Combination therapy for treating cancer

Abstract

The present invention relates to a combination of a PARP inhibitor and Omomyc, a functional equivalent variant thereof, a conjugate comprising Omomyc or said functional equivalent variant, a polynucleotide encoding said polypeptide, a vector comprising said polynucleotide, and a cell capable of secreting said polypeptide or said conjugate. The invention also relates to a pharmaceutical composition comprising the combination of the invention and its medical use, in particular its use in the treatment of cancer.

Description

Technical Field

[0001] The present invention relates to the field of cancer, and more specifically, to a combination comprising a poly (ADP-ribose) polymerase (PARP) inhibitor and Omomyc and its use in medicine, more specifically, in the prevention and / or treatment of cancer. Background Art

[0002] Cancer is the leading cause of death worldwide, accounting for nearly 10 million deaths in 2020. It is a large group of diseases characterized by the uncontrolled growth of abnormal cells. DNA damage is one of the causative factors in the development of cancer, and mutations in different DNA repair systems increase susceptibility to various cancer types.

[0003] Anticancer drugs have been designed to target a whole set of cancer hallmarks. In the past decade, poly (ADP-ribose) polymerase (PARP) inhibitors became the first drugs to enter the clinic that target the DNA damage response. Four PARP inhibitors (olaparib, rucaparib, niraparib, and talazoparib) have been approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA).

[0004] PARP inhibitors inhibit the catalytic activity of PARP-1 and PARP-2 enzymes, which are involved in base excision repair of single-strand breaks in DNA. PARP inhibition leads to the accumulation of single-strand breaks, ultimately leading to double-strand breaks. In addition to catalytic inhibition, PARP inhibitors also trap the PARP enzyme-DNA complex on single-strand breaks, leading to double-strand breaks. Poly (ADP-ribose) polymerase trapping is believed to be the primary mechanism of anti-tumor activity. PARP inhibition is particularly effective in cells with homologous recombination defects (such as pathogenic breast cancer BRCA-1 or BRCA-2 mutations).

[0005] The PARP inhibitors have shown effectiveness in treating a variety of cancer types, such as ovarian, breast, prostate, pancreatic, and small cell lung cancer, and other indications are being explored in clinical trials.

[0006] In general, cancers with high levels of replication stress and genomic instability resulting from DNA repair defects and / or increased oncogene-induced replication origin firing are particularly sensitive to PARP inhibitors. However, there are still challenges to overcome.

[0007] Resistance to PARP inhibitors and adverse effects are common problems in clinical practice and may limit long-term treatment.

[0008] Therefore, there remains a need in the art to develop new and improved therapeutic regimens for treating cancer. Summary of the invention

[0009] In a first aspect, the present invention relates to a combination comprising:

[0010] i) a first component selected from:

[0011] a) a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof;

[0012] b) a conjugate comprising a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or a functionally equivalent variant thereof;

[0013] c) a polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0014] d) a vector comprising the polynucleotide according to c); and

[0015] e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into the culture medium;

[0016] and

[0017] ii) As a second component of a PARP inhibitor.

[0018] In a second aspect, the present invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of a combination according to the present invention and a pharmaceutically acceptable excipient.

[0019] In a third aspect, the present invention relates to a combination according to the invention or a pharmaceutical composition according to the invention for use in medicine.

[0020] In a fourth aspect, the present invention relates to a combination according to the invention or a pharmaceutical composition according to the invention for use in the prevention and / or treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Bar graphs representing the synergistic combination of Omomyc and Olaparib against three triple negative breast cancer (TNBC) cell lines: (A) MDA-MB-231; (B) SUM149; (C) MX-1.

[0022] Figure 2 .Bar graph representing the synergistic combination of Omomyc and Olaparib in the pancreatic ductal adenocarcinoma (PDAC) MIA-PACA-2 cell line.

[0023] Figure 3 Bar graphs representing the synergistic combination of Omomyc and Talazoparib against three triple negative breast cancer (TNBC) cell lines: (A) MDA-MB-231; (B) SUM149; (C) MX-1.

[0024] Figure 4 . Dot plots representing preliminary in vivo data from the SUM149 CDX model. Statistical significance was determined by one-way ANOVA and Tukey's multiple comparison test (* = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001, **** = p-value < 0.001). DETAILED DESCRIPTION

[0025] The present invention relates to the provision of new therapeutic combinations for the prevention and treatment of cancer.

[0026] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0027] All embodiments disclosed in relation to one aspect of the invention are applicable to the other aspects.

[0028] Combinations and pharmaceutical compositions of the invention

[0029] The definitions provided herewith as well as the definitions provided in each other aspect of the invention are equally applicable throughout the invention.

[0030] The inventors of the present invention have found that, surprisingly, the combination of Omomyc and a PARP inhibitor has a synergistic effect in treating cancer. The inventors have demonstrated that the combination of Omomyc and the PARP inhibitor Olaparib synergistically reduces the viability of triple negative breast cancer cell lines MDA-MB-231, SUM149 and MX-1; and the viability of the MIA-PACA-2 pancreatic ductal adenocarcinoma cell line. This synergistic effect is maintained regardless of the dose ( Figure 1 and Figure 2 ), thereby producing beneficial effects, specifically, the increase in the therapeutic effect of the composition of the present invention relative to each of its components, making it possible to achieve the same results with lower doses of each component, thereby reducing side effects to subjects receiving the composition of the present invention.

[0031] Furthermore, the inventors have found that, surprisingly, Omomyc is able to restore olaparib resistance, thereby enhancing sensitivity to PARP inhibitors and overcoming PARP inhibitor resistance, which is one of the major drawbacks of treatment with PARP inhibitors. The combination of the invention expands the population that may respond to PARP inhibitor-based therapies.

[0032] Therefore, the combination of Omomyc and PARP inhibitors may be an effective therapy for the treatment of BRCA mutant and wild-type tumors, as well as for the treatment of tumors that are resistant to PARP inhibitors.

[0033] Thus, in a first aspect, the present invention relates to a combination comprising:

[0034] i) a first component selected from:

[0035] a) a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof;

[0036] b) a conjugate comprising a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or a functionally equivalent variant thereof;

[0037] c) a polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0038] d) a vector comprising the polynucleotide according to c); and

[0039] e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into the culture medium;

[0040] and

[0041] ii) As a second component of a PARP inhibitor.

[0042] According to the present invention, the expression "combination" represents various combinations of compounds (i) and (ii), for example in a composition formulated as a single preparation, in a combined mixture consisting of separate preparations of each component, such as a "pot mix" that can be combined for joint use as a combined preparation, and the combined use of the single active ingredients when administered in a sequential manner, i.e. one after the other within a reasonably short period (such as a few hours or days) or simultaneously. In the present invention, compound (i) represents a therapeutically effective amount of a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof; or represents a conjugate containing a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; or represents a polynucleotide encoding the polypeptide or the conjugate; or represents a vector comprising the polynucleotide; or represents a cell capable of secreting the polypeptide or the conjugate into the culture medium. In the present invention, compound (ii) represents a therapeutically effective amount of a PARP inhibitor. Preferably, the order of applying compounds (i) and (ii) is not important for practicing the present invention.

[0043] The combination may be a kit of parts, wherein each part is separately formulated and packaged.

[0044] The combination of compound (i) and (ii) can be formulated for simultaneous, separate or sequential administration. Specifically, if the administration of the compound is not simultaneous, it is administered within a time interval close to each other. In addition, the compound can be administered in the same or different dosage forms or by the same or different routes, for example, one compound can be administered orally and another compound can be administered intravenously. Preferably, compound (i) is administered intravenously and compound (ii) is administered orally. In another embodiment, compounds (i) and (ii) are administered intravenously.

[0045] The combination of the two compounds (i) and (ii) can be administered as follows:

[0046] - Combinations as components of the same pharmaceutical preparation, then both compounds are always administered simultaneously.

[0047] - As a combination of two units, each containing one of the substances, giving rise to the possibility of simultaneous, sequential or separate administration.

[0048] In a particular embodiment, compound (i) of the combination according to the invention is administered independently from compound (ii), ie in two units, but simultaneously.

[0049] In another specific embodiment, compound (i) of the combination of the invention is administered first and compound (ii) is administered secondarily, ie compound (ii) is administered separately or sequentially.

[0050] In yet another specific embodiment, compound (ii) of the combination of the invention is administered first and compound (i) is administered secondarily, ie compound (i) is administered separately or sequentially, as defined.

[0051] If administered separately, the compounds (i) and (ii) of the combination of the present invention may be administered within a period of time, for example within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours. In another embodiment, the compounds (i) and (ii) of the combination of the present invention may be administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 days, preferably within 1 day, more preferably within 10 days. In a preferred embodiment, compound (ii) is administered 10 days after the first administration of compound (i). In one embodiment, administration of the first compound is stopped before administration of the second compound is started.

[0052] In another aspect, the invention relates to a combination or pharmaceutical composition comprising a synergistically effective amount of the first component according to the first aspect of the invention and a PARP inhibitor.

[0053] Compound (i) of the combination of the present invention

[0054] In a preferred embodiment, the compound (i) of the present invention is a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof; more preferably a polypeptide comprising the sequence of SEQ ID NO: 1.

[0055] The terms "polypeptide" and "peptide" are used interchangeably herein to refer to polymers of amino acids of any length. The polypeptide of the present invention may comprise modified amino acids and may be interrupted by non-amino acids. In a preferred embodiment, the polypeptide is formed only by amino acids. Preferably, the polypeptide forming item (i) of the combination has a length between 80 and 500 amino acids, more preferably between 80 and 300 amino acids, more preferably between 80 and 250 amino acids, more preferably between 80 and 150 amino acids, even more preferably between 80 and 130 amino acids, preferably between 90 and 130 amino acids, preferably no more than 125 amino acids, more preferably no more than 100 amino acids. In a preferred embodiment, the polypeptide has a length between 90 and 98 amino acids, preferably between 90 and 95 amino acids, more preferably 91 amino acids.

[0056] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. In addition, the term "amino acid" includes D-amino acids and L-amino acids (stereoisomers). Preferably, the amino acid is an L-amino acid.

[0057] The term "natural amino acid" or "naturally occurring amino acid" encompasses the 20 naturally occurring amino acids; those amino acids that are frequently post-translationally modified in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other unusual amino acids, including, but not limited to, 2-aminoadipate, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine.

[0058] As used herein, the term "non-natural amino acid" or "synthetic amino acid" refers to a carboxylic acid or derivative thereof that is substituted with an amine group at position "a" and is structurally related to a natural amino acid. Illustrative, non-limiting examples of modified or uncommon amino acids include 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, aliohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyl-lysine, N-methylvaline, norvaline, norleucine, ornithine, and the like.

[0059] The polypeptides of the present invention may also contain non-amino acid moieties, for example, hydrophobic moieties (various straight chain, branched chain, cyclic, polycyclic or heterocyclic hydrocarbons and hydrocarbon derivatives) linked to the peptide; various protecting groups attached to the termini of the compounds to reduce degradation. Suitable protecting functional groups are described in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991.

[0060] Chemical (non-amino acid) groups present in the polypeptide may be included to improve various physiological properties, such as reduced degradation or clearance; reduced rejection by various cellular pumps, improved various modes of administration, increased specificity, increased affinity, increased stability, bioavailability, solubility, reduced toxicity, etc.

[0061] "Mimetics" include molecules that mimic the chemical structure of a peptide structure and retain the functional properties of the peptide structure. Protocols for designing peptide analogs, derivatives, and mimetics are known in the art.

[0062] In one embodiment, the polypeptide of the present invention is a polypeptide consisting of the sequence SEQ ID NO: 1 or a polypeptide consisting of a functionally equivalent variant of SEQ ID NO: 1, preferably a polypeptide consisting of the sequence SEQ ID NO: 1.

[0063] SEQ ID NO:1 corresponds to

[0064] TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA(SEQ ID NO:1)

[0065] The polypeptide of sequence SEQ ID NO:1 corresponds to the Omomyc protein sequence. The term "Omomyc" as used herein refers to a polypeptide consisting of a mutant form of the bHLHZip domain of Myc carrying E61T, E68I, R74Q and R75N ​​mutations (wherein the numbering of the mutation positions is given relative to the sequence of the Myc region corresponding to amino acids 365-454 of the polypeptide defined under accession number NP_002458 in the NCBI database released on March 15, 2015). The sequence of c-Myc provided under accession number NP_002458 in the NCBI database is shown below (SEQ ID NO:2), wherein the region from which Omomyc is derived is shown underlined:

[0066] 1 MDFFRVVENQ QPPATMPLNV SFTNRNYDLD YDSVQPYFYC DEEENFYQQQ QQSELQPPAP

[0067] 61 SEDIWKKFEL LPTPPLSPSR RSGLCSPSYV AVTPFSLRGD NDGGGGSFST ADQLEMVTEL

[0068] 121 LGGDMVNQSF ICDPDDETFI KNIIIQDCMW SGFSAAAKLV SEKLASYQAARKDSGSPNPA

[0069] 181 RGHSVCSTSS LYLQDLSAAASECIDPSVVF PYPLNDSSSP KSCASQDSSAFSPSSDSLLS

[0070] 241 STESSPQGSP EPLVLHEETP PTTSSDSEEE QEDEEEIDVV SVEKRQAPGK RSESGSPSAG

[0071] 301 GHSKPPHSPL VLKRCHVSTH QHNYAAPPST RKDYPAAKRV KLDSVRVLRQ ISNNRKCTSP

[0072] 361 RSSD TEENVK RRTHNVLERQ RRNELKRSFF ALRDQIPELE NNEKAPKVVI LKKATAYILS

[0073] 421 VQAEEQKLIS EEDLLRKRRE QLKHKLEQLR NSCA(SEQ ID NO:2)

[0074] Omomyc also contains the M2 domain of c-Myc, which has the sequence RQRRNELKRSF (SEQ ID NO: 3) (see Dang and Lee, Mol. Cell. Biol., 1988, 8: 4048-4054) (double underlined above), and which corresponds to the nuclear localization signal.

[0075] Omomyc is characterized in that it shows increased dimerization ability with all three oncogenic Myc proteins (c-Myc, N-Myc and L-Myc). Omomyc can be derived from the bHLHZip domain of any Myc protein known in the art, provided that mutations that lead to tumor suppression are retained. Therefore, Omomyc, which can be used in the present invention, can be derived from any mammalian species, including but not limited to domestic and farm animals (cattle, horses, pigs, sheep, goats, dogs, cats or rodents), primates and humans. Preferably, the Omomyc protein is derived from human Myc protein (accession number NP_002458, published on March 12, 2019).

[0076] The term "Myc" used herein means a family of transcription factors including c-Myc, N-Myc and L-Myc. Myc protein activates the expression of many genes by binding to the consensus sequence CACGTG (enhancer-box sequence or E-box and recruiting histone acetyl-transferase or HAT). However, Myc can also serve as a transcription repressor. By binding to the Miz-1 transcription factor and replacing the p300 coactivator, it inhibits the expression of Miz-1 target genes. Myc also has a direct effect in controlling DNA replication.

[0077] Myc b-HLH-LZ or Myc basic region helix-loop-helix leucine zipper domain represents the region that determines the dimerization of Myc with Max protein and the binding to Myc-target genes. This region corresponds to amino acids 365-454 of human Myc and is characterized by two alpha helices connected by a loop (Nair, SK, and Burley, SK, 2003, Cell, 112: 193-205).

[0078] In a preferred embodiment, the polypeptide of the present invention is a polypeptide comprising, consisting of or consisting essentially of SEQ ID NO: 4 shown below.

[0079] MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA(SEQ ID NO:4)

[0080] In this context, "consisting essentially of" means that the particular molecule will not contain any additional sequences that would alter the activity of SEQ ID NO:4.

[0081] Preferably, the polypeptide consists of SEQ ID NO:4.

[0082] The term "functionally equivalent variant" refers to any polypeptide produced by the insertion or addition of one or more amino acids and / or the deletion of one or more amino acids and / or the conservative substitution of one or more amino acids relative to the polypeptide of SEQ ID NO: 1, and / or any polypeptide produced by chemical modification of the polypeptide of SEQ ID NO: 1 and substantially retaining the tumor suppressive activity of SEQ ID NO: 1. Preferably, the functionally equivalent variant refers to any polypeptide produced by the insertion or addition of one or more amino acids and / or the deletion of one or more amino acids and / or the conservative substitution of one or more amino acids relative to the polypeptide of SEQ ID NO: 1 and substantially retaining the tumor suppressive activity of SEQ ID NO: 1; more preferably, produced by the insertion or addition of one or more amino acids relative to the polypeptide of SEQ ID NO: 1.

[0083] The skilled person will understand that the retention of tumor suppressor activity requires that the variant can dimerize with Myc and / or its obligate partner p21 / p22Max and inhibit Myc activity, can translocate across the cell membrane and can translocate across the nuclear membrane. In certain embodiments, the functionally equivalent variants of the polypeptides of the present invention are less homodimerized than Omomyc, or are not forced to form homodimers through the formation of disulfide bridges. Specifically, the disulfide bridge formation in the homodimeric form of certain embodiments of the polypeptides of the present invention is less than the disulfide bridge formation in the polypeptide Omomyc.

[0084] As used herein, "less homodimerization" refers to a lower ability to form obligate homodimers of a polypeptide of the invention even under reducing conditions. In a preferred embodiment, the ability is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% lower than the ability to form homodimers of Omomyc.

