Composition containing Braf peptide amphiphilic substance and method of use thereof

Abstract

Compounds comprising an albumin-binding domain and a Braf peptide, as well as pharmaceutically acceptable salts thereof, are disclosed herein. Furthermore, methods for inducing an immune response in a subject, and methods for inducing an immune response in a subject by administering such compounds, are disclosed herein.

Description

[Technical Field]

[0001] Sequence List This application is submitted electronically in XML format and includes a sequence listing incorporated herein by reference in its entirety. The XML copy created on 23 May 2024 is named 51026-060WO3_Sequence_Listing_5_23_24.xml and has a size of 46,541 bytes. [Background technology]

[0002] Vaccines are used to stimulate an immune response in an individual to provide protection against and / or treatment of a specific disease. Some vaccines contain antigens to induce an immune response. Immune responses resulting from vaccination have made significant contributions to both human and animal health. Since the invention of the first vaccine in 1796, vaccines have come to be considered the most successful method of preventing many infectious diseases by inducing an immune response in the subject. According to the World Health Organization, immunity now prevents 2 to 3 million deaths annually across all age groups. The goal of vaccination is to generate a strong, sustained immune response that provides long-term protection against infection. However, many vaccines currently do not induce optimal immunity.

[0003] There is still a need to develop new and improved compositions and methods for inducing an immune response in these subjects. [Overview of the Initiative] [Means for solving the problem]

[0004] In one embodiment, the present disclosure provides a compound comprising an albumin-binding domain and a Braf peptide, or a pharmaceutically acceptable salt thereof. In some embodiments, the peptide is a 5-50 amino acid fragment of SEQ ID NO: 1 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50). In some embodiments, the peptide is an amino acid fragment of SEQ ID NO: 1, with 10 to 40 amino acids (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40). In some embodiments, the peptide is an amino acid fragment of SEQ ID NO: 1, with 10 to 30 amino acids (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In some embodiments, the Braf peptide is an amino acid fragment of SEQ ID NO: 1, with 30 amino acids. In some embodiments, the Braf peptide is an amino acid fragment of SEQ ID NO: 1, with 9 amino acids. In some embodiments, the Braf peptide is the 15-amino acid fragment of SEQ ID NO: 1. In some embodiments, the Braf peptide is the 29-amino acid fragment of SEQ ID NO: 1.

[0005] In some embodiments, the Braf peptide comprises a fragment of SEQ ID NO: 1 containing one or more amino acid substitutions. In some embodiments, the Braf peptide comprises a fragment of SEQ ID NO: 1 containing an amino acid substitution at Val occupying amino acid position 600 from the N-terminus of SEQ ID NO: 1. In some embodiments, the amino acid substitution at Val occupying position 600 of SEQ ID NO: 1 is V600K. In some embodiments, the amino acid substitution at Val occupying position 600 of SEQ ID NO: 1 is V600E.

[0006] In some embodiments, the peptide comprises or consists of the amino acid sequence EDLTVKIGDFGLATVKSRWSGSHQFEQLS (SEQ ID NO: 2) or a fragment thereof. In some embodiments, the peptide comprises or consists of the amino acid sequence EDLTVKIGDFGLATKKSRWSGSHQFEQLS (SEQ ID NO: 3) or a fragment thereof. In some embodiments, the peptide comprises or consists of the amino acid sequence EDLTVKIGDFGLATEKSRWSGSHQFEQLS (SEQ ID NO: 4) or a fragment thereof. In some embodiments, the peptide comprises a 9 or 10 amino acid fragment of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the peptide comprises the amino acid sequence FGLATKKSR (SEQ ID NO: 61) or FGLATEKSR (SEQ ID NO: 62). In some embodiments, the peptide comprises a 15 amino acid fragment of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the peptide comprises the amino acid sequence GDFGLATKKSRWSGS (SEQ ID NO: 63) or GDFGLATEKSRWSGS (SEQ ID NO: 64).

[0007] In some embodiments, the peptide optionally includes an N-terminal modification. In some embodiments, the peptide includes an N-terminal modification. In some embodiments, the N-terminal modification is the addition of acetylcysteine. In some embodiments, the N-terminal modification is the addition of a des-aminocysteine ​​homolog. In some embodiments, the des-aminocysteine ​​homolog is 3-mercaptopropionic acid or mercaptoacetic acid. In some embodiments, the N-terminus of the peptide is bound to or ligated to an albumin-binding domain. In some embodiments, the C-terminus of the peptide is bound to or ligated to an albumin-binding domain.

[0008] In some embodiments, the albumin-binding domain contains a lipid. In some embodiments, the lipid is a diacyllipid. In some embodiments, the diacyllipid contains an acyl chain comprising 12-30 hydrocarbon units, 14-25 hydrocarbon units, 16-20 hydrocarbon units, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hydrocarbon units. In some embodiments, the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). In some embodiments, the peptide is the following lipid: [ka] or bonded to a salt thereof or linked by a linker. In some embodiments, the linker is selected from the group consisting of hydrophilic polymers, a series of hydrophilic amino acids, polysaccharides, and oligonucleotides, or combinations thereof. In some embodiments, the linker comprises an "N" polyethylene glycol unit, where N is 24-50 (e.g., 24-45, 24-40, 24-35, 24-30, 30-50, 35-50, 40-50, 45-50, or 30-40). In some embodiments, the linker comprises PEG24-amide-PEG24.

[0009] In another embodiment, the Disclosure provides a method for inducing an immune response in a subject, comprising administering one of the compounds described herein or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the method further comprises administering an adjuvant to the subject. In some embodiments, the compound or a pharmaceutically acceptable salt thereof is administered subcutaneously, intramuscularly, intravenously, or transmucosally. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0010] In another aspect, the present disclosure provides a compound comprising an albumin binding domain and a Braf peptide or a pharmaceutically acceptable salt thereof for use in a method of inducing an immune response in a subject, the method comprising administering to the subject any one of the compounds or pharmaceutically acceptable salts thereof described herein. In some embodiments, the method further comprises administering an adjuvant to the subject. In some embodiments, the compound or pharmaceutically acceptable salt thereof is administered subcutaneously, intramuscularly, intravenously, or transmucosally. In some embodiments, the subject is a mammal.

[0011] In another aspect, the present disclosure provides a compound or a pharmaceutically acceptable salt thereof for use as described herein, wherein the subject is a human.

[0012] In another aspect, the present disclosure provides a pharmaceutical composition comprising any one of the compounds or pharmaceutically acceptable salts thereof described herein and a pharmaceutically acceptable carrier.

