U.S. patent application number 14/638636 was filed with the patent office on 2015-06-25 for compositions comprising a complement inhibitor and an interferon alpha inhibitor.
The applicant listed for this patent is ALEXION PHARMACEUTICALS, INC.. Invention is credited to Cynthia MAGRO.
Application Number | 20150174243 14/638636 |
Document ID | / |
Family ID | 44542525 |
Filed Date | 2015-06-25 |
United States Patent
Application |
20150174243 |
Kind Code |
A1 |
MAGRO; Cynthia |
June 25, 2015 |
COMPOSITIONS COMPRISING A COMPLEMENT INHIBITOR AND AN INTERFERON
ALPHA INHIBITOR
Abstract
The present disclosure relates to, inter alia, compositions
containing an inhibitor of human complement and/or an inhibitor of
interferon alpha, and the use of the compositions in methods for
treating or preventing Degos' disease in a subject. In some
embodiments, the inhibitor is an antibody, or antigen-binding
fragment thereof, that binds to a human complement component C5
protein or to a biologically-active fragment of C5 such as C5a or
C5b. In some embodiments, the inhibitor is an antibody, or an
antigen-binding fragment thereof, that binds to interferon alpha or
to an interferon alpha receptor.
Inventors: |
MAGRO; Cynthia; (New York,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALEXION PHARMACEUTICALS, INC. |
Cheshire |
CT |
US |
|
|
Family ID: |
44542525 |
Appl. No.: |
14/638636 |
Filed: |
March 4, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13582018 |
Nov 16, 2012 |
8999340 |
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PCT/US2011/026602 |
Mar 1, 2011 |
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14638636 |
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61309393 |
Mar 1, 2010 |
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Current U.S.
Class: |
424/135.1 |
Current CPC
Class: |
A61K 38/12 20130101;
C07K 16/18 20130101; C07K 2317/76 20130101; A61P 7/00 20180101;
A61P 37/02 20180101; A61K 39/3955 20130101; A61K 2039/507 20130101;
A61K 38/12 20130101; A61P 31/12 20180101; A61P 9/10 20180101; A61P
1/00 20180101; A61P 17/00 20180101; A61K 2300/00 20130101; A61K
38/1725 20130101; A61K 2300/00 20130101; A61K 45/06 20130101; A61P
7/02 20180101; A61P 37/06 20180101; A61K 38/1725 20130101; A61K
31/245 20130101; A61P 25/00 20180101; A61K 31/245 20130101; C07K
2317/24 20130101; C07K 2317/34 20130101; A61K 2300/00 20130101;
A61P 9/00 20180101; A61P 43/00 20180101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 45/06 20060101 A61K045/06 |
Claims
1. A composition or package comprising: (i) a complement inhibitor
and (ii) an inhibitor of interferon alpha.
2. The composition of claim 1, wherein the complement inhibitor is
an antibody, or antigen-binding fragment thereof, that binds to a
human complement component C5 protein.
3. The composition of claim 2, wherein the antibody, or antigen
binding fragment thereof, inhibits the cleavage of complement
component C5 into fragments C5a and C5b.
4. The composition of claim 2, wherein the antibody, or
antigen-binding fragment thereof, is selected from the group
consisting of a humanized antibody, a recombinant antibody, a
diabody, a chimerized or chimeric antibody, a deimmunized human
antibody, a fully human antibody, a single chain antibody, an Fv
fragment, an Fd fragment, an Fab fragment, an Fab'fragment, and an
F(ab').sub.2 fragment.
5. The composition of claim 1, wherein the antibody is
eculizumab.
6. The composition of claim 1, wherein the antibody is
pexelizumab.
7. The composition of claim 1, wherein the inhibitor of interferon
alpha is an antibody, or an antigen-binding fragment thereof, that
binds to interferon alpha or an interferon alpha receptor.
8. The composition of claim 7, wherein the antibody, or
antigen-binding fragment thereof, that binds to interferon alpha or
an interferon alpha receptor is selected from the group consisting
of a monoclonal antibody, a humanized antibody, a recombinant
antibody, a diabody, a chimerized or chimeric antibody, a
deimmunized human antibody, a fully human antibody, a single chain
antibody, an Fv fragment, an Fd fragment, an Fab fragment, an
Fab'fragment, and an F(ab').sub.2 fragment.
9. The composition of claim 1, wherein the composition is
formulated for parental administration.
10. The composition of claim 1, further comprising an
immunosuppressive agent, an antibiotic or an anti-viral agent.
11. The composition of claim 10, wherein the immunosuppressive
agent is selected from the group consisting of corticosteroids,
phenylbutazone, azathioprine, methotrexate, cyclosporine,
tacrolimus, and mycophenolate mofetil, cyclophosphamide, and an
anti-CD20 agent.
12. The composition of claim 1, further comprising a second
inhibitor of interferon alpha.
13. The package of claim 1, wherein the complement inhibitor and an
inhibitor of interferon alpha are formulated separately.
14. The package of claim 1, wherein the complement inhibitor and an
inhibitor of interferon alpha are formulated together.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/582,018, filed on Nov. 16, 2012, which is a national
stage filing of International Patent Application No.
PCT/US2011/026602, filed on Mar. 1, 2011, which claims priority to
and the benefit of U.S. Provisional Patent Application No.
61/309,393, filed on Mar. 1, 2010, the entire contents of which are
hereby incorporated by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Mar. 4,
2015, is named AXJ-153USDV_Seq_Listing.txt, and is 61,477 bytes in
size.
TECHNICAL FIELD
[0003] The field of the invention is medicine, immunology,
molecular biology, and protein chemistry.
BACKGROUND
[0004] Degos' disease (also known as Kohlmeier disease and
malignant atrophic papulosis (MAP)) is a rare vasculopathy (around
200 reported cases) characterized by thrombosis in small to large
vessels. See, e.g., Lester and Rapini (2009) Curr Opin
Gastroenterol 25:66-73 and Englert et al. (1984) Br Med J 289:576.
Although generally considered to be of unknown etiology, Degos'
disease has been associated with viral infections (e.g., B 19
parvovirus and HIV) and autoimmune disorders such as lupus
erythematosis (LE), dermatomyositis, and primary antiphospholipid
syndrome (APS). See, e.g., Crowson et al. (2002) J Cutan Pathol
29:596-601; Englert et al. (1984), supra; Heymann (2009) J Am Acad
Dermatol 61:505-506; Durie et al. (1969) Arch Dermatol
100(5):575-581; Tsao et al. (1997) J Am Acad Dermatol 36:317-319;
and Requena et al. (1998) J Am Acad Dermatol 38:852-856. Some forms
of Degos' disease may be familial. See, e.g., Katz et al. (1997) J
Am Acad Dermatol 37:480-484 and Penault et al. (2004) Ann Dermatol
Venereol 131:989-993. Degos can occur in patients of any age, yet
it appears to preferentially affect men over women at a ratio of
approximately 3 to 1. See, e.g., Katz et al. (1997), supra; Torrelo
et al. (2002) Br J Dermatol 146:916-918; and Wilson et al. (2007)
Pediatr Dermatol 24(1):18-24.
[0005] Degos can manifest as a benign, purely cutaneous form or as
an aggressive, multiorgan, systemic form, the latter of which is
generally fatal within one to twelve years after diagnosis.
Scheinfeld (2007) Clin Exp Derm 32:483-487. The phenotypic hallmark
of cutaneous Degos' disease is the appearance of one or more
erythematous, reddish-colored papules on the skin, which papules
scar over with white, atrophic centers.
[0006] Death occurs in nearly all patients with the systemic form
of Degos' disease, the patients having an average life expectancy
after systemic involvement of around two to three years. See, e.g.,
Scheinfeld (2007), supra. Patients usually die from intestinal
perforation with or without septic complications; however, death
may alternatively result from intestinal infarction,
cardiopulmonary collapse, and/or neurological infarction and
hemorrhage. Id. See also High et al. (2004) J Am Acad Dermatol
50(6):895-899.
[0007] A standard medical treatment for Degos' disease has not been
defined. Many therapeutic agents have had only marginal and/or
inconsistent success in treating the disease. See, e.g., Scheinfeld
(2007), supra. For example, some Degos patients benefited from
intravenous immunoglobulin therapy, but at present there appears to
be no way of predicting which patients would respond to such
therapy, see, e.g., Dyrsen et al. (2008) J Cutan Pathol 35(Suppl
1):20-25; Zhu et al. (2007) Br J Dermatol 157(1):206-207; and De
Breucker et al. (2008) Acta Clin Belg 63(2):99-102 (Abstract).
[0008] In view of the foregoing, it is clear that there is a need
for new approaches and better methods to treat patients with Degos'
disease.
SUMMARY
[0009] The present disclosure is based, at least in part, on the
discovery by the inventor that an inhibitor of complement, namely
the humanized anti-C5 antibody eculizumab, was highly efficacious
in the treatment of a patient afflicted with the systemic form of
Degos' disease. Accordingly, the disclosure features a variety of
compositions and methods useful for the prevention and treatment of
Degos' disease.
[0010] In one aspect, the disclosure provides a method for treating
a patient afflicted with Degos' disease, the method comprising
administering to a patient afflicted with Degos' disease a
complement inhibitor in an amount sufficient to treat the
disease.
[0011] In another aspect, the disclosure features a method for
treating a patient afflicted with Degos' disease, which method
includes chronically administering to a patient afflicted with
Degos' disease a complement inhibitor in an amount and with a
frequency sufficient to maintain a reduced level of complement
activity in the patient to thereby treat the disease.
[0012] In another aspect, the disclosure features a method for
treating Degos' disease, the method comprising: identifying a
patient as being, or likely to be, afflicted with Degos' disease;
and administering to the patient a complement inhibitor in an
amount sufficient to treat the disease.
[0013] In another aspect, the disclosure features a method for
treating or preventing (e.g., preventing the occurrence of Degos'
disease or preventing the progression of the benign cutaneous form
of Degos' disease to a more advanced, multiorgan and/or systemic
form of the disease). The method includes administering to a
patient in need thereof a complement inhibitor in an amount
sufficient to treat or prevent the disease. In some embodiments,
the inhibitor can be chronically administered in an amount and with
a frequency to maintain a reduced level of complement activation in
the blood of the patient for the duration of the treatment.
[0014] In some embodiments of any of the methods described herein,
the Degos' disease is associated with a B19 parvoviral infection or
human immunodeficiency virus infection. In some embodiments, the
Degos' disease is idiopathic.
[0015] In some embodiments of any of the methods described herein,
the Degos' disease pathologically affects one or more of the
gastrointestinal tract, the central nervous system, and the
cardiovascular system. In some embodiments, the Degos' disease is
multiorgan, systemic Degos' disease. In some embodiments, the
Degos' disease is a cutaneous form of the disease.
[0016] In some embodiments of any of the methods described herein,
the Degos' disease is refractory to at least one therapy selected
from the group consisting of an anti-inflammatory agent, an
anticoagulant, an antithrombotic, and intravenous immunoglobulin.
The anti-inflammatory drug can be, e.g., one selected from the
group consisting of corticosteroids, phenylbutazone, azathioprine,
methotrexate, cyclosporine, tacrolimus, and mycophenolate mofetil.
The anticoagulant or antithrombotic can be, e.g., one selected from
the group consisting of clopidogrel, aspirin, and dipyridamole.
[0017] In some embodiments of any of the methods described herein,
the complement inhibitor can be, e.g., one selected from the group
consisting of a polypeptide, a polypeptide analog, a nucleic acid,
a nucleic acid analog, and a small molecule. In some embodiments,
the complement inhibitor can be, e.g., one selected from the group
consisting of soluble CR1, LEX-CR1, MCP, DAF, CD59, Factor H, cobra
venom factor, FUT-175, complestatin, and K76 COOH.
[0018] In some embodiments of any of the methods described herein,
the complement inhibitor inhibits the expression of a human
complement component protein. In some embodiments, the complement
inhibitor can inhibit the activity of a complement protein such as,
but not limited to, complement component C1s, complement component
C1r, the C3 convertase, the C5 convertase, or C5b-9.
[0019] In some embodiments of any of the methods described herein,
the complement inhibitor inhibits the cleavage of human complement
component C5, C4, C3, or C2. For example, a complement inhibitor
can inhibit the cleavage of complement component C5 into fragments
C5a and C5b.
[0020] In some embodiments, the complement inhibitor is an antibody
or antigen-binding fragment thereof that binds to a human
complement component protein (e.g., a C5 protein). In some
embodiments, the antibody or antigen-binding fragment thereof binds
to the alpha chain of C5 protein. In some embodiments, the antibody
or antigen-binding fragment thereof binds to the beta chain of C5.
In some embodiments, the antibody or antigen-binding fragment
thereof binds to the alpha chain of human complement component C5,
and wherein the antibody (i) inhibits complement activation in a
human body fluid, (ii) inhibits the binding of purified human
complement component C5 to either human complement component C3b or
human complement component C4b, and (iii) does not bind to the
human complement activation product free C5a. In some embodiments,
the antibody binds to the human complement component C5 protein
comprising or consisting of the amino acid sequence depicted in any
one of SEQ ID NOs:1-26. In some embodiments, the inhibitor is an
antibody or antigen-binding fragment thereof that binds to
complement component C5 fragment C5b.
[0021] In some embodiments, the antibody can be a monoclonal
antibody. In some embodiments, the antibody or antigen-binding
fragment thereof can be one selected from the group consisting of a
humanized antibody, a recombinant antibody, a diabody, a chimerized
or chimeric antibody, a deimmunized human antibody, a fully human
antibody, a single chain antibody, an Fv fragment, an Fd fragment,
an Fab fragment, an Fab' fragment, and an F(ab').sub.2
fragment.
[0022] In some embodiments of any of the methods described herein,
the complement inhibitor is eculizumab or pexelizumab.
[0023] In yet another aspect, the disclosure features an article of
manufacture, which contains: a container comprising a label; and a
composition comprising a complement inhibitor, wherein the label
indicates that the composition is to be administered to a human
having, suspected of having, or at risk for developing, Degos'
disease. The inhibitor can be, e.g., an antibody or antigen-binding
fragment thereof that binds to a human complement component C5
protein. The inhibitor can be, e.g., an antibody or antigen-binding
fragment thereof that binds to a fragment of human complement
component C5 protein such as C5a or C5b.
[0024] In some embodiments, the article of manufacture includes one
or more additional active agents such as, but not limited to, one
or more anti-inflammatory agents, anticoagulants, or antithrombotic
agents.
[0025] The inventor also discovered that the Degos' patient
described herein had elevated levels of interferon alpha levels in
serum as well as within the biopsied skin tissue. While not being
bound by any particular theory or mechanism of action, as
interferon alpha upregulates adaptive and innate immunity,
potentiating the effects of any antigenic trigger, and
administration of exogenous interferon alpha has been reported as a
cause of cutaneous thrombosis and ulceration, the inventor believes
that inhibiting interferon alpha is a useful strategy for treating
Degos' disease.
[0026] Accordingly, in another aspect, the disclosure features a
method for treating a patient afflicted with Degos' disease, the
method comprising administering to a patient afflicted with Degos'
disease an inhibitor of interferon alpha in an amount sufficient to
treat the disease.
[0027] In another aspect, the disclosure features a method for
treating a patient afflicted with Degos' disease, the method
comprising chronically administering to a patient afflicted with
Degos' disease an inhibitor of interferon alpha in an amount and
with a frequency sufficient to maintain a reduced level of
interferon alpha activity in the patient to thereby treat the
disease.
[0028] In another aspect, the disclosure features a method for
treating Degos' disease, which method includes: identifying a
patient as being, or likely to be, afflicted with Degos' disease;
and administering to the patient an inhibitor of interferon alpha
in an amount sufficient to treat the disease.
[0029] In another aspect, the disclosure features a method for
treating or preventing (e.g., preventing the occurrence of Degos'
disease or preventing the progression of the benign cutaneous form
of Degos' disease to a more advanced, multiorgan and/or systemic
form of the disease). The method includes administering to a
patient in need thereof an inhibitor of interferon alpha in an
amount sufficient to treat or prevent the disease. In some
embodiments, the inhibitor can be chronically administered in an
amount and with a frequency to maintain a reduced level of
interferon alpha expression or activity in the blood of the patient
for the duration of the treatment.
[0030] In some embodiments, the inhibitor of interferon alpha can
be, e.g., one selected from the group consisting of a polypeptide,
a polypeptide analog, a nucleic acid, a nucleic acid analog, and a
small molecule. The inhibitor can, e.g., inhibit the expression of
interferon alpha or an interferon alpha receptor by a cell. The
inhibitor can, e.g., inhibit the activity of interferon alpha or an
interferon alpha receptor protein.
[0031] In some embodiments, the inhibitor of interferon alpha binds
to interferon alpha. In some embodiments, the inhibitor of
interferon alpha binds to an interferon alpha receptor. For
example, in some embodiments, the inhibitor of interferon alpha is
an antibody (or an antigen-binding fragment thereof) that binds to
interferon alpha or to an interferon alpha receptor. The antibody
can be a monoclonal antibody. The antibody or antigen-binding
fragment thereof can be, e.g., one selected from the group
consisting of a humanized antibody, a recombinant antibody, a
diabody, a chimerized or chimeric antibody, a deimmunized human
antibody, a fully human antibody, a single chain antibody, an Fv
fragment, an Fd fragment, an Fab fragment, an Fab' fragment, and an
F(ab').sub.2 fragment.
[0032] In yet another aspect, the disclosure features an article of
manufacture containing: a container comprising a label; and a
composition comprising an inhibitor of interferon alpha, wherein
the label indicates that the composition is to be administered to a
human having, suspected of having, or at risk for developing,
Degos' disease. The inhibitor of interferon alpha can be, e.g., any
inhibitor of interferon alpha described herein such as an antibody
or antigen-binding fragment thereof that binds to interferon alpha
or to an interferon receptor.
[0033] In some embodiments, the article of manufacture includes one
or more additional active agents such as, but not limited to, an
anti-inflammatory agent, an anticoagulant, or an antithrombotic
agent.
[0034] In some embodiments, the methods described herein can
include administration (either as a single agent or in combination
with a complement inhibitor and/or an interferon alpha inhibitor)
of a B cell-targeted therapy. For example, the disclosure features
a method for treating or preventing Degos' disease, the method
comprising administering to a patient having, suspected of having,
or at risk for developing, Degos' disease a therapeutically
effective amount of a B cell-targeted therapy. The B cell-targeted
therapy can be, e.g., an anti-CD20 binding agent such as, but not
limited to, anti-CD20 antibodies. Exemplary therapeutic anti-CD20
antibodies, which are approved for clinical use or are in clinical
development, that can be used in the methods described herein
include, without limitation, rituximab (Biogen Idec),
.sup.90Y-ibritumomab tiuxetan (Biogen Idec), .sup.131I-tositumomab
(GlaxoSmithKline), ofatumumab (Genmab), TRU-015 (Trubion),
veltuzumab (IMMU-106; Immunomedics), ocrelizumab (Roche), and
AME-133v (Applied Molecular Evolution). See, e.g., Levene et al.
(2005), supra; Burge et al. (2008) Clin Ther 30(10):1806-1816;
Kausar et al. (2009) Expert Opin Biol Ther 9(7):889-895;
Morschhauser et al. (2009) J Clin Oncol 27(20):3346-3353; and
Milani and Castillo (2009) Curr Opin Mol Ther 11(2):200-207.
[0035] In another example, any of the methods described herein,
e.g., methods in which a complement inhibitor and/or an interferon
alpha inhibitor is administered to a Degos' disease patient, the
methods can also include administering a B cell-targeted therapy
such as an anti-CD20 antibody. "Polypeptide," "peptide," and
"protein" are used interchangeably and mean any peptide-linked
chain of amino acids, regardless of length or post-translational
modification. The complement component proteins described herein
(e.g., complement component C2, C3, C4, or C5 proteins) can contain
or be wild-type proteins or can be variants that have not more than
50 (e.g., not more than one, two, three, four, five, six, seven,
eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) conservative
amino acid substitutions. Conservative substitutions typically
include substitutions within the following groups: glycine and
alanine; valine, isoleucine, and leucine; aspartic acid and
glutamic acid; asparagine, glutamine, serine, and threonine;
lysine, histidine and arginine; and phenylalanine and tyrosine.
[0036] The human complement component proteins described herein
also include "antigenic peptide fragments" of the proteins, which
are shorter than full-length, immature (pre-pro) proteins, but
retain at least 10% (e.g., at least 10%, at least 15%, at least
20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 50%, at least 55%, at least 60%, at least 70%, at least 80%,
at least 90%, at least 95%, at least 98%, at least 99%, at least
99.5%, or 100% or more) of the ability of the full-length protein
to induce an antigenic response in a mammal. For example, an
antigenic peptide fragment of C5 protein can be any fragment of the
protein, which is less than the full-length immature protein and
retains at least 10% of the ability of the full-length protein to
induce an antigenic response in a mammal. Antigenic peptide
fragments of a complement component protein include terminal as
well internal deletion variants of the protein. Deletion variants
can lack one, two, three, four, five, six, seven, eight, nine, ten,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid segments (of
two or more amino acids) or non-contiguous single amino acids.
Antigenic peptide fragments can be at least 6 (e.g., at least 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,
350, 400, 450, 500, or 600 or more) amino acid residues in length
(e.g., at least 6 contiguous amino acid residues in any one of SEQ
ID NOS:1-11). In some embodiments, an antigenic peptide fragment of
a human complement component protein is less than 500 (e.g., less
than 450, 400, 350, 325, 300, 275, 250, 225, 200, 190, 180, 170,
160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60,
50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34,
33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10, 9, 8, or 7) amino acid residues in
length (e.g., less than 500 contiguous amino acid residues in any
one of SEQ ID NOs:1-11). In some embodiments, an antigenic peptide
fragment of a full-length, immature human complement component
protein (prepro-C5 protein) is at least 6, but less than 500, amino
acid residues in length.
[0037] In some embodiments, the human complement component C5
protein can have an amino acid sequence that is, or is greater
than, 70 (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100) % identical to the human C5 protein having the amino acid
sequence depicted in SEQ ID NO:1.
[0038] Percent (%) amino acid sequence identity is defined as the
percentage of amino acids in a candidate sequence that are
identical to the amino acids in a reference sequence, after
aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity. Alignment for
purposes of determining percent sequence identity can be achieved
in various ways that are within the skill in the art, for instance,
using publicly available computer software such as BLAST, BLAST-2,
ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate
parameters for measuring alignment, including any algorithms needed
to achieve maximal alignment over the full-length of the sequences
being compared can be determined by known methods.
[0039] Amino acid sequences for exemplary human C5 proteins as well
as antigenic peptide fragments thereof are known in the art and are
set forth below.
[0040] As used throughout the present disclosure, the term
"antibody" refers to a whole or intact antibody (e.g., IgM, IgG,
IgA, IgD, or IgE) molecule that is generated by any one of a
variety of methods that are known in the art and described herein.
The term "antibody" includes a polyclonal antibody, a monoclonal
antibody, a chimerized or chimeric antibody, a humanized antibody,
a deimmunized human antibody, and a fully human antibody. The
antibody can be made in or derived from any of a variety of
species, e.g., mammals such as humans, non-human primates (e.g.,
monkeys, baboons, or chimpanzees), horses, cattle, pigs, sheep,
goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats,
and mice. The antibody can be a purified or a recombinant
antibody.
[0041] As used herein, the term "antibody fragment,"
"antigen-binding fragment," or similar terms refer to fragment of
an antibody that retains the ability to bind to an antigen (e.g., a
complement component C5 protein), e.g., a single chain antibody, a
single chain Fv fragment (scFv), an Fd fragment, an Fab fragment,
an Fab' fragment, or an F(ab').sub.2 fragment. An scFv fragment is
a single polypeptide chain that includes both the heavy and light
chain variable regions of the antibody from which the scFv is
derived. In addition, diabodies (Poljak (1994) Structure
2(12):1121-1123; Hudson et al. (1999) J Immunol Methods
23(1-2):177-189, the disclosures of both of which are incorporated
herein by reference in their entirety) and intrabodies (Huston et
al. (2001) Hum. Antibodies 10(3-4):127-142; Wheeler et al. (2003)
Mol Ther 8(3):355-366; Stocks (2004) Drug Discov Today 9(22):
960-966, the disclosures of each of which are incorporated herein
by reference in their entirety) that bind to a complement component
protein (e.g., complement component C5) can be incorporated into
the compositions, and used in the methods, described herein.
[0042] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure pertains. In
case of conflict, the present document, including definitions, will
control. Preferred methods and materials are described below,
although methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
presently disclosed methods and compositions. All publications,
patent applications, patents, and other references mentioned herein
are incorporated by reference in their entirety.
[0043] Other features and advantages of the present disclosure,
e.g., methods for treating or preventing Degos' disease, will be
apparent from the following description, the examples, and from the
claims.
DETAILED DESCRIPTION
[0044] The present disclosure provides compositions containing an
inhibitor of human complement (e.g., an antibody that binds to a
human complement component C5 protein) and methods for using the
compositions to treat or prevent Degos' disease. While in no way
intended to be limiting, exemplary compositions (e.g.,
pharmaceutical compositions and formulations) and methods for using
the compositions are elaborated on below and exemplified in the
working Examples.
The Complement Pathway
[0045] The complement system acts in conjunction with other
immunological systems of the body to defend against intrusion of
cellular and viral pathogens. There are at least 25 complement
proteins, which are found as a complex collection of plasma
proteins and membrane cofactors. The plasma proteins make up about
10% of the globulins in vertebrate serum. Complement components
achieve their immune defensive functions by interacting in a series
of intricate but precise enzymatic cleavage and membrane binding
events. The resulting complement cascade leads to the production of
products with opsonic, immunoregulatory, and lytic functions. A
concise summary of the biologic activities associated with
complement activation is provided, for example, in The Merck
Manual, 16.sup.th Edition.
[0046] The complement cascade progresses via the classical pathway,
the alternative pathway, or the lectin pathway. These pathways
share many components, and while they differ in their initial
steps, they converge and share the same "terminal complement"
components (C5 through C9) responsible for the activation and
destruction of target cells.
