U.S. patent application number 13/720088 was filed with the patent office on 2013-05-16 for dna vaccines encoding heat shock proteins.
The applicant listed for this patent is Pnina Carmi, Irun R. Cohen, Felix Mor, Francisco J. Quintana. Invention is credited to Pnina Carmi, Irun R. Cohen, Felix Mor, Francisco J. Quintana.
Application Number | 20130122083 13/720088 |
Document ID | / |
Family ID | 29550169 |
Filed Date | 2013-05-16 |
United States Patent
Application |
20130122083 |
Kind Code |
A1 |
Cohen; Irun R. ; et
al. |
May 16, 2013 |
DNA VACCINES ENCODING HEAT SHOCK PROTEINS
Abstract
A method of treating a T cell-mediated inflammatory autoimmune
disease by administering to an individual in need thereof an
immunogenic composition comprising a recombinant construct of a
nucleic acid sequence encoding heat shock protein 90 (HSP 90), or
an active fragment thereof, wherein the nucleic acid sequence is
operatively linked to one or more transcription control sequences.
The disease is other than insulin dependent diabetes mellitus
(IDDM) or rheumatoid arthritis. The administering of the
immunogenic composition results in a shift of the immune response
to a Th2 response, thereby treating the disease.
Inventors: |
Cohen; Irun R.; (Rehovot,
IL) ; Quintana; Francisco J.; (Capital Federal,
AR) ; Carmi; Pnina; (Rehovot, IL) ; Mor;
Felix; (Kfar Saba, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cohen; Irun R.
Quintana; Francisco J.
Carmi; Pnina
Mor; Felix |
Rehovot
Capital Federal
Rehovot
Kfar Saba |
|
IL
AR
IL
IL |
|
|
Family ID: |
29550169 |
Appl. No.: |
13/720088 |
Filed: |
December 19, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13293722 |
Nov 10, 2011 |
8361987 |
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13720088 |
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12396401 |
Mar 2, 2009 |
8058254 |
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13293722 |
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10994152 |
Nov 19, 2004 |
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12396401 |
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PCT/IL03/00417 |
May 21, 2003 |
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10994152 |
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60381821 |
May 21, 2002 |
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Current U.S.
Class: |
424/450 ;
424/185.1 |
Current CPC
Class: |
A61K 2039/53 20130101;
A61K 2039/54 20130101; A61P 29/00 20180101; A61K 31/7088 20130101;
A61K 39/00 20130101; A61K 38/1709 20130101; A61K 2039/57 20130101;
A61K 48/00 20130101; A61K 2039/545 20130101; A61K 39/0008 20130101;
A61P 37/06 20180101 |
Class at
Publication: |
424/450 ;
424/185.1 |
International
Class: |
A61K 31/7088 20060101
A61K031/7088 |
Claims
1. A method of treating a T cell-mediated inflammatory autoimmune
disease, which comprises administering to an individual in need
thereof an immunogenic composition comprising a recombinant
construct of a nucleic acid sequence encoding a heat shock protein
90 (HSP 90), or an active fragment thereof, wherein the nucleic
acid sequence is operatively linked to one or more transcription
control sequences, thereby treating the disease, wherein the
disease is other than insulin dependent diabetes mellitus (IDDM) or
rheumatoid arthritis, and wherein the administering of the
immunogenic composition results in a shift of the immune response
to a Th2 response.
2. The method of claim 1, wherein the composition comprises a
delivery vehicle selected from the group consisting of liposomes,
micelles, emulsions and cells.
3. The method of claim 1, wherein the immunogenic composition is
administered to the individual prior to the appearance of disease
symptoms.
4. The method of claim 1, wherein the T cell-mediated inflammatory
autoimmune disease is selected from the group consisting of:
systemic lupus erythematosus, collagen II arthritis, multiple
sclerosis, autoimmune neuritis, psoriasis, Sjogren's disease,
thyroid disease, sarcoidosis, autoimmune uveitis, inflammatory
bowel disease (Crohn's and ulcerative colitis) and autoimmune
hepatitis.
5. The method of claim 1, wherein the T cell-mediated inflammatory
autoimmune disease is inflammatory bowel disease (Crohn's and
ulcerative colitis).
6. The method of claim 1, wherein the immunogenic composition is
administered by a route selected from the group consisting of
intravenous, topical, intradermal, subcutaneous, and
intramuscular.
7. The method of claim 1, wherein the mammalian HSP is human
HSP90.
8. The method of claim 1, wherein the individual is a human.
9. The method of claim 1, comprising the steps of (a) obtaining
cells from the individual; (b) transfecting the cells ex vivo with
the composition comprising the recombinant construct of the nucleic
acid sequence encoding the heat shock protein or the active
fragment thereof; and (c) reintroducing the transfected cells to
the individual.
10. A method of treating a T cell-mediated inflammatory autoimmune
disease, which comprises administering to an individual in need
thereof an immunogenic composition comprising a recombinant
construct of a nucleic acid sequence encoding mammalian heat shock
protein HSP90 (full length), wherein the nucleic acid sequence is
operatively linked to one or more transcription control sequences,
and wherein the immunogenic composition is administered by direct
injection, thereby treating the disease, wherein the disease is
other than insulin dependent diabetes mellitus (IDDM) or rheumatoid
arthritis, and wherein the administering of the immunogenic
composition results in a shift of the immune response to a Th2
response.
11. The method of claim 10, wherein the T cell-mediated
inflammatory autoimmune disease is selected from the group
consisting of: systemic lupus erythematosus, collagen II arthritis,
multiple sclerosis, autoimmune neuritis, psoriasis, Sjogren's
disease, thyroid disease, sarcoidosis, autoimmune uveitis,
inflammatory bowel disease (Crohn's and ulcerative colitis) and
autoimmune hepatitis.
12. The method of claim 10, wherein the T cell-mediated
inflammatory autoimmune disease is inflammatory bowel disease
(Crohn's and ulcerative colitis).
13. The method of claim 10, wherein the composition comprises a
delivery vehicle selected from the group consisting of liposomes,
micelles, emulsions and cells.
14. The method of claim 10, wherein the immunogenic composition is
administered to the individual prior to the appearance of disease
symptoms.
15. The method of claim 10, wherein the immunogenic composition is
administered by a route selected from the group consisting of
intravenous, topical, intradermal, subcutaneous, and
intramuscular.
16. The method of claim 10, comprising the steps of (a) obtaining
cells from the individual; (b) transfecting the cells ex vivo with
the composition comprising the recombinant construct of the nucleic
acid sequence encoding the heat shock protein or the active
fragment thereof; and (c) reintroducing the transfected cells to
the individual.
17. The method of claim 10, wherein the HSP90 is human HSP90.
18. The method of claim 10, wherein the individual is a human.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to recombinant constructs
encoding heat shock proteins or active fragments thereof, effective
in treating T cell mediated diseases including inflammatory
autoimmune diseases by DNA vaccination. The present invention
further relates to compositions and methods for treating T cell
mediated diseases.
BACKGROUND OF THE INVENTION
[0002] While the normal immune system is closely regulated,
aberrations in immune responses are not uncommon. In some
instances, the immune system functions inappropriately and reacts
to a component of the host as if it were, in fact, foreign. Such a
response results in an autoimmune disease, in which the host's
immune system attacks the host's own tissue. T cells, as the
primary regulators of the immune system, directly or indirectly
effect such autoimmune pathologies. T cell-mediated autoimmune
diseases refer to any condition in which an inappropriate T cell
response is a component of the disease. This includes both diseases
directly mediated by T cells, and also diseases in which an
inappropriate T cell response contributes to the production of
abnormal antibodies.
[0003] Numerous diseases are believed to result from autoimmune
mechanisms. Prominent among these are rheumatoid arthritis,
systemic lupus erythematosus, multiple sclerosis, Type I diabetes,
myasthenia gravis and pemphigus vulgaris. Autoimmune diseases
affect millions of individuals world-wide and the cost of these
diseases, in terms of actual treatment expenditures and lost
productivity, is measured in billions of dollars annually.
[0004] Adjuvant arthritis (AA) is an experimental autoimmune
disease that models several features of human rheumatoid arthritis
(1). AA is induced in Lewis rats by immunization with heat killed
Mycobacterium tuberculosis (Mt) suspended in Incomplete Freund's
Adjuvant (IFA) (1). T-cell reactivity against the mycobacterial 65
kDa heat shock protein (HSP65) is involved in the progression of
AA. HSP65-specific T-cells directed against an epitope formed by aa
180-188 (2) cross-react with a self-antigen present in cartilage
(3) and can adoptively transfer AA (4, 5). However, vaccination
with HSP65 or HSP65-peptides can also prevent the development of AA
(6-11). The regulatory properties of HSP65 in AA are thought to
involve the activation of T-cells cross-reactive with the
endogenous 60 kDa heat shock protein (HSP60) (12). This hypothesis
is supported by the finding that immunization with a recombinant
vaccinia virus encoding human HSP60 (about 95% homologous to rat
HSP60) prevents (13) or treats (14) AA. The inventor of the present
invention have recently reported that DNA vaccination with human
HSP60 prevents AA (15). Protection from AA was associated with the
activation of T-cells responding to HSP60 (15). The human hsp60
molecule was formerly designated HSP65, but is now designated HSP60
in view of more accurate molecular weight information; by either
designation, the protein is the same.
[0005] A preferable method for treating autoimmune diseases
includes modulating the immune system of a patient to assist the
patient's natural defense mechanisms. Traditional reagents and
methods used to attempt to regulate an immune response in a patient
also result in unwanted side effects and have limited
effectiveness. For example, immunosuppressive reagents (e.g.,
cyclosporin A, azathioprine, and prednisone) used to treat patients
with autoimmune diseases also suppress the patient's entire immune
response, thereby increasing the risk of infection. In addition,
immunopharmacological reagents used to treat cancer (e.g.,
interleukins) are short-lived in the circulation of a patient and
are ineffective except in large doses. Due to the medical
importance of immune regulation and the inadequacies of existing
immunopharmacological reagents, reagents and methods to regulate
specific parts of the immune system have been the subject of study
for many years.
[0006] EP 262710 of Cohen et al. discloses the use of HSP65, or
fragments thereof for the preparation of compositions for the
alleviation, treatment and diagnosis of autoimmune diseases,
especially arthritic conditions. EP 322990 of Cohen et al.
discloses that a polypeptide having amino acid sequence 172-192 of
HSP65 is capable of inducing resistance to auto-immune arthritis
and similar auto-immune diseases. WO 92/04049 of Boog et al.
discloses peptides derived from Mycobacterium tuberculosis protein
HSP-65 containing at least 7 amino acid residues and inhibits
antigen recognition by T lymphocytes in treatment of arthritis and
organ rejection.
[0007] WO 01/57056 of Karin discloses a method of treating
rheumatoid arthritis. The method comprising the step of expressing
within the individual at least an immunologically recognizable
portion of a cytokine from an exogenous polynucleotide encoding at
least a portion of the cytokine, wherein a level of expression of
the at least a portion of the cytokine is sufficient to induce the
formation of anti-cytokine immunoglobulins which serve for
neutralizing or ameliorating the activity of a respective and/or
cross reactive endogenous cytokine, to thereby treat rheumatoid
arthritis. U.S. Pat. No. 6,316,420 to Karin and coworkers further
discloses DNA cytokine vaccines and use of same for protective
immunity against multiple sclerosis.
[0008] WO 02/16549 of Cohen et al., assigned to the assignee of the
present invention, relates to DNA vaccines useful for the
prevention and treatment of ongoing autoimmune diseases. The
compositions and methods of the invention feature the CpG
oligonucleotide, preferably in a motif flanked by two 5' purines
and two 3' pyrimidines. The vaccines optionally further comprise
DNA encoding a peptide or a polypeptide selected from the group
consisting of Hsp60, p277 or p277 variants. That disclosure is
directed to methods and compositions for the ameliorative treatment
of ongoing autoimmune disease in general and Insulin Dependent
Diabetes Mellitus (IDDM) in particular.
[0009] U.S. Pat. No. 5,993,803 discloses that when HSP60, or
peptides and analogs thereof, are administered in a recipient
subject before transplantation of an organ or tissue, autoimmunity
to HSP60 is down-regulated, resulting in the prevention or
suppression of graft rejection of the transplanted organ or
tissue.
[0010] WO 00/27870 of Naparstek and colleagues discloses a series
of related peptides derived from heat shock proteins HSP65 and
HSP60, their sequences, antibodies, and use as vaccines for
conferring immunity against autoimmune and/or inflammatory
disorders such as arthritis. These peptides are intended by the
inventors to represent the shortest sequence or epitope that is
involved in protection of susceptible rat strains against adjuvant
induced arthritis. These sequences further disclose what the
inventors identify as the common "protective motif".
[0011] There exists a long-felt need for an effective means of
curing or ameliorating T cell mediated inflammatory autoimmune
diseases. None of the background art discloses DNA vaccines
encoding heat shock proteins for treating T cell mediated
inflammatory autoimmune diseases. Such a treatment should ideally
control the inappropriate T cell response, rather than merely
reducing the symptoms.
SUMMARY OF THE INVENTION
[0012] DNA vaccination represents a novel and unexpectedly
effective means of expressing antigen in vivo for the generation of
both humoral and cellular immune responses. The present invention
uses this technology to elicit protective immunity against T
cell-mediated autoimmune diseases. The compositions and methods of
the present invention are effective in any T-cell mediated
inflammatory autoimmune disease including but not limited to:
rheumatoid arthritis, collagen II arthritis, multiple sclerosis,
autoimmune neuritis, systemic lupus erythematosus, psoriasis,
juvenile onset diabetes, Sjogren's disease, thyroid disease,
sarcoidosis, autoimmune uveitis, inflammatory bowel disease
(Crohn's and ulcerative colitis) or autoimmune hepatitis.
[0013] In one aspect, the present invention is related to DNA
vaccines encoding heat shock proteins for treating T cell-mediated
inflammatory autoimmune diseases. According to various specific
embodiments of the present invention, the heat shock proteins are
mammalian heat shock proteins, preferably the full-length heat
shock protein 60 (HSP60), the full-length heat shock protein 70
(HSP70) or the full-length heat shock protein 90 (HSP90). The heat
shock proteins according to the present invention are preferably
human heat shock proteins, however other mammalian heat shock
proteins are within the scope of the present invention. According
to another embodiment, the full-length heat shock protein 70
(HSP70) has the amino acid sequence selected from SEQ ID NO:4--SEQ
ID NO:11. According to another embodiment, the full-length heat
shock protein 70 (HSP70) has the amino acid sequence as set forth
as SEQ ID NO:4. According to another embodiment, the full-length
heat shock protein 90 (HSP90) has the amino acid sequence as set
forth as SEQ ID NO:12.
[0014] According to various additional embodiments of the present
invention, the DNA vaccines encode active fragments of HSP60, HSP70
or HSP90. In certain specific embodiments, the DNA vaccines encode
active fragments of HSP60. Preferred fragments of HSP60 correspond
to amino acids 1-140 of HSP60 (SEQ ID NO:1), amino acids 130-260 of
HSP60 (SEQ ID NO:2) or amino acids 31-50 of HSP60 (SEQ ID
NO:3).
[0015] The treatment with the DNA vaccines of the present invention
provides long-term expression of specific heat shock proteins or
active fragments thereof. Such long-term expression allows for the
maintenance of an effective, but non-toxic, dose of the encoded
polypeptides to treat the disease and limits the frequency of
administration of the therapeutic composition needed to treat an
animal. In addition, because of the lack of toxicity, therapeutic
compositions of the present invention can be used in repeated
treatments.
[0016] In another aspect, the present invention is related to novel
recombinant constructs comprising a nucleic acid sequence encoding
at least part of a heat shock protein being operatively linked to
at least one transcription control element. According to various
embodiments of the present invention, the heat shock protein is a
mammalian heat shock protein, preferably the full-length HSP60, the
full-length HSP70 or the full-length HSP90. The heat shock proteins
according to the present invention are preferably human heat shock
proteins, however other mammalian heat shock proteins are within
the scope of the present invention.
[0017] According to various embodiments of the present invention,
the recombinant constructs encoding active fragments of HSP60,
HSP70 or HSP90. In a preferred embodiment, the recombinant
constructs encode active fragments of HSP60, said active fragments
selected from: amino acids 1-140 of HSP60 (SEQ ID NO:1), amino
acids 130-260 of HSP60 (SEQ ID NO:2) or amino acids 31-50 of HSP60
(SEQ ID NO:3), the nucleic acid sequence being operatively linked
to at least one transcription control element.
[0018] According to various specific embodiments, the constructs of
the present invention comprise at least one transcription control
element selected from the group consisting of: RSV control
sequences, CMV control sequences, retroviral LTR sequences, SV-40
control sequences and .beta.-actin control sequences.
[0019] In another aspect, the present invention is related to an
eukaryotic expression vector comprising the recombinant constructs
of the present invention. According to various embodiments, the
eukaryotic expression vector is selected from pcDNA3,
pcDNA3.1(+/-), pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto,
pCMV/myc/cyto, pCR3.1, pCI, pBK-RSV, pBK-CMV and pTRES.
[0020] Another aspect of the present invention provides a
pharmaceutical composition effective for treating a T cell-mediated
inflammatory autoimmune disease, the composition comprising (a) a
recombinant construct comprising an isolated nucleic acid sequence
encoding a heat shock protein, or an active fragment thereof, the
nucleic acid sequence being operatively linked to one or more
transcription control sequences; and (b) a pharmaceutically
acceptable carrier.
[0021] In one embodiment, the nucleic acid sequence encodes the
full-length HSP60, the full-length HSP70 or the full-length HSP90.
In another embodiment, the nucleic acid sequence encoding an active
fragment of HSP60, HSP70 or HSP90. In a preferred embodiment, the
nucleic acid sequence encoding amino acids 1-140 of human HSP60
(SEQ ID NO:1). In another preferred embodiment, the nucleic acid
sequence encoding amino acids 130-260 of human HSP60 (SEQ ID NO:2).
In yet another preferred embodiment, the nucleic acid sequence
encoding amino acids 31-50 of human HSP60 (SEQ ID NO:3).
[0022] The pharmaceutical compositions comprising the recombinant
constructs according to the present invention may advantageously
comprise liposomes, micelles, emulsions or cells. Still further
embodiments utilize a virus as is known in the art in order to
introduce and express the nucleic acid sequences according to the
present invention in the host cells.
[0023] In another aspect, the present invention is related to a
method of inhibiting or preventing the symptoms of a T-cell
mediated inflammatory autoimmune disease, the method comprising
administering to an individual in need of said treatment,
preferably a human individual, a therapeutic composition comprising
a recombinant construct, said recombinant construct comprising an
isolated nucleic acid sequence encoding a heat shock protein, or a
fragment thereof, thereby inhibiting or preventing the symptoms of
said autoimmune disease.
[0024] In one embodiment, the nucleic acid sequence encodes the
full-length HSP60, the full-length HSP70 or the full-length HSP90.
