The present invention relates to polymeric conjugates of KillerTRAIL to be used in antitumor and/or anti-inflammatory therapy.

Known art

The use of biotechnological drugs in the pharmaceutical sector represents the greatest innovation and therapeutic opportunity of the last decade. The compounds with a peptide and protein structure have a high selectivity and pharmacological activity, properties that make them therapeutic agents of first choice in the treatment of serious diseases such as tumors. However, despite the high pharmacological potential, the clinical use of these therapeutic agents is often limited by their inadequate physico chemical and biopharmaceutical characteristics. The high structural complexity makes the protein macromolecules not very stable and easily inactivated by both physical, chemical and enzymatic processes. These processes can occur during the various stages of formulation development, or during storage or administration. The rapid elimination of these drugs from the systemic circulation, through various mechanisms including glomerular ultrafiltration and hepatic uptake, significantly reduces their bioavailability. The macromolecular character and the high hydrophilicity also prevent absorption through biological membranes and make their administration difficult. Finally, the intrinsic immunological characteristics of these macromolecules, such as immunogenicity and antigenicity, often represent an important limitation for their therapeutic use.

The biopharmaceutical problems that limit the use of protein drugs can be partially overcome by creating formulations that allow to improve their stability and, in some cases, to control their release rate, reduce side effects and optimize their therapeutic dose, with a consequent improvement in patient compliance. Among the techniques used for the formulation of protein drugs currently known is the surface modification of proteins with biocompatible, biodegradable and soluble polymers, such as polyacrylates, polyalkylglycols, polysaccharides, polysialic acid, etc.

Components of thefamily of cytokines Tumor Necrosis Factored as mediators of the immune system and inflammatory response. Their involvement in septic shock, autoimmune diseases and immune reactions related to transplant rejection has in fact been demonstrated (Wiley SR et al., Immunity. 1995; 3 (6): 673-82).

Among the members of TNF, the ligands Apo2 or Apo2L or TRAIL, (all the indicated terms are to be considered synonyms) and KillerTRAIL are mentioned as relevant for the present invention.

The ligand Apo2L wt that induces apoptosis on CD253 is a type of trans-membrane protein II of about 34 kDa, identified in the years 1995/1996 (Pitti RM et al., J. Biol. Chem. 1996; 271 (22) : 12687-90, Wiley SR et al., Immunity. 1995; 3 (6): 673-82). Apo2L or TRAIL is a protein consisting of an amino acid sequence of 281 aa in human form and has a molecular weight of approximately 32.5 kDa. TRAIL is characterized by a transmembrane domain, an extracellular C-terminal domain rich in cysteines and conserved among members of the same family and a cytoplasmic N-terminal domain that instead presents unique characteristics. Crystallographic studies have identified its structure as homotrimeric with three cysteine residues in position 230 of each sub-unit of the homotrimer, coordinated with each other through a Zn atom, which is important for the stability and biological activity of the trimer (Hymowitz SG et al., Molec. Cell. 1999; 4 (4): 563-71 ; Bodmer JL et al., J. Biol. Che., 2000; 275 (27): 20632- 7).

The patent document W02004001009 describes variants of the Apo2L/TRAIL protein and reports the derivatization with polyethylene glycol (PEG) performed on cysteines inserted in the chain of the variants of TRAIL in position 170 or 179 (indicated in the document respectively as PEG- R170C-Apo 2L and PEG -K179C-Apo 2L). PEGylation, which affects the cysteine residues, is considered useful only if carried out on the cysteines inserted in the variants in position 170 and 179 in order to preserve the cysteine in position 230, considered fundamental for the biological activity and therefore not modifiable in order not to modify the homotrimeric structure and the corresponding activity of the protein.

This binding site has been shown to be important for protein activity. In fact, the substitution of cysteine 230 (Cys 230) determines an 8-fold reduction in apoptotic activity. Furthermore, removal of the coordinated metal from the homotrimer cystines causes a 7-fold decrease in affinity for DR5 and a 90-fold reduction in apoptotic activity. Furthermore, after the removal of the coordination Zn, the cysteines can be oxidized and Apo-2L dimers bonded with disulfide bridges are formed, which reduce apoptotic activity. The binding site of the coordination metal is hidden within the structure of the Apo-2L trimer and thus prevents its interaction with the receptor.

As reported in W02004001009 and in the literature prior to it (Flymowitz SG et al., Biochemistry, 2000; 39 (4): 633-40; Bodmer et al., J. Biol. Chem., 2000; 275 (27): 20632-7), the bond with the divalent metal ion appears to be of fundamental importance to maintain the structure of the trimer and the stability of Apo-2L and the consequent biological activity. In document W02004001009, therefore, a derivatization with PEG is carried out on the cysteines inserted in the polypeptide variants of Apo2L in order to maintain the trimerization and evaluate their therapeutic efficacy. The derivatizations with PEG considered particularly interesting are those carried out on the mutations in which the cysteine residues are in position C170 and C179.

The insertion of further cysteines in addition to the naturally present Cys 230, and their corresponding derivatization through PEG actually produce an improvement in the therapeutic activity, which however does not seem to be considered by the inventors to be optimal for use in the pharmaceutical field, as despite some potentially encouraging results, even if obtained by modifying the peptide chain of the native protein, no other research published later has been carried out. In fact, making changes on the native protein chain by substituting one or more amino acids involves the ab origine manipulation of the genetic material. This situation complicates the processing and significantly increases the production costs and the risks of administration (in fact the metabolic characteristics and the potential adverse effects, especially hepatic, of the varied protein are not known). Furthermore, the improvement of the activity obtained through the changes reported in the aforementioned document is not considered such as to justify the risk of use.

Among all the recombinant versions of TRAIL a soluble form bearing a polyhistidine tag is known (Jo M et al., Nat. Med., 2000; 6 (5): 564-7; Nagata S, Nat. Med., 2000; 6 (5): 502-3).

The known compound monomeric human KillerTRAIL and present on the market (http://www.enzoiifesciences.com/ALX-201 -073/kiliertraii- protein-solubie-human-recombinant/) manufactured by E. coli, lends itself to being stabilized and conveyed after conjugation with biopolymers. This variant of KillerTRAIL is active on all human and murine receptors and on the osteoprotegerin receptor.

The ferry and therapeutic potentials of this ligand are manifold. However, the problem of its toxicity remains, linked to the aggregation of the protein after systemic administration, which often prevents the continuation of therapy due to the triggering of an immune response towards the same treatment (Le Blanc HN, Ashkenazi A, Cell. Death Differ., 2003; 10 (1): 66- 75; Pan LQ et al., Sci. Rep., 2015; 5: 14872; Almasan A, Ashkenazi A, Cytokine Growth Factor Rev., 2003; 14 (3-4): 337 -48).

From the above considerations it is clear that the conjugation of KillerTRAIL with a polymer can bring benefits, but the complex structure of this protein and its mechanism of action make this approach difficult to implement.

The inventors have now found polymeric systems that simultaneously shield the protein by protecting it from enzymatic degradation and preventing its aggregation. It has also been unexpectedly found that not only the new polymeric derivatives do not preclude the activity of KillerTRAIL but rather, they reduce its short and long term toxicity. Indeed, it has now been found by the authors that KillerTRAIL, conjugated with biopolymers, not only maintains the therapeutic activity of the native protein, but unexpectedly increases its circulatory half-life and prolongs the administration time.

Summary of the invention

The object of the present invention is therefore a method for obtaining KillerTRAIL polymeric conjugates in which the functionalization of the protein is performed at the level of the cysteine site Cys 230. In this way the targeting and therapeutic potential of this ligand is better exploited, significantly reducing the negative effects on specific tissues of the organism through a drug delivery strategyobtained through polymeric conjugation.

Another object of the present invention is a conjugate of KillerTRAIL which overcomes the disadvantages of the known art and is used for the treatment of tumors and autoimmune diseases.

Still another object of the invention are the pharmaceutical and diagnostic compositions comprising the polymeric conjugate of KillerTRAIL.

A further object of the invention is the use of the conjugate for the diagnosis of tumors and autoimmune pathologies. In fact, the cytokine TRAIL, as well as its receptors, is involved in both tumorigenesis (for example, ductal adenocarcinoma of the pancreas, neuroendocrine tumors, non-small cell lung cancer, large cell carcinoma, adenocarcinoma, carcinoma of the uterine cervix, mesothelioma, rhabdomyosarcoma, gliomas and their subtypes, chronic lymphoid leukemia) and in the development and progression of various autoimmune diseases (e.g., multiple sclerosis, autoimmune thyroiditis, Crohn's disease, active ulcerative colitis, autoimmune encephalomyelitis, atopic dermatitis, systemic lupus erythematosus , rheumatoid arthritis, autoimmune arthritis, autoimmune diabetes) (Lin WW, Hsieh SL, Biochem. Pharmacol., 2011 ; 81 (7): 838-47; Schaefer U et al., Front. Biosci., 2007; 12: 3813) . For this reason, the conjugate of the invention can be advantageously used both for the early diagnosis and in the treatment of pathologies in which the cytokine TRAIL is involved. The radiolabeled conjugate of the invention is therefore considered a useful tool in order to "follow" the same protein to the target, in this case the tumor tissue or the tissue affected by the autoimmune pathology and, to determine the pathological state and the subsequent therapeutic procedure. This is made possible by the presence of the polymer conjugated to the cytokine KillerTRAIL which prevents premature degradation and metabolization, without hindering or compromising targeting and therapeutic effect.

