Combination therapy for cancer treatment

Abstract

The present invention relates to a PARP inhibitor, a combination of Omomyc, a functionally equivalent variant thereof, a conjugate comprising Omomyc or the functionally equivalent variant, a polynucleotide encoding the polypeptide, a vector comprising the polynucleotide, and a combination of cells capable of secreting the polypeptide or conjugate. The present invention also relates to pharmaceutical compositions containing the combinations of the present invention, and their medical use, specifically their use in the treatment of cancer.

Description

[Technical Field]

[0001] This invention relates to the field of cancer, more specifically to combinations comprising poly(ADP-ribose) polymerase (PARP) inhibitors and Omomyc, and to their use in pharmaceuticals, more specifically in the prevention and / or treatment of cancer. Background of the Invention

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

[0003] Anticancer drugs are designed to target the entire panel of cancer traits. Over the past decade, poly(ADP-ribose) polymerase (PARP) inhibitors have been among the first drugs to target the DNA damage response to enter clinical practice. 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 DNA breaks. PARP inhibition leads to the accumulation of single-strand breaks, ultimately resulting in double-strand breaks. In addition to catalytic inhibition, PARP inhibitors capture the PARP enzyme-DNA complex at single-strand breaks, leading to double-strand breaks. Capture of poly(ADP-ribose) polymerase is considered a major mechanism of antitumor activity. PARP inhibition is particularly effective in cells carrying homologous recombination deficiencies, such as pathogenic breast cancer BRCA-1 or BRCA-2 mutations.

[0005] The aforementioned PARP inhibitors have demonstrated efficacy in treating a wide range of cancer types, such as ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, and small cell lung cancer, and are currently undergoing clinical trials to explore further indications.

[0006] In general, cancers with high levels of replication stress and genomic instability due to DNA repair deficiencies, as well as / or increased replication origins induced by oncogenes, are particularly responsive to PARP inhibitors. However, challenges remain to be overcome.

[0007] Drug resistance and adverse effects to PARP inhibitors are frequent problems in clinical practice and can limit long-term treatment.

[0008] Therefore, there remains a need to develop novel and improved therapeutic approaches for cancer treatment using conventional technologies. [Overview of the project]

[0009] Brief summary of the invention In the first embodiment, the present invention is i) A first component selected from the group consisting of the following: a) A polypeptide containing the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cytoacquisition of the polypeptide or its functionally equivalent variant; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) c) vectors containing polynucleotides; and e) Cells that can secrete the polypeptide described in a) or the conjugate described in b) into the culture medium. and ii) The second component is a PARP inhibitor. Regarding combinations that include [this].

[0010] In a second aspect, the present invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of the combination according to the present invention and a pharmaceutically acceptable excipient.

[0011] In a third aspect, the present invention relates to the combination according to the present invention or the pharmaceutical composition according to the present invention for use in medicine.

[0012] In a fourth aspect, the present invention relates to the combination according to the present invention or the pharmaceutical composition according to the present invention for use in the prevention and / or treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] [Figure 1] Bar graph representing the synergistic combination between Omomyc and olaparib against three triple-negative breast cancer (TNBC) cell lines. (A) MDA-MB-231; (B) SUM149; (C) MX-1. [Figure 2] Bar graph representing the synergistic combination between Omomyc and olaparib in the pancreatic ductal adenocarcinoma (PDAC) MIA-PACA-2 cell line. [Figure 3] Bar graph representing the synergistic combination between Omomyc and talazoparib against three triple-negative breast cancer (TNBC) cell lines. (A) MDA-MB-231; (B) SUM149; (C) MX-1. [Figure 4] Dot plot representing preliminary in vivo data from the SUM149 CDX model. Statistical significance was determined via 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 OF THE INVENTION

[0014] The present invention relates to the provision of a new therapeutic combination for the prevention and treatment of cancer.

[0015] 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.

[0016] All embodiments disclosed with respect to one aspect of the present invention are applicable to other aspects.

[0017] Combinations and pharmaceutical compositions of the present invention The definitions provided herein and in any other aspect of the present invention are equally applicable throughout the present invention.

[0018] The inventors have unexpectedly found that the combination of Omomyc and a PARP inhibitor has a synergistic effect in treating cancer. The inventors have shown 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 also the viability of the MIA-PACA-2 pancreatic ductal adenocarcinoma cell line. This synergistic effect is maintained regardless of the dose (Figures 1 and 2), resulting in a beneficial effect, particularly an increase in the therapeutic effect of the composition of the present invention compared to each of its components, so that the same effect can be achieved using lower doses of each component, thereby reducing the side effects on subjects receiving the composition of the present invention.

[0019] Furthermore, the inventors have unexpectedly found that Omomyc can reverse olaparib resistance, thus 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 present invention expands the population that can be responsive to PARP inhibitor-based therapies.

[0020] Therefore, the combination of Omomyc and a PARP inhibitor can be an effective therapy for treating both BRCA mutant and wild-type tumors, as well as tumors resistant to PARP inhibitors.

[0021] Therefore, in the first embodiment, the present invention is i) a) A polypeptide containing the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cytoacquisition of the polypeptide or its functionally equivalent variant; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) c) vectors containing polynucleotides; and e) Cells that can secrete the polypeptide described in a) or the conjugate described in b) into the culture medium. A first component selected from the group consisting of; and ii) The second component is a PARP inhibitor. Regarding combinations that include [this].

[0022] According to the present invention, the term “combination” refers to various combinations of compounds (i) and (ii), for example, in a composition formulated as a single formulation, in a combination mixture consisting of separate formulations of each component, such as a “tank mix” that can be combined for joint use as a combination preparation, and in the combined use of a single active ingredient applied in a sequential manner, i.e., when one is applied after the other over a fairly short period, such as several hours or several days or simultaneous administration. In the present invention, compound (i) refers to a polypeptide containing a therapeutically effective amount of the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof, or a conjugate containing a polypeptide containing 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, or a polynucleotide encoding the polypeptide or conjugate, or a vector containing a polynucleotide, or a cell that can secrete the polypeptide or conjugate into a culture medium. In the present invention, compound (ii) refers to a therapeutically effective amount of a PARP inhibitor. Preferably, the order in which compounds (i) and (ii) are applied is not essential for the present invention to function.

[0023] The combination may be a kit of parts, where each component is individually formulated and packaged.

[0024] The combination of compounds (i) and (ii) can be formulated for simultaneous, separate, or sequential administration. Specifically, if administration is not simultaneous, the compounds are administered in very close proximity to each other. Furthermore, the compounds can be administered in the same or different dosage forms or by the same or different routes of administration; for example, one compound can be administered orally and the other intravenously. Preferably, compound (i) is administered intravenously and compound (ii) is administered orally. In another embodiment, compounds (i) and (ii) are administered intravenously.

[0025] The combination of the two compounds (i) and (ii) is: - As a combination of the same pharmaceutical preparation, and therefore the two compounds are always administered simultaneously, - Each is a combination of two units that gives rise to the possibility of administering one of the substances simultaneously, sequentially, or separately. It can be administered.

[0026] In specific embodiments, compound (i) of the combination of the present invention is administered independently of compound (ii), that is, at the same time but in two units.

[0027] In another specific embodiment, compound (i) of the combination of the present invention is administered first, followed by compound (ii), i.e., compound (ii) is administered separately or sequentially.

[0028] In yet another specific embodiment, compound (ii) of the combination of the present invention is administered first, followed by compound (i), i.e., compound (i) is administered separately or sequentially as defined.

[0029] When administered separately, compounds (i) and (ii) of the combination of the present invention can be administered to each other within a certain time frame, 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 from each other. In another embodiment, compounds (i) and (ii) of the combination of the present invention can be administered to each other 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 from each other, preferably within 1 day from each other, and more preferably within 10 days from each other. 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 discontinued before administration of the second compound is initiated.

[0030] In another embodiment, the present invention relates to a combination or pharmaceutical composition comprising a synergistically effective amount of the first component described in the first embodiment of the present invention and a PARP inhibitor.

[0031] Compound (i) of the combination of the present invention In a preferred embodiment, 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.

[0032] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to polymers of amino acids of any length. Polypeptides of the present invention may contain modified amino acids, which may be interrupted by non-amino acids. In preferred embodiments, polypeptides are formed exclusively of amino acids. Preferably, the polypeptide forming item (i) of the combination has a length of 80 to 500 amino acids, more preferably 80 to 300 amino acids, more preferably 80 to 250 amino acids, more preferably 80 to 150, even more preferably 80 to 130 amino acids, preferably 90 to 130 amino acids, preferably 125 amino acids or less, and more preferably 100 amino acids or less. In preferred embodiments, polypeptides have a length of 90 to 98 amino acids, preferably 90 to 95 amino acids, and more preferably 91 amino acids.

[0033] The term "amino acid" refers to naturally occurring 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. Furthermore, the term "amino acid" includes both D- and L-amino acids (stereoisomers). Preferably, the amino acid is an L-amino acid.

[0034] The term “natural amino acids” or “naturally occurring amino acids” includes 20 naturally occurring amino acids; for example, hydroxyproline, phosphoserine, and phosphothreonine, which are often post-translationally modified in vivo; and other non-ordinary amino acids, including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine.

[0035] As used herein, the terms “non-natural amino acid” or “synthetic amino acid” refer to carboxylic acids or derivatives thereof that are structurally related to natural amino acids by being substituted with an amine group at the position of “a”. Non-exclusive examples of modified or rare amino acids include 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic 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, ariohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline, norvaline, norleucine, and ornithine.

[0036] The polypeptides of the present invention may also include, for example, hydrophobic moieties attached to the peptide (various linear, branched, cyclic, polycyclic, or heterocyclic hydrocarbons and hydrocarbon derivatives); and non-amino acid moieties such as various protecting groups attached to the ends of the compound 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.

[0037] Chemical (non-amino acid) groups present in polypeptides may be included to improve various physiological properties, such as reducing degradation or clearance; reducing repulsion by various cell pumps; improving various administration methods; increasing specificity; increasing affinity; increasing stability; and reducing bioavailability, solubility, and toxicity.

[0038] "Mimetics" include molecules that mimic the chemical structure of a peptide and retain the functional properties of a peptide. Approaches for designing peptide analogs, derivatives, and mimetics are known in the art.

[0039] In one embodiment, the polypeptide of the present invention is a polypeptide consisting of the sequence of SEQ ID NO: 1 or a polypeptide consisting of a functionally equivalent variant of SEQ ID NO: 1, and preferably a polypeptide consisting of the sequence of SEQ ID NO: 1.

[0040] Sequence ID 1 is, TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA(Sequence ID 1) It corresponds to.

[0041] The polypeptide sequence of Sequence ID No. 1 corresponds to the Omomyc protein sequence. The term "Omomyc," as used herein, refers to the polypeptide consisting of mutant versions of the bHLHZip domain of Myc with E61T, E68I, R74Q, and R75N ​​mutations (where the mutation site numbering is given with respect to the sequence of the Myc region corresponding to amino acids 365-454 of the polypeptide defined in the NCBI database, accession number NP_002458, released March 15, 2015). The sequence of c-Myc provided to the NCBI database with accession number NP_002458 is shown below (Sequence ID No. 2), where the region from which Omomyc originates is underlined. [ka]

[0042] Omomyc also contains the M2 domain of c-Myc with the sequence RQRRNELKRSF (sequence number 3) (see Dang and Lee, Mol. Cell. Biol., 1988, 8:4048-4054) (double underlined above), which corresponds to a nuclear localization signal.

[0043] Omomyc is characterized by its increased dimerization ability with all three oncogene Myc proteins (c-Myc, N-Myc, and L-Myc). Omomyc may be derived from the bHLHZip domain of any Myc protein known in the art, provided that the mutation that results in the tumor suppressor effect is conserved. Therefore, Omomyc that can be used in the present invention may be derived from any mammalian species, including but not limited to domesticated animals and livestock (cattle, horses, pigs, sheep, goats, dogs, cats, or rodents), primates, and humans. Preferably, the Omomyc protein is derived from the human Myc protein (accession number NP_002458, released March 12, 2019).

[0044] The term "Myc," as used herein, refers to a family of transcription factors including c-Myc, N-Myc, and L-Myc. Myc proteins activate the expression of many genes through binding to the consensus sequence CACGTG (enhancer box sequence or E-box and recruiting histone acetyl-transferase or HAT). However, Myc can also act as a transcriptional repressor. By binding to the Miz-1 transcription factor and replacing p300 coactivators, it inhibits the expression of Miz-1 target genes. Myc also plays a direct role in the regulation of DNA replication.

[0045] The Myc b-HLH-LZ or helix-loop-helix leucine zipper domain in the Myc basic region refers to the region that determines Myc dimerization with the Max protein and 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).

[0046] In a preferred embodiment, the polypeptide of the present invention is a polypeptide comprising, consisting of, or essentially comprising SEQ ID NO: 4 shown below. MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA(Sequence ID 4)

[0047] In this context, "essentially derived from" means that the defined molecule does not contain any additional sequences that would alter the activity of SEQ ID NO: 4.

[0048] Preferably, the polypeptide consists of Sequence ID No. 4.

[0049] The term "functionally equivalent variant" refers to any polypeptide obtained from 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 with respect to the polypeptide of SEQ ID NO: 1, and / or from the chemical modification of the polypeptide of SEQ ID NO: 1, and which substantially preserves the tumor suppressor activity of SEQ ID NO: 1. Preferably, a functionally equivalent variant refers to any polypeptide obtained from 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 that point to the polypeptide of SEQ ID NO: 1, and which substantially preserves the tumor suppressor activity of SEQ ID NO: 1; more preferably, any polypeptide obtained from the insertion or addition of one or more amino acids with respect to the polypeptide of SEQ ID NO: 1.

[0050] Those skilled in the art will understand that preservation of tumor suppressor activity requires that the variants dimerize with Myc and / or its obligate partner p21 / p22Max, inhibit Myc activity, migrate across the cell membrane, and migrate across the nuclear envelope. In some embodiments, functionally equivalent variants of the polypeptide of the present invention are less homodimerized than Omomyc, or are not forced into homodimerization by disulfide crosslinking. In particular, disulfide crosslinking in the homodimerized form of certain embodiments of the polypeptide of the present invention is less than in polypeptide Omomyc.

[0051] When used herein, "low homodimerization" refers to a lower ability of the polypeptide of the present invention to form obligate homodimers, even under reducing conditions. In preferred embodiments, 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%, and at least 95% lower than the ability of Omomyc to form homodimers.

[0052] Reducing conditions, as used herein, refer to the presence of a reducing agent, which is a compound that donates electrons to another chemical species in a redox reaction. Non-limiting examples of reducing agents include DTT (dithiothreitol), β-mercaptoethanol, or TCEP (tris(2-carboxyethyl)phosphine). The amount of homodimers being the same in vitro, and the differences between functionally equivalent variants and Omomyc, may only exist in cells in the presence of a heterodimerizing partner, where the absence of disulfide allows for potentially higher heterodimer formation.

[0053] As a non-limiting example of thermal denaturation monitored by circular dichroism, several assays may be used to determine the homodimerization of peptides, which may be detected by quantification of folding and thermal stability.

[0054] Preferred functionally equivalent variants include polypeptides that are essentially derived from the polypeptide of SEQ ID NO: 1. In this context, "essentially derived from" means that the defined molecule does not contain any additional sequences that would alter the activity of SEQ ID NO: 1.

[0055] In a preferred embodiment, a functionally equivalent variant of SEQ ID NO: 1 is a polypeptide obtained by the insertion or addition of one or more amino acids with respect to the polypeptide of SEQ ID NO: 1. In a particular embodiment, a functionally equivalent variant is obtained by the insertion of fewer than 10 amino acids, more preferably fewer than 5 amino acids, and more preferably by the insertion of one amino acid. In a preferred embodiment, it is obtained by the insertion of one amino acid, which is methionine.

