U.S. patent application number 11/534124 was filed with the patent office on 2007-01-25 for immunotherapy of autoimmune disorders using antibodies which target b-cells.
This patent application is currently assigned to IMMUNOMEDICS, INC.. Invention is credited to David M. Goldenberg, Hans J. Hansen.
Application Number | 20070020265 11/534124 |
Document ID | / |
Family ID | 22481331 |
Filed Date | 2007-01-25 |
United States Patent
Application |
20070020265 |
Kind Code |
A1 |
Goldenberg; David M. ; et
al. |
January 25, 2007 |
IMMUNOTHERAPY OF AUTOIMMUNE DISORDERS USING ANTIBODIES WHICH TARGET
B-CELLS
Abstract
Antibodies that bind with a B-cell antigen provide an effective
means to treat autoinmune disorders. Antibodies and fragments,
which may be conjugated or naked, are used alone or in multimodal
therapies. The antibodies may be bispecific antibodies which may be
produced recombinantly as fusion proteins, or as hybrid,
polyspecific antibodies.
Inventors: |
Goldenberg; David M.;
(Mendham, NJ) ; Hansen; Hans J.; (Picayune,
MS) |
Correspondence
Address: |
ROSSI, KIMMS & McDOWELL LLP.
P.O. BOX 826
ASHBURN
VA
20146-0826
US
|
Assignee: |
IMMUNOMEDICS, INC.
300 American Road
Morris Plains
NJ
|
Family ID: |
22481331 |
Appl. No.: |
11/534124 |
Filed: |
September 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11104594 |
Apr 13, 2005 |
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11534124 |
Sep 21, 2006 |
|
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09590284 |
Jun 9, 2000 |
7074403 |
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11534124 |
Sep 21, 2006 |
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60138284 |
Jun 9, 1999 |
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Current U.S.
Class: |
424/144.1 ;
424/145.1 |
Current CPC
Class: |
A61P 7/06 20180101; C07K
2317/24 20130101; A61P 37/02 20180101; A61P 7/04 20180101; A61P
13/12 20180101; A61P 25/00 20180101; A61P 19/08 20180101; C07K
16/2896 20130101; A61P 11/00 20180101; A61P 1/04 20180101; Y10S
514/903 20130101; A61P 21/00 20180101; A61K 39/3955 20130101; A61P
21/04 20180101; A61P 3/10 20180101; A61K 47/6813 20170801; A61P
9/00 20180101; C07K 16/28 20130101; A61K 45/06 20130101; C07K
16/2803 20130101; A61K 38/00 20130101; Y10S 514/885 20130101; A61K
47/6849 20170801; A61P 25/28 20180101; A61P 1/16 20180101; A61P
35/00 20180101; A61P 31/12 20180101; A61P 5/14 20180101; A61K
2039/507 20130101; A61K 2039/505 20130101; A61P 37/00 20180101;
A61P 37/06 20180101; C07K 16/2887 20130101; A61P 19/02 20180101;
A61P 29/00 20180101; A61P 19/00 20180101 |
Class at
Publication: |
424/144.1 ;
424/145.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1. A method of treating an autoimmune disease in a subject
comprising administering an anti-CD20 antibody and an
anti-TNF.alpha. antagonist or anti-IL-1 antagonist.
2. A method according to claim 1, wherein said anti-TNF.alpha. or
anti-IL-1 antagonist is administered before said anti-CD20
antibody.
3. A method according to claim 1, wherein said anti-TNF.alpha. or
anti-IL-1 antagonist is administered after said anti-CD20
antibody.
4. A method according to claim 1, wherein said anti-TNF.alpha. or
anti-IL-1 antagonist is administered concurrently with said
anti-CD20 antibody.
5. A method according to claim 1, comprising administering an
anti-CD20 antibody and an anti-TNF.alpha. antagonist.
6. A method according to claim 5, wherein said anti-TNF.alpha.
antagonist is administered before said anti-CD20 antibody.
7. A method according to claim 5, wherein said anti-TNF.alpha.
antagonist is administered after said anti-CD20 antibody.
8. A method according to claim 5, wherein said anti-TNF.alpha.
antagonist is administered concurrently with said anti-CD20
antibody.
9. A method according to claim 1, comprising administering an
anti-CD20 antibody and an anti-IL-1 antagonist.
10. A method according to claim 5, wherein said anti-IL-1
antagonist is administered before said anti-CD20 antibody.
11. A method according to claim 5, wherein said anti-IL-1
antagonist is administered after said anti-CD20 antibody.
12. A method according to claim 5, wherein said anti-IL-1
antagonist is administered concurrently with said anti-CD20
antibody.
13. A method according to claim 1, wherein said anti-TNF.alpha.
antagonist is an anti-TNF.alpha. antibody and said anti-IL-1
antagonist is an anti-IL-1 antagonist is an anti-IL-1 antibody.
14. A method according to claim 1, comprising administering an
anti-CD20 antibody and an anti-TNF.alpha. antibody.
15. A method according to claim 1, comprising administering an
anti-CD20 antibody and an anti-IL-1 antibody.
16. A method according to claim 1, wherein said autoimmune disease
is rheumatoid arthritis.
17. A method according to claim 14, wherein said autoimmune disease
is rheumatoid arthritis.
18. A method according to claim 15, wherein said autoimmune disease
is rheumatoid arthritis.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/104,594, filed Apr. 13, 2005, which is a continuation
of U.S. patent application Ser. No. 09/590,284, filed Jun. 9, 2000,
now U.S. Pat. No. 7,074,403, which claims priority to U.S.
Provisional Application No. 60/138,284, filed Jun. 9, 1999.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to immunotherapeutic methods
for treating autoimmune disorders. In particular, this invention is
directed to methods for treating autoimmune disorders by
administering antibodies that bind to a B-cell antigen, such as the
CD22, CD20, CD19, and CD74 or HLA-DR antigen. The antibodies are
administered alone or in combination, and may be naked or
conjugated to a drug, toxin or therapeutic radioisotope. Bispecific
antibody fusion proteins which bind to the B-cell antigens can be
used according to the present invention, including hybrid
antibodies which bind to more than one B-cell antigen. The present
invention also is directed to multimodal therapeutic methods in
which the antibody administration is supplemented by administration
of other therapeutic modalities.
