Note: Descriptions are shown in the official language in which they were submitted.
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Tln~OR ~-~SO~TAT~n lNL~:~N~r~T~TNG A~TT~N-~ A~nD ~ vS FOR
TAR~ ~ T~R~PElrrIC AGENTS
Throughout this application various publications
are referenced within parentheses. The disclosures of
these publications in their entireties are hereby
incorporated by reference in this application in order to
more fully describe the state of the art to which this
invention pertains.
R~t'RrRnUNI~ OP' T~F~ lNV~':N-llON
The present invention relates generally to
lysosomal and vesicular secretory pathways and, more
particularly, to tumor antigen discovery and to methods
of intracellularly targeting therapeutic agents.
Neoplastic cell transformations, or cancer, is
a disease which results in more than 2.3 million deaths
annually, or greater than 20% of all deaths reported to
the World Health Organization in the industrialized
countries. Neoplastic cell transformations manifests as
a group o~ cells that proliferate outside the normal
growth control mechanisms and can be considered a
collection of many different diseases which differ in
their genetic basis, progression and clinical outcome.
..
The standard methods of treatment for cancer
currently include surgery, radiation therapy, and
chemotherapy using cytotoxic drugs. Such methods can be
effective if treatment is initiated early enough.
However, each therapeutic approach comes with inherent
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problems. Perhaps the most slgnificant o~ these problems
include unacceptable toxic side effects and the lack of
complete surgical removal of the entire neoplastic
growth. An additional problem in treating cancer results
from metastasis of the primary tumor to secondary sites
if treatment is not complete or initiated early before
substantial progression of the disease.
Immunotherapy is one approach to overcome the
lack of specificity inherent in today's current
treatments. In general, immunotherapy of~ers several
advantages which include not only the ability to generate
antibodies to essentially any desired antigen but also
the ability to produce antibodies that exhibit high
specificity and binding affinity to the particular
antigen of interest. This high specificity and binding
a~finity allows specific targeting of therapeutic agents
to essentially all diseased cells in which there is an
identified and specific antigen marker. However, any
cross reactivity of the antibody to other antigens or the
presence of significant quantities of the mar~er antigen
on the surface of non-diseased cells will lead to binding
and the unfortunate targeting of toxic agents to normal
cell types. Thus, the specificity and efficiency of
targeting is the combined function of both the
specificity of the antibody and the reliability of the
antigen marker.
The discovery of such putative antigen markers
generally occurs through a fortuitous observation, or can
result ~rom a labor intensive ef~ort to specifically
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screen and identi~y putative tumor speci~ic antigens.
The latter o~ such ef~orts generally involves either the
generation o~ a panel o~ antibodies against tumor cell
surface antigens and then screening o~ the panel against
tumor and control cells to determine which antibodies may
be signi~icantly reactive and speci~ic ~or a particular
tumor cell type. The percentage o~ those antibodies
screened that are ultimately identi~ied as being reactive
with tumor cell speci~ic markers is usually a very low
percentage.
There are now a number o~ antibodies which
recognize cell sur~ace antigens reported to be
pre~erentially expressed on neoplastic cells. These
antibodies are increasingly being applied in the clinic
as diagnostic tools and as potential therapeutic
treatments. However, even with highly speci~ic
antibodies or antigen markers there still r~m~; n~ at
least one major problem which leads to several side
e~fects and a lower quality o~ e. This problem
results ~rom the toxicity o~ the therapeutic agents that
are conjugated to the tumor speci~ic antibodies. Such
agents generally include radioisotopes which are highly
toxic to all cells which come in contact with the
antibody conjugate and especially to the neighboring
cells around the targeted tumor cell mass. One
possibility to overcome such side e~ects would be to
selectively introduce the toxic agent intracellularly.
Such an intracellular targeting scheme would require not
only the identi~ication o~ a tumor cell speci~ic marker
and generation o~ a highly speci~ic antibody, but also
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that the marker antigen undergo internalization to avoid
toxic side effects to surrounding normal cells.
Lymphocyte marker antigens have been identified
which undergo internallzation from the cell surface.
Many of these lymphocyte antigens, if not all of them,
are cell surface proteins which include cytokine
receptors, T cell receptors, ma}or histocompatibility and
the like. In regard to cells outside of the lymphocyte
lineage, there are relatively very few internalizing
antigens that are known to exist for solid tumors. One
example is the transferrin receptor which naturally
~unctions as a carrier o~ iron between the extracellular
and intracellular environment. Another example is the
mannose-6-phosphate receptor which directs soluble
lysosomal enzymes to prelysosomal compartments. Antigens
of this category which are known to normally cyclize
between different cellular locations also generally
exhibit poor tumor cell specificity.
Of those few antigens that are currently being
evaluated as internalizing antigens for solid tumors,
most if not all were unfortunately discovered by
serendipity. For example, the LeY antigen was initially
characterized as being an altered glycosylation product
found on the cell surface of tumorigenic cells. Other
antigens include lysosomal membrane proteins such as
those belonging to the lamp-l or lamp-3 families. The LeY
antigen is now thought to be an altered glycosylation
product which is primarily associated with lamp-1.
However, because these antigens were discovered
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independently of one another and their full potential
could not be appreciated for the therapeutic benefit of
essentially many different types of cancers.
Thus, there exists a need for the therapeutic
treatment of tumors to enable the consistent and
efficient identification of novel internalizing antigen
markers . Such novel internalizing antigens can be used
to enhance the specificity of immunotherapeutic
approaches The present invention satisfies this need
and provides related advantages as well.