[0056] In another embodiment, a functionally equivalent variant of SEQ ID NO: 1 is a polypeptide obtained from the deletion of one or more amino acids with respect to the polypeptide of SEQ ID NO: 1. In one embodiment, a functionally equivalent variant is obtained from the deletion of fewer than 10 amino acids, more preferably fewer than 5 amino acids, and more preferably from the deletion of one amino acid.

[0057] Preferred functional variants of the targeted peptide exhibit amino acid sequence identity higher than approximately 25% with respect to the peptide of SEQ ID NO: 1, for example, 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of identity between two polypeptides is determined using computer algorithms and methods widely known to those skilled in the art. The identity between two amino acid sequences is preferably determined by using the previously described BLASTP algorithm (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., J. Mol. Biol. 1990; 215: 403-410). In a preferred embodiment, sequence identity is determined by the entire length of the polypeptide of SEQ ID NO: 1, the entire length of the variant, or both.

[0058] Functionally equivalent variants of the polypeptide of the present invention may also include post-translational modifications, such as glycosylation, acetylation, isoprenylation, myristoylation, and proteolytic processing.

[0059] In another embodiment, preferred functional variants of the targeted peptide contain amino acids in which one or more positions within the polypeptide of the present invention are conserved substitutions of amino acids present in the proteins listed above. A “conservative amino acid substitution” is obtained by replacing one amino acid with another having similar structure and / or chemical properties. For example, the following six groups each contain amino acids that are conserved substitutions with respect to one another: 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 those skilled in the art and has been 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).

[0060] In a preferred embodiment, it will be understood that a functionally equivalent variant of Omomyc contains mutations at positions corresponding to the mutations E61T, E68I, R74Q, and R75N ​​found in Omomyc derived from human c-Myc. The locations where the mutations should occur in the functionally equivalent variant can be determined by multiple sequence alignment of different Myc sequences, and can be identified by alignment of positions 61, 68, 74, and 75 in the sequence of Omomyc derived from human c-Myc. In one embodiment, a functionally equivalent variant of Omomyc contains mutations at positions corresponding to the mutations E61T, E68I, R74Q, and R75N ​​found in Omomyc derived from human c-Myc.

[0061] In another embodiment, a functionally equivalent variant of Omomyc contains mutations at positions corresponding to E61, E68, R74, and R75 in the sequence of Omomyc, where E61 is mutated to E61A or E61S, E68 is mutated to E68L, E68M, or E68V, R74 is mutated to R74N, and R75 is mutated to R75Q.

[0062] Multiple sequence alignment is an extension of pairwise alignment, which incorporates more than two sequences at a time. The multiple alignment method aligns all sequences in a given queryset. Preferred multiple sequence alignment programs (and their algorithms) are ClustalW, Clustal2W, or ClustalW XXL (see Thompson et al. (1994) Nucleic Acids Res 22:4673-4680). When c-Myc and variant sequences from different organisms are compared (aligned) as described herein, those skilled in the art can easily identify the positions within the sequences corresponding to the E61T, E68I, R74Q, and R75N ​​positions found in Omomyc and introduce them into Omomyc variant mutations corresponding to the E61T, E68I, R74Q, and R75N ​​mutations found in Omomyc derived from human c-Myc.

[0063] Suitable assays for determining whether a polypeptide can be considered a functionally equivalent variant of Omomyc include, but are not limited to, the following: - Assays that measure the ability of polypeptides to form dimeric complexes with Max and Myc, e.g., assays based on reporter gene expression as described by Soucek et al. (Oncogene, 1998, 17:2463-2472), as well as PLA (protein ligation assay) or co-immunoprecipitation. - Assays that measure the ability of polypeptides to bind to Myc / Max recognition sites (CACGTG sites) in DNA, such as the electrophoretic mobility shift assay (EMSA) described by Soucek et al. (see above). - Assays that measure the ability to respond to Myc-induced transactivation, such as the assay described by Soucek et al. (see above) that is based on the expression of a reporter gene under the control of a Myc / Max-specific DNA binding site. - An assay based on the ability of polypeptides to inhibit the growth of cells expressing the myc oncogene, as described by Soucek et al. (see above). - Assays that measure the ability of polypeptides to enhance myc-induced apoptosis, such as the assay described by Soucek et al. (Oncogene, 1998:17, 2463-2472). Alternatively, any assay commonly known in the art for evaluating apoptosis in cells, such as Hoechst staining, propidium iodide (PI) or annexin V staining, trypan blue, DNA laddering / fragmentation, and TUNEL may be used.

[0064] In a preferred embodiment, a polypeptide is considered a functionally equivalent variant of Omomyc if it exhibits activity that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of that of native Omomyc in one or more of the above assays.

[0065] In a specific embodiment, a functionally equivalent variant of the polypeptide of SEQ ID NO: 1 comprises the polypeptide of SEQ ID NO: 1, where 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 sulfurized amino acid, or a dicarboxyl amino acid or an amide thereof, 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.

[0066] Preferred functionally equivalent variants of SEQ ID NO: 1 having a non-cysteine ​​residue X at position 89 of SEQ ID NO: 1 are disclosed in the following table.

[0067] [Table 1]

[0068] Therefore, in a preferred embodiment, a functionally equivalent variant of the polypeptide of SEQ ID NO: 1 is selected from the group consisting of 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.

[0069] In addition, functionally equivalent variants of Omomyc can also be transduced into cells after the variant has come into contact with the cells. It will be understood that functionally equivalent variants of Omomyc contain a protein transduction domain found in native Omomyc or another functional protein transduction domain.

[0070] In a preferred embodiment, a polypeptide is considered a functionally equivalent variant of SEQ ID NO: 1 if it can transduce target cells with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the efficiency of SEQ ID NO: 1.

[0071] In addition, functionally equivalent variants of Sequence ID No. 1 can also translocate to the nucleus of target tumor cells.

[0072] In a preferred embodiment, a polypeptide is considered a functionally equivalent variant of SEQ ID NO: 1 if it can move to the nucleus of a target tumor cell with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the efficiency of SEQ ID NO: 1.

[0073] A preferred assay for determining whether a polypeptide is a functionally equivalent variant of Sequence ID No. 1 with respect to its ability to traverse the cell membrane and move to the nucleus involves dual labeling of the cell with a polypeptide-specific reagent and a dye that specifically labels the cell nucleus (such as DAPI or Hoechst dye). Detection of the polypeptide of the present invention can be performed by confocal microscopy or fluorescence microscopy.

[0074] In another preferred embodiment, compound (i) of the present invention is a conjugate comprising a polypeptide having 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 its functionally equivalent variant.

[0075] As used herein, the term "conjugate" refers to two or more compounds that are covalently linked together such that the functions of each compound are preserved in the conjugate.

[0076] 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 enriched in hydrophobic amino acids and hydrophobic chemical moieties.

[0077] In a preferred embodiment, the conjugate described in 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 chemical moieties that promote cellular uptake of the polypeptide or a functionally equivalent variant of the polypeptide.

[0078] In one embodiment, the chemical moiety that promotes the uptake of polypeptides into cells is a lipid or a fatty acid.

[0079] Fatty acids are generally molecules that contain a carbon chain having an acidic portion (e.g., a carboxylic acid) at the end of the chain. The carbon chain of a fatty acid may be of any length, however, it is preferable that the carbon chain length 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 derived therefrom. In certain embodiments, the carbon chain length is 4 to 18 carbon atoms in the chain portion of the fatty acid. In certain embodiments, the carbon chain of a fatty acid may contain an odd number of carbon atoms, however, in certain embodiments, an even number of carbon atoms in the chain may be preferred. Fatty acids that contain only single bonds in their carbon chain are called saturated, while fatty acids that contain at least one double bond in their chain are called unsaturated. Fatty acids may be branched, but in preferred embodiments of the present invention, they are 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.

[0080] In a preferred embodiment, the chemical moiety that facilitates cellular uptake of the polypeptide containing the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof is a cell-permeable peptide sequence, in which case the conjugate would comprise a fusion protein comprising the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof and the cell-permeable peptide sequence.

[0081] The term "fusion protein" refers to a protein created by genetic technology, consisting of two or more functional domains derived from different proteins. Fusion proteins may also be obtained by conventional means, for example, by gene expression of the nucleotide sequence encoding the fusion protein in a suitable cell. It will be understood that the cell-permeable peptide refers to a cell-permeable peptide that is different from the cell-permeable peptide that forms part of the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant of SEQ ID NO: 1.

[0082] The term “cell-permeable peptide sequence” is used herein interchangeably with “CPP,” “protein transduction domain,” or “PTD.” This refers to a peptide chain of variable length that directs the transport of proteins within a cell. The process of delivery to the cell generally occurs by endocytosis, although peptides can also be translocated into the cell by direct membrane translocation. CPPs typically have an amino acid composition containing high relative abundances of positively charged amino acids, such as lysine or arginine, or sequences containing an alternating pattern of polar / charged amino acids and nonpolar hydrophobic amino acids.

[0083] Examples of CPPs that can be used in the present invention include, but are not limited to, the CPP found in the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK, SEQ ID NO: 13), the CPP found in the 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), and HIV-1 consisting of amino acids 49-57 (RKKRRQRRR, SEQ ID NO: 16), amino acids 48-60 (GRKKRRQRRRTPQ, SEQ ID NO: 17), and amino acids 47-57 (YGRKKRRQRRR, SEQ ID NO: 18). CPP of TAT protein; CPP of S413-PV peptide (ALWKTLLKKVLKAPKKKRKV; SEQ ID NO: 19), CPP of penetratin (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 (PIRRRKKLRRLK; SEQ ID NO: 23), CPP of PTD-5 (RRQRRTSKLMKR; SEQ ID NO: 24), CPP of FHV coat-(35~49) (RRRRNRTRRNRRRVR; 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), CPP of R9-Tat (GRRRRRRRRRPPQ; SEQ ID NO: 29), CPP of MAP (KLALKLALKLALALKLA; SEQ ID NO: 30), CPP of SBP (MGLGLHLLVLAAALQGAWSQPKKKRKV; SEQ ID NO: 31), CPP of FBP (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), Pep-1 CPP (ac-KETWWETWWTEWSQPKKKRKV-cya; SEQ ID NO: 35), Pep-2 CPP (ac-KETWFETWFTEWSQPKKKRKV-cya; SEQ ID NO: 36), polyarginine sequences with structural RN (where N is 4-17), GRKKRRQRRR sequence (SEQ ID NO: 37), RRRRRRLR sequence (SEQ ID NO: 38), RQRRTS Examples include the KLMKR sequence (sequence number 39); transportan GWTLNSAGYLLGKINLKALAALAKKIL (sequence number 40); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (sequence number 41); RQIKIWFQNRRMKWKK (sequence number 42); YGRKKRRQRRR sequence (sequence number 43); RKKRRQRR sequence (sequence number 44); YARAAARQARA sequence (sequence number 45); THRLPRRRRRR sequence (sequence number 46); and GGRRARRRRRR sequence (sequence number 47).

[0084] In a preferred embodiment, the cell-permeable peptide is not intrinsically contained in SEQ ID NO: 1.

[0085] In a preferred embodiment, the CPP is the 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 the GRKKRRQRRR sequence (SEQ ID NO: 37) or RRRRRRLR (SEQ ID NO: 38). In yet another embodiment, the CPP is the GRKKRRQRRR sequence (SEQ ID NO: 37) or RRRRRRRR (SEQ ID NO: 65).

[0086] In some embodiments, the CPP is as described in WO2019 / 018898, the contents of which are incorporated herein by reference in their entirety.

[0087] In one embodiment, the cell-permeable peptide sequence is fused at the N-terminus of the polypeptide of the present invention or a functionally equivalent variant of the polypeptide. In another embodiment, the cell-permeable peptide is fused at the C-terminus of the polypeptide of the present invention or a functionally equivalent variant of the polypeptide.

[0088] In a preferred embodiment, the conjugate or fusion protein of the combination described in the present invention comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more additional cell-permeable peptides, in addition to the cell-permeable peptide itself found in the polypeptide of SEQ ID NO: 1 or a functionally equivalent variant of said polypeptide.

[0089] A preferred fusion protein of the present invention is the polypeptide Omomyc as defined below. * TAT and Omomyc * Includes LZArg.

[0090] [Table 2]

[0091] Therefore, in a preferred embodiment, the fusion protein is a polypeptide selected from SEQ ID NOs: 11 and 12.

[0092] A suitable assay for determining whether the conjugate preserves Omomyc's cell membrane mobility is, but is not limited to, an assay that measures the conjugate's ability to transduce cells in culture. This assay is based on bringing the conjugate into contact with cultured cells and detecting the presence of the conjugate at its location within the cells.

[0093] In another preferred embodiment, the conjugate of the combination of the present invention further includes an additional nuclear localization signal.

[0094] The term “nuclear localization signal” (NLS), as used herein, refers to an amino acid sequence of approximately 4 to 20 amino acid residues that plays a role in orienting a protein toward 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 some embodiments, the NLS is selected from the group consisting of 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); hnRNP A NLS (NQSSNFGPMKGGNFGGRSSGPYGGGGQYFKPRNQGGY, SEQ ID NO: 53); and rpL23a NLS (VHSHKKKKIRTSPTFTTPKTLRLRRQPKYPRKSAPRRNKLDHY, SEQ ID NO: 54). In one embodiment of the present invention, the nuclear localization signal includes the motif K(K / R)X(K / R).

[0095] In a 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).

[0096] In another preferred embodiment, the NLS may be at the N-terminus or C-terminus of a conjugate or fusion protein comprising the polypeptide of SEQ ID NO: 1 or a functionally equivalent variant thereof.

[0097] Those skilled in the art will understand that the conjugate of the present invention may preferably further comprise a polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof, a cell-permeable peptide sequence, and / or one or more flexible peptides connecting the NLS. Therefore, in a specific embodiment, the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof is directly conjugated to the cell-permeable peptide sequence. In another specific embodiment, the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof is conjugated to the cell-permeable peptide sequence via a flexible peptide. In one embodiment, the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof is directly conjugated to the NLS. In another embodiment, the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof is conjugated to the NLS via a flexible peptide.

[0098] In specific embodiments, the polypeptide of the conjugate described in the present invention is directly ligated to a cell-permeable peptide sequence and NLS.

[0099] In one embodiment, the NLS is one of the NLSs endogenously found in the Myc sequence, for example, the M1 peptide (PAAKRVKLD, SEQ ID NO: 56) or the M2 peptide (RQRRNELKRSF, SEQ ID NO: 57).

[0100] In another embodiment, the additional NLS refers to an NLS distinct from the endogenous NLS found in the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant of SEQ ID NO: 1.

[0101] In a preferred embodiment, the conjugate or fusion protein described in 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, and at least 10 NLS in addition to the endogenous NLS found in the polypeptide of the present invention or a functionally equivalent variant thereof.

[0102] In another specific embodiment, the polypeptide of the conjugate for use described in the present invention is linked to a cell-permeable peptide sequence through a first flexible peptide linker and to the NLS through a second flexible peptide linker.

[0103] As used herein, the terms “flexible peptide,” “spacer peptide,” or “linker peptide” refer to peptides that covalently bond to two proteins or parts but are not part of either polypeptide, and that allow one to move relative to the other without causing substantially adverse effects on the function of either protein or part. Thus, flexible linkers do not affect the tumor trace activity of polypeptide sequences, the cell permeability activity of cell-permeable peptides, or the nuclear localization ability of NLS.

