WO2014141094A1 - Conjugué anticancéreux - Google Patents

Conjugué anticancéreux Download PDF

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Publication number
WO2014141094A1
WO2014141094A1 PCT/IB2014/059679 IB2014059679W WO2014141094A1 WO 2014141094 A1 WO2014141094 A1 WO 2014141094A1 IB 2014059679 W IB2014059679 W IB 2014059679W WO 2014141094 A1 WO2014141094 A1 WO 2014141094A1
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seq
domain
fusion protein
sequence
conjugate
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PCT/IB2014/059679
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English (en)
Inventor
Jerzy Pieczykolan
Wojciech STROZEK
Zbigniew Majka
Anna Maria PIECZYKOLAN
Katarzyna Dorota BUKATO
Marlena Maria GALAZKA
Joanna Adriana JASZCZEWSKA
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Adamed Sp. Z O.O.
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Publication of WO2014141094A1 publication Critical patent/WO2014141094A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the field of therapeutic conjugates comprising a fusion protein, in particular recombinant protein.
  • the invention relates to conjugates of a fusion protein, said fusion protein comprising a sequence derived from a soluble human TRAIL protein linked to the sequence of a short antiblastic peptide acting synergistically with human TRAIL protein, with the molecule of a chemical compound having antiblastic activity, pharmaceutical compositions containing these conjugates as well as their use in therapy, particularly as antitumour agents.
  • WO/2011 /161260 describes fusion proteins comprising a functional fragment of the soluble human TRAIL protein sequence linked with the sequence of a short peptide with proapoptotic activity.
  • WO/2012/093158 describes fusion proteins comprising a functional fragment of the soluble human TRAIL protein sequence linked with the sequence of a short peptide with antiangiogenic activity.
  • WO/2012/143477 describes fusion proteins comprising a functional fragment of the soluble human TRAIL protein sequence linked with the sequence of a short peptide with antiproliferative activity.
  • WO/2013/098755 describes fusion proteins comprising a functional fragment of the soluble human TRAIL protein sequence linked with the sequence of a short cytolytic peptide forming pores in cellular membrane.
  • cancer therapy is chemotherapy.
  • therapies including sequential or simultaneous administration of small molecule and protein therapeutics (Hong Xiang, Oncogene (2002) 21 , 3611 - 3619), aimed to achieving increased proapoptotic activity and cytotoxicity.
  • therapies have not found their use in clinic.
  • Treatment of tumours using chemical compounds, either alone or in mixtures, involves well-known drawbacks of such therapy, such as a need to use high concentrations of a compound due to its poor bioavailability and the resulting systemic toxicity and side effects.
  • linkers with branched structure to which more than one chemical molecule can be attached.
  • linkers with a complex structure that doesn't form loops, which enable connection of more than one molecule of a chemical compound are susceptible to proteolysis.
  • linker molecule in particular, a charged linker - for example amino acid linker
  • linker molecule for example amino acid linker
  • the use of branched linkers for conjugation of chemical compounds requires a multi-step manufacturing process and may also contribute to aggregation of carrier proteins of a conjugate.
  • the present invention proposes to solve this problem and meet this need by providing conjugates of the invention, wherein fusion protein comprising a functional fragment of human TRAIL protein and a short effector peptide with proapoptotic, antiangiogenic or antiproliferative activity, or forming pores in cellular membrane, is attached to at least one chemical molecule with antiblastic activity, which molecule amplifies or supplements the action of the fusion protein.
  • fusion protein comprising a functional fragment of human TRAIL protein and a short effector peptide with proapoptotic, antiangiogenic or antiproliferative activity, or forming pores in cellular membrane
  • at least one chemical molecule with antiblastic activity which molecule amplifies or supplements the action of the fusion protein.
  • the conjugates of the invention exhibit more potent activity than their constituents themselves, i.e. the fusion proteins, the chemical compound with antiblastic activity and the effector peptide, individually or in a mixture.
  • new conjugates overcome natural or induced resistance to TRA
  • the present invention overcomes the limitations of known therapies using low- molecular weight compounds, their mixtures, and/or conjugates with proteins or peptides.
  • the invention improves solubility, reduces toxicity, eliminates side effects and improves pharmacokinetic parameters of the components of a conjugate.
  • the invention solves also the problem of low efficiency of a chemical compound caused by inability of achieving biologically effective doses and resistance to administered chemicals appearing during therapy.
  • the conjugation of a cytostatic compound molecule with an active anticancer protein allows specific targeting of highly toxic molecules to tumours and the concomitant amplification of the apoptotic signal by Apo2L/TRAIL protein receptors.
  • Another advantage is the delivery of a conjugate into the cell via internalization by means of death domain receptors DR and, therefore, circumvention of cell membrane proteins responsible for drug resistance, that prevent chemical molecules from diffusion to the cells. Additionally, the use of a conjugate according to the present invention results in deblocking of apoptotic pathways via an increase of DR4/DR5 expression. The use of a conjugate according to the present invention does not require testing for the presence of surface antigen of the tumor (since expression of TRAIL receptors (death receptors DR4/DR5) is present on all types of tumours).
  • the present invention also overcomes the limitations of the prior art protein- chemical compound conjugates in terms of inefficient concentration of the compound achieved in the target site due to the instability of the branched linker connecting the compound with a protein.
  • long branched linkers are used, the structure of which provides stability in vitro and in vivo.
  • the use of branched linkers allows the attachment of more than one chemical compound molecule to the linker molecule, and thereby formation of a conjugate according to the invention with attached more than three molecules of a chemical compound.
  • Such type of linkers are described for example in US2010/0160409.
  • Another advantage of the present invention is effective, one-step conjugation method of fusion proteins with chemical compounds.
  • Fig. 1 presents tumour volume changes (% of initial stage) in Cr[:SHO-Prkdc scid Hr hr mice burdened with lung cancer A549 treated with the conjugate SEQ. ID No. 24- L7-Z1 of the invention compared to the mixture of the conjugate constituents and to the fusion protein of SEQ. ID No. 24.
  • Fig. 2 presents tumour growth inhibition values (%TGI) in Cr[:SHO-Prkdc scid Hr hr mice burdened with lung cancer A549 treated with conjugate SEQ. ID No. 24-L7- Z1 of the invention compared to the mixture of the conjugate constituents and to the fusion protein of SEQ. ID No. 24.
  • Fig. 3 presents tumour volume changes (% of initial stage) in Cr[:SHO-Prkdc scid Hr hr mice burdened with lung cancer A549 treated with the conjugate SEQ. ID No. 24- L7-Z1 of the invention compared to the mixture of the conjugate constituents and to the fusion protein of SEQ. ID No. 24.
  • Fig. 4 presents tumour growth inhibition values (%TGI) in Cr[:SHO-Prkdc scid Hr hr mice burdened with lung cancer A549 treated with the conjugate SEQ. ID No. 24- L7-Z1 of the invention compared to the mixture of the conjugate constituents and to the fusion protein of SEQ. ID No. 24.
  • Fig. 5 presents tumour volume changes (% of initial stage) in Cr[:SHO-Prkdc scid Hr hr mice burdened with lung cancer H69AR treated with the conjugate SEQ. ID No. 24-L7-Z1 of the invention compared to the mixture of the conjugate constituents and to the fusion protein of SEQ. ID No. 24.
  • Fig. 6 presents tumour growth inhibition values (%TGI) in Cr[:SHO-Prkdc scid Hr hr mice burdened with lung cancer H69AR treated with the conjugate SEQ. ID No. 24-L7-Z1 of the invention compared to the mixture of the conjugate constituents and to the fusion protein of SEQ. ID No. 24.
  • Fig. 7 presents tumour volume changes (% of initial stage) in Cr[:SHO-Prkdc scid Hr hr mice burdened with lung cancer SKOV3 treated with the conjugate SEQ. ID No. 24- L7-Z1 of the invention compared to the mixture of the conjugate constituents and to the fusion protein of SEQ. ID No. 24.
  • Fig. 8 presents tumour growth inhibition values (%TGI) in Cr[:SHO-Prkdc scid Hr hr mice burdened with lung cancer SKOV3 treated with the conjugate SEQ. ID No. 24- L7-Z1 of the invention compared to the mixture of the conjugate constituents and to the fusion protein of SEQ. ID No. 24.
  • the invention relates to a conjugate, said conjugate comprising:
  • a fusion protein comprising domain (a), which is the functional fragment of a sequence of soluble human TRAIL (hTRAIL) protein beginning with an amino acid at a position not lower than hTRAIL95 or a sequence having at least 70% identity with said functional fragment, domain (b) which is the sequence of an effector peptide having proapoptotic, antiangiogenic, antiproliferative or pore forming activity, and conjugation domain (d) for attachment of a chemical compound selected from the group consisting of the sequences Cys Ala Ala Ala Cys Ala Ala Cys and Cys Ala Ala Cys Ala Ala Ala Cys, and
  • domain (b) of the effector peptide in the invention is neither hTRAIL protein nor a part or fragment of hTRAIL protein.
