Note: Descriptions are shown in the official language in which they were submitted.
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CONJUGATED ANTI-PSMA ANTIBODIES
RELATED APPLICATIONS
This application is related to U.S. Provisional Application 60/669,347, filed
April 8,
2005, which is herein,incorporated by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to the development of methods and tools
effective for
treating, preventing, and diagnosing cancer. Specifically, the present
invention is directed to
methods of treating, preventing, and diagnosing cancer comprising using
antibodies which
immunospecifically bind to prostate specific membrane antigen and are
conjugated to a
radioisotope via a MeO-DOTA linkage.
BACKGROUND OF THE INVENTION
Prostate cancer is among the most significant medical problems in the United
States, as
the disease is now the most common malignancy diagnosed in American males. The
American
Cancer Society estimates that for the year 2000, 180,400 new cases of prostate
cancer were
diagnosed witli 31,900 deaths from the disease. Five year survival rates for
patients with prostate
cancer range from 88% for those with localized disease to 29% for those with
metastatic disease.
The rapid increase in the number of cases appears to result in part from an
increase in disease
awareness as well as the widespread use of clinical markers such as the
secreted proteins prostate-
specific antigen (PSA) and prostatic acid phosphatase (PAP) (Chiaroda (1991)
Cancer Res. 51,
2498-2505).
The prostate gland is a site of significant pathology affected by conditions
such as benign
growth (BPH), neoplasia (prostatic cancer) and infection (prostatitis).
Prostate cancer represents
the second leading cause of death from cancer in man (Chiaroda (1991) Cancer
Res. 51, 2498-
2505). However the prostate is the leading site for cancer development in men.
The difference
between these two facts relates to prostatic cancer occurring with increasing
frequency as men
age, especially in the ages beyond sixty at a time when death from other
factors often intervenes.
Also, the spectrum of biologic aggressiveness of prostatic cancer is great, so
that in some men
following detection the tumor remains a latent histologic tumor and does not
become clinically
significant, whereas in the other it progresses rapidly, metastasizes and
kills the patient in a
relatively short two to five year period (Chiaroda (1991) Cancer Res. 51, 2498-
2505; Warner et
al. (1991) Urologic Clinics of North America 18, 25-33).
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In prostate cancer cells, two specific proteins that are made in very high
concentrations
are prostatic acid phosphatase (PAP) and prostate specific antigen (PSA)
(Henttu et al. (1989)
Bioch. Biophys. Res. Comm. 160, 903-908; Nguyen et al. (1990) Clin. Chem. 35,
1450-1455;
Yong et al. (1991) Cancer Res. 51, 3748-3752). These proteins have been
characterized and have
been used to follow response to therapy. With the development of cancer, the
normal architecture
of the gland becomes altered, including loss of the normal duct structure for
the removal of
secretions and thus the secretions reach the serum. Measurement of serum PSA
is suggested as a
potential screening metliod for prostatic cancer. Indeed, the relative amount
of PSA and/or PAP
in the cancer changes as compared to normal or benign tissue.
PAP was one of the earliest serum markers for detecting metastatic spread
(Nguyen et al.
(1990) Clin. Chem. 35, 1450-1455). PAP hydrolyses tyrosine phosphate and has a
broad
substrate specificity. Tyrosine phosphorylation is often increased with
oncogenic transformation.
It has been hypothesized that during neoplastic transformation there is less
phosphatase activity
available to inactivate proteins that are activated by phosphorylation on
tyrosine residues. In
some instances, insertion of phosphatases that have tyrosine phosphatase
activity has reversed the
malignant phenotype.
PSA is a protease and it is not readily appreciated how loss of its activity
correlates witli
cancer development (Henttu et al. (1989) Bioch. Biophys. Res. Comm. 160, 903-
908; Yong et al.
(1991) Cancer Res. 51, 3748-3752). The proteolytic activity of PSA is
inhibited by zinc. Zinc
concentrations are high in the normal prostate and reduced in prostatic
cancer. Possibly the loss
of zinc allows for increased proteolytic activity by PSA. As proteases are
involved in metastasis
and some proteases stimulate mitotic activity, the potentially increased
activity of PSA could be
hypothesized to play a role in the tumors metastases and spread (Liotta (1986)
Cancer Res. 46, 1-
7). Both PSA and PAP are found in prostatic secretions. Both appear to be
dependent on the
presence of androgens for their production and are substantially reduced
following androgen
deprivation.
Prostate-specific membrane antigen (PSMA), which appears to be localized to
the
prostatic membrane, has also been identified as a marker for prostate cancer.
PSMA is expressed
in virtually all prostate cancers (Bostwick et al. (1998) Cancer, 82, 2256-
2261). Although recent
studies have shown that it is also expressed by the small intestine epithelial
(brush-border) cells,
proximal renal tubule cells and salivary glands, the level of expression in
these normal tissue is
100 to 1,000 fold less that in prostate tissue, and these PSMA expressing
normal cells are not
typically exposed to circulating antibodies due to their brush-border/luminal
location (Nanus et al.
(2003) Journal of Urology, 170, S84-S89). In addition, in contrast to other
prostate related
antigens such as PSA, PMSA, which is a type II integral membrane cell surface
protein, it is not
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secreted and, therefore, is an excellent target for monoclonal antibody
therapy (Nanus et al.
(2003) Journal of Urology, 170, S84-S89).
This antigen was identified as the result of generating monoclonal antibodies
to a
prostatic cancer cell, LNCaP (Horoszewicz et al. (1993) Cancer Res., 53, 227-
230). LNCaP is a
cell line established from the lymph node of a hormone refractory, heavily
pretreated patient
(Horoszewicz et al. (1983) Cancer Res. 43, 1809-1818). This cell line was
found to have an
aneuploid human male karyotype. It maintained prostatic differentiation
functionality in that it
produced both PSA and PAP. It possessed an androgen receptor of high affinity
and specificity.
Mice were immunized with LNCaP cells and hybridomas were derived from
sensitized animals.
A monoclonal antibody was derived and was designated 7E11-C5 (Horoszewicz et
al. (1993)
Cancer Res. 53, 227-230). The antibody staining was consistent with a membrane
location and
isolated fractions of LNCaP cell membranes exhibited a strongly positive
reaction with
immunoblotting and ELISA techniques.
This monoclonal antibody was also used for detection of immunoreactive
material in
serum of prostatic cancer patients (Horoszewicz et al. (1993) Cancer Res. 53,
227-230). The
immunoreactivity was detectable in nearly 60% of patients with stage D-2
disease and in a
slightly lower percentage of patients with earlier stage disease, but the
numbers of patients in the
latter group were small. Patients with benign prostatic hyperplasia (BPH) were
negative. Patients
with no apparent disease were negative, but 50 to 60% of patients in
remission, yet with active
stable disease or with progression, demonstrated positive serum reactivity.
Patients with non
prostatic tumors did not show immunoreactivity with 7E11-C5.
The 7E11-C5 monoclonal antibody is now used as a molecular imaging agent and
is the
first and currently the only commercial product targeting PSMA. Prostascint
consists of 7E11-
C5 linked to the radioisotope Indium-111. Due to the selective expression of
PSMA by prostate
cancer cells, Prostascint can image the extent and spread of prostate cancer
using a common
gamma camera. U.S. Patent 5,162,504 discloses and claims the monoclonal
antibody 7E11-C5
and the hyrbirdoma cell line that produces it. U.S. Patents 4,671,958;
4,741,900 and 4,867,973
disclose and claim antibody conjugates, methods for preparing such conjugates,
methods for using
such conjugates for in vivo imaging, testing and therapeutic treatment, and
methods for delivering
radioisotopes by linking them to such antibodies.
There are at least three other anti-PSMA antibodies in the art that have been
conjugated to
radioisotopes and at least one tested for the treatment of prostrate cancer.
J591, J415, and J591 are
monoclonal antibody binds with high affinity to an extracellular epitope of
PSMA and localizes
specifically in PSMA (Smith-Jones et al. (2000) Cancer Res. 60, 5237-5243).
These antibodies
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have been labeled with 131I and 111In via a DOTA linkage. The average DOTA to
antibody ratio
for these antibodies was 5:1, with little apparent loss of immunoreactivity.
Conjugation average
of eight DOTA molecules to J591 resulted in a 20% reduction in
immunoreactivity (Smith-Jones
et al. (2000) Cancer Res. 60, 5237-5243). Thus, the art indicates that there
is an upper limit of
DOTA to antibody ratio without the antibody losing immunoreactivity.
SUMMARY OF THE INVENTION
The invention encompasses novel compositions which comprise an antibody which
immunospecifically binds to prostate specific membrane antigen (PSMA), wherein
said antibody
is conjugated to a radioisotope via a MeO-DOTA linkage (henceforth known as
"conjugated
antibody" or "conjugated antibodies"). In a further embodiment, the ratio of
MeO-DOTA to
antibody is about 9:1 or greater.
The invention also encompasses methods for preventing, treating, or managing
cancer in
a subject which comprises administering an antibody which immunospecifically
binds to prostate
specific membrane antigen (PSMA), wherein said antibody is conjugated to a
radioisotope via a
MeO-DOTA. In a further embodiment, the MeO-DOTA to antibody ratio is about 9:1
or greater.
In an even further embodiment, the MeO-DOTA to antibody ratio is about 9:1, or
greater, with
little, if any, loss of immunoreactivity.
The conjugated antibodies of the invention can be administered in combination
with one
or more other cancer therapies. In particular, the present invention provides
methods of
preventing, treating, or managing cancer in a subject comprising administering
to said subject a
therapeutically or prophylactically effective amount of one or more conjugated
antibodies of the
invention in combination with the administration of a therapeutically or
prophylactically effective
amount of one or more chemotherapies, hormonal therapies, biological
therapies/immunotherapies and/or radiation therapies, other than the
administration of the
conjugated antibody of the invention, and/or in combination with surgery. The
conjugated
antibody of the invention can be administered concurrently to a subject in
separate pharmaceutical
compositions or in the same composition. In addition, it is also contemplated
that the conjugated
antibody of the invention can be administered prior to the administration of
other therapies or
after the administration of other therapies. The therapeutic agents may be
administered to a
subject by the same or different routes of administration.
The invention also includes methods and compositions for the treatment of
cancer in a
mammal, including a human, comprising administering to said mammal an amount
of a cytotoxic
agent, or a pharmaceutical composition comprising an amount of the cytotoxic
agent, that is
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effective in enhancing the binding of a monoclonal antibody to an epitope on
the cytoplasmic
domain of PSMA. In one embodiment, the cytotoxic agent will induce apoptosis
of the malignant
cell, and/or increase permeability, and/or otherwise disrupt the cell
membrane. In another
embodiment, the cytotoxic agent is administered to a mammal prior to or
simultaneously with the
conjugated antibodies of the invention. In this aspect of the invention, the
cytotoxic agent may
disrupt the cancer cell(s), thereby expressing the cytoplasmic domain on the
PSMA antigen.
The invention further provides diagnostic methods to evaluate or diagnose an
individual
with a malignant cell expressing PSMA using the conjugated antibodies of the
invention. In
particular embodiments, the diagnostic methods of the invention provide
methods of imaging and
localizing malignant cells expressing PSMA, methods of diagnosis and prognosis
using tissues
and fluids distal to the primary tumor site (as well as methods using tissues
and fluids of the
primary tumor and tissues and/or using tissues and fluids surrounding the
primary tumor), for
example, whole blood, sputum, urine, serum, fine needle aspirates (i.e.,
biopsies). In other
embodiments, the diagnostic methods of the invention provide methods of
imaging and localizing
metastases and methods of diagnosis and prognosis in vivo. In such
embodiments, primary
tumors are detected using the conjugated antibody of the invention. The
antibodies of the
invention may also be used for immunohistochemical analyses of frozen or fixed
cells or tissue
assays.
