U.S. patent application number 11/443696 was filed with the patent office on 2006-11-23 for diagnostics and therapeutics for macular degeneration-related disorders.
This patent application is currently assigned to University of Iowa Research Foundation. Invention is credited to Gregory S. Hageman, Robert F. Mullins.
Application Number | 20060263819 11/443696 |
Document ID | / |
Family ID | 22742799 |
Filed Date | 2006-11-23 |
United States Patent
Application |
20060263819 |
Kind Code |
A1 |
Hageman; Gregory S. ; et
al. |
November 23, 2006 |
Diagnostics and therapeutics for macular degeneration-related
disorders
Abstract
The invention relates to methods for treating, preventing and
diagnosing macular degeneration-related disorders.
Inventors: |
Hageman; Gregory S.;
(Coralville, IA) ; Mullins; Robert F.;
(Coralville, IA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
University of Iowa Research
Foundation
Iowa City
IA
|
Family ID: |
22742799 |
Appl. No.: |
11/443696 |
Filed: |
May 30, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10419305 |
Apr 18, 2003 |
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11443696 |
May 30, 2006 |
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09845745 |
Apr 30, 2001 |
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10419305 |
Apr 18, 2003 |
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09510230 |
Feb 22, 2000 |
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09845745 |
Apr 30, 2001 |
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60200698 |
Apr 29, 2000 |
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Current U.S.
Class: |
435/6.12 ;
435/23; 435/7.2 |
Current CPC
Class: |
C12Q 2600/158 20130101;
A61K 51/10 20130101; A61P 37/02 20180101; G01N 2800/164 20130101;
G01N 33/564 20130101; G01N 33/6893 20130101; C12Q 1/6883 20130101;
G01N 33/6896 20130101; A61K 38/00 20130101; G01N 2333/4716
20130101; A61P 27/02 20180101; A61K 47/46 20130101 |
Class at
Publication: |
435/006 ;
435/007.2; 435/023 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/567 20060101 G01N033/567; G01N 33/53 20060101
G01N033/53; C12Q 1/37 20060101 C12Q001/37 |
Claims
1. A method for diagnosing, or identifying a predisposition to the
development of, a macular degeneration-related disorder in a
subject, comprising detecting in a biological sample from the
subject an abnormal activity or an abnormal level of at least one
complement pathway associated molecule, or an abnormal cellular
activity mediated by the complement pathway.
2. The method of claim 1, wherein said subject is free of
complement related diseases other than the macular
degeneration-related disorders.
3. The method of claim 1, wherein the detecting further comprises
detecting at least one macular degeneration-associated genetic
marker, drusen-associated phenotypic marker, or drusen-associated
genotypic marker in the subject.
4. The method of claim 1, further comprising examining of the
subject with an ophthalmologic procedure.
5. The method of claim 4, wherein said further examining detects
damages to the choriocapillaris or RPE of said subject.
6. The method of claim 1, wherein said macular degeneration-related
disorder is selected from the group consisting of age-related
macular disorder (AMD), North Carolina macular dystrophy, Sorsby's
fundus dystrophy, Stargardt's disease, pattern dystrophy, Best
disease, dominant drusen, and malattia leventinese.
7. The method of claim 1, wherein said macular degeneration-related
disorder is selected from the group consisting of retinal
detachment, chorioretinal degenerations, retinal degenerations,
photoreceptor degenerations, RPE degenerations,
mucopolysaccharidoses, rod-cone dystrophies, cone-rod dystrophies,
and cone degenerations.
8. The method of claim 1, wherein said biological sample is eye
fluid, urine, blood plasma, serum, or whole blood.
9. The method of claim 1, wherein said abnormal activity is the
presence of an autoantibody.
10. The method of claim 9, wherein the autoantibody is directed
against a complement pathway associated molecule, a RPE protein, a
choroid protein, a retina protein, or a neoantigen.
11. The method of claim 1, wherein the detecting step detects an
abnormal level of a complement pathway molecule.
12. The method of claim 11, wherein said abnormal level is detected
in urine, blood plasma, serum, whole blood sample, or eye fluid
from the subject.
13. The method of claim 11, wherein said complement pathway
associated molecule is haptoglobin, Ig kappa chain, Ig lambda
chain, or Ig gamma chain.
14. The method of claim 11, wherein said complement pathway
molecule is clusterin, C6 or C5b-9 complex.
15. The method of claim 1, wherein the detecting step detects a
variant form of a nucleic acid encoding a complement pathway
associated protein or an autoantigen.
16. The method of claim 15, wherein said nucleic acid is a mRNA,
cDNA, or genomic DNA.
17. The method of claim 15, wherein said variant nucleic acid has a
point mutation, a frameshift mutation, or a deletion relative to
wild type nucleic acid.
18. The method of claim 1, wherein said abnormal activity is
detected by measuring a complement activity in urine, blood plasma,
a serum, whole blood sample, or eye fluid from the subject.
19. The method of claim 18, wherein said complement activity is
detected by a hemolysis assay, T cell proliferative assay, DTH
assay, or an immunological assay.
20. A method for treating or preventing the development of a
macular degeneration in a subject, comprising providing to the
subject an effective amount of a therapeutic agent which modulates
an activity or expression level of at least one complement pathway
associated molecule, or a cellular activity mediated by the
compelement pathway, wherein the subject is suffering from or at
risk of developing a macular degeneration-related disorder.
21. The method of claim 20, wherein the subject has a macular
degeneration-related disorder.
22. The method of claim 20, wherein the subject is at risk of
developing a macular degeneration-related disorder.
23. The method of claim 20, wherein said subject is free of other
complement related diseases.
24. The method of claim 20, wherein said macular
degeneration-related disorder is selected from the group consisting
of age-related macular disorder, North Carolina macular dystrophy,
Sorsby's fundus dystrophy, Stargardt's disease, pattern dystrophy,
Best disease, dominant drusen, and malattia leventinese.
25. The method of claim 20, wherein said macular
degeneration-related disorder is selected from the group consisting
of retinal detachment, chorioretinal degenerations, retinal
degenerations, photoreceptor degenerations, RPE degenerations,
mucopolysaccharidoses, rod-cone dystrophies, cone-rod dystrophies,
and cone degenerations.
26. The method of claim 20, wherein said agent modulates expression
level of a complement pathway associated molecule or a molecule
initiating or triggered by the compelement pathway.
27. The method of claim 20, wherein said complement pathway
associated molecule is anaphylatoxin C3a, anaphylatoxin C5a, C6,
clusterin, haptoglobin, Ig kappa chain, Ig lambda chain, or Ig
gamma chain.
28. The method of claim 20, wherein said agent modulates protein
expression level of said complement pathway associated
molecule.
29. The method of claim 28, furthering comprising detecting said
expression level with urine, blood plasma, serum, whole blood, or
eye fluid from the subject.
30. The method of claim 20; wherein said agent modulates an
enzymatic activity of a complement protein or a complement pathway
associated molecule.
31. The method of claim 30, wherein said enzymatic activity is
catalysis of conversion of C3 into C3a and C3b, conversion of C5
into C5a and C5b, or cleavage of Factor B into Ba and Bb.
32. The method of claim 30, furthering comprising detecting said
activity by a hemolytic assay, T cell proliferative assay, DTH
assay, or an immunological assay.
33. The method of claim 30 wherein said activity is detected with
urine, blood plasma, serum, whole blood, or eye fluid from the
subject.
34. The method of claim 30, wherein said agent modulates a cellular
activity responsive to or mediated by activation of complement
system.
35. The method of claim 34, wherein said cellular activity is cell
lysis.
36. The method of claim 34, furthering comprising detecting said
cellular activity by a hemolysis assay.
37. The method of claim 34, wherein said cellular activity is
detected with urine, blood plasma, serum, whole blood, or eye fluid
from the subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
10/419,305 filed Apr. 18, 2003, which is a continuation application
of application Ser. No. 09/845,745 filed Apr. 30, 2001, which is a
continuation-in-part of application Ser. No. 09/510,230, filed Feb.
22, 2000, and which claims the benefit of Provisional Application
No. 60/200,698 filed Apr. 29, 2000. The full disclosures of these
applications are incorporated herein by reference for all
purposes.
FIELD OF THE INVENTION
[0002] The invention relates in general to therapeutics and
diagnostics for macular degeneration-related disorders or diseases.
The invention finds application in the biomedical sciences.
BACKGROUND OF THE INVENTION
[0003] Macular degeneration is a clinical term that is used to
describe a variety of diseases that are all characterized by a
progressive loss of central vision associated with abnormalities of
Bruch's membrane, the neural retina and the retinal pigment
epithelium. These disorders include very common conditions that
affect older subjects (age-related macular degeneration or AMD) as
well as rarer, earlier-onset dystrophies that in some cases can be
detected in the first decade of life. Other maculopathies include
North Carolina macular dystrophy (Small, et al., 1993), Sorsby's
fundus dystrophy (Capon, et al., 1989), Stargardt's disease
(Parodi, 1994), pattern dystrophy (Marmor and Byers, 1977), Best
disease (Stone, et al., 1992), dominant drusen (Deutman and Jansen,
1970), and radial drusen ("malattia leventinese") (Heon, et al.,
1996).
[0004] Histopathologic studies have documented significant and
widespread abnormalities in the extracellular matrices associated
with the RPE, choroid, and photoreceptors of aged individuals and
of those with clinically-diagnosed AMD (Sarks, 1976; Sarks, et al.,
1988; Bird, 1992a; van der Schaft, et al., 1992; Green and Enger,
1993; Feeney-Burns and Ellersieck, 1985; Young, 1987; Kincaid,
1992). The most prominent extracellular matrix (ECM) abnormality is
drusen, deposits that accumulate between the RPE basal lamina and
the inner collagenous layer of Bruch's membrane (FIG. 1).
[0005] A number of studies have demonstrated that the presence of
macular drusen is a strong risk factor for the development of both
atrophic and neovascular AMD (Bressler, et al., 1994; Bressler, et
al., 1990; Macular Photocoagulation Study). Drusen causes a lateral
stretching of the RPE monolayer and physical displacement of the
RPE from its immediate vascular supply, the choriocapillaris. This
displacement creates a physical barrier that may impede normal
metabolite and waste diffusion between the choriocapillaris and the
retina. It is likely that wastes may be concentrated near the RPE
and that the diffusion of oxygen, glucose, and other nutritive or
regulatory serum-associated molecules required to maintain the
health of the retina and RPE are inhibited. It has also been
suggested that drusen perturb photoreceptor cell function by
placing pressure on rods and cones (Rones, 1937) and/or by
distorting photoreceptor cell alignment (Kincaid, 1992).
[0006] The complement system consists of a group of globulins in
the serum of humans (Hood, L. E. et al. 1984, Immunology, 2d
Edition, The Benjamin/Cummings Publishing Co., Menlo Park, Calif.,
p. 339; See also, U.S. Pat. Nos. 6,087,120 and 5,808,109).
Complement activation plays an important role in the mediation of
immune and allergic reactions (Rapp, H. J. and Borsos, T., 1970,
Molecular Basis of Complement Action, Appleton-Century-Crofts
(Meredith), N.Y.). The activation of complement components leads to
the generation of a group of factors, including chemotactic
peptides that mediate the inflammation associated with
complement-dependent diseases. The activities mediated by activated
complement proteins include lysis of target cells, chemotaxis,
opsonization, stimulation of vascular and other smooth muscle
cells, degranulation of mast cells, increased permeability of small
blood vessels, directed migration of leukocytes, and activation of
B lymphocytes, macrophages and neutrophils (Eisen, H. N., 1974,
Immunology, Harper & Row, Publishers, Inc., Hagerstown, Md., p.
512).
[0007] There are three major pathways of complement activation.
First, the "classical pathway," which is activated by
antibody/antigen binding. Second, the "lectin pathway" or
"collecting pathway," is activated by the binding of acute phase
reactant mannose-binding protein (MBP; or mannose-binding lectin,
MBL) to a complex carbohydrate. Third, the "alternative pathway,"
which involves the recognition of certain polysaccharides (e.g., on
microbial surface) and is activated by the presence of a specific
substrate called C3bB, a complex of complement proteins. See, e.g.,
Cooper, Adv Immunol, 37(-HD-): 151-216, 1985; Fearon & Austen,
J. Exp. Med. 146: 22-33, 1977; Pangburn et al., 266: 16847-53,
1991; Matsushita et al., Microbiol Immunol, 40(12):887-93, 1996;
and Turner et al., Res Immunol, 147(2): 110-5, 1996. The major
classical pathway components are designated C1q, C1r, C1s, C4, C2,
C3, C5, C6, C7, C8, C9. The main alternative pathway components are
designated Factor B, Factor D, Properdin, H and I. In addition to
MBL, the lectin pathway components also include MASP-1 and MASP-2
(Thiel et al., Nature, 386:506-10, 1997). It is also known that
more than one pathway can be involved in a single disease process,
as in Alzheimer's disease (Akiyama et al., Neurobiol Aging,
21:383-421 2000).
[0008] Initiation of the classical pathway begins with antibody
binding to a specific antigen. C1q binds the altered Fc region of
IgG or IgM that has bound antigen. Upon binding, C1r activates C1s
which initiates the activation unit by cleaving a peptide from both
C4 and C2. C1s thus cleaves C4 into C4a and C4b and C2 into C2a and
C2b. C2a binds to C4b forming C4b2a. C4b2a, the C3 convertase, is a
proteolytic enzyme. It cleaves C3 into C3b, which may bind to the
activating surface, and C3a which is released into the fluid phase.
C3 convertase has the ability to cleave many C3 molecules. This
could result in the deposition of a large number of C3b molecules
on the activating surface. However, due to the labile nature of
C3b, very few molecules actually bind. C4b2a3b, the C5 convertase,
is formed when C3 is cleaved. C5 convertase, also an enzyme, can
cleave many C5 molecules into C5a and C5b.
[0009] The alternative pathway provides natural, non-immune defense
against microbial infections. In addition, this pathway amplifies
antibody-antigen reactions. Alternative pathway recognition occurs
in the presence of C3b and an activating substance such as
bacterial lipoprotein, surfaces of certain parasites, yeasts,
viruses and other foreign body surfaces, such as biomaterials. C3b
originates from classical pathway activation and/or from natural
spontaneous hydrolysis of C3. The resulting C3b binds to the
surface of the activating substance. In the presence of magnesium,
Factor B binds to the C3b which is bound to the activating surface.
Factor D then cleaves B, releasing the Ba fragment and forming
C3bBb. Properdin stabilizes the C3bBb complex and protects it from
decay. C3bBbP is the alternative pathway convertase. It also has
the ability to cleave many C3 molecules. Cleavage of C3 results in
the formation of C3bBb3b, the C5 convertase. This enzyme is also
stabilized by P to form C3bBb3bP. C5 convertase can cleave many
molecules of C5 into C5a and C5b.
[0010] Binding of MBL to carbohydrates triggers the lectin pathway.
MBL is structurally related to the complement C1, C1q, and seems to
activate the complement system through an associated serine
protease known as MASP-1 or p100, which is similar to C1r and C1s
of the classical pathway. MBL binds to specific carbohydrate
structures found on the surface of a range of microorganisms,
including bacteria, yeasts, parasitic protozoa and viruses, and
exhibits antibacterial activity through killing mediated by the
terminal, lytic complement components or by promoting phagocytosis.
The level of MBL in plasma is genetically determined, and
deficiency is associated with frequent infections in childhood, and
possibly also in adults. In addition, a further MBL-associated
serine protease (MASP-2) was identified which shows a striking
homology with the previously reported MASP (MASP-1) and the two
C1q-associated serine proteases C1r and C1s (see, e.g., Thiel et
al., Nature, 386:506-10, 1997).
[0011] The membrane attack complex C5b-9 (also termed complement
terminal complex, MAC, or SC5b-9) is common to the complement
pathways (see, e.g., Morgan, Crit Rev Immunol, 19(3):173-98, 1999).
It begins with the cleavage of C5 by C5 convertase generated during
either classical or alternative pathway activation. When C5 is
cleaved, C5a is released into the fluid phase while C5b attaches to
the activating surface at a binding site distinct from that of C3b.
One molecule each of C6 and C7 binds to C5b to form a stable
trimolecular complex to which C8 binds. Then, up to 6 molecules of
C9 can bind to C8 enhancing the effectiveness of the attack complex
to induce membrane damage if the activating surface is a
microorganism.
[0012] The significance of complement activation is not limited to
membrane damage resulting from the attack complex. The active
peptides released in the course of complement activation contribute
to the immune response by increasing vascular permeability and
contraction of smooth muscle, promoting immune adherence,
granulocyte and platelet aggregation, enhancing phagocytosis, and
directing the migration of neutrophils (PMN) and macrophages to the
site of inflammation.
[0013] The cleavage of C3 and C5 results in the release of two
small biologically active peptides, C3a and C5a. The peptides act
as anaphylatoxins. They amplify the immune response by causing the
release of histamine, slow releasing substance of anaphylaxis
(SRS-A), and heparin from basophils and mast cells. These
substances increase capillary permeability and contraction of
smooth muscle resulting in edema and inflammation.
[0014] In addition to its role as an anaphylatoxin, C5a is a potent
chemotactic factor. This mediator causes the directed migration of
leukocytes including dendritic cells and monocytes to the site of
inflammation so these leukocytes will phagocytize and clear immune
complexes, bacteria and viruses from the system.
[0015] In a process known as immune adherence, C3b or C4b deposited
on a soluble immune complex or surface permit binding of complement
receptors on PMN, macrophages, red blood cells and platelets. In
these cases C3b and C5b are considered opsonins as their presence
results in more effective phagocytosis.
[0016] New diagnostics and therapeutics for macular
degeneration-related disorders are needed. For example, there is
currently no reliable biochemical or genetic means in routine use
for diagnosing, e.g., AMD. In addition, there is no therapy
currently in use that significantly slows the degenerative
progression of AMD for the majority of subjects. Current AMD
treatment is limited to laser photocoagulation of the subretinal
neovascular membranes that occur in 10-15% of affected subjects.
The latter may halt the progression of the disease but does not
reverse the dysfunction, repair the damage, or improve vision.
SUMMARY OF THE INVENTION
[0017] The present inventions provides methods for diagnosing, or
identifying a predisposition to the development of, a macular
degeneration-related disorder in a subject by detecting in a
biological sample from the subject an abnormal activity or an
abnormal level of at least one complement pathway associated
molecule, or an abnormal cellular activity mediated by the
complement pathway. In some methods, the subject is free of
complement related diseases other than macular degeneration-related
disorders. In some methods, the detecting step also includes
detecting at least one macular degeneration-associated genetic
marker, drusen-associated phenotypic marker, or drusen-associated
genotypic marker in the subject. In some methods, the detecting
step further examines the subject with an ophthalmologic procedure.
In some methods, the further examining step detects damages to the
choriocapillaris of said subject.
[0018] Macular degeneration-related disorders that can be diagnosed
with methods of the present invention include age-related macular
disorder (AMD), North Carolina macular dystrophy, Sorsby's fundus
dystrophy, Stargardt's disease, pattern dystrophy, Best disease,
dominant drusen, and malattia leventinese. Other diseases or
disorders include retinal detachment, chorioretinal degenerations,
retinal degenerations, photoreceptor degenerations, RPE
degenerations, mucopolysaccharidoses, rod-cone dystrophies,
cone-rod dystrophies, and cone degenerations.
[0019] Samples from the subject that can be used for the
diagnostics of the present invention include eye fluid, urine,
blood plasma, serum, or whole blood. In some methods, the diagnosis
is directed to a serum autoantibody. In some methods, the
autoantibody specifically binds to a complement pathway associated
molecule, a RPE protein, a choroid protein (include proteins of the
Bruch's membrane), a retina protein, circulating molecules or
autoantigens that bind to these ocular tissues, or a neoantigen. In
some methods, the abnormal activity to be detected is an abnormal
level of a complement-pathway molecules. In some methods, the
abnormal level to be detected is the level of complement pathway
associated molecule such as haptoglobin, Ig kappa chain, Ig lambda
chain, or Ig gamma chain. In other methods, the abnormal level to
be detected is the expression level of clusterin, C6 or C5b-9
complex.
[0020] In some methods of the present invention, the abnormal
activity of complement system to be detected is a variant form of a
nucleic acid encoding a complement pathway associated protein. The
nucleic acid can be a mRNA, cDNA, or genomic DNA. The variant
nucleic acid can have a point mutation, a frameshift mutation, or a
deletion relative to the wild type nucleic acid. In some methods,
the variant nucleic acid is detected by measuring levels of
complement pathway associated molecule or complement activities in
urine, blood plasma, serum, whole blood sample, or eye fluid from
the subject. In some methods, the complement activities are
detected by a hemolysis assay, T cell proliferative assay, or an
immunological assay.
