Compounds containing mutant KRAS sequences and lipids, and their use

Abstract

To provide compounds that can be used in effective cancer treatments. [Solution] The present invention provides compounds comprising a KRAS sequence linked to a lipid by a linker, as well as related compositions and methods. In one embodiment, a compound comprising a mutant KRAS sequence and a lipid is provided, wherein the mutant KRAS sequence is conjugated to the lipid by a linker, and (i) the linker comprises one or more polyethylene glycol blocks, (ii) the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and (iii) the mutant KRAS sequence comprises an amino acid sequence selected from a group consisting of specific sequences.

Description

Array List

[0001] This application includes an array listing submitted electronically in ASCII format, the entire listing of which is incorporated herein by reference. The name of the ASCII copy created is _ and its size is _ bytes. [Background technology]

[0002] The RAS protein is a crucial component of cellular signaling pathways. Oncogenic activation of the RAS protein due to mutations has been frequently detected in several types of cancer, including pancreatic cancer. Further development of more effective cancer therapies is needed. [Overview of the Initiative]

[0003] This invention provides compounds that can be used in therapeutic methods.

[0004] In other words, in the first aspect, the present invention is characterized by a compound comprising a mutant KRAS sequence and a lipid, wherein the mutant KRAS sequence is conjugated to the lipid by a linker, and (i) the linker comprises one or more polyethylene glycol blocks, (ii) the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and (iii) the mutant KRAS sequence is YKLVVVGADGVGKSALTI (SEQ ID NO: 23), YKLVV It contains or consists of an amino acid sequence selected from the group consisting of VGAVGVGKSALTI (SEQ ID NO: 24), YKLVVVGARGVGKSALTI (SEQ ID NO: 25), YKLVVVGAAGVGKSALTI (SEQ ID NO: 26), YKLVVVGASGVGKSALTI (SEQ ID NO: 27), YKLVVVGACGVGKSALTI (SEQ ID NO: 28), YKLVVVGATGVGKSALTI (SEQ ID NO: 29), and YKLVVVGAGDVGKSALTI (SEQ ID NO: 30).

[0005] In a second aspect, the present invention is characterized by a compound comprising a mutant KRAS sequence and a lipid, wherein the mutant KRAS sequence is conjugated to the lipid by a linker, and (i) the linker comprises one or more polyethylene glycol blocks, (ii) the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and (iii) the mutant KRAS sequence comprises an amino acid sequence selected from the group consisting of CYKLVVVGADGVGKSALTI (SEQ ID NO: 1), CYKLVVVGAVGVGKSALTI (SEQ ID NO: 2), CYKLVVVGARGVGKSALTI (SEQ ID NO: 3), CYKLVVVGAAGVGKSALTI (SEQ ID NO: 4), CYKLVVVGASGVGKSALTI (SEQ ID NO: 5), CYKLVVVGACGVGKSALTI (SEQ ID NO: 6), CYKLVVVGATGVGKSALTI (SEQ ID NO: 22), and CYKLVVVGAGDVGKSALTI (SEQ ID NO: 7).

[0006] In one embodiment of the first and second aspects of the present invention, the mutant KRAS sequence is conjugated to a linker via a cysteine-maleimide linkage at its N-terminus.

[0007] In other embodiments of the first and second aspects of the present invention, the linker comprises 48 repeating units of polyethylene glycol.

[0008] In further embodiments of the first and second aspects of the present invention, the mutant KRAS sequence has the following structure at its N-terminus [ka] It is conjugated to.

[0009] In a third aspect, the present invention is characterized by a composition comprising one or more compounds of the first and second aspects of the present invention and a pharmaceutically acceptable carrier.

[0010] In one embodiment of the third aspect of the present invention, the composition comprises (1) a compound comprising the amino acid sequence YKLVVVGADGVGKSALTI (SEQ ID NO: 23), (2) a compound comprising the amino acid sequence YKLVVVGAVGVGKSALTI (SEQ ID NO: 24), (3) a compound comprising the amino acid sequence YKLVVVGARGVGKSALTI (SEQ ID NO: 25), (4) a compound comprising the amino acid sequence YKLVVVGAAGVGKSALTI (SEQ ID NO: 26), (5) a compound comprising the amino acid sequence YKLVVVGASGVGKSALTI (SEQ ID NO: 27), (6) a compound comprising the amino acid sequence YKLVVVGACGVGKSALTI (SEQ ID NO: 28), or a compound comprising the amino acid sequence YKLVVVGATGVGKSALTI (SEQ ID NO: 29), and (7) a compound comprising the amino acid sequence YKLVVVGAGDVGKSALTI (SEQ ID NO: 30).

[0011] In one embodiment of a third aspect of the present invention, the composition comprises (1) a compound comprising the amino acid sequence YKLVVVGADGVGKSALTI (SEQ ID NO: 23), (2) a compound comprising the amino acid sequence YKLVVVGAVGVGKSALTI (SEQ ID NO: 24), (3) a compound comprising the amino acid sequence YKLVVVGARGVGKSALTI (SEQ ID NO: 25), (4) a compound comprising the amino acid sequence YKLVVVGAAGVGKSALTI (SEQ ID NO: 26), (5) a compound comprising the amino acid sequence YKLVVVGASGVGKSALTI (SEQ ID NO: 27), (6) a compound comprising the amino acid sequence YKLVVVGACGVGKSALTI (SEQ ID NO: 28), and (7) a compound comprising the amino acid sequence YKLVVVGAGDVGKSALTI (SEQ ID NO: 30).