Still another object is a pharmaceutical or diagnostic kit comprising the KillerTRAIL polymeric conjugate, which can be administered in a hospital setting for the preventive diagnosis of such pathologies.

Further objects, objects and advantages will become apparent from the detailed description of the invention which follows.

Brief description of the figures

Figure 1. Synthesis scheme of mPEG-KillerTRAIL.

Figure 2. Reverse phase HPLC analysis (RP-C18) of the conjugation chemical reaction between KillerTRAIL and mPEG-maleimide at different incubation times (0, 4, 7 h). Key to the synthesis process: A) KillerTRAIL mono-PEGylated; B) KillerTRAIL bi-PEGylated.

Figure 3. SEC analysis of the purified derivative mPEG-KillerTRAIL at a concentration of 1 mg/mL. Legend: A) Refractive index; B) RALS; C) DP viscometer; D) LALS; E) UV.

Figure 4. Mass spectrum of the mPEG-maleimide structure (A, 2119.4 Da) and of the mPEG-KillerTRAIL conjugate (B, 24008.6 Da) by KillerTRAIL mass spectrometry.

Figure 5. SDS-PAGE of the conjugation reactions of KillerTRAIL with PEG 20 kDa as an indicative example). The reaction was performed under reducing (RB) and non-reducing (NRB) conditions. KillerTRAIL was used as a reference standard to monitor the reaction process.

Figure 6. Cell viability inhibition In vitro and relative anti-tumor activity of KillerTRAIL and its derivative mPEG-KillerTRAIL on human colon cancer cells HCT-116 and human keratinocytes NCTC2544.

Figure 7. Pharmacokinetic profile of KillerTRAIL and its derivative mPEG- KillerTRAIL in blood and tissues: KillerTRAIL (A); mPEG-KillerTRAIL (B); plasma concentration of KillerTRAIL and its derivative mPEG-KillerTRAIL at different incubation times (C).

Figure 8. Biodistribution of KillerTRAIL (A) and the derivative mPEG- KillerTRAIL (B) in BALB/c nude mice implanted with HCT 116 tumor cells (3 x 10 ⁶ cells/ml) inoculated subcutaneously to favor the development of the tumor mass . (C) Summary graph of the percentage concentration of native protein accumulated in blood, liver and tumor mass of BALB/c nude mice implanted with HCT116 tumor cells (3 c 10 ⁶ cells/ml) and treated with KillerTRAIL (A) and with the derivative mPEG-KillerTRAIL (B). KillerTRAIL (A) and mPEG-KillerTRAIL (B) were administered in the above-mentioned mouse models intra-peritoneally (ip) with a protein equivalent dose corresponding to 0.04 mg/kg of KillerTRAIL per day for 4 days.

Figure 9. anti-tumor activity In vivo of KillerTRAIL and mPEG-KillerTRAIL in mouse models implanted with HCT116 tumor cells. The in vivo antitumor activity was evaluated by measuring tumor growth for 20 days after treatment of mouse models with KillerTRAIL and mPEG-KillerTRAIL administered ip at the equivalent dose of 0.04 mg/kg/day.

Figure 10. Comparison between the activity data obtained by the present invention (panel A reproduced from Figure 9) and those contained in the patent W02004001009A2 (panel A) relating to the PEGilata Apo-2L R170C variant and (panel B) relating to the PEGilata Apo- 2L K179C.

Figure 11. Mass spectra of the sequences relating to tryptic peptides, the sequences of which are reported in Table 1 , obtained by hydrolysis in trypsin of the KillerTRAIL protein for MS-Digest analysis. Panel A and B: mass spectra of the sequences aa in position 125-130 (Table 1 ); CD panel: mass spectra of the aa sequences in position 80-85 (Table 1 ); Panel EF: mass spectra of the aa sequences in position 56-61 (Table 1 ); GH panel: mass spectra of the aa sequences in position 5-12 (Table 1 ); Panel IJ: mass spectra of the aa sequences in position 170-177 (Table 1 ); Panel KL: mass spectra of the aa sequences in position 111 -118 (Table 1 ); Panel MN: mass spectra of the aa sequences in position 28-38 (Table 1 ); OP panel: mass spectra of the aa sequences in position 39-50 (Table 1 ); QR panel: mass spectra of the aa sequences in position 159-169 (Table 1 ); ST panel: mass spectra of the aa sequences in position 143-155 (Table 1 ); UV panel: mass spectra of the aa sequences at position 85-97 (Table 1 ); Panel WX: mass spectra of the aa sequences at position 162-177 (Table 1 ); WZ panel: mass spectra of the aa sequences at position 20-36 (Table 1 ).

Figure 12. Mass spectra of the sequences relating to tryptic peptides, the sequences of which are shown in Table 4, obtained by hydrolysis of the KillerTRAIL protein in trypsin for MS-Digest analysis.

Figure 13. ESI MS mass spectrum related to the KillerTRAIL sample. Panel B: mass spectrum with high instrumental signal (TIC, Total Ion Current); Panel C: mass spectrum of the instrumental low signal protein (TIC, Total Ion Current); Panel A: molecular weight of the protein obtained following the deconvolution process applied to the mass spectra reported in panels B and C.

Detailed Description of the Invention

With the aim of overcoming the critical problems typical of therapeutic proteins such as rapid elimination, degradation and toxicity deriving from the formation of immune complexes, KillerTRAIL was derivatized in a single process of conjugation with a polymer chain.

Therefore, according to the present invention, the new molecular entity composed of the polyethylene glycol polymer and the single-stranded KillerTRAIL protein, linked by a stable covalent bond of the CS type on the cysteine in position 230 of the KillerTRAIL, is defined as the polymeric conjugate of KillerTRAIL. Tumor Necrosis Factor (KillerTRAIL) Enzo data sheet (htp://www enzoiifesciences.com/fiieadmin/reports/Datasheet-ALX-

Tumor Necrosis Factor (KillerTRAIL) produced by Enzo Life Sciences, Inc. or Apo-2L, TNFSF 10, CD253 has a molecular weight of ~ 24kDa, calculated by the manufacturing company using the Sodium Dodecyl Sulphate - PolyAcrylamide Gel ElectrophoresisSDS orPAGE method (http /www.enzolifesciences.com/fileadmin/reports/Datasheet- ALX-201~073.pdf), and referred to the molecular weight of the commercial monomer. The KillerTRAIL protein is produced using as a cloning vector E. coli. The extracellular domain of the human TRAIL protein, which has the specific sequence constituted by aa 95-281 , is fused in its N-terminal domain with a His-tag and a peptide-structured spacer.

The KillerTRAIL comprises the amino acid sequence aa 95-281 (SEQ.ID.N.

TSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNE KALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQE EIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQ GGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG 1) as official database Expasy.org.

The present invention therefore relates to the realization of a polymeric conjugate of the pro-apoptotic therapeutic protein of the exogenous type, the cytokine KillerTRAIL, which makes it possible to make its tertiary structure more stable. This cytokine induces apoptosis upon binding to pro-apoptotic transmembrane receptors.

The aim achieved by the present invention was to reduce the toxicity problems (hepatic) and low half-life shown by KillerTRAIL through a conjugation on a polymeric backbone at the level of Cys 230. The conjugation was obtained by binding with an alkoxy - preferably methoxy- polyethylene glycol (mPEG) derivatized with a thiol reactive group, preferably a maleimide. The molecular weight of the PEG was preferably of a magnitude comprised between 2-200 kDa, and preferably 10-50 kDa. The process for obtaining the derivative according to the invention involves the thiol of the cysteine in position 230 of the peptide chain of KillerTRAIL with an alkoxy-PEG through the maleimide reactive group of the PEG. The reaction can take place in an aqueous environment in a single step, preferably after the prior purification of the native KillerTRAIL, for example by dialysis against PBS (pH 7.4) containing EDTA to allow the elimination of dithiothreitol (DTT) used as a preservative in the preservation of KillerTRAIL and in the case of the reaction, to avoid the spontaneous formation of disulfide bridges and aggregation between the protein monomers.

The alkoxy-PEG has the alkoxy group in alpha and is preferably the methoxy-PEG (mPEG) and the reactive group towards cysteine is in omega. The PEGs with reactive groups that can be used can be chosen from: PEG- Maleimide or mal-PEG, PEG-orthopyridine disulfide (OPSS-PEG), PEG iodine-acetamide (IA-PEG) and PEG-vinyl sulfone (VS-PEG) . Among these, mal-PEG is preferred which among all guarantees a stable, non- cleavable bond in vivo. Therefore the preferred conjugation group for KillerTRAIL is methoxy-PEG-maleimide conjugated on KillerTRAIL via the thiol -SH group of the amino acid cysteine in position 230.

It is clear that PEGylation can occur using different methods, through a series of conventional reactions. For example, an N-hydroxysuccinimide ester of a PEG (M-NHS-PEG) can be prepared from PEG-monomethyl- ether (which is commercially available from Union Carbide) by reaction with N,N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), according to the method of Buckmann et al. (Buckmann AF, et al., Macromol. Chem. Phys. 1981 ; 182 (5): 1379-84). Furthermore, a PEG terminal hydroxy group can be converted to an amino group, for example by reaction with thionyl bromide to form PEG-Br, followed by aminolysis with excess ammonia to form PEG-NH2. The PEG-NH2 is then conjugated to the protein of interest using standard coupling reagents, such as Woodward's K reagent. Furthermore, a terminal group PEG-CH2OH can be converted into an aldehyde group, for example by oxidation with Mn02. The aldehyde group is conjugated to the protein by reductive alkylation with a reagent such as cyanoborohydride. Alternatively, activated PEGs suitable for use in the present invention can be purchased from a number of vendors. For example, Shearwater Polymers, Inc. (Huntsville, Ala.) sells methoxy-PEG- maleimide, MW 2000, as well as a methoxy-PEG succinimidyl carbonate and methoxy-PEG succinimidyl propionate. In any case, our choice fell on a more site-specific type of bond, as generally the cysteines (and therefore the thiol groups) are in a reduced quantity compared to the other amino acids and in this specific case KillerTRAIL only has a. In fact, it is preferable to avoid random bonds on endogenous proteins.

