U.S. patent application number 10/518701 was filed with the patent office on 2006-03-09 for vaccines for suppressing ige-mediated allergic disease and methods for using the same.
Invention is credited to Sandra Calarota, ArnoldI Levinson, Miguel Otero, DavidB Weiner.
Application Number | 20060052592 10/518701 |
Document ID | / |
Family ID | 30000536 |
Filed Date | 2006-03-09 |
United States Patent
Application |
20060052592 |
Kind Code |
A1 |
Levinson; ArnoldI ; et
al. |
March 9, 2006 |
Vaccines for suppressing ige-mediated allergic disease and methods
for using the same
Abstract
Nucleic acid molecules that encode a protein comprising at least
one epitope of membrane IgE free of epitopes present on the serum
IgE, including proteins that further comprise non-IgE T cell helper
epitope are disclosed. Vaccines, vectors and host cells that
comprise such nucleic acid molecules are disclosed. Isolated
proteins, including haptenized proteins, comprising at least one
epitope of membrane IgE free of epitopes present on the serum IgE,
including proteins that further comprise non-IgE T cell helper
epitope are disclosed. Vaccines that comprise and methods of making
such proteins and antibodies that specifically bind to such
proteins are disclosed. Killed or inactivated cells or particles,
including haptenized killed or inactivated cells or particles, that
comprise a protein comprising at least one epitope of membrane IgE
free of epitopes present on the serum IgE, including proteins that
further comprise non-IgE T cell helper epitope are disclosed.
Vaccines that comprise such killed or inactivated cells or
particles are disclosed. Methods of treating and preventing IgE
mediated allergic disease or condition are disclosed.
Inventors: |
Levinson; ArnoldI; (Radnor,
PA) ; Calarota; Sandra; (Philadelphia, PA) ;
Weiner; DavidB; (Merion, PA) ; Otero; Miguel;
(San Juan, PR) |
Correspondence
Address: |
COZEN O'CONNOR, P.C.
1900 MARKET STREET
PHILADELPHIA
PA
19103-3508
US
|
Family ID: |
30000536 |
Appl. No.: |
10/518701 |
Filed: |
June 20, 2003 |
PCT Filed: |
June 20, 2003 |
PCT NO: |
PCT/US03/19383 |
371 Date: |
September 1, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60390304 |
Jun 20, 2002 |
|
|
|
Current U.S.
Class: |
536/23.1 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 16/00 20130101; A61K 2039/53 20130101; C07K 2317/52 20130101;
C07K 2319/55 20130101; C07K 2318/10 20130101; A61K 39/0005
20130101; A61K 39/08 20130101; A61K 39/00 20130101 |
Class at
Publication: |
536/023.1 ;
514/044 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07H 21/02 20060101 C07H021/02; C07H 21/04 20060101
C07H021/04 |
Claims
1. An isolated nucleic acid molecule that encodes protein
comprising at least one epitope of membrane IgE and being free of
epitopes of serum IgE.
2. The nucleic acid molecule of claim 1 wherein said protein is
membrane IgE or a fragment thereof.
3. The nucleic acid molecule of claim 2 wherein said protein is
membrane IgE.
4. The nucleic acid molecule of claim 1 further comprising coding
sequence encoding of at least one non-IgE helper T cell
epitope.
5. The nucleic acid molecule of claim 4 wherein the coding sequence
encoding of at least one non-IgE helper T cell epitope encodes
tetanus toxoid Th epitope.
6. The nucleic acid molecule of claim 2 wherein said nucleic acid
molecule is a plasmid.
7. The isolated nucleic acid molecule of claim 2 wherein said
nucleic acid molecule is incorporated in a viral vector or a
bacterial cell.
8. A vaccine composition comprising a nucleic acid molecule of
claim 1 and a pharmaceutically acceptable carrier or diluent.
9. A method of treating an individual who has been identified as
being susceptible to an IgE mediated allergic disease or condition
comprising the step of administering to such an individual a
prophylactically effective amount of a vaccine of 8.
10. A method of treating an individual who has been identified as
having an IgE mediated allergic disease or condition comprising the
step of administering to such an individual a therapeutically
effective amount of a vaccine of 8.
11. An isolated protein comprising at least one epitope of membrane
IgE and being free of epitopes of serum IgE.
12. The isolated protein of claim 11 wherein said protein is
membrane IgE or a fragment thereof.
13. The isolated protein of claim 12 wherein said protein is
membrane IgE.
14. The isolated protein of claim 11 further comprising tetanus
toxoid Th epitope.
15. The isolated protein of claim 11 wherein said protein is
haptenized.
16. The vaccine composition comprising an isolated protein of claim
11 and a pharmaceutically acceptable carrier or diluent.
17. The vaccine composition of claim 16 further comprising tetanus
toxoid Th epitope.
18. A vaccine composition comprising killed or inactivated cells or
particles that comprise a protein of claim 11 and a
pharmaceutically acceptable carrier or diluent.
19. The vaccine of claim 18 wherein said killed or inactivated
cells or particles are haptenized.
20. A method of treating an individual who has been identified as
being susceptible to an IgE mediated allergic disease or condition
comprising the step of administering to such an individual a
prophylactically effective amount of a vaccine of claim 16.
21. A method of treating an individual who has been identified as
having an IgE mediated allergic disease or condition comprising the
step of administering to such an individual a therapeutically
effective amount of a vaccine of claim 16.
22. A host cell comprising an isolated nucleic acid molecule that
encodes protein comprising at least one epitope of membrane IgE and
being free of epitopes of serum IgE.
23. The host cell of claim 22 wherein said protein is membrane IgE
or a fragment thereof.
24. The host cell of claim 23 wherein said protein is membrane
IgE.
25. The host cell of claim 22 further comprising coding sequence
encoding of at least one non-IgE helper T cell epitope.
26. The host cell of claim 25 wherein the coding sequence encoding
of at least one non-IgE helper T cell epitope encodes tetanus
toxoid Th epitope.
27. The host cell of claim 22 wherein said nucleic acid molecule is
a plasmid.
28. A method of producing a protein comprising at least one epitope
of membrane IgE and being free of epitopes of serum IgE comprising
culturing a host cell of claim 22 and isolating said protein
expressed thereby.
29. The method of claim 28 wherein the protein is isolated using
antibodies that specifically bind to said protein.
30. Antibodies that specifically bind to a protein of claim 11.
31. The antibodies of claim 30 wherein said antibodies are Mabs,
humanized Mabs, human antibodies, or Fab or (Fab).sub.2 thereof
Description
FIELD OF THE INVENTION
[0001] The present invention relates to vaccines, and to methods
for prophylacticallly and/or therapeutically immunizing individuals
against IgE mediated allergic.