[0012] In another embodiment of a third aspect of the present invention, the composition comprises (1) a compound comprising the amino acid sequence CYKLVVVGADGVGKSALTI (SEQ ID NO: 1), (2) a compound comprising the amino acid sequence CYKLVVVGAVGVGKSALTI (SEQ ID NO: 2), (3) a compound comprising the amino acid sequence CYKLVVVGARGVGKSALTI (SEQ ID NO: 3), (4) a compound comprising the amino acid sequence CYKLVVVGAAGVGKSALTI (SEQ ID NO: 4), (5) a compound comprising the amino acid sequence CYKLVVVGASGVGKSALTI (SEQ ID NO: 5), (6) a compound comprising the amino acid sequence CYKLVVVGACGVGKSALTI (SEQ ID NO: 6), or a compound comprising the amino acid sequence CYKLVVVGATGVGKSALTI (SEQ ID NO: 22), and (7) a compound comprising the amino acid sequence CYKLVVVGAGDVGKSALTI (SEQ ID NO: 7).

[0013] In another embodiment of a third aspect of the present invention, the composition comprises (1) a compound comprising the amino acid sequence CYKLVVVGADGVGKSALTI (SEQ ID NO: 1), (2) a compound comprising the amino acid sequence CYKLVVVGAVGVGKSALTI (SEQ ID NO: 2), (3) a compound comprising the amino acid sequence CYKLVVVGARGVGKSALTI (SEQ ID NO: 3), (4) a compound comprising the amino acid sequence CYKLVVVGAAGVGKSALTI (SEQ ID NO: 4), (5) a compound comprising the amino acid sequence CYKLVVVGASGVGKSALTI (SEQ ID NO: 5), (6) a compound comprising the amino acid sequence CYKLVVVGACGVGKSALTI (SEQ ID NO: 6), and (7) a compound comprising the amino acid sequence CYKLVVVGAGDVGKSALTI (SEQ ID NO: 7).

[0014] In some embodiments, 700 μg of each compound is included in the composition.

[0015] In a further embodiment of a third aspect of the present invention, the composition comprises the following lipids [ka] Or a salt thereof, further comprising a compound consisting of the nucleotide sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8) linked at the 5'-end by a bond or a linker (where X is O or S). In one embodiment, the nucleotide sequence is bound to a lipid. In another embodiment, all internucleoside groups linking the nucleosides of 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8) are phosphorothioates.

[0016] In a fourth aspect, the present invention features a method of treating cancer in a human patient, the method comprising administering to the patient a composition of the third aspect of the present invention.

[0017] In one embodiment of the fourth aspect of the present invention, the method further comprises administering an adjuvant such as an adjuvant comprising a CpG nucleotide sequence (e.g., 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'; SEQ ID NO: 8).

[0018] In some embodiments, 0.1 mg, 0.5 mg, or 2.5 mg of the adjuvant is administered.

[0019] In one embodiment of the fourth aspect of the present invention, the method [Chemical formula] Or a salt thereof, further comprising administering to the patient a compound consisting of the nucleotide sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8) linked at the 5'-end by a bond or a linker (where X is O or S). In one embodiment, the nucleotide sequence is bound to a lipid. In one embodiment, 0.1 mg, 0.5 mg, or 2.5 mg of the compound is administered.

[0020] In another embodiment, all internucleoside groups linking the nucleosides of 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8) are phosphorothioates.

[0021] In one embodiment of the fourth aspect of the present invention, the cancer is pancreatic cancer, lung cancer, or colorectal cancer.

[0022] In a fifth aspect, the present invention relates to (i) any one compound of claims 1 to 5, or any one composition of claims 6 to 8, and (ii) the following lipids [ka] The kit comprises a compound comprising a salt thereof and a nucleotide sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8) linked at the 5' end by a bond or linker. In one embodiment, the nucleotide sequence is bound to a lipid. In another embodiment, all internucleoside groups linking the nucleoside of 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8) are phosphorothioates.

[0023] definition As used herein, "carbonyl" refers to the -C(O)- group.

[0024] As used herein, "carboxylate" refers to a -C(O)-OH group or a salt thereof.

[0025] As used herein, “conjugate” refers to the covalent bonding of a mutant KRAS sequence to a linker, and optionally, further covalent bonding of the linker to a lipid. In various examples, the covalent bond linking the linker to the mutant KRAS sequence or lipid is between a sulfur atom and sp in succinimide. 3The bonds may be covalent between the hybridized carbon atom, between the nitrogen atom and the carbon atom of the carbonyl group, or between the oxygen atom and the phosphorus atom of the thiophosphoryl or phosphoryl group. In various examples, each of the KRAS sequence and the lipid may contain a group for binding to a complementary group in the linker. For example, the KRAS polypeptide may contain a complementary group in the linker, such as a sulfur atom bonded to a succinimide (e.g., a sulfur atom in a cysteine ​​residue). The bond from the sulfur atom in the KRAS polypeptide to the succinimide in the linker may be formed by the reaction of a thiol in the KRAS polypeptide (e.g., a thiol in a cysteine ​​residue) with maleimide in the linker. Alternatively, the KRAS polypeptide may contain a nitrogen atom or a carbonyl group bonded to a carbonyl group or nitrogen atom in the linker, respectively. The bond between the carbonyl group and the nitrogen atom may be formed by the reaction of an amine or carboxylate in the KRAS polypeptide with a carboxylate or amine in the linker, respectively.

