TW202502371A - Combination therapy with braf inhibitors for the treatment of cancer - Google Patents

Abstract

The invention relates to a combination of a BRAF inhibitor with Omomyc, a functionally equivalent variant thereof, a conjugate comprising Omomyc or said functionally equivalent variant, a polynucleotide encoding said polypeptides, a vector comprising said polynucleotide and a cell capable of secreting the polypeptide or the conjugate. The invention also relates to pharmaceutical compositions containing the combination of the invention and to their medical uses, particularly their uses in the treatment of cancer.

Description

Combination therapy with BRAF inhibitors for the treatment of cancer

The present invention relates to the field of cancer, and more specifically, to a combination comprising a v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitor and Omomyc and use thereof in medicine, and more specifically, use thereof in preventing and/or treating cancer.

Cancer is the leading cause of death worldwide, accounting for nearly 10 million deaths in 2020. Cancer is a broad group of diseases characterized by the uncontrolled growth of abnormal cells.

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 signal transducing protein kinases and plays a role in regulating the mitogen-activated protein kinase (MAPK) signaling pathway, which is fundamental for cell proliferation, differentiation, and survival. Dysregulation of the MAPK signaling pathway through somatic mutations that directly overactivate this pathway often leads to cancer.

More than 30 BRAF gene mutations associated with human cancer have been identified in solid tumors. The frequency of BRAF mutations varies widely in human cancers, exceeding 50% in melanoma, 5-10% in colorectal cancer, and less common in ovarian and lung cancer. In 90% of cases, the thymine at nucleotide 1799 is replaced by an adenine. This results in a substitution of valine (V) by glutamine (E) at codon 600 (called V600E). This mutation leads to constitutive activation of BRAF and downstream MAPK signaling pathways. This constitutive activation results in increased cell proliferation and oncogenic activity.

Melanoma is an aggressive skin cancer caused by malignantly transformed melanocytes with increasing morbidity and mortality. The long-term survival rate of patients with metastatic melanoma is <10%, and their median overall survival is <1 year. This emphasizes the need to develop new, effective and more personalized treatments for patients with metastatic melanoma.

Drugs have been developed to treat cancers driven by BRAF mutations. Sorafenib was the first drug to be studied, but clinical trials failed to demonstrate any activity for sorafenib as a monotherapy or in combination with dacarbazine in patients with wild-type and BRAF-mutant metastatic melanoma. In addition, sorafenib does not selectively target mutant BRAF and can therefore cause intolerable off-target side effects. The U.S. Food and Drug Administration (FDA) has approved two BRAF inhibitors, dabrafenib and vemurafenib, for the treatment of advanced melanoma. However, despite the drug's effectiveness, 20% of tumors still become resistant to treatment, and the efficacy of BRAF inhibitor therapy is limited by the emergence of intrinsic and acquired resistance. More effective combination therapies are needed to overcome these limitations.

Therefore, there remains a need in the art to develop new and improved therapeutics for treating cancer that can overcome resistance to BRAF inhibitors, increase their efficacy and/or reduce their side effects.

In a first aspect, the present invention relates to a combination comprising: i) a first component selected from: a) a polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical part that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) a polynucleotide encoding the polypeptide of a) or the conjugate of b); d) a vector comprising the polynucleotide according to c); and e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into a medium; and ii) a second component, which is a BRAF inhibitor.

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.

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

In a fourth aspect, the present invention relates to use of the combination of the present invention or the pharmaceutical composition of the present invention for preventing and/or treating cancer.

The present invention is directed to providing novel therapeutic combinations for the prevention and treatment of cancer.

Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs.

All embodiments disclosed for one aspect of the present invention are also applicable to other aspects.

Combinations and pharmaceutical compositions of the invention The definitions provided herein and in each other aspect of the invention apply equally to the entire invention.

The inventors of the present invention surprisingly found that the combination of Omomyc and BRAF inhibitors has a synergistic effect in the treatment of cancer. The inventors showed that the combination of Omomyc and BRAF inhibitors dabrafenib, conafenib or vemurafenib synergistically reduced the viability of melanoma cell lines SkMel37 (Figures 1-3) and SkMel28 (Figures 4-5). Regardless of the dose, this synergistic effect is maintained, thereby producing beneficial effects, especially the increase in the therapeutic effect of the composition of the present invention relative to each of its components, so that it can achieve the same effect with a lower dose of each component, thereby reducing the side effects of the subjects receiving the composition of the present invention.

Omomyc can also be used to overcome resistance to BRAF inhibitors by enhancing sensitivity to them. The combination of the present invention expands the population that may respond to BRAF inhibitor-based therapies.

Therefore, the combination of Omomyc and BRAF inhibitors could be an effective treatment for both BRAF mutant and wild-type tumors, as well as for tumors that are resistant to BRAF inhibitors.

Therefore, in a first aspect, the present invention relates to a combination comprising: i) a first component selected from: a) a polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical part that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) a polynucleotide encoding the polypeptide of a) or the conjugate of b); d) a vector comprising the polynucleotide according to c); and e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into a medium; and ii) a second component, which is a BRAF inhibitor.

According to the present invention, the term "combination" represents various combinations of compounds (i) and (ii), such as in a composition formulated into a single preparation, in a combined mixture consisting of separate preparations of each component, such as a "tank-mix" which can be combined for joint use as a combined preparation, and in the combined use of single active ingredients in a manner of administration in a sequential manner (i.e. one after the other within a reasonably short time, such as a few hours or days) or simultaneous administration. In the present invention, compound (i) refers to a therapeutically effective amount of a polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, or a conjugate comprising a polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof, or a polynucleotide encoding the polypeptide or the conjugate, or a vector containing the polynucleotide, or a cell capable of secreting the polypeptide or the conjugate into a medium. In the present invention, compound (ii) refers to a therapeutically effective amount of a BRAF inhibitor. Preferably, the order of administration of compounds (i) and (ii) is not essential for the implementation of the present invention.

The combination may be a kit-of-parts, wherein each component is formulated and packaged separately.

The combination of compounds (i) and (ii) can be formulated for simultaneous, separate or sequential administration. In particular, if administration is not simultaneous, the compounds are administered at close times to each other. In addition, the compounds are administered in the same or different dosage forms or by the same or different administration routes, for example, one compound may be administered orally and the other compound may be administered 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 another embodiment, both compounds (i) and (ii) are administered intravenously.

The combination of the two compounds (i) and (ii) can be administered as follows: - as a combination, which is part of the same pharmaceutical preparation, the two compounds are always administered simultaneously. - as a combination of two units, each containing one substance, which can be administered simultaneously, sequentially or separately.

In one specific embodiment, compound (i) of the combination of the invention is administered independently from compound (ii), i.e. as two units but simultaneously.

In another specific embodiment, compound (i) of the combination of the present invention is administered first, and then compound (ii) is administered, i.e., compound (ii) is administered separately or sequentially.

In yet another specific embodiment, compound (ii) of the combination of the invention is administered first and then compound (i), i.e. compound (i) is administered separately or sequentially, as defined.

If administered separately, the compounds (i) and (ii) of the combination of the present invention may be administered at intervals of time from each other, for example within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours from each other. In another embodiment, the compounds (i) and (ii) of the combination of the present invention may be administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 days 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 one embodiment, administration of the first compound is discontinued prior to commencing administration of the second compound.

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

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

The terms "polypeptide" and "peptide" are used interchangeably herein to refer to polymers of amino acids of any length. The polypeptides of the present invention may contain modified amino acids and may be interrupted by non-amino acids. In a preferred embodiment, the polypeptide is formed only by amino acids. Preferably, the length of the polypeptide forming item (i) of the combination is 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 not more than 125 amino acids, and more preferably not more than 100 amino acids. In a preferred embodiment, the length of the polypeptide is 90 to 98 amino acids, preferably 90 to 95 amino acids, and more preferably 91 amino acids.

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

The term "natural amino acid" or "naturally occurring amino acid" includes the 20 naturally occurring amino acids; those amino acids that are commonly modified after translation in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other less common amino acids, including, but not limited to, 2-aminoadipate, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine.

As used herein, the term "non-natural amino acid" or "synthetic amino acid" refers to a carboxylic acid or derivative thereof that is substituted with an amine group at position "a" and is structurally related to a natural amino acid. Illustrative, non-limiting examples of modified or unusual amino acids include 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, lindane, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylaspartamide, hydroxylysine, isomeric hydroxylysine (aliohydroxy lysine), 3-hydroxyproline, 4-hydroxyproline, isoproline, alloleucine, N-methylglycine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline, norvaline, norleucine, ornithine, etc.

The polypeptides of the present invention may also contain non-amino acid moieties, such as hydrophobic moieties (various linear, branched, cyclic, polycyclic or heterocyclic hydrocarbons and hydrocarbon derivatives) attached to the peptides; various protective groups attached to the ends of the compounds to reduce degradation. Suitable protective functional groups are described in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991.

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

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

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

SEQ ID NO: 1 corresponds to

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

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

Omomyc is characterized in that it shows an increased dimerization ability with all three oncogenic Myc proteins (c-Myc, N-Myc and L-Myc). Omomyc can be derived from the bHLHZip domain of any Myc protein known in the art, provided that mutations that produce tumor suppression are retained. Therefore, Omomyc that can be used in the present invention can be derived from any mammalian species, including but not limited to livestock and farm animals (cattle, horses, pigs, sheep, goats, dogs, cats or rodents), primates and humans. Preferably, the Omomyc protein is derived from the human myc protein (Accession No. NP_002458, published on March 12, 2019).

The term "Myc" as used herein refers to a family of transcription factors that include c-Myc, N-Myc, and L-Myc. Myc proteins activate the expression of many genes by binding to the consensus sequence CACGTG (enhancer box sequence or E-box) and recruiting histone acetyltransferases or HATs. However, Myc can also act as a transcriptional repressor. By binding to the Miz-1 transcription factor and displacing the p300 co-promoter, Myc represses the expression of Miz-1 target genes. Myc also has a direct role in controlling DNA replication.

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

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

As used herein, "consisting essentially of" means that the specified molecule does not contain any additional sequence that would alter the activity of SEQ ID NO: 4.

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

The term "functional equivalent variant" refers to any polypeptide produced by inserting or adding one or more amino acids and/or deleting one or more amino acids and/or conservatively substituting one or more amino acids and/or chemically modifying the polypeptide of SEQ ID NO: 1 and substantially retaining the tumor inhibitory activity of SEQ ID NO: 1. Preferably, the functional equivalent variant refers to any polypeptide produced by inserting or adding one or more amino acids and/or deleting one or more amino acids and/or conservatively substituting one or more amino acids and substantially retaining the tumor inhibitory activity of SEQ ID NO: 1, more preferably any polypeptide produced by inserting or adding one or more amino acids relative to the polypeptide of SEQ ID NO: 1.

Those skilled in the art will appreciate that retaining tumor suppressor activity requires that the variant be able to dimerize with Myc and/or its obligate partner p21/p22Max and inhibit Myc activity, that the variant be able to translocate across the cell membrane, and that it be able to be transported across the nuclear membrane. In some embodiments, the homodimerization of the functionally equivalent variants of the polypeptides of the present invention is lower than that of Omomyc, or is not forced to form a homodimer by forming disulfide bonds. Specifically, the disulfide bond formation in the homodimer form of certain embodiments of the polypeptides of the present invention is less than that of the polypeptide Omomyc.

