Combination therapy of omomyc and KRAS inhibitors for treating cancer

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of cancer, and more specifically, to a combination comprising a Kirsten rat sarcoma type 2 virus oncogene homolog (KRAS) inhibitor and Omomyc, and its use in medicine, and more specifically, in the prevention and / or treatment of cancer. Background Technology

[0002] Cancer is a leading cause of death worldwide, with nearly 10 million people dying from it in 2020. Cancer is a large family of diseases characterized by the uncontrolled growth of abnormal cells.

[0003] RAS is a well-known proto-oncogene, and its gain-of-function mutations occur in approximately 30% of human cancers. The most common mutated RAS subtype is KRAS, accounting for 86% of RAS mutations. KRAS-4B is the major subtype in human cancers, present in approximately 90% of pancreatic cancers, 30% to 40% of colon cancers, and 15% to 20% of lung cancers (primarily non-small cell lung cancer (NSCLC)). It is also found in biliary tract malignancies, endometrial cancer, cervical cancer, bladder cancer, liver cancer, myeloid leukemia, and breast cancer.

[0004] The most common mutations in the KRAS gene occur at codons 12, 13, or 61. Over 80% of mutations occur at codon 12, which is usually occupied by a glycine residue. KRAS mutations also occur at codons 63, 117, 119, and 146, but at a lower frequency.

[0005] Currently, a large number of KRAS inhibitors are under development to treat KRAS-mutant cancers by directly targeting mutated KRAS or by targeting key steps required for KRAS activation.

[0006] KRAS-G12C oncoprotein is one of the three most common KRAS mutants in cancer, present in 10% to 20% of all KRAS G12C mutations and approximately 50% of KRAS-driven lung adenocarcinomas. Direct KRAS-G12C inhibitors, such as sotorasib and adagrasib, selectively target KRAS G12C mutations and are used to treat patients with advanced non-small cell lung cancer (NSCLC) harboring KRAS-G12C mutations. Unfortunately, many patients do not respond to KRAS-G12C inhibitor therapy due to inherent or acquired resistance.

[0007] Besides the KRAS-glycine-to-cysteine ​​mutation at codon 12 (KRASG12C), another very common and prevalent mutation in the KRAS oncogene is G12D (glycine-to-aspartic acid mutation) at codon 12. G12D is most commonly found in pancreatic ductal adenocarcinoma, a frustrating disease with an average 5-year survival rate of less than 10% due to difficulty in early diagnosis and a lack of effective treatment. Efforts are underway to optimize the activity of KRAS-G12D inhibitors and overcome their resistance.

[0008] Another type of RAS pathway-targeting therapy is the so-called pan-KRAS inhibitor, which does not distinguish between KRAS mutants and can therefore be used against non-G12C mutants. Regardless of the specific KRAS mutation, pan-KRAS inhibitors are able to target the KRAS pathway. These include direct and indirect pan-KRAS drugs, such as those that interfere with KRAS nucleotide exchange and activation by inhibiting SHP2 or SOS1. SOS1 is a key guanine exchange factor (GEF) in KRAS, binding to and activating GDP-binding RAS family proteins at its catalytic binding site, thereby promoting GDP-GTP exchange. Pan-KRAS SOS1 inhibitors, such as BI-3406 and BAY-293, have attracted particular attention. These molecules bind to SOS1, reducing the formation of active GTP-loaded KRAS. However, BAY-293 has a weaker ability to inhibit cancer cell proliferation.

[0009] Despite recent advances in the availability of small molecules targeting KRAS in clinical settings, only about 30% of patients respond to and benefit from these therapies. Therefore, cancer patients carrying KRAS mutations still represent a significant unmet medical need.

[0010] Therefore, there is still a need to develop new and improved treatments for cancer that can overcome resistance to KRAS inhibitors, increase their efficacy, and / or reduce their side effects within the existing technology. Summary of the Invention

[0011] In a first aspect, the present invention relates to an assembly comprising: i) The first component, which is 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 moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) 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) The second component is a KRAS inhibitor.

[0012] In a second aspect, the present invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of the combination according to the invention and a pharmaceutically acceptable excipient.

[0013] In a third aspect, the present invention relates to the use of combinations or pharmaceutical compositions according to the present invention in medicine.

[0014] In a fourth aspect, the present invention relates to the use of the combinations or pharmaceutical compositions of the present invention for the prevention and / or treatment of cancer. Attached Figure Description

[0015] Figure 1 Synergistic effect of Omomyc with sotorasib (a KRAS G12C inhibitor) in in vitro KRAS G12C-mutant NSCLC and colorectal (CRC) cell lines. Results showed that Omomyc synergized with sotorasib at many different concentrations in all cell lines, significantly reducing the number of viable cells. A. Bar charts show the percentage of viable cells in NSCLC cell lines treated with representative synergistic concentrations of Omomyc and sotorasib. Their associated synergistic scores are shown below each chart. B. Bar charts show the percentage of viable cells in CRC cell lines treated with representative synergistic concentrations of Omomyc and sotorasib. Their associated synergistic scores are shown below each chart. Synergistic scores were calculated using SynergyFinder software. Mean and standard deviation are shown. ** p < 0.01; *** p < 0.001; **** p < 0.0001 。

[0016] Figure 2Synergistic effect of Omomyc with adagrasib (a KRAS G12C inhibitor) in in vitro KRAS G12C-mutant NSCLC and CRC cell lines. Results showed that Omomyc synergized with adagrasib at many different concentrations in all cell lines, significantly reducing the number of viable cells. A. Bar charts show the percentage of viable cells in NSCLC cell lines treated with representative synergistic concentrations of Omomyc and adagrasib. Their associated synergistic scores are shown below each chart. B. Bar charts show the percentage of viable cells in CRC cell lines treated with representative synergistic concentrations of Omomyc and adagrasib. Their associated synergistic scores are shown below each chart. Synergistic scores were calculated using SynergyFinder software. Mean and standard deviation are shown. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0017] Figure 3 Synergistic effect of Omomyc with MRTX1133 (a KRAS G12D inhibitor) in in vitro KRAS G12D-mutated NSCLC and CRC cell lines. Results showed that Omomyc synergized with MRTX1133 at many different concentrations in all cell lines, significantly reducing the number of viable cells. A. Bar charts show the percentage of viable cells in NSCLC cell lines treated with representative synergistic concentrations of Omomyc and MRTX1133. Their associated synergistic scores are shown below each chart. B. Bar charts show the percentage of viable cells in CRC cell lines treated with representative synergistic concentrations of Omomyc and MRTX1133. Their associated synergistic scores are shown below each chart. Synergistic scores were calculated using SynergyFinder software. Mean and standard deviation are shown. * p < 0.05; ** p < 0.01; **** p < 0.0001.

[0018] Figure 4Synergistic effect of Omomyc with BAY-293 [a pan-KRAS (SOS1) inhibitor] in in vitro KRAS-mutant NSCLC and CRC cell lines. Results showed that Omomyc synergized with BAY-293 at many different concentrations in all cell lines, regardless of their KRAS mutation status, significantly reducing the number of viable cells. A. Bar graphs show the percentage of viable cells in NSCLC cell lines treated with representative synergistic concentrations of Omomyc and BAY-293. Their associated synergistic scores are shown below each graph. B. Bar graphs show the percentage of viable cells in CRC cell lines treated with representative synergistic concentrations of Omomyc and BAY-293. Their associated synergistic scores are shown below each graph. Synergistic scores were calculated using SynergyFinder software. Mean and standard deviation are shown. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0019] Figure 5 Synergistic effect of Omomyc with BI-3406 [a pan-KRAS (SOS1) inhibitor] in KRAS-mutant NSCLC cell lines in vitro. Results showed that Omomyc synergized with BI-3406 at many different concentrations in all cell lines, regardless of their KRAS mutation status, significantly reducing the number of viable cells. Bar charts show the percentage of viable cells in NSCLC cell lines treated with representative synergistic concentrations of Omomyc and BI-3406. Their associated synergistic scores are shown below each chart. Synergistic scores were calculated using SynergyFinder software. Mean and standard deviation are shown. * p < 0.05; ** p < 0.01; *** p < 0.001. Detailed Implementation

[0020] This invention relates to providing novel treatment combinations for the prevention and treatment of cancer.

[0021] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art.

[0022] All embodiments disclosed in connection with one aspect of the invention are also applicable to other aspects.

[0023] Combinations and pharmaceutical compositions of the invention The definitions provided herein, and in each other aspect of the invention, also apply to the entire invention.

[0024] The inventors of this invention have surprisingly discovered a synergistic effect of combining Omomyc with KRAS inhibitors in the treatment of cancer. The inventors have demonstrated the synergistic effect of combining Omomyc with KRAS inhibitors, including different KRAS inhibitors targeting the most common specific mutations (G12C and G12D) of the KRAS oncogene and pan-KRAS inhibitors capable of targeting all KRAS mutations by inhibiting the SOS1 (Son of Sevenless 1) molecule (an upstream effector of KRAS). Specifically, the inventors have shown that the combination of Omomyc with the KRAS G12C inhibitor sotoraracil or adarazine synergistically reduces the viability of NSCLC cell lines HCC44, H1792, H23, and H358; and the viability of the colorectal cancer (CRC) cell line SW1463. Figure 1 and Figure 2 The combination of Omomyc and the KRAS G12D inhibitor MRTX1133 also showed synergistic effects in NSCLC cell lines KLA-p53 and KLA-Stk11, as well as the CRC cell line Lim1215. Figure 3 ).

[0025] The inventors also discovered that this synergistic effect is maintained when using KRAS inhibitors that target the KRAS pathway, regardless of specific KRAS mutations. Figure 4 and Figure 5 The combination of Omomyc and the pan-KRAS (SOS1) inhibitors BAY-293 or BI-3406 synergistically reduced the viability of NSCLC cell lines H358, HCC44, and KLA-p53, which are KRASG12C or KRASG12D mutants; as well as the viability of the colorectal cancer (CRC) cell line SW1463.

[0026] This synergistic effect is maintained regardless of dosage. Figures 1-5 This results in beneficial effects, particularly the increased therapeutic effect of the composition of the present invention relative to each of its components, allowing it to achieve the same effect with lower doses of each component, thereby reducing side effects in subjects receiving the composition of the present invention.

[0027] Furthermore, the inventors surprisingly discovered that Omomyc can also reverse KRAS resistance, thereby enhancing sensitivity to KRAS inhibitors and overcoming resistance to KRAS inhibitors, one of the main drawbacks of KRAS inhibitor therapy. The combination of this invention expands the population that may respond to KRAS inhibitor-based therapies.

[0028] Therefore, the combination of Omomyc and KRAS inhibitors could be an effective therapy for treating KRAS-mutant and wild-type tumors, as well as tumors resistant to KRAS inhibitors.

[0029] Therefore, in a first aspect, the present invention relates to an assembly comprising: i) The first component, which is 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 moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) 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) The second component is a KRAS inhibitor.

[0030] According to the present invention, the term "combination" refers to various combinations of compounds (i) and (ii), such as compositions formulated as single agents, combined mixtures consisting of individual agents of each component, such as "tank-mixes" that can be used together as combined agents, and combined use of single active ingredients in a sequential manner (i.e., one after another over a reasonably short period of time, such as hours or days) or simultaneously. 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 said polypeptide or said functionally equivalent variant thereof, or a polynucleotide encoding said polypeptide or said conjugate, or a carrier containing said polynucleotide, or a cell capable of secreting said polypeptide or said conjugate into a medium. In the present invention, compound (ii) refers to a therapeutically effective amount of a KRAS inhibitor. Preferably, the order of administration of compounds (i) and (ii) is not essential for carrying out the present invention.

[0031] The combination can be kit-of-parts, in which each component is individually formulated and packaged.

[0032] The combination of compounds (i) and (ii) can be formulated for simultaneous, separate, or sequential administration. Specifically, if the administration is not simultaneous, the compounds are administered at approximately the same time interval. Furthermore, the compounds can be administered in the same or different dosage forms or via the same or different routes of administration; for example, one compound may be administered orally while another 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 yet another embodiment, both compounds (i) and (ii) are administered intravenously.

[0033] The combination of the two compounds (i) and (ii) can be applied as follows: - As a combination, the combination is part of the same pharmaceutical preparation, and the two compounds are always administered simultaneously.

[0034] - As a combination of two units, each unit contains one substance, which may be applied simultaneously, sequentially, or separately.

[0035] In one specific embodiment, the compound (i) of the combination of the present invention is applied independently of compound (ii), i.e., applied in two units but simultaneously.

[0036] In another specific embodiment, the compound (i) of the present invention is applied first, followed by the compound (ii), i.e., the compound (ii) is applied separately or sequentially.

[0037] In yet another specific embodiment, the compound (ii) of the present invention is applied first, followed by the application of compound (i), i.e., compound (i) is applied separately or sequentially, as defined.

[0038] If applied separately, compounds (i) and (ii) of the present invention can be applied at intervals between each other, for example, at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In another embodiment, compounds (i) and (ii) of the present invention can be applied at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 days, preferably at intervals of 1 day, more preferably at intervals of 10 days. In a preferred embodiment, compound (ii) is applied 10 days after the first application of compound (i). In one embodiment, the first compound is discontinued before the second compound is started.

[0039] On the other hand, the present invention relates to a combination or pharmaceutical composition comprising a synergistically effective amount of a first component according to the first aspect of the present invention and a KRAS inhibitor.

[0040] Compound (i) of the combinations of the invention In a preferred embodiment, the compound (i) 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.

[0041] 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 acid components. In a preferred embodiment, the polypeptide is formed solely of 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 exceeding 125 amino acids, more preferably not exceeding 100 amino acids. In a preferred embodiment, the length of the polypeptide is 90 to 98 amino acids, preferably 90 to 95 amino acids, more preferably 91 amino acids.

[0042] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Furthermore, the term "amino acid" includes D-amino acids and L-amino acids (stereoisomers). Preferably, the amino acid is an L-amino acid.

[0043] The term “natural amino acids” or “naturally occurring amino acids” includes 20 naturally occurring amino acids; those that are typically modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other less common amino acids, including but not limited to 2-aminoadipic acid, hydroxylysine, isodesmosine, valine, leucine, and ornithine.

[0044] As used herein, the term "non-natural amino acid" or "synthetic amino acid" refers to a carboxylic acid or a derivative thereof that has been substituted with an amino group at position "a" and is structurally related to a natural amino acid. Illustrative, non-limiting examples of modified or uncommon amino acids include 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmodium, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, aliohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodestapyrin, alloleucine, N-methylglycine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline, valine, ornithine, ornithine, etc.

[0045] The polypeptides of the present invention may also contain non-amino acid moieties, such as hydrophobic moieties (various straight-chain, branched, cyclic, polycyclic, or heterocyclic hydrocarbons and hydrocarbon derivatives) attached to the peptide; and various protecting groups attached to the end of the compound 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.

[0046] It may include chemical (non-amino acid) groups present in the peptide to improve various physiological properties, such as reducing degradation or clearance rates, reducing the repulsive forces of multiple cell pumps, improving multiple administration methods, increasing specificity, increasing affinity, increasing stability, increasing bioavailability, increasing solubility, and reducing toxicity.

[0047] "Mimetic" refers to molecules that mimic the chemical structure of a peptide while retaining its functional properties. Methods for designing peptide analogs, derivatives, and mimics are known in the art.

[0048] 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.

[0049] SEQ ID NO: 1 corresponds to

[0050] The polypeptide 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 mutation locations are numbered according to the sequence corresponding to amino acid 365-454 of the Myc region of the polypeptide as defined by NCBI accession number NP_002458 published on March 15, 2015). The sequence of c-Myc (SEQ ID NO: 2) provided in the NCBI database with accession number NP_002458 is shown below, where the region from which Omomyc originates is indicated by underline:

[0051] Omomyc also contains the M2 domain of c-Myc, with the sequence RQRRNELKRSF (SEQ ID NO: 3) (see Dang and Lee, Mol.Cell. Biol., 1988, 8:4048-4054) (the double-underlined part above), and it corresponds to the nuclear localization signal.

[0052] Omomyc is characterized by its increased dimerizing 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 the mutation producing the tumor suppressor effect is retained. Therefore, Omomyc used in this 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 number NP_002458, published March 12, 2019).

[0053] The term "Myc" as used in this article refers to the family of transcription factors including c-Myc, N-Myc, and L-Myc. Myc proteins activate the expression of many genes by binding to the concordant sequence CACGTG (an enhancer cassette sequence or E-box and recruiting histone acetyltransferases or HAT). However, Myc can also act as a transcriptional repressor. By binding to the Miz-1 transcription factor and replacing the p300 co-promoter, Myc inhibits the expression of Miz-1 target genes. Myc also plays a direct role in controlling DNA replication.

[0054] The Myc b-HLH-LZ or Myc basic region helical-loop-helical leucine zipper domain refers to the region that determines the dimerization of Myc with Max protein and its binding to Myc target genes. This region corresponds to amino acid 365-454 of human Myc and is characterized by two α-helices linked by a loop (Nair, SK, & Burley, SK, 2003, Cell, 112: 193-205).

[0055] 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.

[0056]

[0057] In this document, “consistently composed of” means that the specified molecule does not contain any additional sequence that would alter the activity of SEQ ID NO: 4.

