U.S. patent application number 11/992194 was filed with the patent office on 2009-05-07 for factor h for the treatment of chronic nephropathies and production thereof.
Invention is credited to Peter Gronski, Bernd Hoppe, Christoph Licht, Christine Skerka, Peter Zipfel.
Application Number | 20090118163 11/992194 |
Document ID | / |
Family ID | 36001069 |
Filed Date | 2009-05-07 |
United States Patent
Application |
20090118163 |
Kind Code |
A1 |
Gronski; Peter ; et
al. |
May 7, 2009 |
Factor H for the Treatment of Chronic Nephropathies and Production
thereof
Abstract
The invention is directed to novel uses of Factor H, in
particular in antibody-independent chronic nephropathies, e.g. in
tubulointerstitial fibrosis (TIF). The invention is further
directed to novel large scale manufacturing processes for Factor
H.
Inventors: |
Gronski; Peter; (Marburg,
DE) ; Licht; Christoph; (Koln, DE) ; Hoppe;
Bernd; (Bonn, DE) ; Zipfel; Peter; (Jena,
DE) ; Skerka; Christine; (Jena, DE) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Family ID: |
36001069 |
Appl. No.: |
11/992194 |
Filed: |
June 13, 2006 |
PCT Filed: |
June 13, 2006 |
PCT NO: |
PCT/EP2006/005631 |
371 Date: |
June 9, 2008 |
Current U.S.
Class: |
514/1.1 ;
530/413 |
Current CPC
Class: |
A61P 13/12 20180101;
A61K 39/3955 20130101; C07K 14/435 20130101; A61P 13/00 20180101;
A61K 38/04 20130101; C07K 16/18 20130101 |
Class at
Publication: |
514/8 ;
530/413 |
International
Class: |
A61K 38/14 20060101
A61K038/14; C07K 1/22 20060101 C07K001/22; A61P 13/12 20060101
A61P013/12 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 19, 2005 |
EP |
05020409.8 |
Claims
1.-13. (canceled)
14. A method of treating antibody-independent chronic nephropathy
not causally associated with proteinuria in a patient in need
thereof, the method comprising administering an effective amount of
Factor H to the patient.
15. The method of claim 14, wherein treatment results in
supraphysiological levels of Factor H in the patient's plasma.
16. The method of claim 14, wherein the patient has a Factor H
defect.
17. The method of claim 16, wherein plasma levels of Factor H at
least 10% above the level of the patient's endogenous defective
Factor H are achieved.
18. The method of claim 16, wherein the Factor H defect is a
mutation resulting in the absence of Factor H from the patient's
plasma.
19. The method of claim 16, wherein the Factor H defect is a
mutation in the regulatory domain of Factor H.
20. The method of claim 19, wherein the Factor H is capable of
binding to membranes.
21. The method of claim 16, wherein the Factor H defect is a
mutation in the recognition domain of Factor H.
22. The method of claim 14, wherein the patient is a human and has
a normal human Factor H concentration.
23. The method of claim 22, wherein plasma levels of Factor H at
least 10% above the level of the patient's endogenous defective
Factor H are achieved.
24. The method of claim 14, wherein the patient suffers from
atypical hemolytic uremic syndrome (aHUS) and/or
membranoproliferative glomerulonephritis type II (MPGN II).
25. The method of claim 24, wherein the patient suffers from
tubulointerstitial fibrosis (TIF) and/or progressive renal
failure.
26. The method of claim 14, wherein the Factor H is a recombinant
Factor H.
27. A process for purifying Factor H comprising: obtaining a sample
of human plasma, contacting the human plasma sample with ethanol,
cooling the sample, and fractionally precipitating the sample,
adsorbing the supernatant of the fractional precipitation on a
heparin affinity chromatography column, and eluting Factor H from
the column separately from antithrombin adsorbed on the column.
28. The process of claim 27 wherein the sample of human plasma is
at least 200 liters.
29. The process of claim 28, wherein the sample of human plasma is
at least 500 liters.
30. The process of claim 29, wherein the sample of human plasma is
at least 2000 liters.
31. The process of claim 26, wherein the Factor H product is at
least 60% pure with respect to contaminating proteins and nucleic
acid molecules.
32. The process of claim 26, wherein the Factor H product is free
from infectious agents.
Description
[0001] The invention relates to the use of Factor H for the
manufacture of a medicament to treat both chronic nephropathies
which are not causally associated with proteinuria and chronic
nephropathies which are causally associated with proteinuria. The
invention also relates to large scale purification methods for
Factor H.