BACKGROUND OF THE INVENTION
[0002] Allergic diseases affect well over 25% of the population in
industrialized nations. They account for a substantial amount of
morbidity and in some cases, mortality. These diseases include
asthma, allergic rhinitis, atopic dermatitis, food allergy, drug
allergy, anaphylaxis, and urticaria, amongst others. Despite the
development of new pharmaceutical agents like inhaled
corticosteroids, non-sedating antihistamines and leukotriene
inhibitors, the most prevalent allergic disorders, namely, asthma
and allergic rhinitis, continue to represent debilitating and
costly conditions, In the U.S. alone, 40 million people suffer from
allergic rhinitis at a cost of over $7 billion dollars. Asthma
sufferers number over 17 million and account for approximately
$10.7 billion dollars in health care-related expenditures.
[0003] The lynchpin of allergic inflammation is the IgE
immunoglobulin molecule. For some time, it has been appreciated
that IgE antibodies specific for environmental allergens bind to
specialized receptors on target cells, called mast cells, that are
distributed along the tissues that line the respiratory tract,
gastrointestinal tract, nerve endings, and blood vessels. The
encounter of such IgE-sensitized mast cells with specific
allergens, e.g. ragweed pollen, bee venom, or latex protein,
triggers the release of many chemical mediators. These, in turn,
engender a characteristic pattern of allergic inflammation in the
involved tissues and cause allergic symptoms like congested runny
nose, itchy eyes, wheezing, shortness of breath, and, in the worst
case scenario, cardiovascular collapse and death.
[0004] In the past decade, extraordinary gains have been made in
our understanding of the cellular and molecular basis of IgE
production and regulation of the allergic inflammatory response. It
is now appreciated that B cells that give rise to IgE secreting
plasma cells do so only with the assistance of so-called helper T
cells that also react with the offending allergen. This assistance
is provided by a physical interaction of the allergen specific T
and B cells and the provision of soluble factors from the T cells
to the B cells that foster their maturation into IgE-secreting
plasma cells. Such helper T cells are called TH2 cells. By
contrast, TH1 cells, another population of helper T cells, inhibit
the activity of allergy-promoting TH2 cells by secreting a myriad
of counter-regulatory molecules that interfere whit TH2 cell
function. It appears that the balance of helper activity in
allergic individuals is skewed towards the TH2 cells, thus favoring
the development of an IgE/allergic inflammatory response.
[0005] Based on this overall mechanistic understanding of the
allergic inflammatory response, a number of strategies have emerged
to treat allergic diseases like allergic rhinitis and asthma.
Several are directed at interdicting the activity of
allergen-specific TH2 cells. These include direct inhibition of TH2
cell activity or augmentation of allergen-specific TH1 cell
activity with resultant indirect inhibitory action of TH2 cells.
Others interfere with IgE-mediated allergic inflammation, e.g.,
prevention of IgE binding the mast cell receptors and interference
with the biochemical signals in allergen-triggered IgE-sensitized
mast cells that lead to the release of inflammatory mediators. All
of these approaches have shown efficacy in short-term animal models
of allergic inflammation including asthma. To date early clinical
trials have provided some evidence for clinical efficacy of some of
these approaches but their addition to the clinical armamentarium
may be limited by toxicity and/or unacceptable expense.
[0006] There remains a need for effective compositions and methods
for preventing and treating IgE mediated allergic disease and
conditions.
SUMMARY OF THE INVENTION
[0007] Aspects of the invention relate to nucleic acid molecules
that encode a protein comprising at least one epitope of membrane
IgE free of epitopes present on the serum IgE, The nucleic acid
molecule may further comprise coding sequences encoding a non-IgE
helper T cell epitope. The nucleic acid molecules are free of
coding sequences encoding epitopes present on the serum IgE. In
some embodiments, the nucleic acid molecules that encode protein
consisting of the membrane or a fragment thereof. In some
embodiments, the nucleic acid molecules that encode isolated
protein consists of the membrane. In some embodiments, the nucleic
acid molecule is a plasmid.
[0008] Aspects of the invention relate to vaccines which comprise
such nucleic acid molecules and a pharmaceutically acceptable
carrier or diluent. Such vaccines are free of epitopes from serum
IgE and free of nucleic acid molecules that contain coding
sequences encoding epitopes present on the serum IgE. Such vaccines
may comprise coding sequences encoding a non-IgE helper T cell
epitope or a peptide that is a non-IgE helper T cell epitope.
[0009] Aspects of the invention relate to vectors that comprise
nucleic acid molecules that encode a protein comprising at least
one epitope of membrane IgE free of epitopes present on the serum
IgE. The vector is free of epitopes from serum IgE and free of
nucleic acid molecules that contain coding sequences encoding
epitopes present on the serum IgE. The vector may further comprise
coding sequences encoding a non-IgE helper T cell epitope or a
peptide that is a non-IgE helper T cell epitope. In some
embodiments, the vector comprises a nucleic acid molecule that
encodes a protein that consists of membrane IgE or a fragment
thereof. In some embodiments, the vector comprises a nucleic acid
molecule that encodes a protein that consists of membrane IgE. In
some embodiments, the vector is a virus or a bacterial cell. In
some embodiments, the vector is a recombinant vaccinia virus.
[0010] Aspects of the invention relate to vaccines which comprise
such vectors and a pharmaceutically acceptable carrier or diluent.
Such vaccines are free of epitopes from serum IgE and free of
nucleic acid molecules that contain coding sequences encoding
epitopes present on the serum IgE. The vaccines may further
comprise coding sequences encoding a non-IgE helper T cell epitope
or a peptide that is a non-IgE helper T cell epitope.
[0011] Aspects of the invention relate to an isolated protein
comprising at least one epitope of membrane IgE free of epitopes
present on the serum IgE. The protein may be a fusion protein which
additionally comprises a non-IgE helper T cell epitope. In some
embodiments, the isolated protein consists of the membrane IgE or a
fragment thereof. In some embodiments, the isolated protein
consists of the membrane IgE. In some embodiments, the protein is
haptenized.
[0012] Aspects of the invention relate to vaccines which comprise
such proteins or fusion proteins and a pharmaceutically acceptable
carrier or diluent. Such vaccines are free of epitopes from serum
IgE and free of nucleic acid molecules that contain coding
sequences encoding epitopes present on the serum IgE and may a
non-IgE helper T cell epitope wither as part of a fusion protein or
as a different peptide or protein.
[0013] Aspects of the invention relate to killed or inactivated
cells or particles that comprise a protein comprising at least one
epitope of membrane IgE free of epitopes present on the serum IgE.