The PEGylated protein can be characterized by SDS-PAGE, gel filtration, NMR, peptide mapping, liquid chromatography-mass spectrophotometry in vitro and biological assays, all methods known to those skilled in the art. The polymeric complex according to the present invention was characterized by SDS-PAGE, colorimetric assays, Gel Filtration/Dimensional Exclusion chromatography with downstream tetradetector (DLS, viscometer, UV/Vis, Refractive index), high performance chromatography, MALDI-TOF mass and in vitro biological assays. The first phase of processing consists in the purification of the protein by dialysis (regenerated cellulose cut-off 10000 Da) to eliminate the solution in which the protein has been stored (Salts, ethylenediaminetetraacetic acid EDTA, dithiothreitol DTT) in which it is marketed. Subsequently, a treatment of the protein solution with 8 mM DTT alone was carried out at room temperature for 1 hour in order to avoid the spontaneous formation of disulfide bridges that would lead to the formation of the protein in the form of a trimer. To maintain the optimal conditions for the conjugate production, the protein solution from the DTT was further purified by desalting chromatography column. Under these experimental conditions the KillerTRAIL protein is found in monomeric form.

At this point the functionalized PEG and the KillerTRAIL protein thus treated and purified, is solubilized in aqueous environment in the presence of a molar excess of PEG (typically in a molar ratio 4/1 during the synthesis phase of the derivative) in buffer, preferred is the phosphate buffer (pH 7.4) and are left under continuous stirring at room temperature, typically for about 7 hours. The reaction can be monitored by high performance chromatography (HPLC) in reverse phase (on column C18) from time t = 0 h and at time t = 7 h.

KillerTRAIL PEGylate is purified for example by gel filtration chromatography to eliminate unreacted PEG. At the end of the reaction process there will be a 1/1 molar ratio between PEG and native protein with an overall reaction yield equal to 70%.

KillerTRAIL PEGylate is then lyophilized and can be stored at a temperature of -20°C for subsequent uses.

The reaction takes place easily in an aqueous environment allowing the selective binding of the PEG with the thiol of the cysteine in position 230 of the peptide chain, by means of the preferred maleimide reactive group.

This reaction can take place in a single step, after the purification of the native KillerTRAIL by dialysis against PBS (pH 7.4) containing 0.05 mM of EDTA to allow the elimination of dithiothreitol (DTT), used as a preservative during the preservation of the protein.

The present invention therefore reports a KillerTRAIL polymeric conjugate capable of preserving at least 5% of the activity of the native protein, more preferably 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120% or 130% of the activity of native, i.e. unconjugated, KillerTRAIL.

The polymer conjugated to the protein preferably has an average molecular weight (MW) between 200 dalton (Da) and 300000 Da, more preferably between 3000-100000 Da, even more preferably between 5000- 80000 Da, even more preferably between 10000-40000 Da , and even more preferably between 10000-30000 Da, and more preferably 20,000 Da.

In preferred forms of the invention the polymer is selected from among those of the following group: poly (ethylene glycol) (PEG) of linear or branched structure (branched), preferably monofunctional, preferably having a molecular weight (MW) average of between 2000 and 40000 Da Da, more preferably between 2000 Da and 30000 Da and even more preferably between 5000 Da and 20000 Da. The most commonly used polyethylene glycols or PEGs are: mPEG-Maleimide 10000 Da, mPEG- Maleimide 20000 Da, mPEG-Maleimide 40000 Da, mPEG -Maleimide branched 10000 Da, mPEG-Maleimide branched 20000 Da and mPEG- Maleimide branched 40000 Da.

The polymeric conjugates obtained, of which the general name will be hereafter for simplicity and not for limitation mPEG-KillerTRAIL, can be used for all those pathologies in which the presence of programmed cell death receptors (Apoptosis), implicated in a series of tumor, immune, angiogenic and turnover bone pathologies was evaluated. KillerT RAIL can bind to five different receptors found on a variety of cell types: four membrane receptors and one soluble. Two of these membrane receptors, TRAIL-R1 /death receptor 4 (DR4) and TRAIL-R2/death receptor 5 (DR5), act as agonist receptors, containing a cytoplasmic death domain through which KillerTRAIL can transmit an apoptotic signal. The other two membrane receptors, TRAIL-R3/c/ecoy receptor 1 (DcR1 ) and TRAIL- R4/ decoy receptor 2 (DcR2), act as antagonist/regulatory receptors, devoid of the death domain. In addition to these four transmembrane receptors, a soluble fifth antagonist receptor osteoprotegerin (OPG), responsible for, has been identified turnover bone. The pathologies to which we mainly refer are various types of solid and liquid tumors. The over-expression of the listed receptors is present in many pathologies listed in table 1 and, without constituting a limitation, these pathologies can be advantageously treated with the mPEG-KillerTRAIL according to the invention: Table 1 : Some pathologies whose cells over express death receptors related to the cytokine KillerTRAIL

Among the most studied we report pancreatic carcinoma (ductal adenocarcinoma of the pancreas and neuroendocrine tumors, which originate from the cells of the islets of Langerhans), non-small cell lung cancer (squamous cell carcinoma, large cell carcinoma and adenocarcinoma that in turn is subdivided into sarcomatous and pulmonary lymphomas), carcinoma of the uterine cervix (squamous cell carcinoma and adenocarcinoma), mesothelioma (affects and differs in various tissues: lung (pleura), heart (pericardium), intestine (peritoneum) and testes (tunica vaginalis), rhabdomyosarcoma (embryonic and alveolar), gliomas and related subtypes (glioblastoma, anaplastic astrocytoma, fibrillary astrocytoma, pilocytic astrocytoma, other astrocytoma, oligodendroglioma, ependymomas), the chronic lymphoid leukemia.

The conjugates produced can be used parenterally, together with vehicles such as water, buffer solutions, physiological solutions, stabilizers i (for example antioxidants), chelating agents (for example ethylenediamino tetracetic acid or EDTA), and can be produced under sterile conditions according to methods known to the skilled in the art. The term parenteral defines the type of administration by injection through the integuments or directly into the circulation (for example the intradermal route, the subcutaneous route, the intramuscular route and the intravenous route).

The conjugates produced can advantageously be administered in useful therapeutic doses, significantly increasing the efficacy of the therapy in terms of duration of the therapeutic effect (effect due to the reduction of clearance renal) and reduction of the formation of immune complexes (due to the steric hindrance of the polymer which prevents the aggregation of multiple protein molecules). The therapeutically useful doses will be defined by the specialist doctor also according to the correspondence of the individual patient. Furthermore, the conjugates produced allow, due to passive targeting phenomena, which exploits the natural tendency of some cells (liver, macrophages) to engulf foreign agents to the organism, the accumulation in the tumor tissue due to the EPR phenomenon ( Enhanced Permeation and Retention) leading to dose decrease in healthy tissues. The conjugation with the PEG also allows the accumulation at the level of diseased tissues thanks to the increase in size but, above all, thanks to the increase in the plasma half-life of KillerTRAIL. The diseased tissue, be it inflamed or tumor, has the peculiarity of having more penetrable blood vessels (fenestrated, to allow the passage of nutrients and those recalled by the inflammatory process) and an amplified retention effect, the latter due to a pH more acid and an absence/loss of lymphatic drainage to retain necessary substances (EPR effect) (Greish K., J. Drug Target., 2007; 15 (7- 8): 457-64). For this reason, the polymeric conjugate is naturally more likely to reach diseased tissue than healthy tissue and compared to the native protein, which degenerates faster, will reach the target site in less quantity and only for a short period of time.

The pharmaceutical compositions comprising the polymeric conjugate PEG-KillerTRAIL, according to the invention, can be prepared by mixing this polymeric conjugate with adjuvants, excipients or stabilizers that meet the requirements for the excipients for pharmaceutical use present on the market (Remington's Pharmaceutical Sciences, 16th edition , Osol, A. ed. (1980)), in the form of lyophilized formulations, aqueous solutions or aqueous suspensions. The carriers, excipients or stabilizers used are preferably selected from: buffers such as Tris, HEPES, phosphate, citrate and other salts of organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; low molecular weight polypeptides (less than approximately 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; sugars such as sucrose, mannitol, trehalose or sorbitol; salt forming counter-ions such as sodium; and/or non-ionic surfactants such as polysorbates or TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Further examples of such carriers include ion exchangers, alumina, stearate of aluminum, lecithin, serum proteins, such as human serum albumin, buffers such as glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone and cellulose-based substances Carriers for topical or gel forms include polysaccharides such as carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylates, ethylene-propylene block polymers, PEGs and waxes. For all the administrations, the known conventional forms are suitably used. Such forms include, for example, microcapsules, nano-capsules, liposomes, patches, inhalation forms, nasal sprays, sublingual tablets and prolonged release preparations.