[0002] The complement system comprising more than 40 different
proteins directly or indirectly mediates attack and elimination of
microbes, foreign particles and altered self cells via three
different pathways of activation (alternative, lectin and classical
pathway; Rother K et al. (Eds) The Complement System. 2.sup.nd
revised edition, 1998; Springer Verlag). This process is highly
restricted in terms of time and space and can discriminate between
self (host cells) and foreign (e.g., microbes).
[0003] Some human diseases are obviously accompanied by an
activation of these cascade-like activation pathways which is
reflected by the occurrence of elevated levels of typical
activation markers comprising the range from early to late
components of the complement system, including inhibitor-protease
complexes. Moreover, the sometimes observed cellular damage is
taken as indicator of at least a local derailment of the complement
system which usually is under tight control. From a quantitative
point of view, proteolytic cleavage of C3 by specific C3
convertases plays a major role for complement activation. These
convertases generate forms of C3b, which represent a potential
component of new C3 convertase molecules, thereby stimulating the
cascade.
[0004] The protection of self-cells and tissue is mediated by
specific regulators or inhibitors, existing in the fluid-phase
and/or in membrane-bound forms. These regulators include complement
receptor 1 (CR1 or CD35: binds C3b and C4b, disassembles C3
convertases and permits C3b/C4b degradation by factor 1), decay
accelerating factor (DAF or CD55: binds C3b and disassembles C3/C5
convertase) and membrane co-factor protein (MCP or CD46: binds C3b
and C4b to permit their degradation by factor I), which all are
exclusively membrane-anchored proteins.
[0005] In addition to the membrane-anchored control proteins, the
attachment of the soluble complement regulator Factor H
(single-chain glycoprotein composed of 20 short consensus repeats,
SCRs; 155 kDa; .about.9.3% carbohydrate) to the polyanionic surface
of self cells represents a potent component for protection of the
cell surface by increasing the inhibitory potential (Jozsi M et
al.; Histol Histopathol 2004; 19:251-8). This protection is mainly
achieved by efficiently reducing the lifetime of the alternative C3
convertase (C3bBb) by both binding to the covalently bound C3b and
displacing Bb (decay acceleration), and catalysing the permanent
inactivation of C3b via proteolytic cleavage by the serine
proteinase Factor I (co-factor activity: generation of, e.g., iC3b,
C3c; Rother K et al. (2.sup.nd revised edition) The Complement
System. 1998, Springer Verlag; p. 28, 34-7). The activity of Factor
H as co-factor for the protease factor I in the outer phase of the
surface layer (approx. 20-140 nm) is facilitated by binding of
Factor H to surface-located proteoglycans by means of the
C-terminal SCR (Jozsi M et al.; Histol Histopathol 2004; 19:251-8).
The protective potential of Factor H limits locally the progression
of the complement cascade. This is of particular importance for
cells which express a low number of the above mentioned
membrane-anchored regulators, or for tissues which completely lack
those endogenous control proteins, such as the kidney glomerular
basement membrane (Hogasen K et al.; J Clin Invest 1995;
95:1054-61).
[0006] Patent EP 0 222 611 B1 comprises the use of Factor H in
immune complex related diseases in which Factor H is only
temporarily decreased, to down-regulate complement activation,
"Factor H and/or Factor I for use in the treatment of a vascular
autoimmune disease", "Factor H and/or Factor I for use in the
treatment of systemic lupus erythematosus, rheumatoid arthritis or
glomerulonephritis", and "A process for preparing a pharmaceutical
composition for use in the treatment of a vascular autoimmune
disease comprising mixing Factor H and/or Factor I with a
pharmaceutically acceptable carrier, diluent or adjuvant". However,
the scope of this patent is unequivocally related to
glomerulonephritis as an Immune complex (IC)-mediated nephropathy
with glomerular deposition/formation of ICs generated outside or
inside the kidney (e.g., Goodpasture-syndrom). In EP 0 222 611 B1
no teaching is comprised on the treatment of antibody-independent
chronic nephropathies like, e.g., tubulointerstitial fibrosis
(TIF), which specifies the formation of fibrous tissue within the
space between the tubuli (interstitium).
[0007] One embodiment of the invention is the use of Factor H for
the manufacture of a medicament to treat antibody independent
chronic nephropathies, which are not causally linked to
proteinuria.