[0085] As used herein, reducing conditions involve the presence of a reducing agent, which is a compound that donates electrons to another chemical in a redox chemical reaction. Exemplary, non-limiting examples of reducing agents are DTT (dithiothreitol), b-mercaptoethanol or TCEP (tris(2-carboxyethyl)phosphine). The amount of homodimers may be identical in vitro, and the difference between functionally equivalent variants and Omomyc is only in the presence of heterodimerization partners in cells, where the absence of disulfides makes it possible to form heterodimers to a higher degree.

[0086] Several assays can be used to determine homodimerization of the peptides, such as thermal denaturation monitored by circular dichroism as an illustrative, non-limiting example, whereby dimerization can be detected by folding and thermal stability quantification.

[0087] Suitable functionally equivalent variants include polypeptides consisting essentially of the polypeptide of SEQ ID NO: 1. In this context, "consisting essentially of" means that the particular molecule will not contain any additional sequences that would alter the activity of SEQ ID NO:1.

[0088] In a preferred embodiment, the functionally equivalent variant of SEQ ID NO: 1 is a polypeptide produced by the insertion or addition of one or more amino acids relative to the polypeptide of SEQ ID NO: 1. In one embodiment, the functionally equivalent variant is produced by the insertion of less than 10 amino acids, more preferably less than 5 amino acids, more preferably by the insertion of one amino acid. In a preferred embodiment, it is produced by the insertion of one amino acid, and the amino acid is methionine.

[0089] In another embodiment, the functionally equivalent variant of SEQ ID NO: 1 is a polypeptide resulting from the deletion of one or more amino acids relative to the polypeptide of SEQ ID NO: 1. In one embodiment, the functionally equivalent variant is resulting from the deletion of less than 10 amino acids, more preferably less than 5 amino acids, more preferably resulting from the deletion of one amino acid.

[0090] Suitable functional variants of targeting peptides are those that show an identity degree of approximately greater than 25% amino acid sequence identity (such as 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) relative to the peptide of SEQ ID NO: 1. The degree of identity between two polypeptides is determined using computer algorithms and methods widely known to those skilled in the art. Preferably, the identity between two amino acid sequences is determined by using the BLASTP algorithm as described above (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 1990; 215: 403-410). In a preferred embodiment, sequence identity is determined over the entire length of the polypeptide of SEQ ID NO: 1 or over the entire length of a variant or both.

[0091] Functionally equivalent variants of the polypeptides of the present invention may also include post-translational modifications, such as glycosylation, acetylation, prenylation, myristoylation, proteolytic processing, and the like.

[0092] In another embodiment, the suitable functional variant of the targeting peptide is a variant wherein one or more positions within the polypeptide of the present invention contain an amino acid which is a conservative replacement of the amino acid present in the above-mentioned protein. "Conservative amino acid replacement" is derived from replacing an amino acid with another amino acid having similar structure and / or chemical properties. For example, the following six groups each contain amino acids that are conservative replacements for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W). The selection of such conservative amino acid substitutions is within the skill of one of ordinary skill in the art and is described, for example, by Dordo et al. (J. Mol. Biol, 1999, 217; 721-739) and Taylor et al. (J. Theor. Biol., 1986, 119: 205-218).

[0093] It should be understood that in a preferred embodiment, the functionally equivalent variants of Omomyc contain mutations at positions corresponding to the mutations E61T, E68I, R74Q and R75N ​​found in Omomyc derived from human c-Myc. The position where the mutation must occur in the functionally equivalent variant can be determined by multiple sequence alignment of different Myc sequences and identified by alignment with those positions corresponding to positions 61, 68, 74 and 75 within the sequence of Omomyc derived from human c-Myc. In one embodiment, the functionally equivalent variants of Omomyc contain mutations at positions corresponding to the mutations E61T, E68I, R74Q and R75N ​​found in Omomyc derived from human c-Myc.

[0094] In another embodiment, a functionally equivalent variant of Omomyc contains mutations at positions corresponding to E61, E68, R74, and R75 within the sequence of Omomyc, wherein E61 has been mutated to E61A or E61S; E68 has been mutated to E68L, E68M, or E68V; R74 has been mutated to R74N; and R75 has been mutated to R75Q.

[0095] Multiple sequence alignment is an expansion of the alignment, to include more than two sequences at a time. Multiple alignment methods can align all sequences in a given query set. Preferred multiple sequence alignment programs (and their algorithms) are ClustalW, Clusal2W or ClustalW XXL (see Thompson et al. (1994) Nucleic Acids Res 22: 4673-4680). Once the sequence of c-Myc from different organisms and the sequence of variants are compared (aligned) as described herein, skilled technicians can easily identify the position corresponding to the position E61T, E68I, R74Q and R75N ​​found in Omomyc in each sequence, and introduce the mutation corresponding to the E61T, E68I, R74Q and R75N ​​mutations found in Omomyc derived from human c-Myc in Omomyc variants.

[0096] Suitable assays for determining whether a polypeptide can be considered a functionally equivalent variant of Omomyc include, but are not limited to:

[0097] - Assays measuring the ability of a polypeptide to form a dimeric complex with Max and Myc, such as the assay based on reporter gene expression described by Soucek et al. (Oncogene, 1998, 17: 2463-2472), as well as PLA (protein ligation assay) or co-immunoprecipitation.

[0098] - an assay that measures the ability of a polypeptide to bind to a Myc / Max recognition site (CACGTG site) within DNA, such as the electrophoretic mobility shift assay (EMSA) described by Soucek et al. (supra).

[0099] - an assay measuring the ability to inhibit Myc-induced transactivation, such as the assay described by Soucek et al. (supra) based on the expression of a reporter gene under the control of a DNA binding site specific for Myc / Max.

[0100] - An assay based on the ability of a polypeptide to inhibit the growth of cells expressing the myc oncogene as described by Soucek et al. (supra).

[0101] -Measurement of the ability of the polypeptide to enhance myc-induced apoptosis, such as the assay described by Soucek et al. (Oncogene, 1998: 17, 2463-2472). In addition, any assay known in the art for assessing apoptosis may be used, such as Hoechst staining, propidium iodide (PI) or annexin V staining, trypan blue, DNA gradient method / fragmentation and TUNEL.

[0102] In a preferred embodiment, a polypeptide is considered to be a functionally equivalent variant of Omomyc if it exhibits at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the activity of native Omomyc in one or more of the above assays.

[0103] In a specific embodiment, a functionally equivalent variant of the polypeptide of SEQ ID NO: 1 comprises a polypeptide of SEQ ID NO: 1, wherein the residue X at position 89 of SEQ ID NO: 1 is not cysteine. Preferably, the residue X at position 89 of SEQ ID NO: 1 is an aliphatic amino acid, or a sulfonated amino acid, or a dicarboxylic acid amino acid or their amides, or an amino acid having two basic groups, or an aromatic amino acid, or a cyclic amino acid, or a hydroxylated amino acid. More preferably, it is an amino acid selected from serine, threonine and alanine, preferably selected from serine and alanine.

[0104] Suitable functionally equivalent variants of SEQ ID NO: 1 are disclosed in the table below, having a residue X at position 89 of SEQ ID NO: 1 which is not cysteine.

[0105]

[0106]

[0107] Thus, in a preferred embodiment, the functionally equivalent variant of the polypeptide of SEQ ID NO: 1 is selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. Preferably, the functionally equivalent variant is SEQ ID NO: 4.

[0108] In addition, functionally equivalent variants of Omomyc are also capable of transducing cells after contacting the variant with the cell. It should be understood that functionally equivalent variants of Omomyc contain a protein transduction domain found in natural Omomyc or another functional protein transduction domain.

[0109] In a preferred embodiment, a polypeptide is considered a functionally equivalent variant of SEQ ID NO: 1 if the polypeptide is able to transduce target cells with an efficiency of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of SEQ ID NO: 1.

[0110] In addition, functionally equivalent variants of SEQ ID NO: 1 are also capable of translocating to the nucleus of target tumor cells.

[0111] In a preferred embodiment, a polypeptide is considered a functionally equivalent variant of SEQ ID NO: 1 if its efficiency in translocating to the nucleus of a target tumor cell is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of that of SEQ ID NO: 1.

[0112] For the ability of polypeptide to cross cell membrane and translocate to cell nucleus, suitable assays for determining whether polypeptide is a functional equivalent variant of SEQ ID NO: 1 include double labeling of cells with reagents specific for the polypeptide and dyes (such as DAPI or Hoechst dyes) that specifically label the nuclei of cells. Detection of polypeptides of the present invention can be performed by confocal microscopy or by fluorescence microscopy.

[0113] In another preferred embodiment, compound (i) of the present invention is a conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or a functionally equivalent variant thereof.

[0114] As used herein, the term "conjugate" refers to two or more compounds that are covalently linked together such that the functionality of each compound is retained in the conjugate.

[0115] The term "chemical moiety" refers to any chemical compound containing at least one carbon atom. Examples of chemical moieties include, but are not limited to, any peptide chain rich in hydrophobic amino acids and hydrophobic chemical moieties.

[0116] In a preferred embodiment, the conjugate according to the invention comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more chemical moieties which promote the cellular uptake of the polypeptide or a functionally equivalent variant of the polypeptide.

[0117] In one embodiment, the chemical moiety that facilitates cellular uptake of the polypeptide is a lipid or a fatty acid.

[0118] Lipid acid is typically a molecule that is included in a carbon chain with an acidic portion (e.g., carboxylic acid) at the end of the chain. The carbon chain of lipid acid can be any length, but preferably the length of the carbon chain is at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more carbon atoms, and any range that can be derived therefrom. In certain embodiments, in the chain portion of lipid acid, the length of the carbon chain is 4 to 18 carbon atoms. In certain embodiments, the fatty acid carbon chain can comprise an odd number of carbon atoms, but, in certain embodiments, it may be preferred that the carbon chain comprises an even number of carbon atoms. The lipid acid that only comprises a single bond in its carbon chain is referred to as saturated, and the lipid acid that comprises at least one double bond in its chain is referred to as unsaturated. The lipid acid can be branched, although in a preferred embodiment of the present invention, it is unbranched. Specific fatty acids include, but are not limited to, linoleic acid, oleic acid, palmitic acid, linolenic acid, stearic acid, lauric acid, myristic acid, arachidic acid, palmitoleic acid, and arachidonic acid.

[0119] In a preferred embodiment, the chemical moiety that promotes the cellular uptake of a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof is a cell penetrating peptide sequence, in which case the conjugate will comprise a fusion protein comprising a polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant thereof and a cell penetrating peptide sequence.

[0120] The term "fusion protein" refers to a protein produced by gene technology, which consists of two or more functional domains derived from different proteins. Fusion proteins can be obtained by conventional means, for example, by gene expression of a nucleotide sequence encoding the fusion protein in a suitable cell. It should be understood that a cell penetrating peptide refers to a cell penetrating peptide that is different from a cell penetrating peptide that forms part of a polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant of SEQ ID NO: 1.

[0121] The term "cell penetrating peptide sequence" is used interchangeably with "CPP", "protein transduction domain" or "PTD" in this specification. It refers to a peptide chain of variable length that directs the transport of proteins within cells. The delivery process into cells usually occurs by endocytosis, but the peptide can also be internalized into cells by direct membrane translocation. CPPs generally have an amino acid composition that contains a high relative abundance of positively charged amino acids such as lysine or arginine, or a sequence having an alternating pattern of polar / charged amino acids and non-polar hydrophobic amino acids.

[0122] Examples of CPPs that can be used in the present invention include, but are not limited to, the CPP found in Drosophila antennapedia protein (RQIKIWFQNRRMKWKK. SEQ ID NO: 13), the CPP found in herpes simplex virus 1 (HSV-1) VP22 DNA-binding protein (DAATATRGRSAASRPTERPRAPARSASRPRRPVE, SEQ ID NO: 14), the CPP of Bac-7 (RRIRPRPPRLPRPRPRPLPFPRPG; SEQ ID NO: 15), the CPP of HIV-1 TAT protein consisting of amino acids 49-57 (RKKRRQRRR, SEQ ID NO: 16), amino acids 48-60 (GRKKRRQRRRTPQ, SEQ ID NO: 17), amino acids 47-57 (YGRKKRRQRRR; SEQ ID NO: 18); the CPP of S413-PV peptide (ALWKTLLKKVLKAPKKKRKV; SEQ ID NO: 19), CPP of penetrating peptide (RQIKWFQNRRMKWKK; SEQ ID NO: 20), CPP of SynB1 (RGGRLSYSRRRFSTSTGR; SEQ ID NO: 21), CPP of SynB3 (RRLSYSRRRF; SEQ ID NO: 22), CPP of PTD-4 (PIRRRKKLRRRK; SEQ ID NO:23), CPP of PTD-5 (RRQRRTSKLMKR; SEQ ID NO:24), CPP of FHV capsid-(35-49) (RRRRRNRTRRNRRRVR; SEQ ID NO:25), CPP of BMV Gag-(7-25) (KMTRAQRRAAARRNRWTAR; SEQ ID NO:26), HTLV-II CPP of Rex-(4-16) (TRRQRTRRARRNR; SEQ ID NO:27), CPP of D-Tat (GRKKRRQRRRPPQ; SEQ ID NO:28), R9-Tat's CPP (GRRRRRRRRRPPQ; SEQ ID NO:29), MAP's CPP (KLALKLALKLALALKLA; SEQ ID NO:30), SBP's CPP (MGLGLHLLVLAAALQGAWSQPKKKRKV; SEQ ID NO:31), FBP's CPP (GALFLGWLGAAGSTMGAWSQPKKKRKV; SEQ ID NO:32), CPP of MPG (ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya;SEQ ID NO:33), CPP of MPG (ENLS) (ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-cya; SEQ ID NO:34), CPP of Pep-1 (ac-KETWWETWWTEWSQPKKKRKV-cya; SEQ ID NO:35), CPP of Pep-2 (ac-KETWFETWFTEWSQPKKKRKV-cya; SEQ ID NO:36), polyarginine sequence with structure RN (wherein N is between 4 and 17), GRKKRRQRRR sequence (SEQ ID NO:37), RRRRRRLR sequence (SEQ ID NO:38), RRQRRTS KLMKR sequence (SEQ ID NO:39); transporter GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:40); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:41). NO: 41); RQIKIWFQNRRMKWKK (SEQ ID NO: 42), YGRKKRRQRRR sequence (SEQ ID NO: 43); RKKRRQRR sequence (SEQ ID NO: 44); YARAAARQARA sequence (SEQ ID NO: 45); THRLPRRRRRR sequence (SEQ ID NO: 46); GGRRARRRRRR sequence (SEQ ID NO: 47). ;

[0123] In a preferred embodiment, the cell penetrating peptide is not the endogenous peptide contained in SEQ ID NO:1.

[0124] In a preferred embodiment, the CPP is a CPP of the HIV-1 TAT protein consisting of amino acids 49-57 (RKKRRQRRR, SEQ ID NO: 16). In another preferred embodiment, the CPP is a GRKKRRQRRR sequence (SEQ ID NO: 37) or RRRRRRLR (SEQ ID NO: 38). In another embodiment, the CPP is a GRKKRRQRRR sequence (SEQ ID NO: 37) or RRRRRRRR (SEQ ID NO: 65).

[0125] In certain embodiments, the CPP is a CPP as described in WO2019 / 018898, the contents of which are incorporated herein by reference in their entirety.

[0126] In one embodiment, the cell penetrating peptide sequence is fused to the N-terminus of the polypeptide of the present invention or a functionally equivalent variant of the polypeptide. In another embodiment, the cell penetrating peptide is fused to the C-terminus of the polypeptide of the present invention or a functionally equivalent variant of the polypeptide.

[0127] In a preferred embodiment, the conjugate or fusion protein of the combination according to the present invention comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more additional cell penetrating peptides in addition to the cell penetrating peptide itself found in the polypeptide of SEQ ID NO: 1 or a functionally equivalent variant of said polypeptide.

[0128] Suitable fusion proteins of the present invention include the polypeptides Omomyc*TAT and Omomyc*LZArg as defined below:

[0129]

[0130] Thus, in a preferred embodiment, the fusion protein is a polypeptide selected from SEQ ID NO: 11 and 12.

[0131] Suitable assays for determining whether a conjugate retains the cell membrane translocation ability of Omomyc include, but are not limited to, assays that measure the ability of the conjugate to transduce cells in culture. The assay is based on contacting the conjugate with cultured cells and detecting the presence of the conjugate at an intracellular location.

[0132] In another preferred embodiment, the conjugate of the combination of the invention further comprises an additional nuclear localization signal.

[0133] The term "nuclear localization signal" (NLS) used herein refers to an amino acid sequence of about 4-20 amino acid residues in length that is used to direct a protein to the nucleus. Typically, nuclear localization sequences are rich in basic amino acids, and exemplary sequences are well known in the art (Gorlich D. (1998) EMBO 5.17:2721-7). In certain embodiments, the NLS is selected from SV40 large T antigen NLS (PKKKRKV, SEQ ID NO:48); nucleoplasmin NLS (KRPAATKKAGQAKKKK, SEQ ID NO:49); CBP80 NLS (RRRHSDENDGGQPHKRRK, SEQ ID NO:50); HIV-I Rev protein NLS (RQARRNRRRWE, SEQ ID NO:51); HTLV-I Rex (MPKTRRRPRRSQRKRPPT, SEQ ID NO:52); hnRNPANLS (NQSSNFGPMKGGNFGGRSSGPYGGGGQYFKPRNQGGY, SEQ ID NO:53); rpL23a NLS (VHSHKKKKIRTSPTFTTPKTLRLRRQPKYPRKSAPRRNKLDHY, SEQ ID NO:54). In one embodiment of the invention, the nuclear localization signal comprises the motif K(K / R)X(K / R).

