Combination therapy using BRAF inhibitors for cancer treatment

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to the field of cancer, more specifically to a combination comprising a v-raf mouse sarcoma virus oncogene homolog B1 (BRAF) inhibitor and Omomyc, and to its use in medicine, more specifically in the prevention and / or treatment of cancer. [Background technology]

[0002] Cancer is the leading cause of death worldwide, accounting for nearly 10 million deaths in 2020. Cancer is a large subset of diseases characterized by the uncontrolled proliferation of abnormal cells.

[0003] BRAF is a well-known proto-oncogene that is mutated in some human cancers. The B-raf protein is a member of the Raf kinase family of growth signaling protein kinases, which play a role in regulating the mitogen-activated protein kinase (MAPK) signaling pathway, which is fundamental to cell proliferation, differentiation, and survival. Cancer generally results from dysregulation of this MAPK signaling pathway through somatic mutations that directly overactivate it.

[0004] More than 30 mutations in the BRAF gene associated with human cancer have been identified in solid tumors. The frequency of BRAF mutations varies widely in human cancers, from over 50% in melanoma to 5-10% in colorectal cancer, and is less common in ovarian and lung cancer. In 90% of cases, thymine is substituted for adenine at nucleotide 1799. As a result, valine (V) at codon 600 is substituted for glutamate (E) (called V600E). This mutation leads to constitutive activation of BRAF and the downstream MAPK signaling pathway. This constitutive activation results in increased cell proliferation and oncogenic activity.

[0005] Melanoma is an aggressive form of skin cancer arising from malignantly cancerous melanin-producing cells, and its incidence and mortality rates are increasing. The long-term survival rate for patients with metastatic melanoma is less than 10%, and the median overall survival is less than one year. This highlights the need to develop novel, effective, and more personalized treatments for patients with metastatic melanoma.

[0006] Drugs are being developed to treat cancers driven by BRAF mutations. Sorafenib was the first drug considered, but clinical trials failed to demonstrate any activity of sorafenib when used as monotherapy or in combination with dacarbazine in patients with wild-type and mutated BRAF metastatic melanoma. Moreover, sorafenib cannot selectively target mutated BRAF, resulting in unbearable off-target side effects. Two BRAF inhibitors, dabrafenib and vemurafenib, have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of end-stage melanoma. However, despite the efficacy of the drugs, 20% of tumors still develop resistance to the treatment, and the therapeutic efficacy of BRAF inhibitor therapy is limited due to the development of endogenous and acquired drug resistance. This clearly indicates the need for more effective combination therapies to overcome these limitations.

[0007] Therefore, there remains a need for cutting-edge technologies to develop novel and improved therapeutic approaches for cancer treatment that can overcome resistance to BRAF inhibitors, increase their efficacy, and / or reduce their adverse effects. [Overview of the project]

[0008] In the first embodiment, the present invention is i) The following group: a) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, b) A conjugate comprising a polypeptide containing the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof, and a chemical moiety that promotes intracellular uptake of the polypeptide or its functionally equivalent variant, c) Polynucleotides encoding the polypeptide of a) or the conjugate of b), d) A vector containing the polynucleotides of c), and e) Cells capable of secreting the polypeptide of a) or the conjugate of b) into the culture medium. A first component selected from, ii) The second component is a BRAF inhibitor and Regarding combinations that include [this].

[0009] 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.

[0010] In a third embodiment, the present invention relates to combinations or pharmaceutical compositions according to the present invention for use in pharmaceuticals.

[0011] In a fourth embodiment, the present invention relates to combinations or pharmaceutical compositions according to the present invention for use in the prevention and / or treatment of cancer. [Brief explanation of the drawing]

[0012] [Figure 1] Bar charts representing combinations of Omomyc and dabrafenib for melanoma cell lineage SkMel37 treated for 5 days with either Omomyc, dabrafenib, or a combination thereof at the indicated concentrations. Relative cell counts were estimated by the AlamarBlue survival test. [Figure 2] A bar chart showing the combinations between Omomyc and encorafenib for melanoma cell lineage SkMel37 treated for 5 days with either Omomyc, encorafenib, or a combination thereof at the indicated concentrations. Relative cell counts were estimated by the AlamarBlue survival test. [Figure 3] A bar chart representing the combination between Omomyc and vemurafenib for melanoma cell line SkMel37 treated for 5 days with any of the indicated concentrations of Omomyc, vemurafenib or their combination. Relative cell numbers were estimated by AlamarBlue viability assay. [Figure 4] A bar chart representing the combination between Omomyc and encorafenib for melanoma cell line SkMel28 treated for 5 days with any of the indicated concentrations of Omomyc, encorafenib or their combination. Relative cell numbers were estimated by AlamarBlue viability assay. [Figure 5] A bar chart representing the combination between Omomyc and vemurafenib for melanoma cell line SkMel28 treated for 5 days with any of the indicated concentrations of Omomyc, vemurafenib or their combination. Relative cell numbers were estimated by AlamarBlue viability assay.

Mode for Carrying Out the Invention

[0013] The present invention relates to the provision of novel therapeutic combinations for cancer prevention and treatment.

[0014] Unless otherwise specified, all technical terms used in this specification have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains.

[0015] All embodiments disclosed in connection with aspects of the present invention are applicable to other aspects as well.

[0016] Combinations and pharmaceutical compositions of the present invention The definitions provided in this aspect together with all other aspects of the present invention are equally applicable to the whole of the present invention.

[0017] The inventors have surprisingly found that combinations of Omomyc and BRAF inhibitors have a synergistic effect in the treatment of cancer. The inventors have shown that combinations of Omomyc with the BRAF inhibitors dabrafenib, encorafenib, or vemurafenib synergistically reduce the survival of melanoma cell lines SkMel37 (Figures 1-3) and SkMel28 (Figures 4-5). This synergistic effect is maintained regardless of dose, resulting in the beneficial effect of increasing the therapeutic effect of the composition of the present invention relative to each component, achieving the same results at lower doses of each component, thereby reducing side effects for subjects receiving the composition of the present invention.

[0018] Omomyc may also be useful in overcoming resistance to BRAF inhibitors by enhancing their sensitivity. The combination of this invention expands the population that can respond to BRAF inhibitor-based therapy.

[0019] Therefore, the combination of Omomyc with a BRAF inhibitor may be an effective therapy for treating both BRAF-mutated tumors and wild-type tumors, as well as for treating tumors resistant to BRAF inhibitors.

[0020] Therefore, in the first embodiment, the present invention is i) The following group: a) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, b) A conjugate comprising a polypeptide containing the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof, and a chemical moiety that promotes intracellular uptake of the polypeptide or its functionally equivalent variant, c) Polynucleotides encoding the polypeptide of a) or the conjugate of b), d) A vector containing the polynucleotides of c), and e) Cells capable of secreting the polypeptide of a) or the conjugate of b) into the culture medium. A first component selected from, ii) The second component is a BRAF inhibitor and Regarding combinations that include [this].

[0021] According to the present invention, the expression “combination” refers to various combinations of compounds (i) and (ii) in a composition formulated as a single formulation, a combined mixture composed of separate formulations of each component, such as a “tank mix” that can be combined for concomitant use as a combined preparation, and in a continuous manner, i.e., in the combined use of a single active ingredient when applied alternately over a moderately short period such as several hours or several days, or in simultaneous administration. In the present invention, compound (i) means a therapeutically effective amount of a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof, or a conjugate comprising a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes the intracellular uptake of the polypeptide or a functionally equivalent variant thereof, or a polynucleotide encoding the polypeptide or the conjugate, or a vector comprising the polynucleotide, or a cell capable of secreting the polypeptide or the conjugate into a culture medium. In the present invention, compound (ii) means a therapeutically effective amount of a BRAF inhibitor. Preferably, the order in which compounds (i) and (ii) are applied is not essential when dealing with the present invention.

[0022] The combination may be a kit-of-parts product in which each component is formulated or packaged individually.

[0023] The combination of compounds (i) and (ii) can be formulated for simultaneous, individual, or sequential administration. In particular, if administration is not simultaneous, the compounds are administered at close time proximity to each other. Furthermore, the compounds can be administered in the same or different forms of dosing, 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, compound (i) is administered intranasally and compound (ii) is administered orally. In yet another embodiment, compounds (i) and (ii) are administered intravenously.

[0024] The combination of the two compounds (i) and (ii) is: - As a combination of the same pharmaceutical product, the two compounds are usually administered simultaneously. - Each substance can be administered as a combination of two units, such that it can be administered simultaneously, sequentially, or individually with the other.

[0025] In certain embodiments, compound (i) of the combination of the present invention is administered independently of compound (ii), that is, in the two units, but simultaneously.

[0026] 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 individually or sequentially.

[0027] 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 individually or sequentially, as defined.

[0028] When administered individually, compounds (i) and (ii) of the combination of the present invention can be administered from each other within a certain period of time, for example, within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours from each other. In another embodiment, compounds (i) and (ii) of the combination of the present invention may be administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 days from each other, preferably within 1 day from each other, 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 a certain embodiment, administration of the first compound is discontinued before administration of the second compound is initiated.

[0029] In another embodiment, the present invention relates to a combination or pharmaceutical composition comprising a synergistic effective amount of a first component according to the first embodiment of the present invention and a BRAF inhibitor.

[0030] 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.

[0031] The terms “polypeptide” and “peptide” are used interchangeably herein to mean a macromolecule of amino acids of any length. The polypeptides of the present invention may contain modified amino acids, which may be interspersed with non-amino acids. In a preferred embodiment, the polypeptide is formed exclusively by 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 amino acids, even more preferably 80 to 130 amino acids, preferably 90 to 130 amino acids, preferably 125 or less amino acids, and more preferably 100 or less amino acids. In a preferred embodiment, the polypeptide has a length of 90 to 98 amino acids, more preferably 90 to 95 amino acids, and more preferably 91 amino acids.

[0032] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and mimics that function in a similar manner to naturally occurring amino acids. Furthermore, the term "amino acid" encompasses both D-amino acids and L-amino acids (stereoisomers). Preferably, the amino acids are L-amino acids.

[0033] The term “natural amino acids” or “naturally occurring amino acids” includes 20 naturally occurring amino acids; amino acids that are often modified post-translation in vivo, such as hydroxyproline, phosphoserine, and phosphothreonine; and other abnormal amino acids, such as, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine.

[0034] As used herein, the terms “unnatural amino acid” or “synthetic amino acid” mean a carboxylic acid or derivative thereof that is structurally related to a natural amino acid and is substituted with an amino group at position “a”. Exemplary and non-exclusive examples of modified amino acids or non-common amino acids include 2-aminoadipic acid, 3-aminoadipic acid, β-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, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyl-lysine, N-methylvaline, norvaline, norleucine, and ornithine.

[0035] The polypeptides of the present invention may include non-amino acid moieties attached to the peptide, such as hydrophobic moieties (various linear, branched, cyclic, polycyclic, or heterocyclic carbohydrates and carbohydrate derivatives); and 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.

[0036] Chemical (non-amino acid) groups present in the polypeptide may be added to improve various physiological properties, such as reduced degradation or clearance; reduced repulsion by various cell pumps; improved modes of administration; increased specificity; increased affinity; increased stability; bioavailability; reduced solubility; and reduced toxicity.

[0037] "Mimics" encompass molecules that mimic the chemical structure of a peptide structure while retaining the functional properties of that peptide structure. Approaches for designing peptide analogs, derivatives, and mimics are known in this field.

[0038] In one embodiment, the polypeptide of the present invention is a polypeptide comprising the sequence of SEQ ID NO: 1, or a polypeptide comprising a functionally equivalent modified version of SEQ ID NO: 1, preferably a polypeptide comprising the sequence of SEQ ID NO: 1.

[0039] Sequence ID 1 is, [ka] It corresponds to.

[0040] The polypeptide sequence of Sequence ID No. 1 corresponds to the Omomyc protein sequence. The term "Omomyc," as used herein, refers to a polypeptide consisting of mutant versions of the bHLHZip domain of Myc having the 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 accession number NP_002458 in the NCBI database, released March 15, 2015). The sequence of c-Myc provided in the NCBI database under accession number NP_002458 is shown below (Sequence ID No. 2), where the region from which Omomyc originates is underlined: [ka]

[0041] Omomyc also contains the M2 domain of c-Myc, which has the sequence RQRRNELKRSF (sequence number 3) (see Dang and Lee, Mol. Cell. Biol., 1988, 8:4048-4054) (double underlined above), and this corresponds to a nuclear localization signal.

[0042] Omomyc is characterized by its increased dimerization ability with all three oncogenic Myc proteins (c-Myc, N-Myc, and L-Myc). Omomyc can be derived from the bHLHZip domain of any Myc protein known in the art, insofar as mutations that result in tumor-suppressive effects are conserved. Therefore, Omomyc that can be used in the present invention may be derived from any mammal, for example, but not limited to, livestock and farm animals (cattle, horses, pigs, goats, sheep, dogs, cats, or rodents), primates, and humans. Preferably, the Omomyc protein may be derived from the human Myc protein (accession number NP_002458, released March 12, 2019).

[0043] 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 by binding to the consensus sequence CACGTG (which recruits the enhancer box sequence or E-box, as well as histone acetyl-transferase or HAT). However, Myc can also function as a transcriptional repressor. It inhibits the expression of Miz-1 target genes by binding to the Miz-1 transcription factor and replacing the p300 coactivator. Myc also plays a direct role in the regulation of DNA replication.

[0044] The Myc b-HLH-LZ or Myc basic region helix-loop-helix leucine zipper domain is 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 α-helices connected by a loop (Nair, SK, & Burley, SK, 2003, Cell, 112:193-205).

[0045] In a preferred embodiment, the polypeptide of the present invention is a polypeptide that contains, consists of, or substantially consists of SEQ ID NO: 4 as shown below. [ka]

[0046] In this context, "substantially derived from" means that the specified molecule does not contain any additional sequences that would alter the activation of Sequence ID No. 4.

[0047] Preferably, the polypeptide is sequence number 4.