  • hTRAIL soluble human TRAIL
  • peptide in accordance with the invention should be understood as a molecule built from plurality of amino acids linked together by means of peptide bonds.
  • peptide according to the invention includes oligopeptides, polypeptides and proteins.
  • amino acid sequences of peptides will be presented in a conventional manner adopted in the art, i.e. 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 conjugate it is meant a single molecule comprising a fusion protein containing two or more proteins or their fragments, covalently linked via peptide bond within their respective peptide chains without additional chemical linkers, with at least one molecule of a chemical compound attached to it, wherein said molecule may be attached directly or by a suitable linker enabling the conjugation (conjugation linker).
  • the fusion protein molecule comprises a conjugation domain (d) of the sequence Cys Ala Ala Ala Cys Ala Ala Cys and Cys Ala Ala Cys Ala Ala Ala Cys (CAAACAAC or CAACAAAC in one-letter notation, respectively), comprising attachment site for the molecule of a chemical compound.
  • the conjugate according to the invention comprises a fusion protein with 3 molecules of a chemical compound attached to said conjugation domain (d) in the fusion protein via a suitable conjugation linker L.
  • the conjugate according to the invention comprises 4 to 9 molecules of a chemical compound attached to said conjugation domain (d) in the fusion protein via an appropriate conjugation linker L.
  • the fusion protein in the conjugate of the invention can have the sequence of the domain (b) attached at the C-terminus and/or N-terminus of domain (a).
  • the fusion protein may comprise at least one domain (b) of the effector peptide attached at the C-terminus and/or N-terminus of the domain (a).
  • Domain (a) of the fusion protein is a fragment of soluble hTRAIL sequence, beginning with an amino acid in the position starting from the position 95 of hTRAIL (hTRAIL95), particularly in the range from amino acid 95 to amino acid 122 of hTRAIL sequence (hTRAIL122) inclusive, and ending with the amino acid 281 of hTRAIL sequence (hTRAIL281 ).
  • designation "hTRAIL” followed by a number means an amino acid having that number in the known hTRAIL sequence.
  • designation "hTRAIL” followed by a range of numbers means an fragment of the known hTRAIL sequence including amino acids in the specified range of numbers in the known sequence hTRAIL (SEQ. ID. No. 1 ) published in GenBank under Accession No. P50591.
  • said domain (a) may be selected from the group consisting of sequences corresponding to hTRAIL95-281 , hTRAIL114-281 , hTRAIL119-281 , hTRAILI 20-281 , hTRAILI 21 -281 and hTRAILI 22-281.
  • hTRAIL95-281 , hTRAIL114-281 , hTRAIL119-281 , hTRAIL120- 281 , hTRAILI 21 -281 and hTRAILI 22-281 represent a fragment of human TRAIL protein starting with amino acid marked with the number 95, 114, 119, 120, 121 and 122 respectively and ending with amino acid marked with a number 281 , in the known sequence of hTRAIL.
  • said domain (a) is the functional fragment of soluble hTRAIL protein sequence beginning with an amino acid at the position in a range from hTRAIL95 to hTRAILI 22, inclusive, and ending with the amino acid at the position hTRAIL281 , or a sequence which has at least in 70%, preferably 85%, identity to said functional fragment (i.e. a homologue of such hTRAIL fragment).
  • a homologue of such hTRAIL fragment i.e. a homologue of such hTRAIL fragment.
  • the definition of the homologue of a fragment of hTRAIL, its activity and affinity, as well as preferred and particularly preferred homologues of hTRAIL fragment of modified sequence with enhanced affinity for the death receptor DR5 in comparison to the death receptor DR4, that is with increased selectivity against DR5/DR4, are described in WO2012072815 (page 9, line 26 to page 11 , line 30).
  • the fusion protein in the conjugate of the may be considered a carrier for the attached compound Z which targets the compound directly to the cancer cell where it can exert its action, depending on its own mechanism of activity, with significantly reduced effect on non-cancer cells.
  • the fusion protein is able to exert its own anticancer activity after arrival and attachment to cancer cell and subsequent cleavage.
  • This anti-cancer activity can be exerted by two components of the fusion protein, i.e. by both domain (a) of the TRAIL fragment and domain (b) of effector peptide. Therefore, the conjugate of the invention can be considered a three-way anti-cancer agent, acting by three mechanisms of elimination of cancer cells, all of them with significantly reduced toxic effects on non-cancer cells.
  • the effector peptide of domain (b) may be a peptide with proapoptotic, antiangiogenic, antiproliferative activity, or pore-forming activity, which acts synergistically with TRAIL protein, thus significantly affecting the ability to eliminate cancer cells.
  • the effector peptide of domain (b) with proapoptotic activity may be a peptide that induces apoptosis directly by activating signalling cascade components of mitochondrial pathway of apoptosis, or by direct induction of mitochondrial apoptosis in cells or inhibits and/or binds intracellular anti-apoptotic factors or also exerts direct destructive effect inside the cell and inhibits the cell cycle, for example by degradation of total cellular RNA or induction of degradative nucleases, thus significantly affecting the ability to eliminate cancer cells.
  • the effector peptide is a short peptide derived from SMAC/DIABLO, a peptide derived from Bid protein or a peptide derived from Nur77 protein.
  • the effector peptide of the fusion protein may be selected from the group consisting of:
  • An effector peptide of domain (b) with antiangiogenic activity can be a peptide which among others complements or enhances TRAIL activity via inhibition of growth factors receptors VEGF (Vascular Endothelial Growth Factor) or PDGF (Platelet-derived growth factor), PEDF (Pigment Epithelium -Derived Factor) receptor, or aminopeptidase N (CD13), thus significantly affecting the ability to eliminate cancer cells.
  • VEGF Vascular Endothelial Growth Factor
  • PDGF Platert-derived growth factor
  • PEDF Progment Epithelium -Derived Factor
  • CD13 aminopeptidase N
  • the effector peptide is a short peptide which is a fragment of growth factor having ability to bind to a receptor of this growth factor and inhibit its activity, while being itself devoid of anti-angiogenic activity, in particular a fragment of human vascular endothelial growth factor VEGF, or a fragment of platelet-derived growth factor PDGF.
  • the effector peptide can be also a fragment of pigment epithelium -derived factor PEDF or a short peptide inhibiting aminopeptidase N/CD13 comprising motifs NGR or RGD.
  • the effector peptide of the fusion protein can be in particular selected from the group consisting of:
  • the effector peptide of domain (b) with antiproliferative activity can be a peptide, which among others complements or enhances TRAIL activity by inhibition of growth of estradiol-dependent cells, inhibition of MAPK kinase signaling pathway, enabling inhibition of cell cycle in G1 phase, for example by inhibition of cycline D1 -CDK4 complex, thus significantly affecting the ability to eliminate cancer cells.
  • the effector is a short DD2 peptide, which is a proline rich C-terminal domain of DOC-2/DAB2 protein, or a p16 peptide or a fragment thereof inhibiting the activity of kinases CDK4 and CDK6, or a fragment of troyan p16 peptide fused to 17-amino acids Antennapedia transporting domain.
  • the effector peptide of a domain (b) of a fusion protein can be in particular selected from the group consisting of: - 34-amino acids fragment of human fetoprotein of SEQ. ID No. 8;
  • the effector peptide of domain (b) with pore-forming activity forms pores in a cellular membrane and can be a peptide, which among others complements or enhances TRAIL activity by destabilization of electrostatic potential of mitochondrial membrane resulting in leakage of cell death factors such as cytochrome C, SMAC/DIABLO and AIF from mitochondria to cytoplasm, thus significantly affecting the ability to eliminate cancer cells.
  • the effector peptide of domain (b) of a fusion protein is a short peptide with a strong positive charge enabling interactions with negatively charged membrane of tumour cells or an amphipathic peptide forming alpha-helical structure.
  • the effector peptide of domain (b) of the fusion protein can be a peptide selected from the group consisting of:
  • the fusion protein comprises domain (c) situated between domain (a) and domain (b) of the fusion protein, said domain (c) comprising the sequence of a protease cleavage site recognized by proteases present in the cell environment, especially in the tumour cell environment, for example such as metalloprotease MMP, urokinase or furin.
  • Sequences recognized by a protease may be selected from a sequence recognized by metalloprotease MMP, such as for example the sequence Pro Leu Gly Leu Ala Gly Glu Pro, or its fragment which with the terminal amino acid of a sequence to which it is attached forms a sequence recognized by metalloprotease MMP; a sequence recognized by urokinase uPA, such as for example the sequence Arg Val Val Arg, or its fragment which with the terminal amino acid of a sequence to which it is attached forms a sequence recognized by urokinase; sequences recognized by furin, such as for example the sequence Arg Gin Pro Arg, Arg Gin Pro Arg Gly, the sequence Arg Lys Lys Arg, or other atypical sites recognized by furin, such as for example those disclosed by M.
