U.S. patent application number 11/911427 was filed with the patent office on 2008-11-20 for conjugate comprising p21 protein for the treatment of cancer.
This patent application is currently assigned to Trojan Technologies Limited. Invention is credited to Agamemnon Epenetos, Christina Kousparou.
Application Number | 20080287357 11/911427 |
Document ID | / |
Family ID | 34611144 |
Filed Date | 2008-11-20 |
United States Patent
Application |
20080287357 |
Kind Code |
A1 |
Epenetos; Agamemnon ; et
al. |
November 20, 2008 |
Conjugate Comprising P21 Protein for the Treatment of Cancer
Abstract
The P21 protein is used as a medicament in the treatment of
cancer. A conjugate comprises a first region comprising the P21
protein, or a homologue or functional fragment thereof; and a
second region comprising a translocation factor.
Inventors: |
Epenetos; Agamemnon;
(London, GB) ; Kousparou; Christina; (London,
GB) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
PO BOX 142950
GAINESVILLE
FL
32614-2950
US
|
Assignee: |
Trojan Technologies Limited
London
GB
|
Family ID: |
34611144 |
Appl. No.: |
11/911427 |
Filed: |
April 18, 2006 |
PCT Filed: |
April 18, 2006 |
PCT NO: |
PCT/GB2006/001407 |
371 Date: |
June 16, 2008 |
Current U.S.
Class: |
514/7.5 ;
435/320.1; 530/350; 536/23.4 |
Current CPC
Class: |
A61K 31/513 20130101;
A61K 38/005 20130101; A61K 31/513 20130101; A61K 38/1709 20130101;
C07K 2319/10 20130101; A61K 45/06 20130101; A61K 31/555 20130101;
A61K 33/24 20130101; A61P 35/00 20180101; A61K 47/64 20170801; C07K
14/43581 20130101; A61K 38/1709 20130101; A61K 31/555 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 33/24 20130101; A61K 2300/00
20130101; A61K 31/337 20130101; A61P 43/00 20180101; A61K 38/1767
20130101; C07K 14/4738 20130101; A61P 31/00 20180101; A61K 31/337
20130101 |
Class at
Publication: |
514/12 ; 530/350;
536/23.4; 435/320.1 |
International
Class: |
A61K 38/00 20060101
A61K038/00; C07K 14/00 20060101 C07K014/00; C12N 15/11 20060101
C12N015/11; A61P 31/00 20060101 A61P031/00; C12N 15/00 20060101
C12N015/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 14, 2005 |
GB |
0507598.1 |
Claims
1-13. (canceled)
14: A conjugate comprising: a) a first region comprising the P21
protein having the amino acid sequence as defined in SEQ ID No. 1,
or a homologue thereof having at least 60% sequence identity
thereto; and b) a second region comprising a translocation
factor.
15: The conjugate according to claim 14, wherein the translocation
factor comprises the homeodomain of antennapedia or a homologue or
functional fragment thereof.
16: The conjugate according to claim 14, wherein the translocation
factor comprises a histone protein or a homologue or functional
fragment thereof.
17: The conjugate according to claim 14, wherein the translocation
factor comprises a TAT protein or a homologue or functional
fragment thereof.
18: The conjugate according to claim 14, wherein the conjugate is a
fusion protein.
19: A nucleic acid encoding the conjugate according to claim
14.
20: An expression vector comprising the nucleic acid of claim 19,
operably linked to a promoter.
21: A composition comprising: (i) a conjugate comprising a) a first
region comprising the P21 protein having the amino acid sequence as
defined in SEQ ID NO:1, or a homologue thereof having at least 60%
sequence identity thereto or a functional fragment thereof; and b)
a second region comprising a translocation factor; and (ii) a
chemotherapeutic drug.
22: A composition comprising the conjugate according to claim 14 in
combination with at least one drug.
23: The composition according to claim 22, wherein the drug is
chemotherapeutic.
24: A method for the treatment of cancer, comprising administering
the conjugate of claim 14 to a subject in need thereof.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the P21 protein and conjugates and
compositions comprising the P21 protein. These products are
particularly useful in the treatment of cancer.
BACKGROUND TO THE INVENTION
[0002] Cancer is one of the major causes of human mortality and has
been the subject of intensive research. It is well known that
almost all malignant tumours result from the transformation of a
single cell into an immortal state, where the cell has lost the
ability to control proliferation.
[0003] Uncontrolled proliferation results from a malfunction in the
control of the cell cycle which, in eukarya, is controlled largely
by cyclins and cyclin-dependent kinases (CDKs). A complex mechanism
of control over the phosphorylation state of the cyclins and CDKs
controls whether cyclin/CDK complexes are able to drive the cell
into the next phase of the cycle. The importance of the
phosphorylation state of cyclin/CDK complexes is reflected by the
fact that a large number of cancers involve mutations in the P53
and P21 tumour suppressor proteins, which are key components in the
control of cyclin/CDK phosphorylation and complex formation. In
particular, P21 blocks cycD/cdk4 complex formation and causes G1
arrest. Reviews detailing the P21 protein and its biological
function may be found in O'Reilly M A, Antioxid Redox Signal. 2005
January-February; 7(1-2):108-18 and Liu G & Lozano G, Cancer
Cell. 2005 February; 7(2):113-4.
