NZ617353B2 - Anticancer fusion protein - Google Patents
Anticancer fusion protein Download PDFInfo
- Publication number
- NZ617353B2 NZ617353B2 NZ617353A NZ61735312A NZ617353B2 NZ 617353 B2 NZ617353 B2 NZ 617353B2 NZ 617353 A NZ617353 A NZ 617353A NZ 61735312 A NZ61735312 A NZ 61735312A NZ 617353 B2 NZ617353 B2 NZ 617353B2
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- New Zealand
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- seq
- sequence
- fusion protein
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- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
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- 125000003831 tetrazolyl group Chemical class 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4715—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
- C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
Abstract
fusion protein comprising: domain (a) which comprises the functional fragment of a soluble hTRAIL protein sequence starting with an amino acid in a position not lower than hTRAIL95, or a homolog of said functional fragment having at least 70% sequence identity; and at least one domain (b) which is the sequence of an effector peptide having anti-proliferative activity against tumour cells, and wherein the sequence of domain (b) is attached at the C-terminus and/or at the N-terminus of domain (a). Also disclosed is the use of said fusion protein for the treatment of cancer. the sequence of an effector peptide having anti-proliferative activity against tumour cells, and wherein the sequence of domain (b) is attached at the C-terminus and/or at the N-terminus of domain (a). Also disclosed is the use of said fusion protein for the treatment of cancer.
Description
Anticancer fusion protein
The invention relates to the field of eutic fusion proteins, especially
recombinant fusion proteins. More ularly, the invention s to fusion
proteins comprising the fragment of a sequence of the soluble human TRAIL
protein and a sequence of an antiproliferative peptide, pharmaceutical
compositions containing them, their use in therapy, especially as anticancer
agents, and to polynucleotide sequences encoding the fusion proteins,
expression vectors containing the polynucleotide sequences, and host cells
containing these expression vectors.
TRAIL n, a member of the cytokines family (Tumor Necrosis Factor-
d Apoptosis ng Ligand), also known as ApoZL (ApoZ-ligand), is a
potent activator of apoptosis in tumor cells and in cells infected by viruses.
TRAIL is a ligand naturally occurring in the body. TRAIL protein, its amino acid
sequence, coding DNA sequences and protein expression systems were disclosed
for the first time in EP0835305A1.
TRAIL protein exerts its anticancer activity by binding to pro-apoptotic surface
TRAIL receptors 1 and 2 (TRAIL-R1/R2) and subsequent activation of these
receptors. These receptors, also known as DR4 and DR5 (death receptor 4 and
death receptor 5), are members of the TNF receptor family and are
overexpressed by different types of cancer cells. Activation of these receptors
can induce al signaling pathway of suppressor gene p53-independent
apoptosis, which by ted caspase-8 leads to the activation of executive
caspases and thereby ation of nucleic acids. Caspase-8 released upon
TRAIL activation may also cause the release of Bid n and thereby indirect
tion of mitochondrial pathway, Bid protein being translocated to
mitochondria, where it stimulates the release of cytochrome c, thus indirectly
amplifying the apoptotic signal from death receptors.
TRAIL acts selectively on tumor cells essentially without ng apoptosis in
healthy cells which are resistant to this protein. Therefore, the enormous
potential of TRAIL was recognized as an anticancer agent which acts on a wide
range of different types of tumor cells, including hematologic malignancies and
solid , while sparing normal cells and exerting potentially relatively little
side effects.
TRAIL protein is a type II membrane protein having the length of 281 amino
acids, and its ellular region comprising amino acid residues 114-281 upon
cleavage by proteases forms soluble sTRAIL molecule of 20 kDa size, which is
also biologically active. Both TRAIL and sTRAIL forms are capable of triggering
apoptosis via ction with TRAIL receptors present on target cells. Strong
antitumor activity and very low ic toxicity of soluble part of TRAIL
molecule was demonstrated using cell lines tests. Also, human clinical studies
with recombinant human soluble TRAIL (rhTRAIL) having amino acid sequence
corresponding to amino acids 114-281 of hTRAIL, known under the INN
dulanermin, showed its good tolerance and absence of dose limiting toxicity.
Fragment of TRAIL shorter than 114-281 is also able to bind with membrane
death receptors and induce apoptosis via these receptors, as recently reported
for recombinant circularly permuted mutant of 122-281hTRAIL for example in EP
1 688 498.
Toxic s of recombinant TRAIL protein on liver cells reported up to now
appear to be associated with the presence of modification, i.e. polyhistidine
tags, while ed TRAIL showed no systemic toxicity.
However, in the course of further research and development it appeared that
many cancer cells showed primary or ed resistance to TRAIL (see for
example W02007/022214). gh the mechanism of resistance to TRAIL has
not been fully understood, it is believed that it may manifest itself at different
levels of TRAIL-induced apoptosis pathway, ranging from the level of cell surface
receptors to the executive caspases within the signaling pathway. This resistance
limits the usefulness of TRAIL as an anticancer agent.
Furthermore, in clinical trials on patients the actual effectiveness of TRAIL as a
monotherapy proved to be low. To overcome this low efficiency and the
ance of tumors to TRAIL, various ation therapies with radio- and
chemotherapeutic agents were designed, which ed in synergistic apoptotic
effect (W02009/002947; A. Almasan and A. Ashkenazi, Cytokine Growth Factor
s 14 (2003) 337-348; RK Srivastava, Neoplasis, Vol 3, No. 6, 2001, 535-
546, Soria JC et al., J. Clin. Oncology, Vol 28, No. 9 (2010), p. 1527-1533). The
use of rhTRAIL for cancer treatment in combination with selected conventional
herapeutic agents (paclitaxel, carboplatin) and onal anti-VEGF
antibodies are described in W02009/140469. However, such a combination
necessarily s well-known deficiencies of conventional chemotherapy or
radiotherapy.
Moreover, the problem connected with TRAIL therapy has proved to be its low
stability and rapid elimination from the body after administration.
Constructed fusion protein containing sequences of angiogenesis inhibitor
vasostatin and TRAIL114-281 linked with a metalloprotease ge site linker
was described as exhibiting apoptosis-inducing effect in tumor cells by A.|. Guo
et al in Chinese Journal of Biochemistry and Molecular Biology 2008, vol. 24(10),
925-930.
Constructed fusion protein containing sequences of angiogenesis inhibitor
iculin and TRAIL114-281 was described as exhibiting apoptosis-inducing
effect in tumor cells in CN1609124A.
CN 1256347C discloses fusion protein composed of gen D5 60-148 and TRAIl
114-281.
Constructed fusion protein containing sequences of enesis inhibitor
kininostatin, vasostatin and tin attached to N- or C-terminus of TRAIL114-
281 linked with linker encoding GGGSGGSG are mentioned in Feng Feng-Yi
“Phase I and Clinical Trial of Rh-Ap02L and Ap02L-Related Experimental Study”,
Ph.D. degree thesis, Chinese Peking Union Medical, 200601;
http: / w23.com/lunwen_957708432.
Constructed fusion protein containing sequences Tumstatin 0 of an
angiogenesis inhibitor tumstatin and 14-281 was described as exhibiting
induction of apoptosis of pancreatic cancer cells by N.Ren et al in Academic
Journal of Second Military Medical University 2008, vol. 28(5), 676-478.
U52005/244370 and corresponding W02004/035794 disclose the construct of
TRAIL95-281 as an effector domain linked by a peptide linker with extracellular
part of another member of TNF family ligands CD40 as a cell surface binding
domain. It is stated that tion of the construct is via binding of its CD40
part.
Shin J.N. et al., Experimental Cell Research, vol. 312, no. 19, 2006, p. 3892-
3898), disclosed constructed fusions proteins of sTRAIL and |L-18 with a matrix
metalloproteinase cleavage site introduced at the ting site as a proform
of TRAIL that can be ted and released in the areas where
metalloproteinases are pathologically produced, such as tumor environment.
Constructs of sTRAIL with IFN-gamma and endostatin were also produced but
neither characterized nor tested.
One of the objectives in cancer therapy is the inhibition of tumor cells
proliferation (growth). Cells with acquired malignant phenotype (due to
mutation, activities of carcinogens or ers of DNA repair) lose their ability
to proper differentiation and acquire the ability to infiltrate. The clones of
tumor cells transcribe mainly genes that are associated with rapid growth and
invasiveness, and tumor cells are characterized, among others, by disturbances
in the control of proliferation.
Beneficial effect of inhibition of tumor cells proliferation in cancer therapy is
known. Attempts are made of the clinical use of substances that inhibit or
te the process of proliferation, both as a cancer therapy and an adjunct
cancer therapy.
Inhibition of tumor cell proliferation can be achieved in various ways, such as for
example described in the review article ,,Hallmarks of Cancer: The Next
tion” (Cell, 2011, 646-674). There are known antiproliferative proteins
used in anticancer therapies - such as trastuzumab - a monoclonal antibody
blocking HER2 used in breast cancer patients with HER2 overexpression. There is
also known an oliferative activity of many proteins that have not yet been
found to be clinically useful in the treatment of human s.
For example, antiproliferative activity of human fetoprotein and its fragments is
well known. Detailed studies of the properties of individual protein domains
revealed the ce of structures located within the no acid region that
is responsible for the growth inhibition of estradiol dependent cells (Mizejewski
et al, Mol. Cell. Endocrinol., 18:15-23, 1996). ylic terminus of this region,
sed of eight consecutive amino acids, is the most ant fragment,
and is able alone to inhibit the growth of cancer cells (Mizejewski 6., Cancer
Biotherapy & Radiopharmaceuticals, 22: 73-98, 2007).
oliferative properties of p21WAF1 protein are also known. Short peptides
based on the amino acid sequence of p21WAF1 exerting comparable ial to
bind and inhibit D1-CDK4 x and thus stop the cell cycle in G1 phase were
synthesized (Ball et al, Current Biology, 7:71-80, 1996).
It is also known that protein DOC-2/DAB2 (Differentially expressed in Ovarian
Cancer-2/Disabled 2) is a powerful inhibitor of proliferation of prostate cancer
cells. It acts by suppressing MAPK kinase transmission pathways by binding to a
number of their respective sub elements (c-Src, Grb2) (Zhou et al, J Biol Chem
276: 27793-27798, 2001, Zhou et al, J Biol Chem, 278: 6936-6941, 2003). Its
essential component is a proline-rich domain t at the carboxy-terminal
DOC-2/DAB2 (Zhou et al, Cancer Res, 66: 8954 - 8958, 2006).
Inhibition of CDK4-cyclin binding by the p16 protein or a fragment thereof is
commonly ed as a ssor of neoplasia (Fahraeus et al, ne, 16:
587-596, 1998).
There is also known influence of kinase ERK on the degree of tumor cell
proliferation (Handra-Luca A., et al, American Journal of Pathology. 2003; 163:
957-967). It is known that a peptide fragment of MEK-1 protein is a ive ERK
kinase substrate, and thus it can serve as its selective inhibitor (Bradley R. et al,
The Journal of Biological Chemistry, 2002, 277, 8741-8748).
It is also known that selective inhibition of Akt kinase ty leads to inhibition
of cell proliferation and tumor cell death (Hennessy B.T, et al, Nature Reviews
Drug Discovery 2005, 4, 988 -1004).
There are also known antiproliferative properties of Phe-Trp-Leu-Arg-Phe-Thr
hexapeptide, consisting in inhibition of the association of E2F and DP and direct
inhibition of E2F binding to DNA (Janin Y. L., Amino Acids, 25: 1 — 40, 2003).
Inhibition of tubulin fibers merisation, preventing sister chromatid
separation in mitosis and causing disorders in the migration of chromosomes also
results in ers of the proliferation process (Xiao et al., J. Cell Mol. Med.,
2010)
2012/057219
Synergistic effect of in protein with the activity of TRAIL protein was
shown (Wang et al., JBC Journal of Biological Chemistry, 284, 3804-3813).
Inhibition of telomerase activity and accumulation in the ondrial
membrane by proteins which are fragments of bee defensin and their analogs is
also known (Iwasaki et al., Biosci. hnol. Biochem., 73:683-687, 2009).
It is also known that lasioglossins, positively charged peptides isolated from the
venom of bee Lasioglossum laticeps, exert cytotoxic activity against tumor cells
(Cerovsky et al., Chembiochem, 2009, 10: 2089-2099).
It is also known that inhibition of RasGAP - Aurora B interactions by e.g. protein
aptamers from the SH3 , exert inhibitory influence on the proliferation of
cancer cells (Pamonsinlapatham P. et al., PLoS ONE 3 (8): e2902, 2008).
The impact of inhibition of cell cycle -dependent kinases e.g. kinase CDK 4, for
example with p16 peptide, which is the fragment of 4A gene product, is
known as well (Derossi D, et al., J Biol Chem. 269:10444-10450, 1994).
There are also known antiproliferative properties of Pep27 protein, the binding
of which by cellular receptors results in phosphorylation of a histidine kinase,
which causes dephosphorylation of the effector factor VncR and consequently
leads to inhibition of autocatalytic pathways and cell death (Dong Gun Lee et
al., Cancer Cell International 2005, 5:21 ).
Many of the antiproliferative substances are tly at different stages of
igations, including clinical trials. However, known therapies aimed at
ting proliferation have many nown disadvantages. For e, there
are adverse effects such as thromboembolic complications, haemoptysis and
lungs bleeding. Many antiproliferative drugs show also poor bioavailability and
toxic side effects.
Safety of anti-antiproliferative drugs is of special importance because of
prolonged use and lack of selectivity of therapy. Strong need for an effective
therapeutic agent and the nature of oncological diseases necessitate a simplified
registration procedure for such group of drugs, ore it is impossible to know
all the side effects and disadvantages of the drug. Although, contrary to the
chemotherapeutics, which are directed to all fast proliferating cells, peptide
antiproliferative drugs are directed at protein kinases and phosphatases
sible for ring cascades of phosphorylation and dephosphorylation of
proteins or at their substrates or other ns engaged in proper course of the
cell cycle, which results in some reduction of the toxicity of therapy. However,
still anticancer therapy directed at inhibiting proliferation while ensuring selec-
tivity t tumor cells is not known. There is therefore a need for new anti-
proliferative anticancer therapies with improved logical characteristics.
The present invention provides a solution of this problem by providing novel
fusion proteins that comprise a domain derived from TRAIL and a short or
peptide domain having antiproliferative activity and not including TRAIL
fragments, wherein the effector peptide potentiates or complements the action
of TRAIL.
Proteins according to the invention are directed selectively to cancer cells,
where the elements of the protein exert their effects, in particular the or
peptide inhibits tumor cells proliferation. Delivery of the proteins of the
invention into the tumor environment allows to minimize toxicity against healthy
cells in the body as well as side effects and to reduce the frequency of
administration. In addition, targeted therapy with the use of proteins according
to the invention allows to avoid the problem of low efficiency of previously
known nonspecific antiproliferative ies caused by high toxicity and by
necessity of stering high doses.
It turned out that in many cases fusion proteins of the invention are more potent
than soluble TRAIL and its variants including a fragment of the sequence. Until
now, known or peptides used in the fusion protein of the invention have
not been used in medicine as such because of unfavorable kinetics, rapid
degradation by nonspecific proteases or accumulation in the body caused by lack
of proper sequence of activation of pathways, which is necessary to enable the
proper action of the effector peptide at target site. Incorporation of the effector
peptides into the fusion protein allows their selective ry to the site where
their action is desirable. rmore, the attachment of the effector peptide
increases the mass of protein, ing in prolonged ife and increased
retention of protein in the tumor and its enhanced efficiency. Additionally, in
many cases, novel fusion ns also overcome natural or induced resistance to TRAIL.
According to a first aspect of the present invention, there is provided a fusion protein
comprising:
- domain (a) which comprises the functional fragment of a soluble
hTRAIL protein sequence starting with an amino acid in a position not
lower than hTRAIL95, or a homolog of said functional fragment having
at least 70% sequence identity; and
- at least one domain (b) which is the sequence of an effector peptide
having anti-proliferative activity against tumour cells,
and wherein the sequence of domain (b) is attached at the C-terminus and/or at the
N-terminus of domain (a).
According to a second aspect of the present invention, there is provideda polynucleotide
sequence, coding the fusion n as defined in the first aspect of the invention.
According to a third aspect of the present invention, there is providedan expression
vector, comprising polynucleotide sequence according theto second aspect of the
invention.
According to a fourth aspect of the t invention, there is provideda host cell,
comprising the expression vector as defined in the third aspect of the invention, wherein
the host cell is not within a human.
According to a fifth aspect of the present invention, there is provideda pharmaceutical
ition, comprising as an active ingredient the fusion protein as d inthe first
aspect of the invention, in combination with a pharmaceutically able r.
According to a sixth aspect of the present invention, there is eduse of an
anti-neoplastic-effective amount of the fusion protein as defined inthe first aspect of the
invention for the manufacture of a medicament for the treatment of cancer diseases in a
mammal.
(9480728_1):KZA
Description of Figures
The invention will now be described indetail with reference to the Figures of the
drawing.
Fig. 1 presents a tic structure of fusion proteins of the invention according to Ex.
1, Ex. 2, Ex. 3, Ex. 4 and Ex. 5.
Fig. 2 presents a schematic structure of fusion proteins of the ion according to Ex.
6, Ex. 7, Ex. 8, Ex. 8A, Ex. 9 and Ex. 10.
Fig. 3 presents a schematic ure of fusion proteins of the invention according to Ex.
11, Ex. 12, Ex. 13, Ex. 14, and Ex. 15.
Fig. 4 presents a schematic structure of fusion proteins of the invention according to Ex.
16, Ex. 17, Ex. 18, Ex. 19, and Ex. 20.
Fig. 5 presents a schematic ure of fusion proteins of the invention according to Ex.
21, Ex. 22, Ex. 23, Ex. 24, and Ex. 25.
Fig. 6A and 6B show circular ism spectra forrhTRAIL95-281 and fusion proteins
of Ex. 1a and Ex. 2a (Fig. 6A), and Ex. 8a and rhTRAIL114-281 (Fig. 6B) expressed in
specific ellipticity.
Fig. 7 presents tumor volume changes (% of initial stage) in Crl:CD1-Foxn1nu mice
burdened with colon cancer HCT116 treated with fusion protein of the invention of Ex.
2a compared to rhTRAIL114-281
Fig. 8 presents the tumor growth inhibition values (%TGI) in Crl:CD1-Foxn1nu 1 mice
burdened with colon cancer HCT116 treated with fusion n of the invention of Ex. 2a
ed to rhTRAIL114-281.
