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  • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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  • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
  • C12Y306/05—Hydrolases acting on acid anhydrides (3.6) acting on GTP; involved in cellular and subcellular movement (3.6.5)
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Definitions

• _ is named _ and is _ bytes in size.
• RAS proteins are essential components of cell signaling pathways. Oncogenic activation of RAS proteins by mutation is frequently detected in several types of cancer, including pancreatic cancer. There continues to be a need for further and more effective cancer treatments.
• the invention provides compounds that can be used in therapeutic methods.
• the invention features a compound including a mutant KRAS sequence and a lipid, where the mutant KRAS sequence is conjugated to the lipid by a linker, and (i) the linker includes one or more polyethylene glycol blocks, (ii) the lipid is 1 ,2-distearoy!-sn-g!ycero-3- phosphoethanolamine (DSPE), and (iii) the mutant KRAS sequence comprises or consists of an amino acid sequence selected from the group consisting of YKLVVVGADGVG
• the invention features a compound including a mutant KRAS sequence and a lipid, where the mutant KRAS sequence is conjugated to the lipid by a linker, and (i) the linker includes 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 consists of an amino acid sequence selected from the group consisting of CYKLVVVGADGVGKSALTI (SEQ ID NO:1 ), C YKLVVVGAVGVGKSALTI (SEQ ID NOS), C YKLVVVGARGVGKSALTI (SEQ ID NO:3), C YKLVVVGAAGVGKSALTI (SEQ ID NO:4), C YKLVVVGASGVGKSALTI (SEQ ID NO:5), C YKLVVVGACGVGKSALTI (SEQ ID NO:6),
• the mutant KRAS sequence is conjugated to the linker through a cysteine-maleimide linkage.
• the linker includes 48 repeat units of polyethylene glycol.
• the mutant KRAS sequence, at its N-terminus is conjugated to the following structure:
• the invention features a composition including one or more compounds of the first and the second aspects of the invention and a pharmaceutically acceptable carrier.
• the composition includes (1 ) a compound including the amino acid sequence YKLVVVGADGVGKSALTI (SEG ID NO:23), (2) a compound including the amino acid sequence YKLVVVGAVGVGKSALTI (SEQ ID NO:24), (3) a compound including the amino acid sequence YKLVVVGARGVGKSALTI (SEQ ID NO:25), (4) a compound including the amino acid sequence YKLVVVGAAGVGKSALTI (SEQ ID NO:26), (5) a compound including the amino acid sequence YKLVVVGASGVGKSALTI (SEQ ID NO:27), (6) a compound including the amino acid sequence YKLVVVGACGVGKSALTI (SEQ ID MQ:28) or a compound including the amino acid sequence YKLVVVGATGVGKSALTI (SEG ID NO:29), and (7) a compound including the amino acid sequence YKLVVVG AG DVG KSALTI (SEQ ID NO:3Q
• the composition includes (1 ) a compound including the amino acid sequence YKLVVVGADGVGKSALTI (SEG ID NQ:23 ⁇ , (2) a compound including the amino acid sequence YKLVVVGAVGVGKSALTI (SEQ ID NG:24), (3) a compound including the amino acid sequence YKLVVVGARGVGKSALTI (SEQ !D NG:25), (4) a compound including the amino acid sequence YKLVVVGAAGVGKSA (5) a compound including the amino acid sequence YKLVVVGASGVGKSALT! ( compound including the amino acid sequence YKLVVVGACGVGKSALTI ( (7) a compound including the amino acid sequence YKLVVVGAGDVGKSALTI (
• the composition includes (1 ) a compound including the amino acid sequence GYKLVVVGADGVGKSALT! (SEQ ID NO:1 ), (2) a compound including the amino acid sequence GYKLVVVGAVGVGKSALT! (SEQ ID NO:2 ⁇ , (3) a compound including the amino acid sequence GYKLVVVGARGVGKSALT! (SEQ !D NO:3), (4) a compound including the amino acid sequence GYKLVVVGAAGVGKSALT! (SEQ ID NO:4 ⁇ , (5) a compound including the amino acid sequence GYKLVVVGASGVGKSALT! (SEQ ID NO:5 ⁇ , (6) a compound including the amino acid sequence GYKLVVVGACGVGKSALT!
