U.S. patent application number 17/122333 was filed with the patent office on 2021-04-29 for humanized anti-bag3 antibodies.
The applicant listed for this patent is BIOUNIVERSA S.R.L.. Invention is credited to Vincenzo DE LAURENZI, Alessandra ROSATI, Gianluca SALA, Maria Caterina TURCO.
Application Number | 20210122818 17/122333 |
Document ID | / |
Family ID | 1000005329191 |
Filed Date | 2021-04-29 |
![](/patent/app/20210122818/US20210122818A1-20210429\US20210122818A1-2021042)
United States Patent
Application |
20210122818 |
Kind Code |
A1 |
TURCO; Maria Caterina ; et
al. |
April 29, 2021 |
HUMANIZED ANTI-BAG3 ANTIBODIES
Abstract
The present invention provides for a humanized anti-BAG3
antibody or a fragment thereof, pharmaceutical compositions
comprising said antibody and its use as a medicament, in particular
for use in the treatment of pancreatic tumours or other pathologies
of an immune, inflammatory, neoplastic, cardiovascular and/or
degenerative nature.
Inventors: |
TURCO; Maria Caterina;
(Avellino (AV), IT) ; ROSATI; Alessandra;
(Baronissi (SA), IT) ; DE LAURENZI; Vincenzo;
(Pescara (PE), IT) ; SALA; Gianluca; (Pescara
(PE), IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOUNIVERSA S.R.L. |
Montoro |
|
IT |
|
|
Family ID: |
1000005329191 |
Appl. No.: |
17/122333 |
Filed: |
December 15, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15770690 |
Apr 24, 2018 |
10889644 |
|
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PCT/EP2016/076384 |
Nov 2, 2016 |
|
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17122333 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/28 20130101;
C07K 2317/24 20130101; A61K 9/0029 20130101; A61K 39/3955 20130101;
C07K 2317/92 20130101; A61K 9/0053 20130101; A61K 45/06 20130101;
A61K 2039/505 20130101; C07K 2317/76 20130101; C07K 16/18 20130101;
A61K 9/0014 20130101; A61P 35/00 20180101; C07K 2317/565 20130101;
C07K 16/30 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/18 20060101 C07K016/18; C07K 16/30 20060101
C07K016/30; A61P 35/00 20060101 A61P035/00; A61K 9/00 20060101
A61K009/00; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101
A61K045/06 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 5, 2015 |
IT |
UB2015A005097 |
Claims
1. A humanized antibody or a fragment thereof which binds to the
BAG3 protein, comprising: a) a heavy chain having the amino acid
sequence of SEQ ID NO: 12 or an amino acid sequence having at least
95% sequence identity thereof, b) a light chain having the amino
acid sequence of SEQ ID NO: 20 or an amino acid sequence having at
least 95% sequence identity thereof.
2. The humanized antibody or a fragment thereof according to claim
1, which is a Fab fragment, a Fab' fragment, a F(ab') fragment, a
Fv fragment, a diabody, a ScFv, a small modular
immunopharmaceutical (SMIP), an affibody, an avimer, a nanobody, a
domain antibody, or single chains.
3. A nucleic acid encoding the antibody or a fragment thereof
according to claim 1.
4. A vector comprising the nucleic acid according to claim 3.
5. A host cell comprising the nucleic acid of claim 3.
6. A pharmaceutical composition comprising at least one antibody or
a fragment thereof according to claim 1 and at least one
pharmaceutically acceptable excipient or carrier.
7. The pharmaceutical composition according to claim 6, in a form
of oral formulation, parenteral formulation, or topical
administration.
8. The pharmaceutical composition according to claim 6, further
comprising antimetabolites, camptothecins, or taxanes.
9. A method for treating a pathological state which involves the
activation of macrophages, selected from the group consisting of:
neoplastic diseases, inflammatory diseases, immune diseases and
degenerative diseases, comprising administering the humanized
antibody or a fragment thereof according to claim 1 to a subject in
need thereof.
10. The method according to claim 9, for treating pancreatic tumor
or bladder tumor.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 15/770,690, filed Apr. 24, 2018; which is a National Stage of
International Application PCT/EP2016/076384, filed Nov. 2, 2016,
published May 11, 2017, under PCT Article 21(2) in English; which
claims the priority of Italian Application No. UB2015A005097, filed
Nov. 5, 2015. The contents of the above-identified applications are
incorporated herein by reference in their entireties.
REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM
[0002] The Sequence Listing is concurrently submitted herewith with
the specification as an ASCII formatted text file via EFS-Web with
a file name of Sequence_Listing_12-31-2020.txt with a creation date
of Dec. 31, 2020, and a size of 39 kilobytes. The Sequence Listing
filed via EFS-Web is part of the specification and is hereby
incorporated in its entirety by reference herein.
DESCRIPTION
[0003] The present invention provides for a humanized anti-BAG3
antibody or a fragment thereof, pharmaceutical compositions
comprising said antibody and its use as a medicament, in particular
for use in the treatment of pancreatic tumours or other pathologies
of an immune, inflammatory, neoplastic, cardiovascular and/or
degenerative nature.
BACKGROUND OF THE INVENTION
[0004] BAG3 protein is a 74 kDa cytoplasmic protein which belongs
to the family of co-chaperons that interact with the ATPase domain
of the protein HSP70 (Heat Shock Protein) through the structural
domain known as the BAG domain (amino acids 110-124). Furthermore,
BAG3 protein contains a WW domain (Trp-Trp), a proline-rich region
(PXXP), and two conserved motifs IPV (Ile-Pro-Val), which can
mediate binding to other proteins. Thanks to the nature of BAG3
protein as an adapter, attributable to the presence of many
functional domains, such protein can therefore interact with
different proteins.
[0005] In humans, bag3 gene expression is constitutive for a few
kinds of normal cells, including myocytes, while mutations thereof
are associated with diseases of the skeletal and cardiac muscles.
Furthermore, BAG3 protein is expressed in many types of primary
tumours or tumour cell lines (lymphoid or myeloid leukemias,
neuroblastoma, pancreatic cancer, thyroid cancer, breast cancer and
prostate cancer, melanoma, osteosarcoma, glioblastoma and tumours
of the kidney, colon, ovary, etc.) (Rosati A. et al., 2011).
[0006] In normal cell types, such as leukocytes, epithelial cells
and glial cells and cells of the retina, bag3 gene expression can
be induced by stressors, such as oxidants, high temperatures, lack
of serum, heavy metals, HIV-1 infections, etc. These findings
indicate that bag3 gene expression regulation is an important
component in the cellular response to stress and is correlated with
the presence of elements that respond to the transcription factor
HSF1 (Heat Shock Transcription Factor), which is activated in
various forms of cellular stress in bag3 gene promoter
(Franceschelli S. et al., 2008).
[0007] Moreover, due to the presence of many protein-protein
interaction domains in the structure thereof, BAG3 protein
influences cell survival in different types of cells, interacting
with different molecular partners. (Rosati A. et al., 2011). The
first mechanism reported in relation to BAG3 anti-apoptotic
activity was identified in osteosarcoma and melanoma cells, where
it was observed that BAG3 protein modulates the activation of
transcription factor NF-kB and cell survival (Ammirante M. et al.,
2010). A different molecular mechanism has been described in
glioblastoma cells, where BAG3 protein cooperates in a positive way
with HSP70 protein to maintain BAX protein in the cytosol and
prevent the translocation thereof into the mitochondria (Festa M.
et al., 2011). Finally, in some tumours, BAG3 has been shown to
regulate proteins that modulate cell adhesion.
