WO2014057436A2 - Anticancer conjugate - Google Patents

Abstract

A conjugate comprises a fusion protein comprising domain (a), which is the functional fragment of a hTRAIL protein sequence, said fragment beginning 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 domain (b) which is the sequence of an immunostimulating effector peptide; and the molecule of a chemical compound Z with antiblastic activity, which is linked to the fusion protein directly or by means ofa conjugation linker L. the conjugate is useful in the treatment of cancer diseases.

Description

Anticancer conjugate

The invention relates to the field of therapeutic conjugates comprising a fusion protein, in particular recombinant protein. Specifically, the invention relates to conjugates of a fusion protein, which comprises a sequence derived from a soluble human TRAIL protein linked to the sequence of a short peptide stimulating the immune system, with the molecule of a chemical compound having antiblastic activity, pharmaceutical compositions containing these conjugates as well as their use in therapy, particularly as antitumor agents.

WO2012072815 describes fusion proteins comprising a functional fragment of the human soluble TRAIL protein sequence linked with the sequence of a short peptide which stimulates the immune system, including various types, variants, fragments and analogs of alpha and gamma interferons, including their mono- and dimeric forms. In animal models of various types of cancers, including TRAIL- resistant tumours, the beneficial effect of such fusion proteins was demonstrated, namely that their use leads to the reduction of tumour cell proliferation and thus to the inhibition of tumour growth. However, this does not ensure the effectiveness in the case of such types of cancer, where the use of a fusion protein comprising TRAIL and an immune system stimulating peptide, such as variants of interferons, does not produce desired effects. This is especially the case when, due to the advanced stage of tumour growth, the inhibition rate is not sufficient for remission of disease symptoms.

The known approach to cancer therapy is chemotherapy. Also described are 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. However, such 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. However, the main drawback of chemotherapy is a systemic toxicity. For this reason, in many cancer indications, effective forms of therapy are lacking. For a more effective therapy it is desirable, therefore, to reduce systemic toxicity while increasing the pharmacological potential of the therapy. For this reason, a multi-component therapy, in particular targeted therapy, to prevent the side effects can be a solution which ensures effective elimination of cancer cells.

Therefore, despite the existence of clinically applied anticancer therapies based on both TRAIL protein and immunostimulating proteins from the interferons family as well as therapies based on chemical compounds, there is still a need for an improved antitumor therapy which would be both effective and selective in vivo with respect to cancer cells. There is still an urgent and unmet need for new anti -cancer agents with higher effectiveness (cytotoxicity) and selectivity. There is also a need for new selective agents with improved stability and pharmacokinetics. The present invention proposes to solve this problem and meet this need by the conjugates of the invention, in which fusion protein comprising a functional fragment of TRAIL protein and a short effector peptide with immune system stimulating activity is attached to a chemical molecule with antiblastic activity, which molecule amplifies or supplements the action of the fusion protein. It has been found that in many cases 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. Additionally, in many cases, new conjugates overcome natural or induced resistance to TRAIL. Furthermore, the addition of a chemical compound increases the weight of the protein, resulting in prolonged half-life, increased retention in the tumour and increased efficiency of the protein.

The present invention overcomes the limitations of known therapies using low- molecular weight compounds, their mixtures, and/or conjugates with proteins or peptides. In particular, 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 emerging during therapy. The conjugation of a cytostatic with an active anti-cancer 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. Delivery of the drug allows to circumvent these limitations and to increase the effectiveness of a therapy.

Description of Figures.

The invention will now be described in detail with reference to the Figures of the drawing.

Fig. 1 presents tumour volume changes (% of initial stage) in Crl:SHO- Prkdc^sad r^hr mice burdened with multidrug resistant human uterine sarcoma MES-SA/Dx5, treated with conjugates of the invention according to Example 3 compared to the conjugate constituents individually, the mixture of the conjugate constituents and the reference compound CPT-1 1 .

Fig. 2 presents the tumour growth inhibition values (%TGI ) in Crl:SHO- Prkdc^sad r^hr mice burdened with multidrug resistant human uterine sarcoma MES-SA/Dx5, treated with conjugates of the invention according to Example 3 on the 29^th day of experiment, compared to the conjugate constituents individually, the mixture of the conjugate constituents and the reference compound CPT-1 1 .

Fig. 3 presents tumour volume changes (% of initial stage) in Crl:SHO- Prkd ^adHi ^ir mice burdened with multidrug resistant human uterine sarcoma MES-SA/Dx5, treated with conjugates of the invention according to Example 3 compared to the conjugate constituents individually, the mixture of the conjugate constituents and the reference compound CPT-1 1 .

Fig. 4 presents the tumour growth inhibition values (%TGI ) in Crl:SHO- Prkd ^adHi ^ir mice burdened with multidrug resistant human uterine sarcoma MES-SA/Dx5, treated with conjugates of the invention according to Example 3, compared to the conjugate constituents individually, the mixture of the conjugate constituents and the reference compound CPT-11.

Detailed Description of the Invention

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 hTRAIL protein beginning 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 domain (b) which is a sequence of an immunostimulating effector peptide,

and

• the molecule of a chemical compound Z with antiblastic activity, which is bound to the fusion protein directly or via a conjugation linker L.

The sequence of the domain (b) in the fusion protein, which is the constituent of the conjugate of the invention, is attached at the C-terminus and/or N-terminus of domain (a).

It should be understood that domain (b) of the immunostimulating effector peptide in the fusion protein of the invention is neither hTRAIL protein nor a part or fragment of hTRAIL protein.

The term "the functional soluble fragment of a sequence of soluble 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.

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 peptide bonds. Thus, the term "peptide" according 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, 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.

In accordance with the present invention, by the conjugate it is meant a single molecule of 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 this molecule may be bound directly or by an appropriate linker enabling the conjugation (conjugation linker).

Preferably, the conjugate according to the invention is a fusion protein molecule with attached one molecule of a chemical compound, wherein the molecule is attached via an appropriate conjugation linker L, and the fusion protein molecule comprises a conjugation domain (d), comprising an attachment site for the molecule of a chemical compound.

The fusion protein in the conjugate of the invention may comprise at least one domain (b) of the effector peptide attached to the C-terminus or N-terminus of the domain (a).

In a particular embodiment, domain (a) of the fusion protein is a fragment of hTRAIL sequence, beginning with an amino acid in the position starting from the position 95 of hTRAIL (hTRAIL95), particularly in a 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 ).

In particular, said domain (a) may be selected from the group consisting of sequences corresponding to hTRAIL95-281 , hTRAIL114-281 , hTRAIL116-281 , hTRAILI 20-281 , hTRAILI 21 -281 and hTRAILI 22-281. It will be evident to those skilled in the art that hTRAIL95-281 , hTRAIL114-281 , hTRAIL116-281 , hTRAILI 20- 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, 116, 119, 120, 121 and 122 respectively and ending with amino acid marked with a number 281 , in the known sequence of hTRAIL (SEQ. No. 1 ) published in GenBank under Accession No P50591.

Thus, the skilled person will appreciate that the term hTRAIL with a number or a range of numbers refers to an amino acid or amino acid sequence in the position designated with this number in a known sequence of hTRAIL.

In a particular embodiment, said domain (a) is the homologue of a functional fragment of soluble hTRAIL protein sequence beginning with an amino acid at the position in a range from hTRAIL95 to hTRAIL122 inclusive, and ending with amino acid an amino acid at the position hTRAIL281 , or a homologue of such hTRAIL fragment the sequence of which is at least in 70%, preferably in 85%, identical to this fragment.

In another particular embodiment, said domain (a) is the homologue of a functional fragment of soluble hTRAIL protein sequence beginning with an amino acid in the position not lower than hTRAIL95, and ending with an amino acid hTRAIL281 , the sequence of which is at least in 70%, preferably in 85%, identical to this fragment.

It will be appreciated by a skilled person that 70% sequence homologues of hTRAIL are known in the prior art.

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 immunostimulating effector peptide of domain (b) may be a cytokine peptide which among others intensely stimulates human monocytes to produce TRAIL protein, thus significantly affecting the ability to eliminate cancer cells.

In one embodiment of the invention, the cytokine is interferon, in particular interferon alpha, including interferon alpha 2b or interferon gamma, their fragments, variants and analogues, both in monomeric and dimeric form.

In one of the embodiments of the invention, the effector peptide of the fusion protein is selected from the group consisting of:

- an interferon alpha 2b fragment set forth as SEQ. No. 2; - an interferon gamma fragment set forth as SEQ. No. 3; - an interferon gamma pseudodimer set forth as SEQ. No. 4;

- an interferon alpha 2b pseudodimer set forth as SEQ. No. 5; and

- a consensus sequence of interferon alpha set forth as SEQ. No. 6.

In one of the embodiments of the invention, domain (a) and domain (b) are linked by means of domain (c) situated between them, 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. Gordon et all. in Inf. and Immun, 1995, 63, No. 1 ,p. 82-87, or its fragment which with the terminal amino acid of a sequence to which it is attached forms a sequence recognized by furin; and their combinations.

In one of the embodiments of the invention, 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.

Preferably, in one embodiment, domain (c) recognized by proteases overexpressed 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.

In the case of absence of the domain (c), 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 fusion protein 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.

For a skilled person it is apparent that the fusion protein in the conjugate of the invention is a carrier protein for the molecule of a chemical compound with antiblastic activity, and in addition to its own therapeutic function acts also as a carrier of a chemical compound directly to the target cell, where it is released.

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.

At the same time, the structure of conjugate ensures that after cleavage by proteases receptor binding properties are unchanged comparing to the unconjugated forms of conjugate constituents.

As stated above, the molecule of a chemical compound Z with antiblastic activity may be attached to the fusion protein 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 bond. Preferably, the compound is linked to the fusion protein via a stable bond, particularly preferably a covalent bond.

Preferably, the molecule of a chemical compound Z with antiblastic activity may be attached to the protein via formation of a stable bond, in particular a covalent bond, directly between the fusion protein and the chemical compound, or between 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 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.

Alternatively, if the molecule of a chemical compound Z is attached via a conjugation linker L, 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.

In one embodiment of the invention, between domain (a) and domain (b) of a fusion protein additionally and independently of domain (c) there is incorporated conjugation domain (d), said domain (b) comprising a sequence destined for attachment (conjugation) of the molecule of a chemical compound to the fusion protein via formation of a stable bond. This sequence contains functional groups capable of forming a bond with a chemical compound, such as sulfhydryl (SH), amino, or carboxyl groups.

This variant 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 variant is also sterically beneficial.

Preferably, conjugation domain (d) having the sequence enabling the attachment of the molecule of a chemical compound to the fusion protein contains cysteine residues with sulfhydryl groups, and therefore in the following description may be also referred to as "a cysteine linker".

The presence of a 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 capable to react with sulfhydryl group. By "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 a number of molecules of the fusion protein (homodimers and homotrimers), said disulfide bonds being necessary for the activity of the protein.

As a sequence of conjugation domain (d) may be used, for example, known sequence suitable for attachment of a PEG molecule to peptides, referenced also as pegylation linker or PEG-linker.

Said conjugation domain (d) comprising a sequence for attachment of the molecule of a chemical compound to a fusion protein can be for example known pegylation sequence Ala Ser Gly Cys Gly Pro Glu (ASGCGPE in a one-letter convention), known pegylation sequence Ala Ala Cys Ala Ala (AACAA in a one- letter convention), or known pegylation sequence Ser Gly Gly Cys Gly Gly Ser (SGGCGGS in a one-letter convention). Such sequences are described as pegylation sequences used in fusion proteins disclosed in WO2012072815.

Preferably, the fusion proteins of the conjugate of the invention possess a free cysteine residue only in the conjugation domain (d).

Particularly preferably, the fusion proteins which are constituents of the conjugates of the invention possess one free cysteine residue in the conjugation domain (d). The use of conjugation domain (d) having one cysteine residue, in the absence of other free cysteine residues within the domains of the fusion protein, enables the attachment of preferably one molecule of a chemical compound.

This allows indirect qualitative determination of the composition of the conjugate product on the basis of its quantitative analysis, i.e. on the basis of the increase of the protein weight, which typically is determined by mass spectroscopy. This approach ensures the homogeneity of the produced conjugate, which in such a case possess always 1 molecule of the fusion protein per 1 molecule of the chemical compound attached at a strictly determined location.

In yet another embodiment of the conjugate, the conjugation domain (d) of the fusion protein does not contain any cysteine residue. Conjugation domain (d) of the fusion protein may not contain a cysteine residue, for example, if a domain (a) or (b) of the fusion protein possess a free cysteine residue allowing the attachment of the molecule of a chemical compound. In such a case, the conjugation domain (d) may act as a spacer. Domain (d) of the fusion protein can not contain a cysteine residue also in the case when for the attachment of a chemical compound functional groups other than a sulfhydryl group are used, such as for example amino group.

In one of the embodiments, the protein of the invention comprises both domain or domains (c) and conjugation domain (d).

In a preferred embodiment, conjugation domain (d) is located between two domains (c), in particular between two domains (c), which are selected from the protease cleavage site and a combination of protease cleavage sites, in particular, the sequence recognized by metalloprotease MMP, such as described above, and the sequence recognized by urokinase uPa, such as described above, and combinations of MMP/uPa.

