Note: Descriptions are shown in the official language in which they were submitted.
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ANTAGONIST ANTI-CD40 MONOCLONAL ANTIBODIES AND
METHODS FOR THEIR USE
FIELD OF THE INVENTION
This invention relates to antibodies capable of binding to the CD40 cell
surface antigen, thereby blocking C4BP-mediated CD40 signaling, and methods of
using the antibodies, including methods for treatment of diseases mediated by
C4BP
stimulation of CD40 signaling on CD40-expressing cells.
BACKGROUND OF THE INVENTION
CD40 is a cell-surface antigen that is related to the human nerve growth
factor
(NGF) receptor, tumor necrosis factor-a (TNF-a) receptor, and Fas. Expression
of
this member of the TNF receptor family was first characterized on B
lymphocytes, but
recent findings show that it is broadly expressed on a number of cell types.
In the
hernatopoietic system, CD40-expressing cells include CD34+ hematopoietic
progenitors, B cell progenitors, mature B lymphocytes (both normal and
malignant),
plasma cells, monocytes, dendritic cells, eisonophils, basophils, and some T
lymphocytes. Non-hematopoietic CD40-expressing cells include endothelial
cells,
epithelial cells, and fibroblasts. CD40 expression also occurs on synovial
membranes
in rheumatoid arthritis, activated platelets, inflamed vascular smooth muscle
cells, and
dermal fibroblasts. It is expressed at low levels on vascular endothelial
cells and is
up-regulated in areas of local inflammation. Given its broad expression
pattern,
CD40 likely plays a more general role in immune regulation by mediating
interactions
between T-cells and B-cells as well as other cell types. See, for example,
Kooten and
Banchereau (1997) Frontiers ifa Bioscieraces 2:1-11.
The effect of activation of CD40 signaling mediated by binding of its ligand,
CD40L or CD154, to the CD40 receptor depends upon the cell type involved.
Thus,
activation of CD40 signaling on B cells stimulates B cell proliferation and
differentiation, antibody production, isotype switching, and B cell memory
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generation, while actmation of LD40 signaling on dendritic cells and monocytes
leads
to expression of costimulatory molecules and secretion of inflammatory
cytokines
(e.g., IL-8, IL-12, and TNF-alpha), providing for efficient activation of T
lymphocytes. Both agonist anti-CD40 monoclonal antibodies and oligomeric
soluble
CD40L can stimulate the CD40 signaling pathway (see, for example,
International
Publication WO 00/75348 and U.S. Patent No. 6,087,329). The overall effect of
these
signals is to markedly enhance T-cell priming and, for CD40-expressing
dendritic
cells, to favor the generation of Thl cellular immune responses.
The complement cascade plays a critical role in in vivo innate immune
responses. See, for example, William Paul (1993) Fundamental Imrraunology (3rd
ed.,
Raven Press), pp. 924-929. During the complement cascade, antigen-bound IgG
initiates a series of hydrolytic reactions that ultimately creates a membrane
attack
complex (MAC) that punctures bacterial cell membranes. C4 is a critical
component
in this pathway. C4, which is normally inactive in the serum, can be cleaved
by Cl
that is present in the antigen-antibody complex. C 1 cleaves C4 into the C4a
and C4b
fragments. C4a is a small soluble protein that acts as a weak anaphylotoxin.
C4b
binds the surface of the bacterial cell membrane arid can either act
independently as an
opsonin, or it caaz bind C3b to form the C3 convertase of the classical
complement
fixation pathway. The generation of target bound C4b is an inefficient process
and
only 5%-10% of C4b becomes substrate bound. Serum regulatory proteins are
responsible for clearing the excess C4b.
C4b binding protein (C4BP) is one of the of the serum regulatory proteins that
binds C4b. Synthesized by liver cells and activated monocytes, it is a co-
factor in the
Factor-I-mediated catabolism of C4b and C3b. See, for example, Dahlback and
Hildebrand (1983) Biochem. J. 209:857; Lappin and Whaley (1990) Bioclaem. J.
271:
767; Kusada-Funakoshi et al. (1991) Biochem. Med. Metab. Biol. 45:350; Blom et
al.
(2001) J. Biol. Chem. 276:27136; Blom et al. (2001) J. Imnaunol. 166:6764;
Blom et
al. (2003) Mol. Immunol. 399:547. In circulation, C4BP is present in three
isoforms
that are based on differing combinations of the alpha (701cDa) and beta
(45kDa) chains.
The predominant isoform is an octamer of 7 alpha subunits and 1 beta subunit
(oc7(31)
(Pardo-Manuel et al. (1990) Proc. Natl. Acad. Sci. USA 87:4529; Kask et al.
(2002)
Biochemistry 41:9348). Reportedly, C4BP is also capable of binding CD40, and
in
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vitro binding to CD40 on B cells can activate these cells in a manner that
mimics
CD40L binding (Brodeur et al. (2003) Immunity 18:837). Moreover, this in vitro
activity is correlated with the ability of C4BP to bind the CD40 protein
directly in a
region of the molecule that does not competitively inhibit binding for the
CD40 ligand.
CD40 stimulation is a key mediator for autoimmune diseases. C4BP may directly
induce CD40 signaling, and thereby contribute to initiation and progression of
autoimmune and inflammatory diseases. C4BP can also additively or
synergistically
work with CD40 ligand to exacerbate CD40 stimulation and contribute to disease
initiation and progression. Thus, binding of C4BP to the C4BP binding site on
CD40
may transduce aberrant or undesirable signals to cells expressing CD40.
Blocking of CD40 engagment and activation has the potential to suppress
antibody and cell-mediated immune responses. Anti-CD40 antagonist antibodies
could be used to treat autoimmune disorders such as systemic lupus, psoriasis,
multiple sclerosis, inflammatory bowel disease, and rheumatoid arthritis. Such
antibodies could also be used to prevent rejection of organ and tissue grafts
by
suppressing autoimmune response, to treat lymphomas by depriving malignant B
lymphocytes of the activating signal provided by CD40, and to deliver toxins
to
CD40-bearing cells in a specific manner. Two types of anti-CD40 antagonist
monoclonal antibodies can block CD40 activation: 1) those that block CD40
ligand-
mediated CD40 signaling, and 2) those that block C4BP-mediated CD40 signaling.
Given the putative role of C4BP in the CD40 activation pathway, there is a
need for interfering with C4BP-mediated CD40 signaling so that diseases
associated
with C4BP-CD40 binding interaction can be treated.
BRIEF STJMMARY OF THE INVENTION
Compositions and methods for inhibiting CD40-directed activities that are
mediated via the binding of C4BP to CD40 are provided, as are methods for
treating
CD40-associated diseases that are mediated via this C4BP-CD40 binding
interaction.
The compositions of the invention include anti-CD40 antibodies, or antigen-
binding
fragments thereof, that have the following characteristics: 1) are free of
significant
CD40 agonist activity when bound to CD40 antigen on CD40-expressing cells; and
2)
are capable of specifically binding to CD40 antigen expressed on the surface
of cells,
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wherein this binding to CD40 antigen blocks C4BP-mediated CD40 signaling of
these
cells, thereby inhibiting one or more CD40-directed activities. These anti-
CD40
antibodies or antigen-binding fragments thereof exhibit a strong single-site
binding
affinity for the CD40 cell-surface antigen. In some embodiments, the
antibodies of
the invention exhibit a dissociation equilibrium constant (KD) for CD40 of at
least 10-5
M, at least 3 X 10-5 M, preferably at least 10-6 M to 10-~ M, more preferably
at least
10-8 M to about 10-12 M. Suitable monoclonal antibodies have human constant
regions; preferably they also have wholly or partially humanized framework
regions;
and most preferably are fully human antibodies or antigen-binding fragments
thereof.
Compositions also include hybridoma cell lines producing these antibodies or
antigen-
binding fragments thereof, and pharmaceutical compositions comprising these
antibodies or antigen-binding fragments thereof.
Methods for inhibiting a CD40-directed activity of a CD40-expressing cell
comprise contacting the cell with an effective amount of an anti-CD40 antibody
of the
invention, or an effective amount of an antigen-binding fragment thereof. CD40-
directed activities that can be inhibited include, but are not limited to,
cell growth and
proliferation, cell differentiation, antibody production, cell memory
generation,
isotype switching, intercellular adhesion, secretion of cytokines, secretion
of
metalloproteases, and expression of cell adhesion molecules. The anti-CD40
antibodies of the invention can be used to treat CD40-associated diseases that
are
mediated via C4BP stimulation of a CD40-directed activity, including, but not
limited
to, cancers, including B cell-related cancers and solid tumors, and diseases
or
disorders having an autoimmune and/or inflammatory component, including organ
and tissue transplant rejections. Methods for identifying antibodies that have
antagonist activity toward CD40 and that interfere with C4BP-mediated CD40
signaling in CD40-expressing cells are also provided.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to anti-CD40 antibodies and methods for
their use. These anti-CD40 antibodies, and antigen-binding fragments thereof,
are
capable of specifically binding to a CD40 antigen expressed on the surface of
a cell,
and are free of significant CD40 agonist activity when bound to CD40 antigen.
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Binding of these anti-CD40 antibodies, or antigen binding fragments thereof,
to CD40
antigen on CD40-expressing cells effectively blocks C4b binding protein (C4BP)-
mediated CD40 signaling of these cells. By "C4BP-mediated CD40 signaling" is
intended the stimulation of CD40 signaling that occurs when C4BP binds to CD40
antigen expressed on the surface of a cell. By "blocks" or "blocking" is
intended the
partial or complete inhibition of the CD40 signaling that would normally
result from
the binding of C4BP to its binding site on CD40 expressed on the surface of a
cell in
the absence of an antagonist such as the anti-CD40 antibodies of the present
invention. Blocking of this CD40 signaling provides a means for inhibiting,
either
partially (i.e., reduction in an activity) or completely (i.e., prevention of
an activity),
one or more CD40-directed activities that results from C4BP-mediated CD40
signaling. Inhibition of these CD40-directed activities can advantageously be
used for
treating CD40-associated diseases, including, but not limited to, cancers,
such as B
cell-related cancers and solid tumors, and diseases or disorders that have an
autoimmune and/or inflammatory component, including organ and tissue
transplant
rejection, as noted herein below.
The following terms appear throughout the invention disclosure and are
further defined herein below.
"Tumor," as used herein, refers to all neoplastic cell growth and
proliferation,
whether malignant or benign, and all pre-cancerous and cancerous cells and
tissues.
The terms "cancer" and "cancerous" refer to or describe the physiological
condition in mammals that is typically characterized by unregulated cell
growth.
Examples of cancer include, but are not limited to, lymphoma and leukemia, and
solid
tumors.
"Antibodies" and "immunoglobulins" (Igs) are glycoproteins having the same
structural characteristics. While antibodies exhibit binding specificity to an
antigen,
immunoglobulins include both antibodies and other antibody-like molecules that
lack
antigen specificity. Polypeptides of the latter kind are, for example,
produced at low
levels by the lymph system and at increased levels by myelomas.
The term "antibody" is used in the broadest sense and covers fully assembled
antibodies, antibody fragments that can bind antigen ( e.g., Fab, F(ab')2, Fv,
single
chain antibodies, diabodies), and recombinant peptides comprising the
foregoing.
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The term "monoclonal antibody" as used herein refers to an antibody obtained
from a population of substantially homogeneous antibodies, i.e., the
individual
antibodies comprising the population are identical except for possible
naturally
occurring mutations that may be present in minor amounts.
"Native antibodies" and "native immunoglobulins" are usually
heterotetrameric glycoproteins of about 150,000 daltons, composed of two
identical
light (L) chains and two identical heavy (H) chains. Each light chain is
linked to a
heavy chain by one covalent disulfide bond, while the number of disulfide
linkages
varies among the heavy chains of different immunoglobulin isotypes. Each heavy
and
light chain also has regularly spaced intrachain disulfide bridges. Each heavy
chain
has at one end a variable domain (VH) followed by a number of constant
domains.
Each light chain has a variable domain at one end (VL) and a constant domain
at its
other end; the constant domain of the light chain is aligned with the first
constant
domain of the heavy chain, and the light chain variable domain is aligned with
the
variable domain of the heavy chain. Particular amino acid residues are
believed to
form an interface between the light and heavy-chain variable domains.
The term "variable" refers to the fact that certain portions of the variable
domains
differ extensively in sequence among antibodies and are used in the binding
and
specificity of each particular antibody for its particular antigen. However,
the
variability is not evenly distributed throughout the variable domains of
antibodies. It
is concentrated in three segments called complementarity determining regions
(CDRs)
or hypervariable regions both in the light chain and the heavy-chain variable
domains.
The more highly conserved portions of variable domains are termed the
framework
(FR) regions. The variable domains of native heavy and light chains each
comprise
four FR regions, largely adopting a /3-pleated sheet configuration, connected
by three
CDRs, which form loops connecting, and in some cases forming part of, the [3-
pleated
sheet. The CDRs in each chain are held together in close proximity by the FR
regions
and, with the CDRs from the other chain, contribute to the formation of the
antigen-
binding site of antibodies (see Kabat et al. (1991) NIHPubI. No. 91-3242, Vol.
I,
pages 647-669). The constant domains are not involved directly in binding an
antibody to an antigen but exhibit various effector functions, such as Fc
receptor
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(r'clt) binding, participation of the antibody in antibody-dependent cellular
toxicity,
initiation of complement dependent cytotoxicity, and mast cell degranulation.
The term "hypervariable region" refers to the amino acid residues of an
antibody that are responsible for antigen binding. The hypervariable region
comprises
amino acid residues from a complementarity determining region (i.e., residues
24-34
(Ll), 50-56 (L2), and 89-97 (L3) in the light-chain variable domain and 31-35
(H1),
50-65 (H2), and 95-102 (H3) in the heavy-chain variable domain; Kabat et al.
(1991)
Sequences of Proteins of Immunological Irate~est (5th ed.; Public Health
Service,
National Institute of Health, Bethesda, MD) and/or those residues from a
"hypervariable loop" (i.e., residues 26-32 (L1),.50-52 (L2), and 91-96 (L3) in
the
light-chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the
heavy-chain variable domain; Clothia and Lesk (1987) J. Mol. Biol. 196:901).
"Framework" or "FR" residues are those variable domain residues other than the
hypervariable region residues.
"Antibody fragments" comprise a portion of an intact antibody, preferably the
antigen-binding or variable region of the intact antibody. Examples of
antibody
fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear
antibodies
(Zapata et al. (1995) Pr~oteih Eng. 10:1057); single-chain antibody molecules;
and
multispecific antibodies formed from antibody fragments. Papain digestion of
antibodies produces two identical antigen-binding fragments, termed "Fab"
fragments,
each with a single antigen-binding site and a residual "Fc" fragment, whose
name
reflects its ability to crystallize readily. Pepsin treatment yields an
F(ab')2 fragment
that has two antigen-combining sites capable of cross-linking antigen.
"Fv" is the minimum antibody fragment that contains a complete antigen
recognition and binding site.1n a two-chain Fv species, this region consists
of a dimer
of one heavy- and one light-chain variable domain in tight, non-covalent
association.
In a single-chain Fv species, one heavy-and one light-chain variable domain
can be
covalently linked by flexible peptide linker such that the light and heavy
chains can
associate in a "dimeric" structure analogous to that in a two-chain Fv
species. It is in
this configuration that the three CDRs of each variable domain interact to
define an
antigen-binding site on the surface of the VH-VL dimer. Collectively, the six
CDRs
confer antigen-binding specificity to the antibody. However, even a single
variable
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domain (or half of an Fv comprising only three CDRs specific for an antigen)
has the
ability to recognize and bind antigen, although at a lower affinity than the
entire
binding site.
The Fab fragment also contains the constant domain of the light chain and the
first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab'
fragments by the addition of a few residues at the carboxy terminus of the
heavy-
chain CH1 domain including one or more cysteines from the antibody hinge
region.
Fab'-SH is the designation herein for Fab' in which the cysteine residues) of
the
constant domains bear a free thiol group. F(ab')2 antibody fragments
originally were
produced as pairs of Fab' fragments that have hinge cysteines between them.
Other
chemical couplings of antibody fragments are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate
species can be assigned to one of two classes, called kappa (K) and lambda
(7~), based
on the amino acid sequences of their constant domains.
Depending on the amino acid sequence of the constant domain of the heavy
chains, immunoglobulins comprise different classes. There are five major
classes of
human immunoglobulins: IgA, IgD, IgE, IgG, and IgM. In humans, these classes
are
further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4,
IgAl, and
IgA2. The heavy-chain constant domains that correspond to the different
classes of
immunoglobulins are termed alpha, delta, epsilon, gamma, and mu, respectively.
The
subunit structures and three-dimensional configurations of different classes
of
immunoglobulins are well known. Different isotypes have different effector
functions. For example, human IgGl and IgG3 isotypes have antibody-dependent
cell-mediated cytotoxicity (ADCC) activity.
The word "label" when used herein refers to a compound or composition that
is conjugated directly or indirectly to the antibody so as to generate a
"labeled"
antibody. The label may be detectable by itself (e.g., radioisotope labels or
fluorescent
labels) or, in the case of an enzymatic label, may catalyze chemical
alteration of a
substrate compound or composition that is detectable. Radionuclides that can
serve as
detectable labels include, for example, I-131, I-123, I-125, Y-90, Re-188, Re-
186, At-
211, Cu-67, Bi-212, and Pd-109. The label might also be a non-detectable
entity such
as a toxin.
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The term "antagonist" is used in the broadest sense, and includes any molecule
that partially or fully blocks, inhibits, or neutralizes a biological activity
of a target
protein molecule disclosed herein or the transcription or translation thereof.
Antagonist antibodies are antibodies that bind a cell-associated antigen
without
transducing a signal to the cell. A signal may include any biochemical
reaction that
results in a change in the cell's state including inducing proliferation
and/or survival,
inducing apoptosis, inducing phosphorylation of other proteins, inducing
release of
calcium stores, and inducing cytokine secretion.
"Carriers" as used herein include pharmaceutically acceptable carriers,
excipients, or stabilizers that are nontoxic to the cell or mammal being
exposed
thereto at the dosages and concentrations employed. Often the physiologically
acceptable carrier is an aqueous pH buffered solution. Examples of
physiologically
acceptable carriers include buffers such as phosphate, citrate, and other
organic acids;
antioxidants including ascorbic acid; low molecular weight (less than about 10
amino
acid residues) polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino
acids
such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose, or
dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol;
salt-
forming counterions such as sodium; and/or nonionic surfactants such as TWEEN,
polyethylene glycol (PEG), and Pluronics. Administration "in combination with"
one
or more further therapeutic agents includes simultaneous (concurrent) and
consecutive
administration in any order.
A "host cell," as used herein, refers to a microorganism or a eukaryotic cell
or
cell line cultured as a unicellular entity that can be, or has been, used as a
recipient for
a recombinant vector or other transfer polynucleotides, and include the
progeny of the
original cell that has been transfected. It is understood that the progeny of
a single cell
may not necessarily be completely identical in morphology or in genomic or
total
DNA complement as the original parent, due to natural, accidental, or
deliberate
mutation.
"Human effector cells" are leukocytes that express one or more FcRs and
perform effector functions. Preferably, the cells express at least FcyRIII and
carry out
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antibody-dependent cell-mediated cyotoxicity (ADCC) effector function.
Examples of
human leukocytes that mediate ADCC include peripheral blood mononuclear cells
(PBMC), natural killer (hTK) cells, monocytes, macrophages, eosinophils, and
neutrophils, with PBMCs and NK cells being preferred. Antibodies that have
ADCC
activity are typically of the IgGl or IgG3 isotype. In addition to isolating
IgG1 and
IgG3 antibodies, ADCC-mediating antibodies can be synthesized by engineering a
variable region from a non-ADCC antibody or variable region fragment to an
IgGl or
IgG3 isotype constant region.
The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to
the Fc region of an antibody. The preferred FcR is a native-sequence human
FcR.
Moreover, a preferred FcR is one that binds an IgG antibody (a gamma receptor)
and
includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including
allelic
variants and alternatively spliced forms of these receptors. FcyRII receptors
include
FcyRnA_ (an "activating receptor") and FcyRIIB (an "inhibiting receptor"),
which
have similar amino acid sequences that differ primarily in the cytoplasmic
domains
thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based
activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcyRIIB
contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its
cytoplasmic
domain (see Daeron (1997) Annu. Rev. Immuraol. 15:203). FcRs are reviewed in
Ravetch and Kinet (1991) Annu. Rev. Immunol 9:457; Capel et al. (1994)
Immunomethods 4:25; and de Haas et al. (1995) J. Lab. Clin. Med. 126:330.
Other
FcRs, including those to be identified in the future, are encompassed by the
term
"FcR" herein. The term also includes the neonatal receptor, FcRn, which is
responsible for the transfer of maternal IgGs to the fetus (Guyer et al.
(1976) J.
Immurtol. 117:587, and Kim et al. (1994) J. Imrnunol. 24:249).
There are a number of methods for synthesizing human antibodies. For
example, secreting cells can be immortalized by infection with the Epstein-
Barr virus
(EBV). However, EBV-infected cells are difficult to clone and usually produce
only
relatively low yields of immunoglobulin (James and Bell (1987) J. Immunol.
Methods
100:5). Eventually, the immortalization of human B cells may be achieved by
introducing a defined combination of transforming genes. Such a possibility is
highlighted by a recent demonstration that the expression of the telomerase
catalytic
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subunit together with the SV40 large oncoprotein and an oncogenic allele of H-
ras
resulted in the tumorigenic conversion of normal human epithelial and
fibroblast cells
(Hahn et al. (1999) Nature 400:464). Transgenic animals (e.g., mice), upon
immunization, can be capable of producing a repertoire of human antibodies in
the
absence of endogenous immunoglobulin production (Jakobovits et al. (1993)
Nature
362:255; Lonberg and Huszar (1995) Irat. Rev. Irnmunol. 13:65; Fishwild et al.
(1996)
Nat. Biotechnol. 14:845; Mendez et al. (1997) Nat. Genet. 15:146; Green et al.
(1999)
J. Immunol. Methods 231:11; Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA
97:722-727; reviewed in Little et al. (2000) Immunol. Today 21: 364). For
example, it
has been described that the homozygous deletion of the antibody heavy chain
joining
region (JH) gene in chimeric and germ-line mutant mice results in complete
inhibition
of endogenous antibody production (Jakobovits et al. (1993) Proc. Natl. Acad.
Sci.
USA 90:2551). Transfer of the human germ-line immunoglobulin gene array in
such
germ-line mutant mice results in the production of human antibodies upon
antigen
challenge (Jakobovits et al. (1993) Nature 362:255). Mendez et al. (1997)
(Nature
Genetics 15:146) have generated a line of transgenic mice that, when
challenged with
an antigen, generates high affinity fully human antibodies. This was achieved
by
germ-line integration of megabase human-heavy chain and light-chain loci into
mice
with deletion into endogenous JH segment as described above. These mice
(XenoMouse~ II technology (Abgenix; Fremont, California)) harbor 1,020 kb of
human heavy-chain locus containing approximately 66 VH genes, complete DH and
JH
regions, and three different constant regions, and also 800 kb of human x
locus
containing 32 VK genes, JK segments, and C~ genes. The antibodies produced in
these
mice closely resemble that seen in humans in all respects, including gene
rearrangement, assembly, and repertoire. The human antibodies are
preferentially
expressed over endogenous antibodies due to deletion in endogenous segment
that
prevents gene rearrangement in the marine locus. Such mice may be immunized
with
an antigen of particular interest.
Sera from such immunized animals may be screened for antibody reactivity
against the antigen of interest. Lymphocytes may be isolated from lymph nodes
or
spleen cells and may further be selected for B cells by selecting for CD138-
negative
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and CD19-positive cells. In one aspect, such B cell cultures (BCCs) may be
fused to
myeloma cells to generate hybridomas as detailed above.
In another aspect, such B cell cultures may be screened further for reactivity
against the initial antigen, preferably. Such screening includes ELISA with
the
target/antigen protein, a competition assay with known antibodies that bind
the
antigen of interest, and in vitno binding to transiently transfected CHO or
other cells
that express the target antigen.
The anti-CD40 antibodies of the invention and methods for their use are
described in more detail below.
Antagonist Anti-CD40 Antibodies
The anti-CD40 antibodies of the present invention, and antigen-binding
fragments thereof, preferably have antagonist activity on CD40, and hence are
referred to herein as "antagonist" anti-CD40 antibodies. More specifically,
the
antagonist anti-CD40 antibodies of the invention specifically bind to CD40
antigen on
the surface of CD40-expressing cells, whereby this binding blocks C4BP-
mediated
CD40 signaling. Blocking of this signaling process results in inhibition of
one or more
CD40-directed activities that are initiated when an agonist, such as C4BP,
binds to the
CD40 cell surface antigen.