[0008] The presence of cytoplasmic BAG3 protein has also been
described in many different cellular systems and has been
associated, not only with various tumours, but also in pathologies
in general related to cell survival.
[0009] Furthermore, patent application n. WO2011/067377 describes
extracellular BAG3 protein, secreted by some cell types, as a
biochemical marker in serum, which is highly specific for the
diagnosis of certain pathological conditions, such as cardiac
pathologies and pancreatic tumour.
[0010] It has recently been reported that BAG3 protein is expressed
in 346/346 patients with pancreatic ductal adenocarcinoma (PDAC)
and is released by the cells of the pancreatic tumour, but such
protein is not expressed in either the surrounding non-neoplastic
tissues or in a normal pancreas; likewise, it has been reported
that the levels of BAG3 expression are related to patient survival.
The results of the study demonstrate that the use of specific siRNA
molecules for BAG3 mRNA can silence bag3 gene expression and induce
cell death, confirming that BAG3 protein is an important survival
factor for pancreatic tumour cells and that the down-regulation
thereof, when combined with gemcitabine, may contribute to the
eradication of the tumor cells (Rosati A. et al., 2012).
[0011] Moreover, in a recent paper we have reported that
PDAC-released BAG3 binds macrophages inducing their activation and
the secretion of PDAC supporting factors. We have also identified
IFITM-2 as a BAG3 receptor and showed that it signals through PI3K
and the p38 MAPK pathways. Finally, we have showed that the use of
a mouse monoclonal anti-BAG3 antibody results in reduced tumor
growth and prevents metastasis formation in three different mouse
models. We have therefore identified a paracrine loop involved in
PDAC growth and metastatic spreading, and showed that an anti-BAG3
antibody has therapeutic potential (Rosati A. et al., Nat. Commun.
2015)
[0012] Conventional chemotherapy treatments for tumour pathologies,
as well as treatments of inflammatory and immune diseases with
corticosteroids or NSAIDs (non-steroidal anti-inflammatory drugs)
pose numerous drawbacks linked to side effects and are not, at
present, definitive means of treating such pathologies.
[0013] There is therefore an evident need for a new and improved
therapeutic treatment which has the advantage of being highly
specific and having few or no side effects, as compared with the
conventional, commonly known therapies used for the treatment of
diseases of an inflammatory, immune, and neoplastic nature
described in the present invention.
Definitions
[0014] Unless otherwise defined, all terms of art, notations and
other scientific terminology used herein are intended to have the
meanings commonly understood by those persons skill in the art to
which this disclosure pertains. In some cases, terms with commonly
understood meanings are defined herein for clarity and/or for ready
reference; thus, the inclusion of such definitions herein should
not be construed to represent a substantial difference over what is
generally understood in the art.
[0015] The term "antibody" as used herein includes "fragments" or
"derivatives", which have at least one antigen binding site of the
antibody and/or show the same biological activity.
[0016] An antibody preferably comprises at least one heavy
immunoglobulin chain and at least one light immunoglobulin chain.
An immunoglobulin chain comprises a variable domain and optionally
a constant domain. A variable domain may comprise complementarity
determining regions (CDRs), e.g. a CDR1, CDR2 and/or CDR3 region,
and framework regions.
[0017] The term "humanized antibody" refers to an antibody of human
origin, whose hypervariable region has been replaced by the
homologous region of non-human monoclonal antibodies.
[0018] The term "chimeric antibody" refers to an antibody
containing portions derived from different antibodies.
[0019] The term "recombinant antibody" refers to an antibody
obtained using recombinant DNA methods.
[0020] The term "scFv fragment" (single chain variable fragment)
refers to immunoglobulin fragments only capable of binding with the
antigen concerned. ScFv fragments can also be synthesised into
dimers (diabodies), trimers (triabodies) and tetramers
(tetrabodies) using peptide linkers.
[0021] The terms "Fab fragment" (antigen-binding fragment) and
"Fab2 fragment" refer to immunoglobulin fragments consisting of a
light chain linked to the Fc fragment of the adjacent heavy chain,
and such fragments are monovalent antibodies. When the Fab portions
are in pairs, the fragment is called Fab2.
[0022] The term "hybridoma" refers to a cell producing monoclonal
antibodies.
[0023] The term "monospecific antibodies" refers to antibodies that
all have affinity for the same antigen.
[0024] The term "multispecific antibodies" refers to antibodies
that have affinity for several antigens.
[0025] The term "bispecific antibody" refers to an antibody that
has affinity for two different antigens.
DESCRIPTION OF THE FIGURES
[0026] FIG. 1. ELISA showing the screening for antibody binding
activity with a coating using Peptide 2 (BAG3 fragment).
[0027] FIG. 2. ELISA showing the screening for antibody binding
activity with a coating using recombinant full length BAG3
protein.
[0028] FIG. 3. Flow cytometric evaluation of the ability of the
antibody variants to block BAG3 binding to macrophage surface.
[0029] FIG. 4. Effect of humanized AC-2 antibody variants on tumor
growth in vivo.
[0030] FIG. 5. Effect of humanized AC-2 antibody variant H2L4 on
activated fibroblasts within the tumor mass.
[0031] FIG. 6. Quantification of the AC-2 H2L4 effect on activated
fibroblasts number.
DISCLOSURE OF THE INVENTION
[0032] It has been surprisingly found by the inventors that
specific BAG3 inhibitors are able to induce tumor regression. In
particular, the humanized anti-BAG3 antibodies, tested showed a
particular affinity for BAG3 protein and can be used in therapy as
BAG3 inhibitors, as they block the interaction between BAG3 protein
and its receptor on the macrophage surfaces. Furthermore, the
experimental data reported in the application demonstrates that
said anti-BAG3 antibodies are particularly effective in reducing
the activation of fibroblasts and therefore that they can be used
in the treatment of all the pathologies wherein fibroblasts are
highly activated, such as neoplastic diseases, inflammatory
diseases, immune diseases and/or degenerative diseases.
[0033] Therefore, a first embodiment of the present invention
relates to an antibody or fragment thereof which binds to the BAG3
protein and which comprises:
a) a heavy chain amino acid sequence as encoded by SEQ ID NO: 12 or
at least the variable domain thereof or an amino acid sequence
having a sequence identity of at least 80% thereof, and b) a light
chain amino acid sequence as encoded by SEQ ID NO: 20 or at least
the variable domain thereof or an amino acid sequence having a
sequence identity of at least 80% thereof. As used herein,
"sequence identity" between two polypeptide sequences, indicates
the percentage of amino acids that are identical between the
sequences, preferably over the entire length of the amino acid
sequences as encoded by SEQ ID NO: 12 and SEQ ID NO: 20. Preferred
polypeptide sequences of the invention have a sequence identity of
at least 85%, more preferably 90%, even more preferably 93%, 95%,
96%, 97%, 98% or 99%.
[0034] In a preferred embodiment of the present invention said
amino acid sequence having a sequence identity of at least 80% with
respect to SEQ ID N. 12 is selected from SEQ ID N. 14, SEQ ID N: 16
or SEQ ID N. 18.
[0035] In a further preferred embodiment said amino acid sequence
having a sequence identity of at least 80% with respect to SEQ ID
N. 20 is selected from SEQ ID N. 22, SEQ ID N: 24 or SEQ ID N.
26.
[0036] In a preferred embodiment the antibody of the present
invention is the antibody wherein the heavy chain amino acid
sequence is encoded by SEQ ID NO. 18 and the light chain amino acid
sequence is encoded by SEQ ID NO 22 or SEQ ID N. 26.
