WO2024243540A1

WO2024243540A1 - Compositions containing braf peptide amphiphiles and methods of use thereof

Info

Publication number: WO2024243540A1
Application number: PCT/US2024/031061
Authority: WO
Inventors: Peter C. DEMUTH; Christopher M. HAQQ; Martin P. STEINBUCK; Pashtoon KASI
Original assignee: Elicio Therapeutics, Inc.; Cornell University
Priority date: 2023-05-25
Filing date: 2024-05-24
Publication date: 2024-11-28

Abstract

Disclosed herein are compounds including an albumin-binding domain and a Braf peptide, as well as pharmaceutically acceptable salts thereof. Furthermore, disclosed herein are methods for inducing an immune response in a subject, and methods of administering such compounds to induce an immune response in a subject.

Description

COMPOSITIONS CONTAINING BRAF PEPTIDE AMPHIPHILES AND METHODS OF USE THEREOF

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created May 23, 2024, is named 51026-060W03_Sequence_Listing_5_23_24.xml and is 46,541 bytes in size.

BACKGROUND OF THE INVENTION

Vaccines are used to stimulate an immune response in an individual to provide protection against and/or treatment for a particular disease. Some vaccines include an antigen to induce an immune response. Immune responses as a result of vaccination have made an enormous contribution 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 for preventing many infectious diseases by provoking an immune response in a subject. According to the World Health Organization, immunization currently prevents 2-3 million deaths every year across all age groups. The purpose of vaccination is to generate a strong and lasting immune response providing long-term protection against infection. However, many vaccines do not currently induce optimal immunity.

There remains a need to develop new and improved compositions and methods for inducing immune responses in a subject thereof.

SUMMARY OF THE INVENTION

In an aspect, the disclosure provides a compound including an albumin-binding domain and a Braf peptide, or a pharmaceutically acceptable salt thereof. In some embodiments, the peptide is a 5 to 50 amino acid (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) fragment of SEQ ID NO: 1 . In some embodiments, the peptide is 10 to 40 (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) amino acid fragment of SEQ ID NO: 1 . In some embodiments, the peptide is 10 to 30 (e.g., 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30) amino acid fragment of SEQ ID NO: 1 . In some embodiments, the Braf peptide is a 30 amino acid fragment of SEQ ID NO: 1 . In some embodiments, the Braf peptide is a 9 amino acid fragment of SEQ ID NO: 1 . In some embodiments, the Braf peptide is a 15 amino acid fragment of SEQ ID NO: 1 . In some embodiments, the Braf peptide a 29 amino acid fragment of SEQ ID NO: 1.

In some embodiments, the Braf peptide includes a fragment of SEQ ID NO: 1 including one or more amino acid substitutions. In some embodiments, the Braf peptide includes a fragment of SEQ ID NO: 1 , wherein the fragment includes an amino acid substitution at the Vai occupying amino acid position 600 from N-terminus of SEQ ID NO: 1 . In some embodiments, the amino acid substitution at the Vai occupying position 600 of SEQ ID NO: 1 is V600K. In some embodiments, the amino acid substitution at the Vai occupying position 600 of SEQ ID NO: 1 is V600E.

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

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 an 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 bonded or linked to the albumin-binding domain. In some embodiments, the C-terminus of the peptide is bonded or linked to the albumin-binding domain.

In some embodiments, the albumin-binding domain includes a lipid. In some embodiments, the lipid is a diacyl lipid. In some embodiments, the diacyl lipid includes acyl chains including 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 bonded or linked by a linker to the following lipid:

or a salt thereof. In some embodiments, the linker is selected from the group consisting of a hydrophilic polymer, a string of hydrophilic amino acids, a polysaccharide, and an oligonucleotide, or a combination thereof. In some embodiments, the linker includes "N" polyethylene glycol units, wherein N is between 24-50 (e.g., between 24 and 45, 24 and 40, 24 and 35, 24 and 30, 30 and 50, 35 and 50, 40 and 50, 45 and 50, or 30 and 40. In some embodiments, the linker includes PEG24-amido-PEG24.

In another aspect, the disclosure provides a method of inducing an immune response in a subject including administering any one of the compounds described herein or pharmaceutically acceptable salt thereof to the subject. In some embodiments, the method further includes 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. In some embodiments, the subject is a human. In another aspect, the disclosure provides a compound or pharmaceutically acceptable salt thereof including an albumin-binding domain and a Braf peptide for use in a method of inducing an immune response in a subject, wherein the method includes administering any one of the compounds described herein, or a pharmaceutically acceptable salt thereof, to the subject. In some embodiments, the method further includes 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.

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

In another aspect, the disclosure provides a pharmaceutical composition including any one of the compounds described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

In another aspect, the disclosure provides a kit including any one of the compounds described herein or a pharmaceutically acceptable salt thereof or the pharmaceutical composition described herein and instructions for administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing of an amphiphile (AMP) conjugated to a PEG-48 linker which is 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. The amino acids that differ between the human and heteroclitic sequences are marked with an asterisk.

FIG. 2A and FIG. 2B are graphs showing the splenocyte IFNy ELISpot responses of C57BL/6 mice that were administered a vaccine including a soluble or amphiphilic peptides as shown in FIG. 1 and were restimulated with either syngeneic or heteroclitic peptides after two doses (FIG. 2A) and three doses (FIG. 2B).

FIG. 3 is a graph showing the splenocyte IFNy ELISpot responses of Balb/c mice that were administered a vaccine including amphiphilic peptides as shown in FIG. 1 after 3 doses when coadministered with an adjuvant comprising an amphiphile conjugated to CpG7909.

FIG. 4A and FIG. 4B are graphs showing the splenocyte post-dose 2 IFNy co-culture ELISpot responses of C57BI6 mice that were administered a vaccine of a soluble (SOL) or amphiphile (AMP) V600E peptide (FIG. 4A) and V600K peptide (FIG. 4B) at a concentration of 20 nmol peptide and 10 nmol adjuvant.

FIG. 5A and FIG. 5B are graphs showing the splenocyte post-dose 3 IFNy co-culture ELISpot responses of C57BI6 mice that were administered a vaccine of a soluble (SOL) or amphiphile (AMP) V600E peptide (FIG. 5A) and V600K peptide (FIG. 5B) at a concentration of 20 nmol peptide and 10 nmol adjuvant.

FIG. 6A-FIG. 6C are graphs showing the concentration of Granzyme B, IN Fy, TNFa, GM-CSF, and IL2 from splenocytes 7 days after dose 3 for C57BL/6J mice that were administered a vaccine of a soluble (SOL) or amphiphile (AMP) V600E peptide (FIG. 6A), V600K peptide (FIG. 6B), or V600E peptide and V600K peptide (FIG. 6C) at a concentration of 20 nmol peptide and 10 nmol adjuvant. FIG. 7A and FIG. 7B are a series of graphs showing the splenocyte IFNy co-culture ELISpot analysis for Granzyme B post-dose 3 of C57BI6 mice that were administered a vaccine of a soluble (SOL) or amphiphile (AMP) V600E peptide (FIG. 7A) and V600K peptide (FIG. 7B) at a concentration of 20 nmol peptide and 10 nmol adjuvant.