[0058] Preferably, the polypeptide consists of SEQ ID NO: 4.

[0059] The term "functionally equivalent variant" refers to any polypeptide that, relative to the polypeptide of SEQ ID NO: 1, involves the insertion or addition of one or more amino acids and / or the deletion of one or more amino acids and / or the conservative substitution of one or more amino acids and / or is generated by chemical modification of the polypeptide of SEQ ID NO: 1, and substantially retains the tumor-suppressive activity of SEQ ID NO: 1. Preferably, the functionally equivalent variant refers to any polypeptide that, relative to the polypeptide of SEQ ID NO: 1, involves the insertion or addition of one or more amino acids and / or the deletion of one or more amino acids and / or the conservative substitution of one or more amino acids, and substantially retains the tumor-suppressive activity of SEQ ID NO: 1; more preferably, any polypeptide generated by the insertion or addition of one or more amino acids relative to the polypeptide of SEQ ID NO: 1.

[0060] Those skilled in the art will understand that retaining tumor suppressor activity requires the variant to dimerize with Myc and / or its specific chaperones p21 / p22Max and inhibit Myc activity, and requires the variant to be able to translocate across the cell membrane and be able to transport across the nuclear membrane. In some embodiments, functionally equivalent variants of the peptides of the present invention exhibit less homodimerization than Omomyc, or are not forced to become homodimers by forming disulfide bonds. Specifically, certain embodiments of the peptides of the present invention exhibit fewer disulfide bonds in their homodimeric forms than the peptide Omomyc.

[0061] As used herein, "lower homodimerization" refers to a lower ability to form specific homodimers of the polypeptides of the present invention even under reducing conditions. In a preferred embodiment, this ability is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% lower than the homodimer-forming ability of Omomyc.

[0062] As used herein, reduction conditions involve the presence of a reducing agent, which is a compound that donates electrons to another chemical substance in a redox chemical reaction. Illustrative, non-limiting examples of reducing agents are DTT (dithiothreitol), β-mercaptoethanol, or TCEP (tris(2-carboxyethyl)phosphine). The amount of homodimer may be the same in vitro, and the difference between functionally equivalent variants and Omomyc exists only in cells with a heterodimerizing chaperone, where the absence of disulfide bonds makes heterodimer formation more likely.

[0063] Several experiments can be used to determine the homodimerization of peptides, illustrative but not limiting examples of monitoring thermal denaturation by circular dichroism, and thus dimerization can be quantitatively detected by folding and thermal stability.

[0064] Suitable functionally equivalent variants include polypeptides that consist essentially of the polypeptide of SEQ ID NO: 1. In this document, “consistently of” means that the specified molecule does not contain any additional sequence that would alter the activity of SEQ ID NO: 1.

[0065] In a preferred embodiment, the functionally equivalent variant of SEQ ID NO: 1 is a polypeptide generated by inserting or adding one or more amino acids relative to the polypeptide of SEQ ID NO: 1. In one embodiment, the functionally equivalent variant is generated by inserting fewer than 10 amino acids, more preferably fewer than 5 amino acids, and even more preferably by inserting one amino acid. In a preferred embodiment, it is generated by inserting one methionine amino acid.

[0066] In another embodiment, the functionally equivalent variant of SEQ ID NO: 1 is a polypeptide generated by deleting one or more amino acids relative to the polypeptide of SEQ ID NO: 1. In one embodiment, the functionally equivalent variant is generated by deleting fewer than 10 amino acids, more preferably fewer than 5 amino acids, and more preferably by deleting one amino acid.

[0067] A suitable functional variant of the targeting peptide is a variant having a degree of amino acid sequence identity greater than 25% relative to the peptide of SEQ ID NO: 1, for example, 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of identity between the two peptides is determined using computer algorithms and methods well known to those skilled in the art. Preferably, the identity between the two amino acid sequences is determined using the previously described BLASTP algorithm (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 1990;215: 403-410). In a preferred embodiment, sequence identity is determined over the entire length of the peptide of SEQ ID NO: 1, or over the entire length of the variant, or both.

[0068] Functional equivalent variants of the peptides of the present invention may also include post-translational modifications, such as glycosylation, acetylation, isopentenylation, myristylation, proteolytic processing, etc.

[0069] In another embodiment, a suitable functional variant of the targeting peptide is a variant in which one or more sites within the polypeptide of the present invention contain a conserved substitution of an amino acid that is present in the aforementioned protein. A “conserved 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 six groups each contain amino acids that are conserved substitutions for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W). Choosing such a conserved amino acid substitution is within the skill of a person of ordinary skill 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).

[0070] It should be understood that, in a preferred embodiment, the functionally equivalent variant of Omomyc contains mutations at the positions of E61T, E68I, R74Q, and R75N, which are found in Omomyc corresponding to human c-Myc. The positions where said mutations must occur in the functionally equivalent variant can be determined by multiple sequence alignment of different Myc sequences and identified by alignment of those positions 61, 68, 74, and 75 in the Omomyc sequence corresponding to human c-Myc. In one embodiment, the functionally equivalent variant of Omomyc contains mutations at the positions of E61T, E68I, R74Q, and R75N, which are found in Omomyc corresponding to human c-Myc.

[0071] In another embodiment, the functionally equivalent variant of Omomyc contains mutations at the E61, E68, R74, and R75 positions in the corresponding 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.

[0072] Multiple sequence alignment is an extension of pairwise alignment, involving more than two sequences at a time. Multiple sequence alignment methods compare all sequences in a given query set. Preferred multiple sequence alignment procedures (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, those skilled in the art can readily identify the positions in each sequence corresponding to E61T, E68I, R74Q, and R75N ​​found in Omomyc, and introduce mutations into Omomyc variants corresponding to the E61T, E68I, R74Q, and R75N ​​mutations found in Omomyc derived from human c-Myc.

[0073] Appropriate tests for determining whether a peptide can be considered a functionally equivalent variant of Omomyc include, but are not limited to: - Assays that measure the ability of a peptide to form a dimer complex with Max and Myc, such as reporter gene expression-based assays described by Soucek et al. (Oncogene, 1998, 17: 2463-2472), as well as PLA (protein linkage assay) or immunoprecipitation.

[0074] - An assay that measures the ability of a peptide to bind to the Myc / Max recognition site (CACGTG site) in DNA, such as the electrophoretic mobility assay (EMSA) described by Soucek et al. (see above).

[0075] - Assays that measure the ability to inhibit Myc-induced transactivation, such as the Myc / Max-specific DNA binding site-controlled reporter gene expression assay described by Soucek et al. (see above).

[0076] - Experiments based on the ability of peptides to inhibit the growth of cells expressing the myc proto-oncogene, as described by Soucek et al. (see above).

[0077] - Assays that measure the ability of peptides to enhance myc-induced apoptosis, such as those described by Soucek et al. (Oncogene, 1998: 17, 2463-2472). Alternatively, any assay commonly known in the art for assessing apoptosis in cells can be used, such as Hoechst staining, propidium iodide (PI) staining or annexin V staining, trypan blue, DNA laddering / fragmentation assays, and TUNEL assays.

[0078] In a preferred embodiment, if the activity of a polypeptide is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of that of natural Omomyc in one or more of the above-described tests, then the polypeptide is considered a functionally equivalent variant of Omomyc.

[0079] In one specific embodiment, a functionally equivalent variant of the polypeptide of SEQ ID NO: 1 contains the polypeptide of SEQ ID NO: 1, wherein residue X at position 89 of SEQ ID NO: 1 is not cysteine. Preferably, residue X at position 89 of SEQ ID NO: 1 is an aliphatic amino acid or a sulfurized amino acid or a dicarboxylic acid or its amide, or an amino acid having two basic groups, or an aromatic amino acid, or a cyclic amino acid, or a hydroxylated amino acid. More preferably, it is an amino acid selected from serine, threonine, and alanine, and most preferably an amino acid selected from serine and alanine.

[0080] Suitable functionally equivalent variants of SEQ ID NO: 1 having a residue X that is not cysteine ​​at position 89 of SEQ ID NO: 1 are disclosed in the table below.

[0081]

[0082] 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.

[0083] Furthermore, functionally equivalent variants of Omomyc are also capable of transducing cells upon contact with the cells. It should be understood that these functionally equivalent variants of Omomyc include the protein transduction domain found in natural Omomyc or another functional protein transduction domain.

[0084] In a preferred embodiment, if the polypeptide can transduce target cells with an efficiency of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of that of SEQ ID NO: 1, then the polypeptide is considered a functionally equivalent variant of SEQ ID NO: 1.

[0085] In addition, the functionally equivalent variant of SEQ ID NO: 1 can also be transported to the nucleus of target tumor cells.

[0086] In a preferred embodiment, if the polypeptide can be transported to the nucleus of the target tumor cell with an efficiency of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of that of SEQ ID NO: 1, then the polypeptide is considered to be a functionally equivalent variant of SEQ ID NO: 1.

[0087] A suitable assay for determining whether a polypeptide is a functionally equivalent variant of SEQ ID NO: 1, based on its ability to be transported across the cell membrane and into the cell nucleus, includes double labeling of the cells with a polypeptide-specific reagent and a dye specifically for labeling the cell nucleus (e.g., DAPI or Hoechst dye). The polypeptides of the present invention can be detected by confocal microscopy or fluorescence microscopy.

[0088] In another preferred embodiment, the compound (i) of the present invention is 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 said polypeptide or a functionally equivalent variant thereof.

[0089] As used herein, the term "coupler" refers to two or more compounds covalently linked together so that the function of each compound is retained in the coupler.

[0090] The term "chemical moiety" refers to any compound containing at least one carbon atom. Examples of chemical moiety include, but are not limited to, any peptide chain rich in hydrophobic amino acids and hydrophobic chemical moiety.

[0091] In a preferred embodiment, the conjugate according to the invention comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more chemical moieties that promote cellular uptake of the polypeptide or a functionally equivalent variant of the polypeptide.

[0092] In one embodiment, the chemical component of the polypeptide that promotes cellular uptake is a lipid or fatty acid.

[0093] Fatty acids are typically molecules comprising carbon chains with an acidic portion (e.g., a carboxylic acid) at the end of the chain. The carbon chain of a fatty acid can be of any length; however, it is preferred that the length of the carbon chain be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms, and any range derived therefrom. In some embodiments, the length of the carbon chain in the fatty acid chain portion is 4 to 18 carbon atoms. In some embodiments, the fatty acid carbon chain can contain an odd number of carbon atoms; however, in some embodiments, it is preferred that the chain has an even number of carbon atoms. Fatty acids containing only single bonds in their carbon chain are called saturated fatty acids, while fatty acids containing at least one double bond in their chain are called unsaturated fatty acids. Fatty acids can be branched, but in a preferred embodiment of the invention, the fatty acids are unbranched. Specific fatty acids include, but are not limited to, linoleic acid, oleic acid, palmitic acid, linolenic acid, stearic acid, lauric acid, myristic acid, arachidic acid, palmitoleic acid, and arachidonic acid.

[0094] In a preferred embodiment, the chemical portion that promotes cellular uptake of the polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof is a cell-penetrating peptide sequence. In this case, the conjugate will comprise a fusion protein containing the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof and a cell-penetrating peptide sequence.

[0095] The term "fusion protein" refers to a protein produced by genetic technology that consists of two or more functional domains derived from different proteins. Fusion proteins can be obtained by conventional methods, such as by gene expression encoding the nucleotide sequence of the fusion protein in suitable cells. It will be understood that the cell-penetrating peptide refers to a cell-penetrating peptide that is different from a cell-penetrating peptide forming part of the polypeptide of SEQ ID NO: 1 or a functionally equivalent variant of SEQ ID NO: 1.

[0096] The term "cell-penetrating peptide sequence" is used interchangeably with "CPP," "protein transduction domain," or "PTD" in this specification. It refers to a variable-length peptide chain that guides the transport of proteins into the cell. This transport of the peptide into the cell typically occurs via endocytosis, but it can also be internalized into the cell via direct membrane transport. CPPs typically have the following amino acid composition: containing a high relative abundance of positively charged amino acids (e.g., lysine or arginine), or a sequence containing alternating patterns of polar / charged and nonpolar hydrophobic amino acids.

[0097] Examples of CPPs that can be used in this invention include, but are not limited to, the CPP found in the mutant antennal leg protein of Drosophila (RQIKIWFQNRRMKWKK, SEQ ID NO: 13); the CPP found in the herpes simplex virus 1 (HSV-1) VP22 DNA-binding protein (DAATATRGRSAASRPTERPRAPARSASRPRRPVE, SEQ ID NO: 14); the CPP of Bac-7 (RRIRPRPPRLPRPRPRPLPLPFPRPG; SEQ ID NO: 15); the CPP of the HIV-1 TAT protein composed of amino acids 49-57 (RKKRRQRRR, SEQ ID NO: 16); the CPP of the HIV-1 TAT protein composed of amino acids 48-60 (GRKKRRQRRRTPQ, SEQ ID NO: 17); and the CPP of the HIV-1 TAT protein composed of amino acids 47-57 (YGRKKRRQRRR; SEQ ID NO: 18). CPP of S413-PV peptide (ALWKTLLKKVLKAPKKKRKV; SEQ ID NO: 19); CPP of penetratin peptide (RQIKWFQNRRMKWKK; SEQ ID NO: 20); CPP of SynB1 (RGGRLSYSRRRFSTSTGR; SEQ ID NO: 21); CPP of SynB3 (RRLSYSRRRF; SEQ ID NO: 22); CPP of PTD-4 (PIRRRKKLRRLK; SEQ ID NO: 23); CPP of PTD-5 (RRQRRTSKLMKR; SEQ ID NO: 24); CPP of FHVCoat- (35-49) (RRRRNRTRRNRRRVR; SEQ ID NO: 25); CPP of BMV Gag- (7-25) (KMTRAQRRAAARRNRWTAR; SEQ ID NO: 26); HTLV-II CPP of Rex-(4-16) (TRRQRTRRARRNR; SEQ ID NO: 27); CPP of D-Tat (GRKKRRQRRRPPQ; SEQ ID NO: 28); CPP of R9-Tat (GRRRRRRRRRPPQ; SEQ ID NO: 29); CPP of MAP (KLALKLALKLALALKLA; SEQ ID NO: 30); CPP of SBP (MGLGLHLLVLAAALQGAWSQPKKKRKV; SEQ ID NO: 31); CPP of FBP (GALFLGLGAAGSTMGAWSQPKKKRKV;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 having structural RN (where N is 4 to 17); GRKKRRQRRR sequence (SEQ ID NO: 37), RRRRRRLR sequence (SEQ ID NO: 38), RRQRRTS KLMKR sequence (SEQ ID NO: 39); Transportant 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: 41); NO: 44); YARAAARQARA sequence (SEQ ID NO: 45); THRLPRRRRRR sequence (SEQ ID NO: 46); GGRRARRRRRR sequence (SEQ ID NO: 47). ;

[0098] In a preferred embodiment, the cell-penetrating peptide is not an endogenous peptide included in SEQ ID NO: 1.

[0099] In one preferred embodiment, the CPP is the CPP of the HIV-1 TAT protein composed of amino acids 49-57 (RKKRRQRRR, SEQ ID NO: 16). In another preferred embodiment, the CPP is the GRKKRRQRRR sequence (SEQ ID NO: 37) or RRRRRRLR (SEQ ID NO: 38). In yet another embodiment, the CPP is the GRKKRRQRRR sequence (SEQ ID NO: 37) or RRRRRRRR (SEQ ID NO: 65).

[0100] In some implementations, the CPP is the CPP as described in WO2019 / 018898, the contents of which are incorporated herein by reference in their entirety.

[0101] 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.

[0102] In a preferred embodiment, the conjugate or fusion protein according to the invention comprises, in addition to the self-cell-penetrating peptide found in the polypeptide of SEQ ID NO: 1 or a functionally equivalent variant of the polypeptide, 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.

[0103] Suitable fusion proteins of this invention include the peptides Omomyc*TAT and Omomyc*LZArg as defined below:

[0104] Therefore, in a preferred embodiment, the fusion protein is a polypeptide selected from SEQ ID NO: 11 and SEQ ID NO: 12.

[0105] Suitable assays for determining whether a conjugate retains Omomyc's cell membrane translocation capacity include, but are not limited to, assays that measure the conjugate's ability to transduce cultured cells. These assays are based on contacting the conjugate with cultured cells and detecting the presence of the conjugate at a location within the cells.

[0106] In another preferred embodiment, the conjugate of the present invention further includes additional nuclear localization signals.

[0107] As used herein, the term “nuclear localization signal” (NLS) refers to an amino acid sequence of approximately 4–20 amino acid residues that guides a protein into the cell nucleus. Typically, NLS 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-1 Rev protein NLS (RQARRNRRRWE, SEQ ID NO: 51); HTLV-1 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 invention, the nuclear localization signal comprises the motif K(K / R)X(K / R).

[0108] In a more preferred embodiment, the nuclear location signal is selected from PKKKRKV (SEQ ID NO:48), PAAKRVKLD (SEQ ID NO:56) and KRPAATKKAGQ AKKKK (SEQ ID NO:49).

[0109] In another preferred embodiment, NLS may be an N-terminus or C-terminus of a conjugate of a polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof, or a fusion protein.

