U.S. patent application number 10/176284 was filed with the patent office on 2002-12-05 for tumor associated internalizing antigens and methods for targeting therapeutic agents.
This patent application is currently assigned to IXSYS, INC.. Invention is credited to Huse, William D., Watkins, Jeffry D..
Application Number | 20020182214 10/176284 |
Document ID | / |
Family ID | 24218229 |
Filed Date | 2002-12-05 |
United States Patent
Application |
20020182214 |
Kind Code |
A1 |
Huse, William D. ; et
al. |
December 5, 2002 |
Tumor associated internalizing antigens and methods for targeting
therapeutic agents
Abstract
The invention provides a method of reducing the proliferation 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 administering to an individual
containing a neoplastic cell population a cytotoxic or cytostatic
binding agent specifically reactive with an aberrantly expressed
vesicular membrane 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 specific 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.
Inventors: |
Huse, William D.; (Del Mar,
CA) ; Watkins, Jeffry D.; (Encinitas, CA) |
Correspondence
Address: |
CAMPBELL & FLORES LLP
4370 LA JOLLA VILLAGE DRIVE
7TH FLOOR
SAN DIEGO
CA
92122
US
|
Assignee: |
IXSYS, INC.
|
Family ID: |
24218229 |
Appl. No.: |
10/176284 |
Filed: |
June 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10176284 |
Jun 18, 2002 |
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09328128 |
Jun 8, 1999 |
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6420126 |
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09328128 |
Jun 8, 1999 |
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08555684 |
Nov 13, 1995 |
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6348194 |
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Current U.S.
Class: |
424/146.1 ;
424/155.1 |
Current CPC
Class: |
C07K 16/30 20130101;
A61P 35/00 20180101; C07K 2317/77 20130101; C07K 16/40 20130101;
C07K 16/28 20130101 |
Class at
Publication: |
424/146.1 ;
424/155.1 |
International
Class: |
A61K 039/395 |
Claims
What is claimed is:
1. A method of reducing the proliferation of a neoplastic cell,
comprising 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.
2. The method of claim 1, wherein said neoplastic cell specific
internalizing antigen is selected from the group consisting of
lamp-2 and limp II families of lysosomal integral membrane
proteins.
3. The method of claim 1, wherein said neoplastic cell specific
internalizing antigen is selected from the group consisting of
p110, vacuolar-H.sup.+-ATPase, acetyl CoA:.alpha.-glucosaminide
N-acetyltransferase, prosaposin, procathepsin L receptor and
lysosomal acid phosphatase.
4. The method of claim 1, wherein said cytotoxic or cytostatic
binding agent is an antibody specific to the lumenal domain of the
neoplastic cell specific internalizing antigen.
5. A method of intracellular targeting a cytotoxic or cytostatic
agent to a neoplastic cell population, comprising administering to
an individual containing a neoplastic cell population a cytotoxic
or cytostatic binding agent specifically reactive with an
aberrantly expressed vesicular membrane associated neoplastic cell
specific internalizing antigen expressed by said neoplastic cell
population, wherein said cytotoxic or cytostatic binding agent is
bound by said neoplastic cell specific internalizing antigen and is
internalized into the intracellular compartment.
6. The method of claim 5, wherein said neoplastic cell specific
internalizing antigen is selected from the group consisting of
lamp-2 and limp II families of lysosomal integral membrane
proteins.
7. The method of claim 5, wherein said neoplastic cell specific
internalizing antigen is selected from the group consisting of
p110, vacuolar-H.sup.+-ATPase, acetyl CoA:.alpha.-glucosaminide
N-acetyltransferase, prosaposin, procathepsin L receptor and
lysosomal acid phosphatase.
8. The method of claim 5, wherein said cytotoxic or cytostatic
binding agent is an antibody specific to the lumenal domain of the
neoplastic cell specific internalizing antigen.