[0021] The present invention also provides methods for treating or
preventing the development of a macular degeneration in a subject
suffering from or at risk of developing a macular
degeneration-related disorder. The methods comprise administering
to the subject an effective amount of a therapeutic agent which
modulates an activity or level of at least one complement pathway
associated molecule, or a cellular activity mediated by the
compelement pathway. In some methods, the subject has a macular
degeneration-related disorder. In other methods, the subject is at
risk of developing a macular degeneration-related disorder. In some
methods, the subject is free of complement-related diseases other
than macular degeneration-related disorders.
[0022] The diseases or disorders that can be treated with the
methods of the present invention include age-related macular
disorder, North Carolina macular dystrophy, Sorsby's fundus
dystrophy, Stargardt's disease, pattern dystrophy, Best disease,
dominant drusen, and malattia leventinese. They also include
retinal detachment, chorioretinal degenerations, retinal
degenerations, photoreceptor degenerations, RPE degenerations,
mucopolysaccharidoses, rod-cone dystrophies, cone-rod dystrophies,
and cone degenerations.
[0023] In some methods, the therapeutic agent modulates level of a
complement pathway associated molecule. In some methods, the
complement pathway associated molecule whose level is to be
modulated is anaphylatoxin C3a, anaphylatoxin C5a, C6, clusterin,
haptoglobin, Ig kappa chain, Ig lambda chain, or Ig gamma chain. In
some methods, the agent modulates protein level of said complement
pathway associated molecule. Some methods further comprise
detecting the level with urine, blood plasma, serum, whole blood,
or eye fluid from the subject.
[0024] In some methods of the present invention, the therapeutic
agents modulates an enzymatic activity of a complement protein or a
complement pathway associated molecule. In some methods, the
enzymatic activity to be modulated is catalysis of conversion of C3
into C3a and C3b, conversion of C5 into C5a and C5b, or cleavage of
Factor B into Ba and Bb. Some methods further comprise detecting
the enzymatic activity by a hemolytic assay or an immunological
assay. In some methods, the enzymatic activity is detected with
urine, blood plasma, serum, whole blood, or eye fluid from the
subject.
[0025] In some methods of the present invention, the therapeutic
agents modulates a cellular activity responsive to or mediated by
the activated complement system. In some methods, the cellular
activity to be modulated is cell lysis. Some methods further
comprise detecting the cellular activity by, e.g., a hemolysis
assay. In some methods, the cellular activity is detected with
urine, blood plasma, serum, whole blood, or eye fluid from the
subject.
BRIEF DESCRIPTION OF THE DRAWING
[0026] FIG. 1 is a schematic representation of the retina and
choroid, as seen in (A) histologic section, and (B) retinal neurons
shown diagrammatically. A, amacrine cells; B, bipolar cells; BM,
Bruch's membrane; C, cone cells; CC, choriocapillaris; ELM,
external limiting membrane; G, ganglion cells; GCL, ganglion cell
layer; H, horizontal cells; ILM, inner limiting membrane; INL,
internal nuclear layer; IPM, interphotoreceptor matrix; IS, inner
segments of rods and cones; IPL, internal plexiform layer; NFL,
nerve fiber layer; ONL, outer nuclear layer; OPL, outer plexiform
layer; OS, outer segments of rods and cones; PE, pigment
epithelium; PRL, photoreceptor layer; PT, photorecptor cell
terminals; R, rod cells; ST, stroma vascularis of choroid.
[0027] FIG. 2 shows distribution and intensity of C5b-9 in the
RPE-choroid of human donor eyes.
[0028] FIG. 3 shows capture ELISA measurements of C5b-9 levels in
cytosolic and membrane fractions of isolated human RPE cells from
four human donor eyes.
[0029] FIG. 4 shows capture ELISA measurements of C5b-9 levels in
cytosolic and membrane fractions of human chorioids from 5 donors.
The membrane-associated fractions consistently exhibit the highest
levels of C5b-9 in these preparations, indicating that a
significant proportion of complexes are inserted into plasma
membranes of resident and/or transient choroidal cells.
[0030] FIG. 5 provides confirmation of capture ELISA results using
standard ELISA methodology. Tissues from the following 5 donors (2
of them with AMD) were employed in these experiments:
TABLE-US-00001 364-00 78 CM 409-00 10 CF 457-00 89 CF AMD 243-00 80
CF 239-00 80 CF AMD with CNV (choroidal neovascularization)
DETAILED DESCRIPTION
[0031] The present invention provides methods for diagnosis of
macular degeneration-related disorders, and for prevention and
treatment of such disorders. The invention is predicated in part on
the discovery that the complement system is locally active,
especially at the RPE-choroid interface in macular
degeneration-related disorders. The methods work by detecting an
abnormal activity or level associated with at least one complement
pathway associated molecule. The presence of abnormal complement
activity or abnormal levels in a biological sample from a subject
can be indicative of the existence of, or a predisposition to
developing, various macular degeneration-related disorders. Such
disorders or disease include, e.g., age-related macular disorder
(AMD), North Carolina macular dystrophy, Sorsby's fundus dystrophy,
Stargardt's disease, pattern dystrophy, Best disease, dominant
drusen, and malattia leventinese. Other macular
degeneration-related ocular diseases that can be diagnosed or
treated with the methods include, e.g., retinal detachment,
chorioretinal degenerations, retinal degenerations, photoreceptor
degenerations, RPE degenerations, mucopolysaccharidoses, rod-cone
dystrophies, cone-rod dystrophies, and cone degenerations.
[0032] The methods are suitable for large scale screening of a
population of subjects for the presence of these macular
degeneration-related disorders, optionally, in conjunction with
additional biochemical and/or genetic markers of other disorders
that may reside in the subjects. The methods are also suitable for
monitoring subjects who have previously been diagnosed with a
macular degeneration-related disorder, particularly their response
to treatment. Methods of analyzing abnormal complement activities
or abnormal levels can be performed in combination, optionally in
further combination with detecting other genetic, phenotypic, or
genotypic markers correlated with macular degeneration-related
disorders or drusen-associated diseases, as described by WO
00/52479. Optionally, analysis of phenotypic markers can be
combined with polymorphic analysis of genes encoding complement
pathway molecules for polymorphisms correlated with the macular
degeneration-related disorders.
[0033] The following sections provide guidance for making and using
the compositions of the invention, and for carrying out the methods
of the invention.
I. Definitions
[0034] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which this invention pertains. The
following references provide one of skill with a general definition
of many of the terms used in this invention: Singleton et al.,
DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THE
CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988);
and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY
(1991). Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, the preferred methods and materials are
described. The following definitions are provided to assist the
reader in the practice of the invention.
[0035] The term "agent" includes any substance, molecule, element,
compound, entity, or a combination thereof. It includes, but is not
limited to, e.g., protein, oligopeptide, small organic molecule,
polysaccharide, polynucleotide, and the like. It can be a natural
product, a synthetic compound, or a chemical compound, or a
combination of two or more substances. Unless otherwise specified,
the terms "agent", "substance", and "compound" can be used
interchangeably.
[0036] The term "agonist" is an agent that enhances or upregulates
(e.g., potentiates or supplements) the production or activity of a
gene product. An agonist can also be a compound which increases the
interaction of a gene product, molecule or cell with another gene
product, molecule or cell, e.g., of a gene product with another
homologous or heterologous gene product, or of a gene product with
its receptor. A preferred agonist is a compound which enhances or
increases binding or activation of a transcription factor to an
upstream region of a gene and thereby activates the gene. Any agent
that activates gene expression, e.g., by increasing RNA or protein
synthesis or decreasing RNA or protein turnover, or gene product
activity may be an agonist whether the agent acts directly on the
gene or gene product or acts indirectly, e.g., upstream in the gene
regulation pathway. Agonists may be RNAs, peptides, antibodies and
small molecules, or a combination thereof.
[0037] The term "antagonist" is an agent that downregulates (e.g.,
suppresses or inhibits) the production or activity of a gene
product. Such an antagonist can be an agent which inhibits or
decreases the interaction between a gene product, molecule or cell
and another gene product, molecule or cell. A preferred antagonist
is a compound which inhibits or decreases binding or activation of
a transcription factor to an upstream region of a gene and thereby
blocks activation of the gene. Any agent that inhibits gene
expression or gene product activity may be an antagonist whether
the agent acts directly on the gene or gene product or acts
indirectly, e.g., upstream in the gene regulation pathway. An
antagonist can also be a compound that downregulates expression of
a gene or which reduces the amount of gene product present, e.g.,
by decreasing RNA or protein synthesis or increasing RNA or protein
turnover. Antagonists may be RNAs, peptides, antibodies and small
molecules, or a combination thereof.
[0038] The term "antibody" or "immunoglobulin" is used to include
intact antibodies and binding fragments thereof. Typically,
fragments compete with the intact antibody from which they were
derived for specific binding to an antigen fragments including
separate heavy chains, light chains Fab, Fab', F(ab')2, Fabc, and
Fv. Fragments are produced by recombinant DNA techniques, or by
enzymatic or chemical separation of intact immunoglobulins. The
term "antibody" also includes one or more immunoglobulin chain that
are chemically conjugated to, or expressed as, fusion proteins with
other proteins. The term "antibody" also includes bispecific
antibody. A bispecific or bifunctional antibody is an artificial
hybrid antibody having two different heavy/light chain pairs and
two different binding sites. Bispecific antibodies can be produced
by a variety of methods including fusion of hybridomas or linking
of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin.
Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148,
1547-1553 (1992).
[0039] The term "antisense molecules" include antisense or sense
oligonucleotides comprising a single-stranded nucleic acid sequence
(either RNA or DNA) capable of binding to target mRNA (sense) or
DNA (antisense) sequences for a specific protein (e.g., a
complement pathway molecule). The ability to derive an antisense or
a sense oligonucleotide, based upon a cDNA sequence encoding a
given protein is described in, e.g., Stein and Cohen (Cancer Res.
48:2659, 1988) and van der Krol et al. (BioTechniques 6:958,
1988).
[0040] The term "complement activity" broadly encompasses the
biochemical and physiological activities associated with the
complement system, individual complement pathway associated
molecules, as well as genes encoding these molecules. Therefore,
complement activities include, e.g., structure and expression of a
gene encoding a complement pathway molecule, biochemical activity
(e.g., enzymatic or regulatory) of a complement pathway molecule,
cellular activities that initiate or result from activation of the
complement system, and presence of serum autoantibodies against
complement pathway molecules.
[0041] The term "complement components" or "complement proteins"
refers to the molecules that are involved in activation of the
complement system. The classical pathway components include, e.g.,
C1q, C1r, C1s, C4, C2, C3, C5, C6, C7, C8, C9, and C5b-9 complex
(membrane attack complex: MAC). The alternative pathway components
include, e.g., Factor B, Factor D, Properdin, H and I. The main
lectin pathway component is mannose-binding protein (MBP).
[0042] The terms "complement pathway associated molecules,"
"complement pathway molecules," and "complement pathway associated
proteins" are used interchangeably and refer to the various
molecules that play a role in complement activation and the
downstream cellular activities mediated by, responsive to, or
triggered by the activated complement system. They include
initiators of complement pathways (i.e., molecules that directly or
indirectly triggers the activation of complement system), molecules
that are produced or play a role during complement activation
(e.g., complement proteins/enzymes such as C3, C5, C5b-9, Factor B,
MASP-1, and MASP-2), complement receptors or inhibitors (e.g.,
clusterin, vitronectin, CR1, or CD59), and molecules regulated or
triggered by the activated complement system (e.g., membrane attack
complex-inhibitory factor, MACIF; see, e.g., Sugita et al., J
Biochem, 106:589-92, 1989). Thus, in addition to complement
proteins noted above, complement pathway associated molecules also
include, e.g., C3/C5 convertase regulators (RCA) such as complement
receptor type 1 (also termed CR1 or CD35), complement receptor type
2 (also termed CR2 or CD21), membrane cofactor protein (MCP or
CD46), and C4bBP; MAC regulators such as vitronectin, clusterin
(also termed "SP40,40"), CRP, CD59, and homologous restriction
factor (HRF); immunoglobulin chains such as Ig kappa, Ig lambda, or
Ig gamma); C1 inhibitor; and other proteins such as CR3, CR4
(CD11b/18), and DAF (CD 55).
[0043] A "detectable label" refers to an atom (e.g., radionuclide),
molecule (e.g., fluorescein), or complex, that is or can be used to
detect (e.g., due to a physical or chemical property) the presence
of another molecule. The term "label" also refers to covalently
bound or otherwise associated molecules (e.g., a biomolecule such
as an enzyme) that act on a substrate to produce a detectable atom,
molecule or complex. Detectable labels suitable for use in the
present invention include any composition detectable by
spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical, chemical means and the like.
[0044] The term "drusen" refers to deposits that accumulate between
the RPE basal lamina and the inner collagenous layer of Bruch's
membrane (see, e.g., van der Schaft et al., Ophthalmol. 99: 278-86,
1992; Spraul et al. Arch. Ophthalmol. 115: 267-73, 1997; and
Mullins et al., Histochemical comparison of ocular "drusen" in
monkey and human, In M. LaVail, J. Hollyfield, and R. Anderson
(Eds.), in Degenerative Retinal Diseases (pp. 1-10). New York:
Plenum Press, 1997). Hard drusen are small distinct deposits
comprising homogeneous eosinophilic material and are usually round
or hemispherical, without sloped borders. Soft drusen are larger,
usually not homogeneous, and typically contain inclusions and
spherical profiles. Some drusen may be calcified. The term "diffuse
drusen," or "basal linear deposit," is used to describe amorphous
material which forms a layer between the inner collagenous layer of
Bruch's membrane and the retinal pigment epithelium (RPE). This
material can appear similar to soft drusen histologically, with the
exception that it is not mounded.
[0045] The term "drusen-associated disease," or "drusen-associated
disorder," refers to any disease in which formation of drusen or
drusen-like extracellular disease plaque takes place, and for which
drusen or drusen-like extracellular disease plaque causes or
contributes thereto or represent a sign thereof. Drusen-associated
disease or disorder primarily includes macular degeneration-related
disorders wherein drusen is present. But it also encompasses
non-ocular age-related diseases with extracellular disease plaques
such as amyloidosis, elastosis, dense deposit disease, and/or
atherosclerosis. The term also includes glomerulonephritis (e.g.,
membranous and post-streptococcal/segmental which have associated
ocular drusen).
[0046] The term "epitope" or "antigenic determinant" refers to a
site on an antigen to which B and/or T cells respond. B-cell
epitopes can be formed both from contiguous amino acids or
noncontiguous amino acids juxtaposed by tertiary folding of a
protein. Epitopes formed from contiguous amino acids are typically
retained on exposure to denaturing solvents whereas epitopes formed
by tertiary folding are typically lost on treatment with denaturing
solvents. An epitope typically includes at least 3, and more
usually, at least 5 or 8-10 amino acids in a unique spatial
conformation. Methods of determining spatial conformation of
epitopes include, for example, x-ray crystallography and
2-dimensional nuclear magnetic resonance. See, e.g., Epitope
Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn
E. Morris, Ed. (1996). Antibodies that recognize the same epitope
can be identified in a simple immunoassay showing the ability of
one antibody to block the binding of another antibody to a target
antigen. T-cells recognize continuous epitopes of about nine amino
acids for CD8 cells or about 13-15 amino acids for CD4 cells. T
cells that recognize the epitope can be identified by in vitro
assays that measure antigen-dependent proliferation, as determined
by 3H-thymidine incorporation by primed T cells in response to an
epitope (Burke et al., J. Inf. Dis. 170, 1110-19 (1994)), by
antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et
al., J. Immunol. 156, 3901-3910) or by cytokine secretion.
[0047] The term "fusion protein" refers to a composite polypeptide,
i.e., a single contiguous amino acid sequence, made up of two (or
more) distinct, heterologous polypeptides which are not normally
fused together in a single amino acid sequence. Thus, a fusion
protein can include a single amino acid sequence that contains two
entirely distinct amino acid sequences or two similar or identical
polypeptide sequences, provided that these sequences are not
normally found together in the same configuration in a single amino
acid sequence found in nature. Fusion proteins can generally be
prepared using either recombinant nucleic acid methods, i.e., as a
result of transcription and translation of a recombinant gene
fusion product, which fusion comprises a segment encoding a
polypeptide of the invention and a segment encoding a heterologous
polypeptide, or by chemical synthesis methods well known in the
art.
[0048] The term "macular degeneration-related disorder" refers to
any of a number of conditions in which the retinal macula
degenerates or becomes dysfunctional, e.g., as a consequence of
decreased growth of cells of the macula, increased death or
rearrangement of the cells of the macula (e.g., RPE cells), loss of
normal biological function, or a combination of these events.
Macular degeneration results in the loss of integrity of the
histoarchitecture of the cells and/or extracellular matrix of the
normal macula and/or the loss of function of the cells of the
macula. Examples of macular degeneration-related disorder include
AMD, North Carolina macular dystrophy, Sorsby's fundus dystrophy,
Stargardt's disease, pattern dystrophy, Best disease, dominant
drusen, and malattia leventinese (radial drusen). The term also
encompasses extramacular changes that occur prior to, or following
dysfunction and/or degeneration of the macula. Thus, the term
"macular degeneration-related disorder" also broadly includes any
condition which alters or damages the integrity or function of the
macula (e.g., damage to the RPE or Bruch's membrane). For example,
the term encompasses retinal detachment, chorioretinal
degenerations, retinal degenerations, photoreceptor degenerations,
RPE degenerations, mucopolysaccharidoses, rod-cone dystrophies,
cone-rod dystrophies and cone degenerations.
[0049] The terms "modulation", "alteration", "modulate", or "alter"
are used interchangeably herein to refer to both upregulation
(i.e., activation or stimulation (e.g., by agonizing or
potentiating) and downregulation (i.e., inhibition or suppression
(e.g., by antagonizing, decreasing or inhibiting)) of an activity
or a biological process (e.g., complement process). "Modulates" or
"alters" is intended to describe both the upregulation or
downregulation of a process. A process which is upregulated by a
certain stimulant may be inhibited by an antagonist to that
stimulant. Conversely, a process that is downregulated by a certain
stimulant may be inhibited by an antagonist to that stimulant.
[0050] By "randomized" is meant that each nucleic acid and peptide
consists of essentially random nucleotides and amino acids,
respectively. Since generally these random peptides (or nucleic
acids, discussed below) are chemically synthesized, they may
incorporate any nucleotide or amino acid at any position. The
synthetic process can be designed to generate randomized proteins
or nucleic acids, to allow the formation of all or most of the
possible combinations over the length of the sequence, thus forming
a library of randomized proteinaceous test agents. The library can
be fully randomized, with no-sequence preferences or constants at
any position.
[0051] "Specific binding" between two entities means an affinity of
at least 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9 M-1, or 10.sup.10
M-1. Affinities greater than 10.sup.8 M-1 are preferred.
[0052] A "subject" includes both humans and other animals
(particularly mammals) and other organisms that receive either
prophylactic or therapeutic treatment.
[0053] The term "test agent" as used herein describes any molecule,
e.g., protein, oligopeptide, small organic molecule,
polysaccharide, polynucleotide, etc., that can be screened for
their capability of directly or indirectly altering the
bioactivities of a complement pathway molecule.
[0054] A "variant" refers to a polypeptide amino acid sequence that
is altered by one or more amino acid residues relative to the wild
type sequence, or a polynucleotide sequence that is altered by one
or more nucleotide residue relative to the wild type sequence.
Unless otherwise specified, the term "analog" can be used
interchangeably with "variant". A variant can be an allelic
variant, a species variant, or an induced variant. The variant can
have "conservative" changes, wherein a substituted amino acid has
similar structural or chemical properties (e.g., replacement of
leucine with isoleucine). Alternatively, a variant can have
"nonconservative" changes (e.g., replacement of glycine with
tryptophan). Analogous minor variations can also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues can be substituted, inserted, or deleted
without abolishing biological or immunological activity can be
found using computer programs well known in the art, for example,
LASERGENE.TM. software.