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

As used herein, reducing conditions involve the presence of a reducing agent, which is a compound that donates electrons to another chemical in a redox chemical reaction. Illustrative, non-limiting examples of reducing agents are dithiothreitol (DTT), b-hydroxyethanol, or TCEP (tris(2-carboxyethyl)phosphine). The amount of homodimers may be the same in vitro, and the difference between functionally equivalent variants and Omomyc only exists in cells in the presence of heterodimerization partners, where the absence of disulfide bonds makes the formation of heterodimers more likely.

Several assays can be used to determine homodimerization of peptides, with the illustrative but non-limiting example of monitoring thermal denaturation by circular dichroism, whereby dimerization can be quantitatively detected by folding and thermal stability.

Suitable functionally equivalent variants include polypeptides consisting essentially of the polypeptide of SEQ ID NO: 1. In the present context, "consisting essentially of" means that the specified molecule does not contain any additional sequence that would alter the activity of SEQ ID NO: 1.

In a preferred embodiment, the functional equivalent variant of SEQ ID NO: 1 is a polypeptide produced by inserting or adding one or more amino acids relative to the polypeptide of SEQ ID NO: 1. In one embodiment, the functional equivalent variant is produced by inserting less than 10 amino acids, more preferably less than 5 amino acids, and more preferably by inserting one amino acid. In a preferred embodiment, it is produced by inserting one methionine amino acid.

In another embodiment, the functionally equivalent variant of SEQ ID NO: 1 is a polypeptide produced by deleting one or more amino acids relative to the polypeptide of SEQ ID NO: 1. In one embodiment, the functionally equivalent variant is produced by deleting less than 10 amino acids, more preferably less than 5 amino acids, and more preferably by deleting one amino acid.

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

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

In another embodiment, a suitable functional variant of the targeting peptide is a variant in which one or more positions within the polypeptide of the present invention contain an amino acid that is a conservative substitution of an amino acid present in the above-mentioned protein. "Conservative amino acid substitution" is produced by replacing one amino acid with another amino acid having similar structure and/or chemical properties. For example, the following 6 groups respectively contain amino acids that are conservative substitutions between each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamine (E); 3) aspartamide (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). Selection of such conservative amino acid substitutions is within the skill of the art and is generally known in the art, as 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).

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

In another embodiment, the functionally equivalent variant of Omomyc comprises mutations at positions corresponding to E61, E68, R74 and R75 in the Omomyc sequence, wherein 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.

Multiple sequence alignment is an extension of pairwise alignment to include more than two sequences at a time. Multiple sequence alignment methods align all sequences in a given query. Preferred multiple sequence alignment programs (and their algorithms) are ClustalW, Clusal2W, or ClustalW XXL (see Thompson et al. (1994) Nucleic Acids Res 22:4673-4680). When c-Myc sequences and variant sequences from different organisms are compared (aligned) as described herein, one of ordinary skill in the art can readily identify positions in each sequence that correspond to positions E61T, E68I, R74Q, and R75N found in Omomyc, and introduce mutations in the Omomyc variants that correspond to the E61T, E68I, R74Q, and R75N mutations found in Omomyc derived from human c-Myc.

Suitable assays for determining whether a polypeptide can be considered a functionally equivalent variant of Omomyc include, but are not limited to: - Assays measuring the ability of the polypeptide to form a dimeric complex with Max and Myc, such as the assay based on reporter gene expression described in Soucek et al. (Oncogene, 1998, 17: 2463-2472), and PLA (protein ligation assay) or immunoprecipitation. - Assays measuring the ability of the polypeptide to bind to the Myc/Max recognition site (CACGTG site) in DNA, such as the electrophoretic mobility shift assay (EMSA) described in Soucek et al. (see above). - Assays measuring the ability to inhibit Myc-induced transactivation, such as the assay based on reporter gene expression under the control of the Myc/Max-specific DNA binding site described in Soucek et al. (see above). - Assays based on the ability of the polypeptide to inhibit the growth of cells expressing the myc proto-oncogene, as described by Soucek et al. (supra). - Assays measuring the ability of the polypeptide to enhance myc-induced apoptosis, such as the assay described by Soucek et al. (Oncogene, 1998: 17, 2463-2472). In addition, any assay generally known in the art for assessing apoptosis in cells may be used, such as Hoechst staining, propidium iodide (PI) staining or Annexin V staining, trypan blue, DNA laddering/fragmentation and TUNEL.

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

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

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

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

In addition, the functionally equivalent variant of Omomyc can also transduce cells after the variant is contacted with the cells. It should be understood that the functionally equivalent variant of Omomyc contains a protein transduction domain found in natural Omomyc or another functional protein transduction domain.

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

In addition, functionally equivalent variants of SEQ ID NO: 1 are also capable of being transported to the nuclei of target tumor cells.

In a preferred embodiment, if the polypeptide can be transported to the nucleus of target tumor cells with an efficiency of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of SEQ ID NO: 1, then the polypeptide is considered to be a functionally equivalent variant of SEQ ID NO: 1.

A suitable test for determining whether a polypeptide is a functionally equivalent variant of SEQ ID NO: 1 for its ability to be transported across the cell membrane and into the cell nucleus comprises double labeling of cells with a specific reagent for the polypeptide and a dye that specifically labels the cell nucleus (e.g., DAPI or Hoechst dye). The polypeptide of the present invention can be detected by confocal microscopy or fluorescence microscopy.

In another preferred embodiment, the 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 cellular uptake of the polypeptide or a functionally equivalent variant thereof.

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

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

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 cellular uptake of the polypeptide or a functionally equivalent variant of the polypeptide.

In one embodiment, the chemical moiety that promotes cellular uptake of the polypeptide is a lipid or a fatty acid.

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

In a preferred embodiment, the chemical part of the polypeptide containing the sequence SEQ ID NO: 1 or its functional equivalent variant that promotes cellular uptake is a cell penetrating peptide sequence. In this case, the conjugate will include a fusion protein, which contains the polypeptide containing SEQ ID NO: 1 or its functional equivalent variant and the cell penetrating peptide sequence.

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

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

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

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

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

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

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

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

Suitable fusion proteins of the present invention include the polypeptides Omomyc*TAT and Omomyc*LZArg defined as follows:

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

Suitable assays for determining whether a conjugate retains the cell membrane trafficking ability of Omomyc include, but are not limited to, assays that measure the ability of the conjugate to transduce cultured cells. Such assays are based on contacting the conjugate with cultured cells and detecting the presence of the conjugate at a location within the cell.

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

As used herein, the term "nuclear localization signal" (NLS) refers to an amino acid sequence of about 4-20 amino acid residues in length that is used to guide proteins into the cell 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 SV40 large T antigen NLS (PKKKRKV, SEQ ID NO: 48); Nucleoplasmin NLS (KRPAATKKAGQ AKKKK, 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); rpL23a NLS (VHSHKKKKKKIRTSPTFTTPKTLRLRRQPKYPRKSAPRRNKLDHY, SEQ ID NO: 54). In one embodiment of the present invention, the nuclear localization signal comprises the motif K(K/R)X(K/R).

In a more preferred embodiment, the nuclear localization signal is selected from PKKKRKV (SEQ ID NO:48), PAAKRVKLD (SEQ ID NO:56) and KRPAATKKAGQ AKKKK (SEQ ID NO:49).

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

A person skilled in the art will appreciate that it may be desirable for the conjugate of the present invention to further comprise one or more flexible peptides, which are linked to the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof, the cell-penetrating peptide sequence and/or the NLS. Therefore, in a specific embodiment, the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof is directly linked to the cell-penetrating peptide sequence. In another specific embodiment, the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof is linked to the cell-penetrating peptide sequence via a flexible peptide. In one embodiment, the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof is directly linked to the NLS. In another embodiment, the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof is linked to the NLS via a flexible peptide.

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

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

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

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

In another specific embodiment, the polypeptide of the conjugate for use according to the present invention is linked to the cell penetrating peptide sequence via a first flexible peptide linker and is linked to the NLS via a second flexible peptide linker.

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

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

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

Additional illustrative, non-limiting examples of tags for isolating or purifying conjugates or polypeptides or fusion proteins containing SEQ ID NO: 1 or variants thereof include Arg tags, FLAG tags (DYKDDDDK; SEQ ID NO: 59), Strep tags (WSHPQFEK, SEQ ID NO: 60), epitopes that can be recognized by antibodies, such as c-myc tags (recognized by anti-c-myc antibodies), HA tags (YPYDVPDYA, SEQ ID NO: 61), V5 tags (GKPIPNPLLGLDST, SEQ ID NO: 62), SBP tag, S tag, calcitonin 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, 60:523-525), amino acid sequence, such as AHGHRP (SEQ ID NO: 63) or PIHDHDHPHLVIHSGMTCXXC (SEQ ID NO: 64), β-galactosidase, etc.

If desired, the tag can be used to isolate or purify the fusion protein.

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

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

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

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

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

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

As used herein, the term "vector" refers to a nucleic acid sequence comprising the necessary sequences so that the polypeptide encoded by the polynucleotide of the present invention is produced after transcription and translation of the sequence in a cell. The sequence is operably linked to additional segments that enable it to replicate autonomously in the host cell of interest. Preferably, the vector is an expression vector, which is defined as a vector that, in addition to the region that replicates autonomously in the host cell, also comprises a region that is operably linked to the nucleic acid of the present invention and is capable of enhancing the expression of the nucleic acid product according to the present invention. The vector of the present invention can be obtained by technical means well known in the art.

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

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

Preferably, the vector comprises a polynucleotide of the present invention operably associated with a sequence that regulates the expression of the polynucleotide of the present invention. The regulatory sequence used in the present invention may be a nuclear promoter or enhancer sequence and/or other regulatory sequence that increases the expression of a heterologous nucleic acid sequence. In principle, any promoter may be used in the present invention as long as the promoter is compatible with the cell in which the polynucleotide is to be expressed. Therefore, promoters suitable for implementing the present invention include but are not necessarily limited to constitutive promoters, such as derivatives of eukaryotic viral genomes, such as polyoma viruses, adenoviruses, SV40, CMV, avian sarcoma viruses, hepatitis B viruses, metallothionein gene promoters, herpes simplex virus thymidine kinase gene promoters, LTR regions of retroviruses, immunoglobulin gene promoters, actin gene promoters, and the like. The invention relates to promoters of the invention, such as EF-1α gene promoters, and induced promoters in which protein expression is dependent on the addition of molecules or exogenous signals, such as the tetracycline system, the NFκB/UV light system, the Cre/Lox system and heat shock gene promoters, the regulatable RNA polymerase II promoters described in WO/2006/135436 and tissue-specific promoters.