9. A method of reducing the growth of a tumor through the
intracellular targeting of a cytotoxic agent, comprising
administering to an individual containing or suspected of
containing a tumor a cytotoxic or cytostatic binding agent
specifically reactive with an aberrantly expressed vesicular
membrane associated neoplastic cell specific internalizing antigen
expressed by cells of said tumor wherein said cytotoxic or
cytostatic binding agent is bound by said neoplastic cell specific
internalizing antigen and is internalized into the intracellular
compartment.
10. The method of claim 9, wherein said neoplastic cell specific
internalizing antigen is selected from the group consisting of
lamp-2 and limp II families of lysosomal integral membrane
proteins.
11. The method of claim 9, wherein said neoplastic cell specific
internalizing antigen is selected from the group consisting of
p110, vacuolar-H.sup.+-ATPase, acetyl CoA:.alpha.-glucosaminide
N-acetyltransferase, prosaposin, procathepsin L receptor and
lysosomal acid phosphatase.
12. The method of claim 9, wherein said therapeutic binding agent
further comprises a cytotoxic or cytostatic binding agent.
13. The method of claim 9, wherein said cytotoxic or cytostatic
binding agent is an antibody specific to the lumenal domain of the
neoplastic cell specific internalizing antigen.
Description
[0001] 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.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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 of 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.
[0004] 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 problems. Perhaps the most significant of 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.
[0005] Immunotherapy is one approach to overcome the lack of
specificity inherent in today's current treatments. In general,
immunotherapy offers 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 affinity 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 marker
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.
[0006] The discovery of such putative antigen markers generally
occurs through a fortuitous observation, or can result from a labor
intensive effort to specifically screen and identify putative tumor
specific antigens. The latter of such efforts generally involves
either the generation of a panel of antibodies against tumor cell
surface antigens and then screening of the panel against tumor and
control cells to determine which antibodies may be significantly
reactive and specific for a particular tumor cell type. The
percentage of those antibodies screened that are ultimately
identified as being reactive with tumor cell specific markers is
usually a very low percentage.
[0007] There are now a number of antibodies which recognize cell
surface antigens reported to be preferentially 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 specific antibodies or
antigen markers there still remains at least one major problem
which leads to several side effects and a lower quality of life.
This problem results from the toxicity of the therapeutic agents
that are conjugated to the tumor specific 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 effects would be to selectively
introduce the toxic agent intracellularly. Such an intracellular
targeting scheme would require not only the identification of a
tumor cell specific marker and generation of a highly specific
antibody, but also that the marker antigen undergo internalization
to avoid toxic side effects to surrounding normal cells.
[0008] Lymphocyte marker antigens have been identified which
undergo internalization 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, major
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 functions as a carrier of
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.
[0009] 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 Le.sup.y
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-1 or lamp-3 families. The Le.sup.y
antigen is now thought to be an altered glycosylation product which
is primarily associated with lamp-1. However, because these
antigens were discovered independently of one another and their
full potential could not be appreciated for the therapeutic benefit
of essentially many different types of cancers.
[0010] 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 THE INVENTION
[0011] The invention provides a method of reducing the
proliferation 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 administering to an individual containing a neoplastic
cell population a cytotoxic or cytostatic binding agent
specifically reactive with an aberrantly expressed vesicular
membrane 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 specific 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.
DETAILED DESCRIPTION OF THE INVENTION
[0012] This invention is directed to novel methods for targeting
immunoconjugates to neoplastic cell populations. The methods rely
on the identification and utilization of 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
effects to neighboring cells is significantly reduced.
[0013] 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 significantly elevated on the cell surface
in neoplastic cells of the same lineage. Although surface
expression of these lysosomal membrane proteins is elevated in the
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.
[0014] As used herein, the term "neoplastic cell" is intended to
mean a cell that exhibits an abnormal morphological or
proliferative phenotype. In vitro such cells are characterized by
anchorage independent cell growth and loss of contact inhibition
whereas in vivo such cells can be characterized by, for example, an
abnormal new growth of tissue, the cells of which can tend to
invade surrounding tissue and metastasize to other body sites.