SUMMARY OF T~ lNv~:Nl,ON
The invention provides a method of reducing the
proli~eration of a neoplastic cell. The method consists
of contacting the neoplastic cell with a cytotoxic or
cytostatic binding agent specifically reactive with an
aberrantly expressed vesicular membrane associated
neoplastic cell specific internalizing antigen. The
neoplastic cell specific internalizing anitgen can be
selected from the group consisting of lamp-2 and limp II
families of lysosomal integral membrane proteins. Also
provided is a method of intracellular targeting of a
cytotoxic or cytostatic agent to a neoplastic cell
~ population. The method consists of a~m; n; ~tering to an
individual containing a neoplastic cell population a
cytotoxic or cytostatic binding agent specifically
reactive with an aberrantly expressed vesicular membrane
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associated neoplastic cell specific internalizing antigen
that is expressed by the neoplastic cell population,
wherein the cytotoxic or cytostatic binding agent is
bound by the neoplastic cell speci~ic internalizing
antigen and is internalized into the intracellular
compartment. A method of reducing tumor growth through
the intracellular targeting of a cytotoxic agent is also
provided.
D~TATT-~n DES~TPTTON OF T~ INv~:N-llON
This invention is directed to novel methods for
targeting immunoconjugates to neoplastic cell
populations. The methods rely on the identi~ication and
utilization o~ neoplastic cell specific internalizing
antigens to achieve high specificity for the target cell
population. One advantage of the methods is that they
employ cell surface antigens which undergo
internalization of the bound immunoconjugate into the
cytoplasm. This internalization provides greater
specificity and therapeutic efficacy since toxic side
e~ects to neighboring cells is significantly reduced.
In one embodiment, lysosomal membrane proteins
are used to specifically target toxic antibody conjugates
to neoplastic cell populations. The lysosomal membrane
proteins can be found expressed on the plasma membrane at
very low levels in normal cell populations but become
signi~icantly elevated on the cell surface in neoplastic
cells o~ the same lineage. Although sur~ace expression
of these lysosomal membrane proteins is elevated in the
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neoplastic phenotype, these proteins are still
internalized and recycled as observed in normal cells for
the endocytic pathway. These two characteristics not
only confer high specificity to the therapeutic approach
but also result in greater efficacy since toxic agents
can be employed which have a direct and specific effect
on cell viability.
As used herein, the term "neoplastic celln is
intended to mean a cell that exhibits an abnormal
morphological or proli~erative phenotype. In vitro such
cells are characterized by anchorage independent cell
growth and loss o~ contact inhibition whereas in vivo
such cells can be characterized by, for example, an
abnormal new growth o~ tissue, the cells of which can
tend to invade surrounding tissue and metastasize to
other body sites.
As used herein, the term " neoplastic cell
speci~ic internalizing antigen" is intended to mean a
class of proteins which are membrane associated and
preferentially localized in non-neoplastic cells to the
lysosomal or other intracellular vesicular compartments.
However, in neoplastic cells, such internalizing antigens
are expressed at elevated levels on the plasma membrane
compared to non-neoplastic cells and as such are
neoplastic cell speci~ic antigens. Thus, such antigens
can also be described as aberrantly expressed membrane
associated vesicular antigens. These antigens are
aberrantly expressed on the plasma membrane of neoplastic
cells and are capable of undergoing internalization.
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Vesicular compartments can include, for example,
membranous subcellular organelles and structures such as
the endoplasmic reticulum, the golgi apparatus,
lysosomes, endosomes, coated pits and caveolae. Membrane
as60ciation includes, for example, integral membrane
proteins as well as peripheral membrane associated
proteins. Proteins which are anchored to the membrane
through glycolipid modification and the like are also
included within the definition of lysosomal membrane
associated proteins.
The definition of neoplastic cell specific
internalizing antigen is intended to include those known
lysosomal membrane glycoproteins which can be classi~ied
into the general categories known by those skilled in the
1~ art as lamp-2 and limp II. Cla-1 is a specific example
of a limp II family member. The initial classification
of these molecules has been described in Fukuda, M. J.
Rlol . ~hem. 266:21327-21330, (1991). Lysosomal membrane
associated proteins which are not yet, or have not been
categorized into these classifications are also intended
to be included within the definition of a neoplastic cell
specific internalization antigen. Such molecules
include, for example, pllO, vacuolar-H'-ATPase, acetyl
CoA:~-glucosaminide N-acetyltransferase, prosaposin,
2~ procathepsin L receptor and lysosomal acid phosphatase.
Further, lysosomal proteins which are subsequently
identified to be in these lysosomal protein families as
well as other lysosomal or vesicular families, including
for example, the lamp-1 and lamp-3 families of lysosomal
membrane glycoproteins are also intended to be included
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within the definition of the term so lony as the molecule
exhibits the characteristics of being a membrane
associated protein expressed at elevated levels on the
plasma membrane in neoplastic cells and can be
internalized.
Thus, the specific tumor associated antigens
known as lamp-1, CD63 (ME491), C0-029, and L6 are
excluded from neoplastic cell specific internalizing
antigen as defined above. Similarly, known soluble
lysosomal enzymes are also excluded from the definition.
As used herein, the term "binding agent" refers
to a molecule which exhibits specific binding activity
toward a neoplastic cell specific internalizing antigen.
Such a binding molecule can include a variety of
different types o~ molecules including, for example,
macromolecules and small organic molecules. The type of
binding agent selected will depend on the need. Small
molecule binding agents can include, for example,
receptor ligands, antagonists and agonists.