FIG. 8 is a graph showing the lung lymphocyte post-dose 3 IFNy co-culture ELISpot responses of C57BI6 mice that were administered a vaccine of a soluble (SOL) or amphiphile (AMP) combination of V600E peptide and V600K peptide at a concentration of 20 nmol of each peptide and 20 nmol adjuvant.

FIG. 9A-FIG. 9C are graphs showing the splenocyte post-dose 3 IFNy co-culture ELISpot responses of C57BI6 mice that were administered a vaccine of a soluble (SOL) or amphiphile (AMP) V600E peptide (FIG. 9A), V600K peptide (FIG. 9B), and the combination of V600E peptide and V600K peptide (FIG. 9C) at a concentration of 5 nmol of each peptide and 10 nmol adjuvant.

FIG. 10A-FIG. 10C are graphs showing the splenocyte post-dose 5 IFNy co-culture ELISpot responses of C57BI6 mice that were administered a vaccine of a soluble (SOL) or amphiphile (AMP) V600E peptide (FIG. 10A), V600K peptide (FIG. 10B), and the combination of V600E peptide and V600K peptide (FIG. 10C) at a concentration of 5 nmol of each peptide and 10 nmol adjuvant.

FIG. 11 is a graph showing the splenocyte IFNy co-culture ELISpot analysis for Granzyme B post-dose 3 of C57BI6 mice that were administered a vaccine of a soluble (SOL) or amphiphile (AMP) V600E peptide and V600K peptide at a concentration of 5 nmol of each peptide and 10 nmol adjuvant.

FIG. 12A-FIG. 12C are graphs showing the concentration of Granzyme B, IFNy, TNFa, GM-CSF, and IL2 from splenocytes 7 days after dose 5 for C57BL/6J mice that were administered a vaccine of a soluble (SOL) or amphiphile (AMP) V600E peptide (FIG. 12A), V600K peptide (FIG. 12B), or V600E peptide and V600K peptide (FIG. 12C) at a concentration of 5 nmol of each peptide and 10 nmol adjuvant.

FIG. 13A-FIG. 13C are a series of graphs showing the cytokine FluoroSpot analysis for splenocytes 7 days after dose 5 for C57BL/6J mice that were administered a vaccine of a soluble (SOL) or amphiphile (AMP) V600E peptide (FIG. 13A), V600K peptide (FIG. 13B), or V600E peptide and V600K peptide (FIG. 13C) at a concentration of 5 nmol of each peptide and 10 nmol adjuvant.

FIG. 14A and FIG. 14B are graphs showing the percentage of cells having IFNy and TNFa, cytokines in CD4+ cells (FIG. 14A) and CD8+ cells (FIG. 14B) collected 7 days after dose 5 for C57BL/6J mice that were administered a vaccine of a soluble (SOL) or amphiphile (AMP) V600E peptide and V600K peptide at a concentration of 10 nmol of each peptide and 10 nmol adjuvant (FIG. 14A) or at a concentration of 20 nmol of each peptide and 20 nmol adjuvant (FIG. 14B).

Definitions

Terms used in the claims and specification are defined as set forth below unless otherwise specified.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, "about" will be understood by persons of ordinary skill and will vary to some extent depending on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill given the context in which it is used, "about" will mean up to plus or minus 10% of the particular value.

As used herein, the term "adjuvant" refers to a compound that, with a specific immunogen or antigen, will augment or otherwise alter or modify the resultant immune response. Modification of the immune response includes intensification or broadening the specificity of either or both antibody and cellular immune responses. Modification of the immune response can also mean decreasing or suppressing certain antigen-specific immune responses. In certain embodiments, the adjuvant is a cyclic dinucleotide.

"Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e. , a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified polypeptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes.

An "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (an amino acid sequence of a starting polypeptide) with a second, different "replacement" amino acid residue. An "amino acid insertion" refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, the present larger "peptide insertions," can be made, e.g., by insertion of about three to about five or even up to about ten, fifteen, or twenty amino acid residues. The inserted residue(s) may be naturally occurring or non-naturally occurring as disclosed above. An "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

As used herein, "amphiphile" or "amphiphilic" refers to a conjugate comprising a hydrophilic head group and a hydrophobic tail, thereby forming an amphiphilic conjugate. In some embodiments, an amphiphile conjugate comprises a peptide, and one or more hydrophobic lipid tails.

A polypeptide or amino acid sequence "derived from" a designated polypeptide or protein or a "polypeptide fragment" refers to the origin of the polypeptide. Preferably, the polypeptide or amino acid sequence which is derived or is a fragment of is from a particular sequence that has an amino acid sequence that is essentially identical to that sequence or a portion thereof, wherein the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the sequence. Polypeptides derived from or that are fragments of another polypeptide may have one or more mutations relative to the starting polypeptide, e.g., one or more amino acid residues which have been substituted with another amino acid residue or which has one or more amino acid residue insertions or deletions.

A polypeptide can comprise an amino acid sequence which is not naturally occurring. Such variants necessarily have less than 100% sequence identity or similarity with the starting molecule. In a preferred embodiment, the variant will have an amino acid sequence from about 75% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide, more preferably from about 80% to less than 100%, more preferably from about 85% to less than 100%, more preferably from about 90% to less than 100% (e.g., 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferably from about 95% to less than 100%, e.g., over the length of the variant molecule.

In one embodiment, there is one amino acid difference between a starting polypeptide sequence and the sequence derived therefrom. Identity or similarity with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) with the starting amino acid residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

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

As used herein, the term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect.

The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Amounts effective for this use will depend upon the severity of the disorder being treated and the general state of the patient's own immune system.

As used herein, "immune cell" is a cell of hematopoietic origin and that plays 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 particular embodiments, the immune cell is T cell.

As used herein, "immune response" refers to a response made by the immune system of an organism to a substance, which includes but is not limited to foreign or self proteins. Three general types of "immune response" include mucosal, humoral, and cellular immune responses. For example, the immune response can include the activation, expansion, and/or increased proliferation of an immune cell. An immune response may also include at least one of the following: cytokine production, T cell activation and/or proliferation, granzyme or perforin production, activation of antigen presenting cells or dendritic cells, antibody production, inflammation, developing immunity, developing hypersensitivity to an antigen, the response of antigen-specific lymphocytes to antigen, clearance of an infectious agent, and transplant or graft rejection.

The terms "inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to the stimulation of an immune response (i.e., either passive or adaptive) to a particular antigen (e.g., a peptide (e.g., the Braf peptide)). The term "induce" as used with respect to inducing complement dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) refer to the stimulation of particular direct cell killing mechanisms.

