U.S. patent application number 17/491715 was filed with the patent office on 2022-03-24 for variants of tissue inhibitor of metalloproteinase type three (timp-3), compositions and methods.
This patent application is currently assigned to AMGEN INC.. The applicant listed for this patent is AMGEN INC.. Invention is credited to Vishnu CHINTALGATTU, Randal R. KETCHEM, TaeWeon LEE, Jason C. O'NEILL, Jennitte LeAnn STEVENS.
Application Number | 20220089689 17/491715 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-24 |
United States Patent
Application |
20220089689 |
Kind Code |
A1 |
O'NEILL; Jason C. ; et
al. |
March 24, 2022 |
VARIANTS OF TISSUE INHIBITOR OF METALLOPROTEINASE TYPE THREE
(TIMP-3), COMPOSITIONS AND METHODS
Abstract
There are disclosed TIMP-3 muteins, variants and derivatives,
nucleic acids encoding them, and methods of making and using
them.
Inventors: |
O'NEILL; Jason C.; (Brier,
WA) ; KETCHEM; Randal R.; (Snohomish, WA) ;
LEE; TaeWeon; (Palo Alto, CA) ; CHINTALGATTU;
Vishnu; (Union City, CA) ; STEVENS; Jennitte
LeAnn; (Thousand Oaks, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AMGEN INC. |
Thousand Oaks |
CA |
US |
|
|
Assignee: |
AMGEN INC.
Thousand Oaks
CA
|
Appl. No.: |
17/491715 |
Filed: |
October 1, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15547730 |
Jul 31, 2017 |
11149078 |
|
|
PCT/US2015/046992 |
Aug 26, 2015 |
|
|
|
17491715 |
|
|
|
|
62042574 |
Aug 27, 2014 |
|
|
|
International
Class: |
C07K 14/81 20060101
C07K014/81 |
Claims
1.-12. (canceled)
13. A method of treating a condition in which matrix
metalloproteases (MMPs) and/or other proteinases that are inhibited
or inhibitable by Tissue Inhibitor of Metalloproteinase Type Three
(TIMP-3) play a causative or exacerbating role, comprising
administering to an individual afflicted with such a condition an
amount of a composition comprising a TIMP-3 mutein sufficient to
treat the condition, wherein the TIMP-3 mutein comprises a mature
region that is at least 90% identical in amino acid sequence to the
mature region of TIMP-3 set forth in SEQ ID NO:2 and: (a) two or
more pairs of mutations selected from K45N/V47T; K50N/V52T;
P56N/G58T; H78N/Q80T; K94N/E96T; or D110N/K112T; (b) one or more
pairs of mutations selected from K45N/V47T; K50N/V52T; P56N/G58T;
H78N/Q80T; K94N/E96T; or D110N/K112T; and an additional mutation
selected from R138T; G173T, or both R138T and G173T; or (c) the
mutations of (a) or (b) further comprising the mutation F57N.
14. The method of 13, wherein the condition is an inflammatory
conditions, osteoarthritis, myocardial ischemia, reperfusion
injury, or progression to congestive heart failure.
15. The method of claim 13, wherein the condition is asthma,
chronic obstructive pulmonary disease (COPD), idiopathic pulmonary
fibrosis (IPF), inflammatory bowel disease, psoriasis, myocarditis,
inflammation related to atherosclerosis, or an arthritic
conditions.
16. The method of claim 13, wherein the condition is rheumatoid
arthritis, psoriatic arthritis, viral myocarditis, dystrophic
epidermolysis bullosa, osteoarthritis, pseudogout, rheumatoid
arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis,
periodontal disease, ulceration, wound healing after surgery,
restenosis, emphysema, Paget's disease of bone, osteoporosis,
scleroderma, pressure atrophy of bone or tissues as in bedsores,
cholesteatoma, abnormal wound healing, pauciarticular rheumatoid
arthritis, polyarticular rheumatoid arthritis, systemic onset
rheumatoid arthritis, enteropathic arthritis, reactive arthritis,
SEA Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome),
dermatomyositis, psoriatic arthritis, vasculitis, myolitis,
polymyolitis, dermatomyolitis, osteoarthritis, polyarteritis
nodossa, Wegener's granulomatosis, arteritis, polymyalgia
rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis,
sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque
psoriasis, guttate psoriasis, inverse psoriasis, pustular
psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis,
atherosclerosis, lupus, Still's disease, Systemic Lupus
Erythematosus (SLE), myasthenia gravis, inflammatory bowel disease,
ulcerative colitis, Crohn's disease, Celiac disease (nontropical
Sprue), enteropathy associated with seronegative arthropathies,
microscopic or collagenous colitis, eosinophilic gastroenteritis,
pouchitis resulting after proctocolectomy and ileoanal anastomosis,
pancreatitis, insulin-dependent diabetes mellitus, mastitis,
cholecystitis, cholangitis, pericholangitis, multiple sclerosis
(MS), asthma, extrinsic asthma, intrinsic asthma,
hyperresponsiveness of the airways, chronic obstructive pulmonary
disease (COPD), chronic bronchitis, emphysema, Acute Respiratory
Disorder Syndrome (ARDS), respiratory distress syndrome, cystic
fibrosis, pulmonary hypertension, pulmonary vasoconstriction, acute
lung injury, allergic bronchopulmonary aspergillosis,
hypersensitivity pneumonia, eosinophilic pneumonia, bronchitis,
allergic bronchitis bronchiectasis, tuberculosis, hypersensitivity
pneumonitis, occupational asthma, asthma-like disorders, sarcoid,
reactive airway disease (or dysfunction) syndrome, byssinosis,
interstitial lung disease, hyper-eosinophilic syndrome, rhinitis,
sinusitis, parasitic lung disease, airway hyperresponsiveness
associated with viral-induced conditions, Guillain-Barre disease,
Graves' disease, Addison's disease, Raynaud's phenomenon,
autoimmune hepatitis, graft versus host disease (GVHD), cerebral
ischemia, traumatic brain injury, neuropathy, myopathy, spinal cord
injury, or amyotrophic lateral sclerosis (ALS).
17. The method of claim 13, wherein the condition is vascular
plaque stabilization, vasculopathy, neointima formation, acute lung
injury, or acute respiratory distress syndrome.
18.-20. (canceled)
21. The method of claim 13, wherein the TIMP-3 mutein comprises a
group of mutations selected from: (i) K45N/V47T, P56N/G58T, Q126N,
and R138T; (ii) K45N/V47T, P56N/G58T, K94N/E96T, and R138T; (iii)
K45N/V47T, P56N/G58T, R138T and G173T; (iv) K45N/V47T, K94N/E96T,
D110N/K112T, and F57N; (v) K45N/V47T, K94N/E96T, F57N and R138T;
(vi) K45N/V47T, H78N/Q80T, K94N/E96T, R138T, and G173T; (vii)
K45N/V47T, K94N/E96T, D110N/K112T, and R138T; (viii) K45N/V47T,
K94N/E96T, D110N/K112T, and G173T; (ix) K45N/V47T, K94N/E96T, R138T
and G173T; (x) K94N/E96T, D110N/K112T, K45S, F57N, and R138T; (xi)
H78N/Q80T, K94N/E96T, K45S, F57N and R138T; (xii) K50N/V52T,
P56N/G58T, K94N/E96T, D110N/K112T, R138T; (xiii) K50N/V52T,
H78N/Q80T, K94N/E96T, R138T and G173T; (xiv) K50N/V52T, K94N/E96T,
D110N/K112T, and R138T; (xv) K50N/V52T, K94N/E96T, D110N/K112T,
R138T and G173T; (xvi) K50N/V52T, K94N/E96T, R138T and G173T;
(xvii) K50N/V52T, Q126N, R138T, and G173T; (xviii) P56N/G58T,
H78N/Q80T, K94N/E96T, and R138T; (xix) P56N/G58T, K94N/E96T, Q126N
and R138T; (xx) P56N/G58T, K94N/E96T, D110N/K112T, and R138T; (xxi)
P56N/G58T, H78N/Q80T, K94N/E96T, and G173T; (xxii) P56N/G58T,
Q126N, R138T, and G173T; (xxiii) H78N/Q80T, K94N/E96T, R138T and
G173T; (xxiv) H78N/Q80T, K94N/E96T, D110N/K112T, and R138T; (xxv)
K50N/V52T, D110N/K112T, R138T and G173T; (xxvi) K45N/V47T,
D110N/K112T, R138T and G173T; (xxvii) H78N/Q80T, D110N/K112T, R138T
and G173T; (xxviii) K45N/V47T, K50N/V52T, H78N/Q80T, R138T; (xxix)
K45N/V47T, H78N/Q80T, D110N/K112T, and G173T; (xxxx) K45N/V47,
H78N/Q80T, R138T and G173T; (xxxi) K50N/V52T, H78N/Q80T, K94N/E96T,
and G173T; (xxxii) K50N/V52T, H78N/Q80T, D110N/K112T, and R138T;
(xxxiii) K45N/V47T, K50N/V52T, H78N/Q80T, and D110N/K112T; (xxxiv)
K50N/V52T, H78N/Q80T, R138T and G173T; (xxxv) K45N/V47T, H78N/Q80T,
R138T and G173T; (xxxvi) K45N/V47T, H78N/Q80T, and D110N/K112T, and
R138T; (xxxvii) K45N/V47T, K50N/V52T, H78N/Q80T, D110N/K112T, and
G173T; (xxxviii) K45N/V47T, K50N/V52T, H78N/Q80T, and R138T and
G173T; (xxxix) K45N/V47T, K50N/V52T, H78N/Q80T, K94N/E96T, and
G173T; (xl) K45N/V47T, H78N/Q80T, K94N/E96T, R138T and G173T; (xli)
K50N/V52T, H78N/Q80T, K94N/E96T, R138T and G173T; (xlii) K45N/V47T,
H78N/Q80T, and D110N/K112T, R138T and G173T; (xliii) K50N/V52T,
H78N/Q80T, D110N/K112T, R138T and G173T; or (xliv) K45N/V52T,
K50N/V52T, H78N/Q80T, D110N/K112T, and R138T.
22. The method of claim 21, wherein the TIMP-3 mutein comprises an
amino acid sequence of any one of SEQ ID NOs: 3-26.
23. The method of claim 13, wherein the TIMP-3 mutein is fused or
conjugated to a moiety that extends half-life of a polypeptide.
24. The method of claim 23, wherein the TIMP-3 mutein is fused to
an antibody, an Fc portion of an antibody, the heavy chain or light
chain of an antibody, or human serum albumin.
25. The method of claim 23, wherein the TIMP-3 mutein is conjugated
to polyethylene glycol.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION BY
REFERENCE
[0001] This application is a divisional application of U.S.
application Ser. No. 15/547,730, filed Jul. 31, 2017; which is a
national stage application under 35 U.S.C. .sctn. 371 of
International Application No. PCT/US2015/046992, having an
international filing date of Aug. 26, 2015; which claims the
benefit of U.S. Provisional Application No. 62/042,574, filed Aug.
27, 2014, all of which are incorporated by reference herein in
their entireties.
[0002] Incorporated by reference in its entirety is a
computer-readable nucleotide/amino acid sequence listing submitted
concurrently herewith and identified as follows: ASCII text file
named "A-1919-US-PCD_SeqListing.txt," 154,331 bytes, created Sep.
27, 2021.
FIELD OF THE INVENTION
[0003] The present invention relates in general to
metalloproteinase inhibitors. In particular, the invention relates
to tissue inhibitor of metalloproteinase 3 ("TIMP-3") and novel,
useful variants, muteins and derivatives thereof.
BACKGROUND OF THE INVENTION
[0004] Connective tissues and articular cartilage are maintained in
dynamic equilibrium by the opposing effects of extracellular matrix
synthesis and degradation. Degradation of the matrix is brought
about primarily by the enzymatic action of metalloproteinases,
including matrix metalloproteinases (MMPs) and
disintegrin-metalloproteinases with thrombospondin motifs
(ADAMTSs). While these enzymes are important in many natural
processes (including development, morphogenesis, bone remodeling,
wound healing and angiogenesis), disregulation of these enzymes
leading to their elevated levels are believed to play a detrimental
role in degradative diseases of connective tissue, including
rheumatoid arthritis and osteoarthritis, as well as in cancer and
cardiovascular conditions.
[0005] Endogenous inhibitors of metalloproteinases include plasma
alpha2-macroglobulin and tissue inhibitors of metalloproteinases
(TIMPs), of which there are four known to be encoded in the human
genome. TIMP-3 inhibits all the major cartilage-degrading
metalloproteases, and multiple lines of evidence indicate that it
protects cartilage. Addition of the protein to cartilage-explants
prevents cytokine-induced degradation, and intra-articular
injection reduces cartilage damage in the rat medial meniscal tear
model of osteoarthritis.
[0006] Dysregulation of MMPs also occurs in congestive heart
failure and is thought to play a role in numerous proinflammatory
processes. However, development of TIMP-3 as a therapeutic
inhibitor of MMP activity has been hampered by challenges in
production of recombinant protein and short half-life of
recombinant forms of TIMP-3. In particular, the serum half-life of
TIMP-3 following intravenous administration in rats is less than
sixty minutes, and such a short residence time negatively impacts
the ability to maintain a therapeutically useful concentration at a
disease site. Accordingly, there is a need in the art for forms of
TIMP-3 that exhibit favorable production, purification and
pharmacokinetic/pharmacodynamic properties.
SUMMARY OF THE INVENTION
[0007] The invention provides TIMP-3 polypeptides having
advantageous properties, e.g., enhanced pharmacokinetic or
pharmacodynamics properties (such as half-life), improved
expression levels compared to native TIMP-3, reduced affinity to
non-targets (e.g., scavenger receptors), and/or reduced dependence
on heparin for production.
[0008] In some embodiments, the invention provides a TIMP-3
polypeptide fused to one or more half-life extending moieties or
chemically modified with one or more half-life extending moieties.
For example, in some aspects, the invention provides a fusion
protein comprising TIMP-3 (or a fragment thereof) fused to the Fc
domain of an isolated antibody at the N- or C-terminus of TIMP-3.
The Fc domain may be fused to TIMP-3 (or a fragment thereof) via
the N- or C-terminus of the Fc moiety. The Fc domain may be
monomeric or heterodimeric. The invention also contemplates a
TIMP-3 polypeptide (or a fragment thereof) fused to human serum
albumin or a full antibody (at the N- or C-terminus of the heavy
chain or light chain). In some aspects, the chemical modification
to TIMP-3 (or a fragment thereof) to extend half-life includes
conjugation to polyethylene glycol (PEG).
[0009] In certain embodiments, the TIMP-3 protein carries mutations
in the native sequence resulting in improved half-life; such TIMP-3
mutations are described herein as, e.g., "TIMP-3 muteins." In
various aspects, the TIMP-3 protein is at least 90% identical in
amino acid sequence to the mature region of TIMP-3 set forth in SEQ
ID NO:2, wherein the mutein has at least one mutation that
introduces at least one N-linked glycosylation site. In an
additional embodiment, the TIMP-3 mutein has two, three, or four
new N-linked glycosylation sites; in a still further embodiment,
the number of N-linked glycosylation sites introduced is five, six,
seven, eight, nine, ten, eleven or twelve. In each mutein, it is
further contemplated that addition of one or more new N-linked
glycosylation cites does not substantially diminish the
metalloproteinase inhibitory activity of the native molecule.
[0010] Also embodied within the invention is a TIMP-3 mutein having
a mature region that is at least 90% identical in amino acid
sequence to the mature region of TIMP-3 set forth in SEQ ID NO:2,
having at least one mutation, the mutation being selected from the
group consisting of K45N, V47T, K50N, V52T, P56N, F57N, G58T, H78N,
Q80T, K94N, E96T, D110N, K112T, R138T, and G173T. Additional
embodiments include a TIMP-3 mutein having two or more pairs of
mutations selected from the group consisting of K45N/V47T,
K50N/V52T, P56N/G58T, H78N/Q80T, K94N/E96T, and D110N/K112T; and a
TIMP-3 mutein having one or more pairs of mutations selected from
the group consisting of K45N/V47T, K50N/V52T, P56N/G58T, H78N/Q80T,
K94N/E96T, and D110N/K112T, and an additional mutation that is
selected from the group consisting of R138T, G173T, and both R138T
and G173T. Further embodiments include muteins having any of the
aforementioned combinations of mutation and in addition the
mutation F57N.
[0011] In one embodiment of the invention, at least one N-linked
glycosylation site is introduced in a region of the TIMP-3 amino
acid sequence selected from the group consisting of: the region
consisting of amino acids 44-59; the region consisting of amino
acids 77-81; the region consisting of amino acids 93-97; the region
consisting of amino acids 109-112; the region consisting of amino
acids 137-139; the region consisting of amino acids 172-174; and
combinations thereof. In an additional embodiment, the TIMP-3
mutein has two, three, four, or five N-linked glycosylation sites;
in a still further embodiment, the number of N-linked glycosylation
sites introduced is four, five, six, seven, eight, nine, ten,
eleven or twelve.
[0012] One embodiment of the invention provides TIMP-3 muteins
K45N, V47T, P56N, G58T, Q126N, R138T (SEQ ID NO:3); K45N, V47T,
P56N, G58T, K94N, E96T, R138T (SEQ ID NO:4); K45N, V47T, P56N,
G58T, R138T, G173T (SEQ ID NO:5); K45N, V47T, F57N, K94N, E96T,
D110N, K112T (SEQ ID NO:6); K45N, V47T, F57N, K94N, E96T, R138T
(SEQ ID NO:7); K45N, V47T, H78N, Q80T, K94N, E96T, R138T, G173T
(SEQ ID NO:8); K45N, V47T, K94N, E96T, D110N, K112T, R138T (SEQ ID
NO:9); K45N, V47T, K94N, E96T, D110N, K112T, G173T (SEQ ID NO:10);
K45N, V47T, K94N, E96T, R138T, G173T (SEQ ID NO:11); K45S, F57N,
K94N, E96T, D110N, K112T, R138T (SEQ ID NO:12); K45S, F57N, H78N,
Q80T, K94N, E96T, R138T (SEQ ID NO:13); K50N, V52T P56N, G58T,
K94N, E96T, D110N, K112T, R138T (SEQ ID NO:14); K50N, V52T, H78N,
Q80T, K94N, E96T, R138T, G173T (SEQ ID NO:15); K50N, V52T, K94N,
E96T, D110N, K112T, R138T (SEQ ID NO:16); K50N, V52T, K94N, E96T,
D110N, K112T, R138T, G173T (SEQ ID NO:17); K50N, V52T, K94N, E96T,
R138T, G173T (SEQ ID NO:18); K50N, V52T, Q126N, R138T, G173T (SEQ
ID NO:19); P56N, G58T, H78N, Q80T, K94N, E96T, R138T (SEQ ID
NO:20); P56N, G58T K94N, E96T, Q126N, R138T (SEQ ID NO:21); P56N,
G58T, K94N, E96T, D110N, K112T, R138T (SEQ ID NO:22); P56N, G58T,
H78N, Q80T, K94N, E96T, G173T (SEQ ID NO:23); P56N, G58T, Q126N,
R138T, G173T (SEQ ID NO:24); H78N, Q80T, K94N, E96T, R138T, G173T
(SEQ ID NO:25); and H78N, Q80T, K94N, E96T, D110N, K112T, R138T
(SEQ ID NO:26).
[0013] Further embodiments include TIMP-3 muteins K50N/V52T,
D110N/K112T, R138T, G173T; K45N/V47T, D110N/K112T, R138T, G173T;
H78N/Q80T, D110N/K112T, R138T, G173T; K45N/V47T, K50N/V52T,
H78N/A80T, R138T; K45N/V47T, H78N/Q80T, D110N/K112T, G173T;
K45N/V47T, H78N/Q80T, R138T, G173T; K50N/V52T, H78N/Q80T,
K94N/E96T, G173T; K50N/V52T, H78N/Q80T, D110N/K112T, R138T;
K45N/V47T, K50N/V52T, H78N/Q80T, D110N/K112T; K50N/V52T, H78N/Q80T,
R138T, G173T; K45N/V47T, H78N/Q80T, R138T, G173T; K45N/V47T,
H78N/Q80T, D110N/K112T, R138T; K45N/V47T, K50N/V52T, H78N/Q80T,
D110N/K112T, G173T; K45N/V47T, K50N/V52T, H78N/Q80T, R138T, G173T;
K45N/V47T, K50N/V52T, H78N/Q80T, K94N/E96T, G173T; K45N/V47T,
H78N/Q80T, K94N/E96T, R138T, G173T; K50N/V52T, H78N/Q80T,
K94N/E96T, R138T, G173T; K45N/V47T, H78N/Q80T, D110N/K112T, R138T,
G173T; K50N/V52T, H78N/Q80T, D110N/K112T, R138T, G173T; and
K45N/V52T, K50N/V52T, H78N/Q80T, D110N/K112T, R138T.
[0014] The invention further provides a TIMP-3 mutein comprising
(or consisting of) the amino acid sequence set forth in SEQ ID NOs:
3-26 and 51-60, as well as a nucleic acid comprising nucleotide
sequence encoding the amino acid sequence of any one of SEQ ID NOs:
3-26 and 51-60.
[0015] In one aspect, the invention provides a nucleic acid (e.g.,
an isolated nucleic acid) that encodes a TIMP-3 mutein according to
any one of the aforementioned TIMP-3 muteins. Other aspects of the
invention are an expression vector comprising the nucleic acid; a
host cell (e.g., an isolated host cell) transformed or transfected
with the expression vector; and a method of producing a recombinant
TIMP-3 mutein comprising culturing the transformed or transfected
host cell of under conditions promoting expression of the TIMP-3
mutein, and recovering the TIMP-3 mutein.
[0016] The invention also provides a nucleic acid comprising (or
consisting of) the nucleic acid sequence set forth in SEQ ID NOs:
27-50 and 61-70.
[0017] Further provided is a composition comprising the TIMP-3
mutein described herein, as well as a method of treating a
condition in which matrix metalloproteases (MMPs) and/or other
proteinases that are inhibited or inhibitable by TIMP-3 play a
causative or exacerbating role, comprising administering to an
individual afflicted with such a condition, an amount of such
composition sufficient to treat the condition.
[0018] In one embodiment, the condition is selected from the group
consisting of inflammatory conditions, osteoarthritis, acute
myocardial infarction, cardiac ischemia (including myocardial
ischemia), reperfusion injury, and progression to chronic heart
failure (e.g., congestive heart failure). In various aspects, the
condition is vascular plaque stabilization, vasculopathy, or
neointima formation. In another embodiment, the condition is
selected from the group consisting of acute lung injury, acute
respiratory distress syndrome, asthma, chronic obstructive
pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF),
inflammatory bowel disease (for example, ulcerative colitis,
Crohn's disease, and celiac disease), psoriasis, myocarditis
including viral myocarditis, inflammation related to
atherosclerosis, and arthritic conditions including rheumatoid
arthritis and psoriatic arthritis.
[0019] In a further embodiment, the condition is selected from the
group consisting of dystrophic epidermolysis bullosa,
osteoarthritis, pseudogout, rheumatoid arthritis including juvenile
rheumatoid arthritis, ankylosing spondylitis, scleroderma,
periodontal disease, ulceration including corneal, epidermal, or
gastric ulceration, wound healing after surgery, restenosis,
emphysema, Paget's disease of bone, osteoporosis, scleroderma,
pressure atrophy of bone or tissues as in bedsores, cholesteatoma,
abnormal wound healing, rheumatoid arthritis, pauciarticular
rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic
onset rheumatoid arthritis, ankylosing spondylitis, enteropathic
arthritis, reactive arthritis, SEA Syndrome (Seronegativity,
Enthesopathy, Arthropathy Syndrome), dermatomyositis, psoriatic
arthritis, scleroderma, systemic lupus erythematosus, vasculitis,
myolitis, polymyolitis, dermatomyolitis, osteoarthritis,
polyarteritis nodossa, Wegener's granulomatosis, arteritis,
polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary
sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis,
plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular
psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis,
atherosclerosis, lupus, Still's disease, Systemic Lupus
Erythematosus (SLE), myasthenia gravis, inflammatory bowel disease,
ulcerative colitis, Crohn's disease, Celiac disease (nontropical
Sprue), enteropathy associated with seronegative arthropathies,
microscopic or collagenous colitis, eosinophilic gastroenteritis,
or pouchitis resulting after proctocolectomy and ileoanal
anastomosis, pancreatitis, insulin-dependent diabetes mellitus,
mastitis, cholecystitis, cholangitis, pericholangitis, multiple
sclerosis (MS), asthma (including extrinsic and intrinsic asthma as
well as related chronic inflammatory conditions, or
hyperresponsiveness, of the airways), chronic obstructive pulmonary
disease (COPD. i.e., chronic bronchitis, emphysema), Acute
Respiratory Disorder Syndrome (ARDS), respiratory distress
syndrome, cystic fibrosis, pulmonary hypertension, pulmonary
vasoconstriction, acute lung injury, allergic bronchopulmonary
aspergillosis, hypersensitivity pneumonia, eosinophilic pneumonia,
bronchitis, allergic bronchitis bronchiectasis, tuberculosis,
hypersensitivity pneumonitis, occupational asthma, asthma-like
disorders, sarcoid, reactive airway disease (or dysfunction)
syndrome, byssinosis, interstitial lung disease, hyper-eosinophilic
syndrome, rhinitis, sinusitis, and parasitic lung disease, airway
hyperresponsiveness associated with viral-induced conditions (for
example, respiratory syncytial virus (RSV), parainfluenza virus
(PIV), rhinovirus (RV) and adenovirus), Guillain-Barre disease,
Graves' disease, Addison's disease, Raynaud's phenomenon,
autoimmune hepatitis, graft versus host disease (GVHD), cerebral
ischemia, traumatic brain injury, multiple sclerosis, neuropathy,
myopathy, spinal cord injury, and amyotrophic lateral sclerosis
(ALS).
DESCRIPTION OF THE FIGURES
[0020] FIG. 1 is a reproduction of an SDS-PAGE gel illustrating the
amount of a TIMP-3 fusion protein, N-TIMP-3 (AA 1-144) fused to Fc
portion of an antibody ("TIMP-3-Fc"), produced in the presence of
varying amounts of heparin. Lane #1-4 contained Fc standards
("STD"): Lane #1 contained 100 ng human Fc; Lane #2 contained 250
ng human Fc; Lane #3 contained 500 ng human Fc; and Lane #4
contained 1000 ng human Fc. Lanes #5-9 contained 10 .mu.L samples
from culture media from CHOK1 cells expressing TIMP-3-Fc grown in
the absence of heparin (Lane #5), in the presence of 500 mg/L
heparin (Lane #6), in the presence of 250 mg/L heparin (Lane #7),
in the presence of 100 mg/L heparin (Lane #8), or in the presence
of 50 mg/L heparin (Lane #9).
[0021] FIG. 2 is a reproduction of an SDS-PAGE gel illustrating the
amount of a TIMP-3 mutein fused to Fc portion of an antibody
produced in the absence of heparin. Lane #1-4 contained Fc
standards ("STD"): Lane #1 contained 100 ng human Fc; Lane #2
contained 250 ng human Fc; Lane #3 contained 500 ng human Fc; and
Lane #4 contained 1000 ng human Fc. Lane #5 represented a host cell
control sample. Lanes #6-9 contained 10 .mu.L samples from culture
media of CHOK1 cells expressing TIMP-3 mutein-Fc: TIMP-3
[K45N/V47T/K94N/E96T/D110N/K112T/G173T]-FcG1 fusion (Lane #6),
TIMP-3 [K45N/V47T/K94N/E96T/D110N/K112T/G173T]-IgG1Fc+EPKSS fusion
(Lane #7), TIMP-3 [H78N/Q80T/K94N/E96T/D110N/K112T/R138T]-FcG1
fusion (Lane #8), or TIMP-3
[H78N/Q80T/K94N/E96T/D110N/K112T/R138T]-IgG1Fc+EPKSS fusion (Lane
#9).
[0022] FIG. 3 is a reproduction of an SDS-PAGE gel illustrating the
amount of a native N-TIMP-3 fused to human serum albumin (HSA)
produced in the presence and absence of heparin. Lane #1-4
contained Fc standards ("STD"): Lane #1 contained 100 ng human Fc;
Lane #2 contained 250 ng human Fc; Lane #3 contained 500 ng human
Fc; and Lane #4 contained 1000 ng human Fc. Lane #5 represented a
host cell control sample. Lanes #6-11 contained 10 .mu.L samples
from different pools of culture media of CHOK1-expressing
TIMP-3-HSA: Pool 1 cultured with heparin (Lane #6), Pool 2 cultured
with heparin (Lane #7), Pool 3 cultured with heparin (Lane #8),
Pool 1 cultured without ("wo") heparin (Lane #9), Pool 2 cultured
without heparin (Lane #10), Pool 3 cultured without heparin (Lane
#11).
[0023] FIG. 4 is an illustration of the three dimensional structure
of TIMP-3 associated with TACE, RAP, and LPR-1. TIMP-3 lysines at
positions 22 and 110 are labeled. See also Wisniewska et al., J.
Mol. Biol., 381, 1307-1319 (2008).
[0024] FIG. 5 contains two line graphs illustrating pharmacokinetic
properties of TIMP-3 [K45S, F56N], comparing fluorescent area/total
area (%) or fluorescent area/total area (% of time 0) (y-axis) to
days post infarction (x-axis).
[0025] FIG. 6 contains two line graphs illustrating pharmacokinetic
properties of TIMP-3 [H78N, Q80T, K94N, E96T, D110N, K112T, R138T]
(SEQ ID NO:26), comparing fluorescent area/total area (%) or
fluorescent area/total area (% of time 0) (y-axis) to days post
infarction (x-axis).
[0026] FIG. 7 is a line graph illustrating ejection fraction (%)
(y-axis) observed over time (day, x-axis) post-myocardial
infarction following administration of TIMP-3 polypeptides.
Triangle=full length TIMP-3 (30 mg); Open square=N-terminal domain
of TIMP-3 (N-TIMP3) (30 mg); Closed square=N-TIMP3 (30 mg),
Circle=control (saline).
[0027] FIGS. 8A-8C are bar graphs illustrating improved cardiac
function and reduced cardiac remodeling mediated by TIMP-3 [H78N,
Q80T, K94N, E96T, D110N, K112T, R138T] (SEQ ID NO:26) (referred to
as "TIMP3v82" in figure) following myocardial infarction in rats.