By "CD40 antigen," "CD40 cell surface antigen," "CD40 receptor," or "CD40"
is intended a transmembrane glycoprotein that belongs to the tumor necrosis
factor
(TNF) receptor family (see, for example, TJ.S. Patent Nos. 5,674,492 and
4,708,871;
Stamenkovic et al. (1989) EMBO 8:1403; Clark (1990) Tissue Antigens 36:33;
Barclay et al. (1997) Tlae Leucocyte Antigen Facts Book (2d ed.; Academic
Press, San
Diego)). Two isoforms of human CD40, encoded by alternatively spliced
transcript
variants of this gene, have been identified. The first isoform (also known as
the "long
isoform" or "isoform 1 ") is expressed as a 277-amino-acid precursor
polypeptide (first
reported as GenBank Accession No. CAA43045, and identified as isoform 1 in
GenBank Accession No. NP_001241; encoded by GenBank Accession Nos. X60592
and NM 001250)), which has a signal sequence represented by the first 19
residues.
The second isoform (also known as the "short isoform" or "isoform 2") is
expressed
as a 203-amino-acid precursor polypeptide (GenBank Accession No. NP_690593;
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encoded by GenBank Accession No. NM 152854), which also has a sigilal sequence
represented by the first 19 residues. The precursor polypeptides of these two
isoforms
of human CD40 share in common their first 165 residues. The precursor
polypeptide
of the short isoform is encoded by a transcript variant that lacks a coding
segment,
which leads to a translation frame shift; the resulting CD40 isoform contains
a shorter
and distinct C-terminus (residues 166-203 of GenBank Accession No. NP 690593)
from that contained in the long isoform of CD40 (C-terminus shown in residues
166-
277 of GenBank Accession No. CAA43045 and GenBank Accession No.
NP_001241). For purposes of the present invention, the term "CD40 antigen,"
"CD40
cell surface antigen," "CD40 receptor," or "CD40" encompasses both the short
and
long isoforms of CD40.
The CD40 receptor is displayed on the surface of a variety of cell types, as
described elsewhere herein. By "displayed on the surface" and "expressed on
the
surface" is intended that all or a portion of the CD40 antigen is exposed to
the exterior
of the cell. The displayed or expressed CD40 antigen may be fully or partially
glycosylated.
By "C4b binding protein" or "C4BP" is intended a soluble peptide or any
fragment thereof including at least a portion of an alpha subunit (GenBank
Accession
No. NP_000706, encoded by GenBank Accession No. NM 000715; Kask et al.
(2002) Biochemistry 41:9349) or a portion of a beta subunit (GenBank Accession
No.
NP 000707, encoded by GenBank Accession No. NM 000716; Webb et al. (2003)
Eur. J. Biochem. 270:93). The term "C4BP" as used herein may include
individual
alpha or beta subunits or larger heteromers comprising these subunits such as
the
three serum isoforms: x7(31 (the predominant isoform in serum), a7(30, and
a6[31.
By the "C4BP binding site on CD40" is intended the region of the CD40 antigen
where any portion of any C4BP subunit binds. See, for example, Brodeur et al.
(2003) Immunity 18:837, herein incorporated by reference in its entirety. The
binding
site may comprise a linear determinant on CD40 or it may comprise a binding
domain
formed by discontiguous amino acids that form a C4BP binding site via
secondary or
tertiary conformation, or a combination thereof. In some instances, the C4BP
binding
site interacts exclusively with a C4BP alpha subunit.
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When C4BP binds to the CD40 antigen on CD40-expressing cells, it serves as
an agonist of CD40 signaling, and is thus said to have CD40 agonist activity.
By
"agonist activity" is intended that the substance functions as an agonist. An
agonist
combines with a receptor on a cell and initiates a reaction or activity that
is similar to
or the same as that initiated by the receptor's natural ligand, for example,
CD40L in
the case of CD40. An agonist of CD40, such as C4BP, induces any or all of, but
not
limited to, the following activities that occur when CD40 transduces a signal:
proliferation and differentiation of antigen presenting cells (ADCs); B cell
antibody
production; intercellular adhesion; B cell memory generation; B cell isotype
switching
(especially IgE isotype switching in the presence of IL-4); upregulation of
cell-surface
expression of MHC Class II, CD54, CD95, and CD80/86 in APCs; upregulation of
bcl-xL, A20, diacylglycerol kinase a, p38, and c-myc gene expression; nuclear
translocation of NF~cB; secretion of cytokines such as IL-2, IL-4, IL-5, IL-6,
IL-8, IL-
10, IL-12, and TNFa; secretion of metalloproteases such as MMP-I/collagenase
and
MMP-9/gelatinase B; and expression of cell adhesion molecules such as E-
selectin,
VCAM-1, and ICAM-1. For purposes of the present invention, such activities are
referred to as "CD40-directed activities," and the induction of such
activities by the
binding of C4BP to its binding site on CD40 is referred to as "C4BP-mediated
CD40
signaling."
The anti-CD40 antibodies of the invention, and antigen-binding fragments
thereof, have antagonist activity with respect to their binding interaction
with CD40.
This antagonist activity results from the anti-CD40 antibodies ability to
block C4BP-
mediated CD40 signaling when these antibodies bind to CD40 or to send a
negative
signal through CD40. By "antagonist activity" is intended that the substance
functions as an antagonist. An antagonist anti-CD40 antibody of the invention
prevents or reduces induction of any one or more of the CD40-directed
activities
induced by binding of the CD40 receptor to an agonist ligand, particularly
C4BP. The
antagonist anti-CD40 antibodies of the invention may reduce induction of any
one or
more of the CD40-directed activities induced by the binding of C4BP to CD40 by
5%,
10%, 15%, 20%, 25%, 30%, 35%, preferably 40%, 45%, 50%, 55%, 60%, more
preferably 70%, 80%, 85%, and most preferably 90%, 95%, 99%, or 100%. Methods
for measuring anti-CD40 antibody and C4BP binding specificity and antagonist
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activity are known to one of skill in the art and include, but are not limited
to,
standard competitive binding assays, assays for dendritic cell induction of T
cell
proliferation, assays for monitoring immunoglobulin secretion by B cells, B
cell
proliferation assays, Banchereau-Like-B cell proliferation assays, T cell
helper assays
for antibody production, co-stimulation of B cell proliferation assays, and
assays for
upregulation of APC activation markers. See, for example, such assays
disclosed in
Brodeur et al. (2003) Immunity 18:837, WO 00175348, and U.S. Patent No.
6,087,329, herein incorporated by reference. In some embodiments, binding to
CD40
displayed on the surface of human cells blocks C4BP-mediated CD40 signaling,
resulting in inhibition of proliferation and differentiation of these human
cells. Thus,
the antagonist anti-CD40 antibodies of the invention include those antibodies
that can
exhibit antagonist activity toward normal and neoplastic human cells
expressing the
cell-surface CD40 antigen.
The antagonist activity of the anti-CD40 antibodies of the present invention
is
manifested via the blocking of C4BP-mediated CD40 signaling, for example, by
competitive or steric interference with the binding of C4BP to its binding
site on
CD40. By "competitively inhibit" is intended that the anti-CD40 antibody, or
antigen-binding fragment thereof, binds the same C4BP binding site on CD40, or
at
least a portion thereof, as native soluble C4BP, thereby inhibiting the
binding of
C4BP to its binding site on CD40. By "sterically inhibit" or "steric
inhibition" is
intended that the anti-CD40 antibody, or antigen-binding fragment thereof,
binds
outside, or at least partially outside, the C4BP binding site on CD40, wherein
the
antibody still inhibits C4BP or fragment thereof from binding to CD40 by
steric
interference or disruption of the structure of the C4BP binding site on CD40,
or some
combination thereof. In some embodiments, binding of the antagonist anti-CD40
antibody or antigen-binding fragment thereof to the CD40 antigen prevents CD40
signal transduction when C4BP ligates to CD40 antigen. One of skill could
determine
whether an antibody competitively or sterically interferes with the binding of
C4BP to
CD40, or prevents CD40 signal transduction with the binding of C4BP to its
binding
site on CD40, using standard methods well known in the art.
When the antagonist anti-CD40 antibodies of the invention bind CD40
displayed on the surface of CD40-expressing cells, such as normal and
neoplastic
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human B cells, and human dendritic cells, the antibodies are free of
significant agonist
activity when bound to CD40. By "significant" agonist activity is intended an
agonist
activity of at least 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%,
95%, or 100% greater than the agonist activity induced by a neutral substance
or
negative control as measured in an assay of a CD40-expressing cell's response.
Preferably, "significant" agonist activity is an agonist activity that is at
least 2-fold
greater or at least 3-fold greater than the agonist activity induced by a
neutral
substance or negative control as measured in an assay of a B cell response.
Thus, for
example, where the B cell response of interest is B cell proliferation,
"significant"
agonist activity would be induction of a level of B cell proliferation that is
at least 2-
fold greater or at least 3-fold greater than the level of B cell proliferation
induced by a
neutral substance or negative control. In one embodiment, a non-specific
immunoglobulin, for example IgGl, that does not bind to CD40 serves as the
negative
control. A substance "free of significant agonist activity" would exhibit an
agonist
activity of not more than about 25% greater than the agonist activity induced
by a
neutral substance or negative control, preferably not more than about 20%
greater,
15% greater, 10% greater, 5% greater, 1% greater, 0.5% greater, or even not
more
than about 0.1% greater than the agonist activity induced by a neutral
substance or
negative control. For purposes of the present invention, the agonist activity
of the
anti-CD40 antibodies of the invention is measured in an assay of a B cell
response.
Such assays axe well known in the art and include, but are not limited to,
assays for
monitoring irnmunoglobulin secretion by B cells, B cell proliferation assays,
Banchereau-Like-B cell proliferation assays, co-stimulation of B cell
proliferation
assays. See, for example, such assays disclosed in Brodeur et al. (2003)
Immuhity
18:837, WO 00/75348, and U.S. Patent No. 6,087,329, herein incorporated by
reference. In one embodiment of the invention, the antagonist anti-CD40
antibody is
free of significant agonist activity in one B cell response. In another
embodiment of
the invention, the antagonist anti-CD40 antibody is free of significant
agonist activity
in assays of more than one B cell response (e.g., proliferation and
differentiation, or
proliferation, differentiation, and antibody production).
In some embodiments, the antagonist anti-CD40 antibodies of the invention
are fully human anti-CD40 monoclonal antibodies of the IgGl isotype produced
from
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a hybridoma cell line. These cell lines are created using splenocytes from
immunized
mice, including mice obtained using XenoMouse° technology (Abgenix;
Fremont,
California), such as described in U.S. Patent No. 6,075,181 and PCT
International
Publication No. WO 94/02602. The spleen cells are fused with the mouse myeloma
SP2/0 cells (Sierra BioSource). The resulting hybridomas are sub-cloned
several
times to create the monoclonal cell lines. Other antibodies of the invention
may be
prepared similarly using mice transgenic for human immunoglobulin loci or by
other
methods known in the art and/or described herein. The human anti-CD40
monoclonal
antibodies of the invention specifically bind CD40, for example, human CD40,
though they may also specifically bind to a non-human sequence that has an
epitope
that the human anti-CD40 antibody recognizes.
In alternative embodiments, marine antibodies to CD40 can be humanized, for
example, using methods described in U.S. Patent No. 5,766,886 or U.S. Patent
No.
6,180,370. In addition, phage display libraries of human antibodies can be
screened
against C4BP to identify anti-CD40 antibodies having the binding
characteristics
described herein.
In addition to antagonist activity, some anti-CD40 antibodies of this
invention
have another mechanism of action, for example, antibodies having antibody-
dependent cell-mediated cytotoxicity (ADCC). Alternatively, the variable
regions of
the anti-CD40 antibodies can be expressed on another antibody isotype that has
ADCC activity. It is also possible to conjugate native forms, recombinant
forms, or
antigen-binding fragments of the anti-CD40 antibodies to a cytotoxic toxin.
Such
antagonist anti-CD40 antibodies are useful in targeting, for example, CD40-
expressing neoplastic cells, for example, malignant B cells or CD40-expressing
neoplastic cells of a solid tumor.
In some embodiments, the antagonist anti-CD40 antibodies of the invention,
or antigen-binding fragments thereof, bind soluble CD40 in ELISA-type assays;
in
other embodiments, the antibodies or antigen binding fragments thereof inhibit
binding of C4BP to cell-surface CD40, and thereby displace the pre-bound C4BP,
as
determined by flow cytometric assays. Suitable antagonist anti-CD40 antibodies
or
antigen-binding fragments thereof for use in the methods of the present
invention
exhibit a strong single-site binding affinity for the CD40 cell-surface
antigen. In
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some embodiments, the antibodies of the invention exhibit a dissociation
equilibrium
constant (KD) for CD40 of at least 10-5 M, at least 3 X 10-5 M, preferably at
least 10-6
M to 10-~ M, more preferably at least 10-$ M to about 10-12 M, measured using
a
standard assay such as BiacoreTM. Biacore analysis is known in the art and
details are
provided in the BIAapplicatiohs Haradbook. Methods described in WO 01/27160
can
be used to modulate the binding affinity.
Production of Antagonist Anti-CD40 Antibodies
The antagonist anti-CD40 antibodies of the invention include antibodies that
specifically recognize the CD40 cell surface antigen, including polyclonal
antibodies,
monoclonal antibodies, single-chain antibodies, and fragments thereof such as
Fab,
F(ab')2, F~, and other fragments that retain the antigen binding function of
the parent
anti-CD40 antibody. Of particular interest to the methods of the present
invention axe
those anti-CD40 antibodies that block C4BP-mediated CD40 cell signaling and
are
free of significant agonist activity when bound to CD40. These antibodies can
be
produced using any antibody production method known to those of skill in the
art, and
include antagonist anti-CD40 antibodies, and antigen-binding fragments
thereof, that
block C4BP-mediated CD40 signaling and which are recombinantly produced.
Polyclonal sera may be prepared by conventional methods. In general, a
solution containing the CD40 antigen is first used to immunize a suitable
animal,
preferably a mouse, rat, rabbit, or goat. Rabbits or goats axe preferred for
the
preparation of polyclonal sera due to the volume of serum obtainable, and the
availability of labeled anti-rabbit and anti-goat antibodies.
Polyclonal sera can be prepared in a transgenic animal, preferably a mouse
bearing human immunoglobulin loci. In a preferred embodiment, Sf~ cells
expressing
CD40 are used as the immunogen. Immunization can also be performed by mixing
or
emulsifying the antigen-containing solution in saline, preferably in an
adjuvant such
as Freund's complete adjuvant, and injecting the mixture or emulsion
parenterally
(generally subcutaneously or intramuscularly). A dose of 50-200 ~,g/injection
is
typically sufficient. hnmunization is generally boosted 2-6 weeks later with
one or
more inj ections of the protein in saline, preferably using Freund's
incomplete
adjuvant. One may alternatively generate antibodies by ira vitro immunization
using
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methods known in the art, which for the purposes of this invention is
considered
equivalent to ih vivo immunization.
Polyclonal antisera are obtained by bleeding the immunized animal into a
glass or plastic container, incubating the blood at 25°C for one hour,
followed by
incubating at 4°C for 2-18 hours. The serum is recovered by
centrifugation (e.g.,
1,000 x g for 10 minutes). About 20-50 ml per bleed may be obtained from
rabbits.
Production of the Sf 9 (Spodoptera frugiperda) cells is disclosed in U.S.
Patent No. 6,004,552, incorporated herein by reference. Briefly, sequences
encoding
human CD40 are recombined into a baculovirus using transfer vectors. The
plasmids
are co-transfected with wild-type baculovirus DNA into Sf 9 cells. Recombinant
baculovirus- infected Sf 9 cells are identified and clonally purified.
Preferably the antibody is monoclonal in nature. By "monoclonal antibody" is
intended an antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the population are
identical
except for possible naturally occurring mutations that may be present in minor
amounts. The term is not limited regarding the species or source of the
antibody. The
term encompasses whole immunoglobulins as well as fragments such as Fab,
F(ab')2,
Fv, and others that retain the antigen-binding function of the antibody.
Monoclonal
antibodies are highly specific, being directed against a single antigenic
site, i.e., the
CD40 cell surface antigen in the present invention. Furthermore, in contrast
to
conventional (polyclonal) antibody preparations that typically include
different
antibodies directed against different determinants (epitopes), each monoclonal
antibody is directed against a single determinant on the antigen. The modifier
"monoclonal" indicates the character of the antibody as being obtained from a
substantially homogeneous population of antibodies, and is not to be construed
as
requiring production of the antibody by any particular method. For example,
the
monoclonal antibodies to be used in accordance with the present invention may
be
made by the hybridoma method first described by Kohler et al. (1975) Nature
256:495, or may be made by recombinant DNA methods (see, e.g., U.S. Patent No.
4,816,567). The "monoclonal antibodies" may also be isolated from phage
antibody
libraries using the techniques described in, for example, Clackson et al.
(1991) Nature
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352:624-628; Marks et al. (1991) J. Mol. Biol. 222:581-597; and U.S. Patent
No.
5,514,548.
By "epitope" is intended the part of an antigenic molecule to which an
antibody is produced and to which the antibody will bind. Epitopes can
comprise
linear amino acid residues (i.e., residues within the epitope are arranged
sequentially
one after another in a linear fashion), nonlinear amino acid residues
(referred to herein
as "nonlinear epitopes"; these epitopes are not arranged sequentially), or
both linear
and nonlinear amino acid residues.
Monoclonal antibodies can be prepared using the method of Kohler et al.
(1975) Nature 256:495-496, or a modification thereof. Typically, a mouse is
immunized with a solution containing an antigen. Immunization can be performed
by
mixing or emulsifying the antigen-containing solution in saline, preferably in
an
adjuvant such as Freund's complete adjuvant, and injecting the mixture or
emulsion
parenterally. Any method of immunization known in the art may be used to
obtain
the monoclonal antibodies of the invention. After immunization of the animal,
the
spleen (and optionally, several large lymph nodes) are removed and dissociated
into
single cells. The spleen cells may be screened by applying a cell suspension
to a plate
or well coated with the antigen of interest. The B cells expressing membrane
bound
immunoglobulin specific for the antigen bind to the plate and are not rinsed
away.
Resulting B cells, or all dissociated spleen cells, are then induced to fuse
with
myeloma cells to form hybridomas, and are cultured in a selective medium. The
resulting cells are plated by serial dilution and are assayed for the
production of
antibodies that specifically bind the antigen of interest (and that do not
bind to
unrelated antigens). The selected monoclonal antibody (mAb)-secreting
hybridomas
are then cultured either ih vitro (e.g., in tissue culture bottles or hollow
fiber reactors),
or in vivo (as ascites in mice).
Where the antagonist anti-CD40 antibodies of the invention are to be prepared
using recombinant DNA methods (i.e., recombinantly produced antagonist anti-
CD40
antibodies), the DNA encoding the monoclonal antibodies is readily isolated
and
sequenced using conventional procedures (e.g., by using oligonucleotide probes
that
are capable of binding specifically to genes encoding the heavy and light
chains of
marine antibodies). The hybridoma cells described herein serve as a preferred
source
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of such DNA. Once isolated, the DNA may be placed into expression vectors,
which
are then transfected into host cells such as E. coli cells, simian COS cells,
Chinese
Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce
imrnunoglobulin protein, to obtain the synthesis of monoclonal antibodies in
the
recombinant host cells. Review articles on recombinant expression in bacteria
of
DNA encoding the antibody include Skerra et al. (1993) Curs. Opinion in
Immunol.
5:256 and Phickthun (1992) Inamunol. Revs. 130:151.
Alternatively, antibody can be produced in a cell line such as a CHO cell
line,
as disclosed in U.S. Patent Nos. 5,545,403; 5,545,405; and 5,998,144;
incorporated
herein by reference. Briefly the cell line is transfected with vectors capable
of
expressing a light chain and a heavy chain, respectively. By transfecting the
two
proteins on separate vectors, chimeric antibodies can be produced. Another
advantage is the correct glycosylation of the antibody. In another embodiment,
the
antagonist anti-CD40 antibody or antigen-binding fragment thereof can be
produced
in CHO cells using the GS gene expression system (Lonza Biologics, Portsmouth,
New Hampshire), which uses glutamine synthetase as a marker. See, also U.S.
Patent
Nos. 5,122,464; 5,591,639; 5,658,759; 5,770,359; 5,827,739; 5,879,936;
5,891,693;
and 5,981,216; the contents of which are herein incorporated by reference in
their
entirety.
Monoclonal antibodies to CD40 are known in the art. See, for example, the
sections dedicated to B cell antigen in McMichael, ed. (1987; 1989) Leukocyte
Typing
III and IV (Oxford University Press, New York); U.S. Patent Nos. 5,674,492;
5,874,082; 5,677,165; 6,056,959; International Publication Nos. WO 00/63395,WO
02/28905, and WO 02/28904; U.S. Patent Application Publication Nos. US
200210142358 A1 and 2003/0059427; the antagonist anti-CD40 antibodies
disclosed
in provisional applications entitled "Antagonist Anti-CD40 Monoclonal
Antibodies
and Methods for Their Use, " filed November 4, 2003, November 26, 2003, and
April
27, 2004, and assigned U.S. Patent Application Nos. 60/517,337 (Attorney
Docket
No. PP20107.001 (035784/258442)), 60/525,579 (Attorney Docket No. PP20107.002
(035784/271525)), and 60/565,710 (Attorney Docket No. PP20107.003
(035784/277214)), respectively, and International Patent Application No.
PCT/LTS2004/037152 (Attorney Docket No. PP20107.004 (035784/282916)), also
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entitled "Antagonist Anti-CD40 Monoclonal Antibodies and Methods for Theif
Use, "
filed November 4, 2004; Gordon et al. (1988) J. Immunol. 140:1425; Valle et
al.
(1989) Eur. J. IrnmurZOl. 19:1463; Clark et al. (1986) PNAS 83:4494; Paulie et
al.
(1989) J. Inanaunol. 142:590; Gordon et al. (1987) Eu~. J. Irnnzunol. 17:1535;
Jabara et
al. (1990) J. Exp. Med. 172:1861; Zhang et al. (1991) J. Immunol. 146:1836;
Gascan
et al. (1991) J. Inzmunol. 147:8; Banchereau et al. (1991) Clin. Immunol.
Spectf~um
3:8; and Banchereau et al. (1991) Science 251:70; all of which are herein
incorporated
by reference. Of particular interest to the present invention are the
antagonist anti-
CD40 antibodies disclosed herein that bind at or near the C4BP binding site on
CD40,
thereby blocking CD40-mediated CD40 signaling, which can be the result of the
antagonist anti-CD40 antibodies' ability to competitively or sterically
inhibit the
binding of C4BP to its binding site on C4BP or their ability to prevent CD40
signal
transduction with the binding of C4BP to its binding site on CD40.
The term "CD40-antigen epitope" as used herein refers to a molecule that is
capable of immunoreactivity with the anti-CD40 monoclonal antibodies of this
invention, excluding the CD40 antigen itself. CD40-antigen epitopes may
comprise
proteins, protein fragments, peptides, carbohydrates, lipids, and other
molecules, but
for the purposes of the present invention are most commonly proteins, short
oligopeptides, oligopeptide mimics (i e, organic compounds which mimic the
antibody binding properties of the CD40 antigen), or combinations thereof.
Suitable
oligopeptide mimics are described, inter alia, in PCT application US 91/04282.
Additionally, the antag~nist anti-CD40 antibodies of the invention include
chimeric anti-CD40 antibodies and humanized anti-CD40 antibodies; such
chimeric
anti-CD40 antibodies and humanized anti-CD40 antibodies of the invention bind
at or
near the C4BP binding site on CD40, thereby blocking C4BP-mediated CD40
signaling. By "chimeric" antibodies is intended antibodies that are most
preferably
derived using recombinant deoxyribonucleic acid techniques and which comprise
both human (including immunologically "related" species, e.g., chimpanzee) and
non-
human components. Thus, the constant region of the chimeric antibody is most
preferably substantially identical to the constant region of a natural human
antibody;
the variable region of the chimeric antibody is most preferably derived from a
non-
human source and has the desired antigenic specificity to the CD40 cell-
surface
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antigen. The non-human source can be any vertebrate source that can be used to
generate antibodies to a human CD40 cell-surface antigen or material
comprising a
human CD40 cell-surface antigen. Such non-human sources include, but are not
limited to, rodents (e.g., rabbit, rat, mouse, etc.; see, for example, U.S.
Patent No.
4,816,567, herein incorporated by reference) and non-human primates (e.g., Old
World Monkey, Ape, etc.; see, for example, U.S. Patent Nos. 5,750,105 and
5,756,096; herein incorporated by reference). Rituxan~ is an example of a
chimeric
antibody with a marine variable region and a human constant region. As used
herein,
the phrase "immunologically active" when used in reference to chimeric anti-
CD40
antibodies means a chimeric antibody that binds CD40, particularly human CD40.
By "humanized" is intended forms of anti-CD40 antibodies that contain
minimal sequence derived from non-human immunoglobulin sequences. For the most
part, humanized antibodies are human immunoglobulins (recipient antibody) in
which
residues from a hypervariable region (also known as complementarity
determining
region or CDR) of the recipient are replaced by residues from a hypervariable
region
of a non-human species (donor antibody) such as mouse, rat, rabbit, or
nonhuman
primate having the desired specificity, affinity, and capacity. The phrase
"complementarity determining region" refers to amino acid sequences that
together
define the binding affinity and specificity of the natural Fv region of a
native
immunoglobulin binding site. See, e.g., Chothia et al ( 1987) J. Mol. Biol.