The invention further provides a novel, efficient method to conjugate
antibodies which
immunospecifically bind to prostate specific membrane antigen (PSMA) with MeO-
DOTA. In
addition, the invention further provides an efficient method to complex the
conjugated antibody
with a radioisotope.
The invention also provides pharmaceutical compositions comprising one or more
monoclonal antibodies of the invention either alone or in combination with one
or more other
agents useful for cancer therapy.
In another embodiment, kits comprisingthe pharmaceutical compositions or
diagnostic
reagents of the invention are provided.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a Coomassie-stained one-dimensional 4 to 20% SDS-PAGE gel of eight
lots of 7E11-
C5 conjugates (conjugated to a radioisotope via a MeO-DOTA linkage) prepared
under conditions
as set forth in Table 1. Arrows indicate heavy chain (50 kDA) and light chain
(25 kDA).
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Figure 2 (A) is size exclusion chromatogram of 7E11-C5 antibody and (B) a
representative
chromatogram of its MeO-DOTA conjugate (lot 200402495-5/8). Relative peak
areas of
unmodified protein and conjugates are summarized in Table 3.
Figure 3A-D are size exclusion chromatograms of different complexations of MeO-
DOTA-
Cyt351, 200402495-29, under the indicated condition. Relative peak areas of
unmodified protein
and conjugates are summarized in Table 5.
Figure 4 is a size exclusion chromatogram of a high specific activity
conjugate preparation at 30
minutes. Relative peak areas of unmodified protein and conjugates are
summarized in Table 6.
DETAILED DESCRIPTION
Radiation is an effective cancer treatment, but it is difficult to direct and
can be
devastating to nontargeted parts of the body. Indeed, treatments are often
limited by its non-
selectivity, resulting in toxicity on the normal tissues. Monoclonal
antibodies, or fragments
thereof, on the other hand, are adept at selectively targeting diseased cells.
The ability of
antibodies to exploit antigenic differences between normal and malignant
tissues and to exact a
variety of antitumor responses offers significant advantages to conventional
forms of therapy.
However, antibodies alone often have inadequate therapeutic effectiveness.
Conjugating a radioisotope to a monoclonal antibody, or fragments thereof, can
solve
both problems. Monoclonal antibodies are highly specific and can be used as
vehicles to deliver
substances to specific target sites. Thus, the conjugation of a monoclonal
antibody to a
radioisotope is an effective way to target specific cells, thus reducing the
side effects of
radiotherapy.
The invention described relates to compositions comprising an antibody, or
fragments
thereof, which immunospecifically binds to prostate specific membrane antigen
(PSMA),
wherein said antibody, or fragment(s) thereof, is conjugated to a radioisotope
via a MeO-DOTA
linkage. In one embodiment of the invention, the anti-PSMA monoclonal antibody
is 7E11-C5.
In another embodiment the conjugated 7E11-C5 antibody is complexed with 1 77
Lu. In a preferred
embodiment, the conjugated 7E11-C5 is complexed with "'Lu at a ratio of
isotope to antibody of
about 9:1 or greater. The invention also discloses methods of using,
formulating and making the
antibodies of the invention.
Conjugated antibodies
The invention comprises antibodies, or fragments thereof, which
immunospecifically bind
to prostate specific membrane antigen (PSMA), wherein said antibody is
conjugated to a
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radioisotope via a MeO-DOTA linkage. MeO-DOTA, a-(5-isothiocyanato-2-
methoxyphenyl)-
1,4,7,10-teraazacyclododeczane-1,4,7,10-tretraacetic (Dow Chemical Company) is
a bifunctional
chelant. A bifunctional chelant is a molecule that has, in addition to
chelating functionality, the
ability to be conjugated (linked) to a biotargeting molecule (e.g. monoclonal
antibody). MeO-
DOTA provides metal complexes with high stability, thereby reducing the
incidence of
background during imaging procedures or damage to non-targeted tissues in
radioimmunotherapy.
In one embodiment of the invention, the antibody is 7E11-C5. In another
embodiment the MeO-
DOTA linked antibody is complexed to a radioisotope which is selected from the
group consisting
of3H, 14C, 1fiF,19F > 31P> 32P> 35s> 131I> 125I> 123I> 64Cu> 187Re, 1 IlIn>
9oY> 99Tc, 177Lu. In another
embodiment the antibody comprises 7E11-C5 conjugated to 177 Lu via MeO-DOTA.
In a
preferred embodiment the MeO-DOTA to antibody ratio is about 9:1 or greater.
An antibody is a heterotetrameric glycoprotein composed of two identical light
(L) chains
and two identical heavy (H) chains (an IgM antibody consists of five of the
basic heterotetramer
unit along with an additional polypeptide called J chain, and therefore
contain ten antigen binding
sites, while secreted IgA antibodies can polymerize to form polyvalent
assemblages comprising
two to five of the basic four chain units along with J chain). The L chain
from any vertebrate
species can be assigned to one of two clearly distinct types, called kappa and
lambda, based on the
amino acid sequences of their constant domains and the methods of the current
invention include
the use of antibodies with either a kappa or lambda L chain. Depending on the
amino acid
sequence of the constant domain of their heavy chains (CH), immunoglobulins
can be assigned to
different classes or isotypes. There are five classes of immunoglobulins: IgA,
IgD, IgE, IgG, and
IgM, having heavy chains designated alpha, delta, epsilon, gamma and mu,
respectively. The
gamma and alpha classes are further divided into subclasses on the basis of
relatively minor
differences in CH sequence and function, e.g., humans express the following
subclasses: IgGI,
IgG2, IgG3, IgG4, IgAl and IgA2. The methods of the present invention include
the use of
antibodies, including monoclonal antibodies, from any of the above classes
and/or subclasses.
As used herein, the term "variable" refers to the fact that certain segments
of the variable
domains differ extensively in sequence among antibodies. The variable domain
mediates antigen
binding and defines specificity of a particular antibody for its particular
antigen. However, the
variability is not evenly distributed across the 110-amino acid span of the
variable domains.
Instead, the variable regions consist of relatively invariant stretches called
framework regions
(FR) of about fifteen to thirty amino acids separated by shorter regions of
extreme variability
called "hypervariable regions" that are each about nine to twelve amino acids
long. The variable
domains of native heavy and light chains each comprise four framework regions,
largely adopting
a beta-sheet configuration, connected by three hypervariable regions, which
form loops
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connecting, and in some cases forming part of, the beta-sheet structure. The
hypervariable
regions in each chain are held together in close proximity by the framework
region and, with the
hypervariable regions from the other chain, contribute to the formation of the
antigen-binding site
of antibodies (see Kabat et al. (1991) Sequences of Proteins of Immunological
Interest, Public
Health Service, National Institutes of Health). The constant domains are not
involved directly in
binding an antibody to an antigen, but exhibit various effector functions,
such as participation of
the antibody in antibody dependent cellular cytotoxicity (ADCC).
The term "hypervariable region" when used herein refers to the amino acid
residues of an
antibody which are responsible for antigen-binding. The hypervariable region
generally
coinprises amino acid residues from a "complementarity determining region" or
"CDR" which
contributes to the specificity of the antibody.
The term "antibodies or fragments thereof' as used herein refers to antibodies
or
fragments tliereof that specifically bind to a PSMA polypeptide or a fragment
of a PSMA
polypeptide and do not specifically bind to other non-PSMA polypeptides.
Preferably, antibodies
or fragments that immunospecifically bind to a PSMA polypeptide or fragment
thereof do not
non-specifically cross-react with other antigens (e.g., binding cannot be
competed away with a
non-PSMA protein, e.g., BSA in an appropriate immunoassay). Antibodies or
fragments that
immunospecifically bind to an PSMA polypeptide can be identified, for example,
by
immunoassays or other techniques known to those of skill in the art.
Antibodies of the invention
include, but are not limited to, synthetic antibodies, monoclonal antibodies,
recombinantly
produced antibodies, intrabodies, diabodies, multispecific antibodies
(including bi-specific:
antibodies), human antibodies, humanized antibodies, chimeric antibodies,
single-chain Fvs
(scFv) (including bi-specific scfvs), single chain antibodies, Fab' fragments,
F(ab')2 fragments,
disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and
epitope-binding fragments
of any of the above. In particular, antibodies of the present invention
include immunoglobulin
molecules and immunologically active portions of immunoglobulin molecules,
i.e., molecules that
contain an antigen binding site that immunospecifically binds to an PSMA
antigen (e.g., one or
more complementarity determining regions (CDRs) of an anti-PSMA antibody).
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 and includes antibody fragments as defined herein. Monoclonal
antibodies are
highly specific, being directed against a single antigenic site. Furthermore,
in contrast to
polyclonal antibody preparations which include different antibodies directed
against different
deterininants (epitopes), each monoclonal antibody is directed against a
single determinant on the
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antigen. In addition to their specificity, the monoclonal antibodies are
advantageous in that they
may be synthesized uncontaminated by other antibodies. The modifier
"monoclonal" is not to be
construed as requiring production of the antibody by any particular method.
For example, the
monoclonal antibodies useful in the present invention may be prepared by the
hybridoma
methodology first described by Kohler et al. (1975) Nature 256, 495 or may be
made using
recombinant DNA metliods in bacterial, eukaryotic animal or plant cells (see
U.S. Patent
4,816,567). The "monoclonal antibodies" may also be isolated from phage
antibody libraries
using the techniques described in Clackson et al. (1991) Nature 352:624-628
and Marks et al.
(1991) J. Mol. Biol. 222, 581-597, for example.
As used herein, an "intact" antibody is one which comprises an antigen-binding
site as
well as a CL and at least heavy chain constant domains, CHl and CH2 and CH3.
The constant
domains may be native sequence constant domains (e.g., human native sequence
constant
domains) or amino acid sequence variant thereof. Preferably, the intact
antibody has one or more
effector functions.
An "antibody fragment" comprises a portion of an intact antibody, preferably
the antigen
binding CDR or variable region of the intact antibody. Examples of antibody
fragments include
Fab, Fv, Fab' and F(ab')2 fragments; diabodies; linear antibodies (see U.S.
Patent 5,641,870 and
Zapata et al. (1995) Protein Eng. 8, 1057-1062); single-chain antibody
molecules; and
multispecific antibodies formed from antibody fragments.
Papain digestion of antibodies produces two identical antigen-binding
fragments, called
"Fab" fragments, and a residual "Fc" fragment, a designation reflecting the
ability to crystallize
readily. The Fab fragment consists of an entire L chain along with the
variable region domain of
the H chain (VH), and the first constant domain of one heavy chain (CHl). Each
Fab fragment is
monovalent with respect to antigen binding, i.e., it has a single antigen-
binding site. Pepsin
treatment of an antibody yields a single large F(ab')2 fragment which roughly
corresponds to two
disulfide linked Fab fragments having divalent antigen-binding activity and is
still capable of
cross-linking antigen. Fab' fragments differ from Fab fragments by having
additional few
residues at the carboxy terminus of the CHI domain including one or more
cysteines from the
antibody hinge region. Fab'-SH is the designation herein for Fab' in which the
cysteine residue(s)
of the constant domains bear a free thiol group. F(ab') 2 antibody fragments
originally were
produced as pairs of Fab' fragments which have hinge cysteines between them.
