U.S. patent application number 15/212833 was filed with the patent office on 2017-01-05 for non-natural ribonuclease conjugates as cytotoxic agents.
The applicant listed for this patent is Quintessence Biosciences, Inc.. Invention is credited to Thomas Burke, Peter A. Leland, Laura E. Strong.
Application Number | 20170000859 15/212833 |
Document ID | / |
Family ID | 35375962 |
Filed Date | 2017-01-05 |
United States Patent
Application |
20170000859 |
Kind Code |
A1 |
Strong; Laura E. ; et
al. |
January 5, 2017 |
NON-NATURAL RIBONUCLEASE CONJUGATES AS CYTOTOXIC AGENTS
Abstract
The present invention is directed toward the delivery of a toxic
protein to pathogenic cells, particularly cancer cells. In
preferred embodiments, the toxic protein is a ribonuclease that has
been modified to make it toxic to target cells and that can be
conjugated to a target cell-specific delivery vector, such as an
antibody, for delivery to pathogenic cells.
Inventors: |
Strong; Laura E.;
(Stoughton, WI) ; Leland; Peter A.; (Fitchburg,
WI) ; Burke; Thomas; (Madison, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Quintessence Biosciences, Inc. |
Madison |
WI |
US |
|
|
Family ID: |
35375962 |
Appl. No.: |
15/212833 |
Filed: |
July 18, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14252475 |
Apr 14, 2014 |
9393319 |
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15212833 |
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13923008 |
Jun 20, 2013 |
8697065 |
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14252475 |
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11105041 |
Apr 13, 2005 |
8470315 |
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13923008 |
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60561609 |
Apr 13, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/14 20180101;
A61P 35/00 20180101; A61K 47/6867 20170801; C07K 16/3061 20130101;
C12N 9/96 20130101; C12N 9/22 20130101; A61K 38/465 20130101; A61K
47/6851 20170801; C12N 9/12 20130101; A61P 43/00 20180101; C07K
16/2803 20130101; A61K 47/6815 20170801; C07K 16/18 20130101; C12Y
301/27005 20130101 |
International
Class: |
A61K 38/46 20060101
A61K038/46; C12N 9/96 20060101 C12N009/96; A61K 47/48 20060101
A61K047/48; C12N 9/22 20060101 C12N009/22 |
Claims
1. A composition comprising a non-natural human ribonuclease one
(human RNase I) conjugated to a cell- or disease-specific targeting
moiety, wherein said ribonuclease is configured to kill said cell
or degrade pathogenic RNA.
2. The composition of claim 1, wherein said non-natural human
ribonuclease one has a variant sequence that disrupts binding to
the ribonuclease inhibitor.
3. The composition of claim 1, wherein said non-natural human
ribonuclease one has a variant sequence compared to a natural
ribonuclease one selected from the group consisting of: N88C/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/R4C, G38R, R39D, L86E, N88R, G89D, R91D, V118C/ and R4C, K7A,
L86E, N88R, G89D, R91D, V118C.
4. The composition of claim 1, wherein said cell is a cancer cell,
virally infected cell, or immune system cell.
5. The composition of claim 1, wherein said pathogenic RNA is of
viral origin.
6. The composition of claim 1, wherein said ribonuclease is
conjugated to said cell- or disease-specific targeting moiety by a
linker.
7. The composition of claim 6, wherein said linker is attached to a
non-native cysteine of said ribonuclease.
8. The composition of claim 1, wherein said targeting moiety
comprises an antigen binding protein or immunoglobulin.
9. The composition of claim 8, wherein said immunoglobulin
comprises a human or humanized antibody.
10. The composition of claim 8, wherein said immunoglobulin
comprises an antibody fragment.
11. The composition of claim 1, wherein said targeting moiety
comprises a receptor ligand.
12. The composition of claim 1, wherein said targeting moiety
comprises a small molecule.
13. The composition of claim 12, wherein said small molecule
comprises a lipid or carbohydrate.
14. The composition of claim 1, wherein said targeting moiety
comprises an engineered non-natural protein.
15. The composition of claim 1, wherein said targeting moiety
comprises a polymer.
16. The composition of claim 1, wherein said targeting moiety is
conjugated to said ribonuclease within a loop region of said
ribonuclease corresponding to amino acids 84-95 of bovine
ribonuclease A.
17. The composition of claim 1, wherein said ribonuclease and said
targeting moiety comprise a fusion protein.
18. A composition comprising a nucleic acid molecule that encodes
the composition of claim 1.
19. A method for killing a cell comprising the step of exposing a
cell to the composition of claim 1.
20. The method of claim 19, wherein said cell is a cancer cell
virally infected cell, or immune system cell.
21. The composition of claim 10, wherein said antibody fragment is
an Fc fragment.
Description
[0001] The present application is a continuation of U.S. patent
application Ser. No. 14/252,475, filed Apr. 14, 2014, which is
divisional of U.S. patent application Ser. No. 13/923,008, filed
Jun. 20, 2013, now U.S. Pat. No. 8,697,065, issued Apr. 15, 2014,
which is a divisional of U.S. patent application Ser. No.
11/105,041, filed Apr. 13, 2005, now U.S. Pat. No. 8,470,315,
issued Jun. 25, 2013, which claims priority to U.S. Provisional
Application Ser. No. 60/561,609 filed Apr. 13, 2004, each of which
are herein incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention is directed toward the delivery of a
toxic protein to pathogenic cells, particularly cancer cells. In
preferred embodiments, the toxic protein is a ribonuclease that has
been modified to make it toxic to target cells and that can be
conjugated to a target cell-specific delivery vector, such as an
antibody, for delivery to pathogenic 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.
SUMMARY OF THE INVENTION
[0004] The present invention relates to the use (e.g., therapeutic
use, diagnostic use, research use) of proteins to target cancers
and other diseases and conditions (e.g., viral or pathogen
infections) where selectively killing pathogenic cells is desired.
