U.S. patent application number 15/076960 was filed with the patent office on 2016-07-14 for complement pathway inhibitors binding to c5 and c5a without preventing the formation of c5b.
The applicant listed for this patent is Genentech, Inc.. Invention is credited to Michael Fung, Mason Lu, Cecily R.Y. Sun, William N.C. Sun.
Application Number | 20160200805 15/076960 |
Document ID | / |
Family ID | 23214536 |
Filed Date | 2016-07-14 |
United States Patent
Application |
20160200805 |
Kind Code |
A1 |
Fung; Michael ; et
al. |
July 14, 2016 |
Complement Pathway Inhibitors Binding To C5 And C5a Without
Preventing The Formation Of C5b
Abstract
The invention relates to inhibitors that bind to C5 and C5a, but
which do not prevent the activation of C5 and do not prevent
formation of or inhibit the activity of C5b. One example of such an
inhibitor molecule is the monoclonal antibody designated MAb137-26,
which binds to a shared epitope of human C5 and C5a. These
inhibitors may be used to inhibit the activity of C5a in treating
diseases and conditions mediated by excessive or uncontrolled
production of C5a. The inhibitor molecules are also useful for
diagnostic detection of the presence/absence or amount of C5 or
C5a.
Inventors: |
Fung; Michael;
(Gaithersburg, MD) ; Lu; Mason; (Houston, TX)
; Sun; William N.C.; (Shanghai, CN) ; Sun; Cecily
R.Y.; (Shanghai, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
23214536 |
Appl. No.: |
15/076960 |
Filed: |
March 22, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14551705 |
Nov 24, 2014 |
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15076960 |
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13759687 |
Feb 5, 2013 |
8907072 |
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14551705 |
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13482328 |
May 29, 2012 |
8372404 |
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13759687 |
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12011058 |
Jan 24, 2008 |
8206716 |
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13482328 |
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10222464 |
Aug 17, 2002 |
7432356 |
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12011058 |
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60313137 |
Aug 17, 2001 |
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Current U.S.
Class: |
424/133.1 ;
424/139.1; 530/387.3; 530/387.9; 530/391.1 |
Current CPC
Class: |
A61P 13/12 20180101;
C07K 2317/94 20130101; C07K 2317/92 20130101; A61P 7/00 20180101;
A61P 35/00 20180101; C07K 16/18 20130101; A61P 11/00 20180101; C07K
2317/34 20130101; G01N 33/6893 20130101; A61P 9/10 20180101; A61P
31/04 20180101; C07K 2317/24 20130101; A61P 29/00 20180101; A61K
39/3955 20130101; C07K 2317/33 20130101; A61P 25/00 20180101; A61P
37/00 20180101; C07K 2317/21 20130101; C07K 2317/76 20130101; G01N
33/6863 20130101; A61P 17/06 20180101; C07K 2317/569 20130101; A61K
2039/505 20130101 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Claims
1-17. (canceled)
18. An isolated antibody or an antigen-binding fragment thereof
that binds to the same epitope of human C5 as the monoclonal
antibody 137-26 produced from the hybridoma deposited with the ATCC
and designated PTA-3650.
19. The antibody or antigen binding fragment thereof of claim 18,
wherein the antibody or antigen-binding fragment binds to human C5
and C5a, but does not prevent the activation of C5 and does not
prevent formation of or inhibit the activity of C5b.
20. The antibody or antigen binding fragment thereof of claim 18,
wherein the antibody or antigen-binding fragment binds an epitope
having the amino acid sequence of EQRAARISLGPR.
21. The antibody or antigen binding fragment thereof of claim 18,
wherein the antibody or the antigen-binding fragment thereof
inhibits human neutrophil activation with at least equal potency as
the monoclonal antibody 137-26 produced from the hybridoma
deposited with the ATCC and designated PTA-3650.
22. The antibody or antigen binding fragment of claim 21, wherein
the inhibition of human neutrophil activation is determined by
measuring the expression of CD11b or the oxidative burst in
neutrophils, induced by opsonized Escherichia coli in
lepirudin-treated whole human blood.
23. The antibody or antigen binding fragment of claim 19, wherein
the antibody or antigen-binding fragment binds to free human C5a
with equal or better affinity than to human C5.
24. The antibody or antigen binding fragment of claim 19, wherein
the antibody or antigen-binding fragment inhibits the binding of
human C5a to human C5a receptor.
25. The antibody or antigen binding fragment of claim 18, wherein
the antibody is a monoclonal antibody.
26. The antibody or antigen binding fragment thereof of claim 18,
wherein the fragment is selected from the group consisting of Fab,
F(ab').sub.2, Fv, and single chain Fv.
27. The antibody or antigen binding fragment thereof of claim 18,
wherein the antibody is a chimeric, deimmunized, humanized or a
human antibody.
28. The antibody or antigen binding fragment thereof of claim 18,
wherein the antibody or antigen binding fragment comprises a
polymer that increases the half life of circulation of the
antibody.
29. The antibody or antigen binding fragment thereof of claim 18,
wherein the polymer is polyethylene glycol having an average
molecular weight between (i) 1,000 and 40,000; (ii) 2,000 and
20,000; or (iii) 3,000 and 12,000.
30. A pharmaceutical composition comprising the antibody or
fragment thereof according to claim 18, and a pharmacologically
acceptable carrier, excipient, stabilizer, or diluent.
31. A method of inhibiting the activity of human C5a comprising
contacting human C5a with an antibody or antigen binding fragment
thereof, wherein the antibody or antigen binding fragment binds to
the same epitope on human C5 and human C5a as the monoclonal
antibody 137-26 produced by the hybridoma deposited with the ATCC
and designated PTA-3650.
32. A method of treating a complement mediated disease or condition
comprising administering to a subject in need thereof an antibody
or antigen binding fragment thereof, wherein the antibody or
antigen binding fragment binds to the same epitope on human C5 and
human C5a as the monoclonal antibody 137-26 produced by the
hybridoma deposited with the ATCC and designated PTA-3650.
33. The method of claim 31, wherein the antibody or antigen-binding
fragment binds an epitope having the amino acid sequence of
EQRAARISLGPR.
34. The method of claim 31, wherein the antibody or antigen binding
fragment binds to free human C5a with equal or better affinity than
to human C5.
35. The method of claim 31, wherein the antibody or antigen binding
fragment inhibits the binding of human C5a to human C5a
receptor.
36. The method of claim 31, wherein the antibody is a monoclonal
antibody.
37. The method of claim 31, wherein the antibody is chimeric,
deimmunized, or humanized, or a human antibody.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/011,058 filed Jan. 24, 2008, which is a continuation of U.S.
application Ser. No. 10/222,464 filed Aug. 17, 2002, now U.S. Pat.
No. 7,432,356, which claims priority to U.S. provisional
application No. 60/313,137 filed Aug. 17, 2001, the disclosure of
each of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to inflammation inhibitors which bind
to complement C5 and C5a without inhibiting the formation of C5b
and C5b-9 membrane attack complexes (MAC).
BACKGROUND OF THE INVENTION
[0003] The complement system plays a central role in the clearance
of immune complexes and in immune responses to infectious agents,
foreign antigens, virus-infected cells and tumor cells. However,
inappropriate or excessive activation of the complement system can
lead to harmful, and even potentially life-threatening,
consequences due to severe inflammation and resulting tissue
destruction. These consequences are clinically manifested in
various disorders including septic shock, myocardial as well as
intestinal ischemia/reperfusion injury, graft rejection, organ
failure, nephritis, pathological inflammation and autoimmune
diseases. Sepsis, for example, is a major cause of mortality
resulting in over 200,000 deaths per year in the United States
alone. Despite the major advances in the past several years in the
treatment of serious infections, the incidence and mortality from
sepsis continues to rise. Therefore, inhibition of excessive or
uncontrolled activation of the complement cascade could provide
clinical benefit to patients with such diseases and conditions.