Macromolecules can include, for example, peptide,
polypeptide and protein, nucleic acids encoding
polypeptide binding agents, lectins, carbohydrate and
lipids. It is understood that the term includes
fragments and domains o~ the agent so long as binding
function is retained. Similarly, the boundaries of the
~ domains are not critical so long as binding activity is
maintained. In the specific example where the binding
agent is a peptide, polypeptide or protein, such binding
proteins can include monomeric or multimeric species.
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Heteromeric binding proteins are a specific example of
multimeric binding proteins. It is understood that when
referring to multimeric binding proteins that the term
includes fragments of the su~units so long as assembly of
the polypeptides and binding function of the assembled
complex is retained. Heteromeric binding proteins
include, for example, antibodies and fragments thereof
such as Fab and F(ab') 2 portions, T cell receptors,
integrins, hormone receptors and transmitter receptors.
The terms "cytotoxic" and "cytostatic" when
used to characterize a binding agent is intended to mean
that the binding agent exhibits or has been modified to
exhibit cytotoxic or cytostatic properties. ~or example,
there are many known toxins or drugs which are known by
those skilled in the art to exhibit these properties.
Speci~ic examples of cytotoxic and cytostatic agents
include, for example, pokeweed antiviral protein, abrin,
ricin and each of their A chains, doxorubicin,
cisplastin, Iodine-131, Yttrium-90, Rhenium-188, Bismuth-
212, Taxol, 5-Fluorouracil VP-16, Bleomycin,
methotrexate, vindesine, adriamycin, vincristine,
vinblastine, BCNU, mitomycin and cyclophosphamide and
certain cytokines such as TNF-~ and TNF-~. Thus,
cytotoxic or cytostatic agents can include, for example,
radionuclides, chemotherapeutic drugs, proteins and
lectins. Any of these agents can be attached to a
binding agent for the cytoplasmic targeting of
therapeutic agents to neoplastic cells.
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As used herein, the term "specifically
reactive" when used in re~erence to a binding agent
refers to the discriminatory binding o~ the binding agent
to an aberrantly expressed vesicular membrane associated
neoplastic cell specific internalizing antigen. For such
binding to be discriminating, the binding agent will not
substantially cross react, or can be made not to
substantially cross react with other surface markers
which are not the particular neoplastic cell speci~ic
internalization antigen. Speci~ic reactivity can include
binding properties such as binding specificity, binding
a~finity and binding avidity.
The invention provides a method of reducing the
proliferation of a neoplastic cell. The method consists
o~ contacting the neoplastic cell with a cytotoxic or
cytostatic binding agent which is speci~ically reactive
with a neoplastic cell speci~ic internalizing antigen.
Among the many phenotypes associated with
neoplasia, a large percentage result ~rom the
deregulation of the cell cycle which leads to enhanced
proliferative phenotype. Apart from the altered
expression or activity of the regulatory proteins
involved in such cell cycle control, there are relatively
~ew pronounced molecular changes which are known.
Although some differences, such as altered glycosylation,
have been observed on neoplastic cells, in general there
are very ~ew molecular markers that are specifically
expressed on the surface of the neoplastic cell and
there~ore available for immunotherapeutic targeting. In
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contrast, many antigens which have been characterized in
regard to tumor specific expression have been found to be
only modestly selective between neoplastic and normal
phenotypes. For these reasons, it is extremely laborious
to find antigens which are specific to neoplastic cells.
It has now been observed by the inventors that
one consequence of neoplastic transformation is a
metabolic imbalance which results in altered sorting and
trafficking of intracellular membrane proteins of the
lysosomal and endocytic pathways. ~ysosomal proteins are
either soluble or membrane-associated. Both types of
proteins are synthesized on the rough endoplasmic
reticulum similar to other proteins of the secretory
pathway. Following synthesis they are then transported,
or sorted, to the lysosome. The same is true for other
proteins of the endocytic pathway. The term ~'sorted" is
used when discussing the accurate transport of these
proteins to the lysosome since other alternative pathways
exist. Such other pathways include, for example,
transport to the plasma membrane or secretion into the
extracellular space.
The sorti~g o~ either soluble or membrane
lysosomal proteins occurs by very distinct mechanisms.
For example, soluble lysosomal enzymes are sorted from
the Golgi apparatus to the lysosomes through association
of phosphorylated carbohydrate moieties with a specific
receptor. These phosphorylated moieties are high
mannose-type asparagine-linked oligosaccharide
modifications of the protein core. Accordingly, the
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receptor ~or these oligosaccharide structures is the
mannose 6-phosphate receptor (MPR). Recognition of
mannose 6-phosphate by MPR occurs in the lumen of the
Golgi apparatus. Bound by MPR, the soluble lysosomal
enzymes are sorted to prelysosomal compartments where
they are then released due to the acidic environment.
Although the MPR directs soluble lysosomal enzymes to
lysosomes, it is not itself a lysosomal membrane protein.
Once dissociated in the prelysosomal compartment, it
recycles to the Golgi complex. Some MPR can also be
~ound on the plasma membrane where it ~unctions to
deliver lysosomal enzymes ~rom the extracellular
environment bac~ to the lysosomes through the endocytic
pathway. The subcellular localization of MPR is in large
part unchanged with neoplastic trans~ormation.