In another embodiment, the nucleic acid sequence encoding the
full-length HSP70 has the nucleic acid sequence as set forth as SEQ
ID NO:13. In another embodiment, the nucleic acid sequence encoding
the full-length HSP90 has the nucleic acid sequence as set forth as
SEQ ID NO:14.
[0025] According to various embodiments, the compositions and
methods of the present invention are effective in any T-cell
mediated inflammatory autoimmune disease such as: rheumatoid
arthritis, collagen II arthritis, multiple sclerosis, autoimmune
neuritis, systemic lupus erythematosus, psoriasis, juvenile onset
diabetes, Sjogren's disease, thyroid disease, sarcoidosis,
autoimmune uveitis, inflammatory bowel disease (Crohn's and
ulcerative colitis) or autoimmune hepatitis.
[0026] The present invention is particularly exemplified by the
animal disease model of adjuvant arthritis (AA), a T cell-mediated
autoimmune disease that serves as an experimental model for
rheumatoid arthritis. This model is intended as a non-limitative
example used for illustrative purposes of the principles of the
invention
[0027] In one embodiment, the therapeutic composition of the
present invention is administered to an individual at risk of
developing a T-cell mediated inflammatory autoimmune disease, thus
serving as a preventive treatment. In another embodiment, the
therapeutic composition of the present invention is administered to
an individual during the initial stages of the disease or after the
appearance of disease symptoms.
[0028] According to another aspect, the present invention provides
a method for treating a T cell-mediated inflammatory autoimmune
disease comprising the steps of (a) obtaining cells from an
individual; (b) transfecting the cells ex vivo with a recombinant
construct comprising an isolated nucleic acid sequence encoding a
heat shock protein, or a fragment thereof, the nucleic acid
sequence being operatively linked to one or more transcription
control sequences; and (c) reintroducing the transfected cells to
the individual.
[0029] According to another aspect, the present invention provides
a method for treating a T cell-mediated inflammatory autoimmune
disease comprising the steps of (a) obtaining cells from an
individual; (b) infecting the cells ex vivo with a virus comprising
a recombinant construct comprising an isolated nucleic acid
sequence encoding a heat shock protein, or a fragment thereof, the
nucleic acid sequence being operatively linked to one or more
transcription control sequences; and (c) reintroducing the infected
cells to the individual.
[0030] According to another aspect, the present invention provides
a method for treating a T cell-mediated inflammatory autoimmune
disease comprising administering to an individual in need thereof a
therapeutic composition comprising (a) a fragment of mammalian
HSP60 having amino acid sequence selected from SEQ ID NO:1, SEQ ID
NO:2 and SEQ ID NO:3; and (b) a pharmaceutically acceptable
carrier.
[0031] According to another aspect, the present invention provides
a method of treating arthritis, said method comprising
administering to an individual in need thereof a therapeutic
composition comprising (a) a fragment of mammalian HSP60 having
amino acid sequence Lys Phe Gly Ala Asp Ala Arg Ala Leu Met Leu Gln
Gly Val Asp Leu Leu Ala Asp Ala corresponding to amino acid residus
31-50 of human HSP60 (denoted as SEQ ID NO:3); and (b) a
pharmaceutically acceptable carrier, thereby treating arthritis.
According to various embodiments, the carrier comprises a delivery
vehicle that delivers the fragment to the individual.
[0032] According to another aspect, the present invention provides
a method of treating arthritis, said method comprising the steps of
(a) obtaining cells from an individual; (b) exposing the cells ex
vivo with an active amount of a fragment of mammalian HSP60 having
amino acid sequence corresponding to amino acids 31-50 of HSP60
(denoted as SEQ ID NO:3); and (c) reintroducing the exposed cells
to the individual, thereby treating arthritis. In a preferred
embodiment the cells are autologous T cells. In another preferred
embodiment, the mammalian HSP60 is human HSP60.
[0033] These and further embodiments will be apparent from the
detailed description and examples that follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1: Prevention of AA by vaccination with pI and pII. A.
Time course of AA. Rats were vaccinated in the quadriceps three
times (on days -40, -26-12 relative to AA induction) with 150 .mu.g
of pcDNA3, pI, pII, pIII, pIV or pV. On day 0, AA was induced by
injecting 1 mg of Mt suspended in 100 .mu.l of IFA, and arthritis
scores were assessed every two or three days starting at day 10.
Bars show the mean.+-.SEM assessment of disease severity. B. Leg
swelling measured at day 26 after AA induction.
[0035] FIG. 2: pHSP60 and pI-vaccination activate T-cell responses
to Hu3. A. Proliferative responses (Stimulation Index, SI) to
overlaping peptides corresponding to the first 260 aa of HSP60.
Rats vaccinated were killed and LNC were collected on day 26 after
induction of AA. B. Dose-response to Hu3 of pHSP60, pHSP65 and
pcDNA3-vaccinated rats. Rats vaccinated were killed and LNC were
collected on day 26 after induction of AA. C. Dose-response to Hu3
of pI, pII and pcDNA3-vaccinated rats. Rats vaccinated were killed
and LNC were collected on day 26 after induction of AA.
[0036] FIG. 3: Prevention of AA by vaccination with Hu3. A. Time
course of AA. Rats were vaccinated once (on day -7 relative to AA
induction) with 100 .mu.g of Hu3, Hu12, Mt3 or PBS in 100 .mu.l of
IFA, or left unvaccinated (AA); and AA was induced on day 0 and
arthritis scores were assessed. B. Leg swelling measured on day 26
after AA induction.
[0037] FIG. 4: Prevention of AA by transfer of Con A-activated
splenocytes from Hu3-vaccinated rats. A. Time course of AA. Rats
were vaccinated once (on day -7 relative to AA induction) with 100
.mu.g of Hu3 or Mt3 in 100 .mu.l of IFA and AA was induced on day
O, Splenocytes were collected on day 26 after induction of AA,
activated for 48 hr with Con A and transferred iv to naive rats.
Three days later, AA was induced in the recipients and arthritis
scores were assessed. B. Leg swelling measured on day 26 after AA
induction.
[0038] FIG. 5: T-cell responses after DNA vaccination. Lewis rats
were vaccinated with pI, pII or pcDNA3 and AA was induced.
Twenty-six days later, LNC were collected, and the proliferative
responses to (A) PPD, (B) HSP65, Mt176-190, Mt3, HSP60 and Hu3 were
studied.
[0039] FIG. 6: Cytokine secretion after DNA vaccination. Lewis rats
were vaccinated with pI, pII or pcDNA3 and AA was induced.
Twenty-six days later, LNC were collected, stimulated in vitro with
PPD, (B) HSP65, Mt176-190, Mt3, HSP60 and Hu3 and the supernatants
were tested after 72 hr for the amounts of secreted (A) INFy, (B)
IL-10 or (C) TGF.beta.1.
[0040] FIG. 7: T-cell responses after DNA vaccination. Lewis rats
were vaccinated with Hu3, Mt3, Hu12 or PBS and AA was induced.
Twenty-six days later, LNC were collected, and the proliferative
responses to (A) PPD, (B) HSP65, Mt176-190, Mt3, HSP60 and Hu3 were
studied.
[0041] FIG. 8: Cytokine secretion after DNA vaccination. Lewis rats
were vaccinated with Hu3, Mt3, Hu12 or PBS, and AA was induced.
Twenty-six days later, LNC were collected, stimulated in vitro with
PPD, (B) HSP65, Mt176-190, Mt3, HSP60 and Hu3 and the supernatants
were tested after 72 hr for the amounts of secreted (A) INF.gamma.,
(B) IL-10 or (C) TGF.beta.1.
[0042] FIG. 9: Modulation of AA by preimmunization with pHSP60 or
pHSP65. A. Time course of AA. Rats were vaccinated in the
quadriceps three times (on days -40, -26-12 relative to AA
induction) with 150 .mu.g of pcDNA3, pMBP, pHSP60 or pHSP65, or
left untreated as controls (AA). On day 0 AA was induced by
injecting 1 mg of Mycobacterium tuberculosis (Mt) suspended in 100
.mu.l of IFA, and arthritis scores were assessed every two or three
days starting at day 8 after Mt injection. B. Leg swelling measured
at day 26 after AA induction.
[0043] FIG. 10: Proliferative responses to HSP60, HSP65 and PPD in
pHSP60--, pMBP and pcDNA3-vaccinated animals. Female Lewis rats
were vaccinated in the quadriceps three times (on days -40, -26-12
relative to AA induction) with 150 .mu.g of pHSP60, pMBP or pcDNA3,
beginning 5 days after i.m. injection of 200 .mu.l of cardiotoxin
10 .mu.M. A group was left untreated as a control. Animals were
killed and LNC were collected on day -1 prior to the induction of
AA.
[0044] FIG. 11: T-cell responses in AA rats vaccinated with pcDNA3.
Animals vaccinated with pcDNA3 were killed on day 26 after
induction of AA and their LNC were collected and stimulated in
vitro for 72 hrs in the presence of different concentrations of
antigen. The release of IFN.gamma. and IL-10 was studied.
[0045] FIG. 12: Effect of vaccination on cytokine secretion.
Animals vaccinated with pHSP60, pHSP65 or Hu3 were killed on day 26
after induction of AA and their LNC were collected. The cells were
stimulated for 72 hrs in the presence of different antigens, and
the proliferation (a) or the release of IFN.gamma. (b), IL-10 (c)
and TGF.beta.1 (d) were studied and are illustrated.
[0046] FIG. 13: Inhibition of AA by preimmunization with pHSP70 or
pHSP90. A. Time course of AA. B. Maximal arthritis score. C. Day of
onset. D. Difference in leg swelling measured at day 26 after AA
induction.
[0047] FIG. 14. Humoral response in DNA vaccinated rats. A.
Antibodies to HSP70 in pHSP70-immunized rats. B. Antibodies to
HSP90 in pHSP90-- immunized rats. The day of induction of AA was
considered day 0.
[0048] FIG. 15. T-cell response to the immunizing antigen in
DNA-vaccinated rats. A. Proliferative response to HSP70 in
pHSP70-immunized rats. The results are presented as the mean.+-.SD
of the stimulation index (SI) in quadruplicate cultures. B.
Proliferative response to HSP90 in pHSP90-- immunized rats. The
results are presented as the mean.+-.SD of the SI in quadruplicate
cultures.
[0049] FIG. 16. Cytokine secretion in response to stimulation with
the immunizing antigen in DNA-vaccinated rats. Draining lymph node
cells were stimulated for 72 hrs in the presence of HSP70 or HSP90,
and the content of IFN.gamma. (A) or IL-10 (B) was determined in
the supernatants by capture ELISA.
[0050] FIG. 17: Effect of DNA vaccination on T-cell proliferation
following AA induction. The results are expressed as the percent
change in proliferation relative to the responses of untreated
rats, 26 after the induction of AA.
[0051] FIG. 18: Effect of DNA vaccination on cytokine secretion
following AA induction. DLN cells were stimulated for 72 hrs in the
presence of HSP70 or HSP90, and the content of IFN.gamma. (A),
IL-10 (B) or TGF.beta.1 (C) was determined in the supernatants by
capture ELISA.
DETAILED DESCRIPTION OF THE INVENTION
[0052] According to the present invention it is now disclosed that
it is possible to treat or prevent T cell-mediated inflammatory
autoimmune diseases by using DNA vaccines encoding a heat shock
protein, or active fragments thereof. The compositions and methods
of the present invention are effective in any T-cell mediated
inflammatory autoimmune disease including but not limited to:
rheumatoid arthritis, collagen II arthritis, multiple sclerosis,
autoimmune neuritis, systemic lupus erythematosus, psoriasis,
juvenile onset diabetes, Sjogren's disease, thyroid disease,
sarcoidosis, autoimmune uveitis, inflammatory bowel disease
(Crohn's and ulcerative colitis) or autoimmune hepatitis.
[0053] The present invention is based in part on studies of the
role of DNA vaccines encoding a heat shock protein, or fragments
thereof in adjuvant induced arthritis in experimental rats.
Specifically, the present invention is based on the unexpected
discovery that certain DNA constructs encoding specific heat shock
proteins, such as HSP60, HSP70 or HSP90, or active fragments
thereof are useful in decreasing the symptoms associated with
arthritis. The protective effect of these DNA constructs was
reflected for example by a significant reduction in ankle
swelling.
[0054] It is now disclosed that it is possible to treat or prevent
T cell-mediated diseases by using DNA vaccines encoding mammalian
heat shock proteins or active fragments thereof. The present
invention is based in part on studies of the role of the immune
response to HSP60 in adjuvant induced arthritis in experimental
rats, using DNA vaccines encoding human HSP60, human HSP70, human
HSP90 or active fragments thereof. The results led to the
identification of novel constructs encoding at least part of the
HSP60 sequence that could effectively suppress AA. In addition,
specific HSP60 fragments were found to be effective in suppressing
AA. The immune effects associated with specific DNA or peptide
suppression of AA were complex and included enhanced T-cell
proliferation to a variety of disease-associated antigens.
Effective vaccination with HSP60 DNA fragments or the HSP60 peptide
led to up-regulation of IFN.gamma. secretion to HSP60 and,
concomitantly to down-regulation of IFN.gamma. secretion to
mycobacterial HSP65 epitopes. There were also variable changes in
the profiles of IL-10 secretion to those antigens. The production
of TGF.beta.1, however, was enhanced to both HSP60 and HSP65
epitopes. The regulation of AA might be due to the induction of
regulatory T-cells directed to HSP60, secreting both Th1 and Th2
cytokines that shifted the immune response towards mycobacterial
antigens to a Th2 non-pathogenic response.
[0055] The present invention provides an effective method of DNA
vaccination for T cell-mediated inflammatory autoimmune diseases,
which avoids many of the problems associated with the previously
suggested methods of treatment. By vaccinating, rather than
passively administering heterologous antibodies, the host's own
immune system is mobilized to suppress the autoaggressive T cells.
Thus, the suppression is persistent and may involve any and all
immunological mechanisms in effecting that suppression. This
multi-faceted response is more effective than the uni-dimensional
suppression achieved by passive administration of monoclonal
antibodies or extant-derived regulatory T cell clones.
[0056] In one aspect, the present invention is related to novel
recombinant constructs comprising a nucleic acid sequence
corresponding to mammalian heat shock proteins, the nucleic acid
sequence being operatively linked to at least one transcription
control element. Preferably, the recombinant constructs of the
present invention correspond to human heat shock proteins. However,
recombinant constructs corresponding to the rat or mouse heat shock
proteins may also be used in the present invention.
[0057] The nucleic acid sequence corresponding to mammalian heat
shock proteins may include DNA, RNA, or derivatives of either DNA
or RNA. An isolated nucleic acid sequence encoding heat shock
proteins can be obtained from its natural source, either as an
entire (i.e., complete) gene or a portion thereof. A nucleic acid
molecule can also be produced using recombinant DNA technology
(e.g., polymerase chain reaction (PCR) amplification, cloning) or
chemical synthesis. Nucleic acid sequences include natural nucleic
acid sequences and homologues thereof, including, but not limited
to, natural allelic variants and modified nucleic acid sequences in
which nucleotides have been inserted, deleted, substituted, and/or
inverted in such a manner that such modifications do not
substantially interfere with the nucleic acid molecule's ability to
encode a functional heat shock protein or an active fragment
thereof.
[0058] A nucleic acid sequence homologue can be produced using a
number of methods known to those skilled in the art (see, for
example, Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Labs Press, 1989). For example, nucleic acid
sequences can be modified using a variety of techniques including,
but not limited to, classic mutagenesis techniques and recombinant
DNA techniques, such as site-directed mutagenesis, chemical
treatment of a nucleic acid molecule to induce mutations,
restriction enzyme cleavage of a nucleic acid fragment, ligation of
nucleic acid fragments, polymerase chain reaction (PCR)
amplification and/or mutagenesis of selected regions of a nucleic
acid sequence, synthesis of oligonucleotide mixtures and ligation
of mixture groups to "build" a mixture of nucleic acid molecules
and combinations thereof. Nucleic acid molecule homologues can be
selected from a mixture of modified nucleic acids by screening for
the function of the protein encoded by the nucleic acid.
[0059] The present invention includes a nucleic acid sequence
operatively linked to one or more transcription control sequences
to form a recombinant molecule. The phrase "operatively linked"
refers to linking a nucleic acid sequence to a transcription
control sequence in a manner such that the molecule is able to be
expressed when transfected (i.e., transformed, transduced or
transfected) into a host cell. Transcription control sequences are
sequences which control the initiation, elongation, and termination
of transcription. Particularly important transcription control
sequences are those which control transcription initiation, such as
promoter, enhancer, operator and repressor sequences. Suitable
transcription control sequences include any transcription control
sequence that can function in at least one of the recombinant cells
of the present invention. A variety of such transcription control
sequences are known to those skilled in the art. Preferred
transcription control sequences include those which function in
animal, bacteria, helminth, insect cells, and preferably in animal
cells. More preferred transcription control sequences include, but
are not limited to RSV control sequences, CMV control sequences,
retroviral LTR sequences, SV-40 control sequences and .beta.-actin
control sequences as well as other sequences capable of controlling
gene expression in eukaryotic cells. Additional suitable
transcription control sequences include tissue-specific promoters
and enhancers (e.g., T cell-specific enhancers and promoters).
Transcription control sequences of the present invention can also
include naturally occurring transcription control sequences
naturally associated with a gene encoding a heat shock protein of
the present invention.
[0060] The present invention is further related to an expression
vector comprising the recombinant constructs of the present
invention. Suitable eukaryotic expression vector is for example:
pcDNA3, pcDNA3.1(+/-), pZeoSV2(+/-), pSecTag2, pDisplay,
pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pCI, pBK-RSV, pBK-CMV, pTRES
or their derivatives.
[0061] According to the present invention, a host cell can be
transfected in vivo (i.e., in an animal) or ex vivo (i.e., outside
of an animal). Transfection of a nucleic acid molecule into a host
cell can be accomplished by any method by which a nucleic acid
molecule can be inserted into the cell. Transfection techniques
include, but are not limited to, transfection, electroporation,
microinjection, lipofection, adsorption, and protoplast fusion.
Preferred methods to transfect host cells in vivo include
lipofection and adsorption.
[0062] It may be appreciated by one skilled in the art that use of
recombinant DNA technologies can improve expression of transfected
nucleic acid molecules by manipulating, for example, the number of
copies of the nucleic acid molecules within a host cell, the
efficiency with which those nucleic acid molecules are transcribed,
the efficiency with which the resultant transcripts are translated,
and the efficiency of post-translational modifications. Recombinant
techniques useful for increasing the expression of nucleic acid
molecules of the present invention include, but are not limited to,
operatively linking nucleic acid molecules to high-copy number
plasmids, integration of the nucleic acid molecules into one or
more host cell chromosomes, addition of vector stability sequences
to plasmids, substitutions or modifications of transcription
control signals (e.g., promoters, operators, enhancers),
substitutions or modifications of translational control signals
(e.g., ribosome binding sites, Shine-Dalgarno sequences),
modification of nucleic acid molecules of the present invention to
correspond to the codon usage of the host cell, and deletion of
sequences that destabilize transcripts.