[0048] The term "functionally equivalent variant" means any polypeptide resulting 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 any polypeptide resulting from the chemical modification of the polypeptide of SEQ ID NO: 1 and substantially preserving the tumor suppressor activity of SEQ ID NO: 1. Preferably, a functionally equivalent variant means any polypeptide resulting from the insertion or addition of one or more amino acids and / or the deletion of one or more amino acids, and / or any polypeptide resulting from the conservative substitution of one or more amino acids with respect to the polypeptide of SEQ ID NO: 1 and substantially preserving the tumor suppressor activity of SEQ ID NO: 1, more preferably any polypeptide resulting from the insertion or addition of one or more amino acids with respect to the polypeptide of SEQ ID NO: 1.

[0049] Those skilled in the art will understand that preserving tumor suppressor activity requires that the variant be able to dimerize with Myc and / or its essential partner p21 / p22Max, inhibit Myc activity, translocate across the cell membrane, and translocate across the nuclear envelope. In some embodiments, functionally equivalent variants of the polypeptide of the present invention are not as homodimerized as Omomyc, or are not forced into homodimerization by disulfide bond formation. In particular, disulfide bridge formation in the homodimerized form of certain embodiments of the polypeptide of the present invention is less pronounced in polypeptide Omomyc.

[0050] "Less homodimerization," as used herein, refers to a lower ability to form homodimers of the polypeptide of the present invention, even under reducing conditions. In preferred embodiments, this ability is less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, less than 85%, less than 90%, and less than 95% of the ability to form homodimers of Omomyc.

[0051] As used herein, reducing conditions relate to the presence of a reducing agent, i.e., a compound that donates electrons to another chemical species in a redox reaction. Exemplary, non-limiting examples of reducing agents are DTT (dithiothreitol), β-mercaptoethanol, or TCEP (tris(2-carboxyethyl)phosphine). It is possible that the amount of homodimers is the same in vitro, and that the difference between functionally equivalent variants and Omomyc exists only in cells in the presence of a heterodimerizing partner such that the absence of disulfide allows for a higher potential formation of heterodimers.

[0052] As an exemplary and non-limiting example, several assays may be used to identify the homodimerization of peptides by thermal denaturation monitored by circular dichroism, and thus dimerization may be detected by quantification of folding and thermal stability.

[0053] Preferred functionally equivalent variants include polypeptides substantially derived from the polypeptide of SEQ ID NO: 1. In this context, "substantially derived from" means that the specified molecule does not contain any additional sequences that would alter the activation of SEQ ID NO: 1.

[0054] In a preferred embodiment, a functionally equivalent variant of SEQ ID NO: 1 is a polypeptide resulting from the insertion or addition of one or more amino acids to the polypeptide of SEQ ID NO: 1. In a particular embodiment, the functionally equivalent variant is resulting from the insertion of fewer than 10 amino acids, more preferably fewer than 5 amino acids, and more preferably from the insertion of one amino acid. In a preferred embodiment, the functionally equivalent variant is resulting from the insertion of one amino acid, which is methionine.

[0055] In another embodiment, a functionally equivalent variant of SEQ ID NO: 1 is a polypeptide resulting from the deletion of one or more amino acids with respect to the polypeptide of SEQ ID NO: 1. In one embodiment, such a functionally equivalent variant results 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.

[0056] Preferred functional variants of the targeting peptide exhibit some degree of identity with respect to the peptide of SEQ ID NO: 1, such as amino acid sequence identity of more than approximately 25%, e.g., 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 the two polypeptides is determined using computer algorithms and methods widely known to those skilled in the art. Preferably, the identity between two amino acid sequences is identified by using the BLASTP algorithm described above (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, the sequence identity is identified over the entire length of the polypeptide of SEQ ID NO: 1, or over the entire length of the variant, or both.

[0057] 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.

[0058] In another embodiment, preferred functional variants of the targeting peptide include 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 mentioned above. A “conservative amino acid substitution” is achieved by substituting one amino acid with another amino acid having similar structure and / or chemical properties. For example, each of the following six groups includes amino acids that are conserved substitutions of each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W). The selection of such conservative amino acid substitutions is within the art of 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).

[0059] 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 human c-Myc-derived Omomyc. The positions where mutations must occur in such a functionally equivalent variant can be identified by multiple sequence alignments of various Myc sequences, as well as by alignments of positions 61, 68, 74, and 75 in the sequence of human c-Myc-derived Omomyc. In one embodiment, a functionally equivalent variant of Omomyc contains mutations at positions corresponding to the mutations E61T, E68I, R74Q, and R75N ​​found in human c-Myc-derived Omomyc.

[0060] In another embodiment, a functionally equivalent variant of Omomyc includes 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.

[0061] Multiple sequence alignment is an extension of pairwise alignment for incorporating more than two sequences at a time. Multiple alignment methods align all sequences in a given query set. 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). Once c-Myc sequences from different organisms or variants are compared (aligned) as described herein, a skilled technician can easily identify the positions within each sequence corresponding to the E61T, E68I, R74Q, and R75N ​​positions found in Omomyc, and can introduce mutations into Omomyc variants corresponding to the E61T, E68I, R74Q, and R75N ​​mutations found in human c-Myc-derived Omomyc.

[0062] Preferred assays for determining whether a polypeptide can be considered a functionally equivalent variant of Omomyc include, but are not limited to, the following: - Assays to measure the polypeptide's ability to form dimeric complexes with Max and Myc, such as those based on reporter gene expression as described in 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 the Myc / Max recognition site (CACGTG site) in DNA, such as electrophoretic mobility shift analysis (EMSA) described by Soucek et al. (see above), - Assays that measure the ability to suppress Myc-induced transactivation, such as assays based on the expression of a reporter gene under the control of a Myc / Max-specific DNA binding site, as described by Soucek et al. (see above). - Assays based on the ability of polypeptides to inhibit the proliferation of cells expressing the Myc tumor gene, as described by Soucek et al. (see above), - Assays measuring the ability of polypeptides to enhance Myc-induced apoptosis, e.g., the assay described by Soucek et al. (Oncogene, 1998:17, 2463 - 2472). Furthermore, any assay commonly known in the art for evaluating apoptosis in cells, e.g., Hoechst staining, propidium iodide (PI) or annexin V staining, trypan blue, DNA ladder / fragmentation, and TUNEL, etc. You can use it.

[0063] In a preferred embodiment, a polypeptide is considered a functionally equivalent variant of Omomyc if it exhibits at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the activity of the original Omomyc in one or more of the above assays.

[0064] In certain embodiments, functionally equivalent variants of the polypeptide of SEQ ID NO: 1 include the polypeptide of SEQ ID NO: 1 in which residue X at position 89 of SEQ ID NO: 1 is not cysteine. Preferably, residue X at position 89 of SEQ ID NO: 1 is an aliphatic amino acid, or a sulfured amino acid, or a dicarboxyl amino acid or its amide, 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, and preferably an amino acid selected from serine and alanine.

[0065] 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 table below.

[0066] [Table 1]

[0067] 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.

[0068] Furthermore, functionally equivalent variants of Omomyc can transduce cells after the variant has come into contact with them. It will be understood that functionally equivalent variants of Omomyc may contain a protein transduction domain found in natural Omomyc or another functional protein transduction domain.

[0069] In a preferred embodiment, a polypeptide is considered a functionally equivalent variant of SEQ ID NO: 1 if it can transduce target cells as efficiently as SEQ ID NO: 1 by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0070] Furthermore, functionally equivalent variants of SEQ ID NO: 1 can also translocate to the nucleus of target tumor cells.

[0071] In a preferred embodiment, a polypeptide is considered a functionally equivalent variant of SEQ ID NO: 1 if it can efficiently translocate to the nucleus of target tumor cells to the same extent as SEQ ID NO: 1, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0072] Regarding its ability to translocate across the cell membrane to the nucleus, a suitable assay for determining whether a polypeptide is a functionally equivalent variant of Sequence ID No. 1 is a dual-labeling method of cells using a reagent specific to the polypeptide and a dye that specifically labels the nucleus of the cell (e.g., DAPI or Hoechst dye). Detection of the polypeptide of the present invention can be performed by confocal microscopy or fluorescence microscopy.

[0073] In another preferred embodiment, compound (i) of the present invention is a conjugate comprising a polypeptide containing the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof, and a chemical moiety that promotes the intracellular uptake of the polypeptide or its functionally equivalent variant.

[0074] As used herein, the term "conjugate" means two or more compounds that are covalently bonded to each other in such a way that the function of each compound is maintained.

[0075] The term "chemical moiety" refers to any chemical compound that contains at least one carbon atom. Examples of chemical moieties, but not limited to these, include any peptide chain enriched with hydrophobic amino acids and hydrophobic chemical moieties.

[0076] In a preferred embodiment, the conjugate according to the present invention comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more chemical moieties that promote intracellular uptake of the polypeptide or a functionally equivalent variant of the polypeptide.

[0077] In one embodiment, the chemical portion that promotes intracellular uptake of the polypeptide is a lipid or a fatty acid.

[0078] 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 length of the carbon chain is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms, and any range that can be derived from these. In certain embodiments, the length of the carbon chain is 4 to 18 carbon atoms in the chain portion of the fatty acid. In certain embodiments, the carbon chain of the 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 the carbon chain are called saturated, while fatty acids that contain at least one double bond in the carbon chain are called unsaturated. The fatty acid may be branched, but in preferred embodiments of the present invention, it is unsaturated. Specific fatty acids, though not limited to these, include linoleic acid, oleic acid, palmitic acid, linolenic acid, stearic acid, lauric acid, myristic acid, arachidic acid, palmitoleic acid, and arachidonic acid.

[0079] In a preferred embodiment, the chemical portion that facilitates the intracellular uptake of the polypeptide containing the sequence of SEQ ID NO: 1 or a functionally equivalent variant is a cell membrane-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 and the cell membrane-permeable peptide sequence.

[0080] The term "fusion protein" refers to a protein produced by genetic technology, consisting of two or more functional domains derived from different proteins. Fusion proteins can be obtained by conventional methods, such as gene expression of the nucleotide sequence encoding the fusion protein in a suitable cell. It will be understood that the cell membrane permeable peptide in question is a cell membrane permeable peptide that is different from the cell membrane permeable peptide that forms part of the polypeptide containing SEQ ID NO: 1 or part of a functionally equivalent variant of SEQ ID NO: 1.

[0081] The term “cell membrane permeable peptide sequence” is used herein interchangeably with “CPP,” “protein transduction domain,” or “PTD.” It refers to peptide chains of varying lengths that direct the transport of proteins within a cell. While the intracellular delivery process is generally induced by endocytosis, the peptide can also be internalized 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 alternating patterns of polar amino acids / charged amino acids and nonpolar hydrophobic amino acids.

[0082] Examples of CPPs that can be used in the present invention are not limited to these, but include the CCP found in Drosophila antennapedia protein (RQIKIWFQNRRMKWKK, SEQ ID NO: 13), the CCP found in herpes simplex virus 1 (HSV-1) VP22 DNA-binding protein (DAATATRGRSAASRPTERPRAPARSASRPRRPVE, SEQ ID NO: 14), the CPP of Bac-7 (RRIRPRPPRLPRPRPRPLPFPRPG; SEQ ID NO: 15), 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), CPP of 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;CPP of SEQ ID NO: 33), CPP of MPG(ENLS)(ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-cya; SEQ ID NO: 34), CPP of Pep-1(ac-KETWWETWWTEWSQPKKKRKV-cya; SEQ ID NO: 35), CPP of Pep-2(ac-KETWFETWFTEWSQPKKKRKV-cya; SEQ ID NO: 36), polyarginine sequence having structure RN (where N is between 4 and 17), GRKKRRQRRR sequence (SEQ ID NO: 37), RRRRRRLR sequence (SEQ ID NO: 38), RRQ Examples include the RRTSKLMKR 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).

[0083] In a preferred embodiment, the cell membrane-permeable peptide is not the endogenous peptide included in SEQ ID NO: 1.

[0084] 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).

[0085] In some embodiments, the CPP is the CPP described in WO2019 / 018898, the contents of which are incorporated herein by reference in their entirety.

[0086] In one embodiment, the cell membrane-permeable peptide sequence is fused at the N-terminus of the polypeptide of the present invention or a functionally equivalent variant thereof. In another embodiment, the cell membrane-permeable peptide is fused at the C-terminus of the polypeptide of the present invention or a functionally equivalent variant thereof.

[0087] In a preferred embodiment, the conjugate or fusion protein of the combination according to the present invention comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more additional cell membrane permeable peptides, in addition to its own cell membrane permeable peptide found in the polypeptide of SEQ ID NO: 1 or a functionally equivalent variant of said polypeptide.

[0088] A preferred fusion protein of the present invention is polypeptide Omomyc, as defined below. * TAT and Omomyc * Includes LZArg.

[0089] [Table 2]

[0090] Therefore, in a preferred embodiment, the fusion protein is a polypeptide selected from SEQ ID NOs: 11 and 12.

[0091] Suitable assays for determining whether a conjugate preserves Omomyc's ability to translocate to the cell membrane include, but are not limited to, assays that measure the conjugate's ability to transduce cells in culture. This assay is based on contacting the conjugate with cultured cells and detecting the presence of the conjugate in the intracellular location.

[0092] In another preferred embodiment, the conjugate of the combination of the present invention further includes an additional nuclear localization signal.

[0093] The term “nuclear localization signal” (NLS), as used herein, refers to an amino acid sequence of approximately 4 to 20 amino acid residues in length that helps direct 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).

[0094] 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 KRPAATKKAGQAKKKK (SEQ ID NO: 49).

[0095] In another preferred embodiment, the NLS may be 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.

[0096] Those skilled in the art will understand that it may be desirable for the conjugate of the present invention to further comprise one or more flexible peptides that connect a polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant to a cell membrane permeable peptide sequence and / or NLS. Therefore, in certain embodiments, the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant is directly conjugated to the cell membrane permeable peptide sequence. In another particular embodiment, the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant is conjugated to the cell membrane permeable peptide sequence by a flexible peptide. In one embodiment, the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant is directly conjugated to the NLS. In another embodiment, the polypeptide comprising SEQ ID NO: 1 or a functionally equivalent variant is conjugated to the NLS by a flexible peptide.

[0097] In certain embodiments, the polypeptide of the conjugate according to the present invention is directly ligated to a cell membrane-permeable peptide sequence and NLS.

[0098] In one embodiment, the NLS is one of the NLSs that appear endogenously in the Myc sequence, such as the M1 peptide (PAAKRVKLD, SEQ ID NO: 56) or the M2 peptide (RQRRNELKRSF, SEQ ID NO: 57).