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

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 endothelial cells, primary cultures of cells isolated from different organs (preferably cells isolated from pancreatic islets), hepatocytes, leukocytes including monocytes, interstitial cells, umbilical cord or adult (skin, lung, kidney and liver), osteoclasts, chondrocytes and other connective tissue cells. Established cell lines, such as Jurkat T cells, NIH-3T3, CHO, Cos, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 cells, C2C12 myoblasts and W138 cells, are also suitable. Those skilled in the art will appreciate that cells that can secrete the polypeptides of the present invention into a medium to form microparticles or microcapsules can be found, so that the cells have a longer life in patients. Materials suitable for forming the microparticle targets of the present invention include any biocompatible polymer material that allows continuous secretion of the therapeutic product and serves as a cell support. Therefore, the biocompatible polymer material can be, for example, a thermoplastic polymer or a hydrogen polymer. Among the thermoplastic polymers we have acrylic acid, acrylamide, 2-aminoethyl methacrylate, poly(tetrafluoroethylene-hexafluoropropylene) copolymer, (7-coumaryl) ethyl methacrylate, N-isopropylacrylamide, polyacrylic acid, polyacrylamide, polyamide, poly(amino)-p-xylene, poly(chloroethyl vinyl ether), polycaprolactone, poly(caprolactone-trimethylene carbonate) copolymer, poly(urea carbonate) urethane, poly(carbonate) urethane, polyethylene, polyethylene and acrylamide copolymer, polyethylene glycol, polyethylene glycol methacrylate, poly(p- terephthalate, poly(4-hydroxybutyl acrylate), poly(hydroxyethyl methacrylate), poly(N-2-hydroxypropyl methacrylate), poly(lactic-glycolic acid), poly(L-lactic acid), poly(γ-methyl-L-glutamic acid), poly(methyl methacrylate), poly(propylene fumarate), poly(propylene oxide), polypyridine, polystyrene, poly(tetrafluoroethylene), polyurethane, polyvinyl alcohol, ultra-high molecular weight polyethylene, 6-(p-vinylbenzamide) hexanoic acid, N-vinylbenzyl-D-maltamide, and copolymers comprising more than one of the above polymers. Among the hydrogel-type polymers we have natural materials such as alginate, agarose, collagen, starch, hyaluronic acid, bovine serum albumin, cellulose and its derivatives, pectin, chondroitin sulfate, fibroin and silk protein, as well as synthetic hydrogels such as Sepharose® and Sephadex®.

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

As used herein, "BRAF" (E.C. 2.7.11.1) refers to the serine/threonine protein kinase B-raf, also known as the proto-oncogene B-Raf, p94, or v-Raf murine sarcoma viral oncogene homolog B1. BRAF is a member of the RAF family of serine/threonine kinases (A-RAF, B-RAF, and C-RAF), which are part of the mitogen-activated protein kinase (MAPK) signaling pathway. BRAF is the most potent activator of MEK kinase, a downstream effector of RAF. In response to upstream activation signals, RAF recruitment to membrane-associated RAS leads to a cascade of downstream events (ERK signaling), including phosphorylation of MEK1 and MEK2 by RAF. The sequence of the human BRAF protein corresponds to sequence P15056 in the UniProt database (entry version 254 as of May 3, 2023).

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.

BRAF is frequently mutated in human cancers. Common activating mutations in the BRAF gene are BRAF wild-type expansion, BRAF deletion, and mutations in the BRAF gene (which are 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, aspartate at position 594, glycine at position 596, leucine at position 597, threonine at position 599, and lysine at position 601). Preferably, the mutation is valine at position 600. In a more preferred embodiment, the mutation is selected from 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 V600E mutation.

As used herein, "BRAF inhibitor" refers to any compound that can cause a decrease in BRAF activity, especially a decrease in the activity of BRAF mutants, including compounds that prevent BRAF gene expression, especially those that prevent BRAF mutant gene expression, and compounds that cause a decrease in BRAF mRNA or protein levels. BRAF inhibitors mainly inhibit the catalytic activity of the BRAF enzyme.

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

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

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

Nucleic acids contained in a sample (e.g., cells or tissue prepared from a subject) are first extracted according to standard methods, such as using lytic enzymes or chemical solutions, or by extraction with nucleic acid-binding resins according to the manufacturer's instructions.

If mRNA is measured in a biological sample, the biological sample can be treated to physically, mechanically or chemically disrupt the tissue or cell structure, thereby releasing intracellular components into an aqueous or organic solution to prepare the nucleic acid for further analysis. The nucleic acid is extracted from the sample by procedures known to those of ordinary skill in the art or commercially available. RNA is then extracted from frozen or fresh samples by any typical method in the art, such as Sambrook, J., et al., 2001. Molecular cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, N.Y., Vol. 1-3. Preferably, care is taken to avoid RNA degradation during the extraction process.

Expression levels can be determined using mRNA obtained from formalin-fixed, paraffin-embedded tissue samples. Archival pathology samples or biopsy samples can be first dewaxed and then mRNA isolated therefrom. An exemplary dewaxing method includes washing the paraffin-treated sample with an organic solvent such as xylene. The dewaxed sample can be rehydrated with an aqueous solution of a lower alcohol. Suitable lower alcohols include, for example, methanol, ethanol, propanol, and butanol. For example, the dewaxed sample can be rehydrated by continuous washing with a lower alcohol solution of reduced concentration. Alternatively, the sample is dewaxed 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 a resected tumor. In a specific embodiment, samples can be obtained from fresh tumor tissue or from OCT-embedded frozen tissue.

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

Relative gene expression quantification can be calculated based on the comparative cycle threshold (Ct) method, using a housekeeping gene as an endogenous control and a commercially available RNA control as a calibrator. The final result is determined according to the formula 2- ^{(Δ Ct sample -} Δ ^{Ct calibrator)} , where the Δ Ct values of the calibrator and sample are determined by subtracting the Ct value of the target gene from the value of the control gene.

Suitable methods for determining whether an inhibitor acts by reducing BRAF protein levels include quantification by conventional means, such as using antibodies that have the ability to specifically bind to the protein encoded by the BRAF gene (or a fragment thereof containing an antigenic determinant) and then quantifying the resulting antibody-antigen complex.

The antibodies used in these tests can be, for example, polyclonal antibody sera, fusion tumor supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' and F(ab')2, ScFv, bispecific antibodies (diabodies), triabodies, tetrabodies and humanized antibodies. At the same time, the antibodies can be labeled or unlabeled. Illustrative but non-exclusive examples of labels that can be used include radioisotopes, enzymes, fluorophores, chemiluminescent reagents, enzymatic substrates or cofactors, enzyme inhibitors, particles, coloring agents, etc. A variety of known assays using unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies) can be used in the present invention; these techniques include Western blot or Western transfer, enzyme-bound immunosorbent assay (ELISA), radioimmunoassay (RIA), competitive EIA (enzyme immunoassay), double antibody sandwich ELISA (DAS-ELISA), immunocytochemistry and immunohistochemistry techniques, techniques based on the use of biochips or protein microarrays containing specific antibodies or assays based on colloid precipitation such as test strips. Other methods for detecting and quantifying the levels of proteins of interest include affinity analysis, binding ligand assays, and other techniques.

Alternatively, BRAF protein levels can be determined by constructing a tissue microarray (TMA) containing collected subject samples and determining the expression level of the corresponding protein by immunohistochemistry. The intensity of immunostaining can be evaluated by two or more different pathologists and scored using a uniform and well-defined cut-off criteria to maintain the reproducibility of the method. Discrepancies can be resolved by simultaneous re-evaluation. In brief, considering the expression of each marker in tumor cells and the specific cut-off value, the results of immunostaining can be recorded as negative expression (0) versus positive expression, low expression (1+) versus intermediate expression (2+) and high expression (3+). As a general standard, the cut-off value is selected to promote reproducibility and, where possible, translation of biological events. Alternatively, immunostaining intensity can be assessed by using imaging techniques and automated methods, such as those disclosed in Rojo, M.G. et al. (Folia Histochem. Cytobiol. 2009; 47: 349-54) or Mulrane, L. et al. (Expert Rev. Mol. Diagn. 2008; 8: 707-25).

Alternatively, in another embodiment, BRAF protein levels are measured by Western blotting, which detects proteins previously resolved by gel electrophoresis under denaturing conditions and immobilized on a membrane (usually nitrocellulose) by incubation with specific antibodies and a visualization system (e.g., chemiluminescence).

BRAF inhibitors will 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 100% and all ranges from 5% to 100%. Suitable methods for determining whether an inhibitor acts by reducing BRAF activity include any method that allows 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 known prior art method may be used, such as Millipore's commercial kit B-Raf kinase assay kit based on detection of MEK1 phosphorylation. Methods for detecting BRAF activity are known in the art and include, but are not limited to, active BRAF pull-down assays, whole cell lysate immunoblotting, enzyme-linked immunosorbent assays (ELISAs) 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). Assays for determining enzyme activity are known to those of ordinary skill in the art and include, but are not limited to, initial rate assays, process curve assays, transient kinetic assays, and relaxation assays. Continuous assays for enzyme activity include, but are not limited to, spectrophotometry, fluorescence assays, calorimetric assays, chemiluminescence, light scattering, and microscale thermopheresis assays. Discontinuous assays for enzyme activity include, but are not limited to, radioassays and chromatographic assays. As known to those of ordinary skill in the art, factors that may affect enzyme activity include salt concentration, temperature, pH, and substrate concentration.

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

As used herein, a "small compound" refers to a molecule that modulates a biological process, in this case inhibiting the catalytic activity of BRAF. The compound may be natural or artificial.

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

In a preferred embodiment, the BRAF inhibitor useful in the present invention is selected from Table 1. Table 1: BRAF inhibitors suitable for use according to the present invention I Sorafenib (Bayer/Onyx), SB-590885 (GlaxoSmithKline), GDC-0879 (Genentech), PLX-4720 (Plexxikon), vemurafenib (Plexxikon/Hoffman La Roche), dabrafenib (GlaxoSmithKline), conafenib (LGX818), XL281 (Exelisis), BI882370 (Boehringer Ingelheim), AZ-628 (AstraZeneca), LY3009120 (Eli Lilly), TAK-632 (Takeda/Millennium), MLN2480 (Takeda/Millennium), CCT-196969 (ICR/Basilea), CCT-241161 (ICR/Basilea), BGB659 (Beigene), BGB283 (Beigene), RAF709 (Novartis), RAF265 (Novartis), PLX7904 (Plexxikon), PLX8394 (Plexxikon), CEP-32496 (Ambit Bio./Ignyta), belvarafenib / HM95573 (Hanmi Pharmaceutical/Genentech), LXH254 (Novartis) II Interfering RNA specific for the BRAF sequence III Antisense oligonucleotides specific for the BRAF sequence IV Ribozymes or DNA enzymes specific for the BRAF sequence V Inhibitory antibodies that specifically bind to BRAF and inhibit BRAF activity VI Aptamers and spiegelmers.

In a preferred embodiment, the BRAF inhibitor is selected from the compounds listed in Item I of Table I or a pharmaceutically acceptable salt thereof; more preferably, selected from SB-590885, GDC-0879, PLX-4720, vemurafenib, dabrafenib, connefenib, XL281, BI882370, AZ-628, LY3009120, ak-632, MLN2480, CCT-196969, CCT-241161, BGB659, BGB283, RAF709, RAF265, PLX7904, PLX8394, CEP-32496, belvarafenib, LXH254 and a pharmaceutically acceptable salt thereof.

In another embodiment, the BRAF inhibitor is selected from sorafenib, SB-590885, GDC-0879 and PLX-4720; preferably, selected from SB-590885, GDC-0879 and PLX-4720.

In another embodiment, the BRAF inhibitor is selected from vemurafenib, dabrafenib, contrafenib, XL281 and BI882370.

In another embodiment, the BRAF inhibitor is selected from AZ-628, LY3009120, TAK-632, MLN2480, CCT-196969, CCT-241161, BGB659, BGB283, RAF709, RAF265, PLX7904, PLX8394, CEP-32496, belvarafenib, and LXH254.

In a preferred embodiment, the BRAF inhibitor is selected from vemurafenib, dabrafenib, contrafenib, PLX-4720, BI882370, PLX7904 and PLX8394.

