U.S. patent application number 13/383676 was filed with the patent office on 2012-11-08 for masp isoforms as inhibitors of complement activation.
This patent application is currently assigned to RIGSHOSPITALET. Invention is credited to Peter Garred, Tina Hummelshoj Glue, Mikkel-Ole Skjodt.
Application Number | 20120282285 13/383676 |
Document ID | / |
Family ID | 43449884 |
Filed Date | 2012-11-08 |
United States Patent
Application |
20120282285 |
Kind Code |
A1 |
Garred; Peter ; et
al. |
November 8, 2012 |
MASP ISOFORMS AS INHIBITORS OF COMPLEMENT ACTIVATION
Abstract
The present invention relates to novel ficolin-associated
polypeptides, and polypeptides derived from these
ficolin-associated polypeptides for the use in the treatment of
conditions associated with inflammation, apoptosis, autoimmunity,
coagulation, thrombotic or coagulopathic related diseases, as well
as the use as biomarkers. The present invention further relates to
anti-bodies recognising such novel ficolin-associated polypeptides,
and polypeptides derived thereof, nucleic acid molecules encoding
such polypeptides, vectors and host cells used in the production of
the polypeptides.
Inventors: |
Garred; Peter;
(Charlottenlund, DK) ; Glue; Tina Hummelshoj;
(Soborg, DK) ; Skjodt; Mikkel-Ole; (Frederiksberg
C, DK) |
Assignee: |
RIGSHOSPITALET
Copenhagen O
DK
SYDDANSK UNIVERSITET
Odense M
DK
KOBENHAVNS UNIVERSITET
Copenhagen K
DK
|
Family ID: |
43449884 |
Appl. No.: |
13/383676 |
Filed: |
July 16, 2010 |
PCT Filed: |
July 16, 2010 |
PCT NO: |
PCT/EP2010/060279 |
371 Date: |
July 13, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61311049 |
Mar 5, 2010 |
|
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|
Current U.S.
Class: |
424/185.1 ;
435/254.2; 435/320.1; 435/325; 435/346; 435/348; 435/352; 435/353;
435/357; 435/358; 435/365; 435/366; 435/369; 435/6.11; 435/69.1;
435/7.9; 435/7.92; 436/501; 514/12.2; 514/18.9; 514/21.2; 530/350;
530/387.9; 530/395; 536/23.5 |
Current CPC
Class: |
A61P 17/00 20180101;
A61P 31/10 20180101; A61P 3/06 20180101; A61P 29/00 20180101; A61P
9/10 20180101; A61P 31/12 20180101; A61P 1/16 20180101; A61P 37/06
20180101; A61P 31/16 20180101; A61P 11/00 20180101; A61P 21/04
20180101; A61P 5/14 20180101; A61P 7/06 20180101; A61P 31/18
20180101; A61P 11/02 20180101; A61P 17/06 20180101; A61P 1/02
20180101; A61P 13/00 20180101; A61P 3/10 20180101; A61P 37/00
20180101; A61P 43/00 20180101; A61P 33/04 20180101; A61P 33/06
20180101; A61P 35/00 20180101; A61P 37/08 20180101; A61P 9/00
20180101; A61P 13/08 20180101; A61P 25/28 20180101; A61P 11/06
20180101; A61P 15/00 20180101; A61P 25/00 20180101; A61P 31/04
20180101; A61P 33/00 20180101; A61P 3/04 20180101; A61P 17/02
20180101; A61P 27/02 20180101; A61P 1/00 20180101; A61P 1/04
20180101; A61P 7/02 20180101; A61P 19/02 20180101; C07K 14/472
20130101; A61P 1/18 20180101; A61P 31/20 20180101; A61P 37/02
20180101; A61P 13/12 20180101 |
Class at
Publication: |
424/185.1 ;
530/350; 530/395; 530/387.9; 536/23.5; 514/21.2; 514/12.2;
514/18.9; 435/320.1; 435/348; 435/254.2; 435/325; 435/366; 435/352;
435/358; 435/369; 435/365; 435/353; 435/357; 435/346; 435/69.1;
436/501; 435/7.9; 435/7.92; 435/6.11 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C07K 16/18 20060101 C07K016/18; C12N 15/12 20060101
C12N015/12; A61P 29/00 20060101 A61P029/00; A61P 43/00 20060101
A61P043/00; C12Q 1/68 20060101 C12Q001/68; A61P 37/00 20060101
A61P037/00; C12N 5/10 20060101 C12N005/10; C12N 1/19 20060101
C12N001/19; C12N 5/12 20060101 C12N005/12; C12P 21/02 20060101
C12P021/02; G01N 33/566 20060101 G01N033/566; C07K 14/435 20060101
C07K014/435; A61K 39/00 20060101 A61K039/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 17, 2009 |
EP |
09165770.0 |
Oct 1, 2009 |
EP |
09171941.9 |
Claims
1. An isolated ficolin-associated polypeptide.
2. (canceled)
3. The polypeptide according to claim 1, wherein said polypeptide
is capable of associating with mannose-binding lectin (MBL), with
any one of ficolin-1, ficolin-2, or ficolin-3, with any one of C1q,
lung surfactant proteins SP-A and/or SP-D, and intracellular
collagen-like defence molecules, such as CL-L1, or with a specific
acceptor protein, such as a specific receptor.
4-6. (canceled)
7. The polypeptide according to claim 1, wherein said polypeptide
comprises the amino acid sequence 20-297 of SEQ NO:3, or a
functional variant thereof.
8. The polypeptide according to claim 1, wherein said polypeptide
comprises the amino acid sequence 20-380 of SEQ NO:1 or a
functional variant thereof.
9. The polypeptide according to claim 1, wherein said polypeptide
comprises the amino acid sequence 16-296 of SEQ ID NO:9 or a
functional variant thereof.
10. (canceled)
11. The polypeptide according to claim 1, wherein said polypeptide
is N-linked glycosylated at one or two amino acids corresponding to
a position selected from 49 and 178 of SEQ NO:1.
12-15. (canceled)
16. A polypeptide comprising the amino acid sequence of SEQ ID NO:4
or variants or immunologic fragment thereof.
17-20. (canceled)
21. An antibody that specifically binds a polypeptide according to
claim 1.
22. An isolated nucleic acid molecule encoding a polypeptide of
claim 1.
23. (canceled)
24. A vector comprising an isolated nucleic acid molecule according
to claim 1.
25. A host cell comprising a vector according to claims 24.
26-28. (canceled)
29. A method for producing an isolated ficolin-associated
polypeptide, said method comprising cultivating a host cell as
defined in claim 25 in an appropriate growth medium under
conditions allowing expression of the polynucleotide construct and
recovering the resulting ficolin-associated polypeptide from the
culture medium.
30. A composition comprising the polypeptide as defined in claim
1.
31. A pharmaceutical composition comprising the polypeptide as
defined in claim 1.
32. A method for detecting a ficolin-associated polypeptide in a
biological sample, said method comprising: a) obtaining a
biological sample; b) contacting said biological sample with an
antibody according to claim 21; and c) detecting complexes of said
antibody and said polypeptide, if any; as an indication of the
presence of said polypeptide in said sample.
33. A polypeptide as defined in claim 1 for use as a
medicament.
34-44. (canceled)
45. A method for the treatment of any indication associated with
inflammation, apoptosis and/or autoimmunity such as for the
treatment of any autoimmune conditions such as Addison's disease,
autoimmune hemolytic anemia, autoimmune thyroiditis, Crohn's
disease, Graves' disease, Guillain-Barre syndrome, systemic lupus
erythematosus (SLE), lupus nephritis, multiple sclerosis,
myasthenia gravis, psoriasis, primary biliary cirrhosis, rheumatoid
arthritis and uveitis, asthma, atherosclerosis, Type I diabetes,
psoriasis, various allergies, or for the treatment of any
inflammatory disorder selected from the group consisting of
appendicitis, peptic ulcer, gastric ulcer, duodenal ulcer,
peritonitis, pancreatitis, ulcerative colitis, pseudomembranous
colitis, acute colitis, ischemic colitis, diverticulitis,
epiglottitis, achalasia, cholangitis, cholecystitis, hepatitis,
Crohn's disease, enteritis, Whipple's disease, allergy, immune
complex disease, organ ischemia, reperfusion injury, organ
necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia,
hyperpyrexia, eosinophilic granuloma, granulomatosis, sarcoidosis
septic abortion, epididymitis, vaginitis, prostatitis, urethritis,
bronchitis, emphysema, rhinitis, pneumonitis,
pneumotransmicroscopicsilicovolcanoconiosis, alvealitis,
bronchiolitis, pharyngitis, pleurisy, sinusitis, influenza,
respiratory syncytial virus infection, HIV infection, hepatitis B
virus infection, hepatitis C virus infection, disseminated
bacteremia, Dengue fever, candidiasis, malaria, filariasis,
amebiasis, hydatid cysts, burns, dermatitis, dermatomyositis,
sunburn, urticaria, warts, wheals, vasulitis, angiitis,
endocarditis, arteritis, atherosclerosis, thrombophlebitis,
pericarditis, myocarditis, myocardial ischemia, periarteritis
nodosa, rheumatic fever, Alzheimer's disease, coeliac disease,
congestive heart failure, adult respiratory distress syndrome,
meningitis, encephalitis, multiple sclerosis, cerebral infarction,
cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia,
spinal cord injury, paralysis, uveitis, arthritides, arthralgias,
osteomyelitis, fasciitis, Paget's disease, gout, periodontal
disease, rheumatoid arthritis, synovitis, myasthenia gravis,
thyroiditis, systemic lupus erythematosis, Goodpasture's syndrome,
Behcet's syndrome, allograft rejection, graft-versus-host disease,
Type I diabetes, ankylosing spondylitis, Berger's disease, Reiter's
syndrome and Hodgkin's disease, keratitis, Type 2 diabetes, cystic
fibrosis, myocardial infarction, reperfusion injury, stroke,
dermatomyositis, metabolic syndrome, systemic inflammatory response
syndrome, sepsis, multiple organ failure, disseminated
intravascular coagulation, anaphylactic shock. Vascular
complication and nephropathy associated with type 1 and/or type 2
diabetes, meningitis, bacterial septicaemia, complicated malaria,
atypic haemolytic uremic syndrome, haemolytic uremic syndrome, age
related macular degeneration, paroxysmal nocturnal hemoglobinuria,
snake venom bite, burn injury, and complications to organ
transplantations, or for the treatment of any inflammatory disorder
selected from the group consisting of organ ischemia, reperfusion
injury, organ necrosis, vasulitis, endocarditis, atherosclerosis,
thrombophlebitis, pericarditis, myocarditis, myocardial ischemia,
periarteritis nodosa, rheumatic fever, congestive heart failure,
adult respiratory distress syndrome, cerebral infarction, cerebral
embolism, or vascular complications and nephropathy associated with
type 1 and/or type 2 diabetes, or for the treatment of any
indications associated with coagulation, thrombotic or
coagulopathic related diseases including inflammatory response and
chronic thromboembolic diseases or disorders associated with fibrin
formation including vascular disorders such as thrombosis, such as
deep venous thrombosis, arterial thrombosis, post surgical
thrombosis, coronary artery bypass graft (CABG), percutaneous
transdermal coronary angioplastry (PTCA), platelet deposition
stroke, tumor growth, tumor metastasis, angiogenesis, thrombolysis,
atherosclerosis, restenosis, such as arteriosclerosis and/or
restenosis following angioplasty, acute and chronic indications
such as inflammation, sepsis, septic shock, septicemia,
hypotension, adult respiratory distress syndrome (ARDS), systemic
inflammatory response syndrome (SIRS), disseminated intravascular
coagulopathy (DIC), pulmonary embolism, pathological platelet
deposition, myocardial infarction, or the prophylactic treatment of
mammals with atherosclerotic vessels at risk for thrombosis,
venoocclusive disease following peripheral blood progenitor cell
(PBPC) transplantation, hemolytic uremic syndrome (HUS), and
thrombotic thrombocytopenic purpura (TTP) and rheumatic fever,
inflammatory response and chronic thromboembolic diseases or
disorders associated with fibrin formation, for preventing the
occurrence of thromboembolic complications in identified high risk
patients, such as those undergoing surgery or those with congestive
heart failure, for the treatment of a medical condition associated
with the heart, or for the treatment of a medical condition
associated with a deficiency in a ficolin-associated polypeptide;
the method comprising administering a therapeutically or
prophylactically effective amount of a polypeptide as defined in
claim 1 to a subject in need thereof.
46-53. (canceled)
54. A method of detecting the presence of a nucleic acid encoding a
ficolin-associated polypeptide in a biological sample, the method
comprising a) obtaining a biological sample; b) contacting said
biological sample with a nucleic acid probe according to claim 22;
and c) detecting complexes of said a nucleic acid probe and said
nucleic acid encoding said ficolin-associated polypeptide, if any;
as an indication of the presence of said nucleic acid encoding said
polypeptide in said sample.
55. A method for diagnosing a disorder associated with aberrant
expression of a ficolin-associated polypeptide, comprising
obtaining a biological sample from a patient and measuring the
expression in said biological sample of said ficolin-associated
polypeptide, wherein increased or decreased expression of said
ficolin-associated polypeptide in said biological sample compared
to a control indicates that said patient suffers from a disorder
associated with aberrant expression of a ficolin-associated
polypeptide.
56-63. (canceled)
64. Use of a polypeptide as defined in claim 1 as a biomarker in
blood and tissue for diagnosis and/or prognosis of an autoimmune,
metabolic and/or inflammatory condition such as Addison's disease,
autoimmune hemolytic anemia, autoimmune thyroiditis, Crohn's
disease, Graves' disease, Guillain-Barre syndrome, systemic lupus
erythematosus (SLE), lupus nephritis, multiple sclerosis,
myasthenia gravis, psoriasis, primary biliary cirrhosis, rheumatoid
arthritis and uveitis, asthma, atherosclerosis, Type I diabetes,
psoriasis, various allergies, or for the treatment of any
inflammatory disorder selected from the group consisting of
appendicitis, peptic ulcer, gastric ulcer, duodenal ulcer,
peritonitis, pancreatitis, ulcerative colitis, pseudomembranous
colitis, acute colitis, ischemic colitis, diverticulitis,
epiglottitis, achalasia, cholangitis, cholecystitis, hepatitis,
Crohn's disease, enteritis, Whipple's disease, allergy, immune
complex disease, organ ischemia, reperfusion injury, organ
necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia,
hyperpyrexia, eosinophilic granuloma, granulomatosis, sarcoidosis,
septic abortion, epididymitis, vaginitis, prostatitis, urethritis,
bronchitis, emphysema, rhinitis, pneumonitis,
pneumotransmicroscopicsilicovolcanoconiosis, alvealitis,
bronchiolitis, pharyngitis, pleurisy, sinusitis, influenza,
respiratory syncytial virus infection, HIV infection, hepatitis B
virus infection, hepatitis C virus infection, disseminated
bacteremia, Dengue fever, candidiasis, malaria, filariasis,
amebiasis, hydatid cysts, burns, dermatitis, dermatomyositis,
sunburn, urticaria, warts, wheals, vasulitis, angiitis,
endocarditis, arteritis, atherosclerosis, thrombophlebitis,
pericarditis, myocarditis, myocardial ischemia, periarteritis
nodosa, rheumatic fever, Alzheimer's disease, coeliac disease,
congestive heart failure, adult respiratory distress syndrome,
meningitis, encephalitis, multiple sclerosis, cerebral infarction,
cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia,
spinal cord injury, paralysis, uveitis, arthritides, arthralgias,
osteomyelitis, fasciitis, Paget's disease, gout, periodontal
disease, rheumatoid arthritis, synovitis, myasthenia gravis,
thyroiditis, systemic lupus erythematosis, Goodpasture's syndrome,
Behcet's syndrome, allograft rejection, graft-versus-host disease,
Type I diabetes, ankylosing spondylitis, Berger's disease, Reiter's
syndrome and Hodgkin's disease, keratitis, Type 2 diabetes, cystic
fibrosis, myocardial infarction, reperfusion injury, stroke,
dermatomyositis, metabolic syndrome, systemic inflammatory response
syndrome, sepsis, multiple organ failure, disseminated
intravascular coagulation, anaphylactic shock. Vascular
complication and nephropathy associated with type 1 and/or type 2
diabetes, meningitis, bacterial septicaemia, complicated malaria,
atypic haemolytic uremic syndrome, haemolytic uremic syndrome, age
related macular degeneration, paroxysmal nocturnal hemoglobinuria,
snake venom bite, burn injury, and complications to organ
transplantations, or for the treatment of any inflammatory disorder
selected from the group consisting of organ ischemia, reperfusion
injury, organ necrosis, vasulitis, endocarditis, atherosclerosis,
thrombophlebitis, pericarditis, myocarditis, myocardial ischemia,
periarteritis nodosa, rheumatic fever, congestive heart failure,
adult respiratory distress syndrome, cerebral infarction, cerebral
embolism, or vascular complications and nephropathy associated with
type 1 and/or type 2 diabetes, or for the treatment of any
indications associated with coagulation, thrombotic or
coagulopathic related diseases including inflammatory response and
chronic thromboembolic diseases or disorders associated with fibrin
formation including vascular disorders such as thrombosis, such as
deep venous thrombosis, arterial thrombosis, post surgical
thrombosis, coronary artery bypass graft (CABG), percutaneous
transdermal coronary angioplastry (PTCA), platelet deposition
stroke, tumor growth, tumor metastasis, angiogenesis, thrombolysis,
atherosclerosis, restenosis, such as arteriosclerosis and/or
restenosis following angioplastry, acute and chronic indications
such as inflammation, sepsis, septic shock, septicemia,
hypotension, adult respiratory distress syndrome (ARDS), systemic
inflammatory response syndrome (SIRS), disseminated intravascular
coagulopathy (DIC), pulmonary embolism, pathological platelet
deposition, myocardial infarction, or the prophylactic treatment of
mammals with atherosclerotic vessels at risk for thrombosis,
venoocclusive disease following peripheral blood progenitor cell
(PBPC) transplantation, hemolytic uremic syndrome (HUS), and
thrombotic thrombocytopenic purpura (TTP) and rheumatic fever,
inflammatory response and chronic thromboembolic diseases or
disorders associated with fibrin formation, for preventing the
occurrence of thromboembolic complications in identified high risk
patients, such as those undergoing surgery or those with congestive
heart failure, for the treatment of a medical condition associated
with the heart, or for the treatment of a medical condition
associated with a deficiency in a ficolin-associated polypeptide.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel ficolin-associated
polypeptides, and polypeptides derived from these
ficolin-associated polypeptides for the use in the treatment of
conditions associated with inflammation, apoptosis, autoimmunity,
coagulation, thrombotic or coagulopathic related diseases, as well
as the use as biomarkers. The present invention further relates to
antibodies recognising such novel ficolin-associated polypeptides,
and polypeptides derived thereof, nucleic acid molecules encoding
such polypeptides, vectors and host cells used in the production of
the polypeptides.
BACKGROUND OF THE INVENTION
[0002] Activation of the complement system (C) is accomplished via
three different initiation pathways: The alternative (AP), the
classical (CP), or the lectin pathway (LCP). AP activation occurs
on foreign surfaces and is caused by a slow, spontaneous hydrolysis
of C3 and the activity of the factors properdin, factor B and
factor D to form the functional C3 convertase C3bBb. AP also
functions as an amplification pathway (the amplification loop) of
the two other pathways. Recently it has been shown that the
alternative convertase assembly may also be initiated by
non-covalent attachment of properdin to some target surfaces. CP
activation on the other hand is initiated when C1q binds to
immunoglobulins in complex with antigens, which triggers the
activation of the C1q-associated serine proteases C1r and C1s. C1s
cleaves and activates C4 and C2 to form the CP C3 convertase C4b2a.
The LCP is activated when mannose-binding lectin (MBL) or ficolins
binds to restricted patterns of carbohydrates or acetylated
compounds e.g. on the surface of microorganisms or when exposed on
dying host cells. Upon binding to the ligand the associated serine
protease MASP-2 activates and cleaves C4 and C2 to form the LCP C3
convertase C4b2a. The function of MASP-1 has been suggested to
involve a stabilization of MASP-2 cleavage of C2 and also direct
low grade cleavage of C3. Yet other studies relate the function and
activity of MASP-1 and MASP-2 to a coagulation system cross-talk
involving prothrombin, fibrinogen and factor XIII. Using MASP1/3
knockout mice it was recently demonstrated that MASP-1 in fact
contributes to the complement activity. The exact function of the
most recently discovered MBL associated serine protease MASP-3 has
yet to be elucidated. Studies indicating that MASP-3 associates
with a limited range of MBL oligomers and that MASP-3 and the small
MBL-associated protein (sMAP) are involved in regulation or
inhibition of MBL dependent LCP complement activation have been
reported.
[0003] MASP-1 and -3 are derived from the same MASP1/3 gene
(present on chromosome 3g27-q28) through differential splicing.
They contain an identical A-chain except for 15 C-terminal
residues. The A chain is comprised of two CUB (C1r/C1s, Urchin-EGF,
Bone morphogenetic protein) domains separated by an EGF (Epidermal
Growth Factor) domain and followed by two CCP domains (complement
control protein). The B-chain including the serine protease domain
is different for MASP-1 and MASP-3. The MASP-2 and sMAP are also
derived from the same gene (present on chromosome 1p36-p36.2) where
sMAP is a truncated form lacking the serine protease domain and a
major part of the A-chain. The MASP1/3 gene has been shown to be
polymorphic, but the functional importance of this is still poorly
understood. However, there is some evidence that polymorphisms in
the MASP2/sMAP gene are associated with increased risk of
infections. Expression of the MASPs is localized to liver
hepatocytes, but a recent study described that human MASP-3 mRNA
(as the only MASP-mRNA) was expressed in a broad range of
tissues.
OBJECT OF THE INVENTION
[0004] It is an object of embodiments of the invention to provide
polypeptides suitable for the treatment of conditions associated
with inflammation, apoptosis, autoimmunity, coagulation, and/or
thrombotic or coagulopathic related diseases. The polypeptides of
the invention may further be suitable as biomarkers for the
diagnosis and/or prognosis of these indications as well as for
malignant diseases, such as cancers.
SUMMARY OF THE INVENTION
[0005] It has been found by the present inventor(s) that novel
polypeptides that associate with the recognition molecules of the
lectin complement pathway as well as polypeptides, such as
fragments derived thereof may be used in the treatment of specific
medical conditions associated with inflammation, apoptosis,
autoimmunity, coagulation, and/or thrombotic or coagulopathic
related diseases.
[0006] So, in a first aspect the present invention relates to an
isolated ficolin-associated polypeptide.
[0007] In a second aspect the present invention relates to a
polypeptide comprising the amino acid sequence of SEQ ID NO:4 or
variants or immunologic fragment thereof.
[0008] In a third aspect the present invention relates to an
antibody that specifically binds a polypeptide according to the
invention.
[0009] In a fourth aspect the present invention relates to an
isolated nucleic acid molecule encoding a polypeptide according to
the invention.
[0010] In a further aspect the present invention relates to an
isolated nucleic acid molecule comprising a nucleotide sequence
that is at least 70% identical to the sequence of SEQ NO:2.
[0011] In a further aspect the present invention relates to a
vector comprising an isolated nucleic acid molecule encoding a
polypeptide according to the invention.
[0012] In a further aspect the present invention relates a host
cell comprising a vector comprising an isolated nucleic acid
molecule encoding a polypeptide according to the invention.
[0013] In a further aspect the present invention relates a method
for producing the polypeptide according to the invention, the
method comprising cultivating a cell according to the invention in
an appropriate growth medium under conditions allowing expression
of the polynucleotide construct and recovering the resulting
polypeptide from the culture medium.
[0014] In a further aspect the present invention relates a
composition comprising a polypeptide according to the
invention.
[0015] In a further aspect the present invention relates a
pharmaceutical composition comprising a polypeptide according to
the invention.
[0016] In a further aspect the present invention relates a method
for detecting a polypeptide according to the present invention in a
biological sample, the method comprising: [0017] a) obtaining a
biological sample; [0018] b) contacting the biological sample with
an antibody according to the invention; and [0019] c) detecting
complexes of the antibody and the polypeptide, if any;
[0020] as an indication of the presence of the polypeptide in the
sample.
[0021] In a further aspect the present invention relates a
polypeptide according to the invention for use as a medicament.
[0022] In a further aspect the present invention relates to the use
of a polypeptide according to the present invention; for the
preparation of a medicament.
[0023] In a further aspect the present invention relates to a
polypeptide according to the present invention for the treatment of
any indications associated with inflammation, apoptosis and/or
autoimmunity.
[0024] In a further aspect the present invention relates to a
polypeptide according to the present invention for the treatment of
any indications associated with coagulation, thrombotic or
coagulopathic related diseases.
[0025] In a further aspect the present invention relates to a
polypeptide according to the present invention for preventing the
occurrence of thromboembolic complications in identified high risk
patients, such as those undergoing surgery or those with congestive
heart failure.
[0026] In a further aspect the present invention relates to a
polypeptide according to the present invention for the treatment of
medical condition associated with the heart.
[0027] In a further aspect the present invention relates to a
polypeptide according to the present invention for the treatment of
a medical condition associated with a deficiency in a
ficolin-associated polypeptide.
[0028] In a further aspect the present invention relates to a
method for the treatment of any indication associated with
inflammation, apoptosis and/or autoimmunity; the method comprising
administering a therapeutically or prophylactically effective
amount of a polypeptide according to the invention to a subject in
need thereof.
[0029] In a further aspect the present invention relates to a
method for the treatment of any indication associated with
coagulation, thrombotic or coagulopathic related diseases; the
method comprising administering a therapeutically or
prophylactically effective amount of a polypeptide according to the
present invention to a subject in need thereof.
[0030] In a further aspect the present invention relates to a
method for preventing the occurrence of thromboembolic
complications in identified high risk patients, such as those
undergoing surgery or those with congestive heart failure; the
method comprising administering a therapeutically or
prophylactically effective amount of a polypeptide according to the
present invention to a subject in need thereof.
[0031] In a further aspect the present invention relates to a
method for the treatment of a medical condition associated with the
heart; the method comprising administering a therapeutically or
prophylactically effective amount of a polypeptide according to the
present invention to a subject in need thereof.
[0032] In a further aspect the present invention relates to a
method for the treatment of a medical condition associated with a
deficiency in a ficolin-associated polypeptide; the method
comprising administering a therapeutically or prophylactically
effective amount of a polypeptide according to the present
invention to a subject in need thereof.
[0033] In a further aspect the present invention relates to a
nucleic acid probe capable of hybridizing under stringent
conditions to a nucleic acid sequence encoding a polypeptide
according to the present invention.
[0034] In a further aspect the present invention relates to a
method of detecting the presence of a nucleic acid encoding a
polypeptide according to the present invention in a biological
sample, the method comprising [0035] a) obtaining a biological
sample; [0036] b) contacting the biological sample with a nucleic
acid probe according to the present invention; and [0037] c)
detecting complexes of the a nucleic acid probe and the nucleic
acid encoding the polypeptide, if any;
[0038] as an indication of the presence of the nucleic acid
encoding the polypeptide in the sample.
[0039] In a further aspect the present invention relates to a
method for diagnosing a disorder associated with aberrant
expression of a ficolin-associated polypeptide, comprising
obtaining a biological sample from a patient and measuring the
expression in the biological sample of the ficolin-associated
polypeptide, wherein increased or decreased expression of the
ficolin-associated polypeptide in the biological sample compared to
a control indicates that the patient suffers from a disorder
associated with aberrant expression of a ficolin-associated
polypeptide.
[0040] In a further aspect the present invention relates to an
isolated composition comprising the combination of a polypeptide
according to the present invention together with one or more
proteins selected from Ficolin-1, 2, 3, mannose-binding lectin
(MBL), C1q, lung surfactant proteins SP-A and/or SP-D, and
intracellular collagen-like defence molecules, such as CLL-11.
[0041] In a further aspect the present invention relates to a
composition comprising a polypeptide according to the present
invention, which is a pharmaceutical composition.
[0042] In a further aspect the present invention relates to a
pharmaceutical composition according to the present invention for
use as a medicament.
[0043] In a further aspect the present invention relates to the use
of a composition according to the present invention; for the
preparation of a medicament.
[0044] In a further aspect the present invention relates to a
pharmaceutical composition according to the present invention for
the treatment of any indications associated with inflammation,
apoptosis and/or autoimmunity.
[0045] In a further aspect the present invention relates to a
pharmaceutical composition according to the present invention for
the treatment of any indication as defined herein.
[0046] In a further aspect the present invention relates to a
method for the treatment of any indication as defined herein, the
method comprising simultaneously or sequentially administering a
therapeutically or prophylactically effective amount of a
polypeptide according to the present invention and one or more
proteins selected from Ficolin-1, 2, 3, and mannose-binding lectin
(MBL), C1q, lung surfactant proteins SP-A and/or SP-D, and
intracellular collagen-like defence molecules, such as CLL-11.
[0047] In a further aspect the present invention relates to the use
of a polypeptide according to the present invention as a biomarker
in the blood and tissue for the diagnosis and/or prognosis of a
malignant disease, such as a cancer disease, such as brain tumors,
liver tumors and tumors in the reproductive tract.
[0048] In a further aspect the present invention relates to the use
of a polypeptide according to the present invention as a biomarker
in blood and tissue for diagnosis and/or prognosis of an
autoimmune, metabolic and/or inflammatory condition as defined
herein.
LEGENDS TO THE FIGURES
[0049] FIG. 1: Alternative transcription of the MASP-1 gene.
Alternative transcription of the MASP1 gene was detected in liver
cDNA. The MASP1, MASP3, and FAP transcripts were amplified using a
common forward primer located in exon 6 and specific reverse
primers located in exon 12 (MASP1), exon 11 (MASP3), and exon 8a
(FAP). MASP1 generates a fragment of 500 bp, MASP3 generates a
fragment of 506 by and FAP generates a fragment of 309 bp.
[0050] FIG. 2: Alternative splicing of the MASP1 gene. MASP1 is
generated by splicing out of 8a and exon 11, which both contain a
stop codon sequence (marked with black boxes). The MASP1 sequence
contains a stop codon in exon 17. MASP3 is generated by splicing
out of exon 8a and FAP is generated if no splicing out of exon 8a
occurs. The FAP protein contains the two CUB domains, the EFG
domain and the first CCP1 domain.
[0051] FIG. 3: Tissue expression of the FAP fragment. The tissue
distributions of the MASP-1, MASP3, and FAP genes were investigated
in cDNA panels from Clontech. MASP-1, MASP-3, and FAP transcripts
were amplified using a common forward primer and specific reverse
primers. GADPH was used as reference gene. All three genes were
highly expressed in the liver, and additionally, FAP was strongly
expressed in heart tissue (marked with black arrows). Minor
expression of the FAP gene was detected in brain, colon, prostate,
skeletal muscle, and small intestine (marked with white
arrows).
[0052] FIG. 4: Alignment of MASP-1, MASP-3, and FAP. The protein
sequences of MASP-1, MASP-3, and FAP were aligned using the BioEdit
Software. MASP-1 and MASP-3 contain different C-terminal serine
protease domains whereas FAP does not contain any serine protease
domain. Instead the protein contains 17 new amino acids in the
C-terminal region.
[0053] FIG. 5: cDNA sequence and corresponding protein sequence of
FAP. The cDNA sequence is shown in the upper row and the
corresponding protein sequence is shown below. Exons regions are
divided by black vertical lines. Amino acids believed to be
involved in the binding to MBL/ficolins are marked with
light-yellow boxes.
[0054] FIG. 6: MASP-1 complement activation. Human MBL were
incubated with increased amount of MASP-1. MASP-1 were able to
activate both the C3 and C4 complement proteins.
[0055] FIG. 7: MASP-2 complement activation. Human MBL were
incubated with increased amount of MASP-2. MASP-2 were able to
strongly activate both the C3 and C4 complement proteins.
[0056] FIG. 8: MASP-3 inhibition of the complement. Human MBL were
incubated with increased amount of MASP-3. MASP-3 were able to
inhibit the activation of both the C3 and C4 complement
proteins.
[0057] FIG. 9: Immunoprecipitation. Immunoprecipitation of serum
Ficolin/MBL with mAb anti-MBL 131-11, anti-Ficolin-2 clone 219, and
anti-Ficolin-3 clone 334. Followed by Dynal magnetic bead
separation, SDS-PAGE, Western blot and biotin labeled
anti-MASP-1/MASP-3 clone 8B3 as signal antibody.