[0037] In a preferred embodiment, the heavy chain amino acid
sequence or at least the variable domain thereof or an amino acid
sequence having a sequence identity of at least 80% thereof,
comprises the CDRs regions having the following amino acid
composition: H-CDR1 comprises the amino acids GFNIKDTYMY (SEQ ID N.
3), H-CDR2 comprises the amino acids GVDPANGNTRYDPKFQG (SEQ ID N.
4), H-CDR3 comprises the amino acids DGAMDY (SEQ ID N. 5) and the
light chain amino acid sequence or at least the variable domain
thereof or an amino acid sequence having a sequence identity of at
least 80% thereof, comprises the CDRs regions having the following
amino acid composition: L-CDR1 comprises the amino acids
KSSQSLLYSSNQKNYLA (SEQ ID N. 6), L-CDR2 comprises the amino acids
WASTRES (SEQ ID N. 7) and L-CDR3 comprises the amino acids
QQYYTYPLT (SEQ ID N. 8).
[0038] A further embodiment of the present invention, is an
antibody or a fragment thereof which binds to the BAG3 protein and
which comprises:
a) a heavy chain nucleotide sequence as encoded by SEQ ID NO: 11 or
at least the variable domain thereof or a nucleotide sequence
having a sequence identity of at least 80% thereof, and b) a light
chain nucleotide sequence as encoded by SEQ ID NO: 19 or at least
the variable domain thereof or a nucleotide sequence having a
sequence identity of at least 80% thereof. As used herein,
"sequence identity" between two nucleotide sequences, indicates the
percentage of nucleotides that are identical between the sequences,
preferably over the entire length of the nucleotide sequences as
encoded by SEQ ID NO: 11 and SEQ ID NO: 19. Preferred nucleotide
sequences of the invention have a sequence identity of at least
85%, more preferably 90%, even more preferably 93%, 95%, 96%, 97%,
98% or 99%.
[0039] In a preferred embodiment of the present invention said
nucleotide sequence having a sequence identity of at least 80% with
respect to SEQ ID N. 11 is selected from SEQ ID N. 13, SEQ ID N: 15
or SEQ ID N. 17.
[0040] In a further preferred embodiment said amino acid sequence
having a sequence identity of at least 80% with respect to SEQ ID
N. 19 is selected from SEQ ID N. 21, SEQ ID N: 23 or SEQ ID N.
25.
[0041] In a preferred embodiment the antibody of the present
invention is the antibody wherein the heavy chain amino acid
sequence is encoded by SEQ ID NO. 17 and the light chain amino acid
sequence is encoded by SEQ ID NO 21 or SEQ ID N. 25.
[0042] The antibody or fragments thereof according to the present
invention may be any antibody of natural and/or synthetic origin,
e.g. an antibody of mammalian origin. Preferably, the constant
domain, if present, is a human constant domain. The variable domain
is preferably a mammalian variable domain, e.g. a humanized or a
human variable domain.
[0043] Antibodies or fragments thereof according to the invention
may be polyclonal or monoclonal antibodies. Monoclonal antibodies
are preferred. In particular the antibodies of the present
invention are preferably selected from the group consisting of
recombinant antibodies, humanized or fully human antibodies,
chimeric antibodies, multispecific antibodies, in particular
bispecific antibodies, or fragments thereof.
[0044] Monoclonal antibodies may be produced by any suitable method
such as that of Kohler and Milstein (1975) or by recombinant DNA
methods. Monoclonal antibodies may also be isolated from phage
antibody libraries using techniques described in Clackson et al.
(1991). Humanized forms of the antibodies may be generated
according to the methods known in the art, (Kettleborough C. A. et
al., 1991), such as chimerization or CDR grafting. Alternative
methods for the production of humanized antibodies are well known
in the art and are described in, e.g., EP 0239400 and WO 90/07861.
Human antibodies can also be derived by in vitro methods. Suitable
examples include but are not limited to phage display, yeast
display, and the like.
[0045] According the present invention "chimeric antibody" relates
to antibodies comprising polypeptides from different species, such
as, for example, mouse and human. The production of chimeric
antibodies is described, for example, in WO 89/09622.
[0046] The term antibody includes "fragments" or "derivatives",
which have at least one antigen binding site of the antibody.
[0047] According to a preferred embodiment the antibody or fragment
thereof may be a Fab fragment, a Fab' fragment, a F(ab') fragment,
a Fv fragment, a diabody, a ScFv, a small modular
immunopharmaceutical (SMIP), an affibody, an avimer, a nanobody, a
domain antibody and/or single chains.
[0048] The antibody of the invention may be preferably of the IgG1,
IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE
antibody-type. It will be appreciated that antibodies that are
generated need not initially possess such an isotype but, rather
the antibody as generated can possess any isotype and that the
antibody can be isotype-switched.
[0049] A further embodiment of the present invention is a vector
comprising the nucleic acid coding for the antibody of the
invention. Said vector is selected from a phage, a plasmid, a viral
or a retroviral vector. Preferably, the vector of the invention is
an expression vector wherein the nucleic acid molecule is
operatively linked to one or more control sequences allowing the
transcription and optionally the expression in prokatiotic and/or
eukaryotic host cells.
[0050] A further embodiment of the present invention is a host
comprising the vector of the invention, selected from a prokaryotic
or eukaryotic cell, preferably a mammalian or a human cell, or a
non-human transgenic animal.
[0051] A further embodiment of the present invention is a method
for the preparation of the antibody or a fragment thereof disclosed
above, comprising culturing the host of the invention under
conditions that allow synthesis of said antibody and recovering
said antibody from said culture.
[0052] A further embodiment is an antibody or a fragment thereof
obtained by the method disclosed above.
[0053] Preferably, the antibodies of fragments thereof according to
the present invention are humanized antibodies.
[0054] A further embodiment of the present invention is the use of
the aforesaid antibody or a fragment thereof as medicament,
preferably in the treatment of a particular pathological state
which involves the activation of macrophages. Such pathological
state can be chosen from: neoplastic diseases, inflammatory
diseases, immune diseases or degenerative diseases.
[0055] Preferably, such neoplastic diseases may be either
pancreatic tumour or bladder tumor, more preferably pancreatic
tumour.
[0056] Preferably, said inflammatory diseases can be chosen from
diseases related to inflammation of the skin, nerves, bones, blood
vessels or connective tissues, and more preferably, psoriasis,
arthritis, neuritis or connectivitis.
[0057] Preferably, said immune diseases can be chosen from
autoimmune diseases such as rheumatic diseases, connective tissue
diseases, neuromuscular diseases, endocrine diseases,
gastrointestinal diseases, haematologic diseases, skin diseases or
vasculitis, and more preferably, rheumatoid arthritis, multiple
sclerosis, connectivitis, lupus erythematosus, endometriosis or
ulcerative colitis.
[0058] Preferably, said degenerative diseases can be chosen from
neurodegenerative diseases and muscular degenerative diseases, and
more preferably Alzheimer's disease, Parkinson's disease, or
muscular dystrophy.
[0059] A further aim of the present invention is a pharmaceutical
composition comprising the aforesaid antibody or fragment thereof
in association with at least one pharmaceutically acceptable
excipient or carrier.
[0060] A further embodiment of the present invention is the use of
said composition as a medicament.
[0061] A preferred embodiment of the present invention is the use
of the composition in the treatment of neoplastic diseases and
diseases of an inflammatory, immune and/or degenerative nature,
preferably neoplastic disease, selected from pancreatic tumor or
bladder tumor.
[0062] The composition of the present invention can be formulated
in a form suitable for oral administration or in a form suitable
for parenteral or topical administration.
[0063] In a preferred embodiment of the present invention, said
oral form can be chosen from the following: tablets, capsules,
solutions, suspensions, granules and oily capsules.