As used herein, a subject "in need of prevention," "in need of treatment," or "in need thereof," refers to one, who by the judgment of an appropriate medical practitioner (e.g., a doctor, a nurse, or a nurse practitioner 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 comprising an amphiphilic ligand conjugate).

The term "in vivo" refers to processes that occur in a living organism.

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

As used herein, the terms "linked," "operably linked," "fused," or "fusion," are used interchangeably. These terms refer to the joining together of two more elements or components or domains, by an appropriate means including chemical conjugation or recombinant DNA technology. Methods of chemical conjugation (e.g., using heterobifunctional crosslinking agents) are known in the art as are methods of recombinant DNA technology.

The term "lipid" refers to a biomolecule that is soluble in nonpolar solvents and insoluble in water. Lipids are often described as hydrophobic or amphiphilic molecules which allows them to form structures such as vesicles or membranes in aqueous environments. Lipids include fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids (including cholesterol), prenol lipids, saccharolipids, and polyketides. In some embodiments, the lipid suitable for the amphiphilic ligand conjugates of the disclosure binds to human serum albumin under physiological conditions. In some embodiments, the lipid suitable for the amphiphilic ligand conjugates of the disclosure inserts into a cell membrane under physiological conditions. In some embodiments, the lipid binds albumin and inserts into a cell membrane under physiological conditions. In some embodiments, the lipid is a diacyl lipid. In some embodiments, the diacyl lipid includes at least 12 carbons. In some embodiments, the diacyl lipid includes 12-30 hydrocarbon units, 14-25 hydrocarbon units, or 16-20 hydrocarbon units. In some embodiments, the diacyl lipid includes 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbons.

"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 , 1991 ; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985); and Cassol etal., 1992; Rossolini et al., Mai. Cell. Probes 8:91 -98, 1994). For arginine and leucine, modifications at the second base can also be conservative. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene. Polynucleotides of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide can also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms. In some embodiments, the polypeptides of the invention are encoded by a nucleotide sequence. Nucleotide sequences of the invention can be useful for a number of applications, including: cloning, gene therapy, protein expression and purification, mutation introduction, DNA vaccination of a host in need thereof, antibody generation for, e.g., passive immunization, PCR, primer and probe generation, and the like.

As used herein, "parenteral administration," "administered parenterally," and other grammatically equivalent phrases, refer to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid and intrasternal injection and infusion.

As generally used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benef it/risk ratio.

The term “pharmaceutically acceptable salt,” as used herein, means any pharmaceutically acceptable salt of a conjugate, oligonucleotide, or peptide disclosed herein. Pharmaceutically acceptable salts of any of the compounds and nucleic acid sequences described herein may include those that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and are commensurate with 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 in Pharmaceutical Salts: Properties, Selection, and Use (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting a free base group with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. References to the compounds, nucleic acids, conjugates, oligonucleotides, or polypeptides in the claims and elsewhere herein optionally include pharmaceutically acceptable salts thereof unless otherwise indicated or not applicable.

As used herein, the term “physiological conditions” refers to the in vivo condition of a subject. In some embodiments, physiological condition refers to a neutral pH (e.g., pH between 6-8).

As used herein, the term “peptide” refers to a polymer having 30 or fewer amino acid residues. "Polypeptide," "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

As used herein, the term "subject" or "mammal" or "patient" includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat a subject with a disease or condition. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, mice, horses, pigs, cows, chickens, amphibians, reptiles, etc.

The term "sufficient amount" or "amount sufficient to" means an amount sufficient to produce a desired effect, e.g., an amount sufficient to reduce the diameter of a tumor.

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 the cell surface. There are several subsets of T cells, including, but not limited to, T helper cells ( a.k.a. TH cells or CD4⁺ T cells) and subtypes, including TH, TH2, TH3, TH17, TH9, and TFH cells, cytotoxic T cells (i.e., Tc cells, CD8⁺ T cells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memory T cells and subtypes, including central memory T cells (TCM cells), effector memory T cells (TEM and TEMRA cells), and resident memory T cells (TRM cells), regulatory T cells (a.k.a. Treg cells or suppressor T cells) and subtypes, including CD4⁺ FOXP3+ T_reg cells, CD4+FOXP3- Treg cells, Tr1 cells, Th3 cells, and T_reg17 cells, natural killer T cells (a.k.a. NKT cells), mucosal associated invariant T cells (MAITs), and gamma delta T cells (yb T cells), including Vy9/V52 T cells. Any one or more of the aforementioned or unmentioned T cells may be the target cell type for a method of use of the invention.

The terms "treat," "treating," and "treatment," as used herein, refer to therapeutic or preventative measures described herein. The methods of "treatment" employ administration to a subject, in need of such treatment, a peptide and an albumin-binding domain of the present disclosure. In some embodiments, Braf peptide conjugated to an albumin-binding domain is administered to a subject in need of an enhanced immune response against a particular antigen or a subject who ultimately may acquire such a disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

As used herein, "vaccine" refers to a formulation which contains an amphiphilic construct described herein, optionally combined with an adjuvant, which is in a form that is capable of being administered to a vertebrate and which induces a protective immune response sufficient to induce immunity to prevent and/or ameliorate a disease or condition and/or to reduce at least one symptom of a disease or condition. Typically, the vaccine comprises a conventional saline or buffered aqueous solution medium in which a composition as described herein is suspended or dissolved. In this form, a composition as described herein is used to prevent, ameliorate, or otherwise treat an infection or disease. Upon introduction into a host, the vaccine provokes an immune response including, but not limited to, the inducing a protective immune response to induce immunity to prevent and/or ameliorate a disease or condition and/or to reduce at least one symptom of a disease or condition.

DETAILED DESCRIPTION

Peptides

Described herein are compounds including a peptide, wherein the peptide is a Braf peptide. The peptide is conjugate to an albumin-binding domain optionally by way of a linker.

In some embodiments, the peptide is a fragment of a Braf polypeptide having the amino acid sequence of:

MAALSGGGGGGAEPGQALFNGDMEPEAGAGAGAAASSAADPAIPEEVWNIKQMIKLT QEHIEALLDKFGGEHNPPSIYLEAYEEYTSKLDALQQREQQLLESLGNGTDFSVSSSAS MDTVTSSSSSSLSVLPSSLSVFQNPTDVARSNPKSPQKPIVRVFLPNKQRTVVPARCGV TVRDSLKKALMMRGLIPECCAVYRIQDGEKKPIGWDTDISWLTGEELHVEVLENVPLTTH NFVRKTFFTLAFCDFCRKLLFQGFRCQTCGYKFHQRCSTEVPLMCVNYDQLDLLFVSKF FEHHPIPQEEASLAETALTSGSSPSAPASDSIGPQILTSPSPSKSIPIPQPFRPADEDHRN QFGQRDRSSSAPNVHINTIEPVNIDDLIRDQGFRGDGGSTTGLSATPPASLPGSLTNVKA LQKSPGPQRERKSSSSSEDRNRMKTLGRRDSSDDWEIPDGQITVGQRIGSGSFGTVYK GKWHGDVAVKMLNVTAPTPQQLQAFKNEVGVLRKTRHVNILLFMGYSTKPQLAIVTQW CEGSSLYHHLHIIETKFEMIKLIDIARQTAQGMDYLHAKSIIHRDLKSNNIFLHEDLTVKIGD FGLATVKSRWSGSHQFEQLSGSILWMAPEVIRMQDKNPYSFQSDVYAFGIVLYELMTG QLPYSNINNRDQIIFMVGRGYLSPDLSKVRSNCPKAMKRLMAECLKKKRDERPLFPQILA SIELLARSLPKIHRSASEPSLNRAGFQTEDFSLYACASPKTPIQAGGYGAFPVH (SEQ ID NO: 1 ).

