U.S. patent application number 15/844391 was filed with the patent office on 2018-11-22 for use of complement pathway inhibitors to treat ocular diseases.
The applicant listed for this patent is GENENTECH, INC.. Invention is credited to Sek Chung FUNG, Zhengbin YAO.
Application Number | 20180334495 15/844391 |
Document ID | / |
Family ID | 38023865 |
Filed Date | 2018-11-22 |
United States Patent
Application |
20180334495 |
Kind Code |
A1 |
FUNG; Sek Chung ; et
al. |
November 22, 2018 |
USE OF COMPLEMENT PATHWAY INHIBITORS TO TREAT OCULAR DISEASES
Abstract
The present invention relates to the treatment of ocular
diseases and conditions by administering a complement pathway
inhibitor, particularly an alternative pathway inhibitor. Ocular
diseases include age-related macular degeneration, diabetic M
retinopathy, and ocular angiogenesis. One embodiment comprises the
administration of an anti-Factor D antibody in the form of a whole
antibody, a Fab fragment or a single domain antibody. Other
complement component inhibitors that may be useful in the present
method include Factor H or inhibitors that block the action of
properdin, factor B, factor Ba, factor Bb, C2, C2a, C3a, C5, C5a,
C5b, C6, C7, C8, C9, or C5b-9.
Inventors: |
FUNG; Sek Chung;
(Gaithersburg, MD) ; YAO; Zhengbin; (Berwyn,
PA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
GENENTECH, INC. |
South San Francisco |
CA |
US |
|
|
Family ID: |
38023865 |
Appl. No.: |
15/844391 |
Filed: |
December 15, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14270848 |
May 6, 2014 |
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15844391 |
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12092346 |
Oct 9, 2008 |
8753625 |
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PCT/US2006/043103 |
Nov 4, 2006 |
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14270848 |
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60733763 |
Nov 4, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 9/10 20180101; A61P
3/10 20180101; A61P 27/00 20180101; A61K 39/395 20130101; A61K
38/00 20130101; A61P 37/02 20180101; A61K 2039/505 20130101; A61P
43/00 20180101; C07K 16/18 20130101; A61P 9/00 20180101; A61K
2300/00 20130101; C12N 15/113 20130101; A61K 38/1709 20130101; A61P
27/04 20180101; A61P 37/06 20180101; A61K 39/3955 20130101; A61K
45/06 20130101; A61P 27/02 20180101 |
International
Class: |
C07K 16/18 20060101
C07K016/18; C12N 15/113 20100101 C12N015/113; A61K 45/06 20060101
A61K045/06; A61K 38/17 20060101 A61K038/17; A61K 39/395 20060101
A61K039/395 |
Claims
1.-27. (canceled)
28. A method for preventing or ameliorating an ocular disease that
involves complement activation, comprising administering an
effective amount of a complement inhibitor to a subject in need
thereof.
29. The method of claim 28, wherein the ocular disease is selected
from the group consisting of macular degeneration, diabetic
retinopathy, and ocular angiogenesis.
30. The method of claim 28, wherein the subject requires inhibition
of ocular neovascularization that affects the choroid, retinal
pigmented epithelium, or retinal tissue.
31. The method of claim 28, wherein the complement inhibitor
inhibits the alternative complement pathway.
32. The method of claim 28, wherein the complement inhibitor is
Factor H, or a functional peptide thereof or a peptidomimetic
thereof.
33. The method according to claim 28, wherein the complement
inhibitor is an antibody or an antigen-binding fragment
thereof.
34. The method according to claim 33 wherein the antibody or the
antigen-binding fragment thereof specifically binds to Factor D,
properdin, Factor B, Factor Ba, Factor Bb, C2, C2a, C3a, C5, C5a,
C5b, C6, C7, C8, C9 or C5b-9.
35. The method according to claim 33, wherein the antibody is
monoclonal antibody 166-32 produced from the hybridoma deposited
with the ATCC and designated HB 12476.
36. The method according to claim 33, wherein the antibody or the
antigen-binding fragment thereof specifically binds to the same
epitope as monoclonal antibody 166-32 produced from the hybridoma
deposited with the ATCC and designated HB 12476.
37. The method according to claim 33, wherein the antibody is a
humanized monoclonal antibody derived from monoclonal antibody
166-32 produced from the hybridoma deposited with the ATCC and
designated HB 12476.
38. The method according to claim 33, wherein the antibody or the
antigen-binding fragment thereof specifically binds to complement
component C5a.
39. The method according to claim 38, wherein the antibody is
137-26 produced from the hybridoma deposited with the ATCC and
designated PTA-3650.
40. The method according to claim 38, wherein the antibody or the
antigen-binding fragment thereof binds to the same epitope as
monoclonal antibody 137-26 produced from the hybridoma deposited
with the ATCC and designated PTA-3650.
41. The method of claim 28, wherein the complement inhibitor is
administered by intraocular administration, intravitreal
administration, or subconjunctival administration, parenteral
administration, intradermal administration, intramuscular
administration, intraperitoneal administration, intravenous
administration, subantaneous administration, intranasal
administration, oral administration, enteral administration,
topical administration, intrathecal administration,
intraventricular administration, epidural, inhalation, a
biocompatible or bioerodable sustained release implant, or
implantation of an infusion pump.
42. The method according to claim 28, wherein the complement
inhibitor is administered in an eye wash solution, an eye ointment,
an eye shield or an eye drop solution.
43. The method according to claim 28, further comprising the step
of administering an immunomodulatory compound, an immunosuppressive
compound, an anti-inflammatory compound, or an anti-angiogenic
compound, to said subject.
44. The method of claim 28, further comprising administering to the
subject an anti-angiogenesis therapy targeting vascular endothelial
growth factor (VEGF).
45. The method of claim 28, further comprising administering to the
subject a steroid.
46. A method of preventing or ameliorating an ocular disease that
involves complement activation, comprising administering an siRNA
specific for a complement pathway protein to a subject in need
thereof.
47. A method of preventing or ameliorating an ocular disease that
involves complement activation, comprising administering a nucleic
acid encoding a complement inhibitor to a subject in need
thereof.
48. The method of claim 29, wherein the macular degeneration is
age-related macular degeneration.
49. The method claim 48, wherein the age-related macular
degeneration is the dry form of age-related macular degeneration.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 14/270,848, filed May 6, 2014, now abandoned, which is a
continuation of U.S. application Ser. No. 12/092,346, filed Oct. 9,
2008, now U.S. Pat. No. 8,753,625, which is a National Stage
application of International Application No. PCT/US2006/043103,
filed Nov. 4, 2006, which claims the benefit of U.S. Provisional
Application No. 60/733,763 filed Nov. 4, 2005, the disclosure of
each of which is incorporated by reference herein in their
entireties.
FIELD OF THE INVENTION
[0002] This invention relates to the inhibition of the complement
pathway, particularly Factor D, in patients suffering from ocular
related conditions and diseases associated with complement
activation such as age-related macular degeneration, diabetic
retinopathy.
BACKGROUND OF THE INVENTION
[0003] Macular degeneration is a clinical term that is used to
describe a family of diseases that are characterized by a
progressive loss of central vision associated with abnormalities of
the Bruch's membrane, the choroid, the neural retina and/or the
retinal pigment epithelium. In the center of the retina is the
macula lutea, which is about 1/3 to 1/2 cm. in diameter. The macula
provides detailed vision, particularly in the center (the fovea),
because the cones are higher in density. Blood vessels, ganglion
cells, inner nuclear layer and cells, and the plexiform layers are
all displaced to one side (rather than resting above the ones),
thereby allowing light a more direct path to the cones. Under the
retina is the choroid, a collection of blood vessels embedded
within a fibrous tissue, and the pigmented epithelium (PE), which
overlays the choroid layer. The choroidal blood vessels provide
nutrition to the retina (particularly its visual cells). The
choroid and PE are found at the posterior of the eye.
[0004] The retinal pigment epithelial (RPE) cells, which make up
the PE, produce, store and transport a variety of factors that are
responsible for the normal function and survival of photoreceptors.
These multifunctional cells transport metabolites to the
photoreceptors from their blood supply, the chorio capillaris of
the eye. RPE cells also function as macrophages, phagocytizing the
tips of the outer segments of rods and cones, which are produced in
the normal course of cell physiology. Various ions, proteins and
water move between the RPE cells and the interphotoreceptor space,
and these molecules ultimately effect the metabolism and viability
of the photoreceptors.
[0005] Age-related macular degeneration (AMD), the most prevalent
macular degeneration, is associated with progressive loss of visual
acuity in the central portion of the visual field, changes in color
vision, and abnormal dark adaptation and sensitivity. Two principal
clinical manifestations of AMD have been described as the dry, or
atrophic, form, and the wet, or exudative, form. The dry form is
associated with atrophic cell death of the central retina or
macula, which is required for fine vision used for activities such
as reading, driving or recognizing faces. About 10-20% of these dry
AMD patients progress to the second form of AMD, known as wet
AMD.
[0006] Wet (neovascular/exudative) AMD is caused by abnormal growth
of blood vessels behind the retina under the macula and vascular
leakage, resulting in displacement of the retina. hemorrhage and
scar formation. This results in a deterioration of sight over a
period of months to years. however, patients can suffer a rapid
loss of vision. All wet AMD cases are originated from advanced dry
AMD. The wet form accounts for 85% of blindness due to AMD. In wet
AMD, as the blood vessels leak fluid and blood, scar tissue is
formed that destroys the central retina.
[0007] The most significant risk factors for the development of
both forms are age and the deposition of drusen, abnormal
extracellular deposits, behind the retinal pigment epithelium.
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. Drusen are the
hallmark deposits associated with AMD. The biogenesis of drusen
involves RPE dysfunction, impaired digestion of photoreceptor outer
segments, and subsequent debris accumulation. Drusen contain
complement activators, inhibitors, activation-specific complement
fragments, and terminal pathway components, including the membrane
attack complex (MAC or C5b-9), which suggests that focal
concentration of these materials may produce a powerful chemotactic
stimulus for leukocytes acting via a complement cascade
(Killingsworth, et al., (2001) Exp Eye Res 73, 887-96). recent
studies have implicated local inflammation and activation of the
complement cascade in their formation (Bok D. Proc Natl Acad Sci
(USA). 2005; 102: 7053-4; Hademan G S, et al. Prog Retin Eye Res.
2001; 73: 887-96).
[0008] Wet AMD is associated with choroidal neovascularization
(CNV) and is a complex biological process. Pathogenesis of new
chloroidal vessel formation is poorly understood, but such factors
as inflammation, ischemia, and local production of angiogenic
factors are thought to be important. Although inflammation has been
suggested as a playing a role, the role of complement has not been
explored. A preliminary study of CNV has been shown to be caused by
complement activation in a mouse model (Bora P S, J Immunol. 2005;
174: 491-497).
[0009] The complement system is a crucial component of the innate
immunity against microbial infection and comprises a group of
proteins that are normally present in the serum in an inactive
state. These proteins are organized in three activation pathways:
the classical, the lectin, and the alternative pathways (V. M.
Holers, In Clinical Immunology: Principles and Practice, ed. R. R.
Rich, Mosby Press; 1996, 362-391). Molecules on the surface of
microbes can activate these pathways resulting in the formation of
protease complexes known as C3-convertases. The classical pathway
is a calcium magnesium-dependent cascade, which is normally
activated by the formation of antigen-antibody complexes. It can
also be activated in an antibody-independent manner by the binding
of C-reactive protein complexed with ligand and by many pathogens
including gram-negative bacteria. The alternative pathway is a
magnesium-dependent cascade which is activated by deposition and
activation of C3 on certain susceptible surfaces (e.g. cell wall
polysaccharides of yeast and bacteria, and certain biopolymer
materials).