  • metalloprotease MMP such as for example the sequence Pro Leu Gly Leu Ala Gly Glu Pro, or its fragment which with the terminal amino acid of a sequence to which it is attached forms a sequence recognized by metal
  • the protease cleavage site can be the combination of a sequence recognized by metalloprotease MMP and/or a sequence recognized by urokinase uPA and/or the sequence recognized by furin, located next to each other in any order.
  • domain (c) recognized by proteases over- expressed in tumour environment is the sequence Arg Val Val Arg Pro Leu Gly Leu Ala Gly, or the sequence Pro Leu Gly Leu Ala Gly Arg Val Val Arg.
  • proteases metalloprotease MMP, urokinase uPA and furin are overexpressed in the tumour environment.
  • the presence of the sequence recognized by proteases enables cleavage of the domain (a) from the domain (b) of the fusion protein, i.e. the release of the functional domain (b), and thus accelerates its activation.
  • the activation of the effector domain (b) may also occur in a non-specific manner by cutting off domain (a) from domain (b) of the invention by lysosomal enzymes.
  • 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.
  • the release in the environment of the tumour enables the action of individual constituents of the conjugate, but only in the tumour environment.
  • the constituents of the fusion protein and the chemical compound of the conjugate tested individually on animals and in clinical trials showed high systemic toxicity and the need of high doses administration due to the inability of obtaining high local concentration selectively in the tumour environment, this resulting in lack of efficacy of a therapy.
  • conjugate ensures that after cleavage by proteases receptor binding properties are unchanged comparing to the unconjugated forms of conjugate constituents.
  • the molecule of a chemical compound Z with antiblastic activity may be attached to the fusion protein via its conjugation domain (d) directly or by means of a conjugation linker L.
  • the chemical compound Z with antiblastic activity may be attached to the fusion protein via formation of a complex or via a stable chemical bond.
  • the compound is attached to the fusion protein via a stable chemical bond, particularly preferably a covalent bond.
  • the molecule of a chemical compound Z with antiblastic activity may be attached to the protein via formation of a stable chemical bond, in particular a covalent bond, directly between the conjugation domain (d) of the fusion protein and the chemical compound, or between the conjugation domain (d) of the fusion protein and the chemical compound via a conjugation linker L.
  • the covalent bond between the fusion protein and the chemical compound may be also formed as a result of a chemical reaction directly with the reactive free amino, carboxyl, or sulfhydryl groups present in the fusion protein, depending on the structure of the chemical compound and on the type of functional groups in the chemical compound capable of reacting with any of these groups.
  • a linker molecule is first attached to the chemical compound, and then chemical compound with attached conjugation linker is reacted with reactive free amino group in the fusion protein.
  • fusion protein comprises, additionally and independently of domain (c), conjugation domain (d) of a sequence destined for attachment (conjugation) of the molecule of a chemical compound to the fusion protein via formation of a stable bond.
  • This domain contains three functional sulfhydryl (SH) groups capable of forming a bond with a chemical compound.
  • domain (d) is situated between domain (a) and domain (b) of the fusion protein.
  • conjugation domain (d) enables the attachment of the chemical compound in a specified amount and in a specified location of the protein. Since the conjugation domain (d) may also act as a spacer (a steric linker), which facilitates the correct folding of the protein product, this is also sterically beneficial.
  • Conjugation domain (d) having the sequence enabling the attachment of the molecule of a chemical compound to the fusion protein contains three cysteine residues with sulfhydryl groups, and therefore in the following description may be also referred to as "a cysteine linker".
  • cysteine residue enables the attachment of the molecule of a chemical compound by reaction of free sulfhydryl group present in the cysteine residue of the fusion protein with functional groups of the compound capable to react with sulfhydryl group.
  • free cysteine residue it is meant a cysteine residue which is not involved in the formation of disulfide bonds within the same molecule of the fusion protein or between more molecules of the fusion protein (homodimers and homotrimers), said disulfide bonds being necessary for the activity of the protein.
  • sequence of conjugation domain (d), depending on amino acid composition of the domains (a), (b), (c) or (e) of the conjugate of the invention which are attached to it, optionally may be flanked with sequences of flexible steric linkers, especially glycine and glycine-serine linkers, such as for example, one glycine residue Gly or Gly Gly, Gly Gly Gly or Gly Ser Gly, fragments and combinations thereof.
  • flexible steric linkers especially glycine and glycine-serine linkers, such as for example, one glycine residue Gly or Gly Gly, Gly Gly Gly or Gly Ser Gly, fragments and combinations thereof.
  • cysteine linkers and their use as trimer structure stabilizing domains are described in WO/2011 /161260, WO/2012/093158, WO/2012/143477, P-397167, WO2013/080147; WO2013/098755 and WO2013098755.
  • the fusion proteins of the conjugate of the invention possess at least one free cysteine residue in the conjugation domain (d) and most preferably 3 free cysteine residue in the conjugation domain (d).
  • the anticancer conjugate comprising domain (d) (cysteine linker) with 3 free cysteine residues is the most preferable embodiment of the present invention.
  • domain (d) cysteine linker
  • cysteine linker domain (d) (cysteine linker) with 3 free cysteine residues
  • conjugation domain (d) - cysteine linker has three cysteine residues.
  • cysteine linkers containing less than 3 cysteine residues such as for example fragments of domain (d) used in the invention, although theoretically conceivable, are less recommended.
  • cysteine linkers containing more than 3 cysteine residues are less recommended since are unstable and cause difficulties during expression of the fusion proteins in a bacterial system.
  • the increase of the number of cysteine residues increases the probability of reaction between two adjacent cysteine residues, which causes blocking of the binding site of a chemical compound molecule. Oxidation of cysteines may further lead to rupture of the protein chain formed at the site of a cysteine linker domain.
  • a domain (d) comprising 3 cysteine residues in the absence of any other free cysteine residues within properly spatially arranged fusion protein domains, allows attachment of at least one chemical compound molecule.
  • 3 chemical compound molecules are attached.
  • This approach ensures the homogeneity of the conjugate, which in such a case always possess strictly determined number of chemical compound molecules attached at a strictly determined location per 1 molecule of the fusion protein. That is, 3 molecules of a chemical compound are attached to one molecule of the fusion protein in the case of direct attachment of a chemical compound (without the use of a linker L) or in the case of using a simple, unbranched linker L, i.e. the one able to bind only one molecule of a chemical compound.
  • branched linkers L i.e. such linkers which are able to bind at least 2, preferably 3 molecules of a chemical compound.
  • domains (a) and (b) of a fusion protein are selected so that they do not comprise free cysteine residues, i.e. they are not involved in formation of proper structures of domain (a) and (b) proteins. Furthermore, the domains are selected so that the cysteines involved in formation of proper structures of a protein are practically sterically not available, and thus in practice they cannot react with chemical compounds (Z) moieties or with the moieties of linkers with attached chemical compounds (L-Z). Therefore, the use of domain (d) comprising sites for attachment of a chemical compound assures lack of interference in protein domains structure - and thus conjugation of a chemical compound doesn't influence the activity of protein domains (a) and (b).
  • domain (c) is located in such a way that after cleavage of the conjugate the conjugation domain (d) is disconnected from domain (b).
  • the fusion proteins may contain a neutral sequence or sequences of a flexible steric glycine or glycine-serine linker (spacer).
  • spacer steric glycine or glycine-serine linker
  • flexible linker may be selected from the group consisting of sequences Gly Gly Gly, Gly Ser Gly Gly Gly, Gly Gly Gly Ser, and Xaa Gly Gly Ser, wherein Xaa designates any amino acid or is absent.
  • a transporting domain (e) may be attached to domain (b) of the effector peptide of the fusion protein being a constituent of a conjugate of the invention.
  • Domain (e) may be selected form the group consisting of:
  • polyhistidine sequence transporting through the cell membrane consisting of 6, 7 , 8, 9, 10 or 11 histidine residues (His/H);
  • e3 a PD4 transporting sequence (protein transduction domain 4) Tyr Ala Arg Ala Ala Ala Arg Gin Ala Arg Ala /YARAAARQARA;
  • Domain (e) may be attached to N- or C-terminus of domain (b).
  • the sequence of a fusion protein in the conjugate of the invention is selected from the group consisting of SEQ. ID No. 20; SEQ. ID No. 21 ; SEQ. ID No. 22; SEQ. ID No. 23; SEQ. ID No. 24; SEQ. ID No. 25; SEQ. ID No. 26; SEQ. ID No. 27; SEQ. ID No. 28; SEQ. ID No. 29; SEQ. ID No. 30; SEQ. ID No. 31 ; SEQ. ID No. 32; SEQ. ID No. 33; SEQ. ID No. 34; SEQ. ID No. 35; SEQ. ID No. 36; SEQ. ID No. 37; SEQ. ID No. 38; SEQ. ID No. 39; SEQ. ID No. 40; SEQ. ID No. 41 , and SEQ. ID No. 42.