In a preferred embodiment, the BRAF inhibitor is selected from dabrafenib, vemurafenib, connefenib, sorafenib, GDC-0879 and PLX-4720; preferably, it is selected from dabrafenib, vemurafenib, connefenib, GDC-0879 and PLX-4720. More preferably, the BRAF inhibitor is selected from dabrafenib, vemurafenib and connefenib; even more preferably, the BRAF inhibitor is dabrafenib.

In another embodiment, the BRAF inhibitor is not sorafenib.

In a preferred embodiment, the BRAF inhibitor is an inhibitor targeting a mutation of BRAF, preferably, the inhibitor targets BRAF V600E; more preferably, the BRAF inhibitor is selected from sorafenib, GDC-0879, vemurafenib, dabrafenib, AZ-628, LY3009120, BGB283 and PLX7904.

All compounds listed in Item I of Table 1 also include pharmaceutically acceptable salts, solvates, polymorphs or cocrystals thereof. Prodrugs of these compounds are also included.

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

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

According to the present invention, the term "solvate" is understood to mean any solid form of the above compound having another molecule linked to it via a non-covalent bond. Examples of solvates include hydrates and alcoholates, preferably C1-C6 alcoholates, such as methanolates.

According to the present invention, the term "polymorph" is to be understood as a specific crystalline form of a compound which is capable of crystallizing in different forms.

As used herein, the term "co-crystal" is understood to mean a crystal structure composed of a BRAF inhibitor and at least one other component.

In another embodiment, the BRAF inhibitor is selected from the compounds listed in entries II, III, IV, V and VI of Table I.

As used herein, the term "interfering RNA" or "iRNA" refers to an RNA molecule capable of silencing the expression of BRAF, particularly BRAF mutant protein or any gene required for BRAF function. For this purpose, iRNAs are generally double-stranded oligonucleotides of at least 30 base pairs in length, and they preferably contain about 25, 24, 23, 22, 21, 20, 19, 18 or 17 RNA base pairs. Several different types of molecules have been effectively used in iRNA technology, including small interfering RNA (siRNA) (sometimes referred to as short interfering RNA or silencing RNA), microRNA (miRNA), miRNAs are generally different from siRNAs because they are processed from single-stranded RNA precursors and they only partially complement the target mRNA and short hairpin RNA (shRNA).

Small interfering RNA (siRNA) agents are able to inhibit target gene expression through interfering RNA. siRNA can be chemically synthesized, or can be obtained through in vitro transcription, or can be synthesized in the target cell. Typically, siRNA consists of double-stranded RNA with a length of 15 to 40 nucleotides and may contain 3' and/or 5' overhangs with a length of 1 to 6 nucleotides. The length of the overhang is independent of the overall length of the siRNA molecule. siRNA acts through post-transcriptional degradation or silencing of the target messenger.

siRNA may be named shRNA (short hairpin RNA), which is characterized by the antiparallel strands forming the siRNA being linked by a loop or hairpin region. siRNA consists of a short antisense sequence (19 to 25 nucleotides), followed by a loop of 5 to 9 nucleotides and a sense strand. shRNA may be encoded by a plasmid or a virus, in particular a retrovirus, more particularly a retrovirus and under the control of a promoter such as the U6 promoter of RNA polymerase III.

The siRNA used in the context of the present invention is substantially homologous to BRAF mRNA, particularly to mutant BRAF mRNA or its protein encoding genomic sequence. The term "substantially homologous" is understood to mean that the siRNA has a sequence that is sufficiently complementary or similar to the target mRNA, so that the siRNA can cause mRNA degradation through RNA interference. Suitable interfering siRNAs include siRNAs formed from RNA, and siRNAs containing chemically different modifications, such as: - siRNAs in which the linkages between nucleotides differ from those found in nature, such as phosphorothioate linkages; - conjugates of chain RNA with functional reagents such as fluorophores; - modifications of the ends of the RNA strands, in particular the 3' end by attachment of different functional hydroxyl groups at the 2'-position; - sugar-modified nucleotides, such as O-alkylated radicals at the 2'-position, such as 2'-O-methylribose or 2'-O-fluororibose; - alkali-modified nucleotides, such as halogenated alkalis (e.g. 5-bromouracil and 5-iodouracil), alkylated alkalis (e.g. 7-methyl-guanosine).

The siRNA and shRNA used in the context of the present invention can be obtained using a range of techniques known to those of ordinary skill in the art. For example, siRNA can be chemically synthesized from protected ribonucleoside phosphoramidites in a conventional DNA/RNA synthesizer. Alternatively, siRNA can be produced by plasmids and viral vectors through recombinant dicer, wherein one or more siRNA single-stranded or double-stranded coding regions are under the effective control of an RNA polymerase III promoter. RNase Dicer processes shRNA into siRNA in cells.

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

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, particularly sc-36368.

In another embodiment, the inhibitor is an "antisense oligonucleotide" specific for BRAF, i.e., a molecule whose sequence is complementary to the mRNA encoding BRAF, i.e., a molecule complementary to the cDNA coding strand. The antisense oligonucleotide may be complementary to the entire coding region, or to the same region including the coding region and the 5' and 3' non-translated regions. The antisense oligonucleotide may be composed of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length. The antisense oligonucleotide may be obtained by chemical synthesis or enzyme binding reaction known to those skilled in the art. For example, the antisense oligonucleotides may also contain modified nucleotides that increase the biological stability of the double-stranded DNA-RNA complex formed between the antisense oligonucleotide and the target polynucleotide, such as phosphorothioate derivatives, peptide nucleic acids, and acridine substituted oligonucleotides. Modified oligonucleotides that can be used to prepare antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetyl-citosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethyl-aminomethyluracil, dihydrouracil, β-D-galactosylguanosine, inosine, N6-isoprenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5 ... ne), N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, antisense nucleic acids can be produced biologically using a nucleic acid expression vector in which the antisense-directed nucleic acid is replicated.

Another group of compounds that may form part of the present invention are catalytically active nucleic acids known as ribozymes. "Ribozymes" comprise a catalytic region and a second region whose sequence is complementary to the target nucleic acid and confers substrate specificity to the ribozyme. Interaction between the ribozyme and its substrate, through hybridization and ligation between the target nucleic acid and the complementary region of the ribozyme, results in activation of the catalytic region, initiating inter- or intramolecular cleavage of the target nucleic acid. The basic considerations for ribozyme design are well known to those of ordinary skill in the art (see, e.g., Doherty and Doudna (Annu. Ref. Biophys. Biomolstruct. 2000; 30:457-75).

Another class of compounds that may form part of the compositions of the invention include inhibitory antibodies. According to the present invention, the term "inhibitory antibody" is understood to mean an antibody that binds to any BRAF, preferably to a mutant BRAF protein, causing inhibition of its catalytic activity.

Antibodies can be prepared using any method known to those of ordinary skill in the art. Thus, polyclonal antibodies are prepared by immunizing 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 that can bind to BRAF are identified, antibodies that can inhibit BRAF activity, particularly BRAF mutant activity, are selected using the above assays for determining BRAF activity. Suitable antibodies in the present invention include complete antibodies, fragments "Fab", "F(ab´)2"y"Fab", Fv, scFv, diabodies and bispecific antibodies comprising an antigen-binding variable region and a constant region.

Other compounds capable of inhibiting BRAF expression that may form part of the combination of the present invention include aptamers and spiegelmers. "Aptamers and spiegelmers" are single-stranded or double-stranded D-nucleic acids or L-nucleic acids that specifically bind to proteins, resulting in changes in the biological activity of the protein (BRAF, especially mutant BRAF protein). Aptamers and spiegelmers are 15 to 80 nucleotides in length, preferably 20 to 50 nucleotides in length.

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, in particular a conjugate of a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a BRAF inhibitor.

In some embodiments, the conjugation between components (i) and (ii) is achieved through a non-cleavable linker. In some embodiments, the conjugation between components (i) and (ii) is achieved through a cleavable linker. Exemplary non-cleavable linkers and cleavable linkers are described in US8088387, US8142784, WO2013075048, US6630579, US8512707, US9120854, US9023351, US20160095938, US9446146, WO2005009369, US57730 01, US6214345, US10111954, US8153768, US7829531, US20160082119, WO2018218004, US8568728, WO2015057699, US20170182181, US9198979, the contents of each of which are fully incorporated herein by reference.

In another embodiment, the combination of the first aspect of the present invention is not a combination comprising the following: i) a first component selected from: a) a polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical part that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; and ii) a second component, which is sorafenib.

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

As used in the present invention, the term "pharmaceutical composition" refers to a formulation suitable for administering a predetermined dose of one or more therapeutic agents to cells, cell populations, organs, tissues or animals with uncontrolled cell division (such as cancer).

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

As used herein, the expression "pharmaceutically effective amount" is understood as an amount capable of providing a therapeutic effect, which can be determined by a person skilled in the art by conventional means. The amount of Omomyc polypeptide, its functionally equivalent variant, conjugate, fusion protein, polynucleotide, vector, cell or BRAF inhibitor that can be combined in the pharmaceutical composition of the present invention will vary depending on the subject and the specific mode of administration. Those skilled in the art will appreciate that dosages may also be determined under the guidance of 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.

The appropriate dose of the active ingredient or ingredients in the pharmaceutical composition will depend on the type of cancer being treated, the severity and course of the disease, whether the composition is administered for prevention or treatment purposes, previous treatments, the patient's clinical history and response to the peptide or polypeptide, and the judgment of the attending physician.

The amount of the polypeptide, functionally equivalent variant, fusion protein, conjugate, polynucleotide, vector or cell containing the sequence SEQ ID NO: 1 is suitable for administration to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, the appropriate dosage level is generally about 0.01 to 500 mg per kilogram of patient body weight per day, which can be administered in a single dose or multiple doses. Preferably, the dosage level is about 0.1 to about 250 mg/kg per day; more preferably, about 0.5 to about 100 mg/kg per day.

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

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

A suitable dosage level may be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Within this range, the dosage may be 0.05 to 0.5 mg/kg, 0.5 to 5 mg/kg or 5 to 50 mg/kg per day. For oral administration, the composition is preferably provided in the form of tablets containing 1.0 to 1000 mg of active ingredient, in particular in the form of tablets containing 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0 and 1000.0 mg of active ingredient, for symptomatic adjustment of the dosage to be treated. The compound can be administered in a regimen of 1 to 4 times a day, preferably 1 or 2 times a day.

In one embodiment, the combination or the combination can be administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, or seven times a week. In one embodiment, the combination or the combination can be administered once a week. In another embodiment, the combination or the combination can be administered twice a week. In another embodiment, the combination or the combination can be administered four times a week. In another preferred embodiment, the first component of the combination or the combination is administered four times a week, and the second component of the combination or the combination is administered once a week. In another embodiment, the first component of the combination or the combination is administered twice a week, and the second component of the combination or the combination is administered once a week. The two compounds can be administered simultaneously or sequentially. When the compounds are administered sequentially, administration of the first compound is discontinued before administration of the second compound is started.