[0004] The complement system is composed of a group of proteins
that are normally present in the serum in an inactive state.
Activation of the complement system encompasses mainly two distinct
pathways, designated the classical and the alternative pathways (V.
M. Holers, In Clinical Immunology: Principles and Practice, ed. R.
R. Rich, Mosby Press; 1996, 363-391). The classical pathway is a
calcium/magnesium-dependent cascade, which is normally activated by
the formation of antigen-antibody complexes. It can also be
activated in an antibody-independent manner by the binding of
C-reactive protein, complexed with ligand, and by many pathogens
including gram-negative bacteria. The alternative pathway is a
magnesium-dependent cascade which is activated by deposition and
activation of C3 on certain susceptible surfaces (e.g. cell wall
polysaccharides of yeast and bacteria, and certain biopolymer
materials).
[0005] Recent studies have shown that complement can also be
activated through the lectin pathway, which involves the initial
binding of mannose-binding lectin and the subsequent activation of
C2 and C4, which are common to the classical pathway (Matsushita,
M. et al., J. Exp. Med. 176: 1497-1502 (1992); Suankratay, C. et
al., J. Immunol. 160: 3006-3013 (1998)). Accumulating evidence
indicates that the alternative pathway participates in the
amplification of the activity of both the classical pathway and the
lectin pathway (Suankratay, C., ibid; Farries, T. C. et al., Mol.
Immunol. 27: 1155-1161 (1990)). Activation of the complement
pathway generates biologically active fragments of complement
proteins, e.g. C3a, C4a and C5a anaphylatoxins and C5b-9 membrane
attack complexes (MAC), which mediate inflammatory responses
through involvement of leukocyte chemotaxis, activation of
macrophages, neutrophils, platelets, mast cells and endothelial
cells, increased vascular permeability, cytolysis, and tissue
injury.
[0006] Complement C5a is one of the most potent proinflammatory
mediators of the complement system. C5a is the activated form of
C5. Complement C5 (190 kD, molecular weight) is present in human
serum at approximately 80 pig/ml (Kohler, P. F. et al., J. Immunol.
99: 1211-1216 (1967)). It is composed of two polypeptide chains,
.alpha. and .beta., with approximate molecular weights of 115 kD
and 75 kD, respectively (Tack, B. F. et al., Biochemistry 18:
1490-1497 (1979)). Biosynthesized as a single-chain promolecule, C5
is enzymatically cleaved into a two-chain structure during
processing and secretion. After cleavage, the two chains are held
together by at least one disulphide bond as well as noncovalent
interactions (Ooi, Y. M. et al., J. Immunol. 124: 2494-2498
(1980)).
[0007] Primary amino acid structures of human and murine C5 were
obtained from cDNA sequencing data (Wetsel, R A. et al.,
Biochemistry 27: 1474-1482 (1988); Haviland, D. L. et al., J.
Immunol. 146: 362-368 (1991): Wetsel, R A. et al., Biochemistry 26:
737-743 (1987)). The deduced amino acid sequence of precursor human
pre-pro-C5 has 1676 amino acids. The .alpha.- and .beta.-chains of
mature C5 have 999 and 655 amino acids, respectively. C5 is
glycosylated in the C5 .alpha.-chain, in particular the asparagine
at residue 64.
[0008] C5 is cleaved into the C5a and C5b fragments during
activation of the complement pathways. The convertase enzymes
responsible for C5 activation are multi-subunit complexes of C4b,
C2a, and C3b for the classical pathway and of (C3b).sub.2, Bb, and
P for the alternative pathway (Goldlust, M. B. et al., J. Immunol.
113: 998-1007 (1974); Schreiber, R D. et al., Proc. Natl. Acad.
Sci. 75: 3948-3952 (1978)). C5 is activated by cleavage at position
74-75 (Arg-Leu) in the .alpha.-chain. After activation, the 11.2
kD, 74 amino acid peptide C5a from the amino-terminus portion of
the .alpha.-chain is released. This C5a peptide shares similar
anaphylatoxin properties with those exhibited by C3a, but is 100
times more potent, on a molar basis, in eliciting inflammatory
responses. Both C5a and C3a are potent stimulators of neutrophils
and monocytes (Schindler, R. et al., Blood 76: 1631-1638 (1990);
Haeffner-Cavaillon, N. et al., J. Immunol. 138: 794-700 (1987);
Cavaillon, J. M. et al., Eur. J. Immunol. 20: 253-257 (1990)).
Furthermore, C3a receptor was recently shown to be important for
protection against endotoxin-induced shock in a mouse model
(Kildsgaard, J. et al., J. Immunol. 165: 5406-5409 (2000)).
[0009] In addition to its anaphylatoxic properties, C5a induces
chemotactic migration of neutrophils (Ward, P. A. et al., J.
Immunol. 102: 93-99 (1969)), eosinophils (Kay, A. B. et al.,
Immunol. 24: 969-976 (1973)), basophils (Lett-Brown, M. A. et al.,
J. Immunol. 117: 246-252 1976)), and monocytes (Snyderman, R. et
al., Proc. Soc. Exp. Biol. Med. 138: 387-390 1971)). The activity
of C5a is regulated by the plasma enzyme carboxypeptidase N (E.C.
3.4.12.7) that removes the carboxy-terminal arginine from C5a
forming the C5a des Arg derivative (Goetzl, E. J. et al., J. Clin.
Invest. 53: 591-599 (1974)). On a molar basis, human G5a des Arg
exhibits only 1% of the anaphylactic activity (Gerard, C. et al.,
Proc. Natl. Acad. Sci. 78: 1833-1837 (1981)) and polymorphonuclear
chemotactic activity as unmodified C5a (Chenoweth, D. E. et al.,
Mol. Immunol. 17: 151-161 (1980)). Both C5a and C5b-9 activate
endothelial cells to express adhesion molecules essential for
sequestration of activated leukocytes, which mediate tissue
inflammation and injury (Foreman, K. E. et al., J. Clin. Invest.
94: 1147-1155 (1994); Foreman, K. E. et al., Inflammation 20: 1-9
(1996); Rollins, S. A. et al., Transplantation 69: 1959-1967
(2000)). C5a also mediates inflammatory reactions by causing smooth
muscle contraction, increasing vascular permeability, inducing
basophil and mast cell degranulation and inducing release of
lysosomal proteases and oxidative free radicals (Gerard, C. et al.,
Ann. Rev. Immunol. 12: 775-808 (1994)). Furthermore, C5a modulates
the hepatic acute-phase gene expression and augments the overall
immune response by increasing the production of TNF.alpha..
IL-1.beta., IL-6, and IL-8 (Lambris, J. D. et al., In: The Human
Complement System in Health and Disease, Volanakis, J. E. ed.,
Marcel Dekker, New York, pp. 83-118).
[0010] The human C5a receptor (C5aR) has been cloned (Gerard, N. P.
et al., Nature 349: 614-617 (1991); Boulay, F. et al., Biochemistry
30: 2993-2999 (1991)). It belongs to a superfamily of
seven-transmembrane-domain, G protein-coupled receptors. C5aR is
expressed on neutrophils, monocytes, basophils, eosinophils,
hepatocytes, lung smooth muscle and endothelial cells, and renal
glomerular tissues (Van-Epps, D. E. et al., J. Immunol. 132:
2862-2867 (1984); Haviland, D. L. et al., J. Immunol. 154:1861-1869
(1995); Wetsel, R. A., Immunol. Lett. 44: 183-187 (1995); Buchner,
R. R. et al., J. Immunol. 155: 308-315 (1995); Chenoweth, D. E. et
al., Proc. Natl. Acad. Sci. 75: 3943-3947 (1978); Zwirner, J. et
al., Mol. Immunol. 36:877-884 (1999)). The ligand-binding site of
C5aR is complex and consists of at least two physically separable
binding domains. One binds the C5a amino terminus (amino acids
1-20) and disulfide-linked core (amino acids 21-61), while the
second binds the C5a carboxy-terminal end (amino acids 62-74)
(Wetsel, R A., Curr. Opin. Immunol. 7: 48-53 (1995)).