[0010] The alternative pathway participates in the amplification of
the activity of both the classical pathway and lectin pathway
(Suankratay, C., ibid; Farries, T. C. et al., Mol. Immunol. 27:
1155-1161(1990). Activation of the complement pathway generates
biologically active fragments of complement proteins, e.g. C3a, C4a
and C5a anaphylatoxins and C5b-9 membrane attack complexes (MAC),
which mediate inflammatory responses through involvement of
leukocyte chemotaxis, activation of macrophages, neutrophils,
platelets, mast cells and endothelial cells, increased vascular
permeability, cytolysis, and tissue injury.
[0011] Factor D may be a suitable target for the inhibition of this
amplification of the complement pathways because its plasma
concentration in humans is very low (1.8 .mu.g/ml), and it has been
shown to be the limiting enzyme for activation of the alternative
complement pathway (P. H. Lesavre and H. J. Muller-Eberhard. J.
Exp. Med., 1978; 148: 1498-1510; J. E. Volanakis et al., New Eng.
J. Med., 1985; 312: 395-401). The inhibition of complement
activation has been demonstrated to be effective in treating
several disease indications using animal models and in ex vivo
studies, e.g. systemic lupus erythematosus and glomerulonephritis
(Y. Wang et al., Proc. Natl. Acad. Sci.; 1996, 93: 8563-8568).
[0012] Using single-nucleotide polymorphism (SNP) analysis of AMD
patients, a Factor H genetic variant (Y402H) was found to be highly
associated with increased incidence of AMD (Zareparsi S, Branham
KEH, Li M, et al. Am J Hum Genet. 2005; 77: 149-53; Hains J L, et
al. Sci 2005; 208: 419-21). Persons who are either homozygous or
heterozygous for this point mutation of Factor H gene may account
for 50% of AMD cases. Factor H is the key soluble inhibitor of the
alternative complement pathway (Rodriguez de Cordoba S, et al. Mol
Immunol 2004; 41: 335-67). It binds to C3b and thus accelerates the
decay of the alternative pathway C3-convertase (C3bBb) and acts as
a co-factor for the Factor I-mediated proteolytic inactivation of
C3b. Histochemical staining studies show that there is similar
distribution of Factor H and MAC at the RPE-choroid interface.
Significant amounts of deposited MAC at this interface found in AMD
patients indicate that the Factor H haplotype (Y402H) may have
attenuated complement inhibitory function. It is speculated that
Factor H (Y402H) may have a lower binding affinity for C3b.
Therefore, it is not as effective as wild type Factor H in
inhibiting the activation of the alternative complement pathway.
This puts RPE and choroids cells at sustained risk for alternative
pathway-medicated complement attack.
[0013] It had been shown that lack of Factor H in plasma causes
uncontrolled activation of the alternative pathway with consumption
of C3 and often other terminal complement components such as C5. In
keeping with this finding, plasma levels of Factor H are known to
decrease with smoking, a known risk Factor for AMD
(Esparza-Gordillo J, et al., Immunogenetics. 2004; 56: 77-82).
[0014] Currently, there is no proven medical therapy for dry AMD,
and no treatments available for advanced dry AMD. In selected cases
of wet AMD, a technique known as laser photocoagulation may be
effective for sealing leaky or bleeding blood vessels.
Unfortunately, laser photocoagulation usually does not restore lost
vision, but merely slows, and in some cases, prevents further loss.
Recently, photodynamic therapy has shown to be effective in
stopping abnormal blood vessel growth in about one third of wet AMD
patients when treated early. In Visdyne Photodynamic Therapy (PDT),
a dye is injected into the patient's eye, it accumulates in the
area of vessel leakage in the retina and, when exposed to a low
power laser, it reacts sealing off the leaking vessels. In addition
to these two laser techniques, there are several anti-angiogenesis
therapies targeting vascular endothelial growth Factor (VEGF) being
developed for the treatment of wet AMD. However, only 10% treated
patients show vision improvement.
[0015] In view of these inadequate treatments for wet AMD and the
total lack of treatments available for advanced dry AMD, there is a
clear need for the development of new treatments for this serious
disease. Our invention provides a novel approach to treating this
serious disease.
SUMMARY OF THE INVENTION
[0016] The present invention relates to complement inhibitors for
the treatment of ocular related conditions or diseases, such as
age-related macular degeneration (AMD), diabetic retinopathy,
ocular angiogenesis (such as ocular neovascularization affecting
choroidal, corneal, or retinal tissue), and the ocular conditions
involving complement activation. treatment of AMD includes both the
dry and we forms of AMD.
[0017] The complement inhibitors of the present invention include,
but are not limited to, those inhibiting the alternative complement
pathway, such as Factor D, properdin, Factor B, Factor Ba, and
Factor Bb, and the classical complement pathway, such as C3a, C5,
C5a, C5b, C6, C7, C8, C9 and C5b-9. The present invention also
includes the use of complement inhibitors in combination with other
agents, such as anti-angiogenic agents and anti-inflammatory agents
such as steroids.
[0018] Another embodiment of the present invention relates to the
use of C5aR and C3aR inhibitors, such as antibodies and derived
fragments and signal domain constructs, as well as small molecule
compounds.
[0019] Another embodiment of the present invention relates to the
use of recombinant soluble CR1 (TP10) and its derived proteins; use
of C3 inhibiting molecules (such as Compstatin, a peptidomimetic
and binds and inhibits C3 activation); siRNAs that block the
synthesis of C3, C5, FD, factor P, factor B
[0020] these inhibitors can be, but not limited to, small molecule
chemical compounds, nucleotides, peptides, proteins,
peptidomimetics and antibodies.
[0021] Another embodiment of the present invention includes the use
of human Factor H purified from human blood or recombinant human
Factor H administered to patients intraocularly or by any other
clinically effective route.
[0022] Antibodies of the present invention include whole
immunoglobulins, scFv, Fab, Fab', Fv, F(ab')2, or dAb. Domain
antibodies comprise either a VH domain or a VL domain.
[0023] One embodiment of the present invention is the use of a
monoclonal antibody which binds to Factor D and blocks its ability
to activate the alternative complement pathway. Such antibodies are
described in WO 01/70818 and US 20020081293, which are incorporated
here by reference, such as monoclonal antibody 166-32 produced from
the hybridoma deposited with the American Type Culture Collection
(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S., on Feb.
24, 1998, and designated HB12476. The present invention also
includes antibodies that specifically bind to the same epitope as
monoclonal antibody 166-32. Monoclonal antibodies of the present
invention may also include the humanized antibodies of co-pending
application No. 60/856,505 filed Nov. 2, 2006, which is
incorporated herein by reference.
[0024] One embodiment of the present invention is the use of a
monoclonal antibody which binds to complement component C5a. Such
antibodies include antibody 137-26 produced from the hybridoma
deposited with the ATCC and designated PTA-3650, and any antibody
that specifically binds to the same epitope as 137-26.
[0025] According to the present invention, the complement pathway
inhibitor may be administered by (a) parenteral administration; (b)
biocompatible or bioerodable sustained release implant; (c)
implantation of an infusion pump; or (d) local administration, such
as subconjunctival administration or by intravitreal
administration. The complement inhibitor may also be administered
by parenteral administration selected from oral administration,
enteral administration and topical administration. Topical
administration may include an eye wash solution, an eye ointment,
an eye shield or an eye drop solution.
[0026] In addition the complement inhibitor of the present
invention may be administered in combination with a
immunomodulatory or immunosuppressive compound.
[0027] Another embodiment of the present invention relates to the
administration of nucleic acid constructs that are capable of
expressing the complement pathway inhibitors for gene therapy.
[0028] Another embodiment of the present invention includes a
method for screening for complement inhibitors that are useful in
the treatment of AMD comprising the use of an AMD model in
senescent Ccl-2 or Ccr-2-deficient mice. These mice manifest
similar histopathological changes found in human dry and wet AMDs.
These mice may be treated with complement inhibitors or Factor H
intravitreally. Histological examination may be performed to
determine protection from AMD development in mice treated with the
agents to be tested.
DETAILED DESCRIPTION OF THE INVENTION
[0029] This invention is not limited to the particular methodology,
protocols, cell lines, vectors, or reagents described herein
because they may vary. Further, the terminology used herein is for
the purpose of describing particular embodiments only and is not
intended to limit the scope of the present invention. As used
herein and in the appended claims, the singular forms "a", "an",
and "the" include plural reference unless the context clearly
dictates otherwise, e.g., reference to "a host cell" includes a
plurality of such host cells.
[0030] Unless defined otherwise, all technical and scientific terms
and any acronyms used herein have the same meanings as commonly
understood by one of ordinary skill in the art in the field of the
invention. Although any methods and materials similar or equivalent
to those described herein can be used in the practice of the
present invention, the exemplary methods, devices, and materials
are described herein.
[0031] All patents and publications mentioned herein are
incorporated herein by reference to the extend allowed by law for
the purpose of describing and disclosing the proteins, enzymes,
vectors, host cells, and methodologies reported therein that might
be used with the present invention. However, nothing herein is to
be construed as an admission that the invention is not entitled to
antedate such disclosure by virtue of prior invention.
Definitions
[0032] The term "amino acid sequence variant" refers to
polypeptides having amino acid sequences that differ to some extent
from a native sequence polypeptide. Ordinarily, amino acid sequence
variants will possess at least about 70% homology, or at least
about 80%, or at least about 90% homology to the native
polypeptide. The amino acid sequence variants possess
substitutions, deletions, and/or insertions at certain positions
within the amino acid sequence of the native amino acid
sequence.
[0033] The term "identity" or "homology" is defined as the
percentage of amino acid residues in the candidate sequence that
are identical with the residue of a corresponding sequence to which
it is compared, after aligning the sequences and introducing gaps,
if necessary to achieve the maximum percent identity for the entire
sequence, and not considering any conservative substitutions as
part of the sequence identity. Neither N- or C-terminal extensions
nor insertions shall be construed as reducing identity or homology.
Methods and computer programs for the alignment are well known in
the art. Sequence identity can be readily calculated by known
methods, including but not limited to those described in
(Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing: Informatics and
Genome projects, Smith, D. W., ed., Academic Press, New York, 1993;
Computer, Analysis of Sequence Data, Part I, Griffin, A. M., and
Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press,
1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M Stockton Press, New York, 1991; and Carillo, 30 H., and
Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to
determine identity are designed to give the largest match between
the sequences tested. Computer program methods to determine
identity between two sequences include, but are not limited to, the
GCG program package (Devereux, J., et al., Nucleic Acids Research
12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et
al., J Molec. Biol. 215: 403-410 (1990). The BLAST X program is
publicly available from NCBI and other sources (BLASTManual,
Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Atschul, S.,
et al., J. Mol. Biol. 215: 403-410 (1990). The wall-known Smith
Waterman algorithm may also be used to determine identity.
[0034] The term "antibody" herein is used in the broadest sense and
specifically covers intact monoclonal antibodies, polyclonal
antibodies, multispecific antibodies (e.g., bispecific antibodies)
formed from at least two intact antibodies, and antibody fragments,
so long as they exhibit the desired biological activity.
[0035] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. In contrast to
polyclonal antibody preparations which include different antibodies
directed against different determinants (epitopes), each monoclonal
antibody is directed against a single determinant on the antigen.
The modifier "monoclonal" indicates the character of the antibody
as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler et al,
Nature, 256:495 (1975), or may be made by recombinant DNA methods
(see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the
techniques described in Clackson et al., Nature, 352:624-628 (1991)
and Marks et al., J. Mol. Biol., 222:581-597 (1991), for
example.
[0036] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad.
Sci. USA, 81:6851-6855 (1984).
[0037] "Antibody fragments" comprise a portion of an intact
antibody comprising the antigen-binding or variable region thereof.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and
Fv fragments; diabodies; linear antibodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragment(s).
[0038] An "intact" antibody is one which comprises an
antigen-binding variable region as well as a light chain constant
domain (C.sub.L) and heavy chain constant domains, C.sub.H1,
C.sub.H2 and C.sub.H3. The constant domains may be native sequence
constant domains (e.g. human native sequence constant domains) or
amino acid sequence variant thereof. The intact antibody may have
one or more effector functions.