[0013] In another aspect, the present disclosure provides a kit comprising any one of the compounds or pharmaceutically acceptable salts thereof or the pharmaceutical composition described herein and instructions for administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] [Figure 1] FIG. 1 is a drawing of an amphiphilic substance (AMP) conjugated to a PEG-48 linker conjugated to a mutant Braf peptide, which is a 9, 15, or 29 amino acid fragment of the amino acid sequence of SEQ ID NO: 4. The human sequence and the heteroclitic sequence (SEQ ID NO: 65) are shown. Amino acids that differ between the human sequence and the heteroclitic sequence are marked with asterisks. [Figure 2A-2B]Figures 2A and 2B are graphs showing the IFNγ ELISpot responses of splenocytes from C57BL / 6 mice re-stimulated with either a syngeneic peptide or a heteroclitic peptide after administration of the vaccine containing the soluble or amphiphilic peptide shown in Figure 1, after 2 doses (Figure 2A) and after 3 doses (Figure 2B). [Figure 3] Figure 3 is a graph showing the IFNγ ELISpot response of splenocytes from Balb / c mice after 3 doses of the vaccine containing the amphiphilic peptide shown in Figure 1 when co-administered with an adjuvant containing an amphiphilic substance conjugated to CpG7909. [Figure 4A-4B] Figures 4A and 4B are graphs showing the IFNγ co-culture ELISpot responses of splenocytes from C57Bl6 mice after dose 2 of vaccines of the V600E peptide (Figure 4A) and V600K peptide (Figure 4B) of soluble (SOL) or amphiphilic substance (AMP), administered at concentrations of 20 nmol of peptide and 10 nmol of adjuvant. [Figure 5A-5B] Figures 5A and 5B are graphs showing the IFNγ co-culture ELISpot responses of splenocytes from C57Bl6 mice after dose 3 of vaccines of the V600E peptide (Figure 5A) and V600K peptide (Figure 5B) of soluble (SOL) or amphiphilic substance (AMP), administered at concentrations of 20 nmol of peptide and 10 nmol of adjuvant. [Figure 6A-6C] Figures 6A - 6C are graphs showing the concentrations of granzyme B, INFγ, TNFα, GM-CSF, and IL2 from splenocytes of C57BL / 6J mice 7 days after dose 3 of vaccines of the V600E peptide (Figure 6A), V600K peptide (Figure 6B), or V600E peptide and V600K peptide (Figure 6C) of soluble (SOL) or amphiphilic substance (AMP), administered at concentrations of 20 nmol of peptide and 10 nmol of adjuvant. [Figures 7A-7B]Figures 7A and 7B are a series of graphs showing ELISpot analysis of granzyme B in splenocyte IFNγ co-cultures of C57Bl6 mice after dose 3, administered with soluble (SOL) or amphiphilic (AMP) V600E peptide (Figure 7A) and V600K peptide (Figure 7B) vaccines at concentrations of 20 nmol of peptide and 10 nmol of adjuvant. [Figure 8] Figure 8 is a graph showing the ELISpot response of pulmonary lymphocytes in IFNγ co-culture after dose 3 in C57Bl6 mice administered a vaccine consisting of a combination of soluble (SOL) or amphiphilic (AMP) V600E peptide and V600K peptide at concentrations of 20 nmol of each peptide and 20 nmol of adjuvant. [Figures 9A-9C] Figures 9A to 9C are graphs showing the ELISpot response in splenocytes co-cultured with IFNγ after dose 3 in C57Bl6 mice administered vaccines consisting of soluble (SOL) or amphiphilic (AMP) V600E peptide (Figure 9A), V600K peptide (Figure 9B), and a combination of V600E peptide and V600K peptide (Figure 9C) at concentrations of 5 nmol of each peptide and 10 nmol of adjuvant. [Figure 10A-10C] Figures 10A to 10C are graphs showing the ELISpot response of splenocytes in IFNγ co-culture after dose 5 in C57Bl6 mice administered vaccines consisting of soluble (SOL) or amphiphilic (AMP) V600E peptide (Figure 10A), V600K peptide (Figure 10B), and a combination of V600E peptide and V600K peptide (Figure 10C) at concentrations of 5 nmol of each peptide and 10 nmol of adjuvant. [Figure 11] Figure 11 is a graph showing the ELISpot analysis of granzyme B in splenocyte IFNγ co-cultures of C57Bl6 mice after dose 3, administered with soluble (SOL) or amphiphilic (AMP) V600E peptide and V600K peptide vaccines at concentrations of 5 nmol of each peptide and 10 nmol of adjuvant. [Figure 12A-12C]Figures 12A to 12C are graphs showing the concentrations of granzyme B, IFNγ, TNFα, GM-CSF, and IL2 from splenocytes 7 days after dose 5 in C57BL / 6J mice administered a vaccine of soluble (SOL) or amphiphilic (AMP) V600E peptide (Figure 12A), V600K peptide (Figure 12B), or V600E peptide and V600K peptide (Figure 12C) at concentrations of 5 nmol of each peptide and 10 nmol of adjuvant. [Figure 13A-13C] Figures 13A–13C are a series of graphs showing cytokine FluoroSpot analysis of splenocytes 7 days after dose 5 in C57BL / 6J mice administered a vaccine of soluble (SOL) or amphiphilic (AMP) V600E peptide (Figure 13A), V600K peptide (Figure 13B), or V600E peptide and V600K peptide (Figure 13C) at concentrations of 5 nmol of each peptide and 10 nmol of the adjuvant. [Figures 14A-14B] Figures 14A and 14B are graphs showing the percentage of cells containing IFNγ, TNFα, and cytokines in CD4+ cells (Figure 14A) and CD8+ cells (Figure 14B) collected 7 days after dose 5 of C57BL / 6J mice that were administered a vaccine of soluble (SOL) or amphiphilic (AMP) V600E peptide and V600K peptide at concentrations of 10 nmol of each peptide and 10 nmol of adjuvant (Figure 14A) or 20 nmol of each peptide and 20 nmol of adjuvant (Figure 14B). [Modes for carrying out the invention]

[0015] definition Unless otherwise specified, terms used in the claims and specification are defined as follows:

[0016] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless otherwise explicitly indicated by the context.

[0017] Where used herein, “approximately” is to be understood by those skilled in the art and varies to some extent depending on the context in which it is used. Where there is a use of a term that is not obvious to those skilled in the art considering the context in which the term is used, “approximately” means within plus or minus 10% of a given value.

[0018] As used herein, the term “adjuvant” refers to a compound that enhances, or otherwise alters or modifies, an immune response using a particular immunogen or antigen. Modification of the immune response includes enhancing or expanding the specificity of either or both antibody and cellular immune responses. Modification of the immune response may also mean reducing or suppressing a particular antigen-specific immune response. In certain embodiments, the adjuvant is a cyclic dinucleotide.

[0019] "Amino acids" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as later modified amino acids, such as hydroxyproline, γ-carboxyglutamate, and phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., hydrogen, a carboxyl group, an amino group, and a carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified polypeptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimes refer to chemical compounds that have a different structure from the general chemical structure of amino acids, but function similarly to naturally occurring amino acids. Amino acids may be referred herein by either a commonly known three-letter symbol or a one-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred by a commonly accepted one-letter code.

[0020] "Amino acid substitution" refers to replacing at least one existing amino acid residue in a given amino acid sequence (the amino acid sequence of the starting polypeptide) with a second, different "substituting" amino acid residue. "Amino acid insertion" refers to the incorporation of at least one additional amino acid into a given amino acid sequence. Insertions typically consist of the insertion of one or two amino acid residues, but larger "peptide insertions" today can be performed by the insertion of, for example, about 3 to about 5 amino acid residues, or even up to about 10, 15, or 20 amino acid residues. The inserted residues may be naturally occurring or not, as disclosed above. "Amino acid deletion" refers to the removal of at least one amino acid residue from a given amino acid sequence.

[0021] As used herein, “amphiphilic substance” or “amphiphilic” refers to a conjugate comprising a hydrophilic head group and a hydrophobic tail, thereby forming an amphiphilic conjugate. In some embodiments, the amphiphilic substance conjugate comprises a peptide and one or more hydrophobic lipid tails.

[0022] A polypeptide or amino acid sequence "derived" from a specified polypeptide or protein or "polypeptide fragment" refers to the origin of the polypeptide. Preferably, a polypeptide or amino acid sequence that is derived from or a fragment thereof of a particular sequence having an amino acid sequence essentially identical to that sequence or a part thereof, has a portion consisting of at least 10 to 20 amino acids, preferably at least 20 to 30 amino acids, more preferably at least 30 to 50 amino acids, or otherwise can be identified to those skilled in the art as having its origin in the sequence. A polypeptide that is derived from or a fragment thereof of another polypeptide may have one or more amino acid residues with one or more mutations relative to the starting polypeptide, for example, substitution with another amino acid residue or insertion or deletion of one or more amino acid residues.

[0023] Polypeptides may contain amino acid sequences that do not exist in nature. Such variants will always have less than 100% sequence identity or similarity to the starting molecule. In preferred embodiments, the variant has, for example, about 75% to less than 100% amino acid sequence identity or similarity, more preferably about 80% to less than 100%, more preferably about 85% to less than 100%, more preferably about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and most preferably about 95% to less than 100% amino acid sequence identity or similarity over the length of the variant molecule.

[0024] In one embodiment, there is a one-amino acid difference between the starting polypeptide sequence and the sequence derived therefrom. This sequence identity or similarity is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., the same residue) to the starting amino acid residue after the sequences have been aligned to achieve the greatest percentage sequence identity and gaps have been introduced as necessary.

[0025] As used herein, the term “cytotoxic T lymphocyte (CTL) response” refers to an immune response induced by cytotoxic T cells. CTL responses are primarily mediated by CD8+ T cells.

[0026] As used herein, the terms “effective dose” or “effective dosage” are defined as an amount sufficient to achieve, or at least partially achieve, the desired effect.

[0027] The term "therapeutic dose" is defined as the amount sufficient to cure or at least partially halt the disease and its complications in a patient who already has the disease. The amount effective for this use depends on the severity of the disorder being treated and the general state of the patient's own immune system.

[0028] As used herein, “immune cells” are cells of hematopoietic origin that play a role in the immune response. Immune cells include lymphocytes (e.g., B cells and T cells), natural killer cells, and myeloid cells (e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes). In certain embodiments, the immune cell is a T cell.

[0029] As used herein, “immune response” refers to the response made by the immune system of an organism to a substance, including but not limited to foreign substances or autologous proteins. Three common types of “immune responses” include mucosal immune responses, humoral immune responses, and cellular immune responses. For example, an immune response may include increased activation, expansion, and / or proliferation of immune cells. An immune response may also include at least one of cytokine production, T cell activation and / or proliferation, granzyme or perforin production, activation of antigen-presenting cells or dendritic cells, antibody production, inflammation, the development of immunity, the development of hypersensitivity to an antigen, the response of antigen-specific lymphocytes to an antigen, clearance of infectious agents, and transplant or graft rejection.

[0030] The terms "induction of an immune response" and "enhancement of an immune response" are used interchangeably and refer to the stimulation of an immune response (i.e., passive or adaptive) to a specific antigen (e.g., a peptide (e.g., Braf peptide)).

[0031] The term "induction" as used in relation to the induction of complement-dependent cell-mediated cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) refers to the stimulation of a specific direct cell death mechanism.