[0026] "Lipids" are any organic compounds that are oily to the touch, insoluble in water, and soluble in organic solvents, and generally include fatty acids and their derivatives. An example of a lipid used in this invention is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0027] As used herein, “linker” refers to a monovalent or divalent group that is covalently bonded to a biologically functional group with one valency and to another biologically functional group with the other valency. In one example, the linker links a KRAS sequence to a lipid, as described above. Optionally, such a linker may contain one or more polyethylene glycol blocks. In another example, the linker links the nucleotide sequence of a CpG oligonucleotide to a lipid (for example, -P(X)(OH)-O-CH(CH2NHCO-(CH2) as described herein). 16-CH3)2 or a salt thereof, wherein X is O or S). Such a linker can optionally contain one or more nucleotides, for example, dinucleotides (e.g., GG).

[0028] As used herein, "pharmaceutically acceptable carrier" refers to a vehicle that can suspend or dissolve an active compound and has the properties of being non-toxic and non-inflammatory in the subject (e.g., a human patient) to which it is administered. Further, a pharmaceutically acceptable carrier may contain pharmaceutically acceptable additives such as preservatives, antioxidants, flavorings, emulsifiers, dyes, or excipients that are known or used in the field of pharmaceutical formulations, do not significantly inhibit the therapeutic effect of the biological activity of the active agent, and are non-toxic to the subject.

[0029] As used herein, "phosphoryl" refers to the -P(O)(OR A )(OR B ) group, where R A is H and R B is valence.

[0030] As used herein, "polyethylene glycol" refers to the block -(OCH2CH2) n -, where n is the number of repeating units and is an integer from 2 to 50 (e.g., 24 or 48).

[0031] As used herein, "thiol" refers to the -SH group.

[0032] As used herein, "thiophosphoryl" refers to the -P(S)(OR A )(OR B ) group, where R A is H and R B is valence.

[0033] The terms “treat,” “treatment,” and “treating” refer to therapeutic approaches aimed at reversing, mitigating, improving, inhibiting, slowing, or halting the progression or severity of a condition associated with a disease or disorder, such as cancer. These terms include reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder. A treatment is generally “effective” if one or more symptoms or clinical markers are reduced or a desired response (e.g., a specific immune response) is induced. Alternatively, a treatment is “effective” if the progression of the disease is reduced or halted.

[0034] The present invention offers several advantages. For example, certain compounds of the present invention, including a DSPE site, bind to endogenous albumin in the target of administration, thereby facilitating the delivery of the compound to the target's lymph nodes. This promotes the induction of a therapeutic immune response against the compound's KRAS sequence, leading to effective cancer treatment.

[0035] Other features and advantages of the present invention will become apparent from the following detailed description, drawings, and claims. [Brief explanation of the drawing]

[0036] [Figure 1] Figure 1 shows that amphiphile-KRas (aKRas) combined with amphiphile-CpG (aCpG) activated spleen cells, but soluble KRas combined with aCpG, as well as the untreated, soluble KRas, and polyIC (pIC) control groups, did not. [Figure 2] Figure 2 shows that both aKRas G12R and G12V combined with aCpG activated spleen cells, but soluble KRas G12R and G12V combined with aCpG, as well as the untreated, soluble KRas, and pIC control groups, did not activate them. [Figure 3]Figure 3 shows the KRas-specific CD8+ T cell response in mice immunized with a combination of aKRas G12D and aCpG. This response was not observed with soluble KRas G12D combined with CpG, or with soluble wild-type KRas combined with CpG. [Figure 4] Figure 4 shows the KRas-specific CD8+ T cell response (9mer) and CD8+ or CD4+ T cell response (18mer) in mice immunized with a combination of aKRas G12D and aCpG. This response was not observed in soluble KRas G12D combined with CpG or in untreated controls. [Figure 5] Figure 5 shows that the Amph-KRAS and Amph-CpG vaccines induce a superior endogenous T cell response compared to the soluble KRas vaccine, as indicated by the increased cytokine production by spleen cells in the cytokine bead array. Soluble KRAS G12D = YKLVVVGADGVGKSALTI (SEQ ID NO: 23), Amph-KRAS G12D = DSPE-amide-dPEG24-amide-dPEG24-CYKLVVVGADGVGKSALTI (SEQ ID NO: 1), KRAS G12D 18-mer = YKLVVVGADGVGKSALTI (SEQ ID NO: 23), CBA = cytokine bead array, IFN = interferon; IL = interleukin, KRAS = Kirsten rat sarcoma. [Figure 6] Figure 6 shows the structure of amphifil-peptide G12D 4-21 (SEQ ID NO: 1). [Figure 7] Figure 7 shows the structure of amphiphyl-CpG-7909. All sugars are deoxyribose. All internucleoside links are phosphorothioates. The structure is shown as the sodium salt. [Modes for carrying out the invention]

[0037] The present invention provides compounds that can be used in therapeutic methods. Each compound of the present invention comprises a mutant KRAS sequence (e.g., one of SEQ ID NOs: 1-7 and 22 (see Table 1 below), or one of SEQ ID NOs: 23-30 (see Table 2 below)) and a lipid (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)). These sites (mutant KRAS sequence and lipid) are linked to each other by a linker, for example, a linker comprising one or more polyethylene glycol blocks. In one example, the linker is [ka] In certain cases, a mutant KRAS sequence (e.g., one of sequence numbers 1-7 or 22, or one of sequence numbers 23-30) is bound to a linker via a Cys residue at its N-terminus, and the linker is bound to a lipid, where the linker is... [ka] Therefore, lipids [ka] Or its salt.

[0038] The present invention also provides compositions comprising one or more compounds of the present invention together with a pharmaceutically acceptable carrier or diluent. Optionally, a composition may comprise seven different compounds as described above, where each of the seven different compounds comprises a different sequence selected from SEQ ID NOs: 1-7 and 23 or SEQ ID NOs: 23-30. Compositions comprising a subset of these compounds (e.g., 2, 3, 4, 5, or 6) are also included in the present invention. The different compounds comprising a composition of the present invention may optionally each comprise the same or different KRAS sequences, linkers, and / or lipids.

