U.S. patent application number 12/937331 was filed with the patent office on 2011-06-09 for hyperglycosylated human coagulation factor ix.
This patent application is currently assigned to Novo Nordisk A/S. Invention is credited to Gert Bolt, Claus Kristensen.
Application Number | 20110137011 12/937331 |
Document ID | / |
Family ID | 41217185 |
Filed Date | 2011-06-09 |
United States Patent
Application |
20110137011 |
Kind Code |
A1 |
Bolt; Gert ; et al. |
June 9, 2011 |
HYPERGLYCOSYLATED HUMAN COAGULATION FACTOR IX
Abstract
The invention relates to hyperglycosylated human coagulation
factor IX polypeptides, to processes for preparing said
polypeptides, to pharmaceutical compositions comprising said
polypeptides and to the use of the compounds for the treatment of
diseases alleviated by human coagulation factor IX, in particular,
but not exclusively hemophilia.
Inventors: |
Bolt; Gert; (Vaerlose,
DK) ; Kristensen; Claus; (Niva, DK) |
Assignee: |
Novo Nordisk A/S
Bagsvaerd
DK
|
Family ID: |
41217185 |
Appl. No.: |
12/937331 |
Filed: |
April 21, 2009 |
PCT Filed: |
April 21, 2009 |
PCT NO: |
PCT/EP2009/054707 |
371 Date: |
January 18, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61047605 |
Apr 24, 2008 |
|
|
|
Current U.S.
Class: |
530/322 ;
435/320.1; 435/69.1 |
Current CPC
Class: |
C12N 9/644 20130101;
C12Y 304/21022 20130101; A61P 43/00 20180101; A61P 7/04
20180101 |
Class at
Publication: |
530/322 ;
435/69.1; 435/320.1 |
International
Class: |
C07K 14/745 20060101
C07K014/745; C12P 21/00 20060101 C12P021/00; C12N 15/63 20060101
C12N015/63 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 21, 2008 |
EP |
08103629.5 |
Claims
1. A human factor IX polypeptide analogue having one or more
mutations wherein said mutations result in the introduction of one
or more glycosylation sites in the polypeptide, wherein said
glycosylation comprises N-glycosylation.
2. The polypeptide of claim 1 wherein the one or more mutations
result in no more than a 0, 1, 2, 5, 10, 20, 50 or 100 fold
reduction of proteolytic activity and/or clot activity of the
factor IX polypeptide analogue when compared with the wild-type
polypeptide.
3. The polypeptide of claim 1 wherein the one or more mutations
comprise incorporation of one or more N--X--S/T motifs.
4. The polypeptide of claim 3 wherein the one or more N--X--S/T
motifs are established at positions where the N residue has a
relative side-chain surface accessibility of more than 25% or more
than 50%.
5. The polypeptide of claim 4 wherein said one or more mutations
are selected from the group consisting of: Y1N, S3N+K5S/T,
G4N+L6S/T, F9N+Q11S/T, V10N+G12S/T, G12N+L14S/T, R16N+C18S/T, K22N,
R29N+V31S/T, T35N+R37S/T, R37N, T39N+F41S/T, F41N+K43S/T,
W42N+Q44S/T, Q44N+V46S/T, V46N+G48S/T, D47N+D49S/T, G48N+Q50S/T,
D49N+C51S/T, Q50N+E52S/T, E52N+N54T, S53N+P55S/T, C56S/T,
L57N+G59S/T, G60S/T, S61N+K63S/T, K63N+D65S/T, D65N+N67S/T,
I66N+S68S/T, Y69S/T, Y69N+C71S/T, S68N+E70S/T, E70N+W72S/T,
W72N+P74S/T, P74N+G76S/T, F77N+G79S/T, G79N+N81S/T, K80N+C82S/T,
E83S/T, E83N+D85S/T, L84N+V86S/T, D85N, V86N+C88S/T, T87N+N89S/T,
I90N+N92S/T, K91S/T, I90N+N92S/T, K91N+G93S/T, R94S/T, R94N+E96S/T,
K100N, A103S/T, S102N+D104S/T, A103N+N105S/T, D104N+K106S/T,
V107S/T, K106N+V108S/T, V108N+V110S/T, S110N, E113N+Y115S/T,
G114N+R116S/T, R116N+A118S/T, E119N+Q121S/T, K122S/T,
Q121N+S123S/T, K122N+C124S/T S123N+E125S/T, E125N+A125S/T,
P126N+V128S/T, V128N+F130S/T, P129N+P131S/T, F130N+C132S/T, R134N,
V135N+V137S/T, S136N, S138N, Q139N, T140N+L142S/T, S141N+L143S/T,
K142N, A146N+A148S/T, E147N+V149S/T, A148N+F150S/T, V149N+P515S/T,
F150N+D152S/T, P151N+V153S/T, D152N+D154S/T, V153N+Y155S/T,
D154N+V156S/T, Y155N+N157S/T, V156N, S158N+E160S/T, T159N+A161S/T,
E160N+E162S/T, A161N, E162N+I164S/T, T163N+L165S/T, I164N+D166S/T,
L165N+N167S/T, D166N+I168S/T, I168N+Q170S/T, T169N, Q170N,
S171N+Q173S/T, T172N, Q173N+F175S/T, S174N+N176S/T, F175N+D177S/T,
F178S/T, D177N, F178N+R180S/T, T179N+V181S/T, R180N+V182S/T,
G183+E185S/T, E185N+A187S/T, D186N+K188S/T, K188N+G190S/T,
P189N+Q181S/T, K201N+D203S/T, V202N+A204S/T, D203N+F205S/T,
E213N+W215S/T, E224N+G226S/T, T225N+V227S/T, G226N+K228S/T, K228N,
E239N, E240N+E242S/T, T241N+H243S/T, H243N+E245S/T, K247N+N249S/T,
I251S/T, I251N+I253S/T, R252N+I254S/T, I253N+P255S/T,
P255N+H257S/T, H257N+Y259S/T, N260S/T, A262S/T, A261N+I263S/T,
A262N+N264S/T, I263N+K265S/T, K265N+N267S/T, A266N+H268S/T,
D276N+P278S/T, P278N+V280S/T, E277N+L279S/T, V280N+N282S/T,
Y284S/T, S283N+V285S/T, Y284N, D292N+K294S/T, K293N+Y295S/T, E294N,
F299S/T, I298N+L300S/T, K301N+G303S/T, F302N, G303N+G305S/T,
S304N+Y306S/T, Y306N+S308S/T, R312N+F314S/T, F314N+K316S/T,
H315N+G317S/T, K316N+R138S/T, G317N, R318N+A320S/T, S319N+L321S/T,
L321N+L323S/T, V322N+Q324S/T, Y325N+R327S/T, R327N+P329S/T,
P329N+V331S/T, L330N+D332S/T, D332N+A334S/T, R333N, A334N+C336S/T,
T335N+L337S/T, L337N, R338N, K341N, F342N+I344S/T, T343N+Y345S/T,
Y345N+N347S/T, M348S/T, H354N+G356S/T, E355N+G357S/T,
G357N+D359S/T, R358N, Q362N+D364S/T, E372N+E374S/T, E374N, G375N,
E388N+A390S/T, M391N+G393S/T, K392N+K394S/T, G393N+Y395S/T,
K394N+G396S/T, R403N+V405S/T, I408S/T, K409N+K411S/T, E410N,
K411N+K413S/T, K413N, and combinations thereof.
6. The polypeptide of claim 4, wherein said one or more mutations
are selected from the group consisting of: Y1N, S3N+K5S/T,
G4N+L6S/T, F9N+Q11S/T, V10N+G12S/T, K22N, R37N, Q44N+V46S/T,
V46N+G48S/T, D47N+D49S/T, G48N+Q50S/T, Q50N+E52S/T, E52N+N54T,
S53N+P55S/T, C56S/T, L57N+G59S/T, S61N+K63S/T, I66N+S68S/T, Y69S/T,
S68N+E70S/T, E70N+W72S/T, W72N+P74S/T, P74N+G76S/T, K80N+C82S/T,
L84N+V86S/T, T87N+N89S/T, K91S/T, I90N+N92S/T, K91N+G93S/T,
R94N+E96S/T, K100N, A103S/T, S102N+D104S/T, A103N+N105S/T,
D104N+K106S/T, V107S/T, K106N+V108S/T, V108N+V110S/T,
E113N+Y115S/T, R116N+A118S/T, E119N+Q121S/T, K122S/T,
Q121N+S123S/T, S123N+E125S/T, E125N+A125S/T, P129N+P131S/T, S138N,
T140N+L142S/T, S141N+L143S/T, K142N, A146N+A148S/T, E147N+V149S/T,
A148N+F150S/T, V149N+P515S/T, F150N+D152S/T, P151N+V153S/T,
D152N+D154S/T, V153N+Y155S/T, D154N+V156S/T, Y155N+N157S/T, V156N,
S158N+E160S/T, T159N+A161S/T, E160N+E162S/T, A161N, E162N+I164S/T,
T163N+L165S/T, I164N+D166S/T, L165N+N167S/T, D166N+I168S/T,
I168N+Q170S/T, T169N, Q170N, S171N+Q173S/T, T172N, Q173N+F175S/T,
S174N+N176S/T, F175N+D177S/T, F178S/T, D177N, F178N+R180S/T,
T179N+V181S/T, R180N+V182S/T, G183+E185S/T, E185N+A187S/T,
D186N+K188S/T, K188N+G190S/T, P189N+Q181S/T, K201N+D203S/T,
V202N+A204S/T, D203N+F205S/T, E224N+G226S/T, T225N+V227S/T,
G226N+K228S/T, K228N, E239N, E240N+E242S/T, T241N+H243S/T,
H243N+E245S/T, K247N+N249S/T, I251S/T, R252N+I254S/T,
P255N+H257S/T, H257N+Y249S/T, A262S/T, A261N+I263S/T,
A262N+N264S/T, I263N+K265S/T, K265N+N267S/T, E277N+L279S/T,
V280N+N282S/T, D292N+K294S/T, K293N+Y295S/T, E294N, K301N+G303S/T,
G303N+G305S/T, F314N+K316S/T, K316N+R138S/T, R318N+A320S/T,
L321N+L323S/T, R327N+P329S/T, D332N+A334S/T, R333N, R338N, K341N,
F342N+I344S/T, T343N+Y345S/T, H354N+G356S/T, E355N+G357S/T,
G357N+D359S/T, E372N+E374S/T, E374N, G375N, M391N+G393S/T,
K392N+K394S/T, G393N+Y395S/T, I408S/T, E410N, K413N, and
combinations thereof.
7. The polypeptide of claim 1, wherein said one or more mutations
are selected from the group consisting of: Y1N, K22N, R37N, C56S/T,
G60S/T, Y69S/T, E83S/T, D85N, K91S/T, R94S/T, K100N, A103S/T,
V107S/T, S110N, K122S/T, R134N, S136N, S138N, Q139N, K142N, V156N,
A161N, T169N, Q170N, T172N, F178S/T, D177N, K228N, E239N, I251S/T,
N260S/T, A262S/T, Y284S/T, Y284N, E294N, F299S/T, F302N, G317N,
R333N, L337N, R338N, K341N, M348S/T, G358N, E374N, G375N, I408S/T,
E410N, K413N, and combinations thereof.
