U.S. patent application number 09/316387 was filed with the patent office on 2003-08-07 for methods for amyloid removal using anti-amyloid antibodies.
Invention is credited to HRNCIC, RUDI, SOLOMON, ALAN, WALL, JONATHAN STUART.
Application Number | 20030147882 09/316387 |
Document ID | / |
Family ID | 27667867 |
Filed Date | 2003-08-07 |
United States Patent
Application |
20030147882 |
Kind Code |
A1 |
SOLOMON, ALAN ; et
al. |
August 7, 2003 |
METHODS FOR AMYLOID REMOVAL USING ANTI-AMYLOID ANTIBODIES
Abstract
Methods and related immunoglobulin peptides and fragments
thereof are disclosed that enhance the cell-mediated immune
response of a patient to deposits of amyloid fibrils. These methods
exploit the opsonizing effect of antibodies directed toward amyloid
material or its component parts.
Inventors: |
SOLOMON, ALAN; (KNOXVILLE,
TN) ; HRNCIC, RUDI; (KNOXVILLE, TN) ; WALL,
JONATHAN STUART; (KNOXVILLE, TN) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Family ID: |
27667867 |
Appl. No.: |
09/316387 |
Filed: |
May 21, 1999 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60086198 |
May 21, 1998 |
|
|
|
Current U.S.
Class: |
424/132.1 ;
424/141.1; 424/146.1 |
Current CPC
Class: |
A61P 25/28 20180101;
A61K 38/00 20130101; C07K 16/18 20130101 |
Class at
Publication: |
424/132.1 ;
424/141.1; 424/146.1 |
International
Class: |
A61K 039/395 |
Goverment Interests
[0002] This invention was made with government support under Grant
No. 2 R01 CA 20056, awarded by The National Institutes of Health.
Thus, the government may have certain rights in this invention.
Claims
What is claimed is:
1. A method of treating a patient having an amyloid deposition
disease comprising the step of administering to the patient a) a
therapeutically effective dose of at least one immunoglobulin
polypeptide or a fragments thereof, wherein the immunoglobulin
polypeptide or fragment thereof binds to an amyloid fibril; and b)
a pharmaceutically acceptable carrier.
2. The method of claim 1, wherein the immunoglobulin polypeptide or
fragment thereof is raised against an immunoglobulin
light-chain.
3. The method of claim 1, wherein binding of the immunoglobulin
polypeptide or fragment thereof opsonizes the amyloid fibril.
4. The method of claim 1, wherein the immunoglobulin polypeptide or
fragment thereof is a monoclonal antibody.
5. The method of claim 4, wherein the monoclonal antibody is a
humanized antibody.
6. The method of claim 4, wherein the monoclonal antibody is a
chimeric antibody.
7. The method of claim 6, wherein the chimeric antibody is a
humanized antibody.
8. The method of claim 4, wherein the antibody is a labeled
antibody.
9. The method of claim 4, wherein the monoclonal antibody is
selected from the group consisting of .kappa.1 (57-18H12), .kappa.4
(11-1F4), .lambda.8 (31-8C7), and combinations thereof.
10. An immunoglobulin polypeptide or fragment thereof that binds to
an amyloid fibril and is effective to enhance the cellular immune
response of a patient to remove disease-associated amyloid fibril
deposits.
11. The immunoglobulin polypeptide or fragment thereof of claim 10,
wherein the immunoglobulin polypeptide or fragment thereof is a
monoclonal antibody or fragment thereof.
12. The immunoglobulin or fragment thereof of claim 11, wherein the
monoclonal antibody is a humanized antibody.
13. The immunoglobulin polypeptide or fragment thereof of claim 11,
wherein the monoclonal antibody is a chimeric antibody.
14. The immunoglobulin polypeptide or fragment thereof of claim 13,
wherein the chimeric antibody is a humanized antibody.
15. The immunoglobulin polypeptide or fragment thereof of claim 11,
wherein the antibody is a labeled antibody.
16. The immunoglobulin polypeptide or fragment thereof of claim 11,
wherein the monoclonal antibody is selected from the group
consisting of .kappa.1 (57-18H12), .kappa.4 (11-1F4), .lambda.8
(31-8C7), and combinations thereof.
17. The monoclonal antibody or fragment thereof of claim 16,
wherein the monoclonal antibody is a humanized antibody.
18. The immunoglobulin polypeptide or fragment thereof of claim 10,
wherein the immunoglobulin polypeptide or fragment thereof has been
raised against synthetic amyloid fibrils.
19. A pharmaceutical composition comprising the immunoglobulin
peptide or fragment thereof of claim 10.
20. A nucleic acid molecule which encodes a polypeptide comprising
at least a hypervariable region of the immunoglobulin polypeptide
of claim 10.
21. A host cell comprising a nucleic acid molecule of claim 20.
22. A method of producing an immunoglobulin polypeptide comprising
the step of culturing the host cell of claim 21.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application No. 60/086,198, filed May 21, 1998, which is herein
incorporated by reference in its entirety.
TECHNICAL FIELD
[0003] The present invention generally relates to methods for
treating amyloid-related diseases. Specifically, the present
invention provides therapeutic antibody-related methods to effect
the removal of amyloid fibrils by a patient's own immunophagocytic
system.
BACKGROUND OF THE INVENTION
[0004] Amyloidosis refers to the pathological deposition of
proteins in the form of congophilic, green birefringent fibrils,
when congo red-stained, either dispersed or in the form of
localized amyloidomas. Such deposits are symptomatic of several
diseases, for example Alzheimer's Disease, inflammation-associated
amyloid, type II diabetes, bovine spongiform encephalopathy (BSE),
Creutzfeld-Jakob disease (CJD), scrapie and primary
amyloidosis.