[0058] FIG. 10: FAP interact with Ficolin when bound to acetylated
human serum albumin (AcHSA). Eluted serum Ficolin binding to AcHSA.
Western blot with biotin labelled anti-MASP-1/MASP-3 clone 8B3 as
signal antibody.
[0059] FIG. 11: Kinetics and dissociation constants for interaction
between MASP-1 and MASP-3 and rFicolin-2 (Hummelshoj T et al., Mol.
Immunol., 2007).
[0060] FIG. 12: Alignment of GULF and the 17 unique amino acids of
FAP.
[0061] FIG. 13: Complement activation of C4 in a mannan/MBL ELISA
assay. Mannan coated wells were incubated with or without
recombinant human MBL followed by incubation with MBL homozygous
deficient serum in serial dilutions. The C4 deposition was measured
using polyclonal anti C4c antibodies. Error bars indicate two times
the standard deviations on double determinations of each point on
the curves.
[0062] FIG. 14: Complement activation of C4 in an acetylated
BSA/Ficolin-3 ELISA assay. AcBSA coated wells were incubated with
or without recombinant human Ficolin-3 followed by incubation with
Ficolin-3 homozygous deficient serum in serial dilutions. The C4
deposition was measured using polyclonal anti C4c antibodies. Error
bars indicate two times the standard deviations on double
determinations of each point on the curves.
[0063] FIG. 15: Complement activation of C4 in a mannan/MBL ELISA
assay. Mannan coated wells were incubated with recombinant human
MBL followed by incubation with serial dilutions of rMASP-1 as
serum free medium culture supernatants in one dimension. MBL
homozygous deficient serum was subsequently incubated in serial
dilutions in the second dimension. The C4 deposition was measured
using polyclonal anti C4c antibodies. Error bars indicate two times
the standard deviations on double determinations of each point on
the curves.
[0064] FIG. 16: Complement activation of C4 in an AcBSA/Ficolin-3
ELISA assay. AcBSA coated wells were incubated with recombinant
human Ficolin-3 followed by incubation with serial dilutions of
rMASP-1 as serum free medium culture supernatants in one dimension.
Ficolin-3 homozygous deficient serum was subsequently incubated in
serial dilutions in the second dimension. The C4 deposition was
measured using polyclonal anti C4c antibodies. Error bars indicate
two times the standard deviations on double determinations of each
point on the curves.
[0065] FIG. 17: Complement activation of C4 in a mannan/MBL ELISA.
Mannan coated wells were incubated with recombinant human MBL
followed by incubation with serial dilutions of rMASP-2 as serum
free medium culture supernatants in one dimension. MBL homozygous
deficient serum was subsequently incubated in serial dilutions in
the second dimension. The C4 deposition was measured using
polyclonal anti C4c antibodies. Error bars indicate two times the
standard deviations on double determinations of each point on the
curves.
[0066] FIG. 18: Complement activation of C4 in an AcBSA/Ficolin-3
ELISA assay. AcBSA coated wells were incubated with recombinant
human Ficolin-3 followed by incubation with serial dilutions of
rMASP-2 as serum free medium culture supernatants in one dimension.
Ficolin-3 homozygous deficient serum was subsequently incubated in
serial dilutions in the second dimension. The C4 deposition was
measured using polyclonal anti C4c antibodies. Error bars indicate
two times the standard deviations on double determinations of each
point on the curves.
[0067] FIG. 19: Complement activation of C4 in a mannan/MBL ELISA
assay. Mannan coated wells were incubated with recombinant human
MBL followed by incubation with serial dilutions of rMASP-3 as
serum free medium culture supernatants in one dimension. MBL
homozygous deficient serum was subsequently incubated in serial
dilutions in the second dimension. The C4 deposition was measured
using polyclonal anti C4c antibodies. Error bars indicate two times
the standard deviations on double determinations of each point on
the curves.
[0068] FIG. 20: Complement activation of C4 in an AcBSA/Ficolin-3
ELISA assay. AcBSA coated wells were incubated with recombinant
human Ficolin-3 followed by incubation with serial dilutions of
rMASP-3 as serum free medium culture supernatants in one dimension.
Ficolin-3 homozygous deficient serum was subsequently incubated in
serial dilutions in the second dimension. The C4 deposition was
measured using polyclonal anti C4c antibodies. Error bars indicate
two times the standard deviations on double determinations of each
point on the curves.
[0069] FIG. 21: Tissue distribution of FAP, MASP1 and MASP3. FAP
was expressed much higher in the heart tissue compared to MASP1 and
MASP3. FAP was expressed three times higher in the heart tissue
compared to the FAP expression in liver. Furthermore, a higher FAP
expression was observed in the liver compared to the MASP1 and
MASP3 expression in the liver. Considerable FAP expression was also
detected in brain, skeletal muscle and prostate tissues. The
experiment was performed three times in duplicates. Standard error
of the mean are indicated.
[0070] FIG. 22: Immunohistochemical liver localization of MAP-1
using polyclonal mouse antiserum raised against the 17 FAP specific
C-terminal residues of the Protein. Control staining was negative.
Several different polyclonal antibodies raised against FAP (rabbit
and mouse) showed the same pattern staining.
[0071] FIG. 23: Immunohistochemical analysis of MAP-1 tissue
localization (OM X10). Left panel shows staining with a mAb (12B11)
to MAP-1. Right panel shows the isotype control staining with a
non-related IgG1k mAb. (A-B): Myocardium, (C-D): Skeletal muscle,
(E-F): Liver sample, (G-H): Aortic tissue. Bottom right corner bar
indicates 50 .mu.m on all slides.
[0072] FIG. 24: Immunoprecipitation of MAP-1 and MASP-1/3 serum
complexes. (A) MAP-1 and MASP-1/3 was immunoprecipitated from serum
using mAb 20C4 (anti MAP-1) and mAb 8B3 (anti MASP-1/3, with an
epitope on the common heavy chain). Reduced samples were
electro-blotted and developed with pAb to MAP-1 or biotinylated
mAbs to Ficolin-3 (FCN334) and MBL (Hyb 131-1). (B)
Immunoprecipitation with mAbs to MBL (Hyb 131-11), Ficolin-2
(FCN219) and Ficolin-3 (FCN334) from 1 ml, 300 .mu.l and 100 .mu.l
serum, respectively (Left side). Controls were MAP-1 precipitated
from serum (sMAP-1) and rMAP-1 from culture supernatant (rMAP-1)
using anti MAP-1 mAb 20C4 (right side). The samples were analyzed
by western blotting probed with pAb to MAP-1.
[0073] FIG. 25: Influence of MASP-2 and MAP-1 on MBL and Ficolin-3
mediated complement C4 deposition. The C4 depositions were measured
using a polyclonal antibody to C4 and are given as OD.sub.490-650nm
values. Error bars indicate two times the standard deviation of
double determinations. Approximated concentrations of rMBL,
rFicolin-3. rMAP-1 and rMASP-2 are given in the figure labels. (A)
Reconstitution of the C4 deposition on a mannan coated surface
using MBL deficient serum with rMBL at 400 ng/ml. Control was
without addition of rMBL. (B) Dose dependent effect of rMASP-2 on
the rMBL mediated C4 deposition. (C) Dose dependent effect of
rMAP-1 on the rMBL mediated C4 deposition. (D) Reconstitution of
the C4 deposition on an AcBSA coated surface using Ficolin-3
deficient serum with rFicolin-3 at 400 ng/ml. Control was without
addition of rFicolin-3. (E) Dose dependent effect of rMASP-2 on the
rFicolin-3 mediated C4 deposition. (F) Dose dependent effect of
rMAP-1 on the rFicolin-3 mediated C4 deposition.
[0074] FIG. 26: Influence of MASP-2 and MAP-1 on the complement C4
deposition in a pure system. rMBL on a mannan surface was
preincubated with serial dilutions of rMASP-2 in the first
dimension. Serial dilutions of rMAP-1 were then applied in the
second dimension followed by application of purified C4 at 1
.mu.g/ml. The C4 depositions were measured with a pAb to C4 and are
given as OD.sub.490-650nm values. Error bars indicate two times the
standard deviation of double determinations. Approximated
concentrations of rMAP-1 and rMASP-2 are given in the figure
labels.
[0075] FIG. 27: SDS-PAGE analysis of rMAP-1. Left hand side shows
the immunoblot analysis+/-N-glycosidase F treatment (ENDO-F). Right
side shows the corresponding coomassie staining.
[0076] FIG. 28A, B. Calibration curves. A) Calibration curve
generated by mAb 20C4/mAb-8B3 two-side ELISA with two-fold serial
dilutions of rMAP-1 applied to a MAP-1 depleted pool of normal
human serum (pNHS) or serial dilutions of rMAP-1 diluted in
PBS/0.05%tween/10 mM EDTA. Error bars indicate two times the
standard deviation of eight determinations. B) Immunoblot of serum
depleted of MAP-1, normal human serum and MAP-1 depleted serum
spiked with rMAP-1.
[0077] FIG. 29A-C. MAP-1 serum concentration. A) Serum
concentrations and distribution range of MAP-1 in 100 Danish blood
donors. Mean serum level: 240 ng/ml. Range: 115-466 ng/ml.; B)
Correlation between the MASP-3 and MAP-1 serum levels.; C)
Influence of freezing and thawing of serum. Serum was frozen and
thawed for 8 rounds and the MAP-1 level was measured for each
round. Error bars indicate two times the standard deviation of
double determinations.
[0078] FIG. 30. A) Association levels (in relative O.D. 490-650 nm
units) between MAP-1 and MBL, Ficolin-2 and Ficolin-3, respectively
in 100 Danish blood donors. P values were obtained by
non-parametric two-tailed t-test. B) Correlation between the MAP-1
serum levels and the relative association to MBL, Ficolin-2 and
Ficolin-3 (left hand side). Correlation between the MBL, Ficolin-2
and Ficolin-3 serum levels and the relative association to MAP-1
(right hand side). Correlation p- and r-values were calculated
using the non-parametric spearman rank correlation test.
[0079] FIG. 31A-C. Sucrose gradient ultracentrifugation. A)
Collected fractions (1-27) from serum subjected to a 10-30% sucrose
density gradient. The fractions were analyzed by specific ELISA
for: MAP-1, MASP-3, MBL, Ficolin-2 and -3. The peaks of serum IgM
(19S) and IgG (7S) indicated at the top of the graph. B) Fractions
number 8-23 analyzed by immunoblotting for: MAP-1, MASP-1, MASP-3,
sMAP, MASP-2, MBL, Ficolin-2 and Ficolin-3. C) The fractions 1-27
analyzed by the capacity to activate exogenously applied human C4
on immobilized acetylated BSA (a Ficolin-3 ligand) or mannan (an
MBL ligand).
DETAILED DISCLOSURE OF THE INVENTION
[0080] The present inventors have discovered a novel plasma protein
of 40 kDa associated with the recognition molecules of the lectin
complement pathway and identified this as a new alternative
transcript variant of MASP-1/MASP-3 that in turn corresponds to the
newly discovered plasma protein.
[0081] The novel protein (by the inventors named FAP (Ficolin
Associated Protein) or MAP-1 (MBL/Ficolin associated protein-1))
has been shown by the present inventors to lack an enzyme domain
but to contain the ficolin/MBL binding domain and is thus expected
to be involved in regulation and inhibition of complement and
coagulation functions through competitions and displacement of the
MASPs or alternatively, but not mutually exclusive as a protein
involved in scavenger or signaling functions.
[0082] Uncontrolled activation of the complement system and/or the
coagulation cascade is strongly associated with fatal severe
outcome in variety of diseases ranging from systemic inflammation
and sepsis, through myocardial infarction and autoimmunity.
[0083] Inhibition of coagulation and complement activation has been
shown to be a promising therapeutic tool.
[0084] This present invention describes both a possible novel
inhibitor of complement and coagulation functions. However, the
polypeptides according to the present invention may have other
functions, such as a scavenger and/or a signalling function.
Moreover, it may be used as a new biomarker in several disease
settings, including malignant diseases, autoimmune, metabolic
and/or inflammatory conditions.
[0085] The inventors of the present invention have found a plasma
protein present in vivo named Ficolin Associated Protein (FAP) and
showed that it is primarily associated with the ficolins (FIG. 9),
but it may likely also be associated with mannose-binding lectin.
By searching nucleotide database of NCBI the inventors of the
present invention found a possible transcript variant that
corresponds to a truncated of MASP-1. Based on this sequence,
primers were designed in order to amplify the putative new gene
transcript. Subsequently, using human liver cDNA a new alternative
transcript variant of the MASP-1 gene (FIG. 1) was identified. This
mRNA strain was sequenced and accordingly the amino acid sequence
was determined, which corresponds to the molecular weight of the
observed protein in plasma/serum of 40 kDa (FIG. 5). The new
protein is partly identical to MASP-1 and MASP-3, but lacks a
serine protease domain, but contain a novel exon encoding 17 amino
acids followed by a stop codon. This exon is spliced out in the
MASP1 and MASP3 transcript (FIG. 2). By using a panel of mRNA
expression libraries the present inventors have found evidence that
this protein is strongly expressed in the heart and in the liver,
followed by skeletal muscle (FIG. 3). Weak expression was observed
in the brain, the digestive tract, prostata and in the spleen (FIG.
3). Taqman analysis confirmed the expression in heart and liver
cells. FAP was expressed much higher in the heart tissue compared
to MASP1 and MASP3. FAP was expressed three times higher in the
heart tissue compared to the FAP expression in liver. Furthermore,
a higher FAP expression was observed in the liver compared to the
MASP1 and MASP3 expression in the liver. Considerable FAP
expression was also detected in brain, skeletal muscle and prostate
tissues. The experiment was performed three times in
duplicates.
[0086] The high expression in the heart is very prominent and has
made the present inventors suggest a use of the polypeptides
according to the present invention as a very useful protector
against tissue damage in autoimmune, metabolic and/or inflammatory
conditions, such as medical conditions associated with the
heart.
[0087] The present inventors have established assays to assess
complement activity initiated by ficolins and mannose-binding
lectin and the present inventors have thus been able to show a
possible functional complement inhibition of FAP.
[0088] The present inventors have establishing real time
quantitative assays to measure the exact relative expression level
in different tissues.
[0089] The polypeptides according to the present invention may be
produced by recombinant techniques. Rabbits or mice may be
immunized with a unique 17 amino acid long peptide in order to
obtain FAP polyclonal and monoclonal specific antibodies,
respectively.
[0090] Specific FAP antibodies may be used for quantitative
measurement of FAP and immunohistochemical detection in different
tissues.
[0091] Binding constants between FAP and different binding partners
as described herein may be determined in ELISA and by using surface
plasmon resonance technology (Biacore).
[0092] A FAP specific acceptor protein, such as a specific cell
surface bound receptor may be identified by standard assays known
to the person skilled in the art, such as assays wherein the
protein is bound directly to cells.
[0093] The novel protein Ficolin Associated Protein (FAP) is an
alternative splicing variant of MASP1. The protein lacks the serine
protease domain but it still contains the domains that are involved
in the binding to the initiators of the lectin pathway of the
complement system. Thus, the present inventors expect the protein
to be involved in regulation and inhibition of the function of
MASP-1 and MASP-3 (complement, coagulation functions and other
enzymes substrates) through competitions and displacement of the
MASPs. Alternatively, but not mutually exclusive FAP may function
as scavenger molecule facilitating removal of FAP/MBL/ficolin
complexes bound to endogenous waste material or pathogens.
[0094] Uncontrolled activation of the complement system and the
coagulation cascade are associated with adverse outcome and
functional inhibitors, such as the polypeptides according to the
present invention may be very useful for the control of the
complement system and the coagulation cascade. In addition the
polypeptides according to the present invention may be used in
other settings. Another angle could be to use the protein as
biomarker in different disease settings.
[0095] The protein is unique and may provide the basis for new
drugs and/or new diagnostic tools.
[0096] Polypeptides according to the present invention comprising
the amino acid sequence of SEQ ID NO:4 or an immunologic fragment
or variant thereof may have a specific function associated with
this sequence of amino acids. It is suggested by the present
inventors that such polypeptides may have a function or activity
corresponding to the activity of one or more protein selected from
DNMT1 DNA (cytosine-5-)-methyltransferase 1 (DNMT1), Golgin
subfamily B member 1 (GOLGB1), A-kinase anchor protein 9 (AKAP9),
B- and T-lymphocyte-associated protein) (CD272 antigen), PTB
domain-containing engulfment adapter protein 1 (GULP), and MACRO
domain-containing protein 2.
[0097] In some particular interesting embodiments the polypeptides
according to the present invention have a function or activity
corresponding to the activity of PTB domain-containing engulfment
adapter protein 1 (GULP).
[0098] Definitions
[0099] The term "ficolin-associated polypeptide" as used herein
means any protein or polypeptide comprising the amino acid sequence
20-380 of native human ficolin-associated protein (FAP) (SEQ ID NO:
1) or amino acid sequence of 16-363 of SEQ ID NO:9, functional
variants, functional truncated versions thereof as well as
functional derivatives or conjugates, which polypeptide do not have
complement activity, but posses the ability to compete with MASP-1,
MASP-2, or MASP-3 for binding to ficolin-3, MBL, C1q, lung
surfactant proteins SP-A and/or SP-D and/or CL-L1 (and other
collectin family members). This includes but is not limited to
human ficolin-associated polypeptide (FAP) having SEQ ID NO:1 and
variants thereof.
[0100] The term "ficolin-associated protein (FAP)" as used herein
means proteins that have the amino acid sequence 1-380 (with or
without signal peptide, such as the amino acid sequence 20-380) of
native human FAP (SEQ ID NO: 1), natural allelic variations and
homologous thereof. It also includes proteins with a slightly
modified amino acid sequence, for instance, a modified N- or
C-terminal end including N- or C-terminal amino acid deletions or
additions so long as those proteins substantially retain the
activity of FAP. The term "ficolin-associated protein (FAP)" is
used interchangeable herein with the terms "MAP-1" or "MBL/Ficolin
associated protein-1". "FAP" within the above definition also
includes natural allelic variations that may exist and occur from
one individual to another. The term also includes proteins with
homologous sequence and similar function derived from other species
than human, such as bovine, pig, dog, horse, rat, and mouse. Also,
degree and location of glycosylation or other post-translation
modifications may vary depending on the chosen host cells and the
nature of the host cellular environment.
[0101] The term "MBL-Associated Serine Protease-1" or "MASP-1" as
used herein means proteins that have the amino acid sequence 1-699
(with or without signal peptide, such as the amino acid sequence
20-699) of native human MASP-1 (SEQ ID NO:5), natural allelic
variations and homologous thereof. It is to be understood that the
sequence may be in one or more peptide chains, such as in two
chains, i.e. the heavy and light chains of the native human
protein.
[0102] The term "MBL-Associated Serine Protease-3" or "MASP-3" as
used herein means proteins that have the amino acid sequence 1-728
(with or without signal peptide, such as the amino acid sequence
20-728) of native human MASP-3 (SEQ ID NO:7), natural allelic
variations and homologous thereof. It is to be understood that the
sequence may be in one or more peptide chains, such as in two
chains, i.e. the heavy and light chains of the native human
protein.
[0103] The term "MBL-Associated Serine Protease-2" or "MASP-2" as
used herein means proteins that have the amino acid sequence 1-686
(with or without signal peptide, such as the amino acid sequence
16-686) of native human MASP-2 (SEQ ID NO:9), natural allelic
variations and homologous thereof. It is to be understood that the
sequence may be in one or more peptide chains, such as in two
chains, i.e. the heavy and light chains of the native human
protein.
[0104] The terms "small MBL-associated protein", "sMAP",
"MBL-associated plasma protein of 19 kD" or, "MAp19" as used herein
means proteins that have the amino acid sequence 1-185 (with or
without signal peptide, such as the amino acid sequence 16-185) of
native human sMAP (SEQ ID NO:11), natural allelic variations and
homologous thereof.
[0105] The terms "variant" or "variants", as used herein, is
intended to designate a ficolin-associated polypeptide having the
sequence of SEQ ID NO:1 or a polypeptide comprising the amino acid
sequence of SEQ ID NO:4, wherein one or more amino acids have been
substituted by another amino acid and/or wherein one or more amino
acids have been deleted and/or wherein one or more amino acids have
been inserted in the polypetide and/or wherein one or more amino
acids have been added to the polypeptide. Such addition can take
place either at the N-terminal end or at the C-terminal end or
both. The "variant" or "variants" within this definition still have
functional activity. In some embodiment a variant has 70% sequence
identity with the sequence of SEQ ID NO:1. In some embodiments a
variant has 80% sequence identity with the sequence of SEQ ID NO:1.
In other embodiments a variant has 90% sequence identity with the
sequence of SEQ ID NO:1. In a further embodiment a variant has 95%
sequence identity with the sequence of SEQ ID NO:1.
[0106] In some embodiments a variant has 70% sequence identity with
the sequence of SEQ ID NO:4. In some embodiments a variant has 80%
sequence identity with the sequence of SEQ ID NO:4. In other
embodiments a variant has 90% sequence identity with the sequence
of SEQ ID NO:4. In a further embodiment a variant has 95% sequence
identity with the sequence of SEQ ID NO:4.
[0107] The phrases "functional variant", "functional truncated
versions", and "functional derivatives" as used herein refers to
variants, truncated versions, as well as derivatives of SEQ ID
NO:1, which polypeptides comprises essential sequence parts of SEQ
ID NO:1 and at least posses the ability to compete with MASP-1 or
MASP-3 for binding to the ficolins or MBL without having the
complement activity and/or serine protease activity. It is to be
understood that a ficolin-associated polypeptide may have to or
three features selected from being a both a variant, and/or
truncated and/or a derivative.
[0108] A functional variant of a ficolin-associated polypeptide
encompass those that exhibit at least about 25%, such as at least
about 50%, such as at least about 75%, such as at least about 90%
of the specific activity of wild-type FAP that has been produced in
the same cell type, when tested in the assays as described
herein.
[0109] The term "immunologic fragment" as used herein refers to
fragment of an amino acid sequence that posses essentially the same
functional activities and the same spatial orientation to be
recognized by an antibody. Accordingly a specific antibody will
bind both the polypeptide and immunologic fragments thereof.
[0110] The term "another amino acid" as used herein means one amino
acid that is different from that amino acid naturally present at
that position. This includes but is not limited to amino acids that
can be encoded by a polynucleotide. In some embodiments the
different amino acid is in natural L-form and can be encoded by a
polynucleotide.
[0111] The term "derivative" as used herein, is intended to
designate a ficolin-associated polypeptide exhibiting substantially
the same or improved biological activity relative to wild-type
human FAP, in which one or more of the amino acids of the parent
peptide have been chemically modified, e.g. by alkylation,
PEGylation, acylation, ester formation or amide formation or the
like.
[0112] The term "complement activity" as used herein means the
ability activate the complement system. The complement activity may
be measured with assay as described in the section headed
"Assays".
[0113] The term "mannose-binding lectin (MBL)" as used herein also
means mannan-binding lectin, mannose-binding protein (MBP1), and
mannan-binding protein, which terms may be used
interchangeably.
[0114] The term "capable of associating" as used herein refers to
the ability of the proteins according to the present invention to
specifically bind in solution one or more of the initiators of the
lectin pathway of the complement system or other proteins that may
be involved in the effect of the polypeptide.
[0115] The term "construct" is intended to indicate a
polynucleotide segment which may be based on a complete or partial
naturally occurring nucleotide sequence encoding the polypeptide of
interest. The construct may optionally contain other polynucleotide
segments. In a similar way, the term "amino acids which can be
encoded by polynucleotide constructs" covers amino acids which can
be encoded by the polynucleotide constructs defined above, i.e.
amino acids such as Ala, Val, Leu, Ile, Met, Phe, Trp, Pro, Gly,
Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp and Gln.
[0116] The term "vector", as used herein, means any nucleic acid
entity capable of the amplification in a host cell. Thus, the
vector may be an autonomously replicating vector, i.e. a vector,
which exists as an extra-chromosomal entity, the replication of
which is independent of chromosomal replication, e.g. a plasmid.
Alternatively, the vector may be one which, when introduced into a
host cell, is integrated into the host cell genome and replicated
together with the chromosome(s) into which it has been integrated.
The choice of vector will often depend on the host cell into which
it is to be introduced. Vectors include, but are not limited to
plasmid vectors, phage vectors, viruses or cosmid vectors. Vectors
usually contain a replication origin and at least one selectable
gene, i.e., a gene which encodes a product which is readily
detectable or the presence of which is essential for cell
growth.
[0117] In a further aspect, the invention provides a recombinant
host cell comprising the polynucleotide construct or the vector. In
some embodiments the recombinant host cell is a eukaryotic cell. In
other embodiments the recombinant host cell is of mammalian origin.
In a further embodiment the recombinant host cell is selected from
the group consisting of CHO cells, HEK cells and BHK cells.
[0118] The term "a host cell", as used herein, represent any cell,
including hybrid cells, in which heterologous DNA can be expressed.
Typical host cells includes, but are not limited to insect cells,
yeast cells, mammalian cells, including human cells, such as BHK,
CHO, HEK, and COS cells. In practicing the present invention, the
host cells being cultivated are preferably mammalian cells, more
preferably an established mammalian cell line, including, without
limitation, CHO (e.g., ATCC CCL 61), COS-1 (e.g., ATCC CRL 1650),
baby hamster kidney (BHK) and HEK293 (e.g., ATCC CRL 1573; Graham
et al., J. Gen. Virol. 36:59-72, 1977) cell lines. A preferred BHK
cell line is the tk.sup.- ts13 BHK cell line (Waechter and Baserga,
Proc. Natl. Acad. Sci. USA 79:1106-1110, 1982), hereinafter
referred to as BHK 570 cells. The BHK 570 cell line is available
from the American Type Culture Collection, 12301 Parklawn Dr.,
Rockville, Md. 20852, under ATCC accession number CRL 10314. A
tk.sup.- ts13 BHK cell line is also available from the ATCC under
accession number CRL 1632. Other suitable cell lines include,
without limitation, Rat Hep I (Rat hepatoma; ATCC CRL 1600), Rat
Hep II (Rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), Human
lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1) and DUKX cells
(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980).
Also useful are 3T3 cells, Namalwa cells, myelomas and fusions of
myelomas with other cells.
[0119] In a further aspect, the invention provides a transgenic
animal containing and expressing the polynucleotide construct.
[0120] In a further aspect, the invention provides a transgenic
plant containing and expressing the polynucleotide construct.
[0121] In a further aspect, the invention relates to a method for
producing the ficolin-associated polypeptide of the invention, the
method comprising cultivating a cell comprising the polynucleotide
construct in an appropriate growth medium under conditions allowing
expression of the polynucleotide construct and recovering the
resulting polypeptide from the culture medium.
[0122] As used herein the term "appropriate growth medium" means a
medium containing nutrients and other components required for the
growth of cells and the expression of the nucleic acid sequence
encoding the ficolin-associated polypeptide of the invention.
[0123] In a further aspect, the invention relates to a method for
producing the ficolin-associated polypeptide, the method comprising
recovering the polypeptide from milk produced by the transgenic
animal.
[0124] In a further aspect, the invention relates to a method for
producing the ficolin-associated polypeptide, the method comprising
cultivating a cell of a transgenic plant comprising the
polynucleotide construct, and recovering the polypeptide from the
resulting plant.
[0125] In the present context, the term "treatment" is meant to
include both prevention of an expected condition involving
inappropriate complement activation, such as inflammation and
reperfusion injury and regulation of an already occurring
condition, such as myocardial infarction and stroke with the
purpose of inhibiting or minimising the tissue damage Prophylactic
administration of the ficolin-associated polypeptide according to
the invention is thus included in the term "treatment".
[0126] The term "subject" as used herein is intended to mean any
animal, in particular mammals, such as humans, and may, where
appropriate, be used interchangeably with the term "patient".
[0127] The term "sequence identity" as known in the art, refers to
a relationship between the sequences of two or more polypeptide
molecules or two or more nucleic acid molecules, as determined by
comparing the sequences. In the art, "identity" also means the
degree of sequence relatedness between nucleic acid molecules or
between polypeptides, as the case may be, as determined by the
number of matches between strings of two or more nucleotide
residues or two or more amino acid residues. "Identity" measures
the percent of identical matches between the smaller of two or more
sequences with gap alignments (if any) addressed by a particular
mathematical model or computer program (i.e., "algorithms").
[0128] The term "similarity" is a related concept, but in contrast
to "identity", refers to a sequence relationship that includes both
identical matches and conservative substitution matches. If two
polypeptide sequences have, for example, (fraction (10/20))
identical amino acids, and the remainder are all non-conservative
substitutions, then the percent identity and similarity would both
be 50%. If, in the same example, there are 5 more positions where
there are conservative substitutions, then the percent identity
remains 50%, but the percent similarity would be 75% ((fraction
(15/20))). Therefore, in cases where there are conservative
substitutions, the degree of similarity between two polypeptides
will be higher than the percent identity between those two
polypeptides.
[0129] Conservative modifications to the amino acid sequence of SEQ
ID NO:1 (and the corresponding modifications to the encoding
nucleotides) will produce ficolin-associated polypeptides having
functional and chemical characteristics similar to those of
naturally occurring FAP. In contrast, substantial modifications in
the functional and/or chemical characteristics of a
ficolin-associated polypeptide may be accomplished by selecting
substitutions in the amino acid sequence of SEQ ID NO:1 that differ
significantly in their effect on maintaining (a) the structure of
the molecular backbone in the area of the substitution, for
example, as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk
of the side chain.
[0130] For example, a "conservative amino acid substitution" may
involve a substitution of a native amino acid residue with a
nonnative residue such that there is little or no effect on the
polarity or charge of the amino acid residue at that position.
Furthermore, any native residue in the polypeptide may also be
substituted with alanine, as has been previously described for
"alanine scanning mutagenesis" (see, for example, MacLennan et al.,
1998, Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al., 1998,
Adv. Biophys. 35:1-24, which discuss alanine scanning
mutagenesis).
[0131] Desired amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at
the time such substitutions are desired. For example, amino acid
substitutions can be used to identify important residues of a
ficolin-associated polypeptide, or to increase or decrease the
affinity of a ficolin-associated polypeptide described herein.
[0132] Naturally occurring residues may be divided into classes
based on common side chain properties: [0133] 1) hydrophobic:
norleucine, Met, Ala, Val, Leu, Ile; [0134] 2) neutral hydrophilic:
Cys, Ser, Thr, Asn, Gln; [0135] 3) acidic: Asp, Glu; [0136] 4)
basic: His, Lys, Arg; [0137] 5) residues that influence chain
orientation: Gly, Pro; and [0138] 6) aromatic: Trp, Tyr, Phe.
[0139] For example, non-conservative substitutions may involve the
exchange of a member of one of these classes for a member from
another class. Such substituted residues may be introduced into
regions of the human ficolin-associated polypeptide that are
homologous with non-human ficolin-associated polypeptides, or into
the non-homologous regions of the molecule.
[0140] In making such changes, the hydropathic index of amino acids
may be considered. Each amino acid has been assigned a hydropathic
index on the basis of their hydrophobicity and charge
characteristics, these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0141] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
understood in the art. Kyte et al., J. Mol. Biol., 157:105-131
(1982). It is known that certain amino acids may be substituted for
other amino acids having a similar hydropathic index or score and
still retain a similar biological activity. In making changes based
upon the hydropathic index, the substitution of amino acids whose
hydropathic indices are within 0..+-.2 is preferred, those that are
within .+-.1 are particularly preferred, and those within .+-.0.5
are even more particularly preferred.
[0142] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity, particularly where the biologically functionally
equivalent protein or peptide thereby created is intended for use
in immunological embodiments, as in the present case. The greatest
local average hydrophilicity of a protein, as governed by the
hydrophilicity of its adjacent amino acids, correlates with its
immunogenicity and antigenicity, i.e., with a biological property
of the protein.
[0143] The following hydrophilicity values have been assigned to
amino acid residues: arginine (+3.0); lysine ('3.0); aspartate
(+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine
(+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline
(-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0);
methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine
(-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
In making changes based upon similar hydrophilicity values, the
substitution of amino acids whose hydrophilicity values are within
.+-.2 is preferred, those that are within .+-.1 are particularly
preferred, and those within .+-.0.5 are even more particularly
preferred. One may also identify epitopes from primary amino acid
sequences on the basis of hydrophilicity. These regions are also
referred to as "epitopic core regions."