Other chemical
couplings of antibody fragments are also known.
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The Fc fragment comprises the carboxy-terminal portions of both H chains held
together
by disulfides. The effector functions of antibodies are determined by
sequences in the Fc region,
which region is also the part recognized by Fc receptors (FcR) found on
certain types of cells.
As used herein, "Fv" is the minimum antibody fragment which contains a
complete
antigen-recognition and -binding site. This fragment consists of a dimer of
one heavy- and one
light-chain variable region domain in tight, non-covalent association. From
the folding of these
two domains emanate six hypervariable loops (three loops each from the H and L
chain) that
contribute the amino acid residues for antigen binding and confer antigen
binding specificity to
the antibody. However, even a single variable 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.
As used herein, "Single-chain Fv" also abbreviated as "sFv" or "scFv" are
antibody
fragments that comprise the VH and VL antibody domains connected into a single
polypeptide
chain. Preferably, the sFv polypeptide further comprises a polypeptide linker
between the VH and
VL domains which enables the sFv to form the desired structure for antigen
binding (see
Rosenburg et al. (1994) The Pharmacology of Monoclonal Antibodies, Springer-
Verlag, pp. 269-
315).
As used herein, the term "diabodies" refers to small antibody fragments
prepared by
constructing sFv fragments (see preceding paragraph) with short linkers (about
5 to about 10
residues) between the VH and VL domains such that inter-chain but not intra-
chain pairing of the
V domains is achieved, resulting in a bivalent fraginent, i.e., fragment
having two antigen-binding
sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments
in which the VH
and VL domains of the two antibodies are present on different polypeptide
chains. Diabodies are
described more fully in, for example, WO 93/11161 and Hollinger et al. (1993)
Proc. Natl. Acad.
Sci. USA 90, 6444-6448.
An "isolated antibody" is one which has been identified and separated and/or
recovered
from a component of its natural environment. Contaminant components of its
natural
environment are materials which would interfere with diagnostic or therapeutic
uses for the
antibody, and may include enzymes, hormones, and other proteinaceous or non-
proteinaceous
components. In preferred embodiments, thg antibody will be purified to greater
than 95% by
weight of antibody, and most preferably more than 99% by weight. Isolated
antibody includes the
antibody in situ within recombinant cells since at least one component of the
antibody's natural
environment will not be present. Ordinarily, however, isolated antibody will
be prepared by at
least one purification step.
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In one embodiment of the invention, the conjugated antibody binds to an
epitope on the
cytoplasmic domain of a protein specific to cancer cells (i.e., a cancer cell
marker). In another
embodiment, the conjugated antibody includes, but is not limited to, an
antibody which binds to
an epitope on the cytoplasmic domain of PSMA, including but not limited to,
the 7E11-C5
monoclonal antibody as described in U.S. Patent 5,162,504 which is herein
incorporated by
reference in its entirety. The hybridoma cell line which produces the 7E11-C5
monoclonal
antibody has been deposited with the American Type Culture Collection under
Deposit No.
HB10494.
The conjugated antibodies used in the methods of the invention include
"chimeric"
antibodies in which a portion of the heavy and/or light chain is identical
with or homologous to
corresponding sequences in antibodies derived from a particular species or
belonging to a
particular antibody class or subclass, while the remainder of the chain(s) is
identical with or
homologous to corresponding sequences in antibodies derived from another
species or belonging
to another antibody class or subclass, as well as fragments of such
antibodies, so long as they
exhibit the desired biological activity (see U.S. Patent 4,816,567 and
Morrison et al. (1984) Proc.
Natl. Acad. Sci. USA 81, 6851-6855). Chimeric antibodies of interest herein
include, but are not
limited to "humanized" antibodies comprising variable domain antigen-binding
sequences derived
from a non-human mammal (e.g., murine) and human constant region sequences.
Antibodies of
the invention may also comprise a fully human antibody sequence. In another
embodiment,
antibodies of the invention may be a fully human antibody.
Diagnostic methods
Antibodies, or fragments thereof, of the invention can be used as diagnostic
or detectable
agents. In a preferred embodiment, the antibody, or fragments thereof,
immunospecifically bind to
prostate specific membrane antigen (PSMA), wherein said antibody is conjugated
to a
radioisotope via a MeO-DOTA linkage. Antibodies of the invention can be useful
for monitoring
or prognosing the development or progression of a cancer as part of a clinical
testing procedure,
such as determining the efficacy of a particular therapy. Additionally, such
antibodies can be
useful for monitoring or prognosing the development or progression of
cancerous conditions.
In another embodiment, following initial administration of the conjugated
antibody of the
invention, the cancer cells can be imaged and the relative amount of cancerous
cells determined
by any available means. The invention includes diagnostic methods to detect
cancer and/or assess
the effect therapeutic agents on cancer cells in an organ or body area of a
patient. The present
methods include administration of a composition comprising a detectable amount
of an anti-
PSMA antibody conjugated to a radioisotope via MeO-DOTA to a patient before
and after
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therapy. In a further embodiment, the MeO-DOTA to antibody ratio is about 9:1
or greater. In an
even further embodiment, the MeO-DOTA to antibody ratio is about 9:1, or
greater, with little, if
any, loss of immunoreactivity. Subsequent to administration of the
tlierapeutic agent, an
additional amount of detectable monoclonal antibody can be administered to
determine the
relative amount of cancer cells remaining following treatment. Comparison of
the before and
after treatinent images can be used as a means to assess the efficacy of the
treatment wherein a
decrease in the number of cancer cells imaged following treatment is
indicative of an efficacious
treatment regimen.
As used herein, the term "detectable amount" refers to the amount of labeled
conjugated
antibody which binds to PSMA administered to a patient that is sufficient to
enable detection of
binding of the labeled monoclonal antibody to one or more malignant cancer
cells in a tumor. As
used herein, the term "imaging effective amount" refers to the amount of the
labeled antibody
administered to a patient that is sufficient to enable imaging of binding of
the antibody to one or
more malignant cancer cells in a tumor.
The methods of the invention comprise conjugated antibodies of the invention
which, in
conjunction with non-invasive neuroimaging techniques such as magnetic
resonance spectroscopy
(MRS) or imaging (MRI), or gamma imaging such as positron emission tomography
(PET) or
single-photon emission computed tomography (SPECT), are used to identify and
quantify
abnormal cells in vivo including malignant cells in tumors. The term "in vivo
imaging" refers to
any method which permits the detection of labeled monoclonal antibody as
described above. For
gamma imaging, the radiation emitted from the tumor or area being examined is
measured and
expressed either as total binding, or as a ratio in which total binding in one
tissue is normalized to
(for example, divided by) the total binding in another tissue or the entire
body of the same subject
during the same in vivo imaging procedure. Total binding in vivo is defined as
the entire signal
detected in a tumor or tissue by an in vivo imaging technique without the need
for correction by a
second injection of an identical quantity of labeled compound along with a
large excess of
unlabeled, but otherwise chemically identical compound. As used herein, the
terms "subject" or
"patient" refers to a mammal, preferably a human, and most preferably a human
suspected of
having abnormal cells, including malignant cells in a tumor.
For purposes of in vivo imaging, the type of detection instrument available is
a major
factor in selecting a given label. For instance, radioactive isotopes are
particularly suitable for in
vivo imaging in the methods of the present invention. The type of instrument
used will guide the
selection of the radioisotope. For instance, the radioisotope chosen must have
a type of decay
detectable by a given type of instrument. Another consideration relates to the
half-life of the
radioisotope. The half-life should be long enough so that it is still
detectable at the time of
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maximum uptake by the target, but short enough so that the host does not
sustain deleterious
radiation. The isotopically-labeled monoclonal antibody can be detected using
gamma imaging
where emitted gamma irradiation of the appropriate wavelength is detected.
Methods of gamma
imaging include, but are not limited to, positron emission tomography (PET)
imaging or for single
photon emission computerized tomography (SPECT). Preferably, for SPECT
detection, the
chosen radiolabel will lack a particulate emission, but will produce a large
number of photons.
For PET detection, the radiolabel will be a positron-emitting radioisotope
which will be detected
by the PET camera.
In the present invention, conjugated antibodies are useful for in vivo
detection and
imaging of tumors. These compounds are to be used in conjunction with non-
invasive
neuroimaging techniques such as magnetic resonance spectroscopy (MRS) or
imaging (MRI),
positron emission tomography (PET), and single-photon emission computed
tomography
(SPECT). In accordance with this invention, the conjugated antibody may be
labeled
(complexed) with any acceptable radioisotope. For example, including, but are
not limited to, 3H,
14C, laF>1sF > 31P, 32P, 35S, 131I11251, 123I664Cu, 187Re, "'In, 90Y, 99Tc, "
'Lu using techniques
described below or known in the art.
The diagnostic methods of the present invention may use isotopes detectable by
nuclear
magnetic resonance spectroscopy for purposes of in vivo imaging and
spectroscopy. Elements
particularly useful in magnetic resonance spectroscopy include, but are not
limited to,19F and 13C.
Suitable radioisotopes for purposes of this invention include beta-emitters,
gamma-emitters,
positron-emitters and x-ray emitters. These radioisotopes include, but are not
limited to, "'In,
1311, 123I> 18 F, 11C> 75Br and 76Br.
Suitable stable isotopes for use in Magnetic Resonance Imaging (MRI) or
Spectroscopy
(MRS), according to this invention include, but are not limited to, 19F and
13C. Suitable
radioisotopes for in vitr o identification and quantification of abnormal
cells including tumor cells,
in a tissue biopsy or post-mortem tissue include 1211, laC and 3H. Examples of
these radiolabels
include, but not limited to, 64Cu or'$F for use in PET in vivo imaging, 1231
or'3'I for use in SPECT
imaging in vivo,19F for MRS and MRI and 3H or 14C for in vitro methods.
However, any
conventional method for visualizing diagnostic probes can be utilized in
accordance with this
invention.
Generally, the dosage of the isotopically-labeled monoclonal antibody will
vary
depending on considerations such as age, condition, sex, and extent of disease
in the patient,
contraindications, if any, concomitant therapies and other variables, to be
adjusted by the skilled
artisan. Dosage can vary from 0.001 mg/kg to 1000 mg/kg, preferably 0.1 mg/kg
to 100 mg/kg.
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Administration to the patient may be local or systemic and accomplished
intravenous, intra-
arterial, intra-thecal (via the spinal fluid), intra-cranial or the like.
Administration may also be
intra-dermal or intra-cavitary, depending upon the body site under
examination.
After a sufficient time has elapsed for the labeled monoclonal antibody to
bind with the
abnormal cells, for example thirty minutes to forty-eight hours, the area of
the subject under
investigation is examined by routine imaging techniques such as MRS/MRI,
SPECT, planar
scintillation imaging, PET, and emerging imaging teclmiques, as well. The
exact protocol will
necessarily vary depending upon factors specific to the patient, as noted
above, and depending
upon the body site under examination, method of administration and type of
label used; the
determination of specific procedures would be routine to the skilled artisan.
For tumor imaging,
preferably, the amount (total or specific binding) of the bound isotopically-
labeled monoclonal
antibody is measured and compared (as a ratio) with the amount of isotopically-
labeled
monoclonal antibody bound to the tumor following chemotherapeutic treatment.