196:901-
917; Kabat et al (1991) U. S. Dept. of Health and Human Services, NIH
Publication
No. 91-3242). The phrase "constant region" refers to the portion of the
antibody
molecule that confers effector functions. In previous work directed towards
producing non-immunogenic antibodies for use in therapy of human disease,
mouse
constant regions were substituted by human constant regions. The constant
regions of
the subject humanized antibodies were derived from human immunoglobulins.
However, these humanized antibodies still elicited an unwanted and potentially
dangerous immune response in humans and there was a loss of affinity.
Humanized
anti-CD40 antibodies of the present invention also specifically bind to CD40,
thereby
blocking or inhibiting C4BP-mediated CD40 signaling.
Humanization can be essentially performed following the method of Winter
and co-workers (Jones et al. (1986) Nature 321:522-525; Riechmann et al.
(1988)
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Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), by
substituting rodent or mutant rodent CDRs or CDR sequences for the
corresponding
sequences of a human antibody. See also U.S. Patent Nos. 5,225,539; 5,585,089;
5,693,761; 5,693,762; 5,859,205; herein incorporated by reference. In some
instances, residues within the framework regions of one or more variable
regions of
the human immunoglobulin are replaced by corresponding non-human residues
(see,
for example, U.S. Patent Nos. 5,585,089; 5,693,761; 5,693,762; and 6,180,370).
Furthermore, humanized antibodies may comprise residues that are not found in
the
recipient antibody or in the donor antibody. These modifications are made to
further
refine antibody performance (e.g., to obtain desired affinity). In general,
the
humanized antibody will comprise substantially all of at least one, and
typically two,
variable domains, in which all or substantially all of the hypervariable
regions
correspond to those of a non-human immunoglobulin and all or substantially all
of the
framework regions are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an immunoglobulin
constant region (Fc), typically that of a human immunoglobulin. For further
details
see Jones et al. (1986) Nature 331:522-525; Riechmann et al. (1988) Nature
332:323-
329; and Presta (1992) Cu~~. Op. St~~uct. Biol. 2:593-596; herein incorporated
by
reference. Accordingly, such "humanized" antibodies may include antibodies
wherein
substantially less than an intact human variable domain has been substituted
by the
corresponding sequence from a non-human species. In practice, humanized
antibodies are typically human antibodies in which some CDR residues and
possibly
some framework residues are substituted by residues from analogous sites in
rodent
antibodies. See, for example, U.S. Patent Nos. 5,225,539; 5,585,089;
5,693,761;
5,693,762; 5,859,205. See also U.S. Patent No. 6,180,370, and International
Publication No. WO 01/27160, where humanized antibodies and techniques for
producing humanized antibodies having improved affinity for a predetermined
antigen are disclosed.
The anti-CD40 antibodies of the present invention also include xenogeneic or
modified anti-CD40 antibodies produced in a non-human mammalian host, more
particularly a transgenic mouse, characterized by inactivated endogenous
immunoglobulin (Ig) loci. In such transgenic animals, competent endogenous
genes
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for the expression of light and heavy subunits of host immunoglobulins are
rendered
non-functional and substituted with the analogous human immunoglobulin loci.
These transgenic animals produce human antibodies in the substantial absence
of light
or heavy host immunoglobulin subunits. See, for example, U.S. Patent Nos.
5,877,397
and 5,939,598, herein incorporated by reference. Accordingly, such antibodies
are
fully human anti-CD40 antibodies.
Preferably, fully human antibodies to CD40 are obtained by immunizing
transgenic mice. One such mouse is obtained using Xenomouse~ technology
(Abgenix; Fremont, California), and is disclosed in U.S. Patent Nos.
6,075,181,
6,091,001, and 6,114,598, all of which are incorporated herein by reference.
Thus,
for example, in one embodiment, the human antagonist anti-CD40 antibodies
disclosed herein can be produced by immunizing mice transgenic for the human
IgGI
heavy chain locus and the human K light chain locus with Sf 9 cells expressing
human
CD40. Mice can also be transgenic for other isotypes. Fully human antagonist
anti-
CD40 antibodies of the present invention are also characterized by binding at
or near
the C4BP binding site on CD40, thereby blocking C4BP-mediated CD40 signaling.
Fragments of the antagonist anti-CD40 antibodies of the present invention are
suitable for use in the methods of the invention so long as they retain the
desired
affinity of the corresponding full-length antagonist anti-CD40 antibody and
are
characterized by properties similar to the corresponding full-length
antagonist anti-
CD40 antibody. That is, the fragments will specifically bind a CD40 antigen
expressed on the surface of a cell, for example, human CD40 antigen on the
surface of
a human cell, and are free of significant agonist activity but exhibit
antagonist activity
when bound to the CD40 antigen. Accordingly, binding of such fragments to CD40
on CD40-expressing cells blocks C4BP-mediated CD40 signaling, thereby
inhibiting
one or more CD40-directed activities. Such fragments are referred to herein as
"antigen-binding" fragments, and are suitable for use in any of the methods of
the
present invention.
Suitable antigen-binding fragments of an antibody comprise a portion of a
full-length antibody, generally the antigen-binding or variable region
thereof.
Examples of antibody fragments include, but are not limited to, Fab, F(ab')2,
and Fv
fragments and single-chain antibody molecules. By "Fab" is intended a
monovalent
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antigen-binding fragment of an immunoglobulin that is composed of the light
chain
and part of the heavy chain. By F(ab')2 is intended a bivalent antigen-binding
fragment of an immunoglobulin that contains both light chains and part of both
heavy
chains. By "single-chain Fv" or "sFv" antibody fragments is intended fragments
comprising the VH and VL domains of an antibody, wherein these domains are
present
in a single polypeptide chain. See, for example, U.S. Patent Nos. 4,946,778,
5,260,203, 5,455,030, and 5,856,456, herein incorporated by reference.
Generally,
the Fv polypeptide further comprises a polypeptide linker between the VH and
VL
domains that enables the sFv to form the desired structure for antigen
binding. For a
review of sFv see Pluckthun (1994) in The Pharmacology ofMorzoclonal
Antibodies,
Vol. 113, ed. Rosenburg and Moore (Springer-Verlag, New York), pp. 269-315.
Antigen-binding fragments of the antagonist anti-CD40 antibodies disclosed
herein
can also be conjugated to a cytotoxin to effect killing of target cells, for
example,
taxget cancer cells, as described herein below.
Antibodies or antibody fragments can be isolated from antibody phage
libraries generated using the techniques described in, for example, McCafferty
et al.
(1990) Nature 348:552-554 (1990) and U.S. Patent No. 5,514,548. Clackson et
al.
(1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597
describe the isolation of marine and human antibodies, respectively, using
phage
libraries. Subsequent publications describe the production of high affinity
(nM range)
human antibodies by chain shuffling (Marks et al. (1992) BiolTechnology 10:779-
783), as well as combinatorial infection and in vivo recombination as a
strategy for
constructing very large phage libraries (Waterhouse et al. (1993) Nucleic.
Acids Res.
21:2265-2266). Thus, these techniques are viable alternatives to traditional
monoclonal antibody hybridoma techniques for isolation of monoclonal
antibodies.
Various techniques have been developed for the production of antibody
fragments. Traditionally, these fragments were derived via proteolytic
digestion of
intact antibodies (see, e.g., Morimoto et al. (1992) .Iournal of Biochemical
and
Biophysical Methods 24:107-117 (1992) and Brennan et al. (1985) Science
229:81).
However, these fragments can now be produced directly by recombinant host
cells.
For example, the antibody fragments can be isolated from the antibody phage
libraries
discussed above. Alternatively, Fab'-SH fragments can be directly recovered
from E.
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coli and chemically coupled to form F(ab')2 fragments (Carter et al. (1992)
BiolTecl~yaology 10:163-167). According to another approach, F(ab')Z fragments
can
be isolated directly from recombinant host cell culture. Other techniques for
the
production of antibody fragments will be apparent to the skilled practitioner.
Antagonist anti-CD40 antibodies useful in the methods of the present
invention include the anti-CD40 monoclonal antibodies described herein above
as
well as antibodies differing from these antibodies but retaining the CDRs; and
antibodies with one or more amino acid addition(s), deletion(s), or
substitution(s),
wherein the antagonist activity is measured by the ability of the antibody to
block
C4BP-mediated CD40 signaling, thereby inhibiting one or more CD40-directed
activities. The invention also encompasses de-immunized antagonist anti-CD40
antibodies, which can be produced as described in, for example, International
Publication Nos. WO 98/52976 and WO 0034317; herein incorporated by reference.
In this manner, residues within the antagonist anti-CD40 antibodies of the
invention
are modified so as to render the antibodies non- or less immunogenic to humans
while
retaining their antagonist activity toward human CD40-expressing cells,
particularly
blocking C4BP-mediated CD40 signaling, resulting in inhibition of one or more
CD40-directed activities, wherein such activities are measured by assays noted
elsewhere herein. Also included within the scope of the claims are fusion
proteins
comprising an antagonist anti-CD40 antibody of the invention, or a fragment
thereof,
which fusion proteins can be synthesized or expressed from corresponding
polynucleotide vectors, as is known in the art. Such fusion proteins are
described
with reference to conjugation of antibodies as noted below.
The antibodies of the present invention can have sequence variations produced
using methods described in, for example, Patent Publication Nos. EP 0 983 303
Al,
WO 00/34317, and WO 98/52976, incorporated herein by reference. For example,
it
has been shown that sequences within the CDR can cause an antibody to bind to
MHC Class II and trigger an unwanted helper T cell response. A conservative
substitution can allow the antibody to retain binding activity yet lose its
ability to
trigger an unwanted T cell response. Any such conservative or non-conservative
substitutions can be made using art-recognized methods, such as those noted
elsewhere herein, and the resulting antibodies will fall within the scope of
the
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invention. The variant antibodies can be routinely tested for antagonist
activity,
amity, and specificity using methods described herein.
An antibody produced by any of the methods described above, or any other
method not disclosed herein, will fall within the scope of the invention if
the binding
of the antibody to CD40 antigen blocks C4BP-mediated CD40 signaling. The
antibody can block C4BP-mediated CD40 signaling by any means including, but
not
limited to: preventing binding of C4BP by sterically inhibiting the binding of
C4BP
to its binding site on CD40, competitively inhibiting binding of C4BP by
competing
for at least a portion of the C4BP binding site on CD40, and preventing CD40
signal
transduction when C4BP ligates cell surface CD40. The antagonistic anti-CD40
antibodies of this invention may thus inhibit one or more of the CD40-directed
activities that is induced by the binding of C4BP to its binding site on CD40,
including, but not limited to, the CD40-directed activities disclosed herein,
for
example, immunoglobulin secretion by normal human peripheral B cells
stimulated
by T cells; survival and/or proliferation of normal human peripheral B cells
stimulated
by Jurkat T cells; survival and/or proliferation of normal human peripheral B
cells
stimulated by C4BP-expressing cells or soluble C4BP; "survival" anti-apoptotic
intracellular signals in any cell stimulated by soluble C4BP or solid-phase
C4BP;
CD40 signal transduction in any cell upon ligation with soluble C4BP or solid-
phase
C4BP; and proliferation of human malignant B cells as noted below. Assays for
determining the ability of an antagonist anti-CD40 antibody to inhibit these
CD40-
directed activities can be performed as described in the Examples herein. See,
also,
the assays described in Schultze et al. (1998) P~oc. Natl. Acad. Sci. ZISA
92:8200;
Demon et al. (1998) Pediatr Transplant. 2:6-15; Evans et al. (2000) J.
Immuraol.
164:688; Noelle (1998) Agents Actions Suppl. 49:17-22; Lederman et al. (1996)
Cur
Opira. Hematol. 3:77; Coligan et al. (1991) Current Protocols in Immunology
13:12;
Kwekkeboom et al. (1993) Immunology 79:439; and U.S. Patent Nos. 5,674,492 and
5,847,082; herein incorporated by reference.
A representative assay to detect antagonistic anti-CD40 antibodies that
specifically bind to the CD40 antigen and block C4BP-mediated CD40 signaling
in
the manner identified herein is a "competitive binding assay." Competitive
binding
assays are serological assays in which unknowns axe detected and quantitated
by their
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ability to inhibit the binding of a labeled known ligand, such as C4BP, to its
specific
receptor such as CD40. This is also referred to as a competitive inhibition
assay. In a
representative competitive binding assay, labeled CD40 polypeptide is
precipitated by
candidate antibodies in a sample, for example, in combination with monoclonal
antibodies raised against one or more epitopes of the antibodies of the
invention.
Anti-CD40 antibodies that specifically bind an epitope of interest can be
identified by
screening a series of antibodies prepared against a CD40 protein or fragment
of the
protein comprising the particular epitope of the CD40 protein of interest. For
example, for human CD40, epitopes of interest include epitopes comprising
linear
and/or nonlinear amino acid residues of the short isoform of human CD40 (see
GenBank Accession No. NP_690593, encoded by GenBank Accession No.
NM 152854), or of the long isoform of human CD40 (see GenBank Accession Nos.
CAA43045 and NP_001241, encoded by GenBank Accession Nos. X60592 and
NM 001250). Alternatively, competitive binding assays with previously
identified
suitable antagonist anti-CD40 antibodies could be used to select monoclonal
antibodies comparable to the previously identified antibodies.
Antibodies employed in such immunoassays may be labeled or unlabeled.
Unlabeled antibodies may be employed in agglutination or ELISA; labeled
antibodies
may be employed in a wide variety of assays, employing a wide variety of
labels.
Detection of the formation of an antibody-antigen complex between an anti-CD40
antibody and an epitope of interest can be facilitated by attaching a
detectable
substance to the antibody. Suitable detection means include the use of labels
such as
radionuclides, enzymes, coenzymes, fluorescers, chemiluminescers, chromogens,
enzyme substrates or co-factors, enzyme inhibitors, prosthetic group
complexes, free
radicals, particles, dyes, and the like. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, [3-galactosidase, or
acetylcholinesterase;
examples of suitable prosthetic group complexes include streptavidin/biotin
and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent
material is
luminol; examples of bioluminescent materials include luciferase, luciferin,
and
aequorin; and examples of suitable radioactive material include lash isih 3sS,
or 3H.
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Such labeled reagents may be used in a variety of well-known assays, such as
radioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescent immunoassays,
and the like. See for example, U.S. Patent Nos. 3,766,162; 3,791,932;
3,817,837; and
4,233,402.
Any of the previously described antagonist anti-CD40 antibodies or antigen-
binding fragments thereof may be conjugated prior to use in the methods of the
present invention. Methods for producing conjugated antibodies are known in
the art.
Thus, the anti-CD40 antibody may be labeled using an indirect labeling or
indirect
labeling approach. By "indirect labeling" or "indirect labeling approach" is
intended
that a chelating agent is covalently attached to an antibody and at least one
radionuclide is inserted into the chelating agent. See, for example, the
chelating
agents and radionuclides described in Srivastava and Mease (1991) Nucl. Med.
Bio.
18:589-603, herein incorporated by reference. Suitable labels include
fluorophores,
chromophores, radioactive atoms (particularly 32P and 12s1), electron-dense
reagents,
enzymes, and ligands having specific binding partners. Enzymes are typically
detected by their activity. For example, horseradish peroxidase is usually
detected by
its ability to convert 3,3 ',5,5 '-tetramethylbenzidine (TMB) to a blue
pigment,
quantifiable with a spectrophotometer. "Specific binding partner" refers to a
protein
capable of binding a ligand molecule with high specificity, as for example in
the case
of an antigen and a monoclonal antibody specific therefore. Other specific
binding
partners include biotin and avidin or streptavidin, IgG and protein A, and the
numerous receptor-ligand couples known in the art. It should be understood
that the
above description is not meant to categorize the various labels into distinct
classes, as
the same label may serve in several different modes. For example, lzsl may
serve as a
radioactive label or as an electron-dense reagent. Horseradish peroxidase
(HRP) may
serve as enzyme or as antigen for a monoclonal antibody. Further, one may
combine
various labels for desired effect. For example, monoclonal antibodies and
avidin also
require labels in the practice of this invention: thus, one might label a
monoclonal
antibody with biotin, and detect its presence with avidin labeled with lash or
with an
anti-biotin monoclonal antibody labeled with HRP. Other permutations and
possibilities will be readily apparent to those of ordinary skill in the art,
and are
considered as equivalents within the scope of the instant invention.
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Alternahvely, the anti-LD40 antibody may be labeled using "direct labeling"
or a "direct labeling approach," where a radionuclide is covalently attached
directly to
an antibody (typically via an amino acid residue). Preferred radionuclides are
provided in Srivagtava and Mease (1991) supra. The indirect labeling approach
is
particularly preferred. See also, for example, International Publication Nos.
WO
00/52031 and WO 00/52473, where a linker is used to attach a radioactive label
to
antibodies; and the labeled forms of anti-CD40 antibodies described in U.S.
Patent
No. 6,015,542; herein incorporated by reference.
Further, an antibody (or fragment thereof) may be conjugated to a therapeutic
moiety such as a cytotoxin, a therapeutic agent, or a radioactive metal ion or
radioisotope. A cytotoxin or cytotoxic agent includes any agent that is
detrimental to
cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide,
emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,
colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs thereof.
Therapeutic
agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-
mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine),
alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine
(BSNU)
and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,
streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin),
anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and
anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
Radioisotopes include, but are not limited to, I-131, I-123, I-125, Y-90, Re-
188, Re-
186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111, and the like. The
conjugated antibodies of the invention can be used for modifying a given
biological
response; the drug moiety is not to be construed as limited to classical
chemical
therapeutic agents. For example, the drug moiety may be a protein or
polypeptide
possessing a desired biological activity. Such proteins may include, for
example, a
toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a
protein such
as tumor necrosis factor, interferon-alpha, interferon-beta, nerve growth
factor,
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platelet derived growth factor, tissue plasminogen activator; or, biological
response
modifiers such as, for example, lymphokines, interleukin-1 ("IL-1 "),
interleukin-2
("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating
factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth
factors.
Techniques for conjugating such therapeutic moiety to antibodies are well
known. See, for example, Arnon et al. (1985) "Monoclonal Antibodies for
Immunotargeting of Drugs in Cancer Therapy, " in Monoclonal Antibodies and
Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.), pp. 243-256;
Hellstrom et al.
(1987) "Antibodies for Drug Delivery, " in Controlled Drug Delivery, ed.
Robinson et
al. (2d ed; Marcel Dekker, Tnc.), pp. 623-653; Thorpe (1985) "Antibody
Carriers of
Cytotoxic Agents in Cancer Therapy: A Review, " in Monocloraal Antibodies '84:
Biological and Clinical Applications, ed. Pinchera et al. (Editrice Kurtis,
Milano,
Italy, 1985), pp. 475-506; "Analysis, Results, and Future Prospective of the
Therapeutic Use of Radiolabeled Antibody in Cancer Therapy, " in Monoclonal
Antibodies for Cancer Detection and Therapy, ed. Baldwin et al. (Academic
Press,
New York, 1985), pp. 303-316; and Thorpe et al. (1982) Immunol. Rev. 62:119-
158.
Alternatively, an antibody can be conjugated to a second antibody to form an
antibody heteroconjugate as described in U.S. Patent No. 4,676,980. In
addition,
linkers may be used between the labels and the antibodies of the invention
(see U.S.
Patent No. 4,831,175). Antibodies, or antigen-binding fragments thereof, may
be
directly labeled with radioactive iodine, indium, yttrium, or other
radioactive particle
known in the art (U.S. Patent No. 5,595,721). Treatment may consist of a
combination of treatment with conjugated and nonconjugated antibodies
administered
simultaneously or subsequently (International Publication Nos. W~ 00/52031 and
WO 00/52473).
Variants of Antagonist Anti-CD40 Antibodies
Suitable biologically active variants of the antagonist anti-CD40 antibodies
can be used in the methods of the present invention. Such variants will retain
the
desired binding properties of the parent antagonist anti-CD40 antibody.
Methods for
making antibody variants are generally available in the art.
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For example, amino acid sequence variants of an antagonist anti-CD40
antibody can be prepared by mutations in the cloned DNA sequence encoding the
antibody of interest. Methods for mutagenesis and nucleotide sequence
alterations are
well known in the art. See, for example, Walker and Gaastra, eds. (1983)
Techniques
in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985)
PYOG. Natl. Acad. Sci. USA 82:488; Kunkel et al. (1987) Methods Enzymol.
154:367;
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring
Harbor, New York); U.S. Patent No. 4,873,192; and the references cited
therein;
herein incorporated by reference. Guidance as to appropriate amino acid
substitutions
that do not affect biological activity of the polypeptide of interest may be
found in the
model of Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure
(Natl.
Biomed. Res. Found., Washington, D.C.), herein incorporated by reference.
Conservative substitutions, such as exchanging one amino acid with another
having
similar properties, may be preferred. Examples of conservative substitutions
include,
but axe not limited to, Gly~Ala, Val~Ile~Leu, Asp~Glu, Lys~Arg, Asn~Gln,
and Phe~Trp~Tyr.
In constructing variants of the antagonist anti-CD40 antibody polypeptide of
interest, modifications are made such that variants continue to possess the
desired
activity, i.e., similar binding affinity and are capable of specifically
binding to a
human CD40 antigen expressed on the surface of a human cell thereby blocking
C4BP-mediated CD40 signaling, and being free of significant agonist activity
but
exhibiting antagonist activity when bound to a CD40 antigen on a human CD40-
expressing cell. Obviously, any mutations made in the DNA encoding the variant
polypeptide must not place the sequence out of reading frame and preferably
will not
create complementary regions that could produce secondary mRNA structure. See
EP
Patent Application Publication No. 75,444.
In addition, the constant region of an antagonist anti-CD40 antibody can be
mutated to alter effector function in a number of ways. For example, see U.S.
Patent
No. 6,737,05681 and U.S. Patent Application Publication No. 2004/0132101A1,
which disclose Fc mutations that optimize antibody binding to Fc receptors.
Preferably, variants of a reference antagonist anti-CD40 antibody have amino
acid sequences that have at least 70% or 75% sequence identity, preferably at
least
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80% or 85% sequence identity, more preferably at least 90%, 91%, 92%, 93%, 94%
or 95% sequence identity to the amino acid sequence for the reference
antagonist anti-
CD40 antibody molecule, or to a shorter portion of the reference antibody
molecule.
More preferably, the molecules share at least 96%, 97%, 98% or 99% sequence
identity. For purposes of the present invention, percent sequence identity is
determined using the Smith-Waterman homology search algorithm using an affine
gap search with a gap open penalty of 12 and a gap extension penalty of 2,
BLOSUM
matrix of 62. The Smith-Waterman homology search algorithm is taught in Smith
and Waterman (1981) Adv. Appl. Math. 2:482-489. A variant may, for example,
differ from the reference antagonist anti-CD40 antibody by as few as 1 to 15
amino
acid residues, as few as 1 to 10 amino acid residues, such as 6-10, as few as
5, as few
as 4, 3, 2, or even 1 amino acid residue.
With respect to optimal alignment of two amino acid sequences, the
contiguous segment of the variant amino acid sequence may have additional
amino
acid residues or deleted amino acid residues with respect to the reference
amino acid
sequence. The contiguous segment used for comparison to the reference amino
acid
sequence will include at least 20 contiguous amino acid residues, and may be
30, 40,
50, or more amino acid residues. Corrections for sequence identity associated
with
conservative residue substitutions or gaps can be made (see Smith-Waterman
homology search algorithm).
The precise chemical structure of an anti-CD40 antibody capable of
specifically binding CD40 and retaining antagonist activity, particularly when
bound
to CD40 antigen, depends on a number of factors. As ionizable amino and
carboxyl
groups are present in the molecule, a particular polypeptide may be obtained
as an
acidic or basic salt, or in neutral form. All such preparations that retain
their
biological activity when placed in suitable environmental conditions are
included in
the definition of antagonist anti-CD40 antibodies as used herein. Further, the
primary
amino acid sequence of the polypeptide may be augmented by derivatization
using
sugar moieties (glycosylation) or by other supplementary molecules such as
lipids,
phosphate, acetyl groups and the like. It may also be augmented by conjugation
with
saccharides. Certain aspects of such augmentation are accomplished through
post-
translational processing systems of the producing host; other such
modifications may
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be introduced iya vitro. In any event, such modifications are included in the
definition
of an anti-CD40 antibody used herein so long as the antagonist properties of
the anti-
CD40 antibody are not destroyed. It is expected that such modifications may
quantitatively or qualitatively affect the activity, either by enhancing or
diminishing
the activity of the antibody, in the various assays. Further, individual amino
acid
residues in the chain may be modified by oxidation, reduction, or other
derivatization,
and the antibody may be cleaved to obtain fragments that retain activity. Such
alterations that do not destroy antagonist activity do not remove the antibody
polypeptide sequence from the definition of anti-CD40 antibodies of interest
as used
herein.
The art provides substantial guidance regarding the preparation and use of
variants of antibodies. In preparing the anti-CD40 antibody variants, one of
skill in
the art can readily determine which modifications to the native nucleotide or
amino
acid sequence will result in a variant that is suitable for use as a
therapeutically active
component of a pharmaceutical composition used in the methods of the present
invention.
