr Thr Ile Thr 1 5 10 15 Ile Phe Ile Thr Leu Phe Leu
Leu Ser Val Cys Tyr Ser Ala Thr Val 20 25 30 Thr Phe Phe Lys Val
Lys Trp Ile Phe Ser Ser Val Val Asp Leu Lys 35 40 45 Gln Thr Ile
Ile Pro Asp Tyr Arg Asn Met Ile Gly Gln Gly Ala 50 55 60
<210> SEQ ID NO 38 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <223> OTHER
INFORMATION: LC Signal <400> SEQUENCE: 38 Met Lys Tyr Leu Leu
Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro
Ala Met Ala 20 <210> SEQ ID NO 39 <211> LENGTH: 99
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: LC Variable <400> SEQUENCE: 39
Gly Ser Pro Gly Gln Ser Val Ser Ile Ser Cys Ser Gly Ser Ser Ser 1 5
10 15 Asn Ile Gly Asn Asn Tyr Val Tyr Trp Tyr Gln His Leu Pro Gly
Thr 20 25 30 Ala Pro Lys Leu Leu Ile Tyr Ser Asp Thr Lys Arg Pro
Ser Gly Val 35 40 45 Pro Asp Arg Ile Ser Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala 50 55 60 Ile Ser Gly Leu Gln Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Ala Ser 65 70 75 80 Trp Asp Asp Ser Leu Asp Gly
Pro Val Phe Gly Gly Gly Thr Lys Leu 85 90 95 Thr Val Leu
<210> SEQ ID NO 40 <211> LENGTH: 106 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: LC Constant Region 1 <400> SEQUENCE: 40 Gly Gln
Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20
25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser
Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln
Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr
Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val
Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr
Glu Cys Ser 100 105 <210> SEQ ID NO 41 <211> LENGTH:
583 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: mIgG Heavy Chain IgG 1/3 hybrid
anti-biotin heavy chain - T323A mutant <400> SEQUENCE: 41 Met
Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Phe Arg Gly 1 5 10
15 Val Gln Cys Gln Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
20 25 30 Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe
Ser Leu 35 40 45 Thr Ala Tyr Gly Val Asp Trp Val Arg Gln Pro Pro
Gly Lys Gly Leu 50 55 60 Glu Trp Leu Gly Val Ile Trp Gly Gly Gly
Arg Thr Asn Tyr Asn Ser 65 70 75 80 Gly Leu Met Ser Arg Leu Ser Ile
Arg Lys Asp Asn Ser Lys Ser Gln 85 90 95 Val Phe Leu Thr Met Asn
Ser Leu Gln Thr Asp Asp Thr Ala Lys Tyr 100 105 110 Tyr Cys Val Lys
His Thr Asn Trp Asp Gly Gly Phe Ala Tyr Trp Gly 115 120 125 Gln Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145
150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Leu Lys Thr Pro 225 230 235 240 Leu Gly Asp
Thr Thr His Thr Cys Pro Arg Cys Pro Glu Pro Lys Ser 245 250 255 Cys
Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys 260 265
270 Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp
275 280 285 Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu
Gly Gly 290 295 300 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile 305 310 315 320 Ser Arg Ala Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 325 330 335 Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 340 345 350 Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 355 360 365 Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 370 375 380 Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 385 390
395 400 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr 405 410 415 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu 420 425 430 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp 435 440 445 Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val 450 455 460 Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp 465 470 475 480 Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 485 490 495 Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 500 505 510
Glu Leu Gln Leu Glu Glu Ser Cys Ala Glu Ala Gln Asp Gly Glu Leu 515
520 525 Asp Gly Leu Trp Thr Thr Ile Thr Ile Phe Ile Thr Leu Phe Leu
Leu 530 535 540 Ser Val Cys Tyr Ser Ala Thr Val Thr Phe Phe Lys Val
Lys Trp Ile 545 550 555 560 Phe Ser Ser Val Val Asp Leu Lys Gln Thr
Ile Ile Pro Asp Tyr Arg 565 570 575 Asn Met Ile Gly Gln Gly Ala 580
<210> SEQ ID NO 42 <211> LENGTH: 211 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: GM-CSF - HLA-I fusion peptide <400> SEQUENCE: 42
Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5
10 15 Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu
His 20 25 30 Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu
Ser Arg Asp 35 40 45 Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val
Ile Ser Glu Met Phe 50 55 60 Asp Leu Gln Glu Pro Thr Cys Leu Gln
Thr Arg Leu Glu Leu Tyr Lys 65 70 75 80 Gln Gly Leu Arg Gly Ser Leu
Thr Lys Leu Lys Gly Pro Leu Thr Met 85 90 95 Met Ala Ser His Tyr
Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 100 105 110 Cys Ala Thr
Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys 115 120 125 Asp
Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 130 135
140 Glu Leu Ser Ser Gln Pro Thr Ile Pro Ile Val Gly Ile Ile Ala Gly
145 150 155 160 Leu Val Leu Leu Gly Ala Val Ile Thr Gly Ala Val Val
Ala Ala Val 165 170 175 Met Trp Arg Arg Lys Ser Ser Asp Arg Lys Gly
Gly Ser Tyr Thr Gln 180 185 190 Ala Ala Ser Ser Asp Ser Ala Gln Gly
Ser Asp Val Ser Leu Thr Ala 195 200 205 Cys Lys Val 210 <210>
SEQ ID NO 43 <211> LENGTH: 583 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: mIgG Heavy Chain IgG 1/3 hybrid anti-biotin heavy
chain - E325A, T323A mutant <400> SEQUENCE: 43 Met Glu Phe
Gly Leu Ser Trp Val Phe Leu Val Ala Leu Phe Arg Gly 1 5 10 15 Val
Gln Cys Gln Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala 20 25
30 Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45 Thr Ala Tyr Gly Val Asp Trp Val Arg Gln Pro Pro Gly Lys
Gly Leu 50 55 60 Glu Trp Leu Gly Val Ile Trp Gly Gly Gly Arg Thr
Asn Tyr Asn Ser 65 70 75 80 Gly Leu Met Ser Arg Leu Ser Ile Arg Lys
Asp Asn Ser Lys Ser Gln 85 90 95 Val Phe Leu Thr Met Asn Ser Leu
Gln Thr Asp Asp Thr Ala Lys Tyr 100 105 110 Tyr Cys Val Lys His Thr
Asn Trp Asp Gly Gly Phe Ala Tyr Trp Gly 115 120 125 Gln Gly Thr Thr
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155
160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Leu Lys Thr Pro 225 230 235 240 Leu Gly Asp Thr Thr
His Thr Cys Pro Arg Cys Pro Glu Pro Lys Ser 245 250 255 Cys Asp Thr
Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys 260 265 270 Asp
Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp 275 280
285 Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu Gly Gly
290 295 300 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile 305 310 315 320 Ser Arg Ala Pro Ala Val Thr Cys Val Val Val
Asp Val Ser His Glu 325 330 335 Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His 340 345 350 Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 355 360 365 Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 370 375 380 Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 385 390 395 400
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 405
410 415 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu 420 425 430 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp 435 440 445 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val 450 455 460 Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp 465 470 475 480 Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His 485 490 495 Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 500 505 510 Glu Leu
Gln Leu Glu Glu Ser Cys Ala Glu Ala Gln Asp Gly Glu Leu 515 520 525
Asp Gly Leu Trp Thr Thr Ile Thr Ile Phe Ile Thr Leu Phe Leu Leu 530
535 540 Ser Val Cys Tyr Ser Ala Thr Val Thr Phe Phe Lys Val Lys Trp
Ile 545 550 555 560 Phe Ser Ser Val Val Asp Leu Lys Gln Thr Ile Ile
Pro Asp Tyr Arg 565 570 575 Asn Met Ile Gly Gln Gly Ala 580
<210> SEQ ID NO 44 <211> LENGTH: 183 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: Soluble Flt3-L <400> SEQUENCE: 44 Met Thr Val
Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu Leu 1 5 10 15 Leu
Leu Leu Leu Ser Ser Gly Leu Ser Gly Thr Gln Asp Cys Ser Phe 20 25
30 Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu
35 40 45 Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser
Asn Leu 50 55 60 Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu
Val Leu Ala Gln 65 70 75 80 Arg Trp Met Glu Arg Leu Lys Thr Val Ala
Gly Ser Lys Met Gln Gly 85 90 95 Leu Leu Glu Arg Val Asn Thr Glu
Ile His Phe Val Thr Lys Cys Ala 100 105 110 Phe Gln Pro Pro Pro Ser
Cys Leu Arg Phe Val Gln Thr Asn Ile Ser 115 120 125 Arg Leu Leu Gln
Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp 130 135 140 Ile Thr
Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro 145 150 155
160 Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala
165 170 175 Thr Ala Pro Thr Ala Pro Gln 180 <210> SEQ ID NO
45 <211> LENGTH: 583 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
mIgG Heavy Chain IgG 1/3 hybrid anti-biotin heavy chain <400>
SEQUENCE: 45 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu
Phe Arg Gly 1 5 10 15 Val Gln Cys Gln Val Lys Leu Gln Glu Ser Gly
Pro Gly Leu Val Ala 20 25 30 Pro Ser Gln Ser Leu Ser Ile Thr Cys
Thr Val Ser Gly Phe Ser Leu 35 40 45 Thr Ala Tyr Gly Val Asp Trp
Val Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60 Glu Trp Leu Gly Val
Ile Trp Gly Gly Gly Arg Thr Asn Tyr Asn Ser 65 70 75 80 Gly Leu Met
Ser Arg Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln 85 90 95 Val
Phe Leu Thr Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Lys Tyr 100 105
110 Tyr Cys Val Lys His Thr Asn Trp Asp Gly Gly Phe Ala Tyr Trp Gly
115 120 125 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Leu Lys Thr Pro 225 230
235 240 Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys Pro Glu Pro Lys
Ser 245 250 255 Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro
Lys Ser Cys 260 265 270 Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu
Pro Lys Ser Cys Asp 275 280 285 Thr Pro Pro Pro Cys Pro Arg Cys Pro
Ala Pro Glu Leu Leu Gly Gly 290 295 300 Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile 305 310 315 320 Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 325 330 335 Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 340 345 350
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 355
360 365 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys 370 375 380 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu 385 390 395 400 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr 405 410 415 Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu 420 425 430 Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 435 440 445 Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 450 455 460 Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 465 470 475
480 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
485 490 495 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro 500 505 510 Glu Leu Gln Leu Glu Glu Ser Cys Ala Glu Ala Gln
Asp Gly Glu Leu 515 520 525 Asp Gly Leu Trp Thr Thr Ile Thr Ile Phe
Ile Thr Leu Phe Leu Leu 530 535 540 Ser Val Cys Tyr Ser Ala Thr Val
Thr Phe Phe Lys Val Lys Trp Ile 545 550 555 560 Phe Ser Ser Val Val
Asp Leu Lys Gln Thr Ile Ile Pro Asp Tyr Arg 565 570 575 Asn Met Ile
Gly Gln Gly Ala 580 <210> SEQ ID NO 46 <211> LENGTH:
227 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: IgG Light Chain <400>
SEQUENCE: 46 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu
Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Gly Ser Pro Gly Gln
Ser Val Ser Ile Ser 20 25 30 Cys Ser Gly Ser Ser Ser Asn Ile Gly
Asn Asn Tyr Val Tyr Trp Tyr 35 40 45 Gln His Leu Pro Gly Thr Ala
Pro Lys Leu Leu Ile Tyr Ser Asp Thr 50 55 60 Lys Arg Pro Ser Gly
Val Pro Asp Arg Ile Ser Gly Ser Lys Ser Gly 65 70 75 80 Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala 85 90 95 Asp
Tyr Tyr Cys Ala Ser Trp Asp Asp Ser Leu Asp Gly Pro Val Phe 100 105
110 Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Asn Pro
115 120 125 Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala
Asn Lys 130 135 140 Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro
Gly Ala Val Thr 145 150 155 160 Val Ala Trp Lys Ala Asp Gly Ser Pro
Val Lys Ala Gly Val Glu Thr 165 170 175 Thr Lys Pro Ser Lys Gln Ser
Asn Asn Lys Tyr Ala Ala Ser Ser Tyr 180 185 190 Leu Ser Leu Thr Pro
Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys 195 200 205 Gln Val Thr
His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr 210 215 220 Glu
Cys Ser 225 <210> SEQ ID NO 47 <211> LENGTH: 9203
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polynucleotide <220> FEATURE:
<223> OTHER INFORMATION: Vector 1 <400> SEQUENCE: 47
aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca
60 tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa
ggtggtacga 120 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg
attggacgaa ccactgaatt 180 gccgcattgc agagatattg tatttaagtg
cctagctcga tacataaacg ggtctctctg 240 gttagaccag atctgagcct
gggagctctc tggctaacta gggaacccac tgcttaagcc 300 tcaataaagc
ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360
taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg
420 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
gactcggctt 480 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg
tgagtacgcc aaaaattttg 540 actagcggag gctagaagga gagagatggg
tgcgagagcg tcagtattaa gcgggggaga 600 attagatcgc gatgggaaaa
aattcggtta aggccagggg gaaagaaaaa atataaatta 660 aaacatatag
tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720
gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga
780 tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
gcatcaaagg 840 atagagataa aagacaccaa ggaagcttta gacaagatag
aggaagagca aaacaaaagt 900 aagaccaccg cacagcaagc ggccgctgat
cttcagacct ggaggaggag atatgaggga 960 caattggaga agtgaattat
ataaatataa agtagtaaaa attgaaccat taggagtagc 1020 acccaccaag
gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080
tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct
1140 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
atttgctgag 1200 ggctattgag gcgcaacagc atctgttgca actcacagtc
tggggcatca agcagctcca 1260 ggcaagaatc ctggctgtgg aaagatacct
aaaggatcaa cagctcctgg ggatttgggg 1320 ttgctctgga aaactcattt
gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 1380 atctctggaa
cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440
ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
1500 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
acataacaaa 1560 ttggctgtgg tatataaaat tattcataat gatagtagga
ggcttggtag gtttaagaat 1620 agtttttgct gtactttcta tagtgaatag
agttaggcag ggatattcac cattatcgtt 1680 tcagacccac ctcccaaccc
cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740 tggagagaga
gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgcta 1800
gcttttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata
1860 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
tcaaaatttt 1920 actagtgatt atcggatcaa ctttgtatag aaaagttggg
ctccggtgcc cgtcagtggg 1980 cagagcgcac atcgcccaca gtccccgaga
agttgggggg aggggtcggc aattgaaccg 2040 gtgcctagag aaggtggcgc
ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc 2100 tttttcccga
gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt 2160
ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg
2220 gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg
ctgcagtacg 2280 tgattcttga tcccgagctt cgggttggaa gtgggtggga
gagttcgagg ccttgcgctt 2340 aaggagcccc ttcgcctcgt gcttgagttg
aggcctggcc tgggcgctgg ggccgccgcg 2400 tgcgaatctg gtggcacctt
cgcgcctgtc tcgctgcttt cgataagtct ctagccattt 2460 aaaatttttg
atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg 2520
gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg
2580 cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg
agaatcggac 2640 gggggtagtc tcaagctggc cggcctgctc tggtgcctgg
tctcgcgccg ccgtgtatcg 2700 ccccgccctg ggcggcaagg ctggcccggt
cggcaccagt tgcgtgagcg gaaagatggc 2760 cgcttcccgg ccctgctgca
gggagctcaa aatggaggac gcggcgctcg ggagagcggg 2820 cgggtgagtc
acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg 2880
actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta
2940 cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac
actgagtggg 3000 tggagactga agttaggcca gcttggcact tgatgtaatt
ctccttggaa tttgcccttt 3060 ttgagtttgg atcttggttc attctcaagc
ctcagacagt ggttcaaagt ttttttcttc 3120 catttcaggt gtcgtgacaa
gtttgtacaa aaaagcaggc tgccaccatg gagttcggcc 3180 tgagctgggt
gttcctggtg gccctgttca gaggcgtgca gtgccaggtg aagctgcagg 3240
agagcggccc cggcctggtg gcccccagcc agagcctgag catcacctgc accgtgagcg
3300 gcttcagcct gaccgcctac ggcgtggact gggtgagaca gccccccggc
aagtgcctgg 3360 agtggctggg cgtgatctgg ggcggcggca gaaccaacta
caacagcggc ctgatgagca 3420 gactgagcat cagaaaggac aacagcaaga
gccaggtgtt cctgaccatg aacagcctgc 3480 agaccgacga caccgccaag
tactactgcg tgaagcacac caactgggac ggcggcttcg 3540 cctactgggg
ccagggcacc accgtgaccg tgagcagcgg cggcggcggc agcggcggcg 3600
gcggcagcgg cggcggcggc agcggcagcc ccggccagag cgtgagcatc agctgcagcg
3660 gcagcagcag caacatcggc aacaactacg tgtactggta ccagcacctg
cccggcaccg 3720 cccccaagct gctgatctac agcgacacca agagacccag
cggcgtgccc gacagaatca 3780 gcggcagcaa gagcggcacc agcgccagcc
tggccatcag cggcctgcag agcgaggacg 3840 aggccgacta ctactgcgcc
agctgggacg acagcctgga cggccccgtg ttcggctgcg 3900 gcaccaagct
gaccgtgctg ctgaagaccc ccctgggcga caccacccac acctgcccca 3960
gatgccccga gcccaagagc tgcgacaccc cccccccctg ccccagatgc cccgagccca
4020 agagctgcga cacccccccc ccctgcccca gatgccccga gcccaagagc
tgcgacaccc 4080 cccccccctg ccccagatgc cccgcccccg agctgctggg
cggccccagc gtgttcctgt 4140 tcccccccaa gcccaaggac accctgatga
tcagcagagc ccccgaggtg acctgcgtgg 4200 tggtggacgt gagccacgag
gaccccgagg tgaagttcaa ctggtacgtg gacggcgtgg 4260 aggtgcacaa
cgccaagacc aagcccagag aggagcagta caacagcacc tacagagtgg 4320
tgagcgtgct gaccgtgctg caccaggact ggctgaacgg caaggagtac aagtgcaagg
4380 tgagcaacaa ggccctgccc gcccccatcg agaagaccat cagcaaggcc
aagggccagc 4440 ccagagagcc ccaggtgtac accctgcccc ccagcagaga
cgagctgacc aagaaccagg 4500 tgagcctgac ctgcctggtg aagggcttct
accccagcga catcgccgtg gagtgggaga 4560 gcaacggcca gcccgagaac
aactacaaga ccaccccccc cgtgctggac agcgacggca 4620 gcttcttcct
gtacagcaag ctgaccgtgg acaagagcag atggcagcag ggcaacgtgt 4680
tcagctgcag cgtgatgcac gaggccctgc acaaccacta cacccagaag agcctgagcc
4740 tgagccccga gctgcagctg gaggagagct gcgccgaggc ccaggacggc
gagctggacg 4800 gcctgtggac caccatcacc atcttcatca ccctgttcct
gctgagcgtg tgctacagcg 4860 ccaccgtgac cttcttcaag gtgaagtgga
tcttcagcag cgtggtggac ctgaagcaga 4920 ccatcatccc cgactacaga
aacatgatcg gccagggcgc ctaaacccag ctttcttgta 4980 caaagtggtg
ataatcgaat tctaaaccca gctttcttgt acaaagtggt gataatcgaa 5040
ttccgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
5100 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
atgctattgc 5160 ttcccgtatg gctttcattt tctcctcctt gtataaatcc
tggttgctgt ctctttatga 5220 ggagttgtgg cccgttgtca ggcaacgtgg
cgtggtgtgc actgtgtttg ctgacgcaac 5280 ccccactggt tggggcattg
ccaccacctg tcagctcctt tccgggactt tcgctttccc 5340 cctccctatt
gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc 5400
tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt cctttccatg
5460 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
acgtcccttc 5520 ggccctcaat ccagcggacc ttccttcccg cggcctgctg
ccggctctgc ggcctcttcc 5580 gcgtcttcgc cttcgccctc agacgagtcg
gatctccctt tgggccgcct ccccgcatcg 5640 ggaattcccg cggttcgctt
taagaccaat gacttacaag gcagctgtag atcttagcca 5700 ctttttaaaa
gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct 5760
gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
5820 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
ttcaagtagt 5880 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
ctcagaccct tttagtcagt 5940 gtggaaaatc tctagcagta gtagttcatg
tcatcttatt attcagtatt tataacttgc 6000 aaagaaatga atatcagaga
gtgagaggaa cttgtttatt gcagcttata atggttacaa 6060 ataaagcaat
agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 6120
tggtttgtcc aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta
6180 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
ccatggctga 6240 ctaatttttt ttatttatgc agaggccgag gccgcctcgg
cctctgagct attccagaag 6300 tagtgaggag gcttttttgg aggcctaggg
acgtacccaa ttcgccctat agtgagtcgt 6360 attacgcgcg ctcactggcc
gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta 6420 cccaacttaa
tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg 6480
cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct
6540 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
gctacacttg 6600 ccagcgccct agcgcccgct cctttcgctt tcttcccttc
ctttctcgcc acgttcgccg 6660 gctttccccg tcaagctcta aatcgggggc
tccctttagg gttccgattt agtgctttac 6720 ggcacctcga ccccaaaaaa
cttgattagg gtgatggttc acgtagtggg ccatcgccct 6780 gatagacggt
ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt 6840
tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
6900 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
aacgcgaatt 6960 ttaacaaaat attaacgctt acaatttagg tggcactttt
cggggaaatg tgcgcggaac 7020 ccctatttgt ttatttttct aaatacattc
aaatatgtat ccgctcatga gacaataacc 7080 ctgataaatg cttcaataat
attgaaaaag gaagagtatg agtattcaac atttccgtgt 7140 cgcccttatt
cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 7200
ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
7260 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
caatgatgag 7320 cacttttaaa gttctgctat gtggcgcggt attatcccgt
attgacgccg ggcaagagca 7380 actcggtcgc cgcatacact attctcagaa
tgacttggtt gagtactcac cagtcacaga 7440 aaagcatctt acggatggca
tgacagtaag agaattatgc agtgctgcca taaccatgag 7500 tgataacact
gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc 7560
ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
7620 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
caacaacgtt 7680 gcgcaaacta ttaactggcg aactacttac tctagcttcc
cggcaacaat taatagactg 7740 gatggaggcg gataaagttg caggaccact
tctgcgctcg gcccttccgg ctggctggtt 7800 tattgctgat aaatctggag
ccggtgagcg tgggtctcgc ggtatcattg cagcactggg 7860 gccagatggt
aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 7920
ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
7980 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
tttaatttaa 8040 aaggatctag gtgaagatcc tttttgataa tctcatgacc
aaaatccctt aacgtgagtt 8100 ttcgttccac tgagcgtcag accccgtaga
aaagatcaaa ggatcttctt gagatccttt 8160 ttttctgcgc gtaatctgct
gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 8220 tttgccggat
caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 8280
gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
8340 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
ccagtggcga 8400 taagtcgtgt cttaccgggt tggactcaag acgatagtta
ccggataagg cgcagcggtc 8460 gggctgaacg gggggttcgt gcacacagcc
cagcttggag cgaacgacct acaccgaact 8520 gagataccta cagcgtgagc
tatgagaaag cgccacgctt cccgaagaga gaaaggcgga 8580 caggtatccg
gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 8640
aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
8700 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
cggccttttt 8760 acggttcctg gccttttgct ggccttttgc tcacatgttc
tttcctgcgt tatcccctga 8820 ttctgtggat aaccgtatta ccgcctttga
gtgagctgat accgctcgcc gcagccgaac 8880 gaccgagcgc agcgagtcag
tgagcgagga agcggaagag cgcccaatac gcaaaccgcc 8940 tctccccgcg
cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa 9000
agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
9060 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
aacaatttca 9120 cacaggaaac agctatgacc atgattacgc caagcgcgca
attaaccctc actaaaggga 9180 acaaaagctg gagctgcaag ctt 9203
<210> SEQ ID NO 48 <211> LENGTH: 10862 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polynucleotide <220> FEATURE: <223>
OTHER INFORMATION: Vector 2 <400> SEQUENCE: 48 aatgtagtct
tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60
tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga
120 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa
ccactgaatt 180 gccgcattgc agagatattg tatttaagtg cctagctcga
tacataaacg ggtctctctg 240 gttagaccag atctgagcct gggagctctc
tggctaacta gggaacccac tgcttaagcc 300 tcaataaagc ttgccttgag
tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360 taactagaga
tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 420
aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt
480 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
aaaaattttg 540 actagcggag gctagaagga gagagatggg tgcgagagcg
tcagtattaa gcgggggaga 600 attagatcgc gatgggaaaa aattcggtta
aggccagggg gaaagaaaaa atataaatta 660 aaacatatag tatgggcaag
cagggagcta gaacgattcg cagttaatcc tggcctgtta 720 gaaacatcag
aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 780
tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg
840 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
aaacaaaagt 900 aagaccaccg cacagcaagc ggccgctgat cttcagacct
ggaggaggag atatgaggga 960 caattggaga agtgaattat ataaatataa
agtagtaaaa attgaaccat taggagtagc 1020 acccaccaag gcaaagagaa
gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080 tttgttcctt
gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140
gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
1200 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
agcagctcca 1260 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
cagctcctgg ggatttgggg 1320 ttgctctgga aaactcattt gcaccactgc
tgtgccttgg aatgctagtt ggagtaataa 1380 atctctggaa cagatttgga
atcacacgac ctggatggag tgggacagag aaattaacaa 1440 ttacacaagc
ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500
acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
1560 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
gtttaagaat 1620 agtttttgct gtactttcta tagtgaatag agttaggcag
ggatattcac cattatcgtt 1680 tcagacccac ctcccaaccc cgaggggacc
cgacaggccc gaaggaatag aagaagaagg 1740 tggagagaga gacagagaca
gatccattcg attagtgaac ggatctcgac ggtatcgcta 1800 gcttttaaaa
gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata 1860
atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
1920 actagtatca actttgtata gaaaagttgg gctccggtgc ccgtcagtgg
gcagagcgca 1980 catcgcccac agtccccgag aagttggggg gaggggtcgg
caattgaacc ggtgcctaga 2040 gaaggtggcg cggggtaaac tgggaaagtg
atgtcgtgta ctggctccgc ctttttcccg 2100 agggtggggg agaaccgtat
ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg 2160 ggtttgccgc
cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta 2220
cgggttatgg cccttgcgtg ccttgaatta cttccacctg gctgcagtac gtgattcttg
2280 atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct
taaggagccc 2340 cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg
gggccgccgc gtgcgaatct 2400 ggtggcacct tcgcgcctgt ctcgctgctt
tcgataagtc tctagccatt taaaattttt 2460 gatgacctgc tgcgacgctt
tttttctggc aagatagtct tgtaaatgcg ggccaagatc 2520 tgcacactgg
tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc 2580
gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt
2640 ctcaagctgg ccggcctgct ctggtgcctg gtctcgcgcc gccgtgtatc
gccccgccct 2700 gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc
ggaaagatgg ccgcttcccg 2760 gccctgctgc agggagctca aaatggagga
cgcggcgctc gggagagcgg gcgggtgagt 2820 cacccacaca aaggaaaagg
gcctttccgt cctcagccgt cgcttcatgt gactccacgg 2880 agtaccgggc
gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt 2940
taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg
3000 aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt
tttgagtttg 3060 gatcttggtt cattctcaag cctcagacag tggttcaaag
tttttttctt ccatttcagg 3120 tgtcgtgaca agtttgtaca aaaaagcagg
ctgccaccat ggagttcggc ctgagctggg 3180 tgttcctggt ggccctgttc
agaggcgtgc agtgccaggt gaagctgcag gagagcggcc 3240 ccggcctggt
ggcccccagc cagagcctga gcatcacctg caccgtgagc ggcttcagcc 3300
tgaccgccta cggcgtggac tgggtgagac agccccccgg caagggcctg gagtggctgg
3360 gcgtgatctg gggcggcggc agaaccaact acaacagcgg cctgatgagc
agactgagca 3420 tcagaaagga caacagcaag agccaggtgt tcctgaccat
gaacagcctg cagaccgacg 3480 acaccgccaa gtactactgc gtgaagcaca
ccaactggga cggcggcttc gcctactggg 3540 gccagggcac caccgtgacc
gtgagcagcc ccagcgtgtt ccccctggcc cccagcagca 3600 agagcaccag
cggcggcacc gccgccctgg gctgcctggt gaaggactac ttccccgagc 3660
ccgtgaccgt gagctggaac agcggcgccc tgaccagcgg cgtgcacacc ttccccgccg
3720 tgctgcagag cagcggcctg tacagcctga gcagcgtggt gaccgtgccc
agcagcagcc 3780 tgggcaccca gacctacatc tgcaacgtga accacaagcc
cagcaacacc aaggtggaca 3840 agaaggtgga gctgaagacc cccctgggcg
acaccaccca cacctgcccc agatgccccg 3900 agcccaagag ctgcgacacc
ccccccccct gccccagatg ccccgagccc aagagctgcg 3960 acaccccccc
cccctgcccc agatgccccg agcccaagag ctgcgacacc ccccccccct 4020
gccccagatg ccccgccccc gagctgctgg gcggccccag cgtgttcctg ttccccccca
4080 agcccaagga caccctgatg atcagcagag cccccgaggt gacctgcgtg
gtggtggacg 4140 tgagccacga ggaccccgag gtgaagttca actggtacgt
ggacggcgtg gaggtgcaca 4200 acgccaagac caagcccaga gaggagcagt
acaacagcac ctacagagtg gtgagcgtgc 4260 tgaccgtgct gcaccaggac
tggctgaacg gcaaggagta caagtgcaag gtgagcaaca 4320 aggccctgcc
cgcccccatc gagaagacca tcagcaaggc caagggccag cccagagagc 4380
cccaggtgta caccctgccc cccagcagag acgagctgac caagaaccag gtgagcctga
4440 cctgcctggt gaagggcttc taccccagcg acatcgccgt ggagtgggag
agcaacggcc 4500 agcccgagaa caactacaag accacccccc ccgtgctgga
cagcgacggc agcttcttcc 4560 tgtacagcaa gctgaccgtg gacaagagca
gatggcagca gggcaacgtg ttcagctgca 4620 gcgtgatgca cgaggccctg
cacaaccact acacccagaa gagcctgagc ctgagccccg 4680 agctgcagct
ggaggagagc tgcgccgagg cccaggacgg cgagctggac ggcctgtgga 4740
ccaccatcac catcttcatc accctgttcc tgctgagcgt gtgctacagc gccaccgtga
4800 ccttcttcaa ggtgaagtgg atcttcagca gcgtggtgga cctgaagcag
accatcatcc 4860 ccgactacag aaacatgatc ggccagggcg cctaaaacaa
caacaattgc attcatttta 4920 tgtttcaggt tcagggggag gtgtgggagg
ttttttaaag caagtaaaac ctctacaaat 4980 gtggtacgcg ttaacaacaa
caattgcatt cattttatgt ttcaggttca gggggaggtg 5040 tgggaggttt
tttaaagcaa gtaaaacctc tacaaatgtg gtacgcgtta cccagctttc 5100
ttgtacaaag tggtaaatag atagaacaac aacaattgca ttcatttttg atttcaggtt
5160 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacactga
cggtacgcgt 5220 taacaacaac aattgcattc atttgtagtt tcaggttcag
ggggaggtgt gggaggtttt 5280 ttaaagcaag ttaaacctct aaaatagtgg
tacgcgttac ccagctttct tgtacaaagt 5340 ggacccagct ttcttgtaca
aagtgggccc ctctccctcc ccccccccta acgttactgg 5400 ccgaagccgc
ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt 5460
gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc
5520 taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg
tgaaggaagc 5580 agttcctctg gaagcttctt gaagacaaac aacgtctgta
gcgacccttt gcaggcagcg 5640 gaacccccca cctggcgaca ggtgcctctg
cggccaaaag ccacgtgtat aagatacacc 5700 tgcaaaggcg gcacaacccc
agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa 5760 atggctctcc
tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg 5820
tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa
5880 aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa
cacgatgata 5940 atatggccac aaccatggcc accgacatga gagtgcccgc
ccagctgctg ggcctgctgc 6000 tgctgtggct gagcggcgcc agatgcggca
gccccggcca gagcgtgagc atcagctgca 6060 gcggcagcag cagcaacatc
ggcaacaact acgtgtactg gtaccagcac ctgcccggca 6120 ccgcccccaa
gctgctgatc tacagcgaca ccaagagacc cagcggcgtg cccgacagaa 6180
tcagcggcag caagagcggc accagcgcca gcctggccat cagcggcctg cagagcgagg
6240 acgaggccga ctactactgc gccagctggg acgacagcct ggacggcccc
gtgttcggcg 6300 gcggcaccaa gctgaccgtg ctgggccagc ccaaggccaa
ccccaccgtg accctgttcc 6360 cccccagcag cgaggagctg caggccaaca
aggccaccct ggtgtgcctg atcagcgact 6420 tctaccccgg cgccgtgacc
gtggcctgga aggccgacgg cagccccgtg aaggccggcg 6480 tggagaccac
caagcccagc aagcagagca acaacaagta cgccgccagc agctacctga 6540
gcctgacccc cgagcagtgg aagagccaca gaagctacag ctgccaggtg acccacgagg
6600 gcagcaccgt ggagaagacc gtggccccca ccgagtgcag ctaacaactt
tattatacat 6660 agttgatcaa ttccaacttt attatacata gttgatcaat
tccgataatc aacctctgga 6720 ttacaaaatt tgtgaaagat tgactggtat
tcttaactat gttgctcctt ttacgctatg 6780 tggatacgct gctttaatgc
ctttgtatca tgctattgct tcccgtatgg ctttcatttt 6840 ctcctccttg
tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag 6900
gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
6960 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
ccacggcgga 7020 actcatcgcc gcctgccttg cccgctgctg gacaggggct
cggctgttgg gcactgacaa 7080 ttccgtggtg ttgtcgggga agctgacgtc
ctttccatgg ctgctcgcct gtgttgccac 7140 ctggattctg cgcgggacgt
ccttctgcta cgtcccttcg gccctcaatc cagcggacct 7200 tccttcccgc
ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca 7260
gacgagtcgg atctcccttt gggccgcctc cccgcatcgg gaattcccgc ggttcgcttt
7320 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
aaaagggggg 7380 actggaaggg ctaattcact cccaacgaag acaagatctg
ctttttgctt gtactgggtc 7440 tctctggtta gaccagatct gagcctggga
gctctctggc taactaggga acccactgct 7500 taagcctcaa taaagcttgc
cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga 7560 ctctggtaac
tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtag 7620
tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag
7680 tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata
gcatcacaaa 7740 tttcacaaat aaagcatttt tttcactgca ttctagttgt
ggtttgtcca aactcatcaa 7800 tgtatcttat catgtctggc tctagctatc
ccgcccctaa ctccgcccat cccgccccta 7860 actccgccca gttccgccca
ttctccgccc catggctgac taattttttt tatttatgca 7920 gaggccgagg
ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga 7980
ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg
8040 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
cgccttgcag 8100 cacatccccc tttcgccagc tggcgtaata gcgaagaggc
ccgcaccgat cgcccttccc 8160 aacagttgcg cagcctgaat ggcgaatggg
acgcgccctg tagcggcgca ttaagcgcgg 8220 cgggtgtggt ggttacgcgc
agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 8280 ctttcgcttt
cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 8340
atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
8400 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
tttcgccctt 8460 tgacgttgga gtccacgttc tttaatagtg gactcttgtt
ccaaactgga acaacactca 8520 accctatctc ggtctattct tttgatttat
aagggatttt gccgatttcg gcctattggt 8580 taaaaaatga gctgatttaa
caaaaattta acgcgaattt taacaaaata ttaacgctta 8640 caatttaggt
ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 8700
aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
8760 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
ccttttttgc 8820 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
gtgaaagtaa aagatgctga 8880 agatcagttg ggtgcacgag tgggttacat
cgaactggat ctcaacagcg gtaagatcct 8940 tgagagtttt cgccccgaag
aacgttttcc aatgatgagc acttttaaag ttctgctatg 9000 tggcgcggta
ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 9060
ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
9120 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
cggccaactt 9180 acttctgaca acgatcggag gaccgaagga gctaaccgct
tttttgcaca acatggggga 9240 tcatgtaact cgccttgatc gttgggaacc
ggagctgaat gaagccatac caaacgacga 9300 gcgtgacacc acgatgcctg
tagcaatggc aacaacgttg cgcaaactat taactggcga 9360 actacttact
ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 9420
aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
9480 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
agccctcccg 9540 tatcgtagtt atctacacga cggggagtca ggcaactatg
gatgaacgaa atagacagat 9600 cgctgagata ggtgcctcac tgattaagca
ttggtaactg tcagaccaag tttactcata 9660 tatactttag attgatttaa
aacttcattt ttaatttaaa aggatctagg tgaagatcct 9720 ttttgataat
ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 9780
ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
9840 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
aagagctacc 9900 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
ataccaaata ctgttcttct 9960 agtgtagccg tagttaggcc accacttcaa
gaactctgta gcaccgccta catacctcgc 10020 tctgctaatc ctgttaccag
tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 10080 ggactcaaga
cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 10140
cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
10200 atgagaaagc gccacgcttc ccgaagagag aaaggcggac aggtatccgg
taagcggcag 10260 ggtcggaaca ggagagcgca cgagggagct tccaggggga
aacgcctggt atctttatag 10320 tcctgtcggg tttcgccacc tctgacttga
gcgtcgattt ttgtgatgct cgtcaggggg 10380 gcggagccta tggaaaaacg
ccagcaacgc ggccttttta cggttcctgg ccttttgctg 10440 gccttttgct
cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 10500
cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
10560 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
gttggccgat 10620 tcattaatgc agctggcacg acaggtttcc cgactggaaa
gcgggcagtg agcgcaacgc 10680 aattaatgtg agttagctca ctcattaggc
accccaggct ttacacttta tgcttccggc 10740 tcgtatgttg tgtggaattg
tgagcggata acaatttcac acaggaaaca gctatgacca 10800 tgattacgcc
aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc 10860 tt
10862 <210> SEQ ID NO 49 <211> LENGTH: 9581 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polynucleotide <220> FEATURE: <223>
OTHER INFORMATION: Vector 3 <400> SEQUENCE: 49 aatgtagtct
tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60
tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga
120 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa
ccactgaatt 180 gccgcattgc agagatattg tatttaagtg cctagctcga
tacataaacg ggtctctctg 240 gttagaccag atctgagcct gggagctctc
tggctaacta gggaacccac tgcttaagcc 300 tcaataaagc ttgccttgag
tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360 taactagaga
tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 420
aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt
480 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
aaaaattttg 540 actagcggag gctagaagga gagagatggg tgcgagagcg