FIG. 8A illustrates ejection fraction (% EF, y-axis) detected on
day 3 and day 7 (x-axis) following administration for subjects
treated with vehicle (bar on the left) or TIMP-3 [H78N, Q80T, K94N,
E96T, D110N, K112T, R138T] (bar on the right). FIG. 8B illustrates
end systolic volume (ESV) (y-axis) measured on day 3 and day 7
(x-axis) following administration of vehicle or TIMP-3 [H78N, Q80T,
K94N, E96T, D110N, K112T, R138T]. FIG. 8C illustrates end diastolic
volume (ESV) (y-axis) measured on day 3 and day 7 (x-axis)
following administration of vehicle or TIMP-3 [H78N, Q80T, K94N,
E96T, D110N, K112T, R138T].
[0028] FIG. 9 is an illustration of the three dimensional structure
of TIMP-3 noting the positions of various amino acids.
[0029] FIGS. 10A-10C provide amino acid sequences of TIMP-3
muteins. The series of "X"s included in amino acid sequences
denotes the position of the signal peptide (e.g., amino acids 1-23
of SEQ ID NO: 2).
DETAILED DESCRIPTION OF THE INVENTION
[0030] The invention provides compositions, kits, and methods
relating to TIMP-3 polypeptides, variants, derivatives or muteins.
Also provided are nucleic acids, and derivatives and fragments
thereof, comprising a sequence of nucleotides that encodes all or a
portion of such a TIMP-3 polypeptide, variant, derivative or
mutein, e.g., a nucleic acid encoding all or part of such TIMP-3
polypeptides, variants, derivatives or muteins; plasmids and
vectors comprising such nucleic acids, and cells or cell lines
comprising such nucleic acids and/or vectors and plasmids. The
provided methods include, for example, methods of making,
identifying, or isolating TIMP-3 polypeptides, variants,
derivatives or muteins that exhibit desirable properties.
[0031] Numerous conditions exist in which it would be advantageous
to augment endogenous TIMP-3 in a mammal, or to increase the level
of TIMP-3 in a particular tissue. Accordingly, also provided herein
are methods of making compositions, such as pharmaceutical
compositions, comprising a TIMP-3 polypeptide, variant, derivative
or mutein, and methods for administering a composition comprising a
TIMP-3 polypeptide, variant, derivative or mutein to a subject, for
example, a subject afflicted with a condition in which
dysregulation of matrix metalloproteinase activity results in
excessive or inappropriate remodeling of tissue.
[0032] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. Generally, nomenclatures used in connection with, and
techniques of, cell and tissue culture, molecular biology,
immunology, microbiology, genetics and protein and nucleic acid
chemistry and hybridization described herein are those well-known
and commonly used in the art. The methods and techniques of the
present invention are generally performed according to conventional
methods well known in the art and as described in various general
and more specific references that are cited and discussed
throughout the present specification unless otherwise indicated.
See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual,
2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1989) and Ausubel et al., Current Protocols in Molecular
Biology, Greene Publishing Associates (1992), and Harlow and Lane
Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1990), which are incorporated
herein by reference. Enzymatic reactions and purification
techniques are performed according to manufacturer's
specifications, as commonly accomplished in the art or as described
herein. The terminology used in connection with, and the laboratory
procedures and techniques of, analytical chemistry, synthetic
organic chemistry, and medicinal and pharmaceutical chemistry
described herein are those well-known and commonly used in the art.
Standard techniques can be used for chemical syntheses, chemical
analyses, pharmaceutical preparation, formulation, and delivery,
and treatment of patients.
[0033] The following terms, unless otherwise indicated, shall be
understood to have the following meanings:
[0034] The term "isolated" as used to characterize a molecule
(where the molecule is, for example, a polypeptide, a
polynucleotide, or an antibody) indicates that the molecule by
virtue of its origin or source of derivation (1) is not associated
with naturally associated components that accompany it in its
native state, (2) is substantially free of other molecules from the
same species (3) is expressed by a cell from a different species,
or (4) does not occur in nature without human intervention. Thus, a
molecule that is chemically synthesized, or synthesized in a
cellular system different from the cell from which it naturally
originates, will be "isolated" from its naturally associated
components. A molecule also may be rendered substantially free of
naturally associated components by isolation, using purification
techniques well known in the art. Molecule purity or homogeneity
may be assayed by a number of means well known in the art. For
example, the purity of a polypeptide sample may be assayed using
polyacrylamide gel electrophoresis and staining of the gel to
visualize the polypeptide using techniques well known in the art.
For certain purposes, higher resolution may be provided by using
HPLC or other means well known in the art for purification. In
various embodiments, the invention provides an isolated TIMP-3
polypeptide, variant, derivative or mutein; an isolated nucleic
acid encoding the TIMP-3 polypeptide, variant, derivative or
mutein; and an isolated host cell comprising the nucleic acid or
expression vector or producing the polypeptide, variant, derivative
or mutein.
[0035] The terms "peptide," "polypeptide," and "protein" each
refers to a molecule comprising two or more amino acid residues
joined to each other by peptide bonds. These terms encompass, e.g.,
native and artificial proteins, protein fragments and polypeptide
analogs (such as muteins, variants, and fusion proteins) of a
protein sequence as well as post-translationally, or otherwise
covalently, or non-covalently, modified proteins. A peptide,
polypeptide, or protein may be monomeric or polymeric.
[0036] The term "polypeptide fragment" as used herein refers to a
polypeptide that has an amino-terminal and/or carboxy-terminal
deletion as compared to a corresponding full-length protein.
Fragments can be, for example, at least 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 20, 50, 70, 80, 90, 100, 150 or 200 amino acids in
length. Fragments can also be, for example, at most 1,000, 750,
500, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20,
15, 14, 13, 12, 11, or 10 amino acids in length. A fragment can
further comprise, at either or both of its ends, one or more
additional amino acids, for example, a sequence of amino acids from
a different naturally-occurring protein (e.g., an Fc or leucine
zipper domain) or an artificial amino acid sequence (e.g., an
artificial linker sequence or a tag protein).
[0037] A "variant" or "mutein" of a polypeptide (e.g., a TIMP-3
variant or mutein) comprises an amino acid sequence wherein one or
more amino acid residues are inserted into, deleted from and/or
substituted into the amino acid sequence relative to another
polypeptide sequence. Variants of the invention include fusion
proteins. It will be understood that, unless context dictates
otherwise, features of "polypeptides" or "proteins" described
herein are also attributed to variants, muteins, and
derivatives.
[0038] A "conservative amino acid substitution" is one that does
not substantially change the structural characteristics of the
parent sequence (e.g., a replacement amino acid should not tend to
break a helix that occurs in the parent sequence, or disrupt other
types of secondary structure that characterize the parent sequence
or are necessary for its functionality). Examples of art-recognized
polypeptide secondary and tertiary structures are described in
Proteins, Structures and Molecular Principles (Creighton, Ed., W.
H. Freeman and Company, New York (1984)); Introduction to Protein
Structure (C. Branden and J. Tooze, eds., Garland Publishing, New
York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991),
which are each incorporated herein by reference.
[0039] One way of referring to the degree of similarity of a
variant or mutein to the native protein is by referring to the
percent identity between the two (or more) polypeptide sequences,
or the encoding nucleic acids sequences, being compared. The
"percent identity" of two polynucleotide or two polypeptide
sequences is determined by comparing the sequences using the GAP
computer program (a part of the GCG Wisconsin Package, version 10.3
(Accelrys, San Diego, Calif.)) using its default parameters.
[0040] A "derivative" of a polypeptide is a polypeptide (e.g., a
TIMP-3 polypeptide, variant or mutein) that has been chemically
modified, e.g., via conjugation to another chemical moiety (such
as, for example, polyethylene glycol or albumin, e.g., human serum
albumin), phosphorylation, and/or glycosylation.
[0041] Polynucleotide and polypeptide sequences are indicated using
standard one- or three-letter abbreviations. Unless otherwise
indicated, each polypeptide sequence has an amino terminus at the
left and a carboxy terminus at the right; each single-stranded
nucleic acid sequence, and the top strand of each double-stranded
nucleic acid sequence, has a 5' terminus at the left and a 3'
terminus at the right. A particular polypeptide or polynucleotide
sequence also can be described by explaining how it differs from a
reference sequence. For example, substitutions of amino acids are
designated herein as "n #m" where "n" designates the amino acid
found in the native, full-length polypeptide, "#" designates the
amino acid residue number, and "m" designates the amino acid that
has been substituted.
[0042] The terms "polynucleotide," "oligonucleotide" and "nucleic
acid" are used interchangeably throughout and include DNA molecules
(e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of
the DNA or RNA generated using nucleotide analogs (e.g., peptide
nucleic acids and non-naturally occurring nucleotide analogs), and
hybrids thereof. The nucleic acid molecule can be single-stranded
or double-stranded. In one embodiment, the nucleic acid molecules
of the invention comprise a contiguous open reading frame encoding
a TIMP-3 polypeptide, fragment, variant, derivative or mutein, of
the invention. Nucleic acid sequences encoding TIMP-3 muteins,
variants, or derivatives described herein are set forth in SEQ ID
NOs: 27-50 and 61-70. Nucleotides 1-69 of SEQ ID NOs" 27-50 and
61-70 comprise the TIMP signal sequence. The invention includes a
nucleic acid comprising a nucleotide sequence comprising at least
90% identity (e.g., at least 95% identity or 100% identity) to SEQ
ID NOs: 27-50 and 61-70, as well as SEQ ID NOs: 27-50 and 61-70
lacking nucleotides 1-69.
[0043] Two single-stranded polynucleotides are "the complement" of
each other if their sequences can be aligned in an anti-parallel
orientation such that every nucleotide in one polynucleotide is
opposite its complementary nucleotide in the other polynucleotide,
without the introduction of gaps, and without unpaired nucleotides
at the 5' or the 3' end of either sequence. A polynucleotide is
"complementary" to another polynucleotide if the two
polynucleotides can hybridize to one another under moderately
stringent conditions. Thus, a polynucleotide can be complementary
to another polynucleotide without being its complement.
[0044] A "vector" is a nucleic acid that can be used to introduce
another nucleic acid linked to it into a cell. One type of vector
is a "plasmid," which refers to a linear or circular double
stranded DNA molecule into which additional nucleic acid segments
can be ligated. Another type of vector is a viral vector (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), wherein additional DNA segments can be
introduced into the viral genome. Certain vectors are capable of
autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors comprising a bacterial origin
of replication and episomal mammalian vectors). Other vectors
(e.g., non-episomal mammalian vectors) are integrated into the
genome of a host cell upon introduction into the host cell, and
thereby are replicated along with the host genome. An "expression
vector" is a type of vector that can direct the expression of a
chosen polynucleotide.
[0045] A nucleotide sequence is "operably linked" to a regulatory
sequence if the regulatory sequence affects the expression (e.g.,
the level, timing, or location of expression) of the nucleotide
sequence. A "regulatory sequence" is a nucleic acid that affects
the expression (e.g., the level, timing, or location of expression)
of a nucleic acid to which it is operably linked. The regulatory
sequence can, for example, exert its effects directly on the
regulated nucleic acid, or through the action of one or more other
molecules (e.g., polypeptides that bind to the regulatory sequence
and/or the nucleic acid). Examples of regulatory sequences include
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Further examples of regulatory sequences
are described in, for example, Goeddel, 1990, Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego,
Calif. and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.
[0046] Naturally occurring extracellular proteins typically include
a "signal sequence," which directs the protein into the cellular
pathway for protein secretion and which is not present in the
mature protein. The signal sequence may also be referred to as a
"signal peptide" or "leader peptide" and is enzymatically cleaved
from the extracellular protein. The protein that has been so
processed (i.e., having the signal sequence removed) is often
referred to as "mature" protein. A polynucleotide encoding a
protein or polypeptide of the invention may encode a naturally
occurring signal sequence or a heterologous signal sequence,
numerous of which are known in the art.
[0047] As appreciated by one of skill in the art, recombinant
proteins or polypeptides in accordance with the present embodiments
can be expressed in cell lines, including mammalian cell lines.
Sequences encoding particular proteins can be used for
transformation of a suitable mammalian host cell. Transformation
can be by any known method for introducing polynucleotides into a
host cell, including, for example packaging the polynucleotide in a
virus (or into a viral vector) and transducing a host cell with the
virus (or vector) or by transfection procedures known in the art,
as exemplified by U.S. Pat. Nos. 4,399,216; 4,912,040; 4,740,461;
and 4,959,455 (which patents are hereby incorporated herein by
reference). The transformation procedure used depends upon the host
to be transformed. Methods for introduction of heterologous
polynucleotides into mammalian cells are well known in the art and
include dextran-mediated transfection, calcium phosphate
precipitation, polybrene mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, and direct microinjection of the DNA into nuclei.
[0048] A "host cell" is a cell that can be used to express a
nucleic acid, e.g., a nucleic acid of the invention. A host cell
can be a prokaryote, for example, E. coli, or it can be a
eukaryote, for example, a single-celled eukaryote (e.g., a yeast or
other fungus), a plant cell (e.g., a tobacco or tomato plant cell),
an animal cell (e.g., a human cell, a monkey cell, a hamster cell,
a rat cell, a mouse cell, or an insect cell) or a hybridoma.
Examples of host cells include the COS-7 line of monkey kidney
cells (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), L
cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary
(CHO) cells or their derivatives such as Veggie CHO and related
cell lines which grow in serum-free media (see Rasmussen et al.,
1998, Cytotechnology 28:31) or CHO strain DX-B11, which is
deficient in DHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci.
USA 77:4216-20), HeLa cells, BHK (ATCC CRL 10) cell lines, the
CV1/EBNA cell line derived from the African green monkey kidney
cell line CV1 (ATCC CCL 70) (see McMahan et al., 1991, EMBO J.
10:2821), human embryonic kidney cells such as 293, 293 EBNA or MSR
293, human epidermal A431 cells, human Colo205 cells, other
transformed primate cell lines, normal diploid cells, cell strains
derived from in vitro culture of primary tissue, primary explants,
HL-60, U937, HaK or Jurkat cells.
[0049] Typically, a host cell is a cultured cell that can be
transformed or transfected with a polypeptide-encoding nucleic
acid, which can then be expressed in the host cell. In a "transient
transfection," the nucleic acid is introduced into the host cell by
one of several methods known in the art, and the recombinant
protein is expressed for a finite period of time, typically up to
about four days, before the nucleic acid is lost or degraded, for
example, when the host cell undergoes mitosis. If a "stable
transfection" is desired, the polypeptide-encoding nucleic acid may
be introduced into the host cell along with a nucleic acid encoding
a selectable marker. Use of a selectable marker allows one of skill
in the art to select transfected host cells in which the
polypeptide-encoding nucleic acid is integrated into the host cell
genome in such a way that the polypeptide-encoding nucleic acid is
maintained through mitosis, and can be expressed by progeny
cells.
[0050] The phrase "recombinant host cell" can be used to denote a
host cell that has been transformed or transfected with a nucleic
acid to be expressed. A host cell also can be a cell that comprises
the nucleic acid but does not express it at a desired level unless
a regulatory sequence is introduced into the host cell such that it
becomes operably linked with the nucleic acid. It is understood
that the term host cell refers not only to the particular subject
cell but also to the progeny or potential progeny of such a cell.
Because certain modifications may occur in succeeding generations
due to, e.g., mutation or environmental influence, such progeny may
not, in fact, be identical to the parent cell, but are still
included within the scope of the term as used herein.
[0051] As used herein, "TIMP-3 DNA," "TIMP-3-encoding DNA" and the
like indicate a selected TIMP-3 encoding nucleic acid in which the
TIMP-3 that is expressed therefrom may be either native TIMP-3 or a
TIMP-3 variant or mutein as described herein. Likewise, "TIMP-3,"
"TIMP-3 protein" and "TIMP-3 polypeptide" are used to designate
either a native TIMP-3 protein or a TIMP-3 protein comprising one
or more mutations (i.e., a TIMP-3 polypeptide, variant, derivative
or mutein). A particular mutein of TIMP-3 may be designated by the
mutation or mutations, for example, "K45N" or "K45N TIMP-3" or
"TIMP-3 K45N" or "K45N TIMP-3 polypeptide" indicates a polypeptide
in which the lysine (K) at amino acid 45 of native TIMP-3 has been
substituted with an asparagine (N).
[0052] The term "native TIMP-3" as used herein refers to wild type
TIMP-3. TIMP-3 is expressed by various cells or tissues in a mammal
and is present in the extracellular matrix; the TIMP-3 that is so
expressed is also referred to herein as "endogenous" TIMP-3. The
amino acid sequence of TIMP-3, and the nucleic acid sequence of a
DNA that encodes TIMP-3, are disclosed in U.S. Pat. No. 6,562,596,
issued May 13, 2003, the disclosure of which is incorporated by
reference herein. The amino acid numbering system used in U.S. Pat.
No. 6,562,596 designates the amino acids in the signal (or leader)
peptide with negative numbers, and references the mature protein
(i.e., the protein from which the signal or leader peptide has been
removed) as amino acids 1-188. The numbering systems used herein
refers to TIMP-3 with the first amino acid of the native leader
peptide designated #1; the full-length TIMP-3 thus includes amino
acids 1-211, and the mature form is amino acids 24-211. Those of
ordinary skill in the art readily comprehend the differences in
amino acid numbering that may occur by the use of these different
numbering systems, and can thus easily apply the numbering system
used herein to, for example, a TIMP-3 polypeptide in which the
first amino acid of the mature form is referred to as #1. Thus, for
example, K45N as designated herein would be designated K22N using
the numbering system of U.S. Pat. No. 6,562,596.
[0053] TIMP-3 is formed of two domains, an N-terminal domain
comprising amino acids 24 through 143 of TIMP-3 (i.e., about
two-thirds of the molecule), and the C-terminal domain, which
comprises amino acids 144 through 211. TIMP-3 exhibits complex
disulphide bonds that facilitate formation of the secondary and
tertiary structure TIMP-3. The N-terminal domain of TIMP-3, often
referred to as "N-TIMP-3," has been found to exhibit at least some
of the biological activities of TIMP-3; accordingly, TIMP-3
variants, derivatives and muteins as described herein include
variants, derivatives and muteins of a fragment of TIMP-3 that
comprises the N-terminal domain.
[0054] Native TIMP-3 protein presents several challenges for its
use as a therapeutic molecule. For example, mammalian expression
titers for TIMP-3 protein using standard mammalian expression
techniques are too low to allow sufficient quantities of TIMP-3 to
be produced at a scale that is suitable for a therapeutic protein.
Moreover, the binding of TIMP-3 to extracellular matrix
necessitates the inclusion of heparin (or a similar agent that
reduces binding of TIMP-3 to extracellular matrix) in cell culture
medium, and binding to the Low density lipoprotein Receptor-related
Protein 1 (LRP1) scavenger protein exacerbates the challenge of
secretion of recombinant TIMP-3 into the medium at a level that
allows a production-scale process to be developed. Microbial
production in prokaryotic cells of full-length TIMP-3 has proved
difficult due to incorrect folding of the protein.
[0055] Accordingly, the TIMP-3 variants or muteins of the invention
have been modified to overcome one or more of these challenges.
Polypeptides of the invention include polypeptides that have been
modified in any way and for any reason, for example, to: (1) reduce
susceptibility to proteolysis, (2) reduce susceptibility to
oxidation, (3) reduce the need for agents that inhibit binding of
TIMP-3 to extracellular matrix in cell culture, (4) alter binding
affinities for other moieties, for example scavenger receptors such
as LRP-1, (5) confer or modify other physicochemical or functional
properties, including pharmacokinetics and/or pharmacodynamics, or
(6) facilitate expression and/or purification of recombinant
protein. Analogs include muteins of a polypeptide. For example,
single or multiple amino acid substitutions (e.g., conservative
amino acid substitutions) may be made in the naturally occurring
sequence (e.g., in the portion of the polypeptide outside the
domain(s) forming intermolecular contacts). Consensus sequences can
be used to select amino acid residues for substitution; those of
skill in the art recognize that additional amino acid residues may
also be substituted.
[0056] In one aspect of the invention, there is provided a TIMP-3
mutein or variant that exhibits an increase in expression levels of
the mutein or variant over that observed with native TIMP-3; in
another aspect of the invention the increased expression occurs in
a mammalian cell expression system. Expression levels may be
determined by any suitable method that will allow a quantitative or
semi-quantitative analysis of the amount of recombinant TIMP-3
(native, variant or mutein) in cell culture supernatant fluid,
i.e., conditioned media (CM). In one embodiment, samples or CM are
assessed by Western blot; in another embodiment, CM samples are
assessed using a standard human TIMP-3 ELISA.
[0057] In one embodiment, the increase in expression is observed in
a transient expression system; in another embodiment, the increase
in expression is observed in a stable transfection system. One
embodiment provides a TIMP-3 mutein or variant for which the
increase in expression observed is two-fold (2.times.) greater than
that observed for native TIMP-3; another embodiment provides a
TIMP-3 mutein or variant for which the increase in expression
observed is five-fold (5.times.) greater than that observed for
native TIMP-3. Further embodiments include TIMP-3 muteins or
variants for which the increase in expression is three-fold
(3.times.), four-fold (4.times.) or six-fold (6.times.). In one
embodiment, the expression of the TIMP-3 mutein or variant is
ten-fold (10.times.) greater than that observed with native TIMP-3;
in another embodiment, the observed expression is more than
ten-fold, for example, 20-fold (20.times.) or greater, than that
observed with native TIMP-3.
[0058] In another aspect of the invention, there are provided
TIMP-3 muteins (or variants) that exhibit reduced requirement for
the addition of heparin (or another agent that inhibits binding of
TIMP-3 to extracellular matrix) to cell culture media (i.e.,
heparin independence). The reduction in the amount of heparin (or
other agent) may be described in a semi-quantitative manner, i.e.,
the reduction may be partial, moderate, substantial, or complete.
In another embodiment, the reduction is expressed as a percentage,
for example the amount of heparin (or similar agent) may be reduced
by 10%, 20%, 30%, 40%, 50%, or more (for example by 60%, 70% 80%,
90% or 100%). Examples of TIMP-3 muteins with at least some degree
of heparin independence include, but are not limited to, TIMP-3
K45S, F57N fused to HSA; TIMP-3 K45N/V47T, P56N/G58T, K94N/E96T,
R138T (SEQ ID NO: 4); TIMP-3 K45N/V47T, K94N/E96T, D110N/K112T,
G173T (SEQ ID NO: 9); TIMP-3 H78N/Q80T, K94N/E96T, D110N/K112T,
R138T (SEQ ID NO: 26); and TIMP-3 H78N/Q80T, K94N/E96T,
D110N/K112T, R138T (SEQ ID NO: 26) fused to HSA. N-TIMP-3 fused to
HSA can be produced using reduced levels of heparin. In one
embodiment, there are provided TIMP-3 variants or muteins
comprising inserted glycosylation sites. As is known in the art,
glycosylation patterns can depend on both the sequence of the
protein (e.g., the presence or absence of particular glycosylation
amino acid residues, discussed below), or the host cell or organism
in which the protein is produced. Particular expression systems are
discussed below. The presence, absence, or degree of glycosylation
may be determined by any method that is known to one of skill in
the art, including semiqualitative measures of shifts in molecular
weight (MW) as observed by western blotting or from coomassie
stained SDS-PAGE gels, while quantitative measures can include
utilizing mass spectrophotometer techniques and observation of MW
shifts corresponding to addition of asparagine-linked
glycosylation, or through observation of mass shift with the
removal of asparagine-linked glycosylation by an enzyme such as
Peptide-N-Glycosidase F (PNGase-F; SigmaAldrich, St. Louis,
Mo.).
[0059] Glycosylation of polypeptides is typically either N-linked
or O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tri-peptide
sequences asparagine-X-serine (N X S) and asparagine-X-threonine (N
X T), where X is any amino acid except proline, are the recognition
sequences for enzymatic attachment of the carbohydrate moiety to
the asparagine side chain. Thus, the presence of either of these
tri-peptide sequences in a polypeptide creates a potential
glycosylation site. O-linked glycosylation refers to the attachment
of one of the sugars N-acetylgalactosamine, galactose, or xylose,
to a hydroxyamino acid, most commonly serine or threonine, although
5-hydroxyproline or 5-hydroxylysine may also be used.
[0060] Addition of glycosylation sites to a protein (e.g., TIMP-3)
is conveniently accomplished by altering the amino acid sequence
such that it contains one or more of the above-described
tri-peptide sequences (for N-linked glycosylation sites). The
alteration may also be made by the addition of, or substitution by,
one or more serine or threonine residues to the starting sequence
(for O-linked glycosylation sites). For ease, the protein amino
acid sequence is preferably altered through changes at the DNA
level, particularly by mutating the DNA encoding the target
polypeptide at preselected bases such that codons are generated
that will translate into the desired amino acids.
[0061] Accordingly, N-linked glycosylation sites may be adding by
altering a codon for a single amino acid. For example, codons
encoding N-X-z (where z is any amino acid) can be altered to encode
N-X-T (or N-X-S), or codons encoding y-X-T/S can be altered to
encode N-X-T/S. Alternatively, codons encoding two amino acids can
be simultaneously changed to introduce an N-linked glycosylation
site (for example, codons for y-X-z can be altered to encode
N-X-T/S). In this manner, from one to twelve N-linked glycosylation
sites can be inserted. Glycosylation insertion may also be useful
for expression improvement (see, for example, Enhancing the
Secretion of Recombinant Proteins by Engineering N-Glycosylation
Sites. Liu Y. et al, Amer Inst Chem Eng 2009, pg. 1468).
[0062] In addition to inserting N-linked glycosylation sites into
TIMP-3, any glycosylation sites that are present in native TIMP-3
can be modified, for example in an effort to stabilize the
structure of the molecule. Thus, for example, the A at residue 208
may be substituted with a different residue, such as Y, V, or G.
Additional modifications at the `N-X-T` site at residues 206-208
include substituting F for I at residue 205, or Y for I at residue
205, in combination with one of the aforementioned substitutions at
residue 208.
[0063] Thus, in another embodiment, a sub-set of solvent exposed
sites developed by computational analysis are screened for
N-glycosylation likelihood. For methods involving insertion of
glycosylation sites, an N-glycosylation prediction tool is useful
in selecting sites that may be mutated to facilitate potential
N-linked glycosylation, for example by identifying residues that
could be mutated to form a canonical N-x-T glycosylation site
(where N is asparagine, x is any amino acid and T is threonine). In
a further embodiment, structure based methods are used to identify
all solvent exposed amino acids (including those amino acids with
sidechain exposure >20 .ANG..sup.2). An additional embodiment
includes the mutation of LRP1 interacting lysines on TIMP-3, based
upon the crystal structure of LRP1/RAP (Receptor Associated
Protein) with interacting RAP lysines mapped against TIMP-3.
[0064] Additional combinations are contemplated herein. For
example, any mutation disclosed herein can be made in combination
with a mutation at a lysine residue, wherein the lysine residue is
any lysine in TIMP-3. In one embodiment, a single lysine is
mutated; in another embodiment, two, three, four or five lysine
residues are mutated. In certain embodiments, lysine residues at
amino acid 45 and/or 133 can be mutated. In another example, a
mutation introduces a single N-linked glycosylation site; this
mutation can be made with additional mutations to introduce
additional glycosylation sites, or with other mutations designed to
affect another property of TIMP-3. Contemplated herein are TIMP-3
muteins or variants, that comprise one introduced N-linked
glycosylation site, that comprise two, three or four introduced
N-linked glycosylation sites, and that comprise five or more
introduced N-linked glycosylation sites.
[0065] Particular mutations are shown in FIGS. 1 and 2 of U.S.
application Ser. No. 14/207,178, filed 12 Mar. 2014, and PCT
Application PCT/US2014/026811, filed 13 Mar. 2014, the disclosures
of which are incorporated by reference herein. Those Figures
present an alignment of native, full-length human TIMP-3 and a
mutated form of full-length human TIMP-3 in which the letter "X"
has been substituted for particular amino acids within the
sequence. The signal sequence is underlined; other signal sequences
can be substituted therefore, as described herein.
[0066] The amino acid sequences of selected muteins are presented
herein in the Sequence Listing. Full length protein sequences are
provided. In many instances, a signal sequence is not present in
the sequences set forth for the various muteins in the sequence
listing to facilitate a consistent amino acid residue numbering
system and the understanding of those of skill in the art of the
amino acid designations used herein. The invention includes the
mutein sequences set forth herein further comprising a signal
sequence which is, in various embodiments, the sequence provided in
SEQ ID NO:2 as amino acids 1-23 (i.e., MTPWLGLIVLLGSWSLGDWGAEA).
SEQ ID NO:2 is a representative native TIMP-3 amino acid sequence.
One of skill in the art will appreciate that the signal peptide is
removed during processing of the protein to result in a mature
protein with an N-terminus starting with the amino acid cysteine.
In various embodiments, the N-terminal cysteine is preserved in the
TIMP-3 mutein. One of skill in the art will also appreciate that
between expression of TIMP-3 mutein DNA in a cell and isolation of
the protein, post-translational modification of the protein occurs.
Specific examples of post-translational modifications include
glycosylation (e.g., N-linked glycosylation) and removal of the
signal peptide; further modifications including phosphorylation,
ubiquitination, nitrosylation, methylation, acetylation,
lipidation, proteolysis, and the like also are contemplated.
[0067] It is known that the native TIMP-3 signal sequence can be
used to express TIMP-3 muteins, or another signal sequence can be
substituted. Thus, the amino acid at residue 1 can be M or another
amino acid; the amino acid at residue 2 can be T or another amino
acid, the amino acid at residue 3 can be P or another amino acid,
etc. through amino acid 23. Additionally, a signal sequence can
comprise additional amino acids (i.e., be longer than the signal
sequence of naive TIMP-3), or can comprise fewer amino acids than
23 (i.e., be shorter than the signal sequence of naive TIMP-3).
Regardless of the length of the signal sequence, those of ordinary
skill in the art will be able to utilize the numbering system
herein to prepare the presently disclosed TIMP-3 muteins, as well
as other muteins that could be made.