[0099] In another embodiment, the additional NLS means 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.

[0100] In a preferred embodiment, the conjugate or fusion protein according to the present invention comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, 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.

[0101] In another specific embodiment, the polypeptide conjugate for use according to the present invention is linked to a cell membrane-permeable peptide sequence by a first flexible peptide linker and to the NLS by a second flexible peptide linker.

[0102] As used herein, the terms “flexible peptide,” “spacer peptide,” or “linker peptide” mean a peptide that covalently bonds to two proteins or moieties but is not part of either polypeptide and allows one to move relative to the other without causing any substantial adverse effect on the function of either protein or moiety. Thus, the flexible linker does not affect the tumor tracking activity of the polypeptide sequence, the cell membrane permeability activity of a cell membrane permeable peptide, or the nuclear localization ability of the NLS.

[0103] The flexible peptide contains at least 1 amino acid, at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 12 amino acids, at least 14 amino acids, at least 16 amino acids, at least 18 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, or about 100 amino acids. In some embodiments, the flexible peptide will allow one protein to move relative to another protein in order to increase the solubility of the other protein and / or to improve its activity. A suitable linker region is a polyglycine region, a GPRRRR sequence (SEQ ID NO: 58) consisting of a combination of glycine, proline, and alanine residues.

[0104] In certain embodiments, the conjugate according to the present invention includes a tag bound to the conjugate, or a tag bound to the C-terminal or N-terminal domain of the polypeptide or fusion protein or its variant. Generally, the tag is a peptide or amino acid sequence that can be used in the isolation or purification of the fusion protein. Thus, the tag can bind to one or more ligands, for example, one or more ligands of an affinity matrix such as a high-affinity chromatographic support or beads. An example of such a tag is a high-affinity nickel (Ni) 2+ ) or cobalt (Co 2+ This includes histidine tags (His tags or HT), such as a tag containing six histidine residues (His6 or H6), which can be bound to the column. The His tag has the desirable characteristic of being able to bind to its ligand under conditions that cause it to denature into most proteins and disrupt most protein-protein interactions. Therefore, it can be used to remove a decoy protein tagged with H6 after the disruption of a protein-protein interaction in which the decoy protein is participating.

[0105] Additional exemplary and non-limiting examples of tags useful for isolating or purifying polypeptides or their variants or fusion proteins containing a conjugate or SEQ ID NO: 1 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 K., Appl. Microbiol. Biotechnol. 2003) Examples include amino acid sequences such as 60:523-525), AHGHRP (sequence number 63), or PIHDHDHPHLVIHSGMTCXXC (sequence number 64), and β-galactosidase.

[0106] The tag can be used, if desired, for the isolation or purification of the fusion protein.

[0107] In another preferred embodiment, compound (i) of the present invention is a polynucleotide encoding the polypeptide or fusion protein disclosed above. In another 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 yet another embodiment, compound (i) of the present invention is a polynucleotide encoding a conjugate comprising a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes intracellular uptake of the polypeptide or its functionally equivalent variant, and 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 membrane permeable peptide sequence.

[0108] The terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably to mean polymerized forms of nucleotides of any length. Polynucleotides may include 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 molecules, double-stranded molecules, 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. Nucleic acid molecules of the present invention may include modified nucleic acid molecules in addition to native nucleic acid molecules. As used herein, mRNA means RNA that can be translated in a cell.

[0109] In a preferred embodiment, the polynucleotide of the present invention is mRNA.

[0110] mRNA can be chemically synthesized, obtained by in vitro transcription, or synthesized in vivo in target cells. The nucleotide sequences that form the polynucleotides encoding the conjugate or fusion proteins of the present invention reside in the same correct reading frame for their expression.

[0111] 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, a polypeptide consisting of a functionally equivalent variant of SEQ ID NO: 1, or a polypeptide consisting of SEQ ID NO: 4.

[0112] In another embodiment, component (i) of the combination of the present invention is a vector containing the polynucleotide of the present invention.

[0113] The term “vector,” as used herein, means a nucleic acid sequence comprising a sequence necessary to generate a polypeptide encoded by the polynucleotide of the present invention after transcription and translation in a cell. The sequence is operably ligated to an additional segment that provides self-replication in the host cell of interest. Preferably, the vector is defined as an expression vector comprising a region operably ligated to the nucleic acid of the present invention, in addition to the self-replication region in the host cell, and capable of enhancing the expression of the nucleic acid product according to the present invention. Vectors of the present invention can be obtained by techniques widely known in the art.

[0114] Examples of vectors, but not limited to these, include 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 producer cells. Suitable polynucleotide vectors of the present invention include expression vectors in prokaryotes, such as pUC18, pUC19, pBluescript and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCRl, RP4, phage and "shuttle" vectors, such as pSA3 and pAT28, and expression vectors in yeast, such as 2-micron plasmid type vectors and integration plasmids. Expression vectors in insect cells, such as plasmids, YEP vectors, kinetochore plasmids and the like, expression vectors in plants, such as the pAC series and pVL series vectors, such as the pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series vectors and the like, as well as excellent eukaryotic cell expression vectors based on viral vectors (adenovirus, adenovirus-associated viruses, etc.) The vectors are derived from retroviruses, particularly lentiviruses, as well as nonviral vectors, such as pSilencer4.1-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. In a preferred embodiment, 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.

[0115] The vectors of the present invention can be used to transform, transfect, or infect cells that can be transformed, transfected, or infected by the vectors. These cells may be prokaryotic or eukaryotic cells.

[0116] Preferably, the vector contains the polynucleotide of the present invention operationally bound to a sequence that modulates the expression of the polynucleotide of the present invention. The modulating sequence used in the present invention may be a nuclear promoter or other regulatory sequences that increase the expression of an enhancer sequence and / or a heterologous nucleic acid sequence. In principle, any promoter may be used in the present invention, provided that the promoter is compatible with the cell in which the polynucleotide is to be expressed. Therefore, suitable promoters for realizing the present invention are not necessarily limited to these, but include constituent promoters, such as derivatives of eukaryotic viral genomes, such as polyomavirus, adenovirus, SV40, CMV, aerosarcoma virus, and hepatitis B virus; metallothionein gene promoters; herpesvirus thymidine kinase gene promoters; retroviral LTR regions; immunoglobulin gene promoters; actin gene promoters; EF-1α gene promoters; and inducible promoters, such as tetracycline systems, NFκB / UV light systems, Cre / Lox systems, and heat shock gene promoters, as well as moduloable RNA polymerase II promoters described in WO2006 / 135436, and tissue-specific promoters, in which protein expression depends on the addition of molecules or exogenous signals.

[0117] 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 of the present invention, or the fusion protein of the present invention into a culture medium.

[0118] 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 cells and T cells), mast cells, eosinophils, vascular intima cells, primary cultures of isolated cells from different organs, preferably cells isolated from the islets of Langerhans, hepatocytes, leukocytes, such as mononuclear leukocytes, mesenchymal, umbilical cord, or adult (of skin, lung, kidney, and liver), osteoclasts, chondrocytes, and other connective tissue cells. Cells from established cell lines, such as Jurkat T cells, NIH-3T3, CHO, Cos, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 cells, C2C12 myoblasts, and W138 cells, are also suitable. Those skilled in the art will understand that cells capable of secreting the polypeptides of the present invention into culture media may be found to form microparticles or microcapsules, allowing the cells to live a more useful life in a patient. Suitable materials for forming the microparticle objects of the present invention include any biocompatible polymer material that enables the continuous secretion of therapeutic agents and functions as a support for the cells. Therefore, such biocompatible polymer material may be, for example, a thermoplastic polymer or a hydrogel polymer.Among thermoplastic polymers, the present inventors have identified acrylic acid, acrylamide, 2-aminoethyl methacrylate, poly(tetrafluoroethylene-cohexafluoropropylene), methacrylate-(7-coumaroxy)ethyl ester, N-isopropylacrylamide, polyacrylic acid, polyacrylamide, polyamidoamine, poly(amino)-p-xylylene, 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 The material comprises methacrylate, poly(ethylene terephthalate), poly(4-hydroxybutyl acrylate), poly(hydroxyethyl methacrylate), poly(N-2-hydroxypropyl methacrylate), poly(glycolic acid lactate), poly(L-lactic acid), poly(γ-methyl, L-glutamic acid), 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-malonamide, and copolymers containing two or more of these polymers. Among the hydrogel-type polymers, the present inventors have identified natural materials such as alginates, agarose, collagen, starch, hyaluronic acid, bovine serum albumin, cellulose and its derivatives, pectin, chondroitin sulfate, fibrin and fibroin, as well as synthetic hydrogels, such as Sepharose® and Sephadex®.

[0119] Compound (ii) of the combination of the present invention Compound (ii) of the present invention is a BRAF inhibitor.

[0120] "BRAF" (EC2.7.11.1), as used herein, refers to the serine / threonine protein kinase B-raf, also known as the oncogene B-Raf, p94, or v-raf mouse sarcoma virus oncogene homolog B1. BRAF is a member of the RAF family of serine / threonine kinases (A-RAF, B-RAF, and C-RAF), which is part of the mitogen-activated protein kinase (MAPK) signaling pathway. BRAF is the most potent activator of MEK kinase (a downstream effector of RAF). The recruitment of RAF in membrane-associated RAS in the upstream activation signal leads to a cascade of downstream events (ERK signaling) involving the phosphorylation of MEK1 and MEK2 by RAF. The sequence of the BRAF protein in humans corresponds to the sequence of P15056 in the UniProt database (version 254 of the entry as of May 3, 2023).

[0121] The gene encoding the BRAF protein is named B-Raf proto-oncogene serine / threonine kinase, BRAF, BRAF1, RAFB1, or B-raf. The human BRAF gene (gene ID: 673) is located on chromosome 7q34.

[0122] BRAF is frequently mutated in human cancers. Common activating mutations in the BRAF gene include BRAF wild-type amplification, BRAF deletion, and mutations in valine at position 600, arginine at position 462, isoleucine at position 463, glycine at position 464, glycine at position 466, serine at position 467, glycine at position 469, aspartic acid at position 594, glycine at position 596, leucine at position 597, threonine at position 599, or lysine at position 601. Preferably, the mutation is at valine at position 600. In a more preferred embodiment, the mutation is selected from the group consisting of V600E mutation, V600K mutation, V600D mutation, V600R mutation, R462I mutation, I463S mutation, G464E mutation, G466E mutation, G466V mutation, S467L mutation, G469A mutation, G469R mutation, G469V mutation, D594G mutation, D594N mutation, G596R mutation, L597R mutation, L597V mutation, K601E mutation, K601D mutation, and K601R mutation. In a preferred embodiment, the mutation is the V600E mutation.

[0123] "BRAF inhibitor," as used herein, refers to any compound that can cause a decrease in the activity of BRAF, in particular the activity of BRAF variants, such as compounds that prevent the expression of BRAF genes, especially BRAF variant genes, and compounds that result in a reduction in BRAF mRNA or protein levels. BRAF inhibitors primarily inhibit the catalytic activity of the BRAF enzyme.

[0124] Protein or nucleic acid expression is considered reduced if its level decreases 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 a reference value.

[0125] The reference value refers to the level of protein or nucleic acid in a control group, which may be individuals without a specific disease.

[0126] Preferred methods for determining whether an inhibitor can reduce BRAF mRNA levels include, but are not limited to, standard assays for identifying mRNA expression levels, such as qPCR, RT-PCR, RNA protection analysis, Northern blotting, RNA dot blotting, insight hybridization, and microarray techniques; tag-based methods, such as gene expression linkage analysis (SAGE) including modified forms like LongSAGE and SuperSAGE; microarrays; and fluorescence insight hybridization (FISH) including modified forms like Flow-FISH, qFiSH, and double-fusion FISH (D-FISH). Quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous.

[0127] 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 soluble enzymes or chemical solutions, or extracted with nucleic acid-binding resin according to the manufacturer's instructions for use.

[0128] When mRNA is measured in a biological sample, the biological sample may be treated to physically, mechanically, or chemically disintegrate the tissue or cellular structure, releasing intracellular components into an aqueous or organic solution to prepare nucleic acids for further analysis. Nucleic acids are extracted from the sample by commercially available procedures known to those skilled in the art. The RNA is then extracted by one of the methods typical in the art, e.g., Sambrook, J., et al., 2001. Molecular cloning: A Laboratory Manual, 3 rdExtraction is performed from frozen or fresh samples according to the Cold Spring Harbor Laboratory Press, NY, Vol. 1-3. Preferably, care is taken to avoid RNA degradation during the extraction process.

[0129] Expression levels can be determined using mRNA obtained from formalin-fixed and paraffin-embedded tissue samples. mRNA may be isolated from archived pathological or biopsy samples that 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 continuous washing with a decreasing concentration of lower alcohol solution. Alternatively, the sample is 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 certain embodiments, samples can be obtained from fresh tumor tissue or from OCT-embedded frozen tissue.

[0130] To normalize mRNA expression levels across 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-tumoric cells. Preferably, the control RNA is mRNA derived from a housekeeping gene, which is constitutively expressed and encodes a protein that performs an essential cellular function. 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.

[0131] Relative gene expression quantification can be calculated according to the comparative threshold cycle (Ct) method, using housekeeping genes as endogenous controls and commercially available RNA controls as standard materials. The final results are 2 -(ΔCt試料-ΔCt標準物質) The ΔCt values ​​are determined according to the following formula, where the ΔCt values ​​of the standard substance and the sample are determined by subtracting the Ct value of the target gene from the value of the control gene.

[0132] A preferred method for determining whether an inhibitor functions by reducing BRAF protein levels includes conventional quantification, for example, using an antibody that has the ability to specifically bind to the protein encoded by the BRAF gene (or a fragment thereof containing an antigenic determinant), and subsequent quantification of the resulting antibody-antigen complex.

[0133] 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. Exemplary but non-exclusive examples of markers that may be used include radioisotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, and colorants. A wide variety of well-known assays can be used in the present invention, which utilize unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies). These techniques include, among others, Western blotting or Western transcription, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzyme-linked immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of biochips or protein microarrays containing specific antibodies, or assays based on colloidal precipitation in formats such as dipsticks. Other methods for detecting and quantifying the level of the protein of interest include techniques such as affinity chromatography and binding ligand assays.

