U.S. patent application number 14/697027 was filed with the patent office on 2015-08-27 for methods and compositions for the treatment of cancer.
The applicant listed for this patent is Quintessence Biosciences, Inc.. Invention is credited to John A. Kink, Mark N. Shahan, Laura E. Strong.
Application Number | 20150238575 14/697027 |
Document ID | / |
Family ID | 38957306 |
Filed Date | 2015-08-27 |
United States Patent
Application |
20150238575 |
Kind Code |
A1 |
Kink; John A. ; et
al. |
August 27, 2015 |
METHODS AND COMPOSITIONS FOR THE TREATMENT OF CANCER
Abstract
The present invention is directed toward the delivery of toxic
agents to pathogenic cells, particularly cancer cells. In some
embodiments, the toxic agent is a human ribonuclease or similar
agent that is toxic to cells.
Inventors: |
Kink; John A.; (Madison,
WI) ; Strong; Laura E.; (Stoughton, WI) ;
Shahan; Mark N.; (Madison, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Quintessence Biosciences, Inc. |
Madison |
WI |
US |
|
|
Family ID: |
38957306 |
Appl. No.: |
14/697027 |
Filed: |
April 27, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13661937 |
Oct 26, 2012 |
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14697027 |
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11879279 |
Jul 17, 2007 |
8298801 |
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13661937 |
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60831378 |
Jul 17, 2006 |
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Current U.S.
Class: |
424/94.6 |
Current CPC
Class: |
A61P 35/04 20180101;
A61P 31/14 20180101; A61K 38/465 20130101; A61K 45/06 20130101;
C12N 9/22 20130101; C12Y 301/27005 20130101; A61K 47/10 20130101;
A61N 5/10 20130101; A61P 35/00 20180101; A61K 38/00 20130101 |
International
Class: |
A61K 38/46 20060101
A61K038/46; A61K 47/10 20060101 A61K047/10 |
Claims
1. A method of treating a subject comprising: administering a human
ribonuclease comprising polyethylene glycol to the subject.
2. The method of claim 1, wherein the ribonuclease is a wild type
human ribonuclease (human RNase).
3. The method of claim 1, wherein said subject is a research
subject.
4. The method of claim 1, wherein said subject has a disease.
5. The method of claim 4, wherein said disease is a disease
characterized by aberrant cell growth.
6. The method of claim 5, wherein said disease is cancer.
7. The method of claim 4, wherein said disease is a vascular
disease.
8. The method of claim 4, wherein said disease is an inflammatory
disease.
9. The method of claim 4, wherein said disease is an autoimmune
disease.
10. The method of claim 1, wherein said subject has an
infection.
11. The method of claim 1, wherein said subject has a degenerative
condition.
12. The method of claim 1, wherein the ribonuclease is administered
at 0.01 to 100 mg/kg body weight of the subject per week for one or
more weeks.
13. The method of claim 1, wherein the ribonuclease is administered
at 0.01 to 100 mg/kg body weight of the subject per day for one or
more days.
14. The method of claim 1, wherein the ribonuclease is administered
at 0.01 to 100 mg/kg body weight of the subject per treatment for
one or more treatments.
15. The method of claim 1, wherein the ribonuclease is
co-administered with one or more other medical interventions.
16. The method of claim 15, wherein the co-administration is
simultaneous.
17. The method of claim 15, wherein the co-administration is
sequential.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation U.S. patent
application Ser. No. 13/661,937, filed Oct. 26, 2012, which is a
continuation of U.S. patent application Ser. No. 11/879,279, filed
Jul. 17, 2007, now U.S. Pat. No. 8,298,801 issued Oct. 30, 2012,
which claims priority to U.S. Provisional Patent Application Ser.
No. 60/831,378, filed Jul. 17, 2006, each of which are herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention is directed toward the delivery of
toxic agents to pathogenic cells, particularly cancer cells. In
some embodiments, the toxic agent is a human ribonuclease or
similar agent that is toxic to cells.
BACKGROUND OF THE INVENTION
[0003] The term "chemotherapy" simply means the treatment of
disease with chemical substances. The father of chemotherapy, Paul
Ehrlich, imagined the perfect chemotherapeutic as a "magic bullet;"
such a compound would kill invading organisms or cells without
harming the host. While significant progress has been made in
identifying compounds that kill or inhibit cancer cells and in
identifying methods of directing such compounds to the intended
target cells, the art remains in need of improved anti-cancer
compounds and therapies.
SUMMARY OF THE INVENTION
[0004] The present invention is directed toward the delivery of a
toxic protein or related agent to pathogenic cells, particularly
cancer cells. In some embodiments, the toxic protein is a human
ribonuclease 1 or similar agent that is toxic to target cells.
Experiments conducted during the development of the present
invention showed that human ribonuclease finds use in cancer
therapies, as well as use in research and diagnostic
applications.
[0005] Accordingly, in some embodiments, the present invention
provides wild type ribonuclease 1 (e.g., from human or other
origin) compositions for use in killing cells and degrading RNA or
otherwise providing cytotoxic activity, cytostatic activity, or
cell damaging activity. The present invention also provides variant
ribonuclease 1 having properties similar to wild type ribonuclease
1. The present invention further provides therapies comprising a
ribonuclease 1 in combination with conventional therapies and
optionally a different (e.g., a non-natural or variant)
ribonuclease.
[0006] For example, in some embodiments, the present invention
provides a pharmaceutical composition comprising a wild type human
ribonuclease 1 (human RNase 1) or variant thereof, wherein the
composition is configured to kill a cell or otherwise affect target
cells (e.g., cancer cells). In preferred embodiments, the human
RNase 1 or variant thereof has one or more activities or properties
of wild type human RNase 1 including, but not limited to, the
ability to reduce tumor size in an animal, the ability to degrade
RNA, lack of reduction in weight in animals administered the RNase,
speed and accuracy of protein folding, minimal immunogenicity of
cytotoxicity, and amenity to reduce symptoms of disease (e.g.
cancer). In certain embodiments, the composition further comprises
a non-natural human ribonuclease 1 (e.g., to be co-administered
with the proteins of the present invention). In some embodiments,
the non-natural human ribonuclease 1 has a variant sequence that
disrupts binding to the ribonuclease inhibitor. In certain
preferred embodiments, the non-natural human ribonuclease 1 has a
variant sequence compared to a natural ribonuclease 1 including,
but not limited to, L86E, N88R, G89D, R91D/R4C, L86E, N88R, G89D,
R91D, V118C/L86E, N88C, R91D/R4C, L86E, N88C, R91D, V118C/R4C,
N88C, V118C/K7A, L86E, N88C, R91D/K7A, L86E, N88R, G89D, R91D/R4C,
K7A, L86E, N88C, R91D, V118C and R4C, K7A, L86E, N88R, G89D, R91D,
V118C/G38R R39D L86E N88R G89D R91D and R4C, G89R, S90R, V118C.
[0007] In some embodiments, the composition further comprises a
known therapeutic agent (e.g., a chemotherapy agent or an apparatus
for providing radiation therapy). In some embodiments, the cell is
a cancer cell, a cancer stem cell, a cell associated with an
inflammatory response, a cell associated with an infection (e.g.,
by a virus) or a cell associated with an autoimmune disease.
[0008] The present invention further provides a pharmaceutical
composition comprising a variant human ribonuclease 1 (variant
human RNase 1) having an equivalent or similar activity of a
wild-type human ribonuclease 1, wherein the RNase 1 is configured
to kill a cell or otherwise affect target cells (e.g., cancer
cells).
[0009] The present invention also provides a method for killing a
cell or otherwise providing cytotoxic activity, cytostatic
activity, or cell damaging activity, comprising the step of
exposing a cell to a wild type or variant human ribonuclease (human
RNase 1). In some embodiments, the cell resides in vitro. In other
embodiments, the cell resides in vivo. In some embodiments, the
cell is a cancer cell, a cancer stem cell, a cell associated with
an inflammatory response or a cell associated with an autoimmune
disease. In some embodiments, the cell resides in a subject
suspected of having cancer. In certain embodiments, the method
further comprises the step of providing a known therapeutic agent
to the cell (e.g., a chemotherapeutic agent or radiation).
[0010] The present invention additionally provides a method for
degrading RNA comprising the step of exposing a sample comprising
the RNA to a wild type or variant human ribonuclease (human RNase
1). In some embodiments, the RNA resides in vitro. In other
embodiments, the RNA resides in vivo. In certain embodiments, the
RNA is viral in origin. In other embodiments, the viral RNA resides
in a subject suspected of being infected.
DESCRIPTION OF THE FIGURES
[0011] FIG. 1 shows in vivo activity of RNases in some embodiments
of the present invention.
[0012] FIG. 2 shows in vivo activity of RNases in some embodiments
of the present invention.
[0013] FIG. 3 shows in vivo activity of RNases in some embodiments
of the present invention.
[0014] FIG. 4 shows enzyme activity of RNases in some embodiments
of the present invention.
[0015] FIG. 5 shows pharmacokinetics of RNases in some embodiments
of the present invention.
[0016] FIG. 6 shows in vivo activity of RNases in some embodiments
of the present invention.
[0017] FIG. 7 shows in vivo activity of RNases in some embodiments
of the present invention.
[0018] FIG. 8 shows toxicity testing data in some embodiments of
the present invention.
[0019] FIG. 9 shows in vivo activity of RNases in some embodiments
of the present invention.
[0020] FIG. 10 shows in vivo activity of RNases in some embodiments
of the present invention.
[0021] FIG. 11 shows in vivo activity of RNases in some embodiments
of the present invention.
[0022] FIG. 12 shows in vivo activity of RNases in some embodiments
of the present invention.
[0023] FIG. 13 shows in vivo activity of RNases in some embodiments
of the present invention.
[0024] FIG. 14 shows in vivo activity of RNases in some embodiments
of the present invention.
[0025] FIG. 15 shows amino acid residues in human ribonucleases as
well as sites modified or targeted for modification ("interest
sites") located therein depicted as low interest (-), medium
interest (0), or high interest (+) sites.
DEFINITIONS
[0026] To facilitate an understanding of the present invention, a
number of terms and phrases are defined below:
[0027] As used herein, the term "variant of human ribonuclease 1"
refers to a human ribonuclease 1 that varies from SEQ ID NO:1 by at
least one amino acid, yet remains homologous to wild-type human
ribonuclease 1.
[0028] As used herein, the term "variant of human ribonuclease 1
that retains the activity of ribonuclease 1" refers to a variant of
human ribonuclease 1 that retains enzymatic activities similar to
that (e.g., that has at least 1%, at least 5%, at least 15%, at
least 30%, at least 50%, at least 60%, at least 75%, at least 85%,
at least 95%, or at least 99% of activity retained) associated with
ribonuclease 1. Tests for measuring enzymatic activities are
described herein and are known in the art.
[0029] As used herein, the term "variant of human ribonuclease 1
retaining RNA degradation activity" refers to a variant of human
ribonuclease 1 (e.g., SEQ ID NO:1) that has at least 1%, at least
5%, at least 15%, at least 30%, at least 50%, at least 60%, at
least 75%, at least 85%, at least 95%, or at least 99% of RNA
degradation activity of wild type human ribonuclease 1. RNA
degradation activity may be measured using any suitable assay
including, but not limited to, visualization and quantitation of a
degraded RNA sample using agarose or polyacrylamide gel
electrophoresis. In some preferred embodiments, the variant has one
or a limited number of amino acid substitutions (e.g., conservative
or non-conservative substitutions), additions, or deletions (e.g.,
truncations) compared to wild type enzyme.
[0030] As used herein, the term "variant of human ribonuclease 1
having substantially the same cell killing activity, cytotoxic
activity, cytostatic activity, or cell damaging activity" refers to
a variant of human ribonuclease 1 (e.g., SEQ ID NO:1) that has at
least 30%, at least 50%, at least 60%, at least 75%, at least 85%,
at least 95%, or at least 99% of the cell killing activity,
cytotoxic activity, cytostatic activity, or cell damaging activity
of wild type human ribonuclease 1. For example, in some
embodiments, the activity is the ability to kill or otherwise
affect cancer cells. In other embodiments, it is the ability to
reduce tumor size in animals. In yet other embodiments, the
activity is the ability to reduce symptoms of a disease
characterized by aberrant cell growth (e.g., cancer). Activity may
be measured using any suitable method including, but not limited
to, commercially available cell viability assays, measurement of
tumor size, and commercially available cell proliferation assays.
In some preferred embodiments, the variant has one or a limited
number of amino acid substitutions (e.g., conservative or
non-conservative substitutions), additions, or deletions (e.g.,
truncations) compared to wild type enzyme.
[0031] As used herein, the term "variant of human ribonuclease 1
retaining protein folding properties" refers to a variant of human
ribonuclease 1 (e.g., SEQ ID NO:1) that exhibits similar protein
folding properties as wild type human ribonuclease 1. Protein
folding properties include speed of protein folding and folding of
proper structure (folding that substantially retains the activity
of wild-type ribonuclease 1). In preferred embodiments variants
fold with at least 5%, at least 25%, at least 50%, at least 60%, at
least 75%, at least 85%, at least 95%, or at least 99% or more of
the speed of the wild type protein. Assays for protein folding are
well known in the art and include, but are not limited to,
spectroscopic and enzymatic (e.g. RNA degradation) assays and HPLC.
In some preferred embodiments, the variant has one or a limited
number of amino acid substitutions (e.g., conservative or
non-conservative substitutions), additions, or deletions (e.g.,
truncations) compared to wild type enzyme.