[0008] A missing or significantly reduced function of Factor H,
either due to missing or reduced protein levels of the functionally
active molecule or due to respective gene mutation(s) in molecular
regions which are important to mediate this function by binding of
relevant ligands, has been demonstrated in diseases which finally
harm kidney function like the atypical hemolytic uremic syndrome
(aHUS) or the membranoproliferative glomerulonephritis type II
(MPGN II). Since the glomerular membrane lacks endogenous
regulators, continuous cleavage of C3 occurs at this site,
resulting in deposition of complement activation products,
presumably in formation of a C3 convertase-mediated damage of the
glomerular basement membranes and of epithelial tubulus and
endothelial cells, membrane thickening via deposition of
extracellular matrix and/or components of the complement system
(e.g., C3 split products) and of antibodies, and, consequently, in
defective filtration (proteinuria).
[0009] MPGN II, also termed "dense deposit disease", is a rare
disease which is characterized by complement containing dense
deposits within the basement membrane of the glomerular capillary
wall and followed by capillary wall thickening, mesangial cell
proliferation and glomerular fibrosis (Ault B H; Pediatr Nephrol
2000; 14:1045-53).
[0010] Besides MPGN II, there are two more subtypes called MPGN I
and MPGN III. All three subtypes are characterized by mesangial
cell proliferation and increase in mesangial matrix combined with a
thickening of the glomerular capillary walls (MPGN I: interposition
of mesangial cells and matrix between basement membrane and
endothelial cells resulting in the formation of a double structure;
subendothelial electron dense deposits. MPGN III: subendothelial
and subepithelial electron dense deposits). Deposits in all
subtypes contain C3 and other complement factors. In some patients
combination of MPGN with extrarenal manifestations like
lipodystrophy and retina alterations can be found (Levy Y et al.;
Immunol Res 1998; 18:55-60).
[0011] MPGN mainly affects children and adults (median age at onset
of disease: about 10 years). 50% of the patients present with
nephrotic syndrome, the others with mild proteinuria, 20% with
macrohematuria. 30% of the patients develop hypertension with onset
of disease. Children with MPGN have an unfavourable late prognosis
and develop end stage renal disease (ESRD) after about 8-16 years
(MPGN I: 15.3 years; MPGN II: 8.7 years; MPGN III: 15.9 years;
Schwertz R et al. Acta Paediatr 1996; 85:308-12).
[0012] Recent findings (Klein R J et al. Science 2005 Mar. 10;
10.1126/science.1109557; Haines J L et al. Science 2005 Mar. 10;
10.1126/science.1110359) indicate an association of an increased
risk of age-related macular degeneration (AMD) and a Factor H
variant (tyrosine-histidine change at amino acid 402 in the short
consensus repeat number 7, SCR7). However, the causal relationship
between a missing Factor H function (SCR7 contains binding sites
for heparin, C-reactive protein and M-protein) and AMD has not yet
been proven.
[0013] A possible therapy of Factor H associated aHUS- and MPGN
II-patients is the administration of fresh frozen plasma, based on
a weight-related treatment schedule (10-40 mL per kg of body weight
biweekly). In this therapy the missing functional Factor H is
restored to normal plasma levels. However, in cases where Factor
H-protein is not reduced but is mutant such that it still binds to
cellular membranes but has lost its ability of decay acceleration
and/or co-factor activity to downregulate the complement system,
mutant Factor H is competitively blocking the linkage of
therapeutically added doses of normal Factor H to the membrane.
Therefore, it is insufficient to restore physiological levels of
functional Factor H on a molar bases, but doses raising the levels
of Factor H above normal need to be administered in order to
replace dysfunctional Factor H from the membrane.
[0014] Factor H mutations can be divided as follows: (1) mutations
which cause a block of Factor H secretion, e.g. from liver cells,
resulting in the complete absence of Factor H in plasma, (2)
mutations which result in a defect of Factor H function (a) in the
regulatory domain of the protein (SCRs 14), (b) in the recognition
domain of the protein (SCRs 19-20), or (c) in different parts of
the protein affecting other functions e.g. heparin binding. While
mutations in the recognition domain (2b) prevent Factor H protein
from binding to surfaces, mutations in the regulatory domain (2a)
results in a functionally defective Factor H but renders the
protein capable to bind to surfaces.