[0134] On the other hand, the level of BRAF protein can be identified by constructing a tissue microarray (TMA) containing the assembled target sample and by identifying the expression level of the corresponding protein using immunohistochemical testing 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. Discrepancies can be resolved by simultaneous re-evaluation. Briefly, the results of immunostaining can be recorded as negative expression (0) versus positive expression, as well as low expression (1+) versus moderate expression (2+) and high expression (3+), taking into account expression in tumor cells and specific cutoffs for each marker. As a general criterion, the cutoff is selected to facilitate reproducibility and, where possible, to translate biological events. Alternatively, the immunohistochemical intensity can be evaluated using imaging techniques and automated methods, such as those disclosed in Rojo, MG et al. (Folia Histochem. Cytobiol. 2009; 47: 349-54) or Mulrane, L. et al. (Expert Rev. Mol. Diagn. 2008; 8: 707-25).

[0135] Alternatively, in another specific embodiment, the level of BRAF protein is determined by Western blotting. Western blotting is based on the detection of proteins previously separated by gel electrophoresis under denaturing conditions and immobilized on a membrane, typically nitrocellulose, by incubation and development with a specific antibody (e.g., chemiluminescence).

[0136] BRAF inhibitors can inhibit BRAF 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 further 100%, and the entire range from 5% to 100%. A preferred method for determining whether an inhibitor functions by reducing BRAF activity is any method that allows for the detection of reduced phosphorylation of key downstream effectors of BRAF, such as MEK and ERK1 / 2. To determine whether a compound is a BRAF inhibitor, any method known in the current state of the art, such as the commercially available Millipore B-Raf kinase assay kit, which is based on the detection of MEK1 phosphorylation, can be used.Methods for detecting BRAF activity are known in the art and are not limited to these, but include active BRAF pull-down experiments, whole cell lysate immunoblotting, enzyme-linked immunosorbent assays (ELISA) for phosphorylated forms of MEK, ERK1, and ERK2 (Cope N. et al. 2018. Chembiochem, 19(18):1988-1997; Garbison KE, Heinz BA, Lajiness ME, authors; Weidner JR, Sittampalam GS, editors. Phospho-ERK assays. 2012 May 1 [Updated 2015 Jun 25]. In: Markossian S, Grossman A, Brimacombe K, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004), and luminescence-based immunoassays (Miyamoto K and Sawa (M. 2019. Scientific Reports, 9, Article number: 636) is an example. Assays for determining enzyme activity are known to those skilled in the art and include, but are not limited to, initial rate assays, reaction progress curve assays, transient kinetic assays, and relaxation assays. Sequential assays of enzyme activity include, but are not limited to, spectrophotometric assays, fluorescence assays, calorimetry assays, chemiluminescence assays, light scattering assays, and microscale thermopheresis assays. Discrete assays of enzyme activity include, but are not limited to, 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.

[0137] BRAF inhibitors can be any organic or inorganic molecule, including modified and non-modified nucleic acids, such as antisense nucleic acids, RNA interference (RNAi) agents, such as siRNA, shRNA, or miRNA, peptides, proteins, peptide mimes, receptors, ligands, antibodies, and small organic molecules, which are useful in the treatment of cancer.

[0138] When used herein, "minor chemical compounds" refer to molecules that modulate biological processes, in this case inhibiting the catalytic activity of BRAF. These compounds may be natural or artificial.

[0139] Exemplary BRAF inhibitors are disclosed in Agianian B. and Gavathiotis E. 2018. J Med Chem, 61:5775-5793.

[0140] In a preferred embodiment, a BRAF inhibitor useful in the present invention is selected from Table 1.

[0141] [Table 3]

[0142] In preferred embodiments, the BRAF inhibitor is selected from the group consisting of the compounds listed in item I of Table 1, or pharmaceutically acceptable salts thereof; more preferably, it is selected from the group consisting of SB-590885, GDC-0879, PLX-4720, vemurafenib, dabrafenib, encorafenib, XL281, BI882370, AZ-628, LY3009120, TAK-632, MLN2480, CCT-196969, CCT-241161, BGB659, BGB283, RAF709, RAF265, PLX7904, PLX8394, CEP-32496, berbarafenib, LXH254, and pharmaceutically acceptable salts thereof.

[0143] In another embodiment, the BRAF inhibitor is selected from the group consisting of sorafenib, SB-590885, GDC-0879, and PLX-4720; preferably, it is selected from the group consisting of SB-590885, GDC-0879, and PLX-4720.

[0144] In another embodiment, the BRAF inhibitor is selected from the group consisting of vemurafenib, dabrafenib, encorafenib, XL281, and BI882370.

[0145] In another embodiment, the BRAF inhibitor is selected from the group consisting of AZ-628, LY3009120, TAK-632, MLN2480, CCT-196969, CCT-241161, BGB659, BGB283, RAF709, RAF265, PLX7904, PLX8394, CEP-32496, verbarafenib, and LXH254.

[0146] In a preferred embodiment, the BRAF inhibitor is selected from the group consisting of vemurafenib, dabrafenib, encorafenib, PLX-4720, BI882370, PLX7904, and PLX8394.

[0147] In a preferred embodiment, the BRAF inhibitor is selected from the group consisting of dabrafenib, vemurafenib, encorafenib, sorafenib, GDC-0879, and PLX-4720; preferably, it is selected from the group consisting of dabrafenib, vemurafenib, encorafenib, GDC-0879, and PLX-4720. More preferably, the BRAF inhibitor is selected from the group consisting of dabrafenib, vemurafenib, and encorafenib; even more preferably, it is dabrafenib.

[0148] In another embodiment, the BRAF inhibitor is not sorafenib.

[0149] In a preferred embodiment, the BRAF inhibitor is an inhibitor that targets a mutation in BRAF, preferably BRAF V600E; more preferably, it is selected from the group consisting of sorafenib, GDC-0879, vemurafenib, dabrafenib, AZ-628, LY3009120, BGB283, and PLX7904.

[0150] All compounds listed in item I of Table 1 include their pharmaceutically acceptable salts, solvates, polymorphs, or cocrystals. Prodrugs of these compounds are also included.

[0151] The term "pharmaceutically acceptable" means a substance and / or properties that are acceptable to the patient from a pharmacological / toxicological standpoint, as well as to the pharmacist who manufactures it from a physical / chemical standpoint with respect to its composition, formulation, stability, patient tolerability, and bioavailability.

[0152] The term "pharmaceutically acceptable salt" encompasses salts with pharmaceutically acceptable acids or bases. Pharmaceutically acceptable acids include, but are not limited to, inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, diphosphate, hydrobromic acid, hydroiodic acid, and nitric acid, as well as, but are 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 p-toluenesulfonic acid. Pharmaceutically acceptable bases include hydroxides of alkali metals (e.g., sodium or potassium) and alkaline earth metals (e.g., calcium or magnesium), as well as organic bases such as, but are not limited to, alkylamines, arylalkylamines, and heterocyclic amines.

[0153] The term "solvate" 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 alkoxides, preferably C1-C6 alkoxides, such as methanolates.

[0154] In this invention, the term "polymorph" should be understood as a specific crystalline form of a compound that can crystallize in different forms.

[0155] When used herein, the term "cocrystal" should be understood as a crystalline structure composed of a BRAF inhibitor and at least one other component.

[0156] In another embodiment, the BRAF inhibitor is selected from the group consisting of compounds listed in items II, III, IV, V, and VI of Table 1.

[0157] As used herein, the term " Interfering RNA "iRNA" refers to an RNA molecule capable of silencing the expression of BRAF, particularly BRAF mutant proteins, or any gene required for BRAF function. For this purpose, iRNAs are typically double-stranded oligonucleotides having a length of at least 30 base pairs, more preferably containing 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 processed from single-stranded RNA precursors and are usually different from siRNAs because they are only partially complementary to target mRNAs; and small hairpin RNAs (shRNAs).

[0158] Small interfering RNA (siRNA) agents can inhibit target gene expression by interfering with RNA. 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 a protruding 3' and / or 5' region 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.

[0159] siRNA is sometimes referred to as 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 can be encoded by plasmids or viruses, particularly retroviruses, under the control of promoters such as the U6 promoter for RNA polymerase III.

[0160] The siRNA for use in the context of this invention is substantially homologous to the mRNA of BRAF, in particular to the mRNA of mutant BRAF, or to the genomic sequence encoding its protein. Substantial homology This is understood to mean that the 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: - siRNAs in which the linkage between nucleotides differs from that found in nature, for example, phosphorothioate linkages; - Strand RNA conjugate with functional reagents such as fluorophores; - Modification of the ends of RNA strands, particularly the 3' end, by combination with different hydroxyl functional groups at the 2' position; - Sugar-modified nucleotides, e.g., an O-alkylating group at the 2' position, e.g., 2'-O-methylribose or 2'-O-fluororibose; - Base-modified nucleotides, such as halogenated bases (e.g., 5-bromouracil and 5-iodouracil), alkylated bases (e.g., 7-methyl-guanosine), etc.

[0161] 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 recombinant dicers, 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.

[0162] The BRAF region used as the basis for siRNA design is not limited and may contain a coding sequence region (between the start and stop codons), or it may contain a 5' or 3' untranslated region sequence, preferably a 25-50 nucleotide length sequence at any position 3' relative to the start codon. The procedure for siRNA design includes identifying the sequence motif AA(N19)TT (where N can be any nucleotide in the BRAF sequence) and selecting one that exhibits a high G / C content. If this sequence motif is not found, the sequence motif NA(N21) (where N can be any nucleotide) can be identified.

[0163] In a preferred embodiment, the BRAF inhibitor is siRNA. In a more preferred embodiment, the siRNA is a commercially available siRNA from Santa Cruz Biotechnology, in particular sc-36368.

[0164] In another embodiment, the inhibitor is BRAF-specific Antisense oligonucleotidesAn antisense oligonucleotide is a molecule whose sequence is complementary to the mRNA encoding BRAF, i.e., complementary to the cDNA coding strand. The antisense oligonucleotide may be complementary to the complete coding region, or to its 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 binding reactions 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 bicatenary DNA-RNA complex formed between the antisense oligonucleotide and a target polynucleotide, such as a phosphorothioate derivative, peptide nucleic acid, and acridine-substituted oligonucleotide.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-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylkeuosin, inosine, N6-isopentenyladenine, 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, β-D-mannosylkeuosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, keuosin, 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 are cloned.

[0165] 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 via hybridization, and the coupling between the complementary region of the target nucleic acid and the ribozyme, activation of the catalytic region occurs, inducing intermolecular or intramolecular disruption of the target nucleic acid. Basic considerations for the design of ribozymes are widely known to those skilled in the art (see, e.g., Doherty and Doudna (Annu. Ref. Biophys. Biomolstruct. 2000; 30:457-75)).

[0166] Another type of compound that can form part of the composition of the present invention is an inhibitory antibody. Inhibitory antibodies According to the present invention, "is understood to mean an antibody that binds to BRAF, preferably a mutant BRAF protein, and induces inhibition of its catalytic activity.

[0167] Antibodies may be prepared using any method known to those skilled in the art. 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 BRAF are identified, antibodies capable of inhibiting BRAF activity are selected using the assay described above for the identification of BRAF activity, particularly BRAF variant activity. Suitable antibodies in the present invention include intact antibodies containing antigen-binding variable and constant regions, fragments "Fab", "F(ab')2" and "Fab", Fv, scFv, diabodies, and bispecific antibodies.

[0168] Other compounds that can inhibit BRAF expression and may form part of the composition of the present invention include aptamers and spiegelmers. Aptamers and spiegelmersThe aptamer and spiegelmer are single- or double-stranded, D- or L-nucleotides that specifically bind to the protein and result in a modification of the protein's (BRAF, particularly mutant BRAF proteins) biological activity. The aptamers and spiegelmers are 15 to 80 nucleotides long, preferably 20 to 50 nucleotides long.

[0169] In one embodiment, the combination of the present invention is a conjugate of component (i) and component (ii) of the combination of the present invention, and in particular, a conjugate of a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof and a BRAF inhibitor.

[0170] In some embodiments, the conjugate between components (i) and (ii) is mediated by an inseparable linker. In some embodiments, the conjugate between components (i) and (ii) is mediated by an inseparable linker. Exemplary inseparable and inseparable linkers are US8088387, US8142784, WO2013075048, US6630579, US8512707, US9120854, US9023351, US20160095938, US9446146, WO2005009369, US5773001, US6214 This is described in 345, US10111954, US8153768, US7829531, US20160082119, WO2018218004, US8568728, WO2015057699, US20170182181, and US9198979, the contents of which are incorporated herein by reference in their entirety.

[0171] In another embodiment, a combination of the first aspect of the present invention is i) The following group: a) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, b) A first component selected from a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant, and a conjugate comprising a chemical moiety that promotes intracellular uptake of the polypeptide or its functionally equivalent variant, ii) The second component is sorafenib and It is not a combination that includes this item.

[0172] 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 a pharmaceutically acceptable excipient.

[0173] As used in the present invention, the expression "pharmaceutical composition" refers to a formulation adapted for administering one or more therapeutically useful agents in predetermined doses to cells, cell groups, organs, tissues, or animals in which cell division is uncontrolled, such as cancer.

[0174] The pharmaceutical composition of the present invention contains a pharmaceutically effective amount of the combination according to 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 according to the present invention, a polynucleotide encoding the polypeptide or the conjugate, a vector comprising the polynucleotide, or a cell capable of secreting the polypeptide or the conjugate into a culture medium, and a BRAF 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.

[0175] The expression “pharmaceutically effective amount,” as used herein, is understood to mean an amount capable of providing a therapeutic effect and can be determined by those skilled in the art by commonly used means. The amount of Omomyc polypeptide, its functionally equivalent variants, such conjugates, fusion proteins, polynucleotides, vectors, cells, or BRAF inhibitors that can be combined in pharmaceutical compositions according to the present invention will vary depending on the subject and the specific mode of administration. Those skilled in the art will understand that the dosage can also be determined by the guidance in Goodman and Goldman's The Pharmacological Basis of Therapeutics, Ninth Edition (1996), Appendix II, pp. 1707–1711 and Goodman and Goldman's The Pharmacological Basis of Therapeutics, Tenth Edition (2001), Appendix II, pp. 475–493.