• the composition includes (1 ) a compound including the amino acid sequence GYKLVVVGADGVGKSALT! (SEQ ID NQ:1 ), (2) a compound including the amino acid sequence GYKLVVVGAVGVGKSALT! (SEQ ID NO:2), (3) a compound including the amino acid sequence GYKLVVVGARGVGKSALT! (SEQ ID NQ:3), (4) a compound including the amino acid sequence GYKLVVVGAAGVGKSALT! (SEQ ID NO:4), (5) a compound including the amino acid sequence CYKLVVVGASGVGKSALT! (SEQ ID NO:5), (6) a compound including the amino acid sequence CYKLVVVGACGVGKSALT! (SEQ ID NQ:6), and (7) a compound including the amino acid sequence CYKLVVVGAGDVGKSALT! (SEQ ID NQ:7).
• 700 pg of each compound is included in the composition.
• the composition further includes a compound consisting of the nucleotide sequence 5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’ (SEQ ID NO:8), which, at its 5’ end, is bonded or linked by a linker to the following lipid:
• nucleotide sequence is bonded to the lipid.
• all internucleoside groups connecting the nucleosides in 5’- TCGT CGTTTT GT CGTTTT GT CGTT-3’ are phosphorothioates.
• the invention features a method of treating a cancer in a human patient, the method including administering the composition of the third aspect of the invention to the patient.
• the method further includes
• an adjuvant such as an adjuvant including a CpG nucleotide sequence (e.g., 5’- TCGT CGTTTT GT CGTTTT GTCGTT-3’ ; SEQ ID NO:8).
• an adjuvant including a CpG nucleotide sequence (e.g., 5’- TCGT CGTTTT GT CGTTTT GTCGTT-3’ ; SEQ ID NO:8).
• 0.1 mg, 0.5 mg, or 2.5 mg of the adjuvant is administered.
• the method further includes
• a compound consisting of the nucleotide sequence 5’- TCGTCGTTTTGTCGTTTTGTCGTT-3’ (SEQ ID NO:8), which, at its 5’ end, is bonded or linked by a linker to the following lipid:
• nucleotide sequence is bonded to the lipid.
• 0.1 mg, 0.5 mg, or 2.5 mg of the compound is administered.
• all internucleoside groups connecting the nucleosides in 5’- TCGT CGTTTT GT CGTTTT GT CGTT-3’ are phosphorothioates.
• the cancer is a pancreatic cancer, a lung cancer, or a colorectal cancer.
• the invention features a kit including (i) a compound of any one of claims 1 to 5, or a composition of any one of claims 6-8, and (ii) a compound consisting of the nucleotide sequence 5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’ (SEQ ID NO:8), which, at its 5’ end, is bonded or linked by a linker to the following lipid:
• nucleotide sequence is bonded to the lipid.
• all internucleoside groups connecting the nucleosides in 5’- TCGT CGTTTT GT CGTTTT GT CGTT-3’ are phosphorothioates.
• A“carbonyl,” as used herein, refers to a group -C(O)-.
• A“carboxylate,” as used herein, refers to a group -C(0)-0H or a salt thereof.
• conjugated refers to a covalent bonding of a mutant KRAS sequence to a linker and, optionally, further covalent bonding of the linker to a lipid.
• the covalent bonds connecting the linker to the mutant KRAS sequence or to the lipid may be a covalent bond between a sulfur atom and a s/ ⁇ -hybridized carbon atom in succinimide, between a nitrogen atom and a carbon atom of a carbonyl, or between oxygen atom and the phosphorus atom of a thiophosphoryl or phosphoryl.