[0032] As used herein, the term "variant of human ribonuclease 1
having similar immunogenicity properties" refers to a variant of
human ribonuclease 1 (e.g., SEQ ID NO:1) that, in some embodiments,
exhibits substantially the same or better immunogenicity properties
as wild type human ribonuclease 1. Immunogenicity properties
include toxicity and undesirable immune responses (e.g., cytotoxic
immune response) in animals. In preferred embodiments, variants
exhibit less than 100%, preferably less than 90%, even more
preferably less than 80%, and still more preferably less than 70%
of the toxicity or undesirable immune response of wild type human
ribonuclease 1. The level of toxicity or immunogenicity can be
determined using any suitable method including, but not limited to,
commercially available assays for toxicity and immune response
(e.g., measurement of cytokines or T-cell response). In some
preferred embodiments, the variant has one or a limited number of
amino acid substitutions (e.g., conservative or non-conservative
substitutions), additions, or deletions (e.g., truncations)
compared to wild type enzyme.
[0033] As used herein, the term "interest site" when used in
reference to a ribonuclease refers to a region, subregion, and/or
amino acid residue within the ribonuclease (e.g., human
ribonuclease) that is modified or targeted for modification (e.g.,
for deletion, substitution or other type of mutation to create a
ribonuclease variant). Accordingly, an "interest site" may be
characterized as a "high interest site," a "medium interest site,"
or a "low interest site" based on characteristics of the
ribonuclease described herein (e.g., biologic activity (e.g.,
ribonucleolytic activity, cancer cell killing activity,
oligomerization capacity, etc.)) desired to be retained within the
ribonuclease after modification of the same (e.g., for deletion,
substitution or other type of mutation to create a ribonuclease
variant, etc.). For example, sites that may be of interest are
depicted in FIG. 15. The level of interest in modification of the
residues in the ribonucleases is indicated by the use of the
following symbols: low interest site ("-"), medium interest site
("0"), and high interest site ("+"). In addition, secondary
structure is noted: where "a" or "a#"=alpha helix; "b" or "b#"=beta
sheet. Contemplated sites of the ribonuclease that bind to
substrate RNA are also labeled: "B1" and "B2"=substrate (base)
binding site, "P1"=main active site, and "P2" and "P-1"=substrate
(phosphate) binding sites. Cysteine residues involved in a
disulfide bond are labeled by "disulf." Contact points that have
been identified for the ribonuclease inhibitor are labeled with
"RI". For angiogenin, the putative receptor binding site is labeled
as "Rec."
[0034] The term "heterologous nucleic acid sequence" or
"heterologous gene" are used interchangeably to refer to a
nucleotide sequence which is ligated to a nucleic acid sequence to
which it is not ligated in nature, or to which it is ligated at a
different location in nature. Heterologous DNA is not endogenous to
the cell into which it is introduced, but has been obtained from
another cell. Generally, although not necessarily, such
heterologous DNA encodes RNA and proteins that are not normally
produced by the cell into which it is expressed. Examples of
heterologous DNA include reporter genes, transcriptional and
translational regulatory sequences, selectable marker proteins
(e.g., proteins which confer drug resistance or therapeutic
benefits), etc.
[0035] As used herein, the term "immunoglobulin" or "antibody"
refer to proteins that bind a specific antigen. Immunoglobulins
include, but are not limited to, polyclonal, monoclonal, chimeric,
and humanized antibodies, Fab fragments, F(ab').sub.2 fragments,
and includes immunoglobulins of the following classes: IgG, IgA,
IgM, IgD, IgE, and secreted immunoglobulins (sIg). Immunoglobulins
generally comprise two identical heavy chains and two light chains.
However, the terms "antibody" and "immunoglobulin" also encompass
single chain antibodies and two chain antibodies.
[0036] As used herein, the term "antigen binding protein" refers to
proteins that bind to a specific antigen. "Antigen binding
proteins" include, but are not limited to, immunoglobulins,
including polyclonal, monoclonal, chimeric, and humanized
antibodies; Fab fragments, F(ab').sub.2 fragments, and Fab
expression libraries; and single chain antibodies.
[0037] The term "epitope" as used herein refers to that portion of
an antigen that makes contact with a particular immunoglobulin.
[0038] When a protein or fragment of a protein is used to immunize
a host animal, numerous regions of the protein may induce the
production of antibodies which bind specifically to a given region
or three-dimensional structure on the protein; these regions or
structures are referred to as "antigenic determinants". An
antigenic determinant may compete with the intact antigen (i.e.,
the "immunogen" used to elicit the immune response) for binding to
an antibody.
[0039] The terms "specific binding" or "specifically binding" when
used in reference to the interaction of an antibody and a protein
or peptide means that the interaction is dependent upon the
presence of a particular structure (i.e., the antigenic determinant
or epitope) on the protein; in other words the antibody is
recognizing and binding to a specific protein structure rather than
to proteins in general. For example, if an antibody is specific for
epitope "A," the presence of a protein containing epitope A (or
free, unlabelled A) in a reaction containing labeled "A" and the
antibody will reduce the amount of labeled A bound to the
antibody.
[0040] As used herein, the terms "non-specific binding" and
"background binding" when used in reference to the interaction of
an antibody and a protein or peptide refer to an interaction that
is not dependent on the presence of a particular structure (i.e.,
the antibody is binding to proteins in general rather that a
particular structure such as an epitope).
[0041] As used herein, the term "subject" refers to any animal
(e.g., a mammal), including, but not limited to, humans, non-human
primates, rodents, and the like, which is to be the recipient of a
particular treatment. Typically, the terms "subject" and "patient"
are used interchangeably herein in reference to a human
subject.
[0042] As used herein, the term "subject suspected of having
cancer" refers to a subject that presents one or more symptoms
indicative of a cancer (e.g., a noticeable lump or mass) or is
being screened for a cancer (e.g., during a routine physical). A
subject suspected of having cancer may also have one or more risk
factors. A subject suspected of having cancer has generally not
been tested for cancer. However, a "subject suspected of having
cancer" encompasses an individual who has received a preliminary
diagnosis (e.g., a CT scan showing a mass) but for whom a
confirmatory test (e.g., biopsy and/or histology) has not been done
or for whom the stage of cancer is not known. The term further
includes subjects who once had cancer (e.g., an individual in
remission). A "subject suspected of having cancer" is sometimes
diagnosed with cancer and is sometimes found to not have
cancer.
[0043] As used herein, the term "subject diagnosed with a cancer"
refers to a subject who has been tested and found to have cancerous
cells. The cancer may be diagnosed using any suitable method,
including but not limited to, biopsy, x-ray, blood test, and the
diagnostic methods of the present invention. A "preliminary
diagnosis" is one based only on visual (e.g., CT scan or the
presence of a lump) and antigen tests.
[0044] As used herein, the term "subject at risk for cancer" refers
to a subject with one or more risk factors for developing a
specific cancer. Risk factors include, but are not limited to,
gender, age, genetic predisposition, environmental exposure, and
previous incidents of cancer, preexisting non-cancer diseases, and
lifestyle.
[0045] As used herein, the term "non-human animals" refers to all
non-human animals including, but are not limited to, vertebrates
such as rodents, non-human primates, ovines, bovines, ruminants,
lagomorphs, porcines, caprines, equines, canines, felines, ayes,
etc.
[0046] As used herein, the term "gene transfer system" refers to
any means of delivering a composition comprising a nucleic acid
sequence to a cell or tissue. For example, gene transfer systems
include, but are not limited to, vectors (e.g., retroviral,
adenoviral, adeno-associated viral, and other nucleic acid-based
delivery systems), microinjection of naked nucleic acid,
polymer-based delivery systems (e.g., liposome-based and metallic
particle-based systems), biolistic injection, and the like. As used
herein, the term "viral gene transfer system" refers to gene
transfer systems comprising viral elements (e.g., intact viruses,
modified viruses and viral components such as nucleic acids or
proteins) to facilitate delivery of the sample to a desired cell or
tissue. As used herein, the term "adenovirus gene transfer system"
refers to gene transfer systems comprising intact or altered
viruses belonging to the family Adenoviridae.
[0047] "Amino acid sequence" and terms such as "polypeptide" or
"protein" are not meant to limit the amino acid sequence to the
complete, native amino acid sequence associated with the recited
protein molecule.
[0048] The terms "test compound" and "candidate compound" refer to
any chemical or biological entity, pharmaceutical, drug, and the
like that is a candidate for use to treat or prevent a disease,
illness, sickness, or disorder of bodily function (e.g., cancer).
Test compounds comprise both known and potential therapeutic
compounds. A test compound can be determined to be therapeutic by
screening using the screening methods of the present invention.
[0049] As used herein, the term "sample" is used in its broadest
sense. In one sense, it is meant to include a specimen or culture
obtained from any source, as well as biological and environmental
samples. Biological samples may be obtained from animals (including
humans) and encompass fluids, solids, tissues, and gases.
Biological samples include blood products, such as plasma, serum
and the like. Environmental samples include environmental material
such as surface matter, soil, water and industrial samples. Such
examples are not however to be construed as limiting the sample
types applicable to the present invention.
[0050] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence that comprises coding sequences necessary for the
production of a polypeptide or precursor (e.g., ribonucleases or
ribonuclease conjugates of the present invention). The polypeptide
can be encoded by a full length coding sequence or by any portion
of the coding sequence so long as the desired activity or
functional properties (e.g., enzymatic activity, etc.) of the
full-length or fragment are retained. The term also encompasses the
coding region of a structural gene and the including sequences
located adjacent to the coding region on both the 5' and 3' ends
for a distance of about 1 kb on either end such that the gene
corresponds to the length of the full-length mRNA. The sequences
that are located 5' of the coding region and which are present on
the mRNA are referred to as 5' untranslated sequences. The
sequences that are located 3' or downstream of the coding region
and that are present on the mRNA are referred to as 3' untranslated
sequences. The term "gene" encompasses both cDNA and genomic forms
of a gene. A genomic form or clone of a gene contains the coding
region interrupted with non-coding sequences termed "introns" or
"intervening regions" or "intervening sequences." Introns are
segments of a gene that are transcribed into nuclear RNA (hnRNA);
introns may contain regulatory elements such as enhancers. Introns
are removed or "spliced out" from the nuclear or primary
transcript; introns therefore are absent in the messenger RNA
(mRNA) transcript. The mRNA functions during translation to specify
the sequence or order of amino acids in a nascent polypeptide.
[0051] The term "wild-type" refers to a gene or gene product that
has the characteristics of that gene or gene product when isolated
from a naturally occurring source. A wild-type gene is that which
is most frequently observed in a population and is thus arbitrarily
designed the "normal" or "wild-type" form of the gene. Wild-type
protein may be produced by synthetic methods. Wild-type proteins
include, but are not limited to, forms that include
post-translational modifications such as glycosylation as well as
any preprocessed forms. In contrast, the terms "modified",
"mutant", and "variant" refer to a gene or gene product that
displays modifications in sequence when compared to the wild-type
gene or gene product. It is noted that naturally-occurring mutants
can be isolated; these are identified by the fact that they have
altered nucleic acid or polypeptide sequence when compared to the
wild-type gene or gene product. This is in contrast to synthetic
mutants that are changes made in a sequence through human (or
machine) intervention.
[0052] The term "fragment" as used herein refers to a polypeptide
that has an amino-terminal and/or carboxy-terminal deletion as
compared to the native protein, but where the remaining amino acid
sequence is identical to the corresponding positions in the amino
acid sequence deduced from a full-length cDNA sequence. Fragments
typically are at least 4 amino acids long, preferably at least 20
amino acids long, usually at least 50 amino acids long or longer,
and span the portion of the polypeptide required for intermolecular
binding of the compositions with its various ligands and/or
substrates. In some embodiments, fragments posses an activity of
the native protein.
[0053] As used herein, the term "purified" or "to purify" refers to
the removal of impurities and contaminants from a sample. For
example, antibodies are purified by removal of non-immunoglobulin
proteins; they are also purified by the removal of immunoglobulin
that does not bind an intended target molecule. The removal of
non-immunoglobulin proteins and/or the removal of immunoglobulins
that do not bind an intended target molecule results in an increase
in the percent of target-reactive immunoglobulins in the sample. In
another example, recombinant polypeptides are expressed in host
cells and the polypeptides are purified by the removal of host cell
proteins; the percent of recombinant polypeptides is thereby
increased in the sample.
[0054] The term "expression vector" as used herein refers to a
recombinant DNA molecule containing a desired coding sequence and
appropriate nucleic acid sequences necessary for the expression of
the operably linked coding sequence in a particular host organism.
Nucleic acid sequences necessary for expression in prokaryotes
usually include a promoter, an operator (optional), and a
ribosome-binding site, often along with other sequences. Eukaryotic
cells are known to utilize promoters, enhancers, and termination
and polyadenylation signals.
[0055] As used herein, the term "host cell" refers to any
eukaryotic or prokaryotic cell (e.g., bacterial cells such as E.
coli, yeast cells, mammalian cells, avian cells, amphibian cells,
plant cells, fish cells, and insect cells), whether located in
vitro or in vivo. For example, host cells may be located in a
transgenic animal.
DETAILED DESCRIPTION OF THE INVENTION
[0056] In some embodiments, the present invention provides human
ribonuclease (RNase 1) proteins that are used to treat or cure
diseases, particularly cancer and viral infections. The
compositions also find use in diagnostic applications (e.g.,
associated with drug screening or cancer characterization) and
research applications. RNase 1 can be used as stand alone reagent
or incorporated into general or specific delivery systems such as
polymers, dendrimers, liposomes, polymeric nanoparticles, or block
copolymer micelles. RNase 1 may also be co-administered with other
drugs and compounds. A variety of human RNases are now known (e.g.,
Zhang et al., Nucleic Acids Research, 31:602 (2003); Zhang et al.,
Nucleic Acids Research, 30:1169 (2002), herein incorporated by
reference in their entireties).