[0176] The appropriate dosage of the active ingredient in the pharmaceutical composition will vary depending on the type of cancer being treated, the severity and course of the disease, whether the composition is administered for preventive or therapeutic purposes, the patient's medical history and response to the peptide or polypeptide, and the discretion of the attending physician.

[0177] The amount of polypeptides containing the sequence of Sequence ID No. 1, their functionally equivalent variants, fusion proteins, conjugates, polynucleotides, vectors, or cells is administered to the patient as appropriate, either in a single dose or over a series of treatments. Depending on the type and severity of the disease, an appropriate dose level will generally be about 0.01 to 500 mg / kg (patient's body weight) / day, which can be administered in single doses or multiple doses. Preferably, the dose level will be about 0.1 to about 250 mg / kg / day, more preferably about 0.5 to about 100 mg / kg / day.

[0178] In a preferred embodiment, the amount of the first component is preferably by intranasal administration, preferably administered 4 times a week, in an amount of about 3.75 mg / kg (body weight of the subject) / day. In a preferred embodiment, the amount of the first component is preferably by intranasal administration, preferably administered 4 times a week, in an amount of about 8 mg / m 2 / day to 15 mg / m 2 / day, preferably 10 mg / m 2 / day to 12 mg / m 2 / day, more preferably 11.25 mg / m 2 / day.

[0179] In a preferred embodiment, the amount of the first component is preferably by intravenous administration, preferably administered 2 times a week, in an amount of about 50 mg / kg (body weight of the subject) / day. In a preferred embodiment, the amount of the first component is preferably by intravenous administration, preferably administered 2 times a week, in an amount of about 100 mg / m 2 / day to 200 mg / m 2 / day, preferably 125 mg / m 2 / day to 175 mg / m 2 / day, preferably 140 mg / m 2 / day to 160 mg / m 2 / day, more preferably 150 mg / m 2 / day.

[0180] Suitable dose levels may range from approximately 0.01 mg / kg / day to 250 mg / kg / day, from approximately 0.05 mg / kg / day to 100 mg / kg / day, or from approximately 0.1 mg / kg / day to 50 mg / kg / day. Within this range, doses may range from 0.05 mg / kg / day to 0.5 mg / kg / day, from 0.5 mg / kg / day to 5 mg / kg / day, or from 5 mg / kg / day to 50 mg / kg / day. For oral administration, the composition is preferably provided in the form of tablets containing 1.0 mg to 1000 mg of the active ingredient, particularly 1.0 mg, 5.0 mg, 10.0 mg, 15.0 mg, 20.0 mg, 25.0 mg, 50.0 mg, 75.0 mg, 100.0 mg, 150.0 mg, 200.0 mg, 250.0 mg, 300.0 mg, 400.0 mg, 500.0 mg, 600.0 mg, 750.0 mg, 800.0 mg, 900.0 mg, and 1000.0 mg of the active ingredient, for symptomatic adjustment of the dosage to the patient being treated. The compound may be administered in a dosing regimen of once a day to four times a day, preferably once a day or twice a day.

[0181] 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 consecutively. If the compounds are administered consecutively, administration of the first compound is discontinued before administration of the second compound is initiated.

[0182] 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 more, or longer. Preferably, the duration of treatment is at least four weeks. In another embodiment, the duration of treatment is at least three weeks.

[0183] The amount of BRAF inhibitor may depend on the specific drug used and may be approximately 0.01 mg / kg(body weight) / day to approximately 200 mg / kg(body weight) / day, preferably 0.01 mg / kg(body weight) / day to approximately 150 mg / kg(body weight) / day, more preferably 0.01 mg / kg(body weight) / day to approximately 100 mg / kg(body weight) / day, preferably 0.01 mg / kg(body weight) / day to approximately 75 mg / kg(body weight) / day, 0.01 mg / kg(body weight) / day to approximately 50 mg / kg(body weight) / day, more preferably 0.5 mg / kg(body weight) / day to approximately 50 mg / kg(body weight) / day, preferably approximately 1 mg / kg(body weight) / day to approximately 25 mg / kg(body weight) / day, preferably administered orally once or multiple times daily to achieve the desired therapeutic effect. In another preferred embodiment, the amount of the BRAF inhibitor is preferably administered orally once or more times daily, ranging from about 0.01 mg / kg(body weight) / day to about 40 mg / kg(body weight) / day; preferably 0.01 mg / kg(body weight) / day to about 30 mg / kg(body weight) / day; preferably 0.02 mg / kg(body weight) / day to about 25 mg / kg(body weight) / day; preferably 0.5 mg / kg(body weight) / day to about 15 mg / kg(body weight) / day; preferably 1 mg / kg(body weight) / day to about 10 mg / kg(body weight) / day; preferably 2 mg / kg(body weight) / day to about 10 mg / kg(body weight) / day; preferably 2 mg / kg(body weight) / day to about 5 mg / kg(body weight) / day, for the desired therapeutic effect. In preferred embodiments, the amount of the BRAF inhibitor is preferably administered once a week, more preferably daily, and more preferably orally, approximately 2.5 mg / kg (body weight) / day or 7.5 mg / m². 2 This is the daily dose. In a preferred embodiment, the dose of the BRAF inhibitor is preferably administered once a week, more preferably daily, and more preferably orally, about 0.5 mg / kg (body weight of the subject) / day or 1.5 mg / m². 2This is the daily dose. In a preferred embodiment, the dose of the BRAF inhibitor is preferably administered once a week, more preferably daily, and more preferably orally, about 5 mg / kg (body weight of the subject) / day or 15 mg / m². 2 This is the daily dose. In another preferred embodiment, the dose of the BRAF inhibitor is preferably administered once a week, preferably daily, more preferably orally, about 10 mg / kg (body weight of the subject) / day or 30 mg / m² 2 This is the amount per day. In another preferred embodiment, the amount of the BRAF inhibitor is preferably administered once a week, preferably daily, more preferably orally, about 50 mg / kg (body weight of the subject) / day or 150 mg / m² 2 This is the daily dose. In another preferred embodiment, the dose of the BRAF inhibitor is preferably administered once a week, preferably daily, more preferably orally, about 100 mg / kg (body weight of the subject) / day or 300 mg / m². 2 This is the daily dose. BRAF inhibitors are preferably administered 7 days a week for a period of 4 weeks.

[0184] A pharmaceutical composition according to the present invention, comprising a first component (i) selected from a polypeptide containing SEQ ID NO: 1, a functionally equivalent variant thereof, a fusion protein, a conjugate, a polynucleotide, a vector, or a cell, and a second component (ii) which is a BRAF inhibitor, may be presented as a single formulation (e.g., as a tablet or capsule containing a quantitative amount of each of the components) or, conversely, as separate formulations to be later combined for joint administration, sequential administration, or separate administration. The compositions of the present invention also encompass formulations as parts kits in which the components are formulated separately but packaged in the same container. Those skilled in the art will understand 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 (in tablets or pills), and they may allow their administration via the same route. When the different components of the present invention are formulated separately, the two components may be presented in blisters. Each blister contains the drug that must be consumed within a day. If the drug needs to be administered several times a day, the drug corresponding to each administration can be placed in different sections of the blister pack, preferably with the time at which the drug should be administered recorded in each section of the blister pack. Alternatively, the components of the composition of the present invention can be formulated separately so that different components are administered separately. Thus, the first component can be formulated for intravenous administration and the second component can be formulated as a tablet or capsule for oral administration, or vice versa. The ratios between the components that are part of the combination or pharmaceutical composition according to the present invention can be adjusted by those skilled in the art depending on the antitumor agent used for each particular medical condition and the desired indicator. Accordingly, the present invention envisions compositions in which the ratio between the amount of component (i) and the amount of 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, in particular 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 specific embodiment, the ratio is in the range of 1:1 to 1:1.5, preferably 1:1.3 to 1:1.4, and more preferably 1:1.34. In another specific embodiment, the ratio is in the range of 1:1 to 1:2.8, preferably 1:2.6 to 1:2.7, and 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, and 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. Exceptionally, favorable results are obtained when the ratio of component (i):component (ii), more preferably Omomyc:dabrafenib, Omomyc:vemurafenib, or Omomyc:encorafenib; even more preferably Omomyc:dabrafenib, is 50:1 to 200,000:1; preferably 75:1 to 150,000:1; preferably 100:1 to 125,000:1; preferably 102.78:1 to 105,279 The ratio obtained was in the range of 0.67:1; preferably 105:1 to 100000:1; more preferably 110:1 to 100000:1; preferably 150:1 to 100000:1; more preferably 200:1 to 100000:1; preferably 500:1 to 100000:1; preferably 1000:1 to 100000:1; preferably 5000:1 to 50000:1; preferably 10000:1 to 30000:1.In a preferred embodiment, the ratio of component (i):component (ii), more preferably the ratio of Omomyc:dabrafenib, Omomyc:vemurafenib, or Omomyc:encorafenib; even more preferably the ratio of Omomyc:dabrafenib, is in the range of 205.56:1 to 105279.67:1; more preferably 210:1 to 100000:1; more preferably 102.78:1 to 105279.67:1; and even more preferably 105:1 to 105000:1. These ratios are effective for treating any type of cancer, but more preferably these ratios are obtained when treating cancer selected from the group consisting of melanoma, colorectal cancer, and breast cancer, and more preferably melanoma, preferably melanoma cancer with a BRAF mutation, and more preferably melanoma cancer with a BRAF V600E mutation.

[0185] Preferably, these ratios are w / w ratios.

[0186] The components of the pharmaceutical composition or combination of the present invention can be administered simultaneously. "Simultaneous administration" encompasses the co-administration of two therapeutic agents, regardless of the relative frequency or timing of administration of each agent. Therefore, simultaneous administration includes the co-administration of two therapeutic agents at the same timing and frequency of administration. In addition, simultaneous administration also means the co-administration of two therapeutic agents in which one agent is administered more frequently than the other. In addition, simultaneous administration also means the co-administration of two therapeutic agents in which one agent is administered only once between administrations of the other.

[0187] In one embodiment, component (i) is administered intranasally. In another embodiment, component (i) is administered intravenously. In yet another embodiment, component (i) is administered orally. In yet another embodiment, component (ii) is administered parenterally, particularly intraperitoneally or intravenously.

[0188] In a preferred embodiment, component (i) of the combination or pharmaceutical composition of the present invention is administered intravenously, while the BRAF inhibitor is administered orally. In the case of intravenous administration, the preferred dose of component (i) of the combination or composition of the present invention, preferably the polypeptide or its functionally equivalent variant, fusion protein, or conjugate, is in the range of 0.01 mg / kg to 250 mg / kg, more preferably between 0.1 mg / kg and about 100 mg / kg per day, which can be administered in a single or multiple dose. The preferred dose of the BRAF inhibitor, when administered orally, is between 0.01 mg / kg and 200 mg / kg, preferably between 0.01 mg / kg and 150 mg / kg, more preferably between 0.1 mg / kg and 100 mg / kg, more preferably between 0.5 mg / kg and 100 mg / kg, even more preferably between 10 mg / kg and 100 mg / kg, and most preferably between 50 mg / kg and 100 mg / kg. In another preferred embodiment, the preferred dose of the BRAF inhibitor, when administered orally, is 0.01 mg / kg to 40 mg / kg; preferably 0.01 mg / kg to 30 mg / kg; preferably 0.02 mg / kg to 25 mg / kg; preferably 0.5 mg / kg to 15 mg / kg; preferably 1 mg / kg to 10 mg / kg; preferably 2 mg / kg to 10 mg / kg; preferably 2 mg / kg to 5 mg / kg. Preferably, the BRAF inhibitor is administered 7 days a week for 4 weeks.

[0189] In another embodiment, components (i) and (ii) of the combination or pharmaceutical composition of the present invention are administered intravenously.

[0190] The pharmaceutical compositions of the present invention may also contain one or more additional compounds for the prevention and / or treatment of pathologies in which uncontrolled cell division is present, such as cancer. Such additional compounds, such as 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, angiogenesis inhibitors, antimetastatic agents, and antiproliferative agents.

[0191] 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 intended to be used to incorporate the active ingredient and is acceptable to the patient from a pharmacological / toxicological standpoint, as well as to the pharmacist who manufactures it from a physical / chemical standpoint with respect to composition, formulation, stability, patient tolerability, and bioavailability. The excipient may be a carrier. As used herein, “carrier” means any substance that helps to improve the delivery and efficacy of the active ingredient within the pharmaceutical composition. In a preferred embodiment, the carrier does not allow direct delivery of component (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 water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and one or more combinations thereof. In many cases, it would be preferable to include an isotonic agent, such as sugar, a polyalcohol, such as mannitol, sorbitol, or sodium chloride in the combination or composition. A pharmaceutically acceptable carrier may further include small amounts of auxiliary agents that enhance the shelf life or efficacy of the components forming part of the combination or composition of the present invention, such as wetting agents and emulsifiers, preservatives, and buffering agents. Examples of suitable carriers are well known in the literature (see, for example, Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).Examples of carriers, though not limited to these, include a range of sugars, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; a range of starches, such as corn starch, wheat starch, rice starch, and potato starch; a range of celluloses, such as cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; and a range of fillers, such as gelatin and polyvinylpyrrolidone. Disintegrants, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate, may be added as needed.

[0192] The number and properties of the pharmaceutically acceptable excipients vary depending on the desired dosage form. Pharmaceutically acceptable excipients are known to those skilled in the art (Fauliy Trillo C. (1993) “Tratado de Farmacia Galenica”, Luzan 5, SA Ediciones, Madrid). The composition can be prepared by conventional methods known in the current state of the art ("Remington: The Science and Practice of Pharmacy", 20th edition (2003) Genaro AR, ed., Lippincott Williams & Wilkins, Philadelphia, US).

[0193] In the case of a pharmaceutical composition containing a drug that is a nucleic acid molecule, the nucleic acid molecule may be present in any delivery system known to those skilled in the art, such as nucleic acids, as well as 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 be "naked," for example, as described in Ulmer et al., Science 259:1745-49, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA can be increased by coating the DNA onto biodegradable beads, which are then efficiently transported into cells.