8. The polypeptide of claim 1, wherein said one or more mutations
are selected from the group consisting of: T172N, K228N, I251T,
A262T, and combinations thereof.
9. A hyperglycosylated human factor IX polypeptide analogue,
wherein said polypeptide is glycosylated at one or more positions
other than N157 or N167 relative to wild-type human factor IX
polypeptide.
10. The polypeptide of claim 9 wherein said polypeptide is
glycosylated at one or more positions selected from the group
consisting of: Y1, S3, G4, F9, V10, Q11, G12, R16, K22, R29, T35,
R37, T39, F41, W42, Q44, V46, D47, G48, D49, Q50, E52, S53, N54,
L57, N58, G59, S61, K63, D65, I66, N67, S68, Y69, E70, W72, P74,
F77, G79, K80, N81, E83, L84, D85, V86, T87, N89, I90, K91, N92,
R94, K100, N101, S102, A103, D104, N105, K106, V108, S110, E113,
G114, R116, E119, N120, Q121, K122, S123, E125, P126, V128, P129,
F130, R134, V135, S136, S138, Q139, T140, S141, K142, A146, E147,
A148, V149, F150, P151, D152, V153, D154, Y155, V156, S158, T159,
E160, A161, E162, T163, I164, L165, D166, I168, T169, Q170, S171,
T172, Q173, S174, F175, N176, D177, F178, T179, R180, G183, E185,
D186, K188, P189, K201, V202, D203, E213, E224, T225, G226, K228,
E239, E240, T241, H243, K247, N249, I251, R252, I253, P255, H257,
N258, N260, A261, A262, I263, N264, K265, A266, D276, E277, P278,
V280, N282, S283, Y284, D292, K293, E294, N297, I298, K301, F302,
G303, S304, Y306, R312, F314, H315, K316, G317, R318, S319, L321,
V322, Y325, R327, P329, L330, D332, R333, A334, T335, L337, R338,
K341, F342, T343, Y345, N346, H354, E355, G357, R358, Q362, E372,
E374, G375, E388, M391, K392, G393, K394, R403, N406, K409, E410,
K411, K413, and combinations thereof.
11. The polypeptide of claim 9 wherein said polypeptide is
glycosylated at one or more positions selected from the group
consisting of: T172, K228, N249, N260, and combinations
thereof.
12. The polypeptide of claim 9 wherein the hyperglycosylated
polypeptide comprises at least one glycan.
13. A pharmaceutical composition comprising the polypeptide of
claim 1.
14. A process for preparing the polypeptide analogue of claim 1
comprising the steps of: (a) performing site directed mutagenesis
of a polynucleotide encoding a human factor IX polypeptide to
incorporate one or more N--X--S/T motifs into the polynucleotide,
thereby forming a nucleic acid construct; (b) transfecting the
nucleic acid construct into a producer cell; and (c) purifying of
the polypeptide analogue from the transfected producer cell.
15. A nucleic acid construct encoding the human factor IX
polypeptide analogue of claim 1.
16. A pharmaceutical composition comprising the polypeptide of
claim 9.
17. A process for preparing the polypeptide analogue of claim 1
comprising the steps of: (a) performing site directed mutagenesis
of a polynucleotide encoding a human factor IX polypeptide to
incorporate one or more N--X--S/T motifs into the polynucleotide,
thereby forming a nucleic acid construct; (b) transfecting the
nucleic acid construct into a producer cell; and (c) purifying the
polypeptide analogue from the transfected producer cell.
18. A nucleic acid construct encoding the human factor IX
polypeptide analogue of claim 1.
Description
FIELD OF THE INVENTION
[0001] The invention relates to hyperglycosylated human coagulation
factor IX polypeptides, to processes for preparing said
polypeptides, to pharmaceutical compositions comprising said
polypeptides and to the use of the compounds for the treatment of
diseases alleviated by human coagulation factor IX, in particular,
but not exclusively hemophilia.
BACKGROUND OF THE INVENTION
[0002] Coagulation factor IX (FIX) is a vitamin K-dependent
coagulation factor with structural similarities to factor VII,
prothrombin, factor X, and protein C. The circulating human FIX
zymogen consists of 415 amino acids divided into four distinct
domains comprising an N-terminal gamma-carboxyglutamic acid rich
(Gla) domain, two EGF domains, and a C-terminal trypsin-like serine
protease domain. Activation of FIX occurs by limited proteolysis at
Arg.sup.145-Ala.sup.146 and Arg.sup.180-Val.sup.181 releasing a 35
amino acid activation peptide (Schmidt A E, and Bajaj S P (2003)
Trends in Cardiovascular Medicine 13, 39-45). Wild-type human
coagulation factor IX (SEQ ID NO: 1) has two N-glycosylation sites
(N157 and N167).
[0003] The half-lives of some proteins can be prolonged by adding
N-glycans at amino acid positions that are not glycosylated in the
wild-type protein (reviewed by Sinclair A M, and Elliott, S (2005)
Journal of Pharmaceutical Sciences 94, 1626-1635). N-glycans are
attached to proteins by eukaryotic cells producing the protein. The
cellular N-glycosylation machinery recognizes and glycosylates
N-glycosylation signals (N--X--S/T motifs) in the amino acid chain,
as the nascent protein is translocated from the ribosome to the
endoplasmic reticulum (Kiely et al. (1976) Journal of Biological
Chemistry 251, 5490-5495; Glabe et al. (1980) Journal of Biological
Chemistry 255, 9236-9242). Thus, glycoengineered proteins can be
produced by introducing mutations that add N-glycosylation sites to
the amino acid sequence of the protein. This principle has been
employed to obtain longer-acting second generation erythropoietin
(Aranesp.RTM., Amgen). This kind of glycoengineering is very
attractive in terms of production of the biopharmaceutical, since
the final prolonged protein is secreted to the medium of the
producer cells. Thus, unlike PEGylation, glycoengineering does not
complicate and increase the cost of downstream processing.
Furthermore, hyperglycosylation may shield protein epitopes
(Cheng-Mayer et al. (1999) Journal of Virology 73, 5294-5300) and
reduce aggregation by increasing the solubility of the protein
(Song et al. (2001) FEBS Letters 491, 63-66). In effect,
glycoengineering may also improve recombinant proteins by
decreasing their immunogenicity, thus reducing the risk that
patients develop neutralizing antibodies against the protein.
[0004] Interestingly, the influence of N-glycans on clearance
varies among different proteins. Several proteins are not
influenced by removal or addition of N-glycans. In contrast, some
proteins are cleared faster in the absence of their N-glycans and
as mentioned above, the clearance of some proteins can be delayed
by addition of extra N-glycans (Elliott et al. (2003) Nature
Biotechnology 21, 414-421; Perlman et al. (2003) Journal of
Clinical Endocrinology and Metabolism 88, 3227-3235). The
mechanisms by which N-glycans influence the clearance of some
proteins are unknown and may vary between the proteins. For
follicle stimulating hormone, reduced renal clearance due to
increased size and increased negative charge from sialic acids has
been proposed to explain the delay in clearance induced by extra
N-glycans (Perlman et al. (2003), supra). The proteins that are
known to be prolonged by addition of extra N-glycans are mostly
relatively small proteins, which is in agreement with an effect on
renal clearance. For erythropoietin, however, solid evidence for an
important role of either renal or hepatic clearance remains to be
presented. Intracellular degradation of erythropoietin internalized
by cells in the bone marrow after binding to the erythropoietin
receptor has been suggested as the major mechanism for clearance of
circulating erythropoietin (reviewed by Jelkman, (2002) European
Journal of Haematology 69, 265-274). The affinity of longer-acting
hyperglycosylated erythropoietin to the erythropoietin receptor is
reduced compared to wild-type erythropoietin (Elliott et al. (2004)
Experimental Hematology 32, 1146-1155), and recent evidence
suggests a link between the reduced receptor affinity and slower
receptor mediated degradation, leading to a longer circulatory
half-life (Gross and Lodish, (2006) Journal of Biological Chemistry
281, 2024-2032). Interestingly, the reduced receptor binding by
hyperglycosylated erythropoietin appears to result from the
increased sialic acid content (Elliott et al. (2004) Experimental
Hematology 32, 1146-1155). The in vitro activity of
hyperglycosylated erythropoietin is significantly reduced compared
to wild-type erythropoietin, and it is known to a person skilled in
the art that a considerable reduction in specific activity must be
expected when N-glycans are introduced in proteins at sites that
are not glycosylated in the wild-type protein.
[0005] US 2003/0036181 (Maxygen, Inc.) describes the addition of
glycosylation sites to polypeptides. EP 0640619 (Amgen Inc.)
describes erythropoietin analogs with additional glycosylation
sites. Mimuro et al. (2004) Journal of Thrombosis and Haemostasis,
2, 275-280 describes human coagulation factor IX with the mutation
A262T introducing a N-glycosylation site at amino acid position
260.
[0006] There is thus a great need for providing an improved variant
of human factor IX which demonstrates an increased in vivo
circulatory half-life but without dramatically reducing the
proteolytic activity or clot activity when compared with wild-type
human factor IX.
SUMMARY OF THE INVENTION
[0007] According to a first aspect of the invention there is
provided a human factor IX polypeptide analogue having one or more
mutations wherein said mutations result in the introduction of one
or more glycosylation sites in the polypeptide.
[0008] According to a second aspect of the invention there is
provided a glycosylated human factor IX polypeptide analogue,
wherein said polypeptide is glycosylated at one or more positions
other than N157 or N167 relative to wild-type human factor IX
polypeptide.
[0009] According to a further aspect of the invention there is
provided a method of treating hemophilia which comprises
administering to a patient a therapeutically effective amount of a
polypeptide as defined hereinbefore.
DESCRIPTION OF THE FIGURES
[0010] FIG. 1 describes a Western blot of factor IX proteins in
medium from HEK293F cells transiently transfected with pTS87
encoding wild-type human coagulation factor IX or with pGB071,
pGB072, pGB073, pGB074, or pGB075 encoding hyperglycosylated human
coagulation factor IX variants with one N-glycosylation site not
present in wild-type human coagulation factor IX (Example 2).
[0011] FIG. 2 describes a Western blot of the same media as shown
in FIG. 1 upon PNGase F treatment (Example 2).
[0012] FIG. 3 describes a Western blot of factor IX proteins in
medium from HEK293F cells transiently transfected with pGB022
encoding hyperglycosylated (H) human coagulation factor IX
(FIX-T172N-K228N-I251T-A262T) or pTS87 encoding wild-type (W) human
coagulation factor IX. The media were incubated with or without
PNGase F prior to SDS-PAGE (Example 4).
[0013] FIG. 4 describes the mean FIX antigen concentrations versus
time in plasma of coagulation factor IX knock-out mice injected
with the hyperglycosylated recombinant human coagulation factor IX
variant FIX-T172N-K228N-I251T-A262T (full squares) or recombinant
wild-type human coagulation factor IX (open circles) (Example
7).