[0064] In a further preferred embodiment of the present invention,
said topical form can be chosen from the following: cream,
ointment, ointment, solution, suspension, eye drops, pessary,
nebuliser solution, spray, powder, or gel.
[0065] In a further preferred embodiment of this invention, said
parenteral form can be either an aqueous buffer solution or an oily
suspension.
[0066] Said parenteral administration include administration by
intramuscular, intravenous, intradermal, subcutaneous,
intraperitoneal, intranodal, or intrasplenic means.
[0067] Preferably the pharmaceutical composition according the
present invention comprises a further active principle, selected
from antimetabolites, camptothecins or taxanes.
[0068] More preferably said active principles are selected from:
Gemcitabine, 5-fluorouracil, Irinotecan, Oxaliplatin, Albumin-bound
paclitaxel, Capecitabine, Cisplatin, Paclitaxel, Docetaxel or
Irinotecan liposome.
EXAMPLES
Example 1--Chimerization and Humanization of AC2 Antibody
[0069] AC-2 murine antibody is produced by a hybridoma isolated
from the hybridoma mother clone no PD02009 deposited on the 17 Dec.
2002 at the Centro Biotecnologie Avanzate di Genova and disclosed
in WO03/055908. Total RNA was extracted and RT-PCR performed to
clone and sequence the variable regions of the antibody using
conventional procedures (e.g., by using oligonucleotide probes that
are capable of binding specifically to genes encoding the heavy and
light chains of murine antibodies).
[0070] Based on sequence information of the variable region, heavy
chain and light chain of AC-2 murine antibody (SEQ ID No. 1 and SEQ
ID N. 2 for the amino acid sequences and SEQ ID 9 and SEQ ID N. 10
for the nucleotide sequences), different humanized variants of said
region have been obtained by gene synthesis using standard
procedures.
[0071] Sequences coding for the antibody variants were cloned in
Evi-5 expression vector (Evitria AG, Switzerland) and expressed in
CHO-K1 cells.
[0072] For antibody chimerization, the murine constant regions were
replaced with the human constant regions. One chimeric versions of
the heavy chain (HC) was made in an IgG1 context.
[0073] For antibody humanization, Complementarity Determining
Regions (CDRs) from the murine were grafted into a human antibody
framework.
[0074] Twenty-four humanized versions of the heavy chain (HC) were
made in an IgG1 and LC-kappa context. Each version is characterized
by specific point mutations in the FR.
Example 2--Screening for Antibody Binding Activity
[0075] Chimeric Ab and humanized variants were tested in parallel
for the ability to bind to BAG3, using an indirect ELISA test.
Material and Methods
[0076] 96 well microplate (NUNC Maxisorp) were coated using a
specific sequence (Pep2) within the BAG3 full-length protein (from
aa 385 to aa 399) recognized by the murine antibody (AC-2). The
plate was incubated overnight with 1 .mu.g/ml of Pep2 (50
.mu.l/well) in a phosphate buffer (PBS, pH 7.4). Then, the plate
was washed two times with a detergent solution (0.05% Tween-20 in
PBS), and the blocking of non-specific sites was performed for one
hour at room temperature using, for each well, 150 .mu.l of a
solution of 0.5% fish gelatin (Sigma) in phosphate buffer (PBS, pH
7.4). Plates were washed two times with the washing buffer and then
the antibodies were loaded on. In some of the wells, scalar
dilutions of chimeric and humanized variants of the antibody, 500
ng/ml (50 .mu.l/well) were loaded in duplicates. Antibodies were
diluted in a solution of 0.5% fish gelatin, 0.05% Tween in PBS
(BSA/ITween). The plate was incubated for 2h at room temperature
and then washed for 5 times with washing buffer. Samples were then
incubated 30 minutes at room temperature with 50 .mu.l/well of
HRP-conjugated anti-mouse IgG or HRP-conjugated anti-human IgG as
the secondary antibody. After 6 washes with the washing buffer, the
peroxidase substrate, tetramethylbenzidine (TMB), was added to the
wells (50 .mu.l/well). The colorimetric reaction was blocked after
10 minutes by addition of sulfuric acid 0.5 M (25 .mu.l/well) and
the optic density values (OD) were detected using a
spectrophotometer at the wavelength of 450 nm (FIG. 1). Chimeric
and humanized variants of the antibody were tested also using
plates coated with recombinant BAG3 full-length protein (rBAG3) by
indirect ELISA test using the same protocol described above (FIG.
2).
Results
[0077] Binding of the different antibody variants was detected in
ELISA using peptide 2 as coating (FIG. 1) or recombinant full
length BAG3 (FIG. 2). The following antibody variants showed higher
binding ability and were chosen for further analysis: HIL2, HIL3,
HIL4, H2L2, H2L3, H2L4, H3L2, H3L3, H3L4, H4L2, H4L3, H4L4.
Example 3--Humanized Antibody Variants KD Determination
Materials and Methods
[0078] Binding experiments were performed on Octet Red96 at
25.degree. C. Antibodies were captured on dip and read AHC
(anti-human IgG Fc capture) sensors, followed by binding of Ag (E.
Coli rBAG3) at variable concentrations. Binding of antigen to the
antibodies was monitored in real time to obtain on (ka) and off
(kd) rates. The equilibrium constant (KD) was calculated from the
observed ka and kd.
[0079] Full kinetic analysis was performed using analyte
concentrations from 100 nM to 0. The analysis was performed using
analyte concentrations of 100 nM and run with serial dilutions in
assay buffer, 100, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0 nM. Chi
square (j) analysis was carried out between the actual Sensorgram
(colored line) and the sensorgram generated from the Fortebio Octet
analysis software (red line) to determine the accuracy of the
analysis. 2 value within 1-2 is considered significant (accurate)
and below 1 is highly significant (highly accurate).
[0080] Note: 2.times.8 sensors were used for 1 full kinetic
analysis, sensors were regenerated and used multiple times and
captured with BAG-H-L, BAG-H-L2, BAG-H-L3, BAG-H1-BAG-H2-L2,
BAG-H2-L3, BAG-H2-L4, BAG-H3-L2, BAG-H3-L3, BAG-H3-L4, BAG-H4-L2,
BAG-H4-L3, BAG H4-L4 to perform 13 full kinetics.
Results
[0081] Affinity for the different variants calculated as above is
reported below. The AC-2 murine antibody showed a KD (M) of
10.sup.-12 while all human variants showed a KD (M) of (see Table
1). However, this difference did not impact on variants biological
activity as will be described.
TABLE-US-00001 TABLE 1 Affinity of AC-2 antibody variants e conc.
In Ligand Analyte ka (1/Ms) kd(1/s) K D (M) nM Chi.sup.2 BAG- rBAG3
2.15 .times. 10.sup.5 5.71 .times. 10.sup.-4 2.66 .times. 10.sup.-9
0-50 0.145 H-L BAG- rBAG3 2.23 .times. 10.sup.5 6.35 .times.
10.sup.-4 2.84 .times. 10.sup.-9 0-50 0.174 H1-L2 BAG- rBAG3 2.17
.times. 10.sup.5 6.15 .times. 10.sup.-4 2.83 .times. 10.sup.-9 0-50
0.168 H1-L3 BAG- rBAG3 2.42 .times. 10.sup.5 5.66 .times. 10.sup.-4
2.34 .times. 10.sup.-9 0-50 0.186 H1-L4 BAG- rBAG3 2.46 .times.