[0032] As used herein, “requiring prevention,” “requiring treatment,” or “needing treatment” refers to persons who, in the judgment of an appropriate healthcare professional (e.g., a physician, nurse, or caregiver in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from a given treatment (such as treatment with a composition containing an amphiphilic ligand conjugate).

[0033] The term "in vivo" refers to processes that occur in living organisms.

[0034] The term "in vitro" refers to processes that occur outside of a living organism, such as in a test tube, flask, or culture plate.

[0035] As used herein, the terms “conjugated,” “operably conjugated,” “fused,” and “fused” are interchangeable. These terms refer to the conjugation of two or more elements, components, or domains by appropriate means, including chemical conjugation or recombinant DNA technology. Methods of chemical conjugation (e.g., the use of heterobifunctional crosslinking agents) are known in the art, as are methods of recombinant DNA technology.

[0036] The term "lipid" refers to biomolecules that are soluble in nonpolar solvents and insoluble in water. Lipids are often described as hydrophobic or amphiphilic molecules, which allow them to form structures such as vesicles or membranes in an aqueous environment. Lipids include fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids (including cholesterol), prenolipids, saccharolipids, and polyketides. In some embodiments, the lipids suitable for the amphiphilic ligand conjugate of this disclosure bind to human serum albumin under physiological conditions. In some embodiments, the lipids suitable for the amphiphilic ligand conjugate of this disclosure are inserted into the cell membrane under physiological conditions. In some embodiments, the lipids bind to albumin under physiological conditions and are inserted into the cell membrane. In some embodiments, the lipids are diacyllipids. In some embodiments, the diacyllipids contain at least 12 carbon atoms. In some embodiments, the diacyllipids contain 12 to 30 hydrocarbon units, 14 to 25 hydrocarbon units, or 16 to 20 hydrocarbon units. In some embodiments, the diacyllipid contains 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms.

[0037] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, and polymers thereof. Unless specifically limited, this term encompasses nucleic acids containing known analogs of native nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a similar manner to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985); and Cassol et al., 1992; Rossolini et al., Mal. Cell. Probes 8:91-98, 1994). In the case of arginine and leucine, modifications at the second base may also be conserved. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by gene.

[0038] The polynucleotides of the present invention may consist of any polyribonucleotide or polydeoxyribonucleotide that may be unmodified RNA or DNA, or modified RNA or DNA. For example, polynucleotides may consist of hybrid molecules comprising single-stranded and double-stranded DNA, DNA which is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA which is a mixture of single-stranded and double-stranded regions, single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. Furthermore, polynucleotides may consist of triple-stranded regions that include RNA or DNA or both RNA and DNA. Polynucleotides may also contain one or more modified bases or DNA or RNA backbone modified for stability or other reasons. Examples of “modified” bases include unusual bases such as tritylated bases and inosine. Various modifications can be made to DNA and RNA; therefore, “polynucleotide” encompasses chemically, enzymatically, or metabolically modified forms. In some embodiments, the polypeptides of the present invention are encoded by a nucleotide sequence. The nucleotide sequences of the present invention may be useful for many applications, including cloning, gene therapy, protein expression and purification, mutagenesis, DNA vaccination of hosts requiring such sequences, antibody production for passive immunity, PCR, primer and probe production, and the like.

[0039] As used herein, “parenteral administration,” “administered parenterally,” and other grammatically equivalent terms refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, carotid, and intrasternal injections and infusions.

[0040] As used herein, “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form suitable for use in contact with human and animal tissues, organs, and / or bodily fluids, within the bounds of sound medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0041] As used herein, the term “pharmaceutically acceptable salt” means any pharmaceutically acceptable salt of any conjugate, oligonucleotide, or peptide disclosed herein. A pharmaceutically acceptable salt of any of the compounds and nucleic acid sequences described herein may include those that are within the bounds of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, or allergic reactions, and that offer a reasonable benefit / risk ratio. pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and Pharmaceutical Salts: Properties, Selection, and Use, (Eds. PHStahl and CGWermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein, or separately by reacting free base groups with a suitable acid. Typical acid addition salts include acetate, adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, and 2-hydroxyethanesulfonate. Examples include nitrates, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfons, 2-naphthalenesulfons, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectins, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyans, toluenesulfons, undecanoates, valersates, etc.Typical alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and, but are not limited to, non-toxic ammonium, quaternary ammonium, and amine cations, including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine. References to compounds, nucleic acids, conjugates, oligonucleotides, or polypeptides in the claims and elsewhere in this specification include, optionally, pharmaceutically acceptable salts thereof, unless otherwise indicated or applicable.

[0042] As used herein, the term “physiological conditions” refers to the in vivo state of the subject. In some embodiments, physiological conditions refer to a neutral pH (e.g., pH 6–8).

[0043] As used herein, the term "peptide" refers to a polymer having 30 or fewer amino acid residues.

[0044] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding natural amino acids, as well as to natural and non-natural amino acid polymers.

[0045] As used herein, the terms “subject,” “mammal,” or “patient” include any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat a subject having a disease or symptom. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, mice, horses, pigs, cattle, chickens, amphibians, reptiles, etc.

[0046] The terms "sufficient amount" or "amount sufficient to" mean an amount sufficient to produce the desired effect, for example, an amount sufficient to reduce the diameter of a tumor.

[0047] The term "T cell" refers to a type of white blood cell that can be distinguished from other white blood cells by the presence of a T cell receptor on its cell surface. There are several subsets of T cells, including, but not limited to, T helper cells (T H cells or CD4 + T cells, also known as T H cells, T H 2, T H 3, T H 17, T H 9 and T FH cells, cytotoxic T cells (i.e., Tc cells, CD8 + T cells, cytotoxic T lymphocytes, T killer cells, killer T cells), memory T cells and, for example, central memory T cells (T CM cells), effector memory T cells (T EM and T EMRA cells) and resident memory T cells (T RM cells), regulatory T cells (also known as Treg cells or suppressor T cells) and, CD4 + FOXP3 + T reg cells, CD4 + FOXP3 - T reg cells, Tr1 cells, Th3 cells and T reg 17 cells, natural killer T cells (also known as NKT cells), mucosal associated invariant T cells (MAIT) and gamma delta T cells (γδT cells), such as Vγ9 / Vδ2 T cells. Any one or more of the foregoing or unmentioned T cells can be a target cell type in the methods of use of the present invention.

[0048] As used herein, the terms “to treat,” “to treat,” and “treatment” refer to the therapeutic or prophylactic measures described herein. A method of “treatment” involves administering the peptide and albumin-binding domain of the Disclosure to a subject requiring such treatment. In some embodiments, the Braf peptide conjugated to the albumin-binding domain is administered to a subject requiring an enhanced immune response to a particular antigen, or a subject that may eventually acquire such a disorder, in order to prevent, cure, delay, reduce the severity of, or improve one or more symptoms of the disorder or recurrent disorder, or to extend the subject’s survival beyond what would be expected in the absence of such treatment.

[0049] As used herein, “vaccine” means a formulation comprising the amphiphilic construct described herein, and optionally combined with an adjuvant, which is capable of being administered to vertebrates and induces a protective immune response sufficient to induce immunity to prevent and / or improve a disease or condition and / or reduce at least one symptom of the disease or condition. Typically, the vaccine comprises a conventional saline or buffered aqueous medium in which the compositions described herein are suspended or dissolved. In this form, the compositions described herein are used to prevent, improve or treat an infection or disease. Upon introduction into a host, the vaccine induces an immune response, including, but not limited to, the induction of a protective immune response to induce immunity to prevent and / or improve a disease or condition and / or reduce at least one symptom of the disease or condition.

[0050] peptide Compounds comprising a peptide that is a Braf peptide are described herein. The peptide is optionally conjugated to an albumin-binding domain via a linker.

[0051] In some embodiments, the peptide is [ka] This is a fragment of the Braf polypeptide having the following amino acid sequence.

[0052] Braf peptides can be 8-30 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30) amino acid fragments of SEQ ID NO: 1. For example, Braf peptides can be 9 amino acid fragments of SEQ ID NO: 1. In some embodiments, Braf peptides are 15 amino acid fragments of SEQ ID NO: 1. In some embodiments, Braf peptides are 29 amino acid fragments of SEQ ID NO: 1.

[0053] Braf peptides may comprise fragments of SEQ ID NO: 1 containing one or more amino acid substitutions. In some embodiments, a Braf peptide comprises fragments of SEQ ID NO: 1 containing an amino acid substitution at Val, which occupies amino acid position 600 from the N-terminus of SEQ ID NO: 1. For example, a Braf peptide may contain an amino acid substitution at position 600 of SEQ ID NO: 1, where the Val residue is substituted with a Lys residue (V600K) or the Val residue is substituted with a Glu residue (V600E).