Fig. 9 presents tumor volume changes (% of initial stage) in Crl:CD1-Foxn1nu mice
burdened with lung cancer NCI-H460-Luc2 treated with fusion protein of the invention
of Ex. 2a compared to rhTRAIL114-281.
(9480728_1):KZA
Fig. 10 presents the tumor growth inhibition values (%TG|) in Crl:CD1-Foxn1”“ 1
mice burdened with lung cancer NCI-H460-Luc2 treated with fusion protein of
the invention of Ex. 2a compared to rhTRAIL114-281.
Fig. 11 presents tumor volume changes (% of initial stage) in Cr|:SHO-
Prkdc’SCidHrhr mice burdened with colon cancer HCT116 treated with fusion
protein of the invention of Ex. 8"11 compared to rhTRAIL114-281.
Fig. 12 presents the tumor growth inhibition values (%TG|) in Cr|:SHO-
Prkdc’SCidHrhr mice burdened with colon cancer HCT116 treated with fusion
n of the invention of Ex. 8"11 compared to rhTRAIL114-281.
Fig. 11a presents tumor volume s (% of initial stage) in Cr|:SHO-
SCidHrhr mice burdened with colon cancer HCT116 treated with fusion
protein of the invention of Ex. 8b compared to rhTRAIL114-281.
Fig. 12a presents the tumor growth inhibition values (%TG|) in Cr|:SHO-
Prkdc’SCidHrhr mice burdened with colon cancer HCT116 treated with fusion
protein of the invention of Ex. 8b compared to rhTRAIL114-281.
Fig. 13 presents tumor volume changes (% of initial stage) in Cr|:SHO-
Prkdc’SCidHrhr mice burdened with colon cancer SW620 d with fusion
protein of the invention of Ex. 8b compared to rhTRAIL114-281.
Fig. 14 presents the tumor growth inhibition values (%TG|) in Cr|:SHO-
Prkdc’SCidHrhr mice burdened with colon cancer SW620 d with fusion
protein of the invention of Ex. 8b compared to rhTRAIL114-281.
Fig. 15 presents tumor volume changes (% of initial stage) in Cr|:SHO-
Prkdc’SCidHrhr mice burdened with colon cancer Col0205 treated with fusion
protein of the invention of Ex. 8b compared to rhTRAIL114-281.
Fig. 16 presents the tumor growth tion values (%TG|) in Cr|:SHO-
Prkdc’SCidHrhr mice burdened with colon cancer Col0205 treated with fusion
protein of the ion of Ex. 8bcompared to rhTRAIL114-281.
Fig. 17 presents tumor volume changes (% of l stage) in Cr|:SHO-
Prkdc’SCidHrhr mice burdened with liver cancer HepGZ d with fusion
n of the invention of Ex. 8b compared to rhTRAIL114-281.
Fig. 18 ts the tumor growth inhibition values (%TG|) in Cr|:SHO-
SCidHrhr mice burdened with liver cancer HepGZ treated with fusion protein
of the invention of Ex. 8b compared to rhTRAIL114-281.
Fig. 19 presents tumor volume changes (% of initial stage) in Cr|:SHO-
Prkdc’SCidHrhr mice burdened with lung cancer NCI-H460 treated with fusion
protein of the invention of Ex. 8b compared to rhTRAIL114-281.
Fig. 20 presents the tumor growth inhibition values (%TG|) in Cr|:SHO-
Prkdc’SCidHrhr mice burdened with lung cancer NCI-H460 treated with fusion
protein of the invention of Ex. 8b compared to rhTRAIL114-281.
Detailed ption of the Invention
The invention relates to a fusion n sing:
domain (a) which is the functional fragment of a sequence of soluble
hTRAIL n, which fragment begins with an amino acid at a position
not lower than hTRAIL95 or a homolog of said functional fragment having
at least 70% sequence identity, and
at least one domain (b) which is the sequence of an effector peptide
having anti-proliferative activity against tumor cells,
wherein the sequence of the domain (b) is attached at the C-terminus and/or N-
terminus of domain (a).
The term “the functional e fragment of a sequence of e hTRAIL”
should be understood as denoting any such fragment of soluble hTRAIL that is
capable of inducing apoptotic signal in mammalian cells upon binding to its
receptors on the surface of the cells.
It will be also appreciated by a skilled person that the existence of at least 70%
homology of the TRAIL ce is known in the art.
It should be understood that domain (b) of the effector peptide in the fusion
protein of the invention is neither hTRAIL protein nor a part or fragment of
hTRAIL protein.
The term “peptide” in accordance with the invention should be understood as a molecule
built from plurality of amino acids linked together by means of a peptide bond. Thus, the
term “peptide” ing to the invention includes oligopeptides, polypeptides and
proteins.
In the present invention the amino acid sequences of peptides will be presented in a
conventional manner adopted in the art in the direction from N-terminus (N-end) of the
peptide towards its C-terminus (C-end). Any sequence will thus have its N-terminus on
the left side and C-terminus on the right side of its linear presentation.
The fusion protein of the invention incorporates at least one domain (b) of the effector
peptide, attached at the C-terminus and/or or at the N-terminus of domain (a).
In a particular ment, domain (a) is the fragment of hTRAIL ce, beginning
with an amino acid from the range of hTRAIL95 to hTRAIL121, inclusive, and ending
with the amino acid hTRAIL 281.
In particular, domain (a) may be ed from the group consisting of sequences
corresponding to hTRAIL95-281, hTRAIL114-281, hTRAIL119-281, hTRAIL120-281
and hTRAIL121-281. It will be evident to those skilled in the art that 95-281,
hTRAIL114-281, hTRAIL119-281, hTRAIL120-281 and 121-281 represent a
fragment of human TRAIL protein starting with amino acid marked with the number 95,
114, 119, 120 and 121, respectively, and ending with the last amino acid 281, in the
known sequence of hTRAILpublished in k under Accession No. P50591 and as
set forth in SEQ ID NO: 78.
In another particular embodiment, domain (a) is a homolog of the functional fragment of
soluble hTRAIL protein sequence beginning at amino acid position not lower than
hTRAIL95 and ending at amino acid hTRAIL281, the sequence of which is at least in
70%, ably in 85%, identical to al sequence.
In specific variants of this embodiment domain (a) is a homolog of the fragment selected
from the group consisting of ces corresponding to hTRAIL95-281, hTRAIL114-
281, hTRAIL116-281, hTRAIL120-281 and hTRAIL121-281.
(9480728_1):KZA
It should be understood that a homolog of the hTRAIL fragment is a
variation/modification of the amino acid sequence of this fragment, wherein at
least one amino acid is changed, including 1 amino acid, 2 amino acids, 3 amino
acids, 4 amino acids, 5 amino acids, 6 amino acids, and not more than 15% of
amino acids, and wherein a fragment of the ed ce has preserved
functionality of the hTRAIL sequence, i.e. the ability of binding to cell surface
death receptors and inducing apoptosis in mammalian cells. Modification of the
amino acid sequence may include, for example, substitution, deletion and/or
addition of amino acids.
ably, the homolog of hTRAIL fragment having modified sequence shows a
modified affinity to the death receptors DR4 (TRAIL-R1) or DR5 (TRAIL-R2) in
comparison with the native fragment of hTRAIL.
The term "modified affinity" refers to an increased ty and/or affinity with
altered receptor selectivity.
Preferably, the homolog of the fragment of hTRAIL having modified sequence
shows increased ty to the death receptors DR4 and DR5 compared to native
fragment of hTRAIL.
ularly preferably, the homolog of fragment of hTRAIL having modified
sequence shows increased affinity to the death receptor DR5 in comparison with
the death receptor DR4, i.e. an increased selectivity DR5/DR4.
Also preferably, the homolog of nt of hTRAIL having modified sequence
shows an increased selectivity s the death receptors DR4 and/or DR5 in
relation to the affinity towards the receptors DR1 (TRAIL-R3) and/or DRZ (TRAIL-
R4).
Modifications of hTRAIL resulting in sed affinity and/or selectivity towards
the death receptors DR4 and DR5 are known to those skilled in the art, for
example from the publication Tur V, van der Sloot AM, Reis CR, Szegezdi E, Cool
RH, Samali A, Serrano L, Quax WJ. DR4-selective tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) variants obtained by ure-based . J.
Biol. Chem. 2008 Jul 18;283(29):20560-8, which describes the D218H mutation
having increased ivity towards DR4, or Gasparian ME, Chernyak BV, Dolgikh
2012/057219
DA, Yagolovich AV, Popova EN, Sycheva AM, Moshkovskii SA, Kirpichnikov MP.
Generation of new TRAIL mutants DR5-A and DR5-B with improved selectivity to
death receptor 5, Apoptosis. 2009 Jun;14(6):778-87, which describes the D269H
mutation having a reduced affinity towards DR4. hTRAIL mutants resulting in
increased affinity towards one receptor ed from the DR4 and DR5
comparing with DR1 and DR2 receptors and increased ty towards the
receptor DR5 comparing with DR4 are also described in W02009077857 and
W02009066174.
Suitable mutations are one or more mutations in the positions of native hTRAL
selected from the group consisting of amino acid 131, 149, 159, 193, 199, 201,
204, 204, 212, 215, 218 and 251, in ular, ons involving the
substitution of an amino acid with a basic amino acid such as lysine, histidine or
arginine, or amino acid such as glutamic acid or aspargic acid. Particularly one
or more mutations selected from the group consisting of G131R, G131K, R149I,
R149M, R149N, R149K, S159R, Q193H, Q193K, N199H, N199R, K201H, K201R,
K204E, K204D, K204L, K204Y, K212R, $215E, $215H, $215K, $215D, D218Y,
D218H, K251D, K251E and K251Q, as described in W02009066174, may be
specified.
Suitable mutations are also one or more mutations in the positions of native
hTRAIL selected from the group ting of amino acid 195, 269 and 214,
particularly mutations involving the substitution of an amino acid with a basic
amino acid such as lysine, histidine or arginine. ularly one or more
mutations selected from the group ting of D269H, E195R, and T214R, as
described in W02009077857, may be specified.
In a particular embodiment, the domain (a) which is a g of the fragment
of hTRAIL is selected from D218H mutant of the native TRAIL sequence, as
described in W02009066174, or the Y189N-R191K-Q193R-H264R-I266R-D269H
mutant of the native TRAIL sequence, as described in Gasparian ME et al.
Generation of new TRAIL mutants DR5-A and DR5-B with improved selectivity to
death receptor 5, Apoptosis. 2009 Jun; 14(6): .
According to the invention, the fusion protein comprises as the effector peptide
an anti-proliferative peptide, which has roliferative activity against tumor
cells, i.e. inhibiting effect on tumor cells proliferation.
It should be understood that “tumor cells proliferation” relates to the step of
cell division and growth in a tumor cell cycle and the or peptide has the
anti-proliferative ty with respect to the growth of tumor cells as such.
Therefore, “tumor cells proliferation” inhibiting effect does not encompass
inhibiting proliferation of endothelial cells as a step of angiogenesis. Effector
peptides having anti-angiogenic activity, i.e. activity of inhibiting growth of
endothelial cells are therefore ed from the scope of the effector peptides
according to the invention.
Specifically, effector peptides selected from the group consisting of calreticulin,
tumstatin 183-230, kininogen D5, vasostatin, kininostatin, endostatin and
canstatin are not encompassed by the invention.
According to the invention, the effector peptide can exert its antiproliferative
effect against tumor cells in different ways, such as for example ed from
the following group:
suppression of MAPK s (mitogen-activated protein kinases)
transmission pathways, for example by blocking FGF-Z or (basic
fibroblast growth factor 2 or, also known as bFGF-, FGFZ- or FGF-B
receptor) or DD2 peptide derived from DABZ protein;
inhibition of growth of estradiol dependent cells, for example by human
fetoprotein or its fragment;
ng cell-cycle in G1 phase, such as by inhibition of cyclin D1-CDK4
(cyclin-dependent kinase 4) complex;
enzymatic breakdown of ne, such as by arginine deiminase from
Mycoplasma arginini;
inhibition of cell-cycle kinases, such as inhibition of /6 kinase
(cyclin-dependent kinases), or inhibition of ERK kinases (extracellularsignal-regulated
kinases) activation, or inhibition of Akt kinase (also
known as Protein Kinase B (PKB), a serine/threonine-specific protein
kinase) coactivation;
tion of transcription factor EZF cription factors (TF) in higher
eukaryotes) association with DP ns (also known as transcription
factor DP, EZF dimerisation partner);
inhibition of tubulin fibres association/polymerization;
tion of telomerase activity;
inhibition of RasGAP (GTPase-activator protein for Ras-like s) -
Aurora B kinase interactions or ine kinase tion; and
disturbing ionic balance across the cell membrane.
In one ment of the invention the effector peptide of domain (b) may be a
peptide e of suppressing MAPK kinases transmission pathways. An example
is an analogue of binding domain of FGF-2 receptor which is responsible for the
blockade of FGF-2 receptor and in consequence inhibition of tumor growth. In
particular, such an effector peptide can be a 16-amino acid peptide presented
by SEQ. No. 26 in the attached Sequence g.
Another effector peptide of this embodiment of the invention can be a fragment
of DOC-Z/DABZ protein. In particular, such an effector peptide can be an 18-
amino acid peptide DDZ— a proline-rich domain present on the carboxy terminus
of DOC-Z/DABZ, presented by SEQ. No. 30 in the attached ce Listing,
which participates in ssion of transmission pathways of MAPK kinases by
binding to a number of their respective sub elements (c-Src, Grb2).
In another embodiment of the invention the effector peptide of domain (b) may
be a peptide capable of inhibition of growth of estradiol dependent cells, for
example human fetoprotein or its fragment. In particular, such an or
peptide can be a 34-amino acid fragment of human alpha—fetoprotein presented
by SEQ. No. 27 in the attached Sequence Listing. Another effector peptide of
this embodiment can be an 8-amino acid fragment of human alpha—fetoprotein,
localized on C-terminal fragment of SEQ. No. 27, and presented by SEQ. No. 28
in the attached Sequence Listing.
In another embodiment of the invention the effector peptide of domain (b) may
be a peptide capable of stopping cell-cycle in G1 phase, such as by inhibition of
cyclin D1-CDK4 x. In ular, such an effector peptide can be a trojan
p16 peptide, or its fragment, inhibiting the activity of kinases CDK4 and CDK6. In
particular, such an effector peptide — a fragment of p16|NK4A gene product — is
presented by SEQ. No. 32 in the attached Sequence Listing. Such an or
peptide can be also another fragment of trojan p16 peptide — a fragment of
p16|NK4A gene product fused with a 17-amino-acid transporting domain of
antennapedia (Derossi D, AH Joliot, G Chassaings, A Prochiantz, J Biol Chem.
269:10444-10450,1994), presented as SEQ. No. 33 in the attached Sequence
Listing.
In r embodiment of the invention the effector peptide of domain (b) may
be a e capable of enzymatic breakdown of arginine, such as by arginine
deiminase from asma arginini. In particular, such an effector peptide is
presented by SEQ. No. 31 in the ed Sequence Listing.
In r embodiment of the invention the effector e of domain (b) may
be a peptide capable of inhibition of cell-cycle kinases, such as a CDK4/5
inhibitor. In particular, such an effector peptide can be a nt of p21WAF1
protein, such as a 20-amino acid fragment of p21WAF1 protein presented by
SEQ. No. 29 in the attached ce Listing.
Another effector peptide of this embodiment can be a peptide — inhibitor of ERK
activation. In particular, such an effector peptide can be a fragment of MEK-1
protein, such as presented by SEQ. No. 34 in the attached Sequence Listing.
Another effector peptide of this ment can be a peptide — coactivator of
Akt kinase. In particular, such an effector peptide — an N-terminal fragment of
PH domain of TCL1 protein - is presented by SEQ. No. 35 in the attached
Sequence Listing.
In another embodiment of the invention the effector peptide of domain (b) may
be a peptide capable of inhibition of transcription factor EZF ation with DP
protein. In particular, such an effector peptide — a hexapeptide p-Leu-Arg-
Phe-Thr - is presented by SEQ. No. 36 in the ed Sequence Listing. Another
effector peptide of domain (b) can be a peptide being an analogue of FGF-2
binding domain. In particular, such an effector e — a 8 amino acid peptide
blocking FGF-Z receptor - is presented by SEQ. No. 41 in the ed Sequence
Listing.
In another embodiment of the invention the effector peptide of domain (b) may
be a peptide e of tion of tubulin fibres association/polymerization.
Such an effector e can be a fragment of tubulin responsible for forming of
heterodimers structures, buting to inhibition of tubulin fibers
polymerisation. In particular, such an effector peptide — a no acid frag-
ment of tubulin - is presented by SEQ. No. 37 in the attached Sequence Listing,
and another effector peptide — a 10-amino acid fragment of tubulin - is
presented by SEQ. No. 38 in the attached Sequence Listing.
In another embodiment of the invention the effector peptide of domain (b) may
be a peptide capable of inhibition of telomerase activity. Such an or
peptide can be a peptide based on the sequence of a bee defensin responsible
for telomerase activity inhibition. In particular, such an effector peptide — a 6
amino acid C2 peptide based on the sequence of a bee defensin — is presented by
SEQ. No. 40 in the attached Sequence Listing. Another effector e of this
embodiment can be a peptide lossin present in the bee venom. In
particular, such an effector peptide — lasioglossin LL-Z — is presented by SEQ. No.
42 in the attached Sequence Listing.
In r embodiment of the invention the effector peptide of domain (b) may
be a peptide capable of inhibition of - Aurora B interactions or histidine
kinase activation. In particular, such an effector peptide — a 13-amino acid
peptid g SH3 domain of RasGAP - is presented by SEQ. No. 43 in the
attached Sequence g. Another effector peptide of this embodiment can be
a peptide which after binding by cell receptors causes histidine kinase
phosphorylation, which in turn leads to effector factor VncR dephosphorylation.
In particular, such an effector peptide — an analogue of Pep27 peptide — is
presented by SEQ. No. 44 in the attached Sequence Listing.
In another embodiment of the invention the effector peptide of domain (b) may
be a peptide capable of disturbing ionic balance across the cell membrane. In
particular, such an or peptide melittin — is presented by SEQ. No. 39 in the
attached Sequence Listing.
