U.S. patent application number 10/419565 was filed with the patent office on 2003-10-23 for process for inhibiting complement activation via the alternative pathway.
Invention is credited to Brunden, Kurt R., Gupta-Bansal, Rekha, Parent, James B..
Application Number | 20030198636 10/419565 |
Document ID | / |
Family ID | 25440234 |
Filed Date | 2003-10-23 |
United States Patent
Application |
20030198636 |
Kind Code |
A1 |
Gupta-Bansal, Rekha ; et
al. |
October 23, 2003 |
Process for inhibiting complement activation via the alternative
pathway
Abstract
A process of inhibiting activation of complement via the
alternative pathway, including inhibiting the formation of
complement activation products via the alternative pathway, is
provided.
Inventors: |
Gupta-Bansal, Rekha;
(Twinsburg, OH) ; Brunden, Kurt R.; (Aurora,
OH) ; Parent, James B.; (Cleveland, OH) |
Correspondence
Address: |
Janet M. McNicholas, Ph.D.
McAndrews, Held & Malloy, Ltd.
34th Floor
500 W. Madison Street
Chicago
IL
60661
US
|
Family ID: |
25440234 |
Appl. No.: |
10/419565 |
Filed: |
April 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10419565 |
Apr 21, 2003 |
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09976649 |
Oct 12, 2001 |
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09976649 |
Oct 12, 2001 |
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09138723 |
Aug 24, 1998 |
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6333034 |
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09138723 |
Aug 24, 1998 |
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08918349 |
Aug 26, 1997 |
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Current U.S.
Class: |
424/139.1 |
Current CPC
Class: |
C07K 16/18 20130101;
A61P 29/00 20180101; A61K 38/00 20130101; A61P 37/06 20180101 |
Class at
Publication: |
424/139.1 |
International
Class: |
A61K 039/395 |
Claims
What is claimed is:
1. A process of inhibiting alternative complement activation
comprising inhibiting properdin-induced stabilization of C3
convertase.
2. The process of claim 1 wherein the properdin-induced
stabilization of C3 convertase is inhibited by inhibiting the
binding of properdin to C3b.
3. The process of claim 2 wherein the binding of properdin of C3b
is inhibited by exposing properdin to an effective amount of an
anti-properdin antibody.
4. The process of claim 3 wherein the C3b is in the form of
C3bBb.
5. The process of claim 3 wherein the anti-properdin antibody is a
monoclonal antibody.
6. The process of claim 3 wherein the C3b is located in the plasma
or interstitial fluid of a subject.
7. The process of claim 6 wherein the subject is a human
subject.
8. The process of claim 7 wherein the human subject's complement
has been activated.
9. The process of claim 8 wherein complement activation is via an
acute pathological injury such as myocardial infarction, acute
respiratory distress syndrome, burn injury, stroke, pancreatitis,
cardiopulmonary bypass, or ischemia/reperfusion injury.
10. The process of claim 8 wherein complement activation
contributes to a chronic condition such as multiple sclerosis,
rheumatoid arthritis, myasthenia gravis or Alzheimer's disease.
11. The process of claim 6 wherein the anti-properdin antibody is
administered into the plasma or interstitial fluid of the
subject.
12. A process of inhibiting the adverse effects of alternative
complement pathway activation in a subject comprising administering
to the subject an amount of an anti-properdin agent effective to
selectively inhibit formation of an alternative complement pathway
activation product.
13. The process of claim 12 wherein of the alternative complement
pathway activation product is MAC.
14. The process of claim 12 wherein the alternative complement
pathway activation product is C3a or C5a.
15. The process of claim 12 wherein the anti-properdin agent is an
anti-properdin antibody.
16. The process of claim 12 wherein the anti-properdin agent is an
antigen-binding fragment of an anti-properdin antibody.
17. The process of claim 12 wherein the anti-properdin agent is a
properdin-derived peptide.
18. The process of claim 12 wherein the anti-properdin agent lacks
the ability to activate Fc.gamma. receptors.
19. A process for inhibiting the adverse effects of classical
complement pathway activation in a subject in which the classical
complement pathway is initiated comprising administering to the
subject an amount of an anti-properdin agent effective to
selectively inhibit formation of an alternative complement pathway
activation product.
20. The process of claim 19 wherein the alternative complement
pathway activation product is MAC.
21. The process of claim 19 wherein the alternative complement
pathway activation product is C3a or C5a.
22. The process of claim 19 wherein the anti-properdin agent is an
anti-properdin antibody.
23. The process of claim 19 wherein the anti-properdin agent is an
antigen-binding fragment of an anti-properdin antibody.
24. The process of claim 19 wherein the anti-properdin agent is a
properdin-derived peptide.
25. The process of claim 19 wherein the anti-properdin agent lacks
the ability to activate Fc.gamma. receptors.
26. A process for inhibiting the adverse effects of classical
complement pathway activation in a subject in which the classical
complement pathway is initiated comprising administering to the
subject an amount of an agent that inhibits alternative pathway C3
convertase effective to selectively inhibit formation of an
alternative complement pathway activation product.
27. The process of claim 26 wherein the alternative complement
pathway activation product is MAC.
28. The process of claim 26 wherein the alternative complement
pathway activation product is C3a or C5a.
29. The process of claim 26 wherein the agent is an anti-properdin
antibody.
30. The process of claim 26 wherein the agent is an antigen-binding
fragment of an anti-properdin antibody.
31. The process of claim 26 wherein the agent is a
properdin-derived peptide.
32. The process of claim 26 wherein the agent lacks the ability to
activate Fc.gamma. receptors.
33. A process for performing a medical procedure on a subject
comprising: (a) passing circulating blood from a blood vessel of
the subject, through a conduit, and back to a blood vessel of the
subject, the conduit having a luminal surface comprising a material
capable of causing at least one of complement activation, platelet
activation, leukocyte activation, or platelet-leukocyte adhesion in
the subject's blood; and (b) introducing an anti-properdin agent
into the subject's bloodstream in an amount effective to reduce at
least one of complement activation, platelet activation, leukocyte
activation, or platelet-leukocyte adhesion resulting from passage
of the circulating blood through the conduit, wherein step (a)
occurs before and/or during and/or after step (b).
34. The process of claim 33 wherein the anti-properdin agent
reduces the alternative pathway-dependent conversion of complement
component C3 into complement components C3a and C3b.
35. The process of claim 33 wherein the anti-properdin agent
reduces the alternative pathway-dependent formation of C5b-C9.
36. The process of claim 33 wherein the anti-properdin agent
reduces the alternative pathway-dependent leukocyte activation.
37. The process of claim 33 wherein the anti-properdin agent
specifically binds to properdin and inhibits alternative pathway C3
convertase.
38. The process of claim 33 wherein the medical procedure is an
extracorporeal circulation procedure.
39. The process of claim 38 wherein the extracorporeal circulation
procedure is a cardiopulmonary bypass procedure.
40. The process of claim 33 wherein the anti-properdin agent is an
anti-properdin antibody.
41. The process of claim 33 wherein the anti-properdin agent is an
antigen-binding fragment of an anti-properdin antibody.
42. The process of claim 33 wherein the anti-properdin agent is a
properdin-derived peptide.
43. The process of claim 33 wherein the anti-properdin agent lacks
the ability to activate Fc.gamma. receptors.
44. An article of manufacture comprising packaging material and a
pharmaceutical agent contained within the packaging material,
wherein: (a) the pharmaceutical agent comprises an anti-properdin
agent, the anti-properdin agent being effective for reducing at
least one of complement activation, platelet activation, leukocyte
activation, or platelet adhesion caused by passage of circulating
blood from a blood vessel of a subject, through a conduit, and back
to a blood vessel of the subject, the conduit having a luminal
surface comprising a material capable of causing at least one of
complement activation, platelet activation, leukocyte activation,
or platelet-leukocyte adhesion in the subject's blood; and (b) the
packaging material comprises a label which indicates that the
pharmaceutical agent is for use in association with an
extracorporeal circulation procedure.
45. The article of manufacture of claim 44 wherein the label
indicates that the pharmaceutical agent is for use in association
with a cardiopulmonary bypass procedure.
46. Use of an anti-properdin agent in the preparation of a
medicament for selectively inhibiting formation of an alternative
complement pathway activation product in a subject.
47. The use of claim 46 wherein the alternative complement pathway
activation product is MAC.
48. The use of claim 46 wherein the alternative complement pathway
activation product is C3a or C5a.
49. The use of claim 46 wherein the anti-properdin agent is an
anti-properdin antibody.
50. The use of claim 46 wherein the anti-properdin agent is an
antigen-binding fragment of an anti-properdin antibody.
51. The use of claim 46 wherein the anti-properdin agent is a
properdin-derived peptide.
52. The use of claim 46 wherein the anti-properdin agent lacks the
ability to activate Fc.gamma. receptors.
53. Use of an anti-properdin agent in the preparation of a
medicament for selectively inhibiting formation of an alternative
complement pathway activation product in a subject in which the
classical complement pathway is initiated.
54. The use of claim 53 wherein the alternative complement pathway
activation product is MAC.
55. The use of claim 53 wherein the alternative complement pathway
activation product is C3a or C5a.
56. The use of claim 53 wherein the anti-properdin agent is an
anti-properdin antibody.
57. The use of claim 53 wherein the anti-properdin agent is an
antigen-binding fragment of an anti-properdin antibody.
58. The use of claim 53 wherein the anti-properdin agent is a
properdin-derived peptide.
59. The process of claim 53 wherein the anti-properdin agent lacks
the ability to activate Fc.gamma. receptors.
60. Use of an alternative pathway C3 convertase-inhibiting agent in
the preparation of medicament for selectively inhibiting formation
of an alternative complement pathway activation product in a
subject in which the classical complement pathway is initiated.
61. The use of claim 60 wherein the alternative complement pathway
activation product is MAC.
62. The use of claim 60 wherein the alternative complement pathway
activation product is C3a.
63. The use of claim 60 wherein the agent is an anti-properdin
antibody.
64. The use of claim 60 wherein the agent is an antigen-binding
fragment of an anti-properdin antibody.
65. The use of claim 60 wherein the agent is a properdin-derived
peptide.
66. The use of claim 60 wherein the agent lacks the ability to
activate Fc.gamma. receptors.
67. A pharmaceutical composition comprising (a) an anti-properdin
antibody or an antigen-binding fragment of an anti-properdin
antibody that (1) selectively inhibits formation of an alternative
complement pathway activation product; and (2) does not
substantially activate Fc.gamma. receptors, and (b) a
pharmaceutically acceptable carrier.
68. The pharmaceutical composition of claim 67 wherein the
alternative complement pathway activation product is MAC.
69. The pharmaceutical composition of claim 67 wherein the
alternative complement pathway activation product is C3a or
C5a.
70. The pharmaceutical composition of claim 67 wherein the
anti-properdin antibody is a monoclonal anti-properdin
antibody.
71. The pharmaceutical composition of claim 70 wherein the
monoclonal anti-properdin antibody has the human .gamma.4 IgG
isotype.
72. The pharmaceutical composition of claim 67 wherein the
antigen-binding fragment of the anti-properdin antibody is
(Fab).sub.2.
73. An anti-properdin agent comprising an anti-properdin antibody
or an antigen-binding fragment of an anti-properdin antibody that
(a) selectively inhibits formation of an alternative complement
pathway activation product, and (b) does not substantially activate
Fc.gamma. receptors.
74. The anti-properdin agent of claim 73 wherein the alternative
complement pathway activation product is MAC.
75. The anti-properdin agent of claim 73 wherein the alternative
complement pathway activation product is C3a or C5a.
76. The anti-properdin agent of claim 73 wherein the anti-properdin
agent is a monoclonal antibody.
77. The anti-properdin agent of claim 76 wherein the monoclonal
anti-properdin antibody has the human .gamma.4 IgG isotype.
78. The anti-properdin agent of claim 73 wherein the
antigen-binding fragment of the anti-properdin antibody is
(Fab).sub.2.
79. A method of screening for and selecting an anti-properdin agent
comprising (a) assaying the agent for its ability to inhibit
formation of an alternative complement pathway activation product;
(b) assaying the agent for its ability to activate Fc.gamma.
receptors; and (c) selecting the agent that inhibits the formation
of an alternative complement pathway activation product and does
not substantially activate Fc.gamma. receptors.
80. The method of claim 79 wherein the alternative complement
pathway activation product is MAC.
81. The method of claim 79 wherein the alternative complement
pathway activation product is C3a or C5a.
82. The method of claim 79 wherein the anti-properdin agent is a
monoclonal antibody.
83. The method of claim 82 wherein the monoclonal anti-properdin
antibody has the human .gamma.4 IgG isotype.
84. The method of claim 79 wherein the antigen-binding fragment of
the anti-properdin antibody is (Fab).sub.2.
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 08/918,349 filed Aug. 26, 1997, incorporated
herein by reference.
TECHNICAL FIELD OF THE INVENTION
[0002] The field of this invention is complement activation. More
particularly, the present invention pertains to a process for
inhibiting complement activation via the alternative pathway,
including for inhibiting the formation (i.e., generation or
production) of complement activation products via the alternative
pathway.
BACKGROUND OF THE INVENTION
[0003] The complement system provides an early acting mechanism to
initiate and amplify the inflammatory response to microbial
infection and other acute insults. (Liszewski, M. K. and J. P.
Atkinson, 1993, In Fundamental Immunology, Third Edition. Edited by
W. E. Paul. Raven Press, Ltd. New York). While complement
activation provides a valuable first-line defense against potential
pathogens, the activities of complement that promote a protective
inflammatory response can also represent a potential threat to the
host. (Kalli, K. R., P. Hsu, and D. T. Fearon, 1994, Springer Semin
Immunopathol. 15:417-431; Morgan, B. P., Eur. J. Clinical Investig.