[0110] Those skilled in the art will understand that the conjugates of the present invention may need to further comprise one or more flexible peptides linked to the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof, a cell-penetrating peptide sequence, and / or an NLS. Therefore, in one 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 an NLS. In another embodiment, the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof is linked to an NLS via a flexible peptide.

[0111] In one specific embodiment, the polypeptide of the conjugate according to the invention is directly linked to the cell-penetrating peptide sequence and the NLS.

[0112] In one implementation, the NLS is one of the NLS that occurs endogenously in the Myc sequence, such as the M1 peptide (PAAKRVKLD, SEQ ID NO: 56) or the M2 peptide (RQRRNELKRSF, SEQ ID NO: 57).

[0113] In another embodiment, the additional NLS refers to an NLS that is different from the endogenous NLS found in the polypeptide containing SEQ ID NO: 1 or in a functionally equivalent variant of SEQ ID NO: 1.

[0114] In a preferred embodiment, in addition to the endogenous NLS found in the polypeptide or its functionally equivalent variants of the present invention, the conjugate or fusion protein according to the present invention further 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, or at least 10 NLS.

[0115] In another specific embodiment, the polypeptide of the conjugate according to the present invention is linked to a cell-penetrating peptide sequence via a first flexible peptide linker and to an NLS via a second flexible peptide linker.

[0116] As used herein, the terms “flexible peptide,” “spacer peptide,” or “linker peptide” refer to a peptide that covalently binds to two proteins or portions, but not to either polypeptide, allowing one protein or portion to move relative to the other without materially detrimental to the function of the protein or portion. Therefore, flexible linkers do not affect the tumor-tracking activity of polypeptide sequences, the cell-penetrating activity of cell-penetrating peptides, or the nuclear localization ability of NLS.

[0117] 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 protein solubility and / or improve its activity. Suitable linker regions include polyglycine regions, GPRRRR sequences of glycine, proline, and alanine residues (SEQ ID NO: 58).

[0118] In one 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 a variant thereof. The tag is typically a peptide or amino acid sequence that can be used to isolate or purify the fusion protein. Therefore, the tag is capable of binding one or more ligands, such as one or more ligands of an affinity matrix (e.g., a chromatographic support or beads with high affinity). An example of the tag is a histidine tag (His-tag or HT), such as a tag containing six histidine residues (His6 or H6), which can bind nickel (Ni) with high affinity. 2+ ) or cobalt (Co) 2+ His-tags possess the desired characteristics, allowing them to bind their ligands under conditions of protein denaturation and disruption of most protein-protein interactions. Therefore, His-tags can be used to remove the bait after protein-protein interactions involving the H6-tagged bait protein are disrupted.

[0119] Further illustrative, non-limiting examples of tags used for the isolation or purification of conjugates, peptides, 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 tags, S tags, calmodulin-binding peptides, cellulose-binding domains, chitin-binding domains, glutathione S-transferase tags, maltose-binding proteins, NusA, TrxA, DsbA, Avi tags, etc. (Terpe K., Appl. Microbiol. Biotechnol. 2003, 60:523-525), and amino acid sequences, such as AHGHRP (SEQ ID NO: 1). NO: 63) or PIHDHDHPHLVIHSGMTCXXC (SEQ ID NO: 64), β-galactosidase, etc.

[0120] If needed, the tag can be used to isolate or purify the fusion protein.

[0121] In another preferred embodiment, the compound (i) of the present invention is a polynucleotide encoding the polypeptide or fusion protein disclosed above. In one 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 moiety that promotes cellular uptake of said polypeptide or a functionally equivalent variant thereof; more preferably, it is a polynucleotide encoding a fusion protein containing a polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a cell-penetrating peptide sequence.

[0122] The terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably to refer to a polymer of nucleotides of any length. The polynucleotide may comprise deoxyribonucleotides, ribonucleotides, and / or their analogues. Nucleotides can have any three-dimensional structure and can perform any known or unknown function. The term “polynucleotide” includes, for example, single-stranded, double-stranded, and triple-stranded 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.

[0123] In a preferred embodiment, the polynucleotide of the present invention is mRNA.

[0124] mRNA can be chemically synthesized, obtained through in vitro transcription, or synthesized in vivo in target cells. The nucleotide sequence of the polynucleotide forming the conjugate or fusion protein of this invention is located in the same correct reading frame used for its expression.

[0125] In a preferred embodiment, the component (i) of the present invention is an mRNA encoding a polypeptide consisting of the sequence SEQ ID NO: 1, a polypeptide consisting of a functionally equivalent variant of SEQ ID NO: 1, or a polypeptide consisting of SEQ ID NO: 4.

[0126] In another embodiment, component (i) of the present invention is a carrier containing the polynucleotide of the present invention.

[0127] As used herein, the term "vector" refers to a nucleic acid sequence containing the necessary sequence such that, upon transcription and translation of said sequence in a cell, a polypeptide encoded by the polynucleotide of the present invention is produced. The sequence is operatively linked to an additional fragment that enables it to autonomously replicate in a host cell of interest. Preferably, the vector is an expression vector, defined as a vector comprising, in addition to a region autonomously replicating in a host cell, a region operatively linked to the nucleic acid of the present invention and capable of enhancing the expression of the nucleic acid product according to the present invention. The vectors of the present invention can be obtained by means well known in the art.

[0128] Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells. Suitable vectors containing the polynucleotides of the present invention are vectors derived from expression vectors in prokaryotes, such as pUC18, pUC19, pBluescript and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, bacteriophages and "shuttle" vectors, such as pSA3 and pAT28; expression vectors in yeast, such as 2-micron plasmid vectors, integrative plasmids, YEP vectors, centromere plasmids and analogues; expression vectors in insect cells, such as the pAC and pVL series vectors; expression vectors in plants, such as pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series vectors and analogues; and viral vectors (adenoviruses, adenovirus-associated viruses, and retroviruses, especially lentiviruses) and non-viral vectors (e.g., pSilencer) in higher eukaryotic cells. 4.1-CMV (Ambion), pcDNA3, pcDNA3.1 / hyg, pHCMV / Zeo, pCR3.1, pEFl / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAXl, pZeoSV2, pCI, pSVL, pKSV-10, pBPV-1, pML2d, and pTDT1) expression vectors. In a preferred embodiment, the polynucleotides of the present invention are contained in a vector selected from pEGFP retroviral vector or pBabe retroviral vector and pTRIPZ lentiviral vector or pSLIK lentiviral vector.

[0129] 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 or eukaryotic cells.

[0130] Preferably, the vector comprises a polynucleotide of the present invention that is operatively bound to a sequence regulating 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 sequences that increase the expression of heterologous nucleic acid sequences. In principle, any promoter can be used in the present invention, provided that the promoter is compatible with the cell on which the polynucleotide is to be expressed. Therefore, promoters suitable for implementing the present invention include, but are not limited to, constitutive promoters, such as derivatives of eukaryotic viral genomes, such as polyomavirus, adenovirus, SV40, CMV, avian sarcoma virus, hepatitis B virus, metallothionein gene promoter, herpes simplex virus thymidine kinase gene promoter, LTR region of retrovirus, immunoglobulin gene promoter, actin gene promoter, EF-1α gene promoter, and inducible promoters in which protein expression depends on the addition of a molecular or exogenous signal, such as the tetracycline system, NFκB / UV light system, Cre / Lox system and heat shock gene promoter, the regulated RNA polymerase II promoter and tissue-specific promoters described in WO / 2006 / 135436.

[0131] In another embodiment, component (i) of the present invention is a cell capable of secreting the polypeptide or conjugate of the present invention, preferably the polypeptide or fusion protein of the present invention, into a medium.

[0132] 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) cells, 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 understand that cells capable of secreting the polypeptides of the present invention into a medium to form microparticles or microcapsules can be found, resulting in a longer cell lifespan in the patient. Suitable materials for forming the microparticle targets of the present invention include any biocompatible polymeric material that allows for continuous secretion of therapeutic products and serves as a cell support. Therefore, the biocompatible polymer material can be, for example, a thermoplastic polymer or a hydrogen polymer. Among thermoplastic polymers, we have acrylic acid, acrylamide, 2-aminoethyl methacrylate, poly(tetrafluoroethylene-hexafluoropropylene) copolymer, (7-coumaroxy)methacrylate ethyl acid, N-isopropylacrylamide, polyacrylic acid, polyacrylamide, poly(amino)-p-xylene, poly(chloroethyl vinyl ether), polycaprolactone, poly(caprolactone-trimethylene carbonate) copolymer, poly(urea carbonate) carbamate, poly(carbonate) carbamate, polyethylene, polyethylene and acrylamide copolymer, polyethylene glycol, polyethylene glycol methacrylate, and poly... Polyethylene terephthalate, poly(4-hydroxybutyl acrylate), poly(hydroxyethyl methacrylate), poly(N-2-hydroxypropyl methacrylate), polylactic-co-glycolic acid, poly(L-lactic acid), poly(γ-methyl-L-glutamic acid), poly(methyl methacrylate), poly(propylene glycol 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 one or more of the above polymers. In hydrogel 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 fibroin, as well as synthetic hydrogels such as Sepharose® and Sephadex®.

[0133] Compound (ii) of the combinations of the invention The compound (ii) in the combination of the present invention is a KRAS inhibitor.

[0134] RAS proteins are members of the small GTPase protein family and act as binary switches in the signal transduction of most growth factor receptors, including EGFR, MET, or KIT. The RAS subfamily includes four proteins encoded by three different genes (KRAS, HRAS, and NRAS).

[0135] As used in this article, "KRAS" (or GTPase KRas, EC 3.6.5.2) refers to the protein encoded by the oncogene homolog of Kirsten rat sarcoma 2 virus, also known as "CK-RAS", "K-RAS2A", "K-RAS2B", "K-RAS4A", "K-RAS4B", "KI-RAS", "KRAS1", "KRAS2", "K-ras", "KRAS proto-oncogene, GTPase", "c-Ki-ras", "K-Ras 2", "K-Ras", "Kirsten rat sarcoma virus" or "Kirsten rat sarcoma virus". KRAS The gene is an oncogene that encodes a small GTPase transducer called KRAS, which converts the nucleotide guanosine triphosphate (GTP) to guanosine diphosphate (GDP). The balance between nucleotide hydrolysis and exchange determines the level of active KRAS in the cell. Limited by GDP, KRAS is in a "off" state. When GDP is exchanged for GTP, typically in response to growth factors and facilitated by guanine-nucleotide exchangers (GEFs) such as SOS1 / SOS2, KRAS cycles back to its activated "on" state. In this form, KRAS activates effector pathways, including the MAPK and PI3K pathways, to promote cell proliferation and survival. When GTP is hydrolyzed to GDP, KRAS returns to the off state, a process catalyzed by GTPase activating proteins (GAPs) such as NF1. In the oncogene form of KRAS, it is primarily kept in the active-on state because its inherent GTPase function and the GTP hydrolysis by enzymes such as GAPs are disrupted.

[0136] The KRAS gene has been identified as a homolog of Kirsten's rat sarcoma virus, which causes malignant transformation of rodent cells. The human KRAS gene (gene ID: 3845) is located on chromosome 12p12.1 and is encoded by 6 exons.

[0137] The sequence of the human KRAS protein corresponds to sequence P01116 in the UniProt database (entry version 255 as of May 3, 2023). Two isotypes, P01116-1 and P01116-2, were generated through alternative splicing, differing in the C-terminal region encoded by two alternative exons (IVA and IVB). Isotype P01116-1 was selected as the canonical sequence and is also known as 2A or K-Ras4A. Isotype P01116-2 is also known as 2B or K-Ras4B.

[0138] KRAS is the most frequently mutated oncogene in humans: more than 80% of pancreatic cancers, more than 30% of colorectal cancers, bile duct cancers, and lung adenocarcinomas contain activating mutations in the KRAS gene.

[0139] Common activating mutations in the KRAS gene are KRAS wild-type amplifications. Mutations in the KRAS gene can be at position 12 (glycine), position 13 (glycine), or position 61 (glutamine). In a more preferred embodiment, the mutation is selected from G12S, G12V, G12D, G13D, G12C, G12R, G12F, G12I, G12A, G13C, G13R, or Q61L mutations. In a preferred embodiment, the mutation is a G12C mutation. In a preferred embodiment, the mutation is a G12D mutation.

[0140] As used herein, “KRAS inhibitor” means any compound that can cause a decrease in the activity of one or more KRAS proteins, preferably a decrease in the activity of mutant KRAS proteins, including those compounds that inhibit KRAS gene expression and those compounds that cause a decrease in KRAS mRNA or protein levels. As used herein, the term “KRAS inhibitor” means a compound that inhibits the catalytic activity of the KRAS enzyme by covalently or reversibly binding directly to KRAS or a KRAS mutant; it also means a compound that inhibits upstream KRAS activators or targets the interaction between KRAS and said upstream KRAS activators to inhibit downstream KRAS signaling pathways.

[0141] In a preferred embodiment, the KRAS inhibitor can be a direct inhibitor or an indirect inhibitor. Direct KRAS inhibitors are those that directly inhibit KRAS proteins (preferably mutant KRAS proteins) by reversibly or irreversibly binding to them. Indirect KRAS inhibitors are those that act indirectly and do not bind to KRAS proteins (preferably mutant KRAS proteins), acting on key steps required for KRAS activation, such as by targeting and inhibiting the interaction between KRAS and related proteins.

[0142] The expression of a nucleic acid or protein is considered reduced when the level of its expression is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% (i.e., non-existent) relative to a reference value.

[0143] Reference values ​​refer to the levels of proteins or nucleic acids in control subjects, who may be subjects without a specific disease.

[0144] Suitable methods for determining whether an inhibitor can reduce KRAS mRNA levels include, but are not limited to, standard assays for determining mRNA expression levels, such as qPCR, RT-PCR, RNA protection assays, Northern blotting, RNA dot blotting, in situ hybridization, microarray techniques, tag-based methods such as gene expression serial analysis (SAGE) (including variants such as LongSAGE and SuperSAGE), microarrays, and fluorescence in situ hybridization (FISH) (including variants such as Flow-FISH, qFiSH, and dual-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.

[0145] First, the nucleic acids contained in the sample (e.g., cells or tissues prepared from the subject) are extracted using standard methods, such as lysins or chemical solutions, or by extracting with nucleic acid-binding resin according to the manufacturer's instructions.

[0146] If mRNA is to be measured in a biological sample, the sample can be treated to physically, mechanically, or chemically disrupt tissue or cellular structures, thereby releasing intracellular components into an aqueous or organic solution to prepare nucleic acids for further analysis. Nucleic acids are then extracted from the sample using procedures known to or commercially available to those skilled in the art. These nucleic acids are then processed using 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, NY, Vol.1-3, Extraction of RNA from frozen or fresh samples. Preferably, care is taken to avoid RNA degradation during extraction.

[0147] Expression levels can be determined using mRNA obtained from formalin-fixed, paraffin-embedded tissue samples. Archived pathological or biopsy samples can be dewaxed first, and then mRNA can be isolated from them. An exemplary dewaxing method involves 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 can be 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 resected tumors. In one specific embodiment, samples can be obtained from fresh tumor tissue or from frozen tissue embedded in OCT.

[0148] To standardize mRNA expression values ​​across different samples, the expression levels of the mRNA of interest in the test sample can be compared with those of the control RNA. As used in this paper, " Control RNA "Control RNA" refers to RNA whose expression level remains unchanged or changes only in a limited amount in tumor cells relative to non-tumorigenic cells. Preferably, the control RNA is mRNA derived from housekeeping genes that encode proteins constitutively expressed and performing basic cellular functions. Examples of housekeeping genes used in this invention include β-2-microglobulin, ubiquitin, 18-S ribosomal protein, cyclophilin, IPO8, HPRT, GAPDH, PSMB4, tubulin, and β-actin.

[0149] Relative gene expression quantification can be calculated using the comparison cycle threshold (Ct) method, with housekeeping genes as endogenous controls and commercially available RNA controls as calibrators. According to Formula 2... -（ΔCt样本-ΔCt校准物） The final result is determined by subtracting the Ct value of the target gene from the value of the control gene.

[0150] Appropriate methods for determining whether an inhibitor works by reducing KRAS protein levels include quantification using conventional methods, such as using antibodies that have the ability to specifically bind to the protein encoded by the KRAS gene (or fragments containing antigenic determinants therein), and subsequently quantifying the resulting antibody-antigen complex.

[0151] The antibodies used in these experiments can be, for example, polyclonal serum, hybridoma supernatant or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' and F(ab')2, ScFv, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and humanized antibodies. These antibodies may or may not be labeled. Illustrative but non-exclusive examples of labels that may be used include radioisotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, colorants, etc. A variety of well-known assays can be used in this invention, employing unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies); these techniques include Western blotting or Western transfer, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemistry and immunohistochemistry techniques, and assays based on the use of biochips or protein microarrays containing specific antibodies or in the form of test strips based on colloidal precipitation. Other methods for detecting and quantifying the levels of proteins of interest include techniques such as affinity chromatography and ligand binding assays.