[0015] Supplementation of Factor H via FFP infusion needs to
achieve normal range plasma levels only in case (1) (=missing
plasma Factor H) and (2b) (=mutations affecting Factor H binding).
Supra-normal plasma Factor H levels, however, are required in cases
(2a) and (2c) (=mutations affecting Factor H function while Factor
H binding is unaffected) since endogenous inactive Factor H
molecules compete with infused active Factor H molecules for
surface binding sites.
[0016] One aspect of the current invention is therefore to treat
antibody independent chronic nephropathies like aHUS or MPGN II
with doses of functional Factor H which lead to supraphysiological
plasma concentrations of the newly added Factor H as compared to
natural concentrations of Factor H. Preferentially, the
concentration of Factor H is increased by more than 10% above the
treated patients' normal plasma levels. More preferentially, the
concentration of Factor H is increased by more than 50%, even more
preferentially by more than 100% or even more preferentially by
more than 200% and most preferentially by more than 300% of the
treated patients' normal plasma levels
[0017] Another embodiment of the invention is the use of Factor H
for the manufacture of a medicament to treat antibody independent
chronic nephropathies, which are causally linked to
proteinuria.
[0018] Prospective, randomized clinical trials have indicated that
insufficient glomerular filtration of proteins is linked to
proteinuria and is a major risk for the onset and progression of
both interstitial fibrosis and progressive renal failure (Jerums G
et al. Kidney Int Suppl 1997; 63:87-92). Little is known about the
mechanisms responsible for the spread of tissue injury from the
glomerular to the tubular compartment in diseases and how
interstitial fibrosis is induced has not been addressed.
[0019] Activation of complement via the alternative pathway has
been shown to be involved at the site of proximal tubular
epithelial cells (reviewed in Tang S et al. Kidney Blood Press Res.
2002; 25:120-6), a mechanism known to be essentially
antibody-independent.
[0020] Another more recent publication substantiates the antibody
independent activation of the alternative pathway of complement as
concomitant of acute tubular necrosis (Thurman J M et al. Kidney
Int 2005; 67:524-30).
[0021] It has been demonstrated that protein overload in the
absence of antibody deposition is associated with the activation of
complement components on the apical membrane of proximal tubules.
The proposed mechanism involved augmented intrarenal levels of
ammonia (ammoniagenesis), a nucleophile which can activate C3,
including the terminal complement cascade (reviewed in: I-Hong Hsu
S, Couser W G. J Am Soc Nephrol 2003; 14:186-91). Various natural
and artificial complement inhibitors, like soluble complement
receptor 1, CR1, decay accelerating factor, DAF, and other
molecules are discussed as potential therapeutic targets for
pharmacologic intervention (see same review). However, the use of
Factor H is not mentioned.
[0022] Thus the use of Factor H as a therapeutic for the protection
of cellular membranes lacking endogenous membrane-anchored
regulators is new and has not yet been investigated in in-vitro or
in-vitro models. In general, patients with antibody independent
chronic nephropathies which are causally associated with
proteinuria, who benefit from Factor H have normal Factor H levels.
The therapeutic effect is preferentially achieved by increasing the
Factor H concentration to supraphysiological levels. The reason why
this specific aspect has not been investigated so far may be due to
the function of Factor H as a protease-associated co-factor, which
is not consumed like a substrate.
[0023] One embodiment of the present invention is providing Factor
H for the treatment of chronic nephropathies, which are causally
associated with proteinuria, the generation of which is independent
from antibody-mediated IC formation. Proteinuria can be primarily
caused by alterations of structural proteins involved in the
cellular mechanism of filtration. However, the subsequent presence
of plasma proteins is thought to promote complement-mediated,
IC-independent cellular damage which apparently happens in the
absence of endogenous membrane regulators (e.g., CR1, DAF) despite
normal levels of Factor H. The pathophysiological causes of
proteinuria can be divided in the following major groups: (1)
genetically determined disturbances of the structures which form
the "glomerular filtration unit" like the glomerular basement
membrane, the podocytes, or the slit diaphragm, (2) inflammatory
processes, either directly caused by autoimmune processes or
indirectly induced by microbes, (3) damage of the glomeruli caused
by agents, or (4) as the final result of progressive
tubulointerstitial injury finally resulting in the loss of function
of the entire nephron. More specifically this invention relates to
the use of Factor H to treat TIF on the level of the tubular
epithelial cells where proteinuria induces the cascade of events
(inflammation and fibrosis) which finally results in TIF. Doses of
Factor H which lead to supraphysiological plasma concentrations of
the newly added Factor H as compared to natural concentrations of
Factor H are preferred embodiments of the present invention.