II. Abnormal Complement Activity in Macular Degeneration-Related
Disorders
A. Complement Pathway Molecules in Drusen and Macular
Degeneration-Related Disorders
[0055] The complement system and complement components are involved
in various immune processes. For example, complement C5b-9 complex,
also termed the terminal complex or the membrane attack complex
(MAC), plays an important role in cell death by inducing membrane
permeability damages. As detailed below in the Examples, the
present inventor has discovered that the complement process is
associated with the development of drusen and the etiology of
macular degeneration-related disorders. Numerous complement pathway
proteins including the MAC are found to be associated with drusen,
Bruch's membrane, the basal surface of the RPE, and/or the sub-RPE
space by immunohistochemical and biochemical studies (Tables 1 and
2). Analyses of drusen composition have revealed the presence of
components of the complement system, such as complements 3, 5 and
9, C5b-9 terminal complexes, and C-reactive protein (CRP; a serum
protein that plays a role in complement activation and
immunomodulation; Volanakis, Ann N Y Acad Sci, 389:235-50; 1982;
and Kilpatricket al., J. Immunol., 134: 3364, 1985). The present
inventor also discovered that other molecules which are involved in
the complement process are also present in drusen, e.g., regulators
of complement system including CR1 (Ng et al., Clin Exp Immunol.
71:481-5, 1988) and CR2 (Mold et al., J Immunol. 140:1923-9, 1988),
clusterin (a complement inhibitor which binds to complement C7,
C8b, and C9; Tschopp et al., J. Immunol. 151:2159-65, 1993),
vitronectin (also termed "complement S-protein," a complement
inhibitor which binds to CSb-7 and C9; Milis et al., Clin Exp
Immunol. 92:114-9, 1993), and gp330/megalin (Bachinsky et al., Am J
Pathol. 143:598-611, 1993).
[0056] Additional complement pathway-associated molecules localized
in Bruch's membrane and/or drusen include C3d, C6, C7, C8, C9,
Factor D, Factor H, Factor I, Factor B, clusterin, and mannose
binding protein. Further, some complement pathway-associated
molecules such as CD21, CD35, CD55/decay accelerating factor, and
CD59/protectin, are present in the basal surface of the RPE.
[0057] In addition, data from differential gene expression analyses
indicate a significant downregulation of complement pathway
molecules (e.g., complement 6, clusterin) and up-regulation of a
number of immune system-associated molecules (including Ig mu,
lambda, J, and kappa chains) in the RPE/choroid of AMD donors, as
compared to age-matched controls (see, e.g., Example 5).
[0058] Another indicator of abnormal complement activity is the
presence or increased levels of autoantibodies against various
macular degeneration-associated autoantigens. Some autoantibodies
have been detected in the sera of AMD subjects (Guerne et al.,
Ophthalmology, 1991. 98: 602-7; Penfold et al., Clin. Exp.
Ophthalmol., 1990. 228: 270-4). Further macular
degeneration-associated autoantigens identified by the present
inventors include complement pathway molecules and various proteins
from RPE, choroid, and retina. As discussed in the Examples,
autoantibodies against these macular degeneration-associated
autoantigens were found in serum of patients with macular
degeneration-related disorders (e.g., AMD and Malattia
Leventinese). Examples of autoantibodies against complement pathway
associated molecules include autoantibodies against vitronectin
(Example 10). Examples of autoantibodies against RPE, choroid, or
retina proteins include autoantibodies against .beta. crystallin
(A2, A3, A4, and S), calreticulin, 14-3-3 protein epsilon,
serotransferrin, albumin, keratin, pyruvate carboxylase, villin 2
(Example 11), as well as a number of other proteins (Example
12).
[0059] As discussed in the Examples, infra, detection of
autoantibodies against complement pathway molecules or against RPE,
choroid, or retina components provides another means for diagnosing
and treating macular degeneration-related disorders (e.g., AMD). In
addition, the specific genetic loci that cause macular
degeneration-related disorders (e.g., AMD) can be identified by
further analysis and identification of the various macular
degeneration-associated antoantigens.
[0060] Taken together, these data indicate that the complement
system plays an important role in drusen development and the
etiology of macular degeneration-related disorders (e.g., AMD).
B. Correlation Between Complement Activity in the RPE-Choroid
Interface and Macular Degeneration
[0061] Significantly, the present inventors also discovered that a
number of messengers for the complement pathway associated
molecules detected in drusen and Bruch's membrane are produced
locally by specific ocular cells (see, e.g., Examples 3 and 4).
These molecules include, e.g., complements 3, 5 and 9, CRP,
immunoglobulin lambda and kappa light chains, Factor X, HLA-DR,
apolipoprotein A, apolipoprotein E, amyloid A, and vitronectin. For
example, C3 and C5 are synthesized by the RPE as are APP,
clusterin, and Factor H. A number of other complement components
that are not synthesized by the RPE, such as C9 and MASP-1, are
synthesized by adjacent choroidal and/or retinal cells and could
therefore contribute to complement activation in Bruch's membrane.
These data indicate a role for locally produced complement
components in the activation of complement in Bruch's membrane and,
possibly, in the etiology of macular degeneration-related
disorders.
[0062] The present inventor has also discovered that there is a
strong correlation between intensity and distribution of complement
components (e.g., C5b-9 complex) in the RPE-choroid (especially in
the interface) and AMD (see, e.g., Example 2). Significantly,
intense labeling of the entire choriocapillaris (endothelium,
pericytes, and associated extracellular matrix) was observed in
donors with AMD (9 of 10 donors), as compared to older, age-matched
donors without a diagnosis of AMD (2 of 10 donors). When combined
with the observation that C5b-9 complexes are associated with RPE
and choroidal cell membranes, these data indicate that the
choriocapillaris of AMD subjects can be under more rigorous attack
than that of individuals without AMD. Also, the distribution of
immunoreactive C5b-9 and detectable levels of C5b-9 in the samples
from AMD donors indicate that complement pathway inhibitors such as
clusterin, vitronectin CD56 and CD55 may fail to suppress the
terminal pathway, thereby permitting formation of MAC.
[0063] Complement-mediated damage to the choriocapillaris can lead
to abnormal responses by the choroid (e.g., inflammation, cytokine
secretion, neovascularization) and/or choriocapillaris cell death.
These events, in turn, can lead to further dysfunction and death of
surrounding cells, including the RPE and choroid, and the
biogenesis of drusen. Indeed, the present inventors have discovered
that MAC is inserted into the cell membranes of both choroidal and
RPE cells (see, e.g., Examples 2 and 3). Similar processes are also
active in other diseases, including atherosclerosis and Alzheimer
disease.
[0064] These data also provide evidence that Bruch's membrane can
serve as an unusual activating surface for complement in its
physiologically "normal" state, and that activated C5b-9 is poorly
cleared from Bruch's membrane compared with other structures in the
healthy choroid. It is clear that complement activation occurs at
the RPE-choroid interface chronically, and that a strong
correlation between intensity and distribution of C5b-9 associated
with the choriocapillaris and AMD exists. As discussed below, the
present invention provides novel diagnostics and therapeutics for
macular degeneration-related disorders in accordance with such
discovery.
III. Diagnostics: Abnormal Complement Activity in Macular
Degeneration-Related Disorders
[0065] The present invention provides methods for diagnosing, or
determining a predisposition to development of, a macular
degeneration-related disorder by detecting abnormal levels or
abnormal activities of complement pathway associated molecules, or
abnormal cellular activities associated with complement pathways.
The complement pathway-associated molecules include initiators of
complement pathways, i.e., any molecule which directly or
indirectly triggers the activation of complement system through any
of the three complement pathways, such as autoantigens,
autoantibodies, immune complexes, or MBL. They also include
molecules that are produced or play a role during complement
activation, e.g., complement proteins/enzymes (e.g., C3, C5, C5b-9,
FactorB, MASP-1, and MASP-2) and receptors or inhibitors (e.g.,
vitronectin, CR1, and vitronectin). Further, the complement pathway
associated molecules that can be diagnosed with methods of the
present invention also include molecules that regulated by the
activated complement system (e.g., MACIF). Cellular activities
regulated by the activated complement system include, e.g., cell
damage resulting from the C5b-9 attack complex, vascular
permeability changes, contraction and migration of smooth muscle
cells, T cell proliferation, immune adherence, aggregation of
dendritic cells, monocytes, granulocyte and platelet, phagocytosis,
migration and activation of neutrophils (PMN) and macrophages. The
diagnostic methods of the present invention encompass detection of
abnormality in any of these complement pathway associated molecules
or cellular activities. Further, the diagnostic methods of the
present invention are also directed to detecting abnormal levels of
activities of molecules that are directly up-regulated or
down-regulated by the complement system.
[0066] Typically, a diagnostic test works by comparing a measured
level of at least one complement pathway molecule (expression level
or a biochemical activity) in a subject with a baseline level
determined in a control population of subjects unaffected by a
macular degeneration-related disorder. If the measured level does
not differ significantly from baselines levels in a control
population, the outcome of the diagnostic test is considered
negative. On the other hand, if there is a significant departure
between the measured level in a subject and baseline levels in
unaffected subjects, it signals a positive outcome of the
diagnostic test, and the subject is considered to have an abnormal
level or activity of that complement pathway molecule.
[0067] A departure is considered significant if the measured value
falls outside the range typically observed in unaffected subjects
due to inherent variation between subjects and experimental error.
For example, in some methods, a departure can be considered
significant if a measured level does not fall within the mean plus
one standard deviation of levels in a control population.
Typically, a significant departure occurs if the difference between
the measured level and baseline levels is at least 20%, 30%, or
40%. Preferably, the difference is by at least 50% or 60%. More
preferably, the difference is more than at least 70% or 80%. Most
preferably, the difference is by at least 90%. The extent of
departure between a measured value and a baseline value in a
control population also provides an indicator of the probable
accuracy of the diagnosis, and/or of the severity of the disease
being suffered by the subject.
[0068] Various biological samples from a subject can be used for
the detection, e.g., samples obtained from any organ, tissue, or
cells, as well as blood, urine, or other bodily fluids (e.g., eye
fluid). For some diagnostic methods, a preferred sample is eye
fluid. For some other methods, a preferred tissue sample is whole
blood and products derived therefrom, such as plasma and serum.
Blood samples can be obtained from blood-spot taken from, for
example, a Guthrie card. Other sources of tissue samples are skin,
hair, urine, saliva, semen, feces, sweat, milk, amniotic fluid,
liver, heart, muscle, kidney and other body organs. Others sources
of tissue are cell lines propagated from primary cells from a
subject. Tissue samples are typically lysed to release the protein
and/or nucleic acid content of cells within the samples. The
protein or nucleic acid fraction from such crude lysates can then
be subject to partial or complete purification before analysis.
[0069] In some methods, multiple diagnostic tests for multiple
markers are performed on the same subject. Typically, multiple
tests are performed on different aliquots of the same biological
sample. However, multiple assays can also be performed on separate
samples from the same tissue source, or on multiple samples from
different tissue sources. For example, a test for one marker can be
performed on a plasma sample, and a test for a second marker on a
whole blood sample. In some methods, multiple samples are obtained
from the same subject at different time points. In such methods,
the multiple samples are typically from the same tissue, for
example, all serum.
A. Diagnosing Abnormal Levels of Complement Pathway Molecules
[0070] The present invention provides methods for detecting
abnormal levels of complement pathway associated molecules that are
indicative of the presence or a predisposition to development of a
macular degeneration-related disorder. Either abnormal levels of
complement pathway-associated proteins or abnormal levels of mRNAs
encoding the complement pathway-associated proteins can be
detected. For example, abnormal levels of complement proteins
(e.g., C6, C3, C5, C6) or other complement pathway molecules (e.g.,
clusterin, CRP, or Ig chains) could be indicative of a disease
state or a predisposition to developing a macular
degeneration-related disorder (e.g., AMD). Either abnormal mRNA
levels or abnormal protein levels can be detected. The abnormal
expression can be either upregulation or downregulation.
[0071] To detect abnormal protein levels of the complement pathway
associated molecules, various immunohistochemical and biochemical
assays can be used. For example, levels of the
complement-components or split products generated in the activation
of the alternative or classical pathway can be measured as
described, e.g., in Buyon et al., Arthritis Rheum, 35:1028-37, 1992
(e.g., plasma levels of Ba, Bb, SC5b-9, and C4d); Langlois et al.,
J Allergy Clin Immunol, 83:11-6, 1989 (serum levels of C3a, C4a,
C5a, C1rC1s-C1-inhibitor complex, and terminal C complex C5b-9),
and Caraher et al., J Endocrinol, 162:143-53, 1999. These methods
can be readily employed to detect body fluid concentration of any
complement pathway associated protein in a subject suspected to
have or to develop a specific macular degeneration-related
disorder. As controls, expression levels of the complement pathway
associated protein are also measured for subjects who respectively
have or do not have the specific macular degeneration-related
disorder.
[0072] For mRNAs encoding complement pathway molecules, there are a
number of methods available for detecting abnormal levels in a
biological sample from a subject. Nucleic acids obtained from a
biological sample of the subject can be amplified first.
Amplification techniques are known to those of skill in the art and
include, but are not limited to cloning, polymerase chain reaction
(PCR), polymerase chain reaction of specific alleles (ASA), ligase
chain reaction (LCR), nested polymerase chain reaction, self
sustained sequence replication (Guatelli, J. C. et al., 1990, Proc.
Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification
system (Kwoh, D. Y. et al., 1989, Proc. Natl. Acad. Sci. USA
86:1173-1177), and Q-Beta Replicase (Lizardi, P. M. et al., 1988,
Bio/Technology 6:1197).
[0073] To determine gene expression level of a given complement
pathway associated molecule, methods routinely practiced in the
art, such as a differential display procedure, Northern analysis,
RT-PCR, and DNA probe arrays, can be employed to detect the
expression level of a given complement pathway associated in a
subject. Fingerprint profiles of expression levels of a number of
complement pathway associated molecules, or of expression levels of
a given complement pathway associated molecule in a population of
control subjects with or without macular degeneration-related
disorders, can be generated using methods described in the art,
e.g., WO99/23254; and Cronin et al. Human Mutation 7:244, 1996.
B. Diagnosing Variant form of Nucleic Acids Encoding Complement
Pathway Molecules or Autoantigens
[0074] The present invention provides methods for diagnosing, or
determining a predisposition to development of, a macular
degeneration-related disorder by detecting a variant form of at
least one nucleic acid molecule encoding a complement pathway
associated molecule or an autoantigen (e.g., RPE-proteins,
choroidal proteins, retinal proteins, or autoantigens from other
tissues that bind to the ocular tissues). The nucleic acids can be,
e.g., genomic DNA, cDNA, or mRNA. Compared to the wild-type nucleic
acid sequence, the variant nucleic acid can have point mutations,
frameshift mutations, or deletions. In some methods, the variant
nucleic acid can have the wild-type sequence except for a single
nucleotide polymorphism.
[0075] A variety of means are currently available for detecting
variant genes or nucleic acids. For example, many methods are
available for detecting specific alleles at human polymorphic loci.
For example, single nucleotide polymorphism in complement pathway
genes can be detected as described, e.g., in Mundy et al., U.S.
Pat. No. 4,656,127; Cohen et al., French Patent 2,650,840; and
WO91/02087). Additional procedures for assaying polymorphic sites
in DNA have been described in Komher, J. S. et al., Nucl. Acids.
Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. Acids Res. 18:3671
(1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990);
Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.)
88:1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164
(1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P. et
al., Anal. Biochem. 208:171-175 (1993)).
[0076] Other suitable techniques to detect variant nucleic acids
encoding oligonucleotide ligation assay (OLA), as described, e.g.,
in U.S. Pat. No. 4,998,617 and Landegren, U. et al. ((1988) Science
241:1077-1080); or selective oligonucleotide hybridization as
described, e.g., in Saiki et al. (1986) Nature 324:163); and Saiki
et al (1989) Proc. Natl. Acad. Sci USA 86:6230). For mutations that
produce premature termination of protein translation, the protein
truncation test (PTT) offers an efficient diagnostic approach
(Roest, et. al., (1993) Hum. Mol. Genet. 2:1719-21; van der Luijt,
et. al., (1994) Genomics 20:1-4). Mismatched bases in RNA/RNA or
RNA/DNA or DNA/DNA heteroduplexes can be detected as described,
e.g., in Myers, et al., Science 230:1242, 1985.
[0077] In addition, a variety of sequencing reactions can be used
to directly sequence the allele. Exemplary sequencing reactions
include those based on techniques developed by Maxim and Gilbert
((1977) Proc. Natl. Acad Sci USA 74:560) or Sanger (Sanger et al
(1977) Proc. Nat. Acad. Sci USA 74:5463). It is also contemplated
that any of a variety of automated sequencing procedures can be
utilized when performing the subject assays (see, for example
Biotechniques (1995) 19:448), including sequencing by mass
spectrometry (see, for example PCT publication WO 94/16101; Cohen
et al. (1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993)
Appl Biochem Biotechnol 38:147-159).
[0078] Any cell type or tissue can be utilized to obtain nucleic
acid samples for use in the diagnostics described herein. For
example, a DNA sample is obtained from blood, a bodily fluid (e.g.,
secretion from the eye), urine, or saliva. In some methods, samples
obtained from the eyes are preferred because test results obtained
from a sample from the eye evidence that an abnormal expression or
activity is likely due to an ocular dysfunction, e.g., macular
degeneration, thereby providing a more rapid and accurate
diagnostic test for macular degeneration-related disorders. In some
methods, nucleic acid tests can be performed on dry samples (e.g.
hair or skin). The diagnostic methods can also be performed in situ
directly upon tissue sections (fixed and/or frozen) of subject
tissue obtained from biopsies or resections, such that no nucleic
acid purification is necessary. Nucleic acid reagents may be used
as probes and/or primers for such in situ procedures (see, e.g.,
Nuovo et al., 1992, PCR in situ hybridization: protocols and
applications, Raven Press, NY).
[0079] In addition to methods which focus primarily on the
detection of one nucleic acid sequence, profiles may also be
assessed in such detection schemes. Fingerprint profiles may be
generated as described.
[0080] The present invention also provides kits for detecting a
predisposition for developing a macular degeneration-related
disorder. This kit may contain one or more oligonucleotides,
including 5' and 3' oligonucleotides that hybridize 5' and 3' to at
least one complement pathway molecule. The assay kits and
diagnostic methods can also employ labeled oligonucleotides to
allow ease of identification in the assays. Examples of labels
which may be employed include radio-labels, enzymes, fluorescent
compounds, streptavidin, avidin, biotin, magnetic moieties, metal
binding moieties, antigen or antibody moieties, and the like.
C. Diagnosing Abnormal Activity of Complement System
[0081] The present invention also provides methods for diagnosing,
or determining a predisposition to developing a macular
degeneration-related disorder by detecting an abnormal bioactivity
of the complement system. The abnormal activities to be detected
can be that which triggers the activation of each of the three
complement pathways. The abnormal activity can also be that of
individual molecules which are produced during activation of the
three complement pathways. The abnormal complement activities to be
detected can also be any of the cellular activities displayed by
the activated complement system. Thus, abnormal complement
activities to be detected with the present invention encompass,
e.g., increased or decreased enzymatic or regulatory function of a
complement pathway protein such as C3a, C5a, C5b-9 complex,
vitronectin, or CR1, serum presence or increased level of
autoantibodies against complement component, abnormal cellular
activities mediated by activated complement system such as lysis of
target cells (e.g., RPE and choroidal cells), chemotaxis,
opsonization, stimulation of vascular and other smooth muscle
cells, degranulation of mast cells, increased permeability of small
blood vessels, initiation of inflammatory processes, directed
migration and activation of leukocytes, and activation of B
lymphocytes, macrophages, dendritic cells, and neutrophils.
[0082] With respect to each complement bioactivity, abnormality
refers to a difference between that activity detected in a
biological sample (e.g., blood) from a test subject and the average
value of the activity detected in a population of control subjects
without macular degeneration-related disorders. Preferably, the
difference is by at least 20%, 30%, or 40%. More preferably, the
difference is more than at least 50%, 60%, 70%, or 80%. Most
preferably, the difference is by at least 90%.
[0083] 1. Detecting Complement Activation
[0084] Various methods can be used to measure activities of
complement pathway molecules and activation of the complement
system (see, e.g., U.S. Pat. No. 6,087,120; and Newell et al., J
Lab Clin Med, 100:437-44, 1982). For example, the complement
activity can be monitored by (i) measurement of inhibition of
complement-mediated lysis of red blood cells (hemolysis); (ii)
measurement of ability to inhibit cleavage of C3 or C5; and (iii)
inhibition of alternative pathway mediated hemolysis.