[0004] 2. Background
[0005] Antibodies against the CD20 antigen have been investigated
for the therapy of B-cell lymphomas. For example, a chimeric
anti-CD20 antibody, designated as "IDEC-C2B8," has activity against
B-cell lymphomas when provided as unconjugated antibodies at
repeated injections of doses exceeding 500 mg per injection.
Maloney et al., Blood 84:2457 (1994); Longo, Curr. Opin. Oncol.
8:353 (1996). About 50 percent of non-Hodgkin's patients, having
the low-grade indolent form, treated with this regimen showed
responses. Therapeutic responses have also been obtained using
.sup.131I-labeled B1 anti-CD-20 murine monoclonal antibody when
provided as repeated doses exceeding 600 mg per injection. Kaminski
et al., N. Engl. J. Med. 329:459 (1993); Press et al., N. Engl. J.
Med. 329:1219 (1993); Press et al., Lancet 346:336 (1995). However,
these antibodies, whether provided as unconjugated forms or
radiolabeled forms, have shown only modest activity in patients
with the more prevalent and lethal form of B-cell lymphoma, the
intermediate or aggressive type.
[0006] Autoimmune diseases are a class of diseases associated with
a B-cell disorder. Examples include immune-mediated
thrombocytopenias, such as acute idiopathic thrombocytopenic
purpura and chronic idiopathic thrombocytopenic purpura, myasthenia
gravis, lupus nephritis, lupus erythematosus, and rheumatoid
arthritis. The most common treatments are corticosteroids and
cytotoxic drugs, which can be very toxic. These drugs also suppress
the entire immune system, can result in serious infection, and have
adverse affects on the liver and kidneys. Other therapeutics that
have been used to treat Class III autoimmune diseases to date have
been directed against T-cells and macrophages. A need remains for
more effective methods of treating autoimmune diseases,
particularly Class III autoimmune diseases.
SUMMARY OF THE INVENTION
[0007] Accordingly, it is an object of the present invention to
provide a method for treating autoimmune diseases using antibody to
a B-cell antigen.
[0008] It is another object of the invention is to use
comparatively low doses of a naked antibody to a B-cell antigen,
preferably to CD22 antigen, or a combination of naked antibodies to
a CD22 antigen and another B-cell antigen, preferably CD20 and/or
CD74.
[0009] Yet another object of the invention is to use a combination
of one or more naked antibodies to B-cell antigens and/or
antibodies to B-cell antigens which are conjugated to drugs, toxins
or therapeutic radioisotopes.
[0010] It is a further object of this invention to provide
multimodal methods for treatment of autoimmune diseases in which a
naked or conjugated antibody to a B-cell antigen is supplemented
with the administration of other therapeutic modalities, such as
those directed against T-cells, plasma cells and macrophages.
[0011] These and other objects are achieved, in accordance with one
embodiment of the present invention, by the provision of a method
of treating an autoimmune disease, comprising the step of
administering to a subject having an autoimmune disease an antibody
to a B-cell antigen and a pharmaceutically acceptable carrier.
[0012] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
DETAILED DESCRIPTION
1. Overview
[0013] B-cell clones that bear autoantibody Ig-receptors are
present in normal individuals. Autoimmunity results when these
B-cells become overactive, and mature to plasma cells that secrete
autoantibody. In accordance with the present invention, autoimmune
disorders can be treated by administering an antibody that binds to
a B-cell antigen, such as the CD22, CD20, CD19, and CD74 or HLA-DR
antigen. In one embodiment, comparatively low doses of an entire,
naked antibody or combination of entire, naked antibodies are used.
In other embodiments, conjugates of such antibodies with drugs,
toxins or therapeutic radioisotopes are useful. Bispecific antibody
fusion proteins which bind to the B-cell antigens can be used
according to the present invention, including hybrid antibodies
which bind to more than one B-cell antigen. Preferably the
bispecific and hybrid antibodies additionally target a T-cell,
plasma cell or macrophage antigen. The present invention also is
directed to multimodal therapeutic methods in which the antibody
administration is supplemented by administration of other
therapeutic modalities.
2. Definitions
[0014] In the description that follows, and in documents
incorporated by reference, a number of terms are used extensively.
The following definitions are provided to facilitate understanding
of the invention.
[0015] A structural gene is a DNA sequence that is transcribed into
messenger RNA (mRNA) which is then translated into a sequence of
amino acids characteristic of a specific polypeptide.
[0016] A promoter is a DNA sequence that directs the transcription
of a structural gene. Typically, a promoter is located in the 5'
region of a gene, proximal to the transcriptional start site of a
structural gene. If a promoter is an inducible promoter, then the
rate of transcription increases in response to an inducing agent.
In contrast, the rate of transcription is not regulated by an
inducing agent when the promoter is a constitutive promoter.
[0017] An isolated DNA molecule is a fragment of DNA that is not
integrated in the genomic DNA of an organism. For example, a cloned
antibody gene is a DNA fragment that has been separated from the
genomic DNA of a mammalian cell. Another example of an isolated DNA
molecule is a chemically synthesized DNA molecule that is not
integrated in the genomic DNA of an organism.
[0018] An enhancer is a DNA regulatory element that can increase
the efficiency of transcription, regardless of the distance or
orientation of the enhancer relative to the start site of
transcription.
[0019] Complementary DNA (cDNA) is a single-stranded DNA molecule
that is formed from a mRNA template by the enzyme reverse
transcriptase. Typically, a primer complementary to portions of
mRNA is employed for the initiation of reverse transcription. Those
skilled in the art also use the term "cDNA" to refer to a
double-stranded DNA molecule consisting of such a single-stranded
DNA molecule and its complementary DNA strand.
[0020] The term expression refers to the biosynthesis of a gene
product. For example, in the case of a structural gene, expression
involves transcription of the structural gene into mRNA and the
translation of mRNA into one or more polypeptides.
[0021] A cloning vector is a DNA molecule, such as a plasmid,
cosmid, or bacteriophage that has the capability of replicating
autonomously in a host cell. Cloning vectors typically contain one
or a small number of restriction endonuclease recognition sites at
which foreign DNA sequences can be inserted in a determinable
fashion without loss of an essential biological function of the
vector, as well as a marker gene that is suitable for use in the
identification and selection of cells transformed with the cloning
vector. Marker genes typically include genes that provide
tetracycline resistance or ampicillin resistance.