[0104] Flexible peptides contain at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, at least ten amino acids, at least twelve amino acids, at least fourteen amino acids, at least sixteen amino acids, at least eighteen 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 some embodiments, the flexible peptide will allow the movement of one protein relative to another protein in order to increase the solubility of the protein and / or improve its activity. A preferred linker region is a polyglycine region, which is a GPRRRR sequence (SEQ ID NO: 58) of a combination of glycine, proline, and alanine residues.

[0105] In specific embodiments, the conjugate described in the present invention comprises a tag attached to the conjugate or C-terminal or N-terminal domain of the polypeptide or its fusion protein or variant. The tag is generally a peptide or amino acid sequence that can be used in the isolation or purification of the fusion protein. Therefore, the tag can bind to one or more ligands, for example, one or more ligands of an affinity matrix such as a chromatography support or beads having high affinity. An example of the tag is a histidine tag (His tag or HT), for example, a tag containing six histidine residues (His6 or H6), which has high affinity to nickel (Ni 2+ ) or cobalt (Co 2+ The His tag can be bound to a column. The His tag has the desirable property of being able to bind to its ligand under conditions that denature most proteins and disrupt most protein-protein interactions. Therefore, it can be used to remove H6-tagged bait proteins after disruption of bait-involved protein-protein interactions.

[0106] Non-limiting examples of tags useful for isolating or purifying conjugates, polypeptides containing SEQ ID NO: 1, their variants, or fusion proteins include: Arg-tag, FLAG-tag (DYKDDDDK; SEQ ID NO: 59), Strep-tag (WSHPQFEK, SEQ ID NO: 60), antibody-recognizable epitopes such as c-myc-tag (recognized by anti-c-myc antibody), HA-tag (YPYDVPDYA, SEQ ID NO: 61), V5-tag (GKPIPNPLLGLDST, SEQ ID NO: 62), SBP-tag, S-tag, calmodulin-binding peptide, cellulose-binding domain, chitin-binding domain, glutathione S-transferase-tag, maltose-binding protein, NusA, TrxA, DsbA, Avi-tag, etc. (Terpe Examples include amino acid sequences (K., Appl. Microbiol. Biotechnol. 2003, 60:523-525), such as AHGHRP (SEQ ID NO: 63) or PIHDHDHPHLVIHSGMTCXXC (SEQ ID NO: 64), and β-galactosidase.

[0107] The tag may be used, if desired, for the isolation or purification of the fusion protein.

[0108] In another preferred embodiment, compound (i) of the present invention is a polynucleotide encoding a polypeptide or fusion protein disclosed above. In a preferred embodiment, compound (i) of the present invention is a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof. In another embodiment, compound (i) of the present invention is a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that facilitates cellular uptake of the polypeptide or its functionally equivalent variant, more preferably a polynucleotide encoding a fusion protein between a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof and a cell-permeable peptide sequence.

[0109] The terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably to refer to polymeric forms of nucleotides of any length. Polynucleotides may contain deoxyribonucleotides, ribonucleotides, and / or analogues thereof. 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-helix 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 native nucleic acid molecules, the nucleic acid molecules of the present invention may also include modified nucleic acid molecules. As used herein, mRNA refers to RNA that can be translated in a cell.

[0110] In a preferred embodiment, the polynucleotide of the present invention is mRNA.

[0111] mRNA can be chemically synthesized, obtained by in vitro transcription, or synthesized in vivo in target cells. The nucleotide sequences forming the polynucleotides encoding the conjugate or fusion protein of the present invention are in the same correct reading frame for their expression.

[0112] In a preferred embodiment, component (i) of the combination of the present invention is an mRNA encoding a polypeptide consisting of the sequence of 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.

[0113] In another embodiment, component (i) of the combination of the present invention is a vector containing the polynucleotide of the present invention.

[0114] The term “vector,” as used herein, refers to a nucleic acid sequence containing the required sequence such that, after transcription and translation in a cell, the polypeptide encoded by the polynucleotide of the present invention is produced. The sequence is operably ligated to an additional segment that provides its self-replication in the host cell of interest. Preferably, the vector is an expression vector, which is defined as a vector that, in addition to the self-replication region in the host cell, contains a region operably ligated to the nucleic acid of the present invention and can enhance the expression of the nucleic acid product described in the present invention. The vector of the present invention can be obtained by techniques widely known in the art.

[0115] Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensers, liposome-encapsulated DNA or RNA expression vectors, and certain eukaryotic cells, such as producing cells. Suitable vectors containing polynucleotides of the present invention include expression vectors in prokaryotes, e.g., pUC18, pUC19, pBluescript and their derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCRl, RP4, phage and "shuttle" vectors, e.g., pSA3 and pAT28; expression vectors in yeast, e.g., 2-micron plasmid-type vectors, embedded plasmids, YEP vectors, centromere plasmids and analogues; expression vectors in insect cells, e.g., pAC series and pVL series vectors; expression vectors in plants, e.g., series pIBI vectors, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE and analogues; and expression vectors in higher eukaryotic cells based on viral vectors (adenoviruses, adenovirus-related viruses, and retroviruses, and especially lentiviruses) and non-viral vectors, e.g., pSilencer 4.1-CMV(Ambion), pcDNA3, pcDNA3.1 / hyg, pHCMV / Zeo, pCR3.1, pEFl / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAXl, pZeoSV2, pCI, pSVL, pKSV-10, pBPV-1, pML2d, and pTDT1 are vectors derived from these. In preferred embodiments, the polynucleotides of the present invention are contained in vectors selected from the group consisting of pEGFP or pBabe retroviral vectors and pTRIPZ or pSLIK lentiviral vectors.

[0116] The vectors of the present invention may be used to transform, transfect, or infect cells that can be transformed, transfected, or infected by the vectors. The cells may be prokaryotic or eukaryotic.

[0117] The vector preferably comprises the polynucleotide of the present invention operationally bound to a sequence that modulates the expression of the polynucleotide of the present invention. The regulatory sequence used in the present invention may be a nuclear promoter, or alternatively, other regulatory sequences that increase the expression of enhancer sequences and / or heterologous nucleic acid sequences. In principle, any promoter can be used in the present invention, provided that the promoter is compatible with the cell in which the polynucleotide is expressed. Therefore, suitable promoters for realizing the present invention are not necessarily limited to, but include constitutive promoters, such as derivatives of eukaryotic viral genomes, such as polyomavirus, adenovirus, SV40, CMV, aerosarcoma virus, hepatitis B virus, metallothionein gene promoter, herpes simplex virus thymidine kinase gene promoter, retroviral LTR region, immunoglobulin gene promoter, actin gene promoter, EF-1 alpha gene promoter, as well as inductive promoters, such as tetracycline systems, NFκB / UV light systems, Cre / Lox systems, and heat shock gene promoters, the moduloable RNA polymerase II promoter described in WO / 2006 / 135436, and tissue-specific promoters.

[0118] In another embodiment, component (i) of the combination of the present invention is a cell capable of secreting the polypeptide or conjugate of the present invention, preferably the polypeptide or fusion protein of the present invention, into a culture medium.

[0119] Suitable cells capable of secreting the polypeptides of the present invention include, but are not limited to, cardiomyocytes, adipocytes, endothelial cells, epithelial cells, lymphocytes (B and T cells), mast cells, eosinophils, vascular intima cells, isolated cells from different organs, preferably islets of Langerhans, hepatocytes, leukocytes, e.g., mononuclear leukocytes, mesenchymal cells, primary cultures of cells isolated from the umbilical cord or from adults (skin, lung, kidney and liver), osteoclasts, chondrocytes and other connective tissue cells. Cells from established strains, e.g., Jurkat T cells, NIH-3T3, CHO, Cos, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 cells, C2C12 myoblasts and W138 cells are also suitable. Those skilled in the art will recognize that cells capable of secreting the polypeptides of the present invention into culture media may be found to form microparticles or microcapsules, so that the cells have a longer useful life in patients. Suitable materials for forming the microparticles of the present invention include any biocompatible polymer material that allows for the continuous secretion of therapeutic products and acts as a support for cells. Therefore, the biocompatible polymer material may be, for example, a thermoplastic polymer or a hydrogen polymer.Among thermoplastic polymers, acrylic acid, acrylamide, 2-aminoethyl methacrylate, poly(tetrafluoroethylene-cohexafluoropropylene), methacrylate-(7-coumaroxy)ethyl ester acid, N-isopropylacrylamide, polyacrylic acid, polyacrylamide, polyamidoamine, poly(amino)-p-xylene, poly(chloroethyl vinyl ether), polycaprolactone, poly(caprolactone-co-trimethylene carbonate), poly(carbonate urea)urethane, poly(carbonate)urethane, polyethylene, polyethylene and acrylamide copolymer, polyethylene glycol, polyethylene glycol methacrylate There are copolymers containing poly(ethylene terephthalate), poly(4-hydroxybutyl acrylate), poly(hydroxyethyl methacrylate), poly(N-2-hydroxypropyl methacrylate), poly(glycolic acid lactate), poly(L-lactic acid), poly(gamma-methyl,L-glutamate), poly(methyl methacrylate), poly(propylene fumarate), poly(propylene oxide), polypyrrole, polystyrene, poly(tetrafluoroethylene), polyurethane, polyvinyl alcohol, ultra-high molecular weight polyethylene, 6-(p-vinylbenzamide)-hexanoate Np-vinylbenzyl-D-maltonamide, and two or more of the above polymers. Among the hydrogel-type polymers are alginates, agarose, collagen, starch, hyaluronic acid, bovine serum albumin, cellulose and their derivatives, pectin, chondroitin sulfate, fibrin and fibroin, and synthetic hydrogels, such as natural materials like Sepharose® and Sephadex®.

[0120] Compound (ii) of the combination of the present invention Compound (ii) of the combination of the present invention is a PARP inhibitor.

[0121] "PARP" (EC2.4.2.30), as used herein, refers to poly(ADP-ribose) polymerases, also known as NAD+ADP-ribosyltransferase or poly(ADP-ribose) synthase, a family of enzymes that play a crucial role in maintaining DNA integrity as part of the base excision pathway of DNA repair. PAR enzymes catalyze the translocation of the ADP-ribose moiety from cellular NAD+ to nucleoproteins, forming ADP-ribose polymers. This led to the first inhibitors being structural analogs of NAD+, which block NAD+ binding and thereby inhibit PARP activity. PARPs share catalytic and scaffolding properties. The PARP superfamily in humans has 17 known members. PARP1, PARP2, tankylase 1, tankylase 2, and vPARP are thought to have roles in DNA repair, but PARP1 accounts for more than 90% of cellular PARP activity. Therefore, in a preferred embodiment, the PARP is selected from the group consisting of PARP1 and / or PARP2.

[0122] PARP1 plays a role in the repair of single-stranded DNA (ssDNA) breaks. PARP1 has three domains responsible for DNA binding, self-modification, and catalyst formation. DNA breaks lead to the recruitment and binding of PARP1 to the site of damage, as well as the formation of long, branched poly(ADP-ribose) (PAR) strands, due to increased catalytic activity. PAR has a net negative charge that promotes the recruitment of DNA repair proteins involved in the base excision repair pathway to the site of DNA damage, and facilitates the removal of PARP1 from the damage site, allowing access to other repair proteins. Furthermore, PARP1 is involved in homologous recombination and non-homologous end joining pathways. The sequence of the human PARP1 protein corresponds to sequence P09874 in the Uniprot database (as of October 12, 2022, entry version 259).

[0123] PARP2 participates in base excision repair simultaneously with PARP1, but its function is still under investigation. While PARP2 is substantially different from PARP1 in its domain structure, it shares considerable structural homology with the catalytic domain of PARP1. The sequence of the human PARP2 protein corresponds to the sequence Q9UGN5 in the Uniprot database (as of October 12, 2022, entry version 202).

[0124] When used herein, "PARP inhibitor" refers to any compound that can cause a decrease in the activity of PARP, specifically the activity of PARP1 and PARP2, and includes compounds that prevent the expression of PARP genes, specifically the PARP1 and PARP2 genes, as well as compounds that result in a reduction in the mRNA or protein levels of PARP, specifically the mRNA or protein levels of PARP1 and / or PARP2. PARP inhibitors primarily inhibit the catalytic activity of the PARP1 and PARP2 enzymes. Therefore, in preferred embodiments, PARP inhibitors are selected from the group consisting of PARP1 inhibitors, PARP2 inhibitors, and inhibitors of both PARP1 and PARP2.

[0125] Protein or nucleic acid expression is considered reduced if 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%, or at least 100% (i.e., absent) relative to the reference value.

[0126] The reference value refers to the level of protein or nucleic acid in a control subject that may not have a specific disease.

[0127] Preferred methods for determining whether an inhibitor can reduce the mRNA levels of PARP, specifically the mRNA levels of PARP1 and / or PARP2, include, but are not limited to, standard assays for determining mRNA expression levels, such as qPCR, RT-PCR, RNA protection analysis, Northern blotting, RNA dot blotting, insight hybridization, microarray techniques, tag-based methods, such as sequential gene expression analysis (SAGE) including modifiers like LongSAGE and SuperSAGE, microarrays, and fluorescence insight hybridization (FISH) including modifiers like Flow-FISH, qFiSH, and double-fusion FISH (D-FISH). Preferably, quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous.

[0128] Nucleic acids contained in a sample (e.g., cells or tissues prepared from a subject) are first extracted according to standard methods, for example, using lytic enzymes or chemical solutions, or by nucleic acid binding resin according to the manufacturer's instructions.

[0129] In cases where mRNA is measured in a biological sample, the biological sample may be processed to physically, mechanically, or chemically disrupt the tissue or cellular structure and release intracellular components into an aqueous or organic solution to prepare nucleic acids for further analysis. Nucleic acids are extracted from the sample by procedures known to those skilled in the art and commercially available. RNA is then extracted from a frozen or fresh sample by any of the typical methods in the art, such as Sambrook, J. et al., 2001. Molecular cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, NY, Vols. 1-3. Preferably, care is taken to avoid the degradation of RNA during the extraction process.

[0130] Expression levels can be determined using mRNA obtained from formalin-fixed, paraffin-embedded tissue samples. mRNA may be isolated from archived pathological or biopsy samples, which are first deparaffinized. An exemplary deparaffinization method involves washing the paraffinized sample with an organic solvent such as xylene. The deparaffinized sample can be rehydrated with an aqueous solution of a lower alcohol. Suitable lower alcohols include, for example, methanol, ethanol, propanol, and butanol. The deparaffinized sample may be rehydrated, for example, by sequential washing with decreasing concentrations of lower alcohol solutions. Alternatively, the sample may be deparaffinized and rehydrated simultaneously. The sample is then lysed, and RNA is extracted from the sample. Samples can also be obtained from fresh tumor tissue, such as excised tumors. In specific embodiments, samples can be obtained from fresh tumor tissue or from OCT-embedded frozen tissue.

[0131] To normalize mRNA expression levels between different samples, it is possible to compare the expression level of the target mRNA in the test sample with that of the control RNA. control RNA When used herein, "control RNA" refers to RNA whose expression level is either unchanged or only partially changed in tumor cells compared to non-tumorogenic cells. Preferably, the control RNA is mRNA derived from a housekeeping gene, which encodes a protein that is constitutively expressed and performs essential cellular functions. Preferred housekeeping genes for use in the present invention include β-2-microglobulin, ubiquitin, 18-S ribosomal protein, cyclophyllin, IPO8, HPRT, GAPDH, PSMB4, tubulin, and β-actin.

[0132] Relative gene expression quantification may be calculated according to the comparative threshold cycle (Ct) method, using housekeeping genes as endogenous controls and commercially available RNA controls as calibrators. The final result is given by Equation 2. -(ΔCt試料-ΔCt較正物質)The ΔCt values ​​of the calibration material and sample are determined according to the following formula, where the ΔCt values ​​of the calibration material and sample are determined by subtracting the Ct value of the target gene from the value of the control gene.