[0054] The peptide may consist of or contain the amino acid sequence (SEQ ID NO: 2) of EDLTVKIGDFGLATVKSRWSGSHQFEQLS or a fragment thereof. The peptide may consist of or contain the amino acid sequence (SEQ ID NO: 3) of EDLTVKIGDFGLATKKSRWSGSHQFEQLS or a fragment thereof. The peptide may consist of or contain the amino acid sequence (SEQ ID NO: 4) of EDLTVKIGDFGLATEKSRWSGSHQFEQLS or a fragment thereof.

[0055] The peptide may consist of or contain the amino acid sequence EDLTVKIGDFGLATVKSRWSGSHQFEQLS (SEQ ID NO: 2) or a fragment thereof, or may have a length of 5 to 60 amino acids (e.g., 5, 6, 7, 89, 10, 11, 12, 13, 15, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60). The peptide may consist of or contain the amino acid sequence (SEQ ID NO: 3) of EDLTVKIGDFGLATKKSRWSGSHQFEQLS or a fragment thereof, or may have a length of 5 to 60 amino acids (e.g., 5, 6, 7, 89, 10, 11, 12, 13, 15, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60). The peptide may consist of or contain the amino acid sequence (SEQ ID NO: 4) of EDLTVKIGDFGLATEKSRWSGSHQFEQLS or a fragment thereof, or may have a length of 5 to 60 amino acids (e.g., 5, 6, 7, 89, 10, 11, 12, 13, 15, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60).

[0056] In some embodiments, the Braf peptide contains a 9-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide contains a 10-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide contains an 11-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide contains a 12-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide contains a 13-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide contains a 14-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide contains a 15-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide contains a 16-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide contains a 17-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide contains an 18-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide contains a 19-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide comprises a 20-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide comprises a 21-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide comprises a 22-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide comprises a 23-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide comprises a 24-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide comprises a 25-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide comprises a 26-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide comprises a 27-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the Braf peptide comprises a 28-amino acid fragment of SEQ ID NO: 2, 3, or 4. In some embodiments, the 9-amino acid fragment of SEQ ID NO: 3 or 4 consists of or includes the sequence FGLATKKSR (SEQ ID NO: 61) or FGLATEKSR (SEQ ID NO: 62).In some embodiments, the 15-amino acid fragment of SEQ ID NO: 3 or 4 consists of or includes the sequence GDFGLATKKSRWSGS (SEQ ID NO: 63) or GDFGLATEKSRWSGS (SEQ ID NO: 64).

[0057] In some embodiments, the peptide includes an N-terminal modification. In some embodiments, the N-terminal modification is the addition of cysteine. In some embodiments, the N-terminal modification is the addition of acetylcysteine. In some embodiments, the N-terminal modification is the addition of a desaminocysteine ​​homolog. In some embodiments, the desaminocysteine ​​homolog is 3-mercaptopropionic acid or mercaptoacetic acid. In some embodiments, the N-terminus of the peptide is bound to or ligated to an albumin-binding domain. In some embodiments, the C-terminus of the peptide is bound to or ligated to an albumin-binding domain.

[0058] amphiphilic peptides The amphiphilic peptide includes an albumin-binding domain, for example, a peptide conjugated to a lipid. In some embodiments, the amphiphilic peptide optionally includes a Braf peptide conjugated to an albumin-binding domain, for example, a lipid, via a linker.

[0059] Lipids The compounds described herein include, as herein, Braf peptides conjugated to an albumin-binding domain. In some embodiments, the albumin-binding domain is a lipid. The lipid may be linear, branched, or cyclic.

[0060] Preferred lipids include, but are not limited to, straight-chain unsaturated and saturated fatty acids, branched saturated and unsaturated fatty acids, fatty acid derivatives such as fatty acid esters, fatty acid amides, and fatty acid thioesters, diacyl lipids, cholesterol, cholesterol derivatives, and steroid acids such as bile acids and lipid A, or combinations thereof, and include fatty acids having an aliphatic tail of 3 to 30 carbon atoms.

[0061] In certain embodiments, the lipid is a diacyllipid or a two-tailed lipid. In some embodiments, the tail of the diacyllipid contains about 12 to about 30 (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) carbon atoms. In some embodiments, the tail of the diacyllipid contains about 14 to about 25 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) carbon atoms. In some embodiments, the tail of the diacyllipid contains about 16 to about 20 (e.g., 17, 18, or 19) carbon atoms. In some embodiments, the diacyllipid contains 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms.

[0062] The carbon tail of a diacyllipid can be saturated, unsaturated, or a combination thereof. The tail can be attached to the head group via an ester bond, amide bond, thioester bond, or a combination thereof. In certain embodiments, the diacyllipid is a phosphatelipid, glycolipid, sphingolipid, or a combination thereof.

[0063] In some embodiments, the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0064] In some embodiments, the Braf peptide is derived from the following lipids: [ka] Alternatively, it may be bonded to the salt or linked by a linker.

[0065] Braf peptides can be directly bound to lipids, or they can be linked to lipids via linkers.

[0066] References to lipids and amphiphilic substances containing lipids in this specification should be understood to include their pharmaceutically acceptable salts.

[0067] Linker In some embodiments, the compound includes an albumin-binding domain, for example, a Braf peptide linked to a lipid by a linker. The linker may be a hydrophilic polymer, a series of hydrophilic amino acids, polysaccharides, and oligonucleotides, or a combination thereof. The linker can reduce or prevent the albumin-binding domain's ability to insert into the plasma membrane of cells, for example, cells in tissue adjacent to the injection site. The linker can also reduce or prevent the amphiphilic peptide sequence's ability to nonspecifically associate with extracellular matrix proteins at the administration site. To efficiently transport the amphiphilic Braf peptide to lymph nodes, it should remain soluble. To enhance the solubility of the amphiphilic Braf peptide, a polarity-blocking linker may be included between the Braf peptide and the albumin-binding domain to which it is conjugated.

[0068] The length and composition of the linker can be adjusted based on the selected albumin-binding domain and peptide. For example, in certain embodiments, the polynucleotide itself may be polar enough to ensure solubility; for example, a polynucleotide with a nucleotide length of 10, 15, 20 or more. Therefore, in some embodiments, no additional linker is required. However, in certain cases, it may be desirable to include a linker that mimics the effect of a polar oligonucleotide. The linker can be used as part of any of the albumin-binding domain conjugates described herein, e.g., lipid-oligonucleotide conjugates and lipid-peptide conjugates that reduce cell membrane insertion / preferential splitting onto albumin.

[0069] Suitable linkers include, but are not limited to, oligonucleotides containing a range of nucleic acids, e.g., the oligonucleotides mentioned above; hydrophilic polymers, e.g., but are not limited to, poly(ethylene glycol) (MW: 500 Da to 20,000 Da), polyacrylamide (MW: 500 Da to 20,000 Da), polyacrylic acid; a range of hydrophilic amino acids such as serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or combinations thereof; but are not limited to, polysaccharides containing dextran (MW: 1,000 Da to 2,000,000 Da), or combinations thereof. The hydrophobic albumin-binding domain and the linker / peptide (e.g., Braf peptide) are covalently bonded. The covalent bond may be an indestructible bond or a cleavable bond. Non-cleavable bonds may include amide bonds or phosphate bonds, while cleavable bonds may include disulfide bonds, acid-cleavable bonds, ester bonds, anhydride bonds, biodegradable bonds, or enzymatically cleavable bonds.

[0070] In some embodiments, the linker is one or more ethylene glycol (EG) units, more preferably two or more EG units (i.e., polyethylene glycol (PEG)). For example, in some embodiments, the compound comprises a Braf peptide linked by polyethylene glycol (PEG) molecules or derivatives or analogs thereof, and a hydrophobic albumin-binding domain.

[0071] In some embodiments, the compounds described herein then include a hydrophobic albumin-binding domain, for example, a Braf peptide linked to lipid-linked PEG. The exact number of PEG units depends on the albumin-binding domain and cargo, but typically the linker has about 1 to about 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, It may have 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100). In some embodiments, the linker may be a PEG linker having about 20 to about 80, about 30 to about 70, or about 40 to about 60 PEG units. In some embodiments, the number of PEG units is 24 to 50 units (e.g., 24 to 45, 24 to 40, 24 to 35, 24 to 30, 30 to 50, 35 to 50, 40 to 50, and 45 to 50 units). In some embodiments, the linker has approximately 45 to 55 PEG units. For example, in some embodiments, the linker has 48 PEG units. In some embodiments, the linker includes a PEG4-amide-PEG4 linker.

[0072] As described above, in some embodiments, the linker is an oligonucleotide comprising a series of nucleic acids. In some embodiments, the compounds described herein then include a hydrophobic albumin-binding domain, for example, a Braf peptide linked to a series of nucleic acids linked to lipids. The linker can be any sequence, for example, the sequence of the oligonucleotide may be a random sequence or a sequence particularly selected for its molecular or biochemical properties (e.g., highly polar). In some embodiments, the linker comprises 20 or more sequences of one or more adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analogs thereof. In some embodiments, the linker consists of a series of adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analogs thereof.