Preferentially the concentration of Factor H is increased by more
than 10% above the patients' individual normal plasma levels. More
preferentially the concentration of Factor H is increased by more
than 50%, even more preferentially by more than 100% or even more
preferentially by more than 200% and most preferentially by more
than 300% of the patients' individual normal plasma levels.
[0024] Factor H can be obtained from human plasma or serum or
recombinantly. "Factor H" as used in the present invention
comprises proteins that have the amino acid sequence of native
human Factor H. It also comprises proteins with a slightly modified
amino acid sequence, for instance, a modified N-terminal end
including N-terminal amino acid deletions or additions so long as
those proteins substantially retain the activity of Factor H.
"Factor H" within the above definition also comprises natural
allelic variations that may exist and occur from one individual to
another. "Factor H" within the above definition further comprises
of Factor H. Such variants differ in one or more amino acid
residues from the wild type sequence. Examples of such differences
may include truncation of the N- and/or C-terminus by one or more
amino acid residues (e.g. 1 to 10 amino acid residues), or addition
of one or more extra residues at the N- and/or C-terminus, e.g.
addition of a methionine residue at the N-terminus, as well as
conservative amino acid substitutions, i.e. substitutions performed
within groups of amino acids with similar characteristics, e.g. (1)
small amino acids, (2) acidic amino acids, (3) polar amino acids,
(4) basic amino acids, (5) hydrophobic amino acids, (6) aromatic
amino acids. Examples of such conservative substitutions are shown
in the following table.
TABLE-US-00001 (1) Alanine Glycine (2) Aspartic acid Glutamic acid
(3a) Asparagine Glutamine (3b) Serine Threonine (4) Arginine
Histidine Lysine (5) Isoleucine Leucine Methionine Valine (6)
Phenylalanine Tyrosine Tryptophane
[0025] "Functional Factor H" as used in this invention comprises
Factor H molecules displaying activity either in solution and/or on
cellular surfaces like decay acceleration of alternative C3
convertase and/or co-factor activity, catalyzing the permanent
proteolysis of C3b by Factor I.
[0026] The term "recombinant" means, for example, that the variant
has been produced in a host organism by genetic engineering
techniques.
The host cells of the invention may be employed in a method of
producing human Factor H. The method comprises: a) culturing host
cells of the invention under conditions such that human Factor H is
expressed; and b) optionally recovering human Factor H from the
host cells or from the culture medium.
[0027] Degree and location of glycosylation or other
post-translation modifications may vary depending on the chosen
host cells and the nature of the host cellular environment. When
referring to specific amino acid sequences, posttranslational
modifications of such sequences are encompassed in this
application.
[0028] The production of recombinant proteins at high levels in
suitable host cells, requires the assembly of the above-mentioned
modified cDNAs into efficient transcriptional units together with
suitable regulatory elements in a recombinant expression vector,
that can be propagated in various expression systems according to
methods known to those skilled in the art. Efficient
transcriptional regulatory elements could be derived from viruses
having animal cells as their natural hosts or from the chromosomal
DNA of animal cells. Preferably, promoter-enhancer combinations
derived from the Simian Virus 40, adenovirus, BK polyoma virus,
human cytomegalovirus, or the long terminal repeat of Rous sarcoma
virus, or promoter-enhancer combinations including strongly
constitutively transcribed genes in animal cells like beta-actin or
GRP78 can be used. In order to achieve stable high levels of mRNA
transcribed from the cDNAs, the transcriptional unit should contain
in its 3'-proximal part a DNA region encoding a transcriptional
termination-polyadenylation sequence. Preferably, this sequence is
derived from the Simian Virus 40 early transcriptional region, the
rabbit beta-globin gene, or the human tissue plasminogen activator
gene.
[0029] The cDNAs are then integrated into the genome of a suitable
host cell line for expression of the Factor H. Preferably this cell
line should be an animal cell-line of vertebrate origin in order to
ensure correct folding, Gla-domain synthesis, disulfide bond
formation, asparagine-linked glycosylation, O-linked glycosylation,
and other post-translational modifications as well as secretion
into the cultivation medium. Examples of other post-translational
modifications are hydroxylation and proteolytic processing of the
nascent polypeptide chain. Examples of cell lines that can be used
are monkey COS-cells, mouse L-cells, mouse C127-cells, hamster
BHK-21 cells, human embryonic kidney 293 cells, and preferentially
hamster CHO-cells. Due to its complex post-translational
modifications recombinant Factor H is preferably expressed in human
cell lines.