The killed or inactivated cell or particles are free of epitopes
from serum
[0014] IgE and free of nucleic acid molecules that contain coding
sequences encoding epitopes present on the serum IgE. The killed or
inactivated cells or particles may further comprise coding
sequences encoding a non-IgE helper T cell epitope or a peptide
that is a non-IgE helper T cell epitope. In some embodiments, the
killed or inactivated cells or particles comprise membrane IgE
protein or a fragment thereof and/or nucleic acid molecules that
contain coding sequences encoding membrane IgE protein or a
fragment thereof. In some embodiments, the killed or inactivated
cells or particles comprise membrane IgE protein and/or nucleic
acid molecules that contain coding sequences encoding membrane IgE
protein. In some embodiments, the killed or inactivated cells or
particles is a killed or inactivated B cell. In some embodiments,
the killed or inactivated cells or particles is haptenized.
[0015] Aspects of the invention relate to vaccines which comprise
such killed or inactivated cells or particles and a
pharmaceutically acceptable carrier or diluent. Such vaccines are
free of epitopes from serum IgE and free of nucleic acid molecules
that contain coding sequences encoding epitopes present on the
serum IgE. IgE. The vaccine may further comprise coding sequences
encoding a non-IgE helper T cell epitope or a peptide that is a
non-IgE helper T cell epitope.
[0016] Aspects of the present invention relate to methods of
treating an individual suffering from IgE mediated allergic disease
or condition. The method comprises administering to such an
individual a therapeutically effective amount of a vaccine of the
invention.
[0017] Aspects of the present invention relate to methods of
preventing IgE mediated allergic disease or condition in an
individual. The method comprises administering to an individual a
therapeutically effective amount of a vaccine of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 diagrams the construction of a gene construct
encoding membrane IgE. FIG. 1 shows the cloning of human membrane
bound IgE by RT-PCR including a schematic representation of the
secretory and two membrane human IgE isoforms and the RT-PCR
strategy to amplify membrane bound long form IgE.
[0019] FIG. 2 diagrams the construction of a gene construct
encoding membrane IgE. FIG. 2 shows the construction of the vector
insert for expression of a membrane IgE fused to tetanus toxoid
including a schematic representation of the a membrane IgE-tetanus
toxoid fusion protein expression cassette and PCR amplification
with specific primer set.
[0020] FIG. 3 shows in vitro expression data of a membrane IgE
construct.
[0021] FIG. 4 shows in vitro expression data of membrane IgE
constructs.
[0022] FIG. 5 shows a diagram of the cloning of membrane IgE
tetanus toxoid sequences and 6.times.HisTag into the pVAX
vector.
[0023] FIG. 6 shows the nucleotide and amino acid sequences of an
membrane IgE tetanus toxoid construct.
[0024] FIG. 7 is a table of synthetic peptides used as antigens in
in vitro assays.
[0025] FIG. 8 shows in vitro assay data. Splenocytes from naive,
vector-only immunized and mlge-TT construct immunized mice were
isolated and stimulated with the synthetic IgE peptide antigens in
FIG. 7 and interferon gamma production was assessed.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0026] As used herein, the terms "membrane IgE" and mIgE are used
interchangeably and meant to refer, unless further more
specifically identified as being the short or long form, either of
the two membrane bound forms of IgE (the membrane bound short form
m/sIgE and the membrane bound long form m/lIgE) as distinguished
from the secreted isoform, serum IgE (sIgE). FIG. 1 includes
schematic diagrams showing the three isoforms.
[0027] As used herein, the term "target protein" is meant to refer
to a protein which comprises at least one epitope of membrane IgE
and is free of any serum IgE epitopes. In some embodiments, the
target protein is a membrane IgE or fragment thereof. In some
embodiments, the target protein is the long form of the membrane
IgE or fragment thereof. In some embodiments, the target protein is
the short form of membrane IgE or fragment thereof. The target
protein preferably includes a non-IgE helper T cell epitope. The
preferred non-IgE helper T cell epitope is a helper T cell epitope
from tetanus toxoid. In some embodiments, the target protein is a
fusion protein comprising at least one epitope of membrane IgE and
a non-IgE helper T cell epitope, free of any serum IgE epitopes
wherein the between one or more epitopes of membrane IgE and the
non-IgE helper T cell epitope is a proteolytic cleavage site.
[0028] As used herein, the term "genetic construct" refers to the
DNA or RNA molecules that comprise a nucleotide sequence, which
encodes a target protein or immunomodulating protein. The coding
sequence includes initiation and termination signals operably
linked to regulatory elements including a promoter and
polyadenylation signal capable of directing expression in the cells
of the individual to whom the nucleic acid molecule is
administered.
[0029] As used herein, the term "expressible form" refers to gene
constructs which contain the necessary regulatory elements operable
linked to a coding sequence that encodes a target protein or an
immunomodulating protein, such that when present in the cell of the
individual, the coding sequence will be expressed.
[0030] The present invention provides vaccines and methods for
prophylactically and/or therapeutically immunizing individuals
against IgE mediated allergic by targeting IgE-expressing B cells.
By immunizing the allergic patient to components of IgE that are
exclusively expressed on the B cell membrane (membrane IgE), this
population of B cells is eradicated or at least substantially
reduce in number, thus removing the source of IgE production.
[0031] B cell membrane IgE structure differs from that of IgE in
the serum (which finds its way to the surface of mast cells) by
containing an additional protein fragment that is found in its
cytoplasmic, transmembrane, and membrane-proximal extracellular
domains. Accordingly, it should be possible to generate an immune
response against components of membrane IgE that will not react
with serum IgE. Such an immune response will not be competed by in
the serum and will not target IgE sitting on mast cells. Therefore,
patients will not be at risk for potentially deleterious mast cell
release reactions. Importantly, these membrane IgE components
differ significantly from the B cell membrane constituents of IgG
and IgM. Therefore, B cells expressing these immunoglobulin
isotypes, which represent critical players in normal host defense,
will not be targeted.
[0032] According to the invention, immune responses to IgE bearing
B cells are elicited. These immune responses make it possible for
the allergic host to scuttle not only IgE bearing B cells in the
extant immune repertoire but also those that might develop in the
future. This approach has important advantages over a monoclonal
anti-IgE antibody, which targets serum IgE. The latter biologic
agent requires multiple injections of extremely costly
compositions, and its use will likely be limited to a small subset
of allergic individuals. By contrast, the immunotherapeutic
strategy employed in the present invention requires limited patient
encounters and will be very inexpensive to mass-produce.
Accordingly, it should enjoy widespread use amongst populations of
allergic patients.