[0073] In some embodiments, the nucleic acid series comprises 1 to 50 nucleic acid residues. In some embodiments, the nucleic acid series comprises 5 to 30 nucleic acid residues. In some embodiments, the linker comprises one or more guanines, for example, 1 to 10 guanines.

[0074] In some embodiments, the linker is an oligonucleotide comprising a series of nucleic acids. In some embodiments, the amphiphilic Braf peptide then comprises a hydrophobic albumin-binding domain, e.g., a p53 peptide or mutant p53 peptide linked to a series of amino acids linked to a lipid. The linker may have any amino acid sequence; for example, the sequence of the oligonucleotide may be a random sequence or a sequence selected for its molecular or biochemical properties (e.g., high flexibility). In some embodiments, the linker comprises a series of glycine residues to form a polyglycine linker. In some embodiments, the linker is (Gly) nThe amino acid sequence includes n, where n can be 2 to 20 residues. Examples of polyglycine linkers include, but are not limited to, GGG, GGGA (SEQ ID NO: 8), GGGG (SEQ ID NO: 9), GGGAG (SEQ ID NO: 10), GGGAGG (SEQ ID NO: 11), GGGAGGG (SEQ ID NO: 12), GGAG (SEQ ID NO: 13), GGSG (SEQ ID NO: 14), AGGG (SEQ ID NO: 15), SGGG (SEQ ID NO: 16), GGAGGA (SEQ ID NO: 17), GGSGGS (SEQ ID NO: 18), GGAGGAGGA (SEQ ID NO: 19), GGSSGGGGS (SEQ ID NO: 20), GGAGGAGGAGGA (SEQ ID NO: 21), GGSGGSGGSGGS (SEQ ID NO: 22), GGAGGGAG (SEQ ID NO: 23), GGSGGGSG (SEQ ID NO: 24), GGAGGGAGGGAG (SEQ ID NO: 25), GGSGGGSGGGSG (SEQ ID NO: 26), GGGGAGGGGAGGGGA (SEQ ID NO: 27), and GGGGSGGGGSGGGGS (SEQ ID NO: 28).

[0075] Conjugation methods Compounds containing Braf peptides and albumin-binding domains are described herein.

[0076] The peptide may be modified with an N-terminal cysteine, acetylcysteine, sulfydryl, transcyclooctene, cyclooctin, azide, or alkyne for conjugation with the Braf peptide and its albumin-binding domain. In some embodiments, the peptide is modified with a C-terminal cysteine, azide, or alkyne for conjugation with the Braf peptide and its albumin-binding domain. In some embodiments, an internal cysteine ​​or lysine of the peptide is used for conjugation with the albumin-binding domain.

[0077] The Braf peptide and albumin-binding domain can be bound to or linked to a linker. In some embodiments, the linker contains a functional group. In some embodiments, the functional group has the ability to conjugate to the peptide. For example, the Braf peptide may be bound to the linker, and the linker may be modified with a functional group. In some embodiments, the albumin-binding domain may be linked to the linker, and the linker may be modified with a functional group. In some embodiments, the linker may be a PEG linker.

[0078] In some embodiments, the Braf peptide is conjugated to the albumin-binding domain and / or linker via a reaction between a dithio group and a free thiol group.

[0079] Adjuvant In some embodiments, the pharmaceutical compositions described herein may be administered with one or more adjuvants. An adjuvant refers to a substance that causes stimulation of the immune system. In this regard, adjuvants are used to enhance the immune response to peptides. Adjuvants may be administered to a subject before, in combination with, or after administration of the compositions described herein. In some embodiments, additional adjuvants are administered to a subject in combination with a Braf peptide conjugated to an albumin-binding domain described herein. In some embodiments, the adjuvant may be conjugated to an albumin-binding domain, for example, a lipid. Adjuvants include, but are not limited to, lipids (e.g., monophosphoryl lipid A (MPLA)), alum (e.g., aluminum hydroxide, aluminum phosphate); Freund's adjuvant; saponins purified from the bark of the Q. saponaria tree, e.g., QS21 (a glycolipid that elutes to the 21st peak by HPLC fractionation; Antigenics, Inc., Worcester, Mass.); poly[di(carboxylatofenoxy)phosphazene] (PCPP polymer; Virus Research Institute, USA), Flt3 ligand, leishmania elongation factor (purified leishmania protein; Corixa Corporation, Seattle, Wash.), ISCOMS (an immunostimulatory complex containing mixed saponins and lipids that forms virus-sized particles with pores capable of holding antigens; CSL, Melbourne, Australia), Pam3Cys, SB-AS4 (SmithKline containing alum and MPL). Nonionic block copolymers that form micelles, such as Beecham adjuvant series #4 (SBB, Belgium), CRL1005 (these include linear hydrophobic polyoxypropylene chains adjacent to polyoxyethylene chains) (Vaxcel, Inc., Norcross, Ga), and Montanide IMS (e.g., IMS 1312, aqueous nanoparticles combined with the soluble immunostimulant Seppic), as well as CDN (cyclic dinucleotide).

[0080] Adjuvants can be Toll-like receptor (TLR) ligands. Adjuvants acting via TLR3 include, but are not limited to, double-stranded RNA. Adjuvants acting via TLR4 include, but are not limited to, lipopolysaccharide derivatives, such as monophosphoryl lipid A (MPLA; Ribi ImmunoChem Research, Inc. Hamilton, Mont.), muramyl dipeptide (MDP; Ribi), and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland). Adjuvants acting via TLR5 include, but are not limited to, flagellin. Adjuvants acting via TLR7 and / or TLR8 include single-stranded RNA, oligoribonucleotides (ORNs), and synthetic low molecular weight compounds such as imidazoquinolineamines (e.g., imiquimod (R-837), reximod (R-848)). Adjuvants acting via TLR9 include viral or bacterial DNA, or synthetic oligodeoxynucleotides (ODNs), such as CpG ODNs. For example, CpG ODNs may have sequences such as TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 5), 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 6), or 5'-TCGTCGTTTTCGGCGCGCGCCG-3' (SEQ ID NO: 7). CpG binding can be any phosphorothioate bond.

[0081] Another class of adjuvants consists of phosphorothioate-containing molecules, such as phosphorothioate nucleotide analogs and nucleic acids containing phosphorothioate backbone bonds.

[0082] Pharmaceutical composition Pharmaceutical compositions of this disclosure comprising a Braf peptide conjugated to an albumin-binding domain are described herein. In addition to a therapeutic amount of the Braf peptide conjugated to an albumin-binding domain described herein, the pharmaceutical compositions may contain pharmaceutically acceptable carriers or excipients that can be formulated by methods known to those skilled in the art. This also includes pharmaceutically acceptable salts of the components as described herein.

[0083] The acceptable carriers and excipients in the pharmaceutical compositions of Braf peptides conjugated to albumin-binding domains described herein are nontoxic to the recipient at the doses and concentrations used. In certain embodiments, the formulation material is for subcutaneous (sc) and / or intravenous (iv) administration. In some embodiments, administration is by inhalation or intranasal administration. In some embodiments, the formulation material is administered intraperitoneally, topically, or orally. In some embodiments, the pharmaceutical composition may include formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolality by weight, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or osmosis of the composition.In some embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, methionine, sodium sulfite, or sodium bisulfite); buffering agents (e.g., borates, bicarbonates, Tris-HCl, citrates, HEPES, TAE, phosphates, or other organic acids); bulking agents (e.g., mannitol, glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, sucrose, mannose, or dextrin); proteins (e.g., human serum albumin, gelatin, dextran, and immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; and salt-forming counterions. Sodium, etc.); Preservatives (e.g., hexamethonium chloride, octadecyldimethylbenzylammonium chloride, resorcinol, and benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); Solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); Sugar alcohols (e.g., mannitol or sorbitol); Suspending agents; Surfactants or wetting agents (e.g., polysorbates such as Pluronics, PEG, sorbitan esters, polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyroxapal); Stability enhancers (e.g., sucrose or sorbitol); Isotonic agents (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); Delivery vehicles; Diluents; Excipients and / or pharmaceutical adjuvants (Remington's Pharmaceutical Sciences, 18th Edition, Argennaro, ed., Mack Publishing Company (1995) is one example.In some embodiments, the optimal pharmaceutical composition can be determined by those skilled in the art, for example, depending on the intended route of administration, delivery method, and desired dose. See, for example, Remington's Pharmaceutical Sciences mentioned above. In some embodiments, such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the amphiphilic conjugate.

[0084] In some embodiments, the main vehicle or carrier in a pharmaceutical composition containing the Braf peptide conjugated to the albumin-binding domain described herein may actually be either aqueous or non-aqueous. For example, in some embodiments, a suitable vehicle or carrier may be water for injection, saline, or artificial cerebrospinal fluid, but other materials common in compositions for parenteral administration may be supplemented. In some embodiments, saline includes isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin is a further exemplary vehicle. In some embodiments, the pharmaceutical composition includes Tris buffer at about pH 7.0–8.5, or acetate buffer at about pH 4.0–5.5, and therefore may further include sorbitol or a suitable substitute. In some embodiments, a composition containing the Braf peptide conjugated to the albumin-binding domain described herein may be prepared for storage by mixing a selected composition having the desired purity with any formulation in the form of a lyophilized cake or aqueous solution (Remington's Pharmaceutical Sciences, cited above). Furthermore, in some embodiments, compositions comprising the Braf peptide conjugated to the albumin-binding domain described herein can be formulated as lyophilized products using appropriate excipients such as sucrose.