[0039] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody.
Examples of antibody effector functions include C1q binding;
complement dependent cytotoxicity; Fc receptor binding;
antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis;
down regulation of cell surface receptors (e.g. B cell receptor;
BCR), etc.
[0040] Depending on the amino acid sequence of the constant domain
of their heavy chains, intact antibodies can be assigned to
different "classes". There are five-major classes of intact
antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may
be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2,
IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that
correspond to the different classes of antibodies are called
.alpha., .delta., , .gamma., and .mu., respectively. The subunit
structures and three-dimensional configurations of different
classes of immunoglobulins are well known.
[0041] "Antibody-dependent cell-mediated cytotoxicity" (ADCC)
refers to a cell-mediated reaction in which nonspecific cytotoxic
cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK)
cells, neutrophils, and macrophages) recognize bound antibody on a
target cell and subsequently cause lysis of the target cell. The
primary cells for mediating ADCC, NK cells, express Fc.gamma.RIII
only, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIII. FcR expression on hematopoietic cells. To access
ADCC activity of a molecule of interest, an in vitro ADCC assay,
such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may
be performed. Useful effector cells for such assays include
peripheral blood mononuclear cells (PBMC) and Natural Killer (NK)
cells. Alternatively, or additionally, ADCC activity of the
molecule of interest may be assessed in vivo, e.g., in a animal
model. Several such models are available.
[0042] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions both in the light chain and the heavy chain
variable domains. These hypervariable regions are also called
complementarity determining regions or CDRs. The more highly
conserved portions of variable domains are called the framework
regions (FRs). The variable domains of native heavy and light
chains each comprise four FRs, largely adopting a .beta.-sheet
configuration, connected by three hypervariable regions, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The hypervariable regions in each chain are
held together in close proximity by the FRs and, with the
hypervariable regions from the other chain, contribute to the
formation of the antigen-binding site of antibodies (see Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)).
[0043] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. the hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L30 in the light
chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in
the heavy chain variable domain; Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (e.g., residues 2632 (L1),
50-52 (L2) and 91-96 (L3) in the light chain variable domain and
26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable
domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987).
"Framework Region" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined.
[0044] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-binding sites
and is still capable of cross-linking antigen.
[0045] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab+-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear at least one free thiol
group. F(ab').sub.2 antibody fragments originally were produced as
pairs of Fab' fragments which have hinge cysteins between them.
Other chemical couplings of antibody fragments are also known.
[0046] The "light chains" of antibodies from any vertebrate species
can be assigned to one of two clearly distinct types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequences
of their constant domains.
[0047] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and antigen-binding site. This region
consists of a dimer of one heavy chain and one light chain variable
domain in tight, non-covalent association. It is in this
configuration that the three hypervariable regions of each variable
domain interact to define an antigen-binding site on the surface of
the V.sub.H. V.sub.L dimer. Collectively, the six hypervariable
regions confer antigen-binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising
only three hypervariable regions specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site.
[0048] "Single-chain Fv" or "scFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the scFv to form the
desired structure for antigen binding. For a review of scFv see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994). Anti-ErbB2 antibody scFv fragments are described in
WO93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No.
5,587,458.
[0049] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which comprise a variable heavy domain
(V.sub.H) connected to a variable light domain (V.sub.l) in the
same polypeptide chain (V.sub.HV.sub.L). By using a linker that is
too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in, for example, EP 404,097; WO
93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993).
[0050] A "single-domain antibody" is synonymous with "dAb" and
refers to an immunoglobulin variable region polypeptide wherein
antigen binding is effected by a single variable region domain. A
"single-domain antibody" as used herein, includes i) an antibody
comprise heavy chain variable domain (VH), or antigen binding
fragment thereof, which forms an antigen binding site independently
of any other variable domain, ii) an antibody comprising a light
chain variable domain (VL), or antigen binding fragment thereof,
which forms an antigen binding site independently of any other
variable domain, iii) an antibody comprising a VH domain
polypeptide linked to another VH or a VL domain polypeptide (e.g.,
VH-VH or VHx-VL), wherein each V domain forms an antigen binding
site independently of any other variable domain, and iv) an
antibody comprising VL domain polypeptide linked to another VL
domain polypeptide (VL-VL), wherein each V domain forms an antigen
binding site independently or any other variable domain. As used
herein, the VL domain refers to both the kappa and lambda forms of
the light chains.
[0051] "Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. Humanized antibodies are human
immunoglobulins wherein the hypervariable regions are replaced by
residues from a hypervariable region of a non-human species, such
as mouse, rat, rabbit or nonhuman primate having the desired
specificity, affinity, and capacity. In some instances, framework
region (FR) residues of the human immunoglobulin are replaced by
corresponding non-human residues. Furthermore, humanized antibodies
may comprise residues that are not found in the human antibody or
in the non-human antibody. These modifications are made to further
refine antibody performance. In general, the humanized antibody
will comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the
hypervariable loops correspond to those of a non-human
immunoglobulin and all or substantially all of the FRs are those of
a human immunoglobulin sequence. The humanized antibody optionally
also will comprise at least a portion of an immunoglobulin constant
region. (Fc), typically that of a human immunoglobulin. Examples of
humanization technology may be found in, e.g., Queen et al. U.S.
Pat. Nos. 5,585,089, 5,693,761; 5,693,762; and 6,180,370, which are
incorporated herein by reference.
Antibody Generation
[0052] The antibodies of the present invention may be generated by
any suitable method known in the art. The antibodies of the present
invention may comprise polyclonal antibodies. Methods of preparing
polyclonal antibodies are known to the skilled artisan (Harlow, et
al., Antibodies: a Laboratory Manual, (Cold spring Harbor
Laboratory Press, 2nd ed. (1988), which is hereby incorporated
herein by reference in its entirety).
[0053] For example, antibodies may be generated by administering an
immunogen comprising the antigen of interest to various host
animals including, but not limited to, rabbits, mice, rats, etc.,
to induce the production of sera containing polyclonal antibodies
specific for the antigen. The administration of the immunogen may
entail one or more injections of an immunizing agent and, if
desired, an adjuvant. Various adjuvants may be used to increase the
immunological response, depending on the host species, and include
but are not limited to, Freund's (complete and incomplete), mineral
gels such as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and Corynebacterium parvum. Additional examples of
adjuvants which may be employed include the MPL-TDM adjuvant
(monophosphoryl lipid A, synthetic trehalose dicorynomycolate).
Immunization protocols are well known in the art in the art and may
be performed by any method that elicits an immune response in the
animal host chosen. Adjuvants are also well known in the art.
[0054] Typically, the immunogen (with or without adjuvant) is
injected into the mammal by multiple subcutaneous or
intraperitoneal injections, or intramuscularly or through IV. The
immunogen may include an antigenic polypeptide, a fusion protein or
variants thereof. Depending upon the nature of the polypeptides
(i.e., percent hydrophobicity, percent hydrophilicity, stability,
net charge, isoelectric point etc.), it may be useful to conjugate
the immunogen to a protein known to be immunogenic in the mammal
being immunized. Such conjugation includes either chemical
conjugation by derivatizing active chemical function groups to both
the immunogen and the immunogenic protein to be conjugated such
that a covalent bond is formed, or through fusion-protein based
methodology, or other methods known to the skilled artisan.
Examples of such immunogenic proteins include, but are not limited
to, keyhole limpet hemocyanin, ovalbumin, serum albumin, bovine
thyroglobulin, soybean trypsin inhibitor, and promiscuous T helper
peptides. Various adjuvants may be used to increase the
immunological response as described above.
[0055] The antibodies useful in the present invention comprise
monoclonal antibodies. Monoclonal antibodies may be prepared using
hybridoma technology, such as those described by Kohler and
Milstein, Nature, 256:495 (1975) and U.S. Pat. No. 4,37,110, by
Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring
Harbor Laboratory Press, 2.sup.nd ed. (1988), by Hammerling, et
al., Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, N.Y.,
(1981)), or other methods known to the artisan. Other examples of
methods which may be employed for producing monoclonal antibodies
include, but are not limited to, the human B-cell hybridoma
technique (Kosbor et al., 1983, Immunology today 4:72; Cole et al.,
1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the
EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies
And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies
may be of any immunoglobulin class including IgG, IgM, IgE, IgA,
IgD and any subclass thereof. The hybridoma producing the
antibodies of this invention may be cultivated in vitro or in
vivo.
[0056] Using typical hybridoma techniques, a host such as a mouse,
a humanized mouse, a mouse with a human immune system, hamster,
rabbit, camel or any other appropriate host animal, is typically
immunized with an immunogen to elicit lymphocytes that produce or
are capable of producing antibodies that will specifically bind to
the antigen of interest. Alternatively, lymphocytes may be
immunized in vitro with the antigen.
[0057] Generally, in making antibody-producing hybridomas, either
peripheral blood lymphocytes ("PBLs") are used if cells of human
origin are desired, or spleen cells or lymph node cells are used if
non-human mammalian sources are desired. The lymphocytes are then
fused with an immortalized call line using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell (Goding,
Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986), pp. 59-103). Immortalized call lines are usually
transformed mammalian cells, particularly myeloma cells of rodent,
bovine or human origin. Typically, a rat or mouse myeloma cell line
is employed. The hybridoma cells may be cultured in a suitable
culture medium that preferably contains one or more substances that
inhibit the growth or survival of the unfused, immortalized cells.
For example, if the parental cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the culture
medium for the hybridomas typically will include hypoxanthine,
aminopterin, and thymidine ("HAT medium"), substances that prevent
the growth of HGPRT-deficient cells.
[0058] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Va. human myeloma
and mouse-human heteromycloma cell lines may also be used for the
production of human monoclonal antibodies (Kozbor, J. Immunol.,
133:3001 (1984); Brodeur el al., Monoclonal Antibody Production
Techniques and Applications, Marcel Dekker, Inc., New York, (1987)
pp. 51-63).
[0059] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against the immunogen. The binding specificity of
monoclonal antibodies produced by the hybridoma cells is determined
by, e.g., immunoprecipitation or by an in vitro binding assay, such
as radioimmunoassay (RIA) or enzyme-linked immunoadsorbant assay
(ELISA). Such techniques are known in the art and within the skill
of the artisan. The binding affinity of the monoclonal antibody
can, for example, be determined by a Scatchard analysis (Munson et
al., Anal. biochem., 107:220 (1980)).
[0060] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, supra). Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's medium
and RPMI-1640. The monoclonal antibodies secreted by the subclones
may be isolated or purified from the culture medium by conventional
immunoglobulin purification procedures such as, e.g., protein
A-sepharose, hydroxyapatite chromatography, gel exclusion
chromatography, gel electrophoresis, dialysis, or affinity
chromatography.
[0061] A variety of methods exist in the art for the production of
monoclonal antibodies and thus, the invention is not limited to
their sole production in hydridomas. For example, the monoclonal
antibodies may be made by recombinant DNA methods, such as those
described in U.S. Pat. No. 4,816,567. In this context, the tem
"monoclonal antibody" refers to an antibody derived from a single
eukaryotic, phage, or prokaryotic clone. The DNA encoding the
monoclonal antibodies of the invention can be readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies, or
such chains from human, humanized, or other sources). The hydridoma
cells of the invention serve as a preferred source of such DNA.
Once isolated, the DNA may be placed into expression vectors, which
are then transformed into host cells such as NS0 cells, Simian COS
cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do
not otherwise produce immunoglobulin protein, to obtain the
synthesis of monoclonal antibodies in the recombinant host cells.
The DNA also may be modified, for example, by substituting the
coding sequence for human heavy and light chain constant domains in
place of the homologous murine sequences (U.S. Pat. No. 4,816,567;
Morrison et al, supra) or by covalently joining to the
immunoglobulin coding sequence all or part of the coding sequence
for a non-immunoglobulin polypeptide. Such a non-immunoglobulin
polypeptide can be substituted for the constant domains of an
antibody of the invention, or can be substituted for the variable
domains of one antigen-combining site of an antibody of the
invention to create a chimeric bivalent antibody.
