U.S. patent application number 12/454772 was filed with the patent office on 2009-12-10 for bag3 nucleotide and protein sequences to be used in research, diagnostics and therapy for cell death-involving diseases, and for modulation of cell survival and/or death.
Invention is credited to ARTURO LEONE, MARIA CATERINA TURCO.
Application Number | 20090306192 12/454772 |
Document ID | / |
Family ID | 8184857 |
Filed Date | 2009-12-10 |
United States Patent
Application |
20090306192 |
Kind Code |
A1 |
LEONE; ARTURO ; et
al. |
December 10, 2009 |
BAG3 nucleotide and protein sequences to be used in research,
diagnostics and therapy for cell death-involving diseases, and for
modulation of cell survival and/or death
Abstract
The present invention provides BAG3 nucleotide and protein
sequences to be used in research, diagnostics and therapy for
modulation of cell survival and/or death, in particular in
leukemias, other neoplasias and apoptosis-involving diseases. More
particularly the invention refers to the use of specific
antisense-based constructs and peptide-specific polyclonal and
monoclonal antibodies in leukemias, other neoplasias and cell
death-involving diseases.
Inventors: |
LEONE; ARTURO; (NAPOLI,
IT) ; TURCO; MARIA CATERINA; (AVELLINO, IT) |
Correspondence
Address: |
ABELMAN, FRAYNE & SCHWAB
666 THIRD AVENUE, 10TH FLOOR
NEW YORK
NY
10017
US
|
Family ID: |
8184857 |
Appl. No.: |
12/454772 |
Filed: |
May 21, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10500665 |
Jun 28, 2004 |
7537760 |
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PCT/EP02/14802 |
Dec 30, 2002 |
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12454772 |
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Current U.S.
Class: |
514/44R ;
435/375; 435/6.16; 536/23.5 |
Current CPC
Class: |
C07K 14/4747 20130101;
G01N 2510/00 20130101; G01N 33/57426 20130101; A61K 38/00
20130101 |
Class at
Publication: |
514/44.R ;
435/375; 435/6; 536/23.5 |
International
Class: |
A61K 31/711 20060101
A61K031/711; C12N 5/06 20060101 C12N005/06; C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 28, 2001 |
EP |
01830834.6 |
Claims
1.-29. (canceled)
30. A method for modulating apoptosis in a cell by regulating BAG-3
expression, which comprises using isolated polynucleotides or
oligonucleotides that recognize or modulate BAG3 protein expression
and wherein said polynucleotides or oligonucleotides are selected
from the group of nucleic acid sequences consisting of SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, and fragments and a complementary sequence
thereof.
31. The method according to claim 30, wherein said isolated
polynucleotides or oligonucleotides have at least 90% homology to
BAG3 SEQ ID NO:1 or fragments or a complementary sequence
thereof.
32. The method according to claim 30, wherein said cell is a
primary cell.
33. The method according to claim 30, wherein said polynucleotides
or oligonucleotides consist of SEQ ID NO:9, SEQ ID NO:10 or SEQ ID
NO:11 or a sequence encoding a peptide of SEQ ID NO:15, SEQ ID
NO:6, SEQ ID NO:17 or SEQ ID NO:18.
34. The method according to claim 30, wherein said isolated
polynucleotides or oligonucleotides that recognize or modulate BAG3
protein expression are comprised in a vector.
35. The method according to claim 30 comprising genetically
engineering a host cell with isolated polynucleotides or
oligonucleotide/s that recognize or modulate BAG3 protein
expression, said polynucleotides or oligonucleotides being selected
from the group of nucleic acid sequences consisting of SEQ ID NO:
1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:10, and SEQ ID NO:11.
36. The method according to claim 35, wherein said polynucleotides
or oligonucleotides are in operative association with a regulatory
sequence that controls expression of said polynucleotides or
oligonucleotides in the host cell.
37. The method according to claim 31, wherein said isolated
polynucleotides or oligonucleotides with at least 90% homology to
BAG3 SEQ ID NO:1 or fragments or a complementary sequence thereof,
encode a peptide with SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17, or SEQ ID NO:18 and modulate apoptosis in
primary cells.
38. A therapeutic method comprising the method for modulating
apoptosis in a cell according to claim 30 for the treatment of a
disease involving excessive or defective apoptosis, said
therapeutic method comprising administering to a subject in need
thereof an effective amount of a polynucleotide or oligonucleotide
selected from the group of nucleic acid sequences consisting of SEQ
ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:10 and SEQ ID NO:11, or a sequence encoding a peptide of SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18 and fragments and
complementary sequences thereof.
39. The therapeutic method according to claim 38, wherein said
disease involving excessive or defective apoptosis is selected from
the group consisting of primary leukemias, acute or chronic tissue
damage, such as heart, kidney, brain or other organ ischaemia,
HIV-related damage of brain or other tissues, skeletal muscle
disorders, transplantation rejection; chronic degenerative
disorders such as Parkinson's disease, amyotrophic lateral
sclerosis and others; and neoplastic, autoimmune and other diseases
involving excessive or defective apoptosis; tissue repair or wound
healing, treatment of surgical incisions, and ulcers, such as
stomach or diabetic ulcers.
40. The therapeutic method according to claim 38, wherein said
subject in need thereof is under chemotherapeutic treatment.
41. A method for detecting the presence of the nucleotide sequence
SEQ ID NO:1 or parts thereof in a sample, said method comprising
the steps of contacting the sample with a polynucleotide or
oligonucleotide that binds to it and forms a complex with the
nucleotide or parts thereof under conditions sufficient to form the
complex, and detecting said complex.
42. The method according to claim 41, wherein said part of SEQ ID
NO:1 to which said polynucleotide or oligonucleotide binds is
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ
ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11,
or is a sequence encoding a peptide of SEQ ID NO:15, SEQ ID NO:16,
SEQ ID NO:17 and SEQ ID NO:18.
43. A kit for identifying or diagnosing a disease involving
excessive or defective apoptosis comprising at least one
polynucleotide or nucleotide selected from the group consisting of
SEQ ID NOS:1, 3, 5, 7, 9, 10 and 11 or a sequence encoding a
peptide of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID
NO:18 fragments and a complementary sequence thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention provides BAG3 nucleotide and protein
sequences to be used in research, diagnostics and therapy for cell
death-involving diseases, and for modulation of cell survival
and/or death.
[0002] More particularly the invention refers to the use of
specific antisense-based constructs and peptide-specific polyclonal
and monoclonal antibodies in leukemias, other neoplasias and cell
death-involving diseases.
BACKGROUND
[0003] Cell death by apoptosis is largely responsible for control
of tissue homeostastis, differentiative and immune processes.
Alterations in the apoptosis program are implied in acute and
chronic tissue damages (heart, kidney, brain or other tissue
ischaemia, chronic degenerative disorders such as Parkinson's
disease, amyotrophic lateral sclerosis and others, etc.),
characterized by excessive apoptosis, and neoplastic, autoimmune
and other diseases involving insufficient apoptosis. Furthermore,
since antineoplastic compounds mainly act by inducing apoptosis in
cancer cells, molecules involved in the apoptotic response
determine neoplastic cell sensitivity or resistance to therapy.
Biochemical components and/or regulators of the apoptotic pathways
can be targets for modulating therapies, some of which have shown
efficacy in preclinical models and are now in human clinical
trials. Furthermore, apoptosis-involved molecules can represent
diagnostic tools in a range of diseases and reagents for laboratory
work (1).
[0004] BAG3 is member of the BAG protein family, involved in
co-chaperone activity for intracellular protein folding (2).
Although BAG3 displays homology with the other members of the BAG
family in some portions, like the BAG domain, other parts of its
nucleotide and protein sequences are unique (2-4). These
BAG3-specific, unique portions have been utilised by us for the
invention here described.
[0005] In the following there are reported the BAG3 nucleotide and
peptide sequences; the underlined parts correspond to parts which
are considered particularly relevant for the present invention.
BAG3 nucleotide sequence (SEQ ID NO: 1): reference: NCBI PubMed, XM
055575 Homo sapiens BCL2-associated athanogene 3 (BAG3), mRNA
gi|16156810|ref|XM.sub.--055575.1|[16156810]
TABLE-US-00001 1 gcggagctcc gcatccaacc ccgggccgcg gccaactttt
ttggactgga ccagaagttt 61 ctagccggcc agttgctacc tccctttatc
tcctccttcc cctctggcag cgaggaggct 121 atttccagac acttccaccc
ctctctggcc acgtcacccc cgcctttaat tcataaaggt 181 gcccggcgcc
ggcttcccgg acacgtcggc ggcggagagg ggcccacggc ggcggcccgg 241
ccagagactc ggcgcccgga gccagcgccc cgcacccgcg ccccagcggg cagaccccaa
301 cccagcatga gcgccgccac ccactcgccc atgatgcagg tggcgtccgg
caacggtgac 361 cgcgaccctt tgccccccgg atgggagatc aagatcgacc
cgcagaccgg ctggcccttc 421 ttcgtggacc acaacagccg caccactacg
tggaacgacc cgcgcgtgcc ctctgagggc 481 cccaaggaga ctccatcctc
tgccaatggc ccttcccggg agggctctag gctgccgcct 541 gctagggaag
gccaccctgt gtacccccag ctccgaccag gctacattcc cattcctgtg 601
ctccatgaag gcgctgagaa ccggcaggtg caccctttcc atgtctatcc ccagcctggg
661 atgcagcgat tccgaactga ggcggcagca gcggctcctc agaggtccca
gtcacctctg 721 cggggcatgc cagaaaccac tcagccagat aaacagtgtg
gacaggtggc agcggcggcg 781 gcagcccagc ccccagcctc ccacggacct
gagcggtccc agtctccagc tgcctctgac 841 tgctcatcct catcctcctc
ggccagcctg ccttcctccg gcaggagcag cctgggcagt 901 caccagctcc
cgcgggggta catctccatt ccggtgatac acgagcagaa cgttacccgg 961
ccagcagccc agccctcctt ccaccaagcc cagaagacgc actacccagc gcagcagggg
1021 gagtaccaga cccaccagcc tgtgtaccac aagatccagg gggatgactg
ggagccccgg 1081 cccctgcggg cggcatcccc gttcaggtca tctgtccagg
gtgcatcgag ccgggagggc 1141 tcaccagcca ggagcagcac gccactccac
tccccctcgc ccatccgtgt gcacaccgtg 1200 gtcgacaggc ctcagcagcc
catgacccat cgagaaactg cacctgtttc ccagcctgaa 1261 aacaaaccag
aaagtaagcc aggcccagtt ggaccagaac tccctcctgg acacatccca 1321
attcaagtga tccgcaaaga ggtggattct aaacctgttt cccagaagcc cccacctccc
1381 tctgagaagg tagaggtgaa agttccccct gctccagttc cttgtcctcc
tcccagccct 1441 ggcccttctg ctgtcccctc ttcccccaag agtgtggcta
cagaagagag ggcagccccc 1501 agcactgccc ctgcagaagc tacacctcca
aaaccaggag aagccgaggc tcccccaaaa 1561 catccaggag tgctgaaagt
ggaagccatc ctggagaagg tgcaggggct ggagcaggct 1621 gtagacaact
ttgaaggcaa gaagactgac aaaaagtacc tgatgatcga agagtatttg 1681
accaaagagc tgctggccct ggattcagtg gaccccgagg gacgagccga tgtgcgtcag
1741 gccaggagag acggtgtcag gaaggttcag accatcttgg aaaaacttga
acagaaagcc 1801 attgatgtcc caggtcaagt ccaggtctat gaactccagc
ccagcaacct tgaagcagat 1861 cagccactgc aggcaatcat ggagatgggt
gccgtggcag cagacaaggg caagaaaaat 1921 gctggaaatg cagaagatcc
ccacacagaa acccagcagc cagaagccac agcagcagcg 1981 acttcaaacc
ccagcagcat gacagacacc cctggtaacc cagcagcacc gtagcctctg 2041
ccctgtaaaa atcagactcg gaaccgatgt gtgctttagg gaattttaag ttgcatgcat
2101 ttcagagact ttaagtcagt tggtttttat tagctgcttg gtatgcagta
acttgggtgg 2161 aggcaaaaca ctaataaaag ggctaaaaag gaaaatgatg
cttttcttct atattcttac 2221 tctgtacaaa taaagaagtt gcttgttgtt
tcagaagttt aaccccgttg cttgttctgc 2281 agccctgtct acttgggcac
ccccaccacc tgttagctgt ggttgtgcac tgtcttttgt 2341 agctctggac
tggaggggta gatggggagt caattaccca tcacataaat atgaaacatt 2401
tatcagaaat gttgccattt taatgagatg attttcttca tctcataatt aaaatacctg
2461 actttagaga gagtaaaatg tgccaggagc cataggaata tctgtatgtt
ggatgacttt 2521 aatgctacat ttt
BAG3 aminoacidic sequence (SEQ ID NO: 2): reference: NCBI PubMed,
XM 055575 Homo sapiens BCL2-associated athanogene 3 (BAG3), mRNA
gi|16156810|ref|XM.sub.--055575.1|[16156810]
TABLE-US-00002 MSAATHSPMMQVASGNGDRDPLPPGWEIKIDPQTGWPFFVDHNSRTTTWN
DPRVPSEGPKETPSSANGPSREGSRLPPAREGHPVYPQLRPGYIPIPVLH
EGAENRQVHPFHVYPQPGMQRFRTEAAAAAPQRSQSPLRGMPETTQPDKQ
CGQVAAAAAAQPPASHGPERSQSPAASDCSSSSSSASLPSSGRSSLGSHQ
LPRGYISIPVIHEQNVTRPAAQPSFHQAQKTHYPAQQGEYQTHQPVYHKI
QGDDWEPRPLRAASPFRSSVQGASSREGSPARSSTPLHSPSPIRVHTVVD
RPQQPMTHRETAPVSQPENKPESKPGPVGPELPPGHIPIQVIRKEVDSKP
VSQKPPPPSEKVEVKVPPAPVPCPPPSPGPSAVPSSPKSVATEERAAPST
APAEATPPKPGEAEAPPKHPGVLKVEAILEKVQGLEQAVDNFEGKKTDKK
YLMIEEYLTKELLALDSVDPEGRADVRQARRDGVRKVQTILEKLEQKAID
VPGQVQVYELQPSNLEADQPLQAIMEMGAVAADKGKKNAGNAEDPHTETQ
QPEATAAATSNPSSMTDTPGNPAAP
[0006] BAG3 protein is known to be expressed in some cell lines,
such as HeLa and A2058, and, as far as normal primary human cells
are concerned, in skeletal muscle, heart, ovary and other types of
normal cells (2-5). BAG3 expression has also been detected in human
pancreas tumour cells (6).