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

The amount of BRAF inhibitor depends on the specific agent used, and can be about 0.01 mg to about 200 mg per kg of subject body weight per day, preferably 0.01 mg to about 150 mg, more preferably 0.01 mg to about 100 mg, preferably 0.01 mg to about 75 mg, 0.01 mg to about 50 mg, more preferably 0.5 mg to about 50 mg, and preferably from about 1 mg to about 25 mg, once or more per day to achieve the desired therapeutic effect, preferably orally. In another preferred embodiment, the amount of the BRAF inhibitor is about 0.01 mg to about 40 mg per kg of subject weight per day; preferably 0.01 mg to about 30 mg; preferably 0.02 mg to about 25 mg; preferably 0.5 mg to about 15 mg; preferably 1 mg to about 10 mg; preferably 2 mg to about 10 mg; preferably 2 mg to about 5 mg, once or more per day, preferably orally administered to obtain the desired therapeutic effect. In a preferred embodiment, the amount of the BRAF inhibitor is about 2.5 mg per kg of subject weight per day, or 7.5 mg/m ² per day, preferably administered once a week, more preferably once a day, and more preferably orally administered. In a preferred embodiment, the amount of the BRAF inhibitor is about 0.5 mg per kilogram of the subject's body weight per day, or 1.5 mg/m ² per day, preferably once a week, more preferably once a day, and more preferably orally. In a preferred embodiment, the amount of the BRAF inhibitor is about 5 mg per kilogram of the subject's body weight per day, or 15 mg/m ² per day, preferably once a week, more preferably once a day, and more preferably orally. In another preferred embodiment, the amount of the BRAF inhibitor is about 10 mg per kilogram of the subject's body weight per day, or 30 mg/m ² per day, preferably once a week, more preferably once a day, and more preferably orally. In another preferred embodiment, the amount of the BRAF inhibitor is about 50 mg per kg of the subject's body weight per day, or 150 mg/m ² per day, preferably once a week, more preferably once a day, and more preferably orally. In another preferred embodiment, the amount of the BRAF inhibitor is about 100 mg per kg of the subject's body weight per day, or 300 mg/m ² per day, preferably once a week, more preferably once a day, and more preferably orally. The BRAF inhibitor is preferably administered 7 days per week, preferably for 4 weeks.

The pharmaceutical composition according to the present invention contains a first component (i) and a second component (ii), wherein the first component is selected from the polypeptide containing SEQ ID NO: 1 according to the present invention, its functionally equivalent variant, fusion protein, conjugate, polynucleotide, vector or cell, and the second component is a BRAF inhibitor, and the pharmaceutical composition can exist as a single preparation (for example, as a tablet or capsule containing a fixed amount of each component), or in another aspect, can exist as separate preparations to be combined subsequently for combined, sequential or separate administration. The composition of the present invention also includes a preparation in the form of a kit, in which each component is separately formulated but packaged in the same container. It will be appreciated by those skilled in the art that the formulations of the different components of the pharmaceutical composition according to the present invention may be similar, in other words, similarly formulated (in tablets or pills), which allows them to be administered by the same route. In the case where the different components of the present invention are separately formulated, the two components may be present in a blister. Each blister contains the medication that must be taken that day. If the medication must be administered several times a day, the medication corresponding to each administration may be placed in different parts of the blister, preferably recording the time they should be administered in each part of the blister. Alternatively, the components of the composition of the present invention may be formulated in different ways so that the different components are administered in different ways. Thus, it is possible to formulate the first component into a dosage form for intravenous administration and the second component into a tablet or capsule for oral administration, or vice versa. A person skilled in the art can adjust the ratio between the components of the combination or pharmaceutical composition of the present invention according to the antitumor agent used in each specific case and the desired indication. Thus, the present invention contemplates a composition in which the ratio between the amounts of component (i) and component (ii) may range from 50:1 to 1:50, in particular from 40:1 to 1:40, in particular from 30:1 to 1:30, in particular from 20:1 to 1:20, from 1:10 to 10:1, or from 5:1 to 1:5. In a more specific embodiment, the ratio ranges from 1:1 to 1:5, preferably from 1:1 to 1:3. In a more specific embodiment, the ratio ranges from 1:1 to 1:1.5, preferably from 1:1.3 to 1:1.4, and more preferably from 1:1.34. In another specific embodiment, the ratio ranges from 1:1 to 1:2.8, preferably from 1:2.6 to 1:2.7, and more preferably from 1:2.67. In another specific embodiment, the ratio ranges from 30:1 to 5:1, preferably from 30:1 to 8:1, more preferably from 25:1 to 15:1, and more preferably from 20:1 to 10:1. In a preferred embodiment, the ratio ranges from 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. When the ratio of component (i): component (ii), preferably Omomyc: dabrafenib, Omomyc: vemurafenib, or Omomyc: conafenib; even more preferably, the ratio of Omomyc: dabrafenib ranges from 50:1 to 200000:1; preferably 75:1 to 150000:1; preferably 100:1 to 125000:1; preferably 102.78:1 to 105279.67:1; Very good results were obtained with a preferred ratio of 105:1 to 100,000:1; more preferably 110:1 to 100,000:1; more preferably 150:1 to 100,000:1; more preferably 200:1 to 100,000:1; more preferably 500:1 to 100,000:1; more preferably 1,000:1 to 100,000:1; more preferably 5,000:1 to 50,000:1; and more preferably 10,000:1 to 30,000:1. In a preferred embodiment, the ratio of component (i): component (ii) is preferably Omomyc: dabrafenib, Omomyc: vemurafenib or Omomy: conafenib; even more preferably, the ratio of Omomyc: dabrafenib ranges from 205.56:1 to 105279.67:1; more preferably 210:1 to 100000:1; more preferably 102.78:1 to 105279.67:1; even more preferably 105:1 to 105000:1. While these ratios are effective for treating any type of cancer, more preferably, these ratios are achieved when treating a cancer selected from melanoma, colorectal cancer, and breast cancer, and even more preferably when treating melanoma, preferably melanoma with a BRAF mutation, more preferably melanoma with a BRAF V600E mutation.

Preferably, these ratios are by weight.

The pharmaceutical compositions or components of the combinations of the present invention can be administered simultaneously. "Simultaneous administration" includes co-administration of two therapeutic agents without regard to the relative frequency or time of administration of each agent. Thus, simultaneous administration includes co-administration of two therapeutic agents at the same time and with the same frequency of administration. In addition, simultaneous administration refers to co-administration of two therapeutic agents, wherein one agent is administered more frequently than the other agent or agents. In addition, simultaneous administration refers to co-administration of two therapeutic agents, wherein one agent is administered only once during the administration period of the other agent or agents.

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

In a preferred embodiment, the combination or component (i) of the pharmaceutical composition of the present invention is administered intravenously, and the BRAF inhibitor is administered orally. For intravenous administration, the preferred dosage of the combination or component (i) of the composition of the present invention, the preferred dosage of the polypeptide or its functionally equivalent variant, fusion protein or conjugate ranges from 0.01 to 250 mg/kg, which can be administered in a single dose or multiple doses, more preferably 0.1 to about 100 mg/kg per day. The preferred dosage of the BRAF inhibitor for oral administration is 0.01 to 200 mg/Kg, preferably 0.01 to 150 mg/Kg, more preferably 0.1 to 100 mg/Kg, more preferably 0.5 to 100 mg/Kg, even more preferably 10 to 100 mg/kg, and most preferably 50 to 100 mg/Kg. In another preferred embodiment, the preferred dosage of the BRAF inhibitor for oral administration 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.

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

The pharmaceutical composition of the present invention may also contain one or more additional compounds for the prevention and/or treatment of pathological conditions in which uncontrolled cell division is present (e.g. cancer). Such additional compounds, such as antitumor agents, may form part of the pharmaceutical composition as independent entities. In a preferred embodiment, the combination or pharmaceutical composition of the present invention comprises one or more antitumor agents selected from cytotoxic agents, antiangiogenic agents, anti-metastatic agents and anti-proliferative agents.

The pharmaceutical composition of the present invention also contains one or several additional pharmaceutically acceptable excipients. A "pharmaceutically acceptable excipient" is understood to be a therapeutically inactive substance that is allegedly used to incorporate the active ingredient and is acceptable to patients from a pharmacological/toxicological point of view and to pharmaceutical chemists who manufacture it from a physical/chemical point of view in terms of composition, formulation, stability, patient acceptance and bioavailability. The excipient may be a carrier. As used herein, "carrier" refers to any substance used to improve the delivery and effectiveness of the active ingredient in the pharmaceutical composition. In a preferred embodiment, the carrier does not allow components (i) and/or (ii) to be delivered directly to the cytoplasm of the cell, i.e. the carrier cannot fuse with the plasma membrane of the target cell. Examples of pharmaceutically acceptable carriers include one or more of the following: water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof. In many cases, it is preferred to include an isotonic agent in the combination or composition, such as a sugar, a polyol such as mannitol, sorbitol, or sodium chloride. Pharmaceutically acceptable carriers may also include a small amount of auxiliary substances, such as a wetting agent or emulsifier, a preservative, or a buffer, which may increase the shelf life or effectiveness of the components that constitute the composition or a part of the composition of the present invention. 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). Non-limiting examples of carriers are a series of sugars, such as lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol; a series of starches, such as corn starch, wheat starch, rice starch and potato starch; a series of celluloses, such as cellulose, methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethylcellulose; and a series of fillers such as gelatin and polyvinylpyrrolidone. In some cases, a disintegrant, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate, may be added.

The amount and nature of the pharmaceutically acceptable excipients depend on the desired dosage form. Pharmaceutically acceptable excipients are known to those of ordinary skill in the art (Faulí y Trillo C. (1993) "Tratado de Farmacia Galénica", Luzán 5, S.A. Ediciones, Madrid). The compositions can be prepared by conventional methods known in the art ("Remington: The Science and Practice of Pharmacy", 20th edition (2003) Genaro A.R., ed., Lippincott Williams & Wilkins, Philadelphia, US).

For pharmaceutical compositions comprising an agent that is a nucleic acid molecule, the nucleic acid molecule can be present in any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acids, and bacterial, viral, and mammalian expression systems, such as the recombinant expression constructs provided herein. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA can also be "naked," as described, for example, in Ulmer et al., Science 259:1745-49, 1993, and summarized by Cohen, Science 259:1691-1692, 1993. Uptake of naked DNA can be increased by coating the DNA onto biodegradable beads that are efficiently transported into cells.

Nucleic acid molecules can be delivered to cells according to any of several methods described in the art (see, e.g., Akhtar et al., Trends Cell Bio. 2:139 (1992); Delivery Strategies for Antisense Oligonucleotide Therapeutics, 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); U.S. Patent No. 6,395,713; International Patent Application Publication No. WO 94/02595); Selbo et al., Int. J. Cancer 87:853-59 (2000); Selbo et al., Tumour Biol. 23:103-12 (2002); U.S. Patent Application Publication Nos. 2001/0007666 and 2003/077829). Such delivery methods known to those of ordinary skill in the art include, but are not limited to, encapsulation in liposomes by iontophoresis, or by incorporation into other vehicles such as biodegradable polymers; hydrogels; cyclodextrins (see, e.g., Gonzalez et al., Bioconjug. Chem. 10: 1068-74 (1999); Wang et al., International Application Publication Nos. WO 03/47518 and WO 03/46185); poly(lactic-co-glycolic) acid (PLGA) and PLCA microspheres (also useful for delivering peptides and polypeptides and other substances) (see, e.g., U.S. Patent No. 6,447,796; U.S. Patent Application Publication No. 2002/130430); biodegradable nanocapsules; and bioadhesive microspheres, or via protein carriers (International Application Publication No. WO 00/53722). In another embodiment, the nucleic acid molecules can also be formulated or complexed with polyethyleneimine and its derivatives, such as polyethyleneimine-polyethylene glycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethylene glycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives (see also, for example, U.S. Patent Application Publication No. 2003/0077829).