[0015] As used herein, the term "neoplastic cell specific
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 specific 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. 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 association 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.
[0016] The definition of neoplastic cell specific internalizing
antigen is intended to include those known lysosomal membrane
glycoproteins which can be classified into the general categories
known by those skilled in the 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. Biol. Chem. 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, p110,
vacuolar-H.sup.+-ATPase, acetyl CoA:.alpha.-glucosaminide
N-acetyltransferase, prosaposin, 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 within the
definition of the term so long 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.
[0017] Thus, the specific tumor associated antigens known as
lamp-1, CD63 (ME491), CO-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.
[0018] 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 of 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 of 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. 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 subunits 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').sub.2 portions, T cell receptors, integrins, hormone
receptors and transmitter receptors.
[0019] 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. For example, there are many known toxins or
drugs which are known by those skilled in the art to exhibit these
properties. Specific 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-.alpha. and TNF-.beta.. 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.
[0020] As used herein, the term "specifically reactive" when used
in reference to a binding agent refers to the discriminatory
binding of 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 specific internalization antigen. Specific
reactivity can include binding properties such as binding
specificity, binding affinity and binding avidity.
[0021] The invention provides a method of reducing the
proliferation of a neoplastic cell. The method consists of
contacting the neoplastic cell with a cytotoxic or cytostatic
binding agent which is specifically reactive with a neoplastic cell
specific internalizing antigen.
[0022] Among the many phenotypes associated with neoplasia, a large
percentage result from 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 few pronounced molecular
changes which are known. Although some differences, such as altered
glycosylation, have been observed on neoplastic cells, in general
there are very few molecular markers that are specifically
expressed on the surface of the neoplastic cell and therefore
available for immunotherapeutic targeting. In 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.
[0023] 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.
Lysosomal 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.
[0024] The sorting of 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 receptor for 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 found on the
plasma membrane where it functions to deliver lysosomal enzymes
from the extracellular environment back to the lysosomes through
the endocytic pathway. The subcellular localization of MPR is in
large part unchanged with neoplastic transformation.
[0025] Although it has been observed that several soluble lysosomal
enzymes are secreted at elevated levels in neoplastic cells, by
virtue of their very different sorting mechanism there have been no
generalizations that sorting, much less sorting of membrane
proteins of the lysosomal and endocytic compartments are a result
of neoplastic transformation. In contrast to the MPR-dependent
sorting of soluble lysosomal enzymes, lysosomal membrane proteins
are sorted through a MPR-independent mechanism. Recognition of a
specific cytoplasmic amino acid sequence directs these
transmembrane proteins to the lysosome. Although the presence of
minor levels of 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 cytoplasmic
recognition sequence or to more physiologically relevant events
such as those requiring exocytosis (Fukuda, M., J. Biol. Chem.
266:21327-21330 (1991)).
[0026] 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. Biol. 117:311-325
(1992)).
[0027] 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 lysosomal membrane proteins to
particular cell lineages or to particular cell phenotypes.
[0028] As stated previously, it has now been observed that one
consequence of neoplastic transformation is a metabolic imbalance
which results in altered sorting and trafficking of membrane
proteins of the lysosomal and endocytic pathways. This metabolic
imbalance leads to the increased expression of these normally
intracellular proteins on the plasma membrane surface 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 find their way back to endocytic
vesicles.
[0029] This aberrant sorting phenotype has two characteristics that
can be advantageously exploited in the neoplastic cell for the
beneficial targeting of therapeutic agents. First, the lysosomal
membrane proteins are specifically expressed on the plasma membrane
of neoplastic cells at high and discriminatory levels compared to
normal cells. This characteristic allows for the specific targeting
of therapeutic agents through binding agents which specifically
recognize the lysosomal membrane protein or other protein of 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 of toxins which
function intra-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.
[0030] 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 defined 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 of 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.