tcagtattaa gcgggggaga 600 attagatcgc gatgggaaaa aattcggtta
aggccagggg gaaagaaaaa atataaatta 660 aaacatatag tatgggcaag
cagggagcta gaacgattcg cagttaatcc tggcctgtta 720 gaaacatcag
aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 780
tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg
840 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
aaacaaaagt 900 aagaccaccg cacagcaagc ggccgctgat cttcagacct
ggaggaggag atatgaggga 960 caattggaga agtgaattat ataaatataa
agtagtaaaa attgaaccat taggagtagc 1020 acccaccaag gcaaagagaa
gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080 tttgttcctt
gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140
gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
1200 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
agcagctcca 1260 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
cagctcctgg ggatttgggg 1320 ttgctctgga aaactcattt gcaccactgc
tgtgccttgg aatgctagtt ggagtaataa 1380 atctctggaa cagatttgga
atcacacgac ctggatggag tgggacagag aaattaacaa 1440 ttacacaagc
ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500
acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
1560 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
gtttaagaat 1620 agtttttgct gtactttcta tagtgaatag agttaggcag
ggatattcac cattatcgtt 1680 tcagacccac ctcccaaccc cgaggggacc
cgacaggccc gaaggaatag aagaagaagg 1740 tggagagaga gacagagaca
gatccattcg attagtgaac ggatctcgac ggtatcgcta 1800 gcttttaaaa
gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata 1860
atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
1920 actagtatca actttgtata gaaaagttgg gctccggtgc ccgtcagtgg
gcagagcgca 1980 catcgcccac agtccccgag aagttggggg gaggggtcgg
caattgaacc ggtgcctaga 2040 gaaggtggcg cggggtaaac tgggaaagtg
atgtcgtgta ctggctccgc ctttttcccg 2100 agggtggggg agaaccgtat
ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg 2160 ggtttgccgc
cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta 2220
cgggttatgg cccttgcgtg ccttgaatta cttccacctg gctgcagtac gtgattcttg
2280 atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct
taaggagccc 2340 cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg
gggccgccgc gtgcgaatct 2400 ggtggcacct tcgcgcctgt ctcgctgctt
tcgataagtc tctagccatt taaaattttt 2460 gatgacctgc tgcgacgctt
tttttctggc aagatagtct tgtaaatgcg ggccaagatc 2520 tgcacactgg
tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc 2580
gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt
2640 ctcaagctgg ccggcctgct ctggtgcctg gtctcgcgcc gccgtgtatc
gccccgccct 2700 gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc
ggaaagatgg ccgcttcccg 2760 gccctgctgc agggagctca aaatggagga
cgcggcgctc gggagagcgg gcgggtgagt 2820 cacccacaca aaggaaaagg
gcctttccgt cctcagccgt cgcttcatgt gactccacgg 2880 agtaccgggc
gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt 2940
taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg
3000 aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt
tttgagtttg 3060 gatcttggtt cattctcaag cctcagacag tggttcaaag
tttttttctt ccatttcagg 3120 tgtcgtgaca agtttgtaca aaaaagcagg
ctgccaccat gtggctgcag agcctgctgc 3180 tgctgggcac cgtggcctgc
agcatcagcg cccccgccag aagccccagc cccagcaccc 3240 agccctggga
gcacgtgaac gccatccagg aggccagaag actgctgaac ctgagcagag 3300
acaccgccgc cgagatgaac gagaccgtgg aggtgatcag cgagatgttc gacctgcagg
3360 agcccacctg cctgcagacc agactggagc tgtacaagca gggcctgaga
ggcagcctga 3420 ccaagctgaa gggccccctg accatgatgg ccagccacta
caagcagcac tgccccccca 3480 cccccgagac cagctgcgcc acccagatca
tcaccttcga gagcttcaag gagaacctga 3540 aggacttcct gctggtgatc
cccttcgact gctgggagcc cgtgcaggag taaaacaaca 3600 acaattgcat
tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca 3660
agtaaaacct ctacaaatgt ggtacgcgtt aacaacaaca attgcattca ttttatgttt
3720 caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta
caaatgtggt 3780 acgcgttacc cagctttctt gtacaaagtg gtaaatagat
agaacaacaa caattgcatt 3840 catttttgat ttcaggttca gggggaggtg
tgggaggttt tttaaagcaa gtaaaacctc 3900 tacactgacg gtacgcgtta
acaacaacaa ttgcattcat ttgtagtttc aggttcaggg 3960 ggaggtgtgg
gaggtttttt aaagcaagtt aaacctctaa aatagtggta cgcgttaccc 4020
agctttcttg tacaaagtgg acccagcttt cttgtacaaa gtgggcccct ctccctcccc
4080 cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt
ttgtctatat 4140 gttattttcc accatattgc cgtcttttgg caatgtgagg
gcccggaaac ctggccctgt 4200 cttcttgacg agcattccta ggggtctttc
ccctctcgcc aaaggaatgc aaggtctgtt 4260 gaatgtcgtg aaggaagcag
ttcctctgga agcttcttga agacaaacaa cgtctgtagc 4320 gaccctttgc
aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc 4380
acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat
4440 agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc
tgaaggatgc 4500 ccagaaggta ccccattgta tgggatctga tctggggcct
cggtgcacat gctttacatg 4560 tgtttagtcg aggttaaaaa aacgtctagg
ccccccgaac cacggggacg tggttttcct 4620 ttgaaaaaca cgatgataat
atggccacaa ccatggccac cgtgctggcc cccgcctgga 4680 gccccaccac
ctacctgctg ctgctgctgc tgctgagcag cggcctgagc ggcacccagg 4740
actgcagctt ccagcacagc cccatcagca gcgacttcgc cgtgaagatc agagagctga
4800 gcgactacct gctgcaggac taccccgtga ccgtggccag caacctgcag
gacgaggagc 4860 tgtgcggcgg cctgtggaga ctggtgctgg cccagagatg
gatggagaga ctgaagaccg 4920 tggccggcag caagatgcag ggcctgctgg
agagagtgaa caccgagatc cacttcgtga 4980 ccaagtgcgc cttccagccc
ccccccagct gcctgagatt cgtgcagacc aacatcagca 5040 gactgctgca
ggagaccagc gagcagctgg tggccctgaa gccctggatc accagacaga 5100
acttcagcag atgcctggag ctgcagtgcc agcccgacag cagcaccctg ccccccccct
5160 ggagccccag acccctggag gccaccgccc ccaccgcccc ccagcccccc
ctgctgctgc 5220 tgctgctgct gcccgtgggc ctgctgctgc tggccgccgc
ctggtgcctg cactggcaga 5280 gaaccagaag aagaaccccc agacccggcg
agcaggtgcc ccccgtgccc agcccccagg 5340 acctgctgct ggtggagcac
taacaacttt attatacata gttgatcaat tccaacttta 5400 ttatacatag
ttgatcaatt ccgataatca acctctggat tacaaaattt gtgaaagatt 5460
gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
5520 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
ataaatcctg 5580 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
caacgtggcg tggtgtgcac 5640 tgtgtttgct gacgcaaccc ccactggttg
gggcattgcc accacctgtc agctcctttc 5700 cgggactttc gctttccccc
tccctattgc cacggcggaa ctcatcgccg cctgccttgc 5760 ccgctgctgg
acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa 5820
gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
5880 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
gcctgctgcc 5940 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
acgagtcgga tctccctttg 6000 ggccgcctcc ccgcatcggg aattcccgcg
gttcgcttta agaccaatga cttacaaggc 6060 agctgtagat cttagccact
ttttaaaaga aaagggggga ctggaagggc taattcactc 6120 ccaacgaaga
caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg 6180
agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
6240 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
agagatccct 6300 cagacccttt tagtcagtgt ggaaaatctc tagcagtagt
agttcatgtc atcttattat 6360 tcagtattta taacttgcaa agaaatgaat
atcagagagt gagaggaact tgtttattgc 6420 agcttataat ggttacaaat
aaagcaatag catcacaaat ttcacaaata aagcattttt 6480 ttcactgcat
tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct 6540
ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
6600 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
cgcctcggcc 6660 tctgagctat tccagaagta gtgaggaggc ttttttggag
gcctagggac gtacccaatt 6720 cgccctatag tgagtcgtat tacgcgcgct
cactggccgt cgttttacaa cgtcgtgact 6780 gggaaaaccc tggcgttacc
caacttaatc gccttgcagc acatccccct ttcgccagct 6840 ggcgtaatag
cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 6900
gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
6960 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
ttcccttcct 7020 ttctcgccac gttcgccggc tttccccgtc aagctctaaa
tcgggggctc cctttagggt 7080 tccgatttag tgctttacgg cacctcgacc
ccaaaaaact tgattagggt gatggttcac 7140 gtagtgggcc atcgccctga
tagacggttt ttcgcccttt gacgttggag tccacgttct 7200 ttaatagtgg
actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 7260
ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
7320 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
gcacttttcg 7380 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
atacattcaa atatgtatcc 7440 gctcatgaga caataaccct gataaatgct
tcaataatat tgaaaaagga agagtatgag 7500 tattcaacat ttccgtgtcg
cccttattcc cttttttgcg gcattttgcc ttcctgtttt 7560 tgctcaccca
gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 7620
gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
7680 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
tatcccgtat 7740 tgacgccggg caagagcaac tcggtcgccg catacactat
tctcagaatg acttggttga 7800 gtactcacca gtcacagaaa agcatcttac
ggatggcatg acagtaagag aattatgcag 7860 tgctgccata accatgagtg
ataacactgc ggccaactta cttctgacaa cgatcggagg 7920 accgaaggag
ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 7980
ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
8040 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
tagcttcccg 8100 gcaacaatta atagactgga tggaggcgga taaagttgca
ggaccacttc tgcgctcggc 8160 ccttccggct ggctggttta ttgctgataa
atctggagcc ggtgagcgtg ggtctcgcgg 8220 tatcattgca gcactggggc
cagatggtaa gccctcccgt atcgtagtta tctacacgac 8280 ggggagtcag
gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 8340
gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
8400 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
tcatgaccaa 8460 aatcccttaa cgtgagtttt cgttccactg agcgtcagac
cccgtagaaa agatcaaagg 8520 atcttcttga gatccttttt ttctgcgcgt
aatctgctgc ttgcaaacaa aaaaaccacc 8580 gctaccagcg gtggtttgtt
tgccggatca agagctacca actctttttc cgaaggtaac 8640 tggcttcagc
agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca 8700
ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
8760 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
gatagttacc 8820 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
acacagccca gcttggagcg 8880 aacgacctac accgaactga gatacctaca
gcgtgagcta tgagaaagcg ccacgcttcc 8940 cgaagagaga aaggcggaca
ggtatccggt aagcggcagg gtcggaacag gagagcgcac 9000 gagggagctt
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 9060
ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
9120 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
acatgttctt 9180 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
gcctttgagt gagctgatac 9240 cgctcgccgc agccgaacga ccgagcgcag
cgagtcagtg agcgaggaag cggaagagcg 9300 cccaatacgc aaaccgcctc
tccccgcgcg ttggccgatt cattaatgca gctggcacga 9360 caggtttccc
gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac 9420
tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
9480 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
agcgcgcaat 9540 taaccctcac taaagggaac aaaagctgga gctgcaagct t 9581
<210> SEQ ID NO 50 <211> LENGTH: 9746 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polynucleotide <220> FEATURE: <223> OTHER
INFORMATION: Vector 4 <400> SEQUENCE: 50 aatgtagtct
tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60
tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga
120 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa
ccactgaatt 180 gccgcattgc agagatattg tatttaagtg cctagctcga
tacataaacg ggtctctctg 240 gttagaccag atctgagcct gggagctctc
tggctaacta gggaacccac tgcttaagcc 300 tcaataaagc ttgccttgag
tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360 taactagaga
tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 420
aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt
480 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
aaaaattttg 540 actagcggag gctagaagga gagagatggg tgcgagagcg
tcagtattaa gcgggggaga 600 attagatcgc gatgggaaaa aattcggtta
aggccagggg gaaagaaaaa atataaatta 660 aaacatatag tatgggcaag
cagggagcta gaacgattcg cagttaatcc tggcctgtta 720 gaaacatcag
aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 780
tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg
840 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
aaacaaaagt 900 aagaccaccg cacagcaagc ggccgctgat cttcagacct
ggaggaggag atatgaggga 960 caattggaga agtgaattat ataaatataa
agtagtaaaa attgaaccat taggagtagc 1020 acccaccaag gcaaagagaa
gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080 tttgttcctt
gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140
gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
1200 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
agcagctcca 1260 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
cagctcctgg ggatttgggg 1320 ttgctctgga aaactcattt gcaccactgc
tgtgccttgg aatgctagtt ggagtaataa 1380 atctctggaa cagatttgga
atcacacgac ctggatggag tgggacagag aaattaacaa 1440 ttacacaagc
ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500
acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
1560 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
gtttaagaat 1620 agtttttgct gtactttcta tagtgaatag agttaggcag
ggatattcac cattatcgtt 1680 tcagacccac ctcccaaccc cgaggggacc
cgacaggccc gaaggaatag aagaagaagg 1740 tggagagaga gacagagaca
gatccattcg attagtgaac ggatctcgac ggtatcgcta 1800 gcttttaaaa
gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata 1860
atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
1920 actagtatca actttgtata gaaaagttgg gctccggtgc ccgtcagtgg
gcagagcgca 1980 catcgcccac agtccccgag aagttggggg gaggggtcgg
caattgaacc ggtgcctaga 2040 gaaggtggcg cggggtaaac tgggaaagtg
atgtcgtgta ctggctccgc ctttttcccg 2100 agggtggggg agaaccgtat
ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg 2160 ggtttgccgc
cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta 2220
cgggttatgg cccttgcgtg ccttgaatta cttccacctg gctgcagtac gtgattcttg
2280 atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct
taaggagccc 2340 cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg
gggccgccgc gtgcgaatct 2400 ggtggcacct tcgcgcctgt ctcgctgctt
tcgataagtc tctagccatt taaaattttt 2460 gatgacctgc tgcgacgctt
tttttctggc aagatagtct tgtaaatgcg ggccaagatc 2520 tgcacactgg
tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc 2580
gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt
2640 ctcaagctgg ccggcctgct ctggtgcctg gtctcgcgcc gccgtgtatc
gccccgccct 2700 gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc
ggaaagatgg ccgcttcccg 2760 gccctgctgc agggagctca aaatggagga
cgcggcgctc gggagagcgg gcgggtgagt 2820 cacccacaca aaggaaaagg
gcctttccgt cctcagccgt cgcttcatgt gactccacgg 2880 agtaccgggc
gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt 2940
taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg
3000 aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt
tttgagtttg 3060 gatcttggtt cattctcaag cctcagacag tggttcaaag
tttttttctt ccatttcagg 3120 tgtcgtgaca agtttgtaca aaaaagcagg
ctgccaccat gaccgtgctg gcccccgcct 3180 ggagccccac cacctacctg
ctgctgctgc tgctgctgag cagcggcctg agcggcaccc 3240 aggactgcag
cttccagcac agccccatca gcagcgactt cgccgtgaag atcagagagc 3300
tgagcgacta cctgctgcag gactaccccg tgaccgtggc cagcaacctg caggacgagg
3360 agctgtgcgg cggcctgtgg agactggtgc tggcccagag atggatggag
agactgaaga 3420 ccgtggccgg cagcaagatg cagggcctgc tggagagagt
gaacaccgag atccacttcg 3480 tgaccaagtg cgccttccag ccccccccca
gctgcctgag attcgtgcag accaacatca 3540 gcagactgct gcaggagacc
agcgagcagc tggtggccct gaagccctgg atcaccagac 3600 agaacttcag
cagatgcctg gagctgcagt gccagcccga cagcagcacc ctgccccccc 3660
cctggagccc cagacccctg gaggccaccg cccccaccgc cccccagtaa aacaacaaca
3720 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt
taaagcaagt 3780 aaaacctcta caaatgtggt acgcgttaac aacaacaatt
gcattcattt tatgtttcag 3840 gttcaggggg aggtgtggga ggttttttaa
agcaagtaaa acctctacaa atgtggtacg 3900 cgttacccag ctttcttgta
caaagtggta aatagataga acaacaacaa ttgcattcat 3960 ttttgatttc
aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac 4020
actgacggta cgcgttaaca acaacaattg cattcatttg tagtttcagg ttcaggggga
4080 ggtgtgggag gttttttaaa gcaagttaaa cctctaaaat agtggtacgc
gttacccagc 4140 tttcttgtac aaagtggacc cagctttctt gtacaaagtg
ggcccctctc cctccccccc 4200 ccctaacgtt actggccgaa gccgcttgga
ataaggccgg tgtgcgtttg tctatatgtt 4260 attttccacc atattgccgt
cttttggcaa tgtgagggcc cggaaacctg gccctgtctt 4320 cttgacgagc
attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa 4380
tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac
4440 cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc
aaaagccacg 4500 tgtataagat acacctgcaa aggcggcaca accccagtgc
cacgttgtga gttggatagt 4560 tgtggaaaga gtcaaatggc tctcctcaag
cgtattcaac aaggggctga aggatgccca 4620 gaaggtaccc cattgtatgg
gatctgatct ggggcctcgg tgcacatgct ttacatgtgt 4680 ttagtcgagg
ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg 4740
aaaaacacga tgataatatg gccacaacca tggccaccgt gctggccccc gcctggagcc
4800 ccaccaccta cctgctgctg ctgctgctgc tgagcagcgg cctgagcgcc
cccgccagaa 4860 gccccagccc cagcacccag ccctgggagc acgtgaacgc
catccaggag gccagaagac 4920 tgctgaacct gagcagagac accgccgccg
agatgaacga gaccgtggag gtgatcagcg 4980 agatgttcga cctgcaggag
cccacctgcc tgcagaccag actggagctg tacaagcagg 5040 gcctgagagg
cagcctgacc aagctgaagg gccccctgac catgatggcc agccactaca 5100
agcagcactg cccccccacc cccgagacca gctgcgccac ccagatcatc accttcgaga
5160 gcttcaagga gaacctgaag gacttcctgc tggtgatccc cttcgactgc
tgggagcccg 5220 tgcaggagcc caccaccacc cccgccccca gaccccccac
ccccgccccc accatcgcca 5280 gccagcccct gagcctgaga cccgaggcct
gcagacccgc cgccggcggc gccgtgcaca 5340 ccagaggcct ggacttcgcc
tgcgacatct acatctgggc ccccctggcc ggcacctgcg 5400 gcgtgctgct
gctgagcctg gtgatcaccc tgtactgcaa ccacagaaac agaagaagag 5460
tgtgcaagtg ccccagaccc gtggtgaaga gcggcgacaa gcccagcctg agcgccagat
5520 acgtgtaaca actttattat acatagttga tcaattccaa ctttattata
catagttgat 5580 caattccgat aatcaacctc tggattacaa aatttgtgaa
agattgactg gtattcttaa 5640 ctatgttgct ccttttacgc tatgtggata
cgctgcttta atgcctttgt atcatgctat 5700 tgcttcccgt atggctttca
ttttctcctc cttgtataaa tcctggttgc tgtctcttta 5760 tgaggagttg
tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc 5820
aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
5880 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
gctggacagg 5940 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
gggaagctga cgtcctttcc 6000 atggctgctc gcctgtgttg ccacctggat
tctgcgcggg acgtccttct gctacgtccc 6060 ttcggccctc aatccagcgg
accttccttc ccgcggcctg ctgccggctc tgcggcctct 6120 tccgcgtctt
cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca 6180
tcgggaattc ccgcggttcg ctttaagacc aatgacttac aaggcagctg tagatcttag
6240 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac
gaagacaaga 6300 tctgcttttt gcttgtactg ggtctctctg gttagaccag
atctgagcct gggagctctc 6360 tggctaacta gggaacccac tgcttaagcc
tcaataaagc ttgccttgag tgcttcaagt 6420 agtgtgtgcc cgtctgttgt
gtgactctgg taactagaga tccctcagac ccttttagtc 6480 agtgtggaaa
atctctagca gtagtagttc atgtcatctt attattcagt atttataact 6540
tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta
6600 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
tgcattctag 6660 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
tggctctagc tatcccgccc 6720 ctaactccgc ccatcccgcc cctaactccg
cccagttccg cccattctcc gccccatggc 6780 tgactaattt tttttattta
tgcagaggcc gaggccgcct cggcctctga gctattccag 6840 aagtagtgag
gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt 6900
cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
6960 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
aatagcgaag 7020 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
gaatggcgaa tgggacgcgc 7080 cctgtagcgg cgcattaagc gcggcgggtg
tggtggttac gcgcagcgtg accgctacac 7140 ttgccagcgc cctagcgccc
gctcctttcg ctttcttccc ttcctttctc gccacgttcg 7200 ccggctttcc
ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 7260
tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
7320 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
agtggactct 7380 tgttccaaac tggaacaaca ctcaacccta tctcggtcta
ttcttttgat ttataaggga 7440 ttttgccgat ttcggcctat tggttaaaaa
atgagctgat ttaacaaaaa tttaacgcga 7500 attttaacaa aatattaacg
cttacaattt aggtggcact tttcggggaa atgtgcgcgg 7560 aacccctatt
tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 7620
accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
7680 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
acccagaaac 7740 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
cgagtgggtt acatcgaact 7800 ggatctcaac agcggtaaga tccttgagag
ttttcgcccc gaagaacgtt ttccaatgat 7860 gagcactttt aaagttctgc
tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 7920 gcaactcggt
cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 7980
agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
8040 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
aggagctaac 8100 cgcttttttg cacaacatgg gggatcatgt aactcgcctt
gatcgttggg aaccggagct 8160 gaatgaagcc ataccaaacg acgagcgtga
caccacgatg cctgtagcaa tggcaacaac 8220 gttgcgcaaa ctattaactg
gcgaactact tactctagct tcccggcaac aattaataga 8280 ctggatggag
gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 8340
gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
8400 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
gtcaggcaac 8460 tatggatgaa cgaaatagac agatcgctga gataggtgcc
tcactgatta agcattggta 8520 actgtcagac caagtttact catatatact
ttagattgat ttaaaacttc atttttaatt 8580 taaaaggatc taggtgaaga
tcctttttga taatctcatg accaaaatcc cttaacgtga 8640 gttttcgttc
cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 8700
tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
8760 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
tcagcagagc 8820 gcagatacca aatactgttc ttctagtgta gccgtagtta
ggccaccact tcaagaactc 8880 tgtagcaccg cctacatacc tcgctctgct
aatcctgtta ccagtggctg ctgccagtgg 8940 cgataagtcg tgtcttaccg
ggttggactc aagacgatag ttaccggata aggcgcagcg 9000 gtcgggctga
acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 9060
actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag agagaaaggc
9120 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
agcttccagg 9180 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
cacctctgac ttgagcgtcg 9240 atttttgtga tgctcgtcag gggggcggag
cctatggaaa aacgccagca acgcggcctt 9300 tttacggttc ctggcctttt
gctggccttt tgctcacatg ttctttcctg cgttatcccc 9360 tgattctgtg
gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 9420
aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
9480 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
ttcccgactg 9540 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
ctcactcatt aggcacccca 9600 ggctttacac tttatgcttc cggctcgtat
gttgtgtgga attgtgagcg gataacaatt 9660 tcacacagga aacagctatg
accatgatta cgccaagcgc gcaattaacc ctcactaaag 9720 ggaacaaaag
ctggagctgc aagctt 9746 <210> SEQ ID NO 51 <211> LENGTH:
8189 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polynucleotide
<220> FEATURE: <223> OTHER INFORMATION: Vector 5
<400> SEQUENCE: 51 aatgtagtct tatgcaatac tcttgtagtc
ttgcaacatg gtaacgatga gttagcaaca 60 tgccttacaa ggagagaaaa
agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120 tcgtgcctta
ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180
gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
240 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
tgcttaagcc 300 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
cgtctgttgt gtgactctgg 360 taactagaga tccctcagac ccttttagtc
agtgtggaaa atctctagca gtggcgcccg 420 aacagggact tgaaagcgaa
agggaaacca gaggagctct ctcgacgcag gactcggctt 480 gctgaagcgc
gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540
actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
600 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
atataaatta 660 aaacatatag tatgggcaag cagggagcta gaacgattcg
cagttaatcc tggcctgtta 720 gaaacatcag aaggctgtag acaaatactg
ggacagctac aaccatccct tcagacagga 780 tcagaagaac ttagatcatt
atataataca gtagcaaccc tctattgtgt gcatcaaagg 840 atagagataa
aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900
aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
960 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
taggagtagc 1020 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
agagcagtgg gaataggagc 1080 tttgttcctt gggttcttgg gagcagcagg
aagcactatg ggcgcagcgt caatgacgct 1140 gacggtacag gccagacaat
tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200 ggctattgag
gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260
ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
1320 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
ggagtaataa 1380 atctctggaa cagatttgga atcacacgac ctggatggag
tgggacagag aaattaacaa 1440 ttacacaagc ttaatacact ccttaattga
agaatcgcaa aaccagcaag aaaagaatga 1500 acaagaatta ttggaattag
ataaatgggc aagtttgtgg aattggttta acataacaaa 1560 ttggctgtgg
tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620
agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
1680 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
aagaagaagg 1740 tggagagaga gacagagaca gatccattcg attagtgaac
ggatctcgac ggtatcgcta 1800 gcttttaaaa gaaaaggggg gattgggggg
tacagtgcag gggaaagaat agtagacata 1860 atagcaacag acatacaaac
taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt 1920 actagtgatt
atcggatcaa ctttgtatag aaaagttggg ctccggtgcc cgtcagtggg 1980
cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg
2040 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
tggctccgcc 2100 tttttcccga gggtggggga gaaccgtata taagtgcagt
agtcgccgtg aacgttcttt 2160 ttcgcaacgg gtttgccgcc agaacacagg
taagtgccgt gtgtggttcc cgcgggcctg 2220 gcctctttac gggttatggc
ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg 2280 tgattcttga
tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt 2340
aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg
2400 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct
ctagccattt 2460 aaaatttttg atgacctgct gcgacgcttt ttttctggca
agatagtctt gtaaatgcgg 2520 gccaagatct gcacactggt atttcggttt
ttggggccgc gggcggcgac ggggcccgtg 2580 cgtcccagcg cacatgttcg
gcgaggcggg gcctgcgagc gcggccaccg agaatcggac 2640 gggggtagtc
tcaagctggc cggcctgctc tggtgcctgg tctcgcgccg ccgtgtatcg 2700
ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc
2760 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg
ggagagcggg 2820 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc
ctcagccgtc gcttcatgtg 2880 actccacgga gtaccgggcg ccgtccaggc
acctcgatta gttctcgagc ttttggagta 2940 cgtcgtcttt aggttggggg
gaggggtttt atgcgatgga gtttccccac actgagtggg 3000 tggagactga
agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt 3060
ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc
3120 catttcaggt gtcgtgacaa gtttgtacaa aaaagcaggc tgccaccatg
atcgagacct 3180 acaaccagac cagccccaga agcgccgcca ccggcctgcc
catcagcatg aagatcttca 3240 tgtacctgct gaccgtgttc ctgatcaccc
agatgatcgg cagcgccctg ttcgccgtgt 3300 acctgcacag aagactggac
aagatcgagg acgagagaaa cctgcacgag gacttcgtgt 3360 tcatgaagac
catccagaga tgcaacaccg gcgagagaag cctgagcctg ctgaactgcg 3420
aggagatcaa gagccagttc gagggcttcg tgaaggacat catgctgaac aaggaggaga
3480 ccaagaagga gaacagcttc gagatgccca gaggcgagga ggacagccag
atcgccgccc 3540 acgtgatcag cgaggccagc agcaagacca ccagcgtgct
gcagtgggcc gagaagggct 3600 actacaccat gagcaacaac ctggtgaccc
tggagaacgg caagcagctg accgtgaaga 3660 gacagggcct gtactacatc
tacgcccagg tgaccttctg cagcaacaga gaggccagca 3720 gccaggcccc
cttcatcgcc agcctgtgcc tgaagagccc cggcagattc gagagaatcc 3780
tgctgagagc cgccaacacc cacagcagcg ccaagccctg cggccagcag agcatccacc
3840 tgggcggcgt gttcgagctg cagcccggcg ccagcgtgtt cgtgaacgtg
accgacccca 3900 gccaggtgag ccacggcacc ggcttcacca gcttcggcct
gctgaagctg taaacccagc 3960 tttcttgtac aaagtggtga taatcgaatt
cacccagctt tcttgtacaa agtggtgata 4020 atcgaattcc gataatcaac
ctctggatta caaaatttgt gaaagattga ctggtattct 4080 taactatgtt
gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc 4140
tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
4200 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
tgtttgctga 4260 cgcaaccccc actggttggg gcattgccac cacctgtcag
ctcctttccg ggactttcgc 4320 tttccccctc cctattgcca cggcggaact
catcgccgcc tgccttgccc gctgctggac 4380 aggggctcgg ctgttgggca
ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt 4440 tccatggctg
ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt 4500
cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
4560 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
ccgcctcccc 4620 gcatcgggaa ttcccgcggt tcgctttaag accaatgact
tacaaggcag ctgtagatct 4680 tagccacttt ttaaaagaaa aggggggact
ggaagggcta attcactccc aacgaagaca 4740 agatctgctt tttgcttgta
ctgggtctct ctggttagac cagatctgag cctgggagct 4800 ctctggctaa
ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 4860
agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
4920 gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc
agtatttata 4980 acttgcaaag aaatgaatat cagagagtga gaggaacttg
tttattgcag cttataatgg 5040 ttacaaataa agcaatagca tcacaaattt
cacaaataaa gcattttttt cactgcattc 5100 tagttgtggt ttgtccaaac
tcatcaatgt atcttatcat gtctggctct agctatcccg 5160 cccctaactc
cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat 5220
ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
5280 cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg
ccctatagtg 5340 agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
tcgtgactgg gaaaaccctg 5400 gcgttaccca acttaatcgc cttgcagcac
atcccccttt cgccagctgg cgtaatagcg 5460 aagaggcccg caccgatcgc
ccttcccaac agttgcgcag cctgaatggc gaatgggacg 5520 cgccctgtag
cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 5580
cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
5640 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
cgatttagtg 5700 ctttacggca cctcgacccc aaaaaacttg attagggtga
tggttcacgt agtgggccat 5760 cgccctgata gacggttttt cgccctttga
cgttggagtc cacgttcttt aatagtggac 5820 tcttgttcca aactggaaca
acactcaacc ctatctcggt ctattctttt gatttataag 5880 ggattttgcc
gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg 5940
cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
6000 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
tcatgagaca 6060 ataaccctga taaatgcttc aataatattg aaaaaggaag
agtatgagta ttcaacattt 6120 ccgtgtcgcc cttattccct tttttgcggc
attttgcctt cctgtttttg ctcacccaga 6180 aacgctggtg aaagtaaaag
atgctgaaga tcagttgggt gcacgagtgg gttacatcga 6240 actggatctc
aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 6300
gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
6360 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
actcaccagt 6420 cacagaaaag catcttacgg atggcatgac agtaagagaa
ttatgcagtg ctgccataac 6480 catgagtgat aacactgcgg ccaacttact
tctgacaacg atcggaggac cgaaggagct 6540 aaccgctttt ttgcacaaca
tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 6600 gctgaatgaa
gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 6660
aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
6720 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
ttccggctgg 6780 ctggtttatt gctgataaat ctggagccgg tgagcgtggg
tctcgcggta tcattgcagc 6840 actggggcca gatggtaagc cctcccgtat
cgtagttatc tacacgacgg ggagtcaggc 6900 aactatggat gaacgaaata
gacagatcgc tgagataggt gcctcactga ttaagcattg 6960 gtaactgtca
gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 7020
atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
7080 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
cttcttgaga 7140 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
aaaccaccgc taccagcggt 7200 ggtttgtttg ccggatcaag agctaccaac
tctttttccg aaggtaactg gcttcagcag 7260 agcgcagata ccaaatactg
ttcttctagt gtagccgtag ttaggccacc acttcaagaa 7320 ctctgtagca
ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 7380
tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
7440 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
cgacctacac 7500 cgaactgaga tacctacagc gtgagctatg agaaagcgcc
acgcttcccg aagagagaaa 7560 ggcggacagg tatccggtaa gcggcagggt
cggaacagga gagcgcacga gggagcttcc 7620 agggggaaac gcctggtatc
tttatagtcc tgtcgggttt cgccacctct gacttgagcg 7680 tcgatttttg
tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 7740
ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
7800 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
ctcgccgcag 7860 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
gaagagcgcc caatacgcaa 7920 accgcctctc cccgcgcgtt ggccgattca
ttaatgcagc tggcacgaca ggtttcccga 7980 ctggaaagcg ggcagtgagc
gcaacgcaat taatgtgagt tagctcactc attaggcacc 8040 ccaggcttta
cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca 8100
atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
8160 aagggaacaa aagctggagc tgcaagctt 8189 <210> SEQ ID NO 52
<211> LENGTH: 8069 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <220> FEATURE: <223> OTHER INFORMATION:
Vector 6 <400> SEQUENCE: 52 aatgtagtct tatgcaatac tcttgtagtc
ttgcaacatg gtaacgatga gttagcaaca 60 tgccttacaa ggagagaaaa
agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120 tcgtgcctta
ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180
gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
240 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
tgcttaagcc 300 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
cgtctgttgt gtgactctgg 360 taactagaga tccctcagac ccttttagtc
agtgtggaaa atctctagca gtggcgcccg 420 aacagggact tgaaagcgaa
agggaaacca gaggagctct ctcgacgcag gactcggctt 480 gctgaagcgc
gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540
actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
600 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
atataaatta 660 aaacatatag tatgggcaag cagggagcta gaacgattcg
cagttaatcc tggcctgtta 720 gaaacatcag aaggctgtag acaaatactg
ggacagctac aaccatccct tcagacagga 780 tcagaagaac ttagatcatt
atataataca gtagcaaccc tctattgtgt gcatcaaagg 840 atagagataa
aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900
aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
960 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
taggagtagc 1020 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
agagcagtgg gaataggagc 1080 tttgttcctt gggttcttgg gagcagcagg
aagcactatg ggcgcagcgt caatgacgct 1140 gacggtacag gccagacaat
tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200 ggctattgag
gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260
ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
1320 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
ggagtaataa 1380 atctctggaa cagatttgga atcacacgac ctggatggag
tgggacagag aaattaacaa 1440 ttacacaagc ttaatacact ccttaattga
agaatcgcaa aaccagcaag aaaagaatga 1500 acaagaatta ttggaattag
ataaatgggc aagtttgtgg aattggttta acataacaaa 1560 ttggctgtgg
tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620
agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
1680 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
aagaagaagg 1740 tggagagaga gacagagaca gatccattcg attagtgaac
ggatctcgac ggtatcgcta 1800 gcttttaaaa gaaaaggggg gattgggggg
tacagtgcag gggaaagaat agtagacata 1860 atagcaacag acatacaaac
taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt 1920 actagtgatt
atcggatcaa ctttgtatag aaaagttggg ctccggtgcc cgtcagtggg 1980
cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg
2040 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
tggctccgcc 2100 tttttcccga gggtggggga gaaccgtata taagtgcagt
agtcgccgtg aacgttcttt 2160 ttcgcaacgg gtttgccgcc agaacacagg
taagtgccgt gtgtggttcc cgcgggcctg 2220 gcctctttac gggttatggc
ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg 2280 tgattcttga
tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt 2340
aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg
2400 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct
ctagccattt 2460 aaaatttttg atgacctgct gcgacgcttt ttttctggca
agatagtctt gtaaatgcgg 2520 gccaagatct gcacactggt atttcggttt
ttggggccgc gggcggcgac ggggcccgtg 2580 cgtcccagcg cacatgttcg
gcgaggcggg gcctgcgagc gcggccaccg agaatcggac 2640 gggggtagtc
tcaagctggc cggcctgctc tggtgcctgg tctcgcgccg ccgtgtatcg 2700
ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc
2760 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg
ggagagcggg 2820 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc
ctcagccgtc gcttcatgtg 2880 actccacgga gtaccgggcg ccgtccaggc
acctcgatta gttctcgagc ttttggagta 2940 cgtcgtcttt aggttggggg
gaggggtttt atgcgatgga gtttccccac actgagtggg 3000 tggagactga
agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt 3060
ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc
3120 catttcaggt gtcgtgacaa gtttgtacaa aaaagcaggc tgccaccatg
agcaccgaga 3180 gcatgatcag agacgtggag ctggccgagg aggccctgcc
caagaagacc ggcggccccc 3240 agggcagcag aagatgcctg ttcctgagcc
tgttcagctt cctgatcgtg gccggcgcca 3300 ccaccctgtt ctgcctgctg
cacttcggcg tgatcggccc ccagagagag gagttcccca 3360 gagacctgag
cctgatcagc cccctggccc aggccgtggc ccacgtggtg gccaaccccc 3420
aggccgaggg ccagctgcag tggctgaaca gaagagccaa cgccctgctg gccaacggcg
3480 tggagctgag agacaaccag ctggtggtgc ccagcgaggg cctgtacctg
atctacagcc 3540 aggtgctgtt caagggccag ggctgcccca gcacccacgt
gctgctgacc cacaccatca 3600 gcagaatcgc cgtgagctac cagaccaagg
tgaacctgct gagcgccatc aagagcccct 3660 gccagagaga gacccccgag
ggcgccgagg ccaagccctg gtacgagccc atctacctgg 3720 gcggcgtgtt
ccagctggag aagggcgaca gactgagcgc cgagatcaac agacccgact 3780
acctggactt cgccgagagc ggccaggtgt acttcggcat catcgccctg taaacccagc
3840 tttcttgtac aaagtggtga taatcgaatt cacccagctt tcttgtacaa
agtggtgata 3900 atcgaattcc gataatcaac ctctggatta caaaatttgt
gaaagattga ctggtattct 3960 taactatgtt gctcctttta cgctatgtgg
atacgctgct ttaatgcctt tgtatcatgc 4020 tattgcttcc cgtatggctt
tcattttctc ctccttgtat aaatcctggt tgctgtctct 4080 ttatgaggag
ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga 4140
cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
4200 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