[0030] The recombinant expression vector encoding the corresponding
cDNAs can be introduced into an animal cell line in several
different ways. For instance, recombinant expression vectors can be
created from vectors based on different animal viruses. Examples of
these are vectors based on baculovirus, vaccinia virus, adenovirus,
and preferably bovine papilloma virus.
[0031] The transcription units encoding the corresponding DNAs can
also be introduced into animal cells together with another
recombinant gene which may function as a dominant selectable marker
in these cells in order to facilitate the isolation of specific
cell clones which have integrated the recombinant DNA into their
genome. Examples of this type of dominant selectable marker genes
are Tn5 amino glycoside phosphotransferase, conferring resistance
to geneticin (G418), hygromycin phosphotransferase, conferring
resistance to hygromycin, and puromycin acetyl transferase,
conferring resistance to puromycin. The recombinant expression
vector encoding such a selectable marker can reside either on the
same vector as the one encoding the cDNA of the desired protein, or
it can be encoded on a separate vector which is simultaneously
introduced and integrated into the genome of the host cell,
frequently resulting in a tight physical linkage between the
different transcription units.
[0032] Other types of selectable marker genes, which can be used
together with the cDNA of the desired protein, are based on various
transcription units encoding dihydrofolate reductase (dhfr). After
introduction of this type of gene into cells lacking endogenous
dhfr-activity, preferentially CHO-cells (DUKX-B11, DG-44) it will
enable these to grow in media lacking nucleosides. An example of
such a medium is Ham's F12 without hypoxanthine, thymidin, and
glycine. These dhfr-genes can be introduced together with the
coagulation factor cDNA transcriptional units into CHO-cells of the
above type, either linked on the same vector or on different
vectors, thus creating dhfr-positive cell lines producing
recombinant protein.
[0033] If the above cell lines are grown in the presence of the
cytotoxic dhfr-inhibitor methotrexate, new cell lines resistant to
methotrexate will emerge. These cell lines may produce recombinant
protein at an increased rate due to the amplified number of linked
dhfr and the desired protein's transcriptional units. When
propagating these cell lines in increasing concentrations of
methotrexate (1-10000 nM), new cell lines can be obtained which
produce the desired protein at very high rate.
[0034] The above cell lines producing the desired protein can be
grown on a large scale, either in suspension culture or on various
solid supports. Examples of these supports are micro carriers based
on dextran or collagen matrices, or solid supports in the form of
hollow fibres or various ceramic materials. When grown in cell
suspension culture or on micro carriers the culture of the above
cell lines can be performed either as a bath culture or as a
perfusion culture with continuous production of conditioned medium
over extended periods of time. Thus, according to the present
invention, the above cell lines are well suited for the development
of an industrial process for the production of the desired
recombinant proteins
[0035] The recombinant protein, which accumulates in the medium of
secreting cells of the above types, can be concentrated and
purified by a variety of biochemical and chromatographic methods,
including methods utilizing differences in size, charge,
hydrophobicity, solubility, specific affinity, etc. between the
desired protein and other substances in the cell cultivation
medium.
[0036] An example of such purification is the adsorption of the
recombinant protein to a monoclonal antibody, which is immobilised
on a solid support. After desorption, the protein can be further
purified by a variety of chromatographic techniques based on the
above properties.
[0037] It is preferred to purify Factor H of the present invention
to .gtoreq.60% purity, more preferably .gtoreq.80% purity, and
particularly preferred is a pharmaceutically pure state that is
greater than 95% pure with respect to contaminating macromolecules,
particularly other proteins and nucleic acids, and free of
infectious and pyrogenic agents.
[0038] All of the potential purification procedures cited in EP 0
222 611 B1 are typical laboratory methodologies exclusively
developed for purification of a single protein from human plasma or
serum, disregarding the technology established in praxis by
industry which is usually based on multicomponent-use with a focus
on albumin, immunoglobulins and clotting factors. Industrial
scale-procedures adapted to already established routine process
routes are not yet existing.