[0134] In an even more preferred embodiment, the nuclear localization signal is selected from the group consisting of PKKKRKV (SEQ ID NO: 48), PAAKRVKLD (SEQ ID NO: 56) and KRPAATKKAGQ AKKKK (SEQ ID NO: 49).

[0135] In another preferred embodiment, the NLS may be at the N-terminus or C-terminus of the conjugate or fusion protein comprising the polypeptide of SEQ ID NO: 1 or a functionally equivalent variant thereof.

[0136] The skilled person will appreciate that it may be desirable that the conjugate of the present invention further comprises one or more flexible peptides that are linked to the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant thereof, the cell penetrating peptide sequence and / or the NLS. Thus, in a specific embodiment, the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant thereof is directly linked to the cell penetrating peptide sequence. In another specific embodiment, the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant thereof is linked to the cell penetrating peptide sequence via a flexible peptide. In one embodiment, the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant thereof is directly linked to the NLS. In another embodiment, the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant thereof is linked to the NLS via a flexible peptide.

[0137] In a specific embodiment, the polypeptide of the conjugate according to the invention is directly linked to the cell penetrating peptide sequence and the NLS.

[0138] In one embodiment, the NLS is one of the NLSs that occurs endogenously in the Myc sequence, such as the M1 peptide (PAAKRVKLD, SEQ ID NO: 56) or the M2 peptide (RQRRNELKRSF, SEQ ID NO: 57).

[0139] In another embodiment, the additional NLS represents a NLS different from the endogenous NLS found in the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant of SEQ ID NO: 1.

[0140] In a preferred embodiment, the conjugate or fusion protein according to the invention comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 NLSs in addition to the endogenous NLS found in the polypeptide of the invention or its functionally equivalent variant.

[0141] In another specific embodiment, the polypeptide of the conjugate used according to the invention is linked to the cell penetrating peptide sequence via a first flexible peptide linker and to the NLS via a second flexible peptide linker.

[0142] As used herein, the term "flexible peptide", "spacer peptide" or "linker peptide" refers to a peptide that covalently binds two proteins or moieties but is not part of either polypeptide, allowing one to move relative to the other without causing substantial adverse effects on the function of the proteins or moieties. Thus, the flexible linker does not affect the tumor tracking activity of the polypeptide sequence, the cell penetrating activity of the cell penetrating peptide, or the nuclear localization ability of the NLS.

[0143] The flexible peptide comprises at least 1 amino acid, at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 12 amino acids, at least 14 amino acids, at least 16 amino acids, at least 18 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids or about 100 amino acids. In certain embodiments, the flexible peptide will allow one protein to move relative to another protein to increase the solubility and / or improve its activity. Suitable linker regions include poly-glycine regions, the GPRRRR sequence (SEQ ID NO: 58) of a combination of glycine, proline and alanine residues.

[0144] In a specific embodiment, the conjugate according to the invention comprises a tag that is bound to the C-terminal or N-terminal domain of the conjugate or the polypeptide or fusion protein or variant thereof. The tag can generally be used to separate or purify the peptide or amino acid sequence of the fusion protein. Thus, the tag is able to bind with high affinity to one or more ligands, such as one or more ligands of an affinity matrix (such as a chromatography support or beads). An example of the tag is a histidine tag (His-tag or HT), such as a tag comprising 6 histidine residues (His6 or H6), which can bind with high affinity to nickel (Ni 2+ ) or cobalt (Co 2+ ) column. The His-tag has the desirable characteristic that it can bind its ligand under conditions that denature most proteins and disrupt most protein-protein interactions. Therefore, it can be used to remove bait proteins tagged with H6 after the protein-protein interactions in which the bait has participated have been disrupted.

[0145] Additional exemplary, non-limiting examples of tags that can be used to isolate or purify a conjugate or a polypeptide comprising SEQ ID NO: 1 or a variant or fusion protein thereof include Arg-tags, FLAG-tags (DYKDDDDK; SEQ ID NO: 59), Strep-tags (WSHPQFEK, SEQ ID NO: 60), epitopes that can be recognized by antibodies such as c-myc-tags (recognized by anti-c-myc antibodies), HA-tags (YPYDVPDYA, SEQ ID NO: 61), V5-tags (GKPIPNPLLGLDST, SEQ ID NO: 62), SBP-tags, S-tags, calmodulin binding peptides, cellulose binding domains, chitin binding domains, glutathione S-transferase-tags, maltose binding protein, NusA, TrxA, DsbA, Avi-tags, etc. (Terpe K., Appl. Microbiol. Biotechnol. 2003, 60: 523-525), amino acid sequences such as AHGHRP (SEQ ID NO: 63) or PIHDHDHPHLVIHSGMTCXXC (SEQ ID NO: 64), β-galactosidase, etc.

[0146] If necessary, the tag can be used to isolate or purify the fusion protein.

[0147] In another preferred embodiment, the compound (i) of the present invention is a polynucleotide encoding the above-mentioned polypeptide or fusion protein. In a preferred embodiment, the compound (i) of the present invention is a polynucleotide encoding a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof. In another embodiment, the compound (i) of the present invention is a polynucleotide encoding a conjugate, the conjugate containing a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety, the chemical moiety promoting the cellular uptake of the polypeptide or its functionally equivalent variant; more preferably, it is a polynucleotide encoding a fusion protein between a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a cell penetrating peptide sequence.

[0148] The terms "polynucleotide", "nucleic acid" and "nucleic acid molecule" are used interchangeably to represent a polymeric form of nucleotides of any length. The polynucleotide may contain deoxyribonucleotides, ribonucleotides and / or their analogs. Nucleotides may have any three-dimensional structure and may perform any known or unknown function. The term "polynucleotide" includes, for example, single-stranded, double-stranded and triple-helical molecules, genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. In addition to natural nucleic acid molecules, nucleic acid molecules of the present invention may also include modified nucleic acid molecules. mRNA used herein means RNA that can be translated in cells.

[0149] In a preferred embodiment, the polynucleotide of the invention is mRNA.

[0150] The mRNA may be chemically synthesized, may be obtained by means of in vitro transcription, or may be synthesized in vivo in the target cell.The nucleotide sequences forming the polynucleotide encoding the conjugate or fusion protein of the invention are in the same correct reading frame for its expression.

[0151] In a preferred embodiment, component (i) of the combination of the present invention is an mRNA encoding a polypeptide consisting of the sequence SEQ ID NO: 1 or a polypeptide consisting of a functionally equivalent variant of SEQ ID NO: 1 or a polypeptide consisting of SEQ ID NO:4.

[0152] In another embodiment, component (i) of the combination of the invention is a vector comprising the polynucleotide of the invention.

[0153] The term "vector" used herein means such a nucleic acid sequence: it comprises the necessary sequence, thereby producing a polypeptide encoded by the polynucleotide of the present invention after transcription and translation of the sequence in the cell. The sequence is operably linked to another section, and the other section provides its autonomous replication in the host cell of interest. Preferably, the vector is an expression vector, which is defined as a vector containing a region operably linked to the nucleic acid of the present invention and capable of enhancing the expression of the nucleic acid product according to the present invention in addition to the region of autonomous replication in the host cell. The vector of the present invention can be obtained by technology widely known in the art.

[0154] Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors combined with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells. Suitable vectors comprising the polynucleotides of the present invention are vectors derived from expression vectors in prokaryotes such as pUC18, pUC19, pBluescript and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, bacteriophages, and "shuttle" vectors such as pSA3 and pAT28, expression vectors in yeast such as vectors of the 2-micron plasmid type, integrating plasmids, YEP vectors, centromeric plasmids and the like, expression vectors in insect cells such as vectors of the pAC series and the pVL series, expression vectors in plants such as vectors of the pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series and the like, and based on viral vectors (adenovirus, adenovirus-related viruses and retroviruses and in particular lentiviruses) and non-viral vectors such as pSilencer In a preferred embodiment, the polynucleotide of the present invention is contained in a vector selected from the group consisting of pEGFP or pBabe retroviral vectors and pTRIPZ or pSLIK lentiviral vectors.

[0155] The vector of the present invention can be used to transform, transfect or infect cells that can be transformed, transfected or infected by the vector. The cells can be prokaryotic cells or eukaryotic cells.

[0156] The vector preferably comprises a polynucleotide of the present invention operably combined with a sequence regulating the expression of the polynucleotide of the present invention. The regulatory sequence used in the present invention can be a nuclear promoter, or alternatively, an enhancer sequence and / or other regulatory sequences that increase the expression of a heterologous nucleic acid sequence. Generally speaking, any promoter can be used in the present invention, as long as the promoter is compatible with the cell in which the polynucleotide is to be expressed. Therefore, promoters suitable for implementing the present invention include, but are not necessarily limited to, constitutive promoters (such as derivatives of eukaryotic viral genomes, for example, polyoma virus, adenovirus, SV40, CMV, avian sarcoma virus, hepatitis B virus), metallothionein gene promoters, herpes simplex virus thymidine kinase gene promoters, LTR regions of retroviruses, immunoglobulin gene promoters, actin gene promoters, EF-1α gene promoters, and inducible promoters (wherein protein expression is dependent on the addition of molecules or exogenous signals, such as the tetracycline system, NFκB / ultraviolet system, Cre / Lox system, and heat shock gene promoters), regulatable RNA polymerase II promoters and tissue-specific promoters described in WO 2006 / 135436.

[0157] In another embodiment, component (i) of the combination of the invention is a cell, which is capable of secreting the polypeptide of the invention or the conjugate of the invention, preferably the polypeptide of the invention or the fusion protein of the invention, into the culture medium.

[0158] Suitable cells capable of secreting the polypeptide of the present invention include, but are not limited to, primary cultures of isolated cells of myocardial cells, adipocytes, endothelial cells, epithelial cells, lymphocytes (B and T cells), mast cells, eosinophils, endothelial cells, different organs, preferably isolated from cells of the islets of Langerhans, hepatocytes, leukocytes (including mononuclear leukocytes), interstitial, umbilical cord or adults (skin, lung, kidney and liver), osteoclasts, chondrocytes and other connective tissue cells. Established cell lines such as Jurkat T cells, NIH-3T3, CHO, Cos, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 cells, C2C12 myoblasts and W138 cells are also suitable. It will be appreciated by those skilled in the art that cells capable of secreting the polypeptide of the present invention in the culture medium may be found to form micron particles or micron capsules, so that the cells have a greater service life in patients. Materials suitable for forming the micron particle target of the present invention include any biocompatible polymeric material that allows continuous secretion of the therapeutic product and serves as a support for the cell. Thus, the biocompatible polymeric material may be, for example, a thermoplastic polymer or a hydropolymer. Among the thermoplastic polymers, we have acrylic acid, acrylamide, 2-aminoethyl methacrylate, poly(tetrafluoroethylene-co-hexafluoropropylene), methacrylate-(7-pyroalkyloxy)ethyl ester acid, N-isopropylacrylamide, polyacrylic acid, polyacrylamide, polyamidoamine, poly(amino)-p-xylene, poly(chloroethyl vinyl ether), polycaprolactone, poly(caprolactone-co-trimethyl carbonate), poly(urea carbonate) urethane, poly(carbonate) urethane, polyethylene, polyethylene and acrylamide copolymers, polyethylene glycol, polyethylene glycol methacrylate, poly(p-phenylene)ene Ethylene glycol dicarboxylate, poly(4-hydroxybutyl acrylate), poly(hydroxyethyl methacrylate), poly(N-2-hydroxypropyl methacrylate), poly(lactic-glycolic acid), poly(L-lactic acid), poly(γ-methyl, L-glutamate), poly(methyl methacrylate), poly(propylene fumarate), poly(propylene oxide), polypyrrole, polystyrene, poly(tetrafluoroethylene), polyurethane, polyvinyl alcohol, polyethylene of ultrahigh molecular weight, 6-(p-vinylbenzamide)-hexanoic acid N-p-vinylbenzyl-D-malonamide (maltonamide) and copolymers containing more than one of said polymers. Among the polymers of the hydrogel type we have natural substances such as alginate, agarose, collagen, starch, hyaluronic acid, bovine serum albumin, cellulose and its derivatives, pectin, chondroitin sulfate, fibrin and fibroin, as well as synthetic hydrogels such as and

[0159] Compound (ii) of the combination of the present invention

[0160] Compound (ii) of the combination of the present invention is a PARP inhibitor.

[0161] As used herein, "PARP" (EC 2.4.2.30) means poly(ADP-ribose) polymerase, also known as NAD+ ADP-ribosyltransferase or poly(ADP-ribose) synthase, and is a family of enzymes that play a key role in maintaining DNA integrity as part of the base excision pathway of DNA repair. PAR enzymes catalyze the transfer of ADP-ribose moieties from cellular NAD+ to nuclear proteins that form ADP-ribose polymers, which results in the first inhibitor being a structural analog of NAD+ that blocks NAD+ binding, thereby inhibiting PARP activity. PARPs have both enzymatic and scaffolding properties. The human PARP superfamily has 17 known members. PARP1, PARP2, anchor polymerase 1, anchor polymerase 2, and vPARP are believed to play a role in DNA repair, but PARP1 accounts for more than 90% of cellular PARP activity. Therefore, in a preferred embodiment, the PARP is selected from PARP1 and / or PARP2.

[0162] PARP1 plays a role in the repair of single-stranded DNA (ssDNA) breaks. PARP1 has three domains responsible for DNA binding, self-modification, and catalysis. DNA cleavage leads to the recruitment of PARP1 and binding to the damage site, accompanied by an increase in its catalytic activity and the formation of long branched poly (ADP-ribose) (PAR) chains. PAR has a net negative charge, which promotes the recruitment of DNA repair proteins involved in the base excision repair pathway to the DNA damage site and promotes the removal of PARP1 from the damage site, thereby allowing access to other repair proteins. In addition, PARP1 has been implicated in homologous recombination and non-homologous end joining pathways. The sequence of the PARP1 protein in humans corresponds to the sequence sequence P09874 in the Uniprot database (version 259 of this entry as of October 12, 2022).

[0163] PARP2 participates in base excision repair together with PARP1, but its function is still under investigation. PARP2 is substantially different from PARP1 in its domain structure, but has significant structural homology with the catalytic domain of PARP1. The sequence of the PARP2 protein in humans corresponds to the sequence of Q9UGN5 in the Uniprot database (version 202 of this entry as of October 12, 2022).

[0164] As used herein, "PARP inhibitor" refers to any compound that can cause a decrease in PARP activity, especially a decrease in the activity of PARP1 and PARP2, including those compounds that prevent PARP gene expression, especially those compounds that prevent PARP1 and PARP2 gene expression, and compounds that cause a decrease in PARP mRNA or protein levels, especially those compounds that cause a decrease in PARP1 and / or PARP2 mRNA or protein levels. PARP inhibitors mainly inhibit the catalytic activity of PARP1 and PARP2 enzymes. Therefore, in a preferred embodiment, the PARP inhibitor is selected from the group consisting of PARP1 inhibitors, PARP2 inhibitors, and PARP1 and PARP2 inhibitors.

[0165] Expression of a protein or nucleic acid is considered reduced when its level is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., absent) relative to a reference value.

[0166] A reference value represents the level of a protein or nucleic acid in a control subject, which may be a subject not suffering from a particular disease.

[0167] Suitable methods for determining whether an inhibitor is able to reduce PARP mRNA levels, in particular PARP1 and / or PARP2 mRNA levels, include, but are not limited to, standard assays for determining mRNA expression levels such as qPCR, RT-PCR, RNA protection assays, Northern blots, Northern dot blots, in situ hybridization, microarray technology, tag-based methods such as serial analysis of gene expression (SAGE) (including variants such as LongSAGE and SuperSAGE), microarrays, fluorescence in situ hybridization (FISH) (including variants such as Flow-FISH, qFiSH and double fusion FISH (D-FISH)), etc. Preferably, quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous.

[0168] Nucleic acids contained in a sample (eg, cells or tissue prepared from a subject) are first extracted according to standard methods (eg, using lytic enzymes or chemical solutions), or with a nucleic acid binding resin according to the manufacturer's instructions.

[0169] If mRNA is measured in a biological sample, the biological sample can be treated to physically, mechanically or chemically disrupt tissue or cell structure, release intracellular components into an aqueous or organic solution, thereby preparing nucleic acids for further analysis. Nucleic acids are extracted from the sample by procedures known to the skilled person and commercially available. RNA is then extracted from frozen or fresh samples by any typical method in the art, for example, Sambrook, J., et al., 2001. Molecular cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, NY, volumes 1-3. Preferably, care is taken to avoid degradation of RNA during the extraction process.