[0004] The treatment of cancer usually involves a combination of
surgical procedures, radiotherapy and chemotherapy (including
immunotherapy). Despite significant advances in cancer therapies in
recent years, there remains a constant need for the development of
improved therapies. Ideally, a cancer therapy will specifically
target and destroy cancerous cells only.
[0005] A further problem that needs to be addressed is drug
resistance resulting from chemotherapy. The continued
administration of a cytotoxic drug can cause a tumour, that was
initially sensitive to the drug, to become increasingly drug
resistant such that the drug loses its therapeutic efficacy.
Methods of improving the sensitivity of cancerous cells to drugs
are therefore also required.
SUMMARY OF THE INVENTION
[0006] This invention is based on the finding that the P21 protein
is useful in the treatment of cancer. In particular, a synergistic
effect is observed when the P21 protein is administered together
with at least one other agent used in cancer therapy.
[0007] According to a first aspect of the invention, a conjugate
comprises:
[0008] (a) a first region comprising the P21 protein, or a
homologue or functional fragment thereof; and
[0009] (b) a second region comprising a translocation factor.
[0010] According to a second aspect of the invention, a composition
comprises a conjugate comprising a first region comprising the P21
protein, or a homologue or functional fragment thereof, and a
second region comprising a translocation factor, in combination
with at least one drug.
[0011] According to a third aspect of the invention, a conjugate or
composition according to the invention may be used as a medicament,
particularly for the treatment of cancer.
[0012] According to a fourth aspect of the invention, the P21
protein or a homologue or functional fragment thereof may be used
as a medicament, particularly for the treatment of cancer.
DESCRIPTION OF THE DRAWINGS
[0013] The invention is described with reference to the following
drawings, wherein:
[0014] FIG. 1 illustrates the cytotoxicity of antennapedia/P21
fusion protein administered to the ovarian carcinoma cell line
SKOV-3 and the osteosarcoma line SAOS-2, which were grown in
96-well ELISA dishes in conditions which resemble the situation in
the human body;
[0015] FIG. 2 illustrates the cytotoxicity of the antennapedia/P21
fusion protein and its synergistic effect with cisplatin, and
SKOV-3 cells;
[0016] FIG. 3 illustrates the cytotoxicity of the antennapedia/P21
fusion protein in combination with cisplatin and taxol, on SKOV-3
cells, indicating that the antennapedia/P21 fusion, cisplatin and
taxol act synergistically when used in combination;
[0017] FIG. 4 illustrates the clearance from the blood of purified,
radiolabelled antennapedia protein (4A) and antennapedia-P21 fusion
protein (4B) in tumour-free mice;
[0018] FIG. 5 illustrates the organ-to-blood ratios and the protein
accumulation in various organs, for the antennapedia protein (5A)
and antennapedia-P21 fusion protein (5B);
[0019] FIG. 6 illustrates the reduction in the growth of tumours
using antennapedia/P21 fusion protein;
[0020] FIG. 7 is a Kaplan-Meier survival curve indicating the
survival of mice is greatest when receiving the antennapedia/P21
fusion protein at its highest concentration;
[0021] FIG. 8 demonstrates a synergistic effect in vivo when using
the antennapedia/P21 fusion protein, taxol and cisplatin.
[0022] FIG. 9 illustrates the Kaplan-Meier survival curve for mice
receiving the antennapedia/P21 fusion protein and chemotherapy, it
is clear that the best survival benefit is demonstrated by the
animals receiving both the fusion protein and supplementary
chemotherapy;
[0023] FIG. 10 illustrates the Kaplan-Meier survival curves for
mice receiving the antennapedia/P21 fusion and animals also
receiving cisplatin and taxol; and
[0024] FIG. 11 shows the Kaplan-Meier survival curves for mice
receiving the antennapedia/P21 fusion and an additional
chemotherapeutic.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The invention utilises the P21 protein as a therapeutic
agent. The P21 protein may be used as a therapeutic agent alone or
in combination with other therapeutic agents. When used in
combination with other therapeutic agents, a surprising synergy is
observed. In a preferred embodiment, the P21 protein is attached to
a translocation factor, to enable the P21 protein to enter a target
cell.
[0026] Prior to the present invention, it was not appreciated that
P21 could be used extensively in cancer therapy. Most cancers
result from mutations in the P53 gene, which is therefore the
obvious, and most favoured, target when designing cancer
therapeutics. The P21 protein, which functions further down the
apoptotic cascade, has not been considered as a therapeutic agent.
The present invention bypasses P53 and allows the direct delivery
of active P21, for the treatment of cancer.
[0027] The administration of P21 and a chemotherapeutic provides a
surprising improvement in anti-cancer therapy.