[0152] On the other hand, KRAS protein levels can be determined by constructing a tissue microarray (TMA) containing assembled subject samples and measuring the expression level of the corresponding protein using immunohistochemistry. Immunostaining intensity can be evaluated by two or more different pathologists using a uniform and well-defined cut-off criterion to maintain method reproducibility. Differences can be addressed by simultaneous re-evaluation. In short, considering the expression of each marker in tumor cells and a specific cut-off value, immunostaining results can be recorded as negative expression (0) versus positive expression, low expression (1+) versus moderate expression (2+), and high expression (3+). As a general standard, cut-off values ​​are selected to promote reproducibility and, where possible, translate biological events. Alternatively, the intensity of immunostaining can be assessed using imaging techniques and automated methods, such as those disclosed in Rojo, MG et al. (Folia Histochem. Cytobiol. 2009; 47: 349-54) or Mullane, L. et al. (Expert Rev.Mol. Diagn. 2008; 8: 707-25).

[0153] Alternatively, in another specific implementation, KRAS protein levels are tested using Western blotting. Western blotting detects proteins that have previously been resolved by gel electrophoresis and immobilized on a membrane (typically nitrocellulose) under denaturing conditions by incubation with a specific antibody and a imaging system (e.g., chemiluminescence).

[0154] KRAS inhibitors can inhibit KRAS 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%, as well as the entire range from 5% to 100%. Appropriate methods for determining whether an inhibitor works by reducing KRAS activity include any method that allows for the detection of reduced phosphorylation of key downstream effectors of KRAS, such as extracellular signal-regulated kinases (ERKs). Methods for detecting KRAS activity are known in the art, including but not limited to KRAS pull-down assays, Ras-binding domain (RBD) pull-down assays; Western blotting, immunoblotting, immunoassays, and immunofluorescence to detect the phosphorylation status of downstream KRAS effectors, nucleotide exchange, transcriptome assays, and detection of protein-protein interactions (e.g., but not limited to KRAS-CRAF) (Canon J. et al. 2019. Nature, 575:217-223; Kim D. et al. 2023 May 31. Nature, doi: 10.1038 / s41586-023-06123-3. Epubahead of print. PMID: 37258666; Hallin J. et al. 2020. Cancer Discov, 10(1):54-71). Assays for determining enzyme activity are known to those skilled in the art, including but not limited to initial rate assays, process curve assays, transient kinetic assays, and relaxation assays. Continuous assays of enzyme activity include, but are not limited to, spectrophotometry, fluorescence assays, calorimetric assays, chemiluminescence assays, light scattering assays, and microscale thermopheresis assays. Discontinuous assays of enzyme activity include, but are not limited to, radiometric assays and chromatographic assays. As is known to those skilled in the art, factors that may affect enzyme activity include salt concentration, temperature, pH, and substrate concentration.

[0155] KRAS inhibitors can be any organic or inorganic molecule, including modified and unmodified nucleic acids such as antisense nucleic acids, RNA interference (RNAi) agents such as siRNA, shRNA or miRNA, peptides, proteins, peptide mimics, receptors, ligands, antibodies and small organic molecules used to treat cancer.

[0156] As used herein, "small compound" refers to a molecule that modulates biological processes, thereby inhibiting the catalytic activity of KRAS. This compound can be natural or synthetic.

[0157] In a preferred embodiment, the KRAS inhibitors used in this invention are selected from Table 1.

[0158]

[0159] In a preferred embodiment, the KRAS inhibitor is a mutation-specific KRAS inhibitor. A “mutation-specific KRAS inhibitor,” “mutation-selective KRAS inhibitor,” “allele-selective KRAS inhibitor,” or “allele-specific KRAS inhibitor” is a KRAS inhibitor that targets a specific variant allele of KRAS.

[0160] In a preferred embodiment, the KRAS inhibitor is a mutation-specific KRAS inhibitor targeting KRAS G12C. These compounds covalently bind to cysteine ​​12 in GDP-KRAS G12C, acting as an inactive state-selective KRAS drug and thus being a direct, irreversible inhibitor. In a more preferred embodiment, the KRAS inhibitor is selected from the compounds listed in Item I of Table I or their pharmaceutically acceptable salts; more preferably, it is selected from sotoraracil and adapalene.

[0161] In a preferred embodiment, the KRAS inhibitor is a mutation-specific KRAS inhibitor targeting KRAS G12D. MRTX1133 and BI-KRASG12D1-3 can reversibly bind directly to KRAS G12D. In a more preferred embodiment, the KRAS inhibitor is selected from the compounds listed in Item II of Table 1 or their pharmaceutically acceptable salts; more preferably, it is MRTX1133.

[0162] In a more preferred embodiment, the KRAS inhibitor is a mutation-specific KRAS inhibitor targeting KRAS G13C. These inhibitors are direct inhibitors of the binding to this allele. In a more preferred embodiment, the KRAS inhibitor is selected from the compounds listed in Item III of Table 1 or their pharmaceutically acceptable salts; more preferably, it is RAS(ON) G13C.

[0163] In one preferred embodiment, the inhibitor is a KRAS G12C inhibitor. In another preferred embodiment, the inhibitor is a KRAS G12D inhibitor.

[0164] Mutation-selective KRAS G12C inhibitors target a subset (approximately 13.6%) of all KRAS-driven cancers. A comprehensive approach to conquering KRAS-driven cancers requires a broad library of KRAS drugs.

[0165] In a preferred embodiment, the KRAS inhibitor is a pan-KRAS inhibitor.

[0166] As used herein, the term "pan-KRAS inhibitor" refers to a KRAS inhibitor that targets a broad range of KRAS alterations without distinguishing between KRAS mutants. Therefore, pan-KRAS inhibitors can target the KRAS pathway regardless of the specific KRAS mutation. Pan-KRAS inhibitors have the potential to target a broad patient population, including KRAS G12D, KRAS G12V, KRAS G13D, KRASG12R, and KRASG12A mutants or wild-type amplified cancers, as well as cancers with acquired resistance to KRAS G12C inhibitors.

[0167] Pan-KRAS inhibitors can be either direct or indirect pan-KRAS inhibitors.

[0168] Direct pan-KRAS inhibitors are compounds that lock KRAS in an inactive state by forming bonds with one or more KRAS proteins or with mutated amino acids in KRAS proteins.

[0169] According to reports, direct pan-KRAS inhibitors have shown activity against a wide range of KRAS-driven cell lines, including KRASG12C, KRASG12D, KRASG12V, and KRASG13D-driven cells, while HRAS-mutant and NRAS-mutant cell lines have not shown sensitivity (Hofmann MH et al. Expanding the reach of precisiononcology by drugging all KRAS mutants. 2022. Cancer Discov, 12(4): 924-937).

[0170] Exemplary, non-limiting direct pan-KRAS inhibitors are: RSC-1255, targeting pan-RAS; BI-Pan-KRAS1-4 inhibitors, targeting pan-KRAS, KRAS G12D / V, and KRAS wild-type; BI-pan-KRASdegrader1, targeting pan-KRAS, KRAS G12C / D / V / A, KRAS G13C, KRAS A146T / P, KRAS Q61E / P, and KRAS wild-type; and RMC-6236, targeting pan-RAS, KRAS G12D / V, KRAS G13D, KRAS Q61K, and RRAS wild-type.

[0171] In a preferred embodiment, the KRAS inhibitor is a direct pan-KRAS inhibitor. In a more preferred embodiment, the KRAS inhibitor is selected from the compounds listed in Item IV of Table 1 or their pharmaceutically acceptable salts.

[0172] Indirect pan-KRAS inhibitors are pathway inhibitors that inhibit upstream KRAS activators. Preferably, indirect pan-KRAS inhibitors interfere with KRAS nucleotide exchange and activation by inhibiting SHP2 or GEF SOS1.

[0173] In a preferred embodiment, the KRAS inhibitor is an indirect pan-KRAS SHP2 inhibitor. The SHP2 inhibitor stabilizes the autoinhibitory conformation of the enzyme, thereby disrupting SOS1-mediated KRAS nucleotide exchange. In a more preferred embodiment, the KRAS inhibitor is selected from the compounds listed in Item V of Table 1 or their pharmaceutically acceptable salts.

[0174] As used in this article, "SHP2 inhibitor" refers to any compound that can reduce SHP2 activity, including those that inhibit SHP2 gene expression and those that reduce SHP2 mRNA or protein levels. SHP2 inhibitors primarily inhibit the catalytic activity of the SHP2 enzyme.

[0175] As used herein, “SHP2” refers to tyrosine protein phosphatase non-receptor type 11 (PTN11), also known as protein tyrosine phosphatase 1D (PTP-1D), phosphatase-2 containing the Src homology 2 domain (SHP2), or protein tyrosine phosphatase 2C (PTP-2C). The sequence of human SHP2 corresponds to sequence Q06124 in the UniProt database (entry version 255 as of May 3, 2023).

[0176] In a more preferred embodiment, the KRAS inhibitor is an indirect pan-KRAS SOS1 inhibitor. The inhibitor of GEF SOS1 blocks the interaction between SOS1 and KRAS-GDP, preventing nucleotide exchange and GTP loading of KRAS. In a more preferred embodiment, the KRAS inhibitor is selected from the compounds listed in Item VI of Table 1 or their pharmaceutically acceptable salts; preferably selected from BAY-293 and BI-3406.

[0177] As used herein, "SOS1 inhibitor" refers to any compound that can reduce SOS1 activity, including those that prevent SOS1 gene expression and those that reduce SOS1 mRNA or protein levels. SOS1 inhibitors primarily inhibit the catalytic activity of the SOS1 enzyme.

[0178] As used herein, “SOS1” refers to the Son of sevenless homolog 1. The sequence of the human SOS1 protein corresponds to sequence Q07889 (entry version 238 as of May 3, 2023).

[0179] In a preferred embodiment, the KRAS inhibitor is selected from sotoprazine, adapalene, MRTX1133, BAY-293, and BI-3406; preferably, it is selected from sotoprazine, adapalene, and MRTX1133.

[0180] All compounds listed in Items I through VI of Table 1 also include their pharmaceutically acceptable salts, solvates, polymorphs, or cocrystals. Prodrugs of these compounds are also included.

[0181] In a more preferred embodiment, the KRAS inhibitor is selected from the compounds listed in items I, II, III, IV, V and VI of Table 1 or their pharmaceutically acceptable salts; preferably the compounds listed in items I, II, III, IV, V and VI of Table 1; more preferably the compounds listed in items I, II, III and IV of Table 1; and even more preferably the compounds listed in items I, II and III of Table 1.

[0182] The term "pharmaceutically acceptable" refers to those properties and / or substances that are acceptable to patients from a pharmacological / toxicological perspective and to the chemists producing the drug from a physical / chemical perspective regarding composition, formulation, stability, patient acceptability, and bioavailability.

[0183] The term "pharmaceutically acceptable salt" includes salts formed with pharmaceutically acceptable acids or alkalis. Pharmaceutically acceptable acids include inorganic acids and organic acids, such as, but not limited to, hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid, hydroiodic acid, and nitric acid; and such organic acids include, 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 alkalis include alkali metal (e.g., sodium or potassium) hydroxides and alkaline earth metal (e.g., calcium or magnesium) hydroxides and organic alkalis, such as, but not limited to, alkylamines, arylalkylamines, and heterocyclic amines.

[0184] According to the present invention, the term "solvent" should be understood to refer to any solid form of the aforementioned compound having another molecule connected to it via a non-covalent bond. Examples of solvates include hydrates and alcohols, preferably C1-C6 alcohols, such as methanols.

[0185] According to the present invention, the term "polymorph" should be understood as a specific crystalline form of a compound that is capable of crystallizing in different forms.

[0186] As used herein, the term "cocrystal" should be understood as a crystal structure consisting of a KRAS inhibitor and at least one other component. Interfering RNA "iRNA" or "KRAS RNA" refers to an RNA molecule capable of silencing one or more KRAS proteins, particularly KRAS mutant proteins, or the expression of any gene required for KRAS function. For this purpose, iRNAs are typically double-stranded oligonucleotides of at least 30 base pairs in length, and more preferably contain about 25, 24, 23, 22, 21, 20, 19, 18, or 17 ribonucleic acid base pairs. Several different types of molecules have been effectively used in iRNA technology, including small interfering RNA (siRNA) (sometimes called short interfering RNA or silencing RNA), microRNAs (miRNA), which are generally different from siRNAs because they are processed from single-stranded RNA precursors and are only partially complementary to target mRNAs and short hairpin RNAs (shRNAs).

[0187] Small interfering RNA (siRNA) agents inhibit the expression of target genes by interfering with RNA. siRNAs can be chemically synthesized, obtained through in vitro transcription, or synthesized in vivo within target cells. Typically, siRNAs consist of a double-stranded RNA of 15 to 40 nucleotides in length and may contain 3' and / or 5' overhangs of 1 to 6 nucleotides in length. The length of the overhangs is independent of the total length of the siRNA molecule. siRNAs function through post-transcriptional degradation or silencing of target messengers.

[0188] siRNA, also known as shRNA (short hairpin RNA), is characterized by antiparallel strands linked by loops or hairpin regions. 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 can be encoded by plasmids or viruses, particularly retroviruses, and more specifically, by promoters such as the U6 promoter of RNA polymerase III.

[0189] In the context of this invention, siRNA is substantially homologous to KRAS mRNA, particularly to mutant KRAS mRNA or its protein-coding genomic sequence. The term " Substantially homologous "This is understood to mean that the siRNA has a sequence that is fully complementary to or similar to the target mRNA, enabling the siRNA to induce mRNA degradation through RNA interference. Suitable siRNAs that induce interference include siRNAs formed from RNA, as well as siRNAs containing different chemical modifications, such as:" -siRNA, in which the linkages between nucleotides differ from those naturally occurring, such as phosphate thioester linkages; - Conjugates of stranded RNA with functional reagents such as fluorophores; - Modifications at the ends of the RNA chain, especially modifications at the 3' end by binding 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; - Base-modified nucleotides, such as halogenated bases (e.g., 5-bromouracil and 5-iodouracil) and alkylated bases (e.g., 7-methyl-guanosine).

[0190] The siRNA and shRNA used in the context of this invention can be obtained using a range of techniques known to those skilled in the art. For example, siRNA can be chemically synthesized from protected ribonucleoside phosphoramide in a conventional DNA / RNA synthesizer. Alternatively, siRNA can be produced from plasmids and viral vectors via recombinant dicer, wherein the coding regions of one or more siRNA strands are effectively controlled by the RNA polymerase III promoter. RNase dicer processes shRNA into siRNA in cells.

[0191] The KRAS region, which forms the basis for siRNA design, is not restrictive and can contain a coding sequence region (between the start and stop codons) or, alternatively, a sequence from the 5' or 3' untranslated region, preferably 25 to 50 nucleotides in length, and located anywhere at the 3' position of the start codon. The siRNA design procedure involves recognizing the sequence motif AA(N19)TT, where N can be any nucleotide in the KRAS sequence, and selecting those exhibiting high G / C content. If the sequence motif is not found, there is a sequence motif NA(N21) that can be recognized, where N can be any nucleotide.

[0192] In a preferred embodiment, the KRAS inhibitor is siRNA. In a more preferred embodiment, the siRNA is a commercially available siRNA from Santa Cruz Biotechnology, particularly sc-35731.

[0193] In another implementation, the inhibitor is a KRAS-specific " Antisense oligonucleotidesAn antisense oligonucleotide is a molecule whose sequence is complementary to the mRNA encoding KRAS, i.e., complementary to the cDNA coding strand. Antisense oligonucleotides can be complementary to the complete coding region or to the same region including the coding region and the 5' and 3' untranslated regions. Antisense oligonucleotides can consist of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides. Antisense oligonucleotides can be obtained through chemical synthesis or enzymatic conjugation reactions known to those skilled in the art. For example, antisense oligonucleotides may also contain additional antisense oligonucleotides and target polynucleotides. Modified nucleotides, such as phosphate thioester derivatives, peptide nucleic acids, and acridine-substituted oligonucleotides, enhance the biostability of double-stranded DNA-RNA complexes formed between acids. 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-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylguanosine, and myoglobin. Glycosides, N6-isopentene adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcitosine, 5-methylcitosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylguanosine (ne), 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentene adenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl uracil-5-oxyacetate, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, antisense nucleic acids can be biologically generated using expression vectors in which antisense directed nucleic acids are cloned.

[0194] Another group of compounds that may form part of this invention are catalytically active nucleic acids called ribozimes. A ribozime comprises a catalytic region and a second region, the sequence of which is complementary to that of the target nucleic acid and imparts substrate specificity to the ribozime. Through hybridization and linkage between the target nucleic acid and the complementary region of the ribozime, the interaction between the ribozime and its substrate leads to activation of the catalytic region, triggering intermolecular or intramolecular breakage of the target nucleic acid. The basic considerations for ribozime design are well known to those skilled in the art (see, for example, Doherty and Doudna (Annu. Ref. Biophys. Biomolstruct. 2000; 30:457-75).

[0195] Another class of compounds that can form part of the compositions of the present invention includes inhibitory antibodies. According to the present invention, the term "..." Inhibitory antibodies "It is understood to refer to antibodies that bind to any KRAS protein, preferably to a mutant KRAS protein, and cause inhibition of its catalytic activity."