For example, in some embodiments, the present invention relates to
the production and delivery of a cytotoxic protein to pathogenic
cells such as tumor cells or virus-infected cells. The ribonuclease
can also be used to degrade pathogenic RNA outside of the cell. In
some preferred embodiments, the present invention provides the use
of ribonuclease proteins (e.g., human ribonuclease proteins) that
are altered in their amino acid sequence (i.e., non-natural) to
make them cytotoxic. In some embodiments, these mutated proteins
are specifically delivered to pathogenic cells by conjugation to
targeting vectors (e.g., human or humanized protein) that are
specific for or at least partially selective for the pathogenic
target cells. Such targeting vectors include, but are not limited
to, antibodies, receptors, ligands, peptides, nucleic acids,
lipids, polymers, small molecules, and synthetic compounds. In some
embodiments, mutant ribonuclease genes are delivered as DNA or RNA
via expression vectors. The ribonuclease genes may be expressed
alone, or may be expressed as chimerical conjugates of the
ribonuclease gene with a cell-specific targeting moiety.
[0005] The present invention also provides methods comprising the
delivery of the cytotoxic ribonucleases under conditions that
minimize or eliminate the human immune response against the
proteins and delivery vectors. This present invention further
provides methods for selective inhibition of cellular growth and/or
viral replication in target cells through the action of the mutated
ribonucleases.
[0006] Thus, in some embodiments, the present invention provides a
novel family of proteins for treating, characterizing, or
understanding disease. In some embodiments, the compositions of the
present invention are used therapeutically, alone or in combination
with other compounds or interventions (e.g., to augment existing
therapies for treatment of human cancers).
[0007] Thus, in some embodiments, the present invention provides a
composition comprising a non-natural ribonuclease (e.g., human
ribonuclease) conjugated to a cell-specific targeting moiety,
wherein the ribonuclease is configured to kill the cell. In some
embodiments, the non-natural human ribonuclease comprises a
non-natural human ribonuclease one (RNase 1). Examples of suitable
non-natural human RNase 1 compounds include, but are not limited
to, those having a variant sequence compared to a natural
ribonuclease one as shown in Table 1.
TABLE-US-00001 TABLE 1 N88C 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 R4C, K7A, L86E, N88R, G89D, R91D,
V118C
The present invention further provides variants of such sequences.
Exemplary variants are provided in Tables 2 and 3, below, as well
as in Example 2. Additional variants that have the desired function
are also within the scope of the invention.
TABLE-US-00002 TABLE 2 Human ribonuclease I amino acid
modifications for increased cytotoxicity Amino Acid Amino Acid
Position Identity Amino Acids Substitution 7 Lysine (K) Glycine
(G), Alanine (A), Aspartatic acid (D), Glutamatic acid (E),
Phenylalanine (F), Tryptophan (W) 85 Arginine (R) Aspartatic acid
(D), Glutamatic acid (E), Phenylalanine (F), Tryptophan (W),
Glycine (G), Alanine (A) 86 Leucine (L) Aspartatic acid (D),
Glutamatic acid (E), Phenylalanine (F), Lysine (K), Arginine (R),
Tryptophan (W) 87 Threonine (T) Leucine (L), Phenylalanine (F),
Tyrosine (Y), Tryptophan (W) 88 Asparagine (N) Lysine (K), Arginine
(R), Leucine (L), Aspartic acid (D), Glutamatic acid (E),
Phenylalanine (F), Tyrosine (Y), Tryptophan (W) 89 Glycine (G)
Lysine (K), Arginine (R), Leucine (L), Aspartic acid (D),
Glutamatic acid (E), Phenylalanine (F), Tyrosine (Y), Tryptophan
(W) 90 Serine (S) Phenylalanine (F), Tryptophan (W), Aspartatic
acid (D), Glutamatic acid (E), Lysine (K), Arginine (R) 91 Arginine
(R) Aspartatic acid (D), Glutamatic acid (E), Phenylalanine (F),
Tryptophan (W) 92 Tyrosine (Y) Glycine (G), Alanine (A), Lysine
(K), Arginine (R), Aspartic acid (D), Glutamatic acid (E) 93
Proline (P) Leucine (L), Phenylalanine (F), Tyrosine (Y),
Tryptophan (W), Lysine (K), Arginine (R), Aspartic acid (D),
Glutamatic acid (E) 94 Asparagine (N) Lysine (K), Arginine (R),
Leucine (L), Aspartic acid (D), Glutamatic acid (E), Phenylalanine
(F), Tyrosine (Y), Tryptophan (W)
TABLE-US-00003 TABLE 3 Amino Acid Amino Acid Position Identity
Amino Acids Substitution 1 Lysine (K) Cysteine (C) 2 Glutamic acid
(E) Cysteine (C) 3 Serine (S) Cysteine (C) 4 Arginine (R) Cysteine
(C) 5 Alanine (A) Cysteine (C) 6 Lysine (K) Cysteine (C) 116 Valine
(V) Cysteine (C) 117 Proline (P) Cysteine (C) 118 Valine (V)
Cysteine (C) 119 Histidine (H) Cysteine (C) 120 Phenylalanine (F)
Cysteine (C) 121 Aspartate (D) Cysteine (C)
[0008] In some embodiments, a plurality of different ribonucleases
and/or targeting moieties are provided in a composition (e.g., a
kit, a pharmaceutical preparation, etc.)
[0009] The present invention is not limited by the nature of or
location of the target cell. In some embodiments, the cell is a
cancer cell, a cell that expresses a marker associated with viral
infection, a cell that is associated with an inflammatory response,
and a cell is associated with an autoimmune disease (e.g., a cell
expressing markers or otherwise characterized as aberrantly failing
to undergo cell death or presenting autoantigens). In some
embodiments, the cell resides in vitro (e.g., in culture). In other
embodiments, the cell resides in vivo (e.g., in a tissue, as a
transplanted cell, in a test animal, in a subject suspected of or
diagnosed as having a disease or condition--e.g., cancer).
[0010] In some preferred embodiments, the ribonuclease is
conjugated to the cell-specific targeting moiety by a linker. The
present invention is not limited by the nature of the linker.
Linkers suitable for use with the present invention include, but
are not limited to, linkers attached to a non-native cysteine of
the ribonuclease, non-cleavable linkers, cleavable linker, and
linkers attached within a loop region of the ribonuclease
corresponding to amino acids 84-95 of bovine ribonuclease A.
[0011] In some embodiments, the ribonuclease is made as a fusion
protein with a disease-specific protein, such as an antibody or
antibody fragment. Those skilled in the art recognize that the
fusion can be created using cDNA and standard molecular biology
techniques.