[0039] The compositions of the present invention can be used in methods for inducing an immune response to the KRAS sequence of a compound. Therefore, the compositions can be collectively referred to as immunogenic or vaccine compositions. Accordingly, the compositions may optionally contain or be administered together with one or more adjuvants. For example, the adjuvants used may include, for instance, the following lipids. [ka] Alternatively, it may comprise a salt thereof (where X is S) and a CpG oligonucleotide linked at its 5' end by a bond or linker (e.g., 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'; SEQ ID NO: 8; CpG-7909). Preferably, X is S.

[0040] CpG oligonucleotides may be directly bound to lipids. Alternatively, the linker bound to the CpG oligonucleotide and the lipid may be GG. In CpG oligonucleotides, all internucleoside groups in the compound are phosphorothioates (for example, all internucleoside groups in the compound may be phosphorothioates).

[0041] The compounds and compositions of the present invention can be used in therapeutic methods. In particular, the KRAS sequence of the compounds can induce an immune response to KRAS expressed in several cancer cells. Accordingly, the present invention provides a method for treating a target cancer by administering one or more of the compounds or compositions of the present invention to a subject (e.g., a human patient). The present invention also includes a method for inducing an immune response to KRAS in a subject (e.g., a human patient) by administering one or more of the compounds or compositions of the present invention to the subject. In various examples, the cancer is selected from the group consisting of pancreatic cancer, lung cancer, and colorectal cancer. Optionally, the method of the present invention may further include the step of administering the compounds or compositions of the present invention in combination with a second (or further) different therapeutic approach.

[0042] Furthermore, the present invention provides a kit in which each comprises, for example, a first container containing one or more compounds of the present invention, and optionally a second container containing an adjuvant such as an adjuvant as described herein.

[0043] [Table 1]

[0044] In each of the "Amph-peptides" in Table 1, the lipid poly(ethylene glycol) moiety was conjugated to the peptide via maleimide (MAL)-cysteine ​​coupling using the following synthon: DSPE-amide-dPEG24-amide-dPEG24-MAL

[0045] [Table 2]

[0046] The compounds of the present invention may be prepared from a mutant KRAS polypeptide, a linker precursor, and a lipid using methods known in the art. In one embodiment, the linker precursor is initially linked to the lipid, for example, as follows. [ka] Amidation reaction conditions are known in the art, and typical amidation conditions include, for example, the use of reagents such as EDC / DMAP, HATU / HOAt, or HBTU / HOAt. Alternatively, the carboxylate may be substituted with O-succinimide ester, pentrafluorophenyl ester, or tetrafluorophenyl ester. Next, the reaction product may be reacted with the mutant KRAS polypeptide, for example, as shown below. [ka] The 1,4-addition reaction may be carried out in a suitable solvent, such as a polar organic solvent or water.

[0047] Alternatively, the compound of the present invention may be prepared as follows. The linker precursor may be initially linked to the lipid, for example, as follows. [ka] Next, the product may be reacted with an amine group (e.g., an N-terminal amine) in the mutant KRAS polypeptide to produce the compound of the present invention. [ka]

[0048] CpG oligonucleotides may be directly bound to lipids or linked by linkers. These compounds can be produced using conventional phosphoramidite chemistry known in the art. In some examples, CpG oligonucleotides or CpG oligonucleotides bound to GG at the 5' end can be produced using the following compounds. [ka] The intermediate may be produced by reacting with the lipid, where oxidation (for example, phosphite oxidation methods known in the art, for example, sulfurizing agents, for example, 3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazol-5-thion) and hydrolysis of the cyanoethyl group are performed. [ka] Alternatively, a compound may be formed from the salt thereof and a CpG oligonucleotide linked at its 5' end by a bond or linker (where X is O or S).

[0049] Administration The dose of each KRAS peptide administered may range from 100 to 5000 μg per peptide (for example, 700 μg / peptide, which would result in a peptide dose of 4900 g for a group of seven peptides).

[0050] When the adjuvant is administered together with the KRAS peptide, the adjuvant dose can be as high as 0.48 mg / kg on a body weight basis. An exemplary dosing regimen is shown in Table 3 below.

[0051] [Table 3]

[0052] Examples are provided below to better illustrate the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. [Examples]

[0053] Example 1: Amphifil KRas G12D triggers an immune response. The efficacy of soluble-KRas (KRas) or amphifil-KRas (aKRas) (Figure 6) mutant sequences, and amphifil-CpG adjuvant (aCpG) in inducing an immune response was investigated. In this experiment, five times the dose of aCpG as amphifil-CKRas was used, as escalation studies of CpG suggested that this dose was more effective.

[0054] The experimental design involved immunizing C57BL / 6 mice with the listed compounds, with the following four groups (n=5 in each group). 1. KRas G12D + aCpG 2. aKRas G12D + aCpG 3. KRas G12D + pIC 4. Non-immunization

[0055] Poly-integrated ICs (pICs) were used as the benchmark adjuvant control.

[0056] The adjuvant was dissolved in H2O solution, similar to the peptide stock solution. The final injector was diluted with 1X PBS (phosphate-buffered saline).

[0057] The aKRas peptide is a mutant sequence with a G12D substitution (wild-type amino acids 4-21 -> YKLVVVGA G The 18-mer sequence of GVGKSALTI (SEQ ID NO: 9) was used. 20 μg was used per 100 μl for each injection.