[0011] C5a plays important roles in inflammation and tissue injury.
In cardiopulmonary bypass and hemodialysis, C5a is formed as a
result of activation of the alternative complement pathway when
human blood makes contact with the artificial surface of the
heart-lung machine or kidney dialysis machine (Howard, R. J. et
al., Arch. Surg. 123: 1496-1501 (1988); Kirklin, J. K. et al., J.
Cardiovasc. Surg. 86: 845-857 (1983); Craddock, P. R. et al., N.
Engl. J. Med. 296: 769-774 (1977)). C5a causes increased capillary
permeability and edema, bronchoconstriction, pulmonary
vasoconstriction, leukocyte and platelet activation and
infiltration to tissues, in particular the lung (Czermak, B. J. et
al., J. Leukoc. Biol. 64: 40-48 (1998)). Administration of an
anti-C5a monoclonal antibody was shown to reduce cardiopulmonary
bypass and cardioplegia-induced coronary endothelial dysfunction
(Tofukuji, M. et al., J. Thorac. Cardiovasc. Surg. 116: 1060-1068
(1998)).
[0012] C5a is also involved in acute respiratory distress syndrome
(ARDS) and multiple organ failure (MOF) (Hack, C. E. et al., Am. J.
Med. 1989: 86: 20-26; Hammerschmidt D E et al. Lancet 1980; 1:
947-949; Heideman M. et al. J. Trauma 1984; 4: 1038-1043). C5a
augments monocyte production of two important pro-inflammatory
cytokines, TNF.alpha. and IL-1. C5a has also been shown to play an
important role in the development of tissue injury, and
particularly pulmonary injury, in animal models of septic shock.
(Smedegard G et al. Am. J. Pathol. 1989; 135: 489-497). In sepsis
models using rats, pigs and non-human primates, anti-C5a antibodies
administered to the animals before treatment with endotoxin or E.
coli resulted in decreased tissue injury, as well as decreased
production of IL-6 (Smedegard, G. et al., Am. J. Pathol. 135:
489-497 (1989); Hopken, U. et al., Eur. J. Immunol. 26: 1103-1109
(1996); Stevens, J. H. et al., J. Clin. Invest. 77: 1812-1816
(1986)). More importantly, blockade of C5a with anti-C5a polyclonal
antibodies has been shown to significantly improve survival rates
in a caecal ligation/puncture model of sepsis in rats (Czermak, B.
J. et al., Nat. Med. 5: 788-792 (1999)). This model shares many
aspects of the clinical manifestation of sepsis in humans. (Parker,
S. J. et al., Br. J. Surg. 88: 22-30 (2001)). In the same sepsis
model, anti-C5a antibodies were shown to inhibit apoptosis of
thymocytes (Guo, R. F. et al., J. Clin. Invest. 106: 1271-1280
2000)) and prevent MOF (Huber-Lang, M. et al., J. Immunol. 166:
1193-1199 (2001)). Anti-C5a antibodies were also protective in a
cobra venom factor model of lung injury in rats, and in immune
complex-induced lung injury (Mulligan, M. S. et al. J. Clin.
Invest. 98: 503-512 (1996)). The importance of C5a in immune
complex-mediated lung injury was later confirmed in mice (Bozic, C.
R. et al., Science 26: 1103-1109 (1996)).
[0013] C5a is found to be a major mediator in myocardial
ischemia-reperfusion injury. Complement depletion reduced
myocardial infarct size in mice (Weisman, H. F. et al., Science
249: 146-151 (1990)), and treatment with anti-C5a antibodies
reduced injury in a rat model of hindlimb ischemia-reperfusion
(Bless, N. M. et al., Am. J. Physiol. 276: L57-L63 (1999)).
Reperfusion injury during myocardial infarction was also markedly
reduced in pigs that were retreated with a monoclonal anti-C5a IgG
(Amsterdam, E. A. et al., Am. J. Physiol. 268:H448-H457 (1995)). A
recombinant human C5aR antagonist reduces infarct size in a porcine
model of surgical revascularization (Riley, R D. et al., J. Thorac.
Cardiovasc. Surg. 120: 350-358 (2000)).
[0014] Complement levels are elevated in patients with rheumatoid
arthritis and systemic lupus erythematosus. C5a levels correlate
with the severity of the disease state (Jose, P. J. et al., Ann.
Rheum. Dis. 49: 747-752 (1989); Porcel, J. M. et al., Clin.
Immunol. Immunopathol. 74: 283-288 (1995)). Therefore, inhibition
of C5a and/or C5a receptor (C5aR) could be useful in treating these
chronic diseases.
[0015] C5aR expression is upregulated on reactive astrocytes,
microglia, and endothelial cells in an inflamed human central
nervous system (Gasque, P. et al., Am. J. Pathol. 150: 31-41
(1997)). G5a might be involved in neurodegenerative diseases, such
as Alzheimer disease (Mukherjee, P. et al., J. Neuroimmunol. 105:
124-130 (2000)). Activation of neuronal C5aR may induce apoptosis
(Farkas I et al. J. Physiol. 1998: 507: 679-687). Therefore,
inhibition of C5a and/or C5aR could also be useful in treating
neurodegenerative diseases.
[0016] Psoriasis is now known to be a T cell-mediated disease
(Gottlieb, E. L. et al., Nat. Med. 1: 442-447 (1995)). However,
neutrophils and mast cells may also be involved in the pathogenesis
of the disease (Terui, T. et al., Exp. Dermatol. 9: 1-10; 2000);
Werfel, T. et al., Arch. Dermatol. Res. 289: 83-86 (1997)). High
levels of C5a des Arg are found in psoriatic scales, indicating
that complement activation is involved. T cells and neutrophils are
chemo-attracted by C5a (Nataf, S. et al., J. Immunol. 162:
4018-4023 (1999); Tsuji, R. F. et al., J. Immunol. 165: 1588-1598
(2000); Cavaillon, J. M. et al., Eur. J. Immunol. 20: 253-257
(1990)). Therefore C5a could be an important therapeutic target for
treatment of psoriasis.
[0017] Immunoglobulin G-containing immune complexes (IC) contribute
to the pathophysiology in a number of autoimmune diseases, such as
systemic lupus erthyematosus, rheumatoid arthritis, Goodpasture's
syndrome, and hypersensitivity pneumonitis (Madaio, M. P., Semin.
Nephrol. 19: 48-56 (1999); Korganow, A. S. et al., Immunity 10:
451-459 (1999); Bolten, W. K., Kidney Int. 50: 1754-1760 (1996);
Ando, M. et al., Curr. Opin. Pulm. Med. 3: 391-399 (1997)). The
classical animal model for the inflammatory response in these IC
diseases is the Arthus reaction, which features the infiltration of
polymorphonuclear cells, hemorrhage, and plasma exudation (Arthus,
M., C. R. Soc. Biol. 55: 817-824 (1903)). Recent studies show that
C5aR deficient mice are protected from tissue injury induced by IC
(Kohl, J. et al., Mol. Immunol. 36: 893-903 (1999); Baumann, U. et
al., J. Immunol. 164: 1065-1070 (2000)). The results are consistent
with the observation that a small peptidic anti-C5aR antagonist
inhibits the inflammatory response caused by IC deposition
(Strachan, A. J. et al., J. Immunol. 164: 6560-6565 (2000)).
Together with its receptor, C5a plays an important role in the
pathogenesis of IC diseases. Inhibitors of C5a and C5aR could be
useful to treat these diseases.
[0018] WO01/15731A1 discusses compositions and methods of treatment
of sepsis using antibodies to C5a. These antibodies react only with
the N-terminal region of the C5a peptide and do not cross-react
with C5.