DETAILED DESCRIPTION OF THE INVENTION
[0014] According to a first aspect of the invention there is
provided a human factor IX polypeptide analogue having one or more
mutations wherein said mutations result in the introduction of one
or more glycosylation sites in the polypeptide.
[0015] According to a second aspect of the invention there is
provided a glycosylated human factor IX polypeptide analogue,
wherein said polypeptide is glycosylated at one or more positions
other than N157 or N167 relative to wild-type human factor IX
polypeptide.
[0016] It has been surprisingly found that the hyperglycosylated
polypeptide analogues of the present invention demonstrate
prolonged circulatory half-life of coagulation factor IX to reduce
the dosing frequency and/or increase the exposure of the
therapeutic peptide.
[0017] The term "protein", "polypeptide" and "peptide" as used
herein means a compound composed of at least five constituent amino
acids connected by peptide bonds. The constituent amino acids may
be from the group of the amino acids encoded by the genetic code
and they may be natural amino acids which are not encoded by the
genetic code, as well as synthetic amino acids. Natural amino acids
which are not encoded by the genetic code are e.g. hydroxyproline,
y-carboxyglutamate, ornithine, phosphoserine, D-alanine and
D-glutamine. Synthetic amino acids comprise amino acids
manufactured by chemical synthesis, i.e. D-isomers of the amino
acids encoded by the genetic code such as D-alanine and D-leucine,
Aib (a-aminoisobutyric acid), Abu (a-aminobutyric acid), Tle
(tert-butylglycine), .beta.-alanine, 3-aminomethyl benzoic acid and
anthranilic acid.
[0018] The term "analogue" as used herein referring to a
polypeptide means a modified peptide wherein one or more amino acid
residues of the peptide have been substituted by other amino acid
residues and/or wherein one or more amino acid residues have been
deleted from the peptide and or wherein one or more amino acid
residues have been added to the peptide. Such addition or deletion
of amino acid residues can take place at the N-terminal of the
peptide and/or at the C-terminal of the peptide. All amino acids
for which the optical isomer is not stated are to be understood to
mean the L-isomer.
[0019] In one embodiment, the one or more mutation results in the
introduction of one or more N-glycosylation sites in the
polypeptide.
[0020] In one embodiment, the one or more mutation results in an
increased molecular weight of the factor IX polypeptide analogue
when compared with the wild-type polypeptide.
[0021] In one embodiment, the one or more mutation results in a
more acidic isoelectric point for the factor IX polypeptide
analogue when compared with the wild-type polypeptide.
[0022] In one embodiment, the one or more mutation results in no
more than a 0, 1, 2, 5, 10, 20, 50 or 100 fold reduction of
proteolytic activity and/or clot activity of the factor IX
polypeptide analogue when compared with the wild-type polypeptide.
In a further embodiment, the one or more mutation results in no
more than a 0 fold reduction of proteolytic activity and/or clot
activity of the factor IX polypeptide analogue when compared with
the wild-type polypeptide.
[0023] The cellular N-glycosylation machinery glycosylates
asparagine residues in N--X--S/T motifs, and potential
N-glycosylation sites can be introduced in proteins by amino acid
alterations establishing such motifs. Thus, in one embodiment, the
mutation within the polypeptide incorporates one or more N--X--S/T
motifs. In a further embodiment, the one or more N--X--S/T motifs
will preferably be established at positions where the residue to be
an "N" has a relative side-chain surface accessibility of more than
25%.
[0024] It will be appreciated that surface accessibilities for the
heavy chain and EGF2 domain may be calculated from published
crystallographic data (1RFN, Hopfner et al. (1999) Structure with
Folding and Design 7, 989-96), while calculations on the GLA and
EGF1 domains may be based on a homology model built from the
crystal (and partially modelled) structure of porcine FIXa (1PFX,
Brandstetter et al. (1995) Proceedings of the National Acedemy of
Science of the USA 92, 9796-9800). In addition, the residues in the
activation peptide, for which no structural information is
available, were considered important. Therefore, in a further
embodiment, the residue having a relative side-chain surface
accessibility of more than 25% may be selected from one or more of
the following:
[0025] Y1, S3, G4, F9, V10, Q11, G12, R16, K22, R29, T35, R37, T39,
F41, W42, Q44, V46, D47, G48, D49, Q50, E52, S53, N54, L57, N58,
G59, S61, K63, D65, I66, N67, S68, Y69, E70, W72, P74, F77, G79,
K80, N81, E83, L84, D85, V86, T87, N89, I90, K91, N92, R94, K100,
N101, S102, A103, D104, N105, K106, V108, S110, E113, G114, R116,
E119, N120, Q121, K122, S123, E125, P126, V128, P129, F130, R134,
V135, S136, S138, Q139, T140, S141, K142, A146, E147, A148, V149,
F150, P151, D152, V153, D154, Y155, V156, S158, T159, E160, A161,
E162, T163, I164, L165, D166, I168, T169, Q170, S171, T172, Q173,
S174, F175, N176, D177, F178, T179, R180, G183, E185, D186, K188,
P189, K201, V202, D203, E213, E224, T225, G226, K228, E239, E240,
T241, H243, K247, N249, I251, R252, I253, P255, H257, N258, N260,
A261, A262, I263, N264, K265, A266, D276, E277, P278, V280, N282,
S283, Y284, D292, K293, E294, N297, I298, K301, F302, G303, S304,
Y306, R312, F314, H315, K316, G317, R318, S319, L321, V322, Y325,
R327, P329, L330, D332, R333, A334, T335, L337, R338, K341, F342,
T343, Y345, N346, H354, E355, G357, R358, Q362, E372, E374, G375,
E388, M391, K392, G393, K394, R403, N406, K409, E410, K411, and
K413.
[0026] The above mentioned amino acid positions within wild-type
human factor IX therefore represent potential targets for
introduction of N-glycosylation sites.
[0027] Thus, in one embodiment, the one or more mutations,
targeting the N-residue having a relative side-chain surface
accessibility of more than 25%, may be selected from the group
consisting of: Y1N, S3N+K5S/T, G4N+L6S/T, F9N+Q11S/T, V10N+G12S/T,
G12N+L14S/T, R16N+C18S/T, K22N, R29N+V31S/T, T35N+R37S/T, R37N,
T39N+F41S/T, F41N+K43S/T, W42N+Q44S/T, Q44N+V46S/T, V46N+G48S/T,
D47N+D49S/T, G48N+Q50S/T, D49N+C51S/T, Q50N+E52S/T, E52N+N54T,
S53N+P55S/T, C56S/T, L57N+G59S/T, G60S/T, S61N+K63S/T, K63N+D65S/T,
D65N+N67S/T, I66N+S68S/T, Y69S/T, Y69N+C71S/T, S68N+E70S/T,
E70N+W72S/T, W72N+P74S/T, P74N+G76S/T, F77N+G79S/T, G79N+N81S/T,
K80N+C82S/T, E83S/T, E83N+D85S/T, L84N+V86S/T, D85N, V86N+C88S/T,
T87N+N89S/T, I90N+N92S/T, K91S/T, I90N+N92S/T, K91N+G93S/T, R94S/T,
R94N+E96S/T, K100N, A103S/T, S102N+D104S/T, A103N+N105S/T,
D104N+K106S/T, V107S/T, K106N+V108S/T, V108N+V110S/T, S110N,
E113N+Y115S/T, G114N+R116S/T, R116N+A118S/T, E119N+Q121S/T,
K122S/T, Q121N+S123S/T, K122N+C124S/T S123N+E125S/T, E125N+A125S/T,
P126N+V128S/T, V128N+F130S/T, P129N+P131S/T, F130N+C132S/T, R134N,
V135N+V137S/T, S136N, S138N, Q139N, T140N+L142S/T, S141N+L143S/T,
K142N, A146N+A148S/T, E147N+V149S/T, A148N+F150S/T, V149N+P515S/T,
F150N+D152S/T, P151N+V153S/T, D152N+D154S/T, V153N+Y155S/T,
D154N+V156S/T, Y155N+N157S/T, V156N, S158N+E160S/T, T159N+A161S/T,
E160N+E162S/T, A161N, E162N+I164S/T, T163N+L165S/T, I164N+D166S/T,
L165N+N167S/T, D166N+I168S/T, I168N+Q170S/T, T169N, Q170N,
S171N+Q173S/T, T172N, Q173N+F175S/T, S174N+N176S/T, F175N+D177S/T,
F178S/T, D177N, F178N+R180S/T, T179N+V181S/T, R180N+V182S/T,
G183+E185S/T, E185N+A187S/T, D186N+K188S/T, K188N+G190S/T,
P189N+Q181S/T, K201N+D203S/T, V202N+A204S/T, D203N+F205S/T,
E213N+W215S/T, E224N+G226S/T, T225N+V227S/T, G226N+K228S/T, K228N,
E239N, E240N+E242S/T, T241N+H243S/T, H243N+E245S/T, K247N+N249S/T,
I251S/T, I251N+I253S/T, R252N+I254S/T, I253N+P255S/T,
P255N+H257S/T, H257N+Y259S/T, N260S/T, A262S/T, A261N+I263S/T,
A262N+N264S/T, I263N+K265S/T, K265N+N267S/T, A266N+H268S/T,
D276N+P278S/T, P278N+V280S/T, E277N+L279S/T, V280N+N282S/T,
Y284S/T, S283N+V285S/T, Y284N, D292N+K294S/T, K293N+Y295S/T, E294N,
F299S/T, I298N+L300S/T, K301N+G303S/T, F302N, G303N+G305S/T,
S304N+Y306S/T, Y306N+S308S/T, R312N+F314S/T, F314N+K316S/T,
H315N+G317S/T, K316N+R138S/T, G317N, R318N+A320S/T, S319N+L321S/T,
L321N+L323S/T, V322N+Q324S/T, Y325N+R327S/T, R327N+P329S/T,
P329N+V331S/T, L330N+D332S/T, D332N+A334S/T, R333N, A334N+C336S/T,
T335N+L337S/T, L337N, R338N, K341N, F342N+I344S/T, T343N+Y345S/T,
Y345N+N347S/T, M348S/T, H354N+G356S/T, E355N+G357S/T,
G357N+D359S/T, R358N, Q362N+D364S/T, E372N+E374S/T, E374N, G375N,
E388N+A390S/T, M391N+G393S/T, K392N+K394S/T, G393N+Y395S/T,
K394N+G396S/T, R403N+V405S/T, I408S/T, K409N+K411S/T, E410N,
K411N+K413S/T, and K413N.
[0028] In a further embodiment, the one or more N--X--S/T motifs
will preferably be established at positions where the residue to be
an "N" has a relative side-chain surface accessibility of more than
50%. Thus, in one embodiment, the residue having a relative
side-chain surface accessibility of more than 50% may be selected
from one or more of the following:
[0029] Y1, S3, G4, F9, V10, Q11, K22, R37, Q44, V46, D47, G48, Q50,
E52, S53, N54, L57, G59, S61, I66, N67, S68, E70, W72, P74, K80,
L84, T87, N89, I90, K91, R94, K100, N101, S102, A103, D104, N105,
K106, V108, E113, R116, E119, N120, Q121, S123, E125, P129, S138,
T140, S141, K142, A146, E147, A148, V149, F150, P151, D152, V153,
D154, Y155, V156, S158, T159, E160, A161, E162, T163, I164, L165,
D166, I168, T169, Q170, S171, T172, Q173, S174, F175, N176, D177,
F178, T179, R180, G183, E185, D186, K188, P189, K201, V202, D203,
E224, T225, G226, K228, E239, E240, T241, H243, K247, N249, R252,
P255, H257, N260, A261, A262, I263, K265, E277, V280, D292, K293,
E294, K301, G303, F314, K316, R318, L321, R327, D332, R333, R338,
K341, F342, T343, H354, E355, G357, E372, E374, G375, M391, K392,
G393, N406, E410, and K413.