  • the fusion protein in a conjugate of the invention is selected from the group consisting of SEQ. ID No. 22, SEQ. ID No. 23, SEQ. ID No. 24, and SEQ. ID No. 29.
  • Methods of synthesis of fusion proteins useful as carriers in the conjugate according to the present invention are apparent to the skilled in the art as such and are described in WO/2011 /161260, WO/2012/093158, WO/2012/143477 and in WO2013/098755.
  • the fusion protein in the conjugate of the invention will have a dual function.
  • the fusion protein acts as a carrier for a chemical compound with antiblastic activity, which delivers the compound to the tumour.
  • the chemical compound Z with antiblastic activity attached to the fusion protein in the conjugate is a compound with antiblastic activity against cells and is selected from the group consisting of compounds having the activity of topoisomerase inhibitors, degrading DNA, nucleoside analogues, DNA intercalators, tubulin polymerization inhibitors, proteasome inhibitors, plant alkaloids, activators of protein kinase, compounds causing the disintegration of lysosomes, depolymerization of actin F or inhibition of actin G polymerization, or binding of actin subunits 1 and 3, compounds interacting with the mitochondrial membrane, inducers of apoptosis (saponins or calcineurin inhibitors), protozoicides, antifungal and antiviral compounds, which have been found toxic to the cancer cells (usually without any indication of the mechanism of action), antibiotics (eg.
  • anthracycline, antiblastic inhibitors of VEGF/VEGFR-1 loop, inhibitors of cell cycle in phase G2 and M, inhibitors of sodium-proton antiporters (NHE3), inhibitors of VEGF binding to its receptors, inhibitors of serine protease, NES inhibitors (nuclear export signal) responsible for proteins transport from nucleus to cytoplasm, blockers of activation of NF- ⁇ in TNF pathway, inhibitors of DNA methyltransferase, molecules interacting with cellular cytoskeleton, compounds cross-linking/alkylating DNA, molecules binding with DNA small groove, compounds activating apoptosis via pathway of caspase 8 and effector caspases 3/7.
  • Antiblastic activity should be understood in accordance with the dictionary definition, namely as an antagonistic effect on the growth of cells, resulting in cell death, regardless of mechanism of action.
  • a chemical compound Z with an antiblastic activity is also simply referred to as a chemical compound.
  • the chemical compound with the topoisomerase I inhibitor activity may be selected from the group of camptothecin derivatives, such as for example SN38, irinotecan, afletecan, belotecan, topotecan, Amonafide (AS1413), or exatecan.
  • camptothecin derivatives such as for example SN38, irinotecan, afletecan, belotecan, topotecan, Amonafide (AS1413), or exatecan.
  • the chemical compound with DNA degrading activity may be in particular selected from the group consisting of esperamicin, spiramycin, cycloheximide, azithromycin, clarithromycin, roxithromycin, erythromycin, and josamycin, calichaemycin gamma 1 , esperamycin A1 or Zalypsis® (PM1004)
  • the chemical compound - a nucleoside analog may be in particular 4-methyl- amine-1 -(B-D-arabinofuranosyl)pyrimidin -2(1 H)-one.
  • the chemical compound with an activity of a DNA intercalator can be especially selected from the group consisting of N-(1 -nitroacridin-9-yl)-propyl-1 ,3-diamine, doxorubicin, and ethidium bromide, pancratistatine (PST), ascididemine, ascididemine derivatives, ledoxantrone (CI-958), sulphachinoxaline, xk469, amrubicine, dactinomicine, hycantone, and C1311.
  • the chemical compound with an activity of tubulin polymerization inhibitor may be particularly selected from the group consisting of deacetylcolchicine, docetaxel, cryptophycin, ixabepilon, epothilone B, 4B-amino-4'-0-4- deoxydemethylepipodophylotoxin, ustiloxine A, and rizoxine, ombrabuliny, eribuline, BNC105, pateamine A, zampanolide, peloruside A, peloruside B, diazonamide A, scleritodermine A, and ansamitocine P-3,
  • the chemical compound of the proteasome inhibitor activity can be in particular selected from the group consisting of salinosporamine A, bortezomibe, disulphiram, carphilzomib, ONX 0912, CEP- 18770, and MLN9708.
  • the chemical compound being plant alkaloid can be in particular selected from the group consisting of paclitaxel, hemiasterline, docetaxel, vinblastine, vincristine, vindesine, colchicine, podophyllotoxin, hemiasterline A, hemiasterline B, and hemiasterline C.
  • the chemical compound with protein C kinase activity can be in particular selected from the group consisting of bryostatin, chelerythrine, nimbolide, epigallocatechin gallate, curcumin, allicin, capsaicin, eugenol, cinnamic aldehyde, and farnesol.
  • the chemical compound with liposome disintegration activity can be in particular selected from the group consisting of kahalide F, ouabaine, digitoxin, and 2"- oxovorusharin.
  • the chemical compound with activity of actin F depolymerisation, inhibition of polymerisation of actin G, or inhibition of binding to actin subunits 1 and 3 can be in particular selected from the group consisting of bistramide A, hypocreline A, staurosporine, and verbascozide.
  • the chemical compound interacting with mitochondrial membrane can be in particular selected from the group consisting of brefeldine A; chlorokine; azithromycine, and clarithromycine.
  • the chemical compound acting as cytotoxic antibiotic can be in particular selected from the group consisting of anthracycline and antiblastic antibiotics, preferably from the group consisting of momensin methyl ester and momensin.
  • the chemical compound from saponins group which also act as inducer of apoptosis mediated by nitrogen oxide, can be in particular akebia saponin D (hederagenin 3-0-alpha-L-arabinopyranosyl-28-beta-D-glucopyranosyl(1 ⁇ 6)- beta-D-glucopyranoside).
  • the chemical compound with calcineurin inhibitor activity being also an apoptosis inhibitor, can be in particular selected from the group consisting of eudistalbin A, 6-methyleudistomidin C, eudistomin E, eudistomin C, OSW-1 , calphostine C, ursolic acid, and 14-deoxyandrographolide.
  • the chemical compound with VEGF/VEGFR- 1 loop inhibitors activity can be in particular aplidine (dihydrodidemnine B),
  • the chemical compound with cell cycle inhibition activity in phase G2 and M can be in particular discodermolide.
  • the chemical compound with sodium-proton antiporter (NHE3) activity can be in particular selected from the group consisting of squlamine and saxitoxine.
  • the chemical compound with serine protease inhibitor activity can be in particular symplocamide A.
  • the chemical compound with NES inhibitors (nuclear export signal) activity responsible for protein transport from nucleus to cytoplasm can be in particular calistatine A.
  • the chemical compound with NF- ⁇ pathway activation blockers TNF pathway can be in particular celastrol.
  • the chemical compound with DNA methyltransferase inhibitor activity can be in particular psammapline G.
  • the chemical compound interacting with cellular cytoskeleton can be in particular spisulosine.
  • the chemical compound with DNA cross-linking/alkylating activity can be in particular nimustine.
  • the chemical compound with DNA small groove activity can be in particular selected from the group consisting of trabactedine (Yondelis®) and trabactedine analogue PM01183.
  • the chemical compound with apoptosis activating activity on caspase 8 and effector caspases 3/7 can be in particular selected from the group consisting of amlodipine and azelnidipine.
  • the chemical compound with antiblastic activity is selected from the group consisting of the compounds Z1 to Z66 presented in Table 1.
  • Z is selected from Z1 (camptothecin derivative SN38), Z5, 122, and Z43.
  • the chemical compound depending on its structure, can be attached to the fusion protein directly.
  • a functional group of the chemical compound is condensed with a suitable reactive group of the fusion protein.
  • the reactive group of the fusion protein can be a primary N-terminal amino group.
  • chemical compounds having carboxyl group or derivative thereof that react with amine group of the fusion protein including those selected from tubulysine D (Z20), hemiasterlin (Z24) and momensin ethyl ester (Z1 1 ).
  • Such a solution is less favorable, due to the fact that lysine-derived free amine residues of the fusion protein can participate in the condensation reaction with carboxylic acid residues of the chemical compound.
  • Reactive functional group of the fusion protein can also be carboxyl group, present at the C-terminal position of the protein.
  • such a variant is possible for chemical compounds having amino or hydroxyl group or a derivative thereof, reactive with carboxyl groups of the fusion protein and in the side chains of aspartic and glutamic acid.
  • Such solution is less preferred, due to the fact that the free carboxylic acid residues of the fusion protein derived from aspartic acid and glutamic acid residues may enter the condensation reaction with functional groups of the chemical compound.
