CN117377683A - New antigenic peptide and application thereof in treating BRAF gene mutation related diseases - Google Patents

Abstract

The present disclosure provides an isolated neoantigen peptide comprising a tumor-specific neoepitope capable of binding to MHC-1 to form an MHC-neoantigen complex, a neoantigen binding peptide, a T cell comprising a TCR or CAR comprising the neoantigen binding peptide, and its use for immunotherapy of cancer, particularly cancer with BRAF G469V mutations.

Description

New antigenic peptide and application thereof in treating BRAF gene mutation related diseases

Technical Field

The present application relates generally to immunotherapeutic peptides, nucleic acids encoding said peptides, peptide binders and their use in immunotherapy, for example of cancer, and in particular to neoantigenic peptides and their use for the treatment of BRAF gene mutation related diseases.

Background

Lung cancer has so far been the leading cause of cancer death in men and women, accounting for almost 25% of all cancer deaths. Lung cancer is generally divided into two major types, known as small cell lung cancer and non-small cell lung cancer. Non-small cell lung cancer (NSCLC) is more common than small cell lung cancer, accounting for 85% of all lung cancers. In recent years, clinical application of immunotherapy typified by immunosuppressant (ICI) anti-PD-1/PD-L1 antibodies has brought long-term survival hope to patients with advanced NSCLC. Recent data show that five-year survival rates of patients with advanced NSCLC treated with PD-1 antibody in the first line have reached 23.2% and that patients with PD-1 antibody in the second line have reached 16% and 2-3 times higher than conventional treatment.

Unfortunately, studies to date have shown that advanced NSCLC carrying genetic mutations rarely benefit from ICI immunotherapy for complex reasons, and that the lack of neoantigens in genetically mutated patients is considered one of the main reasons for the inability to stimulate an effective immune response in the body. How to enhance the capability of gene mutation NSCLC patients to respond to ICI, so that the gene mutation NSCLC patients can still benefit from subsequent immunotherapy after targeted therapy drug resistance, has important significance for improving five-year survival rate of advanced NSCLC patients, and is an important problem to be solved in clinical practice.

The potent anti-tumor immunity of humans is associated with the presence of T cells directed against a new antigen of cancer, a class of HLA-binding peptides generated by tumor-specific mutations, and the data presented suggest that recognition of such new antigens is a major factor in clinical immunotherapy activity. Massively parallel whole exome sequencing has been used to detect all mutations within tumors to predict neoantigens. Neoantigen vaccination can expand the existing population of neoantigen-specific T cells and induce new cancer-specific T cells. Thus, neoantigens have emerged as potential ideal targets for anti-tumor immune responses.

The BRAF gene is a protooncogene found on chromosome 7, has abundant amino acids as shown in NCBI reference sequence np_001365403.1, and becomes an oncogene after mutation. Although BRAF mutations and their relationship to cancer were not discovered until 2002, studies conducted hereafter have identified hundreds of different types of mutations that may be associated with cancer. BRAF mutations are classified according to the newly proposed classification scheme into class 1 (kinase activated, codon 600), class 2 (kinase activated, non-codon 600) and class 3 (kinase impaired) (Lokhandwala, p.m., tseng, LH., rodriguez, e.et al, clinical mutational profiling and categorization of BRAF mutations in melanomas using next generation sequencing. Bmc Cancer 19,665 (2019)).

However, mutation does not mean successful identification of a new antigen that is immunogenic, let alone the ability to induce antigen-specific cytotoxic T Cells (CTLs) that recognize and lyse cancer or tumor cells. In fact, only a very low proportion of mutations lead to the production of interesting neoantigenic peptides.

Thus, there remains a need in the art to develop additional cancer therapeutics.

Disclosure of Invention

In one aspect, an isolated neoantigenic peptide is provided, wherein the isolated neoantigenic peptide comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO. 19 (QRIGSGSFVT) or a functionally equivalent variant of SEQ ID NO. 19.

In some embodiments, the isolated neoantigenic peptide comprises a tumor-specific neoepitope capable of binding MHC-1 to form an MHC-neoantigen complex.

In some embodiments, the functionally equivalent variant hybridizes to SEQ ID NO:19, or comprises one or two amino acid changes compared to SEQ ID No. 19.

In some embodiments, the amino acid change is a conservative amino acid substitution.

In some embodiments, the isolated neoantigenic peptide is linked to one or more additional neoantigenic peptides, optionally via a linker (e.g., a polyglycine or polyserine linker).

In some embodiments, the isolated neoantigenic peptides are about 10 to 30 amino acids in length, such as about 10 to 20 amino acids in length, such as 10, 11, 12, 13, 14, and 15 amino acids in length.

In some embodiments, the isolated neoantigenic peptide binds MHC-1 with a binding affinity of about 500nM or less, e.g., about 250nM or less, or about 50nM or less.