[0030] The above mentioned amino acid positions within wild-type
human factor IX therefore represent potential targets for
introduction of N-glycosylation sites.
[0031] Thus, in one embodiment, the one or more mutations,
targeting the N-residue having a relative side-chain surface
accessibility of more than 50%, may be selected from the group
consisting of:
[0032] Y1N, S3N+K5S/T, G4N+L6S/T, F9N+Q11S/T, V10N+G12S/T, K22N,
R37N, Q44N+V46S/T, V46N+G48S/T, D47N+D49S/T, G48N+Q50S/T,
Q50N+E52S/T, E52N+N54T, S53N+P55S/T, C56S/T, L57N+G59S/T,
S61N+K63S/T, I66N+S68S/T, Y69S/T, S68N+E70S/T, E70N+W72S/T,
W72N+P74S/T, P74N+G76S/T, K80N+C82S/T, L84N+V86S/T, T87N+N89S/T,
K91S/T, I90N+N92S/T, K91N+G93S/T, R94N+E96S/T, K100N, A103S/T,
S102N+D104S/T, A103N+N105S/T, D104N+K106S/T, V107S/T,
K106N+V108S/T, V108N+V110S/T, E113N+Y115S/T, R116N+A118S/T,
E119N+Q121S/T, K122S/T, Q121N+S123S/T, S123N+E125S/T,
E125N+A125S/T, P129N+P131S/T, S138N, T140N+L142S/T, S141N+L143S/T,
K142N, A146N+A148S/T, E147N+V149S/T, A148N+F150S/T, V149N+P515S/T,
F150N+D152S/T, P151N+V153S/T, D152N+D154S/T, V153N+Y155S/T,
D154N+V156S/T, Y155N+N157S/T, V156N, S158N+E160S/T, T159N+A161S/T,
E160N+E162S/T, A161N, E162N+I164S/T, T163N+L165S/T, I164N+D166S/T,
L165N+N167S/T, D166N+I168S/T, I168N+Q170S/T, T169N, Q170N,
S171N+Q173S/T, T172N, Q173N+F175S/T, S174N+N176S/T, F175N+D177S/T,
F178S/T, D177N, F178N+R180S/T, T179N+V181S/T, R180N+V182S/T,
G183+E185S/T, E185N+A187S/T, D186N+K188S/T, K188N+G190S/T,
P189N+Q181S/T, K201N+D203S/T, V202N+A204S/T, D203N+F205S/T,
E224N+G226S/T, T225N+V227S/T, G226N+K228S/T, K228N, E239N,
E240N+E242S/T, T241N+H243S/T, H243N+E245S/T, K247N+N249S/T,
I251S/T, R252N+I254S/T, P255N+H257S/T, H257N+Y249S/T, A262S/T,
A261N+I263S/T, A262N+N264S/T, I263N+K265S/T, K265N+N267S/T,
E277N+L279S/T, V280N+N282S/T, D292N+K294S/T, K293N+Y295S/T, E294N,
K301N+G303S/T, G303N+G305S/T, F314N+K316S/T, K316N+R138S/T,
R318N+A320S/T, L321N+L323S/T, R327N+P329S/T, D332N+A334S/T, R333N,
R338N, K341N, F342N+I344S/T, T343N+Y345S/T, H354N+G356S/T,
E355N+G357S/T, G357N+D359S/T, E372N+E374S/T, E374N, G375N,
M391N+G393S/T, K392N+K394S/T, G393N+Y395S/T, I408S/T, E410N, and
K413N.
[0033] It will be appreciated that it is more preferable to
introduce N-glycosylation sites at locations already holding a N at
position 1 or a S/T at position 3. Therefore, in one embodiment,
the one or more N--X--S/T motifs will preferably be established at
positions where the residue to be an "N" is selected from one or
more of the following:
[0034] Y1, K22, R37, N54, N58, N67, N81, D85, N89, N92, K100, N101,
N105, S110, N120, R134, S136, S138, Q139, K142, V156, A161, T169,
Q170, T172, N176, D177, K228, E239, N249, N258, N260, N282, Y284,
E294, N297, F302, G317, R333, L337, R338, K341, N346, R358, E374,
G375, N406, E410, and K413.
[0035] The above mentioned amino acid positions within wild-type
human factor IX therefore represent potential targets for
introduction of N-glycosylation sites.
[0036] Thus, in one embodiment, the one or more mutations,
targeting a residue already holding a N at position 1 or a S/T at
position 3, may be selected from the group consisting of:
[0037] Y1N, K22N, R37N, C56S/T, G60S/T, Y69S/T, E83S/T, D85N,
K91S/T, R94S/T, K100N, A103S/T, V107S/T, S110N, K122S/T, R134N,
S136N, S138N, Q139N, K142N, V156N, A161N, T169N, Q170N, T172N,
F178S/T, D177N, K228N, E239N, I251S/T, N260S/T, A262S/T, Y284S/T,
Y284N, E294N, F299S/T, F302N, G317N, R333N, L337N, R338N, K341N,
M348S/T, G358N, E374N, G375N, I408S/T, E410N, and K413N.
[0038] In a further embodiment, the one or more mutations are
selected from one or more of the following:
[0039] T172N, K228N, I251T and A262T.
[0040] Thus, the one or more glycosylation positions may be
selected from:
[0041] T172, K228, N249 and N260.
[0042] In one embodiment, the one or more mutations do not comprise
A262T as a single mutation within the factor IX polypeptide.
[0043] In one embodiment, the hyperglycosylated polypeptide
comprises at least one glycan.
[0044] Peptides and pharmaceutical compositions according to the
present invention may be used in the treatment of diseases
alleviated by administration of human coagulation factor IX, such
as a bleeding disorder e.g. hemophilia, a blood disease,
hemarthrosis, hematomas, mucocutaneous bleeding, inherited blood
disease, familial bleeding disorder, familial blood disease or
factor replacement therapy. In one embodiment, the disease
alleviated by administration of human coagulation factor IX is
hemophilia, such as hemophilia B or Christmas disease.
[0045] Thus according to a further aspect of the invention there is
provided a method of treating hemophilia which comprises
administering to a patient a therapeutically effective amount of a
polypeptide as defined hereinbefore.
[0046] There is also provided a polypeptide as defined hereinbefore
for use in the treatment of hemophilia.
[0047] There is also provided the use of a polypeptide as defined
hereinbefore in the manufacture of a medicament for the treatment
of hemophilia.
[0048] There is also provided a pharmaceutical composition
comprising a polypeptide as defined hereinbefore for use in the
treatment of hemophilia.
[0049] The term "treatment" and "treating" as used herein means the
management and care of a patient for the purpose of combating a
condition, such as a disease or a disorder. The term is intended to
include the full spectrum of treatments for a given condition from
which the patient is suffering, such as administration of the
active compound to alleviate the symptoms or complications, to
delay the progression of the disease, disorder or condition, to
alleviate or relief the symptoms and complications, and/or to cure
or eliminate the disease, disorder or condition as well as to
prevent the condition, wherein prevention is to be understood as
the management and care of a patient for the purpose of combating
the disease, condition, or disorder and includes the administration
of the active peptides to prevent the onset of the symptoms or
complications. The patient to be treated is preferably a mammal, in
particular a human being, but it may also include animals, such as
dogs, cats, cows, sheep and pigs. It is to be understood, that
therapeutic and prophylactic (preventive) regimes represent
separate aspects of the present invention.
[0050] A "therapeutically effective amount" of a peptide as used
herein means an amount sufficient to cure, alleviate or partially
arrest the clinical manifestations of a given disease and its
complications. An amount adequate to accomplish this is defined as
"therapeutically effective amount". Effective amounts for each
purpose will depend on the type and severity of the disease or
injury as well as the weight and general state of the subject. It
will be understood that determining an appropriate dosage may be
achieved using routine experimentation, by constructing a matrix of
values and testing different points in the matrix, which is all
within the ordinary skills of a trained physician or
veterinary.
[0051] According to a further aspect of the invention there is
provided a process for preparing a polypeptide analogue as
hereinbefore defined which comprises the steps of:
[0052] (a) site directed mutagenesis of human factor IX to
incorporate one or more N--X--S/T motifs into DNA encoding human
factor IX;
[0053] (b) transfection of the resultant nucleic construct into a
producer cell; and
[0054] (c) purification of the polypeptide analogue from the
culture medium of the transfected producer cells.
[0055] According to a further aspect of the invention there is
provided a nucleic acid construct encoding the human factor IX
polypeptide analogue as hereinbefore defined.
[0056] As used herein the term "nucleic acid construct" is intended
to indicate any nucleic acid molecule of cDNA, genomic DNA,
synthetic DNA or RNA origin. The term "construct" is intended to
indicate a nucleic acid segment which may be single- or
double-stranded, and which may be based on a complete or partial
naturally occurring nucleotide sequence encoding a peptide of
interest. The construct may optionally contain other nucleic acid
segments.
[0057] A nucleic acid construct 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 peptide by hybridization using synthetic
oligonucleotide probes in accordance with standard techniques (cf.
J. Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual, 2d
edition, Cold Spring Harbor, N.Y.) and by introducing the relevant
mutations as it is known in the art.
[0058] A nucleic acid construct of the invention may also be
prepared synthetically by established standard methods, e.g. the
phosphoamidite method described by Beaucage and Caruthers,
Tetrahedron Letters 22, 1859-1869 (1981), or the method described
by Matthes et al., EMBO Journal 3, 801-805 (1984). According to the
phosphoamidite method, oligonucleotides are synthesized, e.g. in an
automatic DNA synthesizer, purified, annealed, ligated and cloned
in suitable vectors.
[0059] 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 synthetic,
genomic or cDNA origin (as appropriate), the fragments
corresponding to various parts of the entire nucleic acid
construct, in accordance with standard techniques.
[0060] The nucleic acid construct may also be prepared by
polymerase chain reaction using specific primers, for instance as
described in U.S. Pat. No. 4,683,202 or Saiki et al., Science 239,
487-491 (1988).
[0061] In one embodiment, the nucleic acid construct of the
invention is a DNA construct which term will be used exclusively in
the following for convenience. The statements in the following may
also read on other nucleic acid constructs of the invention with
appropriate adaptions as it will be clear for a person skilled in
the art.
[0062] In one embodiment, the present invention relates to a
recombinant vector comprising a DNA construct of the invention. The
recombinant vector into which the DNA construct of the invention is
inserted 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.
[0063] The vector may be an expression vector in which the DNA
sequence encoding the peptide of the 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
concert for their intended purposes, e.g. transcription initiates
in a promoter and proceeds through the DNA sequence coding for the
peptide.