[0133] A preferred method for determining whether an inhibitor acts by reducing PARP protein levels, specifically PARP1 and / or PARP2 protein levels, includes quantification by conventional methods, for example, using an antibody having the ability to specifically bind to the PARP gene, specifically the protein (or a fragment thereof containing an antigenic determinant) encoded by the PARP1 and / or PARP2 gene, followed by quantification of the resulting antibody-antigen complex.

[0134] The antibodies used in these assays may be, for example, polyclonal serum, hybridoma supernatant or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' and F(ab')2, ScFv, diabodies, triabodies, tetrabodies, and humanized antibodies. At the same time, the antibodies may or may not be labeled. Examples of markers that can be used, but are not limited to, include radioisotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, and colorants. There are many well-known assays that can be used in the present invention using unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies), among others, which include Western blotting or Western transfer, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzyme-linked immunoassay), DAS-ELISA (double enzyme sandwich ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of biochips or protein microarrays containing specific antibodies, or colloidal precipitation assays in formats such as dipsticks. Other methods for detecting and quantifying the level of a target protein include techniques such as affinity chromatography and binding ligand assays.

[0135] On the other hand, the determination of PARP protein levels, specifically PARP1 and / or PARP2 proteins, can be performed by constructing a tissue microarray (TMA) containing the assembled target sample and determining the expression levels of the corresponding proteins by immunohistochemical techniques. Immunostaining intensity can be evaluated by two or more different pathologists and scored using uniform and clear cutoff criteria to maintain the reproducibility of the method. Inconsistencies can be resolved by simultaneous re-evaluation. Briefly, the results of immunostaining can be recorded as negative expression (0) versus positive expression, and low expression (1+) versus moderate (2+) and high (3+) expression, taking into account the expression and specific cutoffs in tumor cells for each marker. As a general criterion, cutoffs are chosen to facilitate reproducibility and, where possible, to paraphrase biological events. Alternatively, the immunohistochemical staining intensity can be evaluated 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).

[0136] Alternatively, in another specific embodiment, the levels of PARP proteins, specifically PARP1 and / or PARP2 proteins, are determined by Western blotting. Western blotting is based on the detection of previously separated proteins by gel electrophoresis, immobilized on a membrane, generally nitrocellulose, under denaturing conditions, by incubation and development with a specific antibody (e.g., chemiluminescence).

[0137] PARP inhibitors can inhibit PARP activity, specifically 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 the entire range of 5% to 100%. A preferred method for determining whether an inhibitor acts by reducing PARP activity is any method that allows detection of the consumption of nicotinamide adenine dinucleotide (NAD+) of the substrate, or the formation of any of these three products, namely, a polymer of ADP-ribose (pADPr or PAR), nicotinamide, and adenine. Methods for detecting PARP activity, specifically PARP1 activity, are known in the art and 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, March 1, 2004; 326(1):78-86; and Yelamos J. et al., Am J Cancer Res. 2011; 1(3):328-346. Assays for determining enzyme activity are known to those skilled in the art and are not limited to those disclosed in the art, but include initial rate assays, progress curve assays, transient kinetic assays, and relaxation assays. Continuous assays of enzyme activity are not limited to those disclosed in the art, but include spectrophotometric assays, fluorescence assays, calorimetry assays, chemiluminescence assays, light scattering assays, and microscale thermophoresis assays. Discontinuous assays of enzyme activity are not limited to those disclosed in the art, but include radiometric assays and chromatographic assays. As those skilled in the art will understand, factors that may affect enzyme activity include salt concentration, temperature, pH, and substrate concentration.

[0138] PARP inhibitors can be any organic or inorganic molecule, including modified and unmodified nucleic acids, such as antisense nucleic acids, RNA interference (RNAi) agents useful in the treatment of cancer, such as siRNA, shRNA or miRNA, peptides, proteins, peptidomimetics, receptors, ligands, antibodies, and small organic molecules.

[0139] In a preferred embodiment, a PARP inhibitor useful in the present invention is selected from Table 1.

[0140] [Table 3]

[0141] " small chemical compounds As used herein, "a molecule that modulates a biological process, in this case inhibits the catalytic activity of PARP. This compound may be natural or artificial."

[0142] In a preferred embodiment, the PARP inhibitor is selected from the group consisting of olaparib, lucaparib, veliparib, niraparib, talazoparib, pamiparib, fluzoparib, and iniparib; preferably, it is selected from the group consisting of olaparib, lucaparib, veliparib, niraparib, talazoparib, pamiparib, and fluzoparib; more preferably, it is selected from the group consisting of olaparib, lucaparib, veliparib, niraparib, and talazoparib.

[0143] In a preferred embodiment, the PARP inhibitor is olaparib, which is a potent oral inhibitor of PARP1 and PARP2.

[0144] In another embodiment, the PARP inhibitor is talazoparib. Talazoparib is an oral PARP inhibitor.

[0145] The terms olaparib, lucaparib, veliparib, niraparib, thalazoparib, pamiparib, fluzoparib, and iniparib also include their pharmaceutically acceptable salts, solvates, polymorphs, or cocrystals.

[0146] In a more preferred embodiment, the PARP inhibitor is selected from the group consisting of compounds listed in item I, II, or III of Table 1, or pharmaceutically acceptable salts thereof, preferably the compounds listed in items I, II, and III of Table 1.

[0147] In a more preferred embodiment, the PARP inhibitor is selected from the group consisting of compounds listed in items I and II of Table 1, or pharmaceutically acceptable salts thereof, preferably the compounds listed in items I and II of Table 1.

[0148] The term "pharmaceutically acceptable" refers to properties and / or substances that are acceptable to the patient from a pharmacological / toxicological perspective, and acceptable to the pharmaceutical chemist manufacturing them from a physical / chemical perspective with respect to composition, formulation, stability, patient tolerance, and bioavailability.

[0149] The term "pharmaceutically acceptable salt" encompasses salts with pharmaceutically acceptable acids or bases. Examples of pharmaceutically acceptable acids include inorganic acids, such as, but not limited to, hydrochloric acid, sulfuric acid, phosphoric acid, diphosphate, hydrobromic acid, hydroiodic acid, and nitric acid, as well as organic acids, such as, but 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, cyclohexylsulfamic acid (cyclamic acid), or both p-toluenesulfonic acid. Examples of pharmaceutically acceptable bases include alkali metal (e.g., sodium or potassium) and alkaline earth metal (e.g., calcium or magnesium) hydroxides, as well as organic bases, such as, but not limited to, alkylamines, arylalkylamines, and heterocyclic amines.

[0150] The term "solvate" as used in this invention should be understood to mean any solid form of the above compound having another molecule attached thereto via non-covalent bonds. Examples of solvates include hydrates and alkolates, preferably C1-C6 alkolates, such as methanelates.

[0151] The term "polymorph" as used in this invention should be understood as a compound-specific crystalline form that can crystallize in different forms.

[0152] When used herein, the term “cocrystal” should be understood as a crystal structure consisting of a PARP inhibitor and at least one other component. Interfering RNA The term "iRNA" refers to an RNA molecule capable of silencing the expression of PARP, specifically PARP1 and / or PARP2, or any gene required for PARP function. For this purpose, iRNAs are typically 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 are effectively used in iRNA technology, including small interfering RNAs (siRNAs), sometimes known as short interfering RNAs or silencer RNAs; microRNAs (miRNAs), which are usually different from siRNAs because they are processed from single-stranded RNA precursors and they are only partially complementary to target mRNAs; and small hairpin RNAs (shRNAs).

[0153] Small interfering RNA (siRNA) agents can inhibit target gene expression by RNA interference. siRNA may be chemically synthesized, obtained by in vitro transcription, or synthesized in vivo in target cells. Typically, siRNA consists of double-stranded RNA 15–40 nucleotides long and may contain protruding 3' and / or 5' regions 1–6 nucleotides long. The length of the protruding region is independent of the total length of the siRNA molecule. siRNA acts by post-transcriptional degradation or silencing of target messengers.

[0154] siRNA is sometimes named shRNA (small hairpin RNA) because the antiparallel strands that make up the siRNA are connected by loop or hairpin regions. siRNA consists of a short antisense sequence (19-25 nucleotides) followed by a 5-9 nucleotide loop, and a sense strand. shRNA may be encoded by plasmids or viruses, specifically by retroviruses, more specifically by retroviruses under the control of a promoter such as the U6 promoter for RNA polymerase III.

[0155] For use within the context of this invention, the siRNA is substantially homologous to the mRNA of PARP, specifically the mRNA of PARP1 and / or PARP2, or its protein-coding genome sequence. Substantial homology The term "siRNA" is understood to mean that an siRNA has a sequence that is sufficiently complementary or similar to the target mRNA, so that the siRNA may be able to induce mRNA degradation through RNA interference. Suitable siRNAs for inducing interference include siRNAs formed by RNA, and siRNAs containing chemically different modifications, for example, -siRNA in which the linkage between nucleotides differs from that found in nature, such as phosphorothioate linkages; - Functional reagents, e.g., standard RNA conjugates with fluorophores; Modification of the RNA chain end, specifically the 3' end, by combination with different functional hydroxyl groups at the -2' position; - Sugar-modified nucleotides, e.g., O-alkylated radicals at the 2' position, e.g., 2'-O-methylribose or 2'-O-fluororibose; - Base-modified nucleotides, e.g., halogenated bases (e.g., 5-bromouracil and 5-pseudracil), alkylated bases (e.g., 7-methyl-guanosine) These are some examples.

[0156] siRNA and shRNA for use in the context of the present invention may be obtained using a set of techniques known to those skilled in the art. For example, siRNA may be chemically synthesized from protected ribonucleoside phosphoramidites in a conventional DNA / RNA synthesizer. Alternatively, siRNA may be produced from plasmids and viral vectors by a recombinant dicer, where the coding regions of one or more siRNA strands are under the operational control of an RNA polymerase III promoter. The RNase dicer processes shRNA into siRNA in cells.

[0157] The PARP region considered the basis for siRNA design is non-limiting and may contain a coding sequence region (between the start and stop codons), or alternatively, preferably 25–50 nucleotides in length and at any position 3' relative to the start codon, containing a sequence from the 5' or 3' untranslated region. 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 one that exhibits a high G / C content. If the aforementioned sequence motif is not found, it is possible to identify the sequence motif NA(N21) (where N can be any nucleotide).

[0158] In a preferred embodiment, the PARP inhibitor is siRNA. In a more preferred embodiment, the siRNA is a commercially available siRNA from Santa Cruz Biotechnology, specifically sc-29437 or sc-106356.

[0159] In another embodiment, the inhibitor is PARP-specific Antisense oligonucleotides"An antisense oligonucleotide is a molecule whose sequence is complementary to the mRNA encoding PARP, specifically PARP1 and / or PARP2, i.e., complementary to the cDNA coding strand. The antisense oligonucleotide may be complementary to the complete coding region, or to the same region including both the coding region and the 5' and 3' untranslated regions. The antisense oligonucleotide may have a length of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides. The antisense oligonucleotide may be obtained by chemical synthesis or by enzymatic linkage reactions, which are widely known to those skilled in the art. For example, the antisense oligonucleotide may further contain modified nucleotides that increase its biological stability, or the stability of the double catenary DNA-RNA complex formed between the antisense oligonucleotide and the target polynucleotide, e.g., phosphorothioate derivatives, peptide nucleic acids, and acridine-substituted oligonucleotides.Modified oligonucleotides that can be used for the preparation of antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethyl-aminomethyluracil, dihydrouracil, beta-D-galactosyl quosine, inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, and 5-methylcytosine. Examples include N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueucine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queucine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetate methyl ester, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, antisense nucleic acids may be biologically produced using an expression vector from which antisense-directed nucleic acids have been cloned.

[0160] Another group of compounds that may form part of the present invention are catalytically active nucleic acids known as ribozymes. RibozymeThe ribozyme comprises a catalytic region and a second region whose sequence is complementary to the target nucleic acid and which confers substrate specificity to the ribozyme. Following the interaction between the ribozyme and its substrate by hybridization, and the coupling between the complementary regions of the target nucleic acid and the ribozyme, activation of the catalytic region occurs, causing intramolecular or intermolecular cleavage of the target nucleic acid. Basic considerations for the design of ribozymes are widely known to those skilled in the art (e.g., Doherty and Doudna (see Annu. Ref. Biophys. Biomolstruct. 2000; 30: 457-75)).

[0161] Another type of compound that may form part of the composition of the present invention includes an inhibitory antibody. inhibitory antibody The term "antibody" is understood, according to the present invention, to mean an antibody that binds to PARP, specifically PARP1 and / or PARP2, and causes inhibition of its catalytic activity.

[0162] Antibodies may be prepared using any method known to those skilled in the art. For example, polyclonal antibodies are prepared by immunization of animals with the protein to be inhibited. Monoclonal antibodies can be prepared using the method described by Kohler, Milstein et al. (Nature, 1975, 256:495). Once antibodies capable of binding to PARP, specifically PARP1 and / or PARP2, are identified, antibodies capable of inhibiting PARP activity are selected using the aforementioned assay for determining PARP activity. Suitable antibodies in this invention include intact antibodies containing antigen-binding variable and constant regions, fragments "Fab", "F(ab')2" and "Fab'", Fv, scFv, diabodies, and bispecific antibodies.

[0163] Other compounds that can inhibit PARP expression and may form part of the composition of the present invention include aptamers and spiegelmers. Aptamers and SpiegelmersAptamers and Spiegelmers are single- or double-stranded D- or L-nucleotides that specifically bind to proteins, resulting in modifications to the biological activity of the proteins (PARPs, specifically PARPI and / or PARP2). Aptamers and Spiegelmers are 15 to 80 nucleotides long, preferably 20 to 50 nucleotides long.

[0164] In one embodiment, the combination of the present invention is a conjugate between component (i) and component (ii) of the combination of the present invention, specifically a conjugate between a polypeptide containing the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof and a PARP inhibitor.

[0165] In some embodiments, the conjugation between components (i) and (ii) is via a non-disconnectable linker. In some embodiments, the conjugation between components (i) and (ii) is via a disconnectable linker. Exemplary non-disconnectable and disconnectable linkers are US8088387, US8142784, WO2013075048, US6630579, US8512707, US9120854, US9023351, US20160095938, US9446146, WO2005009369, US5773001, US621 The contents of 4345, US10111954, US8153768, US7829531, US20160082119, WO2018218004, US8568728, WO2015057699, US20170182181, and US9198979 are described herein, and the contents of each of these, in whole, are incorporated herein by reference.

[0166] In another embodiment, the present invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of the combination of the present invention together with pharmaceutically acceptable excipients.

[0167] When used in the present invention, the term "pharmaceutical composition" refers to a formulation adapted for administering one or more therapeutically useful drugs in a predetermined dose to cells, groups of cells, organs, tissues, or animals whose cell division is uncontrolled, such as cancer cells.

[0168] The pharmaceutical composition of the present invention contains a pharmaceutically effective amount of the combination described in the present invention and a pharmaceutically active carrier. The pharmaceutical composition of the present invention comprises a polypeptide comprising the sequence of SEQ ID NO: 1, a functionally equivalent variant thereof, a conjugate described in the present invention, a polynucleotide encoding the polypeptide or conjugate, a vector comprising a polynucleotide, or a cell capable of secreting the polypeptide or conjugate into a 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 of the present invention are as defined above.