[0047] The classical complement pathway is typically initiated by
antibody recognition of, and binding to, an antigenic site on a
target cell. The alternative pathway can be antibody independent,
and can be initiated by certain molecules on pathogen surfaces.
Additionally, the lectin pathway is typically initiated with
binding of mannose-binding lectin (MBL) to high mannose substrates.
These pathways converge at the point where complement component C3
is cleaved by an active protease (which is different in each
pathway) to yield C3a and C3b. Other pathways activating complement
attack can act later in the sequence of events leading to various
aspects of complement function.
[0048] C3a is an anaphylatoxin. C3b binds to bacterial and other
cells, as well as to certain viruses and immune complexes, and tags
them for removal from the circulation. (C3b in this role is known
as opsonin.) The opsonic function of C3b is generally considered to
be the most important anti-infective action of the complement
system. Patients with genetic lesions that block C3b function are
prone to infection by a broad variety of pathogenic organisms,
while patients with lesions later in the complement cascade
sequence, i.e., patients with lesions that block C5 functions, are
found to be more prone only to Neisseria infection, and then only
somewhat more prone.
[0049] C3b also forms a complex with other components unique to
each pathway to form classical or alternative C5 convertase, which
cleaves C5 into C5a and C5b. C3 is thus regarded as the central
protein in the complement reaction sequence since it is essential
to both the alternative and classical pathways. This property of
C3b is regulated by the serum protease Factor I, which acts on C3b
to produce iC3b. While still functional as opsonin, iC3b cannot
form an active C5 convertase.
[0050] C5 is a 190 kDa beta globulin found in normal serum at a
concentration of approximately 75 .mu.g/mL (0.4 .mu.M). C5 is
glycosylated, with about 1.5 to 3 percent of its mass attributed to
carbohydrate. Mature C5 is a heterodimer of a 999 amino acid 115
kDa alpha chain that is disulfide linked to a 655 amino acid 75 kDa
beta chain. C5 is synthesized as a single chain precursor protein
product of a single copy gene (Haviland et al. (1991) J Immunol
146:362-368). The cDNA sequence of the transcript of this gene
predicts a secreted pro-C5 precursor of 1658 amino acids along with
an 18 amino acid leader sequence (see, e.g., U.S. Pat. No.
6,355,245).
[0051] The pro-C5 precursor is cleaved after amino acids 655 and
659, to yield the beta chain as an amino terminal fragment (amino
acid residues +1 to 655 of the above sequence) and the alpha chain
as a carboxyl terminal fragment (amino acid residues 660 to 1658 of
the above sequence), with four amino acids (amino acid residues
656-659 of the above sequence) deleted between the two.
[0052] C5a is cleaved from the alpha chain of C5 by either
alternative or classical C5 convertase as an amino terminal
fragment comprising the first 74 amino acids of the alpha chain
(i.e., amino acid residues 660-733 of the above sequence).
Approximately 20 percent of the 11 kDa mass of C5a is attributed to
carbohydrate. The cleavage site for convertase action is at, or
immediately adjacent to, amino acid residue 733 of the above
sequence. A compound that would bind at, or adjacent, to this
cleavage site would have the potential to block access of the C5
convertase enzymes to the cleavage site and thereby act as a
complement inhibitor.
[0053] C5 can also be activated by means other than C5 convertase
activity. Limited trypsin digestion (see, e.g., Minta and Man
(1997) J Immunol 119:1597-1602 and Wetsel and Kolb (1982) Jlmmunol
128:2209-2216) and acid treatment (Yamamoto and Gewurz (1978)
Jlmmunol 120:2008 and Damerau et al. (1989) Molec Immunol
26:1133-1142) can also cleave C5 and produce active C5b.
[0054] Cleavage of C5 releases C5a, a potent anaphylatoxin and
chemotactic factor, and leads to the formation of the lytic
terminal complement complex, C5b-9. C5a and C5b-9 also have
pleiotropic cell activating properties, by amplifying the release
of downstream inflammatory factors, such as hydrolytic enzymes,
reactive oxygen species, arachidonic acid metabolites and various
cytokines.
[0055] C5b combines with C6, C7, and C8 to form the C5b-8 complex
at the surface of the target cell. Upon binding of several C9
molecules, the membrane attack complex (MAC, C5b-9, terminal
complement complex--TCC) is formed. When sufficient numbers of MACs
insert into target cell membranes the openings they create (MAC
pores) mediate rapid osmotic lysis of the target cells. Lower,
non-lytic concentrations of MACs can produce other effects. In
particular, membrane insertion of small numbers of the C5b-9
complexes into endothelial cells and platelets can cause
deleterious cell activation. In some cases activation may precede
cell lysis.
[0056] As mentioned above, C3a and C5a are anaphylatoxins. These
activated complement components can trigger mast cell
degranulation, which releases histamine from basophils and mast
cells, and other mediators of inflammation, resulting in smooth
muscle contraction, increased vascular permeability, leukocyte
activation, and other inflammatory phenomena including cellular
proliferation resulting in hypercellularity. C5a also functions as
a chemotactic peptide that serves to attract pro-inflammatory
granulocytes to the site of complement activation.
[0057] C5a receptors are found on the surfaces of bronchial and
alveolar epithelial cells and bronchial smooth muscle cells. C5a
receptors have also been found on eosinophils, mast cells,
monocytes, neutrophils, and activated lymphocytes.
Compositions
[0058] The compositions described herein can contain an inhibitor
of human complement. Any compound which binds to or otherwise
blocks the generation and/or activity of any of the human
complement components may be utilized in accordance with the
present disclosure. For example, an inhibitor of complement can be,
e.g., a small molecule, a nucleic acid or nucleic acid analog, a
peptidomimetic, or a macromolecule that is not a nucleic acid or a
protein. These agents include, but are not limited to, small
organic molecules, RNA aptamers, L-RNA aptamers, Spiegelmers,
antisense compounds, double stranded RNA, small interfering RNA,
locked nucleic acid inhibitors, and peptide nucleic acid
inhibitors. In some embodiments, a complement inhibitor may be a
protein or protein fragment.
[0059] In some embodiments, the compositions contain antibodies
specific to a human complement component. Some compounds include
antibodies directed against complement components C1, C2, C3, C4,
C5 (or a fragment thereof; see below), C6, C7, C8, C9, Factor D,
Factor B, Factor P, MBL, MASP-1, or MASP-2, thus preventing the
generation of the anaphylatoxic activity associated with C5a and/or
preventing the assembly of the membrane attack complex (MAC)
associated with C5b. In some embodiments, the inhibitor of
complement inhibits the activity and/or assembly of the C5b-9
complex. For example, in some embodiments, the inhibitor is an
antibody or an antigen-binding fragment thereof that binds to one
of C6, C7, C8, C9, or C5b to thus prevent the assembly and/or
activity of the MAC.
[0060] The compositions can also contain naturally occurring or
soluble forms of complement inhibitory compounds such as CR1,
LEX-CR1, MCP, DAF, CD59, Factor H, cobra venom factor, FUT-175,
complestatin, and K76 COOH. Other compounds which may be utilized
to bind to or otherwise block the generation and/or activity of any
of the human complement components include, but are not limited to,
proteins, protein fragments, peptides, small molecules, RNA
aptamers including ARC 187 (which is commercially available from
Archemix Corporation, Cambridge, Mass.), L-RNA aptamers,
spiegelmers, antisense compounds, serine protease inhibitors,
molecules which may be utilized in RNA interference (RNAi) such as
double stranded RNA including small interfering RNA (siRNA), locked
nucleic acid (LNA) inhibitors, peptide nucleic acid (PNA)
inhibitors, etc.
[0061] In some embodiments, the complement inhibitor inhibits the
activation of complement. For example, the complement inhibitor can
bind to and inhibit the complement activation activity of C1 (e.g.,
C1q, C1r, or C1s) or the complement inhibitor can bind to and
inhibit (e.g., inhibit cleavage of) C2, C3, or C4. In some
embodiments, the inhibitor inhibits formation or assembly of the C3
convertase and/or C5 convertase of the alternative and/or classical
pathways of complement. In some embodiments, the complement
inhibitor inhibits terminal complement formation, e.g., formation
of the C5b-9 membrane attack complex. For example, an antibody
complement inhibitor may include an anti-C5 antibody. Such anti-C5
antibodies may directly interact with C5 and/or C5b, so as to
inhibit the formation of and/or physiologic function of C5b.
[0062] In some embodiments, the compositions described herein can
contain an inhibitor of human complement component C5 (e.g., an
antibody, or antigen-binding fragment thereof, that binds to a
human complement component C5 protein or a biologically-active
fragment thereof such as C5a or C5b). As used herein, an "inhibitor
of complement component C5" is any agent that inhibits: (i) the
expression, or proper intracellular trafficking or secretion by a
cell, of a complement component C5 protein; (ii) the activity of C5
cleavage fragments C5a or C5b (e.g., the binding of C5a to its
cognate cellular receptors or the binding of C5b to C6 and/or other
components of the terminal complement complex; see above); (iii)
the cleavage of a human C5 protein to form C5a and C5b; or (iv) the
proper intracellular trafficking of, or secretion by a cell, of a
complement component C5 protein. Inhibition of complement component
C5 protein expression includes: inhibition of transcription of a
gene encoding a human C5 protein; increased degradation of an mRNA
encoding a human C5 protein; inhibition of translation of an mRNA
encoding a human C5 protein; increased degradation of a human C5
protein; inhibition of proper processing of a pre-pro human C5
protein; or inhibition of proper trafficking or secretion by a cell
of a human C5 protein. Methods for determining whether a candidate
agent is an inhibitor of human complement component C5 are known in
the art and described herein.
[0063] An inhibitor of human complement component C5 can be, e.g.,
a small molecule, a polypeptide, a polypeptide analog, a nucleic
acid, or a nucleic acid analog.
[0064] "Small molecule" as used herein, is meant to refer to an
agent, which has a molecular weight of less than about 6 kDa and
most preferably less than about 2.5 kDa. Many pharmaceutical
companies have extensive libraries of chemical and/or biological
mixtures comprising arrays of small molecules, often fungal,
bacterial, or algal extracts, which can be screened with any of the
assays of the application. This application contemplates using,
among other things, small chemical libraries, peptide libraries, or
collections of natural products. Tan et al. described a library
with over two million synthetic compounds that is compatible with
miniaturized cell-based assays (J Am Chem Soc (1998)
120:8565-8566). It is within the scope of this application that
such a library may be used to screen for inhibitors of human
complement component C5. There are numerous commercially available
compound libraries, such as the Chembridge DIVERSet. Libraries are
also available from academic investigators, such as the Diversity
set from the NCI developmental therapeutics program. Rational drug
design may also be employed. For example, rational drug design can
employ the use of crystal or solution structural information on the
human complement component C5 protein. See, e.g., the structures
described in Hagemann et al. (2008) J Biol Chem 283(12):7763-75 and
Zuiderweg et al. (1989) Biochemistry 28(1):172-85. Rational drug
design can also be achieved based on known compounds, e.g., a known
inhibitor of C5 (e.g., an antibody, or antigen-binding fragment
thereof, that binds to a human complement component C5
protein).
[0065] Peptidomimetics can be compounds in which at least a portion
of a subject polypeptide is modified, and the three dimensional
structure of the peptidomimetic remains substantially the same as
that of the subject polypeptide. Peptidomimetics may be analogues
of a subject polypeptide of the disclosure that are, themselves,
polypeptides containing one or more substitutions or other
modifications within the subject polypeptide sequence.
Alternatively, at least a portion of the subject polypeptide
sequence may be replaced with a non-peptide structure, such that
the three-dimensional structure of the subject polypeptide is
substantially retained. In other words, one, two or three amino
acid residues within the subject polypeptide sequence may be
replaced by a non-peptide structure. In addition, other peptide
portions of the subject polypeptide may, but need not, be replaced
with a non-peptide structure. Peptidomimetics (both peptide and
non-peptidyl analogues) may have improved properties (e.g.,
decreased proteolysis, increased retention or increased
bioavailability). Peptidomimetics generally have improved oral
availability, which makes them especially suited to treatment of
disorders in a human or animal. It should be noted that
peptidomimetics may or may not have similar two-dimensional
chemical structures, but share common three-dimensional structural
features and geometry. Each peptidomimetic may further have one or
more unique additional binding elements.
[0066] Nucleic acid inhibitors can be used to decrease expression
of an endogenous gene, e.g., a gene encoding human complement
component C5. The nucleic acid antagonist can be, e.g., an siRNA, a
dsRNA, a ribozyme, a triple-helix former, an aptamer, or an
antisense nucleic acid. siRNAs are small double stranded RNAs
(dsRNAs) that optionally include overhangs. For example, the duplex
region of an siRNA is about 18 to 25 nucleotides in length, e.g.,
about 19, 20, 21, 22, 23, or 24 nucleotides in length. The siRNA
sequences can be, in some embodiments, exactly complementary to the
target mRNA. dsRNAs and siRNAs in particular can be used to silence
gene expression in mammalian cells (e.g., human cells). See, e.g.,
Clemens et al. (2000) Proc Natl Acad Sci USA 97:6499-6503; Billy et
al. (2001) Proc Natl Acad Sci USA 98:14428-14433; Elbashir et al.
(2001) Nature 411:494-8; Yang et al. (2002) Proc Natl Acad Sci USA
99:9942-9947, and U.S. Patent Application Publication Nos.
20030166282, 20030143204, 20040038278, and 20030224432. Anti-sense
agents can include, for example, from about 8 to about 80
nucleobases (i.e. from about 8 to about 80 nucleotides), e.g.,
about 8 to about 50 nucleobases, or about 12 to about 30
nucleobases. Anti-sense compounds include ribozymes, external guide
sequence (EGS) oligonucleotides (oligozymes), and other short
catalytic RNAs or catalytic oligonucleotides which hybridize to the
target nucleic acid and modulate its expression. Anti-sense
compounds can include a stretch of at least eight consecutive
nucleobases that are complementary to a sequence in the target
gene. An oligonucleotide need not be 100% complementary to its
target nucleic acid sequence to be specifically hybridizable. An
oligonucleotide is specifically hybridizable when binding of the
oligonucleotide to the target interferes with the normal function
of the target molecule to cause a loss of utility, and there is a
sufficient degree of complementarity to avoid non-specific binding
of the oligonucleotide to non-target sequences under conditions in
which specific binding is desired, i.e., under physiological
conditions in the case of in vivo assays or therapeutic treatment
or, in the case of in vitro assays, under conditions in which the
assays are conducted. Hybridization of antisense oligonucleotides
with mRNA (e.g., an mRNA encoding a human C5 protein) can interfere
with one or more of the normal functions of mRNA. The functions of
mRNA to be interfered with include all key functions such as, for
example, translocation of the RNA to the site of protein
translation, translation of protein from the RNA, splicing of the
RNA to yield one or more mRNA species, and catalytic activity which
may be engaged in by the RNA. Binding of specific protein(s) to the
RNA may also be interfered with by antisense oligonucleotide
hybridization to the RNA. Exemplary antisense compounds include DNA
or RNA sequences that specifically hybridize to the target nucleic
acid, e.g., the mRNA encoding a human complement component C5
protein. The complementary region can extend for between about 8 to
about 80 nucleobases. The compounds can include one or more
modified nucleobases.
[0067] Modified nucleobases may include, e.g., 5-substituted
pyrimidines such as 5-iodouracil, 5-iodocytosine, and
C.sub.5-propynyl pyrimidines such as C.sub.5-propynylcytosine and
C.sub.5-propynyluracil. Other suitable modified nucleobases
include, e.g., 7-substituted-8-aza-7-deazapurines and
7-substituted-7-deazapurines such as, for example,
7-iodo-7-deazapurines, 7-cyano-7-deazapurines,
7-aminocarbonyl-7-deazapurines. Examples of these include
6-amino-7-iodo-7-deazapurines, 6-amino-7-cyano-7-deazapurines,
6-amino-7-aminocarbonyl-7-deazapurines,
2-amino-6-hydroxy-7-iodo-7-deazapurines,
2-amino-6-hydroxy-7-cyano-7-deazapurines, and
2-amino-6-hydroxy-7-aminocarbonyl-7-deazapurines. See, e.g., U.S.
Pat. Nos. 4,987,071; 5,116,742; and 5,093,246; "Antisense RNA and
DNA," D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (1988); Haselhoff and Gerlach (1988) Nature
334:585-59; Helene, C. (1991) Anticancer Drug D 6:569-84; Helene
(1992) Ann NY Acad Sci 660:27-36; and Maher (1992) Bioassays
14:807-15.
[0068] Aptamers are short oligonucleotide sequences that can be
used to recognize and specifically bind almost any molecule,
including cell surface proteins. The systematic evolution of
ligands by exponential enrichment (SELEX) process is powerful and
can be used to readily identify such aptamers. Aptamers can be made
for a wide range of proteins of importance for therapy and
diagnostics, such as growth factors and cell surface antigens.
These oligonucleotides bind their targets with similar affinities
and specificities as antibodies do. See, e.g., Ulrich (2006) Handb
Exp Pharmacol 173:305-326.
[0069] In some embodiments, the inhibitor of human C5 is an
antibody, or antigen-binding fragment thereof, which binds to a
human complement component C5 protein. (Hereinafter, the antibody
may sometimes be referred to as an "anti-C5 antibody.")
[0070] In some embodiments, the anti-C5 antibody binds to an
epitope in the human pro-C5 precursor protein. For example, the
anti-C5 antibody can bind to an epitope in the human complement
component C5 protein comprising, or consisting of, the amino acid
sequence depicted in SEQ ID NO:1 (NCBI Accession No. AAA51925 and
Haviland et al., supra).
[0071] An "epitope" refers to the site on a protein (e.g., a human
complement component C5 protein) that is bound by an antibody.
"Overlapping epitopes" include at least one (e.g., two, three,
four, five, or six) common amino acid residue(s).
[0072] In some embodiments, the anti-C5 antibody binds to an
epitope in the human pro-C5 precursor protein lacking the leader
sequence. For example, the anti-C5 antibody can bind to an epitope
in the human complement component C5 protein comprising, or
consisting of, the amino acid sequence depicted in SEQ ID NO:2,
which is a human C5 protein lacking the amino terminal leader
sequence.
[0073] In some embodiments, the anti-C5 antibody can bind to an
epitope in the alpha chain of the human complement component C5
protein. For example, the anti-C5 antibody can bind to an epitope
within, or overlapping with, a protein having the amino acid
sequence depicted in SEQ ID NO:3, which is the human complement
component C5 alpha chain protein. Antibodies that bind to the alpha
chain of C5 are described in, for example, Ames et al. (1994) J
Immunol 152:4572-4581.
[0074] In some embodiments, the anti-C5 antibody can bind to an
epitope in the beta chain of the human complement component C5
protein. For example, the anti-C5 antibody can bind to an epitope
within, or overlapping with, a protein having the amino acid
sequence depicted in SEQ ID NO:4, which is the human complement
component C5 beta chain protein. Antibodies that bind to the C5
beta chain are described in, e.g., Moongkarndi et al. (1982)
Immunobiol 162:397; Moongkarndi et al. (1983) Immunobiol 165:323;
and Mollnes et al. (1988) Scand J Immunol 28:307-312.
[0075] In some embodiments, the anti-C5 antibody can bind to an
epitope within, or overlapping with, an antigenic peptide fragment
of a human complement component C5 protein. For example, the
anti-C5 antibody can bind to an epitope within, or overlapping
with, an antigen peptide fragment of a human complement component
C5 protein, the fragment containing, or consisting of, the
following amino acid sequence:
TABLE-US-00001 (SEQ ID NO: 5) VIDHQGTKSSKCVRQKVEGSS or (SEQ ID NO:
6) KSSKC.
[0076] In some embodiments, the anti-C5 antibody can bind to an
epitope within, or overlapping with, a fragment of a human
complement component C5 protein, the fragment containing, or
consisting of, any one of the following amino acid sequences (which
are exemplary antigenic fragments of SEQ ID NO:1):
TABLE-US-00002 (SEQ ID NO: 7)
NFSLETWFGKEILVKTLRVVPEGVKRESYSGVTLDPRGIYGTISRRKEFP
YRIPLDLVPKTEIKRILSVKGLLVGEILSAVLSQEGINILTHLPKGSAEA
ELMSVVPVFYVFHYLETGNHWNIFHSD; (SEQ ID NO: 8)
SESPVIDHQGTKSSKCVRQKVEGSSSHLVTFTVLPLEIGLHNINFSLETW
FGKEILVKTLRVVPEGVKRESYSGVTLDPRGIYGTISRRKEFPYRIPLDL
VPKTEIKRILSVKGLLVGEILSAVLSQEGINILTHLPKGSAEAELMSVVP
VFYVFHYLETGNHWNIFHSDPLIEKQKLKKKLKEGMLSIMSYRNADYSYS; (SEQ ID NO: 9)
SHKDMQLGRLHMKTLLPVSKPEIRSYFPES; (SEQ ID NO: 10)
SHKDMQLGRLHMKTLLPVSKPEIRSYFPESWLWEVHLVPRRKQLQFALPD
SLTTWEIQGIGISNTGICVADTVKAKVFKDVFLEMNIPYSVVRGEQIQLK
GTVYNYRTSGMQFCVKMSAVEGICTSESPVIDHQGTKSSKCVRQKVEGSS
SHLVTFTVLPLEIGLHNINFSLETWFGKEILVKTLRVVPEGVKRESYSGV
TLDPRGIYGTISRRKEFPYRIPLDLVPKTEIKRILSVKGLLVGEILSAVL
SQEGINILTHLPKGSAEAELMSVVPVFYVFHYLETGNHWNIFHSDPLIEK
QKLKKKLKEGMLSIMSYRNADYSYS; and (SEQ ID NO: 11)
DHQGTKSSKCVRQKVEG.
[0077] Additional exemplary antigenic fragments of human complement
component C5 are disclosed in, e.g., U.S. Pat. No. 6,355,245, the
disclosure of which is incorporated herein by reference.
[0078] In some embodiments, the anti-C5 antibody specifically binds
to a human complement component C5 protein (e.g., the human C5
protein having the amino acid sequence depicted in SEQ ID NO:1).
The terms "specific binding" or "specifically binds" refer to two
molecules forming a complex (e.g., a complex between an antibody
and a complement component C5 protein) that is relatively stable
under physiologic conditions. Typically, binding is considered
specific when the association constant (K.sub.a) is higher than
10.sup.6 M.sup.-1. Thus, an antibody can specifically bind to a C5
protein with a K.sub.a of at least (or greater than) 10.sup.6
(e.g., at least or greater than 10.sup.7, 10.sup.8, 10.sup.9,
10.sup.10, 10.sup.1110.sup.12, 10.sup.13, 10.sup.14, or 10.sup.15
or higher) M.sup.-1. Examples of antibodies that specifically bind
to a human complement component C5 protein are described in, e.g.,
U.S. Pat. No. 6,355,245, the disclosure of which is incorporated
herein by reference in its entirety.
[0079] Methods for determining whether an antibody binds to a
protein antigen and/or the affinity for an antibody to a protein
antigen are known in the art. For example, the binding of an
antibody to a protein antigen can be detected and/or quantified
using a variety of techniques such as, but not limited to, Western
blot, dot blot, plasmon surface resonance method (e.g., BIAcore
system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway,
N.J.), or enzyme-linked immunosorbent assays (ELISA). See, e.g.,
Harlow and Lane (1988) "Antibodies: A Laboratory Manual" Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Benny K.
C. Lo (2004) "Antibody Engineering: Methods and Protocols," Humana
Press (ISBN: 1588290921); Borrebaek (1992) "Antibody Engineering, A
Practical Guide," W.H. Freeman and Co., NY; Borrebaek (1995)
"Antibody Engineering," 2.sup.nd Edition, Oxford University Press,
NY, Oxford; Johne et al. (1993) J Immunol Meth 160:191-198; Jonsson
et al. (1993) Ann Biol Clin 51:19-26; and Jonsson et al. (1991)
Biotechniques 11:620-627. See also U.S. Pat. No. 6,355,245.
[0080] In some embodiments, the anti-C5 antibody can crossblock
binding of another antibody that binds to an epitope within, or
overlapping with, a human complement component C5 protein. In some
embodiments, the anti-C5 antibody can crossblock binding of an
antibody that binds to an epitope within, or overlapping with, a
peptide fragment of a human complement component C5 protein. The
peptide fragment can be a fragment of a human complement component
C5 protein having the amino acid sequence depicted in any one of
SEQ ID NOS:1-11. For example, the peptide fragment can contain, or
consist of, the following amino acid sequence:
TABLE-US-00003 (SEQ ID NO: 5) VIDHQGTKSSKCVRQKVEGSS.
[0081] As used herein, the term "crossblocking antibody" refers to
an antibody that lowers the amount of binding of anti-C5 antibody
to an epitope on a complement component C5 protein relative to the
amount of binding of the anti-C5 antibody to the epitope in the
absence of the antibody. Suitable methods for determining whether a
first antibody crossblocks binding of a second antibody to an
epitope are known in the art. For example, crossblocking antibodies
can be identified by comparing the binding of the 5G1.1 anti-C5
monoclonal antibody (produced by the hybridoma cell line ATCC
designation HB-11625; see U.S. Pat. No. 6,355,245) in the presence
and absence of a test antibody. Decreased binding of the 5G1.1
antibody in the presence of the test antibody as compared to
binding of the 5G1.1 antibody in the absence of the test antibody
indicates the test antibody is a crossblocking antibody.
[0082] Methods for identifying the epitope to which a particular
antibody (e.g., an anti-C5 antibody) binds are also known in the
art. For example, the binding epitope of an anti-C5 antibody can be
identified by measuring the binding of the antibody to several
(e.g., three, four, five, six, seven, eight, nine, 10, 15, 20, or
30 or more) overlapping peptide fragments of a complement component
C5 protein (e.g., several overlapping fragments of a protein having
the amino acid sequence depicted in any one of SEQ ID NOs:1-11).