[0170] Using mRNA obtained from formalin-fixed, paraffin-embedded tissue samples, expression levels can be determined. mRNA can be isolated from archived pathology samples or biopsy samples that are first deparaffinized. An exemplary deparaffinization method comprises washing paraffinized samples with organic solvents such as xylene. The deparaffinized sample can be rehydrated with an aqueous solution of lower alcohols. Suitable lower alcohols, for example, include methanol, ethanol, propanol and butanol. For example, by continuous washing with lower alcohol solutions of decreasing concentrations, the deparaffinized sample can be rehydrated. Alternatively, the sample is deparaffinized and rehydrated simultaneously. Then the sample is cracked and RNA is extracted from the sample. Samples can also be obtained from fresh tumor tissues such as excised tumors. In a specific embodiment, samples can be obtained from fresh tumor tissues or from frozen tissues embedded in OCT.

[0171] In order to normalize mRNA expression values ​​between different samples, the expression level of the relevant mRNA in the test sample can be compared with the expression level of the control RNA. As used herein, "control RNA" refers to such RNA: its expression level in tumor cells does not change or changes only in a limited amount relative to non-tumorigenic cells. Preferably, the control RNA is an mRNA derived from a housekeeping gene, and it encodes a protein that is constitutively expressed and performs basic cellular functions. Preferred housekeeping genes for use in the present invention include beta-2-microglobulin, ubiquitin, 18-S ribosomal protein, cyclophilin, IPO8, HPRT, GAPDH, PSMB4, tubulin and beta-actin.

[0172] Relative gene expression quantification can be calculated according to the comparative threshold cycle (Ct) method using housekeeping genes as endogenous controls and commercial RNA controls as calibrators. -(ΔCt样品-ΔCt校准物) Final results were determined where the ΔCt values ​​for the calibrators and samples were determined by subtracting the Ct value of the target gene from the Ct value of the control gene.

[0173] Suitable methods for determining whether an inhibitor acts by reducing PARP protein levels, in particular PARP1 and / or PARP2 protein levels, include quantification by conventional methods, for example, using antibodies having the ability to specifically bind to proteins encoded by PARP genes, in particular PARP1 and / or PARP2 genes (or fragments thereof containing antigenic determinants), and then quantifying the resulting antibody-antigen complex.

[0174] The antibodies to be used in these assays may be, for example, polyclonal sera, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' and F(ab')2, ScFv, diabodies, tribodies, tetrabodies and humanized antibodies. At the same time, the antibodies may be labeled or unlabeled. Exemplary, but non-exclusive examples of markers that can be used include radioisotopes, enzymes, fluorophores, chemiluminescent agents, enzyme substrates or cofactors, enzyme inhibitors, particles, colorants, etc. There are a variety of well-known assays that can be used in the present invention, which use unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies); these techniques include Western blotting or protein transfer, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemistry and immunohistochemistry techniques, techniques based on the use of biochips or protein microarrays including specific antibodies, or assays based on colloidal precipitation (in the form of dipsticks, for example). Other methods for detecting and quantifying the levels of a protein of interest include affinity chromatography, binding ligand assays, and the like.

[0175] On the other hand, the determination of the levels of PARP proteins, in particular PARP1 and / or PARP2 proteins, can be performed as follows: a tissue microarray (TMA) containing assembled subject samples is constructed and the expression levels of the corresponding proteins are determined by immunohistochemistry. The intensity of the immunostaining can be evaluated by two or more different pathologists and scored using uniform and clear cutoff criteria to maintain the reproducibility of the method. Discrepancies can be resolved by simultaneous re-evaluation. In short, the results of the immunostaining can be recorded as negative expression (0) relative to positive expression, and low expression (1+) relative to medium (2+) and high (3+) expression, taking into account the expression in tumor cells and the specific cutoff value of each marker. As a general criterion, the cutoff value is selected to promote reproducibility and, where possible, translate biological events. Alternatively, immunostaining intensity can be assessed by using imaging techniques and automated methods such as those disclosed by Rojo, MG et al. (Folia Histochem. Cytobiol. 2009; 47: 349-54) or Mulrane, L. et al. (Expert Rev. Mol. Diagn. 2008; 8: 707-25).

[0176] Alternatively, in another specific embodiment, the levels of PARP proteins, in particular PARP1 and / or PARP2 proteins, are determined by Western blotting. Western blotting is based on the detection of specific proteins previously resolved by gel electrophoresis under denaturing conditions and immobilized on a membrane (usually nitrocellulose) by incubation with an antibody-specific visualization system (e.g. a chemiluminescent agent).

[0177] PARP inhibitors may inhibit PARP activity, particularly PARP1 and / or PARP2 activity, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or even 100%, and all ranges between 5% and 100%. Suitable methods for determining whether an inhibitor acts by reducing PARP activity include any method that allows for the detection of consumption of the substrate nicotinamide adenine dinucleotide (NAD+) or the formation of any of the three products, i.e., polymers of ADP-ribose (pADPr or PAR), nicotinamide, and protons. Methods for detecting PARP activity, in particular PARP1 activity, are known in the art and include, but are not limited to, those disclosed in Shah G. et al., Methods in Molecular Biology. Vol. 780, 2011, pp. 3-34; Putt KS et al., Anal Biochem. 2004 Mar 1; 326(1):78-86; Yelamos J. et al., Am J Cancer Res. 2011; 1(3):328-346. Assays for determining enzyme activity are known to the skilled person and include, but are not limited to, initial rate assays, progress curve assays, transient kinetic assays, and relaxometry. Continuous assays of enzyme activity include, but are not limited to, spectrophotometry, fluorescence, calorimetry, chemiluminescence, light scattering, and microscale thermal analysis. Discontinuous assays of enzyme activity include, but are not limited to, radiometric assays and chromatographic assays. As will be appreciated by the skilled artisan, factors that may affect enzyme activity include salt concentration, temperature, pH, and substrate concentration.

[0178] PARP inhibitors can be any organic or inorganic molecule, including modified and unmodified nucleic acids such as antisense nucleic acids, RNA interference (RNAi) agents such as siRNA, shRNA or miRNA, peptides, proteins, peptide mimetics, receptors, ligands, antibodies, and small organic molecules that can be used to treat cancer.

[0179] In a preferred embodiment, the PARP inhibitors useful in the present invention are selected from Table 1.

[0180]

[0181] The “ Small chemical compounds " denotes a molecule that modulates a biological process (in the present case, inhibits the catalytic activity of PARP). Such compounds may be natural or artificial.

[0182] In a preferred embodiment, the PARP inhibitor is selected from olaparib, rucaparib, veliparib, niraparib, talazopanib, pamiparib, fluzoparib and iniparib; preferably selected from olaparib, rucaparib, veliparib, niraparib, talazopanib, pamiparib and fluzoparib; more preferably selected from olaparib, rucaparib, veliparib, niraparib and talazopanib.

[0183] In a preferred embodiment, the PARP inhibitor is olaparib. Olaparib is a potent oral inhibitor of PARP1 and PARP2.

[0184] In another embodiment, the PARP inhibitor is talazopanib. Talazopanib is an oral PARP inhibitor.

[0185] The terms olaparib, rucaparib, veliparib, niraparib, talazoparib, pamiparib, fluzoparib and iniparib also include pharmaceutically acceptable salts, solvates, polymorphs or cocrystals thereof.

[0186] In a more preferred embodiment, the PARP inhibitor is selected from the compounds listed in item I, II or III of Table I or pharmaceutically acceptable salts thereof; preferably the compounds listed in item I, II and III of Table I.

[0187] In a more preferred embodiment, the PARP inhibitor is selected from the compounds listed in items I and II of Table I or pharmaceutically acceptable salts thereof; preferably the compounds listed in items I and II of Table I.

[0188] The term "pharmaceutically acceptable" refers to those properties and / or materials that are acceptable to patients from a pharmacological / toxicological point of view, and to manufacturing medicinal chemists from a physical / chemical point of view with respect to composition, formulation, stability, patient acceptance and bioavailability.

[0189] The term "pharmaceutically acceptable salt" includes a salt formed with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acid includes an inorganic acid, such as and not limited to hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid, hydroiodic acid and nitric acid, and an organic acid, such as and not limited to citric acid, fumaric acid, maleic acid, malic acid, mandelic acid, ascorbic acid, oxalic acid, succinic acid, tartaric acid, benzoic acid, acetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, cyclohexylaminosulfonic acid (cyclamic acid) or p-toluenesulfonic acid. Pharmaceutically acceptable bases include alkali metals (such as sodium or potassium) and alkaline earth metals (such as calcium or magnesium) hydroxides and organic bases, such as and not limited to alkylamines, arylalkylamines and heterocyclic amines.

[0190] The term "solvate" according to the present invention is understood to mean any solid form of the above compounds having another molecule attached to it by non-covalent bonding. Examples of solvates include hydrates and alcoholates, preferably C1-C6 alcoholates, such as methanolates.

[0191] The term "polymorph" according to the present invention is to be understood as a specific crystalline form of a compound which can crystallize in different forms.

[0192] As used herein, the term "co-crystal" is understood to mean a crystal structure consisting of a PARP inhibitor and at least one other component. Interfering RNA " or "iRNA" refers to an RNA molecule capable of silencing the expression of PARP, particularly PARP1 and / or PARP2, or the expression of any gene required for PARP function. For this purpose, iRNAs are generally double-stranded oligonucleotides having a length of at least 30 base pairs, and they more preferably contain about 25, 24, 23, 22, 21, 20, 19, 18 or 17 ribonucleic acid base pairs. Several different types of molecules have been effectively used in iRNA technology, including small interfering RNA (siRNA) (sometimes referred to as short interfering RNA or silencer RNA), micro RNA (miRNA) (generally distinguished from siRNA because they are processed from single-stranded RNA precursors and are shown to be only partially complementary to the target mRNA) and short hairpin RNA (shRNA).

[0193] Small interfering RNA (siRNA) reagents can inhibit target gene expression by interfering RNA. siRNA can be chemically synthesized, or can be obtained by in vitro transcription, or can be synthesized in vivo in target cells. Generally, siRNA is composed of double-stranded RNA with a length of 15 to 40 nucleotides, and can contain a protruding region on the 3' and / or 5' side with a length of 1 to 6 nucleotides. The length of the protruding region is independent of the total length of the siRNA molecule. siRNA plays a role by post-transcriptional degradation or silencing of the target messenger.

[0194] siRNA can present shRNA (short hairpin RNA) characteristics, because the antiparallel strands forming siRNA are connected by loops or hairpin regions. siRNA is composed of a short antisense sequence (19 to 25 nucleotides), a subsequent loop of 5-9 nucleotides and a sense strand. shRNA can be encoded by a plasmid or virus, particularly a retrovirus, more particularly a retrovirus, and is controlled by a promoter such as the U6 promoter of RNA polymerase III.

[0195] The siRNA used in the context of the present invention is substantially homologous to PARP mRNA, in particular PARP1 and / or PARP2 mRNA, or the genomic sequence encoding the protein thereof. The term "substantially homologous" is understood to mean that the siRNA has a sequence that is sufficiently complementary or similar to the target mRNA, such that the siRNA may be able to cause mRNA degradation by RNA interference. Suitable siRNAs that cause interference include siRNAs formed from RNA, as well as siRNAs containing different chemical modifications, such as:

[0196] - siRNAs where the linkages between nucleotides are different from those occurring in nature, such as phosphorothioate linkages;

[0197] -stranded RNA is conjugated to a functional agent (e.g., a fluorophore);

[0198] - Modification of the termini of the RNA chain, in particular the 3' termini, by combination with different functional hydroxyl groups at the 2'-position;

[0199] - sugar-modified nucleotides, such as O-alkylated residues at the 2'-position, for example 2'-O-methylribose or 2'-O-fluororibose;

[0200] - base-modified nucleotides, such as halogenated bases (eg 5-bromouracil and 5-iodouracil), alkylated bases (eg 7-methyl-guanosine).

[0201] Using a series of techniques known to those skilled in the art, the siRNA and shRNA used in the context of the present invention can be obtained. For example, siRNA can be synthesized from protected ribonucleoside phosphoramidite chemosynthesis in a conventional DNA / RNA synthesizer. Alternatively, siRNA can be produced from plasmids and viral vectors by recombinant dicer, wherein the coding region of one or more siRNA chains is controlled by the operation of RNA polymerase III promoter. RNase Dicer processes shRNA into siRNA in cells.

[0202] The PARP region used as the basis for siRNA design is not limiting and may contain the region of the coding sequence (between the start codon and the stop codon), or alternatively, may contain sequence from the 5' or 3' untranslated region, preferably 25 to 50 nucleotides in length and anywhere in the 3' position relative to the start codon. The procedure for siRNA design includes identifying the sequence motif AA(N19)TT, where N can be any nucleotide in the PARP sequence, and selecting those nucleotides that exhibit a high G / C content. If such a sequence motif is not found, it is possible to identify the sequence motif NA(N21), where N can be any nucleotide.

[0203] In a preferred embodiment, the PARP inhibitor is siRNA. In a more preferred embodiment, the siRNA is a commercial siRNA from Santa Cruz Biotechnology, in particular sc-29437 or sc-106356.

[0204] In another embodiment, the inhibitor is an "antisense oligonucleotide" specific for PARP, i.e. a molecule whose sequence is complementary to an mRNA encoding PARP, in particular to PARP1 and / or PARP2, i.e. to a cDNA coding strand. The antisense oligonucleotide may be complementary to the entire coding region, or to a region comprising the coding region and the 5' and 3' untranslated regions. The antisense oligonucleotide may consist of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length. The antisense oligonucleotide may be obtained by chemical synthesis or by an enzymatic binding reaction widely known to those skilled in the art. For example, the antisense oligonucleotide may further contain modified nucleotides that improve its biological stability or the stability of the double-stranded (bicatenary) DNA-RNA complex formed between the antisense oligonucleotide and the target polynucleotide, such as phosphorothioate derivatives, peptide nucleic acids and acridine-substituted oligonucleotides. Modified oligonucleotides that can be used to prepare antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetyl-cytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethyl-aminomethyluracil, dihydrouracil, β-D-galactosyl Q nucleoside, inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine , 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl Q nucleoside, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-oxyacetic acid, pseudouracil, Q nucleoside, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil and 2,6-diaminopurine. Alternatively, antisense nucleic acids can be produced biologically using expression vectors in which antisense directional nucleic acids have been cloned.

[0205] Another group of compounds that may form part of the present invention are catalytically active nucleic acids known as ribozymes." Ribozyme" comprises a catalytic region and a second region, the sequence of which is complementary to the target nucleic acid and confers substrate specificity to the ribozyme. After the ribozyme interacts with its substrate through hybridization and coupling between the target nucleic acid and the complementary region of the ribozyme, activation of the catalytic region occurs, thereby initiating inter-molecular or intra-molecular cleavage of the target nucleic acid. The basic considerations for ribozyme design are widely known to those skilled in the art (see, for example, Doherty and Doudna (Annu. Ref. Biophys. Biomolstruct. 2000; 30: 457-75).

[0206] Another class of compounds that may form part of the compositions of the present invention include inhibitory antibodies. Inhibitory antibodies ” is understood to mean an antibody that binds to PARP, in particular to PARP1 and / or PARP2, thereby inhibiting its catalytic activity.

[0207] Antibodies can be prepared using any method known to those skilled in the art. Thus, polyclonal antibodies are prepared by immunizing animals with the protein to be inhibited. Monoclonal antibodies can be prepared using the methods described by Kohler, Milstein et al. (Nature, 1975, 256:495). Once antibodies capable of binding to PARP are identified, those capable of inhibiting PARP activity, particularly PARP1 and / or PARP2, will be selected using the assays described above for determining PARP activity. Suitable antibodies in the present invention include complete antibodies, fragments "Fab", "F(ab')2", "Fab'", Fv, scFv, diabodies and bispecific antibodies comprising antigen binding variable and constant regions.

[0208] Other compounds capable of inhibiting PARP expression that may form part of the compositions of the invention include aptamers and spiegelmers. " Aptamers and spiegelmers "Aptamers" are single-stranded or double-stranded D- or L-nucleic acids that specifically bind to the protein, thereby causing a change in the biological activity of the protein (PARP, in particular PARPI and / or PARP2). Aptamers and spiegelmers are 15 to 80 nucleotides in length, and preferably 20 to 50 nucleotides in length.

[0209] In one embodiment, the combination of the invention is a conjugate between component (i) and component (ii) of the combination of the invention, in particular a conjugate between a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a PARP inhibitor.

[0210] In certain embodiments, the conjugation between components (i) and (ii) is via a non-cleavable linker. In certain embodiments, the conjugation between components (i) and (ii) is via a cleavable linker. Exemplary non-cleavable linkers and cleavable linkers are described in US8088387, US8142784, WO2013075048, US6630579, US8512707, US9120854, US9023351, US20160095938, US9446146, WO2005009369, US5773001, US6214345, US10111954, US8153768, US7829531, US20160082119, WO2018218004, US8568728, WO2015057699, US20170182181, US9198979, the contents of which are incorporated herein by reference in their entirety.

[0211] In another aspect, the present invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of the combination of the present invention and a pharmaceutically acceptable excipient.

[0212] As used in the present invention, the expression "pharmaceutical composition" relates to a preparation that has been adapted for administering a predetermined dose of one or several useful therapeutic agents to cells, cell populations, organs, tissues or animals with uncontrolled cell division, such as cancer.