Thus, in one embodiment of the fusion protein of the invention, the protease cleavage site is a combination of the sequence recognized by metalloprotease MMP and the sequence recognized by urokinase uPA, in any order, separated by sequence (d) of the conjugation domain.

It should be understood that in the case when the fusion protein has both the conjugation domain (d) containing a sequence for conjugation with the molecule of a chemical compound and the domains (c) of the cleavage site between the domains (a) and (b), then two domains (c) are located in such a way that after cleavage of the construct the conjugation domain (d) is disconnected from the domains (a) and (b). These two domains (c) may contain both single protease cleavage site and combinations thereof, as defined above. In other words, if the fusion protein contains both conjugation domain (d) and cleavage site domains (c), then domain (d) is located between the domains (c). The invention does not comprise such a variant in which domain (d) would be located between domain (c) and domain (a) or between domain (c) and domain (b), that is the variant wherein after cleavage of the construct domain (d) with attached chemical compound molecule remains attached to domain (a) or domain (b).

Besides main functional elements of the fusion protein (i.e. domains (a) and (b)), the cleavage site domain(s) and the conjugation domains, the fusion proteins of the invention may contain a neutral sequence or sequences of a flexible steric glycine-serine 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 produced by the process of its overexpression in the host cells.

In particular, flexible linker may be selected from the group consisting of sequences Gly Ser Gly Gly Gly, Gly Gly Gly Ser and Xaa Gly Gly Ser, wherein Xaa designates any amino acid or is absent.

A detailed description of the construction of the above mentioned representative fusion proteins, serving as a carrier protein in the conjugate of the invention, is presented in WO/2012/072815 in Figures 1 to 3 and in the specific embodiments.

In a preferred embodiment of the invention, the sequence of a fusion protein is selected from the group consisting of 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, and SEQ. No. 23.

More preferably, the sequence of a fusion protein being a carrier in a conjugate molecule is selected from the group consisting of SEQ. No. 20, SEQ. No. 21 ; SEQ. No. 22, and SEQ. No. 23.

Methods of synthesis of fusion proteins useful as carriers in the conjugate according to the present invention (the transformation and overexpression procedure of fusion proteins, nucleotide sequences and useful vectors and host cells) are apparent to the skilled in the art as such and are described in WO2012072815, along with the specific embodiments.

The fusion protein in the conjugate of the invention will have a dual function. In addition to antitumor activity, such as described for the fusion proteins in WO2012072815, 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).

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.

As used herein, 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 SN38, for example, irinotecan, afletecan, belotecan, or topotecan.

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.

The chemical compound - a nucleoside analog, may be in particular selected from the group consisting of pyrimidine nucleoside analogs with a free NH₂ group at the position 4 of the heterocyclic ring, such as cytarabine, gemcitabine, dectabine, 1 -(3-C-ethynyl-6-D-ribo-pentofuranosyl)cytosine (ECyd), 1 -(2-deoxy- 2-fluoro-4-thio-6-D-arabino-pentofuranosyl)cytosine (4'-thio-FAC), 1 -(2-C-cyano- 2-deoxy-1 -6-D-arabino-pentofuranosyl)cytosine (CNDAC), [1 -(2-deoxy-2- methylene-6-D-erythro-pentofuranosyl)cytosine] (DMDC), emtricitabine (FTC), lamivudine, zalcitabine (ddC), apricitabine (ATC) and 4-methylamino-1 -(β-D- arabinofuranosyl)-pyrimidin-(1 H)-one, and purine nucleoside analogues containing free NH₂ the position 6 or 2 of a heterocyclic ring, such as clofarabine, fludarabine, vidarabine and entecavir. 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.

The chemical compound with an activity of DNA and RNA synthesis inhibitor RNA can be especially selected from the group consisting of: melphalan, chlorambucil, cyclophosphamide, busulfan, and streptozotocin.

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, 46-amino-4'-0-4- deoxydemethylepipodophylotoxin, ustiloxine A, and rizoxine.

The chemical compound of the proteasome inhibitor activity can be in particular selected from the group consisting of salinosporamine A, 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 activity can be in particular selected from the group consisting of brefeldin A, hypocreline A, staurosporine, and verbascozide.

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 1 -deoxyandrographolide.

Preferably, the chemical compound with antiblastic activity is selected from the group consisting of the compounds Z1 to Z24 presented in Table 1.

Table 1

The chemical compound, depending on its structure, can be attached to the fusion protein directly. In particular, in such embodiment, a functional group of the chemical compound is condensed to a suitable reactive group of the fusion protein. The fusion protein reactive group can be a primary N-terminal amino group. Particularly, such a variant is possible for 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 (Z11 ). Such a solution, however, is less favourable, 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. In particular, 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.

In another preferred embodiment of the conjugate, the chemical compound Z is attached to the fusion protein by conjugation linker L. By 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. It is also possible to use for coupling of chemical compounds to proteins cleavage sites recognized by proteases overproduced in the tumour environment such as MMP2/9 (as described for example in US7803903). Conjugation method used 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.

Methods for modification of amino acid residues of peptides and proteins as well as introduction of specific functional groups that can be used to attach chemical molecules and other linkers used for the conjugation of mono- and poly- functional linkers are known in the art and exemplified by G. Hermanson in „Bioconjugate techniques" Academic Press Inc. 2nd Ed. (2008), or by S. S. Wong in„Chemistry of protein conjugation and cross-linking" CRC Press 1991 . Preferably, 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.

Preferably, 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.

Preferably, the maleimide linker may comprise polyethoxylated (PEG) moiety attached to the maleimide moiety. By polyethoxylated moiety are meant moieties which contain more than two oxyethylene units, as well as oligoethoxylated moieties. 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. Especially useful are 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. The values of these parameters depends on the structure of polyethylene glycol molecules: the chain length, linearity, the degree of branching, type and amount of binding sites and glycol molecules attached. [Delgado C, Francis GE, Fisher D. The uses and properties of PEG-linked proteins. Crit Rev Ther Drug Carrier Syst 1992; 9 (3-4): 249-304]. The use of polyoxyethylated moiety in the linker for conjugation of the chemical compound does not affect the interaction of the conjugate components with receptors. Maleimide linker provides a stable connection of the chemical compound with the fusion protein so that the chemical compound is not released outside the tumour environment. Furthermore, preferably, a conjugation linker should not affect the conformation, activity and receptor specificity of the protein to which it is attached. Examples of preferred maleimide linkers for conjugation to chemical compounds are shown below by formulas L1 to L12 in Table 2. In these formulas, the dashed line indicates the point of attachment to the chemical compound. Before conjugation, linker has a hydroxy group (linkers L1 to L10 and L12) or hydrogen atom (linker L1 1 ) in the place of the dashed line. Table 2

Li o ho^°^o ^ ^

An important feature of the linker for conjugation of the chemical compound should be a stable connection chemical compound - linker under physiological conditions, beside the tumour environment. In the case of different 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. These disadvantages have been overcome by using previously unknown maleimide linker represented by the formula L7. This linker, due to the presence of para-aminobenzoic acid (PABA) residues, is characterized by a particular stability in vitro and in vivo when used as a component of a conjugate, . Therefore, the present invention relates to the compound 4-[({trans-4-[(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)methyl]cyclohexyl}carbonyl)amino]benzoic acid represented by the following structure:

The present invention relates also to a method of attaching a chemical compound to a peptide or protein to form a conjugate, wherein the chemical compound is attached to the peptide or protein by means of 4-[({trans-4-[(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)methyl]cyclohexyl}carbonyl)amino]benzoic acid as a conjugation linker.

By the method of the invention, previously synthesized 4-[({trans-4-[(2,5-dioxo- 2, 5-dihydro-1 H-pyrrol-1 -yl)methyl]cyclohexyl}carbonyl)amino]benzoic acid is attached to the chemical compound by coupling hydroxy or amino group of a chemical compound to carboxyl group, to form corresponding ester or amide derivative of 4-[({trans-4-[(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)methyl]cyclo- hexyl}carbonyl)amino] benzoic acid, respectively. Then, the resulting ester or amide derivative of the chemical compound is coupled to the sulfhydryl group of the peptide or protein, optionally after prior reduction of disulfide bonds of the peptide or protein.

For coupling 4-[({trans-4-[(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)methyl]cyclo- hexyl}carbonyl)amino] benzoic acid to chemical compounds containing amino group or carboxyl group to form the corresponding amide or ester, methods can be applied which are known per se and are described, for example, in monographs G. Hermanson w„Bioconjugate techniques" Academic Press Inc. 2nd Ed. (2008), and S. S. Wong„Chemistry of protein conjugation and cross-linking" CRC Press 1991.

Analogously, for coupling a chemical compound to a protein or peptide by means of a maleimide linker via reaction of maleimide group from the linker attached to a chemical compound, methods can be used which are known per se and are described, for example, in monographs G. Hermanson w „Bioconjugate techniques" Academic Press Inc. 2nd Ed. (2008) i S. S. Wong „Chemistry of protein conjugation and cross-linking" CRC Press 1991.

4-[({trans-4-[(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)methyl]cyclohexyl}carbonyl)- amino] benzoic acid (linker L7) can be obtained according to the following scheme:

Detailed description of the synthesis of linker L7 is shown below in the Examples.

An advantage of the linker L7 is that it can be produced as an individual molecule and then attached to the chemical compound in one chemical reaction, without the need for gradual building of the linker structure directly on the chemical compound.

In a preferred embodiment, the chemical compound Z is attached through the linker L to the conjugation domain (d) of the fusion protein. Preferably, domain (d) comprises free cysteine residues, which are not involved in the formation of a proper fusion protein structure, and are capable of reacting via sulfhydryl groups with linker L groups, preferably with its maleimide residues, to form a thioether bond. In the absence of the conjugation domain (d) in the conjugate, it is also possible to attach the chemical compound through the linker L to free sulfhydryl groups of the fusion protein.

In such cases, the ratio chemical compound/fusion protein can take values >1 .

The conjugate is prepared by attaching the moiety chemical compound Z - linker L to the fusion protein.

Joining (conjugating) the fusion proteins and analysis of the resulting conjugates of the chemical compounds can be carried out according to procedures known in the art as such (Morpurgo, M, et al. (1996) Bioconjugate Chem. 7, 363-368, Wong SS et al. , Chemistry of Protein Conjugation And Cross-Linking. CRC Press, 1991 ; Hermanson GT, Bioconjugate Techniques. Academic Press, Inc. , 2^nd Ed. (2008); Ellman, G.L. A colorimetric method for determining low concentrations of mercaptans (1958) Arch. Biochem. Biophys. 74, 443-450; Hassur, SM et al. UV shadowing-A new and convenient method for the location of ultraviolet- absorbing species in polyacrylamide gels. Analytical Biochemistry, Volume 59, Issue 1 , May 1974, Pages 162-164), and described below in details.

Preparation of the compounds for conjugation with the fusion protein - formation of moieties chemical compound Z - linker L.

The chemical compounds are obtained from commercial sources or by chemical synthesis methods known in the art.

In the case when the chemical compound Z is attached via a linker L, first prepared is the moiety chemical compound Z - linker L.

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.

Generally, in the practice of the present invention, 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). For this purpose, a typically used coupling agent is dicyclohexylcarbodiimide, optionally in the presence of a catalytic amount of dimethylaminopyridine (DMAP), in a suitable solvent such as methylene chloride, at ambient temperature for 48 hours. Following this procedure the following moieties chemical compound Z - linker L can be obtained: L1 -Z1 , L1 -Z2, L1 -Z3, L9-Z4, L1 - Z5, L1 -Z6, L1 -Z7, L1 -Z8, L9-Z8, L1 -Z9, L9-Z9, L1 -Z10, L9-Z10, L1 -Z11 , L9-Z11 , L1 -Z12, L9-Z12, L1 -Z13, L9-Z13, L1 -Z14, L9-Z14, L1 -Z15, L9-Z15, L1 -Z16, L9-Z16, L9-Z17, L1 -Z17, L1 -Z18, L9-Z18, L1 -Z19, L9-Z19, L10-Z20, L9-Z21 , L1 -Z22, L9- Z22, L9-Z23, L1 -Z23, L1 -Z24, L9-Z24, L9-Z6, L7-Z6, L12-Z1 , and L7-Z1. Alternatively, other coupling agents known in peptide chemistry can be used. It is also possible to carry out the synthesis in a sequential manner, i.e. by direct building of consecutive structural blocks of a linker so as to obtain its final form (Method B). Toward this end, suitable building elements are used, and, if required, also those having suitable protecting groups, known to those skilled in the art. An overview of such protecting groups can be found in Wuts, P. G. M. and Greene, T. W. (2006) Greene's Protective Groups in Organic Synthesis, Fourth Edition, John Wiley & Sons, Inc., Hoboken, NJ, USA. A suitable amino protecting group may be tert-butoxycarbonyl group. Each of the steps can be performed using the above-mentioned coupling technique amine reagent/hydroxy reagent - carboxylic acid. Alternatively, 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. Following this procedure the following moieties chemical compound Z - linker L can be obtained: L3-Z1 , L4-Z1 , L6-Z1 , L8-Z1 , and L6-Z6.