[0033] According to some embodiments of the invention, the target
protein is delivered to an individual to elicit an immune response
against the B cells include delivering the target protein using
nucleic acid molecules that encode the target protein. When the
nucleic acid molecules that encode the target protein are taken up
by cells of the individual the nucleotide sequences that encode the
target protein is expressed in the cells and the proteins are
thereby delivered to the individual. Aspects of the invention
include methods of delivering the coding sequences of the target
protein on an isolated nucleic acid molecule, such as a plasmid or
as part of recombinant vaccines.
[0034] According to some embodiments of the invention, the target
protein is delivered to an individual to elicit an immune response
against the B cells that include the target protein by delivering
the target protein as a protein. Aspects of the invention include
methods of delivering the target protein as a protein/peptide, as a
haptenized protein/peptide, as a cell or particle that comprises
the protein/peptide, or as a haptenized cell or particle that
comprises the protein/peptide.
[0035] According to some aspects of the present invention,
compositions and methods are provided which prophylactically and/or
therapeutically immunize an individual against a pathogen or
abnormal, disease-related cells. The vaccine may be any type of
vaccine such as, a subunit vaccine, a cell vaccine, a recombinant
vaccine or a nucleic, acid or DNA vaccine. By delivering the target
protein or nucleic acid molecules that encode the target protein,
the immune response induced by the vaccine may be modulated.
[0036] Regardless of the modality, compositions useful in the
invention generally comprise a non IgE helper T cell epitope to
provide T cell to induce an effective immune response, either as
part of the target protein and/or as a separate protein. If the non
IgE helper T cell epitope is part of a fusion protein that is the
target protein, the fusion protein may preferably contain
proteolytic cleavage sites between the membrane IgE epitope and the
non IgE helper T cell epitope. The non-IgE helper T cell epitope is
preferably tetanus toxoid helper T cell epitope. If the vaccine is
provided and a nucleic acid molecule, a nucleotide sequence is
provided that encodes a non IgE helper T cell epitope, preferably
tetanus toxoid helper T cell epitope. Thus, some aspects of the
invention comprise nucleic acid molecule that encode the target
protein and a non IgE helper T cell epitope. Some aspects of the
invention relate to composition comprising two nucleic acid
molecules, one that encodes the target protein and one that encodes
a non IgE helper T cell epitope. If the vaccine is provided and a
protein based vaccine, a protein is provided that comprises a non
IgE helper T cell epitope, preferably tetanus toxoid helper T cell
epitope. Thus, some aspects of the invention comprise the target
proteins that comprise a non IgE helper T cell epitope. Some
aspects of the invention relate to composition comprising two
protein molecules, the target protein and a non IgE helper T cell
epitope.
[0037] According to the present invention, the membrane IgE serves
as a target against which a protective and therapeutic immune
response can be induced. Specifically, vaccines are provided which
induce an immune response against the membrane IgE. The vaccines of
the invention include, but are not limited to, the following
vaccine technologies: [0038] 1) DNA vaccines, i.e. vaccines in
which DNA that encodes at least an epitope from membrane IgE is
administered to an individual's cells where the epitope is
expressed and serves as a target for an immune response; [0039] 2)
infectious vector mediated vaccines such as recombinant adenovirus,
vaccinia, Salmonella, and BCG wherein the vector carries genetic
information that encodes at least an epitope of membrane IgE such
that when the infectious vector is administered to an individual,
the epitope is expressed and serves as a target for an immune
response; [0040] 3) killed or inactivated vaccines which a)
comprise either killed cells or inactivated viral particles that
display at least an epitope of the membrane IgE and b) when
administered to an individual serves as a target for an immune
response; [0041] 3) haptenized killed or inactivated vaccines which
a) comprise either killed cells or inactivated viral particles that
display at least an epitope of membrane IgE, b) are haptenized to
be more immunogenic and c) when administered to an individual
serves as a target for an immune response; [0042] 4) subunit
vaccines which are vaccines that include protein molecules that
include at least an epitope membrane IgE; and [0043] 5) haptenized
subunit vaccines which are vaccines that a) include protein
molecules that include at least an epitope membrane IgE and b) are
haptenized to be more immunogenic.
[0044] The present invention relates to administering to an
individual a protein or nucleic acid molecule that comprises or
encodes, respectively, the target protein, which includes at least
one epitope from the membrane IgE, against which an therapeutic and
prophylactic immune response can be induced. Epitopes are generally
at least 6-8 amino acids in length. The vaccines of the invention
therefore comprise proteins which are at least, or nucleic acids
which encode at least 6-8 amino acids in length from membrane IgE.
In some embodiments the target protein contains at least 6 mIgE
amino acid sequences. In some embodiments the target protein
contains at least 10 mIgE amino acid sequences. In some embodiments
the target protein contains at least 15 mIgE amino acid sequences.
In some embodiments the target protein contains at least 20 mIgE
amino acid sequences. In some embodiments the target protein
contains at least 25 mIgE amino acid sequences. In some embodiments
the target protein contains at least 30 mIgE amino acid sequences.
In some embodiments the target protein contains at least 35 mIgE
amino acid sequences. In some embodiments the target protein
contains at least 40 mIgE amino acid sequences. In some embodiments
the target protein contains at least 45 mIgE amino acid sequences.
In some embodiments the target protein contains at least 50 mIgE
amino acid sequences. In some embodiments the target protein
contains at least 55 mIgE amino acid sequences. In some embodiments
the target protein contains at least 60 mIgE amino acid sequences.
In some embodiments the target protein contains at least 65 mIgE
amino acid sequences. In some embodiments the target protein
contains at least 70 mIgE amino acid sequences. In some embodiments
the target protein contains at least 75 mIgE amino acid sequences.
In some embodiments the target protein contains full length mIgE
amino acid sequences. It is intended that fragment of mIgE can be
used as the part of or as the target protein which comprise any
size fragment of the group of fragments from more than 6 up to full
length which include at least one epitope. With the required T cell
help induced by a non-IgE epitope, such fragments will induce
immune response to eliminate B cells. Immune response include CTL
responses and/or antibody production. In some embodiments, the
antibodies so produced may be isolated.
[0045] Nucleic acid molecules encoding a target protein comprising
a membrane IgE epitope may be delivered using any of several well
known technologies including DNA injection (also referred to as DNA
vaccination), recombinant vectors such as recombinant adenovims,
recombinant adenovirus associated virus and recombinant
vaccinia.
[0046] DNA vaccines are described in U.S. Pat. Nos. 5,593,972,
5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859,
5,703,055, 5,676,594, and the priority applications cited therein,
which are each incorporated herein by reference. In addition to the
delivery protocols described in those applications, alternative
methods of delivering DNA are described in U.S. Pat. Nos. 4,945,050
and 5,036,006, which are both incorporated herein by reference.