In the specific embodiments of the fusion protein of the t ion, the
effector peptide is selected from the group ting of:
- SEQ. No.26 (16-amino acids peptide blocking FGF-2 receptor),
- SEQ. No.27 (a fragment of alpha-fetoprotein),
- SEQ. No.28 (a C-terminal fragment of alpha-fetoprotein),
- SEQ. No.29 (a fragment of p21WAF1 protein),
- SEQ. No.30 (a DD2 peptide from DAC-2/DAB-2 protein),
- SEQ. No.31 (an arginine deiminase),
- SEQ. No.32 (a fragment of p16 e),
- SEQ. No.33 (a fragment of p16 peptide fused with a 17-amino-acid transporting
domain of antennapedia),
- SEQ. No.34 (a fragment of MEK-1),
- SEQ. No.35 (a fragment of PH domain of TCL1 protein),
- SEQ. No.36 (a hexapeptide inhibitor of E2F),
- SEQ. No.37 (an inhibitor of tubulin polymerisation),
- SEQ. No.38 (an inhibitor of tubulin polymerisation),
- SEQ. No.39 (melittin),
- SEQ. No.40 (synthetic C2 telomerase inhibitor),
- SEQ. No.41 (an 8-amino acids inhibitor of interactions with FGF-2R),
- SEQ. No.42 (lassioglossin LL-2),
- SEQ. No.43 (an inhibitor of Aurora R627 kinase), and
- SEQ. No.44 (an analog of Pep27).
Upon binding to TRAIL receptors present on the e of cancer cells, the
fusion n will exert a double effect. Domain (a), that is a functional
fragment of TRAIL or its homolog with preserved functionality, will exert its
known agonistic activity, i.e. binding to death ors on the cell surface and
activation of extrinsic pathway of apoptosis. The effector peptide of the domain
(b) of the fusion protein will be able to potentially exert its action intracellularly
in parallel to the activity of TRAIL domain by inhibition if proliferation of tumor
cells.
If the fusion protein comprises a cleavage sequence recognized by a protease,
the effector peptide could previously be d from the fragment of TRAIL by
metalloproteinases or ases overexpressed in the tumor environment.
In the fusion protein of the invention, antitumor effect of TRAIL could
potentially be enhanced by activation of other elements that affect proliferation
of cells, such as for example inhibition of growth of estradiol dependent cells,
the inhibition of cyclin D1-CDK4 complex, suppression of MAPK kinases
ission pathways, enzymatic breakdown of arginine, /6 kinase
inhibition, inhibition of ERK kinase activation, inhibition of Akt kinase
coactivation, inhibition of transcription factor EZF association with DP proteins,
inhibition of tubulin fibres association, inhibition of telomerase activity,
inhibition of - Aurora B interactions or histidine kinase activation.
In one of the embodiments of the invention, domain (a) and domain (b) are
linked by at least one domain (c) sing the sequence of a cleavage site
recognized by ses present in the cell environment, especially in the tumor
cell environment. The e of the domain (a) with the domain (b) by at least
one domain (c) means that between domains (a) and (b) more than one domain
(c) may be present, in particular one or two domains (c).
The protease cleavage site can be selected from:
- a sequence recognized by metalloprotease MMP, in particular (Pro Leu Gly Leu
Ala Gly Glu Pro/PLGLAGEP) designated as SEQ. No.45, or (Pro Leu Gly Ile Ala Gly
Glu GE) or (Pro Leu Gly Leu Ala Gly GluPro /PLGLAGEP);
- a ce recognized by urokinase uPA, in particular Arg Val Val Arg (RVVR)
designated as SEQ. No. 46 or a fragment thereof, which with the last amino acid
of the sequence to which is attached forms SEQ. No.46,
and their combinations.
WO 43477
In one of the embodiments of the invention, the protease cleavage site is a
ation of the sequence recognized by metalloprotease MMP and a
sequence recognized by urokinase uPA, located next to each other in any order.
In one embodiment, domain (c) is a combination of MMP/uPA, such as SEQ. No.
45/SEQ. No. 46, or a combination of uPA/MMP, such as SEQ. No. 46/SEQ. No. 45.
Proteases metalloprotease MMP and urokinase uPA are overexpressed in the
tumor environment. The presence of the sequence ized by the protease
enables the cleavage of domain (a) from domain (b), i.e. the e of the
or domain (b) and thus its activation.
The presence of the protease cleavage site, by allowing quick release of the
effector peptide, increases the chances of transporting the peptide to the place
of its action before random degradation of the fusion protein by proteases
present in the cell occurs.
Additionally, a transporting domain (d) may be attached to domain (b) of the
effector peptide of the fusion protein of the ion.
Domain (d) may be for example selected from the group consisting of:
(d1) a ginine sequence transporting through the cell membrane, consisting
of 6, 7, 8, 9, 10 or 11 Arg residues,
(d2) a fragment of antennapedia protein domain (SEQ. No. 48) as a domain
transporting through the cell membrane,
(d3) another fragment of apedia protein domain (SEQ. No. 49) as a
domain transporting through the cell ne,
and combinations thereof.
The combination of domains (d1) (d2) and (d3) may comprise, in particular, the
combination of (d1)/(d2), (d1)/(d3) or (d1)/(d2)/(d3).
Furthermore, the combination of domains (d1), (d2) and (d3) may include
domains located next to each other and connected to one end of domain (b)
and/or domains linked to different ends of domain (b).
It should be understood that in the case when the fusion protein has both the
orting domain (d) attached to domain (b) and domain (c) of the cleavage
site between domains (a) and (b), then domain (c) is located in such a manner
that after cleavage of the construct transporting domain (d) s attached to
domain (b). In other words, if the fusion protein contains both the transporting
domain (d) and the cleavage site domain (c), then domain (d) is located between
domain (b) and domain (c), or is located at the end of domain (b) opposite to
the place of attachment of domain (d).
The ion does not comprise such a variant in which domain (d) is located
between domain (c) and domain (a), that is the case when after cleavage of the
construct orting domain remains attached to the TRAIL domain.
Translocation domain constituting a fragment of antennapedia protein domain
(SEQ. No. 48) as well as another fragment of apedia protein (SEQ.
No. 49) is capable of ocation through the cell membranes (Derossi D, AH
Joliot, G Chassaings, A antz, J Biol Chem. 269:10444-10450 (1994) and can
be used to introduce the effector e to the tumor cell compartments.
The sequence (d1) transporting trough the cell nes may be any sequence
known in the art consisting of several arginine residues, translocating the
effector peptide trough the cell membrane to the cytoplasm of target cell (D.,
Hea, H., Yangb, Q., Lina, H., Huang, Arg9-peptide facilitates the alization
of an anti-CEA immunotoxin and potentiates its specific cytotoxicity to target
cells, The international Journal of Biochemistry & Cell Biology 37 (2005) 192—
205; Shiroh Futaki et al JBC, Vol. 276, No. 8, Issue of February 23, pp. 5836—
5840, 2001 ).
Other useful cell penetrating peptides are described in F. Said Hassane et al
Cell. Mol. Life Sci. DOI 10.1007/5000180186-0.
Apart from the main functional elements of the fusion protein and the cleavage
site domain(s), the fusion proteins of the invention may contain a neutral
sequence/sequences of a flexible steric glycine-cysteine-alanine linker (spacer).
Such linkers/spacers are well known and described in the literature. Their
incorporation into the sequence of the fusion protein is intended to provide the
correct folding of proteins ed by the s of its overexpression in the
host cells.
In particular, the flexible steric linker may be SEQ. No.47, which is a
combination of cysteine and alanine residues. In another embodiment the
flexible steric linker may be a ation of glycine and serine residues such as
for example a fragment Gly Gly Gly Ser Gly / GGGSG or any fragment f
acting as steric linker, for example Gly Gly Gly/GGG.
In other embodiment, the flexible steric linker may be any combination of
linkers consisting of SEQ. No.47 and glycine and serine residues, such as for
example a fragment Gly Gly Gly Ser Gly /GGGSG or any fragment thereof acting
as a steric linker, for example a fragment Gly Gly Gly /GGG. In such case the
steric linker may be a combination of e, cysteine and alanine residues,
such as for example Cys Ala Ala Cys Ala Ala Ala Cys Gly Gly Gly / CAACAAACGGG.
In other embodiment, the flexible steric linker may be a sequence Gly Gly Gly
Cys Ala Ala Ala Cys Ala Ala Cys Gly Ser Gly / GGGCAAACAACGSG (SEQ. No.77) or
any combination f.
In one embodiment, the flexible steric linker may be also selected from single
amino acid residues, such as single cysteine residue.
Particular embodiment of the invention are fusion proteins selected from the
group ting of the proteins represented by SEQ. No. 1, SEQ. No. 4, SEQ. No.
, and SEQ. No. 6 which comprise as the antiproliferative effector peptide the
34-amino acid fragment of human fetoprotein represented by SEQ. No. 27.
Other ic embodiment of the invention are fusion proteins selected from
the group consisting of the proteins represented by SEQ. No. 2, SEQ. No. 3 and
SEQ. No. 7 which comprise as the antiproliferative effector peptide the 8-amino
acid fragment of human fetoprotein represented by SEQ. No. 28.
Other specific embodiment of the ion are fusion proteins selected from
the group consisting of the proteins represented by SEQ. No. 8 and SEQ. No. 9,
which comprise as the effector e the peptide derived from p21WAF
represented by SEQ. No. 29.
Other specific embodiment of the invention is the fusion protein represented by
SEQ. No. 10, which comprises as the or peptide a 16-amino acid analogue
of domain binding FGF-Z receptor represented by SEQ. No. 26.
Other specific embodiment of the invention is the fusion represented by SEQ.
No. 11, which comprises as the effector peptide DD2 from DOC-2/DAB2 protein
ented by SEQ. No. 30.
Other specific ment of the invention is the fusion protein represented by
SEQ. No. 12, which comprises as the effector peptide an arginine deiminase from
Mycoplasma arginini ented by SEQ. No. 31.
Other specific embodiment of the invention is the fusion n represented by
SEQ. No. 13, which comprises as the effector peptide a fragment of p16 peptide
ented by SEQ. No. 32.
Other specific embodiment of the invention is the fusion protein represented by
SEQ. No. 13, which comprises as the effector peptide a fragment of p16 peptide
fused with a 17-amino-acid transporting domain of apedia represented by
SEQ. No. 33.
Other specific embodiment of the invention is the fusion represented by SEQ.
No. 14, which comprises as the or peptide a fragment of MEK-1 protein
represented by SEQ. No. 34.
Other specific embodiment of the invention is the fusion protein represented by
SEQ. No. 15, which comprises as the effector e an N-terminal fragment of
PH domain of TCL1 protein ented by SEQ. No. 35.
Other specific embodiment of the he invention is the fusion protein represented
by SEQ. No. 16, which comprises as the effector peptide a hexapeptide Phe-Trp-
Leu-Arg-Phe-Thr represented by SEQ. No. 36.
Other specific embodiment of the invention is the fusion protein represented by
SEQ. No. 17, which comprises as the effector peptide a 13-amino acid fragment
of tubulin represented by SEQ. No. 37.
Other specific embodiment of the invention is the fusion protein represented by
SEQ. No. 18, which comprises as the effector peptide a 10-amino acid fragment
of tubulin represented by SEQ. No. 39.
Other specific embodiment of the invention is the fusion protein represented by
SEQ. No. 19, which comprises as the effector peptide melittin represented by
SEQ. No. 39.
Other specific embodiment of the invention is the fusion protein represented by
SEQ. No. 20, which comprises as the effector e a 6-amino acid peptide C2
based on sequence of bee defensin represented by SEQ. No. 40.
Other specific embodiment of the ion is the fusion protein represented by
SEQ. No. 21, which comprises as the effector peptide the 8-amino acid peptide
binding to FGF-2 ligand represented by SEQ. No. 41.
Other specific ment of the invention is the fusion n represented by
SEQ. No. 22, which comprises as the effector peptide the 15-amino acid peptide
lasioglossin LL2 represented by SEQ. No. 42.
Other specific embodiment of the invention is the fusion protein ented by
SEQ. No. 23, which ses as the effector peptide the 13-amino acid peptide
binding to SH3 domain of RasGAP represented by SEQ. No. 43.
Other specific embodiment of the invention is the fusion protein represented by
SEQ. No. 25, which comprises as the effector peptide the ue of Pep27
e ented by SEQ. No. 44.
A detailed description of the structure of representative fusion proteins
mentioned above are shown in Figures 1 to 5, and in the Examples ted
below.
In accordance with the present invention, by the fusion protein it is meant a
single protein molecule containing two or more proteins or fragments thereof,
covalently linked via peptide bond within their respective peptide chains,
without onal chemical linkers.
The fusion protein can also be atively described as a protein construct or a
chimeric protein. According to the present invention, the terms “construct” or
“chimeric protein”, if used, should be understood as referring to the fusion
protein as defined above.
For a person skilled in the art it will be apparent that the fusion protein thus
defined can be synthesized by known methods of chemical synthesis of peptides
and proteins.
The fusion protein can be synthesized by methods of chemical peptide synthesis,
especially using the techniques of peptide synthesis in solid phase using suitable
resins as carriers. Such techniques are conventional and known in the art, and
described inter alia in the monographs, such as for example zky and
Bodanszky, The Practice of Peptide Synthesis, 1984, Springer- Verlag, New York,
Stewart et al., Solid Phase Peptide Synthesis, 2nd Edition, 1984, Pierce Chemical
Company.
The fusion protein can be synthesized by the methods of chemical synthesis of
peptides as a continuous protein. Alternatively, the dual fragments
(domains) of n may be synthesized separately and then combined er
in one continuous peptide via a peptide bond, by sation of the amino
terminus of one peptide fragment from the carboxyl terminus of the second
peptide. Such techniques are conventional and well known.
For cation of the structure of the resulting peptide known methods of the
analysis of amino acid composition of peptides may be used, such as high
resolution mass spectrometry technique to determine the lar weight of
the peptide. To confirm the peptide sequence n sequencers can also be
used, which sequentially degrade the peptide and identify the sequence of
amino acids.
ably, however, the fusion n of the invention is a recombinant
protein, generated by methods of gene expression of a cleotide sequence
encoding the fusion protein in host cells.
A further aspect of the invention is the polynucleotide sequence, particularly
DNA sequence ng a fusion protein as defined above.
Preferably, the polynucleotide sequence, particularly DNA, ing to the
invention, encoding the fusion protein as d above, is a sequence optimized
for expression in E. coli.
r aspect of the invention is also an expression vector ning the
polynucleotide sequence, particularly DNA sequence of the invention as defined
above.
r aspect of the invention is also a host cell comprising an expression
vector as defined above.
A preferred host cell for expression of fusion proteins of the invention is an E.
coli cell.
Methods for generation of recombinant proteins, including fusion proteins, are
well known. In brief, this que ts in generation of polynucleotide
molecule, for example DNA molecule encoding the amino acid sequence of the
target protein and directing the sion of the target protein in the host.
Then, the target protein encoding polynucleotide molecule is incorporated into
an appropriate expression vector, which ensures an efficient expression of the
polypeptide. Recombinant expression vector is then introduced into host cells
for transfection/transformation, and as a result a transformed host cell is
produced. This is followed by a culture of transformed cells to overexpress the
target protein, purification of obtained proteins, and optionally cutting off by
cleavage the tag sequences used for expression or purification of the protein.
Suitable techniques of expression and purification are described, for example in
the monograph Goeddel, Gene sion Technology, Methods in Enzymology
185, Academic Press, San Diego, CA (1990), and A. Staron et al., Advances
Mikrobiol., 2008, 47, 2, 995.
Cosmids, plasmids or modified viruses can be used as expression vectors for the
introduction and replication of DNA sequences in host cells. Typically plasmids
are used as expression vectors. Suitable plasmids are well known and
cially available.
sion vector of the invention comprises a polynucleotide molecule encoding
the fusion n of the invention and the necessary regulatory sequences for
ription and translation of the coding sequence incorporated into a suitable
host cell. Selection of regulatory sequences is dependent on the type of host
cells and can be easily carried out by a person skilled in the art. Examples of
such regulatory ces are transcriptional promoter and enhancer or RNA
polymerase binding sequence, ribosome g sequence, ning the
ription initiation signal, inserted before the coding sequence, and
transcription terminator sequence, inserted after the coding sequence.
Moreover, ing on the host cell and the vector used, other sequences may
be introduced into the expression vector, such as the origin of replication,
additional DNA restriction sites, enhancers, and sequences ng induction of
transcription.
The expression vector will also comprise a marker gene sequence, which confers
defined phenotype to the transformed cell and enables specific selection of
transformed cells. Furthermore, the vector may also contain a second marker
sequence which allows to guish cells transformed with recombinant plasmid
containing inserted coding sequence of the target protein from those which have
taken up the plasmid without insert. Most often, typical antibiotic resistance
markers are used, however, any other reporter genes known in the field may be
used, whose presence in a cell (in vivo) can be easily determined using
autoradiography techniques, spectrophotometry or bio- and chemi-
luminescence. For example, ing on the host cell, reporter genes such as
B-galactosidase, B-glucuronidase, luciferase, chloramphenicol acetyltransferase
or green fluorescent protein may be used.
Furthermore, the expression vector may contain signal sequence, transporting
proteins to the appropriate cellular compartment, e.g. periplasma, where
folding is facilitated. Additionally a ce encoding a label/tag, such as
HisTag attached to the N-terminus or GST attached to the C-terminus, may be
present, which facilitates subsequent purification of the n produced using
the principle of affinity, via affinity chromatography on a nickel column.
Additional sequences that protect the protein against proteolytic degradation in
the host cells, as well as sequences that increase its solubility may also be
present.
Auxiliary t attached to the sequence of the target protein may block its
activity, or be detrimental for another reason, such as for example due to
toxicity. Such element must be removed, which may be accomplished by
enzymatic or chemical cleavage. In particular, a six-histidine tag HisTag or other
markers of this type ed to allow n purification by affinity
chromatography should be removed, because of its described effect on the liver
toxicity of soluble TRAIL protein. Heterologous expression systems based on
various well-known host cells may be used, including prokaryotic cells:
bacterial, such as Escherichia coli or Bacillus subtilis, yeasts such as
Saccharomyces cervisiae or Pichia pastoris, and eukaryotic cell lines (insect,
mammalian, plant).
Preferably, due to the ease of culturing and genetic manipulation, and a large
amount of obtained product, the E. coli sion system is used. Accordingly,
the polynucleotide sequence containing the target sequence encoding the fusion
protein of the invention will be optimized for expression in E. coli, i.e. it will
contain in the coding sequence codons optimal for expression in E. coli, selected
from the possible sequence variants known in the state of art. Furthermore, the
expression vector will n the above described elements le for E. coli
attached to the coding sequence.