[0213] The pharmaceutical composition of the present invention contains a pharmaceutically effective amount of a combination according to the present invention and a pharmaceutically active carrier. The pharmaceutical composition of the present invention contains a polypeptide comprising the sequence SEQ ID NO: 1, a functionally equivalent variant thereof, a conjugate according to the present invention, a polynucleotide encoding the polypeptide or the conjugate, a vector comprising the polynucleotide, or a cell capable of secreting the polypeptide or the conjugate into the culture medium and a PARP inhibitor. Suitable functionally equivalent variants of the polypeptide of SEQ ID NO: 1, suitable conjugates, fusion proteins, polynucleotides, vectors or cells for use in the pharmaceutical composition according to the present invention are as defined above.

[0214] The expression "pharmaceutically effective amount" used herein is understood to be an amount capable of providing a therapeutic effect, and it can be determined by a person skilled in the art by conventional means. The amount of Omomyc polypeptides, functionally equivalent variants thereof, conjugates, fusion proteins, polynucleotides, vectors, cells or PARP inhibitors that can be combined in a pharmaceutical composition according to the present invention will vary depending on the subject and the specific mode of administration. Those skilled in the art will appreciate that dosages can also be determined using guidance from the following documents: Goodman and Goldman's The Pharmacological Basis of Therapeutics, Ninth Edition (1996), Appendix II, pages 1707-1711, and Goodman and Goldman's The Pharmacological Basis of Therapeutics, Tenth Edition (2001), Appendix II, pages 475-493.

[0215] The appropriate dosage of the active ingredient in the pharmaceutical composition will depend on the type of cancer to be treated, the severity and course of the disease, whether the composition is for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the peptide or polypeptide, and the discretion of the attending physician.

[0216] The amount of a polypeptide comprising the sequence SEQ ID NO: 1, a functionally equivalent variant thereof, a fusion protein, a conjugate, a polynucleotide, a vector or a cell is appropriately administered to the patient in one or a series of treatments. Depending on the type and severity of the disease, an appropriate dosage level will generally be about 0.01 to 500 mg / kg of patient body weight per day, which can be administered in a single or multiple doses. Preferably, the dosage level will be about 0.1 to about 250 mg / kg per day; more preferably about 0.5 to about 100 mg / kg per day.

[0217] In a preferred embodiment, the amount of the first component is about 3.75 mg / kg subject body weight per day, preferably administered 4 times a week, preferably intranasally. In a preferred embodiment, the amount of the first component is about 8 to 15 mg / m 2 , preferably 10 to 12 mg / m 2 , more preferably 11.25 mg / m 2 , preferably administered 4 times a week, preferably intranasally.

[0218] In a preferred embodiment, the amount of the first component is about 50 mg / kg of subject body weight per day, preferably administered twice a week, preferably intravenously. In a preferred embodiment, the amount of the first component is about 100 to 200 mg / m 2 , preferably 125 to 175 mg / m 2, preferably 140 to 160 mg / m 2 , more preferably 150 mg / m 2 , preferably administered twice a week, preferably intravenously.

[0219] Suitable dosage levels may be about 0.01 to 250 mg / kg per day, about 0.05 to 100 mg / kg per day, or about 0.1 to 50 mg / kg per day. Within this range, the dosage may be 0.05 to 0.5, 0.5 to 5, or 5 to 50 mg / kg per day. For oral administration, the composition is preferably provided in tablet form containing 1.0 to 1000 mg of active ingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0 and 1000.0 mg of active ingredient, the dosage being determined by the symptoms of the patient to be treated. The compounds may be administered on a regimen of 1 to 4 times per day, preferably once or twice per day.

[0220] In one embodiment, the combination or composition can be used once a week, twice a week, three times a week, four times a week, five times a week, six times a week or seven times a week. In one embodiment, the combination or composition can be used once a week. In another embodiment, the combination or composition can be used twice a week. In another embodiment, the combination or composition can be used four times a week. In another preferred embodiment, the first component of the combination or composition is used four times a week, and the second component of the combination or composition is used once a week. In another embodiment, the first component of the combination or composition is used twice a week, and the second component of the combination or composition is used once a week. Two compounds can be administered concomitantly or sequentially. When the compound is administered sequentially, before the second compound is started, the administration of the first compound is stopped.

[0221] The duration of treatment can be at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks or longer. Preferably, the duration of treatment is at least four weeks. In another embodiment, the duration of treatment is at least three weeks.

[0222] The amount of the PARP inhibitor depends on the specific agent used and can be from about 0.01 mg / kg to about 200 mg / kg, preferably 0.01 mg / kg to about 150 mg / kg, more preferably 0.01 mg / kg to about 100 mg / kg, preferably 0.01 mg / kg to about 75 mg / kg, 0.01 mg / kg to about 50 mg / kg, more preferably 0.5 mg / kg to about 50 mg / kg, and preferably about 1 mg / kg to about 25 mg / kg of the subject's body weight per day, one or more times per day to achieve the desired therapeutic effect. In a preferred embodiment, the amount of the PARP inhibitor is about 2.5 mg / kg of the subject's body weight per day or 7.5 mg / kg per day. 2 , preferably once a week, more preferably orally. In a preferred embodiment, the amount of the PARP inhibitor is about 0.5 mg / kg of subject body weight per day or 1.5 mg / m per day. 2 , preferably once a week, more preferably orally. In a preferred embodiment, the amount of the PARP inhibitor is about 5 mg / kg of subject body weight per day or 15 mg / m 2 , preferably once a week, more preferably orally. In another preferred embodiment, the amount of the PARP inhibitor is about 10 mg / kg of subject body weight per day, or 30 mg / m per day. 2 , preferably once a week, more preferably orally. In another preferred embodiment, the amount of the PARP inhibitor is about 50 mg / kg of subject body weight per day, or 150 mg / m per day. 2 , preferably once a week, more preferably orally. In another preferred embodiment, the amount of the PARP inhibitor is about 100 mg / kg of subject body weight per day, or 300 mg / m 2 , preferably once a week, more preferably orally. The PARP inhibitor is preferably administered 6 days a week, preferably in 4 weeks.

[0223] The pharmaceutical composition according to the invention containing a first component (i) (which is selected from a polypeptide comprising SEQ ID NO: 1, a functionally equivalent variant thereof, a fusion protein, a conjugate, a polynucleotide, a vector or a cell according to the invention) and a second component (ii) (which is a PARP inhibitor) can be presented as a single preparation (e.g., as a tablet or capsule containing a fixed amount of each component), or on the other hand, can be presented as separate preparations to be combined later for joint, sequential or separate administration. The composition of the invention also includes preparations as a kit of parts, in which the components are formulated separately but packaged in the same container. It will be understood by those skilled in the art that the preparations of the different components in the pharmaceutical composition according to the invention can be similar, in other words, formulated similarly (tablets or pills), which allows them to be administered by the same route. In the case where the different components of the invention are formulated separately, the two components can be presented in blisters. Each blister contains a drug that must be consumed in a day. If the drug must be administered several times a day, the drugs corresponding to each administration can be placed in different parts of the blister, preferably at each part of the blister, the time at which they should be administered in a day is recorded. Alternatively, the components of the composition of the invention can be formulated differently so that the different components are administered differently. Thus, it is possible to formulate the first component for intravenous administration and the second component for oral administration as tablets or capsules, or vice versa. The ratio between the components as the components of the combination or pharmaceutical composition according to the present invention can be adjusted by the technician according to the antitumor agent used in each specific case and the desired indication. Therefore, the present invention contemplates such a composition, wherein the ratio between the amount of component (i) and component (ii) can be in the range of 50:1 to 1:50, particularly 40:1 to 1:40, particularly 30:1 to 1:30, particularly 20:1 to 1:20, 1:10 to 10:1 or 5:1 to 1:5. In a more specific embodiment, the ratio between the amounts is in the range of 1:1 to 1:5, preferably in the range of 1:1 to 1:3. In a more preferred embodiment, the ratio is in the range of 1:1 to 1:1.5, preferably 1:1.3 to 1:1.4, more preferably 1:1.34. In another preferred embodiment, the ratio is in the range of 1:1 to 1:2.8, preferably 1:2.6 to 1:2.7, more preferably 1:2.67. In another specific embodiment, the ratio between the amounts is in the range of 30:1 to 5:1, preferably 30:1 to 8:1, more preferably 25:1 to 15:1, more preferably 20:1 to 10:1. In a preferred embodiment, the ratio between the amounts is in the range of 20:1 to 1:20. In one embodiment, the ratio is 20:1. In another embodiment, the ratio is 1:20. In another embodiment, the ratio is 10:1. Preferably, these ratios are weight / weight ratios.In a preferred embodiment, these ratios are the ratios of Omomyc:Olaparib. Although these ratios are effective for treating any type of cancer, more preferably, these ratios are obtained when treating a cancer selected from triple negative breast cancer and pancreatic ductal adenocarcinoma.

[0224] The present invention also contemplates compositions wherein the ratio between the amounts of component (i) and component (ii), more preferably the ratio of Omomyc: Tarazopanib, may be in the range of 900,000:1 to 1:900,000, particularly 800,000:1 to 1:800,000, particularly 700,000:1 to 1:700,000, particularly 600,000:1 to 1:600,000, particularly 500,000:1 to 1:500,000, particularly is 400,000:1 to 1:400,000, in particular 300,000:1 to 1:300,000, in particular 200,000:1 to 1:200,000, in particular 150,000:1 to 1:150,000, in particular 100,000:1 to 1:100,000, in particular 75,000:1 to 1:75,000, in particular 50,000:1 to 1:50,000, in particular 30,000:1 to 1:30,000, In particular, 25,000:1 to 1:25,000, in particular 15,000:1 to 1:15,000, in particular 10,000:1 to 1:10,000, in particular 5,000:1 to 1:5,000, in particular 3,500:1 to 1:3,500, in particular 2,000:1 to 1:2,000, in particular 1,800:1 to 1:1,800, in particular 1,600:1 to 1:1,600, in particular 1,500:1 to 1; 1 to 1:90, in particular 75:1 to 1:75, in particular 60:1 to 1:60, in particular 55:1 to 1:55.

[0225] Particularly good results have been obtained when the ratio of component (i):component (ii), more preferably the ratio of Omomyc:Talazoparib, is in the range of 1:1 to 900,000:1, preferably 1:50 to 900,000:1, preferably 50:1 to 900,000:1, preferably 100:1 to 500,000:1, preferably 100:1 to 250,000:1, preferably 200:1 to 100,000: 1. Although these ratios are effective for treating any type of cancer, more preferably these ratios are obtained when treating triple negative breast cancer.

[0226] Preferably, these ratios are weight / weight ratios.

[0227] The components of the pharmaceutical composition or combination of the present invention can be administered simultaneously. "Simultaneous administration" encompasses the co-administration of two therapeutic agents, regardless of the relative frequency or timing of administration of the various agents. Thus, simultaneous administration encompasses the co-administration of two therapeutic agents at the same time with the same frequency of administration. Additionally, simultaneous administration refers to the co-administration of two therapeutic agents, wherein one agent is administered more frequently than the other agent. Additionally, simultaneous administration refers to the co-administration of two therapeutic agents, wherein one agent is administered only once during the administration of the other agent.

[0228] In one embodiment, component (i) is administered intranasally, in another embodiment, component (i) is administered intravenously. In another embodiment, component (ii) is administered orally. In another embodiment, component (ii) is administered parenterally, particularly intraperitoneally or intravenously.

[0229] In a preferred embodiment, component (i) of the combination or pharmaceutical composition of the invention is administered intravenously, while the PARP inhibitor is administered orally. For intravenous administration, the preferred dose of component (i) of the combination or composition of the invention, preferably the polypeptide or its functionally equivalent variant, fusion protein or conjugate, is in the range of 0.01 to 250 mg / kg, which can be administered in a single or multiple doses, more preferably 0.1 to about 100 mg / kg per day. The preferred dose of the PARP inhibitor for oral administration is 0.01 to 200 mg / kg, preferably 0.01 to 150 mg / Kg, more preferably between 0.1 and 100 mg / kg, more preferably 0.5 to 100 mg / kg, even more preferably 10 to 100 mg / kg, most preferably 50 to 100 mg / kg. Preferably, the PARP inhibitor is administered 6 days a week for 4 weeks.

[0230] In another embodiment, components (i) and (ii) of the combination or pharmaceutical composition of the invention are administered intravenously.

[0231] The pharmaceutical composition of the present invention may also contain one or more additional compounds for preventing and / or treating disorders in which there is uncontrolled cell division, such as cancer. The additional compounds (such as anti-tumor agents) may form a part of the pharmaceutical composition as an independent entity. In a preferred embodiment, the combination or pharmaceutical composition of the present invention comprises one or more anti-tumor agents selected from cytotoxic agents, anti-angiogenic agents, anti-metastatic agents and anti-proliferative agents.

[0232] The pharmaceutical composition of the present invention also contains one or more other pharmaceutically acceptable excipients. "Pharmaceutically acceptable excipient" is understood to be a therapeutically inactive substance for incorporating active ingredients, and it is acceptable to patients from a pharmacological / toxicological perspective, and is acceptable to manufacturing pharmaceutical chemists from a physical / chemical perspective about composition, formulation, stability, patient acceptance and bioavailability. The excipient can be a carrier. "Carrier" used herein refers to any substance used to improve the delivery and effectiveness of the active ingredient in the pharmaceutical composition. In a preferred embodiment, the carrier does not allow component (i) and / or (ii) to be directly delivered to the cytoplasm of the cell, i.e., the carrier cannot fuse with the plasma membrane of the target cell. The example of a pharmaceutically acceptable carrier includes one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. In many cases, isotonic agents, for example, sugars, polyols such as mannitol, sorbitol or sodium chloride will preferably be included in the combination or composition. Pharmaceutically acceptable carriers may further include trace amounts of auxiliary substances, such as wetting agents or emulsifiers, preservatives or buffers, which improve the shelf life or effectiveness of the components constituting the combination of the present invention or a part of the composition. The example of a suitable carrier is well known in the literature (see, for example, Remington's Pharmaceutical Sciences, 19th edition, Mack Publishing Company, Easton, PA, 1995). The example of a carrier is not limited to a series of sugars such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol; a series of starches such as corn starch, wheat starch, rice starch and potato starch; a series of celluloses such as cellulose, methylcellulose, sodium carboxymethylcellulose and hydroxypropyl methylcellulose; and a series of fillers such as gelatin and polyvinyl pyrrolidone. In some cases, disintegrants such as cross-linked polyvinyl pyrrolidone, agar, alginic acid or sodium alginate may be added.

[0233] The number and nature of pharmaceutically acceptable excipients depend on the desired dosage form. The pharmaceutically acceptable excipients are known to those skilled in the art (Faulíy Trillo C. (1993) "Tratado de Farmacia Galénica", Luzán 5, SAEdiciones, Madrid). The composition can be prepared by conventional methods known in the art ("Remington: The Science and Practice of Pharmacy", 20th edition (2003) Genaro A. R., ed., Lippincott Williams & Wilkins, Philadelphia, US).

[0234] For pharmaceutical compositions comprising medicaments as nucleic acid molecules, the nucleic acid molecules can be present in any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acids and bacterial, viral and mammalian expression systems, such as, for example, recombinant expression constructs provided herein. The technology of incorporating DNA into such expression systems is well known to those of ordinary skill in the art. The DNA can also be "naked", such as described in Ulmer et al., Science 259: 1745-49, 1993, and Cohen, Science 259: 1691-1692, 1993. By coating the DNA on biodegradable beads (the beads are effectively transported into cells), the uptake of naked DNA can be increased.

[0235] Nucleic acid molecules can be delivered into cells according to any of several methods described in the art (see, e.g., Akhtar et al., Trends Cell Bio. 2:139 (1992); Delivery Strategies for Antisense Oligonucleotide Therapeutics, Akhtar, ed., 1995; Maurer et al., Mol. Membr. Biol. 16:129-40 (1999); Hofland and Huang, Handb. Exp. Pharmacol. 137:165-92 (1999); Lee et al., ACSSymp. Ser. 752:184-92 (2000); U.S. Pat. No. 6,395,713; Int. Pat. App. Pub. No. WO 94 / 02595); Selbo et al., Int. J. Cancer 87:853-59 (2000); Selbo et al., Tumor Pharmacol. 114:115-127 (2001); Biol. 23: 103-12 (2002); U.S. Patent Application Publication Nos. 2001 / 0007666, and 2003 / 077829). Such delivery methods known to those skilled in the art include, but are not limited to, liposomal encapsulation, by iontophoresis, or by incorporation into other vehicles such as biodegradable polymers, hydrogels, cyclodextrins (see, e.g., Gonzalez et al., Bioconjug. Chem. 10:1068-74 (1999); Wang et al., International Application Publication Nos. WO 03 / 47518 and WO 03 / 46185), polylactic-co-glycolic acid (PLGA) and PLCA microspheres (which can also be used to deliver peptides and polypeptides and other substances) (see, e.g., U.S. Pat. No. 6,447,796; U.S. Patent Application Publication No. 2002 / 130430), biodegradable nanocapsules and bioadhesive microspheres, or by proteinaceous carriers (International Application Publication No. WO 00 / 53722). In another embodiment, the nucleic acid molecule can also be formulated or complexed with polyethyleneimine and its derivatives such as polyethyleneimine-polyethylene glycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethylene glycol-tri-N-acetylgalactosamine (PEI-PEG-tri-GAL) derivatives (see also, for example, U.S. Patent Application Publication No. 2003 / 0077829).