24:219-228). For example, C3 and C5 proteolytic products recruit
and activate neutrophils. These activated cells are indiscriminate
in their release of destructive enzymes and may cause organ damage.
In addition, complement activation may cause the deposition of
lytic complement components on nearby host cells as well as on
microbial targets, resulting in host cell lysis. The growing
recognition of the importance of complement-mediated tissue injury
in a variety of disease states underscores the need for effective
complement inhibitory drugs. No approved drugs that inhibit
complement damage currently exist.
[0004] Complement can be activated through either of two distinct
enzymatic cascades, referred to as the classical and alternative
pathways. (Liszewski, M. K. and J. P. Atkinson, 1993, In
Fundamental Immunology, Third Edition. Edited by W. E. Paul. Raven
Press, Ltd. New York). The classical pathway is usually triggered
by antibody bound to a foreign particle and thus requires prior
exposure to that particle for the generation of specific antibody.
There are four plasma proteins specifically involved in the
classical pathway: C1, C2, C4 and C3. The interaction of C1 with
the Fc regions of IgG or IgM in immune complexes activates a C1
protease that can cleave plasma protein C4, resulting in the C4a
and C4b fragments. C4b can bind another plasma protein, C2. The
resulting species, C4b2, is cleaved by the C1 protease to form the
classical pathway C3 convertase, C4b2a. Addition of the C3 cleavage
product, C3b, to C3 convertase leads to the formation of the
classical pathway C5 convertase, C4b2a3b.
[0005] In contrast to the classical pathway, the alternative
pathway is spontaneously triggered by foreign or other abnormal
surfaces (bacteria, yeast, virally infected cells, or damaged
tissue) and is therefore capable of an immediate response to an
invading organism (Liszewski, M. K. and J. P. Atkinson, 1993, In
Fundamental Immunology, Third Edition. Edited by W. E. Paul. Raven
Press, Ltd. New. York). There are four plasma proteins directly
involved in the alternative pathway: C3, factors B and D, and
properdin (also called factor P). The initial interaction that
triggers the alternative pathway is not completely understood.
However, it is thought that spontaneously activated C3 [sometimes
called C3(H.sub.2O)] binds factor B, which is then cleaved by
factor D to form a complex [C3(H.sub.2O)Bb] with C3 convertase
activity. The resulting convertase proteolytically modifies C3,
producing the C3b fragment, which can covalently attach to the
target and then interact with factors B and D and form the
alternative pathway C3 convertase, C3bBb. The alternative pathway
C3 convertase is stabilized by the binding of properdin. Properdin
extends its half-life six-to ten-fold (Liszewski, M. K. and J. P.
Atkinson, 1993, In Fundamental Immunology, Third Edition. Edited by
W. E. Paul. Raven Press, Ltd. New York). However, properdin binding
is not required to form a functioning alternative pathway C3
convertase (Schreiber, R. D., M. K. Pangburn, P. H. Lesavre and H.
J. Muller-Eberhard, 1978, Proc. Natl. Acad. Sci. USA 75:3948-3952;
Sissons, J. G., M. B. Oldstone and R. D. Schreiber, 1980, Proc.
Natl. Acad. Sci. USA 77:559-562). Since the substrate for the
alternative pathway C3 convertase is C3, C3 is therefore both a
component and a product of the reaction. As the C3 convertase
generates increasing amounts of C3b, an amplification loop is
established (Liszewski, M. K. and J. P. Atkinson, 1993, In
Fundamental Immunology, Third Edition. Edited by W. E. Paul. Raven
Press, Ltd. New York). In as much as the classical pathway also may
generate C3b, that C3b can bind factor B and thereby engage the
alternative pathway. This allows more C3b to deposit on a target.
For example, as described above, the binding of antibody to antigen
initiates the classical pathway. If antibodies latch on to
bacteria, the classical pathway generates C3b, which couples to
target pathogens. However, it has been suggested that from 10% to
90% of the subsequent C3b deposited may come from the alternative
pathway (Liszewski, M. K. and J. P. Atkinson, 1993, In Fundamental
Immunology, Third Edition. Edited by W. E. Paul. Raven Press, Ltd.
New York). The actual contribution of the alternative pathway to
the formation of additional C3b subsequent to classical pathway
initiation has not been clearly quantified and thus remains
unknown. Addition of C3b to the C3 convertase leads to the
formation of the alternative pathway C5 convertase, C3bBbC3b.
[0006] Both the classical and alternative pathways converge at C5,
which is cleaved to form products with multiple proinflammatory
effects. The converged pathway has been referred to as the terminal
complement pathway. C5a is the most potent anaphylatoxin, inducing
alterations in smooth muscle and vascular tone, as well as vascular
permeability. It is also a powerful chemotaxin and activator of
both neutrophils and monocytes. C5a-mediated cellular activation
can significantly amplify inflammatory responses by inducing the
release of multiple additional inflammatory mediators, including
cytokines, hydrolytic enzymes, arachidonic acid metabolites and
reactive oxygen species. C5 cleavage leads to the formation of
C5b-9, also known as the membrane attack complex (MAC). There is
now strong evidence that MAC may play an important role in
inflammation in addition to its role as a lytic pore-forming
complex (Liszewski, M. K. and J. P. Atkinson, 1993, In Fundamental
Immunology, Third Edition. Edited by W. E. Paul. Raven Press, Ltd.
New York).
[0007] Complement activation has been implicated as contributing to
a variety of disease states and conditions, as well as
complications from a variety of medical procedures (see references
cited infra) such as: myocardial infarction; ischemia/reperfusion
injury; stroke; acute respiratory distress syndrome (ARDS); sepsis;
burn injury; complications resulting from extracorporeal
circulation (ECC) including most commonly from cardiopulmonary
bypass (CPB) but also from hemodialysis or plasmapheresis or
plateletpheresis or leukophereses or extracorporeal membrane
oxygenation (ECMO) or heparin-induced extracorporeal LDL
precipitation (HELP); use of radiographic contrast media;
transplant rejection; rheumatoid arthritis; multiple sclerosis;
myasthenia gravis; pancreatitis; and Alzheimer's disease. There is
still no effective complement inhibitory drug available for routine
clinical use despite the significant medical need for such
agents.
[0008] The ability to specifically inhibit only the pathway causing
a particular pathology without completely shutting down the immune
defense capabilities of complement would be highly desirable. Based
upon the available clinical data, it appears that in most acute
injury settings, complement activation is mediated predominantly by
the alternative pathway (Moore, F. D. 1994, Advan. Immunol.
56:267-299; Bjornson, A. B., S. Bjornson and W. A. Altemeier, 1981
Ann. Surg. 194:224-231: Gelfand, J. A., M. Donelan, and J. F.
Burke, 1983, Ann. Surg. 198:58-62). These findings suggest that it
would be advantageous to specifically inhibit alternative
pathway-mediated tissue damage in a variety of acute injury
settings, for example, in myocardial infarction, ARDS, reperfusion
injury, stroke, thermal burns, and post-cardiopulmonary bypass
inflammation. This would leave the classical pathway intact to
handle immune complex processing and to aid in host defense against
infection. As essential components of the alternative pathway,
factors B and D are attractive targets for specific inhibition of
the alternative pathway. Because of its non-essential role,
properdin, however, would not be expected to be a suitable target
for such intervention.
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention provides a process of inhibiting
alternative pathway complement activation. The process includes the
step of inhibiting properdin-induced stabilization of C3
convertase. Properdin-induced stabilization of C3 convertase is
inhibited by inhibiting the binding of properdin to C3b or C3bBb.
The binding of properdin to C3b is inhibited by exposing properdin
to an effective amount of an antibody against properdin.
[0010] A process of the present invention is particularly useful in
inhibiting complement activation via the alternative pathway in
vivo in subjects, including humans, suffering from an acute or
chronic pathological injury such as, but not limited to, myocardial
infarction, acute respiratory distress syndrome, burn injury,
stroke, multiple sclerosis, rheumatoid arthritis, Alzheimer's
disease or ischemia/reperfusion injury. In vivo inhibition of
complement activation is accomplished by administering the
anti-properdin antibody to the subject. Pharmaceutical compositions
containing anti-properdin antibodies are also provided.
[0011] The present invention provides, in one aspect, a process of
inhibiting the adverse effects of alternative complement pathway
activation in a subject by administering to the subject an amount
of an anti-properdin agent effective to selectively inhibit
formation (i.e., generation or production) of a complement
activation product via the alternative complement pathway.
Formation of such alternative pathway-dependent activation products
refers to the generation or production of such products by
complement activation, which products when generated or produced
can be detected and include alternative pathway-dependent C3a, C5a,
and/or C5b-9 (MAC) products. An anti-properdin agent according to
the invention blocks properdin as described herein and selectively
inhibits the formation of alternative complement pathway activation
products. Such agents include an anti-properdin antibody, an
antigen-binding fragment of an anti-properdin antibody, and a
properdin-derived peptide. Preferably, the anti-properdin agent
does not substantially activate Fc.gamma. receptors and/or the
classical complement pathway.
[0012] The present invention provides, in another aspect, a process
for inhibiting the adverse effects of classical complement pathway
activation in a subject in which the classical complement pathway
is initiated by administering to the subject an amount of an
anti-properdin agent effective to selectively inhibit formation of
an alternative complement pathway activation product (e.g.,
alternative pathway-dependent C3a, C5a, MAC).
[0013] The present invention provides, in another aspect, a process
for inhibiting the adverse effects of classical complement pathway
activation in a subject in which the classical complement pathway
is initiated by administering to the subject an amount of an agent
that inhibits the alternative pathway C3 convertase effective to
selectively inhibit formation of a complement activation product
via the alternative complement pathway (e.g. alternative
pathway-dependent C3a, C5a, MAC).
[0014] The present invention, in another aspect, provides a process
for performing a medical procedure on a subject comprising: (a)
passing circulating blood from a blood vessel of the subject,
through a conduit and back to a blood vessel of the subject, the
conduit having a luminal surface comprising a material capable of
causing at least one of complement activation, platelet activation,
leukocyte activation, or platelet-leukocyte adhesion in the
subject's blood; and (b) introducing an anti-properdin agent into
the subject's bloodstream in an amount effective to reduce at least
one of complement activation, platelet activation, leukocyte
activation, or platelet-leukocyte adhesion resulting from passage
of the circulating blood through the conduit, wherein step (a)
occurs before and/or during and/or after step (b). Preferably, the
anti-properdin agent reduces the alternative pathway-dependent
conversion of complement component C3 into complement components
C3a and C3b, and/or the alternative pathway-dependent formation of
C5b-C9, and/or the alternative pathway-dependent leukocyte
activation. Medical procedures including therapeutic procedures
according to the invention include extracorporeal circulation
procedures, including for example, cardiopulmonary bypass (CPB)
procedures.
[0015] The present invention provides, in another aspect, an
article of manufacture comprising packaging material and a
pharmaceutical agent (i.e., pharmaceutical composition) contained
within the packaging material, wherein: (a) the pharmaceutical
agent comprises an anti-properdin agent, the anti-properdin agent
being effective for reducing at least one of complement activation,
platelet activation, leukocyte activation, or platelet adhesion
caused by passage of circulating blood from a blood vessel of a
subject, through a conduit, and back to a blood vessel of the
subject, the conduit having a luminal surface comprising a material
capable of causing at least one of complement activation, platelet
activation, leukocyte activation, or platelet-leukocyte adhesion in
the subject's blood; and (b) the packaging material comprises a
label which indicates that the pharmaceutical agent is for use in
association with an extracorporeal circulation procedure. Articles
of manufacture according to the invention include labels that
indicate that the anti-properdin agents are for use in association
with a cardiopulmonary bypass procedure.
[0016] The invention provides a use of an anti-properdin agent in
the preparation of a medicament for selectively inhibiting
formation of complement activation products via the alternative
complement pathway in a subject in need thereof. Also provided is a
use of an anti-properdin agent in the preparation of a medicament
for selectively inhibiting formation of complement activation
products via the alternative complement pathway in a subject in
which the classical complement pathway is initiated. Additionally
provided is a use of an alternative pathway C3
convertase-inhibiting agent in the preparation of a medicament for
selectively inhibiting formation of complement activation products
via the alternative complement pathway in a subject in which the
classical complement pathway is initiated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] In the drawings, which form a portion of the
specification:
[0018] FIG. 1 shows binding of human properdin to C3b.
[0019] FIG. 2 shows the dose-dependent inhibition of properdin
binding to C3b using an anti-properdin monoclonal antibody.
[0020] FIG. 3 shows the dose-dependent inhibition of properdin
binding to C3bBb using an anti-properdin monoclonal antibody.
[0021] FIG. 4 shows the effects of human serum containing
complement components on the deposition of the membrane attack
complex (MAC).
[0022] FIG. 5 shows the inhibition of membrane attack complex (MAC)
deposition caused by an anti-properdin monoclonal antibody.
[0023] FIG. 6 shows the effects on an anti-properdin monoclonal
antibody on rabbit erythrocyte lysis.
[0024] FIG. 7 shows the inhibition of the formation of alternative
complement pathway activation products, including C3a and MAC,
using an anti-properdin monoclonal antibody in a tubing loop model
of cardiopulmonary bypass (CPB).
[0025] FIG. 8 shows the lack of Fc.gamma. receptor activation as
detected by lack of superoxide generation, using an F(ab).sub.2
fragment preparation of an anti-properdin monoclonal antibody with
properdin.
[0026] FIG. 9 shows the inhibition of the formation of alternative
complement pathway activation products, including MAC, initiated by
heparin-protamine complexes using an anti-properdin monoclonal
antibody.
[0027] FIG. 10 shows the inhibition of the formation of alternative
complement pathway activation products, including MAC, initiated by
ovalbumin/anti-ovalbumin immune complexes using an anti-properdin
monoclonal antibody.
[0028] FIG. 11 shows the inhibition of the formation of alternative
pathway-dependent complement and leukocyte activation products,
including inhibition of C3a, MAC or elastase-antitrypsin complex
product formation, using an anti-properdin agent in an ex vivo
model of cardiopulmonary bypass (CPB).