10.sup.5 6.47 .times. 10.sup.-4 2.63 .times. 10.sup.-9 0-50 0.514
H2-L2 BAG- rBAG3 2.25 .times. 10.sup.5 4.47 .times. 10.sup.-4 1.98
.times. 10.sup.-9 0-50 0.283 H2-L3 BAG- rBAG3 2.33 .times. 10.sup.5
4.75 .times. 10.sup.-4 2.04 .times. 10.sup.-9 0-50 0.298 H2-L4 BAG-
rBAG3 1.93 .times. 10.sup.5 3.45 .times. 10.sup.-4 1.78 .times.
10.sup.-9 0-50 0.128 H3-L2 BAG- rBAG3 2.05 .times. 10.sup.5 3.35
.times. 10.sup.-4 1.61 .times. 10.sup.-9 0-50 0.128 H3-L3 BAG-
rBAG3 2.29 .times. 10.sup.5 3.91 .times. 10.sup.-4 1.70 .times.
10.sup.-9 0-50 0.270 H3-L4 BAG- rBAG3 1.92 .times. 10.sup.5 3.90
.times. 10.sup.-4 2.02 .times. 10.sup.-9 0-50 0.185 H4-L2 BAG-
rBAG3 2.02 .times. 10.sup.5 4.03 .times. 10.sup.-4 1.98 .times.
10.sup.-9 0-50 0.193 H4-L3 BAG- rBAG3 2.33 .times. 10.sup.5 4.01
.times. 10.sup.-4 1.72 .times. 10.sup.-9 0-50 0.240 H4-L4 indicates
data missing or illegible when filed
Example 4--Evaluation of the Ability of the Antibody Variants to
Block BAG3 Binding to Macrophage Surface
[0082] BAG3 binds to the surface of macrophages and we have shown
that binding is specifically inhibited by the murine anti-BAG AC-2
antibody that sequesters BAG3 protein. Here we tested the ability
of chimeric and humanized variants of the antibody to block binding
to the macrophage surface.
Materials and Methods
[0083] J774 A.1 cells (1.times.106/ml) were incubated with blocking
solution (PBS containing 5% FBS/0.1% NaN3) and with FcR blocking
mouse (Miltenyi Biotec-cod. 130-092-575) (1 .mu.l/1.times.106
cells) for 30 minutes at 4.degree. C. Then, 1.times.105 cells were
incubated with FITC-rBAG3 protein (40 .mu.g/ml) alone or in
presence of anti-BAG3 mouse antibody (AC2) (3200 .mu.g/ml) or
murine IgG1 (3200 .mu.g/ml), or with the chimeric and humanized
variants of the antibody (3200 .mu.g/ml), or human IgG (3200
.mu.g/ml) in blocking solution, for 30 minutes on ice. After
incubation, cells were washed with PBS and analyzed by flow
cytometry.
Results
[0084] Flow cytometric evaluation of fluorescent BAG3 binding to
the surface of J774 A.1 macrophage cell line. Data are reported as
mean fluorescence intensity (FIG. 3). On the basis of these
results, the H4L2, H4L3, H4L4 and H2LA humanized antibody variants
were chosen.
Example 5--Evaluation of the Ability of Humanized AC-2 Antibody
Variants to Block Primary Monocyte Activation
[0085] The H4L2, H4L3, H4L4 and H2L4 humanized antibody variants
were selected, purified by means of Protein A capture (HiTrap
Protein A HP, GE Healthcare), and further tested for their ability
block activation of primary human monocytes.
[0086] We have previously shown that BAG3 can activate primary
human macrophages. Here we use IL6 secretion as a read out of
macrophage activation to show the ability of the H4L2, H4L3, and
H4L4 humanized antibody variants to block BAG3 dependent monocyte
activation.
Materials and Methods
[0087] Buffy Coats (CompoFlex.RTM. Triple "Top and Top"
System--Fresenius Kabi) were obtained from healthy donors and
stored overnight at 4.degree. C. PBMC were isolated on standard
Ficoll-Hypaque density gradients. Mononuclear cells were isolated
and subjected to sequential washes with 50 ml of PBS IX in order to
eliminate platelets and erythrocytes contaminations; (I: at 1100 g
for 20 mins; II: at 800 g for 10 mins; III: at 400 g for 10 mins;
IV: at 300 g for 10 mins; V and VI: at 100 g for 10 mins). All
centrifugations were made with the brake off. Cells were then
checked under the microscope and if contaminations were still
present, an additional centrifugation at 100 g for 10 mins was
performed.
[0088] PBMC were then plated in 96 well microplates
(2.times.10.sup.6 cells/ml) and cultured in RPMI without glutamine
(100 .mu.l/well) without FBS and with FcR Blocking Reagent human (1
.mu.l every 1 million of cells) (Miltenyi 130-059-901) for 16 hours
(overnight).
[0089] The day after, cells were treated for 6h with with rBAG3
(0.5 .mu.g/ml). mAb blocking assays were performed incubating the
rBAG3 together with different concentrations of AC-2 or the
humanized variants.
[0090] Treatment was performed without changing the media to the
cells, but adding 100 .mu.l of RPMI without FBS to each well
containing molecules described above. All molecules were added to
100 .mu.l of RPMI without FBS in a sterile tube and molecules were
added at twice of their final concentration to the cells. Molecules
were pre-incubated for 30 minutes at room temperature in the tubes
and then added to the cells. Pre-incubation is necessary for mAb
blocking assays.
[0091] After treatment cell culture medium was collected after
centrifugation at 400 g for 5 minutes. Surnatants were analyzed for
IL-6 contents in duplicate at 1:10 dilution with a human ELISA IL-6
Kit (eBioscience, San Diego, Calif.). IL-6 concentration were
evaluated by comparing the OD of the sample with that of a standard
curve of recombinant IL-6.
Results
[0092] Results reported in Table 2 are expressed as % of inhibition
of IL-6 production induced by rBAG3 by the different antibodies.
All variants show inhibitory ability comparable to the murine AC2
anti BAG3 antibody. H4L2 and H4L4 were chosen for further testing
in vivo, for their ability block tumor growth in a xenograft tumor
model.
TABLE-US-00002 TABLE 2 % inhibition of BAG3 dependent IL6 secretion
AC2 120x AC2 60x AC2 30x 85.9% 53.4% 13.1% 74.4% 31.8% 18.6% H4L2
120x H4L260x H4L2 30x 80.3% 47.4% 14.2% 71.0% 27.4% 2.0% H4L3 120x
H4L3 60x H4L3 30x 80.4% 51.2% 25.9% 73.2% 42.3% 20.4% H4L4 120x
H4L4 60x H4L4 30x 84.3% 46.4% 3.15% 75.0% 41.2% 7.7% H2L4 120x H2L4
60x H2L4 30x 83.0% 52.0% 2.14% 73.7% 53.2% 25.9% B12 120x B12 60x
B12 30x 1.3% 0.7% 3.2% 0.1% 3.1% 0.7%
Example 6--Effect of Humanized AC-2 Antibody Variants on Tumor
Growth In Vivo
[0093] Based on in vitro experiments we choose variant H4L2 and
H2L4 for the subsequent in vivo validation.
Materials and Methods
[0094] Mia-Paca 2 cells (2.times.10W) were suspended in PBS (200
.mu.l) and injected into the right flank of female CD1 mice (6
weeks old; Charles River, Italy). After 10 days mice were divided
in four arms consisting of 7 (control) or 6 (treated) mice each in
which tumor volume average ranged 80-100 mm3. Control group
received vehicle (PBS) i.p. injection every 48 hrs whilst treated
groups received i.p. injection every 48 hrs of murine (AC2) or
humanized H2L4, H4L2 anti-BAG3 mAbs at the dose of 20 mg/kg in PBS
for 5 weeks. Animals were weighted and tumor volume measured by
caliper (D.times.d.sup.2/2) once weekly.