KillerTRAIL polymeric conjugates to be used for in vivo administration must be sterile. The sterility process is easily achieved by filtration through sterile filter membranes (0.22 pm), before or after lyophilization and relative reconstitution. The polymeric conjugates of KillerTRAIL are normally stored in anhydrous form, for example lyophilized, or in solution if used for systemic administration. If in lyophilized form, KillerTRAIL polymer conjugates are typically formulated in combination with other excipients used for their reconstitution (cryoprotectants such as, for example, mannitol, trehalose, sucrose, sorbitol, etc.) and with an appropriate diluent at the time of use. An example of a liquid formulation is a sterile, clear, colorless, unpreserved solution filled in a single-dose vial for subcutaneous injection.

The formulations can for example be placed in a container provided with a sterile access port, for example a bag or a vial of intravenous solution having a cap that can be pierced by a hypodermic injection needle. The formulations are preferably administered as repeated intravenous (iv), subcutaneous (sc), intramuscular (im) injections or infusions or as aerosols suitable for intra-nasal or intra-pulmonary delivery.

As regards the use of the invention for diagnostic purposes, it is possible to foresee the same synthesis with the use of the same radio protein labeled with ¹²⁵ l (Xiang H et al., Drug Metab-Dispos., 2004; 32 (11 ) : 1230-8; Duiker EW. Et al., Br. J. Pharmacol., 2012; 165 (7): 2203-12) or ¹⁸⁸ Re / ¹⁸⁸ W (Wang XG. Et al., Ther. Deliv., 2014 ; 5 (2): 139-47). In addition to the substances listed above, the radio-labeled PEGylated protein is added to the formulation to be administered so that it can be followed through the classic diagnostic imaging techniques used for functional analyzes, nuclear magnetic resonance associated with positron emission tomography and computed axial tomography associated with positron emission tomography (MRN/PET and CT/PET respectively). Also according to what is reported in the literature (Xiang H. et al., Drug Metab. Dispos., 2004; 32 (11 ): 1230-8), the presence of the radioisotope can increase the stability in plasma and the therapeutic efficacy and this, reasonably, leads us to think that the invention in question, already in itself improving safety (fewer side effects due to the formation of aggregates and rapid metabolism), stability and efficacy, can further increase the positive characteristics of this drug, especially in the context of safety in therapy. The compositions comprising KillerTRAIL may further comprise additional therapeutic agents such as: 5-fluorouracil, CPT-11 , (Ashkenazi A et al., J. Clin. Invest., 1999; 104 (2): 155-62) and/or be administered in combination with ionizing radiation (Chinnaiyan AM et al., Proc. Natl. Acad. Sci. USA, 2000; 97 (4): 1754-9) and/or diagnostic agents if radiolabelled, such as ¹²⁵ l, ¹⁸⁸ Re, ¹⁸⁸ W and related combinations.

It is important to highlight that, according to the literature reported in the previous paragraphs, the Apo-2L TRAIL protein and consequently also the KillerTRAIL variant, are proteins that exist in a trimeric form and as such act determining the therapeutic effect. However, the invention relates to the direct PEGylation of the single KillerTRAIL chain, resulting in the obtaining of a new macromolecular entity containing the PEG polymer (various molecular weights as indicated above) and a single protein chain. This new invention has proved to be an improvement over both the unconjugated trimeric KillerTRAIL and the PEGylated variants (R170C Apo-2l-PEG and K179C Apo-el-PEG) reported in patent W02004001009A2. The improvement is to be understood on various points of view listed below:

1 ) No changes to the native KillerTRAIL chain are imposed.

2) The dosage used is significantly lower.

3) The therapeutic efficacy is maintained or increased even for a longer time.

4) Low or no mortality of the animal models used. For those pathologies for which the positive effects of this therapy have been ascertained, the advantages of the invention are seen primarily in the increase in the plasma half-life of KillerTRAIL, due to the presence of the mPEG polymer which manages to protect the protein from degradation due to enzymes plasma. This leads to an increase in therapeutic activity over time and therefore to a decrease in doses or to an extension of the time elapsing between one dose and the next.

The decrease in the concentration per single dose or the decrease in the number of doses required, reduce the toxic effect of KillerTRAIL (hepatotoxicity, toxicity at the injection site and serious side effects due to the increase in stimulation of TNF receptors even in healthy tissues, such as MOF, multiple organ failure) and also increases compliance patient. All these positive effects improve the therapeutic approach of the previously listed disabling diseases.

The following examples are to be considered illustrative and not limitative of the scope of the invention.

EXAMPLES

Materials

The KillerTRAIL was purchased by Enzo Lifescience (Florence, Italy). mPEG-maleimide (methoxy-N (maleimide (polyethylene glycol)) (MW 2-200 kDa) was purchased from Iris Biotech srl (Germany). Ethylene- diamino-tetraacetic acid (EDTA), phosphate buffer solution (PBS), kit Bicinconinic acid assay, kits TRAIL ELISA, picrylsulfonic acid, 5,5'-dithiobis (2-nitrobenzoate), acrylamide, bisacrylamide, ammonium persulfate, TEMED, Glycine, Coomassie Blu Brilliant, Bromophenol Blue, LC resins have been purchased from Sigma-Aldrich SpA (Steinheim, Germany). The dialysis membranes used in the experimental phase were purchased from Spectrum Laboratories Inc. (Eindhoven, The Netherlands). All aqueous solutions were prepared using deionized water (MilliQ-grade, 18.2 MSZ) obtained using the Millipore MilliQ system (MA, USA). All the solvents and the remaining reagents used were selected from those specific for HPLC or with a degree of purity from those available in the catalog from Sigma- Aldrich (St. Louis, MO, USA) or by Carlo Erba ( Milan, Ml, Italy).

The conjugates produced were characterized by high performance chromatography both by the inverse phase technique (C18 RP-HPLC), in which the protein was found to be stable, and by the gel filtration technique (GF-HPLC), with the aim to monitor the progress of the reaction, determine its completion, quantify the yield and purify the derivatives obtained, according to what is reported in the following section.

The products were analyzed by various types of colorimetric assays (BCA, Habeeb, Bradford), to determine the concentration of the protein in solution and by assays at known concentration, to determine the PEG interference on these assays.

The same conjugates were analyzed by molecular exclusion chromatography combined with four detectors positioned in series {Light scattering, refractive index, viscometer and UV/Vis photodiode spectrometer), in order to obtain information on the molecular weight and on the arrangement in space of the conjugate and the protein. Thanks to this method, data on the stability of the conjugated protein in solution and the purity obtained after dialysis were obtained.

In order to know the exact molecular weight, the conjugates were subjected to analysis by MALDI/TOF mass spectroscopy.

Evaluations by circular dichroism (CD) analysis allowed to highlight any changes in the molecular arrangements of the protein (tertiary structure) in the presence of PEG. This was monitored to assess whether the presence of PEG at that position of the amino acid chain would interfere excessively with the spatial conformation of the protein, completely preventing recognition by the receptor site.

Electrophoresis studies were carried out on the conjugates to confirm the chromatographic evaluations, under denaturing conditions, in order to prove the absence of dimers, trimers or protein aggregates in general. The stability of the products was evaluated by comparing it with the stability of the protein in a buffered solution at predetermined times and at room temperature. The analyzes performed at physiological (pH 7.4, PBS) and pathological (pH 5, PBS) pH, are predictive of the behavior of the product during storage. Stability was also evaluated on biological fluids (plasma), with the aim of predicting the behavior of the drug in the bloodstream.

Subsequently, the products were tested in vitro on HTC116 liver cancer cells to obtain data on cytotoxic therapeutic activity (by cell viability assay with 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide or MTT) of the products compared to the native protein.

Analyses were developed in vivo on the derivatives described above, on mouse models ( xenographed) implanted with human hepatic carcinoma cells to determine the improvement of pharmacokinetic parameters, biodistribution and therapeutic antitumor activity in terms of tumor reduction and increased product survival compared to native protein.

Example 1 : Synthesis of the mPEG-KillerTRAIL conjugate

Having ascertained that the native KillerTRAIL protein can be identified and monitored by RPC18-HPLC, we moved on to the synthesis of the derivative which involved the use of the maleimide group of PEG for the selective binding with the thiol of the cysteine in position 230 of the above protein. The molar ratios taken into consideration are all those possible starting from 10/1 to ending at 0.01/1 between the mPEG-MAL/KillerTRAIL reagents, and vice versa. The reaction is maintained at pH 7.4 and the pH measurements were performed using pH indicator papers (Whatman ^® , Sigma Aldrich SpA, Steinheim, Germany).

Below is the reaction scheme (Figure 1 ).

Example 2: Colorimetric analysis using standard assays: Determination of the primary amino groups of the KillerTRAIL derivatives (Habeeb assay). The quantitative determination of the amino groups of the mPEG- KillerTRAIL conjugate was calculated by Habeeb's colorimetric assay with 2,4,6-trinitrobenzenesulphonic acid (TNBS). TNBS is made to react under basic conditions with the amino groups of the samples to be analyzed and the chromophore that is formed is determined by spectrophotometry at the maximum wavelength (Amax) of 420 nm.

The samples to be analyzed were prepared by dissolving the compounds in 975 mI of 0.1 M borate buffer, pH 9.3. 25 mI of TNBS (1% w/v in DMF) were subsequently added to each sample. The absorption at 420 nm of the sample is directly proportional to the concentration of amino groups whose concentration was previously calculated with a calibration line obtained from a solution with known concentrations of glycyl-glycine (Gly- Gly) or monomethoxy-PEG -amino (1T1-PEG-NH2).

Example 3: Chromatographic analyzes.