[0058] The aCpG sequence used was the CpG1826 sequence (5'-tccatgacgttcctgacgtt-3'; SEQ ID NO: 11), with guanine added to the 5' end of the CpG1826 sequence (5'-tccatgacgttcctgacgtt-3' (SEQ ID NO: 10)), at a concentration of 5 nmol per 100 μl injection. CpG1826 is the optimal sequence for mice, while CpG7909 is optimal for humans and exhibits low activity in mice. CpG1826 and CpG7909 belong to the same CpG class (Class B) and generally have similar activity profiles in their respective species.

[0059] 50 μg of pIC was used for each 100 μl injection.

[0060] Primer immunization was administered subcutaneously (sc) to the tail base (day 0), followed by a single booster dose two weeks later (day 14). Eight days after the booster dose (day 21), ELISpot analysis of IFNγ in spleen cells was performed using a standard protocol. Spleen cells (10 6 Cells / well are treated with 5 μg / well of G12D sequence 18mer(aa4-21)-> YKLVVVGA D It was activated with GVGKSALTI (SEQ ID NO: 1). As shown in Figure 1, aKRas combined with aCpG activated spleen cells, but soluble KRas combined with aCpG, as well as the untreated, soluble KRas, and pIC control groups, did not activate them.

[0061] Example 2: Amphifil KRas G12R and G12V induce an immune response. The efficacy of additional soluble-KRas (KRas) or amphifil-KRas (aKRas) mutant sequences, and amphifil-CpG adjuvant (aCpG) or soluble-CpG (CpG) in inducing an immune response was investigated.

[0062] The experimental design involved immunizing C57BL / 6 mice with the listed compounds, with the following seven groups (n=5 in each group). 1. KRas G12R + CpG 2. KRas G12V + CpG 3. KRas G12R + pIC 4. KRas G12V + pIC 5. aKRas G12R + aCpG 6. aKRas G12V + aCpG 7. Non-immunization

[0063] Dissolve the adjuvant in H2O, similar to the peptide stock solution. Dilute the final injector with 1X PBS.

[0064] aKras peptide is a mutant sequence with a G12R / V substitution (wild-type amino acids 4-21 -> YKLVVVGA G The 18-mer sequence of GVGKSALTI (SEQ ID NO: 9) was used. 20 μg was used per 100 μl for each injection.

[0065] The aCpG sequence used was the CpG1826 sequence (5'-tccatgacgttcctgacgtt-3' (SEQ ID NO: 10) with guanine added to the 5' end (5'-gg tccatgacgttcctgacgtt-3'; SEQ ID NO: 11), at a concentration of 5 nmol per 100 μl injection.

[0066] 50 μg of pIC was used for each 100 μl injection.

[0067] Primer immunization was administered subcutaneously (sc) to the base of the tail, followed by a single booster dose two weeks later (day 14).

[0068] Seven days after booster dose administration (day 21), ELISpot analysis of IFNγ in spleen cells was performed using a standard protocol. 6 Cells / well) are treated with 5 μg / well of G12V sequence 18mer(aa4-21)-> YKLVVVGA V GVGKSALTI (sequence number 2), or G12R sequence 18mer (aa4-21) -> YKLVVVGA R It was activated by one of the GVGKSALTI (SEQ ID NO: 3) sequences.

[0069] As shown in Figure 2, both aKRas G12R and G12V combined with aCpG activated spleen cells, but neither soluble KRas G12R nor G12V combined with aCpG, nor the untreated, soluble KRas, and pIC control groups activated them.

[0070] Example 3: Amphifil KRas G12D is CD8 + evokes the immune response of T cells In wild-type B6 mice, KRas-specific CD8 + Inducing a T cell response is difficult. B6 mice transfected with the human HLA gene A2.1 were immunized with soluble or amphifyl-conjugate KRas G12D antigen and an adjuvant (CpG or aCpG). The A2.1 allele is known to mount a response to KRas peptides in humans.

[0071] The experimental design involved immunizing B6 HLA-A2.1 Tg mice with the listed compounds, comprising the following four groups (n=5 in each group). 1. KRas wild type + CpG 2. KRas 12D + CpG 3. aKRas 12D + aCpG 4. Non-immunization

[0072] The adjuvant was dissolved in H2O, similar to the peptide stock solution. The final injection was diluted with 1X PBS.

[0073] The aKRas peptide is a mutant sequence with a G12D substitution (wild-type amino acids 4-21 -> YKLVVVGA G The 18-mer sequence amino acids 4-21 of GVGKSALTI (SEQ ID NO: 9) were used. 20 μg was used per 100 μl for each injection.

[0074] The aCpG sequence used was the CpG1826 sequence (5'-tccatgacgttcctgacgtt-3' (SEQ ID NO: 10) with guanine added to the 5' end (5'-gg tccatgacgttcctgacgtt-3'; SEQ ID NO: 11), at a concentration of 5 nmol per 100 μl injection.

[0075] The vaccine was administered subcutaneously to both sides of the tail base of female mice at a concentration of 50 μl per side. Two booster doses were given at two-week intervals.

[0076] Intracellular cytokine staining (ICS) analysis of IFNγ in peripheral blood mononuclear cells (PBMCs) and ELISpot analysis of IFNγ in spleen cells were performed using a standard protocol, 7 days after the second and third booster doses, respectively.

[0077] PBMCs / spleen cells (10 6 Cells / well and 2x10 6 Cells (per well) were activated with either 5 μg / well of SEQ ID NO: 1 or one of SEQ ID NOs: 12-20, as shown below. JPEG2026067877000019.jpg67165

[0078] The specific mutant peptide is administered only to mice immunized with the corresponding 18mer.

[0079] Figure 3 shows KRas-specific CD8 in mice immunized with a combination of aKRas G12D and aCpG. +It exhibits a T cell response. This response was not observed with soluble KRas G12D combined with CpG, nor with soluble wild-type KRas combined with CpG.