Each of the different overlapping peptides is then bound to a
unique address on a solid support, e.g., separate wells of a
multi-well assay plate. Next, the anti-C5 antibody is interrogated
by contacting it to each of the peptides in the assay plate for an
amount of time and under conditions that allow for the antibody to
bind to its epitope. Unbound anti-C5 antibody is removed by washing
each of the wells. Next, a detectably-labeled secondary antibody
that binds to the anti-C5 antibody, if present in a well of the
plate, is contacted to each of the wells, and unbound secondary
antibody is removed by washing steps. The presence or amount of the
detectable signal produced by the detectably-labeled secondary
antibody in a well is an indication that the anti-C5 antibody binds
to the particular peptide fragment associated with the well. See,
e.g., Harlow and Lane (supra), Benny K. C. Lo (supra), and U.S.
Patent Application Publication No. 20060153836, the disclosure of
which is incorporated by reference in its entirety. A particular
epitope to which an antibody binds can also be identified using
BIAcore chromatographic techniques (see, e.g., Pharmacia
BIAtechnology Handbook, "Epitope Mapping," Section 6.3.2, (May
1994); and Johne et al. (1993) J Immunol Methods 160:20191-8).
[0083] The anti-C5 antibodies described herein can have activity in
blocking the generation or activity of the C5a and/or C5b active
fragments of a complement component C5 protein (e.g., a human C5
protein). Through this blocking effect, the anti-C5 antibodies
inhibit, e.g., the proinflammatory effects of C5a and the
generation of the C5b-9 membrane attack complex (MAC) at the
surface of a cell. Anti-C5 antibodies that have the ability to
block the generation of C5a are described in, e.g., Moongkarndi et
al. (1982) Immunobiol 162:397 and Moongkarndi et al. (1983)
Immunobiol 165:323.
[0084] In some embodiments, an anti-C5 antibody, or antigen-binding
fragment thereof, can reduce the ability of a C5 protein to bind to
human complement component C3b (e.g., C3b present in an AP or CP C5
convertase complex) by greater than 50 (e.g., greater than 55, 60,
65, 70, 75, 80, 85, 90, or 95 or more) %. In some embodiments, upon
binding to a C5 protein, the anti-C5 antibody or antigen-binding
fragment thereof can reduce the ability of the C5 protein to bind
to complement component C4b (e.g., C4b present in a CP C5
convertase) by greater than 50 (e.g., greater than 55, 60, 65, 70,
75, 80, 85, 90, or 95 or more) %. Methods for determining whether
an antibody can block the generation or activity of the C5a and/or
C5b active fragments of a complement component C5 protein, or
binding to complement component C4b or C3b, are known in the art
and described in, e.g., U.S. Pat. No. 6,355,245 and Wurzner et al.
(1991) Complement Inflamm 8:328-340.
[0085] In some embodiments, an anti-C5 antibody binds to an
amino-terminal region of the alpha chain of a complement component
C5 protein, but does not bind to free C5a. Epitopes for an anti-C5
antibody within the amino-terminal region of the alpha chain
include, e.g., epitopes within the human sequence
VIDHQGTKSSKCVRQKVEGSS (SEQ ID NO:5).
[0086] In some embodiments, the composition comprises, and/or the
antibody is, eculizumab (Soliris.RTM.; Alexion Pharmaceuticals,
Inc., Cheshire, Conn.). (See, e.g., Kaplan (2002) Curr Opin
Investig Drugs 3(7):1017-23; Hill (2005) Clin Adv Hematol Oncol
3(11):849-50; and Rother et al. (2007) Nature Biotechnology
25(11):1256-1488.)
[0087] In some embodiments, the composition comprises, and/or the
antibody is, pexelizumab (Alexion Pharmaceuticals, Inc., Cheshire,
Conn.). See, e.g., Whiss (2002) Curr Opin Investig Drugs
3(6):870-7; Patel et al. (2005) Drugs Today (Barc) 41(3):165-70;
and Thomas et al. (1996) Mol Immunol 33(17-18):1389-401.
[0088] In some embodiments, the C5 inhibitor is an antibody that
binds to C5a (sometimes referred to herein as "an anti-05a
antibody"). In some embodiments, the antibody binds to C5a, but not
to full-length C5. As discussed above, the proform of C5, a 1676
amino acid residue precursor protein, is processed by a series of
proteolytic cleavage events. The first 18 peptides (numbered -18 to
-1) constitute a signal peptide that is cleaved from the precursor
protein. The remaining 1658 amino acid protein is cleaved in two
places to form the alpha and beta chains. The first cleavage event
occurs between amino acid residues 655 and 656. The second cleavage
occurs between amino acid residues 659 to 660. The two cleavage
events result in the formation of three distinct polypeptide
fragments: (i) a fragment comprising amino acids 1 to 655, which is
referred to as the beta chain; (ii) a fragment comprising amino
acids 660 to 1658, which is referred to as the alpha chain; and
(iii) a tetrapeptide fragment consisting of amino acids 656 to 659.
The alpha chain and the beta chain polypeptide fragments are
connected to each other via disulfide bond and constitute the
mature C5 protein. The CP or AP C5 convertase activates mature C5
by cleaving the alpha chain between residues 733 and 734, which
results in the liberation of C5a fragment (amino acids 660 to 733).
The remaining portion of mature C5 is fragment C5b, which contains
the residues 734 to 1658 of the alpha chain disulfide bonded to the
beta chain.
[0089] In vivo, C5a is rapidly metabolized by a serum enzyme,
carboxypeptidase B, to a 73 amino acid form termed "C5a des-Arg,"
which has lost the carboxyterminal arginine residue. Accordingly,
in some embodiments, an antibody that binds to C5a also binds to
desarginated C5a. In some embodiments, an antibody that binds to
C5a does not bind to desarginated C5a.
[0090] In some embodiments, the C5 inhibitor is an antibody that
binds to a neoepitope present in C5a, i.e., an epitope that becomes
exposed upon the liberation of C5a from the alpha chain fragment of
mature C5. Antibodies that bind to C5a (e.g., a neo-epitope present
in C5a) are known in the art as are methods for producing such
antibodies. For example, an antibody that binds to C5a can have the
binding specificity of a C5a neoepitope specific antibody described
in any one of, e.g., PCT Publication No. WO 01/15731; Ames et al.
(1994) J Immunol 152(9):4572-4581; Inoue (1989) Complement Inflamm
6(3):219-222; and U.S. Pat. No. 6,866,845. In another example, an
antibody that binds to C5a can have the binding specificity of a
commercial C5a neoepitope-specific antibody such as, but not
limited to, sc-52633 (Santa Cruz Biotechnology, Inc., Santa Cruz,
Calif.), 152-1486 (BD Pharmingen/BD Biosciences), ab11877 (Abcam,
Cambridge, Mass.), and HM2079 (clone 2952; HyCult Biotechnology,
the Netherlands). In some embodiments, an antibody that binds to
C5a can crossblock the binding of any of the aforementioned C5a
neoepitope-specific antibodies.
[0091] In some embodiments, the C5 inhibitor can be an antibody
that binds to a mammalian (e.g., human) C5a protein. For example,
the antibody can bind to a human C5a protein having the following
amino acid sequence:
TABLE-US-00004 (SEQ ID NO: 12)
TLQKKIEEIAAKYKHSVVKKCCYDGACVNNDETCEQRAARISLGPRCIKA
FTECCVVASQLRANISHKDMQLGR.
The antibody can bind to human C5a at an epitope within or
overlapping with the amino acid sequence:
TABLE-US-00005 (SEQ ID NO: 13) CCYDGACVNNDETCEQRAAR; (SEQ ID NO:
14) KCCYDGACVNNDETCEQR; (SEQ ID NO: 15) VNNDETCEQR; (SEQ ID NO: 16)
VNNDET; (SEQ ID NO: 17) AARISLGPR; (SEQ ID NO: 18)
CCYDGACVNNDETCEQRAA; (SEQ ID NO: 19) CCYDGACVNNDETCEQRA; (SEQ ID
NO: 20) CCYDGACVNNDETCEQR; (SEQ ID NO: 21) CCYDGACVNNDETCEQ; (SEQ
ID NO: 22) CCYDGACVNNDETCE; (SEQ ID NO: 23) CYDGACVNNDETCEQRAAR;
(SEQ ID NO: 24) YDGACVNNDETCEQRAAR (SEQ ID NO: 25)
CYDGACVNNDETCEQRAAR.
In some embodiments, an antibody can bind to a human C5a protein or
fragment thereof containing an amino acid sequence that contains,
or consists of, at least four (e.g., at least four, five, six,
seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, or 17 or more)
consecutive amino acids depicted in any one of SEQ ID NOs:12-25.
Additional C5a protein fragments to which an antibody described
herein can bind and methods for generating suitable C5a-specific
antigen combining sites are set forth in, e.g., U.S. Pat. No.
4,686,100, the disclosure of which is incorporated herein by
reference in its entirety.
[0092] In some embodiments, the binding of an antibody to C5a can
inhibit the biological activity of C5a. Methods for measuring C5a
activity include, e.g., chemotaxis assays, RIAs, or ELISAs (see,
e.g., Ward and Zvaifler (1971) J Clin Invest 50(3):606-16 and
Wurzner et al. (1991) Complement Inflamm 8:328-340). In some
embodiments, the binding of an antibody to C5a can inhibit the
interaction between C5a and C5aR1. Suitable methods for detecting
and/or measuring the interaction between C5a and C5aR1 (in the
presence and absence of an antibody) are known in the art and
described in, e.g., Mary and Boulay (1993) Eur J Haematol
51(5):282-287; Kaneko et al. (1995) Immunology 86(1):149-154;
Giannini et al. (1995) J Biol Chem 270(32):19166-19172; and U.S.
Patent Application Publication No. 20060160726. For example, the
binding of detectably labeled (e.g., radioactively labeled) C5a to
C5aR1-expressing peripheral blood mononuclear cells can be
evaluated in the presence and absence of an antibody. A decrease in
the amount of detectably-labeled C5a that binds to C5aR1 in the
presence of the antibody, as compared to the amount of binding in
the absence of the antibody, is an indication that the antibody
inhibits the interaction between C5a and C5aR1. In some
embodiments, the binding of an antibody to C5a can inhibit the
interaction between C5a and C5L2 (see below). Methods for detecting
and/or measuring the interaction between C5a and C5L2 are known in
the art and described in, e.g., Ward (2009) J Mol Med 87(4):375-378
and Chen et al. (2007) Nature 446(7132):203-207 (see below).
[0093] In some embodiments, the C5 inhibitor is an antibody that
binds to C5b (sometimes referred to herein as "an anti-05b
antibody"). In some embodiments, the antibody binds to C5b, but
does not bind to full-length C5. The structure of C5b is described
above and also detailed in, e.g., Muller-Eberhard (1985) Biochem
Soc Symp 50:235-246; Yamamoto and Gewurz (1978) J Immunol
120(6):2008-2015; and Haviland et al. (1991), supra. As described
above, C5b combines with C6, C7, and C8 to form the C5b-8 complex
at the surface of the target cell. Protein complex intermediates
formed during the series of combinations include C5b-6 (including
C5b and C6), C5b-7 (including C5b, C6, and C7), and C5b-8
(including C5b, C6, C7, and C8). Upon binding of several C9
molecules, the membrane attack complex (MAC, C5b-9 terminal
complement complex (TCC)) is formed. When sufficient numbers of
MACs insert into target cell membranes, the openings they create
(MAC pores) mediate rapid osmotic lysis of the target cells.
[0094] In some embodiments, the binding of an antibody to C5b can
inhibit the interaction between C5b and C6. In some embodiments,
the binding of the antibody to C5b can inhibit the assembly or
activity of the C5b-9 MAC-TCC. In some embodiments, the binding of
an antibody to C5b can inhibit complement-dependent cell lysis
(e.g., in vitro and/or in vivo). Suitable methods for evaluating
whether an antibody inhibits complement-dependent lysis include,
e.g., hemolytic assays or other functional assays for detecting the
activity of soluble C5b-9. For example, a reduction in the
cell-lysing ability of complement in the presence of an antibody
can be measured by a hemolysis assay described by Kabat and Mayer
(eds.), "Experimental Immunochemistry, 2.sup.nd Edition," 135-240,
Springfield, Ill., CC Thomas (1961), pages 135-139, or a
conventional variation of that assay such as the chicken
erythrocyte hemolysis method as described in, e.g., Hillmen et al.
(2004) N Engl J Med 350(6):552.
[0095] Antibodies that bind to C5b as well as methods for making
such antibodies are known in the art. See, e.g., U.S. Pat. No.
6,355,245. Commercially available anti-C5b antibodies are available
from a number of vendors including, e.g., Hycult Biotechnology
(catalogue number: HM2080; clone 568) and Abcam.TM. (ab46151 or
ab46168).
[0096] In some embodiments, the C5 inhibitor is an antibody that
binds to a mammalian (e.g., human) form of C5b. For example, the
antibody can bind to a portion of a human C5b protein having the
following amino acid sequence:
TABLE-US-00006 (SEQ ID NO: 4)
QEQTYVISAPKIFRVGASENIVIQVYGYTEAFDATISIKSYPDKKFSYSS
GHVHLSSENKFQNSAILTIQPKQLPGGQNPVSYVYLEVVSKHFSKSKRMP
ITYDNGFLFIHTDKPVYTPDQSVKVRVYSLNDDLKPAKRETVLTFIDPEG
SEVDMVEEIDHIGIISFPDFKIPSNPRYGMWTIKAKYKEDFSTTGTAYFE
VKEYVLPHFSVSIEPEYNFIGYKNFKNFEITIKARYFYNKVVTEADVYIT
FGIREDLKDDQKEMMQTAMQNTMLINGIAQVTFDSETAVKELSYYSLEDL
NNKYLYIAVTVIESTGGFSEEAEIPGIKYVLSPYKLNLVATPLFLKPGIP
YPIKVQVKDSLDQLVGGVPVILNAQTIDVNQETSDLDPSKSVTRVDDGVA
SFVLNLPSGVTVLEFNVKTDAPDLPEENQAREGYRAIAYSSLSQSYLYID
WTDNHKALLVGEHLNIIVTPKSPYIDKITHYNYLILSKGKIIHFGTREKF
SDASYQSINIPVTQNMVPSSRLLVYYIVTGEQTAELVSDSVWLNIEEKCG
NQLQVHLSPDADAYSPGQTVSLNMATGMDSWVALAAVDSAVYGVQRGAKK
PLERVFQFLEKSDLGCGAGGGLNNANVFHLAGLTFLTNANADDSQENDEP CKEIL.
In some embodiments, the antibody can bind to a portion of a human
C5b protein having the following amino acid sequence:
TABLE-US-00007 (SEQ ID NO: 26)
LHMKTLLPVSKPEIRSYFPESWLWEVHLVPRRKQLQFALPDSLTTWEIQG
IGISNTGICVADTVKAKVFKDVFLEMNIPYSVVRGEQIQLKGTVYNYRTS
GMQFCVKMSAVEGICTSESPVIDHQGTKSSKCVRQKVEGSSSHLVTFTVL
PLEIGLHNINFSLETWFGKEILVKTLRVVPEGVKRESYSGVTLDPRGIYG
TISRRKEFPYRIPLDLVPKTEIKRILSVKGLLVGEILSAVLSQEGINILT
HLPKGSAEAELMSVVPVFYVFHYLETGNHWNIFHSDPLIEKQKLKKKLKE
GMLSIMSYRNADYSYSVWKGGSASTWLTAFALRVLGQVNKYVEQNQNSIC
NSLLWLVENYQLDNGSFKENSQYQPIKLQGTLPVEARENSLYLTAFTVIG
IRKAFDICPLVKIDTALIKADNFLLENTLPAQSTFTLAISAYALSLGDKT
HPQFRSIVSALKREALVKGNPPIYRFWKDNLQHKDSSVPNTGTARMVETT
AYALLTSLNLKDINYVNPVIKWLSEEQRYGGGFYSTQDTINAIEGLTEYS
LLVKQLRLSMDIDVSYKHKGALHNYKMTDKNFLGRPVEVLLNDDLIVSTG
FGSGLATVHVTTVVHKTSTSEEVCSFYLKIDTQDIEASHYRGYGNSDYKR
IVACASYKPSREESSSGSSHAVMDISLPTGISANEEDLKALVEGVDQLFT
DYQIKDGHVILQLNSIPSSDFLCVRFRIFELFEVGFLSPATFTVYEYHRP
DKQCTMFYSTSNIKIQKVCEGAACKCVEADCGQMQEELDLTISAETRKQT
ACKPEIAYAYKVSITSITVENVFVKYKATLLDIYKTGEAVAEKDSEITFI
KKVTCTNAELVKGRQYLIMGKEALQIKYNFSFRYIYPLDSLTWIEYWPRD
TTCSSCQAFLANLDEFAEDIFLNGC.
In some embodiments, the antibody can bind to human C5b protein or
fragment thereof containing an amino acid sequence that contains,
or consists of, at least four (e.g., at least four, five, six,
seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
or more) consecutive amino acids depicted in SEQ ID NO:4 or SEQ ID
NO:26.
[0097] Additional exemplary sub-fragments of human C5b or C5a to
which a C5 inhibitor antibody can bind are disclosed in, e.g., U.S.
Pat. No. 6,355,245, the disclosure of which is incorporated herein
by reference.
[0098] In some embodiments, the inhibitor is an antibody that
specifically binds to a C5a polypeptide (e.g., the human C5a
polypeptide having the amino acid sequence depicted in SEQ ID
NO:12). In some embodiments, the inhibitor is an antibody that
specifically binds to a C5b polypeptide. Methods for determining
whether a particular agent is an inhibitor of human complement
component C5 are described herein and are known in the art. For
example, the concentration and/or physiologic activity of C5a and
C5b in a body fluid can be measured by methods well known in the
art. Methods for measuring C5a concentration or activity include,
e.g., chemotaxis assays, RIAs, or ELISAs (see, e.g., Ward and
Zvaifler (1971) J Clin Invest 50(3):606-16 and Wurzner et al.
(1991) Complement Inflamm 8:328-340). For C5b, hemolytic assays or
assays for soluble C5b-9 as discussed herein can be used. Other
assays known in the art can also be used. Using assays of these or
other suitable types, candidate agents capable of inhibiting human
complement component C5 such as an anti-C5 antibody, can be
screened in order to, e.g., identify compounds that are useful in
the methods described herein and determine the appropriate dosage
levels of such compounds.
[0099] Methods for detecting inhibition of expression of mRNA or
protein (e.g., inhibition of human C5 protein expression or
expression of an mRNA encoding human C5 protein) are well known in
the art of molecular biology and include, e.g., Northern blot and
RT-PCR (or quantitative RT-PCR) techniques for mRNA and for protein
detection, Western blot, dot blot, or ELISA techniques. See, e.g.,
Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual,
2.sup.nd Edition," Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
[0100] Methods for determining whether a candidate compound
inhibits the cleavage of human C5 into forms C5a and C5b are known
in the art and described in, e.g., Moongkarndi et al. (1982)
Immunobiol 162:397; Moongkarndi et al. (1983) Immunobiol 165:323;
Isenman et al. (1980) J Immunol 124(1):326-31; Thomas et al. (1996)
Mol Immunol 33(17-18):1389-401; and Evans et al. (1995) Mol Immunol
32(16):1183-95.
[0101] Inhibition of human complement component C5 can also reduce
the cell-lysing ability of complement in a subject's body fluids.
Such reductions of the cell-lysing ability of complement present
can be measured by methods well known in the art such as, for
example, by a conventional hemolytic assay such as the hemolysis
assay described by Kabat and Mayer (eds), "Experimental
Immunochemistry, 2.sup.nd Edition," 135-240, Springfield, Ill., CC
Thomas (1961), pages 135-139, or a conventional variation of that
assay such as the chicken erythrocyte hemolysis method as described
in, e.g., Hillmen et al. (2004) N Engl J Med 350(6):552.
[0102] In some embodiments, the compositions described herein can
contain an inhibitor of interferon alpha. Any compound which binds
to or otherwise blocks the generation and/or activity of interferon
alpha may be utilized in accordance with the present disclosure.
For example, an inhibitor of interferon alpha can be, e.g., a small
molecule, a nucleic acid or nucleic acid analog (e.g., an siRNA, a
dsRNA, a ribozyme, a triple-helix former, an aptamer), a
peptidomimetic, or a macromolecule that is not a nucleic acid or a
protein. These agents include, but are not limited to, small
organic molecules, RNA aptamers, L-RNA aptamers, Spiegelmers,
antisense compounds, double stranded RNA, small interfering RNA,
locked nucleic acid inhibitors, and peptide nucleic acid
inhibitors. In some embodiments, an inhibitor of interferon alpha
may be a protein or protein fragment. In some embodiments, the
inhibitor of interferon alpha is an inhibitor of the receptor
(interferon alpha receptor) to which interferon alpha binds. The
human interferon alpha receptor is described in, e.g., Novick et
al. (1994) Cell 77(3):391-400; Chill et al. (2003) Structure
11(7):791-802; and Uze et al. (2007) Curr Top Microbiol Immunol
316:71-95. In some embodiments, the inhibitor of interferon alpha
binds to interferon alpha or its receptor and inhibits the
interaction between interferon alpha and its receptor.
[0103] In some embodiments, the inhibitor of interferon alpha is an
antibody, or antigen-binding fragment thereof, which binds to an
interferon alpha protein. (Hereinafter, the antibody may sometimes
be referred to as an "anti-interferon alpha antibody.") Exemplary
anti-interferon alpha antibodies are known in the art and described
in, e.g., U.S. patent application publication nos. 20090324605,
20070059309, and 20080160030; U.S. Pat. Nos. 7,087,726 and
4,423,147, the disclosures of each of which are incorporated herein
by reference in their entirety.
[0104] Additional exemplary anti-interferon alpha antibodies that
can be used in the compositions and methods described herein
include, e.g., MEDI-545 (MDX-1103; AstraZeneca/Medimmune).
Methods for Producing an Antibody
[0105] Suitable methods for producing an antibody (e.g., an anti-C5
antibody or an anti-interferon alpha antibody), or antigen-binding
fragments thereof, in accordance with the disclosure are known in
the art (see, e.g., U.S. Pat. No. 6,355,245) and described herein.
For example, monoclonal anti-C5 antibodies may be generated using
complement component C5-expressing cells, a C5 polypeptide, or an
antigenic fragment of C5 polypeptide, as an immunogen, thus raising
an immune response in animals from which antibody-producing cells
and in turn monoclonal antibodies may be isolated. The sequence of
such antibodies may be determined and the antibodies or variants
thereof produced by recombinant techniques. Recombinant techniques
may be used to produce chimeric, CDR-grafted, humanized and fully
human antibodies based on the sequence of the monoclonal antibodies
as well as polypeptides capable of binding to human complement
component C5. Similarly, monoclonal anti-interferon alpha
antibodies may be generated using an interferon alpha polypeptide,
or an antigenic fragment of interferon alpha polypeptide, as an
immunogen, thus raising an immune response in animals from which
antibody-producing cells and in turn monoclonal antibodies may be
isolated.
[0106] Moreover, antibodies derived from recombinant libraries
("phage antibodies") may be selected using antigenic polypeptides
such as a complement component protein or interferon alpha, as bait
to isolate the antibodies or polypeptides on the basis of target
specificity. The production and isolation of non-human and chimeric
antibodies are well within the purview of the skilled artisan.
[0107] Recombinant DNA technology can be used to modify one or more
characteristics of the antibodies produced in non-human cells.
Thus, chimeric antibodies can be constructed in order to decrease
the immunogenicity thereof in diagnostic or therapeutic
applications. Moreover, immunogenicity can be minimized by
humanizing the antibodies by CDR grafting and, optionally,
framework modification. See, U.S. Pat. Nos. 5,225,539 and
7,393,648, the contents of each of which are incorporated herein by
reference.
[0108] Antibodies can be obtained from animal serum or, in the case
of monoclonal antibodies or fragments thereof, produced in cell
culture. Recombinant DNA technology can be used to produce the
antibodies according to established procedure, including procedures
in bacterial or preferably mammalian cell culture. The selected
cell culture system preferably secretes the antibody product.
[0109] In another embodiment, a process for the production of an
antibody disclosed herein includes culturing a host, e.g., E. coli
or a mammalian cell, which has been transformed with a hybrid
vector. The vector includes one or more expression cassettes
containing a promoter operably linked to a first DNA sequence
encoding a signal peptide linked in the proper reading frame to a
second DNA sequence encoding the antibody protein. The antibody
protein is then collected and isolated. Optionally, the expression
cassette may include a promoter operably linked to polycistronic
(e.g., bicistronic) DNA sequences encoding antibody proteins each
individually operably linked to a signal peptide in the proper
reading frame.
[0110] Multiplication of hybridoma cells or mammalian host cells in
vitro is carried out in suitable culture media, which include the
customary standard culture media (such as, for example Dulbecco's
Modified Eagle Medium (DMEM) or RPMI 1640 medium), optionally
replenished by a mammalian serum (e.g. fetal calf serum), or trace
elements and growth sustaining supplements (e.g. feeder cells such
as normal mouse peritoneal exudate cells, spleen cells, bone marrow
macrophages, 2-aminoethanol, insulin, transferrin, low density
lipoprotein, oleic acid, or the like). Multiplication of host cells
which are bacterial cells or yeast cells is likewise carried out in
suitable culture media known in the art. For example, for bacteria
suitable culture media include medium LE, NZCYM, NZYM, NZM,
Terrific Broth, SOB, SOC, 2 xYT, or M9 Minimal Medium. For yeast,
suitable culture media include medium YPD, YEPD, Minimal Medium, or
Complete Minimal Dropout Medium.
[0111] In vitro production provides relatively pure antibody
preparations and allows scale-up production to give large amounts
of the desired antibodies. Techniques for bacterial cell, yeast,
plant, or mammalian cell cultivation are known in the art and
include homogeneous suspension culture (e.g. in an airlift reactor
or in a continuous stirrer reactor), and immobilized or entrapped
cell culture (e.g. in hollow fibers, microcapsules, on agarose
microbeads or ceramic cartridges).