[0057] Previous experiments (See Cancer Res., 64:4870 [2004]) did
not demonstrate a reduction in tumor volume with administration of
human RNase 1 (human pancreatic RNase). In contrast, the present
invention demonstrates that human RNase 1 and variants having
similar activities and properties are effective in reducing tumor
volume in animals.
[0058] In some embodiments, it is contemplated that RNase 1 is able
to make chemotherapy or radiation resistant cells susceptible to
standard or lower levels of treatment so that lower doses are
effective and side effects reduced. In addition, RNase 1 is
contemplated to provide benefit when used in combination with
radiotherapy or other interventions, including but not limited to
antibodies, small molecules, or gene therapy.
[0059] In some embodiments, the compositions and methods of the
present invention are used to treat diseased cells, tissues,
organs, or pathological conditions and/or disease states in a
subject organism (e.g., a mammalian subject including, but not
limited to, humans and veterinary animals), or in in vitro and/or
ex vivo cells, tissues, and organs. In this regard, various
diseases and pathologies are amenable to treatment or prophylaxis
using the present methods and compositions. A non-limiting
exemplary list of these diseases and conditions includes, but is
not limited to, breast cancer, prostate cancer, lymphoma, skin
cancer, pancreatic cancer, colon cancer, melanoma, malignant
melanoma, ovarian cancer, brain cancer, primary brain carcinoma,
head-neck cancer, glioma, glioblastoma, liver cancer, bladder
cancer, non-small cell lung cancer, head or neck carcinoma, breast
carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung
carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma,
bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon
carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid
carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal
carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal
cortex carcinoma, malignant pancreatic insulinoma, malignant
carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant
hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic
leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia,
chronic myelogenous leukemia, chronic granulocytic leukemia, acute
granulocytic leukemia, hairy cell leukemia, neuroblastoma,
rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential
thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma,
soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia,
and retinoblastoma, and the like, T and B cell mediated autoimmune
diseases; inflammatory diseases; infections; hyperproliferative
diseases; AIDS; degenerative conditions, vascular diseases, and the
like. In some embodiments, the cancer cells being treated are
metastatic. In other embodiments, the RNases of the present
invention target cancer stem cells.
[0060] In some embodiments, the present invention contemplates the
use of wild type human RNase 1. Wild type human RNase 1 include all
forms of the enzyme including, but not limited to, polypeptides
having the amino acid sequence of wild type RNase 1 encoded by
nucleic acids of different sequences, as well as glycosylated,
lipidated, and other modifications including covalent and
non-covalent modifications.
[0061] The present invention further contemplates variants of wild
type human RNase 1 (e.g., SEQ ID NO:1). A modified peptide can be
produced in which the nucleotide sequence encoding the polypeptide
has been altered, such as by substitution, deletion, addition
(e.g., insertion), modifications (e.g., addition of additional
molecules such as polyethylene glycol (PEG)) or post translational
modifications (e.g., glycosylation). In some embodiments,
insertions are insertions of large peptide segments (e.g.,
targeting proteins). Insertions have been made into proteins to
incorporate additional functionality, such as recognition sequences
(e.g., Characterization of an Adenovirus Vector Containing a
Heterologous Peptide Epitope in the HI Loop of the Fiber Knob, J.
Virology, 1998, 72, 1844). Additionally, the insertion may replace
certain amino acids within the protein. The amino acids to be
replaced may be selected to be similar in nature to sequences where
insertions of amino acids can be placed, including unstructured or
loop regions (e.g., Transplantation of a 17-amino acid a-helical
DNA-binding domain into an antibody molecule confers
sequence-dependent DNA recognition, PNAS, 1995, 92, 5214). The
insertion may be another copy of the ribonuclease protein, either
inserted at one of the termini or into the length of the first
sequence. In other embodiments, variant RNase 1 of the present
invention comprises a modified amino acid. In particularly
preferred embodiments, these modifications do not significantly
reduce the enzymatic activity or other desired activity or property
of the modified human RNase 1. In other words, construct "X" can be
evaluated in order to determine whether it is a member of the genus
of modified or variant RNase 1 of the present invention as defined
functionally.
[0062] Preferred variants maintain at least 1%, at least 5%, at
least 15%, at least 30%, at least 50%, preferably at least 60%,
even more preferably at least 75%, still more preferably at least
85%, yet more preferably at least 95% and most preferably at least
99% of the activity of wild type human ribonuclease 1. In some
embodiments, the activity is enzymatic (e.g., degradation of RNA)
activity. In other embodiments, activity is killing of cells (e.g.,
cancer cells). In other embodiments, preferred variants have
similar properties as the wild type human RNase 1. For example,
exemplary preferred properties include, but are not limited to, the
speed of protein folding and ease of manufacturability, and low
immunogenicity or toxicity in animals or lack of weight loss in
animals administered the RNase 1. It should be noted that, in some
embodiments, one of the activities or properties is rendered less
desirable, but another is rendered desirable, such that, overall,
the enzyme is useful (e.g., increased toxicity is traded for
greater efficacy or vice versa).
[0063] In preferred embodiments, expression of RNase 1 or variants
(e.g., recombinant expression) is greater than 50, and preferably
greater than 75 mg/L. RNase 1 may be expressed in any suitable
expression system. Bacterial and eukaryotic expression systems are
available for production of recombinant proteins. In preferred
embodiments, proteins are expressed at high levels to aid in
purification and to obtain large quantities of protein for animal
studies, clinical studies, or therapeutic manufacture and sale. In
some embodiments, heterologous RNase 1 is expressed in vivo (e.g.,
via transfected nucleic acid constructs provided by transplantation
of engineered ex vivo cells, gene therapy, generation of transgenic
animals, etc.).
[0064] Preferred RNase 1 and variants thereof of the present
invention exhibit tumor size reduction activity in animals. The
present invention is not limited to a particular mechanism. Indeed,
an understanding of the mechanism is not necessary to practice the
present invention. Nonetheless, it is contemplated that one or more
of several factors contribute to the activity of RNase 1 enzymes
including, but not limited to, charge of the protein, pore forming
ability, angiogenic effects, proper dosing considerations, etc.
[0065] Moreover, as described above, variant forms of human RNase 1
are also contemplated as being equivalent to those peptides and DNA
molecules that are set forth in more detail herein. For example, it
is contemplated that isolated replacement of a leucine with an
isoleucine or valine, an aspartate with a glutamate, a threonine
with a serine, or a similar replacement of an amino acid with a
structurally related amino acid (i.e., conservative mutations) will
not have a major effect on the biological activity of the resulting
molecule. Accordingly, some embodiments of the present invention
provide variants of human RNase 1 disclosed herein containing
conservative replacements. Conservative replacements are those that
take place within a family of amino acids that are related in their
side chains. Genetically encoded amino acids can be divided into
four families: (1) acidic (aspartate, glutamate); (2) basic
(lysine, arginine, histidine); (3) nonpolar (alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan); and (4) uncharged polar (glycine, asparagine,
glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine,
tryptophan, and tyrosine are sometimes classified jointly as
aromatic amino acids. In similar fashion, the amino acid repertoire
can be grouped as (1) acidic (aspartate, glutamate); (2) basic
(lysine, arginine, histidine), (3) aliphatic (glycine, alanine,
valine, leucine, isoleucine, serine, threonine), with serine and
threonine optionally be grouped separately as aliphatic-hydroxyl;
(4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide
(asparagine, glutamine); and (6) sulfur-containing (cysteine and
methionine) (e.g., Stryer ed., Biochemistry, pg. 17-21, 2nd ed, WH
Freeman and Co., 1981). Whether a change in the amino acid sequence
of a peptide results in a functional polypeptide can be readily
determined by assessing the ability of the variant peptide to
function in a fashion similar to the wild-type protein. Peptides
having more than one replacement can readily be tested in the same
manner.
[0066] More rarely, a variant includes "nonconservative" changes
(e.g., replacement of a glycine with a tryptophan). Analogous minor
variations can also include amino acid deletions or insertions, or
both. Guidance in determining which amino acid residues can be
substituted, inserted, or deleted without abolishing biological
activity can be found using computer programs (including, but not
limited to, FADE (Mitchell et al., (2004). Molec. Simul. 30,
97-106); MAPS (Ban et al., Proceedings of the 8th Annual
International Conference on Research in Computational Molecular
Biology, 2004, 205-212), SYBYL (Tripos, Inc, St. Louis, Mo.); and
PyMOL (available on the Internet web sit of sourceforge)).
[0067] Crystal structures of RNase 1 are described, for example in
Pous et al. (Acta Crystallogr D Biol Crystallogr. 2001; 57,
498-505) and Pous et al. (J Mol Biol. 2000; 303, 49-60) and serve
as the basis for selection of changes. In addition, crystal
structures are available for other human pancreatic ribonucleases,
including eosinophil derived neurotoxin (EDN, RNase 2; Swaminathan
et al, Biochemistry, 2002, 41, 3341-3352, Mosimann et al J. Mol.
Biol., 1996, 260, 540-552; Iyer et al J Mol Biol, 2005, 347,
637-655), eosinophil cationic protein (ECP, RNase 3; Mohan et al
Biochemistry 2002; 41, 12100-12106; Boix et al Biochemistry, 1999,
38, 16794-16801; Mallorqui-Fernandez et al J. Mol. Biol, 2000, 300,
1297-1307), RNase 4 (Terzyan et al, 1999, 285, 205-214) and
angiogenin (RNase 5; Leonidas et al J. Mol. Biol. 1999, 285,
1209-1233; Leonidas et al Protein Sci., 2001, 10, 1669-1676;
Papageorgiou et al EMBO J., 1997, 16, 5162-5177; Shapiro et al J.
Mol. Biol., 2000, 302, 497-519).
[0068] The amino acid sequences for additional ribonuclease gene
family members have been determined, including RNase 6 (Rosenberg,
et al, Nucleic Acids Research, 1996, 24, 3507-3513), RNase 7
(Harder et al, J. Biol. Chem., 2002, 277, 46779-46784), and RNase 8
(Zhang et al, Nucleic Acids Research, 2002, 30, 1169-1175).
[0069] Variants may be produced by methods such as directed
evolution or other techniques for producing combinatorial libraries
of variants, described in more detail below. In still other
embodiments of the present invention, the nucleotide sequences of
the present invention may be engineered in order to alter a human
RNase 1 coding sequence including, but not limited to, alterations
that modify the cloning, processing, localization, secretion,
and/or expression of the gene product. For example, mutations may
be introduced using techniques that are well known in the art
(e.g., site-directed mutagenesis to insert new restriction sites,
alter glycosylation patterns, or change codon preference, etc.). In
some embodiments, changes are made in the nucleic acid sequence
encoding a polypeptide of the present invention in order to
optimize codon usage to the organism that the gene is expressed
in.
[0070] Exemplary variants are described below, including, but not
limited to, substitutions, truncations, chimeras, etc. The present
invention is not limited to these particular variants. Both
variants in the active site and substrate-binding region and away
from the active site are contemplated to be within the scope of the
present invention. Variants may be selected based on, for example,
experimental data, computer modeling, and by rational design by
comparison to other ribonucleases. Activities may be tested using
assay to select the variants with the desired properties (see e.g.,
Raines et al., J. Biol. Chem, 273, 34134 (1998); Fisher et al.,
Biochemistry 37:12121 (1998); Guar et al., J. Biol. Chem.,
276:24978 (2001); Bosch, et al., Biochemistry, 43:2167 (2004); Lin,
J. Biol. Chem., 245:6726 (1970); Bal et al, Eur. J. Biochem.,
245:465 (1997); Guar et al., Mol. Cell. Biochem., 275:95 (2005);
Benito et al., Protein Eng., 15:887 (2002); Ribo et al., Biol.
Chem. Hoppe-seyler, 375:357 (1994); DiGaetano et al., Biochem. J.,
358:241 (2001); Trautwein et al., FEBS Lett., 281:277 (1991);
Curran et al., Biochemistry 32:2307 (1993); Sorrentino et al.,
Biochemistry 42:10182 (2003); herein incorporated by reference in
their entireties).
[0071] Exemplary amino acid locations for modification in the
production of variants are provided in FIG. 15. One or more sites
may be modified, as desired. Amino acid residues for human
ribonucleases are provided in FIG. 5. The present invention
provides a ranking of the utility for modification of each amino
acid (e.g., as represented by interest in modifying (e.g., so as to
result in a functional ribonuclease (e.g., comprising a desired
property (e.g., cancer cell killing and/or ribonucleolytic
activity). The amino acids are labeled in FIG. 15 as follows:
[0072] low interest (-),
[0073] medium interest (0), or
[0074] high interest (+).
[0075] It will be appreciated that one or more modification sites
may be used. Preferably, the selected sites are high interest
sites. However, one or more medium interest or low interest sites
may be used as desired and appropriate for the intended
application. It should be noted that, in some embodiments, human
RNase is produced (e.g., in vitro, in vivo or ex vivo) in such a
way that a methionine (e.g., that is not part of wild type human
RNase) is incorporated as the first amino acid of the protein
(e.g., via the methods used to produce the protein (e.g.,
recombinant human ribonuclease (e.g., produced in E. coli))). Thus,
in some embodiments, the numbering of amino acid residues depicted
in FIG. 15 may be off by a numerical value of one (e.g., if a
methionine is incorporated into the protein, then the numbering of
the amino acid residues of the human RNases shown in FIG. 15 is off
by 1 (i.e., because a methionine is incorporated in position 1, the
numbering of the amino acids depicted in FIG. 15 will be short by
one, e.g., the residue number 10 would actually be residue number
11 because of the methionine incorporated at position 1)).
Similarly, the positions depicted in FIG. 15 may also be applied to
corresponding numerical positions other related ribonucleases.