[0012] The present invention is not limited by the nature of the
cell-specific targeting moiety. Targeting moieties include, but are
not limited to, immunoglobulins (e.g., antibodies, humanized or
partially humanized antibodies, antibody fragments, etc.),
proteins, peptides, receptor ligands, aptamers, small molecules,
nucleic acid molecules, lipids, etc.
[0013] In some embodiments, the components of the composition
(e.g., the ribonuclease, the cell specific-targeting moieity) are
provided to a cell, alone or together via an expression vector,
such that the components are produce within a cell of a subject or
produced within a cell provided to the subject (e.g., through ex
vivo transfection followed by transplantation).
[0014] The present invention further provides methods of killing
cell using any of the compositions discussed herein.
DESCRIPTION OF FIGURES
[0015] FIG. 1A shows a graph demonstrating the growth inhibiting
effect of QBI-119 on tumor volume (A549 cells) over a number of
days, and FIG. 1B shows a graph depicting the lack of toxicity of
QBI-119.
[0016] FIG. 2A shows a graph demonstrating the growth inhibiting
effect of QBI-119 on tumor volume (Bx-PC-3 cells) over a number of
days, and FIG. 2B shows a graph depicting the lack of toxicity of
QBI-119.
DEFINITIONS
[0017] To facilitate an understanding of the present invention, a
number of terms and phrases are defined below:
[0018] 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, IbE, 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.
[0019] 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.
[0020] The term "epitope" as used herein refers to that portion of
an antigen that makes contact with a particular immunoglobulin.
[0021] 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.
[0022] 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.
[0023] 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).
[0024] 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.
[0025] 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 or increased PSA level)
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 people 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.
[0026] 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 (e.g., PSA).
[0027] 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.
[0028] 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.
[0029] 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.
[0030] "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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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. In contrast,
the terms "modified", "mutant", and "variant" refer to a gene or
gene product that displays modifications in sequence and or
functional properties (i.e., altered characteristics) 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 characteristics 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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
[0039] The present invention is directed toward the delivery of a
toxic protein to pathogenic cells, particularly cancer cells. The
toxic protein may also have benefit when delivered to diseased
areas without being toxic to the diseased cells. In preferred
embodiments, the toxic protein is a ribonuclease that has been
modified to make it toxic to target cells and that can be
conjugated to a target cell-specific delivery vector, such as an
antibody, for delivery to pathogenic cells.
[0040] Preferred embodiments of the present invention are based on
the conversion of naturally occurring ribonucleases (e.g., human
ribonucleases) into toxic 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. These ribonucleases can be used as stand
alone reagents or they may be incorporated into general or specific
delivery systems such as polymers, dendrimers, liposomes, polymeric
nanoparticles, or block copolymer micelles. A feature of these
proteins is that they are proteins that have been engineered to be
toxic to the cells to which they are delivered. This feature
provides a toxin conjugate that is less susceptible to naturally
occurring inhibitors of the toxin. Another feature is that their
starting point was preferably a natural protein (e.g., a natural
human protein) and not a non-natural (e.g., non-human) protein that
had to be modified (e.g., humanized) significantly to escape the
immune system. One embodiment of this invention is to combine these
protein toxins with antibodies (e.g., humanized or human
antibodies) for targeting to specific pathogenic cells. These
protein conjugates make it much less likely that when used in vivo,
they will induce side effects or an immune response.
[0041] In some preferred embodiments, the present invention
provides conjugates of the EVADE human ribonuclease (Quintessence
Biosciences, Madison, Wis.) with a targeting component. As such,
the EVADE human ribonucleases exhibit improved efficacy compared to
the amphibian ribonucleases because the specific ribonucleolytic
activity is higher and the likelihood of side effects and inducing
a human immune response is lower. In addition, binding to the
native inhibitor, ribonuclease inhibitor, is disrupted for the
EVADE ribonucleases. By degrading cellular RNA in target cells, the
EVADE ribonucleases inhibit the cellular growth of the tumors and
also enhances the anti-cancer effects of conventional therapies,
including chemotherapy and radiation. It is also contemplated that
EVADE human ribonucleases are not retained in the human kidney, as
are amphibian ribonucleases. Renal toxicity of the amphibian
ribonucleases is dose limiting in mice and humans. With
conventional chemotherapy, it is often a problem that membrane
based drug pumps can eliminate the small molecule anti-cancer drugs
from the cancerous cell. This requires that higher doses of the
toxic drugs be used. Like the amphibian ribonuclease, it is
expected that the EVADE ribonucleases are able to make these
resistant cells susceptible to standard levels of treatment so that
lower doses are effective and side effects reduced. In addition,
the EVADE ribonucleases are contemplated to provide benefit when
used in combination with radiotherapy or other convention
interventions.
[0042] 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.
[0043] In some preferred embodiments, the ribonuclease or
ribonuclease conjugates 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 4 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-00004 TABLE 4 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-4
H-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) 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)
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) 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) 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-piperidinopiperidino)carbonyloxy]-
Company 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)
Streptozocin ZANOSAR Pharmacia & Upjohn (streptozocin
2-deoxy-2- Company [[(methylnitrosoamino)carbonyl]amino]- a(and
b)-D-glucopyranose and 220 mg citric acid anhydrous) 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)
[0044] A current and still developing approach to cancer therapy
involves using cancer cell-specific reagents to target a malignant
tumor. 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 evade immune surveillance in humans.
[0045] Ribonuclease (RNase) proteins have been tested as human
therapeutics because they selectively target 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. 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
making a ribonuclease toxic to human cells, especially cancer
cells. 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.
[0046] The second approach is to mutate mammalian ribonucleases so
that they maintain high levels of ribonucleolytic activity but are
not significantly inhibited by 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 I 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.
[0047] 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 human immune response.
Therapeutic proteins that elicit immune responses in humans are
always problematic and often unacceptable. The present invention
provides ribonuclease conjugates that are derived from mutated
RNases that exhibit low immunogenicity and side effects and still
maintain high ribonucleolytic activity resulting in cancer-specific
toxicity.
[0048] Certain preferred embodiments of the present invention are
described below. While these embodiments are illustrated with
variant human ribonuclease proteins and antibody targeting
moieties, the present invention is not so limited.
Therapeutic Antibodies and Delivery of Cytotoxins
[0049] 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.