  • Reactive functional group of the fusion protein may also be a sulfhydryl group present in the cysteine side chain. Since sulfhydryl groups are often linked by disulfide bonds (-S-S-), as a part of secondary or tertiary structure of the protein, before joining the chemical compound it is advantageous to carry out their reduction, so as to make sulfhydryl groups available for conjugation with reactive groups.
  • sulfhydryl groups are often linked by disulfide bonds (-S-S-), as a part of secondary or tertiary structure of the protein, before joining the chemical compound it is advantageous to carry out their reduction, so as to make sulfhydryl groups available for conjugation with reactive groups.
  • the chemical compound Z is attached to the fusion protein by a conjugation linker L.
  • conjugation linker L it is meant a compound containing reactive functional groups capable of attaching to the specific chemical functional groups (primary amino groups, carboxyl groups, sulfhydryl groups) on proteins or other molecules.
  • Linkers useful for coupling compounds to peptides/proteins are known, and are described for example in Bioconjugate Techniques, Hermanson, G. T. , Academic Press, Inc. , 2nd Ed. (2008), Chemistry of Protein Conjugation and Cross-Linking Wong, S.S. , Ph. D. , Published by CRC Press, Inc. , 1991, DL1 -9636889A1 , U. Beyer et al. , Chemical Monthly, 128, 91 , 1997, Greenfield R.S. et al. , Cancer Res. , 50, 6600, 1990, Kaneko T. et al. , Bioconjugate Chem. , 2, 133, 1991 .
  • Linkers sensitive to acidic environment are described in EP0495265B. Cleavage sites recognized by proteases overproduced in the tumour environment such as MMP2/9 can also be used for coupling of chemical compounds (as described for example in US7803903).
  • Conjugation method used in the invention which comprises selection of a suitable conjugation linker L, suitable domain (d) comprising site of attachment of a chemical compound and a method of conjugation determines the number of molecules of a chemical compound to be attached to the protein (molar ratio) which is possible to achieve without formation of aggregates or conjugate precipitates. It determines also selectivity and efficiency of drug release.
  • conjugation linker L is a linker reacting with the free sulfhydryl group of a cysteine residue, in particular, the free cysteine residue of domain (d) to give a thioether bond.
  • conjugation linker L reacting with the free sulfhydryl group of the cysteine residue is maleimide linker, that is a linker comprising a maleimide moiety.
  • Maleimide linkers are known in the art.
  • the maleimide linker may comprise polyethoxylated (PEG) moiety attached to the maleimide moiety.
  • PEG polyethoxylated
  • PEG molecules useful for the use in the linker may be selected from linear and branched PEG molecules.
  • Polyethoxylated linker for conjugation of the chemical compound should contain at least two oxyethylene groups.
  • linear PEG molecules composed of 2 to 8 monomers, preferably 2 to 4 monomers.
  • the use of a domain containing polyoxyethylene group may change pharmacokinetic and pharmacodynamic parameters.
  • a conjugation linker should not affect the conformation, activity and receptor specificity of the protein to which it is attached.
  • Another useful linker comprises active ester moiety, in which carboxyl group of the linker is activated with N-hydroxysuccinimide to provide appropriate N-hydroxysuccinimide ester directly reacting with chemical compound to form chemical compound-linker moieties, as for example linker L12 presented below in Table 2.
  • linker L1 to L14 examples of preferred maleimide linkers for conjugation to chemical compounds are shown below by formulas L1 to L14 in Table 2.
  • the dashed line indicates the point of attachment to the chemical compound.
  • linker Before conjugation, linker has a hydroxy group (linkers L1 to L10, L12 and L14) or hydrogen atom (linker L1 1 ) in the place of the dashed line.
  • Linker can be in particular selected from the group consisting of L1 , L2, L3, L4, L5, L6, L7, L12 and L14.
  • Particularly preferred linker is L7.
  • connection chemical compound - linker for conjugation of the chemical compound should be stability of the connection chemical compound - linker under physiological conditions, outside the tumour environment.
  • maleimide linkers containing for example amino acid residues
  • their degradation by esterases and other enzymes was observed, resulting in the release of the molecules of a chemical compound outside the environment of the tumour, and thereby reduction of the effectiveness of the conjugate and increased systemic toxicity.
  • Linker L7 can be obtained according to the following scheme:
  • linker L7 is shown below in the Examples.
  • the chemical compound Z is attached through the linker L to the conjugation domain (d) of the fusion protein.
  • domain (d) comprises free cysteine residues not involved in the formation of a proper fusion protein structure, and capable of reacting via sulfhydryl groups with linker L groups, preferably with its maleimide residues, to form a thioether bond.
  • the ratio chemical compound/fusion protein can take values > 1 .
  • the conjugate is prepared by attaching the moiety chemical compound Z - linker L (L-Z) to the fusion protein.
  • the chemical compounds are obtained from commercial sources or using chemical synthesis methods known in the art.
  • Suitable moieties chemical compound-linker can be prepared using conventional, routine transformations used in organic synthesis, in particular reactions of coupling amino or hydroxyl groups with carboxyl groups in the presence of suitable coupling agents. Such typical reactions are described, for example, in El-Faham, A. and Albericio, F. (2010) Peptide-Coupling Reagents, in Amino Acids, Peptides and Proteins in Organic Chemistry: Building Blocks, Catalysis and Coupling Chemistry, Volume 3 (ed A. B. Hughes), Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany.
  • chemical compound-linker linkage is prepared by a coupling reaction of a suitable compound having amino or hydroxyl group with carboxyl group of the linker, if it is available (Method A).
  • a suitable compound having amino or hydroxyl group with carboxyl group of the linker if it is available
  • typically coupling agent is dicyclohexylcarbodiimide, optionally in the presence of a catalytic amount of dimethylaminopyridine (DMAP), and coupling is carried in a suitable solvent such as methylene chloride, at ambient temperature for 48 hours.
  • DMAP dimethylaminopyridine
  • Exemplary moiety chemical compound Z-linker L (L-Z moieties) that can be obtained by this method is L1 -Z1.
  • linker can be activated by N-hydroxy- succinimide and in a form of an appropriate N-hydroxysuccinimide ester reacted with chemical compound to form chemical compound-linker moiety.
  • a reaction can be performed, for example, by mixing appropriate N-hydroxy- succinimide activated linker with appropriate chemical in an inert solvent, such as methylene chloride, in a room temperature.
  • each of the steps can be performed using the above- mentioned coupling technique amine reagent/hydroxy reagent - carboxylic acid.
  • a suitable carboxylic acid and coupling agent instead of a suitable carboxylic acid and coupling agent, corresponding acid chloride derived from carboxylic acid can be used, in the presence of a suitable base, typically triethylamine, in the reaction with hydroxy or amine reagent.
  • a suitable base typically triethylamine
  • moieties chemical compound Z - linker L in which the linker is selected form L3, L4, L6, L7 and L8 shown in Table 2, and the chemical compound is any compound from the group of compounds from Z1 to Z66 shown in Table 1 can be obtained.
  • Representative moieties chemical compound - linker L (moieties L-Z) obtainable by this method are L3-Z1 , L4-Z1 , L5-Z1 , and L7-Z1.
  • the first step is the addition of the acid chloride - alkyl chloroformate, which is then converted to the corresponding carbamate derivative and subjected to further reaction to join the subsequent linker structural components as described for Method B, to obtain its final form.
  • conjugation proteins represented by specific sequences from SEQ. ID No. 20 to SEQ. ID No. 42 set forth in the attached sequence listing, conjugation linkers represented by the formulas L1 to L14 in Table 2 and chemical compounds with antiblastic activity represented by the formulas Z1 to Z66 in Table 1.
  • conjugate of the invention are combinations of any of the fusion proteins, conjugation linkers and chemical compounds described above, wherein the conjugate comprises 3 moieties chemical compound- Z-linker L (L-Z moiety) attached to the fusion protein through three cysteine residues of conjugation domain (d) of the fusion protein.
  • the combination can therefore be particularly selected from the following group: SEQ. ID No 24-L1 -Z1 , SEQ. ID No 24-L2-Z1 , SEQ. ID No 24-L3-Z1 , SEQ. ID No 24-L4- Z1 , SEQ. ID No 24-L5-Z1 , SEQ. ID No 24-L7-Z1 , SEQ. ID No 24-L6-Z5, SEQ. ID No 24- L7-Z5, SEQ. ID No 24-L7-Z22, SEQ. ID No 24-L12-Z43 and SEQ. ID No 24-L14-Z1 , wherein the conjugate comprises 3 moieties chemical compound- Z-linker L (L-Z moiety) attached to the fusion protein through three cysteine residues of conjugation domain (d) of the fusion protein.
  • 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 receptors on the cell surface and activation of the extrinsic pathway of apoptosis.
  • the domain (b) After internalization of the fusion protein comprising proapoptotic, antiangiogenic, antiproliferative or pore-forming in cellular membrane effector peptide, the domain (b) will be able to potentially exert its action intracellular ⁇ in parallel to the activity of TRAIL domain.