[0005] Amyloidoses are generally categorized into three groups:
major systemic amyloidoses, major localized amyloidoses, and
miscellaneous amyloidoses. Major systemic amyloidoses include:
chronic inflammatory conditions (e.g., tuberculosis, osteomyelitis,
etc.); non-infectious conditions such as juvenile rheumatoid
arthritis, ankylosing spondylitis and Crohn's disease, etc.;
familial Mediterranean Fever, plasma cell dyscrasia (primary
amyloidosis) and various familial polyneuropathies and
cardiomyopathies. Major localized amyloidoses include: chronic
dialysis usually for greater than 8 years, Alzheimer's disease,
Down syndrome, Hereditary cerebral hemorrhage (Dutch), and
non-traumatic cerebral hemorrhage of the elderly. Miscellaneous
amyloidoses include: familial polyneuropathy (Iowa), familial
amyloidosis (Finnish), hereditary cerebral hemorrhage (Icelandic),
CJD, Medullary carcinoma of the thyroid, atrial amyloid, and
diabetes mellitus (insulinomas). Other amyloidoses include those
referenced in Louis W. Heck, "The Amyloid Diseases" in Cecil's
Textbook of Medicine 1504-6 (W. B. Saunders & Co.,
Philadelphia, Pa.; 1996).
[0006] Transmissible spongiform encephalopathies which cause CJD
and Gerstmann-Strssler-Scheinker (GSS) disease are described by B.
Chesebro et al., "Transmissible Spongiform Encephalopathies: A
Brief Introduction" in Field's Virology 2845-49 (3rd Edition; Raven
Publishers, Philadelphia, Pa.; 1996) and in D. C. Gajdusek,
"Infectious amyloids: Subacute Spongiform Encephalopathies as
Transmissible Cerebral Amyloidoses," 2851-2900 in Fields Virology
(1996). Many of these diseases are likely mediated by prions, an
infectious protein. See S. B. Prusiner, "Prions" in Fields Virology
2901-50 (1996) and the references contained therein. The inherited
forms of amyloidoses as described on Online Mendelian Inheritance
in Man (OMIM) "www.ncbi.nlm.nih.gov/htbin-post/Omim/dispmim?" Each
of the above is incorporated herein by reference.
[0007] Very rarely do patients with clinically proven amyloidosis
spontaneously achieve complete remission, perhaps because the
amyloid fibrils themselves are non-immunogenic. Various therapies
for amyloidosis have been investigated, such as high-dose
chemotherapy, steroids, iodinated doxorubicin, and stem cell
replacement therapy. However, in only one type of amyloid disease,
Familial-Mediterranean amyloidosis, has drug treatment (with
colchicine) been shown to be effective.
[0008] The use of monoclonal antibodies (mAbs) to induce or
modulate the immunological removal of an otherwise unrecognized
entity is known. mAbs have been successfully used in treating
non-Hodgkins lymphoma and breast cancer, for example.
[0009] Previously, a variety of studies have characterized
antibodies that bind to amyloid proteins or amyloid fibrils. See,
for example, U.S. Pat. Nos. 5,714,471; 5,693,478; 5,688,651;
5,652,092; 5,593,846; 5,536,640; 5,385,915; 5,348,963; 5,270,165;
5,262,332; 5,262,303; 5,164,295; and 4,782,014. In addition,
several publications have suggested that anti-amyloid antibodies
might be useful for studying the progression of beta-amyloidosis
and for various therapeutic options. See, for example, Bellottii et
al., Scand. J. Immunol. (1992) 36(4):607-615; Bellotti et al., Ren.
Fail. (1993) 15(3):365-371; Walker et al. J. Neuropathol. Exp.
Neurol. (1994) 53(4):377-383; and Bickel et al., Bioconjug. Chem.
(1994) 5(2):119-125. However, no therapeutic antibody has been
demonstrated to halt or reverse the deposition of amyloid fibrils
in a patient. Thus, a need exists for a method for treating
amyloidoses using antibody formulations containing antibodies that
bind to amyloid fibrils.
SUMMARY OF THE INVENTION
[0010] The present inventors have discovered new methods of
treating amyloid-related diseases and conditions. These methods
exploit the opsonizing effect of mAbs directed toward the protein
constituents of amyloid.
[0011] The present invention includes a method of treating a
patient having an amyloid-associated disease comprising the step of
administering to the patient a therapeutically effective dose of at
least one immunoglobulin polypeptide, or fragments thereof,
together with a pharmaceutically acceptable carrier; wherein the
immunoglobulin polypeptide or fragment thereof, may be a
substantially purified immunoglobulin polypeptide that binds to a
human amyloid fibril, wherein binding of the polypeptide opsonizes
the amyloid fibril.
[0012] In particular, the present invention relates to the use of
any one of, or a combination of, the three monoclonal antibodies
discussed below. These antibodies have general anti-amyloid binding
properties and provide an extrinsic opsonizing reagent that
activates a patient's own cellular immune clearance mechanism.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIGS. 1A and 1B. FIGS. 1A and 1B are reproduced photographs
of a Balb/c mouse just after an injection of amyloid is made (1A)
and 14 days after the injection (1B). The injection site was shaved
to better illustrate the "hump" caused by the injection of the
amyloid material.
[0014] FIGS. 2A-2B. FIGS. 2A and 2B are reproduced photographs of
human neutrophils (multi-lobed nuclei) adhering to human amyloid
opsonized in vitro.
[0015] FIGS. 3A-3D. FIGS. 3A-3D are reproduced photographs of
immunohistochemically stained amyloid-laden tissue samples (20X
magnification). FIG. 3A is a tissue sample from a patient with
.kappa.1 amyloidosis stained with Congo red; the amyloid deposits,
viewed under polarized light, appear as blue-green particles. FIG.
3B is a tissue sample stained with alkaline phosphatase after
labeling with anti-.kappa.I (57-18-H12) mAb. FIG. 3C is a tissue
sample stained as in FIG. 3B, but with anti-.kappa.IV (11-1F4) mAb.
FIG. 3D is a tissue sample stained as in FIG. 3B, but with
anti-.lambda.VIII (31-8c7) mAb.
[0016] FIG. 4. FIG. 4 is a reproduced photograph showing a
fluoresceinated (FITC) .kappa.4 mAb bound to human amyloid
implanted into a Balb/c mouse. The mAb was injected into the thigh
of the mouse. The amyloidoma was excised 72 hours post injection
and viewed using an epifluorescence microscope (20X
magnification).