In one aspect, polynucleotides encoding the novel antigenic peptides described herein are provided.

In one aspect, there is provided a vector comprising a polynucleotide described herein.

In some embodiments, the vector is selected from the group consisting of a plasmid, a cosmid, an RNA formulated in a particle, a self-amplifying RNA (SAM), a SAM formulated in a particle, or a viral vector.

In some embodiments, the viral vector is an alphavirus vector, a Venezuelan Equine Encephalitis (VEE) viral vector, a sindbis viral vector, a semliki forest viral vector, a simian or human cytomegalovirus vector, a lymphocytic choriomeningitis viral vector, a retrovirus vector, a lentivirus vector, an adenovirus vector, or a combination thereof.

In one aspect, there is provided a composition comprising an isolated neoantigenic peptide described herein, optionally in the form of an in vivo delivery system, such as nanoparticle encapsulation, virus-like particles, liposomes, or any combination thereof.

In one aspect, provided are compositions comprising the polynucleotides and/or vectors described herein, optionally in the form of an in vivo delivery system, such as a virus, virus-like particle, plasmid, bacterial plasmid, nanoparticle, or any combination thereof.

In some embodiments, the composition further comprises at least one modulator of a checkpoint molecule or an immunomodulatory agent, or a nucleic acid encoding the modulator or immunomodulatory agent, or a vector comprising a nucleic acid encoding the modulator or immunomodulatory agent.

In some embodiments, the modulator of the checkpoint molecule is selected from: (a) Agonists of members of the tumor necrosis factor receptor superfamily, preferably agonists of CD27, CD40, 0X40, GITR or CD 137; (b) Antagonists of PD-1, PD-L1, CD274, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3, VISTA, or antagonists of members of the B7-CD28 superfamily, preferably antagonists of CD28 or ICOS, or antagonists of its ligands; or wherein the immunomodulator is a T cell growth factor, preferably IL-2, IL-12 or IL-15.

In some embodiments, the composition further comprises one or more adjuvants.

In one aspect, there is provided a T Cell Receptor (TCR) capable of binding a neoantigenic peptide described herein or an MHC-I-peptide complex comprising a neoantigenic peptide described herein.

In one aspect, there is provided a chimeric antigen receptor comprising: (i) a T cell activating molecule; (ii) a transmembrane region; and (iii) an antigen recognition moiety capable of binding to a neoantigenic peptide described herein or an MHC-peptide complex comprising a neoantigenic peptide described herein.

In one aspect, there is provided a T cell comprising a T cell receptor or chimeric antigen receptor described herein.

In some embodiments, the T cell is a T cell isolated from a population of T cells from a subject that has been incubated with an antigen presenting cell (e.g., an artificial antigen presenting cell) and a neoantigen peptide described herein for a time sufficient to activate the T cell.

In some embodiments, the T cell is a cd8+ T cell or a cytotoxic T cell.

In one aspect, there is provided a method for activating tumor-specific T cells, the method comprising: (a) isolating a population of T cells from a subject; and (b) incubating the isolated population of T cells with antigen presenting cells (e.g., artificial antigen presenting cells) and the neoantigenic peptides described herein for a time sufficient to activate the T cells.

In one aspect, a modified cd8+ T cell transfected or transduced with a nucleic acid encoding a TCR described herein or a chimeric antigen receptor described herein is provided.

In one aspect, provided are compositions comprising T cells described herein, activated tumor-specific T cells produced by the methods described herein, and/or modified cd8+ T cells described herein.

In one aspect, a method of treating or preventing a BRAF mutation related cancer in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of an isolated neoantigenic peptide described herein, a polynucleotide described herein, a vector described herein, a composition described herein, a T cell described herein, an activated tumor-specific T cell produced by a method described herein, and/or a modified cd8+ T cell described herein.

In one aspect, a method of inhibiting the growth of a tumor cell bearing a BRAF mutation is provided, the method comprising administering to a subject a therapeutically effective amount of an isolated neoantigenic peptide described herein, a polynucleotide described herein, a vector described herein, a composition described herein, a T cell described herein, an activated tumor-specific T cell produced by a method described herein, and/or a modified cd8+ T cell described herein.

In some embodiments, the BRAF mutation is a BRAF G469V mutation.

Drawings

In the accompanying drawings, embodiments of the present disclosure are shown by way of example. It is to be expressly understood that the description and drawings are only for the purpose of illustration and aid in understanding and are not intended as a definition of the limits of the invention.

Fig. 1 shows HLA I ELISpot results for BRAF G469V donors.

Fig. 2 shows candidate peptide predictions for BRAF G469V mutation sites. In the predictive model, a high ranking score indicates high binding affinity.