In a specific embodiment, when the composition or combination according to the present invention comprises nucleic acids (DNA, RNA, siRNA, antisense oligonucleotides, ribozymes, aptamers and spiegelmers), the pharmaceutical composition can be formulated as a composition for gene therapy; as an illustration and not limitation, the pharmaceutical composition can contain a viral vector or a non-viral vector comprising a suitable polynucleotide or gene construct. As an illustration and not limitation, the vector can be a viral vector, such as a vector based on a retrovirus, adenovirus, etc., or a non-viral vector such as ADN-liposomes, ADN-polymers, ADN-polymer-liposome complexes, etc. [See "Nonviral Vectors for Gene Therapy" by Huang, Hung and Wagner, Academic Press (1999)]. The vector containing the corresponding polynucleotide or gene construct can be directly administered to a subject by conventional methods. Alternatively, the vector can be used to transform, transfect or infect cells in vitro, such as mammalian cells, including humans, which are then implanted into the human or animal body to achieve the desired therapeutic effect. For administration to humans or animals, the cells will be formulated in a suitable medium that does not adversely affect cell viability.

The combination or pharmaceutical composition of the present invention can be administered by any type of suitable route, for example by oral route, topical route, by inhalation or parenteral route, and thus pharmaceutically acceptable formulations necessary for the formulation of the desired dosage form are included. Other routes of administration may be rectal, intracisternal or intravaginal administration. The preferred route of administration of the combination or pharmaceutical composition is the intravenous route for component (i) and the oral route for component (ii).

"Oral route" is understood to mean that the pharmaceutical composition enters the organism after being swallowed. In a specific embodiment, the pharmaceutical composition of the present invention may be in a dosage form suitable for administration via the oral route, whether solid or liquid. Dosage forms suitable for administration by the oral route may be tablets, capsules, syrups or solutions, and may contain any conventional excipients known in the art, such as binders, such as syrup, gum arabic, gelatin, sorbitol or polyvinyl pyrrolidone; fillers, such as lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; compression lubricants, such as magnesium stearate; disintegrants, such as starch, polyvinyl pyrrolidone, sodium hydroxyacetate starch or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulfate. Solid oral compositions can be prepared by conventional methods of mixing, filling or compression. Repeated blending operations can be used to distribute the active agent throughout those compositions using large amounts of fillers. Such operations are routine in the art. Tablets can be prepared, for example, by wet granulation or dry granulation and optionally coated according to methods known in conventional pharmaceutical practice, in particular with an enteric coating.

In another aspect, "topical route" is understood as administration by non-systemic routes, and includes external application of the pharmaceutical composition of the present invention on the epidermis, in the mouth, and instillation of the composition into the ears, eyes, and nose, and wherein the composition does not significantly enter the bloodstream. Topical or transdermal administration forms of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches.

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

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

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

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

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

As used herein, the term "parenteral" includes administration via the intravenous route, the intraperitoneal route, the intramuscular route, or the subcutaneous route. Subcutaneous, intramuscular, and intravenous dosage forms of parenteral administration are generally preferred. In one embodiment, the combination or pharmaceutical composition is administered intravenously.

In one embodiment, the combination or pharmaceutical composition of the present invention can be suitable for parenteral administration thereof, such as a sterile solution, suspension or lyophilized product in the form of an appropriate dosage unit. Combinations or pharmaceutical compositions suitable for injection thereof include sterile aqueous solutions (when they are soluble in water), or dispersions and sterile powders for the extemporaneous preparation of sterile injection solutions or dispersions. For intravenous administration, some suitable carriers include saline solutions buffered with phosphate (PBS). In all cases, the combination or composition must be sterile and must have fluidity for easy injection. The combination or composition must be stable under preparation and storage conditions and must prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, pharmaceutically acceptable polyols such as glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the desired particle size for the dispersion, and by the use of a surfactant. The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In most cases, it is preferred to include an isotonic agent, such as a sugar; a polyol such as mannitol, sorbitol; or sodium chloride in the composition. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption, such as aluminum and gelatin monostearate.

Injectable sterile solutions can be prepared by mixing the required amount of the active compound into a suitable solvent containing one or a combination of the above-mentioned ingredients, as required, and then sterilizing by filtering through a sterile membrane. Generally, dispersions are prepared by mixing the active compound into a sterile carrier containing a basic dispersion medium and the remaining required ingredients listed above. For sterile powders for preparing injectable sterile solutions, the preferred preparation methods are vacuum drying and freeze drying, which produce a powder with the active ingredient and any desired additional ingredients from a previously filtered sterile solution.

The compositions or pharmaceutical compositions of the invention may be suitably administered by pulse infusion, for example, using a reduced dose of the composition. Preferably, the dose is administered by injection, more preferably by intravenous or subcutaneous injection, depending in part on whether the administration is acute or chronic.

Alternatively, as described above, the different components of the composition are administered in different ways.

Thus, in one embodiment, the combination or component (i) of the combination, preferably the polypeptide or functionally equivalent variant or conjugate of the invention, is administered intravenously, while the BRAF inhibitor is administered orally.

In another embodiment, components (i) and (ii) are administered intravenously.

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

The dosage form of the composition for intranasal and intrapulmonary administration is preferably a liquid, a suspension or a solid. A suspension is a liquid formulation containing solid particles dispersed in a liquid carrier. The dosage form is preferably metered. For example, a metered droplet/spray refers to a dispenser comprising a droplet/spray delivering a droplet/spray containing a metered dose (predetermined amount) of the composition used according to the present invention.

In the case of intranasal administration routes, one preferred dosage form includes nasal drops. Drops are deposited primarily in the back of the nose and thus move rapidly into the nasopharynx. The problem with drops is often how to precisely control the amount of drug, which is particularly important for the administration of the composition.

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

Another type of intranasal dosage form is the nasal aerosol. Nasal aerosols differ from nasal sprays in the method by which the composition is dispensed: in an aerosol, the compound is dispensed by high pressure and released through a valve. In a spray, the compound is dispensed by pushing hard through a micropump bucket, and the pressure in the vial is similar to atmospheric pressure. Aerosols have similar advantages to sprays.

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

Liquid intranasal dosage forms generally have higher concentrations than corresponding intravenous dosage forms due to the limited volume of the composition that can be effectively delivered to the nasal mucosa. When the substance is poorly soluble or unstable in liquid form, powders can be used to administer the composition of the present invention. Other advantages of powders are that they do not require preservatives and generally have higher stability than liquid formulations. The main limitation of the use of intranasal powders is their irritation to the nasal mucosa.

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

Another dosage form for intrapulmonary administration is an inhalation spray. Inhalation sprays are usually water-based and do not contain any propellant. They are delivered to the lungs by oral inhalation.

Nebulized inhalation solutions and suspensions can also be used to deliver conjugates via the intrapulmonary route. Nebulized inhalation solutions and suspensions are typically water-based formulations containing the compositions according to the present invention. Nebulized inhalation solutions and suspensions deliver the compositions to the lungs via oral inhalation to produce systemic effects and are used with a nebulizer.

Dry powder inhalation is an alternative to aerosol inhalation. The composition is usually contained in a capsule for manual loading or in an inhaler. The dry powder is usually delivered to the lungs by inhalation through the mouth from the inhaler. The dry powder used herein can be formulated as pure. Pure preparations contain only the drug or essentially only the drug, for example as a sprayable dry powder. The dry powder used herein can also be formulated with a carrier such as lactose.

Intrapulmonary dosage forms are preferably metered, i.e., delivered to the lungs in a predetermined amount.

In the context of the present invention, devices for intranasal delivery 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. Intranasal delivery devices can be filled with a single dose or multiple doses of an intranasal formulation.

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

Alternatively, a dry powder inhaler may be used to deliver the composition intrapulmonary. A dry powder inhaler provides a single dose or multiple doses of the powder.

Another type of device used for intrapulmonary delivery is a nebulizer, which includes ultrasonic nebulizers and air jet nebulizers. In ultrasonic nebulizers, ultrasonic waves are created in the ultrasonic nebulizer chamber by the vibration of ceramic piezoelectric transistors when electrically excited. This generates an aerosol cloud on the surface of the solution. Aerosols produced by air jet nebulizers are produced when compressed air is forced through an orifice. Liquid can be drawn back from a vertical nozzle (Bernoulli effect) to mix with the air jet, which is then atomized using a baffle to promote the formation of the aerosol cloud.

In one embodiment, the components of the combination or pharmaceutical composition of the present invention are prepared with a carrier that will protect the components, particularly component (i), from rapid elimination from the body, such as controlled release formulations including implants and microcapsule administration systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyethylene glycol acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing the formulations are clear to those of ordinary skill in the art. These materials can also be purchased from Alza Corporation and Nova Pharmaceuticals, Inc.

Sustained release compositions also include crystalline preparations suspended in a suitable preparation that can keep the crystals suspended. These preparations can produce a sustained release effect when injected subcutaneously or intraperitoneally. Other compositions also include component (i) and/or component (ii) encapsulated in liposomes. Liposomes containing these components are prepared by known methods, such as Epstein et al., Proc. Natl. Acad. Sci. USA, (1985) 82:3688-3692; Hwang et al., Proc. Natl. Acad. Sci. USA, (1980) 77:4030-4034; EP 52,322; EP 36,676; EP 88,046; EP 143,949. In a preferred embodiment, component (i) and/or component (ii) are contained in liposomes, preferably both components are contained in liposomes, and more preferably are contained in the same liposome.

Despite the fact that Omomyc, its functional equivalent variants, conjugates and fusion proteins of the present invention can be transported across biological membranes, Omomyc, any functional equivalent variants, conjugates, polynucleotides, vectors or cells can also be formulated into nanoparticles. Nanoparticles can help maintain the integrity of the components in the biological fluid until they reach the target organ. In addition, in the case where the composition contains component (ii) or other anti-tumor agents, the encapsulation of the composition can reduce the secondary effects caused by the anti-tumor agents. In addition, the nanoparticles can also be modified to include portions that allow the nanoparticles to target organs of interest. In this way, the combination or component (i) of the composition of the present invention will be delivered to the vicinity of the target organ, promoting the entry of component (i) into the cells where its biological activity is required.

Thus, in another embodiment, component (i) of the combination or composition of the present invention forms part of a nanoparticle. In another embodiment, both components of the combination or composition of the present invention form part of a nanoparticle, preferably, both components are within the same nanoparticle.

As used herein, the term "nanoparticle" refers to any material with a size range of 1-1000 nm. In some embodiments, the size of the nanoparticle ranges from 2-200 nm, preferably from 2-150 nm, and even more preferably from 2-100 nm. Nanoparticles that can be used in the present invention include nanoscale materials, such as lipid-based nanoparticles, superparamagnetic nanoparticles, nanoshells, semiconductor nanocrystals, quantum dots, polymer-based nanoparticles, silicon-based nanoparticles, silica-based nanoparticles, metal-based nanoparticles, fullerenes, and nanotubes. Molecules can be embedded in the nanoparticle matrix or adsorbed on its surface, preferably, the molecules are embedded in the nanoparticles.

In a preferred embodiment, the nanoparticles are liposomes.