[0022] An expression vector is a DNA molecule comprising a gene
that is expressed in a host cell. Typically, gene expression is
placed under the control of certain regulatory elements, including
constitutive or inducible promoters, tissue-specific regulatory
elements, and enhancers. Such a gene is said to be "operably linked
to" the regulatory elements.
[0023] A recombinant host may be any prokaryotic or eukaryotic cell
that contains either a cloning vector or expression vector. This
term also includes those prokaryotic or eukaryotic cells that have
been genetically engineered to contain the cloned gene(s) in the
chromosome or genome of the host cell.
[0024] As used herein, antibody encompasses naked antibodies and
conjugated antibodies and antibody fragments, which may be
monospecific or multispecific. It includes both polyclonal and
monoclonal antibodies, as well as certain recombinant antibodies,
such as chimeric and humanized antibodies and fusion proteins.
[0025] A chimeric antibody is a recombinant protein that contains
the variable domains and complementary determining regions derived
from a rodent antibody, while the remainder of the antibody
molecule is derived from a human antibody.
[0026] Humanized antibodies are recombinant proteins in which
murine complementarity determining regions of a monoclonal antibody
have been transferred from heavy and light variable chains of the
murine immunoglobulin into a human variable domain.
[0027] Human antibodies are antibodies that either are isolated
from humans and then grown out in culture or are made using animals
whose immune systems have been altered so that they respond to
antigen stimulation by producing human antibodies.
[0028] As used herein, a therapeutic agent is a molecule or atom,
which is conjugated to an antibody moiety to produce a conjugate
which is useful for therapy. Examples of therapeutic agents include
drugs, toxins, enzymes, hormones, cytokines, immunomodulators,
boron compounds and therapeutic radioisotopes. Preferred
therapeutic radioisotopes include beta, alpha, and Auger emitters,
with a kev range of 80-500 kev. Exemplary therapeutic radioisotopes
include .sup.198Au, .sup.32P, .sup.125I, .sup.131I, .sup.90Y,
.sup.186Re, .sup.188Re, .sup.67Cu, and .sup.211At.
[0029] A naked antibody is an entire antibody which is not
conjugated with a therapeutic agent. Naked antibodies include both
polyclonal and monoclonal antibodies, as well as certain
recombinant antibodies, such as chimeric and humanized
antibodies.
[0030] A conjugated antibody is an antibody or antibody fragment
that is conjugated to a therapeutic agent.
[0031] A multispecific antibody is an antibody which can bind
simultaneously to at least two targets which are of different
structure, e.g., two different antigens, two different epitopes on
the same antigen, or a hapten and/or an antigen or epitope. One
specificity would be for a B-cell antigen or epitope.
[0032] A bispecific antibody is an antibody which can bind
simultaneously to two targets which are of different structure.
Bispecific antibodies (bsAb) and bispecific antibody fragments
(bsFab) have at least one arm that specifically binds to a B-cell
antigen or epitope and at least one other arm that specifically
binds a targetable conjugate.
[0033] A fusion protein is a recombinantly produced antigen-binding
molecule in which two or more different single-chain antibody or
antibody fragment segments with the same or different specificities
are linked. A variety of bispecific fusion proteins can be produced
using molecular engineering. In one form, the bispecific fusion
protein is monovalent, consisting of, for example, a scFv with a
single binding site for one antigen and a Fab fragment with a
single binding site for a second antigen. In another form, the
bispecific fusion protein is divalent, consisting of, for example,
an IgG with two binding sites for one antigen and two scFv with two
binding sites for a second antigen.
3. Production of Monoclonal Antibodies, Humanized Antibodies,
Primate Antibodies and Human Antibodies
[0034] Anti-CD20, anti-CD22, anti-CD19, and anti-CD74 antibodies
are known generally to those of skill in the art. See, for example,
Ghetie et al., Cancer Res. 48:2610 (1988); Hekman et al., Cancer
Immunol. Immunother. 32:364 (1991); Kaminski et al., N. Engl. J.
Med. 329:459 (1993); Press et al., N. Engl. J. Med. 329:1219
(1993); Maloney et al., Blood 84:2457 (1994); Press et al., Lancet
346:336 (1995); Longo, Curr. Opin. Oncol. 8:353 (1996). More
particularly, rodent monoclonal antibodies to CD22, CD20, CD19, or
CD74 antigens can be obtained by methods known to those skilled in
the art. See generally, for example, Kohler and Milstein, Nature
256:495 (1975), and Coligan et al. (eds.), CURRENT PROTOCOLS IN
IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991)
["Coligan"]. Briefly, monoclonal antibodies can be obtained by
injecting mice with a composition comprising the antigen, verifying
the presence of antibody production by removing a serum sample,
removing the spleen to obtain B-lymphocytes, fusing the
B-lymphocytes with myeloma cells to produce hybridomas, cloning the
hybridomas, selecting positive clones which produce antibodies to
the antigen that was injected, culturing the clones that produce
antibodies to the antigen, and isolating the antibodies from the
hybridoma cultures.
[0035] Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography. See, for example, Coligan at pages
2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et al.,
"Purification of Immunoglobulin G (IgG)," in METHODS IN MOLECULAR
BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).
[0036] Suitable amounts of well-characterized antigen for
production of antibodies can be obtained using standard techniques.
As an example, CD22 can be immunoprecipitated from B-lymphocyte
protein using the deposited antibodies described by Tedder et al.,
U.S. Pat. No. 5,484,892 (1996).
[0037] Alternatively, CD22, CD20, CD19, or CD74 antigen proteins
can be obtained from transfected cultured cells that overproduce
the antigen of interest. Expression vectors that comprise DNA
molecules encoding each of these proteins can be constructed using
published nucleotide sequences. See, for example, Wilson et al., J.