• each of the KRAS sequence and the lipid may contain a group for bonding to a complementary group in the linker.
• the KRAS polypeptide may include a sulfur atom (e.g., the sulfur atom in a cysteine residue) bonded to a complementary group, e.g., succinimide, in the linker.
• the bond from the sulfur atom in the KRAS polypeptide to the succinimide in the linker may be formed by a reaction of a thiol (e.g., a thiol in a cysteine residue) in the KRAS polypeptide and a maleimide in the linker.
• the KRAS polypeptide may include a nitrogen atom or a carbonyl group bonded to a carbonyl group or a nitrogen atom, respectively, in the linker.
• the bond between the carbonyl group and the nitrogen atom may be formed by a reaction between an amine or carboxylate in the KRAS polypeptide and a carboxylate or an amine, respectively, in the linker.
• A“lipid” is any of a group of organic compounds that are greasy to the touch, insoluble in water, and soluble in organic solvents, and in general includes fatty acids and their derivatives.
• a lipid used in the invention is 1 ,2-distearoyi-sn-giycero-3-phosphoethanolamine (DSPE).
• A“linker,” as used herein, refers to a monovalent or divalent group, in which one valency is covalently bonded to one biologically functional group, and the other valency is covalently bonded to another biologically functional group.
• a linker connects a KRAS sequence to a lipid, as noted above.
• such a linker can include one or more polyethylene glycol blocks.
• a linker connects a nucleotide sequence of, e.g., a CpG oligonucleotide, to a lipid (e.g., - P(X)(0H)-0-CH(CH2NHG0-(CH2)I6-CH3)2, or a sait thereof, where X is O or S, as described herein).
• a lipid e.g., - P(X)(0H)-0-CH(CH2NHG0-(CH2)I6-CH3
• Such linkers can optionally include one or more nucleotides, for example, a dinucleotide (e.g., GG).
• a dinucleotide e.g., GG
• A“pharmaceutically acceptable carrier,” as used herein, refers to a vehicle capable of suspending or dissolving an active compound, and having the properties of being nontoxic and non-inflammatory in a subject (e.g., a human patient) to whom it is administered.
• a pharmaceutically acceptable carrier may include a pharmaceutically acceptable additive, such as a preservative, antioxidant, fragrance, emulsifier, dye, or excipient known or used in the field of drug formulation and that does not significantly interfere with the therapeutic effectiveness of the biological activity of the active agent, and that is non-toxic to the subject.
• A“phosphoryl,” as used herein, refers to a group -P(0)(OR A )(OR B ), where R A is H, and R B is a valency.
• A“polyethylene glycol,” as used herein, refers to a block -(OCH2CH2)n-, where n is a number of repeat units and is an integer from 2 to 50 (e.g., 24 or 48).
• A“thiol,” as used herein, refers to a group -SH.
• A“thiophosphoryl,” as used herein, refers to a group -P(S)(OR A )(OR B ), where R A is H, and R B is a valency.
• treat refers to therapeutic approaches in which the goal is to reverse, alleviate, ameliorate, inhibit, slow down, or stop the progression or severity of a condition associated with a disease or disorder, e.g., cancer. These terms include reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder.
• Treatment is generally“effective” if one or more symptoms or clinical markers are reduced, or if a desired response (e.g., a specific immune response) is induced. Alternatively, treatment is“effective” if the progression of a disease is reduced or halted.
• certain compounds of the invention bind to endogenous albumin in subjects to whom they are administered, which enhances delivery of the compounds to the lymph nodes of the subjects. This facilitates the induction of a therapeutic immune response against the KRAS sequences of the compounds, leading to effective cancer treatment.
• FIG. 1 shows that amphiphile-KRas (aKRas), in combination with amphiphile-CpG (aCpG), activated splenocytes, while soluble KRas, in combination with aCpG, and the untreated and soluble KRas and polylC (pIC) control groups, did not.