[0050] 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-00005 TABLE 2 Antibody Mode of Action Product Antibody
Target Blockade Ligand binding ERBITUX EGF receptor HUMAX-EGFR EGF
receptor Complement Dependent Cytotoxicity RITUXAN CD20 HUMAX-CD20
CD20 CAMPATH-1H CD52 Antibody dependent cell-mediated RIXTUXAN CD20
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 conjugate ZEVALIN
CD20 BEXXAR CD20 Toxin-mediated killing by conjugate MYLOTARG CD33
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 Agents MAb
Graft versus Host Disease Anti-infective, virus MAb Human
metapneumovirus Cytostatic agent MAb Platelet derived growth factor
Cancer growth and metastosis Preclinical MAb Human beta
hydroxylases Treatment of autoimmune disease MAb Medi 507 Mixed
lymphocyte responses 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 by conjugate Preclinical MAb Various by
Immunogen and taxane derivatives Toxin-mediated killing by
conjugate Cantuzumab Can Ag receptor by mertansine immunogen
conjugate Toxin-mediated killing by conjugate Phase II MAb CD56
maytansinoid conjugate Toxin for mitosis inhibition MAb maitansine
various conjugate Toxin-mediated killing by conjugate Preclinical
MAb Antigen on squamous cell cytotoxic drug cancer (Immunogen) DM1
conjugate
[0051] Any of the targeting antibodies or agents used in these
products may also be employed by the compositions and methods of
the present invention.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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: [0057] 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. [0058] 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. [0059] 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. [0060] 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. [0061] 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.
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.).
[0066] Ribonucleases that evade ribonuclease inhibitor protein 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.
Non-Natural Ribonuclease Polynucleotides
[0067] As described above, a new family of non-natural ribonuclease
proteins that have been discovered. This family was identified by
structure-function analys for ribonuclease sequence with desired
cytotoxic activities. Accordingly, the present invention provides
nucleic acids encoding these novel non-natural ribonucleases,
homologs, and variants (e.g., mutations and polyporphisms). In some
embodiments, the present invention provides polynucleotide
sequences encoding any of the amino acid sequences listed in Tables
1-3. The present invention also provides nucleic acid that are
capable of hybridizing to such nucleic acid sequences under
conditions of low to high stringency as long as the polynucleotide
sequence capable of hybridizing encodes a protein that retains a
biological activity of a ribonuclease (e.g., cytotoxic activity).
The above nucleic acid molecules may also be associated with coding
sequences of targeting molecules (e.g., antibodies) such that the
produced amino acid sequence is a fusion between the ribonuclease
and the targeting molecule.
[0068] In still other embodiments of the present invention, the
nucleotide sequences of the present invention may be engineered in
order to alter a ribonuclease coding sequence for a variety of
reasons, including but not limited to, alterations which modify the
cloning, processing 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, to change codon preference, etc.). It is
contemplated that it is possible to modify the structure of a
peptide having a function (e.g., ribonuclease function) for such
purposes as increasing activity of the ribonuclease (e.g.,
cytotoxic activity). Such modified peptides are considered
functional equivalents of peptides having an activity of a
ribonuclease as defined herein. A modified peptide can be produced
in which the nucleotide sequence encoding the polypeptide has been
altered, such as by substitution, deletion, or addition. In
particularly preferred embodiments, these modifications do not
significantly reduce the cytotoxic activity of the modified
ribonuclease. In other words, construct "X" can be evaluated in
order to determine whether it is a member of the genus of modified
or variant ribonucleases of the present invention as defined
functionally, rather than structurally. In preferred embodiments,
the activity of a variant ribonuclease is evaluated by any known
screening method, including those described herein expressly or by
reference.
[0069] Moreover, variant forms of ribonucleases, as shown in Tables
2 and 3, are provided. Further variations of these compositions are
contemplated, including structural and functional equivalents. 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 ribonucleases 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, 2.sup.nd ed, WH Freeman and Co., 1981).
Whether a change in the amino acid sequence of a peptide results in
a functional homolog can be readily determined by assessing the
ability of the variant peptide to function in a fashion similar to
the reference protein. Peptides having more than one replacement
can readily be tested in the same manner.
[0070] 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 (e.g., LASERGENE
software, DNASTAR Inc., Madison, Wis.).
[0071] As described in more detail below, variants may be produced
by methods such as directed evolution or other techniques for
producing combinatorial libraries of variants. In still other
embodiments of the present invention, the nucleotide sequences of
the present invention may be engineered in order to alter a
ribonuclease coding sequence including, but not limited to,
alterations that modify the cloning, processing, localization,
secretion, and/or expression of the gene product.
Non-Natural Ribonuclease Polypeptides
[0072] Non-natural ribonuclease polypeptides are described in
Tables 1-3. The present invention also provides fragments, fusion
proteins or functional equivalents of these ribonuclease
proteins.
[0073] The polynucleotides of the present invention may be employed
for producing polypeptides by recombinant techniques. Thus, for
example, the polynucleotide may be included in any one of a variety
of expression vectors for expressing a polypeptide. In some
embodiments of the present invention, vectors include, but are not
limited to, chromosomal, nonchromosomal and synthetic DNA sequences
(e.g., derivatives of SV40, bacterial plasmids, phage DNA;
baculovirus, yeast plasmids, vectors derived from combinations of
plasmids and phage DNA, and viral DNA such as vaccinia, adenovirus,
fowl pox virus, and pseudorabies). It is contemplated that any
vector may be used as long as it is replicable and viable in the
host.
[0074] 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 the invention 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.
[0075] 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), 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.
[0076] 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 or ampicillin
resistance in E. coli).
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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, 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.
[0081] 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).
[0082] 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.
[0083] 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, 246I,
1722-1741.).
[0084] The present invention further contemplates methods of
generating sets of combinatorial mutants of the present
ribonuclease proteins and ribonuclease conjugates. Library are
screened to generate, for example, novel ribonuclease or
ribonuclease conjugate variants with improved properties (e.g.,
cytotoxicity against target cells, cell targeting, low systemic
toxicity, stability, clearance, and improved storage, handling, and
administration).
[0085] In some embodiments of the combinatorial mutagenesis
approach of the present invention, the amino acid sequences for a
population of ribonuclease variants or other related proteins are
aligned, preferably to promote the highest homology possible. Such
a population of variants can include, for example, ribonuclease
homologs from one or more species, or ribonuclease variants from
the same species but which differ due to mutation. Amino acids that
appear at each position of the aligned sequences are selected to
create a degenerate set of combinatorial sequences.