[0062] The antibodies may be monovalent antibodies. Methods for
preparing monovalent antibodies are well known in the art. For
example, one method involves recombinant expression of
immunoglobulin light chain and modified heavy chain. The heavy
chain is truncated generally at any point in the Fc region so as to
prevent heavy chain cross-linking. Alternatively, the relevant
cysteine residues are substituted with another amino acid residue
or are deleted so as to prevent cross-linking.
[0063] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain
constant region and the CH1 domain of the heavy chain.
[0064] For some uses, including in vivo use of antibodies in humans
and in vitro detection assays, it may be preferable to use
chimeric, humanized, or human antibodies. A chimeric antibody is a
molecule in which different portions of the antibody are derived
from different animal species, such as antibodies having a variable
region derived from a murine monoclonal antibody and a human
immunoglobulin constant region. Methods for producing chimeric
antibodies are known in the art. See e.g., Morrison, Science
229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et
al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos.
5,807,715; 4,816,567; and 4,816,397, which are incorporated herein
by reference in their entirety.
[0065] Humanized antibodies are antibody molecules generated in a
non-human species that bind the desired antigen having one or more
complementarity determining regions (CDRs) from the non-human
species and framework (FR) regions from a human immunoglobulin
molecule. Often, framework residues in the human framework regions
will be substituted with the corresponding residue from the CDR
donor antibody to alter, preferably improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interaction of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. (See, e.g., Queen et al., U.S.
Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which
are incorporated herein by reference in their entireties).
Antibodies can be humanized using a variety of techniques known in
the art including, for example, CDR-grafting (EP 239,400; PCT
publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and
5,585,089), veneering or resurfacing (EP 592,106; EP 519,596;
Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et
al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS
91:969-973 (1994)), and chain shuffling (U.S. Pat. No.
5,565,332).
[0066] Generally, a humanized antibody has one or more amino acid
residues introduced into it from a source that is non-human. These
non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable
domain. Humanization can be essentially performed following the
methods of Winter and co-workers (Jones et al., Nature 321:522-525
(1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et
al., Science, 239:1534-1536 (1988), by substituting rodent CDRs or
CDR sequences for the corresponding sequences of a human antibody.
Accordingly, such "humanized" antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possible some FR residues are substituted from
analogous sites in rodent antibodies.
[0067] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. See also, U.S. Pat. Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO
98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and
WO 91/10741; each of which is incorporated herein by reference in
its entirety. The techniques of Cole, et al., and Boerder et al.,
are also available for the preparation of human monoclonal
antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy,
Alan R. Riss, (1985); and Boerner et al., J. Immunol.,
147(1):86-95, (1991)).
[0068] Human antibodies can also be single-domain antibodies having
a VH or VL domain that functions independently of any other
variable domain. These antibodies are typically selected from
antibody libraries expressed in phage. These antibodies and methods
for isolating such antibodies are described in U.S. Pat. Nos.
6,595,142; 6,248,516; and applications US20040110941 and
US20030130496 all of which are incorporated herein by
reference.
[0069] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring which express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained
from the immunized, transgenic mice using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar,
Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO
96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;
5,885,793; 5,916,771; and 5,939,598, which are incorporated by
reference herein in their entirety. In addition, companies such as
Abgenix, Inc. (Freemont, Calif.), Genpharm (San Jose, Calif.), and
Medarex, Inc. (Princeton, N.J.) can be engaged to provide human
antibodies directed against a selected antigen using technology
similar to that described above.
[0070] Also human MAbs could be made by immunizing mice
transplanted with human peripheral blood leukocytes, splenocytes or
bone marrows (e.g., Trioma techniques of XTL). Completely human
antibodies which recognize a selected epitope can be generated
using a technique referred to as "guided selection." In this
approach a selected non-human monoclonal antibody, e.g., a mouse
antibody, is used to guide the selection of a completely human
antibody recognizing the same epitope. (Jespers et al.,
Bio/technology 12:899-903 (1988)).
[0071] Further, antibodies to the polypeptides of the invention
can, in turn, be utilized to generate anti-idiotype antibodies that
"mimic" polypeptides of the invention using techniques well known
to those skilled in the art. (See, e.g., Greenspan & Bona,
FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol.
147(8):2429-2438 (1991)). For example, antibodies which bind to and
competitively inhibit polypeptide multimerization and/or binding of
a polypeptide of the invention to a ligand can be used to generate
anti-idiotypes that "mimic" the polypeptide multimerization and/or
binding domain and, as a consequence, bind to and neutralize
polypeptide and/or its ligand. Such neutralizing anti-idiotypes or
Fab fragments of such anti-idiotypes can be used in therapeutic
regimens to neutralize polypeptide ligand. For example, such
anti-idiotypic antibodies can be used to bind a polypeptide of the
invention and/or to bind its ligands/receptors, and thereby block
its biological activity.
[0072] The antibodies of the present invention may be bispecific
antibodies. Bispecific antibodies are monoclonal, preferably human
or humanized, antibodies that have binding specificities for at
least two different antigens. in the present invention, one of the
binding specificities may be directed towards Factor D, the other
may be for any other antigen, and preferably for a cell-surface
protein, receptor, receptor subunit, tissue-specific antigen,
virally derived protein, virally encoded envelope protein,
bacterially derived protein, or bacterial surface protein, etc.
Bispsecific antibodies may also comprise two or more single-domain
antibodies.
[0073] Methods for making bispecific antibodies are well known.
Traditionally, the recombinant production of bispecific antibodies
is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificites (Milstein and Cuello, Nature, 305:537-539
(1983). Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published May 13,
1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
[0074] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It may have the
first heavy-chain constant region (CH1) containing the site
necessary for light-chain binding present in at least one of the
fusions. DNAs encoding the immunoglobulin heavy-chain fusions and,
if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transformed into a suitable
host organism. For further details of generating bispecific
antibodies sec, for example Suresh et al., Meth. In Enzym., 121:210
(1986).
[0075] Heteroconjugate antibodies are also contemplated by the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells (U.S.
Pat. No. 4,676,980). It is contemplated that the antibodies may be
prepared in vitro using known methods in synthetic protein
chemistry, including those involving cross-linking agents. For
example, immunotoxins may be constructed using a disulfide exchange
reaction or by forming a thioester bond. Examples of suitable
reagents for this purpose include iminothiolate and
methyl-4-mercaptobutyrimidate and those disclosed, for example, in
U.S. Pat. No. 4,676,980. In addition, one can generate
single-domain antibodies to IL-33. Examples of this technology have
been described in WO9425591 for antibodies derived from Camelidae
heavy chain Ig, as well in US20030130496 describing the isolation
of single domain fully human antibodies from phage libraries.
Generation of Monoclonal Antibodies (MABS)
[0076] In one embodiment of the invention, monoclonal antibodies,
such as anti-Factor D, can be raised by immunizing rodents (e.g.,
mice, rats, hamsters and guinea pigs) with either native Factor D
purified from human plasma or urine, or recombinant Factor D or its
fragments expressed by either eukaryotic or prokaryotic systems.
Other animals can be used for immunization, e.g., non-human
primates, transgenic mice expressing human immunoglobulins and
severe combined immunodeficient (SCID) mice transplanted with human
B lymphocytes. Hybridomas can be generated by conventional
procedures by fusing b lymphocytes from the immunized animals with
myeloma cells (e.g.. Sp2/0 and NS0), as described by G. Kohler and
C. Milstein (Nature, 1975: 256: 495-497).
[0077] In addition, monoclonal antibodies ca be generated by
screening of recombinant single-chain Fv or Fab libraries from
human B lymphocytes in phage-display systems. The specificity of
the MAbs to a given antigen can be tested by enzyme linked
immunosorbent assay (ELISA), Western immunoblotting, or other
immunochemical techniques. The inhibitory activity of the
antibodies on complement activation can be assessed by hemolytic
assays using unsensitized rabbit or guinea pig red blood cells
(RBCs) for the alternative pathway, and using sensitized chicken or
sheep RBCs for the classical pathway. The hybridomas in the
positive wells are cloned by limiting dilution. The antibodies are
purified for characterization for specificity to the antigen, such
as Factor D, by the assays well known in the art.
[0078] One can also create single peptide chain binding molecules
in which the heavy and light chain Fv regions are connected. Single
chain antibodies ("ScFv") and the method of their construction are
described in U.S. Pat. No. 4,946,778. Alternatively, Fab can be
constructed and expressed by similar means (M. J. Evans et al., J.
Immunol. Meth., 1995; 184: 123-138). All of the wholly and
partially human antibodies are less immunogenic than wholly murine
MAbs, and the fragments and single chain antibodies are also less
immunogenic. All these types of antibodies are therefore less
likely to evoke an immune or allergic response. Consequently, they
are better suited for in vivo administration in humans than wholly
animal antibodies, especially when repeated or long-term
administration is necessary. In addition, the smaller size of the
antibody fragment may help improve tissue bioavailability, which
may be critical for better dose accumulation in acute disease
indications.
[0079] In one preferred embodiment of the invention, a chimeric
Fab, having animal (mouse) variable regions and human constant
regions is used therapeutically. The Fab is preferred because it is
smaller than a whole immunoglobulin and may provide better tissue
permeation; as monovalent molecule, there is less chance of
immunocomplexes and aggregates forming; nd it can be produced in a
micorbial system, which can more easily be sealed-up than a
mammalian system.
Applications of the Complement Pathway Inhibitors
[0080] The complement inhibitors, such as antibodies and their
binding fragments, can be administered to subjects in an
appropriate pharmaceutical formulation by a variety of routes,
including, but not limited, intravenous infusion, intravenous bolus
injection, and intraperitoneal, intradermal, intramuscular,
subcutaneous, intranasal, intratracheal, intraspinal, intracranial,
and oral routes. Such administration enables them to bind to
endogenous antigen, such as Factor D and thus inhibit the
generation of C3b, C3a and C5a anaphylatoxins, and C5b-9.
[0081] The estimated preferred dosage of such antibodies and
molecules is between 10 and 500 .mu.g/ml of serum. The actual
dosage can be determined in clinical trials following the
conventional methodology for determining optimal dosages, i.e.,
administering various dosages and determining which is most
effective.
[0082] The complement pathway inhibitors can function to inhibit in
vivo complement activation and/or the alternative complement
pathway and inflammatory manifestations that accompany it, such as
recruitment and activation of macrophages, neutrophils, platelets,
and mast cells, edema, and tissue damage. These inhibitors can be
used for treatment of diseases or conditions that are mediated by
excessive or uncontrolled activation of the complement system.
[0083] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of greater multispecificity.
Multispecific antibodies may be specific for different epitopes of
a chosen antigen or may be specific for both the antigen as well as
for a heterologous epitope, such as a heterologous polypeptide or
solid support material. See, e.g., PCT publications WO 93/17715; WO
92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648;
5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553
(1992).
[0084] Antibodies useful in the present invention may be described
or specified in terms of the epitope(s) or portion(s) of a
complement pathway component, such as Factor D, which they
recognize or specifically bind. The epitope(s) or polypeptide
portion(s) may be specified as described herein, e.g., by
N-terminal and C-terminal positions, by size in contiguous amino
acid residues.
[0085] Antibodies useful in the present invention may also be
described or specified in terms of their cross-reactivity.
Antibodies the bind complement pathway component polypeptides,
which have at least 95%, at least 90%, at least 85%, at least 80%,
at least 75%, at least 70%, at least 65%, at least 60%, at least
55%, and at least 50% identity (as calculated using methods known
in the art and described herein) to IL-13 are also included in the
present invention. Anti-Factor D antibodies may also bind with a KD
of less than about 10-7 M, less than about 10-6 M, or less than
about 10-5 M to other proteins, such as Factor D antibodies from
species other than that against which the anti-Factor D antibody is
directed.