[0028] As used herein, the term "protein" refers to a polymer
molecule comprising a plurality of amino acid residues linked via
the peptide linkage, as will be appreciated by one skilled in the
art. Peptides and polypeptides are encompassed with the term
"protein".
[0029] The human P21 protein is defined herein as SEQ ID NO. 1. SEQ
ID NO.1: SEPAGDVRQN PCGSKACRRL FGPVDSEQLS RDCDALMAGC IQEARERwNF
DFVTETPLEG DFAWERVRGL GLPKLYLPTG PRRGRDELGG GRRPGTSPAL LQGTAEEDHV
DLSLSCTLVP RSGEQAEGSP GGPGDSQGRK RRQTSMTDFY HSKRRLIFSK RKP
[0030] The annotated human P21 sequence can be found in the
SWISSPROT database with the primary accession number P38936 (Entry
Name CDN1A_HUMAN). Although the human sequence (SEQ ID NO. 1) is
preferred, a P21 protein from any species may be used according to
the invention, for example the Mus musculus protein (SWISSPROT
primary accession number P39689) or the Felis silvestris catus
(Cat) protein (SWISSPROT primary accession number 019002). A
polynucleotide encoding a P21 protein is also within the scope of
the invention.
[0031] Functional variants (i.e. homologues) and fragments of the
human P21 protein (SEQ ID No. 1) are also included within the
invention. For example, proteins with high levels (e.g. greater
than 60%, preferably greater than 70%, more preferably greater than
80% and most preferably greater than 90%, e.g. greater than 95%) of
sequence similarity to SEQ ID NO. 1 are within the scope of the
present invention. The term "similarity" is known in the art. The
term refers to a comparison between amino acid sequences, and takes
into account not only identical amino acids in corresponding
positions, but also functionally similar amino acids in
corresponding positions. Similarity between polypeptide sequences
therefore indicates functional similarity, in addition to sequence
similarity.
[0032] Levels of similarity between amino acid sequences can be
calculated using known methods. Publicly available computer-based
methods for determining similarity include the BLASTP, BLASTN and
FASTA programs, the BLASTX program available from NCBI, and the Gap
program from Genetics Computer Group, Madison Wis. Levels of
similarity referred to herein may, for example, be determined using
the Gap program, with a Gap penalty of 12 and a gap length penalty
of 4.
[0033] Variants or fragments of P21 may be produced using standard
recombinant DNA techniques such as site-directed mutagenesis, as
will be apparent to the skilled person based on conventional
protein technology. Fragments or homologues of the P21 protein
should retain the function of the native P21 protein, i.e. should
be a "functional" fragment or homologue. The function that must be
retained is the ability to act as a tumour-suppressor protein, and
block cycD/CDK4 complex formation, and therefore cause G1 arrest of
a eukaryotic cell. Tests for cell division and viability are
well-known in the art, for example the CellTiter 96.RTM. and
CytoTox 96.RTM. assays available from Promega Corp., Wisconsin
USA.
[0034] In a preferred embodiment, the P21 protein is associated
with a translocation factor, forming a conjugate. As used herein,
the term "conjugate" refers to a chimeric molecule formed from a
translocation factor and a P21 protein. The conjugate is therefore
a hybrid molecule not found together in their natural form.
Preferably, the formation of a conjugate does not reduce or
otherwise adversely affect the ability of the P21 protein or the
translocation factor to function as intended.
[0035] As used herein, the term "translocation" refers to the
delivery of a protein across a cell membrane. It will be apparent
to one skilled in the art that a translocation factor is required
to deliver the P21 protein to a target cell. As used herein, the
term "translocation factor" refers to any moiety that has the
ability to translocate across a cell membrane, i.e. an outer cell
membrane. When the P21 protein is associated with the translocation
factor in a conjugate, P21 is also translocated across the cell
membrane.
[0036] The translocation factor will preferably be a protein. A
number of proteins with the ability to translocate cell membranes
are known in the art, including histone proteins, the herpes
simplex virus VP22 protein and HIV tat domain. The tat protein from
HIV-type I comprises 86 amino acids encoded by two exons. The first
72 amino acids are encoded by exon 1 and exhibit full
trans-activating activity. A cluster of basic amino acid residues
in this region are known to be able to translocate across a cell
membrane. This cluster, contained within amino acid residues 37-72,
is within the scope of the invention. More specifically, amino
acids 49-58 are a preferred translocation factor as disclosed by
Vives et al., J. Biol. Chem: 272 (1997); 25: pp 16010-16017, which
is incorporated herein by reference. Amino acids 49-58 of HIV TAT
are listed below, as SEQ ID NO. 2:
TABLE-US-00001 SEQ ID NO. 2: RKKRRQRRR
[0037] However, any fragment or homologue of the HIV-type 1 tat
protein, that retains the ability to translocate a cell membrane,
is within the scope of the invention.