Although it has been observed that several
soluble lysosomal enzymes are secreted at elevated levels
in neoplastic cells, by virtue of their very di~erent
sorting mechanism there have been no generalizations that
sorting, much less sorting o~ membrane proteins o~ the
lysosomal and endocytic compartments are a result of
neoplastic transformation. In contrast to the MPR-
dependent sorting o~ soluble lysosomal enzymes, lysosomal
membrane proteins are sorted through a MPR-independent
- 25 mechanism. Recognition o~ a speci~ic cytoplasmic amino
acid sequence directs these transmembrane proteins to the
lysosome. Although the presence o~ minor levels o~
lysosomal membrane proteins has been observed on the
plasma membrane, this observation has been attributed to
either the assumption of a saturable receptor to the
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cytoplasmic recognition sequence or to more
physiologically relevant events such as those requiring
exocytosis (Fukuda, M., J. Riol. ~hem. 266:21327-21330
(1991) ) .
The saturable receptor hypothesis has been put
forth as an attempt to explain why in normal cells
variable levels of plasma membrane expression can be
observed with certain lysosomal membrane proteins. Some
support for this hypothesis has now been provide by
artificially overexpressing transfected cDNAs encoding
lysosomal membrane proteins or by mutating the
cytoplasmic recognition sequence (Harter and Mellman, J.
Cell. B;o7. 117:311-325 (1992)).
In the cases of more physiological relevance,
it has been observed that lysosomal proteins can be
detected on the surface following platelet activation and
cytotoxic T cell exocytosis of granule contents during
specific interaction with target cells. The normal
occurrence of lysosomal membrane proteins on the plasma
membrane therefore appears to be a consequence of
membrane fusion. However, the presence of these proteins
on the surface can also be to provide protection for the
plasma membrane from the actions of the released
lysosomal acid hydrolyses. Nevertheless, in either of
the above cases, what is observed is a low and variable
level of expression of lysosomal membrane proteins on the
surface in normal cells. Because of this variability,
there has been no direct correlation of the presence of
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lysosomal membrane proteins to particular cell lineages
or to particular cell phenotypes.
As stated previously, it has now been observed
that one conse~uence of neoplastic trans~ormation is a
metabolic imbalance which results in altered sorting and
tra~icking o~ membrane proteins o~ the lysosomal and
endocytic pathways. This metabolic imbalance leads to
the increased expression of these normally intracellular
proteins on the plasma membrane sur~ace of the neoplastic
cell. These relocalized lysosomal membrane proteins are,
however, not permanently localized to the plasma
membrane. Instead, they still exhibit normal cyclization
properties and can be internalized where they ~ind their
way back to endocytic vesicles.
This aberrant sorting phenotype has two
characteristics that can be advantageously exploited in
the neoplastic cell ~or the bene~icial targeting o~
therapeutic agents. First, the lysosomal membrane
proteins are speci~ically expressed on the plasma
membrane o~ neoplastic cells at high and discriminatory
levels compared to normal cells. This characteristic
allows ~or the speci~ic targeting o~ therapeutic agents
through binding agents which speci~ically recognize the
lysosomal membrane protein or other protein o~ the
endocytic pathway. The second characteristic is that
since these otherwise aberrantly located lysosomal or
vesicular membrane proteins are essentially normal, they
internalize the binding agent. This internalization not
only allows the use o~ toxins which ~unction intra-
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16
cellularly, but, will also significantly decrease the
toxic effects to neighboring cells when using pleiotropic
or ablative agents such as radioisotopes. It is these
properties which lead to the terminology of these
lysosomal and other endocytic membrane proteins as being
neoplastic cell specific internalizing antigen.
The methods of the invention target cytotoxic
or cytostatic agents to neoplastic cells through the use
of binding agents which are specific to the neoplastic
cell specific internalizing antigens. As de~ined above,
such binding agents can be essentially any molecule,
including peptide, polypeptide and protein or other
macromolecules or binding compounds which exhibit
specific binding activity toward the neoplastic cell
specific internalizing antigen. In the specific case
where the neoplastic cell specific internalizing antigen
is a transmembrane protein, the binding agent will be
reactive to the lumenal domain o~ the protein since this
is the domain that will be exposed to the extracellular
environment once the lysosomal or other endocytic
vesicles have fused with the plasma membrane.
Cytotoxic or cytostatic agents are attached to
the binding agents by a variety of methods known in the
art. Attachment, coupliny or conjugation can be
accomplished by, for example, covalent bond formation;
however, other means known in the art can be equally
applied as well. Essentially any type of coupling
methodology will work so long as conditions are used to
maintain the ~unctions of both of the binding agent and
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the cytotoxic or cytostatic agent. Such methods are well
known in the art and are described in, ~or example,
Harlow et al. (~nt;bo~;es: A r~horatory Manual, Cold
Spring Harbor ~lg88)). For example, the covalent bond
5 can be ~ormed by way of carbodiimide, glutaraldehyde,
heterobifunctional cross-linkers, and homobi~unctional
cross-linkers. The cross-linking o~ proteins can
additionally be accomplished by using reactive groups
within the individual protein such as carbohydrate,
10 disul~ide, carboxyl or amino groups. Coupling can be
accomplished by oxidation or reduction of the native
protein, or treatment with an enzyme, ~or example.
Similarly, numerous di~erent cytotoxic and
cytostatic agents are known by those skilled in the art.
15 Selection o~ which cytotoxic or cytostatic agent to use
will depend on the need and will be known, or can be
determined by those skilled in the art. For example,
cisplatin based regimens are utilized ~or ovarian,
esophageal cancer, head and neck cancer, non-small cell
20 lung cancer and testicular cancer.