[0063] According to yet another aspect of the present invention
there is provided a pharmaceutical composition suitable for
effecting the above methods of the present invention. The
composition includes a recombinant construct including an isolated
nucleic acid sequence encoding a heat shock protein or a fragment
thereof, the nucleic acid sequence being operatively linked to one
or more transcription control sequences, and a pharmaceutically
acceptable carrier.
[0064] In one embodiment of the invention, the composition is
useful for treating a T cell-mediated inflammatory autoimmune
disease such as multiple sclerosis, rheumatoid arthritis, collagen
II arthritis, autoimmune neuritis, systemic lupus erythematosus,
psoriasis, juvenile onset diabetes, Sjogren's disease, thyroid
disease, sarcoidosis, autoimmune uveitis, inflammatory bowel
disease (Crohn's and ulcerative colitis) or autoimmune
hepatitis.
[0065] The therapeutic composition of the invention is administered
to an individual in need of said treatment. According to still
further features in the described preferred embodiments the
individual is selected from the group consisting of humans, dogs,
cats, sheep, cattle, horses and pigs.
[0066] In another embodiment of the present invention, a
therapeutic composition further comprises a pharmaceutically
acceptable carrier. As used herein, a "carrier" refers to any
substance suitable as a vehicle for delivering a nucleic acid
sequence of the present invention to a suitable in vivo site. As
such, carriers can act as a pharmaceutically acceptable excipient
of a therapeutic composition containing a nucleic acid molecule of
the present invention. Preferred carriers are capable of
maintaining a nucleic acid molecule of the present invention in a
form that, upon arrival of the nucleic acid molecule to a cell, the
nucleic acid molecule is capable of entering the cell and being
expressed by the cell. Carriers of the present invention include:
(1) excipients or formularies that transport, but do not
specifically target a nucleic acid molecule to a cell (referred to
herein as non-targeting carriers); and (2) excipients or
formularies that deliver a nucleic acid molecule to a specific site
in an animal or a specific cell (i.e., targeting carriers).
Examples of non-targeting carriers include, but are not limited to
water, phosphate buffered saline, Ringer's solution, dextrose
solution, serum-containing solutions, Hank's solution, other
aqueous physiologically balanced solutions, oils, esters and
glycols. Aqueous carriers can contain suitable auxiliary substances
required to approximate the physiological conditions of the
recipient, for example, by enhancing chemical stability and
isotonicity.
[0067] Suitable auxiliary substances include, for example, sodium
acetate, sodium chloride, sodium lactate, potassium chloride,
calcium chloride, and other substances used to produce phosphate
buffer, Tris buffer, and bicarbonate buffer. Auxiliary substances
can also include preservatives, such as thimerosal, m- and
o-cresol, formalin and benzol alcohol. Preferred auxiliary
substances for aerosol delivery include surfactant substances
non-toxic to an animal, for example, esters or partial esters of
fatty acids containing from about six to about twenty-two carbon
atoms. Examples of esters include, caproic, octanoic, lauric,
palmitic, stearic, linoleic, linolenic, olesteric, and oleic acids.
Other carriers can include metal particles (e.g., gold particles)
for use with, for example, a biolistic gun through the skin.
Therapeutic compositions of the present invention can be sterilized
by conventional methods.
[0068] Targeting carriers are herein referred to as "delivery
vehicles". Delivery vehicles of the present invention are capable
of delivering a therapeutic composition of the present invention to
a target site in an animal. A "target site" refers to a site in an
animal to which one desires to deliver a therapeutic composition.
For example, a target site can be a cancer cell, a tumor, or a
lesion caused by an infectious agent, or an area around such cell,
tumor or lesion, which is targeted by direct injection or delivery
using liposomes or other delivery vehicles. Examples of delivery
vehicles include, but are not limited to, artificial and natural
lipid-containing delivery vehicles. Natural lipid-containing
delivery vehicles include cells and cellular membranes. Artificial
lipid-containing delivery vehicles include liposomes and micelles.
A delivery vehicle of the present invention can be modified to
target to a particular site in an animal, thereby targeting and
making use of a nucleic acid molecule of the present invention at
that site. Suitable modifications include manipulating the chemical
formula of the lipid portion of the delivery vehicle and/or
introducing into the vehicle a compound capable of specifically
targeting a delivery vehicle to a preferred site, for example, a
preferred cell type. Specifically targeting refers to causing a
delivery vehicle to bind to a particular cell by the interaction of
the compound in the vehicle to a molecule on the surface of the
cell. Suitable targeting compounds include ligands capable of
selectively (i.e., specifically) binding another molecule at a
particular site. Examples of such ligands include antibodies,
antigens, receptors and receptor ligands. For example, an antibody
specific for an antigen found on the surface of a cancer cell can
be introduced to the outer surface of a liposome delivery vehicle
so as to target the delivery vehicle to the cancer cell. Tumor cell
ligands include ligands capable of binding to a molecule on the
surface of a tumor cell. Manipulating the chemical formula of the
lipid portion of the delivery vehicle can modulate the
extracellular or intracellular targeting of the delivery vehicle.
For example, a chemical can be added to the lipid formula of a
liposome that alters the charge of the lipid bilayer of the
liposome so that the liposome fuses with particular cells having
particular charge characteristics.
[0069] According to one embodiment, fat emulsions may be used as a
vehicle for DNA vaccines. Two examples of such emulsions are the
available commercial fat emulsions known as Intralipid and
Lipofundin. "Intralipid" is a registered trademark of Kabi
Pharmacia, Sweden, for a fat emulsion for intravenous nutrition,
described in U.S. Pat. No. 3,169,094. "Lipofundin" is a registered
trademark of B. Braun Melsungen, Germany. Both contain soybean oil
as fat (100 or 200 g in 1,000 ml distilled water: 10% or 20%,
respectively). Egg-yolk phospholipids are used as emulsifiers in
Intralipid (12 g/l distilled water) and egg-yolk lecithin in
Lipofundin (12 g/l distilled water). Isotonicity results from the
addition of glycerol (25 g/l) both in Intralipid and
Lipofundin.
[0070] According to another embodiment, the delivery vehicle of the
present invention may be a liposome. A liposome is capable of
remaining stable in an animal for a sufficient amount of time to
deliver a nucleic acid sequence of the present invention to a
preferred site in the animal. A liposome of the present invention
is preferably stable in the animal into which it has been
administered for at least about 30 minutes, more preferably for at
least about 1 hour and even more preferably for at least about 24
hours.
[0071] A liposome of the present invention comprises a lipid
composition that is capable of fusing with the plasma membrane of
the targeted cell to deliver a nucleic acid molecule into a cell.
Preferably, the transfection efficiency of a liposome of the
present invention is about 0.5 microgram (.mu.g) of DNA per 16
nanomole (nmol) of liposome delivered to about 10.sup.6 cells, more
preferably about 1.0 .mu.g of DNA per 16 nmol of liposome delivered
to about 10.sup.6 cells, and even more preferably about 2.0 .mu.g
of DNA per 16 nmol of liposome delivered to about 10.sup.6
cells.
[0072] A preferred liposome of the present invention is between
about 100 and 500 nanometers (nm), more preferably between about
150 and 450 nm and even more preferably between about 200 and 400
nm in diameter.
[0073] Suitable liposomes for use with the present invention
include any liposome. Preferred liposomes of the present invention
include those liposomes standardly used in, for example, gene
delivery methods known to those of skill in the art. More preferred
liposomes comprise liposomes having a polycationic lipid
composition and/or liposomes having a cholesterol backbone
conjugated to polyethylene glycol.
[0074] Complexing a liposome with a nucleic acid sequence of the
present invention can be achieved using methods standard in the
art. A suitable concentration of a nucleic acid molecule of the
present invention to add to a liposome includes a concentration
effective for delivering a sufficient amount of nucleic acid
molecule to a cell such that the cell can produce sufficient heat
shock protein to regulate effector cell immunity in a desired
manner. Preferably, from about 0.1 .mu.g to about 10 .mu.g of
nucleic acid sequence of the present invention is combined with
about 8 nmol liposomes, more preferably from about 0.5 .mu.g to
about 5 .mu.g of nucleic acid molecule is combined with about 8
nmol liposomes, and even more preferably about 1.0 .mu.g of nucleic
acid molecule is combined with about 8 nmol liposomes.
[0075] According to another embodiment, the delivery vehicle
comprises a recombinant cell vaccine. Preferred recombinant cell
vaccines of the present invention include cell vaccines, in which
allogeneic (i.e., cells derived from a source other than a patient,
but that are histiotype compatible with the patient) or autologous
(i.e., cells isolated from a patient) cells are transfected with
recombinant molecules contained in a therapeutic composition,
irradiated and administered to a patient by, for example,
intradermal, intravenous or subcutaneous injection. Therapeutic
compositions to be administered by cell vaccine, include
recombinant molecules of the present invention without carrier.
[0076] In order to treat an animal with disease, a therapeutic
composition of the present invention is administered to the animal
in an effective manner such that the composition is capable of
treating that animal from disease. For example, a recombinant
molecule, when administered to an animal in an effective manner, is
able to stimulate effector cell immunity in a manner that is
sufficient to alleviate the disease afflicting the animal.
According to the present invention, treatment of a disease refers
to alleviating a disease and/or preventing the development of a
secondary disease resulting from the occurrence of a primary
disease. An effective administration protocol (i.e., administering
a therapeutic composition in an effective manner) comprises
suitable dose parameters and modes of administration that result in
treatment of a disease. Effective dose parameters and modes of
administration can be determined using methods standard in the art
for a particular disease. Such methods include, for example,
determination of survival rates, side effects (i.e., toxicity) and
progression or regression of disease. In particular, the
effectiveness of dose parameters and modes of administration of a
therapeutic composition of the present invention when treating
cancer can be determined by assessing response rates. Such response
rates refer to the percentage of treated patients in a population
of patients that respond with either partial or complete
remission.
[0077] In accordance with the present invention, a suitable single
dose size is a dose that is capable of treating an animal with
disease when administered one or more times over a suitable time
period. Doses can vary depending upon the disease being treated.
Doses of a therapeutic composition of the present invention
suitable for use with direct injection techniques can be used by
one of skill in the art to determine appropriate single dose sizes
for systemic administration based on the size of an animal. A
suitable single dose of a therapeutic composition is a sufficient
amount of heat shock protein-encoding recombinant sequence to
reduce, and preferably eliminate, the T-cell mediated autoimmune
disease following transfection of the recombinant molecules into
cells. A preferred single dose of heat shock protein-encoding
recombinant molecule is an amount that, when transfected into a
target cell population leads to the production of from about 250
femtograms (fg) to about 1 .mu.g, preferably from about 500 fg to
about 500 picogram (pg), and more preferably from about 1 pg to
about 100 pg of a heat shock protein or fragment thereof per
transfected cell.
[0078] A preferred single dose of heat shock protein-encoding
recombinant molecule complexed with liposomes, is from about 100
.mu.g of total DNA per 800 nmol of liposome to about 2 mg of total
recombinant molecules per 16 micromole (.mu.mol) of liposome, more
preferably from about 150 .mu.g per 1.2 .mu.mol of liposome to
about 1 mg of total recombinant molecules per 8 .mu.mol of
liposome, and even more preferably from about 200 .mu.g per 2
.mu.mol of liposome to about 400 .mu.g of total recombinant
molecules per 3.2 .mu.mol of liposome.
[0079] A preferred single dose of heat shock protein-encoding
recombinant molecule in a non-targeting carrier to administer to an
animal, is from about 100 .mu.g to about 4 mg of total recombinant
molecules, more preferably from about 150 .mu.g to about 3 mg of
total recombinant molecules, and even more preferably from about
200 .mu.g to about 2 mg of total recombinant molecules.
[0080] It will be obvious to one of skill in the art that the
number of doses administered to an animal is dependent upon the
extent of the disease and the response of an individual patient to
the treatment. Thus, it is within the scope of the present
invention that a suitable number of doses includes any number
required to cause regression of a disease. A preferred protocol is
monthly administrations of single doses (as described above) for up
to about 1 year. A preferred number of doses of a therapeutic
composition comprising heat shock protein-encoding recombinant
molecule in a non-targeting carrier or complexed with liposomes is
from about 1 to about 10 administrations per patient, preferably
from about 2 to about 8 administrations per patient, and even more
preferably from about 3 to about 5 administrations per person.
Preferably, such administrations are given once every 2 weeks until
signs of remission appear, then once a month until the disease is
gone.
[0081] A therapeutic composition is administered to an animal in a
fashion to enable expression of the administered recombinant
molecule of the present invention into a curative protein in the
animal to be treated for disease. A therapeutic composition can be
administered to an animal in a variety of methods including, but
not limited to, local administration of the composition into a site
in an animal, and systemic administration.
[0082] Therapeutic compositions to be delivered by local
administration include: (a) recombinant molecules of the present
invention in a non-targeting carrier (e.g., as "naked" DNA
molecules, such as is taught, for example in Wolff et al., 1990,
Science 247, 1465-1468); and (b) recombinant molecules of the
present invention complexed to a delivery vehicle of the present
invention. Suitable delivery vehicles for local administration
comprise liposomes or emulsions. Delivery vehicles for local
administration can further comprise ligands for targeting the
vehicle to a particular site.
[0083] Therapeutic compositions useful in systemic administration
include recombinant molecules of the present invention complexed to
a targeted delivery vehicle of the present invention. Suitable
delivery vehicles for use with systemic administration comprise
liposomes comprising ligands for targeting the vehicle to a
particular site. Systemic administration is particularly
advantageous when organs, in particular difficult to reach organs
(e.g., heart, spleen, lung or liver) are the targeted sites of
treatment.
[0084] Preferred methods of systemic administration, include
intravenous injection, aerosol, oral and percutaneous (topical)
delivery. Intravenous injections can be performed using methods
standard in the art. Aerosol delivery can also be performed using
methods standard in the art (see, for example, Stribling et al.,
Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, which is
incorporated herein by reference in its entirety). Oral delivery
can be performed by complexing a therapeutic composition of the
present invention to a carrier capable of withstanding degradation
by digestive enzymes in the gut of an animal. Examples of such
carriers include plastic capsules or tablets, such as those known
in the art. Topical delivery can be performed by mixing a
therapeutic composition of the present invention with a lipophilic
reagent (e.g., DMSO) that is capable of passing into the skin.
[0085] Suitable embodiments, single dose sizes, number of doses and
modes of administration of a therapeutic composition of the present
invention useful in a treatment method of the present invention are
disclosed in detail herein.
[0086] A therapeutic composition of the present invention is also
advantageous for the treatment of autoimmune diseases in that the
composition suppresses the harmful stimulation of T cells by
autoantigens (i.e., a "self", rather than a foreign antigen). Heat
shock protein-encoding recombinant molecules in a therapeutic
composition, upon transfection into a cell, produce a heat shock
protein or a fragment thereof that reduces the harmful activity of
T cells involved in an autoimmune disease. A preferred therapeutic
composition for use in the treatment of autoimmune disease
comprises heat shock protein-encoding recombinant molecule of the
present invention. A more preferred therapeutic composition for use
in the treatment of autoimmune disease comprises heat shock
protein-encoding recombinant molecule combined with a non-targeting
carrier of the present invention, preferably saline or phosphate
buffered saline.
[0087] A single dose of heat shock protein-encoding nucleic acid
molecule in a non-targeting carrier to administer to an animal to
treat an autoimmune disease is from about 0.1 .mu.g to about 200
.mu.g of total recombinant molecules per kilogram (kg) of body
weight, more preferably from about 0.5 .mu.g to about 150 .mu.g of
total recombinant molecules per kg of body weight, and even more
preferably from about 1 .mu.g to about 10 .mu.g of total
recombinant molecules per kg of body weight.
[0088] The number of doses of heat shock protein-encoding
recombinant molecule in a non-targeting carrier to be administered
to an animal to treat an autoimmune disease is an injection about
once every 6 months, more preferably about once every 3 months, and
even more preferably about once a month.
[0089] A preferred method to administer a therapeutic composition
of the present invention to treat an autoimmune disease is by
direct injection. Direct injection techniques are particularly
important in the treatment of an autoimmune disease. Preferably, a
therapeutic composition is injected directly into muscle cells in a
patient, which results in prolonged expression (e.g., weeks to
months) of a recombinant molecule of the present invention.
Preferably, a recombinant molecule of the present invention in the
form of "naked DNA" is administered by direct injection into muscle
cells in a patient.
[0090] It is to be noted that the compositions and methods of the
present invention do not include the obligatory presence of the CpG
motif disclosed in WO 02/16549, in DNA vaccines suitable for the
treatment of ongoing autoimmune diseases.
[0091] The following examples are presented in order to more fully
illustrate certain embodiments of the invention. They should in no
way, however, be construed as limiting the broad scope of the
invention. One skilled in the art can readily devise many
variations and modifications of the principles disclosed herein
without departing from the scope of the invention.
EXAMPLES
Materials and Methods
Animals
[0092] Female Lewis rats were raised and maintained under
pathogen-free conditions in the Animal Breeding Center of The
Weizmann Institute of Science. One- to two-month old rats were used
for DNA vaccination and peptide-vaccination experiments. The
experiments were performed under the supervision and guidelines of
the Animal Welfare Committee.
Antigens and Adjuvants
[0093] Peptides were synthesized as previously described (15). The
HSP60 peptides used in these studies are listed in Table I. Two
HSP65 peptides were also used: Mt176-190, EESNTFGLQLELTEG (16) and
Mt3, AYDEEARRGLERGLNALADA. Purified recombinant HSP65 was
generously provided by Prof. Ruurd van der Zee (Institute of
Infectious Diseases and Immunology, Faculty of Veterinary Medicine,
Utrecht, The Netherlands). Recombinant HSP60 was prepared in our
laboratory as described (11). M. tuberculosis Strain H37Ra and
incomplete Freund's adjuvant (IFA) were purchased from Difco
(Detroit, Mich., USA). Tuberculin purified protein derivative (PPD)
was provided by the Statens Seruminstitut (Copenhagen, Denmark).
Ovalbumin (OVA) and Concanavalin A (Con A) were purchased from
Sigma (Rehovot, Israel).
DNA Plasmids
[0094] The vector containing the human hsp60 gene (pHSP60) has been
described (17). The construct encoding Mycobacterium leprae HSP65
(pHSP65) was kindly provided by Dr. Douglas Lowrie (Medical
Research Council, London, UK). Five fragments of the human hsp60
gene were amplified by PCR from hsp60 cDNA in pGEM (Promega,
Madison, Wis., USA) using specific oligonucleotides containing
restriction sites for the enzymes BamHI (oligonucleotide 5') or
HindIII (oligonucleotide 3'), and cloned into the pcDNA3 vector
(Invitrogen, NV, Leek, The Netherlands) using standard molecular
biology techniques (Table II). The 5' oligonucleotide also included
an ATG sequence necessary for protein translation. The plasmids
were sequenced to confirm correct insertion of the cDNA and
transcribed in vitro to check that they are functional (data not
shown).