[0064] 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.
[0065] The DNA sequence encoding the peptide of the invention may
also, if necessary, be operably connected to a suitable terminator,
such as the human growth hormone terminator (Palmiter et al., op.
cit.) or (for fungal hosts) the TPI1 (Alber and Kawasaki, op. cit.)
or ADH3 (McKnight et al., op. cit.) terminators. The vector may
further comprise elements such as polyadenylation signals (e.g.
from SV40 or the adenovirus 5 Elb region), transcriptional enhancer
sequences (e.g. the SV40 enhancer) and translational enhancer
sequences (e.g. the ones encoding adenovirus VA RNAs).
[0066] The recombinant vector of the invention may further comprise
a DNA sequence enabling the vector to replicate in the host cell in
question.
[0067] The vector may also comprise a selectable marker, e.g. a
gene the product of which complements a defect in the host cell,
such as the gene coding for dihydrofolate reductase (DHFR) or the
Schizosaccharomyces pombe TPI gene (described by P. R. Russell,
Gene 40, 125-130 (1985)), or one which confers resistance to a
drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol,
neomycin, hygromycin or methotrexate. For filamentous fungi,
selectable markers include amdS, pyrG, argB, niaD and sC.
[0068] According to a further aspect of the invention there is
provided a cell transfected with a vector as hereinbefore defined.
In one embodiment, the cell is a eukaryotic cell (e.g. a eukaryotic
producer cell). In a further embodiment, the cell is a mammalian
cell, such as a Chinese Hamster Ovary (CHO) cell (e.g CHO-K1) or a
human embryonal kidney (HEK) cell (e.g. HEK293).
[0069] According to a further aspect of the invention, there is
provided a pharmaceutical formulation comprising a polypeptide as
hereinbefore defined.
[0070] The formulation may further comprise a buffer system,
preservative(s), tonicity agent(s), chelating agent(s), stabilizers
and surfactants. In one embodiment 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 one embodiment of the invention the
pharmaceutical formulation is an aqueous solution.
[0071] 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.
[0072] In one embodiment the pharmaceutical formulation is a
freeze-dried formulation, whereto the physician or the patient adds
solvents and/or diluents prior to use.
[0073] In one embodiment the pharmaceutical formulation is a dried
formulation (e.g. freeze-dried or spray-dried) ready for use
without any prior dissolution.
[0074] In one embodiment the invention relates to a pharmaceutical
formulation comprising an aqueous solution of a peptide of the
present invention, and a buffer, wherein said peptide is present in
a concentration from 0.1-100 mg/ml, and wherein said formulation
has a pH from about 2.0 to about 10.0.
[0075] In one embodiment 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.
[0076] In one 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.
[0077] In one embodiment of the invention the formulation further
comprises a pharmaceutically acceptable preservative. In one
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.
[0078] In one embodiment of the invention the preservative is
present in a concentration from 0.1 mg/ml to 20 mg/ml. In one
embodiment of the invention the preservative is present in a
concentration from 0.1 mg/ml to 5 mg/ml. In one embodiment of the
invention the preservative is present in a concentration from 5
mg/ml to 10 mg/ml. In one 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, 20.sup.th edition, 2000.
[0079] In one embodiment of the invention the formulation further
comprises an isotonic agent. In one 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
one embodiment 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 one embodiment 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 one embodiment, the sugar or sugar alcohol
concentration is between about 1 mg/ml and about 150 mg/ml. In one
embodiment of the invention the isotonic agent is present in a
concentration from 1 mg/ml to 50 mg/ml. In one embodiment of the
invention the isotonic agent is present in a concentration from 1
mg/ml to 7 mg/ml. In one embodiment of the invention the isotonic
agent is present in a concentration from 8 mg/ml to 24 mg/ml. In
one 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, 20.sup.th edition, 2000.
[0080] In one embodiment of the invention the formulation further
comprises a chelating agent. In one embodiment of the invention the
chelating agent is selected from salts of
ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic
acid, and mixtures thereof. In one embodiment of the invention the
chelating agent is present in a concentration from 0.1 mg/ml to 5
mg/ml. In one embodiment of the invention the chelating agent is
present in a concentration from 0.1 mg/ml to 2 mg/ml. In one
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, 20.sup.th edition, 2000.
[0081] In one 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, 20.sup.th edition, 2000.
[0082] 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.
[0083] 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 one embodiment, 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 mixtures
thereof) 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 one
embodiment 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 one 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.
[0084] In one 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
mixtures thereof) 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.
[0085] In one embodiment of the invention the formulation further
comprises a stabilizer selected from the group of high molecular
weight polymers or low molecular compounds. In one 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.
[0086] 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.
[0087] In one embodiment of the invention the formulation further
comprises a surfactant. In one 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.
[0088] 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, 20.sup.th
edition, 2000.
[0089] 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.
[0090] Pharmaceutical compositions containing a peptide of 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.
[0091] 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.
[0092] 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.
[0093] 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 peptide of the present invention, 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.
[0094] Compositions of the current invention are useful in the
formulation of solids, semisolids, powder and solutions for
pulmonary administration of a peptide of the present invention,
using, for example a metered dose inhaler, dry powder inhaler and a
nebulizer, all being devices well known to those skilled in the
art.
[0095] 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 and nanoparticles.
[0096] 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).
[0097] 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 peptide of the present
invention in the form of a nasal or pulmonal spray. As a still
further option, the pharmaceutical compositions containing the
peptide of the present 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.
[0098] The invention will now be described with reference to the
following non-limited Examples:
EXAMPLES
Example 1
Generation of Expression Constructs Encoding hyperglycosylated
Human Coagulation Factor IX with One N-glycosylation Site not
Present in Wild-Type Human Factor IX
[0099] Constructs encoding hyperglycosylated human coagulation
factor IX were generated by site-directed mutagenesis of pTS87
consisting of pTT5 with an insert encoding wild-type human
coagulation factor IX. Thus, pTS87 encodes human coagulation factor
IX with N-glycosylation sites at amino acid residues 157 and 167.
pTS87 was mutated with the QuikChange Site-Directed Mutagenesis kit
(Stratagene) as recommended by the manufacturer using the forward
and reverse primer pairs shown in Table 1. The plasmids generated
by the mutagenesis procedure were transformed into competent E.
coli, and plasmids from selected clones screened for mutations by
DNA sequencing. This way the constructs pGB071, pGB072, pGB073,
pGB074, and pGB075 described in Table 2 each encoding human
coagulation factor IX with one N-glycosylation site not present in
wild-type human coagulation factor IX were established.
TABLE-US-00001 TABLE 1 Mutagenesis primers (altered nucleotides
underlined) and the amino acid alterations induced by the mutations
Amino acid Mutagenesis alteration primer pairs T172N Forward
primer: 5'-GGATAACATCACTCAAAGCAACCA ATCATTTAATGACTTCACTCGGG-3'
Reverse primer: 5'-CCCGAGTGAAGTCATTAAATGATT
GGTTGCTTTGAGTGATGTTATCC-3' K228N Forward primer:
5'-GCCCACTGTGTTGAAACTGGTG TTAATATTACAGTTGTCGCAGG-3' Reverse primer:
5'-CCTGCGACAACTGTAATATTAA CACCAGTTTCAACACAGTGGG-3' I251T Forward
primer: 5'-CAGAGCAAAAGCGAAATGTGAC TCGAATTATTCCTCACCACAAC-3' Reverse
primer: 5'-GTTGTGGTGAGGAATAATTCGA GTCACATTTCGCTTTTGCTCTG-3' A262T
Forward primer 5'-CCACAACTACAATGCAACTATTAA
TAAGTACAACCATGACATTGCCC-3' Reverse primer:
5'-GGGCAATGTCATGGTTGTACTTAT TAATAGTTGCATTGTAGTTGTGGC-3' T343N-
Forward primer: Y345T 5'-GTCTTCGATCTACAAAGTTCAACAT
CACTAACAACATGTTCTGTGCTGGC-3' Reverse primer:
5'-GCCAGCACAGAACATGTTGTTAGTG ATGTTGAACTTTGTAGATCGAAGAC-3
TABLE-US-00002 TABLE 2 Amino acid alterations relative to wild-type
human coagulation factor IX and potential N-glycosylation sites in
wild-type or hyperglycosylated human coagulation factor IX encoded
by constructs Amino acid Construct alterations 157 167 172 228 249
260 343 pTS87 -- + + - - - - - pGB071 T172N + + + - - - - pGB072
K228N + + - + - - - pGB073 I251T + + - - + - - pGB073 A262T + + - -
- + - pGB075 T343N- + + - - - - + Y345T
Example 2
Transient Expression in Mammalian HEK293 Cells of hyperglycosylated
Human Coagulation Factor IX with One N-glycosylation Site not
Present in Wild-Type Human Factor IX
[0100] Suspension adapted human embryonal kidney (HEK293F) cells
(Freestyle, Invitrogen) were transfected with the pTS87 expression
plasmid encoding wild-type human coagulation factor IX or the
pGB071, pGB072, pGB073, pGB074, and pGB075 constructs encoding
hyperglycosylated human coagulation factor IX per manufacturer's
instructions. Briefly, 30 .mu.g of each plasmid was incubated for
20 min with 40 .mu.l 293fectin (Invitrogen) and added to
3.times.10.sup.7 cells in a 125 ml Erlenmeyer flask with serum free
Freestyle 293 Expression Medium supplemented with vitamin K. The
transfected cells were incubated in a shaking incubator (37.degree.
C., 8% CO.sub.2 and 125 rpm). Four days after transfection, the
cells were pelleted, and the serum free medium was harvested. The
cells were resuspended in Dulbecco's MEM (Invitrogen) supplemented
with fetal calf serum and vitamin K, and incubated in a shaking
incubator. The next day, the cells were pelleted, and the medium
containing serum was harvested.
[0101] Samples of the harvested serum free media were incubated 1 h
at 37.degree. C. with or without peptide: N-glycosidase F (PNGase
F) and loaded on SDS-PAGE gels and electrophoresed. After
electrophoresis, the proteins in the gel were transferred to a PVDF
membrane by electroblotting. Coagulation factor IX on the membrane
was visualized by sequential incubation of the membrane with rabbit
anti-FIX antibody and IRDye 680 conjugated goat anti-rabbit IgI
(LI-COR). Reading was carried out with an Odyssey scanner (LI-COR)
at 680 nm. Readings of membranes are shown in FIG. 1 (samples
incubated with PNGase F) and FIG. 2 (samples incubated with PNGase
F). The electrophoretic mobilities of the coagulation factor IX
variants secreted by cells transfected with pGB071, pGB072, pGB073,
pGB074, or pGB075 were reduced compared to the wild-type
coagulation factor IX secreted by cells transfected with pTS87
(FIG. 1). PNGase F removes N-linked glycans, and the mobilities of
the mutated and the wild-type coagulation factor IX proteins were
equalized upon PNGase F treatment (FIG. 2), demonstrating that the
different electrophoretic mobilities of the wild-type and variant
FIX proteins prior to PNGase F treatment is related to the
N-glycans. Thus, hyperglycosylated human coagulation factor IX was
indeed produced by cells transfected with pGB071, pGB072, pGB073,
pGB074, or pGB075. This demonstrates the utilization of the
N-glycosylation sites introduced at amino acid position 172, 228,
249, 260, and 343 in the 5 FIX variants, respectively.