[0144] A skilled artisan will be able to determine suitable
variants of the polypeptide as set forth in SEQ ID NO:1 using well
known techniques. For identifying suitable areas of the molecule
that may be changed without destroying activity, one skilled in the
art may target areas not believed to be important for activity. For
example, when similar polypeptides with similar activities from the
same species or from other species are known, one skilled in the
art may compare the amino acid sequence of a ficolin-associated
polypeptide to such similar polypeptides. With such a comparison,
one can identify residues and portions of the molecules that are
conserved among similar polypeptides. It will be appreciated that
changes in areas of a ficolin-associated polypeptide that are not
conserved relative to such similar polypeptides would be less
likely to adversely affect the biological activity and/or structure
of the ficolin-associated polypeptide. One skilled in the art would
also know that, even in relatively conserved regions, one may
substitute chemically similar amino acids for the naturally
occurring residues while retaining activity (conservative amino
acid residue substitutions). Therefore, even areas that may be
important for biological activity or for structure may be subject
to conservative amino acid substitutions without destroying the
biological activity or without adversely affecting the polypeptide
structure.
[0145] Additionally, one skilled in the art can review
structure-function studies identifying residues in similar
polypeptides that are important for activity or structure. In view
of such a comparison, one can predict the importance of amino acid
residues in a ficolin-associated polypeptide that correspond to
amino acid residues that are important for activity or structure in
similar polypeptides. One skilled in the art may opt for chemically
similar amino acid substitutions for such predicted important amino
acid residues of ficolin-associated polypeptides and other
polypeptides of the invention.
[0146] One skilled in the art can also analyze the
three-dimensional structure and amino acid sequence in relation to
that structure in similar polypeptides. In view of that
information, one skilled in the art may predict the alignment of
amino acid residues of a ficolin-associated polypeptide with
respect to its three dimensional structure. One skilled in the art
may choose not to make radical changes to amino acid residues
predicted to be on the surface of the protein, since such residues
may be involved in important interactions with other molecules.
Moreover, one skilled in the art may generate test variants
containing a single amino acid substitution at each desired amino
acid residue. The variants can then be screened using activity
assays as described herein. Such variants could be used to gather
information about suitable variants. For example, if one discovered
that a change to a particular amino acid residue resulted in
destroyed, undesirably reduced, or unsuitable activity, variants
with such a change would be avoided. In other words, based on
information gathered from such routine experiments, one skilled in
the art can readily determine the amino acids where further
substitutions should be avoided either alone or in combination with
other mutations.
[0147] A number of scientific publications have been devoted to the
prediction of secondary structure. See Moult J., Curr. Op. in
Biotech., 7(4):422-427 (1996), Chou et al., Biochemistry,
13(2):222-245 (1974); Chou et al., Biochemistry, 113(2):211-222
(1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol,
47:45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:251-276 and
Chou et al., Biophys. J., 26:367-384 (1979). Moreover, computer
programs are currently available to assist with predicting
secondary structure. One method of predicting secondary structure
is based upon homology modeling. For example, two polypeptides or
proteins, which have a sequence identity of greater than 30%, or
similarity greater than 40% often have similar structural
topologies. The recent growth of the protein structural data base
(PDB) has provided enhanced predictability of secondary structure,
including the potential number of folds within a polypeptide's or
protein's structure. See Holm et al., Nucl. Acid. Res.,
27(1):244-247 (1999). It has been suggested (Brenner et al., Curr.
Op. Struct. Biol., 7(3):369-376 (1997)) that there are a limited
number of folds in a given polypeptide or protein and that once a
critical number of structures have been resolved, structural
prediction will gain dramatically in accuracy.
[0148] Additional methods of predicting secondary structure include
"threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87
(1997); Sippl et al., Structure, 4(1):15-9 (1996)), "profile
analysis" (Bowie et al., Science, 253:164-170 (1991); Gribskov et
al., Meth. Enzymol., 183:146-159 (1990); Gribskov et al., Proc.
Nat. Acad. Sci., 84(13):4355-4358 (1987)), and "evolutionary
linkage" (See Home, supra, and Brenner, supra).
[0149] Identity and similarity of related polypeptides can be
readily calculated by known methods. Such methods include, but are
not limited to, those described in Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.
and Devereux, J., eds., M. Stockton Press, New York, 1991; and
Carillo et al., SIAM J. Applied Math., 48:1073 (1988).
[0150] Preferred methods to determine identity and/or similarity
are designed to give the largest match between the sequences
tested. Methods to determine identity and similarity are described
in publicly available computer programs. Preferred computer program
methods to determine identity and similarity between two sequences
include, but are not limited to, the GCG program package, including
GAP (Devereux et al., Nucl. Acid. Res., 12:387 (1984); Genetics
Computer Group, University of Wisconsin, Madison, Wis.), BLASTP,
BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215:403-410
(1990)). The BLASTX program is publicly available from the National
Center for Biotechnology Information (NCBI) and other sources
(BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894;
Altschul et al., supra). The well known Smith Waterman algorithm
may also be used to determine identity.
[0151] Certain alignment schemes for aligning two amino acid
sequences may result in the matching of only a short region of the
two sequences, and this small aligned region may have very high
sequence identity even though there is no significant relationship
between the two full length sequences. Accordingly, in a preferred
embodiment, the selected alignment method (GAP program) will result
in an alignment that spans at least 50 contiguous amino acids of
the target polypeptide.
[0152] For example, using the computer algorithm GAP (Genetics
Computer Group, University of Wisconsin, Madison, Wis.), two
polypeptides for which the percent sequence identity is to be
determined are aligned for optimal matching of their respective
amino acids (the "matched span", as determined by the algorithm). A
gap opening penalty (which is calculated as 3.times. the average
diagonal; the "average diagonal" is the average of the diagonal of
the comparison matrix being used; the "diagonal" is the score or
number assigned to each perfect amino acid match by the particular
comparison matrix) and a gap extension penalty (which is usually
1/10 times the gap opening penalty), as well as a comparison matrix
such as PAM 250 or BLOSUM 62 are used in conjunction with the
algorithm. A standard comparison matrix (see Dayhoff et al., Atlas
of Protein Sequence and Structure, vol. 5, supp. 3 (1978) for the
PAM 250 comparison matrix; Henikoff et al., Proc. Natl. Acad. Sci
USA, 89:10915-10919 (1992) for the BLOSUM 62 comparison matrix) is
also used by the algorithm.
[0153] Preferred parameters for a polypeptide sequence comparison
include the following:
[0154] Algorithm: Needleman et al., J. Mol. Biol, 48:443-453
(1970); Comparison matrix: BLOSUM 62 from Henikoff et al., Proc.
Natl. Acad. Sci. USA, 89:10915-10919 (1992); Gap Penalty: 12, Gap
Length Penalty: 4, Threshold of Similarity: 0.
[0155] The GAP program is useful with the above parameters. The
aforementioned parameters are the default parameters for
polypeptide comparisons (along with no penalty for end gaps) using
the GAP algorithm.
[0156] Preferred parameters for nucleic acid molecule sequence
comparisons include the following: Algorithm: Needleman et al., J.
Mol Biol., 48:443-453 (1970); Comparison matrix: matches=+10,
mismatch=0, Gap Penalty: 50, Gap Length Penalty: 3.
[0157] The GAP program is also useful with the above parameters.
The aforementioned parameters are the default parameters for
nucleic acid molecule comparisons.
[0158] Other exemplary algorithms, gap opening penalties, gap
extension penalties, comparison matrices, thresholds of similarity,
etc. may be used, including those set forth in the Program Manual,
Wisconsin Package, Version 9, September, 1997. The particular
choices to be made will be apparent to those of skill in the art
and will depend on the specific comparison to be made, such as DNA
to DNA, protein to protein, protein to DNA; and additionally,
whether the comparison is between given pairs of sequences (in
which case GAP or BestFit are generally preferred) or between one
sequence and a large database of sequences (in which case FASTA or
BLASTA are preferred).
[0159] Preparation of Ficolin-Associated Polypeptides and Other
Polypeptides of the Invention
[0160] The invention also relates to a method of preparing human
Ficolin-associated polypeptides and other polypeptides of the
invention as mentioned above. The Ficolin-associated polypeptides
and other polypeptides of the invention described herein may be
produced by means of recombinant nucleic acid techniques. In
general, a cloned wild-type FAP nucleic acid sequence is modified
to encode the desired protein. This modified sequence is then
inserted into an expression vector, which is in turn transformed or
transfected into host cells. Higher eukaryotic cells, in particular
cultured mammalian cells, are preferred as host cells. The complete
amino acid and nucleotide sequences for human FAP is given by SEQ
ID NO:1 and SEQ ID NO:2.
[0161] The amino acid sequence alterations may be accomplished by a
variety of techniques. Modification of the nucleic acid sequence
may be by site-specific mutagenesis. Techniques for site-specific
mutagenesis are well known in the art and are described in, for
example, Zoller and Smith (DNA 3:479-488, 1984) or "Splicing by
extension overlap", Horton et al., Gene 77, 1989, pp. 61-68. Thus,
using the nucleotide and amino acid sequences of FAP, one may
introduce the alteration(s) of choice. Likewise, procedures for
preparing a DNA construct using polymerase chain reaction using
specific primers are well known to per-sons skilled in the art (cf.
PCR Protocols, 1990, Academic Press, San Diego, Calif., USA).
[0162] The polypeptides of the present invention can also comprise
non-naturally occurring amino acid residues. Non-naturally
occurring amino acids include, without limitation, beta-alanine,
desaminohistidine, trans-3-methylproline, 2,4-methanoproline,
cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,
allo-threonine, methylthreonine, hydroxyethylcys-teine,
hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic
acid, thiazolidine carboxylic acid, dehydroproline, 3- and
4-methylproline, 3,3-dimethylproline, tert-leucine, nor-valine,
2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and
4-fluorophenylalanine. Several methods are known in the art for
incorporating non-naturally occurring amino acid residues into
polypeptides. For example, an in vitro system can be employed
wherein nonsense mutations are suppressed using chemically
aminoacylated suppressor tRNAs. Methods for synthesizing amino
acids and aminoacylating tRNA are known in the art. Transcription
and translation of plasmids containing nonsense mutations is
carried out in a cell-free system comprising an E. coli S30 extract
and commercially available enzymes and other reagents. Polypeptides
are purified by chromatography. See, for example, Robertson et al.,
J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol.
202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et
al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second
method, translation is carried out in Xenopus oocytes by
microinjection of mutated mRNA and chemically aminoacylated
suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-8,
1996). Within a third method, E. coli cells are cul-tured in the
absence of a natural amino acid that is to be replaced (e.g.,
phenylalanine) and in the presence of the desired non-naturally
occurring amino acid(s) (e.g., 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
The non-naturally occurring amino acid is incorporated into the
polypeptide in place of its natural counterpart. See, Koide et al.,
Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues
can be converted to non-naturally occurring species by in vitro
chemical modification. Chemical modification can be combined with
site-directed mutagenesis to further expand the range of
substitutions (Wynn and Richards, Protein Sci. 2:395-403,
1993).
[0163] The nucleic acid construct encoding the Ficolin-associated
polypeptides and other polypeptides of the invention of the
invention may suitably be of genomic or cDNA origin, for instance
obtained by preparing a genomic or cDNA library and screening for
DNA sequences coding for all or part of the polypeptide by
hybridization using synthetic oligonucleotide probes in accordance
with standard techniques (cf. Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd. Ed. Cold Spring Harbor Labora-tory, Cold
Spring Harbor, N.Y., 1989).
[0164] The nucleic acid construct encoding a Ficolin-associated
polypeptide may also be prepared synthetically by established
standard methods, e.g. the phosphoamidite method described by
Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869,
or the method described by Matthes et al., EMBO Journal 3 (1984),
801-805. According to the phosphoamidite method, oligonucleotides
are synthesised, e.g. in an automatic DNA synthesiser, purified,
annealed, ligated and cloned in suitable vectors. The DNA sequences
encoding the human Ficolin-associated polypeptides and other
polypeptides of the invention may also be prepared by polymerase
chain reaction using specific primers, for instance as described in
U.S. Pat. No. 4,683,202, Saiki et al., Science 239 (1988), 487-491,
or Sambrook et al., supra.
[0165] Furthermore, the nucleic acid construct may be of mixed
synthetic and genomic, mixed synthetic and cDNA or mixed genomic
and cDNA origin prepared by ligating fragments of syn-thetic,
genomic or cDNA origin (as appropriate), the fragments
corresponding to various parts of the entire nucleic acid
construct, in accordance with standard techniques.
[0166] The nucleic acid construct is preferably a DNA construct.
DNA sequences for use in producing Ficolin-associated polypeptides
and other polypeptides according to the present invention will
typically encode a pre-pro polypeptide at the amino-terminus of FAP
to obtain proper posttranslational processing and secretion from
the host cell.
[0167] The DNA sequences encoding the human Ficolin-associated
polypeptides and other polypeptides according to the present
invention are usually inserted into a recombinant vector which may
be any vector, which may conveniently be subjected to recombinant
DNA procedures, and the choice of vector will often depend on the
host cell into which it is to be introduced. Thus, the vector may
be an autonomously replicating vector, i.e. a vector, which exists
as an extrachromosomal entity, the replication of which is
independent of chromosomal replication, e.g. a plasmid.
Alternatively, the vector may be one which, when introduced into a
host cell, is integrated into the host cell genome and replicated
together with the chromosome(s) into which it has been
integrated.
[0168] The vector is preferably an expression vector in which the
DNA sequence encoding the human Ficolin-associated polypeptides and
other polypeptides according to the present invention is operably
linked to additional segments required for transcription of the
DNA. In general, the expression vector is derived from plasmid or
viral DNA, or may contain elements of both. The term, "operably
linked" indicates that the segments are arranged so that they
function in conceit for their intended purposes, e.g. transcription
initiates in a promoter and proceeds through the DNA sequence
coding for the polypeptide.
[0169] Expression vectors for use in expressing Ficolin-associated
polypeptides and other polypeptides according to the present
invention will comprise a promoter capable of directing the
transcription of a cloned gene or cDNA. The promoter may be any DNA
sequence, which shows transcriptional activity in the host cell of
choice and may be derived from genes encoding proteins either
homologous or heterologous to the host cell.
[0170] Examples of suitable promoters for directing the
transcription of the DNA encoding the human Ficolin-associated
polypeptide in mammalian cells are the SV40 promoter (Subramani et
al., Mol. Cell Biol. 1 (1981), 854-864), the MT-1 (metallothionein
gene) promoter (Palmiter et al., Science 222 (1983), 809-814), the
CMV promoter (Boshart et al., Cell 41:521-530, 1985) or the
adenovirus 2 major late promoter (Kaufman and Sharp, Mol. Cell.
Biol, 2:1304-1319, 1982).
[0171] An example of a suitable promoter for use in insect cells is
the polyhedrin promoter (U.S. Pat. No. 4,745,051; Vasuvedan et al.,
FEBS Lett. 311, (1992) 7-11), the P10 promoter (J. M. Vlak et al.,
J. Gen. Virology 69, 1988, pp. 765-776), the Autographa californica
polyhedrosis virus basic protein promoter (EP 397 485), the
baculovirus immediate early gene 1 promoter (U.S. Pat. No.
5,155,037; U.S. Pat. No. 5,162,222), or the baculovirus 39K
delayed-early gene promoter (U.S. Pat. No. 5,155,037; U.S. Pat. No.
5,162,222).
[0172] Examples of suitable promoters for use in yeast host cells
include promoters from yeast glycolytic genes (Hitzeman et al., J.
Biol. Chem. 255 (1980), 12073-12080; Alber and Kawasaki, J. Mol.
Appl. Gen. 1 (1982), 419-434) or alcohol dehydrogenase genes (Young
et al., in Genetic Engineering of Microorganisms for Chemicals
(Hollaender et al, eds.), Plenum Press, New York, 1982), or the
TPI1 (U.S. Pat. No. 4,599,311) or ADH2-4c (Russell et al., Nature
304 (1983), 652-654) promoters.
[0173] Examples of suitable promoters for use in filamentous fungus
host cells are, for instance, the ADH3 promoter (McKnight et al.,
The EMBO J. 4 (1985), 2093-2099) or the tpiA promoter. Examples of
other useful promoters are those derived from the gene encoding A.
oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A.
niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A.
niger or A. awamori glucoamylase (gluA), Rhizomucor miehei lipase,
A. oryzae alkaline protease, A. oryzae triose phosphate isomerase
or A. nidulans acetamidase. Preferred are the TAKA-amylase and gluA
promoters. Suitable promoters are mentioned in, e.g. EP 238 023 and
EP 383 779.
[0174] The DNA sequences encoding the human Ficolin-associated
polypeptides and other polypeptides according to the present
invention may also, if necessary, be operably connected to a
suitable terminator, such as the human growth hormone terminator
(Palmiter et al., Science 222, 1983, pp. 809-814) or the TPI1
(Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp. 419-434) or
ADH3 (McKnight et al., The EMBO J. 4, 1985, pp. 2093-2099)
terminators. Expression vectors may also contain a set of RNA
splice sites located downstream from the promoter and upstream from
the insertion site for the FAP sequence itself. Preferred RNA
splice sites may be obtained from adenovirus and/or immunoglobulin
genes. Also contained in the expression vectors is a
polyadenylation signal located downstream of the insertion site.
Particularly preferred polyadenylation signals include the early or
late polyadenylation signal from SV40 (Kaufman and Sharp, ibid.),
the polyadenylation signal from the adenovirus 5 Elb region, the
human growth hormone gene terminator (DeNoto et al. Nucl. Acids
Res. 9:3719-3730, 1981) or the polyadenylation signal from the
human FAP gene or the bovine FAP gene. The expression vectors may
also include a noncoding viral leader sequence, such as the
adenovirus 2 tripartite leader, located between the promoter and
the RNA splice sites; and enhancer sequences, such as the SV40
enhancer.
[0175] To direct the human Ficolin-associated polypeptides and
other polypeptides of the present invention into the secretory
pathway of the host cells, a secretory signal sequence (also known
as a leader sequence, prepro sequence or pre sequence) may be
provided in the recombinant vector. The secretory signal sequence
is joined to the DNA sequences encoding the human
Ficolin-associated polypeptides and other polypeptides according to
the present invention in the correct reading frame. Secretory
signal sequences are commonly positioned 5' to the DNA sequence
encoding the peptide. The secretory signal sequence may be that,
normally associated with the protein or may be from a gene encoding
another secreted protein.
[0176] For secretion from yeast cells, the secretory signal
sequence may encode any signal peptide, which ensures efficient
direction of the expressed human Ficolin-associated polypeptides
and other polypeptides according to the present invention into the
secretory pathway of the cell. The signal peptide may be naturally
occurring signal peptide, or a functional part thereof, or it may
be a synthetic peptide. Suitable signal peptides have been found to
be the alpha-factor signal peptide (cf. U.S. Pat. No. 4,870,008),
the signal peptide of mouse salivary amylase (cf. O. Hagenbuchle et
al., Nature 289, 1981, pp. 643-646), a modified carboxypeptidase
signal peptide (cf. L. A. Valls et al., Cell 48, 1987, pp.
887-897), the yeast BAR1 signal peptide (cf. WO 87/02670), or the
yeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-Mitani
et al., Yeast 6, 1990, pp. 127-137).
[0177] For efficient secretion in yeast, a sequence encoding a
leader peptide may also be inserted downstream of the signal
sequence and upstream of the DNA sequence encoding the human
Ficolin-associated polypeptides and other polypeptides according to
the present invention. The function of the leader peptide is to
allow the expressed peptide to be directed from the endoplasmic
reticulum to the Golgi apparatus and further to a secretory vesicle
for secretion into the culture medium (i.e. exportation of the
human Ficolin-associated polypeptides and other polypeptides
according to the present invention across the cell wall or at least
through the cellular membrane into the periplasmic space of the
yeast cell). The leader peptide may be the yeast alpha-factor
leader (the use of which is described in e.g. U.S. Pat. No.
4,546,082, U.S. Pat. No. 4,870,008, EP 16 201, EP 123 294, EP 123
544 and EP 163 529). Alternatively, the leader peptide may be a
synthetic leader peptide, which is to say a leader peptide not
found in nature. Synthetic leader peptides may, for instance, be
constructed as described in WO 89/02463 or WO 92/11378.
[0178] For use in filamentous fungi, the signal peptide may
conveniently be derived from a gene encoding an Aspergillus sp.
amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase
or protease or a Humicola lanuginosa lipase. The signal peptide is
preferably derived from a gene encoding A. oryzae TAKA amylase, A.
niger neutral alpha-amylase, A. niger acid-stable amylase, or A.
niger glucoamylase. Suitable signal peptides are disclosed in, e.g.
EP 238 023 and EP 215 594.
[0179] For use in insect cells, the signal peptide may conveniently
be derived from an insect gene (cf. WO 90/05783), such as the
lepidopteran Manduca sexta adipokinetic hormone precursor signal
peptide (cf. U.S. Pat. No. 5,023,328).
[0180] The procedures used to ligate the DNA sequences coding for
the human Ficolin-associated polypeptides and other polypeptides
according to the present invention, the promoter and optionally the
terminator and/or secretory signal sequence, respectively, and to
insert them into suitable vectors containing the information
necessary for replication, are well known to persons skilled in the
art (cf., for instance, Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, N.Y., 1989).
[0181] Methods of transfecting mammalian cells and expressing DNA
sequences introduced in the cells are described in e.g. Kaufman and
Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J.
Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl.
Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978),
725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603,
Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al.,
EMBO J. 1 (1982), 841-845.
[0182] Cloned DNA sequences are introduced into cultured mammalian
cells by, for example, calcium phosphate-mediated transfection
(Wigler et al., Cell 14:725-732, 1978; Corsaro and Pearson, Somatic
Cell Genetics 7:603-616, 1981; Graham and Van der Eb, Virology
52d:456-467, 1973) or electroporation (Neumann et al., EMBO J.
1:841-845, 1982). To identify and select cells that express the
exogenous DNA, a gene that confers a selectable phenotype (a
selectable marker) is generally introduced into cells along with
the gene or cDNA of interest. Preferred selectable markers include
genes that confer resistance to drugs such as neomycin, hygromycin,
and methotrexate. The selectable marker may be an amplifiable
selectable marker. A preferred amplifiable selectable marker is a
dihydrofolate reductase (DHFR) sequence. Selectable markers are
reviewed by Thilly (Mammalian Cell Technology, Butterworth
Publishers, Stoneham, Mass., incorporated herein by reference). The
person skilled in the art will easily be able to choose suitable
selectable markers.
[0183] Selectable markers may be introduced into the cell on a
separate plasmid at the same time as the gene of interest, or they
may be introduced on the same plasmid. If on the same plasmid, the
selectable marker and the gene of interest may be under the control
of different promoters or the same promoter, the latter arrangement
producing a dicistronic message. Constructs of this type are known
in the art (for example, Levinson and Simonsen, U.S. Pat. No.
4,713,339). It may also be advantageous to add additional DNA,
known as "carrier DNA," to the mixture that is introduced into the
cells.
[0184] After the cells have taken up the DNA, they are grown in an
appropriate growth me-dium, typically 1-2 days, to begin expressing
the gene of interest. As used herein the term "appropriate growth
medium" means a medium containing nutrients and other components
required for the growth of cells and the expression of the human
Ficolin-associated polypeptide of interest. Media generally include
a carbon source, a nitrogen source, essential amino acids,
essential sugars, vitamins, salts, phospholipids, protein and
growth factors. Drug selection is then applied to select for the
growth of cells that are expressing the selectable marker in a
stable fashion. For cells that have been transfected with an
amplifiable selectable marker the drug concentration may be
increased to select for an increased copy number of the cloned
sequences, thereby in-creasing expression levels. Clones of stably
transfected cells are then screened for expres-sion of the human
Ficolin-associated polypeptide of interest.
[0185] The host cell into which the DNA sequences encoding the
human Ficolin-associated polypeptides and other polypeptides
according to the present invention is introduced may be any cell,
which is capable of producing the posttranslational modified human
polypeptides and includes yeast, fungi and higher eucaryotic
cells.
[0186] Examples of mammalian cell lines for use in the present
invention are the COS-1 (ATCC CRL 1650), baby hamster kidney (BHK)
and 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72,
1977) cell lines. A preferred BHK cell line is the tk-ts13 BHK cell
line (Waechter and Baserga, Proc. Natl. Acad. Sci. USA
79:1106-1110, 1982, incorporated herein by reference), hereinafter
referred to as BHK 570 cells. The BHK 570 cell line has been
deposited with the American Type Culture Collection, 12301 Parklawn
Dr., Rockville, Md. 20852, under ATCC accession number CRL 10314. A
tk-ts13 BHK cell line is also available from the ATCC under
accession number CRL 1632. In addition, a number of other cell
lines may be used within the present invention, including Rat Hep I
(Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL
1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469
(ATCC CCL 9.1), CHO (ATCC CCL 61) and DUKX cells (Urlaub and
Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980).
[0187] Examples of suitable yeasts cells include cells of
Saccharomyces spp. or Schizosaccharomyces spp., in particular
strains of Saccharomyces cerevisiae or Saccharomyces kluyveri.
Methods for transforming yeast cells with heterologous DNA and
producing heterologous poly-peptides there from are described, e.g.
in U.S. Pat. No. 4,599,311, U.S. Pat. No. 4,931,373, U.S. Pat. Nos.
4,870,008, 5,037,743, and U.S. Pat. No. 4,845,075, all of which are
hereby incorporated by reference. Transformed cells are selected by
a phenotype determined by a selectable marker, commonly drug
resistance or the ability to grow in the absence of a particular
nutrient, e.g. leucine. A preferred vector for use in yeast is the
POT1 vector disclosed in U.S. Pat. No. 4,931,373. The DNA sequences
encoding the human Ficolin-associated polypeptides and other
polypeptides according to the present invention may be preceded by
a signal sequence and optionally a leader sequence, e.g. as
described above. Further examples of suitable yeast cells are
strains of Kluyveromyces, such as K. lactis, Hansenula, e.g. H.
polymorpha, or Pichia, e.g. P. pastoris (cf. Gleeson et al., J.
Gen. Microbiol. 132, 1986, pp. 3459-3465; U.S. Pat. No.
4,882,279).
[0188] Examples of other fungal cells are cells of filamentous
fungi, e.g. Aspergillus spp., Neurospora spp., Fusarium spp. or
Trichoderma spp., in particular strains of A. oryzae, A. nidulans
or A. niger. The use of Aspergillus spp. for the expression of
proteins is described in, e.g., EP 272 277, EP 238 023, EP 184 438
The transformation of F. oxysporum may, for instance, be carried
out as described by Malardier et al., 1989, Gene 78: 147-156. The
transformation of Trichoderma spp. may be performed for instance as
described in EP 244 234.
[0189] When a filamentous fungus is used as the host cell, it may
be transformed with the DNA construct of the invention,
conveniently by integrating the DNA construct in the host
chromosome to obtain a recombinant host cell. This integration is
generally considered to be an advantage as the DNA sequence is more
likely to be stably maintained in the cell. Integration of the DNA
constructs into the host chromosome may be performed according to
conventional methods, e.g. by homologous or heterologous
recombination.
[0190] Transformation of insect cells and production of
heterologous polypeptides therein may be performed as described in
U.S. Pat. No. 4,745,051; U.S. Pat. No. 4,879,236; U.S. Pat. Nos.
5,155,037; 5,162,222; EP 397,485) all of which are incorporated
herein by reference. The insect cell line used as the host may
suitably be a Lepidoptera cell line, such as Spodoptera frugiperda
cells or Trichoplusia ni cells (cf. U.S. Pat. No. 5,077,214).
Culture conditions may suitably be as described in, for instance,
WO 89/01029 or WO 89/01028, or any of the aforementioned
references.
[0191] The transformed or transfected host cell described above is
then cultured in a suitable nutrient medium under conditions
permitting expression of the human Ficolin-associated polypeptide
after which all or part of the resulting peptide may be recovered
from the culture. The medium used to culture the cells may be any
conventional medium suitable for growing the host cells, such as
minimal or complex media containing appropriate supplements.
Suitable media are available from commercial suppliers or may be
prepared according to published recipes (e.g. in catalogues of the
American Type Culture Collection). The human Ficolin-associated
polypeptide produced by the cells may then be recovered from the
culture medium by conventional procedures including separating the
host cells from the medium by centrifugation or filtration,
precipitating the proteinaqueous components of the supernatant or
filtrate by means of a salt, e.g. ammonium sulphate, purification
by a variety of chromatographic procedures, e.g. ion exchange
chromatography, gelfiltration chromatography, affinity
chromatography, or the like, dependent on the type of polypeptide
in question.
[0192] Transgenic animal technology may be employed to produce the
Ficolin-associated polypeptides and other polypeptides of the
invention. It is preferred to produce the proteins within the
mammary glands of a host female mammal. Expression in the mammary
gland and subsequent secretion of the protein of interest into the
milk overcomes many difficulties encountered in isolating proteins
from other sources. Milk is readily collected, available in large
quantities, and biochemically well characterized. Furthermore, the
major milk proteins are present in milk at high concentrations
(typically from about 1 to 15 g/l).
[0193] From a commercial point of view, it is clearly preferable to
use as the host a species that has a large milk yield. While
smaller animals such as mice and rats can be used (and are
preferred at the proof of principle stage), it is preferred to use
livestock mammals including, but not limited to, pigs, goats, sheep
and cattle. Sheep are particularly preferred due to such factors as
the previous history of transgenesis in this species, milk yield,
cost and the ready availability of equipment for collecting sheep
milk (see, for example, WO 88/00239 for a comparison of factors
influencing the choice of host species). It is generally desirable
to select a breed of host animal that has been bred for dairy use,
such as East Friesland sheep, or to introduce dairy stock by
breeding of the transgenic line at a later date. In any event,
animals of known, good health status should be used.
[0194] To obtain expression in the mammary gland, a transcription
promoter from a milk protein gene is used. Milk protein genes
include those genes encoding caseins (see U.S. Pat. No. 5,304,489),
beta lactoglobulin, a lactalbumin, and whey acidic protein. The
beta lactoglobulin (BLG) promoter is preferred. In the case of the
ovine beta lactoglobulin gene, a region of at least the proximal
406 by of 5' flanking sequence of the gene will generally be used,
although larger portions of the 5' flanking sequence, up to about 5
kbp, are preferred, such as a .about.4.25 kbp DNA segment
encompassing the 5' flanking promoter and non coding portion of the
beta lactoglobulin gene (see Whitelaw et al., Biochem. J. 286: 31
39 (1992)). Similar fragments of promoter DNA from other species
are also suitable.
[0195] Other regions of the beta lactoglobulin gene may also be
incorporated in constructs, as may genomic regions of the gene to
be expressed. It is generally accepted in the art that constructs
lacking introns, for example, express poorly in comparison with
those that contain such DNA sequences (see Brinster et al., Proc.
Natl. Acad. Sci. USA 85: 836 840 (1988); Palmiter et al., Proc.
Natl. Acad. Sci. USA 88: 478 482 (1991); Whitelaw et al.,
Transgenic Res. 1: 3 13 (1991); WO 89/01343; and WO 91/02318, each
of which is incorporated herein by reference). In this regard, it
is generally preferred, where possible, to use genomic sequences
containing all or some of the native introns of a gene encoding the
protein or polypeptide of interest, thus the further inclusion of
at least some introns from, e.g, the beta lactoglobulin gene, is
preferred. One such region is a DNA segment that provides for
intron splicing and RNA polyadenylation from the 3' non coding
region of the ovine beta lactoglobulin gene. When substituted for
the natural 3' non coding sequences of a gene, this ovine beta
lactoglobulin segment can both enhance and stabilize expression
levels of the protein or polypeptide of interest. Within other
embodiments, the region surrounding the initiation ATG of the FAP
sequence is replaced with corresponding sequences from a milk
specific protein gene. Such replacement provides a putative tissue
specific initiation environment to enhance expression. It is
convenient to replace the entire FAP pre pro and 5' non coding
sequences with those of, for example, the BLG gene, although
smaller regions may be replaced.
[0196] For expression of Ficolin-associated polypeptides and other
polypeptides according to the present invention in transgenic
animals, a DNA segment encoding FAP is operably linked to
additional DNA segments required for its expression to produce
expression units. Such additional segments include the above
mentioned promoter, as well as sequences that provide for
termination of transcription and polyadenylation of mRNA. The
expression units will further include a DNA segment encoding a
secretory signal sequence operably linked to the segment encoding
modified FAP. The secretory signal sequence may be a native FAP
secretory signal sequence or may be that of another protein, such
as a milk protein (see, for example, von Heijne, Nucl. Acids Res.
14: 4683 4690 (1986); and Meade et al., U.S. Pat. No. 4,873,316,
which are incorporated herein by reference).