DETAILED DESCRIPTION OF THE INVENTION
[0029] I. The Invention
[0030] Properdin is one of three unique plasma proteins that are
directly involved in the alternative pathway. Together with factor
D and factor B, all three are potential targets for the development
of therapeutic agents to inhibit the alternative pathway. As set
forth below, properdin is shown for the first time herein to be a
suitable target for a process of inhibiting complement activation
via the alternative pathway. Properdin is also shown for the first
time herein to be a suitable target even when the classical
complement pathway has been initiated.
[0031] Factor D is a serine proteinase with only a single known
natural substrate: factor B bound to C3b (Volanakis, J. E., S. R.
Barnum, and J. M. Kilpatrick, 1993, Methods in Enzymol. 223:82-97).
The serum concentration of factor D, 2 .mu.g/ml, is the lowest of
any complement protein (Liszewski, M. K. and J. P. Atkinson, 1993,
In Fundamental Immunology, Third Edition. Edited by W. E. Paul.
Raven Press, Ltd. New York). Factor D is known to be the rate
limiting enzyme for the alternative pathway and therefore a
suitable target for therapeutic methods of inhibition complement
activation via the alternative pathway. We believe, however, there
are several reasons why inhibitors of factor D may not be ideal
therapeutic agents for inhibiting complement activation. Firstly,
serine proteinases are also critically involved in the coagulation
and fibrinolytic systems, and it has proven difficult to identify
specific inhibitors of factor D (Kilpatrick, J. M., 1996, IBC's
Second Annual Conference on Controlling the Complement System for
Novel Drug Development, Conference Binder; Whitty, A., 1996, IBC's
Second Annual Conference on Controlling the Complement System for
Novel Drug Development, Conference Binder). Secondly, factor D is a
small protein (24.4 kDa) and is rapidly reabsorbed, and catabolized
by the kidney with a fractional catabolic rate of 60% per hour. The
steady state serum concentration of factor D is maintained by a
correspondingly high rate of synthesis. Therefore, it may be
difficult to inhibit factor D activity in patient serum for
prolonged periods without complicated drug dosing regimes. Thirdly,
factor D is synthesized by adipocytes and there is evidence from
studies with genetically obese mice that factor D may have a
regulatory role in fat metabolism (White, R. T., D. Damm, N.
Hancock, B. S. Rosen, B. B. Lowell, P. Usher, J. S. Flier, and B.
M. Speigelman., 1992, J. Biol. Chem. 267:9210-9213). Therefore,
inhibition of factor D may have detrimental secondary effects on
patients that are not directly related to complement
inhibition.
[0032] Factor B plays a key role in the alternative pathway since
it provides the catalytic subunit, Bb, for the C3 convertase, C3bBb
(Liszewski, M. K. and J. P. Atkinson, 1993, In Fundamental
Immunology, Third Edition. Edited by W. E. Paul. Raven Press, Ltd.
New York). Therefore, factor B also would appear to be another
suitable target for therapeutic methods of inhibiting complement
activation via the alternative pathway. Alone, factor B is a
zymogen with no known catalytic activity, but after binding C3b,
the factor B serine proteinase can be activated by cleavage by
factor D. Based on this, it should be possible to develop specific
inhibitors of factor Bb catalytic activity as therapeutic agents to
inhibit the alternative pathway. However, similar to the experience
with factor D, it has proven difficult to identify inhibitors of
factor Bb proteinase activity that do not also inhibit serine
proteinases involved in blood coagulation hemostasis (Whitty, A.,
1996, IBC's Second Annual Conference on Controlling the Complement
System for Novel Drug Development, Conference Binder). In any case,
we believe factor B is probably not the best target for the
development of therapeutic agents to inhibit the alternative
pathway. Factor B is an abundant serum protein (-210 .mu.g/ml)
(Clardy, C. W., 1994, Infect. Immun. 62:4539-4555; Liszewski, M. K.
and J. P. Atkinson, 1993, In Fundamental Immunology, Third Edition.
Edited by W. E. Paul. Raven Press, Ltd. New York) and it would
probably require a correspondingly high concentration of an
inhibitor of factor B to effectively block activation of the
alternative pathway. Monoclonal antibodies to human factor B,
however, have been prepared and tested for their in vitro ability
to block alternative complement pathway activation by endotoxin
(LPS) (Clardy, C. W. 1994, Infect Immun. 62:4539-4555). One of the
four monoclonal anti-factor B antibodies tested was able to
effectively block alternative pathway activation. The other three
antibodies tested failed to block despite having affinities that
were similar to the blocking antibody. In three other studies of
anti-factor B monoclonal antibodies that were cited by Clardy, et
al., supra (1994), two monoclonals increased factor B activity by
stabilizing the alternative pathway convertase, one increased
factor B activity by enhancing binding of B to C3b, three decreased
factor B activity by destabilizing the convertase and two decreased
factor B activity by blocking binding of factor B to C3b.
[0033] Properdin plays a role in the regulation of the alternative
pathway by virtue of its ability to bind and stabilize the
inherently labile C3 and C5 convertase complexes (C3bBb and
C3bBbC3b) (Nolan, K. F. and K. B. M. Reid, 1993, Methods Enzymol.
223:35-47), although the exact mechanism of C3 convertase
stabilization is unknown (Daoudaki, M. E., J. D. Becherer and J. D.
Lambris, 1988. J. Immunol. 140:1577-1580). Binding of properdin to
these complexes may result in a decreased rate of dissociation of
the Bb catalytic subunit and may also protect the complexes from
degradation by the negative regulatory proteins, factors I and H.
However, properdin is not required for functional C3 convertase
activity (Schreiber, R. D., M. K. Pangburn, P. H. Lesavre and H. J.
Muller-Eberhard, 1978, Proc. Natl. Acad. Sci. USA 75:3948-3952;
Sissons, J. G., M. B. Oldstone and R. D. Schreiber, 1980, Proc.
Natl. Acad. Sci. USA 77:559-562; Pangburn, M. K. and H. J.
Muller-Eberhard, 1984, Springer Semin. Immunopathol. 7:163-192).
The concentration of properdin in normal human plasma was
determined to be 4.3-5.7 .mu.g/ml, making it one of the least
abundant complement proteins (Nolan, K. F. and K. B. M. Reid, 1993,
Methods Enzymol. 223:35-47). In early studies, the plasma
concentration of properdin was reported to be in the 20-25 mg/ml
range; however, this estimate was based on an incorrect molar
extinction coefficient for the protein. It is now known that the
true plasma concentration of properdin is significantly lower
(Nolan, K. F. and K. B. M. Reid, 1993, Methods Enzymol. 223:35-47).
Human monocytes, neutrophils and T lymphocytes synthesize properdin
(Schwaeble, W., W. G. Dippold, M. K. Schafer, H. Pohla, D. Jones,
B. Luttig, E. Weihe, H. P. Huemer, M. P. Dierich, and K. B. M.
Reid, 1993, J. Immunol. 151:2521-2528; Schwaeble, W., U.
Wirthmuller, B. Dewald, M. Thelen, M. K. Schafer, P. Eggelton, K.
Whaley, and K. B. M. Reid, 1996, Molec. Immunol. 33(1):48;
Schwaeble, W., H. P. Huemer, J. Most, M. P. Dierich, M. Strobel, C.
Claus, K. B. M. Redi and H. W. Ziegler-Heitbrock, 1994, J. Eur.
Biochem. 219:759-764). Unlike most other complement proteins,
properdin does not appear to be synthesized by the liver. Properdin
is stored in granules of human neutrophils and
physiologically-relevant levels of TNF, Il-8, fMLP and C5a induce
its rapid secretion (Schwaeble, W., U. Wirthmuller, B. Dewald, M.
Thelen, M. K. Schafer, P. Eggelton, K. Whaley, and K. B. M. Reid,
1996, Molec. Immunol. 33(1):48). In a human monocyte cell line, TNF
and IL-1 enhanced both the abundance of properdin mRNA as well as
secretion of the protein (Schwaeble, W., H. P. Huemer, J. Most, M.
P. Dierich, M. Strobel, C. Claus, K. B. M. Redi and H. W.
Ziegler-Heitbrock, 1994, J. Eur. Biochem. 219:759-764).
[0034] According to the present invention, of the three complement
proteins that are uniquely involved in the alternative pathway,
properdin is the most attractive target for development of a
pathway-specific complement inhibitor to treat acute inflammatory
disorders. As demonstrated hereinafter, an anti-properdin antibody
that prevents binding of properdin to C3 convertase totally
inhibits activation of the alternative pathway. Therefore, as
described for the first time herein, properdin is required for
normal activation of the alternative pathway. Because properdin is
demonstrated herein to play a central role in complement
activation, including in conditions involving initiation of the
classical complement pathway, anti-properdin agents may be screened
and selected that are unexpectedly effective in processes of
selectively and potently inhibiting alternative complement pathway
activation, including processes for inhibiting the formation of
complement activation products via the alternative pathway.
[0035] II. Process of Inhibiting Alternative Pathway Activation of
Complement
[0036] In one aspect, a process of the present invention provides
for inhibition of complement activation via the alternative
pathway, including for inhibiting the formation of complement
activation products via the alternative pathway (e.g., MAC
formation).
[0037] It is not well understood how properdin interacts with C3
convertase, although the primary binding specificity of properdin
has been shown to be directed towards C3b, (Nolan, K. F. and K. B.
M. Reid, 1993, Methods Enzymol. 223:35-47). The properdin binding
site on C3b has been localized to residues 1402-1435 in the alpha
chain of C3, as judged by peptide inhibition studies (Daoudaki, M.
E., J. D. Becherer and J. D. Lambris, 1988. J. Immunol.
140:1577-1580). The analysis of overlapping peptides indicates that
the site could be further refined to residues 1424-1432 (Alzenz,
J., J. D. Becherer, I. Esparza, M. E. Daoudaki, D. Avita, S.
Oppermann, and J. D. Lambris, 1989, Complement Inflamm. 6:307-314).
There is also evidence that properdin binds to factor B and this
interaction appears to take place through sites on both the Ba and
Bb portions of the molecule. Although properdin binds cell-bound
C3b, the binding is significantly enhanced with cell-bound C3bBb,
suggesting that binding sites of both C3b and Bb may contribute to
the interaction of properdin with the convertase complex (Farries,
T. C., P. J. Lachmann, and R. A. Harrison, 1988, Biochem.
252:47-54). Thus, properdin binding to C3b can be inhibited whether
the C3b is unconjugated or conjugated to factor B to form C3bBb
(alternative pathway C3 convertase). As shown in detail hereinafter
in the Examples, anti-properdin antibodies can be screened and
identified that block properdin binding to both C3b and C3bBb.
[0038] In accordance with a process of the present invention,
properdin binding to C3b is inhibited by exposing properdin, in the
presence of C3b, to an effective amount of an anti-properdin
antibody, preferably a blocking antibody and most preferably a
blocking antibody that lacks the ability to active the Fc.gamma.
receptor upon binding to properdin, as described herein. Means for
determining an effective amount of an antibody are well known in
the art. The anti-properdin antibody can be a polyclonal or
monoclonal antibody. The use of monoclonal antibodies is preferred.
According to the invention, blocking antibodies against properdin
have been identified and can be obtained from commercial sources
(e.g., Quidel) or prepared using techniques well known in the art.
Anti-properdin agents that are effective according to the
invention, are preferably antibodies that selectively block
alternative pathway activation, including blocking formation of
complement activation products via the alternative pathway.
However, in addition to such blocking antibodies, other blocking
agents such as blocking peptides that are demonstrated to
substantially or totally inhibit the alternative pathway-dependent
formation of C3a, C5a and/or MAC after initiation of the
alternative pathway or classical pathway or both are similarly
contemplated by the invention as described herein.
[0039] Polyclonal antibodies against properdin can be prepared by
immunizing an animal with properdin or an immunogenic portion
thereof. Means for immunizing animals for the production of
antibodies are well known in the art. By way of example, a mammal
can be injected with an inoculum that includes properdin. Properdin
can be included in an inoculum alone or conjugated to a carrier
protein such as keyhole limpet hemocyanin (KLH). Properdin can be
suspended, as is well known in the art, in an adjuvant to enhance
its immunogenicity. Sera containing immunologically active
antibodies are then produced from the blood of such immunized
animals using standard procedures well known in the art.
[0040] The identification of antibodies that immunoreact
specifically with properdin is made by exposing sera suspected of
containing such antibodies to properdin to form a conjugate between
antibodies and properdin. The existence of the conjugate is then
determined using standard procedures well known in the art.
[0041] Properdin can also be used to prepare monoclonal antibodies
against properdin and used as a screening assay to identify such
monoclonal antibodies. Monoclonal antibodies are produced from
hybridomas prepared in accordance with standard techniques such as
that described by Kohler et al. (Nature, 256:495, 1975). Briefly, a
suitable mammal (e.g., BALB/c mouse) is immunized by injection with
properdin. After a predetermined period of time, splenocytes are
removed from the mouse and suspended in a cell culture medium. The
splenocytes are then fused with an immortal cell line to form a
hybridoma. The formed hybridomas are grown in cell culture and
screened for their ability to produce a monoclonal antibody against
properdin.
[0042] The inhibition of properdin binding to C3b is associated
with inhibition of complement activation via the alternative
pathway. As shown in detail hereinafter in the Examples,
anti-properdin agents, preferably anti-properdin antibodies not
only blocked properdin binding to C3b and C3bBb, but also blocked
formation of products of the alternative pathway, including C5b-9,
the Membrane-Attack Complex (MAC), which complex is the final
end-product of complement activation. Still further, the data in
the Examples show that anti-properdin agents, preferably
anti-properdin antibodies, also block alternative pathway-dependent
erythrocyte lysis mediated by MAC. Still further, the data in the
Examples show that anti-properdin agents, preferably anti-properdin
antibodies and their antigen-binding fragments such as F(ab).sub.2,
are effective in inhibiting alternative pathway complement
activation in models of cardiopulmonary bypass and in conditions of
classical pathway complement activation.