In another embodiment, the conjugated antibodies of the invention can be used
for
diagnosis and prognosis by using tissues and fluids distal to the primary
tumor site (as well as
methods using tissues and fluids of the primary tumor and/or tissue and fluids
surrounding the
tumor). Antibodies of the invention can be used to assay PMSA levels in a
biological sample
using classical immunohistological methods as known to those of skill in the
art (e.g., see
Jalkanen et al. (1985) J. Cell. Biol. 101, 976-985; and Jalkanen et al. (1987)
J. Cell. Biol. 105,
3087-3096). Other antibody-based methods useful for detecting protein gene
expression include
immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in the art
and include
enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine
(121I,121I), carbon ('4C),
sulfur (35S), tritium (3H), indium (121In), and technetium (99Tc).
In still further embodiments, the present invention provides diagnostic kits,
including
both immunodetection and imaging kits, for use with the immunodetection and
imaging methods
described above.
Methods of Treatment
The invention encompasses a method for treating cancer which comprises a
malignant
cell expressing PSMA in a patient in need thereof comprising administering a
conjugated
antibody which specifically binds to PSMA expressed by a malignant cell. In
one embodiment,
the conjugated antibody binds to a cytoplasmic epitope on the PSMA. In another
embodiment,
the conjugated antibody includes, but is not limited to, the 7E11-C5
monoclonal antibody as
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described in U.S. Patent 5,162,504 herein incorporated by reference in its
entirety. The
liybridoma cell line which produces the 7E 11 -C5 monoclonal antibody has been
deposited with
the American Type Culture Collection under Deposit No. HB 10494. In a
preferred embodiment,
the antibody is conjugated with a radioisotope via a MeO-DOTA linkage. In an
even further
embodiment, the MeO-DOTA to antibody ratio is about 9:1 or greater. In a
preferred
embodiment, the MeO-DOTA to antibody ratio is about 9:1, or greater, with
little, if any, loss of
immunoreactivity.
The amount of the conjugated antibody composition of the invention which will
be
effective in the treatment, prevention or management of cancer can be
determined by standard
research techniques. For example, the dosage of the composition which will be
effective in the
treatment, prevention or management of cancer can be determined by
administering the
composition to an animal model such as, e.g., the animal models disclosed
herein or known to
those skilled in the art. In addition, in vitro assays may optionally be
employed to help identify
optimal dosage ranges.
Selection of the preferred effective dose can be determined (e.g., via
clinical trials) by a
skilled artisan based upon the consideration of several factors which will be
known to one of
ordinary skill in the art. Such factors include the disease to be treated or
prevented, the symptoms
involved, the patient's body mass, the patient's immune status and other
factors known by the
skilled artisan to reflect the accuracy of administered pharmaceutical
compositions.
The precise dose to be employed in the formulation will also depend on the
route of
administration, and the seriousness of the cancer, and should be decided
according to the
judgment of the practitioner and each patient's circumstances. Effective doses
may be
extrapolated from dose-response curves derived from in vitro or animal model
test systems.
For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to
100 mg/kg
of the patient's body weight. Preferably, the dosage administered to a patient
is between 0.1
mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10
mg/kg of the
patient's body weight. Generally, human and humanized antibodies have a longer
half-life within
the human body than antibodies from other species due to the immune response
to the foreign
polypeptides. Thus, lower dosages of human or humanized antibodies and less
frequent
administration is often possible.
In specific embodiments, patients with prostate cancer are administered an
effective
amount of one or more conjugated antibodies of the invention. In another
embodiment, the
antibodies of the invention can be administered in combination with an
effective amount of one or
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more other agents useful for prostate cancer therapy including but not limited
to: external-beam
radiation therapy, interstitial implantation of radioisotopes (i.e.,'ZSI,
palladium, iridium),
leuprolide or other LHRH agonists, non-steroidal antiandrogens (flutamide,
nilutamide,
bicalutamide), steroidal antiandrogens (cyproterone acetate), the combination
of leuprolide and
flutamide, estrogens such as DES, chlorotrianisene, ethinyl estradiol,
conjugated estrogens U.S.P.,
DES-diphosphate, radioisotopes, such as strontium-89, the combination of
external-beam
radiation therapy and strontium-89, second-line hormonal therapies such as
aminoglutethimide,
hydrocortisone, flutamide withdrawal, progesterone, and ketoconazole, low-dose
prednisone, or
other chemotherapy regimens reported to produce subjective improvement in
symptoms and
reduction in PSA, PAP and/or PMSA levels including docetaxel, paclitaxel,
estramustine/docetaxel, estramustine/etoposide, estramustine/vinblastine, and
estramustine/paclitaxel.
Given the invention, certain preferred embodiments will encompass the
administration of
lower dosages in combination treatment regimens than dosages recommended for
the
administration of single agents.
The invention provides for any method of administrating lower doses of known
prophylactic or therapeutic agents than previously thought to be effective for
the prevention,
treatment, management or amelioration of cancer. Preferably, lower doses of
known anti-cancer
therapies are administered in combination with lower doses of conjugated
monoclonal antibodies
of the invention.
The invention also includes methods and compositions for the treatment of
cancer in a
mammal, including a human, comprising administering to said mammal an amount
of a cytotoxic
agent, or a pharmaceutical composition comprising an amount of the cytotoxic
agent, that is
effective in enhancing the binding of an antibody to an epitope of PSMA, for
instance a
cytoplasmic epitope. Said cytotoxic agent can be administered prior to or
simultaneously with
said antibody. In a preferred embodiment, said antibody binds to an epitope on
the cytoplasmic
domain of PSMA. In a further embodiment, said antibody is conjugated with MeO-
DOTA. In a
specific embodiment said antibody is 7E11-C5 and is conjugated to MeO-DOTA. In
another
embodiment, the cytotoxic agent will induce apoptosis of the malignant cell,
and/or increase
permeability, and/or otherwise disrupt the cell membrane. In a further aspect
of the invention, the
cytotoxic agent is administered to a mammal prior to or simultaneously with
the conjugated
antibodies of the invention. In this aspect of the invention, the cytotoxic
agent disrupts the cancer
cell(s), thereby expressing the cytoplasmic domain on the PSMA antigen.
Exposure of the
cytoplasmic domain allows for targeting of the remaining cancer cells with the
conjugated
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antibody of the invention. (See, co-pending U.S. application 60/643,589 filed
on January 14,
2005, which is herein incorporated by reference).
Examples of chemotherapeutic agents, include but are not limited to, BCNU,
cisplatin,
gemcitabine, hydroxyurea, paclitaxel, temozomide, topotecan, fluorouracil,
vincristine,
vinblastine, procarbazine, dacarbazine, altretamine, cisplatin, methotrexate,
mercaptopurine,
thioguanine, fludarabine phosphate, cladribine, pentostatin, fluorouracil,
cytarabine, azacitidine,
vinblastine, vincristine, etoposide, teniposide, irinotecan, docetaxel,
doxorubicin, daunorubicin,
dactinomycin, idarubicin, plicamycin, adriamycin, mitomycin, bleomycin,
tamoxifen, flutamide,
leuprolide, goserelin, aminoglutethimide, anastrozole, amsacrine,
asparaginase, mitoxantrone,
mitotane and amifostine.
In another embodiment, the therapeutic method comprises administration of the
conjugated antibody of the invention in combination with radiation for the
treatment of cancer. In
particular, the radiation is designed to disrupt the cell membrane of the
cancer cell to expose
PSMA, for instance the cytoplasmic domain of PSMA. Once the cytoplasmic domain
is exposed,
an antibody can bind to PSMA. In a preferred embodiment, said antibody binds
to an epitope on
the cytoplasmic domain of PSMA. In a further embodiment, said antibody is
conjugated with
MeO-DOTA. In a specific embodiment said antibody is 7E11-C5 and is conjugated
to MeO-
DOTA. The methods of the invention are also designed to induce apoptosis (cell
death) in cancer
cells, reduce the incidence or number of metastases, and reduce tumor size.
Tumor cell resistance
to radiotherapy agents represents a major problem in clinical oncology. Thus,
in the context of
the present invention, it also is contemplated that combination therapy with
such an antibody
could be used on radiation resistant tumors to improve the efficacy of the
radiation therapy. (See,
co-pending U.S. application 60/643,589 filed on January 14, 2005, which is
herein incorporated
by reference).
Types of cancer that can be treated the methods of the invention include solid
tumors.
Examples of solid tumors include, but are not limited to, endothelial cell
carcinoma. Examples of
endothelial cell carcinoma include, but are not limited to, renal cell
carcinoma, colon carcinoma,
transitional cell carcinoma, lung carcinoma, breast carcinoma and prostatic
adenocarcinoma.
Examples of renal cell carcinoma include, but are not limited to, clear cell
carcinoma,
papillary carcinoma, chromophobe carcinoma, collecting duct carcinoma and
unclassified
carcinoma. Examples of lung carcinoma include, but are not limited to,
adenocarcinoma, alveolar
cell carcinoma, squamous cell carcinoma, large cell and small cell carcinoma.
Examples of breast
carcinoma include, but are not limited to, adenocarcinoma, ductal carcinoma in
situ, lobular
carcinoma in situ, invasive ductal carcinoma, medullary carcinoma and mucinous
carcinoma.
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Another example of solid tumor treatable by the methods of the invention
includes
endothelial cell sarcoma. In one embodiment, the sarcoma is a soft tissue
sarcoma. Metatstatic
tumors are also treatable.
Method of Making the Antibodies of the Invention
U.S. patents 5,435,990 and 5,652,361 disclose methods of making and using MeO-
DOTA
and other bifunctional chelators. U.S. patents 5,435,990 and 5,652,361 are
herein incorporated by
reference in their entireties for all purposes.
The modification of antibodies for the addition of MeO-DOTA, in one
embodiment, may
be accomplished by formation of a covalent linkage with an amino acid residue
of the protein and
a functional group of the bifunctional chelator which is capable of binding
proteins. MeO-DOTA
could also be attached to the antibody via a linker molecule. Examples of
linker molecules useful
for conjugating MeO-DOTA to a polypeptide are disclosed in, for example,
DeNardo et al. (1998)
Clin Cancer Res. 4, 2483-2490; Peterson et al. (1999) Bioconjug. Chem. 10, 553-
557; and
Zimmerman et al. (1999) Nucl. Med. Biol. 26, 943-950, which are hereby
incorporated by
reference in their entirety.
U.S. Patents 5,652,361 and 5,756,065, disclose chelating agents that may be
conjugated
to antibodies. The method by which the disclosed chelating agents are
conjugated are also
disclosed in U.S. Patents 5,652,361 and 5,756,065. Any suitable process that
results in the
formation of the conjugates of this invention is within the scope of this
invention. However, one
important aspect for an efficient reaction is the antibody to conjugate ratio.
Once the conjugate
and the antibody are purified and ready for conjugating both the antibody and
are added to a
reaction mixture. The conjugate to antibody ratio should be approximately 5:1,
10:1, 30:1, 40:1,
50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 150:1, 200:1, 250:1, respectively. The pH
of the reaction
mixture should be about 10 to about 5, preferably about 9 to about 8. More
preferably the pH
should be about 8.5. The conjugation reaction should be carried out at a
temperature range from
about 20 C to about 37 C. Up to 5, 6, 7, 8, 9, 10, 11, 12 or more MeO-DOTA
chelates could be
bound to the antibody following the described protocol. In one embodiment, the
MeO-DOTA to
antibody ratio is about 9:1 or greater. In another embodiment, the MeO-DOTA to
antibody ratio
is about 9:1, or greater, with little loss, if any, of immunoreactivity.