The Braf peptide may be a 8 to 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 fragment of SEQ ID NO: 1 . For example, the Braf peptide may be a 9 amino acid fragment of SEQ ID NO: 1 . In some embodiments, the Braf peptide is a 15 amino acid fragment of SEQ ID NO: 1 . In some embodiments, the Braf peptide a 29 amino acid fragment of SEQ ID NO: 1. The Braf peptide may include a fragment of SEQ ID NO: 1 comprising one or more amino acid substitutions. In some embodiments, the Braf peptide comprises a fragment of SEQ ID NO: 1 , wherein the fragment comprises an amino acid substitution at the Vai occupying amino acid position 600 from N- terminus of SEQ ID NO: 1 . For example, the Braf peptide may include an amino acid substitution at position 600 of SEQ ID NO: 1 wherein the Vai residue is substituted for a Lys residue (V600K) or wherein the Vai residue is substituted for a Glu residue (V600E).

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

The peptide may consist of or comprise the amino acid sequence of EDLTVKIGDFGLATVKSRWSGSHQFEQLS (SEQ ID NO: 2) or a fragment thereof and have a length of 5 to 60 (e.g. 5, 6, 7, 8 9, 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) amino acids. The peptide may consist of or comprise an amino acid sequence of EDLTVKIGDFGLATKKSRWSGSHQFEQLS (SEQ ID NO: 3) or a fragment thereof and have a length of 5 to 60 (e.g. 5, 6, 7, 8 9, 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) amino acids. The peptide may consist of or comprise an amino acid sequence of EDLTVKIGDFGLATEKSRWSGSHQFEQLS (SEQ ID NO: 4) or a fragment thereof and have a length of 5 to 60 (e.g. 5, 6, 7, 8 9, 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) amino acids.

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

In some embodiments, the peptide comprises an N-terminal modification. In some embodiments, the N-terminal modification is the addition of a cysteine. In some embodiments, the N-terminal modification is the addition of an 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 bonded or linked to the albumin-binding domain. In some embodiments, the C-terminus of the peptide is bonded or linked to the albumin-binding domain.

Amphiphilic Peptides

Amphiphilic peptides include a peptide and conjugated to an albumin-binding domain, e.g., a lipid. In some embodiments, the amphiphilic peptide includes a Braf peptide conjugated to an albumin-binding domain, e.g., a lipid, optionally by way of a linker.

Lipids

The compounds described herein include herein a Braf peptide that are conjugated to an albumin-binding domain. In some embodiments, the albumin-binding domain is a lipid. The lipid can be linear, branched, or cyclic.

Examples of preferred lipids include, but are not limited to, fatty acids with aliphatic tails of 3-30 carbons including, but not limited to, linear unsaturated and saturated fatty acids, branched saturated and unsaturated fatty acids, and fatty acids 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, Lipid A or combinations thereof.

In certain embodiments, the lipid is a diacyl lipid or two-tailed lipid. In some embodiments, the tails in the diacyl lipid contain from about 12 to about 30 carbons (e.g., 13 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29). In some embodiments the tails in the diacyl lipid contain about 14 to about 25 carbons (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24). In some embodiments, the tails of the diacyl lipid contain from about 16 to about 20 carbons (e.g., 17, 18, or 19). In some embodiments, the diacyl lipid comprises 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbons.

The carbon tails of the diacyl lipid can be saturated, unsaturated, or combinations thereof. The tails can be coupled to the head group via ester bond linkages, amide bond linkages, thioester bond linkages, or combinations thereof. In a particular embodiment, the diacyl lipids are phosphate lipids, glycolipids, sphingolipids, or combinations thereof. In some embodiments, the lipid is 1 ,2-distearoy!-sr?-g!ycero-3-phosphoethanolamine (DSPE).

In some embodiments, the Brat peptide bonded or linked by a linker to the following lipid:

or a salt thereof.

The Braf peptide may be directly bonded to the lipid. Alternatively, the Braf peptide may be linked to the lipid through a linker.

Reference to lipids herein, as well as amphiphiles including the lipid, is to be understood as including pharmaceutically acceptable salts thereof.

Linkers

In some embodiments, the compound includes a Braf peptide linked to an albumin-binding domain, e.g., a lipid, by a linker. The linker may be a hydrophilic polymer, a string of hydrophilic amino acids, a polysaccharide, and an oligonucleotide, or a combination thereof. The linker may reduce or prevent the ability of the albumin-binding domain to insert into the plasma membrane of cells, such as cells in the tissue adjacent to the injection site. The linker can also reduce or prevent the ability of the amphiphilic peptide sequence from non-specifically associating with extracellular matrix proteins at the site of administration. For the amphiphilic Braf peptide to be trafficked efficiently to the lymph node, it should remain soluble. A polar block linker may be included between the Braf peptide and the albuminbinding domain to which it is conjugated to increase solubility of the amphiphilic Braf peptide.

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

Suitable linkers include, but are not limited to, oligonucleotides such as those discussed above, including a string of nucleic acids, a hydrophilic polymer including but not limited to polyethylene glycol) (MW: 500 Da to 20,000 Da), polyacrylamide (MW: 500 Da to 20,000 Da), polyacrylic acid; a string of hydrophilic amino acids such as serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or combinations thereof; polysaccharides, including but not limited to, dextran (MW: 1 ,000 Da to 2,000,000 Da), or combinations thereof. The hydrophobic albuminbinding domain and the linker/peptide (e.g., the Braf peptide) are covalently linked. The covalent bond may be a non-cleavable linkage or a cleavable linkage. The non-cleavable linkage can include an amide bond or phosphate bond, and the cleavable linkage can include a disulfide bond, acid-cleavable linkage, ester bond, anhydride bond, biodegradable bond, or enzyme-cleavable linkage.

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 includes a Braf peptide and a hydrophobic albumin-binding domain linked by a polyethylene glycol (PEG) molecule or a derivative or analog thereof.