[0007] BAG3 expression had not been reported in other types of
primary normal or neoplastic cells before the results here reported
for the first time.
[0008] Some findings describe that transfection of cells of the
human cell line HeLa (5) or of the murine cell line 32D (7) with
BAG3 hyperexpressing constructs can modestly increase cell
apoptosis induced by Bax microinjection or via Fas (5), or by IL-3
deprivation (7), respectively.
[0009] Generically antibodies for BAG3 have been described in
WO00/14106 and WO95/25125, however there has not been characterized
any immunogenic site specific for them. Ref.s 4-6 describe
polyclonal antibodies specific for the carbossi-terminal region of
BAG3 protein starting from amino acid 306 specifically. Liao
describes a rabbit polyclonal anti-BAG3 antibody against the 196
amino acids of the C-terminal portion of BAG3. Lee describes a
polyclonal antibody against the amino acid region encompassing the
portion 306-575. Dong describes a polyclonal antibody against the
two amino acid regions 2 and 8.
[0010] Patent abstract of Japan publication 10327872 describes uses
of BAG3 for diagnosis, prophylaxis and therapy of pathologies
relating to apoptosis, however there has not been characterized any
immunogenic site or any specific antibody, moreover test, in
particular in humans, are absent.
[0011] Before results here reported for the first time, BAG3
expression had not been proved to influence apoptosis in human
primary cells, either normal, neoplastic or affected by other types
of pathologies. Furthermore, BAG3 downmodulation by reagents, such
as oligonucleotides, that can be used in primary cells, and its
effects on cell apoptosis had never been reported.
SUMMARY OF THE INVENTION
[0012] The present invention refers to BAG3 protein (SEQ ID NO: 2)
and corresponding nucleotide sequence (SEQ ID NO: 1) and parts of
them (indicated by underlining inside the above mentioned long
sequences).
[0013] Objects of the present invention are therefore the uses of
BAG3 polypeptides and polynucleotides codifying it and parts of
them in research, diagnostics and therapy for modulating primary
cell survival and/or death, particularly in human leukemias and
other neoplasias or cell death-involving diseases.
[0014] There are considered within the scope of the invention in
that BAG3-related: sense or antisense oligonucleotides; monoclonal
or polyclonal antibodies that specifically recognise one or more
BAG3-specific epitopes: in particular:
TABLE-US-00003 SEQ ID NO 15: DRDPLPPGWEIKIDPQ; SEQ ID NO 16:
SSPKSVATEERAAPS; SEQ ID NO 17: DKGKKNAGNAEDPHT; SEQ ID NO 18:
NPSSMTDTPGNPAAP;
primers for PCR; nucleotide sequences for analysis of DNA or RNA;
the polypeptide and polynucleotide sequences encoding them,
including recombinant DNA molecules, cloned genes or degenerate
variants thereof, especially naturally occurring variants such as
allelic variants.
[0015] Reagents and compositions for the uses described in the
present invention additionally include vectors, including
expression vectors, viruses, etc., containing BAG3-specific
sequences; cells genetically engineered to contain such sequences
and cells genetically engineered to express such sequences.
Reagents additionally include the complement of any of the
nucleotide sequences recited above.
[0016] Compositions for the uses described in the present invention
may further comprise an acceptable carrier, such as
pharmaceutically acceptable carrier.
[0017] BAG3-based uses described in the present invention include
also methods for preventing, treating or ameliorating a medical
condition, which comprises administering to a human or other animal
subject a therapeutically effective amount of a composition
comprising BAG3-based reagents. Examples are methods for
preventing, treating or ameliorating: acute or chronic tissue
damages, such as heart, kidney, brain or other organ ischaemia,
HIV-related damage of brain or other tissues, skeletal muscle
disorders, transplantation rejection; chronic degenerative
disorders such as Parkinson's disease, amyotrophic lateral
sclerosis and others, etc.; and neoplastic, autoimmune and other
diseases involving excessive or defective apoptosis; tissue repair
or wound healing, treatment of surgical incisions, and ulcers, such
as stomach or diabetic ulcers; etc.
[0018] BAG3-based uses described in the present invention relate
also to reagents and methods for detecting the presence of BAG3
nucleotide sequence or protein, or parts of them. Such methods can,
for example, be utilised as part of prognostic and diagnostic
and/or prognostic evaluation of disorders as recited above and for
the identification of subjects exhibiting a predisposition to such
conditions. Furthermore, the invention include BAG3-related uses
for evaluating the efficacy of drugs, and monitoring the progress
of patients, involved in clinical trials for the treatment of
disorders as recited above.
[0019] BAG3-related uses of the present invention include also
reagents and/or methods for the identification of compounds that
modulate the expression or the activity of BAG3. Such reagents or
methods can be utilised, for example, for the identification of
compounds that can ameliorate symptoms of disorders as recited
above. Such methods can include, but are not limited to, assays for
identifying compounds and other substances that interact with
(e.g., bind to) BAG3 protein or nucleotide sequence or parts of
them.
[0020] The invention also includes methods for detecting the
presence of the nucleotide sequence SEQ ID NO: 1 or of the protein
SEQ ID NO: 2 or parts of them in a sample, in particular at least a
part identified as SEQ ID NO: 3, 4, 5, 6, 7, 8, 15, 16, 17, 18;
said method comprising the steps of: contacting the sample with a
compound that binds to and forms a complex with the nucleotide or
the protein in sufficient conditions to form the complex, and
detecting said complex. The expert in the field is able to select
the suitable conditions to perform the method.
[0021] The invention also includes methods for detecting a compound
that binds to the protein SEQ ID NO: 2 or parts of it in a sample,
in particular at least a part identified as SEQ ID NO: 4, 6, 8, 15,
16, 17, 18; said method comprising the steps of: contacting the
compound with the protein or its part/s in sufficient conditions to
form the complex compound/protein or its part/s, and detecting said
complex. The expert in the field is able to select the suitable
conditions to perform the method. The invention also includes
methods for the treatment of disorders as recited above which may
involve the administration of such compounds to individuals
exhibiting symptoms or tendencies related to disorders as recited
above. In addition, the invention encompasses methods for treating
diseases or disorders as recited above by administering compounds
and other substances that modulate the overall activity of BAG3 and
related molecules. Compounds and other substances can effect such
modulation either on the level of gene expression or protein
activity.
[0022] The diagnostic, prognostic or therapeutic compositions for
the BAG3-related use related to the present invention are also
presently valuable for veterinary applications. Particularly
domestic animals and thoroughbred horses, in addition to humans,
are desired patients for such applications.
[0023] The invention further refers to a kit for identification and
diagnosis comprising the polyclonal or monoclonal antibodies
identified in the following description or nucleotide sequence SEQ
ID NO: 1 or the protein SEQ ID NO: 2 or parts of them, in
particular at least a part identified as SEQ ID NO: 3, 4, 5, 6, 7,
8, 15, 16, 17, 18; or the antisense and nonsense oligos identified
as SEQ ID NO: 9, 10, 11, 12, 13, 14, or functionally equivalents of
the above identified sequences.
[0024] BAG3-based uses described in the present invention relate
also to reagents and/or methods and/or kits for laboratory work or
research.
[0025] Further objects of the invention will become evident from
the following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 shows the expression of BAG3 mRNA (A panel) and
protein (B panel) in primary cells from leukemia patients.
[0027] FIG. 2 shows the BAG3 downmodulation ability of anti-BAG3
antisense oligodeoxynucleotides in primary cells from leukemia
patients.
[0028] FIG. 3 shows the stimulation of mitochondrial cytochrome c
release by anti-BAG3 antisense oligodeoxynucleotides in primary
cells from leukemia patients.
[0029] FIG. 4 shows the stimulation of caspase activity by
anti-BAG3 antisense oligodeoxynucleotides in primary cells from
leukemia patients.
[0030] FIG. 5 shows the enhancement on annexin V binding by
anti-BAG3 antisense oligodeoxynucleotides in primary cells from
leukemia patients.
[0031] FIG. 6 shows the stimulation of primary B-CLL (B chronic
lymphocytic leukemia) cell apoptosis by anti-BAG3 antisense
oligodeoxynucleotides.
[0032] FIG. 7 shows the stimulation of primary ALL (acute
lymphoblastic leukemia) cell apoptosis by anti-BAG3 antisense
oligodeoxynucleotides.
[0033] FIG. 8 shows the BAG3 downmodulation ability of anti-BAG3
antisense oligodeoxynucleotides in human U937 cells.
[0034] FIG. 9 shows the stimulation of stress-induced apoptosis in
cells of the human myeloid leukemia line U937 by anti-BAG3
antisense oligodeoxynucleotides.
[0035] FIG. 10 shows the stimulation of stress-induced apoptosis in
human normal peripheral blood primary lymphocytes (A panel) or
monocytes (B panel) by anti-BAG3 antisense
oligodeoxynucleotides.
[0036] FIG. 11 shows the expression of BAG-3 protein and its
modulation by antisense oligonucleotides, as detected in Western
blotting (A) or intracellular immunofluorescence (B).
[0037] FIG. 12 shows the effect of BAG3-specific antisense
oligonucleotides or AraC on ALL cell apoptosis.
[0038] Table 1 describes the effect of anti-BAG3 antisense
oligodeoxynucleotides on apoptosis in cells of the human
osteosarcoma line SAOS.