[0043] The present findings are surprising and unexpected in view
of the existing literature. For example, Schreiber et al.
demonstrated that the alternative pathway could be functionally
assembled by mixing all of the alternative pathway proteins except
properdin; it was concluded that properdin is not required for
alternative pathway initiation and amplification. (Schreiber, R.
D., M. K. Pangburn, P. H. Lesavre and H. J. Muller-Eberhard, 1978,
Proc. Natl. Acad. Sci. USA 75:3948-3952). Moreover, alternative
pathway activation initiated by measles virus-infected HeLa cells
in the absence of IgG was the same in the absence or presence of
properdin. (Sissons, J. G., M. B. Oldstone and R. D. Schreiber,
1980, Proc. Natl. Acad. Sci. USA 77:559-562). These prior findings
have led to the generally accepted hypothesis that "properdin is
not an essential component for the activation of the pathway, but
its presence does result in more rapid amplification of bound C3b"
[emphasis added] (Pangburn, M. K. and H. J. Muller-Eberhard, 1984,
Springer Semin. Immunopathol. 7:163-192). These references
effectively teach away from the identification of properdin as a
target for alternative pathway intervention. In contrast, according
to the present invention, properdin is now identified as an
essential component of and required for alternative complement
pathway activation. Thus, a process of the present invention
relates to the selective inhibition of alternative pathway by an
anti-properdin agent. Such an agent surprisingly and effectively
blocks the alternative complement cascade, including in conditions
involving initiation of the classical complement pathway.
[0044] III. Process of Treating Pathological Injury
[0045] The ability to inhibit complement activation using a process
of the present invention provides a therapeutic regimen for
treatment of patients having clinical symptoms in which complement
activation is deleterious. The complement system has been
implicated as contributing to the pathogenesis of numerous acute
and chronic disease states and conditions, as well as complications
from a variety of medical procedures, including myocardial
infarction (Moroko, P. R., C. B. Carpenter, M. Chiarello, M. C.
Fishbein, P. Radva, J. D. Knostman, and S. L. Hale, 1978, Lab
Invest. 48:43-47; Kilgore, K. S., G. S. Friedrichs, J. W.
Homeister, and B. R. Lucchesi, 1994, Cardiovasc. Res. 28:437-44;
Weisman, H. F., T. Bartow, M. K. Leppo, H. C. Marsh, G. R. Carson,
M. F. Concino, M. P. Boyle, K. H. Roux, M. L. Weisfeldt; and D. T.
Fearon, 1990, Science 249:146-151; Schafer, P. J., D. Mathey, F.
Hugo, and S. Bhaki, 1986, J. Immunol. 137:1945-1949), stroke
(Kaczorowski, S. L., J. K. Schiding, C. A. Toth and P. M. Kochanek,
1995, J. Cereb. Blood Flow Metab. 15:860-864; Morgan, B. P., P.
Gasque, S. K. Singhrao, and S. J. Piddlesden, 1997, Exp. Clin.
Immunogenet. 14: 19-23; Vasthare, U. S., R. H. Rosenwasser, F. C.
Barone, and R. F. Tuma, 1993, FASEB J. 7:A424-429) ARDS (Mulligan,
M. S., C. W. Smith, D. C. Anderson, R. F. Todd, M. Miyaska, T.
Tamatani, T. B. Issekuts, and P. A. Ward, 1993, J. Immunol.
150:2401-2406; Solomkin, J. S., L. A. Cotta, P. S. Satoh, J. M.
Hurst, and R. D. Nelson, 1985, Surgery 97:668-678; Mulligan, M. S.,
C. G. Yeh, A. R. Rudolph, and P. A. Ward, 1992, J. Immunol.
148:1479-1485; Anner, H., R. P. Kaufman, L Kobzik, C. R. Valeri, D.
Shepro, and H. B. Hechtman, 1987, Ann. Surgery 206:642-648; Zilow,
G., A. Sturm, U. Rother; and M. Kirschfink, 1990, Clin. Exp.
Immunol. 79:151-157), reperfusion injury (Hsu, P., R. Simpson, T.
F. Lindsay, T. Hebell, L. Kobzik, F. D. Moore, D. T. Fearon, and H.
B. Hechtman, 1993, Clin. Res. 41:233A; Lindsay, T. F., J. Hill, F.
Ortiz, A. Rudolph, C. R. Valeri, H. B. Hechtman, and F. D. Moore,
1992, Ann. Surg. 216: 677-683; Rubin, B. B., A. Smith, S. Liauw, D.
Isenman, A. D. Romaschin and P. M. Walker, 1990, Am. J. Physiol.
259:H525-H531; Hill, J., T. F. Lindsay, F. Ortiz, C G Yeh, H. B.
Hechtman, and F. D. Moore, 1992, J. Immunol 149:1723-1729;
Mulligan, M. S., E. Schmid, B. Beck-Schimmer, G. O. Till, H. P.
Friedl, R. B. Brauer, T. E. Hugli, M. Miyasaka, R. L. Warner, K. J.
Johnson, and P. A. Ward, 1996, J. Clin. Invest. 98:503-512;
Pemberton, M., G. Anderson, V. Vetvicka, D. E. Justus, and G. D.
Ross, 1993, J. Immunol. 150:5104-5113), sepsis/septic shock (Hack,
C. E., J. H. Nuijens, R. J. F. Felt-Bersma, W. O. Schreuder, A. J.
M. Eerenberg-Belmer, J. Paardekooper, W. Bronsveld, and L. G.
Thijs, 1989, Am J Med 86:20-26; Bengston, A., and M. Heideman,
1988, Arch. Surg. 23:645:649; Stevens, J. H., P. O'Hanley, J. M.
Shapiro, F. G. Mihn, P. S. Satoh, J. A. Collins and T. A. Raffin,
1986, J. Clin. Invest. 77:1812-1816; Wakabayashi, G., J. A.
Gelfand, W. K. Jung, R. J. Connolly, J. F. Burke, and C. A.
Dinarello, 1991, J. Clin. Invest. 87:1925-1935), thermal burns
(Solomkin, J. S., R. D. Nelson, D. E. Chenoweth, L. D. Solem and R.
L. Simmons, 1984, Ann. Surg. 200, 742-746; Bjornson, A. B., S.
Bjornson and W. A. Altemeier, 1981, Ann. Surg. 194:224-231;
Gelfand, J. A., M. Donelan, and J. F. Burke, 1983, Ann. Surg.
198:58-62; Gelfand, J. A., M. Donelan, and J. F. Burke, 1982, J.
Clin. Invest. 70:1170-1176), post-cardiopulmonary bypass
inflammation (Salama, A., F. Hugo, D. Heinrich, R. Hoge, R. Miller,
V. Keifel, C. Muller-Eckhardt, and S. Bakdi, 1988, N. Engl. J. Med.
318:408-414; Chenoweth, D. E., S. W. Cooper, T. E. Hugli, R. W.
Stewart, E. H. Blackstone and J. W. Kirklin, 1981, N. Engl. J. Med.
304:497-503; Moore, F. D., K. G. Warner, S. Assoussa, C. R. Valeri,
and S. F. Khuri, 1987, Ann. Surg. 208:95-103; Rinder, C. S., H. M.
Rinder, B. R. Smith, J. C. K. Fitch, M. J. Smith, J. B. Tracey, L.
A. Matis, S. P. Squinto, and S. A. Rollins, 1995, J. Clin Invest.
96:1564-1572), hemodialysis (Hakim, R. M., J. Breillatt, J. M.
Lazarus, and F. K. Port, 1984, New Eng. J. Med. 311:878-882), use
of radiographic contrast media (Arroyaue, C. M., and E. M. Tan,
1977, Clin. Exp. Immunol. 29:89-94), transplant rejection (Pruitt,
S. K., W. M. Baldwin, H. C. Marsh, S. Lin, C. G. Yeh, and R. R.
Bollinger, 1991, Transplantation 52:868-873; Leventhal, J. R., A.
P. Dalmasso, J. W. Cromwell, J. L. Platt, C. J. Bolman, and A. J.
Matas, 1993, Transplantation 55:857-865; Dalmasso, A. P., G. M.
Vercelloti, R. J. Fischel, R. J. Bolman, F. H. Bach and J. L.
Platt, 1992, Am. J. Pathol. 140:1157-1166; Xia, W., D. T. Fearon,
F. D. Moore, F. J. Schoen, F. Ortiz and R. L. Kirkman, 1992,
Transplant Proc. 24:479-480), rheumatoid arthritis (Mollnes, T. E.,
T. Lea, O. J. Mellbye, J. Pahle, O. Grand, and M. Harboe, 1986,
Arth. Rheum. 29:715-721; Morgan, B. P., R. H. Daniels, and B. D.
Williams, 1988, Clin. Exp. Immunol. 73:473-478), multiple sclerosis
(Linington, C., B. P. Morgan, N. J. Scolding, S. Piddlesden, and P.
Wilkins, 1989, Brain. 112:895-911; Sanders, M. E., C. L. Koski, D.
Robbins, M. L. Shin, M. M. Frank, and K. A. Joiner, 1986, J.
Immunol. 135:4456), myasthenia gravis (Biesecker, G. and C. M.
Gomez, 1989, J. Immunol. 142:2654-9; Nakano, S., and A. G. Engel.
1993 Neurology 43:1167-72), pancreatitis (Roxvall, L., A. Bengtson,
and M. Heideman, 1989, J. Surg. Res. 47:138-143; Roxvall, L., A.
Bentston, and M. Heidman, 1990 Arch. Surg. 125:918-921) and
Alzheimer's disease (Eikelenboom, P., C. E. Hack, J. M. Rozemuller,
and F. C. Stam, 1989, Virchows Arch. (Cell pathol.) 56:259-62;
Rogers, J. N. R. Cooper, and S. Webster, 1992, Proc. Natl. Acad.
Sci. USA 89:10016-20). While complement may not be the only cause
of the pathogenesis in these conditions, it is nevertheless a major
pathological mechanism and represents an effective point for
clinical control in many of these disease states.
[0046] Complement activation products have been detected in
biological fluids or diseased tissues isolated from patients with
many of the aforementioned conditions, and a correlation between
the severity of the clinical indication with the abundance of
complement activation products has been demonstrated for some
diseases (Zilow, G., A. Sturm, U. Rother, and M. Kirschfink, 1990,
Clin. Exp. Immunol. 79:151-157; Hack, C. E., J. H. Nuijens, R. J.
F. Felt-Bersma, W. O. Schreuder, A. J. M. Eerenberg-Belmer, J.
Paardekooper, W. Bronsveld, and L. G. Thijs, 1989, Am. J. Med.
86:20-26; Gelfand, J. A., M. Donelan, and J. F. Burke, 1983, Ann.
Surg. 198:58-62). The most compelling evidence directly implicating
complement in the pathogenesis of a diverse group of human diseases
comes from studies using art accepted animal models of such
diseases. In such animal models, removal of classical
pathway-activating antibodies (Fischel, R. J., R. M. Bolman, J. L.
Platt, K. S. Naharian, F. H. Bach, and J. J. Matas, 1990, Trans.
Proc. 22:1077-1083; Platt, J. L., R. J. Fischel, A. J. Matas, S. A.
Reif, R. M. Bolman., and F. H. Bach, 1991, Transplantation
52:214-230), depletion of complement by cobra venom factor (Moroko,
P. R., C. B. Carpenter, M. Chiarello, M. C. Fishbein, P. Radva, J.
D. Knostman, and S. L. Hale, 1978, Lab. Invest. 48:43-47; Mulligan,
M. S., C. W. Smith, D. C. Anderson, R. F. Todd, M. Miyaska, T.
Tamatani, T. B. Issekuts, and P. A. Ward, 1993, J. Immunol.
150:2401-2406; Gelfand, J. A., M. Donelan, and J. F. Burke, 1983,
Ann. Surg. 198:58-62; Leventahal, J. R., A. P. Dalmasso, J. W.
Cromwell, J. L. Platt, C. J. Bolman, and A. J. Matas, 1993,
Transplantation 55:857-865), inhibition of complement activity
(Weisman, H. F., T. Bartow, M. K. Leppo, H. C. Marsh, G. R. Carson,
M. F. Concino, M. P. Boyle, K. H. Roux, M. L. Weisfeldt, and D. T.
Fearon, 1990, Science 249:146-151; Mulligan, M. S., C. G. Yeh, A.
R. Rudolph, and P. A. Ward, 1992, J. Immunol. 148:1479-1485;
Pemberton, M., G. Anderson, V. Vetvicka, D. E. Justus, and G. D.
Ross, 1993, J. Immunol. 150:5104-5113), or testing in animals
genetically deficient in specific complement components (Gelfand,
J. A., M. Donelan, and J. F. Burke, 1983, Ann. Surg. 198:58-62;
Watson, W. C., and A. C. Townes, 1985, J. Exp. Med. 162:1878-1883),
have all been shown to abrogate or delay pathogenesis.
[0047] Inherited deficiencies have been recognized in humans for
nearly every complement component (Liszewski, M. K. and J. P.
Atkinson, 1993, Fundamental Immunology, Third Edition. Edited by W.
E. Paul. Raven Press, Ltd. New York). Deficiencies of components of
the same pathway cause similar clinical problems. Classical pathway
component deficiencies (C1, C4, C2) commonly cause infections by a
variety of pyrogenic organisms and immune complex diseases, as does
deficiency of C3. Alternative pathway component deficiencies (P, D)
often results in Neisserial infections. There is no evidence that
properdin deficiency causes increased susceptibility to immune
complex disease or to infections with organisms other than
Neisseria. No homozygous deficiencies in Factor B have been
described (Morgan, B. P. and M. J. Walport, 1991, Immunology Today
12:301-306).