Methods of Thera~y Using the Antagonist Anti-CD40 Antibodies of the Invention
As the antagonist anti-CD40 antibodies provide a means for blocking C4BP-
mediated CD40 signaling, they can be used to inhibit one or more CD40-directed
activities as noted herein above. Thus the present invention provides a method
for
inhibiting a CD40-directed activity in a CD40-expressing cell, where the
method
comprises contacting the cell with an amount of an antagonist anti-CD40
antibody of
the invention effective to block C4BP-mediated CD40 signaling. As previously
noted, blocking of this signaling process can be the result of competitive
inhibition or
steric inhibition of the binding of C4BP to its binding site on CD40, or
prevention of
CD40 signal transduction with the binding of C4BP to its binding site on CD40,
so
long as binding of the anti-CD40 antibody to CD40 prevents C4BP-mediated CD40
signaling.
The antagonist anti-CD40 antibodies disclosed herein can be used to treat
patients having a disease mediated by C4BP stimulation of CD40 signaling on
CD40
expressing cells. By "CD40-expressing cell" is intended any cell type that
expresses
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the CD40 cell surface antigen, particularly B cells and other APCs, including
dendritic cells, and can be normal or malignant CD40-expressing cells. Methods
for
detecting CD40 expression in cells are well known in the art and include, but
are not
limited to, PCR techniques, immunohistochemistry, flow cytometry, Western
blot,
ELISA, and the like.
By "malignant B cell" is intended any neoplastic B cell, including but not
limited to B cells derived from lymphomas including low-, intermediate-, and
high-
grade B cell lymphomas, immunoblastic lymphomas, non-Hodgkin's lymphomas,
Hodgkin's disease, Epstein-Barr Virus (EBV) induced lymphomas, and AIDS-
related
lymphomas, as well as B cell acute lymphoblastic leukemias (ALLs), myelomas,
chronic lymphocytic leukemias (CLLs), acute myeloblastic leukemias, and the
like.
In other embodiments, the CD40-expressing cell is a solid tumor cell. By "CD40-
expressing solid tumor cell" is intended any malignant or pre-malignant cell
of a solid
tumor that expresses the CD40 cell-surface antigen. For purposes of the
present
invention, cancerous and precancerous or pre-malignant cells that express the
CD40
antigen are referred to as "CD40-expressing neoplastic cells." Further, where
CD40
ligand (CD40L) and C4BP act synergistically via CD40 activation, the anti-CD40
antibodies of the invention can be used to block C4BP-mediated CD40 signaling,
thereby having an effect on diseases that are mediated by CD40/CD40L
engagement.
"Treatment" is herein defined as the application or administration of an
antagonist anti-CD40 antibody or antigen-binding fragment thereof to a
patient, or
application or administration of an antagonist anti-CD40 antibody or fragment
thereof
to an isolated tissue or cell line from a patient, where the patient has a
disease, a
symptom of a disease, or a predisposition toward a disease, where the purpose
is to
cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect
the disease,
the symptoms of the disease, or the predisposition toward the disease. By
"treatment"
is also intended the application or administration of a pharmaceutical
composition
comprising the antagonist anti-CD40 antibodies or fragments thereof to a
patient, or
application or administration of a pharmaceutical composition comprising the
anti-
CD40 antibodies or fragments thereof to an isolated tissue or cell line from a
patient,
who has a disease, a symptom of a disease, or a predisposition toward a
disease,
where the purpose is to cure, heal, alleviate, relieve, alter, remedy,
ameliorate,
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improve, or affect the disease, the symptoms of the disease, or the
predisposition
toward the disease.
Therapy with at least one antagonist anti-CD40 antibody (or antigen-binding
fragment thereof) of the present invention causes a physiological response
that is
beneficial with respect to treatment of diseases associated with C4BP
stimulation of
CD40 signaling on CD40-expressing cells in a human, referred to herein as CD40-
associated diseases. Such CD40-associated diseases include, but are not
limited to,
hyperproliferative disorders, pre-malignant conditions, which may lead to
cancers,
cancers, including B cell-related cancers and solid tumors comprising CD40-
expressing neoplastic cells, and autoimmune and/or inflammatory diseases.
Thus, the
antagonist anti-CD40 antibodies of the invention could be used to treat
autoimmune
and/or inflammatory diseases such as systemic lupus, psoriasis, multiple
sclerosis,
inflammatory bowel disease (Crohn's disease), rheumatoid arthritis, and
rejection of
organ and tissue transplants, by suppressing autoimmune response, to treat
lymphomas by depriving malignant B lymphocytes of the activating signal
provided
by CD40, and to deliver toxins to CD40-bearing cells in a specific manner.
Thus, for example, the antagonist anti-CD40 antibodies of the invention find
use in the treatment of non-Hodgkin's lymphomas related to abnormal,
uncontrollable
B cell proliferation or accumulation. For purposes of the present invention,
such
lymphomas will be referred to according to the Wo~kih~ Formulation
classification
scheme, that is those B cell lymphomas categorized as low grade, intermediate
grade,
and high grade (see "The Non-Hodgkin's Lymphoma Pathologic Classification
Project," (1982) Cahce~ 49:2112). Thus, low-grade B cell lymphomas include
small
lyrnphocytic, follicular small-cleaved cell, and follicular mixed small-
cleaved and
large cell lymphomas; intermediate-grade lymphomas include follicular large
cell,
diffuse small cleaved cell, diffuse mixed small and large cell, and diffuse
large cell
lymphomas; and high-grade lymphomas include large cell immunoblastic,
lymphoblastic, and small non-cleaved cell lymphomas of the Burkitt's and non-
Burkitt's type.
It is recognized that the antagonist anti-CD40 antibodies of the invention are
useful in the therapeutic treatment of B cell lymphomas that are classified
according
to the Revised European and American Lymphoma Classification (REAL) system.
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Such B cell lymphomas include, but are not limited to, lymphomas classified as
precursor B cell neoplasms, such as B lymphoblastic leukemia/lymphoma;
peripheral
B cell neoplasms, including B cell chronic lymphocytic leukemia/small
lymphocytic
lymphoma, lymphoplasmacytoid lymphoma/immunocytoma, mantle cell lymphoma
(MCL), follicle center lymphoma (follicular) (including diffuse small cell,
diffuse
mixed small and large cell, and diffuse large cell lymphomas), marginal zone B
cell
lymphoma (including extranodal, nodal, and splenic types), hairy cell
leukemia,
plasmacytomal myeloma, diffuse large cell B cell lymphoma of the subtype
primary
mediastinal (thymic), Burkitt's lymphoma, and Burkitt's like high grade B cell
lymphoma; acute leukemias; acute lymphocytic leukemias (ALLs); myeloblastic
leukemias; acute myelocytic leukemias; promyelocytic leukemia; myelomonocytic
leukemia; monocytic leukemia; erythroleukemia; granulocytic leukemia (chronic
myelocytic leukemia); chronic lymphocytic leukemia (CLL); polycythemia vera;
multiple myeloma; Waldenstrom's macroglobulinemia; heavy chain disease; and
unclassifiable low-grade or high-grade B cell lymphomas.
The antagonist anti-CD40 antibodies of the invention may be useful in
preventing further tumor outgrowths arising during therapy, and can be useful
in the
treatment of subjects having low-grade B cell lymphomas, particularly those
subjects
having relapses following standard chemotherapy. Low-grade B cell lymphomas
are
more indolent than the intermediate- and high-grade B cell lymphomas and are
characterized by a relapsing/remitting course. Thus, treatment of these
lymphomas is
improved using the antagonist anti-CD40 antibodies of the invention, as
relapse
episodes can be reduced in number and severity.
Solid tumors that comprise CD40-expressing neoplastic cells include, but are
not limited to, ovarian, lung (for example, non-small cell lung cancer of the
squamous
cell carcinoma, adenocarcinoma, and large cell carcinoma types, and small cell
lung
cancer), breast, colon, kidney (including, for example, renal cell
carcinomas), bladder
(for example, urinary bladder carcinoma), liver (including, for example,
hepatocellular carcinomas), gastric, cervical, prostate, nasopharyngeal,
thyroid (for
example, thyroid papillary carcinoma), and skin cancers such as melanoma, and
sarcomas (including, for example, osteosarcomas and Ewing's sarcomas).
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When administered to a subject having a cancer comprising CD40-expressing
neoplastic cells, the antagonist anti-CD40 antibodies of the invention, and
antigen-
binding fragments thereof, can provide anti-tumor activity. By "anti-tumor
activity" is
intended a reduction in the rate of CD40-expressing neoplastic cell
proliferation or
accumulation, and hence a decline in growth rate of an existing tumor or in a
tumor
that arises during therapy, and/or destruction of existing neoplastic (tumor)
cells or
newly formed neoplastic cells, and hence a decrease in the overall size of a
tumor
during therapy.
Thus, the present invention provides methods for treating a cancer comprising
CD40-expressing cells, such as the B cell lymphomas and solid tumors, wherein
a
therapeutically effective amount of an antagonist anti-CD40 antibody of the
present
invention, or antigen-binding fragment thereof, is administered to a subject
having the
cancer. Administration of these antibodies, or antigen-binding fragment
thereof,
promotes a positive therapeutic response. By "positive therapeutic response"
with
respect to cancer treatment is intended an improvement in the disease in
association
with the anti-tumor activity of these antibodies or fragments thereof, and/or
an
improvement in the symptoms associated with the disease. Thus, for example, a
positive therapeutic response would refer to one or more of the following
improvements in the disease: (1) a reduction in tumor size; (2) a reduction in
the
number of cancer (i.e., neoplastic) cells; (3) an increase in neoplastic cell
death; (4)
inhibition of neoplastic cell survival; (4) inhibition (i.e., slowing to some
extent,
preferably halting) of tumor growth; (5) inhibition (i.e., slowing to some
extent,
preferably halting) of cancer cell infiltration into peripheral organs; (6)
inhibition (i.e.,
slowing to some extent, preferably halting) of tumor metastasis; (7) the
prevention of
further tumor outgrowths; (~) an increased patient survival rate; and (9) some
extent
of relief from one or more symptoms associated with the cancer. Such
therapeutic
responses may be further characterized as to degree of improvement. Thus, for
example, an improvement in the disease may be characterized as a complete
response.
By "complete response" is intended an absence of clinically detectable disease
with
normalization of any previously abnormal radiographic studies, bone marrow,
and
cerebrospinal fluid (CSF). Such a response must persist for at least one month
following treatment according to the methods of the invention. Alternatively,
an
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improvement in the cancer may be categorized as being a partial response. By
"partial response" is intended at least about a 50% decrease in all measurable
tumor
burden (i.e., the number of tumor cells present in the subject) in the absence
of new
lesions and persisting for at least one month. Such a response is applicable
to
measurable tumors only.
Tumor response can be assessed for changes in tumor morphology (i.e.,
overall tumor burden, tumor size, and the like) using screening techniques
such as
magnetic resonance imaging (MRI) scan, x-radiographic imaging, computed
tomographic (CT) scan, bioluminescent imaging, for example, luciferase
imaging,
bone scan imaging, and tumor biopsy sampling including bone marrow aspiration
(BMA). In addition to these positive therapeutic responses, the subject
undergoing
therapy with the antagonist anti-CD40 antibody or antigen-binding fragment
thereof
may experience the beneficial effect of an improvement in the symptoms
associated
with the disease. Thus for B cell tumors, the subj ect may experience a
decrease in the
so-called B symptoms, i.e., night sweats, fever, weight loss, and/or
urticaria.
The antagonist anti-CD40 antibodies described herein may also find use in the
treatment of other CD40-associated diseases where blocking of C4BP-mediated
CD40
signaling results in inhibition of one or more CD40-directed activities, and
for which
such inhibition results in an improvement in the disease, or at least reduces
one or
more undesirable symptoms of the disease. Thus, where C4BP-mediated CD40
signaling is associated with an undesirable immune response or process ih
vivo, such
as occurs with diseases or disorders having an autoimmune and/or inflammatory
component, an antagonist anti-CD40 antibody of the invention can be
administered to
an at-risk subject or subject in need of treatment for one or more of these
CD40-
associated diseases in order to block C4BP-mediated CD40 signaling, thereby
inhibiting or preventing the symptoms associated with the respective CD40-
associated
disease.
Inflammatory diseases are characterized by inflammation and tissue
destruction, or a combination thereof. "Inflammatory disease" includes any
inflammatory immune-mediated process where the initiating event or target of
the
immune response involves non-self antigen(s), including, for example,
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alloantigens, xenoantigens, viral antigens, bacterial antigens, unknown
antigens,
or allergens.
Further, for purposes of the present invention, the term "inflammatory
disease(s)" includes "autoimmune disease(s)." As used herein, the term
"autoimmunity" is generally understood to encompass inflammatory immune-
mediated processes involving "self' antigens. In autoimmune diseases, self
antigens)
trigger host immune responses.
Also, the present invention includes treatment of inflammation associated with
tissue transplant rejection. "Transplant rejection" or "graft rejection"
refers to any
host-mounted immune response against a graft including but not limited to HLA
antigens, blood group antigens, and the like.
The invention can also be used to treat graft versus host disease, such as
that
associated with bone marrow transplantation, for example. In such graft versus
host
disease, the donor bone marrow includes lymphocytes and cells that mature into
lymphocytes. The donor's lymphocytes recognize the recipient's antigens as non-
self
and mount an inflammatory immune response. Hence, as used herein, "graft
versus
host disease" or "graft versus host reaction" refers to any T cell mediated
immune
response in which donor lymphocytes react to the host's antigens.
Thus, the antagonist anti-CD40 antibodies and antigen-binding fragments
thereof described herein can be used in accordance with the methods of the
invention
to treat autoimmune and/or inflammatory disorders including, but not limited
to,
systemic lupus erythematosus (SLE), discoid lupus, lupus nephritis,
sarcoidosis,
inflammatory arthritis, including juvenile arthritis, rheumatoid arthritis,
psoriatic
arthritis, Reiter's syndrome, ankylosing spondylitis, and gouty arthritis,
rejection of an
organ or tissue transplant, hyperacute, acute, or chronic rej ection andlor
graft versus
host disease, multiple sclerosis, hyper IgE syndrome, polyarteritis nodosa,
primary
biliary cirrhosis, inflammatory bowel disease, Crohn's disease, celiac's
disease
(gluten-sensitive enteropathy), autoimmune hepatitis, pernicious anemia,
autoimmune
hemolytic anemia, psoriasis, scleroderma, myasthenia gravis, autoimmune
thrombocytopenic purpura, autoimmune thyroiditis, Grave's disease, Hasimoto's
thyroiditis, immune complex disease, chronic fatigue immune dysfunction
syndrome
(CFIDS), polymyositis and dermatomyositis, cryoglobulinemia, thrombolysis,
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caxdiomyopathy, pemphigus vulgaris, pulmonary interstitial fibrosis, Type I
and Type
II diabetes mellitus, type 1, 2, 3, and 4 delayed-type hypersensitivity,
allergy or
allergic disorders, unwanted/unintended immune responses to therapeutic
proteins
(see for example, U.S. Patent Application No. US 2002/0119151 and Koren, et
al.
(2002) Cuf~~. Pharm. BiotecTznol. 3:349-60), asthma, Churg-Strauss syndrome
(allergic granulomatosis), atopic dermatitis, allergic and irritant contact
dermatitis,
urtecaria, IgE-mediated allergy, atherosclerosis, vasculitis, idiopathic
inflammatory
myopathies, hemolytic disease, Alzheimer's disease, chronic inflammatory
demyelinating polyneuropathy, and the like. In some other embodiments, the
antagonistic anti-CD40 antibodies of the invention are useful in treating
pulmonary
inflammation including but not limited to lung graft rejection, asthma,
sarcoidosis,
emphysema, cystic fibrosis, idiopathic pulmonary fibrosis, chronic bronchitis,
allergic
rhinitis and allergic diseases of the lung such as hypersensitivity
pneumonitis,
eosinophilic pneumonia, bronchiolitis obliterans due to bone marrow and/or
lung
transplantation or other causes, graft atherosclerosis/graft phlebosclerosis,
as well as
pulmonary fibrosis resulting from collagen, vascular, and autoimmune diseases
such
as rheumatoid arthritis and lupus erythematosus.
By "anti-inflammatory activity" is intended a reduction or prevention of
inflammation. Therapy with at least one anti-CD40 antibody or antigen-binding
fragment thereof as defined elsewhere herein causes a physiological response
that is
beneficial with respect to treatment of an autoimmune disease and/or
inflammatory
disease, where the disease involves cells expressing the CD40 antigen. It is
recognized that the methods of the invention may be useful in preventing
phenotypic
change in cells such as proliferation, activation, and the like.
By "positive therapeutic response" with respect to an autoimmune disease
and/or inflammatory disease is intended an improvement in the disease in
association
with the anti-inflammatory activity of these antibodies or antigen-binding
fragments
thereof, and/or an improvement in the symptoms associated with the disease.
That is,
an anti-proliferative effect, the prevention of further proliferation of the
CD40-
expressing cell, a reduction in the inflammatory response including but not
limited to
reduced secretion of inflammatory cytokines, adhesion molecules, proteases,
immunoglobulins (in instances where the CD40 bearing cell is a B cell),
combinations
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thereof, and the like, increased production of anti-inflammatory proteins, a
reduction
in the number of autoreactive cells, an increase in immune tolerance,
inhibition of
autoreactive cell survival, and/or a decrease in one or more symptoms mediated
by
stimulation of CD40-expressing cells can be observed. Such positive
therapeutic
responses are not limited to the route of administration and may comprise
administration to the donor, the donor tissue (such as for example organ
perfusion),
the host, any combination thereof, and the like.
For subj ects undergoing therapy for an autoimmune and/or inflammatory
disease, clinical response can be assessed using screening techniques such as
magnetic resonance imaging (MRI) scan, x-radiographic imaging, computed
tomographic (CT) scan, flow cytometry or fluorescence-activated cell sorter
(FACS)
analysis, histology, gross pathology, and blood chemistry, including but not
limited to
changes detectable by ELISA, RIA, chromatography, and the like. In addition to
these positive therapeutic responses, the subject undergoing therapy with the
antagonist anti-CD40 antibody or antigen-binding fragment thereof may
experience
the beneficial effect of an improvement in the symptoms associated with the
disease.
By "therapeutically effective dose or amount" is intended an amount of
antagonist anti-CD40 antibody or antigen-binding fragment thereof that, when
administered, brings about a positive therapeutic response with respect to
treatment of
a subject with a CD40-associated disease. In some embodiments of the
invention, a
therapeutically effective dose of the antagonist anti-CD40 antibody or
fragment
thereof is in the range from about 0.003 mg/kg to about 50 mg/kg, from about
0.01
mg/kg to about 40 mg/kg, from about 0.01 mg/kg to about 30 mg/kg, from about
0.1
mg/kg to about 30 mg/kg, from about 1 mglkg to about 30 mg/kg, from about 3
mg/kg
to about 30 mg/kg, from about 3 mg/kg to about 25 mglkg, from about 3 mg/kg to
about 20 mg/kg, from about 5 mg/kg to about 15 mg/kg, or from about 7 mg/kg to
about 12 mg/kg. It is recognized that the method of treatment may comprise a
single
administration of a therapeutically effective dose or multiple administrations
of a
therapeutically effective dose of the antagonist anti-CD40 antibody or antigen-
binding
fragment thereof.
A further embodiment of the invention is the use of antagonist anti-CD40
antibodies for diagnostic monitoring of protein levels in tissue as part of a
clinical
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testing procedure, e.g., to determine the efficacy of a given treatment
regimen.
Detection can be facilitated by coupling the antibody to a detectable
substance.
Examples of detectable substances include various enzymes, prosthetic groups,
fluorescent materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include horseradish
peroxidase,
alkaline phosphatase, (3-galactosidase, or acetylcholinesterase; examples of
suitable
prosthetic group complexes include streptavidin/biotin and avidinlbiotin;
examples of
suitable fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride
or
phycoerythrin; an example of a luminescent material includes luminol; examples
of
bioluminescent materials include luciferase, luciferin, and aequorin; and
examples of
suitable radioactive material include lzsh lsy~ ssS~ or 3H.
The antagonist anti-CD40 antibodies can be used in combination with known
chemotherapeutics, cytokines, and/or other monoclonal antibodies, including
other
antagonist anti-CD40 antibodies having a different mode of action, for the
treatment
of CD40-associated diseases. For example, the antagonist anti-CD40 antibodies
of
the invention can be used in combination with cytokines such as interleukin-2.
In
another embodiment, the anti-CD40 antibodies of the invention can be used in
combination with, for example, other monoclonal antibodies, such as rituximab
(B7EC-C2B8; Rituxan~; mEC Pharmaceuticals Corp., San Diego, California) for
treatment of a B cell lymphoma. In yet other embodiments, the anti-CD40
antibodies
of the invention can be used in combination with anti-CD40 monoclonal
antibodies
that block CD40L-mediated CD40 signaling. Such a combination would potentially
be useful for treating autoimmune diseases and/or inflammatory diseases,
including,
but not limited to, organ and tissue transplant rejection. Where the subject
is
undergoing transplantation of a tissue or organ, the antagonist anti-CD40
antibodies
can be used in combination with other therapeutic agents that inhibit
rejection of the
transplanted tissue/organ. Such therapeutic agents include, but are not
limited to,
corticosteroids, cyclosporine, and azathioprine. Where multiple therapeutic
agents axe
used in combination, the individual agents can be administered sequentially,
in either
order, or simultaneously (i.e., concurrently or within the same time frame).
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In this manner, where a subject is being treated for a B cell-related cancer,
including, but not limited to, those disclosed herein above, the antagonist
anti-CD40
antibodies described herein, or antigen-binding fragments thereof, are
administered in
combination with at least one other cancer therapy, including, but not limited
to,
surgery or surgical procedures (e.g. splenectomy, hepatectomy,
lymphadenectomy,
leukophoresis, bone marrow transplantation, and the like); radiation therapy;
chemotherapy, optionally in combination with autologous bone marrow
transplant,
where suitable chemotherapeutic agents include, but are not limited to,
fludarabine or
fludarabine phosphate, chlorambucil, vincristine, pentostatin, 2-
chlorodeoxyadenosine
(cladribine), cyclophosphamide, doxorubicin, prednisone, and combinations
thereof,
for example, anthracycline-containing regimens such as CAP (cyclophosphamide,
doxorubicin plus prednisone), CHOP (cyclophosphamide, vincristine, prednisone
plus
doxorubicin), VAD (vincritsine, doxorubicin, plus dexamethasone), MP
(melphalan
plus prednisone), and other cytotoxic and/or therapeutic agents used in
chemotherapy
such as mitoxantrone, daunorubicin, idarubicin, asparaginase, and
antimetabolites,
including, but not limited to, cytarabine, methotrexate, 5-fluorouracil
decarbazine, 6-
thioguanine, 6-mercaptopurine, and nelarabine; other anti-cancer monoclonal
antibody therapy (for example, alemtuzumab (Campath~) or other anti-CD52
antibody targeting the CD52 cell-surface glycoprotein on malignant B cells;
rituximab
(Rituxan ), the fully human antibody HuMax-CD20, R-1594, llVIMIJ-106, TRU-015,
AME-133, tositumomab/I-131 tositumomab (Bexxar~), ibritumomab tiuxetan
(Zevalin~), or any other therapeutic anti-CD20 antibody targeting the CD20
antigen
on malignant B cells; anti-CD19 antibody (for example, MT103, a bispecific
antibody); anti-CD22 antibody (for example, the humanized monoclonal antibody
epratuzumab); bevacizumab (Avastin~) or other anti-cancer antibody targeting
human vascular endothelial growth factor; anti-CD22 antibody targeting the
CD22
antigen on malignant B cells (for example, the monoclonal antibody BL-22, an
alphaCD22 toxin); a M-CSF antibody targeting macrophage colony stimulating
factor; antibodies targeting the receptor activator of nuclear factor-kappaB
(RANK)
and its ligand (RANI~I,), which are overexpressed in multiple myeloma; anti-
CD23
antibody targeting the CD23 antigen on malignant B cells (for example, IDEC-
152);
anti-CD80 antibody targeting the CD80 antigen (for example, iDEC-114); anti-
CD38
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antibody targeting the CD38 antigen on malignant B cells; antibodies targeting
major
histocompatibility complex class II receptors (anti-MHC antibodies) expressed
on
malignant B cells; other anti-CD40 antibodies targeting the CD40 antigen on
malignant B cells (for example, SGN-40; and other antagonist anti-CD40
antibodies,
such as CHIR-12.12 and CHIR-5.9, and antigen-binding fragments thereof, that
block
CD40L-mediated CD40 signaling on CD40-expressing cells, as disclosed in
International Patent Application No. PCT/LTS2004/037152 (Attorney Docket No.