[0031] Cytotoxic or cytostatic agents are attached to the binding
agents by a variety of methods known in the art. Attachment,
coupling 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 functions of both of the binding agent and the cytotoxic or
cytostatic agent. Such methods are well known in the art and are
described in, for example, Harlow et al. (Antibodies: A Laboratory
Manual, Cold Spring Harbor (1988)). For example, the covalent bond
can be formed by way of carbodiimide, glutaraldehyde,
heterobifunctional cross-linkers, and homobifunctional
cross-linkers. The cross-linking of proteins can additionally be
accomplished by using reactive groups within the individual protein
such as carbohydrate, disulfide, carboxyl or amino groups. Coupling
can be accomplished by oxidation or reduction of the native
protein, or treatment with an enzyme, for example.
[0032] Similarly, numerous different cytotoxic and cytostatic
agents are known by those skilled in the art. Selection of 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 for ovarian,
esophageal cancer, head and neck cancer, non-small cell lung cancer
and testicular cancer.
[0033] In non-small cell lung cancer, cisplatin combined with vinca
alkaloids and mitomycin CMVP resulted in a 77% major response rate.
Platinum containing complexes represent the most important group of
agents now in use for cancer treatment. They can be curative in
combination therapy for testicular and ovarian cancers and play a
central role in the treatment of lung, head and neck, and bladder
cancers members of this group have desirable pharmacologic action
including synergy in 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.
[0034] 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 specifically
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.
[0035] For cytotoxic agents which result in non-specific
destruction of the cell, such as radioactive isotopes, once bound
to the neoplastic cell specific internalizing antigen they will
immediately exert their effect on inhibiting cell proliferation.
Internalization of such cytotoxic agents, however, reduces the
ablative effects 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 effective. These antigens will undergo a
sufficient rate of internalization and therefore are not reliant
upon secondary mechanisms such as the steady-state recycling of
plasma membrane to deliver the cytotoxic or cytostatic agent to the
cytoplasm.
[0036] The methods of the invention utilize aberrantly expressed
vesicular membrane associated neoplastic cell specific
internalizing antigens. Such antigens include, for example, those
known lysosomal membrane proteins which are categorized in the
lamp-2 or limp II families as well as those categorized in these
families and other families which are subsequently identified as
having substantially the same characteristics of neoplastic cell
specific internalizing antigen as defined previously. Further,
other membrane proteins of the endocytic pathway which exhibit, or
are found to exhibit the characteristics described previously are
also considered to be useful in the methods of the invention. Such
other proteins include, for example, p110, vacuolar-H.sup.+-ATPase,
acetyl CoA:.alpha.-glucosaminide N-acetyltransferase, prosaposin,
procathepsin L receptor and lysosomal acid phosphatase. Thus, the
invention provides for a method of reducing the proliferation of a
neoplastic cell by contacting the cell with a cytotoxic or
cytostatic binding agent that is specifically reactive with any of
the above neoplastic cell specific internalizing antigens.
[0037] The invention also provides a method for intracellular
targeting of a cytotoxic or cytostatic 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 specifically reactive with an aberrantly expressed vesicular
membrane associated neoplastic cell specific internalizing antigen
expressed by the neoplastic cell population, wherein said cytotoxic
or cytostatic binding agent is bound by the neoplastic cell
specific internalizing antigen and is internalized into the
intracellular compartment.
[0038] The invention also provides for a method of reducing the
growth of a tumor through the intracellular targeting of a
cytotoxic or cytostatic agent. The method consists of administering
to an individual containing, or suspected of containing a tumor, a
cytotoxic or cytostatic binding agent specifically reactive with an
aberrantly expressed vesicular membrane associated neoplastic cell
specific internalizing antigen expressed by cells of the tumor
wherein the cytotoxic or cytostatic binding agent is bound by the
neoplastic cell specific internalizing antigen and is internalized
into the intracellular compartment.
[0039] The methods described previously for reducing the
proliferation of a neoplastic cell are applicable for in vitro
diagnosis of neoplastic cells or the testing of various agents
applicable to both the ex-vivo and in vivo targeting of therapeutic
agents to neoplastic cell populations. Such methods are useful, for
example, for testing the cytotoxic or cytostatic effects of
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 equally 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 of the neoplastic
cell population or tumor mass expressing the targeted internalizing
antigen.