[0236] In a specific embodiment, when the composition or combination according to the present invention comprises nucleic acid (DNA, RNA, siRNA, antisense oligonucleotide, ribozyme, aptamer and spiegelmers), the pharmaceutical composition can be formulated as a composition for gene therapy; as an illustration and not limitation, the pharmaceutical composition can contain a viral or non-viral vector comprising a suitable polynucleotide or gene construct. As an illustration and not limitation, the vector can be a viral vector, for example based on retrovirus, adenovirus, etc. or non-viral such as ADN-liposome, ADN-polymer, ADN-polymer-liposome complex, etc. [see "Nonviral Vectors for Gene Therapy", Huang, Hung and Wagner, Academic Press (1999)]. The vector containing the corresponding polynucleotide or gene construct can be directly administered to the subject by conventional methods. Alternatively, the vector can be used for ex vivo transformation, transfection or infection of cells, such as mammalian cells, including humans, which are then implanted into the human body or animal to obtain the desired therapeutic effect. For administration to humans or animals, the cells will be formulated in a suitable culture medium that has no adverse effect on cell viability.

[0237] The combination or pharmaceutical composition of the present invention can be administered by any type of suitable route, such as by oral route, topical route, by inhalation or parenteral route, thereby including pharmaceutically acceptable excipients required for the formulation of the desired dosage form. Other routes of administration may be rectal, intracisternal or intravaginal. The preferred route of administration of the combination or pharmaceutical composition is the intravenous route. Alternatively, component (i) may be administered intravenously and component (ii) may be administered orally.

[0238] "Oral route" is understood to be a pharmaceutical composition that is integrated into the organism after swallowing. In a particular embodiment, the pharmaceutical composition of the present invention may be a dosage form suitable for its administration by the oral route, whether it is solid or liquid. The dosage form suitable for administration by the oral route may be a tablet, capsule, syrup or solution, and may contain any conventional excipient known in the art, such as a binder, for example, syrup, gum arabic, gelatin, sorbitol or polyvinyl pyrrolidone; a filler, for example, lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; a lubricant for compression, for example, magnesium stearate; a disintegrant, for example, sodium glycolate of starch, polyvinyl pyrrolidone, starch or microcrystalline cellulose; or a pharmaceutically acceptable wetting agent such as sodium lauryl sulfate. The solid oral composition may be prepared by conventional processes of mixing, filling or compression. Repeated mixing operations may be used to distribute the active agent completely in those compositions using a large amount of fillers. The operations are conventional operations in the art. The tablets can be prepared, for example, by means of wet or dry granulation and optionally coated according to processes known in normal pharmaceutical practice, in particular with an enteric coating.

[0239] On the other hand, "topical route" is understood to be administration by non-systemic routes and includes external application of the pharmaceutical composition of the present invention on the epidermis, in the oral cavity, and instillation of the composition into the ear, eye and nose, where it does not significantly enter the bloodstream. Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches.

[0240] Ophthalmic preparations, ear drops and eye drops are also considered to be within the scope of the present invention. In addition, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of compounds to the body. Such dosage forms can be prepared by dissolving or dispersing the compounds in a suitable medium. Absorption enhancers can also be used to increase the flux of the compounds through the skin. The rate can be controlled by providing a rate-controlled film or by dispersing the compounds in a polymer matrix or gel.

[0241] In one embodiment, the combination or pharmaceutical composition is administered systemically.

[0242] "Systemic route" is understood to mean administration by the oral route, intravenous route, intraperitoneal route and intramuscular route. The amounts of components (i) and (ii) required for a therapeutic or prophylactic effect will naturally vary depending on the compound chosen, the nature and severity of the disease being treated and the patient. Preferably, the combination or pharmaceutical composition is administered orally.

[0243] In another embodiment, the combination or pharmaceutical composition is administered intranasally. In a preferred embodiment, intranasal administration is performed by instillation or nasal inhalation.

[0244] "Inhalation" is understood to mean administration by the intranasal route and by oral inhalation. Dosage forms suitable for such administration, such as formulations in aerosols or metered dose inhalers, can be prepared by conventional techniques. In one embodiment, the administration route is the intranasal route.

[0245] The term "parenteral" as used herein includes administration by intravenous, intraperitoneal, intramuscular or subcutaneous routes. Subcutaneous, intramuscular and intravenous dosage forms for parenteral administration are generally preferred. In one embodiment, the combination or pharmaceutical composition is administered intravenously.

[0246] In one embodiment, the combination or pharmaceutical composition of the present invention may be suitable for its parenteral administration, such as a sterile solution, suspension or lyophilized product in the form of an appropriate dosage unit. Combinations or pharmaceutical compositions suitable for its injection purposes include sterile aqueous solutions (when they are soluble in water), or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For administration by intravenous route, some suitable carriers include phosphate-buffered saline solutions (PBS). In all cases, the combination or composition must be sterile and must be fluid to the extent that it is easy to inject. It must be stable under preparation and storage conditions, and must be protected from the contamination activities of microorganisms such as bacteria and fungi. The carrier may be a solvent or a dispersion medium containing, for example, water, ethanol, pharmaceutically acceptable polyols such as glycerol, propylene glycol, liquid polyethylene glycol and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by means of using a coating such as lecithin, by means of maintaining the desired particle size (in the case of a dispersion) and by means of using a surfactant. Prevention of the action of microorganisms can be achieved with the help of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In most cases, it will be preferred to include isotonic agents, for example, sugars; polyalcohols such as mannitol, sorbitol; or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents that delay absorption (for example, aluminum monostearate and gelatin).

[0247] Injectable sterile solutions can be prepared as follows: the required amount of active compound is mixed with one or a combination of the aforementioned ingredients required in a suitable solvent, followed by sterilization by filtration through a sterile membrane. Typically, dispersions are prepared by mixing the active compound into a sterile vehicle containing a basic dispersion medium and the remaining ingredients required from those previously listed. In the case of sterile powders for the preparation of injectable sterile solutions, preferred preparation processes are vacuum drying and lyophilization, which produce a powder containing the active ingredient plus any desired additional ingredients from its previously filtered sterile solution.

[0248] The combination or pharmaceutical composition of the invention may be suitably administered by means of pulse infusion, for example, a gradually decreasing dose of the composition. Preferably, the dose is administered by means of an injection, more preferably intravenous or subcutaneous injection, depending in part on whether the administration is acute or chronic.

[0249] Alternatively, as described above, the different components of the composition are applied differently.

[0250] Thus, in one embodiment, component (i) of the combination or composition, preferably a polypeptide or functionally equivalent variant or conjugate of the invention, is administered intravenously, while the PARP inhibitor is administered orally.

[0251] In another embodiment, both components (i) and (ii) are administered intravenously.

[0252] In another embodiment, component (i) of the combination or composition, preferably the polypeptide or a functionally equivalent variant thereof, or a conjugate of the composition is administered intranasally or by inhalation.

[0253] The dosage form of the composition intended for intranasal and intrapulmonary administration is preferably a liquid, suspension or solid. A suspension is a liquid preparation containing solid particles dispersed in a liquid vehicle. The dosage form is preferably metered. For example, metered drops / sprays mean that the dispenser comprising the drops / sprays delivers droplets / sprays containing a metered dose (predetermined amount) of the composition for use according to the present invention.

[0254] In the context of intranasal administration, a preferred dosage form includes nasal drops. The droplets are mainly deposited in the back of the nose and thus quickly exit into the nasopharynx. A problem with drops is often how to accurately control the dose of the drug, which is particularly important for the administration of the composition.

[0255] Another intranasal dosage form that can be used to administer the pharmaceutical composition of the present invention is a nasal spray. Nasal sprays typically contain a conjugate in a solution or mixture of an excipient (e.g., preservative, viscosity modifier, emulsifier, buffer) dissolved or suspended in a non-pressurized dispenser. Nasal sprays have several advantages, including the compactness, convenience, simplicity of use, and accuracy of the delivered dose of 25 to 200 pL of a delivery device. They are deposited in the front of the nose and are slowly removed into the nasopharynx by mucociliary removal. The nasal spray used herein can be a liquid or a suspension.

[0256] Another intranasal dosage form is a nasal aerosol. Nasal aerosols differ from nasal sprays in the method of dispensing the composition: in an aerosol, the compound is dispensed due to excess pressure and released through a valve. In a spray, the compound is dispensed due to the exit force generated by a micropump barrel, while the pressure in the vial is similar to atmospheric pressure. Aerosols have similar advantages to sprays.

[0257] The compositions according to the invention may alternatively and preferably be administered via nasal emulsions, ointments, gels, pastes or creams. These are highly viscous solutions or suspensions for application to the nasal mucosa.

[0258] Due to the limited volume of the composition that can be effectively delivered to the nasal mucosa, liquid intranasal dosage forms usually have a higher concentration than corresponding intravenous dosage forms. When the material becomes insoluble or unstable in liquid form, powders can be used to apply the composition of the present invention. Other advantages of powders are that they do not require preservatives and usually have higher stability compared with liquid preparations. The main limitation of intranasal powder application is relevant to its irritation to the nasal mucosa.

[0259] A dosage form in the context of intrapulmonary administration is an inhalation aerosol. An inhalation aerosol is usually packaged under pressure and contains a composition according to the invention, which is released into the respiratory tract, particularly the lungs, after the valve system is activated. The released aerosol is a colloid of fine solid particles (suspension) or liquid droplets (solution) in air or other gases. Therefore, the aerosol can be a solution or a suspended aerosol. The liquid droplets or solid particles preferably have a diameter of less than 100 pm, more preferably less than 10 pm, and most preferably less than 1 pm.

[0260] Another dosage form in the context of intrapulmonary administration is an inhalation spray. Inhalation sprays are typically water-based formulations and do not contain any propellants. The conjugate is delivered to the lungs by oral inhalation.

[0261] Nebulized inhalation solutions and suspensions can also be used to deliver the conjugate via the intrapulmonary route. Nebulized inhalation solutions and suspensions are typically water-based formulations containing the composition according to the invention. Nebulized inhalation solutions and suspensions deliver the composition to the lungs by oral inhalation to produce a systemic effect and are used with a nebulizer.

[0262] Dry powder inhalation is an alternative to aerosol inhalation. The composition is usually included in a capsule for manual loading or in an inhaler. Dry powders are usually delivered to the lungs by oral inhalation from an inhaler. The dry powder used herein can be formulated as pure. Pure preparations contain separate or quasi-separate medicaments, for example as spray-dried powders. The dry powder used herein can also be formulated with a carrier such as lactose.

[0263] Intrapulmonary dosage forms are preferably metered, ie, delivered to the lung in a predetermined amount.

[0264] Devices for intranasal delivery in the context of the present invention include spray pump systems, pipettes for delivering liquid drops, metered dose spray pumps, nasal pressurized metered dose inhalers, powder spray systems, breath-actuated powder inhalers, and nasal powder insufflators. The intranasal delivery device can be filled with a single dose amount or multiple dose amounts of the intranasal formulation.

[0265] Using the intrapulmonary route, the conjugate can be administered with a metered dose inhaler. A metered dose inhaler (MDI) provides a fine mist of the conjugate, typically with an aerodynamic particle size of less than 5 pm.

[0266] A dry powder inhaler may alternatively be used to deliver the composition to the lungs. Dry powder inhalers provide the powder in the form of a single dose or a multi-dose powder.

[0267] Another device for intrapulmonary delivery is a nebulizer, including ultrasound and air jet nebulizer. In an ultrasonic nebulizer, ultrasound waves are formed in an ultrasonic atomizer chamber by a ceramic piezoelectric crystal, which vibrates when electrically excited. This can produce an aerosol cloud at the surface of the solution. The aerosol produced by the air jet nebulizer is produced when compressed air is forced through an orifice. Liquid can be discharged from a vertical nozzle (Bernoulli effect) to mix with an air jet atomized using a baffle, thereby promoting the formation of an aerosol cloud.

[0268] In one embodiment, each component of the combination or pharmaceutical composition of the present invention is prepared with a carrier that protects the components, particularly component (i), from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated administration systems. Biodegradable biocompatible polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The method for preparing the preparation will be clear to those skilled in the art. The material can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.

[0269] The sustained release composition also includes a crystal preparation suspended in a suitable formulation that can keep the crystals in suspension. These preparations can produce a sustained release effect when they are injected subcutaneously or intraperitoneally. Other compositions also include components (i) and / or (ii) captured in liposomes. Liposomes containing such components are prepared by known methods, such as Epstein et al., Proc. Natl. Acad. Sci. USA, (1985) 82: 3688-3692; Hwang et al., Proc. Natl. Acad. Sci. USA, (1980) 77: 4030-4034; EP 52,322; EP 36,676; EP 88,046; EP143,949. In a preferred embodiment, components (i) and / or (ii) are contained in liposomes, preferably both components are contained in liposomes, more preferably in the same liposomes.

[0270] Despite the fact that Omomyc, its functional equivalent variants, conjugates and fusion proteins of the present invention are capable of transmembrane transfer, it is possible to formulate Omomyc, any of its functional equivalent variants, conjugates, polynucleotides, vectors or cells in nanoparticles. The nanoparticles can help maintain the integrity of the components in the biological fluid until it reaches the target organ. In addition, in the case of a composition comprising component (ii) or other anti-tumor agents, the encapsulation of the composition can reduce the side effects caused by the anti-tumor agent. In addition, the nanoparticles can also be modified to include portions that allow the nanoparticles to be targeted to an organ of interest. In this way, component (i) of the combination or composition of the present invention will be delivered near the target organ, thereby promoting component (i) to enter the interior of the cell where its biological activity is required.

[0271] Thus, in another embodiment, component (i) of the combination or composition of the invention is provided to form part of a nanoparticle. In another embodiment, both components of the combination or composition of the invention are provided to form part of a nanoparticle, preferably both components are provided within the same nanoparticle.

[0272] The term "nanoparticle" as used herein means any material with a size within the range of 1-1,000nm. In certain embodiments, the nanoparticle has a size within the range of 2-200nm, preferably within the range of 2-150nm, and even more preferably within the range of 2-100nm. The nanoparticles that can be used in the present invention include, such as nanoparticles based on lipids, superparamagnetic nanoparticles, nanoshells, semiconductor nanocrystals, quantum dots, nanoparticles based on polymers, nanoparticles based on silicon, nanoparticles based on silicon dioxide, nanoparticles based on metals, fullerenes and nanotubes and other nanoscale materials. Molecules can be embedded in the nanoparticle matrix, or can be adsorbed on its surface, preferably molecules are embedded in the nanoparticles.

[0273] In a preferred embodiment, the nanoparticles are liposomes.

[0274] Targeted delivery can be achieved by adding ligands, and the ability of the delivery of its contents of the nanoparticles will not be compromised. Considering that this will achieve delivery to specific cells, tissues and organs. The targeting specificity of the delivery system based on ligands is based on the distribution of ligand receptors on different cell types. The targeting ligand can be combined with the nanoparticles non-covalently or covalently, and can be conjugated to the nanoparticles by a variety of methods discussed herein.

[0275] Examples of proteins or peptides that can be used for targeting nanoparticles include transferrin, lactoferrin, TGF-β, nerve growth factor, albumin, HIV Tat peptide, RGD peptide, and insulin, among others.

[0276] It should be understood that the formulation of the present invention in nanoparticles is not intended or is not intended solely to promote the entry of components (i) and / or (ii) into the cell interior, but also to protect components (i) and / or (ii) from degradation and / or to promote targeting of the nanoparticles to relevant organs.

[0277] In one embodiment, the nanoparticles can be composed of a biodegradable polymer such as poly(butyl cyanoacrylate) (PBCA). Examples of elemental nanoparticles include carbon nanoparticles and iron oxide nanoparticles, which can then be coated with oleic acid (OA)-Pluronic(R). In this approach, a drug (e.g., a hydrophobic or water-insoluble drug) is loaded into the nanoparticles. Other nanoparticles are made of silicon dioxide.

[0278] Nanoparticles can be formed from any useful polymer. Examples of polymers include biodegradable polymers such as poly(butylcyanoacrylate), poly(lactide), poly(glycolide), poly-s-caprolactone, poly(butylene succinate), poly(ethylene succinate), and poly(p-dioxanone); poly(ethylene glycol); poly-2-hydroxyethyl methacrylate (poly(HEMA)); copolymers such as polylactide-co-glycolide, poly(lactide)-poly(ethylene glycol), poly(poly(ethylene glycol) cyanoacrylate-hexadecyl cyanoacrylate) copolymer, and poly[HEMA-methacrylic acid] copolymer; proteins such as fibrinogen, collagen, gelatin, and elastin; and polysaccharides such as pullulan, amylose, and chitosan.

[0279] Other nanoparticles include solid lipid nanoparticles (SLN). Examples of lipid molecules of solid lipid nanoparticles include stearic acid and modified stearic acid, such as stearic acid-PEG 2000; soy lecithin; and emulsifying wax. Solid lipid nanoparticles may optionally include other components, including surfactants, such as Epicuron (R) 200, poloxamer 188 (Pluronic (R) F68), Brij 72, Brij 78, polysorbate 80 (Tween 80); and salts, such as sodium taurocholate. Agents may be introduced into solid lipid nanoparticles by a variety of methods discussed for liposomes, such methods may further include high pressure homogenization and dispersion of microemulsions.