Binding of a therapeutic radioactive isotope to the bifunctional chelator
(complexing)
may be accomplished either with the bifuntional chelator, MeO-DOTA, that is
not complex to an
antibody or, alternatively, a composition consisting of MeO-DOTA conjugated to
an antibody.
The complexing reaction should be accomplished at a pH of about 7 to about 4,
preferably about 5
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to about 6. More preferably the pH should be about 5.5. The complexing
reaction may be
accomplished in an acetate buffer (e.g. sodium acetate, potassium acetate) at
a molar
concentration of at least about 50 mM, 100 mM, 150 mM, 200 mM, or 250 mM or
more.
Preferably the molar concentration should be about 100 mM. The complexing
reaction should
carried out at a temperature range from about 20 C to about 37 C. The percent
of radionucleotide
complexed with DOTA should be between about 80% and about 100%, preferably,
between about
87% to about 95%.
The radioisotope that can be complexed with MeO-DOTA include, but are not
limited to,
3 H, 14C, 18 F,19F , 31P, 32P, 35S, 1311, 1251, 1231, 64Cu, 187Re
, lllj11> 90Y> 99Tc, 177Lu or any radioisotope
suitable for imaging.
Pharmaceutical Compositions
The compositions of the invention include bulk drug compositions useful in the
manufacture of pharmaceutical compositions (e.g., impure or non-sterile
compositions) and
pharmaceutical compositions (i.e., compositions that are suitable for
administration to a subject or
patient) which can be used in the preparation of unit dosage forms. Such
compositions comprise
a prophylactically or therapeutically effective amount of a prophylactic
and/or therapeutic agent
disclosed herein or a combination of those agents and a pharmaceutically
acceptable carrier.
Preferably, compositions of the invention comprise a prophylactically or
therapeutically effective
amount of one or more antibodies of the invention and a pharmaceutically
acceptable carrier or an
agent that reduces expression (e.g., antisense oligonucleotides) and a
pharmaceutically acceptable
carrier. In a further embodiment, the composition of the invention further
comprises an additional
therapeutic, e.g., anti-cancer, agent.
In a specific embodiment, the term "pharmaceutically acceptable" means
approved by a
regulatory agency of the Federal or a state government or listed in the U.S.
Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more particularly in
humans. The
term "carrier" refers to a diluent or vehicle with which the therapeutic is
administered. Such
pharmaceutical carriers can be sterile liquids, such as water and oils,
including those of petroleum,
animal, vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and
the like. Water is a preferred carrier when the pharmaceutical composition is
administered
intravenously. Saline solutions and aqueous dextrose and glycerol solutions
can also be employed
as liquid carriers, particularly for injectable solutions. Suitable
pharmaceutical excipients include
starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica
gel, sodium stearate,
glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,
propylene, glycol, water,
ethanol and the like. The composition, if desired, can also contain minor
amounts of wetting or
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emulsifying agents, or pH buffering agents. These compositions can take the
form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders, sustained-release
formulations and the
like.
Generally, the ingredients of compositions of the invention are supplied
either separately
or mixed together in unit dosage form, for example, as a dry lyophilized
powder or water free
concentrate in a hermetically sealed container such as an ampoule or sachette
indicating the
quantity of active agent. Where the composition is to be administered by
infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical grade
water or saline. Where
the composition is administered by injection, an ampoule of sterile water for
injection or saline
can be provided so that the ingredients may be mixed prior to administration.
The compositions of the invention can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with anions such as
those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those
formed with cations such as
those derived from sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
Various delivery systems are known and can be used to administer the antibody
of the
invention or the combination of a conjugated antibody of the invention and a
prophylactic agent
or therapeutic agent useful for preventing or treating cancer, e.g.,
encapsulation in liposomes,
microparticles, microcapsules, recombinant cells capable of expressing the
antibody or antibody
fragment, receptor-mediated endocytosis (see for example, Wu and Wu (1987) J.
Biol. Chem.
262, 4429-4432). Methods of administering a prophylactic or therapeutic agent
of the invention
include, but are not limited to, parenteral administration (e.g., intradermal,
intramuscular,
intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g.,
intranasal, inhaled,
and oral routes). In a specific embodiment, prophylactic or therapeutic agents
of the invention are
administered intramuscularly, intravenously, or subcutaneously. The
prophylactic or therapeutic
agents may be administered by any convenient route, for example by infusion or
bolus injection,
by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa,
rectal and intestinal
mucosa, etc.) and may be administered together with other biologically active
agents.
Administration can be systemic or local.
In a specific embodiment, it may be desirable to administer the prophylactic
or
therapeutic agents of the invention locally to the area in need of treatment;
this may be achieved
by, for example, and not by way of limitation, local infusion (such as limb
perfusion), by
injection, or by means of an implant, said implant being of a porous, non-
porous, or gelatinous
material, including membranes, such as sialastic membranes, or fibers.
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Kits
The invention provides a pharmaceutical pack or kit comprising one or more
containers
filled with an monoclonal antibody of the invention. In one embodiment the kit
comprises an
anti-PSMA antibody conjugated to a radioisotope via MeO-DOTA to a patient
before and after
therapy. In a further embodiment, the MeO-DOTA to antibody ratio is about 9:1
or greater. In an
even further embodiment, the MeO-DOTA to antibody ratio is about 9:1, or
greater, with little, if
any, loss of immunoreactivity. Additionally, one or more other prophylactic or
therapeutic agents
useful for the treatment of a cancer can also be included in the
pharmaceutical pack or kit. The
invention also provides a pharmaceutical pack or kit comprising one or more
containers filled
with one or more of the ingredients of the pharmaceutical compositions of the
invention.
Optionally associated with such container(s) can be a notice in the form
prescribed by a
governmental agency regulating the manufacture, use or sale of pharmaceuticals
or biological
products, which notice reflects approval by the agency of manufacture, use or
sale for human
administration.
The present invention provides kits that can be used in the above methods. In
one
embodiment, a kit comprises one or more a monoclonal antibodies of the
invention. In another
embodiment, a kit further comprises one or more other prophylactic or
therapeutic agents useful
for the treatment of cancer, in one or more containers. In one embodiment, the
antibody of the
invention is an antibody which immunospecifically binds to prostate specific
membrane antigen
(PSMA), wherein said antibody is conjugated to a radioisotope via a MeO-DOTA
linkage. In a
specific embodiment said antibody is 7E11-C5 conjugated to'7'Lu via MeO-DOTA.
In another
embodiment, the conjugated 7E 11 -C5 is complexed with 17Lu at a ratio of
isotope to antibody of
about 9:1 or greater. In certain embodiments, the other prophylactic or
therapeutic agent is a
chemotherapeutic. In other embodiments, the prophylactic or therapeutic agent
is a biological or
hormonal therapeutic.
Without further description, it is believed that one of ordinary skill in the
art can, using
the preceding description and the following illustrative exainples, make and
utilize the
compounds of the present invention and practice the claimed methods. The
following working
examples describe embodiments of the present invention, and are not to be
construed as limiting
in any way the remainder of the disclosure.
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EXAMPLES
Example 1. Isolation and Characterization of 7E11-C5
1.1 Cell Lines and Tissues
The LNCaP cell line was established from a metastatic lesion of human
prostatic
carcinoma. The LNCaP cells grow readily in vitro (up to 8 x105 cells/cm2 ;
doubling time, 60
hours), form clones in semisolid media, and show an aneuploid (modal number,
76 to 91) human
male karyotype with several marker chromosomes. The malignant properties of
LNCaP cells are
maintained. Athymic nude mice develop tumors at the injection site (volume-
doubling time, 86
hours). Functional differentiation is preserved; both cultures and tumor
produce a prostate acid
phosphatase (PAP) and prostate specific antigen (PSA). High-affinity specific
androgen receptors
are present in the cytosol and nuclear fractions of cells in culture and in
tumors. Estrogen
receptors are demonstrable in the cytosol. The model is hormonally responsive.
In vitro, a-
dihydro-testosterone modulates cell growth and stimulates acid phosphatase
production. In vivo,
the frequency of tumor development and the mean time of tumor appearance are
significantly
different for either sex. LNCaP cells, therefore, meet criteria of a versatile
model for
immunological studies of human prostatic cancer in the laboratory.
Seven malignant cell lines of human origin were obtained from the Memorial
Sloan-
Kettering Institute and included: DU-145 and PC-3 derived from prostatic
cancer; MCF-7,
derived from pleural effusion of scirrhous carcinoma of the breast (Soule et
al. (1973) J. Natl.
Cancer Inst. 51, 1409-1416); MeWo, malignant melanoma; RT-4, transitional cell
carcinoma;
HT-29, adenoma of the colon and A209, rhabdomyosarcoma. Four other cell lines
(two
malignant and two normal), isolated and established at Roswell Park Memorial
Institute were also
used: TT, thyroid medullary carcinoma, pancreatic cancer, BG-9 and MLD - both
normal diploid
neonatal foreskin fibroblast (see Horoszewicz et al. (1978) Infect. Immun. 19,
720-726; Chen et
al. (1982) Human Pancreatic Adenocarcinoma 18, 24-32; Leong et al. (1982)
Advances in
Thyroid Neoplasia 1984, 95-108). All of the above cell lines were routinely
maintained in RPMI
medium 1640 (Roswell Park Memorial Institute) supplemented with 10% heat
inactivated fetal
bovine serum, 1 mM L-glutamine, and 50 gg/ml of penicillin and streptomycin
(Gibco).
Fresh normal, benign and malignant prostate cancer tissues were obtained
either from the
Department of Surgery or the Department of Pathology at Roswell Park Memorial
Institute. The
tissues were quick frozen in M-1 embedding matrix (Lipshaw Corp) and stored at
-80 C.
1.2. Immunization and Cell Fusion
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Ten week old male Balb/c mice (West Seneca Laboratoryreceived intraperitoneal
injections (2 x 107 cells/0.2ml) of washed (three times in RPMI medium 1640)
live LNCaP cells
suspended in RPMI medium 1640, at monthly intervals for 3 months. Three days
before fusion,
the mice received an intraperitoneal challenge of 2 x 107 cells in RPMI medium
1640 and an
intravenous injection of the plasma membrane isolated from 1 X 10$ LNCaP
cells. Cell fusion
was carried out using a modification of the procedure developed by Kohler and
Milstein (1975)
Nature 256, 495-497. Mouse splenocytes (1 x 108 cells) were fused in HyBRL-
Prep 50%
polyethylene glycol 1450 (Bethesda Research Laboratories) with 5 x 10' mouse
myeloma cells
(P3 x 63Ag8.653). Fused cells were distributed to ten 96-well culture plates
(Falcon, Oxnard,
CA) and grown in hypoxanthine/aminopterin/thymidine (HAT) medium at 37 C with
7.5% CO2
in a hunlid atmosphere. Fourteen days later, supernatants were assayed for
binding activity to
plasma membrane isolate from LNCaP cells and MLD (normal human fibroblasts)
using the
Enzyme Linked Immunosorbent Assay (ELISA) with anti-mouse IgG 0-galactosidase
linked
F(ab')2 fragment from sheep (Amersham Corp.) or goat anti-mouse IgG
horseradish peroxidase
conjugate (Bio-Rad Laboratories) in a primary screen. Dried membrane isolate
(400 ng/well)
instead of whole LNCaP cells was used in the primary screening process because
of poor
attachment of the LNCaP cells to the plastic wells. To circumvent this
problem, immunofiltration
on a disposable microfold system (V&P Scientific) using whole LNCaP cells was
used as a
confirmatory assay as described in Section 1.5. In addition, the dot-
immunobinding assay on
nitrocellulose membrane (Section 1.4) was used to screen the supernatants of
hybridomas for
reactivity against LNCaP cell cytosol (100,000 x g supernatants) and crude
plasma membrane
preparation. To determine the specificity spectrum of the cultures showing
reactivity with the
plasma membranes and/or whole LNCaP cells, the culture fluids were further
tested by ELISA on
a panel of an additional nine viable, normal and neoplastic human cells lines
as described in
Section 1.5.