In some embodiments, compounds described herein contain a Braf peptide linked to PEG which is in turn linked to a hydrophobic albumin-binding domain, e.g., a lipid. The precise number of PEG units depends on the albumin-binding domain and the cargo, however, typically, a linker can have between about 1 and 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, 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 between about 20 and about 80, between about 30 and about 70, or between about 40 and about 60 PEG units. In some embodiments, the number of PEG units is between 24 and 50 units (e.g., between 24 and 45, 24 and 40, 24 and 35, 24 and 30, 30 and 50, 35 and 50, 40 and 50, and 45 and 50 units). In some embodiments, the linker has between about 45 and 55 PEG units. For example, in some embodiments, the linker has 48 PEG units. In some embodiments, the linker includes a PEG4-amido-PEG4 linker.

As discussed above, in some embodiments, the linker is an oligonucleotide which includes a string of nucleic acids. In some embodiments, the compounds described herein include a Braf peptide linked to a string of nucleic acids, which is in turn linked to a hydrophobic albumin-binding domain, e.g., a lipid. The linker can be any sequence, for example, the sequence of the oligonucleotide can be a random sequence, or a sequence specifically chosen for its molecular or biochemical properties (e.g., highly polar). In some embodiments, the linker includes 20 one or more series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analog thereof. In some embodiments, the linker consists of a series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analog thereof.

In some embodiments, the string of nucleic acids includes between 1 and 50 nucleic acid residues. In some embodiments, the string of nucleic acids includes between 5 and 30 nucleic acid residues. In some embodiments, the linker includes one or more guanines, for example between 1 -10 guanines.

In some embodiments, the linker is an oligonucleotide that includes a string of amino acids. In some embodiments, the amphiphilic Braf peptide which is in turn linked to a hydrophobic albumin-binding domain, e.g., a lipid. The linker can have any amino acid sequence, for example, the sequence of the oligonucleotide can be a random sequence, or a sequence chosen for its molecular or biochemical properties (e.g., high flexibility). In some embodiments, the linker includes a series of glycine residue to form a polyglycine linker. In some embodiments, the linker includes an amino acid sequence of (Gly)n, wherein n may be between 2 and 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), GGSGGSGGS (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).

Methods of Conjugation

Described herein are compounds including a Braf peptide and an albumin-binding domain.

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

The Braf peptide and the albumin-binding domain may be bonded or linked to a linker. In some embodiments, the linker includes a functional group. In some embodiments, the functional group is capable of conjugating to a peptide. For example, the Braf peptide may be bound to a linker, wherein the linker is modified with a functional group. In some embodiments, the albumin-binding domain may be linked to a linker, wherein the linker is modified with a functional group. In some embodiments, the linker may be a PEG linker.

In some embodiments, the Braf peptide is conjugated to the albumin-binding domain and/or linker by way of a reaction between a dithio group and a free thiol group.

Adjuvants

In some embodiments, a pharmaceutical composition described herein may be administered with one or more adjuvants. An adjuvant refers to a substance that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to the peptide. An adjuvant may be administered to a subject before, in combination with, or after administration of the compositions described herein. In some embodiments, an additional adjuvant is administered to the subject in combination with the Braf peptide conjugated to an albumin-binding domain described herein. In some embodiments, an adjuvant may be conjugated to an albumin-binding domain, e.g., a lipid. The adjuvant may be without limitation 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 such as QS21 (a glycolipid that elutes in the 21st peak with HPLC fractionation; Antigenics, Inc., Worcester, Mass.); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA), Flt3 ligand, Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.), ISCOMS (immunostimulating complexes which contain mixed saponins, lipids and form virus-sized particles with pores that can hold an antigen; CSL, Melbourne, Australia), Pam3Cys, SB-AS4 (SmithKline Beecham adjuvant system #4 which contains alum and MPL; SBB, Belgium), non-ionic block copolymers that form micelles such as CRL 1005 (these contain a linear chain of hydrophobic polyoxypropylene flanked by chains of polyoxyethylene, Vaxcel, Inc., Norcross, Ga.), and Montanide IMS (e.g., IMS1312, water-based nanoparticles combined with a soluble immunostimulant, Seppic), and CDNs (cyclic dinucleotides).

Adjuvants may be toll-like receptor (TLR) ligands. Adjuvants that act through TLR3 include without limitation double-stranded RNA. Adjuvants that act through TLR4 include without limitation derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPLA; Ribi ImmunoChem Research, Inc., Hamilton, Mont.) and muramyl dipeptide (MDP; Ribi) andthreonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland). Adjuvants that act through TLR5 include without limitation flagellin. Adjuvants that act through TLR7 and/or TLR8 include single-stranded RNA, oligoribonucleotides (ORN), synthetic low molecular weight compounds such as imidazoquinolinamines (e.g., imiquimod (R-837), resiquimod (R-848)). Adjuvants 5 acting through TLR9 include DNA of viral or bacterial origin, or synthetic oligodeoxynucleotides (ODN), such as CpG ODN. For example, the CpG ODN may have a sequence of TCGTCGTTTTGTCGTTTTGTCGTT-3’ (SEQ ID NO: 5), 5’-TGACTGTGAACGTTCGAGATGA-3’ (SEQ ID NO: 6), or 5’- TCGTCGTTTTCGGCGCGCGCCG-3’ (SEQ ID NO: 7). The linkages of the CpG may be all phosphorothioate linkages.

Another adjuvant class is phosphorothioate containing molecules such as phosphorothioate nucleotide analogs and nucleic acids containing phosphorothioate backbone linkages.

Pharmaceutical Compositions

Described herein are pharmaceutical compositions of the disclosure including a Braf peptide and conjugated to an albumin-binding domain. In addition to a therapeutic amount of the Braf peptide and conjugated to an albumin-binding domain described herein, the pharmaceutical compositions may contain a pharmaceutically acceptable carrier or excipient, which can be formulated by methods known to those skilled in the art. Pharmaceutically acceptable salts of the components are also included, as described herein.

Acceptable carriers and excipients in the pharmaceutical compositions of the Braf peptide conjugated to an albumin-binding domain described herein are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material(s) are for subcutaneous (s.c.) and/or intravenous (i.v.) administration. In some embodiments, administration is by inhalation or intranasal administration. In some embodiments, the formulation material(s) intraperitoneal, topical, or oral administration. In some embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In some embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, methionine, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCI, citrates, HEPES, TAE, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, sucrose, mannose or dextran); proteins (such as human serum albumin, gelatin, dextran, and immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as hexamethonium chloride, octadecyldimethylbenzyl ammonium chloride, resorcinol, and benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company (1995). In some embodiments, the optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In some embodiments, such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the amphiphilic conjugate.

In some embodiments, the primary vehicle or carrier in a pharmaceutical composition, including the Braf peptide conjugated to an albumin-binding domain described herein can be either aqueous or non-aqueous in nature. For example, in some embodiments, a suitable vehicle or carrier can be water for injection, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In some embodiments, the saline includes isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In some embodiments, pharmaceutical compositions include Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute therefore. In some embodiments, a composition including the Braf peptide conjugated to an albumin-binding domain described herein can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, in some embodiments, the composition including the Braf peptide conjugated to an albuminbinding domain described herein can be formulated as a lyophilizate using appropriate excipients such as sucrose.