• aKRas amphiphile-KRas
• aCpG amphiphile-CpG
• pIC polylC
• Figure 2 shows that both aKRas G12R and G12V, in combination with aCpG, activated splenocytes, while neither soluble KRas G12R nor G12V in combination with aCpG, and the untreated and soluble KRas and pIC control groups, did not.
• Figure 3 shows a KRas-specific CD8 + T-cell response for mice immunized with the combination of aKRas G12D and aCpG, but not for soluble KRas G12D in combination with CpG or soluble wild-type KRas in combination with CpG.
• Figure 4 shows a KRas-specific CD8 + T-cell response (9mers) and CD8 + or CD4 + T-cell response (18mer) for mice immunized with the combination of aKRas G12D and aCpG, but not for soluble KRas G12D in combination with CpG or the untreated control.
• YKLVVVGADGVGKSALTI SEQ ID NO: 23
• Amph-KRAS G12D DSPE-amido-dPEG24-amido- dPEG24-CYKLVVVGADGVGKSALTI (SEQ ID NO: 1 );
• KRAS G12D 18-mer YKLVVVGADGVGKSALTI (SEQ ID NO:23);
• Figure 6 shows the structure of the amphiphile-peptide G12D 4-21 (SEQ ID NO:1 ).
• Figure 7 shows the structure of amphiphile-CpG-7909. All sugars are deoxyribose. All internucleoside linkages are phosphorothioate. The structure is presented as the sodium salt.
• the invention provides compounds that can be used in therapeutic methods.
• the compounds of the invention each include a mutant KRAS sequence (e.g., any one of SEQ ID NOS:1 -7 and 22; see Table 1 , below, or any one of SEQ ID NOS:23-30, see Table 2, below) and a lipid (e.g., 1 ,2-distearoy!-sn- giycero-3-phosphoethanolamine (DSPE)).
• a linker for example, a linker including one or more polyethylene glycol blocks.
• the linker is:
• a mutant KRAS sequence (e.g., any one of SEQ ID NOS:1 -7 or 22, or any one of SEQ ID NOS:23-30) is bonded at its N-terminus through a Cys residue to a linker, and the linker is bonded to a lipid, where the linker is:
• compositions that include one or more compounds of the invention, together with a pharmaceutically acceptable carrier or diluent.
• the compositions can include seven different compounds as described above, where each of the seven different compounds includes a different sequence selected from SEQ ID NOS:1 -7 and 23 or SEQ ID NOS:23-30.
• Compositions including subsets (e.g., 2, 3, 4, 5, or 6) of these compounds are also included in the invention.
• Different compounds included in the compositions of the invention can optionally each include the same or different KRAS sequences, linkers, and/or lipids.
• compositions of the invention can be used in methods to induce immune responses to the KRAS sequences of the compounds. Accordingly, the compositions can be referred to as immunogenic or vaccine compositions.
• the compositions can thus optionally include or be administered with one or more adjuvants.
• the adjuvant used can include a CpG oligonucleotide (e.g., 5’- TCGTCGTTTTGTCGTTTTGTCGTT-3’; SEQ ID NO:8; CpG-7909) that, at its 5’ end, is bonded or linked by a linker to a lipid, such as the following:
• X is O or S.
• X is S.
• the CpG oligonucleotide may be directly bonded to the lipid.
• the linker bonded to the CpG oligonucleotide and to the lipid may be GG.
• all internucleoside groups are phosphorothioates (e.g., all internucleoside groups in the compound may be
• the compounds and compositions of the invention can be used in therapeutic methods.
• the KRAS sequences of the compounds can induce an immune response to KRAS, which is expressed in certain cancer cells.
• the invention provides methods of treating cancer in a subject (e.g., a human patient) by administering one or more compounds or compositions of the invention to the subject.
• the invention also includes methods of inducing an immune response against KRAS in a subject (e.g., a human patient) by administering one or more compounds or compositions of the invention to the subject.
• the cancer is selected from the group consisting of pancreatic cancer, lung cancer, and colorectal cancer.