PP20107.004 (0357841282916)), also entitled "Antagonist Anti-CD40 Monocloraal
Antibodies and Methods for Them Use," filed November 4, 2004)); and antibodies
targeting tumor necrosis factor-related apoptosis-inducing ligand receptor 1
(TRAIL-
Rl) (for example, the agonistic human monoclonal antibody HGS-ETRl) and
TRAIL-R2 expressed on a number of solid tumors and tumors of hematopoietic
origin); small molecule-based cancer therapy, including, but not limited to,
microtubule and/or topoisomerase inhibitors (for example, the mitotic
inhibitor
dolastatin and dolastatin analogues; the tubulin-binding agent T900607; XLl
19; and
the topoisomerase inhibitor aminocamptothecin), SDX-105 (bendamustine
hydrochloride), ixabepilone (an epothilone analog, also referred to as BMS-
247550),
protein kinase C inhibitors, for example, midostaurin ((PKC-412, CGP 41251, N-
benzoylstaurosporine), pixantrone, eloxatin (an antineoplastic agent), ganite
(gallium
nitrate), Thalomid~ (thalidomide), immunomodulatory derivatives of thalidomide
(for
example, revlimid (formerly revimid)), AffinitakTM (antisense inhibitor of
protein
kinase C-alpha), SDX-101 (R-etodolac, inducing apoptosis of malignant
lymphocytes), second-generation purine nucleoside analogs such as clofarabine,
inhibitors of production of the protein Bcl-2 by cancer cells (for example,
the
antisense agents oblimersen and Genasense~), proteasome inhibitors (for
example,
VelcadeTM (bortezomib)), small molecule kinase inhibitors (for example, CHIR-
258),
small molecule VEGF inhibitors (for example, ZD-6474), small molecule
inhibitors
of heat shock protein (HSP) 90 (for example, 17-AAG), small molecule
inhibitors of
histone deacetylases (for example, hybrid/polar cytodifferentiation HPC)
agents such
as suberanilohydroxamic acid (SARA), and FR-901228) and apoptotic agents such
as
Trisenox~ (arsenic trioxide) and Xcytrin~ (motexafin gadolinium); vaccine
/immunotherapy-based cancer therapies, including, but not limited to, vaccine
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approaches (for example, Id-KLH, oncophage, vitalethine), personalized
immunotherapy or active idiotype immunotherapy (for example, MyVaX
Personalized hnmunotherapy, formally designated GTOP-99), Promune~ (CpG 7909,
a synthetic agonist for toll-like receptor 9 (TLR9)), interferon-alpha
therapy,
interleukin-2 (IL-2) therapy, IL-12 therapy, IL-15 therapy, and IL-21 therapy;
steroid
therapy; or other cancer therapy; where the additional cancer therapy is
administered
prior to, during, or subsequent to the antagonist anti-CD40 antibody therapy.
Where a subject is being treated for a solid tumor comprising CD40-
expressing neoplastic cells, including, but not limited to, the solid tumors
disclosed
herein above, the antagonist anti-CD40 antibodies described herein, or antigen-
binding fragments thereof, can be administered in combination with at least
one other
cancer therapy, including, but not limited to, surgery, radiation therapy,
chemotherapy, cytokine therapy, or other monoclonal antibody intended for use
in
treatment of the solid tumor of interest, where the additional cancer therapy
is
administered prior to, during, or subsequent to the anti-CD40 antibody
therapy.
Thus, where the combined therapies comprise administration of an antagonist
anti-CD40 antibody or antigen-binding fragment thereof in combination with
administration of another therapeutic agent, as with chemotherapy, radiation
therapy,
other anti-cancer antibody therapy, small molecule-based cancer therapy, or
vaccine/immunotherapy-based cancer therapy, the methods of the invention
encompass coadministration, using sepaxate formulations or a single
pharmaceutical
formulation, or and consecutive administration in either order. Where the
methods of
the present invention comprise combined therapeutic regimens, these therapies
can be
given simultaneously, i.e., the antagonist anti-CD40 antibody or antigen-
binding
fragment thereof is administered concurrently or within the same time frame as
the
other cancer therapy (i.e., the therapies are going on concurrently, but the
antagonist
anti-CD40 antibody or antigen-binding fragment thereof is not administered
precisely
at the same time as the other cancer therapy). Alternatively, the antagonist
anti-CD40
antibody of the present invention or antigen-binding fragment thereof may also
be
administered prior to or subsequent to the other cancer therapy. Sequential
administration of the different cancer therapies may be performed regardless
of
whether the treated subject responds to the first course of therapy to
decrease the
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possibility of remission or relapse. Where the combined therapies comprise
administration of the antagonist anti-CD40 antibody or antigen-binding
fragment
thereof in combination with administration of a cytotoxic agent, preferably
the
antagonist anti-CD40 antibody or antigen-binding fragment thereof is
administered
prior to administering the cytotoxic agent.
In some embodiments of the invention, the subject has a B-cell related cancer
and the antagonist anti-CD40 antibodies described herein, or antigen-binding
fragments thereof, are administered in combination with chemotherapy, and
optionally in combination with autologous bone marrow transplantation, wherein
the
antibody and the chemotherapeutic agents) may be administered sequentially, in
either order, or simultaneously (i.e., concurrently or within the same time
frame).
Examples of suitable chemotherapeutic agents include, but are not limited to,
fludarabine or fludarabine phosphate, chlorambucil, vincristine, pentostatin,
2-
chlorodeoxyadenosine (cladribine), cyclophosphamide, doxorubicin, prednisone,
and
combinations thereof, for example, anthracycline-containing regimens such as
CAP
(cyclophosphamide, doxorubicin plus prednisone), CHOP (cyclophosphamide,
vincristine, prednisone plus doxorubicin), VAD (vincritsine, doxorubicin, plus
dexamethasone), MP (melphalan plus prednisone), and other cytotoxic and/or
therapeutic agents used in chemotherapy such as mitoxantrone, daunorubicin,
idarubicin, asparaginase, and antimetabolites, including, but not limited to,
cytarabine,
methotrexate, 5-fluorouracil decarbazine, 6-thioguanine, 6-mercaptopurine, and
nelarabine. In some embodiments, the antagonist anti-CD40 antibody disclosed
herein, or an antigen-binding fragment thereof, is administered prior to
treatment with
the chemotherapeutic agent. In alternative embodiments, the antagonist anti-
CD40
antibody or antigen-binding fragment thereof is administered after treatment
with the
chemotherapeutic agent. In yet other embodiments, the chemotherapeutic agent
is
administered simultaneously with the antagonist anti-CD40 antibody or antigen
binding fragment thereof.
Thus, for example, in some embodiments, the antagonist anti-CD40 antibody
or antigen-binding fragment thereof is administered to a subject with a B cell-
related
cancer in combination with fludarabine or fludarabine phosphate. In one such
embodiment, the antagonist anti-CD40 antibody or antigen-binding fragment
thereof
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is administered prior to administration of fludarabine or fludarabine
phosphate. In
alternative embodiments, the antagonist anti-CD40 antibody or antigen-binding
fragment thereof is administered after treatment with fludarabine or
fludarabine
phosphate. In yet other embodiments, the fludarabine or fludarabine phosphate
is
administered simultaneously with the antagonist anti-CD40 antibody or antigen-
binding fragment thereof.
In other embodiments of the invention, chlorambucil, an alkylating drug, is
administered to a subject with a B cell-related cancer in combination with an
antagonist anti-CD40 antibody described herein or an antigen-binding fragment
thereof. In one such embodiment, the antagonist anti-CD40 antibody or antigen-
binding fragment thereof is administered prior to administration of
chlorambucil. In
alternative embodiments, the antagonist anti-CD40 antibody or antigen-binding
fragment thereof is administered after treatment with chlorambucil. In yet
other
embodiments, the chlorambucil is administered simultaneously with the
antagonist
anti-CD40 antibody or antigen-binding fragment thereof.
In yet other embodiments, anthracycline-containing regimens such as CAP
(cyclophosphamide, doxorubicin plus prednisone) and CHOP (cyclophosphamide,
vincristine, prednisone plus doxorubicin) may be combined with administration
of an
antagonist anti-CD40 antibody described herein or antigen-binding fragment
thereof.
In one such embodiment, the antagonist anti-CD40 antibody or antigen-binding
fragment thereof is administered to a subject with a B cell-related cancer
prior to
administration of anthracycline-containing regimens. In other embodiments, the
antagonist anti-CD40 antibody or antigen-binding fragment thereof is
administered to
the subject after treatment with anthracycline-containing regimens. In yet
other
embodiments, the anthracycline-containing regimen is administered to the
subject
simultaneously with the antagonist anti-CD40 antibody or antigen-binding
fragment
thereof.
In alternative embodiments, an antagonist anti-CD40 antibody described
herein or an antigen-binding fragment thereof is administered to a subject
with a B
cell-related cancer in combination with alemtuzumab (Campath~; distributed by
Berlex Laboratories, Richmond, California). Alemtuzumab is a recombinant
humanized monoclonal antibody (Campath-1H) that targets the CD52 antigen
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expressed on malignant B cells. In one such embodiment, the antagonist anti-
CD40
antibody or antigen-binding fragment thereof is administered prior to
administration
of alemtuzumab. In other embodiments, the antagonist anti-CD40 antibody or
antigen-binding fragment thereof is administered after treatment with
alemtuzumab.
In yet other embodiments, the alemtuzumab is administered simultaneously with
the
antagonist anti-CD40 antibody or antigen-binding fragment thereof.
In alternative embodiments, an antagonist anti-CD40 antibody described
herein or antigen-binding fragment thereof is administered to a subj ect with
a B cell-
related cancer in combination with a therapeutic anti-CD20 antibody targeting
the
CD20 antigen on malignant B cells, for example, rituximab (Rituxan ), the
fully
human antibody HuMax-CD20, R-1594, IIVIMU-106, TRU-015, AME-133,
tositumomab/I-131 tositumomab (Bexxar~), or ibritumomab tiuxetan (Zevalin~).
In
one such embodiment, the antagonist anti-CD40 antibody or antigen-binding
fragment
thereof is administered to the subject prior to administration of the anti-
CD20
antibody. In other embodiments, the antagonist anti-CD40 antibody or antigen-
binding fragment thereof is administered to the subject after treatment with
the anti-
CD20 antibody. In yet other embodiments, the anti-CD20 antibody is
administered to
the subject simultaneously with the antagonist anti-CD40 antibody or antigen-
binding
fragment thereof.
~ther examples of monoclonal antibodies intended for treatment of B cell-
related cancers that can be used in combination with the anti-CD40 antibodies
of the
present invention include, but are not limited to, other antagonist anti-CD40
antibodies that block CD40L-mediated CD40 signaling, including, for example,
the
fully human monoclonal antibodies CH1R-12.12 and CHIR-5.9, as disclosed in
International Patent Application No. PCT/LTS2004/037152 (Attorney Docket No.
PP20107.004 (035784/282916)), also entitled "Antagonist Anti-CD40 Monoclonal
Antibodies and Methods fog Them Use," filed November 4, 2004)).
In alternative embodiments, an antagonist anti-CD40 antibody described
herein or antigen-binding fragment thereof is administered to a subject with a
B cell-
related cancer in combination with a small molecule-based cancer therapy,
including,
but not limited to, microtubule and/or topoisomerase inhibitors (for example,
the
mitotic inhibitor dolastatin and dolastatin analogues; the tubulin-binding
agent
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T900607; XL119; and the topoisomerase inhibitor aminocamptothecin), SDX-105
(bendamustine hydrochloride), ixabepilone (an epothilone analog, also referred
to as
BMS-247550), protein kinase C inhibitors, for example, midostaurin ((PKC-412,
CGP 41251, N-benzoylstaurosporine), pixantrone, eloxatin (an antineoplastic
agent),
ganite (gallium nitrate), Thalomid~ (thalidomide), immunomodulatory
derivatives of
thalidomide (for example, revlimid (formerly revimid)), AffinitakTM (antisense
inhibitor of protein kinase C-alpha), SDX-101 (R-etodolac, inducing apoptosis
of
malignant lymphocytes), second-generation purine nucleoside analogs such as
clofarabine, inhibitors of production of the protein Bcl-2 by cancer cells
(for example,
the antisense agents oblimersen and Genasense~), proteasome inhibitors (for
example,
VelcadeTM (bortezomib)), small molecule kinase inhibitors (for example, CHIR-
258),
small molecule VEGF inhibitors (for example, ZD-6474), small molecule
inhibitors
of heat shock protein (HSP) 90 (for example, 17-AAG), small molecule
inhibitors of
histone deacetylases (for example, hybrid/polar cytodifferentiation HPC)
agents such
as suberanilohydroxamic acid (SAHA), and FR-901228) and apoptotic agents such
as
Trisenox (arsenic trioxide) and Xcytrin~ (motexafin gadolinium). In one such
embodiment, the antagonist anti-CD40 antibody or antigen-binding fragment
thereof
is administered to the subject prior to administration of the small molecule-
based
cancer therapy. In other embodiments, the antagonist anti-CD40 antibody or
antigen-
binding fragment thereof is administered to the subj ect after treatment with
the small
molecule-based cancer therapy. In yet other embodiments, the small molecule-
based
cancer therapy is administered to the subject simultaneously with the
antagonist anti-
CD40 antibody or antigen-binding fragment thereof.
In yet other embodiments, an antagonist anti-CD40 antibody described herein
or an antigen-binding fragment thereof can be administered to a subj ect with
a B cell-
related cancer in combination with vaccine/immunotherapy-based cancer therapy,
including, but not limited to, vaccine approaches (for example, Id-KLH,
oncophage,
vitalethine), personalized immunotherapy or active idiotype immunotherapy (for
example, MyVax~ Personalized Immunotherapy, formally designated GTOP-99),
Promune~ (CpG 7909, a synthetic agonist for toll-like receptor 9 (TLR9)),
interferon-
alpha therapy, interleukin-2 (IL-2) therapy, IL-12 therapy, IL-15 therapy, or
IL-21
therapy; or steroid therapy. In one such embodiment, the antagonist anti-CD40
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antibody or antigen-binding fragment thereof is administered to the subject
prior to
administration of the vaccine/immunotherapy-based cancer therapy. In other
embodiments, the antagonist anti-CD40 antibody or antigen-binding fragment
thereof
is administered to the subject after treatment with the vaccine/immunotherapy-
based
cancer therapy. In yet other embodiments, the vaccine/immunotherapy-based
cancer
therapy is administered to the subject simultaneously with the antagonist anti-
CD40
antibody or antigen-binding fragment thereof.
In one such embodiment, an antagonist anti-CD40 antibody described herein
or an antigen-binding fragment thereof can be used in combination with IL-2.
IL-2,
an agent known to expand the number of natural killer (NK) effector cells in
treated
patients, can be administered prior to, or concomitantly with, the antagonist
anti-
CD40 antibody of the invention or antigen-binding fragment thereof. Where the
antagonist anti-CD40 antibody of the invention, or antigen-binding fragment
thereof,
has antibody-dependent cell-mediated cytotoxicity (ADCC) as another mode of
action, the expanded number of NIA effector cells with IL-2 administration may
lead
to enhanced ADCC activity of the administered antagonist anti-CD40 antibody or
antigen-binding fragment thereof. In other embodiments, IL-21 serves as the
immunotherapeutic agent to stimulate NK cell activity when administered in
combination with an antagonist anti-CD40 antibody described herein or an
antigen-
binding fragment thereof.
In some embodiments of the invention, the subject has a solid tumor
comprising CD40-expressing neoplastic cells, and the anti-CD40 antibodies
described
herein, or antigen-binding fragments thereof, are administered to this subject
in
combination with chemotherapy or cytokine therapy, wherein the antibody and
the
chemotherapeutic agents) or cytokine(s) may be administered sequentially, in
either
order, or simultaneously (i.e., concurrently or within the same time frame).
Examples
of suitable chemotherapeutic agents for subjects having a solid tumor
comprising
CD40-expressing neoplastic cells include, but are not limited to, CPT-11
(Irinotecan),
which can be used, for example, in treating colorectal cancer and non-small
cell lung
cancer; gemcitabine, which can be used, for example, in treating lung cancer,
breast
cancer, and epithelial ovarian cancer; and other chemotherapeutic agents
suitable for
treatment of solid tumors. Cytokines of interest include, but are not limited
to, alpha
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interferon, gamma interferon, interleukin-2 (IL-2), IL-12, IL-15, and IL-21,
granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony
stimulating factor (G-CSF), or biologically active variants of these
cytokines.
In other embodiments of the invention, the anti-CD40 antibodies described
herein, or antigen-binding fragments thereof, are administered to a subject
with a solid
tumor comprising CD40-expressing neoplastic cells in combination with other
monoclonal antibodies intended for treatment of the solid tumor. Thus, for
example,
where the subj ect is undergoing treatment for a breast cancer comprising CD40-
expressing carcinoma cells, therapy could include administration of effective
amounts
of an antagonist anti-CD40 antibody described herein, or antigen-binding
fragment
thereof, in combination with administration of effective amounts of Herceptin~
(Genentech, Inc., San Francisco, California), which targets the Her2 receptor
protein
on Her2+ breast cancer cells. Similarly, where the subject is undergoing
treatment for
colorectal cancer comprising CD40-expressing carcinoma cells, therapy could
include
administration of effective amounts of an antagonist anti-CD40 antibody
described
herein, or antigen-binding fragment thereof, in combination with
administration of
effective amounts of the humanized monoclonal antibody AvastinTM (also known
as
bevacizumab; Genentech, Inc., San Francisco, California), which binds to and
inhibits
vascular endothelial growth factor (VEGF), a protein that plays a critical
role in tumor
angiogenesis. Other examples of monoclonal antibodies intended for treatment
of
solid tumors that can be used in combination with the anti-CD40 antibodies of
the
present invention include, but are not limited to, anti-EGFR antibody
targeting the
epidermal growth factor receptor (for example, IMC-C225 (ImClone Systems, New
York, New York) (see, for example, Mendelsohn and Baselga (2000) Oncogene
19:6550-6565 and Solbach et al. (2002) Int. J. Cancer 101:390-394); anti-IGF-1
receptor antibody, targeting the IGF-1 receptor protein (see, for example,
Maloney et
al. (2003) Cartce~ Res. 63:5073-5083 and Hailey et al. (2002) Mol. Cancer.
They.
1:1349-1353; anti-MUC1 antibody, targeting the tumor-associated antigen MUC1;
anti-a5,~1, anti-av~i5, and anti-av~i3, targeting these respective integrins,
which
regulate cell adhesion and signaling processes involved in cell proliferation
and
survival (see, for example, Laidler et al. (2000) Acta Biochirnica Polonica
47(4):1159-1170 and Cruet-Hennequart et al. (2003) Oncogene 22(11):1688-1702);
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anti-P-cadherin antibody, targeting this cadherin family member (see, for
example,
copending U.S. Patent Application 20030194406); anti-VE-cadherin antibody,
targeting angiogenic-related function of this endothelial cell-specific
adhesion
molecule (see, for example, Liao et al. (2002) Cancer Res. 62:2567-2575); and
other
antagonist anti-CD40 antibodies that block CD40L-mediated CD40 signaling,
including, for example, the fully human monoclonal antibodies CHIR-12.12 and
CHIR-5.9, as disclosed in International Patent Application No.
PCT/LTS2004/037152
(Attorney Docket No. PP20107.004 (035784/282916)), also entitled "Antagonist
Ahti-
CD40 MorZOClofaal Ayatibodies arad Methods for Their Use," filed November 4,
2004)).
Combination therapies are also contemplated for subjects having a CD40-
associated disease that comprises an autoimmune and/or inflammatory component.
In
this manner, where a subject is being treated for an autoimmune and/or
inflammatory
disease, including but not limited to the diseases disclosed herein, the
antagonist anti-
CD40 antibodies of the invention that target C4BP-mediated CD40 signaling, or
antigen-binding fragments thereof, can be administered in combination with any
known therapies for autoimmune and inflammatory diseases, including any agent
or
combination of agents that are known to be useful, or which have been used or
are
currently in use, for treatment of autoimmune and inflammatory diseases. Such
therapies and therapeutic agents include, but are not limited to, surgery or
surgical
procedures (e.g. splenectomy, lymphadenectomy, thyroidectomy, plasmaphoresis,
leukophoresis, cell, tissue, or organ transplantation, intestinal procedures,
organ
perfusion, and the like), radiation therapy, therapy such as steroid therapy
and non-
steroidal therapy, hormone therapy, cytokine therapy, therapy with
dermatological
agents (for example, topical agents used to treat skin conditions such as
allergies,
contact dermatitis, and psoriasis), immunosuppressive therapy, and other anti-
inflammatory monoclonal antibody therapy, and the like. In this manner, the
antagonist anti-CD40 antibodies described herein, or antigen-binding fragments
thereof, are administered in combination with at least one other therapy,
including,
but not limited to, surgery, organ perfusion, radiation therapy, steroid
therapy, non-
steroidal therapy, antibiotic therapy, antifungal therapy, hormone therapy,
cytokine
therapy, therapy with dermatological agents (for example, topical agents used
to treat
skin conditions such as allergies, contact dermatitis, and psoriasis),
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immunosuppressive therapy, other anti-inflammatory monoclonal antibody
therapy,
combinations thereof, and the like.
Where the methods of the present invention comprise combined therapeutic
regimens for a subject having an autoirnmune disease and/or inflammatory
disease,
these therapies can be given simultaneously, i.e., the antagonist anti-CD40
antibody
or antigen-binding fragment thereof is administered concurrently or within the
same
time frame as the other therapy (i.e., the therapies are going on
concurrently, but the
anti-CD40 antibody or antigen-binding fragment thereof is not administered
precisely
at the same time as the other therapy). Alternatively, the antagonist anti-
CD40
antibody of the present invention or antigen-binding fragment thereof may also
be
administered prior to or subsequent to the other therapy. Sequential
administration of
the different therapies may be performed regardless of whether the treated
subj ect
responds to the first course of therapy to decrease the possibility of
remission or
relapse.
In some embodiments of the invention, the antagonist anti-CD40 antibodies
described herein, or antigen-binding fragments thereof, are administered in
combination with immunosuppressive drugs or anti-inflammatory drugs, wherein
the
antibody and the therapeutic agents) may be administered sequentially, in
either
order, or simultaneously (i.e., concurrently or within the same time frame).
Examples
of suitable immunosuppressive drugs that can be administered in combination
with
the antagonistic anti-CD40 antibodies of the invention include, but are not
limited to,
methotrexate, cyclophosphamide, mizoribine, chlorambucil, cyclosporine, such
as, for
example, aerosolized cyclosporine (see, U.S. Patent Application Publication
No.
US20020006901, herein incorporated by reference in its entirety), tacrolimus
(FK506;
ProGrafrM), mycophenolate mofetil, and azathioprine (6-mercaptopurine),
sirolimus
(rapamycin), deoxyspergualin, leflunomide and its malononitriloamide analogs;
and
immunosuppressive proteins, including, for example, anti-CTLA4 antibodies and
Ig
fusions, anti-B lymphocyte stimulator antibodies (e.g., LYMPHOSTAT-BTM) and Ig
fusions (BLyS-Ig), anti-CD80 antibodies and etanercept (Enbrel~), as well as
anti-T
cell antibodies such as anti-CD3 (OKT3), anti-CD4, and the like. Examples of
suitable anti-inflammatory agents include, but are not limited to,
corticosteroids such
as, for example, clobetasol, halobetasol, hydrocortisone, triamcinolone,
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betamethasone, fluocinole, fluocinonide, prednisone, prednisolone,
methylprednisolone; non-steroidal anti-inflammatory drugs (NSAIDs) such as,
for
example, sulfasalazine, medications containing mesalamine (known as 5-ASA
agents), celecoxib, diclofenac, etodolac, fenprofen, flurbiprofen, ibuprofen,
ketoprofen, meclofamate, meloxicam , nabumetone, naproxen, oxaprozin,
piroxicam,
rofecoxib, salicylates, sulindac, and tolinetin; anti-inflammatory antibodies
such as
adalimumab (IiUMIRA~, a TNF-a antagonist) and infliximab (Remicade , a TNF-a
antagonist), and the like. In other embodiments, a subject receiving treatment
for an
autoimmune and/or inflammatory disease is administered the anti-CD40
antibodies of
the present invention, or suitable antigen-binding fragment thereof, in
combination
with other antagonist anti-CD40 antibodies that target CD40L-mediated CD40
signaling on CD40-expressing cells , for example, the antagonist anti-CD40
antibodies CHIR-12.12 and CHIR-5.9, and antigen-binding fragments thereof, as
disclosed in International Patent Application No. PCT/LTS2004/037152 (Attorney
Docket No. PP20107.004 (035784/282916)), also entitled "Antagonist Anti-CD40
Monoelonal Antibodies and Methods for Their Use," filed November 4, 2004)).
Transplant rejection and graft versus host disease can be hyperacute
(humoral), acute (T cell mediated), or chronic (unknown etiology), or a
combination
thereof. Thus, the antagonistic anti-CD40 antibodies of the invention are used
in
some embodiments to prevent and/or ameliorate rejection and/or symptoms
associated
with hyperacute, acute, and/or chronic transplant rejection of any tissue,
including,
but not limited to, liver, kidney, pancreas, pancreatic islet cells, small
intestine, lung,
heart, corneas, skin, blood vessels, bone, heterologous or autologous bone
marrow,
and the like. Graft tissues may be obtained from any donor and transplanted
into any
recipient host, and thus the transplant procedure may comprise transplanting
animal
tissue to humans (e.g., xenografts), transplanting tissue from one human to
another
human (e.g., allografts), and/or transplanting tissue from one part of a
human's body
to another (e.g., autografts). Treatment with the antibodies of the invention
may also
reduce transplantation sequelae such as fever, anorexia, hemodynaxnic
abnormalities,
leukopenia, white cell infiltration of the transplanted organ/tissue, as well
as
opportunistic infections.