[0080] Example 4: Analysis at high antigen concentrations and different dosing intervals The antigen concentration was increased from 20 μg to 50 μg, and the study was conducted with a dosing interval of every other week (bw) or every week (w).

[0081] The experimental design involved immunizing C57BL / 6 mice with the listed compounds, with the following five groups (n=5 in each group). 1. KRas 12D + CpG1826 (bw) 2. KRas 12D + CpG1826 (w) 3. aKRas 12D + aCpG1826 (bw) 4. aKRas 12D + aCpG1826 (w) 5. Non-immunization

[0082] The adjuvant was dissolved in H2O, similar to the peptide stock solution. The final injection was diluted with 1X PBS.

[0083] The aKRas peptide is a mutant sequence with a G12D substitution (wild-type amino acids 4-21 -> YKLVVVGA G The 18-mer sequence of GVGKSALTI (SEQ ID NO: 9) was used. 50 μg was used per 100 μl for each injection.

[0084] The aCpG sequence used was the CpG1826 sequence (5'-tccatgacgttcctgacgtt-3' (SEQ ID NO: 10) with guanine added to the 5' end (5'-gg tccatgacgttcctgacgtt-3'; SEQ ID NO: 11), at a concentration of 5 nmol per 100 μl injection.

[0085] Primer immunization was administered subcutaneously (sc) to the base of the tail, followed by a single booster dose two weeks later.

[0086] Seven days after booster dose administration, ELISpot analysis of IFNγ and CBA (cytometric bead array) analysis of IL6, IL10, IL12, TNFα, IFNγ, and MCP1 were performed on spleen cells using a standard protocol. 6 Cells (per well) were activated with either 5 μg / well of SEQ ID NO: 1 or one of SEQ ID NOs: 12-21, as shown below. JPEG2026067877000020.jpg74165

[0087] The specific mutant peptide was administered only to mice immunized with the corresponding 18mer.

[0088] Separate plates were prepared for either short (9-mer and 10-mer) or long (17-mer and 18-mer) peptide stimulation. Cells from "non-immunized" control mice were stimulated with all stimulants.

[0089] Figure 4 shows KRas-specific CD8 in mice immunized with a combination of aKRas G12D and aCpG. + T cell response (9mer) and CD8 + or CD4 + The study exhibits a T cell response (18mer). This response was not observed with soluble KRas G12D combined with CpG or with untreated controls.

[0090] Example 5: Further analysis of KRAS amphifil As described in the above examples, the potential immunogenicity of mutant KRAS peptide-amfil vaccines with aCpG adjuvant has been demonstrated in the C57BL / 6 mouse model. This study and further studies are summarized in Table 4 and show that, due to species differences in MHC binding, mice have historically had a lower immunological response to mutant KRAS peptides than humans. Subcutaneous administration of mutant KRAS Amph-peptides (G12D (Figure 6), G12R, and G12V) is effective in inducing mouse KRAS-specific spleen T cells identified by ELISPOT and cytokine bead arrays (Figures 1, 2, 4, and 5). • Mutant KRAS Amph-peptides (G12D, G12R, and G12V) combined with aCpG as an adjuvant are more effective in inducing KRAS-specific spleen T cells than mutant KRAS peptides combined with soluble CpG or polyinosinic:polycytidylic acid (poly IC) as an adjuvant (Figures 1, 2, and 5), and As demonstrated through effector cytokine responses upon restimulation with long (18-mer) or minimal (9-mer) peptide epitopes (Figure 4), mutant KRAS Amph-peptide (G12D) combined with aCpG is more effective than soluble KRAS peptide combined with soluble CpG in inducing both CD4 and CD8 T cell responses.

[0091] [Table 4] JPEG2026067877000022.jpg121162

[0092] Other Embodiments While the present invention has been described in relation to its particular embodiments, further modifications are possible, and it can be understood that this application is generally intended to cover any variations, uses, or adaptations of the present invention in accordance with the principles of the present invention, including any deviations from the disclosure herein that are within the scope of known or customary practice in the art to which the invention belongs and that can be applied to the essential features set forth herein.

[0093] All publications, patents, and patent applications are incorporated herein by whole to the same extent that each individual publication, patent, or patent application is specifically and individually indicated so as to be incorporated by whole.

[0094] Some embodiments of the present invention are described in the following numbered paragraphs.

[0095] 1. A compound comprising a mutant KRAS sequence and a lipid, wherein the mutant KRAS sequence is conjugated to the lipid by a linker, and (i) the linker comprises one or more polyethylene glycol blocks, (ii) the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and (iii) the mutant KRAS sequence is YKLVVVGADGVGKSALTI (SEQ ID NO:23), YKLVVVGAVGVGKSALTI (SEQ ID NO:24), YKLVVVGARGVGKSALTI (SEQ ID NO:25), YKLVVVGAAGVGKSALTI (SEQ ID NO:26), YKLVVVGASGVGKSALTI (SEQ ID NO:27), YKLVVVGACGVGKSALTI (SEQ ID NO:28), YKLVVVGATGVGKSALTI (SEQ ID NO:29), and YKLVVVGAGDVGKSALTI A compound comprising or consisting of an amino acid sequence selected from the group consisting of (SEQ ID NO:30).