Exp. Med. 173:137 (1991); Wilson et al., J. Immunol. 150:5013
(1993). As an illustration, DNA molecules encoding CD22 can be
obtained by synthesizing DNA molecules using mutually priming long
oligonucleotides. See, for example, Ausubel et al., (eds.), CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, pages 8.2.8 to 8.2.13 (1990)
["Ausubel"]. Also, see Wosnick et al., Gene 60:115 (1987); and
Ausubel et al. (eds.), SHORT PROTOCOLS IN MOLECULAR BIOLOGY, 3rd
Edition, pages 8-8 to 8-9 (John Wiley & Sons, Inc. 1995).
Established techniques using the polymerase chain reaction provide
the ability to synthesize genes as large as 1.8 kilobases in
length. Adang et al., Plant Molec. Biol. 21:1131 (1993); Bambot et
al., PCR Methods and Applications 2:266 (1993); Dillon et al., "Use
of the Polymerase Chain Reaction for the Rapid Construction of
Synthetic Genes," in METHODS IN MOLECULAR BIOLOGY, Vol. 15: PCR
PROTOCOLS: CURRENT METHODS AND APPLICATIONS, White (ed.), pages
263-268, (Humana Press, Inc. 1993).
[0038] In a variation of this approach, monoclonal antibody can be
obtained by fusing myeloma cells with spleen cells from mice
immunize with a murine pre-B cell line stably transfected with cDNA
which encodes the antigen of interest. See Tedder et al., U.S. Pat.
No. 5,484,892 (1996).
[0039] One example of a suitable murine anti-CD22 monoclonal
antibody is the LL2 (formerly EPB-2) monoclonal antibody, which was
produced against human Raji cells derived from a Burkitt lymphoma.
Pawlak-Byczkowska et al., Cancer Res. 49:4568 (1989). This
monoclonal antibody has an IgG.sub.2.alpha. isotype, and the
antibody is rapidly internalized into lymphoma cells. Shih et at,
Int. J. Cancer 56:538 (1994). Immunostaining and in vivo
radioimmunodetection studies have demonstrated the excellent
sensitivity of LL2 in detecting B-cell lymphomas. Pawlak-Byczkowska
et al., Cancer Res. 49:4568 (1989); Murthy et al., Eur. J. Nucl.
Med. 19:394 (1992). Moreover, .sup.99mTc-labeled LL2-Fab' fragments
have been shown to be useful in following upstaging of B-cell
lymphomas, while .sup.131I-labeled intact LL2 and labeled LL2
F(ab').sub.2 fragments have been used to target lymphoma sites and
to induce therapeutic responses. Murthy et al., Eur. J. Nucl. Med.
19:394, (1992); Mills et al., Proc. Am. Assoc. Cancer Res. 34:479
(1993) [Abstract 2857]; Baum et al., Cancer 73 (Suppl. 3):896
(1994); Goldenberg et al., J. Clin. Oncol. 9:548 (1991).
Furthermore, Fab' LL2 fragments conjugated with a derivative of
Pseudomonas exotoxin has been shown to induce complete remissions
for measurable human lymphoma xenografts growing in nude mice.
Kreitman et al., Cancer Res. 53:819 (1993). An example of an
anti-CD74 antibody is the LL1 antibody.
[0040] In an additional embodiment, an antibody of the present
invention is a chimeric antibody in which the variable regions of a
human antibody have been replaced by the variable regions of a
rodent anti-CD22 antibody. The advantages of chimeric antibodies
include decreased immunogenicity and increased in vivo
stability.
[0041] Techniques for constructing chimeric antibodies are well
known to those of skill in the art. As an example, Leung et al.,
Hybridoma 13:469 (1994), describe how they produced an LL2 chimera
by combining DNA sequences encoding the V.sub..kappa. and V.sub.H
domains of LL2 monoclonal antibody with respective human .kappa.
and IgG, constant region domains. This publication also provides
the nucleotide sequences of the LL2 light and heavy chain variable
regions, V.sub..eta. and V.sub.H, respectively.
[0042] In another embodiment, an antibody of the present invention
is a subhuman primate antibody. General techniques for raising
therapeutically useful antibodies in baboons may be found, for
example, in Goldenberg et al., international patent publication No.
WO 91/11465 (1991), and in Losman et al., Int. J. Cancer 46: 310
(1990).
[0043] In yet another embodiment, an antibody of the present
invention is a "humanized" monoclonal antibody. That is, mouse
complementarity determining regions are transferred from heavy and
light variable chains of the mouse immunoglobulin into a human
variable domain, followed by the replacement of some human residues
in the framework regions of their murine counterparts. Humanized
monoclonal antibodies in accordance with this invention are
suitable for use in therapeutic methods. General techniques for
cloning murine immunoglobulin variable domains are described, for
example, by the publication of Orlandi et al., Proc. Nat'l Acad.
Sci. USA 86: 3833 (1989). Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al.,
Nature 321:522 (1986), Riechmann et al., Nature 332:323 (1988),
Verhoeyen et al., Science 239:1534 (1988), Carter et al., Proc.
Nat'l Acad. Sci. USA 89:4285 (1992), Sandhu, Crit. Rev. Biotech.
12:437 (1992), and Singer et al., J. Immun. 150:2844 (1993). The
publication of Leung et al., Mol. Immunol. 32:1413 (1995),
describes the construction of humanized LL2 antibody.
[0044] In another embodiment, an antibody of the present invention
is a human monoclonal antibody. Such antibodies are obtained from
transgenic mice that have been "engineered". to produce specific
human antibodies in response to antigenic challenge. In this
technique, elements of the human heavy and light chain locus are
introduced into strains of mice derived from embryonic stem cell
lines that contain targeted disruptions of the endogenous heavy
chain and light chain loci. The transgenic mice can synthesize
human antibodies specific for human antigens, and the mice can be
used to produce human antibody-secreting hybridomas. Methods for
obtaining human antibodies from transgenic mice are described by
Green et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature
368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994).