[0102] Samples of the harvested media containing serum were
analyzed for coagulation factor IX activity by activated partial
thromboplastin time (APTT) assay in an ACL 9000 clotting instrument
(Instrumentation Laboratory) using reagent from the same
manufacturer. The same media were also analyzed for coagulation
factor IX antigen content by enzyme-linked immunosorbent assay, and
the specific activities of the coagulation factor IX proteins in
the media were calculated by combining the ELISA and clot assay
data as shown in Table 3. The specific activities of the
coagulation factor IX variants with extra N-glycosylation sites at
position 172, 228, 249, or 260 were equal to or moderately reduced
compared to the specific activity of wild-type human coagulation
factor IX. Thus, N-glycans at these positions were not particularly
harmful for the clot activity of human coagulation factor IX. In
contrast, the specific activity of the FIX-T343N-Y354T variant was
dramatically reduced compared to the specific activity of wild-type
human coagulation factor IX.
TABLE-US-00003 TABLE 3 Characterization of wild-type and
hyperglycosylated human coagulation factor IX in medium from
transiently transfected HEK293 cells Factor IX Factor IX activity
antigen Specific Construct Variant (APTT assay) (ELISA) activity
pTS87 Wild-type 0.356 U/ml 5585 ng/ml 0.06 mU/ng FIX pGB071
FIX-T172N 0.258 U/ml 4879 ng/ml 0.05 mU/ng pGB072 FIX-K228N 0.295
U/ml 8565 ng/ml 0.03 mU/ng pGB071 FIX-I251T 0.222 U/ml 4072 ng/m
0.05 mU/ng pGB071 FIX-A262T 0.206 U/ml 8228 ng/m 0.03 mU/ng pGB071
FIX-T343N- 0.007 U/ml 46636 ng/m <0.01 mU/ng Y345T
Example 3
Generation of Expression Constructs Encoding hyperglycosylated
Human Coagulation Factor IX with More than One N-glycosylation Site
not Present in Wild-Type Human Factor IX
[0103] Constructs encoding hyperglycosylated human coagulation
factor IX were generated by site-directed mutagenesis of pTS87
consisting of pTT5 with an insert encoding wild-type human
coagulation factor IX. Thus, pTS87 encodes human coagulation factor
IX with N-glycosylation sites at amino acid residues 157 and 167.
pTS87 was mutated with the QuikChange Multi Site-Directed
Mutagenesis kit (Stratagene) as recommended by the manufacturer
using the primers shown in Table 4. The plasmids generated by the
mutagenesis procedure were transformed into competent E. coli, and
plasmids from selected clones screened for mutations by DNA
sequencing. This way the constructs pGB019, pGB020, pGB021 and
pGB022 described in Table 5 each encoding human coagulation factor
IX with at least two N-glycosylation site not present in wild-type
human coagulation factor IX were established.
[0104] The hyperglycosylated human coagulation factor IX encoding
nucleotide sequence in pGB022 was subcloned by insertion into the
Eco RI site of pMPSVHE to create the plasmid pGB024.
TABLE-US-00004 TABLE 4 Mutagenesis primers (altered nucleotides
underlined) and the amino acid alterations induced by the mutations
Amino acid Mutagenesis alteration primer T172N
5'-GGATAACATCACTCAAAGC AACCAATCATTTAATGAC-3' (SEQ ID NO: 2) K228N
5'-GTGTTGAAACTGGTGTTAA TATTACAGTTGTCGCAGG-3' (SEQ ID NO: 3) I251T
5'-GCAAAAGCGAAATGTGACT CGAATTATTCCTCACCAC-3' (SEQ ID NO: 4) A262T
5'-CCACAACTACAATGCAACTA TTAATAAGTACAACCATGAC-3' (SEQ ID NO: 5)
TABLE-US-00005 TABLE 5 Amino acid alterations relative to wild-type
human coagulation factor IX and potential N-glycosylation sites in
wild-type or hyperglycosylated human coagulation factor IX encoded
by constructs Amino acid Construct alterations 157 167 172 228 249
260 pTS87 -- + + - - - - pGB019 T172N + K228N + + + + - - pGB020
T172N + A262T + + + - - + pGB021 T172N + + + + + - + K228N + A262T
pGB022 T172N + + + + + + + K228N + I251T + A262T pGB024 T172N + + +
+ + + + K228N + I251T + A262T
Example 4
Transient Expression in Mammalian HEK293 Cells of hyperglycosylated
Human Coagulation Factor IX with More than One N-glycosylation Site
not Present in Wild-Type Human Factor IX
[0105] Suspension adapted human embryonal kidney (HEK293F) cells
(Freestyle, Invitrogen) were transfected with the pTS87 expression
plasmid encoding wild-type human coagulation factor IX or the
pGB022 construct encoding hyperglycosylated human coagulation
factor IX per manufacturer's instructions. Briefly, 30 .mu.g of
each plasmid was incubated for 20 min with 40 .mu.l 293fectin
(Invitrogen) and added to 3.times.10.sup.7 cells in a 125 ml
Erlenmeyer flask with Freestyle 293 Expression Medium supplemented
with vitamin K. The transfected cells were incubated in a shaking
incubator (37.degree. C., 8% CO.sub.2 and 125 rpm). Medium
harvested 4 days after transfection was incubated 1 h at 37.degree.
C. with or without peptide: N-glycosidase F (PNGase F) and was
loaded on SDS-PAGE gels and electrophoresed. After electrophoresis,
the proteins in the gel were transferred to a PVDF membrane by
electroblotting. Coagulation factor IX on the membrane was
visualized by sequential incubation of the membrane with rabbit
anti-FIX antibody and HRP-conjugated swine anti-rabbit IgG antibody
(DAKO) followed by incubation with ECL Western Blotting Detection
Reagent (Amersham Biosciences). Reading was carried out with a
Las-1000 Luminescent image analyzer (Fujifilm) and is shown in FIG.
3. The electrophoretic mobility of the coagulation factor IX
secreted by cells transfected with pGB022 was reduced compared to
the wild-type coagulation factor IX secreted by cell transfected
with pTS87. PNGase F removes N-linked glycans, and the mobilities
of the mutated and wild-type coagulation factor IX proteins upon
PNGase F treatment demonstrate that the different electrophoretic
mobilities of the two proteins prior to PNGase F treatment is
related to the N-glycans. Thus, hyperglycosylated human coagulation
factor IX was indeed produced by cells transfected with pGB022.
Example 5
Determination of the Clot Activity of hyperglycosylated Human
Coagulation Factor IX with More than One N-glycosylation Site not
Present in Wild-Type Human Factor IX Transiently Expressed in
Mammalian HEK293 Cells
[0106] Suspension adapted human embryonal kidney (HEK293F) cells
(Freestyle, Invitrogen) were transfected with the pTS87 expression
plasmid encoding wild-type human coagulation factor IX or the
pGB022 construct encoding hyperglycosylated human coagulation
factor IX per manufacturer's instructions. Briefly, 30 pg of each
plasmid was incubated 20 min with 40 .mu.l 293fectin (Invitrogen)
and added to 3.times.10.sup.7 cells in a 125 ml Erlenmeyer flask
with Freestyle 293 Expression Medium (Invitrogen) supplemented with
vitamin K. The transfected cells were incubated in a shaking
incubator (37.degree. C., 8% CO.sub.2 and 125 rpm). Two days after
transfection, the medium was replaced with Dulbecco's MEM
(Invitrogen) supplemented with fetal calf serum and vitamin K. The
cultures were incubated in a shaking incubator two days more, and
medium was harvested. The media were analyzed for coagulation
factor IX activity by activated partial thromboplastin time (APTT)
assay in an ACL 9000 clotting instrument (Instrumentation
Laboratory) using reagent from the same manufacturer. The media
were also analyzed for coagulation factor IX antigen content by
enzyme-linked immunosorbent assay, and the specific activities of
the coagulation factor IX proteins in the media were calculated by
combining the ELISA and clot assay data as shown in Table 6. The
specific activity of the hyperglycosylated coagulation factor IX
variant with extra N-glycosylation sites at position 172, 228, 249,
and 260 encoded by pGB022 was moderately reduced compared to the
specific activity of wild-type human coagulation factor IX. Thus,
the presence of up to 4 extra N-glycans at selected amino acid
positions are not particularly harmful--for the clot activity of
human coagulation factor IX.
TABLE-US-00006 TABLE 6 Characterization of wild-type and
hyperglycosylated human coagulation factor IX in medium from cells
transiently transfected with pTS87 and pGB022, respectively Factor
IX Factor IX activity antigen Specific Construct (APTT assay)
(ELISA) activity pTS87 0.232 U/ml 2260 ng/ml 0.10 mU/ng pGB022
0.065 U/ml 1700 ng/ml 0.04 mU/ng
Example 6
Generation of Stable Mammalian Cell Lines Expressing
hyperglycosylated Human Coagulation Factor IX
[0107] Chinese hamster ovary (CHO-K1) cells were co-transfected
with pSV2-neo containing the neomycin resistance gene and pGB024
encoding the hyperglycosylated FIX-T172N-K228N-I251T-A262T variant
using FuGENE 6 Transfection Reagent (Roche). Transfected cells were
selected with 600 .mu.g/ml G418 and cloned by limiting dilution.
Resistant clones were screened by testing cell culture supernatants
for coagulation factor IX antigen by ELISA, and 12 coagulation
factor IX producing cell lines were expanded. Medium from tissue
culture flasks with the 12 clones was analysed for coagulation
factor IX antigen by ELISA and for coagulation factor IX activity
by activated partial thromboplastin time (APTT) assay in an ACL
9000 clotting instrument (Instrumentation Laboratory) using reagent
from the same manufacturer. The specific activities of the
coagulation factor IX proteins in the media were calculated by
combining the ELISA and clot assay data as shown in Table 7. Clone
15 was seeded in roller bottles, and the hyperglycosylated
FIX-T172N-K228N-I251T-A262T variant was purified from medium
harvested from the roller bottles.