Vectors and Host Cells
[0086] In another aspect, the present invention provides vector
constructs comprising a nucleotide sequence encoding the antibodies
of the present invention and a host cell comprising such a vector.
Standard techniques for cloning and transformation may be used in
the preparation of cell lines expressing the antibodies of the
present invention.
[0087] Recombinant expression vectors containing a nucleotide
sequence encoding the antibodies of the present invention can be
prepared using well known techniques. The expression vectors
include a nucleotide sequence operably linked to suitable
transcriptional or translational regulatory nucleotide sequences
such as those derived from mammalian, microbial, viral, or insect
genes. Examples of regulatory sequences include transcriptional
promoters, operators, enhancers, mRNA ribosomal binding sites,
and/or other appropriate sequences which control transcription and
translation initiation and termination. Nucleotide sequences are
"operably linked" when the regulatory sequences functionally
relates to the nucleotide sequence of the appropriate polypeptide.
Thus, a promotor nucleotide sequence is operably linked to, e.g.,
the antibody heavy chain sequence if the promoter nucleotide
sequence controls the transcription of the appropriate nucleotide
sequence.
[0088] In addition, sequences encoding appropriate signal peptides
that are not naturally associated with antibody heavy an/or light
chain sequences can be incorporated into expression vectors. For
example, a nucleotide sequence for a signal peptide (secretory
leader) may be fused in-frame to the polypeptide sequence so that
the antibody is secreted to the periplasmic space or into the
medium. A signal peptide that is functional in the intended host
cells enhances extracellular secretion of the appropriate antibody.
The signal peptide may be cleaved from the polypeptide upon
secretion of antibody from the cell. Examples of such secretory
signals are well known and include, e.g., those described in U.S.
Pat. No. 5,698,435, U.S. Pat. No. 5,698,417, and U.S. Pat. No.
6,204,023.
[0089] Host cells useful in the present invention include but are
not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., Baculovirus) containing antibody coding
sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3
cells) harboring recombinant expression construct containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
[0090] The vector may be a plasmid vector, a single or
double-stranded phage vector, or a single or double-stranded RNA or
DNA viral vector. Such vectors may be introduced into cells as
polynucleotides by well known techniques for introducing DNA and
RNA into cells. The vectors, in the case of phage and viral vectors
also may be introduced into cells as packaged or encapsulated virus
by well known techniques for infection and transduction. Viral
vectors may be replication competent or replication defective. In
the later case, viral propagation generally will occur only in
complementing host cells. Cell-free translation systems may also be
employed to produce the protein using RNAs derived from the present
DNA constructs. Such vectors may include the nucleotide sequence
encoding the constant region of the antibody molecule (see, e.g.,
PCT Publication WO 86/05807; PCT Publication WO 89/01036; U.S. Pat.
No. 5,122,464) and the variable domain of the antibody may be
cloned into such a vector for expression of the entire heavy or
light chain.
[0091] Prokaryotes useful as host cells in the present invention
include gram negative or gram positive organisms such as E. coli,
and B. subtilis. Expression vectors for use in prokaryotic host
cells generally comprise one or more phenotypic selectable marker
genes. A phenotypic selectable marker gene is, for example, a gene
encoding a protein that confers antibiotic resistance or that
supplies an autotrophic requirement. Examples of useful expression
vectors for prokaryotic host cells include those derived from
commercially available plasmids such as the pKK223-3 (Pharmacia
Fine Chemicals, Uppsala, Sweden) pGEM1 (Promega Biotec, Madison,
Wis., USA), and the pET (Novagen, Madison, Wis., USA) and pRSET
(Invitrogen Corporation, Carlsbad, Calif., USA) series of vectors
(Studier, F. W., J. Mol. Biol. 219: 37 (1991); Schoepfer, R. Gene
124: 83 (1993)). promoter sequences commonly used for recombinant
prokaryotic host cell expression vectors include T7, (Rosenberg, et
al. Gene 56, 125-135 (1987)), .beta.-lactamase (penicillinase),
lactose promoter system (Chang et al., Nature 275:615, (1987); and
Goeddel et al., Nature 281:544, (1979)), tryptophan (trp) promoter
system (Goeddel et al., Nucl. Acids Res. 8:4057, (1980)), and tac
promotor (Sambrook et al., 1980, Molecular Cloning, A Laboratory
Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.)
[0092] Yeasts useful in the present invention include those from
the genus Saccharomyces, Pichia, Actinomycetes and Kluyveromyces.
Yeast vectors will often contain an origin of replication sequence
from a 2.mu. yeast plasmid, an autonomously replicating sequence
(ARS), a promoter region, sequences for polyadenylation, sequences
for transcription termination, and a selectable marker gene.
Suitable promoter sequences for yeast vectors include, among
others, promoters for methallothionein, 3-phosphoglycerate kinase
(Hitzeman et al., J. Biol. Chem. 255:2073, (19080)) or other
glycolytic enzymes (Holland et al., Biochem. 17:4900, (1978)) such
as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,
pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate
isomerase, 3-phosphoglycerate mutase, pyruvate kinase,
triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase. Other suitable vectors and promoters for use in yeast
expression are further described in Fleer et al., Gene, 107:285-195
(1991). Other suitable promoters and vectors for yeast and yeast
transformation protocols are well known in the art. Yeast
transformation protocols are well known. One such protocol is
described by Hinnen et al., Proc. Natl. Acad. Sci., 75:1929 (1978).
The Hinnen protocol selects for Trp+ transformants in a selective
medium.
[0093] Mammalian or insect host cell culture systems may also be
employed to express recombinant antibodies, e.g., Baculovirus
systems for production of heterologous proteins. In an insect
system, Antographa californica nuclear polyhedrosis virus (AcNPV)
may be used as a vector to express foreign genes. The virus grows
in Spodoptera frugiperda cells. The antibody coding sequence may be
cloned individually into non-essential regions (for example the
polyhedrin gene) of the virus and placed under control of an AcNPV
promotor (for example the polyhedrin promoter).
[0094] NSD or Chinese hamster ovary (CHO) cells for mammalian
expression of the antibodies of the present invention may be used.
Transcriptional and translational control sequences for mammalian
host cell expression vectors may be excised from viral genomes.
Commonly used promoter sequences and enhancer sequences are derived
from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human
cytomegalovirus (CMV). DNA sequences derived from the SV40 viral
genome may be used to provide other genetic elements for expression
of a structural gene sequence in a mammalian host cell, e.g., SV40
origin, early and late promoter, enhancer, splice, and
polyadenylation sites. Viral early and late promoters are
particularly useful because both are easily obtained from a viral
genome as a fragment which may also contain a viral origin of
replication. Exemplary expression vectors for use in mammalian host
cells are commercially available.
Polynucleotides Encoding Antibodies
[0095] The invention further provides polynucleotides or nucleic
acids, e.g., DNA, comprising a nucleotide sequence encoding an
antibody of the invention and fragments thereof. Exemplary
polynucleotides include those encoding antibody chains comprising
one or more of the amino acid sequences described herein. The
invention also encompasses polynucleotides that hybridize under
stringent or lower stringency hybridization conditions to
polynucleotides that encode an antibody of the present
invention.
[0096] The polynucleotides may be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. For example, if the nucleotide sequence of the antibody is
known, a polynucleotide encoding the antibody may be assembled from
chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly,
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody, annealing and
ligating of those oligonucleotides, and then amplification of the
ligated oligonucleotides by PCR.
[0097] Alternatively, a polynucleotide encoding an antibody may be
generated from nucleic acid from a suitable source. If a clone
containing a nucleic acid encoding a particular antibody is not
available, but the sequences of the antibody molecule is known, a
nucleic acid encoding the immunoglobulin may be chemically
synthesized or obtained from a suitable source (e.g., an antibody
cDNA library, or a cDNA library generated from, or nucleic acid,
preferably poly A+ RNA, isolated from, any tissue or cells
expressing the antibody, such as hybridoma cells selected to
express an antibody of the invention) by PCR amplification using
synthetic primers hybridizable to the 3' and 5' ends of the
sequence or by cloning using an oligonucleotide probe specific for
the particular gene sequence to identify, e.g., a cDNA clone from a
cDNA library that encodes the antibody. Amplified nucleic acids
generated by PCR may then be cloned into replicable cloning vectors
using any method well known in the art.
[0098] Once the nucleotide sequence and corresponding amino acid
sequence of the antibody is determined, the nucleotide sequence of
the antibody may be manipulated using methods well known in the art
for the manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site directed mutagenesis, PCR, etc. (see, for example,
the techniques described in Sambrook et al., 1990, Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds.,
1998, Current Protocols in Molecular Biology, John Wiley and Sons,
N.Y., which are both incorporated by reference herein in their
entireties), to generate antibodies having a different amino acid
sequence, for example to create amino acid substitutions,
deletions, and/or insertions.
[0099] In a specific embodiment, the amino acid sequence of the
heavy and/or light chain variable domains may be inspected to
identify the sequences of the CDRs by well known methods, e.g., by
comparison to known amino acid sequences of other heavy and light
chain variable regions to determine the regions of sequence
hypervariability. Using routine recombinant DNA techniques, one or
more of the CDRs may be inserted within framework regions, e.g.,
into human framework regions to humanize a non-human antibody, as
described supra. The framework regions may be naturally occurring
or consensus framework regions, and preferably human framework
regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479
(1998) for a listing of human framework regions). Preferably, the
polynucleotide generated by the combination of the framework
regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one
or more amino acid substitutions may be made within the framework
regions, and, preferably, the amino acid substations improve
binding of the antibody to its antigen. Additionally, such methods
may be used to make amino acid substitutions or deletions of one or
more variable region cysteine residues participating in an
intrachain disulfide bond to generate antibody molecules lacking
one or more intrachain disulfide bonds. Other alterations to the
polynucleotide are encompassed by the present invention and within
the skill of the art.
[0100] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., Proc Natl. Acad. Sci.
81:851-855 (1984); Neuberger et al., Nature 312:604-608 (19840;
Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a
mouse antibody molecule of appropriate antigen specificity together
with genes from a human antibody molecule of appropriate biological
activity can be used. As described supra, a chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine MAb and a human immunoglobulin constant region, e.g.,
humanized antibodies.
[0101] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science
242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA
85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can
be adapted to produce single chain antibodies. Single chain
antibodies are formed by linking the heavy and light chain
fragments of the Fv region via an amino acid bridge, resulting in a
single chain polypeptide. Techniques for the assembly of functional
Fv fragments in E. coli may also be used (Skerra et al., Science
242:1038-1041 (1988)).
Methods of Producing Antibodies
[0102] The antibodies of the invention can be produced by any
method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or preferably, by recombiant
expression techniques.
[0103] Recombinant expression of an antibody of the invention, or
fragment, derivative or analog thereof, (e.g., a heavy or light
chain of an antibody of the invention or a single chain antibody of
the invention), requires construction of an expression vector
containing a polynucleotide that encodes the antibody or a fragment
of the antibody. Once a polynucleotide encoding an antibody
molecule has been obtained, the vector for the production of the
antibody may be produced by recombinant DNA technology. An
expression vector is constructed containing antibody coding
sequences and appropriate transcriptional and translational control
signals. These methods include, for example, in vitro recombinant
DNA techniques, synthetic techniques, and in vivo genetic
recombination.
[0104] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an antibody of the invention.
In one aspect of the invention, vectors encoding both the heavy and
light chains may be co-expressed in the host cell for expression of
the entire immunoglobulin molecule, as detailed below.
[0105] A variety of host-expression vector system may be utilized
to express the antibody molecules of the invention as described
above. Such host-expression systems represent vehicles by which the
coding sequences of interest may be produced and subsequently
purified, but also represent cells which may, when transformed or
transacted with the appropriate nucleotide coding sequences,
express an antibody molecule of the invention in situ. Bacterial
cells such as E. coli, and eukaryotic cells are commonly used for
the expression of a recombinant antibody molecule, especially for
the expression of whole recombinant antibody molecule. For example,
mammalian cells such as Chinese hamster ovary cells (CHO), in
conjunction with a vector such as the major intermediate early gene
promoter element from human cytomegalovirus is an effective
expression system for antibodies (Foecking et al., Gene 45:101
(1986); Cockett et al., Bio/Technology 8:2 (1990)).