gctgctggac 4260 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
tcggggaagc tgacgtcctt 4320 tccatggctg ctcgcctgtg ttgccacctg
gattctgcgc gggacgtcct tctgctacgt 4380 cccttcggcc ctcaatccag
cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc 4440 tcttccgcgt
cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc 4500
gcatcgggaa ttcccgcggt tcgctttaag accaatgact tacaaggcag ctgtagatct
4560 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
aacgaagaca 4620 agatctgctt tttgcttgta ctgggtctct ctggttagac
cagatctgag cctgggagct 4680 ctctggctaa ctagggaacc cactgcttaa
gcctcaataa agcttgcctt gagtgcttca 4740 agtagtgtgt gcccgtctgt
tgtgtgactc tggtaactag agatccctca gaccctttta 4800 gtcagtgtgg
aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata 4860
acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg
4920 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
cactgcattc 4980 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
gtctggctct agctatcccg 5040 cccctaactc cgcccatccc gcccctaact
ccgcccagtt ccgcccattc tccgccccat 5100 ggctgactaa ttttttttat
ttatgcagag gccgaggccg cctcggcctc tgagctattc 5160 cagaagtagt
gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg 5220
agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
5280 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
cgtaatagcg 5340 aagaggcccg caccgatcgc ccttcccaac agttgcgcag
cctgaatggc gaatgggacg 5400 cgccctgtag cggcgcatta agcgcggcgg
gtgtggtggt tacgcgcagc gtgaccgcta 5460 cacttgccag cgccctagcg
cccgctcctt tcgctttctt cccttccttt ctcgccacgt 5520 tcgccggctt
tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 5580
ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
5640 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
aatagtggac 5700 tcttgttcca aactggaaca acactcaacc ctatctcggt
ctattctttt gatttataag 5760 ggattttgcc gatttcggcc tattggttaa
aaaatgagct gatttaacaa aaatttaacg 5820 cgaattttaa caaaatatta
acgcttacaa tttaggtggc acttttcggg gaaatgtgcg 5880 cggaacccct
atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 5940
ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
6000 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
ctcacccaga 6060 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
gcacgagtgg gttacatcga 6120 actggatctc aacagcggta agatccttga
gagttttcgc cccgaagaac gttttccaat 6180 gatgagcact tttaaagttc
tgctatgtgg cgcggtatta tcccgtattg acgccgggca 6240 agagcaactc
ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 6300
cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
6360 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
cgaaggagct 6420 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
cttgatcgtt gggaaccgga 6480 gctgaatgaa gccataccaa acgacgagcg
tgacaccacg atgcctgtag caatggcaac 6540 aacgttgcgc aaactattaa
ctggcgaact acttactcta gcttcccggc aacaattaat 6600 agactggatg
gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 6660
ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
6720 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
ggagtcaggc 6780 aactatggat gaacgaaata gacagatcgc tgagataggt
gcctcactga ttaagcattg 6840 gtaactgtca gaccaagttt actcatatat
actttagatt gatttaaaac ttcattttta 6900 atttaaaagg atctaggtga
agatcctttt tgataatctc atgaccaaaa tcccttaacg 6960 tgagttttcg
ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 7020
tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
7080 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
gcttcagcag 7140 agcgcagata ccaaatactg ttcttctagt gtagccgtag
ttaggccacc acttcaagaa 7200 ctctgtagca ccgcctacat acctcgctct
gctaatcctg ttaccagtgg ctgctgccag 7260 tggcgataag tcgtgtctta
ccgggttgga ctcaagacga tagttaccgg ataaggcgca 7320 gcggtcgggc
tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 7380
cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagagagaaa
7440 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
gggagcttcc 7500 agggggaaac gcctggtatc tttatagtcc tgtcgggttt
cgccacctct gacttgagcg 7560 tcgatttttg tgatgctcgt caggggggcg
gagcctatgg aaaaacgcca gcaacgcggc 7620 ctttttacgg ttcctggcct
tttgctggcc ttttgctcac atgttctttc ctgcgttatc 7680 ccctgattct
gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag 7740
ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
7800 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
ggtttcccga 7860 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
tagctcactc attaggcacc 7920 ccaggcttta cactttatgc ttccggctcg
tatgttgtgt ggaattgtga gcggataaca 7980 atttcacaca ggaaacagct
atgaccatga ttacgccaag cgcgcaatta accctcacta 8040 aagggaacaa
aagctggagc tgcaagctt 8069 <210> SEQ ID NO 53 <211>
LENGTH: 10067 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <220> FEATURE: <223> OTHER INFORMATION:
Vector 7 <400> SEQUENCE: 53 aatgtagtct tatgcaatac tcttgtagtc
ttgcaacatg gtaacgatga gttagcaaca 60 tgccttacaa ggagagaaaa
agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120 tcgtgcctta
ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180
gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
240 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
tgcttaagcc 300 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
cgtctgttgt gtgactctgg 360 taactagaga tccctcagac ccttttagtc
agtgtggaaa atctctagca gtggcgcccg 420 aacagggact tgaaagcgaa
agggaaacca gaggagctct ctcgacgcag gactcggctt 480 gctgaagcgc
gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540
actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
600 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
atataaatta 660 aaacatatag tatgggcaag cagggagcta gaacgattcg
cagttaatcc tggcctgtta 720 gaaacatcag aaggctgtag acaaatactg
ggacagctac aaccatccct tcagacagga 780 tcagaagaac ttagatcatt
atataataca gtagcaaccc tctattgtgt gcatcaaagg 840 atagagataa
aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900
aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
960 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
taggagtagc 1020 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
agagcagtgg gaataggagc 1080 tttgttcctt gggttcttgg gagcagcagg
aagcactatg ggcgcagcgt caatgacgct 1140 gacggtacag gccagacaat
tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200 ggctattgag
gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260
ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
1320 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
ggagtaataa 1380 atctctggaa cagatttgga atcacacgac ctggatggag
tgggacagag aaattaacaa 1440 ttacacaagc ttaatacact ccttaattga
agaatcgcaa aaccagcaag aaaagaatga 1500 acaagaatta ttggaattag
ataaatgggc aagtttgtgg aattggttta acataacaaa 1560 ttggctgtgg
tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620
agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
1680 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
aagaagaagg 1740 tggagagaga gacagagaca gatccattcg attagtgaac
ggatctcgac ggtatcgcta 1800 gcttttaaaa gaaaaggggg gattgggggg
tacagtgcag gggaaagaat agtagacata 1860 atagcaacag acatacaaac
taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt 1920 actagtatca
actttgtata gaaaagttgg gctccggtgc ccgtcagtgg gcagagcgca 1980
catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
2040 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
ctttttcccg 2100 agggtggggg agaaccgtat ataagtgcag tagtcgccgt
gaacgttctt tttcgcaacg 2160 ggtttgccgc cagaacacag gtaagtgccg
tgtgtggttc ccgcgggcct ggcctcttta 2220 cgggttatgg cccttgcgtg
ccttgaatta cttccacctg gctgcagtac gtgattcttg 2280 atcccgagct
tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc 2340
cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct
2400 ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt
taaaattttt 2460 gatgacctgc tgcgacgctt tttttctggc aagatagtct
tgtaaatgcg ggccaagatc 2520 tgcacactgg tatttcggtt tttggggccg
cgggcggcga cggggcccgt gcgtcccagc 2580 gcacatgttc ggcgaggcgg
ggcctgcgag cgcggccacc gagaatcgga cgggggtagt 2640 ctcaagctgg
ccggcctgct ctggtgcctg gtctcgcgcc gccgtgtatc gccccgccct 2700
gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg
2760 gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg
gcgggtgagt 2820 cacccacaca aaggaaaagg gcctttccgt cctcagccgt
cgcttcatgt gactccacgg 2880 agtaccgggc gccgtccagg cacctcgatt
agttctcgag cttttggagt acgtcgtctt 2940 taggttgggg ggaggggttt
tatgcgatgg agtttcccca cactgagtgg gtggagactg 3000 aagttaggcc
agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg 3060
gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg
3120 tgtcgtgaca agtttgtaca aaaaagcagg ctgccaccat gagcaccgag
agcatgatca 3180 gagacgtgga gctggccgag gaggccctgc ccaagaagac
cggcggcccc cagggcagca 3240 gaagatgcct gttcctgagc ctgttcagct
tcctgatcgt ggccggcgcc accaccctgt 3300 tctgcctgct gcacttcggc
gtgatcggcc cccagagaga ggagttcccc agagacctga 3360 gcctgatcag
ccccctggcc caggccgtgg cccacgtggt ggccaacccc caggccgagg 3420
gccagctgca gtggctgaac agaagagcca acgccctgct ggccaacggc gtggagctga
3480 gagacaacca gctggtggtg cccagcgagg gcctgtacct gatctacagc
caggtgctgt 3540 tcaagggcca gggctgcccc agcacccacg tgctgctgac
ccacaccatc agcagaatcg 3600 ccgtgagcta ccagaccaag gtgaacctgc
tgagcgccat caagagcccc tgccagagag 3660 agacccccga gggcgccgag
gccaagccct ggtacgagcc catctacctg ggcggcgtgt 3720 tccagctgga
gaagggcgac agactgagcg ccgagatcaa cagacccgac tacctggact 3780
tcgccgagag cggccaggtg tacttcggca tcatcgccct gtaaacccag ctttcttgta
3840 caaagtggtg ataatcgaat tctaaataga tagaacaaca acaattgcat
tcatttttga 3900 tttcaggttc agggggaggt gtgggaggtt ttttaaagca
agtaaaacct ctacactgac 3960 ggtacgcgtt aacaacaaca attgcattca
tttgtagttt caggttcagg gggaggtgtg 4020 ggaggttttt taaagcaagt
taaacctcta aaatagtggt acgcgttacc cagctttctt 4080 gtacaaagtg
gacccagctt tcttgtacaa agtgggcccc tctccctccc ccccccctaa 4140
cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc
4200 caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg
tcttcttgac 4260 gagcattcct aggggtcttt cccctctcgc caaaggaatg
caaggtctgt tgaatgtcgt 4320 gaaggaagca gttcctctgg aagcttcttg
aagacaaaca acgtctgtag cgaccctttg 4380 caggcagcgg aaccccccac
ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata 4440 agatacacct
gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga 4500
aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt
4560 accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat
gtgtttagtc 4620 gaggttaaaa aaacgtctag gccccccgaa ccacggggac
gtggttttcc tttgaaaaac 4680 acgatgataa tatggccaca accatggcca
ccgtgctggc ccccgcctgg agccccacca 4740 cctacctgct gctgctgctg
ctgctgagca gcggcctgag cggcggcggc ggcagcggca 4800 agcccatccc
caaccccctg ctgggcctgg acagcaccgg cggcggcggc agccaggtga 4860
agctgcagga gagcggcccc ggcctggtgg cccccagcca gagcctgagc atcacctgca
4920 ccgtgagcgg cttcagcctg accgcctacg gcgtggactg ggtgagacag
ccccccggca 4980 agtgcctgga gtggctgggc gtgatctggg gcggcggcag
aaccaactac aacagcggcc 5040 tgatgagcag actgagcatc agaaaggaca
acagcaagag ccaggtgttc ctgaccatga 5100 acagcctgca gaccgacgac
accgccaagt actactgcgt gaagcacacc aactgggacg 5160 gcggcttcgc
ctactggggc cagggcacca ccgtgaccgt gagcagcggc ggcggcggca 5220
gcggcggcgg cggcagcggc ggcggcggca gcggcagccc cggccagagc gtgagcatca
5280 gctgcagcgg cagcagcagc aacatcggca acaactacgt gtactggtac
cagcacctgc 5340 ccggcaccgc ccccaagctg ctgatctaca gcgacaccaa
gagacccagc ggcgtgcccg 5400 acagaatcag cggcagcaag agcggcacca
gcgccagcct ggccatcagc ggcctgcaga 5460 gcgaggacga ggccgactac
tactgcgcca gctgggacga cagcctggac ggccccgtgt 5520 tcggctgcgg
caccaagctg accgtgctgc ccaccaccac ccccgccccc agacccccca 5580
cccccgcccc caccatcgcc agccagcccc tgagcctgag acccgaggcc tgcagacccg
5640 ccgccggcgg cgccgtgcac accagaggcc tggacttcgc ctgcgacatc
tacatctggg 5700 cccccctggc cggcacctgc ggcgtgctgc tgctgagcct
ggtgatcacc ctgtactgca 5760 accacagaaa cagaagaaga gtgtgcaagt
gccccagacc cgtggtgaag agcggcgaca 5820 agcccagcct gagcgccaga
tacgtgtaac aactttatta tacatagttg atcaattcca 5880 actttattat
acatagttga tcaattccga taatcaacct ctggattaca aaatttgtga 5940
aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
6000 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
ccttgtataa 6060 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
gtcaggcaac gtggcgtggt 6120 gtgcactgtg tttgctgacg caacccccac
tggttggggc attgccacca cctgtcagct 6180 cctttccggg actttcgctt
tccccctccc tattgccacg gcggaactca tcgccgcctg 6240 ccttgcccgc
tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc 6300
ggggaagctg acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg
6360 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
cccgcggcct 6420 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
cctcagacga gtcggatctc 6480 cctttgggcc gcctccccgc atcgggaatt
cccgcggttc gctttaagac caatgactta 6540 caaggcagct gtagatctta
gccacttttt aaaagaaaag gggggactgg aagggctaat 6600 tcactcccaa
cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca 6660
gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
6720 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
gtaactagag 6780 atccctcaga cccttttagt cagtgtggaa aatctctagc
agtagtagtt catgtcatct 6840 tattattcag tatttataac ttgcaaagaa
atgaatatca gagagtgaga ggaacttgtt 6900 tattgcagct tataatggtt
acaaataaag caatagcatc acaaatttca caaataaagc 6960 atttttttca
ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt 7020
ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
7080 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
cgaggccgcc 7140 tcggcctctg agctattcca gaagtagtga ggaggctttt
ttggaggcct agggacgtac 7200 ccaattcgcc ctatagtgag tcgtattacg
cgcgctcact ggccgtcgtt ttacaacgtc 7260 gtgactggga aaaccctggc
gttacccaac ttaatcgcct tgcagcacat ccccctttcg 7320 ccagctggcg
taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 7380
tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
7440 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
gctttcttcc 7500 cttcctttct cgccacgttc gccggctttc cccgtcaagc
tctaaatcgg gggctccctt 7560 tagggttccg atttagtgct ttacggcacc
tcgaccccaa aaaacttgat tagggtgatg 7620 gttcacgtag tgggccatcg
ccctgataga cggtttttcg ccctttgacg ttggagtcca 7680 cgttctttaa
tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct 7740
attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
7800 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
taggtggcac 7860 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
ttctaaatac attcaaatat 7920 gtatccgctc atgagacaat aaccctgata
aatgcttcaa taatattgaa aaaggaagag 7980 tatgagtatt caacatttcc
gtgtcgccct tattcccttt tttgcggcat tttgccttcc 8040 tgtttttgct
cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 8100
acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
8160 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
cggtattatc 8220 ccgtattgac gccgggcaag agcaactcgg tcgccgcata
cactattctc agaatgactt 8280 ggttgagtac tcaccagtca cagaaaagca
tcttacggat ggcatgacag taagagaatt 8340 atgcagtgct gccataacca
tgagtgataa cactgcggcc aacttacttc tgacaacgat 8400 cggaggaccg
aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 8460
tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
8520 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
ttactctagc 8580 ttcccggcaa caattaatag actggatgga ggcggataaa
gttgcaggac cacttctgcg 8640 ctcggccctt ccggctggct ggtttattgc
tgataaatct ggagccggtg agcgtgggtc 8700 tcgcggtatc attgcagcac
tggggccaga tggtaagccc tcccgtatcg tagttatcta 8760 cacgacgggg
agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 8820
ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
8880 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
ataatctcat 8940 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
tcagaccccg tagaaaagat 9000 caaaggatct tcttgagatc ctttttttct
gcgcgtaatc tgctgcttgc aaacaaaaaa 9060 accaccgcta ccagcggtgg
tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 9120 ggtaactggc
ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt 9180
aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
9240 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
caagacgata 9300 gttaccggat aaggcgcagc ggtcgggctg aacggggggt
tcgtgcacac agcccagctt 9360 ggagcgaacg acctacaccg aactgagata
cctacagcgt gagctatgag aaagcgccac 9420 gcttcccgaa gagagaaagg
cggacaggta tccggtaagc ggcagggtcg gaacaggaga 9480 gcgcacgagg
gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 9540
ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
9600 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
ttgctcacat 9660 gttctttcct gcgttatccc ctgattctgt ggataaccgt
attaccgcct ttgagtgagc 9720 tgataccgct cgccgcagcc gaacgaccga
gcgcagcgag tcagtgagcg aggaagcgga 9780 agagcgccca atacgcaaac
cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg 9840 gcacgacagg
tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 9900
gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
9960 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
acgccaagcg 10020 cgcaattaac cctcactaaa gggaacaaaa gctggagctg
caagctt 10067 <210> SEQ ID NO 54 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<223> OTHER INFORMATION: H7 heavy chain leader <400>
SEQUENCE: 54 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu
Phe Arg Gly 1 5 10 15 Val Gln Cys <210> SEQ ID NO 55
<211> LENGTH: 118 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
anti-biotin murine vH with inserted Cys for inter-domain linkage
<400> SEQUENCE: 55 Gln Val Lys Leu Gln Glu Ser Gly Pro Gly
Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr Ala Tyr 20 25 30 Gly Val Asp Trp Val Arg
Gln Pro Pro Gly Lys Cys Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp
Gly Gly Gly Arg Thr Asn Tyr Asn Ser Gly Leu Met 50 55 60 Ser Arg
Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80
Thr Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Lys Tyr Tyr Cys Val 85
90 95 Lys His Thr Asn Trp Asp Gly Gly Phe Ala Tyr Trp Gly Gln Gly
Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> SEQ ID NO
56 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <220> FEATURE: <223> OTHER INFORMATION: linker
<400> SEQUENCE: 56 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser 1 5 10 15 <210> SEQ ID NO 57 <211>
LENGTH: 99 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<220> FEATURE: <223> OTHER INFORMATION: Light Chain
Variable (human lambda variable) <400> SEQUENCE: 57 Gly Ser
Pro Gly Gln Ser Val Ser Ile Ser Cys Ser Gly Ser Ser Ser 1 5 10 15
Asn Ile Gly Asn Asn Tyr Val Tyr Trp Tyr Gln His Leu Pro Gly Thr 20
25 30 Ala Pro Lys Leu Leu Ile Tyr Ser Asp Thr Lys Arg Pro Ser Gly
Val 35 40 45 Pro Asp Arg Ile Ser Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala 50 55 60 Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ser 65 70 75 80 Trp Asp Asp Ser Leu Asp Gly Pro Val
Phe Gly Cys Gly Thr Lys Leu 85 90 95 Thr Val Leu <210> SEQ ID
NO 58 <211> LENGTH: 61 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
IgG3 hinge for greater accessibility to FcyR <400> SEQUENCE:
58 Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys Pro
1 5 10 15 Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys
Pro Glu 20 25 30 Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg
Cys Pro Glu Pro 35 40 45 Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro
Arg Cys Pro 50 55 60 <210> SEQ ID NO 59 <211> LENGTH:
283 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: IgG1 CH2, CH3 Tm and cytoplasmic
tail (T256A) <400> SEQUENCE: 59 Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Met Ile Ser
Arg Ala Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 35 40 45 Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 50 55
60 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
65 70 75 80 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro 85 90 95 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu 100 105 110 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn 115 120 125 Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile 130 135 140 Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 145 150 155 160 Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 165 170 175 Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 180 185
190 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
195 200 205 Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys Ala Glu
Ala Gln 210 215 220 Asp Gly Glu Leu Asp Gly Leu Trp Thr Thr Ile Thr
Ile Phe Ile Thr 225 230 235 240 Leu Phe Leu Leu Ser Val Cys Tyr Ser
Ala Thr Val Thr Phe Phe Lys 245 250 255 Val Lys Trp Ile Phe Ser Ser
Val Val Asp Leu Lys Gln Thr Ile Ile 260 265 270 Pro Asp Tyr Arg Asn
Met Ile Gly Gln Gly Ala 275 280 <210> SEQ ID NO 60
<211> LENGTH: 598 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
scFv-anti-biotin-G3hinge-IgG1-Tm (598 ORF1) <400> SEQUENCE:
60 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Phe Arg Gly
1 5 10 15 Val Gln Cys Gln Val Lys Leu Gln Glu Ser Gly Pro Gly Leu
Val Ala 20 25 30 Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser
Gly Phe Ser Leu 35 40 45 Thr Ala Tyr Gly Val Asp Trp Val Arg Gln
Pro Pro Gly Lys Cys Leu 50 55 60 Glu Trp Leu Gly Val Ile Trp Gly
Gly Gly Arg Thr Asn Tyr Asn Ser 65 70 75 80 Gly Leu Met Ser Arg Leu
Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln 85 90 95 Val Phe Leu Thr
Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Lys Tyr 100 105 110 Tyr Cys
Val Lys His Thr Asn Trp Asp Gly Gly Phe Ala Tyr Trp Gly 115 120 125
Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 130
135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Pro Gly Gln Ser Val
Ser 145 150 155 160 Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn
Asn Tyr Val Tyr 165 170 175 Trp Tyr Gln His Leu Pro Gly Thr Ala Pro
Lys Leu Leu Ile Tyr Ser 180 185 190 Asp Thr Lys Arg Pro Ser Gly Val
Pro Asp Arg Ile Ser Gly Ser Lys 195 200 205 Ser Gly Thr Ser Ala Ser
Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp 210 215 220 Glu Ala Asp Tyr
Tyr Cys Ala Ser Trp Asp Asp Ser Leu Asp Gly Pro 225 230 235 240 Val
Phe Gly Cys Gly Thr Lys Leu Thr Val Leu Leu Lys Thr Pro Leu 245 250
255 Gly Asp Thr Thr His Thr Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys
260 265 270 Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser
Cys Asp 275 280 285 Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys
Ser Cys Asp Thr 290 295 300 Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro 305 310 315 320 Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 325 330 335 Arg Ala Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp 340 345 350 Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 355 360 365 Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 370 375
380 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
385 390 395 400 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys 405 410 415 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr 420 425 430 Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu Thr 435 440 445 Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu 450 455 460 Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 465 470 475 480 Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 485 490 495
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 500
505 510 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Glu 515 520 525 Leu Gln Leu Glu Glu Ser Cys Ala Glu Ala Gln Asp Gly
Glu Leu Asp 530 535 540 Gly Leu Trp Thr Thr Ile Thr Ile Phe Ile Thr
Leu Phe Leu Leu Ser 545 550 555 560 Val Cys Tyr Ser Ala Thr Val Thr
Phe Phe Lys Val Lys Trp Ile Phe 565 570 575 Ser Ser Val Val Asp Leu
Lys Gln Thr Ile Ile Pro Asp Tyr Arg Asn 580 585 590 Met Ile Gly Gln
Gly Ala 595 <210> SEQ ID NO 61 <211> LENGTH: 24
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<223> OTHER INFORMATION: L1 light chain leader/signal
<400> SEQUENCE: 61 Met Ala Thr Asp Met Arg Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu 1 5 10 15 Leu Trp Leu Ser Gly Ala Arg Cys
20 <210> SEQ ID NO 62 <211> LENGTH: 118 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <223>
OTHER INFORMATION: anti-biotin vH <400> SEQUENCE: 62 Gln Val
Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ala Tyr 20
25 30 Gly Val Asp Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Leu 35 40 45 Gly Val Ile Trp Gly Gly Gly Arg Thr Asn Tyr Asn Ser
Gly Leu Met 50 55 60 Ser Arg Leu Ser Ile Arg Lys Asp Asn Ser Lys
Ser Gln Val Phe Leu 65 70 75 80 Thr Met Asn Ser Leu Gln Thr Asp Asp
Thr Ala Lys Tyr Tyr Cys Val 85 90 95 Lys His Thr Asn Trp Asp Gly
Gly Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser
Ser 115 <210> SEQ ID NO 63 <211> LENGTH: 94 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <223>
OTHER INFORMATION: CH1 <400> SEQUENCE: 63 Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 1 5 10 15 Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 20 25 30 Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 35 40
45 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
50 55 60 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val 65 70 75 80 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu 85 90 <210> SEQ ID NO 64 <211> LENGTH: 61
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: IgG3 hinge <400> SEQUENCE: 64
Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys Pro 1 5
10 15 Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
Glu 20 25 30 Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys
Pro Glu Pro 35 40 45 Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg
Cys Pro 50 55 60 <210> SEQ ID NO 65 <211> LENGTH: 286
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: IgG1 CH2, CH3 Tm and cytoplasmic
tail <400> SEQUENCE: 65 Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile
Ser Arg Ala Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70
75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195
200 205 Lys Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys
Ala 210 215 220 Glu Ala Gln Asp Gly Glu Leu Asp Gly Leu Trp Thr Thr
Ile Thr Ile 225 230 235 240 Phe Ile Thr Leu Phe Leu Leu Ser Val Cys
Tyr Ser Ala Thr Val Thr 245 250 255 Phe Phe Lys Val Lys Trp Ile Phe
Ser Ser Val Val Asp Leu Lys Gln 260 265 270 Thr Ile Ile Pro Asp Tyr
Arg Asn Met Ile Gly Gln Gly Ala 275 280 285 <210> SEQ ID NO
66 <211> LENGTH: 578 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
Summary (578 ORF2a) <400> SEQUENCE: 66 Met Glu Phe Gly Leu
Ser Trp Val Phe Leu Val Ala Leu Phe Arg Gly 1 5 10 15 Val Gln Cys
Gln Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala 20 25 30 Pro
Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu 35 40
45 Thr Ala Tyr Gly Val Asp Trp Val Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60 Glu Trp Leu Gly Val Ile Trp Gly Gly Gly Arg Thr Asn Tyr
Asn Ser 65 70 75 80 Gly Leu Met Ser Arg Leu Ser Ile Arg Lys Asp Asn
Ser Lys Ser Gln 85 90 95 Val Phe Leu Thr Met Asn Ser Leu Gln Thr
Asp Asp Thr Ala Lys Tyr 100 105 110 Tyr Cys Val Lys His Thr Asn Trp
Asp Gly Gly Phe Ala Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Val Thr
Val Ser Ser Pro Ser Val Phe Pro Leu Ala 130 135 140 Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 145 150 155 160 Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 165 170
175 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
180 185 190 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu 195 200 205 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr 210 215 220 Lys Val Asp Lys Lys Val Glu Leu Lys Thr
Pro Leu Gly Asp Thr Thr 225 230 235 240 His Thr Cys Pro Arg Cys Pro
Glu Pro Lys Ser Cys Asp Thr Pro Pro 245 250 255 Pro Cys Pro Arg Cys
Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro 260 265 270 Cys Pro Arg
Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys 275 280 285 Pro
Arg Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 290 295
300 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Ala Pro Glu
305 310 315 320 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys 325 330 335 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys 340 345 350 Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu 355 360 365 Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys 370 375 380 Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 385 390 395 400 Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 405 410 415
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 420
425 430 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln 435 440 445 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly 450 455 460 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln 465 470 475 480 Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn 485 490 495 His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Glu Leu Gln Leu Glu 500 505 510 Glu Ser Cys Ala
Glu Ala Gln Asp Gly Glu Leu Asp Gly Leu Trp Thr 515 520 525 Thr Ile
Thr Ile Phe Ile Thr Leu Phe Leu Leu Ser Val Cys Tyr Ser 530 535 540
Ala Thr Val Thr Phe Phe Lys Val Lys Trp Ile Phe Ser Ser Val Val 545
550 555 560 Asp Leu Lys Gln Thr Ile Ile Pro Asp Tyr Arg Asn Met Ile
Gly Gln 565 570 575 Gly Ala <210> SEQ ID NO 67 <400>
SEQUENCE: 67 000 <210> SEQ ID NO 68 <400> SEQUENCE: 68
000 <210> SEQ ID NO 69 <211> LENGTH: 99 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <223>
OTHER INFORMATION: LC Variable <400> SEQUENCE: 69 Gly Ser Pro
Gly Gln Ser Val Ser Ile Ser Cys Ser Gly Ser Ser Ser 1 5 10 15 Asn
Ile Gly Asn Asn Tyr Val Tyr Trp Tyr Gln His Leu Pro Gly Thr 20 25
30 Ala Pro Lys Leu Leu Ile Tyr Ser Asp Thr Lys Arg Pro Ser Gly Val
35 40 45 Pro Asp Arg Ile Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser
Leu Ala 50 55 60 Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr
Tyr Cys Ala Ser 65 70 75 80 Trp Asp Asp Ser Leu Asp Gly Pro Val Phe
Gly Gly Gly Thr Lys Leu 85 90 95 Thr Val Leu <210> SEQ ID NO
70 <211> LENGTH: 106 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION: LC
Constant Region 1 <400> SEQUENCE: 70 Gly Gln Pro Lys Ala Asn
Pro Thr Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln
Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45
Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn 50
55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp
Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly
Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100
105 <210> SEQ ID NO 71 <211> LENGTH: 229 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <223>
OTHER INFORMATION: Summary (229 ORF2b) <400> SEQUENCE: 71 Met
Ala Thr Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu 1 5 10
15 Leu Trp Leu Ser Gly Ala Arg Cys Gly Ser Pro Gly Gln Ser Val Ser
20 25 30 Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr
Val Tyr 35 40 45 Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu
Leu Ile Tyr Ser 50 55 60 Asp Thr Lys Arg Pro Ser Gly Val Pro Asp
Arg Ile Ser Gly Ser Lys 65 70 75 80 Ser Gly Thr Ser Ala Ser Leu Ala
Ile Ser Gly Leu Gln Ser Glu Asp 85 90 95 Glu Ala Asp Tyr Tyr Cys
Ala Ser Trp Asp Asp Ser Leu Asp Gly Pro 100 105 110 Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala 115 120 125 Asn Pro
Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala 130 135 140
Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala 145
150 155 160 Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Ala
Gly Val 165 170 175 Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys
Tyr Ala Ala Ser 180 185 190 Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp
Lys Ser His Arg Ser Tyr 195 200 205 Ser Cys Gln Val Thr His Glu Gly
Ser Thr Val Glu Lys Thr Val Ala 210 215 220 Pro Thr Glu Cys Ser 225
<210> SEQ ID NO 72 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <223> OTHER
INFORMATION: GM-CSF signal sequence <400> SEQUENCE: 72 Met
Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10
15 Ser <210> SEQ ID NO 73 <211> LENGTH: 127 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <223>
OTHER INFORMATION: wild type GM-CSF sequence <400> SEQUENCE:
73 Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His Val
1 5 10 15 Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg
Asp Thr 20 25 30 Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser
Glu Met Phe Asp 35 40 45 Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg
Leu Glu Leu Tyr Lys Gln 50 55 60 Gly Leu Arg Gly Ser Leu Thr Lys
Leu Lys Gly Pro Leu Thr Met Met 65 70 75 80 Ala Ser His Tyr Lys Gln
His Cys Pro Pro Thr Pro Glu Thr Ser Cys 85 90 95 Ala Thr Gln Ile
Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp 100 105 110 Phe Leu
Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 115 120 125
<210> SEQ ID NO 74 <400> SEQUENCE: 74 000 <210>
SEQ ID NO 75 <211> LENGTH: 26 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <223> OTHER
INFORMATION: FLT3L signal <400> SEQUENCE: 75 Met Ala Thr Val
Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu 1 5 10 15 Leu Leu
Leu Leu Leu Ser Ser Gly Leu Ser 20 25 <210> SEQ ID NO 76
<211> LENGTH: 210 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
FLT3L <400> SEQUENCE: 76 Gly Thr Gln Asp Cys Ser Phe Gln His
Ser Pro Ile Ser Ser Asp Phe 1 5 10 15 Ala Val Lys Ile Arg Glu Leu
Ser Asp Tyr Leu Leu Gln Asp Tyr Pro 20 25 30 Val Thr Val Ala Ser
Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu 35 40 45 Trp Arg Leu
Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val 50 55 60 Ala
Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile 65 70
75 80 His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu
Arg 85 90 95 Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr
Ser Glu Gln 100 105 110 Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln
Asn Phe Ser Arg Cys 115 120 125 Leu Glu Leu Gln Cys Gln Pro Asp Ser
Ser Thr Leu Pro Pro Pro Trp 130 135 140 Ser Pro Arg Pro Leu Glu Ala
Thr Ala Pro Thr Ala Pro Gln Pro Pro 145 150 155 160 Leu Leu Leu Leu
Leu Leu Leu Pro Val Gly Leu Leu Leu Leu Ala Ala 165 170 175 Ala Trp
Cys Leu His Trp Gln Arg Thr Arg Arg Arg Thr Pro Arg Pro 180 185 190
Gly Glu Gln Val Pro Pro Val Pro Ser Pro Gln Asp Leu Leu Leu Val 195
200 205 Glu His 210 <210> SEQ ID NO 77 <211> LENGTH:
144 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: Summary (144 ORF3a) <400>
SEQUENCE: 77 Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala
Cys Ser Ile 1 5 10 15 Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr
Gln Pro Trp Glu His 20 25 30 Val Asn Ala Ile Gln Glu Ala Arg Arg
Leu Leu Asn Leu Ser Arg Asp 35 40 45 Thr Ala Ala Glu Met Asn Glu
Thr Val Glu Val Ile Ser Glu Met Phe 50 55 60 Asp Leu Gln Glu Pro
Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 65 70 75 80 Gln Gly Leu
Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met 85 90 95 Met
Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 100 105
110 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys
115 120 125 Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val
Gln Glu 130 135 140 <210> SEQ ID NO 78 <211> LENGTH:
236 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: Summary (236 ORF3b) <400>
SEQUENCE: 78 Met Ala Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr
Tyr Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly
Thr Gln Asp Cys Ser 20 25 30 Phe Gln His Ser Pro Ile Ser Ser Asp
Phe Ala Val Lys Ile Arg Glu 35 40 45 Leu Ser Asp Tyr Leu Leu Gln
Asp Tyr Pro Val Thr Val Ala Ser Asn 50 55 60 Leu Gln Asp Glu Glu
Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala 65 70 75 80 Gln Arg Trp
Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln 85 90 95 Gly
Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys 100 105
110 Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile
115 120 125 Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu
Lys Pro 130 135 140 Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu
Leu Gln Cys Gln 145 150 155 160 Pro Asp Ser Ser Thr Leu Pro Pro Pro
Trp Ser Pro Arg Pro Leu Glu 165 170 175 Ala Thr Ala Pro Thr Ala Pro
Gln Pro Pro Leu Leu Leu Leu Leu Leu 180 185 190 Leu Pro Val Gly Leu
Leu Leu Leu Ala Ala Ala Trp Cys Leu His Trp 195 200 205 Gln Arg Thr
Arg Arg Arg Thr Pro Arg Pro Gly Glu Gln Val Pro Pro 210 215 220 Val
Pro Ser Pro Gln Asp Leu Leu Leu Val Glu His 225 230 235 <210>
SEQ ID NO 79 <211> LENGTH: 183 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: wild type FLT3L sequence with transmembrane deleted
<400> SEQUENCE: 79 Met Thr Val Leu Ala Pro Ala Trp Ser Pro
Thr Thr Tyr Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Ser Ser Gly Leu
Ser Gly Thr Gln Asp Cys Ser Phe 20 25 30 Gln His Ser Pro Ile Ser
Ser Asp Phe Ala Val Lys Ile Arg Glu Leu 35 40 45 Ser Asp Tyr Leu
Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu 50 55 60 Gln Asp
Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln 65 70 75 80
Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly 85
90 95 Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys
Ala 100 105 110 Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr
Asn Ile Ser 115 120 125 Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val
Ala Leu Lys Pro Trp 130 135 140 Ile Thr Arg Gln Asn Phe Ser Arg Cys
Leu Glu Leu Gln Cys Gln Pro 145 150 155 160 Asp Ser Ser Thr Leu Pro
Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala 165 170 175 Thr Ala Pro Thr
Ala Pro Gln 180 <210> SEQ ID NO 80 <400> SEQUENCE: 80
000 <210> SEQ ID NO 81 <211> LENGTH: 26 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <220> FEATURE: <223> OTHER
INFORMATION: FLT3L signal <400> SEQUENCE: 81 Met Ala Thr Val
Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu 1 5 10 15 Leu Leu
Leu Leu Leu Ser Ser Gly Leu Ser 20 25 <210> SEQ ID NO 82
<211> LENGTH: 127 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
wild type GM-CSF sequence <400> SEQUENCE: 82 Ala Pro Ala Arg
Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His Val 1 5 10 15 Asn Ala
Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp Thr 20 25 30
Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe Asp 35