MODES OF CARRYING OUT THE INVENTION
[0017] General Description
[0018] The present invention utilizes immunoglobulin polypeptides
to modulate and to enhance the degradation and removal of undesired
deposits of amyloid fibrils in a host or patient. It is envisioned
that the invention will be used, for example, to treat humans
suffering from a disease or condition characterized by an undesired
deposition of amyloid fibrils. Without intending to be bound by any
particular mechanism of action, it is believed that the
administration of immunoglobulin peptides according to the present
invention opsonize the deposited amyloid fibrils in a patient
suffering from amyloidosis, thereby assisting in their removal from
the patient by the patients' own immune system. It is believed that
the patient's immune system alone is unable to remove the amyloid
fibrils in conditions modulated by amyloid fibrils without such a
therapeutic intervention, presumably because the amyloid fibrils
are themselves relatively non-immunogenic.
[0019] To treat a patient with amyloidosis, a therapeutically
effective dose of immunoglobulin polypeptide or fragment thereof
according to the present invention is administered together with a
pharmaceutically suitable carrier or excipient. Upon the binding or
adhering of such immunoglobulin polypeptides to undesired deposits
of amyloid fibrils, the latter are believed to be opsonized.
[0020] Single or multiple administrations of the compositions of
the present invention can be carried out in dosages and by
administration protocols known to those skilled in the art for the
administration of other therapeutic antibody products. These
parameters may be selected and/or optimized by the physician
treating a particular patient.
[0021] Preferably, a therapeutically effective dose of a
pharmaceutical formulation of the present invention should deliver
a quantity of anti-amyloid immunoglobulin polypeptide sufficient to
substantially inhibit the undesired deposition of amyloid fibrils
or to substantially inhibit the rate of any undesired deposition of
amyloid fibrils. More preferably, the formulations should reduce
the overall burden of deposited amyloid fibrils in a patient.
Further, administration of such formulations should begin shortly
after diagnosis of amyloidosis and continue until symptoms are
substantially abated and for a period thereafter. In well
established cases of disease, loading doses followed by maintenance
doses may be required.
[0022] Definitions
[0023] The terms "peptide," "polypeptide" or "protein" are used
interchangeably herein. The term "substantial identity," when
referring to polypeptides, indicates that the polypeptide or
protein in question is at least about 30% identical to an entire
naturally occurring protein or a portion thereof, usually at least
about 70% identical, and preferably at least about 95%
identical.
[0024] As used herein, the terms "isolated," "substantially pure"
and "substantially homogenous" are used interchangeably and
describe a protein that has been separated from components which
naturally accompany it. A substantially purified protein will
typically comprise over about 85% to 90% of a protein sample, more
usually about 95%, and preferably will be over about 99% pure.
Protein purity or homogeneity may be indicated by a number of means
well known in the art, such a polyacrylamide gel electrophoresis of
a protein sample, followed by visualizing a single polypeptide band
on a polyacrylamide gel upon staining. For certain purposes high
resolution will be needed and HPLC or a similar means for
purification utilized.
[0025] Proteins may be purified to substantial homogeneity by
standard techniques well known in the art, including selective
precipitation with such substances as ammonium sulfate, column
chromatography, immunopurification methods, and others. See, for
instance, Scopes, Protein Purification: Principles and Practice,
Springer-Verlag: New York (1982), which is incorporated herein by
reference.
[0026] Antibody purification techniques are well known in the art.
Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor (1988), 288-318, which is
incorporated herein by reference, describes, for example,
purification using ammonium sulfate precipitation, caprlic acid,
DEAE, hydroxyapatite chromatography, gel filtration chromatography,
protein A beads, and immunoaffinity.
[0027] Nucleic acids, as used herein, may be DNA or RNA. When
referring to nucleic acids, the term "substantial identity"
indicates that the sequences of two nucleic acids, or designated
portions thereof, when optimally aligned and compared, are
identical, with appropriate nucleotide insertions or deletions, in
at least about 80% of the nucleotides, usually at least about 90%
to 95%, and more preferably at least about 98% to 99.5% of the
nucleotides.
[0028] Alternatively, substantial nucleic acid sequence identity
exists when a nucleic acid segment will hybridize under selective
hybridization conditions, to a complement of another nucleic acid
strand.
[0029] "Substantially complementary" similarly means that one
nucleic acid hybridizes selectively to, or is identical to, another
nucleic acid. Typically, selective hybridization will occur when
there is at least about 55% identity over a stretch of at least
14-25 nucleotides, preferably at least about 65% identity, more
preferably at least about 75%, and most preferably at least about
90% identity. See M. Kanehisa Nucleic Acids Res. 12:203 (1984),
which is incorporated herein by reference.
[0030] Stringent hybridization conditions will typically include
salt concentrations of less than about 1 M, more usually less than
about 500 mM and preferably less than about 200 mM. Temperature
conditions will typically be greater than 22.degree. C., typically
greater than about 30.degree. C. and preferably in excess of about
37.degree. C. As other factors may dramatically affect the
stringency of hybridization, including base composition and size of
the complementary strands, presence of organic solvents and extent
of base mismatching, the combination of parameters is more
important than the absolute measure of any one alone.
[0031] "Isolated" or "substantially pure," when referring to
nucleic acids, refer to those that have been purified away from
other cellular components or other contaminants, e.g., other
cellular nucleic acids or proteins, by standard techniques,
including alkaline/SDS treatment, CsCl banding, column
chromatography, and others well known in the art. See, F. Ausubel,
et al., ed. Current Protocols in Molecular Biology, Greene
Publishing and Wiley-Interscience, New York (1987), incorporated
herein by reference.
[0032] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
instance, a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the sequence.
Generally, operably linked means that the nucleic acid sequences
being linked are contiguous and, where necessary to join two
protein coding regions, contiguous and in reading frame.