[0085] In some embodiments, pharmaceutical compositions may be selected for parenteral delivery. The preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art.

[0086] In some embodiments, the formulation components are present at the administration site at an acceptable concentration. In some embodiments, buffers are used to maintain the composition at a physiological pH or slightly lower, typically within a pH range of about 5 to about 8.

[0087] In some embodiments, when parenteral administration is intended, the therapeutic composition may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the Braf peptide conjugated to the albumin-binding domain described herein, in a pharmaceutically acceptable vehicle. In some embodiments, the vehicle for parenteral injection is sterile distilled water formulated as a sterile isotonic solution in which the Braf peptide conjugated to the albumin-binding domain described herein is appropriately preserved. In some embodiments, the preparation may involve the formulation of the desired molecule using drugs such as injectable microspheres, bio-erosive particles, polymer compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, which can provide controlled or sustained release of the product and can then be delivered via depot injection. In some embodiments, hyaluronic acid may also be used, which may have the effect of promoting the duration of action in circulation. In some embodiments, an implantable drug delivery device may be used to introduce the desired molecule.

[0088] The pharmaceutical composition can be administered, for example, in a therapeutically effective dose to induce an immune response. The therapeutically effective dose of the Braf peptide conjugated to the albumin-binding domain described herein, contained in the pharmaceutical preparation, can be determined by those skilled in the art so that the dose (e.g., within the range of 0.01 to 100 mg / kg body weight) induces an immune response in the subject.

[0089] Vectors can be used in in vivo nucleic acid delivery vehicles, including, but are not limited to, retroviral vectors, adenovirus vectors, poxvirus vectors (e.g., vaccinia virus vectors, e.g., modified vaccinia ankara (MVA)), adeno-associated virus vectors, and alphavirus vectors. In some embodiments, the vector may include an internal ribosome entry site (IRES) that enables the expression of the peptides described herein. Other vehicles and methods for nucleic acid delivery are described in U.S. Patents No. 5,972,707, No. 5,697,901, and No. 6,261,554, each of which is incorporated herein by reference in whole. Other methods for manufacturing pharmaceutical compositions are described, for example, in U.S. Patent Nos. 5,478,925, 8,603,778, 7,662,367, and 7,892,558, all of which are incorporated herein by reference in their entirety.

[0090] In some embodiments, the pharmaceutical compositions described herein may be administered together with one or more adjuvants.

[0091] Route of administration, dosage, and timing The pharmaceutical compositions of this disclosure, containing a Braf peptide conjugated to an albumin-binding domain as described herein, can be formulated for parenteral administration, subcutaneous administration, intravenous administration, intramuscular administration, intranasal administration, or inhalation. In some embodiments, the therapeutic agent is formulated for transmucosal administration. In some embodiments, the therapeutic agent is formulated for buccal administration. In some embodiments, the therapeutic agent is formulated for sublingual administration. Methods for administering therapeutic proteins are known in the art. For example, see U.S. Patent Nos. 6,174,529, 6,613,332, 8,518,869, 7,402,155, and 6,591,129, as well as U.S. Patent Publication No. 20140051634, International Publication No. 1993000077, and U.S. Patent Publication No. 20110184145, in which such disclosures are incorporated by reference in their entirety.

[0092] One or more of these methods can be used to administer the pharmaceutical composition of the present invention containing the Braf peptide conjugated to an albumin-binding domain. For injectable formulations, various effective pharmaceutical carriers are known in the art. See, for example, Pharmaceuticals and Pharmacy Practice, JBLippincott Company, Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986). The dosage of the pharmaceutical composition of the present invention depends on the route of administration and factors including the physical characteristics of the subject, such as age, weight, and overall health status. Typically, the amount of the Braf peptide conjugated to an albumin-binding domain described herein contained in a single dose may be an amount that effectively induces an immune response in the subject without inducing significant toxicity. The pharmaceutical compositions of the present invention may contain a Braf peptide conjugated to an albumin-binding domain as described herein in doses ranging from 0.001 to 500 mg (e.g., 0.01, 0.05, 0.1, 0.2, 0.3, 0.5, 0.7, 0.8, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 50 mg, 100 mg, 250 mg, or 500 mg), and in more specific embodiments, in doses ranging from about 0.1 to about 100 mg. The dose can be adjusted by a clinician according to different parameters of the target.

[0093] The pharmaceutical composition of the present invention, containing a Braf peptide conjugated to an albumin-binding domain, can be administered to subjects requiring it, for example, once a day, once a week, once a month, once every six months, once a year or more (for example, 1 to 10 times or more), or when medically necessary.

[0094] Methods for inducing an immune response This disclosure provides a method for inducing an immune response to Braf peptides in a subject. The method comprises administering one of the compounds described herein to the subject.

[0095] In some embodiments, the disclosure provides a method for inducing an immune response to a Braf peptide in a subject by administering one of the Braf peptides conjugated to an albumin-binding domain to the subject, and further administering an adjuvant to the subject. In some embodiments, the Braf peptide conjugated to an albumin-binding domain can be administered without one or more additional adjuvants.

[0096] In some embodiments, the method involves administering to a subject a therapeutically effective amount of the Braf peptide conjugated to an albumin-binding domain as described herein. In some embodiments, the Braf peptide conjugated to an albumin-binding domain is administered substantially simultaneously. In some embodiments, the Braf peptide conjugated to an albumin-binding domain is administered separately.

[0097] In some embodiments, one or more of the administered components are pharmaceutically acceptable salts of the indicated components as described herein.

[0098] In some embodiments, the Disclosure provides methods for inducing an immune response to a peptide in a subject by subcutaneous administration of any one of the compounds or pharmaceutically acceptable salts described herein to the subject. In some embodiments, the Disclosure provides methods for inducing an immune response to a peptide in a subject by intramuscular, subcutaneous, intravenous, intraperitoneal, topical, oral / buccal, sublingual, transmucosal, nasal, or inhalation administration of the peptide to the subject.

[0099] In some embodiments, the subject is a mammal. For example, the subject may be a human.

[0100] kit The kit may include a Braf peptide conjugated to an albumin-binding domain as disclosed herein and instructions for use. The kit may include, in a suitable container, a Braf peptide conjugated to an albumin-binding domain, one or more controls, and various buffers, reagents, enzymes, and other standard components well known in the art. In some embodiments, the kit further includes an adjuvant. Thus, in some embodiments, the Braf peptide conjugated to an albumin-binding domain is in a vial. In some embodiments, the Braf peptide conjugated to an albumin-binding domain and the adjuvant are in separate vials. In some embodiments, the Braf peptide and the adjuvant are in the same vial. In some embodiments, the Braf peptide and the adjuvant are in separate vials.

[0101] The container may include at least one vial, well, test tube, flask, bottle, syringe, or other container means in which the Braf peptide conjugated to an albumin-binding domain can be placed and, if applicable, appropriately divided. If additional components are provided, the kit may include additional containers in which this compound can be placed. The kit may also include means for containing the Braf peptide conjugated to an albumin-binding domain and any other reagent containers tightly sealed for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the desired vial is held. The container and / or kit may include labels with instructions for use and / or warnings.

[0102] In some embodiments, the Disclosure provides a pharmaceutical product comprising a composition comprising a Braf peptide conjugated to an albumin-binding domain, an optional pharmaceutically acceptable carrier, and a kit comprising a package insert including instructions for administering the pharmaceutical product alone or in combination with the composition comprising an adjuvant and an optional pharmaceutically acceptable carrier of the elements, to treat, slow the progression of, or prevent a disease or condition, wherein the Braf peptide conjugated to an albumin-binding domain optionally comprises a linker.

[0103] In some embodiments, the Disclosure provides a kit comprising a container comprising a composition comprising a Braf peptide conjugated to an albumin-binding domain, an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administering the composition vaccine to a subject, wherein the Braf peptide is conjugated to an albumin-binding domain and optionally comprises a linker. In some embodiments, the kit further comprises an adjuvant and instructions for administering the adjuvant.

[0104] In some embodiments of the kit, one or more of the kit's components are pharmaceutically acceptable salts of components as described herein. [Examples]

[0105] The following examples are intended to illustrate, not limit, and this disclosure is provided to those skilled in the art to explain how the compositions and methods described herein can be used, prepared, and evaluated. The examples are intended purely to illustrate the present disclosure and are not intended to limit the scope of what the inventors consider to be their invention.

[0106] Example 1. Ability of Braf V600E peptide to induce immune response in C57BL / 6J mice. The objective of this experiment was to establish whether an immune response could be induced by AMP-conjugated Braf peptide.