Routes of administration include, but are not limited to,
intramuscular, intransally, intraperitoneal, intradermal,
subcutaneous, intravenous, intraarterially, intraoccularly and oral
as well as topically, transdermally, by inhalation or suppository
or to mucosal tissue such as by lavage to vaginal, rectal,
urethral, buccal and sublingual tissue. Preferred routes of
administration include to mucosal tissue, intramuscular,
intraperitoneal, intradermal and subcutaneous injection. Genetic
constructs may be administered by means including, but not limited
to, traditional syringes, needleless injection devices, or
"microprojectile bombardment gone guns".
[0047] When taken up by a cell, the genetic construct(s) may remain
present in the cell as a functioning extracbromosomal molecule
and/or integrate into the cell's chromosomal DNA. DNA may be
introduced into cells where it remains as separate genetic material
in the form of a plasmid or plasmids. Alternatively, linear DNA
that can integrate into the chromosome may be introduced into the
cell. When introducing DNA into the cell, reagents that promote DNA
integration into chromosomes may be added. DNA sequences that are
useful to promote integration may also be included in the DNA
molecule. Alternatively, RNA may be administered to the cell. It is
also contemplated to provide the genetic construct as a linear
minichromosome including a centromere, telomeres and an origin of
replication. Gene constructs may remain part of the genetic
material in attenuated live microorganisms or recombinant microbial
vectors that live in cells. Gene constructs may be part of genomes
of recombinant viral vaccines where the genetic material either
integrates into the chromosome of the cell or remains
extrachromosomal. Genetic constructs include regulatory elements
necessary for gene expression of a nucleic acid molecule. The
elements include: a promoter, an initiation codon, a stop codon,
and a polyadenylation signal. In addition, enhancers are often
required for gene expression of the sequence that encodes the
target protein or the immunomodulating protein. It is necessary
that these elements be operable linked to the sequence that encodes
the desired proteins and that the regulatory elements are operably
in the individual to whom they are administered.
[0048] Initiation codons and stop codon are generally considered to
be part of a nucleotide sequence that encodes the desired protein.
However, it is necessary that these elements are functional in the
individual to whom the gene construct is administered. The
initiation and termination codons must be in frame with the coding
sequence.
[0049] Promoters and polyadenylation signals used must be
functional within the cells of the individual.
[0050] Examples of promoters useful to practice the present
invention, especially in the production of a genetic vaccine for
humans, include but are not limited to promoters from Simian Virus
40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human
Immunodeficiency Virus (MV) such as the BIV Long Terminal Repeat
(LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as
the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous
Sarcoma Virus (RSV) as well as promoters from human genes such as
human Actin, human Myosin, human Hemoglobin, human muscle creatine
and human metalothionein.
[0051] Examples of polyadenylation signals useful to practice the
present invention, especially in the production of a genetic
vaccine for humans, include but are not limited to SV40
polyadenylation signals and LTR polyadenylation signals. In
particular, the SV40 polyadenylation signal, which is in pCEP4
plasmid (Invitrogen, San Diego Calif.), referred to as the SV40
polyadenylation signal, is used.
[0052] In addition to the regulatory elements required for DNA
expression, other elements may also be included in the DNA
molecule. Such additional elements include enhancers. The enhancer
may be selected from the group including but not limited to: human
Actin, human Myosin, human Hemoglobin, human muscle creatine and
viral enhancers such as those from CMV, RSV and EBV.
[0053] Genetic constructs can be provided with mammalian origin of
replication in order to maintain the construct extrachromosomally
and produce multiple copies of the construct in the cell. Plasmids
pCEP4 and pREP4 from Invitrogen (San Diego, Calif.) contain the
Epstein Barr virus origin of replication and nuclear antigen EBNA-1
coding region which produces high copy episomal replication without
integration. Plasmids pVAX is a useful vector.
[0054] In some preferred embodiments, the IgE signal peptide is
included as part of the target protein.
[0055] In some preferred embodiments related to immunization
applications, nucleic acid molecule(s) are delivered which include
nucleotide sequences that encode a target protein, the
immunomodulating protein and, additionally, genes for proteins
which further enhance the immune response against such target
proteins. Examples of such genes are those which encode other
cytokines and lymphokines such as alpha-interferon,
gamma-interferon, platelet derived growth factor (PDGF), TNF,
GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, II-4, IL-6,
IL-10, IL-12 and IL-15 including IL-15 having the signal sequence
deleted and optionally including the signal peptide from IgE.
[0056] An additional element may be added which serves as a target
for cell destruction if it is desirable to eliminate cells
receiving the genetic construct for any reason. A herpes thymidine
kinase (tk) gene in an expressible form can be included in the
genetic construct. The drug gangcyclovir can be administered to the
individual and that drug will cause the selective killing of any
cell producing tk, thus, providing the means for the selective
destruction of cells with the genetic construct.
[0057] In order to maximize protein production, regulatory
sequences may be selected which are well suited for gene expression
in the cells the construct is administered into. Moreover, codons
may be selected which are most efficiently transcribed in the cell.
One having ordinary skill in the art can produce DNA constructs
that are functional in the cells.
[0058] One method of the present invention comprises the steps of
administering nucleic acid molecules intramuscularly, intranasally,
intraperatoneally, subcutaneously, intradermally, or topically or
by lavage to mucosal tissue selected from the group consisting of
inhalation, vaginal, rectal, urethral, buccal and sublingual.
[0059] In some embodiments, the nucleic acid molecule is delivered
to the cells in conjunction with administration of a polynucleotide
function enhancer or a genetic vaccine facilitator agent.
Polynucleotide function enhancers are described in U.S. Ser. No.
08/008,342 filed Jan. 26, 1993, U.S. Ser. No. 08/029,336 filed Mar.
11, 1993, U.S. Ser. No. 08/125,012 filed Sep. 21, 1993, and
International Application Serial Number PCT/IJ94/00899 filed Jan.
26, 1994, which are each incorporated herein by reference. Genetic
vaccine facilitator agents are described in U.S. Ser. No. 0021,579
filed Apr. 1, 1994, which is incorporated herein by reference. The
co-agents winch are administered in conjunction with nucleic acid
molecules may be administered as a mixture with the nucleic acid
molecule or administered separately simultaneously, before or after
administration of nucleic acid molecules. In addition, other agents
which may function transfecting agents and/or replicating agents
and/or inflammatory agents and which may be co-administered with a
GVF include growth factors, cytokines and lymphokines such as
.alpha.-interferon, gamma-interferon, GM-CSF, platelet derived
growth factor (PDGF), TNF, epidermal growth factor (EGF), ILA,
IL-2, IL-4, IL-6, IL-10, IL-12 and IL-15 as well as fibroblast
growth factor, surface active agents such as immune-stimulating
complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog
including monophosphoryl Lipid A (WL), muramyl peptides, quinone
analogs and vesicles such as squalene and squalene, and hyaluronic
acid may also be used administered in conjunction with the genetic
construct In some embodiments, an immunomodulating protein may be
used as a GVF.