[0095] Plasmid DNA was prepared in large scale and injected after
pretreatment with cardiotoxin (Sigma, Rehovot, Israel). Briefly,
rats were vaccinated in the quadriceps three times (on days -40,
-26-12 relative to AA induction) with 150 .mu.g of pcDNA3, pI, pII,
pIII, pIV or pV. Endotoxin levels were checked by Limulus
Amoebocyte Lysate and found always to be under acceptable levels
for in vivo use (less than 0.02 EU/.mu.g DNA). AA was induced 12
days after the last injection of DNA. The empty vector pcDNA3 was
used as a DNA vaccination control.
AA Induction and Assessment
[0096] AA was induced using 1 mg per rat of heat-killed Mt strain
H37Ra (Difco). The day of AA induction was designated as day 0, and
disease severity was assessed by direct observation of all 4 limbs
in each animal. A relative score between 0 and 4 was assigned to
each limb, based on the degree of joint inflammation, redness and
deformity; thus the maximum possible score for an individual animal
was 16. Arthritis was also quantified by measuring hind limb
diameter with a caliper. Measurements were taken on the day of AA
induction and 26 days later, and they are presented as the
mean.+-.SEM of the difference between the two values. The person
who scored the disease was blinded to the identity of the
groups.
T-Cell Proliferation
[0097] Popliteal and inguinal lymph node cells (LNC) taken 26 days
after the induction of AA were cultured in quadruplicates in 200
.mu.l round bottom microtiter wells (Costar Corp., Cambridge, USA)
at 2.times.10.sup.5 cells per well with or without antigen. The
T-cell mitogen Concanavalin A (Con A) was used as a positive
control for T-cell proliferation. Cultures were incubated for 96
hrs at 37.degree. C. in a humidified atmosphere of 5% CO.sub.2.
T-cell responses were detected by the incorporation of
[methyl-.sup.3H]-thymidine (Amersham, Buckinghamshire, UK; 1
.mu.Ci/well), which was added to the wells for the last 18 hours.
The stimulation index (SI) was computed as the ratio of the mean
c.p.m. of antigen- or mitogen-containing wells to control wells
cultured with medium alone.
Transfer of Cells
[0098] Spleen cells were prepared from peptide-vaccinated rats 26
days after the induction of AA. The splenocytes (10.sup.7 cells per
ml) were activated with 2.5 .mu.g/ml of Con A for 48 hr at
37.degree. C. in a humidified atmosphere of 5% CO.sub.2. The cells
were washed with sterile PBS and injected iv into naive rats
(5.times.10.sup.7 cells per rat). Three days after the transfer of
the splenocytes, AA was induced.
Cytokine Assays
[0099] Supernatants were collected after 72 hrs of stimulation with
each of the antigens tested. Rat IL-10 and IFN.gamma. were
quantitated in culture supernatants by enzyme-linked immunosorbent
assay (ELISA) using Pharmingen's OPTEIA kit (Pharmingen, San Diego,
USA). Rat TGF.beta.1 was quantified using the TGF.beta.1
E.sub.max.RTM. ImmunoAssay System (Promega, Madison, USA) according
to the manufacturer's instructions. Cytokine levels in supernatants
are expressed as pg/ml based on calibration curves constructed
using recombinant cytokines as standards. The lower limits of
detection for the experiments described in this paper were 15 pg/ml
for TGF.beta.1, IL-10 and IFN.gamma..
Statistical Significance
[0100] The InStat 2.01 program was used for statistical analysis.
Student's t-test and the Mann-Whitney test were carried out to
assay significant differences between the different experimental
groups.
Example 1
HSP60 DNA Fragments Inhibit AA
[0101] To learn whether fragments of HSP60 DNA could inhibit AA,
the cDNA corresponding to the human hsp60 gene was divided into
five fragments, each with a 30 pb overlap, and each was cloned into
the pcDNA3 vector (Table II). In this way, five constructs
corresponding to HSP60-derived fragments were generated overlapping
by 10 aa: pI, aa 1-140; pII, aa 130-260; pIII, aa 250-410, pIV, aa
400-470 and pV, aa 460-540 (Table II). Lewis rats were vaccinated
with one of the HSP60 fragment constructs or with pcDNA3 as a
control, and AA was induced. FIG. 1A shows that the rats vaccinated
with pI or pII manifested significantly decreased arthritis
compared to rats vaccinated with constructs pIII, pIV or pV or with
control pcDNA3. The protective effect of the vaccination with
constructs pI and pII was also reflected by a significant reduction
in ankle swelling (FIG. 1B), and by a reduction of the mean maximal
score, which was lower in those rats vaccinated with pI and pII
(p=0.0007 and p=0.0003, respectively, compared to rats vaccinated
with pcDNA3).
Example 2
In Vitro Proliferation of LNC Isolated from pHSP60- or
PI-Vaccinated Rats in Response to Various HSP60 Peptides
[0102] To learn whether the suppression of AA by DNA vaccination
with pHSP60, pI or pII was associated with T-cell reactivity to a
specific HSP60 epitope, the proliferation (Stimulation Index, SI)
of LNC isolated from pHSP60-vaccinated rats was studied in response
to a panel of overlapping peptides spanning the region of human
HSP60 encoded by pI and pII (aa 1-275; Table I). Controls were LNC
prepared from rats vaccinated with pcDNA3 or pHSP65 and challenged
with Mt suspended in IFA to induce AA. FIG. 2A shows that only
peptide Hu3 (aa 31-50 of human HSP60) induced a significant
response in LNC taken from pHSP60-vaccinated rats; cells prepared
from pHSP65 or pcDNA-vaccinated rats did not respond to Hu3. Note
that the sequence of the HSP60 protein in the region 31-50 is
identical in rat and human HSP60; thus Hu3 is a self-peptide (Table
III). FIG. 2B shows a dose-dependant proliferative response to Hu3
using LNC isolated from pHSP60-vaccinated rats. No significant
responses to the control peptide Hu12 (aa 166-185 of human HSP60)
were detected. To confirm these results, T-cell proliferative
responses were studied in LNC taken 26 days after the induction of
AA from rats vaccinated with pI, pII or pcDNA3. FIG. 2C shows that
Hu3, but not its mycobacterial homologue Mt3, triggered a
significant proliferation of LNC taken from pI-immunized rats, but
not by LNC from rats vaccinated with pII or pcDNA3. Non of the
HSP60 peptides was specifically recognized by LNC taken from
pII-immunized rats. In summary, these results show that pHSP60--
and pI-vaccinated rats manifested up-regulated T-cell responses to
the Hu3 peptide of HSP60.
Example 3
Peptide Hu3 Inhibits AA
[0103] To establish a link between the immune response to Hu3 and
prevention of AA, rats were vaccinated with Hu3, or with its
mycobacterial counterpart Mt3 (Table III) or with Hu12 (Table I) as
controls. Each rat received a single i.p. dose of 100 .mu.g of
peptide in IFA seven days (day -7) before the induction of AA (day
0). FIG. 3A shows that the Hu3-vaccinated rats developed
significantly decreased disease compared to non-immunized rats or
rats vaccinated with PBS, Hu12 or Mt3. This reduction in AA was
also reflected by a significant reduction in ankle swelling
(14.2.+-.4.7 in Hu3-vaccinated rats vs 32.2.+-.3.5 in
PBS/IFA-vaccinated rats, p=0.02; FIG. 3B). Therefore, vaccination
with Hu3 prevents AA.
Example 4
Adoptive Transfer of Peptide-Induced Regulation
[0104] To learn whether the protection triggered by Hu3-vaccination
could be adoptively transferred by activated T-cells, splenocytes
prepared from Hu3 vaccinated rats were stimulated in vitro for 2
days with the T-cell mitogen Con A, washed, and injected iv
(5.times.10.sup.7 cells per rat) into naive rats. Only the
recipients of cells taken from Hu3-vaccinated rats were protected
against the subsequent induction of AA (FIGS. 4A and 4B). No
protection was seen in rats that had received Con A activated cells
from Mt3 injected rats. Thus, inhibition of AA by vaccination with
Hu3 could be adoptively transferred by activated T-cells.
Example 5
In Vitro Proliferation of Lnc Isolated from PI- or PII-Vaccinated
Rats in Response to Various Mycobacterial Antigens, HSP60 or its
Hu3 Peptide
[0105] To study the mechanism associated with the inhibition of AA
by DNA vaccination with pI or pII, the T-cell responses of
immunized rats was analysed 26 days after the induction of AA. The
LNC were stimulated in vitro with a collective of mycobacterial
antigens known to be associated with AA: HSP65, PPD, Mt176-90
(which contains the 180-188 epitope of HSP65). The immune response
directed to mammalian HSP60, its regulatory peptide Hu3 and the
HSP60-derived peptide Hu12 as a control was studied as well. OVA
was included as a control antigen. None of the experimental groups
showed significant responses to OVA or Hu12, and they did not
differ in their response to Con A (data not shown). Nevertheless,
inhibition of AA by DNA vaccination with the pI or pII constructs
was associated with the up-regulation of the T-cell proliferative
responses directed against the panel of mycobacterial antigens
(PPD, HSP65 and Mt176-190) (FIG. 5A). FIG. 5B depicts the
proliferative responses to HSP60 and its Hu3 peptide. It can be
seen that both pI and pII induced significant T-cell responses to
HSP60, however, only LNC from pI-vaccinated rats manifested
reactivity to Hu3.
Example 6
Cytokine Secretion by LNC Taken from Rats Vaccinated with pI, pH or
pcDNA3
[0106] Cytokine secretion by LNC taken from rats vaccinated with
pI, pII or pcDNA3 was determined. Inhibition of AA by DNA
vaccination with pI was associated with a decrease in IFN.gamma.
secretion (FIG. 6A), and an increase in IL-10 and TGF.beta.1
secretion upon stimulation with PPD, HSP65 or Mt176-190 (FIGS. 6B
and 6C).
[0107] LNC from pII-vaccinated rats also showed a decrease in
IFN.gamma. secretion upon stimulation with PPD, HSP65 and Mt176-190
(FIG. 6A), however IL-10 secretion was only detected after
activation with HSP65 while TGF.beta.1 secretion was only detected
following activation with Mtl 76-190 or PPD (FIGS. 6B and 6C). Note
that cells from both pI or pII-immunized rats secreted detectable
amounts of TGF.beta.1 upon activation with Mt3. Thus, protection
from AA by DNA vaccination with pI or pII was associated with
decreased IFN.gamma. secretion and a concomitant increase in IL-10
and/or TGF.beta.1 secretion upon stimulation with the mycobacterial
antigens PPD, HSP65 or Mt176-190 (FIGS. 6A, 6B and 6C).
[0108] In addition to the responses to mycobacterial antigens, the
effects of DNA vaccination with HSP60 fragments on the responses to
HSP60 and Hu3 was studied. IFN.gamma. was not secreted in response
to HSP60 or Hu3 by the LNC of either the pI or pII-immunized rats
(FIG. 6A). LNC taken from pI immunized rats secreted both IL-10 and
TGF.beta.1 in response to HSP60 or Hu3 (FIGS. 6B and 6C). LNC cells
taken from pII-vaccinated rats, in contrast, secreted TGF.beta.1
upon activation with HSP60, but IL-10 was not detected. Therefore,
both pI or pII vaccination induced the secretion of TGF.beta.1 in
response to HSP60. However only pI triggered the secretion of
IL-10.
Example 7
In Vitro Proliferation of LNC Isolated from Hu3-Vaccinated Rats in
Response to Various Mycobacterial Antigens or HSP60
[0109] The T-cell responses after induction of AA in rats that had
been vaccinated with peptides Hu3, Hu12 or Mt3 was examined. All
three peptides were immunogenic; significant and specific T-cell
responses could be detected in the immunized rats to each immunogen
(FIG. 7A). However, only the LNC taken from Hu3-vaccinated rats
showed up-regulated T-cell proliferative responses to the
mycobacterial antigens PPD, HSP65 and Mt176-190 (FIG. 7B).
Furthermore, vaccination with Hu3 led to the induction of a
specific response to HSP60 (FIG. 7B). None of the experimental
groups showed significant responses to OVA, and they did not differ
in their response to Con A (data not shown).
Example 8
Cytokine Secretion by LNC Taken from Rats Vaccinated with Hu3
[0110] Vaccination with Hu3 led to a reduction in IFN.gamma.
secretion (FIG. 8A), and to a concomitant increase in the secretion
of IL-10 (FIG. 8B) and TGF.beta.1 (FIG. 8C) upon stimulation with
PPD, HSP65 or Mt176-190. Hu3-vaccination also led to the induction
of T-cells that secreted IFN.gamma., IL-10 and TGF.beta.1 in
response to Hu3. The response to whole HSP60 was predominantly
TGF.beta.1.
Example 9
Human pHPS60 is More Effective than Mycobacterial pHSP65 in
Inhibiting AA
[0111] The effects on AA of vaccination with DNA encoding human
pHSP60 compared to mycobacterial HSP65 was examined. The construct
encoding the full-length human HSP60 (pHSP60) has more than 97%
percent identity at the amino acid level with its rat counterpart.
A construct encoding for the full-length HSP65 of Mycobacterium was
used as well. Two control constructs were used: an empty vector
(pcDNA3), and a construct encoding murine Myelin Basic Protein
(pMBP). FIG. 9a shows that vaccination with pcDNA3 or pMBP did not
have any effect on AA. In contrast, rats immunized with pHSP60 or
pHSP65 manifested a significantly milder arthritis. Inhibition of
AA was also reflected as a diminished swelling of the ankle, as
shown in FIG. 9b. It can be seen that pHSP60 was more effective
than pHSP65 in modulating the autoimmune process. The difference
between pHSP60 and pHSP65 was statistically significant with regard
to the maximal AA index (2.25.+-.0.65 vs. 7.67.+-.1.83, p=0.02),
and leg swelling (10.64.+-.3.43 vs. 27.5.+-.6.35, p=0.03).
Example 10
In Vitro Proliferation of LNC Isolated from Rats after DNA
Vaccination with pHSP60
[0112] The immune response induced by pHSP60-vaccination alone was
studied before disease induction. Spleen cells were prepared 10
days after the administration of the third dose of the DNA-vaccine,
and the proliferative response upon in vitro stimulation with
different antigens was studied. FIG. 10 shows that
pHSP60-vaccination induced a significant proliferative response to
HSP60, but not to HSP65, MBP or PPD, while cells from pMBP-treated
rats only proliferated in response to MBP. These HSP60-specific
T-cells secreted low amounts of both IL-10 (22.+-.5 pg/ml) and
IFN.gamma. (80.+-.15 pg/ml) upon stimulation in vitro with HSP60
(data not shown). No cytokine release was detected when splenocytes
from pcDNA3-treated animals were stimulated with HSP60. No
significant differences were seen between the different
experimental groups neither in T-cell proliferation nor in
cytokine-release in response to stimulation with Con A (data not
shown). Thus pHSP60 vaccination induced a low, but specific T-cell
response to HSP60; the immune response elicited by pHsp60
vaccination is capable of affecting the immune reactions that
characterize AA.
Example 11
Cytokine Secretion in AA Rats Vaccinated with pHSP60 pHSP65 or
Hu3/IFA
[0113] Twenty-six days after the induction of AA, LNC were prepared
from untreated rats, or animals that had been treated with pcDNA3
or PBS/IFA. LNC were stimulated in vitro with a collective of
antigens previously known to be targeted or associated with AA:
HSP60, HSP65, PPD, P176-90 (which contains the 180-188 epitope of
HSP65 (3)) and Hu3 described here. Hu12 and OVA were included as
control antigens. The results were essentially the same whether the
AA was induced in untreated rats, or in rats pre-treated with
injections of PBS/IFA or pcDNA3. FIG. 11 depicts the results
obtained with LNC isolated from pcDNA3-treated animals, showing the
cytokines released to the culture medium. LNC from pcDNA3-treated
animals showed a strong proliferative response to PPD, and low but
significant responses to HSP65 and P176-90, while no proliferation
was detected after stimulation with HSP60, Hu3 or Hu12. Although
the proliferative response to P176-90 was quite low, stimulation
with this peptide led to the release of IFN.gamma. to at least the
same levels as those achieved by stimulation with PPD. IFN.gamma.
was secreted to a lower extent in response to HSP65, while no
secretion was detected upon stimulation with HSP60, Hu3 or Hu12.
IL-10 and TGF.beta.1 were detected only upon activation with PPD.
Thus, induction of AA activates T-cells that almost exclusively
secrete IFN.gamma. in response to activation with mycobacterial
antigens, and that do not appear to recognize HSP60 or its peptides
Hu3 and Hu12.
[0114] FIG. 12A depicts the proliferative response and cytokine
secretion of cells isolated from animals treated with pHSP60,
pHSP65 or Hu3/IFA expressed as the percent change in reactivity
relative to the proliferation seen using cells from their
respective controls (pcDNA3 or PBS/IFA-treated animals). Animals
protected from AA showed increased proliferative responses to
mycobacterial antigens (PPD, HSP65 and P176-90). The increase in
the proliferation to HSP65 and PPD was stronger in pHSP60-treated
animals. Also the response to mammalian HSP60 was up-regulated
throughout all the groups, but this effect was more marked in
pHSP60 vaccinated animals. Moreover, at day 26 after AA induction,
the response to HSP60 was significantly higher than that detected
at the end of the immunization protocol (Stimulation Index,
SI=2.4.+-.0.39 vs. SI=4.44.+-.0.37 respectively, p<0.05). In
addition, Hu3 was only recognized by animals immunized with pHSP60,
or with Hu3 itself, while there were no responses directed towards
Hu12. Cells from HU12/IFA-treated rats proliferated upon
stimulation with HU12 (data not shown). None of the experimental
groups showed significant responses to OVA, and they did not differ
in their response to Con A (data not shown). Thus, modulation of AA
by vaccination with pHSP60, pHSP65 or Hu3 is accompanied by the
up-regulation of T-cell proliferative responses to both self- and
mycobacterial antigens. The phenotype of these augmented cellular
responses has been characterized in terms of cytokine release
profiles.
[0115] As shown in FIG. 12B, Secretion of IFN.gamma. was increased
in LNC from: A, pHSP60-treated rats stimulated with HSP60, Hu3 or
Hu12; B, pHSP65-treated rats stimulated with HSP60 and C, in
Hu3-treated rats stimulated with Hu3. Thus, modulation of AA is
associated with a reduction in IFN.gamma. release to P176-90 and an
increase in the release of IFN.gamma. to HSP60. As shown in FIG.