[0068] Certain substitutions are envisioned in the mature form of
TIMP-3, and are designated herein as "n #m" where "n" designates
the amino acid found in the native, full-length TIMP-3, "#"
designates the amino acid residue number, and "m" designates the
amino acid that has been substituted. Thus, for example, "K45N"
indicates that the lysine (K) at amino acid 45 has been substituted
with asparagine (N). The mutated forms of human TIMP-3 exemplified
herein comprise the following mutations (alone, or in combination):
K45N; V47T; K50N; V52T H78N; K94N; E96T; D110N; K112T; R138T; and
G173T. Combinations of these mutations are also contemplated, and
can include from two to twelve (i.e., 2, 3, 4, 5, 6, 7 8, 9, 10, 11
or 12) of the afore-mentioned substitutions. For example, in one
embodiment, the TIMP-3 mutein comprises (or consists of) amino
acids 24-211 of SEQ ID NO:2 having the following substitutions:
H78N, Q80T, K94N, E96T, D110N, K112T, and R138T.
[0069] Specific combinations of mutations include K50N/V52T,
D110N/K112T, R138T, G173T; K45N/V47T, D110N/K112T, R138T, G173T;
H78N/Q80T, D110N/K112T, R138T, G173T; K45N/V47T, K50N/V52T,
H78N/A80T, R138T; K45N/V47T, H78N/Q80T D110N/K112T, G173T;
K45N/V47T, H78N/Q80T, R138T, G173T; K50N/V52T, H78N/Q80T,
K94N/E96T, G173T; K50N/V52T, H78N/Q80T, D110N/K112T, R138T;
K45N/V47T, K50N/V52T, H78N/Q80T, D110N/K112T; K50N/V52T, H78N/Q80T,
R138T, G173T; K45N/V47T, H78N/Q80T, R138T, G173T; K45N/V47T,
H78N/Q80T, D110N/K112T, R138T; K45N/V47T, K50N/V52T, H78N/Q80T,
D110N/K112T, G173T; K45N/V47T, K50N/V52T, H78N/Q80T, R138T, G173T;
K45N/V47T, K50N/V52T, H78N/Q80T, K94N/E96T, G173T; K45N/V47T,
H78N/Q80T, K94N/E96T, R138T, G173T; K50N/V52T, H78N/Q80T,
K94N/E96T, R138T, G173T; K45N/V47T, H78N/Q80T, D110N/K112T, R138T,
G173T; K50N/V52T, H78N/Q80T, D110N/K112T, R138T, G173T; and
K45N/V52T, K50N/V52T, H78N/Q80T, D110N/K112T, R138T.
[0070] Additional combinations include K50N/V52T, D110N/K112T,
R138T, G173T; K45N/V47T, D110N/K112T, R138T, G173T; H78N/Q80T,
D110N/K112T, R138T, G173T; K45N/V47T, K50N/V52T, H78N/A80T, R138T;
K45N/V47T, H78N/Q80T D110N/K112T, G173T; K45N/V47T, H78N/Q80T,
R138T, G173T; K50N/V52T, H78N/Q80T, K94N/E96T, G173T; K50N/V52T,
H78N/Q80T, D110N/K112T, R138T; K45N/V47T, K50N/V52T, H78N/Q80T,
D110N/K112T; K50N/V52T, H78N/Q80T, R138T, G173T; K45N/V47T,
H78N/Q80T, R138T, G173T; K45N/V47T, H78N/Q80T, D110N/K112T, R138T;
K45N/V47T, K50N/V52T, H78N/Q80T, D110N/K112T, G173T; K45N/V47T,
K50N/V52T, H78N/Q80T, R138T, G173T; K45N/V47T, K50N/V52T,
H78N/Q80T, K94N/E96T, G173T; K45N/V47T, H78N/Q80T, K94N/E96T,
R138T, G173T; K50N/V52T, H78N/Q80T, K94N/E96T, R138T, G173T;
K45N/V47T, H78N/Q80T, D110N/K112T, R138T, G173T; K50N/V52T,
H78N/Q80T, D110N/K112T, R138T, G173T; and K45N/V47T, K50N/V52T,
H78N/Q80T, D110N/K112T, R138T.
[0071] In various embodiments, the mutein is K45N, V47T, P56N,
G58T, Q126N, R138T (SEQ ID NO:3); K45N, V47T, P56N, G58T, K94N,
E96T, R138T (SEQ ID NO:4); K45N, V47T, P56N, G58T, R138T, G173T
(SEQ ID NO:5); K45N, V47T, F57N, K94N, E96T, D110N, K112T (SEQ ID
NO:6); K45N, V47T, F57N, K94N, E96T, R138T (SEQ ID NO:7); K45N,
V47T, H78N, Q80T, K94N, E96T, R138T, G173T (SEQ ID NO:8); K45N,
V47T, K94N, E96T, D110N, K112T, R138T (SEQ ID NO:9); K45N, V47T,
K94N, E96T, D110N, K112T, G173T (SEQ ID NO:10); K45N, V47T, K94N,
E96T, R138T, G173T (SEQ ID NO:11); K45S, F57N, K94N, E96T, D110N,
K112T, R138T (SEQ ID NO:12); K45S, F57N, H78N, Q80T, K94N, E96T,
R138T (SEQ ID NO:13); K50N, V52T P56N, G58T, K94N, E96T, D110N,
K112T, R138T (SEQ ID NO:14); K50N, V52T, H78N, Q80T, K94N, E96T,
R138T, G173T (SEQ ID NO:15); K50N, V52T, K94N, E96T, D110N, K112T,
R138T (SEQ ID NO:16); K50N, V52T, K94N, E96T, D110N, K112T, R138T,
G173T (SEQ ID NO:17); K50N, V52T, K94N, E96T, R138T, G173T (SEQ ID
NO:18); K50N, V52T, Q126N, R138T, G173T (SEQ ID NO:19); P56N, G58T,
H78N, Q80T, K94N, E96T, R138T (SEQ ID NO:20); P56N, G58T K94N,
E96T, Q126N, R138T (SEQ ID NO:21); P56N, G58T, K94N, E96T, D110N,
K112T, R138T (SEQ ID NO:22); P56N, G58T, H78N, Q80T, K94N, E96T,
G173T (SEQ ID NO:23); P56N, G58T, Q126N, R138T, G173T (SEQ ID
NO:24); H78N, Q80T, K94N, E96T, R138T, G173T (SEQ ID NO:25).
[0072] The TIMP-3 variants, muteins or derivative have an amino
acid sequence that is quite similar to that of native TIMP-3. In
one embodiment, a TIMP-3 variant, mutein or derivative will be at
least 85% identical to native TIMP-3; in another embodiment, a
TIMP-3 variant, mutein or derivative will be at least 90% identical
to native TIMP-3; in another embodiment, a TIMP-3 variant, mutein
or derivative will be at least 95% identical to native TIMP-3. In
further embodiments, a TIMP-3 variant, mutein or derivative is at
least 96% identical, 97% identical, 98% identical or 99% identical
to native TIMP-3. As used herein, the percent identities refer to a
comparison of the mature, full-length variant, mutein or derivative
to the mature, full-length form of native TIMP-3, i.e., TIMP-3
lacking a signal peptide (amino acids 24 through 211 of TIMP-3).
Those of skill in the art will readily understand that a similar
comparison can be made between a variant, mutein or derivative of
the N-terminal domain of TIMP-3 and the N-terminal domain of native
TIMP-3.
[0073] Similarity can also be expressed by the number of amino
acids that differ between a mutein or variant and a native TIMP-3.
For example, a TIMP-3 variant or mutein can vary from native TIMP-3
by one amino acid, two amino acids, three amino acids, four amino
acids, five amino acids, six amino acids, seven amino acids, eight
amino acids, nine amino acids, or ten amino acids. A variant or
mutein that differs from native TIMP-3 at ten amino acids will be
about 95% identical to native TIMP-3. In further embodiments, a
TIMP-3 variant or mutein differs from native mature TIMP-3 at 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids
[0074] Additional changes can be made in a nucleic acid encoding a
TIMP-3 polypeptide (either native, mutein, variant or derivative)
to facilitate expression. For example, the signal peptide of native
TIMP-3 can be substituted with a different signal peptide.
[0075] Other derivatives of TIMP-3 polypeptides within the scope of
this invention include covalent or aggregative conjugates of TIMP-3
polypeptides, or fragments thereof, with other proteins or
polypeptides, such as by expression of recombinant fusion proteins
comprising heterologous polypeptides fused to the N-terminus or
C-terminus of a TIMP-3 polypeptide. For example, the conjugated
peptide may be a heterologous signal (or leader) peptide, e.g., the
yeast alpha-factor leader, or a peptide such as an epitope tag.
Those of ordinary skill in the art understand that a heterologous
signal peptide may differ in length from the native TIMP-3 signal
peptide, but can correctly identify the location of muteins with
respect to the amino acid sequence of mature TIMP-3 by aligning the
N-terminal cysteine residues of TIMP-3 polypeptides produced using
a heterologous signal peptide.
[0076] TIMP-3 polypeptide-containing fusion proteins can comprise
peptides added to facilitate purification or identification of the
TIMP-3 polypeptide (e.g., poly-His). Another tag peptide is the
FLAG.RTM. peptide described in Hopp et al., Bio/Technology 6:1204,
1988, and U.S. Pat. No. 5,011,912. The FLAG.RTM. peptide is highly
antigenic and provides an epitope reversibly bound by a specific
monoclonal antibody (mAb), enabling rapid assay and facile
purification of expressed recombinant protein. Reagents useful for
preparing fusion proteins in which the FLAG.RTM. peptide is fused
to a given polypeptide are commercially available (Sigma, St.
Louis, Mo.).
[0077] In various embodiments, the TIMP-3 polypeptide described
herein (e.g., any one of the TIMP-3 muteins described herein) is
fused to a moiety that extends the half-life of the polypeptide in
vivo. Exemplary moieties include, but are not limited to, an
antibody (e.g., IgG) or a fragment thereof (e.g., the Fc portion of
an antibody such as an IgG) or albumin (e.g., human serum albumin).
Alternatively or in addition, the TIMP-3 polypeptide comprises an
albumin binding domain or fatty acid that binds albumin when
administered in vivo. An example of an albumin binding domain is
"albu-tag," a moiety derived from on 4-(p-iodophenyl)-butanoic acid
(Dumelin et al., Angew Chem Int Ed Engl 47:3196-3201 (2008)). The
moiety may be fused to the N-terminus of the TIMP-3 polypeptide or
fused to the C-terminus, and the moiety itself may be in any
orientation (i.e., connected by the moiety N- or C-terminus).
Optionally, the moiety is attached to the TIMP-3 polypeptide via a
linker, such as a flexible peptide linker (e.g., a linker
comprising 1-10 or 2-4 glycines, for example, four glycines, or
EPKSS (SEQ ID NO: 75)). Examples of fusion partners for the TIMP-3
polypeptides described herein include, but are not limited to human
serum albumin of SEQ ID NO: 71, human FcG1 of SEQ ID NO: 72,
Fc-mono of SEQ ID NO: 73, and human Fc-mono Ndel5 of SEQ ID NO: 74.
The invention contemplates fusion proteins comprising any of the
muteins described herein (e.g., SEQ ID NOs: 3-26) fused to any of
the fusion partners described herein (e.g., SEQ ID NOs: 71-74).
[0078] Covalent modifications are also considered derivatives of
the TIMP-3 polypeptides and are included within the scope of this
invention, and are generally, but not always, done
post-translationally. For example, several types of covalent
modifications of the TIMP-3 are introduced into the molecule by
reacting specific amino acid residues of the antigen binding
protein with an organic derivatizing agent that is capable of
reacting with selected side chains or the N- or C-terminal
residues.
[0079] Cysteinyl residues most commonly are reacted with
alpha-haloacetates (and corresponding amines), such as chloroacetic
acid or chloroacetamide, to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteinyl residues also are
derivatized by reaction with bromotrifluoroacetone,
alpha-bromo-beta-(5-imidozoyl)propionic acid, chloroacetyl
phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl
2-pyridyl disulfide, p-chloromercuri benzoate,
2-chloromercuri-4-nitrophenol, or
chloro-7-nitrobenzo-2-oxa-1,3-diazole. Accordingly, in one aspect
of the invention, cysteinyl residues are added to the native TIMP-3
sequence, for example by altering selected codon(s) to encode Cys.
Such Cys substitution can be made in regions of TIMP-3 that are
shown to be important for expression, folding or other properties
as shown herein.
[0080] The number of carbohydrate moieties on the proteins of the
invention can be increased by chemical or enzymatic coupling of
glycosides to the protein. These procedures are advantageous in
that they do not require production of the protein in a host cell
that has glycosylation capabilities for N- and O-linked
glycosylation. Depending on the coupling mode used, the sugar(s)
may be attached to (a) arginine and histidine, (b) free carboxyl
groups, (c) free sulfhydryl groups such as those of cysteine, (d)
free hydroxyl groups such as those of serine, threonine, or
hydroxyproline, (e) aromatic residues such as those of
phenylalanine, tyrosine, or tryptophan, or (f) the amide group of
glutamine. These methods are described in WO 87/05330 published
Sep. 11, 1987, and in Aplin and Wriston, 1981, CRC Crit. Rev.
Biochem., pp. 259-306.
[0081] Removal of carbohydrate moieties present on the starting
recombinant protein may be accomplished chemically or
enzymatically. Chemical deglycosylation requires exposure of the
protein to the compound trifluoromethanesulfonic acid, or an
equivalent compound. This treatment results in the cleavage of most
or all sugars except the linking sugar (N-acetylglucosamine or
N-acetylgalactosamine), while leaving the polypeptide intact.
Chemical deglycosylation is described by Hakimuddin et al., 1987,
Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981, Anal.
Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on
polypeptides can be achieved by the use of a variety of endo- and
exo-glycosidases as described by Thotakura et al., 1987, Meth.
Enzymol. 138:350. Glycosylation at potential glycosylation sites
may be prevented by the use of the compound tunicamycin as
described by Duskin et al., 1982, J. Biol. Chem. 257:3105.
Tunicamycin blocks the formation of protein-N-glycoside
linkages.
[0082] Another type of covalent modification of the antigen binding
protein comprises linking the protein to various nonproteinaceous
polymers, including, but not limited to, various polyols such as
polyethylene glycol (e.g., PEG approximately 40 kD, 30 kD, 20 kD,
10, kD, 5 kD, or 1 kD in size), polypropylene glycol or
polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
Other useful polymers include, but are not limited to,
monomethoxy-polyethylene glycol, dextran, hydroxyethyl starch,
cellulose, poly-(N-vinyl pyrrolidone)-polyethylene glycol,
propylene glycol homopolymers, a polypropylene oxide/ethylene oxide
co-polymer, polysialic acid (PSA), polyoxyethylated polyols (e.g.,
glycerol) and polyvinyl alcohol, as well as mixtures of any of the
foregoing. In one aspect, the TIMP-3 polypeptide of the invention
is a PEGylated peptide. In addition, as is known in the art, amino
acid substitutions may be made in various positions within the
protein to facilitate the addition of such polymers.
[0083] In various aspects, the modifications to the native TIMP-3
amino acid sequence to arrive at the TIMP-3 variant, mutein, or
derivative of the invention does not substantially diminish native
TIMP-3 activity. For example, the TIMP-3 variant, mutein, or
derivative preferably inhibits one or more matrix
metalloproteinases (e.g., MMP-2, MMP-9, and/or MMP-13), inhibits
one or more aggrecanases (ADAMS) (e.g., ADAMTS4 and/or ADAMTSS),
inhibits tumor-necrosis factor alpha (TNF-alpha)-converting enzyme
(TACE), inhibits TNF-alpha production in vitro or in vivo, inhibits
extracellular matrix degradation, and/or inhibits inflammation.
Exemplary methods of characterizing the activity of a TIMP-3
polypeptide are provided in the Examples. Optionally, the TIMP-3
variant, mutein, or derivative exhibits at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, at least about 90%, at least about
95%, or about 100% of any one of the activities associated with
native TIMP-3, including the activities set forth above.
Alternatively or in addition, the TIMP-3 variant, mutein, or
derivative optionally exhibits no more than a 10-fold decrease, no
more than a 5-fold decrease, or no more than a 2-fold decrease in
activity (e.g., MMP-2 or MMP-9 inhibition) compared to native
TIMP-3.
[0084] Expression of TIMP-3 Polypeptides
[0085] Any expression system known in the art can be used to make
the recombinant polypeptides of the invention. In general, host
cells are transformed with a recombinant expression vector that
comprises DNA encoding a desired TIMP-3 polypeptide (including
TIMP-3 muteins or variants). Among the host cells that may be
employed are prokaryotes, yeast or higher eukaryotic cells.
Prokaryotes include gram negative or gram positive organisms, for
example E. coli or bacilli. Higher eukaryotic cells include insect
cells and established cell lines of mammalian origin. Examples of
suitable mammalian host cell lines include the COS-7 line of monkey
kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L
cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese
hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell
lines, and the CVI/EBNA cell line derived from the African green
monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan
et al., 1991, EMBO J. 10: 2821. Appropriate cloning and expression
vectors for use with bacterial, fungal, yeast, and mammalian
cellular hosts are described by Pouwels et al. (Cloning Vectors: A
Laboratory Manual, Elsevier, New York, 1985).
[0086] Mammalian cell expression can provide advantages for the
production of TIMP-3 polypeptides, in facilitating folding and
adoption of conformation that closely resembles that of native
TIMP-3. Numerous mammalian cell expression systems are known in the
art, and/or are commercially available; the latter includes systems
such as Gibco.RTM. Freedom.RTM. CHO-S.RTM. (a product designed for
ease of use with all aspects of cloning and expression of
recombinant proteins in Chinese Hamster Ovary (CHO)-derived
suspension culture; ProBioGen, Life Technologies; Carlsbad,
Calif.), GS Gene Expression System.TM. (a transfection system
designed to provide development of high-yielding, stable,
cGMP-compatible mammalian cell lines; Lonza Biologics, Slough, UK),
PER.C6.RTM. technology (a package of tools designed to facilitate
the large-scale production of recombinant proteins, utilizing a
continuously dividing set of cells derived from a single,
immortalized human cell; Crucell, Leiden, The Netherlands), or
immortalized amniocyte cells such as CAP and CAP-T (human
cell-based expression systems for the expression and production of
complex proteins; Cevec, Cologne, Germany).
[0087] Additional cell expression systems include systems such as
the Selexis SUREtechnology Platform.TM. (a technology platform that
can be applied to a variety of cell lines to facilitate development
cell lines for the production of recombinant proteins; Selexis
Inc., Switzerland); ProFection.RTM. Mammalian Transfection Systems
(a transfection system that provides high-efficiency transfections
of cells for the production of recombinant proteins; Promega,
Madison Wis.); the Expi293.TM. Expression System (a high-density
mammalian transient protein expression system, Life Technologies,
Grand Island, N.Y.); and MaxCyte.RTM. VLX.TM. and STX.TM. Transient
Transfection Systems (a scalable transfection system for use in the
production of recombinant proteins, including antibodies; MaxCyte,
Gaithersurg, Md.). Those of skill in the art are further aware of
other expression systems, such as techniques originally described
by Wigler et al. (Cell 1979:777) and additional techniques that are
described, for example, by the National Research Council of Canada
on their website.
[0088] Various vessels are known in the art to be suitable for the
culture of transformed cells and production of recombinant
proteins. These include 24-deep well plates, 250 ml and 1 L
shakeflasks; and various bioreactors of various sizes, for example,
2 L, 5 L, 10 L, 30 L, 100 L, 1000 L, 10000 L and larger
Bioreactors. Other suitable vessels for cell culture are known in
the art and can also be used as described herein.
[0089] Cell culture media formulations are well known in the art;
typically, a culture medium provides essential and non-essential
amino acids, vitamins, energy sources, lipids, and trace elements
required by the cell for minimal growth and/or survival, as well as
buffers, and salts. A culture medium may also contain supplementary
components that enhance growth and/or survival above the minimal
rate, including, but not limited to, hormones and/or other growth
factors, particular ions (such as sodium, chloride, calcium,
magnesium, and phosphate), buffers, vitamins, nucleosides or
nucleotides, trace elements (inorganic compounds usually present at
very low final concentrations), amino acids, lipids, and/or glucose
or other energy source; as described herein, cell-cycle inhibitors
can be added to a culture medium. In certain embodiments, a medium
is advantageously formulated to a pH and salt concentration optimal
for cell survival and proliferation. In certain embodiments, the
medium is a feed medium that is added after the beginning of the
cell culture. In certain embodiments, the cell culture medium is a
mixture of a starting nutrient solution and any feed medium that is
added after the beginning of the cell culture.
[0090] Various tissue culture media, including defined culture
media, are commercially available, for example, any one or a
combination of the following cell culture media can be used:
RPMI-1640 Medium, RPMI-1641 Medium, Dulbecco's Modified Eagle's
Medium (DMEM), Minimum Essential Medium Eagle, F-12K Medium, Ham's
F12 Medium, Iscove's Modified Dulbecco's Medium, McCoy's 5A Medium,
Leibovitz's L-15 Medium, and serum-free media such as EX-CELL.TM.
300 Series (JRH Biosciences, Lenexa, Kans.), among others.
Serum-free versions of such culture media are also available. Cell
culture media may be supplemented with additional or increased
concentrations of components such as amino acids, salts, sugars,
vitamins, hormones, growth factors, buffers, antibiotics, lipids,
trace elements and the like, depending on the requirements of the
cells to be cultured and/or the desired cell culture
parameters.
[0091] The transformed cells can be cultured under conditions that
promote expression of the polypeptide, and the polypeptide
recovered by conventional protein purification procedures.
[0092] One such purification procedure includes the use of affinity
chromatography as well as other methods that are known in the art.
One method to isolate TIMP-3 parent or TIMP-3 muteins from
mammalian supernatants is to utilize a TIMP-3 that is fused to a
carboxy-terminal 6.times.-Histidine tag in combination a
6.times.-Histidine affinity Ni-Sepharose resin (for example,
Immobilized Metal Affinity Chromatography (IMAC); general
procedures are known in the art, and reagents for, and examples of
such procedures are outlined by QIAGEN, Germantown, Md. and GE
Healthcare, Pittsburgh, Pa.). Cation exchange chromatography (e.g.
SP-HP Sepharose.RTM., GE Healthcare) can be utilized to further
isolate TIMP-3 post IMAC elution, or as an alternative strategy
without the use of IMAC to capture TIMP-3 from mammalian
supernatants (elution of TIMP-3 and muteins thereof occurs with the
use of a sodium chloride gradient at neutral pH). Size Exclusion
Chromatography (e.g., Superdex 200.RTM., GE Healthcare, (mobile
phase example: 10 mM Na.sub.2HPO.sub.4, 1.8 mM KH2PO4, 137 mM NaCl,
2.7 mM KCl)) is a general strategy that can be used to further
isolate TIMP-3 or muteins thereof (in combination with an IMAC
process or ion exchange chromatography. These and other methods are
known in the art; see for example, Protein Purification:
Principles: High Resolution Methods, and Applications, Third
Edition (2012, John Wiley and Sons; Hoboken, N.J.).
[0093] The amount of polypeptide (native TIMP-3 or a TIMP-3 mutein
or variant) can be determined by any suitable, quantitative or
semi-quantitative method that will allow analysis of the amount of
recombinant TIMP-3 (native, variant or mutein) in cell culture
supernatant fluid, i.e., conditioned media (CM). Suitable
qualitative or semi-quantitative methods include Western Blot and
Coomassie stained SDS PAGE gels. Quantitative measurements could
include use of an enzyme immunoassay such as a human TIMP-3 ELISA
(R&D Systems Inc., Minneapolis, Minn.), or ForteBio Octet.RTM.
(Pall ForteBio Corp, Menlo Park, Calif.) with antibody mediated
capture of TIMP-3, or direct UV (ultraviolet) absorbance (280 nm)
measurements on purified TIMP-3.
[0094] Thus, the effects of a particular mutation in TIMP-3 can be
evaluated by comparing the amount of recombinant mutein made to the
amount of native protein made under similar culture conditions. A
TIMP-3 mutein or variant can be expressed at levels that are
1.times., 2.times., 3.times., 4.times., 5.times., 10.times. or
greater than levels observed for native TIMP-3. If desired, the
specific productivity of a particular transformed or transfected
cell line can be determined to allow comparison or the specific
productivity for various forms of TIMP-3. Specific productivity, or
qP, is expressed in picograms of recombinant protein per cell per
day (pg/c/d), and can be readily determined by applying methods
known in the art to quantitated the cells in a culture and the
above-mentioned methods of quantifying recombinant protein.
[0095] Uses for TIMP-3 Polypeptides
[0096] TIMP-3 polypeptides, variants, muteins or derivatives can be
used, for example, in assays, or they can be employed in treating
any condition in which a greater level of TIMP-3 activity is
desired (i.e., conditions in which matrix metalloproteases (MMPs)
and/or other proteinases that are inhibited or inhibitable by
TIMP-3 play a causative or exacerbating role), including but not
limited to inflammatory conditions, osteoarthritis, and other
conditions in which excessive or inappropriate MMP activity occurs
(for example, myocardial ischemia, reperfusion injury,
vasculopathy, neointima formation, and during the progression to
chronic heart failure (e.g., congestive heart failure)).
Inflammatory conditions include asthma, chronic obstructive
pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF),
inflammatory bowel disease (for example, ulcerative colitis,
Crohn's disease, and celiac disease), psoriases, myocarditis
including viral myocarditis, inflammation related to
atherosclerosis, and arthritic conditions including rheumatoid
arthritis, psoriatic arthritis, and the like.
[0097] The TIMP-3 polypeptide, variant, mutein or derivative
compositions described herein modify the pathogenesis and provide a
beneficial therapy for diseases or conditions characterized by
matrix degradation and/or inflammation, i.e., those in which
metalloproteinases play a deleterious role. The compositions may be
used alone or in conjunction with one or more agents used in
treating such conditions. Accordingly, the present TIMP-3
polypeptide, variant, mutein or derivative compositions may be
useful in the treatment of any disorder where excessive matrix loss
(degradation) is caused by metalloproteinase activity. The
inventive TIMP-3 variant, mutein or derivative compositions are
useful, alone or in combination with other drugs, in the treatment
of various disorders linked to the overproduction of collagenase,
gelatinase, aggrecanase, or other matrix-degrading or
inflammation-promoting enzyme(s), including dystrophic
epidermolysis bullosa, osteoarthritis, pseudogout, rheumatoid
arthritis including juvenile rheumatoid arthritis, ankylosing
spondylitis, scleroderma, periodontal disease, ulceration including
corneal, epidermal, or gastric ulceration, wound healing after
surgery, and restenosis. Other pathological conditions in which
excessive collagen and/or proteoglycan degradation may play a role
and thus where TIMP-3 polypeptide, variant, mutein or derivative
compositions can be applied, include emphysema, Paget's disease of
bone, osteoporosis, scleroderma, pressure atrophy of bone or
tissues as in bedsores, cholesteatoma, and abnormal wound healing.
Additional conditions that are, directly or indirectly, a result of
decreased amounts of TIMP-3 or increased amounts of
metalloproteases (for example, in myocardial ischemia, reperfusion
injury, and during the progression to congestive heart failure) may
also be treated with the presently described compositions, either
alone or in conjunction with other drugs commonly used to treat
individuals afflicted with such conditions. The compositions
described herein are useful for vascular plaque stabilization and
inhibition of vascular neointima formation. TIMP-3 polypeptide,
variant, mutein or derivative compositions can additionally be
applied as an adjunct to other wound healing promoters, e.g., to
modulate the turnover of collagen during the healing process.
[0098] Many metalloproteinases also exhibit pro-inflammatory
activity; accordingly, additional embodiments include methods of
treating inflammation and/or autoimmune disorders, wherein the
disorders include, but are not limited to, cartilage inflammation,
and/or bone degradation, arthritis, rheumatoid arthritis,
pauciarticular rheumatoid arthritis, polyarticular rheumatoid
arthritis, systemic onset rheumatoid arthritis, ankylosing
spondylitis, enteropathic arthritis, reactive arthritis, SEA
Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome),
dermatomyositis, psoriatic arthritis, scleroderma, systemic lupus
erythematosus, vasculitis, myolitis, polymyolitis, dermatomyolitis,
osteoarthritis, polyarteritis nodossa, Wegener's granulomatosis,
arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary
biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome,
psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis,
pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic
dermatitis, atherosclerosis, lupus, Still's disease, Systemic Lupus
Erythematosus (SLE), myasthenia gravis, inflammatory bowel disease,
ulcerative colitis, Crohn's disease, Celiac disease (nontropical
Sprue), enteropathy associated with seronegative arthropathies,
microscopic or collagenous colitis, eosinophilic gastroenteritis,
or pouchitis resulting after proctocolectomy and ileoanal
anastomosis, pancreatitis, insulin-dependent diabetes mellitus,
mastitis, cholecystitis, cholangitis, pericholangitis, multiple
sclerosis (MS), asthma (including extrinsic and intrinsic asthma as
well as related chronic inflammatory conditions, or
hyperresponsiveness, of the airways), chronic obstructive pulmonary
disease (COPD. i.e., chronic bronchitis, emphysema), Acute
Respiratory Disorder Syndrome (ARDS), respiratory distress
syndrome, cystic fibrosis, pulmonary hypertension, pulmonary
vasoconstriction, acute lung injury, allergic bronchopulmonary
aspergillosis, hypersensitivity pneumonia, eosinophilic pneumonia,
bronchitis, allergic bronchitis bronchiectasis, tuberculosis,
hypersensitivity pneumonitis, occupational asthma, asthma-like
disorders, sarcoid, reactive airway disease (or dysfunction)
syndrome, byssinosis, interstitial lung disease, hyper-eosinophilic
syndrome, rhinitis, sinusitis, and parasitic lung disease, airway
hyperresponsiveness associated with viral-induced conditions (for
example, respiratory syncytial virus (RSV), parainfluenza virus
(PIV), rhinovirus (RV) and adenovirus), Guillain-Barre disease,
Graves' disease, Addison's disease, Raynaud's phenomenon,
autoimmune hepatitis, GVHD, and the like. TIMP-3 polypeptides,
variants, muteins or derivatives also have application in cases
where decreased relative levels of TIMP-3 (i.e., a decrease in the
ratio of endogenous TIMP-3 to metalloproteases, which may be a
result of decreased amounts of TIMP-3 or increased amounts of
metalloproteases) are associated with pathological effects, for
example, in myocardial ischemia, reperfusion injury, and during the
progression to chronic heart failure.