[0060] The pharmaceutical compositions according to the present
invention comprise about 1 nanogram to about 2000 micrograms of
DNA. In some preferred embodiments, pharmaceutical compositions
according to the present invention comprise about 5 nanogram to
about 1000 micrograms of DNA. In some preferred embodiments, the
pharmaceutical compositions contain about 10 nanograms to about 800
micrograms of DNA. In some preferred embodiments, the
pharmaceutical compositions contain about 0.1 to about 500
micrograms of DNA. In some preferred embodiments, the
pharmaceutical compositions contain about 1 to about 350 micrograms
of DNA. In some preferred embodiments, the pharmaceutical
compositions contain about 25 to about 250 micrograms of DNA. In
some preferred embodiments, the pharmaceutical compositions contain
about 100 to about 200 microgram DNA.
[0061] The pharmaceutical compositions according to the present
invention are formulated according to the mode of administration to
be used. In cases where pharmaceutical compositions are injectable
pharmaceutical compositions, they are sterile, pyrogen free and
particulate free. An isotonic formulation is preferably used.
Generally, additives for isotonicity can include sodium chloride,
dextrose, mannitol, sorbitol and lactose. In some cases, isotonic
solutions such as phosphate buffered saline are preferred.
Stabilizers include gelatin and albumin. In some embodiments, a
vasoconstriction agent is added to the formulation.
[0062] According to some aspects of the present invention, DNA or
RNA that encodes a target protein is introduced into the cells of
tissue of an individual where it is expressed, thus producing the
encoded proteins. The DNA or RNA sequences encoding the target
protein and one or both immunomodulating proteins are linked to
regulatory elements necessary for expression in the cells of the
individual. Regulatory elements for DNA expression include a
promoter and a polyadenylation signal. In addition, other elements,
such as a Kozak region, may also be included in the genetic
construct.
[0063] The nucleic acid molecule(s) may be provided as plasmid DNA,
the nucleic acid molecules of recombinant vectors or as part of the
genetic material provided in an attenuated vaccine or inactivated
or killed particle or cell vaccine.
[0064] The manufacture and use of subunit vaccines are well known.
One having ordinary skill in the art can isolate the nucleic acid
molecule that encode target protein. Once isolated, the nucleic
acid molecule can be inserted it into an expression vector using
standard techniques and readily available starting materials.
[0065] In addition to producing these proteins by recombinant
techniques, automated peptide synthesizers may also be employed to
produce the target protein of the invention. Such techniques are
well known to those having ordinary skill in the art and are useful
if derivatives which have substitutions not provided for in
DNA-encoded protein production.
[0066] In some embodiments, the protein that makes up a subunit
vaccine or the cells or particles of a killed or inactivated
vaccine may be haptenized to increase immunogenicity. In some
cases, the haptenization is the conjugation of a larger molecular
structure to the target protein. In some cases, cells from the
patient are killed and haptenized as a means to make an effective
vaccine product. In cases in which other cells, such as bacteria or
eukaryotic cells which are provided with the genetic information to
make and display the target protein are killed and used as the
active vaccine component, such cells are haptenized to increase
immunogenicity. Haptenization is well known and can be readily
performed.
[0067] Methods of haptenizing cells are described in Berd et al.
May 1991 Cancer Research 51:2731-2734, which are incorporated
herein by reference. Additional haptenization protocols are
disclosed in Miller et al. 1976J. Immunol. 117(5:1):1591-1526.
[0068] Haptenization compositions and methods which may be adapted
to be used to prepare haptenized target protein according to the
present invention include those described in the following U.S.
Patents which are each incorporated herein by reference: U.S. Pat.
No. 5,037,645 issued Aug. 6, 1991 to Strahilevitz; U.S. Pat. No.
5,112,606 issued May 12, 1992 to Shiosaka et al.; U.S. Pat. No.
4,526,716 issued Jul. 2, 1985 to Stevens; U.S. Pat. No. 4,329,281
issued May 11, 1982 to Christenson et al.; and U.S. Pat. No.
4,022,878 issued May 10, 1977 to Gross. Peptide vaccines and
methods of enhancing immunogenicity of peptides which may be
adapted to modify ST immunogens of the invention are also described
in Francis et al. 1989 Methods of Enzymol. 178:659-676, which is
incorporated herein by reference. Sad et al. 1992 Immunolology
76:599-603, which is incorporated herein by reference, teaches
methods of making immunotherapeutic vaccines by conjugating
gonadotropin releasing hormone to diphtheria toxoid. Target protein
may be similarly conjugated to produce an immunotherapeutic vaccine
of the present invention. MacLean et al. 1993 Cancer Immunol.
Immunother. 36:215-222, which is incorporated herein by reference,
describes conjugation methodologies for producing immunotherapeutic
vaccines which may be adaptable to produce an immunotherapeutic
vaccine of the present invention. The hapten is keyhole limpet
hemocyanin which may be conjugated to target protein.
[0069] Vaccines according to some aspects of the invention comprise
a pharmaceutically acceptable carrier in combination with target
protein. Pharmaceutical formulations are well known and
pharmaceutical compositions comprising such proteins may be
routinely formulated by one having ordinary skill in the art.
Suitable pharmaceutical carriers are described in Remington's
Pharmaceutical Sciences, A. Osol, a standard reference text in this
field, which is incorporated herein by reference. The present
invention relates to an injectable pharmaceutical composition that
comprises a pharmaceutically acceptable carrier and a target
protein target protein is preferably sterile and combined with a
sterile pharmaceutical carrier.
[0070] In some embodiments, for example, the target protein can be
formulated as a solution, suspension, emulsion or lyophilized
powder in association with a pharmaceutically acceptable vehicle.
Examples of such vehicles are water, saline, Ringer's solution,
dextrose solution, and 5% human serum albumin. Liposomes and
nonaqueous vehicles such as fixed oils may also be used. The
vehicle or lyophilized powder may contain additives that maintain
isotonicity (e.g., sodium chloride, mannitol) and chemical
stability (e.g., buffers and preservatives). The formulation is
sterilized by commonly used techniques.
[0071] An injectable composition may comprise the target protein in
a diluting agent such as, for example, sterile water,
electrolytes/dextrose, fatty oils of vegetable origin, fatty
esters, or polyols, such as propylene glycol and polyethylene
glycol. The injectable must be sterile and free of pyrogens.
[0072] The vaccines of the present invention may be administered by
any means that enables the target protein to be presented to the
body's immune system for recognition and induction of an
immunogenic response. Pharmaceutical compositions may be
administered parenterally, i.e., intravenous, subcutaneous,
intramuscular.