[0086] In a preferred embodiment of the present invention, the
combinatorial ribonuclease library is produced by way of a
degenerate library of genes encoding a library of polypeptides
which each include at least a portion of potential ribonuclease
protein sequences. For example, a mixture of synthetic
oligonucleotides can be enzymatically ligated into gene sequences
such that the degenerate set of potential ribonuclease sequences
are expressible as individual polypeptides, or alternatively, as a
set of larger fusion proteins (e.g., for phage display) containing
the set of ribonuclease sequences therein.
[0087] There are many ways by which the library of potential
ribonuclease homologs and variants can be generated from a
degenerate oligonucleotide sequence. In some embodiments, chemical
synthesis of a degenerate gene sequence is carried out in an
automatic DNA synthesizer, and the synthetic genes are ligated into
an appropriate gene for expression. The purpose of a degenerate set
of genes is to provide, in one mixture, all of the sequences
encoding the desired set of potential ribonuclease sequences. The
synthesis of degenerate oligonucleotides is well known in the art
(See e.g., Narang, Tetrahedron Lett., 39:3 9 [1983]; Itakura et
al., Recombinant DNA, in Walton (ed.), Proceedings of the 3rd
Cleveland Symposium on Macromolecules, Elsevier, Amsterdam, pp
273-289 [1981]; Itakura et al., Annu. Rev. Biochem., 53:323 [1984];
Itakura et al., Science 198:1056 [1984]; Ike et al., Nucl. Acid
Res., 11:477 [1983]). Such techniques have been employed in the
directed evolution of other proteins (See e.g., Scott et al.,
Science 249:386-390 [1980]; Roberts et al., Proc. Natl. Acad. Sci.
USA 89:2429-2433 [1992]; Devlin et al., Science 249: 404-406
[1990]; Cwirla et al., Proc. Natl. Acad. Sci. USA 87: 6378-6382
[1990]; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and
5,096,815, each of which is incorporated herein by reference).
[0088] It is contemplated that the ribonuclease nucleic acids can
be utilized as starting nucleic acids for directed evolution. These
techniques can be utilized to develop ribonuclease variants having
desirable properties.
[0089] In some embodiments, artificial evolution is performed by
random mutagenesis (e.g., by utilizing error-prone PCR to introduce
random mutations into a given coding sequence). This method
requires that the frequency of mutation be finely tuned. As a
general rule, beneficial mutations are rare, while deleterious
mutations are common. This is because the combination of a
deleterious mutation and a beneficial mutation often results in an
inactive enzyme. The ideal number of base substitutions for
targeted gene is usually between 1.5 and 5 (Moore and Arnold, Nat.
Biotech., 14, 458-67 [1996]; Leung et al., Technique, 1:11-15
[1989]; Eckert and Kunkel, PCR Methods Appl., 1:17-24 [1991];
Caldwell and Joyce, PCR Methods Appl., 2:28-33 (1992); and Zhao and
Arnold, Nuc. Acids. Res., 25:1307-08 [1997]). After mutagenesis,
the resulting clones are selected for desirable activity (e.g.,
screened for ribonuclease activity and/or cytotoxicity). Successive
rounds of mutagenesis and selection are often necessary to develop
enzymes with desirable properties. It should be noted that,
preferably, only the useful mutations are carried over to the next
round of mutagenesis.
[0090] In other embodiments of the present invention, the
polynucleotides of the present invention are used in gene shuffling
or sexual PCR procedures (e.g., Smith, Nature, 370:324-25 [1994];
U.S. Pat. Nos. 5,837,458; 5,830,721; 5,811,238; 5,733,731; all of
which are herein incorporated by reference). Gene shuffling
involves random fragmentation of several mutant DNAs followed by
their reassembly by PCR into full length molecules. Examples of
various gene shuffling procedures include, but are not limited to,
assembly following DNase treatment, the staggered extension
process
(STEP), and random priming in vitro recombination. In the DNase
mediated method, DNA segments isolated from a pool of positive
mutants are cleaved into random fragments with DNaseI and subjected
to multiple rounds of PCR with no added primer. The lengths of
random fragments approach that of the uncleaved segment as the PCR
cycles proceed, resulting in mutations in present in different
clones becoming mixed and accumulating in some of the resulting
sequences. Multiple cycles of selection and shuffling have led to
the functional enhancement of several enzymes (Stemmer, Nature,
370:398-91 [1994]; Stemmer, Proc. Natl. Acad. Sci. USA, 91,
10747-51 [1994]; Crameri et al., Nat. Biotech., 14:315-19 [1996];
Zhang et al., Proc. Natl. Acad. Sci. USA, 94:4504-09 [1997]; and
Crameri et al., Nat. Biotech., 15:436-38 [1997]).
[0091] A wide range of techniques are known in the art for
screening gene products of combinatorial libraries made by point
mutations, and for screening cDNA libraries for gene products
having a certain property. Such techniques will be generally
adaptable for rapid screening of the gene libraries generated by
the combinatorial mutagenesis or recombination of ribonuclease
homologs. The most widely used techniques for screening large gene
libraries typically comprises cloning the gene library into
replicable expression vectors, transforming appropriate cells with
the resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates relatively easy isolation of the vector encoding the
gene whose product was detected.
EXAMPLES
Example 1
Exemplary Embodiments
[0092] The following Example describes a number of exemplary
embodiments of the compositions and methods of the present
invention.
[0093] Ribonuclease:
[0094] The EVADE family of ribonucleases comprises several members,
based on human ribonuclease one (RNase I, also known as human
pancreatic ribonuclease, hpRNase or hRNase). Some of the EVADE
ribonucleases have been assigned the following numbers (QBI-#####)
and the single letters and numbers describe the amino acid changes.
For example, N88C refers to a substitution of Cysteine (C) for
Asparagine (N) at position 88.