[0197] Construction of expression units for use in transgenic
animals is conveniently carried out by inserting a FAP sequence
into a plasmid or phage vector containing the additional DNA
segments, although the expression unit may be constructed by
essentially any sequence of ligations. It is particularly
convenient to provide a vector containing a DNA segment encoding a
milk protein and to replace the coding sequence for the milk
protein with that of a FAP variant; thereby creating a gene fusion
that includes the expression control sequences of the milk protein
gene. In any event, cloning of the expression units in plasmids or
other vectors facilitates the amplification of the FAP sequence.
Amplification is conveniently carried out in bacterial (e.g. E.
coli) host cells, thus the vectors will typically include an origin
of replication and a selectable marker functional in bacterial host
cells. The expression unit is then introduced into fertilized eggs
(including early stage embryos) of the chosen host species.
Introduction of heterologous DNA can be accomplished by one of
several routes, including microinjection (e.g. U.S. Pat. No.
4,873,191), retroviral infection (Jaenisch, Science 240: 1468 1474
(1988)) or site directed integration using embryonic stem (ES)
cells (reviewed by Bradley et al., Bio/Technology 10: 534 539
(1992)). The eggs are then implanted into the oviducts or uteri of
pseudopregnant females and allowed to develop to term. Offspring
carrying the introduced DNA in their germ line can pass the DNA on
to their progeny in the normal, Mendelian fashion, allowing the
development of transgenic herds. General procedures for producing
transgenic animals are known in the art (see, for example, Hogan et
al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold
Spring Harbor Laboratory, 1986; Simons et al., Bio/Technology 6:
179 183 (1988); Wall et al., Biol. Reprod. 32: 645 651 (1985);
Buhler et al., Bio/Technology 8: 140 143 (1990); Ebert et al.,
Bio/Technology 9: 835 838 (1991); Krimpenfort et al.,
Bio/Technology 9: 844 847 (1991); Wall et al., J. Cell. Biochem.
49: 113 120 (1992); U.S. Pat. No. 4,873,191; U.S. Pat. No.
4,873,316; WO 88/00239, WO 90/05188, WO 92/11757; and GB 87/00458).
Techniques for introducing foreign DNA sequences into mammals and
their germ cells were originally developed in the mouse (see, e.g.,
Gordon et al., Proc. Natl. Acad. Sci. USA 77: 7380 7384 (1980);
Gordon and Ruddle, Science 214: 1244 1246 (1981); Palmiter and
Brinster, Cell 41: 343 345 (1985); Brinster et al., Proc. Natl.
Acad. Sci. USA 82: 4438 4442 (1985); and Hogan et al. (ibid.)).
These techniques were subsequently adapted for use with larger
animals, including livestock species (see, e.g., WO 88/00239, WO
90/05188, and WO 92/11757; and Simons et al., Bio/Technology 6: 179
183 (1988)). To summarise, in the most efficient route used to date
in the generation of transgenic mice or livestock, several hundred
linear molecules of the DNA of interest are injected into one of
the pro nuclei of a fertilized egg according to established
techniques. Injection of DNA into the cytoplasm of a zygote can
also be employed.
[0198] Production in transgenic plants may also be employed.
Expression may be generalised or directed to a particular organ,
such as a tuber (see, Hiatt, Nature 344:469 479 (1990); Edelbaum et
al., J. Interferon Res. 12:449 453 (1992); Sijmons et al.,
Bio/Technology 8:217 221 (1990); and EP 0 255 378).
[0199] FAP Purification
[0200] The Ficolin-associated polypeptides and other polypeptides
of the invention may be recovered from cell culture medium or milk.
The Ficolin-associated polypeptides and other polypeptides of the
present invention may be purified by a variety of procedures known
in the art including, but not limited to, chromatography (e.g., ion
exchange, affinity, hydrophobic, chromatofocusing, and size
exclusion), electrophoretic procedures (e.g., preparative
isoelectric focusing (IEF), differential solubility (e.g., ammonium
sulfate precipitation), or extraction (see, e.g., Protein
Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers,
New York, 1989). Preferably, they may be purified by affinity
chromatography on an anti-FAP antibody column. Additional
purification may be achieved by conventional chemical purification
means, such as high performance liquid chromatography. Other
methods of purification, including barium citrate precipitation,
are known in the art, and may be applied to the purification of the
novel Ficolin-associated polypeptides and other polypeptides
described herein (see, for example, Scopes, R., Protein
Purification, Springer-Verlag, N.Y., 1982).
[0201] For therapeutic purposes it is preferred that the
Ficolin-associated polypeptides and other polypeptides of the
invention are substantially pure. Thus, in a preferred embodiment
of the invention the and other polypeptides of the invention is
purified to at least about 90 to 95% homogeneity, preferably to at
least about 98% homogeneity. Purity may be assessed by e.g. gel
electrophoresis and amino-terminal amino acid sequencing.
[0202] The term "isolated polypeptide" refers to a polypeptide of
the present invention that (1) has been separated from at least
about 50 percent of polynucleotides, lipids, carbohydrates or other
materials (i.e., contaminants) with which it is naturally
associated. Preferably, the isolated polypeptide is substantially
free from any other contaminating polypeptides or other
contaminants that are found in its natural environment, which would
interfere with its therapeutic, diagnostic, prophylactic or
research use.
[0203] The term "microorganism" as used herein refers to bacteria,
fungi, archaea, protists; microscopic plants and animals (such as
green algae or plankton), the planarian and amoeba. Included within
this definition are pathogenic microorganisms.
[0204] Assays
[0205] A General Procedure for SDS-PAGE and Western Blotting:
[0206] Electrophoresis was performed on 10% or 4-12% (w/v) Bis-Tris
Polyacrylamide-gels with discontinuous buffers using the
NuPAGE.RTM. system (Invitrogen) as recommended by the manufacture.
Western blotting was performed using polyvinylidene difluoride
membranes (PVDF-HyBond, GE-healthcare, Hilleroed, Denmark, cat. no.
RPN303F), 2 .mu.g/ml of biotin labeled primary monoclonal antibody
and secondary visualization by HRP conjugated streptavidin (P0397,
Dako, Glostrup, Denmark) diluted to 1:1500 in PBS, 0.05% Tween20.
The membranes were developed with 0.04% 3-amino-9-ethylcarbazole
(Sigma-aldrich, Broenby, Denmark, cat. no. A5754-100G) in acetone
and 0.015% H.sub.2O.sub.2 in 50 mM sodium acetate buffer pH 5.
[0207] Co-immunoprecipitation:
[0208] Immunoprecipitation of mannose binding lectin (MBL) serum
complexes: 1 ml of normal human serum was diluted 1:1 in TBS (10 mM
Tris, 140 mM NaCl, pH 7.5) and incubated end over end for 1 hour at
4.degree. C. with 5 .mu.g of the MBL specific mouse monoclonal
antibody Hyb 131-11 (Bioporto, Gentofte, Denmark).
[0209] Immunoprecipitation of Ficolin-2 serum complexes: 0.5 ml of
normal human serum was diluted 1:1 in TBS (10 mM Tris, 140 mM NaCl,
pH 7.5) and incubated end over end for 1 hour at 4.degree. C. with
5 .mu.g of the Ficolin-2 specific mouse monoclonal antibody Hyb 219
(Munthe-Fog L, et al.
[0210] Immunoprecipitation of Ficolin-3 serum complexes: 0.2 ml of
normal human serum was diluted 1:1 in TBS (10 mM Tris, 140 mM NaCl,
pH 7.5) and incubated end over end for 1 hour at 4.degree. C. with
5 .mu.g of the Ficolin-3 specific mouse monoclonal antibody Hyb 334
(Munthe-Fog L, et al.
[0211] Immune complex precipitation was conducted with sheep anti
mouse IgG conjugated magnetic dynal beads (Dynal-Invitrogen, Cat.
No. 112.02D): After incubation with serum and primary antibodies
(as above) 5.times.10.sup.7 sheep anti mouse conjugated magnetic
dynal beads were added and incubated for 30 min 4.degree. C. The
beads were magnetically separated and washed for three times with
TBS-tween-Ca.sup.2+ (10 mM Tris, 140 mM NaCl, 0.05% tween, 5 mM
CaCl.sub.2, pH 7.5) and finally boiled in SDS-loading buffer and
analyzed by SDS-PAGE and western blotting with biotin labeled
monoclonal antibody mAb-8B3 (reacting with an epitope on the heavy
chain/A-chain shared by MASP-1 and -3).
[0212] Immunoaffinity purification of FAP:10 mg of mAb-8B3
(reacting with an epitope on the heavy chain/A-chain shared by FAP,
MASP-1 and -3) or 10 mg of rabbit polyclonal anti FAP antibodies
were conjugated to CNBr activated sepharose as recommended by the
manufacturer (GE-healthcare, Hilleroed, Denmark, cat. no.
17-0430-01) and packed onto a column.
[0213] Purification from serum: 150 ml of a pool of normal human
serum was diluted 1:1 with TBS+0.5 M NaCl+10 mM EDTA (10 mM Tris,
640 mM NaCl, 10 mM EDTA, pH 7.5) and loaded on the columns
described above. The columns were washed with 1 l of TBS+0.5 M
NaCl+10 mM EDTA and 1 ml fractions were eluted with 1 M
Glycine-HCl, pH 2.5 and analyzed by SDS-PAGE and western blotting
with biotin labeled monoclonal antibody mAb-8B3.
[0214] Purification of recombinant FAP: 2-3 l of culture
supernatant (from CHO serum free medium/Gibco-Invitrogen, cat. no.
12651-014) from Chinese hamster ovarian cells (CHO cells)
expressing recombinant FAP (rFAP) was loaded on the antibody
columns described above. The columns were washed with 1.5 l of
TBS+0.5 M NaCl+10 mM EDTA and 1 ml fractions were eluted with 1 M
Glycine-HCl, pH 2.5. The eluted fractions were analyzed by SDS-PAGE
and coomassie staining.
[0215] Recombinant expression of FAP: Full-length cDNA inserted
into the pcDNA5/FRT vector (Invitrogen, cat. no. V6010-20) was
ordered from Genscript (Genscript, New Jersey, USA) and
co-transfected with the pOG44 vector (Invitrogen, cat. no.
V6005-20) into the CHO Flp-In cell line (Invitrogen, cat. no.
R758-07) and selected and cloned as recommended by the manufacturer
(Invitrogen). The cells were grown in Freestyle CHO serum free
medium (Invitrogen, cat. no. 12651-014) and culture supernatants
were harvested and analyzed.
[0216] Production of mono- and polyclonal antibodies:A peptide
construct (ordered from Genscript, New Jersey, USA) of the FAP
specific 17 C-terminal residues were coupled onto the toxoid form
of tetanus and diphtheria using the cysteine coupling method with
m-Maleimidobenzoyl-N-hydroxysuccinimide ester as recommended by the
manufacturer (Thermo Fisher Scientific/Pierce, Ill., USA).
[0217] Six mice and two rabbits were each immunized three times
(with 14 days intervals) with 25 jig antigen adsorbed onto
Al(OH).sub.3 and Freunds incomplete adjuvant. The polyclonal
antibody titers were assessed using ELISA with the different FAP
peptides coupled to a protein carrier. Polyclonal rabbit antiserum
(.apprxeq.10 ml) was harvested 14 days after the first, second and
third immunization.
[0218] Two mice were used for production of monoclonal antibodies.
Four days prior to the fusion the mice received an intravenous
injection of 25 .mu.g antigen. The fusion was conducted as
described elsewhere (Kohler, G. and C. Milstein. 1975. Continuous
cultures of fused cells secreting antibody of predefined
specificity. Nature 256:495-497).
[0219] Clones were selected by differential ELISA screening against
peptides coupled to different protein carriers.
[0220] Functional complement assays:Ficolin-3 and MBL homozygous
defect sera were used to investigate the function of FAP.
[0221] Ficolin-3 assay:Maxisorp plates (NUNC, Roskilde, Denmark,
cat. no. 439454) were coated with acetylated bovine serum albumin
at 5 .mu.g/ml for 12 hours at 4.degree. C. in coating buffer (15 mM
Na.sub.2CO.sub.3, 35 mM NaHCO.sub.3, pH 9.5). After
blocking/washing four times in barbital/tween buffer (4 mM
barbital, 145 mM NaCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2, pH
7.4+0.05% Tween), recombinant human Ficolin-3 was added at 500
ng/ml I barbital/tween buffer and incubated for 1.5 hours at
20.degree. C. with shaking. After washing the plates twice in
barbital/tween buffer, recombinant FAP, human MASP-1, -2 or -3 as
serum free medium culture supernatants were added in serial
dilutions in the 1.sup.st dimension on separate plates and
incubated for 1 hour at 20.degree. C. with shaking. After washing
the plates twice in barbital/tween buffer, Ficolin-3 or MASP-2
deficient serum were added in serial dilutions in the 2.sup.nd
dimension on the plates and incubated for 30 min at 37.degree. C.
After washing the plates four times in barbital/tween buffer the
deposition of complement factor C4 was measured by incubation for 1
hour at 20.degree. C. with polyclonal rabbit antibodies to human
C4c (Dako, Glostrup, Denmark cat. no Q0369) diluted at 1:2000,
followed by four washing steps and incubation with horseradish
peroxidase conjugated swine anti rabbit antibodies (Dako, Glostrup,
Denmark cat. no P0399) for 45 min at 20.degree. C. The signal was
obtained by the plates were developed with 1000/well of
Ortho-phenylene-diamine (OPD) (0.4 mg/ml) dissolved in citrate
buffer (35 mM citric acid, 65 mM Na.sub.2PO.sub.4, pH 5) with
0.12%o (v/v) H.sub.2O.sub.2. The enzyme reaction was stopped with 1
M H.sub.2SO.sub.4 and optical density (OD) levels were measured at
490 nm-650 nm using a V-max Kinetic-reader (Molecular Devices).
[0222] Mannose-Binding Lectin assay:Maxisorp plates (NUNC,
Roskilde, Denmark, cat. no. 439454) were coated with mannan
(Sigma-aldrich, Broenby, Denmark, cat. no. M7504-1G) at 10 .mu.g/ml
for 12 hours at 4.degree. C. in coating buffer (15 mM
Na.sub.2CO.sub.3, 35 mM NaHCO.sub.3, pH 9.5). After
blocking/washing four times in barbital/tween buffer (4 mM
barbital, 145 mM NaCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2, pH
7.4+0.05% Tween) recombinant human Mannose-Binding Lectin was added
at 0.5 .mu.g/ml I barbital/tween buffer and incubated for 1.5 hours
at 20.degree. C. with shaking. After washing the plates twice in
barbital/tween buffer, recombinant FAP, human MASP-1, -2 or -3 as
serum free medium culture supernatants were added in serial
dilutions in the 1.sup.st dimension on separate plates and
incubated for 1 hour at 20.degree. C. with shaking. After washing
the plates twice in barbital/tween buffer, MBL or MASP-2 deficient
serum were added in serial dilutions in the 2.sup.nd dimension on
the plates and incubated for 45 min at 37.degree. C. After washing
the plates four times in barbital/tween buffer the deposition of
complement factor C4 was measured by incubation for 1 hour at
20.degree. C. with polyclonal rabbit antibodies to human C4c (Dako,
Glostrup, Denmark cat. no Q0369) diluted at 1:2000, followed by
four washing steps and incubation with horseradish peroxidase
conjugated swine anti rabbit antibodies (Dako, Glostrup, Denmark
cat. no P0399) for 45 min at 20.degree. C. The signal was obtained
by the plates were developed with 100 .mu.l/well of
Ortho-phenylene-diamine (OPD) (0.4 mg/ml) dissolved in citrate
buffer (35 mM citric acid, 65 mM Na.sub.2PO.sub.4, pH 5) with 0.12%
(v/v) H.sub.2O.sub.2. The enzyme reaction was stopped with 1 M
H.sub.2SO.sub.4 and optical density (OD) levels were measured at
490 nm-650 nm using a V-max Kinetic-reader (Molecular Devices).
[0223] Genotyping assay: Different genotyping assays may be
conducted where the genotype is determined in individuals using
biological assays. Different kind of assays could be used such as:
[0224] Hybridization-based methods [0225] Dynamic allele-specific
hybridization [0226] Molecular beacons [0227] SNP microarrays
[0228] Enzyme-based methods [0229] Restriction fragment length
polymorphism [0230] PCR-based methods [0231] Flap endonuclease
[0232] Primer extension [0233] 5'-nuclease [0234] Oligonucleotide
ligase assay [0235] Other post-amplification methods based on
physical properties of DNA [0236] Single strand conformation
polymorphism [0237] Temperature gradient gel electrophoresis [0238]
Denaturing high performance liquid chromatography [0239]
High-Resolution Melting of the entire amplicon [0240] SNPlex [0241]
Sequencing
[0242] Administration and Pharmaceutical Compositions
[0243] Combination Treatments
[0244] The ficolin-associated polypeptide as defined in the present
specification may be administered simultaneously or sequentially
with one or more proteins selected from Ficolin-1, 2, 3, and
mannose-binding lectin (MBL). The factors may be supplied in
single-dosage form wherein the single-dosage form contains both
compounds, or in the form of a kit-of-parts comprising a
preparation of a ficolin-associated polypeptide as a first unit
dosage form and a preparation of the one or more other compound as
a second unit dosage form. Whenever a first or second or third,
etc., unit dose is mentioned throughout this specification this
does not indicate the preferred order of administration, but is
merely done for convenience purposes.
[0245] By "simultaneous" dosing of a preparation of a
ficolin-associated polypeptide and a preparation of one or more
other compound is meant administration of the compounds in
single-dosage form, or administration of a first agent followed by
administration of a second agent with a time separation of no more
than 15 minutes, preferably 10, more preferred 5, more preferred 2
minutes. Either factor may be administered first.
[0246] By "sequential" dosing is meant administration of a first
agent followed by administration of a second agent with a time
separation of more than 15 minutes. Either of the two unit dosage
form may be administered first. Preferably, both products are
injected through the same intravenous access.
[0247] Another object of the present invention is to provide a
pharmaceutical formulation comprising a ficolin-associated
polypeptide which is present in a serum/plasma concentration from 0
mg/ml to 1 mg/ml, and wherein the formulation has a pH from 2.0 to
10.0. The formulation may further comprise a buffer system,
preservative(s), tonicity agent(s), chelating agent(s), stabilizers
and surfactants. In some embodiments of the invention the
pharmaceutical formulation is an aqueous formulation, i.e.
formulation comprising water. Such formulation is typically a
solution or a suspension. In a further embodiment of the invention
the pharmaceutical formulation is an aqueous solution. The term
"aqueous formulation" is defined as a formulation comprising at
least 50% w/w water. Likewise, the term "aqueous solution" is
defined as a solution comprising at least 50%w/w water, and the
term "aqueous suspension" is defined as a suspension comprising at
least 50% w/w water.
[0248] In other embodiments the pharmaceutical formulation is a
freeze-dried formulation, whereto the physician or the patient adds
solvents and/or diluents prior to use.
[0249] In other embodiments the pharmaceutical formulation is a
dried formulation (e.g. freeze-dried or spray-dried) ready for use
without any prior dissolution.
[0250] In a further aspect the invention relates to a
pharmaceutical formulation comprising an aqueous solution of a
ficolin-associated polypeptide, and a buffer, wherein the
ficolin-associated polypeptide is present in a serum/plasma
concentration from 0-1 mg/ml or above, and wherein the formulation
has a pH from about 2.0 to about 10.0.
[0251] In a other embodiments of the invention the pH of the
formulation is selected from the list consisting of 2.0, 2.1, 2.2,
2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,
3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8,
4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,
6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,
7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and
10.0.
[0252] In a further embodiment of the invention the buffer is
selected from the group consisting of sodium acetate, sodium
carbonate, citrate, glycylglycine, histidine, glycine, lysine,
arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate,
sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine,
tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric
acid, aspartic acid or mixtures thereof. Each one of these specific
buffers constitutes an alternative embodiment of the invention.
[0253] In a further embodiment of the invention the formulation
further comprises a pharmaceutically acceptable preservative. In a
further embodiment of the invention the preservative is selected
from the group consisting of phenol, o-cresol, m-cresol, p-cresol,
methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,
2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzyl
alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid,
imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol,
ethyl p-hydroxybenzoate, benzethonium chloride, chlorphenesine
(3p-chlorphenoxypropane-1,2-diol) or mixtures thereof. In a further
embodiment of the invention the preservative is present in a
concentration from 0.1 mg/ml to 20 mg/ml. In a further embodiment
of the invention the preservative is present in a concentration
from 0.1 mg/ml to 5 mg/ml. In a further embodiment of the invention
the preservative is present in a concentration from 5 mg/ml to 10
mg/ml. In a further embodiment of the invention the preservative is
present in a concentration from 10 mg/ml to 20 mg/ml. Each one of
these specific preservatives constitutes an alternative embodiment
of the invention. The use of a preservative in pharmaceutical
compositions is well-known to the skilled person. For convenience
reference is made to Remington: The Science and Practice of
Pharmacy, 19.sup.th edition, 1995.
[0254] In a further embodiment of the invention the formulation
further comprises an isotonic agent. In a further embodiment of the
invention the isotonic agent is selected from the group consisting
of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an
amino acid (e.g. L-glycine, L-histidine, arginine, lysine,
isoleucine, aspartic acid, tryptophan, threonine), an alditol (e.g.
glycerol (glycerine), 1,2-propanediol (propyleneglycol),
1,3-propanediol, 1,3-butanediol)polyethyleneglycol (e.g. PEG400),
or mixtures thereof. Any sugar such as mono-, di-, or
polysaccharides, or water-soluble glucans, including for example
fructose, glucose, mannose, sorbose, xylose, maltose, lactose,
sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin,
soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na
may be used. In some embodiments the sugar additive is sucrose.
Sugar alcohol is defined as a C4-C8 hydrocarbon having at least one
--OH group and includes, for example, mannitol, sorbitol, inositol,
galactitol, dulcitol, xylitol, and arabitol. In some embodiments
the sugar alcohol additive is mannitol. The sugars or sugar
alcohols mentioned above may be used individually or in
combination. There is no fixed limit to the amount used, as long as
the sugar or sugar alcohol is soluble in the liquid preparation and
does not adversely effect the stabilizing effects achieved using
the methods of the invention. In some embodiments, the sugar or
sugar alcohol concentration is between about 1 mg/ml and about 150
mg/ml. In a further embodiment of the invention the isotonic agent
is present in a concentration from 1 mg/ml to 50 mg/ml. In a
further embodiment of the invention the isotonic agent is present
in a concentration from 1 mg/ml to 7 mg/ml. In a further embodiment
of the invention the isotonic agent is present in a concentration
from 8 mg/ml to 24 mg/ml. In a further embodiment of the invention
the isotonic agent is present in a concentration from 25 mg/ml to
50 mg/ml. Each one of these specific isotonic agents constitutes an
alternative embodiment of the invention. The use of an isotonic
agent in pharmaceutical compositions is well-known to the skilled
person. For convenience reference is made to Remington: The Science
and Practice of Pharmacy, 19.sup.th edition, 1995.
[0255] In a further embodiment of the invention the formulation
further comprises a chelating agent. In a further embodiment of the
invention the chelating agent is selected from salts of
ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic
acid, and mixtures thereof. In a further embodiment of the
invention the chelating agent is present in a concentration from
0.1 mg/ml to 5 mg/ml. In a further embodiment of the invention the
chelating agent is present in a concentration from 0.1 mg/ml to 2
mg/ml. In a further embodiment of the invention the chelating agent
is present in a concentration from 2 mg/ml to 5 mg/ml. Each one of
these specific chelating agents constitutes an alternative
embodiment of the invention. The use of a chelating agent in
pharmaceutical compositions is well-known to the skilled person.
For convenience reference is made to Remington: The Science and
Practice of Pharmacy, 19.sup.th edition, 1995.
[0256] In a further embodiment of the invention the formulation
further comprises a stabilizer. The use of a stabilizer in
pharmaceutical compositions is well-known to the skilled person.
For convenience reference is made to Remington: The Science and
Practice of Pharmacy, 19.sup.th edition, 1995.
[0257] More particularly, compositions of the invention are
stabilized liquid pharmaceutical compositions whose therapeutically
active components include a polypeptide that possibly exhibits
aggregate formation during storage in liquid pharmaceutical
formulations. By "aggregate formation" is intended a physical
interaction between the polypeptide molecules that results in
formation of oligomers, which may remain soluble, or large visible
aggregates that precipitate from the solution. By "during storage"
is intended a liquid pharmaceutical composition or formulation once
prepared, is not immediately administered to a subject. Rather,
following preparation, it is packaged for storage, either in a
liquid form, in a frozen state, or in a dried form for later
reconstitution into a liquid form or other form suitable for
administration to a subject. By "dried form" is intended the liquid
pharmaceutical composition or formulation is dried either by freeze
drying (i.e., lyophilization; see, for example, Williams and Polli
(1984) J. Parenteral Sci. Technol. 38:48-59), spray drying (see
Masters (1991) in Spray-Drying Handbook (5th ed; Longman Scientific
and Technical, Essez, U.K.), pp. 491-676; Broadhead et al. (1992)
Drug Devel. Ind. Pharm. 18:1169-1206; and Mumenthaler et al. (1994)
Pharm. Res. 11:12-20), or air drying (Carpenter and Crowe (1988)
Cryobiology 25:459-470; and Roser (1991) Biopharm. 4:47-53).
Aggregate formation by a polypeptide during storage of a liquid
pharmaceutical composition can adversely affect biological activity
of that polypeptide, resulting in loss of therapeutic efficacy of
the pharmaceutical composition. Furthermore, aggregate formation
may cause other problems such as blockage of tubing, membranes, or
pumps when the polypeptide-containing pharmaceutical composition is
administered using an infusion system.
[0258] The pharmaceutical compositions of the invention may further
comprise an amount of an amino acid base sufficient to decrease
aggregate formation by the polypeptide during storage of the
composition. By "amino acid base" is intended an amino acid or a
combination of amino acids, where any given amino acid is present
either in its free base form or in its salt form. Where a
combination of amino acids is used, all of the amino acids may be
present in their free base forms, all may be present in their salt
forms, or some may be present in their free base forms while others
are present in their salt forms. In some embodiments, amino acids
to use in preparing the compositions of the invention are those
carrying a charged side chain, such as arginine, lysine, aspartic
acid, and glutamic acid. Any stereoisomer (i.e., L, D, or DL
isomer) of a particular amino acid (e.g. glycine, methionine,
histidine, imidazole, arginine, lysine, isoleucine, aspartic acid,
tryptophan, threonine and mixtures thereof) or combinations of
these stereoisomers, may be present in the pharmaceutical
compositions of the invention so long as the particular amino acid
is present either in its free base form or its salt form. In some
embodiments the L-stereoisomer is used. Compositions of the
invention may also be formulated with analogues of these amino
acids. By "amino acid analogue" is intended a derivative of the
naturally occurring amino acid that brings about the desired effect
of decreasing aggregate formation by the polypeptide during storage
of the liquid pharmaceutical compositions of the invention.
Suitable arginine analogues include, for example, aminoguanidine,
ornithine and N-monoethyl L-arginine, suitable methionine analogues
include ethionine and buthionine and suitable cysteine analogues
include S-methyl-L cysteine. As with the other amino acids, the
amino acid analogues are incorporated into the compositions in
either their free base form or their salt form. In a further
embodiment of the invention the amino acids or amino acid analogues
are used in a concentration, which is sufficient to prevent or
delay aggregation of the protein.
[0259] In a further embodiment of the invention methionine (or
other sulphuric amino acids or amino acid analogous) may be added
to inhibit oxidation of methionine residues to methionine sulfoxide
when the polypeptide acting as the therapeutic agent is a
polypeptide comprising at least one methionine residue susceptible
to such oxidation. By "inhibit" is intended minimal accumulation of
methionine oxidized species over time. Inhibiting methionine
oxidation results in greater retention of the polypeptide in its
proper molecular form. Any stereoisomer of methionine (L, D, or DL
isomer) or combinations thereof can be used. The amount to be added
should be an amount sufficient to inhibit oxidation of the
methionine residues such that the amount of methionine sulfoxide is
acceptable to regulatory agencies. Typically, this means that the
composition contains no more than about 10% to about 30% methionine
sulfoxide. Generally, this can be achieved by adding methionine
such that the ratio of methionine added to methionine residues
ranges from about 1:1 to about 1000:1, such as 10:1 to about
100:1.
[0260] In a further embodiment of the invention the formulation
further comprises a stabilizer selected from the group of high
molecular weight polymers or low molecular compounds. In a further
embodiment of the invention the stabilizer is selected from
polyethylene glycol (e.g. PEG 3350), polyvinyl alcohol (PVA),
polyvinylpyrrolidone, carboxy-/hydroxycellulose or derivates
thereof (e.g. HPC, HPC-SL, HPC-L and HPMC), cyclodextrins,
sulphur-containing substances as monothioglycerol, thioglycolic
acid and 2-methylthioethanol, and different salts (e.g. sodium
chloride). Each one of these specific stabilizers constitutes an
alternative embodiment of the invention.
[0261] The pharmaceutical compositions may also comprise additional
stabilizing agents, which further enhance stability of a
therapeutically active polypeptide therein. Stabilizing agents of
particular interest to the present invention include, but are not
limited to, methionine and EDTA, which protect the polypeptide
against methionine oxidation, and a nonionic surfactant, which
protects the polypeptide against aggregation associated with
freeze-thawing or mechanical shearing.
[0262] In a further embodiment of the invention the formulation
further comprises a surfactant. In a further embodiment of the
invention the surfactant is selected from a detergent, ethoxylated
castor oil, polyglycolyzed glycerides, acetylated monoglycerides,
sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block
polymers (eg. poloxamers such as Pluronic.RTM. F68, poloxamer 188
and 407, Triton X-100), polyoxyethylene sorbitan fatty acid esters,
polyoxyethylene and polyethylene derivatives such as alkylated and
alkoxylated derivatives (tweens, e.g. Tween-20, Tween-40, Tween-80
and Brij-35), monoglycerides or ethoxylated derivatives thereof,
diglycerides or polyoxyethylene derivatives thereof, alcohols,
glycerol, lectins and phospholipids (eg. phosphatidyl serine,
phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl
inositol, diphosphatidyl glycerol and sphingomyelin), derivates of
phospholipids (eg. dipalmitoyl phosphatidic acid) and
lysophospholipids (eg. palmitoyl lysophosphatidyl-L-serine and
1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline,
serine or threonine) and alkyl, alkoxyl(alkyl ester), alkoxy(alkyl
ether)- derivatives of lysophosphatidyl and phosphatidylcholines,
e.g. lauroyl and myristoyl derivatives of lysophosphatidylcholine,
dipalmitoylphosphatidylcholine, and modifications of the polar head
group, that is cholines, ethanolamines, phosphatidic acid, serines,
threonines, glycerol, inositol, and the positively charged DODAC,
DOTMA, DCP, BISHOP, lysophosphatidylserine and
lysophosphatidylthreonine, and glycerophospholipids (eg.
cephalins), glyceroglycolipids (eg. galactopyransoide),
sphingoglycolipids (eg. ceramides, gangliosides),
dodecylphosphocholine, hen egg lysolecithin, fusidic acid
derivatives- (e.g. sodium tauro-dihydrofusidate etc.), long-chain
fatty acids and salts thereof C6-C12 (eg. oleic acid and caprylic
acid), acylcarnitines and derivatives, N.sup..alpha.-acylated
derivatives of lysine, arginine or histidine, or side-chain
acylated derivatives of lysine or arginine, N.sup..alpha.-acylated
derivatives of dipeptides comprising any combination of lysine,
arginine or histidine and a neutral or acidic amino acid,
N.sup..alpha.-acylated derivative of a tripeptide comprising any
combination of a neutral amino acid and two charged amino acids,
DSS (docusate sodium, CAS registry no [577-11-7]), docusate
calcium, CAS registry no [128-49-4]), docusate potassium, CAS
registry no [7491-09-0]), SDS (sodium dodecyl sulphate or sodium
lauryl sulphate), sodium caprylate, cholic acid or derivatives
thereof, bile acids and salts thereof and glycine or taurine
conjugates, ursodeoxycholic acid, sodium cholate, sodium
deoxycholate, sodium taurocholate, sodium glycocholate,
N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, anionic
(alkyl-aryl-sulphonates) monovalent surfactants, zwitterionic
surfactants (e.g. N-alkyl-N,N-dimethylammonio-1-propanesulfonates,
3-cholamido-1-propyldimethylammonio-1-propanesulfonate, cationic
surfactants (quaternary ammonium bases) (e.g.
cetyl-trimethylammonium bromide, cetylpyridinium chloride),
non-ionic surfactants (eg. Dodecyl .beta.-D-glucopyranoside),
poloxamines (eg. Tetronic's), which are tetrafunctional block
copolymers derived from sequential addition of propylene oxide and
ethylene oxide to ethylenediamine, or the surfactant may be
selected from the group of imidazoline derivatives, or mixtures
thereof. Each one of these specific surfactants constitutes an
alternative embodiment of the invention.