40 45 Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys
Gln 50 55 60 Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu
Thr Met Met 65 70 75 80 Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr
Pro Glu Thr Ser Cys 85 90 95 Ala Thr Gln Ile Ile Thr Phe Glu Ser
Phe Lys Glu Asn Leu Lys Asp 100 105 110 Phe Leu Leu Val Ile Pro Phe
Asp Cys Trp Glu Pro Val Gln Glu 115 120 125 <210> SEQ ID NO
83 <211> LENGTH: 99 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
CD8alpha transmembrane and cytoplasmic domain <400> SEQUENCE:
83 Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
1 5 10 15 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
Ala Ala 20 25 30 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala
Cys Asp Ile Tyr 35 40 45 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly
Val Leu Leu Leu Ser Leu 50 55 60 Val Ile Thr Leu Tyr Cys Asn His
Arg Asn Arg Arg Arg Val Cys Lys 65 70 75 80 Cys Pro Arg Pro Val Val
Lys Ser Gly Asp Lys Pro Ser Leu Ser Ala 85 90 95 Arg Tyr Val
<210> SEQ ID NO 84 <211> LENGTH: 183 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: Summary (183 ORF4a) <400> SEQUENCE: 84 Met Thr
Val Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu Leu 1 5 10 15
Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly Thr Gln Asp Cys Ser Phe 20
25 30 Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu
Leu 35 40 45 Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala
Ser Asn Leu 50 55 60 Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg
Leu Val Leu Ala Gln 65 70 75 80 Arg Trp Met Glu Arg Leu Lys Thr Val
Ala Gly Ser Lys Met Gln Gly 85 90 95 Leu Leu Glu Arg Val Asn Thr
Glu Ile His Phe Val Thr Lys Cys Ala 100 105 110 Phe Gln Pro Pro Pro
Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser 115 120 125 Arg Leu Leu
Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp 130 135 140 Ile
Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro 145 150
155 160 Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu
Ala 165 170 175 Thr Ala Pro Thr Ala Pro Gln 180 <210> SEQ ID
NO 85 <211> LENGTH: 252 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
Summary for CYAGEN (253 ORF4b) <400> SEQUENCE: 85 Met Ala Thr
Val Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu 1 5 10 15 Leu
Leu Leu Leu Leu Ser Ser Gly Leu Ser Ala Pro Ala Arg Ser Pro 20 25
30 Ser Pro Ser Thr Gln Pro Trp Glu His Val Asn Ala Ile Gln Glu Ala
35 40 45 Arg Arg Leu Leu Asn Leu Ser Arg Asp Thr Ala Ala Glu Met
Asn Glu 50 55 60 Thr Val Glu Val Ile Ser Glu Met Phe Asp Leu Gln
Glu Pro Thr Cys 65 70 75 80 Leu Gln Thr Arg Leu Glu Leu Tyr Lys Gln
Gly Leu Arg Gly Ser Leu 85 90 95 Thr Lys Leu Lys Gly Pro Leu Thr
Met Met Ala Ser His Tyr Lys Gln 100 105 110 His Cys Pro Pro Thr Pro
Glu Thr Ser Cys Ala Thr Gln Ile Ile Thr 115 120 125 Phe Glu Ser Phe
Lys Glu Asn Leu Lys Asp Phe Leu Leu Val Ile Pro 130 135 140 Phe Asp
Cys Trp Glu Pro Val Gln Glu Pro Thr Thr Thr Pro Ala Pro 145 150 155
160 Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu
165 170 175 Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
Thr Arg 180 185 190 Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala
Pro Leu Ala Gly 195 200 205 Thr Cys Gly Val Leu Leu Leu Ser Leu Val
Ile Thr Leu Tyr Cys Asn 210 215 220 His Arg Asn Arg Arg Arg Val Cys
Lys Cys Pro Arg Pro Val Val Lys 225 230 235 240 Ser Gly Asp Lys Pro
Ser Leu Ser Ala Arg Tyr Val 245 250 <210> SEQ ID NO 86
<211> LENGTH: 261 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
mCD40L modified to stop cleavage <400> SEQUENCE: 86 Met Ile
Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15
Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20
25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His
Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu
Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly
Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys Ser
Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn Lys Glu
Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110 Met Pro Arg Gly Glu
Glu Asp Ser Gln Ile Ala Ala His Val Ile Ser 115 120 125 Glu Ala Ser
Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130 135 140 Tyr
Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 145 150
155 160 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val
Thr 165 170 175 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe
Ile Ala Ser 180 185 190 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg
Ile Leu Leu Arg Ala 195 200 205 Ala Asn Thr His Ser Ser Ala Lys Pro
Cys Gly Gln Gln Ser Ile His 210 215 220 Leu Gly Gly Val Phe Glu Leu
Gln Pro Gly Ala Ser Val Phe Val Asn 225 230 235 240 Val Thr Asp Pro
Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 245 250 255 Gly Leu
Leu Lys Leu 260 <210> SEQ ID NO 87 <211> LENGTH: 261
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: Summary (261 ORF5) <400>
SEQUENCE: 87 Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala
Ala Thr Gly 1 5 10 15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu
Leu Thr Val Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu
Phe Ala Val Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp
Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile
Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn
Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp
Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105
110 Met Pro Arg Gly Glu Glu Asp Ser Gln Ile Ala Ala His Val Ile Ser
115 120 125 Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu
Lys Gly 130 135 140 Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu
Asn Gly Lys Gln 145 150 155 160 Leu Thr Val Lys Arg Gln Gly Leu Tyr
Tyr Ile Tyr Ala Gln Val Thr 165 170 175 Phe Cys Ser Asn Arg Glu Ala
Ser Ser Gln Ala Pro Phe Ile Ala Ser 180 185 190 Leu Cys Leu Lys Ser
Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205 Ala Asn Thr
His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210 215 220 Leu
Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn 225 230
235 240 Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser
Phe 245 250 255 Gly Leu Leu Lys Leu 260 <210> SEQ ID NO 88
<211> LENGTH: 221 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
mTNFalpha modified to stop cleavage <400> SEQUENCE: 88 Met
Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala 1 5 10
15 Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe
20 25 30 Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr
Leu Phe 35 40 45 Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg
Glu Glu Phe Pro 50 55 60 Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala
Gln Ala Val Ala His Val 65 70 75 80 Val Ala Asn Pro Gln Ala Glu Gly
Gln Leu Gln Trp Leu Asn Arg Arg 85 90 95 Ala Asn Ala Leu Leu Ala
Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 100 105 110 Val Val Pro Ser
Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 115 120 125 Lys Gly
Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 130 135 140
Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 145
150 155 160 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu
Ala Lys 165 170 175 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe
Gln Leu Glu Lys 180 185 190 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg
Pro Asp Tyr Leu Asp Phe 195 200 205 Ala Glu Ser Gly Gln Val Tyr Phe
Gly Ile Ile Ala Leu 210 215 220 <210> SEQ ID NO 89
<211> LENGTH: 221 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
Summary (221 ORF6) <400> SEQUENCE: 89 Met Ser Thr Glu Ser Met
Ile Arg Asp Val Glu Leu Ala Glu Glu Ala 1 5 10 15 Leu Pro Lys Lys
Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe 20 25 30 Leu Ser
Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe 35 40 45
Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro 50
55 60 Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Ala His
Val 65 70 75 80 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu
Asn Arg Arg 85 90 95 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu
Arg Asp Asn Gln Leu 100 105 110 Val Val Pro Ser Glu Gly Leu Tyr Leu
Ile Tyr Ser Gln Val Leu Phe 115 120 125 Lys Gly Gln Gly Cys Pro Ser
Thr His Val Leu Leu Thr His Thr Ile 130 135 140 Ser Arg Ile Ala Val
Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 145 150 155 160 Ile Lys
Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 165 170 175
Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 180
185 190 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp
Phe 195 200 205 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu
210 215 220 <210> SEQ ID NO 90 <211> LENGTH: 244
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: wild-type <400> SEQUENCE: 90
Met Asp Pro Asn Arg Ile Ser Glu Asp Gly Thr His Cys Ile Tyr Arg 1 5
10 15 Ile Leu Arg Leu His Glu Asn Ala Asp Phe Gln Asp Thr Thr Leu
Glu 20 25 30 Ser Gln Asp Thr Lys Leu Ile Pro Asp Ser Cys Arg Arg
Ile Lys Gln 35 40 45 Ala Phe Gln Gly Ala Val Gln Lys Glu Leu Gln
His Ile Val Gly Ser 50 55 60 Gln His Ile Arg Ala Glu Lys Ala Met
Val Asp Gly Ser Trp Leu Asp 65 70 75 80 Leu Ala Lys Arg Ser Lys Leu
Glu Ala Gln Pro Phe Ala His Leu Thr 85 90 95 Ile Asn Ala Thr Asp
Ile Pro Ser Gly Ser His Lys Val Ser Leu Ser 100 105 110 Ser Trp Tyr
His Asp Arg Gly Trp Ala Lys Ile Ser Asn Met Thr Phe 115 120 125 Ser
Asn Gly Lys Leu Ile Val Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr 130 135
140 Ala Asn Ile Cys Phe Arg His His Glu Thr Ser Gly Asp Leu Ala Thr
145 150 155 160 Glu Tyr Leu Gln Leu Met Val Tyr Val Thr Lys Thr Ser
Ile Lys Ile 165 170 175 Pro Ser Ser His Thr Leu Met Lys Gly Gly Ser
Thr Lys Tyr Trp Ser 180 185 190 Gly Asn Ser Glu Phe His Phe Tyr Ser
Ile Asn Val Gly Gly Phe Phe 195 200 205 Lys Leu Arg Ser Gly Glu Glu
Ile Ser Ile Glu Val Ser Asn Pro Ser 210 215 220 Leu Leu Asp Pro Asp
Gln Asp Ala Thr Tyr Phe Gly Ala Phe Lys Val 225 230 235 240 Arg Asp
Ile Asp <210> SEQ ID NO 91 <400> SEQUENCE: 91 000
<210> SEQ ID NO 92 <211> LENGTH: 26 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <223> OTHER
INFORMATION: FLT3L signal <400> SEQUENCE: 92 Met Ala Thr Val
Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu 1 5 10 15 Leu Leu
Leu Leu Leu Ser Ser Gly Leu Ser 20 25 <210> SEQ ID NO 93
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <223> OTHER INFORMATION: Linker
<400> SEQUENCE: 93 Gly Gly Gly Gly Ser 1 5 <210> SEQ ID
NO 94 <211> LENGTH: 14 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <220> FEATURE: <223> OTHER INFORMATION: V5
epitope tag for flow detection <400> SEQUENCE: 94 Gly Lys Pro
Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 1 5 10 <210> SEQ
ID NO 95 <211> LENGTH: 118 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
anti-biotin murine vH with inserted Cys for intralinkage
<400> SEQUENCE: 95 Gln Val Lys Leu Gln Glu Ser Gly Pro Gly
Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr Ala Tyr 20 25 30 Gly Val Asp Trp Val Arg
Gln Pro Pro Gly Lys Cys Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp
Gly Gly Gly Arg Thr Asn Tyr Asn Ser Gly Leu Met 50 55 60 Ser Arg
Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80
Thr Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Lys Tyr Tyr Cys Val 85
90 95 Lys His Thr Asn Trp Asp Gly Gly Phe Ala Tyr Trp Gly Gln Gly
Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> SEQ ID NO
96 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <220> FEATURE: <223> OTHER INFORMATION: Linker
<400> SEQUENCE: 96 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser 1 5 10 15 <210> SEQ ID NO 97 <211>
LENGTH: 99 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<220> FEATURE: <223> OTHER INFORMATION: LC Variable
<400> SEQUENCE: 97 Gly Ser Pro Gly Gln Ser Val Ser Ile Ser
Cys Ser Gly Ser Ser Ser 1 5 10 15 Asn Ile Gly Asn Asn Tyr Val Tyr
Trp Tyr Gln His Leu Pro Gly Thr 20 25 30 Ala Pro Lys Leu Leu Ile
Tyr Ser Asp Thr Lys Arg Pro Ser Gly Val 35 40 45 Pro Asp Arg Ile
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala 50 55 60 Ile Ser
Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser 65 70 75 80
Trp Asp Asp Ser Leu Asp Gly Pro Val Phe Gly Cys Gly Thr Lys Leu 85
90 95 Thr Val Leu <210> SEQ ID NO 98 <211> LENGTH: 99
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: CD8alpha transmembrane and
cytoplasmic domain <400> SEQUENCE: 98 Pro Thr Thr Thr Pro Ala
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 1 5 10 15 Ala Ser Gln Pro
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 20 25 30 Gly Gly
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 35 40 45
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 50
55 60 Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg Arg Arg Val Cys
Lys 65 70 75 80 Cys Pro Arg Pro Val Val Lys Ser Gly Asp Lys Pro Ser
Leu Ser Ala 85 90 95 Arg Tyr Val <210> SEQ ID NO 99
<211> LENGTH: 244 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
Summary (244 ORF7a) <400> SEQUENCE: 99 Met Asp Pro Asn Arg
Ile Ser Glu Asp Gly Thr His Cys Ile Tyr Arg 1 5 10 15 Ile Leu Arg
Leu His Glu Asn Ala Asp Phe Gln Asp Thr Thr Leu Glu 20 25 30 Ser
Gln Asp Thr Lys Leu Ile Pro Asp Ser Cys Arg Arg Ile Lys Gln 35 40
45 Ala Phe Gln Gly Ala Val Gln Lys Glu Leu Gln His Ile Val Gly Ser
50 55 60 Gln His Ile Arg Ala Glu Lys Ala Met Val Asp Gly Ser Trp
Leu Asp 65 70 75 80 Leu Ala Lys Arg Ser Lys Leu Glu Ala Gln Pro Phe
Ala His Leu Thr 85 90 95 Ile Asn Ala Thr Asp Ile Pro Ser Gly Ser
His Lys Val Ser Leu Ser 100 105 110 Ser Trp Tyr His Asp Arg Gly Trp
Ala Lys Ile Ser Asn Met Thr Phe 115 120 125 Ser Asn Gly Lys Leu Ile
Val Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr 130 135 140 Ala Asn Ile Cys
Phe Arg His His Glu Thr Ser Gly Asp Leu Ala Thr 145 150 155 160 Glu
Tyr Leu Gln Leu Met Val Tyr Val Thr Lys Thr Ser Ile Lys Ile 165 170
175 Pro Ser Ser His Thr Leu Met Lys Gly Gly Ser Thr Lys Tyr Trp Ser
180 185 190 Gly Asn Ser Glu Phe His Phe Tyr Ser Ile Asn Val Gly Gly
Phe Phe 195 200 205 Lys Leu Arg Ser Gly Glu Glu Ile Ser Ile Glu Val
Ser Asn Pro Ser 210 215 220 Leu Leu Asp Pro Asp Gln Asp Ala Thr Tyr
Phe Gly Ala Phe Lys Val 225 230 235 240 Arg Asp Ile Asp <210>
SEQ ID NO 100 <211> LENGTH: 381 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: Summary (381aa ORF7b) <400> SEQUENCE: 100 Met
Ala Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu 1 5 10
15 Leu Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly Gly Gly Gly Ser Gly
20 25 30 Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Gly
Gly Gly 35 40 45 Gly Ser Gln Val Lys Leu Gln Glu Ser Gly Pro Gly
Leu Val Ala Pro 50 55 60 Ser Gln Ser Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr 65 70 75 80 Ala Tyr Gly Val Asp Trp Val Arg
Gln Pro Pro Gly Lys Cys Leu Glu 85 90 95 Trp Leu Gly Val Ile Trp
Gly Gly Gly Arg Thr Asn Tyr Asn Ser Gly 100 105 110 Leu Met Ser Arg
Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln Val 115 120 125 Phe Leu
Thr Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Lys Tyr Tyr 130 135 140
Cys Val Lys His Thr Asn Trp Asp Gly Gly Phe Ala Tyr Trp Gly Gln 145
150 155 160 Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
Gly Gly 165 170 175 Gly Ser Gly Gly Gly Gly Ser Gly Ser Pro Gly Gln
Ser Val Ser Ile 180 185 190 Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly
Asn Asn Tyr Val Tyr Trp 195 200 205 Tyr Gln His Leu Pro Gly Thr Ala
Pro Lys Leu Leu Ile Tyr Ser Asp 210 215 220 Thr Lys Arg Pro Ser Gly
Val Pro Asp Arg Ile Ser Gly Ser Lys Ser 225 230 235 240 Gly Thr Ser
Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu 245 250 255 Ala
Asp Tyr Tyr Cys Ala Ser Trp Asp Asp Ser Leu Asp Gly Pro Val 260 265
270 Phe Gly Cys Gly Thr Lys Leu Thr Val Leu Pro Thr Thr Thr Pro Ala
275 280 285 Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
Leu Ser 290 295 300 Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
Ala Val His Thr 305 310 315 320 Arg Gly Leu Asp Phe Ala Cys Asp Ile
Tyr Ile Trp Ala Pro Leu Ala 325 330 335 Gly Thr Cys Gly Val Leu Leu
Leu Ser Leu Val Ile Thr Leu Tyr Cys 340 345 350 Asn His Arg Asn Arg
Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val 355 360 365 Lys Ser Gly
Asp Lys Pro Ser Leu Ser Ala Arg Tyr Val 370 375 380 <210> SEQ
ID NO 101 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <220> FEATURE: <223> OTHER INFORMATION:
Synthetic peptide <400> SEQUENCE: 101 Gln Met Gln Gly Val Asn
Cys Thr Val Ser Ser 1 5 10 <210> SEQ ID NO 102 <211>
LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<220> FEATURE: <223> OTHER INFORMATION: CpG <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(1)..(2) <223> OTHER INFORMATION: g-phosphorothioate
<220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (25)..(29) <223> OTHER INFORMATION:
g-phosphorothioate <400> SEQUENCE: 102 ggaaccgtat cggcgatatc
ggttgggggg 30 <210> SEQ ID NO 103 <211> LENGTH: 30
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <220> FEATURE:
<223> OTHER INFORMATION: CpG <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (1)..(2) <223>
OTHER INFORMATION: g-phosphorothioate <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION:
(25)..(29) <223> OTHER INFORMATION: g-phosphorothioate
<400> SEQUENCE: 103 ggaaccgtat gcggcatatc ggttgggggg 30
<210> SEQ ID NO 104 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <223> OTHER
INFORMATION: Synthetic peptide <400> SEQUENCE: 104 Val Arg
Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro 1 5 10 <210> SEQ ID
NO 105 <211> LENGTH: 6 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 105 Met Tyr Pro Pro Pro Tyr 1 5
<210> SEQ ID NO 106 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 106 Tyr Met Asn Met 1
<210> SEQ ID NO 107 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 107 Pro Tyr Ala Pro 1
<210> SEQ ID NO 108 <211> LENGTH: 183 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown: OX40L sequence
<400> SEQUENCE: 108 Met Glu Arg Val Gln Pro Leu Glu Glu Asn
Val Gly Asn Ala Ala Arg 1 5 10 15 Pro Arg Phe Glu Arg Asn Lys Leu
Leu Leu Val Ala Ser Val Ile Gln 20 25 30 Gly Leu Gly Leu Leu Leu
Cys Phe Thr Tyr Ile Cys Leu His Phe Ser 35 40 45 Ala Leu Gln Val
Ser His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val 50 55 60 Gln Phe
Thr Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln 65 70 75 80
Lys Glu Asp Glu Ile Met Lys Val Gln Asn Asn Ser Val Ile Ile Asn 85
90 95 Cys Asp Gly Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln
Glu 100 105 110 Val Asn Ile Ser Leu His Tyr Gln Lys Asp Glu Glu Pro
Leu Phe Gln 115 120 125 Leu Lys Lys Val Arg Ser Val Asn Ser Leu Met
Val Ala Ser Leu Thr 130 135 140 Tyr Lys Asp Lys Val Tyr Leu Asn Val
Thr Thr Asp Asn Thr Ser Leu 145 150 155 160 Asp Asp Phe His Val Asn
Gly Gly Glu Leu Ile Leu Ile His Gln Asn 165 170 175 Pro Gly Glu Phe
Cys Val Leu 180 <210> SEQ ID NO 109 <211> LENGTH: 193
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown:
CD27L sequence <400> SEQUENCE: 109 Met Pro Glu Glu Gly Ser
Gly Cys Ser Val Arg Arg Arg Pro Tyr Gly 1 5 10 15 Cys Val Leu Arg
Ala Ala Leu Val Pro Leu Val Ala Gly Leu Val Ile 20 25 30 Cys Leu
Val Val Cys Ile Gln Arg Phe Ala Gln Ala Gln Gln Gln Leu 35 40 45
Pro Leu Glu Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His 50
55 60 Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro
Ala 65 70 75 80 Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys
Gly Gln Leu 85 90 95 Arg Ile His Arg Asp Gly Ile Tyr Met Val His
Ile Gln Val Thr Leu 100 105 110 Ala Ile Cys Ser Ser Thr Thr Ala Ser
Arg His His Pro Thr Thr Leu 115 120 125 Ala Val Gly Ile Cys Ser Pro
Ala Ser Arg Ser Ile Ser Leu Leu Arg 130 135 140 Leu Ser Phe His Gln
Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro 145 150 155 160 Leu Ala
Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu 165 170 175
Pro Ser Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg 180
185 190 Pro <210> SEQ ID NO 110 <211> LENGTH: 288
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 110 Met Gly His Thr Arg Arg Gln Gly Thr Ser
Pro Ser Lys Cys Pro Tyr 1 5 10 15 Leu Asn Phe Phe Gln Leu Leu Val
Leu Ala Gly Leu Ser His Phe Cys 20 25 30 Ser Gly Val Ile His Val
Thr Lys Glu Val Lys Glu Val Ala Thr Leu 35 40 45 Ser Cys Gly His
Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile 50 55 60 Tyr Trp
Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp 65 70 75 80
Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr 85
90 95 Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu
Gly 100 105 110 Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala
Phe Lys Arg 115 120 125 Glu His Leu Ala Glu Val Thr Leu Ser Val Lys
Ala Asp Phe Pro Thr 130 135 140 Pro Ser Ile Ser Asp Phe Glu Ile Pro
Thr Ser Asn Ile Arg Arg Ile 145 150 155 160 Ile Cys Ser Thr Ser Gly
Gly Phe Pro Glu Pro His Leu Ser Trp Leu 165 170 175 Glu Asn Gly Glu
Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp 180 185 190 Pro Glu
Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met 195 200 205
Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg 210
215 220 Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe
Pro 225 230 235 240 Asp Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile
Ser Val Asn Gly 245 250 255 Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys
Phe Ala Pro Arg Cys Arg 260 265 270 Glu Arg Arg Arg Asn Glu Arg Leu
Arg Arg Glu Ser Val Arg Pro Val 275 280 285 <210> SEQ ID NO
111 <211> LENGTH: 329 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 111 Met Asp Pro Gln
Cys Thr Met Gly Leu Ser Asn Ile Leu Phe Val Met 1 5 10 15 Ala Phe
Leu Leu Ser Gly Ala Ala Pro Leu Lys Ile Gln Ala Tyr Phe 20 25 30
Asn Glu Thr Ala Asp Leu Pro Cys Gln Phe Ala Asn Ser Gln Asn Gln 35
40 45 Ser Leu Ser Glu Leu Val Val Phe Trp Gln Asp Gln Glu Asn Leu
Val 50 55 60 Leu Asn Glu Val Tyr Leu Gly Lys Glu Lys Phe Asp Ser
Val His Ser 65 70 75 80 Lys Tyr Met Gly Arg Thr Ser Phe Asp Ser Asp
Ser Trp Thr Leu Arg 85 90 95 Leu His Asn Leu Gln Ile Lys Asp Lys
Gly Leu Tyr Gln Cys Ile Ile 100 105 110 His His Lys Lys Pro Thr Gly
Met Ile Arg Ile His Gln Met Asn Ser 115 120 125 Glu Leu Ser Val Leu
Ala Asn Phe Ser Gln Pro Glu Ile Val Pro Ile 130 135 140 Ser Asn Ile
Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser Ile 145 150 155 160
His Gly Tyr Pro Glu Pro Lys Lys Met Ser Val Leu Leu Arg Thr Lys 165
170 175 Asn Ser Thr Ile Glu Tyr Asp Gly Val Met Gln Lys Ser Gln Asp
Asn 180 185 190 Val Thr Glu Leu Tyr Asp Val Ser Ile Ser Leu Ser Val
Ser Phe Pro 195 200 205 Asp Val Thr Ser Asn Met Thr Ile Phe Cys Ile
Leu Glu Thr Asp Lys 210 215 220 Thr Arg Leu Leu Ser Ser Pro Phe Ser
Ile Glu Leu Glu Asp Pro Gln 225 230 235 240 Pro Pro Pro Asp His Ile
Pro Trp Ile Thr Ala Val Leu Pro Thr Val 245 250 255 Ile Ile Cys Val
Met Val Phe Cys Leu Ile Leu Trp Lys Trp Lys Lys 260 265 270 Lys Lys
Arg Pro Arg Asn Ser Tyr Lys Cys Gly Thr Asn Thr Met Glu 275 280 285
Arg Glu Glu Ser Glu Gln Thr Lys Lys Arg Glu Lys Ile His Ile Pro 290
295 300 Glu Arg Ser Asp Glu Ala Gln Arg Val Phe Lys Ser Ser Lys Thr
Ser 305 310 315 320 Ser Cys Asp Lys Ser Asp Thr Cys Phe 325
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 111
<210> SEQ ID NO 1 <211> LENGTH: 399 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: membrane IgG-1 heavy chain <400> SEQUENCE: 1 Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145
150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Glu
Leu Gln Leu Glu Glu Ser Cys 325 330 335 Ala Glu Ala Gln Asp Gly Glu
Leu Asp Gly Leu Trp Thr Thr Ile Thr 340 345 350 Ile Phe Ile Thr Leu
Phe Leu Leu Ser Val Cys Tyr Ser Ala Thr Val 355 360 365 Thr Phe Phe
Lys Val Lys Trp Ile Phe Ser Ser Val Val Asp Leu Lys 370 375 380 Gln
Thr Ile Ile Pro Asp Tyr Arg Asn Met Ile Gly Gln Gly Ala 385 390 395
<210> SEQ ID NO 2 <211> LENGTH: 330 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: secreted IgG-1 heavy chain <400> SEQUENCE: 2 Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145
150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 330 <210> SEQ ID NO 3 <211> LENGTH: 295
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: fragment secreted IgG-1 heavy chain
<400> SEQUENCE: 3 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu 165 170 175 Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 180 185 190 Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 195 200 205 Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 210 215
220 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
225 230 235 240 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser 245 250 255 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser 260 265 270 Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser 275 280 285 Leu Ser Leu Ser Pro Gly Lys
290 295 <210> SEQ ID NO 4 <211> LENGTH: 377 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
IgG-3 heavy chain constant region <400> SEQUENCE: 4 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro
Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro
Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu
Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150
155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val
Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg
Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275
280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro
Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser
Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser Leu Ser
Leu Ser Pro Gly Lys 370 375 <210> SEQ ID NO 5 <211>
LENGTH: 184 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<220> FEATURE: <223> OTHER INFORMATION: mGM-CSF HLA
derived TM and SHORTENED cytoplasmic domain <400> SEQUENCE: 5
Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5
10 15 Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu
His 20 25 30 Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu
Ser Arg Asp 35 40 45 Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val
Ile Ser Glu Met Phe 50 55 60 Asp Leu Gln Glu Pro Thr Cys Leu Gln
Thr Arg Leu Glu Leu Tyr Lys 65 70 75 80 Gln Gly Leu Arg Gly Ser Leu
Thr Lys Leu Lys Gly Pro Leu Thr Met 85 90 95 Met Ala Ser His Tyr
Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 100 105 110 Cys Ala Thr
Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys 115 120 125 Asp
Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 130 135
140 Glu Leu Ser Ser Gln Pro Thr Ile Pro Ile Val Gly Ile Ile Ala Gly
145 150 155 160 Leu Val Leu Leu Gly Ala Val Ile Thr Gly Ala Val Val
Ala Ala Val 165 170 175 Met Trp Arg Arg Lys Ser Ser Asp 180
<210> SEQ ID NO 6 <211> LENGTH: 261 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: CD40L <400> SEQUENCE: 6 Met Ile Glu Thr Tyr Asn
Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15 Leu Pro Ile Ser
Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30 Ile Thr
Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45
Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50
55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu
Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly
Phe Val Lys 85 90 95 Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys
Glu Asn Ser Phe Glu 100 105 110 Met Gln Lys Gly Asp Gln Asn Pro Gln
Ile Ala Ala His Val Ile Ser 115 120 125 Glu Ala Ser Ser Lys Thr Thr
Ser Val Leu Gln Trp Ala Glu Lys Gly 130 135 140 Tyr Tyr Thr Met Ser
Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 145 150 155 160 Leu Thr
Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 165 170 175
Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 180
185 190 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg
Ala 195 200 205 Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln
Ser Ile His 210 215 220 Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala
Ser Val Phe Val Asn 225 230 235 240 Val Thr Asp Pro Ser Gln Val Ser
His Gly Thr Gly Phe Thr Ser Phe 245 250 255 Gly Leu Leu Lys Leu 260
<210> SEQ ID NO 7 <211> LENGTH: 261 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: non-cleavable CD40L <400> SEQUENCE: 7 Met Ile
Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15
Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20
25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His
Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu
Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly
Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys Ser
Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn Lys Glu
Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110 Met Pro Arg Gly Glu
Glu Asp Ser Gln Ile Ala Ala His Val Ile Ser 115 120 125 Glu Ala Ser
Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130 135 140 Tyr
Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 145 150
155 160 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val
Thr 165 170 175 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe
Ile Ala Ser 180 185 190 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg
Ile Leu Leu Arg Ala 195 200 205 Ala Asn Thr His Ser Ser Ala Lys Pro
Cys Gly Gln Gln Ser Ile His 210 215 220 Leu Gly Gly Val Phe Glu Leu
Gln Pro Gly Ala Ser Val Phe Val Asn
225 230 235 240 Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe
Thr Ser Phe 245 250 255 Gly Leu Leu Lys Leu 260 <210> SEQ ID
NO 8 <211> LENGTH: 233 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION: TNF
<400> SEQUENCE: 8 Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu
Leu Ala Glu Glu Ala 1 5 10 15 Leu Pro Lys Lys Thr Gly Gly Pro Gln
Gly Ser Arg Arg Cys Leu Phe 20 25 30 Leu Ser Leu Phe Ser Phe Leu
Ile Val Ala Gly Ala Thr Thr Leu Phe 35 40 45 Cys Leu Leu His Phe
Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro 50 55 60 Arg Asp Leu
Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser 65 70 75 80 Ser
Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro 85 90
95 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu
100 105 110 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val
Pro Ser 115 120 125 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe
Lys Gly Gln Gly 130 135 140 Cys Pro Ser Thr His Val Leu Leu Thr His
Thr Ile Ser Arg Ile Ala 145 150 155 160 Val Ser Tyr Gln Thr Lys Val
Asn Leu Leu Ser Ala Ile Lys Ser Pro 165 170 175 Cys Gln Arg Glu Thr
Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu 180 185 190 Pro Ile Tyr
Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 195 200 205 Ser
Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 210 215
220 Gln Val Tyr Phe Gly Ile Ile Ala Leu 225 230 <210> SEQ ID
NO 9 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
CD40L piece <400> SEQUENCE: 9 Met Ile Glu Thr Tyr Asn Gln Thr
Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15 Leu Pro Ile Ser Met Lys
Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30 Ile Thr Gln Met
Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45 Arg Leu
Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60
Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser 65
70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe
Val Lys 85 90 95 Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu
100 105 <210> SEQ ID NO 10 <211> LENGTH: 141
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: TNF piece <400> SEQUENCE: 10
Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 1 5
10 15 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln
Leu 20 25 30 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln
Val Leu Phe 35 40 45 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu
Leu Thr His Thr Ile 50 55 60 Ser Arg Ile Ala Val Ser Tyr Gln Thr
Lys Val Asn Leu Leu Ser Ala 65 70 75 80 Ile Lys Ser Pro Cys Gln Arg
Glu Thr Pro Glu Gly Ala Glu Ala Lys 85 90 95 Pro Trp Tyr Glu Pro
Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 100 105 110 Gly Asp Arg
Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 115 120 125 Ala
Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 130 135 140
<210> SEQ ID NO 11 <211> LENGTH: 233 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: mTNF-a <400> SEQUENCE: 11 Met Ser Thr Glu Ser
Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala 1 5 10 15 Leu Pro Lys
Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe 20 25 30 Leu
Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe 35 40
45 Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro
50 55 60 Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Ser Gly
Ser Gly 65 70 75 80 Ser Gly Ser Gly Ser Gly Glu Pro Val Ala His Val
Val Ala Asn Pro 85 90 95 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn
Arg Arg Ala Asn Ala Leu 100 105 110 Leu Ala Asn Gly Val Glu Leu Arg
Asp Asn Gln Leu Val Val Pro Ser 115 120 125 Glu Gly Leu Tyr Leu Ile
Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly 130 135 140 Cys Pro Ser Thr
His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala 145 150 155 160 Val
Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro 165 170
175 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu
180 185 190 Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp
Arg Leu 195 200 205 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe
Ala Glu Ser Gly 210 215 220 Gln Val Tyr Phe Gly Ile Ile Ala Leu 225
230 <210> SEQ ID NO 12 <211> LENGTH: 113 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <223>
OTHER INFORMATION: HC Constant Region 3 from IgG1 <400>
SEQUENCE: 12 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr
Gln Lys Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser 100 105
110 Cys <210> SEQ ID NO 13 <211> LENGTH: 144
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: native GM-CSF
<400> SEQUENCE: 13 Met Trp Leu Gln Ser Leu Leu Leu Leu Gly
Thr Val Ala Cys Ser Ile 1 5 10 15 Ser Ala Pro Ala Arg Ser Pro Ser
Pro Ser Thr Gln Pro Trp Glu His 20 25 30 Val Asn Ala Ile Gln Glu
Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp 35 40 45 Thr Ala Ala Glu
Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe 50 55 60 Asp Leu
Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 65 70 75 80
Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met 85
90 95 Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr
Ser 100 105 110 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu
Asn Leu Lys 115 120 125 Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp
Glu Pro Val Gln Glu 130 135 140 <210> SEQ ID NO 14
<211> LENGTH: 235 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
Flt3L <400> SEQUENCE: 14 Met Thr Val Leu Ala Pro Ala Trp Ser
Pro Thr Thr Tyr Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Ser Ser Gly
Leu Ser Gly Thr Gln Asp Cys Ser Phe 20 25 30 Gln His Ser Pro Ile
Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu 35 40 45 Ser Asp Tyr
Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu 50 55 60 Gln
Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln 65 70
75 80 Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln
Gly 85 90 95 Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr
Lys Cys Ala 100 105 110 Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val
Gln Thr Asn Ile Ser 115 120 125 Arg Leu Leu Gln Glu Thr Ser Glu Gln
Leu Val Ala Leu Lys Pro Trp 130 135 140 Ile Thr Arg Gln Asn Phe Ser
Arg Cys Leu Glu Leu Gln Cys Gln Pro 145 150 155 160 Asp Ser Ser Thr
Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala 165 170 175 Thr Ala
Pro Thr Ala Pro Gln Pro Pro Leu Leu Leu Leu Leu Leu Leu 180 185 190
Pro Val Gly Leu Leu Leu Leu Ala Ala Ala Trp Cys Leu His Trp Gln 195
200 205 Arg Thr Arg Arg Arg Thr Pro Arg Pro Gly Glu Gln Val Pro Pro
Val 210 215 220 Pro Ser Pro Gln Asp Leu Leu Leu Val Glu His 225 230
235 <210> SEQ ID NO 15 <211> LENGTH: 13 <212>
TYPE: RNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <220> FEATURE:
<223> OTHER INFORMATION: Kozak sequence <400> SEQUENCE:
15 gccgccrcca ugg 13 <210> SEQ ID NO 16 <211> LENGTH:
377 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: IgG-3 heavy chain constant region
E213Q <400> SEQUENCE: 16 Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70
75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His
Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro
Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr
Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp
Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190
Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195
200 205 Val Asp Gly Val Gln Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr
Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp
Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315
320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu
325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
Arg Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370
375 <210> SEQ ID NO 17 <211> LENGTH: 377 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <223>
OTHER INFORMATION: IgG-3 heavy chain constant region P221L
<400> SEQUENCE: 17 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys
Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro
Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro
Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro
Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Leu Arg Glu Glu 210
215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu
His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys 245 250 255
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260
265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly
Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser
Leu Ser Leu Ser Pro Gly Lys 370 375 <210> SEQ ID NO 18