[0112] Large quantities of the desired antibodies can also be
obtained by multiplying mammalian cells in vivo. For this purpose,
hybridoma cells producing the desired antibodies are injected into
histocompatible mammals to cause growth of antibody-producing
tumors. Optionally, the animals are primed with a hydrocarbon,
especially mineral oils such as pristane (tetramethyl-pentadecane),
prior to the injection. After one to three weeks, the antibodies
are isolated from the body fluids of those mammals. For example,
hybridoma cells obtained by fusion of suitable myeloma cells with
antibody-producing spleen cells from Balb/c mice, or transfected
cells derived from hybridoma cell line Sp2/0 that produce the
desired antibodies are injected intraperitoneally into Balb/c mice
optionally pre-treated with pristane. After one to two weeks,
ascitic fluid is taken from the animals.
[0113] The foregoing, and other, techniques are discussed in, for
example, Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat.
No. 4,376,110; Harlow and Lane, Antibodies: a Laboratory Manual,
(1988) Cold Spring Harbor, the disclosures of which are all
incorporated herein by reference. Techniques for the preparation of
recombinant antibody molecules are described in the above
references and also in, e.g.: WO97/08320; U.S. Pat. No. 5,427,908;
U.S. Pat. No. 5,508,717; Smith (1985) Science 225:1315-1317;
Parmley and Smith (1988) Gene 73:305-318; De La Cruz et al. (1988)
Journal of Biological Chemistry 263:4318-4322; U.S. Pat. No.
5,403,484; U.S. Pat. No. 5,223,409; WO88/06630; WO92/15679; U.S.
Pat. No. 5,780,279; U.S. Pat. No. 5,571,698; U.S. Pat. No.
6,040,136; Davis et al. (1999) Cancer Metastasis Rev 18(4):421-5;
and Taylor et al. (1992) Nucleic Acids Research 20: 6287-6295;
Tomizuka et al. (2000) Proc Natl Acad Sci USA 97(2): 722-727, the
contents of each of which are incorporated herein by reference in
their entirety.
[0114] The cell culture supernatants are screened for the desired
antibodies by, e.g., immunoblotting, by an enzyme immunoassay, e.g.
a sandwich assay or a dot-assay, or a radioimmunoassay.
[0115] For isolation of the antibodies, the immunoglobulins in the
culture supernatants or in the ascitic fluid may be concentrated,
e.g., by precipitation with ammonium sulfate, dialysis against
hygroscopic material such as polyethylene glycol, filtration
through selective membranes, or the like. If necessary and/or
desired, the antibodies are purified by the customary
chromatography methods, for example gel filtration, ion-exchange
chromatography, chromatography over DEAE-cellulose and/or (immuno-)
affinity chromatography, e.g. affinity chromatography with one or
more surface polypeptides derived from a complement component
C5-expressing cell line, or with Protein-A or -G.
[0116] Another embodiment provides a process for the preparation of
a bacterial cell line secreting antibodies directed against a
protein (e.g., a complement protein or interferon alpha) in a
suitable mammal. A phage display library produced from the
immunized rabbit is constructed and panned for the desired
antibodies in accordance with methods well known in the art (such
as, e.g., the methods disclosed in the various references
incorporated herein by reference).
[0117] Hybridoma cells secreting the monoclonal antibodies are also
disclosed. The preferred hybridoma cells are genetically stable,
secrete monoclonal antibodies described herein of the desired
specificity, and can be expanded from deep-frozen cultures by
thawing and propagation in vitro or as ascites in vivo.
[0118] In another embodiment, a process is provided for the
preparation of a hybridoma cell line secreting monoclonal
antibodies against a protein of interest (e.g., C5 protein or
interferon alpha). In that process, a suitable mammal, for example
a Balb/c mouse, is immunized with one or more polypeptide antigens
of interest or antigenic fragments thereof. Antibody-producing
cells of the immunized mammal are grown briefly in culture or fused
with cells of a suitable myeloma cell line. The hybrid cells
obtained in the fusion are cloned, and cell clones secreting the
desired antibodies are selected. Methods for preparing a hybridoma
cell line include immunizing Balb/c mice by injecting
subcutaneously and/or intraperitoneally an immunogenic composition
containing the protein of interest (or an immunogenic fragment
thereof) several times, e.g., four to six times, over several
months, e.g., between two and four months. Spleen cells from the
immunized mice are taken two to four days after the last injection
and fused with cells of the myeloma cell line PAI in the presence
of a fusion promoter, preferably polyethylene glycol. Preferably,
the myeloma cells are fused with a three- to twenty-fold excess of
spleen cells from the immunized mice in a solution containing about
30% to about 50% polyethylene glycol of a molecular weight around
4000. After the fusion, the cells are expanded in suitable culture
media as described supra, supplemented with a selection medium, for
example HAT medium, at regular intervals in order to prevent normal
myeloma cells from overgrowing the desired hybridoma cells.
[0119] The antibodies and fragments thereof can be "chimeric."
Chimeric antibodies and antigen-binding fragments thereof comprise
portions from two or more different species (e.g., mouse and
human). Chimeric antibodies can be produced with mouse variable
regions of desired specificity spliced into human constant domain
gene segments (for example, U.S. Pat. No. 4,816,567). In this
manner, non-human antibodies can be modified to make them more
suitable for human clinical application (e.g., methods for treating
or preventing Degos' disease in a human subject).
[0120] The monoclonal antibodies of the present disclosure include
"humanized" forms of the non-human (e.g., mouse) antibodies.
Humanized or CDR-grafted mAbs are particularly useful as
therapeutic agents for humans because they are not cleared from the
circulation as rapidly as mouse antibodies and do not typically
provoke an adverse immune reaction. Methods of preparing humanized
antibodies are generally well known in the art. For example,
humanization can be essentially performed following the method of
Winter and co-workers (see, e.g., Jones et al. (1986) Nature
321:522-525; Riechmann et al. (1988) Nature 332:323-327; and
Verhoeyen et al. (1988) Science 239:1534-1536), by substituting
rodent CDRs or CDR sequences for the corresponding sequences of a
human antibody. Also see, e.g., Staelens et al. (2006) Mol Immunol
43:1243-1257. In some embodiments, humanized forms of non-human
(e.g., mouse) antibodies are human antibodies (recipient antibody)
in which hypervariable (CDR) region residues of the recipient
antibody are replaced by hypervariable region residues from a
non-human species (donor antibody) such as a mouse, rat, rabbit, or
non-human primate having the desired specificity, affinity, and
binding capacity. In some instances, framework region residues of
the human immunoglobulin are also replaced by corresponding
non-human residues (so called "back mutations"). In addition, phage
display libraries can be used to vary amino acids at chosen
positions within the antibody sequence. The properties of a
humanized antibody are also affected by the choice of the human
framework. Furthermore, humanized and chimerized antibodies can be
modified to comprise residues that are not found in the recipient
antibody or in the donor antibody in order to further improve
antibody properties, such as, for example, affinity or effector
function.
[0121] Fully human antibodies are also provided in the disclosure.
The term "human antibody" includes antibodies having variable and
constant regions (if present) derived from human germline
immunoglobulin sequences. Human antibodies can include amino acid
residues not encoded by human germline immunoglobulin sequences
(e.g., mutations introduced by random or site-specific mutagenesis
in vitro or by somatic mutation in vivo). However, the term "human
antibody" does not include antibodies in which CDR sequences
derived from the germline of another mammalian species, such as a
mouse, have been grafted onto human framework sequences (i.e.,
humanized antibodies). Fully human or human antibodies may be
derived from transgenic mice carrying human antibody genes
(carrying the variable (V), diversity (D), joining (J), and
constant (C) exons) or from human cells. For example, it is now
possible to produce transgenic animals (e.g., mice) that are
capable, upon immunization, of producing a full repertoire of human
antibodies in the absence of endogenous immunoglobulin production.
See, e.g., Jakobovits et al. (1993) Proc Natl Acad Sci USA 90:2551;
Jakobovits et al. (1993) Nature 362:255-258; Bruggemann et al.
(1993) Year in Immunol 7:33; and Duchosal et al. (1992) Nature
355:258. Transgenic mice strains can be engineered to contain gene
sequences from unrearranged human immunoglobulin genes. The human
sequences may code for both the heavy and light chains of human
antibodies and would function correctly in the mice, undergoing
rearrangement to provide a wide antibody repertoire similar to that
in humans. The transgenic mice can be immunized with the target
protein to create a diverse array of specific antibodies and their
encoding RNA. Nucleic acids encoding the antibody chain components
of such antibodies may then be cloned from the animal into a
display vector. Typically, separate populations of nucleic acids
encoding heavy and light chain sequences are cloned, and the
separate populations then recombined on insertion into the vector,
such that any given copy of the vector receives a random
combination of a heavy and a light chain. The vector is designed to
express antibody chains so that they can be assembled and displayed
on the outer surface of a display package containing the vector.
For example, antibody chains can be expressed as fusion proteins
with a phage coat protein from the outer surface of the phage.
Thereafter, display packages can be screened for display of
antibodies binding to a target.
[0122] In addition, human antibodies can be derived from
phage-display libraries (Hoogenboom et al. (1991) JMo1 Biol
227:381; Marks et al. (1991) JMo1 Biol, 222:581-597; and Vaughan et
al. (1996) Nature Biotech 14:309 (1996)). Synthetic phage libraries
can be created which use randomized combinations of synthetic human
antibody V-regions. By selection on antigen fully human antibodies
can be made in which the V-regions are very human-like in nature.
See, e.g., U.S. Pat. Nos. 6,794,132, 6,680,209, 4,634,666, and
Ostberg et al. (1983), Hybridoma 2:361-367, the contents of each of
which are incorporated herein by reference in their entirety.
[0123] For the generation of human antibodies, also see Mendez et
al. (1998) Nature Genetics 15:146-156 and Green and Jakobovits
(1998) J Exp Med 188:483-495, the disclosures of which are hereby
incorporated by reference in their entirety. Human antibodies are
further discussed and delineated in U.S. Pat. Nos. 5,939,598;
6,673,986; 6,114,598; 6,075,181; 6,162,963; 6,150,584; 6,713,610;
and 6,657,103 as well as U.S. Patent Publication Nos. 2003-0229905
A1, 2004-0010810 A1, US 2004-0093622 A1, 2006-0040363 A1,
2005-0054055 A1, 2005-0076395 A1, 2005-0287630 A1. See also
International Publication Nos. WO 94/02602, WO 96/34096, and WO
98/24893, and European Patent No. EP 0 463 151 B1. The disclosures
of each of the above-cited patents, applications, and references
are hereby incorporated by reference in their entirety.
[0124] In an alternative approach, others, including GenPharm
International, Inc., have utilized a "minilocus" approach. In the
minilocus approach, an exogenous Ig locus is mimicked through the
inclusion of pieces (individual genes) from the Ig locus. Thus, one
or more V.sub.H genes, one or more D.sub.H genes, one or more
J.sub.H genes, a mu constant region, and a second constant region
(preferably a gamma constant region) are formed into a construct
for insertion into an animal. This approach is described in, e.g.,
U.S. Pat. Nos. 5,545,807; 5,545,806; 5,625,825; 5,625,126;
5,633,425; 5,661,016; 5,770,429; 5,789,650; and 5,814,318;
5,591,669; 5,612,205; 5,721,367; 5,789,215; 5,643,763; 5,569,825;
5,877,397; 6,300,129; 5,874,299; 6,255,458; and 7,041,871, the
disclosures of which are hereby incorporated by reference. See also
European Patent No. 0 546 073 B1, International Patent Application
Publication Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO
92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO
97/13852, and WO 98/24884, the disclosures of each of which are
hereby incorporated by reference in their entirety. See further
Taylor et al. (1992) Nucleic Acids Res 20: 6287; Chen et al. (1993)
Int. Immunol. 5: 647; Tuaillon et al. (1993) Proc Natl Acad Sci USA
90: 3720-4; Choi et al. (1993) Nature Genetics 4: 117; Lonberg et
al. (1994) Nature 368: 856-859; Taylor et al. (1994) International
Immunology 6: 579-591; Tuaillon et al. (1995) J Immunol 154:
6453-65; Fishwild et al. (1996) Nature Biotechnology 14: 845; and
Tuaillon et al. (2000) Eur J Immunol. 10: 2998-3005, the
disclosures of each of which are hereby incorporated by reference
in their entirety.
[0125] In certain embodiments, de-immunized antibodies or
antigen-binding fragments thereof are provided. De-immunized
antibodies or antigen-binding fragments thereof are antibodies that
have been modified so as to render the antibody or antigen-binding
fragment thereof non-immunogenic, or less immunogenic, to a given
species (e.g., to a human). De-immunization can be achieved by
modifying the antibody or antigen-binding fragment thereof
utilizing any of a variety of techniques known to those skilled in
the art (see, e.g., PCT Publication Nos. WO 04/108158 and WO
00/34317). For example, an antibody or antigen-binding fragment
thereof may be de-immunized by identifying potential T cell
epitopes and/or B cell epitopes within the amino acid sequence of
the antibody or antigen-binding fragment thereof and removing one
or more of the potential T cell epitopes and/or B cell epitopes
from the antibody or antigen-binding fragment thereof, for example,
using recombinant techniques. The modified antibody or
antigen-binding fragment thereof may then optionally be produced
and tested to identify antibodies or antigen-binding fragments
thereof that have retained one or more desired biological
activities, such as, for example, binding affinity, but have
reduced immunogenicity. Methods for identifying potential T cell
epitopes and/or B cell epitopes may be carried out using techniques
known in the art, such as, for example, computational methods (see
e.g., PCT Publication No. WO 02/069232), in vitro or in silico
techniques, and biological assays or physical methods (such as, for
example, determination of the binding of peptides to MHC molecules,
determination of the binding of peptide:MHC complexes to the T cell
receptors from the species to receive the antibody or
antigen-binding fragment thereof, testing of the protein or peptide
parts thereof using transgenic animals with the MHC molecules of
the species to receive the antibody or antigen-binding fragment
thereof, or testing with transgenic animals reconstituted with
immune system cells from the species to receive the antibody or
antigen-binding fragment thereof, etc.). In various embodiments,
the de-immunized antibodies described herein include de-immunized
antigen-binding fragments, Fab, Fv, scFv, Fab' and F(ab').sub.2,
monoclonal antibodies, murine antibodies, engineered antibodies
(such as, for example, chimeric, single chain, CDR-grafted,
humanized, fully human antibodies, and artificially selected
antibodies), synthetic antibodies and semi-synthetic
antibodies.
[0126] In some embodiments, a recombinant DNA comprising an insert
coding for a heavy chain variable domain and/or for a light chain
variable domain of an antibody (e.g., an anti-C5 antibody or an
anti-interferon alpha antibody) is produced and transfected into a
host cell for expression of the antibody. The term DNA includes
coding single stranded DNAs, double stranded DNAs consisting of
said coding DNAs and of complementary DNAs thereto, or these
complementary (single stranded) DNAs themselves.
[0127] Furthermore, a DNA encoding a heavy chain variable domain
and/or a light chain variable domain of anti-C5 antibodies can be
enzymatically or chemically synthesized to contain the authentic
DNA sequence coding for a heavy chain variable domain and/or for
the light chain variable domain, or a mutant thereof. A mutant of
the authentic DNA is a DNA encoding a heavy chain variable domain
and/or a light chain variable domain of the above-mentioned
antibodies in which one or more amino acids are deleted, inserted,
or exchanged with one or more other amino acids. Preferably said
modification(s) are outside the CDRs of the heavy chain variable
domain and/or of the light chain variable domain of the antibody in
humanization and expression optimization applications. The term
mutant DNA also embraces silent mutants wherein one or more
nucleotides are replaced by other nucleotides with the new codons
coding for the same amino acid(s). The term mutant sequence also
includes a degenerate sequence. Degenerate sequences are degenerate
within the meaning of the genetic code in that an unlimited number
of nucleotides are replaced by other nucleotides without resulting
in a change of the amino acid sequence originally encoded. Such
degenerate sequences may be useful due to their different
restriction sites and/or frequency of particular codons which are
preferred by the specific host, particularly E. coli, to obtain an
optimal expression of the heavy chain murine variable domain and/or
a light chain murine variable domain.
[0128] The term mutant is intended to include a DNA mutant obtained
by in vitro mutagenesis of the authentic DNA according to methods
known in the art.
[0129] For the assembly of complete tetrameric immunoglobulin
molecules and the expression of chimeric antibodies, the
recombinant DNA inserts coding for heavy and light chain variable
domains are fused with the corresponding DNAs coding for heavy and
light chain constant domains, then transferred into appropriate
host cells, for example after incorporation into hybrid
vectors.
[0130] Another embodiment pertains to recombinant DNAs coding for a
recombinant polypeptide wherein the heavy chain variable domain and
the light chain variable domain are linked by way of a spacer
group, optionally comprising a signal sequence facilitating the
processing of the antibody in the host cell and/or a DNA sequence
encoding a peptide facilitating the purification of the antibody
and/or a cleavage site and/or a peptide spacer and/or an agent. The
DNA coding for an agent is intended to be a DNA coding for the
agent useful in diagnostic or therapeutic applications. Thus, agent
molecules which are toxins or enzymes, especially enzymes capable
of catalyzing the activation of prodrugs, are particularly
indicated. The DNA encoding such an agent has the sequence of a
naturally occurring enzyme or toxin encoding DNA, or a mutant
thereof, and can be prepared by methods well known in the art.
[0131] Accordingly, the monoclonal antibodies or antigen-binding
fragments of the disclosure can be naked antibodies or
antigen-binding fragments that are not conjugated to other agents,
for example, a therapeutic agent or detectable label.
Alternatively, the monoclonal antibody or antigen-binding fragment
can be conjugated to an agent such as, for example, a cytotoxic
agent, a small molecule, a hormone, an enzyme, a growth factor, a
cytokine, a ribozyme, a peptidomimetic, a chemical, a prodrug, a
nucleic acid molecule including coding sequences (such as
antisense, RNAi, gene-targeting constructs, etc.), or a detectable
label (e.g., an NMR or X-ray contrasting agent, fluorescent
molecule, etc.). In certain embodiments, an anti-C5 antibody or
antigen-binding fragment (e.g., Fab, Fv, single-chain scFv, Fab',
and F(ab').sub.2) is linked to a molecule that increases the
half-life of the antibody or antigen-binding fragment (see
above).
[0132] Several possible vector systems are available for the
expression of cloned heavy chain and light chain genes in mammalian
cells. One class of vectors relies upon the integration of the
desired gene sequences into the host cell genome. Cells which have
stably integrated DNA can be selected by simultaneously introducing
drug resistance genes such as E. coli gpt (Mulligan and Berg (1981)
Proc Natl Acad Sci USA, 78:2072) or Tn5 neo (Southern and Berg
(1982) Mol Appl Genet 1:327). The selectable marker gene can be
either linked to the DNA gene sequences to be expressed, or
introduced into the same cell by co-transfection (Wigler et al.
(1979) Cell 16:77). A second class of vectors utilizes DNA elements
which confer autonomously replicating capabilities to an
extrachromosomal plasmid. These vectors can be derived from animal
viruses, such as bovine papillomavirus (Sarver et al. (1982) Proc
Natl Acad Sci USA, 79:7147), polyoma virus (Deans et al. (1984)
Proc Natl Acad Sci USA 81:1292), or SV40 virus (Lusky and Botchan
(1981) Nature 293:79).
[0133] Since an immunoglobulin cDNA is comprised only of sequences
representing the mature mRNA encoding an antibody protein,
additional gene expression elements regulating transcription of the
gene and processing of the RNA are required for the synthesis of
immunoglobulin mRNA. These elements may include splice signals,
transcription promoters, including inducible promoters, enhancers,
and termination signals. cDNA expression vectors incorporating such
elements include those described by Okayama and Berg (1983) Mol
Cell Biol 3:280; Cepko et al. (1984) Cell 37:1053; and Kaufman
(1985) Proc Natl Acad Sci USA 82:689.
[0134] In the therapeutic embodiments of the present disclosure,
bispecific antibodies are contemplated. Bispecific antibodies are
monoclonal, preferably human or humanized, antibodies that have
binding specificities for at least two different antigens. In the
present case, one of the binding specificities is for a human
complement protein or interferon alpha protein, and the other one
is for any other antigen.
[0135] Methods for making bispecific antibodies are within the
purview of those skilled in the art. Traditionally, the recombinant
production of bispecific antibodies is based on the co-expression
of two immunoglobulin heavy-chain/light-chain pairs, where the two
heavy chains have different specificities (Milstein and Cuello
(1983) Nature 305:537-539). Antibody variable domains with the
desired binding specificities (antibody-antigen combining sites)
can be fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy-chain constant
domain, including at least part of the hinge, C.sub.H2, and
C.sub.H3 regions. DNAs encoding the immunoglobulin heavy-chain
fusions and, if desired, the immunoglobulin light chain, are
inserted into separate expression vectors, and are co-transfected
into a suitable host organism. For further details of illustrative
currently known methods for generating bispecific antibodies see,
e.g., Suresh et al. (1986) Methods in Enzymology 121:210; PCT
Publication No. WO 96/27011; Brennan et al. (1985) Science 229:81;
Shalaby et al., J Exp Med (1992) 175:217-225; Kostelny et al.
(1992) Jlmmunol 148(5):1547-1553; Hollinger et al. (1993) Proc Natl
Acad Sci USA 90:6444-6448; Gruber et al. (1994) Jlmmunol 152:5368;
and Tutt et al. (1991) J Immunol 147:60. Bispecific antibodies also
include cross-linked or heteroconjugate antibodies. Heteroconjugate
antibodies may be made using any convenient cross-linking methods.
Suitable cross-linking agents are well known in the art, and are
disclosed in U.S. Pat. No. 4,676,980, along with a number of
cross-linking techniques.
[0136] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. See, e.g., Kostelny et al. (1992)
Jlmmunol 148(5):1547-1553. The leucine zipper peptides from the Fos
and Jun proteins may be linked to the Fab' portions of two
different antibodies by gene fusion. The antibody homodimers may be
reduced at the hinge region to form monomers and then re-oxidized
to form the antibody heterodimers. This method can also be utilized
for the production of antibody homodimers. The "diabody" technology
described by Hollinger et al. (1993) Proc Natl Acad Sci USA
90:6444-6448 has provided an alternative mechanism for making
bispecific antibody fragments. The fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) by a linker which is too short to allow pairing between the
two domains on the same chain. Accordingly, the VH and VL domains
of one fragment are forced to pair with the complementary VL and VH
domains of another fragment, thereby forming two antigen-binding
sites. Another strategy for making bispecific antibody fragments by
the use of single-chain Fv (scFv) dimers has also been reported.
See, e.g., Gruber et al. (1994) J Immuno1152:5368. Alternatively,
the antibodies can be "linear antibodies" as described in, e.g.,
Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, these
antibodies comprise a pair of tandem Fd segments
(V.sub.H--C.sub.H1-V.sub.H--C.sub.H1) which form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific.
[0137] The disclosure also embraces variant forms of bispecific
antibodies such as the tetravalent dual variable domain
immunoglobulin (DVD-Ig) molecules described in Wu et al. (2007) Nat
Biotechnol 25(11):1290-1297. The DVD-Ig molecules are designed such
that two different light chain variable domains (VL) from two
different parent antibodies are linked in tandem directly or via a
short linker by recombinant DNA techniques, followed by the light
chain constant domain. Methods for generating DVD-Ig molecules from
two parent antibodies are further described in, e.g., PCT
Publication Nos. WO 08/024188 and WO 07/024715, the disclosures of
each of which are incorporated herein by reference in their
entirety.
Pharmaceutical Compositions and Formulations
[0138] The compositions containing a complement inhibitor (e.g., an
inhibitor of human complement component C5 such as an anti-C5
antibody or antigen-binding fragment thereof or an inhibitor of
interferon alpha) can be formulated as a pharmaceutical
composition, e.g., for administration to a subject to treat Degos'
disease. The pharmaceutical compositions will generally include a
pharmaceutically acceptable carrier. As used herein, a
"pharmaceutically acceptable carrier" refers to, and includes, any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like that are physiologically compatible. The compositions can
include a pharmaceutically acceptable salt, e.g., an acid addition
salt or a base addition salt (see, e.g., Berge et al. (1977) J
Pharm Sci 66:1-19).
[0139] The compositions can be formulated according to standard
methods. Pharmaceutical formulation is a well-established art, and
is further described in, e.g., Gennaro (2000) "Remington: The
Science and Practice of Pharmacy," 20.sup.th Edition, Lippincott,
Williams & Wilkins (ISBN: 0683306472); Ansel et al. (1999)
"Pharmaceutical Dosage Forms and Drug Delivery Systems," 7.sup.th
Edition, Lippincott Williams & Wilkins Publishers (ISBN:
0683305727); and Kibbe (2000) "Handbook of Pharmaceutical
Excipients American Pharmaceutical Association," 3.sup.rd Edition
(ISBN: 091733096X). In some embodiments, a composition can be
formulated, for example, as a buffered solution at a suitable
concentration and suitable for storage at 2-8.degree. C. In some
embodiments, a composition can be formulated for storage at a
temperature below 0.degree. C. (e.g., -20.degree. C. or -80.degree.
C.).
[0140] The pharmaceutical compositions can be in a variety of
forms. These forms include, e.g., liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form depends,
in part, on the intended mode of administration and therapeutic
application. For example, compositions containing an anti-C5
antibody intended for systemic or local delivery can be in the form
of injectable or infusible solutions. Accordingly, the compositions
can be formulated for administration by a parenteral mode (e.g.,
intravenous, subcutaneous, intraperitoneal, or intramuscular
injection). "Parenteral administration," "administered
parenterally," and other grammatically equivalent phrases, as used
herein, refer to modes of administration other than enteral and
topical administration, usually by injection, and include, without
limitation, intravenous, intranasal, intraocular, pulmonary,
intramuscular, intraarterial, intrathecal, intracapsular,
intraorbital, intracardiac, intradermal, intrapulmonary,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural,
intracerebral, intracranial, intracarotid and intrasternal
injection and infusion (see below).