[0039] Table 2 describes the protective effect of BAG3
hyperexpression on stress-induced apoptosis in the human cell line
293.
[0040] Table 3 describes the effect of BAG3 hyperexpression on the
growth of human neoplastic (osteosarcoma) cells xenografted in nude
mice.
[0041] Table 4 shows the results of the ELISA tests performed to
verify the binding of hybridoma mother clone supernatants to
MAP-BAG3 constructs.
[0042] FIG. 13 shows the binding of the polyclonal antibodies
AC-BAG3-2 and AC-BAG3-1 to lysates from HeLa or primary leukemia
cells (A) and of the hybridoma mother clones (AC-1, AC-2, AC-3,
AC-4) supernatants to cell lysates from HeLa cells (B), as detected
by Western blotting.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The polynucleotidic and aminoacidic fragments that are
considered particularly relevant for the present invention and are
comprised inside SEQ ID NO: 1 and 2, are indicated in the
following, such sequences are relevant because are specific of BAG3
and not shared with any other known sequence of other BAG genes or
proteins:
TABLE-US-00004 SEQ ID NO: 3: gcggagctcc gcatccaacc ccgggccgcg
gccaactttt ttggactgga ccagaagttt ctagccggcc agttgctacc tccctttatc
tcctccttcc cctctggcag cgaggaggct atttccagac acttccaccc ctctctggcc
acgtcacccc cgcctttaat tcataaaggt gcccggcgcc ggcttcccgg acacgtcggc
ggcggagagg ggcccacggc ggcggcccgg ccagagactc ggcgcccgga gccagcgccc
cgcacccgcg ccccagcggg cagaccccaa cccagcatga gcgccgccac ccactcgccc
atgatgcagg tggcgtccgg caacggtgac SEQ ID NO: 4:
MSAATHSPMMQVASGNGDRDPLPPGWEIKIDPQTG SEQ ID NO: 5: gtgcc ctctgagggc
cccaaggaga ctccatcctc tgccaatggc ccttcccggg agggctctag gctgccgcct
gctagggaag gccaccctgt gtacccccag ctccgaccag gctacattcc cattcctgtg
ctccatgaag gcgctgagaa ccggcaggtg caccctttcc atgtctatcc ccagcctggg
atgcagcgat tccgaactga ggcggcagca gcggctcctc agaggtccca gtcacctctg
cggggcatgc cagaaaccac tcagccagat aaacagtgtg gacaggtggc agcggcggcg
gcagcccagc ccccagcctc ccacggacct gagcggtccc agtctccagc tgcctctgac
tgctcatcct catcctcctc ggccagcctg ccttcctccg gcaggagcag cctgggcagt
caccagctcc cgcgggggta catctccatt ccggtgatac acgagcagaa cgttacccgg
ccagcagccc agccctcctt ccaccaagcc cagaagacgc actacccagc gcagcagggg
gagtaccaga cccaccagcc tgtgtaccac aagatccagg gggatgactg ggagccccgg
cccctgcggg cggcatcccc gttcaggtca tctgtccagg gtgcatcgag ccgggagggc
tcaccagcca ggagcagcac gccactccac tccccctcgc ccatccgtgt gcacaccgtg
gtcgacaggc ctcagcagcc catgacccat cgagaaactg cacctgtttc ccagcctgaa
aacaaaccag aaagtaagcc aggcccagtt ggaccagaac tccctcctgg acacatccca
attcaagtga tccgcaaaga ggtggattct aaacctgttt cccagaagcc cccacctccc
tctgagaagg tagaggtgaa agttccccct gctccagttc cttgtcctcc tcccagccct
ggcccttctg ctgtcccctc ttcccccaag agtgtggcta cagaagagag ggcagccccc
agcactgccc ctgcagaagc tacacctcca aaaccaggag aagccgaggc tcccccaaaa
catccaggag SEQ ID NO: 6:
NDPRVPSEGPKETPSSANGPSREGSRLPPAREGHPVYPQLRPGYIPIPVL
HEGAENRQVHPFHVYPQPGMQRFRTEAAAAAPQRSQSPLRGMPETTQPDK
QCGQVAAAAAAQPPASHGPERSQSPAASDCSSSSSSASLPSSGRSSLGSH
QLPRGYISIPVIHEQNVTRPAAQPSFHQAQKTHYPAQQGEYQTHQPVYHK
IQGDDWEPRPLRAASPFRSSVQGASSREGSPARSSTPLHSPSPIRVHTVV
DRPQQPMTHRETAPVSQPENKPESKPGPVGPELPPGHIPIQVIRKEVDSK
PVSQKPPPPSEKVEVKVPPAPVPCPPPSPGPSAVPSSPKSVATEERAAPS
TAPAEATPPKPGEAEAPPKHPGVLKVEAILEKVQGLEQAVDNFEG SEQ ID NO: 7
attgatgtcc caggtcaagt ccaggtctat gaactccagc ccagcaacct tgaagcagat
cagccactgc aggcaatcat ggagatgggt gccgtggcag cagacaaggg caagaaaaat
gctggaaatg cagaagatcc ccacacagaa acccagcagc cagaagccac agcagcagcg
acttcaaacc ccagcagcat gacagacacc cctggtaacc cagcagcacc gtagcctctg
ccctgtaaaa atcagactcg gaaccgatgt gtgctttagg gaattttaag ttgcatgcat
ttcagagact ttaagtcagt tggtttttat tagctgcttg gtatgcagta acttgggtgg
aggcaaaaca ctaataaaag ggctaaaaag gaaaatgatg cttttcttct ataftcttac
tctgtacaaa taaagaagtt gcttgttgtt tcagaagttt aaccccgttg cttgttctgc
agccctgtct acttgggcac ccccaccacc tgttagctgt ggttgtgcac tgtcttttgt
agctctggac tggaggggta gatggggagt caattaccca tcacataaat atgaaacatt
tatcagaaat gttgccattt taatgagatg attttcttca tctcataatt aaaatacctg
actttagaga gagtaaaatg tgccaggagc cataggaata tctgtatgtt ggatgacttt
aatgctacat ttt SEQ ID NO: 8:
ELQPSNLEADQPLQAIMEMGAVAADKGKKNAGNAEDPHTETQQPEATAAA
TSNPSSMTDTPGNPAAP
[0044] The experiments performed in our laboratories indicate for
the first time that specific antisense oligonucleotides are able to
modulate, in human primary cells and human cell lines, the levels
of BAG3 protein; these antisense oligos modulate also the survival
and/or death, either spontaneous or in response to therapy, of
human primary cells and human cell lines. Experiments with primary
cells, that are the target of diagnostic and therapeutic
applications, are particularly relevant, and the results were not
predictable from data obtained with cell lines, since stable cell
lines and primary cells are differently sensitive to modulators of
cell survival and/or death (14-18); furthermore, the effect of BAG3
protein downmodulation on cell survival and/or death, either in
cell lines or primary cells, were not reported before, nor were
predictable from data concerning BAG3 hyperexpression, since
several examples have been reported, in which the overexpression of
a protein (i.e. Bcl-2 family proteins) can protect cells from
pro-apoptotic insults, but its downmodulation does not stimulate
apoptosis (19-21); finally, BAG3 downmodulation has been obtained
with specific antisense oligonucleotides, that can be used for
research, diagnosis and/or therapy, and their effectiveness was not
predictable before the experimental work, since not all antisense
oligonucleotides against a specific mRNA display comparable
activities when introduced in a cell, and furthermore some
antisense molecules can exert unpredicted, not desired effects,
such as citotoxicity (22-23).
[0045] BAG3 modulation is able to influence the development of a
human tumour in vivo; these results are necessary for in vivo
applications, are absolutely required for proving the biological
activity of a gene and/or protein and the effects of its modulation
in pluricellular organisms, and cannot be extrapolated in this
respect from results in vitro (1).
[0046] Based on the apoptosis-modulating effect of the antisense
according to the invention, a panel of polyclonal and monoclonal
antibodies raised against peptide constructs (MAP-BAG3-peptides)
has been designed to: map different BAG3 epitopes and/or domains;
relate them to the functional activity of BAG3 (i.e., modulation of
cell survival); relate them to specific biochemical interaction
with molecular partners and/or formation of complexes; target them
to neutralize (antagonistic antibodies) or trigger (agonistic
antibodies) BAG3 functional activity.
Identification of BAG3 Expression in Human Primary Leukemia Cells
and Effectiveness of Specific Antisense Oligonucleotides in
Modulating BAG3 Levels and Cell Survival and/or Death.
[0047] We analysed by PCR the expression of BAG3 mRNA in primary
cells from B-CLL patients. BAG3 mRNA was detectable in such cells,
and its levels appeared to be enhanced by treatment with a
chemotherapeutic compound, fludarabine phosphate (FIG. 1, A panel).
[0048] To explore the levels of BAG3 protein, we first used a
polyclonal antibody according to the teaching of the patent appl
WO95/25125. Such antibody appeared to bind with a low avidity BAG3
protein from primary leukemic cells and had therefore to be used in
condition of high resolution (high antibody concentration, long
incubation times, etc.). Therefore we decided to produce novel
polyclonal antibodies by using a different approach, i.e. using a
Multiple Antigen Peptide (MAP) prepared in a single synthesis by
the solid-phase method described in ref. 24-26. Such approach
allows to improve the immunogenecity of the antigenic peptides and
obtain particularly efficient antibodies. (24-26) This is of high
relevance for detecting proteins expressed in low amounts, as
usually happens for many relevant proteins in physiologic or
pathologic conditions in primary cells. The kind of MAP used, here
as for the subsequent production of hybridomas (see below), was an
octa-branching MAP consisting of a core matrix made up of three
levels of lysine and eight amino terminals for anchoring peptide
antigens. In this case, we used the peptide NPSSMTDTPGNPAAP (SEQ ID
NO: 18), corresponding to the last 15 aminoacids of the
carboxyterminal region of BAG3 protein. For obtaining policlonal
antibodies, two rabbits were immunised with 4 boosts (a boost every
2 week) of MAP-BAG3-4 (400 micrograms for each boost); the serum
was finally tested against MAP-BAG3-4 in ELISA test and verified to
be positive. We named the two polyconal antibodies, obtained from
the two rabbits, AC-BAG3-1 and AC-BAG3-2: both recognised the
carboxyterminal region of BAG3 protein and were efficient in
detecting BAG3 protein, either in Western blotting or in
immunofluorescence, in primary cells, as shown in FIGS. 1,2,11,13.
[0049] With this BAG3-specific antibodies we analysed by
immunofluorescence the expression of BAG3 protein, that was
detectable in primary cells from B-CLL patients and whose levels
appeared to be enhanced by treatment fludarabine (FIG. 1, B panel).
In a comprehensive investigation of 18 different B-CLL specimens,
13 displayed detectable levels of BAG3 protein, and in 11 of these
BAG3 levels were upregulated by treatment with fludarabine.
[0050] These findings for the first time demonstrate that BAG3
expression can be detected in primary leukemic cells and modulated
by therapy. Such results disclose a diagnostic and/or prognostic
use, not shown before, of BAG3-detecting reagents in leukemias.
[0051] To be able to modulate BAG3 expression, we constructed the
following BAG-3-based antisense oligonucleotides:
TABLE-US-00005 antisense 1: TGCATCATGG GCGAGTGGGT GGCGG, (SEQ ID
NO: 9) antisense 2: GCTCATGCTG GGTTGGGGTC TG, (SEQ ID NO: 10)
antisense 3: ATTAAAGGCG GGGGTGACGT GG, (SEQ ID NO: 11)
and control nonsense:
TABLE-US-00006 nonsense 1: TTATATTCTATTATATTTATGAACTCC, (SEQ ID NO:
12) nonsense 2: CCTCGTAACCACCG ACCTCAAT, (SEQ ID NO: 13) nonsense
3: GCTTATGGAG GATTGAGGTT GG. (SEQ ID NO: 14)
[0052] Other oligonucleotides can be constructed, functionally
analogues to the ones mentioned before, in particular the
oligonucleotides can be constructed based on sequences indicated as
SEQ ID NO: 3, 5, 7.