• the methods of the invention can further include administering a compound or composition of the invention in combination with a second (or further) different approach to treatment.
• kits that each contain, for example, a first vessel that includes one or more compounds of the invention, optionally together with a second vessel that includes an adjuvant, such as an adjuvant as described herein.
• the lipid polyethylene glycol) moiety was conjugated via a maleimide (MAL) -cysteine coupling to the peptide using the following synthon :
• a compound of the invention may be prepared from a mutant KRAS polypeptide, a linker precursor, and a lipid using methods known in the art.
• a linker precursor is first linked to a lipid, e.g., as follows:
• Amidation reaction conditions are known in the art, for example, typical amidation conditions include the use of reagents, such as EDC/DMAP, HATU/HOAt, or HBTU/HOAt.
• reagents such as EDC/DMAP, HATU/HOAt, or HBTU/HOAt.
• the carboxylate may be replaced with an O-succinimide ester, pentrafluorophenyl ester, or tetrafluorophenyl ester.
• the product of this reaction may then be reacted with a mutant KRAS polypeptide, e.g., as follows:
• the 1 ,4-addition reaction may be carried out in an appropriate solvent, e.g., a polar organic solvent or water.
• a linker precursor may be first linked to a lipid, e.g., as follows:
• the product may then be reacted with an amine group (e.g., N-terminal amine) in the mutant KRAS polypeptide to produce a compound of the invention:
• an amine group e.g., N-terminal amine
• a CpG oligonucleotide may be bonded directly or linked by a linker to the lipid. These compounds may be produced using the ordinary phosphoramidite chemistry known in the art. In some examples, the CpG oligonucleotide or CpG oligonucleotide that, at its 5’ end, is bonded to GG may be reacted with the following compound:
• oligonucleotide that, at its 5’ end, is bonded or linked by a linker to the lipid:
• each KRAS peptide administered can be in the range of 100 to 5000 pg per peptide (e.g., 700 pg/peptide, which would yield a peptide dose of 4900 pg for a group of 7 peptides).
• the dose of the adjuvant can be, on a body weight basis, as high as 0.48 mg/kg. Exemplary dosing regimens are shown below in Table 3.
• PolylC (pIC) was used as a benchmark adjuvant control.
• the adjuvant, as well as peptide stock, solutions were dissolved in H2O. Final injections were diluted with 1 X PBS (phosphate buffered saline).
• the aKRas peptides used were 18mer sequences of mutant sequences with a G12D substitution (amino acids 4-21 of wild-type - YKLVVVGAGGVGKSALTI (SEQ ID NO:9)). 20pg in 10OmI were used for each injection.
• the aCpG sequence used was the CpG1826 sequence (5’-tccatgacgttcctgacgtt-3’; SEQ ID NO:10) with two guanines added at the 5’ end (5’-gg tccatgacgttcctgacgtt-3’; SEQ ID NO:1 1 ) at a concentration of 5nmol for each 10OmI injection.
• CpG1826 is an optimal mouse sequence while CpG7909 is optimal for humans and poorly active in mice.
• CpG1826 and CpG7909 are in the same CpG class (class B) and generally have similar activity profiles in their respective species.
• Primer immunizations were given subcutaneously (s.c.) into the tail base (day 0) with one booster dose after 2 weeks (day 14). ELISpot analysis for IFNy on splenocytes was performed, using standard protocols, 8 days after booster dose administration (day 21 ). Splenocytes (10 6 cells/well) were activated with 5pg/well of G12D sequence 18mer (aa4-21 ) ® ⁇ YKLVVVGADGVGKSALTI (SEQ ID NO:1 ).
• aKRas in combination with aCpG activated splenocytes while soluble KRas in combination with aCpG, and the untreated and soluble KRas and pIC control groups, did not.
• KRas soluble-KRas
• aKRas amphiphile-KRas mutant sequences
• aCpG amphiphile-CpG adjuvant
• CpG soluble CpG
• the adjuvant as well as peptide stock solutions are dissolved in H2O. Final injections are diluted with 1 X PBS.