Results
[0095] FIG. 4 reports tumor fold change at week intervals of
control (PBS) or animals treated with the indicated antibodies. The
ratio is between the average volume at the indicated time point and
the average volume at day 0. While tumors of control treated
animals show a 4 fold increase, treated animals only increase by
2.5 fold.
Example 7--Impact of Humanized AC-2 H2L4 Variant on Activated
Fibroblast Number within the Tumor Mass
[0096] Results obtained on tumor growth prompted us to further
investigate changes in tumor microenvironment. One of the major
player in tumor development are fibroblasts associated to the tumor
mass.
Materials and Methods
[0097] At the end of the experiment described in example 6 tumors
were paraffin embedded and sections analysed by immunofluorescence
using an anti-.alpha.sma antibody (A2547, Sigma-Aldrich, at 1:350).
Nuclei were counterstained with 1 .mu.g ml.sup.-1 Hoechst 33342
(Molecular Probes, Oregon, USA). Images were acquired in sequential
scan mode by using the same acquisitions parameters (laser
intensities, gain photomultipliers, pinhole aperture,
objective.times.40, zoom 1) when comparing experimental and control
material. Leica Confocal Software and ImageJ were used for data
analysis (% of .alpha.-sma positive area).
Results
[0098] FIG. 5 show representative images from 2 PBS-treated and 2
AC-2 H2L4 treated tumors of .alpha.-sma staining. The presence of
activated fibroblasts associated to the masses grown in the control
animals is very high as demonstrated by .alpha.-sma protein
positivity. Instead, AC-2 H2L4 treated animals are negative. In
FIG. 6 we have reported a quantification of .alpha.-sma positive
area by comparing control (PBS-treated) and AC-2 H2L4 treated
samples.
[0099] These data strongly support the evidence that humanized
anti-BAG3 antibodies have an effect on fibroblasts that have an
important role not only in neoplastic diseases but also in other
pathological processes such as inflammation (Kalluri, 2016).
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Sequence CWU 1
1
261115PRTMus musculus 1Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Thr Ala Ser Gly
Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met Tyr Trp Val Lys Gln Arg Pro
Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly Gly Val Asp Pro Ala Asn Gly
Asn Thr Arg Tyr Asp Pro Lys Phe 50 55 60Gln Gly Lys Ala Thr Leu Thr
Ala Asp Thr Ser Ser Ser Thr Ala Tyr65 70 75 80Leu Gln Leu Ser Ser
Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Asp Gly
Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110Val Ser
Ser 1152113PRTMus musculus 2Asp Ile Val Met Ser Gln Ser Pro Ser Ser
Leu Ala Val Ser Val Gly1 5 10 15Glu Lys Val Thr Met Ser Cys Lys Ser
Ser Gln Ser Leu Leu Tyr Ser 20 25 30Ser Asn Gln Lys Asn Tyr Leu Ala
Trp His Gln Gln Lys Pro Gly Gln 35 40 45Ser Pro Lys Leu Leu Ile Tyr
Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Thr Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Val
Lys Ala Glu Asp Leu Ala Ile Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr Thr
Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105
110Lys310PRTMus musculus 3Gly Phe Asn Ile Lys Asp Thr Tyr Met Tyr1
5 10417PRTMus musculus 4Gly Val Asp Pro Ala Asn Gly Asn Thr Arg Tyr
Asp Pro Lys Phe Gln1 5 10 15Gly56PRTMus musculus 5Asp Gly Ala Met
Asp Tyr1 5617PRTMus musculus 6Lys Ser Ser Gln Ser Leu Leu Tyr Ser
Ser Asn Gln Lys Asn Tyr Leu1 5 10 15Ala77PRTMus musculus 7Trp Ala
Ser Thr Arg Glu Ser1 589PRTMus musculus 8Gln Gln Tyr Tyr Thr Tyr
Pro Leu Thr1 59345DNAMus musculussource1..345/mol_type="genomic
DNA" /organism="mouse" 9gaggtccagc tgcagcagag cggtgccgaa ctggtgaagc
caggagcatc cgtcaaactg 60tcttgtacag catccgggtt taacattaag gacacctaca
tgtattgggt gaaacagagg 120ccagagcagg gcctggaatg gatcggcgga
gtggaccccg ctaacgggaa tacacgatac 180gatcctaagt tccagggaaa
agccaccctg acagctgaca cttccagctc taccgcatat 240ctgcaactga
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300gctatggact actggggtca ggggacttcc gtcactgtct cgagc 34510339DNAMus
musculussource1..339/mol_type="genomic DNA" /organism="mouse"
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cagcacaagg 180gagtctggcg tgccagaccg gttcactggc tcaggctccg
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atctactatt gccagcagta ttacacttat 300cctctgacat ttggagcagg
gactaaactg gaactgaag 339111416DNAHomo
sapienssource1..1416/mol_type="other DNA" /organism="Homo sapiens"
11gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc
60cagtgtcagg tgcagctggt ccagagcggg gcagaggtga agaaaccagg tgccagcgtg
120aaggtctctt gcaaagccag tggcttcaac atcaaggaca catacatgta
ttgggtgcga 180caggcccctg gccagggtct ggaatggatg ggcggcgtgg
accccgcaaa tggaaatact 240agatacgatc ctaaatttca gggaagggtg
accatgacac gggacacttc aacctcgacg 300gtctatatgg agctgtccag
cctgagatcc gaagatacag ccgtgtacta ttgtgcccgc 360gacggggcta
tggattactg gggccaggga actctggtga ccgtctcgag cgctagcaca
420aagggcccta gtgtgtttcc tctggctccc tcttccaaat ccacttctgg
tggcactgct 480gctctgggat gcctggtgaa ggattacttt cctgaacctg
tgactgtctc atggaactct 540ggtgctctga cttctggtgt ccacactttc
cctgctgtgc tgcagtctag tggactgtac 600tctctgtcat ctgtggtcac
tgtgccctct tcatctctgg gaacccagac ctacatttgt 660aatgtgaacc
acaaaccatc caacactaaa gtggacaaaa aagtggaacc caaatcctgt
720gacaaaaccc acacctgccc accttgtcct gcccctgaac tgctgggagg
accttctgtg 780tttctgttcc cccccaaacc aaaggatacc ctgatgatct
ctagaacccc tgaggtgaca 840tgtgtggtgg tggatgtgtc tcatgaggac
cctgaggtca aattcaactg gtacgtggat 900ggagtggaag tccacaatgc
caaaaccaag cctagagagg aacagtacaa ttcaacctac 960agagtggtca
gtgtgctgac tgtgctgcat caggattggc tgaatggcaa ggaatacaag
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141612445PRTHomo sapiens 12Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met Tyr Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Val Asp Pro Ala Asn
Gly Asn Thr Arg Tyr Asp Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120
125Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala145 150 155 160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu225 230 235
240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360
365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln 405 410 415Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 435 440 445131416DNAHomo
sapienssource1..1416/mol_type="other DNA" /organism="Homo sapiens"
13gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc
60cagtgtcagg tgcagctggt ccagtctgga gctgaggtga agaaaccagg agcctccgtg
120aaggtctctt gcaaagccag tggcttcaac atcaaggaca catacatgta
ttgggtgcga 180caggcccctg gccagggtct ggaatggatg ggcggcgtgg
accccgcaaa tggaaatact 240agatacgatc ctaaatttca aggcagggtg
accctgacac gggacacttc aacctcgacg 300gtctatatgg agctgtccag
cctgagatcc gaagatacag cagtgtacta ttgtgggcgc 360gacggtgcta
tggactactg gggccaggga actctggtga ccgtctcgag cgctagcaca
420aagggcccta gtgtgtttcc tctggctccc tcttccaaat ccacttctgg