[0053] There are within the scope of the present invention the
nucleotide and peptide sequences that show functional equivalence
with the ones identified in the description or that have a homology
of at least 75%, preferably at least 80% homology, more preferably
at least 90% homology, more preferably at least 95% homology, even
more preferably at least 98% homology to at least one of the
sequences mentioned in the description.
[0054] Administering of antisense, but not of nonsense,
oligonucleotides to human primary leukemic cells ex vivo resulted
in a downmodulation of BAG3 protein levels. Representative results
are shown in FIG. 2; analogous results were obtained in experiments
with three different B-CLL specimens. These findings disclose the
use, not shown before, of BAG3 antisense oligonucleotides for
affecting BAG3 protein levels in primary (in this case neoplastic,
and specifically leukemic) cells. We then analysed whether
antisense oligonucleotides, by downmodulating BAG3 protein levels,
could affect cell apoptosis. Primary cells from leukemia patients
were incubated with or without antisense or control
oligonucleotides and/or fludarabine, and different events of
apoptosis: mitochondrial cytochrome c release (8), caspase 3
activation (9), annexin V binding (10) and appearance of
hypodiploid elements (11) were analysed. A comprehensive analysis
of 15 B-CLL samples indicated that administering of antisense, but
not of nonsense, oligonucleotides to the cells resulted in
stimulation of mitochondrial cytochrome c release (FIG. 3), caspase
activity (FIG. 4), annexin V binding (FIG. 5) and appearance of
hypodiploid elements (FIG. 6). Apoptosis stimulation was even more
amplified by the addition of fludarabine (FIG. 6). Furthermore, in
4 of 4 ALL specimens analysed, the pro-apoptotic effect of the
antisense oligonucleotides alone was particularly remarkable, since
the percentage of hypodiploid elements reached >60% of the cells
(similar to the value obtained with the chemotherapeutic compound
AraC) (FIG. 7).
[0055] Therefore we demonstrate for the first time that
downmodulation of BAG3 protein levels by administration of BAG3
antisense oligonucleotides to different types of human primary
leukemia cells can stimulate apoptosis. The pro-apoptotic effect is
remarkable when the antisense oligonucleotides are administered
alone and can be synergic with that of different chemotherapeutic
compounds.
[0056] These findings disclose the possible use, not shown before,
of BAG3-modulating reagents, such as antisense oligonucleotides,
for modulating survival and/or death in human primary cells, in
this case neoplastic, and specifically leukemic. They also indicate
the possible use of such reagents in synergy with other drugs.
Additional results were obtained by using human cells of different
types: osteosarcoma cells of the SAOS line, in which we detected a
remarkable pro-apoptotic effect of the antisense oligonucleotides
alone (table 1); and myeloid cells of the U937 line, in which BAG3
antisense could enhance apoptosis induced by stress (FIG. 9).
Particularly, the enhancement of stress-induced apoptosis in U937
cells suggested to us to verify whether BAG3-based reagents could
interfere also with stress effects in human primary cells.
Therefore we administered the antisense or control oligonucleotides
to human normal peripheral blood lymphocytes or monocytes ex vivo,
treated with the stress inducers diethylmaleate (DEM) and
2-Methoxymethylestradiol (2-ME). Antisense, but not control, oligo,
highly enhanced cell apoptosis in these cells (FIG. 10). These
findings for the first time demonstrate that stress effects on
human primary cells (in this case, normal cells, and specifically
lymphocytes and monocytes from peripheral blood) can be modulated
by BAG3-based reagents.
[0057] We investigated whether protection from cell death could be
obtained with BAG3-based reagents. Therefore we transfected 293
cells with a BAG3-hyperexpressing construct and verified the effect
on stress-induced apoptosis. Transfection with the BAG3 construct
resulted in protection from stress-induced apoptosis (table 2).
[0058] The above described results indicate for the first time
that: 1) BAG3 is expressed in human primary leukemic cells; 2) BAG3
protein levels, and spontaneous or therapy-induced death of human
primary cells, can be modulated by using specific antisense
oligonucleotides.
[0059] It is worthy of note that this is the first reported
observation that specific BAG3 antisense oligonucleotides are able
to enhance human primary cell apoptosis. It has been previously
shown that the overexpression of BAG-3 in transfected cell lines
could partially protect them from apoptosis induced via Fas or
growth factor deprivation (5,7). Our invention was not predictable
from such previous observation, for three reasons: 1) stable cell
lines and primary cells are differently sensitive to modulators of
cell survival and/or death (14-18); 2) several examples have been
reported, in which the overexpression of a protein (i.e. Bcl-2
family proteins) can protect cells from pro-apoptotic insults, but
its downmodulation does not stimulate apoptosis (19-21); 3) not all
antisense oligonucleotides against a specific mRNA display
comparable activities when introduced in a cell; furthermore, some
antisense molecules can exert unpredicted, not desired effects,
such as citotoxicity (22-23). Therefore, the properties of the
specific antisense oligonucleotide sequences used by us could not
be predicted before our experimental work.
[0060] This is the first reported observation of BAG3 expression in
human primary leukemic cells. This was not predictable from
previous results described in stable cell lines and primary cells
other than leukemia cells. Indeed: a) cell lines are no longer
subjected to the environmental influences of a pluricellular
organisms, and furthermore and more importantly are selected for
their survival in culture: therefore they usually differ in gene
expression and/or levels of particular proteins from primary cells,
even when belonging to the same type; b) different cell types,
either from lines or primary cells, differ in gene expression
and/or levels of particular proteins (14-19).
[0061] Finally, since we have demonstrated that the modulation of
BAG3 protein levels can modulate cell survival and/or death in
primary cells, also polynucleotides and corresponding codified
polypeptides indicated as SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 15, 16,
17, 18 and constructs comprising them dare una definizione minima
di costrutto (hyperexpressing constructs, either in plasmid or
other vectors; naked DNA; etc.) that positively modulate such
levels are relevant in this functional activity. Particularly, we
have demonstrated that the functional effect is specific of BAG3
and not shared with other BAG proteins, and therefore the SEQ ID NO
3, 5 and 7 are identified as particularly relevant for the
functional effect (i.e., modulation of cell survival and/or
death).
Demonstration that BAG3 Modulation can Influence Tumour Development
In Vivo.
[0062] Hyperexpression of BAG3 had been reported to suppress
apoptosis in cell lines in vitro, since the overexpression of BAG-3
in transfected cell lines could partially protect them from
apoptosis induced via Fas or growth-factor deprivation (5,7). These
results did not allow to predict the effect of BAG3 hyperexpression
on tumour development in vivo. Indeed, tumours in vivo are
subjected to the environmental influences of a pluricellular
organisms, and molecules that have a specific activity in vitro can
fail their effects or show different activities in vivo: therefore
effects in vivo cannot be extrapolated from results in vitro and a
specific experimental work in vivo is required (1). We transfected
cells of the human osteosarcoma cell line Saos with a
BAG3-overexpressing plasmid vector and obtained a mass culture of
stably transfected cells. Wild type cells, the transfected culture
and a control, void vector-transfected culture were injected in
three different sites (back, left and right sides) in nude mice.
Wild type and control cells did not give rise to any tumour, while
BAG3-hyperexpressing cells developed detectable tumours,
demonstrating that BAG3 modulation can influence the development of
a tumour in vivo (table 3).
Design and Construction of a Panel of Polyclonal and Monoclonal
Antibodies
[0063] The antibodies were raised against peptide constructs
(MAP-BAG3-peptides) to recognise and/or trigger the following
defined BAG3 epitopes and/or domains
TABLE-US-00007 SEQ ID NO 15: DRDPLPPGWEIKIDPQ; SEQ ID NO 16:
SSPKSVATEERAAPS; SEQ ID NO 17: DKGKKNAGNAEDPHT; SEQ ID NO 18:
NPSSMTDTPGNPAAP
of functional importance in human primary cells and other cell
types of different origins.
[0064] The above described antisense oligonucleotides were all able
to downmodulate the levels of BAG3 protein. This is relevant for
the consequent modulation of cell death, here reported for the
first time.
[0065] The functional activity of BAG3 in modulating cell survival
and/or death can rely on biochemical interactions of specific BAG3
epitopes and/or domains indicated as SEQ ID NO 16, 16, 17, 18 with
molecular partners involved in survival/death pathway (2). Indeed,
a variety of such partners have been detected for BAG proteins in
general and BAG3 in particular (2-5); such interactions can
potentially involve different parts of the molecule, such as, in
addition to the BAG domain, the WW domain, the SER-rich part, the
PRO-rich part, etc. (described in 2-5). In view of the functional
activity of BAG3 in modulating cell survival, it is important to be
able to map different BAG3 epitopes and/or domains: this can allow
to: 1) relate such epitopes to the functional activity of BAG3; 2)
identify the site(s) of interactions with known partners, as well
as new sites of interactions with still undescribed partners; 3)
interfere with the formation of complexes with molecular partners;
4) block or mimic the interaction with these partners, leading to
modulation of BAG3 functional activity.
[0066] To produce effective tools able to explore the above
mentioned issues, we decided to obtain polyclonal and monoclonal
antibodies against specific peptides representing spatially
distinct portions of BAG3 protein. Such polyclonal and monoclonal
antibodies are desirable to: map different BAG3 epitopes and/or
domains; relate them to the functional activity of BAG3 (i.e.,
modulation of cell survival); relate them to specific biochemical
interaction with molecular partners and/or formation of complexes;
target them to neutralize (antagonistic antibodies) or trigger
(agonistic antibodies) BAG3 functional activity.
[0067] We identified the following, spatially distinct BAG3-derived
peptides:
(SEQ ID NO 15): DRDPLPPGWEIKIDPQ;
(SEQ ID NO 16): SSPKSVATEERAAPS;
(SEQ ID NO 17): DKGKKNAGNAEDPHT;
[0068] (SEQ ID NO 18): NPSSMTDTPGNPMP. Such peptide corresponded to
one we used for raising the polyclonal antibodies (see above). Its
use is here aimed at obtaining monoclonal antibodies against the
carboxyterminal part of BAG3 (indeed, only polyclonal antibodies
against such part have been so far: see ref. 4-6). Furthermore, its
use is in addition, and not alternative, to that of the other three
peptides (SEQ ID N. 15, 16 and 17).
[0069] We used these peptides to obtain separate monoclonal
antibodies against each one of the four peptides.
[0070] All four peptides are specific of BAG3 protein and are not
shared with other any protein, including other BAG proteins.
[0071] For immunizing the animals, we decided to use MAPs (Multiple
Antigenic Peptides) (24-26). As described in the previous section,
the construction of MAPs allows to significantly enhance the
immunogenicity of the antigenic peptides and to obtain particularly
efficient antibodies. This is of relevance for detecting proteins
expressed in low amounts, as usually happens for many relevant
proteins in physiologic or pathologic conditions in primary cells.
Following this approach, we obtained the following unique map
constructs:
TABLE-US-00008 (SEQ ID NO 15) MAP-BAG3-1: nh2-DRDPLPPGWEIKIDPQ-MAP
containing (SEQ ID NO 16) MAP-BAG3-2: nh2-SSPKSVATEERAAPS-MAP
containing (SEQ ID NO 17) MAP-BAG3-3: nh2-DKGKKNAGNAEDPHT-MAP
containing (SEQ ID NO 18) MAP-BAG3-4: nh2-NPSSMTDTPGNPAAP-MAP
containing
[0072] The production of the polyclonal antibodies has been
described above. In this respect, as well as for the production of
monoclonal antibodies, MAP constructs are to be considered unique
and different from the simple peptides alone, since their ability
to elicit immunogenic responses in the animal is different form
that of the peptides used alone (24-26).