• the aKras peptides used were 18mer sequences of mutant sequences with G12R/V substitutions (SEQ amino acids 4-21 of wild-type - YKLVVVGAGGVGKSALTI (SEQ ID NO:9)). 20pg in 10OmI were used for each injection.
• the aCpG sequence used was the CpG1826 sequence (5’-tccatgacgttcctgacgtt-3’; SEQ ID NO:10) with two guanines added at the 5’ end (5’-gg tccatgacgttcctgacgtt-3’; SEQ ID NO:1 1 ) at a concentration of 5nmol for each 10OmI injection.
• Primer immunizations were given subcutaneously (s.c.) into the tail base with one booster dose after 2 weeks (d14).
• ELISpot analysis for IFNy on splenocytes was performed, using standard protocols, 7 days after booster dose administration (d21 ).
• Splenocytes (10 6 cells/well) were activated with 5pg/well of either: the G12V sequence 18mer (aa4-21 ) - YKLVVVGAVGVGKSALTI (SEQ ID NO:2) or the G12R sequence 18mer (aa4-21 ) ® ⁇ YKLVVVGARGVGKSALTI (SEQ ID NO:3).
• both aKRas G12R and G12V in combination with aCpG activated splenocytes while neither soluble KRasG12R nor G12V in combination with aCpG, and the untreated and soluble KRas and pIC control groups, did not.
• B6 mice transgenic for the human HLA gene, A2.1 were immunized either with the soluble or amphiphile- conjugated forms of the KRas G12D antigen, as well as adjuvant (CpG or aCpG).
• the A2.1 allele is known to mount responses to the KRas peptides in humans.
• the adjuvant as well as peptide stock solutions were dissolved in H2O. Final injections were diluted with 1 X PBS.
• the aKRas peptides used were 18mer sequences of mutant sequences with a G12D substitution (amino acids 4-21 of wild-type - YKLVVVGAGGVGKSALTI (SEQ ID NO:9)). 20pg in 10OmI were used for each injection.
• the aCpG sequence used was the CpG1826 sequence (5’-tccatgacgttcctgacgtt-3’; SEQ ID NO:10) with two guanines added at the 5’ end (5’-gg tccatgacgttcctgacgtt-3’; SEQ ID NO:1 1 ) at a concentration of 5nmol for each 10OmI injection.
• the vaccine was administered subcutaneously to female mice bilaterally at the tail base at 50pl per side. Two booster doses were given in two-week intervals.
• ICS Intracellular cytokine staining
• PBMCs peripheral blood mononuclear cells
• ELISpot analysis for IFNy on splenocytes was performed, using standard protocols, 7 days after the second and third booster dose, respectively.
• PBMCs/Splenocytes (10 6 cells/well and 2x10 6 cells/well) were activated with 5pg/well of either SEQ ID NO:1 or one of SEQ ID NOS:12-20 as shown below:
• Figure 3 shows a KRas-specific CD8 + T-cell response for mice immunized with the combination of aKRas G12D and aCpG, but not for soluble KRas G12D in combination with CpG or soluble wild-type KRas in combination with CpG.
• the concentration of antigen was increased from 20pg to 50pg and bi-weekly (bw) or weekly (w) dose intervals were tested.
• the adjuvant as well as peptide stock solutions were dissolved in H2O. Final injections were diluted with 1 X PBS.
• the aKRas peptides used were 18mer sequences of mutant sequences with a G12D substitution (amino acids 4-21 of wild-type - YKLVVVGAGGVGKSALTI (SEQ ID NO:9)). 50pg in 10OmI were used for each injection.
• the aCpG sequence used was the CpG1826 sequence (5’-tccatgacgttcctgacgtt-3’; SEQ ID NO:10) with two guanines added at the 5’ end (5’-gg tccatgacgttcctgacgtt-3’; SEQ ID NO:1 1 ) at a concentration of 5nmol for each 10OmI injection.