Fig. 3 shows ELISpot results for 35 BRAF G469V peptides.

Fig. 4 shows ELISpot results for 4 BRAF G469V peptides.

Figure 5 shows ELISpot results for healthy donors.

Figure 6 shows ELISpot results for 16 healthy donors.

FIG. 7 shows ELISPot results for 4 HLA A A0201 healthy donors.

FIG. 8 shows a plasmid map of the MSCV plasmid encoding HLA A A0201 and BRAF-19 peptides.

Detailed Description

For the purposes of promoting an understanding of the principles of the disclosure, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe the same. However, it should be understood that it is not intended to limit the scope of the present disclosure.

In the present disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined herein are more fully described by reference to the entire specification.

As used herein, the singular terms one (a), one (an) and the plural reference are included unless the context clearly dictates otherwise. Unless otherwise indicated, nucleic acids are written in a 5 'to 3' direction from left to right; the amino acid sequence is written from left to right in the amino to carboxyl direction. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary depending upon the context in which one of ordinary skill in the art uses.

As used herein, the term "consisting essentially of" in the context of amino acid sequences means the recited amino acid sequences and additional 1, 2, 3, 4, or 5 amino acids at the N-or C-terminus thereof.

Unless the context requires otherwise, the terms "comprise," "include," and "comprise," or the like, are intended to imply a non-exclusive inclusion, such that an element or feature listed is not limited to only those elements or features listed or stated but may include other elements or features not listed or stated.

The term "optional" or "optionally" means that the subsequently described event, circumstance or replacement may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

As used herein, the term "about" when referring to a given value, e.g., parameter, quantity, etc., is intended to encompass variations in the given value, e.g., +/-10% or less, +/-5% or less, +/-1% or less, and +/-0.1% or less, from the given value.

As used herein, the terms "patient," "individual," and "subject" are used in the context of any mammalian recipient or composition disclosed herein. Accordingly, the methods and compositions disclosed herein may have medical and/or veterinary applications. In a preferred form, the mammal is a human.

As used herein, the term "sequence identity" is intended to include the number of exact nucleotide or amino acid matches, taking into account the proper alignment using standard algorithms, taking into account the degree to which sequences are identical over a comparison window. Thus, the "percent sequence identity" is calculated by: comparing the two optimally aligned sequences over a comparison window, determining the number of positions at which the same nucleobase (e.g., A, T, C, G) occurs in the two sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, "sequence identity" may be understood to mean the "percentage of matches" calculated by the DNASIS computer program (Windows version 2.5; available from Hitachi software engineering Co., of san Francisco, calif.).

New antigens/peptides

As used herein, the term "neoantigen" or "neoantigenic peptide" refers to a class of tumor antigens derived from tumor-specific changes in proteins. New antigens include, but are not limited to, tumor antigens, which result from, for example, substitution of protein sequences, frame shift mutations, in-frame deletions, insertions, and tumor-specific overexpression of polypeptides. The term "tumor-specific change" refers to a change that is not present in a very cancer cell but is found in a cancer or tumor cell.

Because of the technical difficulties of identifying neoantigens, selecting optimized antigens, and producing neoantigens for use in vaccines or immunogenic compositions, neoantigens are rarely used in cancer vaccines or immunogenic compositions.

MHC binding neoantigens are a dynamic process and do not undergo chemical changes during transport. Since the level of activation of T cells is determined by the concentration of successful MHC-neoantigen complexes, the task of finding active neoantigens is shifted to recognition of stable MHC-peptide complexes. The accuracy of predictive algorithms for identifying stable MHC-peptide complexes is driven by the use of validated, combined and non-combined peptide neural network-based learning methods.

Sequencing methods are used to identify tumor-specific mutations. Any suitable sequencing method may be used, such as Next Generation Sequencing (NGS) techniques. "NGS" may include all new high throughput sequencing techniques that read nucleic acid templates in random parallel along the entire genome by dividing the entire genome into small fragments. Such NGS techniques (massively parallel sequencing techniques) are capable of delivering nucleic acid sequence information of a whole genome, an exome, a transcriptome (all transcribed sequences of a genome), or a methylation group (all methylated sequences of a genome) in a very short time.

Herein, the present inventors predicted a neoantigenic peptide sequence of patients with BRAF G469V mutation by NGS and machine learning methods and confirmed the sequence of the amino acid sequence as set forth in SEQ ID NO:19 is capable of activating cd8+ cells, indicating its anti-tumor function in patients with BRAF G469V mutations.

The BRAF G469V mutation results from a single nucleotide change (c.1406g > T), resulting in a glycine (G) at position 469 being substituted with a valine (V) amino acid. BRAF G469V was present in 0.07% of AACR GENIE cases, including lung adenocarcinoma, colon adenocarcinoma, bladder urothelial carcinoma, primary unknown cancer, and primary unknown cancer.