[0141] The compositions can be formulated as a solution,
microemulsion, dispersion, liposome, or other ordered structure
suitable for stable storage at high concentration. Sterile
injectable solutions can be prepared by incorporating an antibody
described herein in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by filter sterilization. Generally, dispersions
are prepared by incorporating an inhibitor of human complement
(e.g., an anti-C5 antibody) and/or an inhibitor of interferon alpha
described herein into a sterile vehicle that contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, methods for
preparation include vacuum drying and freeze-drying that yield a
powder of the antibody described herein plus any additional desired
ingredient from a previously sterile-filtered solution thereof. The
proper fluidity of a solution can be maintained, for example, by
the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. Prolonged absorption of injectable compositions can be
brought about by including in the composition a reagent that delays
absorption, for example, monostearate salts and gelatin.
[0142] In certain embodiments, the complement inhibitor (e.g., an
anti-C5 antibody or antigen-binding fragment thereof) or inhibitor
of interferon alpha can be prepared with a carrier that will
protect the compound against rapid release, such as a controlled
release formulation, including implants and microencapsulated
delivery systems. Biodegradable, biocompatible polymers can be
used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic
acid, collagen, polyorthoesters, and polylactic acid. Many methods
for the preparation of such formulations are known in the art. See,
e.g., J. R. Robinson (1978) "Sustained and Controlled Release Drug
Delivery Systems," Marcel Dekker, Inc., New York.
[0143] In some embodiments, an inhibitor described herein can be
formulated in a composition suitable for intrapulmonary
administration (e.g., for administration via nebulizer) to a mammal
such as a human. Methods for preparing such compositions are well
known in the art and described in, e.g., U.S. Patent Application
Publication No. 20080202513; U.S. Pat. Nos. 7,112,341 and
6,019,968; and PCT Publication Nos. WO 00/061178 and WO 06/122257,
the disclosures of each of which are incorporated herein by
reference in their entirety. Dry powder inhaler formulations and
suitable systems for administration of the formulations are
described in, e.g., U.S. Patent Application Publication No.
20070235029, PCT Publication No. WO 00/69887; and U.S. Pat. No.
5,997,848.
[0144] In some embodiments, an inhibitor of human complement (e.g.,
an anti-C5 antibody or antigen-binding fragment thereof) or
inhibitor of interferon alpha described herein can be modified,
e.g., with a moiety that improves its stabilization and/or
retention in circulation, e.g., in blood, serum, or other tissues.
The stabilization moiety can improve the stability, or retention
of, the antibody by at least 1.5 (e.g., at least 2, 5, 10, 15, 20,
25, 30, 40, or 50 or more) fold.
[0145] The nucleic acid inhibitors of human complement described
herein (e.g., an anti-sense nucleic acid or siRNA) can be
incorporated into a gene construct to be used as a part of a gene
therapy protocol to deliver nucleic acids that can be used to
express and produce agents within cells. Expression constructs of
such components may be administered in any biologically effective
carrier, e.g. any formulation or composition capable of effectively
delivering the component gene to cells in vivo. Approaches include
insertion of the subject gene in viral vectors including
recombinant retroviruses, adenovirus, adeno-associated virus,
lentivirus, and herpes simplex virus-1 (HSV-1), or recombinant
bacterial or eukaryotic plasmids. Viral vectors can transfect cells
directly; plasmid DNA can be delivered with the help of, for
example, cationic liposomes (lipofectin) or derivatized (e.g.,
antibody conjugated), polylysine conjugates, gramicidin S,
artificial viral envelopes or other such intracellular carriers, as
well as direct injection of the gene construct or CaPO.sub.4
precipitation carried out in vivo. (See also, "Ex vivo Approaches,"
below.) Examples of suitable retroviruses include pLJ, pZIP, pWE
and pEM which are known to those skilled in the art (see, e.g.,
Eglitis et al. (1985) Science 230:1395-1398; Danos and Mulligan
(1988) Proc Natl Acad Sci USA 85:6460-6464; Wilson et al. (1988)
Proc Natl Acad Sci USA 85:3014-3018; Armentano et al. (1990) Proc
Natl Acad Sci USA 87:6141-6145; Huber et al. (1991) Proc Natl Acad
Sci USA 88:8039-8043; Ferry et al. (1991) Proc Natl Acad Sci USA
88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van
Beusechem et al. (1992) Proc Natl Acad Sci USA 89:7640-7644; Kay et
al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc
Natl Acad Sci USA 89:10892-10895; Hwu et al. (1993) J Immunol
150:4104-4115; U.S. Pat. Nos. 4,868,116 and 4,980,286; PCT
Publication Nos. WO89/07136, WO89/02468, WO89/05345, and
WO92/07573). Another viral gene delivery system utilizes
adenovirus-derived vectors (see, e.g., Berkner et al. (1988)
BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434;
and Rosenfeld et al. (1992) Cell 68:143-155). Suitable adenoviral
vectors derived from the adenovirus strain Ad type 5 d1324 or other
strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are known to
those skilled in the art. Yet another viral vector system useful
for delivery of the subject gene is the adeno-associated virus
(AAV). See, e.g., Flotte et al. (1992) Am J Respir Cell Mol Biol
7:349-356; Samulski et al. (1989) J Virol 63:3822-3828; and
McLaughlin et al. (1989) J Virol 62: 1963-1973.
[0146] In some embodiments, more than one (e.g., two, three, four,
five, six, seven, eight, nine, or 10 or more) inhibitor(s) (e.g.,
one or more inhibitors of human C5) can be co-formulated. For
example, a C5-specific siRNA and an anti-C5 antibody can be
formulated together.
[0147] In some embodiments, an inhibitor of human complement (e.g.,
an inhibitor of human complement such as an anti-C5 antibody or
antigen-binding fragment thereof) or inhibitor of interferon alpha
described herein can be formulated with one or more additional
active agents useful for treating Degos' disease or ameliorating a
symptom thereof. For example, an anti-C5 antibody can be formulated
with an immunosuppressive agent. Immunosuppressive agents include,
e.g., corticosteroids, phenylbutazone, azathioprine, methotrexate,
cyclosporine, tacrolimus, and mycophenolate mofetil,
cyclophosphamide, and an anti-CD20 agent such as rituximab
(Rituxan.TM.; Biogen Idec, Cambridge, Mass.). In some embodiments,
the inhibitor human complement can be formulated for administration
to a subject along with intravenous immunoglobulin therapy (IVIG),
red cell transfusion, plasmapheresis, or with plasma exchange. See,
e.g., Dyrsen et al. (2008), supra and Zhu et al. (2007), supra.
[0148] In some embodiments, the inhibitor of human complement can
be formulated for joint therapy (e.g., simultaneous or concurrent)
with an antithrombotic agent and/or an anticoagulant such as, but
not limited to, clopidogrel, aspirin, dipyridamole, warfarin
(Coumadin), heparin, phenindione, fondaparinux, idraparinux, and
thrombin inhibitors (e.g., argatroban, lepirudin, bivalirudin, or
dabigatran). An inhibitor of human complement (e.g., an anti-C5
antibody) can also be formulated for use with a fibrinolytic agent
(e.g., ancrod, .epsilon.-aminocaproic acid, antiplasmin-a.sub.1,
prostacyclin, and defibrotide) for the treatment of Degos'
disease.
[0149] In some embodiments, e.g., where the Degos' disease is
associated with an infection, the inhibitor human complement and/or
inhibitor of interferon alpha can be formulated with one or more
agents for use in treating an infection. For example, a C5
inhibitor can be formulated with an antibiotic or an anti-viral
agent.
[0150] When the inhibitor of human complement is to be used in
combination with a second active agent, or when two or more
inhibitors of human complement are to be used (e.g., an anti-C5
antibody and an anti-factor B antibody), the agents can be
formulated separately or together. For example, the respective
pharmaceutical compositions can be mixed, e.g., just prior to
administration, and administered together or can be administered
separately, e.g., at the same or different times (see below).
[0151] Likewise, when the inhibitor of interferon alpha (e.g., an
anti-interferon alpha antibody) is to be used in combination with a
second active agent, or when two or more inhibitors of interferon
alpha are to be used, the agents can be formulated separately or
together.
[0152] As described above, a composition can be formulated such
that it includes a therapeutically effective amount of an inhibitor
of human complement (e.g., an anti-C5 antibody or antigen-binding
fragment thereof) or the composition can be formulated to include a
sub-therapeutic amount of the inhibitor and a sub-therapeutic
amount of one or more additional active agents such that the
components in total are therapeutically effective for treating
Degos' disease. In some embodiments, a composition can be
formulated to include two or more inhibitors of human complement,
each at sub-therapeutic doses, such that the inhibitors in total
are at a concentration that is therapeutically effective for
treating Degos' disease. A composition can be formulated such that
it includes a therapeutically effective amount of an inhibitor of
interferon alpha (e.g., an anti-interferon alpha antibody or
antigen-binding fragment thereof) or the composition can be
formulated to include a sub-therapeutic amount of the inhibitor and
a sub-therapeutic amount of one or more additional active agents
such that the components in total are therapeutically effective for
treating Degos' disease. In some embodiments, a composition can be
formulated to include two or more inhibitors of interferon alpha,
each at sub-therapeutic doses, such that the inhibitors in total
are at a concentration that is therapeutically effective for
treating Degos' disease. In some embodiments, the composition
includes an inhibitor of human complement and an inhibitor of
interferon alpha. Methods for determining a therapeutically
effective dose (e.g., a therapeutically effective dose of an
anti-C5 antibody or an anti-interferon antibody) are known in the
art and described herein.
Methods for Treatment
[0153] The above-described compositions are useful in, inter alia,
methods for treating or preventing Degos' disease in a subject
(e.g., a human). The compositions can be administered to a subject,
e.g., a human subject, using a variety of methods that depend, in
part, on the route of administration. The route can be, e.g.,
intravenous injection or infusion (IV), subcutaneous injection
(SC), intraperitoneal (IP), or intramuscular injection.
[0154] Administration can be achieved by, e.g., local infusion,
injection, or by means of an implant. The implant can be of a
porous, non-porous, or gelatinous material, including membranes,
such as sialastic membranes, or fibers. The implant can be
configured for sustained or periodic release of the composition to
the subject. (See, e.g., U.S. Patent Publication No. 20080241223;
U.S. Pat. Nos. 5,501,856; 4,863,457; and 3,710,795; EP488401; and
EP 430539, the disclosures of each of which are incorporated herein
by reference in their entirety.) The composition can be delivered
to the subject by way of an implantable device based on, e.g.,
diffusive, erodible, or convective systems, e.g., osmotic pumps,
biodegradable implants, electrodiffusion systems, electroosmosis
systems, vapor pressure pumps, electrolytic pumps, effervescent
pumps, piezoelectric pumps, erosion-based systems, or
electromechanical systems.
[0155] A suitable dose of an inhibitor of human complement (e.g.,
an anti-C5 antibody or fragment thereof) or an inhibitor of
interferon alpha (e.g., an anti-interferon alpha antibody), which
dose is capable of treating or preventing Degos' disease in a
subject, can depend on a variety of factors including, e.g., the
age, sex, and weight of a subject to be treated and the particular
inhibitor compound used. For example, a different dose of a siRNA
specific for human C5 may be required to treat a subject with
Degos' disease as compared to the dose of an anti-C5 antibody
required to treat the same patient. Other factors affecting the
dose administered to the subject include, e.g., the type or
severity of Degos' disease. For example, a subject having a
cutaneous form of Degos' disease may require administration of a
different dosage of the inhibitor than a subject with a systemic
form of Degos' disease. Other factors can include, e.g., other
medical disorders concurrently or previously affecting the subject,
the general health of the subject, the genetic disposition of the
subject, diet, time of administration, rate of excretion, drug
combination, and any other additional therapeutics that are
administered to the subject. It should also be understood that a
specific dosage and treatment regimen for any particular subject
will depend upon the judgment of the treating medical practitioner
(e.g., doctor or nurse).
[0156] The inhibitor can be administered as a fixed dose, or in a
milligram per kilogram (mg/kg) dose. In some embodiments, the dose
can also be chosen to reduce or avoid production of antibodies or
other host immune responses against one or more active agents in
the composition. While in no way intended to be limiting, exemplary
dosages of a complement inhibitor (e.g., an anti-C5 antibody)
and/or an inhibitor of interferon alpha include, e.g., 1-100
.mu.g/kg, 0.5-50 .mu.g/kg, 0.1-100 .mu.g/kg, 0.5-25 .mu.g/kg, 1-20
.mu.g/kg, and 1-10 .mu.g/kg, 1-100 mg/kg, 0.5-50 mg/kg, 0.1-100
mg/kg, 0.5-25 mg/kg, 1-20 mg/kg, and 1-10 mg/kg. Exemplary dosages
of an antibody described herein include, without limitation, 0.1
.mu.g/kg, 0.5 .mu.g/kg, 1.0 .mu.g/kg, 2.0 .mu.g/kg, 4 .mu.g/kg, and
8 .mu.g/kg, 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 4 mg/kg,
and 8 mg/kg.
[0157] In some embodiments, a human can be intravenously
administered an anti-C5 antibody (e.g., eculizumab) at a dose of
about 900 mg about every 12 (e.g., about every 10, 11, 13, 14, 15,
16, 17, 18, 19, 20, 21, 28, 30, 42, or 49 or more) days. See, e.g.,
Hill et al. (2005) Blood 106(7):2559.
[0158] In some embodiments, a human can be intravenously
administered an anti-C5 antibody (e.g., eculizumab) at a dose of
about 600 (e.g., about 625, 650, 700, 725, 750, 800, 825, 850, 875,
900, 925, 950, or 1,000 or more) mg every week, optionally, for two
or more (e.g., three, four, five, six, seven, or eight or more)
weeks. Following the initial treatment, the human can be
administered the antibody at a dose of about 900 mg about every 14
(e.g., about every 15, 16, 17, 18, 19, 20, 21, 28, 30, 42, or 49 or
more) days, e.g., as a maintenance dose. See, e.g., Hillmen et al.
(2004) N Engl J Med. 350(6):552-9 and Dmytrijuk et al. (2008) The
Oncologist 13(9):993.
[0159] A pharmaceutical composition can include a therapeutically
effective amount of an inhibitor of human complement component C5
(e.g., an anti-C5 antibody or antigen-binding fragment thereof)
and/or a therapeutically effective amount of an inhibitor of
interferon alpha. Such effective amounts can be readily determined
by one of ordinary skill in the art based, in part, on the effect
of the administered inhibitor, or the combinatorial effect of the
antibody and one or more additional active agents, if more than one
agent is used. A therapeutically effective amount of an inhibitor
of human complement (e.g., an anti-C5 antibody) can also vary
according to factors such as the disease state, age, sex, and
weight of the individual, and the ability of the antibody (and one
or more additional active agents) to elicit a desired response in
the individual, e.g., amelioration of at least one condition
parameter, e.g., amelioration of at least one symptom of Degos'
disease. For example, a therapeutically effective amount of an
inhibitor of human complement (e.g., an anti-C5 antibody) can
inhibit (lessen the severity of or eliminate the occurrence of)
and/or prevent Degos' disease, and/or any one of the symptoms of
Degos' disease known in the art or described herein. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the composition are outweighed by the
therapeutically beneficial effects.
[0160] The terms "therapeutically effective amount" or
"therapeutically effective dose," or similar terms used herein are
intended to mean an amount of an agent (e.g., an inhibitor of human
complement) that will elicit the desired biological or medical
response (e.g., an improvement in one or more symptoms of Degos'
disease). In some embodiments, a composition described herein
contains a therapeutically effective amount of an inhibitor of
human complement component C5. In some embodiments, a composition
described herein contains a therapeutically effective amount of an
antibody, or antigen-binding fragment thereof, which binds to a
complement component C5 protein. In some embodiments, the
composition contains two or more (e.g., three, four, five, six,
seven, eight, nine, 10, or 11 or more) different inhibitors of
human complement such that the composition as a whole is
therapeutically effective. For example, a composition can contain
an antibody that binds to a human C5 protein and a siRNA that binds
to, and promotes the degradation of, an mRNA encoding a human C5
protein, wherein the antibody and siRNA are each at a concentration
that when combined are therapeutically effective. In some
embodiments, the composition contains the inhibitor and one or more
second active agents such that the composition as a whole is
therapeutically effective. For example, the composition can contain
an antibody that binds to a human C5 protein and another agent
useful for treating or preventing Degos' disease.
[0161] In some embodiments, a composition described herein contains
a therapeutically effective amount of an anti-inteferon alpha
antibody or an antigen-binding fragment thereof.
[0162] Toxicity and therapeutic efficacy of such compositions can
be determined by known pharmaceutical procedures in cell cultures
or experimental animals. These procedures can be used, e.g., for
determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD.sub.50/ED.sub.50. Compositions, or
complement inhibitors (e.g., anti-C5 antibodies) of the
compositions, that exhibit high therapeutic indices are preferred.
While compositions that exhibit toxic side effects may be used,
care should be taken to design a delivery system that targets such
compounds to the site of affected tissue and to minimize potential
damage to normal cells and, thereby, reduce side effects.
[0163] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such inhibitors lies generally within a range
of circulating concentrations of the inhibitor(s) (e.g., an anti-C5
antibody, an anti-interferon alpha antibody, or antigen-binding
fragments thereof) that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized. For
an inhibitor of human complement component C5 (e.g., an anti-C5
antibody) used as described herein (e.g., for treating or
preventing Degos' disease), the therapeutically effective dose can
be estimated initially from cell culture assays. Levels of the
complement inhibitor in, e.g., the plasma of treated humans or
animals may be measured, for example, by high performance liquid
chromatography.
[0164] In some embodiments, the required dose of an inhibitor of
human complement (e.g., an anti-C5 antibody) can be determined
based on the concentration in the subject's blood of the complement
protein to which the inhibitor is directed. For example, a subject
having a higher concentration of circulating human C5 protein
levels may require a higher dose of a human C5 inhibitor than a
subject having lower levels of circulating human C5. Methods for
determining the concentration of human complement in a
blood-derived fluid sample from a subject are known in the art. For
example, a method for determining serum C5 levels is described in,
e.g., Rawal et al. (1998) J Biol Chem 273(27):16828-16835.
[0165] A "subject," as used herein, can be any mammal. For example,
a subject can be a human, a non-human primate (e.g., monkey,
baboon, or chimpanzee), a horse, a cow, a pig, a sheep, a goat, a
dog, a cat, a rabbit, a guinea pig, a gerbil, a hamster, a rat, or
a mouse. In some embodiments, the subject is an infant (e.g., a
human infant).
[0166] In some embodiments, the subject is one who is refractory to
one or more additional therapeutic agents that were administered to
treat Degos' disease. "Resistance" to a therapy, "refractory" to
therapy, and like grammatical phrases, as used herein, refer to a
patient's clinical state of being, in which there is a reduction in
the effectiveness of a given therapy in treating or curing a given
disorder (e.g., Degos' disease) or a reduction in the effectiveness
of the treatment in ameliorating one or more symptoms associated
with the disorder. For example, the therapeutic benefits of IVIg to
a patient afflicted with Degos' disease may diminish over time such
that the disease remains or progresses even with IVIg therapy. See,
e.g., De Breucker et al. (2008), supra.
[0167] As used herein, a subject "in need of prevention," "in need
of treatment," or "in need thereof," refers to one, who by the
judgment of an appropriate medical practitioner (e.g., a doctor, a
nurse, or a nurse practitioner in the case of humans; a
veterinarian in the case of non-human mammals), would reasonably
benefit from a given treatment (such as treatment with a
composition comprising an inhibitor of human complement or an
inhibitor of interferon alpha).
[0168] As used herein, a subject "at risk for developing Degos'
disease" is a subject having one or more (e.g., two, three, four,
five, six, seven, or eight or more) risk factors for developing the
disorder. Risk factors for Degos' disease are well known in the art
of medicine and include, e.g., a predisposition to develop the
condition, i.e., a family history of the condition or a genetic
status associated with Degos' disease such as, e.g., a Protein S
deficiency. See, e.g., Gileberte et al. (2005) Br J Dermatol
153(3):666-7. Risk factors for Degos' disease also include those
conditions that are associated with Degos' disease such as, but not
limited to, viral infections (e.g., HIV or B19 parvovirus
infections), a procoagulant state (e.g., Factor V Leiden), or an
autoimmune disease such as LE, dermatomyositis, scleroderma, and
antiphospholipid syndrome. Hereinafter, such manifestations of
Degos' disease may be, where appropriate, referred to as, e.g.,
"infection-associated Degos' disease" or "autoimmune-associated
Degos' disease." From the above it will be clear that subjects "at
risk for developing Degos' disease" are not all the subjects within
a species of interest.
[0169] A subject "suspected of having Degos' disease" is one having
one or more (e.g., two, three, four, five, six, seven, eight, nine,
or 10 or more) symptoms of the condition. Symptoms of this
condition are known to those of skill in the art of medicine and
include, e.g., skin lesions (e.g., one or more papules that are
raised and skin- or rose-colored, which papules progress to
depressed scars with white centers and surrounding erythema and
telangiectasias), gastrointestinal bleeding, vomiting,
enterocutaneous fistula, neurological symptoms (e.g., facial and
acral paraesthesia, headaches, dizziness, aphagia, paraplegia, gaze
palsy, epilepsy, memory loss, or altered sensation), strokes,
diplopia, ptosis, visual-field defects, weakness, shortness of
breath, and chest pain. In some embodiments, Degos' disease is
associated with the presence in the patients of anticardiolipin
antibodies, the lupus anticoagulant, antiphospholipid antibodies,
or vascular IgA deposition. See, e.g., Englert et al. (1984),
supra; Crowson et al. (2002), supra; and Grattan and Burton (1991)
Semin Dermatol 10(3):152-159. From the above it will be clear that
subjects "suspected of having Degos' disease" are not all the
subjects within a species of interest. In some embodiments, the
methods can include identifying the subject as one having,
suspected of having, or at risk for developing, Degos' disease.
Suitable methods for identifying the subject are known in the art.
For example, Ackerman describes a histological method for
positively identifying a Degos' disease-associated cutaneous
lesion. Am J Dermatopathol (1985) 7(2):105-7. As described above,
Degos' disease can be associated with the presence in the patients
of anticardiolipin antibodies, the lupus anticoagulant, and/or
antiphospholipid antibodies. Diagnostic histological methods are
also exemplified in the working examples. Suitable methods for
detecting the presence of these antibodies in the blood of a
patient are known in the art and described in, e.g., Caux et al.
(1994) Ann Dermatol Venereol 121:537-542. In some embodiments,
Degos' disease can be associated with IgA deposition within the
cutaneous vasculature. Methods for detecting such deposition are
known in the art. See, e.g., Crowson et al. (2002), supra.
[0170] In addition, as described in the working examples, Degos'
disease can be associated with prominent vascular C5b-9 MAC
deposition as well as elevated serum C reactive protein and factor
VIII levels. Methods for detecting each of these parameters are
exemplified herein.
[0171] In some embodiments, the composition can be administered to
a subject prophylactically to prevent the progression of the benign
form of Degos' disease to the systemic, multiorgan form of the
disease. For example, a subject who has a cutaneous form of Degos'
disease can be administered a composition described herein to
prevent or lessen the likelihood of development in the patient of
the lethal, systemic form of Degos' disease. Similarly, a subject
who has a Degos' disease-associated B19 parvoviral infection, an
HIV infection, or an autoimmune disease can be administered a
composition described herein to prevent or lessen the likelihood of
the development of Degos' disease in the patient.
[0172] The term "preventing" is art-recognized, and when used in
relation to a condition, is well understood in the art, and
includes administration of a composition which reduces the
frequency of, or delays the onset of, symptoms of a medical
condition in a subject relative to a subject which does not receive
the composition. Thus, prevention of Degos' disease includes, for
example, slowing the progression of the benign form of Degos'
disease to the systemic, multiorgan form of the disease in a
population of patients receiving a prophylactic treatment relative
to an untreated control population, and/or reducing the severity
and/or delaying the onset of the one or more symptoms of the
disease in a treated population versus an untreated control
population, e.g., by a statistically and/or clinically significant
amount.
[0173] In some embodiments, the inhibitor of human complement
(e.g., an anti-C5 antibody or antigen-binding fragment thereof)
and/or the inhibitor of interferon alpha can be administered to a
subject as a monotherapy. Alternatively, as described above, the
inhibitor can be administered to a subject as a combination therapy
with another treatment, e.g., another treatment for Degos' disease.
For example, the combination therapy can include administering to
the subject (e.g., a human patient) one or more additional agents
(e.g., anti-coagulants or anti-inflammatory agents) that provide a
therapeutic benefit to the subject who has, or is at risk of
developing, Degos disease. In some embodiments, the inhibitor of
human complement (e.g., an anti-C5 antibody) or inhibitor of
interferon alpha and the one or more additional active agents are
administered at the same time. In other embodiments, the inhibitor
is administered first in time and the one or more additional active
agents are administered second in time. In some embodiments, the
one or more additional active agents are administered first in time
and the inhibitor is administered second in time.
[0174] The inhibitor of human complement or inhibitor of interferon
alpha can replace or augment a previously or currently administered
therapy. For example, upon treating with an anti-C5 antibody or
antigen-binding fragment thereof, administration of the one or more
additional active agents can cease or diminish, e.g., be
administered at lower levels. In some embodiments, administration
of the previous therapy can be maintained. In some embodiments, a
previous therapy will be maintained until the level of inhibitor of
human complement (e.g., an anti-C5 antibody) or inhibitor of
interferon alpha reaches a level sufficient to provide a
therapeutic effect. The two therapies can be administered in
combination.
[0175] Monitoring a subject (e.g., a human patient) for an
improvement in Degos' disease, as defined herein, means evaluating
the subject for a change in a disease parameter, e.g., an
improvement in one or more symptoms of the disease. For example, a
medical practitioner can examine the extent of vascular C5b-9 MAC
deposition before and after treatment using a complement inhibitor
described herein. Such symptoms include any of the symptoms of
Degos' disease described herein. In some embodiments, the
evaluation is performed at least 1 hour, e.g., at least 2, 4, 6, 8,
12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13
days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10
weeks, 13 weeks, 20 weeks or more, after an administration. The
subject can be evaluated in one or more of the following periods:
prior to beginning of treatment; during the treatment; or after one
or more elements of the treatment have been administered.