[0076] In some embodiments, the desired residues for modification
(e.g., deletion, mutation, etc.) in human ribonucleases (e.g.,
hRNase 1) are selected to avoid disruption of the tertiary
structure and/or stability of the ribonuclease. In some
embodiments, these residues are on the surface of the protein
(e.g., residues generally exposed to solvent (e.g., water or
buffer)). For example, in some embodiments, the types of residues
that are modified include, but are not limited to, amino acids that
appear disordered in crystal structures, residues that contact the
ribonuclease inhibitor protein, and amino acids not involved in
tertiary structures (e.g., alpha helices and beta sheets), amino
acids in loop regions between structures (e.g. alpha helices and
beta sheets) as well as amino acids towards the end of the protein
(the N- and C-termini). In some embodiments, additional amino acid
residues are added to either the N- or C-terminus (e.g., to
generate a RNase analogue and/or for conjugation of a water-soluble
polymer).
[0077] Experiments conducted during the development of the present
invention involved the formation of multiple variants of the human
RNase 1. Such variants include changes of residues that have been
described as binding sites for single and/or double stranded RNA.
The enzymatic activity of these variants is provided below. Despite
the range in enzymatic activity displayed by each RNase 1 variant,
they were all active in xenograft models of non-small cell lung
cancer. Thus, in addition to change made outside of the region
attributed to enzyme activity, change may also be made in the
region.
TABLE-US-00001 TABLE 1 Enzymatic Activity RNase (kcat/Km; M-1s-1)
Wild type human RNase 1 2.97 .times. 10.sup.7 L86E/N88R/G89D/R91D
RNase 1 1.17 .times. 10.sup.7 G38R/R39D/L86E/N88R/G89D/R91D RNase 1
4.90 .times. 10.sup.6 R4C/G38R/R39D/L86E/N88R/G89D/R91D/V118C 1.28
.times. 10.sup.5 RNase 1 R4C/K7A/G38R/R39D/L86E/N88R/G89D/R91D/
1.69 10.sup.4 V118C RNase 1
[0078] The G38R/R39D/L86E/N88R/G89D/R91D RNase 1 and
L86E/N88R/G89D/R91D RNase 1 displayed tumor growth inhibition in
this model. Results are shown in FIG. 1 and Table 2.
TABLE-US-00002 TABLE 2 (% TGI) Starting Final (Final - (Final -
volume volume start) start)/Control % TGI Vehicle 73 975 902 1 0
L86E/N88R/G89D/ 84 624 540 0.60 40 R91D RNase 1 G38R/R39D/L86E/ 76
723 647 0.72 28 N88R/G89D/ R91D RNase 1 cisplatin 79 369 290 0.32
68
[0079] The R4C/G38R/R39D/L86E/N88R/G89D/R91DN118C RNase 1 variant
impacted tumor growth in the xenograft model. Results are shown in
FIG. 2 and Table 3.
TABLE-US-00003 TABLE 3 (% TGI) (Final - Starting Final (Final -
start)/ volume volume start) Control % TGI Vehicle 118 2071 1953 1
0 R4C/G38R/R39D/ 108 1130 1022 0.52 48 L86E/N88R/G89D/ R91D/V118C
RNase 1
[0080] Despite having significantly lower enzymatic activity than
the wild type RNase 1, the R4C/K7A/G38R/R39D/L
86E/N88R/G89D/R91D/V118C RNase 1 variant demonstrated measurable
tumor growth inhibition. Results are shown in FIG. 3 and Table
4.
TABLE-US-00004 TABLE 4 (% TGI) Starting Final (Final - (Final -
start)/ volume volume start) Control % TGI Vehicle 75 1407 1332 1 0
R4C/K7A/G38R/ 74 552 478 0.36 36 R39D/L86E/ N88R/G89D/ R91D/V118C
RNase 1
[0081] Thus, the present invention provides a broad array of
ribonucleases that find use in the methods and compositions of the
invention. Indeed, the examples above provide variants in the
substrate and active sites that might have been expected to
interfere with desired activities and properties of the protein.
However, empirical data demonstrated that these variants
substantially retain desired activities and properties of wild-type
RNase 1.
[0082] In some embodiments, RNase 1 or a variant thereof is
delivered to a target cell using complementation. For example, in
some embodiments, two or more fragments of RNase 1 are delivered
separately to a cell. The fragments re-associate to form a
functional enzyme. In some embodiments, two protein fragments are
delivered. In other embodiments, vectors comprising nucleic acids
encoding fragments of RNase 1 are introduced into a cell or
organism separately.
[0083] Suitable fragments for delivery by complementation may be
determined by screening fragments (e.g., in a cell culture assay)
for activity. Preferred fragments are those that rapidly
re-associate to form a functional enzyme. Enzyme activity can be
determined using any suitable method, including, but not limited
to, those disclosed herein.
[0084] In some embodiments, the present invention utilizes
digestion of RNases to produce S-peptide and S-protein (See, e.g.,
Hamachi et al., Bioorg Med Chem Lett 9, 1215-1218 (1999); Goldberg
and Baldwin, Proc Natl Acad Sci, 96, 2019-2024 (1999); Asai et al.,
J Immun Meth, 299, 63-76 (2005); Backer et al., J Cont Release, 89,
499-511 (2003); Backer et al., Bioconj Chem, 15, 1021-1029 (2004)).
For example, digestion of bovine RNase A by subtilisin results
primarily in two fragments due to cleavage between A1a20 and Ser21.
The shorter fragment (amino acids 1-20) is referred to as
S-peptide, whereas the longer fragment (amino acids 20-124) is
referred to as S-protein. The two fragments bind tightly at neutral
pH and are sometime referred to as RNase S or RNase S'. RNase S is
an active ribonuclease. The S-peptide-S-protein interaction has
been used for affinity purification as well as in tertiary docking
systems to target imaging agents or drugs. Thus, in some
embodiments, the present invention provides S-peptide-S-protein for
human ribonucleases.
[0085] In some embodiments, the present invention provides a
composition comprising a plurality of human RNases (e.g., hRNase1).
In some embodiments, the plurality of RNases comprise monomers,
dimers, trimers, and/or higher order complexes (i.e., oligomers) of
hRNases.
Combination Therapies
[0086] In some preferred embodiments, the RNase 1 or RNase 1
variants of the present invention are co-administered with other
medical interventions, either simultaneously or sequentially. For
example, for cancer therapy, any oncolytic agent that is routinely
used in a cancer therapy may be co-administered with the
compositions and methods of the present invention. For example, the
U.S. Food and Drug Administration maintains a formulary of
oncolytic agents approved for use in the United States.
International counterpart agencies to the U.S.F.D.A. maintain
similar formularies. Table 5 provides a list of exemplary
antineoplastic agents approved for use in the U.S. Those skilled in
the art will appreciate that the "product labels" required on all
U.S. approved chemotherapeutics describe approved indications,
dosing information, toxicity data, and the like, for the exemplary
agents. It is contemplated, that in some cases, co-administration
with the compositions of the present invention permits lower doses
of such compounds, thereby reducing toxicity.
TABLE-US-00005 TABLE 5 Aldesleukin PROLEUKIN Chiron Corp.,
(des-alanyl-1, serine-125 human Emeryville, CA interleukin-2)
Alemtuzumab CAMPATH Millennium and (IgG1.kappa. anti CD52 antibody)
ILEX Partners, LP, Cambridge, MA Alitretinoin PANRETIN Ligand
(9-cis-retinoic acid) Pharmaceuticals, Inc., San Diego CA
Allopurinol ZYLOPRIM GlaxoSmithKline, (1,5-dihydro-4H-pyrazolo[3,4-
Research Triangle d]pyrimidin-4-one monosodium salt) Park, NC
Altretamine HEXALEN US Bioscience, West
(N,N,N',N',N'',N'',-hexamethyl-1,3,5- Conshohocken, PA
triazine-2,4,6-triamine) Amifostine ETHYOL US Bioscience
(ethanethiol, 2-[(3-aminopropyl)amino]-, dihydrogen phosphate
(ester)) Anastrozole ARIMIDEX AstraZeneca
(1,3-Benzenediacetonitrile, a,a,a',a'- Pharmaceuticals, LP,
tetramethyl-5-(1H-1,2,4-triazol-1- Wilmington, DE ylmethyl))
Arsenic trioxide TRISENOX Cell Therapeutic, Inc., Seattle, WA
Asparaginase ELSPAR Merck & Co., Inc., (L-asparagine
amidohydrolase, type EC-2) Whitehouse Station, NJ BCG Live TICE BCG
Organon Teknika, (lyophilized preparation of an attenuated Corp.,
Durham, NC strain of Mycobacterium bovis (Bacillus Calmette-Gukin
[BCG], substrain Montreal) Bevacizumab AVASTIN Genentech bexarotene
capsules TARGRETIN Ligand (4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-
Pharmaceuticals pentamethyl-2-napthalenyl) ethenyl] benzoic acid)
bexarotene gel TARGRETIN Ligand Pharmaceuticals Bleomycin BLENOXANE
Bristol-Myers Squibb (cytotoxic glycopeptide antibiotics Co., NY,
NY produced by Streptomyces verticillus; bleomycin A.sub.2 and
bleomycin B.sub.2) Capecitabine XELODA Roche (5'-deoxy-5-fluoro-N-
[(pentyloxy)carbonyl]-cytidine) Carboplatin PARAPLATIN
Bristol-Myers Squibb (platinum, diammine [1,1-
cyclobutanedicarboxylato(2-)-0,0']-,(SP-4- 2)) Carmustine BCNU,
BICNU Bristol-Myers Squibb (1,3-bis(2-chloroethyl)-1-nitrosourea)
Carmustine with Polifeprosan 20 Implant GLIADEL WAFER Guilford
Pharmaceuticals, Inc., Baltimore, MD Celecoxib CELEBREX Searle (as
4-[5-(4-methylphenyl)-3- Pharmaceuticals,
(trifluoromethyl)-1H-pyrazol-1-yl] England benzenesulfonamide)
Cetuximab ERBITUX ImClone/BMS Chlorambucil LEUKERAN GlaxoSmithKline
(4-[bis(2chlorethyl)amino]benzenebutanoic acid) Cisplatin PLATINOL
Bristol-Myers Squibb (PtCl.sub.2H.sub.6N.sub.2) Cladribine
LEUSTATIN, 2-CDA R.W. Johnson (2-chloro-2'-deoxy-b-D-adenosine)
Pharmaceutical Research Institute, Raritan, NJ Cyclophosphamide
CYTOXAN, NEOSAR Bristol-Myers Squibb (2-[bis(2-chloroethyl)amino]
tetrahydro- 2H-13,2-oxazaphosphorine 2-oxide monohydrate)
Cytarabine CYTOSAR-U Pharmacia & Upjohn
(1-b-D-Arabinofuranosylcytosine, Company
C.sub.9H.sub.13N.sub.3O.sub.5) cytarabine liposomal DEPOCYT Skye
Pharmaceuticals, Inc., San Diego, CA Dacarbazine DTIC-DOME Bayer
AG, (5-(3,3-dimethyl-1-triazeno)-imidazole-4- Leverkusen,
carboxamide (DTIC)) Germany Dactinomycin, actinomycin D COSMEGEN
Merck (actinomycin produced by Streptomyces parvullus,
C.sub.62H.sub.86N.sub.12O.sub.16) Darbepoetin alfa ARANESP Amgen,
Inc., (recombinant peptide) Thousand Oaks, CA daunorubicin
liposomal DANUOXOME Nexstar ((8S-cis)-8-acetyl-10-[(3-amino-2,3,6-
Pharmaceuticals, Inc., trideoxy-a-L-lyxo-hexopyranosyl)oxy]-
Boulder, CO 7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-
methoxy-5,12-naphthacenedione hydrochloride) Daunorubicin HCl,
daunomycin CERUBIDINE Wyeth Ayerst, ((1S,3S)-3-Acetyl-1,2,3,4,6,11-
Madison, NJ hexahydro-3,5,12-trihydroxy-10-methoxy-
6,11-dioxo-1-naphthacenyl 3-amino-2,3,6-
trideoxy-(alpha)-L-lyxo-hexopyranoside hydrochloride) Denileukin
diftitox ONTAK Seragen, Inc., (recombinant peptide) Hopkinton, MA