Accordingly, in a preferred embodiment of the invention a polynucleotide
ce comprising a sequence encoding a fusion protein of the invention,
optimized for expression in E. coli is ed from the group of polynucleotide
sequences consisting of:
SEQ. N0. 50; SEQ. N0. 51; SEQ. N0. 52, SEQ. N0. 53; SEQ. N0. 54; SEQ. N0. 55;
SEQ. N0. 56; SEQ. N0. 57; SEQ. N0. 58; SEQ. N0. 59; SEQ. N0. 60, and SEQ. N0.
61; SEQ. N0. 62 SEQ. N0. 63; SEQ. N0. 64; SEQ. N0. 65; SEQ. N0. 66, SEQ. N0. 67;
SEQ. N0. 68; SEQ. N0. 69; SEQ. N0. 70; SEQ. N0. 71; SEQ. N0. 72; SEQ. N0. 73;
SEQ. N0. 74 and SEQ. N0. 76.
which encode a fusion protein having an amino acid ce corresponding to
amino acid sequences selected from the group consisting of amino acid
sequences, respectively:
SEQ. No. 1; SEQ. No. 2; SEQ. No. 3; SEQ. No. 4; SEQ. No. 5; SEQ. No. 6;
SEQ. No.7; SEQ. No. 8; SEQ. No. 9; SEQ. No. 10; SEQ. No. 11; SEQ. No. 12;
SEQ. No. 13; SEQ. No. 14; SEQ. No. 15; SEQ. No. 16; SEQ. No. 17; SEQ. No. 18;
SEQ. No. 19; SEQ. No. 20; SEQ. No. 21; SEQ. No. 22; SEQ. No. 23; SEQ. No. 24;
SEQ. No. 25 and SEQ. No. 75.
In a preferred embodiment, the invention provides also an expression vector
suitable for transformation of E. coli, comprising the cleotide sequence
selected from the group of polynucleotide sequences SEQ. No. 50 to SEQ. No. 74
and SEQ. No. 76 indicated above, as well as E. coli cell transformed with such an
expression vector.
Transformation, i.e. uction of a DNA sequence into bacterial host cells,
ularly E. coli, is usually performed on the competent cells, prepared to
take up the DNA for example by treatment with calcium ions at low temperature
(4°C), and then ting to the heat-shock (at 37-42°C) or by electroporation.
Such techniques are well known and are usually determined by the manufacturer
of the sion system or are described in the literature and manuals for
laboratory work, such as Maniatis et al., Molecular Cloning. Cold Spring Harbor,
N.Y., 1982).
The procedure of overexpression of fusion proteins of the invention in E. coli
expression system will be further described below.
The invention also provides a pharmaceutical composition containing the fusion
protein of the invention as defined above as an active ingredient and a suitable
pharmaceutically acceptable carrier, t and conventional auxiliary
components. The pharmaceutical composition will contain an effective amount
of the fusion protein of the invention and pharmaceutically acceptable auxiliary
components dissolved or dispersed in a carrier or diluent, and preferably will be
in the form of a pharmaceutical composition formulated in a unit dosage form or
formulation containing a plurality of doses. Pharmaceutical forms and methods
of their formulation as well as other ents, carriers and diluents are
known to the skilled person and described in the literature. For e, they
are described in the monograph ton's Pharmaceutical Sciences, ed. 20,
2000, Mack Publishing Company, Easton, USA.
The terms "pharmaceutically acceptable r, diluent, and auxiliary
ingredient" comprise any solvents, dispersion media, surfactants, antioxidants,
stabilizers, vatives (e.g. antibacterial agents, antifungal agents),
isotonizing , known in the art. The pharmaceutical ition of the
invention may contain various types of rs, diluents and excipients,
depending on the chosen route of administration and desired dosage form, such
as liquid, solid and aerosol forms for oral, parenteral, inhaled, topical, and
whether that selected form must be sterile for administration route such as by
injection. The preferred route of administration of the pharmaceutical
composition according to the invention is parenteral, including injection routes
such as intravenous, intramuscular, subcutaneous, intraperitoneal, intratumoral,
or by single or continuous intravenous ons.
In one embodiment, the pharmaceutical composition of the invention may be
stered by injection directly to the tumor. In another embodiment, the
ceutical composition of the invention may be administered intravenously.
In yet another embodiment, the pharmaceutical composition of the invention
can be administered subcutaneously or intraperitoneally. A pharmaceutical
composition for parenteral administration may be a solution or dispersion in a
pharmaceutically acceptable aqueous or non-aqueous medium, buffered to an
appropriate pH and isoosmotic with body fluids, if necessary, and may also
contain antioxidants, buffers, bacteriostatic agents and soluble substances,
which make the composition compatible with the s or blood of recipient.
Other components, which may included in the composition, are for example
water, alcohols such as ethanol, polyols such as glycerol, ene glycol, liquid
polyethylene glycol, lipids such as triglycerides, vegetable oils, liposomes.
Proper fluidity and the les size of the substance may be provided by
g nces, such as lecithin, and surfactants, such as hydroxypropyl
celulose polysorbates, and the like.
Suitable isotonizing agents for liquid parenteral itions are, for example,
sugars such as glucose, and sodium chloride, and combinations thereof.
Alternatively, the pharmaceutical composition for administration by injection or
infusion may be in a powder form, such as a lyophilized powder for
reconstitution immediately prior to use in a le carrier such as, for
example, sterile pyrogen-free water.
The pharmaceutical composition of the invention for parenteral administration
may also have the form of nasal stration, ing solutions, sprays or
aerosols. Preferably, the form for intranasal stration will be an aqueous
solution and will be isotonic or ed o maintain the pH from about 5.5 to
about 6.5, so as to maintain a character similar to nasal secretions. Moreover, it
will contain preservatives or stabilizers, such as in the well-known intranasal
preparations.
The composition may contain various antioxidants which delay oxidation of one
or more components. Furthermore, in order to prevent the action of
microorganisms, the composition may contain various antibacterial and anti
fungal agents, including, for example, and not limited to, parabens,
chlorobutanol, himerosal, sorbic acid, and similar known nces of this type.
In general, the pharmaceutical composition of the invention can include, for
example at least about 0.01 wt% of active ingredient. More particularly, the
composition may contain the active ingredient in the amount from 1% to 75% by
weight of the composition unit, or for example from 25% to 60% by weight, but
not limited to the ted values. The actual amount of the dose of the
composition according to the present invention administered to patients,
including man, will be determined by physical and physiological factors, such as
body weight, severity of the condition, type of disease being treated, previous
or concomitant therapeutic interventions, the t and the route of
administration. A suitable unit dose, the total dose and the concentration of
active ingredient in the composition is to be ined by the treating
physician.
The composition may for example be administered at a dose of about 1
microgram/kg of body weight to about 1000 mg/kg of body weight of the
t, for example in the range of 5 mg/kg of body weight to 100 mg/kg of
body weight or in the range of 5 mg/kg of body weight to 500 mg/kg of body
weight. The fusion protein and the compositions containing it t anticancer
or antitumor and can be used for the treatment of cancer diseases. The
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invention also provides the use of the fusion protein of the invention as defined
above for treating cancer diseases in s, including humans. The invention
also provides a method of treating neoplastic/cancer diseases in s,
including humans, comprising administering to a t in need of such
treatment an eoplasticc/anticancer effective amount of the fusion protein
of the invention as defined above, optionally in the form of riate
pharmaceutical composition.
The fusion protein of the invention can be used for the treatment of hematologic
malignancies, such as leukaemia, granulomatosis, a and other
hematologic malignancies. The fusion protein can also be used for the treatment
of solid tumors, such as breast cancer, lung cancer, including non-small cell lung
cancer, colon cancer, pancreatic cancer, ovarian cancer, bladder cancer,
prostate cancer, kidney cancer, brain cancer, and the like. Appropriate route of
administration of the fusion protein in the treatment of cancer will be in
particular parenteral route, which consists in administering the fusion protein of
the invention in the form of injections or infusions, in the composition and form
appropriate for this administration route. The invention will be described in
more detail in the following general procedures and es of specific fusion
proteins.
General procedure for overexpression of the fusion protein
Preparation of a plasmid
Amino acid sequence of the target fusion protein was used as a template to
3generate a DNA sequence encoding it, comprising codons optimized for
expression in Escherichia coli. Such a procedure allows to increase the efficiency
of a further step of target protein synthesis in Escherichia coli. Resulting
nucleotide ce was then automatically synthesized. Additionally, the
cleavage sites of restriction enzymes Ndel (at the 5'-end of leading strand) and
Xho| (at the 3'-end of g ) were added to the resulting gene ng
the target protein. These were used to clone the gene into the vector pET28a
(Novagen). They may be also be used for cloning the gene encoding the protein
to other vectors. Target protein expressed from this construct can be optionally
equipped at the N-terminus with a polyhistidine tag (six histidines), preceded by
a site ized by thrombin, which subsequently served to its purification via
affinity chromatography. Some target were expressed without any tag, in
particular without histidine tag, and those were subsequently purified on SP
Sepharose. The correctness of the resulting construct was confirmed firstly by
restriction analysis of isolated ds using the enzymes Ndel and Xhol,
followed by automatic cing of the entire reading frame of the target
protein. The primers used for sequencing were complementary to the sequences
of T7 er (5'-TAATACGACTCACTATAGG-3') and T7 terminator (5'-
GCTAG'I'I'A'I'I'GCTCAGCGG-3') present in the vector. Resulting plasmid was used
for overexpression of the target fusion protein in a commercial E. coli strain,
which was transformed according to the manufacturer's recommendations.
Colonies obtained on the selection medium (LB agar, kanamycin 50 ug/ml, 1%
glucose) were used for preparing an overnight culture in LB liquid medium
mented with kanamycin (50 ug/ml) and 1% glucose. After about 15h of
growth in shaking incubator, the cultures were used to inoculate the appropriate
culture.
Overexpression and purification of fusion proteins - general procedure A
LB medium with kanamycin (30 ug/ml) and 100 (M zinc e was inoculated
with overnight culture. The culture was incubated at 37°C until the l
density (OD) at 600 nm reached 0.60-0.80. Then IPTG was added to the final
concentration in the range of 0.25 -1mM. After incubation (3.5 - 20h) with
g at 25°C the culture was centrifuged for 25 min at 6,000 g. Bacterial
pellets were resuspended in a buffer containing 50 mM KH2P04, 0.5 M NaCl, 10
mM imidazole, pH 7.4. The sion was ted on ice for 8 minutes (40%
amplitude, 15-second pulse, 10 s interval). The resulting extract was ied by
centrifugation for 40 minutes at 20000 g, 4°C. Ni-Sepharose (GE Healthcare)
resin was pre-treated by bration with buffer, which was used for
preparation of the bacterial cells extract. The resin was then incubated
overnight at 4°C with the supernatant obtained after centrifugation of the
extract. Then it was loaded into chromatography column and washed with 15 to
50 volumes of buffer 50 mM KH2P04, 0.5 M NaCl, 20 mM imidazole, pH 7.4. The
obtained protein was eluted from the column using imidazole gradient in 50 mM
KHzPO4 buffer with 0.5 M NaCl, pH 7.4. Obtained fractions were analyzed by SDS-
PAGE. Appropriate fractions were combined and dialyzed ght at 4°C
against 50 mM Tris buffer, pH 7.2, 150 mM NaCl, 500 mM L-arginine, 0.1 mM
ZnSO4, 0.01% Tween 20, and at the same time Histag, if present, was cleaved
with thrombin (1:50). After the cleavage, thrombin was separated from the
target fusion protein expressed with His tag by purification using Benzamidine
SepharoseTM resin. Purification of target fusion proteins expressed without
Histag was med on SP Sepharose. The purity of the product was analyzed
by GE electrophoresis (Maniatis et al, Molecular Cloning. Cold Spring
, NY, 1982).
pression and purification of fusion ns - general procedure B
LB medium with kanamycin (30 ug/ml) and 100 uM zinc sulfate was inoculated
with overnight culture. Cultures were incubated at 37°C until optical density
(OD) at 600 nm reached 0.60-0.80. Then IPTG was added to the final
concentration in the range 0.5 -1mM. After 20h incubation with shaking at 25°C
the culture was centrifuged for 25 min at 6000 g. Bacterial cells after
pression were disrupted in a French Press in a buffer containing 50 mM
KHzPO4, 0.5 M NaCl, 10 mM imidazole, 5mM beta-mercaptoethanol, 0.5mM PMSF
(phenylmethylsulphonyl fluoride), pH 7.8. Resulting extract was clarified by
centrifugation for 50 minutes at 8000 g. The Ni-Sepharose resin was incubated
overnight with the obtained supernatant. Then the resin with bound protein was
packed into the chromatography column. To wash-out the fractions containing
non-binding proteins, the column was washed with 15 to 50 volumes of buffer 50
mM KHzPO4, 0.5 M NaCl, 10 mM imidazole, 5mM ercaptoethanol, 0.5mM
PMSF (phenylmethylsulphonyl fluoride), pH 7.8. Then, to wash-out the majority
of proteins binding specifically with the bed, the column was washed with a
buffer ning 50 mM KHZPO4, 0.5 M NaCl, 500 mM imidazole, 10% glycerol,
0.5mM PMSF, pH 7.5. Obtained fractions were analyzed by SDS-PAGE tis et
al, Molecular Cloning. Cold Spring Harbor, NY, 1982). The ons containing
the target protein were combined and, if the protein was expressed with
histidine tag, cleaved with in (1U per 4 mg of protein, 8h at 16°C) to
remove polyhistidine tag. Then the fractions were ed against formulation
buffer (500 mM L-arginine, 50 mM Tris, 2.5 mM ZnSO4, pH 7.4).
Further in this description proteins originally expressed with histidine tag that
was subsequently removed are designated as a) at the Ex. No.. Proteins that
were originally expressed t histidine tag are designated as b) at the Ex.
No..
Example 1. The fusion protein of SEQ. No. 1
The protein of SEQ. No. 1 is a fusion protein having the length of 203 amino
acids and the mass of 23.3 kDa, in which at the N-terminus of the sequence
TRAIL114-281 a 34-amino acid fragment of human fetoprotein (SEQ. No. 27) is
attached as an effector peptide. Between the effector peptide and the sequence
of TRAIL there is incorporated a sequence of cleavage site recognized by
ase uPA (SEQ. No. 46) due to which the effector peptide will undergo
cleavage in the tumor environment.
ure of the fusion protein is shown schematically in Fig. 1 and its amino
acid sequence and the DNA encoding sequence comprising codons optimized for
expression in E. coli are, tively, SEQ. No. 1 and SEQ. No. 50 as shown in
the attached Sequence g.
The amino acid sequence SEQ. No. 1 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 50. A plasmid
containing the coding sequence of DNA was generated and overexpression of the
fusion protein was d out in accordance with the general procedures
described above. Overexpression was performed according to the general
procedure A, using E. coli BL21 (DE3) or Tuner(DE3)pLysS strains from Novagen.
The protein was separated by electrophoresis in accordance with the general
procedure bed above.
e 2. The fusion protein of SEQ. No. 2
The protein of SEQ. No. 2 is a fusion protein having the length of 178 amino
acids and the mass of 20.5 kDa, in which at the N-terminus of the sequence
TRAIL114-281 a 8-amino acid fragment of human fetoprotein (SEQ. No. 28) is
attached as an effector peptide. Between the or peptide and the sequence
of TRAIL there is incorporated a sequence of ge site recognized by
urokinase uPA (SEQ. No. 46) due to which the effector peptide will undergo
cleavage in the tumor environment.
Structure of the fusion protein is shown schematically in Fig. 1 and its amino
acid sequence and the DNA encoding sequence comprising codons optimized for
expression in E. coli are, respectively, SEQ. No. 2 and SEQ. No. 51 as shown in
the attached Sequence Listing.
The amino acid sequence SEQ. No. 2 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 51. A plasmid
containing the coding sequence of DNA was generated and overexpression of the
fusion n was carried out in accordance with the general procedures
described above. Overexpression was performed according to the l
procedure B, using E. coli BL21 (DE3) strain from Novagen. The protein was
separated by ophoresis in accordance with the general ure
described above.
Example 3. The fusion protein of SEQ. No. 3
The protein of SEQ. No. 3 is a fusion protein having the length of 179 amino
acids and the mass of 20.5 kDa, in which at the inus of the sequence
TRAIL121-281 a 8-amino acid fragment of human fetoprotein (SEQ. No. 28) is
attached as an effector peptide. n the effector peptide and the sequence
of TRAIL there are orated sequentially next to each other sequences of
cleavage sites recognized by metalloprotease MMP (SEQ. No. 45) and urokinase
uPA (SEQ. No. 46) due to which the effector peptide will undergo cleavage in
the tumor environment.
Structure of the fusion protein is shown schematically in Fig. 1 and its amino
acid sequence and the DNA encoding ce sing codons optimized for
expression in E. coli are, respectively, SEQ. No. 3 and SEQ. No. 52 as shown in
the attached Sequence Listing.
The amino acid sequence SEQ. No. 3 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 52. A plasmid
containing the coding sequence of DNA was generated and overexpression of the
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fusion protein was carried out in accordance with the l procedures
described above. Overexpression was performed according to the general
procedure A, using E. coli BL21 (DE3) strain from Novagen. The n was
separated by electrophoresis in accordance with the general procedure
described above.
Example 4. The fusion protein of SEQ. No. 4
The protein of SEQ. No. 4 is a fusion protein having the length of 204 amino
acids and the mass of 23.2 kDa, in which at the C-terminus of the sequence
TRAIL121-281 a 34-amino acid nt of human fetoprotein (SEQ. No. 27) is
attached as an effector peptide. Between the effector peptide and the sequence
of TRAIL there are incorporated sequentially next to each other sequences of
cleavage sites recognized by metalloprotease MMP (SEQ. No. 45) and urokinase
uPA (SEQ. No. 46) due to which the effector peptide will undergo cleavage in
the tumor environment.
Structure of the fusion protein is shown tically in Fig. 1 and its amino
acid sequence and the DNA encoding sequence comprising codons optimized for
expression in E. coli are, respectively, SEQ. No. 4 and SEQ. No. 53 as shown in
the attached Sequence g.