[0194] Nucleic acid molecules can be delivered into cells by any of the methods described in this art (e.g., Akhtar et al., Trends Cell Bio. 2:139 (1992); Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995; Maurer et al., Mol. Membr. Biol. 16:129-40 (1999); Hofland and Huang, Handb. Exp. Pharmacol. 137:165-92 (1999); Lee et al., ACS Symp. Ser. 752:184-92 (2000); US6,395,713;WO 94 / 02595); Selbo et al., Int. J. Cancer 87:853-59 (2000); Selbo et al., Tumour Biol. 23:103-12 (2002); see US2001 / 0007666 and US2003 / 077829). Such delivery methods known to those skilled in the art include, but are not limited to, iontophoresis, or encapsulation in liposomes by incorporation into other vehicles, such as biodegradable polymers; hydrogels; cyclodextrins (see, e.g., Gonzalez et al., Bioconjug. Chem. 10:1068-74 (1999); Wang et al., International Publication WO 03 / 47518 and WO 03 / 46185); poly(lactic acid-coglycolic acid) acids (PLGA) and PLCA microspheres (also useful for the delivery of peptides and polypeptides and other substances) (see, e.g., US6,447,796; US2002 / 130430); biodegradable nanocapsules and bioadherent microspheres, or by protein vectors (International Publication WO 00 / 53722).In another embodiment, the nucleic acid molecule may 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 also, e.g., US2003 / 0077829).

[0195] In certain embodiments, if a composition or combination according to 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; the pharmaceutical composition may, but is not limited to, contain a viral or nonviral vector containing a suitable polynucleotide or gene construct. The vector may, but is not limited to, be viral, for example, based on retroviruses, adenoviruses, etc., or nonviral, for example, based on ADN-liposomes, ADN-polymers, ADN-polymer-liposome complexes, etc. [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, such as human cells, which would then be transplanted into a human or animal to obtain the desired therapeutic effect. For administration to a human or animal, the cells would be formulated in a suitable medium that would not adversely affect cell survival.

[0196] The combination or pharmaceutical composition of the present invention can be administered by any type of preferred route, such as by oral, topical, inhalation, or parenteral routes, which include pharmaceutically acceptable excipients necessary for formulation into the desired dosage form. Other routes of administration may be transrectal, intracisional, or vaginal. The preferred routes of administration of the combination or pharmaceutical composition are intravenous for component (i) and oral for component (ii).

[0197] The “oral route” is understood as a pharmaceutical composition that is taken up by an organism after swallowing. In certain embodiments, the pharmaceutical compositions of the present invention may be in a dosage form suitable for oral administration, whether solid or liquid. Suitable dosage forms for oral administration may be tablets, capsules, syrups, or solutions, and may also include any conventional excipients known in the art, e.g., binders, e.g., syrups, 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 processes of mixing, filling, or compression. By using repeated mixing operations, the active agent can be completely distributed into those compositions using large amounts of fillers. This procedure is customary in the art. Tablets can be prepared, for example, by wet or dry granulation and, optionally, by coating them with enteric coatings according to processes known in general pharmaceutical practice.

[0198] 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 by topical administration, oral administration, and dropwise administration of the compositions into the ears, eyes, and noses, such that they do not enter the bloodstream. Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches.

[0199] Ophthalmic formulations, ear drops, and eye drops are also conceivable to fall within the scope of the present invention. Furthermore, the present invention also conceivable the use of transdermal patches, which have the added advantage of providing controlled delivery of the compound to the body. Such a dosage form can be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers may also be used to increase the skin penetration of the compound. The rate can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0200] In one embodiment, the combination or pharmaceutical composition is administered systemically.

[0201] "Systemic routes" are understood as administration via oral, intravenous, intraperitoneal, and intramuscular routes. The amounts of components (i) and (ii) required for therapeutic or preventive effects will naturally vary depending on the selected compounds, the nature and severity of the disease being treated, and the patient. Preferably, the combination or pharmaceutical composition is administered orally.

[0202] In another embodiment, the combination or pharmaceutical composition is administered intranasally. In a preferred embodiment, intranasal administration is carried out by drip administration or intranasal inhalation.

[0203] "Inhalation" is understood as administration via the intranasal route and administration by oral inhalation. Suitable drug formulations for such administration, such as aerosols or formulations in metered-dose inhalers, can be prepared by prior art. In one embodiment, the route of administration is the intranasal route.

[0204] As used herein, the term “parenteral” includes administration via intravenous, intraperitoneal, intramuscular, or subcutaneous routes. Subcutaneous, intramuscular, and intravenous parenteral administrations are generally preferred. In some embodiments, the combination or pharmaceutical composition is administered intravenously.

[0205] In one embodiment, the combinations or pharmaceutical compositions of the present invention can be adapted for parenteral administration, for example, in appropriate dosing unit forms, such as sterile solutions, suspensions, or lyophilized products. Suitable combinations or pharmaceutical compositions for injection use include sterile aqueous solutions (if soluble in water), or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For administration via intravenous routes, some suitable carriers include phosphate-buffered aqueous saline (PBS). In all cases, the combinations or compositions must be sterile and fluid enough to be easily injected. They must be stable under preparation and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carriers may be solvents or dispersion media containing, for example, water, ethanol, pharmaceutically acceptable polyols, such as glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, maintaining the required particle size in the case of a dispersion, and by using surfactants. Prevention of microbial contamination can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thiomersal, etc. In most cases, it would be preferable to include isotonic agents, such as sugars; polyalcohols, such as mannitol, sorbitol, etc.; or sodium chloride in the composition. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin.

[0206] The injectable sterile solution can be prepared by adding the required amount of the active compound to a suitable solvent along with one or a combination of the aforementioned raw materials, and, if necessary, by subsequent sterilization by filtration through a sterile membrane. Generally, the dispersion is prepared by adding the active compound to a sterile vehicle containing a basic dispersion medium and the remainder of the required raw materials listed above. In the case of sterile powders for the preparation of injectable sterile solutions, preferred preparation processes are vacuum drying and freeze-drying, thereby forming a powder with the active raw material and any desired additional raw materials from the previously filtered sterile solution.

[0207] The combination or pharmaceutical composition of the present invention can be suitably administered, for example, by pulse infusion with decreasing doses of the composition. Preferably, the dose is administered by injection, more preferably intravenous or subcutaneous injection, depending in part whether the administration is urgent or sustained.

[0208] Alternatively, as described above, the different components of the composition may be administered separately.

[0209] Accordingly, in one embodiment, component (i) of the combination or composition of the present invention, preferably a polypeptide or a functionally equivalent variant or conjugate, is administered intravenously, while the BRAF inhibitor is administered orally.

[0210] In another embodiment, both components (i) and (ii) are administered intravenously.

[0211] In another embodiment, component (i) of the combination or composition, preferably a polypeptide or functionally equivalent variant or conjugate of the composition, is administered intranasally or by inhalation.

[0212] 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 metered. For example, a metered drop / spray means that a dispenser containing the drop / spray delivers the drop / spray containing a metered dose (a predetermined amount) of the composition for use according to the present invention.

[0213] One preferred form of administration in relation to intranasal administration is nasal drops. The drops adhere mostly to the posterior part of the nose and are therefore rapidly removed into the nasal pharynx. A concern with drops is often how to precisely control the dose of the drug, which is particularly important for the administration of the composition.

[0214] Another intranasal administration 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, buffers) in a non-pressurized dispenser. Nasal sprays have several advantages, such as the compactness of the delivery device, convenience, ease of use, and accuracy in delivering dosages from 25 pL to 200 pL. Nasal sprays are applied to the posterior portion of the nose and slowly removed to the nasal pharynx by mucociliary clearance. When used herein, nasal sprays may be liquids or suspensions.

[0215] Another form of intranasal medication is nasal aerosol. Nasal aerosols differ from spray nasal drops in the method of dispensing the composition; in aerosols, the compound is dispensed through a valve by excessive pressure and release. In sprays, the compound is dispensed by being forcibly released by a micropump bucket, while the pressure in the vial is similar to atmospheric pressure. Aerosols have similar advantages to sprays.

[0216] Alternatively, the compositions according to this specification may be administered preferably by means of a nasal emulsion, ointment, gel, paste, or cream. These are highly viscous solutions or suspensions applied to the nasal mucosa.

[0217] Due to the limited amount of composition that can be efficiently delivered to the nasal mucosa, liquid intranasal formulations typically have higher concentrations than their corresponding intravenous formulations. If a substance is poorly soluble or insoluble in liquid form, powder formulations can be used to administer the composition of the present invention. Further advantages of powder formulations are that they do not require preservatives and generally have higher stability compared to liquid formulations. The main limitation regarding the application of intranasal powder formulations relates to their irritating effect on the nasal mucosa.

[0218] One form of drug administration in relation to intrapulmonary administration is inhalation aerosol. Inhalation aerosols are typically packaged under pressure and contain compositions according to the present invention, which are released into the respiratory tract, particularly the lungs, when a valve system is activated. The released aerosol is a colloid (suspension) or droplet (solution) of solid particulate matter in the air or another gas. Thus, the aerosol can be a solution aerosol or a suspension aerosol. The droplet 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.

[0219] One form of medication in relation to intrapulmonary administration is the inhalation spray. Inhalation sprays are typically water-based and contain no propellant. They deliver the conjugate to the lungs by oral inhalation.

[0220] Atomized inhalation solutions and suspensions may also be used to deliver the conjugate via an intrapulmonary route. These atomized inhalation solutions and suspensions are typically water-based formulations containing the composition according to the present invention. These atomized inhalation solutions and suspensions are used to deliver the composition to the lungs by oral inhalation for systemic effects, as well as using a nebulizer.

[0221] Dry powder inhalation is an alternative to aerosol inhalation. The composition is typically contained in a capsule or in an inhaler for manual loading. Dry powder is typically delivered to the lungs by oral inhalation using an inhaler. When used herein, dry powder can be formulated in a pure form. A neat formulation contains only the drug or only a placebo, for example, as a spray dry powder. When used herein, dry powder can also be formulated with a carrier such as lactose.

[0222] Intrapulmonary medication is preferably measured and delivered to the lungs in a predetermined amount.

[0223] Devices for intranasal delivery in connection with the present invention include spray pump systems, pipettes for delivering drops, metered-dose spray pumps, nasal pressurized metered-dose inhalers, powder spray systems, breath-actuated powder inhalers, and nasal powder insufflators. These intranasal delivery devices may be filled with single-dose or multi-dose intranasal formulations.

[0224] By using an intrapulmonary route, the conjugate can be administered by a metered-dose inhaler. Metered-dose inhalers (MDIs) generally provide a fine mist of the conjugate with an aerodynamic particle size of less than 5 pm.

[0225] Dry powder inhalers can be used as an alternative method for delivering compositions into the lungs. Dry powder inhalers present the powder medication as a single-dose or multi-dose powder.

[0226] Another device for intrapulmonary delivery is atomizers such as ultrasonic atomizers and air jet atomizers. In ultrasonic atomizers, ultrasonic waves are generated in the atomizer chamber by a ceramic piezoelectric crystal that vibrates when electrically excited. This generates an aerosol cloud at the surface of the solution. Aerosols generated by air jet atomizers are produced when compressed air is forced through an outlet. Liquid can be drawn from a vertical nozzle to mix with an atomized air jet using a baffle to facilitate the formation of an aerosol cloud (Bernoulli effect).

[0227] In one embodiment, each component of the combination or pharmaceutical composition of the present invention is prepared using a carrier that protects the component, particularly component (i), from rapid elimination from the body, such as in controlled-release formulations including implantation and microencapsulation administration systems. Biodegradable and biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. The process for preparing such formulations will be apparent to those skilled in the art. The materials can also be commercially obtained from Alza Corporation and Nova Pharmaceuticals, Inc.

[0228] The sustained-release composition also includes preparations of crystals suspended in a suitable formulation that can maintain the crystals in a suspended state. These preparations may produce a sustained-release effect when injected via a subcutaneous or intraperitoneal route. 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, etc. In a preferred embodiment, components (i) and / or (ii) are contained in liposomes, preferably both components are contained in liposomes, more preferably the same liposome.

[0229] Despite the fact that Omomyc, any of 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 in nanoparticles. Such nanoparticles may contribute to preserving the integrity of the component in biological fluids until it reaches the target organ. Furthermore, in the case of a composition containing component (ii) or other antitumor agents, encapsulation of the composition may reduce the secondary effects caused by the antitumor agent. In addition, the nanoparticles may be modified to include a portion that enables the nanoparticles to target the organ of interest. In this manner, component (i) of the combination or composition of the present invention is delivered near the target organ and facilitates access of component (i) into the interior of cells where biological activity is required.

[0230] Accordingly, in another embodiment, component (i) of the combination or composition of the present invention is provided to form part of a nanoparticle. In another embodiment, both components of the combination or composition of the present invention are provided to form part of a nanoparticle, preferably both components are provided within the same nanoparticle.

[0231] As used herein, the term “nanoparticles” means 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. Examples of 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 incorporated into a nanoparticle matrix or adsorbed onto its surface, and preferably, molecules can be incorporated into the nanoparticles.

[0232] In a preferred embodiment, the nanoparticles are liposomes.

[0233] Targeted delivery can be achieved by adding a ligand without sacrificing the nanoparticle's ability to deliver its contents. This allows for delivery to specific cells, tissues, and organs. The target specificity of ligand-based delivery systems is based on the distribution of ligand receptors to different cell types. Targeting ligands may be associated with nanoparticles non-covalently or covalently, and may also be conjugated to nanoparticles by various methods described herein.

[0234] 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, insulin, and others.

[0235] While the formulations of the present invention in nanoparticles are not intended, or merely not intended, to facilitate access of components (i) and / or (ii) into the interior of cells, it will be understood that they are intended to protect components (i) and / or (ii) from degradation and / or to facilitate the targeting of the nanoparticles to the organ of interest.

[0236] In one embodiment, the nanoparticles may be made from a biodegradable polymer, such as poly(butylcyanoacrylate) (PBCA). Examples of elemental nanoparticles include carbon nanoparticles and iron oxide nanoparticles, which can be coated with oleic acid (OA)-Pluronic(R). In this approach, the drug (e.g., a hydrophobic or water-insoluble drug) is loaded into the nanoparticles. Other nanoparticles are made from silica.

[0237] Nanoparticles can be formed from any useful polymer. Examples of polymers include biodegradable polymers such as poly(butylcyanoacrylate), poly(lactide), poly(glycolide), poly-s-caprolactone, poly(butylene succinate), poly(ethylene succinate), and poly(p-dioxanone); poly(ethylene glycol); poly-2-hydroxyethyl methacrylate (poly(HEMA)); copolymers such as poly(lactide-co-glycolide), poly(lactide)-poly(ethylene glycol), poly(poly(ethylene glycol)cyanoacrylate-cohexadecylcyanoacrylate), and poly[HEMA-co-methacrylic acid]; proteins such as fibrinogen, collagen, gelatin, and elastin; and polysaccharides such as amylopectin, amylose, and chitosan.