[0040] 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 of the original
growth can occur.
[0041] Cytotoxic or cytostatic binding agents can be administered
in many possible formulations, including pharmaceutically
acceptable media. In the case of a short peptide, the peptide can
be conjugated to a carrier, for example, in order to increase its
stability within the circulatory system. Antibodies are
advantageous for use as a binding agent since they are naturally
long-lived proteins of the circulatory system. Antibodies can be
produced in a variety of mammals and then genetically engineered to
resemble proteins of human origin and in this way avoid endogenous
host defense mechanisms. Methods for humanizationof antibodies are
well known in the art and are described, for example, in Winter and
Harris, Immunol. Today, 14:243-246 (1993); Winter and Harris, Tips,
14:139-143 (1993) and Couto et al. Cancer Res., 55:1717-1722
(1995).
[0042] The cytotoxic or cytostatic binding agents are administered
by conventional methods, in dosages which are sufficient to effect
binding of the neoplastic cell specific internalizing antigen. Such
dosages are known or can be easily determined by those skilled in
the art. Administration can be accomplished by, for example,
intravenous, interperiential or subcutaneous injection.
Administration can be performed in a variety of different regimes
which include single high dose administration or repeated small
dose administration or a combination of both. The dosing will
depend on the type of neoplasia, progression of the disease and
overall health of the individual and will be known or can be
determined by those skilled in the art.
[0043] The cytotoxic or cytostatic binding agent can be
administered to an individual either singly or in a cocktail
containing two or more cytotoxic or cytostatic binding agents,
other therapeutic agents, compositions, or the like, including, for
example, immunosuppressive agents, potentiators and side-effect
relieving agents. Immunosuppressive agents include for 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, for example, monensin, ammonium chloride and
chloroquine. All of these agents are administered in generally
accepted efficacious dose ranges such as those disclosed in the
Physician Desk Reference, 41st Ed. (1987), Publisher Edward R.
Barnhart, New Jersey.
[0044] The cytotoxic or cytostatic binding agents can be formulated
into, for example, injectable or topical preparation for
administration. Parenteral formulations are known and are suitable
for use in the invention, preferably for i.m. or i.v.
administration. Formulations containing therapeutically effective
amounts of the cytotoxic or cytostatic binding agents can be
sterile liquid solutions, liquid suspensions or lyophilized
versions and can additionally contain stabilizers or excipients for
example. Therapeutically effective doses of the cytotoxic or
cytostatic binding agents can be, for example, in a range of from
about less then 0.01 mg/kg to about greater than 10 mg/kg body
weight of the treated individual administered over several days to
two weeks by daily intravenous infusion.
[0045] The cytotoxic or cytostatic binding agents can be formulated
into topical preparations for local therapy by including it. The
cytotoxic or cytostatic binding agents can be formulated into
topical preparations for local therapy by including it in a
dermatological vehicle. The amount of agent to be administered will
depend upon the vehicle selected, the clinical condition of the
patient, the systemic toxicity and the stability of the anti-T cell
immunotoxin in the formulation. Suitable vehicles include for
example, gels or water-in-oil emulsions using mineral oils,
petrolatum and the like.
[0046] 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 containing or derivatives thereof.
Ordinarily, an aqueous aerosol is made by formulating an aqueous
solution or suspension of the cytotoxic or cytostatic binding
agents together with conventional pharmaceutically acceptable
carriers and stabilizers. The carriers and stabilizers will vary
depending upon the requirements for the particular binding agent
but include, for example, nonionic surfactants (Tweens, Pluronics,
or polyethylene glycol), innocuous proteins like serum albumin,
sorbitan esters, oleic acid, lecithin, amino acids such as glycine,
buffers, salts, sugars or sugar alcohols. The formulations also can
include mucolytic agents as well as bronchodilating agents. The
formulations will be sterile. Aerosols generally will be prepared
from isotonic solutions. The particles optionally include normal
lung surfactants.