[0196] Antibodies can be prepared using any method known to those skilled in the art. Therefore, polyclonal antibodies are prepared by immunizing animals with the protein that needs to be inhibited. Monoclonal antibodies can be prepared using the method described by Kohler, Milstein et al. (Nature, 1975, 256: 495). Once an antibody capable of binding to the KRAS protein is identified, an antibody that can inhibit KRAS activity, particularly KRAS mutant activity, using the assays described above for determining KRAS activity is selected. Suitable antibodies in this invention include complete antibodies comprising antigen-binding variable and constant regions, fragments “Fab”, “F(ab´)2”y“Fab’”, Fv, scFv, diabodies, and bispecific antibodies.

[0197] Other compounds capable of inhibiting KRAS expression that can form part of the combination of the present invention include aptamers and spiegelmers. Aptamers and spiegelmers "A aptamer is a single-stranded or double-stranded D- or L-nucleotide that specifically binds to a protein, causing an alteration in the protein's (KRAS protein, especially mutant KRAS protein) biological activity. The aptamer and spiegelmer are 15 to 80 nucleotides in length, preferably 20 to 50 nucleotides in length."

[0198] 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, particularly a conjugate of a polypeptide containing the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof with a KRAS inhibitor.

[0199] In some embodiments, the coupling between components (i) and (ii) is achieved through a non-pyrolytic joint. In some embodiments, the coupling between components (i) and (ii) is achieved through a pyrolytic joint. Exemplary non-disintegrable and disintegrable joints are described in US8088387, US8142784, WO2013075048, US6630579, US8512707, US9120854, US9023351, US20160095938, US9446146, WO2005009369, US5773001, US6214345, US10111954, US8153768, US7829531, US20160082119, WO2018218004, US8568728, WO2015057699, US20170182181, and US9198979, the contents of each of which are incorporated herein by reference in their entirety.

[0200] On the other hand, the present invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of the combination of the present invention and a pharmaceutically acceptable excipient.

[0201] When used in this 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 in which cell division is uncontrolled (e.g., cancer).

[0202] The pharmaceutical compositions of the present invention comprise pharmaceutically effective amounts of the combination of the present invention and a pharmaceutically active carrier. The pharmaceutical compositions of the present invention comprise a polypeptide containing the sequence SEQ ID NO: 1, a functionally equivalent variant thereof, a conjugate according to the present invention, a polynucleotide encoding said polypeptide or said conjugate, a carrier comprising said polynucleotide, or a cell capable of secreting said polypeptide or said conjugate into a medium, and a KRAS inhibitor. Suitable functionally equivalent variants of the polypeptide of SEQ ID NO: 1, suitable conjugates, fusion proteins, polynucleotides, carriers, or cells used in the pharmaceutical compositions according to the present invention are as defined above.

[0203] As used herein, the term "pharmaceuticalally effective amount" is understood to mean an amount that provides a therapeutic effect, which can be determined by those skilled in the art using conventional methods. The amounts of composable Omomyc peptides, their functionally equivalent variants, conjugates, fusion proteins, polynucleotides, carriers, cells, or KRAS inhibitors in the pharmaceutical compositions according to the invention will vary depending on the subject and the specific route of administration. Those skilled in the art will understand that dosage can 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.

[0204] The appropriate dosage of one or more active ingredients in a pharmaceutical composition will depend on the type of cancer to be treated, the severity and course of the disease, whether the composition is intended for prevention or treatment, prior treatment, the patient's clinical history and response to peptides or polypeptides, and the judgment of the attending physician.

[0205] The amount of the polypeptide containing the sequence SEQ ID NO:1, its functionally equivalent variants, fusion proteins, conjugates, polynucleotides, carriers, or cells is suitable for administration to a patient in a single dose or as part of a series of treatments. Depending on the type and severity of the disease, a suitable dose level is typically from about 0.01 to 500 mg per kilogram of patient body weight per day, and can be administered in single or multiple doses. Preferably, the dose level is from about 0.1 to about 250 mg / kg per day; more preferably, from about 0.5 to about 100 mg / kg per day.

[0206] In a preferred embodiment, the first component is administered at approximately 3.75 mg per kilogram of the subject's body weight per day, preferably four times a week, preferably intranasally. In another preferred embodiment, the first component is administered at approximately 8 to 15 mg / m² per day. 2 Preferred concentration: 10 to 12 mg / m³ 2 More preferably 11.25 mg / m 2 It is recommended to apply it four times a week, preferably intranasally.

[0207] In a preferred embodiment, the first component is administered at approximately 50 mg per kilogram of the subject's body weight per day, preferably twice weekly, preferably intravenously. In another preferred embodiment, the first component is administered at approximately 100 to 200 mg / m² per day.2 Preferred concentration: 125 to 175 mg / m³ 2 Preferred concentration: 140 to 160 mg / m³ 2 More preferably 150 mg / m 2 It is preferred to administer twice a week, preferably intravenously.

[0208] Suitable dosage levels may be from about 0.01 to 250 mg / kg daily, from about 0.05 to 100 mg / kg daily, or from about 0.1 to 50 mg / kg daily. Within this range, the dosage may be from 0.05 to 0.5 mg / kg, 0.5 to 5 mg / kg, or 5 to 50 mg / kg daily. For oral administration, the composition is preferably provided in tablet form containing 1.0 to 1000 mg of active ingredient, particularly in tablet form 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 the patient being treated. The compound can be administered 1 to 4 times daily, preferably 1 or 2 times daily.

[0209] In one embodiment, the combination or composition may be applied once a week, twice a week, three times a week, four times a week, five times a week, six times a week, or seven times a week. In one embodiment, the combination or composition may be applied once a week. In another embodiment, the combination or composition may be applied twice a week. In another embodiment, the combination or composition may be applied four times a week. In another preferred embodiment, the first component of the combination or composition is applied four times a week, and the second component of the combination or composition is applied once a week. In another embodiment, the first component of the combination or composition is applied twice a week, and the second component of the combination or composition is applied once a week. The two compounds may be applied simultaneously or sequentially. When the compounds are applied sequentially, the application of the first compound is stopped before the application of the second compound begins.

[0210] 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.

[0211] The dosage of the KRAS inhibitor depends on the specific reagent used and can be from about 0.01 mg to about 200 mg per kilogram of subject body weight per day, preferably from 0.01 mg to about 150 mg, more preferably from 0.01 mg to about 100 mg, preferably from 0.01 mg to about 75 mg, 0.01 mg to about 50 mg, more preferably from 0.5 mg to about 50 mg, preferably from about 1 mg to about 25 mg, even more preferably from about 5 mg to about 20 mg, more preferably from about 5 mg / kg to about 20 mg, more preferably from about 8 mg / kg to about 18 mg, more preferably from about 10 mg / kg to about 18 mg, once or more daily to achieve the desired therapeutic effect, preferably orally. In another preferred embodiment, the dosage of the KRAS inhibitor is about 2.5 mg per kilogram of subject body weight per day, or 7.5 mg / m² per day. 2 Preferably, it is administered once weekly, more preferably once daily, and even more preferably orally. In a preferred embodiment, the amount of KRAS inhibitor is approximately 0.5 mg per kilogram of subject body weight per day, or 1.5 mg / m² per day. 2 Preferably, it is administered once weekly, more preferably once daily, and even more preferably orally. In a preferred embodiment, the amount of KRAS inhibitor is approximately 5 mg per kilogram of the subject's body weight per day, or 15 mg / m² per day. 2 Preferably, it is administered once weekly, more preferably once daily, and even more preferably orally. In another preferred embodiment, the amount of KRAS inhibitor is approximately 10 mg per kilogram of the subject's body weight per day, or 30 mg / m² per day. 2 Preferably, it is administered once weekly, more preferably once daily, and even more preferably orally. In another preferred embodiment, the amount of KRAS inhibitor is approximately 50 mg per kilogram of the subject's body weight per day, or 150 mg / m² per day. 2 Preferably, it is administered once weekly, more preferably once daily, and even more preferably orally. In another preferred embodiment, the amount of KRAS inhibitor is approximately 100 mg per kilogram of the subject's body weight per day, or 300 mg / m² per day. 2 It is preferred to administer once a week, more preferably once a day, and even more preferably orally. KRAS inhibitors are preferably administered 7 days a week, preferably for 4 weeks.

[0212] The pharmaceutical composition according to the invention comprises a first component (i) and a second component (ii), wherein the first component is selected from polypeptides containing SEQ ID NO:1 according to the invention, their functionally equivalent variants, fusion proteins, conjugates, polynucleotides, carriers, or cells, and the second component is a KRAS inhibitor. The pharmaceutical composition may be present as a single formulation (e.g., as a tablet or capsule containing a fixed amount of each component), or, on the other hand, as separate formulations for subsequent combination for combined, sequential, or separate administration. The compositions of the invention also include formulations in the form of kit components, wherein the components are formulated separately but packaged in the same container. Those skilled in the art will understand that the formulations of different components in the pharmaceutical composition according to the invention may be similar, in other words, similarly formulated (as tablets or pills), which allows them to be administered via the same route. In the case where the different components of the invention are formulated separately, both components may be present in a blister pack. Each blister pack contains the amount of medication to be taken that day. If the medication must be administered several times a day, the medication corresponding to each administration can be placed in different portions of the blister pack, preferably with the time of administration recorded in each portion of the blister pack. Alternatively, the components of the compositions of the present invention can be formulated in different ways so that different components can be administered in different ways. Thus, it is possible to dispense the first group into a dosage form for intravenous administration, while dispensing the second group into tablets or capsules for oral administration, and vice versa. Those skilled in the art can adjust the ratio between the components of the combinations or pharmaceutical compositions of the present invention according to the antitumor agent used in each specific case and the desired indication. Therefore, the present invention contemplates compositions in which the ratio between the amounts of component (i) and component (ii) can range from 50:1 to 1:50, particularly 40:1 to 1:40, particularly 30:1 to 1:30, particularly 20:1 to 1:20, 1:10 to 10:1, or 5:1 to 1:5. In a more specific embodiment, the ratio ranges from 1:1 to 1:5, preferably 1:1 to 1:3. In a more preferred embodiment, the ratio ranges from 1:1 to 1:1.5, preferably 1:1.3 to 1:1.4, more preferably 1:1.34. In another preferred embodiment, the ratio 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 between the quantities ranges from 30:1 to 5:1, preferably from 30:1 to 8:1, more preferably from 25:1 to 15:1, and even more preferably from 20:1 to 10:1. In a preferred embodiment, the ratio between the quantities ranges from 20:1 to 1:20. In one embodiment, the ratio is 20:1. In another embodiment, the ratio is 1:20. In yet another embodiment, the ratio is 10:1.When the ratio of component (i) to component (ii) is preferably 1:1 to 2,000,000:1; more preferably 2:1 to 2,000,000:1; more preferably 3:1 to 2,000,000:1; more preferably 3:1 to 1,905,13 6:1; more preferably 6:1 to 1,000,000:1; more preferably 8:1 to 800,000:1; more preferably 24:1 to 500,000:1; more preferably 29:1 to 500,000:1; more preferably 36:1 to 100,000:1; more preferably 48:1 to 50,000:1; more preferably 143:1 to 10,000:1; more preferably 268:1 to 5,000:1; more preferably 889:1 to 1000:1.

[0213] In a preferred embodiment, the ratio of component (i) to component (ii), more preferably the ratio of Omomyc to Sotorasi, ranges from 8:1 to 2,000,000:1; preferably 8:1 to 1,905,136:1; more preferably 8:1 to 1,900,000:1; even more preferably 29:1 to 245,824:1; even more preferably 29:1 to 240,000:1; even more preferably 3,969:1 to 245,824:1; even more preferably 4,000:1 to 245,000:1; even more preferably 7,682:1 to 127,009:1; even more preferably 8,000:1 to 120,000:1. While these ratios are effective for treating any type of cancer, they are more preferably obtained when treating cancers selected from non-small cell lung cancer or colorectal cancer, even more preferably when the cancer has a KRAS mutation, and more preferably when it has a KRAS G12C mutation. In a preferred embodiment, in non-small cell lung cancer, preferably non-small cell lung cancer with a KRAS mutation, more preferably non-small cell lung cancer with a KRAS G12C mutation, the ratio of component (i):component (ii), more preferably the Omomyc:sotorasi ratio, ranges from 25:1 to 300,000:1, more preferably 29:1 to 245,824:1; preferably 29:1 to 240,000:1, more preferably 3,500:1 to 250,000:1; preferably 3,969:1 to 245,824:1; preferably 4,000:1 to 240,000:1; preferably 25:1 to 120:1; preferably 29:1 to 114:1. In a preferred embodiment, in colorectal cancer, preferably colorectal cancer with a KRAS mutation, more preferably colorectal cancer with a KRAS G12C mutation, the ratio of component (i):component (ii), more preferably the ratio of Omomyc:sotorasi is in the range of 7,000:1 to 130,000:1, more preferably 7,500:1 to 128,000:1; more preferably 7,682:1 to 127,009:1; more preferably 8,000:1 to 125,000:1.

[0214] In a preferred embodiment, the ratio of component (i):component (ii), more preferably the ratio of Omomyc:adaraci, ranges from 30:1 to 2,000,000:1; more preferably 35:1 to 1,900,000:1; more preferably 36:1 to 1,813,190:1; even more preferably 38:1 to 1,500,000:1; even more preferably 40:1 to 1,500,000:1. While these ratios are effective for treating any type of cancer, they are more preferably obtained when treating cancers selected from non-small cell lung cancer or colorectal cancer, and even more preferably when the cancer has a KRAS mutation, more preferably a KRAS G12C mutation. In a preferred embodiment, in non-small cell lung cancer, preferably non-small cell lung cancer with a KRAS mutation, more preferably non-small cell lung cancer with a KRAS G12C mutation, the ratio of component (i):component (ii), more preferably Omomyc:adaraci, ranges from 30:1 to 2,000,000:1; more preferably 35:1 to 1,900,000:1; more preferably 36:1 to 1,813,190:1; even more preferably 38:1 to 1,500,000:1; even more preferably 40:1 to 1,500,000:1. The myc:adarasi ratio ranges from 100:1 to 2,000,000:1, more preferably from 125:1 to 2,000,000:1; more preferably from 140:1 to 1,900,000:1; more preferably from 143:1 to 1,813,190:1; more preferably from 260:1 to 1,900,000:1; more preferably from 268:1 to 1,813,190:1. More preferably 260:1 to 1,800,000:1; more preferably 140:1 to 18,000:1; more preferably 143:1 to 17,679:1; more preferably 140:1 to 17,000:1; more preferably 3,500:1 to 925,000:1; more preferably 3,683:1 to 906,595:1; more preferably 3,500:1 to 900,000:1. In a preferred embodiment, in colorectal cancer, preferably colorectal cancer with a KRAS mutation, more preferably colorectal cancer with a KRAS G12C mutation, the ratio of component (i):component (ii), more preferably the ratio of Omomyc:adalacil, ranges from 1,000:1 to 300,000:1, more preferably 2,000:1 to 300,000:1; more preferably 3,000:1 to 250,000:1; more preferably 3,500:1 to 250,000:1; more preferably 3,508:1 to 226,649:1; and even more preferably 3,600:1 to 225,000:1.

[0215] In a preferred embodiment, the ratio of component (i):component (ii), more preferably the ratio of Omomyc:MRTX1133, ranges from 500:1 to 2,000,000:1; preferably from 800:1 to 2,800,000:1; more preferably from 889:1 to 1,778,133:1; and more preferably from 900:1 to 1,700,000:1. While these ratios are effective for treating any type of cancer, they are more preferably obtained when treating cancers selected from non-small cell lung cancer or colorectal cancer, and even more preferably when the cancer has a KRAS mutation, and more preferably when it has a KRAS G12D mutation. In a preferred embodiment, in non-small cell lung cancer, preferably non-small cell lung cancer with a KRAS mutation, more preferably non-small cell lung cancer with a KRAS G12D mutation, the ratio of component (i):component (ii), more preferably the ratio of Omomyc:MRTX1133, ranges from 10,000:1 to 500,000:1; more preferably 12,000:1 to 480,000:1; more preferably 14,000:1 to 460,000:1; more preferably 14,340:1 to 455,932:1; even more preferably 14,500:1 to 450,000:1; even more preferably 15,000:1 to 440,000:1. In a preferred embodiment, in colorectal cancer, preferably colorectal cancer with a KRAS mutation, more preferably colorectal cancer with a KRAS G12D mutation, the ratio of component (i):component (ii), more preferably Omomyc:MRTX1133, ranges from 500:1 to 2,000,000:1, more preferably 800:1 to 1,800,000:1; more preferably 889:1 to 1,778,133:1; even more preferably 900:1 to 1,700,000:1; even more preferably 950:1 to 1,600,000:1.