In some embodiments, the pharmaceutical composition may be selected for parenteral delivery. The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art.

In some embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In some embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. In some embodiments, when parenteral administration is contemplated, a therapeutic composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution including the Braf peptide conjugated to an albumin-binding domain described herein in a pharmaceutically acceptable vehicle. In some embodiments, a vehicle for parenteral injection is sterile distilled water in which a Braf peptide conjugated to an albumin-binding domain described herein is formulated as a sterile, isotonic solution, properly preserved. In some embodiments, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection. In some embodiments, hyaluronic acid can also be used, and can have the effect of promoting sustained duration in the circulation. In some embodiments, implantable drug delivery devices can be used to introduce the desired molecule.

The pharmaceutical composition may be administered in therapeutically effective amount such as to induce an immune response. The therapeutically effective amount of the Braf peptide conjugated to an albumin-binding domain described herein included in the pharmaceutical preparations may be determined by one of skill in art, such that the dosage (e.g., a dose within the range of 0.01 -100 mg/kg of body weight) induces an immune response in the subject.

Vectors may be used as in vivo nucleic acid delivery vehicle include, but are not limited to, retroviral vectors, adenoviral vectors, poxviral vectors (e.g., vaccinia viral vectors, such as Modified Vaccinia Ankara (MVA)), adeno-associated viral vectors, and alphaviral vectors. In some embodiments, a vector can include internal ribosome entry site (IRES) that allows the expression of peptides described herein. Other vehicles and methods for nucleic acid delivery are described in US Patent Nos. 5,972,707, 5,697,901 , and 6,261 ,554, each of which is incorporated by reference herein in its entirety. Other methods of producing pharmaceutical compositions are described in, e.g., US Patent Nos. 5,478,925, 8,603,778, 7,662,367, and 7,892,558, all of which are incorporated by reference herein in their entireties.

In some embodiments, a pharmaceutical composition described herein may be administered with one or more adjuvants.

Routes, Dosage, and Timing of Administration

Pharmaceutical compositions of the disclosure that contain the Braf peptide conjugated to an albumin-binding domain described herein as the therapeutic agent may 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 of administering therapeutic proteins are known in the art. See, for example, US Patent Nos. 6,174,529, 6,613,332, 8,518,869, 7,402,155, and 6,591 ,129, and US Patent Application Publication Nos. US20140051634, WO1993000077, and US20110184145, the disclosures of which are incorporated by reference in their entireties. One or more of these methods may be used to administer a pharmaceutical composition of the invention that contains a Braf peptide conjugated to an albumin-binding domain. For injectable formulations, various effective pharmaceutical carriers are known in the art. See, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott 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 compositions of the invention depends on factors including the route of administration and the physical characteristics, e.g., age, weight, general health, of the subject. Typically, the amount of a Braf peptide conjugated to an albumin-binding domain described herein contained within a single dose may be an amount that effectively induces an immune response in the subject without inducing significant toxicity. A pharmaceutical composition of the invention may include a dosage of a Braf peptide conjugated to an albumin-binding domain described herein 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 a more specific embodiment, about 0.1 to about 100 mg. The dosage may be adapted by the clinician in accordance with the different parameters of the subject.

Pharmaceutical compositions of the invention that contain a Braf peptide conjugated to an albumin-binding domain may be administered to a subject in need thereof, for example, one or more times (e.g., 1 -10 times or more) daily, weekly, monthly, biannually, annually, or as medically necessary.

Methods of Inducing an Immune Response

The disclosure provides methods of inducing an immune response against the Braf peptide in a subject. The method includes administering any one of the compounds described herein to the subject.

In some embodiments, the disclosure provides a method of inducing an immune response against the Braf peptide in subject by administering any one of the Braf peptide conjugated to an albuminbinding domain to the subject and further administering an adjuvant to the subject. In some embodiments, the Braf peptide conjugated to an albumin-binding domain may be administered without one or more additional adjuvants.

In some embodiments, the method includes administering to the subject a therapeutically effective amount of the Braf peptide conjugated to an albumin-binding domain 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.

In some embodiments, one or more of the components administered is a pharmaceutically acceptable salt of the indicated component, as described herein.

In some embodiments, the disclosure provides a method of inducing an immune response against the peptide in a subject by administering any one of the compounds or pharmaceutically acceptable salts described herein subcutaneously to the subject. In some embodiments, the disclosure provides a method of inducing an immune response against the peptide in a subject by administering the peptide intramuscularly, subcutaneously, intravenously, intraperitoneally, topically, orally/buccally, sublingually, transmucosally, intranasally, or by inhalation to the subject.

In some embodiments, the subject is a mammal. For the example, the subject may be a human. Kits

A kit can include the Brat peptide conjugated to an albumin-binding domains disclosed herein and instructions for use. The kits 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 ingredients well known in the art. In some embodiments, the kits further include an adjuvant. Accordingly, 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 adjuvant are in the same vial. In some embodiments, the Braf peptide and the adjuvant are in separate vials.

The container can include at least one vial, well, test tube, flask, bottle, syringe, or other container means, into which the Braf peptide conjugated to an albumin-binding domain and in some instances, suitably aliquoted. When an additional component is provided, the kit can contain additional containers into which this compound may be placed. The kits can also include a means for containing the Braf peptide conjugated to an albumin-binding domain and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Containers and/or kits can include labeling with instructions for use and/or warnings.

In some embodiments, the disclosure provides a kit including a medicament including a composition including a Braf peptide conjugated to an albumin-binding domain, an optional pharmaceutically acceptable carrier, and a package insert including instructions for administration of the medicament alone or in combination with a composition including an adjuvant and an optional pharmaceutically acceptable carrier, for treating, delaying progression of, or preventing a disease or condition, wherein the Braf peptide conjugated to an albumin-binding domain optionally includes a linker.

In some embodiments, the disclosure provides a kit including a container including a composition including a Braf peptide conjugated to an albumin-binding domain, an optional pharmaceutically acceptable carrier, and a package insert including instructions for administration of composition vaccine in a subject, wherein the Braf peptide is conjugate to an albumin-binding domain and optionally includes a linker. In some embodiments, the kit further includes an adjuvant and instructions for administration of the adjuvant.

In some embodiments of the kits, one of more of the components of the kits is a pharmaceutically acceptable salt of the component as described herein.

EXAMPLES

The following examples, which are intended to illustrate, rather than limit, the disclosure, are put forth to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated. The examples are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their invention. Example 1. Ability of Braf V600E peptides to elicit an immune response in C57BL/6J mice

The purpose of this experiment was to establish if immune responses can be elicited with AMP- conjugated Braf peptides.