[0095] Amino Acid Sequence for Bovine Ribonuclease A
TABLE-US-00006 1 10 20 30 40 KETAAAKFE RQHMDSSTSA ASSSNYCNQM
MKSRNLTKDR CKPVNTFVHE 50 60 70 80 90 SLADVQAVCS QKNVACKNGQ
TNCYQSYSTM SITDCRETGS SKYPNCAYKT 100 110 120 TQANKHIIVA CEGNPYVPVH
FDASV
[0096] Amino Acid Sequence for Human Pancreatic Ribonuclease I
TABLE-US-00007 1 10 20 30 40 KESRAKKFQ RQHMDSDSSP SSSSTYCNQM
MRRRNMTQGR CKPVNTFVHE 50 60 70 80 90 PLVDVQNVCF QEKVTCKNGQ
GNCYKSNSSM HITDCRLTNG SRYPNCAYRT 100 110 120 SPKERHIIVA CEGSPYVPVH
FDASVEDST
TABLE-US-00008 Amino Acid Three Letter Abbreviations Single Letter
Abbreviations Alanine Ala A Arginine Arg R Asparagine Asn N
Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid
Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L
Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P
Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine
Val V
TABLE-US-00009 QBI-50101 N88C RNase I QBI-50109 L86E, N88R, G89D,
R91D RNase I QBI-50110 R4C, L86E, N88R, G89D, R91D, V118C RNase I
QBI-50111 L86E, N88C, R91D RNase I QBI-50112 R4C, L86E, N88C, R91D,
V118C RNase I QBI-50118 R4C, N88C, V118C RNase I QBI-50125 K7A,
L86E, N88C, R91D RNase I QBI-50126 K7A, L86E, N88R, G89D, R91D
RNase I QBI-50127 R4C, K7A, L86E, N88C, R91D, V118C RNase I
QBI-50128 R4C, K7A, L86E, N88R, G89D, R91D, V118C RNase I
[0097] In some embodiments, EVADE ribonucleases are conjugated to
molecules that accelerate their targeting to and uptake by diseased
cells (e.g., cancer, viruses, autoimmune diseases). This type of
modification extends the utility and enhances the efficacy of the
EVADE ribonucleases. An EVADE ribonuclease that has been modified
to carry a Cys at any amino acid can be readily adapted for use in
this conjugation strategy. Amino acids in the loop region
corresponding to amino acids 84-95 of bovine ribonuclease A are of
particular interest for conjugation. A preferred conjugation
partner is an antibody that binds to a cell-specific epitope (e.g.
a cancer marker). The antibody is cross-linked to the EVADE
ribonuclease via a non-cleavable or a cleavable cross-linker. A
non-cleavable chemical cross-linker may include
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Alternatively,
a cleavable linker may be used, with the cleavage occurring as a
result of enzymatic activity (e.g., protease or a lactamase) or a
change in environment (e.g., reducing or acidic environment). A
number of chemistries are available for conjugation to an antibody,
including an aldehyde (generated by oxidation of carbohydrate), an
amine (present on the lysine side chains), or a thiol (particularly
useful for conjugation to Fab' fragment or scFv).
TABLE-US-00010 Cross-linker Ribonuclease Antibody category Examples
thiol of Cys of thiol of Fab' bifunctional thiol BMB (8.0 .ANG.)
EVADE ribonuclease fragment BMDP (10.2 .ANG.) BMOE (10.9 .ANG.)
BM(PEO).sub.3 (14.7 .ANG.) BM(PEO).sub.4 (17.8 .ANG.) thiol of Cys
of non-native Cys of bifunctional thiol See above EVADE
ribonuclease single chain (scFv) thiol of Cys of Thiol of mAb
bifunctional thiol See above EVADE ribonuclease (introduced by
reaction of small molecule with amines of mAb; e.g with
iminothiolane) thiol of Cys of Amine of mAb thiol, amine sulfo-GMBS
(6.8 .ANG.) EVADE ribonuclease heterobifunctional sulfo-SIAB (10.6
.ANG.) sulfo-EMCS (9.4 .ANG.) thiol of Cys of Aldehyde of mAb
thiol, aldehyde BMPH (8.1 .ANG.) EVADE ribonuclease
heterobifunctional EMCH (11.82 .ANG.) KMUH (19 .ANG.)
M.sub.2C.sub.2H MPBH (17.9 .ANG.) thiol of Cys of Amine of antibody
Thiol, amine PDPH EVADE ribonuclease heterobifunctional cleavable
linker
[0098] A cleavable cross-linker of particular interest is made by
incorporation of a protease sensitive peptide into the
cross-linker. The protease, in some embodiments, is selected from a
wide variety of naturally occurring enzymes, including endosomal
and lysosomal proteases. Cathepsin B (a lysosomal cysteine protease
of the papain family) expression is elevated in some cancerous
cells, especially at the invasive edge of the tumor. Cathepsin B
preferentially cleaves the Arg-Arg dipeptide, but is promiscuous in
its substrate recognition. Furin is a cellular endoprotease that
catalyzes the proteolytic maturation of proteins in the secretory
pathway. Furin localizes predominately to the trans golgi network,
but does travel to many cellular compartments, including endosomes,
lysosomes, secretory granules, and the cell surface.
[0099] An alternative strategy is to use a cross linker that is
sensitive to .beta.-lactamase and administer the .beta.-lactamase
concomitantly with the targeted EVADE ribonuclease. There are
several additional types of linkers that can be cleaved such as
peptide bonds, disulfide bonds, hydrazones, and phosphodiesters.
The present invention is not limited to the linkers discussed
herein. One skilled in the art will appreciate that a variety of
linkers will find use with the present invention.
Conjugating Antibodies:
[0100] Many different antibodies may be conjugated the EVADE
ribonuclease to generate conjugates of the present invention. The
CEA antigen is one of many known protein antigens that are
over-expressed on the surface of cancer cells and has been used
previously to create targeted therapeutics. Another antigen is
CD33, which is present on acute myeloid leukemia cells. Acute
myeloid leukemia (AML) is a cancer that may be treated by an
anti-CD33 strategy. MYLOTARG is a CD33 antibody-calicheamicin
conjugate approved for treatment of AML. CD22 is a cell surface
receptor found on B-cells which can also be used for antibody-based
therapeutics.
[0101] The targeted EVADE ribonucleases may also be made using
antibodies against other cancer cell antigens. A variety of
antibodies may be amenable to such a conjugation strategy. Among
these, the preferred antigens of interest are: [0102]
over-expressed on cancer cells relative to normal cells [0103]
internalized by the cell (to facilitate ribonuclease entry into the
cytosol) [0104] recognized by a monoclonal antibody
[0105] The following examples describe conjugates of humanized M195
(huM195), an antibody specific for CD33 (Immunotoxin Resistance in
Multidrug Resistant Cells. Cancer Res., 2003, 63, 72-79.) and an
EVADE ribonuclease QBI-50112 (R4C, L86E, N88C, R91D, V118C RNase
I). The linkers are varied and include stable and cleavable
linkers.