4. Production of Bispecific Antibodies
[0045] The present invention also may employ a bispecific antibody
(bsAb) or antibody fragment (bsFab) having at least one arm that
specifically binds to a B-cell antigen and at least one other arm
that specifically binds a targetable conjugate. The targetable
conjugate comprises a carrier portion which comprises or bears at
least one epitope recognized by at least one arm of the bispecific
antibody or antibody fragment. In a preferred embodiment, the
epitope is a hapten. In an alternative embodiment, the epitope is a
part of the carrier. Examples of recognizable haptens include, but
are not limited to, chelators, such as DTPA, fluorescein
isothiocyanate, vitamin B-12 and other moieties to which specific
antibodies can be raised. The carrier portion also may be
conjugated to a variety of agents. Examples of conjugated agents
include, but are not limited to, metal chelate complexes, drugs,
toxins and other effector molecules, such as cytokines,
lymphokines, chemokines, immunomodulators, radiosensitizers,
asparaginase, carboranes and radioactive halogens. Additionally,
enzymes useful for activating a prodrug or increasing the
target-specific toxicity of a drug can be conjugated to the
carrier. Thus, the use of bispecific antibodies and fragments which
have at least one arm that specifically binds a targetable
conjugate allows a variety of therapeutic and diagnostic
applications to be performed without raising new bsAb for each
application.
[0046] The present invention encompasses antibodies and antibody
fragments. The antibody fragments are antigen binding portions of
an antibody, such as F(ab')2, F(ab)2, Fab', Fab, and the like. The
antibody fragments bind to the same antigen that is recognized by
the intact antibody. For example, an anti-CD22 monoclonal antibody
fragment binds to an epitope of CD22. The bsAb of the present
invention include, but are not limited to, IgG.times.IgG,
IgG.times.F(ab')2, IgG.times.Fab', IgG.times.scFv,
F(ab')2.times.F(ab')2, Fab'.times.F(ab')2, Fab'.times.Fab',
Fab'.times.scFv and scFv.times.scFv bsmabs. Also, species such as
scFv.times.IgG.times.scFv and Fab'.times.IgG.times.Fab',
scFv.times.F(ab')2.times.scFv and Fab'.times.F(ab')2.times.Fab' are
included.
[0047] The term "antibody fragment" also includes any synthetic or
genetically engineered protein that acts like an antibody by
binding to a specific antigen to form a complex. For example,
antibody fragments include isolated fragments, "Fv" fragments,
consisting of the variable regions of the heavy and light chains,
recombinant single chain polypeptide molecules in which light and
heavy chain variable regions are connected by a peptide linker
("sFv proteins"), and minimal recognition units consisting of the
amino acid residues that mimic the hypervariable region.
5. Production of Fusion Proteins
[0048] Another method for producing bsAbs is to engineer
recombinant fusion proteins linking two or more different
single-chain antibody or antibody fragment segments with the needed
dual specificities. See, e.g., Coloma et al., Nature Biotech.
15:159-163, 1997. A variety of bispecific fusion proteins can be
produced using molecular engineering. In one form, the bispecific
fusion protein is monovalent, consisting of, for example, a scFv
with a single binding site for one antigen and a Fab fragment with
a single binding site for a second antigen. In another form, the
bispecific fusion protein is divalent, consisting of, for example,
an IgG with two binding sites for one antigen and two scFv with two
binding sites for a second antigen.
[0049] Functional bispecific single-chain antibodies (bscAb), also
called diabodies, can be produced in mammalian cells using
recombinant methods. See, e.g., Mack et al., Proc. Natl. Acad.
Sci., 92: 7021-7025, 1995. For example, bscAb are produced by
joining two single-chain Fv fragments via a glycine-serine linker
using recombinant methods. The V light-chain (VL) and V heavy-chain
(VH) domains of two antibodies of interest are isolated using
standard PCR methods. The VL and VH cDNA's obtained from each
hybridoma are then joined to form a single-chain fragment in a
two-step fusion PCR. The first PCR step introduces the (Gly4-Ser1)3
linker, and the second step joins the VL and VH amplicons. Each
single chain molecule is then cloned into a bacterial expression
vector. Following amplification, one of the single-chain molecules
is excised and sub-cloned into the other vector, containing the
second single-chain molecule of interest. The resulting bscAb
fragment is subcloned into an eukaryotic expression vector.
Functional protein expression can be obtained by transfecting the
vector into chinese hamster ovary cells. Recombinant methods can be
used to produce a variety of fusion proteins.
6. Coupling of Antibodies to Lipid Emulsions
[0050] Long-circulating sub-micron lipid emulsions, stabilized with
poly(ethylene glycol)-modified phosphatidylethanolamine (PEG-PE),
can be used as drug carriers for the antibodies of the present
invention. The emulsions are composed of two major parts: an oil
core, e.g., triglyceride, stabilized by emulsifiers, e.g.,
phospholipids. The poor emulsifying properties of phospholipids can
be enhanced by adding a biocompatible co-emulsifier such as
polysorbate 80. In a preferred embodiment, the antibody is
conjugated to the surface of the lipid emulsion globules with a
poly(ethylene glycol)-based, heterobifunctional coupling agent,
poly(ethylene glycol)-vinylsulfone-N-hydroxy-succinimidyl ester
(NHS-PEG-VS).
[0051] The submicron lipid emulsion is prepared and characterized
as described. Lundberg, J. Pharm. Sci., 83:72 (1993); Lundberg et
al., Int. J. Pharm., 134:119 (1996). The basic composition of the
lipid emulsion is triolein:DPPC:polysorbate 80, 2:1:0.4 (w/w). When
indicated, PEG-DPPE is added into the lipid mixture at an amount of
2-8 mol % calculated on DPPC.
[0052] The coupling procedure starts with the reaction of the NHS
ester group of NHS-PEG-VS with the amino group of distearoyl
phosphatidyl-ethanolamine (DSPE). Twenty-five .mu.mol of NHS-PEG-VS
are reacted with 23 .mu.mol of DSPE and 50 .mu.mol triethylamine in
1 ml of chloroform for 6 hours at 40.degree. C. to produce a
poly(ethylene glycol) derivative of phosphatidyl-ethanolamide with
a vinylsulfone group at the distal terminus of the poly(ethylene
glycol) chain (DSPE-PEG-VS). For antibody conjugation, DSPE-PEG-VS
is included in the lipid emulsion at 2 mol % of DPPC. The
components are dispersed into vials from stock solutions at
-20.degree. C., the solvent is evaporated to dryness under reduced
pressure. Phosphate-buffered saline (PBS) is added, the mixture is
heated to 50.degree. C., vortexed for 30 seconds and sonicated with
a MSE probe sonicator for 1 minute. Emulsions can be stored at
4.degree. C., and preferably are used for conjugation within 24
hours.