Dexrazoxane ZINECARD Pharmacia & Upjohn
((S)-4,4'-(1-methyl-1,2-ethanediyl)bis-2,6- Company
piperazinedione) Docetaxel TAXOTERE Aventis
((2R,3S)-N-carboxy-3-phenylisoserine, N- Pharmaceuticals, Inc.,
tert-butyl ester, 13-ester with 5b-20-epoxy- Bridgewater, NJ
12a,4,7b,10b,13a-hexahydroxytax-11-en- 9-one 4-acetate 2-benzoate,
trihydrate) Doxorubicin HCl ADRIAMYCIN, Pharmacia & Upjohn
(8S,10S)-10-[(3-amino-2,3,6-trideoxy-a-L- RUBEX Company
lyxo-hexopyranosyl)oxy]-8-glycolyl-
7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-
methoxy-5,12-naphthacenedione hydrochloride) doxorubicin ADRIAMYCIN
PFS Pharmacia & Upjohn INTRAVENOUS Company INJECTION
doxorubicin liposomal DOXIL Sequus Pharmaceuticals, Inc., Menlo
park, CA dromostanolone propionate DROMOSTANOLONE Eli Lilly &
Company, (17b-Hydroxy-2a-methyl-5a-androstan-3- Indianapolis, IN
one propionate) Dromostanolone propionate MASTERONE Syntex, Corp.,
Palo INJECTION Alto, CA Elliott's B Solution ELLIOTT'S B Orphan
Medical, Inc SOLUTION Epirubicin ELLENCE Pharmacia & Upjohn
((8S-cis)-10-[(3-amino-2,3,6-trideoxy-a-L- Company
arabino-hexopyranosyl)oxy]-7,8,9,10-
tetrahydro-6,8,11-trihydroxy-8- (hydroxyacetyl)-1-methoxy-5,12-
naphthacenedione hydrochloride) Epoetin alfa EPOGEN Amgen, Inc
(recombinant peptide) Erlotinib (N-(3-ethynylphenyl)-6,7-bis(2-
TARCEVA Genentech/OSI methoxyethoxy)quinazolin-4-amine)
Estramustine EMCYT Pharmacia & Upjohn
(estra-1,3,5(10)-triene-3,17-diol(17(beta))-, Company
3-[bis(2-chloroethyl)carbamate] 17- (dihydrogen phosphate),
disodium salt, monohydrate, or estradiol 3-[bis(2-
chloroethyl)carbamate] 17-(dihydrogen phosphate), disodium salt,
monohydrate) Etoposide phosphate ETOPOPHOS Bristol-Myers Squibb
(4'-Demethylepipodophyllotoxin 9-[4,6-O-
(R)-ethylidene-(beta)-D-glucopyranoside], 4'-(dihydrogen
phosphate)) etoposide, VP-16 VEPESID Bristol-Myers Squibb
(4'-demethylepipodophyllotoxin 9-[4,6-0-
(R)-ethylidene-(beta)-D-glucopyranoside]) Exemestane AROMASIN
Pharmacia & Upjohn (6-methylenandrosta-1,4-diene-3,17-dione)
Company Filgrastim NEUPOGEN Amgen, Inc (r-metHuG-CSF) floxuridine
(intraarterial) FUDR Roche (2'-deoxy-5-fluorouridine) Fludarabine
FLUDARA Berlex Laboratories, (fluorinated nucleotide analog of the
Inc., Cedar Knolls, antiviral agent vidarabine, 9-b-D- NJ
arabinofuranosyladenine (ara-A)) Fluorouracil, 5-FU ADRUCIL ICN
Pharmaceuticals, (5-fluoro-2,4(1H,3H)-pyrimidinedione) Inc.,
Humacao, Puerto Rico Fulvestrant FASLODEX IPR Pharmaceuticals,
(7-alpha-[9-(4,4,5,5,5-penta Guayama, Puerto fluoropentylsulphinyl)
nonyl]estra-1,3,5- Rico (10)-triene-3,17-beta-diol) Gefitinib
(N-(3-chloro-4-fluoro-phenyl)-7- IRESSA AstraZeneca methoxy-
6-(3-morpholin-4-ylpropoxy)quinazolin-4- amine) Gemcitabine GEMZAR
Eli Lilly (2'-deoxy-2',2'-difluorocytidine monohydrochloride
(b-isomer)) Gemtuzumab Ozogamicin MYLOTARG Wyeth Ayerst (anti-CD33
hP67.6) Goserelin acetate ZOLADEX IMPLANT AstraZeneca (acetate salt
of [D- Pharmaceuticals Ser(But).sup.6,Azgly.sup.10]LHRH;
pyro-Glu-His- Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro- Azgly-NH2 acetate
[C.sub.59H.sub.84N.sub.18O.sub.14.cndot.(C.sub.2H.sub.4O.sub.2).sub.x
Hydroxyurea HYDREA Bristol-Myers Squibb Ibritumomab Tiuxetan
ZEVALIN Biogen IDEC, Inc., (immunoconjugate resulting from a
Cambridge MA thiourea covalent bond between the monoclonal antibody
Ibritumomab and the linker-chelator tiuxetan [N-[2-
bis(carboxymethyl)amino]-3-(p- isothiocyanatophenyl)-propyl]-[N-[2-
bis(carboxymethyl)amino]-2-(methyl)- ethyl]glycine) Idarubicin
IDAMYCIN Pharmacia & Upjohn (5,12-Naphthacenedione,
9-acetyl-7-[(3- Company amino-2,3,6-trideoxy-(alpha)-L-lyxo-
hexopyranosyl)oxy]-7,8,9,10-tetrahydro-
6,9,11-trihydroxyhydrochloride, (7S-cis)) Ifosfamide IFEX
Bristol-Myers Squibb (3-(2-chloroethyl)-2-[(2-
chloroethyl)amino]tetrahydro-2H-1,3,2- oxazaphosphorine 2-oxide)
Imatinib Mesilate GLEEVEC Novartis AG, Basel,
(4-[(4-Methyl-1-piperazinyl)methyl]-N-[4- Switzerland
methyl-3-[[4-(3-pyridinyl)-2- pyrimidinyl]amino]-phenyl]benzamide
methanesulfonate) Interferon alfa-2a ROFERON-A Hoffmann-La Roche,
(recombinant peptide) Inc., Nutley, NJ Interferon alfa-2b INTRON A
Schering AG, Berlin, (recombinant peptide) (LYOPHILIZED Germany
BETASERON) Irinotecan HCl CAMPTOSAR Pharmacia & Upjohn
((4S)-4,11-diethyl-4-hydroxy-9-[(4- Company
piperidinopiperidino)carbonyloxy]- 1H-pyrano[3', 4':6,7]
indolizino[1,2-b] quinoline- 3,14(4H,12H) dione hydrochloride
trihydrate) Letrozole FEMARA Novartis
(4,4'-(1H-1,2,4-Triazol-1-ylmethylene) dibenzonitrile) Leucovorin
WELLCOVORIN, Immunex, Corp., (L-Glutamic acid,
N[4[[(2amino-5-formyl- LEUCOVORIN Seattle, WA 1,4,5,6,7,8
hexahydro4oxo6- pteridinyl)methyl]amino]benzoyl], calcium salt
(1:1)) Levamisole HCl ERGAMISOL Janssen Research
((-)-(S)-2,3,5,6-tetrahydro-6- Foundation, phenylimidazo [2,1-b]
thiazole Titusville, NJ monohydrochloride
C.sub.11H.sub.12N.sub.2S.cndot.HCl) Lomustine CEENU Bristol-Myers
Squibb (1-(2-chloro-ethyl)-3-cyclohexyl-1- nitrosourea)
Meclorethamine, nitrogen mustard MUSTARGEN Merck
(2-chloro-N-(2-chloroethyl)-N- methylethanamine hydrochloride)
Megestrol acetate MEGACE Bristol-Myers Squibb
17.alpha.(acetyloxy)-6-methylpregna-4,6- diene-3,20-dione
Melphalan, L-PAM ALKERAN GlaxoSmithKline (4-[bis(2-chloroethyl)
amino]-L- phenylalanine) Mercaptopurine, 6-MP PURINETHOL
GlaxoSmithKline (1,7-dihydro-6H-purine-6-thione monohydrate) Mesna
MESNEX Asta Medica
(sodium 2-mercaptoethane sulfonate) Methotrexate METHOTREXATE
Lederle Laboratories (N-[4-[[(2,4-diamino-6-
pteridinyl)methyl]methylamino]benzoyl]- L-glutamic acid)
Methoxsalen UVADEX Therakos, Inc., Way
(9-methoxy-7H-furo[3,2-g][1]-benzopyran- Exton, Pa 7-one) Mitomycin
C MUTAMYCIN Bristol-Myers Squibb mitomycin C MITOZYTREX SuperGen,
Inc., Dublin, CA Mitotane LYSODREN Bristol-Myers Squibb
(1,1-dichloro-2-(o-chlorophenyl)-2-(p- chlorophenyl) ethane)
Mitoxantrone NOVANTRONE Immunex (1,4-dihydroxy-5,8-bis[[2-[(2-
Corporation hydroxyethyl)amino]ethyl]amino]-9,10- anthracenedione
dihydrochloride) Nandrolone phenpropionate DURABOLIN-50 Organon,
Inc., West Orange, NJ Nofetumomab VERLUMA Boehringer Ingelheim
Pharma KG, Germany Oprelvekin NEUMEGA Genetics Institute, (IL-11)
Inc., Alexandria, VA Oxaliplatin ELOXATIN Sanofi Synthelabo,
(cis-[(1R,2R)-1,2-cyclohexanediamine- Inc., NY, NY N,N']
[oxalato(2-)-O,O'] platinum) Paclitaxel TAXOL Bristol-Myers Squibb
(5.beta.,20-Epoxy-1,2a,4,7.beta.,10.beta.,13a-
hexahydroxytax-11-en-9-one 4,10-diacetate 2-benzoate 13-ester with
(2R,3S)-N- benzoyl-3-phenylisoserine) Pamidronate AREDIA Novartis
(phosphonic acid (3-amino-1- hydroxypropylidene) bis-, disodium
salt, pentahydrate, (APD)) Pegademase ADAGEN Enzon
((monomethoxypolyethylene glycol (PEGADEMASE Pharmaceuticals, Inc.,
succinimidyl) 11-17-adenosine BOVINE) Bridgewater, NJ deaminase)
Pegaspargase ONCASPAR Enzon (monomethoxypolyethylene glycol
succinimidyl L-asparaginase) Pegfilgrastim NEULASTA Amgen, Inc
(covalent conjugate of recombinant methionyl human G-CSF
(Filgrastim) and monomethoxypolyethylene glycol) Pentostatin NIPENT
Parke-Davis Pharmaceutical Co., Rockville, MD Pipobroman VERCYTE
Abbott Laboratories, Abbott Park, IL Plicamycin, Mithramycin
MITHRACIN Pfizer, Inc., NY, NY (antibiotic produced by Streptomyces
plicatus) Porfimer sodium PHOTOFRIN QLT Phototherapeutics, Inc.,
Vancouver, Canada Procarbazine MATULANE Sigma Tau
(N-isopropyl-.mu.-(2-methylhydrazino)-p- Pharmaceuticals, Inc.,
toluamide monohydrochloride) Gaithersburg, MD Quinacrine ATABRINE
Abbott Labs (6-chloro-9-(1-methyl-4-diethyl-amine)
butylamino-2-methoxyacridine) Rasburicase ELITEK Sanofi-Synthelabo,
(recombinant peptide) Inc., Rituximab RITUXAN Genentech, Inc.,
(recombinant anti-CD20 antibody) South San Francisco, CA
Sargramostim PROKINE Immunex Corp (recombinant peptide) Sorafenib
NEXAVAR Bayer/Onyx Streptozocin ZANOSAR Pharmacia & Upjohn
(streptozocin 2-deoxy-2- Company
[[(methylnitrosoamino)carbonyl]amino]- a(and b)-D-glucopyranose and
220 mg citric acid anhydrous) Sunitnib malate SUTENT Pfizer Talc
SCLEROSOL Bryan, Corp., (Mg.sub.3Si.sub.4O.sub.10(OH).sub.2)
Woburn, MA Tamoxifen NOLVADEX AstraZeneca
((Z)2-[4-(1,2-diphenyl-1-butenyl) Pharmaceuticals
phenoxy]-N,N-dimethylethanamine 2-
hydroxy-1,2,3-propanetricarboxylate (1:1)) Temozolomide TEMODAR
Schering (3,4-dihydro-3-methyl-4-oxoimidazo[5,1-
d]-as-tetrazine-8-carboxamide) teniposide, VM-26 VUMON
Bristol-Myers Squibb (4'-demethylepipodophyllotoxin 9-[4,6-0-
(R)-2-thenylidene-(beta)-D- glucopyranoside]) Testolactone TESLAC
Bristol-Myers Squibb (13-hydroxy-3-oxo-13,17-secoandrosta-1,4-
dien-17-oic acid [dgr]-lactone) Thioguanine, 6-TG THIOGUANINE
GlaxoSmithKline (2-amino-1,7-dihydro-6H-purine-6- thione) Thiotepa
THIOPLEX Immunex (Aziridine, 1,1',1''- Corporation
phosphinothioylidynetris-, or Tris (1- aziridinyl) phosphine
sulfide) Topotecan HCl HYCAMTIN GlaxoSmithKline
((S)-10-[(dimethylamino) methyl]-4-ethyl-
4,9-dihydroxy-1H-pyrano[3',4':6,7] indolizino [1,2-b]
quinoline-3,14- (4H,12H)-dione monohydrochloride) Toremifene
FARESTON Roberts (2-(p-[(Z)-4-chloro-1,2-diphenyl-1- Pharmaceutical
butenyl]-phenoxy)-N,N- Corp., Eatontown, NJ dimethylethylamine
citrate (1:1)) Tositumomab, I 131 Tositumomab BEXXAR Corixa Corp.,
Seattle, (recombinant murine immunotherapeutic WA monoclonal
IgG.sub.2a lambda anti-CD20 antibody (I 131 is a
radioimmunotherapeutic antibody)) Trastuzumab HERCEPTIN Genentech,
Inc (recombinant monoclonal IgG.sub.1 kappa anti- HER2 antibody)
Tretinoin, ATRA VESANOID Roche (all-trans retinoic acid) Uracil
Mustard URACIL MUSTARD Roberts Labs CAPSULES Valrubicin,
N-trifluoroacetyladriamycin- VALSTAR Anthra --> Medeva
14-valerate ((2S-cis)-2-[1,2,3,4,6,11-hexahydro-
2,5,12-trihydroxy-7 methoxy-6,11-dioxo- [[4
2,3,6-trideoxy-3-[(trifluoroacetyl)-
amino-.alpha.-L-lyxo-hexopyranosyl]oxyl]-2-
naphthacenyl]-2-oxoethyl pentanoate) Vinblastine, Leurocristine
VELBAN Eli Lilly
(C.sub.46H.sub.56N.sub.4O.sub.10.cndot.H.sub.2SO.sub.4) Vincristine
ONCOVIN Eli Lilly
(C.sub.46H.sub.56N.sub.4O.sub.10.cndot.H.sub.2SO.sub.4) Vinorelbine
NAVELBINE GlaxoSmithKline (3',4'-didehydro-4'-deoxy-C'-
norvincaleukoblastine [R-(R*,R*)-2,3- dihydroxybutanedioate
(1:2)(salt)]) Zoledronate, Zoledronic acid ZOMETA Novartis
((1-Hydroxy-2-imidazol-1-yl- phosphonoethyl) phosphonic acid
monohydrate)
[0087] In other embodiments, the RNase 1 compositions of the
present invention are used in combination with variant human RNase
proteins (See e.g., U.S. patent application Ser. No. 11/105,041,
herein incorporated by reference in its entirety) that are toxic to
cells.