[0096] 2. A compound comprising a mutant KRAS sequence and a lipid, wherein the mutant KRAS sequence is conjugated to the lipid by a linker, and (i) the linker comprises one or more polyethylene glycol blocks, (ii) the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and (iii) the mutant KRAS sequence comprises or comprises an amino acid sequence selected from the group consisting of CYKLVVVGADGVGKSALTI (SEQ ID NO: 1), CYKLVVVGAVGVGKSALTI (SEQ ID NO: 2), CYKLVVVGARGVGKSALTI (SEQ ID NO: 3), CYKLVVVGAAGVGKSALTI (SEQ ID NO: 4), CYKLVVVGASGVGKSALTI (SEQ ID NO: 5), CYKLVVVGACGVGKSALTI (SEQ ID NO: 6), CYKLVVVGATGVGKSALTI (SEQ ID NO: 22), and CYKLVVVGAGDVGKSALTI (SEQ ID NO: 7).

[0097] 3. The compound of paragraph 1 or 2, wherein the above-mentioned mutant KRAS sequence is conjugated to a linker via a cysteine-maleimide linkage at its N-terminus.

[0098] 4. The linker is one of the compounds from paragraphs 1 to 3, wherein the linker contains 48 repeating units of polyethylene glycol.

[0099] 5. The above mutant KRAS sequence has the following structure at its N-terminus. [ka] A compound from paragraph 1 or 2 that is conjugated to it.

[0100] 6. A composition comprising one or more compounds from paragraphs 1 to 5, and a pharmaceutically acceptable carrier.

[0101] 7. The composition of paragraph 6, wherein the composition comprises (1) a compound containing the amino acid sequence YKLVVVGADGVGKSALTI (SEQ ID NO: 23), (2) a compound containing the amino acid sequence YKLVVVGAVGVGKSALTI (SEQ ID NO: 24), (3) a compound containing the amino acid sequence YKLVVVGARGVGKSALTI (SEQ ID NO: 25), (4) a compound containing the amino acid sequence YKLVVVGAAGVGKSALTI (SEQ ID NO: 26), (5) a compound containing the amino acid sequence YKLVVVGASGVGKSALTI (SEQ ID NO: 27), (6) a compound containing the amino acid sequence YKLVVVGACGVGKSALTI (SEQ ID NO: 28), or a compound containing the amino acid sequence YKLVVVGATGVGKSALTI (SEQ ID NO: 29), and (7) a compound containing the amino acid sequence YKLVVVGAGDVGKSALTI (SEQ ID NO: 30).

[0102] 8. The composition of paragraph 6, wherein the composition comprises (1) a compound containing the amino acid sequence CYKLVVVGADGVGKSALTI (SEQ ID NO: 1), (2) a compound containing the amino acid sequence CYKLVVVGAVGVGKSALTI (SEQ ID NO: 2), (3) a compound containing the amino acid sequence CYKLVVVGARGVGKSALTI (SEQ ID NO: 3), (4) a compound containing the amino acid sequence CYKLVVVGAAGVGKSALTI (SEQ ID NO: 4), (5) a compound containing the amino acid sequence CYKLVVVGASGVGKSALTI (SEQ ID NO: 5), (6) a compound containing the amino acid sequence CYKLVVVGACGVGKSALTI (SEQ ID NO: 6), or a compound containing the amino acid sequence CYKLVVVGATGVGKSALTI (SEQ ID NO: 22), and (7) a compound containing the amino acid sequence CYKLVVVGAGDVGKSALTI (SEQ ID NO: 7).

[0103] 9. The above composition contains the following lipids [ka] A composition from any one of paragraphs 6 to 8, further comprising a salt thereof and a compound consisting of the nucleotide sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8) linked at its 5' end by a bond or linker (where X is O or S).

[0104] 10. The composition of paragraph 6, wherein the above composition contains 700 μg of each compound.

[0105] 11. A method for treating cancer in a human patient, comprising the step of administering to the patient one of the compositions described in paragraphs 6 to 10.

[0106] 12. The method of paragraph 11, further comprising the step of administering an adjuvant.

[0107] 13. The method of paragraph 12, wherein the adjuvant contains a CpG nucleotide sequence.

[0108] 14. The method of paragraph 13, wherein the above CpG nucleotide sequence includes 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8).

[0109] 15. Administer 0.1 mg, 0.5 mg, or 2.5 mg of the above adjuvant, using one of the methods described in paragraphs 12-14.

[0110] 16. The above method applies to the following lipids. [ka] The method of paragraph 10 further comprises administering to the patient a compound comprising the nucleotide sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8) (where X is O or S), which is bonded or linked at its 5' end to or by a linker, or a salt thereof.

[0111] 17. The composition of paragraph 9 or the method of paragraph 16, wherein the above nucleotide sequence is bound to the above lipid.

[0112] 18. The above method applies to the following lipids. [ka] Alternatively, the method of paragraph 16 or 17, which involves administering 0.1 mg, 0.5 mg, or 2.5 mg of the above compound (where X is O or S), comprising a salt thereof and the nucleotide sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8) linked at the 5' end by a bond or linker.

[0113] 19. The cancer described above is pancreatic cancer, lung cancer, or colorectal cancer, in any one of the methods described in paragraphs 11-16 or 18.

[0114] 20. Any one of the methods described in paragraphs 11-16 or 18, wherein all internucleoside groups linking the nucleoside of 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8) are phosphorothioates.

[0115] 21. (i) any one compound from paragraphs 1 to 5, or any one composition from paragraphs 6 to 8, and (ii) the following lipids [ka] A kit comprising a salt thereof and a compound consisting of the nucleotide sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8) linked at the 5' end by a bond or linker (where X is O or S).

[0116] Other embodiments are described in the following claims.