Targeted delivery can be achieved by adding ligands without compromising the ability of the nanoparticles to deliver their contents. It is envisioned that this will enable delivery to specific cells, tissues, and organs. The targeting specificity of ligand-based delivery systems is based on the distribution of ligand receptors on different types of cells. Targeting ligands can be non-covalently or covalently bound to nanoparticles and can be conjugated to nanoparticles by a variety of methods described herein.

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

It should be understood that the nanoparticle formulation of the present invention is not intended to promote component (i) and/or component (ii) to enter the interior of the cell or is not only intended to promote component (i) and/or component (ii) to enter the interior of the cell, but rather to protect component (i) and/or component (ii) from degradation and/or promote the targeting of nanoparticles to organs of interest.

In one example, the nanoparticles can be made from a biodegradable polymer such as polybutylcyanoacrylate (PBCA). Examples of elemental nanoparticles include carbon nanoparticles and iron oxide nanoparticles, which can then be coated with oleic acid (OA)-Pluronic(R). In this approach, drugs (e.g., hydrophobic drugs or water-insoluble drugs) are loaded into the nanoparticles. Other nanoparticles are made from silica.

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-co-hexadecyl cyanoacrylate, and poly[HEMA-co-methacrylic acid]; proteins such as fibrinogen, collagen, gelatin, and elastin; and polysaccharides such as branched starch, linear starch, and chitosan.

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

The nanoparticles may also include nanosized micelles. The micelles may be formed from any polymer described herein. Exemplary polymers for forming micelles include block copolymers such as poly(ethylene glycol) and poly(ε-caprolactone). (For example, a PEO-b-PCL block copolymer comprising a polymer of ε-caprolactone and α-methoxy-ω-hydroxy-poly(ethylene glycol)).

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

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

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

Therapeutic Uses of the Invention In one aspect, the present invention relates to the use of the combination or drug composition of the present invention for medicine.

In another aspect, the present invention relates to use of the combination or pharmaceutical composition of the present invention for preventing and/or treating cancer.

In another aspect, the present invention relates to the combination or pharmaceutical composition of the present invention for preparing a medicament for preventing and/or treating cancer.

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

In a preferred embodiment, the preventive or therapeutic method according to the present invention comprises directly using a combination or composition comprising a polypeptide, wherein the combination or composition comprises a polypeptide containing Omomyc, a functionally equivalent variant thereof, a conjugate or a fusion protein. Therefore, in a preferred embodiment, the preventive or therapeutic method according to the present invention does not involve the administration of a nucleic acid encoding a polypeptide containing Omomyc or a functionally equivalent variant thereof or a fusion protein, or the administration of a vector encoding the nucleic acid or a cell containing the nucleic acid.

"Prevention" is understood as administering the combination or compositions of the invention at the initial or early stages of the disease, or also preventing its onset.

The term "treatment" is used to indicate the administration of a combination or compositions of the present invention before or after the onset of clinical symptoms to control the progression of a disease. Control of disease progression is understood to be a beneficial or desired clinical outcome, including but not limited to alleviation of symptoms, shortening of the course of the disease, stabilization of pathological conditions (particularly avoiding additional damage), delay of disease progression, improvement of pathological conditions, and remission (partial and complete). Control of disease progression also includes prolonging survival compared to the expected survival without treatment. In a preferred embodiment, control of disease progression is measured by the healthy lung/thorax volume ratio. In another embodiment, control of disease progression is measured by a reduction in tumor volume. In another embodiment, control of disease progression is measured by a reduction in tumor cell viability.

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

In one embodiment, the cancer includes but is not limited to leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, leukemia), acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), hairy cell leukemia, polycythemia vera, lymphomas (e.g., Hodgkin's or non-Hodgkin's), AIDS-related leukemias, Waldenstrom's macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, mendotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovialoma, mesothelioma, Ewing's tumor, tumor), leiomyosarcoma, rhabdomyosarcoma, Kaposi's sarcoma, colon cancer, pancreatic cancer, breast cancer, gallbladder cancer, esophageal cancer, ovarian cancer, prostate cancer, oral cancer (including squamous cell carcinoma), basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, teratoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, intraepithelial neoplasia (including Bowen's disease and Paget's disease), disease), neuroglioma, glioma, astrocytoma, glioblastoma multiforme (GBM, also called glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, neurothecoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

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

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

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

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

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

In some embodiments, the cancer is a solid tumor, such as a sarcoma, carcinoma, or lymphoma. Solid tumors generally contain abnormal tissue masses that typically do not include cysts or fluid areas. In some embodiments, the cancer is selected from 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, ovarian epithelial cancer, ovarian cancer (ovarian carcinoma or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatobiliary carcinoma; synovial sarcoma of soft tissue and bone; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing sarcoma; anaplastic thyroid carcinoma; adrenal cortical carcinoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal/gastric (GIST) cancer; lymphoma; squamous cell carcinoma of the head and neck (SCCHN); salivary gland cancer; glioma or brain cancer; neurofibromatosis-1-related malignant peripheral nerve sheath tumor (MPNST); Waldenstrom’s macroglobulinemia; or medulloblastoma.

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

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 cancer. In some embodiments, the cancer is ovarian epithelial 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 hepatocholangiocarcinoma. In some embodiments, the cancer is synovial sarcoma of soft tissue and bone. In some embodiments, the cancer is rhabdomyosarcoma. In some embodiments, the cancer is osteosarcoma. In some embodiments, the cancer is undifferentiated thyroid carcinoma. In some embodiments, the cancer is adrenocortical carcinoma. In some embodiments, the cancer is pancreatic cancer or pancreatic ductal carcinoma. In some embodiments, the cancer is pancreatic adenocarcinoma. In some embodiments, the cancer is glioma. In some embodiments, the cancer is malignant peripheral nerve sheath tumor (MPNST). In some embodiments, the cancer is neurofibromatosis-1 associated MPNST. In some embodiments, the cancer is Waldenstrom's macroglobulinemia. In some embodiments, the cancer is medulloblastoma.

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

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

In a preferred embodiment, the cancer is selected from 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, cholangiocarcinoma, glioblastoma, multiple myeloma, lymphoma. More preferably, the cancer is selected from breast cancer, ovarian cancer and prostate cancer, even more preferably, it is breast cancer or ovarian cancer, more preferably, it is breast cancer.

In a more preferred embodiment, the cancer is selected from melanoma, colorectal cancer and breast cancer.

In a preferred embodiment, the cancer is a BRAF mutant cancer, i.e. a cancer with a BRAF mutation. More preferably, it is a BRAF V600E mutant cancer.

In another embodiment, the mutation in BRAF is selected from BRAF wild-type expansion, BRAF deletion, mutation in the BRAF gene (i.e., mutations at 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, aspartate at position 594, glycine at position 596, leucine at position 597, threonine at position 599, lysine at position 601). Preferably, the mutation is at valine at position 600. In a more preferred embodiment, the mutation is selected from 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 BRAF mutation is V600E mutation.

In another embodiment, the cancer to be treated is a wild-type BRAF cancer.

In a preferred embodiment, the cancer is melanoma cancer. The term "melanoma" refers to malignant melanoma, a type of skin cancer that develops from melanocytes. More preferably, the melanoma has a BRAF mutation.

In a preferred embodiment, the cancer is colorectal cancer, preferably colorectal cancer with a BRAF mutation.

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

In a preferred embodiment, the cancer is triple negative breast cancer (TNBC). The term triple negative breast cancer refers to cancer cells that are immunohistochemically defined as lacking expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), i.e., the cells are negative in all 3 tests. This is a highly aggressive subtype of breast cancer that generally has a relatively poor clinical outcome, earlier recurrence, and a higher tendency to visceral metastasis compared to other types of breast cancer.

In another embodiment, the cancer is pancreatic cancer, particularly pancreatic ductal adenocarcinoma.

In another embodiment, the cancer is glioblastoma.

Glioblastoma, also called glioblastoma and grade IV astrocytoma, is the most common and most aggressive cancer that begins in the brain.

In another embodiment, the cancer is lung cancer.

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

As used herein, the term non-small cell lung cancer (NSCLC) refers to a heterogeneous group of diseases that are grouped together because their prognosis and management are largely similar and based on the World Health Organization/International Association for the Study of Lung Cancer histological classification (Travis WD et al. Histological typing of lung and pleural tumours. 3rd ed. Berlin: Springer-Verlag, 1999) include: (I) Squamous cell carcinoma (SCC), which accounts for 30% to 40% of NSCLC, begins in the larger respiratory tract but grows more slowly, which means that the size of these tumors can vary at diagnosis. (ii) Adenocarcinoma, the most common subtype of NSCLC, accounting for 50% to 60% of NSCLCs, begins near the gas exchange surfaces of the lungs and includes a subtype, bronchoalveolar carcinoma, which may respond differently to treatment. (iii) Large cell carcinoma, a fast-growing form that grows near the surface of the lung. It is primarily a diagnosis of exclusion and is often reclassified as squamous cell carcinoma or adenocarcinoma when more investigation is done. (iv) Adenosquamous carcinoma, a cancer that contains two types of cells: squamous cells (thin, flat cells that line some organs) and adenoid cells. (v) Carcinomas with pleomorphic, sarcomatoid, or sarcomatous components. This is a group of rare tumors that reflect a continuum of histological heterogeneity and epithelial and mesenchymal differentiation. (vi) Carcinoid tumors are slow-growing neuroendocrine lung tumors that arise from cells that release hormones in response to stimuli provided by the nervous system. (vii) Salivary gland carcinomas arise from salivary gland cells located in the large airways of the lungs. (viii) Unclassified cancers include cancers that do not fall into any of the above lung cancer categories.

In a specific embodiment, NSCLC is selected from lung squamous cell carcinoma, lung large cell carcinoma and lung adenocarcinoma.

As used herein, the term small cell lung cancer (SCLC) refers to a proliferation of small cells with unique and strict morphological features that contain dense neurocrine granules, giving this tumor an endocrine/paraneoplastic syndrome. Most cases occur in the larger airways (primary and secondary bronchi). These cancers grow rapidly and spread early in the course of the disease.

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

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

In one embodiment, the cancer is a primary tumor. As used herein, the term "primary tumor" refers to a tumor that originates in the location or organ where it is located and has not metastasized to that location from another location.

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

In one embodiment, the cancer is a solid tumor.

In another embodiment, the cancer is a cancer that is resistant to a BRAF inhibitor.

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

All combinations of the compounds of the invention and cancer types are included in the invention.

In some embodiments, the combinations or compositions of the invention produce an inhibition of tumor growth. In some embodiments, the combinations or compositions of the invention reduce tumor size (e.g., volume or mass) by at least 5%, 10%, 25%, 50%, 75%, 90%, or 99% relative to the size of the tumor before treatment. In some embodiments, the combinations or compositions of the invention reduce the amount of tumor in a patient by at least 5%, 10%, 25%, 50%, 75%, 90%, or 99% relative to the amount of tumor before treatment.

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

Combinations or compositions for use in the prevention and/or treatment of cancer can be administered in any amount and any route of administration that is effective in treating cancer or reducing the severity of cancer. The exact amount required varies from subject to subject, depending on the species, age and general condition of the subject, the severity of the disease or condition, the specific agent, its mode of administration, etc. For ease of administration and dosage uniformity, the compounds of the present invention are preferably formulated in dosage unit form. As used herein, the expression "dose unit form" refers to physically discrete units of a dose suitable for the patient to be treated. However, it should be understood that the total daily dosage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of reasonable medical judgment. The specific effective dosage level for any particular patient or organism will depend upon a variety of factors, including the condition being treated and the severity of that condition; the activity of the specific compound employed; the specific ingredient employed; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or concomitantly with the specific compound employed, and similar factors well known in the medical arts.