[0088] A current and still developing approach to cancer therapy
involves using cancer cell-specific reagents to target a malignant
tumor, although the proteins of the present invention may be used
without targeting reagents. These toxic reagents can be produced by
attaching a toxic payload to a cell-specific delivery vector. Over
the past few years, a wide variety of tumor-specific targeting
proteins, including antibodies, antibody fragments, and ligands for
cell surface receptors have been developed and clinically tested.
These targeting molecules have been conjugated to several classes
of therapeutic toxins such as small molecule drugs, enzymes,
radioisotopes, protein toxins, and other toxins for specific
delivery to patients. While these efforts have made meaningful
inroads to treat cancers, significant challenges lie ahead to
develop more effective toxins, to create more robust and specific
delivery systems, and to design therapeutic proteins and protein
vectors that avoid a detrimental immune response in humans.
[0089] Ribonuclease (RNase) proteins have been tested as human
therapeutics because they have some selectivity for tumor cells;
this has been demonstrated most clearly with an RNase from Rana
pipiens early embryos. Rana pipiens is a species of leopard frogs
and its embryonic RNase is distantly related to the more highly
conserved bovine and human pancreatic ribonucleases. In mammalian
cells, pancreatic-type ribonucleases, such as RNase A, are
secretory enzymes that catalyze the degradation of RNA to
ribonucleotides and their activity is inhibited by binding to
ribonuclease inhibitor (RI), a ubiquitous cytosolic protein. The
present invention is not limited to a particular mechanism. Indeed,
an understanding of the mechanism is not necessary to practice the
present invention. Nonetheless, it is contemplated that
ribonuclease inhibitor binds exceptionally tight to pancreatic-type
RNases, abating their activity and thereby making them non-toxic to
normal or cancer cells. If the RNase activity is inhibited, the
cellular RNA is undamaged and the cell remains viable. In normal
cells the ribonuclease activity is tightly controlled, but if
ribonuclease activity is uncontrolled, the ribonucleolytic activity
destroys cellular RNA and kills the cell. There are two main
approaches to diminishing the binding of ribonucleases to the
ribonuclease inhibitor protein. The first approach is to select a
ribonuclease that is evolutionarily distant to humans and is not
inhibited by human ribonuclease inhibitor protein. The frog (Rana
pipiens) ribonuclease, when placed in a human cell, is not strongly
inhibited by RI and its RNase activity destroys cellular RNA and
kills the target cell. This has been the approach with a specific
Rana pipiens RNase called Ranpirnase. Ranpirnase is generic name of
the pharmaceutical that is described and claimed in U.S. Pat. No.
5,559,212 and that is presently known by the registered trademark
ONCONASE.
[0090] The second approach is to mutate mammalian ribonucleases so
that they have diminished binding to the human ribonuclease
inhibitor. These mutated enzymes provide high levels of
ribonucleolytic activity within cancer cells because of disruption
of binding to RI. This unregulated activity is particularly lethal
to cancer cells. This mutation approach has been demonstrated with
the mammalian proteins bovine RNase A and human RNase 1 and is
described in U.S. Pat. Nos. 5,389,537 and 6,280,991, the
disclosures of which are herein incorporated by reference in their
entireties. Surprisingly, the present invention demonstrates that
wild type human ribonuclease 1 and equivalent variants have the
ability to kill cancer cells.
[0091] An ideal protein candidate for cancer therapy would be more
toxic to tumor cells compared to non-cancerous cells and would be
targetable to a specific tumor. This candidate should have few side
effects and should not stimulate a detrimental human immune
response. Therapeutic proteins that elicit detrimental immune
responses in humans are often problematic and sometimes
unacceptable. Experiments conducted during the course of
development of the present invention demonstrated that human RNase
1 exhibited significant anti-tumor activity in mouse xenografts
while exhibiting minimal toxicity. Accordingly, in some
embodiments, the present invention provides RNase 1 proteins, alone
or in combination with other therapeutic agents, for use in killing
cancer cells or degrading toxic RNA.
[0092] Certain preferred embodiments of the present invention are
described below. While the present invention is illustrated with
human RNase 1 proteins, the present invention is not limited to the
use of RNase 1 of human origin. The present invention contemplates
the use of homologs of RNase 1 from any organism and engineered
proteins.
Genetic Therapy
[0093] In some embodiments, RNase 1 or a variant thereof is
provided as a nucleic acid encoding the RNase. Introduction of
molecules carrying genetic information into cells is achieved by
any of various methods including, but not limited to, directed
injection of naked DNA constructs, bombardment with gold particles
loaded with said constructs, and macromolecule mediated gene
transfer using, for example, liposomes, biopolymers, and the like.
Preferred methods use gene delivery vehicles derived from viruses,
including, but not limited to, adenoviruses, retroviruses, vaccinia
viruses, and adeno-associated viruses. Because of the higher
efficiency as compared to retroviruses, vectors derived from
adenoviruses are the preferred gene delivery vehicles for
transferring nucleic acid molecules into host cells in vivo.
Adenoviral vectors have been shown to provide very efficient in
vivo gene transfer into a variety of solid tumors in animal models
and into human solid tumor xenografts in immune-deficient mice.
Examples of adenoviral vectors and methods for gene transfer are
described in PCT publications WO 00/12738 and WO 00/09675 and U.S.
Pat. Nos. 6,033,908, 6,019,978, 6,001,557, 5,994,132, 5,994,128,
5,994,106, 5,981,225, 5,885,808, 5,872,154, 5,830,730, and
5,824,544, each of which is herein incorporated by reference in its
entirety.
[0094] Vectors may be administered to subject in a variety of ways.
For example, in some embodiments of the present invention, vectors
are administered into tumors or tissue associated with tumors using
direct injection. In other embodiments, administration is via the
blood or lymphatic circulation (See e.g., PCT publication 99/02685
herein incorporated by reference in its entirety). Exemplary dose
levels of adenoviral vector are preferably 10.sup.8 to 10.sup.11
vector particles added to the perfusate.
Therapeutic Antibodies and Delivery of Cytotoxins
[0095] Antibodies are glycoprotein molecules produced by white
blood cells (B-lymphocytes) of the immune system and their function
is to recognize and bind to matter harmful to the organism. Once an
antigen is marked by an antibody, it is destroyed by other
components of the immune system. A typical organism makes millions
of different antibodies, each designed to bind a specific epitope
(or antigenic determinant) on the foreign antigen. Antibodies
naturally combine specificity (the ability to exquisitely
discriminate diverse harmful molecules) and affinity (the ability
to tightly lock onto those targets) with the ability to recruit
effector functions of the immune system such as antibody- and
complement-mediated cytolysis and antibody-dependent cell-mediated
cytotoxicity (ADCC). Many new therapeutic approaches involving
antibodies have succeeded in potentiating the natural antibody
functions to treat or cure diseases.
[0096] Alternatively, a "toxic payload" (such as a radioactive
element or a toxin) attached to the antibody can be accurately
delivered to the pathogenic target. The following table lists the
mechanisms of some cancer therapeutic antibodies, including three
antibody conjugates that carry a toxic payload for lymphomas and
leukemias. (Drug Discovery Today, Vol. 8, No. 11 Jun. 2003). Two of
the conjugates, ZEVALIN and BEXXAR, carry radioactive iodine as the
toxin and the third, MYLOTARG, carries a cytotoxic antitumor
antibiotic, calicheaminin which is isolated from a bacterial
fermentation. The Mylotarg antibody binds specifically to the CD33
antigen which is expressed on the surface of leukemic blasts that
are found in more than 80% of patients with acute myeloid leukemia
(AML). The antibody in this conjugate has approximately 98.3% of
its amino acid sequences derived from human origins.
TABLE-US-00006 TABLE 6 Antibody Mode of Action Product Antibody
Target Blockade Ligand ERBITUX EGF receptor binding HUMAX-EGFR EGF
receptor Complement RITUXAN CD20 Dependent Cytotoxicity HUMAX-CD20
CD20 CAMPATH-1H CD52 Antibody dependent RIXTUXAN CD20 cell-mediated
cytotoxicity HUMAX-CD20 CD20 HERCEPTIN Her-2/neu HUMAX-EGFR EGF
receptor Apoptosis induction Various IdiotypeB cell tumors
Disruption signaling 2C4 Her-2/neu (PERTUZUMAB) Inhibition
angiogenesis AVASTIN VEGF Targeted radiolysis ZEVALIN CD20
conjugate BEXXAR CD20 Toxin-mediated killing MYLOTARG CD33 by
conjugate Antagonist activity MDX-010 CTLA4 Agonist activity
Various CD40, CD137 Antagonist activity Preclinical MAb Epithelial
cell receptor protein tyrosine kinase (EphA2) Antagonist activity
Phase II Mab alpha 5 beta 3 integrin (receptor) Antagonist activity
Phase I bispecific CD19/CD3 single chain monoclonal antibody
Antagonist activity Preclinical MAb Interleukin 9 Antagonist
activity RespiGam Respiratory syncytial virus Polyclonal Antibody
Antagonist activity Phase II MAb CD2 Catalytic Activity Mab Cocaine
cleavage Anti-infective, bacteria Mab bacteria Immunosuppressive
Mab Graft versus Host Disease Agents Anti-infective, virus Mab
Human metapneumovirus Cytostatic agent Mab Platelet derived growth
factor Cancer growth and Preclinical MAb Human beta hydroxylases
metastosis Treatment of MAb Medi 507 Mixed lymphocyte autoimmune
responses disease Anti-infective, virus Polyclonal antibody
cytomegalovirus Anti-idiotype antibody Mab Neu-glycolyl-GM3
ganglioside Prodrug carrier Mab Immungen's CC 1065 prodrugs
Toxin-mediated killing Preclinical MAb Various by Immunogen by
conjugate and taxane derivatives Toxin-mediated killing Cantuzumab
Can Ag receptor by by conjugate mertansine immunogen conjugate
Toxin-mediated killing Phase II MAb CD56 by conjugate maytansinoid
conjugate Toxin for mitosis MAb maitansine various inhibition
conjugate Toxin-mediated killing Preclinical MAb Antigen on
squamous cell by conjugate cytotoxic drug cancer (Immunogen) DM1
conjugate
[0097] Any of the targeting antibodies or agents used in these
products may also be employed by the compositions and methods of
the present invention.
[0098] Generally, the most specific method for targeting toxins is
the use of monoclonal antibodies or antibody fragments that are
designed to recognize surface antigens specific to tumor cells.
Because normal cells lack the surface antigens, they are not
targeted and killed by the toxin conjugate. Whole antibodies have
two domains: a variable domain that gives the antibody its affinity
and binding specificity and a constant domain that interacts with
other portions of the immune system to stimulate immune responses
in the host organism. The variable domain is composed of the
complementarity determining regions (CDRs), which bind to the
antibody's target, and a framework region that anchors the CDRs to
the rest of the antibody and helps maintain CDR shape. The six
CDR's in each antibody differ in length and sequence between
different antibodies and are mainly responsible for the specificity
(recognition) and affinity (binding) of the antibodies to their
target markers.
[0099] The functions of antibodies are reflected in their
characteristic three-dimensional structure, which is ultimately
determined by the primary sequence of amino acids and how those
amino acids fold into a functional 3-dimensional protein chain. A
step in developing therapeutic monoclonal antibodies is to
simultaneously optimize biochemical and cellular functions for
anti-cancer performance and still keep the protein as humanlike as
possible to minimize any anti-antibody human immune response.
[0100] Monoclonal antibodies were originally produced in mice, but
when they are used in human therapeutic applications, they present
formidable obstacles. Mouse antibodies are recognized as foreign by
the human immune system and thus they provoke the Human Anti-Mouse
Antibody or HAMA reaction. The HAMA reaction alters the mouse
monoclonal effectiveness and can cause severe adverse symptoms in
the recipient. Furthermore, mouse antibodies are simply not as
effective as human antibodies in mediating the human immune system
to destroy the malignant cells. For these reasons, it is often
desired to design monoclonal antibodies that are as humanlike as
possible but still maintain optimal biochemical, immunological, and
therapeutic performance.
[0101] There are several factors that influence whether a
therapeutic antibody will induce an immune response in the human
host. These include the efficiency of uptake by an APC (antigen
presenting cell) via pinocytosis, receptor-mediated endocytosis, or
phagocytosis. The efficiency of uptake is in turn influenced by the
route of administration, the solubility (or aggregation) of the
protein, its receptor binding specificity, and whether the protein
is recognized by class II major histocompatibility complex (MHC)
molecules, T-cell receptors (TCR), and B-cell receptors (BCR). One
of the most straightforward ways to evade the human immune response
is to make the therapeutic protein sequence and structure as
humanlike as possible.
[0102] Two main approaches have emerged to produce human or
humanized therapeutic monoclonal antibodies, either used alone as a
therapeutic or as a carrier for a toxin. These include 1)
`humanizing` mouse or other non-human antibodies to make them
compatible with the human immune system and 2) producing fully
human antibodies in transgenic mice or by using genetic engineering
methods in the laboratory. The processes have produced several
categories of monoclonal antibodies. These include mouse,
chimaeric, humanized and human antibodies. They are described
briefly below: [0103] 1. Murine Monoclonal antibodies from mice and
rats: The original Kohler and Milstein technology from 1975
provided mouse monoclonal antibodies using a hybridoma technology.