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In some embodiments, the antagonistic anti-CD40 antibodies of the invention
may be used alone or in combination with immunosuppressive drugs to treat
and/or
prevent transplant rejection such as hyperacute, acute, andlor chronic
rejection andlor
graft versus host disease. Thus, in some embodiments where the antagonistic
anti-
s CD40 antibodies of the invention are used to treat graft rejection, the
antibodies may
used in combination with suitable immunosuppressive drugs, including, but not
limited, to methotrexate; cyclophosphamide; mizoribine; chlorambucil;
cyclosporine,
such as, for example, aerosolized cyclosporine (see, U.S. Patent Application
Publication No. US20020006901, herein incorporated by reference in its
entirety),
tacrolimus (FK506; ProGrafr"~, mycophenolate mofetil, and azathioprine (6-
mercaptopurine), sirolimus (rapamycin), deoxyspergualin, leflunomide and its
malononitriloamide analogs; immunosuppressive proteins, including, for
example,
anti-CTLA antibodies and Ig fusions, anti-B lymphocyte stimulator antibodies
(e.g.,
LYNII'HOSTAT-BTM) and Ig fusions (BLyS-Ig), anti-CD80 antibodies and
etanercept
(Enbrel~), as well as anti-T cell antibodies such as anti-CD3 (OKT3), anti-
CD4, and
the like; or other antagonist anti-CD40 antibodies that target CD40L-mediated
CD40
signaling on CD40-expressing cells, for example, the CHIR-12.12 or CHIR-5.9
antibody or antigen-binding fragment thereof.
As such, it is specifically contemplated that the compositions and methods of
the invention are used in combination with other drugs to further improve
symptoms
and outcomes in transplant recipients, such as those receiving lung grafts,
for
example. Thus, in some embodiments, the antagonistic anti-CD40 antibodies of
the
invention are used to treat transplant rejection (such as, for example
hyperacute,
acute, and/or chronic rejection or graft versus host disease in lung
transplant
recipients) alone or in combination with parenterally and/or non-parenterally
administered cyclosporine, including for example oral cyclosporine, injectable
cyclosporine, aerosolized (e.g., inhaled) cyclosporine, and combinations
thereof. In
some embodiments where at least a component of the therapy is aerosolized
cyclosporine, the cyclosporine is delivered to the lung of the recipient by
inhalation of
cyclosporine in aerosol spray form using, for example, a pressurized delivery
device
or nebulizer. The cyclosporine may be administered in either dry powder or wet
form.
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In some other embodiments, the antagonistic anti-CD40 antibodies of the
invention may be used alone or in combination with immunosuppressive drugs to
treat
and/or prevent rheumatoid arthritis. Thus in some embodiments where the
antagonistic anti-CD40 antibodies of the invention axe used to treat
rheumatoid
arthritis, the antibodies may used in combination with suitable
immunosuppressive
drugs, including, but not limited to, methotrexate, cyclophosphamide,
mizoribine,
chlorambucil, cyclosporine, tacrolimus (FK506; PROGRAFT"~, mycophenolate
mofetil, and azathioprine (6-mercaptopurine), sirolimus (rapamycin),
deoxyspergualin, leflunomide and its malononitriloamide analogs;
immunosuppressive proteins, including, for example, anti-CTLA antibodies and
Ig
fusions, anti-B lymphocyte stimulator antibodies (e.g., LYMPHOSTAT-BTM) and Ig
fusions (BLyS-Ig), anti-CD20 antibodies (e.g. RITUXAN~); the fully human
antibody HuMax-CD20, R-1594, IMMU-106, TRU-015, AME-133, tositumomab/I-
131, tositumomab (Bexxar~), ibritumomab tituxetan (Zevalin~); anti-CD80
antibodies, and etanercept (ENBREL~), as well as anti-T cell antibodies such
as anti-
CD3 (OKT3), anti-CD4, and the like; or other antagonist anti-CD40 antibodies
that
target CD40L-mediated CD40 signaling on CD40-expressing cells, for example,
the
CHIR-12.12 or CHIR-5.9 antibody or antigen-binding fragment thereof. As
discussed
above, treatment effectiveness may be assessed using any means and includes,
but is
not limited to, effectiveness as measured by clinical responses defined by the
American College of Rheumatology criteria, the European League of Rheumatism
criteria, or any other criteria. See for example, Felson et al. (1995)
Arthritis. Rheum.
38:727-35 and van Gestel et al. (1996) Arthritis Rheum. 39:34-40.
In yet other embodiments, the antagonistic anti-CD40 antibodies of the
invention may be used alone or in combination with immunosuppressive drugs to
treat
and/or prevent multiple sclerosis. Thus in some embodiments where the
antagonistic
anti-CD40 antibodies of the invention are used to treat multiple sclerosis,
the
antibodies may used in combination with suitable immunosuppressive drugs,
including, but not limited to, methotrexate, cyclophosphamide, mizoribine,
chlorambucil, cyclosporine, tacrolimus (FK506; PROGR.AFTM), mycophenolate
mofetil, and azathioprine (6-mercaptopurine), sirolimus (rapamycin),
deoxyspergualin, leflunomide and its malononitriloamide analogs;
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immunosuppressive proteins, including, for example, anti-CTLA antibodies and
Ig
fusions, anti-B lymphocyte stimulator antibodies (e.g., LYMPHOSTAT-BTU and Ig
fusions (BLyS-Ig), anti-CD20 antibodies (e.g., RITUXAN~); the fully human
antibody HuMax-CD20, R-1594, IMMU-106, TRU-015, AME-133, tositumomab/I-
131, tositumomab (Bexxar~), ibritumomab tituxetan (Zevalin~); anti-CD80
antibodies, and etanercept (ENBREL~), as well as anti-T cell antibodies such
as anti-
CD3 (OKT3), anti-CD4, and the like; or other antagonist anti-CD40 antibodies
that
target CD40L-mediated CD40 signaling on CD40-expressing cells, for example,
the
CHIR-12.12 or CHIR-5.9 antibody or antigen-binding fragment thereof.
Further, combination therapy with two or more therapeutic agents and an
antagonist anti-CD40 antibody described herein can also be used for treatment
of
disease states comprising stimulated CD40-expressing cells, for example, B
cell-
related cancers, solid tumors, and autoimmune and/or inflammatory disorders.
Without being limiting, examples include triple combination therapy, where two
chemotherapeutic agents axe administered in combination with an antagonist
anti-
CD40 antibody described herein, and where a chemotherapeutic agent and another
anti-cancer monoclonal antibody (for example, alemtuzumab, rituximab, anti-
CD23
antibody, or another antagonist anti-CD40 antibody such as CHIR-12.12 or CHIR-
5.9
that targets CD40L-mediated CD40 signaling) are administered in combination
with
an antagonist anti-CD40 antibody described herein. Examples of such
combinations
include, but are not limited to, combinations of fludarabine,
cyclophosphaxnide, and
the antagonist anti-CD40 antibody, of the invention, or an antigen-binding
fragment
thereof; combinations of fludarabine, an anti-CD20 antibody, for example,
rituximab
(I2ituxan~; IDEC Pharmaceuticals Corp., San Diego, California), and the
antagonist
anti-CD40 antibody of the invention or an antigen-binding fragment thereof;
and
combinations of fludarabine, another antagonist anti-CD40 antibody that
targets
CD40L-mediated CD40 signaling, for example, CHIR-12.12 or CHIR 5.9, and the
antagonist anti-CD40 antibody of the invention or an antigen-binding fragment
thereof that targets C4BP-mediated CD40 signaling.
The antagonist anti-CD40 antibodies described herein can further be used to
provide reagents, e.g., labeled antibodies that can be used, for example, to
identify
cells expressing CD40. This can be very useful in determining the cell type of
an
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unknown sample. Panels of monoclonal antibodies can be used to identify tissue
by
species and/or by organ type. In a similar fashion, these anti-CD40 antibodies
can be
used to screen tissue culture cells for contamination (i.e., screen for the
presence of a
mixture of CD40-expressing and non-CD40 expressing cells in a culture).
Pharmaceutical Formulations and Modes of Administration
The antagonist anti-CD40 antibodies of this invention are administered at a
concentration that is therapeutically effective to prevent or treat CD40-
associated
diseases such as autoimmunity, hypersensitivity, inflammation, auto-antibody
production, organ or tissue transplant rejection, graft versus host disease,
and CD40-
expressing cancers such as the B-cell lymphomas and solid tumors. To
accomplish
this goal, the antibodies may be formulated using a variety of acceptable
excipients
known in the axt. Typically, the antibodies are administered by injection,
either
intravenously or intraperitoneally. Methods to accomplish this administration
are
known to those of ordinary skill in the art. It may also be possible to obtain
compositions which may be topically or orally administered, or which may be
capable
of transmission across mucous membranes.
Intravenous administration occurs preferably by infusion over a period of
about 1 to about 10 hours, more preferably over about 1 to about 8 hours, even
more
preferably over about 2 to about 7 hours, still more preferably over about 4
to about 6
hours, depending upon the anti-CD40 antibody being administered. The initial
infusion with the pharmaceutical composition may be given over a period of
about 4
to about 6 hours with subsequent infusions delivered more quickly. Subsequent
infusions may be administered over a period of about 1 to about 6 hours,
including,
for example, about 1 to about 4 hours, about 1 to about 3 hours, or about 1 to
about 2
hours.
A pharmaceutical composition of the invention is formulated to be compatible
with its intended route of administration. Examples of possible routes of
administration include parenteral, (e.g., intravenous (IV), intramuscular
(IM),
intradermal, subcutaneous (SC), or infusion), oral and pulmonary (e.g.,
inhalation),
nasal, transdermal (topical), transmucosal, and rectal administration.
Solutions or
suspensions used for parenteral, intradermal, or subcutaneous application can
include
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the following components: a sterile diluent such as water for injection,
saline solution,
fixed oils, polyethylene glycols, glycerin, propylene glycol or other
synthetic
solvents; antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants
such as ascorbic acid or sodium bisulfate; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates or
phosphates and
agents for the adjustment of tonicity such as sodium chloride or dextrose. pH
can be
adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
The
parenteral preparation can be enclosed in ampoules, disposable syringes, or
multiple
dose vials made of glass or plastic.
The anti-CD40 antibodies are typically provided by standard technique within
a pharmaceutically acceptable buffer, for example, sterile saline, sterile
buffered
water, propylene glycol, combinations ~f the foregoing, etc. Methods for
preparing
parenterally administrable agents are described in Remington's Pharmaceutical
Sciences (18th ed.; Mack Publishing Company, Eaton, Pennsylvania, 1990),
herein
incorporated by reference. See also, for example, International Publication
No. WO
98/56418, which describes stabilized antibody pharmaceutical formulations
suitable
for use in the methods of the present invention.
The amount of at least one antagonist anti-CD40 antibody or antigen-binding
fragment thereof to be administered is readily determined by one of ordinary
skill in
the art without undue experimentation. Factors influencing the mode of
administration and the respective amount of at least one antagonist anti-CD40
antibody (or antigen-binding fragment thereof) include, but are not limited
to, the
particular disease undergoing therapy, the severity of the disease, the
history of the
disease, and the age, height, weight, health, and physical condition of the
individual
undergoing therapy. Similarly, the amount of antagonist anti-CD40 antibody or
antigen-binding fragment thereof to be administered will be dependent upon the
mode
of administration and whether the subject will undergo a single dose or
multiple doses
of this anti-tumor agent. Generally, a higher dosage of anti-CD40 antibody or
antigen-binding fragment thereof is preferred with increasing weight of the
patient
undergoing therapy. The dose of anti-CD40 antibody or antigen-binding fragment
thereof to be administered is in the range from about 0.003 mg/kg to about 50
mg/kg,
preferably in the range of 0.01 mg/kg to about 40 mg/kg. Thus, for example,
the dose
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can be 0.01 mglkg, 0.03 mg/kg, 0.1 mglkg, 0.3 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5
,mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 5 mg/kg, 7 mg/kg, 10 mg/kg, 15 mg/kg, 20
mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mglkg, or 50 mg/kg.
In another embodiment of the invention, the method comprises administration
of multiple doses of antagonist anti-CD40 antibody or antigen-binding fragment
thereof. The method may comprise administration of 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 15,
20, 25, 30, 35, 40, or more therapeutically effective doses of a
pharmaceutical
composition comprising an antagonist anti-CD40 antibody or antigen-binding
fragment thereof. The frequency and duration of administration of multiple
doses of
the pharmaceutical compositions comprising anti-CD40 antibody or antigen-
binding
fragment thereof is dependent upon the disease, state of the disease, and
medical
history of the subject undergoing treatment. Moreover, treatment of a subject
with a
therapeutically effective amount of an antibody can include a single treatment
or,
preferably, can include a series of treatments. In a preferred example, a
subject is
treated with antagonist anti-CD40 antibody or antigen-binding fragment thereof
in the
range of between about 0.1 to 20 mg/kg body weight, once per week for between
about 1 to 10 weeks, preferably between about 2 to 8 weeks, more preferably
between
about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
Treatment
may occur annually to prevent relapse or upon indication of relapse. It will
also be
appreciated that the effective dosage of antibody or antigen-binding fragment
thereof
used for treatment may increase or decrease over the course of a particular
treatment.
Changes in dosage may result and become apparent from the results of
diagnostic
assays as described herein.
Thus, in one embodiment, the dosing regimen includes a first administration
of a therapeutically effective dose of at least one anti-CD40 antibody or
antigen-
binding fragment thereof on days 1, 7, 14, and 21 of a treatment period. In
another
embodiment, the dosing regimen includes a first administration of a
therapeutically
effective dose of at least one anti-CD40 antibody or antigen-binding fragment
thereof
on days 1, 2, 3, 4, 5, 6, and 7 of a week in a treatment period. Further
embodiments
include a dosing regimen having a first administration of a therapeutically
effective
dose of at least one anti-CD40 antibody or antigen-binding fragment thereof on
days
l, 3, 5, and 7 of a week in a treatment period; a dosing regimen including a
first
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administration of a therapeutically effective dose of at least one anti-CD40
antibody
or antigen-binding fragment thereof on days 1 and 3 of a week in a treatment
period;
and a preferred dosing regimen including a first administration of a
therapeutically
effective dose of at least one anti-CD40 antibody or antigen-binding fragment
thereof
on day 1 of a week in a treatment period. The treatment period may comprise 1
week,
2 weeks, 3 weeks, a month, 3 months, 6 months, or a year. Treatment periods
may be
subsequent or separated from each other by a day, a week, 2 weeks, a month, 3
months, 6 months, or a year.
In some embodiments, the therapeutically effective doses of antagonist anti-
CD40 antibody or antigen-binding fragment thereof ranges from about 0.003
mg/kg to
about 50 mg/kg, from about 0.01 mg/kg to about 40 mg/kg, from about 0.01 mg/kg
to
about 30 mg/kg, from about 0.1 mg/kg to about 30 mglkg, from about 0.5 mg/kg
to
about 30 mg/kg, from about 1 mg/kg to about 30 mg/kg, from about 3 mg/kg to
about
30 mg/kg, from about 3 mg/kg to about 25 mg/kg, from about 3 mg/kg to about 20
mg/kg, from about 5 mg/kg to about 15 mglkg, or from about 7 mg/kg to about 12
mglkg. Thus, for example, the dose of any one antagonist anti-CD40 antibody or
antigen-binding fragment thereof can be 0.003 mg/kg, 0.01 mglkg, 0.03 mg/kg,
0.1
mg/kg, 0.3 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg,
5
mg/kg, 7 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mglkg, 35 mglkg, 40
mg/kg, 45 mglkg, 50 mg/kg, or other such doses falling within the range of
about
0.003 mg/kg to about 50 mg/kg. The same therapeutically effective dose of an
antagonist anti-CD40 antibody or antigen-binding fragment thereof can be
administered throughout each week of antibody dosing. Alternatively, different
therapeutically effective doses of an antagonist anti-CD40 antibody or antigen-
binding fragment thereof can be used over the course of a treatment period.
In other embodiments, the initial therapeutically effective dose of an
antagonist anti-CD40 antibody or antigen-binding fragment thereof as defined
elsewhere herein can be in the lower dosing range (i.e., about 0.003 mg/kg to
about 20
mg/kg) with subsequent doses falling within the higher dosing range (i.e.,
from about
20 mg/kg to about 50 mg/kg).
In alternative embodiments, the initial therapeutically effective dose of an
antagonist anti-CD40 antibody or antigen-binding fragment thereof as defined
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elsewhere herein can be in the upper dosing range (i.e., about 20 mg/kg to
about 50
mg/kg) with subsequent doses falling within the lower dosing range (i.e.,
0.003 mg/kg
to about 20 mg/kg). Thus, in one embodiment, the initial therapeutically
effective
dose of the antagonist anti-CD40 antibody or antigen-binding fragment thereof
is
about 20 mg/kg to about 35 mg/kg, including about 20 mg/kg, about 25 mg/kg,
about
30 mg/kg, and about 35 mg/lcg, and subsequent therapeutically effective doses
of the
antagonist anti-CD40 antibody or antigen binding fragment thereof are about 5
mg/kg
to about 15 mg/kg, including about 5 mg/kg, 8 mg/kg, 10 mg/kg, 12 mg/kg, and
about
mg/kg.
10 In some embodiments of the invention, antagonist anti-CD40 antibody therapy
is initiated by administering a "loading dose" of the antibody or antigen-
binding
fragment thereof to the subject in need of antagonist anti-CD40 antibody
therapy. By
"loading dose" is intended an initial dose of the antagonist anti-CD40
antibody or
antigen-binding fragment thereof that is administered to the subject, where
the dose of
15 the antibody or antigen-binding fragment thereof administered falls within
the higher
dosing range (i.e., from about 20 mg/kg to about 50 mg/kg). The "loading dose"
can
be administered as a single administration, for example, a single infusion
where the
antibody or antigen-binding fragment thereof is administered IV, or as
multiple
administrations, for example, multiple infusions where the antibody or antigen-
binding fragment thereof is administered IV, so long as the complete "loading
dose" is
administered within about a 24-hour period. Following administration of the
"loading
dose, " the subject is then administered one or more additional
therapeutically
effective doses of the antagonist anti-CD40 antibody or antigen-binding
fragment
thereof. Subsequent therapeutically effective doses can be administered, for
example,
according to a weekly dosing schedule, or once every two weeks, once every
three
weeks, or once every four weeks. In such embodiments, the subsequent
therapeutically effective doses generally fall within the lower dosing range
(i.e., 0.003
mg/kg to about 20 mglkg).
Alternatively, in some embodiments, following the "loading dose," the
subsequent therapeutically effective doses of the antagonist anti-CD40
antibody or
antigen-binding fragment thereof are administered according to a "maintenance
schedule, " wherein the therapeutically effective dose of the antibody or
antigen-
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binding fragment thereof is administered once a month, once every 6 weeks,
once
every two months, once every 10 weeks, once every three months, once every 14
weeks, once every four months, once every 18 weeks, once every five months,
once
every 22 weeks, once every six months, once every 7 months, once every 8
months,
once every 9 months, once every 10 months, once every 11 months, or once every
12
months. In such embodiments, the therapeutically effective doses of the
antagonist
anti-CD40 antibody or antigen-binding fragment thereof fall within the lower
dosing
range (i.e., 0.003 mg/kg to about 20 mg/kg), particularly when the subsequent
doses
are administered at more frequent intervals, for example, once every two weeks
to
once every month, or within the higher dosing range (i.e., from about 20 mg/kg
to
about 50 mg/kg), particularly when the subsequent doses are administered at
less
frequent intervals, for example, where subsequent doses are administered about
one
month to about 12 months apart.
The antagonist anti-CD40 antibodies present in the pharmaceutical
compositions described herein for use in the methods of the invention may be
native
or obtained by recombinant techniques, and may be from any source, including
mammalian sources such as, e.g., mouse, rat, rabbit, primate, pig, and human.
Preferably such polypeptides are derived from a human source, and more
preferably
are recombinant, human proteins from hybridoma cell lines.
The pharmaceutical compositions useful in the methods of the invention may
comprise biologically active variants of the antagonist anti-CD40 antibodies
of the
invention. Such variants should retain the desired biological activity of the
reference
antagonist anti-CD40 antibody such that the pharmaceutical composition
comprising
the variant antibody has the same therapeutic effect as the pharmaceutical
composition comprising the reference antagonist anti-CD40 antibody when
administered to a subject. That is, the variant anti-CD40 antibody will serve
as a
therapeutically active component in the pharmaceutical composition in a manner
similar to that observed for the reference antagonist anti-CD40 antibody.
Methods are
available in the art for determining whether a variant anti-CD40 antibody
retains the
desired biological activity, and hence serves as a therapeutically active
component in
the pharmaceutical composition. Biological activity of antibody variants can
be
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measured using assays specifically designed for measuring activity of the
reference
antagonist anti-CD40 antibody, including assays described in the present
invention.
Any pharmaceutical composition comprising an antagonist anti-CD40
antibody or antigen-binding fragment thereof having the binding properties
described
herein as the therapeutically active component can be used in the methods of
the
invention. Thus liquid, lyophilized, or spray-dried compositions comprising
one or
more of the antagonist anti-CD40 antibodies of the invention, or antigen-
binding
fragment thereof, may be prepared as an aqueous or nonaqueous solution or
suspension for subsequent administration to a subject in accordance with the
methods
of the invention. Each of these compositions will comprise at least one of the
antagonist anti-CD40 antibodies of the present invention, or an antigen-
binding
fragment thereof, as a therapeutically or prophylactically active component.
By
"therapeutically or prophylactically active component" is intended the anti-
CD40
antibody or antigen-binding fragment thereof is specifically incorporated into
the
composition to bring about a desired therapeutic or prophylactic response with
regard
to treatment, prevention, or diagnosis of a disease or condition within a
subject when
the pharmaceutical composition is administered to that subject. Preferably the
pharmaceutical compositions comprise appropriate stabilizing agents, bulking
agents,
or both to minimize problems associated with loss of protein stability and
biological
activity during preparation and storage.
Formulants may be added to pharmaceutical compositions comprising an
antagonist anti-CD40 antibody of the invention or antigen-binding fragment
thereof.
These formulants may include, but are not limited to, oils, polymers,
vitamins,
carbohydrates, amine acids, salts, buffers, albumin, surfactants, or bulking
agents.
Preferably carbohydrates include sugar or sugar alcohols such as mono-, di-,
or
polysaccharides, or water soluble glucans. The saccharides or glucans can
include
fructose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran,
pullulan,
dextrin, a, /3, and y cyclodextrin, soluble starch, hydroxyethyl staxch, and
carboxymethylcellulose, or mixtures thereof. "Sugar alcohol" is defined as a
C4 to C8
hydrocarbon having a hydroxyl group and includes galactitol, inositol,
mannitol,
xylitol, sorbitol, glycerol, and arabitol. These sugars or sugar alcohols may
be used
individually or in combination. The sugar or sugar alcohol concentration is
between
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1.0% and 7% w/v., more preferably between 2.0% and 6.0% w/v. Preferably amino
acids include levorotary (L) forms of carnitine, arginine, and betaine;
however, other
amino acids may be added. Preferred polymers include polyvinylpyrrolidone
(PVP)
with an average molecular weight between 2,000 and 3,000, or polyethylene
glycol
(PEG) with an average molecular weight between 3,000 and 5,000. Surfactants
that
can be added to the formulation are shown in EP Nos. 270,799 and 268,110.
Additionally, antibodies can be chemically modified by covalent conjugation
to a polymer to increase their circulating half life, for example. Preferred
polymers,
and methods to attach them to peptides, are shown in U.S. Patent Nos.
4,766,106;
4,179,337; 4,495,285; and 4,609,546; which are all hereby incorporated by
reference
in their entireties. Preferred polymers are polyoxyethylated polyols and
polyethylene
glycol (PEG). PEG is soluble in water at room temperature and has the general
formula: R(O--CH2 --CH2)" O--R where R can be hydrogen, or a protective group
such as an alkyl or alkanol group. Preferably, the protective group has
between 1 and
8 carbons, more preferably it is methyl. The symbol n is a positive integer,
preferably
between 1 and 1,000, more preferably between 2 and 500. The PEG has a
preferred
average molecular weight between 1,000 and 40,000, more preferably between
2,000
and 20,000, most preferably between 3,000 and 12,000. Preferably, PEG has at
least
one hydroxy group, more preferably it is a terminal hydroxy group. It is this
hydroxy
group which is preferably activated to react with a free amino group on the
inhibitor.
However, it will be understood that the type and amount of the reactive groups
may be
varied to achieve a covalently conjugated PEG/antibody of the present
invention.
Water-soluble polyoxyethylated polyols are also useful in the present
invention.