[0019] WO86/05692 discusses the treatment of adult respiratory
distress syndrome (ARDS) with an antibody specific for C5a or the
des Arg derivative thereof. It also discloses the treatment of
sepsis by administering this antibody. This antibody was produced
in response to the C5a des Arg derivative because it is more
immunogenic, but will elicit antibodies cross reactive with C5a.
U.S. Pat. No. 5,853,722 discusses anti-C5 antibodies that block the
activation of C5 and thus, the formation of C5a and C5b.
[0020] U.S. Pat. No. 6,074,642 discusses the use of anti-C5
antibodies to treat glomerulonephritis. These antibodies also block
the generation of C5a and C5b, inhibiting the effect of both C5a
and the formation of C5b-9. U.S. Pat. No. 5,562,904 discusses
anti-C5 antibodies that completely block the formation of MAC.
[0021] In other discussions of anti-C5 antibodies, the antibodies
disclosed block activation of C5 and its cleavage to form C5a and
C5b (Vakeva, A. P. et al., Circulation 97:2259-2267 (1998); Thomas,
T. C. et al., Mol. Immunol. 33:1389-1401 (1996); Wang, Y. et al.,
Proc Natl Acad Sci. 93:8563-8568 (1996); Kroshus, T. et al.,
Transplantation 60:1194-1202 (1995); Frei, Y. et al., Mol. Cell
Probes 1:141-149 (1987)).
[0022] Monoclonal antibodies cross-reactive with C5, C5a, or C5a
des Arg have been reported (Schulze, M. et al., Complement 3: 25-39
(1986); Takeda, J. et al., J. Immunol. Meth. 101: 265-270 (1987);
Inoue, K., Complement Inflamm. 6: 219-222 (1989). It has also been
reported that monoclonal antibodies cross-reactive with C5 and C5a
inhibited C5a-mediated ATP release from guinea pig platelets (Klos,
A. et al., J. Immunol. Meth. 111: 241-252 (1988); Oppermann, M. et
al., Complement Inflamm. 8: 13-24 (1991)).
SUMMARY OF THE INVENTION
[0023] C5 activation normally results in cleavage of C5 to C5a and
C5b. The inhibitor molecules of the present invention bind to C5
and C5a with high affinity, do not inhibit C5 activation, and do
not prevent the formation of or inhibit the activity of C5b. One
example of such an inhibitor is the monoclonal antibody designated
MAb 137-26, which binds to a shared epitope on human C5 and C5a.
The hybridoma that produces the monoclonal antibody 137-26 has been
deposited at the American Type Culture Collection, 10801 University
Blvd., Manassas, Va. 20110-2209, under Accession No. PTA-3650 on
Aug. 17, 2001.
[0024] The inhibitor molecules of the invention also include: (i)
other antibodies or fragments thereof, peptides, oligonucleotides,
or peptidomimetics that bind to C5 and C5a with high affinity, but
do not inhibit C5 activation, and do not prevent the formation of
or inhibit the activity of C5b, or (ii) any antibody that binds to
the same epitope as the monoclonal antibody 137-26. Antibody
fragments include Fab, F(ab').sub.2, Fv, or single chain Fv, and
monoclonal antibodies and fragments of the invention include
chimeric, deimmunized, humanized or human antibodies and fragments,
and other forms acceptable for human use. The inhibitor molecules
may be included as part of a pharmaceutical composition.
[0025] The inhibitor molecules of the invention are useful for
treatment of diseases and conditions involving excessive or
uncontrolled production of C5a, or for diagnostic use in detecting
the presence of, or quantitation, C5a.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1 shows the binding of MAb 137-26 (anti-C5a, closed
squares) and MAb 137-76 (anti-C5 .beta.-chain, open circles) to
purified human C5a in ELISA. An isotype-matched irrelevant
monoclonal antibody was used as negative control. The Y-axis
represents the reactivity of the MAbs with C5a expressed as optical
density (OD) at 450 nm and the X-axis represents the concentration
of the MAbs. MAb 137-26 reacted with human C5a, whereas MAb 137-76
and the irrelevant control antibody did not.
[0027] FIG. 2 shows the binding of MAb 137-26 (closed squares) and
MAb 137-76 (open circles) to purified human C5 in ELISA. An
isotype-matched irrelevant monoclonal antibody was used as negative
control. The Y-axis represents the reactivity of the MAbs with C5
expressed as optical density (OD) at 450 nm and the X-axis
represents the concentration of the monoclonal antibodies. Both MAb
137-26 and MAb 137-76 reacted with human C5, whereas the irrelevant
antibody did not.
[0028] FIG. 3 shows the inability of anti-C5a MAb 137-26 to inhibit
complement-mediated hemolysis of sensitized chicken red blood cells
via the classical pathway (CP). Anti-C2 MAb 175-62 effectively
inhibited the hemolysis. An isotype-matched irrelevant monoclonal
antibody had no effect. The Y-axis represents the percentage of
hemolysis inhibition, as further described in the text. The X-axis
represents the concentration of the monoclonal antibodies.
[0029] FIG. 4 shows the inability of anti-C5a MAb 137-26 to inhibit
complement-mediated hemolysis of rabbit red blood cells via the
alternative pathway (AP). Anti-factor D MAb 166-32 effectively
inhibited the hemolysis. An isotype-matched irrelevant monoclonal
antibody had no effect. The Y-axis represents the percentage
hemolysis inhibition, as further described in the text. The X-axis
represents the concentration of monoclonal antibodies.
[0030] FIG. 5 shows the inhibition of the binding of radioiodinated
(.sup.125I)-human C5a to purified human neutrophils. The positive
control, purified recombinant human C5a (rHuC5a), inhibited the
binding. An isotype-matched irrelevant monoclonal antibody did not
show any effect. The Y-axis represents the percentage inhibition of
.sup.125I-C5a binding, as further described in the text. The X-axis
represents the concentration of the competing agents.
[0031] FIG. 6 shows the binding epitope of MAb 137-26 on human C5a
mapped by overlapping synthetic peptides on cellulose membrane.
[0032] FIG. 7A shows CD11b expression on human neutrophils
stimulated by opsonized E. coli in a lepirudin anti-coagulated
whole blood model. Anti-C5/C5a MAb137-26 (closed circles) inhibited
CD11b expression more effectively than anti-C5a MAb561 (Dr. Jurg
Kohl, closed squares) and anti-C5 MAb137-30 (closed triangles). The
latter antibody inhibited C5 activation. The irrelevant MAb (closed
inverted triangles) had no effect. The Y-axis represents mean
fluorescence intensity (MFI) measured by immunofluorocytometry. The
X-axis represents the concentration of the antibodies (.mu.g/ml)
T-0=baseline whole blood sample at time 0 min. T-10=whole blood
incubated only with PBS for 10 min without E. coli. Other samples,
with or without inhibitors, had E. coli added.
[0033] FIG. 7B shows CD11b expression on human neutrophils
stimulated by opsonized E. coli in a lepirudin anti-coagulated
whole blood model. Anti-C5/C5a MAb 137-26 (closed circles)
inhibited CD11b expression more effectively than a peptidic C5aR
antagonist (Dr. Stephen Taylor) (closed squares). The irrelevant
peptide had no effect (closed inverted triangles). The Y-axis
represents mean fluorescence intensity (MFI) measured by
immunofluorocytometry. The X-axis represents the concentration of
the antibodies/peptide (.mu.g/ml). T-0=baseline whole blood sample
at time 0 min. T-10=whole blood incubated only with PBS for 10 min
without E. coli. Other samples, with or without inhibitors, had E.
coli added.