[0048] Inactivation of the alternative complement pathway is
particularly useful in subjects where activation is associated with
the pathological effects of disease states. As set forth
hereinbefore, complement activation is known to be associated with
a large and diverse group of disease states. Complement activation
via the alternative pathway is particularly prominent in states of
acute injury.
[0049] The complement-induced fulminant meningococcal septicemia in
patients with systemic meningococcal disease is likely caused
predominantly by activation of the alternative pathway (Brandtzaeg
et al., Journal of Infectious Disease, 173:647-55, 1996). In
patients with adult respiratory distress syndrome (ARDS),
complement activation occurred only via the alternative pathway for
the first 48 hours (Zilow et al., J. Exp. Immunology, 79 151-57,
1990). An agent that suppresses complement activation via both
pathways has been used to treat post-ischemic myocardial
inflammation and necrosis in animal models of cardiovascular
disease (Weisman et al., Science, 249:146-71, 1990). According to
Weisman et al., the acute tissue injury associated with numerous
autoimmune diseases is the result of complement activation.
Complement induced tissue injury is found in immune complex-induced
vasculitis, glomerulonephritis, hemolytic anemia, myasthenia
gravis, type II collagen-induced arthritis, and ischemia.
[0050] The role of the alternative complement pathway in inducing
ischemic cardiac damage during reperfusion has also been reported
(Amsterdam et al., Amer. J. Physiol., 268: H448-H457, 1995).
Mulligan et al. 1992, J. Immunol. 148:1479-1485 reported that
complement activation plays a role in a variety of tissue injuries
including glycogen-induced peritonitis, lung and dermal injury
after intra-alveolar or intra-dermal deposition of IgG immune
complexes, acute lung injury resulting from intravascular
activation of complement after injection of cobra venom factor, and
acute skin and lung injury after thermal trauma.
[0051] A variety of medical procedures utilize extracorporeal
circulation (ECC), including hemodialysis, plasmapheresis,
plateletpheresis, leukophereses, extracorporeal membrane
oxygenation (ECMO), heparin-induced extracorporeal LDL
precipitation (HELP) and most commonly cardiopulmonary bypass
(CPB). These procedures expose blood or blood products to foreign
surfaces that may alter normal cellular function and hemostasis.
For example, it is well-established that CPB often leads to complex
inflammatory responses that result in post-surgical complications,
generally termed "post-perfusion syndrome". Among these
postoperative events are respiratory failure, bleeding disorders,
renal dysfunction and, in the most severe cases, multiple organ
failure (Wan, S., J-L. LeClerc, and J-L. Vincent, 1997, Chest
112:676-692). The primary suspected cause of these CPB-related
problems is inappropriate activation of complement during the
bypass procedure (Chenoweth, K., S. Cooper, T. Hugli, R. Stewart,
E. Blackstone, and J. Kirklin, 1981, N. Engl. J. Med. 304:497-503;
P. Haslam, P. Townsend, and M. Branthwaite, 1980, Anaesthesia
25:22-26; J. K. Kirklin, S. Westaby, E. Blackstone, J. W. Kirklin,
K. Chenoweth, and A. Pacifico, 1983, J. Thorac. Cardiovasc. Surg.
86:845-857; Moore, F. D., K. G. Warner, B. A. Assousa, C. R.
Valeri, and S. F. Khuri, 1988, Ann. Surg. 208:95-103; J. Steinberg,
D. Kapelanski, J. Olson, and J. Weiler, 1993, J. Thorac.
Cardiovasc. Surg. 106:1901-1918). While it appears that blood
contact with the tubing and oxygenator surfaces of the CPB circuit
results in activation of the alternative complement pathway
(Chenoweth, K., S. Cooper, T, Hugli, R. Stewart, E. Blackstone, and
J. Kirklin, 1981, N. Engl. J. Med. 304:497-503; Velthuis, H., P. G.
M. Jansen, C. E. Hack, L. Eijsan, and C. R. H. Wildevuur, 1996,
Ann. Thorac. Surg. 61:1153-1157), there is also evidence that the
classical complement pathway is activated during CPB (Wachtfogel,
Y. T., P. C. Harpel, L. H. Edmunds, Jr. and R. W. Colman, 1989,
Blood 73:468-471). Moreover, the classical complement cascade is
initiated after the termination of CPB due to the addition of
protamine to a patient's blood. Protamine is utilized clinically to
bind and remove the heparin that is added as an anti-coagulant
during surgery. The heparin-protamine complexes cause significant
activation of the classical complement pathway (Steinberg, J., D.
Kapelanski, J. Olson, and J. Weiler, 1993 J. Thorac. Cardiovasc.
Surg. 106:1901-1918), further contributing to post-perfusion
syndrome.
[0052] Activated complement species, particularly the
anaphylotoxins C3a and C5a, are known to elicit a variety of
inflammatory responses from many cell types. For example, C5a can
up-regulate cell adhesion molecule expression on neutrophils, and
can also invoke lysosomal enzyme and free radical release from both
neutrophils and monocytes (Chenoweth, D. and T. Hugli, 1978, Proc.
Natl. Acad. Sci. USA 75:3943-3947; Fletcher, M. P., G. Stakl, and
J. Longhurst, 1993, Am. J. Physiol. 265:H1750-H1761). Likewise, C5a
can activate platelets, rendering them incapable of normal clotting
function (Foreman, K. E., A. A. Vaporciyan, B. K. Bonish, M. L.
Jones, K. J. Johnson, M. M. Glovsky, S. M. Eddy, and P. A. Ward,
1994, J. Clin. Invest. 94:1147-1155). Finally, the terminal
activated complement product, C5b-9 (membrane-attack complex), can
also affect platelet and endothelial cell function (Foreman, K. E.,
A. A. Vaporciyan, B. K. Bonish, M. L. Jones, K. L. Johnson, M. M.
Glovsky, S. M. Eddy, and P. A. Ward, 1994, J. Clin. Invest.
94:1147-1155; Hattori, R., K. K. Hamilton, R. D. Fugate, R. P.
McEver, and P. J. Sims, 1989, J. Biol. Chem. 264:9053-9060). It is
the actions of these complement species on neutrophils, platelets
and other circulatory cells that likely lead to the various
problems that arise after CPB.
[0053] Recently, there has been direct experimental evidence that
complement activation is, in fact, responsible for many of the
changes involving dysfunction of the immune and hemostatic systems
seen after CPB. Using soluble complement receptor type 1 (sCR1),
which prevents activation of both the classical and alternative
complement pathways, Gillinov, A. M., P. A. DeValeria, J. A.
Winkelstein, I. Wilson, W. E. Curtis, D. Shaw, C. G. Yeh, A. R.
Rudolph, W. A. Baumgartner, A. Herskowitz, and D. E. Cameron,
(1993, Ann. Thorac. Surg. 55:619-624) demonstrated that inhibiting
complement activation improved pulmonary vascular resistance in
pigs undergoing a CPB procedure. Utilizing an ex vivo model of
simulated CPB, Rinder et al. (1995, supra) showed that addition of
a monoclonal antibody to C5 significantly reduced the neutrophil
and platelet activation that occurred during the bypass procedure.
The. C5 antibody blocks the cleavage of C5 in both the classical
and alternative complement pathways, and thus prevents the
production of both the membrane-attack complex and the
anaphylotoxin, C5a. The use of such an anti-C5 monoclonal antibody
for reducing complement, platelet or leukocyte activation or
platelet--leukocyte adhesion resulting from passage of a patient's
blood via extracorporeal circulation is described in WO95/25540
[PCT/US95/03614].
[0054] IV. Pharmaceutical Compositions
[0055] A process of the present invention can thus be used to
inhibit complement activation via the alternative pathway,
including to inhibit the formation of complement activation
products via the alternative pathway, in patients by administering
to a patient in need of complement inactivation an effective
inhibiting amount of an anti-properdin agent, preferably an
anti-properdin antibody or antigen-binding domain thereof.
Preferably, the anti-properdin agent, most preferably an
anti-properdin antibody or antigen-binding domain thereof, is
administered in the form of a pharmaceutical composition.
[0056] Such a pharmaceutical composition comprises a
therapeutically effective amount of an anti-properdin agent,
preferably an anti-properdin antibody formulated together with one
or more non-toxic pharmaceutically acceptable carriers are
disclosed. As used herein, the term "pharmaceutically acceptable
carrier" means a non-toxic, insert solid, semi-solid or liquid
filler, diluent, encapsulating material or formulation auxiliary of
any type. Some examples of materials which can serve as
pharmaceutically acceptable carriers are sugars such as lactose,
glucose and sucrose; starches such as corn starch and potato
starch; cellulose and its derivatives such as sodium carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; powdered
tragacanth; malt; gelatin; talc; excipients such as cocoa butter
and suppository waxes; oils such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil;
glycols such as propylene glycol; esters such as ethyl oleate and
ethyl laurate; agar; buffering agents such as magnesium hydroxide
and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic
saline; Ringer's solution; ethyl alcohol; and phosphate buffer
solutions, as well as other non-toxic compatible lubricants such as
sodium lauryl sulfate and magnesium stearate, as well as coloring
agents, releasing agents, coating agents, sweetening, flavoring and
perfuming agents, preservatives and antioxidants can also be
present in the composition, according to the judgment of the
formulator.
[0057] The pharmaceutical compositions of this invention can be
administered to humans and other animals orally, rectally,
parenterally, intracisternally, intravaginally, intraperitoneally,
transdermally, topically (as by powders, ointments, or drops),
bucally, or as an oral or nasal spray.
[0058] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. In addition to the active
compounds, the liquid dosage forms may contain inert diluents
commonly used in the art such as, for example, water or other
solvents, solubilizing agents and emulsifiers such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
dimethylformamide, oils, (in particular, cottonseed, groundnut,
corn, germ, olive, castor, and sesame oils), glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid
esters of sorbitan, and mixtures thereof. Besides inert diluents,
the oral composition can also include adjuvants such as wetting
agents, emulsifying and suspending agents, sweetening, flavoring,
and perfuming agents.
[0059] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in preparation of injectables.
[0060] The injectables formulations can be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0061] In order to prolong the effect of a drug, it is often
desirable to slow the absorption of the drug from subcutaneous or
intramuscular injection. This may be accomplished by the use of a
liquid suspension of crystalline or amorphous material with poor
water solubility. The rate of absorption of the drug then depends
upon its rate of dissolution which in turn, may depend upon crystal
size and crystalline form. Alternatively, delayed absorption of a
parenterally administered drug is accomplished by dissolving or
suspending the drug in an oil vehicle. Injection depot forms are
made by forming micorencapsule matrices of the drug, in
biodegradable polymers such as
polylactide-polylactide-polyglycolide. Depending upon the ratio of
drug to polymer and the nature of the particular polymer employed,
the rate of drug release can be controlled. Examples of other
biodegradable polymers include poly (orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes of microemulsions which are
compatible with body tissues.
[0062] Compositions for rectal or vaginal administration are
preferably suppositories which can be prepared by mixing the
compounds of this invention with suitable non-irritating excipients
or carries such as cocoa butter, polyethylene glycol or a
suppository wax which are solid at ambient temperature but liquid
at body temperature and therefore melt in the rectum or vaginal
cavity and release the active compound.
[0063] Solid compositions of a similar type may be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like.
[0064] The active compounds can also be in micro-encapsulated form
with one or more excipients as noted above. The solid dosage forms
of tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings well known in the pharmaceutical formulating
art. In such solid dosage forms the active compound may be admixed
with at least one inert diluent such as sucrose, lactose or starch.
Such dosage forms may also comprise, as is normal practice,
additional substances other inert diluents, e.g., tableting
lubricants and other tableting acids such as magnesium stearate and
microcrystalline cellulose. In the case of capsules, tablets and
pills, the dosage forms may also comprise buffering agents. They
may optionally contain opacifying agents and can also be of a
composition that they release the active ingredient(s) only, or
preferentially, in a certain part of the intestinal tract,
optionally, in a delayed manner. Examples of embedding compositions
which can be used include polymeric substances and waxes.
[0065] Dosage forms for topical or transdermal administration of a
compound of this invention include ointments, pastes, creams,
lotions, gels, powders, solutions, sprays, inhalants or patches.
The active component is admixed under sterile conditions with a
pharmaceutically acceptable carrier and any needed preservatives or
buffers as may be required. Ophthalmic formulation, ear drops, eye
ointments, powders and solutions are also contemplated as being
within the scope of this invention.
[0066] The ointments, pastes, creams and gels may contain, in
addition to an active compound of this invention, excipients such
as animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0067] Powders and sprays can contain, in addition to the compounds
of this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicaters and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants such as chloroflurohydrocarbons.
[0068] Transdermal patches have the added advantages of providing
controlled delivery of a compound to the body. Such dosage forms
can be made by dissolving or dispensing the compound in the proper
medium. Absorption enhancers can also be used to increase the flux
of the compound across the skin. The rate can be controlled by
either providing a rate controlling membrane or by dispersing the
compound in a polymer matrix or gel.
[0069] The Examples that follow illustrate preferred embodiments of
the present invention and are not limiting of the specification and
claims in any way.
EXAMPLE 1
[0070] C3b-Properdin Binding
[0071] Polystyrene microtiter plates were coated with human C3b
(0.5 .mu.g/50 .mu.l per well) (Calbiochem, San Diego, Calif., Cat.
No. 204860) in Veronal buffered saline (VBS): (5 mM diethyl
barbiturate, 120 mM NaCl, 5 mM MgCl.sub.2, 5 mM EGTA) overnight at
4.degree. C. After aspirating the C3b solution, wells were blocked
with VBS containing 0.5% human serum albumin (HSA) (Sigma Chemical
Company, St. Louis, Mo., Cat. No. A9511) for 2 hours at room
temperature. Wells without C3b coating served as background
controls. Aliquots of human properdin (or factor P) (Advanced
Research Technology, San Diego, Calif., Cat. No. A139) at varying
concentrations in blocking solution were added to the wells.