Evaluating can include evaluating the need for further treatment,
e.g., evaluating whether a dosage, frequency of administration, or
duration of treatment should be altered. It can also include
evaluating the need to add or drop a selected therapeutic modality,
e.g., adding or dropping any of the treatments for Degos' disease
described herein.
[0176] Ex Vivo Approaches.
[0177] Where the inhibitor of human complement or inhibitor of
interferon alpha is a polypeptide (e.g., an antibody) or a nucleic
acid (e.g., an siRNA or anti-sense nucleic acid), an ex vivo
strategy for treating or preventing Degos disease can involve
transfecting or transducing one or more cells obtained from a
subject with a polynucleotide encoding the protein or nucleic acid.
For example, the cells can be transfected with a single vector
encoding a heavy and light chain of an anti-C5 antibody or the
cells can be transfected with a first vector encoding a heavy chain
and a second vector encoding a light chain of the antibody.
[0178] The transfected or transduced cells are then returned to the
subject. The cells can be any of a wide range of types including,
without limitation, hemopoietic cells (e.g., bone marrow cells,
macrophages, monocytes, dendritic cells, T cells, or B cells),
fibroblasts, epithelial cells, endothelial cells, keratinocytes, or
muscle cells. Such cells can act as a source (e.g., sustained or
periodic source) of the complement inhibitor (e.g., the anti-C5
antibody or antigen-binding fragment thereof, or anti-C5 siRNA) or
inhibitor of interferon alpha for as long as they survive in the
subject. In some embodiments, the vectors and/or cells can be
configured for inducible or repressible expression of the antibody
(see, e.g., Schockett et al. (1996) Proc Natl Acad Sci USA 93:
5173-5176 and U.S. Pat. No. 7,056,897.)
[0179] Preferably, the cells are obtained from the subject
(autologous), but can potentially be obtained from a subject of the
same species other than the subject (allogeneic).
[0180] Suitable methods for obtaining cells from a subject and
transducing or transfecting the cells are known in the art of
molecular biology. For example, the transduction step can be
accomplished by any standard means used for ex vivo gene therapy,
including calcium phosphate, lipofection, electroporation, viral
infection (see above), and biolistic gene transfer. See, e.g.,
Sambrook et al. (supra) and Ausubel et al. (1992) "Current
Protocols in Molecular Biology," Greene Publishing Associates.
Alternatively, liposomes or polymeric microparticles can be used.
Cells that have been successfully transduced can be selected, for
example, for expression of the coding sequence or of a drug
resistance gene.
[0181] Kits
[0182] The disclosure also features articles of manufacture or
kits, which include a container with a label; and a composition
containing one or more (e.g., one, two, three, four, five, six,
seven, eight, nine, or 10 or more) inhibitor(s) of human complement
(e.g., an anti-C5 antibody or antigen-binding fragment thereof)
and/or one or more (e.g., one, two, three, four, five, six, seven,
eight, nine, or 10 or more) inhibitors of interferon alpha (e.g.,
an anti-interferon alpha antibody). The label indicates that the
composition is to be administered to a subject (e.g., a human)
having, suspected of having, or at risk for developing, Degos'
disease. The kit can, optionally, include a means for administering
the composition to the subject. For example, the kits can include
one or more syringes.
[0183] In some embodiments, the kits can contain two or more (e.g.,
three, four, five, six, seven, eight, nine, or 10 or more)
different types of inhibitors of human complement. For example, a
kit can contain an anti-C5 antibody (or antigen-binding fragment
thereof) and a siRNA that binds to an mRNA that encodes a human C5
protein. In some embodiments, the kit can contain an anti-C5
antibody, a siRNA, and a small molecule inhibitor of
complement.
[0184] In some embodiments, the kits can further include one or
more additional active agents such as any of those described
herein. For example, the kits can include one or more
anti-coagulants, anti-thrombotic agents, or anti-inflammatory
agents. The kits can also include one or more B cell-targeted
therapies such as an anti-CD20 antibody. The kits can optionally
include one or more means for detecting the presence of
anti-cardiolipin antibodies, the lupus anticoagulant,
antiphospholipid antibodies, and/or vascular IgA deposition.
[0185] The following examples are intended to illustrate, not
limit, the invention.
Examples
Example 1
Materials and Methods
[0186] Routine light microscopy.
[0187] Five-micron thick sections of paraffin embedded, formalin
fixed tissue were stained with hematoxylin and eosin and examined
by conventional light microscospy.
[0188] Immunohistochemical assessment for MxA.
[0189] The slides for use in detecting MxA protein were placed in
Tissue Tek Slide Holders and staining dishes (Miles, Elkhart, Ill.)
and submersed in 200 mL of EDTA buffer, pH 8.0 (Zymed Laboratories,
South San Francisco, Calif.). Following a 30-minute incubation with
the primary antibodies, staining was performed using the
commercially available Vision BioSystems Define Kit adhering to the
protocol. Incubation with the primary antibodies was conducted
using the following dilutions: anti-MxA antibody 1:1600. A
semi-qualitative assessment was made in regards to the extent of
staining based on an approximate percentage of cells stained for a
particular marker as well as the distribution of staining across
the slide. Slides were cover-slipped using the Shandon
Consul.RTM.-Mount Histology (Product No. 9990440) system on a
Consul automated cover-slipper (ThermoShandon, Pittsburgh, Pa.).
See Magro et al. (2009) J Cutan Pathol (November 4, electronic
publication ahead of print; PMID: 19891658).
[0190] Immunofluorescence Studies on Tissue.
[0191] The following immunofluorescence studies (as described in
Crowson and Magro (1996) Human Pathol 27:15-19) were performed on
skin biopsy material obtained in Michael's transport medium and
subsequently stored at -30.degree. C. Direct immunofluorescence
(DIF) studies for immunoglobulin G (IgG), IgA, IgM, fibrin, and
complement component C3 (DAKO, Carpinteria, Calif., USA) were
performed on lesional skin by the overlay of fluorescein-conjugated
primary antibodies upon individual biopsy sections. An indirect
immunofluorescence (IF) methodology with a fluorescein-conjugated
rabbit anti-mouse antibody was used to detect the presence of C5b-9
MAC (DAKO), by way of its binding to a primary anti-05b-9 antibody
initially contacted to a biopsy section.
[0192] Electron Microscopy.
[0193] Skin biopsy tissue was placed in glutaraldehyde fixative for
ultrastructural examination.
[0194] Cell Culture for Indirect Immunofluorescence and Western
Blot Studies.
[0195] Neonatal human dermal blood microvascular endothelial cells,
HMVEC-dBlNeo (Lonza, Walkersville, Md.), were cultured at
37.degree. C. in complete medium (EGM.RTM.-2 Basal Medium
supplemented with EGM.RTM.-2 MV BulletKit.RTM. reagents and 5%
Premium FBS) (Lonza) with 5% CO.sub.2. The cells were cultured for
a time sufficient to reach 75% to 80% confluency in the culture
flasks. The cells were then washed with warm phosphate-buffered
saline (PBS) and harvested using a 1.times. trypsin-EDTA solution
(Sigma-Aldrich, St. Louis, Mo.). After an additional wash with PBS,
the cells were re-plated in pre-warmed complete medium onto tissue
culture chamber slides (Nunc Lab-Tek.TM. Chamber Slide System, 2
wells on Permanox.TM., Sigma-Aldrich) at a density of
2.times.10.sup.4 cells/mL. The chamber slides were cultured for 24
hours at 37.degree. C. with 5% CO.sub.2 after which time the media
was discarded. Following removal of the media, the slides of each
chamber were removed, rinsed in PBS, and then air dried in
combination with paper towels to wick away excess PBS. The dried
slides were then stored at -80.degree. C. in an airtight container
until examined using immunofluorescence microscopy.
[0196] Western Blot Studies.
[0197] The cultured HMVEC-dBlNeo cells were lysed using a lysis
buffer consisting of 0.05M sodium fluoride, 1% Triton X-100, 50 mM
Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, and a 1:200 dilution of Calbiochem
Protease Inhibitor Cocktail Set III (EMD Chemicals, Inc.,
Gibbstown, N.J.) for 30 minutes at 4.degree. C. with occasional
vortexing. The lysates were then cleared of nuclei and other
insoluble material by microcentrifugation. Next, sodium dodecyl
sulfate-polyacrylamide gel (SDS-PAGE) loading buffer containing
.beta.-mercaptoethanol (5% final concentration) was added to the
cell lysate (1:4 dilution of the 5.times. loading buffer into
lysate) prior to boiling for two minutes. Lysates were then
resolved by electrophoresis through an 8% to 16% gradient Tris-HCl
SDS-PAGE gels (BioRad, Hercules, Calif.) and transferred to Whatman
Protran.TM. nitrocellulose membranes (Sigma-Aldrich). Membranes
were incubated for six hours at room temperature with a blocking
solution of 3% non-fat milk in Tris-buffered saline with 0.1%
Tween-20 (TBS-T) on an orbital shaker, and then fitted into a
Miniblotter 25 blotting manifold (Immunetics, Cambridge, Mass.).
Miniblotter wells were filled with either patient or healthy
control serum diluted 1:250 in TBS-T containing 0.2% bovine serum
albumin, and the Miniblotter rocked gently overnight at 4.degree.
C. on an orbital shaker. The sera were then removed, the wells
washed twice with TBS-T, and the membranes removed from the
apparatus. Following additional extensive washing with TBS-T, the
membranes were stained for two hours at room temperature with a
1:20,000 dilution of horseradish peroxidase-conjugated goat
anti-human Ig(heavy and light chain) secondary antibody
(SouthernBiotech, Birmingham, Ala.). After extensive washing of the
membranes with TBS-T, antibody complexes were detected using
Supersignal.RTM. West Pico Chemiluminescent Substrate (Thermo
Scientific, Rockford, Ill.), with chemilluminescent bands
visualized on HyBlot CL.TM. autoradiography film (Denville
Scientific, Inc., Metuchen, N.J.).
[0198] Indirect Immunofluorescence Studies to Detect the Presence
of Anti-Endothelial Cell Antibodies.
[0199] Serum samples were diluted to a dilution factor of 1:100 and
incubated with human cutaneous endothelial cells. Antibody binding
was detected using a fluorescein-conjugated goat anti-human IgG
(diluted 1:100 in PBS; Caltag, Burlingame, Calif.).
Fluorescent-antibody complexes were visualized using an Olympus
microscope and images were recorded with a digital camera.
Example 2
Results
[0200] Clinical history. Over a two-year period, a 43-year old
previously healthy male developed asymptomatic small cutaneous
lesions initially defined by raised papular lesions eventuating
into depressed white scars. On 23 Jul. 2009, the patient entered
the emergency room with a three-day history of intermittent, low
grade fevers accompanied by severe abdominal pain and green tinged
vomiting. An exploratory laparotomy was performed--a small segment
of bowel was removed. Both the skin and bowel specimens showed
classic changes associated with Degos' Disease. No anticardiolipin
antibodies, anti-beta 2 glycoprotein antibodies, or lupus
anticoagulant was detected. Additional laboratory tests confirmed
abnormally elevated serum factor VIII levels (199 above normal),
elevated von Willebrand factor (VWF) levels (217 above normal), and
increased vascular endothelial cell growth factor (VEGF) levels.
While the complement component proteins C2, C3 and C4 were
determined to be within normal levels, C5 levels were elevated.
[0201] A thoracocentesis was performed on the patient to relieve
the pneumothorax. The patient was started on intravenous gamma
globulin and Lovenox.RTM. at 490 mg daily. During a follow up visit
on 12 Aug. 2009, the patient had lost 15 pounds due to decreased
appetite attributable to persistent abdominal pain. A respiratory
exam of the patient demonstrated decreased breath sounds in the
bibasilar regions. The patient's C reactive protein, VWF, and VEGF
levels remained elevated at 6.51, 177, and 971, respectively. After
four treatments of IVIG there was some improvement in the patient's
skin lesions, although the patient continued to suffer from
abdominal pain. In October 2009, the patient developed chest pain
and shortness of breath, which necessitated hospital admission. The
patient quickly decompensated hemodynamically and was placed in the
medical intensive care unit. The patient was noted to have an
ejection fraction of 14% associated with a large pericardial and
pleural effusion. A pericardial biopsy was obtained that confirmed
a severe thrombotic microangiopathy consistent with Degos' disease.
Vascular C5b-9 deposition was also observed. Eculizumab was then
administered to the patient. An almost immediate improvement in the
patient's condition occurred. For example, the patient's ejection
fraction improved dramatically. Within 24 hours of receiving the
eculizumab, the patient was extubated and transferred to the
medical ward. Over the next several weeks, the patient was
symptomatically better and there was an objective improvement in
the appearance of his skin lesions.
[0202] Pathology. Three sets of skin biopsies were obtained from
the patient. The time at which these biopsies were performed was
temporally associated with variations in treatment intervention.
The first set was obtained from the patient prior to the
administration of either IVIG or eculizumab to the patient and
included two evolutionary phases of his skin lesions. One biopsy
was from a raised papular lesion revealing striking mesenchymal
mucin deposition, a morphologic finding that led to an initial
erroneous diagnosis of tumid lupus erythematosus two years
previously. Examination of the vasculature revealed abnormalities
including endothelial cell swelling, necrosis, and detachment with
focal mural and luminal fibrin deposition. A biopsy of the classic
Degos' disease associated porcelain depressed scarred papule
demonstrated prominent epidermal thinning and dermal fibrosis with
vascular drop out. The vessels demonstrated an extensive occlusive
thrombotic microangiopathy with endothelial cell
degeneration/necrosis and endothelial cell sloughing into the
vascular lumen. All of the biopsies revealed a dearth of
inflammatory cells. The bowel specimen showed an extensive
obliterative vasculopathy affecting larger caliber arteries of the
submucosa.
[0203] Several weeks after commencing IVIG, another skin biopsy was
obtained, analysis of which revealed persistent cutaneous ischemic
changes including mucin, fibrosis and epithelial thinning. However,
there was a reduction in the extent of active vascular injury.
Vessels containing significant luminal and mural fibrin deposition
were not observed.
[0204] An additional set of biopsies (the third set) was obtained
from the patient two weeks after administration of eculizumab.
Analysis of the biopsied tissue revealed superficial fibroplasias
with vascular drop out. The vessels were thickened reflecting
basement membrane zone reduplication. Only rare vessels exhibited
endothelial cell damage and thrombosis.
[0205] Interferon Alpha Assessment in Tissue Samples.
[0206] There was extensive expression of MxA protein in endothelial
cells, inflammatory cells, and epidermal keratinocytes.
[0207] C5b-9 Immunofluorescence Studies.
[0208] Indirect immunofluorescence studies revealed extensive
deposits of C5b-9 within the cutaneous vasculature of the first set
of biopsies prior to commencing IVIG and or eculizumab. The
deposition pattern was both intraluminal and mural. The extent of
deposition was very prominent involving several vessels throughout
the dermis. Although IVIG reduced C5b-9 deposition to some degree,
the extent of C5b-9 deposition observed in biopsies obtained from
the patient following eculizumab therapy was markedly diminished.
Only three vessels showed mural C5b-9 deposition.
[0209] Electron Microscopy.
[0210] Ultrastructural analysis of the biopsied tissue obtained
pre-treatment showed extensive tubular reticular structures in the
epidermal keratinocytes and in endothelial cells throughout the
dermis. The lining endothelial cells showed profound degenerative
changes with detachment of the cells from the vessel lumens. The
vascular basement membrane zones were reduplicated and also showed
by collagen deposition.
[0211] Antiendothelial Cell Antibody Assay.
[0212] Patient serum obtained prior to eculizumab treatment and
serum obtained two weeks following eculizumab treatment were
incubated with cutaneous endothelial cells and
fluorescein-conjugated human anti-IgG antibodies. A granular
nuclear decoration was observed in most of the endothelial cells in
the pre- and post-eculizumab treatment samples. No staining was
observed when the patient serum was contacted to human umbilical
vein endothelial cells.
[0213] Western Blot Studies Using Endothelial Cell Lysates.
[0214] The pre- and post-treatment patient serum was also used in a
Western blot analysis to identify the protein or proteins to which
the human-anti-human antibodies in the patient serum bind. As
described above, the pre- and post-treatment serum was incubated
with a membrane containing the size-resolved endothelial cell
proteins and any binding of antibodies to the proteins of the
membrane was detected by way of a fluorescein-conjugated secondary
antibody. A distinct band of immunoreactivity corresponding to a
molecular weight of 92 kDa was observed. A similar band of
reactivity was not identified in normal control cases or in Western
blots of lysates of human umbilical vein endothelial cells.
[0215] Assessment of Serum Interferon Alpha Levels.
[0216] Interferon alpha levels in the peripheral blood of the
patient were very high--measuring 20.56, which was comparable to
patients having lupus erythematosus and several fold higher than
interferon alpha levels in blood from healthy patients.
[0217] Discussion.
[0218] The patient described herein developed multifocal
intestinal, pericardial, myocardial and cutaneous ischemia,
attributable to prominent endothelial cell injury. The patient's
symptoms were consistent with Degos' Disease. Using cutaneous
endothelial cells as substrate, there was evidence of
anti-endothelial cell antibodies using indirect immunofluorescence
and Western blot techniques. A dominant antigenic epitope was
isolated based on Western Blot analysis, although its identity is
unclear. In view of the finding that the anti-endothelial cell
antibodies were not reactive with human umbilical vein cells, it is
likely that the antiendothelial cell antibodies observed in this
patient were organ selective, whereby the immunogenicity to the
selected endothelial cell based epitope is site dependent.
[0219] The studies described herein implicate complement-mediated
endothelial injury as the likely effector mechanism in the Degos'
disease patient treated with eculizumab. There was a dramatic
objective clinical and pathological response to the administration
of the drug.
[0220] Interferon alpha expression was markedly increased in the
patient's serum as well as within his tissue. Interferon alpha is
known to upregulate adaptive and innate immunity, potentiating the
effects of any antigenic trigger. The administration of exogenous
interferon alpha has been reported as a cause of cutaneous
thrombosis and ulceration. The patient's interferon alpha signature
in the peripheral blood was remarkably similar to that observed in
patients with SLE although he had no specific clinical features of
systemic lupus erythematosus. The findings described herein support
a conclusion that inhibition of interferon alpha in patients with
Degos' disease can be useful for treating the disease.
[0221] In conclusion, eculizumab defines an important therapeutic
modality to treat Degos' disease that is otherwise fatal. The exact
trigger to the activation of the complement cascade sequence is
unclear. While anti-endothelial cell antibodies (e.g.,
autoantibodies that bind to a 92 kDa antigen present on endothelial
cells) provoking activation of the classic complement cascade
sequence may be causative of C5b-9 deposition, the role of epitope
spreading in the natural course of tissue injury precludes
establishing a direct causal effect of these antibodies.
Nonetheless at some point in the patient's clinical course
additional B cell targeted therapies to reduce the production of
these antibodies would be useful for the treatment of Degos'
disease provided that the autoantibodies persist.
[0222] While the present disclosure has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the disclosure. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present disclosure. All such
modifications are intended to be within the scope of the
disclosure.