[0216] In a preferred embodiment, the ratio of component (i):component (ii), more preferably the Omomyc:BAY-293 ratio, ranges from 10:1 to 10,000:1; preferably 20:1 to 8,000:1; more preferably 24:1 to 6,105:1; and even more preferably 25:1 to 6,000:1. While these ratios are effective for treating any type of cancer, they are more preferably obtained when treating cancers selected from non-small cell lung cancer or colorectal cancer, and even more preferably when the cancer has a KRAS mutation, more preferably a KRAS G12C mutation or a KRAS G12D mutation. In a preferred embodiment, in non-small cell lung cancer, preferably non-small cell lung cancer with a KRAS mutation, more preferably non-small cell lung cancer with a KRAS G12 mutation or a KRAS G12D mutation, the ratio of component (i) to component (ii), more preferably the ratio of Omomyc to BAY-293, ranges from 20:1 to 10,000:1, more preferably 24:1 to 6,500:1; more preferably 24:1 to 6,105:1; more preferably 25:1 to 6,000:1; even more preferably 24:1 to 3,500:1; more preferably 24:1 to 3,052:1; even more preferably 25:1 to 3,000:1. In a preferred embodiment, in colorectal cancer, preferably colorectal cancer with KRAS mutation, more preferably colorectal cancer with KRAS G12C mutation or KRAS G12D mutation, the ratio of component (i):component (ii), more preferably Omomyc:BAY-293, ranges from 40:1 to 800:1, more preferably 48:1 to 763:1, and even more preferably 50:1 to 700:1.

[0217] In a preferred embodiment, the ratio of component (i):component (ii), more preferably the ratio of Omomyc:BI-3406, ranges from 1:1 to 10,000:1; more preferably 2:1 to 8,000:1; more preferably 3:1 to 6,000:1; even more preferably 3:1 to 5,922:1; even more preferably 3:1 to 5,500:1. While these ratios are effective for treating any type of cancer, they are more preferably obtained when treating cancers selected from non-small cell lung cancer or colorectal cancer, even more preferably when the cancer has a KRAS mutation, and even more preferably when it has a KRAS G12C mutation or a KRAS G12D mutation. In a preferred embodiment, in non-small cell lung cancer, preferably non-small cell lung cancer with KRAS mutation, more preferably non-small cell lung cancer with KRAS G12C mutation or KRAS G12D mutation, the ratio of component (i):component (ii), more preferably Omomyc:BI-3406, ranges from 6:1 to 6,000:1, more preferably 6:1 to 5,922:1; even more preferably 6:1 to 1,000:1; even more preferably 6:1 to 500:1; even more preferably 6:1 to 400:1; even more preferably 6:1 to 385:1; even more preferably 6:1 to 350:1.

[0218] Preferably, these ratios are by weight.

[0219] The pharmaceutical compositions or components of the present invention can be administered simultaneously. "Simultaneous administration" includes the co-administration of two therapeutic agents, regardless of the relative frequency or timing of their respective administration. Therefore, simultaneous administration includes co-administering two therapeutic agents at the same time and with the same frequency. Furthermore, simultaneous administration refers to the co-administration of two therapeutic agents in which one agent is administered more frequently than the other one or more agents. Additionally, simultaneous administration refers to the co-administration of two therapeutic agents in which one agent is administered only once during the administration of the other one or more agents.

[0220] 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 yet another embodiment, component (ii) is administered parenterally, particularly intraperitoneally or intravenously.

[0221] In a preferred embodiment, component (i) of the combination or pharmaceutical composition of the present invention is administered intravenously, while the KRAS inhibitor is administered orally. For intravenous administration, the preferred dose of component (i) of the combination or composition of the present invention, preferably, the preferred dose range of the polypeptide or its functionally equivalent variant, fusion protein or conjugate, is 0.01 to 250 mg / kg, which can be administered in single or multiple doses, more preferably from 0.1 to about 100 mg / kg daily. The preferred dose of the KRAS 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 one embodiment, the orally administered KRAS inhibitor is administered at a dose of 1 mg / kg to 25 mg / kg, more preferably 3 mg / kg to 20 mg / kg, more preferably 5 mg / kg to 20 mg / kg, more preferably 8 mg / kg to 18 mg / kg, and even more preferably 10 mg / kg to 18 mg / kg. Preferably, the KRAS inhibitor is administered 7 days a week for 4 weeks.

[0222] In another embodiment, the components (i) and (ii) of the combination or pharmaceutical composition of the present invention are administered intravenously.

[0223] The pharmaceutical compositions of the present invention may also contain one or more additional compounds for the prevention and / or treatment of pathological states in which uncontrolled cell division (e.g., cancer) occurs. These additional compounds, such as antitumor agents, may form part of the pharmaceutical composition as a separate entity. 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, antimetastatic agents, and antiproliferative agents.

[0224] The pharmaceutical compositions of the present invention also contain one or more other pharmaceutically acceptable excipients. A “pharmaceutically acceptable excipient” is understood to be a therapeutically inactive substance claimed to be incorporated into the active ingredient and, from a pharmacological / toxicological point of view, acceptable to the patient and, from a physical / chemical point of view, acceptable to the medicinal chemist who manufactures it in terms of composition, formulation, stability, patient acceptability, and bioavailability. The excipient may be a carrier. As used herein, “carrier” means any substance used to improve the delivery and efficacy of the active ingredient in the pharmaceutical composition. In a preferred embodiment, the carrier does not allow the direct delivery of components (i) and / or (ii) to the cytoplasm of the cell; that is, 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, such as sugar, polyols such as mannitol, sorbitol, or sodium chloride, in the combination or composition. Pharmaceutically acceptable carriers may also contain small amounts of excipients, such as wetting agents or emulsifiers, preservatives, or buffers, which can improve the shelf life or effectiveness of the components forming part of the combination or composition of the present invention. Examples of suitable carriers are well known in the literature (see, for example, Remington's Pharmaceutical Sciences, 19th 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 hydroxypropyl methylcellulose; and a series of fillers, such as gelatin and polyvinylpyrrolidone. In some cases, disintegrants, such as croscarmellose, agar, alginate, or sodium alginate, may be added.

[0225] The quantity and properties of pharmaceutically acceptable excipients depend on the desired dosage form. Pharmaceutically acceptable excipients are known to those skilled in the art (Faulí y Trillo C. (1993) “Tratado de Farmacia Galénica”, Luzán 5, SA Ediciones, Madrid). The composition can be prepared by conventional methods known in the prior art (“Remington: The Science and Practice of Pharmacy”, 20th edition (2003) Genaro AR, ed., Lippincott Williams & Wilkins, Philadelphia, US).

[0226] For pharmaceutical compositions comprising agents as nucleic acid molecules, the nucleic acid molecules can be present in any of a variety of delivery systems known to those skilled 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 skilled in the art. DNA can also be “naked,” as described, for example, in Ulmer et al., Science 259:1745-49, 1993, and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA can be increased by coating DNA onto biodegradable beads that are efficiently transported into cells.

[0227] Nucleic acid molecules can be delivered into cells according to any of the several methods described in the art (see, for example, 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 skilled in the art include, but are not limited to, encapsulation in liposomes via iontophoresis, or incorporation into other vehicles such as biodegradable polymers; hydrogels; cyclodextrins (see, for example, Gonzalez et al., Bioconjug. Chem. 10: 1068-74 (1999); Wang et al., International Application Publications WO 03 / 47518 and WO 03 / 46185); poly(lactic acid-co-glycolic acid) copolymers (PLGA) and PLCA microspheres (which can also be used to deliver peptides and polypeptides and other substances) (see, for example, 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 molecule may also be formulated or compounded 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).

[0228] In one specific embodiment, when the composition or combination according to the 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; by way of illustration and not limitation, the pharmaceutical composition may contain a viral or non-viral vector comprising a suitable polynucleotide or gene construct. By way of illustration and not limitation, the vector may be a viral vector, such as a vector based on retroviruses, adenoviruses, etc., or a non-viral vector such as ADN-liposomes, ADN-polymers, ADN-polymer-liposome complexes, etc. [see “Nonviral Vectors for Gene Therapy”, eds. 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, such as mammalian cells including human cells, in vitro, and then implanted into a human or animal to obtain the desired therapeutic effect. For administration to a human or animal, the cells will be formulated in a suitable medium that does not adversely affect cell viability.

[0229] The combination or pharmaceutical composition of the present invention can be administered via any suitable route, such as oral, local, inhalation, or parenteral route, thus including pharmaceutically acceptable excipients necessary for the formulation of the desired dosage form. Other routes of administration may include rectal, intracisional, or vaginal administration. Preferred routes of administration for the combination or pharmaceutical composition are intravenous administration for component (i) and oral administration for component (ii). Alternatively, the combination or pharmaceutical composition may be administered via intravenous administration.

[0230] "Oral route" is understood as the pharmaceutical composition entering the body after swallowing. In a specific embodiment, the pharmaceutical composition of the present invention can be a dosage form suitable for oral administration, whether it is solid or liquid. Dosage forms suitable for oral administration can be tablets, capsules, syrups, or solutions, and can contain any conventional excipients known in the art, such as binders, such as syrups, gum arabic, gelatin, sorbitol, or polyvinylpyrrolidone; fillers, such as lactose, sugar, corn starch, calcium phosphate, sorbitol, or glycine; compression lubricants, such as magnesium stearate; disintegrants, such as starch, polyvinylpyrrolidone, sodium glycolate 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 mixing operations can be used to completely distribute the active agent in those compositions using a large amount of filler. Such operations are conventional in the art. Tablets can be prepared by, for example, wet granulation or dry granulation, and optionally coated according to methods known in conventional pharmaceutical practice, particularly enteric coating.

[0231] On the other hand, "local route" is understood to mean administration via a non-systemic route and includes applying the pharmaceutical composition of the invention topically to the epidermis, in the mouth, and by instilling the composition into the ear, eye, and nose, wherein the composition does not significantly enter the bloodstream. Topical or transdermal dosage forms of the compounds of the invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches.

[0232] Ophthalmic preparations, ear drops, and eye drops are also considered to be within the scope of this invention. Furthermore, the invention contemplates the use of transdermal patches, which offer the additional advantage of controlled delivery of compounds 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 across the skin. The rate can be controlled by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0233] In one embodiment, the combination or pharmaceutical composition is administered systemically.

[0234] "Systemic route" is understood to mean administration via oral, intravenous, intraperitoneal, and intramuscular routes. The amounts of components (i) and (ii) required for therapeutic or preventative effects will naturally vary depending on the compounds selected, the nature and severity of the disease to be treated, and the patient. Preferably, the combination or pharmaceutical composition is administered orally.

[0235] In another embodiment, the combination or pharmaceutical composition is administered intranasally. In a preferred embodiment, intranasal administration is performed by instillation or nasal inhalation.

[0236] "Inhalation" is understood to mean administration via the intranasal route and oral inhalation. Suitable dosage forms for this administration, such as formulations in aerosols or metered-dose inhalers, can be prepared using conventional techniques. In one embodiment, the route of administration is intranasal.

[0237] As used herein, the term "parenteral" includes administration via intravenous, intraperitoneal, intramuscular, or subcutaneous routes. Subcutaneous, intramuscular, and intravenous formulations for parenteral administration are generally preferred. In one embodiment, the combination or pharmaceutical composition is administered intravenously.

[0238] In one embodiment, the combination or pharmaceutical composition of the present invention may be suitable for parenteral administration, for example, as a sterile solution, suspension, or lyophilized product in appropriate dose units. Combination or pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (when they are soluble in water), or dispersions and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. For intravenous administration, some suitable carriers include phosphate-buffered saline (PBS). In all cases, the composition or formulation must be sterile and must have easily injectable flowability. The composition or formulation must be stable under preparation and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium, including, for example, water, ethanol, pharmaceutically acceptable polyols such as glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. Suitable flowability can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size of the dispersion, and by using surfactants. Microbial action can be prevented by a variety of antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In most cases, it is preferable to include an isotonic agent, such as sugar; a polyol, such as mannitol or sorbitol; or sodium chloride in the composition. Extended absorption of injectable compositions can be achieved by including agents that delay absorption, such as aluminum and gelatin monostearate.

[0239] Injectable sterile solutions can be prepared, as needed, by incorporating the desired amount of the active compound into a suitable solvent containing one or a combination of the aforementioned components, followed by sterilization through sterile membrane filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile carrier containing a basic dispersion medium and the remaining desired components listed above. For sterile powders used to prepare injectable sterile solutions, a preferred preparation method is vacuum drying and lyophilization, which yields a powder containing the active ingredient and any desired additional components from a previously filtered sterile solution.

[0240] The combination or pharmaceutical composition of the present invention can 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.

[0241] Alternatively, as described above, the different components of the composition may be applied in different ways.

[0242] Therefore, in one embodiment, the component (i) of the combination or composition, preferably the polypeptide or functionally equivalent variant or conjugate of the present invention, is administered intravenously, while the KRAS inhibitor is administered orally.

[0243] In another embodiment, components (i) and (ii) are administered intravenously.

[0244] In another embodiment, the component (i) of the combination or composition, preferably a polypeptide or a functionally equivalent variant thereof or a conjugate of the composition, is administered intranasally or by inhalation.

[0245] The dosage form of the composition for intranasal and intrapulmonary administration is preferably a liquid, suspension, or solid. A suspension is a liquid formulation containing solid particles dispersed in a liquid carrier. The dosage form is preferably metered. For example, metered droplets / sprays refer to droplets / sprays containing a metered dose (predetermined amount) of the composition used according to the invention delivered by a dispenser comprising droplets / sprays.

[0246] In cases of intranasal administration, a preferred dosage form includes nasal drops. Drops deposit primarily in the back of the nose and thus rapidly migrate into the nasopharynx. A common challenge with drops is precisely controlling the dosage of the medication, which is particularly important for the administration of the composition.

[0247] Another intranasal dosage form for administering the pharmaceutical compositions of the present invention is a nasal spray. Nasal sprays typically contain a conjugate dissolved or suspended in a mixture of solution or excipients (e.g., preservatives, viscosity modifiers, emulsifiers, buffers) in a non-pressurized dispenser. Nasal sprays offer several advantages, including simplicity and convenience of delivery, ease of use, and accuracy of dosage delivery (25 pL to 200 pL). They deposit in the anterior nasal cavity and are slowly introduced into the nasopharynx by mucociliary clearance. The nasal sprays used herein may be liquids or suspensions.

[0248] Another intranasal dosage form is the nasal aerosol. The difference between nasal aerosols and nasal sprays lies in the method of compound distribution: in aerosols, the compound is distributed under higher pressure and released through a valve. In sprays, the compound is distributed by forceful propulsion from a micropump bucket, with the pressure in the vial similar to atmospheric pressure. Aerosols offer similar advantages to sprays.

[0249] Alternatively, the compositions according to the invention can preferably be applied as nasal emulsions, ointments, gels, pastes, or creams. These are highly viscous solutions or suspensions applied to the nasal mucosa.

[0250] Because of the limited volume of compositions that can be effectively delivered to the nasal mucosa, liquid intranasal dosage forms typically have higher concentrations than their corresponding intravenous dosage forms. When a substance has poor solubility or is unstable in liquid form, powders can be used to administer the compositions of the present invention. Other advantages of powders are that they do not require preservatives and generally have greater stability compared to liquid formulations. The main limitation to the application of intranasal powders is their irritant effect on the nasal mucosa.

[0251] One dosage form for intrapulmonary administration is an inhaled aerosol. Inhaled aerosols are typically packaged under pressure and contain a composition according to the invention, which is released into the respiratory tract, particularly the lungs, upon activation of a valve system. The released aerosol is a colloidal mixture 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.

[0252] Another dosage form for intrapulmonary administration is inhaled sprays. Inhaled sprays are typically water-based and do not contain any propellant. They deliver the conjugate to the lungs via oral inhalation.

[0253] 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 compositions according to the invention. Nebulized inhalation solutions and suspensions deliver the composition to the lungs via oral inhalation to produce a systemic effect and are used in conjunction with a nebulizer.

[0254] Dry powder inhalation is an alternative to aerosol inhalation. The composition is typically contained in capsules or inhalers for manual loading. The dry powder is usually delivered to the lungs via oral inhalation. The dry powder used herein can be formulated as a pure form. Pure formulations contain only the drug or essentially only the drug, such as as a spray dry powder. The dry powder used herein can also be formulated with a carrier such as lactose.

[0255] Preferably, the intrapulmonary dosage form is metered, i.e., delivered to the lungs in a predetermined amount.

[0256] In the context of this invention, devices for intranasal delivery include a spray pump system, a suction tube for delivering drops, a metered-dose spray pump, a nasal pressurized metered-dose inhaler, a powder spray system, a respiratory-actuated powder inhaler, and a nasal powder blower. Intranasal delivery devices may be filled with single-dose or multi-dose intranasal formulations.

[0257] The conjugate can be administered via an intrapulmonary route using a metered-dose inhaler (MDI). A metered-dose inhaler (MDI) delivers a fine mist of the conjugate, which typically has an aerodynamic particle size of less than 5 pm.

[0258] Alternatively, a dry powder inhaler can be used to deliver the composition intrapulmonaryly. Dry powder inhalers deliver single- or multiple-dose powders.

[0259] Another type of device used for intrapulmonary delivery includes nebulizers, such as ultrasonic nebulizers and air jet nebulizers. In an ultrasonic nebulizer, ultrasound waves are generated in the ultrasonic nebulizer chamber by the vibration of a ceramic piezoelectric transistor when electrically excited. This creates an aerosol cloud on the surface of the solution. The aerosol produced by an air jet nebulizer is generated 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, and then atomized using a baffle to promote aerosol cloud formation.