[0033] Techniques for nucleic acid manipulation, such as subcloning
nucleic acid sequences encoding polypeptides into expression
vectors, labelling probes, DNA hybridization, and so on are
described generally, for example in Sambrook et al., (1989)
Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold
Spring Harbor Laboratory, or Ausubel et al., ed. (1987) op. cit.,
both of which are incorporated herein by reference.
[0034] "Expression vectors," "cloning vectors," or "vectors" are
often plasmids or other nucleic acid molecules that are able to
replicate in a chosen host cell. Expression vectors may replicate
autonomously, or they may replicate by being inserted into a genome
of the host cell, by methods well known in the art. Vectors that
replicate autonomously will have an origin of replication or
autonomous replicating sequence ("ARS") that is functional in the
chosen host cell(s). Often, it is desirable for a vector to be
usable in more than one host cell, e.g., in E. coli for cloning and
construction, and in a mammalian cell for expression.
[0035] Mammalian cell lines are often used as host cells for the
expression of polypeptides derived from eukaryotes. Propagation of
mammalian cells in culture is per se well known. See, Tissue
Culture, Academic Press, Kruse and Patterson, ed. (1973),
incorporated herein by reference. Host cell lines may also include
such organisms as bacteria (e.g., E. coli or B. subtilis), yeast,
filamentous fungi, plant cells, or insect cells, among others.
[0036] "Transformation" refers to the introduction of vectors
containing the nucleic acids of interest directly into host cells
by well-known methods. Transformation methods, which vary depending
on the type of host cell, include electroporation; transfection
employing calcium chloride, rubidium chloride calcium phosphate,
DEAE-dextran, or other substances; microprojectile bombardment;
lipofection; infection (where the vector is an infectious agent);
and other methods. See generally, Sambrook et al., (1989) op. cit.
Reference to cells into which the nucleic acids described above
have been introduced is meant to also include the progeny of such
cells.
[0037] As used herein, "immunoglobulin polypeptide" refers to
molecules that are derived from native immunoglobulins (e.g.,
antibodies) that have specific immunoreactive activity against a
particular target, e.g., against amyloid fibrils. Antibodies are
typically tetramers of immunoglobulin polypeptides. As used herein,
the term "antibody" also refers to a protein consisting of one or
more polypeptides substantially encoded by immunoglobulin genes.
Immunoglobulin genes include those coding for the light chains,
which may be of the kappa or lambda types, and those coding for the
heavy chains. Heavy chain types are alpha, gamma, delta, epsilon
and mu. The carboxy terminal portions of immunoglobulin heavy and
light chains are constant regions, while the amino terminal
portions are encoded by the myriad immunoglobulin variable region
genes. The variable regions of an immunoglobulin are the portions
that provide antigen recognition specificity. In particular, the
specificity resides in the complementarity determining regions
("CDRs"), also known as hypervariable regions, of the
immunoglobulins.
[0038] The immunoglobulins may exist in a variety of fragment forms
including, for example, Fv, Fab, F(ab"), F(ab').sub.2, SvFv and
other fragments, as well as single chains (e.g., Huston, et al.,
Proc. Nat. Acad. Sci. U.S.A., 85:5879-5883 (1988) and Bird, et al.,
Science 242:423-426 (1988), which are incorporated herein by
reference). (See, generally, Hood, et al., "Immunology," Benjamin,
N.Y., 2nd ed. (1984), and Hunkapiller and Hood, Nature, 323:15-16
(1986), which are incorporated herein by reference). Single-chain
antibodies, in which genes for a heavy chain and a light chain are
combined into a single coding sequence, may also be used.
Immunoglobulin polypeptide also encompasses a truncated
immunoglobulin chain, for example, a chain containing less constant
region domains than in the native polypeptide. Such truncated
polypeptides can be produced by standard methods such as
introducing a stop codon into the gene sequence 5' of the domain
sequences to be deleted. The truncated polypeptides can then be
assembled into truncated antibodies. Antibodies as used herein also
include bispecific antibodies which can be produced such as by the
methods described in the following references: Glennie et al., J.
Immunol., 139:2367-2375 (1987); Segal et al., Biologic Therapy of
Cancer Therapy of Cancer Updates 2(4):1-12 (1992); and Shalaby et
al., J. Exp. Med. 175:217-225 (1992).
[0039] "Monoclonal antibodies" may be obtained by various
techniques familiar to those skilled in the art. Briefly, spleen
cells from an animal immunized with a desired antigen are
immortalized, commonly by fusion with a myeloma cell (see Kohler
and Milstein, Eur. J. Immunol. 6:511-519 (1976)). Alternative
methods of immortalization include transformation with Epstein Barr
Virus, oncogenes, or retroviruses, or other methods well known in
the art. Colonies arising from single immortalized cells are
screened for production of antibodies of the desired specificity
and affinity for the antigen, and yield of the monoclonal
antibodies produced by such cells may be enhanced by various
techniques, including injection into the peritoneal cavity of a
vertebrate host.
[0040] Monospecific and bispecific immunoglobulins may also be
produced by recombinant techniques in prokaryotic or eukaryotic
host cells.
[0041] "Chimeric" antibodies are encoded by immunoglobulin genes
that have been genetically engineered so that the light and heavy
chain genes are composed of immunoglobulin gene segments belonging
to different species. For example, the variable (V) segments of the
genes from a mouse monoclonal antibody may be joined to human
constant (C) segments. Such a chimeric antibody is likely to be
less antigenic to a human than antibodies with mouse constant
regions as well as mouse variable regions.
[0042] As used herein, the term chimeric antibody also refers to an
antibody that includes an immunoglobulin that has a human-like
framework and in which any constant region present has at least
about 85%-90%, and preferably about 95% polypeptide sequence
identity to a human immunoglobulin constant region, a so-called
"humanized" immunoglobulin (see, for example, PCT Publication WO
90/07861, which is incorporated herein by reference). Hence, all
parts of such a "humanized" immunoglobulin, except possibly the
complementarity determining regions (CDRs), are substantially
identical to corresponding parts of one or more native human
immunoglobulin sequences. Where necessary, framework residues may
also be replaced with those within or across species especially if
certain framework residues are found to affect the structure of the
CDRs. A chimeric antibody may also contain truncated variable or
constant regions.