[0047] It is understood that modifications which do not
substantially affect the activity of the various embodiments of
this invention are also included within the definition of the
invention provided herein. Accordingly, the following examples are
intended to illustrate but not limit the present invention.
EXAMPLE I
Altered Trafficking of Membrane-Associated Lysosomal Proteins
[0048] This example shows that membrane-associated lysosomal
proteins are mistargeted to the plasma membrane in carcinoma
cells.
[0049] Demonstration of the mistargeting of membrane-associated
lysosomal proteins to the cell surface was shown using two
different approaches. In the first approach, monoclonal antibodies
specific for different membrane-associated lysosomal proteins
(Table 1) are utilized in an ELISA format with either normal or
carcinoma cells obtained from a variety of human tissues (Table 2).
In order to measure only the antigen present on the cell surface,
cells are plated in 96-well microtiter dishes one day prior to
determining antibody reactivity. On the next day, cells are rinsed
with phosphate-buffered saline (PBS), and incubated with dilutions
of each antibody in 1% bovine serum albumin (BSA) in PBS for 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 staining kit, Pierce) for 15 minutes. Unbound complex is
removed with several PBS washes. All antibody incubations and wash
steps are performed at 4.degree. C. After the final PBS wash the
binding of antibody is detected by the addition of
o-phenylenediamine hydrochloride. The reaction is terminated by the
addition of 2.5 M H.sub.2SO.sub.4 and the formation of product is
measured by adsorption at 492 nm. In order to measure non-specific
binding of the secondary antibodies and avidin complexes one sample
is incubated without primary antibody.
[0050] 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 containing 10.sup.6 cells/ml of culture media are
incubated for one hour with 10 .mu.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
(FITC)-labeled anti-mouse IgG antibody for one hour. The cells are
washed and analyzed for antibody binding by fluorescent activated
cell sorting (FACS). All incubations and washes are performed at
4.degree. C. Reactivity is expressed as the binding ratio of the
test antibody (mean fluorescent intensity) versus the control
antibody. Analysis of the results of the above assays will reveal
significant and discriminatory binding on the carcinoma cell lines
with little binding on the normal human cells.
EXAMPLE 2
Lysosomal Proteins Mistargeted to the Plasma Membrane are
Internalized
[0051] This example demonstrates that membrane-associated lysosomal
proteins expressed on the cell surface are rapidly internalized to
an acidic vesicular compartment.
[0052] 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
of 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 for 15 minutes at room temperature. The
cells are rinsed twice with PBS and incubated one hour with primary
antibody at 10 .mu.g/ml in 1% BSA in PBS. As a positive control for
plasma membrane localization, some cells are incubated with
anti-transferrin receptor antibody. The cells are then washed with
PBS, incubated with FITC-labeled anti-mouse IgG antibody for one
hour and washed with PBS again. Little non-specific binding of
FITC-labeled secondary antibody is observed in samples in which the
primary antibody is omitted from the incubations. The coverslips
are mounted in Fluoromount-G, to minimize florescent quenching, and
examined with a Zeiss microscope equipped with epifluorescent
optics and a Zeiss Plan 100.times. (NA 1.3) oil objective lens.
[0053] The intracellular localization of these same antigens is
examined as described above with minor modifications. Specifically,
0.1% digitonin is included in all buffers used for antibody
dilutions and cell washes. Digitonin selectively interacts with
cholesterol, preserving the structure of most membranes while
permeabilizing them sufficiently to allow the introduction of
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 examined for co-localization with the
antibodies against the membrane-associated lysosomal proteins.
[0054] 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. Specifically, cells which have been
seeded on coverslips one day prior are chilled on ice for 30-60
minutes to inhibit endocytosis. The cells are then washed with
ice-cold PBS and 10 .mu.g/ml antibody diluted in ice-cold cell
culture media is added and incubated with the cells for one hour.