[0073] Dosage varies depending upon known factors such as the
pharmacodynamic characteristics of the particular agent, and its
mode and route of administration; age, health, and weight of the
recipient; nature and extent of symptoms, kind of concurrent
treatment, frequency of treatment, and the effect desired. An
amount of immunogen is delivered to induce a protective or
therapeutically effective immune response. Those having ordinary
skill in the art can readily determine the range and optimal dosage
by route methods.
[0074] Target proteins including target proteins that are fusion
proteins can be produced by recombinant technology wherein nucleic
acid molecules that encode the target protein are constructed and
inserted into expression vectors such as plasmids or viral vectors.
The expression vectors contain regulatory elements that function in
host cells. When the expression vectors are incorporated into host
cells, the target protein is expressed by the host cell. The
protein may be isolated using standard techniques including, for
example, immunocolumns that include antibodies that specifically
bind to the target protein. Antibodies that specifically bind to
the target protein can be generated using standard techniques
including production of monoclonal antibodies by hybridoma
technology.
[0075] In addition to uses in protein purification, such
antibodies, including Mabs, humanized Mabs, human antibodies, and
Fab and F(ab).sub.2 fragments thereof may be used in passive
immunity therapy as therapeutic compounds to be administered to
patients as an alternative to or in conjunction with the vaccines
described herein. Such compositions may be routinely formulated and
administered by those skilled in the art following the teachings
generally disclosed herein.
EXAMPLE 1
Strategy for Development of DNA Vaccine for Allergy
[0076] There are two forms of IgE, one is secreted (slgE) and the
other is membrane-bound (mIgE) forms (FIG. 1). The mIgE form has
two isoforms, short (m/s IgE) and long (m/l Ige) forms. The m/l lgE
form was cloned by RT-PCR amplification method. Here after, m/l IgE
form will be designated as "mIgE".
[0077] Total RNA was extracted from a human myeloid cell line
SKO-007 (ATCC # CRL-8033-1) that secretes IgE and is HLA A2
positive. The first cDNA was generated by reverse transcription
using oligo-dT, random hexamer, or specific primers for mIgE gene.
The mIgE fragment was generated by PCR amplification using specific
primer set mIgEH3.S1 (5'-CCC AAG CTT ATG GAC TGG ACC TGG ATC CTC
TTC TTG GTG GCA GCA GCC ACG CGA GTC CAC TCC CAT GGG CTG GCT GGC GGC
TCC GCG C; SEQ ID NO:1) and mIgEXho.ASI (5'CCG CTC GAG CGT GGG GCT
GGA GGA CGT TGG; SEQ ID NO:2) (FIG. 2). To enhance protein
expression level, huIgE leader sequence was fused to 5' end of mIgE
fragment (FIG. 2). Moreover, to enhance immune response in vivo,
tetanus toxoid Th epitope (TTTh) was fused to mIgE by proteolytic
cleavage site. The sequence for proteolytic cleavage site followed
by tetanus toxoid Th epitope was generated by overlapping PCR using
synthetic oligos, mIgEXho.S1 (5'-CCG CTC GAG AGA AAC GAG CTG TCG
TAG GAT CCG ATC CAA ATT ATT TAA GGA CTG ATT CTG ATA AAG ATA GAT TTT
TAC AAA CCA TGG; SEQ ID NO:3), mIgEEco.ASI (5'-CCG GAA TTC TTA ATT
CTG TTA AAC AGT TTT ACC ATG GTT TGT AAA AAT CTA TCT TTA TCA GAA TCA
GTC CTT AAA TAA TTT GGA TCG G; SEQ ID NO:4). The complete human
mIgE fused to TTh was constructed by overlapping PCR of mIgE and
TTTh fragments, and then cloned into pcDNA3.1V5/His plasmid. The
final construct was named as "pcHu-mIgE".
EXAMPLE 2
In Vitro Protein Expression
1. In Vitro Transcription/Translation and
Immunoprecipitation/Western Blot Analysis.
[0078] Two .mu.g of plasmid DNA was transcribed/translated in a
single tube using TNT-T7 coupled Transcription/Translation System
(Promega) according to the Manufacturer's protocol. The reaction
was immunoprecipitated with monoclonal anti-6.times.His (C-term) Ab
along with Protein G-Sepharose beads for overnight. The protein was
resolved on 15% of SDS-PAGE and Western blot analyzed with
polyclonal anti-6.times.His Ab. The blot was developed with an ECL
Chemiluminescent detection Kit (Amersham). The synthesized protein
size was about 20 kD which is close to the predicted protein size
(FIG. 3).
2. Protein Expression in Mammalian Cells by Transfection.
[0079] Two .mu.g of plasmid DNA was transfected to RD cells using
DATAP transfection reagent according to the manufacturer's
suggestion (Roche). Five days after transfection, cellular proteins
were harvested by freezing/thaw method. Hundred .mu.g of total
cellular protein was resolved on 15% of SDS-PAGE and Western blot
analyzed using polyclonal anti-6.times.His (C-term) Ab. The blot
was developed with an ECL Chemiluminescent detection Kit
(Amersham). The synthesized protein size was about 20 kD which is
comparable to the size of the in vitro translated protein (FIG.
4).
EXAMPLE 3
Functional Analysis of a Vaccine for IgE Producing B Cells
[0080] M-IgE-TT was constructed as described in the FIGS. 5 and 6
and used to immunize HLA-A2 mice. HLA-A2 mice are transgenic mice
which express the human MHC haplotype A2 facilitating testing of
the concept of immunization against the IgE molecule and targeting
the portion of the IgE molecule that is expressed only on producer
cells. Theses epitopes are not present in cells that bind IgE.
Synthetic peptides shown in FIG. 7 were made to use as antigens in
in vitro assays. As shown in FIG. 8 a strong cellular response is
induced by M-IgE-TT that targets relevant peptide epitopes
expressed by IgE producing cells. The construct was highly
effective at inducing these epitope specific responses.