[0043] The term "framework region," as used herein, refers to those
portions of immunoglobulin light and heavy chain variable regions
that are relatively conserved (i.e., other than the CDRs) among
different immunoglobulins in a single species, as defined by Kabat,
et al., (1987); Sequences of Proteins of Immunologic Interest, 4th
Ed., U.S. Dept. Health and Human Services, which is incorporated
herein by reference). As used herein, a "human-like framework
region" is a framework region that in each existing chain comprises
at least about 70 or more amino acid residues, typically 75 to 85
or more residues, identical to those in a human immunoglobulin.
[0044] Human constant region DNA sequences can be isolated in
accordance with well-known procedures from a variety of human
cells, but preferably from immortalized B-cells. The variable
regions or CDRs for producing the chimeric immunoglobulins of the
present invention may be similarly derived from monoclonal
antibodies capable of binding to the human type amyloid, and will
be produced in any convenient mammalian system, including mice,
rats, rabbits, human cell lines, or other vertebrates capable of
producing antibodies by well-known methods. Variable regions or
CDRs may be produced synthetically, by standard recombinant
methods, including polymerase chain reaction ("PER") or through
phage-display libraries. For phage display methods, see for
example, McCafferty et al., Nature 348:552-554 (1990); Clackson et
al., Nature 352:624-628 and Marks et al., Biotechnology
11:1145-1149 (1993). Suitable prokaryotic systems such as bacteria,
yeast and phage may be employed.
[0045] Suitable source cells for the DNA sequences and host cells
for immunoglobulin expression and secretion can be obtained from a
number of sources, such as the American Type Culture Collection
("Catalogue of Cell Lines and Hybridomas," Fifth edition (1985)
Rockville, Md., U.S.A., which is incorporated herein by
reference).
[0046] In addition to the chimeric and "humanized" immunoglobulins
specifically described herein, other substantially identical
modified immunoglobulins can be readily designed and manufactured
utilizing various recombinant DNA techniques well known to those
skilled in the art. In general, modifications of the genes may be
readily accomplished by a variety of well-known techniques, such as
PCR and site-directed mutagenesis (see, Gillman and Smith, Gene
8:81-97 (1979) and S. Roberts et al., Nature 328:731-734 (1987),
both of which are incorporated herein by reference).
[0047] Alternatively, polypeptide fragments comprising only a
portion of the primary immunoglobulin structure may be produced.
For example, it may be desirable to produce immunoglobulin
polypeptide fragments that possess one or more immunoglobulin
activities in addition to, or other than, antigen recognition
(e.g., complement fixation).
[0048] Immunoglobulin genes, in whole or in part, may also be
combined with functional regions from other genes (e.g., enzymes),
or with other molecules such as toxins, labels and targeting
moieties to produce fusion proteins (e.g., "immunotoxins") having
novel properties. In these cases of gene fusion, the two components
are present within the same polypeptide chain. Alternatively, the
immunoglobulin or fragment thereof may be chemically bonded to the
toxin or label by any of a variety of well-known chemical
procedures. For example, when the label or cytotoxic agent is a
protein and the second component is an intact immunoglobulin, the
linkage may be by way of heterobifunctional cross-linkers, e.g.,
SPDP, carbodiimide, glutaraldehyde, or the like.
[0049] Suitable labels include, for example, radionuclides,
enzymes, substrates, cofactors, inhibitors, fluorescers,
chemiluminescers, magnetic particles. See, for examples of patents
teaching the use of such labels, U.S. Pat. Nos. 3,817,837;
3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and
4,366,241, all of which are incorporated by reference.
[0050] Immunotoxins, including single chain molecules, may also be
produced by recombinant means. Production of various immunotoxins
is well-known with the art, and methods can be found, for example
in "Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet,"
Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic
Press, pp. 168-190 (1982); E. Vitetta, Science (1987)
238:1098-1104; and G. Winter and C. Milstein, Nature (1991)
349:293-299; all incorporated herein by reference.
[0051] Additional techniques for preparing immunoglobulins and
immunoglobulin fragments are described in V. S. Malik et al,
Antibody Techniques (Academic Press, 1994); C. A. K. Borrebaeck,
Antibody Engineering: Breakthroughs in Molecular Biology (Oxford
Univ. Press, 1995); and P. J. Delves et al., Antibody Production:
Essential Techniques (John Wiley & Sons, 1997), which are
incorporated herein by reference.
[0052] "Opsonize", as used herein, refers to the binding of an
immunoglobulin polypeptide to a particular target, particularly
epitopes found on deposits of amyloid fibrils, such that the
antibody and targets together are recognized as "foreign" by the
host's cellular immune system. In other words the binding of the
immunoglobulin of the present invention enhances the
phagocytization of the amyloid fibrils.
[0053] "Amyloidosis", as used herein, is intended to refer to any
condition that is characterized by the presence of amyloid
material. Such material may be in the form of an amyloidoma or more
disperse amyloid deposits or fibrils.
[0054] Pharmaceutical Compositions
[0055] The pharmaceutical compositions for therapeutic treatment
according to the present invention are intended for parenteral,
oral or local administration. Preferably, the pharmaceutical
compositions are administered parenterally, e.g., intravenously,
subcutaneously, intradermally, or intramuscularly. As the blood
brain barrier is impermeable to IgG (see U. Bickel et al., 1994
Bioconjug. Chem. 5: 119-25), delivery of antibodies to overcome the
blood-brain barrier (BBB) may be achieved through liposomal or
micellar delivery of the antibody to the desired site.
Alternatively, the agents of this invention can be delivered
directly into the cerebrospinal fluid (see for example L. C. Walker
et al., 1994 J. Neuropathol. Exp. Neurol. 53: 377-83). For other
delivery mechanisms, refer to P. M. Friden, 1996 U.S. Pat. No.
5,527,527 and W. M. Pardridge, 1991 U.S. Pat. No. 5,004,697. All of
the above documents are incorporated herein by reference.