1.3. Isolation of Plasma Membrane-Enriched Fraction
Plasma membrane-enriched fractions were obtained from LNCaP cells and normal
human
diploid fibroblast strain MLD by modification of published methods (Kartner et
al. (1977) J.
Membrane Biol. 36, 191-211). Briefly, MLD cells in roller bottles or LNCaP
cells in plastic
culture flasks were gently rinsed 4 times with phosphate buffered saline
(PBS). The cells were
then rinsed once with hypotonic lysing buffer (3 mM Hepes
[hydroxyethylpiperazine-
ethanesulfonic acid] (pH 7.0), 0.3 mM MgCIZ, 0.5 mM CaC12) and the buffer
discarded. Fresh
lysing buffer (5-25 ml) was added to each bottle or flask and the cells
allowed to swell for 30
minutes at room temperature. The swollen cells were removed from the surface
and disrupted by
manual shaking. The progress of disruption was monitored by phase microscopy
of a sample
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droplet. Gentle trituration (8-10 times) with a 10 ml pipette was used to
complete disruption of the
LNCaP cells. Vigorous shaking and pipetting were necessary to completely break-
up the MLD
cells. Phenyl-methylsulfonyl fluoride (PMSF, 0.5 mM) (Calbiochem) was added to
minimize
proteolysis. The disrupted cell suspensions were centrifuged at 100 x g to
remove nuclei and
incompletely disrupted cell clumps. The nuclei pellet was washed once with the
lysing buffer and
after centrifugation the supernatant was combined with the first supernatant
and centrifuged at
3,000 x g for 10 minutes, at 4 C. The pellet consisting of mitochondria and
debris was discarded
and the supernatant designated as membrane lysate was layered over a
discontinuous density
gradient composed of 15 ml each of 10, 30 and 38% sucrose (w/v) and
centrifuged at 60,000 x g
for 2-1/2 hours in an SW 25.2 rotor (Beckinan). Material banding at the
interface between 10%
and 30% sucrose layers was removed by aspiration, washed free of sucrose using
lysing buffer
and pelleted by centrifugation at 36,000 x g for 60 minutes. Pellets were
resuspended in PBS and
aliquots taken for assay of protein and the enzyme phosphodiesterase-I
(EC3.1.3.35) as a marker
for plasma membranes. The 10/30 plasma membrane isolate was used in the
screening assays for
the hybridoma supernatants. All fractions were dispensed and stored as single-
use aliquots at -
90 C.
1.4. Dot-Immunobinding Assay
The dot-immunobinding assay was used to screen large numbers of supernatants
of
hybridomas producing monoclonal antibodies (Hawkes et al. (1982) Anal.
Biochem. 119, 142-
147). The crude plasma membrane isolate, the 10/30 plasma membrane isolate
and/or the cytosol
fractions containing the cellular antigen were dotted (1-3 l) on a washed
nitrocellulose filter
paper grid (Bio-Rad). The protein concentration of the "antigen" ranged from
between 0.1 to 0.1
mg/ml. After thorough drying, the filter was washed in Tris Buffered Saline
(TBS, 50 mM Tris-
HCI, 200 mM NaC1, pH 7.4). Treatment of the filter paper with 3% (w/v) bovine
serum albumin
(Sigma, St. Louis, MO) in TBS for 15 minutes at room temperature resulted in
the blockage of
nonspecific antibody binding sites on the filter and on the walls of the
plastic vessel used to carry
out the reaction. The filter paper was then incubated with hybridoma
supernatant or purified
monoclonal antibody (2-20 l) for 60 minutes in several changes of TBS, the
blocking step was
repeated. A second antibody (F(ab')2 goat anti-mouse IgG) conjugated to
horseradish peroxidase
(Bio-Rad) (diluted 1:1000 in blocking solution) was added and incubation was
carried out at 37 C
for 120 minutes. After washing with TBS the peroxidase activity was developed
with 4-chloro-l-
napthol (0.6 mg/ml in TBS, Merck Inc.) and hydrogen peroxide (0.01% v/v). A
positive reaction
appeared as a blue colored dot against the white filter background.
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Immunoblotting of cytosol and membrane fractions indicated that the soluble
cytosol
fraction of LNCaP cells was not reactive, while sedimentable (approximately
105,000 x g)
membrane associated fractions gave strongly positive spots with monoclonal
antibody 7E11.
1.5. Enzyme Linked Immunosorbent Assay (ELISA)
The enzyme linked immunosorbent assay (ELISA) was used for general enzyme
immunoassay of antigen and screening for monoclonal antibody production.
Target cells were
seeded (2-30 x 104 cells/ml) on microtiter plates (Falcon) 4-7 days before
assay. Nonspecific
binding sites on the plates were blocked with 1% (w/v) swine gelatin in a
special media (FL),
formulated to keep the cells viable. The FL media consisted of Dulbecco's
Modified Eagle
Medium (Gibco) supplemented with 15 mM Hepes, 0.3% NaCI, 10 mM NaN3 and swine
gelatin
(1% for blocking or 0.3% for washing) pH 7.2-7.4. Hybridoma culture fluids (50
1 per well)
were added and incubation was carried out at 37 C. for 60 minutes. The plates
were washed 4
times with FL media and F(ab')2 goat anti-mouse IgG conjugated to horseradish
peroxidase
(1:1250 in 0.3% swine gelatin, 0.O1M PBS) was used in place of the (3-
galactoxidase conjugate.
After washing, 100 l of substrate (25 ml of 0.1 M citrate buffer pH 5.0, 10
l of 30% H202 and
10 mg of a-phenylenediamine (Sigma) was added to each well. The plate was
incubated for 30
minutes in the dark, and the reaction stopped with 50 l of 2 N HZS04/well.
The absorbance was
determined at 490 nm using the Bio-Tek EIA reader.
Dried crude plasma membrane isolates from LNCaP cells or dried cells were used
initially in the primary screening procedure of hybridoma culture fluids.
Approximately 400 ng
of membrane protein in 50 1 of buffer (S3 mM Hepes, 0.3 mM MgC12, 0.5 mM
CaC12) was dried
(35 C overnight) in 96 well flat bottom microtiter plates (Falcon).
Nonspecific binding sites on
the plates were blocked with 1% swine gelatin in PBS containing 0.1% NaN3.
With the exception
of the wash buffer consisting of 0.O1M Hepes and 0.2 M of PMSF in saline (pH
7.6), all other
reagents used were as described above for cell surface enzyme immunoassay.
LNCaP cells attach poorly to plastic wells and detach from the plastic surface
during the
ELISA procedure. To circumvent this problem immunofiltration on a disposable
microfold
system (V&P Scientific) using whole LNCaP cells was employed as a confirmatory
assay for the
dried membrane assay. After nonspecific binding sites on the disposable
microfold system was
blocked with 5% human serum albumin (HSA) in PBS, 2.5 x 104 LNCaP cells in 100
l of 5%
HSA buffer was deposited on the filter discs with vacuum. After washing the
filters with 0.3%
gelatin in 0.01M phosphate buffer, the plates were processed as described
above. However, after
incubation with substrate, the reaction mixture from each well (100 l) was
transferred to %z area
Costar plates (Costar) before spectrophotometric determination on the EIA
reader. Hybridomas
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were detected in approximately 500 culture wells. 206 hybridomas were
successfully expanded
and on primary ELISA screen, 126 reacted with partially purified LNCaP
membranes, 92 reacted
with intact LNCaP cells 76 reacted with normal human fibroblast-cells and
membrane
preparations. Further screening by ELISA and by immunoperoxidase staining on a
panel of
additional 11 viable, normal and neoplastic cell lines and by immunoblotting
of cytosol and
membranes fractions from LNCaP cells narrowed the field of two cloned
hybridoma cell lines of
particular interest, including MAb 7E11 and 9H10.
Hybridoma cultures showing specificity restricted to the LNCaP cells and
membranes
were cloned by limiting dilution and subcloned in agarose (see e.g., Schreier
et al.(1980)
Hybridoma Techniques pp. 11-15, Cold Spring Harbor Laboratory Press). Stable
cultures of
antibody-producing hybridomas were expanded in complete media (RPMI 1640 media
supplemented with 10% (w/v) heat-inactivated fetal bovine serum, 100 U/ml
penicillin, 100 g/ml
streptomycin, and 10 g/ml insulin) and cryopreserved. After cloning, two
stable monoclonal
hybridoma cell lines were obtained and designated as 7E11-C5 and 9H10-A4
respectively.
Exhausted culture fluids and mouse ascites fluids were the source of
antibodies used for
further studies. Ascites fluid from mice carrying the hybridoma cell line was
used to obtain large
quantities of monoclonal antibodies. Hybridoma cells for ascites fluid
production were washed 2
times with RPMI 1640 medium and resuspended at a density of 1-5 x 107
cells/ml. Using a 20-
gauge needle, 0.2 ml of the cell suspension was injected into the peritoneal
cavity of female nude
mice. Pristane was not routinely used to precondition the animals. Ascites
fluid containing high
titers of antibodies was regularly harvested four to five weeks after
injection with the hybridoma
cells.
1.6. Isotyping of Monoclonal Antibodies
Monoclonals 7E11-C5 and 9H10-A4 are of the IgGI subclass, as determined by
double
diffusion gel precipitation with isotype specific antisera (Miles). Consistent
with this finding
were observations that Protein A conjugated with either fluoroscein or
horseradish peroxidase
(BIO-RAD) failed to react with smears of LNCaP cells following incubation with
either
monoclonal.
Example 2. Conjugation of 7EC11-C5
Conjugates were prepared at pH 8 and 9 in 0.1 M HEPES buffer, at chelant to
protein
ratios of 50 and 200, at 20 and 37 C for four hours. Antibody 7E1 1-C5
concentration was 0.022
mM. Conjugates were purified by gel filtration chromatography. Table 1
summarizes the lot
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numbers and reaction conditions. As Table 2 indicates average loading values
from 0.2 to 5.3
were observed. Increasing molar ratios, temperature and pH facilitated the
conjugation reaction.
Table 1. Lot numbers of 7E11-C5 conjugates and conditions of preparations.
Lot number Conditions
200402495-5/1 pH=B, 20 C, r=50, HEPES
200402495-5/2 pH=8, 37 C, r=50, HEPES
200402495-5/3 pH=8, 20 C, r=200, HEPES
200402495-5/4 pH=8, 37 C, r=200, HEPES
200402495-5/5 pH=9, 20 C, r=50, HEPES
200402495-5/6 pH=9, 37 C, r=50, HEPES
200402495-5/7 pH=9, 20 C, r=200, HEPES
200402495-5/8 pH=9, 37 C, r=200, HEPES
Table 2. Masses & average loading value of MeO-DOTA conjugated to antibodies.