[0073] Monoclonal antibodies (not yet reported in literature) were
highly required, either in general because of the high specificity
and homogeneity of such reagents, but also in particular in view of
our results demonstrating the apoptosis-modulating properties of
BAG3 protein in primary cells. Indeed BAG proteins, including BAG3,
interact with several molecular partners (2-5,7), and monoclonal
antibodies are required to map the protein epitopes involved in
interacting with specific partners, thereby leading to effects on
cell survival and/or death. Furthermore, monoclonal antibodies can
display agonistic or antagonistic properties respect to the
biological functions of a protein, and this is of relevance for the
potential application in modulating BAG3 activity in cell survival
and/or death.
[0074] For obtaining the monoclonal antibodies, we followed
standard procedures, already performed in our laboratory (12).
Specifically: [0075] nine Balb/c female mice of 4 weeks were
immunised with 4 boosts (a boost every 2 week) of the four MAP-BAG3
together (200 micrograms each, i.e. 800 micrograms of total
protein/mouse/boost). Spleens were then obtained and fused with
myeloma cells (NS0) to obtain monoclonal antibodies mother clones.
These were tested against each of the four MAP-BAG3 in ELISA test
(see table 4).
[0076] We produced: [0077] nine murine monoclonal antibody mother
clones (AC-1, AC-2, AC-3, AC-4, AC-5, AC-6, AC-7, AC-8, AC-9)
obtained from mice immunised with the four MAP-BAG3 together. The
nine mother clones are presently being subcloned to obtain
hybridomas against each one of the four MAP-BAG3 constructs. The
ELISA tests of the antibodies produced by the nine mother clones
are presented in table 4. Importantly, the ELISA tests demonstrate
that the mother clones contains hybridomas able to recognise each
one of the four MAP-BAG3 used. Therefore, the nine mother clones
already contain several specific hybridomas, each of whom can
recognise one of the four epitopes represented in the MAPs and can
hence be used to map one BAG3 epitope and interfere with its
functional interactions and activities; the monospecific hybridomas
are presently being separated by subcloning procedures. [0078] The
detection, by Western blot analysis, of BAG3 protein in lysates
from the cell line HeLa and primary leukemia cells are shown in
FIG. 13. Specifically: [0079] the antibodies from the nine
monoclonal mother clones did recognise the four MAP-BAG3 constructs
in ELISA test (table 4); [0080] four of them have been as yet
tested, with positive results, in Western blot with HeLa lysates
(FIG. 13, B panel).
[0081] In conclusion, the nine murine monoclonal antibody mother
clones (AC-1, AC-2, AC-3, AC-4, AC-5, AC-6, AC-7, AC-8, AC-9)
contain hybridomas specific for each one of the four MAP-BAG3
constructs, and are able to identify spatially distinct parts of
BAG3 molecule, in particular the mother clone AC-1 was no PD02009
deposited on the 17/12/2002 at the Centro Biotecnologie Avanzate di
Genova They can therefore be used to: map different BAG3 epitopes
and/or domains; relate them to the functional activity of BAG3
(i.e., modulation of cell survival); relate them to specific
biochemical interaction with molecular partners and/or formation of
complexes; target them to neutralize (antagonistic antibodies) or
trigger (agonistic antibodies) BAG3 functional activity.
[0082] The original features of these results are: [0083] the
downmodulating effect of antisense oligos constitutes the original
rationale, not predictable before, leading to the necessity of
mapping and triggering BAG3 epitopes that mediate the mechanism of
apoptosis modulation. This constituted the premise for the
production of a panel of antibodies raised-against different region
of BAG3 protein; [0084] a panel of nine monoclonal-producing mother
clones (AC-1; AC-2; AC-3; AC-4; AC-5; AC-6; AC-7; AC-8; AC-9) have
been obtained and can be used to: map different BAG3 epitopes
and/or domains; relate them to the functional activity of BAG3
(i.e., modulation of cell survival); relate them to specific
biochemical interaction with molecular partners and/or formation of
complexes; target them to neutralize (antagonistic antibodies) or
trigger (agonistic antibodies) BAG3 functional activity; [0085] two
polyclonal antibodies (AC-BAG3-1 and AC-BAG3-2), able to reveal the
presence of BAG3 protein in human primary leukemias and its
modulated expression of BAG3 protein by specific antisense
oligodeoxynucleotides, have been obtained.
[0086] Within the scope of the present invention, BAG3-protein, the
corresponding polynucleotide, corresponding parts of them and
corresponding antisense oligonucleotides can be used for research,
diagnostic and therapeutic purposes for example in leukemias, other
neoplasias and cell death-involving diseases, and for modulation of
cell survival and/or death. BAG3-based reagents include in a
non-limitative manner, oligonucleotides, primers, probes,
(poly)peptides or protein, polyclonal or monoclonal antibodies,
etc., and any other reagent able to detect or modulate BAG3
expression.
[0087] Findings illustrated in the present invention and obtained
with the described BAG3-based reagents could be obtained with
modified reagents with equivalent activities. These latter are
therefore considered equivalent to those illustrated in the present
invention.
[0088] Particularly, as far as protein or its parts, or peptides,
are concerned, are considered equivalent: [0089] naturally
occurring (poly)peptides or proteins, that are (poly)peptides or
proteins produced by cells that have not been genetically
engineered and specifically contemplates various (poly)peptides or
proteins arising from post-translational modifications of the
(poly)peptide or protein including, but not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation and acylation; [0090] derivatives, that are
(poly)peptides or proteins chemically modified by such techniques
as ubiquitination, labeling (e.g., with radionuclides,
fluorochromes or various enzymes), pegylation (derivatization with
polyethylene glycol) and insertion or substitution by chemical
synthesis of amino acids such as ornithine, which do not normally
occur in human proteins; [0091] recombinant variants, that are
(poly)peptides or proteins differing from naturally occurring
(poly)peptides or proteins by amino acid insertions, deletions, and
substitutions, created using recombinant DNA techniques. Guidance
in determining which amino acid residues may be replaced, added or
deleted without abolishing activities of interest, such as cellular
trafficking, may be found by comparing the sequence of the
particular polypeptide with that of homologous peptides and
minimizinig the number of amino acid sequence changes made in
regions of high homology.
[0092] Preferably, amino acid substitutions are the result of
replacing one amino acid with another amino acid having similar
structural and/or chemical properties, i.e., conservative amino
acid replacements. Amino acid substitutions may be made on the
basis of similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues involved. For example, nonpolar (hydrophobic) amino
acids include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan, and methionine; polar neutral amino
acids include glycine, serine, threonine, cysteine, tyrosine,
asparagine, and glutamine; positively charged (basic) amino acids
include arginine, lysine, and histidine; and negatively charged
(acidic) amino acids include aspartic acid and glutamic acid. The
variation allowed may be experimentally determined by
systematically making insertions, deletions, or substitutions of
amino acids in a polypeptide molecule using recombinant DNA
techniques and assaying the resulting recombinant variants for
activity.
[0093] Alternatively, where alteration of function is desired,
insertions, deletions or non-conservative alterations can be
engineered to produce altered (poly)peptides or proteins. Such
alterations can, for example, alter one or more of the biological
functions or biochemical characteristics. For example, such
alterations may change (poly)peptide or protein characteristics
such as ligand-binding affinities, interchain affinities, or
degradation/turnover rate. Further, such alterations can be
selected so as to generate (poly)peptides or proteins that are
better suited for expression, scale up and the like in the host
cells chosen for expression. For example, cysteine residues can be
deleted or substituted with another amino acid residue in order to
eliminate disulfide bridges.
[0094] Substantially equivalent can be either nucleotide or amino
acid sequences, for example a mutant sequence, that varies from a
reference sequence by one or more substitutions, deletions, or
additions, the net effect of which does not result in an adverse
functional dissimilarity between the reference and subject
sequences. Typically, such a substantially equivalent sequence
varies from the reference one by no more than about 20%, i.e. the
number of individual residue substitutions, additions, and/or
deletions in a substantially equivalent sequence; as compared to
the corresponding reference sequence, divided by the total number
of residues in the substantially equivalent sequence is about 0.2
or less. Such a sequence is said to have 80% sequence identity to
the listed sequence. In one embodiment, a substantially equivalent,
mutant, sequence of the invention varies from a listed sequence by
no more than 10% (90% sequence identity); in a variation of this
embodiment, by no more than 5% (95% sequence identity); and in a
further variation of this embodiment, by no more than 2% (98%
sequence identity). Compared to aminoacidic identity, substantially
equivalent nucleotide sequence(s) of the invention can have lower
percent sequence identities, taking into account, for example, the
redundancy or degeneracy of the genetic code. For the purposes of
the present invention, sequences having substantially equivalent
biological activity and substantially equivalent expression
characteristics are considered substantially equivalent. For the
purposes of determining equivalence, truncation of the mature
sequence (e.g., via a mutation, which creates a spurious stop
codon) should be disregarded.
[0095] Nucleic acid sequences encoding such substantially
equivalent sequences, sequences of the recited percent identities
can routinely be isolated and identified via standard hybridization
procedures well known to those of skill in the art.
[0096] Where desired, an expression vector may be designed to
contain a signal or leader sequence which will direct the
polypeptide through the membrane of a cell. Such a sequence may be
naturally present or provided from heterologous protein sources by
recombinant DNA techniques.
[0097] Recombinant variants encoding these same or similar
(poly)peptides or proteins may be synthesized or selected by making
use of the redundancy in the genetic code. Various codon
substitutions, such as the silent changes, which produce various
restriction sites, may be introduced to optimize cloning into a
plasmid or viral vector or expression in a particular prokaryotic
or eukaryotic system. Mutations in the polynucleotide sequence may
be reflected in the polypeptide or domains of other peptides added
to the polypeptide to modify the properties of any part of the
polypeptide, to change characteristics such as ligand-binding
affinities, interchain affinities, or degradation/turnover
rate.
[0098] Parts of the BAG3-related nucleotide or aminoacid sequence
may be fused to carrier molecules such as immunoglobulins for many
purposes, including increasing the valency of protein binding
sites.
[0099] Reagents based on species homologs of BAG3 are considered
equivalent respect to the uses illustrated in the present
invention.
[0100] The sequences falling within the scope of the present
invention are not limited to the specific sequences herein
described, but also include allelic variations thereof. The present
invention will be illustrated by the following examples, figures
and tables which are not to be considered as limiting the scope of
the invention.
DETAILED DESCRIPTION OF THE FIGURES AND TABLES
[0101] FIG. 1--Expression of BAG3 mRNA and Protein in Primary cells
from leukemia patients. Leukemic cells were isolated from B-CLL
patients' peripheral blood specimens by centrifugation through
Ficoll-Hypaque (13) and cultured for 24 hours in RPMI 1640 medium
supplemented with 10% fetal calf serum (10% FCS-RPMI), without or
with fludarabine phosphate. A panel: cell mRNA was the extracted
and BAG3 expression was verified by PCR (GAPDH expression is shown
for comparative purpose); B panel: cells were permabilised and
analysed by indirect immunofluorescence with the polyclonal
antibody AC-BAG3-1. A=control rabbit Ig; b=cells incubated with
control medium and analysed with anti-BAG3; c=cells incubated with
fludarabine and analysed with anti-BAG3.
[0102] FIG. 2--Downmodulation of BAG3 protein levels by anti-BAG3
antisense oligodeoxynucleotides. Leukemic cells were isolated from
B-CLL patients' peripheral blood specimens by centrifugation
through Ficoll-Hypaque and cultured for 20 hours without (b) or
with BAG3 antisense (b+.alpha.) or control nonsense (b+.nu.)
phosphorothioate oligodeoxynucleotides (5 microM) described in the
text. Then cells were permabilised and analysed by indirect
immunofluorescence with the polyclonal antibody AC-BAG3-1.
a=control rabbit Ig.
[0103] FIG. 3--Effect of anti-BAG3 antisense oligodeoxynucleotides
on mitochondrial cytochrome c release in B-CLLs. Leukemic cells
were isolated from B-CLL patients' peripheral blood specimens by
centrifugation through Ficoll-Hypaque and cultured for the
indicated times without or with the BAG3 antisense or control
nonsense phosphorothioate oligodeoxynucleotides (5 microM)
described in the text. Then cell extracts were obtained and
mitochondrial cytochrome c release was analysed according to ref.