High performance liquid chromatography (HPLC)

The chromatographic analyses were performed with a High Performance Liquid Chromatography system (Jasco, Tokyo, Japan), consisting of a PU-1580 binary pump system, a MD 1510 diode array detector (DAD) (Tokyo, Japan). A Jupiter C18 Phenomenex analytical column (250 c 4.6 mm, 5pm) was used for the RP-HPLC analysis. The eluents and buffer solutions were filtered with Millipore systems (Benford, MA, USA) equipped with 0.22 pm filters and were sonicated with the ultrasonic system Ultrasonic Cleaner mod. 5210 Branson (Dambury, USA) for at least 30 minutes before their use in chromatographic analysis.

The protein solutions of KillerTRAIL and derivatives were analyzed by RP-C18 HPLC and the elution profile was obtained by spectrophotometric determination at Amax of 280 nm.

The mobile phase used for the evaluation of both proteins was H2O and CH3CN both containing 0.05% (v/v) of TFA. The chromatographic separation was carried out by gradient elution using the following mobile phases: H2O/CH3CN: at time (t) t = 0-90% (v/v) H2O and 10% (v/v ) CH3CN; at = 25 minutes 10% (v/v) H2O and 90% (v/v) of CH3CN; at = 30 minutes 90% (v/v) H2O and 10% (v/v) of CH3CN. The KillerTRAIL conjugation reaction was monitored until the final conjugation product was obtained (Figure 2).

Example 4: Gel Permeation Chromatography/Molecular Exclusion Chromatography (GPC/SEC).

The GPC/SEC technique was used to determine the molecular weight (MW) and MW distribution for different macromolecules, proteins, natural and synthetic polymers. The instrument used for this type of study was the Viscotek GPC/SEC TDAmax (Malvern Company, Viscotek Corp, Houston, USA), a system equipped with a Tetra Detector (TDA) including a Refractive Index (Rl) which provides a accurate determination of molecular weight and specific increase of the refractive index (dn/dc), a detector UV/Vis (programmed for analysis at Amax of 280 nm) that allows to study the chemical composition of the derivatives, a detector differential viscosimetric that provides a measure of the intrinsic viscosity of the derivatives and allows to determine the size, conformation and molecular structure of the derivatives, a double angle Light Scattering detector, one at 7° {Low Angle Light Scattering or LALS) to measure the Absolute MW also of the largest macromolecules, and one at 90° {Right Angle Light Scattering or RALS) to measure the absolute MW of small molecules. The GPCmax features an in line degassing system to remove dissolved gases, allow for optimum pump performance, and improve baseline on all detectors.

The instrument is equipped with a double piston pump, a thermostatted autosampler, which has been equipped with two columns in series A2500 and A6000M (30 cm, 8 mm ID), and an automatic eluent sensor that stops the flow if it detects that the solvent supply is low avoiding damage to the columns.

The eluents used were ultra-pure water (90% v/v) and CH3CN (10% v/v) at a flow rate of 0.7 ml/min. Two standards reference (PEG and Dextran) were used to calibrate the instrument. The data obtained were processed using Omni SEC 4.7 software (Malvern Instruments, Ltd, Worcestershire, UK) and showed the presence of the PEGylated derivative of KillerTRAIL (Figure 3). The data concerning the molecular weight, the polydispersion index, the hydrodynamic radius, and the diffraction increase of the monoPEGylated derivative of KillerTRAIL and its relative PEG residues are reported in Table (Table 2).

Table 2. Analysis of the chemical-physical properties of mPEG-KillerTRAIL by means of the GPCmax SEC tetradetector Viscotek viscometer.

Peak Mw Mw Rh(w RALS lals RV (Dalton /Mn ) Area Area

(ml) s) (nm) (mV/ml (mV/ml

) ) mPEG- 64,19 15,188 1 ,22 9,012 16.28 9.81 0147

KillerTRAI 6 4

L residues 30,67 20.62 1 ,25 4,097 0.38 0.37 0,14 of 9 5 7

PEGylatio n

Peak RV: retention volume.

Mw: molecular weight.

Mw/Mn: polydispersion index. Rh: Hydrodynamic radius.

RALS: right angle light scattering (90°measurament angle).

LALS: low angle light scattering (7°measurement angle) dn/dc: differential increase of the refractive index. Example 5: UV-Vis spectroscopy The first preliminary evaluation of the KillerTRAIL protein was performed spectrophotometrically using a UV/Vis Lamda 25 Perkin-Elmer spectrophotometer (Northwolk, CT, USA) thus determining its known concentration. Starting from these considerations, a calibration line was constructed (y = 4 ^* 10 ⁸ x +0.0213; R ² = 0.9973), to be used as a reference system for analyzing the derivatives of the native protein. The data reported in the literature (Expasy Data-protein, SIB Bioinformatics Resource Portal), showed that the molar extinction coefficient e of the protein fraction 95-281 is 27390 (Abs 0.1% (1 g/l) = 1 .274, assuming that all cysteine residues are in reduced form, A = 280nm).

Example 6: MALDI-TOF mass spectrometry ( Matrix-Assisted Laser Desorption Ionization -Time of Flight).

MALDI-TOF mass spectrometry is an analytical technique that allows to very accurately measure the molecular weight of macromolecules of biological interest and to determine their identity based on the mass/charge ratio, and has been used to characterize derivatives of KillerTRAIL. For analytical purposes only, the mPEG-MAL 2kDa polymer and the respective conjugate with the KillerTRAIL protein were analyzed. The obtained results showed that the KillerTRAIL protein was conjugated to the 2kDa mPEG chains through the maleimide residue leading to the formation of a stable final product with a yield of about 90% (Figure 4).

The spectra were produced with REFLEX instrumentation (AB Sciex 4800 plus MALDI-TOF Analyzer) equipped with an ion source SCOUT capable of operating in positive linear mode. The ions generated by a pulsed UV laser beam (nitrogen laser, Amax = 337 nm) were accelerated to 25 kV. A saturated solution of synapinic acid in acetonitrile/water (1 : 1 v/v) was used as a matrix and mixed with the samples dissolved in 0.1% (v/v) aqueous solution of TFA in a ratio of 1 : 1 v/v .

The data obtained from the MALDI-TOF analysis demonstrated the presence of the PEGylated conjugate with the protein under examination. The analysis was carried out on the m-PEG-MAL 2kDa polymer (Figure 4A) whose molecular weight was 2119.4 Da and on the conjugate (Figure 4B) mPEG-KillerTRAIL thus obtaining the absolute molecular weight of 24008.6 Da.

Example 7: Electrophoretic analysis SDS PAGE (Polyacrylamide gel electrophoresis).

Electrophoresis is an analytical technique that allows to separate charged molecules, on the basis of the different mobility in a given electric field. The proteins were denatured with sodium dodecyl sulfate (SDS), an anionic detergent that binds with great affinity to proteins (one molecule of SDS every two years or so) and gives them a net charge proportional to mass. In this way the effective charge of the polypeptide chains is negligible.

Since all proteins are denatured and have the same mass/charge ratio, the mobility of the molecules depends solely on the molecular weight and porosity of the gel, which acts as a molecular sieve.

A gel was constructed for the electrophoretic run of the protein samples, consisting of a 12% (w/v) running gel and a 4% (w/v) stacking gel. The instrument used was a Mini-PROTEAN ^® Electrophoresis System (Bio- Rad, Laboratories, Inc., USA) equipped with a vertical cell, powered by a power supply. The percentage of polyacrylamide of 10% (w/v) in the gel was used because it is suitable for the resolution of the protein derivatives to be analyzed. For the electrophoretic run, 10 pg of protein were loaded into a final buffer volume of 15 mI. Each sample was denatured with 15 mI of sample buffer consisting of: TRIS 250 mM, pH6.8; 2% (v/v) SDS, 25% (v/v) of b-mercaptoethanol as a reducing agent to break any sulphide bridges present; bromophenol blue 0.1% (w/v); 10% (w/v) of glycerol. The samples diluted in the sample buffer, before being loaded on the gel, were completely denatured by heat treatment at 100°C for at least 5 minutes. The electrophoretic run was carried out at a constant voltage of 100 mV for at least 1 h in a run buffer consisting of: 1 mM TRIS, pH 8.3; glycine 960 mM; SDS 0.1% (w/v); water. At the end of the stroke, to observe the separation of the proteins, the gel was immersed in a dye solution consisting of: 0.1% (w/v) Coomassie Brillant Blue, 40% (v/v) ethanol, 10% (v/v) acetic acid. The acid-ethanol mixture is intended to precipitate the proteins on the gel and fix the added dye there. Finally, the gel was bleached to remove excess dye and highlight only the remaining protein bound. The obtained polyacrylamide gel was then analyzed using a Molecular Imageroptical system ^® (Chemi Doc TM, Biorad Laboratories Inc., USA). The Horizon 58 electrophoretic chamber (Life Techonologies Horizontal Gel Electrophoresis System, USA) was used for the electrophoretic evaluation.

The synthesis of KillerTRAIL derivatives was monitored by SDS- PAGE. This method highlighted the formation of the mPEG-KillerTRAIL conjugate (Figure 5) confirming the previously reported chromatographic data (HPLC and mass spectrometry), thus highlighting the formation of a therapeutic protein with a macromolecular structure with potential therapeutic activity.

Example 8: Analysis of biological activity in vitro of free protein and conjugated protein.

Cytotoxicity studies were conducted on all synthesized conjugates. Furthermore, biological activity and residual activity studies were performed on the KillerTRAIL conjugate alone.