[0034] FIG. 7C shows oxidative burst of human neutrophils
stimulated by opsonized E. coli in a lepirudin anti-coagulated
whole blood model. Both anti-C5/C5a MAb137-26 (closed circles) and
anti-C5 MAb137-30 (closed triangles) inhibited oxidative burst more
effectively than anti-C5a MAb561 (closed squares). The irrelevant
antibody (closed inverted triangles) had no effect. The Y-axis
represents mean fluorescence intensity (MFI) measured by
immunofluorocytometry. The X-axis represents the concentration of
the antibodies (.mu.g/ml). T-0=baseline whole blood sample at time
0 min. T-10 whole blood incubated only with PBS for 10 min without
E. coli. Other samples, with or without inhibitors, had E. coli
added.
[0035] FIG. 7D shows oxidative burst of human neutrophils
stimulated by opsonized E. coli in a lepirudin anti-coagulated
whole blood model. Anti-C5/C5a MAb137-26 was more effective than a
peptidic C5aR antagonist (closed square) in inhibiting oxidative
burst. The irrelevant peptide had no effect. The Y-axis represents
mean fluorescence intensity (MFI) measured by
immunofluorocytometry. The X-axis represents the concentration of
the antibodies in nM. T-0=baseline whole blood sample at time 0
min. T-10=whole blood incubated only with PBS for 10 min without E.
coli. Other samples, with or without inhibitors, had E. coli
added.
[0036] FIG. 8 shows the MAC-mediated killing of Neisseria
meningitides in a lepirudin anti-coagulated whole blood model. The
bacteria were effectively killed by incubation with the human whole
blood in the presence of anti-C5/C5a MAb137-26 (closed circles), an
irrelevant MAb (closed triangles), or PBS (open squares). In
contrast, the bacteria were not killed when the whole blood was
treated with anti-C5 MAb137-30 (open diamonds) which inhibited C5
activation and thus MAC formation. The Y-axis represents colony
forming units (CFU) per 100 .mu.l of whole blood incubated for 24
hours at 37.degree. C. on blood agar. The X-axis represents
different time points of blood sample collection from the whole
blood culture experiment.
DETAILED DESCRIPTION
1. Advantages of the Invention
[0037] The inhibitors of the invention, including monoclonal
antibody MAb137-26, are advantageous over known monoclonal antibody
inhibitors for treating complement-mediated inflammation and tissue
damage. MAb 137-26 is capable of binding to C5 before it is
activated. After C5 is activated to form C5a, the antibody can
neutralize C5a, which is an anaphylatoxin. Normally, once C5a is
formed, it rapidly binds to C5aR on cells, thereby triggering the
signal transduction cascade leading to inflammation. MAb137-26 does
not inhibit the cleavage of C5 to form C5a and C5b, but C5a remains
bound to MAb137-26 after it is produced, and inhibits binding of
C5a to C5aR. The formation of C5b-9, however, is not affected, and,
inasmuch as C5b-9 is needed for MAC formation, which is involved in
killing bacteria, maintaining production of C5b-9 is important for
a protective immune response.
[0038] MAb 137-26 can effectively neutralize the inflammatory
effects of C5a, but still allow other components of the complement
cascade, including C3 and C5b-9, to mediate anti-bacterial
functions. This pharmacological property is exceptionally important
with respect to the treatment of bacterial sepsis, chronic IC
diseases, and psoriasis.
2. Making and Using the Invention
[0039] A. Monoclonal Antibodies
[0040] Monoclonal antibodies may be made using the hybridoma method
first described by Kohler et al., Nature, 256:495 (1975), or may be
made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
[0041] In the hybridoma method, a mouse or other appropriate host
animal is immunized as hereinabove described to elicit lymphocytes
that produce or are capable of producing antibodies that will
specifically bind to the protein used for immunization. Animals can
also be immunized with DNA constructs to express the encoding
proteins in vivo for inducing specific antibodies.
[0042] Alternatively, lymphocytes may be immunized in vitro.
Lymphocytes then are fused with myeloma cells using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)).
[0043] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0044] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these myeloma cell lines are murine myeloma lines,
such as those derived from MOPC-21 and MPC-11 mouse tumors
available from the Salk Institute Cell Distribution Center, San
Diego, Calif. USA, and SP2/0 or X63-Ag8-653 cells available from
the American Type Culture Collection, Rockville, Md. USA. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal
Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987)). The mouse myeloma cell line NS0 may
also be used (European Collection of Cell Cultures, Salisbury,
Wiltshire UK).
[0045] Culture medium in which hybridoma cells are grown is assayed
for production of monoclonal antibodies directed against the
antigen. The binding specificity of monoclonal antibodies produced
by hybridoma cells may be determined by immunoprecipitation or by
an in vitro binding assay, such as radioimmunoassay (RIA) or
enzyme-linked immunoabsorbent assay (ELISA).
[0046] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal.
[0047] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose*, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0048] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (Innis M. et al. In PCR
Protocols. A Guide to Methods and Applications, Academic, San
Diego, Calif. (1990), Sanger, F. S., et al. Proc. Nat. Acad. Sci.
74:5463-5467 (1977)). The hybridoma cells serve as a source of such
DNA. Once isolated, the DNA may be placed into expression vectors,
which are then transfected into host cells such as E. coli cells,
simian COS cells. Chinese hamster ovary (CHO) cells, or myeloma
cells that do not otherwise produce immunoglobulin protein, to
obtain the synthesis of monoclonal antibodies in the recombinant
host cells. Recombinant production of antibodies will be described
in more detail below.
[0049] In a further embodiment, antibodies or antibody fragments
can be isolated from antibody phage libraries generated using the
techniques described in McCafferty. et al., Nature 348:552-554
(1990). Clackson, et al., Nature 352:624-628 (1991) and Marks, et
al., J. Mol. Biol. 222:581-597 (1991) describe the isolation of
murine and human antibodies, respectively, using phage libraries.
Subsequent publications describe the production of high affinity
(nM range) human antibodies by chain shuffling (Marks, et al.,
Bio/Technology 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse, et al., Nuc. Acids. Res.
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0050] The DNA also may be modified, for example, by substituting
the coding sequence for human heavy- and light-chain constant
domains in place of the homologous murine sequences (U.S. Pat. No.
4,816,567; Morrison, et al., Proc. Nat. Acad. Sci. USA 81:6851
(1984)), or by covalently joining to the immunoglobulin coding
sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide.
[0051] Typically, such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody, or they are
substituted for the variable domains of one antigen-combining site
of an antibody to create a chimeric bivalent antibody comprising
one antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0052] Another alternative is to use electrical fusion rather than
chemical fusion to form hybridomas. This technique is well
established. Instead of fusion, one can also transform a B-cell to
make it immortal using, for example, an Epstein Barr Virus, or a
transforming gene. (See, e.g., "Continuously Proliferating Human
Cell Lines Synthesizing Antibody of Predetermined Specificity,"
Zurawaki, V. R. et al. in Monoclonal Antibodies, ed. by Kennett R.
H. et al., Plenum Press, N.Y. 1980, pp 19-33.)
[0053] B. Humanized and Human Antibodies
[0054] A humanized antibody has one or more amino acid residues
introduced into it from a source, which is non-human. These
non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable
domain. Humanization can be essentially performed following the
method of Winter and co-workers (Jones et al., Nature 321:522-525
(1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen, et
al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or
CDR sequences for the corresponding sequences of a human antibody.
Accordingly, in such "humanized" antibodies, a substantially less
than intact human variable domain has been substituted by the
corresponding sequence from a nonhuman species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possibly some FR residues are substituted by
residues from analogous sites in rodent antibodies.
[0055] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework (FR) for the
humanized antibody (Sims et al., J. Immunol., 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains.
The same framework may be used for several different humanized
antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285
(1992); Presta et al., J. Immunol., 151:2623 (1993)).
[0056] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to a
preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
CDR residues are directly and most substantially involved in
influencing antigen binding.