[0169] When used herein, the term "pharmaceutical effective dose" is understood to mean the amount capable of providing a therapeutic effect, which can be determined by those skilled in the art by commonly used means. The amount of Omomyc polypeptide, its functionally equivalent variants, conjugates, fusion proteins, polynucleotides, vectors, cells, or PARP inhibitors that can be combined in the pharmaceutical compositions described in the present invention will vary depending on the target and the specific method of administration. Those skilled in the art will recognize that the dosage may also be determined using guidance from Goodman and Goldman's The Pharmacological Basis of Therapeutics, 9th edition (1996), Appendix II, pp. 1707–1711 and Goodman and Goldman's The Pharmacological Basis of Therapeutics, 10th edition (2001), Appendix II, pp. 475–493.

[0170] The appropriate dosage of one or more active agents in the pharmaceutical composition depends on the type of cancer being treated, the severity and course of the disease, whether the composition is administered for prophylactic or therapeutic purposes, previous therapies, the patient's medical history and response to the peptide or polypeptide, and the discretion of the attending physician.

[0171] The amount of the polypeptide comprising the sequence of SEQ ID NO:1, its functionally equivalent variants, fusion proteins, conjugates, polynucleotides, vectors or cells is preferably administered to the patient over one treatment or a series of treatments. Depending on the type and severity of the disease, the appropriate dosage level is generally about 0.01 - 500 mg per kg of the patient's body weight per day, which can be administered in single or multiple doses. Preferably, the dosage level is about 0.1 - about 250 mg / kg per day, more preferably about 0.5 - about 100 mg / kg per day.

[0172] In a preferred embodiment, the amount of the first component is preferably administered 4 times a week, preferably intranasally, at about 3.75 mg / kg per day for the body weight of the subject. In a preferred embodiment, the amount of the first component is preferably administered 4 times a week, preferably intranasally, at about 8 - 15 mg / m 2 preferably 10 - 12 mg / m 2 more preferably 11.25 mg / m 2 per day.

[0173] In a preferred embodiment, the amount of the first component is preferably administered 2 times a week, preferably intravenously, at about 50 mg / kg per day for the body weight of the subject. In a preferred embodiment, the amount of the first component is at about 100 - 200 mg / m per day 2 preferably 125 - 175 mg / m 2 preferably 140 - 160 mg / m 2 more preferably 150 mg / m 2 per day, and is preferably administered 2 times a week, preferably intravenously.

[0174] Suitable dosage levels may be approximately 0.01–250 mg / kg per day, approximately 0.05–100 mg / kg per day, or approximately 0.1–50 mg / kg per day. Within this range, the dosage may be 0.05–0.5, 0.5–5, or 5–50 mg / kg per day. For oral administration, the composition is provided in the form of tablets containing, preferably, 1.0 to 1000 milligrams of the active ingredient, specifically 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 milligrams of the active ingredient, for symptomatic adjustment of the dosage for the patient being treated. The compound may be administered 1 to 4 times per day, preferably in a regimen of once or twice per day.

[0175] In one embodiment, the combination or composition may be administered once, twice, three, four, five, six, or seven times a week. In another embodiment, the combination or composition may be administered once a week. In yet another embodiment, the combination or composition may be administered twice a week. In yet another embodiment, the combination or composition may be administered four times a week. In yet another preferred embodiment, the first component of the combination or composition is administered four times a week, and the second component of the combination or composition is administered once a week. In yet another embodiment, the first component of the combination or composition is administered twice a week, and the second component of the combination or composition is administered once a week. Both compounds may be administered simultaneously or sequentially. If the compounds are administered sequentially, administration of the first compound is discontinued before initiating administration of the second compound.

[0176] The duration of treatment may 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.

[0177] The amount of PARP inhibitor depends on the specific drug used and may be approximately 0.01 mg / kg to approximately 200 mg / kg per day, preferably 0.01 mg / kg to approximately 150 mg / kg, more preferably 0.01 mg / kg to approximately 100 mg / kg, preferably 0.01 mg / kg to approximately 75 mg / kg, 0.01 mg / kg to approximately 50 mg / kg, more preferably 0.5 mg / kg to approximately 50 mg / kg, preferably approximately 1 mg / kg to approximately 25 mg / kg, once or more per day, based on the subject's body weight, in order to obtain the desired therapeutic effect. In a preferred embodiment, the amount of PARP inhibitor may be approximately 2.5 mg / kg per day, or 7.5 mg / m² per day, preferably administered once a week, more preferably orally. 2 In a preferred embodiment, the amount of the PARP inhibitor is preferably administered once a week, more preferably orally, at a dose of about 0.5 mg / kg or 1.5 mg / m² per day based on the subject's body weight. 2 In a preferred embodiment, the amount of the PARP inhibitor is preferably administered once a week, more preferably orally, at a dose of about 5 mg / kg or 15 mg / m² per day based on the subject's body weight. 2 In another preferred embodiment, the amount of the PARP inhibitor is preferably administered once a week, more preferably orally, at a dose of about 10 mg / kg or 30 mg / m² per day based on the subject's body weight. 2 In another preferred embodiment, the amount of the PARP inhibitor is preferably administered once a week, more preferably orally, at a rate of about 50 mg / kg or 150 mg / m² per day based on the subject's body weight. 2In another preferred embodiment, the amount of the PARP inhibitor is preferably administered once a week, more preferably orally, at a rate of about 100 mg / kg or 300 mg / m² per day based on the subject's body weight. 2 The PARP inhibitor is preferably administered 6 days a week, preferably for 4 weeks.

[0178] A pharmaceutical composition according to the present invention, comprising a polypeptide containing SEQ ID NO: 1 described in the present invention, a functionally equivalent variant thereof, a fusion protein, a conjugate, a polynucleotide, a vector, or a cell, a first component (i) selected from these, and a second component (ii) which is a PARP inhibitor, may be prepared as a single formulation (for example, as a tablet or capsule containing one of each component in a fixed amount), or on the other hand, as separate formulations to be later combined for joint, sequential, or separate administration. The compositions of the present invention also include formulations as kits of parts, where the components are formulated separately but packaged in the same container. Those skilled in the art will recognize that the formulations of the different components in the pharmaceutical composition according to the present invention may be similar, in other words, they may be formulated similarly (into tablets or pills), which would allow for their administration via the same route. In cases where the different components of the present invention are formulated separately, the two components may be present in a blister pack. Each blister pack contains the drug to be taken during the day. If a drug needs to be administered several times a day, the drugs corresponding to each administration can be placed in different sections of the blister, preferably with the number of times they should be administered recorded in each section of the blister. Alternatively, the components of the composition of the present invention can be formulated differently so that different components are administered differently. For example, a first component may be formulated for its intravenous administration, and a second component may be formulated as a tablet or capsule for its oral administration, or vice versa. The ratios between the components that make up the combination or pharmaceutical composition described in the present invention can be adjusted by those skilled in the art depending on the antitumor agent used in each specific case and the desired indication. For example, the present invention envisions a composition in which the ratio between the amounts of component (i) and component (ii) may be in the range of 50:1 to 1:50, in particular 40:1 to 1:40, in particular 30:1 to 1:30, in particular 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 quantities is in the range of 1:1 to 1:5, preferably 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 quantities 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 quantities 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 w / w ratios. In a preferred embodiment, these ratios are Omomyc:olaparib ratios. These ratios are validated to treat any type of cancer, but more preferably, these ratios are obtained when treating cancers selected from the group consisting of triple-negative breast cancer and pancreatic ductal adenocarcinoma.

[0179] The present invention relates to a ratio between the amounts of component (i) and component (ii), more preferably the ratio of Omomyc:thalazoparib, such that 900,000:1~1:900,000, particularly 800,000:1~1:800,000, particularly 700,000:1~1:700,000, particularly 600,000:1~1:600,000, particularly 500,000:1~1:500,000, particularly 400,000: 1~1:400,000, especially, 300,000:1~1:300,000, especially, 200,000:1~1:200,000, especially, 150,000:1~1:150,000, especially, 100,000:1~1:100,000, especially, 75,000:1~1:75,000, especially, 50,000:1~1:50,000, especially, 30,000:1~1:30,000, especially, 25,000:1~ 1:25,000, especially 15,000:1~1:15,000, especially 10,000:1~1:10,000, especially 5,000:1~1:5,000, especially 3,500:1~1:3,500, especially 2,000:1~1:2,000, especially 1,800:1~1:1,800, especially 1,600:1~1:1,600, especially 1,500:1~1;1,500, especially 1,000:1~1:1 Compositions that may fall within the range of ,000, particularly 850:1~1:850; particularly 800:1~1:800; particularly 500:1~1:500; particularly 300:1~1:300; particularly 200:1~1:200; particularly 150:1~1:150; particularly 100:1~1:100; particularly 90:1~1:90; particularly 75:1~1:75; particularly 60:1~1:60; and particularly 55:1~1:55 are also assumed.

[0180] Exceptionally good results were obtained when the ratio of component (i):component (ii), more preferably Omomyc:talazoparib, was 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, and preferably 200:1 to 100,000:1. These ratios are validated for treating any type of cancer, but more preferably these ratios are obtained when treating triple-negative breast cancer.

[0181] Preferably, these ratios are w / w ratios.

[0182] The components of the pharmaceutical composition or combination of the present invention can be administered simultaneously. "Simultaneous administration" includes the co-administration of two therapeutic agents, regardless of the relative frequency or timing of administration of the individual agents. Therefore, simultaneous administration includes the co-administration of two therapeutic agents at the same time and with the same frequency of administration. In addition, simultaneous administration refers to the co-administration of two therapeutic agents in which one agent is administered more frequently than the other. In addition, simultaneous administration refers to the co-administration of two therapeutic agents in which one agent is administered only once between the administrations of the other agent.

[0183] 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, specifically intraperitoneally or intravenously.

[0184] In a preferred embodiment, component (i) of the combination or pharmaceutical composition of the present 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 present invention, preferably the polypeptide or a functionally equivalent variant thereof, fusion protein, or conjugate, is in the range of 0.01 to 250 mg / kg, which can be administered as a single or multiple dose, more preferably at 0.1 to about 100 mg / kg per day. For oral administration, the preferred dose of the PARP inhibitor is 0.01 to 200 mg / kg, preferably 0.01 to 150 mg / kg, more preferably 0.1 to 100 mg / kg, more preferably 0.5 to 100 mg / kg, even more preferably 10 to 100 mg / kg, and most preferably 50 to 100 mg / kg. Preferably, the PARP inhibitor is administered 6 days a week for 4 weeks.

[0185] In another embodiment, components (i) and (ii) of the combination or pharmaceutical composition of the present invention are administered intravenously.

[0186] The pharmaceutical compositions of the present invention may also contain one or more additional compounds for the prevention and / or treatment of conditions in which uncontrolled cell division is present, such as cancer. The additional compounds, for example, antitumor agents, may form part of the pharmaceutical composition as independent entities. In preferred embodiments, the combination or pharmaceutical composition of the present invention comprises one or more antitumor agents selected from the group consisting of cytotoxic agents, anti-angiogenic agents, anti-metastatic agents and anti-proliferative agents.

[0187] The pharmaceutical compositions of the present invention also contain one or more additional pharmaceutically acceptable excipients. A “pharmaceutically acceptable excipient” is understood to be a therapeutically inert substance that is used to incorporate the active ingredient, is acceptable to the patient from a pharmacological / toxicological standpoint, and is acceptable to the pharmaceutical chemist who manufactures it from a physical / chemical standpoint with respect to composition, formulation, stability, patient tolerance, and bioavailability. An excipient may be a carrier. As used herein, “carrier” means any substance that serves to improve the delivery and efficacy of the active ingredient in the pharmaceutical composition. In a preferred embodiment, the carrier does not allow direct delivery of components (i) and / or (ii) to the cytoplasm of a cell; i.e., the carrier cannot fuse with the plasma membrane of the target cell. Examples of pharmaceutically acceptable carriers include one or more of the following: water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and combinations thereof. In many cases, it is preferable that the combination or composition contains an isotonic agent, such as a sugar, a polyhydric alcohol, such as mannitol, sorbitol, or sodium chloride. A pharmaceutically acceptable carrier may further contain small amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the components forming part of the combination or composition of the present invention. Examples of suitable carriers are well known in the literature (see, for example, Remington's Pharmaceutical Sciences, 19th edition, Mack Publishing Company, Easton, PA, 1995). Examples of carriers include, but are not limited to, a range of saccharides, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; a range of starches, e.g., corn starch, wheat starch, rice starch, and potato starch; a range of celluloses, e.g., cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropyl methylcellulose; and a range of fillers, e.g., gelatin and polyvinylpyrrolidone.In some cases, a disintegrant, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate, may be added.

[0188] The number and characteristics of pharmaceutically acceptable excipients depend on the desired dosage form. Pharmaceutically acceptable excipients are known to those skilled in the art (Fauli y Trillo C. (1993) "Tratado de Pharmaacia Galenica", Luzan 5, SAEdiciones, Madrid). The compositions can be prepared by conventional methods known in the prior art ("Remington: Science and Practice of Pharmacy", 20th edition (2003) edited by Genaro AR, Lippincott Williams & Wilkins, Philadelphia, US).

[0189] With regard to pharmaceutical compositions containing a drug that is a nucleic acid molecule, the nucleic acid molecule may be present in any of the various delivery systems known to those skilled in the art, including nucleic acids and bacterial, viral, and mammalian expression systems, such as recombinant expression constructs provided herein. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. The DNA may also be “naked,” as described, for example, in Ulmer et al., Science 259:1745-49, 1993 and outlined in Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA can be increased by coating the DNA onto biodegradable beads, which are efficiently transported to cells.

[0190] Nucleic acid molecules may be delivered to cells according to one of several methods described in the Art (e.g., Akhtar et al., Trends Cell Bio. 2:139 (1992); Delivery Strategies for Antisense Oligonucleotide Therapeutics, edited by Akhtar, 1995; Maurer et al., Mol. Membr. Bio. 16:129-40 (1999); Hofland and Huang, Handb. Exp. Pharmacol. 137:165-92 (1999); Lee et al., ACS Symp. Ser. 752:184-92 (2000); U.S. Patent No. 6,395,713; International Patent Application Publication No. WO94 / 02595; Selbo et al., Int. J. Cancer 87:853-59 (2000); Selbo et al., Tumour Biol. 23:103-12 (2002); see U.S. Patent Application Publications 2001 / 0007666 and 2003 / 077829). Such delivery methods known to those skilled in the art include, but are not limited to, iontophoresis, or encapsulation in liposomes by other vehicles, such as biodegradable polymers; hydrogels; cyclodextrins (see, e.g., Gonzalez et al., Bioconjug. Chem. 10:1068-74 (1999); Wang et al., International Patent Publications WO03 / 47518 and WO03 / 46185); poly(lactic acid-coglycolic acid) (PLGA) and PLCA microspheres (also useful for the delivery of peptides and polypeptides and other substances) (see, e.g., U.S. Patent No. 6,447,796; U.S. Patent Publication No. 2002 / 130430); biodegradable nanocapsules; and bioadhesive microspheres, or by protein vectors (International Patent Publication No. WO00 / 53722).In another embodiment, nucleic acid molecules 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-triGAL) derivatives (see, for example, U.S. Patent Application Publication 2003 / 0077829).

[0191] In specific embodiments, if the composition or combination described in the present invention contains nucleic acids (DNA, RNA, siRNA, antisense oligonucleotides, ribozymes, aptamers, and Spiegelmers), the pharmaceutical composition may be formulated as a composition intended for use in gene therapy, and, as an example, but not limited to, the pharmaceutical composition may contain a viral or nonviral vector containing a suitable polynucleotide or gene construct. As an example, but not limited to, the vector may be, for example, a virus based on a retrovirus, an adenovirus, or a nonviral, such as an ADN-liposome, an ADN-polymer, or an ADN-polymer-liposome complex [see "Nonviral Vectors for Gene Therapy," edited by Huang, Hung, and Wagner, Academic Press (1999)]. The vector containing the corresponding polynucleotide or gene construct may be administered directly to a subject by conventional methods. Alternatively, the vector may be used ex vivo to transform, transfect, or infect cells, such as mammalian cells including human cells, which are then implanted in the human body or animals to obtain the desired therapeutic effect. For administration to the human body or animals, the cells are formulated in a suitable culture medium that does not have a detrimental effect on cell viability.