tggcactgct 480gctctgggat gcctggtgaa ggattacttt cctgaacctg
tgactgtctc atggaactct 540ggtgctctga cttctggtgt ccacactttc
cctgctgtgc tgcagtctag tggactgtac 600tctctgtcat ctgtggtcac
tgtgccctct tcatctctgg gaacccagac ctacatttgt 660aatgtgaacc
acaaaccatc caacactaaa gtggacaaaa aagtggaacc caaatcctgt
720gacaaaaccc acacctgccc accttgtcct gcccctgaac tgctgggagg
accttctgtg 780tttctgttcc cccccaaacc aaaggatacc ctgatgatct
ctagaacccc tgaggtgaca 840tgtgtggtgg tggatgtgtc tcatgaggac
cctgaggtca aattcaactg gtacgtggat 900ggagtggaag tccacaatgc
caaaaccaag cctagagagg aacagtacaa ttcaacctac 960agagtggtca
gtgtgctgac tgtgctgcat caggattggc tgaatggcaa ggaatacaag
1020tgtaaagtct caaacaaggc cctgcctgct ccaattgaga aaacaatctc
aaaggccaag 1080ggacagccta gggaacccca ggtctacacc ctgccacctt
caagagagga aatgaccaaa 1140aaccaggtgt ccctgacatg cctggtcaaa
ggcttctacc cttctgacat tgctgtggag 1200tgggagtcaa atggacagcc
tgagaacaac tacaaaacaa ccccccctgt gctggattct 1260gatggctctt
tctttctgta ctccaaactg actgtggaca agtctagatg gcagcagggg
1320aatgtctttt cttgctctgt catgcatgag gctctgcata accactacac
tcagaaatcc 1380ctgtctctgt ctcccgggaa atgatagtaa aagctt
141614445PRTHomo sapiens 14Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met Tyr Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Val Asp Pro Ala Asn
Gly Asn Thr Arg Tyr Asp Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Leu
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Asp
Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120
125Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala145 150 155 160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu225 230 235
240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360
365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln 405 410 415Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 435 440 445151416DNAHomo
sapienssource1..1416/mol_type="other DNA" /organism="Homo sapiens"
15gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc
60cagtgtcagg tgcagctggt ccagtctgga gctgaggtga agaaaccagg agcctccgtg
120aaggtctctt gcacagctag tggcttcaac atcaaggaca cttacatgta
ttgggtgaaa 180caggcccctg gccagggtct ggaatggatt ggcggcgtgg
accccgcaaa cgggaatacc 240agatacgatc ctaagtttca aggcaaagcc
accctgacaa gggacacttc aacctcgacg 300gtgtatatgg agctgtccag
cctgaggtcc gaagatacag cagtgtacta ttgtgggcgg 360gacggtgcta
tggactactg gggccaggga actctggtga ccgtctcgag cgctagcaca
420aagggcccta gtgtgtttcc tctggctccc tcttccaaat ccacttctgg
tggcactgct 480gctctgggat gcctggtgaa ggattacttt cctgaacctg
tgactgtctc atggaactct 540ggtgctctga cttctggtgt ccacactttc
cctgctgtgc tgcagtctag tggactgtac 600tctctgtcat ctgtggtcac
tgtgccctct tcatctctgg gaacccagac ctacatttgt 660aatgtgaacc
acaaaccatc caacactaaa gtggacaaaa aagtggaacc caaatcctgt
720gacaaaaccc acacctgccc accttgtcct gcccctgaac tgctgggagg
accttctgtg 780tttctgttcc cccccaaacc aaaggatacc ctgatgatct
ctagaacccc tgaggtgaca 840tgtgtggtgg tggatgtgtc tcatgaggac
cctgaggtca aattcaactg gtacgtggat 900ggagtggaag tccacaatgc
caaaaccaag cctagagagg aacagtacaa ttcaacctac 960agagtggtca
gtgtgctgac tgtgctgcat caggattggc tgaatggcaa ggaatacaag
1020tgtaaagtct caaacaaggc cctgcctgct ccaattgaga aaacaatctc
aaaggccaag 1080ggacagccta gggaacccca ggtctacacc ctgccacctt
caagagagga aatgaccaaa 1140aaccaggtgt ccctgacatg cctggtcaaa
ggcttctacc cttctgacat tgctgtggag 1200tgggagtcaa atggacagcc
tgagaacaac tacaaaacaa ccccccctgt gctggattct 1260gatggctctt
tctttctgta ctccaaactg actgtggaca agtctagatg gcagcagggg
1320aatgtctttt cttgctctgt catgcatgag gctctgcata accactacac
tcagaaatcc 1380ctgtctctgt ctcccgggaa atgatagtaa aagctt
141616445PRTHomo sapiens 16Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Thr Ala Ser
Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met Tyr Trp Val Lys Gln Ala
Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Gly Val Asp Pro Ala Asn
Gly Asn Thr Arg Tyr Asp Pro Lys Phe 50 55 60Gln Gly Lys Ala Thr Leu
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Asp
Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120
125Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala145 150 155 160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu225 230 235
240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln 405 410 415Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445171416DNAHomo
sapienssource1..1416/mol_type="other DNA" /organism="Homo sapiens"
17gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc
60cagtgtgagg tgcagctggt ccagagtggg gcagaagtga agaaaccagg tgccacagtg
120aagatctcat gcaaagtctc cggcttcaac attaaggaca cttacatgta
ttgggtgcag 180caggcccccg gcaagggtct ggagtggatg ggcggcgtgg
accccgctaa cggcaatacc 240agatacgatc ctaagtttca aggacgggtg
accatcacag ctgacactag caccgatacg 300gcatatatgg agctgtccag
cctgagatct gaagatacag cagtgtacta ttgtgccagg 360gacggggcta
tggattactg gggccaggga actctggtga ccgtctcgag cgctagcaca
420aagggcccta gtgtgtttcc tctggctccc tcttccaaat ccacttctgg
tggcactgct 480gctctgggat gcctggtgaa ggattacttt cctgaacctg
tgactgtctc atggaactct 540ggtgctctga cttctggtgt ccacactttc
cctgctgtgc tgcagtctag tggactgtac 600tctctgtcat ctgtggtcac
tgtgccctct tcatctctgg gaacccagac ctacatttgt 660aatgtgaacc
acaaaccatc caacactaaa gtggacaaaa aagtggaacc caaatcctgt
720gacaaaaccc acacctgccc accttgtcct gcccctgaac tgctgggagg
accttctgtg 780tttctgttcc cccccaaacc aaaggatacc ctgatgatct
ctagaacccc tgaggtgaca 840tgtgtggtgg tggatgtgtc tcatgaggac
cctgaggtca aattcaactg gtacgtggat 900ggagtggaag tccacaatgc
caaaaccaag cctagagagg aacagtacaa ttcaacctac 960agagtggtca
gtgtgctgac tgtgctgcat caggattggc tgaatggcaa ggaatacaag
1020tgtaaagtct caaacaaggc cctgcctgct ccaattgaga aaacaatctc
aaaggccaag 1080ggacagccta gggaacccca ggtctacacc ctgccacctt
caagagagga aatgaccaaa 1140aaccaggtgt ccctgacatg cctggtcaaa
ggcttctacc cttctgacat tgctgtggag 1200tgggagtcaa atggacagcc
tgagaacaac tacaaaacaa ccccccctgt gctggattct 1260gatggctctt
tctttctgta ctccaaactg actgtggaca agtctagatg gcagcagggg
1320aatgtctttt cttgctctgt catgcatgag gctctgcata accactacac
tcagaaatcc 1380ctgtctctgt ctcccgggaa atgatagtaa aagctt
141618445PRTHomo sapiens 18Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys Val Ser
Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met Tyr Trp Val Gln Gln Ala
Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Gly Val Asp Pro Ala Asn
Gly Asn Thr Arg Tyr Asp Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Ile
Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120
125Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala145 150 155 160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu225 230 235
240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360
365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln 405 410 415Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 435 440 44519741DNAHomo
sapienssource1..741/mol_type="other DNA" /organism="Homo sapiens"
19gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc
60cagtgtgaca tcgtgatgac acagtcacct gattccctgg cagtcagtct gggcgagaga
120gccactatta actgcaagtc cagccagtct ctgctgtact ctagtaacca