[0085] The two most commonly used techniques are hemolytic assays
(see, e.g., Baatrup et al., Ann Rheum Dis, 51:892-7, 1992) and
immunological assays (see, e.g., Auda et al., Rheumatol Int, 10:
185-9, 1990). The hemolytic techniques measure the functional
capacity of the entire sequence-either the classical or alternative
pathway. Immunological techniques measure the protein concentration
of a specific complement component or split product. Other assays
that can be employed to detect complement activation or measure
activities of complement components in the methods of the present
invention include, e.g., T cell proliferation assay (Chain et al.,
J Immunol Methods, 99:221-8, 1987), and delayed type
hypersensitivity (DTH) assay (Forstrom et al., 1983, Nature
303:627-629; Hallidayet al., 1982, in Assessment of Immune Status
by the Leukocyte Adherence Inhibition Test, Academic, New York pp.
1-26; Koppi et al., 1982, Cell. Immunol. 66:394-406; and U.S. Pat.
No. 5,843,449).
[0086] In hemolytic techniques, all of the complement components
must be present and functional. Therefore hemolytic techniques can
screen both functional integrity and deficiencies of the complement
system (see, e.g., Dijk et al., J Immunol Methods 36: 29-39, 1980;
Minh et al., Clin Lab Haematol. 5:23-34 1983; and Tanaka et al., J
Immunol 86: 161-170, 1986). To measure the functional capacity of
the classical pathway, sheep red blood cells coated with hemolysin
(rabbit IgG to sheep red blood cells) are used as target cells
(sensitized cells). These Ag-Ab complexes activate the classical
pathway and result in lysis of the target cells when the components
are functional and present in adequate concentration. To determine
the functional capacity of the alternative pathway, rabbit red
blood cells are used as the target cell (see, e.g., U.S. Pat. No.
6,087,120).
[0087] The hemolytic complement measurement is applicable to detect
deficiencies and functional disorders of complement proteins, e.g.,
in the blood of a subject, since it is based on the function of
complement to induce cell lysis, which requires a complete range of
functional complement proteins. The so-called CH50 method, which
determines classical pathway activation, and the AP50 method for
the alternative pathway have been extended by using specific
isolated complement proteins instead of whole serum, while the
highly diluted test sample contains the unknown concentration of
the limiting complement component. By this method a more detailed
measurement of the complement system can be performed, indicating
which component is deficient.
[0088] Immunologic techniques employ polyclonal or monoclonal
antibodies against the different epitopes of the various complement
components (e.g., C3, C4 an C5) to detect, e.g., the split products
of complement components (see, e.g., Hugli et al., Immunoassays
Clinical Laboratory Techniques 443-460, 1980; Gorski et al., J
Immunol Meth 47: 61-73, 1981; Linder et al., J Immunol Meth 47:
49-59, 1981; and Burger et al., J Immunol 141: 553-558, 1988).
Binding of the antibody with the split product in competition with
a known concentration of labeled split product could then be
measured. Various assays such as radio-immunoassays, ELISA's, and
radial diffusion assays are available to detect complement split
products.
[0089] The immunologic techniques provide a high sensitivity to
detect complement activation, since they allow measurement of
split-product formation in blood from a test subject and control
subjects with or without macular degeneration-related disorders.
Accordingly, in some methods of the present invention, diagnosis of
a macular degeneration-related disorder is obtained by measurement
of abnormal complement activation through quantification of the
soluble split products of complement components (e.g., C3a, C4a,
C5a, and the C5b-9 terminal complex) in blood plasma from a test
subjects. The measurements can be performed as described, e.g., in
Chenoweth et al., N Engl J Med 304: 497-502, 1981; and Bhakdi et
al., Biochim Biophys Acta 737: 343-372, 1983. Preferably, only the
complement activation formed in vivo is measured. This can be
accomplished by collecting a biological sample from the subject
(e.g., serum) in medium containing inhibitors of the complement
system, and subsequently measuring complement activation (e.g.,
quantification of the split products) in the sample.
[0090] 2. Detecting Autoantibodies to Macular
Degeneration-Associated Autoantigens or Immune Complexes
[0091] Abnormal presence or increased level of autoantibodies
against tissue specific antigens, neoantigens, and/or complement
pathway molecules can also be an indicator of a predisposition of
development of macular degeneration-related disorders. Accordingly,
the present invention provides methods for diagnosing, or
determining a predisposition to development of, a macular
degeneration-related disorder by detecting autoantibodies against
macular degeneration-associated autoantigens. The diagnostic
methods of the present invention are also directed to detecting in
a subject circulating immune complexes that can also be indicative
of a macular degeneration-related disorder.
[0092] As discussed above, autoantibodies against various macular
degeneration-associated autoantigens were found in serum from
subjects with macular degeneration-related disorders. Such
autoantigens include complement pathway molecules and various
autoantigens from RPE, choroid, and retina. Thus, diagnosis can be
directed to serum autoantibodies against macular
degeneration-associated autoantigens such as vitronectin, .beta.
crystallin, calreticulin, serotransferrin, keratin, pyruvate
carboxylase, C1, and villin 2. For example, in some methods, a
blood sample (e.g., serum) from a test subject can be examined for
specific binding to these known autoantigens. In some methods, the
sample is examined for specific binding to any autoantigens from
the ocular tissues (e.g., RPE, choroid) using proteins extracted
from the ocuclar tissues. For example, proteins extracted from
ocular tissues from non-human animals (e.g., rat) or from deceased
human beings can be used to screen for autoantibodies against
ocular autoantigens in a serum from the subject.
[0093] In some methods, the diagnosis also include detection of
autoantibodies against neoantigens. Neoantigens are antigens
resulting from modification and/or crosslinking of existing
molecules by various processes such as oxidation. Examples of
neoantigens include neoantigens associated with oxidized LDL in
atherosclerosis (Reaven et al., Adv Exp Med Biol, 366(-HD-):
113-28, 1994; Kita et al., Ann N Y Acad Sci, 902(-HD-):95-100,
2000), or oxidation-derived complex in other diseases (Ratnoff et
al., Am J Reprod Immunol, 34:72-9 1995; and Debrock et al., FEBS
Lett, 376:243-6, 1995). Further, detection of autoantibodies
against autoantigens from other tissues can be indicative of a
systemic nature of that macular degeneration-related disorder.
[0094] A number of biochemical or immunochemical techniques can be
readily employed to detect autoantibodies in a biological sample
from a subject. For example, techinques routinely praticed in the
art such as immunoprecipitation or radioimmune assays are suitable
for detecting autoantibodies in a serum sample. Various other
methods for detection of autoantibodies against complement proteins
or complement regulatory proteins have been described in the art.
For example, Pinter et al. described detection of autoantibodies
against two complement regulatory molecules expressed in the
membrane of human cells (CD46 and CD59) in sera from subjects with
multiple sclerosis (J Neurovirol, 6 Suppl 2:S42-6, 2000). Strife et
al. described detection of serum autoantibodies to C3 convertases
C3bBb and C3bBbP in membranoproliferative glomerulonephritis (J
Pediatr. 116:S98-102, 1990). Ravelli et al. disclosed
autoantibodies to complement C1q in subjects with pediatric-onset
systemic lupus erythematosus (Clin Exp Rheumatol. 15:215-9, 1997).
As discussed in the Examples, infra, these methods can be readily
applied to detect autoantibodies against of complement components,
e.g., in the serum from a subject suspected to have an macular
degeneration-related disorder.
[0095] Significance of circulating immune complexes are well
documented in the art. For example, the causative mechanism for
glomerulonephritis is typically the deposit of circulating immune
complexes in the kidney (see, e.g., U.S. Pat. No. 6,074,642).
Circulating immune complexes as a result of activation and
consumption of individual complement components have also been
shown in many other human diseases occurs (see, e.g., U.S. Pat. No.
5,221,616). Detection of circulating immune complexes also can be
of diagnostic value in macular degeneration related disorders. A
number of assays are routinely practiced to detect circulating
immune complexes in a subject, e.g., as described in
Tomimori-Yamashita et al., Lepr Rev, 70(3):261-71, 1999
(antibody-based enzyme-linked immunosorbent assay); Krapf et al., J
Clin Lab Immunol, 21(4): 183-7, 1986 (fluorescence linked
immunosorbent assay); Kazeem et al., East Afr Med J, 67
(6):396-403, --1990 (laser immunonephelometry); and Rodrick et al.,
J Clin Lab Immunol, 7(3): 193-8, 1982 (Protein A-glass fiber filter
assay, PA-GFF, and polyethylene glycol insolubilization assay).
Each of these well known assays can be employed to detect
circulating immune complexes for the methods of the present
invention.
D. Additional Tests for Diagnosing Macular Degeneration-Related
Disorders
[0096] If a diagnostic test described above gives a positive
outcome, the subject is, at minimum, identified as being
susceptible to or at risk of a macular degeneration-related
disorder. The subject is then typically subject to further tests or
screening. For example, the present inventors have found that there
is a correlation between macular degeneration and the distribution
of the C5b-9 complex in choriocapillaris. Thus, the additional
tests or screening can include examination of the function or
physical integrity of an ocular tissue of the subject's eyes (e.g.,
choriocapillaris) by one of the ophthalmologic procedures described
below. The additional tests or screening can also include analyses
of additional complement pathway molecules that have not already
been tested. The additional tests can also include examination of
the presence of macular degeneration-associated genetic markers,
drusen-associated phenotypic markers, or drusen-associated
genotypic markers that often correlate with macular
degeneration-related disorders, as discussed below.
[0097] Macular degeneration-associated genetic markers are genetic
loci which are shown to be correlated with a risk of developing a
macular degeneration-related disorder. Such markers have been
described, e.g., in WO 00/52479, and include, e.g., 1p21-q13, for
recessive Stargardt's disease or fundus flavi maculatus (Allikmets
et al. Science 277:1805-1807, 1997); 1q25-q31, for recessive AMD
(Klein et al., Arch. Ophthalmol. 116:1082-1088, 1988); 2p16, for
dominant radial macular drusen, dominant Doyne honeycomb retinal
degeneration, or Malattia Leventinese (Edwards et al., Am. J.
Ophthalmol. 126:417-424, 1998); 6p21.2-cen, for dominant macular
degeneration, adult vitelloform (Felbor et al. Hum. Mutat.
10:301-309, 1997); 6p21.1 for dominant cone dystrophy (Payne et al.
Hum. Mol. Genet. 7:273-277, 1998); 6q, for dominant cone-rod
dystrophy (Kelsell et al. Am. J. Hum. Genet. 63:274-279, 1998);
6q11-q15, for dominant macular degeneration, Stargardt's-like
disease (Griesinger et al., Am. J. Hum. Genet. 63:A30, 1998);
6q14-q16.2, for dominant macular degeneration, North Carolina Type
(Robb et al., Am. J. Ophthalmol. 125:502-508, 1998); 6q25-q26,
dominant retinal cone dystrophy 1
((http://www3.ncbi.nlm.nih.gov/omim, (1998)); 7p21-p15, for
dominant cystoid macular degeneration (Inglehearn et al., Am. J.
Hum. Genet. 55:581-582, 1994); 7q31.3-32, for dominant tritanopia,
protein: blue cone opsin (Fitzgibbon et al., Hum. Genet. 93:79-80,
1994); 11p12-q13, for dominant macular degeneration, Best type
(bestrophin) (Marquardt et al., Hum. Mol. Genet. 7:1517-1525,
1998); 13q34, for dominant macular degeneration, Stargardt type
(Zhang et al., Arch. Ophthalmol. 112:759-764, 1994); 16p12.1, for
recessive Batten disease (Munroe et al., Am. J. Hum. Genet.
61:310-316, 1997); 17p, for dominant areolar choroidal dystrophy
(Lotery, A. J. et al., Ophthalmol. Vis. Sci. 37:1124, 1996);
17p13-p12, for dominant cone dystrophy, progressive (Small et al.,
Am. J. Ophthalmol. 121:13-18, 1996); 17q, for cone rod dystrophy
(Klystra, J. A. et al., Can. J. Ophthalmol. 28:79-80, 1993);
18q21.1-q21.3, for cone-rod dystrophy, de Grouchy syndrome
(Manhant, S. et al., Am. J. Hum. Genet. 57:A96, 1995; Warburg, M.
et al., Am. J. Med. Genet. 39:288-293, 1991); 19q13.3, for dominant
cone-rod dystrophy; recessive, dominant and `de novo` Leber
congenital amaurosis; dominant RP; protein: cone-rod otx-like
photoreceptor homeobox transcription factor (Li et al., Proc. Natl.
Acad. Sci USA 95:1876-1881, 1998); 22q12.1-q13.2, for dominant
Sorsby's fundus dystrophy, tissue inhibitors of metalloproteases-3
(TIMP3) (Felbor et al., Am. J. Hum. Genet. 60:57-62, 1997); and
Xp11.4, for X-linked cone dystrophy (Seymour et al., Am. J. Hum.
Genet. 62:122-129, 1998).
[0098] Drusen-associated phenotypic or genotypic markers that
correlate with macular degeneration-related disorders or drusen
associated disorders have been described in WO 00/52479. Examples
of drusen-associated phenotypic markers include: RPE dysfunction
and/or death, immune mediated events, dendritic cell activation,
migration and differentiation, extrusion of the dendritic cell
process into the sub RPE space (e.g. by detecting the presence or
level of a dendritic cell marker such as CD68, CD1a and S100), the
presence of geographic atrophy or disciform scars, the presence of
choroidal neovascularization and/or choroidal fibrosis, especially
in the macula. Examples of drusen-associated genotypic markers
include mutant genes and/or a distinct pattern of differential gene
expression. Genes expressed by dysfunctional and/or dying RPE cells
include: HLA-DR, CD68, vitronectin, apolipoprotein E, clusterin and
S-100. Genes expressed by choroidal and RPE cells in AMD include
heat shock protein 70, death protein, proteasome, Cu/Zn superoxide
dismutase, cathepsins, and death adaptor protein RAIDD. Other
markers involved in immune mediated events associated with drusen
formation include: autoantibodies (e.g. directed against drusen,
RPE and/or retina components), leukocytes, dendritic cells,
myofibroblasts, type VI collagen, and a cadre of chemokines and
cytokines. In addition to complement proteins, other molecules
associated with drusen include: immunoglobulins, amyloid A, amyloid
P component, HLA-DR, fibrinogen, Factor X, prothrombin, C reactive
protein (CRP) apolipoprotein A, apolipoprotein E, antichymotrypsin,
thrombospondin, and vitronectin. Markers of drusen associated
dendritic cells include: CD1a, CD4, CD14, CD68, CD83, CD86, and
CD45, PECAM, MMP14, ubiquitin, and FGF. Important dendritic
cell-associated accessory molecules that participate in T cell
recognition include ICAM-1, LFA1, LFA3, and B7, IL-1, IL-6, IL-12,
TNF.alpha., GM-CSF and heat shock proteins. Markers associated with
dendritic cell expression include: colony stimulating factor,
TNF.alpha., and IL-1. Markers associated with dendritic cell
proliferation include: GM-CSF, IL-4, IL-3, SCF, FLT-3 and
TNF.alpha.. Markers associated with dendritic cell differentiation
include IL-10, M-CSF, IL-6 and IL-4. Markers of fibrosis include: a
decrease in BIG H3, increase in .beta.1-integrin, increase in
collagen (e.g. collagen 6 .alpha.2 and collagen 6 .alpha.3),
increase in elastin, and an increase in human metallo elastase
(HME).
[0099] The other phenotypic or genotypic markers can be detected
with assays described above, e.g., detection of the identity,
expression level, or activities of the gene, mRNA transcript, or
encoded protein. Some markers can also be detected by one or more
ophthalmologic procedures, such as fundus fluorescein angiography
(FFA), indocyanine green angiography (ICG), fundus ophthalmoscopy
or photography (FP), electroretinogram (ERG), electrooculogram
(EOG), visual fields, scanning laser ophthalmoscopy (SLO), visual
acuity measurements, dark adaptation measurements or other standard
method. Ophthalmologic procedures have been used to evaluate
patients with various macular degeneration-related disorders. For
example, Spraul et al. (Klin Monatsbl Augenheilkd, 21:141-8, 1998)
described the use of optical coherence tomography for evaluation of
patients with AMD; Kohno et al. (Bull Soc Belge Ophtalmol,
259(-HD-):81-8, 1995) reports detection of choroidal
neovascularization in age-related macular degeneration using
subtraction methods in indocyanine green angiography; Kuck et al.
(Retina, 13:36-9, 1993) discussed examination of patients with
exudative age-related macular degeneration and clinical signs of
subretinal neovascular membranes were examined by scanning laser
fluorescein angiography; Kaluzny et al. (Klin Oczna, 101:355-9,
1999) and Yuzawa et al. (Eur J Ophthalmol, 2:115-21, 1992)
described the use of indocyanine green (ICG) angiography in
diagnosis of occult choroidal neovascularization in age-related
macular degeneration; Lubinski et al. (Klin Oczna, 100:263-8, 1998)
reported evaluation of foveal cone function in healthy subjects and
patients with different macular diseases with foveal cone
electroretinogram (FCERG, a type of focal ERG); and Kakehashi et
al. (Jpn J Ophthalmol, 40:116-22, 19960 discussed differential
diagnosis of macular breaks using the scanning laser ophthalmoscope
(SLO). All these procedures can be used in conjunction with the
diagnostic methods of the present invention. For instance, fundus
autofluorescein angiography can be used for identifying defects at
the level of the RPE (see, e.g., Delori et al., Invest Ophthalmol,
14:487-92, 1975; Holz et al., Graefes Arch Clin Exp Ophthalmol,
237:145-52, 1999; and Delori et al., Invest Ophthalmol Vis Sci,
36:718-29, 1995).
[0100] Further tests or screening can also include monitoring for
clinical symptoms of a macular degeneration-related disorder, which
include presence of drusen, retinal pigmentary changes, and
includes early stages of degeneration of the macula in which vision
has not been significantly affected ("dry" macular degeneration),
atrophic macular degeneration, and exudative disease in which
neovascularization is prevalent ("wet" macular degeneration).
Further screening can also include analyses of family history for
related family members with macular degeneration-related disorders,
and/or genetic analyses of polymorphisms associated with macular
degeneration-related disorders (as described above). As a result of
one or more of these additional tests, the initial diagnosis based
on abnormal complement activities or expression levels can be
confirmed (or otherwise), and the particular type of macular
degeneration-related disorder affecting a subject can be
identified.
IV. Therapeutics: Prevention and Treatment of Macular
Degeneration-Related Disorders
[0101] The present invention provides methods for treating or
preventing macular degeneration-related disorders in a subject by
administering to the subject therapeutic agents that modulate
activity of the complement system. Detrimental nonspecific
activation of the complement system, or unfavorable activation by
the alternative pathway, can be prevented or treated by therapeutic
agents of the invention. For example, as discussed above, the
choriocapillaris was implicated as a target of MAC attack in AMD
patients, and MAC is present in choroidal and RPE cell membranes.
Accordingly, in some methods, therapeutic agents are directed to
prevention or alleviation of damages to the choriocapillaris and/or
RPE caused by the C5b-9 complex. In some methods, the treatment is
directed to inhibition of the formation of the C5b-9
membrane-attack complex using, e.g., inhibitors such as vitronectin
or clusterin, or a monoclonal antibody to the complement component
C8 alpha subunit (see, e.g., Abraha et al., A; Biochem J,
264:933-6, 1989).
[0102] The therapeutics of the present invention are directed to
complement pathway associated molecules as well as cellular
activities regulated by the activated complement system. Thus,
targets of the therapeutic agents of the present invention can
include any of the initiators of complement pathways (e.g.,
autoantibodies), molecules produced during complement activation,
molecules produced or differentially regulated as a result of
complement activation, regulators of complement pathways, and
molecules regulated by the activated complement system (e.g.,
MACIF). The therapeutic agents can also be used to modulate
cellular activities (biologic or immune functions) directly or
indirectly mediated by the complement system. Thus, the therapeutic
agents of the invention can be directed to cellular activities such
as lysis of target cells, chemotaxis, opsonization, stimulation of
vascular cells, degranulation of mast cells, increased permeability
of small blood vessels, directed migration of leukocytes, and
activation of B lymphocytes, macrophages, dendritic cells,
monocytes, and neutrophils. These cellular functions can be either
antagonized or agonized with therapeutic agents of the present
invention.