[0056] Thus, the invention provides compositions for parenteral
administration which comprise a solution of the anti-amyloid
immunoglobulin polypeptide dissolved or suspended in a
pharmaceutically acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers may be used, e.g., water, buffered
water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
These compositions may be sterilized by conventional, well known
sterilization techniques, or may be sterile filtered. The resulting
aqueous solutions may be packaged for use as is, or lyophilized,
the lyophilized preparation being combined with a sterile solution
prior to administration. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions, such as pH adjusting and
buffering agents, tonicity adjusting agents, wetting agents and the
like, for example, sodium acetate, sodium lactate, sodium chloride,
potassium chloride, calcium chloride, sorbitan monolaurate,
triethanolamine oleate, etc.
[0057] The concentration of anti-amyloid immunoglobulin
polypeptides of the invention in the pharmaceutical formulations
can vary widely, i.e., from less than about 1%, usually at or at
least about 10-15% to as much as 50% or more by weight, and will be
selected primarily by fluid volumes, viscosities, etc., in an
accordance with the particular mode of administration selected.
[0058] Without undue experimentation, one of ordinary skill in the
art could determine the quantity of immunoglobulin polypeptides
that would be effective in adequately opsonizing an amyloidoma.
Amounts effective for this use will depend on, e.g., the nature of
the anti-amyloid immunoglobulin polypeptide composition, the manner
of administration, the stage and severity of the disease being
treated, the weight and general state of health of the patient, and
the judgment of the prescribing physician. A typical single dose of
0.5 mg/kg could generally be used. It must be kept in an mind that
the anti-amyloid immunoglobulin polypeptide and peptide
compositions derived therefrom may be employed in serious disease
states, that is, life-threatening or potentially life-threatening
situations. In such cases it is possible and may be felt desirable
by the treating physician to administer substantial excesses of
these compositions. Thus, human anti-amyloid monoclonal antibodies
or substantially human anti-amyloid receptor monoclonal antibodies
of the invention are most preferred under these circumstances.
[0059] Treatment of humans with amyloidosis according to the
present invention could also be applied to animals susceptible to
amyloidosis, such as cows or chickens. Thus, references to human
patients herein apply also to non-human patients.
[0060] The immunoglobulin polypeptides, as defined herein, are
preferably anti-amyloid mAbs directed toward an amyloidoma or
components or precursors thereof. The mAbs can be raised against
IgLC variable region domains or, preferably, against the IgLC
subsets .kappa.1, .kappa.4, .lambda.8, or combinations thereof. The
administration to humans of immunoglobulin polypeptides that are
substantially non-human may elicit anti-antibody responses. Thus,
it may be desirable to prepare anti-IgLC immunoglobulin
polypeptides of the present invention which are substantially
human. By "substantially human" is meant an antibody or binding
fragment thereof comprised of amino acid sequences which are at
least about 50% human in origin, at least 70 to 80% more preferred,
and about 95-99% or more human most preferred, particularly for
repeated administrations over a prolonged period as may be
necessary to treat established cases of amyloidosis. As used
herein, human antibody is meant to include antibodies of entirely
human origin as well as those which are substantially human, unless
the context indicates otherwise.
[0061] Monoclonal antibodies can also be raised against synthetic
amyloid fibrils. Recombinant light chain, variable region peptides
are isolated and purified in vitro using standard techniques.
Synthetic fibrils are then prepared from the peptides using
techniques such as those described by Wall et al., "In vitro
Immunoglobulin Light Chain Fibrillogenesis," METHODS IN ENZYMOLOGY,
Vol. 309 (In Press). Antibodies are then raised against the
synthetic fibrils using standard immunization techniques, typically
in mice or rabbits. Monoclonal cell lines secreting anit-fibril
antibodies are produced using standard hybridoma techniques.
[0062] The anti-amyloid immunoglobulin polypeptides of the
invention may be prepared by any of a number of well-known
techniques. For instance, they may be prepared by immunizing an
animal with purified or partially purified human amyloid. The
animals immunized can be any one of a variety of species which are
capable of immunologically recognizing epitopes characteristic of
the human type amyloid extracellular domain, such as murine,
lagomorph, equine, etc.
[0063] Monoclonal antibodies of the invention may be prepared by
immortalizing cells comprising nucleic acid sequences which encode
immunoglobulin polypeptides or portions thereof that bind
specifically to antigenic determinants characteristic of the
extracellular domain of the human type amyloid. The immortalization
process can be carried out by hybridoma fusion techniques, by viral
transformation of antibody-producing lymphocytes, recombinant DNA
techniques, or by techniques that combine cell fusion, viral
transformation and/or recombinant DNA methodologies. Immunogens to
raise the monoclonal antibodies include synthetic amyloid fibrils
as described, for example by, A. Lomakin et al., 1997 Proc. Nat'l
Acad. Sci. USA 94: 7942-7, which is incorporated herein by
reference.
[0064] As the generation of human anti-amyloid monoclonal
antibodies may be difficult with conventional immortalization
techniques, it may be desirable to first make non-human antibodies
and then transfer via recombinant DNA techniques the antigen
binding regions of the non-human antibodies, e.g., the Fab,
complementarity determining regions (CDRs) or hypervariable
regions, to human constant regions (Fc) or framework regions as
appropriate to produce substantially human molecules. Such methods
are generally known in the art and are described in, for example,
U.S. Pat. No. 4,816,397, PCT publication WO 90/07861, and EP
publications 173494 and 239400, wherein each is incorporated herein
by reference. However, completely human antibodies can be produced
in transgenic animals. The desired human immunoglobulin genes or
gene segments can be isolated, for example by PCR from human B
cells, the DNA cloned into appropriate vectors for expression in
eukaryotic cells and the cloned DNA introduced into animals to
produce transgenics. Animals suitable for the production of
transgenics expressing human immunoglobulin include mice, rats,
rabbits and pigs with rodents of transgenics that express human
immunoglobulins should preferably have one or more of their
endogenous immunoglobulin loci inactivated or "knocked-out" to
facilitate identification and isolation of the human antibodies
(See e.g., Lonberg, et al. Nature 368:856-859 (1994)).