[0038] Specific amino acid motifs with the ability to translocate
are described in WO03/002598, which is incorporated herein by
reference. According to the current invention, it is preferred that
the homeodomain of the Drosophila antennapedia protein is used as
the translocation factor. Functional variants and homologues of the
antennapedia homeodomain may also be used, provided that they
maintain the ability to translocate. A full description of the
antennapedia homeodomain translocation factor is given in
WO-A-99/11809, the content of which is incorporated herein by
reference. The homeodomain of the Antp gene is shown in SEQ ID No.
3.
TABLE-US-00002 SEQ ID NO. 3: RKRGRQTYTR YQTLELEKEF HFNRYLTRRR
RIEIAHALCL TERQIKIWFQ NRRMKWKKEN
[0039] For the avoidance of doubt, the amino acid sequence of the
Drosophila antennapedia protein contains a motif of approximately
60 amino acids, known as the homeodomain, which has the ability to
function as a translocation factor. The full-length protein,
homeodomain or any homologue or fragment (of the protein or
homeodomain) that maintains the ability to translocate, may be used
as a translocation factor in the present invention. A homologue
preferably comprises a region with a high level (eg greater than
60%, preferably greater than 70%, more preferably greater than 80%
and most preferably greater than 90%, e.g. 95% or more) of sequence
similarity to the Drosophila antennapedia homeodomain.
[0040] Although the Drosophila homeodomain and antennapedia
full-length protein are preferred, one skilled in the art will
realise that functional homologues may originate from any organism.
Antennapedia homologues have been found in the majority of
multicellular organisms and are well conserved. For example, the
human and drosophila homeodomains differ by only one conservative
amino acid substitution. Homologues produced artificially, for
example using site-directed mutagenesis, are also within the scope
of the invention. The ability of a naturally-occurring or
artificial sequence to translocate the membrane may be tested by
routine methods that are well-known in the art.
[0041] Variants of the homeodomain which retain the ability to
translocate the membrane have been reported in the art and are
within the scope of the invention. For example, EP-B-0 485 578 to
CNRS discloses homeopeptides comprising the helix 3 sequence of
pAntp, and these are incorporated herein by reference.
[0042] WO97/12912 also to CNRS discloses the actual sequence of the
helix 3 of pAntp, and variants thereof. These also are incorporated
herein be reference. In particular, helix 3 is said to have the
sequence:
TABLE-US-00003 SEQ ID NO. 4: Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-
Arg-Arg-Met-Lys-Trp-Lys-Lys
[0043] The variants are said to have the sequence:
TABLE-US-00004 SEQ ID NO. 5: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-
X12-X13-X14-X15-X16 or SEQ ID NO. 6:
X16-X15-X14-X13-X12-X11-X10-X9-X8-X7- X6-X5-X4-X3-X2-X1
[0044] wherein each X represents an .alpha.-amino acid, with X6
representing tryptophan; said peptide comprising between 6 and 10
hydrophobic amino acids.
[0045] Other variants are disclosed in for example, Gehring W
(1987) Homeo Boxes in the Study of Development. Science 236
1245-1252 discloses a homeodomain of 62 amino acids, i.e. with glu
at position 0 and lys at position 61. Bloch-Gallego E et al (1993)
Antennapedia Homeobox Peptide Enhances Growth and Branching of
Embryonic Chicken Motoneurons In Vitro. The Journal of Cell Biology
120(2) 485-492 discloses a mutant called pAntp40P2 that was still
able to translocate through the motoneuron membrane and to reach
the nucleus. In this mutant the leucine and threonine residues in
positions 40 and 41 were replaced by two proline residues. Le Roux
et a; (1993) Neurotropic activity of the Antennapedia homeodomain
depends on its specific DNA-binding properties. Proc. Natl. Acad.
Sci. 90 9120-9124 discloses two mutants pAntp 50A and pAntp 40P2 as
shown in FIG. 2 which retain the ability to translocate through the
neuronal membrane. Schutze-Redelmeier et al (1996) supra disclose
that a 16 amino acid C-terminal (third helix) segment has been used
to address oligonucleotides and oligopeptides to the cytoplasm and
nuclei of cells culture.
[0046] All of the references listed above are hereby incorporated
wherein by reference. It is preferred that a homeodomain of about
60 residues is used.
[0047] It is preferred that the P21 protein and translocation
factor are covalently linked. The covalent linkage may be in the
form of a chemical linker molecule. More preferably, the P21
protein and translocation factor are produced as a single fusion
protein. In the fusion protein, the P21 protein and translocation
factor may be in any order. It is preferred that the translocation
factor is located at the amino terminal end of the P21 protein.
Methods of producing fusion proteins are well known in the art,
using standard recombinant nucleic acid procedures, as described in
Sambrook et al, Molecular Cloning: A Laboratory Manual (Cold Spring
Harbour Laboratory Press), the content of which is incorporated
herein by reference. Nucleic acids encoding a fusion protein are
within the scope of the invention, as are expression vectors
comprising the nucleic acid encoding the fusion protein and at
least one promoter region.