12C, the study of IL-10 secretion revealed that LNC from animals
protected from AA (either by DNA or peptide vaccination) showed
increased secretion of IL-10 in response to in vitro stimulation
with mycobacterial antigens. In cells from all the groups protected
we found secretion of IL-10 in response to stimulation with PPD,
HSP65 (pHSP60 and pHSP65-treated animals) or P176-90 (pHSP60-- and
Hu3-treated rats). Release of IL-10 upon stimulation with HSP60 and
its peptides was not uniform and only found in cells from pHSP60 or
pHSP65-vaccinated animals. Therefore protection was associated with
the induction of IL-10 secretors responsive to mycobacterial
antigens (PPD or P180-90) and, in DNA vaccinated animals, to
mammalian HSP60. The analysis of TGF.beta.1 release from LNC taken
from all the groups protected from AA, (either by DNA or peptide
vaccination), showed an increase in the secretion of TGF.beta.1 in
response to stimulation with mycobacterial antigens and HSP60 or
its peptides (FIG. 12D). However, this effect was stronger in
DNA-treated rats.
[0116] All together, the results suggest that modulation of AA by
treatment with specific DNA or peptide is associated with three
observations. A, decreased secretion of IFN.gamma. upon stimulation
with the HSP65 peptide P178-190. B, the induction HSP60-specific
T-cells that secrete IFN.gamma.. C, the appearance of IL-10 and
TGF.beta.1 secretors specific for mycobacterial antigens and
HSP60.
Example 12
Inhibition of AA by DNA Vaccines Encoding Human HSP70 and Human
HSP90
[0117] The full-length cDNA of human HSP70 (pHSP70) or human HSP90
(pHSP90) were cloned into the pcDNA3 vector. The gene constructs
were found to be functional in an in vitro
transcription/translation system (data not shown). Rats were
immunized with pHSP90 and pHSP70 following the same scheme of
vaccination used for pHSP60, and 12 days after the last injection
of DNA, AA was induced. FIG. 13A shows that in rats vaccinated with
pHSP70 or pHSP90 there was a significant inhibition of AA
Inhibition of AA was also seen as a reduction in the maximal score
(FIG. 13B), leg swelling (FIG. 13C) and a significant delay in the
mean day of disease onset (FIG. 13D).
[0118] In order to gain some insight into the mechanism that
mediates prevention of AA by treatment with pHSP70 or pHSP90, the
induction of antibodies of the IgG isotype to the antigen encoded
by the vector was studied. FIGS. 14A and 14B depict the results
obtained for pHSP70- and pHSP90-treated rats respectively.
Vaccination with the DNA constructs induced specific antibodies,
indicating that both constructs are immunogenic; and the humoral
response induced is up-regulated upon induction of AA.
Example 13
Cytokine Secretion and In Vitro Proliferation of LNC Isolated from
AA Rats Vaccinated with pHSP70 or pHSP90
[0119] FIGS. 15A and 15B shows that draining lymph node DLN cells
from immunized animals showed a dose response proliferation upon
activation with the protein encoded by the immunizing vector.
Furthermore, the analysis of the cytokines released in response to
antigen-specific stimulation revealed that cells taken from
pHSP90-immunized animals secreted both IL-10 and IFN.gamma. in
response to activation with HSP90 (FIGS. 16A and 16B). There was no
IFN.gamma. or IL-10 secretion when cells from pHSP70-treated
animals were stimulated with HSP70 (data not shown).
[0120] The immune response to a panel of antigens in DLN cells
isolated from pcDNA3-, pHSP70- or pHSP90-treated rats was detected
26 days after the induction of AA. The results are shown in FIG. 17
as the percentage of the proliferation observed in untreated rats.
Vaccination with pHSP70 or pHSP90 led to a significant
up-regulation in the proliferative response to HSP65, HSP71 and
PPD, and this increase in the immune response was stronger in
pHSP70 vaccinated rats. To further characterize the nature of the
up-regulated immune response in pHSP70- and pHSP90-vaccinated,
cytokine secretion in response to in vitro stimulation with the
same AA-related antigens was detected. IFN.gamma. secretion in
response to stimulation with HSP65, its epitope P176-188 or PPD was
down-regulated in animals treated with pHSP70 or pHSP90 (FIG. 18A).
In addition, IL-10 secretion was up-regulated upon in vitro
activation with PPD, HSP71, HSP65 or HSP60, both in pHSP70- and
pHSP90-vaccinated animals (FIG. 18B). Finally, TGF.beta.1 release
upon stimulation with HSP65 and its peptide P176-80 was increased
in cells taken from pHSP90-vaccinated animals, and there was a
slight but significant release of in response to stimulation with
PPD in cells from pHSP70-treated animals (FIG. 18C). Note that the
decrease in IFN.gamma. secretion and the concomitant increase in
IL-10 and TGF.beta.1 release was stronger in the group treated with
pHSP90; in this group AA was inhibited even more effectively than
in pHSP70-immunized rats.
TABLE-US-00001 TABLE I Overlapping peptides of human HSP60, region
1-275 Pep- tide Position Sequence Hu1 1-20 (SEQ ID NO: 15)
MLRLPTVFRQMRPVSRVLAP Hu2 16-35 (SEQ ID NO: 16) RVLAPHLTRAYAKDVKFGAD
Hu3 31-50 (SEQ ID NO: 3) KFGADARALMLQGVDLLADA Hu4 46-65 (SEQ ID NO:
17) LLADAVAVTMGKGRTVIIE Hu5 61-80 (SEQ ID NO: 18)
TVIIEQSWGSPKVTKDGVTV Hu6 76-95 (SEQ ID NO: 19) DGVTVAKSIDLKDKYKNIGA
Hu7 91-110 (SEQ ID NO: 20) KNIGAKLVQDVANNTNEEAG Hu6 106-125 (SEQ ID
NO: 21) NEEAGKGTTTATVLARSIAK Hu9 121-140 (SEQ ID NO: 22)
RSIAKEGFEKISKGANPVEI Hu10 136-155 (SEQ ID NO: 23)
NPVEIRRGVMLAVDAVIAEL Hu11 151-170 (SEQ ID NO: 24)
VIAELKKQSKPVTTPEEIAQ Hu12 166-185 (SEQ ID NO: 25)
EEIAQVATISANGDKEIGNI Hu13 181-199 (SEQ ID NO: 26)
EIGNIISDAMKKVGRKGVI Hu14 195-214 (SEQ ID NO: 27)
RKGVITVKDGKTLNDELEII Hu15 210-229 (SEQ ID NO: 28)
ELEIIEGMKFDRGYISPYFI Hu16 225-244 (SEQ ID NO: 29)
SPYFINTSKGQKCEFQDAYV Hu17 240-259 (SEQ ID NO: 30)
QDAYVLLSEKKISSIQSIVP Hu18 255-275 (SEQ ID NO: 31)
QSIVPALEIANAHRKPLVIIA
TABLE-US-00002 TABLE II Plasmids constructed containing overlapping
fragments of the hsp60 gene Plasmid Position Corresponding Peptides
included pI 1-140 Hu1-Hu9 (a.k.a. P1-P9; SEQ ID NO: 1) pII 130-260
Hu10-Hu18 (a.k.a. P10-P18; SEQ ID NO: 2) pIII 250-410 NS pIV
400-470 NS pIV 460-540 NS The position is expressed as amino acid
residue numbers. NS = Not synthesized as individual peptides.
TABLE-US-00003 TABLE III Comparison of human HSP60, rat HSP60 and
mycobacterial HSP65 in the region corresponding to the Hu3
sequence.sup.a, b. H. sapiens 31 KFGADARALMLQGVDLLADA 50 (SEQ ID
NO: 3) R. norvergicus 31 KFGADARALMLQGVDLLADA 50 (SEQ ID NO: 35) M.
tuberculosis 5 AYDEEARRGLERGLNALADA 24 (SEQ ID NO: 32) .sup.aH.
sapiens, Homo sapiens; R. norvergicus, Rattus norvergicus; M.
tuberculosis, Mycobacterium tuberculosis. .sup.bResidues sharing
identity with the corresponding sequence of human HSP60 are shown
in bold, and conserved substitutions are shown as underlined
residues.
[0121] Peptides were synthesized as previously described (15). The
HSP60 peptides used in these studies are listed in Table I. Two
HSP65 peptides were also used: Mt176-190, EESNTFGLQLELTEG (SEQ ID
NO:33) (16) and Mt3, AYDEEARRGLERGLNALADA (SEQ ID NO:34). Purified
recombinant HSP65 was generously provided by Prof. Ruurd van der
Zee (Institute of Infectious Diseases and Immunology, Faculty of
Veterinary Medicine, Utrecht, The Netherlands). Recombinant HSP60
was prepared in our laboratory as described (11). M. tuberculosis
Strain H37Ra and incomplete Freund's adjuvant (IFA) were purchased
from Difco (Detroit, Mich., USA). Tuberculin purified protein
derivative (PPD) was provided by the Statens Seruminstitut
(Copenhagen, Denmark). Ovalbumin (OVA) and Concanavalin A (Con A)
were purchased from Sigma (Rehovot, Israel).
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cells. Cell Stress Chaperones 5:452. [0134] 13. Lopez-Guerrero, J.
A., J. P. Lopez-Bote, M. A. Ortiz, R. S. Gupta, E. Paez, and C.
Bernabeu. 1993. Modulation of adjuvant arthritis in Lewis rats by
recombinant vaccinia virus expressing the human 60-kilodalton heat
shock protein. Infect Immun 61:4225. [0135] 14. Lopez-Guerrero, J.
A., M. A. Ortiz, E. Paez, C. Bernabeu, and J. P. Lopez-Bote. 1994.
Therapeutic effect of recombinant vaccinia virus expressing the
60-kd heat-shock protein on adjuvant arthritis. Arthritis Rheum
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Cohen. 2002 Inhibition of adjuvant arthritis by a DNA vaccine
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Sequence CWU 1
1
351140PRTHomo sapiens 1Met Leu Arg Leu Pro Thr Val Phe Arg Gln Met
Arg Pro Val Ser Arg 1 5 10 15 Val Leu Ala Pro His Leu Thr Arg Ala
Tyr Ala Lys Asp Val Lys Phe 20 25 30 Gly Ala Asp Ala Arg Ala Leu
Met Leu Gln Gly Val Asp Leu Leu Ala 35 40 45 Asp Ala Val Ala Val
Thr Met Gly Pro Lys Gly Arg Thr Val Ile Ile 50 55 60 Glu Gln Gly
Trp Gly Ser Pro Lys Val Thr Lys Asp Gly Val Thr Val 65 70 75 80 Ala
Lys Ser Ile Asp Leu Lys Asp Lys Tyr Lys Asn Ile Gly Ala Lys 85 90
95 Leu Val Gln Asp Val Ala Asn Asn Thr Asn Glu Glu Ala Gly Asp Gly
100 105 110 Thr Thr Thr Ala Thr Val Leu Ala Arg Ser Ile Ala Lys Glu
Gly Phe 115 120 125 Glu Lys Ile Ser Lys Gly Ala Asn Pro Val Glu Ile
130 135 140 2131PRTHomo sapiens 2Lys Ile Ser Lys Gly Ala Asn Pro
Val Glu Ile Arg Arg Gly Val Met 1 5 10 15 Leu Ala Val Asp Ala Val
Ile Ala Glu Leu Lys Lys Gln Ser Lys Pro 20 25 30 Val Thr Thr Pro
Glu Glu Ile Ala Gln Val Ala Thr Ile Ser Ala Asn 35 40 45 Gly Asp
Lys Glu Ile Gly Asn Ile Ile Ser Asp Ala Met Lys Lys Val 50 55 60
Gly Arg Lys Gly Val Ile Thr Val Lys Asp Gly Lys Thr Leu Asn Asp 65
70 75 80 Glu Leu Glu Ile Ile Glu Gly Met Lys Phe Asp Arg Gly Tyr
Ile Ser 85 90 95 Pro Tyr Phe Ile Asn Thr Ser Lys Gly Gln Lys Cys
Glu Phe Gln Asp 100 105 110 Ala Tyr Val Leu Leu Ser Glu Lys Lys Ile
Ser Ser Ile Gln Ser Ile 115 120 125 Val Pro Ala 130 320PRTHomo
sapiens 3Lys Phe Gly Ala Asp Ala Arg Ala Leu Met Leu Gln Gly Val
Asp Leu 1 5 10 15 Leu Ala Asp Ala 20 4641PRTHomo sapiens 4Met Ala
Lys Ala Ala Ala Ile Gly Ile Asp Leu Gly Thr Thr Tyr Ser 1 5 10 15
Cys Val Gly Val Phe Gln His Gly Lys Val Glu Ile Ile Ala Asn Asp 20
25 30 Gln Gly Asn Arg Thr Thr Pro Ser Tyr Val Ala Phe Thr Asp Thr
Glu 35 40 45 Arg Leu Ile Gly Asp Ala Ala Lys Asn Gln Val Ala Leu
Asn Pro Gln 50 55 60 Asn Thr Val Phe Asp Ala Lys Arg Leu Ile Gly
Arg Lys Phe Gly Asp 65 70 75 80 Pro Val Val Gln Ser Asp Met Lys His
Trp Pro Phe Gln Val Ile Asn 85 90 95 Asp Gly Asp Lys Pro Lys Val
Gln Val Ser Tyr Lys Gly Glu Thr Lys 100 105 110 Ala Phe Tyr Pro Glu
Glu Ile Ser Ser Met Val Leu Thr Lys Met Lys 115 120 125 Glu Ile Ala
Glu Ala Tyr Leu Gly Tyr Pro Val Thr Asn Ala Val Ile 130 135 140 Thr
Val Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp 145 150
155 160 Ala Gly Val Ile Ala Gly Leu Asn Val Leu Arg Ile Ile Asn Glu
Pro 165 170 175 Thr Ala Ala Ala Ile Ala Tyr Gly Leu Asp Arg Thr Gly
Lys Gly Glu 180 185 190 Arg Asn Val Leu Ile Phe Asp Leu Gly Gly Gly
Thr Phe Asp Val Ser 195 200 205 Ile Leu Thr Ile Asp Asp Gly Ile Phe
Glu Val Lys Ala Thr Ala Gly 210 215 220 Asp Thr His Leu Gly Gly Glu
Asp Phe Asp Asn Arg Leu Val Asn His 225 230 235 240 Phe Val Glu Glu
Phe Lys Arg Lys His Lys Lys Asp Ile Ser Gln Asn 245 250 255 Lys Arg
Ala Val Arg Arg Leu Arg Thr Ala Cys Glu Arg Ala Lys Arg 260 265 270
Thr Leu Ser Ser Ser Thr Gln Ala Ser Leu Glu Ile Asp Ser Leu Phe 275
280 285 Glu Gly Ile Asp Phe Tyr Thr Ser Ile Thr Arg Ala Arg Phe Glu
Glu 290 295 300 Leu Cys Ser Asp Leu Phe Arg Ser Thr Leu Glu Pro Val
Glu Lys Ala 305 310 315 320 Leu Arg Asp Ala Lys Leu Asp Lys Ala Gln
Ile His Asp Leu Val Leu 325 330 335 Val Gly Gly Ser Thr Arg Ile Pro
Lys Val Gln Lys Leu Leu Gln Asp 340 345 350 Phe Phe Asn Gly Arg Asp
Leu Asn Lys Ser Ile Asn Pro Asp Glu Ala 355 360 365 Val Ala Tyr Gly
Ala Ala Val Gln Ala Ala Ile Leu Met Gly Asp Lys 370 375 380 Ser Glu
Asn Val Gln Asp Leu Leu Leu Leu Asp Val Ala Pro Leu Ser 385 390 395
400 Leu Gly Leu Glu Thr Ala Gly Gly Val Met Thr Ala Leu Ile Lys Arg
405 410 415 Asn Ser Thr Ile Pro Thr Lys Gln Thr Gln Ile Phe Thr Thr