[0280] Nanoparticles can also include nano-sized micelles. Micelles can be formed from any polymer described herein. Exemplary polymers for forming micelles include block copolymers, such as poly (ethylene glycol) and poly (ε-caprolactone). (For example, PEO-b-PCL block copolymers, including polymers of ε-caprolactone and α-methoxy-ω-hydroxy-poly (ethylene glycol)).

[0281] In certain embodiments, the properties of the nanoparticles are altered by coating with a surfactant. Any biocompatible surfactant can be used, for example, polysorbate surfactants such as polysorbate 20, 40, 60 and 80 (Tween 80); Epicuron (R) 200; poloxamer surfactants such as 188 (Pluronic (R) F68) poloxamer 908 and 1508; and Brij surfactants such as Brij 72 and Brij 78.

[0282] The nanoparticles can optionally be modified to include hydrophilic polymer groups (e.g., poly(ethylene glycol) or poly(propylene glycol)), for example, by covalently attaching the hydrophilic polymer groups to the surface, or using polymers containing such hydrophilic polymer groups (e.g., poly[methoxypoly(ethylene glycol)cyanoacrylate-co-hexadecylcyanoacrylate]). The nanoparticles can optionally be cross-linked, which can be particularly useful for protein-based nanoparticles.

[0283] In another embodiment, the pharmaceutical composition of the present invention is a nanoemulsion. "Nanoemulsion" as used herein refers to a colloidal dispersion of droplets (or particles) wherein at least some of the droplets have a diameter in the nanometer size range. The nanoemulsion is composed of an oil rich in omega-3, -6 or -9 fatty acids in an aqueous phase and is thermodynamically stabilized by an amphiphilic surfactant (which constitutes an interfacial surface film), is produced using a high shear microfluidization process, and typically has a droplet diameter in the range of about 80-220 nm.

[0284] Therapeutic uses of the invention

[0285] In one aspect, the invention relates to a combination or pharmaceutical composition of the invention for use in medicine.

[0286] In another aspect, the present invention relates to a combination or pharmaceutical composition of the present invention for use in the prevention and / or treatment of cancer.

[0287] In another aspect, the present invention relates to a combination or pharmaceutical composition of the present invention for use in the preparation of a medicament for the prevention and / or treatment of cancer.

[0288] In another aspect, the present invention also relates to a method for preventing and / or treating cancer, which comprises administering a therapeutically effective amount of the combination or pharmaceutical composition of the present invention to a subject in need thereof.

[0289] In a preferred embodiment, the method for prevention or treatment according to the present invention involves the direct use of a combination or composition containing a polypeptide comprising Omomyc, a functionally equivalent variant thereof, a conjugate or a fusion protein. Thus, in a preferred embodiment, the method for prevention or treatment according to the present invention does not involve the administration of a nucleic acid encoding a polypeptide comprising Omomyc or a functionally equivalent variant thereof or a fusion protein, or the administration of a vector encoding the nucleic acid or a cell comprising the nucleic acid.

[0290] "Prevention" is understood as administering the combinations or compositions of the invention at the initial or early stages of a disease, or also preventing its onset.

[0291] The term "treatment" refers to the administration of a combination or composition of the invention before or after clinical signs have appeared to control the progression of the disease. Control of disease progression is understood to be a beneficial or desired clinical outcome, which includes, but is not limited to, a reduction in symptoms, a shortening of the duration of the disease, a stabilization of the pathological condition (particularly avoiding additional damage), a delay in the progression of the disease, an improvement in the pathological condition, and alleviation (partial and complete). Control of disease progression also involves an extension of survival compared to the expected survival if treatment is not applied. In a preferred embodiment, control of disease progression is measured as a healthy lung / thorax volume ratio. In another embodiment, control of disease progression is measured as a reduction in tumor volume. In another embodiment, control of disease progression is measured as a reduction in tumor cell viability.

[0292] The term "cancer" refers to a disease characterized by uncontrolled cell division (or increased survival or resistance to apoptosis), the ability of the cells to invade other adjacent tissues (invasion) or to spread through lymphatic and blood vessels to other areas of the body where the cells would not normally be located (metastasis). Tumors are classified as benign or malignant depending on whether they can spread by invasion and metastasis: benign tumors are tumors that cannot spread by invasion or metastasis, i.e., they can only grow locally; whereas malignant tumors are tumors that can spread by invasion and metastasis. The method according to the invention can be used to treat both localized and malignant tumors.

[0293] In one embodiment, cancer includes, but is not limited to, leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute granulocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), hairy cell leukemia, polycythemia vera, lymphoma (e.g., Hodgkin's disease, Hodgkin's disease or non-Hodgkin's disease), AIDS-related leukemia, Waldenstrom's macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, mendotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma , Kaposi's sarcoma, colon cancer, pancreatic cancer, breast cancer, bile duct cancer, esophageal cancer, ovarian cancer, prostate cancer, oral cancer (including squamous cell carcinoma), basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct cancer, teratoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilman's tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, upper The following are examples of cancers that may be treated: melanoma, intraepithelial neoplasms (including Bowen's disease and Paget's disease), glioma, neuroglioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0294] In certain embodiments, the cancer is a glioma, an astrocytoma, a glioblastoma multiforme (GBM, also known as a glioblastoma), a medulloblastoma, a craniopharyngioma, an ependymoma, a pinealoma, a hemangioblastoma, an acoustic neuroma, an oligodendroglioma, a schwannoma, a neurofibrosarcoma, a meningioma, a melanoma, a neuroblastoma, or a retinoblastoma.

[0295] In certain embodiments, the cancer is an acoustic neuroma, an astrocytoma (e.g., grade I - pilocytic astrocytoma, grade II - low-grade astrocytoma, grade III - anaplastic astrocytoma, or grade IV - glioblastoma (GBM)), a chordoma, a CNS lymphoma, a craniopharyngioma, a brainstem glioma, an ependymoma, a mixed glioma, an optic nerve glioma, a subependymoma, a medulloblastoma, a meningioma, a metastatic brain tumor, an oligodendroglioma, a pituitary tumor, a primitive neuroectodermal (PNET) tumor, or a schwannoma. In certain embodiments, the cancer is a type that is more common in children than in adults, such as a brainstem glioma, a craniopharyngioma, an ependymoma, a juvenile pilocytic astrocytoma (JPA), a medulloblastoma, an optic nerve glioma, a pineal tumor, a primitive neuroectodermal tumor (PNET), or a rhabdomyosarcoma. In certain embodiments, the patient is an adult. In certain embodiments, the patient is a child or a pediatric patient.

[0296] In another embodiment, cancers include, but are not limited to, mesothelioma, hepatobiliary (liver and bile duct) cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, gastrointestinal cancer (gastric cancer, colorectal cancer, and duodenal cancer), uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, Adenocarcinoma, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvic cancer, non-Hodgkin lymphoma, spinal cord axial tumors, brain stem glioma, pituitary adenoma, adrenal cortical carcinoma, gallbladder cancer, multiple myeloma, bile duct cancer, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the foregoing cancers.

[0297] In certain embodiments, the cancer is selected from hepatocellular carcinoma, ovarian cancer, ovarian epithelial cancer, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatobiliary cancer; synovial sarcoma of soft tissue and bone; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; anaplastic thyroid cancer; adrenocortical adenoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / gastric (GIST) cancer; lymphoma; squamous cell carcinoma of the head and neck (SCCHN); salivary gland cancer; glioma, or brain cancer; neurofibromatosis-1-associated malignant peripheral nerve sheath tumor (MPNST); Waldenstrom's macroglobulinemia; or medulloblastoma.

[0298] In certain embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial cancer, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), hepatobiliary carcinoma, synovial sarcoma of soft tissue and bone, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid cancer, adrenocortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST), Waldenstrom's macroglobulinemia, or medulloblastoma.

[0299] In certain embodiments, the cancer is a solid tumor, such as a sarcoma, carcinoma, or lymphoma. Solid tumors generally include abnormal tissue masses that generally do not include cysts or fluid areas. In certain embodiments, the cancer is selected from renal cell carcinoma or kidney cancer; hepatocellular carcinoma (HCC) or hepatoblastoma or liver cancer; melanoma; breast cancer; colorectal cancer or colorectal cancer; colon cancer; rectal cancer; anal cancer; lung cancer, such as non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC); ovarian cancer, ovarian epithelial cancer, ovarian cancer, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer ; hepatobiliary carcinoma; synovial sarcoma of soft tissue and bone; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing sarcoma; anaplastic thyroid cancer; adrenocortical carcinoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / gastric (GIST) cancer; lymphoma; squamous cell carcinoma of the head and neck (SCCHN); salivary gland cancer; glioma, or brain cancer; neurofibromatosis-1-associated malignant peripheral nerve sheath tumor (MPNST); Waldenstrom's macroglobulinemia; or medulloblastoma.

[0300] In certain embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial cancer, ovarian cancer, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), hepatobiliary carcinoma, synovial sarcoma of soft tissue and bone, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid cancer, adrenocortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST), Waldenstrom's macroglobulinemia, or medulloblastoma.

[0301] In certain embodiments, the cancer is hepatocellular carcinoma (HCC). In certain embodiments, the cancer is hepatoblastoma. In certain embodiments, the cancer is colon cancer. In certain embodiments, the cancer is rectal cancer. In certain embodiments, the cancer is ovarian cancer or ovarian cancer. In certain embodiments, the cancer is ovarian epithelial cancer. In certain embodiments, the cancer is fallopian tube cancer. In certain embodiments, the cancer is papillary serous cystadenocarcinoma. In certain embodiments, the cancer is uterine papillary serous carcinoma (UPSC). In certain embodiments, the cancer is hepatobiliary cancer. In certain embodiments, the cancer is synovial sarcoma of soft tissue and bone. In certain embodiments, the cancer is rhabdomyosarcoma. In certain embodiments, the cancer is osteosarcoma. In certain embodiments, the cancer is anaplastic thyroid cancer. In certain embodiments, the cancer is adrenocortical carcinoma. In certain embodiments, the cancer is pancreatic cancer or pancreatic ductal carcinoma. In certain embodiments, the cancer is pancreatic adenocarcinoma. In certain embodiments, the cancer is glioma. In certain embodiments, the cancer is a malignant peripheral nerve sheath tumor (MPNST). In certain embodiments, the cancer is a neurofibromatosis-1 associated MPNST. In certain embodiments, the cancer is Waldenstrom's macroglobulinemia. In certain embodiments, the cancer is a medulloblastoma.

[0302] In certain embodiments, the cancer is a virus-related cancer, including human immunodeficiency virus (HIV)-associated solid tumors, human papillomavirus (HPV)-16-positive incurable solid tumors, and adult T-cell leukemia, which is caused by human T-cell leukemia virus type 1 (HTLV-I) and is a highly aggressive form of CD4+ T-cell leukemia characterized by clonal integration of HTLV-I in leukemic cells (see https: / / clinicaltrials.gov / ct2 / show / study / NCT02631746); and Virus-associated tumors in gastric cancer, nasopharyngeal cancer, cervical cancer, vaginal cancer, vulvar cancer, head and neck squamous cell carcinoma, and Merkel cell carcinoma (see https: / / clinicaltrials.gov / ct2 / show / study / NCT02488759; see also https: / / clinicaltrials.gov / ct2 / show / study / NCT0240886; https: / / clinicaltrials.gov / ct2 / show / NCT02426892).

[0303] Other cancers will be known to those of ordinary skill in the art.

[0304] In a preferred embodiment, the cancer is selected from breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, lung cancer, colorectal cancer, stomach / gastric cancer, endometrial / uterine / cervical cancer, bladder cancer, head and neck cancer, leukemia, sarcoma, bile duct cancer, glioblastoma, multiple myeloma, lymphoma. More preferably, the cancer is selected from breast cancer, ovarian cancer and prostate cancer, even more preferably, it is breast cancer or ovarian cancer, more preferably, it is breast cancer.

[0305] In certain embodiments, the cancer is melanoma cancer.

[0306] In a preferred embodiment, the cancer is breast cancer. The term "breast cancer" refers to any malignant proliferative disorder of breast cells, most commonly from the lining of the milk ducts or the lobules that supply the ducts with milk. Cancers that originate in the ducts are called ductal carcinomas, while cancers that originate in the lobules are called lobular carcinomas.

[0307] In a preferred embodiment, the cancer is triple negative breast cancer (TNBC). The term triple negative breast cancer represents the following fact: cancer cells are defined on immunohistochemistry by the lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), i.e., the cells are negative in all 3 tests. It is a highly malignant subtype of breast cancer, which is usually associated with relatively poor clinical outcomes, earlier recurrence and high visceral metastasis tendency compared to other breast cancer types. These cancers tend to be more common in women under 40 years old (which are blacks or have BRCA1 mutations). Thus, in a preferred embodiment, the cancer is a cancer with BRCA1 mutations, preferably a TNBC with BRCA1 mutations. In another embodiment, the cancer is a cancer with BRCA1 wild type, preferably a TNBC with BRCA1 wild type.

[0308] As used herein, "BRCA1" means early-onset breast cancer 1 and is part of a complex that repairs double-strand breaks in DNA. BRCA1 functions in the same related DNA repair pathway as PARP1 / PARG. The sequence of the BRCA1 protein in humans corresponds to sequence P38398 in the Uniprot database (version 267 of this entry as of October 12, 2022).

[0309] In another embodiment, the cancer is a cancer with a BRCA2 mutation.

[0310] As used herein, "BRCA2" means early-onset breast cancer 2 and is part of a complex that repairs double-strand breaks in DNA. The sequence of the BRCA2 protein in humans corresponds to sequence P51587 in the Uniprot database (version 234 of this entry as of October 12, 2022).

[0311] If BRCA1 or BRCA2 itself is damaged due to a BRCA mutation in breast cells, the damaged DNA cannot be properly repaired and this increases the risk of breast and ovarian cancer.

[0312] More preferably, the cancer is a cancer having a BRCA1 mutation, a BRCA2 mutation, or both a BRCA1 and BRCA2 mutation.

[0313] In another embodiment, the cancer is a BRCA1 and / or BRCA2 wild-type cancer.

[0314] In another preferred embodiment, the cancer is pancreatic cancer, in particular pancreatic ductal adenocarcinoma.

[0315] In another embodiment, the cancer is glioblastoma.

[0316] "Glioblastoma" (also called glioblastoma and grade IV astrocytoma) is the most common and most aggressive cancer that begins in the brain.

[0317] In another embodiment, the cancer is lung cancer.

[0318] The term "lung cancer" or "lung tumor" refers to the physiological condition in mammals characterized by unregulated cell growth in lung tissue. The term lung cancer is intended to refer to any cancer of the lung, and includes non-small cell lung cancer and small cell lung cancer. In one embodiment, the lung cancer is non-small cell lung cancer (NSCLC). In another embodiment, the lung cancer is small cell lung cancer (SCLC).

[0319] The term non-small cell lung cancer (NSCLC) as used herein refers to a group of heterogeneous diseases that are grouped together because their prognosis and treatment are generally similar, and include according to the World Health Organization / International Association for the Study of Lung Cancer histological classification (Travis WD et al. Histological typing of lung and pleural tumours. Berlin: Springer-Verlag, 3rd edition 1999):

[0320] (i) Squamous cell carcinoma (SCC), which accounts for 30% to 40% of NSCLC, begins in the larger breathing tubes but grows more slowly, meaning that these tumors vary in size by diagnosis.

[0321] (ii) Adenocarcinoma is the most common subtype of NSCLC, accounting for 50% to 60% of NSCLCs. It begins near the gas exchange surfaces of the lungs and includes a subtype, bronchioloalveolar carcinoma, which may respond differently to treatment.

[0322] (iii) Large cell carcinoma is a fast-growing form that grows near the surface of the lung. It is primarily a diagnosis of exclusion, and when more research is done it is often reclassified as either squamous cell carcinoma or adenocarcinoma.

[0323] (iv) Adenosquamous carcinoma is a type of cancer that involves two types of cells: squamous cells (thin, flat cells that line certain organs) and glandular cells.

[0324] (v) Carcinomas with pleomorphic, sarcomatoid, or sarcomatous elements. This is a rare group of tumors that reflects histologic heterogeneity and a continuum of epithelial and mesenchymal differentiation.

[0325] (vi) Carcinoid tumors are a slow-growing neuroendocrine lung tumor that begins in cells that release hormones in response to stimulation provided by the nervous system.

[0326] (vii) Salivary gland cancer begins in the cells of the salivary glands, which are located in the large airways of the lungs.

[0327] (viii) Unclassified cancers include cancers that do not belong to any of the aforementioned cancer categories.

[0328] In a specific embodiment, the NSCLC is selected from the group consisting of squamous cell carcinoma of the lung, large cell carcinoma of the lung, and adenocarcinoma of the lung.

[0329] The term small cell lung cancer (SCLC) as used herein refers to the proliferation of small cells with unique and strict morphological features, which contain dense neurosecretory granules that provide the tumor with an associated endocrine / paraneoplastic syndrome. Most cases occur in the larger airways (primary and secondary bronchi). These cancers grow rapidly and spread early in the course of the disease.