[0048] The conjugate comprising the P21 protein and a translocation
factor can be included in a composition additionally containing at
least one drug. Preferably, the drug is used in cancer therapy. In
a preferred embodiment, the drug is a chemotherapeutic, for
example, cisplatin or taxol. However, other drugs that are used in
cancer therapy may also be combined with a conjugate according to
the invention, for example anti-inflammatory drugs, antibodies
(including monoclonal antibodies), immunomodulating drugs, hormones
and hormone antagonists, antibacterials, antifungals and
antivirals. Non-limiting examples are given below.
(1) Cytotoxic Drugs/Cytostatics: alkylating agents (e.g.
cyclophosphamide, melphalan etc.), cytotoxic antibiotics
(doxorubicin, epirubicin, bleomycin, mitomycin etc.),
antimetabolites (methotrexate, capecitabine, gemcitabine,
fluorouracil, vinca alkaloids and etoposide (vinblastine,
vincristine etc.), platinum compounds (carboplatin, cisplatin,
oxaliplatin), taxanes (paclitaxel, docetaxel etc), topoisomerase I
inhibitors (irinotecan, topotecan etc.). (2) Immune Response
Modifiers: antiproliferative immunosuppressants, corticosteroids.
(3) Immunomodulators: interferons, interleukins. (4) Monoclonals:
transtuzumab, rituximab, alemtuzumab. (5) Antibacterial drugs:
penicillins, cephalosporins, cephamycins, tetracyclins, macrolides,
aminoglycosides, other antibacterials (chloramphenicol, fusidic
acid, vancomycin etc). (6) Hormones: thyroid hormones, oestrogens,
progesterones, androgens, and all their antagonists. (7) Others:
vitamins, non-steroidal anti-inflammatory drugs (e.g. celecoxib,
rofecoxib etc), vaccines, antisera, antifungal drugs, antiviral
drugs and steroids.
[0049] The P21 protein or a homologue or functional fragment
thereof, or a conjugate as described above may be used in the
treatment of cancer. Preferably, a composition comprising the P21
protein or a conjugate as described above and an additional drug is
used in the treatment of cancer.
[0050] A pharmaceutical composition containing the active component
may be in any suitable form. It is preferred that the P21 protein
or conjugate is in a form suitable for transdermal or intravenous
administration. Suitable pharmaceutically-acceptable buffers,
excipients, diluents etc. may also be present, as well be
appreciated by the skilled person. The composition may be in a form
intended for administration directly to the diseased tissue or
maybe in a form that is targeted indirectly to the diseased
tissue.
[0051] Dosage levels of the order of from about 0.1 mg to about 25
mg per kilogram of body weight per day are useful in the treatment
of the above-indicated conditions (about 8 mg to about 2 g per
patient per day). For example, the patient may be effectively
treated by the administration of from about 0.25 to 12.5 mg of the
P21 compound per kilogram of body weight per day (about 20 mg to
about 1 mg per patient per day).
[0052] The amount of active ingredient that may be combined with
the carrier materials to produce a single dosage form will vary
depending upon the host treated and the particular mode of
administration. Dosage unit forms will generally contain between
from about 1 mg to about 500 mg of active ingredient.
[0053] It will be understood, however, that the specific dose level
for any particular patient will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, sex, diet, time of administration,
route of administration, rate of excretion, drug combination and
the severity of the particular disease undergoing therapy.
[0054] In addition the P21 compound can be delivered to the site of
the disease by mechanical means, or targeted to the site of the
disease through the use of systemic targeting technologies such as
liposomes (with or without chemical modification that provides them
with affinity for the diseased tissue), antibodies, aptamers,
lectins, or chemical ligands with affinity for aspects of the
diseased tissue that are less abundant or not present on normal
tissue.
[0055] The invention is further described by the following
Examples.
EXAMPLE 1
[0056] Bacterial plasmid clones were constructed for the expression
of the P21 protein alone or as a fusion to the antennapedia
peptide. Expression of the ANTP-P21 fusion (the "fusion protein")
was maximal three hours post induction, after which protein
purification under denaturing conditions with Guanidinium
Hydrochloride and Urea followed. During renaturation and refolding,
protein precipitation was observed. This suggested that various
buffers needed to be tested. Five buffers were tried; 20 mM
Tris-base/0.5 M NaCl/0.1% Tween-20 pH 8 kept the protein in
solution.
[0057] The production of the fusion protein allowed a cell
translocation experiment to be performed, to test the ability of
the antennapedia-containing fusion protein to translocate across
biological membranes. Two cell lines were used in this experiment,
ASPC1 and HeLa (ASPC1--human pancreatic adenocarcinoma, HeLa--human
cervical adenocarcinoma).The protein was incubated for 10 and 60
min on cells in various dilutions at 37.degree. C. Translocation of
the fusion protein was evident in both cell lines, even after only
ten minutes of incubation. P21 protein alone did not appear to
translocate across cells. Protein internalisation was even evident
after incubation at 4.degree. C., suggesting an energy-independent
mechanism of translocation not involving classical endocytosis.