[0099] Based on the ability of TIMP-3 to inhibit connective tissue
degradation, TIMP-3 polypeptides, variants, muteins or derivatives
have application in cases where inhibition of angiogenesis is
useful, e.g., in preventing or retarding tumor development, and the
prevention of the invasion of parasites. For example, in the field
of tumor invasion and metastasis, the metastatic potential of some
particular tumors correlates with the increased ability to
synthesize and secrete collagenases, and with the inability to
synthesize and secrete significant amounts of a metalloproteinase
inhibitor. The presently disclosed TIMP-3 proteins also have
therapeutic application in inhibiting tumor cell dissemination
during removal of primary tumors, during chemotherapy and radiation
therapy, during harvesting of contaminated bone marrow, and during
shunting of carcinomatous ascites. Diagnostically, correlation
between absence of TIMP-3 production in a tumor specimen and its
metastatic potential is useful as a prognostic indicator as well as
an indicator for possible prevention therapy.
[0100] MMPs also act on the basal lamina and tight junction
proteins in the brain, as part of the pathway for opening the
blood-brain barrier (BBB), facilitating the entrance of cells and
soluble mediators of inflammation into the brain. Accordingly, the
present compositions and methods are useful in the treatment of
disorders of the nervous system characterized by excessive or
inappropriate permeabilization of the BBB. Additionally,
degradation of matrix proteins around neurons can result in loss of
contact and cell death; thus, the disclosed TIMP-3 compositions may
protect nerve cells from damage by preserving the basement membrane
surrounding nerve cells. The inventive TIMP-3 compositions are
useful in treating or ameliorating the neuroinflammatory response
to injury, for example, cerebral ischemia, or for traumatic brain
injury. The compositions disclosed herein will also be useful in
the treatment of neurodegenerative diseases where inflammation is
an underlying cause of the disease, for example, multiple
sclerosis, as well as in treatment of various forms of neuropathy
and/or myopathy, spinal cord injury, and amyotrophic lateral
sclerosis (ALS). Accordingly, uses of the inventive compositions
may involve co-administration with BDNF, NT-3, NGF, CNTF, NDF, SCF,
or other nerve cell growth or proliferation modulation factors. In
addition, the present compositions and methods may be applicable
for cosmetic purposes, in that localized inhibition of connective
tissue breakdown may alter the appearance of tissue.
[0101] TIMP-3 polypeptides, variants, muteins or derivatives may be
employed in an in vitro procedure, or administered in vivo to
augment endogenous TIMP-3 activity and/or enhance a TIMP-3-induced
biological activity. The inventive TIMP-3 polypeptides, variants,
muteins or derivative may be employed in vivo under circumstances
in which endogenous TIMP-3 is downregulated or present at low
levels. Disorders caused or exacerbated (directly or indirectly) by
TIMP-3-inhibitable proteinases, examples of which are provided
herein, thus may be treated. In one embodiment, the present
invention provides a therapeutic method comprising in vivo
administration of a TIMP-3 polypeptide, variant, mutein, or
derivative or a nucleic acid encoding the TIMP3 polypeptide,
variant, mutein, or derivative (for example, present in an
expression vector, such as a viral vector (e.g., adenoviral,
retroviral, or adeno-associated viral vector) to a mammal in need
thereof in an amount effective for increasing a TIMP-3-induced
biological activity. In another embodiment, the invention provides
a therapeutic method comprising in vivo administration of a TIMP-3
polypeptide, variant, mutein or derivative to a mammal in need
thereof in an amount effective for elevating endogenous levels of
TIMP-3.
[0102] For example, the invention provides a method of treating a
disorder, such as any one of the disorders described above,
comprising administering to a subject in need thereof an amount of
TIMP-3 variant, mutein or derivative effective to treat the
disorder. The invention further provides use of the TIMP-3 variant,
mutein or derivative described herein in the treatment of a
disorder, such as any one of the disorders described above, as well
as use of the TIMP-3 variant, mutein or derivative in the
preparation of a medicament for the treatment of a disorder. It
will be appreciated that "treating" and "treatment" refers to any
reduction in the severity and/or onset of symptoms associated with
a disorder. Any degree of protection from, or amelioration of, a
disorder or symptom associated therewith is beneficial to a
subject, such as a human patient.
[0103] Included in the invention is a method of inhibiting cardiac
extracellular matrix (ECM) degradation and/or adverse remodeling,
optionally associated myocardial infarction (e.g., acute myocardial
infarction). The method comprises administering to a subject in
need thereof a therapeutically effective amount of TIMP-3 variant,
mutein or derivative, thereby inhibiting ECM degradation and/or
adverse remodeling. Complete inhibition is not required in the
context of the invention; any degree of reduction in ECM
degradation and/or adverse cardiac remodeling is contemplated. ECM
homeostasis is disrupted in the hours following infarction, causing
ECM instability and adverse cardiac remodeling. Adverse cardiac
remodeling results in structural and functional changes in the
heart, such as ventricular wall thinning, left ventricular dilation
(LV EDV increase), systolic and diastolic dysfunction (% ejection
fraction (EF) decrease), infarct expansion and, ultimately, heart
failure. Maintaining ECM homeostasis (in whole or in part) reduces
the severity of tissue damage and improves cardiac function.
Accordingly, the method in one aspect is performed as soon as
possible after it has been determined that a subject is at risk for
myocardial infarction (or any of the disorders described herein) or
as soon as possible after myocardial infarction is detected. For
example, in at least one embodiment, the TIMP-3 variant, mutein or
derivative is administered within 1, 2, 3, 4, 5, 6, 7, 8, 12, or 24
hours of myocardial infarction. Optionally, administration of the
TIMP-3 variant, mutein or derivative results in at least a 3%, at
least a 5%, at least a 10%, or at least a 15% improvement in
ejection fraction (compared to EF in a subject not administered the
TIMP-3 variant, mutein or derivative) following myocardial
infarction, and/or an improvement in cardiac output, and/or a
reduction in left ventricular wall thinning, and/or increase or
maintenance of end-systolic volume or end-diastolic volume.
[0104] In another aspect, the present invention provides TIMP-3
polypeptides, variants, muteins or derivatives having improved
half-life in vivo. In one embodiment, the half-life of a TIMP-3
mutein is at least twice that of native TIMP-3; in another
embodiment, the half-life is at least three times, four times, five
times, six times, eight times or ten times greater than that of
native TIMP-3. Alternatively or in addition, the TIMP-3 variant,
mutein or derivative has a half-life that is at least 0.5 hours
longer, at least 1 hour longer, at least 1.5 hour longer, at least
2 hours longer, at least 3 hours longer, at least 6 hours longer,
at least 8 hours longer, at least 10 hours longer, at least 12
hours longer, or at least 24 hours longer than native TIMP-3 (e.g.,
SEQ ID NO: 2 or amino acids 1-144 of SEQ ID NO: 2). In one
embodiment, the half-life is determined in a non-human mammal; in
another embodiment, the half-life is determined in a human subject.
In various embodiments, the TIMP-3 mutein, variant, or derivative
has a half-life of at least two hours, at least three hours, at
least four hours, at least five hours, or more, e.g., up to 24
hours, up to 18 hours, up to 13 hours, or up to 12 hours. Further
embodiments provide a TIMP-3 mutein or variant that has a half-life
of at least one day in vivo (e.g., when administered to a human
subject). In one embodiment, the TIMP-3 polypeptides, variants,
muteins or derivatives have a half-life of at least three days. In
another embodiment, the TIMP-3 polypeptides, variants, muteins or
derivatives have a half-life of four days or longer or five days or
longer. In another embodiment, the TIMP-3 polypeptides, variants,
muteins or derivatives have a half-life of eight days or longer.
Systemic half-life can be measured (e.g., in plasma) or local, in
situ half-life can be measured (e.g., in cardiac tissue or tissue
adjacent to local administration sites).
[0105] In another embodiment, the TIMP-3 polypeptide, variants, or
muteins is derivatized or modified such that it has a longer
half-life as compared to the underivatized or unmodified TIMP-3
binding protein. The derivatized polypeptide can comprise any
molecule or substance that imparts a desired property to the
polypeptide, such as increased half-life in a particular use. The
derivatized polypeptide can comprise, for example, a detectable (or
labeling) moiety (e.g., a radioactive, colorimetric, antigenic or
enzymatic molecule, a detectable bead (such as a magnetic or
electrodense (e.g., gold) bead), or a molecule that binds to
another molecule (e.g., biotin or streptavidin)), a therapeutic or
diagnostic moiety (e.g., a radioactive, cytotoxic, or
pharmaceutically active moiety), or a molecule that increases the
suitability of the polypeptide for a particular use (e.g.,
administration to a subject, such as a human subject, or other in
vivo or in vitro uses).
[0106] In one such example, the polypeptide is derivatized with a
ligand that specifically binds to articular cartilage tissues, for
example as disclosed in WO2008063291 and/or Rothenfluh et al.,
Nature Materials 7:248 (2008). Examples of molecules that can be
used to derivatize a polypeptide include albumin (e.g., human serum
albumin) and polyethylene glycol (PEG). Albumin-linked and
PEGylated derivatives of polypeptides can be prepared using
techniques well known in the art. In one embodiment, the
polypeptide is conjugated or otherwise linked to transthyretin
(TTR) or a TTR variant. The TTR or TTR variant can be chemically
modified with, for example, a chemical selected from the group
consisting of dextran, poly(n-vinyl pyurrolidone), polyethylene
glycols, propropylene glycol homopolymers, polypropylene
oxide/ethylene oxide co-polymers, polyoxyethylated polyols and
polyvinyl alcohols (US Pat. App. No. 20030195154).
[0107] Compositions
[0108] The invention includes pharmaceutical compositions
comprising effective amounts of polypeptide products (i.e., TIMP-3
polypeptides, variants, muteins or derivatives) of the invention
together with pharmaceutically acceptable diluents, preservatives,
solubilizers, emulsifiers, adjuvants and/or carriers useful in
TIMP-3 therapy (i.e., conditions in which increasing the endogenous
levels of TIMP-3 or augmenting the activity of endogenous TIMP-3
are useful). Such compositions include diluents of various buffer
content (e.g., Tris-HCl, acetate, phosphate), pH and ionic
strength; additives such as detergents and solubilizing agents
(e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic
acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl
alcohol) and bulking substances (e.g., lactose, mannitol); covalent
attachment of polymers such as polyethylene glycol to the protein
(as discussed supra, see, for example U.S. Pat. No. 4,179,337
hereby incorporated by reference); incorporation of the material
into particulate preparations of polymeric compounds such as
polylactic acid, polyglycolic acid, etc. or into liposomes. Such
compositions will influence the physical state, stability, rate of
in vivo release, and rate of in vivo clearance of TIMP-3 binding
proteins. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed.
(1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712
which are herein incorporated by reference.
[0109] Generally, an effective amount of the present polypeptides
will be determined by the age, weight and condition or severity of
disease of the recipient. See, Remingtons Pharmaceutical Sciences,
supra, at pages 697-773, herein incorporated by reference.
Typically, a dosage of between about 0.001 g/kg body weight to
about 1 g/kg body weight (or 1 mg-1000 mg), may be used, but more
or less, as a skilled practitioner will recognize, may be used. For
local (i.e., non-systemic) applications, such as topical or
intra-articular applications, the dosing may be between about 0.001
g/cm.sup.2 to about 1 g/cm.sup.2. In the context of reducing or
inhibiting ECM degradation and/or adverse cardiac tissue
remodeling, a direct injection (or series of injections that
constitute a single administration) into myocardium optionally
comprises 1 mg-50 mg of TIMP-3 polypeptide (e.g., 3 mg-40 mg, 5
mg-30 mg, or 10 mg-25 mg). Dosing may be one or more times daily,
or less frequently, and may be in conjunction with other
compositions as described herein. An administration of TIMP-3
variant, mutein or derivative may be applied one, two, three, four,
five, six, or seven days a week as needed. Alternatively, the
TIMP-3 variant, mutein or derivative is administered once a week,
once every two weeks, once every three weeks, or once every four
weeks (once monthly). In various embodiments, the treatment regimen
comprises a single administration of TIMP-3 polypeptide; for
example, intervention following or during myocardial infarction may
comprise a single administration directly into the heart,
optionally during a surgical procedure. It should be noted that the
present invention is not limited to the dosages recited herein.
[0110] As is understood in the pertinent field, pharmaceutical
compositions comprising the molecules of the invention are
administered to a subject in a manner appropriate to the
indication. Pharmaceutical compositions may be administered by any
suitable technique, including but not limited to parenterally,
topically, locally or by inhalation. If injected, the
pharmaceutical composition can be administered, for example, via
intravenous, intramuscular, intralesional, intraperitoneal or
subcutaneous routes, by bolus injection, or continuous
infusion.
[0111] Localized administration, e.g., at a site of disease or
injury is contemplated, as are transdermal delivery and sustained
release from implants or patches. Other alternatives include
eyedrops; oral preparations including pills, syrups, lozenges or
chewing gum; and topical preparations such as lotions, gels,
sprays, and ointments. For example, localized administration to
joints or the musculoskeletal systems includes periarticular,
intra-articular, intrabursal, intracartilaginous, intrasynovial and
intratendinous administration. Administration to the respiratory
system includes intrapulmonary, intraplural, intrapulmonary,
intratracheal, intrasinal and intrabronchial delivery, and can be
facilitated, for example, by an inhaler or a nebulizer. Intrathecal
delivery and other methods that are useful to introduce
compositions into the brain and/or nervous system are also
contemplated herein, for example, epidural, intradural or
peridural, administration, as well as perineural, intracaudal,
intracerebral, intracisternal, and intraspinal administration.
[0112] Further examples of local administration include delivery to
tissue in conjunction with surgery or another medical procedure.
For example, a pharmaceutical composition of the invention can be
administered to heart tissue during surgery that is performed to
treat or ameliorate cardiac symptoms, or during a procedure such as
cardiac catheterization (for example, percutaneous coronary
intervention or angioplasty). Delivery may be via intracoronary,
intracardial, intramyocardial, epicardial, and/or transendocardial
route, for example, and may be guided by endocardial angiography or
electromechanical maps of the area of the heart to be injected, or
by the use of other techniques, such as magnetic resonance imaging
(MRI). Compositions can also be delivered via inclusion in a
cardiac patch, intracoronary catheter or in the coating of a stent
or other device useful in cardiac conditions. An example of a
suitable delivery device is described in U.S. Provisional Patent
Application No. 62/037,743, filed Aug. 15, 2014, which is hereby
incorporated by reference in its entirety.
[0113] In addition to eye drops, the use of ointments, creams or
gels to administer the present compositions to the eye is also
contemplated. Direct administration to the interior of the eye may
be accomplished by periocular, conjunctival, intracorneal,
subconjunctival, subtenons, retrobulbar, intraocular, and/or
intravitreal injection or administration. These and other
techniques are discussed, for example, in Gibaldi's Drug Delivery
Systems in Pharmaceutical Care (2007, American Society of
Healthe-Sytem Pharmacists, Bethesda, Md.).
[0114] A plurality of agents act in concert in order to maintain
the dynamic equilibrium of the extracellular matrix and tissues. In
treatment of conditions where the equilibrium is skewed, one or
more of the other agents may be used in conjunction with the
present polypeptides. These other agents may be co-administered or
administered in seriatim, or a combination thereof. Generally,
these other agents may be selected from the list consisting of the
metalloproteinases, serine proteases, inhibitors of matrix
degrading enzymes, intracellular enzymes, cell adhesion modulators,
and factors regulating the expression of extracellular matrix
degrading proteinases and their inhibitors. While specific examples
are listed below, one skilled in the art will recognize other
agents performing equivalent functions, including additional
agents, or other forms of the listed agents (such as those produced
synthetically, via recombinant DNA techniques, and analogs and
derivatives).
[0115] Other degradation inhibitors may also be used if increased
or more specific prevention of extracellular matrix degradation is
desired. Inhibitors may be selected from the group consisting of
alpha.sub.2 macroglobulin, pregnancy zone protein, ovostatin,
alpha.sub.1-proteinase inhibitor, alpha.sub.2-antiplasmin,
aprotinin, protease nexin-1, plasminogen activator inhibitor
(PAI)-1, PAI-2, TIMP-1, TIMP-2, and TIMP-4. Others may be used, as
one skilled in the art will recognize.
[0116] Intracellular enzymes may also be used in conjunction with
the present polypeptides. Intracellular enzymes also may affect
extracellular matrix degradation, and include lysozomal enzymes,
glycosidases and cathepsins.
[0117] Cell adhesion modulators may also be used in combination
with the present polypeptides. For example, one may wish to
modulate cell adhesion to the extracellular matrix prior to,
during, or after inhibition of degradation of the extracellular
matrix using the present polypeptides. Cells which have exhibited
cell adhesion to the extracellular matrix include osteoclasts,
macrophages, neutrophils, eosinophils, killer T cells and mast
cells. Cell adhesion modulators include peptides containing an
"RGD" motif or analog or mimetic antagonists or agonists.
[0118] Factors regulating expression of extracellular matrix
degrading proteinases and their inhibitors include cytokines, such
as IL-1 and TNF-alpha, TGF-beta, glucocorticoids, and retinoids.
Other growth factors effecting cell proliferation and/or
differentiation may also be used if the desired effect is to
inhibit degradation of the extracellular matrix using the present
polypeptides, in conjunction with such cellular effects. For
example, during inflammation, one may desire the maintenance of the
extracellular matrix (via inhibition of enzymatic activity) yet
desire the production of neutrophils; therefore one may administer
G-CSF. Other factors include erythropoietin, interleukin family
members, SCF, M-CSF, IGF-I, IGF-II, EGF, FGF family members such as
KGF, PDGF, and others. One may wish additionally the activity of
interferons, such as interferon alpha's, beta's, gamma's, or
consensus interferon. Intracellular agents include G-proteins,
protein kinase C and inositol phosphatases. The use of the present
polypeptides may provide therapeutic benefit with one or more
agents involved in inflammation therapy.
[0119] Cell trafficking agents may also be used. For example,
inflammation involves the degradation of the extracellular matrix,
and the movement, or trafficking of cells to the site of injury.
Prevention of degradation of the extracellular matrix may prevent
such cell trafficking. Use of the present polypeptides in
conjunction with agonists or antagonists of cell
trafficking-modulation agents may therefore be desired in treating
inflammation. Cell trafficking-modulating agents may be selected
from the list consisting of endothelial cell surface receptors
(such as E-selectins and integrins); leukocyte cell surface
receptors (L-selectins); chemokines and chemoattractants. For a
review of compositions involved in inflammation, see Carlos et al.,
Immunol. Rev. 114: 5-28 (1990), which is herein incorporated by
reference.
[0120] Moreover, compositions may include neu differentiation
factor, "NDF," and methods of treatment may include the
administration of NDF before, simultaneously with, or after the
administration of TIMP-3. NDF has been found to stimulate the
production of TIMP-2, and the combination of NDF, TIMP-1, -2 and/or
-3 may provide benefits in treating tumors.
[0121] Polypeptides of the invention may be "labeled" by
association with a detectable marker substance (e.g., radiolabeled
with .sup.125I, or labeled with a fluorophore such as
AlexaFluor.RTM. [LifeTechnologies, Grand Island N.Y.]) or IR dyes
[DyLight 800 NHS ester, Thermo Scientific] to provide reagents
useful in detection and quantification of TIMP-3 in solid tissue
and fluid samples such as blood or urine. Nucleic acid products of
the invention may also be labeled with detectable markers (such as
radiolabels and non-isotopic labels such as biotin) and employed in
hybridization processes to identify relevant genes, for
example.
[0122] As described above, the present TIMP-3 polypeptide, variant,
mutein or derivative compositions have wide application in a
variety of disorders. Thus, another embodiment contemplated herein
is a kit including the present compositions and optionally one or
more of the additional compositions described above for the
treatment of a disorder involving the degradation of extracellular
matrix. An additional embodiment is an article of manufacture
comprising a packaging material and a pharmaceutical agent within
said packaging material, wherein said pharmaceutical agent contains
the present polypeptide(s), variant(s), mutein(s) or derivative(s)
and wherein said packaging material comprises a label which
indicates a therapeutic use for TIMP-3. In some aspects, the
article of manufacture comprises TIMP-3 polypeptide, variant,
mutein or derivative in a desired amount (e.g., 1-1000 mg, 1-100
mg, 1-50 mg, or any of the other amounts disclosed herein). In one
embodiment, the pharmaceutical agent may be used for an indication
selected from the group consisting of: cancer, inflammation,
arthritis (including osteoarthritis and the like), dystrophic
epidermolysis bullosa, periodontal disease, ulceration, emphysema,
bone disorders, scleroderma, wound healing, erythrocyte
deficiencies, cosmetic tissue reconstruction, fertilization or
embryo implant modulation, and nerve cell disorders. This article
of manufacture may optionally include other compositions or label
descriptions of other compositions.
[0123] The following examples are provided for the purpose of
illustrating specific embodiments or features of the instant
invention and do not limit its scope.
EXAMPLES
Example 1
[0124] This Example describes a method used to determine the
effects, if any, of a mutation or mutations in TIMP-3 on expression
in a mammalian expression system. This Example describes a general
vector and host cell system, numerous vector and host cell systems
are known in the art, described herein, and are suitable for
determination of the effects, if any, of particular mutations in a
TIMP-3 sequence on the expression of recombinant protein.
[0125] In general, a TIMP-3-encoding DNA is ligated into an
expression vector under conventional conditions (i.e., the TIMP-3
encoding DNA is operably linked to other sequences in the vector so
as to be expressible), and suitable mammalian cells are transformed
or transfected with the vector. The transformed or transfected
cells are cultured under appropriate conditions, and the
recombinant protein is expressed and the amount evaluated, either
qualitatively/semi-quantitatively, for example by Western blot or
SDS=PAGE, or more quantitatively using an assay such as an ELSA
(R&D Systems, Minneapolis Minn.) or ForteBio Octet.RTM. (Pall
ForteBio Corp, Menlo Park, Calif.) In this manner, the effects of
various mutations on the ability of mammalian cells to express a
TIMP-3 protein, mutein or variant can be determined.
[0126] If the mutation or mutations were made to introduce N-linked
glycosylation sites into a TIMP-3 polypeptide, or to enhance the
native glycosylation site, it may be desirable to evaluate the
presence and/or degree of glycosylation. Cells are transformed or
transfected as described previously and semi-quantitative measures
(e.g. western blots) can be used to determine if N-linked
glycosylation was not successfully incorporated, partially
incorporated, or fully incorporated.
Example 2
[0127] This Example describes a method used to determine whether a
mutation or mutations in TIMP-3 resulted in increased heparin
independence. Cells are transformed or transfected and cultured in
the presence or absence of heparin. The heparin can be added in
varying amounts, to develop a semi-quantitative notion of the
degree of heparin dependence. The amounts of TIMP-3 protein, mutein
or variant expressed under various conditions is then determined,
and a comparison is made to determine whether a particular mutation
has any effect on whether or not heparin is required for release of
the TIMP-3 protein, mutein or variant from the extracellular
matrix, or whether the amount or heparin required is reduced.
[0128] Using the method described above, the heparin dependence of
various TIMP-3 muteins was determined. CHOK1 cells were stably
transfected (selected with puromycin) to produce TIMP-3 or TIMP-3
muteins. When cell viability reached greater than 90%, the cells
were seeded into a production media and cultured in the presence or
absence of heparin (up to 500 ug/mL) for six days. The amounts of
TIMP-3 protein was determined via SDS-PAGE (4-20% Tris-Glycine;
Non-reduced+Iodoacetamide). Representative data is illustrated in
FIGS. 1-3.
[0129] In the absence of heparin, expression of a fusion protein
comprising an TIMP-3 fragment fused to an Fc was not detected in
culture media. FIG. 1, Lane 5. As the amount of heparin supplied to
the culture media increased, the amount of fusion protein detected
in the culture media correspondingly increased. FIG. 1, Lanes
6-9.
[0130] Introduction of glycosylation sites affected heparin
dependence of TIMP-3 muteins. TIMP-3 variants
[H78N/Q80T/K94N/E96T/D110N/K112T/R138T] and
[K45N/V47T/K94N/E96T/D110N/K112T/G173T] fused to an Fc were
produced in CHOK1 cells in the absence of heparin. Expression of
the TIMP-3 muteins was detected after six days of incubation,
indicating a reduced dependence on heparin. Compare FIG. 2 with
FIG. 1. The expression estimates in mg/mL (ForteBio Protein A) are
set forth in Table 1.
TABLE-US-00001 TABLE 1 Expression TIMP-3 Mutein Lane Estimate
[K45N/V47T/K94N/E96T/D110N/K112T/G173T]- 6 95 FcG1
[K45N/V47T/K94N/E96T/D110N/K112T/G173T]- 7 89 IgG1Fc + EPKSS fusion
[H78N/Q80T/K94N/E96T/D110N/K112T/R138T]- 8 113 FcG1
[H78N/Q80T/K94N/E96T/D110N/K112T/R138T]- 9 117 IgG1Fc + EPKSS
fusion
[0131] Heparin dependence is also affected by the choice of fusion
partner. Unlike the Fc fusion, fusion of a native TIMP-3 fragment
(AA 1-144) to human serum albumin (HSA) reduced the dependence on
heparin. FIG. 3 illustrates the robust expression of the
N-TIMP-3-HSA fusion. Similarly, fusion of TIMP-3 variant
[F57N/K45S] to HSA resulted in strong expression of the protein in
the absence of heparin, whereas fusion to Fc (instead of HSA) did
not abrogate heparin dependence.
[0132] This Example demonstrates that TIMP-3 muteins described
herein exhibit reduced dependence on heparin for production in
culture media.
Example 3
[0133] This Example describes MMP Inhibition Assays in which MMP
activity is measured by using fluorimetric methods; other methods
are known in the art. For example, fluorescence signal is increased
upon cleaving a quenched MMP subtype 5-FAM/QXL 520 fluorescence
resonance energy transfer (FRET) peptide substrate by an activated
MMP subtype or subtype specific catalytic domain. FRET peptides are
available for a number of different MMP, for example, from Anaspec
(Fremont, Calif.) or R&D Systems (Minneapolis, Minn.). The
TIMP-3 proteins used herein may be either native TIMP-3 or TIMP-3
mutein, variant or derivative; the proteins to be tested are
referred to as test molecules.
[0134] For MMP2 activity assay, human pro-MMP2 (Anaspec, Fremont,
Calif.) is activated with 1 mM 4-aminophenylmercuric acetate (APMA,
Anaspec, Fremont, Calif.) for 1 hour at 37.degree. C. before
incubating with MMP2 sensitive 5-FAM/QXL 520 FRET peptide in assay
buffer provided by the vendor against various concentrations of
test molecules in a black 384-well Optiplate (PerkinElmer, Waltham,
Mass.) at 37.degree. C. After 2 hours of incubation, fluorescence
signal from the reaction plate is measured at excitation (490 nm)
and emission (520 nm) on EnVision multilabel microplate reader
(PerkinElmer, Waltham, Mass.). Data in relative fluorescence unit
(RFU) is plotted against tested test molecule concentrations in
GraphPad Prism 5.0 (GraphPad, San Diego, Calif.) to estimate half
maximal inhibition constant (IC50).
[0135] For MMP9 activity measurement, a catalytic domain of human
MMP9 (Anaspec, Fremont, Calif.) is incubated with MMP9 sensitive
5-FAM/QXL 520 FRET peptide and various concentrations of test
molecules in a black 384-well Optiplate (PerkinElmer, Waltham,
Mass.) at 37.degree. C. After 2 hours of incubation, fluorescence
signal is measured at excitation (490 nm) and emission (520 nm) on
EnVision multilabel microplate reader (PerkinElmer, Waltham,
Mass.). Data in relative fluorescence unit (RFU) is plotted against
tested test molecule concentrations in GraphPad Prism 5.0
(GraphPad, San Diego, Calif.) to estimate half maximal inhibition
constant (IC50).
[0136] For MMP13 activity, test molecules are titrated in assay
buffer (20 mM Tris, 10 mM CaCl.sub.2, 10 uM ZnCl.sub.2, 0.01% Brij
35 (Calbiochem/EMD, San Diego, Calif.), pH 7.5) and added to black
polystyrene 96 or 384 well assay plate (Griener Bio-One, Germany).
Active MMP13 (Calbiochem/EMD) is diluted in assay buffer and added
to the test molecule titration and incubated for 10 minutes at room
temperature in a final volume of 50 microL. Alternatively,
pro-MMP-13 (R & D Systems, Minneapolis, Minn.) is activated
with APMA for 2 hours at 37 degrees C., and used in the assay. A
fluorogenic substrate such as Mca-PLGL-Dpa-AR-NH2 Fluorogenic MMP
Substrate or Mca-KPLGL-Dpa-AR-NH2 Fluorogenic Peptide Substrate (R
& D Systems) is prepared, and added to the MMP-13
enzyme/huTIMP-3/test molecule solution. MMP-13 activity is measured
kinetically, for example for 20 minutes using Molecular Devices
fluorescent plate reader (or equivalent).
[0137] The effect of the molecules being tested may be expressed as
percent of expected maximum TIMP-3 inhibition of MMP enzymatic
activity. Alternatively, a quantitative evaluation of MMP
inhibitory activity may not be necessary; rather, individual test
molecules can be evaluated as to whether they inhibit MMP or not.
Those of ordinary skill in the art recognize that the parameters
outlined herein can be varied by the application of routine
experimentation. For example, preliminary experiments are performed
using previously tested TIMP-3 and other materials to determine an
appropriate concentration of an MMP or pro-MPP. Similarly, the type
and appropriate concentration of substrate can also be determined.
Thus, for example, MMP can be titrated and compared to a previously
tested batch of MMP to optimize the assay parameters. Additionally,
those of ordinary skill in the art can utilize similar assays to
evaluate the effects, if any, or various TIMP-3 mutations on
ability to of a TIMP-3 mutein or variant to inhibit other MMPs
including TNF alpha converting enzyme (TACE).