In non-small cell lung cancer, cisplatin
combined with vinca alkaloids and mitomycin CMVP resulted
in a 77~ major response rate. Platinum containing
v complexes represent the most important group o~ agents
25 now in use ~or cancer treatment. They can be curative in
- combination therapy ~or testicular and ovarian cancers
and play a central role in the treatment of lung, head
and neck, and bladder cancers members o~ this group have
desirable pharmacologic action including synergy in
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18
combination with antimetabolites and radiation therapy
but differ significantly in their patterns of toxicity
and pharmacokinetics Thus, the cytotoxic or cytostatic
agents can range from small organic molecules to large
biologically active proteins and other macromolecules.
To reduce proliferation of a neoplastic cell,
the cytotoxic or cytostatic agent and the binding agent
are attached, or conjugated, to one another to produce a
cytotoxic or cytostatic binding agent. The binding agent
is chosen so as to be speci~ically reactive with one or
more neoplastic cell specific internalizing antigens
present on the surface of the neoplastic cell. These
therapeutic binding agents are then placed in contact
with the neoplastic cells and allowed to bind the
neoplastic cell specific internalizing antigen. Once
bound, the cytotoxic or cytostatic binding agent will be
internalized through the endocytic pathway.
For cytotoxic agents which result in non-
speci~ic destruction of the cell, such as radioactive
isotopes, once bound to the neoplastic cell specific
internalizing antigen they will immediately exert their
ef~ect on inhibiting cell proliferation. Internalization
o~ such cytotoxic agents, however, reduces the ablative
ef~ects on neighboring, normal cells. In contrast, when
using cytotoxic or cytostatic agents which are effective
only once they become internalized, such as toxins which
ribosylate the protein synthesis machinery, targeting of
neoplastic cell specific internalizing antigens ensures
that such agents will enter the cytoplasm and become
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19
e~ective. These antigens will undergo a su~icient rate
of internalization and there~ore are not reliant upon
secondary mechanisms such as the steady-state recycling
o~ plasma membrane to deliver the cytotoxic or cytostatic
agent to the cytoplasm.
The methods o~ the invention utilize aberrantly
expressed vesicular membrane associated neoplastic cell
speci~ic internalizing antigens. Such antigens include,
~or example, those known lysosomal membrane proteins
which are categorized in the lamp-2 or limp II ~amilies
as well as those categorized in these ~amilies and other
~amilies which are subsequently identified as having
substantially the same characteristics o~ neoplastic cell
speci~ic internalizing antigen as de~ined previously.
Further, other membrane proteins o~ the endocytic pathway
which exhibit, or are found to exhibit the
characteristics described previously are also considered
to be useful in the methods o~ the invention. ~uch other
proteins include, ~or example, pllO, vacuolar-H~-ATPase,
acetyl CoA:~-glucosaminide N-acetyltrans~erase,
prosaposin, procathepsin L receptor and lysosomal acid
phosphatase. Thus, the invention provides for a method
o~ reducing the proli~eration o~ a neoplastic cell by
contacting the cell with a cytotoxic or cytostatic
binding agent that is speci~ically reactive with any of
the above neoplastic cell speci~ic internalizing
~ antigens.
The invention also provides a method for
intracellular targeting o~ a cytotoxic or cytostatic
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agent to a neoplastic cell population. The method
comprises administering to an individual containing a
neoplastic cell population, or suspected of containing a
neoplastic cell population, a cytotoxic or cytostatic
binding agent speci~ically reactive with an aberrantly
expressed vesicular membrane associated neoplastic cell
speci~ic internalizing antigen expressed by the
neoplastic cell population, wherein said cytotoxic or
cytostatic binding agent is bound by the neoplastic cell
speci~ic internalizing antigen and is internalized into
the intraceliular compartment.
The invention also provides for a method o~
reducing the growth of a tumor through the intracellular
targeting o~ a cytotoxic or cytostatic agent. The method
consists o~ administering to an individual containing, or
suspected o~ containing a tumor, a cytotoxic or
cytostatic binding agent specifically reactive with an
aberrantly expressed vesicular membrane associated
neoplastic cell speci~ic internalizing antigen expressed
by cells o~ the tumor wherein the cytotoxic or cytostatic
binding agent is bound by the neoplastic cell speci~ic
internalizing antigen and is internalized into the
intracellular compartment.
The methods described previously ~or reducing
the proli~eration of a neoplastic cell are applicable ~or
in vi tro diagnosis o~ neoplastic cells or the testing of
various agents applicable to both the ex-vivo and in vivo
targeting o~ therapeutic agents to neoplastic cell
populations. Such methods are use~ul, ~or example, ~or
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testing the cytotoxic or cytostatic ef~ects o~ specific
agents in vitro, bone marrow purging ex vivo and for
inhibiting the growth of single or small populations of
metastatic cells at secondary tumor sites. Similarly,
the methods describe previously are e~ually applicable
for inhibiting proliferation and/or viability of larger
neoplastic cell populations or solid tumors, for example.
In these specific examples, the cytotoxic or cytostatic
binding agent is administered in a therapeutically
effective dose so as to circulate and bind the neoplastic
cell specific internalizing antigen which is specific for
the tumor cell of interest. Once bound, the cytotoxic or
cytostatic agent will be internalized to specifically
reduce the growth o~ the neoplastic cell population or
tumor mass expressing the targeted internalizing antigen.