In the case when chemical compound - linker moiety is a carbonic acid derivative, i.e. when a linker fragment is connected with the rest of the compound by carbamate bond, it is required due to the instability of the corresponding carbamic acid to use the latter approach, based on the sequential attachment of consecutive linker components (Method C). In this case, 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. Following this procedure the following moieties chemical compound Z - linker L can be obtained: L2-Z1 , L5-Z1.

Using respective methods indicated above the moieties chemical compound - linker L represented by the following structures in Table 3 below were obtained.

Table 3.

Specific embodiments of the conjugate of the invention are combinations of fusion proteins represented by specific sequences SEQ. No. 7 to SEQ. No. 23 set forth in the attached sequence listing, chemical compounds with antiblastic activity represented by the formulas Z1 to Z24 in Table 2, and conjugation linkers represented by the formulas L1 to L12 in Table 1.

Particularly preferred embodiments of the conjugate of the invention are combinations of fusion proteins, conjugation linkers and chemical compounds presented below. It should be understood that in these embodiments, the conjugate comprises a specific sequence of the fusion protein (SEQ. No. 20, SEQ. No. 21 , SEQ. No. 22, SEQ. No. 23) with attached moiety chemical compound- Z- linker L. The site of the attachment of the moiety chemical compound- Z-linker L to the fusion protein in each case is determined by cysteine residue of domain (d) of the fusion protein. The conjugate can therefore be particularly selected from the following group:

SEQ. No. 20-L1 -Z1 SEQ. No. 21 -L1 -Z1 SEQ. No. 22-L1 -Z1 SEQ. No. 23-L1 -Z1 SEQ. No. 20-L2-Z1 SEQ. No. 21 -L2-Z1 SEQ. No. 22-L2-Z1 SEQ. No. 23-L2-Z1 SEQ. No. 20-L3-Z1 SEQ. No. 21 -L3-Z1 SEQ. No. 22-L3-Z1 SEQ. No. 23-L3-Z1 SEQ. No. 20-L4-Z1 SEQ. No. 21 -L4-Z1 SEQ. No. 22-L4-Z1 SEQ. No. 23-L4-Z1 SEQ. No. 20-L5-Z1 SEQ. No. 21 -L5-Z1 SEQ. No. 22-L5-Z1 SEQ. No. 23-L5-Z1 SEQ. No. 20-L6-Z1 SEQ. No. 21 -L6-Z1 SEQ. No. 22-L6-Z1 SEQ. No. 23-L6-Z1 SEQ. No.20- L7^■Z1; SEQ. No.21-L7-Z1; SEQ. No.22-L7-Z1; SEQ. No.23-L7-Z1;

SEQ. No. 20- L8 ^■Z1; SEQ. No.21-L8-Z1; SEQ. No.22-L8-Z1; SEQ. No.23-L8-Z1;

SEQ. No. 20- L1 ^■Z2; SEQ. No.21-L1-Z2; SEQ. No.22-L1-Z2; SEQ. No.23-L1-Z2;

SEQ. No. 20- L1 ^■Z3; SEQ. No.21-L1-Z3; SEQ. No.22-L1-Z3; SEQ. No.23-L1-Z3;

SEQ. No. 20- L9 ^■14; SEQ. No.21-L9-Z4; SEQ. No.22-L9-Z4; SEQ. No.23-L9-Z4;

SEQ. No. 20- L1 ^■15; SEQ. No.21-L1-Z5; SEQ. No.22-L1-Z5; SEQ. No.23-L1-Z5;

SEQ. No. 20- L1 ^■16; SEQ. No.21-L1-Z6; SEQ. No.22-L1-Z6; SEQ. No.23-L1-Z6;

SEQ. No. 20- L1 ^■Z7; SEQ. No.21-L1-Z7; SEQ. No.22-L1-Z7; SEQ. No.23-L1-Z7;

SEQ. No. 20- L1 ^■18; SEQ. No.21-L1-Z8; SEQ. No.22-L1-Z8; SEQ. No.23-L1-Z8;

SEQ. No. 20- L9 ^■18; SEQ. No.21-L9-Z8; SEQ. No.22-L9-Z8; SEQ. No.23-L9-Z8;

SEQ. No. 20- L1 ^■19; SEQ. No.21-L1-Z9; SEQ. No.22-L1-Z9; SEQ. No.23-L1-Z9;

SEQ. No. 20- L9 ^■19; SEQ. No.21-L9-Z9; SEQ. No.22-L9-Z9; SEQ. No.23-L9-Z9;

SEQ. No. 20- L1 ^■Z10; SEQ. No.21-L1-Z10; SEQ. No.22-L1-Z10; SEQ. No.23-L1- Z10;

SEQ. No. 20- L9 ^■Z10; SEQ. No.21-L9-Z10; SEQ. No.22-L9-Z10; SEQ. No.23-L9-Z10;

SEQ. No. 20- L1 -Z11; SEQ. No.21-L1-Z11; SEQ. No.22-L1-Z11; SEQ. No.23-L1-Z11;

SEQ. No. 20- L9 ^■Z11; SEQ. No.21-L9-Z11; SEQ. No.22-L9-Z11; SEQ. No.23-L9-Z11;

SEQ. No. 20- L1 ^■Z12; SEQ. No.21-L1-Z12; SEQ. No.22-L1-Z12; SEQ. No.23-L1-Z12;

SEQ. No. 20- L9 ^■Z12; SEQ. No.21-L9-Z12; SEQ. No.22-L9-Z12; SEQ. No.23-L9-Z12;

SEQ. No. 20- L1 ^■Z13; SEQ. No.21-L1-Z13; SEQ. No.22-L1-Z13; SEQ. No.23-L1-Z13;

SEQ. No. 20- L9 ^■Z13; SEQ. No.21-L9-Z13; SEQ. No.22-L9-Z13; SEQ. No.23-L9-Z13;

SEQ. No. 20- L1 ^■Z14; SEQ. No.21-L1-Z14; SEQ. No.22-L1-Z14; SEQ. No.23-L1-Z14;

SEQ. No. 20- L9 ^■Z14; SEQ. No.21-L9-Z14; SEQ. No.22-L9-Z14; SEQ. No.23-L9-Z14;

SEQ. No. 20- L1 ^■Z15; SEQ. No.21-L1-Z15; SEQ. No.22-L1-Z15; SEQ. No.23-L1-Z15; SEQ. No.20-L9-Z15; SEQ. No.21-L9-Z15; SEQ. No.22-L9-Z15; SEQ. No.23-L9-Z15;

SEQ. No.20-L1-Z16; SEQ. No.21-L1-Z16; SEQ. No.22-L1-Z16; SEQ. No.23-L1-Z16;

SEQ. No.20-L9-Z16; SEQ. No.21-L9-Z16; SEQ. No.22-L9-Z16; SEQ. No.23-L9-Z16;

SEQ. No.20-L9-Z17; SEQ. No.21-L9-Z17; SEQ. No.22-L9-Z17; SEQ. No.23-L9-Z17; SEQ. No.20-L1-Z17; SEQ. No.21-L1-Z17; SEQ. No.22-L1-Z17; SEQ. No.23-L1-Z17;

SEQ. No.20-L1-Z18; SEQ. No.21-L1-Z18; SEQ. No.22-L1-Z18; SEQ. No.23-L1-Z18;

SEQ. No.20-L9-Z18; SEQ. No.21-L9-Z18; SEQ. No.22-L9-Z18; SEQ. No.23-L9-Z18;

SEQ. No.20-L1-Z19; SEQ. No.21-L1-Z19; SEQ. No.22-L1-Z19; SEQ. No.23-L1-Z19;

SEQ. No.20-L9-Z19; SEQ. No.21-L9-Z19; SEQ. No.22-L9-Z19; SEQ. No.23-L9-Z19; SEQ. No.20-L10-Z20; SEQ. No.21-L10-Z20; SEQ. No.22-L10-Z20; SEQ. No.23-L10- Z20;

SEQ. No.20-L9-Z21; SEQ. No.21-L9-Z21; SEQ. No.22-L9-Z21; SEQ. No.23-L9-Z21;

SEQ. No.20-L1-Z22; SEQ. No.21-L1-Z22; SEQ. No.22-L1-Z22; SEQ. No.23-L1-Z22;

SEQ. No.20-L9-Z22; SEQ. No.21-L9-Z22; SEQ. No.22-L9-Z22; SEQ. No.23-L9-Z22; SEQ. No.20-L9-Z23; SEQ. No.21-L9-Z23; SEQ. No.22-L9-Z23; SEQ. No.23-L9-Z23;

SEQ. No.20-L1-Z23; SEQ. No.21-L1-Z23;SEQ. No.22-L1-Z23; SEQ. No.23-L1-Z23;

SEQ. No.20-L1-Z24; SEQ. No.21-L1-Z24; SEQ. No.22-L1-Z24; SEQ. No.23-L1-Z24;

SEQ. No.20-L9-Z24; SEQ. No.21-L9-Z24; SEQ. No.22-L9-Z24; SEQ. No.23-L9-Z24;

SEQ. No. 20-L11-Z24; SEQ. No. 21-L11-Z24; SEQ. No. 22-L11-Z24; SEQ. No. 23- L11-Z24; SEQ. No.20-L6-Z6; SEQ. No.21-L6-Z6; SEQ. No.22-L6-Z6; SEQ. No.23- L6-Z6; SEQ. No.20-L7-Z6; SEQ. No.21-L7-Z6; SEQ. No.22-L7-Z6; SEQ. No.23-L7- Z6; SEQ. No 20-L9-Z6; SEQ. No 21-L9-Z6; SEQ. No 22-L9-Z6; SEQ. No 23-L9-Z6; SEQ. No 20-L12-Z1; SEQ. No 21-L12-Z1; SEQ. No 22-L12-Z1 and SEQ. No 23-L12- Z1. Upon binding to TRAIL receptors present on the surface of cancer cells, the conjugate 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 receptors on the cell surface and activation of the extrinsic pathway of apoptosis. After internalization of the fusion protein comprising immunostimulating peptide, the domain (b) will be able to potentially exert its action intracellular^ in parallel to the activity of TRAIL domain. In this way, anti-cancer activity of TRAIL can be potentiated by activation of other elements and mechanisms, such as stimulation of B cells to produce antibodies, stimulation of caspase 7 and 8 expression, or stimulation of overexpression of TRAIL.

Additionally, the chemical compound conjugated with the fusion protein will exert antiblastic activity consisting of 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.

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.

The terms "pharmaceutically acceptable carrier, diluent, and 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.

In one embodiment, the pharmaceutical composition of the invention may be administered by injection directly to the tumour. In another embodiment, the pharmaceutical 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 tissues or blood of recipient. Other components, which may included in the composition, are for example water, alcohols such as ethanol, polyols such as glycerol, propylene glycol, liquid polyethylene glycol, lipids such as triglycerides, vegetable oils, liposomes. Proper fluidity and the particles size of the substance may be provided by coating substances, such as lecithin, and surfactants, such as hydroxypropyl- celulose polysorbates, and the like.

Suitable isotonicity agents for liquid parenteral compositions 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 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. Preferably, 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. 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 antifungal agents, including, for example, and not limited to, parabens, chlorobutanol, thimerosal, sorbic acid, and similar known substances of this type.

In general, 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. The 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.

The invention will be described in more detail in the following general procedures and examples of specific conjugates.

The fusion proteins constituting carriers in the conjugates of the invention were prepared as described in the publication WO2012072815, in embodiments, respectively, of Ex. 1 to Ex. 17.

Example 1 : Preparation of the moiety chemical compound Z - linker L

1.1. Preparation of L1 -Z1 moiety Method A described in the description above was used.

To dry methylene chloride (10 ml) (4S)-4, 1 1 -diethyl-4,9-dihydroxy-1 H-pyrano- [3',4':6,7]indolizino[1 ,2-b]qunolin-3, 14(4H, 12H)dione (Z1 ) (200 mg, 0.51 mmol), 6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanoic acid (L1 ) (1 13 mg, 0.53 mmol) and DMAP (6.5 mg, 0.05 mmol) is added, the mixture is cooled to 0°C and dicyclohexylcarbodiimide (121 mg, 0.59 mmol) dissolved in methylene chloride (5 ml) is added, while stirring vigorously. The mixture is stirred at room temperature for 48 hours. Then, the precipitated dicyclohexylurea is filtered off, the solvent evaporated and the residue purified on a silica gel column eluting with ethyl acetate - methanol 10 : 1 to obtain (S)-4, 1 1 -diethyl-4-hydroxy- 3, 14-dioxo-3,4, 12, 14-tetrahydro-1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-9- yl 6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -ylo)hexanoate L1 -Z1 as a powder, yield 155 mg (52%). MS: 586.2 (M+1 ).

1.2. Preparation of L3-Z1 moiety

Method B described in the description above was used.