Tyr Ser 420 425 430 Asp Asn Gln Pro Gly Val Leu Ile Gln Val Tyr Glu
Gly Glu Arg Ala 435 440 445 Met Thr Lys Asp Asn Asn Leu Leu Gly Arg
Phe Glu Leu Ser Gly Ile 450 455 460 Pro Pro Ala Pro Arg Gly Val Pro
Gln Ile Glu Val Thr Phe Asp Ile 465 470 475 480 Asp Ala Asn Gly Ile
Leu Asn Val Thr Ala Thr Asp Lys Ser Thr Gly 485 490 495 Lys Ala Asn
Lys Ile Thr Ile Thr Asn Asp Lys Gly Arg Leu Ser Lys 500 505 510 Glu
Glu Ile Glu Arg Met Val Gln Glu Ala Glu Lys Tyr Lys Ala Glu 515 520
525 Asp Glu Val Gln Arg Glu Arg Val Ser Ala Lys Asn Ala Leu Glu Ser
530 535 540 Tyr Ala Phe Asn Met Lys Ser Ala Val Glu Asp Glu Gly Leu
Lys Gly 545 550 555 560 Lys Ile Ser Glu Ala Asp Lys Lys Lys Val Leu
Asp Lys Cys Gln Glu 565 570 575 Val Ile Ser Trp Leu Asp Ala Asn Thr
Leu Ala Glu Lys Asp Glu Phe 580 585 590 Glu His Lys Arg Lys Glu Leu
Glu Gln Val Cys Asn Pro Ile Ile Ser 595 600 605 Gly Leu Tyr Gln Gly
Ala Gly Gly Pro Gly Pro Gly Gly Phe Gly Ala 610 615 620 Gln Gly Pro
Lys Gly Gly Ser Gly Ser Gly Pro Thr Ile Glu Glu Val 625 630 635 640
Asp 5641PRTHomo sapiens 5Met Ala Lys Ala Ala Ala Ile Gly Ile Asp
Leu Gly Thr Thr Tyr Ser 1 5 10 15 Cys Val Gly Val Phe Gln His Gly
Lys Val Glu Ile Ile Ala Asn Asp 20 25 30 Gln Gly Asn Arg Thr Thr
Pro Ser Tyr Val Ala Phe Thr Asp Thr Glu 35 40 45 Arg Leu Ile Gly
Asp Ala Ala Lys Asn Gln Val Ala Leu Asn Pro Gln 50 55 60 Asn Thr
Val Phe Asp Ala Lys Arg Leu Ile Gly Arg Lys Phe Gly Asp 65 70 75 80
Pro Val Val Gln Ser Asp Met Lys His Trp Pro Phe Gln Val Ile Asn 85
90 95 Asp Gly Asp Lys Pro Lys Val Gln Val Ser Tyr Lys Gly Glu Thr
Lys 100 105 110 Ala Phe Tyr Pro Glu Glu Ile Ser Ser Met Val Leu Thr
Lys Met Lys 115 120 125 Glu Ile Ala Glu Ala Tyr Leu Gly Tyr Pro Val
Thr Asn Ala Val Ile 130 135 140 Thr Val Pro Ala Tyr Phe Asn Asp Ser
Gln Arg Gln Ala Thr Lys Asp 145 150 155 160 Ala Gly Val Ile Ala Gly
Leu Asn Val Leu Arg Ile Ile Asn Glu Pro 165 170 175 Thr Ala Ala Ala
Ile Ala Tyr Gly Leu Asp Arg Thr Gly Lys Gly Glu 180 185 190 Arg Asn
Val Leu Ile Phe Asp Leu Gly Gly Gly Thr Phe Asp Val Ser 195 200 205
Ile Leu Thr Ile Asp Asp Gly Ile Phe Glu Val Lys Ala Thr Ala Gly 210
215 220 Asp Thr His Leu Gly Gly Glu Asp Phe Asp Asn Arg Leu Val Asn
His 225 230 235 240 Phe Val Glu Glu Phe Lys Arg Lys His Lys Lys Asp
Ile Ser Gln Asn 245 250 255 Lys Arg Ala Val Arg Arg Leu Arg Thr Ala
Cys Glu Arg Ala Lys Arg 260 265 270 Thr Leu Ser Ser Ser Thr Gln Ala
Ser Leu Glu Ile Asp Ser Leu Phe 275 280 285 Glu Gly Ile Asp Phe Tyr
Thr Ser Ile Thr Arg Ala Arg Phe Glu Glu 290 295 300 Leu Cys Ser Asp
Leu Phe Arg Ser Thr Leu Glu Pro Val Glu Lys Ala 305 310 315 320 Leu
Arg Asp Ala Lys Leu Asp Lys Ala Gln Ile His Asp Leu Val Leu 325 330
335 Val Gly Gly Ser Thr Arg Ile Pro Lys Val Gln Lys Leu Leu Gln Asp
340 345 350 Phe Phe Asn Gly Arg Asp Leu Asn Lys Ser Ile Asn Pro Asp
Glu Ala 355 360 365 Val Ala Tyr Gly Ala Ala Val Gln Ala Ala Ile Leu
Met Gly Asp Lys 370 375 380 Ser Glu Asn Val Gln Asp Leu Leu Leu Leu
Asp Val Ala Pro Leu Ser 385 390 395 400 Leu Gly Leu Glu Thr Ala Gly
Gly Val Met Thr Ala Leu Ile Lys Arg 405 410 415 Asn Ser Thr Ile Pro
Thr Lys Gln Thr Gln Ile Phe Thr Thr Tyr Ser 420 425 430 Asp Asn Gln
Pro Gly Val Leu Ile Gln Val Tyr Glu Gly Glu Arg Ala 435 440 445 Met
Thr Lys Asp Asn Asn Leu Leu Gly Arg Phe Glu Leu Ser Gly Ile 450 455
460 Pro Pro Ala Pro Arg Gly Val Pro Gln Ile Glu Val Thr Phe Asp Ile
465 470 475 480 Asp Ala Asn Gly Ile Leu Asn Val Thr Ala Thr Asp Lys
Ser Thr Gly 485 490 495 Lys Ala Asn Lys Ile Thr Ile Thr Asn Asp Lys
Gly Arg Leu Ser Lys 500 505 510 Glu Glu Ile Glu Arg Met Val Gln Glu
Ala Glu Lys Tyr Lys Ala Glu 515 520 525 Asp Glu Val Gln Arg Glu Arg
Val Ser Ala Lys Asn Ala Leu Glu Ser 530 535 540 Tyr Ala Phe Asn Met
Lys Ser Ala Val Glu Asp Glu Gly Leu Lys Gly 545 550 555 560 Lys Ile
Ser Glu Ala Asp Lys Lys Lys Val Leu Asp Lys Cys Gln Glu 565 570 575
Val Ile Ser Trp Leu Asp Ala Asn Thr Leu Ala Glu Lys Asp Glu Phe 580
585 590 Glu His Lys Arg Lys Glu Leu Glu Gln Val Cys Asn Pro Ile Ile
Ser 595 600 605 Gly Leu Tyr Gln Gly Ala Gly Gly Pro Gly Pro Gly Gly
Phe Gly Ala 610 615 620 Gln Gly Pro Lys Gly Gly Ser Gly Ser Gly Pro
Thr Ile Glu Glu Val 625 630 635 640 Asp 6641PRTHomo sapiens 6Met
Ala Thr Ala Lys Gly Ile Ala Ile Gly Ile Asp Leu Gly Thr Thr 1 5 10
15 Tyr Ser Cys Val Gly Val Phe Gln His Gly Lys Val Glu Ile Ile Ala
20 25 30 Asn Asp Gln Gly Asn Arg Thr Thr Pro Ser Tyr Val Ala Phe
Thr Asp 35 40 45 Thr Glu Arg Leu Ile Gly Asp Ala Ala Lys Asn Gln
Val Ala Met Asn 50 55 60 Pro Gln Asn Thr Val Phe Asp Ala Lys Arg
Leu Ile Gly Arg Lys Phe 65 70 75 80 Asn Asp Pro Val Val Gln Ala Asp
Met Lys Leu Trp Pro Phe Gln Val 85 90 95 Ile Asn Glu Gly Gly Lys
Pro Lys Val Leu Val Ser Tyr Lys Gly Glu 100 105 110 Asn Lys Ala Phe
Tyr Pro Glu Glu Ile Ser Ser Met Val Leu Thr Lys 115 120 125 Leu Lys
Glu Thr Ala Glu Ala Phe Leu Gly His Pro Val Thr Asn Ala 130 135 140
Val Ile Thr Val Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr 145
150 155 160 Lys Asp Ala Gly Val Ile Ala Gly Leu Asn Val Leu Arg Ile
Ile Asn 165 170 175 Glu Pro Thr Ala Ala Ala Ile Ala Tyr Gly Leu Asp
Lys Gly Gly Gln 180 185 190 Gly Glu Arg His Val Leu Ile Phe Asp Leu
Gly Gly Gly Thr Phe Asp 195 200 205 Val Ser Ile Leu Thr Ile Asp Asp
Gly Ile Phe Glu Val Lys Ala Thr 210 215 220 Ala Gly Asp Thr His Leu
Gly Gly Glu Asp Phe Asp Asn Arg Leu Val 225 230 235 240 Ser His Phe
Val Glu Glu Phe Lys Arg Lys His Lys Lys Asp Ile Ser 245 250 255 Gln
Asn Lys Arg Ala Val Arg Arg Leu Arg Thr Ala Cys Glu Arg Ala 260 265
270 Lys Arg Thr Leu Ser Ser Ser Thr Gln Ala Asn Leu Glu Ile Asp Ser
275 280 285 Leu Tyr Glu Gly Ile Asp Phe Tyr Thr Ser Ile Thr Arg Ala
Arg Phe 290 295 300 Glu Glu Leu Cys Ala Asp Leu Phe Arg Gly Thr Leu
Glu Pro Val Glu 305 310 315 320 Lys Ala Leu Arg Asp Ala Lys Met Asp
Lys Ala Lys Ile His Asp Ile 325 330 335 Val Leu Val Gly Gly Ser Thr
Arg Ile Pro Lys Val Gln Arg Leu Leu 340 345 350 Gln Asp Tyr Phe Asn
Gly Arg Asp Leu Asn Lys Ser Ile Asn Pro Asp 355 360 365 Glu Ala Val
Ala Tyr Gly Ala Ala Val Gln Ala Ala Ile Leu Met Gly 370 375 380 Asp
Lys Ser Glu Lys Val Gln Asp Leu Leu Leu Leu Asp Val Ala Pro 385 390
395 400 Leu Ser Leu Gly Leu Glu Thr Ala Gly Gly Val Met Thr Ala Leu
Ile 405 410 415 Lys Arg Asn Ser Thr Ile Pro Thr Lys Gln Thr Gln Ile
Phe Thr Thr 420 425 430 Tyr Ser Asp Asn Gln Pro Gly Val Leu Ile Gln
Val Tyr Glu Gly Glu 435 440 445 Arg Ala Met Thr Lys Asp Asn Asn Leu
Leu Gly Arg Phe Asp Leu Thr 450 455 460 Gly Ile Pro Pro Ala Pro Arg
Gly Val Pro Gln Ile Glu Val Thr Phe 465 470 475 480 Asp Ile Asp Ala
Asn Gly Ile Leu Asn Val Thr Ala Thr Asp Lys Ser 485 490 495 Thr Gly
Lys Val Asn Lys Ile Thr Ile Thr Asn Asp Lys Gly Arg Leu 500 505 510
Ser Lys Glu Glu Ile Glu Arg Met Val Leu Asp Ala Glu Lys Tyr Lys 515
520 525 Ala Glu Asp Glu Val Gln Arg Glu Lys Ile Ala Ala Lys Asn Ala
Leu 530 535 540 Glu Ser Tyr Ala Phe Asn Met Lys Ser Val Val Ser Asp
Glu Gly Leu 545 550 555 560 Lys Gly Lys Ile Ser Glu Ser Asp Lys Asn
Lys Ile Leu Asp Lys Cys 565 570 575 Asn Glu Leu Leu Ser Trp Leu Glu
Val Asn Gln Leu Ala Glu Lys Asp 580 585 590 Glu Phe Asp His Lys Arg
Lys Glu Leu Glu Gln Met Cys Asn Pro Ile 595 600 605 Ile Thr Lys Leu
Tyr Gln Gly Gly Cys Thr Gly Pro Ala Cys Gly Thr 610 615 620 Gly Tyr
Val Pro Gly Arg Pro Ala Thr Gly Pro Thr Ile Glu Glu Val 625 630 635
640 Asp 7639PRTHomo sapiens 7Met Ser Ala Arg Gly Pro Ala Ile Gly
Ile Asp Leu Gly Thr Thr Tyr 1 5 10 15 Ser Cys Val Gly Val Phe Gln
His Gly Lys Val Glu Ile Ile Ala Asn 20 25 30 Asp Gln Gly Asn Arg
Thr Thr Pro Ser Tyr Val Ala Phe Thr Asp Thr 35 40 45 Glu Arg Leu
Ile Gly Asp Ala Ala Lys Asn Gln Val Ala Met Asn Pro 50 55 60 Thr
Asn Thr Ile Phe Asp Ala Lys Arg Leu Ile Gly Arg Lys Phe Glu 65
70 75 80 Asp Ala Thr Val Gln Ser Asp Met Lys His Trp Pro Phe Arg
Val Val 85 90 95 Ser Glu Gly Gly Lys Pro Lys Val Gln Val Glu Tyr
Lys Gly Glu Thr 100 105 110 Lys Thr Phe Phe Pro Glu Glu Ile Ser Ser
Met Val Leu Thr Lys Met 115 120 125 Lys Glu Ile Ala Glu Ala Tyr Leu
Gly Gly Lys Val His Ser Ala Val 130 135 140 Ile Thr Val Pro Ala Tyr
Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys 145 150 155 160 Asp Ala Gly
Thr Ile Thr Gly Leu Asn Val Leu Arg Ile Ile Asn Glu 165 170 175 Pro
Thr Ala Ala Ala Ile Ala Tyr Gly Leu Asp Lys Lys Gly Cys Ala 180 185
190 Gly Gly Glu Lys Asn Val Leu Ile Phe Asp Leu Gly Gly Gly Thr Phe
195 200 205 Asp Val Ser Ile Leu Thr Ile Glu Asp Gly Ile Phe Glu Val
Lys Ser 210 215 220 Thr Ala Gly Asp Thr His Leu Gly Gly Glu Asp Phe
Asp Asn Arg Met 225 230 235 240 Val Ser His Leu Ala Glu Glu Phe Lys
Arg Lys His Lys Lys Asp Ile 245 250 255 Gly Pro Asn Lys Arg Ala Val
Arg Arg Leu Arg Thr Ala Cys Glu Arg 260 265 270 Ala Lys Arg Thr Leu
Ser Ser Ser Thr Gln Ala Ser Ile Glu Ile Asp 275 280 285 Ser Leu Tyr
Glu Gly Val Asp Phe Tyr Thr Ser Ile Thr Arg Ala Arg 290 295 300 Phe
Glu Glu Leu Asn Ala Asp Leu Phe Arg Gly Thr Leu Glu Pro Val 305 310
315 320 Glu Lys Ala Leu Arg Asp Ala Lys Leu Asp Lys Gly Gln Ile Gln
Glu 325 330 335 Ile Val Leu Val Gly Gly Ser Thr Arg Ile Pro Lys Ile
Gln Lys Leu 340 345 350 Leu Gln Asp Phe Phe Asn Gly Lys Glu Leu Asn
Lys Ser Ile Asn Pro 355 360 365 Asp Glu Ala Val Ala Tyr Gly Ala Ala
Val Gln Ala Ala Ile Leu Ile 370 375 380 Gly Asp Lys Ser Glu Asn Val
Gln Asp Leu Leu Leu Leu Asp Val Thr 385 390 395 400 Pro Leu Ser Leu
Gly Ile Glu Thr Ala Gly Gly Val Met Thr Pro Leu 405 410 415 Ile Lys
Arg Asn Thr Thr Ile Pro Thr Lys Gln Thr Gln Thr Phe Thr 420 425 430
Thr Tyr Ser Asp Asn Gln Ser Ser Val Leu Val Gln Val Tyr Glu Gly 435
440 445 Glu Arg Ala Met Thr Lys Asp Asn Asn Leu Leu Gly Lys Phe Asp
Leu 450 455 460 Thr Gly Ile Pro Pro Ala Pro Arg Gly Val Pro Gln Ile
Glu Val Thr 465 470 475 480 Phe Asp Ile Asp Ala Asn Gly Ile Leu Asn
Val Thr Ala Ala Asp Lys 485 490 495 Ser Thr Gly Lys Glu Asn Lys Ile
Thr Ile Thr Asn Asp Lys Gly Arg 500 505 510 Leu Ser Lys Asp Asp Ile
Asp Arg Met Val Gln Glu Ala Glu Arg Tyr 515 520 525 Lys Ser Glu Asp
Glu Ala Asn Arg Asp Arg Val Ala Ala Lys Asn Ala 530 535 540 Leu Glu
Ser Tyr Thr Tyr Asn Ile Lys Gln Thr Val Glu Asp Glu Lys 545 550 555
560 Leu Arg Gly Lys Ile Ser Glu Gln Asp Lys Asn Lys Ile Leu Asp Lys
565 570 575 Cys Gln Glu Val Ile Asn Trp Leu Asp Arg Asn Gln Met Ala
Glu Lys 580 585 590 Asp Glu Tyr Glu His Lys Gln Lys Glu Leu Glu Arg
Val Cys Asn Pro 595 600 605 Ile Ile Ser Lys Leu Tyr Gln Gly Gly Pro
Gly Gly Gly Ser Gly Gly 610 615 620 Gly Gly Ser Gly Ala Ser Gly Gly
Pro Thr Ile Glu Glu Val Asp 625 630 635 8654PRTHomo sapiens 8Met
Lys Leu Ser Leu Val Ala Ala Met Leu Leu Leu Leu Ser Ala Ala 1 5 10
15 Arg Ala Glu Glu Glu Asp Lys Lys Glu Asp Val Gly Thr Val Val Gly
20 25 30 Ile Asp Leu Gly Thr Thr Tyr Ser Cys Val Gly Val Phe Lys
Asn Gly 35 40 45 Arg Val Glu Ile Ile Ala Asn Asp Gln Gly Asn Arg
Ile Thr Pro Ser 50 55 60 Tyr Val Ala Phe Thr Pro Glu Gly Glu Arg
Leu Ile Gly Asp Ala Ala 65 70 75 80 Lys Asn Gln Leu Thr Ser Asn Pro
Glu Asn Thr Val Phe Asp Ala Lys 85 90 95 Arg Leu Ile Gly Arg Thr
Trp Asn Asp Pro Ser Val Gln Gln Asp Ile 100 105 110 Lys Phe Leu Pro
Phe Lys Val Val Glu Lys Lys Thr Lys Pro Tyr Ile 115 120 125 Gln Val
Asp Ile Gly Gly Gly Gln Thr Lys Thr Phe Ala Pro Glu Glu 130 135 140
Ile Ser Ala Met Val Leu Thr Lys Met Lys Glu Thr Ala Glu Ala Tyr 145
150 155 160 Leu Gly Lys Lys Val Thr His Ala Val Val Thr Val Pro Ala
Tyr Phe 165 170 175 Asn Asp Ala Gln Arg Gln Ala Thr Lys Asp Ala Gly
Thr Ile Ala Gly 180 185 190 Leu Asn Val Met Arg Ile Ile Asn Glu Pro
Thr Ala Ala Ala Ile Ala 195 200 205 Tyr Gly Leu Asp Lys Arg Glu Gly
Glu Lys Asn Ile Leu Val Phe Asp 210 215 220 Leu Gly Gly Gly Thr Phe
Asp Val Ser Leu Leu Thr Ile Asp Asn Gly 225 230 235 240 Val Phe Glu
Val Val Ala Thr Asn Gly Asp Thr His Leu Gly Gly Glu 245 250 255 Asp
Phe Asp Gln Arg Val Met Glu His Phe Ile Lys Leu Tyr Lys Lys 260 265
270 Lys Thr Gly Lys Asp Val Arg Lys Asp Asn Arg Ala Val Gln Lys Leu
275 280 285 Arg Arg Glu Val Glu Lys Ala Lys Arg Ala Leu Ser Ser Gln
His Gln 290 295 300 Ala Arg Ile Glu Ile Glu Ser Phe Tyr Glu Gly Glu
Asp Phe Ser Glu 305 310 315 320 Thr Leu Thr Arg Ala Lys Phe Glu Glu