These have been used therapeutically. In 1986, the first approved
use of mouse monoclonals was for transplant patients whose immune
system was suppressed to avoid organ rejection. Rodent antibodies
tend to provoke strong Human anti-Murine Antibody (HAMA) immune
responses that restrict their usefulness for repeated application
in the same patient. [0104] 2. Chimaeric Antibodies: These are
mutated antibodies in which the entire variable regions of a
functional mouse antibody are joined to human constant regions.
These antibodies have human effector functions from the constant
(Fc regions) such as activating complement and recruiting immune
cells. These chimaeric antibodies also reduce the immunogenicity
(HAMA) caused by the mouse constant region. [0105] 3. Humanized/CDR
grafted/Reshaped antibodies: These antibodies are more humanlike
than chimaeric antibodies because only the complementarity
determining regions from the mouse antibody variable regions are
combined with framework regions from human variable regions.
Because these antibodies are more human-like than chimaeric
antibodies, it is expected they could be designed to be less
immunogenic when given to human in recurring therapeutic doses.
Using computer modeling software to guide the humanization of
murine antibodies or random shuffling of sequences followed by
screening, it is possible to design an antibody that retains most
or all of the binding affinity and specificity of the murine
antibody but which is >90% human. [0106] 4. Human antibodies
from immune donors: Some antibodies have been rescued from immune
human donors using either Epstein Barr Virus transformation of
B-cells or by PCR cloning and phage display. By definition these
antibodies are completely human in origin. [0107] 5. Fully human
antibodies from phage libraries: Synthetic phage libraries have
been created which use randomized combinations of synthetic human
antibody V-regions. By panning these libraries against a target
antigen, these so called `fully human antibodies` are assumed to be
very human but possibly more diverse than natural antibodies.
[0108] 6. Fully human antibodies from transgenic mice: Transgenic
mice have been created that have functional human immunoglobulin
germline genes sequences. These transgenic mice produce human-like
antibodies when immunized.
[0109] The human antibodies produced by methods 4, 5, and 6 are
typically most desired because they produce a starting antibody
that contains no mouse or otherwise "foreign" protein sequences
that should stimulate an immune response in human patient. This
approach (in 4, 5, and 6) also can bypass the challenge of
substituting mouse CDR regions into human frameworks that often
alters the 3-dimensional structure of the variable region, thereby
changing the antibody's binding and specificity. This approach (in
4, 5, 6) successfully produced an anti-CD3 antibody. The murine
version elicited neutralizing antibodies after a single dose in all
patients tested, while a humanized version was only immunogenic in
25% of patients following multiple injections.
[0110] Besides making monoclonal antibodies as human-like as
possible in the primary sequence to escape the human immune
response, several other approaches make antibodies less immunogenic
and more therapeutically effective are available. One approach is
to covalently modify the antibody surface with reagents such as
polyethylene glycol (PEG) to suppress its antigenicity and improve
its solubility. These biochemical modifications also can have
several other benefits such as reduced toxicity, increased
bioavailability, and improved efficacy. Another approach is to use
antibody fragments in which the potentially antigenic parts of the
mouse antibody, such as the constant region, have been removed.
This approach typically works only when the regulatory components
within the antibody constant region are not required for
therapeutic efficacy. Neither of these approaches has proven
completely satisfactory, which has driven the humanization effort
to produce `the ideal` antibody candidate mentioned above.
[0111] In addition to antibody delivery vectors, toxic molecules
can be delivered to cancer cells using several other specific and
non-specific vectors including peptides, polymers, dendrimers,
liposomes, polymeric nanoparticles, and block copolymer micelles.
For example, peptides that bind to the leutinizing
hormone-releasing hormone have been used to target a small molecule
toxin, camptothecin, to ovarian cancer cells (Journal of Controlled
Release, 2003, 91, 61-73).
[0112] Ribonucleases such as RNase 1 are effective toxins in human
cells, particularly against cancer cells. The following references,
each of which is herein incorporated by reference in its entirety,
describe some chemical conjugates of ribonucleases to targeting
proteins (including proteins and antibodies): Newton et al. (2001),
Blood 97(2): 528-35, Hursey et al. (2002) Leuk Lymphoma 43(5):
953-9, Rybak et al., (1991) Journal of Biological Chemistry
266(31): 21202-7, Newton et al. (1992) Journal of Biological
Chemistry 267(27): 19572-8, Jinno and Ueda (1996) Cancer Chemother
Pharmacol 38: 303-308, Yamamura et al. (2002) Eur J Surg 168:
49-54, Jinno et al. (1996) Life Sci 58: 1901-1908, Suzuki et al.
(1999) Nature Biotechnology 17(3): 265-70, Rybak et al. (1992),
Cell Biophys 21(1-3): 121-38, Jinno et al. (2002) Anticancer Res.
22: 4141-4146.
[0113] Due to the minimal side effects seen thus far for human
ribonuclease 1, the ribonuclease itself could be used to target
drugs to diseased cells, such as cancer cells.
[0114] In particular, some embodiments of the present invention
provide recombinant constructs comprising one or more of the
sequences as broadly described above. In some embodiments of the
present invention, the constructs comprise a vector, such as a
plasmid or viral vector, into which a sequence of RNase 1 has been
inserted, in a forward or reverse orientation. In still other
embodiments, the heterologous structural sequence is assembled in
appropriate phase with translation initiation and termination
sequences. In preferred embodiments of the present invention, the
appropriate DNA sequence is inserted into the vector using any of a
variety of procedures. In general, the DNA sequence is inserted
into an appropriate restriction endonuclease site(s) by procedures
known in the art.
[0115] Large numbers of suitable vectors are known to those of
skill in the art, and are commercially available. Such vectors
include, but are not limited to, the following vectors: 1)
Bacterial--pQE70, pQE60, pQE-9 (Qiagen), pet 22b, pet26b, pet 30b
(Novagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS,
pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3,
pKK233-3, pDR540, pRIT5 (Pharmacia); and 2) Eukaryotic--pWLNEO,
pSV2CAT, pOG44, PXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL
(Pharmacia). Any other plasmid or vector may be used as long as
they are replicable and viable in the host. In some preferred
embodiments of the present invention, mammalian expression vectors
comprise an origin of replication, a suitable promoter and
enhancer, and also any necessary ribosome binding sites,
polyadenylation sites, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
non-transcribed sequences. In other embodiments, DNA sequences
derived from the SV40 splice, and polyadenylation sites may be used
to provide the required non-transcribed genetic elements.
[0116] In certain embodiments of the present invention, the DNA
sequence in the expression vector is operatively linked to an
appropriate expression control sequence(s) (promoter) to direct
mRNA synthesis. Promoters useful in the present invention include,
but are not limited to, the LTR or SV40 promoter, the E. coli lac
or trp, the phage lambda PL and PR, T3 and T7 promoters, and the
cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV)
thymidine kinase, and mouse metallothionein-I promoters and other
promoters known to control expression of gene in prokaryotic or
eukaryotic cells or their viruses. In other embodiments of the
present invention, recombinant expression vectors include origins
of replication and selectable markers permitting transformation of
the host cell (e.g., dihydrofolate reductase or neomycin resistance
for eukaryotic cell culture, or tetracycline, kanamycin, or
ampicillin resistance in E. coli).
[0117] In some embodiments of the present invention, transcription
of the DNA encoding the polypeptides of the present invention by
higher eukaryotes is increased by inserting an enhancer sequence
into the vector. Enhancers are cis-acting elements of DNA, usually
about from 10 to 300 bp that act on a promoter to increase its
transcription. Enhancers useful in the present invention include,
but are not limited to, the SV40 enhancer on the late side of the
replication origin by 100 to 270, a cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers.
[0118] In other embodiments, the expression vector also contains a
ribosome-binding site for translation initiation and a
transcription terminator. In still other embodiments of the present
invention, the vector may also include appropriate sequences for
amplifying expression.
[0119] In a further embodiment, the present invention provides host
cells containing the above-described constructs. In some
embodiments of the present invention, the host cell is a higher
eukaryotic cell (e.g., a mammalian or insect cell). In other
embodiments of the present invention, the host cell is a lower
eukaryotic cell (e.g., a yeast cell). In still other embodiments of
the present invention, the host cell can be a prokaryotic cell
(e.g., a bacterial cell). Specific examples of host cells include,
but are not limited to, Escherichia coli, Salmonella typhimurium,
Bacillus subtilis, and various species within the genera
Pseudomonas, Streptomyces, and Staphylococcus, as well as
Saccharomycees cerivisiae, Schizosaccharomycees pombe, Drosophila
S2 cells, Spodoptera Sf9 cells, Chinese hamster ovary (CHO) cells,
COS-7 lines of monkey kidney fibroblasts, (Gluzman, Cell 23:175
[1981]), C127, 3T3, 293, 293T, HeLa and BHK cell lines.
[0120] The constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. In some embodiments, introduction of the construct into
the host cell can be accomplished by calcium phosphate
transfection, DEAE-Dextran mediated transfection, transformation,
or electroporation (See e.g., Davis et al., Basic Methods in
Molecular Biology, [1986]). Alternatively, in some embodiments of
the present invention, the polypeptides of the invention can be
synthetically produced by conventional peptide synthesizers.
[0121] Proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the present invention. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described by
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989).
[0122] In some embodiments of the present invention, following
transformation of a suitable host strain and growth of the host
strain to an appropriate cell density, the selected promoter is
induced by appropriate means (e.g., temperature shift or chemical
induction) and cells are cultured for an additional period. In
other embodiments of the present invention, cells are typically
harvested by centrifugation, disrupted by physical or chemical
means, and the resulting crude extract retained for further
purification. In still other embodiments of the present invention,
microbial cells employed in expression of proteins can be disrupted
by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing
agents.
[0123] The polypeptides of the present invention may also be
chemically synthesized (Gutte, B. and Merrifield, R. B. The
synthesis of ribonuclease A. J. Biol. Chem. 1971, 2461,
1722-1741).
Pharmaceutical Compositions
[0124] The present invention further provides pharmaceutical
compositions (e.g., comprising the cell killing compositions
described above). The pharmaceutical compositions of the present
invention may be administered in a number of ways depending upon
whether local or systemic treatment is desired and upon the area to
be treated. Administration may be topical (including ophthalmic and
to mucous membranes including vaginal and rectal delivery),
pulmonary (e.g., by inhalation or insufflation of powders or
aerosols, including by nebulizer; intratracheal, intranasal,
epidermal and transdermal), oral or parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal or intramuscular injection or infusion; or
intracranial, e.g., intrathecal or intraventricular,
administration.
[0125] Pharmaceutical compositions and formulations for topical
administration may include transdermal patches, ointments, lotions,
creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily
bases, thickeners and the like may be necessary or desirable.
[0126] Compositions and formulations for oral administration
include powders or granules, suspensions or solutions in water or
non-aqueous media, capsules, sachets or tablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable.
[0127] Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous
solutions that may also contain buffers, diluents and other
suitable additives such as, but not limited to, penetration
enhancers, carrier compounds and other pharmaceutically acceptable
carriers or excipients.
[0128] Pharmaceutical compositions of the present invention
include, but are not limited to, solutions, emulsions, and
liposome-containing formulations. These compositions may be
generated from a variety of components that include, but are not
limited to, preformed liquids, self-emulsifying solids and
self-emulsifying semisolids.
[0129] The pharmaceutical formulations of the present invention,
which may conveniently be presented in unit dosage form, may be
prepared according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0130] The compositions of the present invention may be formulated
into any of many possible dosage forms such as, but not limited to,
tablets, capsules, liquid syrups, soft gels, suppositories, and
enemas. The compositions of the present invention may also be
formulated as suspensions in aqueous, non-aqueous or mixed media.
Aqueous suspensions may further contain substances that increase
the viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0131] In one embodiment of the present invention the
pharmaceutical compositions may be formulated and used as foams.
Pharmaceutical foams include formulations such as, but not limited
to, emulsions, microemulsions, creams, jellies and liposomes. While
basically similar in nature these formulations vary in the
components and the consistency of the final product.
[0132] The preferred method of administration is by intravenous or
IP injection. It is alternatively possible to use injection into
the tumor to be treated. In some embodiments, administration is
continued as an adjuvant treatment for an additional period (e.g.,
several days to several months).
[0133] The compositions of the present invention may additionally
contain other adjunct components conventionally found in
pharmaceutical compositions. Thus, for example, the compositions
may contain additional, compatible, pharmaceutically-active
materials such as, for example, antipruritics, astringents, local
anesthetics or anti-inflammatory agents, or may contain additional
materials useful in physically formulating various dosage forms of
the compositions of the present invention, such as dyes, flavoring
agents, preservatives, antioxidants, opacifiers, thickening agents
and stabilizers. However, such materials, when added, should not
unduly interfere with the biological activities of the components
of the compositions of the present invention. The formulations can
be sterilized and, if desired, mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings and/or aromatic substances and the like which
do not deleteriously interact with the active pharmaceutical agents
of the formulation.