To a suspension of (4S)-4, 1 1 -diethyl-4,9-dihydroxy-1 H-pyrano[3',4':6,7]- indolizino[1 ,2-b]quinolin-3, 14(4H, 12H)dione (Z1 ) (150 mg, 0.38 mmol) in dry methylene chloride (10 ml), trans-4-Boc-aminomethylcyclohexanecarboxylic acid (100 mg, 0.40 mmol), (benzotriazol-1 -yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) (180 mg, 0.4 mmol) and triethylamine (0.1 1 ml, 0.80 mmol) are added, with stirring. The mixture is stirred at room temperature for 24 hours. Solvent is then evaporated, and the residue containing 4, 1 1 -diethyl-4- hydroxy-3, 14-dioxo-3,4, 12, 14-tetrahydro-1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]- quinolin-9-yl (1 R,4S)-(S)-4-(((tert-butoxycarbonyl)-amino)methyl)cyclo- hexanecarboxylate (Formula 1 ) is washed with water, dried and used without further purification in the next step, yield 240 mg (100%).

Formula 1

4, 1 1 -Diethyl-4-hydroxy-3, 14-dioxo-3,4, 12, 14-tetrahydro-1 H-pyrano[3',4':6,7]- indolizino[1 ,2-b]quinolin (1 R,4S)-(S)-4-(((tert-butoxycarbonyl)amino)- methyl)cyclohexanecarboxylate obtained as described above (150 mg, 0.24 mmol) is suspended in dry methylene chloride (6 ml), cooled in ice and trifluoroacetic acid (0.8 ml) is added with stirring. The mixture is stirred at room temperature for 3 hours, The solvent is then evaporated and the residue treated with aqueous sodium bicarbonate solution, and extracted three times with ethyl acetate. The organic layer is dried and evaporated to give 4, 1 1 -diethyl-4- hydroxy-3, 14-dioxo-3,4, 12, 14-tetrahydro-1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]- quinolin-9-yl (1 R,4S)-(S)-4-(aminomethyl)cyclohexanecarboxylate (Formula 2), yield 130 mg (90%).

Formula 2

4, 1 1 -Diethyl-4-hydroxy-3, 14-dioxo-3,4, 12, 14-tetrahydro-1 H-pyrano-

[3^',4^':6,7]indolizino[1 ,2-b]quinolin-9-yl (1 R,4S)-(S)-4-(aminomethyl)cyclohexane- carboxylate (Formula 2) (59 mg, 0.25 mmol) is dissolved in dry methylene chloride (10 ml), and trans-4-((2,5-dioxo)-2H-pyrrol-1 (5H)-yl)methyl)cyclo- hexanecarboxylic acid (59 mg, 0.25 mmol) and (benzotriazol-l -yloxy)tris- (dimethylamino)phosphonium hexafluorophosphate (BOP) (1 10 mg, 0.25 mmol) and triethylamine (0.07 ml) are added, with stirring. The mixture is stirred at room temperature for 24 hours, then the solvent is evaporated, and the residue is purified on a silica gel column eluting with ethyl acetate - methanol 10 : 1 to obtain 4, 1 1 -diethyl-4-hydroxy-3, 14-dioxo-3,4, 12, 14-tetrahydro-1 H-pyrano- [3',4':6,7]indolizino[1 ,2-b]quinolin-9-yl (1 R,4S)-(S)-4-(((1 R,4R)-4-((2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -ylo)methyl)cyclohexanecarboxamide)methyl)cyclohexane- carboxylate (L3-Z1 ) (Formula 3), yield 100 mg (55%). MS: 773.23 (M+23).

Formula 3

1.3. Preparation of L4-Z1 moiety Method B described in the description above was used. To the solution of 4, 1 1 -diethyl-4-hydroxy-3, 14-dioxo-3,4, 12, 14-tetrahydro- 1 H- pyrano[3^',4^':6,7]indolizino[1 ,2-b]quinolin-9-yl (1 R,4S)-(S)-4-(aminomethyl)- cyclohexanecarboxylate (170 mg, 0.33 mmol) in dry methylene chloride, 15- (Boc-amino)-4,7, 10, 13-tetraoxapentadecanoic acid (120 mg, 0.33 mmol), (benzotriazol-1 -yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) (150 mg, 0.34 mmol) and triethylamine (0.09 ml) are added. The mixture is stirred at room temperature for 24 hours, then the solvent is evaporated, and the residue is purified on a silica gel column eluting with ethyl acetate - methanol 5 : 1 , to obtain (S)-4, 1 1 -diethyl-4-hydroxy-3, 14-dioxo-3,4, 12, 14- tetrahydro-1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-9-yl (1 R,4S)-4-(21 ,21 - dimethyl-3, 19-dioxo-6,9, 12, 15,20-pentaoxa-2, 18-diazadocosyl)- cyclohexanecarboxylate (Formula 4), yield 280 mg (96%).

Formula 4

The resulting product is dissolved in anhydrous dichloromethane (5 ml), solution is cooled down to 0°C, trifluoroacetic acid (0.5 ml) is added, and the reaction mixture is stirred for 4 hours. Solvent is then evaporated, aqueous sodium bicarbonate is added solution to the residue and the whole is extracted with ethyl acetate. The organic extract is dried, evaporated and the residue is purified on a silica gel column in methanol, to obtain (S)-4, 1 1 -diethyl-4-hydroxy- 3, 14-dioxo-3,4, 12, 14-tetrahydro- 1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-9- yl (1 R,4S)-4-(17-amino-3-oxo-6,9, 12, 15-tetraoxa-2-azaheptadecyl)cyclohexane- carboxylate (Formula 5), yield 200 mg (80%).

Formula 5

The resulting product is dissolved in dry methylene chloride (10 ml), to the stirred solution trans-4-((2,5-dioxo-2,5dihydro-pyrrol-1 -yl)methyl)cyclohexane- carboxylic acid (0.62 mg, 0.26 mmol), (benzotriazol-1 -yloxy)tris(dimethyl- amino)phosphonium hexafluorophosphate (BOP) (1 10 mg, 0.26 mmol) and triethylamine (0.06 ml) are added. The mixture is stirred at room temperature for 24 hours, then the solvent is evaporated, and the residue is purified on a silica gel column eluting with ethyl acetate - methanol 5 : 1 , to obtain (S)-4, 1 1 - diethyl-4-hydroxy-3, 1 -dioxo-3,4, 12, 1 -tetrahydro-1 H-pyrano[3',4':6,7]- indolizino[1 ,2-b]quinolin-9-yl (1 R,4S)-4-(1 -((1 R,4R)-4-((2,5-dioxo-2,5-dihydro-1 H- pyrrol-1 -yl)methyl)cyclohexyl)-1 , 17-dioxo-5,8, 1 1 , 14-tetraoxa-2, 18- diazanonadekan- 19-yl)cyclohexanecarboxylate L4-Z1 (Formula 6), yield 1 10 mg (42%). MS: 1020.37 (M+23).

Formula 6

1.4. Preparation of L5-Z1 moiety

Method C described in the description above was used.

To a stirred solution of (S)-4, 1 1 -diethyl-4-hydroxy-3, 14-dioxo-3,4, 12, 14- tetrahydro-1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-9-yl piperazine-1 - carboxylate of Formula 7 (60 mg, 0.12 mmol)

Formula 7 in dry methylene chloride (6 ml) 15-Boc-amino)-4,7, 10, 13-tetraoxapenta- decanoic acid (43 mg, 0.12 mmol), (benzotriazol-l -yloxy)tris(dimethylamino)- phosphonium hexafluorophosphate (BOP) (57 mg, 0.13 mmol) and triethylamine (0.035 ml, 0.26) are added. The mixture is stirred at room temperature for 24 hours, then the solvent is evaporated, and the residue is purified on a silica gel column eluting with ethyl acetate - methanol 5: 1 , to obtain (S)-4, 1 1 -diethyl-4- hydroxy-3, 14-dioxo-3,4, 12, 14-tetrahydro- 1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]- quinolin-9-yl 4-[3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]- ethoxy]ethoxy]propanoyl]piperazine-1 -carboxylate [or 4-(2,2-dimethyl-4-oxo- 3,8, 1 1 , 1 , 17-pentaoxa-5-azaicosan-20-oyl)piperazine-1 -carboxylate] (Formula 8), yield 99 mg (99%).

Formula 8 The compound of Formula 8, obtained as described above, is dissolved in dry methylene chloride, cooled in an ice bath and trifluoroacetic acid (0,3 ml) is added with stirring. The mixture is stirred for 5 hours, the volatile components are evaporated and the residue dissolved in dry methylene chloride (10 ml). To the resulting solution of (S)-4, 1 1 -diethyl-4-hydroxy-3, 14-dioxo-3,4, 12, 14-tetra- hydro-1 H-pyrano[3^',4^':6,7]indolizino[1 ,2-b]quinolin-9-yl 4-[3- [2-[2-[2-(2-amino- ethoxy)ethoxy]ethoxy]ethoxy]propanoyl]piperazine-1 -carboxylate triethylamine (0.062 ml, 0.45 mmol) and trans-4-((2,5-dioxo-2,5-dihydro-pyrrol-1 -ylo)methyl)- cyclohexanecarboxyl chloride (38 mg, 0.15 mmol) are added. The mixture is stirred at room temperature for 4 hours, The solvent is evaporated and the residue purified on a silica gel column eluting with ethyl acetate - methanol 5 : 2, to obtain (S)-4, 1 1 -diethyl-4-hydroxy-3, 14-dioxo-3,4, 12, 14-tetrahydro- 1 H- pyrano[3^',4^':6,7]indolizino[1 ,2-b]quinolin-9-yl 4-(1 -((1 R,4R)-4-((2,5-dioxo-2, 5- dihydro-1 H-pyrrol-1 -ilo)methyl)cyclohexyl)-1 -oxo-5,8, 1 1 , 14-tetraoxa-2-aza- heptadeca-17-oyl)piperazine-1 -carboxylate (Formula 9), yield 90 mg (82%). MS: 993.33 (M+23).

Formula 9

1.5. Preparation of L2-Z1 moiety

Method C described in the description above was used. Suspension of (4S)-4, 1 1 -diethyl-4,9-dihydroxy-1 H-pyrano[3',4':6,7]indolizino[1 ,2- b]quinolin-3, 14(41-1, 12H)dione (Z1 ) (200 mg, 0.51 mmol) in dry methylene chloride (10 ml) is cooled to 0°C and 1 -chloroethyl chloroformate (1 10 mg, 0.76 mmol), followed by diisopropylethylamine (98 mg, 0.76 mmol) are added with vigorous stirring. The mixture is stirred at room temperature for 48 hours. Solvent is then evaporated, the residue is washed with water and dried in a desiccator. The crude product is crystallized from ethyl acetate to give (S)-1 - chloroethyl 4, 1 1 -diethyl-4-hydroxy-3, 14-dioxo-3,4, 12, 14-tetrahydro- 1 H- pyrano[3^',4^':6,7]indolizino[1 ,2-b]quinolin-9-yl carbonate (Formula 10) as yellow crystals, yield 220 mg (88%).

Formula 10

To a stirred solution of the compound of Formula 10 (220 mg, 0,44 mmol) in tetrahydrofuran (10 ml), solution of 1 -Boc-piperazine (98 mg, 0.52 mmol) in tetrahydrofuran (1 ml) was added, followed by anhydrous potassium carbonate (400 mg). The mixture is stirred at room temperature for 24 hours, then the solvent is evaporated, and the residue is purified on a silica gel column in ethyl acetate. (S)-1 ,4-piperazinedicarboxylate 1 -tert-butyl 4-(4, 1 1 -diethyl-4-hydroxy- 3, 14-dioxo-3,4, 12, 14-tetrahydro-1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-9- yl (Formula 1 1 ) is obtained as yellow oil, yield 210 mg (81 %).

Formula 1 1

The solution of the compound of Formula 1 1 (210 mg, 0.35 mmol) in dry methylene chloride (10 ml) is cooled to 0°C and trifluoroacetic acid (0,5 ml) is added with stirring. The resulting solution is stirred for 8 hours and then the solvents are evaporated. 4-(4, 1 1 -Diethyl-4-hydroxy-3, 14-dioxo-3,4, 12, 14- tetrahydro-1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-9-yl (S)-piperazine-l - carboxylate is obtained as an oil (Formula 12)

Formula 12

It is dissolved in dry methylene chloride (8 ml) and a solution 6-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)hexanoyl chloride (83 mg, 0,36 mmol) in methylene chloride (2 ml), followed by triethylamine (72 mg, 0,72 mmol) are added with stirring. The mixture is stirred for 24 hours, then the solution is evaporated and the residue is purified on a silica gel column with ethyl acetate - methanol 10 : 1 , to obtain 4, 1 1 -diethyl-4-hydroxy-3, 14-dioxo-3,4, 12, 14-tetrahydro-1 H- pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-9-yl (S)-4-(6-(2,5-dioxo-2,5-dihydro- 1 H-pyrrol-1 -ilo)hexanoyl)piperazine-1 -carboxylate (Formula 13) as a pale yellow glassy substance, yield 58 mg (40%). MS: 698.25 (M+1 ).

Formula 13

Example 2. Synthesis of L7 linker 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)cyclohexane- carboxylic acid was formed. The mixture was then heated under reflux to complete the cyclization of the trans-4-(N-maleimidmethyl)cyclohexane-1 - carboxylic acid, and the mixture is concentrated under reduced pressure to remove acetic acid. The crude product was purified by flash column chromatography (SiO2-CHCl₃/MeOH, eluting with gradient 99: 1 -> 90: 10. The product was obtained as a cream solid, yield 435 mg (58%), ESI-ITMS: m/z (%): 236 (M - H)-.