5 groups of 10 C57BL/6J mice each were administered a vaccine including the components of Table 2. 5 mice in each group were taken down after two doses and the remaining 5 mice were taken down after three doses as described in Table 1 .

Table 1. Summary of Vaccine Administration in Mice

The amount of peptide-antigen used was 5 nmol per injection. Because of low solubility of some AMP-peptides, all AMP-peptide stocks were co-solubilized with AMP-CpG. To achieve this, lyophilized AMP-peptides were dissolved in 50% t-butanol first, followed by the addition of 1 :1 molar ratio of AMP- CpG and subsequent lyophilization. The obtained powder was then resuspended in 1X PBS to a concentration of 1 mg/ml. The vaccine components are described in Table 2. AMP-vaccine stocks were further diluted to their final concentrations using 1X PBS such that each injection contained 5 nmol AMP- antigen and 5 nmol AMP-adjuvant.

Soluble peptide stock solutions were prepared in 1 .1 X PBS at a concentration of 0.5 and 0.45 mg/ml, respectively and further diluted with 1X PBS such that the final concentration was 5 nmol/100 pL injection. Soluble adjuvant stock solutions (CpG) were prepared in limulus amebocyte lysate (LAL) H2O such that the final concentration was 5 nmol/100 pL injection.

The immunizations were administered subcutaneously (SC) into the tail base of female B6 mice, bilaterally with 50 pL per side. A booster dose was given at roughly 2-week intervals. SC injections ensured that the vaccine was optimally delivered into lymph nodes via natural lymph drainage, and biweekly injections were determined to be optimal in immune response.

Table 2. Vaccine Components

NMS - N-Methylsuccinimide

ELISpot analysis for IFNy was performed on splenocytes 7 days after dose 2, with 0.2x10⁶ cells/well and 2 pg/ml of each peptide, and after dose 3, with 0.2x10⁶ cells/well and 2 pg/ml of each peptide. Splenocytes were activated with the Braf peptides described in Table . IFNy plates were stimulated overnight. The results of this analysis are shown in FIG. 2A and FIG. 2B.

Table 1. Re-Stimulation Peptides

Stim #1 = syngeneic stim; Stim #2 = heteroclitic stim

Immunization with AMP-conjugated, but not with soluble, Braf V600E 29mer elicited a significant immune response in mice after dose 2, which was further potentiated after administration of a third dose. This immune response was not detected with a 15mer version of the AMP-peptide.

Example 2. Ability of Braf V600E peptides to elicit an immune response in Balb/c mice

The purpose of this experiment was to establish if immune responses could be elicited with any of the AMP-Braf peptides in Balb/c mice that express MHC haplotype d.

4 groups of 10 mice each were administered a vaccine including the components of Table 5. 5 mice in each group were taken down after two doses and the remaining 5 mice were taken down after three doses as shown in Table 4. Table 4. Summary of Vaccine Administration in Mice

The amount of peptide-antigen used was 5 nmol per injection. Because of low solubility of some AMP-peptides, all AMP-peptide stocks were co-solubilized with AMP-CpG. To achieve this, lyophilized AMP-peptides were dissolved in 50% t-butanol first, followed by the addition of 1 :1 molar ratio of AMP- CpG and subsequent lyophilization. The obtained powder was then resuspended in 1 X PBS to a concentration of 1 mg/ml. The vaccine components are described in Table 5. AMP-vaccine stocks were further diluted to their final concentrations using 1 X PBS such that each injection contained 5 nmol AMP- antigen and 5 nmol AMP-adjuvant.

Table 5. Vaccine Components

ELISpot analysis for IFNy was performed on splenocytes 7 days after dose 3, with 0.2x10⁶ cells/well and 2 pg/ml of each peptide. Splenocytes were activated with the Braf peptides described in Table . IFNy plates were stimulated overnight. The results of this analysis are shown in FIG. 3. Table 6. Re-Stimulation Peptides

Stim #1 = syngeneic stim; Stim #2 = heteroclitic stim Immunization with AMP-conjugated Brat V600E 29mer elicited a significant immune response in

Balb/c mice after dose 3. This immune response was not detected with 9mer or 15mer version of the AMP-peptide.

Example 3. Effects of a combination Braf vaccine containing both V600E and V600K peptides The purpose of this experiment was to evaluate a combination Braf vaccine that contains both the

29-mer for the V600E and V600K peptide sequences. This experiment was designed to determine if there was immunodominance of one peptide over the other and if epitopes were promiscuous among the two mutations.

7 groups of mice were each administered a vaccine including the components of Table 8. A set of mice in each group were taken down after two doses and the remaining mice were taken down after three doses as shown in Table 7.

Table 7. Summary of Vaccine Administration in Mice

Previous dose finding studies have determined that the antigen to adjuvant ratio is optimal at 2:1 . Therefore, 20 nmol of antigen and 10 nmol of AMP-CpG were used per injection. Because of low solubility of some AMP-peptides, all AMP-peptide stocks were co-solubilized with AMP-CpG. To achieve this, lyophilized AMP-peptides were dissolved in 50% t-butanol first, followed by the addition of 2:1 molar ratio of AMP-CpG and subsequent lyophilization. The obtained powder was then resuspended in 1X PBS to a concentration of 1 mg/ml. The vaccine components are described in Table 8. AMP-vaccine stocks were further diluted to their final concentrations using 1 X PBS such that each injection contained 20 nmol AMP-antigen and 20 nmol AMP-adjuvant.

Soluble peptides were prepared in 1 .1 X PBS at a concentration of 1 mg/ml and further diluted with 1 X PBS such that the final concentration of the soluble peptide was 20 nmol/100 pL injection. The soluble adjuvant solutions (CpG) were prepared in limulus amebocyte lysate (LAL) H2O and further diluted with 1 X PBS such that the final concentration was 20 nmol/100 pL injection.

Table 8. Vaccine Components

An Intracellular Stain (ICS) assay for GzmB was performed on splenocytes (1 x10⁶ cells/well) 7 days post dose 3 (FIGS. 7A and 7B). Cells were also stained for CD4, CD8 and CD3 as described in Table 9 ICS samples were activated overnight (in the presence of Brefeldin A and Monensin) with peptide pools listed in Table 10 with 2 pg/ml of each peptide.

Table 9. Antibodies used for ICS

Table 10. Re-Stimulation Peptides

ELISpot analysis for IFNy was performed on splenocytes 7 days post dose 2 and 3, as shown in FIGS. 4A, 4B, 5A and 5B. Splenocytes were activated with peptide pools listed in Table 10 with 0.2x10⁶ cells/well and 2 pg/ml of each peptide. IFNy plates were stimulated overnight.

Luminex analysis was conducted on splenocytes 7 days post dose 3 (FIGS. 6A-C) with 1 x10⁶ cells/well which were activated with 2 pg/ml of peptide pools listed in Table 10. The cells were stimulated overnight. Supernatant was tested with mouse cytokine/chemokine magnetic bead kit (Table 11 ) for the simultaneous quantification of the following analytes: GM-CSF, IL2, TNFa, I NFy, Granzyme B. ELISpot analysis for Granzyme B was performed on splenocytes 7 days post dose 3 only, as shown in FIG. 7A and FIG. 7B.