[0057] Alternatively, the skilled researcher can 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. Such transgenic mice are
available from Abgenix, Inc., Fremont, Calif., and Medarex, Inc.,
Annandale, N.J. It has been described that the homozygous deletion
of the antibody heavy-chain joining region (JH) gene in chimeric
and germ-line mutant mice results in complete inhibition of
endogenous antibody production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies upon antigen challenge. See,
e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993);
Jakobovits et al., Nature 362:255-258 (1993); Bruggermann et al.,
Year in Immunol. 7:33 (1993); and Duchosal et al. Nature 355:258
(1992). Human antibodies can also be derived from phage-display
libraries (Hoogenboom et al., J. Mol. Biol. 227:381 (1991); Marks
et al., J. Mol. Biol. 222:581-597 (1991); Vaughan, et al., Nature
Biotech 14:309 (1996)).
[0058] C. Deimmunized Antibodies
[0059] Deimmunized antibodies are antibodies in which the potential
T cell epitopes have been eliminated, as described in International
Patent Application PCT/GB98/01473. Therefore, immunogenicity in
humans is expected to be eliminated or substantially reduced when
they are applied in vivo.
[0060] Additionally, antibodies can be chemically modified by
covalent conjugation to a polymer to increase their circulating
half-life, for example. Preferred polymers, and methods to attach
them to peptides, are shown in U.S. Pat. Nos. 4,766,106; 4,179,337;
4,495,285; and 4,609,546, which are all hereby incorporated by
reference in their entireties. Preferred polymers are
polyoxyethylated polyols and polyethylene glycol (PEG). PEG is
soluble in water at room temperature and has a preferred average
molecular weight between 1000 and 40,000, more preferably between
2000 and 20,000, most preferably between 3,000 and 12,000.
[0061] D. Generation of Anti-C5/C5a MAbs
[0062] Antibodies of the present invention may be generated by
traditional hybridoma techniques well known in the art. Briefly,
mice are immunized with C5 purified from human sera as an immunogen
emulsified in complete Freund's adjuvant, and injected
subcutaneously or intraperitoneally in amounts ranging from 10-100
.mu.g. Ten to fifteen days later, the immunized animals are boosted
with additional C5 emulsified in incomplete Freund's adjuvant. Mice
are periodically boosted thereafter on a weekly to bi-weekly
immunization schedule.
[0063] For each fusion, single cell suspensions were prepared from
the spleen of an immunized mouse and used for fusion with SP2/0
myeloma cells. SP2/0 cells (1.times.10.sup.8) and spleen cells
(1.times.10.sup.8) were fused in a medium containing 50%
polyethylene glycol (M.W. 1450) (Kodak, Rochester, N.Y.) and 5%
dimethylsulfoxide (Sigma Chemical Co., St. Louis, Mo.). The cells
were then adjusted to a concentration of 1.7.times.10.sup.5 spleen
cells/ml of the suspension in DMEM medium (Gibco, Grand Island,
N.Y.), supplemented with 5% fetal bovine serum and HAT (10 mM
sodium hypoxanthine, 40 .mu.M aminopterin, and 1.6 mM thymidine).
Two hundred and fifty microliters of the cell suspension were added
to each well of about fifty 96-well microtest plates. After about
ten days culture supernatants were withdrawn for screening for
reactivity with purified human C5 by ELISA.
[0064] Wells of Immulon.RTM. II (Dynatech Laboratories, Chantilly,
Va.) microtest plates were coated overnight with human C5 at 0.1
.mu.g/ml (50 .mu.l/well). The non-specific binding sites in the
wells were then saturated by incubation with 200 .mu.l of 5% BLOTTO
(non-fat dry milk) in phosphate-buffered saline (PBS) for one hour.
The wells were then washed with PBST buffer (PBS containing 0.05%
TWEEN.RTM. 20). Fifty microliters of culture supernatant from each
fusion well were added to the coated well together with 50 .mu.l of
BLOTTO for one hour at room temperature. The wells were washed with
PBST. The bound antibodies were then detected by reaction with
diluted horseradish peroxidase (HRP) conjugated goat anti-mouse IgG
(Fc specific) (Jackson ImmunoResearch Laboratories, West Grove,
Pa.) for one hour at room temperature. The wells were then washed
with PBST. Peroxidase substrate solution containing 0.1% 3,3,5,5,
tetramethyl benzidine (Sigma, St. Louis, Mo.) and 0.003% hydrogen
peroxide (Sigma, St. Louis, Mo.) in 0.1 M sodium acetate pH 6.0 was
added to the wells for color development for 30 minutes. The
reaction was terminated by addition of 50 .mu.l of 2M
H.sub.2SO.sub.4 per well. The optical density (00) was read at 450
nm with an ELISA reader (Dynatech Laboratories, Chantilly,
Va.).
[0065] Hybridomas in wells positive for C5 reactivity were
single-cell cloned by a limiting dilution method. Monoclonal
hybridomas were then expanded and culture supernatants collected
for purification by protein A chromatography. The purified
antibodies were then characterized for reactivity with human C5 and
C5a by ELISA, for determination of affinity and kinetic binding
constants by BIAcore, for effects on complement-mediated hemolysis
via both the classical and the alternative pathways, and for
inhibition of .sup.125I-C5a binding to purified human
neutrophils.
[0066] Antibodies may also be selected by panning a library of
human scFv for those which bind C5 (Griffiths et. al., EMBO J.
12:725-734 (1993)). The specificity and activity of specific clones
can be assessed using known assays (Griffiths et. al.; Clarkson et.
al., Nature. 352: 642-648 (1991)). After a first panning step, one
obtains a library of phage containing a plurality of different
single chain antibodies displayed on phage having improved binding
for C5. Subsequent panning steps provide additional libraries with
higher binding affinities. When avidity effects are a problem,
monovalent phage display libraries may be used in which less than
20%, less than 10%, or less than 1% of the phage display more than
one copy of an antibody on the surface of the phage. Monovalent
display can be accomplished with the use of phagemid and helper
phage. Suitable phage include M13, fl and fd filamentous phage.
Fusion protein display with virus coat proteins is also known and
may be used in this invention.
[0067] MAb137-26, which binds both C5 and C5a with comparable
affinity, was further characterized. MAb 137-26 does not inhibit C5
activation, but inhibits with very high potency the binding of C5a
to C5aR on purified human neutrophils. Experiments demonstrating
these properties are further explained in the Examples below.
[0068] To screen for antibodies which bind to a particular epitope
on the antigen of interest (e.g., those which block binding of any
of the antibodies disclosed herein to C5), a routine cross-blocking
assay such as that described in Antibodies, A Laboratory Manual,
Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can
be performed. Alternatively, epitope mapping, e.g. as described in
Champe et al., J. Biol. Chem. 270:1388-1394 (1995), can be
performed to determine whether the antibody binds an epitope of
interest.
[0069] E. Making Other Inhibitors of the Invention
[0070] Other molecules suitable for use in the invention can be
isolated or screened from compound libraries by conventional means,
for example, by determining whether they bind to C5/C5a, and then
doing a functional screen to determine if they inhibit the activity
of C5b. An automated system for generating and screening a compound
library is described in U.S. Pat. Nos. 5,901,069 and 5,463,564.
More focused approaches involve a competitive screen against the
MAb 137-26, or making a three-dimensional model of the binding
site, and then making a family of molecules, which fit the model.
These are then screened for those with optimal binding
characteristics. In addition, other molecules may be identified by
competition assay, or a functional screen for inhibitors with the
same properties as MAb137-26.
[0071] F. Using the Inhibitors of the Invention
[0072] The molecules of the present invention can be administered
by any of a number of routes and are administered at a
concentration that is therapeutically effective in the indication
or for the purpose sought. To accomplish this goal, the antibodies
may be formulated using a variety of acceptable excipients known in
the art. Typically, the antibodies are administered by injection.
Methods to accomplish this administration are known to those of
ordinary skill in the art. It may also be possible to obtain
compositions which may be topically or orally administered, or
which may be capable of transmission across mucous membranes.
[0073] The dosage and mode of administration will depend on the
individual and the agent to be administered. The dosage can be
determined by routine experimentation in clinical trials or
extrapolation from animal models in which the antibody was
effective.