[0107] Five groups of 10 C57BL / 6J mice were administered a vaccine containing the components shown in Table 2. As shown in Table 1, five mice from each group were removed after two doses, and the remaining five mice were removed after three doses.

[0108] [Table 1]

[0109] The amount of peptide antigen used was 5 nmol per injection. Due to the low solubility of some AMP-peptides, all AMP-peptide stock solutions were co-solubilized with AMP-CpG. To achieve this, lyophilized AMP-peptides were first dissolved in 50% t-butanol, then AMP-CpG was added in a 1:1 molar ratio, and the mixture was then lyophilized. The resulting powder was then resuspended in 1×PBS to a concentration of 1 mg / ml. The vaccine components are listed in Table 2. Furthermore, the AMP-vaccine stock solutions were diluted using 1×PBS to their final concentrations so that each injection contained 5 nmol of AMP-antigen and 5 nmol of AMP-adjuvant.

[0110] The soluble peptide stock solution was prepared in 1.1×PBS at concentrations of 0.5 mg / ml and 0.45 mg / ml, respectively, and further diluted with 1×PBS to a final concentration of 5 nmol / 100 μL injection solution. The soluble adjuvant stock solution (CpG) was prepared in horseshoe crab hemocyte extract (LAL)H2O to a final concentration of 5 nmol / 100 μL injection solution.

[0111] The immunizer was administered subcutaneously (SC) at a rate of 50 μL per side bilaterally into the tail base of female B6 mice, with booster doses given approximately every two weeks. SC injection ensured optimal delivery of the vaccine into the lymph nodes via natural lymphatic drainage, and bi-weekly injections were determined to be optimal for the immune response.

[0112] [Table 2]

[0113] 0.2 × 10 6 Doses of 2,0.2 × 10¹ cells / well and 2 μg / ml of each peptide. 6 ELISpot analysis for IFNγ was performed on splenocytes 7 days after dose 3 of each peptide at 2 μg / ml per well. Splenocytes were activated with the Braf peptides listed in Table 3. IFNy plates were stimulated overnight. The results of this analysis are shown in Figures 2A and 2B.

[0114] [Table 3]

[0115] Immunization with the insoluble AMP conjugate Braf V600E 29-mer induced a significant immune response in mice after dose 2, which was further enhanced after administration of a third dose. This immune response was not detected with the 15-mer version of the AMP peptide.

[0116] Example 2. Ability of Braf V600E peptide to induce immune response in Balb / c mice. The objective of this experiment was to establish whether an immune response could be induced by any of the AMP-Braf peptides in Balb / c mice expressing MHC haplotype d.

[0117] Four groups of 10 mice each were administered a vaccine containing the components shown in Table 5. As shown in Table 4, five mice from each group were removed after two doses, and the remaining five mice were removed after three doses.

[0118] [Table 4]

[0119] The amount of peptide antigen used was 5 nmol per injection. Due to the low solubility of some AMP-peptides, all AMP-peptide stock solutions were co-solubilized with AMP-CpG. To achieve this, lyophilized AMP-peptides were first dissolved in 50% t-butanol, then AMP-CpG was added in a 1:1 molar ratio, and the mixture was then lyophilized. The resulting powder was then resuspended in 1×PBS to a concentration of 1 mg / ml. The vaccine components are listed in Table 5. Furthermore, the AMP-vaccine stock solutions were diluted using 1×PBS to their final concentrations so that each injection contained 5 nmol of AMP-antigen and 5 nmol of AMP-adjuvant.

[0120] The immunizer was administered subcutaneously (SC) at a dose of 50 μL per side bilaterally into the tail base of female B6 mice. Booster doses were given approximately every two weeks. SC injection ensured optimal delivery of the vaccine into the lymph nodes via natural lymphatic drainage, and bi-weekly injections were determined to be optimal for the immune response.

[0121] [Table 5]

[0122] 0.2 × 10 6 ELISpot analysis for IFNγ was performed on splenocytes 7 days after dose 3 of each peptide at 2 μg / ml per well. Splenocytes were activated with the Braf peptides listed in Table 6. IFNy plates were stimulated overnight. The results of this analysis are shown in Figure 3.

[0123] [Table 6]

[0124] Immunization with the AMP conjugate Braf V600E 29mer induced a significant immune response in Balb / c mice after 3 doses. This immune response was not detected with the 9-mer or 15-mer versions of the AMP peptide.

[0125] Example 3. Efficacy of a combination Braf vaccine containing both V600E and V600K peptides. The objective of this experiment was to evaluate a combination Braf vaccine containing 29-mers of both the V600E and V600K peptide sequences. The experiment was designed to determine whether immunodominance of one peptide over the other exists and whether the epitopes are random between the two mutations.

[0126] Seven groups of mice were each administered a vaccine containing the components listed in Table 8. As shown in Table 7, a set of mice from each group was removed after two doses, and the remaining mice were removed after three doses.

[0127] [Table 7]

[0128] Previous dose-finding studies determined that an antigen-to-adjuvant ratio of 2:1 was optimal. Therefore, 20 nmol of antigen and 10 nmol of AMP-CpG were used per injection. Due to the low solubility of some AMP-peptides, all AMP-peptide stock solutions were co-solubilized with AMP-CpG. To achieve this, lyophilized AMP-peptides were first dissolved in 50% t-butanol, then AMP-CpG was added in a 2:1 molar ratio, and the mixture was lyophilized. The resulting powder was then resuspended in 1×PBS to a concentration of 1 mg / ml. The vaccine components are listed in Table 8. Furthermore, the AMP-vaccine stock solutions were diluted using 1×PBS to their final concentrations so that each injection contained 20 nmol of AMP-antigen and 20 nmol of AMP-adjuvant.

[0129] The soluble peptide was prepared in 1.1×PBS at a concentration of 1 mg / ml, and then further diluted with 1×PBS to a final concentration of 20 nmol / 100 μL injection solution. The soluble adjuvant stock solution (CpG) was prepared in horseshoe crab hemocyte extract (LAL) H2O, and then further diluted with 1×PBS to a final concentration of 20 nmol / 100 μL injection solution.

[0130] The immunizer was administered subcutaneously (SC) at a dose of 50 μL per side bilaterally into the tail base of female B6 mice. Booster doses were given approximately every two weeks. SC injection ensured optimal delivery of the vaccine into the lymph nodes via natural lymphatic drainage, and bi-weekly injections were determined to be optimal for the immune response.

[0131] [Table 8]

[0132] Splenocytes (1 × 10) 7 days after dose 3 6 Intracellular staining (ICS) assays for GzmB were performed on cells (1 per well) (Figures 7A and 7B). Cells were also stained for CD4, CD8, and CD3 as described in Table 9. ICS samples were activated overnight with 2 μg / ml of each peptide from the peptide pool listed in Table 10 (in the presence of brefeldin A and monensin).

[0133] [Table 9]

[0134] [Table 10]

[0135] As shown in Figures 4A, 4B, 5A, and 5B, ELISpot analysis for IFNγ was performed on splenocytes 7 days after doses 2 and 3. Splenocytes were analyzed using 0.2 × 10¹⁶ peptide pools listed in Table 10. 6Cells were activated with 1 cell / well and 2 μg / ml of each peptide. IFNy plates were stimulated overnight.

[0136] Table 10 lists 1×10⁶ peptides activated in a 2 μg / ml peptide pool. 6 Luminex analysis was performed on splenocytes 7 days after dose 3 at a cell / well rate (Figures 6A-C). Cells were stimulated overnight. The supernatant was tested with a mouse cytokine / chemokine magnetic bead kit for simultaneous quantification of analytes such as GM-CSF, IL2, TNFα, INFγ, and granzyme B (Table 11). As shown in Figures 7A and 7B, ELISpot analysis for granzyme B was performed exclusively on splenocytes 7 days after dose 3.

[0137] [Table 11]

[0138] Example 4. Efficacy of the V600E and V600K peptide combination vaccine. This experiment was conducted to evaluate the optimal concentrations of V600E and V600K 29mer peptides in the 2-peptide vaccine in mice. In addition, the study determined whether five extended dosing schedules of bi-weekly doses increased the immune response to the BRAF antigen.

[0139] Each of the nine groups of 130 mice was administered a vaccine containing the components listed in Tables 13-15. The administration to the mice in each group is shown in Table 12.

[0140] [Table 12]

[0141] Immunization was administered subcutaneously (SC) at a dose of 50 μL per side bilaterally into the tail base of female B6 mice on days 1, 14, 29, 42, and 55.

[0142] [Table 13]

[0143] [Table 14]

[0144] [Table 15]

[0145] The antigen-to-adjuvant ratios for the tested V600E and V600K combinations were 1:4 (2.5 nmol:10 nmol), 1:1 (10 nmol:10 nmol), 2:1 (20 nmol:10 nmol), and 2:1 (40 nmol:20 nmol).