Table 11. Luminex Kit

Example 4. Effects of V600E and V600K peptide combination vaccines

This experiment was performed to evaluate the optimal concentrations of the V600E and V600K 29mer peptides in a 2-peptide vaccine in mice. Additionally, the study determined if an extended dose regimen of 5 bi-weekly doses would increase the immune response against the BRAF antigens.

130 mice in 9 groups were each administered a vaccine including the components of Tables 13- 15. The mice in each group were dosed as shown in Table 12.

Table 12. Summary of Vaccine Administration in Mice

The immunizations were administered subcutaneously (SC) into the tail base of female B6 mice, bilaterally with 50 pL per side on Days 1 , 14, 29, 42, and 55. Table 13. Vaccine Components

Table 14. Summary of AMP-vaccine components

Table 15. Summary of SOL-vaccine components

The combined antigen of V600E and V600K to adjuvant ratios tested 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). Spleens and lungs were collected from immunized mice. The cells were counted and diluted to the correct concentration for each assay. The cells in the assays listed in Table 16 were stimulated with the peptide pools described in Table 17. Re-stimulation peptide pools were prepared by combining the peptides from

Table 17 in complete media at a 2X concentration of 4 pg/ml per peptide. Table 16. Peptide Stimulation Scheme

Table 17. Re-Stimulation Peptides

^Amutated amino acids are highlighted in bold.

ELISpot IFNy analysis was performed on splenocytes 7 days post dose 3 and 5 (FIG. 9A-FIG. 9C and FIG. 10A-FIG. 10C) and lung-resident lymphocytes 7 days post dose 3 (FIG. 8) with 0.1 x10⁶ cells/well and a peptide concentration of 2 pg/ml. ELISpot GzmB analysis was performed on splenocytes 7 days post dose 3 and 5 with 0.1 x10⁶ cells/well and a peptide concentration of 2 pg/ml (FIG. 11 ).

FluoroSpot IFNy/TNFa/IL2 analysis was performed on splenocytes 7 days post dose 3 and 5 and lung-resident lymphocytes 7 days post dose 3 with 0.1 x10⁶ cells/well and a peptide concentration of 2 ig/ml (FIG. 13A-FIG. 13C). Intracellular Stain Flow Cytometry IFNy/TNFa analysis was performed on splenocytes 7 days post dose 3 and 5 and lung-resident lymphocytes 7 days post dose 3 (FIG. 14A and FIG.14B). The antibody panel used in this experiment is described in Table .

Table 18. Antibodies used for ICS (IFNy/TNFa) Flow Cytometry

Intracellular Stain Flow Cytometry GzmB/Perforin analysis was performed on splenocytes 7 days post dose 3 and 5. The antibody panel used in this experiment is described in Table . Table 19. Antibodies used for ICS (GzmB/Perforin) Flow Cytometry

Luminex analysis of GzmB/IFNY/TNFa/GM-CSF/IL2/IL4/IL10/sFasL was performed on splenocytes 7 days post dose 3 and 5 and lung-resident lymphocytes 7 days post dose 3 (FIG. 12A-FIG. 12C).

After 3 doses of V600K and V600E 29mer peptides significant immune responses can be detected at the 5 nmol+5 nmol antigen + 10 nmol adjuvant dose. The strongest responses are observed with the 20 nmol+20 nmol antigen + 20 nmol adjuvant dose. However, after 5 doses the highest vaccine dose (20 nmol+20 nmol antigen + 20 nmol adjuvant dose) yielded attenuated responses compared to the 5 nmol+5 nmol antigen + 10 nmol adjuvant dose, which yields the highest responses. In most assays the 10nmol+10 nmol antigen + 10 nmol adjuvant dose showed no improvement over the 5 nmol+5 nmol antigen + 10 nmol adjuvant dose.

Other Embodiments

Various modifications and variations of the described compositions, methods, and uses of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

What is claimed is:

Claims

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

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

3. The compound or pharmaceutically acceptable salt thereof of claim 2, wherein the peptide is 10 to 40 amino acid fragment of SEQ ID NO: 1 .

4. The compound or pharmaceutically acceptable salt thereof of claim 3, wherein the peptide is 10 to 30 amino acid fragment of SEQ ID NO: 1 .

5. The compound or pharmaceutically acceptable salt thereof of claim 3, wherein the Braf peptide is a 30 amino acid fragment of SEQ ID NO: 1 .

6. The compound or pharmaceutically acceptable salt thereof of claim 4, wherein the Braf peptide is a 9 amino acid fragment of SEQ ID NO: 1 .

7. The compound or pharmaceutically acceptable salt thereof of claim 4, wherein the Braf peptide is a 15 amino acid fragment of SEQ ID NO: 1 .

8. The compound or pharmaceutically acceptable salt thereof of claim 4, wherein the Braf peptide a 29 amino acid fragment of SEQ ID NO: 1 .

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

10. The compound or pharmaceutically acceptable salt thereof of claim 9, wherein the Braf peptide comprises a fragment of SEQ ID NO: 1 , wherein the fragment comprises an amino acid substitution at the Vai occupying amino acid position 600 from N-terminus of SEQ ID NO: 1 .

11 . The compound or pharmaceutically acceptable salt thereof of claim 10, wherein the amino acid substitution at the Vai occupying position 600 of SEQ ID NO: 1 is V600K.

12. The compound or pharmaceutically acceptable salt thereof of claim 11 , wherein the amino acid substitution at the Vai occupying position 600 of SEQ ID NO: 1 is V600E.

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

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

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

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

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

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

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

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

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

22. The compound or pharmaceutically acceptable salt thereof of claim 21 , wherein the N-terminal modification is the addition of a des-aminocysteine homolog.

23. The compound or pharmaceutically acceptable salt thereof of claim 22, wherein the des- aminocysteine homolog is 3-mercaptopropionic acid or mercaptoacetic acid.

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

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

26. The compound or pharmaceutically acceptable salt thereof of claim 25, wherein the diacyl lipid comprises acyl chains 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.

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

28. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 -27, wherein the peptide is bonded or linked by a linker to the following lipid:

or a salt thereof.

29. The compound or pharmaceutically acceptable salt thereof of claim 28, wherein the linker is selected from the group consisting of a hydrophilic polymer, a string of hydrophilic amino acids, a polysaccharide, and an oligonucleotide, or a combination thereof.

30. The compound or pharmaceutically acceptable salt thereof of claim 29, wherein the linker comprises "N" polyethylene glycol units, wherein N is between 24-50.

31 . The compound or pharmaceutically acceptable salt thereof of claim 30, wherein the linker comprises PEG24-amido-PEG24.

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

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

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

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

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

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

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

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

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

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

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

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