[0074] The antibodies of the present invention may be used in the
treatment of diseases and conditions mediated by excessive or
uncontrolled production of C5a. Evidence of the utility of the
inhibitors of the invention in treating these diseases and
conditions is set forth below.
Example 1
Reactivity of MAb137-26 with Human C5 and C5a
[0075] MAb 137-26 was tested for reactivity with purified human C5
and recombinant C5a (Sigma, St. Louis, Mo.). The procedures of the
ELISA are described above. MAb 137-26 binds to C5a in a
dose-dependent manner with high potency (FIG. 1). Another anti-G5
MAb 137-76, specific for the .beta.-chain of human C5, does not
bind C5a, because C5a resides on the .alpha.-chain of C5. An
isotype-matched irrelevant antibody used as a negative control also
does not react with C5a. On the other hand, both MAbs 137-26 and
137-76 bind to C5 (FIG. 2).
[0076] The affinity equilibrium constant and the binding kinetic
constants (association and dissociation) of MAb 137-26 with C5a and
C5 were also determined by a BIAcore instrument (Pharmacia
Biosensor AB. Uppsala, Sweden). All the binding measurements were
performed in HEPES-buffered saline (HBS) (10 mM HEPES, pH 7.4, 150
mM NaCl, 3.4 mM EDTA, 0.005% Surfactant P20) at 25.degree. C. To
measure the binding rate constants of C5 and C5a to MAb137-26,
rabbit anti-mouse IgG(Fc) antibodies were immobilized onto a CM5
sensorchip by amine coupling using N-hydroxysuccinimide and
N-ethyl-N'-(3-diethylaminopropyl)cardodiimide. MAb 37-26 was then
captured onto the coated sensorchip before the injection of C5 at
different concentrations. The data are summarized in Table 1.
MAb137-26 has a high binding affinity for both solution-phase C5a
and G5. The results also indicate that MAb137-26 binds to an
epitope shared by both G5a and G5.
TABLE-US-00001 TABLE 1 Kinetic Constants of C5 and C5a Binding to
MAb137-26 k.sub.on k.sub.off k.sub.D (M.sup.-1 s.sup.-1) (S.sup.-1)
(M) C5 1.42 .times. 10.sup.5 6.97 .times. 10.sup.-5 4.92 .times.
10.sup.-10 C5a 3.7 .times. 10.sup.6 2.25 .times. 10.sup.-4 6.09
.times. 10.sup.-11 k.sub.on, kinetic association constant
k.sub.off, kinetic dissociation constant k.sub.D, equilibrium
dissociation constant = k.sub.off/k.sub.on
Example 2
Complement-Mediated Hemolysis
[0077] To study the effects of MAb 137-26 on the activation of C5
in human serum, the antibody was tested for inhibition of hemolysis
mediated by the classical and the alternative complement
pathways.
[0078] For the classical pathway experiments, chicken RBCs
(5.times.10.sup.7 cells/ml) in gelatin/veronal buffered saline
(GVB.sup.++) containing 0.5 mM MgCI.sub.2 and 0.15 mM CaCI.sub.2
were sensitized with purified rabbit anti-chicken RBC
immunoglobulins at 8 .mu.g/ml (Inter-Cell Technologies, Hoperwell,
N.J.) for 15 minutes at 4.degree. C. The cells were then washed
with GVB.sup.++. The washed cells were re-suspended in the same
buffer at 1.7.times.10.sup.8 cells/ml. In each well of a
round-bottom 96-well microtest plate, 50 .mu.l of normal human
serum (5.2%) was mixed with 50 .mu.l of GVB.sup.++ of serially
diluted MAb137-26 or an anti-C2 MAb 175-62 as a positive control.
Then 30 .mu.l of the washed sensitized chicken RBCs suspension was
added to the wells containing the mixtures. Fifty microliters of
normal human serum (5.2%) was mixed with 80 .mu.l of GVB.sup.++ to
give the serum color background. An isotype-matched anti-HIV-1
gp120 MAb was used as negative control. The final human serum
concentration used was 2%. The mixture was incubated at 37.degree.
C. for 30 minutes. The plate was shaken on a microtest plate shaker
for 15 seconds. The plate was then centrifuged at 300.times.g for 3
minutes. Supernatants (80 .mu.l) were collected and transferred to
wells in a flat-bottom 96-well microtest plates for measurement of
OD at 405 nm by an ELISA plate reader. The percent inhibition of
hemolysis is defined as:
100.times.[(OD.sub.without MAb-OD.sub.serum color
background)-(OD.sub.with MAb-OD.sub.serum color
background)]/(OD.sub.without MAb-OD.sub.serum color
background).
[0079] FIG. 3 shows that MAb137-26 and the irrelevant control MAb
G3-519 do not inhibit the classical pathway hemolysis of sensitized
chicken RBCs, whereas the positive control, anti-C2 MAb 175-62,
effectively inhibits hemolysis. C2 is specifically involved in the
classical complement pathway.
[0080] For the alternative pathway, unsensitized rabbit RBCs were
washed three times with gelatin/veronal-buffered saline
(GVB/Mg-EGTA) containing 2 mM MgCI.sub.2 and 1.6 mM EGTA. EGTA at a
concentration of 10 mM was used to inhibit the classical pathway
(K. Whaley et al., in A. W. Dodds (Ed.), Complement: A Practical
Approach. Oxford University Press, Oxford, 1997, pp. 19-47). The
assay procedures are similar to those of the classical pathway
hemolysis assay described above. The final concentration of human
serum used in the assay was 10%. Anti-factor D MAb 166-32 was used
as positive control. The same isotype-matched irrelevant anti-HIV-1
gp120 MAb described above was used as negative control.
[0081] FIG. 4 shows that MAb137-26 does not inhibit the alternative
pathway hemolysis of unsensitized rabbit RBCs, whereas anti-factor
D MAb166-32 strongly inhibits the hemolysis. Factor D is specific
for the alternative complement pathway. The negative control
antibody has no effect.
[0082] Taken together, the results verify that MAb 137-26 does not
inhibit C5 activation, and therefore the formation of C5a and C5b-9
are not inhibited. MAb137-26 does not inhibit the activation of the
alternative and classical complement pathways.
Example 3
Inhibition of .sup.125I-C5a Binding to Purified Human Neutrophils
by MAb 137-26
[0083] Human neutrophils were purified from human whole blood and
diluted with Dextran T-500/saline solution. The mixture was
incubated at room temperature for about 20 minutes or until a
clearly defined surface layer appeared. This surface layer was
transferred to a 50-ml polypropylene centrifuge tube. Following
centrifugation, the cell pellet was suspended in 30 ml of cold PBSB
(1% BSA in PBS). The cell suspension was layered on top of 10 ml of
Histopaque-1077 (Sigma, St. Louis, Mo.) in a 50-ml polypropylene
centrifuge tube. Following another centrifugation, the cell pellet
was then re-suspended in 20 ml of cold 0.2% NaCl for 30 seconds to
lyse RBCs. Then, 20 ml of cold 1.6% NaCl were added to the cell
suspension, recentrifuged, and the neutrophils were resuspended in
PBSB. The neutrophils were kept on ice until being used for
.sup.125I-C5a binding.