gaaaaattac 180ctggcttggt atcagcagaa gccagggcag ccccctaaac
tgctgatcta ttgggcaagc 240accagggaat ctggagtgcc cgaccggttc
agcggttctg gcagtggaac agattttacc 300ctgacaattt catccctgca
agccgaggac gtggctgtct actattgtca gcagtactat 360acttacccac
tgaccttcgg cggagggacc aagctcgaga tcaaacgtac ggtcgcggcg
420ccttctgtgt tcattttccc cccatctgat gaacagctga aatctggcac
tgcttctgtg 480gtctgtctgc tgaacaactt ctaccctaga gaggccaaag
tccagtggaa agtggacaat 540gctctgcaga gtgggaattc ccaggaatct
gtcactgagc aggactctaa ggatagcaca 600tactccctgt cctctactct
gacactgagc aaggctgatt acgagaaaca caaagtgtac 660gcctgtgaag
tcacacatca ggggctgtct agtcctgtga ccaaatcctt caatagggga
720gagtgctgat agtaaaagct t 74120220PRTHomo sapiens 20Asp Ile Val
Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg
Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30Ser
Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40
45Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr65 70 75 80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr
Cys Gln Gln 85 90 95Tyr Tyr Thr Tyr Pro Leu Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile 100 105 110Lys Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp 115 120 125Glu Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn 130 135 140Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu145 150 155 160Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175Ser
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185
190Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
22021741DNAHomo sapienssource1..741/mol_type="other DNA"
/organism="Homo sapiens" 21gcggccgcca tgaattttgg actgaggctg
attttcctgg tgctgaccct gaaaggcgtc 60cagtgtgaca tcgtgatgac acagtcacct
gattccctgg cagtcagtct gggcgagaga 120gccactatta actgcaagtc
cagccagtct ctgctgtact ctagtaacca gaaaaattat 180ctggcttggc
accagcagaa gccagggcag ccccctaaac tgctgatcta ctgggcaagc
240accagggaat ctggagtgcc cgaccggttc agcggttctg gcagtggaac
agattttacc 300ctgacaattt catccctgca agccgaggac gtggctgtct
actattgtca gcagtactat 360acttatccac tgaccttcgg cggagggacc
aagctcgaga tcaaacgtac ggtcgcggcg 420ccttctgtgt tcattttccc
cccatctgat gaacagctga aatctggcac tgcttctgtg 480gtctgtctgc
tgaacaactt ctaccctaga gaggccaaag tccagtggaa agtggacaat
540gctctgcaga gtgggaattc ccaggaatct gtcactgagc aggactctaa
ggatagcaca 600tactccctgt cctctactct gacactgagc aaggctgatt
acgagaaaca caaagtgtac 660gcctgtgaag tcacacatca ggggctgtct
agtcctgtga ccaaatcctt caatagggga 720gagtgctgat agtaaaagct t
74122220PRTHomo sapiens 22Asp Ile Val Met Thr Gln Ser Pro Asp Ser
Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser
Ser Gln Ser Leu Leu Tyr Ser 20 25 30Ser Asn Gln Lys Asn Tyr Leu Ala
Trp His Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr
Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu
Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr Thr
Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110Lys
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120
125Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu145 150 155 160Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp 165 170 175Ser Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr 180 185 190Glu Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205Ser Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 210 215 22023741DNAHomo
sapienssource1..741/mol_type="other DNA" /organism="Homo sapiens"
23gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc
60cagtgtgaca tcgtgatgac acagtcacct gattccctgg cagtctccct gggcgagaga
120gccactatga gttgcaagtc cagccagtct ctgctgtact ctagtaacca
gaaaaattat 180ctggcttggc accagcagaa gccagggcag ccccctaaac
tgctgatcta ctgggcaagc 240accagggaat ctggagtgcc cgaccggttc
agcggttctg gcagtggaac agattttacc 300ctgacaattt catccctgca
agccgaggac gtggctgtct actattgtca gcagtactat 360acttatccac
tgaccttcgg cggagggacc aagctcgaga tcaagcgtac ggtcgcggcg
420ccttctgtgt tcattttccc cccatctgat gaacagctga aatctggcac
tgcttctgtg 480gtctgtctgc tgaacaactt ctaccctaga gaggccaaag
tccagtggaa agtggacaat 540gctctgcaga gtgggaattc ccaggaatct
gtcactgagc aggactctaa ggatagcaca 600tactccctgt cctctactct
gacactgagc aaggctgatt acgagaaaca caaagtgtac 660gcctgtgaag
tcacacatca ggggctgtct agtcctgtga ccaaatcctt caatagggga
720gagtgctgat agtaaaagct t 74124220PRTHomo sapiens 24Asp Ile Val
Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg
Ala Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30Ser
Asn Gln Lys Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Gln 35 40
45Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr65 70 75 80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr
Cys Gln Gln 85 90 95Tyr Tyr Thr Tyr Pro Leu Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile 100 105 110Lys Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp 115 120 125Glu Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn 130 135 140Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu145 150 155 160Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175Ser
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185
190Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
22025741DNAHomo sapienssource1..741/mol_type="other DNA"
/organism="Homo sapiens" 25gcggccgcca tgaattttgg actgaggctg
attttcctgg tgctgaccct gaaaggcgtc 60cagtgtgaca tcgtgatgac acagtcacct
gattccctgg cagtctccct gggcgagaga 120gccactatga gttgcaagtc
cagccagtct ctgctgtact ctagtaacca gaaaaattat 180ctggcttggc
accagcagaa gccaggacag ccccctaaac tgctgatcta ctgggcaagc
240accagggaat ctggcgtgcc cgaccggttc agcggctctg gaagtgggac
agattttacc 300ctgacaatct catccctgca agccgaggac ctggctatct
actattgtca gcagtactat 360acttatccac tgaccttcgg tgccggcacc
aagctcgaga tcaaacgtac ggtcgcggcg 420ccttctgtgt tcattttccc
cccatctgat gaacagctga aatctggcac tgcttctgtg 480gtctgtctgc
tgaacaactt ctaccctaga gaggccaaag tccagtggaa agtggacaat
540gctctgcaga gtgggaattc ccaggaatct gtcactgagc aggactctaa
ggatagcaca 600tactccctgt cctctactct gacactgagc aaggctgatt
acgagaaaca caaagtgtac 660gcctgtgaag tcacacatca ggggctgtct
agtcctgtga ccaaatcctt caatagggga 720gagtgctgat agtaaaagct t
74126220PRTHomo sapiens 26Asp Ile Val Met Thr Gln Ser Pro Asp Ser
Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Met Ser Cys Lys Ser
Ser Gln Ser Leu Leu Tyr Ser 20 25 30Ser Asn Gln Lys Asn Tyr Leu Ala
Trp His Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr
Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu
Gln Ala Glu Asp Leu Ala Ile Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr Thr
Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile 100 105 110Lys
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120
125Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu145 150 155 160Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp 165 170 175Ser Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr 180 185 190Glu Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205Ser Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 210 215 220
* * * * *