A. General Considerations
[0103] Subjects amenable to treatment include those who are
presently asymptomatic but who are at risk of developing a
symptomatic macular degeneration-related disorder at a later time.
For example, human individuals include those having relatives who
have experienced such a disease, and those whose risk is determined
by analysis of genetic or biochemical markers, by biochemical
methods, or by other assays such as T cell proliferation assay (as
described above).
[0104] Other subjects who are amenable to treatment include
individuals free of known complement related diseases other than
macular degeneration-related disorders. Complement related diseases
or disorders have been described in the art, e.g., in U.S. Pat. No.
6,169,068. Examples of known complement related diseases include:
neurological disorders, multiple sclerosis, stroke, Guillain Barre
Syndrome, traumatic brain injury, Parkinson's disease, disorders of
inappropriate or undesirable complement activation, hemodialysis
complications, hyperacute allograft rejection, xenograft rejection,
interleukin-2 induced toxicity during IL-2 therapy, inflammatory
disorders, inflammation of autoimmune diseases, Crohn's disease,
adult respiratory distress syndrome, thermal injury including burns
or frostbite, post-ischemic reperfusion conditions, myocardial
infarction, balloon angioplasty, post-pump syndrome in
cardiopulmonary bypass or renal bypass, hemodialysis, renal
ischemia, mesenteric artery reperfusion after acrotic
reconstruction, infectious disease or sepsis, immune complex
disorders and autoimmune diseases, rheumatoid arthritis, systemic
lupus erythematosus (SLE), SLE nephritis, proliferative nephritis,
hemolytic anemia, and myasthenia gravis. In addition, other known
complement related disease are lung disease and disorders such as
dyspnea, hemoptysis, ARDS, asthma, chronic obstructive pulmonary
disease (COPD), emphysema, pulmonary embolisms and infarcts,
pneumonia, fibrogenic dust diseases, inert dusts and minerals
(e.g., silicon, coal dust, beryllium, and asbestos), pulmonary
fibrosis, organic dust diseases, chemical injury (due to irritant
gasses and chemicals, e.g., chlorine, phosgene, sulfur dioxide,
hydrogen sulfide, nitrogen dioxide, ammonia, and hydrochloric
acid), smoke injury, thermal injury (e.g., burn, freeze), asthma,
allergy, bronchoconstriction, hypersensitivity pneumonitis,
parasitic diseases, Goodpasture's Syndrome, pulmonary vasculitis,
and immune complex-associated inflammation.
[0105] Genetic markers of risk of developing a macular
degeneration-related disorder have been described above. The
presence of any of these genetic markers in asymptomatic
individuals signifies that a complement pathway-mediated process
leading to a macular degeneration-related disorder is likely
underway, although has not yet progressed so far as to produce
symptoms.
[0106] A biochemical marker can be any of those described in the
previous sections, such as an abnormal activity or abnormal level
of, or an autoantibody against, a complement pathway molecule. If
such a marker is detected, treatment should usually begin shortly
thereafter. If likelihood of developing a macular
degeneration-related disorder is based on relatives having the
disease or detection of a genetic marker, treatment can also be
administered shortly after identification of these risk factors, or
shortly after diagnosis. Alternatively, an individual found to
possess a genetic marker can be left untreated but subjected to
regular monitoring for biochemical or symptomatic changes without
treatment. The decision whether to treat immediately or to monitor
symptoms depends in part on the extent of risk predicted by the
various other marker(s) found in the subject. Once begun, the
treatment is typically continued at intervals for a period of a
week, a month, three months, six months or a year. In some
subjects, treatment is administered for up to the rest of a
subject's life. Treatment can generally be stopped if a biochemical
risk marker disappears.
[0107] In addition to ocular diseases (e.g., AMD), subjects with
other age-related diseases, such as amyloidosis, elastosis, dense
deposit disease, and atherosclerosis, are also amendable to
treatment with the methods of the present invention. Similarly,
subjects with other types of macular degeneration-related
disorders, e.g., membranous and post-streptococcal/segmental
glomerulonephritis can also be treated with the presently claimed
methods.
[0108] The second principal application of the methods lies in
monitoring the condition of subjects receiving treatment for a
macular degeneration-related disorder. A successful treatment
outcome is indicated by return of complement pathway associated
activity, such as expression level, biochemical activity (e.g.,
enzymatic activity of a complement component), or serum
autoantibodies against complement pathway molecules, from abnormal
levels to or toward normal levels. Typically, such methods measure
an initial value for the level of abnormal activity (e.g., abnormal
presence of an autoantibody, abnormal level of complement pathway
molecule) before the subject has received treatment. Repeat
measurements are then made over a period of time. If the initial
level is elevated relative to the mean level in a control
population, a significant reduction in level in subsequent
measurements indicates a positive treatment outcome. Likewise, if
the initial level of an measure marker is reduced relative to the
mean in a control population, a significant increase in measured
levels relative to the initial level signals a positive treatment
outcome. Subsequently measured levels are considered to have
changed significantly relative to initial levels if a subsequent
measured level differs by more than one standard deviation from the
mean of repeat measurements of the initial level. If monitoring
reveals a positive treatment outcome, the same treatment regime can
be continued, or replaced with a treatment regime with a lower
dosage. If monitoring reveals a negative treatment outcome, the
previous treatment regime is typically modified, either by using a
different therapeutic agent or increasing the dosage of the
previous agent.
[0109] In general, the subjects can be treated with a combination
of different therapeutic agents of the present invention. The
treatment can also proceed in conjunction with other known methods
of treating macular degeneration-related disorders, e.g.,
antibiotic treatment as described in U.S. Pat. No. 6,218,368.
[0110] Further, immunosuppression could provide therapeutic effects
in subjects suffering from, or at risk of developing, macular
degeneration-related disorders (e.g., by inhibiting or ameliorating
autoimmune responses). Thus, subjects to be treated with
therapeutic agents of the present invention can also be
administered with immunosuppressive agents such as cyclosporine.
Immunosuppressive agents are agents capable of suppressing immune
responses. These agents include cytotoxic drugs, corticosteriods,
nonsteroidal anti-inflammatory drugs (NSAIDs), specific
T-lymphocyte immunosuppressants, and antibodies or fragments
thereof (see Physicians' Desk Reference, 53rd edition, Medical
Economics Company Inc., Montvale, N.J. (1999). Immunosuppressive
treatment is typically continued at intervals for a period of a
week, a month, three months, six months or a year. In some
patients, treatment is administered for up to the rest of a
patient's life. Treatment can generally be stopped if a biochemical
risk marker disappears. Treatment can sometimes be temporarily
discontinued if the subject is infected with a pathogen for which a
full immune response is needed for clearance.
B. Modulation of Levels of Complement Pathway Molecules
[0111] The present invention provides methods for treating or
preventing the development of macular degeneration-related
disorders by modulating levels of complement pathway molecules.
Levels of either mRNAs encoding the complement pathway associated
proteins or levels of the complement pathway-associated proteins
can be modulated. In some methods, the therapeutics are inhibitors
of the expression of one or more complement components, e.g.,
complement 3, complement C5, or C5b-9 terminal complexes. In some
methods, therapeutics are agents which alter the gene expression of
factors that regulate the expression of one or more complement
components. In some methods, therapeutics are agents which alter
the gene expression of complement pathway molecules that regulate
complement activity or activation, e.g., CR1, CR2, vitronectin, or
clusterin.
[0112] Alteration of the above-noted gene expressions can be
accomplished by a number of regimes, such as (i) modulation of mRNA
synthesis, (ii) modulation of RNA turnover or degradation, (iii)
modulation of translation of mRNA into protein, (iv) modulation of
protein processing or transport, (v) modulation of formation of
protein complex of the complement system (e.g., C3 convertase, C5
convertase, or the terminal complex C5b-9) by blocking inter- or
intra-molecular binding necessary for the formation; and (vi)
modulation of the concentration of complement pathway molecules,
e.g., by targeting and destroying complement components in situ
(e.g., using enzyme-antibody techniques).
[0113] In some methods, the therapeutics of the invention relate to
antisense therapy. By administration or in situ generation of
oligonucleotide molecules which specifically hybridize to the
cellular mRNA and/or genomic DNA encoding one or more complement
pathway molecules, such a therapy functions by inhibiting
expression of that protein, e.g., by inhibiting transcription
and/or translation (see, e.g., Stanley et al., Basic Principles of
Antisense Therapeutics, Springer-Verlag, N.Y., p. 3, July 1998).
The binding can be by conventional base pair complementarity, or,
for example, in the case of binding to DNA duplexes, through
specific interactions in the major groove of the double helix.
[0114] In some methods, the therapeutic agents utilize zinc finger
motif which can be selected to bind diverse nucleic acid sequences
(see, e.g., U.S. Pat. No. 6,140,466). For example, therapeutic
agents which activate or repress a target nucleic acid expression
can be expressed as fusions with zinc finger motifs. Such fusion
proteins are useful for inhibiting, activating or enhancing gene
expression from a zinc finger-nucleotide binding motif containing
promoter or other transcriptional control element, as well as a
structural gene or RNA sequence.
C. Modulation of Complement Activity
[0115] The present invention provides methods of treatment or
prevention of macular degeneration-related disorders by
administering therapeutic agent which modulate bioactivities of the
complement pathway molecules or cellular activities mediated by the
activated complement system. In addition to methods described
herein, methods for administering therapeutic agents to modulate
complement activities in a subject have also been described in the
art. For example, U.S. Pat. No. 5,472,939 describes modulation of
complement mediated activities by administering to a subject CR1 or
its fragment which inhibits C3 convertase activity or C5 convertase
activity.
[0116] Various therapeutic agents are suitable for the present
invention. Some agents are known in the art to be able to modulate
the activities of complement components (see, e.g., U.S. Pat. No.
5,808,109). Many agents have been reported to diminish
complement-mediated activity. Such agents include: amino acids
(Takada, Y. et al. Immunology 1978, 34, 509); phosphonate esters
(Becker, L. Biochem. Biophy. Acta 1967, 147, 289); polyanionic
substances (Conrow, R. B. et al. J. Med. Chem. 1980, 23, 242);
sulfonyl fluorides (Hansch, C.; Yoshimoto, M. J. Med. Chem. 1974,
17, 1160, and references cited therein); polynucleotides (DeClercq,
P. F. et al. Biochem. Biophys. Res. Commun. 1975, 67, 255); pimaric
acids (Glovsky, M. M. et al. J. Immlunol. 1969, 102, 1); porphines
(Lapidus, M. and Tomasco, J. Immunopharmacol. 1981, 3, 137);
several antiinflammatories (Burge, J. J. et al. J. Immunol. 1978,
120, 1625); phenols (Muller-Eberhard, H. J. 1978, in Molecular
Basis of Biological Degradative Processes, Berlin, R. D. et al.,
eds. Academic Press, New York, p. 65); and benzamidines (Vogt, W.
et al Immunology 1979, 36, 138). Some of these agents function by
general inhibition of proteases and esterases. Others are not
specific to any particular intermediate step in the complement
pathway, but, rather, inhibit more than one step of complement
activation. Examples of the latter compounds include the
benzamidines, which block C1, C4 and C5 utilization (see, e.g.,
Vogt et al. Immunol. 1979, 36, 138).
[0117] Additional agents known in the art that can inhibit activity
of complement components include K-76, a fungal metabolite from
Stachybotrys (Corey et al., J. Amer. Chem. Soc. 104: 5551, 1982).
Both K-76 and K-76 COOH have been shown to inhibit complement
mainly at the C5 step (Hong et al., J. Immunol. 122: 2418, 1979;
Miyazaki et al., Microbiol. Immunol. 24: 1091, 1980), and to
prevent the generation of a chemotactic factor from normal human
complement (Bumpers et al., Lab. Clinc. Med. 102: 421, 1983). At
high concentrations of K-76 or K-76 COOH, some inhibition of the
reactions of C2, C3, C6, C7, and C9 with their respective preceding
intermediaries is exhibited. K-76 or K-76 COOH has also been
reported to inhibit the C3b inactivator system of complement (Hong
et al., J. Immunol. 127: 104-108, 1981). Other suitable agents for
practicing methods of the present invention include griseofulvin
(Weinberg, in Principles of Medicinal Chemistry, 2d Ed., Foye, W.
O., ed., Lea & Febiger, Philadelphia, Pa., p. 813, 1981),
isopannarin (Djura et al., Aust. J. Chem. 36: 1057, 1983), and
metabolites of Siphonodictyon coralli-phagum (Sullivan et al.,
Tetrahedron 37: 979, 1981).
D. Macular Degeneration-Related Autoantigens and Tolerance
[0118] As discussed in the Examples, infra, autoantibodies against
various complement pathway molecules and against RPE, choroid, and
retina proteins were found in serum of patients with macular
degeneration-related disorders (e.g., AMD and Malattia
Leventinese). This evidence indicates that autoimmune response play
certain roles in the etiology macular degeneration-related
disorders. Thus, identification of macular degeneration-associated
autoantigens and/or autoantibodies also provides novel means for
treating or preventing macular degeneration-related disorders. For
example, novel therapeutics specifically directed to these
autoantigens can be designed and produced, e.g., by computed-aided
methods (see, e.g., Topper et al., Clin Orthop, -HD-(256):39-43,
1990).
[0119] The presence of macular degeneration-associated autoantigens
and autoantibodies underscores how these molecules can activate the
complement system and subsequent damages to the ocular tissues
(e.g., choriocapillaris in RPE/choroid interface). For example, the
complement system can be activated, e.g., by antigen-antibody
complexes formed by the autoantigens and autoantibodies through the
classic pathway. However, they can also activate the complement
system through the other pathways, as demonstrated by the present
inventors (see, e.g., Example 6). Thus, identification of the
macular degeneration-related autoantigens provides another means of
treating or preventing macular degeneration through induction in a
subject of tolerance to the specific macular degeneration-related
autoantigen. Induction of immunological tolerance is a therapeutic
or preventive method in which a lack of immune responses to certain
antigens is achieved. Induction of tolerance against a given
antigen can be performed as described, e.g., in U.S. Pat. Nos.
6,153,203, 6,103,235, and 5,951,984.
[0120] To induce tolerance, it is to be noted that the nature of
response (i.e., immunogenic or tolerogenic) depends on the dose,
physical form and route of administration of antigen. High or low
doses of an antigen often lead to immunotolerance, whereas
intermediate doses may be immunogenic. Monomeric forms of antigen
are usually tolerogenic, whereas high molecular weight aggregates
are likely to be immunogenic. Oral, nasal, gastric or intravenous
injection of antigen frequently leads to tolerance, whereas
intradermal or intramuscular challenge especially in the presence
of adjuvants favors an immunogenic response. See Marx, Science 252,
27-28 (1991); Trentham et al., Science 261, 1727-1730 (1993);
Metzler & Wraith, International Immunology 5, 1159-1165 (1993);
Cobbold et al., WO90/15152 (1990).
[0121] Identification of macular degeneration-related autoantigens
also provide means for further understanding the genetic nature of
macular degeneration-related disorders. Similar to many other
diseases, mutations in the genes which encode the macular
degeneration-associated autoantigens (e.g., complement pathway
associated proteins, or the RPE autoantigens) can be the genetic
cause of macular degeneration-related disorders. For example, a
number of diseases are due to deficiencies in proteins associated
with the complement pathway, and the deficiency is often due to
mutations in the complement protein. Examples of such disease
include: SLE like symptoms (point mutation in C1q); hereditary
angioedema (mutations and polymorphisms in C1q inhibitor); SLE
(deletions in C2); pyogenic infections (61 bp deletion in exon 18
of C3 gene); membranoproliferative (C3); glomerulonephritis (C3);
partial lipodystrophy (C3); SLE (frameshift in C4a); predisposition
to Neisseria (C6: stop codon insertion leading to truncated gene
product); meningitis and Neisseria infection (Factor P (Properdin):
point mutations; X-linked); autosomal recessive atypical hemolytic
uremic syndrome (Factor H: point mutations); aplastic anemia and
paroxysmal nocturnal hemoglobinuria (PNH) (CD59: deletion in codon
16, also single base pair mutations; and PNH (CD55, deletion point
mutation).
[0122] To identify the genetic causes of macular
degeneration-related disorders, the specific autoantigens
identified, e.g., as described in Examples 9-13, can be subject to
further analysis. For example, the identity and sequence
information of the autoantigens can be revealed by standard amino
acid sequencing procedures (e.g., Current Protocols in Molecular
Biology, Ausubel, F. M. et al., 1999, John Wiley & Sons, Inc.,
New York) as well as other methods for protein identification
(e.g., matrix assisted-laser desorption ionization mass
spectrometry, as disclosed in Example 11). Polynucleotide primers
can be generated and used to clone the genes which encode these
autoantigens with standard techniques routinely practiced in
molecular biology (Sambrook et al., Molecular Cloning A Laboratory
Manual, 3rd Ed., 2000, Cold Spring Harbor Laboratory Press). The
nucleotide sequences of such autoantigens can thus be obtained. The
sequences can be compared with the DNA sequences from the genomic
databases (e.g., GenBank). Any mutation or polymorphism identified
in the autoantigen-encoding sequence relative to a wild type
sequence would indicate that the corresponding gene is a likely
candidate which causes the macular degeneration-related
disorder.
E. Screening for Novel Therapeutics for Macular
Degeneration-Related Disorders
[0123] 1. Test Agents
[0124] The present invention provides methods for prevention or
treatment of diseases or disorders associated with abnormal
complement activity by administering therapeutic agents that
modulate complement activity. In addition to the above-described
agents, therapeutic agents that modulate complement activity can
also be obtained by screening test agents with high throughput
screening techniques.
[0125] Test agents that can be screened with include polypeptides,
beta-turn mimetics, polysaccharides, phospholipids, hormones,
prostaglandins, steroids, aromatic compounds, heterocyclic
compounds, benzodiazepines, oligomeric N-substituted glycines,
oligocarbamates, polypeptides, saccharides, fatty acids, steroids,
purines, pyrimidines, derivatives, structural analogs or
combinations thereof. Usually, test compounds are organic. Some
test compounds are synthetic molecules, and others natural
molecules.
[0126] Test agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds.
Combinatorial libraries can be produced for many types of compound
that can be synthesized in a step-by-step fashion. Large
combinatorial libraries of compounds can be constructed by the
encoded synthetic libraries (ESL) method described in WO 95/12608,
WO 93/06121, WO 94/08051, WO 95/35503 and WO 95/30642. Peptide
libraries can also be generated by phage display methods (see,
e.g., Devlin, WO 91/18980). Libraries of natural compounds in the
form of bacterial, fungal, plant and animal extracts can be
obtained from commercial sources or collected in the field. Known
pharmacological agents can be subject to directed or random
chemical modifications, such as acylation, alkylation,
esterification, amidification to produce structural analogs.
[0127] Combinatorial libraries of peptides or other compounds can
be fully randomized, with no sequence preferences or constants at
any position. Alternatively, the library can be biased, i.e., some
positions within the sequence are either held constant, or are
selected from a limited number of possibilities. For example, in
some cases, the nucleotides or amino acid residues are randomized
within a defined class, for example, of hydrophobic amino acids,
hydrophilic residues, sterically biased (either small or large)
residues, towards the creation of cysteines, for cross-linking,
prolines for SH-3 domains, serines, threonines, tyrosines or
histidines for phosphorylation sites, or to purines.
[0128] The test agents can be naturally occurring proteins or their
fragments. The test agents can also be peptides, e.g., peptides of
from about 5 to about 30 amino acids, with from about 5 to about 20
amino acids being preferred, and from about 7 to about 15 being
particularly preferred. The peptides can be digests of naturally
occurring proteins, random peptides, or "biased" random
peptides.
[0129] The test agents can also be nucleic acids. Nucleic acid test
agents can be naturally occurring nucleic acids, random nucleic
acids, or "biased" random nucleic acids. For example, digests of
prokaryotic or eukaryotic genomes can be similarly used as
described above for proteins.
[0130] Libraries of test agents to be screened can also be
generated based on structural studies of a target complement
pathway molecule. Such structural studies allow the identification
of test agents that are more likely to bind to the target molecule.
The three-dimensional structure of a complement pathway molecules
can be studied in a number of ways, e.g., crystal structure and
molecular modeling. Methods of studying protein structures using
x-ray crystallography are well known in the literature. See
Physical Bio-chemistry, Van Holde, K. E. (Prentice-Hall, New Jersey
1971), pp. 221-239, and Physical Chemistry with Applications to the
Life Sciences, D. Eisenberg & D. C. Crothers (Benjamin
Cummings, Menlo Park 1979). Computer modeling of the structures of
complement pathway molecules provides another means for designing
test compounds for screening modulators of complement system.