EXAMPLE 2
[0058] Expression of the fusion protein was scaled up to purify
sufficient material to perform cytotoxicity assays. The protein was
dialysed in Guanidinium Hydrochloride solution instead of Tris to
prevent it from precipitating. It was expected that, when taken up
by cells, endocellular disulphide isomerases and chaperones would
result in a functional protein. The ovarian carcinoma cell line
SKOV-3 and the osteosarcoma line SAOS-2 were grown in 96-well ELISA
dishes in conditions which resemble the situation in the human body
(tissue culture facility).
[0059] Fusion protein and P21 alone were given to the cells at 50
mg/ml for 24 and 48 hours. The controls included untreated cells to
give the background cell death; cells to which only Guanidinium
Hydrochloride was added; and cells which were totally lysed with
detergent in order to give 100% cell death. The experiment was
terminated at 24 and 48 hours and plates were assessed for
apoptotic cell death. The results indicated that the buffer
(Guanidinium Hydrochloride) used was itself cytotoxic to the cells
after exposure for a prolonged period of time. This background cell
death did not allow for any evidence of cytotoxicity due to the
fusion protein. Therefore, following refolding the proteins were
dialysed in PBS buffer, which is known to be non-toxic to cells.
Any precipitated material was removed by centrifugation.
[0060] The proteins were then applied to the cell cultures.
Significant cell death was observed in two cancer cell lines on
administration of the fusion protein as shown in FIG. 1.
[0061] Optimization experiments for the conditions of cellular
administration were performed. The duration of the administration
was tested, as well as the combination of this cytotoxic agent
(Antp-P21 fusion) with other agents that are known to kill SKOV-3
cells. An agent used in the experiments was cisplatin. This drug
inhibits DNA synthesis by producing intra-strand and inter-strand
crosslinks in the DNA. Protein and RNA synthesis are also inhibited
to a lesser extent. Results indicated a synergistic effect between
the two modes of therapy, and a surprisingly enhanced cytotoxic
effect in the presence of both at relatively low concentrations,
which should mimic the situation in vivo.
[0062] The set of data which demonstrates clearly the killing
ability of the Antp-P21 molecule, as well as its synergistic effect
with the killing agent cisplatin, is shown in FIG. 2.
[0063] The fusion protein was then tested in combination with
cisplatin and taxol. Taxol disrupts the dynamic equilibrium within
the microtubule system and blocks cells in the late G2 phase and M
phase of the cell cycle, inhibiting cell replication. Results
indicate a synergistic effect between the three modes of therapy,
and an enhanced cytotoxic effect in the presence of the drugs along
with the fusion protein at relatively low concentrations.
[0064] Data which demonstrates the killing ability of the fusion
protein together with its synergistic effect with the other killing
agents is shown in FIG. 3. The Antp-P21 fusion protein resulted in
increased cell death when incubated with the cytotoxic agents
cisplatin and taxol. Antp-P21, cisplatin and taxol act
synergistically when used in combination.
EXAMPLE 3
[0065] The kinetics and biodistribution of purified, radiolabelled,
antennapedia protein (alone) in tumour-free mice was tested. This
demonstrates that the protein translocates via the blood to all
tissues of the body. A summary of the results is shown in FIG.
4A.
[0066] The antennapedia protein appeared to have a rapid initial
clearance, but it took more than fifteen hours to clear completely
from the circulation. This behaviour is expected from
positively-charged proteins. This interval should allow sufficient
time for it to exit the blood vessels and accumulate in tissues.
After dissecting out and counting all organs and tissues of the
mice, the organ-to-blood ratios and the protein accumulation in
various organs was calculated; as shown in FIG. 5A.
[0067] The protein did not have an affinity for any particular type
of tissue. It appeared to accumulate in highly vascularised
tissues.
[0068] The kinetics and biodistribution of purified, radiolabelled,
antennapedia-P21 fusion protein in tumour-free mice were then
tested. The results are shown in FIGS. 4B and 5B. The fusion
protein appeared to have a rapid initial clearance, but to take
more than seventeen hours for it to clear completely from the
circulation (FIG. 4B). This behaviour resembled that observed when
the antennapedia protein alone was tested, and was expected from
positively-charged proteins. This interval was expected to allow
sufficient time for it to exit the blood vessels and accumulate in
tissues. After dissecting out and counting all organs and tissues
of the mice, we were able to calculate the organ-to-blood ratios
and to plot the biodistribution of the protein, as illustrated in
FIG. 5B.
[0069] The fusion protein did not have an affinity for any
particular type of tissue, as expected. It appeared to accumulate
in highly vascularised tissues. Experiments were performed which
allowed us to locate the protein in the tissues by staining with
anti-protein antibodies (anti-HIS antibody--Qiagen). These
immunohistochemistry studies were performed on frozen tissue
sections.
[0070] Results showed that the fusion protein accumulated in major
organs, as indicated in Table 1, and was found as a full-length
molecule. The behaviour of the molecule is likely to remain
essentially unaltered in tumour-bearing mice, with the exception of
it accumulating in large amounts in the actual tumour.