Cell viability was assessed by MTT assay. The MTT salt is reduced in the presence of NADH ( nicotinamide adenine of nucleotide hydrogenase) by cellular mitochondrial dehydrogenase, with formation of insoluble formazan crystals (blue in color). The cytotoxicity study of KillerTRAIL derivatives in vitro was evaluated using human colon cancer cells (HCT116) responsive to human keratinocyte cells (NCTC 2544 cells) not responsive to native protein. The cells were incubated (Guaire ^® TS Autoflow Codue water Jacketed-Incubator), in Petri dishes for cell culture (100 mm x 20 mm) at 37°C (5% CO2) using D-MEM supplemented with glutamate, penicillin (100 lU/ml), streptomycin (100 pg/ml) and FBS (10%, v/v).

Having obtained an 80% confluence, the cells were detached with trypsin (2 ml), collected in a centrifuge tube containing 4 ml of culture medium, further washed with 2 ml of PBS to remove the residual cells and finally transferred to a centrifuge tube. The cell suspension obtained was centrifuged at 1200 rpm at room temperature for 5 minutes with a Heraeus Sepatech Megafuge 1.0 (Heraeus Sepatech, Osterode/Harz, Germany). The pellet obtained was resuspended in an appropriate volume of culture medium (2 ml), seeded in 96-well plates (5 c 10 ³ cells/0.1 ml) and incubated for 24 h at 37°C to favor surface adhesion of cell culture plates.

The culture medium was then removed and the HCT116 and NCTC 2544 NCTC 2544 cells were treated for 24, 48 and 72 h with a solution of KillerTRAIL (100 ng/ml) or with the PEGylated conjugate at the same protein concentration, in order to evaluate cell viability following treatment by MTT- test. Each plate presented untreated cells as a negative control.

After 24 h of incubation, a 10% (w/v) solution of tetrazolium salts solubilized in PBS (5 mg/ml) was added to each well and the plates were incubated again for 3 h. The cell culture medium was removed and the formazan salts (precipitated at the bottom of the well after oxidation) were dissolved with 100 pi of a mixture of DMSO/ethanol (1 : 1 v/v), shaking the plates for 20 minutes at 230 rpm (IKA ^® KS 130 control, IKA ^® Werke GmbH & Co., Staufen, Germany).

The solubilized formazan salts were quantified with a spectrophotometer for microplates (BIO-RAD xMark ™, Bio-Rad Laboratories Sri, Segrate (Ml), Italy) at a wavelength of 570 nm and 690 nm. Cell viability was evaluated by measuring the amount of formazan crystals obtained after the oxidation reaction which is directly proportional to the amount of cells still alive after treatment with the compounds (KillerTRAIL and its PEGylated derivative) reported above.

Cell viability was calculated using the equation below:

Cell viability (%) = (AbsT/AbsC) c 100 where, AbsT is the absorbance of the treated cells and AbsC is the absorbance of the control (untreated cells) . The concentration of formazan is directly proportional to the cell viability, which is the result of the average of three different experiments ± the standard deviation. The results obtained show that KillerTRAIL and its derivative mPEG- KillerTRAIL have antitumor activity against HCT116 cells (Figure 6). In particular, mPEG-KillerTRAIL decreases the cell viability of HCT116 cells by 50% at a concentration of 1 .5 ng/ml. A reduction in cell viability of less than 20% was instead obtained by increasing the equivalent amount of KillerTRAIL in the PEGylated product to 2 ng/ml (Figure 6). No effect of KillerTRAIL and its derivative mPEG-KillerTRAIL was observed on NCTC 2544 human keratinocyte cells as this cell line is an untransformed human cell line which is not sensitive to KillerTRAIL (Figure 6).

Example devaluation In vivo of KillerTRAIL and mPEG-KillerTRAIL

The animals used for the experiment are male Sprague Dawley (SD) rats with an average weight of 220-250 g of 4-5 weeks and BALB/c nude mice (20-25g , 4-5 weeks, purchased from Charles River Laboratories (Germany). The animals were kept in the enclosure under continuous monitoring, divided into groups of 6-8, and subjected to 12 h light/dark cycles, with food and water ad libitum for 2 weeks. The procedures involving the use of animals and their welfare have been conducted in accordance with institutional guidelines that comply with national and international laws and policies (European Community Council Directive, of 24 November 1986, 86/609/EEC).

Example 10: Pharmacokinetic studies of KillerTRAIL and mPEG- KillerTRAIL

The pharmacokinetic characteristics of the KillerTRAIL protein and its conjugate were studied in mouse models (rats) after intraperitoneal (ip) administration. The animals were cannulated through the jugular vein one day before the experiments and divided into 6 groups (n = 5): 3 groups used for the treatment with KillerTRAIL and 3 groups for the treatment with mPEG-KillerTRAIL. The animals subdivided into the different groups reported above were administered with a dose of KillerTRAIL equivalent to 10 pg (0.04 mg/kg) or the molar equivalent of PEGylated conjugates of KillerTRAIL in PBS (pH 6.0). The plasma samples taken at different incubation times, after ip administration, were centrifuged and stored at - 80°C until the time of analysis. The amount of KillerTRAIL in rat plasma samples was evaluated using the commercial protein kit ELISA (BioSource International, Inc.). Pharmacokinetic parameters were calculated from plasma concentration profiles by non-compartmental model analysis. Pharmacokinetic data demonstrated that KillerTRAIL is rapidly removed from the systemic circulation after intraperitoneal (ip) administration, while the mPEG-KillerTtrail derivative remains more in the systemic circulation. The half-life (ti/2) of KillerTRAIL and mPEG-KillerTRAIL has been reported in Table 3; and in particular ti/2 of mPEG-KillerTRAIL is 28.3 times greater thani/2 tof KillerTRAIL (Figure 7). All other pharmacokinetic parameters demonstrate that KillerTRAIL PEGylation increases the systemic circulation of the native protein and reduces its metabolism and elimination (Table 3 and Figure 7). mPEG-KillerTRAIL accumulates more in organs and tissues than KillerTRAIL (Figure7). PEGylation increases the distribution of KillerTRAIL in the systemic circulation (Figure 7) and reduces its uptake by the organs of the reticulum endothelial system (RES) such as the liver, spleen, lungs, kidneys (Figure 7).

Table 3. Pharmacokinetic parameters of KillerTRAIL and its derivative mPEG-KillerTRAIL.

Pharmacokinetic parameters

Formulations fl/2 (h) ^a Cmax 7max(h) ^c Vd (ml) ^d AUC

(pg/ml) ^b (pg/mlxh) ^e

KillerTRAIL 1.3±0.12 1 30±0.17 13.00±0.19 11.3±0.05 mPEG- 37.24±0.26 1.42±0.11 - 43.2±0.23 58±0.016

KillerTRAIL at1/2 = plasma half-life. bCmax = maximum concentration. cTmax = maximum time. dVd = volume of distribution. eAUC = area under the curve.

Example 11: Biodistribution in vivo of KillerTRAIL and mPEG- KilleiTRAIL The evaluation of biodistribution was performed on mice implanted

(xenographed) with HCT116 tumor cells (3 c 10 ⁶ cells/ml). Cells were inoculated subcutaneously into BALB/c nude mice. The tumor mass reached its optimal size after one week. The mice were treated for four days with a single dose (administered ip) of KillerTRAIL and its PEGylated conjugate, and were sacrificed 96 h after administration of the compounds. Tumors and organs (liver, lung, kidney, heart, brain, muscle, bone, pancreas, bladder, stomach, small intestine, colon, spleen) were surgically removed, collected, centrifuged and stored at -80°C until the time of analysis. The samples were extracted and the supernatants obtained were analyzed using a kit commercial ELISA for KillerTRAIL (BioSource International, Inc.).

For tumor targeting in vivo studies, guinea pigs were divided into groups of 8 animals and the analyzes were performed using the concentration value of the PEGylated derivatives within the tumor mass at different incubation times as a reference parameter ( 15, 30, 60, 240 min). The experimental analyzes were performed in duplicate both on the control mice and on the treated samples.

The experimental data obtained showed that KillerTRAIL and its derivative mPEG-KillerTRAIL are distributed in different organs and tissues in a time-dependent manner (0 - 240 min) and the distribution kinetics can be used to evaluate the accumulation rate of the native protein and of its PEGylated derivative in tumor tissue (Figure 8). In particular, KillerTRAIL increases the amount of native protein accumulated in the tumor compared to mPEG-KillerTRAIL; while it reduces that of the native protein present in the bloodstream and in the RES organs due to the effect of PEGylation (Figure 8). This type of effect demonstrates a better biopharmaceutical of the mPEG-KillerTRAIL derivative than KillerTRAIL, while the better selectivity for tumor tissue of KillerTRAIL compared to its PEGylated derivative depends on the greater amount of free native protein able to rapidly bind to the KillerTRAIL receptors present in the tumor tissue. These results are in agreement with the data reported in the literature for other therapeutic proteins, monoclonal antibodies, enzymes, biologically active compounds and drugs conjugated to polymeric macromolecules (from Silva Freitas D et al., Bioconjug Chem., 2013; 24 (3): 456-63; da-Silva-Freitas D. et al., Protein Pept. Lett., 2015; 22 (12): 1133-9).

Example 12: Antitumor activity of KillerTRAIL and mPEG-KillerTRAIL.