[0053] Coupling of antibodies to emulsion globules is performed via
a reaction between the vinylsulfone group at the distal PEG
terminus on the surface of the globules and free thiol groups on
the antibody. Vinylsulfone is an attractive derivative for
selective coupling to thiol groups. At approximately neutral pH, VS
will couple with a half life of 15-20 minutes to proteins
containing thiol groups. The reactivity of VS is slightly less than
that of maleimide, but the VS group is more stable in water and a
stable linkage is produced from reaction with thiol groups.
[0054] Before conjugation, the antibody is reduced by 50 mM
2-mercaptoethanol for 10 minutes at 4.degree. C. in 0.2 M Tris
buffer (pH 8.7). The reduced antibody is separated from excess
2-mercaptoethanol with a Sephadex G-25 spin column, equilibrated in
50 mM sodium acetate buffered 0.9% saline (pH 5.3). The product is
assayed for protein concentration by measuring its absorbance at
280 mm (and assuming that a 1 mg/ml antibody solution of 1.4) or by
quantitation of .sup.125I-labeled antibody. Thiol groups are
determined with Aldrithiol.TM. following the change in absorbance
at 343 mm and with cystein as standard.
[0055] The coupling reaction is performed in HEPES-buffered saline
(pH 7.4) overnight at ambient temperature under argon. Excess
vinylsulfone groups are quenched with 2 mM 2-mercaptoethanol for 30
minutes, excess 2-mercaptoethanol and antibody are removed by gel
chromatography on a Sepharose CL-48 column. The immunoconjugates
are collected near the void volume of the column, sterilized by
passage through a 0.45 .mu.m sterile filter, and stored at
4.degree. C.
[0056] Coupling efficiency is calculated using .sup.125I-labeled
antibody. Recovery of emulsions is estimated from measurements of
[.sup.14C]DPPC in parallel experiments. The conjugation of reduced
LL2 to the VS group of surface-grafted DSPE-PEG-VS is very
reproducible with a typical efficiency of near 85%.
7. Therapeutic Use of Antibodies in Simple and Multimodal
Regimens
[0057] The present invention contemplates the use of naked and/or
conjugated antibodies as the primary therapeutic composition for
treatment of autoimmune diseases. Such a composition can contain
polyclonal antibodies or monoclonal antibodies. Preferred
antibodies are anti-CD22 antibodies, such as LL2 antibodies,
including murine LL2 monoclonal antibody, chimeric LL2 antibody,
and humanized LL2 antibody. Antibodies to a single B-cell antigen
or to more than one B-cell antigen may be used. In a preferred
embodiment, bispecific antibodies and fusion proteins which
comprise specificities for more than one B-ell antigen or epitope
are employed.
[0058] For example, a therapeutic composition of the present
invention can contain a mixture of monoclonal naked anti-CD22
antibodies directed to different, non-blocking CD22 epitopes.
Monoclonal antibody cross-inhibition studies have identified five
epitopes on CD22, designated as epitopes A-E. See, for example,
Schwartz-Albiez et al., "The Carbohydrate Moiety of the CD22
Antigen Can Be Modulated by Inhibitors of the Glycosylation
Pathway," in LEUKOCYTE TYPING IV. WHITE CELL DIFFERENTIATION
ANTIGENS, Knapp et al. (eds.), p. 65 (Oxford University Press
1989). As an illustration, the LL2 antibody binds with epitope B.
Stein et al., Cancer Immunol. Immunother. 37:293 (1993).
Accordingly, the present invention contemplates therapeutic
compositions comprising a mixture of monoclonal anti-CD22
antibodies that bind at least two CD22 epitopes. For example, such
a mixture can contain monoclonal antibodies that bind with at least
two CD22 epitopes selected from the group consisting of epitope A,
epitope B, epitope C, epitope D and epitope E.
[0059] Methods for determining the binding specificity of an
anti-CD22 antibody are well-known to those of skill in the art.
General methods are provided, for example, by Mole, "Epitope
Mapping," in METHODS IN MOLECULAR BIOLOGY, VOLUME 10:
IMMUNOCHEMICAL PROTOCOLS, Manson (ed.), pages 105-116 (The Humana
Press, Inc. 1992). More specifically, competitive blocking assays
to determine CD22 epitope specificity are described by Stein et
al., Cancer Immunol. Immunother. 37:293 (1993), and by Tedder et
al., U.S. Pat. No. 5,484,892 (1996).
[0060] The Tedder patent also describes the production of CD22
mutants, which lack one or more immunoglobulin-like domains. These
mutant proteins were used to determine that immunoglobulin-like
domains 1, 2, 3, and 4 correspond with epitopes A, D, B, and C,
respectively. Thus, binding a test antibody with a panel of CD22
proteins lacking particular immunoglobulin-like domain can also
identify CD22 epitope specificity.
[0061] The therapeutic compositions described herein are useful for
treatment of autoimmune diseases, particularly for the treatment of
Class III autoimmune diseases including immune-mediated
thrombocytopenias, such as acute idiopathic thrombocytopenic
purpura and chronic idiopathic thrombocytopenic purpura,
dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic
lupus erythematosus, lupus nephritis, rheumatic fever,
polyglandular syndromes, bullous pemphigoid, diabetes mellitus,
Henoch-Schonlein purpura, post-streptococcal nephritis, erythema
nodosum, Takayasu's arteritis, Addison's disease, rheumatoid
arthritis multiple sclerosis, sarcoidosis, ulcerative colitis,
erythema multiforme, IgA nephropathy, polyarteritis nodosa,
ankylosing spondylitis, Goodpasture's syndrome, thromboangitis
ubiterans, Sjogren's syndrome, primary biliary cirrhosis,
Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic
active hepatitis, polymyositis/dermatomyositis, polychondritis,
pamphigus vulgaris, Wegener's granulomatosis, membranous
nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant
cell arteritis/polymyalgia, pernicious anemia, rapidly progressive
glomerulonephritis and fibrosing alveolitis. In this context, the
therapeutic compositions are used to deplete the blood of normal
B-cells for an extended period.