They include polyoxyethylated sorbitol, polyoxyethylated glucose,
polyoxyethylated
glycerol (POG), and the like. POG is preferred. One reason is because the
glycerol
backbone of polyoxyethylated glycerol is the same backbone occurring naturally
in, for
example, animals and humans in mono-, di-, triglycerides. Therefore, this
branching
would not necessarily be seen as a foreign agent in the body. The POG has a
preferred
molecular weight in the same range as PEG The structure for POG is shown in
Knauf
et al. (1988) J. Bio. Chem. 263:15064-15070, and a discussion of POG/IL-2
conjugates
is found in U.S. Patent No. 4,766,106, both of which are hereby incorporated
by
reference in their entireties.
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Another drug delivery system for increasing circulatory half life is the
liposome. Methods of preparing liposome delivery systems are discussed in
Gabizon
et al. (1982) Cancer Research 42:4734; Cafiso (1981) Biochem. Biophys. Acta
649:129; and Szoka (1980) Anna. Rev. Biophys. Eng. 9:467. Other drug delivery
systems are known in the art and are described in, e.g., Poznansky et al.
(1980) Drug
Delivefy Systems (R.L. Juliano, ed., Oxford, New York), pp. 253; Poznansky
(1984)
Pharrn. Revs. 36:277.
The formulants to be incorporated into a pharmaceutical composition should
provide for the stability of the antagonist anti-CD40 antibody or antigen-
binding
fragment thereof. That is, the antagonist anti-CD40 antibody or antigen-
binding
fragment thereof should retain its physical and/or chemical stability and have
the
desired biological activity, i.e., one or more of the antagonist activities
defined herein
above, including, but not limited to, inhibition of immunoglobulin secretion
by
normal human peripheral B cells stimulated by T cells; inhibition of survival
and/or
proliferation of normal human peripheral B cells stimulated by Jurkat T cells;
inhibition of survival and/or proliferation of normal human peripheral B cells
stimulated by C4BP-expressing cells or soluble C4BP; inhibition of "survival"
anti-
apoptotic intracellular signals in any cell stimulated by soluble C4BP or
solid-phase
C4BP; inhibition of CD40 signal transduction in any cell upon ligation with
soluble
C4BP or solid-phase C4BP; and inhibition of proliferation of human malignant B
cells as noted elsewhere herein.
Methods for monitoring protein stability are well known in the art. See, for
example, Jones (1993) Adv Drug Delivery Rev 10:29-90; Lee, ed. (1991) Peptide
and
Protein Drug Delivery (Marcel Dekker, Inc., New York, New York). Generally,
protein stability is measured at a chosen temperature for a specified period
of time. In
preferred embodiments, a stable antibody pharmaceutical formulation provides
for
stability of the antagonist anti-CD40 antibody or antigen-binding fragment
thereof
when stored at room temperature (about 25°C) for at least 1 month, at
least 3 months,
or at least 6 months, andlor is stable at about 2-8°C for at least 6
months, at least 9
months, at least 12 months, at least 18 months, at least 24 months.
A protein such as an antibody, when formulated in a pharmaceutical
composition, is considered to retain its physical stability at a given point
in time if it
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shows no visual signs (i.e., discoloration or loss of clarity) or measurable
signs (for
example, using size-exclusion chromatography (SEC) or UV light scattering) of
precipitation, aggregation, and/or denaturation in that pharmaceutical
composition.
With respect to chemical stability, a protein such as an antibody, when
formulated in a
pharmaceutical composition, is considered to retain its chemical stability at
a given
point in time if measurements of chemical stability are indicative that the
protein (i.e.,
antibody) retains the biological activity of interest in that pharmaceutical
composition.
Methods for monitoring changes in chemical stability are well known in the art
and
include, but are not limited to, methods to detect chemically altered forms of
the
protein such as result from clipping, using, for example, SDS-PAGE, SEC,
andlor
matrix-assisted laser desorption ionization/time of flight mass spectrometry;
and
degradation associated with changes in molecular charge (for example,
associated
with deamidation), using, for example, ion-exchange chromatography See, for
example, the methods disclosed in International Patent Application No.
PCT/US2004/037152 (Attorney Docket No. PP20107.004 (035784/282916)), also
entitled "Antagonist Anti-CD40 Monoclonal Antibodies and Methods for Them
Use,"
filed November 4, 2004; herein incorporated by reference in its entirety.
An antagonist anti-CD40 antibody or antigen-binding fragment thereof, when
formulated in a pharmaceutical composition, is considered to retain a desired
biological activity at a given point in time if the desired biological
activity at that time
is within about 30%, preferably within about 20% of the desired biological
activity
exhibited at the time the pharmaceutical composition was prepared as
determined in a
suitable assay for the desired biological activity. Assays for measuring the
desired
biological activity of the antagonist anti-CD40 antibodies disclosed herein,
and
antigen-binding fragments thereof, can be performed as described in the
Examples
herein. See also the assays described in Schultze et al. (1998) Proc. Natl.
Acad. Sci.
USA 92:8200-8204; Denton et al. (1998) Pediatr Transplant. 2:6-15; Evans et
al.
(2000) J. Imrraunol. 164:688-697; Noelle (1998) Agents Actions Suppl. 49:17-
22;
Lederman et al. (1996) Curx Opin. Hernatol. 3:77-86; Coligan et al. (1991)
Current
Protocols in Immunology 13:12; Kwekkeboom et al. (1993) Immunology 79:439-444;
and U.S. Patent Nos. 5,674,492 and 5,847,082; herein incorporated by
reference.
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Where the antagonist anti-CD40 antibody is formulated as a liquid
formulation, the liquid pharmaceutical composition is preferably lyophilized
to
prevent degradation and to preserve sterility. Methods for lyophilizing liquid
compositions are known to those of ordinary skill in the art. Just prior to
use, the
composition may be reconstituted with a sterile diluent (Ringer's solution,
distilled
water, or sterile saline, for example) that may include additional
ingredients. Upon
reconstitution, the composition is preferably administered to subjects using
those
methods that are known to those skilled in the art.
Use of Antagonist Anti-CD40 Antibodies in the Manufacture of Medicaments
The present invention also provides for the use of an antagonist anti-CD40
antibody of the invention that blocks C4BP-mediated CD40 signaling, or antigen-
binding fragment thereof, in the manufacture of a medicament for treating a
subject
for a cancer comprising CD40-expressing neoplastic cells, wherein the
medicament is
coordinated with treatment with at least one other cancer therapy. In some
embodiments, the cancer is characterized by neoplastic B cell growth. Such
cancers
include, but are not limited to, the B cell-related cancers discussed herein
above, for
example, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, multiple
myeloma, B cell lymphoma, high-grade B cell lymphoma, intermediate-grade B
cell
lymphoma, low-grade B cell lymphoma, B cell acute lympohoblastic leukemia,
myeloblastic leukemia, Hodgkin's disease, plasmacytoma, follicular lymphoma,
follicular small cleaved lymphoma, follicular large cell lymphoma, follicular
mixed
small cleaved lymphoma, diffuse small cleaved cell lymphoma, diffuse small
lymphocytic lymphoma, prolymphocytic leukemia (PLL), lymphoplasmacytic
lymphoma, marginal zone lymphoma, mucosal associated lymphoid tissue lymphoma,
monocytoid B cell lymphoma, splenic lymphoma, hairy cell leukemia, diffuse
large
cell lymphoma, mediastinal large B cell lymphoma, lymphomatoid granulomatosis,
intravascular lymphomatosis, diffuse mixed cell lymphoma, diffuse large cell
lymphoma, immunoblastic lymphoma, Burkitt's lymphoma, AmS-related lymphoma,
and mantle cell lymphoma. In other embodiments, the cancer is a solid tumor.
Examples of solid tumors comprising CD40-expressing neoplastic cells include,
but
are not limited to, ovarian, lung (for example, non-small cell lung cancer of
the
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squamous cell carcinoma, adenocarcinoma, and large cell carcinoma types, and
small
cell lung cancer), breast, colon, kidney (including, for example, renal cell
carcinomas), bladder, liver (including, for example, hepatocellular
carcinomas),
gastric, cervical, prostate, nasopharyngeal, thyroid (for example, thyroid
papillary
carcinoma), and skin cancers such as melanoma, and sarcomas (including, for
example, osteosarcomas and Ewing's sarcomas).
By "coordinated" in the context of a subject in need of treatment for a cancer
is intended the medicament comprising the antagonist anti-CD40 antibody or
antigen-
binding fragment thereof is to be used either prior to, during, or after
treatment of the
subject with at least one other cancer therapy. Examples of other cancer
therapies for
subjects having a B cell-related cancer include, but are not limited to,
surgery;
radiation therapy; chemotherapy, optionally in combination with autologous
bone
marrow transplant, where suitable chemotherapeutic agents include, but are not
limited to, fludarabine or fludarabine phosphate, chlorambucil, vincristine,
pentostatin, 2-chlorodeoxyadenosine (cladribine), cyclophosphamide,
doxorubicin,
prednisone, and combinations thereof, for example, anthracycline-containing
regimens such as CAP (cyclophosphamide, doxorubicin plus prediusone), CHOP
(cyclophosphamide, vincristine, prednisone plus doxorubicin), VAD
(vincritsine,
doxorubicin, plus dexamethasone), MP (melphalan plus prednisone), and other
cytotoxic and/or therapeutic agents used in chemotherapy such as mitoxantrone,
daunorubicin, idarubicin, asparaginase, and antimetabolites, including, but
not limited
to, cytarabine, methotrexate, 5-fluorouracil decarbazine, 6-thioguanine, 6-
mercaptopurine, and nelarabine; other anti-cancer monoclonal antibody therapy
(for
example, alemtuzumab (Campath~) or other anti-CD52 antibody targeting the CD52
cell-surface glycoprotein on malignant B cells; rituximab (Rituxari ), the
fully human
antibody HuMax-CD20, R-1594, IMMU-106, TRU-015, AME-133, tositumomab/I-
131 tositumomab (Bexxar~), ibritumomab tiuxetan (Zevalin~), or any other
therapeutic anti-CD20 antibody targeting the CD20 antigen on malignant B
cells;
anti-CD19 antibody (for example, MT103, a bispecific antibody); anti-CD22
antibody
(for example, the humanized monoclonal antibody epratuzumab); bevacizumab
(Avastin~) or other anti-cancer antibody targeting human vascular endothelial
growth
factor; anti-CD22 antibody targeting the CD22 antigen on malignant B cells
(for
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example, the monoclonal antibody BL-22, an alphaCD22 toxin); a M-CSF antibody
targeting macrophage colony stimulating factor; antibodies targeting the
receptor
activator of nuclear factor-kappaB (RANK) and its ligand (R.ANKL,), which are
overexpressed in multiple myeloma; anti-CD23 antibody targeting the CD23
antigen
on malignant B cells (for example, IDEC-152); anti-CD38 antibody targeting the
CD38 antigen on malignant B cells; antibodies targeting major
histocompatibility
complex class II receptors (anti-MHC antibodies) expressed on malignant B
cells;
other anti-CD40 antibodies targeting the CD40 antigen on malignant B cells
(for
example, SGN-40; and other antagonist anti-CD40 antibodies, such as CHIR-12.12
and CHIR-5.9, and antigen-binding fragments thereof, that block CD40L-mediated
CD40 signaling on CD40-expressing cells, as disclosed in International Patent
Application No. PCT/US2004/037152 (Attorney Docket No. PP20107.004
(035784/282916)), also entitled "Antagonist Anti-CD40 Monoclonal Antibodies
and
Methods for Their Use," filed November 4, 2004)); and antibodies targeting
tumor
necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-Rl) (for
example, the agonistic human monoclonal antibody HGS-ETRl) expressed on a
number of solid tumors and tumors of hematopoietic origin); small molecule-
based
cancer therapy, including, but not limited to, microtubule and/or
topoisomerase
inhibitors (for example, the mitotic inhibitor dolastatin and dolastatin
analogues; the
tubulin-binding agent T900607; XL119; and the topoisomerase inhibitor
aminocamptothecin), SDX-105 (bendamustine hydrochloride), ixabepilone (an
epothilone analog, also referred to as BMS-247550), protein kinase C
inhibitors, for
example, midostaurin ((PKC-412, CGP 41251, N-benzoylstaurosporine),
pixantrone,
eloxatin (an antineoplastic agent), ganite (gallium nitrate), Thalomid~
(thalidomide),
immunomodulatory derivatives of thalidomide (for example, revlimid (formerly
revimid)), AffinitakTM (antisense inhibitor of protein kinase C-alpha), SDX-
101 (R-
etodolac, inducing apoptosis of malignant lymphocytes), second-generation
purine
nucleoside analogs such as clofarabine, inhibitors of production of the
protein Bcl-2
by cancer cells (for example, the antisense agents oblimersen and Genasense~),
proteasome inhibitors (for example, VelcadeTM (bortezomib)), small molecule
kinase
inhibitors (for example, CHIR-258), small molecule VEGF inhibitors (for
example,
ZD-6474), small molecule inhibitors of heat shock protein (HSP) 90 (for
example, 17-
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AAG), small molecule inhibitors of histone deacetylases (for example,
hybrid/polar
cytodifferentiation HPC) agents such as suberanilohydroxamic acid (SAHA), and
FR-
901228) and apoptotic agents such as Trisenox~ (arsenic trioxide) and Xcytriri
(motexafm gadolinium); vaccine /immunotherapy-based cancer therapies,
including,
but not limited to, vaccine approaches (for example, Id-KLH, oncophage,
vitalethine),
personalized immunotherapy or active idiotype immunotherapy (for example,
MyVax~ Personalized Immunotherapy, formally designated GTOP-99), Promune~
(CpG 7909, a synthetic agonist for toll-like receptor 9 (TLR9)), interferon-
alpha
therapy, interleukin-2 (IL-2) therapy, IL-12 therapy; IL-15 therapy, and IL-21
therapy; steroid therapy; or other cancer therapy; where treatment with the
additional
cancer therapy, or additional cancer therapies, occurs prior to, during, or
subsequent
to treatment of the subject with the medicament comprising the antagonist anti-
CD40
antibody or antigen-binding fragment thereof, as noted herein above.
Examples of other cancer therapies for subjects having a cancer that is a
solid
tumor comprising CD40-expressing neoplastic cells include, but are not limited
to,
surgery; radiation therapy; chemotherapy, where suitable chemotherapeutic
agents
include, but are not limited to, fludarabine or fludarabine phosphate,
chlorambucil,
vincristine, pentostatin, 2-chlorodeoxyadenosine (cladribine),
cyclophosphamide,
doxorubicin, prednisone, and combinations thereof, for example, anthracycline
containing regimens such as CAP (cyclophosphamide, doxorubicin plus
prednisone),
CHOP (cyclophosphamide, vincristine, prednisone plus doxorubicin), VAD
(vincritsine, doxorubicin, plus dexamethasone), MP (melphalan plus
prednisone), and
other cytotoxic and/or therapeutic agents used in chemotherapy such as
mitoxantrone,
daunorubicin, idarubicin, asparaginase, and antimetabolites, including, but
not limited
to, cytarabine, methotrexate, 5-fluorouracil decarbazine, 6-thioguanine, 6-
mercaptopurine, and nelarabine; cytokine therapy, including, but not limited
to, alpha-
interferon therapy, gamma-interferon therapy, therapy with interleukin-2 (IL-
2), IL-
12, IL-15, and IL-21, granulocyte macrophage colony stimulating factor (GM-
CSF),
granulocyte colony stimulating factor (G-CSF), or biologically active variants
of these
cytokines; or other monoclonal antibody intended for use in treatment of the
solid
tumor of interest, for example, Herceptin~ (Genentech, Inc., San Francisco,
California), which targets the Her2 receptor protein on Her2+ breast cancer
cells; the
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humanized monoclonal antibody AvastinTM (also known as bevacizumab; Genentech,
Inc., San Francisco, California), which binds to and inhibits vascular
endothelial
growth factor (VEGF), and has use in treatment of colon cancer; anti-EGFR
antibody
targeting the epidermal growth factor receptor (for example, IMC-C225
(IrnClone
Systems, New York, New York); anti-IGF-1 receptor antibody, targeting the IGF-
1
receptor protein; anti-MUCI antibody, targeting the tumor-associated antigen
MT.JCl;
anti-x5(31, anti-av~35, and anti-av,~3, targeting these respective integrins,
which
regulate cell adhesion and signaling processes involved in cell proliferation
and
survival; anti-P-cadherin antibody, targeting this cadherin family member
(see, for
example, copending U.S. Patent Application Publication No. 20030194406); anti-
VE-
cadherin antibody, targeting angiogenic-related function of this endothelial
cell-
specific adhesion molecule; and other antagonist anti-CD40 antibodies, such as
CHIR-12.12 and CHIR-5.9, and antigen-binding fragments thereof, that block
CD40L-mediated CD40 signaling on CD40-expressing neoplastic cells, as
disclosed
in International Patent Application No. PCT/LJS2004/037152 (Attorney Docket
No.
PP20107.004 (035784/282916)), also entitled "Aratagoraist Anti-CD40 Mo~oclohal
Ayatibodies ahd Methods fog Their Use," filed November 4, 2004)); where
treatment
with the additional cancer therapy, or additional cancer therapies, occurs
prior to,
during, or subsequent to treatment of the subject with the medicament
comprising the
antagonist anti-CD40 antibody or antigen-binding fragment thereof, as noted
herein
above.
Thus, in some embodiments, the present invention provides for the use of the
antagonist anti-CD40 antibody that blocks C4BP-mediated CD40 signaling, or
antigen-binding fragment thereof, in the manufacture of a medicament for
treating a B
cell lymphoma, for example non-Hodgkin's lymphoma, in a subject, wherein the
medicament is coordinated with treatment with at least one other cancer
therapy
selected from the group consisting of chemotherapy, anti-cancer antibody
therapy,
small molecule-based cancer therapy, and vaccine/immunotherapy-based cancer
therapy, wherein the medicament is to be used either prior to, during, or
after
treatment of the subject with the other cancer therapy or, in the case of
multiple
combination therapies, either prior to, during, or after treatment of the
subject with the
other cancer therapies.
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Thus, for example, in some embodiments, the invention provides for the use of
an antagonist anti-CD40 antibody of the invention, or antigen-binding fragment
thereof, in the manufacture of a medicament for treating a B cell lymphoma,
for
example, non-Hodgkin's lymphoma, in a subject, wherein the medicament is
coordinated with treatment with chemotherapy, where the chemotherapeutic agent
is
selected from the group consisting of cytoxan, doxorubicin, vincristine,
prednisone,
and combinations thereof, for example CHOF. In other embodiments, the
invention
provides for the use of an antoagonist anti-CD40 antibody of the invention, or
antigen-binding fragment thereof, in the manufacture of a medicament for
treating a B
cell lymphoma, for example non-Hodgkin's lymphoma, in a subj ect, wherein the
medicament is coordinated with treatment with at least one other anti-cancer
antibody
selected from the group consisting of alemtuzumab (Campath~) or other anti-
CD52
antibody targeting the CD52 cell-surface glycoprotein on malignant B cells;
rituximab
(Rituxari ), the fully human antibody HuMax-CD20, R-1594, nVIMU-106, TRU-015,
AME-133, tositumomab/I-131 tositumomab (Bexxar~), ibritumomab tiuxetan
(Zevalin~), or any other therapeutic anti-CD20 antibody targeting the CD20
antigen
on malignant B cells; anti-CD19 antibody (for example, MT103, a bispecific
antibody); anti-CD22 antibody (for example, the humanized monoclonal antibody
epratuzumab); bevacizumab (Avastin~) or other anti-cancer antibody targeting
human vasculax endothelial growth factor; the fully human monoclonal antibody
CHIR-12.12 or CHIR-5.9, or other antagonist anti-CD40 antibody that blocks
CD40L-mediated CD40 signaling; and any combinations thereof; wherein the
medicament is to be used either prior to, during, or after treatment of the
subject with
the other cancer therapy or, in the case of multiple combination therapies,
either prior
to, during, or after treatment of the subject with the other cancer therapies.
In yet other embodiments, the present invention provides for the use of an
antagonist anti-CD40 antibody of the invention, or antigen-binding fragment
thereof,
in the manufacture of a medicament for treating a B cell lymphoma, for example
non-
Hodgkin's lymphoma, in a subject, wherein the medicament is coordinated with
treatment with at least one other small molecule-based cancer therapy selected
from
the group consisting of microtubule and/or topoisomerase inhibitors (for
example, the
mitotic inhibitor dolastatin and dolastatin analogues; the tubulin-binding
agent
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T900607; XLl 19; and the topoisomerase inhibitor aminocamptothecin), SDX-105
(bendamustine hydrochloride), ixabepilone (an epothilone analog, also referred
to as
BMS-247550), protein kinase C inhibitors, for example, midostaurin ((PKC-412,
CGP 41251, N-benzoylstaurosporine), pixantrone, eloxatin (an antineoplastic
agent),
ganite (gallium nitrate), Thalomid~ (thalidomide), an apoptotic agent such as
Xcytrin~
(motexafin gadolinium), inhibitors of production of the protein Bcl-2 by
cancer cells
(for example, the antisense agents oblimersen and Genasense~), nelarabine, and
any
combinations thereof; wherein the medicament is to be used either prior to,
during, or
after treatment of the subject with the other cancer therapy or, in the case
of multiple
combination therapies, either prior to, during, or after treatment of the
subject with the
other cancer therapies.
In still other embodiments, the present invention provides for the use of an
antagonist anti-CD40 antibody, or antigen-binding fragment thereof, in the
manufacture of a medicament for treating a B cell lymphoma, for example non-
Hodgkin's lymphoma, in a subject, wherein the medicament is coordinated with
treatment with at least one other vaccine/immunotherapy-based cancer therapy
selected from the group consisting of vaccine approaches (for example, Id-KLH,
oncophage, vitalethine), personalized immunotherapy or active idiotype
immunotherapy (for example, MyVax~ Personalized Imrnunotherapy, formally
designated GTOP-99), Promune~ (CpG 7909, a synthetic agonist for toll-like
receptor 9 (TLR9)), interleukin-2 (IL-2) therapy, IL-12 therapy; IL-15
therapy, and
IL-21 therapy, and any combinations thereof; wherein the medicament is to be
used
either prior to, during, or after treatment of the subject with the other
cancer therapy
or, in the case of multiple combination therapies, either prior to, during, or
after
treatment of the subject with the other cancer therapies.
In some embodiments, the present invention provides for the use of the
antagonist anti-CD40 antibody of the invention, or antigen-binding fragment
thereof,
in the manufacture of a medicament for treating a B cell-related leukemia, for
example B-cell acute lymphocytic leukemia (B-ALL), in a subject, wherein the
medicament is coordinated with treatment with at least one other cancer
therapy
selected from the group consisting of chemotherapy and anti-metabolite
therapy,
wherein the medicament is to be used either prior to, during, or after
treatment of the
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subject with the other cancer therapy or, in the case of multiple combination
therapies,
either prior to, during, or after treatment of the subj ect with the other
cancer therapies.
Examples of such embodiments include, but are not limited to, those instances
where
the medicament comprising the antagonist anti-CD40 antibody or antigen-binding
fragment thereof is coordinated with treatment with a chemotherapeutic agent
or anti-
metabolite selected from the group consisting of cytoxan, doxorubicin,
vincristine,
prednisone, cytarabine, mitoxantrone, idarubicin, asparaginase, methotrexate,
6-
thioguanine, 6-mercaptopurine, and combinations thereof; wherein the
medicament is
to be used either prior to, during, or after treatment of the subject with the
other
cancer therapy or, in the case of multiple combination therapies, either prior
to,
during, or after treatment of the subject with the other cancer therapies. In
one such
example, the medicament is coordinated with treatment with cytarabine plus
daunorubicin, cytarabine plus mitoxantrone, and/or cytarabine plus idarubicin;
wherein the medicament is to be used either prior to, during, or after
treatment of the
B-ALL subj ect with the other cancer therapy or, in the case of multiple
combination
therapies, either prior to, during, or after treatment of the subject with the
other cancer
therapies.
In some embodiments, the invention provides for the use of an antagonist anti-
CD40 antibody of the invention that block C4BP-mediated CD40 signaling, or
antigen-binding fragment thereof, in the manufacture of a medicament for
treating a
subject for a solid tumor comprising neoplastic cells expressing CD40 antigen,
wherein the medicament is coordinated with treatment with chemotherapy, where
the
chemotherapeutic agent is selected from the group consisting of CPT-11
(Irinotecan),
which can be used, for example, in treating colorectal cancer and non-small
cell lung
cancer; gemcitabine, which can be used, for example, in treating lung cancer,
breast
cancer, and epithelial ovarian cancer; and other chemotherapeutic agents
suitable for
treatment of solid tumors; where treatment with the additional cancer therapy,
or
additional cancer therapies, occurs prior to, during, or subsequent to
treatment of the
subject with the medicament comprising the antagonist anti-CD40 antibody or
antigen-binding fragment thereof, as noted herein above.