Methods of molecular modeling have been described in the
literature, e.g., U.S. Pat. No. 5,612,894 entitled "System and
method for molecular modeling utilizing a sensitivity factor", and
U.S. Pat. No. 5,583,973 entitled "Molecular modeling method and
system". In addition, protein structures can also be determined by
neutron diffraction and nuclear magnetic resonance (NMR). See,
e.g., Physical Chemistry, 4th Ed. Moore, W. J. (Prentice-Hall, New
Jersey 1972), and NMR of Proteins and Nucleic Acids, K. Wuthrich
(Wiley-Interscience, New York 1986).
[0131] Therapeutic agents of the present invention also include
antibodies that specifically bind to the various complement pathway
molecules (e.g., C5b). Such antibodies can be monoclonal or
polyclonal, and many are described in the art. In addition, methods
for producing antibodies are well known in the art. For example,
the production of non-human monoclonal antibodies, e.g., murine or
rat, can be accomplished by, for example, immunizing the animal
with a given complement component protein or an antigenic fragment
thereof (See Harlow & Lane, Antibodies, A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y.). Such
an immunogen can be obtained from a natural source, by peptides
synthesis or by recombinant expression.
[0132] Humanized forms of mouse antibodies can be generated by
linking the CDR regions of non-human antibodies to human constant
regions by recombinant DNA techniques. See Queen et al., Proc.
Natl. Acad. Sci. USA 86, 10029-10033 (1989) and WO 90/07861. Human
antibodies can be obtained using phage-display methods. See, e.g.,
Dower et al., WO 91/17271; McCafferty et al., WO 92/01047. In these
methods, libraries of phage are produced in which members display
different antibodies on their outer surfaces. Antibodies are
usually displayed as Fv or Fab fragments. Phage displaying
antibodies with a desired specificity are selected by affinity
enrichment to the complement protein or antigenic fragment.
[0133] Human antibodies against a complement pathway molecule can
also be produced from non-human transgenic mammals having
transgenes encoding at least a segment of the human immunoglobulin
locus and an inactivated endogenous immunoglobulin locus. See,
e.g., Lonberg et al., WO93/12227 (1993); Kucherlapati, WO 91/10741
(1991). Human antibodies can be selected by competitive binding
experiments, or otherwise, to have the same epitope specificity as
a particular mouse antibody. Such antibodies are particularly
likely to share the useful functional properties of the mouse
antibodies. Human polyclonal antibodies can also be provided in the
form of serum from humans immunized with an immunogenic agent.
Optionally, such polyclonal antibodies can be concentrated by
affinity purification using the complement component or an
antigenic fragment as an affinity reagent.
[0134] 2. Cell-Free Assays for Detecting Binding Between a Test
Agent and a Complement Pathway Molecule
[0135] Cell-free assays can be used to identify agents which are
capable of interacting with a complement pathway molecule and
modulating its activity and/or interaction with another molecule.
Binding of a test agent to a complement pathway molecule is
determined in a reaction mixture. Binding can be assayed by a
number of methods including, e.g., labeled in vitro protein-protein
binding assays, electrophoretic mobility shift assays, immunoassays
for protein binding, functional assays (e.g., phosphorylation
assays, etc.). See, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;
4,517,288; and 4,837,168, and also Bevan et al., Trends in
Biotechnology 13:115-122, 1995; Ecker et al., Bio/Technology
13:351-360, 1995; and Hodgson, Bio/Technology 10:973-980, 1992. The
test agent can be identified by detecting a direct binding to the
complement component, e.g., co-immunoprecipitation of with the
complement component. The test agent can also be identified by
detecting a signal that indicates that the agent binds to the
complement component, e.g., fluorescence quenching.
[0136] Competition assays provide a suitable format for identifying
test compounds that specifically bind to a complement pathway
molecule. In such formats, test compounds are screened in
competition with a compound already known to bind to the complement
pathway molecule. The known complement-binding agent can be a
synthetic polypeptide. It can also be an antibody which
specifically recognizes the complement pathway molecule. If the
test compound inhibits binding of the compound known to bind the
complement pathway molecule, then the test compound also binds the
complement pathway molecule.
[0137] Numerous types of competitive binding assays are known,
e.g., solid phase direct or indirect radioimmunoassay (RIA), solid
phase direct or indirect enzyme immunoassay (EIA), sandwich
competition assay (see Stahli et al., Methods in Enzymology
9:242-253 (1983)); solid phase direct biotin-avidin EIA (see
Kirkland et al., J. Immunol. 137:3614-361-9 (1986)); solid phase
direct labeled assay, solid phase direct labeled sandwich assay
(see Harlow and Lane, "Antibodies, A Laboratory Manual," Cold
Spring Harbor Press (1988)); solid phase direct label RIA using
1-125 label (see Morel et al., Mol. Immunol. 25(1):7-15 (1988));
solid phase direct biotin-avidin EIA (Cheung et al., Virology
176:546-552 (1990)); and direct labeled RIA (Moldenhauer et al.,
Scand. J. Immunol. 32:77-82 (1990)). Typically, such an assay
involves the use of purified antigen bound to a solid surface or
cells bearing either of these, an unlabelled test immunoglobulin
and a labeled reference immunoglobulin. Competitive inhibition is
measured by determining the amount of label bound to the solid
surface or cells in the presence of the test immunoglobulin.
Usually the test immunoglobulin is present in excess. Antibodies
identified by competition assay (competing antibodies) include
antibodies binding to the same epitope as the reference antibody
and antibodies binding to an adjacent epitope sufficiently proximal
to the epitope bound by the reference antibody for steric hindrance
to occur. Usually, when a competing antibody is present in excess,
it will inhibit specific binding of a reference antibody to a
common antigen by at least 50 or 75%.
[0138] The screening assays can be either in insoluble or soluble
formats. One example of the insoluble assays is to immobilize a
given complement pathway molecule, or a fragment thereof, onto a
solid phase matrix. The solid phase matrix is then put in contact
with test agents for an interval sufficient to allow the test
agents to bind. Following washing away any unbound material from
the solid phase matrix, the presence of the agent bound to the
solid phase allows identification of the agent. The methods can
further include the step of eluting the bound agent from the solid
phase matrix, thereby isolating the agent. Alternatively, other
than immobilizing the complement pathway molecule, the test agents
are bound to the solid matrix and the complement pathway molecule
is then added.
[0139] Soluble assays include some of the combinatory libraries
screening methods and the genetic screening systems described
above. Under the soluble assay formats, neither the test agents nor
the complement pathway molecule are bound to a solid support.
Binding of a complement pathway molecule or fragment thereof to a
test agent can be determined by, e.g., changes in fluorescence of
either the complement pathway molecule or the test agents, or both.
Fluorescence may be intrinsic or conferred by labeling either
component with a fluorophor. Binding can be detected by
fluorescence polarization.
[0140] In some binding assays, either the complement pathway
molecule, the test agent, or a third molecule (e.g., an
anti-complement-antibody) as labeled entities, i.e., covalently
attached or linked to a detectable label or group, or
cross-linkable group, to facilitate identification, detection and
quantification of the polypeptide in a given situation. These
detectable groups can comprise a detectable polypeptide group,
e.g., an assayable enzyme or antibody epitope. Alternatively, the
detectable group can be selected from a variety of other detectable
groups or labels, such as radiolabels (e.g., .sup.125I, .sup.32P,
.sup.35S) or a chemiluminescent or fluorescent group. Similarly,
the detectable group can be a substrate, cofactor, inhibitor or
affinity ligand.
[0141] Some test compounds with specific binding activity to a
given complement pathway molecule (e.g., C5b) identified by such
assays are specific to that complement pathway molecule and can be
used to modify the activity of only that complement pathway
molecule. Other test compounds show specific binding to a plurality
of complement pathway molecules and can be used to modulate the
activity of all of these complement pathway molecule.
[0142] 3. Cell-Based Assays
[0143] An interaction between the test compound and the complement
pathway molecule or between the complement pathway molecule and the
complement pathway molecule binding partner can also be detected
with cell-based assays. For example, a microphysiometer described
in McConnell et al. (1992) Science 257:1906 can be used. Cell based
assays can also be used to identify compounds which modulate
expression of a gene encoding a complement pathway molecule,
modulate translation of a mRNA encoding a complement component, or
which modulate the stability of the complement pathway protein or
its mRNA.
[0144] In some methods, to identify an reagent which modulate
expression of a complement pathway molecule, a cell which is
capable of expressing a complement pathway molecule is incubated
with a test compound and the amount of complement pathway molecule
produced in the cell medium is measured and compared to that
produced from a cell which has not been contacted with the test
compound. Compounds which can be tested include small molecules,
proteins, and nucleic acids. In particular, this assay can be used
to determine the efficacy of antisense or ribozymes to genes
encoding a complement component.
[0145] In other methods, the effect of a test compound on
transcription of a gene encoding a complement pathway molecule is
determined by transfection experiments using a reporter gene
operatively linked to at least a portion of the promoter of a gene
encoding a complement pathway molecule. A promoter region of a gene
can be isolated, e.g., from a genomic library according to methods
known in the art. The reporter gene can be any gene encoding a
protein which is readily quantifiable, e.g., the luciferase or CAT
gene. Such reporter gene are well known in the art.
F. Formulation and Dosages
[0146] 1. Formulations and Modes of Administration
[0147] Therapeutics of the present invention can be formulated in a
conventional manner using one or more physiologically acceptable
carriers or excipients. Thus, the therapeutic agents described
above can be formulated for administration by, for example, eye
drops, injection, inhalation or insufflation (either through the
mouth or the nose) or oral, buccal, parenteral or rectal
administration. Treatment can also follow guidance provided in the
art. For example, pulmonary administration of soluble complement
receptor-1 (sCR1) to treat certain medical conditions (U.S. Pat.
No. 6,169,068), intraocular administration of drugs to treat
macular degeneration (U.S. Pat. No. 5,632,984), and treatment of
macular edema with topical administration of carbonic anhydrase
inhibitors to the eye (U.S. Pat. No. 6,046,223) have all been
described in the art.
[0148] The therapeutic agents of the invention can be formulated
for a variety of modes of administration, including systemic and
topical or localized administration. Techniques and formulations
generally can be found in Remmington's Pharmaceutical Sciences,
Meade Publishing Co., Easton, Pa. The compositions are formulated
as sterile, substantially isotonic and in full compliance with all
Good Manufacturing Practice (GMP) regulations of the U.S. Food and
Drug Administration. A preferred method of administration is an eye
drop. For systemic administration, injection is preferred,
including intramuscular, intravenous, intraperitoneal, and
subcutaneous.
[0149] Preferred methods of administration include, e.g., choroidal
injection, transscleral injection or placing a scleral patch,
selective arterial catheterization, intraocular administration
including transretinal, subconjunctival bulbar, scleral pocket and
scleral cutdown injections. The agent can also be alternatively
administered intravascularly, such as intravenously (IV) or
intraarterially. In choroidal injection and scleral patching, the
clinician uses a local approach to the eye after initiation of
appropriate anesthesia, including painkillers and ophthalmoplegics.
A needle containing the therapeutic compound is directed into the
subject's choroid or sclera and inserted under sterile conditions.
When the needle is properly positioned the compound is injected
into either or both of the choroid or sclera. When using either of
these methods, the clinician can choose a sustained release or
longer acting formulation. Thus, the procedure can be repeated only
every several months or several years, depending on the subject's
tolerance of the treatment and response.
[0150] For injection, the compounds of the invention can be
formulated in liquid solutions, preferably in physiologically
compatible buffers such as Hank's solution or Ringer's solution. In
addition, the compounds can be formulated in solid form and
redissolved or suspended immediately prior to use. Lyophilized
forms are also included.
[0151] For oral administration, the pharmaceutical compositions can
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinized maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulfate). Liquid preparations
for oral administration can take the form of, for example,
solutions, syrups or suspensions, or they can be presented as a dry
product for constitution with water or other suitable vehicle
before use. Such liquid preparations can be prepared by
conventional means with pharmaceutically acceptable additives such
as suspending agents (e.g., sorbitol syrup, cellulose derivatives
or hydrogenated edible fats); emulsifying agents (e.g., lecithin or
acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl
alcohol or fractionated vegetable oils); and preservatives (e.g.,
methyl or propyl-p-hydroxybenzoates or sorbic acid). The
preparations can also contain buffer salts, flavoring, coloring and
sweetening agents as appropriate.
[0152] The therapeutic can be administered alone or in combination
with other molecules known to have a beneficial effect on retinal
attachment or damaged retinal tissue, including molecules capable
of tissue repair and regeneration and/or inhibiting inflammation.
Examples of useful cofactors include basic fibroblast growth factor
(bFGF), LaVail et al. (1998), Invest. Ophthalmol. Vis. Sci.
39:592-602, ciliary neurotrophic factor (CNTF), LaVail et al.
(1998), Invest. Ophthalmol. Vis. Sci. 39:592-602, axokine (a mutein
of CNTF), LaVail et al. (1998), Invest. Ophthalmol. Vis. Sci.
39:592-602, leukemia inhibitory factor (LIF), LaVail et al. (1998),
Invest. Ophthalmol. Vis. Sci. 39:592-602, neutrotrophin 3 (NT-3),
LaVail et al. (1998), Invest. Ophthalmol. Vis. Sci. 39:592-602,
neurotrophin-4 (NT-4), LaVail et al. (1998), Invest. Ophthalmol.
Vis. Sci. 39:592-602, nerve growth factor (NGF), LaVail et al.
(1998), Invest. Ophthalmol. Vis. Sci. 39:592-602, insulin-like
growth factor II, LaVail et al. (1998), Invest. Ophthalmol. Vis.
Sci. 39:592-602, prostaglandin E2, La Vail et al. (1998), Invest.
Ophthalmol. Vis. Sci. 39:581-591, 30 kD survival factor, taurine,
and vitamin A. Other useful cofactors include symptom-alleviating
cofactors, including antiseptics, antibiotics, antiviral and
antifungal agents and analgesics and anesthetics.
[0153] A therapeutic also can be associated with means for
targeting the therapeutics to a desired tissue. For example, in
some methods, a therapeutic agent can be directed to the
choriocapillaris which is implicated to be a target of activated
complement system (e.g., C5b-9 complex) in AMD patients. Useful
targeting molecules can be designed, for example, using the simple
chain binding site technology disclosed, e.g., in U.S. Pat. No.
5,091,513. Thus, by targeted delivery, therapeutic agents are aimed
to prevention or alleviation of damages to the choriocapillaris
caused by the C5b-9 complex. Such effects can be achieved by the
various means described above, e.g., inhibiting complement
activation, stimulating the functions of the complement pathway
inhibitors (e.g., clusterin, vitronectin), or disrupting the
complex already form on the choriocapillaris.
[0154] In clinical settings, a gene delivery system for a gene
therapeutic can be introduced into a subject by any of a number of
methods. For instance, a pharmaceutical preparation of the gene
delivery system can be introduced systemically, e.g., by
intravenous injection, and specific transduction of the protein in
the target cells occurs predominantly from specificity of
transfection provided by the gene delivery vehicle, cell-type or
tissue-type expression due to the transcriptional regulatory
sequences controlling expression of the receptor gene, or a
combination thereof. In other embodiments, initial delivery of the
recombinant gene is more limited with introduction into the animal
being quite localized. For example, the gene delivery vehicle can
be introduced by catheter, See U.S. Pat. No. 5,328,470, or by
stereotactic injection, Chen et al. (1994), Proc. Natl. Acad. Sci.,
USA 91: 3054-3057. A sequence homologous thereto can be delivered
in a gene therapy construct by electroporation using techniques
described, Dev et al. (1994), Cancer Treat. Rev. 20:105-115.
[0155] The pharmaceutical preparation of the gene therapy construct
or compound of the invention can consist essentially of the gene
delivery system in an acceptable diluent, or can comprise a slow
release matrix in which the gene delivery vehicle or compound is
imbedded. Alternatively, where the complete gene delivery system
can be produced intact from recombinant cells, e.g., retroviral
vectors, the pharmaceutical preparation can comprise one or more
cells which produce the gene delivery system.
[0156] The compositions can, if desired, be presented in a pack or
dispenser device which can contain one or more unit dosage forms
containing the active ingredient. The pack can for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device can be accompanied by instructions for
administration.
[0157] 2. Dosages
[0158] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the Ld.sub.50 (the
dose lethal to 50% of the population) and the Ed.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
which exhibit large therapeutic indices are preferred. While
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0159] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
[0160] The practice of the present invention can employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the
art. Such techniques are explained fully in the literature.
Molecular Cloning A Laboratory Manual (1989), 2.sup.nd Ed., ed. by
Sambrook, Fritsch and Maniatis, eds., Cold Spring Harbor Laboratory
Press, Chapters 16 and 17; Hogan et al. (Manipulating the Mouse
Embryo: A Laboratory Manual (1986), Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.; See U.S. Pat. No. 4,683,195; DNA
Cloning, Volumes I and II, Glover, ed., 1985; Oligonucleotide
Synthesis, M. J. Gait, ed., 1984; Nucleic Acid Hybridization, D.
Hames & S. J. Higgins, eds., 1984; Transcription and
Translation, B. D. Hames & S. J. Higgins, eds., 1984; Culture
Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., 1987;
Immobilized Cells And Enzymes, IRL Press, 1986; Perbal (1984), A
Practical Guide To Molecular Cloning; See Methods In Enzymology
(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian
Cells, J. H. Miller and M. P. Calos, eds., Cold Spring Harbor
Laboratory, 1987; Methods In Enzymology, Vols. 154 and 155, Wu et
al., eds., Academic Press Inc., N.Y.; Immunochemical Methods In
Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,
London, 1987; Handbook Of Experimental Immunology, Volumes I-IV, D.
M. Weir and C. C. Blackwell, eds., 1986.
[0161] Many modifications and variations of this invention can be
made without departing from its spirit and scope. The specific
examples described herein are for illustration only and are not
intended to limit the invention in any way.
[0162] All publications, figures, patents and patent applications
cited herein are hereby expressly incorporated by reference for all
purposes to the same extent as if each was so individually
denoted.
EXAMPLES
Example 1
Identification of Complement Pathway Molecules in Drusen, Bruch's
membrane, and Choriocapillaris
[0163] Tissues: Eyes from the human donor repository and CDD,
ranging in age between 45 and 101 years, were processed within four
hours of death. Many of these donors had a documented clinical
diagnosis of AMD (including donors with geographic atrophy,
choroidal neovascularization, and disciform scars in at least one
eye) and one donor was diagnosed with cuticular drusen. Human liver
was obtained within 2 hours of biopsy. RPE cells were isolated with
2% dispase within 5 hours of death and were grown in Coon's F-12
media with 10% fetal bovine serum.
Immunohistochemistry:
[0164] Tissues were fixed for at least two hours in one-half
strength Karnovsky fixative (1/2K; 2% formaldehyde and 2.5%
glutaraldehyde in 100mM cacodylate buffer, pH 7.4, containing
0.025% CaCl.sub.2) prior to washing 3.times.10 min. in 100 mM
cacodylate buffer. Slides were blocked for 15 min. in 0.01M sodium
phosphate (pH 7.4) containing 0.85% NaCl, 1 mM calcium chloride, 1
mM magnesium chloride (PBS/M/C), and 1 mg/ml globulin-free bovine
serum albumin (PBS/M/C/BSA). Sections were then rinsed for 10 min.
in PBS/M/C, incubated in primary antibody diluted in PBS/M/C/BSA,
for one hr., at room temperature. In some cases, sections were
pretreated, prior to blocking, with 0.5% trypsin (Sigma, St. Louis,
Mo.) for 10 min. as specified by the supplier. Following exposure
to primary antibody, sections were rinsed (2.times.10 min.) in
PBS/M/C, incubated in the appropriate fluorescein-conjugated
secondary antibody (often adsorbed against human serum) diluted in
PBS/M/C/BSA (30 min., room temperature), rinsed (2.times.10 min.)
in PBS/M/C, and mounted in Immumount (Shandon, Pittsburgh, Pa.).
Adjacent sections were reacted with secondary antibody alone, as
negative controls. Some sections were pre-treated for 10 min with
0.5% trypsin (Sigma; St. Louis, Mo.), or 0.2-0.02 U/ml
chondroitinase ABC (Seikagaku; Rockville, Md.), for use in
conjunction with antibodies for collagen type IV or various
chondroitin sulfate proteoglycans, respectively. Drusen-containing
tissues from a minimum of five donor eyes were examined for each
antibody.