[0263] The use of a surfactant in pharmaceutical compositions is
well-known to the skilled person. For convenience reference is made
to Remington: The Science and Practice of Pharmacy, 19.sup.th
edition, 1995.
[0264] It is possible that other ingredients may be present in the
peptide pharmaceutical formulation of the present invention. Such
additional ingredients may include wetting agents, emulsifiers,
antioxidants, bulking agents, tonicity modifiers, chelating agents,
metal ions, oleaginous vehicles, proteins (e.g., human serum
albumin, gelatine or proteins) and a zwitterion (e.g., an amino
acid such as betaine, taurine, arginine, glycine, lysine and
histidine). Such additional ingredients, of course, should not
adversely affect the overall stability of the pharmaceutical
formulation of the present invention.
[0265] Pharmaceutical compositions containing a ficolin-associated
polypeptide according to the present invention may be administered
to a patient in need of such treatment at several sites, for
example, at topical sites, for example, skin and mucosal sites, at
sites which bypass absorption, for example, administration in an
artery, in a vein, in the heart, and at sites which involve
absorption, for example, administration in the skin, under the
skin, in a muscle or in the abdomen.
[0266] Topical administration may be a particular advantage in the
treatment of conditions associated with local inflammation, such as
in the treatment of inflammation associated with burn or other
conditions associated with the skin. Accordingly, in some
embodiments administration is by topical administration.
[0267] In some particular embodiments, eye droplets may be used in
conditions associated with the eye, such as keratitis, such as
diffuse lamellar keratitis (DLK).
[0268] Administration of pharmaceutical compositions according to
the invention may be through several routes of administration, for
example, lingual, sublingual, buccal, in the mouth, oral, in the
stomach and intestine, nasal, pulmonary, for example, through the
bronchioles and alveoli or a combination thereof, epidermal,
dermal, transdermal, vaginal, rectal, ocular, for examples through
the conjunctiva, uretal, and parenteral to patients in need of such
a treatment.
[0269] Compositions of the current invention may be administered in
several dosage forms, for example, as solutions, suspensions,
emulsions, microemulsions, multiple emulsion, foams, salves,
pastes, plasters, ointments, tablets, coated tablets, rinses,
capsules, for example, hard gelatine capsules and soft gelatine
capsules, suppositories, rectal capsules, drops, gels, sprays,
powder, aerosols, inhalants, eye drops, ophthalmic ointments,
ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal
ointments, injection solution, in situ transforming solutions, for
example in situ gelling, in situ setting, in situ precipitating, in
situ crystallization, infusion solution, and implants.
[0270] Compositions of the invention may further be compounded in,
or attached to, for example through covalent, hydrophobic and
electrostatic interactions, a drug carrier, drug delivery system
and advanced drug delivery system in order to further enhance
stability of the ficolin-associated polypeptide, increase
bioavailability, increase solubility, decrease adverse effects,
achieve chronotherapy well known to those skilled in the art, and
increase patient compliance or any combination thereof. Examples of
carriers, drug delivery systems and advanced drug delivery systems
include, but are not limited to, polymers, for example cellulose
and derivatives, polysaccharides, for example dextran and
derivatives, starch and derivatives, poly(vinyl alcohol), acrylate
and methacrylate polymers, polylactic and polyglycolic acid and
block co-polymers thereof, polyethylene glycols, carrier proteins,
for example albumin, gels, for example, thermogelling systems, for
example block co-polymeric systems well known to those skilled in
the art, micelles, liposomes, microspheres, nanoparticulates,
liquid crystals and dispersions thereof, L2 phase and dispersions
there of, well known to those skilled in the art of phase behaviour
in lipid-water systems, polymeric micelles, multiple emulsions,
self-emulsifying, self-microemulsifying, cyclodextrins and
derivatives thereof, and dendrimers.
[0271] Compositions of the current invention are useful in the
formulation of solids, semisolids, powder and solutions for
pulmonary administration of the ficolin-associated polypeptide,
using, for example a metered dose inhaler, dry powder inhaler and a
nebulizer, all being devices well known to those skilled in the
art.
[0272] Compositions of the current invention are specifically
useful in the formulation of controlled, sustained, protracting,
retarded, and slow release drug delivery systems. More
specifically, but not limited to, compositions are useful in
formulation of parenteral controlled release and sustained release
systems (both systems leading to a many-fold reduction in number of
administrations), well known to those skilled in the art. Even more
preferably, are controlled release and sustained release systems
administered subcutaneous. Without limiting the scope of the
invention, examples of useful controlled release system and
compositions are hydrogels, oleaginous gels, liquid crystals,
polymeric micelles, microspheres, nanoparticles,
[0273] Methods to produce controlled release systems useful for
compositions of the current invention include, but are not limited
to, crystallization, condensation, co-crystallization,
precipitation, co-precipitation, emulsification, dispersion, high
pressure homogenisation, encapsulation, spray drying,
microencapsulating, coacervation, phase separation, solvent
evaporation to produce microspheres, extrusion and supercritical
fluid processes. General reference is made to Handbook of
Pharmaceutical Controlled Release (Wise, D. L., ed. Marcel Dekker,
New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99:
Protein Formulation and Delivery (MacNally, E. J., ed. Marcel
Dekker, New York, 2000).
[0274] Parenteral administration may be performed by subcutaneous,
intramuscular, intraperitoneal or intravenous injection by means of
a syringe, optionally a pen-like syringe. Alternatively, parenteral
administration can be performed by means of an infusion pump. A
further option is a composition which may be a solution or
suspension for the administration of the ficolin-associated
polypeptide in the form of a nasal or pulmonal spray. As a still
further option, the pharmaceutical compositions containing the
ficolin-associated polypeptide of the invention can also be adapted
to transdermal administration, e.g. by needle-free injection or
from a patch, optionally an iontophoretic patch, or transmucosal,
e.g. buccal, administration.
[0275] The term "stabilized formulation" refers to a formulation
with increased physical stability, increased chemical stability or
increased physical and chemical stability.
[0276] The term "physical stability" of the protein formulation as
used herein refers to the tendency of the protein to form
biologically inactive and/or insoluble aggregates of the protein as
a result of exposure of the protein to thermo-mechanical stresses
and/or interaction with interfaces and surfaces that are
destabilizing, such as hydrophobic surfaces and interfaces.
Physical stability of the aqueous protein formulations is evaluated
by means of visual inspection and/or turbidity measurements after
exposing the formulation filled in suitable containers (e.g.
cartridges or vials) to mechanical/physical stress (e.g. agitation)
at different temperatures for various time periods. Visual
inspection of the formulations is performed in a sharp focused
light with a dark background. The turbidity of the formulation is
characterized by a visual score ranking the degree of turbidity for
instance on a scale from 0 to 3 (a formulation showing no turbidity
corresponds to a visual score 0, and a formulation showing visual
turbidity in daylight corresponds to visual score 3). A formulation
is classified physical unstable with respect to protein
aggregation, when it shows visual turbidity in daylight.
Alternatively, the turbidity of the formulation can be evaluated by
simple turbidity measurements well-known to the skilled person.
Physical stability of the aqueous protein formulations can also be
evaluated by using a spectroscopic agent or probe of the
conformational status of the protein. The probe is preferably a
small molecule that preferentially binds to a non-native conformer
of the protein. One example of a small molecular spectroscopic
probe of protein structure is Thioflavin T. Thioflavin T is a
fluorescent dye that has been widely used for the detection of
amyloid fibrils. In the presence of fibrils, and perhaps other
protein configurations as well, Thioflavin T gives rise to a new
excitation maximum at about 450 nm and enhanced emission at about
482 nm when bound to a fibril protein form. Unbound Thioflavin T is
essentially non-fluorescent at the wavelengths.
[0277] Other small molecules can be used as probes of the changes
in protein structure from native to non-native states. For instance
the "hydrophobic patch" probes that bind preferentially to exposed
hydrophobic patches of a protein. The hydrophobic patches are
generally buried within the tertiary structure of a protein in its
native state, but become exposed as a protein begins to unfold or
denature. Examples of these small molecular, spectroscopic probes
are aromatic, hydrophobic dyes, such as antrhacene, acridine,
phenanthroline or the like. Other spectroscopic probes are
metal-amino acid complexes, such as cobalt metal complexes of
hydrophobic amino acids, such as phenylalanine, leucine,
isoleucine, methionine, and valine, or the like.
[0278] The term "chemical stability" of the protein formulation as
used herein refers to chemical covalent changes in the protein
structure leading to formation of chemical degradation products
with potential less biological potency and/or potential increased
immunogenic properties compared to the native protein structure.
Various chemical degradation products can be formed depending on
the type and nature of the native protein and the environment to
which the protein is exposed. Elimination of chemical degradation
can most probably not be completely avoided and increasing amounts
of chemical degradation products is often seen during storage and
use of the protein formulation as well-known by the person skilled
in the art. Most proteins are prone to deamidation, a process in
which the side chain amide group in glutaminyl or asparaginyl
residues is hydrolysed to form a free carboxylic acid. Other
degradations pathways involves formation of high molecular weight
transformation products where two or more protein molecules are
covalently bound to each other through transamidation and/or
disulfide interactions leading to formation of covalently bound
dimer, oligomer and polymer degradation products (Stability of
Protein Pharmaceuticals, Ahern. T. J. & Manning M. C., Plenum
Press, New York 1992). Oxidation (of for instance methionine
residues) can be mentioned as another variant of chemical
degradation. The chemical stability of the protein formulation can
be evaluated by measuring the amount of the chemical degradation
products at various time-points after exposure to different
environmental conditions (the formation of degradation products can
often be accelerated by for instance increasing temperature). The
amount of each individual degradation product is often determined
by separation of the degradation products depending on molecule
size and/or charge using various chromatography techniques (e.g.
SEC-HPLC and/or RP-HPLC).
[0279] Hence, as outlined above, a "stabilized formulation" refers
to a formulation with increased physical stability, increased
chemical stability or increased physical and chemical stability. In
general, a formulation must be stable during use and storage (in
compliance with recommended use and storage conditions) until the
expiration date is reached.
[0280] In some embodiments of the invention the pharmaceutical
formulation comprising the ficolin-associated polypeptide is stable
for more than 6 weeks of usage and for more than 3 years of
storage. In other embodiments of the invention the pharmaceutical
formulation comprising the ficolin-associated polypeptide is stable
for more than 4 weeks of usage and for more than 3 years of
storage. In a further embodiment of the invention the
pharmaceutical formulation comprising the ficolin-associated
polypeptide is stable for more than 4 weeks of usage and for more
than two years of storage. In an even further embodiment of the
invention the pharmaceutical formulation comprising the
ficolin-associated polypeptide is stable for more than 2 weeks of
usage and for more than two years of storage.
Specific Embodiments of the Invention
[0281] As described above the present invention relates to isolated
ficolin-associated polypeptides as well as polypeptides comprising
the amino acid sequence of SEQ ID NO:4 or variants or immunologic
fragment thereof.
[0282] In some embodiments the polypeptide according to the present
invention is substantially pure.
[0283] In some embodiments the polypeptide according to the present
invention is capable of associating with mannose-binding lectin
(MBL).
[0284] In some embodiments the polypeptide according to the present
invention is capable of associating with any one of ficolin-1,
ficolin-2, or ficolin-3.
[0285] In some embodiments the polypeptide according to the present
invention is capable of associating with any one of C1q, lung
surfactant proteins SP-A and/or SP-D, and intracellular
collagen-like defence molecules, such as CLL-11.
[0286] In some embodiments the polypeptide according to the present
invention is capable of associating with a specific acceptor
protein, such as a specific receptor.
[0287] In some embodiments the polypeptide according to the present
invention comprises the amino acid sequence 20-297 of SEQ NO:3, or
a functional variant thereof.
[0288] In some embodiments the polypeptide according to the present
invention comprises the amino acid sequence 20-380 of SEQ NO:1 or a
functional variant thereof.
[0289] In some embodiments the polypeptide according to the present
invention comprises the amino acid sequence 16-296 of SEQ ID NO:9
or a functional variant thereof.
[0290] In some embodiments the polypeptide according to the present
invention has a molecular mass of about 40 kDa under non-reducing
conditions on an SDS-PAGE.
[0291] In some embodiments the polypeptide according to the present
invention is N-linked glycosylated at one or two amino acids
corresponding to a position selected from 49 and 178 of SEQ
NO:1.
[0292] In some embodiments the polypeptide according to the present
invention is a recombinant protein.
[0293] In some embodiments the polypeptide according to the present
invention is in homodimer form.
[0294] In some embodiments the polypeptide according to the present
invention consists of the amino acid sequence 20-380 of SEQ ID NO
1.
[0295] In some embodiments the polypeptide according to the present
invention comprises the amino acid sequence of SEQ ID NO:4 or
variants or immunologic fragments thereof.
[0296] In some embodiments the polypeptide according to the present
invention consist of SEQ ID NO:4, or variants or immunologic
fragments thereof.
[0297] In some embodiments the polypeptide according to the present
invention mediates phagocytosis of dying or dead cells, such as
apoptotic cells, and/or cellular debris.
[0298] In some embodiments the polypeptide according to the present
invention mediates phagocytosis of a microorganism.
[0299] In some embodiments the antibodies that specifically bind a
polypeptide according to the present invention is a monoclonal
antibody.
[0300] In some embodiments the antibodies that specifically bind a
polypeptide according to the present invention is a polyclonal
antibody.
[0301] In some embodiments the polypeptide according to the present
invention has activity similar to other proteins with sequence
homology, such as the engulfment adapter protein (GULP).
[0302] In some embodiments the isolated nucleic acid molecule
encoding a polypeptide according to the present invention comprises
a nucleotide sequence that is at least 70% identical to the
sequence of SEQ NO:2.
[0303] In some embodiments the host cell according the present
invention is a eukaryotic cell.
[0304] In some embodiments the host cell according the present
invention is of mammalian origin.
[0305] In some embodiments the host cell according to the present
invention is selected from the group consisting of CHO cells, HEK
cells and BHK cells.
[0306] In some embodiments the polypeptide according to the present
invention is for the treatment of any indications associated with
inflammation, apoptosis and/or autoimmunity.
[0307] In some embodiments the polypeptide according to the present
invention is for the treatment of any autoimmune conditions such as
Addison's disease, autoimmune hemolytic anemia, autoimmune
thyroiditis, Crohn's disease, Graves' disease, Guillain-Barre
syndrome, systemic lupus erythematosus (SLE), lupus nephritis,
multiple sclerosis, myasthenia gravis, psoriasis, primary biliary
cirrhosis, rheumatoid arthritis and uveitis, asthma,
atherosclerosis, Type I diabetes, psoriasis, various allergies.
[0308] In some embodiments the polypeptide according to the present
invention is for the treatment of any inflammatory disorder
selected from the group consisting of appendicitis, peptic ulcer,
gastric ulcer, duodenal ulcer, peritonitis, pancreatitis,
ulcerative colitis, pseudomembranous colitis, acute colitis,
ischemic colitis, diverticulitis, epiglottitis, achalasia,
cholangitis, cholecystitis, hepatitis, Crohn's disease, enteritis,
Whipple's disease, allergy, immune complex disease, organ ischemia,
reperfusion injury, organ necrosis, hay fever, sepsis, septicemia,
endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma,
granulomatosis, sarcoidosis, septic abortion, epididymitis,
vaginitis, prostatitis, urethritis, bronchitis, emphysema,
rhinitis, pneumonitis, pneumotransmicroscopicsilicovolcanoconiosis,
alvealitis, bronchiolitis, pharyngitis, pleurisy, sinusitis,
influenza, respiratory syncytial virus infection, HIV infection,
hepatitis B virus infection, hepatitis C virus infection,
disseminated bacteremia, Dengue fever, candidiasis, malaria,
filariasis, amebiasis, hydatid cysts, burns, dermatitis,
dermatomyositis, sunburn, urticaria, waits, wheals, vasulitis,
angiitis, endocarditis, arteritis, atherosclerosis,
thrombophlebitis, pericarditis, myocarditis, myocardial ischemia,
periarteritis nodosa, rheumatic fever, Alzheimer's disease, coeliac
disease, congestive heart failure, adult respiratory distress
syndrome, meningitis, encephalitis, multiple sclerosis, cerebral
infarction, cerebral embolism, Guillame-Barre syndrome, neuritis,
neuralgia, spinal cord injury, paralysis, uveitis, arthritides,
arthralgias, osteomyelitis, fasciitis, Paget's disease, gout,
periodontal disease, rheumatoid arthritis, synovitis, myasthenia
gravis, thyroiditis, systemic lupus erythematosis, Goodpasture's
syndrome, Behcet's syndrome, allograft rejection, graft-versus-host
disease, Type I diabetes, ankylosing spondylitis, Berger's disease,
Reiter's syndrome and Hodgkin's disease, keratitis, Type 2
diabetes, cystic fibrosis, myocardial infarction, reperfusion
injury, stroke, dermatomyositis, metabolic syndrome, systemic
inflammatory response syndrome, sepsis, multiple organ failure,
disseminated intravascular coagulation, anaphylactic shock.
Vascular complication and nephropathy associated with type 1 and/or
type 2 diabetes, meningitis, bacterial septicaemia, complicated
malaria, atypic haemolytic uremic syndrome, haemolytic uremic
syndrome, age related macular degeneration, paroxysmal nocturnal
hemoglobinuria, snake venom bite, burn injury, and complications to
organ transplantations.
[0309] In some embodiments the polypeptide according to the present
invention is for the treatment of any inflammatory disorder
selected from the group consisting of organ ischemia, reperfusion
injury, organ necrosis, vasulitis, endocarditis, atherosclerosis,
thrombophlebitis, pericarditis, myocarditis, myocardial ischemia,
periarteritis nodosa, rheumatic fever, congestive heart failure,
adult respiratory distress syndrome, cerebral infarction, cerebral
embolism. Vascular complications and nephropathy associated with
type 1 and/or type 2 diabetes.
[0310] In some embodiments the polypeptide according to the present
invention is for the treatment of any indications associated with
coagulation, thrombotic or coagulopathic related diseases.
[0311] In some embodiments the polypeptide according to the present
invention is for the treatment of an indication associated with
coagulation, thrombotic or coagulopathic related diseases or
disorders including inflammatory response and chronic
thromboembolic diseases or disorders associated with fibrin
formation including vascular disorders such as thrombosis, such as
deep venous thrombosis, arterial thrombosis, post surgical
thrombosis, coronary artery bypass graft (CABG), percutaneous
transdermal coronary angioplastry (PTCA), platelet deposition
stroke, tumor growth, tumor metastasis, angiogenesis, thrombolysis,
atherosclerosis, restenosis, such as arteriosclerosis and/or
restenosis following angioplastry, acute and chronic indications
such as inflammation, sepsis, septic chock, septicemia,
hypotension, adult respiratory distress syndrome (ARDS), systemic
inflammatory response syndrome (SIRS), disseminated intravascular
coagulopathy (DIC), pulmonary embolism, pathological platelet
deposition, myocardial infarction, or the prophylactic treatment of
mammals with atherosclerotic vessels at risk for thrombosis,
venoocclusive disease following peripheral blood progenitor cell
(PBPC) transplantation, hemolytic uremic syndrome (HUS), and
thrombotic thrombocytopenic purpura (TTP) and rheumatic fever.
[0312] In some embodiments the polypeptide according to the present
invention is for the treatment of an indication associated with
coagulation, thrombotic or coagulopathic related diseases or
disorders including inflammatory response and chronic
thromboembolic diseases or disorders associated with fibrin
formation including vascular disorders such as thrombosis, such as
deep venous thrombosis, arterial thrombosis, post surgical
thrombosis, coronary artery bypass graft (CABG), percutaneous
transdermal coronary angioplastry (PTCA), platelet deposition
stroke, tumor growth, tumor metastasis, angiogenesis, thrombolysis,
atherosclerosis, restenosis, such as arteriosclerosis and/or
restenosis following angioplastry, acute and chronic indications
such as inflammation, pathological platelet deposition, myocardial
infarction, or the prophylactic treatment of mammals with
atherosclerotic vessels at risk for thrombosis, venoocclusive
disease following peripheral blood progenitor cell (PBPC)
transplantation, hemolytic uremic syndrome (HUS), and thrombotic
thrombocytopenic purpura (UP) and rheumatic fever.
[0313] In some embodiments the polypeptide according to the present
invention is for preventing the occurrence of thromboembolic
complications in identified high risk patients, such as those
undergoing surgery or those with congestive heart failure.
[0314] In some embodiments the polypeptide according to the present
invention is for the treatment of a medical condition associated
with the heart.
[0315] In some embodiments the polypeptide according to the present
invention is for the treatment of a medical condition associated
with a deficiency in a ficolin-associated polypeptide.
EXAMPLE 1
[0316] Detection of Alternative Transcription of the MASP1 Gene
[0317] Methods: In order to detect the three transcript variants of
MASP1: MASP1, MASP3 and FAP, specific primers for each variant were
design. PCR was set up with a common forward primer in exon 6
(5'-gcacccagagccacagtg-3') and specific reverse primers: MASP1 in
exon 12 (5'-gccttccagtgtgtgggc-3'), MASP3 in exon 11
(5-gccttccagagtgtggtca-3') and FAP in exon 8a
(5'-cgatctggagagcgaactc-3') (FIG. 1). PCR amplifications were
carried out in 20-.mu.l volumes containing: 50 ng liver cDNA
(Clontech), 0.25 .mu.M of each primer, 2.5 mM MgCl.sub.2, 0.2 mM
dNTP, 50 mM KCl, 10 mM Tris.HCl, pH 8.4, and 0.4 units of Platinum
Taq DNA polymerase (Invitrogen). The PCR reactions were performed
at the following cycling parameters: 10 min 94.degree. C., 30 or 40
cycles (30 sec 94.degree. C., 50 sec 58.degree. C., 90 sec
72.degree. C.), 10 min 72.degree. C. Samples were analysed on 2%
agarose gels.
[0318] Results: Alternative transcription of the MASP1 gene was
detected in liver cDNA. The MASP1, MASP3, and FAP transcripts were
amplified using a common forward primer located in exon 6 and
specific reverse primers located in exon 12 (MASP1), exon 11
(MASP3), and exon 8a (FAP). MASP1 generates a fragment of 500 bp,
MASP3 generates a fragment of 506 by and FAP generates a fragment
of 309 bp.
[0319] Tissue Expression of the FAP Fragment
[0320] Methods: Commercially available human tissue cDNA panels
(Clontech) were investigated for MASP1, MASP3, and FAP expression
with the same PCR assays as described above. Samples were analysed
on 2% agarose gels.
[0321] Results: The tissue distributions of the MASP1, MASP3, and
FAP genes were investigated in cDNA panels from Clontech (FIG. 2).
MASP1, MASP3, and FAP transcripts were amplified using a common
forward primer and specific reverse primers. GADPH was used as
reference gene. All three genes were highly expressed in the liver,
and additionally, FAP was strongly expressed in heart tissue
(marked with black arrows). Minor expression of the FAP gene was
detected in brain, colon, prostate, skeletal muscle, and small
intestine (marked with white arrows).
[0322] DNA Sequencing of the FAPexon8a of 100 Individuals.
[0323] Methods: Direct sequencing of the exon 8a including the
intron-exon boundary of the MASP1/MASP3/FAP gene spanning from
position +44,083 to +44,431 relative to the translation ATG start
site, was performed on genomic DNA templates from 100 healthy
Caucasian individuals. The fragment was amplified by using a single
primer set (forward: 5'-ctgttcttcacactggctg-3', reverse:
5'-ctgctgagatcatgttgttc-3'), where the forward primers contained a
5'-T7 sequence (5'-ttatacgactcacta-3'). PCR amplifications were
carried out in 20-.mu.l volumes containing: 50 ng genomic DNA, 0.25
.mu.M of each primer, 2.5 mM MgCl.sub.2, 0.2 mM dNTP, 50 mM KCl, 10
mM Tris.HCl, pH 8.4, and 0.4 units of Platinum Taq DNA polymerase
(Invitrogen). The PCR reactions were performed at the following
cycling parameters: 2 min 94.degree. C., 15 cycles (30 sec
94.degree. C., 60 sec 64.degree. C., 60 sec 72.degree. C.), 15
cycles (30 sec 94.degree. C., 60 sec 58.degree. C., 60 sec
72.degree. C.), 5 min 72.degree. C. and were sequenced in the
forward direction using the ABI BigDye cycle sequencing terminator
kit (Applied Biosystems, Foster City, Calif.) according to the
protocol using 5'-biotinylated sequence primers. Sequence reactions
were purified on the PyroMark Vacuum Prep Workstation (Biotage)
using streptavidin beads (GenoVision). Sequence analysis was
performed on an ABI Prism 3100 Genetic Analyser (Applied
Biosystems). The resulting DNA sequences were aligned using BioEdit
software, and DNA polymorphisms were confirmed visually from
sequence electropherograms.
[0324] Results: All sequences were aligned using BioEdit software.
No genetic variations in the 100 healthy individuals were observed
in the exon 8a or the exon-intron regions.
EXAMPLE 2
[0325] Immunoprecipitation.
[0326] Specific immunoprecipitation of MAP-1 from serum was
performed with the MAP-1 specific mAb 20C4 (raised against the 17
MAP-1 specific C-terminal peptide) or mAb 8B3, a monoclonal
antibody reacting against the common heavy chain of MASP-1/3 used
as control precipitation antibody. A total of 10 .mu.g of anti
MAP-1 or MASP-1/3 antibody was allowed to bind to sheep anti mouse
or rabbit IgG Dynabeads (M-280, cat. 112.02D/112.04D,
Dynal/Invitrogen). After a washing step the beads were applied to a
pool of normal human serum (diluted 1:1 in TBS) and incubated end
over end for 1 hour at 4.degree. C. After final washing steps and
magnetic separation the beads were boiled in SDS loading buffer and
subjected to SDS-PAGE and western blotting probed with antibodies
to MAP-1, MBL, and Ficolin-3.
[0327] The same precipitation procedure as described above was
performed with mAbs to MBL (Hyb 131-11, Bioporto, Denmark),
Ficolin-2 (FCN219) and Ficolin-3 (FCN334). To compensate for
differences in serum concentrations of MBL, Ficolin-2 and -3 were
precipitated from 1 ml, 300 .mu.l and 100 .mu.l serum,
respectively. Samples were analyzed by SDS-PAGE and western
blotting probed with pAb against MAP-1.
[0328] Immunohistochemistry.
[0329] CHO cells expressing rMAP-1 were grown in culture flasks in
RPMI+10%. Cells were harvested at 80-90% confluence the cells were
harvested and fixed for 24 h in 4% formaldehyde-PBS and
subsequently embedded in paraffin. Six different human liver
tissues and samples from two different myocardial tissues, two
skeleton muscle tissues and two samples obtained from human aorta
were also fixed and paraffin embedded as described above. Sections
of 5 .mu.m slices were obtained with a Leitz Wetzlar microtome and
placed on glass slides and stored at 4.degree. C. until assayed.
Pre-treatments and analyses were performed as described previously.
Primary antibodies were the MAP-1 specific monoclonal antibodies
mAb 12B11 or affinity purified, monospecific rabbit anti-MAP-1 all
diluted to 5 .mu.g/ml. Isotype antibody controls were applied to
the tissues at the same concentration. Secondary antibody was
EnVision.TM. antibody (HRP-anti mouse or HRP-anti rabbit, Dako,
Glostrup, Denmark). Analysis of staining patterns was conducted
under a Leica DMLB2 microscope.
[0330] SDS-PAGE and Western Blotting.
[0331] Electrophoresis was performed on 10% or 4-12% (w/v) Bis-Tris
Polyacrylamide-gels with discontinuous buffers using the
NuPAGE.RTM. system (Invitrogen) essentially as described by the
manufacturer. Western blotting was performed using polyvinylidene
difluoride membranes (PVDF-HyBond, Amersham Bioscience), 2 .mu.g/ml
of primary mAbs and secondary visualization by HRP conjugated
streptavidin (P0397, Dako) diluted to 1:1500 or HRP-Rabbit anti
mouse IgG (PO260, Dako) diluted to 1:1000 in PBS, 0.05% Tween20.
The membranes were developed with 3-amino-9-ethylcarbazole (Sigma)
(0.04% in acetone) and 0.015% H.sub.2O.sub.2 in 50 mM sodium
acetate buffer pH 5.
[0332] Complement Activation Assay.
[0333] The influence of MAP-1 on the MBL and Ficolin-3 mediated
complement factor C4 deposition was assessed essentially as
described previously. Briefly, mannan (MBL ligand) (Sigma-Aldrich
M7504) or acetylated bovine serum albumin (Ficolin-3 ligand) was
immobilized to Maxisorp ELISA plates (Nunc, Denmark) at 10
.mu.g/ml. After washing with, rMBL or rFicolin-3 (0.4 .mu.g/ml) was
added and incubated for 1.5 hour. rMAP-1 or rMASP-2 was applied for
1 hour in two-fold serial dilutions in the first dimension followed
by incubation for 45 min at 37.degree. C. with serial dilutions of
serum deficient of MBL or Ficolin-3 in the second dimension. The C4
deposition was measured using a pAb to C4c (Q0369, Dako,
Glostrup/Denmark).
[0334] In addition we assessed the displacement of MASP-2 with
MAP-1 using a pure system. rMASP-2 was pre-incubated for 45 min at
20.degree. C. in serial dilutions in the first dimension on an
rMBL/mannan matrix as described above followed by incubation with
dilutions of rMAP-1 in the second dimension for 45 min at
20.degree. C. Purified C4 (from Quidel, Calif., USA) was added at a
concentration of 1 .mu.g/ml and incubated for 45 min at 37.degree.
C. Detection was conducted as above.
[0335] Results.
[0336] MAP-1 Co-Precipitates with Ficolin-2, Ficolin-3 and MBL
[0337] To investigate a possible association of MAP-1 with MBL and
Ficolin-3 we precipitated serum complexes using both anti MAP-1
mAb20C4 and a mAb against the common heavy chain of MASP-1 and
MASP-3 (mAb8B3). The precipitates were subsequently analyzed by
western blotting probed with antibodies to MAP-1, MBL, and
Ficolin-3, respectively. We observed pronounced Ficolin-3
co-precipitation bands, but weaker bands were also seen with MBL
(FIG. 24A). The samples wee not probed with antibodies against
Ficolin-2 since they did not work in western blot. We then reversed
the immunoprecipitation using mAbs against MBL, Ficolin-2 and
Ficolin-3 to precipitate 1 ml, 300 .mu.l and 100 .mu.l serum,
respectively, which was perform to adjust for differences in the
serum concentration of MBL (2 .mu.g/ml), Ficolin-2 (5 .mu.g/ml) and
Ficolin-3 (20 .mu.g/ml), respectively. The samples were
subsequently analyzed by western blotting probed with antibodies to
MAP-1. Distinct MAP-1 bands were observed in the precipitates from
Ficolin-2 and -3 and a much weaker band was apparent in the MBL
precipitate, where immunoprecipitated rMAP-1 and serum MAP-1 served
as controls (FIG. 24B).
[0338] MAP-1 Inhibits Complement Activity of the Lectin
Pathway.
[0339] Serum deficient of MBL and Ficolin-3 in combination with
rMBL and rFicolin-3 were used to reconstitute for MBL and Ficolin-3
complement C4 activation activity. Mannan and acetylated BSA served
as ligands for MBL and Ficolin-3, respectively. Both rMBL and
rFicolin-3 were able to initiate C4 deposition in MBL and Ficolin-3
deficient sera, respectively (FIGS. 25A and 25D). Application of
rMASP-2 resulted in a strong positive dose dependent enhancement of
the C4 deposition via both the Ficolin-3 and MBL activation
pathways (FIGS. 25B and 25E), whereas application of rMAP-1
resulted in a pronounced dose dependent inhibition of the C4
deposition via both pathways (FIGS. 25C and 25F).
[0340] In addition we addressed a possible displacement of MASP-2
with MAP-1 using a system of pure components comprising only of
rMBL, rMASP-2, rMAP-1 and purified C4. rMASP-2 was pre-incubated
with mannan/rMBL complexes in serial dilutions. Thereafter, rMAP-1
was added in varying concentrations followed by addition of
purified C4. Application of rMAP-1 to the system clearly resulted
in a dose dependent inhibition of C4 deposition (FIG. 26).