Sequence CWU 1
1
13 1 88 DNA Artificial Sequence Oligonucleotide 1 cccaagctta
tggactggac ctggatcctc ttcttggtgg cagcagccac gcgagtccac 60
tcccatgggc tggctggcgg ctccgcgc 88 2 30 DNA Artificial Sequence
Oligonucleotide 2 ccgctcgagc gtggggctgg aggacgttgg 30 3 87 DNA
Artificial Sequence Oligonucleotide 3 ccgctcgaga gaaacgagct
gtcgtaggat ccgatccaaa ttatttaagg actgattctg 60 ataaagatag
atttttacaa accatgg 87 4 88 DNA Artificial Sequence Oligonucleotide
4 ccggaattct taattctgtt aaacagtttt accatggttt gtaaaaatct atctttatca
60 gaatcagtcc ttaaataatt tggatcgg 88 5 630 DNA Homo sapiens CDS
(1)..(627) sig_peptide (1)..(54) misc_signal (418)..(441) protease
cleavage signal 5 atg gac tgg acc tgg atc ctc ttc ttg gtg gca gca
gcc acg cga gtc 48 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala
Ala Thr Arg Val 1 5 10 15 cac tcc cat ggg ctg gct ggc ggc tcc gcg
cag tcc cag agg gcc ccg 96 His Ser His Gly Leu Ala Gly Gly Ser Ala
Gln Ser Gln Arg Ala Pro 20 25 30 gat agg gtg ctc tgc cac tcc gga
cag cag cag gga ctg ccg aga gca 144 Asp Arg Val Leu Cys His Ser Gly
Gln Gln Gln Gly Leu Pro Arg Ala 35 40 45 gca gga ggc tct gtc ccc
cac ccc cgc tgc cac tgt gga gcc ggg agg 192 Ala Gly Gly Ser Val Pro
His Pro Arg Cys His Cys Gly Ala Gly Arg 50 55 60 gct gac tgg cca
ggt ccc cca gag ctg gac gtg tgc gtg gag gag gcc 240 Ala Asp Trp Pro
Gly Pro Pro Glu Leu Asp Val Cys Val Glu Glu Ala 65 70 75 80 gag ggc
gag gcg ccg tgg acg tgg acc ggc ctc tgc atc ttc gcc gca 288 Glu Gly
Glu Ala Pro Trp Thr Trp Thr Gly Leu Cys Ile Phe Ala Ala 85 90 95
ctc ttc ctg ctc agc gtg agc tac agc gcc gcc ctc acg ctc ctc atg 336
Leu Phe Leu Leu Ser Val Ser Tyr Ser Ala Ala Leu Thr Leu Leu Met 100
105 110 gtg cag cgg ttc ctc tca gcc acg cgg cag ggg agg ccc cag acc
tcc 384 Val Gln Arg Phe Leu Ser Ala Thr Arg Gln Gly Arg Pro Gln Thr
Ser 115 120 125 ctc gac tac acc aac gtc ctc cag ccc cac gcc aga gaa
aaa aga gct 432 Leu Asp Tyr Thr Asn Val Leu Gln Pro His Ala Arg Glu
Lys Arg Ala 130 135 140 gtt gtt ggt tac gat cca aat tat tta agg act
gat tct gat aaa gat 480 Val Val Gly Tyr Asp Pro Asn Tyr Leu Arg Thr
Asp Ser Asp Lys Asp 145 150 155 160 aga ttt tta caa acc atg gta aaa
ctg ttt aac aga att aag aga gaa 528 Arg Phe Leu Gln Thr Met Val Lys
Leu Phe Asn Arg Ile Lys Arg Glu 165 170 175 aaa aga gct gtt gtt ggt
ttt aat aat ttt acc gtt agc ttt tgg ttg 576 Lys Arg Ala Val Val Gly
Phe Asn Asn Phe Thr Val Ser Phe Trp Leu 180 185 190 agg gtt cct aaa
gta tct gct agt cat tta gaa cat cat cat cat cat 624 Arg Val Pro Lys
Val Ser Ala Ser His Leu Glu His His His His His 195 200 205 cat tag
630 His 6 209 PRT Homo sapiens 6 Met Asp Trp Thr Trp Ile Leu Phe
Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser His Gly Leu Ala
Gly Gly Ser Ala Gln Ser Gln Arg Ala Pro 20 25 30 Asp Arg Val Leu
Cys His Ser Gly Gln Gln Gln Gly Leu Pro Arg Ala 35 40 45 Ala Gly
Gly Ser Val Pro His Pro Arg Cys His Cys Gly Ala Gly Arg 50 55 60
Ala Asp Trp Pro Gly Pro Pro Glu Leu Asp Val Cys Val Glu Glu Ala 65
70 75 80 Glu Gly Glu Ala Pro Trp Thr Trp Thr Gly Leu Cys Ile Phe
Ala Ala 85 90 95 Leu Phe Leu Leu Ser Val Ser Tyr Ser Ala Ala Leu
Thr Leu Leu Met 100 105 110 Val Gln Arg Phe Leu Ser Ala Thr Arg Gln
Gly Arg Pro Gln Thr Ser 115 120 125 Leu Asp Tyr Thr Asn Val Leu Gln
Pro His Ala Arg Glu Lys Arg Ala 130 135 140 Val Val Gly Tyr Asp Pro
Asn Tyr Leu Arg Thr Asp Ser Asp Lys Asp 145 150 155 160 Arg Phe Leu
Gln Thr Met Val Lys Leu Phe Asn Arg Ile Lys Arg Glu 165 170 175 Lys
Arg Ala Val Val Gly Phe Asn Asn Phe Thr Val Ser Phe Trp Leu 180 185
190 Arg Val Pro Lys Val Ser Ala Ser His Leu Glu His His His His His
195 200 205 His 7 22 PRT Artificial Sequence Chemically synthesized
peptide 7 Ser Ala Gln Ser Gln Arg Ala Pro Asp Arg Val Leu Cys His
Ser Gly 1 5 10 15 Gln Gln Gln Gly Leu Pro 20 8 22 PRT Artificial
Sequence Chemically synthesized peptide 8 Ala Gly Gly Ser Val Pro
His Pro Arg Cys His Cys Gly Ala Gly Arg 1 5 10 15 Ala Asp Trp Pro
Gly Pro 20 9 15 PRT Artificial Sequence Chemically synthesized
peptide 9 Glu Leu Asp Val Cys Val Glu Glu Ala Glu Gly Glu Ala Pro
Trp 1 5 10 15 10 9 PRT Artificial Sequence Chemically synthesized
peptide 10 Glu Ala Pro Trp Thr Trp Thr Gly Leu 1 5 11 10 PRT
Artificial sequence Chemically synthesized peptide 11 Thr Gly Leu
Cys Ile Phe Ala Ala Leu Phe 1 5 10 12 27 PRT Artificial Sequence
Chemically synthesized peptide 12 Val Gln Arg Phe Leu Ser Ala Thr
Arg Gln Gly Arg Pro Gln Thr Ser 1 5 10 15 Leu Asp Tyr Thr Asn Val
Leu Gln Pro His Ala 20 25 13 27 PRT Artificial Sequence Chemically
synthesized peptide 13 Tyr Asp Pro Asn Tyr Leu Arg Thr Asp Ser Asp
Lys Asp Arg Phe Leu 1 5 10 15 Gln Thr Met Val Lys Leu Phe Asn Arg
Ile Lys 20 25
* * * * *