Step II. trans-4-(N-maleimidmethyl)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. The crude product was purified by flash column chromatography (Si0₂- CHCls/MeOH, eluting with gradient 99:1 -> 90:10. 4-[({trans-4-[(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)methyl]cyclohexyl}carbonyl)amino]benzoic acid was obtained as a white solid, yield 312 mg (48%), ESI-ITMS: m/z (%): 355 (M - H)-, 357 (M + H)+. Example 3. Conjugation of fusion protein with the molecule of a chemical compound molecule provide with a conjugation linker.

The fusion protein of SEQ. No. 20 prepared as described in WO2012072815 Example 14 in concentration of 2 mg/ml was reduced with DTT or TCEP (5- 10mM) 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 KH₂P0₄, 17.53 mM Na₂HP0₄*2H₂0, 116.9mM 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 one free cysteine per one protein molecule was obtained (the result of more than one cysteine residue is obtained due to the presence of cysteine residue in lysozyme used in the protein isolation procedure). Conjugation reaction was carried out as follows. The moiety chemical compound- linker L2-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 L2-Z1 moiety equal 2 times of the concentration of free thiol groups. Conjugation reaction was carried out for 1.5h at 8°C until complete substitution of thiol groups was achieved, as monitored by DTNB reagent (Ellman method). In order to stop the reaction, N- acetyl- L-cysteine was added to a final concentration of 400μΜ. Excess of the compound was removed by overnight dialysis into a buffer (50mM Trizma®Base, 200mM NaCl, 5mM glutathione, 0.1 mM ZnCl₂, 10% v/v glycerol, 80 mM sucrose, pH 8.0)

HPLC-MS analysis of conjugates

In order to confirm the structure, the conjugates were analyzed by HPLC-MS. Samples were prepared by dilution with buffer B to a final protein concentration of 0.2 mg/ml and filtered using a syringe PVDF membrane filter - 0.22 μητι. MS measurements were performed in positive ionization mode, using an internal calibration (sodium formate). During the run, an elution gradient was used (buffer A: ACN, 0.1% formic acid, buffer B: H₂0, 0.1% formic acid). Gradient A:B 5%-70%, 30 min. Separation was achieved using a C8 BioBasic-8 50x2.1 mm column Thermo Scientific, flow 200μΙ/ιηι^'η, 5μΙ sample injection. HPLC-MS analysis allows for precise determination of the degree of substitution of the protein with the linked compound. The difference of molecular weights between the fusion protein (carrier) and the conjugate with a L2-Z1 moiety unambiguously indicates that in accordance with the assumptions that one molecule of the protein per one molecule of L2-Z1 moiety (that is, one molecule of the compound SN38 (Z1 ), linked with L2 linker) is present.

Table 4 shows the results of mass analysis for the carrier (fusion) protein of SEQ. No. 20, and for the conjugate consisting of fusion protein of SEQ. No. 20 linked with compound Z1. Table 4

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 fusion protein of SEQ. 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.

Other conjugates of the invention exemplified in the description above were obtained and analyzed analogously.

Example 4. Determination of an aggregation degree of the fusion carrier proteins and conjugates thereof Steady-state measurement of fluorescence of a dye added to the protein solution was used to detect potential aggregates formed by the fusion proteins and conjugates (set Enzo Life Sciences (# ENZ-51023-KP002). Procedure was followed in accordance with manufacturer's instructions [A novel protein aggregation assay for biologies formulation studies and production QA/QC. Proteostat® fluorescent reagent for microplate-based aggregate quantitation. Dee Shen, Wayne Patton, Enzo Life Sciences, Farmingdale, NY. Peter Banks, BioTek Instruments, Inc. , Winooski, VT. Application Note. Biologies Drug Discovery. AN031811 _02 Rev.03/ 18/ 11].

A solution of lysozyme in monomelic and aggregated forms (50% aggregate, respectively composed of the two forms of the protein) was used as a control (Enzo Life Sciences, # ENZ-51023-KP002). Monoclonal antibody bevacizumab (Avastin ®) was used as an additional negative control.

Percentage of aggregated form of the protein was calculated from the calibration curve (R² = 0.998) of the lysozyme (0.3 mg/ml)fluorescence intensity (RFU), which was generated for samples with varying degrees of aggregation obtained by mixing monomeric form with the aggregated form, while maintaining appropriate final protein concentration.

Table 5. The degree of aggregation of the fusion protein of Example 3 and its conjugates.

In the preparations of fusion protein and the conjugates a degree of aggregation that would affect their functionality was not found. On this basis, it can be concluded that they have suitable stability and solubility, and are suitable for long-term storage without the formation of precipitates. The degree of aggregation of the conjugates did not differ significantly from the degree of aggregation of the fusion proteins. The preparations also didn't cause allergic reactions in laboratory animals

Example 5. Determination of dimensions of the fusion protein of SEQ. 20 and its conjugates with chemical compounds by hydrodynamic light scattering (DLS - Dynamic Light Scattering)

The DLS method allows i.a. hydrodynamic particle measurements, determination of the coefficient of polydispersity, as well as the detection of protein aggregates in solution ('Dynamic Light Scattering. The method and some applications" ed. W.Brown, Oxford University Press, Oxford 1993.; .Berne, R.Pecora, 'Dynamic light scattering", John Wiley t Sons, New York 1976].

All measurements were performed at 25 ° C, in quartz cells using a backscattering (173° ) in Zetasizer Nano, Malvern Instruments Ltd. The final result of the measurement is the average of 6-10 partial measurements. Prior each measurement the samples were filtered through a 0.22 μητι PVDF membrane. A cumulative analysis (measurement is an average over all components of the test sample) and distribution analysis (the data presented are specific to the population of molecules present in the sample) was performed.

All the analyzed preparations are characterized by high polydispersity (impact of the solution components other than the fusion protein/conjugate, including the egg white lysozyme). However, assuming the corresponding protein profile for the tested conjugates relative to the control protein, it can be concluded that the DLS profiles do not reveal drastic differences (comparing conjugates to the corresponding control proteins). Therefore high similarity of hydrodynamic parameters can be assumed for analyzed conjugates with respect to the starting fusion (carrier) protein. Table 6. Hydrodynamic properties of the tested proteins.

Example 6. Tests on cell lines in vitro

Cell lines

Table 7. Adherent cell li

number of cells per

Cell line Cancer type Medium

well (thousands)

Colo 205 human colorectal RPMI + 10% FBS + penicillin +

5 ATCC #CCL-222 cancer streptomycin

HT-29 human colorectal McCoy's + 10% FBS + penicillin

5 ATCC # CCL-2 cancer + streptomycin

human colorectal McCoy's + 10% FBS + penicillin

HCT 1 16 3.0 cancer + streptomycin

ATCC# CCL-247

SW620 human colorectal DMEM + 10% FBS + penicillin +

5 ATCC #CCL-227 cancer streptomycin

LS1034 human colorectal RPMI + 10% FBS + penicillin +

15.0 ATCC# CRL- cancer streptomycin

2158

LS41 1 N human colorectal RPMI + 10% FBS + penicillin +

15.0 ATCC# CRL- cancer streptomycin

2159

LS513 human colorectal RPMI + 10% FBS + penicillin +

15.0 ATCC# CRL- cancer streptomycin

2134 number of cells per

Cell line Cancer type Medium

well (thousands)

Panc-1 human pancreatic DMEM+ 10% FBS + penicillin +

5.0 cancer streptomycin

CLS# 300228

Pane 03.27 human pancreatic RPMI + 10% FBS + penicillin +

5.0 ATCC# CRL- cancer streptomycin

2549

MIA PaCa-2 human pancreatic DMEM+ 10% FBS + penicillin +

3.0 ATCC# CRL- cancer streptomycin

1420

BxPC-3

human pancreatic RPMI + 10% FBS + penicillin +

ATCC #CRL- 4.5 cancer streptomycin

1687

PLC/PRF/5 DMEM+ 10% FBS + penicillin +

human liver cancer 5.0 streptomycin

CLS# 30031 5

SK-HEP-1 RPMI + 10% FBS + penicillin +

human liver cancer 10.0 CLS# 300334 streptomycin

HepG2

human liver MEM + 10% FBS + penicillin +

ATCC # HB- 7 hepatoma streptomycin

8065

MCF-7 MEM + 10% FBS + penicillin +

human breast cancer 4.5 ATCC #HTB-22 streptomycin

MDA-MB-231 DMEM + 10% FBS + penicillin +

human breast cancer 4.5 ATCC # HTB-26 streptomycin

DMEM+ 10% FBS + penicillin +

MDA-MB-435S human breast cancer 4.0 streptomycin

ATCC# HTB-129

NCI- H69AR

human small cell RPMI + 10% FBS + penicillin +

ATCC #CRL- 10 lung cancer streptomycin

1 1351

NCI-H460 MEM + 10% FBS + penicillin +

human lung cancer 2.5 ATCC #HTB-177 streptomycin

NCI-H2122 RPMI + 10% FBS + penicillin +

human lung cancer 6.0 ATCC# CRL- streptomycin

5985

A549 RPMI + 10% FBS + penicillin +

human lung cancer 2.5 streptomycin

ATCC# CCL-185

A549/K1 .5 RPMI + 10% FBS + penicillin +

human lung cancer 2.5 streptomycin

MEM+ 10% FBS + penicillin +

SK-MES-1 human lung cancer 4.0 streptomycin

ATCC# HTB-58

SK-OV-3 human ovarian McCoy's + 10% FBS + penicillin

4 ATCC # HTB-77 cancer + streptomycin

RPMI + 20% FBS + 0,01 mg/ml

NIH: OVCAR-3 human ovarian

insulin + penicillin + 7 ATCC #HTB-161 cancer

streptomycin number of cells per

Cell line Cancer type Medium

well (thousands)

A498 MEM+ 10% FBS + penicillin +

human kidney cancer 3.0

streptomycin

CLS# 3001 1 3

ACHN MEM + 10% FBS + penicillin +

human kidney cancer 4 ATCC #CCL-222 streptomycin

Caki-1 McCoy's + 10% FBS + penicillin

human kidney cancer 3.5

+ streptomycin

ATCC# HTB-46

CAKI 2 McCoy's + 10% FBS + penicillin

human kidney cancer 3.5 ATCC # HTB-47 + streptomycin

MES-SA human uterine McCoy's + 10% FBS + penicillin

3.5 ATCC# CRL- cancer + streptomycin

1976

MES-SA/ Dx5 human uterine McCoy's + 10% FBS + penicillin

4.0 ATCC# CRL- cancer + streptomycin

1977

MES-SA/Mx2 Waymouth's MB/ McCoy's +

human uterine

10% FBS + penicillin + 8.0 ATCC# CRL- cancer

streptomycin

2274

HT144 McCoy's + 10% FBS + penicillin

human skin cancer 7 ATCC # HTB-63 + streptomycin

Hs294T DMEM+ 10% FBS + penicillin +

human skin cancer 4.5

streptomycin

ATCC# HTB-140

HT-1080 human connective MEM+ 10% FBS + penicillin +

2.5 tissue cancer streptomycin

ATCC# CCL-121

0E19 human oesophageal RPMI + 10% FBS + penicillin +

8.0 ECACC# cancer streptomycin

96071721

Table 8. Nonadherent cells:

MTT cytotoxicity test

The test was performed according to the literature descriptions (Cell^'s, JE, (1998). Cell Biology, a Laboratory Handbook, second edition, Academic Press, San Diego; Yang, Y., Koh, LW, Tsai, JH. , (2004); 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 μΐ). Then 100 μΐ 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% C0₂, depending on the medium used, and then to the cells (in 100 μΐ of medium) further 100 μΐ of the medium containing various concentrations of tested conjugates and its constituents were added. After incubation of the cells with tested conjugates and its constituents over the period of further 72 hours, which is equivalent to 3-4 times of cell division, the medium with tested conjugates and its constituents was added with 20 ml of MTT working solution [5 mg/ml], and incubation was continued for 3 h at 37°C in 5% C0₂. Then the medium with MTT solution was removed, and formazan crystals were dissolved by adding 100 μΐ 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 activity of the conjugates and its constituents in nonadherent cell lines. The test is a modification of the MTT wherein formazan formed in the reduction of tetrazolium salt is water- soluble. Cell viability study was carried out after continuous 72-hour incubation of the cells with conjugates and its constituents (seven concentrations of protein, each in triplicates). On this basis IC50 values were determined (as an average of two independent experiments) using the GraphPad Prism 5 software.

The results of in vitro cytotoxicity tests are summarized in Table 9, as IC50 values (ng/ml), which corresponds to a protein concentration at which the cytotoxic effect of conjugates and its constituents is observed at the level of 50% with respect 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 conjugates and its constituents preparations the IC50 limit of 2000 ng/ml was adopted. Conjugates and its constituents with an IC50 value above 2000 were considered inactive. Cells for this test were selected so as to include the tumour cell lines naturally resistant to TRAIL protein (the criterion of natural resistance to TRAIL: IC50 for TRAIL protein > 2000), tumour cell lines sensitive to TRAIL protein and drug resistant cell lines MES-SA/DX5, MES-SA/Mx2, NCI-H69AR, LS1034, LS513 as cancer lines resistant to conventional anticancer medicaments.