[0260] In one embodiment, the components of the combination or pharmaceutical composition of the present invention are prepared using a carrier that protects the components, particularly component (i), from rapid elimination from the body, such as controlled-release formulations comprising implants and microcapsule delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyethylene glycol, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing the formulations are well known to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc.

[0261] Sustained-release compositions also include crystal formulations suspended in a suitable formulation that keeps the crystals suspended. These formulations can produce a sustained-release effect when injected subcutaneously or intraperitoneally. Other compositions also include components (i) and / or components (ii) encapsulated in liposomes. Liposomes containing these components are prepared by known methods, for example, 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; EP36,676; EP 88,046; EP 143,949. In a preferred embodiment, components (i) and / or components (ii) are contained in liposomes, preferably both components are contained in liposomes, more preferably in the same liposome.

[0262] Although Omomyc, its functionally equivalent variants, conjugates, and fusion proteins of the present invention are indeed capable of transmembrane transport, Omomyc, any functionally equivalent variants, conjugates, polynucleotides, carriers, or cells can also be formulated into nanoparticles. Nanoparticles can help maintain the integrity of components in biological fluids until they reach the target organ. Furthermore, in the case where the composition contains component (ii) or other antitumor agents, encapsulation of the composition can reduce secondary effects caused by the antitumor agents. Additionally, nanoparticles can be modified to include portions that allow the nanoparticles to target the organ of interest. In this way, component (i) of the combinations or compositions of the present invention will be delivered to the vicinity of the target organ, facilitating the entry of component (i) into the cellular interior where its biological activity is required.

[0263] Therefore, in another embodiment, component (i) of the combination or composition of the present invention forms part of the nanoparticle. In another embodiment, both components of the combination or composition of the present invention form part of the nanoparticle, preferably, the two components are located within the same nanoparticle.

[0264] 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 nanoparticles ranges from 2-200 nm, preferably from 2-150 nm, and even more preferably from 2-100 nm. Nanoparticles that can be used in this invention include nanoscale materials such as lipid-based nanoparticles, superparamagnetic nanoparticles, nanoshells, semiconductor nanocrystals, quantum dots, polymer-based nanoparticles, silicon-based nanoparticles, silica-based nanoparticles, metal-based nanoparticles, fullerenes, and nanotubes. Molecules may be embedded in the nanoparticle matrix or adsorbed on its surface; preferably, molecules are embedded in the nanoparticles.

[0265] In a preferred embodiment, the nanoparticles are liposomes.

[0266] Targeted delivery can be achieved by adding ligands without compromising the nanoparticles' ability to deliver their contents. This is envisioned to enable delivery to specific cells, tissues, and organs. The targeting specificity of ligand-based delivery systems is based on the distribution of ligand receptors on different cell types. Targeting ligands can bind nonvalently or covalently to nanoparticles and can be coupled to nanoparticles using a variety of methods described herein.

[0267] Examples of proteins or peptides that can be used to target nanoparticles include transferrin, lactoferrin, TGF-β, nerve growth factor, albumin, HIV Tat peptide, RGD peptide, and insulin.

[0268] It should be understood that the nanoparticle formulations of the present invention are not intended to promote the entry of component (i) and / or component (ii) into cells or are not merely intended to promote the entry of component (i) and / or component (ii) into cells, but rather to protect component (i) and / or component (ii) from degradation and / or to promote the targeting of nanoparticles to organs of interest.

[0269] In one example, the nanoparticles can be made from biodegradable polymers such as poly(butyl cyanoacrylate) (PBCA). Examples of elemental nanoparticles include carbon nanoparticles and iron oxide nanoparticles, which can subsequently be coated with oleic acid (OA)-Pluronic®. In this method, a drug (e.g., a hydrophobic or water-insoluble drug) is loaded into the nanoparticles. Other nanoparticles are made of silica.

[0270] Nanoparticles can be formed from any useful polymer. Examples of polymers include biodegradable polymers such as poly(butyl cyanoacrylate), poly(lactide), poly(glycolic acid), poly-s-caprolactone, poly(butylene succinate), poly(ethylene glycol succinate), and poly(p-dioxanone); poly(ethylene glycol); poly-2-hydroxyethyl methacrylate (poly(HEMA)); copolymers such as poly(lactide-co-glycolic acid), 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 amylopectin, amylose, and chitosan.

[0271] Other nanoparticles include solid lipid nanoparticles (SLNs). Examples of lipid molecules in solid lipid nanoparticles include stearic acid and modified stearic acid, such as stearic acid-PEG 2000; soy lecithin; and emulsified waxes. Solid lipid nanoparticles may optionally include other components, including surfactants such as Epicuron® 200, Pluronic® F68, Brij 72, Brij 78, polysorbate 80 (Tween 80); and salts such as sodium taurocholate. Agents can be introduced into solid lipid nanoparticles by a variety of methods discussed for liposomes, which may also include high-pressure homogenization and microemulsion dispersion.

[0272] Nanoparticles may also comprise nanoscale micelles. Micelles can be formed from any polymer described herein. Exemplary polymers for forming micelles include block copolymers, such as poly(ethylene glycol) and poly(ε-caprolactone). (For example, a PEO-b-PCL block copolymer comprising ε-caprolactone and α-methoxy-ω-hydroxy-poly(ethylene glycol)).

[0273] In some embodiments, the properties of the nanoparticles are modified by coating them 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® 200; poloxamer surfactants, such as 188 (Pluronic® F68), poloxamer 908 and poloxamer 1508; and Brij surfactants, such as Brij 72 and Brij 78.

[0274] Nanoparticles can optionally be modified to include hydrophilic polymer groups (e.g., poly(ethylene glycol) or poly(propylene glycol)), for example, by covalently attaching the hydrophilic polymer groups to the surface or by using polymers containing such hydrophilic polymer groups (e.g., poly[methoxy poly(ethylene glycol) cyanoacrylate-co-hexadecyl cyanoacrylate]). Nanoparticles can optionally be cross-linked, which is particularly useful for protein-based nanoparticles.

[0275] 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 droplets have diameters in the nanoscale range. The nanoemulsion consists of an oil rich in ω-3, ω-6, or ω-9 fatty acids in an aqueous phase and is thermodynamically stabilized by an amphiphilic surfactant forming an interfacial film. It is prepared using a high-shear microfluidization process, and the droplet diameter is typically in the range of about 80-220 nm.

[0276] Therapeutic uses of the invention On one hand, the present invention relates to the use of the combination or pharmaceutical composition of the present invention in a pharmaceutical remedy.

[0277] On the other hand, the present invention relates to the use of the combination or pharmaceutical composition of the present invention for the prevention and / or treatment of cancer.

[0278] On the other hand, the present invention relates to the use of the combination or pharmaceutical composition of the present invention in the preparation of a medicament for the prevention and / or treatment of cancer.

[0279] On the other hand, the present invention also relates to a method for preventing and / or treating cancer, the method comprising administering a therapeutically effective amount of the combination or pharmaceutical composition of the present invention to a subject in need.

[0280] In a preferred embodiment, the preventive or therapeutic method according to the invention comprises the direct use of a combination or composition containing 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 invention does not involve the application of a nucleic acid encoding a polypeptide containing Omomyc or a functionally equivalent variant thereof or a fusion protein, or the application of a vector encoding such nucleic acid or cells containing said nucleic acid.

[0281] "Prevention" is understood to mean applying the combination or composition of the present invention at the initial or early stage of a disease, or to prevent its onset.

[0282] The term "treatment" is used to refer to the application of the combinations or compositions of the present invention before or after the onset of clinical symptoms to control disease progression. Control of disease progression is understood as a beneficial or desired clinical outcome, including but not limited to symptom relief, shortened disease duration, stable pathological condition (partially avoiding further damage), delayed disease progression, improved pathological condition, and remission (partial and complete). Control of disease progression also includes prolonged survival compared to expected survival without treatment. In one preferred embodiment, control of disease progression is measured by the healthy lung / thoracic volume ratio. In another embodiment, control of disease progression is measured by a reduction in tumor volume. In yet another embodiment, control of disease progression is measured by a decrease in tumor cell viability.

[0283] The term "cancer" refers to a disease characterized by uncontrolled cell division (or increased cell survival or increased resistance to apoptosis), the ability of said cells to invade other adjacent tissues via lymphatic vessels and blood vessels (invasion), or the spread to other areas of the body where the cells are not normally located (metastasis). Tumors are classified as benign or malignant based on whether they can spread through invasion and metastasis: benign tumors are tumors that cannot spread through invasion or metastasis, i.e., they grow only locally; while malignant tumors are tumors that can spread through invasion and metastasis. The method according to the invention can be used to treat both localized tumors and malignant tumors.

[0284] In one implementation, cancer includes, but is not limited to, leukemia (e.g., acute leukemia, acute lymphoblastic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia). Leukemia, acute promyelocytic leukemia, acute myeloid monocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, hairy cell leukemia, polycythemia vera, lymphoma (e.g., Hodgkin's or non-Hodgkin's), AIDS-related leukemia, Waldenström macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, meningothelioma, lymphangiosarcoma, lymphangioendothelioma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, Kaposi's sarcoma, colon cancer, pancreatic cancer, breast cancer). Adenocarcinoma, biliary tract cancer, esophageal cancer, ovarian cancer, prostate cancer, oral cancer (including squamous cell carcinoma), basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct cancer, 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), glioma, glioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0285] In some implementations, the cancer is glioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

[0286] In some implementations, the cancer is an acoustic neuroma, astrocytoma (e.g., grade I - pilocytic astrocytoma, grade II - low-grade astrocytoma, grade III - anaplastic astrocytoma, or grade IV - glioblastoma (GBM)), chordoma, CNS lymphoma, craniopharyngioma, brainstem glioma, ependymoma, mixed glioma, optic nerve glioma, subependymoma, medulloblastoma, meningioma, metastatic brain tumor, oligodendroglioma, pituitary adenoma, primitive neuroectodermal (PNET) tumor, or schwannoma. In some implementations, the cancer is a type more common in children than in adults, such as brainstem glioma, craniopharyngioma, ependymoma, juvenile pilocytic astrocytoma (JPA), medulloblastoma, optic nerve glioma, pineal tumor, primitive neuroectodermal tumor (PNET), or rhabdoid tumor. In some implementations, the patient is an adult. In some implementation schemes, the patient is a child or a pediatric patient.

[0287] In another implementation, the cancers include, but are not limited to, mesothelioma, hepatobiliary (liver and bile duct) cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin melanoma or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, gastrointestinal cancer (gastric cancer, colorectal cancer, and duodenal cancer), uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine system cancers, thyroid cancer, parathyroid cancer, and so on. 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 or ureteral cancer, renal cell carcinoma, renal pelvis cancer, non-Hodgkin's lymphoma, spinal axis tumor, brainstem glioma, pituitary adenoma, adrenocortical carcinoma, gallbladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the above cancers.

[0288] In some implementations, 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; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing sarcoma; anaplastic thyroid cancer; adrenocortical adenoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / stomach (GIST) cancer; lymphoma; head and neck squamous cell carcinoma (SCCHN); salivary gland cancer; glioma or brain cancer; neurofibromatosis-1-associated malignant peripheral nerve sheath tumor (MPNST); Waldenström macroglobulinemia; or medulloblastoma.

[0289] In some implementation schemes, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial carcinoma, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), cholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, undifferentiated thyroid carcinoma, adrenocortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1-associated malignant peripheral nerve sheath tumor (MPNST), Waldenström macroglobulinemia, or medulloblastoma.

[0290] In some implementations, the cancer is a solid tumor, such as sarcoma, carcinoma, or lymphoma. Solid tumors generally consist of an abnormal mass of tissue that does not typically include cysts or fluid-filled areas. In some implementations, 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. Carcinoma or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatobiliary duct cancer; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing sarcoma; undifferentiated thyroid carcinoma; adrenocortical carcinoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / stomach (GIST) cancer; lymphoma; head and neck squamous cell carcinoma (SCCHN); salivary gland cancer; glioma or brain cancer; neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST); Waldenström macroglobulinemia; or medulloblastoma.

[0291] In some embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial carcinoma, ovarian cancer, fallopian tube cancer, papillary serous cystadenocarcinoma, papillary serous uterine carcinoma (UPSC), cholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, undifferentiated thyroid carcinoma, adrenocortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1-associated malignant peripheral nerve sheath tumor (MPNST), Waldenström 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 tumor. In some embodiments, the cancer is ovarian epithelial carcinoma. In some embodiments, the cancer is fallopian tube cancer. In some embodiments, the cancer is papillary serous cystadenocarcinoma. In some embodiments, the cancer is papillary serous uterine carcinoma (UPSC). In some embodiments, the cancer is hepatobiliary duct cancer. In some embodiments, the cancer is soft tissue and bone synovial sarcoma. In some embodiments, the cancer is rhabdomyosarcoma. In some embodiments, the cancer is osteosarcoma. In some embodiments, the cancer is undifferentiated thyroid carcinoma. In some embodiments, the cancer is adrenocortical carcinoma. In some embodiments, the cancer is pancreatic cancer or pancreatic duct 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 implementations, the cancer is neurofibromatosis-1-associated MPNST. In some implementations, the cancer is Waldenström macroglobulinemia. In some implementations, the cancer is medulloblastoma.

[0292] In some implementations, the cancer is virus-associated cancer, including human immunodeficiency virus (HIV)-associated solid tumors, human papillomavirus (HPV)-16-positive incurable solid tumors, and adult T-cell leukemia caused by human T-cell leukemia virus type I (HTLV-I), a highly aggressive form of CD4+ T-cell leukemia characterized by clonal integration of HTLV-I into leukemia cells (see https: / / clinicaltrials.gov / ct2 / show / study / NCT02631746); as well as virus-associated tumors in gastric cancer, nasopharyngeal carcinoma, 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).

[0293] 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, stomach / gastric cancer, endometrial cancer / uterine cancer / cervical cancer, bladder cancer, head and neck cancer, leukemia, sarcoma, cholangiocarcinoma, glioblastoma, multiple myeloma, and lymphoma. More preferably, the cancer is selected from breast cancer, ovarian cancer, and prostate cancer, and even more preferably, it is breast cancer or ovarian cancer, more preferably, it is breast cancer.

[0294] In a preferred embodiment, the cancer is a KRAS-mutant cancer, i.e., cancer with a KRAS mutation. More preferably, the cancer is a mutant cancer selected from the following: KRAS G12C mutant cancer, KRAS G12D mutant cancer, and KRAS G13C mutant cancer.

[0295] In another implementation, the mutation in KRAS is selected from G12C mutation, G12D mutation, G13C mutation, G12S mutation, G12V mutation, G12R mutation, G12F mutation, G12I mutation, G13D mutation, G13C mutation, G13R mutation and Q61L mutation.

[0296] In another preferred embodiment, the cancer is pancreatic cancer, particularly pancreatic ductal adenocarcinoma.

[0297] In another implementation, the cancer is glioblastoma.

[0298] Glioblastoma, also known as glioblastoma and grade IV astrocytoma, is the most common and most aggressive cancer that begins in the brain.

[0299] In another implementation, the cancer is lung cancer.

[0300] 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, lung cancer is non-small cell lung cancer (NSCLC). In another embodiment, lung cancer is small cell lung cancer (SCLC).

[0301] As used in this article, the term non-small cell lung cancer (NSCLC) refers to a heterogeneous group of diseases grouped together because their prognosis and management are broadly similar, and according to the histological classification of the World Health Organization / International Association for the Study of Lung Cancer (Travis WD et al. Histological typing of lung and pleural tumours. 3rd ed. Berlin: Springer-Verlag, 1999), it includes: (i) Squamous cell carcinoma (SCC), which accounts for 30% to 40% of NSCLC, begins in the larger airways but grows slowly, which means that the size of these tumors can vary at the time of diagnosis.

[0302] (ii) Adenocarcinoma, the most common subtype of NSCLC, accounting for 50% to 60% of NSCLC, begins near the gas exchange surface of the lung and includes a subtype, bronchioloalveolar carcinoma, which may respond differently to treatment.

[0303] (iii) Large cell carcinoma is a rapidly 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 upon further investigation.

[0304] (iv) Adenosquamous carcinoma is a type of cancer that contains two types of cells: squamous cells (thin, flat cells that line up in certain organs) and glandular cells.

[0305] (v) Carcinomas with pleomorphic, sarcomatoid, or sarcomatoid components. This is a rare group of tumors that reflect the continuity of histological heterogeneity and the differentiation of epithelium and mesenchyme.

[0306] (vi) Carcinoid tumors are slow-growing neuroendocrine lung tumors that begin in cells that release hormones in response to stimuli provided by the nervous system.

[0307] (vii) Salivary gland carcinoma, which begins in salivary gland cells located in the large airways of the lungs.

[0308] (viii) Unclassified cancers, including cancers that do not fall under any of the above lung cancer categories.

[0309] In one specific implementation, NSCLC is selected from squamous cell carcinoma of the lung, large cell carcinoma of the lung, and adenocarcinoma of the lung.