The cells are washed with ice-cold PBS and incubated with
FITC-labeled anti-mouse IgG antibody for 30 minutes. It is critical
to maintain the cells at 4.degree. 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 shifted
to 37.degree. C. by placing pre-warmed media on the dish.
Incubations are stopped at various intervals by shifting the cells
back to 4.degree. C. and fixing the cells with 2% paraformaldehyde
in PBS. Internalization to pre-lysosomal compartments is monitored
with antibodies to the transferrin receptor while internalization
to the lysosomal compartment is monitored with cathepsin D
antibodies.
[0055] Analysis of the results of the above procedure will
initially show diffuse surface binding of the antibodies, followed
shortly thereafter by patching or congregation at multiple sites on
the cell surface. With longer incubation times, the patches of
antibodies will cap (localize to a single site on the cell surface)
and begin to be internalized. Depending on the duration of
incubation at 37.degree. C., antibodies will co-localize either
with the pre-lysosomal endocytic marker (transferrin receptor) or
with the lysosomal marker, cathepsin D. Little or no staining is
observed with (1) control primary antibodies, (2) if cells are
incubated with secondary antibody only, or (3) if the cells are
maintained at 4.degree. C. for the duration of the experiment.
[0056] 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
[.sup.3H]thymidine uptake assay.
[0057] 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.degree. C. for 6 hours. Next, 1 .mu.Ci/well of
[.sup.3H]thymidine is added and the cells are incubated for an
additional 6 hours at 37.degree. C. The cells are frozen, thawed,
harvested onto glass filters, and counted in a beta counter.
Inhibition of the incorporation of [.sup.3H]thymidine into cellular
DNA in treated cells, as compared to untreated control cells, is
consistent with the internalization of the antibodies observed by
immunofluorescence.
[0058] To demonstrate 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 shown by ELISA to express little of the targeted
lysosomal membrane proteins on the plasma membrane are more
resistant to the cytotoxic effects of the ricin A-chain
immunoconjugates and display cytotoxicity profiles similar to those
obtained with irrelevant immunoconjugates.
EXAMPLE 3
Treatment of Xenografted Human Carcinomas with Doxorubicin
Immunoconjugates
[0059] This example demonstrates the ability of antibodies to
neoplastic cell specific internalizating antigens to efficiently
target solid tumors in vivo.
[0060] 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 of the cytotoxic agent in the acidic intracellular
environment of the endosomes/lysosomes.
[0061] Human carcinoma lines which are demonstrated to be reactive
with the primary antibody by ELISA, FACS, immunofluorescence, and
the functional 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 for 14-28 days, at which
time the animals 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 of mg doxorubicing/kg body weight. Following treatment,
efficacy is measured as partial regression (decrease in tumor
volume to less than 50% of original volume), complete regression
(not observable for defined time), or cured (not observable for
greater than 10 doubling times). One doubling time is defined as
the time required for tumors in control (untreated) animals to
double in size. Animals treated with optimal doses of 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 of doxorubicin are achieved with the
immunoconjugates of internalizing lysosomal antigens than is
achievable with free doxorubicin.
1TABLE 1 Membrane-associated Lysosomal Protein Targets Class Target
lamp-2 lamp-2 limp II limp II CLA-1 NA p110 NA 64 kDa chloride
channel NA vacuolar-H.sup.+-ATPase NA lysosomal acid
phosphatase
[0062]
2TABLE 2 Cell Lines Analyzed In Vitro Type Tissue Name ATCC No.
carcinoma breast MDA-MB-231 HTB 26 carcinoma colon COLO 205 CCL 222
carcinoma colon DLD-1 CCL 221 carcinoma colon HCT-15 CCL 225
carcinoma colon SW 1417 CCL 238 carcinoma kidney ACHN CRL1611
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 NA
[0063] Although the invention has been described with reference to
the disclosed embodiments, those skilled in the art will readily
appreciate that the specific experiments detailed are only
illustrative of the invention. It should be 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.
* * * * *