The cytotoxic or cytostatic agents are
administered to an individual exhibiting or at risk of
exhibiting cells having a neoplastic phenotype. Definite
clinical diagnosis of neoplasia warrants the
administration of one or more cytotoxic or cytostatic
binding agents to the relevant neoplastic cell specific
antigen. Prophylactic applications are warranted in
cases where there is a genetic disposition to develop
neoplasia or where there is a possibility that secondary
metastasis or recurrence o~ the original growth can
occur.
Cytotoxic or cytostatic binding agents can be
administered in many possible ~ormulations, including
pharmaceutically acceptable media. In the case of a
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short peptide, the peptide can be conjugated to a
carrier, ~or example, in order to increase its stability
within the circulatory system. Antibodies are
advantageous ~or use as a binding agent since they are
naturally long-lived proteins o~ the circulatory system.
Antibodies can be produced in a variety of m~mmA1s and
then genetically engineered to resemble proteins of human
origin and in this way avoid endogenous host de~ense
mechanisms. Methods for "hllmAn~zation" o~ antibodies are
well known in the art and are described, ~or example, in
Winter and Harris, Immunol. Today, 14:243-246 (1993);
Winter and Harris, ~i~, 14:139-143 (1993) and Couto et
al. CAncer Res., 55:1717-1722 (1995).
The cytotoxic or cytostatic binding agents are
administered by conventional methods, in dosages which
are su~icient to e~fect binding o~ the neoplastic cell
specific internalizing antigen. Such dosages are known
or can be easily determined by those skilled in the art.
~; n; stration can be accomplished by, for example,
intravenous, interperiential or subcutaneous injection.
A~m; n; ~tration can be performed in a variety of di~ferent
regimes which include single high dose a~m; ni qtration or
repeated small dose ~m; n; stration or a combination o~
both. The dosing will depend on the type of neoplasia,
progression o~ the disease and overall health o~ the
individual and will be known or can be determined by
those skilled in the art.
The cytotoxic or cytostatic binding agent can
be administered to an individual either singly or in a
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cocktail containlng two or more cytotoxic or cytostatic
binding agents, other therapeutic agents, compositions,
or the like, including, ~or example, immunosuppressive
agents, potentiators and side-e~ect relieving agents.
Immunosuppressive agents include ~or example, prednisone,
DECADRON (Merck, Sharp & Dohme, West Point, PA),
cyclophosphamide, cyclosporine, 6-mercaptopurine,
methotrexate, azathioprine and i.v. gamma globulin or
their combination. Potentiators include, ~or example,
monensin, ammonium chloride and chloroquine. All o~
these agents are administered in generally accepted
e~icacious dose ranges such as those disclosed in the
Physician Desk Reference, 41st Ed. (1987), Publisher
Edward R. Barnhart, New Jersey.
The cytotoxic or cytostatic binding agents can
be ~ormulated into, ~or example, injectable or topical
preparation ~or ~m~ n ~ stration. Parenteral ~ormulations
are known and are suitable ~or use in the invention,
pre~erably ~or i.m. or i.v. administration. Formulations
containing therapeutically e~ective amounts o~ the
cytotoxic or cytostatic binding agents can be sterile
liquid solutions, liquid suspensions or lyophilized
versions and can additionally contain stabilizers or
excipients ~or example. Therapeutically e~ective doses
o~ the cytotoxic or cytostatic binding agents can be, ~or
example, in a range o~ from about less then 0.01 mg/kg to
about greater than 10 mg/kg body weight o~ the treated
individual administered over several days to two weeks by
daily intravenous in~usion.
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24
The cytotoxic or cytostatic binding agents can
be ~ormulated into topical preparations ~or local therapy
by including it. The cytotoxic or cytostatic binding
agents can be formulated into topical preparations ~or
local therapy by including it in a dermatological
vehicle. The amount of agent to be ~mi ni stered will
depend upon the vehicle selected, the clinical condition
o~ the patient, the systemic toxicity and the stability
o~ the anti-T cell ;mmllnotoxin in the ~ormulation.
Suitable vehicles include ~or example, gels or water-in-
oil emulsions using mineral oils, petrolatum and the
like.
Cytotoxic or cytostatic binding agents can also
be administered by aerosol to achieve localized delivery
to the lungs. This is accomplished by preparing an
aqueous aerosol, liposomal preparation or solid particles
cont~;n;ng or derivatives thereo~. Ordinarily, an
a~ueous aerosol is made by ~ormulating an aqueous
solution or suspension o~ the cytotoxic or cytostatic
binding agents together with conventional
pharmaceutically acceptable carriers and stabilizers.
The carriers and stabilizers will vary depending upon the
requirements ~or the particular binding agent but
include, ~or example, nonionic sur~actants (Tweens,
Pluronics, or polyethylene glycol), innocuous proteins
like serum albumin, sorbitan esters, oleic acid,
lecithin, amino acids such as glycine, bu~ers, salts,
sugars or sugar alcohols. The ~ormulations also can
include mucolytic agents as well as bronchodilating
agents. The ~ormulations will be sterile. Aerosols
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generally will be prepared ~rom isotonic solutions. The
particles optionally include normal lung surfactants.
It is understood that modifications which do
not substantially af~ect the activity of the various
embodiments of this invention are also included within
the definition o~ the invention provided herein.
Accordingly, the following examples are intended to
illustrate but not limit the present invention.
EXAMPLE I
0 Al tered Traffi~kina o* M~mhrane-Associated Lvsosn~
Prot~
This example shows that membrane-associated
lysosomal proteins are mistargeted to the plasma membrane
in carcinoma cells.