• Primer immunizations were given subcutaneously (s.c.) into the tail base with one booster dose after 2 weeks.
• ELISpot analysis for IFNy, and CBA (cytometric bead array) analysis for IL6, IL10, IL12, TNFa, IFNy, and MCP1 was performed on splenocytes, using standard protocols, 7 days after booster dose administration.
• Splenocytes (10 6 cells/well) were activated with 5pg/well of either SEQ ID NO:1 or one of SEQ ID NOS:12-21 as shown below:
• Figure 4 shows a KRas-specific CD8 + T-cell response (9mers) and CD8 + or CD4 + T-cell response (18mer) for mice immunized with the combination of aKRas G12D and aCpG, but not for soluble KRas G12D in combination with CpG or the untreated control.
• CBA cytometric bead array
• ELISPOT enzyme-linked immunospot
• IFNy interferon gamma
• IL-6 interleukin 6
• KRAS Kirsten Rat Sarcoma
• poly IC polyinosinic:polycytidylic acid
• ROA route of administration
• SC subcutaneous
• TNFa tumor necrosis factor alpha
• 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-phosphoethanoiamine (DSPE), and (iii) the mutant KRAS sequence comprises or consists of an amino acid sequence selected from the group
• 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-phosphoethanoiamine (DSPE), and (iii) the mutant KRAS sequence comprises or consists of an amino acid sequence selected from the group consisting of CYKLVVVGADGVGKSALTi (SEQ ID NO:1 ), CYKLVVVGAVGVGKSALTI (SEQ ID NO:2),
• CYKLVVVGATGVGKSALTi SEQ ID NO:22
• CYKLVVVGAGDVGKSALTi SEQ ID NO:7
• linker comprises 48 repeat units of polyethylene glycol.
• composition comprising one or more compounds of any one of paragraphs 1 -5 and a pharmaceutically acceptable carrier.
• 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 NG:26), (5) a compound comprising the amino acid sequence YKLVVVGASGVGKSALTi (SEQ ID NG:27), (6) a compound comprising the ammo acid sequence YKLVVVGACGVGKSALTI (SEQ ID NG:2S) or a compound comprising the ammo acid sequence YKLVVVGATGVGKSALTI (SEQ ID NQ:29), and (7) a compound comprising the amino acid sequence YKLVVVGAG
• composition comprises (1 ) a compound comprising the ammo acid sequence CYKLVVVGADGVGKSALT! a compound comprising the ammo acid sequence CYKLVVVGAVGVGKSALTI a compound comprising the ammo acid sequence CYKLVVVGARGVGKSALT!
• a compound comprising the ammo acid sequence CYKLVVVGAAGVGKSALTI a compound comprising the amino acid sequence CYKLVVVGASGVGKSALTI a compound comprising the amino acid sequence CYKLVVVGACGVGKSALTI compound comprising the amino acid sequence C YKLVVVGATGVGKSALTI d (7) a compound comprising the amino acid sequence CYKLVVVGAGDVGKSALTI
• composition of paragraph 6 wherein the composition comprises 700 pg of each compound.
• a method of treating a cancer in a human patient comprising administering the composition of any one of paragraphs 6-10 to the patient.
• the method further comprises administering to the patient a compound consisting of the nucleotide sequence 5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’ (SEQ ID NO:8), which, at its 5’ end, is bonded or linked by a linker to the following lipid:
• composition of paragraph 9 or the method of paragraph 16 wherein the nucleotide sequence is bonded to the lipid 17.
• a kit comprising (i) a compound of any one of paragraphs 1 -5, or a composition of any one of paragraphs 6-8, and (ii) a compound consisting of the nucleotide sequence 5’-
• TCGTCGTTTTGTCGTTTTGTCGTT-3’ (SEQ ID NO:8), which, at its 5’ end, is bonded or linked by a linker to the following lipid:

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