Example 4
[0138] Using standard techniques of molecular biology, nucleic
acids encoding numerous muteins of TIMP-3 were prepared and
expressed in mammalian cells, substantially as described
previously. The effects of the mutations on the expression of the
encoded TIMP-3 muteins were evaluated. The listing of mutations
made includes K45N; V47T; K50N; V52T; P56N; F57N; G58T; H78N; Q80T;
K94N; E96T; D110N; K112T; R138T; G173T; and combinations
thereof
[0139] This Table summarizes expression and MMP inhibition results
obtained with numerous TIMP-3 muteins that did express in mammalian
cells. The increase in the level of expression demonstrating the
fold increase in expression as compared to that observed for
wild-type TIMP-3 is determined either qualitatively through the use
of western blots or SDS-PAGE Coomassie stained gels, or through the
measurement of expression titers as measured using a ForteBio
Octet.RTM. readout using an anti TIMP-3 antibody to capture TIMP-3
(such antibodies are publicly available, for example from EMD
Millipore, Billerica, Mass.: AbCam.RTM., Cambridge, Mass.: or
R&D Systems, Minneapolis, Minn.).
TABLE-US-00002 TABLE 2 Variant # EG HI Titer MMP2 MMP9 TACE K45N,
V47T, P56N, G58T, 4 no + nd nd nd Q126N, R138T (SEQ ID NO: 3) K45N,
V47T, P56N, G58T, 4 Yes ++++ 1 9 none K94N, E96T, R138T (SEQ ID NO:
4) K45N, V47T, P56N, G58T, 4 nd - nd nd nd R138T, G173T (SEQ ID NO:
5) K45N, V47T, F57N, K94N, 4 Yes +++ 2 59 none E96T, D110N, K112T
(SEQ ID NO: 6) K45N, V47T, F57N, K94N, 4 Yes +++ 2 38 none E96T,
R138T (SEQ ID NO: 7) K45N, V47T, H78N, Q80T, 5 nd - nd nd nd K94N,
E96T, R138T, G173T (SEQ ID NO: 8) K45N, V47T, K94N, E96T, 4 nd - nd
nd nd D110N, K112T, R138T (SEQ ID NO: 9) K45N, V47T, K94N, E96T, 4
Yes +++ 2 9 2 D110N, K112T, G173T (SEQ ID NO: 10) K45N, V47T, K94N,
E96T, 4 no + nd nd nd R138T, G173T (SEQ ID NO: 11) K45S, F57N,
K94N, E96T, 4 No +++ 3 74 none D110N, K112T, R138T (SEQ ID NO: 12)
K45S, F57N, H78N, Q80T, 4 no + nd nd nd K94N, E96T, R138T (SEQ ID
NO: 13) K50N, V52T P56N, G58T, 5 Yes +++ 2 19 none K94N, E96T,
D110N, K112T, R138T (SEQ ID NO: 14) K50N, V52T, H78N, Q80T, 5 yes +
nd nd nd K94N, E96T, R138T, G173T (SEQ ID NO: 15) K50N, V52T, K94N,
E96T, 4 Partial +++ 3 10 0.6 D110N, K112T, R138T (SEQ ID NO: 16)
K50N, V52T, K94N, E96T, 5 Partial +++ 5 21 2 D110N, K112T, R138T,
G173T (SEQ ID NO: 17) K50N, V52T, K94N, E96T, 4 no + nd nd nd
R138T, G173T (SEQ ID NO: 18) K50N, V52T, Q126N, R138T, 4 no + nd nd
nd G173T (SEQ ID NO: 19) P56N, G58T, H78N, Q80T, 4 Partial ++++ 2
15 none K94N, E96T, R138T (SEQ ID NO: 20) P56N, G58T K94N, E96T, 4
Yes ++++ 2 22 none Q126N, R138T (SEQ ID NO: 21) P56N, G58T, K94N,
E96T, 4 Yes ++++ 2 20 none D110N, K112T, R138T (SEQ ID NO: 22)
P56N, G58T, H78N, Q80T, 4 nd - nd nd nd K94N, E96T, G173T (SEQ ID
NO: 23) P56N, G58T, Q126N, R138T, 4 no + nd nd nd G173T (SEQ ID NO:
24) H78N, Q80T, K94N, E96T, 4 nd - nd nd nd R138T, G173T (SEQ ID
NO: 25) H78N, Q80T, K94N, E96T, 4 Yes +++ 2 6 0.4 D110N, K112T,
R138T (SEQ ID NO: 26) 1: "# EG" = number of engineered
glycosylation sites 2: "HI" indicates heparin independence, `Yes,`;
No,` or `Partial` as described in the Example 3: "Titer" refers to
relative titer as compared to wild-type: `+`: 1-10 mg/L, `++`:
10-25 mg/L, `+++` 50-100 mg/L, `++++` >100 mg/L 4: "MMP2" lists
the shift in MMP2 inhibitory activity (i.e., fold decrease) as
compared to wild-type 5: "MMP9" lists the shift in MMP9 inhibitory
activity (i.e., fold decrease) as compared to wild-type 6: "TACE"
lists the shift in TACE inhibitory activity (i.e., fold decrease)
as compared to wild-type nd = no data
[0140] Additional studies were conducted to characterize fusion
proteins of the invention using the methods described herein.
TIMP-3 [H78N, Q80T, K94N, E96T, D110N, K112T, R138T] (SEQ ID NO:26)
inhibited MMP2 and MMP9 (EC.sub.50 of 1 nM and 2.5 nM,
respectively). TIMP-3 [H78N, Q80T, K94N, E96T, D110N, K112T, R138T]
(SEQ ID NO:26) fused to HSA, Fc, and IgG maintained inhibitory
activity: HSA fusion, MMP2 EC.sub.50=1.4 nM, MMP9 EC.sub.50=5.1 nM;
Fc fusion, MMP2 EC.sub.50=13.3 nM, MMP9 EC.sub.50=32.9 nM; IgG
fusion, MMP2 EC.sub.50=14 nM, MMP9 EC.sub.50=26 nM. For comparison,
N-TIMP3 exhibited MMP2 EC.sub.50=7.6 nM, MMP9 EC.sub.50=1.7 nM.
Fusion of HSA to N-TIMP3 resulted in MMP2 EC.sub.50=38.7 nM, MMP9
EC.sub.50=36.5 nM, and fusion of Fc resulted in MMP2 EC.sub.50=6.9
nM, MMP9 EC.sub.50=1.6 nM.
[0141] Additional muteins are described, including those shown in
Table 3 below. In the Table, particular mutations are listed in the
heading; an "x" below a particular heading indicates that that
mutation is present. The heading "#Gly" indicates the number of
engineered glycosylation sites, and the heading "Designation"
indicates the combination of mutations contemplated. These muteins
can be made and tested as described herein.
TABLE-US-00003 TABLE 3 K45N, K50N, H78N, K94N, D110N V47T V52T Q80T
E96T K112T R138T G173T #Glyc Designation x x x x 4 K50N/V52T,
D110N/K112T, R138T, G173T x x x x 4 K45N/V47T, D110N/K112T, R138T,
G173T x x x x 4 H78N/Q80T, D110N/K112T, R138T, G173T x x x x 4
K45N/V47T, K50N/V52T, H78N/A80T, R138T x x x x 4 K45N/V47T,
H78N/Q80T D110N/K112T, G173T x x x x 4 K45N/V47T, H78N/Q80T, R138T,
G173T x x x x 4 K50N/V52T, H78N/Q80T K94N/E96T, G173T x x x x 4
K50N/V52T, H78N/Q80T D110N/K112T, R138T x x x x 4 K45N/V47T,
K50N/V52T, H78N/Q80T, D110N/K112T x x x x 4 K50N/V52T, H78N/Q80T,
R138T, G173T x x x x 4 K45N/V47T, H78N/Q80T, R138T, G173T x x x x 4
K45N/V47T, H78N/Q80T, D110N/K112T, R138T x x x x x 5 K45N/V47T,
K50N/V52T, H78N/Q80T, D110N/K112T, G173T x x x x x 5 K45N/V47T,
K50N/V52T, H78N/Q80T, R138T, G173T x x x x x 5 K45N/V47T,
K50N/V52T, H78N/Q80T, K94N/E96T, G173T x x x x x 5 K45N/V47T,
H78N/Q80T, K94N/E96T, R138T, G173T x x x x x 5 K50N/V52T,
H78N/Q80T, K94N/E96T, R138T, G173T x x x x x 5 K45N/V47T,
H78N/Q80T, D110N/K112T, R138T, G173T x x x x x 5 K50N/V52T,
H78N/Q80T, D110N/K112T, R138T, G173T x x x x x 5 K45N/V52T,
K50N/V52T, H78N/Q80T, D110N/K112T, R138T
Example 5
[0142] This Example describes an assay to evaluate the ability of a
TIMP-3 protein to bind to HTB-94.TM. cells (a chondrocytic cell
line available from the American Type Culture Collection, Manassas,
Va.) by fluorescence activated cell sorter (FACS) analysis.
HTB-94.TM. cells express high levels of LRP1 and ECM protein and
are useful for monitoring TIMP-3 mutein binding to cells. HTB-94
cells are cultured in HTB-94 culture medium (high-glucose DMEM
containing 10% fetal bovine serum [FBS] and 2 mM L Glutamine) at 37
C in 5% CO.sub.2. Cells are seeded at a cell density of
2.5.times.10.sup.4 cells/ml in standard tissue culture flasks for
6-12 weeks prior to staining and are passaged every 3-4 days after
removal from flask via trypsinization. Approximately 16 hours prior
to FACS stain, HTB-94 cells are seeded at 100,000 cells per well
onto standard tissue culture 12-well plates in 2 ml volume HTB94
medium and incubated at 37 C in 5% CO.sub.2. Cell are 80-90%
confluent prior to stain.
[0143] After approximately 16 hours, the HTB94 culture medium is
removed from the 12-well plates by aspiration and 1 ml 4 C stain
buffer (phosphate buffered saline [PBS] 2% FBS 0.15% NaN.sub.3) is
applied per well. Cell plates are incubated 1h on ice. Stain buffer
is aspirated and TIMP-3 HIS-Myc tagged proteins (either native
TIMP-3 or TIMP-3 variant) diluted in stain buffer to 80 microg/ml
is added, 0.9 ml/well; the same volume of buffer only is added to a
negative control well. Cell plates are incubated 30 min on ice,
aspirated and washed twice with 1 ml/well stain buffer. After the
second wash buffer is aspirated, and mouse anti-pentaHlS
AlexaFluor488 conjugated antibody (Qiagen, Valencia, Calif.)
diluted in stain buffer to 20 ug/ml is added, 0.9 mL/well. In
parallel, irrelevant mIgG.sub.1 AlexaFluor488 conjugated antibody
(eBioscience, San Diego, Calif.) negative control stain reagent
diluted in stain buffer to 20 microg/ml is added in parallel to a
replicate well stained with known binder TIMP3 HIS-Myc (for
example, K45S, F57N, SEQ ID NO:23).
[0144] Cell plates are incubated 30 min on ice while protected from
light, aspirated and washed twice with 1 ml/well stain buffer.
After the second wash buffer is aspirated, 1 mL per well
cell-dissociation buffer (enzyme-free, PBS, catalog #13151-014;
Life Technologies, Grand Island N.Y.) is added. Cell plates are
incubated 5 min at 37 C, and cells are transferred to 4 ml FACS
tubes. Plate wells are rinsed with 1 ml/well 25 C PBS and the
rinses are added to corresponding FACS tubes containing cells in
cell dissociation buffer. Tubes are centrifuged 5 min at 1000 RPM
to form a cell pellet, and aspirated. Cells are resuspended in 300
microL 4% paraformaldehyde in PBS (PFA) and may be stored at 4 C
protected from light until run on FACS.
[0145] Within two days of TIMP3 staining, 8000 fixed HTB94 cell
events are acquired, for example on a Becton Dickinson FACS Calibur
using FL1 for detecting AlexaFluor488 fluorescence. The forward
scatter (FSC) detector's voltage is set at E00, and the
side-scatter (SSC) detector's voltage is set at 316. Used in
combination, these detectors measure light reflected off of cells
as `forward scatter` and `side scatter,` which allows for the
HTB-94 cell gate to be defined, also referred to as `gated`, and
separated from non-cell material in the tube based on cell size and
granularity. The FL1 detector's voltage is set at 370. Analysis is
done, for example, using FlowJo vX.0.6.
[0146] Using the methods described above, it was determined that
TIMP-3 [K45N, V47T, K94N, E96T, D110N, K112T, G173T] (SEQ ID NO:10)
only weakly bound HTB-94.TM. cells, and TIMP-3 [K45S, F57N, K94N,
E96T, D110N, K112T, R138T] (SEQ ID NO:12), TIMP-3 [K50N, V52T,
K94N, E96T, D110N, K112T, R138T] (SEQ ID NO:16), TIMP-3 [P56N,
G58T, K94N, E96T, D110N, K112T, R138T] (SEQ ID NO:22), and TIMP-3
[H78N, Q80T, K94N, E96T, D110N, K112T, R138T] (SEQ ID NO:26) did
not bind HTB-94.TM. cells.
[0147] It was also determined that TIMP-3 [H78N, Q80T, K94N, E96T,
D110N, K112T, R138T] (SEQ ID NO:26) does not bind LRP1. Reduced
binding to components of the extracellular matrix (as indicated by
reduced binding to HTB-94 cells) and LRP-1 scavenger proteins
simplifies production by reducing reliance on heparin, improves
yield, and increases availability of the molecule in vivo, all of
which address complications in TIMP-3 production and therapy
suffered by prior TIMP-3 polypeptides. Thus, the TIMP-3 muteins
described herein provide unique advantages over previously
identified TIMP-3 polypeptides.
Example 6
[0148] This Example describes pharmacokinetic properties of TIMP-3
muteins described herein.
[0149] Sprague Darley rats with jugular vein cannulation (200 g-300
g, Charles River Labs, San Diego, Calif.) were anesthetized with 5%
isofluorine before administering with TIMP-3 proteins (3-6 mg/kg)
through the jugular vein. Blood samples (0.2 mL) were collected at
each desired time point from 5 minutes to 72 hours in EDTA-treated
syringe tubes and centrifuged for serum and blood cell separation.
The collected serum samples were analyzed by either immunoassays in
Gyros (Gyrolabs, Uppsala, Sweden) with TIMP-3 specific monoclonal
antibody (10A7, Amgen) as capture antibody and anti-penta histidine
antibody (Qiagen, Alameda, Calif.) or anti-Fc antibody (Amgen) as
detection antibody. Additionally, TIMP-3 specific fragment was
quantified via LC-MS/MS method. Serum samples (254) were incubated
with TIMP-3-specific 10A7 capture antibody before trypsin digestion
at 37.degree. C. overnight. TIMP-3 signature peptide fragments
(WDQLTLSQR and TQYLLTGR) were quantified by extrapolating from
standard curves generated with the peptides.
TABLE-US-00004 TABLE 4 Clearance Half-life Vss TIMP-3 polypeptide
(mL/hr/kg) (t.sub.1/2, hr) (mL/kg) TIMP-3 [H78N, Q80T, 48 .+-. 3
3.8 .+-. 0.2 134 .+-. 16 K94N, E96T, D110N, K112T, R138T] TIMP-3
[K45S, F57N] 189 .+-. 23 1.1 .+-. 0.1 71 .+-. 7 TIMP-3 [K45S,
F57N]- 45 .+-. 0 13.6 .+-. 3.5 208 .+-. 20 hetero Fc fusion
[0150] Introduction of N-linked glycosylation sites increased
half-life and Vss and reduced clearance of TIMP-3 muteins. TIMP-3
[H78N, Q80T, K94N, E96T, D110N, K112T, R138T] (SEQ ID NO: 26)
demonstrated substantially improved pharmacokinetic properties
compared to TIMP-3 [K45S, F57N], having fewer N-linked
glycosylation sites. Fusion of a half-life extension moiety (a
hetero Fc fusion) to TIMP-3 [K45S, F57N] substantially improved
pharmacokinetics properties compared to TIMP-3 [K45S, F57N] lacking
the Fc portion.
[0151] Systemic half-life was also determined for TIMP-3 [K45N,
V47T, P56N, G58T, K94N, E96T, R138T] (SEQ ID NO:4) (2.7 hours),
TIMP-3 [K50N, V52T, K94N, E96T, D110N, K112T, R138T, G173T] (SEQ ID
NO:17) (2 hours), TIMP-3 [K45N, V47T, K94N, E96T, D110N, K112T,
G173T] (SEQ ID NO:10) (1.4 hours), and TIMP-3 [K50N, V52T, K94N,
E96T, D110N, K112T, R138T] (SEQ ID NO:16) (2 hours) using similar
methods. The TIMP-3 muteins demonstrated increased system half-life
compared to native TIMP-3 and the N-terminal domain of native
TIMP-3 (both 0.8 hours).
[0152] Area under the curve (AUC; hr*.mu.g/mL) was examined for
TIMP-3 [H78N, Q80T, K94N, E96T, D110N, K112T, R138T] and TIMP-3
[K45S, F57N]. An intravenous bolus of 3 mg/kg of TIMP-3 polypeptide
was administered to rats. AUC for TIMP-3 [H78N, Q80T, K94N, E96T,
D110N, K112T, R138T] was 62.5.+-.4.0 compared to 16.0.+-.1.9 for
TIMP-3 [K45S, F57N]. TIMP-3 [H78N, Q80T, K94N, E96T, D110N, K112T,
R138T] exhibited improvement in clearance and exposure in vivo
compared to TIMP-3 [K45S, F57N].
Example 7
[0153] This Example describes representative in vivo studies of
TIMP-3 polypeptides in clinically-acceptable animal models. TIMP-3
and TIMP-3 muteins were administered to porcine or rat hearts to
determine half-life and effect on cardiac function.
[0154] Mature Yorkshire pigs (25-30 kg) were acclimated and handled
with pre-operative procedures according to IACUC protocol. The
region encompassing the right femoral artery was prepared in a
sterile fashion and the main branch of the femoral artery
surgically exposed. A catheter introducer (6F Input Introducer
Sheath, Medtronic) was positioned and stabilized in the artery, and
the sheath was placed with an initial heparin bolus (4000 units,
IV) followed by an additional bolus every hour (1000 units, IV).
Under fluoroscopic guidance (GE OEC 9600, UT), a coronary
angiography catheter/launcher (5F Launcher guiding catheter,
Medtronic) was placed in the left coronary ostia. An angioplasty
balloon catheter containing an injection lumen (3 mm.times.10 mm
Sprinter OTW balloon catheter, Medtronic) was positioned in the
lower portion of the left anterior descending artery (LAD). The LAD
was occluded by balloon inflation (12 ATM balloon inflation
pressure, Everest 30 Disposable inflation device, Medtronic) and
maintained for 90 minutes. When imaging was performed, IR800 dye
(DyLight 800 NHS ester, Thermo Scientific) labeled TIMP-3 (5 mg)
was slowly infused through the lumen of the balloon occlusion
catheter to the ischemic myocardial region just prior to
reperfusion. The balloon was then deflated, and the catheter system
was disengaged and removed. The femoral artery was ligated and the
incision closed. Post-operative analgesia was facilitated by
buprenorphine (0.05 mg/kg, IM) administered pre-operatively, as
well as a fentanyl patch (25 ug/hr, 72 hr) placed pre-operatively
and three days post-operatively. Additional lidocaine (1 mg/kg, IV)
and amiodarone (200 mg PO) was administered for three days
post-operatively, and aspirin (81 mg PO) was administered each day
until the terminal procedure.
[0155] Full length native TIMP-3 (F-TIMP3) and the N-terminal
TIMP-3 domain (AA 1-144, N-TIMP3) was directly injected into
porcine myocardium following ligation-induced myocardial
infarction. Administration of the polypeptides significantly
improved cardiac function; ejection fraction was improved greater
than 10% compared to saline injection at two weeks post-infarction.
See FIG. 7.
[0156] For imaging procedures, at the designated post I/R time
point (3 hrs, 1 day, 3 days, 7 days, and 14 days), the pigs were
anesthetized with isoflurane (5%) and the LV was harvested. The
entire LV was prepared for analysis. Full circumferential sections
of the apical, mid (2 sections) and base regions were subjected to
whole mount imaging to compute TIMP-3 distribution as a function of
LV area. The sections were placed on ice and immediately subjected
to imaging. The circumferential LV section from each region was
subjected to epi-illumination imaging (Xenogen IVIS, PerkinElmer,
Inc, Mass.). The settings for the imaging system was predicated
upon the IRDye800 spectra (745/800 ex/em), and the signal was
collected over a 0.5 sec exposure window. The digitized images
(Living Image Software, PerkinElmer Inc., Mass.) were subjected to
planimetry (Image J Software, Research Services Branch, Md.) to
determine the total LV circumferential area for that region. For
the mid LV region, whereby duplicate measurements were made, the
averages for both were computed. The final results were expressed
as the area occupied by IR800-TIMP-3, and expressed as a percent of
the total LV regional area. For additional quantitative measures of
TIMP-3 distribution, LV sections (70-100 mg) from each region and
from each sector were subjected to fluorescence/spectroscopy
(Li-Cor Odyssey CLx, Li-Cor Biosciences, Nebr.). Sections from the
harvested organs (.about.100 mg) and plasma samples (200 uL) were
also placed in a 96 well, black walled microplate subjected to
analysis. Following correction for background, the spectroscopic
signal from the sample well plate (20 min) was then normalized to
absolute sample weights (mg) or plasma volume (mL).
[0157] TIMP-3 is rapidly cleared when administered systemically;
the half-life of TIMP-3 when administered intravenously is less
than one hour. Using imaging procedures similar to those described
above, it was determined that the half-life of full length native
TIMP-3 (F-TIMP3) and the N-terminal TIMP-3 domain (AA 1-144,
N-TIMP3) was about 5 days and 3.4 days, respectively, in cardiac
tissue following direct injection after ligation-induced myocardial
infarction. Similarly, a longer cardiac retention of TIMP-3
polypeptides following intracoronary catheter delivery is expected
because TIMP3 has high binding affinity toward extracellular matrix
proteins in the myocardium. Cardiac tissue half-life of TIMP-3
[K45S, F57N] was approximately 3 days in the porcine myocardial
infarct model. See FIG. 5 (t.sub.1/2=y==a*e.sup.(-b*x)). Cardiac
tissue half-life of TIMP-3 [H78N, Q80T, K94N, E96T, D110N, K112T,
R138T] (SEQ ID NO:26) was approximately 5.43 days (i.e., improved
greater than 50-fold longer than intravenous administration). See
FIG. 6.
[0158] Using methods similar to those described above for porcine,
myocardial infarction was instituted in rat hearts and TIMP-3
[H78N, Q80T, K94N, E96T, D110N, K112T, R138T] (SEQ ID NO:26) was
administered to observe the impact on cardiac function. Rats were
administered vehicle (PBS, n=9) or 4 mg of TIMP-3 [H78N, Q80T,
K94N, E96T, D110N, K112T, R138T] (n=8) via myocardial injection.
Ejection fraction (EF %) was measured via echocardiography at day 3
and day 7 post-injection. Animals administered TIMP-3 [H78N, Q80T,
K94N, E96T, D110N, K112T, R138T] demonstrated significantly
enhanced EF on both days compared to control (more than 20%
increase compared to control). See FIG. 8A. End-systolic volume
(ESV) and end-diastolic volume (EDV) are indicators of cardiac
remodeling; left ventricular (LV) remodeling after acute myocardial
infarction is marked by a progressive increase of EDV and ESV
compared to baseline. As illustrated in FIGS. 8B and 8C, TIMP-3
[H78N, Q80T, K94N, E96T, D110N, K112T, R138T] reduced ESV and EDV
compared to control.
[0159] This results described above demonstrate that a
representative TIMP-3 mutein of the invention has increased
half-life compared to native TIMP-3, reduces adverse cardiac
remodeling, and improves cardiac function following acute
myocardial infarction.