Sequence CWU 1
1
2611676PRTHomo sapiens 1Met Gly Leu Leu Gly Ile Leu Cys Phe Leu Ile
Phe Leu Gly Lys Thr 1 5 10 15 Trp Gly Gln Glu Gln Thr Tyr Val Ile
Ser Ala Pro Lys Ile Phe Arg 20 25 30 Val Gly Ala Ser Glu Asn Ile
Val Ile Gln Val Tyr Gly Tyr Thr Glu 35 40 45 Ala Phe Asp Ala Thr
Ile Ser Ile Lys Ser Tyr Pro Asp Lys Lys Phe 50 55 60 Ser Tyr Ser
Ser Gly His Val His Leu Ser Ser Glu Asn Lys Phe Gln 65 70 75 80 Asn
Ser Ala Ile Leu Thr Ile Gln Pro Lys Gln Leu Pro Gly Gly Gln 85 90
95 Asn Pro Val Ser Tyr Val Tyr Leu Glu Val Val Ser Lys His Phe Ser
100 105 110 Lys Ser Lys Arg Met Pro Ile Thr Tyr Asp Asn Gly Phe Leu
Phe Ile 115 120 125 His Thr Asp Lys Pro Val Tyr Thr Pro Asp Gln Ser
Val Lys Val Arg 130 135 140 Val Tyr Ser Leu Asn Asp Asp Leu Lys Pro
Ala Lys Arg Glu Thr Val 145 150 155 160 Leu Thr Phe Ile Asp Pro Glu
Gly Ser Glu Val Asp Met Val Glu Glu 165 170 175 Ile Asp His Ile Gly
Ile Ile Ser Phe Pro Asp Phe Lys Ile Pro Ser 180 185 190 Asn Pro Arg
Tyr Gly Met Trp Thr Ile Lys Ala Lys Tyr Lys Glu Asp 195 200 205 Phe
Ser Thr Thr Gly Thr Ala Tyr Phe Glu Val Lys Glu Tyr Val Leu 210 215
220 Pro His Phe Ser Val Ser Ile Glu Pro Glu Tyr Asn Phe Ile Gly Tyr
225 230 235 240 Lys Asn Phe Lys Asn Phe Glu Ile Thr Ile Lys Ala Arg
Tyr Phe Tyr 245 250 255 Asn Lys Val Val Thr Glu Ala Asp Val Tyr Ile
Thr Phe Gly Ile Arg 260 265 270 Glu Asp Leu Lys Asp Asp Gln Lys Glu
Met Met Gln Thr Ala Met Gln 275 280 285 Asn Thr Met Leu Ile Asn Gly
Ile Ala Gln Val Thr Phe Asp Ser Glu 290 295 300 Thr Ala Val Lys Glu
Leu Ser Tyr Tyr Ser Leu Glu Asp Leu Asn Asn 305 310 315 320 Lys Tyr
Leu Tyr Ile Ala Val Thr Val Ile Glu Ser Thr Gly Gly Phe 325 330 335
Ser Glu Glu Ala Glu Ile Pro Gly Ile Lys Tyr Val Leu Ser Pro Tyr 340
345 350 Lys Leu Asn Leu Val Ala Thr Pro Leu Phe Leu Lys Pro Gly Ile
Pro 355 360 365 Tyr Pro Ile Lys Val Gln Val Lys Asp Ser Leu Asp Gln
Leu Val Gly 370 375 380 Gly Val Pro Val Ile Leu Asn Ala Gln Thr Ile
Asp Val Asn Gln Glu 385 390 395 400 Thr Ser Asp Leu Asp Pro Ser Lys
Ser Val Thr Arg Val Asp Asp Gly 405 410 415 Val Ala Ser Phe Val Leu
Asn Leu Pro Ser Gly Val Thr Val Leu Glu 420 425 430 Phe Asn Val Lys
Thr Asp Ala Pro Asp Leu Pro Glu Glu Asn Gln Ala 435 440 445 Arg Glu
Gly Tyr Arg Ala Ile Ala Tyr Ser Ser Leu Ser Gln Ser Tyr 450 455 460
Leu Tyr Ile Asp Trp Thr Asp Asn His Lys Ala Leu Leu Val Gly Glu 465
470 475 480 His Leu Asn Ile Ile Val Thr Pro Lys Ser Pro Tyr Ile Asp
Lys Ile 485 490 495 Thr His Tyr Asn Tyr Leu Ile Leu Ser Lys Gly Lys
Ile Ile His Phe 500 505 510 Gly Thr Arg Glu Lys Phe Ser Asp Ala Ser
Tyr Gln Ser Ile Asn Ile 515 520 525 Pro Val Thr Gln Asn Met Val Pro
Ser Ser Arg Leu Leu Val Tyr Tyr 530 535 540 Ile Val Thr Gly Glu Gln
Thr Ala Glu Leu Val Ser Asp Ser Val Trp 545 550 555 560 Leu Asn Ile
Glu Glu Lys Cys Gly Asn Gln Leu Gln Val His Leu Ser 565 570 575 Pro
Asp Ala Asp Ala Tyr Ser Pro Gly Gln Thr Val Ser Leu Asn Met 580 585
590 Ala Thr Gly Met Asp Ser Trp Val Ala Leu Ala Ala Val Asp Ser Ala
595 600 605 Val Tyr Gly Val Gln Arg Gly Ala Lys Lys Pro Leu Glu Arg
Val Phe 610 615 620 Gln Phe Leu Glu Lys Ser Asp Leu Gly Cys Gly Ala
Gly Gly Gly Leu 625 630 635 640 Asn Asn Ala Asn Val Phe His Leu Ala
Gly Leu Thr Phe Leu Thr Asn 645 650 655 Ala Asn Ala Asp Asp Ser Gln
Glu Asn Asp Glu Pro Cys Lys Glu Ile 660 665 670 Leu Arg Pro Arg Arg
Thr Leu Gln Lys Lys Ile Glu Glu Ile Ala Ala 675 680 685 Lys Tyr Lys
His Ser Val Val Lys Lys Cys Cys Tyr Asp Gly Ala Cys 690 695 700 Val
Asn Asn Asp Glu Thr Cys Glu Gln Arg Ala Ala Arg Ile Ser Leu 705 710
715 720 Gly Pro Arg Cys Ile Lys Ala Phe Thr Glu Cys Cys Val Val Ala
Ser 725 730 735 Gln Leu Arg Ala Asn Ile Ser His Lys Asp Met Gln Leu
Gly Arg Leu 740 745 750 His Met Lys Thr Leu Leu Pro Val Ser Lys Pro
Glu Ile Arg Ser Tyr 755 760 765 Phe Pro Glu Ser Trp Leu Trp Glu Val
His Leu Val Pro Arg Arg Lys 770 775 780 Gln Leu Gln Phe Ala Leu Pro
Asp Ser Leu Thr Thr Trp Glu Ile Gln 785 790 795 800 Gly Ile Gly Ile
Ser Asn Thr Gly Ile Cys Val Ala Asp Thr Val Lys 805 810 815 Ala Lys
Val Phe Lys Asp Val Phe Leu Glu Met Asn Ile Pro Tyr Ser 820 825 830
Val Val Arg Gly Glu Gln Ile Gln Leu Lys Gly Thr Val Tyr Asn Tyr 835
840 845 Arg Thr Ser Gly Met Gln Phe Cys Val Lys Met Ser Ala Val Glu
Gly 850 855 860 Ile Cys Thr Ser Glu Ser Pro Val Ile Asp His Gln Gly
Thr Lys Ser 865 870 875 880 Ser Lys Cys Val Arg Gln Lys Val Glu Gly
Ser Ser Ser His Leu Val 885 890 895 Thr Phe Thr Val Leu Pro Leu Glu
Ile Gly Leu His Asn Ile Asn Phe 900 905 910 Ser Leu Glu Thr Trp Phe
Gly Lys Glu Ile Leu Val Lys Thr Leu Arg 915 920 925 Val Val Pro Glu
Gly Val Lys Arg Glu Ser Tyr Ser Gly Val Thr Leu 930 935 940 Asp Pro
Arg Gly Ile Tyr Gly Thr Ile Ser Arg Arg Lys Glu Phe Pro 945 950 955
960 Tyr Arg Ile Pro Leu Asp Leu Val Pro Lys Thr Glu Ile Lys Arg Ile
965 970 975 Leu Ser Val Lys Gly Leu Leu Val Gly Glu Ile Leu Ser Ala
Val Leu 980 985 990 Ser Gln Glu Gly Ile Asn Ile Leu Thr His Leu Pro
Lys Gly Ser Ala 995 1000 1005 Glu Ala Glu Leu Met Ser Val Val Pro
Val Phe Tyr Val Phe His 1010 1015 1020 Tyr Leu Glu Thr Gly Asn His
Trp Asn Ile Phe His Ser Asp Pro 1025 1030 1035 Leu Ile Glu Lys Gln
Lys Leu Lys Lys Lys Leu Lys Glu Gly Met 1040 1045 1050 Leu Ser Ile
Met Ser Tyr Arg Asn Ala Asp Tyr Ser Tyr Ser Val 1055 1060 1065 Trp
Lys Gly Gly Ser Ala Ser Thr Trp Leu Thr Ala Phe Ala Leu 1070 1075
1080 Arg Val Leu Gly Gln Val Asn Lys Tyr Val Glu Gln Asn Gln Asn
1085 1090 1095 Ser Ile Cys Asn Ser Leu Leu Trp Leu Val Glu Asn Tyr
Gln Leu 1100 1105 1110 Asp Asn Gly Ser Phe Lys Glu Asn Ser Gln Tyr
Gln Pro Ile Lys 1115 1120 1125 Leu Gln Gly Thr Leu Pro Val Glu Ala
Arg Glu Asn Ser Leu Tyr 1130 1135 1140 Leu Thr Ala Phe Thr Val Ile
Gly Ile Arg Lys Ala Phe Asp Ile 1145 1150 1155 Cys Pro Leu Val Lys
Ile Asp Thr Ala Leu Ile Lys Ala Asp Asn 1160 1165 1170 Phe Leu Leu
Glu Asn Thr Leu Pro Ala Gln Ser Thr Phe Thr Leu 1175 1180 1185 Ala
Ile Ser Ala Tyr Ala Leu Ser Leu Gly Asp Lys Thr His Pro 1190 1195
1200 Gln Phe Arg Ser Ile Val Ser Ala Leu Lys Arg Glu Ala Leu Val
1205 1210 1215 Lys Gly Asn Pro Pro Ile Tyr Arg Phe Trp Lys Asp Asn
Leu Gln 1220 1225 1230 His Lys Asp Ser Ser Val Pro Asn Thr Gly Thr
Ala Arg Met Val 1235 1240 1245 Glu Thr Thr Ala Tyr Ala Leu Leu Thr
Ser Leu Asn Leu Lys Asp 1250 1255 1260 Ile Asn Tyr Val Asn Pro Val
Ile Lys Trp Leu Ser Glu Glu Gln 1265 1270 1275 Arg Tyr Gly Gly Gly
Phe Tyr Ser Thr Gln Asp Thr Ile Asn Ala 1280 1285 1290 Ile Glu Gly
Leu Thr Glu Tyr Ser Leu Leu Val Lys Gln Leu Arg 1295 1300 1305 Leu
Ser Met Asp Ile Asp Val Ser Tyr Lys His Lys Gly Ala Leu 1310 1315
1320 His Asn Tyr Lys Met Thr Asp Lys Asn Phe Leu Gly Arg Pro Val
1325 1330 1335 Glu Val Leu Leu Asn Asp Asp Leu Ile Val Ser Thr Gly
Phe Gly 1340 1345 1350 Ser Gly Leu Ala Thr Val His Val Thr Thr Val
Val His Lys Thr 1355 1360 1365 Ser Thr Ser Glu Glu Val Cys Ser Phe
Tyr Leu Lys Ile Asp Thr 1370 1375 1380 Gln Asp Ile Glu Ala Ser His
Tyr Arg Gly Tyr Gly Asn Ser Asp 1385 1390 1395 Tyr Lys Arg Ile Val
Ala Cys Ala Ser Tyr Lys Pro Ser Arg Glu 1400 1405 1410 Glu Ser Ser
Ser Gly Ser Ser His Ala Val Met Asp Ile Ser Leu 1415 1420 1425 Pro
Thr Gly Ile Ser Ala Asn Glu Glu Asp Leu Lys Ala Leu Val 1430 1435
1440 Glu Gly Val Asp Gln Leu Phe Thr Asp Tyr Gln Ile Lys Asp Gly
1445 1450 1455 His Val Ile Leu Gln Leu Asn Ser Ile Pro Ser Ser Asp
Phe Leu 1460 1465 1470 Cys Val Arg Phe Arg Ile Phe Glu Leu Phe Glu
Val Gly Phe Leu 1475 1480 1485 Ser Pro Ala Thr Phe Thr Val Tyr Glu
Tyr His Arg Pro Asp Lys 1490 1495 1500 Gln Cys Thr Met Phe Tyr Ser
Thr Ser Asn Ile Lys Ile Gln Lys 1505 1510 1515 Val Cys Glu Gly Ala
Ala Cys Lys Cys Val Glu Ala Asp Cys Gly 1520 1525 1530 Gln Met Gln
Glu Glu Leu Asp Leu Thr Ile Ser Ala Glu Thr Arg 1535 1540 1545 Lys
Gln Thr Ala Cys Lys Pro Glu Ile Ala Tyr Ala Tyr Lys Val 1550 1555
1560 Ser Ile Thr Ser Ile Thr Val Glu Asn Val Phe Val Lys Tyr Lys
1565 1570 1575 Ala Thr Leu Leu Asp Ile Tyr Lys Thr Gly Glu Ala Val
Ala Glu 1580 1585 1590 Lys Asp Ser Glu Ile Thr Phe Ile Lys Lys Val
Thr Cys Thr Asn 1595 1600 1605 Ala Glu Leu Val Lys Gly Arg Gln Tyr
Leu Ile Met Gly Lys Glu 1610 1615 1620 Ala Leu Gln Ile Lys Tyr Asn
Phe Ser Phe Arg Tyr Ile Tyr Pro 1625 1630 1635 Leu Asp Ser Leu Thr
Trp Ile Glu Tyr Trp Pro Arg Asp Thr Thr 1640 1645 1650 Cys Ser Ser
Cys Gln Ala Phe Leu Ala Asn Leu Asp Glu Phe Ala 1655 1660 1665 Glu
Asp Ile Phe Leu Asn Gly Cys 1670 1675 21658PRTHomo sapiens 2Gln Glu
Gln Thr Tyr Val Ile Ser Ala Pro Lys Ile Phe Arg Val Gly 1 5 10 15
Ala Ser Glu Asn Ile Val Ile Gln Val Tyr Gly Tyr Thr Glu Ala Phe 20
25 30 Asp Ala Thr Ile Ser Ile Lys Ser Tyr Pro Asp Lys Lys Phe Ser
Tyr 35 40 45 Ser Ser Gly His Val His Leu Ser Ser Glu Asn Lys Phe
Gln Asn Ser 50 55 60 Ala Ile Leu Thr Ile Gln Pro Lys Gln Leu Pro
Gly Gly Gln Asn Pro 65 70 75 80 Val Ser Tyr Val Tyr Leu Glu Val Val
Ser Lys His Phe Ser Lys Ser 85 90 95 Lys Arg Met Pro Ile Thr Tyr
Asp Asn Gly Phe Leu Phe Ile His Thr 100 105 110 Asp Lys Pro Val Tyr
Thr Pro Asp Gln Ser Val Lys Val Arg Val Tyr 115 120 125 Ser Leu Asn
Asp Asp Leu Lys Pro Ala Lys Arg Glu Thr Val Leu Thr 130 135 140 Phe
Ile Asp Pro Glu Gly Ser Glu Val Asp Met Val Glu Glu Ile Asp 145 150
155 160 His Ile Gly Ile Ile Ser Phe Pro Asp Phe Lys Ile Pro Ser Asn
Pro 165 170 175 Arg Tyr Gly Met Trp Thr Ile Lys Ala Lys Tyr Lys Glu
Asp Phe Ser 180 185 190 Thr Thr Gly Thr Ala Tyr Phe Glu Val Lys Glu
Tyr Val Leu Pro His 195 200 205 Phe Ser Val Ser Ile Glu Pro Glu Tyr
Asn Phe Ile Gly Tyr Lys Asn 210 215 220 Phe Lys Asn Phe Glu Ile Thr
Ile Lys Ala Arg Tyr Phe Tyr Asn Lys 225 230 235 240 Val Val Thr Glu
Ala Asp Val Tyr Ile Thr Phe Gly Ile Arg Glu Asp 245 250 255 Leu Lys
Asp Asp Gln Lys Glu Met Met Gln Thr Ala Met Gln Asn Thr 260 265 270
Met Leu Ile Asn Gly Ile Ala Gln Val Thr Phe Asp Ser Glu Thr Ala 275
280 285 Val Lys Glu Leu Ser Tyr Tyr Ser Leu Glu Asp Leu Asn Asn Lys
Tyr 290 295 300 Leu Tyr Ile Ala Val Thr Val Ile Glu Ser Thr Gly Gly
Phe Ser Glu 305 310 315 320 Glu Ala Glu Ile Pro Gly Ile Lys Tyr Val
Leu Ser Pro Tyr Lys Leu 325 330 335 Asn Leu Val Ala Thr Pro Leu Phe
Leu Lys Pro Gly Ile Pro Tyr Pro 340 345 350 Ile Lys Val Gln Val Lys
Asp Ser Leu Asp Gln Leu Val Gly Gly Val 355 360 365 Pro Val Ile Leu
Asn Ala Gln Thr Ile Asp Val Asn Gln Glu Thr Ser 370 375 380 Asp Leu
Asp Pro Ser Lys Ser Val Thr Arg Val Asp Asp Gly Val Ala 385 390 395
400 Ser Phe Val Leu Asn Leu Pro Ser Gly Val Thr Val Leu Glu Phe Asn
405 410 415 Val Lys Thr Asp Ala Pro Asp Leu Pro Glu Glu Asn Gln Ala
Arg Glu 420 425 430 Gly Tyr Arg Ala Ile Ala Tyr Ser Ser Leu Ser Gln
Ser Tyr Leu Tyr 435 440 445 Ile Asp Trp Thr Asp Asn His Lys Ala Leu
Leu Val Gly Glu His Leu 450 455 460 Asn Ile Ile Val Thr Pro Lys Ser
Pro Tyr Ile Asp Lys Ile Thr His 465 470 475 480 Tyr Asn Tyr Leu Ile
Leu Ser Lys Gly Lys Ile Ile His Phe Gly Thr 485 490 495 Arg Glu Lys
Phe Ser Asp Ala Ser Tyr Gln Ser Ile Asn Ile Pro Val 500 505 510 Thr
Gln Asn Met Val Pro Ser Ser Arg Leu Leu Val Tyr Tyr Ile Val 515 520
525 Thr Gly Glu Gln Thr Ala Glu Leu Val Ser Asp Ser Val Trp Leu Asn
530 535 540 Ile Glu Glu Lys Cys Gly Asn Gln Leu Gln Val His Leu Ser
Pro Asp 545 550 555 560 Ala Asp Ala Tyr Ser Pro Gly Gln Thr Val Ser
Leu Asn Met Ala Thr 565 570 575 Gly Met Asp Ser Trp Val Ala Leu Ala
Ala Val Asp Ser Ala Val Tyr 580 585 590
Gly Val Gln Arg Gly Ala Lys Lys Pro Leu Glu Arg Val Phe Gln Phe 595
600 605 Leu Glu Lys Ser Asp Leu Gly Cys Gly Ala Gly Gly Gly Leu Asn
Asn 610 615 620 Ala Asn Val Phe His Leu Ala Gly Leu Thr Phe Leu Thr
Asn Ala Asn 625 630 635 640 Ala Asp Asp Ser Gln Glu Asn Asp Glu Pro
Cys Lys Glu Ile Leu Arg 645 650 655 Pro Arg Arg Thr Leu Gln Lys Lys
Ile Glu Glu Ile Ala Ala Lys Tyr 660 665 670 Lys His Ser Val Val Lys
Lys Cys Cys Tyr Asp Gly Ala Cys Val Asn 675 680 685 Asn Asp Glu Thr
Cys Glu Gln Arg Ala Ala Arg Ile Ser Leu Gly Pro 690 695 700 Arg Cys
Ile Lys Ala Phe Thr Glu Cys Cys Val Val Ala Ser Gln Leu 705 710 715
720 Arg Ala Asn Ile Ser His Lys Asp Met Gln Leu Gly Arg Leu His Met
725 730 735 Lys Thr Leu Leu Pro Val Ser Lys Pro Glu Ile Arg Ser Tyr
Phe Pro 740 745 750 Glu Ser Trp Leu Trp Glu Val His Leu Val Pro Arg
Arg Lys Gln Leu 755 760 765 Gln Phe Ala Leu Pro Asp Ser Leu Thr Thr
Trp Glu Ile Gln Gly Ile 770 775 780 Gly Ile Ser Asn Thr Gly Ile Cys
Val Ala Asp Thr Val Lys Ala Lys 785 790 795 800 Val Phe Lys Asp Val
Phe Leu Glu Met Asn Ile Pro Tyr Ser Val Val 805 810 815 Arg Gly Glu
Gln Ile Gln Leu Lys Gly Thr Val Tyr Asn Tyr Arg Thr 820 825 830 Ser
Gly Met Gln Phe Cys Val Lys Met Ser Ala Val Glu Gly Ile Cys 835 840
845 Thr Ser Glu Ser Pro Val Ile Asp His Gln Gly Thr Lys Ser Ser Lys
850 855 860 Cys Val Arg Gln Lys Val Glu Gly Ser Ser Ser His Leu Val
Thr Phe 865 870 875 880 Thr Val Leu Pro Leu Glu Ile Gly Leu His Asn
Ile Asn Phe Ser Leu 885 890 895 Glu Thr Trp Phe Gly Lys Glu Ile Leu
Val Lys Thr Leu Arg Val Val 900 905 910 Pro Glu Gly Val Lys Arg Glu
Ser Tyr Ser Gly Val Thr Leu Asp Pro 915 920 925 Arg Gly Ile Tyr Gly
Thr Ile Ser Arg Arg Lys Glu Phe Pro Tyr Arg 930 935 940 Ile Pro Leu
Asp Leu Val Pro Lys Thr Glu Ile Lys Arg Ile Leu Ser 945 950 955 960
Val Lys Gly Leu Leu Val Gly Glu Ile Leu Ser Ala Val Leu Ser Gln 965
970 975 Glu Gly Ile Asn Ile Leu Thr His Leu Pro Lys Gly Ser Ala Glu
Ala 980 985 990 Glu Leu Met Ser Val Val Pro Val Phe Tyr Val Phe His
Tyr Leu Glu 995 1000 1005 Thr Gly Asn His Trp Asn Ile Phe His Ser
Asp Pro Leu Ile Glu 1010 1015 1020 Lys Gln Lys Leu Lys Lys Lys Leu
Lys Glu Gly Met Leu Ser Ile 1025 1030 1035 Met Ser Tyr Arg Asn Ala
Asp Tyr Ser Tyr Ser Val Trp Lys Gly 1040 1045 1050 Gly Ser Ala Ser
Thr Trp Leu Thr Ala Phe Ala Leu Arg Val Leu 1055 1060 1065 Gly Gln
Val Asn Lys Tyr Val Glu Gln Asn Gln Asn Ser Ile Cys 1070 1075 1080
Asn Ser Leu Leu Trp Leu Val Glu Asn Tyr Gln Leu Asp Asn Gly 1085
1090 1095 Ser Phe Lys Glu Asn Ser Gln Tyr Gln Pro Ile Lys Leu Gln
Gly 1100 1105 1110 Thr Leu Pro Val Glu Ala Arg Glu Asn Ser Leu Tyr
Leu Thr Ala 1115 1120 1125 Phe Thr Val Ile Gly Ile Arg Lys Ala Phe
Asp Ile Cys Pro Leu 1130 1135 1140 Val Lys Ile Asp Thr Ala Leu Ile
Lys Ala Asp Asn Phe Leu Leu 1145 1150 1155 Glu Asn Thr Leu Pro Ala
Gln Ser Thr Phe Thr Leu Ala Ile Ser 1160 1165 1170 Ala Tyr Ala Leu
Ser Leu Gly Asp Lys Thr His Pro Gln Phe Arg 1175 1180 1185 Ser Ile
Val Ser Ala Leu Lys Arg Glu Ala Leu Val Lys Gly Asn 1190 1195 1200
Pro Pro Ile Tyr Arg Phe Trp Lys Asp Asn Leu Gln His Lys Asp 1205
1210 1215 Ser Ser Val Pro Asn Thr Gly Thr Ala Arg Met Val Glu Thr
Thr 1220 1225 1230 Ala Tyr Ala Leu Leu Thr Ser Leu Asn Leu Lys Asp
Ile Asn Tyr 1235 1240 1245 Val Asn Pro Val Ile Lys Trp Leu Ser Glu
Glu Gln Arg Tyr Gly 1250 1255 1260 Gly Gly Phe Tyr Ser Thr Gln Asp
Thr Ile Asn Ala Ile Glu Gly 1265 1270 1275 Leu Thr Glu Tyr Ser Leu
Leu Val Lys Gln Leu Arg Leu Ser Met 1280 1285 1290 Asp Ile Asp Val
Ser Tyr Lys His Lys Gly Ala Leu His Asn Tyr 1295 1300 1305 Lys Met
Thr Asp Lys Asn Phe Leu Gly Arg Pro Val Glu Val Leu 1310 1315 1320
Leu Asn Asp Asp Leu Ile Val Ser Thr Gly Phe Gly Ser Gly Leu 1325
1330 1335 Ala Thr Val His Val Thr Thr Val Val His Lys Thr Ser Thr
Ser 1340 1345 1350 Glu Glu Val Cys Ser Phe Tyr Leu Lys Ile Asp Thr
Gln Asp Ile 1355 1360 1365 Glu Ala Ser His Tyr Arg Gly Tyr Gly Asn
Ser Asp Tyr Lys Arg 1370 1375 1380 Ile Val Ala Cys Ala Ser Tyr Lys
Pro Ser Arg Glu Glu Ser Ser 1385 1390 1395 Ser Gly Ser Ser His Ala
Val Met Asp Ile Ser Leu Pro Thr Gly 1400 1405 1410 Ile Ser Ala Asn
Glu Glu Asp Leu Lys Ala Leu Val Glu Gly Val 1415 1420 1425 Asp Gln
Leu Phe Thr Asp Tyr Gln Ile Lys Asp Gly His Val Ile 1430 1435 1440
Leu Gln Leu Asn Ser Ile Pro Ser Ser Asp Phe Leu Cys Val Arg 1445
1450 1455 Phe Arg Ile Phe Glu Leu Phe Glu Val Gly Phe Leu Ser Pro
Ala 1460 1465 1470 Thr Phe Thr Val Tyr Glu Tyr His Arg Pro Asp Lys
Gln Cys Thr 1475 1480 1485 Met Phe Tyr Ser Thr Ser Asn Ile Lys Ile
Gln Lys Val Cys Glu 1490 1495 1500 Gly Ala Ala Cys Lys Cys Val Glu
Ala Asp Cys Gly Gln Met Gln 1505 1510 1515 Glu Glu Leu Asp Leu Thr
Ile Ser Ala Glu Thr Arg Lys Gln Thr 1520 1525 1530 Ala Cys Lys Pro
Glu Ile Ala Tyr Ala Tyr Lys Val Ser Ile Thr 1535 1540 1545 Ser Ile
Thr Val Glu Asn Val Phe Val Lys Tyr Lys Ala Thr Leu 1550 1555 1560
Leu Asp Ile Tyr Lys Thr Gly Glu Ala Val Ala Glu Lys Asp Ser 1565
1570 1575 Glu Ile Thr Phe Ile Lys Lys Val Thr Cys Thr Asn Ala Glu
Leu 1580 1585 1590 Val Lys Gly Arg Gln Tyr Leu Ile Met Gly Lys Glu
Ala Leu Gln 1595 1600 1605 Ile Lys Tyr Asn Phe Ser Phe Arg Tyr Ile
Tyr Pro Leu Asp Ser 1610 1615 1620 Leu Thr Trp Ile Glu Tyr Trp Pro
Arg Asp Thr Thr Cys Ser Ser 1625 1630 1635 Cys Gln Ala Phe Leu Ala
Asn Leu Asp Glu Phe Ala Glu Asp Ile 1640 1645 1650 Phe Leu Asn Gly
Cys 1655 3999PRTHomo sapiens 3Thr Leu Gln Lys Lys Ile Glu Glu Ile
Ala Ala Lys Tyr Lys His Ser 1 5 10 15 Val Val Lys Lys Cys Cys Tyr
Asp Gly Ala Cys Val Asn Asn Asp Glu 20 25 30 Thr Cys Glu Gln Arg
Ala Ala Arg Ile Ser Leu Gly Pro Arg Cys Ile 35 40 45 Lys Ala Phe
Thr Glu Cys Cys Val Val Ala Ser Gln Leu Arg Ala Asn 50 55 60 Ile
Ser His Lys Asp Met Gln Leu Gly Arg Leu His Met Lys Thr Leu 65 70
75 80 Leu Pro Val Ser Lys Pro Glu Ile Arg Ser Tyr Phe Pro Glu Ser
Trp 85 90 95 Leu Trp Glu Val His Leu Val Pro Arg Arg Lys Gln Leu
Gln Phe Ala 100 105 110 Leu Pro Asp Ser Leu Thr Thr Trp Glu Ile Gln
Gly Ile Gly Ile Ser 115 120 125 Asn Thr Gly Ile Cys Val Ala Asp Thr