[0106] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modification (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include, but are not limited to, CHO, COS, 293, 3T3, or
myeloma cells.
[0107] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the antibody molecule may be engineered.
Rather than using expression vectors which contain viral origins of
replication, host cells can be transformed with DNA controlled by
appropriate expression control elements; (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. Following the introduction of the foreign
DNA, engineered cells may be allowed to grow for 1-2 days in an
enriched media, and then are switched to a selective media. The
selectable marker its the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines. This method may advantageously be
used to engineer cell lines which express the antibody molecule.
Such engineered cell lines may be particularly useful in screening
and evaluation of compounds that interact directly or indirectly
with the antibody molecule.
[0108] A number of selection systems, may be used, including but
not limited to the herpes simplex virus thymidine kinase (Wigler et
al., Cell 11:273 (1977), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA 48:202 (1992)), and adenine phosphorihosytransferase
(Lowy et al., Cell 22:817 (1980)) genes can be employed in tk,
hgprt or aprt-cells, respectively. Also, antimetabolite resistance
can be used as the basis of selection for the following genes:
dhfr, which confers resistance to methotrexate (Wigler et al.,
Proc. Natl. Acad. Sci., USA 77:357 (1980); O'Hare et al., Proc.
Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance
to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci.
USA 78:2072 (1981)); neo, which confers resistance to the
aminoglycoside G-418 (Wu and Wu, Biotherapy 3:87-95 (1991)); and
hygro, which confers resistance to hygromycin (Santerre et al.,
Gene 30:147 (1984)). Methods commonly known in the art of
recombinant DNA technology may be routinely applied to select the
desired recombinant clone, and such methods are described, for
example, in Ausubel et al. (eds.), Current Protocols in Molecular
Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer
and Expression, A Laboratory Manual, Stockton Press, NY (1980); and
in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in
Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin
et al., J. Mol. Biol. 150:1 (1981), which are incorporated by
reference herein in their entireties.
[0109] The expression levels of an antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, "The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells" (DNA Cloning, Vol.
3. Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of
inhibitor present in culture of host cell will increase the number
of copies of the marker gene. Since the amplified region is
associated with the antibody gene, production of the antibody will
also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
[0110] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes, and is capable of expressing, both heavy and light
chain polypeptides. In such situations, the light chain should be
placed before the heavy chain to avoid an excess of toxic free
heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl.
Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy
and light chains may comprise cDNA or genomic DNA.
[0111] Once an antibody molecule of the invention has been produced
by an animal, chemically synthesized, or recombinantly expressed,
it may be purified by any method known in the art for purification
of an immunoglobulin molecule, for example, by chromatography
(e.g., ion exchange, affinity, particularly by affinity for the
specific antigen after protein A, and size-exclusion
chromatography), centrifugation, differential solubility, or by any
other standard technique for the purification of proteins. In
addition, the antibodies of the present invention or fragments
thereof can be fused to heterologous polypeptide sequences
described herein or otherwise known in the art, to facilitate
purification.
[0112] The present invention encompasses antibodies recombinantly
fused or chemically conjugated (including both convalently and
non-covalently conjugations) to a polypeptide. Fused or conjugated
antibodies of the present invention may be used for ease in
purification. See e.g., Harbor et al., supra, and PCT publication
WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99
(1994); U.S. Pat. No. 5,474,981; Gillies et al., Proc. Natl. Acad.
Sci. 89:1328-1432. (19920; Fell et al., J. Immunol. 146:2446-2452
(1991), which are incorporated by reference in their
entireties.
[0113] Moreover, the antibodies or fragments thereof of the present
invention can be fused to marker sequences, such as a peptide to
facilitate purification. In preferred embodiments, the marker amino
acid sequence is a hexa-histidine peptide, such as the tag provided
in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,
Calif., 91311), among other, many of which are commercially
available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA
86:821-824 (1989), for instance, hexa-histidine provides for
convenient purification of the fusion protein. Other peptide tags
useful for purification include, but are not limited to, the "HA"
tag, which corresponds to an epitope derived from the influenza
hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the
"flag" tag.
Diagnostic Uses for Antibodies
[0114] The antibodies of the invention include derivatives that are
modified, i.e., by the covalent attachment of any type of molecule
to the antibody, such that covalent attachment does not interfere
with binding to the antigen. For example, but not by way of
limitation, the antibody derivatives include antibodies that have
been modified, e.g., by biotinylation, HRP, or any other detectable
moiety.
[0115] Antibodies of the present invention may be used, for
example, but not limited to, to detect Factor D, including both in
vitro and in vivo diagnostic methods. For example, the antibodies
have use in immunoassays for qualitatively and quantitatively
measuring levels of Factor D in biological samples obtained from
the eyes of subjects suffering from ocular conditions or diseases.
Typically immunoassays are described in, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) (incorporated by reference herein in its
entirety).
[0116] As discussed in more detail below, the antibodies of the
present invention may be used either alone or in combination with
other compositions. The antibodies may further be recombinantly
fused to a heterologous polypeptide at the N- or C-terminus or
chemically conjugated (including covalently and non-covalently
conjugations) to polypeptides or other compositions. For example,
antibodies of the present invention may be recombinantly fused or
conjugated to molecules useful as labels in detection assays.
[0117] The present invention further encompasses the use of
antibodies or fragments thereof conjugated to a diagnostic agent
for the detection of the levels of complement pathway components in
the eye of an affected individual. The antibodies can be used
diagnostically to, for example, monitor the development or
progression of an ocular condition or disease as part of a clinical
testing procedure to, e.g., determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling the
antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials,
radioactive materials, positron emitting metals using various
positron emission tomographies, and nonradioactive paramagnetic
metal ions. The detectable substance may be coupled or conjugated
either directly to the antibody (or fragment thereof) or
indirectly, through an intermediate (such as, for example, a linker
known in the art) using techniques known in the art. See, for
example, U.S. Pat. No. 4,741,900 for metal ions which can be
conjugated to antibodies for use as diagnostics according to the
present invention. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyante, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include 125I, 131I, 111In or -99Tc.
[0118] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the target
antigen. Such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
[0119] Labeled antibodies, and derivatives and analogs thereof,
which specifically bind to Factor D can be used for diagnostic
purposes to detect, diagnose, or monitor diseases, disorders,
and/or conditions associated with the aberrant expression and/or
activity of Factor D. The invention provides for the detection of
aberrant expression of Factor D, comprising (a) assaying the
expression of Factor D in cells or body fluid of an individual
using one or more antibodies of the present invention specific to
Factor D and (b) comparing the level of gene expression with a
standard gene expression level, whereby an increase or decrease in
the assayed Factor D expression level compared to the standard
expression level is indicative of aberrant expression.
[0120] Antibodies may be used for detecting the presence and/or
levels of Factor D in a sample, e.g., ocular fluid. The detecting
method may comprise contacting the sample with an anti-Factor D
antibody and determining the amount of antibody that is bound to
the sample.
[0121] The invention provides a diagnostic assay for diagnosing a
disorder, comprising (a) assaying the expression of Factor D in
cells or body fluid of an individual using one or more antibodies
of the present invention and (b) comparing the level of gene
expression with a standard gene expression level, whereby an
increase or decrease in the assayed gene expression level compared
to the standard expression level is indicative of a particular
disorder.
[0122] Antibodies of the invention can be used to assay protein
levels in a biological sample using classical immunohistological
methods known to those of skill in the art (e.g., see Jalkanen, et
al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell.
Bio. 105:3087-3096 (1987)). Other antibody-based methods useful for
detecting protein gene expression include immunoassays, such as the
enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay
(RIA). Suitable antibody assay labels are known in the art and
include enzyme labels, such as, glucose oxidase; radioisotopes,
such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium
(3H), indium (112In), and technetium (99Tc); luminescent labels,
such as luminol; and fluorescent labels, such as fluorescein and
rhodamine, and biotin.
[0123] One aspect of the invention is the detection and diagnosis
of a disease or disorder associated with complement activation in
the eyes of a subject, preferably a mammal and most preferably a
human. In one embodiment, diagnosis comprises: a) taking a sample
from the eye of a patient; b) measuring the level of complement
components, such as C3a or C3b or C5a. Background level can be
determined by various methods including, comparing the amount of
labeled molecule detected to a standard value previously determined
for a particular system.
[0124] In an embodiment, monitoring of the disease or disorder is
carried out by repeating the method for diagnosing the disease or
disease, for example, one month after initial diagnosis, six months
after initial diagnosis, one year after initial diagnosis, etc.
Therapeutic Uses of Complement Pathway Inhibitors
[0125] Complement pathway inhibitors may be administered to a
subject suffering from an ocular disease such as age-related
mascular degeneration. An antibody, with or without a therapeutic
moiety conjugated to it, can be used as a therapeutic. The present
invention is directed to the use of complement pathway inhibitors,
particularly antibodies, comprising administering said inhibitors
to an animal, a mammal, or a human, for treating a ocular disease,
disorder, or condition involving complement pathway activation. The
animal or subject may be an animal in need of a particular
treatment, such as an animal having been diagnosed with a
particular disorder, e.g., one relating to complement. Antibodies
directed against Factor D are useful for inhibiting the alternative
complement pathway and thus inhibiting complement pathway related
disorder or conditions. In particular, the present invention
relates to the treatment of AMD, diabetic retinopathy, and
choroidal neovascularization. For example, by administering a
therapeutically acceptable dose of an antibody, or antibodies, of
the present invention, or a cocktail of the present antibodies, or
in combination with other molecules of varying sources, the effects
of activation of complement pathway components may be reduced or
eliminated in the treated mammal.
[0126] Therapeutic compounds of the invention include, but are not
limited to, antibodies of the invention (including fragments,
analogs and derivatives thereof as described herein)and nucleic
acids encoding antibodies of the invention as described below
(including fragments, analogs and derivatives thereof and
anti-idiotypic antibodies as described herein). The antibodies of
the invention can be used to treat, inhibit or prevent diseases,
disorders or conditions associated with aberrant expression and/or
activity of the complement pathway, particularly the alternative
pathway, and particularly Factor D. The treatment and/or prevention
of diseases, disorders, or conditions associated with aberrant
expression and/or activity of Factor D includes, but is not limited
to, alleviating at least one symptoms associated with those
diseases, disorders or conditions. Antibodies of the invention may
be provided in pharmaceutically acceptable compositions as known in
the art or as described herein.
[0127] The amount of the antibody which will be effective in the
treatment, inhibition and prevention of a disease or disorder
associated with aberrant expression and/or activation of the
complement pathway can be determined by standard clinical
techniques. The antibody can be administered in treatment regimes
consistent with the disease, e.g., a single or a few doses over one
to several days to ameliorate a disease state or periodic doses
over an extended time to prevent ocular diseases or conditions.
[0128] In addition, in vitro assays may optionally be employed to
help identify optimal dosage ranges. The precise dose to be
employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgement of the practitioner
and each patient's circumstances. Effective doses may be
extrapolated from dose-response curves derived from in vitro or
animal model test systems.
[0129] For antibodies, the dosage administered to a patient is
typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
Preferably, the dosage administered to a patient is between 0.1
mg/kg and 20 mg/kg of the patient's body weight, more preferably 1
mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half-life within the human body than
antibodies from other species due to the immune response to the
foreign polypeptides. Thus, lower dosages of human antibodies and
less frequent administration is often possible. Further, the dosage
and frequency of administration of antibodies of the invention may
be reduced by enhancing uptake and tissue penetration (e.g., into
the brain) of the antibodies by modifications such as, for example,
lipidation. In a preferred aspect, the antibody is substantially
purified (e.g., substantially free from substances that limit its
effect or produce undesired side-effects).