[0192] The combination or pharmaceutical composition of the present invention may be administered by any preferred route, for example, by oral, topical, inhalation, or parenteral route, such as by oral, topical, inhalation, or parenteral route, so as to contain pharmaceutically acceptable excipients necessary for the formulation of the desired dosage form. Other routes of administration may be rectal, intracisional, or vaginal. The preferred route of administration of the combination or pharmaceutical composition is within the range of intravenous administration. Alternatively, component (i) may be administered intravenously and component (ii) may be administered orally.

[0193] The “oral route” is understood as the incorporation of a pharmaceutical composition into a living organism after ingestion. In specific embodiments, the pharmaceutical compositions of the present invention may be in dosage forms suitable for their administration by oral route, whether solid or liquid. Dosage forms suitable for their administration by oral route may be tablets, capsules, syrups or liquids, and may contain any conventional excipients known in the art, e.g., binders, e.g., syrup, acacia, gelatin, sorbitol or polyvinylpyrrolidone; fillers, e.g., lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; lubricants for compression, e.g., magnesium stearate; disintegrants, e.g., starch, polyvinylpyrrolidone, sodium starch glycolate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents, e.g., sodium lauryl sulfate. Solid oral compositions can be prepared by conventional process means of mixing, filling or compressing. Repeated mixing operations can be used to completely distribute the active agent into those compositions using high amounts of fillers. The operations described above are conventional methods in the art. Tablets can be prepared, for example, by wet or cyclic granulation, and optionally by coating them with enteric coatings, following known processes in normal pharmaceutical practice.

[0194] On the other hand, “local routes” are understood as administration via non-systemic routes and include external application of the pharmaceutical compositions of the present invention, such as epidermal, oral, and dropwise injection of the compositions into the ears, eyes, and noses, where this 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, liquids, sprays, inhalations, or patches.

[0195] Ophthalmic formulations, ear drops, and eye drops are also intended to be within the scope of the present invention. In addition, the present invention intends to use transdermal patches, which have the additional advantage of providing controlled delivery of compounds to the body. Such dosage forms can be made by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flow of the compound across the skin. The rate can be controlled by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0196] In one embodiment, the combination or pharmaceutical composition is administered systemically.

[0197] "Systemic routes" are understood as administration via oral, intravenous, intraperitoneal, and intramuscular routes. The amounts of components (i) and (ii) required for the therapeutic or prophylactic effect will naturally vary depending on the selected compound, the characteristics and severity of the disease to be treated, and the patient. Preferably, the combination or pharmaceutical composition is administered orally.

[0198] In another embodiment, the combination or pharmaceutical composition is administered intranasally. In a preferred embodiment, intranasal administration is performed by drip infusion or nasal inhalation.

[0199] "Inhalation" is understood as administration via the intranasal route and oral inhalation. Dosage forms suitable for such administration, such as aerosol formulations or metered inhalers, can be prepared by conventional techniques. In some embodiments, the route of administration is the intranasal route.

[0200] As used herein, the term "parenteral" includes administration via intravenous, intraperitoneal, intramuscular, or subcutaneous routes. Subcutaneous, intramuscular, and intravenous dosage forms for parenteral administration are generally preferred. In some embodiments, the combination or pharmaceutical composition is administered intravenously.

[0201] In one embodiment, the combinations or pharmaceutical compositions of the present invention can be adapted for parenteral administration, for example, as sterile solutions, suspensions, or lyophilized products in appropriate dosage unit forms. Combinations or pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if they dissolve in water) or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or suspensions. With regard to its administration via intravenous route, some suitable carriers include phosphate-buffered saline (PBS). In all cases, the combinations or compositions must be sterile and fluid enough to be readily injectable. They must be stable in preparation and under storage conditions and protected from microbial contamination such as bacteria and fungi. The carriers can be solvents or dispersion media, which include, 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 using coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. The prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thiomersal. In most cases, it is preferable that the composition contains isotonic agents, such as sugars; polyhydric alcohols, such as mannitol, sorbitol; or sodium chloride. The extension of absorption of the injectable composition may be caused by the inclusion of absorption-delaying agents, such as aluminum and gelatin monostearate.

[0202] Sterile solutions for injection can be prepared by incorporating the required amount of the active compound with one or a combination of the aforementioned components into a suitable solvent, and, if necessary, subsequently by sterilization by filtration through a sterile membrane. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the remaining components as needed from those listed above. In the case of sterile powders for the preparation of sterile solutions for injection, preferred preparation processes are vacuum drying and freeze-drying, which yield a powder having the active component and any additional desired components from the previously filtered sterile solution.

[0203] The combinations or pharmaceutical compositions of the present invention can preferably be administered by pulse infusion, for example, using a gradually decreasing dose composition. Preferably, the dose is administered by injection, more preferably intravenous or subcutaneous injection, depending in part whether the administration is acute or chronic.

[0204] Alternatively, as mentioned above, different components of the composition are administered differently.

[0205] Therefore, in one embodiment, the component(i) of the combination or composition, preferably the polypeptide of the present invention or a functionally equivalent variant or conjugate, is administered intravenously, while the PARP inhibitor is administered orally.

[0206] In another embodiment, both components (i) and (ii) are administered intravenously.

[0207] In another embodiment, the combination or component (i) of the composition, preferably a polypeptide of the composition or a functionally equivalent variant thereof, or a conjugate, is administered intranasally or by inhalation.

[0208] The dosage form of a 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 a metered form. For example, a metered droplet / spray means that a dispenser containing a droplet / spray delivers a droplet / spray containing a metered dose (a predetermined amount) of the composition for use as described in the present invention.

[0209] One preferred dosage form in situations involving intranasal administration is a nasal spray. The droplet is mostly placed in the posterior part of the nose and thus rapidly removed to the nasal pharynx. A concern with using droplets is often how to precisely control the dose of the drug, which is particularly important for the administration of the composition.

[0210] Another intranasal dosage form in which the pharmaceutical composition of the present invention can be administered is a nasal spray. Nasal sprays typically contain a conjugate dissolved or suspended in a solution or mixture of excipients (e.g., preservatives, viscosity modifiers, emulsifiers, buffering agents) in a non-pressurized dispenser. Nasal sprays have several advantages, including the simplicity of the delivery device, convenience, ease of use, and accuracy of the delivery dose of 25 to 200 pL. They are placed in the anterior part of the nose and slowly cleared to the pharyngeal nasal portion by mucociliary clearance. Nasal sprays used herein may be liquids or suspensions.

[0211] Another intranasal dosage form is the nasal aerosol. Unlike nasal sprays, nasal aerosols differ in the method of dispensing the composition. In aerosols, the compound is dispensed due to excessive pressure and released through a valve. In sprays, the compound is dispensed due to forced extrusion by a micropump bucket, but the pressure in the vial is similar to atmospheric pressure. Aerosols offer similar advantages to sprays.

[0212] The compositions described in the present invention may, alternatively, be administered by means of a nasal emulsion, ointment, gel, paste, or cream. There are also highly viscous solutions or suspensions that are applied to the nasal mucosa.

[0213] Due to the limited volume of the composition that can be efficiently delivered to the nasal mucosa, liquid intranasal dosage forms typically have higher concentrations than their corresponding intravenous dosage forms. If a substance becomes poorly soluble or unstable in liquid form, the composition of the present invention can be administered using a powder. A further advantage of powders is that they do not require preservatives and generally have higher stability compared to liquid formulations. The main limitation to intranasal powder applications concerns their irritating effect on the nasal mucosa.

[0214] One dosage form for intrapulmonary administration is an inhalation aerosol. Inhalation aerosols are typically packaged under pressure and contain the composition described in the present invention, which is released into the airways, particularly the lungs, upon activation of a valve system. The released aerosol is a colloid (suspension) of fine solid particles or droplets (solution) in air or another gas. Therefore, the aerosol may be a solution or a suspension aerosol. The 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.

[0215] Another dosage form for intrapulmonary administration is the inhalation spray. Inhalation sprays are typically water-based and do not contain any aerosols. They deliver the conjugate to the lungs by oral inhalation.

[0216] Aerosol inhalation solutions and suspensions may also be used to deliver the conjugate via an intrapulmonary route. Aerosol inhalation solutions and suspensions are typically aqueous-based formulations containing the compositions described in the present invention. For systemic effects, the aerosol inhalation solutions and suspensions are used with a sprayer, delivered to the lungs by oral inhalation.

[0217] Dry powder inhalation is an alternative to aerosol inhalation. The composition is typically contained in a capsule for manual loading or in an inhaler. The dry powder is typically delivered to the lungs by an inhaler, usually by oral inhalation. The dry powder used herein can be formulated neat. Neat formulations contain, for example, the drug alone or a placebo alone as a spray dry powder. The dry powder used herein can also be formulated with a carrier such as lactose.

[0218] The intrapulmonary dosage form is preferably a metered form, meaning that a predetermined amount is delivered to the lungs.

[0219] Devices for intranasal delivery in the context of the present invention include spray pump systems, pipettes for delivering droplets, metered spray pumps, nasal pressurized metered inhalers, powder spray systems, respiratory-operated powder inhalers, and nasal powder inhalers. The intranasal delivery device may be filled with a single-dose or multi-dose intranasal formulation.

[0220] The conjugate may be administered via an intrapulmonary route using a medium-dose inhaler (MDI). MDIs generally provide a mist of the conjugate with an aerodynamic particle size of less than 5 pm.

[0221] Dry powder inhalers can be used as an alternative for delivering compositions into the lungs. Dry powder inhalers offer powder as a single dose or as a multi-dose powder.

[0222] Another device for intrapulmonary delivery is a nebulizer, including ultrasonic and air jet nebulizers. In an ultrasonic nebulizer, ultrasound is generated in the chamber of the ultrasonic nebulizer by a ceramic piezoelectric crystal that vibrates when electrically excited. This generates an aerosol cloud at the surface of the solution. Aerosols generated by an air jet nebulizer are produced when compressed air is forced through an orifice. The liquid may be discharged from a vertical nozzle (Bernoulli effect) and mixed with an air jet that is atomized using a baffle to facilitate the formation of an aerosol cloud.

[0223] In one embodiment, each component of the combination or pharmaceutical composition of the present invention, such as a controlled-release formulation including an implant and a microencapsulation administration system, is prepared using a carrier that protects the component, specifically component (i), from rapid elimination from the body. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid, can be used. The process for preparing the formulation will be apparent to those skilled in the art. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc.

[0224] The sustained-release compositions also include preparations of crystals suspended in a suitable formulation that can maintain the crystals in the suspension. These preparations may produce a sustained-release effect when 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, e.g., Epstein et al., Proc. Natl. Acad. Sci. USA, (1985) 82:3688-3692; Hwang et al., Proc. Natl. Acad. Sci. USA, (1980) 77:4030-4034; EP52,322; EP36,676; EP88,046; 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 liposome.

[0225] Despite the fact that Omomyc, its functionally equivalent variants, conjugates, and fusion proteins of the present invention can migrate across biological membranes, it is possible to formulate Omomyc, any of its functionally equivalent variants, conjugates, polynucleotides, vectors, or cells within nanoparticles. Nanoparticles can contribute to preserving the integrity of the components in biological fluids until they reach the target organ. Furthermore, in the case of compositions containing component (ii) or other antitumor agents, encapsulation of the composition may reduce the secondary effects caused by the antitumor agent. In addition, nanoparticles can also be modified to include a portion that enables the targeting of the nanoparticles to the organ of interest. In this way, component (i) of the combination or composition of the present invention is delivered proximal to the target organ, facilitating access of component (i) to the inside of cells where its biological activity is required.

[0226] Therefore, in another embodiment, component (i) of the combination or composition of the present invention is provided as part of a nanoparticle. In another embodiment, both components of the combination or composition of the present invention are provided as part of a nanoparticle, preferably both components are provided inside the same nanoparticle.

[0227] As used herein, the term “nanoparticles” refers to any material having dimensions in the range of 1 to 1,000 nm. In some embodiments, the nanoparticles have dimensions in the range of 2 to 200 nm, preferably 2 to 150 nm, and more preferably 2 to 100 nm. Nanoparticles that can be used in the present invention include nanoscale materials such as lipid-based nanoparticles, superparamagnetic nanoparticles, nanoshells, semiconductor nanocrystals, quantum dots, polymer-based nanoparticles, silicon-based nanoparticles, silica-based nanoparticles, metal-based nanoparticles, fullerenes, and nanotubes. Molecules may be embedded in a nanoparticle matrix or adsorbed on its surface, preferably the molecules are embedded in the nanoparticles.

[0228] In a preferred embodiment, the nanoparticles are liposomes.

[0229] Targeted delivery can be achieved by adding ligands without impairing the nanoparticles' ability to deliver their contents. This is intended to enable delivery to specific cells, tissues, and organs. The targeting specificity of ligand-based delivery systems is based on the distribution of ligand receptors on different cell types. Targeted ligands may associate with nanoparticles non-covalently or covalently and can be conjugated to nanoparticles by various methods discussed herein.

[0230] Examples of proteins or peptides that can be used to target nanoparticles include transferrin, lactoferrin, TGF-β, nerve growth factor, albumin, HIV Tat peptide, RGD peptide, and insulin.

[0231] It will be understood that the formulation of the present invention in nanoparticles is not intended, or not intended for, to facilitate access of components (i) and / or (ii) to the inside of a cell, but is intended, or not intended for, to protect components (i) and / or (ii) from degradation and / or to facilitate targeting of nanoparticles to an organ of interest.

[0232] In one example, the nanoparticles may be composed of a biodegradable polymer, such as poly(butylcyanoacrylate) (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 onto the nanoparticles. Other nanoparticles are composed of silica.

[0233] Nanoparticles can be formed from any useful polymer. Examples of polymers include biodegradable polymers, e.g., 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, e.g., poly(lactide-co-glycolide), poly(lactide)-poly(ethylene glycol), poly(poly(ethylene glycol)cyanoacrylate-cohexadecylcyanoacrylate), and poly[HEMA-co-methacrylic acid]; proteins, e.g., fibrinogen, collagen, gelatin, and elastin; and polysaccharides, e.g., amylopectin, amylose, and chitosan.

[0234] Other types of nanoparticles include solid lipid nanoparticles (SLNs). Examples of lipid molecules for solid lipid nanoparticles include stearic acid and modified stearic acid, e.g., stearic acid-PEG2000; soy lecithin; and emulsifying waxes. Solid lipid nanoparticles may optionally contain other components, including surfactants, e.g., Epicuron(R)200, poloxamer 188 (Pluronic(R)F68), Brij 72, Brij 78, polysorbate 80 (Tween 80); and salts, e.g., sodium taurocholate. Drugs can be introduced into solid lipid nanoparticles by many of the methods discussed for liposomes, such methods may further include high-pressure homogenization and dispersion of microemulsions.

[0235] Nanoparticles may also include micelles of nanometer size. Micelles can be formed from any polymer described herein. Exemplary polymers for forming micelles include block copolymers, e.g., poly(ethylene glycol) and poly(ε-caprolactone) (e.g., PEO-b-PCL block copolymers comprising polymers of ε-caprolactone and α-methoxy-ω-hydroxy-poly(ethylene glycol)).