[0330] In an even more preferred embodiment, the lung cancer is an adenocarcinoma, more preferably a KRas-driven lung adenocarcinoma, preferably a cancer associated with a mutation in the KRAS gene. In one embodiment, the mutation in the KRAS gene is a mutation at glycine at position 12, at glycine at position 13, or at glutamine at position 61. In a more preferred embodiment, the mutation is selected from a G12S mutation, a G12V mutation, a G12D mutation, a G13D mutation, a G12C mutation, a G12R mutation, a G12F mutation, a G12I mutation, a G13C mutation, a G13R mutation, or a Q61L mutation. In a preferred embodiment, the mutation is a G12D mutation. In another embodiment, the lung cancer is a KRas GD12 / p53-driven lung cancer, preferably KRas GD12 / p53-driven NSCLC.

[0331] In another preferred embodiment, the cancer to be treated according to the invention is characterized by the expression of increased levels of PARP, in particular PARP1 and / or PARP2. The PARP level, in particular PARP1 and / or PARP2 level, is considered to be increased relative to a reference value when the level of PARP, in particular PARP1 and / or PARP2 in a sample of said cancer shows an increase of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or more. The reference value may be a value corresponding to the expression level of PARP, in particular PARP1 and / or PARP2 in a non-cancerous sample.

[0332] In another embodiment, the cancer is a hormone-dependent cancer. Hormone-dependent cancer means a cancer that is hormone-sensitive. Examples of such cancers include, but are not limited to, cancers of the breast, endometrium, ovary, prostate, testis, thyroid, and osteosarcoma. In a specific embodiment, the cancer is a steroid-dependent cancer, more preferably an estrogen and progesterone cancer. In a more preferred embodiment, the progesterone-dependent cancer is a progesterone-dependent breast cancer. In another embodiment, the cancer is an androgen-dependent cancer, more preferably an androgen-dependent prostate cancer.

[0333] In one embodiment, the cancer is a primary tumor.The term "primary tumor" as used herein refers to a tumor that originates in the site or organ where it resides and has not metastasized to that site from another site.

[0334] In another embodiment, the cancer is cancer metastasis. In the context of the present invention, "metastasis" is understood to be the spread of cancer from the organ where it started to a different organ. It usually occurs through the blood or lymphatic system. When cancer cells spread and form new tumors, the latter are referred to as secondary or metastatic tumors. The cancer cells that form secondary tumors are similar to those of the original tumor. For example, if breast cancer spreads (metastasizes) to the lungs, secondary tumors are formed by malignant breast cancer cells. The disease in the lungs is metastatic breast cancer, and is not lung cancer. The authors of the present invention have also observed that the combination or composition of the present invention can reduce cell proliferation, regardless of whether the cancer shows increased Myc protein expression or activity. In a preferred embodiment, the cancer to be prevented or treated is a Myc-induced cancer.

[0335] In one embodiment, the cancer is a solid tumor.

[0336] In another embodiment, the cancer is a cancer that is resistant to a PARP inhibitor.

[0337] "Resistance" is a reduction in the effectiveness of a drug for treating a disease or condition. As used herein, the expression "cancer resistant" refers to cancers that are resistant (either innate or acquired) to PARP inhibitors. Innate resistance occurs when PARP inhibitors are ineffective from the beginning of treatment due to pre-existing resistance mechanisms. Acquired resistance occurs when PARP inhibitors become ineffective during treatment and after clinical benefit has been observed.

[0338] All combinations of compounds of the invention and cancer types are included in the present invention.

[0339] In certain embodiments, the combination or composition of the present invention produces an inhibition of tumor growth. In certain embodiments, the combination or composition of the present invention reduces tumor size (e.g., volume or mass) by at least 5%, 10%, 25%, 50%, 75%, 90% or 99% relative to the size of the tumor before treatment. In certain embodiments, the combination or composition of the present invention reduces the amount of tumor in the patient by at least 5%, 10%, 25%, 50%, 75%, 90% or 99% relative to the amount of tumor before treatment.

[0340] As used herein, "subject" includes any animal having cancer or showing symptoms of cancer, or at risk of having cancer or showing symptoms of cancer. Suitable subjects (patients) include experimental animals (such as mice, rats, rabbits or guinea pigs), farm animals and domestic animals or pets (such as cats or dogs). Non-human primates are included, and human patients are preferred. Preferably, the subject is a mammal, most preferably a human.

[0341] Combinations or compositions for the prevention and / or treatment of cancer may be administered using any amount and any route of administration that is effective in treating or alleviating the severity of cancer. The exact amount required will vary between subjects, depending on the species, age, and general condition of the subject; the severity of the disease or condition; the specific agent; its mode of administration, etc. The compounds of the present invention are preferably formulated in dosage unit form for ease of administration and dosage uniformity. The expression "dosage unit form" used herein means a physically discrete dosage unit suitable for the patient to be treated. However, it should be understood that the total daily dosage of the compounds and compositions of the present invention is determined by the attending physician within the scope of reasonable medical judgment. The specific effective dosage level for any particular patient or organism will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health, sex, and diet; the administration time, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination or concomitantly with the specific compound used, and similar factors well known in the medical field.

[0342] In a preferred embodiment, component (i) of the present invention, preferably the polypeptide or a functionally equivalent variant thereof or the conjugate, interacts synergistically with the PARP inhibitor of the combination or composition in the treatment of cancer (to achieve a therapeutic effect).

[0343] Specifically, in a more preferred embodiment, the combination or pharmaceutical composition for preventing and / or treating cancer is a combination or pharmaceutical composition wherein the amount of the polypeptide or its functionally equivalent variant or the conjugate is capable of interacting synergistically with the PARP inhibitor in treating cancer.

[0344] The terms "synergistic effect" or "synergistically interact" are used interchangeably. A synergistic effect is an effect that is greater than the additive effect predicted by summing the actual effects of the single agents in vitro. In vivo, a synergistic effect is a physiological effect, and especially a therapeutic effect, that is greater than the additive effect predicted by summing the actual effects of the single agents in vivo.

[0345] Therefore, if two medicaments are used, they provide a measurable physiological effect together, and in particular a therapeutic effect, if the actual effect of the medicaments together is greater than the effect predicted by summing the actual therapeutic effect of a single medicament. Specifically, a synergistic effect is provided in the following cases: the first medicament provides a certain measurable effect alone, the second medicament provides a certain measurable effect alone, and the measurable effect provided by the two medicaments together is greater than the effect provided by the sum of the two single medicaments. More specifically, a synergistic effect is provided in the following cases: the first medicament does not provide a measurable effect alone, the second medicament provides a certain measurable effect alone, and the measurable effect provided by the two medicaments together is greater than the effect provided by the second medicament alone. More specifically, a synergistic effect is provided in the following cases: neither the first agent alone nor the second agent alone provides any measurable effect, but the two medicaments provide a measurable effect together. Because components (i) and (ii) act synergistically, the amount of components (i) and / or (ii) of the combination or composition of the present invention can be less than the amount required for a monotherapy using only one of them as a therapeutic agent. Preferably, in these combinations or compositions, one or the other therapeutic agent may be administered at a dosage of 0.01-1.000 μg / kg body weight / day.

[0346] The amount of therapeutic agent present in the combination or composition may not exceed the amount normally applied in a composition comprising the therapeutic agent as the sole active agent. Preferably, the amount of therapeutic agent in the present composition will be about 50% to 100% of the amount normally present in a composition comprising the medicament as the sole therapeutic active agent. In certain embodiments, the dosage of a therapeutic agent is about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% of the amount normally applied by the medicament. The phrase "normally applied" used herein refers to the amount that the FDA-approved therapeutic agent is approved for quantitative application according to the FDA label insert.

[0347] The combinations or compositions of the invention may also be used in combination with known therapeutic procedures, for example, with chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, hormones or a combination of these.

[0348] All embodiments of the combinations of the invention are also applicable to the methods of treatment of the invention.

[0349] Finished products and test kits

[0350] The present disclosure also provides a manufactured product comprising any of the combinations or pharmaceutical compositions disclosed herein in one or more containers. In certain embodiments, the manufactured product comprises, for example, promotional materials, printed instructions, labels, or package inserts that instruct a user (e.g., a distributor or end user) to combine and / or use the composition of the manufactured product to prevent and / or treat cancer.

[0351] In certain embodiments, the manufactured product comprises, for example, a bottle, a vial, a barrel, a box, a hypodermic syringe, a syringe, or any combination thereof. In certain embodiments, the label indicates the use or administration of the combination or the pharmaceutical composition in the manufactured product according to the methods disclosed herein. In certain aspects, the label suggests, for example, a regimen for use, a regimen for treating, preventing, or ameliorating cancer.

[0352] The contents of all cited references (including literature sources, patents, patent applications, and websites) that may be cited throughout this application are hereby expressly incorporated by reference in their entirety for any purpose, as are the references cited therein.

[0353] ***

[0354] Unless otherwise stated, all terms used herein should be understood as their common meanings known in the art. Other more specific definitions of certain terms used in this application are described below, and are intended to be uniformly applied to the specification and claims, unless otherwise clearly stated definitions provide a wider definition. Throughout the specification and claims, the word "comprising" and the variants of the word are not intended to exclude other technical features, additives, components or steps. In addition, the word "comprising" covers the situation of "consisting of". Other objects, advantages and features of the present invention will become apparent to those skilled in the art after reviewing the specification, or can be learned through the practice of the present invention. In addition, the present invention covers all possible combinations of the specific embodiments described herein.

[0355] In this specification and the appended claims, the singular forms "one", "a", and "the" include plural referents unless the context clearly indicates otherwise. The term "one" (or "a") and the terms "one / kind or more / kind" and "at least one / kind" can be used interchangeably herein. In addition, "and / or" as used herein should be regarded as a specific disclosure of each of two specific features or components, with or without the other. Therefore, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to cover each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). Throughout this specification and claims, the term "about" used in conjunction with a numerical value represents a familiar and acceptable accuracy interval for those skilled in the art. In general, such an accuracy interval is ± 15%. Unless otherwise defined, all technical and scientific terms used in this article have the same meanings as those of ordinary skill in the art related to the present disclosure are generally understood. Units, prefixes and symbols are expressed in the form accepted by their International System of Units (SI). Numerical ranges include numbers that limit the range. Unless otherwise noted, amino acid sequences are written from left to right in the direction of amino to carboxyl. The titles provided herein are not limitations on the various aspects of the present disclosure, and the aspects can be obtained by reference to the entire specification. Therefore, the terms defined below are more fully defined by reference to the specification as a whole.

[0356] The present invention will be described by way of the following examples, which should be regarded as merely illustrative and not limiting of the scope of the invention.

[0357] Example

[0358] Production and purification of Omomyc

[0359] The Omomyc peptide sequence SEQ ID NO:4 (including the N-terminal methionine) was reverse transcribed, codon optimized for expression in E. coli, cloned in the pET3a expression vector (Novagen), and purified from BL21 (DE3) arabinose-inducible strains using a protocol adapted from the Max° purification protocol described in Purification from bacterial strains: J.-F. Naud et al. 2003. J Mol Biol, 326: 1577-1595; F.-O. and Mcduff et al. 2009. J Mol Recognit, 22: 261-269. The purified construct obtained is the polypeptide of SEQ ID NO: 4. The identity of each purified construct was confirmed by mass spectrometry and Western blot analysis. Omomyc was purified by cation exchange chromatography and the purity was confirmed by mass spectrometry analysis, SDS-PAGE and UV spectroscopy.

[0360] Omomyc in combination with PARP inhibitors acts synergistically to reduce the viability of triple-negative breast cancer cells

[0361] Triple negative breast cancer (TNBC) cell lines MDA-MB-231, SUM149 and MX-1 were treated with increasing concentrations of Olaparib or Talazoparib and Omomyc peptide sequence SEQ ID NO: 4 (alone and in combination) for 5 days.

[0362] Then, after performing 3 biological replicates, the synergy score was calculated by SynergyFinder.org.

[0363] The synergy score can be interpreted as the average excess response due to drug interaction. A synergy score close to 0 provides limited confidence in synergy or antagonism. Therefore, when the synergy score is less than -10, the interaction between the two drugs is likely to be antagonistic. At -10 to 10, the interaction between the two drugs is likely to be additive. At greater than 10, the interaction between the two drugs is likely to be synergistic.

[0364] Figure 1 A shows that in MDA-MB-231TNBC cells, Omomyc treatment significantly reduced cell viability. In addition, the inventors have found through microarray analysis that Omomyc treatment significantly downregulated gene selection in MDA-MB-231 cells (data not shown). Specifically, Omomyc treatment reduced the expression of genes and pathways associated with homologous recombination (HR-) and BRCA1 / 2-deficiency. Treatment of these BRCA wild-type cells (MDA-MB-231) with Omomyc and the PARP inhibitor Olaparib revealed that the combination of these drugs has a synergistic effect. It is worth noting that Figure 1 B and Figure 1 C shows that cell lines with BRCA mutations but resistant to PARP inhibitors (SUM149 and MX-1) also exhibited a synergistic response to the combination of Olaparib and Omomyc.

[0365] The combination between Omomyc and talazopanib was effective in MDA-MB-231 cells ( Figure 3 A) and in MX-1 (BRCA1 / 2 mutant) and SUM149 (BRCA1 mutant) ( Figure 3 B and Figure 3 Synergy is shown in C). This confirms that for olaparib and talazoparib, the synergistic output is independent of the mutational profile of the cell line.

[0366] All of the above evidence suggests that the combination of Omomyc and PARP inhibitors is an effective therapy for both BRCA mutant and wild-type tumors, potentially benefiting the entire TNBC population. Importantly, Omomyc was able to reverse olaparib-induced resistance.

[0367] Omomyc in combination with PARP inhibitors acts synergistically to reduce viability of pancreatic ductal adenocarcinoma cells

[0368] Pancreatic ductal adenocarcinoma (PDAC) MIA-PACA-2 cells were treated with increasing concentrations of Olaparib and Omomyc peptide sequence SEQ ID NO: 4 (alone and in combination) for 5 days.

[0369] Then, the synergy score was calculated by SynergyFinder.org after 3 biological replicates as described above.

[0370] Figure 2 The PDDAC BRCA wild-type cell line MIA-PACA-2 also showed strong synergy with the combination of Omomyc and Olaparib. This cell line appears to be particularly resistant to Olaparib ( Figure 2 A), and Omomyc was able to reverse olaparib resistance in these cells.

[0371] Omomyc and PARP inhibitors act synergistically in vivo

[0372] SUM149 cell-derived xenograft (CDX) model mice were used for in vivo studies. Five million cells were inoculated orthotopically in the mammary fat pad per mouse. When tumors reached 100 mm 3 When, mice were randomly divided into 4 treatment groups. Specifically, mice were treated with Omomyc once a week (50 mg / kg, intravenous), treated with Olaparib 6 times a week (50 mg / kg oral gavage), a combination of the two drugs (combo), or treated with vehicle alone for 4 weeks. The relative volume was calculated as the percentage of tumor growth relative to the day of randomization and the start of treatment. At the end point, the mean values ​​of each group were: vehicle 901.8%, Omomyc 663.6%, Olaparib 623.6%, and combination 335.8%.

[0373] Initial in vivo results with SUM149 cell-derived xenografts (CDX) confirmed that this synergistic effect could also be seen in mice. Indeed, after 4 weeks of Omomyc+Olaparib combination treatment, the mean growth reduction between vehicle and the combination was greater than the sum of the growth reductions between vehicle and the monotherapy ( Figure 4 ).

Claims

1. A combination comprising: i) a first component selected from: a) a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or a functionally equivalent variant thereof; c) a polynucleotide encoding the polypeptide of a) or the conjugate of b); d) a vector comprising the polynucleotide according to c); and e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into the culture medium; and ii) As a second component of a PARP inhibitor.

2. The combination according to claim 1, wherein the first component is a polypeptide comprising the sequence SEQ ID NO:

1.

3. The combination according to claim 1, wherein the functionally equivalent variant of SEQ ID NO: 1 is selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO:

10.

4. The combination according to any one of claims 1 or 3, wherein the chemical moiety that promotes cellular uptake of the polypeptide or its functionally equivalent variant is a cell penetrating peptide sequence, and wherein the cell penetrating peptide sequence and the polypeptide or its functionally equivalent variant form a fusion protein.

5. The combination according to any one of claims 1 or 3 to 4, wherein the conjugate additionally comprises an additional nuclear localization signal.

6. The combination according to any one of claims 1 to 5, wherein the PARP inhibitor is selected from olaparib, talazoparib, rucaparib, niraparib, veliparib, pamiparib, fluzoparib and iniparib.

7. The combination according to claim 6, wherein the PARP inhibitor is olaparib.

8. A pharmaceutical composition comprising a pharmaceutically effective amount of the combination according to any one of claims 1 to 7 and a pharmaceutically acceptable excipient.

9. A combination according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 for use in medicine.

10. The combination according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 8 for use in the prevention and / or treatment of cancer.

11. The combination or pharmaceutical composition according to claim 10, wherein the cancer is selected from breast cancer and pancreatic cancer.

12. The combination or pharmaceutical composition according to claim 11, wherein the cancer is selected from triple negative breast cancer and pancreatic ductal adenocarcinoma.

13. The combination or pharmaceutical composition according to any one of claims 10 to 12, wherein the cancer is a cancer resistant to PARP inhibitors.

14. The combination or pharmaceutical composition according to any one of claims 9 to 13, wherein the combination is administered systemically, preferably intravenously.

15. The combination or pharmaceutical composition according to any one of claims 9 to 13, wherein the first component is administered intravenously and the second component is administered orally.

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