Following a 2 hour incubation at room temperature, the wells were
extensively rinsed with VBS.
[0072] C3b-bound properdin was detected by the addition of mouse
monoclonal anti-human properdin antibody (detection antibody)
(Quidel, San Diego, Calif., anti-human properdin monoclonal
antibody P#2, Cat. No. A235) at 1:1000 dilution in blocking
solution, which was allowed to incubate for 1 hour at room
temperature. After washing the plates with VBS, a
peroxidase-conjugated goat anti-mouse antibody (1:1000 dilution in
blocking solution) (Sigma Chemical Company) was added and allowed
to incubate for 1 hour. The plate was again rinsed thoroughly with
VBS, and 100 .mu.l of 3,3',5,5'-tetramethyl benzidine (TMB)
substrate (Kirkegaard & Perry Laboratories, Gaithersburg, Md.,
Cat. No. 50-65-00) was added. After incubation for 10 minutes at
25.degree. C., the reaction of TMB was quenched by the addition of
100 .mu.l of phosphoric acid, and the plate was read at 450 nm in a
microplate reader (e.g., SPECTRA MAX 250, Molecular Devices,
Sunnyvale, Calif.). The estimated K.sub.d of properdin binding to
C3b was based on the concentration of properdin at 50% maximal
binding (Microcal Origin Program).
[0073] The ability of a murine anti-human properdin monoclonal
antibody (blocking antibody) to inhibit C3b-P binding was evaluated
by adding varying concentrations of this antibody (blocking
antibody, Quidel, anti-human properdin monoclonal antibody P#1,
Cat. No. A233) to a constant concentration of properdin (2 nM). The
amount of properdin bound to C3b was detected with the antibody
detection system described above.
[0074] As shown in FIG. 1, human properdin binds to C3b, which has
been immobilized onto microtiter plate wells. The apparent binding
constant from these data, defined as the concentration of properdin
needed to reach half-maximal binding, is approximately 1 nM. When
blocking anti-properdin monoclonal antibody is added along with
properdin in this assay, dose-dependent inhibition of properdin
binding to C3b is observed (FIG. 2). The IC.sub.50 value of 1 nM
indicates that the antibody binds with high-affinity to properdin,
thereby blocking its interaction with C3b.
EXAMPLE 2
[0075] C3b(Bb)-Properdin Binding
[0076] This assay was carried out as described in Example 1 above
with some changes. Briefly, microtiter wells were coated with C3b,
washed, blocked and incubated with various concentrations of normal
human serum (NHS) (Sigma Chemical Company, Cat. No. S1764) diluted
in blocking solution for 2 hours at room temperature. Under the
conditions of this binding assay, factor B in serum will bind solid
phase-bound C3b and, after cleavage by factor D in serum, generates
C3bBb. Properdin is known to bind C3bBb with high affinity
(Farries, T. C., P. J. Lachmann, and R. A. Harrison, 1988, Biochem.
252:47-54). Uncoated wells served as background controls. After
washing, bound-properdin was detected with the anti-properdin
antibody as described in Example 1 above.
[0077] To evaluate the effect of the blocking antibody to inhibit
properdin binding to C3b(Bb), various concentrations of the
blocking antibody described in Example 1 above were added to a
fixed concentrations of serum (4% in blocking solution). The amount
of properdin bound to C3b(Bb) was detected using the detection
system described in Example 1 above.
[0078] In addition to purified properdin, properdin in serum can
bind to immobilized C3b. Since serum also contains factor B, which
binds C3b with a resulting conversion to Bb after cleavage by
factor D, it is likely that serum-derived properdin is binding the
C3bBb complex in this assay format. When the blocking
anti-properdin monoclonal antibody of Example 1 is added with human
serum to C3b-coated wells, there is a dose-dependent inhibition of
properdin binding to C3bBb (FIG. 3). Again, the IC.sub.50 value in
this assay is 1-2 nM, consistent with the results obtained in FIG.
2 described in Example 1 above.
EXAMPLE 3
[0079] Alternative Pathway-Dependent MAC Assay:
[0080] The binding data of Examples 1 and 2 above reveal that the
properdin monoclonal antibody prevents the binding of properdin to
C3b and the functional C3 convertase (C3bBb). Since the literature
suggests that properdin stabilizes the C3 convertase, it was of
interest to us to determine whether the properdin antibody might
appreciably affect the terminal aspects of the alternative
complement cascade. The final end product of this pathway is the
C5b-9 membrane-attack complex (MAC). To analyze the effects of the
properdin antibody on MAC formation via the alternative pathways,
an assay was utilized in which bacterial LPS was used as a
substrate to initiate the alternative complement pathway
cascade.
[0081] Previous studies have demonstrated that lipopolysaccharide
(LPS) from Salmonella typhosa (S. Typhosa) (Sigma Chemical Company,
Cat. No. 6386) serves as a potent substrate for complement
alternative pathway activation (Clardy, C. W., 1994, Infect. Immun.
62:4539-4555). Microtiter wells were coated with LPS (2 .mu.g/50
.mu.l per well) in VBS overnight at 4.degree. C. Uncoated wells
served as background controls. After aspirating the LPS solution,
wells were treated with blocking solution and incubated with
various concentrations of normal human serum. Following a 3 hour
incubation at 37.degree. C., deposited MAC was detected with mouse
anti-human soluble C5b-9 monoclonal antibody (Quidel, Cat. No.
A239) using standard ELISA methodologies essentially as described
in the Examples above. The effect of the blocking antibody on the
MAC formation was evaluated by adding various concentrations of
blocking antibody to a fixed concentration of serum (4% in blocking
solution). The amount of inhibition of soluble C5b-9 formation was
determined using the antibody detection system described in the
Examples above.
[0082] As demonstrated in FIG. 4, addition of increasing amounts of
normal human serum, which contains all of the complement
components, resulted in increased MAC deposition on the LPS
surface. The formation of MAC in this assay could be completely
prevented by the addition of the properdin monoclonal antibody, as
seen in FIG. 5. These data indicate that properdin does not merely
stabilize and alter the kinetics of the alternative pathway, as
suggested in the literature, but demonstrates for the first time
that properdin is in fact necessary for progression of the
cascade.
EXAMPLE 4
[0083] Alternative Pathway--Dependent Hemolysis
[0084] To confirm and extend these results, the properdin antibody
was examined in another assay of the alternative pathway. Rabbit
erythrocytes initiate the alternative complement cascade, and the
resulting formation of MAC causes lysis of these cells. If the
properdin antibody is capable of complete inhibition of the
alternative pathway, then addition of the reagent to rabbit
erythrocytes bathed in human serum should prevent cellular lysis.
This can be assayed by examining the light scattering caused by
intact red blood cells; lysed cells do not diffract light, and
there is a consequent reduction in scattered light. It is well
established that rabbit erythrocytes specifically activate the
complement alternative pathway, with a resulting lysis of the cells
by the C5b-9 complex (Nolan, K. F. and K. B. M. Reid, 1993,
Properdin. Methods Enzymol. 223:35-47). Normal human serum, at
various concentrations in Gelatin Veronal Buffer (GVB) (Advanced
Research Technology) with 5 mM MgCl.sub.2 and 10 mM EGTA, was
incubated with 37.degree. C. with a fixed number of rabbit
erythrocytes (Advanced Research Technology). A progressive decrease
in light scatter (due to lysis of intact cells) was measured at 595
nm as a function of time in a temperature-controlled ELISA plate
reader (Polhill et al., J. Immunol. 121:383:370). To determine the
ability of blocking antibody to inhibit hemolysis of rabbit
erythrocytes, various concentrations of the blocking antibody were
added to a fixed concentration of normal human serum (8%) and the
assay was performed as described above. The data were recorded and
analyzed with a SpectraMax plate reader and software.
[0085] As shown in FIG. 6, addition of serum in the absence of
properdin antibody resulted in lysis of the cells and a dramatic
reduction in light scattering. Addition of increasing
concentrations of the antibody caused a decrement in erythrocyte
lysis, with 30 nM antibody completely blocking MAC-mediated
cellular destruction. These results confirm that monoclonal
antibodies that bind and block properdin interaction with C3
convertase are potent reagents that can completely abrogate the
effects of the alternative complement pathway.
[0086] Skilled artisans recognize and accept that in vitro studies
of complement are representative of and predictive of the in vivo
state of the complement system. By way of example, the use of in
vitro ELISA (enzyme-linked immunosorbent assay) procedures to
detect properdin associated with lipopolysaccharide (LPS) is a
"simple, rapid and reliable method for the assessment of complement
function particularly the detection of complement deficiency
states" (Fredrikson et al., J. Immunol. Meth., 166:263-70, 1993).
The authors conclude that the in vitro technique can be used in
vivo with the same likelihood of success in detecting alternative
complement pathway activation in disease states.
[0087] Similarly, the use of a 34-amino acid peptide to study
properdin binding with C3b using an in vitro hemolysis test was
found to be an appropriate indication of both the role of properdin
during infection and the mechanism of C3 convertase stabilization
(Daoudaki et al., J. of Immun., 140:1577-80, 1988).
[0088] Still further, the standard rabbit erythrocyte hemolysis
assay (described in detail herein in Example 4), which assay is
used to measure alternative complement pathway activity, is
accepted in the art as being the "most convenient assay for the
activity of the human alternative pathway" (Pangburn, Meth. In
Enzymology, 162:639-53, 1988).
EXAMPLE 5
[0089] Cardiopulmonary Bypass: Tubing Loop Model
[0090] To test the effect of a blocking anti-properdin monoclonal
antibody, as described in Examples 1-4 above, on inhibition of
complement activation in cardiopulmonary bypass (CPB), a tubing
loop model of CPB as described by Gong, J., R. Larsson, K. N.
Edkahl, T. E. Mollnes, U. Nilsson, and B. Nilsson, 1996, J. Clin.
Immunol. 16:222-229 was utilized. Whole blood from a healthy donor
was collected into a 7-ml vacutainer tube (Becton Dickinson, San
Jose, Calif.) containing 20 U of heparin/ml of blood. Polyethylene
tubing like that used during CPB (PE 330; I.D., 2.92 mm; O.D., 3.73
mm; Clay Adams, N.J.) was filled with 0.5 ml of the heparinized
human blood and closed into a loop with a short piece of silicon
tubing. Heparinized blood containing 20 mM EDTA (which inactivates
complement) served as a background control. Sample and control
tubing loops were rotated vertically in a water bath for 1 hour at
37.degree. C. After incubation, blood samples were transferred into
1.7 ml siliconized eppendorf tubes which contained 0.5 M EDTA to
give a final EDTA concentration of 20 mM. The samples were
centrifuged (4000.times.g for 5 minutes at 4.degree. C.) and the
plasma was collected. The plasma samples were diluted to 10% with
sample diluent buffer and the amounts of C3a as well as soluble MAC
(sMAC) were determined using ELISA assay kits following the
manufacturer's instructions (Quidel, Catalog Nos. A015 for C3a and
A009 for C5b-9/MAC). For complement inhibition studies, various
concentrations (100-600 nM) of the blocking anti-properdin
monoclonal antibody described in Examples 1-4 were added to the
heparinized blood immediately before circulation for 1 hour at
37.degree. C. After circulation/rotation in a 37.degree. C. water
bath for 1 hour, aliquots were analyzed for soluble MAC and C3a as
described above using ELISA assay kits (Quidel).
[0091] Using this simplified CPB paradigm in which standard CPB
tubing was partially filled with fresh human blood, leaving an
air-blood interface and where the tubing is joined end-to-end with
a silicon sleeve to form a loop, such that this blood-filled loop
is rotated in a heated water bath (37.degree.) to simulate the
movement of blood through a bypass circuit, there is marked
activation of complement during the rotation of the blood in the
tubing. Importantly, the blocking anti-human properdin antibody
causes significant inhibition of this complement activation. This
can be seen in FIG. 7, where the formation of soluble
membrane-attack complex (sMAC) in the loop model is nearly
completely inhibited by 100 nM anti-properdin antibody. Likewise,
the same amount of antibody causes a significant reduction in C3a
formation (FIG. 7).
[0092] This is the first demonstration of the effectiveness of an
agent that selectively inhibits alternative pathway activation in a
model of CPB. In contrast, all prior agents to date that have been
used experimentally to inhibit complement activation in CPB
protocols inhibit both the classical and alternative complement
pathways (e.g., Gillinov, A. M., P. A. DeValeria, J. A.
Winkelstein, I. Wilson, W. E. Curtis, D. Shaw, C. G. Yeh, A. R.
Rudolph, W. A. Baumgartner, A. Herskowitz, and D. E. Cameron, 1993,
Ann. Thorac. Surg. 55:619-624; Rinder, C. S., H. M. Rinder, B. R.
Smith, J. C. K. Fitch, M. J. Smith, J. B. Tracey, L. A. Matis, S.
P. Squinto, and S. A. Rollins, 1995, J. Clin. Invest.
96:1564-1572). Because it has been suggested that both the
classical and alternative pathways are activated during CPB
(Wachtfogel, Y. T., P. C. Harpel, L. H. Edmunds, Jr. and R. W.
Colman, R. W., 1989, Blood 73:468-471), these results with an
alternative pathway targeting agent are particularly
surprising.
EXAMPLE 6
[0093] Blocking Agents: Lack of Fc.gamma. Receptor Activation
[0094] As described in Example 5 above, the blocking anti-properdin
monoclonal antibody potently inhibits soluble MAC and C3a
generation in a tubing loop model of cardiopulmonary bypass. The
proinflammatory agents (C5a, C3a and sMAC) generated by complement
activation are known to activate leukocytes, platelets and
endothelial cells. As a marker for neutrophil activation, serum
levels of neutrophil elastase in whole blood in the tubing loop
model were also determined. As expected, elastase levels in blood
samples incubated in the tubing loop were increased over levels in
control samples. However, release of elastase was not inhibited by
the anti-properdin monoclonal antibody. Rather, there was an
unexpected increase in serum elastase levels with increasing
antibody concentrations. If immune-complexes are generated when the
anti-properdin monoclonal antibody is added to blood containing
properdin, these immune-complexes could interact with Fc.gamma.
receptors on neutrophils resulting in cellular activation.