[0065] The resulting chimeric antibodies or chimeric immunoglobulin
polypeptides that bind to human amyloid are also within the scope
of the present invention. A typical therapeutic chimeric antibody
would be a hybrid protein consisting of the variable (V) or
antigen-binding domain from a mouse immunoglobulin specific for a
human amyloid antigenic determinant, and the constant (C) or
effector domain from a human immunoglobulin, although domains from
other mammalian species may be used for both variable and constant
domains. As used herein, the therm "chimeric antibody" also refers
to antibodies coded for by immunoglobulin genes in which only the
CDRs are transferred from the immunoglobulin that specifically
recognizes the antigenic determinants, the remainder of the
immunoglobulin gene being derived from a human (or other mammalian,
as desired) immunoglobulin gene. As discussed before, this type of
chimeric antibody is referred to as a "humanized" (in the case of a
human immunoglobulin gene being used) antibody. Also considered are
recombinant human antibodies that do not contain sequences of
another species.
[0066] The hypervariable regions of the variable domains of the
anti-amyloid immunoglobulin polypeptides comprise a related aspect
of the invention. The hypervariable regions, or CDRs, in
conjunction with the framework regions (those portions of
immunoglobulin light and heavy chain variable regions that are
relatively conserved among different immunoglobulins in a single
species), enable the anti-amyloid immunoglobulin polypeptides to
recognize and thus bind to human amyloid. The hypervariable regions
can be cloned and sequenced. Once identified, these regions that
confer specific recognition of human amyloid can then be cloned
into a vector for expression in a host as part of another
immunoglobulin molecule or as a fusion protein, e.g., a carrier
molecule which functions to enhance immunogenicity of the cloned
idiotype.
[0067] The anti-amyloid immunoglobulin polypeptides of the
invention will generally be used intact, or as immunogenic
fragments, such Fv, Fab, F(ab').sub.2 fragments. The fragments may
be obtained from antibodies by conventional techniques, such as by
proteolytic digestion of the antibody using, e.g., pepsin, papain
or other proteolytic enzymes, or by recombinant DNA techniques in
which a gene or portion thereof encoding the desired fragment is
cloned or synthesized, and expressed in a variety of hosts.
[0068] Those skilled in the art will realize that "anti-idiotypic"
antibodies can be produced by using a specific immunoglobulin as an
immunogen in accordance with standard techniques. For example,
infection or immunization with an amyloid fibril or fragment
thereof, induces a neutralizing immunoglobulin, which has on its
Fab variable region combining site an image of the amyloid that is
unique to that particular immunoglobulin, i.e., an idiotype.
Immunization with such an anti-amyloid immunoglobulin induces an
anti-idiotype antibody, which has a conformation at its combining
site that mimics the structure of the original amyloid antigen.
These anti-idiotype antibodies may therefore be used instead of the
amyloid antigen. See, for example, Nisonoff (1991) J. Immunol.
147:2429-2438, which is incorporated herein by reference.
[0069] The following working examples specifically point out
preferred embodiments of the present invention, and are not to be
construed as limiting in any way the remainder of the disclosure.
Other generic configurations will be apparent to one skilled in the
art.
EXAMPLE 1
Unassisted Resolution of Human IgLC Amyloid in Murine Host
[0070] Human IgLC amyloid was extracted and purified from infected
organs obtained during an autopsy. The first experiments involved
transplanting 50-200 mg of this amyloid material into a Balb/c
mouse. The amyloid mass, or "amyloidoma," was prepared in sterile
PBS by serial sonication and grinding steps in order to produce a
fine suspension of amyloid fibrils complete with the accessory
molecules found in vivo. This procedure was performed to allow the
amyloid to be injected into the mice through a wide-gauge
hypodermic needle.
[0071] The amyloid material, equivalent to 10% of the body weight
of the animal, was injected into mice (under anesthetic) between
the scapula, which resulted in a large mass being visible (see FIG.
1A). The mouse required 15-18 days to achieve the complete removal
of the amyloidoma (see FIG. 1B), after which the animal appeared
healthy and lived a normal life span. The removal of the amyloidoma
was determined subjectively by the experimenter; by simply
palpating the injection site, an amyloidoma, like a hard pea, can
be easily felt under the skin.
EXAMPLE 2
Involvement of Both Antibody-Meditated and Cellular Immunity in the
Removal of Amyloidomas
[0072] The involvement of anti-amyloid antibodies in the removal of
amyloidomas was shown by screening serum from a mouse previously
injected with amyloid material against a sample of the injected
material. This was done by Western blot analysis using suitable
dilutions of the mouse serum as the primary antibody. It was shown
that there were antibodies to every component of the amyloid
matrix, i.e., every band on the gel was stained by the mouse serum,
even at a 10,000-fold serum dilution (data not shown).
[0073] The involvement of a cellular component was demonstrated by
in vitro neutrophil binding assays (see FIGS. 2A and 2B) and by
using knockout-mutant mouse strains (data not shown). FIGS. 2A and
2B show human neutrophils adhering to human amyloid after the
amyloid was treated with mouse anti-human IgLC mAbs. This shows
that the mouse mAb can bind to human amyloid as well as attract
human neutrophils.
[0074] Studies of knockout-mutant mouse strains further support a
finding of antibody involvement in amyloid removal. First,
scid/scid mice, which lack B and T lymphocytes, were unable to
remove an injected amyloidoma even after three months (data not
shown). Second, CD18 knockout animals were unable to remove the
amyloidoma as rapidly as normal animals. CD18 knockout animals are
97% deficient in CD18, a cell surface integrin found on
granulocyte/macrophage lineages. Although these cells cannot leave
the circulation, the animals are B and T cell competent and can
therefore mount an antibody response. Third, nude mice, which have
no white blood cells, were unable to remove the amyloidoma.