Sequence CWU 1
1
751633DNAHomo sapiens 1atgacccctt ggctcgggct catcgtgctc ctgggcagct
ggagcctggg ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg
ccttctgcaa ctccgacatc 120gtgatccggg ccaaggtggt ggggaagaag
ctggtaaagg aggggccctt cggcacgctg 180gtctacacca tcaagcagat
gaagatgtac cgaggcttca ccaagatgcc ccatgtgcag 240tacatccaca
cggaagcttc cgagagtctc tgtggcctta agctggaggt caacaagtac
300cagtacctgc tgacaggtcg cgtctatgat ggcaagatgt acacggggct
gtgcaacttc 360gtggagaggt gggaccagct caccctctcc cagcgcaagg
ggctgaacta tcggtatcac 420ctgggttgta actgcaagat caagtcctgc
tactacctgc cttgctttgt gacttccaag 480aacgagtgtc tctggaccga
catgctctcc aatttcggtt accctggcta ccagtccaaa 540cactacgcct
gcatccggca gaagggcggc tactgcagct ggtaccgagg atgggccccc
600ccggataaaa gcatcatcaa tgccacagac ccc 6332211PRTHomo sapiens 2Met
Thr Pro Trp Leu Gly Leu Ile Val Leu Leu Gly Ser Trp Ser Leu1 5 10
15Gly Asp Trp Gly Ala Glu Ala Cys Thr Cys Ser Pro Ser His Pro Gln
20 25 30Asp Ala Phe Cys Asn Ser Asp Ile Val Ile Arg Ala Lys Val Val
Gly 35 40 45Lys Lys Leu Val Lys Glu Gly Pro Phe Gly Thr Leu Val Tyr
Thr Ile 50 55 60Lys Gln Met Lys Met Tyr Arg Gly Phe Thr Lys Met Pro
His Val Gln65 70 75 80Tyr Ile His Thr Glu Ala Ser Glu Ser Leu Cys
Gly Leu Lys Leu Glu 85 90 95Val Asn Lys Tyr Gln Tyr Leu Leu Thr Gly
Arg Val Tyr Asp Gly Lys 100 105 110Met Tyr Thr Gly Leu Cys Asn Phe
Val Glu Arg Trp Asp Gln Leu Thr 115 120 125Leu Ser Gln Arg Lys Gly
Leu Asn Tyr Arg Tyr His Leu Gly Cys Asn 130 135 140Cys Lys Ile Lys
Ser Cys Tyr Tyr Leu Pro Cys Phe Val Thr Ser Lys145 150 155 160Asn
Glu Cys Leu Trp Thr Asp Met Leu Ser Asn Phe Gly Tyr Pro Gly 165 170
175Tyr Gln Ser Lys His Tyr Ala Cys Ile Arg Gln Lys Gly Gly Tyr Cys
180 185 190Ser Trp Tyr Arg Gly Trp Ala Pro Pro Asp Lys Ser Ile Ile
Asn Ala 195 200 205Thr Asp Pro 2103188PRTHomo sapiens 3Cys Thr Cys
Ser Pro Ser His Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val
Ile Arg Ala Asn Val Thr Gly Lys Lys Leu Val Lys Glu Gly 20 25 30Asn
Phe Thr Thr Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr Arg 35 40
45Gly Phe Thr Lys Met Pro His Val Gln Tyr Ile His Thr Glu Ala Ser
50 55 60Glu Ser Leu Cys Gly Leu Lys Leu Glu Val Asn Lys Tyr Gln Tyr
Leu65 70 75 80Leu Thr Gly Arg Val Tyr Asp Gly Lys Met Tyr Thr Gly
Leu Cys Asn 85 90 95Phe Val Glu Arg Trp Asp Asn Leu Thr Leu Ser Gln
Arg Lys Gly Leu 100 105 110Asn Tyr Thr Tyr His Leu Gly Cys Asn Cys
Lys Ile Lys Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val Thr Ser
Lys Asn Glu Cys Leu Trp Thr Asp 130 135 140Met Leu Ser Asn Phe Gly
Tyr Pro Gly Tyr Gln Ser Lys His Tyr Ala145 150 155 160Cys Ile Arg
Gln Lys Gly Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170 175Pro
Pro Asp Lys Ser Ile Ile Asn Ala Thr Asp Pro 180 1854188PRTHomo
sapiens 4Cys Thr Cys Ser Pro Ser His Pro Gln Asp Ala Phe Cys Asn
Ser Asp1 5 10 15Ile Val Ile Arg Ala Asn Val Thr Gly Lys Lys Leu Val
Lys Glu Gly 20 25 30Asn Phe Thr Thr Leu Val Tyr Thr Ile Lys Gln Met
Lys Met Tyr Arg 35 40 45Gly Phe Thr Lys Met Pro His Val Gln Tyr Ile
His Thr Glu Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu Asn Leu Thr Val
Asn Lys Tyr Gln Tyr Leu65 70 75 80Leu Thr Gly Arg Val Tyr Asp Gly
Lys Met Tyr Thr Gly Leu Cys Asn 85 90 95Phe Val Glu Arg Trp Asp Gln
Leu Thr Leu Ser Gln Arg Lys Gly Leu 100 105 110Asn Tyr Thr Tyr His
Leu Gly Cys Asn Cys Lys Ile Lys Ser Cys Tyr 115 120 125Tyr Leu Pro
Cys Phe Val Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp 130 135 140Met
Leu Ser Asn Phe Gly Tyr Pro Gly Tyr Gln Ser Lys His Tyr Ala145 150
155 160Cys Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp Tyr Arg Gly Trp
Ala 165 170 175Pro Pro Asp Lys Ser Ile Ile Asn Ala Thr Asp Pro 180
1855188PRTHomo sapiens 5Cys Thr Cys Ser Pro Ser His Pro Gln Asp Ala
Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala Asn Val Thr Gly Lys
Lys Leu Val Lys Glu Gly 20 25 30Asn Phe Thr Thr Leu Val Tyr Thr Ile
Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr Lys Met Pro His Val
Gln Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu Lys
Leu Glu Val Asn Lys Tyr Gln Tyr Leu65 70 75 80Leu Thr Gly Arg Val
Tyr Asp Gly Lys Met Tyr Thr Gly Leu Cys Asn 85 90 95Phe Val Glu Arg
Trp Asp Gln Leu Thr Leu Ser Gln Arg Lys Gly Leu 100 105 110Asn Tyr
Thr Tyr His Leu Gly Cys Asn Cys Lys Ile Lys Ser Cys Tyr 115 120
125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp
130 135 140Met Leu Ser Asn Phe Thr Tyr Pro Gly Tyr Gln Ser Lys His
Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp
Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys Ser Ile Ile Asn Ala
Thr Asp Pro 180 1856188PRTHomo sapiens 6Cys Thr Cys Ser Pro Ser His
Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala Asn
Val Thr Gly Lys Lys Leu Val Lys Glu Gly 20 25 30Pro Asn Gly Thr Leu
Val Tyr Thr Ile Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr Lys
Met Pro His Val Gln Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser Leu
Cys Gly Leu Asn Leu Thr Val Asn Lys Tyr Gln Tyr Leu65 70 75 80Leu
Thr Gly Arg Val Tyr Asn Gly Thr Met Tyr Thr Gly Leu Cys Asn 85 90
95Phe Val Glu Arg Trp Asp Gln Leu Thr Leu Ser Gln Arg Lys Gly Leu
100 105 110Asn Tyr Arg Tyr His Leu Gly Cys Asn Cys Lys Ile Lys Ser
Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu Cys
Leu Trp Thr Asp 130 135 140Met Leu Ser Asn Phe Gly Tyr Pro Gly Tyr
Gln Ser Lys His Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly Gly
Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys Ser
Ile Ile Asn Ala Thr Asp Pro 180 1857188PRTHomo sapiens 7Cys Thr Cys
Ser Pro Ser His Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val
Ile Arg Ala Asn Val Thr Gly Lys Lys Leu Val Lys Glu Gly 20 25 30Pro
Asn Gly Thr Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr Arg 35 40
45Gly Phe Thr Lys Met Pro His Val Gln Tyr Ile His Thr Glu Ala Ser
50 55 60Glu Ser Leu Cys Gly Leu Asn Leu Thr Val Asn Lys Tyr Gln Tyr
Leu65 70 75 80Leu Thr Gly Arg Val Tyr Asp Gly Lys Met Tyr Thr Gly
Leu Cys Asn 85 90 95Phe Val Glu Arg Trp Asp Gln Leu Thr Leu Ser Gln
Arg Lys Gly Leu 100 105 110Asn Tyr Thr Tyr His Leu Gly Cys Asn Cys
Lys Ile Lys Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val Thr Ser
Lys Asn Glu Cys Leu Trp Thr Asp 130 135 140Met Leu Ser Asn Phe Gly
Tyr Pro Gly Tyr Gln Ser Lys His Tyr Ala145 150 155 160Cys Ile Arg
Gln Lys Gly Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170 175Pro
Pro Asp Lys Ser Ile Ile Asn Ala Thr Asp Pro 180 1858188PRTHomo
sapiens 8Cys Thr Cys Ser Pro Ser His Pro Gln Asp Ala Phe Cys Asn
Ser Asp1 5 10 15Ile Val Ile Arg Ala Asn Val Thr Gly Lys Lys Leu Val
Lys Glu Gly 20 25 30Pro Phe Gly Thr Leu Val Tyr Thr Ile Lys Gln Met
Lys Met Tyr Arg 35 40 45Gly Phe Thr Lys Met Pro Asn Val Thr Tyr Ile
His Thr Glu Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu Asn Leu Thr Val
Asn Lys Tyr Gln Tyr Leu65 70 75 80Leu Thr Gly Arg Val Tyr Asp Gly
Lys Met Tyr Thr Gly Leu Cys Asn 85 90 95Phe Val Glu Arg Trp Asp Gln
Leu Thr Leu Ser Gln Arg Lys Gly Leu 100 105 110Asn Tyr Thr Tyr His
Leu Gly Cys Asn Cys Lys Ile Lys Ser Cys Tyr 115 120 125Tyr Leu Pro
Cys Phe Val Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp 130 135 140Met
Leu Ser Asn Phe Thr Tyr Pro Gly Tyr Gln Ser Lys His Tyr Ala145 150
155 160Cys Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp Tyr Arg Gly Trp
Ala 165 170 175Pro Pro Asp Lys Ser Ile Ile Asn Ala Thr Asp Pro 180
1859188PRTHomo sapiens 9Cys Thr Cys Ser Pro Ser His Pro Gln Asp Ala
Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala Asn Val Thr Gly Lys
Lys Leu Val Lys Glu Gly 20 25 30Pro Phe Gly Thr Leu Val Tyr Thr Ile
Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr Lys Met Pro His Val
Gln Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu Asn
Leu Thr Val Asn Lys Tyr Gln Tyr Leu65 70 75 80Leu Thr Gly Arg Val
Tyr Asn Gly Thr Met Tyr Thr Gly Leu Cys Asn 85 90 95Phe Val Glu Arg
Trp Asp Gln Leu Thr Leu Ser Gln Arg Lys Gly Leu 100 105 110Asn Tyr
Thr Tyr His Leu Gly Cys Asn Cys Lys Ile Lys Ser Cys Tyr 115 120
125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp
130 135 140Met Leu Ser Asn Phe Gly Tyr Pro Gly Tyr Gln Ser Lys His
Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp
Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys Ser Ile Ile Asn Ala
Thr Asp Pro 180 18510188PRTHomo sapiens 10Cys Thr Cys Ser Pro Ser
His Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala
Asn Val Thr Gly Lys Lys Leu Val Lys Glu Gly 20 25 30Pro Phe Gly Thr
Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr
Lys Met Pro His Val Gln Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser
Leu Cys Gly Leu Asn Leu Thr Val Asn Lys Tyr Gln Tyr Leu65 70 75
80Leu Thr Gly Arg Val Tyr Asn Gly Thr Met Tyr Thr Gly Leu Cys Asn
85 90 95Phe Val Glu Arg Trp Asp Gln Leu Thr Leu Ser Gln Arg Lys Gly
Leu 100 105 110Asn Tyr Arg Tyr His Leu Gly Cys Asn Cys Lys Ile Lys
Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu
Cys Leu Trp Thr Asp 130 135 140Met Leu Ser Asn Phe Thr Tyr Pro Gly
Tyr Gln Ser Lys His Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly
Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys
Ser Ile Ile Asn Ala Thr Asp Pro 180 18511188PRTHomo sapiens 11Cys
Thr Cys Ser Pro Ser His Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10
15Ile Val Ile Arg Ala Asn Val Thr Gly Lys Lys Leu Val Lys Glu Gly
20 25 30Pro Phe Gly Thr Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr
Arg 35 40 45Gly Phe Thr Lys Met Pro His Val Gln Tyr Ile His Thr Glu
Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu Asn Leu Thr Val Asn Lys Tyr
Gln Tyr Leu65 70 75 80Leu Thr Gly Arg Val Tyr Asp Gly Lys Met Tyr
Thr Gly Leu Cys Asn 85 90 95Phe Val Glu Arg Trp Asp Gln Leu Thr Leu
Ser Gln Arg Lys Gly Leu 100 105 110Asn Tyr Thr Tyr His Leu Gly Cys
Asn Cys Lys Ile Lys Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val
Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp 130 135 140Met Leu Ser Asn
Phe Thr Tyr Pro Gly Tyr Gln Ser Lys His Tyr Ala145 150 155 160Cys
Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170
175Pro Pro Asp Lys Ser Ile Ile Asn Ala Thr Asp Pro 180
18512188PRTHomo sapiens 12Cys Thr Cys Ser Pro Ser His Pro Gln Asp
Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala Ser Val Val Gly
Lys Lys Leu Val Lys Glu Gly 20 25 30Pro Asn Gly Thr Leu Val Tyr Thr
Ile Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr Lys Met Pro His
Val Gln Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu
Asn Leu Thr Val Asn Lys Tyr Gln Tyr Leu65 70 75 80Leu Thr Gly Arg
Val Tyr Asn Gly Thr Met Tyr Thr Gly Leu Cys Asn 85 90 95Phe Val Glu
Arg Trp Asp Gln Leu Thr Leu Ser Gln Arg Lys Gly Leu 100 105 110Asn
Tyr Thr Tyr His Leu Gly Cys Asn Cys Lys Ile Lys Ser Cys Tyr 115 120
125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp
130 135 140Met Leu Ser Asn Phe Gly Tyr Pro Gly Tyr Gln Ser Lys His
Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp
Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys Ser Ile Ile Asn Ala
Thr Asp Pro 180 18513188PRTHomo sapiens 13Cys Thr Cys Ser Pro Ser
His Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala
Ser Val Val Gly Lys Lys Leu Val Lys Glu Gly 20 25 30Pro Asn Gly Thr
Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr
Lys Met Pro Asn Val Thr Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser
Leu Cys Gly Leu Asn Leu Thr Val Asn Lys Tyr Gln Tyr Leu65 70 75
80Leu Thr Gly Arg Val Tyr Asp Gly Lys Met Tyr Thr Gly Leu Cys Asn
85 90 95Phe Val Glu Arg Trp Asp Gln Leu Thr Leu Ser Gln Arg Lys Gly
Leu 100 105 110Asn Tyr Thr Tyr His Leu Gly Cys Asn Cys Lys Ile Lys
Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu
Cys Leu Trp Thr Asp 130 135 140Met Leu Ser Asn Phe Gly Tyr Pro Gly
Tyr Gln Ser Lys His Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly
Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys
Ser Ile Ile Asn Ala Thr Asp Pro 180 18514188PRTHomo sapiens 14Cys
Thr Cys Ser Pro Ser His Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10
15Ile Val Ile Arg Ala Lys Val Val Gly Lys Asn Leu Thr Lys Glu Gly
20 25 30Asn Phe Thr Thr Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr
Arg 35 40 45Gly Phe Thr Lys Met Pro His Val Gln Tyr Ile His Thr
Glu
Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu Asn Leu Thr Val Asn Lys Tyr
Gln Tyr Leu65 70 75 80Leu Thr Gly Arg Val Tyr Asn Gly Thr Met Tyr
Thr Gly Leu Cys Asn 85 90 95Phe Val Glu Arg Trp Asp Gln Leu Thr Leu
Ser Gln Arg Lys Gly Leu 100 105 110Asn Tyr Thr Tyr His Leu Gly Cys
Asn Cys Lys Ile Lys Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val
Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp 130 135 140Met Leu Ser Asn
Phe Gly Tyr Pro Gly Tyr Gln Ser Lys His Tyr Ala145 150 155 160Cys
Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170
175Pro Pro Asp Lys Ser Ile Ile Asn Ala Thr Asp Pro 180
18515188PRTHomo sapiens 15Cys Thr Cys Ser Pro Ser His Pro Gln Asp
Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala Lys Val Val Gly
Lys Asn Leu Thr Lys Glu Gly 20 25 30Pro Phe Gly Thr Leu Val Tyr Thr
Ile Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr Lys Met Pro Asn
Val Thr Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu
Asn Leu Thr Val Asn Lys Tyr Gln Tyr Leu65 70 75 80Leu Thr Gly Arg
Val Tyr Asp Gly Lys Met Tyr Thr Gly Leu Cys Asn 85 90 95Phe Val Glu
Arg Trp Asp Gln Leu Thr Leu Ser Gln Arg Lys Gly Leu 100 105 110Asn
Tyr Thr Tyr His Leu Gly Cys Asn Cys Lys Ile Lys Ser Cys Tyr 115 120
125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp
130 135 140Met Leu Ser Asn Phe Thr Tyr Pro Gly Tyr Gln Ser Lys His
Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp
Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys Ser Ile Ile Asn Ala
Thr Asp Pro 180 18516188PRTHomo sapiens 16Cys Thr Cys Ser Pro Ser
His Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala
Lys Val Val Gly Lys Asn Leu Thr Lys Glu Gly 20 25 30Pro Phe Gly Thr
Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr
Lys Met Pro His Val Gln Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser
Leu Cys Gly Leu Asn Leu Thr Val Asn Lys Tyr Gln Tyr Leu65 70 75
80Leu Thr Gly Arg Val Tyr Asn Gly Thr Met Tyr Thr Gly Leu Cys Asn
85 90 95Phe Val Glu Arg Trp Asp Gln Leu Thr Leu Ser Gln Arg Lys Gly
Leu 100 105 110Asn Tyr Thr Tyr His Leu Gly Cys Asn Cys Lys Ile Lys
Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu
Cys Leu Trp Thr Asp 130 135 140Met Leu Ser Asn Phe Gly Tyr Pro Gly
Tyr Gln Ser Lys His Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly
Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys
Ser Ile Ile Asn Ala Thr Asp Pro 180 18517188PRTHomo sapiens 17Cys
Thr Cys Ser Pro Ser His Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10
15Ile Val Ile Arg Ala Lys Val Val Gly Lys Asn Leu Thr Lys Glu Gly
20 25 30Pro Phe Gly Thr Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr
Arg 35 40 45Gly Phe Thr Lys Met Pro His Val Gln Tyr Ile His Thr Glu
Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu Asn Leu Thr Val Asn Lys Tyr
Gln Tyr Leu65 70 75 80Leu Thr Gly Arg Val Tyr Asn Gly Thr Met Tyr
Thr Gly Leu Cys Asn 85 90 95Phe Val Glu Arg Trp Asp Gln Leu Thr Leu
Ser Gln Arg Lys Gly Leu 100 105 110Asn Tyr Thr Tyr His Leu Gly Cys
Asn Cys Lys Ile Lys Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val
Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp 130 135 140Met Leu Ser Asn
Phe Thr Tyr Pro Gly Tyr Gln Ser Lys His Tyr Ala145 150 155 160Cys
Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170
175Pro Pro Asp Lys Ser Ile Ile Asn Ala Thr Asp Pro 180
18518188PRTHomo sapiens 18Cys Thr Cys Ser Pro Ser His Pro Gln Asp
Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala Lys Val Val Gly
Lys Asn Leu Thr Lys Glu Gly 20 25 30Pro Phe Gly Thr Leu Val Tyr Thr
Ile Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr Lys Met Pro His
Val Gln Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu
Asn Leu Thr Val Asn Lys Tyr Gln Tyr Leu65 70 75 80Leu Thr Gly Arg
Val Tyr Asp Gly Lys Met Tyr Thr Gly Leu Cys Asn 85 90 95Phe Val Glu
Arg Trp Asp Gln Leu Thr Leu Ser Gln Arg Lys Gly Leu 100 105 110Asn
Tyr Thr Tyr His Leu Gly Cys Asn Cys Lys Ile Lys Ser Cys Tyr 115 120
125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp
130 135 140Met Leu Ser Asn Phe Thr Tyr Pro Gly Tyr Gln Ser Lys His
Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp
Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys Ser Ile Ile Asn Ala
Thr Asp Pro 180 18519188PRTHomo sapiens 19Cys Thr Cys Ser Pro Ser
His Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala
Lys Val Val Gly Lys Asn Leu Thr Lys Glu Gly 20 25 30Pro Phe Gly Thr
Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr
Lys Met Pro His Val Gln Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser
Leu Cys Gly Leu Lys Leu Glu Val Asn Lys Tyr Gln Tyr Leu65 70 75
80Leu Thr Gly Arg Val Tyr Asp Gly Lys Met Tyr Thr Gly Leu Cys Asn
85 90 95Phe Val Glu Arg Trp Asp Asn Leu Thr Leu Ser Gln Arg Lys Gly
Leu 100 105 110Asn Tyr Thr Tyr His Leu Gly Cys Asn Cys Lys Ile Lys
Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu
Cys Leu Trp Thr Asp 130 135 140Met Leu Ser Asn Phe Thr Tyr Pro Gly
Tyr Gln Ser Lys His Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly
Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys
Ser Ile Ile Asn Ala Thr Asp Pro 180 18520188PRTHomo sapiens 20Cys
Thr Cys Ser Pro Ser His Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10
15Ile Val Ile Arg Ala Lys Val Val Gly Lys Lys Leu Val Lys Glu Gly
20 25 30Asn Phe Thr Thr Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr
Arg 35 40 45Gly Phe Thr Lys Met Pro Asn Val Thr Tyr Ile His Thr Glu
Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu Asn Leu Thr Val Asn Lys Tyr
Gln Tyr Leu65 70 75 80Leu Thr Gly Arg Val Tyr Asp Gly Lys Met Tyr
Thr Gly Leu Cys Asn 85 90 95Phe Val Glu Arg Trp Asp Gln Leu Thr Leu
Ser Gln Arg Lys Gly Leu 100 105 110Asn Tyr Thr Tyr His Leu Gly Cys
Asn Cys Lys Ile Lys Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val
Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp 130 135 140Met Leu Ser Asn
Phe Gly Tyr Pro Gly Tyr Gln Ser Lys His Tyr Ala145 150 155 160Cys
Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170
175Pro Pro Asp Lys Ser Ile Ile Asn Ala Thr Asp Pro 180
18521188PRTHomo sapiens 21Cys Thr Cys Ser Pro Ser His Pro Gln Asp
Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala Lys Val Val Gly
Lys Lys Leu Val Lys Glu Gly 20 25 30Asn Phe Thr Thr Leu Val Tyr Thr
Ile Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr Lys Met Pro His
Val Gln Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu
Asn Leu Thr Val Asn Lys Tyr Gln Tyr Leu65 70 75 80Leu Thr Gly Arg
Val Tyr Asp Gly Lys Met Tyr Thr Gly Leu Cys Asn 85 90 95Phe Val Glu
Arg Trp Asp Asn Leu Thr Leu Ser Gln Arg Lys Gly Leu 100 105 110Asn
Tyr Thr Tyr His Leu Gly Cys Asn Cys Lys Ile Lys Ser Cys Tyr 115 120
125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp
130 135 140Met Leu Ser Asn Phe Gly Tyr Pro Gly Tyr Gln Ser Lys His
Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp
Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys Ser Ile Ile Asn Ala
Thr Asp Pro 180 18522188PRTHomo sapiens 22Cys Thr Cys Ser Pro Ser
His Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala
Lys Val Val Gly Lys Lys Leu Val Lys Glu Gly 20 25 30Asn Phe Thr Thr
Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr
Lys Met Pro His Val Gln Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser
Leu Cys Gly Leu Asn Leu Thr Val Asn Lys Tyr Gln Tyr Leu65 70 75
80Leu Thr Gly Arg Val Tyr Asn Gly Thr Met Tyr Thr Gly Leu Cys Asn
85 90 95Phe Val Glu Arg Trp Asp Gln Leu Thr Leu Ser Gln Arg Lys Gly
Leu 100 105 110Asn Tyr Thr Tyr His Leu Gly Cys Asn Cys Lys Ile Lys
Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu
Cys Leu Trp Thr Asp 130 135 140Met Leu Ser Asn Phe Gly Tyr Pro Gly
Tyr Gln Ser Lys His Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly
Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys
Ser Ile Ile Asn Ala Thr Asp Pro 180 18523188PRTHomo sapiens 23Cys
Thr Cys Ser Pro Ser His Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10
15Ile Val Ile Arg Ala Lys Val Val Gly Lys Lys Leu Val Lys Glu Gly
20 25 30Asn Phe Thr Thr Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr
Arg 35 40 45Gly Phe Thr Lys Met Pro Asn Val Thr Tyr Ile His Thr Glu
Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu Asn Leu Thr Val Asn Lys Tyr
Gln Tyr Leu65 70 75 80Leu Thr Gly Arg Val Tyr Asp Gly Lys Met Tyr
Thr Gly Leu Cys Asn 85 90 95Phe Val Glu Arg Trp Asp Gln Leu Thr Leu
Ser Gln Arg Lys Gly Leu 100 105 110Asn Tyr Arg Tyr His Leu Gly Cys
Asn Cys Lys Ile Lys Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val
Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp 130 135 140Met Leu Ser Asn
Phe Thr Tyr Pro Gly Tyr Gln Ser Lys His Tyr Ala145 150 155 160Cys
Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170
175Pro Pro Asp Lys Ser Ile Ile Asn Ala Thr Asp Pro 180
18524188PRTHomo sapiens 24Cys Thr Cys Ser Pro Ser His Pro Gln Asp
Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala Lys Val Val Gly
Lys Lys Leu Val Lys Glu Gly 20 25 30Asn Phe Thr Thr Leu Val Tyr Thr
Ile Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr Lys Met Pro His
Val Gln Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu
Lys Leu Glu Val Asn Lys Tyr Gln Tyr Leu65 70 75 80Leu Thr Gly Arg
Val Tyr Asp Gly Lys Met Tyr Thr Gly Leu Cys Asn 85 90 95Phe Val Glu
Arg Trp Asp Asn Leu Thr Leu Ser Gln Arg Lys Gly Leu 100 105 110Asn
Tyr Thr Tyr His Leu Gly Cys Asn Cys Lys Ile Lys Ser Cys Tyr 115 120
125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp
130 135 140Met Leu Ser Asn Phe Thr Tyr Pro Gly Tyr Gln Ser Lys His
Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp
Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys Ser Ile Ile Asn Ala
Thr Asp Pro 180 18525188PRTHomo sapiens 25Cys Thr Cys Ser Pro Ser
His Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala
Lys Val Val Gly Lys Lys Leu Val Lys Glu Gly 20 25 30Pro Phe Gly Thr
Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr
Lys Met Pro Asn Val Thr Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser
Leu Cys Gly Leu Asn Leu Thr Val Asn Lys Tyr Gln Tyr Leu65 70 75
80Leu Thr Gly Arg Val Tyr Asp Gly Lys Met Tyr Thr Gly Leu Cys Asn
85 90 95Phe Val Glu Arg Trp Asp Gln Leu Thr Leu Ser Gln Arg Lys Gly
Leu 100 105 110Asn Tyr Thr Tyr His Leu Gly Cys Asn Cys Lys Ile Lys
Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu
Cys Leu Trp Thr Asp 130 135 140Met Leu Ser Asn Phe Thr Tyr Pro Gly
Tyr Gln Ser Lys His Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly
Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys
Ser Ile Ile Asn Ala Thr Asp Pro 180 18526188PRTHomo sapiens 26Cys
Thr Cys Ser Pro Ser His Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10
15Ile Val Ile Arg Ala Lys Val Val Gly Lys Lys Leu Val Lys Glu Gly
20 25 30Pro Phe Gly Thr Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr
Arg 35 40 45Gly Phe Thr Lys Met Pro Asn Val Thr Tyr Ile His Thr Glu
Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu Asn Leu Thr Val Asn Lys Tyr
Gln Tyr Leu65 70 75 80Leu Thr Gly Arg Val Tyr Asn Gly Thr Met Tyr
Thr Gly Leu Cys Asn 85 90 95Phe Val Glu Arg Trp Asp Gln Leu Thr Leu
Ser Gln Arg Lys Gly Leu 100 105 110Asn Tyr Thr Tyr His Leu Gly Cys
Asn Cys Lys Ile Lys Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val
Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp 130 135 140Met Leu Ser Asn
Phe Gly Tyr Pro Gly Tyr Gln Ser Lys His Tyr Ala145 150 155 160Cys
Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170
175Pro Pro Asp Lys Ser Ile Ile Asn Ala Thr Asp Pro 180
18527651DNAHomo sapiens 27atgacccctt ggctcgggct catcgtgctc
ctgggcagct ggagcctggg ggactggggc 60gccgaggcgt gcacatgctc gcccagccac
ccccaggacg ccttctgcaa ctccgacatc 120gtgatccggg ccaatgtgac
ggggaagaag ctggtaaagg aggggaactt caccacgctg 180gtctacacca
tcaagcagat gaagatgtac cgaggcttca ccaagatgcc ccatgtgcag
240tacatccaca cggaagcttc cgagagtctc tgtggcctta agctggaggt
caacaagtac 300cagtacctgc
tgacaggtcg cgtctatgat ggcaagatgt acacggggct gtgcaacttc
360gtggagaggt gggacaatct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcggtt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65128651DNAHomo
sapiens 28atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaatgtgac ggggaagaag ctggtaaagg
aggggaactt caccacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc ccatgtgcag 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctatgat ggcaagatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcggtt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65129651DNAHomo
sapiens 29atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaatgtgac ggggaagaag ctggtaaagg
aggggaactt caccacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc ccatgtgcag 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta agctggaggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctatgat ggcaagatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcactt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65130651DNAHomo
sapiens 30atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaatgtgac ggggaagaag ctggtaaagg
aggggcccaa cggcacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc ccatgtgcag 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctataat ggcacgatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tcggtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcggtt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65131651DNAHomo
sapiens 31atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaatgtgac ggggaagaag ctggtaaagg
aggggcccaa cggcacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc ccatgtgcag 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctatgat ggcaagatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcggtt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65132651DNAHomo
sapiens 32atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaatgtgac ggggaagaag ctggtaaagg
aggggccctt cggcacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc caatgtgacg 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctatgat ggcaagatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcactt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65133651DNAHomo
sapiens 33atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaatgtgac ggggaagaag ctggtaaagg
aggggccctt cggcacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc ccatgtgcag 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctataat ggcacgatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcggtt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65134651DNAHomo
sapiens 34atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaatgtgac ggggaagaag ctggtaaagg
aggggccctt cggcacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc ccatgtgcag 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctataat ggcacgatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tcggtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcactt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65135651DNAHomo
sapiens 35atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaatgtgac ggggaagaag ctggtaaagg
aggggccctt cggcacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc ccatgtgcag 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctatgat ggcaagatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcactt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65136651DNAHomo
sapiens 36atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccagcgtggt ggggaagaag ctggtaaagg
aggggcccaa cggcacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc ccatgtgcag 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctataat ggcacgatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcggtt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65137651DNAHomo
sapiens 37atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccagcgtggt ggggaagaag ctggtaaagg
aggggcccaa cggcacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc caatgtgacg 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctatgat ggcaagatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcggtt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65138651DNAHomo
sapiens 38atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaaggtggt ggggaagaat ctgacaaagg
aggggaactt caccacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc ccatgtgcag 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctataat ggcacgatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcggtt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65139651DNAHomo
sapiens 39atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaaggtggt ggggaagaat ctgacaaagg
aggggccctt cggcacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc caatgtgacg 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctatgat ggcaagatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcactt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65140651DNAHomo
sapiens 40atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaaggtggt ggggaagaat ctgacaaagg
aggggccctt cggcacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc ccatgtgcag 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctataat ggcacgatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcggtt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65141651DNAHomo
sapiens 41atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaaggtggt ggggaagaat ctgacaaagg
aggggccctt cggcacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc ccatgtgcag 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctataat ggcacgatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcactt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65142651DNAHomo
sapiens 42atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaaggtggt ggggaagaat ctgacaaagg