Fc.gamma. receptors and their activation have been reviewed by
Ravetch, J. V. and J. P. Kinet, 1991, Ann Rev. Immunol. 9:457-492
and Hulett, M. D. and P. M. Hogarth, 1994, Adv. Immunol.
57:1-127.
[0095] Agents according to the present invention that selectively
inhibit alternative complement pathway activation are preferably
agents that do not activate Fc.gamma. receptors, e.g., via immune
complex formation with their antigen. Such agents may be screened
for their ability to activate Fc.gamma. receptors by a variety of
assays known in the art. Since superoxide generation is one of the
classic responses of neutrophils and other phagocytes to activation
via Fc.gamma. receptors (RII), a specific assay for superoxide
release from cells in diluted whole blood was utilized to screen
and evaluate agents and the possible role of Fc.gamma. receptors in
neutrophil activation by such an agent. Monoclonal antibody agents
are generally ineffective aggregating agents upon binding to their
antigen and thus ineffective to activate Fc.gamma. receptors via
immune complex formation. However, Fc.gamma. activation was
detected for a blocking anti-properdin monoclonal antibody as
follows.
[0096] A chemiluminescence assay for superoxide production in
diluted whole blood was performed (Tose, M. F. and A. Itamedani,
1992, Am. J. Clin. Pathol. 97:566-573). Fresh blood was collected
by a finger prick and rapidly diluted 350-fold into phenol red-free
RPMI-1640 media containing Pen-Strep which was buffered with 10 mM
HEPES (pH 7.4) at 37.degree. C. The media also contains 10 .mu.M
lucigenin (Sigma Chemical Co.), a compound which becomes
chemiluminescent when reduced by superoxide. Potential stimulators
of oxidative burst (e.g., PMA or C5a) or buffer controls were added
to duplicate 1 ml samples of the diluted blood-lucigenin-RPMI media
and incubated at 37.degree. C. for 2-3 hours. At regular intervals,
chemiluminescence was monitored by transferring each sample into a
liquid scintillation counter (Packard Model 1900 TR) operated in a
single photon mode to determine the "cpm". Control experiments
demonstrated that the specific "cpm" signal was completely
inhibited by addition of 100 .mu.g/ml superoxide dismutase (Sigma
Chemical Co.) to the samples.
[0097] One important characteristic of the Fc.gamma. receptor (RII)
is that it is activated by binding of polymeric immune-complexes;
monomeric IgG is ineffective. Therefore, blood cells should have to
be exposed simultaneously to properdin and anti-properdin
monoclonal antibody to allow immune-complex formation and
consequent cellular activation via Fc.gamma. receptors. Results
from the chemiluminescence assay demonstrates that superoxide
levels in diluted blood samples are not significantly elevated over
control levels by addition of either 5 .mu.g/ml properdin alone or
100 nM monoclonal antibody alone. However, simultaneous addition of
both 5 .mu.g/ml properdin and 100 nM monoclonal antibody to the
diluted blood samples results in a marked increase in superoxide
generation over control levels, indicating that cell activation
occurs via Fc.gamma. receptors. C5a also activates the neutrophil
oxidative burst response via C5a receptors, and samples containing
10 nM C5a were positive controls in this assay. The anti-human
properdin monoclonal antibody has a binding specificity for the
human protein and does not bind rat properdin. Consistent with this
specificity, incubation of blood samples with 5 .mu.g/ml rat
properdin and 100 nM anti-human monoclonal antibody did not elicit
an increase in superoxide generation over control levels. The
results of this study indicate that neutrophil activation is
mediated via binding of properdin-monoclonal antibody
immune-complexes to Fc.gamma. receptors on cells, resulting in an
increased release of elastase upon addition of the monoclonal
antibody to the tubing loop model.
[0098] There are several potential strategies that can be used in
the design of agents according to the present invention that avoid
Fc.gamma. receptor interactions. For monoclonal antibody agents,
one approach is to select the human .gamma.4 IgG isotype during
construction of a humanized antibody. The .gamma.4 IgG isotype does
not bind Fc.gamma. receptors. Alternatively, a monoclonal antibody
agent can be genetically engineered that lacks the Fc region,
including for example, single chain antibodies and antigen-binding
domains. Yet another approach is to chemically remove the Fc region
of a monoclonal antibody using partial digestion by proteolytic
enzymes, thereby generating, for example, antigen-binding antibody
fragments such as Fab or F(ab).sub.2 fragments. Such
antigen-binding antibody fragments and derivatives are similarly
useful as potent inhibitors of alternative pathway complement
activation.
[0099] In this study, proteolysis was utilized to remove the Fc
region of the blocking anti-properdin monoclonal antibody described
in the Examples above, which is a murine IgG1. Specifically, a
procedure for generating F(ab).sub.2 from murine IgG1 using ficin
digestion (Mariani, M, M. Camagna, L. Tarditi and E. Seccamani,
1991, Mol. Immunol. 28:69-71) was utilized. The progressive
ficin-mediated cleavage of this IgG1 antibody yielded a 116 kD
species corresponding to F(ab).sub.2 and another species at 32 kD
corresponding to the cleaved Fc region. Ficin digestion conditions
were identified which resulted in the generation of F(ab).sub.2 at
high yield and the total absence of any detectable intact IgG band
on Coomassie-stained SDS-PAGE gels.
[0100] The potency of this F(ab).sub.2 fragment as an inhibitor of
complement activation was compared to that of the intact
anti-properdin monoclonal antibody, using the rabbit RBC hemolysis
assay as described in Example 4. The results showed that the
ficin-digested monoclonal antibody preparation containing the
F(ab).sub.2 fragment has essentially the identical inhibitory
activity as the intact monoclonal antibody when both are tested at
3.3 nM (partial inhibition) or 6.7 nM (complete inhibition). In
addition, these anti-properdin agents have essentially equivalent
potency as inhibitors of C3 and sMAC generation in the tubing loop
model of cardiopulmonary bypass described in Example 5.
[0101] As shown in FIG. 8, when the activity of F(ab).sub.2 and the
intact antibody were compared in the superoxide generation assay
using diluted blood, addition of both intact monoclonal antibody
(100 nM) and properdin (5 .mu.g/ml) to the diluted blood samples
resulted in a marked increase in superoxide generation over control
levels. In comparison, superoxide generation in the diluted blood
samples following addition of both F(ab).sub.2 (100 nM) and
properdin (5 .mu.g/ml) was substantially reduced and was similar to
superoxide generation after addition of the F(ab).sub.2 alone (FIG.
8). At later time points (>130 minutes), superoxide generation
in both of the F(ab).sub.2 containing samples was slightly higher
than in the control samples (FIG. 8). However, since the
F(ab).sub.2 preparation was not purified to remove contaminating Fc
or trace amounts of intact monoclonal antibody, small amounts of
such contaminants could generate a small residual response. Fc
contaminants can be removed by standard purification methods if
desired. The results of this study demonstrate that generation of
an antigen-binding fragment such as F(ab).sub.2 can essentially
eliminate activation of blood cells via binding of Fc.gamma.
receptors.
[0102] It is of interest that this type of activation using an
anti-properdin monoclonal antibody has not been noted with other
anti-complement monoclonal antibodies. For example, a monoclonal
antibody to C5 has been shown to block the classical and
alternative complement pathways where they converge at C5 (the
terminal complement pathway) by blocking the cleavage of C5, thus
preventing production of MAC and C5a (WO95/25540 [PCT/US95/03614];
Rinder, et al., supra (1995)).
[0103] According to the present invention, preferred therapeutic
agents may be screened and prepared that lack such Fc.gamma.
receptor activation and are particularly effective in processes for
selective inhibition of the formation of alternative complement
pathway activation products. In addition, agents according to the
present invention are preferably agents that are selective for
their inhibition of formation of alternative pathway activation
products (i.e., do not inhibit classical pathway components) and
that do not activate the classical complement pathway. Immune
complexes, in addition to activating cells via binding to Fc.gamma.
receptors, can trigger the classical pathway of complement
activation by binding to complement component C1. To show that the
classical pathway of complement activation was not activated by the
blocking anti-properdin monoclonal antibody described above,
duplicate samples of normal human serum (NHS) were incubated at
37.degree. C. for 120 minutes with or without 200 nM anti-properdin
monoclonal antibody (30 .mu.g/ml). At 0, 30, 60, and 120 minutes,
50 .mu.l aliquots were removed from the mixture and chelated by the
addition of EDTA to a final concentration of 13 mM in order to stop
all magnesium- and calcium-dependent complement activation. The
concentration of the complement activation product C3a was
determined in all samples using an ELISA kit following the
manufacturer's instructions (Quidel, Catalog No. A015). None of the
samples containing the anti-properdin monoclonal antibody had
elevated C3a levels compared to corresponding control samples.
These results demonstrate that blocking anti-properdin monoclonal
antibody does not trigger activation of the classical pathway.
Furthermore, these results suggest that the structural determinants
on immune complexes recognized by C1 are different than those
recognized by Fc.gamma. receptors. Preferred agents according to
the invention are therefore agents that do not substantially
activate Fc.gamma. receptors or the classical complement pathway as
shown herein.
EXAMPLE 7
[0104] Classical Pathway Activation: Heparin--Protamine
Complexes
[0105] Protamine addition to heparinized blood has been shown to
activate the classical complement pathway (Cavarocchi, N. C., H. V.
Schaff, T. A. Orszulak, H. A. Homburger, W. A. Schnell, and J. R.
Pluth, 1985, Surgery 98:525-531. Thus, complement activation occurs
not only during blood circulation through tubing for a bypass
circuit like that utilized in Example 5 above, but also after the
addition of protamine (to neutralize anticoagulant heparin) at the
end of the CPB procedure. To evaluate the effect of protamine on
generation of soluble MAC, heparinized blood was incubated in tubes
at 37.degree. C. for 60 minutes with 100 .mu.g/ml protamine either
in the absence or presence of a blocking anti-properdin monoclonal
antibody as described in Examples 1-5 above. Samples were processed
and analyzed for sMAC generation using an ELISA kit following the
manufacturer's instructions (Quidel, Catalog No. A009).
[0106] As shown in FIG. 9, the anti-properdin monoclonal antibody
inhibits complement activation initiated by heparin-protamine
complexes, under conditions where fresh heparinized blood was
incubated as described above in tubes at 37.degree. C. for 60
minutes with 100 .mu.g/ml protamine either in the absence of
presence of 13, 66, 200 or 330 nM anti-properdin monoclonal
antibody and as detected by sMAC generation.
[0107] Because complement activation occurs not only during the
movement of blood through a bypass circuit, but also after the
addition of protamine upon completion of CPB, and because this
latter activation involves the classical complement pathway
(Cavarocchi, N. C., H. V. Schaff, T. A. Orszulak, H. A. Homburger,
W. A. Schnell, and J. R. Pluth, 1985, Surgery 98:525-531), it was
unexpected that an alternative pathway-specific anti-properdin
agent would attenuate or substantially inhibit the production of
complement activation products triggered by heparin-protamine
complexes as shown in FIG. 9.
[0108] Although it has been suggested that the alternative pathway
might contribute somewhat to the classical pathway-initiated
production of terminal activation products because the alternative
pathway C3 convertase could, in theory, be assembled from C3b
generated in the classical cascade, there has been a general
paucity of experimental data addressing this hypothesis. A
relatively recent study has more definitively addressed the
question of alternative pathway contribution to the classical
pathway, particularly as it pertains to the role of properdin.
Specifically, Fredrikson et al. (1993) examined the effect of
properdin deficiency on the amount of MAC generated after
activation of the classical pathway. Their results reveal that the
absence of properdin in serum had no effect on classical pathway
production of MAC. In contrast, depletion of classical pathway
components (i.e., C1q, C2, C4) completely abolished MAC generation
in their assay system. These results indicate that inhibiting
properdin action with an anti-properdin agent should have no effect
on the production of activated complement species after initiation
of the classical pathway by protamine-heparin complexes. Further
support for this interpretation is supplied by the work of
Soderstrom, C., J. H. Braconier, D. Danielsson, and A. G. Sjoholm,
1987, J. Infect. Dis. 156:107-112, who showed that serum
bactericidal reactions mediated via the classical complement
pathway were not impaired in properdin-deficient serum. These
results specifically teach that inhibiting properdin action should
have little, if any, effect on the production of complement
activation proteins after initiation of the classical complement
pathway. More generally, these data imply that the alternative
pathway contributes negligibly to the classical pathway-induced
production of complement activation products. This more general
interpretation is supported by the work of Clardy, C. W., 1994,
Infect. Immun. 62:4549-4555, who reported that an antibody to the
alternative pathway-specific component, factor B, had no effect on
classical pathway complement activation.
[0109] In agreement with what is seen during clinical CPB and as
shown above, protamine addition to heparinized human blood causes
significant complement activation, as measured by the production of
sMAC (FIG. 9). Remarkably, addition of the anti-properdin antibody
to the heparinized blood prior to the addition of protamine results
in nearly complete inhibition of sMAC formation (FIG. 9) and
demonstrates that an anti-properdin agent is effective in reducing
the post-perfusion complications associated with CPB since it is
capable of inhibiting both classical and alternative complement
pathways.