The amino acid sequence SEQ. No. 4 of the structure described above was used
as a template to te its coding DNA ce SEQ. No. 53. A plasmid
containing the coding sequence of DNA was generated and overexpression of the
fusion protein was carried out in accordance with the general procedures
described above. Overexpression was performed according to the general
procedure B, using E. coli BL21DE3pLysSRIL strain from gene or Tuner
(DE3) strain from Novagen. The protein was separated by electrophoresis in
ance with the general procedure described above.
Example 5. The fusion protein of SEQ. No. 5
The protein of SEQ. No. 5 is a fusion protein having the length of 230 amino
acids and the mass of 26 kDa, in which at the N-terminus of the sequence
5-281 a 34-amino acid fragment of human fetoprotein (SEQ. No. 27) is
attached as an effector peptide. Between the effector peptide and the sequence
of TRAIL there are incorporated sequentially next to each other sequences of
cleavage sites recognized by urokinase uPA (SEQ. No. 46) and metalloprotease
MMP (SEQ. No. 45) due to which the effector peptide will o cleavage in
the tumor environment.
Structure of the fusion protein is shown schematically in Fig. 1 and its amino
acid sequence and the DNA encoding sequence comprising codons optimized for
expression in E. coli are, respectively, SEQ. No. 5 and SEQ. No. 54 as shown in
the attached Sequence Listing.
The amino acid sequence SEQ. No. 5 of the structure described above was used
as a te to generate its coding DNA sequence SEQ. No. 54. A plasmid
containing the coding ce of DNA was generated and overexpression of the
fusion protein was d out in accordance with the l procedures
bed above. Overexpression was performed according to the general
procedure A, using E. coli Tuner (DE3) strain from Novagen. The protein was
separated by ophoresis in accordance with the general procedure
described above.
Example 6. The fusion protein of SEQ. No. 6
The protein of SEQ. No. 6 is a fusion protein having the length of 238 amino
acids and the mass of 26.7 kDa, in which at the C-terminus of the sequence
TRAIL95-281 a 34-amino acid fragment of human fetoprotein (SEQ. No. 27) is
attached as an effector peptide. Between the effector peptide and the sequence
of TRAIL there are incorporated sequentially next to each other sequences of
cleavage sites recognized by metalloprotease MMP (SEQ. No. 45) and ase
uPA (SEQ. No. 46) due to which the effector peptide will undergo cleavage in the
tumor environment. Between the sequence of TRAIL and the ce of
ge site recognized by metalloprotease MP the fusion protein contains
additionally a flexible cysteine — alanine linker (SEQ. No. 47).
Structure of the fusion protein is shown schematically in Fig. 2 and its amino
acid sequence and the DNA encoding sequence comprising codons zed for
expression in E. coli are, respectively, SEQ. No. 6 and SEQ. No. 55 as shown in
the attached Sequence Listing.
The amino acid sequence SEQ. No. 6 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 55. A d
containing the coding sequence of DNA was generated and pression of the
fusion protein was carried out in accordance with the general procedures
described above. Overexpression was performed according to the general
procedure A, using E. coli Tuner (DE3) strain from Novagen. The protein was
separated by electrophoresis in accordance with the general procedure
described above.
Example 7. The fusion protein of SEQ. No. 7
The protein of SEQ. No. 7 is a fusion protein having the length of 213 amino
acids and the mass of 24.1 kDa, in which at the C-terminus of the sequence
TRAIL95-281 a 8-amino acid fragment of human otein (SEQ. No. 28) is
attached as an effector peptide. Between the effector peptide and the sequence
of TRAIL there are orated sequentially next to each other sequences of
cleavage sites ized by metalloprotease MMP (SEQ. No. 45) and urokinase
uPA (SEQ. No. 46) due to which the effector peptide will undergo cleavage in the
tumor environment. Between the sequence of TRAIL and the sequence of
cleavage site ized by metalloprotease MP the fusion protein contains
additionally a le cysteine — alanine linker (SEQ. No. 47).
ure of the fusion protein is shown schematically in Fig. 2 and its amino
acid sequence and the DNA encoding sequence comprising codons optimized for
expression in E. coli are, respectively, SEQ. No. 7 and SEQ. No. 56 as shown in
the attached Sequence Listing.
The amino acid sequence SEQ. No. 7 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 56. A plasmid
containing the coding sequence of DNA was generated and overexpression of the
fusion protein was carried out in accordance with the general procedures
described above. Overexpression was med according to the general
procedure A, using E. coli Tuner (DE3) strain from Novagen. The protein was
separated by electrophoresis in ance with the general procedure
described above.
Example 8. The fusion protein of SEQ. No. 8
The n of SEQ. No. 8 is a fusion protein having the length of 191 amino
acids and the mass of 23 kDa, in which at the N-terminus of the sequence
21-281 a 20-amino acid nt of peptide derived from p21WAF protein
(SEQ. No. 29) is attached as an effector e. Additionally, at the C-terminus
of the or protein there is attached a fragment of antennapedia n
domain (SEQ. No. 49) as a transporting sequence, which aids in penetration of
the cell membrane and transportation of the fusion protein into the cell.
Between the transporting sequence and the sequence of TRAIL there are
incorporated sequentially next to each other sequences of cleavage sites
recognized by urokinase uPA (SEQ. No. 46) and metalloprotease MMP (SEQ. No.
45) due to which the effector peptide will undergo cleavage in the tumor
environment.
Structure of the fusion protein is shown schematically in Fig. 2 and its amino
acid sequence and the DNA encoding ce comprising codons optimized for
expression in E. coli are, respectively, SEQ. No. 8 and SEQ. No. 57 as shown in
the attached Sequence Listing.
The amino acid sequence SEQ. No. 8 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 57. A plasmid
containing the coding sequence of DNA in two versions, one allowing to express
His tag and a site recognized by thrombin and the second without any tag was
generated and overexpression of the fusion protein was carried out in
accordance with the l procedures described above. pression was
performed according to the general procedure A, using E. coli Tuner (DE3) strain
from Novagen. The protein was separated by electrophoresis in accordance with
the general procedure described above.
Example 8A. The fusion protein of SEQ. No. 75
The protein of SEQ. No. 75 is a fusion protein having the length of 212 amino
acids and the mass of 24,13 kDa, in which at the N-terminus of the sequence
TRAIL120-281 a 20-amino acid fragment of peptide derived from p21WAF protein
(SEQ. No. 29) is attached as an or peptide. Additionally, at the C-terminus
WO 43477 2012/057219
of the effector protein there is attached a fragment of antennapedia protein
domain (SEQ. No. 49) as a transporting sequence, which aids in penetration of
the cell membrane and transportation of the fusion protein into the cell.
Between the transporting sequence and the sequence of TRAIL there are
incorporated sequentially next to each other sequences of ge sites
recognized by urokinase uPA (SEQ. No. 46) and oprotease MMP (SEQ. No.
45) due to which the effector peptide will undergo cleavage in the tumor
environment. Additionally between the metalloprotease cleavage site and the
sequence of TRAIL the fusion protein ns additionally a flexible linker (SEQ.
No. 77).
Structure of the fusion protein is shown schematically in Fig. 2 and its amino
acid sequence and the DNA encoding sequence comprising codons optimized for
expression in E. coli are, tively, SEQ. No. 75 and SEQ. No. 76 as shown in
the attached Sequence g.
The amino acid sequence SEQ. No. 75 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 76. A plasmid
containing the coding sequence of DNA in two versions, one allowing to express
His tag and a site recognized by thrombin and the second without any tag was
generated and overexpression of the fusion protein was carried out in
accordance with the general procedures described above. Overexpression was
performed according to the general procedure A, using E. coli Tuner (DE3) strain
from Novagen. The protein was separated by electrophoresis in accordance with
the general ure described above.
Example 9. The fusion protein of SEQ. No. 9
The protein of SEQ. No. 9 is a fusion n having the length of 231 amino
acids and the mass of 26.5 kDa, in which at the C-terminus of the ce
TRAIL95-281 a 20-amino acid fragment of peptide derived from p21WAF protein
(SEQ. No. 29) is attached as an or peptide. Between the effector peptide
and the sequence of TRAIL there are incorporated sequentially next to each
other sequences of cleavage sites recognized by metalloprotease MMP (SEQ. No.
45) and urokinase uPA (SEQ. No. 46) due to which the effector peptide will
o cleavage in the tumor environment. Between the sequence of TRAIL and
the sequence of cleavage sites the fusion protein ns onally a flexible
cysteine — alanine linker (SEQ. No. 47). Additionally, at the C-terminus of the
effector protein there is attached a fragment of antennapedia protein domain
(SEQ. No. 49) forming C-terminal fragment of entire construct as a transporting
sequence which aids in penetration of the cell membrane and transportation of
the fusion protein into the cell.
Structure of the fusion protein is shown schematically in Fig. 2 and its amino
acid sequence and the DNA encoding sequence comprising codons optimized for
expression in E. coli are, respectively, SEQ. No. 9 and SEQ. No. 58 as shown in
the attached Sequence Listing.
The amino acid sequence SEQ. No. 9 of the ure described above was used
as a template to generate its coding DNA sequence SEQ. No. 58. A plasmid
containing the coding sequence of DNA was ted and overexpression of the
fusion protein was d out in accordance with the general procedures
described above. pression was performed according to the general
procedure A, using E. coli Rosetta (DE3) strain from Novagen. The protein was
separated by electrophoresis in accordance with the general procedure
described above.
Example 10. The fusion protein of SEQ. No. 10
The protein of SEQ. No. 10 is a fusion n having the length of 200 amino
acids and the mass of 22.8 kDa, in which at the N-terminus of the sequence
TRAIL120-281 a 16-amino acid fragment of peptide analogue of domain binding
to FGF-Z receptor (SEQ. No. 26) is attached as an or peptide. Between the
effector peptide and the sequence of TRAIL there are incorporated sequentially
next to each other sequences of cleavage sites ized by urokinase uPA
(SEQ. No. 46) and metalloprotease MMP (SEQ. No. 45) due to which the effector
peptide will undergo ge in the tumor environment. Between the sequence
of TRAIL and the sequence of ge sites the fusion protein contains
additionally a flexible cysteine — alanine linker (SEQ. No. 47). Additionally,
between the sequence of cleavage site and the sequence of flexible linker as
well as n the sequence of flexible linker and TRAIL domain there is
incorporated a linker consisting of two glycine residues aids in ization of
trimeric structure.
ure of the fusion protein is shown tically in Fig. 2 and its amino
acid sequence and the DNA encoding sequence comprising codons zed for
sion in E. coli are, respectively, SEQ. No. 10 and SEQ. No. 59 as shown in
the attached Sequence Listing.
The amino acid ce SEQ. No. 10 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 59. A plasmid
containing the coding sequence of DNA was generated and overexpression of the
fusion protein was carried out in accordance with the general procedures
bed above. Overexpression was performed according to the general
procedure A, using E. coli BL21 (DE3) strain from Novagen. The protein was
separated by electrophoresis in accordance with the l ure
described above.
Example 11. The fusion protein of SEQ. No. 11
The protein of SEQ. No. 11 is a fusion protein having the length of 233 amino
acids and the mass of 26.5 kDa, in which at the C-terminus of the sequence
TRAIL95-281 an 18-amino acid fragment of peptide DD2 derived from DOC-
2/DAB2 (SEQ. No. 30) is attached as an effector peptide. Between the effector
e and the sequence of TRAIL there are incorporated sequentially next to
each other sequences of cleavage sites recognized by metalloprotease MMP
(SEQ. No. 45) and urokinase uPA (SEQ. No. 46) due to which the effector peptide
will undergo cleavage in the tumor environment. The sequence of the effector
peptide has attached at its N-terminus the poly-arginine transporting domain
consisting of 7 Arg residues. Transporting sequence aids in penetration of the
cell membrane and transportation of the fusion protein into the cell. Between
the sequence of TRAIL and the sequence of cleavage sites the fusion protein
contains additionally a flexible cysteine — alanine - glycine linker CAACAAACGGG.
ure of the fusion protein is shown schematically in Fig. 3 and its amino
acid sequence and the DNA encoding sequence comprising codons optimized for
expression in E. coli are, respectively, SEQ. No. 11 and SEQ. No. 60 as shown in
the attached Sequence Listing.
The amino acid sequence SEQ. No. 11 of the structure described above was used
as a te to generate its coding DNA sequence SEQ. No. 60. A d
ning the coding sequence of DNA was generated and overexpression of the
fusion protein was carried out in accordance with the general procedures
described above. Overexpression was performed according to the general
procedure A, using E. coli BL21 (DE3) or DE3)pLysS strains from Novagen.
The protein was ted by electrophoresis in accordance with the general
procedure described above.
Example 12. The fusion protein of SEQ. No. 12
The protein of SEQ. No. 12 is a fusion protein having the length of 590 amino
acids and the mass of 66.7 kDa, in which at the C-terminus of the ce
21-281 an arginine deiminase from Mycoplasma arginini (SEQ. No. 31) is
attached as an effector peptide. Between the effector peptide and the sequence
of TRAIL there are orated sequentially next to each other ces of
cleavage sites recognized by metalloprotease MMP (SEQ. No. 45) and urokinase
uPA (SEQ. No. 46) due to which the effector peptide will o cleavage in the
tumor environment. Between the sequence of TRAIL and the sequence of
metalloprotease cleavage site the fusion protein contains additionally a flexible
linker consisting of glycine and serine residues Gly Gly Ser Gly. Between the
sequence of urokinase cleavage site and the sequence of effector protein the
fusion protein contains additionally a flexible e serine linker
Gly Gly Gly Ser Gly.
Structure of the fusion protein is shown schematically in Fig. 3 and its amino
acid sequence and the DNA encoding sequence comprising codons optimized for
expression in E. coli are, respectively, SEQ. No. 12 and SEQ. No. 61 as shown in
the attached Sequence Listing.
The amino acid sequence SEQ. No. 12 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 61. A plasmid
containing the coding sequence of DNA was generated and overexpression of the
fusion protein was carried out in accordance with the general procedures
described above. Overexpression was performed according to the general
procedure A, using E. coli BL21 (DE3) strain from Novagen. The protein was
separated by electrophoresis in accordance with the l ure
described above.
Example 13. The fusion protein of SEQ. No. 13
The protein of SEQ. No. 13 is a fusion protein having the length of 187 amino
acids and the mass of 21.6 kDa, in which at the N-terminus of the sequence
TRAIL121-281 a 10-amino acid peptide from p16 protein (SEQ. No. 32) is
ed as an effector peptide. Between the effector peptide and the N-
us of TRAIL domain there are incorporated sequentially next to each other
sequences of cleavage sites recognized by urokinase uPA (SEQ. No. 46) and
metalloprotease MMP (SEQ. No. 45) due to which the effector peptide will
undergo cleavage in the tumor environment. The sequence of the effector
peptide has attached at its C-terminus a transporting sequence (SEQ. No. 49)
ting of nt of antennapedia protein domain fragment. Transporting
sequence aids in penetration of the cell membrane and transportation of the
fusion protein into the cell.
Structure of the fusion protein is shown tically in Fig. 3 and its amino
acid sequence and the DNA encoding sequence comprising codons zed for
expression in E. coli are, respectively, SEQ. No. 13 and SEQ. No. 62 as shown in
the attached Sequence Listing.
The amino acid sequence SEQ. No. 13 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 62. A plasmid
containing the coding sequence of DNA was ted and pression of the
fusion protein was carried out in accordance with the general procedures
described above. Overexpression was performed according to the general
procedure B, using E. coli B.21 (DE3) strain from Novagen or BL21DE3pLysSRIL
strain from Stratagene. The protein was separated by electrophoresis in
ance with the general procedure described above.
Example 14. The fusion protein of SEQ. No. 14
The protein of SEQ. No. 14 is a fusion protein having the length of 203 amino
acids and the mass of 23.6 kDa, in which at the C-terminus of the sequence
TRAIL121-281 a 13-amino acid fragment of MEK-1 protein — an tor of ERK
activation (SEQ. No. 34) is attached as an effector e. Between the C-
terminus of TRAIL and the effector peptide domain there are incorporated
sequentially next to each other sequences of cleavage sites recognized by
metalloprotease MMP (SEQ. No. 45) and urokinase uPA (SEQ. No. 46) due to
which the effector peptide will undergo cleavage in the tumor environment. The
sequence of the effector peptide has attached at its N-terminus a transporting
sequence (SEQ. No. 48) consisting of antennapedia protein domain fragment.
Transporting ce aids in penetration of the cell membrane and
transportation of the fusion n into the cell. n the sequence of
TRAIL and the sequence of cleavage sites the fusion protein contains additionally
a flexible glycine —cysteine linker GS.
ure of the fusion protein is shown schematically in Fig. 3 and its amino
acid sequence and the DNA encoding sequence comprising codons optimized for
expression in E. coli are, tively, SEQ. No. 14 and SEQ. No. 63 as shown in
the attached Sequence Listing.
The amino acid sequence SEQ. No. 14 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 63. A d
containing the coding sequence of DNA was ted and overexpression of the
fusion protein was carried out in accordance with the general procedures
described above. Overexpression was performed according to the general
procedure B, using E. coli B.21 (DE3) strain from Novagen or BL21DE3pLysSRIL
strain from Stratagene. The protein was separated by electrophoresis in
accordance with the general procedure described above.
Example 15. The fusion protein of SEQ. No. 15
The protein of SEQ. No. 15 is a fusion protein having the length of 205 amino
acids and the mass of 24 kDa, in which at the C-terminus of the sequence
TRAIL121-281 a 15-amino acid N-terminal nt of PH domain of TCL1 protein
— acting as Akt coactivator (SEQ. No. 35) is attached as an or peptide.
Between the TRAIL domain and the effector peptide there are incorporated
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sequentially next to each other sequences of cleavage sites recognized by
oprotease MMP (SEQ. No. 45) and urokinase uPA (SEQ. No. 46) due to
which the effector peptide will undergo cleavage in the tumor environment. The
sequence of the or peptide has attached at its N-terminus a transporting
sequence (SEQ. No. 48) consisting of fragment of antennapedia protein domain
fragment. orting sequence aids in penetration of the cell membrane and
transportation of the fusion protein into the cell. Between the ce of
TRAIL and the sequence of cleavage sites the fusion protein contains additionally
a flexible glycine —cysteine linker GS.
ure of the fusion protein is shown schematically in Fig. 3 and its amino
acid sequence and the DNA encoding sequence comprising codons optimized for
expression in E. coli are, respectively, SEQ. No. 15 and SEQ. No. 64 as shown in
the attached Sequence Listing.