[0238] 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, such as surfactants, e.g., Epicuron® 200, Poloxamer 188 (Pluronic® F68), Brij72, Brij78, polysorbate 80 (Tween80); and salts, e.g., sodium taurocholate. Drugs can be introduced into solid lipid nanoparticles by several methods described for liposomes, and such methods may further include high-pressure homogenization and dispersion of microemulsions.

[0239] Nanoparticles may also include nanometer-sized micelles. Micelles can be formed from any polymer described herein. Exemplary polymers for forming micelles include block copolymers, such as poly(ethylene glycol) and poly(ε-caprolactone) (e.g., aPEO-bPCL block copolymers containing polymers of ε-caprolactone and α-methoxy-ω-hydroxy-poly(ethylene glycol)).

[0240] In certain embodiments, the properties of nanoparticles are modified by coating them with a surfactant. Any biocompatible surfactant can be used, such as polysorbate surfactants, e.g., polysorbate 20, 40, 60, and 80 (Tween 80); Epicuron® 200; poloxamer surfactants, e.g., 188 (Pluronic® F68), poloxamer 908, and 1508; and Brij surfactants, e.g., Brij 72 and Brij 78.

[0241] Nanoparticles can optionally be modified to include hydrophilic polymer groups (e.g., poly(ethylene glycol) or poly(propylene glycol)) by, for example, covalently bonding hydrophilic polymer groups to their surface, or by using polymers having such hydrophilic polymer groups (e.g., poly[methoxypoly(ethylene glycol)cyanoacrylate-co-hexadecylcyanoacrylate]). Nanoparticles can also optionally be crosslinked, which may be particularly useful in the case of protein-based nanoparticles.

[0242] In another embodiment, the pharmaceutical composition of the present invention is a nanoemulsion. “Nanoemulsion,” as used herein, means a colloidal dispersion of droplets (or particles) such that at least some of the droplets have a diameter in the nanometer range. The nanoemulsion is composed of ω-3, -6, or -9 fatty acid-rich oils in an aqueous phase, and is thermodynamically stabilized by an amphiphilic surfactant that forms an interfacial film, and is typically produced using a high-shear microfluidization process with droplet diameters in the range of about 80–220 nm.

[0243] Therapeutic applications of the present invention In one embodiment, the present invention relates to a combination or pharmaceutical composition of the present invention for use in the medical field.

[0244] 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.

[0245] In another embodiment, the present invention means a combination or pharmaceutical composition of the present invention for the preparation of a pharmacopoeia for the prevention and / or treatment of cancer.

[0246] In another embodiment, the present invention also means a method for the prevention and / or treatment of cancer, comprising the step of administering a therapeutically effective amount of the combination or pharmaceutical composition of the present invention to a subject in need thereof.

[0247] In preferred embodiments, the preventive or therapeutic method according to the present invention involves the direct use of a combination or pharmaceutical 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 method according to the present invention does not involve the administration of a nucleic acid encoding a polypeptide or fusion protein containing Omomyc or a functionally equivalent variant thereof, or a vector encoding said nucleic acid, or cells containing said nucleic acid.

[0248] "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 before its onset.

[0249] The term “treatment” is used to indicate 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 beneficial or desired clinical outcomes, including, but not limited to, symptom relief, reduction of disease duration, fixation of the pathological state (in particular, avoidance of further impairment), delay of disease progression, improvement of the pathological state, and remission (both partial and complete). Control of disease progression also involves an extension of survival compared to the survival expected if treatment had not been applied. In a preferred embodiment, control of disease progression is measured as the healthy lung / thoracic 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 survival.

[0250] The term "cancer" refers to a disease characterized by uncontrolled cell division (or increased resistance to survival or apoptosis), the ability of cells to invade other adjacent tissues (invasion), or the spread of cells through lymphatic vessels and blood vessels to other areas of the body where they are not normally located (metastasis). Depending on whether a tumor can spread by invasion and metastasis, tumors are classified as either benign or malignant. Benign tumors are tumors that cannot spread by invasion or metastasis; that is, they only grow locally, while malignant tumors are tumors that can spread by invasion and metastasis. The method according to the present invention is useful for the treatment of local malignant tumors.

[0251] In one embodiment, cancers include, but are 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 eureka, lymphoma (e.g., Hodgkin's disease or non-Hodgkin's disease), AIDS-related leukemia, Waldenström macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors, such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangiosarcoma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, Kaposi's sarcoma, colon carcinoma, pancreatic cancer, breast cancer, biliary tract cancer, esophageal cancer, ovarian cancer, prostate cancer, oral cancer (e.g., squamous cell carcinoma), basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, teratoma, choriocarcinoma, seminomas, fetal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung carcinoma, Examples include bladder carcinoma, epithelial carcinoma, carcinoma in situ, such as Bowen's disease and Paget's disease, glioma, astrocytoma, glioblastoma multiforme (also known as GBM or gliablastoma), medulloblastoma, craniopharyngioma, ependymal cell tumor, pineal gland tumor, hemangioblastoma, acoustic neuroma, oligodendrone, neurofibroma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0252] In some embodiments, the cancer is a glioma, astrocytoma, glioblastoma multiforme (also known as GBM or gliablastoma), medulloblastoma, craniopharyngioma, ependymal cell tumor, pineal gland tumor, hemangioblastoma, acoustic neuroma, oligodendrone, neurofibroma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

[0253] In some embodiments, the cancer is an acoustic neuroma, astrocytoma (e.g., grade I - pilocytic astrocytoma, grade II - low-grade astrocytoma, grade III - undifferentiated astrocytoma, or grade IV - gliablastoma (GBM)), chordoma, CNS lymphoma, craniopharyngioma, brainstem glioma, ependymal cell tumor, mixed glioma, optic glioma, subependymal cell tumor, medulloblastoma, meningioma, metastatic brain tumor, oligodendrocyte, pituitary tumor, primitive neuroectodermal (PNET) tumor, or neurofibroma. In some embodiments, the cancer is a type that is generally found more in children than in adults, such as brainstem glioma, craniopharyngioma, ependymal cell tumor, juvenile pilarcytic astrocytoma (JPA), medulloblastoma, optic glioma, pineal tumor, primitive neuroectodermal (PNET) tumor, or rhabdoid tumor. In some embodiments, the patient is an adult. In some embodiments, the patient is a child or a pediatric patient.

[0254] In another embodiment, cancers include, but are not limited to, mesothelioma, hepatobiliary (liver and bile ducts), bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, ovarian cancer, colon cancer, rectal cancer, anal cancer, gastric cancer, gastrointestinal cancer (stomach, colorectal, and duodenum), uterine cancer, fallopian tube cancer, endometrial carcinoma, cervical carcinoma, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, Examples include cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, renal cell carcinoma, renal pelvis cancer, non-Hodgkin lymphoma, spinal axial tumor, brainstem glioma, pituitary adenoma, adrenocortical carcinoma, gallbladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or one or more combinations of the aforementioned cancers.

[0255] In some embodiments, the cancer is selected from hepatocellular carcinoma, ovarian cancer, epithelial ovarian cancer, or fallopian tube cancer; papillary serous cystadenocarcinoma or papillary serous carcinoma of the uterus (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatobiliary cancer; synovial sarcoma of soft tissue and bone; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; anaplastic thyroid carcinoma; 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 tumor; neurofibromatosis-1-associated malignant peripheral nerve schwannoma (MPNST); Waldenström macroglobulinemia; or medulloblastoma.

[0256] In some embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, epithelial ovarian cancer, fallopian tube cancer, papillary serous cystadenocarcinoma, papillary serous carcinoma of the uterus (UPSC), hepatobiliary carcinoma, synovial sarcoma of soft tissue and bone, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid carcinoma, adrenocortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1-associated malignant peripheral nerve schwannoma (MPNST), Waldenström macroglobulinemia, or medulloblastoma.

[0257] In some embodiments, cancer is a solid tumor, such as sarcoma, carcinoma, or lymphoma. Solid tumors generally consist of a mass of abnormal tissue that typically does not contain a sac or fluid area. In some embodiments, such cancer is renal cell carcinoma, or kidney cancer; hepatocellular carcinoma (HCC) or hepatoblastoma, or liver cancer; melanoma; breast cancer; colorectal carcinoma, 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, epithelial ovarian cancer, ovarian carcinoma, or fallopian tube cancer; papillary serous cystadenocarcinoma or papillary serous carcinoma of the uterus (UPSC); prostate cancer; testicular cancer; Selected from gallbladder cancer; hepatobiliary cancer; synovial sarcoma of soft tissue and bone; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; anaplastic 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 tumor; neurofibromatosis-1-associated malignant peripheral nerve schwannoma (MPNST); Waldenström macroglobulinemia; or medulloblastoma.

[0258] In some embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, epithelial ovarian cancer, ovarian carcinoma, fallopian tube cancer, papillary serous cystadenocarcinoma, papillary serous carcinoma of the uterus (UPSC), hepatobiliary carcinoma, synovial sarcoma of soft tissue and bone, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid carcinoma, adrenocortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1-associated malignant peripheral nerve schwannoma (MPNST), Waldenström macroglobulinemia, or medulloblastoma.

[0259] 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 epithelial ovarian cancer. In some embodiments, the cancer is fallopian tube cancer. In some embodiments, the cancer is papillary serous cystadenocarcinoma. In some embodiments, the cancer is uterine papillary serous carcinoma (UPSC). In some embodiments, the cancer is intrahepatic 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 anaplastic thyroid cancer. In some embodiments, the cancer is adrenocortical carcinoma. In some embodiments, the cancer is pancreatic cancer, or pancreatic adenocarcinoma. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is malignant peripheral nerve sheath tumor (MPNST). In some embodiments, the cancer is neurofibromatosis-1 associated MPNST. In some embodiments, the cancer is Waldenström macroglobulinemia. In some embodiments, the cancer is medulloblastoma.

[0260] In some embodiments, the cancer is a virus-related cancer, such as a human immunodeficiency virus (HIV)-related solid tumor, a human papillomavirus (HPV)-16 positive incurable solid tumor, and an adult T-cell leukemia, a very aggressive form of CD4+ T-cell leukemia caused by human T-cell leukemia virus type I (HTLV-I) and characterized by clonal integration of HTLV-I in leukemia cells (see https: / / clinicaltrials.gov / ct2 / show / study / NCT02631746); as well as virus-related tumors in gastric cancer, nasopharyngeal cancer, cervical cancer, vaginal cancer, vulvar cancer, squamous cell carcinoma of the head and neck, and Merkel cell carcinoma, etc. (see https: / / clinicaltrials.gov / ct2 / show / study / NCT02488759; also see https: / / clinicaltrials.gov / ct2 / show / study / NCT0240886; https: / / clinicaltrials.gov / ct2 / show / NCT02426892).

[0261] Other cancers will be known to those skilled in the art.

[0262] 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 cancer / uterine cancer / cervical cancer, bladder cancer, head and neck cancer, leukemia, sarcoma, hepatobiliary cancer, 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 still more preferably, breast cancer.

[0263] In a more preferred embodiment, the cancer is selected from the group consisting of melanoma, colorectal cancer, and breast cancer.

[0264] In a preferred embodiment, the cancer is a BRAF-mutated cancer, i.e., a cancer having a BRAF mutation. More preferably, it is a BRAF V600E-mutated cancer.

[0265] In another embodiment, the BRAF mutation is selected from the group of mutations in the BRAF gene that are BRAF wild-type amplification, BRAF deletion, or mutations in valine at position 600, arginine at position 462, isoleucine at position 463, glycine at position 464, glycine at position 466, serine at position 467, glycine at position 469, aspartic acid at position 594, glycine at position 596, leucine at position 597, threonine at position 599, or lysine at position 601. Preferably, the mutation is at valine at position 600. In a more preferred embodiment, the mutation is selected from the group consisting of V600E, V600K, V600D, V600R, R462I, I463S, G464E, G466E, G466V, S467L, G469A, G469R, G469V, D594G, D594N, G596R, L597R, L597V, K601E, K601D, and K601R mutations. In a preferred embodiment, the BRAF mutation is the V600E mutation.

[0266] In another embodiment, the cancer being treated is wild-type BRAF cancer.

[0267] In a preferred embodiment, the cancer is melanoma. The term "melanoma" refers to malignant melanoma, a type of skin cancer that arises from melanin-forming cells. More preferably, the melanoma has a BRAF mutation.

[0268] In a preferred embodiment, the cancer is colorectal cancer. Preferably, it is colorectal cancer having a BRAF mutation.

[0269] In a preferred embodiment, the cancer is breast cancer. The term “breast cancer” refers to any malignant proliferative disorder of mammary cells, most commonly originating from the inner lining of the milk ducts or from the lobules that supply milk to the ducts. Cancer originating from the milk ducts is known as ductal carcinoma, while cancer originating from the lobules is known as lobular carcinoma. Preferably, the breast cancer has a BRAF mutation.

[0270] In a preferred embodiment, the cancer is triple-negative breast cancer (TNBC). The term triple-negative breast cancer is immunohistochemically defined by the fact that cancer cells lack 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. It is a highly malignant subtype of breast cancer, typically associated with relatively poor clinical outcomes, early recurrence, and a higher tendency for visceral metastasis compared to other types of breast cancer.

[0271] In another embodiment, the cancer is pancreatic cancer, and more particularly, pancreatic ductal adenocarcinoma.

[0272] In another embodiment, the cancer is a gliablastoma.

[0273] Gliablastoma, also known as gliablastoma or grade IV astrocytoma, is the most common and aggressive type of cancer that originates in the brain.

[0274] In another embodiment, the cancer is lung cancer.

[0275] The term “lung cancer” or “lung tumor” refers to a physiological condition in mammals characterized by uncontrolled cell proliferation in lung tissue. The term “lung cancer” is intended to mean any cancer of the lung, and includes non-small cell lung cancer and small cell lung cancer. In one embodiment, lung cancer is non-small cell lung cancer (NSCLC). In another embodiment, lung cancer is small cell lung cancer (SCLC).