[0130] Various delivery systems are known and can be used to
administer an antibody of the present invention, including
injection, e.g., encapsulation in liposomes, microparticles,
microcapsules, recombinant cells capable of expressing the
compound, receptor-mediated endocytosis (see, e.g., Wu et al., J.
Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid
as part of a retroviral or other vector, etc.
[0131] The antibody can be administered to the mammal in any
acceptable manner. Methods of introduction include but are not
limited to intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, inhalation and
oral routes. However, for purpose of the present invention, the
preferred route of administration is intraocular.
[0132] Administration can be systemic or local. in addition, it may
be desirable to introduce the therapeutic antibodies or
compositions of the invention into the central nervous system by
any suitable route, including intraventricular and intrathecal
injection; intraventricular injection may be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir.
[0133] In another embodiment, the antibody can be delivered in a
vesicle, in particular a liposome (see Langer, Science
249:1527-1533 (1990); Treat el al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.
317-327; see generally ibid.).
[0134] In yet another embodiment, the antibody can be delivered in
a controlled release system. In one embodiment, a pump may be used
(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201
(1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N.
Engl. J. med. 321:574 (1989)). In another embodiment, polymeric
materials can be used (see Medical Applications of Controlled
Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.
(1974); Controlled Drug Bioavailability, Drug Product Design and
Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger
and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983);
see also Levy et al., Science 228:190 (1985); During et al., Ann.
Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).
In yet another embodiment, a controlled release system can be
placed in proximity of the therapeutic target.
[0135] The present invention also provides pharmaceutical
compositions useful in the present method. Such compositions
comprise a therapeutically effective amount of the antibody, and a
physiologically acceptable carrier. In a specific embodiment, the
term "physiologically acceptable" means approved by a regulatory
agency of the Federal or a state government or listed in the U.S.
Pharmacopeia or other generally recognized pharmacopeia for use in
animals, and more particularly in humans. The term "carrier" refers
to a diluent, adjuvant, excipient, or vehicle with which the
therapeutic is administered. Such physiological carriers can be
sterile liquids, such as water and oils, including those of
petroleum, animal, vegetable or synthetic origin, such as peanut
oil, soybean oil, mineral oil, sesame oil and the like. Water is a
preferred carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical
excipients include starch, glucose, lactose, sucrose, gelatin,
malt, rice, flour, chalk, silica gel, sodium stearate, glycerol
monostearate, talc, sodium chloride, dried skim milk, glycerol,
propylene, glycol, water, ethanol and the like. The composition, if
desired, can also contain minor amounts of wetting or emulsifying
agents, or pH buffering agents. These compositions can take the
form of solutions, suspensions, emulsion, tablets, pills, capsules,
powders, sustained release formulations and the like. The
composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. oral formulation can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, etc. Examples of suitable carriers
are described in "Remington's Pharmaceutical Sciences" by E. W.
Martin. Such compositions will contain an effective amount of the
antibody, preferably in purified form, together with a suitable
amount of carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0136] In one embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0137] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use of sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use of
sale for human administration.
[0138] In addition, the antibodies of the present invention may be
conjugated to various effector molecules such as heterologous
polypeptides, drugs, radionucleotides, or toxins. See, e.g., PCT
publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.
5,314,995; and EP 396,387. An antibody or fragment thereof may be
conjugated to a therapeutic moiety such as a cytotoxin, e.g., a
cytostatic or cytocidal agent, a therapeutic agent or a radioactive
metal ion, e.g., alpha-emitters such as, for example, 213Bi. A
cytotoxin or cytotoxic agent includes any agent that is detrimental
to cells. Examples include paclitaxol, cytochalasin B, gramicidin
D, ethidium bromide, emetine, mitomycin, etoposide, tneoposide,
bincristine, binblastine, colchicin, doxorubicin, daunorubicin,
dihydorxy anthracin dione, mitoxantrone, mithramycin, actinomycin
D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologues
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercapiopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0139] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev. 62:119-58 (1982). Alternatively, an antibody can be
conjugated to a second antibody to form an antibody heterconjugate.
(See, e.g., Segal in U.S. Pat. No. 4,676,980.)
[0140] The conjugates of the invention can be used for modifying a
given biological response, the therapeutic agent or drug moiety is
not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis Factor, .alpha.-interferon, .beta.-interferon, nerve
growth Factor, platelet derived growth Factor, tissue plasminogen
activator, an apoptotic agent, e.g., TNF-.alpha., TNF-.beta., AIM I
(See, International Publication No. WO 97/33899), AIM II (See,
International Publication No. WO 97/34911), Fas Ligand (Takahashi
et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See,
International Publication No. WO 99/23105), a thrombotic agent or
an anti-angiogenic agent, e.g., angiostatin or endostatin; or,
biological response modifiers such as, for example, lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophage colony stimulating Factor
("GM-CSF"), granulocyte colony stimulating Factor ("G-CSF"), or
other growth Factors.
Antibody-Based Gene Therapy
[0141] In a another aspect of the invention, nucleic acids
comprising sequences encoding antibodies or binding fragments
thereof, are administered to treat, inhibit or prevent a disease or
disorder associated with aberrant expression and/or activation of
the complement pathway by way of gene therapy. Gene therapy refers
to therapy performed by the administration to a subject of an
expressed or expressible nucleic acid. In this embodiment of the
invention, the nucleic acids produce their encoded protein that
mediates a therapeutic effect. Any of the methods for gene therapy
available can be used according to the present invention. Exemplary
methods are described below.
[0142] For general reviews of the methods of gene therapy, see
Goldspiel et al., Clinical Pharmacy 12:488-505 (1983); Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol,
Toxicol, 32:573-596 (19930; Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May,
TIBTECH 11(5):155-215 (1993).
[0143] In a one aspect, the compound comprises nucleic acid
sequences encoding an antibody, said nucleic acid sequences being
part of expression vectors that express the antibody or fragments
or chimeric proteins or heavy or light chains thereof in a suitable
host. In particular, such nucleic acid sequences have promoters
operably linked to the antibody coding region, said promoter being
inducible or constitutive, and, optionally, tissue-specific.
[0144] In another particular embodiment, nucleic acid molecules are
used in which the antibody coding sequences and any other desired
sequences are flanked by regions that promote homologous
recombination at a desired site in the genome, thus providing for
intrachromosomal expression of the antibody encoding nucleic acids
(Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935
(1989); Zijistra et al., Nature 342:435-438 (1989). In specific
embodiments, the expressed antibody molecule is a single chain
antibody; alternatively, the nucleic acid sequences include
sequences encoding both the heavy and light chains, or fragments
thereof, of the antibody.
[0145] Delivery of the nucleic acids into a patient may be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in
vitro, then transplanted into the patient. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy.
[0146] In a specific embodiment, the nucleic acid sequences are
directly administered in vivo, where it is expressed to produce the
encoded product. This can be accomplished by any of numerous
methods known in the art, e.g., by constructing them as part of an
appropriate nucleic acid expression vector and administering it so
that they become intracellular, e.g., by infection using defective
or attenuated retrovirals or other viral vectors (see U.S. Pat. No.
4,980,286), or by direct injection of naked DNA, or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating with lipids or cell-surface receptors or transfecting
agents, encapsulating in liposomes, microparticles, or
microcapsules, or by administering them in linkage to a peptide
which is known to enter the nucleus, by administering it in linkage
to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu
and Wu, J. Biol. Chem., 262:4429-4432 (19870) (which can be used to
target cell types specifically expressing the receptors), etc. in
another embodiment, nucleic acid-ligand complexes can be formed in
which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see, e.g., PCT Publications WO
92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221).
Alternatively, the nucleic acid can be introduced intracellularly
and incorporated within host cell DNA for expression, by homologous
recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA
86:8932-8935 (1989); Zijistra et al., Nature 342:435-438
(1989)).
[0147] In a specific embodiment, viral vectors that contain nucleic
acid sequences encoding an antibody of the invention are used. For
example, a retroviral vector can be used (see Miller et al., Meth.
Enzymol. 217:581-599 (1993)). These retroviral vectors contain the
components necessary for the correct packaging of the viral genome
and integration into the host cell DNA. The nucleic acid sequences
encoding the antibody to be used in gene therapy are cloned into
one or more vectors, which facilitates the delivery of the gene
into a patient. More detail about retroviral vectors can be found
in Boesen et al., Biotherapy 6:291-302 (1994), which describes the
use of a retroviral vector to deliver the mdrl gene to
hematopoietic stem cells in order to make the stem cells more
resistant to chemotherapy. Other references illustrating the use of
retroviral vectors in gene therapy are: Clowes et al., J. Clin.
Invest. 93:644-651 (1994): Kiem et al., Blood 83:1467-1473 (1994);
Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and
Grossman and Wilson, Curr. Opin. Gen. and Dev. 3:110-114
(1993).
[0148] Adenoviruses may also be used in the present invention.
Adenoviruses are especially attractive vehicles in the present
invention for delivering antibodies to respiratory epithelia.
Adenoviruses naturally infect respiratory epithelia. Other targets
for adenovirus-based delivery systems are liver, the central
nervous system, endothelial cells, and muscle. Adenoviruses have
the advantage of being capable of infecting non-dividing cells.
Kozarsky and Wilson, Curr. Opin. Gen. Dev. 3:499-503 (1993) present
a review of adenovirus-based gene therapy. Boul et al., Human Gene
Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to
transfer genes to the respiratory epithelia of rhesus monkeys.
Other instances of the use of adenoviruses in gene therapy can be
found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfield
et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin.
Invest. 91:335-234 (1993); PCT Publication WO94/12649; and Wang, et
al., Gene Therapy 2:775-783 (1995). Adeno-associated virus (AAV)
has also been proposed for use in gene therapy (Walsh et al., Proc.
Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. Nos. 5,436,146;
6,632,670; 6,642,051).
[0149] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient.
[0150] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see,
e.g., Loeffier and Bahr, Meth. Enzymol, 217:599-618 (1993); Cohen
et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther.
29:69-92 m (1985) and may be used in accordance with the present
invention, provided that the necessary developmental and
physiological functions of the recipient cells are not disrupted.
The technique should provide for the stable transfer of the nucleic
acid to the cell, so that the nucleic acid is expressible by the
cell and preferably heritable and expressible by its cell
progeny.
[0151] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. Recombinant blood
cells (e.g., hematopoietic stem or progenitor cells) are preferably
administered intravenously. The amount of cells envisioned for use
depends on the desired effect, patient state, etc., and can be
determined by one skilled in the art.
[0152] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include by are not limited to epithelial cells,
endothelial cells, kerathocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as T lymphocytes, B lymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver,
etc.
[0153] In a one embodiment, the cell used for gene therapy is
autotogous to the patient. Nucleic acid sequences encoding an
antibody of the present invention are introduced into the cells
such that they are expressible by the cells or their progeny, and
the recombinant cells are then administered in vivo for therapeutic
effect. In a specific embodiment, stem or progenitor cells are
used. Any stem and/or progenitor cells which can be isolated and
maintained in vitro can potentially be used in accordance with this
embodiment of the present invention (see e.g., PCT Publication WO
94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald,
Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo
Clinic Proc. 61:771 (1986)).
Modulating Complement Pathway Component Expression by SIRNA
[0154] siRNAs have proven useful as a tool in studies of modulating
gene expression where traditional antagonists such as small
molecules or antibodies may be less effective. (Shi Y., Trends in
Genetics 19(10:9-12 (2003)). In vitro synthesized, double stranded
RNAs that are 21 to 23 nucleotides in length can act as interfering
RNAs (iRNAs) and can specifically inhibit gene expression (Fire A.,
Trends in Genetics 391; 806-810 (1999)). These iRNAs act by
mediating degradation of their target RNAs. Since they are under 30
nucleotides in length, they do not trigger a cell antiviral defense
mechanism. Such mechanisms include interferon production, and a
general shutdown of host cell protein synthesis. Practically,
siRNAs can be synthesized and then cloned into DNA vectors. Such
vectors can be transfected and made to express the siRNA at high
levels. The high level of siRNA expression is used to "knockdown"
or significantly reduce the amount of protein produced in a cell,
and thus it is useful in experiments where overexpression of a
protein is believed to be linked to a disorder such as cancer.
siRNAs are useful antagonists to complement pathway proteins by
limiting cellular production of the antigen and inhibit activation
of the complement cascade.