In a preferred embodiment, component (i) of the present invention, the preferred polypeptide or its functionally equivalent variant or conjugate, interacts synergistically with the BRAF inhibitor of the combination or the combination to treat cancer (to achieve a therapeutic effect).

Specifically, in a more preferred embodiment, the combination or pharmaceutical composition for use in preventing and/or treating cancer is a combination or pharmaceutical composition in which the amount of the polypeptide or its functionally equivalent variant or conjugate interacts synergistically with the BRAF inhibitor to treat cancer.

The terms "synergistic effect" or "synergistically interacting" are used interchangeably. A synergistic effect is greater than the additive effect that would be predicted by adding the actual effects of the individual agents in vitro. In vivo, a synergistic effect is a physiological effect, especially a therapeutic effect, that is greater than the additive effect that would be predicted by adding the actual effects of the individual agents in vivo.

Thus, if two agents are administered, they together provide a measurable physiological effect, particularly a therapeutic effect, if the actual effect produced by the two agents together is greater than the effect predicted by the sum of the actual therapeutic effects of the individual agents. Specifically, a synergistic effect is provided when the first agent alone provides some measurable effect, the second agent alone provides some measurable effect, and the measurable effect provided by the two agents together is greater than the effect provided by the sum of the two individual agents. More specifically, a synergistic effect is provided when the first agent alone does not provide a measurable effect, the second agent alone provides some measurable effect, and the measurable effect provided by the two agents together is greater than the effect provided by the second agent alone. More specifically, when neither the first agent alone nor the second agent alone provides any measurable effect, but the two agents together provide a measurable effect, a synergistic effect is provided. Due to the synergistic effect of components (i) and (ii), the amount of component (i) and/or component (ii) in the combination or composition of the present invention may be less than the amount required for a single therapy using only one of them as a therapeutic agent. Preferably, in these combinations or compositions, the dosage of one or the other therapeutic agent may be 0.01-1.000 μg/kg body weight/day.

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 is calculated using the SynergyFinder 3.0 web application from Ianevsky A et al. Nucleic Acids Res. 2022;50(W1):W739-W743.

The amount of the therapeutic agent present in the combination or composition may not exceed the amount normally used in the composition comprising the therapeutic agent as the sole active agent. Preferably, the amount of the therapeutic agent in the composition of the present invention is a range of about 50% to 100% of the amount normally present in the composition comprising the agent as the sole therapeutic active agent. In some embodiments, a therapeutic agent is administered in an amount 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 normally applied to the agent. As used herein, the phrase "normally administered" refers to the amount of the therapeutic agent approved by the FDA according to the FDA label instructions.

The combination or composition of the present invention can also be used in combination with known treatment methods, such as chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, hormones, or a combination thereof.

All embodiments of the combination of the present invention are also applicable to the treatment methods of the present invention.

Articles and Kits The disclosure also provides articles of manufacture comprising any one of the combinations or pharmaceutical compositions disclosed herein in one or more containers. In some embodiments, the articles of manufacture include, for example, a brochure, printed instructions, label, or package insert that instructs a user (e.g., a distributor or end user) to combine and/or use the composition of the article of manufacture to prevent and/or treat cancer.

In some embodiments, the article includes, for example, a bottle, a vial, a cartridge, a box, a syringe, an injector, or any combination thereof. In some embodiments, the label refers to the use or administration of the composition or pharmaceutical composition in the article according to the methods disclosed herein. In some aspects, the label recommends, for example, a regimen for use, treatment, prevention, or amelioration of cancer.

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

Unless otherwise stated, all terms used herein should be understood as the common meanings known in the art. Other more specific definitions of certain terms used in this application are described below, and are intended to be uniformly applied throughout the specification and the scope of the invention, unless the definition explicitly listed otherwise provides a broader definition. Throughout the specification and the scope of the invention, the word "include" and its variants are not intended to exclude other technical features, additives, components or steps. In addition, the word "include" covers the situation of "consisting of ...". By reading the specification, other objects, advantages and features of the present invention will become apparent to those with ordinary knowledge in the relevant technical field, or can be learned by practicing the present invention. In addition, the present invention covers all possible combinations of specific and particular embodiments described herein.

In this specification and the accompanying invention claims, the singular indefinite article terms ("a", "an") and the definite article terms ("the") include plural referents. Unless the context clearly indicates otherwise. The indefinite article term "a" (or "an") and the terms "one or more" and "at least one" can be used interchangeably herein. In addition, "and/or" used herein should be regarded as explicitly disclosing that each of the two specified features or components has or does not have the other. Therefore, the term "and/or" (for example, "A and/or B") used in the phrases herein is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Likewise, the term "and/or" used in a phrase (e.g., "A, B, and/or C") is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). The term "about" used in conjunction with numerical values throughout the specification and the scope of the invention application indicates a precision range familiar to and acceptable to a person of ordinary skill in the art. Typically, this precision range is ±15%. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which this disclosure relates. Units, prefixes, and symbols are expressed in the form accepted by their International System of Units (Système International de Unites) (SI). Numerical ranges include the numbers defining the range. Unless otherwise specified, amino acid sequences are written from left to right in the direction of amine to carboxyl. The titles provided herein are not limitations of the various aspects or aspects of the disclosure, which can be obtained as a whole by reference to the entire specification. Therefore, the terms directly defined below are more completely defined by reference to the entire specification.

The present invention will be described by the following embodiments, which are to be considered as illustrative only and not limiting of the scope of the present invention.

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

Combination of Omomyc and BRAF inhibitors synergistically reduces viability of melanoma cancer cells A human melanoma cell line (SkMel37) with a BRAF mutation (BRAFV600E) was used. Cells were seeded in 96-well plates and treated with increasing concentrations of dabrafenib and Omomyc alone or in combination the next day. After 5 days of treatment, the medium was removed and AlamarBlue™ Cell Viability Assay was added to each well, incubated for 3 hours until metabolism was complete, and fluorescence was read with a microplate reader using an excitation wavelength of 530-560 nm and an emission wavelength of 590 nm.

Synergy was assessed using the SynergyFinder 3.0 web application from Ianevsky A et al. Nucleic Acids Res. 2022;50(W1):W739-W743.

The synergy score can be interpreted as the average excess reaction caused by the drug interaction. When the synergy score is close to 0, the confidence in synergy or antagonism is limited. Therefore, when the synergy score is less than -10, the interaction between the two drugs is likely to be antagonistic. When the synergy score is between -10 and 10, the interaction between the two drugs is likely to be additive. When the synergy score is greater than 10, the interaction between the two drugs is likely to be synergistic.

Figure 1 shows that in melanoma SkMel37 cells, treatment with either Omomyc or Dabrafenib significantly reduced cell viability. Treatment of these cells with a combination of Omomyc and Dabrafenib revealed that the combination of these drugs had a synergistic effect: the ZIP synergy score was 11.02 in the left panel of Figure 1 and 26.55 in the right panel of Figure 1.

Table 2. Dunnett's multiple comparison test of SkMel37 cells Dunnett's multiple comparison test Mean difference 95% confidence interval (CI) of the difference Significance Omomyc 10 µM - Dabrafenib 15.63 nM vs. Omomyc 10 µM -22,65 -44.61 to -0.6851 * Omomyc 10 µM - Dabrafenib 15.63 nM vs. Dabrafenib 15.63 nM -26,27 -48.23 to -4.305 * Omomyc 20 µM - Dabrafenib 500 nM vs. Omomyc 20 µM -55,72 -76.92 to -34.51 *** Omomyc 20 µM - Dabrafenib 500 nM vs. Dabrafenib 500 nM -53,44 -72.41 to -34.47 ***

The same experiments were performed with different combinations of Omomyc and enemafenib or Omomyc and vemurafenib in SkMel37 cells (Figures 2 and 3) and SkMel28 cells (Figures 4 and 5), and synergistic effects were obtained.

without

Figure 1. Bar graph representing the combination of Omomyc and dabrafenib for melanoma cell line SkMel37 treated with Omomyc, dabrafenib or their combination at the indicated concentrations for 5 days. Relative cell number was inferred by AlamarBlue viability assay. Figure 2. Bar graph representing the combination of Omomyc and encorafenib for melanoma cell line SkMel37 treated with Omomyc, encorafenib or their combination at the indicated concentrations for 5 days. Relative cell number was inferred by AlamarBlue viability assay. Figure 3. Bar graph representing the combination of Omomyc and vemurafenib for melanoma cell line SkMel37 treated with Omomyc, vemurafenib or their combination at the indicated concentrations for 5 days. Relative cell numbers were inferred by the AlamarBlue viability assay. Figure 4. Bar graph representing the combination of Omomyc and connefenib for melanoma cell line SkMel28 treated with Omomyc, connefenib or their combination at the indicated concentrations for 5 days. Relative cell numbers were inferred by the AlamarBlue viability assay. Figure 5. Bar graph representing the combination of Omomyc and vemurafenib for melanoma cell line SkMel28 treated with Omomyc, vemurafenib or their combination at the indicated concentrations for 5 days. Relative cell number was estimated by AlamarBlue viability assay.

without

TW202502371A_113121016_SEQL.xml

Claims (17)

A combination comprising: i) a first component selected from: a) a polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical part that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) a polynucleotide encoding the polypeptide of a) or the conjugate of b); d) a vector comprising the polynucleotide according to c); and e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into a medium; and ii) a second component, which is a BRAF inhibitor. A combination as described in claim 1, wherein the first component is a polypeptide containing the sequence SEQ ID NO: 1. The combination as described in claim 1, wherein the functionally equivalent variant of SEQ ID NO: 1 is selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. A combination as described in any one of claims 1 or 3, wherein the chemical part that promotes cellular uptake of the polypeptide or its functionally equivalent variant is a cell penetrating peptide sequence, and wherein the cell penetrating peptide sequence forms a fusion protein with the polypeptide or its functionally equivalent variant. The combination of any one of claims 1 to 3, wherein the conjugate further comprises an additional nuclear localization signal. The combination of any one of claims 1 to 3, wherein the BRAF inhibitor is selected from dabrafenib, vemurafenib and enemafenib. The combination of claim 6, wherein the BRAF inhibitor is dabrafenib. A pharmaceutical composition comprising a pharmaceutically effective amount of the combination as described in any one of claims 1 to 7 and a pharmaceutically acceptable formulation. A use of the combination according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 8 for use in medicine. Use of the combination as described in any one of claims 1 to 7 or the pharmaceutical composition as described in claim 8 in the preparation of a medicament for preventing and/or treating cancer. Use of the first component of the combination as claimed in claim 1 in the preparation of a medicament for use in combination with a BRAF inhibitor for the prevention and/or treatment of cancer. Use of a BRAF inhibitor in the preparation of a medicament for use in combination with the first component of the combination as claimed in claim 1 for preventing and/or treating cancer. The use according to any one of claims 10 to 12, wherein the cancer is selected from melanoma, colorectal cancer and breast cancer. The use as described in claim 13, wherein the cancer is melanoma. The use of any one of claims 10 to 12, wherein the cancer is a cancer with a BRAF mutation. The use according to any one of claims 10 to 12, wherein the cancer is resistant to BRAF inhibitors. The use of any one of claims 10 to 12, wherein the first component is administered intravenously and the BRAF inhibitor is administered orally.