  • anti -cancer activity of TRAIL can be potentiated by activation of other elements and mechanisms, such as for example induction of apoptosis by activation of elements of signaling cascade of mitochondrial apoptosis pathway or inhibition and/or binding intracellular antiapoptotic factors or inhibition of cell cycle by degradation of total cellular RNA or induction of degrading nucleases; or by inhibition of receptors of growth factors VEGF and PDGF, PEDF- receptor, or aminopeptidase N (CD13); or inhibition of growth of estradiol-dependent cells, inhibition of MAPK kinase signaling pathway, resulting in inhibition of cell cycle in phase G1 , for example by inhibition of cycline complex D1 -CDK4 able to inhibition of cell cycle kinases; or destabilization of electrostatic potential of mitochondrial membrane and consequently leak from mitochondrion to cytoplasm cell death factors such as e.g.. cytochrome C, SMAC/DIABLO and AIF, which
  • the chemical compound conjugated with the fusion protein will exert antiblastic activity involving inhibition of topoisomerase I or II, DNA degradation, inhibition of DNA polymerase, DNA intercalation, inhibition of mitosis, inhibition of tubulin polymerization, inhibition of the proteasome, stabilization of microtubules, induction of tubulin aggregation, cell cycle arrest, inhibition of calcineurin, mitochondrial damage, damage to the function of the Golgi apparatus, the inhibition of GTP-dependent interactions of ARF and beta-COP with mitochondrial membrane, activation of protein kinase C, activation of protein kinase C-delta, activation of calcium-dependent apoptosis, inhibition of signal pathway of PI3K-Akt kinase (phosphatidylinositol), damage or permeabilisation of cell membrane, influence on the expression of p53 and p21 protein, hemolysis of red blood cells or induction of nitrogen oxides-dependent apoptosis, VEGF/VEGFR
  • the invention also provides a pharmaceutical composition containing the conjugate of the invention as defined above as an active ingredient and a suitable pharmaceutically acceptable carrier, diluent and conventional auxiliary components.
  • the pharmaceutical composition will contain an effective amount of the conjugate 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 components, carriers and diluents are known to the skilled person and described in the literature. For example, they are described in the monograph Remington's Pharmaceutical Sciences, ed. 20, 2000, Mack Publishing Company, Easton, USA.
  • pharmaceutically acceptable carrier diluent, and auxiliary ingredient
  • auxiliary ingredient comprise any solvents, dispersion media, surfactants, antioxidants, stabilizers, preservatives (e.g. antibacterial agents, antifungal agents), and isotonicity agents, known in the art.
  • the pharmaceutical composition of the invention may contain various types of carriers, 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, or topical administration, 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 infusions.
  • the pharmaceutical composition of the invention may be administered by injection directly to the tumour.
  • the pharmaceutical composition of the invention may be administered intravenously.
  • 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 tissues or blood of recipient.
  • compositions are for example water, alcohols such as ethanol, polyols such as glycerol, propylene glycol, liquid polyethylene glycol, lipids such as triglycerides, vegetable oils, liposomes.
  • alcohols such as ethanol
  • polyols such as glycerol
  • propylene glycol liquid polyethylene glycol
  • lipids such as triglycerides
  • vegetable oils liposomes.
  • liposomes are for example water, alcohols such as ethanol, polyols such as glycerol, propylene glycol, liquid polyethylene glycol, lipids such as triglycerides, vegetable oils, liposomes.
  • lipids such as triglycerides, vegetable oils, liposomes.
  • surfactants such as hydroxypropylcellulose polysorbates, and the like.
  • Suitable isotonicity agents for liquid parenteral compositions are, for example, sugars such as glucose, and sodium chloride, and combinations thereof.
  • 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 suitable carrier such as, for example, sterile pyrogen- free water.
  • the pharmaceutical composition of the invention for parenteral administration may also have the form for nasal administration, including solutions, sprays or aerosols.
  • the form for intranasal administration will be an aqueous solution and will be isotonic or buffered to maintain the pH from about 5.5 to about 6.5, so as to maintain a character similar to nasal secretions.
  • 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 antifungal agents, including, for example, and not limited to, parabens, chlorobutanol, thimerosal, sorbic acid, and similar known substances of this type.
  • the pharmaceutical composition of the invention can include, for example at least about 0.01 wt% of an 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 indicated 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 patient condition and the route of administration.
  • a suitable unit dose, the total dose and the concentration of active ingredient in the composition is to be determined by the treating physician.
  • composition can be, for example, administered at a dose of about 1 microgram/kg of body weight to about 1000 mg/kg of body weight of the patient, 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 conjugate and the compositions containing it exhibit anticancer or antitumor activity and can be used for the treatment of cancer diseases.
  • the invention also provides the use of the conjugate of the invention as defined above for treating cancer diseases in mammals, including humans.
  • the invention also provides a method of treating cancer diseases in a mammal subject, including humans, in need of such treatment, comprising administering to said subject an anticancer effective amount of the conjugate of the invention as defined above, optionally in the form of a suitable pharmaceutical composition.
  • the conjugate of the invention can be used for the treatment of hematologic malignancies, such as leukemia, granulomatosis, myeloma and other hematologic malignancies.
  • the conjugate can also be used for the treatment of solid tumours, 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 conjugate in the treatment of cancer will be in particular parenteral route, which consists in administering the conjugate of the invention in the form of injections or infusions, in the composition and form suitable for this administration route.
  • fusion proteins constituting carriers in the conjugates of the invention were prepared as described in the publication WO2012072815, in embodiments from Ex. 1 to Ex. 17.
  • Other fusion proteins - not described in the prior art - were prepared as shown in the Examples 1A, 1 B and 1C below.
  • Example 1A Fusion protein of SEQ. ID No. 22
  • the protein of SEQ. ID No. 22 is a fusion protein having the length of 224 amino acids, in which domain (a) is a sequence of hTRAIL95-281 , and domain (b) of the effector peptide is a 9-amino acids peptide derived from Nur77 protein (SEQ. ID No. 4), attached at the N-terminus of domain (a). Additionally, domain (b) has at its C-terminus a transporting sequence consisting of 7 arginine residues.
  • domains (b) and (a) there are incorporated sequentially sequences of cleavage site recognized by urokinase (Arg Val Val Arg/RVVR) and metalloprotease MMP (Pro Leu Gly Leu Ala Gly/ PLGLAG), a sequence of steric glycine linker (Gly Gly Gly/GGG), and cysteine linker Cys Ala Ala Ala Cys Ala Ala Cys/CAAACAAC as a conjugation domain (d).
  • urokinase Arg Val Val Arg/RVVR
  • metalloprotease MMP Pro Leu Gly Leu Ala Gly/ PLGLAG
  • Gly Gly Gly/GGG a sequence of steric glycine linker
  • cysteine linker Cys Ala Ala Ala Cys Ala Ala Cys/CAAACAAC Cys Ala Ala Ala Cys/CAAACAAC as a conjugation domain (d).
  • the structure of the fusion protein of the invention is as follows:
  • amino acid sequence and the DNA encoding sequence comprising codons optimized for expression in E. coli are, respectively SEQ. ID No. 22 and SEQ. ID No. 43, as shown in the attached Sequence Listing.
  • the amino acid sequence SEQ. ID No. 22 of the structure described above was used as a template to generate its coding DNA sequence SEQ. ID No. 43.
  • a plasmid containing the coding sequence of DNA was generated and overexpression of the fusion protein was carried out in E. coli Tuner (DE3) strain from Novagen in accordance with the general procedures described in Maniatis et al, Molecular Cloning. Cold Spring Harbor, N.Y. , 1982.
  • Example 1 B Fusion protein of SEQ. ID No. 23
  • the protein of SEQ. ID No. 23 is a fusion protein having the length of 209 amino acids, in which domain (a) is a sequence of hTRAILI 20-281 , and domain (b) of the effector peptide is a 15-amino acids domain BH3 from Bid protein (SEQ. ID No. 3), attached at the N-terminus of domain (a).
  • the amino acid sequence and the DNA encoding sequence comprising codons optimized for expression in E. coli are, respectively SEQ. ID No. 23 and SEQ. ID No. 44, as shown in the attached Sequence Listing.
  • the amino acid sequence SEQ. ID No. 23 of the structure described above was used as a template to generate its coding DNA sequence SEQ. ID No. 44.
  • a plasmid containing the coding sequence of DNA was generated and overexpression of the fusion protein was carried out in E. coli Tuner (DE3) strain from Novagen in accordance with the general procedures described in Maniatis et al, Molecular Cloning. Cold Spring Harbor, N.Y. , 1982.
  • Example 1 C Fusion protein of SEQ. ID No. 29
  • the protein of SEQ. ID No. 29 is a fusion protein having the length of 209 amino acids, in which domain (a) is a sequence of hTRAIL.120-281 , and domain (b) of the effector peptide is an 18-amino acids fragment of PEDF (SEQ. ID No. 7), attached to the N-terminus of domain (a).