EXAMPLE 3
[0341] Determining serum concentration and association properties
of the novel MBL/Ficolin associated protein 1 (MAP-1).
[0342] A full-length non-tagged recombinant constructs of MAP-1 was
generated and stably expressed in CHO-DG44 cells. Specific
monoclonal antibodies against MAP-1 were raised. Also a
quantitative ELISA for MAP-1 serum measurements was established and
the associations between serum MAP-1 and Ficolin-2, -3 and MBL was
examined by ELISA and density gradient fractionation.
[0343] Recombinant Proteins
[0344] Full length constructs of non-tagged human MAP-1 was
expressed in CHO-DG44 cells as described elsewhere (Hummelshoj et
al., Mol Immunol 44, 401-11, 2007; Larsen et al., J Biol Chem 279,
21302-11, 2004; Ma et al., 2009 J Biol Chem, October 9; 284(41))
with the modifications that PowerCHO1 serum-free medium (Lonza,
Vallensbaek/Denmark, www.lonza.com) was used as the expression
medium. We used antibody affinity purification to purify rMAP-1 as
described previously (Skjoedt et al., 2009; Immunobiology, November
23). In brief 15 mg of the anti MAP-1 antibody (mAb 20C4) was
covalently coupled to CNBr activated sepharose essentially as
described by Pfeiffer et al. (Pfeiffer et al., J Immunol Methods
97, 1-9, 1987) and used as the purification matrix. The anti-MAP-1
column was also used to deplete MAP-1 from serum.
[0345] The generation of monoclonal antibodies was done as
described previously (Skjoedt et al., J Biol Chem 285, 8234-43,
2010).
[0346] Electrophoresis was performed on 10% or 4-12% (w/v) Bis-Tris
Polyacrylamide-gels with discontinuous buffers using the
NuPAGE.RTM. system (Invitrogen) as recommended. Western blotting
was performed using polyvinylidene difluoride membranes
(PVDF-HyBond, GE Healthcare). The membranes were developed using 2
.mu.g/ml of primary mAbs and secondary visualization by HRP
conjugated streptavidin diluted to 1:1500 or HRP-Rabbit anti mouse
IgG (P0397/P0260, Dako, Glostrup/Denmark, www.dako.com) with 0.04%
3-amino-9-ethylcarbazole (Sigma-Aldrich, Broendby/Denmark,
www.sigmaaldrich.com) +0.015% H2O2 in 50 mM sodium acetate buffer
pH5 as substrate.
[0347] rMAP-1 was treated with N-glycosidase-F/ENDO-F
(N-glycosidase-F deglycosylation kit, Roche, Mannheim/Germany,
www.roche.com) as recommended and described previously (Skjoedt et
al., 2009). Products were analyzed by SDS-PAGE under reducing
conditions followed by Coomassie staining or western blotting.
[0348] The specificity of the anti-MAP-1 mAb 20C4 has previously
been demonstrated (Skjoedt et al., 2010). The mAb 20C4 was used as
the catching antibody in a quantitative MAP-1 ELISA immobilized at
6 .mu.g/ml to Maxisorb ELISA plates (NUNC.TM., Roskilde/Denmark,
www.nuncbrand.com). Serial dilutions of the calibrator (rMAP-1 or
rMAP-1 spiked in MAP-1 depleted serum) or donor serum samples were
applied in PBS+0.05% Tween20+0.5% bovine serum and 10 mM EDTA.
Detection antibody was biotin labeled mAb 8B3 reacting with the
common chain of MASP-1, -3 and MAP-1 described previously (Skjoedt
et al., 2010; Skjoedt et al., 2009) applied at 3 .mu.g/ml.
[0349] The Ficolin-2 and -3 serum concentrations were determined as
described by Munthe-Fog et al. and Hummelshoj et al. (Hummelshoj et
al., Hum Mol Genet 14, 1651-8, 2005; Munthe-Fog et al., Scand J
Immunol 65, 383-92, 2007; Munthe-Fog et al., Mol Immunol 45,
2660-6, 2008) and the MBL and MASP-3 serum concentrations were
determined as described previously (Skjoedt et al., 2009).
[0350] Development was obtained with Ortho-phenylene-diamine (Dako,
Glostrup/Denmark) and the enzyme reaction was stopped with 1M H2SO4
as recommended. Optical density (OD490 nm-650 nm) levels were
measured using a V-max Kinetic-reader (Molecular Devices,
Sunnyvale/Calif./U.S).
[0351] The relative association between MAP-1 and MBL, Ficolin-2
and -3 was assessed essentially as described previously (Skjoedt et
al., 2009) with the modification that the MAP-1 specific mAb 20C4
was used as capture antibody (coated at 6 .mu.g/ml). Detection mAbs
were biotin-labeled FCN-219 (Ficolin-2 specific) or FCN-334
(Ficolin-3 specific) (24-25), or Hyb 131-11 all applied at 2
.mu.g/ml. The serum samples from the same 100 Danish blood donors
as above were analyzed.
[0352] Normal human serum was subjected to sucrose gradient
separation. 0.75 ml serum was loaded onto 40 ml centrifugation
columns consisting of 10-30% sucrose gradients buffered in 10 mM
Tris, 145 mM NaCl, 3 mM CaCl.sub.2 and human serum albumin at 30
.mu.g/ml. The loaded columns were centrifuged at 150.000.times.g in
vacuum for 24 hours at 4.degree. C. in a L70 Beckmann
ultracentrifuge with a SW28 rotor head. 1.5 ml fractions were
collected from the bottom and analyzed by specific ELISA or
immunoblotting for the following antigens: MAP-1, MASP-1, MASP-2,
MASP-3, sMAP, MBL, Ficolin-2 and Ficolin-3. The peaks of the serum
IgM (19S) and IgG (7S) were also assessed indicating the molecular
surface to mass ratio. Additionally the fractions were analyzed for
the capacity to activate exogenously applied C4. Briefly, the
fractions were applied in serial dilutions to ELISA plates coated
with acetylated BSA (a Ficolin-3 ligand) or mannan (an MBL ligand)
as described previously (Skjoedt et al., 2010) followed by
incubation for 1 hour at 4.degree. C. with shaking. The plates were
then washed and incubated with purified C4 at 1 .mu.g/ml for 1 hour
at 37.degree. C. The C4 deposition was subsequently measured with
polyclonal antibodies to C4c (Q 0369, Dako, Glostrup, Denmark).
[0353] Statistical Analysis
[0354] Statistics (Spearman non-parametric correlation,
non-parametric two-tailed t-test) and MAP-1, MBL, Ficolin-2 and -3
serum levels were calculated using Prism4 software (GraphPad
Software, Inc., La Jolla/Calif./US, www.graphpad.com
[0355] Results
[0356] Purification and Characterization of rMAP-1
[0357] Expression of rMAP-1 in CHO DG44 cells resulted in a high
yield in presence of 150 nM methotrexate (yield: 10-20 .mu.g/ml in
serum free medium). After purification rMAP-1 was analyzed in
SDS-PAGE followed by Coomassie brilliant blue staining or
immunoblotting. The SDS-PAGE/coomassie staining analysis revealed a
band with an estimated reduced molecular mass of .about.45 kDa
(FIG. 27). Deglycosylation of rMAP-1 with N-glycosidase F resulted
in a shift in molecular mass to .about.40 kDa corresponding to the
theoretical mass without signal peptide. This pattern was also
observed with immunblotting using specific antibodies to MAP-1.
[0358] MAP-1 Serum Levels
[0359] We developed a quantitative ELISA to determine the serum
level of MAP-1. The assay was based on the MAP-1 specific mAb 20C4
as capture antibody and a detection antibody (mAb 8B3) that
recognizes the common heavy chain of MASP-1, -3 and MAP-1. Perfect
parallelism was observed between the purified rMAP-1 calibrator and
MAP-1 depleted serum spiked purified MAP-1 at a known concentration
with standard curve (FIG. 28A). We analyzed the serum level of
MAP-1 in 100 Danish blood donors and found a mean of 240 ng/ml with
a range of 115-466 ng/ml (FIG. 29A). We measured the MASP-3 serum
level in the same group as described previously (Skjoedt et al.,
2009) and plotted the MAP-1 and MASP-3 concentration (FIG. 29B). We
found no correlation between the serum concentration of MAP-1 and
MASP-3 although they represent alternative transcripts from the
same gene.
[0360] We assessed the antigen and assay stability in serum and
during freeze-thaw cycles (FIG. 29C). We observed that the
assessment of MAP-1 was very robust regardless of freeze-thaw
cycles.
[0361] Association between MAP-1 and Ficolin-2, -3 and MBL
[0362] In order to measure the interactions between MAP-1 and MBL,
Ficolin-2 and -3, we developed three different ELISAs using mAb
20C4 as capture antibody and probing with biotin labeled mAbs:
FCN-219 (Ficolin-2 specific), FCN-334 (Ficolin-3 specific) or Hyb
131-11 (MBL specific). We analyzed the same 100 donor serum samples
as used for the MAP-1 determinations and assessed the serum
association levels between MAP-1 and Ficolin-2, -3 and MBL given as
relative O.D. 490-650 nm (FIG. 30A). In addition we measured the
serum concentration of MBL, Ficolin-2 and -3 as previously (Skjoedt
et al., 2009).
[0363] We found that MAP-1 exists in complex with MBL, Ficolin-2
and -3. It appears, however, that the major part of MAP-1 is
associated to the ficolins and especially Ficolin-3 (p<0.0001) a
pattern that has also been observed previously for MASP-3 (Skjoedt
et al., 2009).
[0364] We plotted the serum concentrations of MAP-1, MBL, Ficolin-2
and -3 to the relative association levels and found that the
association between MAP-1 and MBL is highly correlated to the MBL
level (Spearman r: 0.92, p<0.0001) (FIG. 30B, top right hand
side). In contrast to this the relative MAP-1 association to
Ficolin-2 and -3 correlates to the serum level of MAP-1 (Spearman
r: 0.45 and 0.61, respectively, p<0.0001, FIG. 30B left hand
side). Although we observed a certain correlation between the MAP-1
concentration and relative association to MBL and the Ficolin-3
concentration to the, the tendencies were less pronounced.
[0365] Density Gradient Fractionation
[0366] In order to investigate the distribution of MAP-1 in
relation to associated molecules and to examine how much appears
non-associated we subjected normal human serum to density
fractionation using a 10-30% sucrose gradient and
ultracentrifugation. Subsequently the collected fractions were
analyzed for MAP-1, MASP-3, MBL, Ficolin-2 and -3 by ELISA (FIG.
31A) and MAP-1, MASP-1, -2 and -3, sMAP, MBL, Ficolin-2 and -3 by
western blotting (FIG. 31B). The results showed that serum MAP-1
was only present in the fractions with the ficolins and MBL
suggesting that MAP-1 does not exist as a non-associated molecule.
The same pattern was observed for sMAP, MASP-1, -2 and -3.
Additionally the data indicate that the majority of MAP-1, sMAP and
MASP-1, -2 and -3 co-localize in the peak fractions of Ficolin-3.
This distribution was also analyzed by size exclusion
chromatography on a sephadex-200 column. An equivalent distribution
pattern of the molecules was observed (data not shown).
[0367] Finally we assessed the capacity of the sucrose gradient
fractions to activate exogenously applied C4. Solid phase mannan
and acetylated BSA were used as ligands for MBL and Ficolin-3,
respectively. We observed two different C4 deposition curves
reflecting the peaks of Ficolin-3 and MBL complexes separated by
the sucrose gradient (FIG. 31C).
[0368] Discussion
[0369] To investigate structural aspects and to establish the serum
level of the novel MBL/Ficolin associated protein 1 (MAP-1), we
expressed non-tagged, recombinant MAP-1 and generated specific
antibodies against it. N-glycosidase F treatment and SDS-PAGE
analysis indicated that MAP-1 is glycosylated resulting in a
molecular mass of .about.45 kDa with N-glycans and .about.40 kDa
after deglycosylation equivalent to the calculated molecular mass
from the deduced amino acid sequence without the signal
peptide.
[0370] We used a monoclonal antibody generated against the MAP-1
specific C-terminal end to establish a quantitative MAP-1 ELISA and
to determine the serum concentration range in 100 healthy Danish
blood donors. We found a relatively low serum concentration (mean:
240 ng/ml, range 115-466 ng/ml) in the donor group compared to the
MASP-3 concentration (mean: 6500 ng/ml). Additionally there was no
correlation between the serum concentrations of the two proteins
suggesting that although the two molecules are differentially
spiced variants of the same gene the regulation of the expression
is different. Recently, a significant difference in the tissue
distribution of MASP-1, -3 and MAP-1 was described (Degn et al.,
2009; Skjoedt et al., 2010). The finding of a major difference in
the serum concentration between MASP-3 and MAP-1 further supports
the notion of a differential regulatory mechanism of the
transcripts variants derived from the MASP1 gene.
[0371] We developed ELISA based assays to assess the relative
association between serum MAP-1 and MBL, Ficolin-2 and -3,
respectively. Additionally we determined the serum concentration of
Ficolin-2, -3 and MBL in order to relate them to the relative
association levels. The results show that MAP-1 is associated
primarily to Ficolin-3 and Ficolin-2 and that the relative
association to MBL appears less pronounced. It could be argued that
this distribution reflects the difference in the mean serum
concentration of MBL, Ficolin-2 and -3. However, although the
MBL-MAP-1 association correlates to the MBL concentration the same
is not evident for Ficolin-2 where the MAP-1 serum concentration
correlates to the association level with Ficolin-2. The relative
association between Ficolin-3 and MAP-1 was highly correlated with
the MAP-1 serum concentration, while a positive correlation to the
Ficolin-3 serum level was very weak. The above findings indicate
that the major association between MAP-1 and Ficolin-2 and -3 is
not simply due to the general higher concentration of Ficolin-2 and
-3. This distribution pattern was further substantiated by analysis
of serum subjected to density gradient separation. We found a clear
tendency that not only MAP-1, but also sMAP, MASP-1, -2 and -3
co-localized with the Ficolin-3 peak fractions. This is a
phenomenon that we have observed previously for MASP-3 (Skjoedt et
al., 2009). The separation of the Ficolin-3 and MBL peak fractions
was also assessed by the capacity to activate exogenously added C4
on acetylated BSA (a Ficolin-3 ligand) and mannan (an MBL ligand).
The C4 deposition on the two different activation surfaces clearly
illustrated the different peak fractions containing MBL or
Ficolin-3 complexes.
[0372] The data from the sucrose gradient density analysis also
indicated that the surface to mass ratio is higher for MBL than for
Ficolin-2 and Ficolin-3, which supports the observations from a
recent study suggesting that MBL has a very loose and open
conformation in the quaternary structure (Jensenius et al., 2009).
However the smaller surface to mass ratio of the ficolins could
also reflect the molecular distribution with associated molecules
such as MAP-1, sMAP and the MASPs. In this respect being more
associated to MAP-1/sMAP/MASPs would result in a higher mass and a
further migration through the density gradient.
[0373] In conclusion, we have shown that MAP-1 is present in low
serum concentrations compared to MASP-3 and that MAP-1 and
circulates in complex predominantly with the ficolins but also to
some degree with MBL. Furthermore we could demonstrate that
Ficolin-3 appears to be the main MAP-1 associated molecule among
the LCP recognition molecules.
TABLE-US-00001 The complete 380 amino acid sequences for human FAP.
(Two potential glycosylation sites identified at amino acid
position 49 and 178 are highlighted). SEQ ID NO: 1
MRWLLLYYALCFSLSKASAHTVELNNMFGQIQSPGYPDSYPSDSEVTWNITVPDGFRIKLYFMHFNLESSYLCE-
YDYVKV 80
ETEDQVLATFCGRETTDTEQTPGQEVVLSPGSFMSITFRSDFSNEERFTGFDAHYMAVDVDECKEREDEELSCD-
HYCHNY 160
IGGYYCSCRFGYILHTDNRTCRVECSDNLFTQRTGVITSPDFPNPYPKSSECLYTIELEEGFMVNLQFEDIFDI-
EDHPEV 240
PCPYDYIKIKVGPKVLGPFCGEKAPEPISTQSHSVLILFHSDNSGENRGWRLSYRAAGNECPELQPPVHGKIEP-
SQAKYF 320
FKDQVLVSCDTGYKVLKDNVEMDTFQIECLKDGTWSNKIPTCKKNEIDLESELKSEQVTE. The
complete cDNA nucleotide sequences for human FAP. SEQ ID NO: 2
atgaggtggctgcttctctattatgctctgtgcttctccctgtcaaaggcttcagcccacaccgtggagctaaa-
caata
tgtttggccagatccagtcgcctggttatccagactcctatcccagtgattcagaggtgacttggaatatcact-
gtccc
agatgggtttcggatcaagctttacttcatgcacttcaacttggaatcctcctacctttgtgaatatgactatg-
tgaag
gtagaaactgaggaccaggtgctggcaaccttctgtggcagggagaccacagacacagagcagactcccggcca-
ggagg
tggtcctctcccctggctccttcatgtccatcactttccggtcagatttctccaatgaggagcgtttcacaggc-
tttga
tgcccactacatggctgtggatgtggacgagtgcaaggagagggaggacgaggagctgtcctgtgaccactact-
gccac
aactacattggcggctactactgctcctgccgcttcggctacatcctccacacagacaacaggacctgccgagt-
ggagt
gcagtgacaacctcttcactcaaaggactggggtgatcaccagccctgacttcccaaacccttaccccaagagc-
tctga
atgcctgtataccatcgagctggaggagggtttcatggtcaacctgcagtttgaggacatatttgacattgagg-
accat
cctgaggtgccctgcccctatgactacatcaagatcaaagttggtccaaaagttttggggcctttctgtggaga-
gaaag
ccccagaacccatcagcacccagagccacagtgtcctgatcctgttccatagtgacaactcgggagagaaccgg-
ggctg
gaggctctcatacagggctgcaggaaatgagtgcccagagctacagcctcctgtccatgggaaaatcgagccct-
cccaa
gccaagtatttcttcaaagaccaagtgctcgtcagctgtgacacaggctacaaagtgctgaaggataatgtgga-
gatgg
acacattccagattgagtgtctgaaggatgggacgtggagtaacaagattcccacctgtaaaaaaaatgaaatc-
gatct ggagagcgaactcaagtcagagcaagtgacagagtga. Minimum sequence of a
ficolin-associated polypeptide comprising the CUB1-EGF- CUB2
domains including a signal peptide of amino acids 1-19. The
sequence corresponds to exon 2 to exon 6. SEQ NO: 3
MRWLLLYYALCFSLSKASAHTVELNNMFGQIQSPGYPDSYPSDSEVTWNITVPDGFRIKLYFMHFNLESSYLCE-
YDYVKV 80
ETEDQVLATFCGRETTDTEQTPGQEVVLSPGSFMSITFRSDFSNEERFTGFDAHYMAVDVDECKEREDEELSCD-
HYCHNY 160
IGGYYCSCRFGYILHTDNRTCRVECSDNLFTQRTGVITSPDFPNPYPKSSECLYTIELEEGFMVNLQFEDIFDI-
EDHPEV 240
PCPYDYIKIKVGPKVLGPFCGEKAPEPISTQSHSVLILFHSDNSGENRGWRLSYRAA. Unique
terminal 17 amino acids of FAP SEQ ID NO: 4 KNEIDLESELKSEQVTE.
Protein sequence of human MASP-1. SEQ ID NO: 5
MRWLLLYYALCFSLSKASAHTVELNNMFGQIQSPGYPDSYPSDSEVTWNITVPDGFRIKLYFMHFNLESSYL
CEYDYVKVETEDQVLATFCGRETTDTEQTPGQEVVLSPGSFMSITFRSDFSNEERFTGFDAHYMAVDVDEC
KEREDEELSCDHYCHNYIGGYYCSCRFGYILHTDNRTCRVECSDNLFTQRTGVITSPDFPNPYPKSSECLYTI
ELEEGFMVNLQFEDIFDIEDHPEVPCPYDYIKIKVGPKVLGPFCGEKAPEPISTQSHSVLILFHSDNSGENRG
WRLSYRAAGNECPELQPPVHGKIEPSQAKYFFKDQVLVSCDTGYKVLKDNVEMDTFQIECLKDGTWSNKIP
TCKIVDCRAPGELEHGLITFSTRNNLTTYKSEIKYSCQEPYYKMLNNNTGIYTCSAQGVWMNKVLGRSLPTC
LPVCGLPKFSRKLMARIFNGRPAQKGTTPWIAMLSHLNGQPFCGGSLLGSSWIVTAAHCLHQSLDPEDPTLR
DSDLLSPSDFKIILGKHWRLRSDENEQHLGVKHTTLHPQYDPNTFENDVALVELLESPVLNAFVMPICLPEGP
QQEGAMVIVSGWGKQFLQRFPETLMEIEIPIVDHSTCQKAYAPLKKKVTRDMICAGEKEGGKDACAGDSGG
PMVTLNRERGQWYLVGTVSWGDDCGKKDRYGVYSYIHHNKDWIQRVTGVRN cDNA sequence
of human MASP-1 SEQ ID NO: 6
GAAGTCAGCCACACAGGATAAAGGAGGGAAGGGAAGGAGCAGATCTTTTCGGTAGGAAGACAGATTTTGT
TGTCAGGTTCCTGGGAGTGCAAGAGCAAGTCAAAGGAGAGAGAGAGGAGAGAGGAAAAGCCAGAGGGAGA
GAGGGGGAGAGGGGATCTGTTGCAGGCAGGGGAAGGCGTGACCTGAATGGAGAATGCCAGCCAATTCCAG
AGACACACAGGGACCTCAGAACAAAGATAAGGCATCACGGACACCACACCGGGCACGAGCTCACAGGCAA
GTCAAGCTGGGAGGACCAAGGCCGGGCAGCCGGGAGCACCCAAGGCAGGAAAATGAGGTGGCTGCTTCTC
TATTATGCTCTGTGCTTCTCCCTGTCAAAGGCTTCAGCCCACACCGTGGAGCTAAACAATATGTTTGGCC
AGATCCAGTCGCCTGGTTATCCAGACTCCTATCCCAGTGATTCAGAGGTGACTTGGAATATCACTGTCCC
AGATGGGTTTCGGATCAAGCTTTACTTCATGCACTTCAACTTGGAATCCTCCTACCTTTGTGAATATGAC
TATGTGAAGGTAGAAACTGAGGACCAGGTGCTGGCAACCTTCTGTGGCAGGGAGACCACAGACACAGAGC
AGACTCCCGGCCAGGAGGTGGTCCTCTCCCCTGGCTCCTTCATGTCCATCACTTTCCGGTCAGATTTCTC
CAATGAGGAGCGTTTCACAGGCTTTGATGCCCACTACATGGCTGTGGATGTGGACGAGTGCAAGGAGAGG
GAGGACGAGGAGCTGTCCTGTGACCACTACTGCCACAACTACATTGGCGGCTACTACTGCTCCTGCCGCT
TCGGCTACATCCTCCACACAGACAACAGGACCTGCCGAGTGGAGTGCAGTGACAACCTCTTCACTCAAAG
GACTGGGGTGATCACCAGCCCTGACTTCCCAAACCCTTACCCCAAGAGCTCTGAATGCCTGTATACCATC
GAGCTGGAGGAGGGTTTCATGGTCAACCTGCAGTTTGAGGACATATTTGACATTGAGGACCATCCTGAGG
TGCCCTGCCCCTATGACTACATCAAGATCAAAGTTGGTCCAAAAGTTTTGGGGCCTTTCTGTGGAGAGAA
AGCCCCAGAACCCATCAGCACCCAGAGCCACAGTGTCCTGATCCTGTTCCATAGTGACAACTCGGGAGAG
AACCGGGGCTGGAGGCTCTCATACAGGGCTGCAGGAAATGAGTGCCCAGAGCTACAGCCTCCTGTCCATG
GGAAAATCGAGCCCTCCCAAGCCAAGTATTTCTTCAAAGACCAAGTGCTCGTCAGCTGTGACACAGGCTA
CAAAGTGCTGAAGGATAATGTGGAGATGGACACATTCCAGATTGAGTGTCTGAAGGATGGGACGTGGAGT
AACAAGATTCCCACCTGTAAAATTGTAGACTGTAGAGCCCCAGGAGAGCTGGAACACGGGCTGATCACCT
TCTCTACAAGGAACAACCTCACCACATACAAGTCTGAGATCAAATACTCCTGTCAGGAGCCCTATTACAA
GATGCTCAACAATAACACAGGTATATATACCTGTTCTGCCCAAGGAGTCTGGATGAATAAAGTATTGGGG
AGAAGCCTACCCACCTGCCTTCCAGTGTGTGGGCTCCCCAAGTTCTCCCGGAAGCTGATGGCCAGGATCT
TCAATGGACGCCCAGCCCAGAAAGGCACCACTCCCTGGATTGCCATGCTGTCACACCTGAATGGGCAGCC
CTTCTGCGGAGGCTCCCTTCTAGGCTCCAGCTGGATCGTGACCGCCGCACACTGCCTCCACCAGTCACTC
GATCCGGAAGATCCGACCCTACGTGATTCAGACTTGCTCAGCCCTTCTGACTTCAAAATCATCCTGGGCA
AGCATTGGAGGCTCCGGTCAGATGAAAATGAACAGCATCTCGGCGTCAAACACACCACTCTCCACCCCCA
GTATGATCCCAACACATTCGAGAATGACGTGGCTCTGGTGGAGCTGTTGGAGAGCCCAGTGCTGAATGCC
TTCGTGATGCCCATCTGTCTGCCTGAGGGACCCCAGCAGGAAGGAGCCATGGTCATCGTCAGCGGCTGGG
GGAAGCAGTTCTTGCAAAGGTTCCCAGAGACCCTGATGGAGATTGAAATCCCGATTGTTGACCACAGCAC
CTGCCAGAAGGCTTATGCCCCGCTGAAGAAGAAAGTGACCAGGGACATGATCTGTGCTGGGGAGAAGGAA
GGGGGAAAGGACGCCTGTGCGGGTGACTCTGGAGGCCCCATGGTGACCCTGAATAGAGAAAGAGGCCAGT
GGTACCTGGTGGGCACTGTGTCCTGGGGTGATGACTGTGGGAAGAAGGACCGCTACGGAGTATACTCTTA
CATCCACCACAACAAGGACTGGATCCAGAGGGTCACCGGAGTGAGGAACTGAATTTGGCTCCTCAGCCCC
AGCACCACCAGCTGTGGGCAGTCAGTAGCAGAGGACGATCCTCCGATGAAAGCAGCCATTTCTCCTTTCC
TTCCTCCCATCCCCCCTCCTTCGGCCTATCCATTACTGGGCAATAGAGCAGGTATCTTCACCCCCTTTTC