[0276] The term non-small cell lung cancer (NSCLC), as used herein, refers to a group of heterogeneous diseases classified together, because their prognosis and management are generally similar. Examples include, according to the histological classification of the World Health Organization / International Society for Lung Cancer (Travis WD et al. Histological typing of lung and pleural tumours. 3rd ed. Berlin: Springer-Verlag, 1999): (i) Squamous cell carcinoma (SCC), which accounts for 30% to 40% of NSCLCs, develops in larger respiratory tracts but grows more slowly, meaning that the size of these tumors changes by the time of diagnosis. (ii) Adenocarcinoma, the most common subtype of NSCLC, accounting for 50% to 60% of NSCLC cases, develops near the gas exchange surface of the lung, and also includes the subtype bronchoalveolar carcinoma, which may have different responses to treatment. (iii) Large cell carcinoma, a rapidly growing form that grows near the surface of the lung. It is primarily diagnosed by exclusion, and if more investigations are completed, it is usually reclassified as squamous cell carcinoma or adenocarcinoma. (iv) Adenosquamous cell carcinoma, a type of cancer that includes two types of cells: squamous cells (thin, flat cells that line certain organs) and adenoid cells. (v) Carcinomas with pleomorphic, sarcomatous, or sarcomatous elements. This is a group of rare tumors that reflect histological heterogeneity and continuity in epithelial and mesenchymal differentiation. (vi) Carcinoid tumors are slow-growing neuroendocrine lung tumors that develop in cells capable of releasing hormones in response to stimuli from the nervous system. (vii) Salivary gland carcinoma, which develops in salivary gland cells located within the large airways of the lungs. (viii) Unclassified carcinomas, including cancers that do not fit into any of the lung cancer categories mentioned above.

[0277] In certain embodiments, the NSCLC is selected from squamous cell carcinoma of the lung, large cell carcinoma of the lung, and adenocarcinoma of the lung.

[0278] The term "small cell lung cancer (SCLC)" as used herein means a proliferation of small cells having distinctive and precise morphological features and containing dense neurosecretory granules that confer related endocrine / paraneoplastic syndromes on this tumor. Most cases occur in the larger airways (primary and secondary bronchi). These cancers grow rapidly and spread early in the course of the disease.

[0279] In an even more preferred embodiment, the lung cancer is an adenocarcinoma, more preferably a BRAF-driven lung adenocarcinoma, preferably a cancer associated with a mutation in the BRAF gene. Any of the mutations disclosed above may be present in the lung cancer.

[0280] In another preferred embodiment, the cancer to be treated according to the invention is characterized by expressing increased levels of BRAF, preferably mutant BRAF protein. The BRAF level is considered increased relative to a reference value when the level of BRAF in a sample of the cancer shows an increase of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or more. The reference value may be a value corresponding to the level of BRAF expression in a non-cancer sample.

[0281] In one embodiment, the cancer is a primary tumor. The term "primary tumor" as used herein means a tumor that has occurred in a location or organ and is present at that location but has not metastasized to another location.

[0282] In another embodiment, the cancer is metastatic cancer. In connection with the present invention, “metastasis” is understood as the transmission of cancer from one organ to another. It 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 the cancer cells of the original tumor. For example, if breast cancer spreads (metastasizes) to the lungs, the secondary tumor is formed of 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.

[0283] In one embodiment, the cancer is a solid tumor.

[0284] In another embodiment, the cancer is resistant to BRAF inhibitors.

[0285] "Resistance" is a decrease in the effectiveness of drug therapy in treating a disease or condition. The expression "cancer resistant to" means, as used herein, cancer that is resistant to a BRAF inhibitor, either spontaneously or acquiredly. Spontaneous resistance occurs when a BRAF inhibitor is ineffective from the start of treatment due to pre-existing resistance mechanisms. Acquired resistance occurs when a BRAF inhibitor becomes ineffective during the course of treatment and after clinical benefits have been observed.

[0286] All combinations of compounds and types of cancer of the present invention are included in the present invention.

[0287] In some embodiments, the combination or composition of the present invention results in the cessation of tumor growth. In some embodiments, the combination or composition of the present invention results in a reduction of tumor size (e.g., volume or mass) of at least 5%, 10%, 25%, 50%, 75%, 90%, or 99% compared to the size of the tumor before treatment. In some embodiments, the combination or composition of the present invention results in a reduction of tumor volume in the patient of at least 5%, 10%, 25%, 50%, 75%, 90%, or 99% compared to the amount of tumor before treatment.

[0288] As used herein, “subject” includes any animal that has cancer, exhibits symptoms of cancer, or is at risk of developing cancer or exhibiting symptoms of cancer. Preferred subjects (patients) include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, and domesticated animals or pets (e.g., cats or dogs). Non-human primates, preferably human patients, are also included. Preferably, the subject is a mammal, most preferably a human.

[0289] The combination or composition for use in the prevention and / or treatment of cancer may be administered using any amount and route of administration that is effective for treating or reducing the severity of cancer. 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, the mode of administration, etc. The compounds of the present invention are preferably formulated in unit dosing form for ease of administration and uniformity of dosage. The expression “unit dosing form,” as used herein, means a single dose of the drug that is physically separated and 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 the bounds of sound medical judgment. The specific effective dose level for any particular patient or organism will depend on a variety of factors, such as the disorder being treated and its severity; the activity of the specific compound used; the specific composition used, the patient's age, weight, overall health, sex, and diet; the timing of administration, the route of administration, and the 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 other factors well known in the medical field.

[0290] 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 BRAF inhibitor of the combination or composition in the treatment of cancer (to achieve a therapeutic effect).

[0291] In particular, 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 the polypeptide or a functionally equivalent variant or conjugate interacts synergistically with a BRAF inhibitor in the treatment of cancer in an amount that is sufficient.

[0292] The terms “synergistic effect” or “synergistically interacting” are used interchangeably. A synergistic effect is an effect that exceeds the additive effect expected by summing the actual effects of individual drugs in vitro. In vivo, a synergistic effect is a physiological effect, and more specifically, a therapeutic effect that exceeds the additive effect expected by summing the actual effects of individual drugs in vivo.

[0293] Therefore, when two drugs are administered, they together provide a measurable physiological effect, particularly a therapeutic effect, if the actual effect of the combined drugs exceeds what would be expected by summing the actual therapeutic effects of the individual drugs. In particular, 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 that exceeds the effect provided by the sum of both individual drugs. More specifically, a synergistic effect is provided when the first drug alone provides no measurable effect, the second drug alone provides some measurable effect, and the two drugs together provide a measurable effect that exceeds 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 the therapeutic agent. Preferably, in these combinations or compositions, a dose of one or the other therapeutic agent between 0.01 and 1,000 μg / kg body weight / day can be administered.

[0294] In a preferred embodiment, the degree of synergy is calculated using the zero interaction potency (ZIP) model (Bhagwan Y. et al. 2015. Comput Struct Biotechnol J, 13: 504-513). In a more preferred embodiment, the ZIP synergy score was calculated using the SynergyFinder 3.0 web application derived from Ianevsky A et al. Nucleic Acids Res. 2022;50(W1):W739-W743.

[0295] The amount of the therapeutic agent present in the combination or composition may be less than or equal to the amount typically administered in a composition containing the therapeutic agent as the sole active agent. Preferably, the amount of the therapeutic agent in the composition will be in the range of about 50% to 100% of the amount typically present in a composition containing the agent as the sole therapeutic active agent. In some embodiments, one therapeutic agent is administered at a dose 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. As used herein, the phrase "typically administered" means the amount of an FDA-approved therapeutic agent approved for administration per FDA label insertion.

[0296] The combinations or compositions of the present invention may be used in combination with known therapeutic processes, such as chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, hormones, or combinations thereof.

[0297] All embodiments of the combinations of the present invention are also applicable to the therapeutic methods of the present invention.

[0298] Manufactured goods and kits This disclosure also provides manufactured articles containing, in one or more containers, any one of the combinations or pharmaceutical compositions disclosed herein. In some embodiments, such manufactured articles include, for example, booklets, printed instructions, labels, or accompanying documents intended for users (e.g., distributors or end users) for combining and / or using the compositions of such manufactured articles for the prevention and / or treatment of cancer.

[0299] In some embodiments, the manufactured article includes, for example, a bottle, vial, cartridge, box, syringe, injector, or any combination thereof. In some embodiments, the label signifies the use or administration of the combination or the pharmaceutical composition in the manufactured article by the method disclosed herein. In some embodiments, the label indicates, for example, a dosage plan for use, a dosage plan for treating, preventing, or improving cancer, etc.

[0300] The contents of all listed references (including references, patents, patent applications, and websites) that can be cited throughout this application are expressly incorporated herein by reference in their entirety, for any purpose whatsoever.

[0301] All terms used herein will be understood in their common sense as known in the art unless otherwise specified. Other more specific definitions of certain terms used herein are described in detail below and are intended to apply uniformly throughout the description and claims unless another explicitly specified definition provides a broader definition. Throughout the description and claims, the word “comprise” and variations thereof are not intended to exclude other technical features, additives, components, or processes, and moreover, the word “comprise” encompasses the case of “consisting of.” Further objects, advantages, and features of the present invention may become apparent to those skilled in the art by careful reading of the description or may be learned through the practice of the invention. Furthermore, the present invention encompasses all possible combinations of the specific embodiments described herein.

[0302] In this specification and the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless explicitly negated in context. The terms “a” (or “an”), as well as the terms “one or more” and “at least one,” are interchangeable in this specification. Furthermore, as used herein, “and / or” is understood to refer to each of two specified features or components, one with or without the other. Thus, when the term “and / or” is used in this specification in a phrase such as “A and / or B,” it is intended to include “A and B,” “A or B,” “A” (only), and “B” (only). Similarly, when the term “and / or” is used in a phrase such as “A, B, and / or C,” it 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 (only); B (only); and C (a only). The term "about," when used in relation to a numerical value throughout this specification and the claims, indicates to those skilled in the art an interval of precision, familiarity, and tolerance. Generally, such an interval of precision is 15% ±. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in which this disclosure relates. Units, prefixes, and symbols are indicated in their forms accepted by their International System of Units (SI). Numerical ranges include the numerical values ​​that define such ranges. Unless otherwise specified, amino acid sequences are written from left to right in the amino in the carboxyl direction. The headings provided herein are not limitations on the various aspects of this disclosure, and they can be obtained by reference to this specification as a whole. Thus, the terms defined immediately thereafter are more fully defined by reference to this specification.

[0303] The present invention is illustrated by the following embodiments, which are to be considered merely illustrative and not to be considered limitations on the scope of the invention. [Examples]

[0304] Preparation 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 Escherichia coli (E. coli), cloned in a pET3a expression vector (Novagen), and purified from the BL21(DE3) arabinose-inducible (Invitrogen®) strain using a protocol adapted from the Max° purification protocol described in 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 cationic exchange chromatography, and its purity was confirmed by mass spectrometry, SDS-PAGE, and ultraviolet spectroscopy.

[0305] Omomyc, when combined with a BRAF inhibitor, acts synergistically to reduce the survival of melanoma cancer cells. BRAF mutation (BRAF V600E Human melanoma cell line (SkMel37) containing ) was used. Cells were seeded in 96-well plates and treated the following day with dabrafenib and Omomyc at increasing concentrations, either alone or in combination. Five days after treatment, the medium was removed, AlamarBlue® cell viability reagent was added to each well, and incubated for 3 hours until metabolized. Fluorescence was read using a microplate reader with excitation between 530–560 nm and emission at 590 nm.

[0306] The synergies were evaluated using the SynergyFinder 3.0 web application, derived from Ianevsky A et al. Nucleic Acids Res. 2022;50(W1):W739-W743.

[0307] The synergy score can be interpreted as the mean overresponse resulting from drug interactions. A synergy score close to 0 gives limited confidence in synergy or antagonism. Therefore, if the synergy score is less than -10, the interaction between the two drugs is likely antagonistic. Between -10 and 10, the interaction between the two drugs is likely additive. If it is greater than 10, the interaction between the two drugs is likely synergistic.

[0308] Figure 1 shows that treatment with omomyc or dabrafenib significantly reduces cell survival in melanoma SkMel37 cells. Treatment of these cells with a combination of omomyc and dabrafenib reveals that the combination of these drugs is synergistic, with a ZIP synergy score of 11.02 in the left panel of Figure 1 and a ZIP synergy score of 26.55 in the right panel of Figure 1.

[0309] [Table 4]

[0310] The same experiment was performed in SkMel37 cells (Figures 2 and 3) or SkMel28 cells (Figures 4 and 5) with different combinations of Omomyc and encorafenib or Omomyc and vemurafenib, and a synergistic effect was observed.

Claims

1. i) The group consisting of the following: a) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, b) A conjugate comprising a polypeptide containing the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof, and a chemical moiety that promotes intracellular uptake of the polypeptide or a functionally equivalent variant thereof. c) Polynucleotides encoding the polypeptide of a) or the conjugate of b), d) A vector containing the polynucleotides of c), and e) Cells that can secrete the polypeptide of a) or the conjugate of b) into the culture medium. A first component selected from, ii) The second component is a BRAF inhibitor and A combination that includes this item.

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 No. 1 is selected from the group consisting of Sequence ID No. 4, Sequence ID No. 5, Sequence ID No. 6, Sequence ID No. 7, Sequence ID No. 8, Sequence ID No. 9, and Sequence ID No.

10.

4. The combination according to any one of claims 1 or 3, wherein the chemical portion that promotes intracellular uptake of the polypeptide or a functionally equivalent modified thereof is a cell membrane permeable peptide sequence, and further, the cell membrane permeable peptide sequence and the polypeptide or a functionally equivalent modified 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 BRAF inhibitor is selected from the group consisting of dabrafenib, vemurafenib, and encorafenib.

7. The combination according to claim 6, wherein the BRAF inhibitor is dabrafenib.

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 medical settings.

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 melanoma, colorectal cancer, and breast cancer.

12. The combination or pharmaceutical composition for use according to claim 11, wherein the cancer is melanoma.

13. The combination or pharmaceutical composition for use according to any one of claims 10 to 12, wherein the cancer is a cancer having a BRAF mutation.

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

15. A combination or pharmaceutical composition for use according to any one of claims 9 to 14, wherein the first component is administered intravenously and the second component is administered orally.

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