The amino acid sequence SEQ. No. 15 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 64. A plasmid
containing the coding sequence of DNA was generated and overexpression of the
fusion protein was carried out in accordance with the general procedures
described above. Overexpression was med according to the general
procedure B, using E. coli B.21 (DE3) or Tuner (DE3) strains from n. The
protein was separated by electrophoresis in accordance with the general
procedure bed above.
Example 16. The fusion protein of SEQ. No. 16
The protein of SEQ. No. 16 is a fusion protein having the length of 183 amino
acids and the mass of 21.2 kDa, in which at the N-terminus of the sequence
TRAIL121-281 a hexapeptide acting as inhibitor of EZF (SEQ. No. 36) is ed
as an effector peptide. n the effector peptide and the TRAIL domain
there are incorporated sequentially next to each other sequences of cleavage
sites recognized by urokinase uPA (SEQ. No. 46) and metalloprotease MMP (SEQ.
No. 45) due to which the effector e will undergo cleavage in the tumor
environment. Additionally, the sequence of the effector peptide has attached at
its C-terminus a transporting sequence (SEQ. No. 49) consisting of fragment of
antennapedia protein domain fragment. Transporting sequence aids in
penetration of the cell membrane and transportation of the fusion protein into
the cell.
Structure of the fusion protein is shown schematically in Fig. 4 and its amino
acid sequence and the DNA ng sequence sing codons optimized for
expression in E. coli are, respectively, SEQ. No. 16 and SEQ. No. 65 as shown in
the ed Sequence g.
The amino acid sequence SEQ. No. 16 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 65. A plasmid
containing the coding sequence of DNA was generated and overexpression of the
fusion protein was carried out in accordance with the general ures
described above. Overexpression was performed according to the general
procedure B, using E. coli B.21 (DE3) or Tuner (DE3) strains from Novagen. The
protein was separated by electrophoresis in accordance with the general
procedure described above.
Example 17. The fusion protein of SEQ. No. 17
The protein of SEQ. No. 17 is a fusion protein having the length of 190 amino
acids and the mass of 22.3 kDa, in which at the N-terminus of the sequence
TRAIL121-281 a 13-amino acid fragment of tubulin (SEQ. No. 37) is attached as
an effector peptide. Between the effector peptide and the N-terminus of TRAIL
domain there are incorporated sequentially next to each other sequences of
cleavage sites recognized by urokinase uPA (SEQ. No. 46) and oprotease
MMP (SEQ. No. 45) due to which the effector e will undergo cleavage in
the tumor nment. Additionally, the sequence of the effector peptide has
attached at its C-terminus a transporting sequence consisting of 6 arginine
residues. Transporting sequence aids in penetration of the cell membrane and
transportation of the fusion protein into the cell.
Structure of the fusion protein is shown schematically in Fig. 4 and its amino
acid sequence and the DNA ng sequence comprising codons optimized for
expression in E. coli are, respectively, SEQ. No. 17 and SEQ. No. 66 as shown in
the attached Sequence Listing.
The amino acid ce SEQ. No. 17 of the structure bed above was used
as a template to generate its coding DNA sequence SEQ. No. 66. A plasmid
containing the coding sequence of DNA was generated and overexpression of the
fusion protein was carried out in accordance with the general procedures
described above. Overexpression was performed according to the general
procedure B, using E. coli B.21 (DE3) or Tuner (DE3) strains from n. The
protein was separated by electrophoresis in accordance with the general
ure described above.
Example 18. The fusion protein of SEQ. No. 18
The protein of SEQ. No. 18 is a fusion protein having the length of 187 amino
acids and the mass of 21.7 kDa, in which at the N-terminus of the sequence
TRAIL121-281 a 10-amino acid nt of tubulin (SEQ. No. 38) is attached as
an effector peptide. Between the effector peptide and the N-terminus of TRAIL
domain there are incorporated sequentially next to each other sequences of
cleavage sites recognized by ase uPA (SEQ. No. 46) and metalloprotease
MMP (SEQ. No. 45) due to which the effector peptide will undergo cleavage in
the tumor environment. Additionally, the sequence of the effector peptide has
ed at its C-terminus a transporting sequence consisting of 6 arginine
residues. Transporting ce aids in penetration of the cell membrane and
transportation of the fusion protein into the cell.
Structure of the fusion protein is shown schematically in Fig. 4 and its amino
acid sequence and the DNA encoding sequence comprising codons optimized for
expression in E. coli are, respectively, SEQ. No. 18 and SEQ. No. 67 as shown in
the attached Sequence Listing.
The amino acid sequence SEQ. No. 18 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 67. A plasmid
ning the coding sequence of DNA was generated and overexpression of the
fusion protein was carried out in accordance with the general procedures
described above. Overexpression was performed ing to the general
procedure B, using E. coli B.21 (DE3) or Tuner (DE3) strains from Novagen. The
protein was separated by electrophoresis in ance with the l
procedure described above.
Example 19. The fusion protein of SEQ. No. 19
The protein of SEQ. No. 19 is a fusion protein having the length of 196 amino
acids and the mass of 22,54 kDa, in which at the N-terminus of the sequence
TRAIL121-281 a melittin (SEQ. No. 39) is attached as an effector peptide.
Between the or peptide and the N-terminus of TRAIL domain there are
incorporated tially next to each other sequences of cleavage sites
recognized by urokinase uPA (SEQ. No. 46) and metalloprotease MMP (SEQ. No.
45) due to which the effector peptide will undergo cleavage in the tumor
environment.
Structure of the fusion protein is shown schematically in Fig. 4 and its amino
acid sequence and the DNA encoding sequence comprising codons zed for
expression in E. coli are, respectively, SEQ. No. 19 and SEQ. No. 68 as shown in
the attached Sequence Listing.
The amino acid ce SEQ. No. 19 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 68. A plasmid
containing the coding sequence of DNA was ted and overexpression of the
fusion n was d out in accordance with the general procedures
described above. Overexpression was performed according to the general
procedure B, using E. coli B.21 (DE3) or Tuner (DE3) strains from Novagen. The
protein was separated by electrophoresis in accordance with the general
ure bed above.
Example 20. The fusion protein of SEQ. No. 20
The n of SEQ. No. 20 is a fusion protein having the length of 184 amino
acids and the mass of 21.4 kDa, in which at the N-terminus of the sequence
TRAIL121-281 a 6-amino acid peptide C2 derived from bee defensin (SEQ. No. 40)
is attached as an effector peptide. Between the effector peptide and the N-
terminus of TRAIL domain there are incorporated sequentially next to each other
sequences of cleavage sites recognized by urokinase uPA (SEQ. No. 46) and
metalloprotease MMP (SEQ. No. 45) due to which the effector peptide will
undergo ge in the tumor environment. Additionally, the sequence of the
effector peptide has attached at its C-terminus a transporting sequence
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consisting of 6 arginine residues. Transporting sequence aids in penetration of
the cell membrane and transportation of the fusion protein into the cell.
Structure of the fusion protein is shown schematically in Fig. 4 and its amino
acid sequence and the DNA ng sequence comprising codons optimized for
expression in E. coli are, respectively, SEQ. No. 20 and SEQ. No. 69 as shown in
the attached Sequence Listing.
The amino acid sequence SEQ. No. 20 of the structure bed above was used
as a template to generate its coding DNA sequence SEQ. No. 69. A plasmid
ning the coding sequence of DNA was generated and overexpression of the
fusion protein was d out in accordance with the general procedures
described above. Overexpression was performed according to the l
procedure B, using E. coli B.21 (DE3) or Tuner (DE3) s from Novagen. The
protein was separated by electrophoresis in accordance with the general
procedure described above.
Example 21. The fusion protein of SEQ. No. 21
The protein of SEQ. No. 21 is a fusion protein having the length of 189 amino
acids and the mass of 21.4 kDa, in which at the N-terminus of the sequence
TRAIL121-281 there are attached two ed sequences of 8-amino acid
peptide binding to FGF-Z ligand (SEQ. No. 41) as an effector peptide. Between
the effector peptides sequences there are incorporated sequentially next to
each other sequences of cleavage sites recognized by urokinase uPA (SEQ. No.
46) and metalloprotease MMP (SEQ. No. 45) due to which the effector peptide
will undergo cleavage in the tumor environment. onally, between the
second effector peptide and the sequence of TRAIL domain there is orated
a linker consisting of two e residues which aids in stabilization of trimeric
structure.
Structure of the fusion protein is shown schematically in Fig. 5 and its amino
acid sequence and the DNA encoding sequence comprising codons optimized for
expression in E. coli are, respectively, SEQ. No. 21 and SEQ. No. 70 as shown in
the attached Sequence Listing.
2012/057219
The amino acid sequence SEQ. No. 21 of the structure bed above was used
as a te to generate its coding DNA sequence SEQ. No. 70. A plasmid
ning the coding ce of DNA was generated and overexpression of the
fusion protein was carried out in accordance with the general procedures
described above. pression was performed according to the general
procedure B, using E. coli B.21 (DE3) or Tuner (DE3) strains from n. The
protein was separated by electrophoresis in accordance with the general
procedure described above.
Example 22. The fusion protein of SEQ. No. 22
The protein of SEQ. No. 22 is a fusion protein having the length of 188 amino
acids and the mass of 21.6 kDa, in which at the N-terminus of the sequence
TRAIL119-281 a 15-amino acid peptide lasioglossin LL2 (SEQ. No. 42) is attached
as an effector peptide. Between the effector peptide sequence and the N-
terminus of TRAIL domain there are incorporated sequentially next to each other
sequences of cleavage sites recognized by urokinase uPA (SEQ. No. 46) and
metalloprotease MMP (SEQ. No. 45) due to which the or peptide will
undergo cleavage in the tumor environment.
Structure of the fusion protein is shown schematically in Fig. 5 and its amino
acid sequence and the DNA encoding sequence comprising codons optimized for
expression in E. coli are, respectively, SEQ. No. 22 and SEQ. No. 71 as shown in
the attached ce Listing.
The amino acid sequence SEQ. No. 22 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 71. A plasmid
containing the coding sequence of DNA was generated and overexpression of the
fusion protein was carried out in ance with the general procedures
described above. Overexpression was performed according to the general
procedure B, using E. coli B.21 (DE3) or Tuner (DE3) strains from Novagen. The
protein was separated by electrophoresis in accordance with the general
procedure described above.
Example 23. The fusion protein of SEQ. No. 23
The protein of SEQ. No. 23 is a fusion protein having the length of 193 amino
acids and the mass of 21.6 kDa, in which at the N-terminus of the sequence
TRAIL121-281 a 13-amino acid peptide acting as an inhibitor of interactions
RasGAP — Aurora B (SEQ. No. 43) is attached as an effector peptide. Between the
effector peptide sequence and the TRAIL domain there are incorporated
sequentially next to each other sequences of cleavage sites recognized by
urokinase uPA (SEQ. No. 46) and metalloprotease MMP (SEQ. No. 45) due to
which the effector peptide will undergo cleavage in the tumor environment.
onally, the sequence of the effector peptide has attached at its C-terminus
a transporting ce consisting of 8 arginine residues. Transporting sequence
aids in penetration of the cell membrane and transportation of the fusion
protein into the cell. Additionally, between the sequence of metalloprotease
cleavage site and the sequence of TRAIL domain there is incorporated a ne
residue which aids in stabilization of trimeric structure.
Structure of the fusion protein is shown schematically in Fig. 5 and its amino
acid sequence and the DNA encoding sequence comprising codons optimized for
expression in E. coli are, respectively, SEQ. No. 23 and SEQ. No. 72 as shown in
the attached Sequence Listing.
The amino acid sequence SEQ. No. 23 of the structure bed above was used
as a template to generate its coding DNA ce SEQ. No. 72. A plasmid
containing the coding sequence of DNA was ted and pression of the
fusion protein was carried out in accordance with the general procedures
described above. Overexpression was performed according to the general
procedure B, using E. coli B.21 (DE3) or Tuner (DE3) s from n. The
protein was separated by electrophoresis in accordance with the l
procedure described above.
e 24. The fusion protein of SEQ. No. 24
The protein of SEQ. No. 24 is a fusion protein having the length of 243 amino
acids and the mass of 27.8 kDa, in which at the C-terminus of the sequence
TRAIL95-281 a 38-amino acid fragment of p16 peptide fused with a 17-amino-
acid transporting domain of antennapedia (SEQ. No. 33) is attached as an
effector peptide. Between the effector peptide sequence and the TRAIL domain
there are incorporated sequentially next to each other sequences of cleavage
sites recognized by metalloprotease MMP (SEQ. No. 45) and urokinase uPA (SEQ.
No. 46) due to which the effector e will undergo cleavage in the tumor
environment. Additionally, n sequence of TRAIL and the sequence of
cleavage site recognized by metalloproteinase MMP there is incorporated a
flexible cysteine-alanine linker (SEQ. No. 47).
ure of the fusion protein is shown schematically in Fig. 5 and its amino
acid sequence and the DNA encoding sequence comprising codons optimized for
expression in E. coli are, respectively, SEQ. No. 24 and SEQ. No. 73 as shown in
the attached Sequence Listing.
The amino acid sequence SEQ. No. 24 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 73. A plasmid
containing the coding sequence of DNA was generated and overexpression of the
fusion protein was carried out in accordance with the general procedures
described above. Overexpression was med according to the general
procedure A, using E. coli Tuner (DE3) strain from Novagen. The protein was
ted by electrophoresis in ance with the general procedure
described above.
e 25. The fusion protein of SEQ. No. 25
The protein of SEQ. No. 25 is a fusion n having the length of 199 amino
acids and the mass of 23.4 kDa, in which at the N-terminus of the sequence
TRAIL120-281 the analogue of Pep27 e (SEQ. No. 44) is ed as the
effector peptide. Between the effector peptide sequence and the N-terminus of
TRAIL domain there are incorporated sequentially next to each other sequences
of cleavage sites recognized by urokinase uPA (SEQ. No. 46) and metalloprotease
MMP (SEQ. No. 45) due to which the effector peptide will undergo cleavage in
the tumor environment.
Structure of the fusion protein is shown schematically in Fig. 5 and its amino
acid sequence and the DNA encoding sequence comprising codons optimized for
expression in E. coli are, respectively, SEQ. No. 25 and SEQ. No. 74 as shown in
the attached Sequence Listing.
The amino acid ce SEQ. No. 25 of the structure described above was used
as a template to generate its coding DNA sequence SEQ. No. 74. A d
containing the coding sequence of DNA was generated and overexpression of the
fusion n was carried out in accordance with the general procedures
described above. Overexpression was med according to the general
procedure B, using E. coli BL21 (DE3) or E. coli Tuner (DE3) strain from Novagen.
The protein was separated by electrophoresis in accordance with the general
procedure described above.
Example 26. Examination of anti-tumor activity of the fusion proteins
Examination of anti-tumor activity of the fusion proteins was carried out in vitro
in a cytotoxicity assay on tumor cell lines and in vivo in mice. For comparison
purposes, rhTRAIL114-281 protein and placebo were used.
1. ement of circular dichroism
Quality of the preparations of fusion proteins in terms of their structures was
determined by circular dichroism (CD) for Ex. 1a, Ex. 2a, and Ex. 8a.
ar dichroism is used for determination of secondary structures and
conformation of proteins. CD method uses optical activity of the protein
structures, manifested in rotating the plane of zation of light and the
appearance of elliptical polarization. CD spectrum of proteins in far ultraviolet
(UV) provides precise data on the conformation of the main polypeptide chain.
Samples of the protein to be analysed, after formulation into a buffer consisting
of 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10% glycerol, 0.1 mM ZnClz, 80 mM
saccharose, 5mM D'I'I', were dialysed in the dialysis bags (Sigma-Aldrich) with
cut-off 12 kDa. Dialysis was med against 100 fold excess (v/v) of buffer
comparing to the protein preparations with stirring for several hours at 4°C.
After dialysis was completed, each preparation was centrifuged (25 000 rpm., 10
min., 4°C) and the appropriate atants were collected. Protein
tration in the samples thus obtained was determined by Bradford method.
Measurement of circular ism for proteins in the concentration range of
0.127 mg/ml was performed on Jasco J-710 spectropolarimeter, in a quartz
cuvette with optical way 0.2 mm or 1 mm. The measurement was performed
under the flow of nitrogen at 7 l/min, which allowed to perform of the
measurement in the wavelength range from 195 to 250 nm. ters of the
measurement: spectral resolution of - 1 nm; half width of the light beam 1 nm;
sensitivity 20 mdeg, the averaging time for one wavelength - 8 s, scan speed 10
nm/min.
The results were presented as the e of three measurements. Circular
dichroism spectra for rhTRAIL114-281 and proteins of Ex. 1a, Ex. 2a and Ex. 8a
are presented in Fig. 6.
Obtained spectra were analyzed numerically in the range of 193-250 nm using
CDPro software. Points for which the e at the photomultiplier exceeded
700 V were omitted, due to too low signal to noise ratio in this wavelength
range.
The data obtained served for calculations of particular secondary structures
content in the analyzed proteins with use of CDPro software (Table 1).
Table 1. Content of secondary structures in the analyzed proteins
n (EXEMEaDl) a-helix 8- sheet Schift Disorder
Ex.1a 0.205 0.6% 44.1% 27.3% 28.0%
Ex.2a 0.092 0.1% 40.8% 24.5% 34.6%
Ex.8a 0.197 4.3% 32.0% 25.5% 38.2%
rhTRA|L* 1.94% 50.97% 7.74% 39.35%
rhTRAIL114-281 0.389 4.9% 33.7% 23.1% 38.3%
* value obtained
on the basis of crystalline structure 1D4V
The l molecule (rhTRAIL114-281) shows CD spectrum characteristic for the
proteins with predominantly type B-sheet structures (sharply outlined icity
minimum at the wavelength of 220 nm). This ms the calculation of secon-
dary structure components, suggesting a marginal number of a-helix elements.
The obtained result is also consistent with data from the l structure of
hTRAIL protein, and characteristic for fusion ns of the invention Ex. 1a
Ex. 2a and Ex. 8a), wherein beta ts constitute 32-44% of their structure.
In the case of all embodiments, dichroism spectra are characterized one
minimum at wavelength 220 nm.
Since the small peptides attached to TRAIL constitute a small portion of the
protein and do not need to create a defined secondary structure, analyzed
proteins should not differ significantly from the starting protein.