[0039] Therefore, another objective of the present invention is to
provide a production procedure for a plasma-derived version of
human Factor H for therapeutic use suitable for large scale. Large
scale with regard to the present invention means a production
procedure based on at least 200 l plasma, preferentially at least
500 l, even more preferentially at least 2000 l human plasma.
Regarding production, the claimed processes starting from human
plasma shall be based on the subfractionation of typical industrial
intermediates obtained by, e.g., the fractional precipitation by
ethanol in the cold (reviewed in Schultze H E, Heremans J F;
Molecular Biology of Human Proteins. Volume I: Nature and
Metabolism of Extracellular Proteins 1966, Elsevier Publishing
Company; p. 236-317). A preferred embodiment of such purification
is the purification of functional Factor H from side fractions of
industrial scale plasma fractionation in such a way that
established and licensed manufacturing processes of plasma
products, which are under control of pharmaceutical regulatory
authorities, like antithrombin (AT) or immunoglobulins are not
affected. The supernatant of the 8% ethanol-precipitate (method of
Cohn et al.; previous citation, p. 251) is one example of a source
of Factor H, originating from industrial scale plasma
fractionation. AT together with Factor H can be adsorbed from this
supernatant by Heparin-based affinity chromatography and Factor H
can be purified fractions of elution which do not contain AT.
Precipitate III (method of Oncley et al.; previous citation, p.
253) or precipitate B (method of Kistler and Nitschmann; previous
citation, p. 253) are other examples of such industrial sources for
Factor H in case adsorption of AT is not routinely carried out.
Starting from those side fractions, purification procedures known
in the art can be used to purify Factor H. They may be based on
precipitation with polyethylene glycol (Nagasawa S, Stroud R M; Mol
Immunol 1980; 17:1365-72), affinity chromatography via immobilized
heparin (citation as before), ion exchange chromatography (Crossley
L G, Porter R R; Biochem J 1980; 191:173-82) and hydrophobic
interaction chromatography (Ripoche J, Al Salihi A, Rousseaux J,
Fontaine M; Biochem J 1984; 221, 89-96).
[0040] Factor H as described in this invention can be formulated
into pharmaceutical preparations for therapeutic use. The purified
protein may be dissolved in conventional physiologically compatible
aqueous buffer solutions to which there may be added, optionally,
pharmaceutical excipients to provide pharmaceutical
preparations.
[0041] Such pharmaceutical carriers and excipients as well as
suitable pharmaceutical formulations are well known in the art (see
for example "Pharmaceutical Formulation Development of Peptides and
Proteins", Frokjaer et al., Taylor & Francis (2000) or
"Handbook of Pharmaceutical Excipients", 3.sup.rd edition, Kibbe et
al., Pharmaceutical Press (2000)). In particular, the
pharmaceutical composition comprising the polypeptide variant of
the invention may be formulated in lyophilized or stable soluble
form. The polypeptide variant may be lyophilized by a variety of
procedures known in the art. Lyophilized formulations are
reconstituted prior to use by the addition of one or more
pharmaceutically acceptable diluents such as sterile water for
injection or sterile physiological saline solution.
[0042] Formulations of the composition are delivered to the
individual by any pharmaceutically suitable means of
administration. Various delivery systems are known and can be used
to administer the composition by any convenient route.
Preferentially the compositions of the invention are administered
systemically. For systemic use, Factor H of the invention is
formulated for parenteral (e.g. intravenous, subcutaneous,
intramuscular, intraperitoneal, intracerebral, intrapulmonar,
intranasal or transdermal) or enteral (e.g., oral, vaginal or
rectal) delivery according to conventional methods. The most
preferential routes of administration are intravenous and
subcutaneous administration. The formulations can be administered
continuously by infusion or by bolus injection. Some formulations
encompass slow release systems.
[0043] The Factor H of the present invention is administered to
patients in a therapeutically effective dose, meaning a dose that
is sufficient to produce the desired effects, preventing or
lessening the severity or spread of the condition or indication
being treated without reaching a dose which produces intolerable
adverse side effects. The exact dose depends on many factors as
e.g. the indication, formulation, mode of administration and has to
be determined in preclinical and clinical trials for each
respective indication.
[0044] The pharmaceutical composition of the invention may be
administered alone or in conjunction with other therapeutic agents.
These agents may be incorporated as part of the same
pharmaceutical.