Leu Asn Met Asp Leu Phe Arg 325 330 335 Ser Thr Met Lys Pro Val Gln
Lys Val Leu Glu Asp Ser Asp Leu Lys 340 345 350 Lys Ser Asp Ile Asp
Glu Ile Val Leu Val Gly Gly Ser Thr Arg Ile 355 360 365 Pro Lys Ile
Gln Gln Leu Val Lys Glu Phe Phe Asn Gly Lys Glu Pro 370 375 380 Ser
Arg Gly Ile Asn Pro Asp Glu Ala Val Ala Tyr Gly Ala Ala Val 385 390
395 400 Gln Ala Gly Val Leu Ser Gly Asp Gln Asp Thr Gly Asp Leu Val
Leu 405 410 415 Leu Asp Val Cys Pro Leu Thr Leu Gly Ile Glu Thr Val
Gly Gly Val 420 425 430 Met Thr Lys Leu Ile Pro Arg Asn Thr Val Val
Pro Thr Lys Lys Ser 435 440 445 Gln Ile Phe Ser Thr Ala Ser Asp Asn
Gln Pro Thr Val Thr Ile Lys 450 455 460 Val Tyr Glu Gly Glu Arg Pro
Leu Thr Lys Asp Asn His Leu Leu Gly 465 470 475 480 Thr Phe Asp Leu
Thr Gly Ile Pro Pro Ala Pro Arg Gly Val Pro Gln 485 490 495 Ile Glu
Val Thr Phe Glu Ile Asp Val Asn Gly Ile Leu Arg Val Thr 500 505 510
Ala Glu Asp Lys Gly Thr Gly Asn Lys Asn Lys Ile Thr Ile Thr Asn 515
520 525 Asp Gln Asn Arg Leu Thr Pro Glu Glu Ile Glu Arg Met Val Asn
Asp 530 535 540 Ala Glu Lys Phe Ala Glu Glu Asp Lys Lys Leu Lys Glu
Arg Ile Asp 545 550 555 560 Thr Arg Asn Glu Leu Glu Ser Tyr Ala Tyr
Ser Leu Lys Asn Gln Ile 565 570 575 Gly Asp Lys Glu Lys Leu Gly Gly
Lys Leu Ser Ser Glu Asp Lys Glu 580 585 590 Thr Met Glu Lys Ala Val
Glu Glu Lys Ile Glu Trp Leu Glu Ser His 595 600 605 Gln Asp Ala Asp
Ile Glu Asp Phe Lys Ala Lys Lys Lys Glu Leu Glu 610 615 620 Glu Ile
Val Gln Pro Ile Ile Ser Lys Leu Tyr Gly Ser Ala Gly Pro 625 630 635
640 Pro Pro Thr Gly Glu Glu Asp Thr Ala Glu Lys Asp Glu Leu 645 650
9643PRTHomo sapiens 9Met Gln Ala Pro Arg Glu Leu Ala Val Gly Ile
Asp Leu Gly Thr Thr 1 5 10 15 Tyr Ser Cys Val Gly Val Phe Gln Gln
Gly Arg Val Glu Ile Leu Ala 20 25 30 Asn Asp Gln Gly Asn Arg Thr
Thr Pro Ser Tyr Val Ala Phe Thr Asp 35 40 45 Thr Glu Arg Leu Val
Gly Asp Ala Ala Lys Ser Gln Ala Ala Leu Asn 50 55 60 Pro His Asn
Thr Val Phe Asp Ala Lys Arg Leu Ile Gly Arg Lys Phe 65 70 75 80 Ala
Asp Thr Thr Val Gln Ser Asp Met Lys His Trp Pro Phe Arg Val 85 90
95 Val Ser Glu Gly Gly Lys Pro Lys Val Arg Val Cys Tyr Arg Gly Glu
100 105 110 Asp Lys Thr Phe Tyr Pro Glu Glu Ile Ser Ser Met Val Leu
Ser Lys 115 120 125 Met Lys Glu Thr Ala Glu Ala Tyr Leu Gly Gln Pro
Val Lys His Ala 130 135 140 Val Ile Thr Val Pro Ala Tyr Phe Asn Asp
Ser Gln Arg Gln Ala Thr 145 150 155 160 Lys Asp Ala Gly Ala Ile Ala
Gly Leu Asn Val Leu Arg Ile Ile Asn 165 170 175 Glu Pro Thr Ala Ala
Ala Ile Ala Tyr Gly Leu Asp Arg Arg Gly Ala 180 185 190 Gly Glu Arg
Asn Val Leu Ile Phe Asp Leu Gly Gly Gly Thr Phe Asp 195 200 205 Val
Ser Val Leu Ser Ile Asp Ala Gly Val Phe Glu Val Lys Ala Thr 210 215
220 Ala Gly Asp Thr His Leu Gly Gly Glu Asp Phe Asp Asn Arg Leu Val
225 230 235 240 Asn His Phe Met Glu Glu Phe Arg Arg Lys His Gly Lys
Asp Leu Ser 245 250 255 Gly Asn Lys Arg Ala Leu Arg Arg Leu Arg Thr
Ala Cys Glu Arg Ala 260 265 270 Lys Arg Thr Leu Ser Ser Ser Thr Gln
Ala Thr Leu Glu Ile Asp Ser 275 280 285 Leu Phe Glu Gly Val Asp Phe
Tyr Thr Ser Ile Thr Arg Ala Arg Phe 290 295 300 Glu Glu Leu Cys Ser
Asp Leu Phe Arg Ser Thr Leu Glu Pro Val Glu 305 310 315 320 Lys Ala
Leu Arg Asp Ala Lys Leu Asp Lys Ala Gln Ile His Asp Val 325 330 335
Val Leu Val Gly Gly Ser Thr Arg Ile Pro Lys Val Gln Lys Leu Leu 340
345 350 Gln Asp Phe Phe Asn Gly Lys Glu Leu Asn Lys Ser Ile Asn Pro
Asp 355 360 365 Glu Ala Val Ala Tyr Gly Ala Ala Val Gln Ala Ala Val
Leu Met Gly 370 375 380 Asp Lys Cys Glu Lys Val Gln Asp Leu Leu Leu
Leu Asp Val Ala Pro 385 390 395 400 Leu Ser Leu Gly Leu Glu Thr Ala
Gly Gly Val Met Thr Thr Leu Ile 405 410 415 Gln Arg Asn Ala Thr Ile
Pro Thr Lys Gln Thr Gln Thr Phe Thr Thr 420 425 430 Tyr Ser Asp Asn
Gln Pro Gly Val Phe Ile Gln Val Tyr Glu Gly Glu 435 440 445 Arg Ala
Met Thr Lys Asp Asn Asn Leu Leu Gly Arg Phe Glu Leu Ser 450 455 460
Gly Ile Pro Pro Ala Pro Arg Gly Val Pro Gln Ile Glu Val Thr Phe 465
470 475 480 Asp Ile Asp Ala Asn Gly Ile Leu Ser Val Thr Ala Thr Asp
Arg Ser 485 490 495 Thr Gly Lys Ala Asn Lys Ile Thr Ile Thr Asn Asp
Lys Gly Arg Leu 500 505 510 Ser Lys Glu Glu Val Glu Arg Met Val His
Glu Ala Glu Gln Tyr Lys 515 520 525 Ala Glu Asp Glu Ala Gln Arg Asp
Arg Val Ala Ala Lys Asn Ser Leu 530 535 540 Glu Ala His Val Phe His
Val Lys Gly Ser Leu Gln Glu Glu Ser Leu 545 550 555 560 Arg Asp Lys
Ile Pro Glu Glu Asp Arg Arg Lys Met Gln Asp Lys Cys 565 570 575 Arg
Glu Val Leu Ala Trp Leu Glu His Asn Gln Leu Ala Glu Lys Glu 580 585
590 Glu Tyr Glu His Gln Lys Arg Glu Leu Glu Gln Ile Cys Arg Pro Ile
595 600 605 Phe Ser Arg Leu Tyr Gly Gly Pro Gly Val Pro Gly Gly Ser
Ser Cys 610 615 620 Gly Thr Gln Ala Arg Gln Gly Asp Pro Ser Thr Gly
Pro Ile Ile Glu 625 630 635 640 Glu Val Asp 10646PRTHomo sapiens
10Met Ser Lys Gly Pro Ala Val Gly Ile Asp Leu Gly Thr Thr Tyr Ser 1
5 10 15 Cys Val Gly Val Phe Gln His Gly Lys Val Glu Ile Ile Ala Asn
Asp 20 25 30 Gln Gly Asn Arg Thr Thr Pro Ser Tyr Val Ala Phe Thr
Asp Thr Glu 35 40 45 Arg Leu Ile Gly Asp Ala Ala Lys Asn Gln Val
Ala Met Asn Pro Thr 50 55 60 Asn Thr Val Phe Asp Ala Lys Arg Leu
Ile Gly Arg Arg Phe Asp Asp 65 70 75 80 Ala Val Val Gln Ser Asp Met
Lys His Trp Pro Phe Met Val Val Asn 85 90 95 Asp Ala Gly Arg Pro
Lys Val Gln Val Glu Tyr Lys Gly Glu Thr Lys 100 105 110 Ser Phe Tyr
Pro Glu Glu Val Ser Ser Met Val Leu Thr Lys Met Lys 115 120 125 Glu
Ile Ala Glu Ala Tyr Leu Gly Lys Thr Val Thr Asn Ala Val Val 130 135
140 Thr Val Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp
145 150 155 160 Ala Gly Thr Ile Ala Gly Leu Asn Val Leu Arg Ile Ile
Asn Glu Pro 165 170 175 Thr Ala Ala Ala Ile Ala Tyr Gly Leu Asp Lys
Lys Val Gly Ala Glu 180 185 190 Arg Asn Val Leu Ile Phe Asp Leu Gly
Gly Gly Thr Phe Asp Val Ser 195 200 205 Ile Leu Thr Ile Glu Asp Gly
Ile Phe Glu Val Lys Ser Thr Ala Gly 210 215 220 Asp Thr His Leu Gly
Gly Glu Asp Phe Asp Asn Arg Met Val Asn His 225 230 235 240 Phe Ile
Ala Glu Phe Lys Arg Lys His Lys Lys Asp Ile Ser Glu Asn 245 250 255
Lys Arg Ala Val Arg Arg Leu Arg Thr Ala Cys Glu Arg Ala Lys Arg 260
265 270 Thr Leu Ser Ser Ser Thr Gln Ala Ser Ile Glu Ile Asp Ser Leu
Tyr 275 280 285 Glu Gly Ile Asp Phe Tyr Thr Ser Ile Thr Arg Ala Arg
Phe Glu Glu 290 295 300 Leu Asn Ala Asp Leu Phe Arg Gly Thr Leu Asp
Pro Val Glu Lys Ala 305 310 315 320 Leu Arg Asp Ala Lys Leu Asp Lys
Ser Gln Ile His Asp Ile Val Leu 325 330 335 Val Gly Gly Ser Thr Arg
Ile Pro Lys Ile Gln Lys Leu Leu Gln Asp 340 345 350 Phe Phe Asn Gly
Lys Glu Leu Asn Lys Ser Ile Asn Pro Asp Glu Ala 355 360 365 Val Ala
Tyr Gly Ala Ala Val Gln Ala Ala Ile Leu Ser Gly Asp Lys 370 375 380
Ser Glu Asn Val Gln Asp Leu Leu Leu Leu Asp Val Thr Pro Leu Ser 385
390 395 400 Leu Gly Ile Glu Thr Ala Gly Gly Val Met Thr Val Leu Ile
Lys Arg 405 410 415 Asn Thr Thr Ile Pro Thr Lys Gln Thr Gln Thr Phe
Thr Thr Tyr Ser 420 425 430 Asp Asn Gln Pro Gly Val Leu Ile Gln Val
Tyr Glu Gly Glu
Arg Ala 435 440 445 Met Thr Lys Asp Asn Asn Leu Leu Gly Lys Phe Glu
Leu Thr Gly Ile 450 455 460 Pro Pro Ala Pro Arg Gly Val Pro Gln Ile
Glu Val Thr Phe Asp Ile 465 470 475 480 Asp Ala Asn Gly Ile Leu Asn
Val Ser Ala Val Asp Lys Ser Thr Gly 485 490 495 Lys Glu Asn Lys Ile
Thr Ile Thr Asn Asp Lys Gly Arg Leu Ser Lys 500 505 510 Glu Asp Ile
Glu Arg Met Val Gln Glu Ala Glu Lys Tyr Lys Ala Glu 515 520 525 Asp
Glu Lys Gln Arg Asp Lys Val Ser Ser Lys Asn Ser Leu Glu Ser 530 535
540 Tyr Ala Phe Asn Met Lys Ala Thr Val Glu Asp Glu Lys Leu Gln Gly
545 550 555 560 Lys Ile Asn Asp Glu Asp Lys Gln Lys Ile Leu Asp Lys
Cys Asn Glu 565 570 575 Ile Ile Asn Trp Leu Asp Lys Asn Gln Thr Ala
Glu Lys Glu Glu Phe 580 585 590 Glu His Gln Gln Lys Glu Leu Glu Lys
Val Cys Asn Pro Ile Ile Thr 595 600 605 Lys Leu Tyr Gln Ser Ala Gly
Gly Met Pro Gly Gly Met Pro Gly Gly 610 615 620 Phe Pro Gly Gly Gly
Ala Pro Pro Ser Gly Gly Ala Ser Ser Gly Pro 625 630 635 640 Thr Ile
Glu Glu Val Asp 645 11679PRTHomo sapiens 11Met Ile Ser Ala Ser Arg
Ala Ala Ala Ala Arg Leu Val Gly Ala Ala 1 5 10 15 Ala Ser Arg Gly
Pro Thr Ala Ala Arg His Gln Asp Ser Trp Asn Gly 20 25 30 Leu Ser
His Glu Ala Phe Arg Leu Val Ser Arg Arg Asp Tyr Ala Ser 35 40 45
Glu Ala Ile Lys Gly Ala Val Val Gly Ile Asp Leu Gly Thr Thr Asn 50
55 60 Ser Cys Val Ala Val Met Glu Gly Lys Gln Ala Lys Val Leu Glu
Asn 65 70 75 80 Ala Glu Gly Ala Arg Thr Thr Pro Ser Val Val Ala Phe
Thr Ala Asp 85 90 95 Gly Glu Arg Leu Val Gly Met Pro Ala Lys Arg
Gln Ala Val Thr Asn 100 105 110 Pro Asn Asn Thr Phe Tyr Ala Thr Lys
Arg Leu Ile Gly Arg Arg Tyr 115 120 125 Asp Asp Pro Glu Val Gln Lys
Asp Ile Lys Asn Val Pro Phe Lys Ile 130 135 140 Val Arg Ala Ser Asn
Gly Asp Ala Trp Val Glu Ala His Gly Lys Leu 145 150 155 160 Tyr Ser
Pro Ser Gln Ile Gly Ala Phe Val Leu Met Lys Met Lys Glu 165 170 175
Thr Ala Glu Asn Tyr Leu Gly His Thr Ala Lys Asn Ala Val Ile Thr 180
185 190 Val Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp
Ala 195 200 205 Gly Gln Ile Ser Gly Leu Asn Val Leu Arg Val Ile Asn
Glu Pro Thr 210 215 220 Ala Ala Ala Leu Ala Tyr Gly Leu Asp Lys Ser
Glu Asp Lys Val Ile 225 230 235 240 Ala Val Tyr Asp Leu Gly Gly Gly
Thr Phe Asp Ile Ser Ile Leu Glu 245 250 255 Ile Gln Lys Gly Val Phe
Glu Val Lys Ser Thr Asn Gly Asp Thr Phe 260 265 270 Leu Gly Gly Glu
Asp Phe Asp Gln Ala Leu Leu Arg His Ile Val Lys 275 280 285 Glu Phe
Lys Arg Glu Thr Gly Val Asp Leu Thr Lys Asp Asn Met Ala 290 295 300
Leu Gln Arg Val Arg Glu Ala Ala Glu Lys Ala Lys Cys Glu Leu Ser 305
310 315 320 Ser Ser Val Gln Thr Asp Ile Asn Leu Pro Tyr Leu Thr Met
Asp Ser 325 330 335 Ser Gly Pro Lys His Leu Asn Met Lys Leu Thr Arg
Ala Gln Phe Glu 340 345 350 Gly Ile Val Thr Asp Leu Ile Arg Arg Thr
Ile Ala Pro Cys Gln Lys 355 360 365 Ala Met Gln Asp Ala Glu Val Ser
Lys Ser Asp Ile Gly Glu Val Ile 370 375 380 Leu Val Gly Gly Met Thr
Arg Met Pro Lys Val Gln Gln Thr Val Gln 385 390 395 400 Asp Leu Phe
Gly Arg Ala Pro Ser Lys Ala Val Asn Pro Asp Glu Ala 405 410 415 Val
Ala Ile Gly Ala Ala Ile Gln Gly Gly Val Leu Ala Gly Asp Val 420 425
430 Thr Asp Val Leu Leu Leu Asp Val Thr Pro Leu Ser Leu Gly Ile Glu
435 440 445 Thr Leu Gly Gly Val Phe Thr Lys Leu Ile Asn Arg Asn Thr
Thr Ile 450 455 460 Pro Thr Lys Lys Ser Gln Val Phe Ser Thr Ala Ala
Asp Gly Gln Thr 465 470 475 480 Gln Val Glu Ile Lys Val Cys Gln Gly
Glu Arg Glu Met Ala Gly Asp 485 490 495 Asn Lys Leu Leu Gly Gln Phe
Thr Leu Ile Gly Ile Pro Pro Ala Pro 500 505 510 Arg Gly Val Pro Gln
Ile Glu Val Thr Phe Asp Ile Asp Ala Asn Gly 515 520 525 Ile Val His
Val Ser Ala Lys Asp Lys Gly Thr Gly Arg Glu Gln Gln 530 535 540 Ile
Val Ile Gln Ser Ser Gly Gly Leu Ser Lys Asp Asp Ile Glu Asn 545 550
555 560 Met Val Lys Asn Ala Glu Lys Tyr Ala Glu Glu Asp Arg Arg Lys
Lys 565 570 575 Glu Arg Val Glu Ala Val Asn Met Ala Glu Gly Ile Ile
His Asp Thr 580 585 590 Glu Thr Lys Met Glu Glu Phe Lys Asp Gln Leu
Pro Ala Asp Glu Cys 595 600 605 Asn Lys Leu Lys Glu Glu Ile Ser Lys
Met Arg Glu Leu Leu Ala Arg 610 615 620 Lys Asp Ser Glu Thr Gly Glu
Asn Ile Arg Gln Ala Ala Ser Ser Leu 625 630 635 640 Gln Gln Ala Ser
Leu Lys Leu Phe Glu Met Ala Tyr Lys Lys Met Ala 645 650 655 Ser Glu
Arg Glu Gly Ser Gly Ser Ser Gly Thr Gly Glu Gln Lys Glu 660 665 670
Asp Gln Lys Glu Glu Lys Gln 675 12732PRTHomo sapiens 12Met Pro Glu
Glu Thr Gln Thr Gln Asp Gln Pro Met Glu Glu Glu Glu 1 5 10 15 Val
Glu Thr Phe Ala Phe Gln Ala Glu Ile Ala Gln Leu Met Ser Leu 20 25
30 Ile Ile Asn Thr Phe Tyr Ser Asn Lys Glu Ile Phe Leu Arg Glu Leu
35 40 45 Ile Ser Asn Ser Ser Asp Ala Leu Asp Lys Ile Arg Tyr Glu
Ser Leu 50 55 60 Thr Asp Pro Ser Lys Leu Asp Ser Gly Lys Glu Leu
His Ile Asn Leu 65 70 75 80 Ile Pro Asn Lys Gln Asp Arg Thr Leu Thr
Ile Val Asp Thr Gly Ile 85 90 95 Gly Met Thr Lys Ala Asp Leu Ile
Asn Asn Leu Gly Thr Ile Ala Lys 100 105 110 Ser Gly Thr Lys Ala Phe
Met Glu Ala Leu Gln Ala Gly Ala Asp Ile 115 120 125 Ser Met Ile Gly
Gln Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu Val 130 135 140 Ala Glu
Lys Val Thr Val Ile Thr Lys His Asn Asp Asp Glu Gln Tyr 145 150 155
160 Ala Trp Glu Ser Ser Ala Gly Gly Ser Phe Thr Val Arg Thr Asp Thr
165 170 175 Gly Glu Pro Met Gly Arg Gly Thr Lys Val Ile Leu His Leu