8.
[0104] FIG. 4--Effect of anti-BAG3 antisense oligodeoxynucleotides
on caspase 3 activity in B-CLLs. Leukemic cells were isolated from
B-CLL patients' peripheral blood specimens by centrifugation
through Ficoll-Hypaque and cultured for the indicated times without
or with the BAG3 antisense or control nonsense phosphorothioate
oligodeoxynucleotides (5 microM) described in the text. Then cell
extracts were obtained and caspase 3 activity was analysed
according to ref. 9.
[0105] FIG. 5--Effect of anti-BAG3 antisense oligodeoxynucleotides
on annexin V binding in B-CLLs Leukemic cells were isolated from
B-CLL patients' peripheral blood specimens by centrifugation
through Ficoll-Hypaque and cultured for 40 hours the indicated
times without or with the BAG3 antisense or control nonsense
phosphorothioate oligodeoxynucleotides (5 microM) described in the
text. Then cell vitality was analysed by propidium iodide
incorporation in non permeabilized cells, while at the same time
annexin V binding was analysed by immunofluorescence according to
ref. 10. A: percentages of alive, apoptotic and dead cells in the
cytogram regions; B: PI--versus Annexin V--staining.
[0106] FIG. 6--Effect of anti-BAG3 antisense oligodeoxynucleotides
on apoptosis in 15 B-CLL specimens Leukemic cells were isolated
from B-CLL patients' peripheral blood specimens by centrifugation
through Ficoll-Hypaque and cultured for 5 days without or with
fludarabine phosphate (2 microgr/ml) and/or the BAG3 antisense or
control nonsense phosphorothioate oligodeoxynucleotides (5 microM)
described in the text. Then cell apoptosis was analysed by cell
permeabilization and PI staining according to ref. 11.
[0107] FIG. 7--Effect of anti-BAG3 antisense oliqodeoxvnucleotides
on ALL cell apoptosis Leukemic cells were isolated from ALL
patients' peripheral blood specimens by centrifugation through
Ficoll-Hypaque and cultured for four days (A panel) or the
indicated times (B panel) without or with cytosine arabinoside
(AraC, 1 microM) and/or the BAG3 antisense or control nonsense
phosphorothioate oligodeoxynucleotides (5 microM) described in the
text. Then cell apoptosis was analysed by cell permeabilization and
PI staining according to ref. 11.
TABLE-US-00009 TABLE 1 Effect of anti-BAG3 antisense
oligodeoxynucleotides on apoptosis in cells of the human
osteosarcoma line SAOS. Table 1 Effect of anti-BAG3 antisense
oligodeoxynucleotides on apoptosis in cells of the human
osteosarcoma line SAOS. Incubation Control Etoposide Topotecan
Oligo medium (5 microM) (40 ng/ml) 17.74* 38.32 36.84 BAG3
antisense 52.38 73.40 68.62 control nonsense 25.84 45.40 41.60 *%
of apoptosis
[0108] Cells of the SAOS line were incubated for 72 h in 10%
FCS-RPMI without or with chemotherapeutic compounds (etoposide or
topotecan) and/or the BAG3 antisense or control nonsense
phosphorothioate oligodeoxynucleotides (5 microM) described in the
text. Then cell apoptosis was analysed by cell permeabilization and
PI staining according to ref. 11.
[0109] FIG. 8--Effect of anti-BAG3 antisense oligodeoxynucleotides
on BAG3 Protein levels in cells of the human myeloid leukemia line
U937 U937 cells were cultured for 24 hours in 10% FCS-RPMI without
or with the BAG3 antisense or control nonsense phosphorothioate
oligodeoxynucleotides (5 microM) described in the text. Then cells
were permeabilised and analysed by indirect immunofluorescence with
AC-BAG3-1.
[0110] FIG. 9--Effect of anti-BAG3 antisense oligodeoxynucleotides
on stress-induced apoptosis in cells of the human myeloid leukemia
line U937 U937 cells were cultured for 40 h without or with
diethylmaleate (DEM, 1.2 microM) and/or the BAG3 antisense or
control nonsense phosphorothioate oligodeoxynucleotides (5 microM)
described in the text. Then cell apoptosis was analysed by cell
permeabilization and PI staining according to ref. 11.
[0111] FIG. 10--Effect of anti-BAG3 antisense oligodeoxynucleotides
on stress-induced apoptosis in normal human peripheral blood
leucocytes Lymphocytes (A panel) and monocytes (B panel) were
obtained from human normal peripheral blood specimens by
centrifugation through a Ficoll-Hypaque 50-72% density gradient and
cultured in 10% FCS-RPMI for 48 hours with or without a combination
of DEM (1.2 microM) and 2ME (20 microM) and/or the or the BAG3
antisense or control nonsense phosphorothioate
oligodeoxynucleotides (5 microM) described in the text. Then cell
apoptosis was analysed by cell permeabilization and PI staining
according to ref. 11.
TABLE-US-00010 TABLE 2 Protective effect of BAG3 hyperexpression on
stress- induced apoptosis in the human cell line 293. Transfected
construct Incubation % of apoptosis Control pcDNA control medium
6.1 .+-. 0.3* DEM + 2ME 32.4 .+-. 1.2 BAG3-pcDNA control medium 5.3
.+-. 0.2 DEM + 2ME 13.4 .+-. 0.5 *mean of duplicates .+-. SD
[0112] Cells of the human line 293 were transfected using a Fugene
(Roche) preparation with a pcDNA construct hyperexpressing BAG3 or
a void control pcDNA. BAG3 protein hyperexpression was verified by
immunofluorescence. Then the cells were incubated for 48 hours in
10% FCS-RPMI with or without a combination of DEM+2ME and apoptosis
was analysed by cell permeabilization and PI staining according to
ref. 11.
TABLE-US-00011 TABLE 3 BAG3 expression influences the growth of
human neoplastic (osteosarcoma) cells xenografted in nude mice #
mouse tumour volume (mm.sup.3) <40 B 65 C <40 2 A <40 B 45
C <40 3 A <40 B .92 C <40 4 A <40 B <40 C <40 5 A
<40 B 65 C <40 Human osteosarcoma cells of the SaOs line (10
.times. 10.sup.6), wild type (A) or stably transfected with a
BAG3-hyperexpressing (B) or a control void (C) vector, were
injected in 6 week-old nu/nu mice; tumour volume was measured every
week. Final results at the end of the 8th week are reported.
[0113] FIG. 11 Expression of BAG3 Protein in ALL cells and its
downmodulation by BAG3-specific antisense oligonucleotides. A--ALL
cells (1.times.10.sup.6/ml) were cultured in 10% FCS-RPMI without
or with control nonsense (TTATATTCTATTATATTTATGMCTCC, SEQ ID NO 12,
nonsense 1) or BAG3-specific antisense (TGCATCATGGGCGAGTGGGTGGCGG,
SEQ ID NO 9, antisense 1) oligonucleotides (5 microM) for 24 hr.
Then cell lysates were obtained and analyzed in Western blot with
anti-BAG3 (AC-BAG3-1; analogous results were obtained with
AC-BAG3-2) or anti-tubulin antibodies. B--ALL cells
(1.times.10.sup.6/ml) were cultured in 10% FCS-RPMI without or with
BAG3-specific antisense (TGCATCATGGGCGAGTGGGTGGCGG, SEQ ID NO 9,
antisense 1) (a) or control nonsense (TTATATTCTATTATATTTATGMCTCC,
SEQ ID NO 12, nonsense 1) (b) oligonucleotides (5 microM) for 24
hr. Then the cells were analyzed by intracellular
immunofluorescence with the anti-BAG3 polyclonal antibody. Negative
controls with a control rabbit antibody preparation are shown on
the left in a and b. Results are representative of experiments with
at least three different ALL samples; comparable results were
obtained using any one of the three antisense or nonsense ODN.
[0114] FIG. 12--Effects of BAG3-specific antisense oligonucleotides
or AraC on ALL cell apoptosis. A--ALL cells (1.times.10.sup.6/ml)
from ten different samples were cultured in 10% FCS-RPMI without or
with control nonsense or BAG3-specific antisense oligonucleotides
(5 microM), or with AraC (10 microM), for 4 days. Then cell
apoptosis was analyzed by propidium iodide incorporation in
permeabilized cells and flow cytometry. Student's t test was
performed to evaluate the difference between apoptosis percentages
detected in control and BAG3 antisense-cultured cells,
respectively.
TABLE-US-00012 TABLE 4 Binding of hybridoma mother clone
supernatants to MAP-BAG3 constructs as detecteds by ELISA test. Map
1 Map 2 Map 3 Map 4 Map 1 Map 2 Map 3 Map 4 Map 1 Map 2 Map 3 Map 4
1/2 1.254 2.475 0.050 0.042 1.808 0.504 0.412 0.424 3.825 0.054
0.053 0.050 1/10 0.345 0.966 0.047 0.042 0.474 0.137 0.128 0.123
3.756 0.053 0.050 0.046 1/2 2.012 1.568 0.047 0.042 1.782 0.666
0.438 0.451 3.747 0.059 0.065 0.062 1/10 0.715 0.460 0.045 0.042
0.608 0.164 0.150 0.149 3.729 0.046 0.051 0.049 1/2 0.044 0.048
0.300 0.046 2.133 0.646 0.547 0.396 3.822 0.047 0.047 0.052 1/10
0.042 0.046 0.109 0.045 0.580 0.154 0.140 0.138 3.556 0.049 0.046
0.048 1/2 AC-4 AC-1 AC-7 1/10 1/2 AC-5 AC-2 AC-8 1/10 1/2 AC-6 AC-3
AC-9 1/10 ELISA test of antibodies produced by the monoclonal
mother clones AC-1, AC-2, AC-3, AC-4, AC-5, AC-6, AC-7, AC-8, AC-9.
Supernatants were obtained from nine hybridoma mother clones (AC-1
to -9) and analysed for their binding to MAP-BAG3 constructs.
[0115] FIG. 13 Binding of BAG3-specific polvclonal and monoclonal
antibodies to proteins from HeLa or Primary acute leukemia cells.
A--Lysates from HeLa or primary acute leukemia cells were analysed
in Western blotting using AC-BAG3-1 (central lanes: 3 and 4) or
AC-BAG3-2 (lanes 1, 2 and 5) antibodies (A). B--Supernatants from
the hybridoma mother clones AC-1 (1), AC-2 (2), AC-3 (3) or AC-4
(4) were analysed for their binding to proteins from HeLa cells in
Western blotting.