TABLE-US-00005 TABLE 1 ORGAN STAINING MUSCLE + COLON ++ SM. INTEST.
+ SPLEEN ++ LIVER ++ HEART + BRAIN - LUNGS - KIDNEYS -
[0071] An experiment was then performed in tumour-bearing animals
which indicated the optimal time of protein accumulation in the
tumour and therefore the time it takes for the fusion protein to
exit the blood circulation and enter the neighboring tissues. This
information is desirable when designing experiments which were to
involve repeated doses and time interval between each dose.
[0072] Radiolabelled fusion protein was administered as a single
dose via the tail vein to tumour-bearing mice and tumours examined
at various time points. Results demonstrated that the optimal time
of tumour localization was three hours after administration. After
this period, staining intensity and therefore protein quantity
decreased. Results are selectively shown in the following
tables:
TABLE-US-00006 TABLE 2 T = 15 MIN SKOV-3 tumour-bearing female nude
mouse injected with 25 .mu.g of I.sup.125-labelled Antennapedia-P21
fusion protein via tail vein. Essentially no staining observed. %
i.d./g tissue 3.2211
TABLE-US-00007 TABLE 3 T = 1 hour SKOV-3 tumour-bearing female nude
mouse injected with 25 .mu.g of I.sup.125-labelled Antennapedia-P21
fusion protein via tail vein. Diffuse staining observed throughout
the cells. % i.d./g tissue 1.7389
TABLE-US-00008 TABLE 4 T = 3 hours SKOV-3 tumour-bearing female
nude mouse injected with 25 .mu.g of I.sup.125-labelled
Antennapedia-P21 fusion protein via tail vein. Nuclear localization
observed. This is the time point at which maximal staining is
observed. % i.d./g tissue 1.4930
TABLE-US-00009 TABLE 5 T = 6 hours SKOV-3 tumour-bearing female
nude mouse injected with 25 .mu.g of I.sup.125-labelled
Antennapedia-P21 fusion protein via tail vein. Staining intensity
decreased. Fusion protein is possibly degraded intracellularly. %
i.d./g tissue 1.1064
[0073] The maximum tolerated dose which can be safely administered
to tumour-bearing mice was then tested. Three different
concentrations of the fusion protein was tested, as well as
controls, both intravenously and intratumourally. Animals were
monitored for signs of distress and toxicity, and measurements of
tumour diameter, mice weight and white blood cell count were
recorded. The concentration of 2.5 mg/ml of fusion protein was
shown to be the highest which can be used without toxicity effects
in a repetitive manner and was therefore used in subsequent
studies.
[0074] SKOV3 ovarian adenocarcinoma-bearing xenografts were used.
The Antennapedia-P21-treated animals were in large groups of 8
animals, and the experiment included controls such as 8 animals
receiving saline solution and 8 animals receiving increasing doses
of the Antennapedia protein only. Six injections were performed on
a weekly basis. Day zero shown in FIG. 6 is the first day of the
injections.
[0075] Results indicated that the fusion protein demonstrated
maximum tumour delay profile. The concentration of 2.5 mg/ml of the
fusion protein, was shown to be the one demonstrating maximum
efficacy. When analysing the results further, a survival profile
which supported further the mode of action of our therapeutic agent
was identified. When grouping animals by tumour size and looking at
animals with small starting tumours separately from animals with
larger starting tumours, it was noted that the benefit of treatment
in small-tumour animals is higher than in animals with larger
tumours. Penetration and therapeutic effect of proteins has been
shown to be improved in smaller tumours. The rationale behind this
is that the interstitial pressure is higher in the core of large
tumours and the environment is more difficult for non-targeted
molecules to penetrate. Therefore, therapeutic benefit was higher
in smaller tumours with lower interstitial pressure.
[0076] This theory is supported by the Kaplan-Meier data. Analysing
all the animals together gives clear survival benefit to the group
receiving the fusion protein at its highest concentration as
indicated in FIG. 7.
[0077] Conventional chemotherapeutic drugs cisplatin and taxol were
added to the therapeutic protocol at standard published doses. In
vitro, a positive effect was seen. Results indicated a synergistic
effect between the three modes of therapy, and an enhanced tumour
growth retardation effect in the presence of the drugs along with
the fusion protein at the chosen concentrations, as indicated by
FIG. 8.
[0078] The Kaplan-Meier survival curve for the animals receiving
the fusion protein and chemotherapy is given in FIG. 9. It is
evident that the best survival benefit was demonstrated by the
animals receiving both the fusion protein and supplementary
chemotherapy.
[0079] When the survival curve of the animals receiving the fusion
protein versus the curve of the animals receiving the fusion
protein plus complementary chemotherapy were compared, in FIG. 10,
it is evident that the addition of chemotherapy increased the
percentage of animals alive at all times. This demonstrated that
results in vitro correlated well with the situation which was
observed in vivo.