The anticancer effect of KillerTRAIL and mPEG-KillerTRAIL was studied in mouse models implanted (xenogratransplanted) with HCT116 tumor cells. After 7 days from inoculation, the mice were treated for 7 days with KillerTRAIL and mPEG-KillerTRAIL (50 or 100 mg/kg/day, dose equivalent to KillerTRAIL). The native protein and its PEGylated derivatives were administered intravenously through the marginal vein of the animal's tail. The volume of the tumor mass was monitored for the next 24 days every 5 days. The volume of the tumor mass was calculated as a function of the longitudinal length (L) and the transverse diameter (W) of the tumor using the following formula:

V = (L x W2)/2

In contrast, the tumor growth inhibition values (TGI) were calculated using the following equation:

TGI = (1 -TV sample/TV control) c 100

Where TV is the tumor volume. The antitumor activity of KillerTRAIL and mPEG-KillerTRAIL was evaluated in mouse models implanted (xenografted) with HCT116 tumor cells. After tumor engraftment and subcutaneous development, the mouse models were treated ip with KillerTRAIL and mPEG-KillerTRAIL at the equivalent dose of 0.04 mg/kg/day. mPEG was used as a negative control. The development and growth of the tumor mass were monitored over time. The data obtained show that mPEG-KillerTRAIL significantly reduces the volume of the tumor mass after 20 days of treatment compared to the same dose of KillerTRAIL (Figure 9). At the same time, mPEG-KillerTRAIL reduces tumor growth by 74% compared to KillerTRAIL which, on the contrary, only reduces tumor mass by 37.5% (Figure 9). The improved in vivo antitumor activity of mPEG-KillerTRAIL compared to KillerTRAIL is linked to an improvement in the biopharmaceutical parameters of the native protein after its conjugation with mPEG. The PEGylation of KillerTRAIL modifies the half-life of the native protein and favors its accumulation in the tumor tissue. These values are similar to those already published in the literature that show the therapeutic advantages of bioactive PEG conjugates and their potential therapeutic applications (Pasut G., Veronese FM. Drugs Today (Bare), 2009; 45 (9): 687-95 ; Pasut G., Veronese FM. J. Control. Release, 2012; 161 (2): 461 -72).

The data reported in the patent document W02004001009 and relating to the PEGylation of the Apo 2L protein on cysteines inserted in the chain in position 170 or 179 show a dosage of 10 mg/kg, administered in two weekly doses for two weeks (therefore an overall quantity of compound equal to 20 mg/kg/week). The authors obtained a reduction in tumor volume increase, after 15 days, compared to the native protein, equal to about 57.6% for the PEGylated R170C-Apo 2L mutant and equal to about 6.2%, for the PEGylated K179C-Apo 2I. The data obtained by us, on the other hand, relate to a 7-day treatment with a pro-die dose of 0.04 mg/kg (therefore 0.21 mg/kg/week, about a tenth of what was used by the authors of the patent document W02004001009 of the aforementioned treatment, the analysis of the mean tumor volume was performed by us for the following 13 days. The data obtained show a reduction of the tumor at 15 days of 56% and at 20 days of 59% for the mPEG-KillerTRAIL conjugate compared to the native protein. It should be noted that the treatment was blocked on the seventh day and that the analysis was not performed beyond the twentieth because the control mice (towards which the reduction at 20 days is 74% for the PEG-KillerTRAIL compared to 37% of the native protein) and the mice treated with native KillerTRAIL died.

Considering these factors, it is evident that the mPEG-KillerTRAIL derivative object of this invention is better than that reported by the state of the art for the following reasons: the dosage used is 10 times lower and administered for a time equivalent to half of that reported in the literature, which presents an extension of the treatment more than 5 days more, the therapeutic yield in terms of reduction of the tumor volume is equivalent if not higher than one of the two conjugates reported in the patent W02004001 009A2, namely the PEG-R170C-Apo 2L, while it is significantly greater (by about 90%) than the second conjugate reported in the patent W02004001 009A2 or the PEG -K179C-Apo 2L.

These results are absolutely unexpected compared to what is reported in the patent W02004001009 where the amino acid chain of Apo2L is modified by introducing a cysteine in position 170 or 179 thanks to the substitution of an arginine and a lysine respectively. This modification, according to the authors of W02004001009, would have been necessary in order to protect the cysteine in position 230, considered fundamental for the trimerization necessary for the activation of the receptor system thanks to the ionic bond with a zinc ion. On the contrary, our studies have shown that the PEGylation on the Cys 230 residue increases the therapeutic action of the protein, without modifying the amino acid chain of the native protein in any way.

Please note that killerTRAIL (the polyhistidinehttp://www.enzolifesciences.com/ALX-201 -073/killertrail- protein-soiubie-human-recombinant/) is found to cause hepatic toxicity (apoptosis) due to tag. At the same time it appears to be much more effective than the Apo2L protein, which in order to achieve the in vitro activity demonstrated on killerTRAIL, must be co-administered with a high dose of doxorubicin, which is also particularly toxic (Jo M et al., Nat. Med. 2000; 6 (1): 564-7). The effectiveness of PEGylation KillerTRAIL is due to the reduction of the hepatotoxic effect reported in the literature, which was confirmed by us by the reduction of the accumulation of KillerTRAIL in the liver tissue (Figure 8C). The in vitro activity study, object of this invention, on HCT 116 tumor cells, sensitive to the therapeutic activity of KillerTRAIL, has in fact highlighted a reduction in the cytotoxicity of the same native protein (KillerTRAIL) (Figure 6), which is however balanced by the greater permanence in the bloodstream after PEGylation, as demonstrated in the related pharmacokinetic studies (Figure 7).

The results obtained from a comparison with C170 and C179 of W02004001009 are absolutely unexpected. The authors of W02004001009 in fact modified the amino acid chain by introducing a cysteine in position 170 or 179 thanks to the substitution of an arginine and a lysine respectively. This modification, according to their studies, was necessary to easily PEGilate the protein while maintaining the cysteine present in position 230 unchanged for trimerization, necessary for the activation of the receptor system thanks to the ionic bond with a zinc ion.

We instead carried out the PEGylation directly on the Cys 230 and unexpectedly this procedure guaranteed an improvement action of the protein. As can clearly be seen from the comparison shown in Figure 10, in fact, the Apo 2L protein modified in position 179 (K179C-Apo 2L), graph C, Figure 10 B of W02004001009 shows an activity comparable to the control and slightly lower than the thick PEGilated protein. The modification of the protein in the amino acid chain canceled the activity of the native protein and the PEGylation had practically no effect. The Apo 2L protein modified in position 170 (R170C-Apo 2L), graph B corresponding to Figure 10 A of the aforementioned document, on the other hand, appears to maintain the activity, even if much lower than the native protein and, in this case, the PEGylation improves its therapeutic characteristics by about 56%. On the other hand, our invention, in which KillerTRAIL is not modified, maintains its activity at significantly lower dosages both in the non-PEGylated and PEGylated form, where the reduction in tumor volume is evident even after 15 days from the end of the treatment. In this case, PEGylation results in increasing its effectiveness by 60% (Figure 9). We must also highlight the differences in the timing of administration: in fact in the aforementioned patent the authors describe a weekly treatment of 20 mg/kg (in protein) divided into two doses, for two weeks, for a total of 40 mg/kg per the entire duration of the experiment. Considering the timing of administration, it appears that the animals were treated every 3 days for the duration of the volumetric monitoring of the tumor. Our treatment modalities instead provided for a daily dosage of 0.04 mg/kg (in protein) for 7 days (therefore equivalent to 0.28 mg/kg in all) and a monitoring of the tumor volume from time 0 (first inoculum) for the following 15 days after the seventh day of treatment, for a total of 21 days of volumetric monitoring. From this comparison the following results: our product is improved in terms of:

1 . increased therapeutic efficacy;

2. noticeable increase in the time of therapeutic activity of the protein not accompanied by the administration of a dose.

It is fair to point out that the treatments did not last longer than 21 days as the control animals and those treated with the native protein did not survive, unlike those treated with the PEGylated protein mPEG-KillerTRAIL. According to what is reported in the invention, the conjugation with the PEG was directed on the only available cysteine, Cys 230, responsible for the trimerization. This procedure allowed not only to PEGilate the single monomer, but also to prevent trimerization. The experimental evidence relates to the MALDI-TOF mass spectroscopy study carried out on the conjugate obtained with PEG2000 Da, in which the absolute molecular weight of PEG (2119.4 Da) and of the monomer mPEG-KillerTRAIL- (24008.6 Da) is evident ( Example 6, Figure 4). In fact, the molecular weight of the single filament of KillerTRAIL corresponds to about 22000 Da, as reported by the manufacturer, as well as by the reference database Expasy.org. The fact that only the single filament has been PEGylated, presents us with a substantial difference with the patent W02004001009, where the PEGylation of the homotrimeric form of KillerTRAIL is reported. This difference is also evident in activity and efficacy in an absolutely unexpected way, as already highlighted above.

Example 13: KillerTRAILCharacterization Sequence KillerTRAIL

The amino acid sequence of KillerTRAIL Enzo Life has been partially solved as shown below:

TSEETISTVQ EKQQNISPLV RERGPQRVAA HITGTRGRSN TLSSPNSKNE KALGRKINSW ESSRSGHSFL SNLHLRNGEL VIHEKGFYYI YSQTYFRFQE EIKENTKNDK QMVQYIYKYT SYPDPILLMK SARNSCWSKD AEYGLYSIYQ GGIFELKEND RIFVSVTNEH LIDMDHEASF FGAFLVG (sequence of KillerTRAIL Protein protein ( soluble) (human), (recombinant)).