2.Tests on cell lines in vitro
Cell lines
Table 2. nt cell lines
number of
cells per
Cell line Cancer type Medium
well
(thousands)
COAl-IO—CZCOS human colorectal RPMI + 10% FBS + penicillin +
cancer streptomycm
#CCL-222
HT-29
human colorectal McCoy , . . .
s + 10% FBS + pemCIllin
ATCC 5
cancer + streptomycm
# CCL-2
DETEES human prostate RPMI + 10% FBS + penicillin +
cancer omycm
# HTB-81
2&3: human te RPMI + 10% FBS + penicillin +
cancer Streptomycm
# CRL-1435
MCF-7
MEM + Lafeflign: pcermculin +. . .
ATCC human breast cancer 4.5
P y
#HTB-22
MDA-MB-231
DMEM + 23:13:; EienmCIllm +. . .
ATCC human breast cancer 4.5
P y
# HTB-26
MDA-MB-4355
human breast cancer DMEM + 10% FBS + m +. . . 4
ATCC# HTB-129
streptomycm
UAAATléEE; human bladder MEM + 10% FBS + penicillin +
cancer StrePtOmycm
# CLR-1749
53-210 human bladder DMEM + 10% FBS + penicillin +
cancer Streptomycm
#CRL-2169
W0 2012/143477
,3210 human colorectal DMEM + 10% FBS + penicillin +
cancer streptomycm
#CCL-227
3}ng human pancreatic RPMI + 10% FBS + penicillin +
cancer streptomycm
#CRL-1 687
9:1082-3 human ovarian McCoy’s + 10% FBS + penicillin
cancer + streptomycm
# HTB-77
NIH: OVCAR-3 RPMI + 20% FBS + 0,01mg/ml
human n
ATCC insulina + penicillin + 7
cancer
#HTB-161 streptomycin
"L‘eTpCGCZ human liver MEM + 10% FBS + penicillin +
hepatoma streptomycm
# HB-8065
Human embrional MEM + 10% FBS + penicillin +
ATCC 4
k'dne' y cells stre tomp yc'n‘
#CLR-1573
ACHN
MEM + Biff)“: pggucfllin +. . .
ATCC human kidney cancer 4
p y
#CCL-222
CAKI 1
ATCC human kidney cancer McCoy’s + 10% FBS + penicillin 3.5
#HTB-46 + omycin
CAKI 2
McCoy’s + 107 FBS + enicillin
ATCC human kidney cancer 3.5
+ streo tom c}: p y ‘
# HTB-47
NCIATchgAR human small cell RPMI + 10% FBS + penicillin +
lung cancer omycm
1351
HT144
human melanoma McCoy , . . .
s + 10% FBS + penic1llin
ATCC 7
cells + streptomycm
# HTB-63
NCI-H460
RPMI + Ez’eFESn: Eilcmm +. . .
ATCC human lung cancer 2.5
p y
A549
RPMI + Ez’eFESn: Eilcmm +. . .
ATCC human lung cancer 2.5
p y
# CCL-185
ATECEZA human uterine McCoy’s + 10% FBS + penicillin
sarcoma + streptomycm
# CRL-1976
MES-SA/Dx5 multidrug-resistant
McCoy , . .
ATCC human uterine S++st1r0e% tFoE: :fiemaum. 4
p y
#CRL-1977 sarcoma
Waymouth’s MB 752/1 +
/MXZ
human uterine s (1 : 1)
ATCC 4
sarcoma + 10% FBS + penicillin +
#CRL-2274
streptomycm.
SK-MES-1 ATCC MEM + 10% FBS + penicillin +
human lung cancer 5
# HTB-58 streptomycin
HCT-116 ATCC human ctal McCoy’s + 10% FBS + penicillin
# CCL-247 cancer + streptomycin
W0 2012/143477
DMEM:F12 + 5% horse plasma +
MCF10A ATCC mammary epithelial 0.5 ug/ml hydrocortisone + 10
# 317 cells ug/ml insuline + 20 ng/ml
growth factor EGF
Panc-1 CLS human pancreatic DMEM + 10% FBS + penicillin +
330228 cancer omycin
Pa2c_%3227 human pancreatic RPMI + 10% FBS + penicillin +
cancer streptomycm
# CRL-2549
PLC/PRF/5 CLS human liver DMEM + 10% FBS + penicillin +
330315 hepatoma streptomycin
LNCaP
human prostate RPMI + 10% FBS + penicillin +
ATCC 4'5
cancer stre tomp ycin
# CRL-1740
SK-Hep-1 human liver
RPMI + 10% FBS + pemc1llin +. . . 10
CLS300334 hepatoma
streptomycm
A498 MEM + 10% FBS + penicillin +
human kidney cancer 3
CLS 300113 streptomycin
HT1080 ATCC MEM + 10% FBS + penicillin +
Human arcoma 3
21 streptomycin
Table 3. Nonadherent cells:
number of
cells per
Cell line Cancer type Medium
well
(thousands)
NCI-H69 human small cell RPMI + 10% FBS + penicillin
ATCC # HTB-119 lung cancer + streptomycin
Jurkat A3 RPMI + 10% FBS + penicillin
human leukaemia 10
ATCC #CRL-2570 + streptomycin
HL60 human leukaemia RPMI + 20% FBS + penicillin
ATCC # CCL-240 + streptomycin
CCRF-CEM human leukaemia RPMI + 20% FBS + penicillin
ATCC # 9 + streptomycin
MTT cytotoxicity test
M'I'I' assay is a colorimetric assay used to e proliferation, viability and
cytotoxicity of cells. It consists in decomposition of a yellow tetrazolium salt
M'I'I' (4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide) to the water-
insoluble purple dye formazan by mitochondrial enzyme succinate-tetrazolium
reductase 1. MIT reduction occurs only in living cells. Data analysis consists in
determining |C50 concentration of the protein (in ng/ml), at which the 50%
reduction in the number of cells occurs in the population treated compared to
control cells. Results were analyzed using GraphPad Prism 5.0 software. The test
was performed according to the ture descriptions (Celis, JE, (1998). Cell
Biology, a Laboratory ok, second edition, Academic Press, San Diego;
Yang, Y., Koh, LW, Tsai, JH., ; Involvement of viral and chemical factors
with oral cancer in Taiwan, Jpn J Clin Oncol, 34 (4), 176-183).
Cell culture medium was diluted to a defined density (104 - 105 cells per 100 pl).
Then 100 pl of appropriately diluted cell suspension was applied to a 96-well
plate in triplicates. Thus prepared cells were incubated for 24 h at 37°C in 5% or
% C02, depending on the medium used, and then to the cells (in 100 pl of
medium) further 100 pl of the medium containing various concentrations of
tested proteins were added. In the case of combination hTRAIL114-281 and
p21WAF effector protein, 100 pl of the medium containing mixture of
114-281 and p21WAF effector protein in molar ratio 1:1 was added. After
incubation of the cells with tested proteins over the period of next 72 hours,
which is lent to 3-4 times of cell division, the medium with the test
n was added with 20 ml of MIT working solution [5 , and incubation
was continued for 3 h at 37°C in 5% C02. Then the medium with WT solution
was removed, and formazan crystals were dissolved by adding 100 pl of DMSO.
After stirring, the absorbance was measured at 570 nm (reference filter 690
nm).
EZ4U cytotoxicity test
EZ4U (Biomedica) test was used for testing cytotoxic ty of the proteins in
nonadherent cell lines. The test is a cation of the MIT method, wherein
formazan formed in the reduction of olium salt is water-soluble. Cell
viability study was carried out after continuous 72-hour incubation of the cells
with protein (seven concentrations of protein, each in cates). On this basis
|C50 values were determined (as an average of two independent experiments)
using the GraphPad Prism 5 software. Control cells were incubated with the
solvent only.
The results of in vitro cytotoxicity tests are summarized as IC50 values (ng/ ml),
which corresponds to the n concentration at which the cytotoxic effect of
fusion proteins is observed at the level of 50% with t to control cells
treated only with solvent. Each experiment represents the average value of at
least two independent experiments performed in triplicates. As a criterion of
lack of activity of protein preparations the IC50 limit of 2000 ng/ml was adopted.
Fusion proteins with an IC50 value above 2000 were considered inactive.
Cells selected for this test included tumor cell lines that are naturally resistant
to TRAIL protein (the criterion of natural resistance to TRAIL: IC50 for TRAIL
protein > 2000), as well as tumor cell lines sensitive to TRAIL protein and
resistant to doxorubicin line MES-SA/DX5 as a cancer line resistant to
conventional anticancer medicaments.
Undifferentiated HUVEC cell line was used as a healthy control cell line for
assessment of the effect/toxicity of the fusion proteins in non-cancer cells.
The results obtained confirm the possibility of overcoming the resistance of the
cell lines to TRAIL by administration of certain fusion ns of the invention to
cells naturally resistant to TRAIL. When fusion proteins of the invention were
administered to the cells sensitive to TRAIL, in some cases a clear and strong
potentiation of the potency of action was observed, which was manifested in
reduced IC50 values of the fusion n compared with IC50 for the TRAIL alone.
Furthermore, cytotoxic activity of the fusion protein of the invention in the cells
resistant to classical anti-cancer medicament doxorubicin was obtained, and in
some cases it was stronger than activity of TRAIL alone.
The IC50 values above 2000 obtained for the ncer cell lines show the ab-
sence of toxic s associated with the use of proteins of the invention for
healthy cells, which indicates potential low systemic ty of the protein.
The s obtained for ation of hTRAIL114-281 and p21WAF or
peptide ting of mixture of hTRAIL114-281 and 20-amino acid p21WAF
derived effector peptide (custom solid phase synthesis) in molar ratio 1:1,
compared with results obtained for fusion protein of Ex. 8b (comprising
hTRAIL121-281 and 20-amino acid p21WAF derived effector peptide) and with
results ed for single molecule of hTRAIL114-281 and single molecule of
p21WAF derived effector peptide revealed the ageous properties of the
fusion protein over its single constituents and combination thereof.
The fusion protein of Ex. 8b overcomes the resistance to TRAIL of A549 cell line.
In the case of TRAIL sensitive cell lines the fusion protein of Ex. 8b reveals higher
cytotoxic activity than single molecules of hTRAIL114-281 and p21WAF derived
peptide.
Determination of cytotoxic activity of selected protein preparations against
extended panel of tumor cell lines
Table 4 presents the results of the tests of cytotoxic activity in vitro for selected
fusion proteins of the ion against a broad panel of tumor cells from
different organs, corresponding to the broad range of most common s.
The experimental results are presented as a mean value i standard deviation
(SD). All calculations and graphs were prepared using the GraphPad Prism 5.0
software.
Obtained |C50 values confirm high cytotoxic ty of fusion proteins and thus
their ial utility in the treatment of cancer.
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WO 43477 74
2. Antitumor effectiveness of fusion proteins in vivo on xenografts
Antitumor activity of protein preparations was tested in a mouse model of
human colon cancer HCT116, human colon cancer Col0205, human colon cancer
model SW620, human liver cancer model HepGZ, and human lung cancer
models NCI-H460 and NCI-H460-Luc2.
ns tested for antitumor ty on xenografts ally expressed with
histidine tag that was subsequently removed are designated as a) at the Ex.
No.. Proteins that were originally expressed without histidine tag are
designated as b) at the Ex. No..
C's—US
The HCT116 (in mice Crl:CD1-Foxn1"“ 1), Col0205, NCI-H460, NCI-H460-Luc2
cells were maintained in RPMI 1640 medium (Hyclone, Logan, UT, USA) mixed
in the ratio of 1:1 with Opti-MEM ((Invitrogen, Cat.22600-134) supplemented
with 10% fetal calf serum and 2 mM glutamine. On the day of mice grafting, the
cells were detached from the support by washing the cells with trypsin
(Invitrogen), then the cells were centrifuged at 1300 rpm, 4°C, 8 min.,
suspended in HBSS buffer (Hanks medium), d and diluted to the
concentration of 25x106 cells/ml.
The HCT116 (in mice Crl:SHO-PrkchddHrhr) were alternatively maintained in
McCoy’s medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal calf
serum and 2 mM glutamine. On the day of mice grafting, the cells were
detached from the support by washing the cells with trypsin (Invitrogen), then
the cells were centrifuged at 1300 rpm, 4°C, 8 min., suspended in HBSS buffer
(Hanks medium), counted and d to the concentration of 25x106 cells/ml.
SW620 cells were maintained in DMEM (HyClone, Logan, UT, USA) mented
with 10% fetal calf serum and 2 mM glutamine. On the day of mice grafting, the
cells were detached from the support by g the cells with trypsin
(Invitrogen), then the cells were centrifuged at 1300 rpm, 4°C, 8 min.,
suspended in HBSS buffer (Hanks medium), counted and diluted to the
concentration of 25x106 cells/ml.
The HepGZcells were maintained in MEM ne, Logan, UT, USA)
supplemented with 10% fetal calf serum and 2 mM ine. On the day of
mice grafting, the cells were detached from the support by washing the cells
with trypsin (Invitrogen), then the cells were centrifuged at 1300 rpm, 4°C, 8
min., suspended in HBSS buffer (Hanks ), counted and diluted to the
concentration of 25x106 ml.
Examination of antitumor activity of proteins of the invention was conducted
on 7-9 ld CD- nude (Crl:CD1-Foxn1"“ 1) or 4-6 ld Crl:SHO-
PrkchddHrhr mice obtained from Charles River Germany. Mice were kept under
specific pathogen-free conditions with free access to food and demineralised
water (ad libitum). All experiments on animals were carried in accordance with
the guidelines: "Interdisciplinary Principles and Guidelines for the Use of
Animals in Research, Marketing and Education" issued by the New York
Academy of Sciences' Ad Hoc Committee on Animal Research and were
approved by the IV Local Ethics Committee on Animal Experimentation in
Warsaw (No. 9).
The course and evaluation of the experiments
Human colon cancer model
Mice CD- nude (Crl:CD1-Foxn1”“ 1 )HCT116 model
On day 0 mice Crl:CD1-Foxn1”“ 1 were grafted subcutaneously (sc) in the right
side with 5x106 of HCT116 cells suspended in 0.2 ml HBSS buffer by means of a
syringe with a 0.5 x25 mm needle (Bogmark). When tumors reached the size of
~ 55-68 mm3 (day 8), mice were randomized to obtain the average size of
tumors in the group of ~ 63 mm3 and assigned to treatment groups. The
treatment groups were administered with the preparations of fusion protein of
the ion of Ex. 2a (10 mg/kg) and rhTRAIL114-281 (10 mg/kg) as a
comparison. The preparations were administered intravenously (i.v.) following
the scheme 10 daily ations with a two-day break after the first 5
ations. When a therapeutic group reached the average tumor size of
~ 1000 mm3, mice were sacrificed by disruption of the spinal cord. The control
group received rhTRAIL114-281.
The experimental s obtained in mice Crl:CD1-Foxn1”“ burdened with
HCT116 colon cancer treated with fusion proteins of the invention of Ex. 2a
and comparatively with rhTRAIL114-281 are shown in Fig. 7 as a diagram of
changes of the tumor volume and in Figure 8 which shows tumor growth
inhibition (%TG|) as the percentage of control.
The mental results obtained in mice Crl:CD1-Foxn1”“ burdened with
HCT116 colon cancer treated with fusion protein of the invention of Ex. 2a and
comparatively with rhTRAIL114-281 are shown in Fig. 7 as a diagram of s
of the tumor volume and in Figure 8 which shows tumor growth inhibition
(%TG|) as the percentage of control.
The results of experiments presented in the graphs in Figures 7 and 8 show that
administration of the fusion protein of the invention of Ex. 2"11 caused tumor
HCT116 growth inhibition, with TGI 71.2% relative to the control on 27th day of
the experiment. For rhTRAIL114-281 used as the comparative reference, a
slight inhibitory effect on tumor cell growth was ed relative to the
control, with TGI at the level of 44%. Thus, fusion proteins of the invention
exert much stronger effect compared to TRAIL alone.
On day 0 mice 1-Foxn1”“ 1 were grafted subcutaneously (sc) in the right
side with 5x106 of HCT116 cells suspended in 0.2 ml HBSS buffer by means of a
syringe with a 0.5 x25 mm needle (Bogmark). When tumors reached the size of
~ 50-110 mm3 (day 23), mice were ized to obtain the average size of
tumors in the group of ~ 85 mm3 and assigned to treatment groups. The
treatment groups were administered with the preparations of fusion protein of
the invention of Ex. 8"11 (10 mg/kg)and L114-281 (10 mg/kg) as a
comparison. The preparations were administered intravenously (i.v.) daily for
ten days. When a therapeutic group reached the average tumor size of
~ 1000 mm3, mice were sacrificed by disruption of the spinal cord. The control
group received rhTRAIL114-281.
The experimental results obtained in mice Crl:CD1-Foxn1”“ burdened with
HCT116 colon cancer treated with fusion proteins of the invention of Ex. 8"11 and
comparatively with rhTRAIL114-281 are shown in Fig. 11 as a diagram of
changes of the tumor volume and in Figure 12 which shows tumor growth
inhibition (%TG|) as the percentage of control.
The experimental results ed in mice Crl:CD1-Foxn1”“ ed with
HCT116 colon cancer treated with fusion protein of the invention of Ex. 8a and
comparatively with rhTRAIL114-281 are shown in Fig. 11 as a diagram of
changes of the tumor volume and in Figure 12 which shows tumor growth
inhibition (%TG|) as the percentage of control.
The results of experiments presented in the graphs in Figures 11 and 12 show
that administration of the fusion protein of the invention of Ex. 8"11 caused
tumor HCT116 growth inhibition, with TGI 53.3 relative to the control on 31th
day of the experiment. For rhTRAIL114-281 used as the comparative reference,
a slight inhibitory effect on tumor cell growth was obtained relative to the
control, with TGI at the level of 21.8%. Thus, fusion proteins of the invention
exert much stronger effect compared to TRAIL alone.
HCT116 model
On day 0 mice O-PrkchddHrhr were grafted subcutaneously (sc) in the
right side with 5x106 of HCT116 cells suspended in 0.1 ml 3:1 mixture of HBSS
bufferzMatrigel by means of a syringe with a 0.5 x25 mm needle (Bogmark).
When tumors reached the size of 71-432 mm3 (day 13), mice were randomized
to obtain the average size of tumors in the group of ~ 180 mm3 and ed to
treatment groups. The treatment groups were administered with the prepara-
tions of fusion proteins of the invention of Ex. 8b (50 mg/kg), and rhTRAIL114-
281 (65 mg/kg) as a comparison against ation buffer (50 mM Trizma
Base, 200 mM NaCl, 5 mM glutathione, 0.1 mM ZnClz, 10% glycerol, 80 mM
saccharose, pH 8.0). The preparations were administered intravenously (i.v.)
ing the schema 10 daily applications with a y break after the first
applications.