In other embodiments, the invention provides for the use of an antagonist anti-
CD40 antibody of the invention, or antigen-binding fragment thereof, in the
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manufacture of a medicament for treating a subject for a solid tumor
comprising
neoplastic cells expressing CD40 antigen, wherein the medicament is
coordinated
with treatment with at least one other anti-cancer antibody selected from the
group
consisting of Herceptin~ (Genentech, Inc., San Francisco, California), which
targets
the Her2 receptor protein on Her2+ breast cancer cells; the humanized
monoclonal
antibody AvastinTM (also known as bevacizumab; Genentech, Inc., San Francisco,
California), which binds to and inhibits vascular endothelial growth factor
(VEGF),
and has use in treatment of colon cancer; anti-EGFR antibody targeting the
epidermal
growth factor receptor (for example, IMC-C225 (ImClone Systems, New York, New
York); anti-IGF-1 receptor antibody, targeting the IGF-1 receptor protein;
anti-MCTC1
antibody, targeting the tumor-associated antigen MUCl; anti-a5~31, anti-av,~5,
and
anti-av,~3, targeting these respective integrins, which regulate cell adhesion
and
signaling processes involved in cell proliferation and survival; anti-P-
cadherin
antibody, targeting this cadherin family member (see, for example, copending
U.S.
Patent Application Publication No. 20030194406); anti-VE-cadherin antibody,
targeting angiogenic-related function of this endothelial cell-specific
adhesion
molecule; and the fully human monoclonal antibody CHIR-12.12 or CHIR-5.9, or
other antagonist anti-CD40 antibody that blocks CD40L-mediated CD40 signaling;
where treatment with the additional cancer therapy, or additional cancer
therapies,
occurs prior to, during, or subsequent to treatment of the subject with the
medicament
comprising the antagonist anti-CD40 antibody or antigen-binding fragment
thereof, as
noted herein above.
The invention also provides for the use of an antagonist anti-CD40 antibody of
the invention, or antigen-binding fragment thereof, in the manufacture of a
medicament for treating a subject for a cancer comprising CD40-expressing
neoplastic cells, for example, a cancer characterized by neoplastic B cell
growth,
including the B cell-related cancers described herein above, or a solid tumor,
wherein
the medicament is used in a subject that has been pretreated with at least one
other
cancer therapy. By "pretreated" or "pretreatment" is intended the subject has
received
one or more other cancer therapies (i.e., been treated with at least one other
cancer
therapy) prior to receiving the medicament comprising the antagonist anti-CD40
antibody or antigen-binding fragment thereof. "Pretreated" or "pretreatment"
includes
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subjects that have been treated with at least one other cancer therapy within
2 years,
within 18 months, within 1 year, within 6 months, within 2 months, within 6
weeks,
within 1 month, within 4 weeks, within 3 weeks, within 2 weeks, within 1 week,
within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, or
even
within 1 day prior to initiation of treatment with the medicament comprising
the
antagonist anti-CD40 antibody or antigen-binding fragment thereof. It is not
necessary that the subject was a responder to pretreatment with the prior
cancer
therapy, or prior cancer therapies. Thus, the subject that receives the
medicament
comprising the antagonist anti-CD40 antibody or antigen-binding fragment
thereof
could have responded, or could have failed to respond (i.e. the cancer was
refractory),
to pretreatment with the prior cancer therapy, or to one or more of the prior
cancer
therapies where pretreatment comprised multiple cancer therapies. Examples of
other
cancer therapies for which a subject can have received pretreatment prior to
receiving
the medicament comprising the antagonist anti-CD40 antibody or antigen-binding
fragment thereof include, but are not limited to, surgery; radiation therapy;
chemotherapy, optionally in combination with autologous bone marrow
transplant,
where suitable chemotherapeutic agents include, but are not limited to, those
listed
herein above; other anti-cancer monoclonal antibody therapy, including, but
not
limited to, those anti-cancer antibodies listed herein above; small molecule-
based
cancer therapy, including, but not limited to, the small molecules listed
herein above;
vaccine/immunotherapy-based cancer therapies, including, but limited to, those
listed
herein above; steroid therapy; other cancer therapy; or any combination
thereof.
"Treatment" in the context of coordinated use of a medicament described
herein with one or more other cancer therapies is herein defined as the
application or
administration of the medicament or of the other cancer therapy to a subj ect,
or
application or administration of the medicament or other cancer therapy to an
isolated
tissue or cell line from a subject, where the subject has a cancer comprising
CD40-
expressing neoplastic cells, for example, a cancer characterized by neoplastic
B cell
growth or a solid tumor, a symptom associated with such a cancer, or a
predisposition
toward development of such a cancer, where the purpose is to cure, heal,
alleviate,
relieve, alter, remedy, ameliorate, improve, or affect the cancer, any
associated
symptoms of the cancer, or the predisposition toward the development of the
cancer.
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The present invention also provides for the use of an antagonist anti-CD40
antibody of the invention that blocks C4BP-mediated CD40 signaling, or antigen-
binding fragment thereof, in the manufacture of a medicament for treating an
autoimmune disease andlor inflammatory disease in a subj ect, wherein the
medicament is coordinated with treatment with at least one other therapy. By
"coordinated" in the context of a subject in need of treatment for an
autoimmune
disease and/or inflammatory disease is intended the medicament is to be used
either
prior to, during, or after treatment of the subj ect with at least one other
therapy.
Examples of other therapies for autoimmune and/or inflammatory diseases
include,
but are not limited to, those described herein above, i.e., surgery or
surgical
procedures (e.g. splenectomy, lymphadenectomy, thyroidectomy, plasmaphoresis,
leukophoresis, cell, tissue, or organ transplantation, organ perfusion,
intestinal
procedures, and the like), radiation therapy, therapy such as steroid therapy
and non-
steroidal therapy, hormone therapy, cytokine therapy, therapy with
dermatological
agents (for example, topical agents used to treat skin conditions such as
allergies,
contact dermatitis, and psoriasis), immunosuppressive therapy, and other anti-
inflammatory monoclonal antibody therapy, and the like, where treatment with
the
additional therapy, or additional therapies, occurs prior to, during, or
subsequent to
treatment of the subject with the medicament comprising the antagonist anti-
CD40
antibody or antigen-binding fragment thereof, as noted herein above. In one
such
embodiment, the present invention provides for the use of an antagonist anti-
CD40
antibody of the invention, or antigen-binding fragment thereof, in the
manufacture of
a medicament for treating an autoimmune disease and/or inflammatory disease in
a
subject, wherein the medicament is coordinated with treatment with at least
one other
therapy as noted herein above.
In some embodiments, the medicament comprising the antagonist anti-CD40
antibody of the invention or antigen-binding fragment thereof is coordinated
with
treatment with two other therapies. Where the medicament comprising the
antagonist
anti-CD40 antibody or antigen-binding fragment thereof is coordinated with two
other
therapies, use of the medicament can be prior to, during, or after treatment
of the
subject with either or both of the other therapies.
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The invention also provides for the use of an antagonist anti-CD40 antibody
that blocks C4BP-mediated CD40 signaling, or antigen-biding fragment thereof,
in
the manufacture of a medicament for treating an autoimmune disease and/or
inflammatory disease in a subject, wherein the medicament is used in a subject
that
has been pretreated with at least one other therapy. By "pretreated" or
"pretreatment"
is intended the subject has been treated with one or more other therapies
prior to
receiving the medicament comprising the antagonist anti-CD40 antibody or
antigen-
binding fragment thereof. "Pretreated" or "pretreatment" includes subjects
that have
been treated with the other therapy, or other therapies, within 2 years,
within 18
months, within 1 year, within 6 months, within 2 months, within 6 weeks,
within 1
month, within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, within 6
days,
within 5 days, within 4 days, within 3 days, within 2 days, or even within 1
day prior
to initiation of treatment with the medicament comprising the antagonist anti-
CD40
antibody or antigen-binding fragment thereof. It is not necessary that the
subj ect was
a responder to pretreatment with the prior therapy, or prior therapies. Thus,
the
subject that receives the medicament comprising the antagonist anti-CD40
antibody or
antigen-binding fragment thereof could have responded, or could have failed to
respond, to pretreatment with the prior therapy, or to one or more of the
prior
therapies where pretreatment comprised multiple therapies.
"Treatment" in the context of coordinated use of a medicament described
herein with one or more other therapies for an autoimmune disease and/or
inflammatory disease is herein defined as the application or administration of
the
medicament or of the other therapy to a subject, or application or
administration of the
medicament or other therapy to an isolated tissue or cell line from a subj
ect, where the
subject has an autoimmune disease and/or inflammatory disease, a symptom
associated with an autoimmune disease and/or inflammatory disease, or a
predisposition toward development of an autoimmune disease and/or inflammatory
disease, where the purpose is to cure, heal, alleviate, relieve, alter,
remedy,
ameliorate, improve, or affect the autoimmune disease and/or inflammatory
disease,
any associated symptoms of the autoimmune disease andlor inflammatory disease,
or
the predisposition toward the development of the autoimmune disease and/or
inflammatory disease.
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The following examples are offered by way of illustration and not by way of
limitation.
EXPERIMENTAL
The following protocols may be used in the examples described below.
ELISA Assay for Irnmuraoglobulin Quantification
The concentrations of human IgM and IgG are estimated by ELISA. 96-well
ELISA plates are coated with 2 ~.g/ml goat anti-human IgG mAb (The Jackson
Laboratory, Bar Harbor, Maine) or with 2 ~g/ml goat anti-human IgM mAb 4102
(Bio Source International, California) in 0.05 M carbonate buffer (pH 9.6), by
incubation for 16 hours at 4°C. Plates are washed 3 times with PBS-0.05
% Tween-
(PBS-Tween) and saturated with BSA for 1 hour. After 2 washes the plates are
incubated for 2 hours at 37°C with different dilutions of the test
samples. After 3
15 washes, bound Ig is detected by incubation for 2 hours at 37°C with
1 ~,g/ml
peroxidase-labeled goat anti-human IgG mAb or goat anti-human IgM mAb. Plates
are washed 4 times, and bound peroxidase activity is revealed by the addition
of O-
phenylenediamine as a substrate. Human IgG or IgM standards (Caltaq,
Burlingame,
California) are used to establish a standard curve for each assay.
Isolation of the Peripheral Blood Mononuclear Cells (PBMC) from Human
Peripheral Blood
20 ml of Ficoll-Paque solution (low endotoxin; Pharmacia) is added per 50 ml
polystyrene tube, in 3 tubes, 30 minutes before adding the blood. The Ficoll-
Paque
solution is warmed up to room temperature. 3 L of bleach in 1:10 dilution is
prepared, and used to wash all the tubes and pipettes contacting the blood.
The blood
is layered on the top of the Ficoll-Paque solution without disturbing the
Ficoll layer,
at 1.5 ml blood/1 ml of Ficoll-Paque. The tubes are centrifuged at 1700 rpm
for 30
minutes at room temperature with the brake on the centrifuge turned off. As
much of
the top layer (plasma) as possible is removed, mininuzing the vacuum in order
to
avoid removing the second layer of solution. The second layer, which contains
the B
and T lymphocytes, is collected using a sterile Pasteur pipette, and placed in
two 50-
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ml polystyrene tubes. The collection is diluted with 3 x the volume of cold
RPMI
with no additives, and the tubes are centrifuged at 1000 RPM for 10 minutes.
The
media is removed by aspiration, and the cells from both 50-ml tubes are
resuspended
in a total of 10 ml cold RPMI (with additives) and transferred to a 15-ml
tube. The
cells are counted using the hemacytometer, then centrifuged at 1000 RPM for 10
minutes. The media is removed and the cells resuspended in 4 ml RPMI. This
fraction contains the PBMC.
Isolation of the B cells fi~orn PBMC
100 ~.1 of Dynabeads (anti-CD 19) are placed in a 5-ml plastic tube. 3 ml of
sterile PBS are added to the beads and mixed, and placed in the magnetic
holder, then
allowed to sit for 2 minutes. The solution is removed using a Pasteur pipette.
3 ml of
sterile PBS are added, mixed, and placed in the magnetic holder, then allowed
to sit
for 2 minutes. This procedure with sterile PBS is repeated one more time for a
total
of 3 washes. The PBMC is added into the beads and incubated, while mixing, for
30
minutes at 40°C. The tube containing the PBMC and beads is placed into
the
magnetic holder for 2 minutes, then the solution is transferred to a new 5-ml
tube in
the magnetic holder. After 2 minutes, the solution is transferred to a new 15-
ml tube.
This step is repeated four more times, and the solutions of the first four
times are
collected in the 15-ml tube, then centrifuged at 1000 RPM for 5 minutes. This
step
produces the pellet for T-cell separation.
100 ~,1 RPMI (with additives) is added to collect the beads, and the solution
is
transferred into a 0.7-ml tube. 10 ~,1 of Dynal Detacha Beads are added into
the
suspension at room temperature, and it is allowed to rotate for 45 minutes.
The
suspension is transferred into a new 5-ml tube and 3-ml of RPMI (with
additives) are
added. The tube is placed in the magnetic holder for 2 minutes. The solution
is
transferred into a new 5-ml tube in the holder for 2 minutes, then to a 15-ml
tube. The
previous step is repeated three more times, collecting the solution in the 15-
ml tube.
The 15-ml tube is centrifuged at 1000 RPM for 10 minutes, and the cells
resuspended
in 10 ml RMPI. The washing step is repeated 2 more times for a total of 3
washes.
The cells are counted before the last centrifugation. This step completes the
B-cell
purification. Cells axe stored in 90% FCS and 10% DMSO and frozen at -
80°C.
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Flow Cytofluo~ometric Assay
Ramos cells (106 cells/sample) are incubated in 100 ~.1 primary antibody (10
~,g/ml in PBS-BSA) for 20 min at 4°C. After 3 washes with PBS-BSA or
HBSS-
BSA, the cells are incubated in 100 ~,1 FITC-labeled F(ab')2 fragments of goat
anti-
human IgG) antibodies (Calta~ for 20 min at 4°C. After 3 washes with
PBS-BSA
and 1 wash with PBS, the cells are resuspended in 0.5-ml PBS. Analyses are
performed with a FACSCAN V (Becton Dickinson, San Jose, California).
Generation of Hyb~idoma Clones
Splenocytes from immunized mice are fused with SP 2/0 or P 3 x 63Ag8.653
marine myeloma cells at a ratio of 10:1 using 50% polyethylene glycol as
previously
described by de Boer et al. (1988) J. Immunol. Meth. 113:143. The fused cells
are
resuspended in complete IIVVIDM medium supplemented with hypoxanthine ( 0.1
mM),
aminopterin ( 0.01 mM), thymidine ( 0.016 mM), and 0.5 ng/ml hIL-6 (Genzyme,
Cambridge, Massachusetts). The fused cells are then distributed between the
wells of
96-well tissue culture plates, so that each well contains 1 growing hybridoma
on
average.
After 10-14 days the supernatants of the hybridoma populations are screened
for specific antibody production. For the screening of specific antibody
production by
the hybridoma clones, the supernatants from each well are pooled and tested
for anti-
CD40 activity specificity by ELISA first. The positives are then used for
fluorescent
cell staining of EBV-transformed B cells as described for the FAGS assay
above.
Positive hybridoma cells are cloned twice by limiting dilution in IMDM/FBS
containing 0.5 ng/ml hIL-6.
Example 1: Production of Anti-CD40 Antibodies
Transgenic mice bearing the human IgGl or IgG2 heavy chain locus and the
human K chain locus (Abgenix y-1 xenomouse) are used to generate anti-CD40
antibodies. SF9 insect cells expressing CD40 extracellular domain axe used as
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immunogen. Mice spleens are fused with the mouse myeloma SP2/0 cells to
generate
antibodies that recognize recombinant CD40 in ELISA. On average approximately
10% of hybridomas produced in Abgenix xenomice may contain mouse lambda light
chain instead of human kappa chain. The antibodies containing mouse light
lambda
chain are selected out. A subset of antibodies that also show binding to cell-
surface
CD40 is selected for further analysis. Stable hybridomas selected during a
series of
subcloning procedures are used for further characterization in binding and
functional
assays. Clones from other hybridomas are further identified as having
antagonistic
activity. Based on their relative antagonist potency and ability to inhibit
C4BP-
mediated CD40 signaling, and thus impact CD40-directed activities, hybridoma
clones are selected for further evaluation.
Example 2: Binding Properties of Selected Hybridomas
Protein A is immobilized onto CMS biosensor chips via amine coupling. Anti-
CD40 monoclonal antibodies, at 1.5 wg/ml, are captured onto the modified
biosensor
surface for 1.5 minutes at 10 ~1/min. Recombinant soluble CD40-his is flowed
over
the biosensor surface at varying concentrations. Antibody and antigen are
diluted in
0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20 (HBS-
EP). Kinetic and affinity constants are determined using the Biaevaluation
software
with a 1:1 interaction model/global fit.
Example 3: Effect of Antagonist Anti-CD40 Antibodies on the CD40/C4BP
Interaction Ira vitro
In some instances, the candidate antibodies will prevent the binding of C4BP
to cell surface CD40 and displace the pre-bound C4BP. Candidate antibodies are
tested for their ability to prevent C4BP binding to CD40 on the surface of a
lymphoma cell line (Ramps). Binding of suitable antagonist anti-CD40
antibodies to
CD40 antigen on these cells prevents the subsequent binding of PE-C4BP, FITC-
C4BP, biotin-C4BP, or Alexfluor-C4BP, as measured by flow cytometric assays.
In a
second set of assays, the candidate antibodies are tested for their ability to
displace
C4BP pre-bound to cell surface CD40.
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Example 4: Antibody Antagonists for CD40-Directed Proliferation
of Human Lymphocytes from Normal Subjects
Engagement of CD40 by C4BP stimulates CD40 signaling, which induces
proliferation of normal human B cells. An antagonist anti-CD40 antibody that
blocks
C4BP-mediated CD40 signaling is expected to inhibit this proliferation.
Candidate antibodies are tested for their ability to inhibit C4BP-mediated
CD40 signaling and subsequent proliferation of PBMC from normal human
subjects.
Soluble C4BP alpha chain subunit and C4BP x7(31 heteromer are used as
agonists.
The proliferation of PBMC is measured by tritiated-thyrnidine incorporation.
The
experiment is performed with multiple donors of PBMC (n>1) to ensure that the
observed inhibition is not a peculiarity of cells from a single donor. A wide
range of
antibody concentrations (0.01 p,g/ml to 100 p,g/ml) is used in these assays.
Generally,
antibodies of interest will interfere with C4BP-mediated CD40 signaling,
thereby
inhibiting CD40-directed proliferation, at 0.1 ~g/ml concentration of
antibodies in
most cases.
In addition to B cells, human PBMC also contain natural killer cells that can
mediate antibody dependent cytotoxicity (ADCC). To clarify the mechanism of
antibody-mediated inhibition of proliferation, assays are performed with B
cells
purified from human PBMC. If antibodies can inhibit CD40-directed
proliferation of
purified B cells that is induced by binding of C4BP to CD40, then the
antagonist
activity of the candidate antibodies, and not the mechanism of ADCC, causes
proliferation inhibition in these assays.
Example 5: Antagonist Antibodies Do Not Induce Strong Proliferation of
Human B Cells from Normal Subjects
C4BP induces normal B cells to proliferate. Binding of agonist anti-CD40
antibodies can provide a similar stimulatory signal for the proliferation of
normal and
malignant B cells. Antibodies with strong B cell stimulatory activity are not
suitable
candidates for therapeutic treatment of B cell lymphomas and autoimmune
disorders.
The candidate antibodies that block C4BP-mediated CD40 signaling are tested
for
their ability to induce proliferation of B cells from normal volunteer donors.
The B
cells purified from normal donor PBMC are cultured with varying concentrations
of
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candidate antibodies (range of 0.001 to 100 ~.g/ml) for a total of 4 days. The
B cell
proliferation is measured by incorporation of tritiated thymidine. While
soluble C4BP
induces vigorous proliferation of B cells, candidate antibodies that are
suitable for
methods of the present invention induce only weak proliferation of normal B
cells.
In addition to B cells, human PBMC contain cell types that bear Fc receptors
(FcR) for IgGl molecules that can provide cross linking of anti-CD40
antibodies
bound to CD40 on B cells. This cross-linking could potentially enhance
stimulatory
activity of anti-CD40 antibodies. To confn~n the lack of B cell stimulatory
activity of
candidate antibodies in the presence of cross-linking cells, proliferation
experiments
are performed with total PBMC containing B cells as well as FcR+ cells.
Generally,
these candidate antibodies even in the presence of FcR-bearing cells do not
stimulate
B cells to proliferate over background proliferation induced by control human
IgGl.
The lack of stimulatory activity by candidate mAbs is further confirmed by
measuring
the PBMC proliferation in response to candidate anti-CD40 antibodies
immobilized
on the plastic surface of culture wells. Taken together these data show that
the
candidate anti-CD40 antibodies do not possess strong B cell stimulatory
properties.
Example 6: Candidate Antibodies Are Able to Kill CD40-Bearing
Target Cells by ADCC
In some instances, the candidate antibodies can kill CD40-bearing target cells
(lymphoma lines andlor solid tumor lines) by the mechanism of ADCC. Antibodies
of the IgGl isotype are expected to have the ability to induce the killing of
target cells
by the mechanism of ADCC. The candidate anti-CD40 antibodies are tested for
their
ability to kill cancer cell lines in ira vitro assays. Two human lymphoma cell
lines
(Ramos and Daudi), one human colon cancer cell line (HCT116), and seven other
carcinoma cell lines, including the ovarian cancer cell lines SKOV3 and HEY,
the
skin squamous cancer cell line A431, the breast cancer cell lines MDA-MB231
and
MDA-MB435, and the lung cancer cell lines NCI-H460 and SK-MES-1 are selected
as target cells for these assays. PBMC or enriched NK cells from normal
volunteer
donors are used as effector cells in these assays.
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Example 7: Candidate Antibodies Do Not Stimulate Proliferation of
Cancer Cells from the Lymph Nodes of NHL Patients
CD40 signaling induces survival and proliferation of lymphoma cells from
NHL patients. As such C4BP may play a role in NHL. Binding of some anti-CD40
antibodies (agonist) can provide a similar stimulatory signal for the
proliferation of
patient cancer cells. As noted above, antibodies with strong B cell
stimulatory activity
are not suitable candidates for therapeutic treatment of B cell lymphomas.
Candidate
antibodies are tested for their ability to induce proliferation of NHL cells
from
patients. The cells isolated from lymph node (LN) biopsies axe cultured with
varying
concentrations of candidate antibodies (range of 0.01 to 300 ~,g/ml) for a
total of 3
days. The cell proliferation is measured by incorporation of tritiated
thymidine.
Generally, candidate mAbs should not induce any proliferation of cancer cells
at any
concentration tested. Antibodies even in the presence of exogenously added IL-
4, a B
cell growth factor, should not induce proliferation of NHL cells. These
results will
indicate whether candidate antibodies are non- agonist anti-CD40 antibodies
and do
not stimulate proliferation in vitro of NHL cells from patients.
Example 8: Candidate Antibodies Can Block C4BP-Induced Proliferation
of Cancer Cells from Non-Hodgkin's Lymphoma Patients
Engagement of CD40 by C4BP may induce proliferation of cancer cells from
NHL patients. Candidate antagonist anti-CD40 antibodies are expected to
inhibit this
proliferation. Candidate anti-CD40 antibodies are tested at varying
concentrations
(0.01 ~,g /ml to 100 ~,g /ml) for their ability to inhibit CD40-directed
proliferation of
NHL cells that is induced by the binding of C4BP to CD40 antigen on these
cells.
NHL cells from patients are cultured in suspension with C4BP in the presence
of IL-
4. The NHL cell proliferation is measured by 3H-thymidine incorporation.
Candidate
antibodies of interest inhibit the proliferation of NHL cells when compared to
the
control in a dose-dependent manner, as the inhibitory effect increases with
increasing
antagonist anti-CD40 antibody concentration.
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Example 9: Effect of Candidate Antibodies on Number of Viable
NHL Cells When Cultured with C4BP-Expressing Cells
Binding of C4BP to CD40 is an alternative method of stimulating CD40-
expressing cells. CD40 signaling is important for B cell survival. This set of
experiments evaluates the effect of candidate anti-CD40 antibodies on NHL cell
numbers at days 7, 10, and 14 in the presence of C4BP. NHL cells from patients
are
cultured in suspension with C4BP in the presence of IL-4. The control human
IgG and
candidate antibodies are added at concentrations of 10 ~,g/ml at day 0 and day
7. The
viable cells under each condition are counted on the specified day. Cell
numbers in
the control group (IgG) generally increases with time. Generally, reduced
numbers of
cells are recovered in the presence of antagonist antibodies as compared to
control
group.
Many modifications and other embodiments of the inventions set forth herein
will come to mind to one skilled in the art to which these inventions pertain
having
the benefit of the teachings presented in the foregoing descriptions and the
associated
drawings. Therefore, it is to be understood that the inventions are not to be
limited to
the specific embodiments disclosed and that modifications and other
embodiments are
intended to be included within the scope of the appended claims disclosed
herein.
Although specific terms are employed herein, they are used in a generic and
descriptive sense only and not for purposes of limitation.
All publications and patent applications mentioned in the specification are
indicative of the level of those skilled in the art to which this invention
pertains. All
publications and patent applications are herein incorporated by reference to
the same
extent as if each individual publication or patent application was
specifically and
individually indicated to be incorporated by reference.
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