Demonstration of the mistargeting of membrane-
associated lysosomal proteins to the cell surface was
shown using two di~erent approaches. In the first
approach, monoclonal antibodies speci~ic ~or different
membrane-associated lysosomal proteins (Table 1) are
utilized in an ELISA format with either normal or
carcinoma cells obtained from a variety o~ human tissues
(Table 2). In order to measure only the antigen present
on the ce~l sur~ace, cells are plated in 96-well
microtiter dishes one day prior to determining antibody
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26
reactivity. On the next day, cells are rinsed with
phosphate-buffered saline (PBS), and incubated with
dilutions o~ each antibody in 1~ bovine serum albumin
~BSA) in PBS ~or one hour. The cells are gently washed
with PBS and incubated an additional one hour with
biotinylated anti-mouse IgG (or other appropriate
specificity). The cells are washed with PBS and then
incubated with biotinylated horseradish peroxidase-avidin
complexes (ABC st~;n;ng kit, Pierce) for 15 minutes.
Unbound complex is removed with several PBS washes. All
antibody incubations and wash steps are performed at 4~C.
After the ~inal PBS wash the binding o~ antibody is
detected by the addition of o-phenylenediamine
hydrochloride. The reaction is terminated by the
addition of 2.5 M H2S04 and the formation of product is
measured by adsorption at 492 nm. In order to measure
non-specific b;n~;ng of the secondary antibodies and
avidin complexes one sample is incubated without primary
antibody.
A second approach is utilized to measure
specific cell surface expression of membrane-associated
lysosomal proteins on non-adherent cells, such as
peripheral blood lymphocytes. Briefly, a suspension
cont~;n;ng lo6 cells/ml of culture media are incubated for
one hour with 10 ~g/ml of either primary antibody or an
isotype-matched control antibody. Cells are pelleted,
washed with 1~ BSA in PBS, and incubated with fluoroscein
isothiocyanate (FIT~)-labeled anti-mouse IgG antibody for
one hour. The cells are washed and analyzed for antibody
binding by fluorescent activated cell sorting (FACS).
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All incubations and washes are performed at 4~C.
Reactivity is expressed as the ~inding ratio of the test
antibody (mean fluorescent intensity) versus the control
antibody. Analysis of the results of the above assays
will reveal signi~icant and discriminatory binding on the
carcinoma cell lines with little binding on the normal
human cells.
Example 2
I.y8080mal Protein~3 Mistaraeted to the Plasma Membrn~ ~e
Tnter~lized
This example demonstrates that membrane-
associated lysosomal proteins expressed on the cell
surface are rapidly internalized to an acidic vesicular
compartment.
To demonstrate the dual localization of
antigens at the plasma membrane and the lysosomes,
double-label immunofluorescence is employed. Initially,
the cell surface localization of antigens obtained by
ELISA and FACS analysis is first confirmed by
immunofluorescence on non-permeabilized cells. Briefly,
monolayers o~ cells are seeded on coverslips one day
prior to use. The next day, cells are rinsed with ice-
cold PBS and fixed with 2~ paraformaldehyde in PBS ~or 15
minutes at room temperature. The cells are rinsed twice
with PBS and incubated one hour with primary antibody at
10 ~g/ml in 1~ BSA in PBS. As a positive control ~or
plasma membrane localization, some cells are incubated
with anti-transferrin receptor antibody. The cells are
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28
then washed with PBS, incubated with FITC-labeled anti-
mouse IgG antibody ~or one hour and washed with PBS
again. Little non-specific binding of FITC-labeled
secondary antibody i8 observed in samples in which the
primary antibody is omitted from the incubations. The
coverslips are mounted in Fluoromount-G, to minimize
~lorescent quenching, and ~m; ned with a Zeiss
microscope e~uipped with epifluorescent optics and a
Zeiss Plan lOOX (NA 1.3) oil ob]ective lens.
The intracellular localization of these same
antigens is ~Am;ned as described above with minor
modifications. Speci~ically, 0.1% digitonin is included
in all buffers used for antibody dilutions and cell
washes. Digitonin selectively interacts with
cholesterol, preserving the structure o~ most membranes
while permeabilizing them sufficiently to allow the
introduction o~ antibodies to intracellular structures.
Permeabilized cells incubated with the test antibodies
will display punctate staining distributed around the
cell nuclei consistent with lysosomal localization. To
further verify this, permeabilized cells are probed with
antibodies against soluble lysosomal proteins (cathepsins
B, D, and L) and ~mine~ ~or co-localization with the
antibodies against the membrane-associated lysosomal
proteins.
To determine internalization of such lysosomal
membrane-associated proteins which have been
redistributed to the plasma membrane, the above described
procedure is used with yet some more minor modifications.
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29
Speci~ically, cells which have been seeded on coverslips
one day prior are chilled on ice ~or 30-60 minutes to
inhibit endocytosis. The cells are then washed with ice-
cold PBS and 10 ~g/ml antibody diluted in ice-cold cell
culture media is added and incubated with the cells ~or
one hour. The cells are washed with ice-cold PBS and
incubated with FITC-labeled anti-mouse IgG antibody ~or
30 minutes. It is critical to maintain the cells at 4~C
for all preceding steps to insure that endocytosis is
completely inhibited. Excess antibody is removed with
additional PBS washes and the cells are then shi~ted to
37~C by placing pre-warmed media on the dish.
Incubations are stopped at various intervals by shi~ting
the cells back to 4~C and ~ixing the cells with 2
para~ormaldehyde in P~S. Internalization to pre-
lysosomal compartments is monitored with antibodies to
the trans~errin receptor while internalization to the
lysosomal compartment is monitored with cathepsin D
antibodies.