  • the structure of the fusion protein of the invention is as follows:
  • the amino acid sequence SEQ. ID No. 29 of the structure described above was used as a template to generate its coding DNA sequence SEQ. ID No. 45.
  • a plasmid containing the coding sequence of DNA was generated and overexpression of the fusion protein was carried out in E. coli Tuner (DE3) strain from Novagen in accordance with the general procedures described in Maniatis et al, Molecular Cloning. Cold Spring Harbor, N.Y. , 1982.
  • Example 2 Preparation of the moiety chemical compound Z - linker L 2.1 . Preparation of L1 -Z1 moiety
  • Step I Trans-4-(aminomethyl)-cyclohexanecarboxylic acid (500 mg, 3.18 mmol) was dissolved in 3 ml of glacial acetic acid, maleic anhydride (31 1 mg, 3.18 mmol) was added and the mixture stirred at room temperature until a white precipitate of trans-4-( ⁇ [(2Z)-3-carboxyprop-2-enoyl]amino ⁇ methyl)cyclohexanecarboxylic acid was formed. The mixture was then heated under reflux to complete the cyclization of the trans-4-(N-maleimidemethyl)cyclohexane-1 -carboxylic acid, and the mixture is concentrated under reduced pressure to remove acetic acid.
  • Step II trans-4-(N-maleimidemethyl)cyclohexane-1 -carboxylic acid (435 mg, 1 .83 mmol) was dissolved in 5 ml of dry DMF and cooled to -20°C, then N- methylmorpholine (0.202 ml, 1 .83 mmol), followed by ethyl chloroformate (0.174 ml, 1 .83 mmol) were added. After 5 min, 4-aminobenzoic acid (264 mg, 1 .92 mmol) was added, and after further 5 minutes the cooling bath was removed and the reaction mixture was allowed to reach room temperature over 1 hour. Then, the mixture was concentrated under reduced pressure to remove DMF.
  • Example 4 Conjugation of fusion protein with the molecule of a chemical compound molecule provide with a conjugation linker.
  • the SEQ. ID No. 24 prepared as described in WO/2012/093158 Example 6 in concentration of 2 mg/ml was reduced with DTT or TCEP (5-1 OmM) in order to increase the availability of thiol groups present on the protein. The reduction reaction was carried out for 30 min-1 .5 h at 4-8°C with occasional stirring.
  • the reduced protein was purified using gel filtration chromatography on a column HiPrep desalting 10/26 (equilibrated with buffer 5.87 mM KH 2 P0 4 , 17.53 mM Na 2 HP0 4 * 2H 2 0, 1 16.9mM sucrose, 200mM NaCl, 5mM EDTA, 10% v/v glycerol, pH 6.6-7.2). After reformulation, the protein concentration was measured using Bradford method and the amount of free thiol groups was determined by Ellman method (Ellman, G.L. (1958) Arch. Biochem. Biophys. 74, 443-450). On average, slightly more than 2-3 cysteines per one protein molecule was obtained.
  • Conjugation reaction was carried out as follows. The moiety chemical compound- linker L7-Z1 was dissolved in dimethylacetamide at a concentration 200 fold higher relative to the amount of free thiol groups. Then, the prepared mixture was diluted 10-fold in a 30% aqueous solution of (2-hydroxyethyl)-6-cyclodextrin. Immediately after preparation, the solution was added to the reduced protein, so as to obtain final concentration of the L7-Z1 moiety equal 2 times of the concentration of free thiol groups. Conjugation reaction was carried out for 1 .5h at 20°C, until complete substitution of thiol groups was achieved, as monitored by DTNB reagent (Ellman method).
  • N -acetyl- L-cysteine was added to a final concentration of 400 ⁇ . Excess of the compound was removed by overnight dialysis into a buffer (5mM NaH2P0 4 , 95mM Na2HP0 4 , 200mM NaCl, 5mM glutathione, 0, 1 mM ZnC , 10% v/v glycerol, 80 mM sucrose, pH 7,0) with addition of 2,5mM (2-hydroxyethyl)-6- cyclodextrin.
  • a buffer 5mM NaH2P0 4 , 95mM Na2HP0 4 , 200mM NaCl, 5mM glutathione, 0, 1 mM ZnC , 10% v/v glycerol, 80 mM sucrose, pH 7,0
  • Table 4 shows the results of mass analysis for the carrier (fusion) protein of SEQ. ID No. 24 and for the conjugates consisting of carrier (fusion) protein of SEQ. ID No. 24 linked with compound Z1 via linkers L7, L1 , L6 and L14, respectively.
  • Electrophoretic analysis In order to confirm the structure, the conjugate was analyzed by SDS-PAGE. Electrophoresis was carried out in the Laemmli system in Mini-PROTEAN Tetra Cell apparatus, using 15% acrylamide gels which then were analyzed using UV shadowing method (according Hassur SM, Analytical Biochemistry, Volume 59, Issue 1 , May 1974, 162-164), followed by Coomassie staining. This analysis confirmed the attachment of the L2-Z1 moiety to the SEQ. ID No. 20 by the observed phenomenon of UV absorption and by differences of obtained bands heights of the fusion carrier protein and the respective conjugate.
  • conjugates of the invention in particular conjugates of the fusion protein of SEQ. ID No. 24 with moieties L7-Z1 , L1 -Z1 , L3-Z1 , L4-Z1 , L5- Z1 , L6-Z5, L7-Z5, L7-Z22 L14-Z1 , and L12-Z43, were obtained and analyzed analogously.
  • Example 5 Determination of the aggregation degree of fusion proteins and conjugates thereof
  • Cell culture medium was diluted to a defined density (10 4 - 10 5 cells per 100 ⁇ ). Then 100 ⁇ 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 10% CO2, depending on the medium used, and then to the cells (in 100 ⁇ of medium) further 100 ⁇ of the medium containing various concentrations of tested conjugates and its constituents were added.
  • the medium with tested conjugates and its constituents was added with 20 ⁇ of MTT working solution [5 mg/ml], and incubation was continued for 3 h at 37° C in 5% CO2. Then the medium with MTT solution was removed, and formazan crystals were dissolved by adding 100 ⁇ of DMSO. After stirring, the absorbance was measured at 570 nm (reference filter 690 nm).
  • results of in vitro cytotoxicity assays for selected conjugates, the fusion protein being its component, a mixture of the components of the conjugate according to the invention against a panel of tumor cells derived from different organs, corresponding to the most frequently occurring cancers, are shown in Table 4 as IC50 values (ng/ml), which corresponds to a concentration of conjugate at which the 50% cytotoxic effect is observed compared to the control cells.
  • IC50 values confirm high cytotoxic activity of conjugates and thus potential utility in treatment.
  • Each experiment represents the average value of at least two independent experiments performed in triplicates.
  • the IC50 limit of 2000 ng/ml was adopted. Conjugates and its constituents 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.
  • IC50 values above 2000 obtained for the non-cancer cell lines show the absence of toxic effects associated with the use of conjugates of the invention for healthy cells, which indicates potential low systemic toxicity of the conjugate.
  • Table 4 presents the results of the tests of cytotoxic activity in vitro for conjugates of the invention against a broad panel of tumor cells from different organs, corresponding to the broad range of most common cancers.
  • the experimental results are presented as a mean value ⁇ standard deviation (SD). All calculations and graphs were prepared using the GraphPad Prism 5.0 software. Obtained IC50 values confirm high cytotoxic activity of conjugates and thus their potential utility in the treatment of cancer.
  • TR114-281 >2000 25.57 0.93 >2000 5.1 1 .74 11 .21 1 .62
  • the human lung cancer A549 cells and human lung cancer were maintained in RPMI1640 medium (human lung cancer A549 and H69AR) or McCoy's medium (SKOV3) supplemented with 10% fetal calf serum in standard conditions: 37 ° C, 5% CO2, 95% humidity.
  • RPMI1640 medium human lung cancer A549 and H69AR
  • McCoy's medium SKOV3
  • 10% fetal calf serum in standard conditions: 37 ° C, 5% CO2, 95% humidity.
  • 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 a 3: 1 mixture of HBSS:Matrigel (BD Bioscience).
  • mice Examination of antitumor activity of proteins of the invention was conducted on 4-6 week-old Crl:SHO-Prkdc sc,d Hr hr 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. 71 /2009).
  • mice were grafted subcutaneously (sc) in the right side with 7x10 6 (Experiments A and B) of A549 cells suspended in 0.1 ml mixture of HBSS:Matrigel.
  • tumours reached the size of 180 mm 3 'Experiment A) or 200 mm 3 (Experiment B) mice were randomized to obtain the similar average size of tumours in the group and assigned to treatment groups.
  • the treatment groups were administered i.v. in a schema q2dx12 (i.e. administration every second day, totally 12 administration, Experiment A) or q2dx6 (i.e. administration every second day, totally 6 administrations, Experiment B) with the preparations of conjugate SEQ. No.