Non-Cleavable Linkers
Maleimide-Hydrazine
[0106] Carbohydrates found in the constant region of an antibody is
oxidized to provide an aldehyde, which is reactive with hydrazine.
The hydrazine of a cross-linker (BMPH, KMUH) is reacted with the
aldehydes (oxidized carbohydrates) to form a hydrazone. The
modified antibody then displays a maleimide, which is reacted with
the free thiol in a protein to form an antibody-protein conjugate.
Thioether formation takes place at neutral pH.
[0107] The carbohydrates of huM195 are oxidized with by treatment
of the antibody with 10 mM sodium periodate at room temperature for
approximately one hour at 4.degree. C. The reaction is performed in
the dark because sodium periodate is light sensitive. A desalting
column (Amersham Biosciences, Sephadex G-25 Fine) is used prior to
use of oxidized huM195 for conjugation. A solution of BMPH is added
to the oxidized huM195, and the reaction allowed to proceed for
30-60 minutes at room temperature. The reaction is then applied to
a desalting column (Amersham Biosciences, Sephadex G-25 Fine)
[0108] Reaction times, ratios of reagents, solution concentrations,
and temperatures may be optimized to increase yield and purity of
the conjugate. Fractions are collected, and their absorbance at 280
nm monitored. Once the maleimide-huM195 containing fractions are
pooled, a solution of the EVADE ribonuclease variant QBI-50112 (20
mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0 (PBS) with 10 mM
EDTA) with a free cysteine residue is added in a one to one ratio
with maleimide-huM195. The reaction is allowed to proceed for 30
minutes and then quenched by the addition of Tris buffer with
cysteine. The conjugated sample is applied to a desalting column
(Amersham Biosciences, Sephadex G-25 Fine) and eluted with buffer
(10 mM sodium phosphate, 150 mM NaCl, pH 7.4). The fractions are
monitored using the 280 nm absorbance. The product-containing
(huM195-QBI-50112) fractions are pooled. Reaction times, ratios of
reagents, solution concentrations, and temperatures may be
optimized to increase yield and purity of the conjugate.
##STR00001##
Maleimide-NHS
[0109] The activated ester (an N-hydroxy succinimide) can be
selectively reacted with amines in the antibody (typically lysine
side chains) without affecting the maleimide. A covalent,
chemically stable amide bond is formed. The modified antibody is
then reacted with the single free thiol in the ribonuclease variant
to form the conjugate. The reaction of the thiol of the
ribonuclease variant with the maleimide is most selective at pH
6.5-7.5.
[0110] A four-fold excess of the crosslinker (EMCS or SMCC; Pierce)
is dissolved in DMF (or DMSO if necessary) and is then added to a
solution of huM195 in buffer (20 mM sodium phosphate buffer, 0.15 M
NaCl, pH 7.0 (PBS)). The reaction is allowed to proceed for 30
minutes at 4.degree. C. The reaction mixture is applied to a
desalting column (Amersham Biosciences, Sephadex G-25 Fine).
Fractions are collected, and their absorbance at 280 nm monitored.
Once the maleimide-huM195 containing fractions are pooled, a
solution of the EVADE ribonuclease variant QBI-50112 (20 mM sodium
phosphate buffer, 0.15 M NaCl, pH 7.0 with 10 mM EDTA) with a free
cysteine residue is added in a one to one ratio with
maleimide-huM195. The reaction is allowed to proceed for 30 minutes
and then quenched by the addition of Tris buffer with cysteine. The
conjugated sample is applied to a desalting column (Amersham
Biosciences, Sephadex G-25 Fine) and eluted with buffer (10 mM
sodium phosphate, 150 mM NaCl, pH 7.4). The fractions are monitored
using the 280 nm absorbance. The product-containing
(huM195-QBI-50112) fractions are pooled. Reaction times, ratios of
reagents, solution concentrations, and temperatures may be
optimized to increase yield and purity of the conjugate.
##STR00002##
.alpha.-Haloacetyl-NHS
[0111] The activated ester (an N-hydroxy succinimide) is
selectively reacted with amines without affecting the haloacetyl.
The optimal pH for the reaction is pH 7-9. The modified antibody is
then reacted with the single free thiol in the ribonuclease variant
to form the conjugate.
[0112] A solution of crosslinker (SBAP or SIAB) in DMSO is added to
huM195 solution (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2) and
allowed to react for approximately 30 minutes. The reaction mixture
is be run over a desalting column (Amersham Biosciences, Sephadex
G-25 Fine) with borate buffer (50 mM sodium borate, pH 8.3, 5 mM
EDTA).
[0113] Fractions are collected, and their absorbance at 280 nm
monitored. A solution of the EVADE ribonuclease variant QBI-50112
(R4C, L86E, N88C, R91D, V118C RNase I) is added, and the reaction
of the single free thiol of the RNase with the haloacetyl sits for
approximately one hour. These reactions are performed in the dark
due to the potential for side products. The reactions are quenched
by the addition of Tris buffer with cysteine. The quenching
reaction is allowed to proceed for 15 minutes at room temperature
in the dark. The conjugated sample is applied to a desalting column
(Amersham Biosciences, Sephadex G-25 Fine) and eluted with buffer
(10 mM sodium phosphate, 150 mM NaCl, pH 7.4 (PBS)). The fractions
are monitored using the 280 nm absorbance. The product-containing
(huM195-QBI-50112) fractions are pooled. Reaction times, ratios of
reagents, solution concentrations, and temperatures may be
optimized to increase yield and purity of the conjugate.
##STR00003##
Cleavable Linkers
[0114] These linkers are peptide-based and are be cleaved by a
human protease. The example describes linkers cleaved by the
protease furin. Furin recognizes Arg-Xaa-Yaa-Arg, where Xaa is
unspecified and Yaa is Lys or Arg. Hydrolysis occurs after the
C-terminal Arg.
[0115] The reactive groups (maleimide, hydrazine, N-hydroxy
succinimide ester, .alpha.-halo acetyl) used in the
protease-sensitive cross-linkers are the same as the commercially
available linkers.
[0116] Peptides are dissolved in ddH.sub.2O and added at a 3-fold
molar excess to a solution of huM195 (0.1 M sodium phosphate, 0.15
M NaCl, pH 7.2) and allowed to react for approximately 30 minutes.