Peptidomimetics and Small Molecules
[0155] It is well-known to those normally skilled in the art that
it is possible to replace peptides with peptidomimetics.
peptidomimetics are generally preferable as therapeutic agents to
peptides owing to their enhanced bioavailability and relative lack
of attack from proteolytic enzymes. Techniques of molecular
modeling may be used to design a peptidomimetics which mimic the
structure of the complement related peptides disclosed herein.
Accordingly, the present invention also provides peptidomimetics
and other lead compounds which can be identified based on the data
obtained from structural analysis of the complement pathway
protein. A potential Factor D analog may be examined through the
use of computer modeling using a docking program such as GRAM,
DOCK, or AUTODOCK. This procedure can include computer fitting of
potential Factor D analogs. Computer programs can also be employed
to estimate the attraction, repulsion, and steric hindrance of an
analog to a potential binding site. Generally the tighter the fit
(e.g., the lower the steric hindrance, and/or the greater the
attractive force) the more potent the potential drug will be since
these properties are consistent with a tighter binding constant.
Furthermore, the more specificity in the design of a potential drug
the more likely that the drug will not interfere with other
properties of the expression system. The will minimize potential
side-effects due to unwanted interactions with other proteins.
[0156] Initially a potential Factor D analog could be obtained by
screening a random peptide library produced by a recombinant
bacteriophage, for example, or a chemical library. An analog ligand
selected in this manner could be then be systematically modified by
computer modeling programs until one or more promising potential
ligands are identified.
[0157] Such computer modeling allows the selection of a finite
number of rational chemical modifications, as opposed to the
countless number of essentially random chemical modifications that
could be made, and of which any one might lead to a useful drug.
Thus through the use of the three-dimensional structure disclosed
herein and computer modeling, a large number of compounds is
rapidly screened and a few likely candidates can be determined
without the laborious synthesis of untold numbers of compounds.
[0158] Once a potential Factor D analog is identified it can be
either selected from a library of chemicals commercially available
from most large chemical companies including Merck, GlaxoWelcome,
Bristol Meyers Squib, Monsanto/Searle, Eli Lilly, Novartis and
Pharmacia UpJohn, or alternatively the potential ligand is
synthesized de novo. As mentioned above, the de novo synthesis of
one or even a relatively small group of specific compounds is
reasonable in the art of drug design.
[0159] Alternatively, based on the molecular structures of the
variable regions of the anti-Factor D antibodies, one could use
molecular modeling and rational molecular design to generate and
screen small molecules which mimic the molecular structures of the
binding region of the antibodies and inhibit the activities of
Factor D. These small molecules can be peptides, peptidomimetics,
oligonucleotides, or organic compounds.
Example
Efficacy of Antibody in a Laser-Induced Choroidal Neoy
Ascularization (CNV) as a Model of Wet AMD
[0160] The efficacy of intraocular injections of an antibody may be
tested in a laser-injury CNV model as described earlier by
Krzystolik M G et al. (Arch Ophthalm. 2002; 120: 338-346). This
model may be used to test the efficacy of any drug candidate for
the prevention and/or amelioration of AMD. This laser induced CNV
model uses argon green laser to induce CNV in the monkey macula.
There is a good correlation between the number of CNV lesions with
significant angiographic leakage.
[0161] There are two phases of the studies: Phase 1, the prevention
phase, involves the initiation of antibody treatment before laser
induction of the CNV and 1 week after exposure to the laser to
inhibit the formation of CNV, which typically appears 2 to 3 weeks
after laser injury. Phase 2, the treatment phase, is initiated on
day 42 (3 weeks after laser injury) when CNV lesions would be
expected in the control eyes from phase 1. Phase 2 assesses the
effect of treatment on attenuating the extent and leakiness of
existing CNV lesions.
[0162] Ten cynomolgus monkeys (Macaca fasicularis) are typically
used in a study of this type. The monkeys are anesthetized for all
procedures with intramuscular injections of, e.g., ketamine
hydrochloride (20 mg/kg); acepromazine maleate (0.125 mg/kg); and
atropine sulfate (0.125 mg/kg). Supplemental anesthesia of 5 to 6
mg/kg of ketamine hydrochloride may be administered as needed. In
addition, 0.5% proparacaine hydrochloride is typically used for
topical anesthesia. Supplemental anesthesia, with intravenous
peutobarbital sodium solution (5 mg/kg), may be administered before
enucleation. Animals are euthanized following the
experimentation.
Antibody Treatment
[0163] The antibody to be tested is administered in a physiological
buffer at a concentration of about e.g., 10 .mu.g/.mu.L. The
control eye is injected with a vehicle consisting of all components
except the antibody to be tested. Intraocular injections of about,
e.g., 50 .mu.L per eye with either antibody or vehicle is performed
on each eye, respectively, through the pars plana using a 30-gauge
needle and tuberculin syringe after instilling topical anesthesia
and 5% providone iodine solution. The antibody is withdrawn from a
vial through a 5-.mu.m filter, and a new (sharp) 30-gauge needle is
used for the intraocular injection. After the injection, a
bacteriocidal ophthalmic ointment such as bacitracin is instilled
in the fornices. The injection sites are typically varied to avoid
trauma to the sclera.
[0164] In phase 1, the right or left eye of each animal is randomly
assigned to receive intraocular injections of antibody at a dose of
about, e.g., 500 .mu.g (50 .mu.L per eye), and this eye is termed
the prevention eye. The dose used may be determined based on a
safety and toxicology study prior to this efficacy study, or by
other clinically appropriate means. The other eye is assigned to
receive intraocular injections of vehicle and is termed the control
eye. Both eyes of each animal typically receive two intraocular
injections with either the antibody to be tested or the vehicle
alone on days 0 and 14 before laser treatment. On day 21, all eyes
undergo an argon green laser photocoagulation to induce CNV
lesions. On day 28, one week after laser induction, the prevention
eye receives another injection of antibody and the control eye
received vehicle. Phase 2 of the study begins on day 42 or 3 weeks
after laser induction, when CNV is expected to have developed.
Following fluorescein angiography on day 42, both eyes of each
animal will receive intraocular injections of antibody at a dose of
about, e.g., 500 .mu.g (50 .mu.L per eye), and this is repeated on
day 56.
Induction of Experimental CNV
[0165] The CNV membranes are induced in the macula of cynomolgus
monkeys with argon green laser burns (Coherent Argon Dye Laser 920;
Coherent Medical Laser, Palo Alto, Calif.) using a slit-lamp and a
plano fundus contact lens. Nine lesions are symmetrically placed in
the macula of each eye by a masked surgeon. The laser variables
include a 50- to 100-.mu.m spot size, 0.1-second duration, and
power ranging from 350 to 700 mW. The power used is determined by
the laser's ability to produce a blister and a small hemorrhage
under the power chosen. If no hemorrhage is noted, an additional
laser spot will be placed adjacent to the first spot following the
same laser procedure. Color photographs and fluorescein angiography
are typically used to detect and measure the extent and leakiness
of the CNV. However, any method capable of measuring laser-induced
CNV and its associated effects may be used.
Ocular Examinations
[0166] The eyes of the animals are checked for relative pupillary
afferent defect and then dilated with 2.5% phenylephrine
hydorchloride and 0.8% tropicamide. Both eyes are examined using
slitlamp biomicroscopy and indirect ophthalmoscopy on days 0, 14,
28, 42, and 56 (before antibody injection); days 1, 15, 29, 43, and
57 (after injection); day 21 (before laser); days 35 and 49
(intermediate days); and day 63 (enucleation and death).
Color Photography and Fluorescein Angiography
[0167] Fundus photography is typically performed on all animals on
the same days as the ocular examination. Photographs may be
obtained with a fundus camera (Canon Fundus CF-60Z; Canon USA Inc,
Lake Success, N.Y.) and 35-mm film, but any photography device may
be used.
[0168] The Imagenet Digital Angiography System (Topcon 501 A and
Imagenet system; Topcon America Corp, Paramus, N.J.) may be used
for fluorescein angiography. Red-free photographs of both eyes is
typically obtained followed by fluorescein angiography using 0.1
mL/kg of body weight of 10% sodium fluorescein (Akorn Inc. Abita
Springs, La.) at a rate of 1 mL/s. Following the fluorescein
injection, a rapid series of images is obtained in the first minute
of the posterior pole of first the right eye and then the left eye.
Additional pairs of images are typically obtained at approximately
1 to 2 and 5 minutes. Between 2 and 5 minutes, two images of the
midperipheral fields (temporal and nasal) are taken of each eye.
Fluorescein angiography is performed at baseline (day 0) and days
7, 14, 29, 42, 49, 57, and 63.
Analysis of Ophthalmic Data
[0169] Photographs and angiograms are evaluated for evidence of
angiographic leakage, hemorrhages, or any other abnormalities. The
fundus hemorrhages are graded based on a grading system with
retinal hemorrhages that involves less than 3 disc areas defined as
grade 1, hemorrhages between 3 and 6 disc areas defined as grade 2,
and hemorrhages of more than 6 disc areas defined as grade 3. The
association of hemorrhages with CNV membranes or the laser
induction site is also assessed. Clinically significant bleeding is
defined as any fundus hemorrhage greater than or equal to a 6-disc
area.
[0170] Ocular inflammation is also assessed using a slit-lamp
biomicroscopy. Anterior chamber and vitreal cells are counted with
a 2-mm slit-lamp at a high magnification and graded using the
schema of the American Academy of Ophthalmology. The CNV lesions
are graded by reviewing fluorescein angiograms performed on days
35, 42, 49, 56, and 63 by experienced examiners, typically two, who
grade by consensus opinion. The CNV lesions are graded according to
the following scheme, using standardized angiographs for
comparison. Grade 1 lesions have no hyperfluorescence. Grade 2
lesions exhibit hyperfluorescence without leakage. Grade 3 lesions
show hyperfluorescence in the early or mid-transit images and late
leakage. Grade 4 lesions show bright hyperfluorescence in the
transit and late leakage beyond the treated areas. Grade 4 lesions
are defined as clinically significant.
[0171] Statistical analysis may be performed using the
Population-Aggregated Panel Data with Generalized Estimating
Equations nd the incidence rate ratio (IRR). The incidence rate is
usually defined as the number of grade 4 lesions that occur during
a given interval divided by the total number of lesions induced. In
phase 1, the IRR refers to the ratio of incidence rate of grade 4
lesions in the prevention eyes to the incidence rate in control
eyes. An IRR of 1 signifies no difference between incidence rates.
A number much smaller than 1 will indicate a reduction in the
incidence of grade 4 lesions in the prevention group vs. control
group. In phase 2, the incidence of grade 4 lesions in the control
eyes vs. the treatment eyes is compared. This means that the
incidence of grade 4 lesions is compared over time in the set of
eyes that are first assigned to the control group but on days 42
and 56 are treated with antibody and become treatment eyes.
Screen for Agents Useful in the Treatment of AMD
[0172] The study and treatment of age-related macular degeneration
(AMD) can be accomplished using a new animal model comprising mice
deficient either in monocyte chemoattractant protein-1 (Ccl-2; also
known as MCP-1) or its cognate C--C chemokine receptor-2 (Ccr-2)
(Ambati, J. et al. Nat Med. 2003 November; 9(11):1390-7. Epob 2003
Oct. 19). These mice develop cardinal features of AMD, including
accumulation of lipofuscin in and drusen beneath the retinal
pigmented epithelium (RPE), photoreceptor atrophy and choroidal
neovascularization (CNV).
[0173] Treatment of these mice with a desired agent may allow
assessment of the efficacy of such an agent for its efficacy in
treating AMD.
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