Analysis of the results oi~ the above procedure
will initially show di~use sur~ace binding o~ the
antibodies, ~ollowed shortly therea~ter by patching or
congregation at multiple sites on the cell sur~ace. With
longer incubation times, the patches o~ antibodies will
cap (localize to a single site on the cell sur~ace) and
begin to be internalized. Depending on the duration o~
incubation at 37~C, antibodies will co-localize either
with the pre-lysosomal endocytic marker (trans~errin
receptor) or with the lysosomal marker, cathepsin D.
Little or no staining is observed with (1) control
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primary antibodies, (2) if cells are incubated with
secondary antibody only, or (3) if the cells are
maintained at 4~C for the duration of the experiment.
A further showing that these antibodies are
being internalized can be obtained through the use of a
functional assay which measures cell proliferation.
Briefly, immunoconjugates of antibody and the toxin ricin
A-chain, which must be internalized to be effective, are
synthesized. Inhibition of proliferation in the presence
of such cytotoxic binding proteins is measured using a
[3H]thymidine uptake assay.
In order to perform this assay, cells are
seeded in a 96-well microtiter plate one day prior to the
experiment. After removing the culture media, titrations
of the antibody ricin A-chain immunoconjugates in culture
media are added to the cells and incubated at 37~C for 6
hours. Next, 1 ~Ci/well of [3H]thymidine is added and the
cells are incubated for an additional 6 hours at 37~C.
The cells are frozen, thawed, harvested onto glass
filters, and counted in a beta counter. Inhibition of
the incorporation of E3H]thymidine into cellular DNA in
treated cells, as compared to untreated control cells, is
consistent with the internalization of the antibodies
observed by immunof~uorescence.
To ~mon~trate the specificity of the
cytotoxicity, carcinoma cells are treated with similar
doses of a control antibody ricin A-chain
immunoconjugate. Moreover, fibroblast cells which are
-
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shown by ELISA to express little of the targeted
lysosomal membrane proteins on the plasma membrane are
more resistant to the cytotoxic ef~ects o~ the ricin A-
chain immunoconjugates and display cytotoxicity pro~iles
similar to those obtained with irrelevant
immunoconjugates.
Example 3
Treat-m-~nt of ~enc~a~Afted ~u~ arc;~m~s w~1-h
Do~l~h;c~n Tmm~ ~conju~ates
This example demonstrates the ability o~
antibodies to neoplastic cell speci~ic internalizating
antigens to ef~iciently target solid tumors in vivo .
The antibodies of Table 1 are conjugated to
doxorubicin, or another suitable cytoxic agent, via an
acid-labile linker. For example, midocaproyl doxorubicin
hydrazone derivatives have been shown to provide suitable
plasma stability while allowing the release o~ the
cytotoxic agent in the acidic intracellular environment
o~ the endosomes/lysosomes.
Human carcinoma lines which are demonstrated to
be reactive with the primary antibody by ELISA, FACS,
immunofluorescence, and the ~unctional internalization
assay, are grown as subcutaneous implants in athymic
mice. In one set of control animals, implants of tumor
lines not reactive with the primary antibody are
established. All tumors are established ~or 14-28 days,
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at which time the ~nim~1s are treated with multiple doses
of ~1) the doxorubicin immunoconjugates, (2) doxorubicin
conjugated to a control antibody, or (3) doxorubicin
alone. Doses are matched on the basis o~ mg
doxorubicing/kg body weight. Following treatment,
e~icacy is measured as partial regression (decrease in
tumor volume to less than 50~ o~ original volume),
complete regression (not observable ~or de~ined time), or
cured ~not observable ~or greater than 10 doubling
times). One doubling time is de~ined as the time
required ~or tumors in control (untreated) ~nlm~l S to
double in size. Animals treated with optimal doses o~
control immunoconjugates or doxorubicin alone will
display fewer partial regressions, complete regressions,
or cures than mice which are treated with the doxorubicin
immunoconjugates utilizing the internalizing antigens
described herein. In addition, higher maximum tolerable
doses o~ doxorubicin are achieved with the
immunoconjugates o~ internalizing lysosomal antigens than
is achievable with ~ree doxorubicin.
Table 1.
Membrane-associated Lysosomal Protein Targets
~l~ T~aet
lamp-2 lamp-2
limp II limp II
CLA-1
NA pllO
NA 64 kDa chloride channel
NA vacuolar-~-ATPase
NA lysosomal acid phosphatase
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Table 2.
Cell Lines Analyzed In Vitro
Ty~e T;ssue ~m~ ATCC No
carcinoma breast MDA-MB-231 HTB 26
carcinoma colon COLO 205 CCL 222
carcinoma colon DLD-1 CCL 221
carcinoma colon ~CT-15 CCL 225
carcinoma colon SW 1417 CCL 238
carcinoma kidney ACHN C~L1611
carcinoma lung A549 CCL 185
carcinoma ovary Caov-4 HTB 76
carcinoma pancreas PANC-1 CRL1469
carcinoma thyroid SW 579 HTB 107
normal colon CCD-18Co CRL1459
normal lung CCD-18Lu CCL 205
normal peripheral blood lymphocytes N~
Although the invention has been described with
re~erence to the disclosed embodiments, those skilled in
the art will readily appreciate that the speci~ic
20 experiments detailed are only illustrative o~ the
invention. It should be understood that various
modi~ications can be made without departing from the
spirit o~ the invention. Accordingly, the invention is
s limited only by the ~ollowing claims.
~ . . . , - ~,