ACTCTCTTTAAAGAGATGGAGCAAGAGAGTGGTCAGAACACAGGCCGAATCCAGGCTCTATCACTTACTA
GTTTGCAGTGCTGGGCAGGTGACTTCATCTCTTCGAACTTCAGTTTCTTCATAAGATGGAAATGCTATAC
CTTACCTACCTCGTAAAAGTCTGATGAGGAAAAGATTAACTAATAGATGCATAGCACTTAACAGAGTGCA
TAGCATACACTGTTTTCAATAAATGCACCTTAGCAGAAGGTCGATGTGTCTACCAGGCAGACGAAGCTCT
CTTACAAACCCCTGCCTGGGTCTTAGCATTGATCAGTGACACACCTCTCCCCTCAACCTTGACCATCTCC
ATCTGCCCTTAAATGCTGTATGCTTTTTTGCCACCGTGCAACTTGCCCAACATCAATCTTCACCCTCATC
CCTAAAAAAGTAAAACAGACAAGGTTCTGAGTCCTGTGGTATGTCCCCTAGCAAATGTAACTAGGAACAT
GCACTAGATGACAGATTGCGGGAGGGCCTGAGAGAAGCAGGGACAGGAGGGAGCCTGGGGATTGTGGTTT
GGGAAGGCAGACACCTGGTTCTAGAACTAGCTCTGCCCTTAGCCCCCTGTATGACCCTATGCAAGTCCTC
CTCCCTCATCTCAAAGGGTCCTCAAAGCTCTGACGATCTAAGATACAATGAAGCCATTTTCCCCCTGATA
AGATGAGGTAAAGCCAATGTAACCAAAAGGCAAAAATTACAATCGGTTCAAAGGAACTTTGATGCAGACA
AAATGCTGCTGCTGCTGCTCCTGAAATACCCACCCCTTTCCACTACGGGTGGGTTCCCAAGGACATGGGA
CAGGCAAAGTGTGAGCCAAAGGATCCTTCCTTATTCCTAAGCAGAGCATCTGCTCTGGGCCCTGGCCTCC
TTCCCTTCTTGGGAAACTGGGCTGCATGAGGTGGGCCCTGGTAGTTTGTACCCCAGGCCCCTATACTCTT
CCTTCCTATGTCCACAGCTGACCCCAAGCAGCCGTTCCCCGACTCCTCACCCCTGAGCCTCACCCTGAAC
TCCCTCATCTTGCAAGGCCATAAGTGTTTTCCAAGCAAAATGCCTCTCCCATCCTCTCTCAGGAAGCTTC
TAGAGACTTTATGCCCTCCAGAGCTCCAAGATATAAGCCCTCCAAGGGATCAGAAGCTCCAAGTTCCTGT
CTTCTGTTTTATAGAAATTGATCTTCCCTGGGGGACTTTAACTCTTGACCTGTATGCAGCTGTTGGAGTA
ATTCCAGGTCTCTTGAAAAAAAAGAGGAAGATAATGGAGAATGAGAACATATATATATATATATTAAGCC
CCAGGCTGAATACTCAGGGACAGCAATTCACAGCCTGCCTCTGGTTCTATAAACAAGTCATTCTACCTCT
TTGTGCCCTGCTGTTTATTCTGTAAGGGGAAGGTGGCAATGGGACCCAGCTCCATCAGACACTTGTCAAG
CTAGCAGAAACTCCATTTTCAATGCCAAAGAAGAACTGTAATGCTGTTTTGGAATCATCCCAAGGCATCC
CAAGACACCATATCTTCCCATTTCAAGCACTGCCTGGGCACACCCCAACATCCCAGGCTGTGGTGGCTCC
TGTGGGAACTACCTAGATGAAGAGAGTATCATTTATACCTTCTAGGAGCTCCTATTGGGAGACATGAAAC
ATATGTAATTGACTACCATGTAATAGAACAAACCCTGCCAAGTGCTGCTTTGGAAAGTCATGGAGGTAAA
AGAAAGACCATTC Protein sequence of human MASP-3. SEQ ID NO: 7
MRWLLLYYALCFSLSKASAHTVELNNMFGQIQSPGYPDSYPSDSEVTWNITVPDGFRIKLYFMHFNLESSYLCE-
YDYVKVETEDQ
VLATFCGRETTDTEQTPGQEVVLSPGSFMSITFRSDFSNEERFTGFDAHYMAVDVDECKEREDEELSCDHYCHN-
YIGGYYCSCRF
GYILHTDNRTCRVECSDNLFTQRTGVITSPDFPNPYPKSSECLYTIELEEGFMVNLQFEDIFDIEDHPEVPCPY-
DYIKIKVGPKV
LGPFCGEKAPEPISTQSHSVLILFHSDNSGENRGWRLSYRAAGNECPELQPPVHGKIEPSQAKYFFKDQVLVSC-
DTGYKVLKDNV
EMDTFQIECLKDGTWSNKIPTCKIVDCRAPGELEHGLITFSTRNNLTTYKSEIKYSCQEPYYKMLNNNTGIYTC-
SAQGVWMNKVL
GRSLPTCLPECGQPSRSLPSLVKRIIGGRNAEPGLFPWQALIVVEDTSRVPNDKWFGSGALLSASWILTAAHVL-
RSQRRDTTVIP
VSKEHVTVYLGLHDVRDKSGAVNSSAARVVLHPDFNIQNYNHDIALVQLQEPVPLGPHVMPVCLPRLEPEGPAP-
HMLGLVAGWGI
SNPNVTVDEIISSGTRTLSDVLQYVKLPVVPHAECKTSYESRSGNYSVTENMFCAGYYEGGKDTCLGDSGGAFV-
IFDDLSQRWVV QGLVSWGGPEECGSKQVYGVYTKVSNYVDWVWEQMGLPQSVVEPQVER cDNA
sequence of human MASP-3 SEQ ID NO: 8
GAAGTCAGCCACACAGGATAAAGGAGGGAAGGGAAGGAGCAGATCTTTTCGGTAGGAAGACAGATTTTGT
TGTCAGGTTCCTGGGAGTGCAAGAGCAAGTCAAAGGAGAGAGAGAGGAGAGAGGAAAAGCCAGAGGGAGA
GAGGGGGAGAGGGGATCTGTTGCAGGCAGGGGAAGGCGTGACCTGAATGGAGAATGCCAGCCAATTCCAG
AGACACACAGGGACCTCAGAACAAAGATAAGGCATCACGGACACCACACCGGGCACGAGCTCACAGGCAA
GTCAAGCTGGGAGGACCAAGGCCGGGCAGCCGGGAGCACCCAAGGCAGGAAAATGAGGTGGCTGCTTCTC
TATTATGCTCTGTGCTTCTCCCTGTCAAAGGCTTCAGCCCACACCGTGGAGCTAAACAATATGTTTGGCC
AGATCCAGTCGCCTGGTTATCCAGACTCCTATCCCAGTGATTCAGAGGTGACTTGGAATATCACTGTCCC
AGATGGGTTTCGGATCAAGCTTTACTTCATGCACTTCAACTTGGAATCCTCCTACCTTTGTGAATATGAC
TATGTGAAGGTAGAAACTGAGGACCAGGTGCTGGCAACCTTCTGTGGCAGGGAGACCACAGACACAGAGC
AGACTCCCGGCCAGGAGGTGGTCCTCTCCCCTGGCTCCTTCATGTCCATCACTTTCCGGTCAGATTTCTC
CAATGAGGAGCGTTTCACAGGCTTTGATGCCCACTACATGGCTGTGGATGTGGACGAGTGCAAGGAGAGG
GAGGACGAGGAGCTGTCCTGTGACCACTACTGCCACAACTACATTGGCGGCTACTACTGCTCCTGCCGCT
TCGGCTACATCCTCCACACAGACAACAGGACCTGCCGAGTGGAGTGCAGTGACAACCTCTTCACTCAAAG
GACTGGGGTGATCACCAGCCCTGACTTCCCAAACCCTTACCCCAAGAGCTCTGAATGCCTGTATACCATC
GAGCTGGAGGAGGGTTTCATGGTCAACCTGCAGTTTGAGGACATATTTGACATTGAGGACCATCCTGAGG
TGCCCTGCCCCTATGACTACATCAAGATCAAAGTTGGTCCAAAAGTTTTGGGGCCTTTCTGTGGAGAGAA
AGCCCCAGAACCCATCAGCACCCAGAGCCACAGTGTCCTGATCCTGTTCCATAGTGACAACTCGGGAGAG
AACCGGGGCTGGAGGCTCTCATACAGGGCTGCAGGAAATGAGTGCCCAGAGCTACAGCCTCCTGTCCATG
GGAAAATCGAGCCCTCCCAAGCCAAGTATTTCTTCAAAGACCAAGTGCTCGTCAGCTGTGACACAGGCTA
CAAAGTGCTGAAGGATAATGTGGAGATGGACACATTCCAGATTGAGTGTCTGAAGGATGGGACGTGGAGT
AACAAGATTCCCACCTGTAAAATTGTAGACTGTAGAGCCCCAGGAGAGCTGGAACACGGGCTGATCACCT
TCTCTACAAGGAACAACCTCACCACATACAAGTCTGAGATCAAATACTCCTGTCAGGAGCCCTATTACAA
GATGCTCAACAATAACACAGGTATATATACCTGTTCTGCCCAAGGAGTCTGGATGAATAAAGTATTGGGG
AGAAGCCTACCCACCTGCCTTCCAGAGTGTGGTCAGCCCTCCCGCTCCCTGCCAAGCCTGGTCAAGAGGA
TCATTGGGGGCCGAAATGCTGAGCCTGGCCTCTTCCCGTGGCAGGCCCTGATAGTGGTGGAGGACACTTC
GAGAGTGCCAAATGACAAGTGGTTTGGGAGTGGGGCCCTGCTCTCTGCGTCCTGGATCCTCACAGCAGCT
CATGTGCTGCGCTCCCAGCGTAGAGACACCACGGTGATACCAGTCTCCAAGGAGCATGTCACCGTCTACC
TGGGCTTGCATGATGTGCGAGACAAATCGGGGGCAGTCAACAGCTCAGCTGCCCGAGTGGTGCTCCACCC
AGACTTCAACATCCAAAACTACAACCACGATATAGCTCTGGTGCAGCTGCAGGAGCCTGTGCCCCTGGGA
CCCCACGTTATGCCTGTCTGCCTGCCAAGGCTTGAGCCTGAAGGCCCGGCCCCCCACATGCTGGGCCTGG
TGGCCGGCTGGGGCATCTCCAATCCCAATGTGACAGTGGATGAGATCATCAGCAGTGGCACACGGACCTT
GTCAGATGTCCTGCAGTATGTCAAGTTACCCGTGGTGCCTCACGCTGAGTGCAAAACTAGCTATGAGTCC
CGCTCGGGCAATTACAGCGTCACGGAGAACATGTTCTGTGCTGGCTACTACGAGGGCGGCAAAGACACGT
GCCTTGGAGATAGCGGTGGGGCCTTTGTCATCTTTGATGACTTGAGCCAGCGCTGGGTGGTGCAAGGCCT
GGTGTCCTGGGGGGGACCTGAAGAATGCGGCAGCAAGCAGGTCTATGGAGTCTACACAAAGGTCTCCAAT
TACGTGGACTGGGTGTGGGAGCAGATGGGCTTACCACAAAGTGTTGTGGAGCCCCAGGTGGAACGGTGAG
CTGACTTACTTCCTCGGGGCCTGCCTCCCCTGAGCGAAGCTACACCGCACTTCCGACAGCACACTCCACA
TTACTTATCAGACCATATGGAATGGAACACACTGACCTAGCGGTGGCTTCTCCTACCGAGACAGCCCCCA
GGACCCTGAGAGGCAGAGTGTGGTATAGGGAAAAGGCTCCAGGCAGGAGACCTGTGTTCCTGAGCTTGTC
CAAGTCTCTTTCCCTGTCTGGGCCTCACTCTACCGAGTAATACAATGCAGGAGCTCAACCAAGGCCTCTG
TGCCAATCCCAGCACTCCTTTCCAGGCCATGCTTCTTACCCCAGTGGCCTTTATTCACTCCTGACCACTT
ATCAAACCCATCGGTCCTACTGTTGGTATAACTGAGCTTGGACCTGACTATTAGAAAATGGTTTCTAACA
TTGAACTGAATGCCGCATCTGTATATTTTCCTGCTCTGCCTTCTGGGACTAGCCTTGGCCTAATCCTTCC
TCTAGGAGAAGAGCATTCAGGTTTTGGGAGATGGCTCATAGCCAAGCCCCTCTCTCTTAGTGTGATCCCT
TGGAGCACCTTCATGCCTGGGGTTTCTCTCCCAAAAGCTTCTTGCAGTCTAAGCCTTATCCCTTATGTTC
CCCATTAAAGGAATTTCAAAAGACATGGAGAAAGTTGGGAAGGTTTGTGCTGACTGCTGGGAGCAGAATA
GCCGTGGGAGGCCCACCAAGCCCTTAAATTCCCATTGTCAACTCAGAACACATTTGGGCCCATATGCCAC
CCTGGAACACCAGCTGACACCATGGGCGTCCACACCTGCTGCTCCAGACAAGCACAAAGCAATCTTTCAG
CCTTGAAATGTATTATCTGAAAGGCTACCTGAAGCCCAGGCCCGAATATGGGGACTTAGTCGATTACCTG
GAAAAAGAAAAGACCCACACTGTGTCCTGCTGTGCTTTTGGGCAGGAAAATGGAAGAAAGAGTGGGGTGG
GCACATTAGAAGTCACCCAAATCCTGCCAGGCTGCCTGGCATCCCTGGGGCATGAGCTGGGCGGAGAATC
CACCCCGCAGGATGTTCAGAGGGACCCACTCCTTCATTTTTCAGAGTCAAAGGAATCAGAGGCTCACCCA
TGGCAGGCAGTGAAAAGAGCCAGGAGTCCTGGGTTCTAGTCCCTGCTCTGCCCCCAACTGGCTGTATAAC
CTTTGAAAAATCATTTTCTTTGTCTGAGTCTCTGGTTCTCCGTCAGCAACAGGCTGGCATAAGGTCCCCT
GCAGGTTCCTTCTAGCTGGAGCACTCAGAGCTTCCCTGACTGCTAGCAGCCTCTCTGGCCCTCACAGGGC
TGATTGTTCTCCTTCTCCCTGGAGCTCTCTCTCCTGAAAATCTCCATCAGAGCAAGGCAGCCAGAGAAGC
CCCTGAGAGGGAATGATTGGGAAGTGTCCACTTTCTCAACCGGCTCATCAAACACACTCCTTTGTCTATG
AATGGCACATGTAAATGATGTTATATTTTGTATCTTTTATATCATATGCTTCACCATTCTGTAAAGGGCC
TCTGCATTGTTGCTCCCATCAGGGGTCTCAAGTGGAAATAAACCCTCGTGGATAACCAAAAAAAAAAAAA
AAAAAAA Protein sequence of human MASP-2 SEQ ID NO: 9
MRLLTLLGLLCGSVATPLGPKWPEPVFGRLASPGFPGEYANDQERRWTLTAPPGYRLRLYFTHFDLELSHLCE
YDFVKLSSGAKVLATLCGQESTDTERAPGKDTFYSLGSSLDITFRSDYSNEKPFTGFEAFYAAEDIDECQVAP
GEAPTCDHHCHNHLGGFYCSCRAGYVLHRNKRTCSALCSGQVFTQRSGELSSPEYPRPYPKLSSCTYSISLE
EGFSVILDFVESFDVETHPETLCPYDFLKIQTDREEHGPFCGKTLPHRIETKSNTVTITFVTDESGDHTGWKI
HYTSTAQPCPYPMAPPNGHVSPVQAKYILKDSFSIFCETGYELLQGHLPLKSFTAVCQKDGSWDRPMPACSI
VDCGPPDDLPSGRVEYITGPGVTTYKAVIQYSCEETFYTMKVNDGKYVCEADGFWTSSKGEKSLPVCEPVC
GLSARTTGGRIYGGQKAKPGDFPWQVLILGGTTAAGALLYDNWVLTAAHAVYEQKHDASALDIRMGTLKRL
SPHYTQAWSEAVFIHEGYTHDAGFDNDIALIKLNNKVVINSNITPICLPRKEAESFMRTDDIGTASGWGLTQ
RGFLARNLMYVDIPIVDHQKCTAAYEKPPYPRGSVTANMLCAGLESGGKDSCRGDSGGALVFLDSETERWF
VGGIVSWGSMNCGEAGQYGVYTKVINYIPWIENIISDF cDNA sequence of human
MASP-2 SEQ ID NO: 10
GGCCAGCTGGACGGGCACACCATGAGGCTGCTGACCCTCCTGGGCCTTCTGTGTGGCTCGGTGGCCACCC
CCTTGGGCCCGAAGTGGCCTGAACCTGTGTTCGGGCGCCTGGCATCCCCCGGCTTTCCAGGGGAGTATGC
CAATGACCAGGAGCGGCGCTGGACCCTGACTGCACCCCCCGGCTACCGCCTGCGCCTCTACTTCACCCAC
TTCGACCTGGAGCTCTCCCACCTCTGCGAGTACGACTTCGTCAAGCTGAGCTCGGGGGCCAAGGTGCTGG
CCACGCTGTGCGGGCAGGAGAGCACAGACACGGAGCGGGCCCCTGGCAAGGACACTTTCTACTCGCTGGG
CTCCAGCCTGGACATTACCTTCCGCTCCGACTACTCCAACGAGAAGCCGTTCACGGGGTTCGAGGCCTTC
TATGCAGCCGAGGACATTGACGAGTGCCAGGTGGCCCCGGGAGAGGCGCCCACCTGCGACCACCACTGCC
ACAACCACCTGGGCGGTTTCTACTGCTCCTGCCGCGCAGGCTACGTCCTGCACCGTAACAAGCGCACCTG
CTCAGCCCTGTGCTCCGGCCAGGTCTTCACCCAGAGGTCTGGGGAGCTCAGCAGCCCTGAATACCCACGG
CCGTATCCCAAACTCTCCAGTTGCACTTACAGCATCAGCCTGGAGGAGGGGTTCAGTGTCATTCTGGACT
TTGTGGAGTCCTTCGATGTGGAGACACACCCTGAAACCCTGTGTCCCTACGACTTTCTCAAGATTCAAAC
AGACAGAGAAGAACATGGCCCATTCTGTGGGAAGACATTGCCCCACAGGATTGAAACAAAAAGCAACACG
GTGACCATCACCTTTGTCACAGATGAATCAGGAGACCACACAGGCTGGAAGATCCACTACACGAGCACAG
CGCAGCCTTGCCCTTATCCGATGGCGCCACCTAATGGCCACGTTTCACCTGTGCAAGCCAAATACATCCT
GAAAGACAGCTTCTCCATCTTTTGCGAGACTGGCTATGAGCTTCTGCAAGGTCACTTGCCCCTGAAATCC
TTTACTGCAGTTTGTCAGAAAGATGGATCTTGGGACCGGCCAATGCCCGCGTGCAGCATTGTTGACTGTG
GCCCTCCTGATGATCTACCCAGTGGCCGAGTGGAGTACATCACAGGTCCTGGAGTGACCACCTACAAAGC
TGTGATTCAGTACAGCTGTGAAGAGACCTTCTACACAATGAAAGTGAATGATGGTAAATATGTGTGTGAG
GCTGATGGATTCTGGACGAGCTCCAAAGGAGAAAAATCACTCCCAGTCTGTGAGCCTGTTTGTGGACTAT
CAGCCCGCACAACAGGAGGGCGTATATATGGAGGGCAAAAGGCAAAACCTGGTGATTTTCCTTGGCAAGT
CCTGATATTAGGTGGAACCACAGCAGCAGGTGCACTTTTATATGACAACTGGGTCCTAACAGCTGCTCAT
GCCGTCTATGAGCAAAAACATGATGCATCCGCCCTGGACATTCGAATGGGCACCCTGAAAAGACTATCAC
CTCATTATACACAAGCCTGGTCTGAAGCTGTTTTTATACATGAAGGTTATACTCATGATGCTGGCTTTGA
CAATGACATAGCACTGATTAAATTGAATAACAAAGTTGTAATCAATAGCAACATCACGCCTATTTGTCTG
CCAAGAAAAGAAGCTGAATCCTTTATGAGGACAGATGACATTGGAACTGCATCTGGATGGGGATTAACCC
AAAGGGGTTTTCTTGCTAGAAATCTAATGTATGTCGACATACCGATTGTTGACCATCAAAAATGTACTGC
TGCATATGAAAAGCCACCCTATCCAAGGGGAAGTGTAACTGCTAACATGCTTTGTGCTGGCTTAGAAAGT
GGGGGCAAGGACAGCTGCAGAGGTGACAGCGGAGGGGCACTGGTGTTTCTAGATAGTGAAACAGAGAGGT
GGTTTGTGGGAGGAATAGTGTCCTGGGGTTCCATGAATTGTGGGGAAGCAGGTCAGTATGGAGTCTACAC
AAAAGTTATTAACTATATTCCCTGGATCGAGAACATAATTAGTGATTTTTAACTTGCGTGTCTGCAGTCA
AGGATTCTTCATTTTTAGAAATGCCTGTGAAGACCTTGGCAGCGACGTGGCTCGAGAAGCATTCATCATT
ACTGTGGACATGGCAGTTGTTGCTCCACCCAAAAAAACAGACTCCAGGTGAGGCTGCTGTCATTTCTCCA
CTTGCCAGTTTAATTCCAGCCTTACCCATTGACTCAAGGGGACATAAACCACGAGAGTGACAGTCATCTT
TGCCCACCCAGTGTAATGTCACTGCTCAAATTACATTTCATTACCTTAAAAAGCCAGTCTCTTTTCATAC
TGGCTGTTGGCATTTCTGTAAACTGCCTGTCCATGCTCTTTGTTTTTAAACTTGTTCTTATTGAAAAAAA
AAAAAAAAAA Protein sequence of human sMAP (MAp19) SEQ ID NO: 11
MRLLTLLGLLCGSVATPLGPKWPEPVFGRLASPGFPGEYANDQERRWTLTAPPGYRLRLYFTHFDLELSHL
CEYDFVKLSSGAKVLATLCGQESTDTERAPGKDTFYSLGSSLDITFRSDYSNEKPFTGFEAFYAAEDIDEC
QVAPGEAPTCDHHCHNHLGGFYCSCRAGYVLHRNKRTCSEQSL cDNA sequence of human
sMAP (MAp19) SEQ ID NO: 12
GGCCAGCTGGACGGGCACACCATGAGGCTGCTGACCCTCCTGGGCCTTCTGTGTGGCTCGGTGGCCACCC
CCTTGGGCCCGAAGTGGCCTGAACCTGTGTTCGGGCGCCTGGCATCCCCCGGCTTTCCAGGGGAGTATGC
CAATGACCAGGAGCGGCGCTGGACCCTGACTGCACCCCCCGGCTACCGCCTGCGCCTCTACTTCACCCAC
TTCGACCTGGAGCTCTCCCACCTCTGCGAGTACGACTTCGTCAAGCTGAGCTCGGGGGCCAAGGTGCTGG
CCACGCTGTGCGGGCAGGAGAGCACAGACACGGAGCGGGCCCCTGGCAAGGACACTTTCTACTCGCTGGG
CTCCAGCCTGGACATTACCTTCCGCTCCGACTACTCCAACGAGAAGCCGTTCACGGGGTTCGAGGCCTTC
TATGCAGCCGAGGACATTGACGAGTGCCAGGTGGCCCCGGGAGAGGCGCCCACCTGCGACCACCACTGCC
ACAACCACCTGGGCGGTTTCTACTGCTCCTGCCGCGCAGGCTACGTCCTGCACCGTAACAAGCGCACCTG
CTCAGAGCAGAGCCTCTAGCCTCCCCTGGAGCTCCGGCCTGCCCAGCAGGTCAGAAGCCAGAGCCAGCCT
GCTGGCCTCAGCTCCGGGTTGGGCTGAGATGGCTGTGCCCCAACTCCCATTCACCCACCATGGACCCAAT
AATAAACCTGGCCCCACCCCAAAAAAAAAAAAAAAAAA DNA primers: SEQ ID NO: 13:
5'-gcacccagagccacagtg-3' SEQ ID NO: 14: 5'-gccttccagtgtgtgggc-3'
SEQ ID NO: 15: 5-gccttccagagtgtggtca-3' SEQ ID NO: 16:
5'-cgatctggagagcgaactc-3' SEQ ID NO: 17: 5'-ctgttcttcacactggctg-3'
SEQ ID NO: 18: 5'-ctgctgagatcatgttgttc-3' SEQ ID NO: 19:
5'-TTATACGACTCACTA-3'
Sequence CWU 1
1
191380PRTHomo sapiens 1Met Arg Trp Leu Leu Leu Tyr Tyr Ala Leu Cys
Phe Ser Leu Ser Lys1 5 10 15Ala Ser Ala His Thr Val Glu Leu Asn Asn
Met Phe Gly Gln Ile Gln 20 25 30Ser Pro Gly Tyr Pro Asp Ser Tyr Pro
Ser Asp Ser Glu Val Thr Trp 35 40 45Asn Ile Thr Val Pro Asp Gly Phe
Arg Ile Lys Leu Tyr Phe Met His 50 55 60Phe Asn Leu Glu Ser Ser Tyr
Leu Cys Glu Tyr Asp Tyr Val Lys Val65 70 75 80Glu Thr Glu Asp Gln
Val Leu Ala Thr Phe Cys Gly Arg Glu Thr Thr 85 90 95Asp Thr Glu Gln
Thr Pro Gly Gln Glu Val Val Leu Ser Pro Gly Ser 100 105 110Phe Met
Ser Ile Thr Phe Arg Ser Asp Phe Ser Asn Glu Glu Arg Phe 115 120
125Thr Gly Phe Asp Ala His Tyr Met Ala Val Asp Val Asp Glu Cys Lys
130 135 140Glu Arg Glu Asp Glu Glu Leu Ser Cys Asp His Tyr Cys His
Asn Tyr145 150 155 160Ile Gly Gly Tyr Tyr Cys Ser Cys Arg Phe Gly
Tyr Ile Leu His Thr 165 170 175Asp Asn Arg Thr Cys Arg Val Glu Cys
Ser Asp Asn Leu Phe Thr Gln 180 185 190Arg Thr Gly Val Ile Thr Ser
Pro Asp Phe Pro Asn Pro Tyr Pro Lys 195 200 205Ser Ser Glu Cys Leu
Tyr Thr Ile Glu Leu Glu Glu Gly Phe Met Val 210 215 220Asn Leu Gln
Phe Glu Asp Ile Phe Asp Ile Glu Asp His Pro Glu Val225 230 235
240Pro Cys Pro Tyr Asp Tyr Ile Lys Ile Lys Val Gly Pro Lys Val Leu
245 250 255Gly Pro Phe Cys Gly Glu Lys Ala Pro Glu Pro Ile Ser Thr
Gln Ser 260 265 270His Ser Val Leu Ile Leu Phe His Ser Asp Asn Ser
Gly Glu Asn Arg 275 280 285Gly Trp Arg Leu Ser Tyr Arg Ala Ala Gly
Asn Glu Cys Pro Glu Leu 290 295 300Gln Pro Pro Val His Gly Lys Ile
Glu Pro Ser Gln Ala Lys Tyr Phe305 310 315 320Phe Lys Asp Gln Val
Leu Val Ser Cys Asp Thr Gly Tyr Lys Val Leu 325 330 335Lys Asp Asn
Val Glu Met Asp Thr Phe Gln Ile Glu Cys Leu Lys Asp 340 345 350Gly
Thr Trp Ser Asn Lys Ile Pro Thr Cys Lys Lys Asn Glu Ile Asp 355 360
365Leu Glu Ser Glu Leu Lys Ser Glu Gln Val Thr Glu 370 375
38021143DNAHomo sapiens 2atgaggtggc tgcttctcta ttatgctctg
tgcttctccc tgtcaaaggc ttcagcccac 60accgtggagc taaacaatat gtttggccag
atccagtcgc ctggttatcc agactcctat 120cccagtgatt cagaggtgac
ttggaatatc actgtcccag atgggtttcg gatcaagctt 180tacttcatgc
acttcaactt ggaatcctcc tacctttgtg aatatgacta tgtgaaggta
240gaaactgagg accaggtgct ggcaaccttc tgtggcaggg agaccacaga
cacagagcag 300actcccggcc aggaggtggt cctctcccct ggctccttca
tgtccatcac tttccggtca 360gatttctcca atgaggagcg tttcacaggc
tttgatgccc actacatggc tgtggatgtg 420gacgagtgca aggagaggga
ggacgaggag ctgtcctgtg accactactg ccacaactac 480attggcggct
actactgctc ctgccgcttc ggctacatcc tccacacaga caacaggacc
540tgccgagtgg agtgcagtga caacctcttc actcaaagga ctggggtgat
caccagccct 600gacttcccaa acccttaccc caagagctct gaatgcctgt
ataccatcga gctggaggag 660ggtttcatgg tcaacctgca gtttgaggac
atatttgaca ttgaggacca tcctgaggtg 720ccctgcccct atgactacat
caagatcaaa gttggtccaa aagttttggg gcctttctgt 780ggagagaaag
ccccagaacc catcagcacc cagagccaca gtgtcctgat cctgttccat
840agtgacaact cgggagagaa ccggggctgg aggctctcat acagggctgc
aggaaatgag 900tgcccagagc tacagcctcc tgtccatggg aaaatcgagc
cctcccaagc caagtatttc 960ttcaaagacc aagtgctcgt cagctgtgac
acaggctaca aagtgctgaa ggataatgtg 1020gagatggaca cattccagat
tgagtgtctg aaggatggga cgtggagtaa caagattccc 1080acctgtaaaa
aaaatgaaat cgatctggag agcgaactca agtcagagca agtgacagag 1140tga
11433297PRTHomo sapiens 3Met Arg Trp Leu Leu Leu Tyr Tyr Ala Leu
Cys Phe Ser Leu Ser Lys1 5 10 15Ala Ser Ala His Thr Val Glu Leu Asn
Asn Met Phe Gly Gln Ile Gln 20 25 30Ser Pro Gly Tyr Pro Asp Ser Tyr
Pro Ser Asp Ser Glu Val Thr Trp 35 40 45Asn Ile Thr Val Pro Asp Gly
Phe Arg Ile Lys Leu Tyr Phe Met His 50 55 60Phe Asn Leu Glu Ser Ser
Tyr Leu Cys Glu Tyr Asp Tyr Val Lys Val65 70 75 80Glu Thr Glu Asp
Gln Val Leu Ala Thr Phe Cys Gly Arg Glu Thr Thr 85 90 95Asp Thr Glu
Gln Thr Pro Gly Gln Glu Val Val Leu Ser Pro Gly Ser 100 105 110Phe
Met Ser Ile Thr Phe Arg Ser Asp Phe Ser Asn Glu Glu Arg Phe 115 120
125Thr Gly Phe Asp Ala His Tyr Met Ala Val Asp Val Asp Glu Cys Lys
130 135 140Glu Arg Glu Asp Glu Glu Leu Ser Cys Asp His Tyr Cys His
Asn Tyr145 150 155 160Ile Gly Gly Tyr Tyr Cys Ser Cys Arg Phe Gly
Tyr Ile Leu His Thr 165 170 175Asp Asn Arg Thr Cys Arg Val Glu Cys
Ser Asp Asn Leu Phe Thr Gln 180 185 190Arg Thr Gly Val Ile Thr Ser
Pro Asp Phe Pro Asn Pro Tyr Pro Lys 195 200 205Ser Ser Glu Cys Leu
Tyr Thr Ile Glu Leu Glu Glu Gly Phe Met Val 210 215 220Asn Leu Gln
Phe Glu Asp Ile Phe Asp Ile Glu Asp His Pro Glu Val225 230 235
240Pro Cys Pro Tyr Asp Tyr Ile Lys Ile Lys Val Gly Pro Lys Val Leu
245 250 255Gly Pro Phe Cys Gly Glu Lys Ala Pro Glu Pro Ile Ser Thr
Gln Ser 260 265 270His Ser Val Leu Ile Leu Phe His Ser Asp Asn Ser
Gly Glu Asn Arg 275 280 285Gly Trp Arg Leu Ser Tyr Arg Ala Ala 290
295417PRTHomo sapiens 4Lys Asn Glu Ile Asp Leu Glu Ser Glu Leu Lys
Ser Glu Gln Val Thr1 5 10 15Glu5699PRTHomo sapiens 5Met Arg Trp Leu
Leu Leu Tyr Tyr Ala Leu Cys Phe Ser Leu Ser Lys1 5 10 15Ala Ser Ala
His Thr Val Glu Leu Asn Asn Met Phe Gly Gln Ile Gln 20 25 30Ser Pro
Gly Tyr Pro Asp Ser Tyr Pro Ser Asp Ser Glu Val Thr Trp 35 40 45Asn
Ile Thr Val Pro Asp Gly Phe Arg Ile Lys Leu Tyr Phe Met His 50 55
60Phe Asn Leu Glu Ser Ser Tyr Leu Cys Glu Tyr Asp Tyr Val Lys Val65
70 75 80Glu Thr Glu Asp Gln Val Leu Ala Thr Phe Cys Gly Arg Glu Thr
Thr 85 90 95Asp Thr Glu Gln Thr Pro Gly Gln Glu Val Val Leu Ser Pro
Gly Ser 100 105 110Phe Met Ser Ile Thr Phe Arg Ser Asp Phe Ser Asn
Glu Glu Arg Phe 115 120 125Thr Gly Phe Asp Ala His Tyr Met Ala Val
Asp Val Asp Glu Cys Lys 130 135 140Glu Arg Glu Asp Glu Glu Leu Ser
Cys Asp His Tyr Cys His Asn Tyr145 150 155 160Ile Gly Gly Tyr Tyr
Cys Ser Cys Arg Phe Gly Tyr Ile Leu His Thr 165 170 175Asp Asn Arg
Thr Cys Arg Val Glu Cys Ser Asp Asn Leu Phe Thr Gln 180 185 190Arg
Thr Gly Val Ile Thr Ser Pro Asp Phe Pro Asn Pro Tyr Pro Lys 195 200
205Ser Ser Glu Cys Leu Tyr Thr Ile Glu Leu Glu Glu Gly Phe Met Val
210 215 220Asn Leu Gln Phe Glu Asp Ile Phe Asp Ile Glu Asp His Pro
Glu Val225 230 235 240Pro Cys Pro Tyr Asp Tyr Ile Lys Ile Lys Val
Gly Pro Lys Val Leu 245 250 255Gly Pro Phe Cys Gly Glu Lys Ala Pro
Glu Pro Ile Ser Thr Gln Ser 260 265 270His Ser Val Leu Ile Leu Phe
His Ser Asp Asn Ser Gly Glu Asn Arg 275 280 285Gly Trp Arg Leu Ser
Tyr Arg Ala Ala Gly Asn Glu Cys Pro Glu Leu 290 295 300Gln Pro Pro
Val His Gly Lys Ile Glu Pro Ser Gln Ala Lys Tyr Phe305 310 315
320Phe Lys Asp Gln Val Leu Val Ser Cys Asp Thr Gly Tyr Lys Val Leu
325 330 335Lys Asp Asn Val Glu Met Asp Thr Phe Gln Ile Glu Cys Leu
Lys Asp 340 345 350Gly Thr Trp Ser Asn Lys Ile Pro Thr Cys Lys Ile
Val Asp Cys Arg 355 360 365Ala Pro Gly Glu Leu Glu His Gly Leu Ile
Thr Phe Ser Thr Arg Asn 370 375 380Asn Leu Thr Thr Tyr Lys Ser Glu
Ile Lys Tyr Ser Cys Gln Glu Pro385 390 395 400Tyr Tyr Lys Met Leu
Asn Asn Asn Thr Gly Ile Tyr Thr Cys Ser Ala 405 410 415Gln Gly Val
Trp Met Asn Lys Val Leu Gly Arg Ser Leu Pro Thr Cys 420 425 430Leu
Pro Val Cys Gly Leu Pro Lys Phe Ser Arg Lys Leu Met Ala Arg 435 440
445Ile Phe Asn Gly Arg Pro Ala Gln Lys Gly Thr Thr Pro Trp Ile Ala
450 455 460Met Leu Ser His Leu Asn Gly Gln Pro Phe Cys Gly Gly Ser
Leu Leu465 470 475 480Gly Ser Ser Trp Ile Val Thr Ala Ala His Cys
Leu His Gln Ser Leu 485 490 495Asp Pro Glu Asp Pro Thr Leu Arg Asp
Ser Asp Leu Leu Ser Pro Ser 500 505 510Asp Phe Lys Ile Ile Leu Gly
Lys His Trp Arg Leu Arg Ser Asp Glu 515 520 525Asn Glu Gln His Leu
Gly Val Lys His Thr Thr Leu His Pro Gln Tyr 530 535 540Asp Pro Asn
Thr Phe Glu Asn Asp Val Ala Leu Val Glu Leu Leu Glu545 550 555
560Ser Pro Val Leu Asn Ala Phe Val Met Pro Ile Cys Leu Pro Glu Gly
565 570 575Pro Gln Gln Glu Gly Ala Met Val Ile Val Ser Gly Trp Gly
Lys Gln 580 585 590Phe Leu Gln Arg Phe Pro Glu Thr Leu Met Glu Ile
Glu Ile Pro Ile 595 600 605Val Asp His Ser Thr Cys Gln Lys Ala Tyr
Ala Pro Leu Lys Lys Lys 610 615 620Val Thr Arg Asp Met Ile Cys Ala
Gly Glu Lys Glu Gly Gly Lys Asp625 630 635 640Ala Cys Ala Gly Asp
Ser Gly Gly Pro Met Val Thr Leu Asn Arg Glu 645 650 655Arg Gly Gln
Trp Tyr Leu Val Gly Thr Val Ser Trp Gly Asp Asp Cys 660 665 670Gly
Lys Lys Asp Arg Tyr Gly Val Tyr Ser Tyr Ile His His Asn Lys 675 680
685Asp Trp Ile Gln Arg Val Thr Gly Val Arg Asn 690 69564353DNAHomo
sapiens 6gaagtcagcc acacaggata aaggagggaa gggaaggagc agatcttttc
ggtaggaaga 60cagattttgt tgtcaggttc ctgggagtgc aagagcaagt caaaggagag
agagaggaga 120gaggaaaagc cagagggaga gagggggaga ggggatctgt
tgcaggcagg ggaaggcgtg 180acctgaatgg agaatgccag ccaattccag
agacacacag ggacctcaga acaaagataa 240ggcatcacgg acaccacacc
gggcacgagc tcacaggcaa gtcaagctgg gaggaccaag 300gccgggcagc
cgggagcacc caaggcagga aaatgaggtg gctgcttctc tattatgctc
360tgtgcttctc cctgtcaaag gcttcagccc acaccgtgga gctaaacaat
atgtttggcc 420agatccagtc gcctggttat ccagactcct atcccagtga
ttcagaggtg acttggaata 480tcactgtccc agatgggttt cggatcaagc
tttacttcat gcacttcaac ttggaatcct 540cctacctttg tgaatatgac
tatgtgaagg tagaaactga ggaccaggtg ctggcaacct 600tctgtggcag
ggagaccaca gacacagagc agactcccgg ccaggaggtg gtcctctccc
660ctggctcctt catgtccatc actttccggt cagatttctc caatgaggag
cgtttcacag 720gctttgatgc ccactacatg gctgtggatg tggacgagtg
caaggagagg gaggacgagg 780agctgtcctg tgaccactac tgccacaact
acattggcgg ctactactgc tcctgccgct 840tcggctacat cctccacaca
gacaacagga cctgccgagt ggagtgcagt gacaacctct 900tcactcaaag
gactggggtg atcaccagcc ctgacttccc aaacccttac cccaagagct
960ctgaatgcct gtataccatc gagctggagg agggtttcat ggtcaacctg
cagtttgagg 1020acatatttga cattgaggac catcctgagg tgccctgccc