[0084] MAb137-26 was serially diluted in 1.5 ml Eppendorf
centrifuged tubes with a binding buffer (1% BSA in RPMI1640 medium)
to give final concentrations ranging from 640 nM to 0.04 nM. Four
microliters of 4 nM .sup.125I-C5a (NEN Life Science Products, Inc.,
Boston, Mass.) were added to 36 .mu.l of diluted MAb 137-26 for
incubation at room temperature for 15 minutes. Purified recombinant
human C5a (Sigma, St. Louis, Mo. was used as positive control,
whereas an isotype-matched irrelevant monoclonal antibody was used
as negative control. For the maximum binding of .sup.125I-C5a, 36
.mu.l of the binding buffer without the antibodies or C5a was used
instead. Fifty microliters of the neutrophil suspension was added
to each tube for incubation on ice. At the end of the 40-minute
incubation period, the mixture from each tube was transferred to
the top of 800 .mu.l of a separation buffer (6% BSA in PBS) in
another Eppendorf tube. The tubes were than centrifuged at
2000.times.g for 3 minutes at room temperature. After the
supernatant was aspirated, the cell pellet was re-suspended in 0.5
ml of de-ionized water to lyse the cells. The cell lysate was then
mixed with 3 ml of Ultima Gold scintillation fluid (Packard
Instrument, Meriden, Conn.) for radioactive counting. The percent
inhibition of .sup.125I-C5a binding is defined as:
[Cpm.sub.max-Cpm.sub.bkg]-[Cpm.sub.ca-Cpm.sub.bkg]/[Cpm.sub.max-Cpm.sub.-
bkg].times.100
where: Cpm.sub.max=maximum count per minute without competing
agents; Cpm.sub.bkg=background cpm without addition of
.sup.125I-C5a; and Cpm.sub.ca=cpm with competing agents.
[0085] FIG. 5 shows the inhibition of the binding of radioiodinated
(.sup.125I)-human C5a to purified human neutrophils. MAb 137-26 is
more potent than unlabeled C5a in inhibiting the binding of
.sup.125I-C5a to purified human neutrophils. The dose for 50%
inhibition (ID50) for MAb 137-26 was 0.45 nM as compared to 30 nM
C5a.
Example 4
Mapping of the Binding Epitope of MAb137-26 on Human C5a by SPOTs
Peptides on Cellulose Membrane
[0086] The binding epitope of MAb 137-26 on human C5a was mapped by
a technique using SPOTs peptides synthesized by Sigma Genosys (the
Woodlands, Tex.). Overlapping peptides (12-mer) encompassing the
entire human C5a were synthesized on a cellulose membrane. In the
assay, the membrane was first treated with a blocking solution
TBSTB (10 mM Tris chloride, 250 mM sodium chloride, 1% bovine serum
albumin and 0.05% TWEEN.RTM. 20) for 1 hour at room temperature to
saturate all the non-specific binding sites. MAb 136-26 at 1
.mu.g/ml in the blocking solution was then added to the membrane
for 1 hour at room temperature. The membrane was then washed
thoroughly with a washing buffer TBST (10 mM Tris chloride, 250 mM
sodium chloride and 0.05% TWEEN.RTM. 20). The membrane was then
treated with HRP-conjugated goat anti-mouse IgG (Fc) antibody
(diluted 1:5,000 in the blocking buffer) (Jackson Immunoresearch,
West Grove, Pa.) for 1 hour at room temperature. The membrane was
then washed again. The binding of MAb137-26 to the individual SPOTs
peptides of C5a was detected by incubation with Supersignal West
Pico chemilluminescent substrate (Pierce, Rockford, Ill.). The
intensity of chemillumescence was then detected by exposure to
Kodak X-OMAT AR film (Rochester, N.Y.). FIG. 6 shows the sequence
of the epitope bound by MAb 137-26.
Example 5
An Ex Vivo Human Whole Blood Model for Studying Complement-Mediated
Inflammation: Effect of MAb137-26 on Neutrophil Activation by E.
coli and on Killing of Neisseria meningitides
[0087] In order to investigate the role of complement in the
complex inflammatory network, all potential cellular and
fluid-phase mediators need to be present and therefore are able to
interact simultaneously. For devising such an experiment condition
in vitro, human whole blood was used. In this model, lepirudin
(REFLUDAN.RTM.), a thrombin specific hirudin analogue, was used as
an anticoagulant instead of heparin. Unlike heparin, lepirudin does
not interfere with complement activation.
[0088] In this model system, MAb 137-26 blocked the inflammatory
effects of C5a formed as a result of complement activation by E.
coli. The antibody did not inhibit MAC-mediated killing of N.
meningitidis. Therefore, MAb 137-26 neutralizes C5a without
inhibiting C5 activation and the subsequent formation of MAC. This
is an important feature of the monoclonal antibodies of the present
invention.
[0089] Whole blood was collected in polypropylene tubes containing
lepirudin (50 .mu.g/ml). The anti-coagulated whole blood was
pre-incubated with PBS or anti-C5 inhibitors for 4 minutes at
37.degree. C. For the studies of CD11b expression and oxidative
burst on neutrophils, opsonized E. coli strain LE392 (ATCC 33572)
was added to the whole blood samples for 10 minutes at 37.degree.
C. The E. coli concentration was 1.times.10.sup.7/ml blood in the
CD11 b experiments and 1.times.10.sup.8/ml in the oxidative burst
experiments. The T-0 baseline sample was processed immediately.
After incubation, 100.mu. of samples was used for measurement of
CD11b expression on neutrophils by immunofluorocytometry. The
oxidative burst of activated neutrophils was measured using the
substrate dihydrorhodamine 123 and performed as described in the
Burst-test procedure (ORPEGEN Pharma, Heidelberg, Germany).
[0090] FIGS. 7A-7D depict the results of the flow cytometric assays
of neutrophil activation for CD11b expression and oxidative burst.
Anti-C5/C5a MAb137-26 inhibited effectively neutrophil activation
induced by E. coli in the human whole blood model of inflammation.
In these assays, MAb137-26 is more potent than anti-C5a MAb561 (Dr.
Jurg Kohl) and a peptidic C5aR antagonist (Dr. Stephen Taylor).
[0091] For the bactericidal assays, N. meningitidis H44/76-1 was
grown overnight on BHI-agar, subcultured and grown into log-phase
for 4 hours. 5000-10000 colony forming units (CFUs) were added to
1.1 ml of lepirudin anti-coagulated whole blood samples
preincubated for 5 minutes with PBS or antibody. At each time
period, 100 .mu.l of whole blood was seeded on microbiological
Petri dishes containing blood agar and incubated for 24 hours at
37.degree. C. Bacterial growth was expressed as CFU/100 .mu.l of
whole blood added. T-0 sample was obtained immediately after adding
the bacteria.
[0092] FIG. 8 show that MAb137-26 did not inhibit MAC-mediated
killing of Neisseria meningitides. In contrast, MAb137-30 inhibited
the killing of Neisseria meningitides by human whole blood. This
antibody inhibits C5 activation.
[0093] It should be understood that the terms and expressions used
in the foregoing sections are exemplary only and not limiting, and
that the scope of the invention is defined only in the claims which
follow, and includes all equivalents of the subject matter of those
claims.
Sequence CWU 1
1
7174PRTArtificial Sequencebinding epitope of MAb137-26 1Thr Leu Gln
Lys Lys Ile Glu Glu Ile Ala Ala Lys Tyr Lys His Ser1 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 Arg65 70
212PRTArtificial Sequencesynthetic peptide for mapping 2Asn Asn Asp
Glu Thr Cys Glu Gln Arg Ala Ala Arg1 5 10 312PRTArtificial
Sequencesynthetic peptide for mapping 3Glu Thr Cys Glu Gln Arg Ala
Ala Arg Ile Ser Leu1 5 10 412PRTArtificial Sequencesynthetic
peptide for mapping 4Glu Gln Arg Ala Ala Arg Ile Ser Leu Gly Pro
Arg1 5 10 512PRTArtificial Sequencesynthetic peptide for mapping
5Ala Ala Arg Ile Ser Leu Gly Pro Arg Cys Ile Lys1 5 10
612PRTArtificial Sequencesynthetic peptide for mapping 6Ile Ser Leu
Gly Pro Arg Cys Ile Lys Ala Phe Thr1 5 10 712PRTArtificial
Sequencesynthetic peptide for mapping 7Gly Pro Arg Cys Ile Lys Ala
Phe Thr Glu Cys Cys1 5 10
* * * * *