Lot #' Condition MassCh Conj. Pure Mass shift: MALDI
arge mAb mAb conj-pure ALVb
massa massa massa
200402495_5/1 pH=8, 20 C, r=50 M'' 150254 150143 111 0.2
2004024955/2 pH=8, 37 C, r=50 M+' 150658 150143 515 0.9
200402495_5/3 pH=8, 20 C, r=200 M+' 150652 150143 509 0.9
200402495_5/4 pH=8, 37 C, r=200 M+' 151616 150143 1473 2.6
200402495_5/5 pH=9, 20 C, r=50 M+' 150587 150143 444 0.8
200402495_5/6 pH=9, 37 C, r=50 MF' 151731 150143 1588 2.8
2004024955/7 pH=9, 20 C, r=200 M+' 151721 150143 1578 2.8
200402495_5/8 pH=9, 37 C, r=200 M+' 153146 150143 3003 5.3
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aMALDI-MS values were computed from the average value of a z*MZ+, z = 1, ...
4, where z is the
charge number and MZ+ is the mass-to-charge ratio of the (multiple charged)
molecular ion,
observed in the analysis of the pure or conjugated monoclonal antibodies.
bALV is the average loading value.
Lysine conjugate. dSingly charged mass.
eDoubly charged mass.
To determine the apparent molecular weight, the protein samples were analyzed
by high
resolution SDS-PAGE (Figure 1). Two distinct protein bands were observed for
each monoclonal
antibody and respective conjugate at approximately 25 and 50 kDa.
To determine protein integrity after conjugation, size exclusion
chromatography was
conducted. Unmodified protein was also analyzed to compare if some aggregation
occur after
conjugation. Relative peak areas of unmodified protein and conjugates are
summarized in Table
3. The chromatogram of the unmodified protein and a representative
chromatogram of a
conjugate are shown in Figure 2. Unmodified 7E11-C5 antibody shows two peaks,
one eluting at
15.53 minutes (area = 1.79%) and the main peak at 18.13 minutes (area =
98.21%). The area of
the minor peak of the conjugates varies from 2.2 to 4.4 %.
Table 3. Relative peak areas 7E11-C5 antibody and MeO-DOTA-conjugates,
determined
from size exclusion chromatograms.
Sample Peak area 1(%) Peak area 2(%)
7E11-C5 1.79 98.21
200402495-5/1 3.52 96.48
200402495-5/2 2.70 97.30
200402495-5/3 4.38 95.62
200402495-5/4 2.02 97.00
200402495-5/5 3.64 96.36
200402495-5/6 2.85 97.15
200402495-5/7 3.88 96.12
200402495-5/8 2.19 97.81
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Example 3. Complexation of Conjugated Antibody
MeO-DOTA-7E1 1-C5 was complexed with 17Lu at 2.25 mg/mL protein concentration
at
pH 5.2 at 20 C in sodium acetate buffer. Buffer concentrations of 50, 100 and
250 mM were
studied. Complexation was monitored using size exclusion chromatography with
radiometric and
iJV detection. Samples were analyzed after 30 min reaction time. Results are
summarized in
Table 4.
Table 4. Effect of NaOAc buffer concentration on complexation
[Buffer] (mM) % Lu complexed
50 97.24
100 98.50
250 90.32
These data indicated that the 100 mM buffer was optimal for complexation.
Therefore
the 100 mM buffer was chosen for the further complexations.
Example 4. Effect of pH on complexation
Complexation was carried out at pH 5.5 and 6 using a 100 mM NaOAc buffer.
Representative chromatograms at pH 5.5 are shown in Figure 3. Samples were
analyzed at 30
minutes and 2 hours. Aggregation was between 3-4 %, determined by UV. Summary
of
complexation at pH 5.5 and 6 is in Table 5.
Table 5. Summary of complexation using 100 mM sodium acetate buffer
Conjugate Lot pH Temp ( C) Time (h) % complexed % Aggregate (UV)
200402495-14 5.2 20 0.5 98.5 3.6
200402495-14 6 20 0.5 76.7 3.1
2.0 77.2 3.0
6 37 0.5 80.1 3.0
2.0 82.5 3.0
2004-02495-29 5.5 20 0.5 97.8 3.4
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Conjugate Lot pH Temp ( C) Time (h) % complexed % Aggregate (UV)
2.0 100 3.4
37 0.5 98.6 3.3
2.0 100 3.3
These data indicate that optimal pH for the complexing reaction is about 5.5.
Example 5. Preparation of high specific activity conjugate
On the basis of complexations carried out using trace amount of 17Lu (enough
"'Lu to
have a good radiometric signal on the HPLC), the following conditions were
selected to prepare
high specific activity material: 100 mM sodium acetate buffer, 37 C, pH 5.5,
protein
concentration 2.5 mg/mL. 0.5 mg protein and 656 mCi of "'Lu was used. The
reaction was
monitor by size exclusion chromatography. Target specific activity was 1
mCi/mg. Table 6
summarizes the results. Calculating with 90 % radioactivity incorporation, the
specific activity of
the complex-conjugate is 1.18 mCi/mg. Maximum incorporation is reached within
30 min.
Aggregation is 3%. Figure 4 shows the chromatogram of the 30 min. time point.
Table 6. Time study of the complexation of Lu-177, high specific activity
preparation
Time (h) % complex % Aggregate (UV)
0.5 89.9 3.0
1 91.8 3.0
2.5 89.8 3.5
Upon review of these data presented above the optimal conditions for
conjugating an
antibody of the invention, preferably 7E11-C5, is incubating said antibody in
0.1M HEPES buffer
at a pH of 8.5 with molar ratio of linker to antibody of approximately 200:1
at 37 C for
approximately 4 hours with slight agitation. The resulting antibody will be
conjugated with molar
ratio of MeO-DOTA to antibody of approximately 9:1 or greater. Optimal
complexing reactions
occur in 100mM Na-Actetate, pH 5.5 at 20 C for 0.5 hours. The resulting
antibody is 1.2mCi/mg
of 7E11-C5 at 90% complexation. A person skilled in the art will recognize
that modifications
and variations of these conditions will slightly vary the actual conjugated
and complexed antibody
composition.
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Example 6: Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500)
Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) is comprised of 7E11C5-3
monoclonal antibody (CYT-351) that is currently used in the manufacture of its
commercial
product ProstaScint . ProstaScint is comprised of CYT-351 conjugated via
periodate oxidation
of the carbohydrate groups located on the heavy chains to the linker-chelator
GYK-DTPA HCl
[glycyl-tyrosyl-(N-E-diethylenetriaminepentaacetic acid)-lysine hydrochloride]
which is
complexed with the gamma emiting radioistope 111In. Anti-PSMA-meO-DOTA
Immunoconjugate is comprised of CYT-351 covalently conjugated to the linker-
chelator meO-
DOTA [a-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraazacyclododecane-
1,4,7,10-
tetraacetic acid].
The CYT-351-meO-DOTA immunoconjugate has been shown to be stable in human
serum thereby decreasing the chance for secondary toxicities as a result of
shed linker and/or
radioisotopes.
Example 7: 7E11C5-3 Monoclonal Antibody (CYT-351)
CYT-351 is a murine IgGl monoclonal antibody secreted by a murine/murine
hybridoma
cell line, which was produced by immunizing BALB/c mice with live LNCaP human
prostatic
adenocarcinoma cells and partially purified LNCaP plasma membranes. The LNCaP
cell line
used to immunize the mice is a well characterized continuous cell line which
was established from
a needle biopsy taken from a lymph node metastasis of human prostatic
adenocarcinoma. LNCaP
cells grow readily in vitro, form clones in semisolid media, show an aneuploid
(modal number 76-
91) human male karyotype with several marker chromosomes and maintain the
malignant
properties of an adenocarcinoma.
The CYT-3 51 hybridoma was established and originally described by
Horoszewicez et al.
(1987) Anticancer Res. 7, 927-936 and U.S. Patents 5,162,504 and 5,578,484).
Spleen cells from
mice immunized with live LNCaP cells were fused with P3X63Ag8.653 murine
myeloma cells.
The cells were cloned twice by limited dilution cloning and a stable
hybridoma, designated
hybridoma 7E11-C5, was expanded and cryopreserved. This clone secreted a
prostate-specific
monoclonal antibody of the IgGl subclass which was originally designated
monoclonal antibody
7E11-C5.
A culture of the CYT-3 51 seed stock was used to establish a 100 vial Master
Cell Bank
(MCB). A single vial of cells was thawed and the cells recovered into a 25 em2
flask containing
basal cell culture medium supplemented with 2.5% FBS (fetal bovine serum). The
cells were
subsequently expanded into 75 cm2 flasks, 150 cmZ flasks, a 500 ml spinner
flask, and finally on
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into a three liter spinner. The cells were harvested and placed into freezing
medium (basal
medium supplemented with 20% FBS and 10% DMSO). The cells were then aliquoted
into 100
vials, each containing approximately 9 x 106 cells and labeled with the
designation 2MM0180-
M001-9M, and subsequently stored in the vapor phase of liquid nitrogen. Ten
vials from the
serum-grown MCB were used for tests to determine if the preparation was
sterile and free of
infectious adventitious agents. The results of these tests demonstrated that
the CYT-351 MCB
was sterile and free of infectious adventitious agents
Example 8: Methoxy-DOTA linker
Methoxy-DOTA (a-(5 -isothiocyanato-2-methoxyphenyl)- 1,4,7,10-
tetraazacyclododecane- 1,4,7, 1 0-tetraacetic acid) is prepared from a purely
synthetic process.
meDOTA and its methods of use and manufacture is disclosed in U.S. Patents
5,435,990 and
5,652,361 both of which are herein incorporated by reference in their
entirety.
Example 9: CYT-351 Manufacturing Process
The cell banks, components, raw materials and manufacturing process used to
produce
CYT-351 intermediate antibody for use in producing Anti-PSMA-meO-DOTA
Immunoconjugate
(CYT-500) are done so in accordance with the GMP manufacturing process.
The growth/production medium for the CYT-351 hybridoma is a defined, serum-
free
media available from HyClone Laboratory (HyQ-CCMTM) and is comprised of 925
basal medium.
Cell culture is performed in an AcuSyst-Xcell hollow fiber bioreactor and pH,
temperature and
oxygen levels monitored throughout the run. Samples are removed to monitor
glucose, lactate
and CYT-351 levels. Media feed is achieved via peristaltic pump. Medium is
perfused through
the bioreactor and the conditioned medium containing CYT-351 is harvested,
clarified by
filtration and stored at 2 to 8 C. The production run typically lasts for 60
to 70 days.
Each CYT-351 harvest is sampled and tested for CYT-351 titer,
immunoreactivity,
endotoxin and bioburden. Prior to purification, pooled harvest samples are
tested minimally for:
CYT-351 concentration, Mycoplasma, sterility and virus by reverse
transcriptase, XC Plaque,
S+L- Focus and in vitf o viral testing.
Harvest and purification of CYT-351 are performed in classified rooms with
appropriate
environmental monitoring to allow for aseptic processing. The CYT-351 harvest
is filtered
through a 0.45 m filter, concentrated to approximately 6 to 12 mg/ml CYT-351
using a Pellicon
tangential-flow ultrafiltration device fitted with a 30 kDa cutoff membrane.
Following
concentration, the concentrated crude CYT-351 product is passed over a
Sephadex G-25 column
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to remove low molecular weight moieties. The G-25 column is equilibrated and
eluted with 0.7
M ammonium sulfate (pH 8.0 to 8.4).
The eluted protein (CYT-351) peak is loaded onto a Protein A affinity column
equilibrated with 0.7 M ammonium sulfate. The loaded Protein A column is
washed with thirty
(30) column volumes of 0.7 M ammonium sulfate followed by a short wash with 55
mM sodium
acetate (pH 7.0 to 8.5). Bound CYT-3 51 is eluted from the Protein A column
witli 55 mM
sodium acetate (pH 4.0 to 4.5) and the pH of the eluted product adjusted to
5.1 to 5.3 with 55 mM
sodium acetate (pH 7.0 to 8.5).