Experimental Confirmation of Mode of Factor H Action
[0045] COL4.alpha.3 knock out mice, mimicking Alport syndrome (AS),
express a defect 03 chain of collagen 4 causing a defect of the
glomerular basement membrane. This results in glomerular
proteinuria and progressive tubulointerstitial fibrosis beginning
4.5 weeks postpartum, and causes renal failure and death after
approximately 10 weeks. According to the "Brenner hypothesis"
[Brenner et al.; N Engl J Med 1982; 307: 652-9] intraluminal
protein is reabsorbed by tubular epithelial cells which become
thereby activated. Activated epithelial cells induce (1)
inflammatory or (2) profibrotic second messenger pathways, or (3)
undergo "epithelial-mesenchymal transition" (EMT) by
themselves.
[0046] As mentioned above, Factor H is the key plasma/humoral
regulator of the activated complement system. Factor H deficiency
is known to cause renal diseases like aHUS or MPGN II. While Factor
H deficiency induces/causes complement activation at the level of
the endothelial cell surface or within the glomerular basement
membrane, the supplementation of Factor H, which can at least in
part pass the glomerular filter, will be able to decrease
complement activation at the level of tubular epithelial cells and
will thereby serve as therapeutic option for chronic/progressive
renal disease caused by glomerular proteinuria.
[0047] The effect of Factor H administration can be tested by
treating COL4.alpha.3 knock out mice with supraphysiological levels
of Factor H purified from mouse serum, starting 4.5 weeks
postpartum (earliest time point possible after weaning of the
mice). Factor H is applied s.c., i.p. or i.m. Results in the
treated group are compared to vehicle (NaCl 0.9%) treated controls.
Mice are treated until they die (group 1), or are sacrificed after
7.5 and 9.5 weeks (group 2). Animals of group 2 are anesthetized,
urine and blood samples are collected, kidneys are rapidly
harvested, one kidney is formaldehyde fixed and used for
immunohistology, and from the other kidney cortex is isolated for
RNA extraction and subsequent real time reverse transcriptase PCR
(RT-PCR) analysis.
[0048] The results show that in a mouse model of chronic glomerular
proteinuria (1) chronic treatment with Factor H mitigates
complement activation on the level of tubular epithelial cells, (2)
reduces activation of inflammatory and profibrotic secondary
pathways launched by tubular epithelial cells, (3) reduces degree
of tubulointerstitial fibrosis, and (4) increases life span of
COL4.alpha.3 knock out mice.
[0049] These results strongly support the use of Factor H for the
treatment of chronic proteinuria, which is the final common key
feature of chronic renal disease in general.
[0050] As already outlined proteinuria is not only indicating an
acute or chronic defect of the function of the glomerular filter
(e.g. nephritic or nephrotic syndrome) but also promotes
progression of chronic renal disease via the induction of
inflammatory and profibrotic processes in the tubular interstitium.
Without exaggeration proteinuria can be seen as the final common
pathway of chronic renal disease (CRD), and reduction of
proteinuria or of the effects which are induced by proteinuria
might proof of the key for efficient treatment of chronic renal
disease.
[0051] Based on the concept that activation of the complement
system on the apical surface of tubular epithelial cells is one of
the major mediators in the pathogenic cascade of events in
proteinuria caused CRD, treatment via Factor H (e.g. infusion of up
to 80 ml FFP/kg body weight/treatment or i.v. or s.c. or i.m.
administration of the Factor H equivalent of 80 ml FFP/kg body
weight) is increasing plasma Factor H above physiological level
(e.g. twofold) and is resulting in (a) the availability of Factor H
on the apical surfaces of tubular epithelial cells which is then
(b) decreasing activation of the complement system at this
side.
[0052] In patients in whom disease is caused by a mutation of the
Factor H gene which results in the expression of a protein with
intact cell binding characteristics while being functionally defect
(e.g. MPGN II based on a mutation of SCR 4 of Factor H: Licht et
al, Kidney Int 2006) treatment via FFP (e.g. infusion of up to
80-120 ml FFP/kg/treatment or i.v. or s.c. or i.m. administration
of the Factor H equivalent of 80-120 ml FFP/kg body weight) aiming
at raising plasma Factor H levels up to two- or threefold is
resulting in competitive binding of intact Factor H molecules to
cell surfaces and subsequent reduction of complement
activation.
[0053] Therapeutic success is indicated by (a) reduction in
complement activation (increased C3, decreased C3b), (b) reduction
(or at least prevention of further increase) of hematuria and
proteinuria, and (c) stabilization--possibly improvement--of renal
function.
* * * * *