Claims

1. A compound comprising a mutant KRAS sequence and a lipid, wherein the mutant KRAS sequence is conjugated to the lipid by a linker, and (i) the linker comprises one or more polyethylene glycol blocks, (ii) the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and (iii) the mutant KRAS sequence consists of an amino acid sequence selected from the group consisting of CYKLVVVGADGVGKSALTI (SEQ ID NO: 1), CYKLVVVGAVGVGKSALTI (SEQ ID NO: 2), CYKLVVVGARGVGKSALTI (SEQ ID NO: 3), CYKLVVVGAAGVGKSALTI (SEQ ID NO: 4), CYKLVVVGASGVGKSALTI (SEQ ID NO: 5), CYKLVVVGACGVGKSALTI (SEQ ID NO: 6), CYKLVVVGATGVGKSALTI (SEQ ID NO: 22), and CYKLVVVGAGDVGKSALTI (SEQ ID NO: 7).

2. The compound according to claim 1, wherein the mutant KRAS sequence consists of an amino acid sequence selected from the group consisting of YKLVVVGADGVGKSALTI (SEQ ID NO: 23), YKLVVVGAVGVGKSALTI (SEQ ID NO: 24), YKLVVVGARGVGKSALTI (SEQ ID NO: 25), YKLVVVGAAGVGKSALTI (SEQ ID NO: 26), YKLVVVGASGVGKSALTI (SEQ ID NO: 27), YKLVVVGACGVGKSALTI (SEQ ID NO: 28), YKLVVVGATGVGKSALTI (SEQ ID NO: 29), and YKLVVVGAGDVGKSALTI (SEQ ID NO: 30).

3. The compound according to claim 1 or 2, wherein the mutant KRAS sequence is conjugated to a linker via a cysteine-maleimide linkage at its N-terminus.

4. The compound according to claim 1 or 2, wherein the linker comprises 48 repeating units of polyethylene glycol.

5. The aforementioned mutant KRAS sequence has the following structure at its N-terminus. 【Chemistry 1】 A compound according to claim 1 or 2, which is conjugated to a compound.

6. A composition comprising one or more compounds according to claim 1 or 2, and a pharmaceutically acceptable carrier.

7. The composition of claim 6, wherein the composition comprises (1) a compound comprising the amino acid sequence YKLVVVGADGVGKSALTI (SEQ ID NO: 23), (2) a compound comprising the amino acid sequence YKLVVVGAVGVGKSALTI (SEQ ID NO: 24), (3) a compound comprising the amino acid sequence YKLVVVGARGVGKSALTI (SEQ ID NO: 25), (4) a compound comprising the amino acid sequence YKLVVVGAAGVGKSALTI (SEQ ID NO: 26), (5) a compound comprising the amino acid sequence YKLVVVGASGVGKSALTI (SEQ ID NO: 27), (6) a compound comprising the amino acid sequence YKLVVVGACGVGKSALTI (SEQ ID NO: 28), or a compound comprising the amino acid sequence YKLVVVGATGVGKSALTI (SEQ ID NO: 29), and (7) a compound comprising the amino acid sequence YKLVVVGAGDVGKSALTI (SEQ ID NO: 30).

8. The composition of claim 6, wherein the composition comprises (1) a compound comprising the amino acid sequence CYKLVVVGADGVGKSALTI (SEQ ID NO: 1), (2) a compound comprising the amino acid sequence CYKLVVVGAVGVGKSALTI (SEQ ID NO: 2), (3) a compound comprising the amino acid sequence CYKLVVVGARGVGKSALTI (SEQ ID NO: 3), (4) a compound comprising the amino acid sequence CYKLVVVGAAGVGKSALTI (SEQ ID NO: 4), (5) a compound comprising the amino acid sequence CYKLVVVGASGVGKSALTI (SEQ ID NO: 5), (6) a compound comprising the amino acid sequence CYKLVVVGACGVGKSALTI (SEQ ID NO: 6), or a compound comprising the amino acid sequence CYKLVVVGATGVGKSALTI (SEQ ID NO: 22), and (7) a compound comprising the amino acid sequence CYKLVVVGAGDVGKSALTI (SEQ ID NO: 7).

9. The above composition is the following lipid 【Chemistry 2】 The composition of claim 6, further comprising a salt thereof and a compound comprising the nucleotide sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8) linked at the 5' end by a bond or linker (where X is O or S).

10. The composition according to claim 6, wherein the composition comprises 700 μg of each compound.

11. The composition according to claim 9, wherein the nucleotide sequence is bound to the lipid.

12. A method for treating cancer in a human patient, comprising the step of administering the composition of claim 6 to the patient.

13. The method of claim 12, further comprising the step of administering an adjuvant.

14. The method of claim 13, wherein the adjuvant comprises a CpG nucleotide sequence.

15. The method of claim 14, wherein the CpG nucleotide sequence comprises 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8).

16. The method of claim 12, comprising administering 0.1 mg, 0.5 mg, or 2.5 mg of the adjuvant.

17. The above method uses the following lipids 【Transformation 3】 The method of claim 12, further comprising the step of administering to the patient a salt thereof and a compound comprising the nucleotide sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8) linked at the 5' end by a bond or linker (where X is O or S).

18. The method of claim 17, wherein the nucleotide sequence is bound to the lipid.

19. The above method uses the following lipids 【Chemistry 4】 The method of claim 17, comprising administering 0.1 mg, 0.5 mg, or 2.5 mg of the compound (where X is O or S) comprising a salt thereof and a nucleotide sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8) linked at the 5' end by a bond or linker.

20. The method of claim 12, wherein the cancer is pancreatic cancer, lung cancer, or colorectal cancer.

21. The method of claim 12, wherein all internucleoside groups linking the nucleoside of 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8) are phosphorothioates.

22. (i) the compound of claim 1 or 2, and (ii) the lipids listed below. 【Transformation 5】 A kit comprising a salt thereof and a compound consisting of the nucleotide sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 8) linked at the 5' end by a bond or linker (where X is O or S).

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