Val Lys Ala Lys Val Phe Lys 130 135 140 Asp Val Phe Leu Glu Met Asn
Ile Pro Tyr Ser Val Val Arg Gly Glu 145 150 155 160 Gln Ile Gln Leu
Lys Gly Thr Val Tyr Asn Tyr Arg Thr Ser Gly Met 165 170 175 Gln Phe
Cys Val Lys Met Ser Ala Val Glu Gly Ile Cys Thr Ser Glu 180 185 190
Ser Pro Val Ile Asp His Gln Gly Thr Lys Ser Ser Lys Cys Val Arg 195
200 205 Gln Lys Val Glu Gly Ser Ser Ser His Leu Val Thr Phe Thr Val
Leu 210 215 220 Pro Leu Glu Ile Gly Leu His Asn Ile Asn Phe Ser Leu
Glu Thr Trp 225 230 235 240 Phe Gly Lys Glu Ile Leu Val Lys Thr Leu
Arg Val Val Pro Glu Gly 245 250 255 Val Lys Arg Glu Ser Tyr Ser Gly
Val Thr Leu Asp Pro Arg Gly Ile 260 265 270 Tyr Gly Thr Ile Ser Arg
Arg Lys Glu Phe Pro Tyr Arg Ile Pro Leu 275 280 285 Asp Leu Val Pro
Lys Thr Glu Ile Lys Arg Ile Leu Ser Val Lys Gly 290 295 300 Leu Leu
Val Gly Glu Ile Leu Ser Ala Val Leu Ser Gln Glu Gly Ile 305 310 315
320 Asn Ile Leu Thr His Leu Pro Lys Gly Ser Ala Glu Ala Glu Leu Met
325 330 335 Ser Val Val Pro Val Phe Tyr Val Phe His Tyr Leu Glu Thr
Gly Asn 340 345 350 His Trp Asn Ile Phe His Ser Asp Pro Leu Ile Glu
Lys Gln Lys Leu 355 360 365 Lys Lys Lys Leu Lys Glu Gly Met Leu Ser
Ile Met Ser Tyr Arg Asn 370 375 380 Ala Asp Tyr Ser Tyr Ser Val Trp
Lys Gly Gly Ser Ala Ser Thr Trp 385 390 395 400 Leu Thr Ala Phe Ala
Leu Arg Val Leu Gly Gln Val Asn Lys Tyr Val 405 410 415 Glu Gln Asn
Gln Asn Ser Ile Cys Asn Ser Leu Leu Trp Leu Val Glu 420 425 430 Asn
Tyr Gln Leu Asp Asn Gly Ser Phe Lys Glu Asn Ser Gln Tyr Gln 435 440
445 Pro Ile Lys Leu Gln Gly Thr Leu Pro Val Glu Ala Arg Glu Asn Ser
450 455 460 Leu Tyr Leu Thr Ala Phe Thr Val Ile Gly Ile Arg Lys Ala
Phe Asp 465 470 475 480 Ile Cys Pro Leu Val Lys Ile Asp Thr Ala Leu
Ile Lys Ala Asp Asn 485 490 495 Phe Leu Leu Glu Asn Thr Leu Pro Ala
Gln Ser Thr Phe Thr Leu Ala 500 505 510 Ile Ser Ala Tyr Ala Leu Ser
Leu Gly Asp Lys Thr His Pro Gln Phe 515 520 525 Arg Ser Ile Val Ser
Ala Leu Lys Arg Glu Ala Leu Val Lys Gly Asn 530 535 540 Pro Pro Ile
Tyr Arg Phe Trp Lys Asp Asn Leu Gln His Lys Asp Ser 545 550 555 560
Ser Val Pro Asn Thr Gly Thr Ala Arg Met Val Glu Thr Thr Ala Tyr 565
570 575 Ala Leu Leu Thr Ser Leu Asn Leu Lys Asp Ile Asn Tyr Val Asn
Pro 580 585 590 Val Ile Lys Trp Leu Ser Glu Glu Gln Arg Tyr Gly Gly
Gly Phe Tyr 595 600 605 Ser Thr Gln Asp Thr Ile Asn Ala Ile Glu Gly
Leu Thr Glu Tyr Ser 610 615 620 Leu Leu Val Lys Gln Leu Arg Leu Ser
Met Asp Ile Asp Val Ser Tyr 625 630 635 640 Lys His Lys Gly Ala Leu
His Asn Tyr Lys Met Thr Asp Lys Asn Phe 645 650 655 Leu Gly Arg Pro
Val Glu Val Leu Leu Asn Asp Asp Leu Ile Val Ser 660 665 670 Thr Gly
Phe Gly Ser Gly Leu Ala Thr Val His Val Thr Thr Val Val 675 680 685
His Lys Thr Ser Thr Ser Glu Glu Val Cys Ser Phe Tyr Leu Lys Ile 690
695 700 Asp Thr Gln Asp Ile Glu Ala Ser His Tyr Arg Gly Tyr Gly Asn
Ser 705 710 715 720 Asp Tyr Lys Arg Ile Val Ala Cys Ala Ser Tyr Lys
Pro Ser Arg Glu 725 730 735 Glu Ser Ser Ser Gly Ser Ser His Ala Val
Met Asp Ile Ser Leu Pro 740 745 750 Thr Gly Ile Ser Ala Asn Glu Glu
Asp Leu Lys Ala Leu Val Glu Gly 755 760 765 Val Asp Gln Leu Phe Thr
Asp Tyr Gln Ile Lys Asp Gly His Val Ile 770 775 780 Leu Gln Leu Asn
Ser Ile Pro Ser Ser Asp Phe Leu Cys Val Arg Phe 785 790 795 800 Arg
Ile Phe Glu Leu Phe Glu Val Gly Phe Leu Ser Pro Ala Thr Phe 805 810
815 Thr Val Tyr Glu Tyr His Arg Pro Asp Lys Gln Cys Thr Met Phe Tyr
820 825 830 Ser Thr Ser Asn Ile Lys Ile Gln Lys Val Cys Glu Gly Ala
Ala Cys 835 840 845 Lys Cys Val Glu Ala Asp Cys Gly Gln Met Gln Glu
Glu Leu Asp Leu 850 855 860 Thr Ile Ser Ala Glu Thr Arg Lys Gln Thr
Ala Cys Lys Pro Glu Ile 865 870 875 880 Ala Tyr Ala Tyr Lys Val Ser
Ile Thr Ser Ile Thr Val Glu Asn Val 885 890 895 Phe Val Lys Tyr Lys
Ala Thr Leu Leu Asp Ile Tyr Lys Thr Gly Glu 900 905 910 Ala Val Ala
Glu Lys Asp Ser Glu Ile Thr Phe Ile Lys Lys Val Thr 915 920 925 Cys
Thr Asn Ala Glu Leu Val Lys Gly Arg Gln Tyr Leu Ile Met Gly 930 935
940 Lys Glu Ala Leu Gln Ile Lys Tyr Asn Phe Ser Phe Arg Tyr Ile Tyr
945 950 955 960 Pro Leu Asp Ser Leu Thr Trp Ile Glu Tyr Trp Pro Arg
Asp Thr Thr 965 970 975 Cys Ser Ser Cys Gln Ala Phe Leu Ala Asn Leu
Asp Glu Phe Ala Glu 980 985 990 Asp Ile Phe Leu Asn Gly Cys 995
4655PRTHomo sapiens 4Gln Glu Gln Thr Tyr Val Ile Ser Ala Pro Lys
Ile Phe Arg Val Gly 1 5 10 15 Ala Ser Glu Asn Ile Val Ile Gln Val
Tyr Gly Tyr Thr Glu Ala Phe 20 25 30 Asp Ala Thr Ile Ser Ile Lys
Ser Tyr Pro Asp Lys Lys Phe Ser Tyr 35 40 45 Ser Ser Gly His Val
His Leu Ser Ser Glu Asn Lys Phe Gln Asn Ser 50 55 60 Ala Ile Leu
Thr Ile Gln Pro Lys Gln Leu Pro Gly Gly Gln Asn Pro 65 70 75 80 Val
Ser Tyr Val Tyr Leu Glu Val Val Ser Lys His Phe Ser Lys Ser 85 90
95 Lys Arg Met Pro Ile Thr Tyr Asp Asn Gly Phe Leu Phe Ile His Thr
100 105 110 Asp Lys Pro Val Tyr Thr Pro Asp Gln Ser Val Lys Val Arg
Val Tyr 115 120 125 Ser Leu Asn Asp Asp Leu Lys Pro Ala Lys Arg Glu
Thr Val Leu Thr 130 135 140 Phe Ile Asp Pro Glu Gly Ser Glu Val Asp
Met Val Glu Glu Ile Asp 145 150 155 160 His Ile Gly Ile Ile Ser Phe
Pro Asp Phe Lys Ile Pro Ser Asn Pro 165 170 175 Arg Tyr Gly Met Trp
Thr Ile Lys Ala Lys Tyr Lys Glu Asp Phe Ser 180 185 190 Thr Thr Gly
Thr Ala Tyr Phe Glu Val Lys Glu Tyr Val Leu Pro His 195
200 205 Phe Ser Val Ser Ile Glu Pro Glu Tyr Asn Phe Ile Gly Tyr Lys
Asn 210 215 220 Phe Lys Asn Phe Glu Ile Thr Ile Lys Ala Arg Tyr Phe
Tyr Asn Lys 225 230 235 240 Val Val Thr Glu Ala Asp Val Tyr Ile Thr
Phe Gly Ile Arg Glu Asp 245 250 255 Leu Lys Asp Asp Gln Lys Glu Met
Met Gln Thr Ala Met Gln Asn Thr 260 265 270 Met Leu Ile Asn Gly Ile
Ala Gln Val Thr Phe Asp Ser Glu Thr Ala 275 280 285 Val Lys Glu Leu
Ser Tyr Tyr Ser Leu Glu Asp Leu Asn Asn Lys Tyr 290 295 300 Leu Tyr
Ile Ala Val Thr Val Ile Glu Ser Thr Gly Gly Phe Ser Glu 305 310 315
320 Glu Ala Glu Ile Pro Gly Ile Lys Tyr Val Leu Ser Pro Tyr Lys Leu
325 330 335 Asn Leu Val Ala Thr Pro Leu Phe Leu Lys Pro Gly Ile Pro
Tyr Pro 340 345 350 Ile Lys Val Gln Val Lys Asp Ser Leu Asp Gln Leu
Val Gly Gly Val 355 360 365 Pro Val Ile Leu Asn Ala Gln Thr Ile Asp
Val Asn Gln Glu Thr Ser 370 375 380 Asp Leu Asp Pro Ser Lys Ser Val
Thr Arg Val Asp Asp Gly Val Ala 385 390 395 400 Ser Phe Val Leu Asn
Leu Pro Ser Gly Val Thr Val Leu Glu Phe Asn 405 410 415 Val Lys Thr
Asp Ala Pro Asp Leu Pro Glu Glu Asn Gln Ala Arg Glu 420 425 430 Gly
Tyr Arg Ala Ile Ala Tyr Ser Ser Leu Ser Gln Ser Tyr Leu Tyr 435 440
445 Ile Asp Trp Thr Asp Asn His Lys Ala Leu Leu Val Gly Glu His Leu
450 455 460 Asn Ile Ile Val Thr Pro Lys Ser Pro Tyr Ile Asp Lys Ile
Thr His 465 470 475 480 Tyr Asn Tyr Leu Ile Leu Ser Lys Gly Lys Ile
Ile His Phe Gly Thr 485 490 495 Arg Glu Lys Phe Ser Asp Ala Ser Tyr
Gln Ser Ile Asn Ile Pro Val 500 505 510 Thr Gln Asn Met Val Pro Ser
Ser Arg Leu Leu Val Tyr Tyr Ile Val 515 520 525 Thr Gly Glu Gln Thr
Ala Glu Leu Val Ser Asp Ser Val Trp Leu Asn 530 535 540 Ile Glu Glu
Lys Cys Gly Asn Gln Leu Gln Val His Leu Ser Pro Asp 545 550 555 560
Ala Asp Ala Tyr Ser Pro Gly Gln Thr Val Ser Leu Asn Met Ala Thr 565
570 575 Gly Met Asp Ser Trp Val Ala Leu Ala Ala Val Asp Ser Ala Val
Tyr 580 585 590 Gly Val Gln Arg Gly Ala Lys Lys Pro Leu Glu Arg Val
Phe Gln Phe 595 600 605 Leu Glu Lys Ser Asp Leu Gly Cys Gly Ala Gly
Gly Gly Leu Asn Asn 610 615 620 Ala Asn Val Phe His Leu Ala Gly Leu
Thr Phe Leu Thr Asn Ala Asn 625 630 635 640 Ala Asp Asp Ser Gln Glu
Asn Asp Glu Pro Cys Lys Glu Ile Leu 645 650 655 521PRTHomo sapiens
5Val Ile Asp His Gln Gly Thr Lys Ser Ser Lys Cys Val Arg Gln Lys 1
5 10 15 Val Glu Gly Ser Ser 20 65PRTHomo sapiens 6Lys Ser Ser Lys
Cys 1 5 7127PRTHomo sapiens 7Asn Phe Ser Leu Glu Thr Trp Phe Gly
Lys Glu Ile Leu Val Lys Thr 1 5 10 15 Leu Arg Val Val Pro Glu Gly
Val Lys Arg Glu Ser Tyr Ser Gly Val 20 25 30 Thr Leu Asp Pro Arg
Gly Ile Tyr Gly Thr Ile Ser Arg Arg Lys Glu 35 40 45 Phe Pro Tyr
Arg Ile Pro Leu Asp Leu Val Pro Lys Thr Glu Ile Lys 50 55 60 Arg
Ile Leu Ser Val Lys Gly Leu Leu Val Gly Glu Ile Leu Ser Ala 65 70
75 80 Val Leu Ser Gln Glu Gly Ile Asn Ile Leu Thr His Leu Pro Lys
Gly 85 90 95 Ser Ala Glu Ala Glu Leu Met Ser Val Val Pro Val Phe
Tyr Val Phe 100 105 110 His Tyr Leu Glu Thr Gly Asn His Trp Asn Ile
Phe His Ser Asp 115 120 125 8200PRTHomo sapiens 8Ser Glu Ser Pro
Val Ile Asp His Gln Gly Thr Lys Ser Ser Lys Cys 1 5 10 15 Val Arg
Gln Lys Val Glu Gly Ser Ser Ser His Leu Val Thr Phe Thr 20 25 30
Val Leu Pro Leu Glu Ile Gly Leu His Asn Ile Asn Phe Ser Leu Glu 35
40 45 Thr Trp Phe Gly Lys Glu Ile Leu Val Lys Thr Leu Arg Val Val
Pro 50 55 60 Glu Gly Val Lys Arg Glu Ser Tyr Ser Gly Val Thr Leu
Asp Pro Arg 65 70 75 80 Gly Ile Tyr Gly Thr Ile Ser Arg Arg Lys Glu
Phe Pro Tyr Arg Ile 85 90 95 Pro Leu Asp Leu Val Pro Lys Thr Glu
Ile Lys Arg Ile Leu Ser Val 100 105 110 Lys Gly Leu Leu Val Gly Glu
Ile Leu Ser Ala Val Leu Ser Gln Glu 115 120 125 Gly Ile Asn Ile Leu
Thr His Leu Pro Lys Gly Ser Ala Glu Ala Glu 130 135 140 Leu Met Ser
Val Val Pro Val Phe Tyr Val Phe His Tyr Leu Glu Thr 145 150 155 160
Gly Asn His Trp Asn Ile Phe His Ser Asp Pro Leu Ile Glu Lys Gln 165
170 175 Lys Leu Lys Lys Lys Leu Lys Glu Gly Met Leu Ser Ile Met Ser
Tyr 180 185 190 Arg Asn Ala Asp Tyr Ser Tyr Ser 195 200 930PRTHomo
sapiens 9Ser His Lys Asp Met Gln Leu Gly Arg Leu His Met Lys Thr
Leu Leu 1 5 10 15 Pro Val Ser Lys Pro Glu Ile Arg Ser Tyr Phe Pro
Glu Ser 20 25 30 10325PRTHomo sapiens 10Ser His Lys Asp Met Gln Leu
Gly Arg Leu His Met Lys Thr Leu Leu 1 5 10 15 Pro Val Ser Lys Pro
Glu Ile Arg Ser Tyr Phe Pro Glu Ser Trp Leu 20 25 30 Trp Glu Val
His Leu Val Pro Arg Arg Lys Gln Leu Gln Phe Ala Leu 35 40 45 Pro
Asp Ser Leu Thr Thr Trp Glu Ile Gln Gly Ile Gly Ile Ser Asn 50 55
60 Thr Gly Ile Cys Val Ala Asp Thr Val Lys Ala Lys Val Phe Lys Asp
65 70 75 80 Val Phe Leu Glu Met Asn Ile Pro Tyr Ser Val Val Arg Gly
Glu Gln 85 90 95 Ile Gln Leu Lys Gly Thr Val Tyr Asn Tyr Arg Thr
Ser Gly Met Gln 100 105 110 Phe Cys Val Lys Met Ser Ala Val Glu Gly
Ile Cys Thr Ser Glu Ser 115 120 125 Pro Val Ile Asp His Gln Gly Thr
Lys Ser Ser Lys Cys Val Arg Gln 130 135 140 Lys Val Glu Gly Ser Ser
Ser His Leu Val Thr Phe Thr Val Leu Pro 145 150 155 160 Leu Glu Ile
Gly Leu His Asn Ile Asn Phe Ser Leu Glu Thr Trp Phe 165 170 175 Gly
Lys Glu Ile Leu Val Lys Thr Leu Arg Val Val Pro Glu Gly Val 180 185
190 Lys Arg Glu Ser Tyr Ser Gly Val Thr Leu Asp Pro Arg Gly Ile Tyr
195 200 205 Gly Thr Ile Ser Arg Arg Lys Glu Phe Pro Tyr Arg Ile Pro
Leu Asp 210 215 220 Leu Val Pro Lys Thr Glu Ile Lys Arg Ile Leu Ser
Val Lys Gly Leu 225 230 235 240 Leu Val Gly Glu Ile Leu Ser Ala Val
Leu Ser Gln Glu Gly Ile Asn 245 250 255 Ile Leu Thr His Leu Pro Lys
Gly Ser Ala Glu Ala Glu Leu Met Ser 260 265 270 Val Val Pro Val Phe
Tyr Val Phe His Tyr Leu Glu Thr Gly Asn His 275 280 285 Trp Asn Ile
Phe His Ser Asp Pro Leu Ile Glu Lys Gln Lys Leu Lys 290 295 300 Lys
Lys Leu Lys Glu Gly Met Leu Ser Ile Met Ser Tyr Arg Asn Ala 305 310
315 320 Asp Tyr Ser Tyr Ser 325 1117PRTHomo sapiens 11Asp His Gln
Gly Thr Lys Ser Ser Lys Cys Val Arg Gln Lys Val Glu 1 5 10 15 Gly
1274PRTHomo sapiens 12Thr Leu Gln Lys Lys Ile Glu Glu Ile Ala Ala
Lys Tyr Lys His Ser 1 5 10 15 Val Val Lys Lys Cys Cys Tyr Asp Gly
Ala Cys Val Asn Asn Asp Glu 20 25 30 Thr Cys Glu Gln Arg Ala Ala
Arg Ile Ser Leu Gly Pro Arg Cys Ile 35 40 45 Lys Ala Phe Thr Glu
Cys Cys Val Val Ala Ser Gln Leu Arg Ala Asn 50 55 60 Ile Ser His
Lys Asp Met Gln Leu Gly Arg 65 70 1320PRTHomo sapiens 13Cys Cys Tyr
Asp Gly Ala Cys Val Asn Asn Asp Glu Thr Cys Glu Gln 1 5 10 15 Arg
Ala Ala Arg 20 1418PRTHomo sapiens 14Lys Cys Cys Tyr Asp Gly Ala
Cys Val Asn Asn Asp Glu Thr Cys Glu 1 5 10 15 Gln Arg 1510PRTHomo
sapiens 15Val Asn Asn Asp Glu Thr Cys Glu Gln Arg 1 5 10 166PRTHomo
sapiens 16Val Asn Asn Asp Glu Thr 1 5 179PRTHomo sapiens 17Ala Ala
Arg Ile Ser Leu Gly Pro Arg 1 5 1819PRTHomo sapiens 18Cys Cys Tyr
Asp Gly Ala Cys Val Asn Asn Asp Glu Thr Cys Glu Gln 1 5 10 15 Arg
Ala Ala 1918PRTHomo sapiens 19Cys Cys Tyr Asp Gly Ala Cys Val Asn
Asn Asp Glu Thr Cys Glu Gln 1 5 10 15 Arg Ala 2017PRTHomo sapiens
20Cys Cys Tyr Asp Gly Ala Cys Val Asn Asn Asp Glu Thr Cys Glu Gln 1
5 10 15 Arg 2116PRTHomo sapiens 21Cys Cys Tyr Asp Gly Ala Cys Val
Asn Asn Asp Glu Thr Cys Glu Gln 1 5 10 15 2215PRTHomo sapiens 22Cys
Cys Tyr Asp Gly Ala Cys Val Asn Asn Asp Glu Thr Cys Glu 1 5 10 15
2319PRTHomo sapiens 23Cys Tyr Asp Gly Ala Cys Val Asn Asn Asp Glu
Thr Cys Glu Gln Arg 1 5 10 15 Ala Ala Arg 2418PRTHomo sapiens 24Tyr
Asp Gly Ala Cys Val Asn Asn Asp Glu Thr Cys Glu Gln Arg Ala 1 5 10
15 Ala Arg 2519PRTHomo sapiens 25Cys Tyr Asp Gly Ala Cys Val Asn
Asn Asp Glu Thr Cys Glu Gln Arg 1 5 10 15 Ala Ala Arg 26925PRTHomo
sapiens 26Leu His Met Lys Thr Leu Leu Pro Val Ser Lys Pro Glu Ile
Arg Ser 1 5 10 15 Tyr Phe Pro Glu Ser Trp Leu Trp Glu Val His Leu
Val Pro Arg Arg 20 25 30 Lys Gln Leu Gln Phe Ala Leu Pro Asp Ser
Leu Thr Thr Trp Glu Ile 35 40 45 Gln Gly Ile Gly Ile Ser Asn Thr
Gly Ile Cys Val Ala Asp Thr Val 50 55 60 Lys Ala Lys Val Phe Lys
Asp Val Phe Leu Glu Met Asn Ile Pro Tyr 65 70 75 80 Ser Val Val Arg
Gly Glu Gln Ile Gln Leu Lys Gly Thr Val Tyr Asn 85 90 95 Tyr Arg
Thr Ser Gly Met Gln Phe Cys Val Lys Met Ser Ala Val Glu 100 105 110
Gly Ile Cys Thr Ser Glu Ser Pro Val Ile Asp His Gln Gly Thr Lys 115
120 125 Ser Ser Lys Cys Val Arg Gln Lys Val Glu Gly Ser Ser Ser His
Leu 130 135 140 Val Thr Phe Thr Val Leu Pro Leu Glu Ile Gly Leu His
Asn Ile Asn 145 150 155 160 Phe Ser Leu Glu Thr Trp Phe Gly Lys Glu
Ile Leu Val Lys Thr Leu 165 170 175 Arg Val Val Pro Glu Gly Val Lys
Arg Glu Ser Tyr Ser Gly Val Thr 180 185 190 Leu Asp Pro Arg Gly Ile
Tyr Gly Thr Ile Ser Arg Arg Lys Glu Phe 195 200 205 Pro Tyr Arg Ile
Pro Leu Asp Leu Val Pro Lys Thr Glu Ile Lys Arg 210 215 220 Ile Leu
Ser Val Lys Gly Leu Leu Val Gly Glu Ile Leu Ser Ala Val 225 230 235
240 Leu Ser Gln Glu Gly Ile Asn Ile Leu Thr His Leu Pro Lys Gly Ser
245 250 255 Ala Glu Ala Glu Leu Met Ser Val Val Pro Val Phe Tyr Val
Phe His 260 265 270 Tyr Leu Glu Thr Gly Asn His Trp Asn Ile Phe His
Ser Asp Pro Leu 275 280 285 Ile Glu Lys Gln Lys Leu Lys Lys Lys Leu
Lys Glu Gly Met Leu Ser 290 295 300 Ile Met Ser Tyr Arg Asn Ala Asp
Tyr Ser Tyr Ser Val Trp Lys Gly 305 310 315 320 Gly Ser Ala Ser Thr
Trp Leu Thr Ala Phe Ala Leu Arg Val Leu Gly 325 330 335 Gln Val Asn
Lys Tyr Val Glu Gln Asn Gln Asn Ser Ile Cys Asn Ser 340 345 350 Leu
Leu Trp Leu Val Glu Asn Tyr Gln Leu Asp Asn Gly Ser Phe Lys 355 360
365 Glu Asn Ser Gln Tyr Gln Pro Ile Lys Leu Gln Gly Thr Leu Pro Val
370 375 380 Glu Ala Arg Glu Asn Ser Leu Tyr Leu Thr Ala Phe Thr Val
Ile Gly 385 390 395 400 Ile Arg Lys Ala Phe Asp Ile Cys Pro Leu Val
Lys Ile Asp Thr Ala 405 410 415 Leu Ile Lys Ala Asp Asn Phe Leu Leu
Glu Asn Thr Leu Pro Ala Gln 420 425 430 Ser Thr Phe Thr Leu Ala Ile
Ser Ala Tyr Ala Leu Ser Leu Gly Asp 435 440 445 Lys Thr His Pro Gln
Phe Arg Ser Ile Val Ser Ala Leu Lys Arg Glu 450 455 460 Ala Leu Val
Lys Gly Asn Pro Pro Ile Tyr Arg Phe Trp Lys Asp Asn 465 470 475 480
Leu Gln His Lys Asp Ser Ser Val Pro Asn Thr Gly Thr Ala Arg Met 485
490 495 Val Glu Thr Thr Ala Tyr Ala Leu Leu Thr Ser Leu Asn Leu Lys
Asp 500 505 510 Ile Asn Tyr Val Asn Pro Val Ile Lys Trp Leu Ser Glu
Glu Gln Arg 515 520 525 Tyr Gly Gly Gly Phe Tyr Ser Thr Gln Asp Thr
Ile Asn Ala Ile Glu 530 535 540 Gly Leu Thr Glu Tyr Ser Leu Leu Val
Lys Gln Leu Arg Leu Ser Met 545 550 555 560 Asp Ile Asp Val Ser Tyr
Lys His Lys Gly Ala Leu His Asn Tyr Lys 565 570 575 Met Thr Asp Lys
Asn Phe Leu Gly Arg Pro Val Glu Val Leu Leu Asn 580 585 590 Asp Asp
Leu Ile Val Ser Thr Gly Phe Gly Ser Gly Leu Ala Thr Val 595 600 605
His Val Thr Thr Val Val His Lys Thr Ser Thr Ser Glu Glu Val Cys 610
615 620 Ser Phe Tyr Leu Lys Ile Asp Thr Gln Asp Ile Glu Ala Ser His
Tyr 625 630 635 640 Arg Gly Tyr Gly Asn Ser Asp Tyr Lys Arg Ile Val
Ala Cys Ala Ser 645 650 655 Tyr Lys Pro Ser Arg Glu Glu Ser Ser Ser
Gly Ser Ser His Ala Val 660 665 670 Met Asp Ile Ser Leu Pro Thr Gly
Ile Ser Ala Asn Glu Glu Asp Leu 675 680 685 Lys Ala Leu Val Glu Gly
Val Asp Gln Leu Phe Thr Asp Tyr Gln Ile 690 695 700 Lys Asp Gly His
Val Ile Leu Gln Leu Asn Ser Ile Pro Ser Ser Asp 705 710 715 720 Phe
Leu Cys Val Arg Phe Arg Ile Phe Glu Leu Phe Glu Val Gly Phe 725 730
735 Leu Ser Pro Ala Thr Phe Thr Val Tyr Glu Tyr His Arg Pro Asp Lys
740 745 750 Gln Cys Thr Met Phe Tyr Ser Thr Ser Asn Ile Lys Ile Gln
Lys Val 755 760 765 Cys Glu Gly Ala Ala Cys Lys Cys Val Glu Ala Asp
Cys Gly Gln Met 770 775 780 Gln Glu Glu Leu Asp Leu Thr Ile Ser Ala
Glu Thr Arg Lys Gln Thr 785 790
795 800 Ala Cys Lys Pro Glu Ile Ala Tyr Ala Tyr Lys Val Ser Ile Thr
Ser 805 810 815 Ile Thr Val Glu Asn Val Phe Val Lys Tyr Lys Ala Thr
Leu Leu Asp 820 825 830 Ile Tyr Lys Thr Gly Glu Ala Val Ala Glu Lys
Asp Ser Glu Ile Thr 835 840 845 Phe Ile Lys Lys Val Thr Cys Thr Asn
Ala Glu Leu Val Lys Gly Arg 850 855 860 Gln Tyr Leu Ile Met Gly Lys
Glu Ala Leu Gln Ile Lys Tyr Asn Phe 865 870 875 880 Ser Phe Arg Tyr
Ile Tyr Pro Leu Asp Ser Leu Thr Trp Ile Glu Tyr 885 890 895 Trp Pro
Arg Asp Thr Thr Cys Ser Ser Cys Gln Ala Phe Leu Ala Asn 900 905 910
Leu Asp Glu Phe Ala Glu Asp Ile Phe Leu Asn Gly Cys 915 920 925
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