EXAMPLE 4
[0080] Following experiments in SKO-V3 tumours, the fusion protein
Antennapedia-P21 was tested in nude mice xenografted with colon
carcinoma RKO-E6 cells. These cells contain a stably integrated
human papilloma virus (HPV) E6 oncogene under the control of the
cytomegalovirus (CMV) promoter. The HPV E6 oncogene causes a
decrease in normal p53 levels and functions, to the extent that
this line lacks appreciable functional p53.
[0081] Lack of p53, blocks downstream p53-mediated transactivation
of target genes, such as the Cdk-inhibitor P21. The expression of
P21 results in the inhibition of Rb phosphorylation, and, thus, the
subsequent expression of E2F-dependent genes is blocked.
[0082] The RKO-E6 cell line has been used frequently to investigate
the effects of p53 loss on cellular parameter such as p53-mediated
transcription and apoptosis. In the experiments described here, it
has been used to study the effects of administering P21 to cells,
therefore by-passing the need for p53 activation. Apoptosis is
measured as tumour cell death and tumour size reduction.
[0083] Nude mice were xenografted with RKO E6 tumours, randomized
into four groups and administered one of the following via tail
vein injection: [0084] i. Phosphate-Buffered Solution (once per
week for 5 weeks) [0085] ii. 2.5 mg/ml Antennapedia-P21 Fusion
(once per week for 5 weeks) [0086] iii. 2 mg 5-fluorouracil/1 mg
Leucovorin, 0.2 mg Oxaliplatin (once per week for 5 weeks) [0087]
iv. 2.5 mg/ml Antennapedia-P21 Fusion+2 mg 5-fluorouracil/1 mg
Leucovorin, 0.2 mg Oxaliplatin (once per week for 5 weeks)
[0088] Chemotherapeutic drugs were administered in doses previously
documented to cause measurable tumour reductions. Tumour regression
was evaluated, which in turn was translated into a survival
benefit.
[0089] Results are shown in FIG. 11. reatment with PBS, Fusion
protein or Chemotherapy alone had little effect on overall
survival, with a median survival in the range of 30 days for all
these groups. A significant improvement in survival was observed in
animals treated with the Fusion in combination to Chemotherapy,
with a median survival of 45 days, demonstrating synergistic
interactions.
[0090] Because a majority of patients who succumb to colorectal
cancer do so to secondary systemic metastatic disease, therapeutic
strategies which could have an effect on metastatic disease are
needed to significantly impact this cancer. The Antennapedia-P21
fusion protein is expected to have such an effect since
administration of it has previously been shown to result in its
accumulation in various tissues, and because its application has
been shown not to be limited by a dose-dependent toxicity.
Sequence CWU 1
1
61163PRTHomo sapiens 1Ser Glu Pro Ala Gly Asp Val Arg Gln Asn Pro
Cys Gly Ser Lys Ala1 5 10 15Cys Arg Arg Leu Phe Gly Pro Val Asp Ser
Glu Gln Leu Ser Arg Asp20 25 30Cys Asp Ala Leu Met Ala Gly Cys Ile
Gln Glu Ala Arg Glu Arg Trp35 40 45Asn Phe Asp Phe Val Thr Glu Thr
Pro Leu Glu Gly Asp Phe Ala Trp50 55 60Glu Arg Val Arg Gly Leu Gly
Leu Pro Lys Leu Tyr Leu Pro Thr Gly65 70 75 80Pro Arg Arg Gly Arg
Asp Glu Leu Gly Gly Gly Arg Arg Pro Gly Thr85 90 95Ser Pro Ala Leu
Leu Gln Gly Thr Ala Glu Glu Asp His Val Asp Leu100 105 110Ser Leu
Ser Cys Thr Leu Val Pro Arg Ser Gly Glu Gln Ala Glu Gly115 120
125Ser Pro Gly Gly Pro Gly Asp Ser Gln Gly Arg Lys Arg Arg Gln
Thr130 135 140Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile
Phe Ser Lys145 150 155 160Arg Lys Pro29PRTHuman immunodeficiency
virus 2Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5360PRTDrosophila
melanogaster 3Arg Lys Arg Gly Arg Gln Thr Tyr Thr Arg Tyr Gln Thr
Leu Glu Leu1 5 10 15Glu Lys Glu Phe His Phe Asn Arg Tyr Leu Thr Arg
Arg Arg Arg Ile20 25 30Glu Ile Ala His Ala Leu Cys Leu Thr Glu Arg
Gln Ile Lys Ile Trp35 40 45Phe Gln Asn Arg Arg Met Lys Trp Lys Lys
Glu Asn50 55 60416PRTDrosophila melanogaster 4Arg Gln Ile Lys Ile
Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys1 5 10
15516PRTArtificialpAntp Helix 3 Variant; between 6 and 10 of the
amino acid residues are hydrophobic. 5Xaa Xaa Xaa Xaa Xaa Trp Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15616PRTArtificialpAntp
Helix 3 Variant; between 6 and 10 of the amino acid residues are
hydrophobic. 6Xaa Xaa Xaa Xaa Xaa Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa1 5 10 15
* * * * *