The results obtained confirm the possibility of overcoming the resistance of the cell lines to TRAIL by administration of certain conjugates of the invention to cells naturally resistant to TRAIL. When conjugates of the invention were administered into the cells sensitive to TRAIL, in some cases a clear and strong potentiation of the potency of action was observed, manifesting in reduced IC50 values of the conjugates compared with IC50 for the constituents of the conjugate when administered individually. Furthermore, cytotoxic activity of the conjugates of the invention in the cells resistant to classical anti-cancer medicament doxorubicin was obtained, and in some cases was stronger than activity of conjugates constituents when administered individually (i.e. fusion proteins and chemical compounds used separately) or in the form of a mixture.

Determination of cytotoxic activity of selected conjugate preparations against a panel of tumour cell lines

Table 9 presents the results of the tests of cytotoxic activity in vitro for selected conjugates of the invention and its constituents against a broad panel of tumour cells from different organs, corresponding to the broad range of most common cancers. Obtained IC50 values confirm high cytotoxic activity of the conjugates and thus their potential utility in the treatment of cancer.

In Table 9, tested preparations are designated with the abbreviation Con. when tested preparation is a conjugate of the invention, and with abbreviation Mix. when tested preparation is a mixture of given constituents of the conjugate. Additionally, in Table 9 and in the whole description and Figures in any designation of a conjugate symbol "-" is used between constituents of this conjugate to illustrate the existence of a chemical bond between them. Symbol "+" between conjugate constituents is used to designate a mixture of individual conjugate constituents without chemical link between them. Table 9. Cytotoxic activity of the proteins and conjugates

Continuous incubation of preparations with cells over 72h (test MTT, ng/ml)

Tested preparation A549 A549/K1.5 NCI-H460 SK-MES-1 NCI-H2122 H69AR

IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD hTRAIU 14-281 >2000 >2000 69.7 2.5 1 1.21 1.62 0.176 0.078 >2000

IFN-a UniProtKB/Swiss- Prot: P01563.1 (24-188) >2000 >2000 193.9 92.1 961 287 >2000

INF-γ Gen Bank:

CAA44325.1 (1 -144) >2000 >2000 146.0 35.7 9.04 2.93 >2000 fusion protein SEQ. No. 20 67.9 20.8 3.24 2.03 3.60 1.14 0.22 0.04 <0,002 >2000

Con. SEQ. No. 20-L2-Z1 23.0 2.5 4.62 3.97 5.35 0.92 1.04 0.15 0.177 0.074 >2000

Con. SEQ. No. 20-L7-Z1 18.68 4.79 4.25 1.05 2.53 0.41 0.49 0.09 0.168 0.070 >2000

Mix. SEQ. No. 20 + Z6 238.3 1 1 1.2 6.01 1.24 2.80 0.98 <0,002 >2000

Mix. SEQ. No. 20 + Z1 61.6 23.9 4.57 2.64 2.66 0.66 0.66 0.08 <0,002 >2000

Con. SEQ. No. 20-L14-Z1 20.64 5.0 6.84 1.05 2.14 0.68 0.051 0.037 >2000

Mix. SEQ. No. 20 + (3xZ1 ) 16.31 2.15 2.67 0.74 1.54 0.44 0.019 0.008 1790 562

Table 9 continuation

Continuous incubation of preparations with cells over 72h (test MTT, ng/ml)

Tested preparation A549 A549/K1.5 NCI-H460 SK-MES-1 NCI-H2122 H69AR

IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD

Con. SEQ. No. 20-L1 -Z2 197.2 46.0 4.19 3.56 0.95 0.26 0.07 <0.005 >2000

Mix. SEQ. No. 20 + Z2 179.3 33.6 0.41 3.43 1.12 0.74 0.15 0.052 0.012 >2000

Con. SEQ. No. 20-L9-Z21 434.0 0.4 0.08

Mix. SEQ. No. 20 + Z21 109.3 0.33 0.14

Con. SEQ. No. 20 -L9-Z4 203.8 26.2 0.12 0.07

Mix. SEQ. No. 20 + Z4 1 19.1 54.5 0.44 0.07

Con. SEQ. No. 20-L9-Z5 146.5 3.45 0.29

Mix. SEQ. No. 20 + Z5 5.04 1.17 1.55 1.79 1.49 0.165 0.065 <0.002 120.3 33.6

Con. SEQ. No. 20-L1 -Z6 135.4 23.7 6.53 0.20 0.232 0.020 <0.002 1390 176

Con. SEQ. No. 20-L9-Z6 140.9 30.3 4.21 0.14 1.94 0.78 <0.002 >2000

Table 9 continuation

Continuous incubation of preparations with cells over 72h (test MTT, ng/ml)

Tested preparation NCI -H69 HCT1 16 HT-29 SW620 Colo205 LS41 1 N

IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD hTRAIU 14-281 >2000 25.57 0.93 >2000 >2000 2.27 0.09 >2000

IFN-a UniProtKB/Swiss- Prot: P01563.1 (24-188) >2000 >2000 >2000 >2000 >2000 >2000

INF-γ GenBank:

CAA44325.1 (1 -144) >2000 >2000 >2000 >2000 >2000 >2000 fusion protein SEQ. No. 20 >2000 1.29 0.49 5.5 2.1 440.4 58.3 1.27 0.16 794 250

Con. SEQ. No. 20-L2-Z1 >2000 5.83 3.65 14.7 3.4 493.4 13.7 3.01 1.66 >2000

Con. SEQ. No. 20-L7-Z1 1989 592 5.06 2.29 56.7 35.7 133.6 21.39 0.73 0.55 441 1 19

Mix. SEQ. No. 20 + Z6 >2000 2.23 0.35 78.1 1 13.36 230.6 40.51 2.07 0.91 752 236

Mix. SEQ. No. 20 + Z1 805 1 16 3.35 1.15 55.01 22.0 101.9 21.88 2.21 1.30 445 97

Con. SEQ. No. 20-L14-Z1 349 149 5.78 2.97 45.8 20.6 42.97 3.03 4.21 0.55 257.6 36.7

Mix. SEQ. No. 20 + (3xZ1 ) 254 1 18 5.38 1.93 29.7 1.7 19.64 3.59 2.41 0.23 183.4 57.6

Table 9 continuation

Continuous incubation of preparations with cells over 72h (test MTT, ng/ml)

Tested preparation NCI -H69 HCT1 16 HT-29 SW620 Colo205 LS41 1 N

IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD

Con. SEQ. No. 20-L1 -Z2 >2000 0.39 0.19 194 91 >2000 2.62 1.50 1751 644

Mix. SEQ. No. 20 + Z2 >2000 0.71 0.24 47.8 16.8 828 286 2.36 0.63 1786 850

Con. SEQ. No. 20-L9-Z21 0.73 0.04

Mix. SEQ. No. 20 + Z21 1.45 0.46

Con. SEQ. No. 20-L9-Z4 0.3 0.03

Mix. SEQ. No. 20 + Z4 1.54 0.39

Con. SEQ. No. 20-L9-Z5 4.12

Mix. SEQ. No. 20 + Z5 79.2 35.8 0.96 0.44 6.53 1.59 16.1 1 5.57 2.60 0.32 40.73 10.8

Con. SEQ. No. 20-L1 -Z6 >2000 3.33 1.12 59.89 22.85 152.4 54.7 2.18 0.42 131 1 101

Con. SEQ. No. 20-L9-Z6 >2000 1.81 0.23 84.93 10.6 916 57 2.62 0.30 1 133 278

Table 9 continuation

Continuous incubation of preparations with cells over 72h (test MTT, ng/ml)

Tested preparation LS1034 LS513 Panc-1 Panc03.27 BxPC-3 MIA PaCa-2

IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD hTRAIU 14-281 208.2 71.6 13.87 >2000 24.3 7.3 7.69 2.61 6.65 1.12

IFN-a UniProtKB/Swiss- Prot: P01563.1 (24-188) >2000 >2000 >2000 195.0 63.4 28.76 16.79 >2000

INF-γ Gen Bank:

CAA44325.1 (1 -144) >2000 >2000 >2000 2.43 0.37 1.63 0.6 >2000 fusion protein SEQ. No. 20 22.76 9.17 8.98 1.25 5.24 1.78 6.83 1.75 1.15 0.25 1.70 0.58

Con. SEQ. No. 20-L2-Z1 24.18 7.58 15.03 5.03 13.76 1.74 5.58 1.71 1.36 0.20 3.21 1.37

Con. SEQ. No. 20-L7-Z1 27.13 13.60 10.78 2.97 7.74 1.84 5.90 1.63 0.812 0.292 1.80 0.98

Mix. SEQ. No. 20 + Z6 30.80 9.21 17.81 1.39 8.53 0.71 6.67 0.97 1.61 0.43 1.15 0.49

Mix. SEQ. No. 20 + Z1 20.92 4.97 10.55 4.27 9.82 3.81 7.64 0.89 1.42 0.89 0.612 0.24

Con. SEQ. No. 20-L14-Z1 30.68 5.25 12.42 4.20 18.04 2.49 8.64 2.36 1.69 0.39 1.97 0.73

Mix. SEQ. No. 20 + (3xZ1 ) 14.94 2.12 8.06 3.40 5.28 1.47 4.15 1.31 0.842 0.277 1.10 0.68

Table 9 continuation

Continuous incubation of preparations with cells over 72h (test MTT, ng/ml)

Tested preparation HepG2 SK-HEP-1 PLC/PRF/5 MDA-MB-435S MCF-7 MDA-MB-231

IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD hTRAIL.1 14-281 >2000 >2000 >2000 >2000 >2000 973 98

IFN-a UniProtKB/Swiss- Prot: P01563.1 (24-188) >2000 >2000 >2000 >2000 >2000 >2000

INF-γ Gen Bank:

CAA44325.1 (1 -144) >2000 >2000 >2000 >2000 >2000 >2000 fusion protein SEQ. No. 20 >2000 57.1 19.9 14.07 8.54 12.38 3.18 339, 1 64,7 20.48 5.50

Con. SEQ. No. 20-L2-Z1 >2000 30.36 6.76 16.1 8.1 12.65 2.17 746 3 22.61 3.80

Con. SEQ. No. 20-L7-Z1 334.0 23.2 1897 692 10.80 7.37 1 1.09 2.36 142.5 50.4 10.55 0.67

Mix. SEQ. No. 20 + Z6 1374 145 4918 21.34 45.94 41.61 16.21 1.36 120.8 54.0 17.84 1.36

Mix. SEQ. No. 20 + Z1 265.2 29.05 4469 12.65 7.95 4.51 12.86 0.98 136.4 48.3 8.74 3.46

Mix. SEQ. No. 20-L14-Z1 2442 958 3376 5.04 1 1.7 8.0 33.45 7.63 137.2 67.9 33.67 6.96

Mix. SEQ. No. 20 + (3xZ1 ) 209 56 1654 3.44 5.36 5.54 12.13 1.04 1 13.4 39.1 1 1.94 2.53

Table 9 continuation

Continuous incubation of preparations with cells over 72h (test MTT, ng/ml)

Tested preparation ACHN Caki-1 Caki-2 A498 MES-SA MES-SA/ Dx5

IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD hTRAIU 14-281 >2000 2,92 0,71 >2000 31 ,45 9,98 >2000 5, 1 1 ,7

IFN-a UniProtKB/Swiss- Prot: P01563.1 (24-188) >2000 >2000 >2000 >2000 >2000 >2000

INF-γ Gen Bank:

CAA44325.1 (1 -144) >2000 >2000 >2000 >2000 >2000 >2000 fusion protein SEQ. No. 20 2.34 0.29 7.15 3.50 253.9 47.4 2.68 0.77 0.55 0.17 0.041 0.02

Con. SEQ. No. 20-L2-Z1 63.61 20.63 1 1.91 2.04 529 72 4.03 0.87 1.66 0.64 0.37 0.13

Con. SEQ. No. 20-L7-Z1 1 1.60 5.20 12.15 3.55 26.00 1 1.22 4.96 0.81 1.48 0.22 0.05 0.03

Mix. SEQ. No. 20 + Z6 10.59 0.18 3.62 1.71 147.6 30.3 4.41 1.12 1.79 0.20 0.189 0.07

Mix. SEQ. No. 20 + Z1 24.41 1 1.00 3.44 0.06 129.9 15.58 4.15 0.25 1.16 0.20 0.059 0.03

Con. SEQ. No. 20-L14-Z1 53.07 4.21 15.16 3.94 96.4 21.8 8.24 0.55 1.66 0.66 0.371 0.05

Mix. SEQ. No. 20 + (3xZ1 ) 10.51 4.38 8.47 2.88 63.89 14.58 2.53 0.51 1.04 0.38 0.044 0.01

Table 9 continuation

Continuous incubation of preparations with cells over 72h (test MTT, ng/ml)

Tested preparation ACHN Caki-1 Caki-2 A498 MES-SA MES-SA/ Dx5

IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD

Con. SEQ. No. 20-L1 -Z2 25.27 10.07 4.04 4.30 >2000 6.28 1.55 1.34 0.43 0.066 0.04