<211> LENGTH: 377 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
IgG-3 heavy chain constant region E224Q <400> SEQUENCE: 18
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys
Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys
Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135
140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Gln 210 215 220 Gln Tyr Asn Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260
265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly
Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser
Leu Ser Leu Ser Pro Gly Lys 370 375 <210> SEQ ID NO 19
<211> LENGTH: 377 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
IgG-3 heavy chain constant region Y226F <400> SEQUENCE: 19
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys
Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys
Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135
140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Phe Asn Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260
265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly
Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser
Leu Ser Leu Ser Pro Gly Lys 370 375 <210> SEQ ID NO 20
<211> LENGTH: 377 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
IgG-3 heavy chain constant region D242N <400> SEQUENCE: 20
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys
Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys
Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135
140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu
210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
Leu His 225 230 235 240 Gln Asn Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile
Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320
Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325
330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg
Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375
<210> SEQ ID NO 21 <211> LENGTH: 377 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: IgG-3 heavy chain constant region N245D <400>
SEQUENCE: 21 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105
110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg
115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro
Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys
Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser
His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln
Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230
235 240 Gln Asp Trp Leu Asp Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp
Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355
360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 <210> SEQ
ID NO 22 <211> LENGTH: 377 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
IgG-3 heavy chain constant region T269A <400> SEQUENCE: 22
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys
Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys
Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135
140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 260
265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly
Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser
Leu Ser Leu Ser Pro Gly Lys 370 375 <210> SEQ ID NO 23
<211> LENGTH: 377 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
IgG-3 heavy chain constant region S314N <400> SEQUENCE: 23
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys
Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys
Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135
140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 165 170 175
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180
185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp
Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val
Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 305
310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe
Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn Arg Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly
Lys 370 375 <210> SEQ ID NO 24 <211> LENGTH: 376
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: IgG-3 heavy chain constant region
S314 del <400> SEQUENCE: 24 Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65
70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr
His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr
Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp
Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys
Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185
190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr
195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu
Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser
Asp Ile Ala Val Glu Trp Glu Ser Gly Gln Pro Glu Asn Asn Tyr 305 310
315 320 Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr 325 330 335 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Ile Phe 340 345 350 Ser Cys Ser Val Met His Glu Ala Leu His Asn
Arg Phe Thr Gln Lys 355 360 365 Ser Leu Ser Leu Ser Pro Gly Lys 370
375 <210> SEQ ID NO 25 <211> LENGTH: 377 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <223>
OTHER INFORMATION: IgG-3 heavy chain constant region F366Y
<400> SEQUENCE: 25 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys
Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro
Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro
Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro
Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210
215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu
His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala
Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr
Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330
335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile
340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Tyr
Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375
<210> SEQ ID NO 26 <211> LENGTH: 221 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: TNF - VRSSSRTPSDKP del <400> SEQUENCE: 26 Met
Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala 1 5 10
15 Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe
20 25 30 Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr
Leu Phe 35 40 45 Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg
Glu Glu Phe Pro 50 55 60 Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala
Gln Ala Val Ala His Val 65 70 75 80 Val Ala Asn Pro Gln Ala Glu Gly
Gln Leu Gln Trp Leu Asn Arg Arg 85 90 95 Ala Asn Ala Leu Leu Ala
Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 100 105 110 Val Val Pro Ser
Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 115 120 125 Lys Gly
Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 130 135
140
Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 145
150 155 160 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu
Ala Lys 165 170 175 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe
Gln Leu Glu Lys 180 185 190 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg
Pro Asp Tyr Leu Asp Phe 195 200 205 Ala Glu Ser Gly Gln Val Tyr Phe
Gly Ile Ile Ala Leu 210 215 220 <210> SEQ ID NO 27
<211> LENGTH: 212 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION: TNF
- FSFLIVAGATTLFCLLHFGVI del <400> SEQUENCE: 27 Met Ser Thr
Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala 1 5 10 15 Leu
Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe 20 25
30 Leu Ser Leu Gly Pro Gln Arg Glu Glu Phe Pro Arg Asp Leu Ser Leu
35 40 45 Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser Ser Arg Thr
Pro Ser 50 55 60 Asp Lys Pro Val Ala His Val Val Ala Asn Pro Gln
Ala Glu Gly Gln 65 70 75 80 Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala
Leu Leu Ala Asn Gly Val 85 90 95 Glu Leu Arg Asp Asn Gln Leu Val
Val Pro Ser Glu Gly Leu Tyr Leu 100 105 110 Ile Tyr Ser Gln Val Leu
Phe Lys Gly Gln Gly Cys Pro Ser Thr His 115 120 125 Val Leu Leu Thr
His Thr Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr 130 135 140 Lys Val
Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr 145 150 155
160 Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly
165 170 175 Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu
Ile Asn 180 185 190 Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gln
Val Tyr Phe Gly 195 200 205 Ile Ile Ala Leu 210 <210> SEQ ID
NO 28 <211> LENGTH: 9 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <220> FEATURE: <223> OTHER INFORMATION: HA tag
seq <400> SEQUENCE: 28 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1
5 <210> SEQ ID NO 29 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <223> OTHER
INFORMATION: FLAG TAG <400> SEQUENCE: 29 Asp Tyr Lys Asp Asp
Asp Asp Lys 1 5 <210> SEQ ID NO 30 <211> LENGTH: 583
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: mIgG Heavy Chain IgG 1/3 hybrid
anti-biotin heavy chain - E325A mutant <400> SEQUENCE: 30 Met
Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Phe Arg Gly 1 5 10
15 Val Gln Cys Gln Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
20 25 30 Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe
Ser Leu 35 40 45 Thr Ala Tyr Gly Val Asp Trp Val Arg Gln Pro Pro
Gly Lys Gly Leu 50 55 60 Glu Trp Leu Gly Val Ile Trp Gly Gly Gly
Arg Thr Asn Tyr Asn Ser 65 70 75 80 Gly Leu Met Ser Arg Leu Ser Ile
Arg Lys Asp Asn Ser Lys Ser Gln 85 90 95 Val Phe Leu Thr Met Asn
Ser Leu Gln Thr Asp Asp Thr Ala Lys Tyr 100 105 110 Tyr Cys Val Lys
His Thr Asn Trp Asp Gly Gly Phe Ala Tyr Trp Gly 115 120 125 Gln Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145
150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Leu Lys Thr Pro 225 230 235 240 Leu Gly Asp
Thr Thr His Thr Cys Pro Arg Cys Pro Glu Pro Lys Ser 245 250 255 Cys
Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys 260 265
270 Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp
275 280 285 Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu
Gly Gly 290 295 300 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile 305 310 315 320 Ser Arg Thr Pro Ala Val Thr Cys Val
Val Val Asp Val Ser His Glu 325 330 335 Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 340 345 350 Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 355 360 365 Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 370 375 380 Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 385 390
395 400 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr 405 410 415 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu 420 425 430 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp 435 440 445 Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val 450 455 460 Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp 465 470 475 480 Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 485 490 495 Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 500 505 510
Glu Leu Gln Leu Glu Glu Ser Cys Ala Glu Ala Gln Asp Gly Glu Leu 515
520 525 Asp Gly Leu Trp Thr Thr Ile Thr Ile Phe Ile Thr Leu Phe Leu
Leu 530 535 540 Ser Val Cys Tyr Ser Ala Thr Val Thr Phe Phe Lys Val
Lys Trp Ile 545 550 555 560 Phe Ser Ser Val Val Asp Leu Lys Gln Thr
Ile Ile Pro Asp Tyr Arg 565 570 575 Asn Met Ile Gly Gln Gly Ala 580
<210> SEQ ID NO 31 <211> LENGTH: 223 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: CD40L-TNFa fusion protein <400> SEQUENCE: 31 Met
Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10
15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe
Leu
20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu
His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His
Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr
Gly Glu Arg Pro Val Ala 65 70 75 80 His Val Val Ala Asn Pro Gln Ala
Glu Gly Gln Leu Gln Trp Leu Asn 85 90 95 Arg Arg Ala Asn Ala Leu
Leu Ala Asn Gly Val Glu Leu Arg Asp Asn 100 105 110 Gln Leu Val Val
Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val 115 120 125 Leu Phe
Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His 130 135 140
Thr Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu 145
150 155 160 Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly
Ala Glu 165 170 175 Ala Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly
Val Phe Gln Leu 180 185 190 Glu Lys Gly Asp Arg Leu Ser Ala Glu Ile
Asn Arg Pro Asp Tyr Leu 195 200 205 Asp Phe Ala Glu Ser Gly Gln Val
Tyr Phe Gly Ile Ile Ala Leu 210 215 220 <210> SEQ ID NO 32
<211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <223> OTHER INFORMATION: HC Signal
<400> SEQUENCE: 32 Met Glu Phe Gly Leu Ser Trp Val Phe Leu
Val Ala Leu Phe Arg Gly 1 5 10 15 Val Gln Cys <210> SEQ ID NO
33 <211> LENGTH: 118 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION: HC
Variable anti-biotin <400> SEQUENCE: 33 Gln Val Lys Leu Gln
Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser
Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ala Tyr 20 25 30 Gly
Val Asp Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40
45 Gly Val Ile Trp Gly Gly Gly Arg Thr Asn Tyr Asn Ser Gly Leu Met
50 55 60 Ser Arg Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln Val
Phe Leu 65 70 75 80 Thr Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Lys
Tyr Tyr Cys Val 85 90 95 Lys His Thr Asn Trp Asp Gly Gly Phe Ala
Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
<210> SEQ ID NO 34 <211> LENGTH: 99 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: HC Constant Region 1 from IgG1 <400> SEQUENCE:
34 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu
<210> SEQ ID NO 35 <211> LENGTH: 61 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: HC Hinge Region from IgG3 <400> SEQUENCE: 35 Leu
Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys Pro 1 5 10
15 Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu
20 25 30 Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
Glu Pro 35 40 45 Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys
Pro 50 55 60 <210> SEQ ID NO 36 <211> LENGTH: 110
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: HC Constant Region 2 from IgG1
<400> SEQUENCE: 36 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100
105 110 <210> SEQ ID NO 37 <211> LENGTH: 63 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <223>
OTHER INFORMATION: HC Transmembrane and Cytoplasmic region from
IgG1 <400> SEQUENCE: 37 Ala Glu Ala Gln Asp Gly Glu Leu Asp
Gly Leu Trp Thr Thr Ile Thr 1 5 10 15 Ile Phe Ile Thr Leu Phe Leu
Leu Ser Val Cys Tyr Ser Ala Thr Val 20 25 30 Thr Phe Phe Lys Val
Lys Trp Ile Phe Ser Ser Val Val Asp Leu Lys 35 40 45 Gln Thr Ile
Ile Pro Asp Tyr Arg Asn Met Ile Gly Gln Gly Ala 50 55 60
<210> SEQ ID NO 38 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <223> OTHER
INFORMATION: LC Signal <400> SEQUENCE: 38 Met Lys Tyr Leu Leu
Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro
Ala Met Ala 20 <210> SEQ ID NO 39 <211> LENGTH: 99
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: LC Variable <400> SEQUENCE:
39
Gly Ser Pro Gly Gln Ser Val Ser Ile Ser Cys Ser Gly Ser Ser Ser 1 5
10 15 Asn Ile Gly Asn Asn Tyr Val Tyr Trp Tyr Gln His Leu Pro Gly
Thr 20 25 30 Ala Pro Lys Leu Leu Ile Tyr Ser Asp Thr Lys Arg Pro
Ser Gly Val 35 40 45 Pro Asp Arg Ile Ser Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala 50 55 60 Ile Ser Gly Leu Gln Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Ala Ser 65 70 75 80 Trp Asp Asp Ser Leu Asp Gly
Pro Val Phe Gly Gly Gly Thr Lys Leu 85 90 95 Thr Val Leu
<210> SEQ ID NO 40 <211> LENGTH: 106 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: LC Constant Region 1 <400> SEQUENCE: 40 Gly Gln
Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20
25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser
Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln
Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr
Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val
Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr
Glu Cys Ser 100 105 <210> SEQ ID NO 41 <211> LENGTH:
583 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: mIgG Heavy Chain IgG 1/3 hybrid
anti-biotin heavy chain - T323A mutant <400> SEQUENCE: 41 Met
Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Phe Arg Gly 1 5 10
15 Val Gln Cys Gln Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
20 25 30 Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe
Ser Leu 35 40 45 Thr Ala Tyr Gly Val Asp Trp Val Arg Gln Pro Pro
Gly Lys Gly Leu 50 55 60 Glu Trp Leu Gly Val Ile Trp Gly Gly Gly
Arg Thr Asn Tyr Asn Ser 65 70 75 80 Gly Leu Met Ser Arg Leu Ser Ile
Arg Lys Asp Asn Ser Lys Ser Gln 85 90 95 Val Phe Leu Thr Met Asn
Ser Leu Gln Thr Asp Asp Thr Ala Lys Tyr 100 105 110 Tyr Cys Val Lys
His Thr Asn Trp Asp Gly Gly Phe Ala Tyr Trp Gly 115 120 125 Gln Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145
150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Leu Lys Thr Pro 225 230 235 240 Leu Gly Asp
Thr Thr His Thr Cys Pro Arg Cys Pro Glu Pro Lys Ser 245 250 255 Cys
Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys 260 265
270 Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp
275 280 285 Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu
Gly Gly 290 295 300 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile 305 310 315 320 Ser Arg Ala Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 325 330 335 Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 340 345 350 Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 355 360 365 Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 370 375 380 Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 385 390
395 400 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr 405 410 415 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu 420 425 430 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp 435 440 445 Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val 450 455 460 Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp 465 470 475 480 Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 485 490 495 Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 500 505 510
Glu Leu Gln Leu Glu Glu Ser Cys Ala Glu Ala Gln Asp Gly Glu Leu 515
520 525 Asp Gly Leu Trp Thr Thr Ile Thr Ile Phe Ile Thr Leu Phe Leu
Leu 530 535 540 Ser Val Cys Tyr Ser Ala Thr Val Thr Phe Phe Lys Val
Lys Trp Ile 545 550 555 560 Phe Ser Ser Val Val Asp Leu Lys Gln Thr
Ile Ile Pro Asp Tyr Arg 565 570 575 Asn Met Ile Gly Gln Gly Ala 580
<210> SEQ ID NO 42 <211> LENGTH: 211 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: GM-CSF - HLA-I fusion peptide <400> SEQUENCE: 42
Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5
10 15 Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu
His 20 25 30 Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu
Ser Arg Asp 35 40 45 Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val
Ile Ser Glu Met Phe 50 55 60 Asp Leu Gln Glu Pro Thr Cys Leu Gln
Thr Arg Leu Glu Leu Tyr Lys 65 70 75 80 Gln Gly Leu Arg Gly Ser Leu
Thr Lys Leu Lys Gly Pro Leu Thr Met 85 90 95 Met Ala Ser His Tyr
Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 100 105 110 Cys Ala Thr
Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys 115 120 125 Asp
Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 130 135
140 Glu Leu Ser Ser Gln Pro Thr Ile Pro Ile Val Gly Ile Ile Ala Gly
145 150 155 160 Leu Val Leu Leu Gly Ala Val Ile Thr Gly Ala Val Val
Ala Ala Val 165 170 175 Met Trp Arg Arg Lys Ser Ser Asp Arg Lys Gly
Gly Ser Tyr Thr Gln 180 185 190 Ala Ala Ser Ser Asp Ser Ala Gln Gly
Ser Asp Val Ser Leu Thr Ala 195 200 205 Cys Lys Val 210 <210>
SEQ ID NO 43 <211> LENGTH: 583 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: mIgG Heavy Chain IgG 1/3 hybrid anti-biotin
heavy chain - E325A, T323A mutant <400> SEQUENCE: 43 Met Glu
Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Phe Arg Gly 1 5 10 15
Val Gln Cys Gln Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala 20
25 30 Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser
Leu 35 40 45 Thr Ala Tyr Gly Val Asp Trp Val Arg Gln Pro Pro Gly
Lys Gly Leu 50 55 60 Glu Trp Leu Gly Val Ile Trp Gly Gly Gly Arg
Thr Asn Tyr Asn Ser 65 70 75 80 Gly Leu Met Ser Arg Leu Ser Ile Arg
Lys Asp Asn Ser Lys Ser Gln 85 90 95 Val Phe Leu Thr Met Asn Ser
Leu Gln Thr Asp Asp Thr Ala Lys Tyr 100 105 110 Tyr Cys Val Lys His
Thr Asn Trp Asp Gly Gly Phe Ala Tyr Trp Gly 115 120 125 Gln Gly Thr
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150
155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Leu Lys Thr Pro 225 230 235 240 Leu Gly Asp Thr
Thr His Thr Cys Pro Arg Cys Pro Glu Pro Lys Ser 245 250 255 Cys Asp
Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys 260 265 270
Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp 275
280 285 Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu Gly
Gly 290 295 300 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile 305 310 315 320 Ser Arg Ala Pro Ala Val Thr Cys Val Val
Val Asp Val Ser His Glu 325 330 335 Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His 340 345 350 Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 355 360 365 Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 370 375 380 Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 385 390 395
400 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
405 410 415 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu 420 425 430 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp 435 440 445 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val 450 455 460 Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp 465 470 475 480 Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His 485 490 495 Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 500 505 510 Glu
Leu Gln Leu Glu Glu Ser Cys Ala Glu Ala Gln Asp Gly Glu Leu 515 520
525 Asp Gly Leu Trp Thr Thr Ile Thr Ile Phe Ile Thr Leu Phe Leu Leu
530 535 540 Ser Val Cys Tyr Ser Ala Thr Val Thr Phe Phe Lys Val Lys
Trp Ile 545 550 555 560 Phe Ser Ser Val Val Asp Leu Lys Gln Thr Ile
Ile Pro Asp Tyr Arg 565 570 575 Asn Met Ile Gly Gln Gly Ala 580
<210> SEQ ID NO 44 <211> LENGTH: 183 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: Soluble Flt3-L <400> SEQUENCE: 44 Met Thr Val
Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu Leu 1 5 10 15 Leu
Leu Leu Leu Ser Ser Gly Leu Ser Gly Thr Gln Asp Cys Ser Phe 20 25
30 Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu
35 40 45 Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser
Asn Leu 50 55 60 Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu
Val Leu Ala Gln 65 70 75 80 Arg Trp Met Glu Arg Leu Lys Thr Val Ala
Gly Ser Lys Met Gln Gly 85 90 95 Leu Leu Glu Arg Val Asn Thr Glu
Ile His Phe Val Thr Lys Cys Ala 100 105 110 Phe Gln Pro Pro Pro Ser
Cys Leu Arg Phe Val Gln Thr Asn Ile Ser 115 120 125 Arg Leu Leu Gln
Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp 130 135 140 Ile Thr
Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro 145 150 155
160 Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala
165 170 175 Thr Ala Pro Thr Ala Pro Gln 180 <210> SEQ ID NO
45 <211> LENGTH: 583 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
mIgG Heavy Chain IgG 1/3 hybrid anti-biotin heavy chain <400>
SEQUENCE: 45 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu
Phe Arg Gly 1 5 10 15 Val Gln Cys Gln Val Lys Leu Gln Glu Ser Gly
Pro Gly Leu Val Ala 20 25 30 Pro Ser Gln Ser Leu Ser Ile Thr Cys
Thr Val Ser Gly Phe Ser Leu 35 40 45 Thr Ala Tyr Gly Val Asp Trp
Val Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60 Glu Trp Leu Gly Val
Ile Trp Gly Gly Gly Arg Thr Asn Tyr Asn Ser 65 70 75 80 Gly Leu Met
Ser Arg Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln 85 90 95 Val
Phe Leu Thr Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Lys Tyr 100 105
110 Tyr Cys Val Lys His Thr Asn Trp Asp Gly Gly Phe Ala Tyr Trp Gly
115 120 125 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Leu Lys Thr Pro 225 230
235 240 Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys Pro Glu Pro Lys
Ser 245 250 255 Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro
Lys Ser Cys 260 265 270 Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu
Pro Lys Ser Cys Asp 275 280 285 Thr Pro Pro Pro Cys Pro Arg Cys Pro
Ala Pro Glu Leu Leu Gly Gly 290 295 300 Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile 305 310 315 320 Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 325 330 335 Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 340 345 350
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 355
360 365 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys
370 375 380 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu 385 390 395 400 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr 405 410 415 Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu 420 425 430 Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp 435 440 445 Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 450 455 460 Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 465 470 475 480
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 485
490 495 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro 500 505 510 Glu Leu Gln Leu Glu Glu Ser Cys Ala Glu Ala Gln Asp
Gly Glu Leu 515 520 525 Asp Gly Leu Trp Thr Thr Ile Thr Ile Phe Ile
Thr Leu Phe Leu Leu 530 535 540 Ser Val Cys Tyr Ser Ala Thr Val Thr
Phe Phe Lys Val Lys Trp Ile 545 550 555 560 Phe Ser Ser Val Val Asp
Leu Lys Gln Thr Ile Ile Pro Asp Tyr Arg 565 570 575 Asn Met Ile Gly
Gln Gly Ala 580 <210> SEQ ID NO 46 <211> LENGTH: 227
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: IgG Light Chain <400>
SEQUENCE: 46 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu
Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Gly Ser Pro Gly Gln
Ser Val Ser Ile Ser 20 25 30 Cys Ser Gly Ser Ser Ser Asn Ile Gly
Asn Asn Tyr Val Tyr Trp Tyr 35 40 45 Gln His Leu Pro Gly Thr Ala
Pro Lys Leu Leu Ile Tyr Ser Asp Thr 50 55 60 Lys Arg Pro Ser Gly
Val Pro Asp Arg Ile Ser Gly Ser Lys Ser Gly 65 70 75 80 Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala 85 90 95 Asp
Tyr Tyr Cys Ala Ser Trp Asp Asp Ser Leu Asp Gly Pro Val Phe 100 105
110 Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Asn Pro
115 120 125 Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala
Asn Lys 130 135 140 Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro
Gly Ala Val Thr 145 150 155 160 Val Ala Trp Lys Ala Asp Gly Ser Pro
Val Lys Ala Gly Val Glu Thr 165 170 175 Thr Lys Pro Ser Lys Gln Ser
Asn Asn Lys Tyr Ala Ala Ser Ser Tyr 180 185 190 Leu Ser Leu Thr Pro
Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys 195 200 205 Gln Val Thr
His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr 210 215 220 Glu
Cys Ser 225 <210> SEQ ID NO 47 <211> LENGTH: 9203
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polynucleotide <220> FEATURE:
<223> OTHER INFORMATION: Vector 1 <400> SEQUENCE: 47
aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca
60 tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa
ggtggtacga 120 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg
attggacgaa ccactgaatt 180 gccgcattgc agagatattg tatttaagtg
cctagctcga tacataaacg ggtctctctg 240 gttagaccag atctgagcct
gggagctctc tggctaacta gggaacccac tgcttaagcc 300 tcaataaagc
ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360
taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg
420 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
gactcggctt 480 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg
tgagtacgcc aaaaattttg 540 actagcggag gctagaagga gagagatggg
tgcgagagcg tcagtattaa gcgggggaga 600 attagatcgc gatgggaaaa
aattcggtta aggccagggg gaaagaaaaa atataaatta 660 aaacatatag
tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720
gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga
780 tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
gcatcaaagg 840 atagagataa aagacaccaa ggaagcttta gacaagatag
aggaagagca aaacaaaagt 900 aagaccaccg cacagcaagc ggccgctgat
cttcagacct ggaggaggag atatgaggga 960 caattggaga agtgaattat
ataaatataa agtagtaaaa attgaaccat taggagtagc 1020 acccaccaag
gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080
tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct
1140 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
atttgctgag 1200 ggctattgag gcgcaacagc atctgttgca actcacagtc
tggggcatca agcagctcca 1260 ggcaagaatc ctggctgtgg aaagatacct
aaaggatcaa cagctcctgg ggatttgggg 1320 ttgctctgga aaactcattt
gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 1380 atctctggaa
cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440
ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
1500 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
acataacaaa 1560 ttggctgtgg tatataaaat tattcataat gatagtagga
ggcttggtag gtttaagaat 1620 agtttttgct gtactttcta tagtgaatag
agttaggcag ggatattcac cattatcgtt 1680 tcagacccac ctcccaaccc
cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740 tggagagaga
gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgcta 1800
gcttttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata
1860 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
tcaaaatttt 1920 actagtgatt atcggatcaa ctttgtatag aaaagttggg
ctccggtgcc cgtcagtggg 1980 cagagcgcac atcgcccaca gtccccgaga
agttgggggg aggggtcggc aattgaaccg 2040 gtgcctagag aaggtggcgc
ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc 2100 tttttcccga
gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt 2160
ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg
2220 gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg
ctgcagtacg 2280 tgattcttga tcccgagctt cgggttggaa gtgggtggga
gagttcgagg ccttgcgctt 2340 aaggagcccc ttcgcctcgt gcttgagttg
aggcctggcc tgggcgctgg ggccgccgcg 2400 tgcgaatctg gtggcacctt
cgcgcctgtc tcgctgcttt cgataagtct ctagccattt 2460 aaaatttttg
atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg 2520
gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg
2580 cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg
agaatcggac 2640 gggggtagtc tcaagctggc cggcctgctc tggtgcctgg
tctcgcgccg ccgtgtatcg 2700 ccccgccctg ggcggcaagg ctggcccggt
cggcaccagt tgcgtgagcg gaaagatggc 2760 cgcttcccgg ccctgctgca
gggagctcaa aatggaggac gcggcgctcg ggagagcggg 2820 cgggtgagtc
acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg 2880
actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta
2940 cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac
actgagtggg 3000 tggagactga agttaggcca gcttggcact tgatgtaatt
ctccttggaa tttgcccttt 3060 ttgagtttgg atcttggttc attctcaagc
ctcagacagt ggttcaaagt ttttttcttc 3120 catttcaggt gtcgtgacaa
gtttgtacaa aaaagcaggc tgccaccatg gagttcggcc 3180 tgagctgggt
gttcctggtg gccctgttca gaggcgtgca gtgccaggtg aagctgcagg 3240
agagcggccc cggcctggtg gcccccagcc agagcctgag catcacctgc accgtgagcg
3300 gcttcagcct gaccgcctac ggcgtggact gggtgagaca gccccccggc
aagtgcctgg 3360 agtggctggg cgtgatctgg ggcggcggca gaaccaacta
caacagcggc ctgatgagca 3420 gactgagcat cagaaaggac aacagcaaga
gccaggtgtt cctgaccatg aacagcctgc 3480 agaccgacga caccgccaag
tactactgcg tgaagcacac caactgggac ggcggcttcg 3540 cctactgggg
ccagggcacc accgtgaccg tgagcagcgg cggcggcggc agcggcggcg 3600
gcggcagcgg cggcggcggc agcggcagcc ccggccagag cgtgagcatc agctgcagcg
3660 gcagcagcag caacatcggc aacaactacg tgtactggta ccagcacctg
cccggcaccg 3720 cccccaagct gctgatctac agcgacacca agagacccag
cggcgtgccc gacagaatca 3780 gcggcagcaa gagcggcacc agcgccagcc
tggccatcag cggcctgcag agcgaggacg 3840 aggccgacta ctactgcgcc
agctgggacg acagcctgga cggccccgtg ttcggctgcg 3900 gcaccaagct
gaccgtgctg ctgaagaccc ccctgggcga caccacccac acctgcccca 3960
gatgccccga gcccaagagc tgcgacaccc cccccccctg ccccagatgc cccgagccca
4020 agagctgcga cacccccccc ccctgcccca gatgccccga gcccaagagc
tgcgacaccc 4080 cccccccctg ccccagatgc cccgcccccg agctgctggg
cggccccagc gtgttcctgt 4140
tcccccccaa gcccaaggac accctgatga tcagcagagc ccccgaggtg acctgcgtgg
4200 tggtggacgt gagccacgag gaccccgagg tgaagttcaa ctggtacgtg
gacggcgtgg 4260 aggtgcacaa cgccaagacc aagcccagag aggagcagta
caacagcacc tacagagtgg 4320 tgagcgtgct gaccgtgctg caccaggact
ggctgaacgg caaggagtac aagtgcaagg 4380 tgagcaacaa ggccctgccc
gcccccatcg agaagaccat cagcaaggcc aagggccagc 4440 ccagagagcc
ccaggtgtac accctgcccc ccagcagaga cgagctgacc aagaaccagg 4500
tgagcctgac ctgcctggtg aagggcttct accccagcga catcgccgtg gagtgggaga
4560 gcaacggcca gcccgagaac aactacaaga ccaccccccc cgtgctggac
agcgacggca 4620 gcttcttcct gtacagcaag ctgaccgtgg acaagagcag
atggcagcag ggcaacgtgt 4680 tcagctgcag cgtgatgcac gaggccctgc
acaaccacta cacccagaag agcctgagcc 4740 tgagccccga gctgcagctg
gaggagagct gcgccgaggc ccaggacggc gagctggacg 4800 gcctgtggac
caccatcacc atcttcatca ccctgttcct gctgagcgtg tgctacagcg 4860
ccaccgtgac cttcttcaag gtgaagtgga tcttcagcag cgtggtggac ctgaagcaga
4920 ccatcatccc cgactacaga aacatgatcg gccagggcgc ctaaacccag
ctttcttgta 4980 caaagtggtg ataatcgaat tctaaaccca gctttcttgt
acaaagtggt gataatcgaa 5040 ttccgataat caacctctgg attacaaaat
ttgtgaaaga ttgactggta ttcttaacta 5100 tgttgctcct tttacgctat
gtggatacgc tgctttaatg cctttgtatc atgctattgc 5160 ttcccgtatg
gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga 5220
ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
5280 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
tcgctttccc 5340 cctccctatt gccacggcgg aactcatcgc cgcctgcctt
gcccgctgct ggacaggggc 5400 tcggctgttg ggcactgaca attccgtggt
gttgtcgggg aagctgacgt cctttccatg 5460 gctgctcgcc tgtgttgcca
cctggattct gcgcgggacg tccttctgct acgtcccttc 5520 ggccctcaat
ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc 5580
gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg
5640 ggaattcccg cggttcgctt taagaccaat gacttacaag gcagctgtag
atcttagcca 5700 ctttttaaaa gaaaaggggg gactggaagg gctaattcac
tcccaacgaa gacaagatct 5760 gctttttgct tgtactgggt ctctctggtt
agaccagatc tgagcctggg agctctctgg 5820 ctaactaggg aacccactgc
ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt 5880 gtgtgcccgt
ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt 5940
gtggaaaatc tctagcagta gtagttcatg tcatcttatt attcagtatt tataacttgc
6000 aaagaaatga atatcagaga gtgagaggaa cttgtttatt gcagcttata
atggttacaa 6060 ataaagcaat agcatcacaa atttcacaaa taaagcattt
ttttcactgc attctagttg 6120 tggtttgtcc aaactcatca atgtatctta
tcatgtctgg ctctagctat cccgccccta 6180 actccgccca tcccgcccct
aactccgccc agttccgccc attctccgcc ccatggctga 6240 ctaatttttt
ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag 6300
tagtgaggag gcttttttgg aggcctaggg acgtacccaa ttcgccctat agtgagtcgt
6360 attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac
cctggcgtta 6420 cccaacttaa tcgccttgca gcacatcccc ctttcgccag
ctggcgtaat agcgaagagg 6480 cccgcaccga tcgcccttcc caacagttgc
gcagcctgaa tggcgaatgg gacgcgccct 6540 gtagcggcgc attaagcgcg
gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 6600 ccagcgccct
agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg 6660
gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
6720 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
ccatcgccct 6780 gatagacggt ttttcgccct ttgacgttgg agtccacgtt
ctttaatagt ggactcttgt 6840 tccaaactgg aacaacactc aaccctatct
cggtctattc ttttgattta taagggattt 6900 tgccgatttc ggcctattgg
ttaaaaaatg agctgattta acaaaaattt aacgcgaatt 6960 ttaacaaaat
attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac 7020
ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
7080 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
atttccgtgt 7140 cgcccttatt cccttttttg cggcattttg ccttcctgtt
tttgctcacc cagaaacgct 7200 ggtgaaagta aaagatgctg aagatcagtt
gggtgcacga gtgggttaca tcgaactgga 7260 tctcaacagc ggtaagatcc
ttgagagttt tcgccccgaa gaacgttttc caatgatgag 7320 cacttttaaa
gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca 7380
actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
7440 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
taaccatgag 7500 tgataacact gcggccaact tacttctgac aacgatcgga
ggaccgaagg agctaaccgc 7560 ttttttgcac aacatggggg atcatgtaac
tcgccttgat cgttgggaac cggagctgaa 7620 tgaagccata ccaaacgacg
agcgtgacac cacgatgcct gtagcaatgg caacaacgtt 7680 gcgcaaacta
ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg 7740
gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
7800 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
cagcactggg 7860 gccagatggt aagccctccc gtatcgtagt tatctacacg
acggggagtc aggcaactat 7920 ggatgaacga aatagacaga tcgctgagat
aggtgcctca ctgattaagc attggtaact 7980 gtcagaccaa gtttactcat
atatacttta gattgattta aaacttcatt tttaatttaa 8040 aaggatctag
gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt 8100
ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
8160 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
cggtggtttg 8220 tttgccggat caagagctac caactctttt tccgaaggta
actggcttca gcagagcgca 8280 gataccaaat actgttcttc tagtgtagcc
gtagttaggc caccacttca agaactctgt 8340 agcaccgcct acatacctcg
ctctgctaat cctgttacca gtggctgctg ccagtggcga 8400 taagtcgtgt
cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 8460
gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
8520 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaagaga
gaaaggcgga 8580 caggtatccg gtaagcggca gggtcggaac aggagagcgc
acgagggagc ttccaggggg 8640 aaacgcctgg tatctttata gtcctgtcgg
gtttcgccac ctctgacttg agcgtcgatt 8700 tttgtgatgc tcgtcagggg
ggcggagcct atggaaaaac gccagcaacg cggccttttt 8760 acggttcctg
gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga 8820
ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
8880 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
gcaaaccgcc 8940 tctccccgcg cgttggccga ttcattaatg cagctggcac
gacaggtttc ccgactggaa 9000 agcgggcagt gagcgcaacg caattaatgt
gagttagctc actcattagg caccccaggc 9060 tttacacttt atgcttccgg
ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca 9120 cacaggaaac
agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga 9180
acaaaagctg gagctgcaag ctt 9203 <210> SEQ ID NO 48 <211>
LENGTH: 10862 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <220> FEATURE: <223> OTHER INFORMATION:
Vector 2 <400> SEQUENCE: 48 aatgtagtct tatgcaatac tcttgtagtc
ttgcaacatg gtaacgatga gttagcaaca 60 tgccttacaa ggagagaaaa
agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120 tcgtgcctta
ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180
gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
240 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
tgcttaagcc 300 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
cgtctgttgt gtgactctgg 360 taactagaga tccctcagac ccttttagtc
agtgtggaaa atctctagca gtggcgcccg 420 aacagggact tgaaagcgaa
agggaaacca gaggagctct ctcgacgcag gactcggctt 480 gctgaagcgc
gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540
actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
600 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
atataaatta 660 aaacatatag tatgggcaag cagggagcta gaacgattcg
cagttaatcc tggcctgtta 720 gaaacatcag aaggctgtag acaaatactg
ggacagctac aaccatccct tcagacagga 780 tcagaagaac ttagatcatt
atataataca gtagcaaccc tctattgtgt gcatcaaagg 840 atagagataa
aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900
aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
960 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
taggagtagc 1020 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
agagcagtgg gaataggagc 1080 tttgttcctt gggttcttgg gagcagcagg
aagcactatg ggcgcagcgt caatgacgct 1140 gacggtacag gccagacaat
tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200 ggctattgag
gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260
ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
1320 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
ggagtaataa 1380 atctctggaa cagatttgga atcacacgac ctggatggag
tgggacagag aaattaacaa 1440 ttacacaagc ttaatacact ccttaattga
agaatcgcaa aaccagcaag aaaagaatga 1500 acaagaatta ttggaattag
ataaatgggc aagtttgtgg aattggttta acataacaaa 1560 ttggctgtgg
tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620
agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
1680 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
aagaagaagg 1740 tggagagaga gacagagaca gatccattcg attagtgaac
ggatctcgac ggtatcgcta 1800 gcttttaaaa gaaaaggggg gattgggggg
tacagtgcag gggaaagaat agtagacata 1860 atagcaacag acatacaaac
taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt 1920 actagtatca
actttgtata gaaaagttgg gctccggtgc ccgtcagtgg gcagagcgca 1980
catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
2040 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
ctttttcccg 2100 agggtggggg agaaccgtat ataagtgcag tagtcgccgt
gaacgttctt tttcgcaacg 2160 ggtttgccgc cagaacacag gtaagtgccg
tgtgtggttc ccgcgggcct ggcctcttta 2220 cgggttatgg cccttgcgtg
ccttgaatta cttccacctg gctgcagtac gtgattcttg 2280 atcccgagct
tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc 2340
cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct
2400 ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt
taaaattttt 2460 gatgacctgc tgcgacgctt tttttctggc aagatagtct
tgtaaatgcg ggccaagatc 2520 tgcacactgg tatttcggtt tttggggccg
cgggcggcga cggggcccgt gcgtcccagc 2580 gcacatgttc ggcgaggcgg
ggcctgcgag cgcggccacc gagaatcgga cgggggtagt 2640 ctcaagctgg
ccggcctgct ctggtgcctg gtctcgcgcc gccgtgtatc gccccgccct 2700
gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg
2760 gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg
gcgggtgagt 2820 cacccacaca aaggaaaagg gcctttccgt cctcagccgt
cgcttcatgt gactccacgg 2880 agtaccgggc gccgtccagg cacctcgatt
agttctcgag cttttggagt acgtcgtctt 2940 taggttgggg ggaggggttt
tatgcgatgg agtttcccca cactgagtgg gtggagactg 3000 aagttaggcc
agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg 3060
gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg
3120 tgtcgtgaca agtttgtaca aaaaagcagg ctgccaccat ggagttcggc
ctgagctggg 3180 tgttcctggt ggccctgttc agaggcgtgc agtgccaggt
gaagctgcag gagagcggcc 3240 ccggcctggt ggcccccagc cagagcctga
gcatcacctg caccgtgagc ggcttcagcc 3300 tgaccgccta cggcgtggac
tgggtgagac agccccccgg caagggcctg gagtggctgg 3360 gcgtgatctg
gggcggcggc agaaccaact acaacagcgg cctgatgagc agactgagca 3420
tcagaaagga caacagcaag agccaggtgt tcctgaccat gaacagcctg cagaccgacg
3480 acaccgccaa gtactactgc gtgaagcaca ccaactggga cggcggcttc
gcctactggg 3540 gccagggcac caccgtgacc gtgagcagcc ccagcgtgtt
ccccctggcc cccagcagca 3600 agagcaccag cggcggcacc gccgccctgg
gctgcctggt gaaggactac ttccccgagc 3660 ccgtgaccgt gagctggaac
agcggcgccc tgaccagcgg cgtgcacacc ttccccgccg 3720 tgctgcagag
cagcggcctg tacagcctga gcagcgtggt gaccgtgccc agcagcagcc 3780
tgggcaccca gacctacatc tgcaacgtga accacaagcc cagcaacacc aaggtggaca
3840 agaaggtgga gctgaagacc cccctgggcg acaccaccca cacctgcccc
agatgccccg 3900 agcccaagag ctgcgacacc ccccccccct gccccagatg
ccccgagccc aagagctgcg 3960 acaccccccc cccctgcccc agatgccccg
agcccaagag ctgcgacacc ccccccccct 4020 gccccagatg ccccgccccc
gagctgctgg gcggccccag cgtgttcctg ttccccccca 4080 agcccaagga
caccctgatg atcagcagag cccccgaggt gacctgcgtg gtggtggacg 4140
tgagccacga ggaccccgag gtgaagttca actggtacgt ggacggcgtg gaggtgcaca
4200 acgccaagac caagcccaga gaggagcagt acaacagcac ctacagagtg
gtgagcgtgc 4260 tgaccgtgct gcaccaggac tggctgaacg gcaaggagta
caagtgcaag gtgagcaaca 4320 aggccctgcc cgcccccatc gagaagacca
tcagcaaggc caagggccag cccagagagc 4380 cccaggtgta caccctgccc
cccagcagag acgagctgac caagaaccag gtgagcctga 4440 cctgcctggt
gaagggcttc taccccagcg acatcgccgt ggagtgggag agcaacggcc 4500
agcccgagaa caactacaag accacccccc ccgtgctgga cagcgacggc agcttcttcc
4560 tgtacagcaa gctgaccgtg gacaagagca gatggcagca gggcaacgtg
ttcagctgca 4620 gcgtgatgca cgaggccctg cacaaccact acacccagaa
gagcctgagc ctgagccccg 4680 agctgcagct ggaggagagc tgcgccgagg
cccaggacgg cgagctggac ggcctgtgga 4740 ccaccatcac catcttcatc
accctgttcc tgctgagcgt gtgctacagc gccaccgtga 4800 ccttcttcaa
ggtgaagtgg atcttcagca gcgtggtgga cctgaagcag accatcatcc 4860
ccgactacag aaacatgatc ggccagggcg cctaaaacaa caacaattgc attcatttta
4920 tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
ctctacaaat 4980 gtggtacgcg ttaacaacaa caattgcatt cattttatgt
ttcaggttca gggggaggtg 5040 tgggaggttt tttaaagcaa gtaaaacctc
tacaaatgtg gtacgcgtta cccagctttc 5100 ttgtacaaag tggtaaatag
atagaacaac aacaattgca ttcatttttg atttcaggtt 5160 cagggggagg
tgtgggaggt tttttaaagc aagtaaaacc tctacactga cggtacgcgt 5220
taacaacaac aattgcattc atttgtagtt tcaggttcag ggggaggtgt gggaggtttt
5280 ttaaagcaag ttaaacctct aaaatagtgg tacgcgttac ccagctttct
tgtacaaagt 5340 ggacccagct ttcttgtaca aagtgggccc ctctccctcc
ccccccccta acgttactgg 5400 ccgaagccgc ttggaataag gccggtgtgc
gtttgtctat atgttatttt ccaccatatt 5460 gccgtctttt ggcaatgtga
gggcccggaa acctggccct gtcttcttga cgagcattcc 5520 taggggtctt
tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc 5580
agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg
5640 gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat
aagatacacc 5700 tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg
atagttgtgg aaagagtcaa 5760 atggctctcc tcaagcgtat tcaacaaggg
gctgaaggat gcccagaagg taccccattg 5820 tatgggatct gatctggggc
ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa 5880 aaaacgtcta
ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata 5940
atatggccac aaccatggcc accgacatga gagtgcccgc ccagctgctg ggcctgctgc
6000 tgctgtggct gagcggcgcc agatgcggca gccccggcca gagcgtgagc
atcagctgca 6060 gcggcagcag cagcaacatc ggcaacaact acgtgtactg
gtaccagcac ctgcccggca 6120 ccgcccccaa gctgctgatc tacagcgaca
ccaagagacc cagcggcgtg cccgacagaa 6180 tcagcggcag caagagcggc
accagcgcca gcctggccat cagcggcctg cagagcgagg 6240 acgaggccga
ctactactgc gccagctggg acgacagcct ggacggcccc gtgttcggcg 6300
gcggcaccaa gctgaccgtg ctgggccagc ccaaggccaa ccccaccgtg accctgttcc
6360 cccccagcag cgaggagctg caggccaaca aggccaccct ggtgtgcctg
atcagcgact 6420 tctaccccgg cgccgtgacc gtggcctgga aggccgacgg
cagccccgtg aaggccggcg 6480 tggagaccac caagcccagc aagcagagca
acaacaagta cgccgccagc agctacctga 6540 gcctgacccc cgagcagtgg
aagagccaca gaagctacag ctgccaggtg acccacgagg 6600 gcagcaccgt
ggagaagacc gtggccccca ccgagtgcag ctaacaactt tattatacat 6660
agttgatcaa ttccaacttt attatacata gttgatcaat tccgataatc aacctctgga
6720 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
ttacgctatg 6780 tggatacgct gctttaatgc ctttgtatca tgctattgct
tcccgtatgg ctttcatttt 6840 ctcctccttg tataaatcct ggttgctgtc
tctttatgag gagttgtggc ccgttgtcag 6900 gcaacgtggc gtggtgtgca
ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc 6960 caccacctgt
cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga 7020
actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
7080 ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct
gtgttgccac 7140 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
gccctcaatc cagcggacct 7200 tccttcccgc ggcctgctgc cggctctgcg
gcctcttccg cgtcttcgcc ttcgccctca 7260 gacgagtcgg atctcccttt
gggccgcctc cccgcatcgg gaattcccgc ggttcgcttt 7320 aagaccaatg
acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 7380
actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt gtactgggtc
7440 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
acccactgct 7500 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
tgtgcccgtc tgttgtgtga 7560 ctctggtaac tagagatccc tcagaccctt
ttagtcagtg tggaaaatct ctagcagtag 7620 tagttcatgt catcttatta
ttcagtattt ataacttgca aagaaatgaa tatcagagag 7680 tgagaggaac
ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 7740
tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
7800 tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat
cccgccccta 7860 actccgccca gttccgccca ttctccgccc catggctgac
taattttttt tatttatgca 7920 gaggccgagg ccgcctcggc ctctgagcta
ttccagaagt agtgaggagg cttttttgga 7980 ggcctaggga cgtacccaat
tcgccctata gtgagtcgta ttacgcgcgc tcactggccg 8040 tcgttttaca
acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 8100
cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
8160 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
ttaagcgcgg 8220 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
cagcgcccta gcgcccgctc 8280 ctttcgcttt cttcccttcc tttctcgcca
cgttcgccgg ctttccccgt caagctctaa 8340 atcgggggct ccctttaggg
ttccgattta gtgctttacg gcacctcgac cccaaaaaac 8400 ttgattaggg
tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 8460
tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
8520 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
gcctattggt 8580 taaaaaatga gctgatttaa caaaaattta acgcgaattt
taacaaaata ttaacgctta 8640 caatttaggt ggcacttttc ggggaaatgt
gcgcggaacc cctatttgtt tatttttcta 8700 aatacattca aatatgtatc
cgctcatgag acaataaccc tgataaatgc ttcaataata 8760 ttgaaaaagg
aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 8820
ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
8880 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
gtaagatcct 8940 tgagagtttt cgccccgaag aacgttttcc aatgatgagc
acttttaaag ttctgctatg 9000 tggcgcggta ttatcccgta ttgacgccgg
gcaagagcaa ctcggtcgcc gcatacacta 9060 ttctcagaat gacttggttg
agtactcacc agtcacagaa aagcatctta cggatggcat 9120 gacagtaaga
gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 9180
acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
9240 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
caaacgacga 9300 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
cgcaaactat taactggcga 9360 actacttact ctagcttccc ggcaacaatt
aatagactgg atggaggcgg ataaagttgc 9420 aggaccactt ctgcgctcgg
cccttccggc tggctggttt attgctgata aatctggagc 9480 cggtgagcgt
gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 9540
tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
9600 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
tttactcata 9660 tatactttag attgatttaa aacttcattt ttaatttaaa
aggatctagg tgaagatcct 9720 ttttgataat ctcatgacca aaatccctta
acgtgagttt tcgttccact gagcgtcaga 9780 ccccgtagaa aagatcaaag
gatcttcttg agatcctttt tttctgcgcg taatctgctg 9840 cttgcaaaca
aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 9900
aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
9960 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
catacctcgc 10020 tctgctaatc ctgttaccag tggctgctgc cagtggcgat
aagtcgtgtc ttaccgggtt 10080 ggactcaaga cgatagttac cggataaggc
gcagcggtcg ggctgaacgg ggggttcgtg 10140 cacacagccc agcttggagc
gaacgaccta caccgaactg agatacctac agcgtgagct 10200 atgagaaagc
gccacgcttc ccgaagagag aaaggcggac aggtatccgg taagcggcag 10260
ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
10320 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
cgtcaggggg 10380 gcggagccta tggaaaaacg ccagcaacgc ggccttttta
cggttcctgg ccttttgctg 10440 gccttttgct cacatgttct ttcctgcgtt
atcccctgat tctgtggata accgtattac 10500 cgcctttgag tgagctgata
ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 10560 gagcgaggaa
gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 10620
tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
10680 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
tgcttccggc 10740 tcgtatgttg tgtggaattg tgagcggata acaatttcac
acaggaaaca gctatgacca 10800 tgattacgcc aagcgcgcaa ttaaccctca
ctaaagggaa caaaagctgg agctgcaagc 10860 tt 10862 <210> SEQ ID
NO 49 <211> LENGTH: 9581 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <220> FEATURE: <223> OTHER INFORMATION:
Vector 3 <400> SEQUENCE: 49 aatgtagtct tatgcaatac tcttgtagtc
ttgcaacatg gtaacgatga gttagcaaca 60 tgccttacaa ggagagaaaa
agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120 tcgtgcctta
ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180
gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
240 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
tgcttaagcc 300 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
cgtctgttgt gtgactctgg 360 taactagaga tccctcagac ccttttagtc
agtgtggaaa atctctagca gtggcgcccg 420 aacagggact tgaaagcgaa
agggaaacca gaggagctct ctcgacgcag gactcggctt 480 gctgaagcgc
gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540
actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
600 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
atataaatta 660 aaacatatag tatgggcaag cagggagcta gaacgattcg
cagttaatcc tggcctgtta 720 gaaacatcag aaggctgtag acaaatactg
ggacagctac aaccatccct tcagacagga 780 tcagaagaac ttagatcatt
atataataca gtagcaaccc tctattgtgt gcatcaaagg 840 atagagataa
aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900
aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
960 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
taggagtagc 1020 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
agagcagtgg gaataggagc 1080 tttgttcctt gggttcttgg gagcagcagg
aagcactatg ggcgcagcgt caatgacgct 1140 gacggtacag gccagacaat
tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200 ggctattgag
gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260
ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
1320 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
ggagtaataa 1380 atctctggaa cagatttgga atcacacgac ctggatggag
tgggacagag aaattaacaa 1440 ttacacaagc ttaatacact ccttaattga
agaatcgcaa aaccagcaag aaaagaatga 1500 acaagaatta ttggaattag
ataaatgggc aagtttgtgg aattggttta acataacaaa 1560 ttggctgtgg
tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620
agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
1680 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
aagaagaagg 1740 tggagagaga gacagagaca gatccattcg attagtgaac
ggatctcgac ggtatcgcta 1800 gcttttaaaa gaaaaggggg gattgggggg
tacagtgcag gggaaagaat agtagacata 1860 atagcaacag acatacaaac
taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt 1920 actagtatca
actttgtata gaaaagttgg gctccggtgc ccgtcagtgg gcagagcgca 1980
catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
2040 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
ctttttcccg 2100 agggtggggg agaaccgtat ataagtgcag tagtcgccgt
gaacgttctt tttcgcaacg 2160 ggtttgccgc cagaacacag gtaagtgccg
tgtgtggttc ccgcgggcct ggcctcttta 2220 cgggttatgg cccttgcgtg
ccttgaatta cttccacctg gctgcagtac gtgattcttg 2280 atcccgagct
tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc 2340
cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct
2400 ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt
taaaattttt 2460 gatgacctgc tgcgacgctt tttttctggc aagatagtct
tgtaaatgcg ggccaagatc 2520 tgcacactgg tatttcggtt tttggggccg
cgggcggcga cggggcccgt gcgtcccagc 2580 gcacatgttc ggcgaggcgg
ggcctgcgag cgcggccacc gagaatcgga cgggggtagt 2640 ctcaagctgg
ccggcctgct ctggtgcctg gtctcgcgcc gccgtgtatc gccccgccct 2700
gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg
2760 gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg
gcgggtgagt 2820 cacccacaca aaggaaaagg gcctttccgt cctcagccgt
cgcttcatgt gactccacgg 2880 agtaccgggc gccgtccagg cacctcgatt
agttctcgag cttttggagt acgtcgtctt 2940 taggttgggg ggaggggttt
tatgcgatgg agtttcccca cactgagtgg gtggagactg 3000 aagttaggcc
agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg 3060
gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg
3120 tgtcgtgaca agtttgtaca aaaaagcagg ctgccaccat gtggctgcag
agcctgctgc 3180 tgctgggcac cgtggcctgc agcatcagcg cccccgccag
aagccccagc cccagcaccc 3240 agccctggga gcacgtgaac gccatccagg
aggccagaag actgctgaac ctgagcagag 3300 acaccgccgc cgagatgaac
gagaccgtgg aggtgatcag cgagatgttc gacctgcagg 3360 agcccacctg
cctgcagacc agactggagc tgtacaagca gggcctgaga ggcagcctga 3420
ccaagctgaa gggccccctg accatgatgg ccagccacta caagcagcac tgccccccca
3480 cccccgagac cagctgcgcc acccagatca tcaccttcga gagcttcaag
gagaacctga 3540 aggacttcct gctggtgatc cccttcgact gctgggagcc
cgtgcaggag taaaacaaca 3600 acaattgcat tcattttatg tttcaggttc
agggggaggt gtgggaggtt ttttaaagca 3660 agtaaaacct ctacaaatgt
ggtacgcgtt aacaacaaca attgcattca ttttatgttt 3720 caggttcagg
gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt 3780
acgcgttacc cagctttctt gtacaaagtg gtaaatagat agaacaacaa caattgcatt
3840 catttttgat ttcaggttca gggggaggtg tgggaggttt tttaaagcaa
gtaaaacctc 3900 tacactgacg gtacgcgtta acaacaacaa ttgcattcat
ttgtagtttc aggttcaggg 3960 ggaggtgtgg gaggtttttt aaagcaagtt
aaacctctaa aatagtggta cgcgttaccc 4020 agctttcttg tacaaagtgg
acccagcttt cttgtacaaa gtgggcccct ctccctcccc 4080 cccccctaac
gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat 4140
gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt
4200 cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc
aaggtctgtt 4260 gaatgtcgtg aaggaagcag ttcctctgga agcttcttga
agacaaacaa cgtctgtagc 4320 gaccctttgc aggcagcgga accccccacc
tggcgacagg tgcctctgcg gccaaaagcc 4380 acgtgtataa gatacacctg
caaaggcggc acaaccccag tgccacgttg tgagttggat 4440 agttgtggaa
agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc 4500
ccagaaggta ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg
4560 tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg
tggttttcct 4620 ttgaaaaaca cgatgataat atggccacaa ccatggccac
cgtgctggcc cccgcctgga 4680 gccccaccac ctacctgctg ctgctgctgc
tgctgagcag cggcctgagc ggcacccagg 4740 actgcagctt ccagcacagc
cccatcagca gcgacttcgc cgtgaagatc agagagctga 4800 gcgactacct
gctgcaggac taccccgtga ccgtggccag caacctgcag gacgaggagc 4860
tgtgcggcgg cctgtggaga ctggtgctgg cccagagatg gatggagaga ctgaagaccg
4920 tggccggcag caagatgcag ggcctgctgg agagagtgaa caccgagatc
cacttcgtga 4980 ccaagtgcgc cttccagccc ccccccagct gcctgagatt
cgtgcagacc aacatcagca 5040 gactgctgca ggagaccagc gagcagctgg
tggccctgaa gccctggatc accagacaga 5100 acttcagcag atgcctggag
ctgcagtgcc agcccgacag cagcaccctg ccccccccct 5160 ggagccccag
acccctggag gccaccgccc ccaccgcccc ccagcccccc ctgctgctgc 5220
tgctgctgct gcccgtgggc ctgctgctgc tggccgccgc ctggtgcctg cactggcaga
5280 gaaccagaag aagaaccccc agacccggcg agcaggtgcc ccccgtgccc
agcccccagg 5340 acctgctgct ggtggagcac taacaacttt attatacata
gttgatcaat tccaacttta 5400 ttatacatag ttgatcaatt ccgataatca
acctctggat tacaaaattt gtgaaagatt 5460 gactggtatt cttaactatg
ttgctccttt tacgctatgt ggatacgctg ctttaatgcc 5520 tttgtatcat
gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg 5580
gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
5640 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
agctcctttc 5700
cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
5760 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
tgtcggggaa 5820 gctgacgtcc tttccatggc tgctcgcctg tgttgccacc
tggattctgc gcgggacgtc 5880 cttctgctac gtcccttcgg ccctcaatcc
agcggacctt ccttcccgcg gcctgctgcc 5940 ggctctgcgg cctcttccgc
gtcttcgcct tcgccctcag acgagtcgga tctccctttg 6000 ggccgcctcc
ccgcatcggg aattcccgcg gttcgcttta agaccaatga cttacaaggc 6060
agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
6120 ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
accagatctg 6180 agcctgggag ctctctggct aactagggaa cccactgctt
aagcctcaat aaagcttgcc 6240 ttgagtgctt caagtagtgt gtgcccgtct
gttgtgtgac tctggtaact agagatccct 6300 cagacccttt tagtcagtgt
ggaaaatctc tagcagtagt agttcatgtc atcttattat 6360 tcagtattta
taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc 6420
agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
6480 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
atgtctggct 6540 ctagctatcc cgcccctaac tccgcccatc ccgcccctaa
ctccgcccag ttccgcccat 6600 tctccgcccc atggctgact aatttttttt
atttatgcag aggccgaggc cgcctcggcc 6660 tctgagctat tccagaagta
gtgaggaggc ttttttggag gcctagggac gtacccaatt 6720 cgccctatag
tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact 6780
gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
6840 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
agcctgaatg 6900 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
gggtgtggtg gttacgcgca 6960 gcgtgaccgc tacacttgcc agcgccctag
cgcccgctcc tttcgctttc ttcccttcct 7020 ttctcgccac gttcgccggc
tttccccgtc aagctctaaa tcgggggctc cctttagggt 7080 tccgatttag
tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac 7140
gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
7200 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
gtctattctt 7260 ttgatttata agggattttg ccgatttcgg cctattggtt
aaaaaatgag ctgatttaac 7320 aaaaatttaa cgcgaatttt aacaaaatat
taacgcttac aatttaggtg gcacttttcg 7380 gggaaatgtg cgcggaaccc
ctatttgttt atttttctaa atacattcaa atatgtatcc 7440 gctcatgaga
caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 7500
tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
7560 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
gtgcacgagt 7620 gggttacatc gaactggatc tcaacagcgg taagatcctt
gagagttttc gccccgaaga 7680 acgttttcca atgatgagca cttttaaagt
tctgctatgt ggcgcggtat tatcccgtat 7740 tgacgccggg caagagcaac
tcggtcgccg catacactat tctcagaatg acttggttga 7800 gtactcacca
gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 7860
tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
7920 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
gccttgatcg 7980 ttgggaaccg gagctgaatg aagccatacc aaacgacgag
cgtgacacca cgatgcctgt 8040 agcaatggca acaacgttgc gcaaactatt
aactggcgaa ctacttactc tagcttcccg 8100 gcaacaatta atagactgga
tggaggcgga taaagttgca ggaccacttc tgcgctcggc 8160 ccttccggct
ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 8220
tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
8280 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
gtgcctcact 8340 gattaagcat tggtaactgt cagaccaagt ttactcatat
atactttaga ttgatttaaa 8400 acttcatttt taatttaaaa ggatctaggt
gaagatcctt tttgataatc tcatgaccaa 8460 aatcccttaa cgtgagtttt
cgttccactg agcgtcagac cccgtagaaa agatcaaagg 8520 atcttcttga
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 8580
gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
8640 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
agttaggcca 8700 ccacttcaag aactctgtag caccgcctac atacctcgct
ctgctaatcc tgttaccagt 8760 ggctgctgcc agtggcgata agtcgtgtct
taccgggttg gactcaagac gatagttacc 8820 ggataaggcg cagcggtcgg
gctgaacggg gggttcgtgc acacagccca gcttggagcg 8880 aacgacctac
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 8940
cgaagagaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
9000 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
ttcgccacct 9060 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
cggagcctat ggaaaaacgc 9120 cagcaacgcg gcctttttac ggttcctggc
cttttgctgg ccttttgctc acatgttctt 9180 tcctgcgtta tcccctgatt
ctgtggataa ccgtattacc gcctttgagt gagctgatac 9240 cgctcgccgc
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 9300
cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
9360 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
gttagctcac 9420 tcattaggca ccccaggctt tacactttat gcttccggct
cgtatgttgt gtggaattgt 9480 gagcggataa caatttcaca caggaaacag
ctatgaccat gattacgcca agcgcgcaat 9540 taaccctcac taaagggaac
aaaagctgga gctgcaagct t 9581 <210> SEQ ID NO 50 <211>
LENGTH: 9746 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polynucleotide
<220> FEATURE: <223> OTHER INFORMATION: Vector 4
<400> SEQUENCE: 50 aatgtagtct tatgcaatac tcttgtagtc
ttgcaacatg gtaacgatga gttagcaaca 60 tgccttacaa ggagagaaaa
agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120 tcgtgcctta
ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180
gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
240 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
tgcttaagcc 300 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
cgtctgttgt gtgactctgg 360 taactagaga tccctcagac ccttttagtc
agtgtggaaa atctctagca gtggcgcccg 420 aacagggact tgaaagcgaa
agggaaacca gaggagctct ctcgacgcag gactcggctt 480 gctgaagcgc
gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540
actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
600 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
atataaatta 660 aaacatatag tatgggcaag cagggagcta gaacgattcg
cagttaatcc tggcctgtta 720 gaaacatcag aaggctgtag acaaatactg
ggacagctac aaccatccct tcagacagga 780 tcagaagaac ttagatcatt
atataataca gtagcaaccc tctattgtgt gcatcaaagg 840 atagagataa
aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900
aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
960 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
taggagtagc 1020 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
agagcagtgg gaataggagc 1080 tttgttcctt gggttcttgg gagcagcagg
aagcactatg ggcgcagcgt caatgacgct 1140 gacggtacag gccagacaat
tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200 ggctattgag
gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260
ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
1320 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
ggagtaataa 1380 atctctggaa cagatttgga atcacacgac ctggatggag
tgggacagag aaattaacaa 1440 ttacacaagc ttaatacact ccttaattga
agaatcgcaa aaccagcaag aaaagaatga 1500 acaagaatta ttggaattag
ataaatgggc aagtttgtgg aattggttta acataacaaa 1560 ttggctgtgg
tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620
agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
1680 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
aagaagaagg 1740 tggagagaga gacagagaca gatccattcg attagtgaac
ggatctcgac ggtatcgcta 1800 gcttttaaaa gaaaaggggg gattgggggg
tacagtgcag gggaaagaat agtagacata 1860 atagcaacag acatacaaac
taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt 1920 actagtatca
actttgtata gaaaagttgg gctccggtgc ccgtcagtgg gcagagcgca 1980
catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
2040 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
ctttttcccg 2100 agggtggggg agaaccgtat ataagtgcag tagtcgccgt
gaacgttctt tttcgcaacg 2160 ggtttgccgc cagaacacag gtaagtgccg
tgtgtggttc ccgcgggcct ggcctcttta 2220 cgggttatgg cccttgcgtg
ccttgaatta cttccacctg gctgcagtac gtgattcttg 2280 atcccgagct
tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc 2340
cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct
2400 ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt
taaaattttt 2460 gatgacctgc tgcgacgctt tttttctggc aagatagtct
tgtaaatgcg ggccaagatc 2520 tgcacactgg tatttcggtt tttggggccg
cgggcggcga cggggcccgt gcgtcccagc 2580 gcacatgttc ggcgaggcgg
ggcctgcgag cgcggccacc gagaatcgga cgggggtagt 2640 ctcaagctgg
ccggcctgct ctggtgcctg gtctcgcgcc gccgtgtatc gccccgccct 2700
gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg
2760 gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg
gcgggtgagt 2820 cacccacaca aaggaaaagg gcctttccgt cctcagccgt
cgcttcatgt gactccacgg 2880 agtaccgggc gccgtccagg cacctcgatt
agttctcgag cttttggagt acgtcgtctt 2940 taggttgggg ggaggggttt
tatgcgatgg agtttcccca cactgagtgg gtggagactg 3000 aagttaggcc
agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg 3060
gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg
3120 tgtcgtgaca agtttgtaca aaaaagcagg ctgccaccat gaccgtgctg
gcccccgcct 3180 ggagccccac cacctacctg ctgctgctgc tgctgctgag
cagcggcctg agcggcaccc 3240
aggactgcag cttccagcac agccccatca gcagcgactt cgccgtgaag atcagagagc
3300 tgagcgacta cctgctgcag gactaccccg tgaccgtggc cagcaacctg
caggacgagg 3360 agctgtgcgg cggcctgtgg agactggtgc tggcccagag
atggatggag agactgaaga 3420 ccgtggccgg cagcaagatg cagggcctgc
tggagagagt gaacaccgag atccacttcg 3480 tgaccaagtg cgccttccag
ccccccccca gctgcctgag attcgtgcag accaacatca 3540 gcagactgct
gcaggagacc agcgagcagc tggtggccct gaagccctgg atcaccagac 3600
agaacttcag cagatgcctg gagctgcagt gccagcccga cagcagcacc ctgccccccc
3660 cctggagccc cagacccctg gaggccaccg cccccaccgc cccccagtaa
aacaacaaca 3720 attgcattca ttttatgttt caggttcagg gggaggtgtg
ggaggttttt taaagcaagt 3780 aaaacctcta caaatgtggt acgcgttaac
aacaacaatt gcattcattt tatgtttcag 3840 gttcaggggg aggtgtggga
ggttttttaa agcaagtaaa acctctacaa atgtggtacg 3900 cgttacccag
ctttcttgta caaagtggta aatagataga acaacaacaa ttgcattcat 3960
ttttgatttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac
4020 actgacggta cgcgttaaca acaacaattg cattcatttg tagtttcagg
ttcaggggga 4080 ggtgtgggag gttttttaaa gcaagttaaa cctctaaaat
agtggtacgc gttacccagc 4140 tttcttgtac aaagtggacc cagctttctt
gtacaaagtg ggcccctctc cctccccccc 4200 ccctaacgtt actggccgaa
gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt 4260 attttccacc
atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt 4320
cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa
4380 tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt
ctgtagcgac 4440 cctttgcagg cagcggaacc ccccacctgg cgacaggtgc
ctctgcggcc aaaagccacg 4500 tgtataagat acacctgcaa aggcggcaca
accccagtgc cacgttgtga gttggatagt 4560 tgtggaaaga gtcaaatggc
tctcctcaag cgtattcaac aaggggctga aggatgccca 4620 gaaggtaccc
cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt 4680
ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg
4740 aaaaacacga tgataatatg gccacaacca tggccaccgt gctggccccc
gcctggagcc 4800 ccaccaccta cctgctgctg ctgctgctgc tgagcagcgg
cctgagcgcc cccgccagaa 4860 gccccagccc cagcacccag ccctgggagc
acgtgaacgc catccaggag gccagaagac 4920 tgctgaacct gagcagagac
accgccgccg agatgaacga gaccgtggag gtgatcagcg 4980 agatgttcga
cctgcaggag cccacctgcc tgcagaccag actggagctg tacaagcagg 5040
gcctgagagg cagcctgacc aagctgaagg gccccctgac catgatggcc agccactaca
5100 agcagcactg cccccccacc cccgagacca gctgcgccac ccagatcatc
accttcgaga 5160 gcttcaagga gaacctgaag gacttcctgc tggtgatccc
cttcgactgc tgggagcccg 5220 tgcaggagcc caccaccacc cccgccccca
gaccccccac ccccgccccc accatcgcca 5280 gccagcccct gagcctgaga
cccgaggcct gcagacccgc cgccggcggc gccgtgcaca 5340 ccagaggcct
ggacttcgcc tgcgacatct acatctgggc ccccctggcc ggcacctgcg 5400
gcgtgctgct gctgagcctg gtgatcaccc tgtactgcaa ccacagaaac agaagaagag
5460 tgtgcaagtg ccccagaccc gtggtgaaga gcggcgacaa gcccagcctg
agcgccagat 5520 acgtgtaaca actttattat acatagttga tcaattccaa
ctttattata catagttgat 5580 caattccgat aatcaacctc tggattacaa
aatttgtgaa agattgactg gtattcttaa 5640 ctatgttgct ccttttacgc
tatgtggata cgctgcttta atgcctttgt atcatgctat 5700 tgcttcccgt
atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta 5760
tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
5820 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
ctttcgcttt 5880 ccccctccct attgccacgg cggaactcat cgccgcctgc
cttgcccgct gctggacagg 5940 ggctcggctg ttgggcactg acaattccgt
ggtgttgtcg gggaagctga cgtcctttcc 6000 atggctgctc gcctgtgttg
ccacctggat tctgcgcggg acgtccttct gctacgtccc 6060 ttcggccctc
aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct 6120
tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
6180 tcgggaattc ccgcggttcg ctttaagacc aatgacttac aaggcagctg
tagatcttag 6240 ccacttttta aaagaaaagg ggggactgga agggctaatt
cactcccaac gaagacaaga 6300 tctgcttttt gcttgtactg ggtctctctg
gttagaccag atctgagcct gggagctctc 6360 tggctaacta gggaacccac
tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt 6420 agtgtgtgcc
cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc 6480
agtgtggaaa atctctagca gtagtagttc atgtcatctt attattcagt atttataact
6540 tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt
ataatggtta 6600 caaataaagc aatagcatca caaatttcac aaataaagca
tttttttcac tgcattctag 6660 ttgtggtttg tccaaactca tcaatgtatc
ttatcatgtc tggctctagc tatcccgccc 6720 ctaactccgc ccatcccgcc
cctaactccg cccagttccg cccattctcc gccccatggc 6780 tgactaattt
tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag 6840
aagtagtgag gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt
6900 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
aaccctggcg 6960 ttacccaact taatcgcctt gcagcacatc cccctttcgc
cagctggcgt aatagcgaag 7020 aggcccgcac cgatcgccct tcccaacagt
tgcgcagcct gaatggcgaa tgggacgcgc 7080 cctgtagcgg cgcattaagc
gcggcgggtg tggtggttac gcgcagcgtg accgctacac 7140 ttgccagcgc
cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 7200
ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
7260 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
gggccatcgc 7320 cctgatagac ggtttttcgc cctttgacgt tggagtccac
gttctttaat agtggactct 7380 tgttccaaac tggaacaaca ctcaacccta
tctcggtcta ttcttttgat ttataaggga 7440 ttttgccgat ttcggcctat
tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 7500 attttaacaa
aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg 7560
aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
7620 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
aacatttccg 7680 tgtcgccctt attccctttt ttgcggcatt ttgccttcct
gtttttgctc acccagaaac 7740 gctggtgaaa gtaaaagatg ctgaagatca
gttgggtgca cgagtgggtt acatcgaact 7800 ggatctcaac agcggtaaga
tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 7860 gagcactttt
aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 7920
gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
7980 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
ccataaccat 8040 gagtgataac actgcggcca acttacttct gacaacgatc
ggaggaccga aggagctaac 8100 cgcttttttg cacaacatgg gggatcatgt
aactcgcctt gatcgttggg aaccggagct 8160 gaatgaagcc ataccaaacg
acgagcgtga caccacgatg cctgtagcaa tggcaacaac 8220 gttgcgcaaa
ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 8280
ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
8340 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
ttgcagcact 8400 ggggccagat ggtaagccct cccgtatcgt agttatctac
acgacgggga gtcaggcaac 8460 tatggatgaa cgaaatagac agatcgctga
gataggtgcc tcactgatta agcattggta 8520 actgtcagac caagtttact
catatatact ttagattgat ttaaaacttc atttttaatt 8580 taaaaggatc
taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 8640
gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
8700 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
cagcggtggt 8760 ttgtttgccg gatcaagagc taccaactct ttttccgaag
gtaactggct tcagcagagc 8820 gcagatacca aatactgttc ttctagtgta
gccgtagtta ggccaccact tcaagaactc 8880 tgtagcaccg cctacatacc
tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 8940 cgataagtcg
tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 9000
gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
9060 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
agagaaaggc 9120 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
cgcacgaggg agcttccagg 9180 gggaaacgcc tggtatcttt atagtcctgt
cgggtttcgc cacctctgac ttgagcgtcg 9240 atttttgtga tgctcgtcag
gggggcggag cctatggaaa aacgccagca acgcggcctt 9300 tttacggttc
ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 9360
tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
9420 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
tacgcaaacc 9480 gcctctcccc gcgcgttggc cgattcatta atgcagctgg
cacgacaggt ttcccgactg 9540 gaaagcgggc agtgagcgca acgcaattaa
tgtgagttag ctcactcatt aggcacccca 9600 ggctttacac tttatgcttc
cggctcgtat gttgtgtgga attgtgagcg gataacaatt 9660 tcacacagga
aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag 9720
ggaacaaaag ctggagctgc aagctt 9746 <210> SEQ ID NO 51
<211> LENGTH: 8189 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <220> FEATURE: <223> OTHER INFORMATION:
Vector 5 <400> SEQUENCE: 51 aatgtagtct tatgcaatac tcttgtagtc
ttgcaacatg gtaacgatga gttagcaaca 60 tgccttacaa ggagagaaaa
agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120 tcgtgcctta
ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180
gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
240 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
tgcttaagcc 300 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
cgtctgttgt gtgactctgg 360 taactagaga tccctcagac ccttttagtc
agtgtggaaa atctctagca gtggcgcccg 420 aacagggact tgaaagcgaa
agggaaacca gaggagctct ctcgacgcag gactcggctt 480 gctgaagcgc
gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540
actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
600 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
atataaatta 660 aaacatatag tatgggcaag cagggagcta gaacgattcg
cagttaatcc tggcctgtta 720 gaaacatcag aaggctgtag acaaatactg
ggacagctac aaccatccct tcagacagga 780 tcagaagaac ttagatcatt
atataataca gtagcaaccc tctattgtgt gcatcaaagg 840 atagagataa
aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900
aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
960 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
taggagtagc 1020 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
agagcagtgg gaataggagc 1080 tttgttcctt gggttcttgg gagcagcagg
aagcactatg ggcgcagcgt caatgacgct 1140 gacggtacag gccagacaat
tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200 ggctattgag
gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260
ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
1320 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
ggagtaataa 1380 atctctggaa cagatttgga atcacacgac ctggatggag
tgggacagag aaattaacaa 1440 ttacacaagc ttaatacact ccttaattga
agaatcgcaa aaccagcaag aaaagaatga 1500 acaagaatta ttggaattag
ataaatgggc aagtttgtgg aattggttta acataacaaa 1560 ttggctgtgg
tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620
agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
1680 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
aagaagaagg 1740 tggagagaga gacagagaca gatccattcg attagtgaac
ggatctcgac ggtatcgcta 1800 gcttttaaaa gaaaaggggg gattgggggg
tacagtgcag gggaaagaat agtagacata 1860 atagcaacag acatacaaac
taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt 1920 actagtgatt
atcggatcaa ctttgtatag aaaagttggg ctccggtgcc cgtcagtggg 1980
cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg
2040 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
tggctccgcc 2100 tttttcccga gggtggggga gaaccgtata taagtgcagt
agtcgccgtg aacgttcttt 2160 ttcgcaacgg gtttgccgcc agaacacagg
taagtgccgt gtgtggttcc cgcgggcctg 2220 gcctctttac gggttatggc
ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg 2280 tgattcttga
tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt 2340
aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg
2400 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct
ctagccattt 2460 aaaatttttg atgacctgct gcgacgcttt ttttctggca
agatagtctt gtaaatgcgg 2520 gccaagatct gcacactggt atttcggttt
ttggggccgc gggcggcgac ggggcccgtg 2580 cgtcccagcg cacatgttcg
gcgaggcggg gcctgcgagc gcggccaccg agaatcggac 2640 gggggtagtc
tcaagctggc cggcctgctc tggtgcctgg tctcgcgccg ccgtgtatcg 2700
ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc
2760 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg
ggagagcggg 2820 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc
ctcagccgtc gcttcatgtg 2880 actccacgga gtaccgggcg ccgtccaggc
acctcgatta gttctcgagc ttttggagta 2940 cgtcgtcttt aggttggggg
gaggggtttt atgcgatgga gtttccccac actgagtggg 3000 tggagactga
agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt 3060
ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc
3120 catttcaggt gtcgtgacaa gtttgtacaa aaaagcaggc tgccaccatg
atcgagacct 3180 acaaccagac cagccccaga agcgccgcca ccggcctgcc
catcagcatg aagatcttca 3240 tgtacctgct gaccgtgttc ctgatcaccc
agatgatcgg cagcgccctg ttcgccgtgt 3300 acctgcacag aagactggac
aagatcgagg acgagagaaa cctgcacgag gacttcgtgt 3360 tcatgaagac
catccagaga tgcaacaccg gcgagagaag cctgagcctg ctgaactgcg 3420
aggagatcaa gagccagttc gagggcttcg tgaaggacat catgctgaac aaggaggaga
3480 ccaagaagga gaacagcttc gagatgccca gaggcgagga ggacagccag
atcgccgccc 3540 acgtgatcag cgaggccagc agcaagacca ccagcgtgct
gcagtgggcc gagaagggct 3600 actacaccat gagcaacaac ctggtgaccc
tggagaacgg caagcagctg accgtgaaga 3660 gacagggcct gtactacatc
tacgcccagg tgaccttctg cagcaacaga gaggccagca 3720 gccaggcccc
cttcatcgcc agcctgtgcc tgaagagccc cggcagattc gagagaatcc 3780
tgctgagagc cgccaacacc cacagcagcg ccaagccctg cggccagcag agcatccacc
3840 tgggcggcgt gttcgagctg cagcccggcg ccagcgtgtt cgtgaacgtg
accgacccca 3900 gccaggtgag ccacggcacc ggcttcacca gcttcggcct
gctgaagctg taaacccagc 3960 tttcttgtac aaagtggtga taatcgaatt
cacccagctt tcttgtacaa agtggtgata 4020 atcgaattcc gataatcaac
ctctggatta caaaatttgt gaaagattga ctggtattct 4080 taactatgtt
gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc 4140
tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
4200 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
tgtttgctga 4260 cgcaaccccc actggttggg gcattgccac cacctgtcag
ctcctttccg ggactttcgc 4320 tttccccctc cctattgcca cggcggaact
catcgccgcc tgccttgccc gctgctggac 4380 aggggctcgg ctgttgggca
ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt 4440 tccatggctg
ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt 4500
cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
4560 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
ccgcctcccc 4620 gcatcgggaa ttcccgcggt tcgctttaag accaatgact
tacaaggcag ctgtagatct 4680 tagccacttt ttaaaagaaa aggggggact
ggaagggcta attcactccc aacgaagaca 4740 agatctgctt tttgcttgta
ctgggtctct ctggttagac cagatctgag cctgggagct 4800 ctctggctaa
ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 4860
agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
4920 gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc
agtatttata 4980 acttgcaaag aaatgaatat cagagagtga gaggaacttg
tttattgcag cttataatgg 5040 ttacaaataa agcaatagca tcacaaattt
cacaaataaa gcattttttt cactgcattc 5100 tagttgtggt ttgtccaaac
tcatcaatgt atcttatcat gtctggctct agctatcccg 5160 cccctaactc
cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat 5220
ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
5280 cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg
ccctatagtg 5340 agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
tcgtgactgg gaaaaccctg 5400 gcgttaccca acttaatcgc cttgcagcac
atcccccttt cgccagctgg cgtaatagcg 5460 aagaggcccg caccgatcgc
ccttcccaac agttgcgcag cctgaatggc gaatgggacg 5520 cgccctgtag
cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 5580
cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
5640 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
cgatttagtg 5700 ctttacggca cctcgacccc aaaaaacttg attagggtga
tggttcacgt agtgggccat 5760 cgccctgata gacggttttt cgccctttga
cgttggagtc cacgttcttt aatagtggac 5820 tcttgttcca aactggaaca
acactcaacc ctatctcggt ctattctttt gatttataag 5880 ggattttgcc
gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg 5940
cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
6000 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
tcatgagaca 6060 ataaccctga taaatgcttc aataatattg aaaaaggaag
agtatgagta ttcaacattt 6120 ccgtgtcgcc cttattccct tttttgcggc
attttgcctt cctgtttttg ctcacccaga 6180 aacgctggtg aaagtaaaag
atgctgaaga tcagttgggt gcacgagtgg gttacatcga 6240 actggatctc
aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 6300
gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
6360 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
actcaccagt 6420 cacagaaaag catcttacgg atggcatgac agtaagagaa
ttatgcagtg ctgccataac 6480 catgagtgat aacactgcgg ccaacttact
tctgacaacg atcggaggac cgaaggagct 6540 aaccgctttt ttgcacaaca
tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 6600 gctgaatgaa
gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 6660
aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
6720 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
ttccggctgg 6780 ctggtttatt gctgataaat ctggagccgg tgagcgtggg
tctcgcggta tcattgcagc 6840 actggggcca gatggtaagc cctcccgtat
cgtagttatc tacacgacgg ggagtcaggc 6900 aactatggat gaacgaaata
gacagatcgc tgagataggt gcctcactga ttaagcattg 6960 gtaactgtca
gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 7020
atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
7080 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
cttcttgaga 7140 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
aaaccaccgc taccagcggt 7200 ggtttgtttg ccggatcaag agctaccaac
tctttttccg aaggtaactg gcttcagcag 7260 agcgcagata ccaaatactg
ttcttctagt gtagccgtag ttaggccacc acttcaagaa 7320 ctctgtagca
ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 7380
tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
7440 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
cgacctacac 7500 cgaactgaga tacctacagc gtgagctatg agaaagcgcc
acgcttcccg aagagagaaa 7560 ggcggacagg tatccggtaa gcggcagggt
cggaacagga gagcgcacga gggagcttcc 7620 agggggaaac gcctggtatc
tttatagtcc tgtcgggttt cgccacctct gacttgagcg 7680 tcgatttttg
tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 7740
ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
7800 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
ctcgccgcag 7860 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
gaagagcgcc caatacgcaa 7920 accgcctctc cccgcgcgtt ggccgattca
ttaatgcagc tggcacgaca ggtttcccga 7980 ctggaaagcg ggcagtgagc
gcaacgcaat taatgtgagt tagctcactc attaggcacc 8040 ccaggcttta
cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca 8100
atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
8160 aagggaacaa aagctggagc tgcaagctt 8189 <210> SEQ ID NO 52
<211> LENGTH: 8069 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <220> FEATURE: <223> OTHER INFORMATION:
Vector 6 <400> SEQUENCE: 52 aatgtagtct tatgcaatac tcttgtagtc
ttgcaacatg gtaacgatga gttagcaaca 60 tgccttacaa ggagagaaaa
agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120 tcgtgcctta
ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180
gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
240 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
tgcttaagcc 300 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
cgtctgttgt gtgactctgg 360 taactagaga tccctcagac ccttttagtc
agtgtggaaa atctctagca gtggcgcccg 420 aacagggact tgaaagcgaa
agggaaacca gaggagctct ctcgacgcag gactcggctt 480 gctgaagcgc
gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540
actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
600 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
atataaatta 660 aaacatatag tatgggcaag cagggagcta gaacgattcg
cagttaatcc tggcctgtta 720 gaaacatcag aaggctgtag acaaatactg
ggacagctac aaccatccct tcagacagga 780 tcagaagaac ttagatcatt
atataataca gtagcaaccc tctattgtgt gcatcaaagg 840 atagagataa
aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900
aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
960 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
taggagtagc 1020 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
agagcagtgg gaataggagc 1080 tttgttcctt gggttcttgg gagcagcagg
aagcactatg ggcgcagcgt caatgacgct 1140 gacggtacag gccagacaat
tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200 ggctattgag
gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260
ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
1320 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
ggagtaataa 1380 atctctggaa cagatttgga atcacacgac ctggatggag
tgggacagag aaattaacaa 1440 ttacacaagc ttaatacact ccttaattga
agaatcgcaa aaccagcaag aaaagaatga 1500 acaagaatta ttggaattag
ataaatgggc aagtttgtgg aattggttta acataacaaa 1560 ttggctgtgg
tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620
agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
1680 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
aagaagaagg 1740 tggagagaga gacagagaca gatccattcg attagtgaac
ggatctcgac ggtatcgcta 1800 gcttttaaaa gaaaaggggg gattgggggg
tacagtgcag gggaaagaat agtagacata 1860 atagcaacag acatacaaac
taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt 1920 actagtgatt
atcggatcaa ctttgtatag aaaagttggg ctccggtgcc cgtcagtggg 1980
cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg
2040 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
tggctccgcc 2100 tttttcccga gggtggggga gaaccgtata taagtgcagt
agtcgccgtg aacgttcttt 2160 ttcgcaacgg gtttgccgcc agaacacagg
taagtgccgt gtgtggttcc cgcgggcctg 2220 gcctctttac gggttatggc
ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg 2280 tgattcttga
tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt 2340
aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg
2400 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct
ctagccattt 2460 aaaatttttg atgacctgct gcgacgcttt ttttctggca
agatagtctt gtaaatgcgg 2520 gccaagatct gcacactggt atttcggttt
ttggggccgc gggcggcgac ggggcccgtg 2580 cgtcccagcg cacatgttcg
gcgaggcggg gcctgcgagc gcggccaccg agaatcggac 2640 gggggtagtc
tcaagctggc cggcctgctc tggtgcctgg tctcgcgccg ccgtgtatcg 2700
ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc
2760 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg
ggagagcggg 2820 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc
ctcagccgtc gcttcatgtg 2880 actccacgga gtaccgggcg ccgtccaggc
acctcgatta gttctcgagc ttttggagta 2940 cgtcgtcttt aggttggggg
gaggggtttt atgcgatgga gtttccccac actgagtggg 3000 tggagactga
agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt 3060
ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc
3120 catttcaggt gtcgtgacaa gtttgtacaa aaaagcaggc tgccaccatg
agcaccgaga 3180 gcatgatcag agacgtggag ctggccgagg aggccctgcc
caagaagacc ggcggccccc 3240 agggcagcag aagatgcctg ttcctgagcc
tgttcagctt cctgatcgtg gccggcgcca 3300 ccaccctgtt ctgcctgctg
cacttcggcg tgatcggccc ccagagagag gagttcccca 3360 gagacctgag
cctgatcagc cccctggccc aggccgtggc ccacgtggtg gccaaccccc 3420
aggccgaggg ccagctgcag tggctgaaca gaagagccaa cgccctgctg gccaacggcg
3480 tggagctgag agacaaccag ctggtggtgc ccagcgaggg cctgtacctg
atctacagcc 3540 aggtgctgtt caagggccag ggctgcccca gcacccacgt
gctgctgacc cacaccatca 3600 gcagaatcgc cgtgagctac cagaccaagg
tgaacctgct gagcgccatc aagagcccct 3660 gccagagaga gacccccgag
ggcgccgagg ccaagccctg gtacgagccc atctacctgg 3720 gcggcgtgtt
ccagctggag aagggcgaca gactgagcgc cgagatcaac agacccgact 3780
acctggactt cgccgagagc ggccaggtgt acttcggcat catcgccctg taaacccagc
3840 tttcttgtac aaagtggtga taatcgaatt cacccagctt tcttgtacaa
agtggtgata 3900 atcgaattcc gataatcaac ctctggatta caaaatttgt
gaaagattga ctggtattct 3960 taactatgtt gctcctttta cgctatgtgg
atacgctgct ttaatgcctt tgtatcatgc 4020 tattgcttcc cgtatggctt
tcattttctc ctccttgtat aaatcctggt tgctgtctct 4080 ttatgaggag
ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga 4140
cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
4200 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
gctgctggac 4260 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
tcggggaagc tgacgtcctt 4320 tccatggctg ctcgcctgtg ttgccacctg
gattctgcgc gggacgtcct tctgctacgt 4380 cccttcggcc ctcaatccag
cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc 4440 tcttccgcgt
cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc 4500
gcatcgggaa ttcccgcggt tcgctttaag accaatgact tacaaggcag ctgtagatct
4560 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
aacgaagaca 4620 agatctgctt tttgcttgta ctgggtctct ctggttagac
cagatctgag cctgggagct 4680 ctctggctaa ctagggaacc cactgcttaa
gcctcaataa agcttgcctt gagtgcttca 4740 agtagtgtgt gcccgtctgt
tgtgtgactc tggtaactag agatccctca gaccctttta 4800 gtcagtgtgg
aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata 4860
acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg
4920 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
cactgcattc 4980 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
gtctggctct agctatcccg 5040 cccctaactc cgcccatccc gcccctaact
ccgcccagtt ccgcccattc tccgccccat 5100 ggctgactaa ttttttttat
ttatgcagag gccgaggccg cctcggcctc tgagctattc 5160 cagaagtagt
gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg 5220
agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
5280 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
cgtaatagcg 5340 aagaggcccg caccgatcgc ccttcccaac agttgcgcag
cctgaatggc gaatgggacg 5400 cgccctgtag cggcgcatta agcgcggcgg
gtgtggtggt tacgcgcagc gtgaccgcta 5460 cacttgccag cgccctagcg
cccgctcctt tcgctttctt cccttccttt ctcgccacgt 5520 tcgccggctt
tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 5580
ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
5640 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
aatagtggac 5700 tcttgttcca aactggaaca acactcaacc ctatctcggt
ctattctttt gatttataag 5760 ggattttgcc gatttcggcc tattggttaa
aaaatgagct gatttaacaa aaatttaacg 5820 cgaattttaa caaaatatta
acgcttacaa tttaggtggc acttttcggg gaaatgtgcg 5880 cggaacccct
atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 5940
ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
6000 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
ctcacccaga 6060 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
gcacgagtgg gttacatcga 6120 actggatctc aacagcggta agatccttga
gagttttcgc cccgaagaac gttttccaat 6180 gatgagcact tttaaagttc
tgctatgtgg cgcggtatta tcccgtattg acgccgggca 6240 agagcaactc
ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 6300
cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
6360 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
cgaaggagct 6420 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
cttgatcgtt gggaaccgga 6480 gctgaatgaa gccataccaa acgacgagcg
tgacaccacg atgcctgtag caatggcaac 6540 aacgttgcgc aaactattaa
ctggcgaact acttactcta gcttcccggc aacaattaat 6600 agactggatg
gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 6660
ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
6720 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
ggagtcaggc 6780 aactatggat gaacgaaata gacagatcgc tgagataggt
gcctcactga ttaagcattg 6840 gtaactgtca gaccaagttt actcatatat
actttagatt gatttaaaac ttcattttta 6900 atttaaaagg atctaggtga
agatcctttt tgataatctc atgaccaaaa tcccttaacg 6960
tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
7020 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
taccagcggt 7080 ggtttgtttg ccggatcaag agctaccaac tctttttccg
aaggtaactg gcttcagcag 7140 agcgcagata ccaaatactg ttcttctagt
gtagccgtag ttaggccacc acttcaagaa 7200 ctctgtagca ccgcctacat
acctcgctct gctaatcctg ttaccagtgg ctgctgccag 7260 tggcgataag
tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 7320
gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
7380 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
aagagagaaa 7440 ggcggacagg tatccggtaa gcggcagggt cggaacagga
gagcgcacga gggagcttcc 7500 agggggaaac gcctggtatc tttatagtcc
tgtcgggttt cgccacctct gacttgagcg 7560 tcgatttttg tgatgctcgt
caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 7620 ctttttacgg
ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 7680
ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
7740 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
caatacgcaa 7800 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
tggcacgaca ggtttcccga 7860 ctggaaagcg ggcagtgagc gcaacgcaat
taatgtgagt tagctcactc attaggcacc 7920 ccaggcttta cactttatgc
ttccggctcg tatgttgtgt ggaattgtga gcggataaca 7980 atttcacaca
ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta 8040
aagggaacaa aagctggagc tgcaagctt 8069 <210> SEQ ID NO 53
<211> LENGTH: 10067 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <220> FEATURE: <223> OTHER INFORMATION:
Vector 7 <400> SEQUENCE: 53 aatgtagtct tatgcaatac tcttgtagtc
ttgcaacatg gtaacgatga gttagcaaca 60 tgccttacaa ggagagaaaa
agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120 tcgtgcctta
ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180
gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
240 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
tgcttaagcc 300 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
cgtctgttgt gtgactctgg 360 taactagaga tccctcagac ccttttagtc
agtgtggaaa atctctagca gtggcgcccg 420 aacagggact tgaaagcgaa
agggaaacca gaggagctct ctcgacgcag gactcggctt 480 gctgaagcgc
gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540
actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
600 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
atataaatta 660 aaacatatag tatgggcaag cagggagcta gaacgattcg
cagttaatcc tggcctgtta 720 gaaacatcag aaggctgtag acaaatactg
ggacagctac aaccatccct tcagacagga 780 tcagaagaac ttagatcatt
atataataca gtagcaaccc tctattgtgt gcatcaaagg 840 atagagataa
aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900
aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
960 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
taggagtagc 1020 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
agagcagtgg gaataggagc 1080 tttgttcctt gggttcttgg gagcagcagg
aagcactatg ggcgcagcgt caatgacgct 1140 gacggtacag gccagacaat
tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200 ggctattgag
gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260
ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
1320 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
ggagtaataa 1380 atctctggaa cagatttgga atcacacgac ctggatggag
tgggacagag aaattaacaa 1440 ttacacaagc ttaatacact ccttaattga
agaatcgcaa aaccagcaag aaaagaatga 1500 acaagaatta ttggaattag
ataaatgggc aagtttgtgg aattggttta acataacaaa 1560 ttggctgtgg
tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620
agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
1680 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
aagaagaagg 1740 tggagagaga gacagagaca gatccattcg attagtgaac
ggatctcgac ggtatcgcta 1800 gcttttaaaa gaaaaggggg gattgggggg
tacagtgcag gggaaagaat agtagacata 1860 atagcaacag acatacaaac
taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt 1920 actagtatca
actttgtata gaaaagttgg gctccggtgc ccgtcagtgg gcagagcgca 1980
catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
2040 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
ctttttcccg 2100 agggtggggg agaaccgtat ataagtgcag tagtcgccgt
gaacgttctt tttcgcaacg 2160 ggtttgccgc cagaacacag gtaagtgccg
tgtgtggttc ccgcgggcct ggcctcttta 2220 cgggttatgg cccttgcgtg
ccttgaatta cttccacctg gctgcagtac gtgattcttg 2280 atcccgagct
tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc 2340
cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct
2400 ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt
taaaattttt 2460 gatgacctgc tgcgacgctt tttttctggc aagatagtct
tgtaaatgcg ggccaagatc 2520 tgcacactgg tatttcggtt tttggggccg
cgggcggcga cggggcccgt gcgtcccagc 2580 gcacatgttc ggcgaggcgg
ggcctgcgag cgcggccacc gagaatcgga cgggggtagt 2640 ctcaagctgg
ccggcctgct ctggtgcctg gtctcgcgcc gccgtgtatc gccccgccct 2700
gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg
2760 gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg
gcgggtgagt 2820 cacccacaca aaggaaaagg gcctttccgt cctcagccgt
cgcttcatgt gactccacgg 2880 agtaccgggc gccgtccagg cacctcgatt
agttctcgag cttttggagt acgtcgtctt 2940 taggttgggg ggaggggttt
tatgcgatgg agtttcccca cactgagtgg gtggagactg 3000 aagttaggcc
agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg 3060
gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg
3120 tgtcgtgaca agtttgtaca aaaaagcagg ctgccaccat gagcaccgag
agcatgatca 3180 gagacgtgga gctggccgag gaggccctgc ccaagaagac
cggcggcccc cagggcagca 3240 gaagatgcct gttcctgagc ctgttcagct
tcctgatcgt ggccggcgcc accaccctgt 3300 tctgcctgct gcacttcggc
gtgatcggcc cccagagaga ggagttcccc agagacctga 3360 gcctgatcag
ccccctggcc caggccgtgg cccacgtggt ggccaacccc caggccgagg 3420
gccagctgca gtggctgaac agaagagcca acgccctgct ggccaacggc gtggagctga
3480 gagacaacca gctggtggtg cccagcgagg gcctgtacct gatctacagc
caggtgctgt 3540 tcaagggcca gggctgcccc agcacccacg tgctgctgac
ccacaccatc agcagaatcg 3600 ccgtgagcta ccagaccaag gtgaacctgc
tgagcgccat caagagcccc tgccagagag 3660 agacccccga gggcgccgag
gccaagccct ggtacgagcc catctacctg ggcggcgtgt 3720 tccagctgga
gaagggcgac agactgagcg ccgagatcaa cagacccgac tacctggact 3780
tcgccgagag cggccaggtg tacttcggca tcatcgccct gtaaacccag ctttcttgta
3840 caaagtggtg ataatcgaat tctaaataga tagaacaaca acaattgcat
tcatttttga 3900 tttcaggttc agggggaggt gtgggaggtt ttttaaagca
agtaaaacct ctacactgac 3960 ggtacgcgtt aacaacaaca attgcattca
tttgtagttt caggttcagg gggaggtgtg 4020 ggaggttttt taaagcaagt
taaacctcta aaatagtggt acgcgttacc cagctttctt 4080 gtacaaagtg
gacccagctt tcttgtacaa agtgggcccc tctccctccc ccccccctaa 4140
cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc
4200 caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg
tcttcttgac 4260 gagcattcct aggggtcttt cccctctcgc caaaggaatg
caaggtctgt tgaatgtcgt 4320 gaaggaagca gttcctctgg aagcttcttg
aagacaaaca acgtctgtag cgaccctttg 4380 caggcagcgg aaccccccac
ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata 4440 agatacacct
gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga 4500
aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt
4560 accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat
gtgtttagtc 4620 gaggttaaaa aaacgtctag gccccccgaa ccacggggac
gtggttttcc tttgaaaaac 4680 acgatgataa tatggccaca accatggcca
ccgtgctggc ccccgcctgg agccccacca 4740 cctacctgct gctgctgctg
ctgctgagca gcggcctgag cggcggcggc ggcagcggca 4800 agcccatccc
caaccccctg ctgggcctgg acagcaccgg cggcggcggc agccaggtga 4860
agctgcagga gagcggcccc ggcctggtgg cccccagcca gagcctgagc atcacctgca
4920 ccgtgagcgg cttcagcctg accgcctacg gcgtggactg ggtgagacag
ccccccggca 4980 agtgcctgga gtggctgggc gtgatctggg gcggcggcag
aaccaactac aacagcggcc 5040 tgatgagcag actgagcatc agaaaggaca
acagcaagag ccaggtgttc ctgaccatga 5100 acagcctgca gaccgacgac
accgccaagt actactgcgt gaagcacacc aactgggacg 5160 gcggcttcgc
ctactggggc cagggcacca ccgtgaccgt gagcagcggc ggcggcggca 5220
gcggcggcgg cggcagcggc ggcggcggca gcggcagccc cggccagagc gtgagcatca
5280 gctgcagcgg cagcagcagc aacatcggca acaactacgt gtactggtac
cagcacctgc 5340 ccggcaccgc ccccaagctg ctgatctaca gcgacaccaa
gagacccagc ggcgtgcccg 5400 acagaatcag cggcagcaag agcggcacca
gcgccagcct ggccatcagc ggcctgcaga 5460 gcgaggacga ggccgactac
tactgcgcca gctgggacga cagcctggac ggccccgtgt 5520 tcggctgcgg
caccaagctg accgtgctgc ccaccaccac ccccgccccc agacccccca 5580
cccccgcccc caccatcgcc agccagcccc tgagcctgag acccgaggcc tgcagacccg
5640 ccgccggcgg cgccgtgcac accagaggcc tggacttcgc ctgcgacatc
tacatctggg 5700 cccccctggc cggcacctgc ggcgtgctgc tgctgagcct
ggtgatcacc ctgtactgca 5760 accacagaaa cagaagaaga gtgtgcaagt
gccccagacc cgtggtgaag agcggcgaca 5820 agcccagcct gagcgccaga
tacgtgtaac aactttatta tacatagttg atcaattcca 5880 actttattat
acatagttga tcaattccga taatcaacct ctggattaca aaatttgtga 5940
aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
6000
aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
6060 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
gtggcgtggt 6120 gtgcactgtg tttgctgacg caacccccac tggttggggc
attgccacca cctgtcagct 6180 cctttccggg actttcgctt tccccctccc
tattgccacg gcggaactca tcgccgcctg 6240 ccttgcccgc tgctggacag
gggctcggct gttgggcact gacaattccg tggtgttgtc 6300 ggggaagctg
acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg 6360
gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
6420 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
gtcggatctc 6480 cctttgggcc gcctccccgc atcgggaatt cccgcggttc
gctttaagac caatgactta 6540 caaggcagct gtagatctta gccacttttt
aaaagaaaag gggggactgg aagggctaat 6600 tcactcccaa cgaagacaag
atctgctttt tgcttgtact gggtctctct ggttagacca 6660 gatctgagcc
tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag 6720
cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
6780 atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt
catgtcatct 6840 tattattcag tatttataac ttgcaaagaa atgaatatca
gagagtgaga ggaacttgtt 6900 tattgcagct tataatggtt acaaataaag
caatagcatc acaaatttca caaataaagc 6960 atttttttca ctgcattcta
gttgtggttt gtccaaactc atcaatgtat cttatcatgt 7020 ctggctctag
ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc 7080
gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
7140 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
agggacgtac 7200 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
ggccgtcgtt ttacaacgtc 7260 gtgactggga aaaccctggc gttacccaac
ttaatcgcct tgcagcacat ccccctttcg 7320 ccagctggcg taatagcgaa
gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 7380 tgaatggcga
atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 7440
cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
7500 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
gggctccctt 7560 tagggttccg atttagtgct ttacggcacc tcgaccccaa
aaaacttgat tagggtgatg 7620 gttcacgtag tgggccatcg ccctgataga
cggtttttcg ccctttgacg ttggagtcca 7680 cgttctttaa tagtggactc
ttgttccaaa ctggaacaac actcaaccct atctcggtct 7740 attcttttga
tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga 7800
tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
7860 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
attcaaatat 7920 gtatccgctc atgagacaat aaccctgata aatgcttcaa
taatattgaa aaaggaagag 7980 tatgagtatt caacatttcc gtgtcgccct
tattcccttt tttgcggcat tttgccttcc 8040 tgtttttgct cacccagaaa
cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 8100 acgagtgggt
tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 8160
cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
8220 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
agaatgactt 8280 ggttgagtac tcaccagtca cagaaaagca tcttacggat
ggcatgacag taagagaatt 8340 atgcagtgct gccataacca tgagtgataa
cactgcggcc aacttacttc tgacaacgat 8400 cggaggaccg aaggagctaa
ccgctttttt gcacaacatg ggggatcatg taactcgcct 8460 tgatcgttgg
gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 8520
gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
8580 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
cacttctgcg 8640 ctcggccctt ccggctggct ggtttattgc tgataaatct
ggagccggtg agcgtgggtc 8700 tcgcggtatc attgcagcac tggggccaga
tggtaagccc tcccgtatcg tagttatcta 8760 cacgacgggg agtcaggcaa
ctatggatga acgaaataga cagatcgctg agataggtgc 8820 ctcactgatt
aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga 8880
tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
8940 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
tagaaaagat 9000 caaaggatct tcttgagatc ctttttttct gcgcgtaatc
tgctgcttgc aaacaaaaaa 9060 accaccgcta ccagcggtgg tttgtttgcc
ggatcaagag ctaccaactc tttttccgaa 9120 ggtaactggc ttcagcagag
cgcagatacc aaatactgtt cttctagtgt agccgtagtt 9180 aggccaccac
ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 9240
accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
9300 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
agcccagctt 9360 ggagcgaacg acctacaccg aactgagata cctacagcgt
gagctatgag aaagcgccac 9420 gcttcccgaa gagagaaagg cggacaggta
tccggtaagc ggcagggtcg gaacaggaga 9480 gcgcacgagg gagcttccag
ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 9540 ccacctctga
cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 9600
aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
9660 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
ttgagtgagc 9720 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
tcagtgagcg aggaagcgga 9780 agagcgccca atacgcaaac cgcctctccc
cgcgcgttgg ccgattcatt aatgcagctg 9840 gcacgacagg tttcccgact
ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 9900 gctcactcat
taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg 9960
aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
10020 cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagctt 10067
<210> SEQ ID NO 54 <211> LENGTH: 19 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <223> OTHER
INFORMATION: H7 heavy chain leader <400> SEQUENCE: 54 Met Glu
Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Phe Arg Gly 1 5 10 15
Val Gln Cys <210> SEQ ID NO 55 <211> LENGTH: 118
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: anti-biotin murine vH with inserted
Cys for inter-domain linkage <400> SEQUENCE: 55 Gln Val Lys
Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser
Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ala Tyr 20 25
30 Gly Val Asp Trp Val Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Leu
35 40 45 Gly Val Ile Trp Gly Gly Gly Arg Thr Asn Tyr Asn Ser Gly
Leu Met 50 55 60 Ser Arg Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser
Gln Val Phe Leu 65 70 75 80 Thr Met Asn Ser Leu Gln Thr Asp Asp Thr
Ala Lys Tyr Tyr Cys Val 85 90 95 Lys His Thr Asn Trp Asp Gly Gly
Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser
115 <210> SEQ ID NO 56 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <220> FEATURE: <223> OTHER
INFORMATION: linker <400> SEQUENCE: 56 Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> SEQ
ID NO 57 <211> LENGTH: 99 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
Light Chain Variable (human lambda variable) <400> SEQUENCE:
57 Gly Ser Pro Gly Gln Ser Val Ser Ile Ser Cys Ser Gly Ser Ser Ser
1 5 10 15 Asn Ile Gly Asn Asn Tyr Val Tyr Trp Tyr Gln His Leu Pro
Gly Thr 20 25 30 Ala Pro Lys Leu Leu Ile Tyr Ser Asp Thr Lys Arg
Pro Ser Gly Val 35 40 45 Pro Asp Arg Ile Ser Gly Ser Lys Ser Gly
Thr Ser Ala Ser Leu Ala 50 55 60 Ile Ser Gly Leu Gln Ser Glu Asp
Glu Ala Asp Tyr Tyr Cys Ala Ser 65 70 75 80 Trp Asp Asp Ser Leu Asp
Gly Pro Val Phe Gly Cys Gly Thr Lys Leu 85 90 95 Thr Val Leu
<210> SEQ ID NO 58 <211> LENGTH: 61
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: IgG3 hinge for greater accessibility
to FcyR <400> SEQUENCE: 58 Leu Lys Thr Pro Leu Gly Asp Thr
Thr His Thr Cys Pro Arg Cys Pro 1 5 10 15 Glu Pro Lys Ser Cys Asp
Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu 20 25 30 Pro Lys Ser Cys
Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro 35 40 45 Lys Ser
Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 50 55 60 <210>
SEQ ID NO 59 <211> LENGTH: 283 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: IgG1 CH2, CH3 Tm and cytoplasmic tail (T256A)
<400> SEQUENCE: 59 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Met Ile Ser Arg Ala Pro
Glu Val Thr Cys Val Val Val Asp 20 25 30 Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 35 40 45 Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 50 55 60 Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 65 70 75 80
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 85
90 95 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu 100 105 110 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn 115 120 125 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile 130 135 140 Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr 145 150 155 160 Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 165 170 175 Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 180 185 190 Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 195 200 205
Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys Ala Glu Ala Gln 210
215 220 Asp Gly Glu Leu Asp Gly Leu Trp Thr Thr Ile Thr Ile Phe Ile
Thr 225 230 235 240 Leu Phe Leu Leu Ser Val Cys Tyr Ser Ala Thr Val
Thr Phe Phe Lys 245 250 255 Val Lys Trp Ile Phe Ser Ser Val Val Asp
Leu Lys Gln Thr Ile Ile 260 265 270 Pro Asp Tyr Arg Asn Met Ile Gly
Gln Gly Ala 275 280 <210> SEQ ID NO 60 <211> LENGTH:
598 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: scFv-anti-biotin-G3hinge-IgG1-Tm
(598 ORF1) <400> SEQUENCE: 60 Met Glu Phe Gly Leu Ser Trp Val
Phe Leu Val Ala Leu Phe Arg Gly 1 5 10 15 Val Gln Cys Gln Val Lys
Leu Gln Glu Ser Gly Pro Gly Leu Val Ala 20 25 30 Pro Ser Gln Ser
Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu 35 40 45 Thr Ala
Tyr Gly Val Asp Trp Val Arg Gln Pro Pro Gly Lys Cys Leu 50 55 60
Glu Trp Leu Gly Val Ile Trp Gly Gly Gly Arg Thr Asn Tyr Asn Ser 65
70 75 80 Gly Leu Met Ser Arg Leu Ser Ile Arg Lys Asp Asn Ser Lys
Ser Gln 85 90 95 Val Phe Leu Thr Met Asn Ser Leu Gln Thr Asp Asp
Thr Ala Lys Tyr 100 105 110 Tyr Cys Val Lys His Thr Asn Trp Asp Gly
Gly Phe Ala Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Val Thr Val Ser
Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly
Gly Ser Gly Ser Pro Gly Gln Ser Val Ser 145 150 155 160 Ile Ser Cys
Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Tyr 165 170 175 Trp
Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ser 180 185
190 Asp Thr Lys Arg Pro Ser Gly Val Pro Asp Arg Ile Ser Gly Ser Lys
195 200 205 Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser
Glu Asp 210 215 220 Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Asp Ser
Leu Asp Gly Pro 225 230 235 240 Val Phe Gly Cys Gly Thr Lys Leu Thr
Val Leu Leu Lys Thr Pro Leu 245 250 255 Gly Asp Thr Thr His Thr Cys
Pro Arg Cys Pro Glu Pro Lys Ser Cys 260 265 270 Asp Thr Pro Pro Pro
Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp 275 280 285 Thr Pro Pro
Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr 290 295 300 Pro
Pro Pro Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 305 310
315 320 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 325 330 335 Arg Ala Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp 340 345 350 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn 355 360 365 Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val 370 375 380 Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu 385 390 395 400 Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 405 410 415 Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 420 425 430
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 435
440 445 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu 450 455 460 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu 465 470 475 480 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys 485 490 495 Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu 500 505 510 Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Glu 515 520 525 Leu Gln Leu Glu
Glu Ser Cys Ala Glu Ala Gln Asp Gly Glu Leu Asp 530 535 540 Gly Leu
Trp Thr Thr Ile Thr Ile Phe Ile Thr Leu Phe Leu Leu Ser 545 550 555
560 Val Cys Tyr Ser Ala Thr Val Thr Phe Phe Lys Val Lys Trp Ile Phe
565 570 575 Ser Ser Val Val Asp Leu Lys Gln Thr Ile Ile Pro Asp Tyr