[0062] Although naked, preferably anti-CD22, antibodies are the
preferred, primary therapeutic compositions for treatment of
autoimmune diseases, the efficacy of such naked antibody therapy
can be enhanced by supplementing the naked antibodies with other
therapies described herein. In such multimodal regimens, the
supplemental therapeutic compositions can be administered before,
concurrently or after administration of the naked, preferably
anti-CD22, antibodies. Multimodal therapy of Class III autoimmune
diseases may comprise co-administration of therapeutics that are
targeted against T-cells, plasma cells or macrophages, such as
antibodies directed against T-cell epitopes, more particularly
against the CD4 and CD5 epitopes. Gamma globulins also may be
co-administered. In some cases, it may be desirable to
co-administer immunosuppressive drugs such as corticosteroids and
possibly also cytotoxic drugs. In this case, lower doses of the
corticosteroids and cytotoxic drugs can be used as compared to the
doses used in conventional therapies, thereby reducing the negative
side effects of these therapeutics. The supplemental therapeutic
compositions can be administered before, concurrently or after
administration of the naked B-cell, preferably anti-CD22,
antibodies.
[0063] In an alternative embodiment, the antibodies to the CD22,
CD20, CD 19, and CD74 or HLA-DR antigen are conjugated to a drug,
toxin, enzyme, hormone, cytokine, immunomodulator, boron compound
or therapeutic radioisotope, or a fusion protein of an antibody and
a toxin may be used. These conjugates and fusion proteins may be
used alone, or in combination with naked B-cell antibodies. In a
further preferred embodiment, an antibody is used that comprises an
arm that is specific for a low-molecular weight hapten to which a
therapeutic agent is conjugated or fused. In this case, the
antibody pretargets the B-cells, and the low-molecular weight
hapten with the attached therapeutic agent is administered after
the antibody has bound to the B-cell targets. Examples of
recognizable haptens include, but are not limited to, chelators,
such as DTPA, fluorescein isothiocyanate, vitamin B-12 and other
moieties to which specific antibodies can be raised.
[0064] Drugs which are known to act on B-cells, plasma cells and/or
T-cells are particularly useful in accordance with the present
invention, whether conjugated to a B-cell antibody, or administered
as a separate component in combination with a naked or conjugated
B-cell antibody. These include methotrexate, phenyl butyrate,
bryostatin, cyclophosphamide, etoposide, bleomycin, doxorubicin,
carmustine, vincristine, procarbazine, dexamethasone, leucovorin,
prednisone, maytansinoids such as DM1, calicheamicin, rapamycin,
leflunomide, FK506, immuran, fludarabine, azathiopine,
mycophenolate, and cyclosporin. Drugs such as immuran,
methotrexate, and fludarabine which act on both B-cells and T-cells
are particularly preferred. Illustrative of toxins which are
suitably employed in accordance with the present invention are
ricin, abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A,
pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas
exotoxin, Pseudomonas endotoxin and RNAses, such as onconase. See,
for example, Pastan et al., Cell 47:641 (1986), and Goldenberg, C
A--A Cancer Journal for Clinicians 44:43 (1994). Other suitable
drugs and toxins are known to those of skill in the art.
[0065] Cytokine agonists and antagonists may also be used in
multimodal therapies according to the present invention. Tumor
necrosis factor alpha (TNF.alpha.) and interleukin-1 (IL-1) are
important in mediating inflammation in rheumatoid arthritis.
Accordingly, anti-TNF.alpha. reagents, such as Infiximab and
Etanercept (Embrel), are useful in multimodal therapy according to
the invention, as well as anti-IL-1 reagents.
[0066] Other useful secondary therapeutics useful in multimodal
therapies are IL-2 and GM-CSF, which may be conjugated with an
anti-B-cell antibody, or combined with a naked anti-B-cell antibody
as a separate component.
[0067] In general, the dosage of administered antibodies will vary
depending upon such factors as the patient's age, weight, height,
sex, general medical condition and previous medical history.
Typically, it is desirable to provide the recipient with a dosage
of antibody component, immunoconjugate or fusion protein which is
in the range of from about 1 pg/kg to 10 mg/kg (amount of
agent/body weight of patient), although a lower or higher dosage
also may be administered as circumstances dictate.
[0068] Administration of antibodies to a patient can be
intravenous, intraarterial, intraperitoneal, intramuscular,
subcutaneous, intrapleural, intrathecal, by perfusion through a
regional catheter, or by direct intralesional injection. When
administering therapeutic proteins by injection, the administration
may be by continuous infusion or by single or multiple boluses.
Intravenous injection provides a useful mode of administration due
to the thoroughness of the circulation in rapidly distributing
antibodies.
[0069] In preferred embodiments, naked anti-B-ell antibodies,
particularly anti-CD22 antibodies, are administered at low protein
doses, such as 20 milligrams to 2 grams protein per dose, given
once, or repeatedly, parenterally. Alternatively, naked antibodies
are administered in doses of 20 to 1000 milligrams protein per
dose, or 20 to 500 milligrams protein per dose, or 20 to 100
milligrams protein per dose.
[0070] The antibodies, alone or conjugated to liposomes, can be
formulated according to known methods to prepare pharmaceutically
useful compositions, whereby the therapeutic proteins are combined
in a mixture with a pharmaceutically acceptable carrier. A
composition is said to be a "pharmaceutically acceptable carrier"
if its administration can be tolerated by a recipient patient.
Sterile phosphate-buffered saline is one example of a
pharmaceutically acceptable carrier. Other suitable carriers are
well-known to those in the art. See, for example, REMINGTON'S
PHARMACEUTICAL SCIENCES, 19th Ed. (1995).
[0071] For purposes of therapy, antibodies are administered to a
patient in a therapeutically effective amount in a pharmaceutically
acceptable carrier. In this regard, a "therapeutically effective
amount" is one that is physiologically significant. An agent is
physiologically significant if its presence results in a detectable
change in the physiology of a recipient patient. In the present
context, an agent is physiologically significant if its presence
results in the inactivation or killing of targeted B-cells.
[0072] Additional pharmaceutical methods may be employed to control
the duration of action of an antibody in a therapeutic application.