The reaction mixture will be run over a desalting column (Amersham
Biosciences, Sephadex G-25 Fine) with borate buffer (50 mM sodium
borate, pH 8.3, 5 mM EDTA). Fractions are collected, and their
absorbance at 280 nm monitored. A solution of the EVADE
ribonuclease variant QBI-112 in 20 mM sodium phosphate, pH 7.0,
containing NaCl (0.15 M) and EDTA (0.01 M) is added. The reaction
proceeds at room temperature with stirring. Under these conditions,
reaction between the maleimide and a thiol is favored by 1000-fold
over reaction between the maleimide and an amine. After 30 min, the
reaction will be quenched by addition of a Tris-HCl buffer
containing cysteine at a concentration 10-fold greater than that of
the peptide substitutent. Peptides bearing the .alpha.-bromo-acetyl
group are conjugated by using identical procedures, with three
exceptions. Reactions are run in a 20 mM MOPS buffer, pH 8.2,
containing NaCl (150 mM) and EDTA (0.1 mM) for 60 min at 37.degree.
C. in the dark. The conjugated sample is applied to a desalting
column (Amersham Biosciences, Sephadex G-25 Fine) and eluted with
buffer (10 mM sodium phosphate, 150 mM NaCl, pH 7.4). The fractions
are monitored using the 280 nm absorbance. The product-containing
(huM195-QBI-50112) fractions are pooled. Reaction times, ratios of
reagents, solution concentrations, and temperatures may be
optimized to increase yield and purity of the conjugate.
##STR00004##
Fusion Proteins
[0117] The cDNA encoding an anti-CEA scFv fragment will be fused to
the 5'-end of the cDNA encoding the EVade ribonuclease QBI-50110
(R4C, L86E, N88R, G89D, R91D, V118C RNase I) RNase. Six glycine
residues will be incorporated between the scFv and the ribonuclease
to minimize hindrance. The cDNA will be sequenced, cloned in E.
coli, and expressed as an insoluble protein sequestered in
inclusion bodies. The inclusion bodies will be denatured and
refolded. After refolding, the fusion will be purified by standard
protein chromatography methods, including size-exclusion and
hydrophobic interactions. Production of the anti-CEA scFv-EVade
fusion may be optimized to increase yield and/or purity.
[0118] The following reference, incorporated herein by reference in
their entireties, describe various ribonucleases, targeting
moieties, conjugation methods, pharmaceutical compositions, and
treatment methods that find use in conjunction with the present
invention: U.S. Pat. Nos. 3,627,876, 4,331,764, 4,882,421,
4,904,469, 5,200,182, 5,270,204, 5,286,487, 5,286,637, 5,359,030,
5,389,537, 5,484,589, 5,529,775, 5,540,925, 5,559,212, 5,562,907,
5,595,734, 5,660,827, 5,702,704, 5,728,805, 5,786,457, 5,840,296,
5,840,840, 5,866,119, 5,955,073, 5,973,116, 6,045,793, 6,051,230,
6,083,477, 6,175,003, 6,183,744, 6,197,528, 6,235,313, 6,239,257,
6,271,369, 6,280,991, 6,290,951, 6,312,694, 6,395,276, 6,399,068,
6,406,897, 6,416,758, 6,423,515, 6,541,619, 6,558,648, 6,649,392,
6,077,499, 4,888,172, 6,653,104, and U.S. Pat. Publ. Nos.
US20020006379A1, US20020037289A1, US20020048550A1, US20020111300A1,
US20020119153A1, US20020187153A1, US20030031669A1, US20030219785A1,
US20020106359A1, and US20030148409A1.
Example 2
Tumor Growth Inhibition by QBI-119
[0119] This Example describes the use of RNase variant QBI-119 for
tumor growth inhibition. QBI-119 is an RNA variant that is R4C,
G38R, R39D, L86E, N88R, G89D, R91D, V118C RNase I. The in vivo
efficacy of this RNase variant was determined using a standard
xenograft model. In particular, A549 non-small human cancer cells
(American Type Culture Collection Number CRL-1687) and Bx-PC-3
pancreatic cells (American Type Culture Collection Number CCL-185)
were employed with athymic nude mice. Approximately
2.times.10.sup.6 cells were implanted into the right rear flank of
5-6 week old male homozygous (nu/nu) nude mice (Harlan). 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 with vehicle (PBS) on the same
dosing schedule as the treatment arm. QBI-119 was dosed at 15 mg/kg
qd.times.5 (five times per week) throughout the course of the
study. 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##
The percent tumor growth inhibition is determined using the
following formula:
Percent tumor growth inhibition ( % TGI ) % TGI = 1 - ( final size
- starting size ) treated ( final size - starting size ) control
.times. 100 ##EQU00002##
The results of this Example are shown in FIGS. 1-2. FIG. 1A shows
the tumor growth inhibition caused by QBI-119 on A549 cells was
significant (64%), while FIG. 1B shows that QBI-119 had no
significant impact on animal weights at this dosage. FIG. 2A shows
the tumor growth inhibition caused by QBI-119 on Bx-PC-3 cells was
significant (60%), while FIG. 1B shows that QBI-119 had no
significant impact on animal weights at this dosage.
[0120] 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
21124PRTBos taurus 1Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His
Met Asp Ser Ser 1 5 10 15 Thr Ser Ala Ala Ser Ser Ser Asn Tyr Cys
Asn Gln Met Met Lys Ser 20 25 30 Arg Asn Leu Thr Lys Asp Arg Cys
Lys Pro Val Asn Thr Phe Val His 35 40 45 Glu Ser Leu Ala Asp Val
Gln Ala Val Cys Ser Gln Lys Asn Val Ala 50 55 60 Cys Lys Asn Gly
Gln Thr Asn Cys Tyr Gln Ser Tyr Ser Thr Met Ser 65 70 75 80 Ile Thr
Asp Cys Arg Glu Thr Gly Ser Ser Lys Tyr Pro Asn Cys Ala 85 90 95
Tyr Lys Thr Thr Gln Ala Asn Lys His Ile Ile Val Ala Cys Glu Gly 100
105 110 Asn Pro Tyr Val Pro Val His Phe Asp Ala Ser Val 115 120
2128PRTHomo sapiens 2Lys 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
* * * * *