Arg Asn 580 585 590 Met Ile Gly Gln Gly Ala 595 <210> SEQ ID
NO 61 <211> LENGTH: 24 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <220> FEATURE: <223> OTHER INFORMATION: L1
light chain leader/signal <400> SEQUENCE: 61 Met Ala Thr Asp
Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu 1 5 10 15 Leu Trp
Leu Ser Gly Ala Arg Cys 20 <210> SEQ ID NO 62 <211>
LENGTH: 118 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<220> FEATURE: <223> OTHER INFORMATION: anti-biotin vH
<400> SEQUENCE: 62 Gln Val Lys Leu Gln Glu Ser Gly Pro Gly
Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr Ala Tyr 20 25 30 Gly Val Asp Trp Val Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp
Gly Gly Gly Arg Thr Asn Tyr Asn Ser Gly Leu Met 50 55 60 Ser Arg
Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80
Thr Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Lys Tyr Tyr Cys Val 85
90 95 Lys His Thr Asn Trp Asp Gly Gly Phe Ala Tyr Trp Gly Gln Gly
Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> SEQ ID NO
63 <211> LENGTH: 94 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION: CH1
<400> SEQUENCE: 63 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly 1 5 10 15 Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val 20 25 30 Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe 35 40 45 Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 50 55 60 Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 65 70 75 80
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 85 90
<210> SEQ ID NO 64 <211> LENGTH: 61 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: IgG3 hinge <400> SEQUENCE: 64 Leu Lys Thr Pro
Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys Pro 1 5 10 15 Glu Pro
Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu 20 25 30
Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro 35
40 45 Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 50 55 60
<210> SEQ ID NO 65 <211> LENGTH: 286 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: IgG1 CH2, CH3 Tm and cytoplasmic tail <400>
SEQUENCE: 65 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Ala Pro
Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105
110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu
Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys Ala 210 215 220 Glu
Ala Gln Asp Gly Glu Leu Asp Gly Leu Trp Thr Thr Ile Thr Ile 225 230
235 240 Phe Ile Thr Leu Phe Leu Leu Ser Val Cys Tyr Ser Ala Thr Val
Thr 245 250 255 Phe Phe Lys Val Lys Trp Ile Phe Ser Ser Val Val Asp
Leu Lys Gln 260 265 270 Thr Ile Ile Pro Asp Tyr Arg Asn Met Ile Gly
Gln Gly Ala 275 280 285 <210> SEQ ID NO 66 <211>
LENGTH: 578 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<220> FEATURE: <223> OTHER INFORMATION: Summary (578
ORF2a) <400> SEQUENCE: 66 Met Glu Phe Gly Leu Ser Trp Val Phe
Leu Val Ala Leu Phe Arg Gly 1 5 10 15 Val Gln Cys Gln Val Lys Leu
Gln Glu Ser Gly Pro Gly Leu Val Ala 20 25 30 Pro Ser Gln Ser Leu
Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu 35 40 45 Thr Ala Tyr
Gly Val Asp Trp Val Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60 Glu
Trp Leu Gly Val Ile Trp Gly Gly Gly Arg Thr Asn Tyr Asn Ser 65 70
75 80 Gly Leu Met Ser Arg Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser
Gln 85 90 95 Val Phe Leu Thr Met Asn Ser Leu Gln Thr Asp Asp Thr
Ala Lys Tyr 100 105 110 Tyr Cys Val Lys His Thr Asn Trp Asp Gly Gly
Phe Ala Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Val Thr Val Ser Ser
Pro Ser Val Phe Pro Leu Ala 130 135 140 Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu 145 150 155 160 Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 165 170 175 Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 180 185 190
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 195
200 205 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr 210 215 220 Lys Val Asp Lys Lys Val Glu Leu Lys Thr Pro Leu Gly
Asp Thr Thr 225 230 235 240 His Thr Cys Pro Arg Cys Pro Glu Pro Lys
Ser Cys Asp Thr Pro Pro 245 250 255 Pro Cys Pro Arg Cys Pro Glu Pro
Lys Ser Cys Asp Thr Pro Pro Pro 260 265 270 Cys Pro Arg Cys Pro Glu
Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys 275 280 285 Pro Arg Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 290 295 300 Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Ala Pro Glu 305 310 315
320 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
325 330 335 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys 340 345 350 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu 355 360 365 Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys 370 375 380 Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys 385 390 395 400 Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 405 410 415 Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 420 425 430 Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
435 440 445 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly 450 455 460 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln 465 470 475 480 Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn 485 490 495 His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Glu Leu Gln Leu Glu 500 505 510 Glu Ser Cys Ala Glu
Ala Gln Asp Gly Glu Leu Asp Gly Leu Trp Thr 515 520 525 Thr Ile Thr
Ile Phe Ile Thr Leu Phe Leu Leu Ser Val Cys Tyr Ser 530 535 540 Ala
Thr Val Thr Phe Phe Lys Val Lys Trp Ile Phe Ser Ser Val Val 545 550
555 560 Asp Leu Lys Gln Thr Ile Ile Pro Asp Tyr Arg Asn Met Ile Gly
Gln 565 570 575 Gly Ala <210> SEQ ID NO 67 <400>
SEQUENCE: 67 000 <210> SEQ ID NO 68 <400> SEQUENCE: 68
000 <210> SEQ ID NO 69 <211> LENGTH: 99 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <223>
OTHER INFORMATION: LC Variable <400> SEQUENCE: 69 Gly Ser Pro
Gly Gln Ser Val Ser Ile Ser Cys Ser Gly Ser Ser Ser 1 5 10 15 Asn
Ile Gly Asn Asn Tyr Val Tyr Trp Tyr Gln His Leu Pro Gly Thr 20 25
30 Ala Pro Lys Leu Leu Ile Tyr Ser Asp Thr Lys Arg Pro Ser Gly Val
35 40 45 Pro Asp Arg Ile Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser
Leu Ala 50 55 60 Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr
Tyr Cys Ala Ser 65 70 75 80 Trp Asp Asp Ser Leu Asp Gly Pro Val Phe
Gly Gly Gly Thr Lys Leu 85 90 95 Thr Val Leu <210> SEQ ID NO
70 <211> LENGTH: 106 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION: LC
Constant Region 1 <400> SEQUENCE: 70 Gly Gln Pro Lys Ala Asn
Pro Thr Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln
Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45
Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn 50
55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp
Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly
Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100
105 <210> SEQ ID NO 71 <211> LENGTH: 229 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <223>
OTHER INFORMATION: Summary (229 ORF2b) <400> SEQUENCE: 71 Met
Ala Thr Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu 1 5 10
15 Leu Trp Leu Ser Gly Ala Arg Cys Gly Ser Pro Gly Gln Ser Val Ser
20 25 30 Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr
Val Tyr 35 40 45 Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu
Leu Ile Tyr Ser 50 55 60 Asp Thr Lys Arg Pro Ser Gly Val Pro Asp
Arg Ile Ser Gly Ser Lys 65 70 75 80 Ser Gly Thr Ser Ala Ser Leu Ala
Ile Ser Gly Leu Gln Ser Glu Asp 85 90 95 Glu Ala Asp Tyr Tyr Cys
Ala Ser Trp Asp Asp Ser Leu Asp Gly Pro 100 105 110 Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala 115 120 125 Asn Pro
Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala 130 135 140
Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala 145
150 155 160 Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Ala
Gly Val 165 170 175 Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys
Tyr Ala Ala Ser 180 185 190 Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp
Lys Ser His Arg Ser Tyr 195 200 205 Ser Cys Gln Val Thr His Glu Gly
Ser Thr Val Glu Lys Thr Val Ala 210 215 220 Pro Thr Glu Cys Ser 225
<210> SEQ ID NO 72 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <223> OTHER
INFORMATION: GM-CSF signal sequence <400> SEQUENCE: 72 Met
Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10
15 Ser <210> SEQ ID NO 73 <211> LENGTH: 127 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <223>
OTHER INFORMATION: wild type GM-CSF sequence <400> SEQUENCE:
73 Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His Val
1 5 10 15 Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg
Asp Thr 20 25 30 Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser
Glu Met Phe Asp 35 40 45 Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg
Leu Glu Leu Tyr Lys Gln 50 55 60 Gly Leu Arg Gly Ser Leu Thr Lys
Leu Lys Gly Pro Leu Thr Met Met 65 70 75 80 Ala Ser His Tyr Lys Gln
His Cys Pro Pro Thr Pro Glu Thr Ser Cys 85 90 95 Ala Thr Gln Ile
Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp 100 105 110 Phe Leu
Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 115 120 125
<210> SEQ ID NO 74 <400> SEQUENCE: 74 000 <210>
SEQ ID NO 75 <211> LENGTH: 26 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <223> OTHER
INFORMATION: FLT3L signal <400> SEQUENCE: 75 Met Ala Thr Val
Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu 1 5 10 15
Leu Leu Leu Leu Leu Ser Ser Gly Leu Ser 20 25 <210> SEQ ID NO
76 <211> LENGTH: 210 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
FLT3L <400> SEQUENCE: 76 Gly Thr Gln Asp Cys Ser Phe Gln His
Ser Pro Ile Ser Ser Asp Phe 1 5 10 15 Ala Val Lys Ile Arg Glu Leu
Ser Asp Tyr Leu Leu Gln Asp Tyr Pro 20 25 30 Val Thr Val Ala Ser
Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu 35 40 45 Trp Arg Leu
Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val 50 55 60 Ala
Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile 65 70
75 80 His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu
Arg 85 90 95 Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr
Ser Glu Gln 100 105 110 Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln
Asn Phe Ser Arg Cys 115 120 125 Leu Glu Leu Gln Cys Gln Pro Asp Ser
Ser Thr Leu Pro Pro Pro Trp 130 135 140 Ser Pro Arg Pro Leu Glu Ala
Thr Ala Pro Thr Ala Pro Gln Pro Pro 145 150 155 160 Leu Leu Leu Leu
Leu Leu Leu Pro Val Gly Leu Leu Leu Leu Ala Ala 165 170 175 Ala Trp
Cys Leu His Trp Gln Arg Thr Arg Arg Arg Thr Pro Arg Pro 180 185 190
Gly Glu Gln Val Pro Pro Val Pro Ser Pro Gln Asp Leu Leu Leu Val 195
200 205 Glu His 210 <210> SEQ ID NO 77 <211> LENGTH:
144 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: Summary (144 ORF3a) <400>
SEQUENCE: 77 Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala
Cys Ser Ile 1 5 10 15 Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr
Gln Pro Trp Glu His 20 25 30 Val Asn Ala Ile Gln Glu Ala Arg Arg
Leu Leu Asn Leu Ser Arg Asp 35 40 45 Thr Ala Ala Glu Met Asn Glu
Thr Val Glu Val Ile Ser Glu Met Phe 50 55 60 Asp Leu Gln Glu Pro
Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 65 70 75 80 Gln Gly Leu
Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met 85 90 95 Met
Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 100 105
110 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys
115 120 125 Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val
Gln Glu 130 135 140 <210> SEQ ID NO 78 <211> LENGTH:
236 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: Summary (236 ORF3b) <400>
SEQUENCE: 78 Met Ala Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr
Tyr Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly
Thr Gln Asp Cys Ser 20 25 30 Phe Gln His Ser Pro Ile Ser Ser Asp
Phe Ala Val Lys Ile Arg Glu 35 40 45 Leu Ser Asp Tyr Leu Leu Gln
Asp Tyr Pro Val Thr Val Ala Ser Asn 50 55 60 Leu Gln Asp Glu Glu
Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala 65 70 75 80 Gln Arg Trp
Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln 85 90 95 Gly
Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys 100 105
110 Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile
115 120 125 Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu
Lys Pro 130 135 140 Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu
Leu Gln Cys Gln 145 150 155 160 Pro Asp Ser Ser Thr Leu Pro Pro Pro
Trp Ser Pro Arg Pro Leu Glu 165 170 175 Ala Thr Ala Pro Thr Ala Pro
Gln Pro Pro Leu Leu Leu Leu Leu Leu 180 185 190 Leu Pro Val Gly Leu
Leu Leu Leu Ala Ala Ala Trp Cys Leu His Trp 195 200 205 Gln Arg Thr
Arg Arg Arg Thr Pro Arg Pro Gly Glu Gln Val Pro Pro 210 215 220 Val
Pro Ser Pro Gln Asp Leu Leu Leu Val Glu His 225 230 235 <210>
SEQ ID NO 79 <211> LENGTH: 183 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: wild type FLT3L sequence with transmembrane deleted
<400> SEQUENCE: 79 Met Thr Val Leu Ala Pro Ala Trp Ser Pro
Thr Thr Tyr Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Ser Ser Gly Leu
Ser Gly Thr Gln Asp Cys Ser Phe 20 25 30 Gln His Ser Pro Ile Ser
Ser Asp Phe Ala Val Lys Ile Arg Glu Leu 35 40 45 Ser Asp Tyr Leu
Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu 50 55 60 Gln Asp
Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln 65 70 75 80
Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly 85
90 95 Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys
Ala 100 105 110 Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr
Asn Ile Ser 115 120 125 Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val
Ala Leu Lys Pro Trp 130 135 140 Ile Thr Arg Gln Asn Phe Ser Arg Cys
Leu Glu Leu Gln Cys Gln Pro 145 150 155 160 Asp Ser Ser Thr Leu Pro
Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala 165 170 175 Thr Ala Pro Thr
Ala Pro Gln 180 <210> SEQ ID NO 80 <400> SEQUENCE: 80
000 <210> SEQ ID NO 81 <211> LENGTH: 26 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <220> FEATURE: <223> OTHER
INFORMATION: FLT3L signal <400> SEQUENCE: 81 Met Ala Thr Val
Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu 1 5 10 15 Leu Leu
Leu Leu Leu Ser Ser Gly Leu Ser 20 25 <210> SEQ ID NO 82
<211> LENGTH: 127 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
wild type GM-CSF sequence <400> SEQUENCE: 82
Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His Val 1 5
10 15 Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp
Thr 20 25 30 Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu
Met Phe Asp 35 40 45 Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu
Glu Leu Tyr Lys Gln 50 55 60 Gly Leu Arg Gly Ser Leu Thr Lys Leu
Lys Gly Pro Leu Thr Met Met 65 70 75 80 Ala Ser His Tyr Lys Gln His
Cys Pro Pro Thr Pro Glu Thr Ser Cys 85 90 95 Ala Thr Gln Ile Ile
Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp 100 105 110 Phe Leu Leu
Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 115 120 125
<210> SEQ ID NO 83 <211> LENGTH: 99 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: CD8alpha transmembrane and cytoplasmic domain
<400> SEQUENCE: 83 Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro
Thr Pro Ala Pro Thr Ile 1 5 10 15 Ala Ser Gln Pro Leu Ser Leu Arg
Pro Glu Ala Cys Arg Pro Ala Ala 20 25 30 Gly Gly Ala Val His Thr
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 35 40 45 Ile Trp Ala Pro
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 50 55 60 Val Ile
Thr Leu Tyr Cys Asn His Arg Asn Arg Arg Arg Val Cys Lys 65 70 75 80
Cys Pro Arg Pro Val Val Lys Ser Gly Asp Lys Pro Ser Leu Ser Ala 85
90 95 Arg Tyr Val <210> SEQ ID NO 84 <211> LENGTH: 183
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: Summary (183 ORF4a) <400>
SEQUENCE: 84 Met Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr
Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly Thr
Gln Asp Cys Ser Phe 20 25 30 Gln His Ser Pro Ile Ser Ser Asp Phe
Ala Val Lys Ile Arg Glu Leu 35 40 45 Ser Asp Tyr Leu Leu Gln Asp
Tyr Pro Val Thr Val Ala Ser Asn Leu 50 55 60 Gln Asp Glu Glu Leu
Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln 65 70 75 80 Arg Trp Met
Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly 85 90 95 Leu
Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala 100 105
110 Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser
115 120 125 Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys
Pro Trp 130 135 140 Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu
Gln Cys Gln Pro 145 150 155 160 Asp Ser Ser Thr Leu Pro Pro Pro Trp
Ser Pro Arg Pro Leu Glu Ala 165 170 175 Thr Ala Pro Thr Ala Pro Gln
180 <210> SEQ ID NO 85 <211> LENGTH: 252 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <223>
OTHER INFORMATION: Summary for CYAGEN (253 ORF4b) <400>
SEQUENCE: 85 Met Ala Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr
Tyr Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Ser Ser Gly Leu Ser Ala
Pro Ala Arg Ser Pro 20 25 30 Ser Pro Ser Thr Gln Pro Trp Glu His
Val Asn Ala Ile Gln Glu Ala 35 40 45 Arg Arg Leu Leu Asn Leu Ser
Arg Asp Thr Ala Ala Glu Met Asn Glu 50 55 60 Thr Val Glu Val Ile
Ser Glu Met Phe Asp Leu Gln Glu Pro Thr Cys 65 70 75 80 Leu Gln Thr
Arg Leu Glu Leu Tyr Lys Gln Gly Leu Arg Gly Ser Leu 85 90 95 Thr
Lys Leu Lys Gly Pro Leu Thr Met Met Ala Ser His Tyr Lys Gln 100 105
110 His Cys Pro Pro Thr Pro Glu Thr Ser Cys Ala Thr Gln Ile Ile Thr
115 120 125 Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp Phe Leu Leu Val
Ile Pro 130 135 140 Phe Asp Cys Trp Glu Pro Val Gln Glu Pro Thr Thr
Thr Pro Ala Pro 145 150 155 160 Arg Pro Pro Thr Pro Ala Pro Thr Ile
Ala Ser Gln Pro Leu Ser Leu 165 170 175 Arg Pro Glu Ala Cys Arg Pro
Ala Ala Gly Gly Ala Val His Thr Arg 180 185 190 Gly Leu Asp Phe Ala
Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly 195 200 205 Thr Cys Gly
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn 210 215 220 His
Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Lys 225 230
235 240 Ser Gly Asp Lys Pro Ser Leu Ser Ala Arg Tyr Val 245 250
<210> SEQ ID NO 86 <211> LENGTH: 261 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: mCD40L modified to stop cleavage <400> SEQUENCE:
86 Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly
1 5 10 15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val
Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val
Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn
Leu His Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys
Asn Thr Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu
Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu
Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110 Met Pro
Arg Gly Glu Glu Asp Ser Gln Ile Ala Ala His Val Ile Ser 115 120 125
Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130
135 140 Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys
Gln 145 150 155 160 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr
Ala Gln Val Thr 165 170 175 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln
Ala Pro Phe Ile Ala Ser 180 185 190 Leu Cys Leu Lys Ser Pro Gly Arg
Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205 Ala Asn Thr His Ser Ser
Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210 215 220 Leu Gly Gly Val
Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn 225 230 235 240 Val
Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 245 250
255 Gly Leu Leu Lys Leu 260 <210> SEQ ID NO 87 <211>
LENGTH: 261 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<220> FEATURE: <223> OTHER INFORMATION: Summary (261
ORF5) <400> SEQUENCE: 87
Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5
10 15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe
Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr
Leu His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu
His Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn
Thr Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile
Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn
Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110 Met Pro Arg
Gly Glu Glu Asp Ser Gln Ile Ala Ala His Val Ile Ser 115 120 125 Glu
Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130 135
140 Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln
145 150 155 160 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala
Gln Val Thr 165 170 175 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala
Pro Phe Ile Ala Ser 180 185 190 Leu Cys Leu Lys Ser Pro Gly Arg Phe
Glu Arg Ile Leu Leu Arg Ala 195 200 205 Ala Asn Thr His Ser Ser Ala
Lys Pro Cys Gly Gln Gln Ser Ile His 210 215 220 Leu Gly Gly Val Phe
Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn 225 230 235 240 Val Thr
Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 245 250 255
Gly Leu Leu Lys Leu 260 <210> SEQ ID NO 88 <211>
LENGTH: 221 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<220> FEATURE: <223> OTHER INFORMATION: mTNFalpha
modified to stop cleavage <400> SEQUENCE: 88 Met Ser Thr Glu
Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala 1 5 10 15 Leu Pro
Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe 20 25 30
Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe 35
40 45 Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe
Pro 50 55 60 Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val
Ala His Val 65 70 75 80 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln
Trp Leu Asn Arg Arg 85 90 95 Ala Asn Ala Leu Leu Ala Asn Gly Val
Glu Leu Arg Asp Asn Gln Leu 100 105 110 Val Val Pro Ser Glu Gly Leu
Tyr Leu Ile Tyr Ser Gln Val Leu Phe 115 120 125 Lys Gly Gln Gly Cys
Pro Ser Thr His Val Leu Leu Thr His Thr Ile 130 135 140 Ser Arg Ile
Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 145 150 155 160
Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 165
170 175 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu
Lys 180 185 190 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr
Leu Asp Phe 195 200 205 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile
Ala Leu 210 215 220 <210> SEQ ID NO 89 <211> LENGTH:
221 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: Summary (221 ORF6) <400>
SEQUENCE: 89 Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala
Glu Glu Ala 1 5 10 15 Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser
Arg Arg Cys Leu Phe 20 25 30 Leu Ser Leu Phe Ser Phe Leu Ile Val
Ala Gly Ala Thr Thr Leu Phe 35 40 45 Cys Leu Leu His Phe Gly Val
Ile Gly Pro Gln Arg Glu Glu Phe Pro 50 55 60 Arg Asp Leu Ser Leu
Ile Ser Pro Leu Ala Gln Ala Val Ala His Val 65 70 75 80 Val Ala Asn
Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 85 90 95 Ala
Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 100 105
110 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe
115 120 125 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His
Thr Ile 130 135 140 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn
Leu Leu Ser Ala 145 150 155 160 Ile Lys Ser Pro Cys Gln Arg Glu Thr
Pro Glu Gly Ala Glu Ala Lys 165 170 175 Pro Trp Tyr Glu Pro Ile Tyr
Leu Gly Gly Val Phe Gln Leu Glu Lys 180 185 190 Gly Asp Arg Leu Ser
Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 195 200 205 Ala Glu Ser
Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 210 215 220 <210> SEQ
ID NO 90 <211> LENGTH: 244 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION:
wild-type <400> SEQUENCE: 90 Met Asp Pro Asn Arg Ile Ser Glu
Asp Gly Thr His Cys Ile Tyr Arg 1 5 10 15 Ile Leu Arg Leu His Glu
Asn Ala Asp Phe Gln Asp Thr Thr Leu Glu 20 25 30 Ser Gln Asp Thr
Lys Leu Ile Pro Asp Ser Cys Arg Arg Ile Lys Gln 35 40 45 Ala Phe
Gln Gly Ala Val Gln Lys Glu Leu Gln His Ile Val Gly Ser 50 55 60
Gln His Ile Arg Ala Glu Lys Ala Met Val Asp Gly Ser Trp Leu Asp 65
70 75 80 Leu Ala Lys Arg Ser Lys Leu Glu Ala Gln Pro Phe Ala His
Leu Thr 85 90 95 Ile Asn Ala Thr Asp Ile Pro Ser Gly Ser His Lys
Val Ser Leu Ser 100 105 110 Ser Trp Tyr His Asp Arg Gly Trp Ala Lys
Ile Ser Asn Met Thr Phe 115 120 125 Ser Asn Gly Lys Leu Ile Val Asn
Gln Asp Gly Phe Tyr Tyr Leu Tyr 130 135 140 Ala Asn Ile Cys Phe Arg
His His Glu Thr Ser Gly Asp Leu Ala Thr 145 150 155 160 Glu Tyr Leu
Gln Leu Met Val Tyr Val Thr Lys Thr Ser Ile Lys Ile 165 170 175 Pro
Ser Ser His Thr Leu Met Lys Gly Gly Ser Thr Lys Tyr Trp Ser 180 185
190 Gly Asn Ser Glu Phe His Phe Tyr Ser Ile Asn Val Gly Gly Phe Phe
195 200 205 Lys Leu Arg Ser Gly Glu Glu Ile Ser Ile Glu Val Ser Asn
Pro Ser 210 215 220 Leu Leu Asp Pro Asp Gln Asp Ala Thr Tyr Phe Gly
Ala Phe Lys Val 225 230 235 240 Arg Asp Ile Asp <210> SEQ ID
NO 91 <400> SEQUENCE: 91 000 <210> SEQ ID NO 92
<211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <223> OTHER INFORMATION: FLT3L signal
<400> SEQUENCE: 92 Met Ala Thr Val Leu Ala Pro Ala Trp Ser
Pro Thr Thr Tyr Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Ser Ser Gly
Leu Ser 20 25
<210> SEQ ID NO 93 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <223> OTHER
INFORMATION: Linker <400> SEQUENCE: 93 Gly Gly Gly Gly Ser 1
5 <210> SEQ ID NO 94 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <223> OTHER
INFORMATION: V5 epitope tag for flow detection <400>
SEQUENCE: 94 Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser
Thr 1 5 10 <210> SEQ ID NO 95 <211> LENGTH: 118
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <220> FEATURE:
<223> OTHER INFORMATION: anti-biotin murine vH with inserted
Cys for intralinkage <400> SEQUENCE: 95 Gln Val Lys Leu Gln
Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser
Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ala Tyr 20 25 30 Gly
Val Asp Trp Val Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Leu 35 40
45 Gly Val Ile Trp Gly Gly Gly Arg Thr Asn Tyr Asn Ser Gly Leu Met
50 55 60 Ser Arg Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln Val
Phe Leu 65 70 75 80 Thr Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Lys
Tyr Tyr Cys Val 85 90 95 Lys His Thr Asn Trp Asp Gly Gly Phe Ala
Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
<210> SEQ ID NO 96 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <223> OTHER
INFORMATION: Linker <400> SEQUENCE: 96 Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> SEQ
ID NO 97 <211> LENGTH: 99 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <223> OTHER INFORMATION: LC
Variable <400> SEQUENCE: 97 Gly Ser Pro Gly Gln Ser Val Ser
Ile Ser Cys Ser Gly Ser Ser Ser 1 5 10 15 Asn Ile Gly Asn Asn Tyr
Val Tyr Trp Tyr Gln His Leu Pro Gly Thr 20 25 30 Ala Pro Lys Leu
Leu Ile Tyr Ser Asp Thr Lys Arg Pro Ser Gly Val 35 40 45 Pro Asp
Arg Ile Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala 50 55 60
Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser 65
70 75 80 Trp Asp Asp Ser Leu Asp Gly Pro Val Phe Gly Cys Gly Thr
Lys Leu 85 90 95 Thr Val Leu <210> SEQ ID NO 98 <211>
LENGTH: 99 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<220> FEATURE: <223> OTHER INFORMATION: CD8alpha
transmembrane and cytoplasmic domain <400> SEQUENCE: 98 Pro
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 1 5 10
15 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
20 25 30 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
Ile Tyr 35 40 45 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu
Leu Leu Ser Leu 50 55 60 Val Ile Thr Leu Tyr Cys Asn His Arg Asn
Arg Arg Arg Val Cys Lys 65 70 75 80 Cys Pro Arg Pro Val Val Lys Ser
Gly Asp Lys Pro Ser Leu Ser Ala 85 90 95 Arg Tyr Val <210>
SEQ ID NO 99 <211> LENGTH: 244 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: Summary (244 ORF7a) <400> SEQUENCE: 99 Met Asp
Pro Asn Arg Ile Ser Glu Asp Gly Thr His Cys Ile Tyr Arg 1 5 10 15
Ile Leu Arg Leu His Glu Asn Ala Asp Phe Gln Asp Thr Thr Leu Glu 20
25 30 Ser Gln Asp Thr Lys Leu Ile Pro Asp Ser Cys Arg Arg Ile Lys
Gln 35 40 45 Ala Phe Gln Gly Ala Val Gln Lys Glu Leu Gln His Ile
Val Gly Ser 50 55 60 Gln His Ile Arg Ala Glu Lys Ala Met Val Asp
Gly Ser Trp Leu Asp 65 70 75 80 Leu Ala Lys Arg Ser Lys Leu Glu Ala
Gln Pro Phe Ala His Leu Thr 85 90 95 Ile Asn Ala Thr Asp Ile Pro
Ser Gly Ser His Lys Val Ser Leu Ser 100 105 110 Ser Trp Tyr His Asp
Arg Gly Trp Ala Lys Ile Ser Asn Met Thr Phe 115 120 125 Ser Asn Gly
Lys Leu Ile Val Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr 130 135 140 Ala
Asn Ile Cys Phe Arg His His Glu Thr Ser Gly Asp Leu Ala Thr 145 150
155 160 Glu Tyr Leu Gln Leu Met Val Tyr Val Thr Lys Thr Ser Ile Lys
Ile 165 170 175 Pro Ser Ser His Thr Leu Met Lys Gly Gly Ser Thr Lys
Tyr Trp Ser 180 185 190 Gly Asn Ser Glu Phe His Phe Tyr Ser Ile Asn
Val Gly Gly Phe Phe 195 200 205 Lys Leu Arg Ser Gly Glu Glu Ile Ser
Ile Glu Val Ser Asn Pro Ser 210 215 220 Leu Leu Asp Pro Asp Gln Asp
Ala Thr Tyr Phe Gly Ala Phe Lys Val 225 230 235 240 Arg Asp Ile Asp
<210> SEQ ID NO 100 <211> LENGTH: 381 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <223> OTHER
INFORMATION: Summary (381aa ORF7b) <400> SEQUENCE: 100 Met
Ala Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu 1 5 10
15 Leu Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly Gly Gly Gly Ser Gly
20 25 30 Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Gly
Gly Gly 35 40 45 Gly Ser Gln Val Lys Leu Gln Glu Ser Gly Pro Gly
Leu Val Ala Pro 50 55 60 Ser Gln Ser Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr 65 70 75 80 Ala Tyr Gly Val Asp Trp Val Arg
Gln Pro Pro Gly Lys Cys Leu Glu
85 90 95 Trp Leu Gly Val Ile Trp Gly Gly Gly Arg Thr Asn Tyr Asn
Ser Gly 100 105 110 Leu Met Ser Arg Leu Ser Ile Arg Lys Asp Asn Ser
Lys Ser Gln Val 115 120 125 Phe Leu Thr Met Asn Ser Leu Gln Thr Asp
Asp Thr Ala Lys Tyr Tyr 130 135 140 Cys Val Lys His Thr Asn Trp Asp
Gly Gly Phe Ala Tyr Trp Gly Gln 145 150 155 160 Gly Thr Thr Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 165 170 175 Gly Ser Gly
Gly Gly Gly Ser Gly Ser Pro Gly Gln Ser Val Ser Ile 180 185 190 Ser
Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Tyr Trp 195 200
205 Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ser Asp
210 215 220 Thr Lys Arg Pro Ser Gly Val Pro Asp Arg Ile Ser Gly Ser
Lys Ser 225 230 235 240 Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu
Gln Ser Glu Asp Glu 245 250 255 Ala Asp Tyr Tyr Cys Ala Ser Trp Asp
Asp Ser Leu Asp Gly Pro Val 260 265 270 Phe Gly Cys Gly Thr Lys Leu
Thr Val Leu Pro Thr Thr Thr Pro Ala 275 280 285 Pro Arg Pro Pro Thr
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 290 295 300 Leu Arg Pro
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 305 310 315 320
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 325
330 335 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
Cys 340 345 350 Asn His Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg
Pro Val Val 355 360 365 Lys Ser Gly Asp Lys Pro Ser Leu Ser Ala Arg
Tyr Val 370 375 380 <210> SEQ ID NO 101 <211> LENGTH:
11 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide <400>
SEQUENCE: 101 Gln Met Gln Gly Val Asn Cys Thr Val Ser Ser 1 5 10
<210> SEQ ID NO 102 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <220> FEATURE: <223> OTHER
INFORMATION: CpG <220> FEATURE: <221> NAME/KEY:
modified_base <222> LOCATION: (1)..(2) <223> OTHER
INFORMATION: g-phosphorothioate <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (25)..(29)
<223> OTHER INFORMATION: g-phosphorothioate <400>
SEQUENCE: 102 ggaaccgtat cggcgatatc ggttgggggg 30 <210> SEQ
ID NO 103 <211> LENGTH: 30 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <220> FEATURE: <223> OTHER INFORMATION:
CpG <220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (1)..(2) <223> OTHER INFORMATION:
g-phosphorothioate <220> FEATURE: <221> NAME/KEY:
modified_base <222> LOCATION: (25)..(29) <223> OTHER
INFORMATION: g-phosphorothioate <400> SEQUENCE: 103
ggaaccgtat gcggcatatc ggttgggggg 30 <210> SEQ ID NO 104
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide <400> SEQUENCE: 104 Val Arg Ser Ser Ser Arg Thr Pro
Ser Asp Lys Pro 1 5 10 <210> SEQ ID NO 105 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic peptide <400>
SEQUENCE: 105 Met Tyr Pro Pro Pro Tyr 1 5 <210> SEQ ID NO 106
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 106 Tyr Met Asn Met 1 <210> SEQ ID NO
107 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 107 Pro Tyr Ala Pro 1 <210> SEQ
ID NO 108 <211> LENGTH: 183 <212> TYPE: PRT <213>
ORGANISM: Unknown <220> FEATURE: <223> OTHER
INFORMATION: Description of Unknown: OX40L sequence <400>
SEQUENCE: 108 Met Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn
Ala Ala Arg 1 5 10 15 Pro Arg Phe Glu Arg Asn Lys Leu Leu Leu Val
Ala Ser Val Ile Gln 20 25 30 Gly Leu Gly Leu Leu Leu Cys Phe Thr
Tyr Ile Cys Leu His Phe Ser 35 40 45 Ala Leu Gln Val Ser His Arg
Tyr Pro Arg Ile Gln Ser Ile Lys Val 50 55 60 Gln Phe Thr Glu Tyr
Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln 65 70 75 80 Lys Glu Asp
Glu Ile Met Lys Val Gln Asn Asn Ser Val Ile Ile Asn 85 90 95 Cys
Asp Gly Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu 100 105
110 Val Asn Ile Ser Leu His Tyr Gln Lys Asp Glu Glu Pro Leu Phe Gln
115 120 125 Leu Lys Lys Val Arg Ser Val Asn Ser Leu Met Val Ala Ser
Leu Thr 130 135 140 Tyr Lys Asp Lys Val Tyr Leu Asn Val Thr Thr Asp
Asn Thr Ser Leu 145 150 155 160 Asp Asp Phe His Val Asn Gly Gly Glu
Leu Ile Leu Ile His Gln Asn 165 170 175 Pro Gly Glu Phe Cys Val Leu
180 <210> SEQ ID NO 109 <211> LENGTH: 193 <212>
TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:
<223> OTHER INFORMATION: Description of Unknown: CD27L
sequence <400> SEQUENCE: 109 Met Pro Glu Glu Gly Ser Gly Cys
Ser Val Arg Arg Arg Pro Tyr Gly 1 5 10 15 Cys Val Leu Arg Ala Ala
Leu Val Pro Leu Val Ala Gly Leu Val Ile 20 25 30 Cys Leu Val Val
Cys Ile Gln Arg Phe Ala Gln Ala Gln Gln Gln Leu 35 40 45 Pro Leu
Glu Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His
50 55 60 Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly
Pro Ala 65 70 75 80 Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp
Lys Gly Gln Leu 85 90 95 Arg Ile His Arg Asp Gly Ile Tyr Met Val
His Ile Gln Val Thr Leu 100 105 110 Ala Ile Cys Ser Ser Thr Thr Ala
Ser Arg His His Pro Thr Thr Leu 115 120 125 Ala Val Gly Ile Cys Ser
Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg 130 135 140 Leu Ser Phe His
Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro 145 150 155 160 Leu
Ala Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu 165 170
175 Pro Ser Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg
180 185 190 Pro <210> SEQ ID NO 110 <211> LENGTH: 288
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 110 Met Gly His Thr Arg Arg Gln Gly Thr Ser
Pro Ser Lys Cys Pro Tyr 1 5 10 15 Leu Asn Phe Phe Gln Leu Leu Val
Leu Ala Gly Leu Ser His Phe Cys 20 25 30 Ser Gly Val Ile His Val
Thr Lys Glu Val Lys Glu Val Ala Thr Leu 35 40 45 Ser Cys Gly His
Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile 50 55 60 Tyr Trp
Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp 65 70 75 80
Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr 85
90 95 Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu
Gly 100 105 110 Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala
Phe Lys Arg 115 120 125 Glu His Leu Ala Glu Val Thr Leu Ser Val Lys
Ala Asp Phe Pro Thr 130 135 140 Pro Ser Ile Ser Asp Phe Glu Ile Pro
Thr Ser Asn Ile Arg Arg Ile 145 150 155 160 Ile Cys Ser Thr Ser Gly
Gly Phe Pro Glu Pro His Leu Ser Trp Leu 165 170 175 Glu Asn Gly Glu
Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp 180 185 190 Pro Glu
Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met 195 200 205
Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg 210
215 220 Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe
Pro 225 230 235 240 Asp Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile
Ser Val Asn Gly 245 250 255 Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys
Phe Ala Pro Arg Cys Arg 260 265 270 Glu Arg Arg Arg Asn Glu Arg Leu
Arg Arg Glu Ser Val Arg Pro Val 275 280 285 <210> SEQ ID NO
111 <211> LENGTH: 329 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 111 Met Asp Pro Gln
Cys Thr Met Gly Leu Ser Asn Ile Leu Phe Val Met 1 5 10 15 Ala Phe
Leu Leu Ser Gly Ala Ala Pro Leu Lys Ile Gln Ala Tyr Phe 20 25 30
Asn Glu Thr Ala Asp Leu Pro Cys Gln Phe Ala Asn Ser Gln Asn Gln 35
40 45 Ser Leu Ser Glu Leu Val Val Phe Trp Gln Asp Gln Glu Asn Leu
Val 50 55 60 Leu Asn Glu Val Tyr Leu Gly Lys Glu Lys Phe Asp Ser
Val His Ser 65 70 75 80 Lys Tyr Met Gly Arg Thr Ser Phe Asp Ser Asp
Ser Trp Thr Leu Arg 85 90 95 Leu His Asn Leu Gln Ile Lys Asp Lys
Gly Leu Tyr Gln Cys Ile Ile 100 105 110 His His Lys Lys Pro Thr Gly
Met Ile Arg Ile His Gln Met Asn Ser 115 120 125 Glu Leu Ser Val Leu
Ala Asn Phe Ser Gln Pro Glu Ile Val Pro Ile 130 135 140 Ser Asn Ile
Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser Ile 145 150 155 160
His Gly Tyr Pro Glu Pro Lys Lys Met Ser Val Leu Leu Arg Thr Lys 165
170 175 Asn Ser Thr Ile Glu Tyr Asp Gly Val Met Gln Lys Ser Gln Asp
Asn 180 185 190 Val Thr Glu Leu Tyr Asp Val Ser Ile Ser Leu Ser Val
Ser Phe Pro 195 200 205 Asp Val Thr Ser Asn Met Thr Ile Phe Cys Ile
Leu Glu Thr Asp Lys 210 215 220 Thr Arg Leu Leu Ser Ser Pro Phe Ser
Ile Glu Leu Glu Asp Pro Gln 225 230 235 240 Pro Pro Pro Asp His Ile
Pro Trp Ile Thr Ala Val Leu Pro Thr Val 245 250 255 Ile Ile Cys Val
Met Val Phe Cys Leu Ile Leu Trp Lys Trp Lys Lys 260 265 270 Lys Lys
Arg Pro Arg Asn Ser Tyr Lys Cys Gly Thr Asn Thr Met Glu 275 280 285
Arg Glu Glu Ser Glu Gln Thr Lys Lys Arg Glu Lys Ile His Ile Pro 290
295 300 Glu Arg Ser Asp Glu Ala Gln Arg Val Phe Lys Ser Ser Lys Thr
Ser 305 310 315 320 Ser Cys Asp Lys Ser Asp Thr Cys Phe 325
* * * * *
References