[0165] For negative controls, sections were exposed to PBS/M/C/BSA
containing: a) no primary antibody; b) 1% (v/v) normal serum;
and/or c) antibodies to irrelevant proteins. In some cases, an
additional control included adsorption of primary antibody to
purified antigen. Positive controls included reaction of antibodies
with the extracellular matrices of sclera, choroid, and vitreous;
retinal and choroidal basal laminae; retinal interphotoreceptor
matrix; and liver. In order to determine the "specificity" of serum
protein accumulation in drusen, drusen-containing sections were
reacted with antibodies to human albumin (Cappel; Malvern, Pa.) and
haptoglobin (Dako; Carpenteria, Calif.). Haptoblobin is a
macromolecular glycoprotein which is the major acute phase reactant
(Putnam, Haptoglobin, In: The Plasma Proteins, Structure, Function,
and Genetic Control, 11, Putnam (ed.), Academic Press, New York,
pp. 1-50, 1975; and Morimatsu et al., J. Biol. Chem.,
266:11833-11837, 1991).
Results
[0166] Reactivities of antibodies with drusen are listed in Table 1
below. In general, all positive antibodies bound to all drusen
phenotypes. Controls confirm all antibody reactivities to be
specific. In addition, the majority of the antibodies utilized
bound to the expected regions of sclera, choroid, RPE, retina,
vitreous, and/or other "control" tissues. TABLE-US-00002 TABLE 1
Drusen associated molecules (DRAMs) ANTIGEN SOURCE DRUSEN .alpha.1
antichymotrypsin Dako + .alpha.1 antitrypsin Dako -/+ .alpha.2
macroglobin Biodesign - aFGF - AKS - Albumin Cappel - Amyloid A
Dako + Amyloid b Dako - to +/- Amyloid P Dako + Amyloid Prec Prot
B-M - Antithrombin III Calb +/- Apo A1 Calb - Apo E Calb + ASPG-1 -
Atrial Natriuretic Factor Chemicon - .beta.2 microglobin B-M +/-
bFGF - Basement Membrane Chemicon - Bovine nas. cart. p. ICN - CD1a
Dako + CD3 Pharm -/+ Dako - CD4 Pharm +/- CD8 Pharm - CD14 Dako +
CD15 Chemicon - CD31 Dako +/- CD44 Various - CD45 Dako + CD68 Dako
+ CD83 + CD86 Dako + CRP Dako - to +/- Calcitonin Dako - Carbonic
Anhydrase - Carc Assoc Ag - cfms/CSF-1 receptor - Chondroitin
sulfate - Chondroitin 0 sulfate - Chondroitin 4 sulfate +
Chondroitin 6 sulfate + Chondroitin sulfate PG Chemicon - Collagen
I Southern Biotech - Collagen II SB - Collagen III SB - Collagen IV
SB, Chemicon - Collagen V SB - Collagen VI - Collagen VII -
Collagen IX - Collagenases C1q Calb -/+ Complement 3 - to + C5 +
C5-C9 complex Calb +/- COS - CRALBP - Cystatin C - Decorin Chemicon
- Elastin Sigma - Entactin - Factor X Dako + Fibrin - Fibrinogen
Dako - to +/- Fibronectin - Fibulin 3 Timpl -/? Fibulin 4 Timpl -/?
FnR - .alpha. Fodrin - .beta. Fodrin -/+ Gangliosides Dev Hyb -
Gelsolin - GFAP - Glucose Transporters 1, 3, 4 - Glycolipid Dev Hyb
- Glycophorin A, C - Haptoglobin Dako +/- (variable) Heckenlively
serum Ag +/- Heparan sulfate (MAB) +/- (MAC) Kimata Hermes - HLA
ABC -/? HLA DR Various + HNK-1 - Heat Shock Prot 70 - HSPG - Human
IgA - Human IgG +/- Hyaluronic Acid - Ig Kappa chain - to +/- Ig
Lambda chain Dako +/- to + Integrin .alpha.2 - Integrin .alpha.3 -
Integrin .alpha.4 - Integrin .alpha.5 - Integrin .alpha.6 -
Integrin .beta.1 - Integrin .beta.2 - Integrin .beta.4 -
Intermediate Filaments - Interphotoreceptor Matrix - IRBP - Keratan
sulfate - Keratin - Laminin - LAMP-1 Dev Hyb - LAMP-2 Dev Hyb -
Link Protein Dev Hyb - Lipoprotein b - to +/- Melanoma Assoc Ag -
Milk mucin core Ag - MMPs - Mitochindrial Ag - N.S. Enolase - Nerve
Growth Factor - NGFR - Neurofibrillary tangles - PG40 (Decorin) -
Phospholipase A2 - Plasminogen# Dako + Plasminogen Act. Inhib.-1 -
Platelet Derived GF - Prealbumin# B-M - to + Prothrombin# +/- S-100
(Bovine) -/? Sialo Cell Surface Ag - Tau - Tenascin - TGFb -
Thrombin Sera +/- Thrombospondin (Gib/AMAC) - to +/- TIMP1 - TIMP2
- TIMP3 + TIMP4 +/- Tubulin - Ubiquitin - to + UPAR Anderson -
Vimentin - Vitronectin Various + VnR - von W Factor - B-M =
Boehringer-Mannheim; Calb = Calbiochem; Gib = Gibco/BRL; Pharm =
Pharmingen; Sera = Sera Labs; Tel = Telios; "-" no reactivity; "+"
consistent positive reactivity; "+/-" weak reactivity; "-/+" very
weak reactivity
Example 2
Identification of Complement Pathway Components Including the
Activated C5b-9 Membrane Attack Complex in Drusen and Bruch's
Membrane
[0167] As demonstrated in Example 1, proteins associated with
cellular and humoral immune processes, including amyloid A
component, amyloid P component, apolipoprotein E, factor X, MHC
class II molecules, vitronectin, and complement proteins (C3, C5
and C5b-9 complex) are prevalent among the drusen-associated
constituents identified. Other complement components, including the
terminal complement complex C5b-9 (the membrane attack complex,
MAC), are also distributed within Bruch's membrane at the
RPE-choroid interface. The presence of widespread terminal C5b-9
complement complexes within Bruch's membrane and drusen indicates
that inappropriate complement activation can occur within the
sub-RPE space. The abnormal process can have injurious effects on
the RPE and/or choroidal cells, promote neovascularization and
microangiopathy (including loss of pericytes and hyperplasia of
endothelial cells), increase blood vessels permeability and/or
promote recruitment of monocytes, thereby contributing to the
pathology of AMD.
[0168] Studies were conducted to examine complement pathway
molecules in eyes derived from donors with and without AMD using
immunohistochemical, ELISA, and Western blot analyses. These
studies were aimed to identification of complement
pathway-associated constituents that are present at the RPE-choroid
interface, to identify the specific complement pathway(s) that is
activated, to determine whether the C5b-9 complex is inserted into
the membranes of local ocular cells, to determine whether
complement components are synthesized by locally by ocular cells,
and to assess the relative amounts of mRNAs coding for complement
constituents in eyes from donors with and without AMD.
[0169] Numerous complement pathway proteins were found to be
associated with drusen, Bruch's membrane, the basal surface of the
RPE, and/or the sub-RPE space using immunohistochemistry (Table 2).
Complement pathway-associated molecules localized to the basal
surface of the RPE include CD21, CD35, CD55/decay accelerating
factor, and CD59/protectin. Complement pathway-associated molecules
localized in Bruch's membrane and/or drusen include C3d, C6, C7,
C8, C9, Factor D, Factor H, Factor I, Factor B, SP40,40
(clusterin), and mannose binding protein, in addition to the
previously described complement components C3, C5 and the terminal
complement complex C5b-9. Complement pathway components C1q, C1
inhibitor, C2, C3a, C4, C5a, and Factor Ba are present within the
choroidal stroma, but do not appear to be major components of
drusen or Bruch's membrane. The presence of many of these
complement pathway-associated components has been confirmed using
ELISA and Western analyses (Table 2). TABLE-US-00003 TABLE 2
Identification of Complement Pathway-Associated Molecules Within
the Human RPE-Choroid Western Western ELISA ELISA
Immunohistochemistry (RPE/Ch) (RPE) (RPE/Ch) (RPE) (Donor Numbers)
Ig .kappa. ND ND ND ND CH+, D+/-(459-00) Ig .lamda. +(205-98)
+(205-98) +(411-99) +(411- CH+, D-(33-99, 459-99) 99) C1q ND
-(411-99) -(325-00, +(411- CH+/-, D-, RPE+, (239-00, 58-00, 294-00)
99, 407-99, 247-99, 86-98, 294-00, 242- 205-98) 00) C1 ND +(86-98,
+(325-00, +/- RPE+, D+/-(242-00) Inhibitor 247-99) 294-00) (242-
00) C2 +(294-00) +(294-00) ND ND CH+. D-/+(294-00) C4 +(294-00)
+(294-00) ND ND CH+, D-/+(294-00) C3 ND -(247- ND ND CH+,
D-(239-00, 58-00, 407-99, 99, 86-98) 247-99, 459-99, 294-99, 97-99)
C3a ND +/-(86-98, ND +(205- CH+, D-(1-97, 459-00, 239-00, 247-99)
98) 242-00) C3d +(294-00, ND +(325-00, ND CH+, D+, RPE+, cores?
(294-00, 325-00) 294-00) 94-00, 404-00, 453-00, 325-00) C5 ND
+/-(205- +(325-00, +(325- CH+/-, D+(242-00, 239-00, 407-99, 98,
411-99) 294-00) 00, 247-00) 294-00) C5a ND +/-(86-98, +(325-00,
+(205- CH+, D-(407-99, 247-99, 86-98, 247-99) 294-00) 98) 242-00,
294-00) C5b-9 ND -(86-98, +(325-00, +(205- CH-/+, D+(all 20 donors)
247-99) 294-00) 98) Clusterin ND +(242-00) -(325-00, +(205-
D+(242-00) 294-00) 98) CD21 ND -- ND ND RPE+/-(239-00, 459-00,
294-00, 242-00, 58-00) CD35 ND -(86-98, ND -(242- RPE +/-(58-00,
294-00, 239-00, 1- 247-99) 00) 97, 459-99, 242-00 CD55 ND -(242-00)
-(325-00, -(242- RPE+(239-00, 459-99) 294-00) 00) CD59 ND -(242-00)
-(325-00, -(242- RPE+, CH+(242-00, 1-97, 459-99, 294-00) 00)
294-00) Factor B +/-(294-00, +(242-00, -/+(325- -(242- CH+, D+/-,
cores? (294-00) 242-00) 294-00) 00, 294- 00) 00) Factor Ba
+(325-00, ND -(325-00, -(242- CH+, D-(294-00, 94-00, 404-00,
294-00) 294-00) 00) 453-00, 325-00, 330-00, 457-00) Factor D ND ND
ND ND DR+/- Factor H ND -(325-00, +/-(325- +(205- CH-/+, D+(242-00,
294-00, 459-00, 294-00) 00, 294- 98, 457-00, 330-00, 325-00, 94-00,
404- 00) 242-00) 00, 453-00) Factor I +/-(294-00) ND +(325-00, ND
DR-/+, CH-/+(294-00, 94-00, 404- 294-00) 00, 453-00, 325-00,
330-00) Mannose +/-(325-00, +/-(325- +/-325- +(242- DR+,
CH+(294-00, 330-00, 457-00, Binding 294-00) 00, 294-00) 00, 294-
00) 325-00, 94-00, 404-00, 453-00) Protein 00) Mannose -(325-00,
-(325-00, ND ND -(294-00) Receptor 294-00) 294-00) Retinal
pigmented epithelium (RPE) and RPE-choroid complexes (RPE/Ch) from
20 donors (five with a diagnosis of AMD and two with probable AMD)
were used for various Western blot, ELISA and immunohistochemical
studies of complement activation pathways. Donor designations are
listed in parentheses in the table and the age, sex (M, male or F,
female), race (C, Caucasian), and disease state (AMD) of each donor
are detailed below. 450-99 30 CF 453-00 61 CM 411-99 67 CF Family
history of AMD 247-99 70 CF 86-98 73 CM 407-99 74 CF AMD and family
history of AMD 58-00 74 CM 330-00 75 CF Macular RPE changes (AMD?)
325-00 77 CF 404-00 77 CF Macular drusen (AMD?) 247-99 79 CF 94-00
80 CM 239-00 80 CF AMD and family history of AMD 459-00 80 CM
294-00 84 CF AMD 33-99 86 CF AMD 457-00 89 CF AMD 205-98 91 CM
242-00 99 CF "D" denotes drusen in the Table; "+" positive labeling
according to the assay; "-" no labeling; "+/-" weak labeling; "-/+"
very weak labeling
Example 3
Determination of the Relationship Between Terminal Complement
Complex Deposition in Bruch's Membrane, Age, and AMD
[0170] Based on the observation that the full spectrum of
complement proteins and inhibitors, as well as activated complement
complexes, are present at the RPE-choroid interface, the
distribution of the CSb-9 terminal complement complex was examined
in 30 human donor eyes ranging in age between less than 1 year and
94 years of age. The donors above the age of 60 were evenly divided
between donors with and without AMD. In summary, a strong
correlation between intensity and distribution of C5b-9 associated
with the choriocapillaris and a diagnosis of AMD was observed.
[0171] Aldehyde-fixed sagittal wedges were infiltrated and embedded
in acrylamide and optimal cutting temperature compound. Frozen
sections were prepared and were labeled with two different
monoclonal antibodies directed against the C5b-9 terminal
complement complex neoantigen.
[0172] Notably, Bruch's membrane also reacted with these antibodies
in most of the donors evaluated. In younger donors, C5b-9 was
observed in the outer collagenous layer of Bruch's membrane, with
relatively little labeling of the inner collagenous layer. Sporadic
labeling to the basal and lateral aspects of the choriocapillaris
was also observed in some younger donors. The distribution of C5b-9
within Bruch's membrane "shifted" toward the inner collagenous
layer in individuals of advanced age. In these individuals, C5b-9
was observed on both sides of the elastic lamina or, in some cases,
solely within the inner collagenous layer. In addition, the
antibodies directed against C5b-9 bound intensely to all drusen
phenotypes in older individuals, as we have described previously
(Mullins et al. FASEB J. 2000). Significantly, intense labeling of
the entire choriocapillaris (endothelium, pericytes, and associated
extracellular matrix) was observed in donors with AMD (9 of 10
donors), as compared to older, age-matched donors without a
diagnosis of AMD (2 of 10 donors) (FIG. 2).
[0173] When combined with the observation that C5b-9 complexes are
associated (most likely inserted into) with RPE and choroidal cell
membranes (see above), these data imply that the choriocapillaris
of AMD subjects may be under more rigorous attack than that of
individuals without AMD. This may be due to an inability of local
cells in individuals with AMD to inhibit and/or defend themselves
against complement activation. The distribution of immunoreactive
C5b-9 and detectable levels of C5b-9 in the samples from AMD donors
implies that complement pathway inhibitors such as clusterin,
vitronectin CD56 and CD55 may fail to suppress the terminal
pathway, thereby permitting formation of MAC. Complement-mediated
damage to the choriocapillaris, whether by direct insertion or due
to bystander effects, may lead to abnormal responses by the choroid
(e.g., inflammation, cytokine secretion, neovascularization) and/or
choriocapillaris cell death. These events, in turn, may lead to
further dysfunction and death of surrounding cells, including the
RPE, and the biogenesis of drusen. Although this represents a
paradigm shift in our thinking about the etiology of AMD, similar
processes are indeed active in other diseases, including
atherosclerosis and Alzheimer disease. These data also provide
evidence that Bruch's membrane may serve as an unusual activating
surface for complement in its physiologically "normal" state,
and/or that activated C5b-9 is poorly cleared from Bruch's membrane
compared with other structures in the healthy choroid. Whatever the
precise pathways and initiating events that are involved, it is
clear that complement activation occurs at the RPE-choroid
interface chronically and that a strong correlation between
intensity and distribution of C5b-9 associated with the
choriocapillaris and a diagnosis of AMD exists.
Example 4
Expression of mRNAs for Complement Pathway-Associated Proteins by
RPE and Choroidal Cells in the Human Eye
[0174] In order to determine whether the complement components
detected in drusen and Bruch's membrane are synthesized remotely
(i.e., the liver) or are produced locally by specific ocular cells,
total RNA was isolated from the neural retina, the RPE-choroid
complex, isolated RPE cells, and liver from human donors. RNA was
reverse-transcribed and the resultant cDNA was used as a template
for PCR. Genomic DNA isolated from human lymphocytes was used to
control for the effects of DNA contamination. Specific complement
mediators evaluated are indicated on Table 3. Notably, C3 and C5
are synthesized by the RPE (see also Mullins et al., FASEB J. 2000)
as are APP, clusterin, and Factor H. A number of other complement
components that are not synthesized by the RPE, such as C9 and
MASP-1, are synthesized by adjacent choroidal and/or retinal cells
and could therefore contribute to complement activation in Bruch's
membrane.
[0175] These gene expression data indicate a role for locally
produced complement components in the activation of complement in
Bruch's membrane and, possibly, in the etiology of early AMD. Some
differences in the apparent expression levels of APP2, C3, and C9
were noted in one donor with AMD, but these data were not verified
in a larger sample set. Instead, quantitative gene expression
studies were initiated to examine differences in expression levels
between donors with and without AMD (see Table 4 and Example 4
below). TABLE-US-00004 TABLE 3 Complement Pathway Constituents
(RT-PCR) Gen. Ret. R/Ch RPE DNA Liver MBL-1 + + + - - MBL-2 (2) - -
- - + MBPC (1) - - - - + CRP-1 - - - - + APP1 - + + - - APP2 - + +
- small MASP1 - + -? - + C3 + + + - + C5 + + + - + C9 + + - - +
HCR1 + + - - + HCR2 +/- +/- + +/- + Factor H + + + - + VN + + - +
Clusterin + + + - CD44 + + - CD63 + + + - + CD68 ? + -
Example 5
Analyses of Complement Pathway-Associated Component Gene Expression
by the RPE and Choroid Using Gene Array Analyses
[0176] In addition to the patterns of gene expression identified by
RT-PCR analyses (above), data derived from gene array analyses have
yielded novel information concerning the expression and abundance
of specific participants in the complement pathway(s) by the RPE
and/or choroid (Table 4). Most significantly, these data confirm
that a majority of molecules involved in complement activation and
inhibition are expressed locally by RPE and choroid cells. They
also provide insight into specific pathways that may be active in
the etiology of AMD.
[0177] In these studies, mRNA was isolated from 4 mm diameter
punches of the equatorial RPE/choroid complex from 78 human donor
eyes between the ages of 2 weeks and 101 years. Thirty-one (31) of
these donors over the age of 50 had a clinical diagnosis of AMD.
The mRNA expression levels of several specific components
associated with the complement pathway were compared between this
group of 31 AMD donors to those of thirty three (33) age-matched
donors without AMD (see Table 4). The local expression of C1q, C1r,
C1s, C2 (low abundance), C3, C4, C5, C6 (low abundance), C7, C8 and
C9 by RPE and/or choroidal cells has been confirmed and quantified
as a function of AMD disease state (Table 4). Moreover, the
expression levels of mRNAs for numerous mediators of complement
pathways, including mannose binding lectin, factor H, clusterin,
vitronectin, and immunoglobulin chain precursors, have been
determined and quantified as a function of AMD disease state (Table
4). In addition, these analyses have revealed the expression of
numerous additional complement pathway-associated molecules by RPE
and/or choroidal cells (Table 4). A number of these molecules are
significantly upregulated (e.g. Ig J chain, Ig .lamda. chain) or
downregulated (e.g. complement 6, clusterin) in individuals with
AMD, as compared to controls. Ongoing studies are being directed
toward verification of these data. TABLE-US-00005 TABLE 4
Complement Pathway Constituents (Gene Expression Array Analyses)
Control .times. 100 mRNA Abundance AMD Human Ig J Chain + 28% Human
Ig .lamda. Chain ++ 34% Human IgG Fc Fragment + 35% HSP 70 + 41%
Complement C2 +/- 48% CRP +/- to + 61% C4 Binding Protein + 61%
Alpha-2-Macroglobulin + 66% Complement C1q +/++ 69% Stress Induced
Phosphoprotein 1 + 70% Heat Shock Protein 75 + 72% Complement C3a
Receptor 1 + 73% Complement C8 Alpha Chain + 75% Complement C3 ++
78% Mannose Binding Lectin + 80% Ficolin 2 ++ 82% Complement C7 +
83% Vitronectin ++ 87% IgG Fc Binding Protein ++ 88% Ig Heavy Chain
C Region a1 ++ 88% Complement C9 + 90% CD59 ++ 90% Factor X ++ 92%
MBL Serine Protease-2 + 92% Complement Component Recep 2 +/- 93%
Complement 8 Gamma Chain ++ 94% Complement C1s ++ 95% Ficolin 3 +
96% Complement C5 + 99% Complement 4a + 100% Ficolin-1 + 101%
Factor H ++ 104% Complement C1q Binding Protein + 107% Complement
C1r + 107% Ig Kappa Variable 1D-8 ++ 133% Clusterin ++ 142%
Complement C6 +/- 185% Abundance (relative signal): 1-1000 (+/-);
1000-50,000 (+); >50,000 = (++)
Example 6
Characterization of Complement Activation Pathway(s)
[0178] Because C5b-9 complexes, if they are present in sufficient
quantities at the RPE-choroid interface, are likely cause serious
damage to RPE and choroidal cells, we have conducted studies to
characterize the mechanism(s) of complement activation in
individuals with AMD. At least three pathways of complement
activation have been described. These include the classical pathway
that is activated via immunoglobulin and C1q, the alternative
pathway that is activated at cell surfaces, and the lectin pathway
in which the acute phase reactant mannose-binding protein (MBP)
activates complement activation along the alternative and/or
classical pathways.