aggggccctt cggcacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc ccatgtgcag 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctatgat ggcaagatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcactt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65143651DNAHomo
sapiens 43atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaaggtggt ggggaagaat ctgacaaagg
aggggccctt cggcacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc ccatgtgcag 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta agctggaggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctatgat ggcaagatgt acacggggct gtgcaacttc
360gtggagaggt gggacaatct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcactt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65144651DNAHomo
sapiens 44atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaaggtggt ggggaagaag ctggtaaagg
aggggaactt caccacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc caatgtgacg 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctatgat ggcaagatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcggtt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65145651DNAHomo
sapiens 45atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaaggtggt ggggaagaag ctggtaaagg
aggggaactt caccacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc ccatgtgcag 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctatgat ggcaagatgt acacggggct gtgcaacttc
360gtggagaggt gggacaatct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcggtt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65146651DNAHomo
sapiens 46atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaaggtggt ggggaagaag ctggtaaagg
aggggaactt caccacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc ccatgtgcag 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctataat ggcacgatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcggtt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65147651DNAHomo
sapiens 47atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaaggtggt ggggaagaag ctggtaaagg
aggggaactt caccacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc caatgtgacg 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctatgat ggcaagatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tcggtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcactt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65148651DNAHomo
sapiens 48atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaaggtggt ggggaagaag ctggtaaagg
aggggaactt caccacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc ccatgtgcag 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta agctggaggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctatgat ggcaagatgt acacggggct gtgcaacttc
360gtggagaggt gggacaatct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcactt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 65149651DNAHomo
sapiens 49atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaaggtggt ggggaagaag ctggtaaagg
aggggccctt cggcacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc caatgtgacg 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctatgat ggcaagatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcactt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac ccccaccacc atcaccatca t 651502391DNAHomo
sapiens 50atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg
ggactggggc 60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa
ctccgacatc 120gtgatccggg ccaaggtggt ggggaagaag ctggtaaagg
aggggccctt cggcacgctg 180gtctacacca tcaagcagat gaagatgtac
cgaggcttca ccaagatgcc caatgtgacg 240tacatccaca cggaagcttc
cgagagtctc tgtggcctta atctgacggt caacaagtac 300cagtacctgc
tgacaggtcg cgtctataat ggcacgatgt acacggggct gtgcaacttc
360gtggagaggt gggaccagct caccctctcc cagcgcaagg ggctgaacta
tacgtatcac 420ctgggttgta actgcaagat caagtcctgc tactacctgc
cttgctttgt gacttccaag 480aacgagtgtc tctggaccga catgctctcc
aatttcggtt accctggcta ccagtccaaa 540cactacgcct gcatccggca
gaagggcggc tactgcagct ggtaccgagg atgggccccc 600ccggataaaa
gcatcatcaa tgccacagac cccgatgcac acaagagtga ggttgctcat
660cgatttaaag atttgggaga agaaaatttc aaagccttgg tgttgattgc
ctttgctcag 720tatcttcagc agtgtccatt tgaagatcat gtaaaattag
tgaatgaagt aactgaattt 780gcaaaaacat gtgttgctga tgagtcagct
gaaaattgtg acaaatcact tcataccctt 840tttggagaca aattatgcac
agttgcaact cttcgtgaaa cctatggtga aatggctgac 900tgctgtgcaa
aacaagaacc tgagagaaat gaatgcttct tgcaacacaa agatgacaac
960ccaaacctcc cccgattggt gagaccagag gttgatgtga tgtgcactgc
ttttcatgac 1020aatgaagaga catttttgaa aaaatactta tatgaaattg
ccagaagaca tccttacttt 1080tatgccccgg aactcctttt ctttgctaaa
aggtataaag ctgcttttac agaatgttgc 1140caagctgctg ataaagctgc
ctgcctgttg ccaaagctcg atgaacttcg ggatgaaggg 1200aaggcttcgt
ctgccaaaca gagactcaag tgtgccagtc tccaaaaatt tggagaaaga
1260gctttcaaag catgggcagt agctcgcctg agccagagat ttcccaaagc
tgagtttgca 1320gaagtttcca agttagtgac agatcttacc aaagtccaca
cggaatgctg ccatggagat 1380ctgcttgaat gtgctgatga cagggcggac
cttgccaagt atatctgtga aaatcaagat 1440tcgatctcca gtaaactgaa
ggaatgctgt gaaaaacctc tgttggaaaa atcccactgc 1500attgccgaag
tggaaaatga tgagatgcct gctgacttgc cttcattagc tgctgatttt
1560gttgaaagta aggatgtttg caaaaactat gctgaggcaa aggatgtctt
cctgggcatg 1620tttttgtatg aatatgcaag aaggcatcct gattactctg
tcgtgctgct gctgagactt 1680gccaagacat atgaaaccac tctagagaag
tgctgtgccg ctgcagatcc tcatgaatgc 1740tatgccaaag tgttcgatga
atttaaacct cttgtggaag agcctcagaa tttaatcaaa 1800caaaattgtg
agctttttga gcagcttgga gagtacaaat tccagaatgc gctattagtt
1860cgttacacca agaaagtacc ccaactgtca actccaactc ttatcgaggt
ctcaagaaac 1920ctaggaaaag tgggcagcaa atgttgtaaa catcctgaag
caaaaagaat gccctgtgca 1980gaagactatc tatccgtggt cctgaaccag
ttatgtgtgt tgcatgagaa aacgccagta 2040agtgacagag tcaccaaatg
ctgcacagaa tccttggtga acaggcgacc atgcttttca 2100gctctggaag
tcgatgaaac atacgttccc aaagagttta cagctaacac attcaccttc
2160catgcagata tatgcacact ttctgagaag gagagacaaa tcaagaaaca
aactgtgctt 2220gttgagctcg tgaaacacaa gcccaaggca acaaaagagc
aactgaaagc tgccatggat 2280gatttcgcag cttttgtaga gaagtgctgc
aaggctgacg ataaggagac ctgctttagc 2340gaggagggta aaaaacttgt
tgcggccagt caggccgcct taggcttatg a 239151773PRTHomo sapiens 51Cys
Thr Cys Ser Pro Ser His Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10
15Ile Val Ile Arg Ala Lys Val Val Gly Lys Lys Leu Val Lys Glu Gly
20 25 30Pro Phe Gly Thr Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr
Arg 35 40 45Gly Phe Thr Lys Met Pro Asn Val Thr Tyr Ile His Thr Glu
Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu Asn Leu Thr Val Asn Lys Tyr
Gln Tyr Leu65 70 75 80Leu Thr Gly Arg Val Tyr Asn Gly Thr Met Tyr
Thr Gly Leu Cys Asn 85 90 95Phe Val Glu Arg Trp Asp Gln Leu Thr Leu
Ser Gln Arg Lys Gly Leu 100 105 110Asn Tyr Thr Tyr His Leu Gly Cys
Asn Cys Lys Ile Lys Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val
Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp 130 135 140Met Leu Ser Asn
Phe Gly Tyr Pro Gly Tyr Gln Ser Lys His Tyr Ala145 150 155 160Cys
Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170
175Pro Pro Asp Lys Ser Ile Ile Asn Ala Thr Asp Pro Asp Ala His Lys
180 185 190Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn
Phe Lys 195 200 205Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln
Gln Cys Pro Phe 210 215 220Glu Asp His Val Lys Leu Val Asn Glu Val
Thr Glu Phe Ala Lys Thr225 230 235 240Cys Val Ala Asp Glu Ser Ala
Glu Asn Cys Asp Lys Ser Leu His Thr 245 250 255Leu Phe Gly Asp Lys
Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr 260 265 270Gly Glu Met
Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu 275 280 285Cys
Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val 290 295
300Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu
Glu305 310 315 320Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg
Arg His Pro Tyr 325 330 335Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala
Lys Arg Tyr Lys Ala Ala 340 345 350Phe Thr Glu Cys Cys Gln Ala Ala
Asp Lys Ala Ala Cys Leu Leu Pro 355 360 365Lys Leu Asp Glu Leu Arg
Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln 370 375 380Arg Leu Lys Cys
Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys385 390 395 400Ala
Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe 405 410
415Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu
420 425 430Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala
Asp Leu 435 440 445Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser
Ser Lys Leu Lys 450 455 460Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys
Ser His Cys Ile Ala Glu465 470 475 480Val Glu Asn Asp Glu Met Pro
Ala Asp Leu Pro Ser Leu Ala Ala Asp 485 490 495Phe Val Glu Ser Lys
Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp 500 505 510Val Phe Leu
Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp 515 520 525Tyr
Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr 530 535
540Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala
Lys545 550 555 560Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro
Gln Asn Leu Ile 565 570 575Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu
Gly Glu Tyr Lys Phe Gln 580 585 590Asn Ala Leu Leu Val Arg Tyr Thr
Lys Lys Val Pro Gln Leu Ser Thr 595 600 605Pro Thr Leu Ile Glu Val
Ser Arg Asn Leu Gly Lys Val Gly Ser Lys 610 615 620Cys Cys Lys His
Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr625 630 635 640Leu
Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro 645 650
655Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg
660 665 670Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val
Pro Lys 675 680 685Glu Phe Thr Ala Asn Thr Phe Thr Phe His Ala Asp
Ile Cys Thr Leu 690 695 700Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln
Thr Val Leu Val Glu Leu705 710 715 720Val Lys His Lys Pro Lys Ala
Thr Lys Glu Gln Leu Lys Ala Ala Met 725 730 735Asp Asp Phe Ala Ala
Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys 740 745 750Glu Thr Cys
Phe Ser Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln 755 760 765Ala
Ala Leu Gly Leu 77052419PRTHomo sapiens 52Cys Thr Cys Ser Pro Ser
His Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala
Ser Val Val Gly Lys Lys Leu Val Lys Glu Gly 20 25 30Pro Asn Gly Thr
Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr
Lys Met Pro His Val Gln Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser
Leu Cys Gly Leu Lys Leu Glu Val Asn Lys Tyr Gln Tyr Leu65 70 75
80Leu Thr Gly Arg Val Tyr Asp Gly Lys Met Tyr Thr Gly Leu Cys Asn
85 90 95Phe Val Glu Arg Trp Asp Gln Leu Thr Leu Ser Gln Arg Lys Gly
Leu 100 105 110Asn Tyr Arg Tyr His Leu Gly Cys Asn Cys Lys Ile Lys
Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu
Cys Leu Trp Thr Asp 130 135 140Met Leu Ser Asn Phe Gly Tyr Pro Gly
Tyr Gln Ser Lys His Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly
Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys
Ser Ile Ile Asn Ala Thr Asp Pro Gly Gly Gly Gly 180 185 190Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 195 200
205Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
210 215 220Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His225 230 235 240Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 245 250 255His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 260 265 270Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 275 280 285Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 290 295 300Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val305 310 315
320Tyr Thr Leu Pro Pro Ser Arg Lys Glu Met Thr Lys Asn Gln Val Ser
325 330 335Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 340 345 350Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 355 360 365Val Leu Lys Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 370 375 380Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met385 390 395 400His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 405 410 415Pro Gly
Lys53227PRTHomo sapiens 53Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120
125Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
130 135 140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Asp Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Asp Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly
Lys22554640PRTHomo sapiens 54Cys Thr Cys Ser Pro Ser His Pro Gln
Asp Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala Lys Val Val
Gly Lys Asn Leu Thr Lys Glu Gly 20 25 30Pro Phe Gly Thr Leu Val Tyr
Thr Ile Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr Lys Met Pro
His Val Gln Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser Leu Cys Gly
Leu Asn Leu Thr Val Asn Lys Tyr Gln Tyr Leu65 70 75 80Leu Thr Gly
Arg Val Tyr Asp Gly Lys Met Tyr Thr Gly Leu Cys Asn 85 90 95Phe Val
Glu Arg Trp Asp Gln Leu Thr Leu Ser Gln Arg Lys Gly Leu 100 105
110Asn Tyr Thr Tyr His Leu Gly Cys Asn Cys Lys Ile Lys Ser Cys Tyr
115 120 125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu Cys Leu Trp
Thr Asp 130 135 140Met Leu Ser Asn Phe Thr Tyr Pro Gly Tyr Gln Ser
Lys His Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly Gly Tyr Cys
Ser Trp Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys Ser Ile Ile
Asn Ala Thr Asp Pro Gln Val Gln Leu 180 185 190Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu 195 200 205Thr Cys Thr
Val Ser Gly Gly Ser Ile Ser Ser Gly Asp Tyr Phe Trp 210 215 220Ser
Trp Ile Arg Gln Leu Pro Gly Lys Gly Leu Glu Trp Ile Gly His225 230
235 240Ile His Asn Ser Gly Thr Thr Tyr Tyr Asn Pro Ser Leu Lys Ser
Arg 245 250 255Val Thr Ile Ser Val Asp Thr Ser Lys Lys Gln Phe Ser
Leu Arg Leu 260 265 270Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr Cys Ala Arg Asp 275 280 285Arg Gly Gly Asp Tyr Ala Tyr Gly Met
Asp Val Trp Gly Gln Gly Thr 290 295 300Thr Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro305 310 315 320Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 325 330 335Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 340 345
350Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
355 360 365Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser 370 375 380Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser385 390 395 400Asn Thr Lys Val Asp Lys Arg Val Glu
Pro Lys Ser Cys Asp Lys Thr 405 410 415His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser 420 425 430Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 435 440 445Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 450 455 460Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala465 470
475 480Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val 485 490 495Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr 500 505 510Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr 515 520 525Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu 530 535 540Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys545 550 555 560Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 565 570 575Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 580 585
590Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
595 600 605Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 610 615
620Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys625 630 635 64055403PRTHomo sapiens 55Cys Thr Cys Ser Pro Ser
His Pro Gln Asp Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala
Lys Val Val Gly Lys Asn Leu Thr Lys Glu Gly 20 25 30Pro Phe Gly Thr
Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr
Lys Met Pro His Val Gln Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser
Leu Cys Gly Leu Asn Leu Thr Val Asn Lys Tyr Gln Tyr Leu65 70 75
80Leu Thr Gly Arg Val Tyr Asp Gly Lys Met Tyr Thr Gly Leu Cys Asn
85 90 95Phe Val Glu Arg Trp Asp Gln Leu Thr Leu Ser Gln Arg Lys Gly
Leu 100 105 110Asn Tyr Thr Tyr His Leu Gly Cys Asn Cys Lys Ile Lys
Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu
Cys Leu Trp Thr Asp 130 135 140Met Leu Ser Asn Phe Thr Tyr Pro Gly
Tyr Gln Ser Lys His Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly
Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys
Ser Ile Ile Asn Ala Thr Asp Pro Glu Ile Val Leu 180 185 190Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr 195 200
205Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Arg Ser Glu Leu Ala Trp
210 215 220Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ser Leu Leu Ile Tyr
Gly Ala225 230 235 240Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser Gly Ser Gly Ser 245 250 255Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu Pro Glu Asp Phe 260 265 270Ala Val Tyr Tyr Cys Gln Gln
Phe Gly Ser Ser Pro Trp Thr Phe Gly 275 280 285Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val 290 295 300Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser305 310 315
320Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
325 330 335Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
Ser Val 340 345 350Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser Ser Thr Leu 355 360 365Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala Cys Glu 370 375 380Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser Phe Asn Arg385 390 395 400Gly Glu
Cys56405PRTHomo sapiens 56Cys Thr Cys Ser Pro Ser His Pro Gln Asp
Ala Phe Cys Asn Ser Asp1 5 10 15Ile Val Ile Arg Ala Lys Val Val Gly
Lys Lys Leu Val Lys Glu Gly 20 25 30Pro Phe Gly Thr Leu Val Tyr Thr
Ile Lys Gln Met Lys Met Tyr Arg 35 40 45Gly Phe Thr Lys Met Pro Asn
Val Thr Tyr Ile His Thr Glu Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu
Asn Leu Thr Val Asn Lys Tyr Gln Tyr Leu65 70 75 80Leu Thr Gly Arg
Val Tyr Asn Gly Thr Met Tyr Thr Gly Leu Cys Asn 85 90 95Phe Val Glu
Arg Trp Asp Gln Leu Thr Leu Ser Gln Arg Lys Gly Leu 100 105 110Asn
Tyr Thr Tyr His Leu Gly Cys Asn Cys Lys Ile Lys Ser Cys Tyr 115 120
125Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp
130 135 140Met Leu Ser Asn Phe Gly Tyr Pro Gly Tyr Gln Ser Lys His
Tyr Ala145 150 155 160Cys Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp
Tyr Arg Gly Trp Ala 165 170 175Pro Pro Asp Lys Ser Ile Ile Asn Ala
Thr Asp Pro Ala Pro Glu Leu 180 185 190Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr 195 200 205Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val 210 215 220Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val225 230 235
240Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
245 250 255Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu 260 265 270Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala 275 280 285Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro 290 295 300Gln Val Thr Thr Leu Pro Pro Ser
Arg Glu Glu Met Asn Lys Thr Gln305 310 315 320Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 325 330 335Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr 340 345 350Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu 355 360
365Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
370 375 380Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser385 390 395 400Leu Ser Pro Gly Lys 40557779PRTHomo sapiens
57Cys Thr Cys Ser Pro Ser His Pro Gln Asp Ala Phe Cys Asn Ser Asp1
5 10 15Ile Val Ile Arg Ala Lys Val Val Gly Lys Lys Leu Val Lys Glu
Gly 20 25 30Pro Phe Gly Thr Leu Val Tyr Thr Ile Lys Gln Met Lys Met
Tyr Arg 35 40 45Gly Phe Thr Lys Met Pro His Val Gln Tyr Ile His Thr
Glu Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu Lys Leu Glu Val Asn Lys
Tyr Gln Tyr Leu65 70 75 80Leu Thr Gly Arg Val Tyr Asp Gly Lys Met
Tyr Thr Gly Leu Cys Asn 85 90 95Phe Val Glu Arg Trp Asp Gln Leu Thr
Leu Ser Gln Arg Lys Gly Leu 100 105 110Asn Tyr Arg Tyr His Leu Gly
Cys Asn Cys Lys Ile Lys Ser Cys Tyr 115 120 125Tyr Leu Pro Cys Phe
Val Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp 130 135 140Met Leu Ser
Asn Phe Gly Tyr Pro Gly Tyr Gln Ser Lys His Tyr Ala145 150 155
160Cys Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala
165 170 175Pro Pro Asp Lys Ser Ile Ile Asn Ala Thr Asp Pro Asp Ala
His Lys 180 185 190Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu
Glu Asn Phe Lys 195 200 205Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr
Leu Gln Gln Cys Pro Phe 210 215 220Glu Asp His Val Lys Leu Val Asn
Glu Val Thr Glu Phe Ala Lys Thr225 230 235 240Cys Val Ala Asp Glu
Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr 245 250 255Leu Phe Gly
Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr 260 265 270Gly
Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu 275 280
285Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val
290 295 300Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn
Glu Glu305 310 315 320Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala
Arg Arg His Pro Tyr 325 330 335Phe Tyr Ala Pro Glu Leu Leu Phe Phe
Ala Lys Arg Tyr Lys Ala Ala 340 345 350Phe Thr Glu Cys Cys Gln Ala
Ala Asp Lys Ala Ala Cys Leu Leu Pro 355 360 365Lys Leu Asp Glu Leu
Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln 370 375 380Arg Leu Lys
Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys385 390 395
400Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe
405 410 415Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His
Thr Glu 420 425 430Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp
Arg Ala Asp Leu 435 440 445Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser
Ile Ser Ser Lys Leu Lys 450 455 460Glu Cys Cys Glu Lys Pro Leu Leu
Glu Lys Ser His Cys Ile Ala Glu465 470 475 480Val Glu Asn Asp Glu
Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp 485 490 495Phe Val Glu
Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp 500 505 510Val
Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp 515 520
525Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr
530 535 540Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr
Ala Lys545 550 555 560Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu
Pro Gln Asn Leu Ile 565 570 575Lys Gln Asn Cys Glu Leu Phe Glu Gln
Leu Gly Glu Tyr Lys Phe Gln 580 585 590Asn Ala Leu Leu Val Arg Tyr
Thr Lys Lys Val Pro Gln Val Ser Thr 595 600 605Pro Thr Leu Val Glu
Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys 610 615 620Cys Cys Lys
His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr625 630 635
640Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro
645 650 655Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val
Asn Arg 660 665 670Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr
Tyr Val Pro Lys 675 680 685Glu Phe Asn Ala Glu Thr Phe Thr Phe His
Ala Asp Ile Cys Thr Leu 690 695 700Ser Glu Lys Glu Arg Gln Ile Lys
Lys Gln Thr Ala Leu Val Glu Leu705 710 715 720Val Lys His Lys Pro
Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met 725 730 735Asp Asp Phe
Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys 740 745 750Glu
Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln 755 760
765Ala Ala Leu Gly Leu His His His His His His 770 77558777PRTHomo
sapiens 58Cys Thr Cys Ser Pro Ser His Pro Gln Asp Ala Phe Cys Asn
Ser Asp1 5 10 15Ile Val Ile Arg Ala Ser Val Val Gly Lys Lys Leu Val
Lys Glu Gly 20 25 30Pro Asn Gly Thr Leu Val Tyr Thr Ile Lys Gln Met
Lys Met Tyr Arg 35 40 45Gly Phe Thr Lys Met Pro His Val Gln Tyr Ile
His Thr Glu Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu Lys Leu Glu Val
Asn Lys Tyr Gln Tyr Leu65 70 75 80Leu Thr Gly Arg Val Tyr Asp Gly
Lys Met Tyr Thr Gly Leu Cys Asn 85 90 95Phe Val Glu Arg Trp Asp Gln
Leu Thr Leu Ser Gln Arg Lys Gly Leu 100 105 110Asn Tyr Arg Tyr His
Leu Gly Cys Asn Cys Lys Ile Lys Ser Cys Tyr 115 120 125Tyr Leu Pro
Cys Phe Val Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp 130 135 140Met
Leu Ser Asn Phe Gly Tyr Pro Gly Tyr Gln Ser Lys His Tyr Ala145 150
155 160Cys Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp Tyr Arg Gly Trp
Ala 165 170 175Pro Pro Asp Lys Ser Ile Ile Asn Ala Thr Asp Pro Gly
Gly Gly Gly 180 185 190Asp Ala His Lys Ser Glu Val Ala His Arg Phe
Lys Asp Leu Gly Glu 195 200 205Glu Asn Phe Lys Ala Leu Val Leu Ile
Ala Phe Ala Gln Tyr Leu Gln 210 215 220Gln Cys Pro Phe Glu Asp His
Val Lys Leu Val Asn Glu Val Thr Glu225 230 235 240Phe Ala Lys Thr
Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 245 250 255Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 260 265
270Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
275 280 285Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro
Asn Leu 290 295 300Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys
Thr Ala Phe His305 310 315 320Asp Asn Glu Glu Thr Phe Leu Lys Lys
Tyr Leu Tyr Glu Ile Ala Arg 325 330 335Arg His Pro Tyr Phe Tyr Ala
Pro Glu Leu Leu Phe Phe Ala Lys Arg 340 345 350Tyr Lys Ala Ala Phe
Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 355 360 365Cys Leu Leu
Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 370 375 380Ser
Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu385 390
395 400Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe
Pro 405 410 415Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp
Leu Thr Lys 420 425 430Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 435 440 445Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 450 455 460Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His465 470 475 480Cys Ile Ala Glu
Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 485 490 495Leu Ala
Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 500 505
510Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
515 520 525Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 530 535 540Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu545 550 555 560Cys Tyr Ala Lys Val Phe Asp Glu Phe
Lys Pro Leu Val Glu Glu Pro 565 570 575Gln Asn Leu Ile Lys Gln Asn
Cys Glu Leu Phe Glu Gln Leu Gly Glu 580 585 590Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 595 600 605Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 610 615 620Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys625 630
635 640Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu
His 645 650 655Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys
Thr Glu Ser 660 665 670Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 675 680 685Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 690 695 700Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala705 710 715 720Leu Val Glu Leu
Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 725 730 735Lys Ala
Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 740 745
750Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
755 760 765Ala Ala Ser Gln Ala Ala Leu Gly Leu 770 77559718PRTHomo
sapiens 59Cys Thr Cys Ser Pro Ser His Pro Gln Asp Ala Phe Cys Asn
Ser Asp1 5 10 15Ile Val Ile Arg Ala Lys Val Val Gly Lys Lys Leu Val
Lys Glu Gly 20 25 30Pro Phe Gly Thr Leu Val Tyr Thr Ile Lys Gln Met
Lys Met Tyr Arg 35 40 45Gly Phe Thr Lys Met Pro His Val Gln Tyr Ile
His Thr Glu Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu
Lys Leu Glu Val Asn Lys Tyr Gln Tyr Leu65 70 75 80Leu Thr Gly Arg
Val Tyr Asp Gly Lys Met Tyr Thr Gly Leu Cys Asn 85 90 95Phe Val Glu
Arg Trp Asp Gln Leu Thr Leu Ser Gln Arg Lys Gly Leu 100 105 110Asn
Tyr Arg Tyr His Leu Gly Cys Asn Gly Gly Gly Gly Asp Ala His 115 120
125Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe
130 135 140Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln
Cys Pro145 150 155 160Phe Glu Asp His Val Lys Leu Val Asn Glu Val
Thr Glu Phe Ala Lys 165 170 175Thr Cys Val Ala Asp Glu Ser Ala Glu
Asn Cys Asp Lys Ser Leu His 180 185 190Thr Leu Phe Gly Asp Lys Leu
Cys Thr Val Ala Thr Leu Arg Glu Thr 195 200 205Tyr Gly Glu Met Ala
Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn 210 215 220Glu Cys Phe
Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu225 230 235
240Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu
245 250 255Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg
His Pro 260 265 270Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys
Arg Tyr Lys Ala 275 280 285Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp
Lys Ala Ala Cys Leu Leu 290 295 300Pro Lys Leu Asp Glu Leu Arg Asp
Glu Gly Lys Ala Ser Ser Ala Lys305 310 315 320Gln Arg Leu Lys Cys
Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe 325 330 335Lys Ala Trp
Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu 340 345 350Phe
Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr 355 360
365Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp
370 375 380Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser
Lys Leu385 390 395 400Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys
Ser His Cys Ile Ala 405 410 415Glu Val Glu Asn Asp Glu Met Pro Ala
Asp Leu Pro Ser Leu Ala Ala 420 425 430Asp Phe Val Glu Ser Lys Asp
Val Cys Lys Asn Tyr Ala Glu Ala Lys 435 440 445Asp Val Phe Leu Gly
Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro 450 455 460Asp Tyr Ser
Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr465 470 475
480Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala
485 490 495Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln
Asn Leu 500 505 510Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly
Glu Tyr Lys Phe 515 520 525Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys
Lys Val Pro Gln Val Ser 530 535 540Thr Pro Thr Leu Val Glu Val Ser
Arg Asn Leu Gly Lys Val Gly Ser545 550 555 560Lys Cys Cys Lys His
Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp 565 570 575Tyr Leu Ser
Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr 580 585 590Pro
Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn 595 600
605Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro
610 615 620Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile
Cys Thr625 630 635 640Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln
Thr Ala Leu Val Glu 645 650 655Leu Val Lys His Lys Pro Lys Ala Thr
Lys Glu Gln Leu Lys Ala Val 660 665 670Met Asp Asp Phe Ala Ala Phe
Val Glu Lys Cys Cys Lys Ala Asp Asp 675 680 685Lys Glu Thr Cys Phe
Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser 690 695 700Gln Ala Ala
Leu Gly Leu Val Asp His His His His His His705 710 71560718PRTHomo
sapiens 60Cys Thr Cys Ser Pro Ser His Pro Gln Asp Ala Phe Cys Asn
Ser Asp1 5 10 15Ile Val Ile Arg Ala Ser Val Val Gly Lys Lys Leu Val
Lys Glu Gly 20 25 30Pro Asn Gly Thr Leu Val Tyr Thr Ile Lys Gln Met
Lys Met Tyr Arg 35 40 45Gly Phe Thr Lys Met Pro His Val Gln Tyr Ile
His Thr Glu Ala Ser 50 55 60Glu Ser Leu Cys Gly Leu Lys Leu Glu Val
Asn Lys Tyr Gln Tyr Leu65 70 75 80Leu Thr Gly Arg Val Tyr Asp Gly
Lys Met Tyr Thr Gly Leu Cys Asn 85 90 95Phe Val Glu Arg Trp Asp Gln
Leu Thr Leu Ser Gln Arg Lys Gly Leu 100 105 110Asn Tyr Arg Tyr His
Leu Gly Cys Asn Gly Gly Gly Gly Asp Ala His 115 120 125Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe 130 135 140Lys
Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro145 150
155 160Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala
Lys 165 170 175Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys
Ser Leu His 180 185 190Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala
Thr Leu Arg Glu Thr 195 200 205Tyr Gly Glu Met Ala Asp Cys Cys Ala
Lys Gln Glu Pro Glu Arg Asn 210 215 220Glu Cys Phe Leu Gln His Lys
Asp Asp Asn Pro Asn Leu Pro Arg Leu225 230 235 240Val Arg Pro Glu
Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu 245 250 255Glu Thr
Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro 260 265
270Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala
275 280 285Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys
Leu Leu 290 295 300Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala
Ser Ser Ala Lys305 310 315 320Gln Arg Leu Lys Cys Ala Ser Leu Gln
Lys Phe Gly Glu Arg Ala Phe 325 330 335Lys Ala Trp Ala Val Ala Arg
Leu Ser Gln Arg Phe Pro Lys Ala Glu 340 345 350Phe Ala Glu Val Ser
Lys Leu Val Thr Asp Leu Thr Lys Val His Thr 355 360 365Glu Cys Cys
His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp 370 375 380Leu
Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu385 390
395 400Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile
Ala 405 410 415Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser
Leu Ala Ala 420 425 430Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn
Tyr Ala Glu Ala Lys 435 440 445Asp Val Phe Leu Gly Met Phe Leu Tyr
Glu Tyr Ala Arg Arg His Pro 450 455 460Asp Tyr Ser Val Val Leu Leu
Leu Arg Leu Ala Lys Thr Tyr Glu Thr465 470 475 480Thr Leu Glu Lys
Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala 485 490 495Lys Val
Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu 500 505
510Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe
515 520 525Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln
Val Ser 530 535 540Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly
Lys Val Gly Ser545 550 555 560Lys Cys Cys Lys His Pro Glu Ala Lys
Arg Met Pro Cys Ala Glu Asp 565 570 575Tyr Leu Ser Val Val Leu Asn
Gln Leu Cys Val Leu His Glu Lys Thr 580 585 590Pro Val Ser Asp Arg
Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn 595 600 605Arg Arg Pro
Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro 610 615 620Lys
Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr625 630
635 640Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val
Glu 645 650 655Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu
Lys Ala Val 660 665 670Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys
Cys Lys Ala Asp Asp 675 680 685Lys Glu Thr Cys Phe Ala Glu Glu Gly
Lys Lys Leu Val Ala Ala Ser 690 695 700Gln Ala Ala Leu Gly Leu Val
Asp His His His His His His705 710 715612391DNAHomo sapiens
61atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg ggactggggc
60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa ctccgacatc
120gtgatccggg ccaaggtggt ggggaagaag ctggtaaagg aggggccctt
cggcacgctg 180gtctacacca tcaagcagat gaagatgtac cgaggcttca
ccaagatgcc caatgtgacg 240tacatccaca cggaagcttc cgagagtctc
tgtggcctta atctgacggt caacaagtac 300cagtacctgc tgacaggtcg
cgtctataat ggcacgatgt acacggggct gtgcaacttc 360gtggagaggt
gggaccagct caccctctcc cagcgcaagg ggctgaacta tacgtatcac
420ctgggttgta actgcaagat caagtcctgc tactacctgc cttgctttgt
gacttccaag 480aacgagtgtc tctggaccga catgctctcc aatttcggtt
accctggcta ccagtccaaa 540cactacgcct gcatccggca gaagggcggc
tactgcagct ggtaccgagg atgggccccc 600ccggataaaa gcatcatcaa
tgccacagac cccgatgcac acaagagtga ggttgctcat 660cgatttaaag
atttgggaga agaaaatttc aaagccttgg tgttgattgc ctttgctcag
720tatcttcagc agtgtccatt tgaagatcat gtaaaattag tgaatgaagt
aactgaattt 780gcaaaaacat gtgttgctga tgagtcagct gaaaattgtg
acaaatcact tcataccctt 840tttggagaca aattatgcac agttgcaact
cttcgtgaaa cctatggtga aatggctgac 900tgctgtgcaa aacaagaacc
tgagagaaat gaatgcttct tgcaacacaa agatgacaac 960ccaaacctcc
cccgattggt gagaccagag gttgatgtga tgtgcactgc ttttcatgac
1020aatgaagaga catttttgaa aaaatactta tatgaaattg ccagaagaca
tccttacttt 1080tatgccccgg aactcctttt ctttgctaaa aggtataaag
ctgcttttac agaatgttgc 1140caagctgctg ataaagctgc ctgcctgttg
ccaaagctcg atgaacttcg ggatgaaggg 1200aaggcttcgt ctgccaaaca
gagactcaag tgtgccagtc tccaaaaatt tggagaaaga 1260gctttcaaag
catgggcagt agctcgcctg agccagagat ttcccaaagc tgagtttgca
1320gaagtttcca agttagtgac agatcttacc aaagtccaca cggaatgctg
ccatggagat 1380ctgcttgaat gtgctgatga cagggcggac cttgccaagt
atatctgtga aaatcaagat 1440tcgatctcca gtaaactgaa ggaatgctgt
gaaaaacctc tgttggaaaa atcccactgc 1500attgccgaag tggaaaatga
tgagatgcct gctgacttgc cttcattagc tgctgatttt 1560gttgaaagta
aggatgtttg caaaaactat gctgaggcaa aggatgtctt cctgggcatg
1620tttttgtatg aatatgcaag aaggcatcct gattactctg tcgtgctgct
gctgagactt 1680gccaagacat atgaaaccac tctagagaag tgctgtgccg
ctgcagatcc tcatgaatgc 1740tatgccaaag tgttcgatga atttaaacct
cttgtggaag agcctcagaa tttaatcaaa 1800caaaattgtg agctttttga
gcagcttgga gagtacaaat tccagaatgc gctattagtt 1860cgttacacca
agaaagtacc ccaactgtca actccaactc ttatcgaggt ctcaagaaac
1920ctaggaaaag tgggcagcaa atgttgtaaa catcctgaag caaaaagaat
gccctgtgca 1980gaagactatc tatccgtggt cctgaaccag ttatgtgtgt
tgcatgagaa aacgccagta 2040agtgacagag tcaccaaatg ctgcacagaa
tccttggtga acaggcgacc atgcttttca 2100gctctggaag tcgatgaaac
atacgttccc aaagagttta cagctaacac attcaccttc 2160catgcagata
tatgcacact ttctgagaag gagagacaaa tcaagaaaca aactgtgctt
2220gttgagctcg tgaaacacaa gcccaaggca acaaaagagc aactgaaagc
tgccatggat 2280gatttcgcag cttttgtaga gaagtgctgc aaggctgacg
ataaggagac ctgctttagc 2340gaggagggta aaaaacttgt tgcggccagt
caggccgcct taggcttatg a 2391621326DNAHomo sapiens 62atgacccctt
ggctcgggct catcgtgctc ctgggcagct ggagcctggg ggactggggc 60gccgaggcgt
gcacatgctc gcccagccac ccccaggacg ccttctgcaa ctccgacatc
120gtgatccggg ccagtgtggt ggggaagaag ctggtaaagg aggggcccaa
tggcacgctg 180gtctacacca tcaagcagat gaagatgtac cgaggcttca
ccaagatgcc ccatgtgcag 240tacatccata cggaagcttc cgagagtctc
tgtggcctta agctggaggt caacaagtac 300cagtacctgc tgacaggtcg
cgtctatgat ggcaagatgt acacggggct gtgcaacttc 360gtggagaggt
gggaccagct caccctctcc cagcgcaagg ggctgaacta tcggtatcac
420ctgggttgta actgcaagat caagtcctgc tactacctgc cttgctttgt
gacttccaag 480aacgagtgtc tctggaccga catgctctcc aatttcggtt
accctggcta ccagtccaaa 540cactacgcct gcatccggca gaagggcggc
tactgcagct ggtaccgagg atgggccccc 600ccggataaaa gcatcatcaa
tgccacagac cccggtggag gtggagacaa aactcacaca 660tgtcccccgt
gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca
720aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt
ggtggtggac 780gtgagccacg aagaccctga ggtcaagttc aactggtacg
tggacggcgt ggaggtgcat 840aatgccaaga caaagccgcg agaggagcag
tacaacagca cgtaccgtgt ggtcagcgtc 900ctcaccgtcc tgcaccagga
ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 960aaagccctcc
cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa
1020ccacaggtgt acaccctgcc cccatcccgg aaggagatga ccaagaacca
ggtcagcctg 1080acctgcctgg tcaaaggctt ctatcccagc gacatcgccg
tggagtggga gagcaatggg 1140cagccggaga acaactacaa gaccacgcct
cccgtgctga agtccgacgg ctccttcttc 1200ctctatagca agctcaccgt
ggacaagagc aggtggcagc aggggaacgt cttctcatgc 1260tccgtgatgc
atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 1320ggtaaa
132663741DNAHomo sapiens 63atggggtcaa ccgccatcct cgccctcctc
ctggctgttc tccaaggagt ctgcgctgac 60aaaactcaca catgtccacc gtgcccagca
cctgaactcc tggggggacc gtcagtcttc 120ctcttccccc caaaacccaa
ggacaccctc atgatctccc ggacccctga ggtcacatgc 180gtggtggtgg
acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc
240gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag
cacgtaccgt 300gtggtcagcg tcctcaccgt cctgcaccag gactggctga
atggcaagga gtacaagtgc 360aaggtctcca acaaagccct cccagccccc
atcgagaaaa ccatctccaa agccaaaggg 420cagccccgag aaccacaggt
gtacaccctg cccccatccc gggaggagat gaccaagaac 480caggtcagcc
tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg
540gagagcaatg ggcagccgga gaacaactac gacaccacgc ctcccgtgct
ggactccgac 600ggctccttct tcctctatag cgacctcacc gtggacaaga
gcaggtggca gcaggggaac 660gtcttctcat gctccgtgat gcatgaggct
ctgcacaacc actacacgca gaagagcctc 720tccctgtctc cgggtaaatg a
741641989DNAHomo sapiens 64atgacccctt ggctcgggct catcgtgctc
ctgggcagct ggagcctggg ggactggggc 60gccgaggcgt gcacatgctc gcccagccac
ccccaggacg ccttctgcaa ctccgacatc 120gtgatccggg ccaaggtggt
ggggaagaat ctgacaaagg aggggccctt cggcacgctg 180gtctacacca
tcaagcagat gaagatgtac cgaggcttca ccaagatgcc ccatgtgcag
240tacatccaca cggaagcttc cgagagtctc tgtggcctta atctgacggt
caacaagtac 300cagtacctgc tgacaggtcg cgtctatgat ggcaagatgt
acacggggct gtgcaacttc 360gtggagaggt gggaccagct caccctctcc
cagcgcaagg ggctgaacta tacgtatcac 420ctgggttgta actgcaagat
caagtcctgc tactacctgc cttgctttgt gacttccaag 480aacgagtgtc
tctggaccga catgctctcc aatttcactt accctggcta ccagtccaaa
540cactacgcct gcatccggca gaagggcggc tactgcagct ggtaccgagg
atgggcccct 600ccggataaaa gcatcatcaa tgccacagac ccccaggtgc
agctgcagga gtcgggccca 660ggactggtga agccttcaca gaccctgtcc
ctcacctgca ctgtctctgg tggctccatc 720agcagtggtg attacttctg
gagctggatc cgccagctcc cagggaaggg cctggagtgg 780attgggcaca
tccataacag tgggaccacc tactacaatc cgtccctcaa gagtcgagtt
840accatatcag tagacacgtc taagaagcag ttctccctga ggctgagttc
tgtgactgcc 900gcggacacgg ccgtatatta ctgtgcgaga gatcgagggg
gtgactacgc ttatggtatg 960gacgtctggg gccaagggac cacggtcacc
gtctcctcag cctccaccaa gggcccatcc 1020gtcttccccc tggcaccctc
ctccaagagc acctctgggg gcacagcggc cctgggctgc 1080ctggtcaagg
actacttccc cgaaccggtg acggtgtcgt ggaactcagg cgccctgacc
1140agcggcgtgc acaccttccc ggctgtccta cagtcctcag gactctactc
cctcagcagc 1200gtggtgaccg tgccctccag cagcttgggc acccagacct
acatctgcaa cgtgaatcac 1260aagcccagca acaccaaggt ggacaagaga
gttgagccca aatcttgtga caaaactcac 1320acatgcccac cgtgcccagc
acctgaactc ctggggggac cgtcagtctt cctcttcccc 1380ccaaaaccca
aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg
1440gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg
cgtggaggtg 1500cataatgcca agacaaagcc gcgggaggag cagtacaaca
gcacgtaccg tgtggtcagc 1560gtcctcaccg tcctgcacca ggactggctg
aatggcaagg agtacaagtg caaggtctcc 1620aacaaagccc tcccagcccc
catcgagaaa accatctcca aagccaaagg gcagccccga 1680gaaccacagg
tgtacaccct gcccccatcc cgggaggaga tgaccaagaa ccaggtcagc
1740ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg
ggagagcaat 1800gggcagccgg agaacaacta caagaccacg cctcccgtgc
tggactccga cggctccttc 1860ttcctctata gcaagctcac cgtggacaag
agcaggtggc agcaggggaa cgtcttctca 1920tgctccgtga tgcatgaggc
tctgcacaac cactacacgc agaagagcct ctccctgtct 1980ccgggtaaa
1989651278DNAHomo sapiens 65atgacccctt ggctcgggct
catcgtgctc ctgggcagct ggagcctggg ggactggggc 60gccgaggcgt gcacatgctc
gcccagccac ccccaggacg ccttctgcaa ctccgacatc 120gtgatccggg
ccaaggtggt ggggaagaat ctgacaaagg aggggccctt cggcacgctg
180gtctacacca tcaagcagat gaagatgtac cgaggcttca ccaagatgcc
ccatgtgcag 240tacatccaca cggaagcttc cgagagtctc tgtggcctta
atctgacggt caacaagtac 300cagtacctgc tgacaggtcg cgtctatgat
ggcaagatgt acacggggct gtgcaacttc 360gtggagaggt gggaccagct
caccctctcc cagcgcaagg ggctgaacta tacgtatcac 420ctgggttgta
actgcaagat caagtcctgc tactacctgc cttgctttgt gacttccaag
480aacgagtgtc tctggaccga catgctctcc aatttcactt accctggcta
ccagtccaaa 540cactacgcct gcatccggca gaagggcggc tactgcagct
ggtaccgagg atgggcccct 600ccggataaaa gcatcatcaa tgccacagac
cccgaaattg tgttgacgca gtctccaggc 660accctgtctt tgtctccagg
ggaaagagcc accctctcct gcagggccag tcagggtatt 720agtagaagcg
aattagcctg gtaccagcag aaacctggcc aggctcccag cctcctcatc
780tatggtgcat ccagcagggc cactggcatc ccagacaggt tcagtggcag
tgggtctggg 840acagacttca ctctcaccat cagcagactg gagcctgaag
attttgcagt gtattactgt 900caacaatttg gtagttcacc gtggacgttc
ggccaaggga ccaaggtgga aatcaaacga 960actgtggctg caccatctgt
cttcatcttc ccgccatctg atgagcagtt gaaatctgga 1020actgctagcg
ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg
1080aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga
gcaggacagc 1140aaggacagca cctacagcct cagcagcacc ctgacgctga
gcaaagcaga ctacgagaaa 1200cacaaagtct acgcctgcga agtcacccat
cagggcctga gctcgcccgt cacaaagagc 1260ttcaacaggg gagagtgt
1278661284DNAHomo sapiens 66atgacccctt ggctcgggct catcgtgctc
ctgggcagct ggagcctggg ggactggggc 60gccgaggcgt gcacatgctc gcccagccac
ccccaggacg ccttctgcaa ctccgacatc 120gtgatccggg ccaaggtggt
ggggaagaag ctggtaaagg aggggccctt cggcacgctg 180gtctacacca
tcaagcagat gaagatgtac cgaggcttca ccaagatgcc caatgtgacg
240tacatccaca cggaagcttc cgagagtctc tgtggcctta atctgacggt
caacaagtac 300cagtacctgc tgacaggtcg cgtctataat ggcacgatgt
acacggggct gtgcaacttc 360gtggagaggt gggaccagct caccctctcc
cagcgcaagg ggctgaacta tacgtatcac 420ctgggttgta actgcaagat
caagtcctgc tactacctgc cttgctttgt gacttccaag 480aacgagtgtc
tctggaccga catgctctcc aatttcggtt accctggcta ccagtccaaa
540cactacgcct gcatccggca gaagggcggc tactgcagct ggtaccgagg
atgggccccc 600ccggataaaa gcatcatcaa tgccacagac cccgcacctg
aactcctggg gggaccgtca 660gtcttcctct tccccccaaa acccaaggac
accctcatga tctcccggac ccctgaggtc 720acatgcgtgg tggtggacgt
gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 780gacggcgtgg
aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg
840taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg
caaggagtac 900aagtgcaagg tctccaacaa agccctccca gcccccatcg
agaaaaccat ctccaaagcc 960aaagggcagc cccgagaacc acaggtgacc
accctgcccc catcccggga ggagatgaac 1020aagacccagg tcagcctgac
ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1080gagtgggaga
gcaatgggca gccggagaac aactacgaca ccacgcctcc cgtgctggac
1140tccgacggct ccttcttcct ctatagcgac ctcaccgtgg acaagagcag
gtggcagcag 1200gggaacgtct tctcatgctc cgtgatgcat gaggctctgc
acaaccacta cacgcagaag 1260agcctctccc tgtctccggg taag
1284672409DNAHomo sapiens 67atgacccctt ggctcgggct catcgtgctc
ctgggcagct ggagcctggg ggactggggc 60gccgaggcgt gcacatgctc gcccagccac
ccccaggacg ccttctgcaa ctccgacatc 120gtgatccggg ccaaggtggt
ggggaagaag ctggtaaagg aggggccctt cggcacgctg 180gtctacacca
tcaagcagat gaagatgtac cgaggcttca ccaagatgcc ccatgtgcag
240tacatccata cggaagcttc cgagagtctc tgtggcctta agctggaggt
caacaagtac 300cagtacctgc tgacaggtcg cgtctatgat ggcaagatgt
acacggggct gtgcaacttc 360gtggagaggt gggaccagct caccctctcc
cagcgcaagg ggctgaacta tcggtatcac 420ctgggttgta actgcaagat
caagtcctgc tactacctgc cttgctttgt gacttccaag 480aacgagtgtc
tctggaccga catgctctcc aatttcggtt accctggcta ccagtccaaa
540cactacgcct gcatccggca gaagggcggc tactgcagct ggtaccgagg
atgggccccc 600ccggataaaa gcatcatcaa tgccacagac cccgatgcac
acaagagtga ggttgctcat 660cggtttaaag atttgggaga agaaaatttc
aaagccttgg tgttgattgc ctttgctcag 720tatcttcagc agtgtccatt
tgaagatcat gtaaaattag tgaatgaagt aactgaattt 780gcaaaaacat
gtgttgctga tgagtcagct gaaaattgtg acaaatcact tcataccctt
840tttggagaca aattatgcac agttgcaact cttcgtgaaa cctatggtga
aatggctgac 900tgctgtgcaa aacaagaacc tgagagaaat gaatgcttct
tgcaacacaa agatgacaac 960ccaaacctcc cccgattggt gagaccagag
gttgatgtga tgtgcactgc ttttcatgac 1020aatgaagaga catttttgaa
aaaatactta tatgaaattg ccagaagaca tccttacttt 1080tatgccccgg
aactcctttt ctttgctaaa aggtataaag ctgcttttac agaatgttgc
1140caagctgctg ataaagctgc ctgcctgttg ccaaagctcg atgaacttcg
ggatgaaggg 1200aaggcttcgt ctgccaaaca gagactcaag tgtgccagtc
tccaaaaatt tggagaaaga 1260gctttcaaag catgggcagt agctcgcctg
agccagagat ttcccaaagc tgagtttgca 1320gaagtttcca agttagtgac
agatcttacc aaagtccaca cggaatgctg ccatggagat 1380ctgcttgaat
gtgctgatga cagggcggac cttgccaagt atatctgtga aaatcaagat
1440tcgatctcca gtaaactgaa ggaatgctgt gaaaaacctc tgttggaaaa
atcccactgc 1500attgccgaag tggaaaatga tgagatgcct gctgacttgc
cttcattagc tgctgatttt 1560gttgaaagta aggatgtttg caaaaactat
gctgaggcaa aggatgtctt cctgggcatg 1620tttttgtatg aatatgcaag
aaggcatcct gattactctg tcgtgctgct gctgagactt 1680gccaagacat
atgaaaccac tctagagaag tgctgtgccg ctgcagatcc tcatgaatgc
1740tatgccaaag tgttcgatga atttaaacct cttgtggaag agcctcagaa
tttaatcaaa 1800caaaattgtg agctttttga gcagcttgga gagtacaaat
tccagaatgc gctattagtt 1860cgttacacca agaaagtacc ccaagtgtca
actccaactc ttgtagaggt ctcaagaaac 1920ctaggaaaag tgggcagcaa
atgttgtaaa catcctgaag caaaaagaat gccctgtgca 1980gaagactatc
tatccgtggt cctgaaccag ttatgtgtgt tgcatgagaa aacgccagta
2040agtgacagag tcaccaaatg ctgcacagaa tccttggtga acaggcgacc
atgcttttca 2100gctctggaag tcgatgaaac atacgttccc aaagagttta
atgctgaaac attcaccttc 2160catgcagata tatgcacact ttctgagaag
gagagacaaa tcaagaaaca aactgcactt 2220gttgagctcg tgaaacacaa
gcccaaggca acaaaagagc aactgaaagc tgttatggat 2280gatttcgcag
cttttgtaga gaagtgctgc aaggctgacg ataaggagac ctgctttgcc
2340gaggagggta aaaaacttgt tgctgcaagt caagctgcct taggcttaca
tcatcatcat 2400catcattga 2409682400DNAHomo sapiens 68atgacccctt
ggctcgggct catcgtgctc ctgggcagct ggagcctggg ggactggggc 60gccgaggcgt
gcacatgctc gcccagccac ccccaggacg ccttctgcaa ctccgacatc
120gtgatccggg ccagtgtggt ggggaagaag ctggtaaagg aggggcccaa
tggcacgctg 180gtctacacca tcaagcagat gaagatgtac cgaggcttca
ccaagatgcc ccatgtgcag 240tacatccata cggaagcttc cgagagtctc
tgtggcctta agctggaggt caacaagtac 300cagtacctgc tgacaggtcg
cgtctatgat ggcaagatgt acacggggct gtgcaacttc 360gtggagaggt
gggaccagct caccctctcc cagcgcaagg ggctgaacta tcggtatcac
420ctgggttgta actgcaagat caagtcctgc tactacctgc cttgctttgt
gacttccaag 480aacgagtgtc tctggaccga catgctctcc aatttcggtt
accctggcta ccagtccaaa 540cactacgcct gcatccggca gaagggcggc
tactgcagct ggtaccgagg atgggccccc 600ccggataaaa gcatcatcaa
tgccacagac cccggtggag gtggagatgc acacaagagt 660gaggttgctc
atcggtttaa agatttggga gaagaaaatt tcaaagcctt ggtgttgatt
720gcctttgctc agtatcttca gcagtgtcca tttgaagatc atgtaaaatt
agtgaatgaa 780gtaactgaat ttgcaaaaac atgtgttgct gatgagtcag
ctgaaaattg tgacaaatca 840cttcataccc tttttggaga caaattatgc
acagttgcaa ctcttcgtga aacctatggt 900gaaatggctg actgctgtgc
aaaacaagaa cctgagagaa atgaatgctt cttgcaacac 960aaagatgaca
acccaaacct cccccgattg gtgagaccag aggttgatgt gatgtgcact
1020gcttttcatg acaatgaaga gacatttttg aaaaaatact tatatgaaat
tgccagaaga 1080catccttact tttatgcccc ggaactcctt ttctttgcta
aaaggtataa agctgctttt 1140acagaatgtt gccaagctgc tgataaagct
gcctgcctgt tgccaaagct cgatgaactt 1200cgggatgaag ggaaggcttc
gtctgccaaa cagagactca agtgtgccag tctccaaaaa 1260tttggagaaa
gagctttcaa agcatgggca gtagctcgcc tgagccagag atttcccaaa
1320gctgagtttg cagaagtttc caagttagtg acagatctta ccaaagtcca
cacggaatgc 1380tgccatggag atctgcttga atgtgctgat gacagggcgg
accttgccaa gtatatctgt 1440gaaaatcaag attcgatctc cagtaaactg
aaggaatgct gtgaaaaacc tctgttggaa 1500aaatcccact gcattgccga
agtggaaaat gatgagatgc ctgctgactt gccttcatta 1560gctgctgatt
ttgttgaaag taaggatgtt tgcaaaaact atgctgaggc aaaggatgtc
1620ttcctgggca tgtttttgta tgaatatgca agaaggcatc ctgattactc
tgtcgtgctg 1680ctgctgagac ttgccaagac atatgaaacc actctagaga
agtgctgtgc cgctgcagat 1740cctcatgaat gctatgccaa agtgttcgat
gaatttaaac ctcttgtgga agagcctcag 1800aatttaatca aacaaaattg
tgagcttttt gagcagcttg gagagtacaa attccagaat 1860gcgctattag
ttcgttacac caagaaagta ccccaagtgt caactccaac tcttgtagag
1920gtctcaagaa acctaggaaa agtgggcagc aaatgttgta aacatcctga
agcaaaaaga 1980atgccctgtg cagaagacta tctatccgtg gtcctgaacc
agttatgtgt gttgcatgag 2040aaaacgccag taagtgacag agtcaccaaa
tgctgcacag aatccttggt gaacaggcga 2100ccatgctttt cagctctgga
agtcgatgaa acatacgttc ccaaagagtt taatgctgaa 2160acattcacct
tccatgcaga tatatgcaca ctttctgaga aggagagaca aatcaagaaa
2220caaactgcac ttgttgagct cgtgaaacac aagcccaagg caacaaaaga
gcaactgaaa 2280gctgttatgg atgatttcgc agcttttgta gagaagtgct
gcaaggctga cgataaggag 2340acctgctttg ccgaggaggg taaaaaactt
gttgctgcaa gtcaagctgc cttaggctta 2400692223DNAHomo sapiens
69atgacccctt ggctcgggct catcgtgctc ctgggcagct ggagcctggg ggactggggc
60gccgaggcgt gcacatgctc gcccagccac ccccaggacg ccttctgcaa ctccgacatc
120gtgatccggg ccaaggtggt ggggaagaag ctggtaaagg aggggccctt
cggcacgctg 180gtctacacca tcaagcagat gaagatgtac cgaggcttca
ccaagatgcc ccatgtgcag 240tacatccaca cggaagcttc cgagagtctc
tgtggcctta agctggaggt caacaagtac 300cagtacctgc tgacaggtcg
cgtctatgat ggcaagatgt acacggggct gtgcaacttc 360gtggagaggt
gggaccagct caccctctcc cagcgcaagg ggctgaacta tcggtatcac
420ctgggttgta acggtggagg tggagatgca cacaagagtg aggttgctca
tcgatttaaa 480gatttgggag aagaaaattt caaagccttg gtgttgattg
cctttgctca gtatcttcag 540cagtgtccat ttgaagatca tgtaaaatta
gtgaatgaag taactgaatt tgcaaaaaca 600tgtgttgctg atgagtcagc
tgaaaattgt gacaaatcac ttcataccct ttttggagac 660aaattatgca
cagttgcaac tcttcgtgaa acctatggtg aaatggctga ctgctgtgca
720aaacaagaac ctgagagaaa tgaatgcttc ttgcaacaca aagatgacaa
cccaaacctc 780ccccgattgg tgagaccaga ggttgatgtg atgtgcactg
cttttcatga caatgaagag 840acatttttga aaaaatactt atatgaaatt
gccagaagac atccttactt ttatgccccg 900gaactccttt tctttgctaa
aaggtataaa gctgctttta cagaatgttg ccaagctgct 960gataaagctg
cctgcctgtt gccaaagctc gatgaacttc gggatgaagg gaaggcttcg
1020tctgccaaac agagactcaa gtgtgccagt ctccaaaaat ttggagaaag
agctttcaaa 1080gcatgggcag tagctcgcct gagccagaga tttcccaaag
ctgagtttgc agaagtttcc 1140aagttagtga cagatcttac caaagtccac
acggaatgct gccatggaga tctgcttgaa 1200tgtgctgatg acagggcgga
ccttgccaag tatatctgtg aaaatcaaga ttcgatctcc 1260agtaaactga
aggaatgctg tgaaaaacct ctgttggaaa aatcccactg cattgccgaa
1320gtggaaaatg atgagatgcc tgctgacttg ccttcattag ctgctgattt
tgttgaaagt 1380aaggatgttt gcaaaaacta tgctgaggca aaggatgtct
tcctgggcat gtttttgtat 1440gaatatgcaa gaaggcatcc tgattactct
gtcgtgctgc tgctgagact tgccaagaca 1500tatgaaacca ctctagagaa
gtgctgtgcc gctgcagatc ctcatgaatg ctatgccaaa 1560gtgttcgatg
aatttaaacc tcttgtggaa gagcctcaga atttaatcaa acaaaattgt
1620gagctttttg agcagcttgg agagtacaaa ttccagaatg cgctattagt
tcgttacacc 1680aagaaagtac cccaagtgtc aactccaact cttgtagagg
tctcaagaaa cctaggaaaa 1740gtgggcagca aatgttgtaa acatcctgaa
gcaaaaagaa tgccctgtgc agaagactat 1800ctatccgtgg tcctgaacca
gttatgtgtg ttgcatgaga aaacgccagt aagtgacaga 1860gtcaccaaat
gctgcacaga atccttggtg aacaggcgac catgcttttc agctctggaa
1920gtcgatgaaa catacgttcc caaagagttt aatgctgaaa cattcacctt
ccatgcagat 1980atatgcacac tttctgagaa ggagagacaa atcaagaaac
aaactgcact tgttgagctc 2040gtgaaacaca agcccaaggc aacaaaagag
caactgaaag ctgttatgga tgatttcgca 2100gcttttgtag agaagtgctg
caaggctgac gataaggaga cctgctttgc cgaggagggt 2160aaaaaacttg
ttgcggccag tcaggccgcc ttaggcttag tcgaccatca tcatcatcat 2220cat
2223702223DNAHomo sapiens 70atgacccctt ggctcgggct catcgtgctc
ctgggcagct ggagcctggg ggactggggc 60gccgaggcgt gcacatgctc gcccagccac
ccccaggacg ccttctgcaa ctccgacatc 120gtgatccggg ccagtgtggt
ggggaagaag ctggtaaagg aggggcccaa tggcacgctg 180gtctacacca
tcaagcagat gaagatgtac cgaggcttca ccaagatgcc ccatgtgcag
240tacatccata cggaagcttc cgagagtctc tgtggcctta agctggaggt
caacaagtac 300cagtacctgc tgacaggtcg cgtctatgat ggcaagatgt
acacggggct gtgcaacttc 360gtggagaggt gggaccagct caccctctcc
cagcgcaagg ggctgaacta tcggtatcac 420ctgggttgta acggtggagg
tggagatgca cacaagagtg aggttgctca tcgatttaaa 480gatttgggag
aagaaaattt caaagccttg gtgttgattg cctttgctca gtatcttcag
540cagtgtccat ttgaagatca tgtaaaatta gtgaatgaag taactgaatt
tgcaaaaaca 600tgtgttgctg atgagtcagc tgaaaattgt gacaaatcac
ttcataccct ttttggagac 660aaattatgca cagttgcaac tcttcgtgaa
acctatggtg aaatggctga ctgctgtgca 720aaacaagaac ctgagagaaa
tgaatgcttc ttgcaacaca aagatgacaa cccaaacctc 780ccccgattgg
tgagaccaga ggttgatgtg atgtgcactg cttttcatga caatgaagag
840acatttttga aaaaatactt atatgaaatt gccagaagac atccttactt
ttatgccccg 900gaactccttt tctttgctaa aaggtataaa gctgctttta
cagaatgttg ccaagctgct 960gataaagctg cctgcctgtt gccaaagctc
gatgaacttc gggatgaagg gaaggcttcg 1020tctgccaaac agagactcaa
gtgtgccagt ctccaaaaat ttggagaaag agctttcaaa 1080gcatgggcag
tagctcgcct gagccagaga tttcccaaag ctgagtttgc agaagtttcc
1140aagttagtga cagatcttac caaagtccac acggaatgct gccatggaga
tctgcttgaa 1200tgtgctgatg acagggcgga ccttgccaag tatatctgtg
aaaatcaaga ttcgatctcc 1260agtaaactga aggaatgctg tgaaaaacct
ctgttggaaa aatcccactg cattgccgaa 1320gtggaaaatg atgagatgcc
tgctgacttg ccttcattag ctgctgattt tgttgaaagt 1380aaggatgttt
gcaaaaacta tgctgaggca aaggatgtct tcctgggcat gtttttgtat
1440gaatatgcaa gaaggcatcc tgattactct gtcgtgctgc tgctgagact
tgccaagaca 1500tatgaaacca ctctagagaa gtgctgtgcc gctgcagatc
ctcatgaatg ctatgccaaa 1560gtgttcgatg aatttaaacc tcttgtggaa
gagcctcaga atttaatcaa acaaaattgt 1620gagctttttg agcagcttgg
agagtacaaa ttccagaatg cgctattagt tcgttacacc 1680aagaaagtac
cccaagtgtc aactccaact cttgtagagg tctcaagaaa cctaggaaaa
1740gtgggcagca aatgttgtaa acatcctgaa gcaaaaagaa tgccctgtgc
agaagactat 1800ctatccgtgg tcctgaacca gttatgtgtg ttgcatgaga
aaacgccagt aagtgacaga 1860gtcaccaaat gctgcacaga atccttggtg
aacaggcgac catgcttttc agctctggaa 1920gtcgatgaaa catacgttcc
caaagagttt aatgctgaaa cattcacctt ccatgcagat 1980atatgcacac
tttctgagaa ggagagacaa atcaagaaac aaactgcact tgttgagctc
2040gtgaaacaca agcccaaggc aacaaaagag caactgaaag ctgttatgga
tgatttcgca 2100gcttttgtag agaagtgctg caaggctgac gataaggaga
cctgctttgc cgaggagggt 2160aaaaaacttg ttgcggccag tcaggccgcc
ttaggcttag tcgaccatca tcatcatcat 2220cat 222371585PRTHomo sapiens
71Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1
5 10 15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu
Gln 20 25 30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val
Thr Glu 35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn
Cys Asp Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr
Val Ala Thr Leu65 70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys
Cys Ala Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His
Lys Asp Asp Asn Pro Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu
Val Asp Val Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr
Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro
Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155
160Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
165 170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys
Ala Ser 180 185 190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln
Lys Phe Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg
Leu Ser Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val Ser
Lys Leu Val Thr Asp Leu Thr Lys225 230 235 240Val His Thr Glu Cys
Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp
Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser
Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280
285Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser
290 295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn
Tyr Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu
Tyr Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr Ser Val Val Leu
Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys
Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val
Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu
Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395
400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro
405 410 415Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu
Gly Lys 420 425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys
Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn
Gln Leu Cys Val Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg
Val Thr Lys Cys Cys Thr Glu Ser465 470
475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu
Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe
His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile
Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val Lys His Lys Pro
Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val Met Asp Asp Phe
Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555 560Ala Asp Asp Lys
Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575Ala Ala
Ser Gln Ala Ala Leu Gly Leu 580 58572227PRTHomo sapiens 72Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170
175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 210 215 220Pro Gly Lys22573217PRTHomo sapiens 73Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10
15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr 35 40 45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu 50 55 60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His65 70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln 100 105 110Pro Arg Glu Pro Gln Val Thr Thr
Leu Pro Pro Ser Arg Glu Glu Met 115 120 125Asn Lys Thr Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145 150 155 160Tyr
Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170
175Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 195 200 205Lys Ser Leu Ser Leu Ser Pro Gly Lys 210
21574212PRTHomo sapiens 74Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu1 5 10 15Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 20 25 30His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu 35 40 45Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr 50 55 60Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn65 70 75 80Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 85 90 95Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 100 105 110Val
Thr Thr Leu Pro Pro Ser Arg Glu Glu Met Asn Lys Thr Gln Val 115 120
125Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
130 135 140Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr
Thr Pro145 150 155 160Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Asp Leu Thr 165 170 175Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val 180 185 190Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu 195 200 205Ser Pro Gly Lys
210755PRTArtificial SequenceLinker 75Glu Pro Lys Ser Ser1 5
* * * * *