TABLE-US-00007 TABLE 7 Characterization of hyperglycosylated human
coagulation factor IX in medium from CHO-K1 cell lines transfected
with pGB024 Factor IX activity FIX antigen Specific Clone (APTT
assay) (ELISA) activity 2 0.019 U/ml 988 ng/ml 0.019 mU/ng 6 0.013
U/ml 1118 ng/ml 0.012 mU/ng 7 0.009 U/ml 662 ng/ml 0.014 mU/ng 8
0.008 U/ml 606 ng/ml 0.013 mU/ng 9 0.014 U/ml 500 ng/ml 0.021 mU/ng
10 0.048 U/ml 960 ng/ml 0.050 mU/ng 11 0.023 U/ml 996 ng/ml 0.023
mU/ng 12 0.011 U/ml 622 ng/ml 0.018 mU/ng 15 0.029 U/ml 722 ng/ml
0.040 mU/ng 16 0.019 U/ml 360 ng/ml 0.052 mU/ng 17 0.009 U/ml 990
ng/ml 0.009 mU/ng 18 0.019 U/ml 526 ng/ml 0.036 mU/ng
Example 7
Comparison of the Pharmacokinetic Properties of hyperglycosylated
Human Coagulation Factor IX with those Wild-Type Human Coagulation
Factor IX
[0108] Hyperglycosylated human coagulation factor IX
(FIX-T172N-K228N-I251T-A262T) and human wild-type coagulation
factor IX (BeneFIX.RTM., Wyeth) were diluted in 10 mM histidine,
0.26 M glycine, 1% sucrose, 0.005 Tween80, pH 6.8 to a
concentration of 0.2 mg/ml (FIX-T172N-K228N-I251T-A262T) or 0.4
mg/ml (wild-type coagulation factor IX). Each compound was
administered via the tail vein of 15 coagulation factor IX
knock-out mice at a dose of 1.0 mg/kg (FIX-T172N-K228N-I251T-A262T)
or 1.5 mg/kg (wild-type coagulation factor IX). At various time
points after administration, 3 mice were anaesthetized and blood
was sampled from the orbital plexus. Three blood samples were drawn
from each mouse and after the third blood sample, the mice were
euthanized by cervical dislocation. Blood samples were stabilized
with sodium-citrate and diluted 5 times in 50 mM HEPES, 150 nM
NaCl, 0.5% bovine serum albumin, pH 7.4. The stabilized and diluted
blood was centrifuged to pellet blood cells. The resulting plasma
samples were analyzed for FIX antigen by ELISA using compound
specific standard curves. The mean FIX antigen concentrations
versus time are shown in FIG. 4. The estimated pharmacokinetic
parameters are listed in Table 8. The data demonstrate prolongation
of the in vivo half-life and reduced clearance of the
hyperglycosylated FIX-T172N-K228N-I251T-A262T variant as compared
to wild-type coagulation factor IX.
TABLE-US-00008 TABLE 8 Pharmacokinetic parameters in coagulation
factor IX knock-out mice determined by ELISA Mean Terminal
residence Compound half-life Clearance time Wild-type FIX (BeneFIX
.RTM., Wyeth) 9 h 81 ml/h/kg 8 h FIX-T172N-K228N-I251T-A262T 22 h
15 ml/h/kg 27 h
[0109] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference in
their entirety and to the same extent as if each reference were
individually and specifically indicated to be incorporated by
reference and were set forth in its entirety herein (to the maximum
extent permitted by law), regardless of any separately provided
incorporation of particular documents made elsewhere herein.
[0110] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention are to be
construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. For
example, the phrase "the compound" is to be understood as referring
to various "compounds" of the invention or particular described
aspect, unless otherwise indicated.
[0111] Unless otherwise indicated, all exact values provided herein
are representative of corresponding approximate values (e.g., all
exact exemplary values provided with respect to a particular factor
or measurement can be considered to also provide a corresponding
approximate measurement, modified by "about," where
appropriate).
[0112] The description herein of any aspect or aspect of the
invention using terms such as "comprising", "having," "including,"
or "containing" with reference to an element or elements is
intended to provide support for a similar aspect or aspect of the
invention that "consists of", "consists essentially of", or
"substantially comprises" that particular element or elements,
unless otherwise stated or clearly contradicted by context (e.g., a
composition described herein as comprising a particular element
should be understood as also describing a composition consisting of
that element, unless otherwise stated or clearly contradicted by
context).
[0113] Preferred features of the invention:
[0114] 1. A human factor IX polypeptide analogue having one or more
mutations wherein said mutations result in the introduction of one
or more glycosylation sites in the polypeptide.
[0115] 2. A polypeptide as defined in clause 1 wherein said
glycosylation comprises N-glycosylation.
[0116] 3. A polypeptide as defined in clause 1 wherein the one or
more mutations result in an increased molecular weight of the
factor IX polypeptide analogue when compared with the wild-type
polypeptide.
[0117] 4. A polypeptide as defined in clause 1 wherein the one or
more mutations result in a more acidic isoelectric point for the
factor IX polypeptide analogue when compared with the wild-type
polypeptide.
[0118] 5. A polypeptide as defined in clause 1 wherein the one or
more mutations result in no more than a 0, 1, 2, 5, 10, 20, 50 or
100 fold reduction of proteolytic activity and/or clot activity of
the factor IX polypeptide analogue when compared with the wild-type
polypeptide.
[0119] 6. A polypeptide as defined in clause 5 wherein the one or
more mutations result in no more than a 0 fold reduction of
proteolytic activity and/or clot activity of the factor IX
polypeptide analogue when compared with the wild-type
polypeptide.
[0120] 7. A polypeptide as defined in clause 1 or clause 2 wherein
said one or more mutations comprise incorporation of one or more
N--X--S/T motifs.
[0121] 8. A polypeptide as defined in clause 7 wherein the one or
more N--X--S/T motifs are established at positions where said
residue intended to be an "N" residue has a relative side-chain
surface accessibility of more than 25%.
[0122] 9. A polypeptide as defined in clause 8 wherein said one or
more mutations are selected from one or more of the following:
[0123] Y1N, S3N+K5S/T, G4N+L6S/T, F9N+Q11S/T, V10N+G12S/T,
G12N+L14S/T, R16N+C18S/T, K22N, R29N+V31S/T, T35N+R37S/T, R37N,
T39N+F41S/T, F41N+K43S/T, W42N+Q44S/T, Q44N+V46S/T, V46N+G48S/T,
D47N+D49S/T, G48N+Q50S/T, D49N+C51S/T, Q50N+E52S/T, E52N+N54T,
S53N+P55S/T, C56S/T, L57N+G59S/T, G60S/T, S61N+K63S/T, K63N+D65S/T,
D65N+N67S/T, I66N+S68S/T, Y69S/T, Y69N+C71S/T, S68N+E70S/T,
E70N+W72S/T, W72N+P74S/T, P74N+G76S/T, F77N+G79S/T, G79N+N81S/T,
K80N+C82S/T, E83S/T, E83N+D85S/T, L84N+V86S/T, D85N, V86N+C88S/T,
T87N+N89S/T, I90N+N92S/T, K91S/T, I90N+N92S/T, K91N+G93S/T, R94S/T,
R94N+E96S/T, K100N, A103S/T, S102N+D104S/T, A103N+N105S/T,
D104N+K106S/T, V107S/T, K106N+V108S/T, V108N+V110S/T, S110N,
E113N+Y115S/T, G114N+R116S/T, R116N+A118S/T, E119N+Q121S/T,
K122S/T, Q121N+S123S/T, K122N+C124S/T S123N+E125S/T, E125N+A125S/T,
P126N+V128S/T, V128N+F130S/T, P129N+P131S/T, F130N+C132S/T, R134N,
V135N+V137S/T, S136N, S138N, Q139N, T140N+L142S/T, S141N+L143S/T,
K142N, A146N+A148S/T, E147N+V149S/T, A148N+F150S/T, V149N+P515S/T,
F150N+D152S/T, P151N+V153S/T, D152N+D154S/T, V153N+Y155S/T,
D154N+V156S/T, Y155N+N157S/T, V156N, S158N+E160S/T, T159N+A161S/T,
E160N+E162S/T, A161N, E162N+I164S/T, T163N+L165S/T, I164N+D166S/T,
L165N+N167S/T, D166N+I168S/T, I168N+Q170S/T, T169N, Q170N,
S171N+Q173S/T, T172N, Q173N+F175S/T, S174N+N176S/T, F175N+D177S/T,
F178S/T, D177N, F178N+R180S/T, T179N+V181S/T, R180N+V182S/T,
G183+E185S/T, E185N+A187S/T, D186N+K188S/T, K188N+G190S/T,
P189N+Q181S/T, K201N+D203S/T, V202N+A204S/T, D203N+F205S/T,
E213N+W215S/T, E224N+G226S/T, T225N+V227S/T, G226N+K228S/T, K228N,
E239N, E240N+E242S/T, T241N+H243S/T, H243N+E245S/T, K247N+N249S/T,
I251S/T, I251N+I253S/T, R252N+I254S/T, I253N+P255S/T,
P255N+H257S/T, H257N+Y259S/T, N260S/T, A262S/T, A261N+I263S/T,
A262N+N264S/T, I263N+K265S/T, K265N+N267S/T, A266N+H268S/T,
D276N+P278S/T, P278N+V280S/T, E277N+L279S/T, V280N+N282S/T,
Y284S/T, S283N+V285S/T, Y284N, D292N+K294S/T, K293N+Y295S/T, E294N,
F299S/T, I298N+L300S/T, K301N+G303S/T, F302N, G303N+G305S/T,
S304N+Y306S/T, Y306N+S308S/T, R312N+F314S/T, F314N+K316S/T,
H315N+G317S/T, K316N+R138S/T, G317N, R318N+A320S/T, S319N+L321S/T,
L321N+L323S/T, V322N+Q324S/T, Y325N+R327S/T, R327N+P329S/T,
P329N+V331S/T, L330N+D332S/T, D332N+A334S/T, R333N, A334N+C336S/T,
T335N+L337S/T, L337N, R338N, K341N, F342N+I344S/T, T343N+Y345S/T,
Y345N+N347S/T, M348S/T, H354N+G356S/T, E355N+G357S/T,
G357N+D359S/T, R358N, Q362N+D364S/T, E372N+E374S/T, E374N, G375N,
E388N+A390S/T, M391N+G393S/T, K392N+K394S/T, G393N+Y395S/T,
K394N+G396S/T, R403N+V405S/T, I408S/T, K409N+K411S/T, E410N,
K411N+K413S/T, and K413N.
[0124] 10. A polypeptide as defined in clause 7 wherein the one or
more N--X--S/T motifs are established at positions where said
residue intended to be an "N" residue has a relative side-chain
surface accessibility of more than 50%.