Lys Glu 180 185 190 Asp Gln Thr Glu Tyr Leu Glu Glu Arg Arg Ile Lys
Glu Ile Val Lys 195 200 205 Lys His Ser Gln Phe Ile Gly Tyr Pro Ile
Thr Leu Phe Val Glu Lys 210 215 220 Glu Arg Asp Lys Glu Val Ser Asp
Asp Glu Ala Glu Glu Lys Glu Asp 225 230 235 240 Lys Glu Glu Glu Lys
Glu Lys Glu Glu Lys Glu Ser Glu Asp Lys Pro 245 250 255 Glu Ile Glu
Asp Val Gly Ser Asp Glu Glu Glu Glu Lys Lys Asp Gly 260 265 270 Asp
Lys Lys Lys Lys Lys Lys Ile Lys Glu Lys Tyr Ile Asp Gln Glu 275 280
285 Glu Leu Asn Lys Thr Lys Pro Ile Trp Thr Arg Asn Pro Asp Asp Ile
290 295 300 Thr Asn Glu Glu Tyr Gly Glu Phe Tyr Lys Ser Leu Thr Asn
Asp Trp 305 310 315 320 Glu Asp His Leu Ala Val Lys His Phe Ser Val
Glu Gly Gln Leu Glu 325 330 335 Phe Arg Ala Leu Leu Phe Val Pro Arg
Arg Ala Pro Phe Asp Leu Phe 340 345 350 Glu Asn Arg Lys Lys Lys Asn
Asn Ile Lys Leu Tyr Val Arg Arg Val 355 360 365 Phe Ile Met Asp Asn
Cys Glu Glu Leu Ile Pro Glu Tyr Leu Asn Phe 370 375 380 Ile Arg Gly
Val Val Asp Ser Glu Asp Leu Pro Leu Asn Ile Ser Arg 385 390 395 400
Glu Met Leu Gln Gln Ser Lys Ile Leu Lys Val Ile Arg Lys Asn Leu 405
410 415 Val Lys Lys Cys Leu Glu Leu Phe Thr Glu Leu Ala Glu Asp Lys
Glu 420 425 430 Asn Tyr Lys Lys Phe Tyr Glu Gln Phe Ser Lys Asn Ile
Lys Leu Gly 435 440 445 Ile His Glu Asp Ser Gln Asn Arg Lys Lys Leu
Ser Glu Leu Leu Arg 450 455 460 Tyr Tyr Thr Ser Ala Ser Gly Asp Glu
Met Val Ser Leu Lys Asp Tyr 465 470 475 480 Cys Thr Arg Met Lys Glu
Asn Gln Lys His Ile Tyr Tyr Ile Thr Gly 485 490 495 Glu Thr Lys Asp
Gln Val Ala Asn Ser Ala Phe Val Glu Arg Leu Arg 500 505 510 Lys His
Gly Leu Glu Val Ile Tyr Met Ile Glu Pro Ile Asp Glu Tyr 515 520 525
Cys Val Gln Gln Leu Lys Glu Phe Glu Gly Lys Thr Leu Val Ser Val 530
535 540 Thr Lys Glu Gly Leu Glu Leu Pro Glu Asp Glu Glu Glu Lys Lys
Lys 545 550 555 560 Gln Glu Glu Lys Lys Thr Lys Phe Glu Asn Leu Cys
Lys Ile Met Lys 565 570 575 Asp Ile Leu Glu Lys Lys Val Glu Lys Val
Val Val Ser Asn Arg Leu 580 585 590 Val Thr Ser Pro Cys Cys Ile Val
Thr Ser Thr Tyr Gly Trp Thr Ala 595 600 605 Asn Met Glu Arg Ile Met
Lys Ala Gln Ala Leu Arg Asp Asn Ser Thr 610 615 620 Met Gly Tyr Met
Ala Ala Lys Lys His Leu Glu Ile Asn Pro Asp His 625 630 635 640 Ser
Ile Ile Glu Thr Leu Arg Gln Lys Ala Glu Ala Asp Lys Asn Asp 645 650
655 Lys Ser Val Lys Asp Leu Val Ile Leu Leu Tyr Glu Thr Ala Leu Leu
660 665 670 Ser Ser Gly Phe Ser Leu Glu Asp Pro Gln Thr His Ala Asn
Arg Ile 675 680 685 Tyr Arg Met Ile Lys Leu Gly Leu Gly Ile Asp Glu
Asp Asp Pro Thr 690 695 700 Ala Asp Asp Thr Ser Ala Ala Val Thr Glu
Glu Met Pro Pro Leu Glu 705 710 715 720 Gly Asp Asp Asp Thr Ser Arg
Met Glu Glu Val Asp 725 730 132445DNAHomo sapiens 13ataaaagccc
aggggcaagc ggtccggata acggctagcc tgaggagctg ctgcgacagt 60ccactacctt
tttcgagagt gactcccgtt gtcccaaggc ttcccagagc gaacctgtgc
120ggctgcaggc accggcgcgt cgagtttccg gcgtccggaa ggaccgagct
cttctcgcgg 180atccagtgtt ccgtttccag cccccaatct cagagcggag
ccgacagaga gcagggaacc 240ggcatggcca aagccgcggc gatcggcatc
gacctgggca ccacctactc ctgcgtgggg 300gtgttccaac acggcaaggt
ggagatcatc gccaacgacc agggcaaccg caccaccccc 360agctacgtgg
ccttcacgga caccgagcgg ctcatcgggg atgcggccaa gaaccaggtg
420gcgctgaacc cgcagaacac cgtgtttgac gcgaagcggc tgattggccg
caagttcggc 480gacccggtgg tgcagtcgga catgaagcac tggcctttcc
aggtgatcaa cgacggagac 540aagcccaagg tgcaggtgag ctacaagggg
gagaccaagg cattctaccc cgaggagatc 600tcgtccatgg tgctgaccaa
gatgaaggag atcgccgagg cgtacctggg ctacccggtg 660accaacgcgg
tgatcaccgt gccggcctac ttcaacgact cgcagcgcca ggccaccaag
720gatgcgggtg tgatcgcggg gctcaacgtg ctgcggatca tcaacgagcc
cacggccgcc 780gccatcgcct acggcctgga cagaacgggc aagggggagc
gcaacgtgct catctttgac 840ctgggcgggg gcaccttcga cgtgtccatc
ctgacgatcg acgacggcat cttcgaggtg 900aaggccacgg ccggggacac
ccacctgggt ggggaggact ttgacaacag gctggtgaac 960cacttcgtgg
aggagttcaa gagaaaacac aagaaggaca tcagccagaa caagcgagcc
1020gtgaggcggc tgcgcaccgc ctgcgagagg gccaagagga ccctgtcgtc
cagcacccag 1080gccagcctgg agatcgactc cctgtttgag ggcatcgact
tctacacgtc catcaccagg 1140gcgaggttcg aggagctgtg ctccgacctg
ttccgaagca ccctggagcc cgtggagaag 1200gctctgcgcg acgccaagct
ggacaaggcc cagattcacg acctggtcct ggtcgggggc 1260tccacccgca
tccccaaggt gcagaagctg ctgcaggact tcttcaacgg gcgcgacctg
1320aacaagagca tcaaccccga cgaggctgtg gcctacgggg cggcggtgca
ggcggccatc 1380ctgatggggg acaagtccga gaacgtgcag gacctgctgc
tgctggacgt ggctcccctg 1440tcgctggggc tggagacggc cggaggcgtg
atgactgccc tgatcaagcg caactccacc 1500atccccacca agcagacgca
gatcttcacc acctactccg acaaccaacc cggggtgctg 1560atccaggtgt
acgagggcga gagggccatg acgaaagaca acaatctgtt ggggcgcttc
1620gagctgagcg gcatccctcc ggcccccagg ggcgtgcccc agatcgaggt
gaccttcgac 1680atcgatgcca acggcatcct gaacgtcacg gccacggaca
agagcaccgg caaggccaac 1740aagatcacca tcaccaacga caagggccgc
ctgagcaagg aggagatcga gcgcatggtg 1800caggaggcgg agaagtacaa
agcggaggac gaggtgcagc gcgagagggt gtcagccaag 1860aacgccctgg
agtcctacgc cttcaacatg aagagcgccg tggaggatga ggggctcaag
1920ggcaagatca gcgaggcgga caagaagaag gtgctggaca agtgtcaaga
ggtcatctcg 1980tggctggacg ccaacacctt ggccgagaag gacgagtttg
agcacaagag gaaggagctg 2040gagcaggtgt gtaaccccat catcagcgga
ctgtaccagg gtgccggtgg tcccgggcct 2100gggggcttcg gggctcaggg
tcccaaggga gggtctgggt caggccccac cattgaggag 2160gtagattagg
ggcctttcca agattgctgt ttttgttttg gagcttcaag actttgcatt
2220tcctagtatt tctgtttgtc agttctcaat ttcctgtgtt tgcaatgttg
aaattttttg 2280gtgaagtact gaacttgctt tttttccggt ttctacatgc
agagatgaat ttatactgcc 2340atcttacgac tatttcttct ttttaataca
cttaactcag gccatttttt aagttggtta 2400cttcaaagta aataaacttt
aaaattcaaa aaaaaaaaaa aaaaa 2445143366DNAHomo sapiens 14gcatgcgtag
gcgcgcggcc gcggcggcgg ctggggaggg ttcttccgga aggttcggga 60ggcttctgga
aaaagcgccg cgcgctgggc gggcccgtcg ctatataagg caggcgcggg
120ggtggcgcgt cagttgcttc agcgtcccgg tgtggctgtg ccgttggtcc
tgtgcggtca 180cttagccaag atgcctgagg aaacccagac ccaagaccaa
ccgatggagg aggaggaggt 240tgagacgttc gcctttcagg cagaaattgc
ccagttgatg tcattgatca tcaatacttt 300ctactcgaac aaagagatct
ttctgagaga gctcatttca aattcatcag atgcattgga 360caaaatccgg
tatgaaagct tgacagatcc cagtaaatta gactctggga aagagctgca
420tattaacctt ataccgaaca aacaagatcg aactctcact attgtggata
ctggaattgg 480aatgaccaag gctgacttga tcaataacct tggtactatc
gccaagtctg ggaccaaagc 540gttcatggaa gctttgcagg ctggtgcaga
tatctctatg attggccagt tcggtgttgg 600tttttattct gcttatttgg
ttgctgagaa agtaactgtg atcaccaaac ataacgatga 660tgagcagtac
gcttgggagt cctcagcagg gggatcattc acagtgagga cagacacagg
720tgaacctatg ggtcgtggaa caaaagttat cctacacctg aaagaagacc
aaactgagta 780cttggaggaa cgaagaataa aggagattgt gaagaaacat
tctcagttta ttggatatcc 840cattactctt tttgtggaga aggaacgtga
taaagaagta agcgatgatg aggctgaaga 900aaaggaagac aaagaagaag
aaaaagaaaa agaagagaaa gagtcggaag acaaacctga 960aattgaagat
gttggttctg atgaggaaga agaaaagaag gatggtgaca agaagaagaa
1020gaagaagatt aaggaaaagt acatcgatca agaagagctc aacaaaacaa
agcccatctg 1080gaccagaaat cccgacgata ttactaatga ggagtacgga
gaattctata agagcttgac 1140caatgactgg gaagatcact tggcagtgaa
gcatttttca gttgaaggac agttggaatt 1200cagagccctt ctatttgtcc
cacgacgtgc tccttttgat ctgtttgaaa acagaaagaa 1260aaagaacaac
atcaaattgt atgtacgcag agttttcatc atggataact gtgaggagct
1320aatccctgaa tatctgaact tcattagagg ggtggtagac tcggaggatc
tccctctaaa 1380catatcccgt gagatgttgc aacaaagcaa aattttgaaa
gttatcagga agaatttggt 1440caaaaaatgc ttagaactct ttactgaact
ggcggaagat aaagagaact acaagaaatt 1500ctatgagcag ttctctaaaa
acataaagct tggaatacac gaagactctc aaaatcggaa 1560gaagctttca
gagctgttaa ggtactacac atctgcctct ggtgatgaga tggtttctct
1620caaggactac tgcaccagaa tgaaggagaa ccagaaacat atctattata
tcacaggtga 1680gaccaaggac caggtagcta actcagcctt tgtggaacgt
cttcggaaac atggcttaga 1740agtgatctat atgattgagc ccattgatga
gtactgtgtc caacagctga aggaatttga 1800ggggaagact ttagtgtcag
tcaccaaaga aggcctggaa cttccagagg atgaagaaga 1860gaaaaagaag
caggaagaga aaaaaacaaa gtttgagaac ctctgcaaaa tcatgaaaga
1920catattggag aaaaaagttg aaaaggtggt tgtgtcaaac
cgattggtga catctccatg 1980ctgtattgtc acaagcacat atggctggac
agcaaacatg gagagaatca tgaaagctca 2040agccctaaga gacaactcaa
caatgggtta catggcagca aagaaacacc tggagataaa 2100ccctgaccat
tccattattg agaccttaag gcaaaaggca gaggctgata agaacgacaa
2160gtctgtgaag gatctggtca tcttgcttta tgaaactgcg ctcctgtctt
ctggcttcag 2220tctggaagat ccccagacac atgctaacag gatctacagg
atgatcaaac ttggtctggg 2280tattgatgaa gatgacccta ctgctgatga
taccagtgct gctgtaactg aagaaatgcc 2340accccttgaa ggagatgacg
acacatcacg catggaagaa gtagactaat ctctggctga 2400gggatgactt
acctgttcag tactctacaa ttcctctgat aatatatttt caaggatgtt
2460tttctttatt tttgttaata ttaaaaagtc tgtatggcat gacaactact
ttaaggggaa 2520gataagattt ctgtctacta agtgatgctg tgatacctta
ggcactaaag cagagctagt 2580aatgcttttt gagtttcatg ttggtttatt
ttcacagatt ggggtaacgt gcactgtaag 2640acgtatgtaa catgatgtta
actttgtggt ctaaagtgtt tagctgtcaa gccggatgcc 2700taagtagacc
aaatcttgtt attgaagtgt tctgagctgt atcttgatgt ttagaaaagt
2760attcgttaca tcttgtagga tctacttttt gaacttttca ttccctgtag
ttgacaattc 2820tgcatgtact agtcctctag aaataggtta aactgaagca
acttgatgga aggatctctc 2880cacagggctt gttttccaaa gaaaagtatt
gtttggagga gcaaagttaa aagcctacct 2940aagcatatcg taaagctgtt
caaaaataac tcagacccag tcttgtggat ggaaatgtag 3000tgctcgagtc
acattctgct taaagttgta acaaatacag atgagttaaa agatattgtg
3060tgacagtgtc ttatttaggg ggaaagggga gtatctggat gacagttagt
gccaaaatgt 3120aaaacatgag gcgctagcag gagatggtta aacactagct
gctccaaggg ttgacatggt 3180cttcccagca tgtactcagc aggtgtgggg
tggagcacac gtaggcacag aaaacaggaa 3240tgcagacaac atgcatcccc
tgcgtccatg agttacatgt gttctcttag tgtccacgtt 3300gttttgatgt
tattcatgga ataccttctg tgttaaatac agtcacttaa ttccttggcc 3360ttaaaa
33661520PRTHomo sapiens 15Met Leu Arg Leu Pro Thr Val Phe Arg Gln
Met Arg Pro Val Ser Arg 1 5 10 15 Val Leu Ala Pro 20 1620PRTHomo
sapiens 16Arg Val Leu Ala Pro His Leu Thr Arg Ala Tyr Ala Lys Asp
Val Lys 1 5 10 15 Phe Gly Ala Asp 20 1719PRTHomo sapiens 17Leu Leu
Ala Asp Ala Val Ala Val Thr Met Gly Lys Gly Arg Thr Val 1 5 10 15
Ile Ile Glu 1820PRTHomo sapiens 18Thr Val Ile Ile Glu Gln Ser Trp
Gly Ser Pro Lys Val Thr Lys Asp 1 5 10 15 Gly Val Thr Val 20
1920PRTHomo sapiens 19Asp Gly Val Thr Val Ala Lys Ser Ile Asp Leu
Lys Asp Lys Tyr Lys 1 5 10 15 Asn Ile Gly Ala 20 2020PRTHomo
sapiens 20Lys Asn Ile Gly Ala Lys Leu Val Gln Asp Val Ala Asn Asn
Thr Asn 1 5 10 15 Glu Glu Ala Gly 20 2120PRTHomo sapiens 21Asn Glu
Glu Ala Gly Lys Gly Thr Thr Thr Ala Thr Val Leu Ala Arg 1 5 10 15
Ser Ile Ala Lys 20 2220PRTHomo sapiens 22Arg Ser Ile Ala Lys Glu
Gly Phe Glu Lys Ile Ser Lys Gly Ala Asn 1 5 10 15 Pro Val Glu Ile
20 2320PRTHomo sapiens 23Asn Pro Val Glu Ile Arg Arg Gly Val Met
Leu Ala Val Asp Ala Val 1 5 10 15 Ile Ala Glu Leu 20 2420PRTHomo
sapiens 24Val Ile Ala Glu Leu Lys Lys Gln Ser Lys Pro Val Thr Thr
Pro Glu 1 5 10 15 Glu Ile Ala Gln 20 2520PRTHomo sapiens 25Glu Glu
Ile Ala Gln Val Ala Thr Ile Ser Ala Asn Gly Asp Lys Glu 1 5 10 15
Ile Gly Asn Ile 20 2619PRTHomo sapiens 26Glu Ile Gly Asn Ile Ile
Ser Asp Ala Met Lys Lys Val Gly Arg Lys 1 5 10 15 Gly Val Ile
2720PRTHomo sapiens 27Arg Lys Gly Val Ile Thr Val Lys Asp Gly Lys
Thr Leu Asn Asp Glu 1 5 10 15 Leu Glu Ile Ile 20 2820PRTHomo
sapiens 28Glu Leu Glu Ile Ile Glu Gly Met Lys Phe Asp Arg Gly Tyr
Ile Ser 1 5 10 15 Pro Tyr Phe Ile 20 2920PRTHomo sapiens 29Ser Pro
Tyr Phe Ile Asn Thr Ser Lys Gly Gln Lys Cys Glu Phe Gln 1 5 10 15
Asp Ala Tyr Val 20 3020PRTHomo sapiens 30Gln Asp Ala Tyr Val Leu
Leu Ser Glu Lys Lys Ile Ser Ser Ile Gln 1 5 10 15 Ser Ile Val Pro
20 3121PRTHomo sapiens 31Gln Ser Ile Val Pro Ala Leu Glu Ile Ala
Asn Ala His Arg Lys Pro 1 5 10 15 Leu Val Ile Ile Ala 20
3220PRTMycobacterium tuberculosis 32Ala Tyr Asp Glu Glu Ala Arg Arg
Gly Leu Glu Arg Gly Leu Asn Ala 1 5 10 15 Leu Ala Asp Ala 20
3315PRTMycobacterium tuberculosis 33Glu Glu Ser Asn Thr Phe Gly Leu
Gln Leu Glu Leu Thr Glu Gly 1 5 10 15 3420PRTMycobacterium
tuberculosis 34Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu Glu Arg Gly
Leu Asn Ala 1 5 10 15 Leu Ala Asp Ala 20 3520PRTRattus norvergicus
35Lys Phe Gly Ala Asp Ala Arg Ala Leu Met Leu Gln Gly Val Asp Leu 1
5 10 15 Leu Ala Asp Ala 20
* * * * *