Mix. SEQ. No. 20 + Z2 64.06 17.51 4.97 2.84 294.1 132.8 4.59 1.25 1.1 1 0.35 0.1 13 0.01

Con. SEQ. No. 20-L9-Z21 0.5 0.21 0.04 0.00

Mix. SEQ. No. 20 + Z21 0.71 0.14 0.25

Con. SEQ. No. 20-L9-Z4 0.19 0.05 0.01 0.01

Mix. SEQ. No. 20 + Z4 0.41 0.34 0.06

Con. SEQ. No. 20-L9-Z5 0.98 0.04

Mix. SEQ. No. 20 + Z5 4.15 0.77 0.325 0.215 12.39 0.85 2.91 0.37 1.79 0.47 0.270 0.14

Con. SEQ. No. 20-L1 -Z6 22.97 14.0 2.56 1.19 248.9 98.1 2.94 0.76 2.18 0.55 0.362 0.15

Con. SEQ. No. 20-L9-Z6 7.39 2.53 3.37 1.86 176.7 75.0 4.06 0.87 1.22 0.52 0.121 0.04

Table 9 continuation

Continuous incubation of preparations with cells over 72h (test MTT, ng/ml)

Tested preparation MES-SA/Mx2 SK-OV-3 NIH: OVCAR-3 HT-144 HS-294T OE19

IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD hTRAIU 14-281 5,79 2,55 >2000 14, 1 1 ,0 >2000 1 ,73 1 ,03 10, 19 0,85

IFN-a UniProtKB/Swiss- Prot: P01563.1 (24-188) >2000 >2000 51 ,54 4, 15 >2000 20,48 7,66 >2000

INF-γ Gen Bank:

CAA44325.1 (1 -144) >2000 >2000 >2000 >2000 >2000 >2000

Fusion protein SEQ. No. 20 0.071 0.046 44.77 1 1.51 0.055 0.01 1 1.95 0.78 1.97 1.38 5.63 1.77

Con. SEQ. No. 20-L2-Z1 0.241 0.120 1862 388 0.134 0.099 3.96 2.20 5.50 4.14 8.07 2.65

Con. SEQ. No. 20-L7-Z1 0.057 0.020 1 1.13 2.52 0.129 0.108 3.14 1.1 1 3.62 2.27 6.43 1.92

Mix. SEQ. No. 20 + Z6 0.516 0.314 212.3 1 10.8 0.301 0.084 3.10 2.55 2.67 1.23 12.44 2.57

Mix. SEQ. No. 20 + Z1 0.102 0.056 18.50 3.97 0.218 0.225 2.00 0.37 2.69 1.25 6.04 1.50

Con. SEQ. No. 20-L14-Z1 0.162 0.099 24.85 5.50 0.103 0.058 3.64 1.50 6.37 4.28 7.53 2.72

Mix. SEQ. No. 20 + (3xZ1 ) 0.164 0.097 14.33 4.41 0.070 0.034 1.59 0.19 2.89 1.76 5.20 2.02

Table 9 continuation

Continuous incubation of preparations with cells over 72h (test MTT, ng/ml)

Tested preparation MES-SA/Mx2 SK-OV-3 NIH: OVCAR-3 HT-144 HS-294T OE19

IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD

Con. SEQ. No. 20-L1 -Z2 0.073 0.055 >2000 0.039 0.048 1.55 0.49 2.45 1.72 5.36 1.69

Mix. SEQ. No. 20 + Z2 0.245 0.025 >2000 0.082 0.069 1.79 0.67 2.68 1.28 6.41 1.79

Con. SEQ. No. 20-L9-Z5 0.03

Mix. SEQ. No. 20 + Z5 0.041 0.01 1 4.16 1.35 0.052 0.015 1.50 1.01 0.794 0.656 0.391 0.09

Con. SEQ. No. 20-L1 -Z6 0.059 0.01 1 175.4 46.1 0.006 0.003 3.1 1 2.09 3.96 1.03 9.28 5.86

Con. SEQ. No. 20-L9-Z6 0.057 0.012 171.2 29.5 0.012 0.005 3.70 2.94 3.28 0.17 7.72 5.58

Table 9 continuation

Continuous incubation of preparations with cells over 72h (test MTT, ng/ml)

Tested preparation HT-1080 HT-1080

IC50 ±SD Protein IC50 ±SD

hTRAIU 14-281 >2000 SEQ. No. 20-L1 -Z2 >2000 321

IFN-a UniProtKB/Swiss- Prot: P01563.1 (24-188) >2000 SEQ. No. 20 + Z2 968

INF-γ Gen Bank:

CAA44325.1 (1 -144) >2000 SEQ. No. 20-L9-Z5 0.90

fusion protein SEQ. No. 20 1441 515 SEQ. No. 20 + Z5 2.80 37.8

Con. SEQ. No. 20-L2-Z1 541 92 SEQ. No. 20-L1 -Z6 244.1 19.7

Con. SEQ. No. 20-L7-Z1 35.2 12.0 SEQ. No. 20-L9-Z6 103.37 344

Mix. SEQ. No. 20 + Z6 1087 344 SEQ. No. 20 + Z6 1087

Mix. SEQ. No. 20 + Z1 44.3 3.6

Con. SEQ. No. 20-L14-Z1 19.15 1.06

Mix. SEQ. No. 20 + (3xZ1 ) 1 1.14 1.09

Table 9 continuation

Continuous incubation of preparations with cells over 72h (test MTT, ng/ml)

Tested preparation HepG2 SK-HEP-1 PLC/PRF/5 MDA-MB-435S MCF-7 MDA-MB-231

IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD

Con. SEQ. No. 20-L1 -Z2 >2000 43.14 21.26 1 15.6 9.1 1 1.82 3.39 >2000 22.79 9.73

Mix. SEQ. No. 20 + Z2 >2000 33.88 13.75 35.52 2.85 10.69 0.85 486.1 127.7 18.97 2.93

Mix. SEQ. No. 20 + Z5 65.85 12.14 57.21 23.55 2.72 1.56 1.14 0.26 33.80 5.41 14.39 3.30

Con. SEQ. No. 20-L1 -Z6 >2000 88.68 33.66 30.17 10.74 16.54 3.68 351.2 59.9 8.69 3.91

Con. SEQ. No. 20-L9-Z6 >2000 48.8 12.27 24.07 14.45 13.79 5.36 426.4 1 16.3 12.73 4.55

LS1034 LS513 Panc-1 Panc03.27 BxPC-3 MIA PaCa-2

Tested preparation

IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD IC50 ±SD

Con. SEQ. No. 20-L1 -Z2 34.1 1 1 1.41 17.07 6.72 10.38 2.87 6.04 3.29 0.587 0.383 2.60 0.91

Mix. SEQ. No. 20 + Z2 24.12 8.05 8.69 0.59 6.67 0.24 6.75 3.54 0.902 0.306 2.10 0.91

Mix. SEQ. No. 20 + Z5 24.34 3.59 1 1.28 4.19 4.64 1.46 0.18 0.1 1 0.168 0.03 0.338 0.106

Con. SEQ. No. 20-L1 -Z6 19.43 1 1.40 16.47 4.43 3.71 1.56 8.97 2.97 0.176 0.1 1 1.43 0.53

Con. SEQ. No. 20-L9-Z6 18.62 5.18 15.19 3.59 2.31 0.16 4.34 0.35 0.575 0.198 0.919 0.467

Example 7. Antitumour effectiveness of conjugates in vivo on xenografts

Antitumour activity of conjugates preparations was tested in a mouse model of multidrug resistant human uterine sarcoma MES-SA/Dx5.

Cells The MES-SA/ Dx5 cells were maintained in McCoy's medium (Gibco) 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 a 3: 1 mixture of HBSS:Matrigel (BD bioscience), counted and diluted to the concentration of 25x10⁶ cells/ml.

Mice

Examination of antitumor activity of proteins of the invention was conducted on 4-6 week-old Crl:SHO-Prkdc^{s 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).

The course and evaluation of the experiments

MES-SA/Dx5cells

On day 1 mice Crl:SHO-Prkdc^sc1dHr^hr were grafted subcutaneously (sc) in the right side with 7x10⁶ (Experiment A) or 5x10⁶ (Experiment B) of MES-SA/Dx5 cells suspended in 0.1 ml mixture of HBSS:Matrigel by means of a syringe with a 0.5 x25 mm needle (Bogmark). When tumours reached the size of 180 mm³ Experiment A) or 200 mm³ (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 q2dx6 schema (i.e. administration every second day, totally 6 administrations) with the preparations of conjugate of Example 3 (SEQ. No. 20-L2-Z1 ) of the invention (60 mg/kg in Experiment A or 100 mg/kg in Experiment B, counted on the protein), wherein the concentration of the linked SN38 compound (Z1 ) amounts to 0,75 mg/kg and 1 ,2 mg/kg, respectively. The conjugate constituents were also administered alone in a dose corresponding to their amount in conjugate. Irinotecan (30 mg/kg i.p. ) was used as a comparative reference, and formulation buffer (i. v. ) (5 mM NaH₂P0₄ ,95 mM Na₂HP0₄, 200 mM NaCl, 5 mM glutathione, 0, 1 mM ZnCl₂, 10% glycerol, 80 mM sucrose, pH 8,0) as a control.

The experimental results obtained in mice Crl:SHO-Prkdc^sc1dHr^hr burdened with MES-SA/Dx5 multidrug resistant human uterine sarcoma treated with conjugate of the invention of Example 3 (SEQ. No. 20-L2-Z1 ) and comparatively with its constituents and with a reference compound are shown in Fig. 1 (Experiment A) and Fig. 3 (Experiment B) as a diagram of changes of the tumour volume and in Fig. 2 (Experiment A) and Fig. 4 (Experiment B) which shows tumour growth inhibition (%TGI ) as the percentage of control.

The results of experiments presented in the graphs in Figures 1 and 2 as well as Figures 3 and 4 show that administration of the conjugate of the invention SEQ. No. 20-L2-Z1 caused MES-SA/Dx5 tumour growth inhibition, with TGI 78% (Experiment A) and 77% (Experiment B) relative to the control on 29^th and 30^th day of the experiment, respectively. For the constituents of the conjugate administered individually, used as the comparative reference, an inhibitory effect on tumour cell growth was obtained relative to the control, with TGI of 59% and -0,4% (for fusion protein of SEQ. No 20 in Experiment A and Experiment B, respectively) and 46% and -0,45 (for compound Z1 in Experiment A and Experiment B, respectively). The mixture of the tested conjugate constituents achieved TGI% values of 58% (Experiment A) and 41 % (Experiment B). TGI% value obtained for the reference compound (irinotecan) was on a level of 63% (Experiment A) and 41 % (Experiment B). Thus, conjugate of Example 3 of the invention wherein the fusion protein of Example 3 and SN38 (Z1 ) compound are covalently linked exert a much stronger tumour inhibition effect compared to the constituents of the conjugate when administered individually, to a mixture of its constituents and to a reference compound.

Tumour size was measured using an electronic calliper, tumour volume was calculated using the formula: (a² x b)/2, where a = shorter diagonal of the 25 tumour (mm) and b = longer diagonal of the tumour (mm). Inhibition of tumour growth was calculated using the formula:

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.

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.

The tested conjugate 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 tested conjugate of the invention.

Claims

Claims

1. A conjugate, which comprises:

• a fusion protein comprising domain (a), which is the functional fragment of a hTRAIL protein sequence, said fragment beginning 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 domain (b) which is the sequence of an immunostimulating effector peptide, and

• the molecule of a chemical compound Z with antiblastic activity, which is linked to the fusion protein directly or by means of a conjugation linker L.

2. The conjugate according to claim 1 , wherein domain (a) of the fusion protein comprises the fragment of hTRAIL sequence which begins with an amino acid in the position from the range 95 to 122, inclusive, of the hTRAIL protein sequence, and ends with the amino acid in the position 281 of the hTRAIL sequence.

3. The conjugate according to claim 1 or 2, wherein domain (a) of the fusion protein is selected from the group consisting of fragments of hTRAIL beginning with an amino acid in a position 95, 116, 120, 121 or 122 of hTRAIL sequence and ending with the amino acid in the position 281 of hTRAIL sequence.

4. The conjugate according to any one of claims 1 to 3, wherein domain (b) of the fusion protein is selected from the group consisting of

- the fragment of interferon alpha 2b set forth as SEQ. No. 2;

- the fragment of interferon gamma set forth as SEQ. No. 3;

- the pseudodimer of interferon gamma set forth as SEQ. No. 4;

- the pseudodimer of interferon alpha 2b set forth as SEQ. No. 5; and

- the consensus sequence of interferon alpha set forth as SEQ. No. 6.

5. The conjugate according to any one of the claims 1 to 4, which comprises incorporated in the fusion protein between domain (a) and domain (b) at least one domain (c) comprising a protease cleavage site selected from the group consisting of a sequence recognized by metalloprotease MMP, a sequence recognized by urokinase uPA, a sequence recognized by furin, and combinations thereof.

6. The conjugate according to claim 5, wherein the sequence recognized by metalloprotease MMP is Pro Leu Gly Leu Ala Gly, and the sequence recognized by urokinase uPA is Arg Val Val Arg.