[0146] Spleens and lungs were collected from immunized mice. Cells were counted and diluted to the precise concentrations required for each assay. Cells for the assays listed in Table 16 were stimulated with the peptide pools described in Table 17. Restimulated peptide pools were prepared by combining peptides from Table 17 in complete medium at a 2× concentration of 4 μg / ml per peptide.

[0147] [Table 16]

[0148] [Table 17]

[0149] 0.1 × 10 6 ELISpot IFNγ analysis was performed on spleen cells (Figures 9A-9C and 10A-10C) 7 days after doses 3 and 5, and on pulmonary resident lymphocytes (Figure 8) 7 days after dose 3, using individual cells / well and a peptide concentration of 2 μg / ml.

[0150] 0.1 × 10 6ELISpot GzmB analysis was performed on splenocytes 7 days after doses 3 and 5 with peptide concentrations of 2 μg / ml and individual cells / well (Figure 11).

[0151] 0.1 × 10 6 FluoroSpot IFNγ / TNFα / IL2 analysis was performed on spleen cells 7 days after doses 3 and 5, and on pulmonary resident lymphocytes 7 days after dose 3, using individual cells / well and a peptide concentration of 2 μg / ml (Figures 13A-13C).

[0152] Intracellular staining flow cytometry IFNγ / TNFα analysis was performed on spleen cells 7 days after doses 3 and 5, and on pulmonary resident lymphocytes 7 days after dose 3 (Figures 14A and 14B). The antibody panel used in this experiment is listed in Table 18.

[0153] [Table 18]

[0154] Intracellular staining flow cytometry GzmB / perforin analysis was performed on splenocytes 7 days after doses 3 and 5. The antibody panel used in this experiment is listed in the table.

[0155] [Table 19]

[0156] Luminex analysis for GzmB / IFNγ / TNFα / GM-CSF / IL2 / IL4 / IL10 / sFasL was performed on spleen cells 7 days after doses 3 and 5, and on pulmonary resident lymphocytes 7 days after dose 3 (Figures 12A-12C).

[0157] After three doses of V600K and V600E 29-mer peptides, a significant immune response could be detected with a dose of 5 nmol + 5 nmol antigen + 10 nmol adjuvant. The strongest response was observed with a dose of 20 nmol + 20 nmol antigen + 20 nmol adjuvant. However, after five doses, the best vaccine dose (20 nmol + 20 nmol antigen + 20 nmol adjuvant) produced a less effective response compared to the 5 nmol + 5 nmol antigen + 10 nmol adjuvant dose, and the strongest response was produced. In most assays, the 10 nmol + 10 nmol antigen + 10 nmol adjuvant dose did not show improvement over the 5 nmol + 5 nmol antigen + 10 nmol adjuvant dose.

[0158] Other Embodiments Various modifications and alterations of the compositions, methods, and uses of the present invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in relation to specific embodiments, it should be understood that the claimed invention should not be excessively limited to such specific embodiments. In fact, various modifications of the described forms for carrying out the present invention which are apparent to those skilled in the art are intended to be within the scope of the invention.

[0159] All publications, patents, and patent applications are incorporated herein by reference in the same way that each individual publication, patent, or patent application is incorporated specifically and individually by reference in the whole.

Claims

1. A compound containing an albumin-binding domain and a Braf peptide, or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the peptide is a 5-50 amino acid fragment of SEQ ID NO:

1.

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, wherein the peptide is a 10-40 amino acid fragment of SEQ ID NO:

1.

4. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein the peptide is a 10-30 amino acid fragment of SEQ ID NO:

1.

5. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein the Braf peptide is the 30-amino acid fragment of SEQ ID NO:

1.

6. The compound according to claim 4 or a pharmaceutically acceptable salt thereof, wherein the Braf peptide is the nine-amino acid fragment of SEQ ID NO:

1.

7. The compound according to claim 4 or a pharmaceutically acceptable salt thereof, wherein the Braf peptide is the 15-amino acid fragment of SEQ ID NO:

1.

8. The compound according to claim 4 or a pharmaceutically acceptable salt thereof, wherein the Braf peptide is the 29-amino acid fragment of SEQ ID NO:

1.

9. The compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, wherein the Braf peptide comprises a fragment of SEQ ID NO: 1 comprising one or more amino acid substitutions.

10. The compound according to claim 9 or a pharmaceutically acceptable salt thereof, comprising a fragment of SEQ ID NO: 1, wherein the Braf peptide includes an amino acid substitution with Val occupying amino acid position 600 from the N-terminus of SEQ ID NO:

1.

11. The compound according to claim 10, or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at Val occupying the 600th position of Sequence ID No. 1 is V600K.

12. The compound according to claim 11 or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at Val occupying position 600 of Sequence ID No. 1 is V600E.

13. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises the amino acid sequence (SEQ ID NO: 2) of EDLTVKIGDFGLATVKSRWSGSHQFEQLS or a fragment thereof.

14. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises the amino acid sequence (SEQ ID NO: 3) of EDLTVKIGDFGLATKKSRWSGSHQFEQLS or a fragment thereof.

15. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises the amino acid sequence (SEQ ID NO: 4) of EDLTVKIGDFGLATEKSRWSGSHQFEQLS or a fragment thereof.

16. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises the 9 or 10 amino acid fragment of SEQ ID NO: 3 or 4.

17. The compound according to claim 16 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises the amino acid sequence FGLATKKSR (SEQ ID NO: 61) or FGLATEKSR (SEQ ID NO: 62).

18. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises the 15-amino acid fragment of SEQ ID NO: 3 or 4.

19. The compound according to claim 18 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises the amino acid sequence GDFGLATKKSRWSGS (SEQ ID NO: 63) or GDFGLATEKSRWSGS (SEQ ID NO: 64).

20. The compound according to any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, wherein the peptide comprises an N-terminal modification.

21. The compound according to claim 20, or a pharmaceutically acceptable salt thereof, wherein the N-terminal modification is the addition of acetylcysteine.

22. The compound according to claim 21 or a pharmaceutically acceptable salt thereof, wherein the N-terminal modification is the addition of a desaminocysteine ​​homolog.

23. The compound according to claim 22 or a pharmaceutically acceptable salt thereof, wherein the desaminocysteine ​​homolog is 3-mercaptopropionic acid or mercaptoacetic acid.

24. The compound according to any one of claims 1 to 23, or a pharmaceutically acceptable salt thereof, wherein the albumin-binding domain contains a lipid.

25. The compound according to claim 24 or a pharmaceutically acceptable salt thereof, wherein the lipid is a diacyl lipid.

26. The compound according to claim 25 or a pharmaceutically acceptable salt thereof, wherein the diacyllipid comprises an acyl chain containing 12 to 30 hydrocarbon units, 14 to 25 hydrocarbon units, 16 to 20 hydrocarbon units, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hydrocarbon units.

27. The compound according to claim 24 or 25, or a pharmaceutically acceptable salt thereof, wherein the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

28. The peptide in question is the following lipid: 【Chemistry 1】 A compound according to any one of claims 1 to 27 or a pharmaceutically acceptable salt thereof, which is bonded to a salt thereof or linked by a linker.

29. The compound according to claim 28 or a pharmaceutically acceptable salt thereof, wherein the linker is selected from the group consisting of a hydrophilic polymer, a series of hydrophilic amino acids, polysaccharides, and oligonucleotides, or a combination thereof.

30. The compound according to claim 29 or a pharmaceutically acceptable salt thereof, wherein the linker contains an "N" polyethylene glycol unit and N is 24 to 50.

31. The compound according to claim 30 or a pharmaceutically acceptable salt thereof, wherein the linker comprises PEG24-amide-PEG24.

32. A method for inducing an immune response in a subject, comprising administering to the subject a compound according to any one of claims 1 to 31 or a pharmaceutically acceptable salt thereof.

33. The method according to claim 31, further comprising administering an adjuvant to the subject.

34. The method according to claim 32, wherein the compound or a pharmaceutically acceptable salt thereof is administered subcutaneously, intramuscularly, intravenously, or transmucosally.

35. The method according to any one of claims 32 to 34, wherein the subject is a mammal.

36. The method according to claim 35, wherein the subject is a human.

37. A compound comprising an albumin-binding domain and a Braf peptide or a pharmaceutically acceptable salt thereof for use in a method for inducing an immune response in a subject, comprising administering to the subject a compound according to any one of claims 1 to 31 or a pharmaceutically acceptable salt thereof.

38. The compound or a pharmaceutically acceptable salt thereof for use according to claim 37, further comprising administering an adjuvant to the subject.

39. The compound or a pharmaceutically acceptable salt thereof for use according to claim 37 or 38, formulated for subcutaneous, intramuscular, intravenous, or transmucosal administration.

40. The compound or a pharmaceutically acceptable salt thereof for use according to any one of claims 37 to 39, wherein the subject is a mammal.

41. The compound or a pharmaceutically acceptable salt thereof for use according to claim 40, wherein the subject is a human.

42. A pharmaceutical composition comprising a compound according to any one of claims 1 to 31 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

43. A kit comprising a compound according to any one of claims 1 to 31 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 42, and instructions for administration.

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