ctatgactac atcaagatca 1080aagttggtcc aaaagttttg gggcctttct
gtggagagaa agccccagaa cccatcagca 1140cccagagcca cagtgtcctg
atcctgttcc atagtgacaa ctcgggagag aaccggggct 1200ggaggctctc
atacagggct gcaggaaatg agtgcccaga gctacagcct cctgtccatg
1260ggaaaatcga gccctcccaa gccaagtatt tcttcaaaga ccaagtgctc
gtcagctgtg 1320acacaggcta caaagtgctg aaggataatg tggagatgga
cacattccag attgagtgtc 1380tgaaggatgg gacgtggagt aacaagattc
ccacctgtaa aattgtagac tgtagagccc 1440caggagagct ggaacacggg
ctgatcacct tctctacaag gaacaacctc accacataca 1500agtctgagat
caaatactcc tgtcaggagc cctattacaa gatgctcaac aataacacag
1560gtatatatac ctgttctgcc caaggagtct ggatgaataa agtattgggg
agaagcctac 1620ccacctgcct tccagtgtgt gggctcccca agttctcccg
gaagctgatg gccaggatct 1680tcaatggacg cccagcccag aaaggcacca
ctccctggat tgccatgctg tcacacctga 1740atgggcagcc cttctgcgga
ggctcccttc taggctccag ctggatcgtg accgccgcac 1800actgcctcca
ccagtcactc gatccggaag atccgaccct acgtgattca gacttgctca
1860gcccttctga cttcaaaatc atcctgggca agcattggag gctccggtca
gatgaaaatg 1920aacagcatct cggcgtcaaa cacaccactc tccaccccca
gtatgatccc aacacattcg 1980agaatgacgt ggctctggtg gagctgttgg
agagcccagt gctgaatgcc ttcgtgatgc 2040ccatctgtct gcctgaggga
ccccagcagg aaggagccat ggtcatcgtc agcggctggg 2100ggaagcagtt
cttgcaaagg ttcccagaga ccctgatgga gattgaaatc ccgattgttg
2160accacagcac ctgccagaag gcttatgccc cgctgaagaa gaaagtgacc
agggacatga 2220tctgtgctgg ggagaaggaa gggggaaagg acgcctgtgc
gggtgactct ggaggcccca 2280tggtgaccct gaatagagaa agaggccagt
ggtacctggt gggcactgtg tcctggggtg 2340atgactgtgg gaagaaggac
cgctacggag tatactctta catccaccac aacaaggact 2400ggatccagag
ggtcaccgga gtgaggaact gaatttggct cctcagcccc agcaccacca
2460gctgtgggca gtcagtagca gaggacgatc ctccgatgaa agcagccatt
tctcctttcc 2520ttcctcccat cccccctcct tcggcctatc cattactggg
caatagagca ggtatcttca 2580cccccttttc actctcttta aagagatgga
gcaagagagt ggtcagaaca caggccgaat 2640ccaggctcta tcacttacta
gtttgcagtg ctgggcaggt gacttcatct cttcgaactt 2700cagtttcttc
ataagatgga aatgctatac cttacctacc tcgtaaaagt ctgatgagga
2760aaagattaac taatagatgc atagcactta acagagtgca tagcatacac
tgttttcaat 2820aaatgcacct tagcagaagg tcgatgtgtc taccaggcag
acgaagctct cttacaaacc 2880cctgcctggg tcttagcatt gatcagtgac
acacctctcc cctcaacctt gaccatctcc 2940atctgccctt aaatgctgta
tgcttttttg ccaccgtgca acttgcccaa catcaatctt 3000caccctcatc
cctaaaaaag taaaacagac aaggttctga gtcctgtggt atgtccccta
3060gcaaatgtaa ctaggaacat gcactagatg acagattgcg ggagggcctg
agagaagcag 3120ggacaggagg gagcctgggg attgtggttt gggaaggcag
acacctggtt ctagaactag 3180ctctgccctt agccccctgt atgaccctat
gcaagtcctc ctccctcatc tcaaagggtc 3240ctcaaagctc tgacgatcta
agatacaatg aagccatttt ccccctgata agatgaggta 3300aagccaatgt
aaccaaaagg caaaaattac aatcggttca aaggaacttt gatgcagaca
3360aaatgctgct gctgctgctc ctgaaatacc cacccctttc cactacgggt
gggttcccaa 3420ggacatggga caggcaaagt gtgagccaaa ggatccttcc
ttattcctaa gcagagcatc 3480tgctctgggc cctggcctcc ttcccttctt
gggaaactgg gctgcatgag gtgggccctg 3540gtagtttgta ccccaggccc
ctatactctt ccttcctatg tccacagctg accccaagca 3600gccgttcccc
gactcctcac ccctgagcct caccctgaac tccctcatct tgcaaggcca
3660taagtgtttt ccaagcaaaa tgcctctccc atcctctctc aggaagcttc
tagagacttt 3720atgccctcca gagctccaag atataagccc tccaagggat
cagaagctcc aagttcctgt 3780cttctgtttt atagaaattg atcttccctg
ggggacttta actcttgacc tgtatgcagc 3840tgttggagta attccaggtc
tcttgaaaaa aaagaggaag ataatggaga atgagaacat 3900atatatatat
atattaagcc ccaggctgaa tactcaggga cagcaattca cagcctgcct
3960ctggttctat aaacaagtca ttctacctct ttgtgccctg ctgtttattc
tgtaagggga 4020aggtggcaat gggacccagc tccatcagac acttgtcaag
ctagcagaaa ctccattttc 4080aatgccaaag aagaactgta atgctgtttt
ggaatcatcc caaggcatcc caagacacca 4140tatcttccca tttcaagcac
tgcctgggca caccccaaca tcccaggctg tggtggctcc 4200tgtgggaact
acctagatga agagagtatc atttatacct tctaggagct cctattggga
4260gacatgaaac atatgtaatt gactaccatg taatagaaca aaccctgcca
agtgctgctt 4320tggaaagtca tggaggtaaa agaaagacca ttc 43537728PRTHomo
sapiens 7Met Arg Trp Leu Leu Leu Tyr Tyr Ala Leu Cys Phe Ser Leu
Ser Lys1 5 10 15Ala Ser Ala His Thr Val Glu Leu Asn Asn Met Phe Gly
Gln Ile Gln 20 25 30Ser Pro Gly Tyr Pro Asp Ser Tyr Pro Ser Asp Ser
Glu Val Thr Trp 35 40 45Asn Ile Thr Val Pro Asp Gly Phe Arg Ile Lys
Leu Tyr Phe Met His 50 55 60Phe Asn Leu Glu Ser Ser Tyr Leu Cys Glu
Tyr Asp Tyr Val Lys Val65 70 75 80Glu Thr Glu Asp Gln Val Leu Ala
Thr Phe Cys Gly Arg Glu Thr Thr 85 90 95Asp Thr Glu Gln Thr Pro Gly
Gln Glu Val Val Leu Ser Pro Gly Ser 100 105 110Phe Met Ser Ile Thr
Phe Arg Ser Asp Phe Ser Asn Glu Glu Arg Phe 115 120 125Thr Gly Phe
Asp Ala His Tyr Met Ala Val Asp Val Asp Glu Cys Lys 130 135 140Glu
Arg Glu Asp Glu Glu Leu Ser
Cys Asp His Tyr Cys His Asn Tyr145 150 155 160Ile Gly Gly Tyr Tyr
Cys Ser Cys Arg Phe Gly Tyr Ile Leu His Thr 165 170 175Asp Asn Arg
Thr Cys Arg Val Glu Cys Ser Asp Asn Leu Phe Thr Gln 180 185 190Arg
Thr Gly Val Ile Thr Ser Pro Asp Phe Pro Asn Pro Tyr Pro Lys 195 200
205Ser Ser Glu Cys Leu Tyr Thr Ile Glu Leu Glu Glu Gly Phe Met Val
210 215 220Asn Leu Gln Phe Glu Asp Ile Phe Asp Ile Glu Asp His Pro
Glu Val225 230 235 240Pro Cys Pro Tyr Asp Tyr Ile Lys Ile Lys Val
Gly Pro Lys Val Leu 245 250 255Gly Pro Phe Cys Gly Glu Lys Ala Pro
Glu Pro Ile Ser Thr Gln Ser 260 265 270His Ser Val Leu Ile Leu Phe
His Ser Asp Asn Ser Gly Glu Asn Arg 275 280 285Gly Trp Arg Leu Ser
Tyr Arg Ala Ala Gly Asn Glu Cys Pro Glu Leu 290 295 300Gln Pro Pro
Val His Gly Lys Ile Glu Pro Ser Gln Ala Lys Tyr Phe305 310 315
320Phe Lys Asp Gln Val Leu Val Ser Cys Asp Thr Gly Tyr Lys Val Leu
325 330 335Lys Asp Asn Val Glu Met Asp Thr Phe Gln Ile Glu Cys Leu
Lys Asp 340 345 350Gly Thr Trp Ser Asn Lys Ile Pro Thr Cys Lys Ile
Val Asp Cys Arg 355 360 365Ala Pro Gly Glu Leu Glu His Gly Leu Ile
Thr Phe Ser Thr Arg Asn 370 375 380Asn Leu Thr Thr Tyr Lys Ser Glu
Ile Lys Tyr Ser Cys Gln Glu Pro385 390 395 400Tyr Tyr Lys Met Leu
Asn Asn Asn Thr Gly Ile Tyr Thr Cys Ser Ala 405 410 415Gln Gly Val
Trp Met Asn Lys Val Leu Gly Arg Ser Leu Pro Thr Cys 420 425 430Leu
Pro Glu Cys Gly Gln Pro Ser Arg Ser Leu Pro Ser Leu Val Lys 435 440
445Arg Ile Ile Gly Gly Arg Asn Ala Glu Pro Gly Leu Phe Pro Trp Gln
450 455 460Ala Leu Ile Val Val Glu Asp Thr Ser Arg Val Pro Asn Asp
Lys Trp465 470 475 480Phe Gly Ser Gly Ala Leu Leu Ser Ala Ser Trp
Ile Leu Thr Ala Ala 485 490 495His Val Leu Arg Ser Gln Arg Arg Asp
Thr Thr Val Ile Pro Val Ser 500 505 510Lys Glu His Val Thr Val Tyr
Leu Gly Leu His Asp Val Arg Asp Lys 515 520 525Ser Gly Ala Val Asn
Ser Ser Ala Ala Arg Val Val Leu His Pro Asp 530 535 540Phe Asn Ile
Gln Asn Tyr Asn His Asp Ile Ala Leu Val Gln Leu Gln545 550 555
560Glu Pro Val Pro Leu Gly Pro His Val Met Pro Val Cys Leu Pro Arg
565 570 575Leu Glu Pro Glu Gly Pro Ala Pro His Met Leu Gly Leu Val
Ala Gly 580 585 590Trp Gly Ile Ser Asn Pro Asn Val Thr Val Asp Glu
Ile Ile Ser Ser 595 600 605Gly Thr Arg Thr Leu Ser Asp Val Leu Gln
Tyr Val Lys Leu Pro Val 610 615 620Val Pro His Ala Glu Cys Lys Thr
Ser Tyr Glu Ser Arg Ser Gly Asn625 630 635 640Tyr Ser Val Thr Glu
Asn Met Phe Cys Ala Gly Tyr Tyr Glu Gly Gly 645 650 655Lys Asp Thr
Cys Leu Gly Asp Ser Gly Gly Ala Phe Val Ile Phe Asp 660 665 670Asp
Leu Ser Gln Arg Trp Val Val Gln Gly Leu Val Ser Trp Gly Gly 675 680
685Pro Glu Glu Cys Gly Ser Lys Gln Val Tyr Gly Val Tyr Thr Lys Val
690 695 700Ser Asn Tyr Val Asp Trp Val Trp Glu Gln Met Gly Leu Pro
Gln Ser705 710 715 720Val Val Glu Pro Gln Val Glu Arg
72584137DNAHomo sapiens 8gaagtcagcc acacaggata aaggagggaa
gggaaggagc agatcttttc ggtaggaaga 60cagattttgt tgtcaggttc ctgggagtgc
aagagcaagt caaaggagag agagaggaga 120gaggaaaagc cagagggaga
gagggggaga ggggatctgt tgcaggcagg ggaaggcgtg 180acctgaatgg
agaatgccag ccaattccag agacacacag ggacctcaga acaaagataa
240ggcatcacgg acaccacacc gggcacgagc tcacaggcaa gtcaagctgg
gaggaccaag 300gccgggcagc cgggagcacc caaggcagga aaatgaggtg
gctgcttctc tattatgctc 360tgtgcttctc cctgtcaaag gcttcagccc
acaccgtgga gctaaacaat atgtttggcc 420agatccagtc gcctggttat
ccagactcct atcccagtga ttcagaggtg acttggaata 480tcactgtccc
agatgggttt cggatcaagc tttacttcat gcacttcaac ttggaatcct
540cctacctttg tgaatatgac tatgtgaagg tagaaactga ggaccaggtg
ctggcaacct 600tctgtggcag ggagaccaca gacacagagc agactcccgg
ccaggaggtg gtcctctccc 660ctggctcctt catgtccatc actttccggt
cagatttctc caatgaggag cgtttcacag 720gctttgatgc ccactacatg
gctgtggatg tggacgagtg caaggagagg gaggacgagg 780agctgtcctg
tgaccactac tgccacaact acattggcgg ctactactgc tcctgccgct
840tcggctacat cctccacaca gacaacagga cctgccgagt ggagtgcagt
gacaacctct 900tcactcaaag gactggggtg atcaccagcc ctgacttccc
aaacccttac cccaagagct 960ctgaatgcct gtataccatc gagctggagg
agggtttcat ggtcaacctg cagtttgagg 1020acatatttga cattgaggac
catcctgagg tgccctgccc ctatgactac atcaagatca 1080aagttggtcc
aaaagttttg gggcctttct gtggagagaa agccccagaa cccatcagca
1140cccagagcca cagtgtcctg atcctgttcc atagtgacaa ctcgggagag
aaccggggct 1200ggaggctctc atacagggct gcaggaaatg agtgcccaga
gctacagcct cctgtccatg 1260ggaaaatcga gccctcccaa gccaagtatt
tcttcaaaga ccaagtgctc gtcagctgtg 1320acacaggcta caaagtgctg
aaggataatg tggagatgga cacattccag attgagtgtc 1380tgaaggatgg
gacgtggagt aacaagattc ccacctgtaa aattgtagac tgtagagccc
1440caggagagct ggaacacggg ctgatcacct tctctacaag gaacaacctc
accacataca 1500agtctgagat caaatactcc tgtcaggagc cctattacaa
gatgctcaac aataacacag 1560gtatatatac ctgttctgcc caaggagtct
ggatgaataa agtattgggg agaagcctac 1620ccacctgcct tccagagtgt
ggtcagccct cccgctccct gccaagcctg gtcaagagga 1680tcattggggg
ccgaaatgct gagcctggcc tcttcccgtg gcaggccctg atagtggtgg
1740aggacacttc gagagtgcca aatgacaagt ggtttgggag tggggccctg
ctctctgcgt 1800cctggatcct cacagcagct catgtgctgc gctcccagcg
tagagacacc acggtgatac 1860cagtctccaa ggagcatgtc accgtctacc
tgggcttgca tgatgtgcga gacaaatcgg 1920gggcagtcaa cagctcagct
gcccgagtgg tgctccaccc agacttcaac atccaaaact 1980acaaccacga
tatagctctg gtgcagctgc aggagcctgt gcccctggga ccccacgtta
2040tgcctgtctg cctgccaagg cttgagcctg aaggcccggc cccccacatg
ctgggcctgg 2100tggccggctg gggcatctcc aatcccaatg tgacagtgga
tgagatcatc agcagtggca 2160cacggacctt gtcagatgtc ctgcagtatg
tcaagttacc cgtggtgcct cacgctgagt 2220gcaaaactag ctatgagtcc
cgctcgggca attacagcgt cacggagaac atgttctgtg 2280ctggctacta
cgagggcggc aaagacacgt gccttggaga tagcggtggg gcctttgtca
2340tctttgatga cttgagccag cgctgggtgg tgcaaggcct ggtgtcctgg
gggggacctg 2400aagaatgcgg cagcaagcag gtctatggag tctacacaaa
ggtctccaat tacgtggact 2460gggtgtggga gcagatgggc ttaccacaaa
gtgttgtgga gccccaggtg gaacggtgag 2520ctgacttact tcctcggggc
ctgcctcccc tgagcgaagc tacaccgcac ttccgacagc 2580acactccaca
ttacttatca gaccatatgg aatggaacac actgacctag cggtggcttc
2640tcctaccgag acagccccca ggaccctgag aggcagagtg tggtataggg
aaaaggctcc 2700aggcaggaga cctgtgttcc tgagcttgtc caagtctctt
tccctgtctg ggcctcactc 2760taccgagtaa tacaatgcag gagctcaacc
aaggcctctg tgccaatccc agcactcctt 2820tccaggccat gcttcttacc
ccagtggcct ttattcactc ctgaccactt atcaaaccca 2880tcggtcctac
tgttggtata actgagcttg gacctgacta ttagaaaatg gtttctaaca
2940ttgaactgaa tgccgcatct gtatattttc ctgctctgcc ttctgggact
agccttggcc 3000taatccttcc tctaggagaa gagcattcag gttttgggag
atggctcata gccaagcccc 3060tctctcttag tgtgatccct tggagcacct
tcatgcctgg ggtttctctc ccaaaagctt 3120cttgcagtct aagccttatc
ccttatgttc cccattaaag gaatttcaaa agacatggag 3180aaagttggga
aggtttgtgc tgactgctgg gagcagaata gccgtgggag gcccaccaag
3240cccttaaatt cccattgtca actcagaaca catttgggcc catatgccac
cctggaacac 3300cagctgacac catgggcgtc cacacctgct gctccagaca
agcacaaagc aatctttcag 3360ccttgaaatg tattatctga aaggctacct
gaagcccagg cccgaatatg gggacttagt 3420cgattacctg gaaaaagaaa
agacccacac tgtgtcctgc tgtgcttttg ggcaggaaaa 3480tggaagaaag
agtggggtgg gcacattaga agtcacccaa atcctgccag gctgcctggc
3540atccctgggg catgagctgg gcggagaatc caccccgcag gatgttcaga
gggacccact 3600ccttcatttt tcagagtcaa aggaatcaga ggctcaccca
tggcaggcag tgaaaagagc 3660caggagtcct gggttctagt ccctgctctg
cccccaactg gctgtataac ctttgaaaaa 3720tcattttctt tgtctgagtc
tctggttctc cgtcagcaac aggctggcat aaggtcccct 3780gcaggttcct
tctagctgga gcactcagag cttccctgac tgctagcagc ctctctggcc
3840ctcacagggc tgattgttct ccttctccct ggagctctct ctcctgaaaa
tctccatcag 3900agcaaggcag ccagagaagc ccctgagagg gaatgattgg
gaagtgtcca ctttctcaac 3960cggctcatca aacacactcc tttgtctatg
aatggcacat gtaaatgatg ttatattttg 4020tatcttttat atcatatgct
tcaccattct gtaaagggcc tctgcattgt tgctcccatc 4080aggggtctca
agtggaaata aaccctcgtg gataaccaaa aaaaaaaaaa aaaaaaa 41379686PRTHomo
sapiens 9Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val
Ala Thr1 5 10 15Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg
Leu Ala Ser 20 25 30Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu
Arg Arg Trp Thr 35 40 45Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu
Tyr Phe Thr His Phe 50 55 60Asp Leu Glu Leu Ser His Leu Cys Glu Tyr
Asp Phe Val Lys Leu Ser65 70 75 80Ser Gly Ala Lys Val Leu Ala Thr
Leu Cys Gly Gln Glu Ser Thr Asp 85 90 95Thr Glu Arg Ala Pro Gly Lys
Asp Thr Phe Tyr Ser Leu Gly Ser Ser 100 105 110Leu Asp Ile Thr Phe
Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr 115 120 125Gly Phe Glu
Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln Val 130 135 140Ala
Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His Leu145 150
155 160Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg
Asn 165 170 175Lys Arg Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe
Thr Gln Arg 180 185 190Ser Gly Glu Leu Ser Ser Pro Glu Tyr Pro Arg
Pro Tyr Pro Lys Leu 195 200 205Ser Ser Cys Thr Tyr Ser Ile Ser Leu
Glu Glu Gly Phe Ser Val Ile 210 215 220Leu Asp Phe Val Glu Ser Phe
Asp Val Glu Thr His Pro Glu Thr Leu225 230 235 240Cys Pro Tyr Asp
Phe Leu Lys Ile Gln Thr Asp Arg Glu Glu His Gly 245 250 255Pro Phe
Cys Gly Lys Thr Leu Pro His Arg Ile Glu Thr Lys Ser Asn 260 265
270Thr Val Thr Ile Thr Phe Val Thr Asp Glu Ser Gly Asp His Thr Gly
275 280 285Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Tyr
Pro Met 290 295 300Ala Pro Pro Asn Gly His Val Ser Pro Val Gln Ala
Lys Tyr Ile Leu305 310 315 320Lys Asp Ser Phe Ser Ile Phe Cys Glu
Thr Gly Tyr Glu Leu Leu Gln 325 330 335Gly His Leu Pro Leu Lys Ser
Phe Thr Ala Val Cys Gln Lys Asp Gly 340 345 350Ser Trp Asp Arg Pro
Met Pro Ala Cys Ser Ile Val Asp Cys Gly Pro 355 360 365Pro Asp Asp
Leu Pro Ser Gly Arg Val Glu Tyr Ile Thr Gly Pro Gly 370 375 380Val
Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr Phe385 390
395 400Tyr Thr Met Lys Val Asn Asp Gly Lys Tyr Val Cys Glu Ala Asp
Gly 405 410 415Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val
Cys Glu Pro 420 425 430Val Cys Gly Leu Ser Ala Arg Thr Thr Gly Gly
Arg Ile Tyr Gly Gly 435 440 445Gln Lys Ala Lys Pro Gly Asp Phe Pro
Trp Gln Val Leu Ile Leu Gly 450 455 460Gly Thr Thr Ala Ala Gly Ala
Leu Leu Tyr Asp Asn Trp Val Leu Thr465 470 475 480Ala Ala His Ala
Val Tyr Glu Gln Lys His Asp Ala Ser Ala Leu Asp 485 490 495Ile Arg
Met Gly Thr Leu Lys Arg Leu Ser Pro His Tyr Thr Gln Ala 500 505
510Trp Ser Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Asp Ala Gly
515 520 525Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Asn Asn Lys Val
Val Ile 530 535 540Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro Arg Lys
Glu Ala Glu Ser545 550 555 560Phe Met Arg Thr Asp Asp Ile Gly Thr
Ala Ser Gly Trp Gly Leu Thr 565 570 575Gln Arg Gly Phe Leu Ala Arg
Asn Leu Met Tyr Val Asp Ile Pro Ile 580 585 590Val Asp His Gln Lys
Cys Thr Ala Ala Tyr Glu Lys Pro Pro Tyr Pro 595 600 605Arg Gly Ser
Val Thr Ala Asn Met Leu Cys Ala Gly Leu Glu Ser Gly 610 615 620Gly
Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu625 630
635 640Asp Ser Glu Thr Glu Arg Trp Phe Val Gly Gly Ile Val Ser Trp
Gly 645 650 655Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr Gly Val Tyr
Thr Lys Val 660 665 670Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile
Ser Asp Phe 675 680 685102460DNAHomo sapiens 10ggccagctgg
acgggcacac catgaggctg ctgaccctcc tgggccttct gtgtggctcg 60gtggccaccc
ccttgggccc gaagtggcct gaacctgtgt tcgggcgcct ggcatccccc
120ggctttccag gggagtatgc caatgaccag gagcggcgct ggaccctgac
tgcacccccc 180ggctaccgcc tgcgcctcta cttcacccac ttcgacctgg
agctctccca cctctgcgag 240tacgacttcg tcaagctgag ctcgggggcc
aaggtgctgg ccacgctgtg cgggcaggag 300agcacagaca cggagcgggc
ccctggcaag gacactttct actcgctggg ctccagcctg 360gacattacct
tccgctccga ctactccaac gagaagccgt tcacggggtt cgaggccttc
420tatgcagccg aggacattga cgagtgccag gtggccccgg gagaggcgcc
cacctgcgac 480caccactgcc acaaccacct gggcggtttc tactgctcct
gccgcgcagg ctacgtcctg 540caccgtaaca agcgcacctg ctcagccctg
tgctccggcc aggtcttcac ccagaggtct 600ggggagctca gcagccctga
atacccacgg ccgtatccca aactctccag ttgcacttac 660agcatcagcc
tggaggaggg gttcagtgtc attctggact ttgtggagtc cttcgatgtg
720gagacacacc ctgaaaccct gtgtccctac gactttctca agattcaaac
agacagagaa 780gaacatggcc cattctgtgg gaagacattg ccccacagga
ttgaaacaaa aagcaacacg 840gtgaccatca cctttgtcac agatgaatca
ggagaccaca caggctggaa gatccactac 900acgagcacag cgcagccttg
cccttatccg atggcgccac ctaatggcca cgtttcacct 960gtgcaagcca
aatacatcct gaaagacagc ttctccatct tttgcgagac tggctatgag
1020cttctgcaag gtcacttgcc cctgaaatcc tttactgcag tttgtcagaa
agatggatct 1080tgggaccggc caatgcccgc gtgcagcatt gttgactgtg
gccctcctga tgatctaccc 1140agtggccgag tggagtacat cacaggtcct
ggagtgacca cctacaaagc tgtgattcag 1200tacagctgtg aagagacctt
ctacacaatg aaagtgaatg atggtaaata tgtgtgtgag 1260gctgatggat
tctggacgag ctccaaagga gaaaaatcac tcccagtctg tgagcctgtt
1320tgtggactat cagcccgcac aacaggaggg cgtatatatg gagggcaaaa
ggcaaaacct 1380ggtgattttc cttggcaagt cctgatatta ggtggaacca
cagcagcagg tgcactttta 1440tatgacaact gggtcctaac agctgctcat
gccgtctatg agcaaaaaca tgatgcatcc 1500gccctggaca ttcgaatggg
caccctgaaa agactatcac ctcattatac acaagcctgg 1560tctgaagctg
tttttataca tgaaggttat actcatgatg ctggctttga caatgacata
1620gcactgatta aattgaataa caaagttgta atcaatagca acatcacgcc
tatttgtctg 1680ccaagaaaag aagctgaatc ctttatgagg acagatgaca
ttggaactgc atctggatgg 1740ggattaaccc aaaggggttt tcttgctaga
aatctaatgt atgtcgacat accgattgtt 1800gaccatcaaa aatgtactgc
tgcatatgaa aagccaccct atccaagggg aagtgtaact 1860gctaacatgc
tttgtgctgg cttagaaagt gggggcaagg acagctgcag aggtgacagc
1920ggaggggcac tggtgtttct agatagtgaa acagagaggt ggtttgtggg
aggaatagtg 1980tcctggggtt ccatgaattg tggggaagca ggtcagtatg
gagtctacac aaaagttatt 2040aactatattc cctggatcga gaacataatt
agtgattttt aacttgcgtg tctgcagtca 2100aggattcttc atttttagaa
atgcctgtga agaccttggc agcgacgtgg ctcgagaagc 2160attcatcatt
actgtggaca tggcagttgt tgctccaccc aaaaaaacag actccaggtg
2220aggctgctgt catttctcca cttgccagtt taattccagc cttacccatt
gactcaaggg 2280gacataaacc acgagagtga cagtcatctt tgcccaccca
gtgtaatgtc actgctcaaa 2340ttacatttca ttaccttaaa aagccagtct
cttttcatac tggctgttgg catttctgta 2400aactgcctgt ccatgctctt
tgtttttaaa cttgttctta ttgaaaaaaa aaaaaaaaaa 246011185PRTHomo
sapiens 11Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val
Ala Thr1 5 10 15Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg
Leu Ala Ser 20 25 30Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu
Arg Arg Trp Thr 35 40 45Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu
Tyr Phe Thr His Phe 50 55 60Asp Leu Glu Leu Ser His Leu Cys Glu Tyr
Asp Phe Val Lys Leu Ser65 70 75 80Ser Gly Ala Lys Val Leu Ala Thr
Leu Cys Gly Gln Glu Ser Thr Asp 85 90 95Thr Glu Arg Ala Pro Gly Lys
Asp Thr Phe Tyr Ser Leu Gly Ser Ser 100
105 110Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe
Thr 115 120 125Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu
Cys Gln Val 130 135 140Ala Pro Gly Glu Ala Pro Thr Cys Asp His His
Cys His Asn His Leu145 150 155 160Gly Gly Phe Tyr Cys Ser Cys Arg
Ala Gly Tyr Val Leu His Arg Asn 165 170 175Lys Arg Thr Cys Ser Glu
Gln Ser Leu 180 18512738DNAHomo sapiens 12ggccagctgg acgggcacac
catgaggctg ctgaccctcc tgggccttct gtgtggctcg 60gtggccaccc ccttgggccc
gaagtggcct gaacctgtgt tcgggcgcct ggcatccccc 120ggctttccag
gggagtatgc caatgaccag gagcggcgct ggaccctgac tgcacccccc
180ggctaccgcc tgcgcctcta cttcacccac ttcgacctgg agctctccca
cctctgcgag 240tacgacttcg tcaagctgag ctcgggggcc aaggtgctgg
ccacgctgtg cgggcaggag 300agcacagaca cggagcgggc ccctggcaag
gacactttct actcgctggg ctccagcctg 360gacattacct tccgctccga
ctactccaac gagaagccgt tcacggggtt cgaggccttc 420tatgcagccg
aggacattga cgagtgccag gtggccccgg gagaggcgcc cacctgcgac
480caccactgcc acaaccacct gggcggtttc tactgctcct gccgcgcagg
ctacgtcctg 540caccgtaaca agcgcacctg ctcagagcag agcctctagc
ctcccctgga gctccggcct 600gcccagcagg tcagaagcca gagccagcct
gctggcctca gctccgggtt gggctgagat 660ggctgtgccc caactcccat
tcacccacca tggacccaat aataaacctg gccccacccc 720aaaaaaaaaa aaaaaaaa
7381318DNAArtificial SequenceDNA primer 13gcacccagag ccacagtg
181418DNAArtificial SequenceDNA Primer 14gccttccagt gtgtgggc
181519DNAArtificial SequenceDNA Primer 15gccttccaga gtgtggtca
191619DNAArtificial SequenceDNA Primer 16cgatctggag agcgaactc
191719DNAArtificial SequenceDNA Primer 17ctgttcttca cactggctg
191820DNAArtificial SequenceDNA Primer 18ctgctgagat catgttgttc
201915DNAArtificial SequenceDNA Primer 19ttatacgact cacta 15
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References