Control release preparations can be prepared through the use of
polymers to complex or adsorb the antibody. For example,
biocompatible polymers include matrices of poly(ethylene-co-vinyl
acetate) and matrices of a polyanhydride copolymer of a stearic
acid dimer and sebacic acid. Sherwood et al., Bio/Technology
10:1446 (1992). The rate of release of an antibody from such a
matrix depends upon the molecular weight of the protein, the amount
of antibody within the matrix, and the size of dispersed particles.
Saltzman et al, Biophys. J. 55:163 (1989); Sherwood et al., supra.
Other solid dosage forms are described in REMINGTON'S
PHARMACEUTICAL SCIENCES, 19th ed. (1995).
[0073] The present invention, thus generally described, will be
understood more readily by reference to the following examples,
which are provided by way of illustration and are not intended to
be limiting of the present invention.
EXAMPLE 1
Treatment of a Patient with Humanized LL2
[0074] A patient undergoes therapy with humanized LL2 monoclonal
antibody. The patient was infused intravenously with 634 mg of
humanized LL2 antibody, and the treatment was repeated 6, 13, and
20 days following this initial treatment. Immediately following the
last dose, the serum value of hLL2 was 389.7 .mu.g/ml, and one
month following the last dose the serum value of hLL2 was 186.5
.mu.g/ml. Normal B-cells in the blood prior to therapy with hLL2
were significantly depleted from the blood 2 months post-therapy,
and there was minimal reappearance of normal B cells five months
post-therapy. The results are shown in the following table.
TABLE-US-00001 TABLE 1 B-cells and T-cells in blood % blood % blood
% blood B-cells T-cells HLA-Dr CD19 CD20 Kappa lambda CD3 (Ia) Day
T4/T8 Flow cytometry 0 1.5 5 5 6 2 38 6 28 hLL2 therapy 34 hLL2
therapy 41 hLL2 therapy 48 hLL2 therapy Flow cytometry 76 1.3 <2
<2 <1 <1 71 6 191 2.0 <2 <2 <1 <1 73 4
EXAMPLE 2
Treatment of a Patient with Chronic Idiopathic Thrombocytopenia
Purpura
[0075] A 50-year-old female with chronic idiopathic
thrombocytopenia purpura has been treated with prednisone, gamma
globulins, and high dose dexamethason, but the disease progresses.
She undergoes splenectomy, which fails to stabilize the disease.
Her platelet count falls to less than 20,000/microliter, and
hemorraghic events increase in frequency. The patient is then
treated with hLL2, 480 mg intravenously each week, for a period of
six weeks. Four weeks after the last dose of hLL2, platelet number
is increased by 100%, and the hemorraghic events become infrequent.
Three months after the last antibody infusion the disease is in
remission.
EXAMPLE 3
Treatment of a Patient with Progressive Rheumatoid Arthritis
[0076] A 60-year-old male, with severe progressive rheumatoid
arthritis of the finger joints, wrists, and elbows, has failed
therapy with methotrexate, and obtains only minor relief when
placed on Enbrel therapy. The patient is then treated with hLL2,
600 mg intravenously each week, for a period of eight weeks. After
3 weeks a 30% improvement in measures of disease activity is
observed, which is maintained for 6 months. The patient is again
treated with hLL2, at the same dose and frequency. The patient
continues to improve, and 6 months after the second hLL2 therapy, a
70% improvement is observed. No human anti-hLL2 antibodies are
observed at any time during, or after the hLL2 therapy. Although
normal B-cells are significantly reduced from the blood, no
infectious complications, or other drug-related toxicity are
observed.
EXAMPLE 4
Treatment of a Patient with Myasthenia Gravis
[0077] A 55-year-old male has failed all conventional therapy for
myasthenia gravis, and is admitted to a neurological intensive
therapy unit. The patient was stabilized by plasma exchange, and
given intravenous immunoglobulin to reduce the titer of
anti-acetylcholine receptor antibody. The patient remained
bedridden, and was then treated with hLL2, 800 mg intravenously
each week, for a period of six weeks. One week after the last dose
of hLL2, a 70% drop in B-lymphocytes is observed, and a significant
drop in the titer of the anti-acetylcholine was observed. Two
months after the last hLL2 dose the patient was mobile, and was
released from the hospital.
EXAMPLE 5
Combination Therapy of Progressive Rheumatoid Arthritis
[0078] Another patient with severe progressive rheumatoid arthritis
of the finger joints, wrists, and elbows, has failed therapy with
methotrexate, and obtains only minor relief when placed on Enbrel
therapy. The patient is then treated with 300 mg each of hLL2 and
Rituximab, intravenously each week, for a period of five weeks.
Significant improvement in measures of disease activity is
observed, which is maintained for 6 months. The patient is again
treated with the same regimen and continues to improve. Six months
after the second course of therapy, additional improvement is
observed. No human anti-hLL2 or anti-Rituximab antibodies are
observed at any time during, or after the therapy. Although normal
B-cells are significantly reduced from the blood, no infectious
complications, or other drug-related toxicity are observed.
EXAMPLE 6
Combination Therapy of Chronic Idiopathic Thrombocytopenia
Purpura
[0079] A patient with chronic idiopathic thrombocytopenia purpura
has been treated with prednisone, gamma globulins, and high dose
dexamethason, but the disease progresses. He undergoes
spleenectomy, which fails to stabilize the disease. The platelet
count falls to less than 20,000/microliter, and hemorraghic events
increase in frequency. This patient is treated with 10 mCi of
90-yttrium-hLL2 and 200 mg of hLL2, followed by 300 mg doses each
of hLL2 and Rituximab, intravenously each week, for a period of six
weeks. Four weeks after the last dose of hLL2 and Rituximab,
platelet number is increased by 150%, and the hemorraghic events
become infrequent. Three months after the last antibody infusion
the disease is in remission.
[0080] Although the foregoing refers to particular preferred
embodiments, it will be understood that the present invention is
not so limited. It will occur to those of ordinary skill in the art
that various modifications may be made to the disclosed embodiments
and that such modifications are intended to be within the scope of
the present invention, which is defined by the following
claims.
[0081] All publications and patent applications mentioned in this
specification are indicative of the level of skill of those in the
art to which the invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application were
specifically and individually indicated to be incorporated by
reference in its entirety.
Sequence CWU 1
1
1 1 15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic linker peptide 1 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser 1 5 10 15
* * * * *