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Sequence CWU 1
1
1812533DNAHomo sapiensCDS(307)..(2034)Human BAG3 gene sequence NCBI
Pub Med Accession Number XM_055575 Homo sapiens BCL2-associated
athanogene 3 (BAG3) 1gcggagctcc gcatccaacc ccgggccgcg gccaactttt
ttggactgga ccagaagttt 60ctagccggcc agttgctacc tccctttatc tcctccttcc
cctctggcag cgaggaggct 120atttccagac acttccaccc ctctctggcc
acgtcacccc cgcctttaat tcataaaggt 180gcccggcgcc ggcttcccgg
acacgtcggc ggcggagagg ggcccacggc ggcggcccgg 240ccagagactc
ggcgcccgga gccagcgccc cgcacccgcg ccccagcggg cagaccccaa 300cccagc
atg agc gcc gcc acc cac tcg ccc atg atg cag gtg gcg tcc 348Met Ser
Ala Ala Thr His Ser Pro Met Met Gln Val Ala Ser1 5 10ggc aac ggt
gac cgc gac cct ttg ccc ccc gga tgg gag atc aag atc 396Gly Asn Gly
Asp Arg Asp Pro Leu Pro Pro Gly Trp Glu Ile Lys Ile15 20 25 30gac
ccg cag acc ggc tgg ccc ttc ttc gtg gac cac aac agc cgc acc 444Asp
Pro Gln Thr Gly Trp Pro Phe Phe Val Asp His Asn Ser Arg Thr 35 40
45act acg tgg aac gac ccg cgc gtg ccc tct gag ggc ccc aag gag act
492Thr Thr Trp Asn Asp Pro Arg Val Pro Ser Glu Gly Pro Lys Glu Thr
50 55 60cca tcc tct gcc aat ggc cct tcc cgg gag ggc tct agg ctg ccg
cct 540Pro Ser Ser Ala Asn Gly Pro Ser Arg Glu Gly Ser Arg Leu Pro
Pro 65 70 75gct agg gaa ggc cac cct gtg tac ccc cag ctc cga cca ggc
tac att 588Ala Arg Glu Gly His Pro Val Tyr Pro Gln Leu Arg Pro Gly
Tyr Ile 80 85 90ccc att cct gtg ctc cat gaa ggc gct gag aac cgg cag
gtg cac cct 636Pro Ile Pro Val Leu His Glu Gly Ala Glu Asn Arg Gln
Val His Pro95 100 105 110ttc cat gtc tat ccc cag cct ggg atg cag
cga ttc cga act gag gcg 684Phe His Val Tyr Pro Gln Pro Gly Met Gln
Arg Phe Arg Thr Glu Ala 115 120 125gca gca gcg gct cct cag agg tcc
cag tca cct ctg cgg ggc atg cca 732Ala Ala Ala Ala Pro Gln Arg Ser
Gln Ser Pro Leu Arg Gly Met Pro 130 135 140gaa acc act cag cca gat
aaa cag tgt gga cag gtg gca gcg gcg gcg 780Glu Thr Thr Gln Pro Asp
Lys Gln Cys Gly Gln Val Ala Ala Ala Ala 145 150 155gca gcc cag ccc
cca gcc tcc cac gga cct gag cgg tcc cag tct cca 828Ala Ala Gln Pro
Pro Ala Ser His Gly Pro Glu Arg Ser Gln Ser Pro 160 165 170gct gcc
tct gac tgc tca tcc tca tcc tcc tcg gcc agc ctg cct tcc 876Ala Ala
Ser Asp Cys Ser Ser Ser Ser Ser Ser Ala Ser Leu Pro Ser175 180 185
190tcc ggc agg agc agc ctg ggc agt cac cag ctc ccg cgg ggg tac atc
924Ser Gly Arg Ser Ser Leu Gly Ser His Gln Leu Pro Arg Gly Tyr Ile
195 200 205tcc att ccg gtg ata cac gag cag aac gtt acc cgg cca gca
gcc cag 972Ser Ile Pro Val Ile His Glu Gln Asn Val Thr Arg Pro Ala
Ala Gln 210 215 220ccc tcc ttc cac caa gcc cag aag acg cac tac cca
gcg cag cag ggg 1020Pro Ser Phe His Gln Ala Gln Lys Thr His Tyr Pro
Ala Gln Gln Gly 225 230 235gag tac cag acc cac cag cct gtg tac cac
aag atc cag ggg gat gac 1068Glu Tyr Gln Thr His Gln Pro Val Tyr His
Lys Ile Gln Gly Asp Asp 240 245 250tgg gag ccc cgg ccc ctg cgg gcg
gca tcc ccg ttc agg tca tct gtc 1116Trp Glu Pro Arg Pro Leu Arg Ala
Ala Ser Pro Phe Arg Ser Ser Val255 260 265 270cag ggt gca tcg agc
cgg gag ggc tca cca gcc agg agc agc acg cca 1164Gln Gly Ala Ser Ser
Arg Glu Gly Ser Pro Ala Arg Ser Ser Thr Pro 275 280 285ctc cac tcc
ccc tcg ccc atc cgt gtg cac acc gtg gtc gac agg cct 1212Leu His Ser
Pro Ser Pro Ile Arg Val His Thr Val Val Asp Arg Pro 290 295 300cag
cag ccc atg acc cat cga gaa act gca cct gtt tcc cag cct gaa 1260Gln
Gln Pro Met Thr His Arg Glu Thr Ala Pro Val Ser Gln Pro Glu 305 310
315aac aaa cca gaa agt aag cca ggc cca gtt gga cca gaa ctc cct cct
1308Asn Lys Pro Glu Ser Lys Pro Gly Pro Val Gly Pro Glu Leu Pro Pro
320 325 330gga cac atc cca att caa gtg atc cgc aaa gag gtg gat tct
aaa cct 1356Gly His Ile Pro Ile Gln Val Ile Arg Lys Glu Val Asp Ser
Lys Pro335 340 345 350gtt tcc cag aag ccc cca cct ccc tct gag aag
gta gag gtg aaa gtt 1404Val Ser Gln Lys Pro Pro Pro Pro Ser Glu Lys
Val Glu Val Lys Val 355 360 365ccc cct gct cca gtt cct tgt cct cct
ccc agc cct ggc cct tct gct 1452Pro Pro Ala Pro Val Pro Cys Pro Pro
Pro Ser Pro Gly Pro Ser Ala 370 375 380gtc ccc tct tcc ccc aag agt
gtg gct aca gaa gag agg gca gcc ccc 1500Val Pro Ser Ser Pro Lys Ser
Val Ala Thr Glu Glu Arg Ala Ala Pro 385 390 395agc act gcc cct gca
gaa gct aca cct cca aaa cca gga gaa gcc gag 1548Ser Thr Ala Pro Ala
Glu Ala Thr Pro Pro Lys Pro Gly Glu Ala Glu 400 405 410gct ccc cca
aaa cat cca gga gtg ctg aaa gtg gaa gcc atc ctg gag 1596Ala Pro Pro
Lys His Pro Gly Val Leu Lys Val Glu Ala Ile Leu Glu415 420 425
430aag gtg cag ggg ctg gag cag gct gta gac aac ttt gaa ggc aag aag
1644Lys Val Gln Gly Leu Glu Gln Ala Val Asp Asn Phe Glu Gly Lys Lys
435 440 445act gac aaa aag tac ctg atg atc gaa gag tat ttg acc aaa
gag ctg 1692Thr Asp Lys Lys Tyr Leu Met Ile Glu Glu Tyr Leu Thr Lys
Glu Leu 450 455 460ctg gcc ctg gat tca gtg gac ccc gag gga cga gcc
gat gtg cgt cag 1740Leu Ala Leu Asp Ser Val Asp Pro Glu Gly Arg Ala
Asp Val Arg Gln 465 470 475gcc agg aga gac ggt gtc agg aag gtt cag
acc atc ttg gaa aaa ctt 1788Ala Arg Arg Asp Gly Val Arg Lys Val Gln
Thr Ile Leu Glu Lys Leu 480 485 490gaa cag aaa gcc att gat gtc cca
ggt caa gtc cag gtc tat gaa ctc 1836Glu Gln Lys Ala Ile Asp Val Pro
Gly Gln Val Gln Val Tyr Glu Leu495 500 505 510cag ccc agc aac ctt
gaa gca gat cag cca ctg cag gca atc atg gag 1884Gln Pro Ser Asn Leu
Glu Ala Asp Gln Pro Leu Gln Ala Ile Met Glu 515 520 525atg ggt gcc
gtg gca gca gac aag ggc aag aaa aat gct gga aat gca 1932Met Gly Ala
Val Ala Ala Asp Lys Gly Lys Lys Asn Ala Gly Asn Ala 530 535 540gaa
gat ccc cac aca gaa acc cag cag cca gaa gcc aca gca gca gcg 1980Glu
Asp Pro His Thr Glu Thr Gln Gln Pro Glu Ala Thr Ala Ala Ala 545 550
555act tca aac ccc agc agc atg aca gac acc cct ggt aac cca gca gca
2028Thr Ser Asn Pro Ser Ser Met Thr Asp Thr Pro Gly Asn Pro Ala Ala
560 565 570ccg tag cctctgccct gtaaaaatca gactcggaac cgatgtgtgc
tttagggaat 2084Pro575tttaagttgc atgcatttca gagactttaa gtcagttggt
ttttattagc tgcttggtat 2144gcagtaactt gggtggaggc aaaacactaa
taaaagggct aaaaaggaaa atgatgcttt 2204tcttctatat tcttactctg
tacaaataaa gaagttgctt gttgtttcag aagtttaacc 2264ccgttgcttg
ttctgcagcc ctgtctactt gggcaccccc accacctgtt agctgtggtt
2324gtgcactgtc ttttgtagct ctggactgga ggggtagatg gggagtcaat
tacccatcac 2384ataaatatga aacatttatc agaaatgttg ccattttaat
gagatgattt tcttcatctc 2444ataattaaaa tacctgactt tagagagagt
aaaatgtgcc aggagccata ggaatatctg 2504tatgttggat gactttaatg
ctacatttt 25332575PRTHomo sapiens 2Met Ser Ala Ala Thr His Ser Pro
Met Met Gln Val Ala Ser Gly Asn1 5 10 15Gly Asp Arg Asp Pro Leu Pro
Pro Gly Trp Glu Ile Lys Ile Asp Pro 20 25 30 Gln Thr Gly Trp Pro
Phe Phe Val Asp His Asn Ser Arg Thr Thr Thr 35 40 45Trp Asn Asp Pro
Arg Val Pro Ser Glu Gly Pro Lys Glu Thr Pro Ser 50 55 60Ser Ala Asn
Gly Pro Ser Arg Glu Gly Ser Arg Leu Pro Pro Ala Arg65 70 75 80Glu
Gly His Pro Val Tyr Pro Gln Leu Arg Pro Gly Tyr Ile Pro Ile 85 90
95Pro Val Leu His Glu Gly Ala Glu Asn Arg Gln Val His Pro Phe His
100 105 110 Val Tyr Pro Gln Pro Gly Met Gln Arg Phe Arg Thr Glu Ala
Ala Ala 115 120 125Ala Ala Pro Gln Arg Ser Gln Ser Pro Leu Arg Gly
Met Pro Glu Thr 130 135 140Thr Gln Pro Asp Lys Gln Cys Gly Gln Val
Ala Ala Ala Ala Ala Ala145 150 155 160Gln Pro Pro Ala Ser His Gly
Pro Glu Arg Ser Gln Ser Pro Ala Ala 165 170 175Ser Asp Cys Ser Ser
Ser Ser Ser Ser Ala Ser Leu Pro Ser Ser Gly 180 185 190 Arg Ser Ser
Leu Gly Ser His Gln Leu Pro Arg Gly Tyr Ile Ser Ile 195 200 205Pro
Val Ile His Glu Gln Asn Val Thr Arg Pro Ala Ala Gln Pro Ser 210 215
220Phe His Gln Ala Gln Lys Thr His Tyr Pro Ala Gln Gln Gly Glu
Tyr225 230 235 240Gln Thr His Gln Pro Val Tyr His Lys Ile Gln Gly
Asp Asp Trp Glu 245 250 255Pro Arg Pro Leu Arg Ala Ala Ser Pro Phe
Arg Ser Ser Val Gln Gly 260 265 270 Ala Ser Ser Arg Glu Gly Ser Pro
Ala Arg Ser Ser Thr Pro Leu His 275 280 285Ser Pro Ser Pro Ile Arg
Val His Thr Val Val Asp Arg Pro Gln Gln 290 295 300Pro Met Thr His
Arg Glu Thr Ala Pro Val Ser Gln Pro Glu Asn Lys305 310 315 320Pro
Glu Ser Lys Pro Gly Pro Val Gly Pro Glu Leu Pro Pro Gly His 325 330