[0179] Based upon the known molecular relationships in each of
these activation pathways, one would predict that, if complement
activation occurs along the classical pathway, detectable levels of
the immunoglobulins IgG and IgM, as well as C1q, should be present
in drusen and Bruch's membrane. On the other hand, if activation
occurs along the alternative pathway, Factors H, I, D, and B would
be detectable, and if the lectin pathway of activation is involved,
mannose-binding protein would be expected to be present.
[0180] C1q--an indicator of the classical pathway--is not a major
drusen constituent, although it is detected in some drusen.
Similarly, other proteins involved in the classical pathway of
complement activation are detected only in the choroid, and not in
Bruch's membrane and/or drusen. Collectively, these data suggest
that the classical pathway may not be the primary pathway involved
in the activation of the complement cascade. However,
immunoglobulin has been identified within drusen using both
biochemical and immunohistochemical approaches (Tables 2 and 4).
Thus, immunoglobulin in the form of immune complexes can trigger
the classical pathway.
[0181] Data collected to date reveal that nearly all of the above
major activators of the alternative pathway are present in the
drusen and/or the RPE. Six proteins--C3, Factor B, Factor D, Factor
H, Factor I and properdin--perform the function of initiation,
recognition, and amplification of the alternative pathway, which
results in the formation of the activator-bound C3/C5 convertase.
The recognition of the alternative pathway activator involves C3b
bound to the cell membrane. Factor H--a fluid phase regulator of
the alternative pathway of complement that functions to prevent
amplification of complement activation by accelerating the decay of
C3 and C5 convertases and by acting as a cofactor for factor
I-mediated cleavage of surface bound C3b--is a major component of
all drusen phenotypes. Further activation of the C3 convertase and
cleavage of C3 into smaller molecules results the generation of C3b
that propagates an amplification loop. An additional cleavage
product from this reaction, the C3d molecule, is also found to be
present in all drusen phenotypes.
[0182] Mannose binding protein, which is believed to be a potent
initiator of the lectin pathway, is also present in all drusen
phenotypes, indicating that the lectin pathway can also be
operative at the RPE-choroid interface.
[0183] The data collected thus far indicate that activation of the
complement cascade at the level of the RPE-choroid interface occurs
via the alternative and/or lectin pathways, although some
activation via the classical pathway can not be ruled out
completely at this point. Whatever the exact mechanism of
activation, these data provide further evidence that local
complement activation occurs at the RPE-choroid interface where it
may induce significant tissue damage and pathology.
Example 7
Biochemical Assessment of Plasma Membrane-Associated C5b-9 in RPE
and Choroid Cells in Human Donor Eyes
[0184] Biochemical studies were conducted in order to determine
whether C5b-9 complexes are inserted into plasma membranes of RPE
and/or choroidal cells in the vicinity of Bruch's membrane.
Isolated RPE cells and the RPE/choroid layers from five donors of
various ages (two with AMD) were used in these experiments. The
samples were homogenized, and plasma membranes were collected using
a sucrose gradient ultracentrifugation procedure. Proteins derived
from these membrane preparations, as well as the cytosolic
supernatant fractions, were tested for the presence of C5b-9
complex by means of ELISA and Western blotting analyses with
anti-complement antibodies. Indirect ELISA as well as capture ELISA
was performed using a commercial C5b-9 detection kit (Quidel, San
Diego).
[0185] The results of these analyses demonstrate that the C5b-9 MAC
complexes are present, and predominant, in the plasma membranes
isolated from the RPE and choroid samples from older donors with
and without AMD (FIGS. 3, 4, and 5). In contrast, cells from a ten
year old donor (409-00) possess almost undetectable C5b-9 levels in
both membrane and cytosolic preparations. The highest levels of
C5b-9 is detected in cells from a donor with neovascular AMD
(239-00), which supports the hypothesis that complement activation
and MAC formation in AMD may be associated with the process of
choroidal neovascularization. These differences in C5b-9 levels and
distribution between these donors were confirmed
immunohistochemically. Intense positive labeling of drusen and
Bruch's membrane, as well as some labeling of the choriocapillaris,
was observed in the neovascular AMD donor. In contrast, the tissue
of the young donor was sporadically, and weakly, positive in some
regions of the choroid near Bruch's membrane. In addition, the
presence of proteins that are part of the C5b-9 complex was
confirmed by Western blot analyses. Positive labeling with
antibodies directed against C6 and C9 in RPE membranes and
membranes derived from the RPE-choroid complex indicates that
membrane insertion of complement complexes does take place in these
tissues, and that this insertion may be injurious to the RPE and/or
choroid.
Example 8
Characterization of Drusen associated with Glomerulonephritis
[0186] Many subjects with membranoproliferative glomerulonephritis
type II (MPGN-II) are characterized by the presence of deposits
within Bruch's membrane that resemble drusen.
Glomerulonephritis-associated drusen appear at a younger age,
however, than do drusen in individuals with AMD. The structure and
composition of drusen in eyes obtained from human donors with two
distinct glomerulopathies, both of which involve complement
deposition in the glomerulus were examined. Eyes obtained from two
human donors diagnosed with membranous and post-streptococcal
glomerulonephritis, respectively, were analyzed histochemically,
immunohistochemically, and ultrastructurally. These characteristics
were compared to those derived from individuals with AMD.
[0187] Subretinal pigment epithelial (RPE) deposits in both types
of glomerulonephritis are numerous and indistinguishable, both
structurally and compositionally, from drusen in donors with AMD.
Glomerulonephritis-associated drusen exhibit sudanophilia, bind
filipin, and react with antibodies directed against vitronectin,
complement C5 and C5b-9 complexes, TIMP-3, and amyloid P component.
Drusen from the membranous GN donor, but not the post-streptococcal
GN donor, react with peanut agglutinin and antibodies directed
against MHC class II antigens and IgG. The ultrastructural
characteristics of these deposits were also identical with those of
AMD-associated drusen.
[0188] These data show that composition and structure of ocular
drusen-associated with membranous and post-streptococcal/segmental
glomerulonephritis are generally similar to drusen in individuals
with AMD. These data support other data which indicate that chronic
complement pathway activation is an important contributory factor
in drusen biogenesis and Bruch's membrane pathogenesis. It appears
that defective complement activation alone may be sufficient to
induce the formation of drusen in Bruch's membrane.
Example 9
Autoantibodies in the Sera of Donors with AMD and/or Drusen
[0189] It has been observed that serum autoantibodies are present
in some AMD subjects. The aim of this study is to determine whether
subjects with AMD and ocular drusen have increased levels of
specific autoantibodies against complement component when compared
to controls without such ocular disorder. The identification of
autoantibodies or mediators of complement system provides a
diagnostic means for the identification of AMD or other macular
degeneration-related disorders.
[0190] In order to address the role of autoantibodies in drusen
biogenesis and AMD, a series of experiments were performed using
enriched drusen preparations in order to identify
anti-drusen/Bruch's membrane/RPE autoantibodies that might be
present in the sera of donors with AMD and/or drusen.
[0191] Protein extracts from an enriched drusen preparation (DR+)
obtained by debridement of Bruch's membrane with a #69 Beaver blade
and from a control (DR-) preparation were prepared using PBS with
proteinase inhibitor cocktail and mild detergent. Proteins were
separated by molecular weight using 10-20% gradient mini SDS gels
(Amresco) and transferred to PVDF membranes for Western blot
analysis. PVDF strips with human retinal proteins from 50 normal
human retinas were also used for detection of any anti-retinal
autoantibodies in the donor sera.
[0192] Sera from the same eight donors described above were
screened. Serum from one AMD donor (#90-98) positively labeled a
band in the RPE (both DR+ and DR-) and RPE/choroid preparations of
approximately 35 kDa. A second band of approximately 60 kDa was
labeled weakly only in the DR+ protein extract. Sera from an AAA
donor (#189-97) reacted with a protein(s) of approximately 53 kDa.
This band labeled in all three protein extracts. There was one band
of approximately 64 kDa that this serum sample labeled only in the
DR+ sample.
[0193] The presence of serum anti-drusen/RPE autoantibodies in
donors with AMD and/or drusen further indicates a possible role for
shared immune-mediated processes in these conditions.
Example 10
Analyses of Autoantibodies in the Sera of Living AMD Subjects
[0194] In order to determine whether the sera of AMD subjects
possesses autoantibodies or alterations in the abundance and/or
mobility of serum proteins, plasma was collected from 20 subjects
with clinically-diagnosed AMD and from 20 unaffected subjects to
serve as controls.
[0195] For some experiments, sera were separated by SDS-PAGE and
proteins were visualized with either silver stain or Coomassie
blue, or (for preparative purposes) proteins were transferred to
PVDF membranes for amino acid sequencing. Abnormalities of serum
proteins were detected in a subset of AMD donors. These differences
included the presence of "additional" bands in the sera of some AMD
subjects (molecular weights of .about.25, 29, 30 and 80 kDa) that
were not present in control donors. Amino acid sequencing of these
molecules revealed N-terminal sequences consistent with haptoglobin
(25 kDa) and immunoglobulin kappa (29 kDa), lambda (30 kDa), and
gamma (80 kDa) chains.
[0196] In a second set of experiments, sera from AMD and control
donors was screened for the presence of auto-antibodies against RPE
and choroid proteins. As an extension of experiments in which
weak-moderate immunoreactivity of drusen in tissue sections was
previously observed, purified vitronectin was electrophoretically
separated and blotted onto PDVF. Because vitronectin had previously
been identified as a drusen associated molecule (as detailed in
Example 1), the sera from AMD subjects was then evaluated for the
presence of anti-vitronectin immunoreactivity. Strong labeling of
both the 65 kDa and 75 kDa vitronectin species was identified in
these sera, indicating that AMD sera contain autoantibodies
directed against at least some drusen-associated molecules and/or
Bruch's membrane constituents.
[0197] As an additional approach toward the identification of AMD
autoantibodies and their targets in ocular tissues, RPE-choroidal
proteins from one donor with large numbers of drusen and a nine
month old donor were separated electrophoretically according to
molecular weight and transferred to nitrocellulose. Proteins were
then immunolabeled with either sera from 3 AMD donors or polyclonal
antiserum directed against vitronectin. The AMD sera reacted with
bands of roughly 65, 150 and 200 kDa only in the sample from the
donor with numerous drusen. These results indicate that age and/or
the presence of drusen leads to an increase in AMD autoantigen.
Example 11
Autoantibodies Directed Against RPE, Retina, and Fetal Eye Proteins
in a Patient with Malattia Leventinese
[0198] Proteins extracted from the neural retinal, isolated RPE
cells, and an entire fetal human eye (96 day) were separated by
two-dimensional gel electrophoresis followed by either (a) transfer
of the separated proteins to PVDF membranes or (b) silver staining
of the 2D gel with a modified solution that is compatible with
Matrix Assisted Laser Desorption Ionization (MALDI) mass
spectrometry analyses.
[0199] Blots were probed with human serum derived from a patient
with the early onset macular dystrophy Malattia leventinese,
followed by detection of immobilized primary antibodies with
alkaline phosphatase-conjugated antibodies directed against human
immunoglobulins, and positively labeled spots were matched with the
corresponding spots on the silver-stained gels. Silver-stained
protein spots corresponding with autoantigens on the Western blots
were excised and digested in a solution containing endoproteinase
Lys-c/Trypsin, and the resultant peptides were analyzed by matrix
assisted-laser desorption ionization mass spectrometry, a technique
that permits the identification of a protein based upon the
molecular weights of its peptides (Wheeler et al., Electrophoresis,
17(3):580-7 1996). MALDI-MS can be used as a complement to internal
amino acid sequencing. In J. Walker (Ed.), The Protein Protocols
Handbook (pp. 541-555, Totowa: Humana Press). This technique
resulted in the identification of a number of autoantigens within
these tissues:
[0200] Seven proteins that have been identified from the fetal eye
tissue are: [0201] (i) #1 and #2--MW=27 KD and 25 KD--beta
crystallin A4 (Slingsby et al., Exp Eye Res, 51:21-6, 1990); [0202]
(ii) #3--MW=25 KD--beta crystallin A2 and trace of beta crystallin
A4 (Slingsby et al., supra); [0203] (iii) #4--MW=26 KD--beta
crystallin A3 (Slingsby et al., supra); [0204] (iv) #5--MW=18
KD--beta crystallin S (Quax-Jeuken et al., EMBO J, 4(10):2597-602,
1-985); [0205] (v) #6--MW=26 KD--beta crystallin A4; and [0206]
(vi) #7--MW=80 KD--78 KD glucose-regulated protein Kiang et al.,
Chin J Physiol, 40:213-9, 1997)
[0207] Six proteins were identified from the retinal protein
extract: [0208] (i) #1. MW=60 KD-calreticulin (Kovacs et al.,
Biochemistry, 37(51):17865-74, 1998 [0209] (ii) #2. MW=33
KD--possibly complement component 1 (a.k.a glycoprotein GC1QBP,
hyaluronan-binding protein; Lynch et al., FEBS Lett,
418(1-2):111-4, 1997) [0210] (iii) #3. MW=29 KD--14-3-3 protein
epsilon (Yamanaka et al., Proc Natl Acad Sci USA, 94:6462-7, 1997)
[0211] (iv) #4. MW=85 KD--serotransferrin (Campbell et al., J Biol
Chem, 252:5996-6001, 1977) [0212] (v) #5. MW=80 KD--albumin [0213]
(vi) #6. MW=75 KD--keratin (Hintner et al., J Invest Dermatol,
93:656-61, 1989)
[0214] Two proteins were identified from the RPE protein extract:
[0215] (i) #1. MW=120 KD--pyruvate carboxylase; and [0216] (ii) #2.
MW=88 KD--hypothetical protein DKFZp762H157.1 (also called villin
2; Burgess et al., J. Immunol., 1992, 149: 1847-1852, and U.S. Pat.
No. 5,773,573).
Example 12
Autoantibodies Directed Against RPE, Choroidal, and Retinal
Proteins in a Patient with AMD
[0217] In a separate set of experiments, the serum from donor
#189-97 (diagnosed with both AMD and AAA) was employed to probe
protein extracts from human choroid (donor 325-00, 77 CF), RPE
(donor 318-00, 67 CM) and retina (donor 294-00, 84 CF, AMD) on
blots following two-dimensional gel electrophoresis, as described
above. Several positively-labeled spots, corresponding to putative
autoantigens, were identified. The characteristics of these protein
spots were as follows:
[0218] Choroidal extract proteins:
[0219] (i) three spots were identified with an approximate MW of 86
KD, PI between 5 and 6;
[0220] (ii) four spots were identified with an approximate MW of 60
KD, PI between 7 and 8;
[0221] (iii) five spots were identified with an approximate MW of
45 KD, PI between 6 and 7;
[0222] (iv) 6 spots were identified with an approximate MW between
30 and 43 KD, PI between 4.5 and 6;
[0223] (v) 2 spots were identified with an approximate MW of 33 and
35 kD, PI an approximate 7.5;
[0224] (vi) 1 spot was identified with MW of 29 KD, PI between 5
and 5.5; and
[0225] (vii) 1 spot was identified with an approximate MW of 25 KD,
PI approximately 7.5.
[0226] RPE Extract Proteins:
[0227] (i) three spots were identified with an approximate MW of 86
KD, PI between 5 and 6;
[0228] (ii) three confluent spots were identified with an
approximate MW of 95-100K, PI 6.5-7;
[0229] (iii) two spots were identified with an approximate MW of 94
KD, PI between 5 and 6;
[0230] (iv) one spot was identified with an approximate MW of 60
KD, PI .about.4.5;
[0231] (v) 2 spots were identified with an approximate MW of 33 and
35 kD, PI.about.7.5; and
[0232] (vi) 5 spots were identified with an approximate MW between
35 and 43 KD, PI between 6 and 7;
[0233] Retinal extract proteins:
[0234] (i) thee confluent spots were identified with an approximate
MW of 95-100K, PI 6.5-7;
[0235] (ii) 2 spots were identified with an approximate MW of 33
and 35 kD, PI.about.7.5;
[0236] (iii) one spot was identified with an approximate MW of
30-33 KD, PI.about.7;
[0237] (iv) several confluent spots were identified with an
approximate MW of 60 KD, PI 4-5;
[0238] (v) one spot was identified with an approximate MW of 28-30
KD, PI 4.5-5; and
[0239] (vi) several spots were identified between 28 and 65 KD with
PI from 4 and 7.5.
Example 13
Additional Serological Tests for Markers in Drusen Biogenesis and
AMD
[0240] Visual acuity measurements, stereo macula photos, and
peripheral photos can be taken at the beginning of the study and
every six months thereafter. Blood and sera can be drawn when
subjects enter the study and every 6-12 months thereafter. DNA can
be prepared from a portion of each blood sample for future genetic
studies. The presence of serum autoantibodies and immune complexes
can be determined using standard protocols. In addition, sera can
be reacted with tissue sections derived from donors with and
without AMD, followed by a secondary antibody that has been
adsorbed against human immunoglobulins. Western blots of
retina/RPE/choroid from AMD and non-AMD donors can also be
incubated with serum samples to identify specific bands against
which autoantibodies react.
[0241] The presence of antibodies directed against the following
proteins (many observed in other age-related conditions and/or
MPGN) can also be determined: type IV collagen, glomerular basement
membrane, neutrophils, cytoplasm (c-ANCA, p-ANCA), C3 convertase
(C3 nephritic factor), alpha-1 anti-trypsin levels (decreased in
MPGN), epsilon 4 allele, apolipoprotein E, GFAP, ANA, serum
senescent cell antigen, S-100, type 2 plasminogen activator,
alpha-1-antichymotrypsin, SP-40,40, endothelial cell, parietal
cell, mitochondria, Jo-1, islet cell, inner ear antigen,
epidermolysis Bullosa Acquista, endomysial IgA, cancer antigen
15-3, phospholipid, neuronal nucleus, cardiolipin, and
ganglioside.
[0242] In addition to autoantibodies against complement components,
sera from the subject can be reacted with tissue sections derived
from donors with and without AMD, followed by a secondary antibody
that has been adsorbed against human immunoglobulins. Western blots
of retina/RPE/choroid from AMD and non-AMD donors can also be
incubated with serum samples to identify specific bands against
which autoantibodies react.
[0243] Further, other than autoantibodies, levels of the following
proteins, additional indicators of autoantibody responses, chronic
inflammation and/or acute phase responses, can be assayed by a
clinical diagnostic laboratory. These can include Bence Jones
protein, serum amyloid A, M components, CRP, mannose binding
protein, serum amyloid A, C3a, C5a, other complement proteins,
coagulation proteins, fibrinogen, vitronectin, CD25, interleukin 1,
interleukin 6, and apolipoprotein E. Serum protein electrophoresis,
lymphocyte transformation, sedimentation rate, and spontaneous,
whole blood, white cell count can also be measured. Other proteins
that provide additional indication of autoantibody responses,
chronic inflammation and/or acute phase responses, can also be
assayed.
* * * * *
References