The sequence experimentally obtained is comparable with the amino acid sequence of TRAIL aa 95-281 (1 SEQ.ID.N. TSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNE KALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQE EIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQ GGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG) as Expasy.org official database.

The amino acid sequence obtained experimentally, and further analyzed by mass analysis present in the commercial product, does not show any difference with the theoretical one of KillerTRAIL.

The average value relative to the theoretical molecular weight (MW) of TRAIL (sequence 95-281 ) was found to be equivalent to 21607.1 Da.

The protein sequence was mapped by MALDI analysis. The tryptic peptide mixtures were sub-digested with chymotrypsin and the identified peptides are highlighted in gray as reported in Table 4. In the tryptic peptide mapping approach, the KillerTRAIL protein was characterized by MALDI analysis with a sequence coverage equal to 97.8%.

Table 4. The table shows the mass values identified in the MALDI spectra and assigned to the corresponding peptides in the KillerTRAIL protein.

¹ PYR GLU: Cyclization of the N-terminal Gin peptide.

The data reported in Table 4 indicate the mass/charge fragments that refer to the molecule weight and the relative amino acid sequences that have been identified by analyzing the KillerT RAIL samples. The identified peptides are highlighted in gray and correspond to the tryptic peptide sequences corresponding to a KillerTRAIL sequence, after digestion of the samples with chymotrypsin and identification by MALDI of tryptic peptides, equivalent to 97.8% of the analyzed sequence. The data reported in Table 4 were obtained by the Mass Mapping technique which is a strategy used for the analysis of the primary structure of proteins. This technique allows, once the spectrum of a peptide mixture has been obtained, to interpret the different mass signals, attributing to the signals present on the spectrum, specific molecular weights related to tryptic peptide sequences, in order to verify and confirm the protein sequence or identify any mutations. The program allows, through the MS-Digest program, to insert the known sequence of the protein and obtain a list of all the possible peptides that can be generated by a hydrolysis of the protein, with an appropriate enzyme, with the relative molecular weights. The MS- Digest program may also present some possible modifications on some peptides such as oxidations or cyclizations if they have been previously set. The set of peptides found contributes to define the sequence “mapping” or the coverage of the amino acid sequence.

The data in Table 4 show the KillerTRAIL specific tryptic peptide sequences, with their relative molecular weight, as a function of the trypsin hydrolysis process. These data are confirmed and in accordance with the mass spectra obtained after digestion (Figures 11 A - 11 Z) which indicate the fragments obtained for the tryptic peptide sequences obtained by mass analysis of KillerTRAIL and the corresponding molecular weights (Table 4).

Hydrolysis of KillerTRAIL in trypsin

The sample was dried using a bench centrifuge (Savant Speed-Vac, Holbrook, NY) at a speed of 12,000 rotations per minute (rpm) for 4 h, at room temperature and subsequently resuspended in ammonium Bicarbonate (50 mM, pH 8.0)m and then digested with 37°C trypsin for 16 hours. The peptide mixture obtained, purified by C18 zip-tip (Sigma-Aldrich, Milan) with a nominal volume of 0.6 mI, according to the specifications reported by the supplier

(https://www.sigmaaldrich.com/catalog/product/mm/ztc18s ? lang = it & region = IT & gelid = CjwKCAiAoOz-BRBdEiwAyuvA63lo4iMhL- FNOcDyl swOzwOhEKoUKAslwkds6BVT- jh79Q6ldqr4oBoCKIMQAvD_BwE) was dried in a bench centrifuge (Savant Speed-Vac, Holbrook, NY) at a speed of 12,000 rpm , at room temperature, for 4h, and then resuspended in 0.2% TFA. Finally, a MALDI-MS analysis of the tryptic digest was performed by MALDI TOF-TOF 4800 Plus mass spectrometer.

Hydrolysis of KillerTRAIL into Chymotrypsintryptic

The peptide mixtures, obtained by hydrolysis in trypsin, were further digested with chymotrypsin in Ammonium Bicarbonate (50 mM, pH 8.0) for 4 hours (E: S 1 :30 w/w) to provide complete coverage of the amino acid sequence to be analyzed and identified by MALDI.

MALDI-MS by KillerTRAIL

MALDI-MS analyzes were performed on a MALDI TOF-TOF (AB Sciex) 4800 plus mass spectrometer and the obtained data analyzed using 4000 Series Explorer v3.5 software.

For the analysis of the KillerTRAIL samples, 0.5 mI of the peptide mixture was mixed with an equal volume of a-cyano-4-hydroxycinnamic acid as matrix (10 mg/ml in 0.2% trifluoro acetic acid (TFA) in 70% of acetonitrile), loaded onto the sample metal plate and air dried.

Mass calibration was performed using the standard mix provided by the manufacturer. MALDI-MS data were acquired over a mass range between 500 and 5600 m/z in positive-ion reflector mode. The data obtained were acquired and processed using the software provided by the manufacturer.

The mass spectra were manually interpreted by associating a specific peptide with each peak (Figure 11 ). The results of the mass spectra were then correlated with the values reported in the MS-Digest software which provides a list of all possible peptides that can be generated by hydrolysis of the protein with an appropriate enzyme with relative molecular weights. The process of assigning each signal to a specific peptide made it possible to obtain the effective coverage of the protein, attributing a number of peptides to each individual sequence to obtain the best mapping of the protein.

The mass spectra of the tryptic peptides obtained relate the intensity with the m/z ratio in a mass range ranging from 500 to 5600 Da. The mass spectra of the individual tryptic peptides and the relative molecular weights were shown in Figure 11 and Table 4.

The identification of the peptides was performed on the basis of the mass values recorded in the spectra (Figure 12) and the data are summarized in Table 4.

Determination of the molecular weight of KillerTRAIL

300 mI of KillerTRAIL sample, equivalent to 833 pmol, was purified by reverse phase chromatography (RP-FIPLC) on a Phenomenex Jupiter C4 column (250 mm x 2.0 mm, pore size 300 A) with a linear gradient 10% to 95% solvent B (0.07% TFA in 95% acetonitrile) for 30 minutes, at a flow rate of 0.2 ml/min using an Agilent Technologies 1100 FIPLC (Agilent Technologies, USA) system connected with a UV-Vis detector.

The chromatographic peak in Figure 12 refers to the analysis of the identified KillerTRAIL protein using an HPLC system connected to a UV-Vis detector at a wavelength of 220 nm. The chromatographic peak corresponding to the elution of a sample of KillerTRAIL protein after drying in a SpeedVac centrifuge and reconstitution in the mobile phase (0.07% of TFA in 95% of vinegar nitrile). The analysis shows that KillerTRAIL has a single peak with a retention time of 18,876 minutes, symmetrical, which is free of impurities and well defined after chromatographic analysis.

The sample eluted by HPLC analysis was then re-dried by bench centrifuge (Savant Speed-Vac, Holbrook, NY) at a rate of 12,000 rotations per minute (rpm) for 4 h, at room temperature, resuspended in 50 pi of ACN 50% v/v and HCOOH 0.2% v/v and analyzed by ESI-MS mass spectrometry using the Q-TOF Premier system (Waters, Milford, MA, USA) with ESI source by direct injection with a calibrated syringe at a flow rate of 10 mI/min. The mass spectrum was acquired in a range from 800 to 2500 m/z. Raw data was processed using MassLynx 4.1 software (Waters, Milford, MA, USA).

In the obtained ESI MS spectrum, the sample showed an experimental molecular weight of 24013.91 ± 0.94 Da which corresponds to the KillerTRAIL protein (theoretical molecular weight: 21607.1 Da) with a mass difference of 2406.81 Da calculated difference between the two species (TRAIL and KillerTRAIL) is represented by the peptide linker and the His tag (Figure 13). The experimental difference of 2406.81 Da obtained by making the difference between the theoretical molecular weight of 21607.1 Da of the known sequence of TRAIL and the experimental molecular weight (24013.39 Da) of KillerTRAIL show that the amino acid sequence of TRAIL is present and stored in the KillerTRAIL (commercial Enzo Life product) and there are also a peptide linker and a His tag sequence that are specific to KillerTRAIL and are not present in the TRAIL. The presence of a peptide linker and His tag is confirmed by the ESI MS mass spectrum for direct injection which shows fragmentation peaks for functional group signals related to the amino acid sequence of TRAIL together with those of the peptide linker and His tag (Figure 13).

The peak in Figure 13A indicates the molecular weight of the protein (equal to 24013.91 Da) relative to KillerTRAIL consisting of the known sequence TRAIL, the Killer peptide and the His tag. The ESI MS analysis indicates that the peak corresponding to the molecular weight 24013.91 Da is the main one obtained from the mass spectrum and therefore characteristic of KillerTRAIL. This mass value is obtained through the deconvolution process of the high (Figure 1 3B) and low (Figure 13C) signal intensity mass spectra. Following enzymatic digestion and LC-MS analysis, the peptide corresponding to KillerTRAIL can in turn be isolated and further fragmented in tandem mass spectrometry into the individual amino acids that compose it. The mass spectra shown in Figure 13B and Figure 13C contain the peptide linker and the His tag. To obtain the linker peptide sequence it is necessary to resort to further tandem mass spectrometry (MS/MS) experiments following enzymatic digestion, isolation of the peptide linker and its subsequent fragmentation in MS/MS. The tandem mass spectrometry allows to fragment a single peptide using an appropriate collision energy which, together with the presence of an inert collision gas (generally Argon), causes the breaking of the peptide bonds providing the amino acid sequence of the peptide of interest.