[0236] In certain embodiments, the properties of the nanoparticles are modified by coating them with a surfactant. Any biocompatible surfactant may be used, for example, polysorbate surfactants, e.g., polysorbate 20, 40, 60, and 80 (Tween 80); Epicuron(R) 200; poloxamer surfactants, e.g., 188 (Pluronic(R) F68) poloxamer 908 and 1508; and Brij surfactants, e.g., Brij 72 and Brij 78.

[0237] Nanoparticles can optionally be modified to include hydrophilic polymer groups (e.g., poly(ethylene glycol) or poly(propylene glycol)) by, for example, covalently attaching hydrophilic polymer groups to their surface, or by using polymers containing such hydrophilic polymer groups (e.g., poly[methoxypoly(ethylene glycol)cyanoacrylate-co-hexadecylcyanoacrylate]). Nanoparticles can optionally be crosslinked, which may be particularly useful for protein-based nanoparticles.

[0238] In another embodiment, the pharmaceutical composition of the present invention is a nanoemulsion. When used herein, “nanoemulsion” means a colloidal dispersion of droplets (or particles) in which at least a portion of the droplets have a diameter in the range of nanometers. The nanoemulsion is composed of an omega-3, -6, or -9 fatty acid-rich oil in an aqueous phase, which is thermodynamically stabilized by an amphiphilic surfactant, which constitutes a surface film at the interface, and is typically produced using a high-shear microfluidization process with droplet diameters in the range of about 80–220 nm.

[0239] Therapeutic use of the present invention In one embodiment, the present invention relates to a combination or pharmaceutical composition of the present invention for use in pharmaceuticals.

[0240] In a further embodiment, the present invention relates to combinations or pharmaceutical compositions of the present invention for use in the prevention and / or treatment of cancer.

[0241] In another embodiment, the present invention refers to combinations or pharmaceutical compositions of the present invention for the preparation of pharmaceuticals for the prevention and / or treatment of cancer.

[0242] In another embodiment, the present invention also refers to a method for the prevention and / or treatment of cancer, comprising administering a therapeutically effective amount of the combination or pharmaceutical composition of the present invention to a subject in need thereof.

[0243] In preferred embodiments, the preventive or therapeutic methods described in the present invention include the direct use of a combination or composition comprising a polypeptide containing Omomyc, a functionally equivalent variant thereof, a conjugate, or a fusion protein. Therefore, in preferred embodiments, the preventive or therapeutic methods described in the present invention do not involve the administration of a nucleic acid encoding a polypeptide containing Omomyc or a functionally equivalent variant or fusion protein thereof, or the administration of a vector encoding said nucleic acid or cells containing said nucleic acid.

[0244] "Prevention" is understood as the administration of the combination or composition of the present invention to prevent the disease from occurring in its early stages or even from having an onset.

[0245] The term “treatment” is used to specify the administration of a combination or composition of the present invention to control disease progression before or after the appearance of clinical signs. Control of disease progression is understood as a beneficial or desired clinical outcome, including, but not limited to, reduction of symptoms, reduction of disease duration, stabilization of the pathological state (in detail, avoidance of additional functional impairment), delay of disease progression, improvement of the pathological state, and remission (both partial and complete). Control of disease progression also includes extension of survival compared to expected survival if treatment were not applied. In a preferred embodiment, control of disease progression is measured as the healthy lung / chest volume ratio. In another embodiment, control of disease progression is measured as a reduction in tumor volume. In yet another embodiment, control of disease progression is measured as a reduction in tumor cell viability.

[0246] The term "cancer" refers to a disease characterized by the ability of cells to invade other adjacent tissues through uncontrolled cell division (or increased survival or apoptosis resistance) (invasion), or by the spread of cells to other areas of the body where they are not normally located, via lymphatic vessels and blood vessels (metastasis). Depending on whether tumors can spread by invasion and metastasis, they are classified as either benign or malignant. Benign tumors are tumors that cannot spread by invasion or metastasis; that is, they grow only locally, while malignant tumors are tumors that can spread by invasion and metastasis. The methods of the present invention are useful for the treatment of localized and metastatic tumors.

[0247] Cancers include, in one embodiment, but not limited to, leukemia (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid 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 or Hodgkin's disease), AIDS-related leukemia, Waldenström hypergammaglobulinemia, multiple myeloma, heavy chain disease, and solid tumors, such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, mendotheliosarcoma, lymphangiosarcoma, intralymphatic sarcoma, synoviomas, mesothelioma, Ewing's disease). Tumors, 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 carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial cancer, renal cell carcinoma, hepatocellular carcinoma, bile duct cancer, teratoma, choriocarcinoma, seminomas, embryonic carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer These include lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, carcinomas in situ including Bowen's disease and Paget's disease, gliomas, astrocytomas, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, Schwann cell tumor, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0248] In some embodiments, the cancer is a glioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, Schwann cell tumor, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

[0249] In some embodiments, the cancer is an acoustic neuroma, astrocytoma (e.g., Grade I - pilocytic astrocytoma, Grade II - low-grade astrocytoma, Grade III - anaplastic astrocytoma, or Grade IV - glioblastoma (GBM)), chordoma, CNS lymphoma, craniopharyngioma, brainstem glioma, ependymoma, mixed glioma, optic glioma, subependymoma, medulloblastoma, meningioma, metastatic brain tumor, oligodendroglioma, pituitary tumor, primitive neuroectodermal (PNET) tumor, or Schwann cell tumor. In some embodiments, the cancer is a type more commonly found in children than in adults, e.g., brainstem glioma, craniopharyngioma, ependymoma, juvenile pilocytic astrocytoma (JPA), medulloblastoma, optic glioma, pineal tumor, primitive neuroectodermal (PNET) tumor, or rhabdoid tumor. In some embodiments, the patient is an adult human. In some embodiments, the patient is a child or a pediatric patient.

[0250] Cancers, in other embodiments, include, but are not limited to, mesothelioma, hepatobiliary (liver and bile duct) cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eyeball, ovarian cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, gastrointestinal (stomach, colorectal, and duodenum) cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, and parathyroid cancer. Cancers of the urethra, adrenal gland cancer, 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 pelvis carcinoma, non-Hodgkin lymphoma, spinal axial tumor, brainstem glioma, pituitary adenoma, adrenocortical carcinoma, gallbladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or any combination of one or more of the aforementioned cancers.

[0251] In some embodiments, cancer is selected from hepatocellular carcinoma, ovarian cancer, ovarian epithelial carcinoma, or fallopian tube cancer; papillary serous cystadenocarcinoma or papillary serous carcinoma of the uterus (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatobiliary carcinoma; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; undifferentiated thyroid cancer; adrenocortical adenoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / stomach (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); Waldenström hypergammaglobulinemia; or medulloblastoma.

[0252] In some embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial carcinoma, fallopian tube cancer, papillary serous cystadenocarcinoma, papillary serous carcinoma of the uterus (UPSC), hepatobiliary cholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, undifferentiated thyroid cancer, adrenocortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1-associated malignant peripheral nerve sheath tumor (MPNST), Waldenström hypergammaglobulinemia, or medulloblastoma.

[0253] In some embodiments, cancer is a solid tumor, such as a sarcoma, carcinoma, or lymphoma. A solid tumor generally consists of an abnormal mass of tissue that typically does not contain cystic or fluid areas. In some embodiments, cancer is 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 carcinoma, ovarian cancer, or fallopian tube cancer; papillary serous cystadenocarcinoma or papillary serous carcinoma of the uterus (UPSC); prostate cancer; testicular cancer; bile duct cancer. Cystic carcinoma; hepatobiliary carcinoma; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; undifferentiated thyroid carcinoma; adrenocortical carcinoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / stomach (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); Waldenström hypergammaglobulinemia; or medulloblastoma (selected from these).

[0254] In some embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial carcinoma, ovarian cancer, fallopian tube cancer, papillary serous cystadenocarcinoma, papillary serous carcinoma of the uterus (UPSC), hepatobiliary cholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, undifferentiated thyroid cancer, adrenocortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1-associated malignant peripheral nerve sheath tumor (MPNST), Waldenström hypergammaglobulinemia, or medulloblastoma.

[0255] In some embodiments, the cancer is hepatocellular carcinoma (HCC). In some embodiments, the cancer is hepatoblastoma. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is rectal cancer. In some embodiments, the cancer is ovarian cancer or ovarian carcinoma. In some embodiments, the cancer is ovarian epithelial carcinoma. In some embodiments, the cancer is fallopian tube cancer. In some embodiments, the cancer is papillary serous cystadenocarcinoma. In some embodiments, the cancer is papillary serous carcinoma of the uterus (UPSC). In some embodiments, the cancer is hepatobiliary cholangiocarcinoma. In some embodiments, the cancer is soft tissue and bone synovial sarcoma. In some embodiments, the cancer is rhabdomyosarcoma. In some embodiments, the cancer is osteosarcoma. In some embodiments, the cancer is undifferentiated thyroid cancer. In some embodiments, the cancer is adrenocortical carcinoma. In some embodiments, the cancer is pancreatic cancer or pancreatic ductal carcinoma. In some embodiments, the cancer is pancreatic adenocarcinoma. In some embodiments, the cancer is glioma. In some embodiments, the cancer is a malignant peripheral nerve sheath tumor (MPNST). In some embodiments, the cancer is neurofibromatosis-1-associated MPNST. In some embodiments, the cancer is Waldenström hypergammaglobulinemia. In some embodiments, the cancer is medulloblastoma.

[0256] In some embodiments, the cancer is a virus-associated cancer, such as human immunodeficiency virus (HIV)-associated solid tumors, human papillomavirus (HPV)-16-positive untreated solid tumors, and CD4+ cancers caused by human T-cell leukemia virus type 1 (HTLV-I) and characterized by the clonal incorporation of HTLV-I into leukemia cells. Adult T-cell leukemia (NCT02426892), a highly aggressive form of T-cell leukemia; as well as virus-associated tumors in gastric cancer, nasopharyngeal cancer, cervical cancer, vaginal cancer, vulvar cancer, squamous cell carcinoma of the head and neck, and Merkel cell carcinoma (see https: / / clinicaltrials.gov / ct2 / show / study / NCT02488759; also see https: / / clinicaltrials.gov / ct2 / show / study / NCT0240886; and https: / / clinicaltrials.gov / ct2 / show / NCT02426892).

[0257] Other cancers are known to those skilled in the art.

[0258] In a preferred embodiment, the cancer is selected from the group consisting of breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, lung cancer, colorectal cancer, gastric cancer, endometrial / uterine / cervical cancer, bladder cancer, head and neck cancer, leukemia, sarcoma, cholangiocarcinoma, glioblastoma, multiple myeloma, and lymphoma. More preferably, the cancer is selected from the list consisting of breast cancer, ovarian cancer, and prostate cancer, even more preferably breast cancer or ovarian cancer, and more preferably breast cancer.

[0259] In some embodiments, the cancer is melanoma.

[0260] In a preferred embodiment, the cancer is breast cancer. The term “breast cancer” most commonly refers to any malignant proliferative disorder of mammary cells originating from the inner wall of the milk duct or the lobules that supply milk to the milk ducts. Cancers originating from the ducts are known as tubular carcinomas, while those originating from the lobules are known as lobular carcinomas.

[0261] In a preferred embodiment, the cancer is triple-negative breast cancer (TNBC). The term triple-negative breast cancer refers to the fact that cancer cells are immunohistochemically defined 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 three tests. This is a highly malignant subtype of breast cancer, typically associated with relatively inferior clinical outcomes, early recurrence, and a high tendency to metastasize to visceral organs compared to other types of breast cancer. These cancers tend to be more common in women under 40 years of age who are Black or have a BRCA1 mutation. Therefore, in a more preferred embodiment, the cancer is a cancer with a BRCA1 mutation, preferably a TNBC with a BRCA1 mutation. In another embodiment, the cancer is a cancer with BRCA1 wild-type, preferably a TNBC with BRCA1 wild-type.

[0262] As used herein, "BRCA1" refers to early-onset breast cancer and is part of a complex that repairs double-strand breaks in DNA. BRCA1 acts in the same related DNA repair pathway as PARP1 / PARG. The sequence of the human BRCA1 protein corresponds to sequence P38398 in the Uniprot database (as of October 12, 2022, entry version 267).

[0263] In another embodiment, the cancer is a cancer having a BRCA2 mutation.

[0264] "BRCA2", as used herein, refers to breast cancer 2, early onset, and is part of a complex that repairs double-strand breaks in DNA. The sequence of the BRCA2 protein in humans corresponds to the sequence P51587 in the Uniprot database (as of October 12, 2022, version 234 of the entry).

[0265] When BRCA1 or BRCA2 itself is damaged by a BRCA mutation in breast-derived cells, the damaged DNA is not properly repaired, which increases the risk of breast and ovarian cancer.

[0266] More preferably, the cancer is a cancer having a mutation in BRCA1, a mutation in BRCA2, or mutations in both BRCA1 and BRCA2.

[0267] In another embodiment, the cancer is a cancer having wild-type BRCA1 and / or BRCA2.

[0268] In another preferred embodiment, the cancer is pancreatic cancer, specifically pancreatic ductal adenocarcinoma.

[0269] In another embodiment, the cancer is glioblastoma.

[0270] "Glioblastoma", also known as glioblastoma multiforme and grade IV astrocytoma, is the most common and most aggressive cancer that starts in the brain.

[0271] In another embodiment, the cancer is lung cancer.

[0272] The term "lung cancer" or "lung tumor" refers to a physiological state in mammals characterized by unregulated cell growth in lung tissue. The term lung cancer means any cancer of the lung, including 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).

[0273] When used herein, the term non-small cell lung cancer (NSCLC) refers to a group of heterogeneous diseases that are classified together because their prognosis and management are broadly identical, and includes the following according to the World Health Organization / International Society for Lung Cancer histological classification (Travis WD et al. Histological typing of lung and pleural tumours. 3rd edition Berlin: Springer-Verlag, 1999): (i) Squamous cell carcinoma (SCC), which accounts for 30% to 40% of NSCLCs, starts in the major respiratory tract but grows slowly, meaning that the size of these tumors varies at the time of diagnosis. (ii) Adenocarcinoma is the most common subtype of NSCLC, accounting for 50%–60% of NSCLCs. It begins near the gas exchange surface of the lung and includes the subtype bronchoalveolar carcinoma, which may have different responses to treatment. (iii) Large cell carcinoma is a rapidly growing form that grows near the surface of the lung. This is primarily a diagnosis of exclusion, and if a more detailed examination is performed, it is usually reclassified as squamous cell carcinoma or adenocarcinoma. (iv) Adenosquamous carcinoma is a type of cancer that contains two types of cells: squamous cells (thin, flat cells that line specific organs) and adenoid cells. (v) Pleomorphic, sarcomatous, or sarcomatous carcinomas. These are a group of rare tumors that reflect histological heterogeneity as well as a continuum in epithelial and mesenchymal differentiation. (vi) Carcinoid tumors are slow-growing neuroendocrine lung tumors that begin in cells capable of releasing hormones in response to stimuli provided by the nervous system. (vii) The type of salivary gland carcinoma begins in the salivary glands located inside the large airways of the lungs. (viii) Unclassified carcinomas include cancers that do not fit into any of the lung cancer categories mentioned above.

[0274] In specific embodiments, NSCLC is selected from squamous cell carcinoma of the lung, large cell carcinoma of the lung, and adenocarcinoma of the lung.

[0275] When used herein, the term small cell lung cancer (SCLC) refers to the proliferation of small cells with unique and rigid morphological characteristics, containing high density of neurosecretory granules that contribute to associated endocrine / paraneoplastic syndromes. Most cases occur in the larger airways (primary and secondary bronchi). These cancers grow rapidly and spread early in the course of the disease.