EXAMPLE 8
[0110] Classical Pathway Activation: Immune Complexes
[0111] The classical complement pathway is typically triggered by
immune complexes, for example, an antibody bound to a foreign
particle, and thus requires prior exposure to that particle for the
generation of specific antibody. There are four plasma proteins
involved in the initial steps of the classical pathway: C1, C2, C4
and C3. The interaction of C1 with the Fc regions of IgG or IgM in
immune complexes activates a C1 protease that can cleave plasma
protein C4, resulting in the C4a and C4b fragments. C4b can bind
another plasma protein, C2. The resulting species, C4b2, is cleaved
by the C1 protease to form the classical pathway C3 convertase,
C4b2a. Addition of the C3 cleavage product, C3b, to the C3
convertase leads to the formation of the classical pathway C5
convertase, C4b2a3b. To evaluate the effect of immune complexes on
the generation of C3a and soluble MAC, immune complexes were
prepared by incubating rabbit anti-ovalbumin IgG (28 mg)
(Biodesign, Kennebunk, Me.) and ovalbumin (0.67 mg) (Sigma Chemical
Company) in 3 ml PBS for 3 day at 4.degree. C. to allow maximum
precipitation. Preliminary experiments demonstrated that this ratio
of reagents corresponds to the equivalence point for the
antigen-antibody reaction. The precipitate was collected by
centrifugation at 15,000 rpm for 5 min at 4.degree. C. and washed 3
times by resuspension in 5 ml of PBS and recentrifugation. The
final precipitate was resuspended in PBS at 1 mg/ml and frozen in
aliquots at -70.degree. C. SDS-PAGE analysis confirmed that the
precipitate contains essentially only antibody and antigen.
[0112] To test the effect of an anti-properdin monoclonal antibody
on conditions of complement activation where the classical pathway
is initiated by immune complexes, triplicate 50 .mu.l samples
containing 90% NHS were incubated at 37.degree. C. in the presence
of 10 .mu.g/ml immune complex (IC) or PBS, and parallel triplicate
samples (+/- IC) also contained 200 nM anti-properdin monoclonal
antibody during the 37.degree. C. incubation. After two hour
incubation at 37.degree. C., 13 mM EDTA was added to all samples to
stop further complement activation and the samples were immediately
cooled to 5.degree. C. The samples were stored at -70.degree. C.
prior to being assayed for complement activation products (C3a and
sC5b-9) using ELISA kits (Quidel, Catalog Nos. A015 and A009)
following the manufacturer's instructions. The results of a sMAC
assay are shown in FIG. 10.
[0113] Surprisingly, the addition of an anti-properdin monoclonal
antibody to the serum prior to the addition of immune complexes
substantially inhibited (e.g., .gtoreq.50%) both C3a and sMAC
formation (approximately 80% for sMAC as shown in FIG. 10). Similar
to the results described in Example 7 above with heparin-protamine
complexes, it was likewise unexpected that an alternative
pathway-specific anti-properdin agent would attenuate or
substantially inhibit the production of activated complement
species after initiation of the classical pathway by immune
complexes as shown in FIG. 10.
EXAMPLE 9
[0114] Cardiopulmonary Bypass: Ex Vivo Extracorporeal
Circulation
[0115] To further confirm the usefulness of anti-properdin agents,
including anti-properdin antibody agents, in reducing complement
activation during CPB and other extracorporeal procedures, which
involve passing circulating blood from a blood vessel of a subject,
through a conduit and back to a blood vessel of the subject,
studies were performed in which freshly-collected human blood was
passed through a circuit that is identical to that which is
typically used during surgical procedures requiring pediatric
extracorporeal circulation. An F(ab).sub.2 anti-properdin agent
prepared as described in Example 6 was used in these studies.
[0116] Pediatric extracorporeal circuits were assembled using a
hollow-fiber pediatric membrane oxygenator (Lilliput oxygenator),
polyvinyl chloride tubing polycarbonate connectors, and a minimally
occlusive roller pump. Oxygenator and circuitry were primed with
400 ml of Plasmalyte. Blood (225 ml) was drawn from a healthy
volunteer into a transfer pack containing 1000 U heparin which was
then added to the extracorporeal circuit. For studies of complement
inhibition during extracorporeal circulation, a F(ab).sub.2
preparation of an anti-properdin monoclonal antibody in PBS (-25
.mu.g/ml of blood) was added to the transfer pack immediately
before addition of blood to the extracorporeal circuit. As blood
was introduced to the reservoir via the prime port, 225 ml of prime
fluid was simultaneously withdrawn distal to the oxygenator outlet
to yield a final circuit volume of 400 ml and a final hematocrit of
20%. Blood was circulated with prime, and complete mixing was
accomplished within 2 minutes. A baseline sample was drawn and
designated as time 0. The circuit was maintained at 37.degree. C.
for 30 minutes, then cooled to 28.degree. C. over five minutes and
maintained at that temperature for 60 minutes, after which it was
rewarmed to 37.degree. C. for an additional 60 minutes. Blood
samples were drawn at multiple times during recirculation. Serum
samples were prepared by immediate centrifugation, and stored at
-70.degree. C. in aliquots until assayed for C3a or sMAC via ELISA
kits following the manufacturer's instructions (Quidel: C3a kit,
Catalog No. A015; sMAC kit, Catalog No. A009) or neutrophil
elastase using an ELISA as described by Brower, M. S. and P. C.
Harpel, 1983, Blood 61:842-849.
[0117] As shown in FIG. 11, an F(ab).sub.2 preparation of a
blocking anti-properdin monoclonal antibody inhibits complement
activation in an ex vivo model of CPB where fresh human blood was
pumped through a pediatric bypass circuit either in the absence
(closed circles) or presence.(open circles) of an F(ab).sub.2
preparation of an anti-properdin monoclonal antibody. As described
above, blood samples were collected at various times during the
bypass procedure and analyzed for sMAC, C3a or elastase-antitrypsin
complexes.
[0118] For this study, the two bypass circuits that were utilized
were connected to a common non-pulsatory pump such that blood flow
was identical in the two systems. Whereas one circuit contained
untreated blood, the other contained blood to which the
anti-properdin agent was added prior to the onset of circulation.
In addition in this study, a F(ab).sub.2 preparation of the
anti-properdin monoclonal antibody was utilized to ensure that
properdin-anti-properdin complexes did not trigger cellular signal
transduction events by binding to Fc.gamma. receptors. This
F(ab).sub.2 antibody fragment was prepared by proteolytic cleavage
with ficin as described in Example 6. Standard proteolytic
methodologies as well as standard recombinant methodologies may be
used to prepare antibody-based proteins, including fragments,
derivatives, single chain antibodies (SCA) and antigen-binding
domains that lack the Fc portion of the immunoglobulin that allows
binding to Fc.gamma. receptors (Janeway, C. and P. Travers, Jr.,
1994, Immunobiology: the Immune System in Health and Disease. pp
3:28-3:30. Garland Publishing, Inc., New York). Alternatively, the
potential binding of therapeutic antibodies Fc.gamma. receptors can
be eliminated, if desirable or necessary, by utilizing antibodies
of the .gamma.4 sub-class of IgG, which do not interact with these
receptors (Janeway, C. and P. Travers, Jr., supra (1994)).
[0119] As demonstrated in FIG. 11, the onset of blood flow in this
ex vivo model of CPB resulted in the rapid production of sMAC and
C3a, with the levels of these activated complement components
increasing as a function of bypass time. Likewise, blood levels of
elastase-antitrypsin complexes, a marker of neutrophil activation
(Finn, A., S. Naik, N. Klein, R. J. Levinsky, S. Strobel, and M.
Elliott, 1993, J. Thorac. Cardiovasc. Surg. 105:234-241), increased
with circulation time in the CPB circuit (FIG. 9). It has been
postulated that neutrophil activation during CPB results from the
binding of complement activation species to these cells (Rinder, C.
S., H. M. Rinder, B. R. Smith, J. C. K. Fitch, M. J. Smith, J. B.
Tracey, L. A. Matis, S. P. Squinto, and S. A. Rollins, S. A., 1995,
J. Clin. Invest. 96:1564-1572; Wan, S., J-L. LeClerc, and J-L.
Vincent, 1997, Chest 112:676-692). The parallel circuit containing
blood treated with the F(ab).sub.2 anti-properdin monoclonal
antibody preparation showed essentially no complement activation,
as revealed by the virtual absence of sMAC and C3a at all bypass
times. Importantly F(ab).sub.2 anti-properdin also caused a
reduction in neutrophil activation, as demonstrated by the
reduction in blood elastase-antitrypsin complex levels (FIG. 11).
These results confirm the results obtained with the tubing loop
model described in Example 5 above. These results further
demonstrate that an anti-properdin agents that lacks Fc.gamma.
receptor activation ability effectively reduces the complement
activation and related cellular inflammatory events that result
from extracorporeal circulation and subsequent protamine
complexation of heparin.
EXAMPLE 10
[0120] Blocking Agents: Screening of Properdin-Derived Peptides
[0121] Several decapeptides of human properdin as described by
Fredrikson, et al., supra (1996) were prepared and tested for their
ability to block the effects of alternative complement pathway
activation as described for anti-properdin antibodies in the
Examples above. Specifically, these properdin-derived peptides were
assayed for their ability to inhibit MAC formation in an ELISA as
described in Example 3. Peptide 1 consisting of amino acids 43-52
of properdin (1158 M.W.), peptide 2 consisting of amino acids 48-57
of properdin (1320 M.W.) and peptide 3 consisting of amino acids
73-82 of properdin (1309 M.W.) each reduced MAC formation in this
assay, with IC.sub.50 values of 268 .mu.M, 335 .mu.M and 242 .mu.M,
respectively. In contrast, peptide 4 consisting of amino acids
218-227 of properdin (1173 M.W.) did not similarly inhibit MAC
formation in this assay (IC.sub.50>600 .mu.M). When these four
peptides were tested as described in Example 2 above, peptides 1, 2
and 3, but not peptide 4, blocked C3bBb binding, with the IC.sub.50
for the 3 blocking peptides in the range of about 400-600
.mu.M.
[0122] In a previous study by Fredrikson, et al., supra (1996) to
characterize a dysfunctional properdin protein from a patient with
Type III properdin deficiency, 87 overlapping decapeptides of human
properdin, including the four peptides described above, were
synthesized. When these peptides assayed at concentrations of 0-200
.mu.g/ml for their ability to compete with the binding of properdin
to C3b coated plates, five peptides designated as 9, 10, 15, 44
(corresponding to peptides 1, 2, 3 and 4 herein) and 81 were
determined by Fredrikson, et al., supra (1996) to compete with
properdin for C3 binding. These peptides were not tested in assays
of complement activation, such as the MAC formation assay described
above.
[0123] Since properdin has now been demonstrated according to the
present invention to be a critical component for activation of the
alternative pathway, anti-properdin agents, including
properdin-derived peptides, may be screened, identified and
selected for their ability to block alternative pathway activation,
as demonstrated herein, for example, by blocking MAC formation.
Since the screening assay showed that inhibition of MAC formation
was essentially complete at the higher peptide concentrations,
properdin was again demonstrated to be required for activation of
the alternative pathway. Anti-properdin agents, including
properdin-derived peptides, may be identified according to the
present invention as effective agents in a process for selectively
inhibiting the generation (i.e., formation or production) of an
alternate complement pathway activation product in a subject in
which either the alternative pathway or the classical pathway has
been initiated, including in subjects with a variety of disease
states and conditions, as well as complications from a variety of
medical procedures, and including subjects with acute and/or
chronic pathological injuries as described and referenced
herein.
EXAMPLE 11
[0124] Blocking Agents: Screening and Identification
[0125] Agents, which selectively block the formation of complement
activation products via the alternative complement pathway,
including preferred anti-human properdin antibodies, may be
obtained and then screened, identified and selected as taught
herein, for their ability to substantially or completely block the
formation or production of alternative complement pathway-dependent
activation products, including in conditions involving initiation
of the classical complement pathway.
[0126] Seven commercially available anti-human properdin monoclonal
antibodies were screened for blocking activity: (1) Quidel
anti-human Factor P#1 (A233); (2) Quidel anti-human Factor P#2
(A235); (3) Dako (Santa Barbara, Calif.) anti human Factor P
(MO837); (4) Serum Institute (Copenhagen, Denmark) anti-human
Factor P (HYB039 Clone 06); (5) Serum Institute anti-human Factor P
(HYB039 Clone 04); (6) Biogenesis (Poole, UK) anti-human Factor P
(Clone 10-18) [same as Quidel #1]; and Biogenesis anti-human Factor
P (Clone 10-24) [same as Quidel #2]. Each of these seven antibodies
were able to bind to properdin with high affinity
(K.sub.D.apprxeq.0.1-1 nM). However, only the Quidel P#1 monoclonal
antibody (and the identical monoclonal antibody (Clone 10-18) from
Biogenesis) completely blocked alternative pathway complement
activation, as detected by complete inhibition of MAC formation.
The Serum Institute HYB039 clone 04 was found to only partially
block and increasing the concentration of this monoclonal antibody
did not achieve complete blocking. This partially blocking
monoclonal antibody and the completely blocking Quidel #1
monoclonal antibody have comparable binding affinities for
properdin (K.sub.D.apprxeq.0.1-0.2 nM). According to the present
invention, agents are therefore effectively screened for
essentially complete, partial or no blocking activity in one or
more assays as described herein, including blocking of C3b binding
(Example 1), blocking of C3bBb binding (Example 2), blocking of
alternative pathway-dependent MAC formation (Examples 3 and 5-7),
blocking of alternative pathway-dependent hemolysis (Example 4),
blocking of alternative pathway-dependent C3a formation (Examples
5-7), or blocking of one or more markers of alternative
pathway-dependent cell activation (Example 7), including markers of
leukocyte activation (e.g., elastase-antitrypsin, CD11b/CD18),
platelet activation (e.g., P-selection, GPIIIa, GPIb (CD45b),
GPIIb) and platelet-leukocyte adhesion. Agents may be further
screened for lack of activation of Fc.gamma. receptors and/or
classical pathway activation (Example 6).
* * * * *