[0134] Dosing is dependent on severity and responsiveness of the
disease state to be treated, with the course of treatment lasting
from several days to several months, or until a cure is effected or
a diminution of the disease state is achieved. Optimal dosing
schedules can be calculated from measurements of drug accumulation
in the body of the patient. The administering physician can easily
determine optimum dosages, dosing methodologies and repetition
rates. Optimum dosages may vary depending on the relative potency
of individual pharmaceutical compositions, and can generally be
estimated based on EC.sub.50s found to be effective in in vitro and
in vivo animal models or based on the examples described herein. In
general, dosage is from 0.01 .mu.g to 100 g per kg of body weight,
for example between 0.1 and 1000 mg per kg of body weight,
preferably between 0.1 and 500 mg/kg of body weight, and still more
preferably between 0.1 and 200 mg/kg of body weight, for a period
of between 1 and 240 minutes (e.g., between 2 and 60 minutes and
preferably between 15 and 45 minutes). Dosages may be administered
as often as need to obtain the desired effect (e.g., reduction of
tumor size or number of cancerous cells), for example once or more
daily to once or more weekly or monthly. In some embodiments, the
compositions are administered weekly at a dose of between 0.1 and
10 mg (e.g., 1 mg) for a period of between 5 and 60 minutes (e.g.,
30 minutes). The treating physician can estimate repetition rates
for dosing based on measured residence times and concentrations of
the drug in bodily fluids or tissues. Following successful
treatment, it may be desirable to have the subject undergo
maintenance therapy to prevent the recurrence of the disease state
once or more daily, to once every 20 years. In some preferred
embodiments, dosages are 0.25-1000 mg/kg daily, weekly, or monthly
to achieve the desired therapeutic effect. In some preferred
embodiments, dosages are 50 mcg/m.sup.2 to 400 mcg/m.sup.2 daily,
weekly, or monthly to achieve the desired therapeutic effect. Drugs
are also sometimes dosed in units of activity per dose as opposed
to amount (weight) of drug.
EXAMPLES
[0135] The following examples serve to illustrate certain preferred
embodiments and aspects of the present invention and are not to be
construed as limiting the scope thereof.
Example 1
Enzymatic Activity Assay
[0136] The enzymatic activity of the wild type human RNase 1 was
determined using a fluorescent assay based on fluorescence
resonance energy transfer (FRET). The substrate for the assay,
5'FAM-ArUAA-3'TAMRA (IDT), is not fluorescent until cleaved.
[0137] In a typical assay, the buffer (160 microliters of 100 mM
NaCl, 100 mM Tris, pH 7.0, 100 microgram/mL BSA) is added to the
wells of a 96-well non-binding surface, black, polystyrene plate.
The RNase (typically 10 microliters of an approximately
2.times.10.sup.-1.degree. M solution) is also added. Substrate (30
microliters of a 1.33 micromolar solution of 5'FAM-ArUAA-3'TAMRA)
is then added to each well, and the samples mixed. The plate is
read on a fluorescent plate reader immediately.
[0138] Control wells are included for F0 (no enzyme) and Fmax
determinations (typically 10 microliters of a 0.1 mg/mL solution of
RNase A per 200 microliter assay). In this example, wild type human
RNase 1 had a k.sub.cat/K.sub.M=2.65.times.10.sup.7
M.sup.-1sec.sup.-1. The results are shown in FIG. 4.
Example 2
Pharmacokinetic Determination in BALB/c Mice
[0139] Sixteen CD-1 mice (5 weeks old, Harlan) were injected with
75 mg per kg of body weight of wild type human RNase 1. Blood was
collected from three mice at each of the following time points:
0.5, 1.0, 1.5, 2, 4, 6, 8, 24, 36, 48 hours. The animals were
rotated for bleeding, leaving as much time as possible between
bleeding times. The blood was allowed to clot for several hours at
4.degree. C. and spun in a microfuge at top speed for 10 minutes.
The serum was then collected and stored at -20.degree. C.
[0140] The serum samples for each timepoint were pooled and the
serum diluted (1:10,000). The enzymatic activity assay described in
the previous example was used to analyze the samples. The slope of
the line for each time point was plotted and is provided FIG.
5.
Example 3
In Vivo Determination of Inhibition of Tumor Growth
[0141] Cells from a non-small cell lung cancer cell line (A549)
were grown in nine T175 flasks in F12K media and 10% fetal calf
serum until the cells were confluent. 4.5.times.10.sup.6 cells (in
100 microliters) were injected into the right rear flank of 4-5
week old male homozygous (nu/nu) nude mice (Harlan, Madison Wis.).
Tumors were allowed to grow to an average size of .gtoreq.75
mm.sup.3 before treatments were initiated. Animals of each tumor
type, with the properly sized tumors, were divided into treatment
groups, including one set of animals treated weekly with vehicle
(phosphate buffered saline, PBS). The vehicle and the test agents
were all administered by intraperitoneal injection. Each animal was
weighed twice a week during treatment. The tumors were measured
twice weekly using calipers. Tumor volume (mm.sup.3) was determined
by using the formula for an ellipsoid sphere:
volume = l .times. w 2 2 ##EQU00001##
Results are shown in FIG. 6 and Table 7. The efficacy of wild type
human RNase 1 is shown relative to cisplatin. The RNase 1 was
administered at 75 mg per kg of body weight of the animal once a
week (75 mg/kg 1.times.wk), while the cisplatin was used at 6 mg/kg
once a week. The value of n represents the number of animals in the
specific treatment arm of the experiment.
TABLE-US-00007 TABLE 7 Starting Final (Final - volume volume (Final
- start) start)/Control % TGI Vehicle 67 376 309 1 0 wt human 68
217 149 0.49 51% RNase 1 (75 mg) cisplatin (6 72 130 58 0.19 81%
mg)
[0142] The percent tumor growth inhibition (% TGI) is calculated
by:
% TGI = 1 - ( final size - starting size ) treated ( final size -
starting size ) control .times. 100 ##EQU00002##
Example 4
In Vivo Determination of Inhibition of Tumor Growth
[0143] This xenograft model was set up as in the previous example
except that a human prostate cancer cell line (DU145) was used. The
efficacy of wild type human RNase 1 is shown relative to docetaxel.
The RNase 1 was administered at 75 mg per kg of body weight of the
animal (75 mg/kg 1.times.wk), while the docetaxel was used at 8
mg/kg once a week. The value of n represents the number of animals
in the specific treatment arm of the experiment. Results are shown
in FIG. 7 and Table 8.
TABLE-US-00008 TABLE 8 Starting Final (Final - (Final - volume
volume start) start)/Control % TGI Vehicle 64 2452 2388 1 0 wt
human RNase 1 61 539 478 0.20 80 Docetaxel 60 249 180 0.08 92
Clinical Chemistry and Complete Blood Count Plus Platelets
[0144] Clinical chemistry and a complete blood count (CBC) plus
platelets were performed on whole blood and serum samples collected
from the mice 24 hours after the last treatment of the prostate
xenograft model above. In all tests, the results for wild type
human RNase 1 and docetaxel were determined not to be significantly
different from the vehicle treated animals (Mann-Whitney U test).
Creatinine was also tested and found not to differ. The levels of
the liver enzymes, gamma glutamyl transferase (GGT) and alanine
transaminase (ALT) in all treatment groups were similar (GGT data
not shown). Results are shown in FIG. 8. The values were determined
for the various treatment groups of the prostate (DU145) xenograft
study for clinical chemistry (A., B.), complete blood count
(C.,D.), and platelets (F.). Abbreviations: TCO.sub.2, amount of
bicarbonate ion (HCO.sub.3.sup.-); Anion Gap,
[Na+K]-[Cl+HCO.sub.3]; ALT, alanine aminotransferase; WBC, white
blood count; RBC, red blood count; HGB, hemoglobin; RBC HGB,
(CHCM.times.RBC.times.MCV)/1000); MCH, mean corpuscular hemoglobin;
MCHC, mean corpuscular hemoglobin concentration; RDW, red cell
distribution width; MPV, mean platelet volume; HCT, hematocrit;
MCV, mean corpuscular volume.
Example 5
In Vivo Determination of Inhibition of Tumor Growth
[0145] The xenograft model of this example was set up as in the
previous example except that a human pancreatic cancer cell line
(BxPC-3) was used. The efficacy of wild type human RNase 1 is shown
relative to gemcite. The RNase 1 was administered at 15 mg per kg
of body weight of the animal five times per week (15 mg/kg
qd.times.5), while the gemcite was used at 60 mg/kg twice a week.
The value of n represents the number of animals in the specific
treatment arm of the experiment. Results are shown in FIG. 9 and
Table 9.
TABLE-US-00009 TABLE 9 Starting Final (Final - (Final - start)/
volume volume start) Control % TGI Vehicle 81 1158 1077 1 0 wt
human RNase 77 899 822 0.76 24 1 Gemcite 78 505 427 0.40 60
Example 6
In Vivo Determination of Inhibition of Tumor Growth
[0146] The xenograft model of this example was set up with the
human non-small cell lung cancer cell line (A549) as in the
previous examples. The efficacy of wild type human RNase 1 is shown
relative to tarceva. The RNase 1 was administered at 75 mg per kg
of body weight of the animal (75 mg/kg 1.times.wk) or 150 mg/kg
1.times.wk. Tarceva was given orally with doses of 50 mg/kg twice a
week. The value of n represents the number of animals in the
specific treatment arm of the experiment. Results are shown in FIG.
10 and Table 10.
TABLE-US-00010 TABLE 10 Starting Final (Final - (Final - start)/
volume volume start) Control % TGI Vehicle 83 886 803 1 0 wt human
RNase 78 378 300 0.37 63 1 (75 mg) wt human RNase 79 355 276 0.34
66 1 (150 mg) Tarceva 84 265 181 0.23 77
Example 7
In Vivo Determination of Inhibition of Tumor Growth
[0147] The xenograft model of this example was set up as in the
previous example except that a human pancreatic cancer cell line
(BxPC-3) was used. The efficacy of wild type human RNase 1 is
shown. The RNase 1 was administered at three different doses: (1)
30 mg per kg of body weight of the animal once per week (30 mg/kg
1.times.wk), (2) 75 mg/kg 1.times.wk, or (3) 150 mg/kg 1.times.wk.
The value of n represents the number of animals in the specific
treatment arm of the experiment. Results are shown in FIG. 11 and
Table 11.
TABLE-US-00011 TABLE 11 Starting Final (Final - (Final - start)/
volume volume start) Control % TGI Vehicle 70 639 569 1 0 wt human
RNase 78 632 554 0.97 3 1 (30 mg) wt human RNase 69 392 323 0.57 43
1 (75 mg) wt human RNase 69 471 402 0.71 29 1 (150 mg)
Example 8
In Vivo Determination of Inhibition of Tumor Growth
[0148] The xenograft model of this example was set up as in the
previous example except that a human prostate cancer cell line
(DU145) was used. The efficacy of wild type human RNase 1 is shown.
The RNase 1 was administered at 75 mg per kg of body weight of the
animal once per week (75 mg/kg 1.times.wk). The value of n
represents the number of animals in the specific treatment arm of
the experiment. Results are shown in FIG. 12 and Table 12.
TABLE-US-00012 TABLE 12 Starting Final (Final - (Final - volume
volume start) start)/Control % TGI Vehicle 63 2732 2669 1 0 wt
human RNase 1 62 669 607 0.23 77% (75 mg)
Example 9
In Vivo Determination of Inhibition of Tumor Growth
[0149] The xenograft model of this example was set up as in the
previous example. The efficacy of wild type human RNase 1 is shown.
The RNase 1 was administered at three different doses: (1) 15 mg
per kg of body weight of the animal once per week (15 mg/kg
1.times.wk), (2) 75 mg/kg 1.times.wk, or (3) 300 mg/kg 1.times.wk.
The value of n represents the number of animals in the specific
treatment arm of the experiment. Results are shown in FIG. 13 and
Table 13.
TABLE-US-00013 TABLE 13 Starting Final (Final - (Final - volume
volume start) start)/Control % TGI Vehicle 117 1256 1139 1 0 wt
human RNase 1 110 1246 1136 1 0 (15 mg) wt human RNase 1 114 559
445 0.39 61% (75 mg) wt human RNase 1 114 766 652 0.57 43% (300
mg)
Example 10
In Vivo Determination of Inhibition of Tumor Growth
[0150] The xenograft model of this example was set up as in the
previous example except that a human breast cancer cell line
(MDA-MB-231) and female mice were used. The efficacy of wild type
human RNase 1 is shown relative to doxorubicin. The RNase 1 was
administered at 75 mg per kg of body weight of the animal once per
week (75 mg/kg 1.times.wk), while the doxorubicin was given at 3
mg/kg once per week. The value of n represents the number of
animals in the specific treatment arm of the experiment. Results
are shown in FIG. 14 and Table 14.
TABLE-US-00014 TABLE 14 Starting Final (Final - (Final - volume
volume start) start)/Control % TGI Vehicle 73 211 138 1 0 wt human
RNase 1 71 121 50 0.36 64% (75 mg) doxorubicin (3 mg) 64 86 22 0.16
84%
[0151] All publications and patents mentioned in the above
specification are herein incorporated by reference as if expressly
set forth herein. Various modifications and variations of the
described method and system of the invention will be apparent to
those skilled in the art without departing from the scope and
spirit of the invention. Although the invention has been described
in connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention that are
obvious to those skilled in relevant fields are intended to be
within the scope of the following claims.
Sequence CWU 1
1
11128PRTHomo sapiens 1Lys Glu Ser Arg Ala Lys Lys Phe Gln Arg Gln
His Met Asp Ser Asp 1 5 10 15 Ser Ser Pro Ser Ser Ser Ser Thr Tyr
Cys Asn Gln Met Met Arg Arg 20 25 30 Arg Asn Met Thr Gln Gly Arg
Cys Lys Pro Val Asn Thr Phe Val His 35 40 45 Glu Pro Leu Val Asp
Val Gln Asn Val Cys Phe Gln Glu Lys Val Thr 50 55 60 Cys Lys Asn
Gly Gln Gly Asn Cys Tyr Lys Ser Asn Ser Ser Met His 65 70 75 80 Ile
Thr Asp Cys Arg Leu Thr Asn Gly Ser Arg Tyr Pro Asn Cys Ala 85 90
95 Tyr Arg Thr Ser Pro Lys Glu Arg His Ile Ile Val Ala Cys Glu Gly
100 105 110 Ser Pro Tyr Val Pro Val His Phe Asp Ala Ser Val Glu Asp
Ser Thr 115 120 125
* * * * *