7. The conjugate according to claim 5 or 6, wherein domain (c) is a combination of the sequence recognized by metalloprotease MMP and the sequence recognized by urokinase uPA located next to each other.

8. The conjugate according to any one of the claims 5 to 7, wherein the fusion protein between two domains (c) additionally comprises a conjugation domain (d).

9. The conjugate according to claim 8, wherein the conjugation domain (d) is selected from the group consisting of Ala Ser Gly Cys Gly Pro Glu, Ala Ala Cys Ala Ala, Ser Gly Gly Cys Gly Gly Ser, and Ser Gly Cys Gly Ser.

10. The conjugate according to any one of claims 5 to 9, wherein the fusion protein between domains (a), (b), (c) and/or (d) additionally comprises a glycine-serine steric linker.

1 1 . The conjugate according to claim 10, wherein the glycine-serine steric linker is selected from the group consisting of Gly Ser Gly Gly Gly, Gly Gly Gly Ser, Xaa Gly Gly Ser, wherein Xaa may be any naturally occurring amino acid, and Gly Gly Ser Gly.

12. The conjugate according to any one of claims 1 to 1 1 , wherein the sequence of the fusion protein is selected from the group consisting of SEQ. No. 7; SEQ. No. 8; SEQ. No. 9; SEQ. No. 10; SEQ. No. 1 1 ; SEQ. No. 12; SEQ. No. 1 3; SEQ. No. 14; SEQ. No. 1 5; SEQ. No. 16; SEQ. No. 17; SEQ. No. 18; SEQ. No. 19; SEQ. No. 20; SEQ. No. 21 ; SEQ. No. 22, and SEQ. No. 23.

13. The conjugate according to any one of claims 1 to 12, wherein the fusion protein is a recombinant protein.

14. The conjugate according to any one of claims 1 to 13, wherein the molecule of the chemical compound Z is linked by means of a conjugation linker L.

15. The conjugate according to claim 14, wherein the conjugation linker L is selected from the group consisting of linkers represented by the following formulas L1 to L12:

16. The conjugate according to any one of claims 1 to 15, which wherein the chemical compound Z is selected from the group consisting of compounds represented by the following formulas Z1 to Z24: 77

78

17. The conjugate according to any one of claims 14 to 16, wherein the conjugation linker L and the chemical compound Z are selected from combinations represented by formulas: L1-Z1; L2-Z1; L3-Z1; L4-Z1; L5-Z1; L6- Z1; L7-Z1; L8-Z1; L1-Z2; L1-Z3; L9-Z4; L1-Z5; L1-Z6; L1-Z7; L1-Z8; L9-Z8; L1- 19; L9-Z9; L1-Z10; L9-Z10; L1-Z11; L9-Z11; L1-Z12; L9-Z12; L1-Z13; L9-Z13; L1-Z14; L9-Z14; L1-Z15; L9-Z15; L1-Z16; L9-Z16; L9-Z17; L1-Z17; L1-Z18; L9- Z18; L1-Z19; L9-Z19; L10-Z20; L9-Z21; L1-Z22; L9-Z22; L9-Z23; L1-Z23; L1- Z24; L9-Z24, L11-Z24, L6-Z6, L7-Z6, L9-Z6 and L12-Z1.

18. The conjugate according to any of claims 1 to 17, wherein the fusion protein is represented by amino acid sequence selected from the group consisting of SEQ. No. 20, SEQ. No. 21, SEQ. No. 22 and SEQ. No. 23, the conjugation linker L is selected from the group consisting of linkers represented by the formulas L1 to L12, and the chemical compound Z is selected from the group consisting of compounds represented by the formulas Z1 to Z24.

19. The conjugate according to any of the claims from 1 to 18 selected from the following group: SEQ. No.20-L1-Z1; SEQ. No.21-L1-Z1; SEQ. No.22-L1-Z1; SEQ. No. 23-L1-Z1; SEQ. No. 20-L2-Z1; SEQ. No. 21-L2-Z1; SEQ. No. 22-L2-Z1; SEQ. No. 23-L2-Z1; SEQ. No. 20-L3-Z1; SEQ. No. 21-L3Z1; SEQ. No. 22-L3-Z1; SEQ. No. 23-L3-Z1; SEQ. No. 20-L4-Z1; SEQ. No. 21-L4-Z1; SEQ. No. 22-L4-Z1; SEQ. No. 23-L4-Z1; SEQ. No. 20-L5-Z1; SEQ. No. 21-L5-Z1; SEQ. No. 22-L5-Z1; SEQ. No. 23-L5-Z1; SEQ. No. 20-L6-Z1; SEQ. No. 21-L6-Z1; SEQ. No. 22-L6-Z1; SEQ. No. 23-L6-Z1; SEQ. No. 20-L7-Z1; SEQ. No. 21-L7-Z1; SEQ. No. 22-L7-Z1; SEQ. No. 23-L7-Z1 SEQ. No. 20-L8-Z1 SEQ. No. 21-L8-Z1 SEQ. No. 22-L8-Z1 SEQ. No. 23-L8-Z1 SEQ. No. 20-L1-Z2 SEQ. No. 21-L1-Z2 SEQ. No. 22-L1-Z2 SEQ. No. 23-L1-Z2 SEQ. No. 20-L1-Z3 SEQ. No. 21-L1-Z3 SEQ. No. 22-L1-Z3 SEQ. No. 23-L1-Z3 SEQ. No. 20-L9-Z4 SEQ. No. 21-L9-Z4 SEQ. No. 22-L9-Z4 SEQ. No. 23-L9-Z4 SEQ. No. 20-L1-Z5 SEQ. No. 21-L1-Z5 SEQ. No. 22-L1-Z5 SEQ. No. 23-L1-Z5 SEQ. No. 20-L1-Z6 SEQ. No. 21-L1-Z6 SEQ. No. 22-L1-Z6 SEQ. No. 23-L1-Z6 SEQ. No. 20-L1-Z7 SEQ. No. 21-L1-Z7 SEQ. No. 22-L1-Z7 SEQ. No. 23-L1-Z7 SEQ. No. 20-L1-Z8 SEQ. No. 21-L1-Z8 SEQ. No. 22-L1-Z8 SEQ. No. 23-L1-Z8 SEQ. No. 20-L9-Z8 SEQ. No. 21-L9-Z8 SEQ. No. 22-L9-Z8 SEQ. No. 23-L9-Z8 SEQ. No. 20-L1-Z9 SEQ. No. 21-L1-Z9 SEQ. No. 22-L1-Z9 SEQ. No. 23-L1-Z9 SEQ. No. 20-L9-Z9 SEQ. No. 21-L9-Z9 SEQ. No. 22-L9-Z9

SEQ. No. 23-L9-Z9; SEQ. No. 20-L1-Z10; SEQ. No. 21-L1-Z10; SEQ. No. 22 L1-Z10; SEQ. No.23-L1-Z10; SEQ. No.20-L9-Z10; SEQ. No.21-L9-Z10; SEQ. No

22- L9-Z10; SEQ. No.23-L9-Z10; SEQ. No. 20-L1-Z11; SEQ. No.21-L1-Z11; SEQ No. 22-L1-Z11; SEQ. No. 23-L1-Z11; SEQ. No. 20-L9-Z11; SEQ. No. 21-L9-Z11 SEQ. No. 22-L9-Z11; SEQ. No. 23-L9-Z11; SEQ. No. 20-L1-Z12; SEQ. No

21- L1-Z12; SEQ. No.22-L1-Z12; SEQ. No. 23-L1-Z12; SEQ. No.20-L9-Z12; SEQ No. 21-L9-Z12; SEQ. No. 22-L9-Z12; SEQ. No. 23-L9-Z12; SEQ. No. 20-L1-Z13 SEQ. No. 21-L1-Z13; SEQ. No. 22-L1-Z13; SEQ. No. 23-L1-Z13; SEQ. No

20- L9-Z13; SEQ. No.21-L9-Z13; SEQ. No. 22-L9-Z13; SEQ. No.23-L9-Z13; SEQ No. 20-L1-Z14; SEQ. No. 21-L1-Z14; SEQ. No. 22-L1-Z14; SEQ. No. 23-L1-Z14 SEQ. No. 20-L9-Z14; SEQ. No. 21-L9-Z14; SEQ. No. 22-L9-Z14; SEQ. No

23- L9-Z14; SEQ. No.20-L1-Z15; SEQ. No. 21-L1-Z15; SEQ. No.22-L1-Z15; SEQ No. 23-L1-Z15; SEQ. No. 20-L9-Z15; SEQ. No. 21-L9-Z15; SEQ. No. 22-L9-Z15 SEQ. No. 23-L9-Z15; SEQ. No. 20-L1-Z16; SEQ. No. 21-L1-Z16; SEQ. No

22- L1-Z16; SEQ. No.23-L1-Z16; SEQ. No. 20-L9-Z16; SEQ. No.21-L9-Z16; SEQ No. 22-L9-Z16; SEQ. No. 23-L9-Z16; SEQ. No. 20-L9-Z17; SEQ. No. 21-L9-Z17 SEQ. No. 22-L9-Z17; SEQ. No. 23-L9-Z17; SEQ. No. 20-L1-Z17; SEQ. No

21- L1-Z17; SEQ. No.22-L1-Z17; SEQ. No. 23-L1-Z17; SEQ. No.20-L1-Z18; SEQ No. 21-L1-Z18; SEQ. No. 22-L1-Z18; SEQ. No. 23-L1-Z18; SEQ. No. 20-L9-Z18 SEQ. No. 21-L9-Z18; SEQ. No. 22-L9-Z18; SEQ. No. 23-L9-Z18; SEQ. No. 20- L1-Z19; SEQ. No.21-L1-Z19; SEQ. No.22-L1-Z19; SEQ. No.23-L1-Z19; SEQ. No.

20- L9-Z19; SEQ. No.21-L9-Z19; SEQ. No. 22-L9-Z19; SEQ. No.23-L9-Z19; SEQ. No. 20-L10-Z20; SEQ. No. 21-L10-Z20; SEQ. No. 22-L10-Z20; SEQ. No. 23- L10-Z20; SEQ. No. 20-L9-Z21; SEQ. No. 21-L9-Z21; SEQ. No. 22-L9-Z21; SEQ. No. 23-L9-Z21; SEQ. No. 20-L1-Z22; SEQ. No. 21-L1-Z22; SEQ. No. 22-L1-Z22; SEQ. No. 23-L1-Z22; SEQ. No. 20-L9-Z22; SEQ. No. 21-L9-Z22; SEQ. No.

22- L9-Z22; SEQ. No.23-L9-Z22; SEQ. No. 20-L9-Z23; SEQ. No.21-L9-Z23; SEQ. No. 22-L9-Z23; SEQ. No. 23-L9-Z23; SEQ. No. 20-L1-Z23; SEQ. No. 21-L1-Z23; SEQ. No. 22-L1-Z23; SEQ. No. 23-L1-Z23; SEQ. No. 20-L1-Z24; SEQ. No. 21- L1-Z24; SEQ. No.22-L1-Z24; SEQ. No.23-L1-Z24; SEQ. No.20-L9-Z24; SEQ. No.

21- L9-Z24; SEQ. No. 22-L9-Z24; SEQ. No. 23-L9-Z24; SEQ. No. 20-L11-Z24; SEQ. No. 21 -L1 1 -Z24; SEQ. No. 22-L1 1 -Z24; SEQ. No. 23-L1 1 -Z24; SEQ. No. 20- L6-Z6; SEQ. No. 21 -L6-Z6; SEQ. No. 22-L6-Z6; SEQ. No. 23-L6-Z6; SEQ. No. 20- L7-Z6; SEQ. No. 21 -L7-Z6; SEQ. No. 22-L7-Z6; SEQ. No. 23-L7-Z6; SEQ. No 20- L9-Z6; SEQ. No 21 -L9-Z6; SEQ. No 22-L9-Z6; SEQ. No 23-L9-Z6; SEQ. No. 20- L12-Z1 ; SEQ. No. 21 -L12-Z1 ; SEQ. No. 22-L12-Z1 , and SEQ. No. 23-L12-Z1 ;

20. A pharmaceutical composition, comprising as an active ingredient the conjugate as defined in any one of claims 1 to 19 in combination with a pharmaceutically acceptable carrier

21 . A pharmaceutical composition as defined in claim 20 in a form for parenteral administration.

24. A conjugate as defined in any of the claims 1 to 19 for use in the treatment of cancer diseases in a mammal, including humans.

25. A method of treatment of cancer diseases in a mammal subject, including human, in a need thereof, comprising administration to said subject of an anti-cancer effective amount of the conjugate as defined in claims 1 to 19 or the pharmaceutical composition as defined in claims 20 or 21 .

26. A chemical compound 4-[({trans-4- [(2,5-dioxo-2,5-dihydro-1 H-pyrrol- 1 - ylo)methyl]cyclohexyl}carbonyl)amine] benzoic acid represented by the formula:

27. A method of linking of a chemical compound to a peptide or protein to form a conjugate, wherein the chemical compound is linked to the peptide or protein by means of 4-[({trans-4- [(2,5-dioxo-2,5-dihydro-1 H-pyrrol- 1 - ylo)methyl]cyclohexyl}carbonyl)amine] benzoic acid as a conjugation linker.