[0075] Furthermore, amyloid that had been incubated with
amyloid-reactive serum from another mouse, when implanted into the
second mouse, was removed within 4 days. In this experiment a
Balb/c mouse was injected with 50 mg HIG amyloid and left for 1
week, after which it was bled by tail-vein clipping. The blood was
spun down at 1500 rpm and the cells removed by aspiration. The
plasma was stored at 4.degree. C. until used. Another preparation
of HIG amyloid (100 mg) was prepared by suspending in sterile PBS
to which was added 1 ml of plasma from the previous mouse. This
preparation was then injected into a second mouse (Balb/c) and the
amyloid was removed in 4 days. Thus, it was concluded that the
process could be sped up by opsonizing the material prior to
injection.
EXAMPLE 3
ELISA Screening of IgLC Subsets
[0076] A systematic study was performed using ELISA techniques to
screen a large number of human extracted amyloid samples using mAbs
raised against the IgLC subsets (.lambda.1, .lambda.2, .lambda.3,
.lambda.4, .lambda.5, .lambda.6, .kappa.1, .kappa.2, .kappa.3,
.kappa.4, free .kappa. and .lambda. and total .kappa. and
.lambda.). Interestingly, it was found that more often than not,
the amyloids tested positive with mAbs specific for their own
subtype, the total .kappa. or .lambda. antibodies and a
.kappa.1(57-18H12), .kappa.4(11-1F4) and .lambda.8(31-8C7) mAb.
These latter three reagents were found to react in a non-subgroup
specific manner, i.e., .kappa.1 reacted with amyloids comprised of
IgLCs other than .kappa.1; and the other two mAbs exhibit the same
quality. This shows that the epitope recognized by these antibodies
may be a general feature of amyloid fibrils, indicating the
possibility of a shared amyloid epitope that can be targeted.
EXAMPLE 4
Immunochemical Staining
[0077] Tissue samples from amyloid patients were stained using
standard immunochemical techniques and a similar binding phenomenon
was observed. FIGS. 3A-3D show that anti-.kappa.1 binds to the
.kappa.1 amyloid and, surprisingly, that the anti-.kappa.4 reacts
with the .kappa.1 amyloid, suggesting an amyloid epitope that these
antibodies may recognize. Additionally, the anti-.kappa.4 reacts
with .lambda.-containing amyloid (not illustrated). This is an
example of cross-isotype reactivity. However, the results from the
ELISA and the immunohistochemistry were not always consistent. This
is likely due to the inherent difference in what you are looking
at, i.e., ELISA is a liquid phase binding assay using extracted
purified amyloid, whereas immunohistochemistry is performed on
fixed tissue sections on a slide.
[0078] Samples of hybridoma cells that secrete anti-.kappa.1
(57-18-H12 (ATCC Acc. No. ______)), anti-.kappa.4 (11-1F4 (ATCC
Acc. No. ______)) and anti-.kappa.8 (31-8c7 (ATCC Acc. No. ______))
monoclonal antibodies were deposited with the American Type Culture
Collection (ATCC) on May 21, 1999 in compliance with the Budapest
Treaty.
EXAMPLE 5
In Vivo Studies of Anti-IgLC Subgroups
[0079] 0.1 mg of one of three antibodies--.kappa.1, .kappa.4, or
.lambda.8, identified above--was injected into the thigh of a mouse
into which amyloid had been introduced in the form of an amyloidoma
as described above. The .kappa.1 and .kappa.4 reagents resulted in
the complete removal by the host of most amyloid fibril species
tested within 7 days (as little as 4 days for certain sources of
amyloid). FIG. 4 shows fluoresceinated .kappa.4 mAb binding to
human amyloid.
[0080] The .lambda.8 reagent, which is reactive in certain
instances in both in vitro studies (above), increased the
resolution of amyloidomas by up to about 10% in in vivo
experiments.
EXAMPLE 6
In Vivo Studies of Anti-IgLC Subgroups
[0081] Human amyloid was isolated from a patient with
inflammation-associated, AA-amyloid and prepared for injection into
Balb/C mice by repeated sonication and grinding in order to permit
its injection into the mouse (see Example 1). Immediately after the
injection of 100 mg of human AA-amyloid extract, the mice were
treated with 100 .mu.g of .kappa.4 mAb, anti-AA mAb, no mAb and
non-specific control mAb (anti-free .kappa.). Complete resolution
of the material was observed with 48 hours in the animals that had
been treated with the .kappa.4 and anti-AA mAbs. In contrast, the
control animals had a large mass of amyloid remaining at the site
of injection.
EXAMPLE 7
Production of Specific Anti-Amyloid Fibril mAbs
[0082] Synthetic amyloid fibrils were prepared in vitro and used as
an immunogen in mice to produce a first generation of anti-amyloid
fibril mAbs. Briefly, recombinant .lambda.6-light chain, variable
region peptides were produced, isolated and purified using a
bacterial expression system and standard protein purification
techniques. Synthetic fibrils were prepared from these peptides by
extended periods of agitation in solution as described, for
example, in Wall et al., "In vitro Immunoglobulin Light Chain
Fibrillogenesis," METHODS IN ENZYMOLOGY, Vol. 309 (In Press), which
is incorporated herein by reference in its entirety. Fibrils were
concentrated by centrifugation at 17,000.times.g for 20 minutes at
room temperature.
[0083] The concentrated fibrils were then used to immunize Balb/c
mice over a period of several weeks. Monoclonal cell lines
secreting anti-fibril antibodies were produced using standard
hybridoma techniques. The resultant antibodies have demonstrable
anti-fibril activity based upon ELISA assays, described in Example
3. These antibodies reacted with 99% of all human IgLC amyloid
extracts tested to date irrespective of the nature of the isotype
or subgroup of the precursor protein when tested by ELISA.
Similarly, the antibodies reacted in an ELISA format with isolated
murine AA-amyloid and synthetic fibrils composed of a peptide
derived from the Alzheimer's protein A.beta. [A.beta.(25-35)].
[0084] It should be understood that the foregoing discussion and
examples merely present a detailed description of certain preferred
embodiments. It therefore should be apparent to those of ordinary
skill in the art that various modifications and equivalents can be
made without departing from the spirit and scope of the invention.
All references, articles and patents identified above are herein
incorporated by reference in their entirety.
* * * * *