335Ile Pro Ile Gln Val Ile Arg Lys Glu Val Asp Ser Lys Pro Val Ser
340 345 350 Gln Lys Pro Pro Pro Pro Ser Glu Lys Val Glu Val Lys Val
Pro Pro 355 360 365Ala Pro Val Pro Cys Pro Pro Pro Ser Pro Gly Pro
Ser Ala Val Pro 370 375 380Ser Ser Pro Lys Ser Val Ala Thr Glu Glu
Arg Ala Ala Pro Ser Thr385 390 395 400Ala Pro Ala Glu Ala Thr Pro
Pro Lys Pro Gly Glu Ala Glu Ala Pro 405 410 415Pro Lys His Pro Gly
Val Leu Lys Val Glu Ala Ile Leu Glu Lys Val 420 425 430 Gln Gly Leu
Glu Gln Ala Val Asp Asn Phe Glu Gly Lys Lys Thr Asp 435 440 445Lys
Lys Tyr Leu Met Ile Glu Glu Tyr Leu Thr Lys Glu Leu Leu Ala 450 455
460Leu Asp Ser Val Asp Pro Glu Gly Arg Ala Asp Val Arg Gln Ala
Arg465 470 475 480Arg Asp Gly Val Arg Lys Val Gln Thr Ile Leu Glu
Lys Leu Glu Gln 485 490 495Lys Ala Ile Asp Val Pro Gly Gln Val Gln
Val Tyr Glu Leu Gln Pro 500 505 510 Ser Asn Leu Glu Ala Asp Gln Pro
Leu Gln Ala Ile Met Glu Met Gly 515 520 525Ala Val Ala Ala Asp Lys
Gly Lys Lys Asn Ala Gly Asn Ala Glu Asp 530 535 540Pro His Thr Glu
Thr Gln Gln Pro Glu Ala Thr Ala Ala Ala Thr Ser545 550 555 560Asn
Pro Ser Ser Met Thr Asp Thr Pro Gly Asn Pro Ala Ala Pro 565 570
5753360DNAHomo sapiensmisc_feature(1)..(360)Specific sequence
comprised inside BAG3 gene sequence 3gcggagctcc gcatccaacc
ccgggccgcg gccaactttt ttggactgga ccagaagttt 60ctagccggcc agttgctacc
tccctttatc tcctccttcc cctctggcag cgaggaggct 120atttccagac
acttccaccc ctctctggcc acgtcacccc cgcctttaat tcataaaggt
180gcccggcgcc ggcttcccgg acacgtcggc ggcggagagg ggcccacggc
ggcggcccgg 240ccagagactc ggcgcccgga gccagcgccc cgcacccgcg
ccccagcggg cagaccccaa 300cccagcatga gcgccgccac ccactcgccc
atgatgcagg tggcgtccgg caacggtgac 360435PRTHomo
sapiensPEPTIDE(1)..(35)Specific sequence comprised inside BAG3
protein 4Met Ser Ala Ala Thr His Ser Pro Met Met Gln Val Ala Ser
Gly Asn1 5 10 15Gly Asp Arg Asp Pro Leu Pro Pro Gly Trp Glu Ile Lys
Ile Asp Pro 20 25 30 Gln Thr Gly 3551105DNAHomo
sapiensmisc_feature(1)..(1105)Specific sequence comprised inside
BAG3 gene sequence 5gtgccctctg agggccccaa ggagactcca tcctctgcca
atggcccttc ccgggagggc 60tctaggctgc cgcctgctag ggaaggccac cctgtgtacc
cccagctccg accaggctac 120attcccattc ctgtgctcca tgaaggcgct
gagaaccggc aggtgcaccc tttccatgtc 180tatccccagc ctgggatgca
gcgattccga actgaggcgg cagcagcggc tcctcagagg 240tcccagtcac
ctctgcgggg catgccagaa accactcagc cagataaaca gtgtggacag
300gtggcagcgg cggcggcagc ccagccccca gcctcccacg gacctgagcg
gtcccagtct 360ccagctgcct ctgactgctc atcctcatcc tcctcggcca
gcctgccttc ctccggcagg 420agcagcctgg gcagtcacca gctcccgcgg
gggtacatct ccattccggt gatacacgag 480cagaacgtta cccggccagc
agcccagccc tccttccacc aagcccagaa gacgcactac 540ccagcgcagc
agggggagta ccagacccac cagcctgtgt accacaagat ccagggggat
600gactgggagc cccggcccct gcgggcggca tccccgttca ggtcatctgt
ccagggtgca 660tcgagccggg agggctcacc agccaggagc agcacgccac
tccactcccc ctcgcccatc 720cgtgtgcaca ccgtggtcga caggcctcag
cagcccatga cccatcgaga aactgcacct 780gtttcccagc ctgaaaacaa
accagaaagt aagccaggcc cagttggacc agaactccct 840cctggacaca
tcccaattca agtgatccgc aaagaggtgg attctaaacc tgtttcccag
900aagcccccac ctccctctga gaaggtagag gtgaaagttc cccctgctcc
agttccttgt 960cctcctccca gccctggccc ttctgctgtc ccctcttccc
ccaagagtgt ggctacagaa 1020gagagggcag cccccagcac tgcccctgca
gaagctacac ctccaaaacc aggagaagcc 1080gaggctcccc caaaacatcc aggag
11056395PRTHomo sapiensPEPTIDE(1)..(395)Specific sequence comprised
inside BAG3 protein 6Asn Asp Pro Arg Val Pro Ser Glu Gly Pro Lys
Glu Thr Pro Ser Ser1 5 10 15Ala Asn Gly Pro Ser Arg Glu Gly Ser Arg
Leu Pro Pro Ala Arg Glu 20 25 30Gly His Pro Val Tyr Pro Gln Leu Arg
Pro Gly Tyr Ile Pro Ile Pro 35 40 45 Val Leu His Glu Gly Ala Glu
Asn Arg Gln Val His Pro Phe His Val 50 55 60Tyr Pro Gln Pro Gly Met
Gln Arg Phe Arg Thr Glu Ala Ala Ala Ala65 70 75 80Ala Pro Gln Arg
Ser Gln Ser Pro Leu Arg Gly Met Pro Glu Thr Thr 85 90 95Gln Pro Asp
Lys Gln Cys Gly Gln Val Ala Ala Ala Ala Ala Ala Gln 100 105 110 Pro
Pro Ala Ser His Gly Pro Glu Arg Ser Gln Ser Pro Ala Ala Ser 115 120
125Asp Cys Ser Ser Ser Ser Ser Ser Ala Ser Leu Pro Ser Ser Gly Arg
130 135 140Ser Ser Leu Gly Ser His Gln Leu Pro Arg Gly Tyr Ile Ser
Ile Pro145 150 155 160Val Ile His Glu Gln Asn Val Thr Arg Pro Ala
Ala Gln Pro Ser Phe 165 170 175His Gln Ala Gln Lys Thr His Tyr Pro
Ala Gln Gln Gly Glu Tyr Gln 180 185 190 Thr His Gln Pro Val Tyr His
Lys Ile Gln Gly Asp Asp Trp Glu Pro 195 200 205Arg Pro Leu Arg Ala
Ala Ser Pro Phe Arg Ser Ser Val Gln Gly Ala 210 215 220Ser Ser Arg
Glu Gly Ser Pro Ala Arg Ser Ser Thr Pro Leu His Ser225 230 235
240Pro Ser Pro Ile Arg Val His Thr Val Val Asp Arg Pro Gln Gln Pro
245 250 255Met Thr His Arg Glu Thr Ala Pro Val Ser Gln Pro Glu Asn
Lys Pro 260 265 270 Glu Ser Lys Pro Gly Pro Val Gly Pro Glu Leu Pro
Pro Gly His Ile 275 280 285Pro Ile Gln Val Ile Arg Lys Glu Val Asp
Ser Lys Pro Val Ser Gln 290 295 300Lys Pro Pro Pro Pro Ser Glu Lys
Val Glu Val Lys Val Pro Pro Ala305 310 315 320Pro Val Pro Cys Pro
Pro Pro Ser Pro Gly Pro Ser Ala Val Pro Ser 325 330 335Ser Pro Lys
Ser Val Ala Thr Glu Glu Arg Ala Ala Pro Ser Thr Ala 340 345 350 Pro
Ala Glu Ala Thr Pro Pro Lys Pro Gly Glu Ala Glu Ala Pro Pro 355 360
365Lys His Pro Gly Val Leu Lys Val Glu Ala Ile Leu Glu Lys Val Gln
370 375 380Gly Leu Glu Gln Ala Val Asp Asn Phe Glu Gly385 390
3957733DNAHomo sapiensmisc_feature(1)..(733)Specific sequence
comprised inside BAG3 gene sequence 7attgatgtcc caggtcaagt
ccaggtctat gaactccagc ccagcaacct tgaagcagat 60cagccactgc aggcaatcat
ggagatgggt gccgtggcag cagacaaggg caagaaaaat 120gctggaaatg
cagaagatcc ccacacagaa acccagcagc cagaagccac agcagcagcg
180acttcaaacc ccagcagcat gacagacacc cctggtaacc cagcagcacc
gtagcctctg 240ccctgtaaaa atcagactcg gaaccgatgt gtgctttagg
gaattttaag ttgcatgcat 300ttcagagact ttaagtcagt tggtttttat
tagctgcttg gtatgcagta acttgggtgg 360aggcaaaaca ctaataaaag
ggctaaaaag
gaaaatgatg cttttcttct atattcttac 420tctgtacaaa taaagaagtt
gcttgttgtt tcagaagttt aaccccgttg cttgttctgc 480agccctgtct
acttgggcac ccccaccacc tgttagctgt ggttgtgcac tgtcttttgt
540agctctggac tggaggggta gatggggagt caattaccca tcacataaat
atgaaacatt 600tatcagaaat gttgccattt taatgagatg attttcttca
tctcataatt aaaatacctg 660actttagaga gagtaaaatg tgccaggagc
cataggaata tctgtatgtt ggatgacttt 720aatgctacat ttt 733867PRTHomo
sapiensPEPTIDE(1)..(67)Specific sequence comprised inside BAG3
protein 8Glu Leu Gln Pro Ser Asn Leu Glu Ala Asp Gln Pro Leu Gln
Ala Ile1 5 10 15Met Glu Met Gly Ala Val Ala Ala Asp Lys Gly Lys Lys
Asn Ala Gly 20 25 30Asn Ala Glu Asp Pro His Thr Glu Thr Gln Gln Pro
Glu Ala Thr Ala 35 40 45Ala Ala Thr Ser Asn Pro Ser Ser Met Thr Asp
Thr Pro Gly Asn Pro 50 55 60Ala Ala Pro65925DNAHomo
sapiensmisc_feature(1)..(25)BAG3-based specific antisense
oligonucleotide 9tgcatcatgg gcgagtgggt ggcgg 251022DNAHomo
sapiensmisc_feature(1)..(22)BAG3-based specific antisense
oligonucleotide 10gctcatgctg ggttggggtc tg 221122DNAHomo
sapiensmisc_feature(1)..(22)BAG3-based specific antisense
oligonucleotide 11attaaaggcg ggggtgacgt gg 221227DNAHomo
sapiensmisc_feature(1)..(27)BAG3-based specific control nonsense
oligonucleotide 12ttatattcta ttatatttat gaactcc 271322DNAHomo
sapiensmisc_feature(1)..(22)BAG3-based specific control nonsense
oligonucleotide 13cctcgtaacc accgacctca at 221422DNAHomo
sapiensmisc_feature(1)..(22)BAG3-based specific control nonsense
oligonucleotide 14gcttatggag gattgaggtt gg 221516PRTHomo
sapiensPEPTIDE(1)..(16)BAG3-protein specific epitope 15Asp Arg Asp
Pro Leu Pro Pro Gly Trp Glu Ile Lys Ile Asp Pro Gln1 5 10
151615PRTHomo sapiensPEPTIDE(1)..(15)BAG3-protein specific epitope
16Ser Ser Pro Lys Ser Val Ala Thr Glu Glu Arg Ala Ala Pro Ser1 5 10
151715PRTHomo sapiensPEPTIDE(1)..(15)BAG3-protein specific epitope
17Asp Lys Gly Lys Lys Asn Ala Gly Asn Ala Glu Asp Pro His Thr1 5 10
151815PRTHomo sapiensPEPTIDE(1)..(15)BAG3-protein specific epitope
18Asn Pro Ser Ser Met Thr Asp Thr Pro Gly Asn Pro Ala Ala Pro1 5 10
15
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