[0276] In a more preferred embodiment, the lung cancer is an adenocarcinoma, more preferably a KRas-driven lung adenocarcinoma, and more 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 position 12, glycine position 13, or glutamine position 61. In a more preferred embodiment, the mutation is selected from the group consisting of G12S mutation, G12V mutation, G12D mutation, G13D mutation, G12C mutation, G12R mutation, G12F mutation, G12I mutation, G13C mutation, G13R mutation, or 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 drive NSCLC.

[0277] In another preferred embodiment, the cancer treated according to the present invention is characterized by expressing increased levels of PARP, specifically PARP1 and / or PARP2. PARP levels, specifically PARP1 and / or PARP2 levels, are considered increased relative to a reference value if the levels of PARP, specifically PARP1 and / or PARP2 in a sample of the cancer show 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 higher. The reference value may correspond to the expression levels of PARP, specifically PARP1 and / or PARP2, in non-cancer samples.

[0278] In another embodiment, cancer is hormone-dependent cancer. Hormone-dependent cancer refers to cancer that is hormone-sensitive. Examples of such cancers, but are not limited to, breast cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, thyroid cancer, and osteosarcoma. In a specific embodiment, cancer is steroid-dependent cancer, more preferably estrogen and progestin-dependent cancer. In a more preferred embodiment, progestin-dependent cancer is progestin-dependent breast cancer. In another embodiment, cancer is androgen-dependent cancer, more preferably androgen-dependent prostate cancer.

[0279] In one embodiment, cancer is a primary tumor. The term “primary tumor,” as used herein, means a tumor that originates in the place or organ in which it is located and has not metastasized to that location from another place.

[0280] In another embodiment, cancer is metastatic cancer. In the context of the present invention, “metastasis” is understood as the propagation of cancer from the organ in which it originated to a different organ. This generally occurs through the blood or lymphatic system. When cancer cells spread and form a new tumor, the latter is called a secondary tumor or metastatic tumor. The cancer cells that form a secondary tumor are similar to those of the original tumor. If breast cancer spreads (metastasizes) to the lungs, for example, the secondary tumor is formed from malignant breast cancer cells. The disease in the lungs is metastatic breast cancer, not lung cancer. The inventors have also observed that combinations or compositions of the present invention can reduce cell proliferation, regardless of whether the cancer exhibits increased expression or activity of the Myc protein. In a preferred embodiment, the cancer to be prevented or treated is Myc-induced cancer.

[0281] In one embodiment, cancer is a solid tumor.

[0282] In another embodiment, the cancer is a cancer resistant to PARP inhibitors.

[0283] "Resistance" refers to a reduction in the effectiveness of a drug in treating a disease or condition. The term "cancer resistance," as used herein, refers to cancer that is resistant to a PARP inhibitor, either spontaneously or acquiredly. Spontaneous resistance occurs when a PARP inhibitor is ineffective from the start of treatment due to pre-existing resistance mechanisms. Acquired resistance occurs when a PARP inhibitor becomes ineffective during the course of treatment and after clinical benefits have been observed.

[0284] All combinations of compounds and types of cancer of the present invention are included in the present invention.

[0285] In some embodiments, the combination or composition of the present invention results in the arrest of tumor growth. In some embodiments, the combination or composition of the present invention results in a reduction of at least 5%, 10%, 25%, 50%, 75%, 90% or 99% in tumor size (e.g., volume or mass) compared to the tumor size before treatment. In some embodiments, the combination or composition of the present invention results in a reduction of at least 5%, 10%, 25%, 50%, 75%, 90% or 99% in the amount of tumor in a patient compared to the amount of tumor before treatment.

[0286] "Subject", as used herein, includes any animal that has cancer, or exhibits symptoms of cancer, or is at risk of having or exhibiting symptoms of cancer. Suitable subjects (patients) include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), livestock, and captive or pet animals (e.g., cats or dogs). Non-human primates, and preferably, human patients are included. Preferably, the subject is a mammal, most preferably, a human.

[0287] Combinations or compositions for use in the prevention and / or treatment of cancer may be administered using any amount and route of any dosage effective for treating cancer or reducing its severity. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease or condition, the specific drug, and the method of administration. The compounds of the present invention are preferably formulated in dosing unit form for ease of administration and uniformity of dosage. As used herein, the expression “dosing unit form” refers to a physically distinct unit of the drug appropriate for the patient being treated. However, it will be understood that the total daily dose of the compounds and compositions of the present invention will be determined by the attending physician within reasonable medical judgment. The specific effective dose level for any particular patient or organism will depend on a variety of factors, including the disorder and its severity being treated; the activity of the specific compound used; the specific composition used; the patient’s age, weight, overall health, sex, and diet; the number of doses, route of administration, and rate of excretion of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors well known in the medical field.

[0288] In a preferred embodiment, component (i) of the present invention, preferably a polypeptide or a functionally equivalent variant thereof, or a conjugate, synergistically interacts with a PARP inhibitor of the combination or composition when treating cancer (to achieve a therapeutic effect).

[0289] Specifically, in a more preferred embodiment, the combination or pharmaceutical composition for use in the prevention and / or treatment of cancer is a combination or pharmaceutical composition in which a polypeptide or a functionally equivalent variant thereof, or a conjugate, interacts synergistically with a PARP inhibitor in the treatment of cancer.

[0290] The terms "synergistic effect" and "synergistic interaction" are used interchangeably. A synergistic effect is greater than the additive effect predicted by summing the actual effects of individual drugs in vitro. In vivo, a synergistic effect is a physiological effect, specifically a therapeutic effect, that is greater than the additive effect predicted by summing the actual effects of individual drugs in vivo.

[0291] Therefore, when two drugs are administered, they together provide a measurable physiological effect, specifically a therapeutic effect, when the actual effects of the drugs together are greater than what would be predicted by summing the actual therapeutic effects of the individual drugs. Specifically, a synergistic effect is provided when the first drug alone provides some measurable effect, the second drug alone provides some measurable effect, and the two drugs together provide a measurable effect greater than the effect provided by the sum of both individual drugs. More specifically, a synergistic effect is provided when the first drug alone does not provide a measurable effect, the second drug alone provides some measurable effect, and the two drugs together provide a measurable effect greater than the effect provided by the second drug alone. Even more specifically, a synergistic effect is provided when neither the first drug alone nor the second drug alone provides any measurable effect, but the two drugs together provide a measurable effect. Since components (i) and (ii) act synergistically, the amounts of components (i) and / or (ii) in the combination or composition of the present invention may be less than those required in monotherapy using only one of them as a therapeutic agent. Preferably, in these combinations or compositions, a dosage of one or the other therapeutic agent between 0.01 and 1,000 μg / kg body weight / day can be administered.

[0292] The amount of therapeutic agent present in a combination or composition may be less than or equal to the amount typically administered in a composition containing that therapeutic agent as the sole active agent. Preferably, the amount of therapeutic agent in the composition is in the range of about 50% to 100% of the amount typically present in a composition containing that agent as the sole therapeutic active agent. In some embodiments, one therapeutic agent is administered at a dosage of about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the amount typically administered for that agent. As used herein, the phrase "typically administered" means the amount approved for administration of an FDA-approved therapeutic agent in accordance with the FDA label insert.

[0293] The combinations or compositions of the present invention may also be used in combination with known therapeutic processes, for example, chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, hormones, or combinations thereof.

[0294] All embodiments of the combinations of the present invention are also applicable to the therapeutic methods of the present invention.

[0295] Products and Kits This disclosure also provides products containing one of the combinations or pharmaceutical compositions disclosed herein in one or more containers. In some embodiments, the product includes, for example, a booklet, printed instructions, label, or accompanying document that instructs the user (e.g., a distributor or end-user) to combine and / or use the composition of the product for the prevention and / or treatment of cancer.

[0296] In some embodiments, the product includes, for example, bottles, vials, cartridges, boxes, syringes, injectors, or any combination thereof. In some embodiments, the label refers to the use or administration of the combination or pharmaceutical composition in the product in accordance with the methods disclosed herein. In some embodiments, the label suggests, for example, a regimen for use, a regimen for treating, preventing or relieving cancer.

[0297] The content of all cited references (including document references, patents, patent applications, and websites) that may be cited throughout this application is expressly incorporated herein by reference in their entirety for any purpose, just as the references cited therein are.

[0298] All terms used herein should be understood in their common sense, known in the art, unless otherwise stated. Further detailed definitions of specific terms used herein are set forth below and are intended to apply uniformly throughout this description and claims unless other expressly provided definitions provide broader definitions. Throughout this description and claims, the word “including” and variations thereof are not intended to exclude other technical features, additives, components, or processes. Furthermore, the word “including” also encompasses instances of “consisting of.” Additional objectives, advantages, and characteristics of the present invention may become apparent to those skilled in the art during the experiments described herein or may be learned through the practice of the present invention. Furthermore, the present invention encompasses the specific embodiments described herein and all possible combinations of those embodiments.

[0299] In this specification and the appended claims, the singular forms “a,” “an,” and “the” include multiple referents unless the context explicitly indicates otherwise. The terms “a” (or “an”), as well as the terms “one or more” and “at least one,” are interchangeable in this specification. Furthermore, “and / or,” when used in this specification, should be understood as a detailed disclosure of each of two specified characteristics or components, with or without the other. Thus, the term “and / or” as used in phrases such as “A and / or B” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to include each of the following embodiments: 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). The term “about” as used in conjunction with numerical values ​​throughout this specification and the claims represents an interval of precision that is familiar to and acceptable to those skilled in the art. Generally, such an interval of precision is ±15%. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in the field to which this disclosure relates. Units, prefixes, and symbols are expressed in their acceptable forms in the International System of Units (SI). Numerical ranges include the number defining the range. Unless otherwise indicated, amino acid sequences are written from left to right, in the direction of amino to carboxyl. The headings provided herein are not different aspects or limitations of this disclosure, which can be obtained by reference to this specification in whole. Thus, the terms defined immediately below are more fully defined in whole by reference to this specification.

[0300] The present invention is described by the following examples, which should be considered merely illustrative and not as limitations on the scope of the invention. [Examples]

[0301] Production and purification of Omomyc The Omomyc peptide sequence of Sequence ID No. 4, containing methionine at the N-terminus, was reverse transcribed, codon-optimized for expression in E. coli, cloned into a pET3a expression vector (Novagen), and purified from the BL21(DE3) arabinose-inducible (Invitrogen®) bacterial strain using a protocol adapted from the Max° purification protocol described by 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 resulting purified construct was the polypeptide of Sequence ID No. 4. The identity of each purified construct was confirmed by mass spectrometry and Western blotting. Omomyc was purified by cation exchange chromatography, and its purity was confirmed by mass spectrometry, SDS-PAGE, and UV spectroscopy.

[0302] Omomyc, when combined with a PARP inhibitor, acts synergistically in reducing the survival rate of triple-negative breast cancer cells. Triple-negative breast cancer (TNBC) cell lines MDA-MB-231, SUM149, and MX-1 were treated for 5 days with escalating concentrations of olaparib or talazoparib and the Omomyc peptide sequence of SEQ ID NO: 4, either alone or in combination.

[0303] Next, the synergy score was calculated using SynergyFinder.org after three biological replicates.

[0304] The synergy score can be interpreted as the mean overresponse resulting from a drug interaction. A synergy score close to 0 gives limited confidence in the synergy or antagonism. Therefore, if the synergy score is less than -10, the interaction between the two drugs may be antagonistic. Between -10 and 10, the interaction between the two drugs may be additive. Above 10, the interaction between the two drugs may be synergistic.

[0305] Figure 1A shows that treatment with Omomyc significantly reduces cell viability in MDA-MB-231 TNBC cells. Furthermore, the inventors discovered through microarray analysis that selected genes were significantly downregulated in MDA-MB-231 cells by treatment with Omomyc (data not shown). Specifically, treatment with Omomyc reduces the expression of homologous recombination (HR-) and BRCA1 / 2-deficiency-related genes and pathways. Treatment of these BRCA wild-type cells (MDA-MB-231) with Omomyc and the PARP inhibitor olaparib demonstrates that the combination of these drugs is synergistic. In particular, Figures 1B and 1C show that cell lines with BRCA mutations but resistance to PARP inhibitors (SUM149 and MX-1) also showed a synergistic response to the combination of olaparib and Omomyc.

[0306] The combination of Omomyc and talazoparib demonstrated synergistic effects in MDA-MB-231 cells (Figure 3A), as well as in MX-1 (BRCA1 / 2 mutant) and SUM149 (BRCA1 mutant) (Figures 3B and 3C). This confirmed that the synergistic output with both olaparib and talazoparib was independent of the cell line's mutation profile.

[0307] All the evidence presented above suggests that the combination of Omomyc and a PARP inhibitor is an effective therapy for treating both BRCA-mutated and wild-type tumors and potentially benefits the entire TNBC population. Importantly, Omomyc can reverse olaparib-driven resistance.

[0308] Omomyc, when combined with a PARP inhibitor, acts synergistically in reducing the survival rate of pancreatic ductal adenocarcinoma cells. Pancreatic ductal adenocarcinoma (PDAC) MIA-PACA-2 cells were treated for 5 days with escalating concentrations of olaparib and the Omomyc peptide sequence of Sequence ID No. 4, both alone and in combination.

[0309] Next, the synergy score was calculated using SynergyFinder.org after three biological replicates as described above.

[0310] Figure 2 shows that the PDDAC BRCA wild-type cell line MIA-PACA-2 also exhibits a potent synergistic effect with the combination of Omomyc and olaparib. This cell line appears to be particularly resistant to olaparib (first graph in Figure 2A), and Omomyc can reverse olaparib resistance in these cells.

[0311] Omomyc, when combined with a PARP inhibitor, acts synergistically in vivo. SUM149 cell-derived xenograft (CDX) model mice were used for in vivo studies. Each mouse was orthotopically inoculated with 5 million cells into the mammary fat pad. The tumor size was 100 mm. 3 Once the mice reached a certain stage, they were randomized into one of four treatment groups. Specifically, the mice were treated for four weeks with either a combination of two drugs (combo) – omomyc once weekly (50 mg / kg, intravenous), olaparib six times weekly (50 mg / kg, forced oral), or vehicle alone. Relative volume was calculated as the percentage of tumor growth from the day of randomization and the start of treatment. At the endpoint, the mean group percentages were 901.8% for vehicle, 663.6% for omomyc, 623.6% for olaparib, and 335.8% for the combination, respectively.

[0312] Preliminary in vivo results using SUM149 cell-derived xenotransplantation (CDX) confirmed that this synergistic effect can also be observed in mice. Indeed, four weeks after the introduction of the Omomyc + olaparib combination therapy, the mean growth reduction between vehicle and combination therapy was greater than the sum of the growth reductions between vehicle and monotherapy (Figure 4).

Claims

1. i) A first component selected from the group consisting of the following: a) A polypeptide containing the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) c) vectors containing the polynucleotides described above; and e) Cells capable of secreting the polypeptide described in a) or the conjugate described in b) into a culture medium, and ii) The second component is a PARP inhibitor. A combination that includes [something].

2. The combination according to claim 1, wherein the first component is a polypeptide containing the sequence of sequence number 1.

3. The combination according to claim 1, wherein a functionally equivalent variant of Sequence ID 1 is selected from the group consisting of Sequence ID 4, Sequence ID 5, Sequence ID 6, Sequence ID 7, Sequence ID 8, Sequence ID 9, and Sequence ID 10.

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

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

6. The combination according to any one of claims 1 to 5, wherein the PARP inhibitor is selected from the group consisting of olaparib, talazoparib, lucaparib, 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 described in 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 pharmaceuticals.

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

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

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

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

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

15. A combination or pharmaceutical composition for use 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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