The Protein A purified material is passed over a DEAE Sepharose column
equilibrated in
55 mM sodium acetate (pH 5.1 to 5.3). This is a passive purification step in
that the CYT-351
passes over the column whereas DNA, albumin and other acidic components bind
to the support.
The CYT-351 peak is then loaded onto a S-Sepharose column equilibrated with 55
mM
sodium acetate (pH 5.1 to 5.3). The column is washed with 10 mM sodium
phosphate buffer (pH
5.9 to 6.1). The bound CYT-351 is eluted with 10 mM phosphate buffered saline
(pH 5.9 to 6.1).
Purified CYT-351 is filtered through a sterile 0.22 m, sampled for Quality
Control testing and
stored at 2 to 8 C until needed for conjugation. Sterile filtered bulk CYT-351
has an approved
shelf life of three years at 2 to 8 C.
Example 10: Manufacturing Process for the Immunoconjugate CYT-500
Prior to conjugation, the purified CYT-351 is passed through a DV-20 (PALL)
virus
removal filter. The commercial manufacturing process for CYT-351, described
above, results in
8.91og viral removal. An additional 5 to 61og viral removal is obtained using
the DV-20 filter,
resulting in approximately 141og removal.
Purified monoclonal antibody CYT-351 is combined with 0.22 m filtered
(cellulose
acetate) meO-DOTA in 0.5 M HEPES (pH 8.85). The linker to CYT-351 ratio is
70:1 with a total
of 6 grams CYT-351 used for the toxicology lot (clinical lots also are 6 gram
CYT-351 scale).
The reaction mixture is incubated for three hours at 35 to 37 C with gentle
stirring. Following
three hours, the reaction mixture is adjusted to 7.0 with 1 M acetic acid to
slow the reaction. The
resultant product was concentrated from approximately 1900 to 300 ml using a
Millipore
Labscale TFF system with one Pelicon XL Biomax 50 filter. The concentrate was
stored
overnight at 2-8 C. The concentrate was chromatographed with 0. 1M sodium
acetate (pH 5.5) on
a 9 x 90 cm Superose 12 column. The main (product) fraction was collected and
concentrated to
approximately 21 mg/ml using a Millipore Labscale TFF system with one Pelicon
XL Biomax 50
filter. This material (CYT-500) was filtered through a 0.22 m filter and
stored at 2 to 8 C.
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The bulk CYT-500 is stored at 2 to 8 C and tested for contaminants before
being released
for use. Released CYT-351 is filtered through a sterile 0.22 m filter and
filled into 10 ml Type 1
borosilicate glass vials and stoppered with presterilized 20 mm stoppers. The
filled, unlabeled
vials are sealed with 20 mm flip-off crimp, visually inspected and sampled for
Quality Control
testing. Vials are placed in trays marked "quarantine" pending release.
Example 11: 7E11-meO-DOTA Serum Stability
An important requirement of antibody-chelating agent immunoconjugates is that
they
form kinetically inert complexes with metals of interest, in this case, 177
Lu. These complexes
must be stable following conjugating to a protein and should stay intact in
vivo to avoid secondary
toxicities. Similarly, loss of lanthanide metals can result in toxic effects,
such as radioactive
doses to the liver and bone. Accordingly, we tested serum stability of 177Lu
labeled CYT-500 and
compared it to11'In-labeled ProstaScint.
Size exclusion chromatography was used to analyze the radioactivity (I77 Lu)
loss from the
complex-conjugate in serum. Uncomplexed 177Lu associates with serum proteins
and tends to
elute with the high molecular weight species, similarly to 177Lu-meO-DOTA-
immunoconjugate
(177 Lu-CYT-500). The fact that serum proteins bind Lu weakly in a non-
specific manner allows
us to differentiate between the serum protein "'Lu complex and 177Lu-CYT-500.
The weak
association between 177Lu and serum proteins can be broken up by DTPA, while
DTPA can not
transchelate the metal from DOTA type chelates.
To determine if one percent metal loss from the complex conjugate can be
measured,
mixtures of 177 Lu-CYT-500and 177Lu-MeO-DOTA were prepared. The radioactivity
in both
17Lu-CYT-500 and'7'Lu-MeO-DOTA was determined by radioactive counting before
mixing
them. Two samples were prepared. In the first sample 7% of the total
radioactivity came from
177 Lu-MeO-DOTA and in the second one 1%. The size exclusion chromatograpliy
analysis
showed 9.8 and 3.0% of the radioactivity eluting as the low molecular weight
component. The
chromatographic method and counting gave the same results within the
experimental error.
177Lu-CYT-500 antibody conjugate was incubated in human serum and before HPLC
analysis DTPA was added to the sample to complex nonspecifically bound Lu. The
results are
tabulated in Table 7. During the two week course of the study insignificant
metal loss was
observed for 177 Lu-CYT-500 (98% at day 0 and 96% at day 15) and minimal metal
loss was
observed for "'In-DTPA-Cyt-351 (98% at day 1 and 91 % at day 15).
Radioactivity associated
with the high molecular weight components of the mixture, determined by size
exclusion
chromatography after addition of DTPA.
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Table 7. Stability in Human Serum
% Radioactivity associated with high mw
Time (day) 17Lu 177Lu-CYT-500 SD 1 lIn-ProstaScint SD
0 0 97.8 0.8 98.4 0.1
1 0 98.8 0.1 97.1 0.1
2 0 98.5 0.4 97.2 0.1
3 0 96.5 0.2 97.1 0.1
4 0 97.7 0.2 96.2 0.6
6 0 97.1 0.7 96.1 0.7
7 0 97.9 0.5 95.1 1.1
8 0 98.2 0.1 94.7 0.6
9 0 97.0 0.5 94.6 1.1
0 98.1 94.2 1.1
0 95.6 0.4 91.0 0.4
Example 12: 7E11-meO-DOTA Acute Toxicity Study in Rats
The purpose of this study was to determine the potential toxicity (including
neurotoxicity)
5 of Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) when administered once by
intravenous injection to male Sprague Dawley rats. Eighty male rats were
randomly assigned to
one of four groups and administered 100 mM sodium acetate buffer (control
article) or Anti-
PSMA-meO-DOTA Immunoconjugate (CYT-500) at 3, 15 or 30 mg/kg once on Study Day
(SD).
Forty rats (10/group) were subjected to a full gross necropsy on SD 4; the
remaining rats were
10 necropsied on SD 15. An additional 27 rats were assigned to one of the
three treated groups
(9/group) and blood was collected at selected timepoints for future
toxicokinetic profiling.
Parameters evaluated included mortality, clinical observations, body weight,
food
consumption, neurotoxicity, oplithalmology, clinical pathology, gross
pathology, absolute and
relative organ weights and histopathology. Treatment with Anti-PSMA-meO-DOTA
15 Immunoconjugate (CYT-500) had no effect on mortality, clinical
observations, body weight, food
consumption, neurotoxicity, ophthalmology, clinical pathology, gross
pathology, absolute and
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relative organ weights and histopathology. Therefore, under the conditions of
this study the
observed no-effect level (NOEL) is at least 30 mg/kg (100x the anticipated
human dose).
Example 13: 7E11-meO-DOTA Acute Toxicity Study in Dogs
The purpose of this study was to determine the potential toxicity of Anti-PSMA-
meO-
DOTA Immunoconjugate (CYT-500) when administered once by intravenous injection
to male
beagle dogs. Twenty four male dogs were randomly assigned to one of four
groups and
administered 100 mM sodium acetate buffer (control article) or Anti-PSMA-meO-
DOTA
Immunoconjugate (CYT-500) at 0.6, 3 or 6 mg/kg once on SD 1. Twelve dogs
(three per group)
were subjected to a full gross necropsy on SD 4; the remaining 12 dogs were
necropsied on SD
15. Parameters evaluated included mortality, clinical observations, body
weights, food
consumption, ophthalmology, cardiology, clinical pathology, gross pathology,
absolute and
relative organ weights, and histopathology.
Treatment with Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) had no effect on
mortality, clinical observations, body weights, food consumption,
ophthalmology, cardiology,
clinical pathology, gross pathology or absolute and relative organ weights.
Test article related
findings consisted of vasculitis of the central veins of the liver in treated
animals. Lesions were
more pronounced in SD 4 animals and, although present, appeared to be
resolving in SD 15
animals. The most severe lesions in SD 4 animals were seen in animals treated
at 3 or 6 mg/kg
(10 and 20x the anticipated human dose, respectively). By SD 15, the lesions
were milder
overall, suggesting that with additional time resolution may be possible. In
conclusion,
intravenous injections of Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) were
generally
well tolerated.
Example 14: 7E11-meO-DOTA Cardiovascular Safety Pharmacology Study
The purpose of this study was to evaluate cardiovascular safety following
intravenous
administration of Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) in male Beagle
dogs.
Seven male dogs were given an intravenous injection of 100 mM sodium acetate
buffer on SD 1,
and Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) at 0.6 mg/kg on SD 8, 3 mg/kg
on
SD 15, and 6 mg/kg on SD 22 and 29. Each dose administration was followed by
at least a one-
week wash-out period. Cardiovascular profiling and body temperature data were
collected via
telemetry following doses on SD 1, 8, 15 and 22. Other parameters evaluated
included mortality,
clinical observations, and body weights.
Treatment with Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) at doses up to 6
mg/kg had no effects on blood pressure, heart rate, electrocardiographic
parameters, body
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WO 2006/110745 PCT/US2006/013473
temperature, body weights or mortality. One animal experienced anaphylaxis
shortly after
administration of a 6 mg/kg dose on SD 22. This animal was removed from the
study and
returned to the stock colony. Symptoms of anaphylaxis were not observed in any
other animals
following both a single and repeat dose at 6 mg/kg. In conclusion, intravenous
injection of Anti-
PSMA-meO-DOTA Immunoconjugate (CYT-500) at doses up to 6mg/kg were generally
well
tolerated.
Example 15: 7E11-meO-DOTA Respiratory Function Study
The purpose of this study was to evaluate respiratory function following
intravenous
administration of Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) in male Beagle
dogs.
Six male dogs were given an intravenous injection of 100 mM sodium acetate
buffer on SD 1, and
Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) at 6 mg/kg on SD 4. Parameters
evaluated included mortality, clinical observations, body weights and
respiratory function
assessment. Respiratory function assessment included respiratory rate,
saturated blood oxygen
levels (SpO2) and end-tidal pressures (ETCOz).
Treatment with Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) had no effect on
mortality, clinical observations, body weight, or respiratory function.
Therefore under the
conditions of this study the no-observed effect-level (NOEL) is at least 6
mg/kg.
Although the present invention has been described in detail, it is understood
that various
modifications can be made without departing from the spirit of the invention.
Accordingly, the
invention is limited only by the following claims. All cited patents, patent
applications and
publications referred to in this application are herein incorporated by
reference in their entirety.
All references cited herein are incorporated herein by reference in their
entirety and for all
purposes to the same extent as if each individual publication or patent or
patent application was
specifically and individually indicated to be incorporated by reference in its
entirety for all
purposes.
Many modifications and variations of the present invention can be made without
departing from its spirit and scope, as will be apparent to those skilled in
the art. The specific
embodiments described herein are offered by way of example only, and the
invention is to be
limited only by the terms of the appended claims along with the full scope of
equivalents to which
such claims are entitled.
37