  • 24-L7-Z1 of the invention (50 mg/kg), calculated on the protein), wherein the concentration of the linked SN38 compound (Z1 ) was 3.7 mg/kg.
  • the conjugate constituents i.e. fusion protein of SEQ. ID No. 24 and chemical compound Z1
  • fusion protein of SEQ. ID No. 24 was administered i.v. alone at 90 mg/kg (Experiment A) and at 50 mg/kg (Experiment B).
  • Formulation buffer (50mM Trisma-base, 200 mM NaCl, 5 mM glutathione, 0.1 mM ZnC , 10% glycerol, 80 mM saccharose, pH 8.0) was administered ; ' .v as a control.
  • Paclitaxel (Paclitaxin, Teva, 12 mg/kg /.v., q1dx5, everyday administration, totally 5 administrations) was used as a comparative reference.
  • the experimental results obtained in mice Crl:SHO-Prkdc sc,d Hr hr burdened with A549 lung cancer treated with conjugate of the invention (SEQ. No. 24-L7-Z1 ) and comparatively with the mixture of its constituents (i.e.
  • fusion protein of SEQ. ID No. 24 and chemical compound Z1 ) as well as with fusion protein of SEQ. ID No. 24 and with a reference compound (paclitaxel) alone are shown in Fig. 1 (Experiment A) and Fig. 3 (Experiment B) as a diagram of changes of the tumour volume.
  • Fig. 2 (Experiment A) and Fig. 4 (Experiment B) show tumour growth inhibition (%TGI) as the percentage of control.
  • TGI% value obtained for the reference compound (paclitaxel) was on the level of 53% (Experiment A) and 61% (Experiment B).
  • conjugate SEQ. ID No. 24-L7-Z1 of the invention exerts a much stronger tumour inhibition effect compared to the mixture of its constituents and the fusion protein of SEQ. ID No. 24 administered in an increased dose.
  • mice On day 1 mice were grafted subcutaneously (sc) in the right side with 5x10 6 of H69AR cells suspended in 0.1 ml mixture of HBSS:Matrigel. When tumours reached the size of 200 mm 3 (day 20), mice were randomized to obtain the similar average size of tumours in the group and assigned to treatment groups.
  • the treatment groups were administered i.v. in a schema q2dx6 (i.e. administration every second day, totally 6 administrations) with the preparations of conjugates SEQ. No. 24-L7-Z1 and SEQ. ID No.
  • 24-L14-Z1 of the invention (50 mg/kg), calculated on the protein), wherein the concentration of the linked SN38 compound (Z1 ) was 3.7 and 11.1 mg/kg, respectively.
  • the conjugate constituents i.e. fusion protein of SEQ. ID No. 24 and chemical compound Z1
  • fusion protein of SEQ. ID No. 24 was administered i.v. alone at a dose of 50 mg/kg.
  • Formulation buffer (50mM Trisma- base, 200 mM NaCl, 5 mM glutathione, 0.1 mM ZnC , 10% glycerol, 80 mM saccharose, pH 8,0) was administered i.v as a control.
  • mice Crl:SHO-Prkdc sc,d Hr hr burdened with H69AR lung cancer treated with conjugates of the invention SEQ. No. 24-L7-Z1 and SEQ. ID No. 24-L14-Z1
  • Fig. 5 shows a diagram of changes of the tumour volume
  • Fig. 6 which shows tumour growth inhibition (%TGI) as the percentage of control.
  • 24-L14-Z1 of the invention exert a much stronger tumour inhibition effect compared to the mixture of its constituents and the fusion protein of SEQ. ID No. 24.
  • mice were grafted subcutaneously (sc) in the right side with 3x10 6 of SKOV3 cells suspended in 0.1 ml mixture of HBSS:Matrigel.
  • tumours reached the size of 166 mm 3 (day 36)
  • mice were randomized to obtain the similar average size of tumours in the group and assigned to treatment groups.
  • the treatment groups were administered i.v. in the schema q2dx6 (i.e. administration every second day, totally 6 administrations) with the preparations of conjugate SEQ. No.
  • 24-L7-Z1 of the invention (50 mg/kg), calculated on the protein), wherein the concentration of the linked SN38 compound (Z1 ) is 3.7 mg/kg.
  • the conjugate constituents i.e. fusion protein of SEQ. ID No. 24 and chemical compound Z1
  • fusion protein of SEQ. ID No. 24 was administered alone i.v. at the dose of 50 mg/kg.
  • Formulation buffer (50mM Trisma-base, 200 mM NaCl, 5 mM glutathione, 0.1 mM ZnC , 10% glycerol, 80 mM saccharose, pH 8,0) was administered ; ' .v as a control.
  • Paclitaxel (Paclitaxin Teva, 12 mg/kg i.v. , q1dx5, everyday administration, totally 5 administrations) and carboplatin (25 mg/kg /.p., q3dx6, every third day administration, totally 6 administrations) combination (as separate administrations) were used as a comparative reference.
  • mice Crl:SHO-Prkdc sc,d Hr hr burdened with SKOV3 lung cancer treated with the conjugate of the invention SEQ. No. 24-L7- Z1
  • the conjugate of the invention SEQ. No. 24-L7- Z1
  • Fig. 7 shows a diagram of changes of the tumour volume
  • Fig. 8 which shows tumour growth inhibition (%TGI) as the percentage of control.
  • TGI [%] (Tumour growth inhibition) (WT/WC) x 100 - 100% wherein WT refers to the average tumour volume in the treatment group, WC refers to the average tumour volume in the control group.

Abstract

L'invention concerne un conjugué anticancéreux qui comprend une protéine de fusion comprenant un domaine (a) qui est le fragment fonctionnel d'une séquence de la protéine TRAIL humaine soluble (hTRAIL) commençant par un acide aminé à une position non inférieure à hTRAIL95 ou une séquence ayant au moins 70 % d'identité avec ledit fragment fonctionnel, le domaine (b) qui est la séquence d'un peptide effecteur ayant une activité proapoptotique, antiangiogénique, antiproliférative ou de formation des pores, et un domaine de conjugaison (d) destiné à la fixation d'un composé chimique choisi dans le groupe consistant en les séquences Cys Ala Ala Ala Cys Ala Ala Cys et Cys Ala Ala Cys Ala Ala Ala Cys, et une molécule d'un composé chimique Z ayant une activité antiblastique, qui est fixée audit domaine de conjugaison (d) de ladite protéine de fusion directement ou par l'intermédiaire d'un lieur de conjugaison L.
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WO2017191252A1 (fr) * 2016-05-04 2017-11-09 Navigo Proteins Gmbh Composés ciblés pour le couplage spécifique d'un site de fractions chimiques comprenant un lieur peptidique
EP3266796A4 (fr) * 2015-03-02 2018-02-21 Chengdu Huachuang Biotechnology Co., Ltd Mutant mur5 de type peptidique pénétrant dans la membrane de trail, son procédé de préparation et son application
US9938323B2 (en) 2014-11-06 2018-04-10 Novartis Ag Amatoxin derivatives and conjugates thereof as inhibitors of RNA polymerase
US10464969B2 (en) 2016-05-05 2019-11-05 Novartis Ag Amatoxin derivatives and conjugates thereof as inhibitors of RNA polymerase
WO2020130838A3 (fr) * 2018-12-21 2020-07-30 Qvq Holding B.V. Anticorps pour la prévention ou le traitement de la candidose
CN112724258A (zh) * 2019-10-29 2021-04-30 深圳市第二人民医院 靶向杀死癌细胞的复合多肽分子及其制备方法

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US9938323B2 (en) 2014-11-06 2018-04-10 Novartis Ag Amatoxin derivatives and conjugates thereof as inhibitors of RNA polymerase
EP3266796A4 (fr) * 2015-03-02 2018-02-21 Chengdu Huachuang Biotechnology Co., Ltd Mutant mur5 de type peptidique pénétrant dans la membrane de trail, son procédé de préparation et son application
WO2017191252A1 (fr) * 2016-05-04 2017-11-09 Navigo Proteins Gmbh Composés ciblés pour le couplage spécifique d'un site de fractions chimiques comprenant un lieur peptidique
CN109310780A (zh) * 2016-05-04 2019-02-05 纳维格蛋白质有限公司 包含肽接头的用于化学部分位点-特异性偶联的靶向化合物
US10464969B2 (en) 2016-05-05 2019-11-05 Novartis Ag Amatoxin derivatives and conjugates thereof as inhibitors of RNA polymerase
US10604547B2 (en) 2016-05-05 2020-03-31 Novartis Ag Amatoxin derivatives and conjugates thereof as inhibitors of RNA polymerase
WO2020130838A3 (fr) * 2018-12-21 2020-07-30 Qvq Holding B.V. Anticorps pour la prévention ou le traitement de la candidose
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