[0310] In even more preferred embodiments, the lung cancer is adenocarcinoma, more preferably KRas-driven lung adenocarcinoma, and most preferably KRAS-mutant adenocarcinoma. In the case of NSCLC, KRAS mutations occur predominantly (95%) at codons 12 (>80%) and 13. The most common codon variation is the KRAS G12C mutation, accounting for approximately 39% of KRAS-mutant NSCLC. Other common mutations include KRAS G12V (18-21%) and KRAS G12D (17-18%) variations. Notably, smokers and non-smokers have different codon variants and mutation profiles in KRAS. Therefore, transition mutations (G>A) are more common in non-smokers, while transversion mutations (G>C or G>T) are more common in former or current smokers. In one preferred embodiment, the KRAS-mutant adenocarcinoma is the KRAS G12D mutation. In another preferred embodiment, the KRAS-mutant adenocarcinoma is the KRAS G12C mutation.

[0311] In one embodiment, the mutation in the KRAS gene is a mutation of glycine at position 12, glycine at position 13, or glutamine at position 61. In a more preferred embodiment, the mutation is selected from G12S, G12V, G12D, G13D, G12C, G12R, G12F, G12I, G13C, G13R, or Q61L mutations. In a preferred embodiment, the mutation is a G12D mutation. In another embodiment, lung cancer is KRas GD12 / p53-driven lung cancer, preferably KRas GD12 NSCLC driven by / p53.

[0312] As used in this article, the term small cell lung cancer (SCLC) refers to the proliferation of small cells with distinctive and strictly defined morphological characteristics, containing dense neurosecretory granules, giving the 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.

[0313] In another implementation, the cancer is colorectal cancer.

[0314] In another preferred embodiment, the cancer to be treated according to the invention is characterized by an increased expression level of one or more KRAS proteins, particularly mutated KRAS proteins. The KRAS level is considered to be increased relative to a reference value when the KRAS level 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 KRAS, particularly mutated KRAS, in a non-cancer sample.

[0315] In one implementation, cancer is a primary tumor. As used herein, the term "primary tumor" refers to a tumor that originates at its location or organ and is not metastasized from another location to that location.

[0316] In another embodiment, the cancer is cancer metastasis. In the context of this invention, "metastasis" is understood as the spread of cancer from its originating organ to different organs. Metastasis typically occurs via the blood or lymphatic system. When cancer cells spread and form new tumors, the latter are referred to as secondary or metastatic tumors. The cancer cells that form secondary tumors are similar to the cancer cells of the original tumor. For example, if breast cancer spreads (metastasizes) to the lungs, the secondary tumor is formed by malignant breast cancer cells. The lung disease is metastatic breast cancer, not lung cancer. The authors of this invention have also observed that the combinations or compositions of this invention are able to reduce cell proliferation regardless of whether the cancer exhibits increased expression or activity of the Myc protein. In a preferred embodiment, the cancer to be prevented or treated is Myc-induced cancer.

[0317] In one implementation scheme, cancer is a solid tumor.

[0318] In another implementation, the cancer is a cancer that is resistant to KRAS inhibitors.

[0319] “Drug resistance” refers to a decrease in the effectiveness of a drug in treating a disease or condition. As used herein, “cancer drug resistance” refers to cancer that exhibits resistance to KRAS inhibitors, whether innate or acquired. Innate resistance occurs when KRAS inhibitors are ineffective from the start of treatment due to pre-existing resistance mechanisms. Acquired resistance occurs when KRAS inhibitors become ineffective during treatment and after clinical benefit has been observed.

[0320] All combinations of compounds and cancer types of the present invention are included in the present invention.

[0321] In some embodiments, the combinations or compositions of the present invention produce an inhibition of tumor growth. In some embodiments, the combinations or compositions of the present invention reduce tumor size (e.g., volume or mass) by at least 5%, 10%, 25%, 50%, 75%, 90%, or 99% relative to pre-treatment tumor size. In some embodiments, the combinations or compositions of the present invention reduce the amount of tumor in a patient by at least 5%, 10%, 25%, 50%, 75%, 90%, or 99% relative to pre-treatment tumor amount.

[0322] As used herein, "subject" includes any animal that has cancer or exhibits symptoms of cancer, or is at risk of developing cancer or exhibiting symptoms of cancer. Suitable subjects (patients) include laboratory 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, with human patients being preferred. Preferably, the subject is a mammal, and most preferably a human.

[0323] Combinations or formulations intended for the prevention and / or treatment of cancer may be administered in any amount and via any route of administration that is effective in treating cancer or reducing its severity. The exact amount required varies from subject to subject, depending on the subject’s species, age and general condition, severity of disease or condition, specific agent, route of administration, etc. For ease of administration and dosage uniformity, the compounds of the present invention are preferably formulated in dose-unit form. As used herein, the expression “dose-unit form” refers to physically discrete units of the agent 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 reasonable medical judgment. The specific effective dose level for any particular patient or organism will depend on a variety of factors, including the condition being treated and its severity; the activity of the specific compound used; the specific ingredients used; the patient’s age, weight, general health condition, sex, and diet; the time of administration, route of administration, and rate of excretion of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors well known in the medical field.

[0324] In a preferred embodiment, the component (i) of the present invention, preferably a polypeptide or a functionally equivalent variant or conjugate thereof, synergistically interacts with a KRAS inhibitor in combination or composition to treat cancer (to achieve a therapeutic effect).

[0325] Specifically, in a more preferred embodiment, the combination or pharmaceutical composition for the prevention and / or treatment of cancer is a combination or pharmaceutical composition in which an amount of a polypeptide or a functionally equivalent variant or conjugate thereof synergistically interacts with a KRAS inhibitor to treat cancer.

[0326] The terms "synergistic effect" or "synergistic interaction" are used interchangeably. A synergistic effect is greater than the additive effect predicted by summing the actual in vitro effects of the individual agents. In vivo, a synergistic effect is a physiological effect, particularly a therapeutic effect, that is greater than the additive effect predicted by summing the actual in vivo effects of the individual agents.

[0327] Therefore, if two agents are administered, and 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, a synergistic effect is provided when neither the first agent alone nor the second agent alone provides any measurable effect, but the two agents together provide a measurable effect. Due to the synergistic effect of components (i) and (ii), the amount of components (i) and / or components (ii) in the combination or composition of the present invention can 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 more therapeutic agents may be 0.01-1.000 µg / kg body weight / day.

[0328] In a preferred embodiment, the degree of synergy is calculated using a zero-interaction power (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 SynergyFinder.org (Zheng, S.; Wang, W.; Aldahdooh, J.; Malyutina, A.; Shadbahr, T.; Tanoli, Z.; Passia, A.; Tang, J. 2022. SynergyFinder Plus: Toward Better Interpretation and Annotation of Drug Combination Screening Datasets. Genomics, Proteomics & Bioinformatics, 20(3):587-596).

[0329] The amount of therapeutic agent present in the combination or composition may not exceed the amount normally administered in a composition containing the therapeutic agent as the sole active agent. Preferably, the amount of therapeutic agent in the composition of the present invention is about 50% to 100% of the amount normally present in a composition containing the agent as the sole active agent. In some embodiments, a therapeutic agent is administered at a dose of about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the normally administered amount of the agent. As used herein, the phrase “normally administered” means the amount of an FDA-approved therapeutic agent approved for administration according to the FDA label instructions.

[0330] The combinations or compositions of the present invention can also be used in combination with known treatment methods, such as chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, hormones, or combinations thereof.

[0331] All embodiments of the present invention are also applicable to the treatment methods of the present invention.

[0332] Articles of manufacture and kits This disclosure also provides articles comprising any of the combinations or pharmaceutical compositions disclosed herein in one or more containers. In some embodiments, the articles include, for example, manuals, printed instructions, labels, or packaging inserts that instruct users (e.g., distributors or end users) to combine and / or use the compositions of the articles to prevent and / or treat cancer.

[0333] In some embodiments, the article includes, for example, a bottle, vial, cartridge, box, syringe, injector, or any combination thereof. In some embodiments, the label refers to the use or administration of the combination or pharmaceutical composition in the article according to the methods disclosed herein. In some aspects, the label suggests, for example, a regimen for use, treatment, prevention, or improvement of cancer.

[0334] All references that may be cited in this application (including references, patents, patent applications and websites) and the references cited therein are hereby expressly incorporated in their entirety by reference for any purpose.

[0335] *** Unless otherwise stated, all terms used herein should be understood to have their ordinary meaning as known in the art. Further more specific definitions of certain terms used in this application are set forth below and are intended to be applied uniformly throughout the specification and claims, unless otherwise expressly listed, which provides a broader definition. Throughout the specification and claims, the word “comprising” and its variations are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “comprising” covers situations where “consisting of”. Other objects, advantages, and features of the invention will become apparent to those skilled in the art upon reading the specification, or may be learned by practicing the invention. Moreover, the invention covers all possible combinations of the specific and particular embodiments described herein.

[0336] In this specification and the accompanying claims, the singular indefinite article terms (“a”, “an”) and the definite article term (“the”) include plural indicators, unless the context clearly indicates otherwise. The indefinite article term “a” (or “an”) and the terms “one or more” and “at least one” are used interchangeably herein. Furthermore, the term “and / or” as used herein should be considered as explicitly disclosing that each of the two specified features or components has or does not have the other. Thus, the term “and / or” (e.g., “A and / or B”) as used in phrases herein is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” (e.g., “A, B, and / or C”) as used in phrases 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 throughout the specification and claims in conjunction with numerical values ​​indicates a range of precision familiar and acceptable to those skilled in the art. Typically, this precision range is ±15%. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Units, prefixes, and symbols are expressed in their forms accepted by the International System of Units (SI). Numerical ranges include the numbers defining the range. Unless otherwise stated, amino acid sequences are written from left to right in the direction from amino to carboxyl. The headings provided herein are not intended to limit any aspect or facet of this disclosure, which can be obtained by referring to the entire specification as a whole. Thus, the terms directly defined below are defined more fully by referring to the entire specification.

[0337] The present invention will be described through the following embodiments, which are considered to be illustrative only and not to limit the scope of the invention.

[0338] Example Production and purification of Omomyc The Omomyc peptide sequence SEQ ID NO: 4, containing methionine at the N-terminus, was codon-optimized using GeneGPS® expression optimization technology for use in Escherichia coli (E. coli). E. coli The gene was expressed in [a specific gene], cloned into a medium copy number plasmid (pPC-OMO-T5) with a kanamycin resistance gene as a selectable marker, and purified from strain BL21(DE3) using a modified Max purification protocol described in J.-F. Naud et al. 2003. J Mol Biol, 326:1577-1595; F.-O. and Mcduff et al. 2009. J Mol Recognit, 22:261-269. The 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 purity was confirmed by mass spectrometry, SDS-PAGE, and UV spectroscopy.

[0339] Experimental details Several KRAS mutant cell lines were used to test the synergistic effect of Omomyc (a MYC inhibitor) with different KRAS inhibitors. Table 2 lists the cell lines used and their KRAS mutations. HCC44, H1792, H23, H358, and SW1463 were commercially available cell lines. KLA-p53 and KLA-Stk11 were derived from transgenic KRAS. G12D A KRAS lung adenocarcinoma (KLA) cell line was used to drive a mouse model of non-small cell lung cancer (NSCLC) and specific knockout of the TP53 (KLA-p53) and STK11 (KLA-Stk11) genes was obtained through CRISPR / Cas9 technology. Lim1215 is a commercially available colorectal cancer (CRC) cell line (originally KRAS wild-type) that has been genetically modified using CRISPR / Cas9 technology to express the KRAS G12D mutation.

[0340]

[0341] Table 1. KRAS mutant cell lines. NSCLC: Non-small cell lung cancer. CRC: Colorectal cancer.

[0342] To assess the potential synergistic effects of Omomyc and different KRAS inhibitors, cells were seeded in 96-well plates. Twenty-four hours post-seeding, cells were treated with increased concentrations of Omomyc (e.g., 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM) and increased concentrations of different KRAS inhibitors. Five days later, AlamarBlue (an indirect method for measuring the metabolic profile of live cells) was added to the wells, and fluorescence at 590 nm was measured using a Spark microplate reader 4–8 hours later. To determine whether the combination of two drugs has a synergistic effect, the ZIP synergy score was calculated using SynergyFinder.org (Zheng, S.; Wang, W.; Aldahdooh, J.; Malyutina, A.; Shadbahr, T.; Tanoli, Z.; Passia, A.; Tang, J. 2022. SynergyFinder Plus: Toward Better Interpretation and Annotation of Drug Combination Screening Datasets. Genomics, Proteomics & Bioinformatics, 20(3):587-596). When the synergy score is ≤10, the interaction between the two drugs is considered antagonistic; when the synergy score is between -10 and 10, the interaction is considered additive; and when the synergy score is >10, the interaction is considered synergistic (Yadav B, Wennerberg K, Aittokallio T, Tang J. 2015. Searching for drug synergy in complex dose-response landscapes using an interaction potency model. Comput Struct Biotechnol J, 13: 504-13). Statistical analysis was performed using Graph-Pad Prism 8. To determine the statistical significance between combination therapy and monotherapy, a one-way ANOVA with Dunnett's post-hoc test was performed.

[0343] Results To determine whether Omomyc synergizes with the KRAS G12C inhibitor, KRAS G12C-mutant NSCLC and CRC cell lines (HCC44, H1792, H23, H358, and SW1463) were treated with increased concentrations of Omomyc and sotoprazine (a KRAS G12C inhibitor). Cell viability was assessed using the AlamarBlue metabolic assay. In all cell lines, the combination of Omomyc and sotoprazine was significantly more effective than either treatment alone and exhibited synergistic effects at many different drug concentrations. Synergistic effects at representative concentrations are shown below. Figure 1 As shown.

[0344] The synergistic effect of Omomyc in combination with another KRASG12C inhibitor, adapalene, was also investigated. Similarly, in KRASG12C-mutant NSCLC and CRC cell lines (HCC44, H1792, H23, H358, and SW1463), the combination of Omomyc and adapalene was significantly more effective than either drug alone, and showed synergistic effects at many different drug concentrations. Synergistic effects at representative concentrations are shown below. Figure 2 As shown.

[0345] To determine whether Omomyc could synergize with KRASG12D inhibitors, KRASG12D-mutant NSCLC and CRC cell lines (KLA-p53, KLA-Stk11, and Lim1215) were treated with a combination of Omomyc and MRTX1133 (a KRASG12D inhibitor). Again, the combination of Omomyc and MRTX1133 significantly outperformed either therapy alone and showed synergistic effects at many different drug concentrations. Synergistic effects at representative concentrations are shown below. Figure 3 As shown.

[0346] The inventors also investigated the synergistic effect of Omomyc with pan-KRAS inhibitors. Examples of these pan-KRAS inhibitors are inhibitors that target the SOS1 molecule, located upstream of KRAS and responsible for KRAS activation. In this regard, the synergistic effect of Omomyc in combination with two pan-KRAS inhibitors, BAY-293 and BI-3406, was tested. To determine whether Omomyc synergizes with BAY-293, KRAS-mutant H358, HCC44, KLA-p53, and SW1463 cell lines were treated with escalating concentrations of Omomyc and BAY-293. To determine whether Omomyc synergizes with BI-3406, KRAS-mutant H358, HCC44, and KLA-p53 cell lines were treated with escalating concentrations of Omomyc and BI-3406. The results again showed that Omomyc can synergize with BAY-293 (…Figure 4 ) and BI-3406 ( Figure 5 The combination exhibits synergistic effects, and is superior to individual treatments, demonstrating synergistic effects at many different drug concentrations.

[0347] Taken together, all these results clearly demonstrate that Omomyc exhibits potent synergistic effects with both direct and indirect KRAS inhibitors across a wide range of concentrations and different cancer types. The combination of Omomyc and KRAS inhibitors represents an effective combination partner, offering an attractive therapeutic concept for most (if not all) KRAS-mutant cancers, regardless of their specific KRAS mutation.

Claims

1. A combination comprising: i) The first component, which is 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 moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) 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) The second component is a KRAS inhibitor.

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

1.

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

10.

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

5. The combination according to any one of claims 1 to 4, wherein the KRAS inhibitor is selected from KRAS G12C inhibitors, KRAS G12D inhibitors and pan-KRAS (SOS1) inhibitors; more preferably, the KRAS inhibitor is selected from KRAS G12C inhibitors and KRAS G12D inhibitors.

6. The combination according to claim 5, wherein the KRAS inhibitor is selected from sotoprazine, adarazine, and MRTX1133.

7. The combination according to claim 5, wherein the KRAS inhibitor is a pan-KRAS (SOS1) inhibitor.

8. The combination according to claim 7, wherein the pan-KRAS (SOS1) inhibitor is selected from BAY-293 and BI-3406.

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

10. Use of the combination of any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 for pharmaceutical purposes.

11. Use of the combination of any one of claims 1 to 8 or the pharmaceutical composition of claim 9 for the prevention and / or treatment of cancer.

12. The combination or pharmaceutical composition for the use of claim 11, wherein the cancer is selected from NSCLC and colorectal cancer.

13. The combination or pharmaceutical composition for the use according to claim 11 or 12, wherein the cancer is a KRAS-mutant cancer.

14. The combination or pharmaceutical composition for use according to any one of claims 11 to 13, wherein the cancer is a cancer resistant to a KRAS inhibitor.

15. A combination or pharmaceutical composition for use according to any one of claims 10 to 14, wherein the first component is administered intravenously and the second component is administered orally.

Images