When a therapeutic group reached the average tumor size of ~ 1000 mm3, mice
were sacrificed by disruption of the spinal cord. The control group received
rhTRAIL114-281.
The mental results obtained in mice Crl:SHO-PrkchddHrhr ed with
HCT116 colon cancer treated with fusion protein of the invention of Ex.8b, and
comparatively with rhTRAIL114-281 are shown in Fig. 11a as a diagram of
changes of the tumor volume, and in Figure 12a which shows tumor growth
tion (%TG|) as the percentage of control.
The results of experiments presented in the graphs in Figures 11a and 12a show
that administration of the fusion n of the invention Ex.8b caused tumor
HCT116 growth inhibition, with TGI 70% relative to the control on 24th day of
the experiment. For L114-281 used as the comparative reference, the
slight inhibitory effect on tumor cell growth was obtained relative to the
control, with TGI at the level of 38%. Thus, fusion protein of the invention
exert much stronger effect ed to rhTRAIL114-281 alone.
SW620 model
On day 0 mice Crl:SHO-PrkchddHrhr were grafted subcutaneously (sc) in the
right side with 5x106 of SW620 cells suspended in 0.1 ml 3:1 mixture of HBSS
bufferzMatrigel by means of a syringe with a 0.5 x25 mm needle (Bogmark).
When tumors d the size of 280-340 mm3 (day 17), mice were randomized
to obtain the average size of tumors in the group of ~ 320 mm3 and assigned to
treatment groups. The treatment groups were administered with the
preparations of fusion proteins of the invention of Ex.8b (40 mg/kg), and
rhTRAIL114-281 (30 mg/kg) as a comparison against formulation buffer (5 mM
NaHzPO4, 95 mM NazHPO4, 200 mM NaCl, 5 mM glutathione, 0.1 mM ZnClz, 10%
glycerol, 80 mM saccharose, pH 8.0). The preparations were administered
intravenously (i.v.) six times every second day. When a eutic group
reached the average tumor size of ~ 1000 mm3, mice were sacrificed by
disruption of the spinal cord. The control group received rhTRAIL114-281.
The experimental results obtained in mice Crl:SHO-PrkdcscidHrhr burdened with
SW620 colon cancer treated with fusion protein of the invention of Ex. 8b, and
comparatively with L114-281 are shown in Fig. 13 as a m of
changes of the tumor , and in Figure 14 which shows tumor growth
inhibition (%TG|) as the tage of l.
The results of experiments presented in the graphs in Figures 13 and 14 show
that administration of the fusion protein of the invention Ex. 8b caused tumor
SW620 growth inhibition, with TGI 44% relative to the control on 31St day of the
experiment. For rhTRAIL114-281 used as the comparative reference, the slight
inhibitory effect on tumor cell growth was obtained relative to the control,
WO 43477 79
with TGI at the level of -9%. Thus, fusion proteins of the ion exert much
stronger effect compared to rhTRAIL114-281 alone.
Col0205 model
On day 0 mice Crl:SHO-PrkchddHrhr were grafted subcutaneously (sc) in the
right side with 5x106 of Col0205 cells suspended in 0.1 ml 3:1 mixture of HBSS
bufferzMatrigel by means of a syringe with a 0.5 x25 mm needle rk).
When tumors reached the size of 108-128 mm3 (day 13), mice were randomized
to obtain the average size of tumors in the group of ~ 115 mm3 and assigned to
treatment groups. The treatment groups were administered with the
preparations of fusion proteins of the invention of Ex.8b (30 mg/kg), and
rhTRAIL114-281 (30 mg/kg) as a ison against formulation buffer (5 mM
4, 95 mM 4, 200 mM NaCl, 5 mM glutathione, 0.1 mM ZnClz, 10%
glycerol, 80 mM saccharose, pH 8.0). The preparations were administered
intravenously (i.v.) six times every second day. When a therapeutic group
reached the average tumor size of ~ 1000 mm3, mice were sacrificed by
disruption of the spinal cord. The control group received rhTRAIL114-281.
The experimental results obtained in mice Crl:SHO-PrkdcscidHrhr burdened with
Col0205 colon cancer treated with fusion protein of the invention of Ex. 8b, and
comparatively with L114-281 are shown in Fig. 15 as a diagram of
changes of the tumor volume, and in Figure 16 which shows tumor growth
inhibition (%TG|) as the percentage of control.
The results of experiments presented in the graphs in s 15 and 16 show
that administration of the fusion protein of the invention Ex. 8b caused tumor
Col0205 growth inhibition, with TGI 97.6 % relative to the control on 33rd day of
the experiment. For rhTRAIL114-281 used as the ative reference, the
slight inhibitory effect on tumor cell growth was obtained relative to the
control, with TGI at the level of 18.8%. Thus, fusion proteins of the invention
exert much stronger effect compared to rhTRAIL114-281 alone.
Liver cancer model
Mice Crl:SHO-PrkchddHrhr
He 62 model
On day 0 mice Crl:SHO-PrkchddHrhr were grafted subcutaneously (sc) in the
right side with 7x106 of HepGZ cells suspended in 0.1 ml 3:1 mixture of HBSS
bufferzMatrigel by means of a syringe with a 0.5 x25 mm needle (Bogmark).
When tumors reached the size of ~ 4 mm3 (day 19), mice were rando-
mized to obtain the average size of tumors in the group of ~ 340 mm3 and
assigned to treatment groups. The treatment groups were stered with
the preparations of fusion protein of the invention of Ex. 8b (30 mg/kg) and
rhTRAIL114-281 (30 mg/kg) as a comparison against formulation buffer (5 mM
NaHzPO4, 95 mM 4, 200 mM NaCl, 5 mM glutatione, 0.1 mM ZnClz, 10%
glycerol, 80 mM saccharose, pH 8.0) as a control. The preparations were
administered intravenously (i.v.) six times every second day. When a
therapeutic group reached the e tumor size of ~ 1000 mm3, mice were
sacrificed by disruption of the spinal cord. The control group received
rhTRAIL114-281.
The experimental results ed in mice O-PrkchddHrhr burdened with
HepGZ liver cancer d with fusion protein of the ion of Ex. 8b and
comparatively with rhTRAIL114-281 are shown in Fig. 17 as a diagram of
changes of the tumor volume, and in Fig. 18 which shows tumor growth
inhibition (%TG|) as the percentage of control.
The results of experiments presented in the graphs in Figures 17 and 18 show
that administration of the fusion proteins of the invention Ex. 8b caused tumor
HepGZ growth inhibition, with TGI 65.7% relative to the control on 33rd day of
the experiment. For rhTRAIL114-281 used as the comparative reference, the
slight inhibitory effect on tumor cell growth was obtained relative to the
control, with TGI at the level of 12.6%. Thus, fusion proteins of the invention
exert much stronger effect compared to L114-281 alone.
Lung cancer model
Mice: Crl:CD1-Foxn1"“ 1
NCI-H460-Luc2 model
On day 0 mice Crl:CD1-Foxn1nu 1were grafted subcutaneously (sc) in the right
side with 5x106 of NCI-H460-Luc2 cells suspended in 0.1 ml HBSS buffer by
means of a syringe with a 0.5 x25 mm needle (Bogmark). When tumors reached
the size of ~ 20-233 mm3 (day 16), mice were randomized to obtain the average
size of tumors in the group of ~ 110 mm3 and assigned to treatment groups. The
treatment groups were administered with the preparations of fusion protein of
the invention of Ex. 2a (20 mg/kg) and rhTRAIL114-281 (10 mg/kg) as a
comparison against formulation buffer f16 (19 mM NaHzPO4, 81 mM 4, 50
mM NaCl, 5 mM glutathione, 0.1 mM ZnClz, 10% glycerol, pH 7.4) as a control.
The preparations were administered intravenously (i.v.) six times every second
day. When a therapeutic group reached the average tumor size of ~ 1000 mm3,
mice were iced by disruption of the spinal cord. The control group
received rhTRAIL114-281.
The experimental results obtained in mice Crl:SHO-PrkchddHrhr burdened with
NCI-H460-Luc2 lung cancer d with fusion protein of the invention of Ex.
2a and comparatively with rhTRAIL114-281 are shown in Fig. 9 as a diagram of
changes of the tumor volume, and in Fig. 10 which shows tumor growth
inhibition (%TG|) as the percentage of control.
The s of experiments presented in the graphs in Figures 9 and 10 show
that administration of the fusion protein of the invention Ex. 2a caused tumor
NCI-H460-Luc2 growth tion, with TGI 81.3% relative to the control on 30th
day of the ment. For rhTRAIL114-281 used as the comparative reference,
a slight inhibitory effect on tumor cell growth was obtained relative to the
control, with TGI at the level of 53.1%. Thus, fusion proteins of the invention
exert much er effect compared to rhTRAIL114-281 alone.
Mice: Crl:SHO-PrkdcscidHrhr
NCI-H460 model
On day 0 mice Crl:SHO-PrkdcscidHrhr were grafted subcutaneously (sc) in the
right side with 5x106 of NCI-H460 cells suspended in 0.1 ml HBSS buffer by
means of a syringe with a 0.5 x25 mm needle (Bogmark). When tumors reached
the size of ~150-178mm3 (day 13), mice were randomized to obtain the
average size of tumors in the group of ~ 160 mm3 and assigned to treatment
groups. The treatment groups were administered with the preparations of
fusion protein of the invention of Ex. 8b TRP5 (30 mg/kg) and rhTRAIL114-281
(30 mg/kg) as a comparison against formulation buffer (5 mM 4, 95 mM
NazHPO4, 200 mM NaCl, 5 mM hione, 0.1 mM ZnClz, 10% glycerol, 80 mM
saccharose, pH 8.0) as a control. The preparations were administered
intravenously (i.v.) six times every second day. When a therapeutic group
reached the average tumor size of ~ 1000 mm3, mice were sacrificed by
disruption of the spinal cord. The l group ed rhTRAIL114-281.
The mental results obtained in mice O-PrkdcscidHrhr burdened with
NCI-H460 lung cancer treated with fusion protein of the ion of Ex.8b and
comparatively with rhTRAIL114-281 are shown in Fig. 19 as a diagram of
changes of the tumor volume, and in Fig. 20 which shows tumor growth
inhibition (%TG|) as the percentage of control.
The results of experiments presented in the graphs in Figures 19 and 20 show
that administration of the fusion n of the invention Ex. 8b caused tumor
NCI-H460 growth inhibition, with TGI 61% relative to the control on 28th day of
the experiment. For rhTRAIL114-281 used as the comparative reference, a
slight inhibitory effect on tumor cell growth was obtained relative to the
control, with TGI at the level of 17.5%. Thus, fusion proteins of the invention
exert much stronger effect compared to rhTRAIL114-281 alone.
The tested fusion proteins did not cause significant side effects manifested by
a decrease in body weight of mice (i.e. less than 10% of the baseline body
weight). This shows low systemic toxicity of the protein.
Claims (27)
1. A fusion protein comprising: - domain (a) which comprises the functional fragment of a soluble hTRAIL protein sequence ng with an amino acid in a position not 5 lower than hTRAIL95, or a homolog of said functional fragment having at least 70% sequence identity; and - at least one domain (b) which is the ce of an effector e having anti-proliferative activity against tumour cells, and wherein the sequence of domain (b) is attached at the C-terminus and/or at 10 the N-terminus of domain (a).
2. The fusion protein ing to claim 1, wherein domain (a) comprises a fragment of soluble hTRAIL protein sequence starting with an amino acid in the range from hTRAIL95 to hTRAIL121, ive, and ending with the amino acid
281. 15 3. The fusion protein ing to claim 1 orclaim 2, wherein domain (a) is selected from the group ting hTRAIL95-of 281, hTRAIL114-281, hTRAIL119-281, hTRAIL120-281, and hTRAIL121-281.
4. The fusion protein according to any one of claims 1 to 3, wherein domain (b) is selected from the group consisting of: 20 - 16-amino acid peptide blocking FGF-2 receptor of SEQ. No. 26; - 34 amino acid nt of human fetoprotein of SEQ. No. 27; - 8-amino acid fragment of human fetoprotein of SEQ. No. 28; - peptide derived from p21 WAF of SEQ. No. 29; - peptide DD2 from DOC-2/DAB2 protein of SEQ. No. 30; 25 - arginine deiminase from Mycoplasma arginini of SEQ. No. 31; - fragment of p16 e of SEQ. No. 32; - fragment of p16 peptide fused with -amino-acida 17 transporting domain of antennapedia of SEQ. No. 33; - fragment of MEK-1 protein of SEQ. No. 34; 30 - N terminal fragment of PH domain of TCL1 protein of SEQ. No. 35; - hexapeptide Phe- Trp-Leu-Arg-Phe-Thr of SEQ. No. 36; - 13-amino acid tubulin fragment of SEQ. No. 37; (9480728_1):KZA - 10-amino acid tubulin fragment of SEQ. No. 38; - melittin of SEQ. No. 39; - 6-amino acid peptide C2 derived from bee defensin of SEQ. No. 40; - 8-amino acid peptide binding to FGF-2 ligand of SEQ. No. 41; 5 - 15-amino acid lasioglossin LL2 peptide of SEQ. No. 42; - 13- amino acid peptide binding to SH3 RasGAP domain of SEQ. No. 43; - analogue of Pep27 peptide of SEQ. No. 44.
5. The fusion protein according to any one of claims 1 to 4, which n domain (a) and domain (b) contains domain (c) comprising a protease cleavagesite, 10 selected from a sequence recognized by metalloprotease MMP, a ce recognized by urokinase uPA, and combinations thereof.
6. The fusion protein according to claim 5, wherein the sequence recognized by metalloprotease MMP is SEQ. No. 45, and the sequence ized by urokinase uPA is SEQ. No. 46. 15
7. The fusion protein according to claim 5 or claim 6, wherein domain (c) is a combination of sequences recognized by metalloprotease MMP and urokinase uPA located next to each other.
8. The fusion protein according to any one of claims 1 to 7, wherein domain (b) is additionally linked with a transporting domain (d) selected from the group 20 consisting of: - (d1) a fragment of apedia protein domain of SEQ. No. 48, - (d2) a fragment of antennapedia protein domain of SEQ. No. 49, - (d3) polyarginine sequence transporting through a cell membrane, consisting of 6, 7, 8, 9, 10 or 11 Arg residues, 25 and combinations f.
9. The fusion protein according to claim 8, wherein the sequence (d) is located at the inus or at the inus of the fusion protein.
10. The fusion protein according to claim 8, n the transporting sequence (d) is located between domains (b) and (c). (9480728_1):KZA
11. The fusion protein according to claim 8, wherein the sequence (d) is located at the C-terminus of the fusion protein.
12. The fusion protein according to any one of claims 5 to 11, which onally comprises a flexible steric linker between domains (a), (b), (c) and/or (d). 5
13. The fusion protein according to claim 11, wherein the flexible steric linker is selected from the group consisting of SEQ. No. 47, sequenceGly Gly Ser, sequence Gly Gly Gly Ser Gly, two glycine residues Gly Gly, cysteine residue Cys, and combinations f.
14. The fusion protein according to claim 1, having the amino acid sequence selected 10 from the group consisting of SEQ. No. 1; SEQ. No. 2; SEQ. No. 3; SEQ. No. 4; SEQ. No. 5; SEQ. No. 6; SEQ. No. 7; SEQ. No. 8; SEQ. No. 9; SEQ. No. 10; SEQ. No. 11; SEQ. No. 12; SEQ. No. 13; SEQ. No. 14, SEQ. No. 15, SEQ. No. 16; SEQ. No. 17; SEQ. No. 18; SEQ. No. 19; SEQ. No. 20; SEQ. No. 21; SEQ. No. 22; SEQ. No. 23; SEQ. No. 24; SEQ. No. 25, and SEQ. No. 75.
15 15. The fusion protein according to any one of the preceding claims, which is a recombinant protein.
16. A polynucleotide sequence, coding the fusion protein as defined in any one of claims 1 to 14.
17. The polynucleotide sequence ing to claim 16, optimized for genetic 20 sion in E. coli.
18. The polynucleotide sequence according to claim 17, selected from the group consisting of SEQ. No. 50; SEQ. No. 51; SEQ. No. 52; SEQ. No. 53; SEQ. No. 54; SEQ. No. 55; SEQ. No. 56; SEQ. No. 57; SEQ. No. 58; SEQ. No. 59; SEQ. No. 60; SEQ. No. 61; SEQ. No. 62; SEQ. No. 63; SEQ. No. 64; SEQ. No. 65; 25 SEQ. No. 66; SEQ. No. 67; SEQ. No. 68; SEQ. No. 69; SEQ. No. 70; SEQ. No. 71; SEQ. No. 72; SEQ. No. 73, SEQ. No. 74, and SEQ. No. 76.
19. An expression , comprising cleotide sequence according to any one of claims 16 to 18. (9480728_1):KZA [Stamp] kza #23clipboard
20. A host cell, comprising the expression vector as defined in claim19, wherein the host cell is not within a human.
21. The host cell according to claim 20, which is an E. coli cell.
22. A pharmaceutical composition, comprising as an active ingredient thefusion 5 protein as d in any one of claims 1 to 15, in combination with a pharmaceutically acceptable carrier.
23. The pharmaceutical composition according to claim 22, in a form for eral stration.
24. The fusion protein as defined in any one of claims 1 to 15,for use in the 10 treatment of neoplastic diseases in mammals, including humans.
25. Use of a fusion protein as defined inany one ofclaims 1 to 15, for the manufacture of a medicament for the treatment of cancer diseases in a mammal.
26. The use of claim 25, n the mammal is a human.
27. A fusion protein ing to claim 1 and substantially as hereinbefore described 15 with reference to any one of the examples. Adamed sp. z o.o. By the Attorneys for the Applicant SPRUSON & FERGUSON Per: (9480728_1):KZA
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PL394618A PL394618A1 (en) | 2011-04-19 | 2011-04-19 | Anticancer fusion protein |
PLPL394618 | 2011-04-19 | ||
PCT/EP2012/057219 WO2012143477A2 (en) | 2011-04-19 | 2012-04-19 | Anticancer fusion protein |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ617353A NZ617353A (en) | 2015-01-30 |
NZ617353B2 true NZ617353B2 (en) | 2015-05-01 |
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