[0125] 11. A polypeptide as defined in clause 10, wherein said one
or more mutations are selected from one or more of the
following:
[0126] Y1N, S3N+K5S/T, G4N+L6S/T, F9N+Q11S/T, V10N+G12S/T, K22N,
R37N, Q44N+V46S/T, V46N+G48S/T, D47N+D49S/T, G48N+Q50S/T,
Q50N+E52S/T, E52N+N54T, S53N+P55S/T, C56S/T, L57N+G59S/T,
S61N+K63S/T, I66N+S68S/T, Y69S/T, S68N+E70S/T, E70N+W72S/T,
W72N+P74S/T, P74N+G76S/T, K80N+C82S/T, L84N+V86S/T, T87N+N89S/T,
K91S/T, I90N+N92S/T, K91N+G93S/T, R94N+E96S/T, K100N, A103S/T,
S102N+D104S/T, A103N+N105S/T, D104N+K106S/T, V107S/T,
K106N+V108S/T, V108N+V110S/T, E113N+Y115S/T, R116N+A118S/T,
E119N+Q121S/T, K122S/T, Q121N+S123S/T, S123N+E125S/T,
E125N+A125S/T, P129N+P131S/T, S138N, T140N+L142S/T, S141N+L143S/T,
K142N, A146N+A148S/T, E147N+V149S/T, A148N+F150S/T, V149N+P515S/T,
F150N+D152S/T, P151N+V153S/T, D152N+D154S/T, V153N+Y155S/T,
D154N+V156S/T, Y155N+N157S/T, V156N, S158N+E160S/T, T159N+A161S/T,
E160N+E162S/T, A161N, E162N+I164S/T, T163N+L165S/T, I164N+D166S/T,
L165N+N167S/T, D166N+I168S/T, I168N+Q170S/T, T169N, Q170N,
S171N+Q173S/T, T172N, Q173N+F175S/T, S174N+N176S/T, F175N+D177S/T,
F178S/T, D177N, F178N+R180S/T, T179N+V181S/T, R180N+V182S/T,
G183+E185S/T, E185N+A187S/T, D186N+K188S/T, K188N+G190S/T,
P189N+Q181S/T, K201N+D203S/T, V202N+A204S/T, D203N+F205S/T,
E224N+G226S/T, T225N+V227S/T, G226N+K228S/T, K228N, E239N,
E240N+E242S/T, T241N+H243S/T, H243N+E245S/T, K247N+N249S/T,
I251S/T, R252N+I254S/T, P255N+H257S/T, H257N+Y249S/T, A262S/T,
A261N+I263S/T, A262N+N264S/T, I263N+K265S/T, K265N+N267S/T,
E277N+L279S/T, V280N+N282S/T, D292N+K294S/T, K293N+Y295S/T, E294N,
K301N+G303S/T, G303N+G305S/T, F314N+K316S/T, K316N+R138S/T,
R318N+A320S/T, L321N+L323S/T, R327N+P329S/T, D332N+A334S/T, R333N,
R338N, K341N, F342N+I344S/T, T343N+Y345S/T, H354N+G356S/T,
E355N+G357S/T, G357N+D359S/T, E372N+E374S/T, E374N, G375N,
M391N+G393S/T, K392N+K394S/T, G393N+Y395S/T, I408S/T, E410N, and
K413N.
[0127] 12. A polypeptide as defined in clause 1, wherein said one
or more mutations are selected from one or more of the
following:
[0128] Y1N, K22N, R37N, C56S/T, G60S/T, Y69S/T, E83S/T, D85N,
K91S/T, R94S/T, K100N, A103S/T, V107S/T, S110N, K122S/T, R134N,
S136N, S138N, Q139N, K142N, V156N, A161N, T169N, Q170N, T172N,
F178S/T, D177N, K228N, E239N, I251S/T, N260S/T, A262S/T, Y284S/T,
Y284N, E294N, F299S/T, F302N, G317N, R333N, L337N, R338N, K341N,
M348S/T, G358N, E374N, G375N, I408S/T, E410N, and K413N.
[0129] 13. A polypeptide as defined in clause 1, wherein said one
or more mutations are selected from one or more of the
following:
[0130] T172N, K228N, I251T and A262T.
[0131] 14. A glycosylated human factor IX polypeptide analogue,
wherein said polypeptide is glycosylated at one or more positions
other than N157 or N167 relative to wild-type human factor IX
polypeptide.
[0132] 15. A polypeptide as defined in clause 14 wherein said
polypeptide is glycosylated at one or more positions selected from
one or more of the following:
[0133] Y1, S3, G4, F9, V10, Q11, G12, R16, K22, R29, T35, R37, T39,
F41, W42, Q44, V46, D47, G48, D49, Q50, E52, S53, N54, L57, N58,
G59, S61, K63, D65, I66, N67, S68, Y69, E70, W72, P74, F77, G79,
K80, N81, E83, L84, D85, V86, T87, N89, I90, K91, N92, R94, K100,
N101, S102, A103, D104, N105, K106, V108, S110, E113, G114, R116,
E119, N120, Q121, K122, S123, E125, P126, V128, P129, F130, R134,
V135, S136, S138, Q139, T140, S141, K142, A146, E147, A148, V149,
F150, P151, D152, V153, D154, Y155, V156, S158, T159, E160, A161,
E162, T163, I164, L165, D166, I168, T169, Q170, S171, T172, Q173,
S174, F175, N176, D177, F178, T179, R180, G183, E185, D186, K188,
P189, K201, V202, D203, E213, E224, T225, G226, K228, E239, E240,
T241, H243, K247, N249, I251, R252, I253, P255, H257, N258, N260,
A261, A262, I263, N264, K265, A266, D276, E277, P278, V280, N282,
S283, Y284, D292, K293, E294, N297, I298, K301, F302, G303, S304,
Y306, R312, F314, H315, K316, G317, R318, S319, L321, V322, Y325,
R327, P329, L330, D332, R333, A334, T335, L337, R338, K341, F342,
T343, Y345, N346, H354, E355, G357, R358, Q362, E372, E374, G375,
E388, M391, K392, G393, K394, R403, N406, K409, E410, K411, and
K413.
[0134] 16. A polypeptide as defined in clause 14 wherein said
polypeptide is glycosylated at one or more positions selected from
one or more of the following:
[0135] Y1, S3, G4, F9, V10, Q11, K22, R37, Q44, V46, D47, G48, Q50,
E52, S53, N54, L57, G59, S61, I66, N67, S68, E70, W72, P74, K80,
L84, T87, N89, I90, K91, R94, K100, N101, S102, A103, D104, N105,
K106, V108, E113, R116, E119, N120, Q121, S123, E125, P129, S138,
T140, S141, K142, A146, E147, A148, V149, F150, P151, D152, V153,
D154, Y155, V156, S158, T159, E160, A161, E162, T163, I164, L165,
D166, I168, T169, Q170, S171, T172, Q173, S174, F175, N176, D177,
F178, T179, R180, G183, E185, D186, K188, P189, K201, V202, D203,
E224, T225, G226, K228, E239, E240, T241, H243, K247, N249, R252,
P255, H257, N260, A261, A262, I263, K265, E277, V280, D292, K293,
E294, K301, G303, F314, K316, R318, L321, R327, D332, R333, R338,
K341, F342, T343, H354, E355, G357, E372, E374, G375, M391, K392,
G393, N406, E410, and K413.
[0136] 17. A polypeptide as defined in clause 14 wherein said
polypeptide is glycosylated at one or more positions selected from
one or more of the following:
[0137] Y1, K22, R37, N54, N58, N67, N81, D85, N89, N92, K100, N101,
N105, S110, N120, R134, S136, S138, Q139, K142, V156, A161, T169,
Q170, T172, N176, D177, K228, E239, N249, N258, N260, N282, Y284,
E294, N297, F302, G317, R333, L337, R338, K341, N346, R358, E374,
G375, N406, E410, and K413.
[0138] 18. A polypeptide as defined in clause 14 wherein said
polypeptide is glycosylated at one or more positions selected from
one or more of the following:
[0139] T172, K228, N249 and N260.
[0140] 19. A polypeptide as defined in any of clauses 14 to 18
wherein the hyperglycosylated polypeptide comprises at least one
glycan.
[0141] 20. A method of treating hemophilia which comprises
administering to a patient a therapeutically effective amount of a
polypeptide as defined in any preceding clauses.
[0142] 21. A polypeptide as defined in any of clauses 1 to 19 for
use in the treatment of hemophilia.
[0143] 22. Use of a polypeptide as defined in any of clauses 1 to
19 in the manufacture of a medicament for the treatment of
hemophilia.
[0144] 23. A pharmaceutical composition comprising a polypeptide as
defined in any of clauses 1 to 19 for use in the treatment of
hemophilia.
[0145] 24. A process for preparing a polypeptide analogue as
defined in any of clauses 1 to 19 which comprises the steps of:
[0146] (a) site directed mutagenesis of human factor IX to
incorporate one or more N--X--S/T motifs into DNA encoding human
factor IX;
[0147] (b) transfection of the resultant nucleic construct into a
producer cell; and
[0148] (c) purification of the polypeptide analogue from the
transfected producer cells.
[0149] 25. A nucleic acid construct encoding the human factor IX
polypeptide analogue as defined in any of clauses 1 to 19.
[0150] 26. A recombinant vector comprising a nucleic construct as
defined in clause 25.
[0151] 27. A cell transfected with a vector as defined in clause
26.
[0152] 28. A cell as defined in clause 27 which is a eukaryotic
producer cell.
[0153] 29. A cell as defined in clause 27 or clause 28 which is a
mammalian cell, such as a Chinese Hamster Ovary (CHO) cell (e.g
CHO-K1) or a human embryonal kidney (HEK) cell (e.g. HEK293F).
[0154] 30. A pharmaceutical formulation comprising a polypeptide as
defined in any of clauses 1 to 19.
Sequence CWU 1
1
51415PRTHuman 1Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn
Leu Glu Arg1 5 10 15Glu Cys Met Glu Glu Lys Cys Ser Phe Glu Glu Ala
Arg Glu Val Phe 20 25 30Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys
Gln Tyr Val Asp Gly 35 40 45Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn
Gly Gly Ser Cys Lys Asp 50 55 60Asp Ile Asn Ser Tyr Glu Cys Trp Cys
Pro Phe Gly Phe Glu Gly Lys65 70 75 80Asn Cys Glu Leu Asp Val Thr
Cys Asn Ile Lys Asn Gly Arg Cys Glu 85 90 95Gln Phe Cys Lys Asn Ser
Ala Asp Asn Lys Val Val Cys Ser Cys Thr 100 105 110Glu Gly Tyr Arg
Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val 115 120 125Pro Phe
Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr 130 135
140Arg Ala Glu Ala Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr
Glu145 150 155 160Ala Glu Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr
Gln Ser Phe Asn 165 170 175Asp Phe Thr Arg Val Val Gly Gly Glu Asp
Ala Lys Pro Gly Gln Phe 180 185 190Pro Trp Gln Val Val Leu Asn Gly
Lys Val Asp Ala Phe Cys Gly Gly 195 200 205Ser Ile Val Asn Glu Lys
Trp Ile Val Thr Ala Ala His Cys Val Glu 210 215 220Thr Gly Val Lys
Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu225 230 235 240Thr
Glu His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His 245 250
255His Asn Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu
260 265 270Leu Glu Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr
Pro Ile 275 280 285Cys Ile Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu
Lys Phe Gly Ser 290 295 300Gly Tyr Val Ser Gly Trp Gly Arg Val Phe
His Lys Gly Arg Ser Ala305 310 315 320Leu Val Leu Gln Tyr Leu Arg
Val Pro Leu Val Asp Arg Ala Thr Cys 325 330 335Leu Arg Ser Thr Lys
Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly 340 345 350Phe His Glu
Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro 355 360 365His
Val Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser 370 375
380Trp Gly Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr
Lys385 390 395 400Val Ser Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr
Lys Leu Thr 405 410 415237DNAArtificial SequenceSynthetic
2ggataacatc actcaaagca accaatcatt taatgac 37337DNAArtificial
SequenceSynthetic 3gtgttgaaac tggtgttaat attacagttg tcgcagg
37437DNAArtificial SequenceSynthetic 4gcaaaagcga aatgtgactc
gaattattcc tcaccac 37540DNAArtificial SequenceSynthetic 5ccacaactac
aatgcaacta ttaataagta caaccatgac 40
* * * * *