In one aspect, the invention provides an isolated neoantigenic peptide, wherein the isolated neoantigenic peptide comprises the amino acid sequence set forth in SEQ ID NO. 19 (QRIGSGSFVT), or a functionally equivalent variant of SEQ ID NO. 19 (QRIGSGSFVT).

The term "isolated" refers to a material that is substantially or essentially free of components that normally accompany the material when it is found in its natural state. Thus, the isolated neoantigenic peptides described herein do not contain some or all of the materials normally associated with peptides in an in situ environment. An "isolated" neoantigenic peptide refers to a neoantigenic epitope that does not include the entire sequence of the antigen from which the epitope is derived.

In some embodiments, the isolated neoantigenic peptide comprises a tumor-specific neoepitope capable of binding MHC-1 to form an MHC-neoantigen complex. The term "tumor-specific neoepitope" refers to a neoepitope that is not present in normal non-cancerous cells but is found in cancer or tumor cells.

In some embodiments, the present disclosure contemplates functionally equivalent variants of the isolated neoantigenic peptides identified herein. The term "functionally equivalent variant" as used herein means a variant polypeptide species having one or more amino acid substitutions, insertions or deletions compared to a reference peptide, provided that the variant retains or substantially retains its specific binding function. In some embodiments, the functionally equivalent variant hybridizes to SEQ ID NO:19 has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or more) sequence identity, or comprises one or two amino acid changes compared to SEQ ID No. 19.

In some embodiments, the amino acid change is a conservative amino acid substitution. "conservative amino acid substitution" refers to an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Amino acid residue families having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of phenylalanine for tyrosine is a conservative substitution. Another example is the substitution of glutamine for glutamic acid or asparagine for aspartic acid, which can be considered a similar substitution, as glutamine and asparagine are the amide derivatives of glutamic acid and aspartic acid, respectively. Methods for identifying nucleotide and amino acid conservative substitutions that do not eliminate peptide function are known to those skilled in the art.

In some embodiments, an isolated neoantigenic peptide as described herein may be linked, optionally through a linker, to one or more additional neoantigenic peptides, preferably to one or more neoantigenic peptides specific for the same tumor or cancer type. It is beneficial to use a combination of different neoantigenic peptides directed against the same tumor or cancer type to induce antigen specific cytotoxic T cells that recognize and lyse tumor cells.

A variety of linkers known in the art (e.g., polyglycine or polyserine linkers) may be used in the present disclosure. For example, short oligopeptides or polypeptide linkers, preferably between 2 and 10 amino acids in length, may form a linkage between the peptides. Glycine-serine doublets provide particularly suitable linkers.

In some embodiments, the isolated neoantigenic peptides are about 10 to 30 amino acids in length, such as about 10 to 20 amino acids in length, such as 10, 11, 12, 13, 14, and 15 amino acids in length. In some embodiments, the isolated neoantigenic peptide is about 10 to about 15 amino acids in length. In some embodiments, the isolated neoantigenic peptide is about 10 to about 11 amino acids in length.

In some embodiments, the isolated neoantigenic peptide binds to class I Major Histocompatibility Complex (MHC). In some embodiments, the isolated neoantigenic peptide binds to MHC class I (MHC-I) with a binding affinity of about 500nM or less. In some embodiments, the isolated neoantigenic peptide binds to MHC-1 with a binding affinity of about 250nM or less. In some embodiments, the isolated neoantigenic peptide binds to MHC-1 with a binding affinity of about 50nM or less. As used herein, the term "major histocompatibility complex" or "MHC" is a cluster of genes that play a role in controlling cellular interactions responsible for physiological immune responses. In humans, MHC complexes are also known as Human Leukocyte Antigen (HLA) complexes. In some embodiments, the MHC complex is HLA class I. In some embodiments, HLA class I may be selected from HLA a0201, HLA a2402, HLA B07, HLA B18, HLA B35, or HLA B44, e.g., HLA a0201. In some embodiments, the neoantigenic peptides described herein have peptide binding specificity common to HLA molecules encoded by two or more HLA alleles.

In some embodiments, the isolated neoantigenic peptide may further comprise modifications that increase in vivo half-life, cell targeting, antigen uptake, antigen processing, MHC-I affinity, MHC stability, antigen presentation, or a combination thereof. In some embodiments, the modification is conjugation to a carrier protein, nanoparticle attachment, nanoparticle encapsulation, conjugation to a ligand, conjugation to an antibody, albumin fusion, fc fusion, cholesterol fusion, pegylation, acylation, amidation, glycosylation, phosphorylation, biotinylation, or addition of an unnatural amino acid.

In some embodiments, provided herein are polynucleotides encoding the novel antigenic peptides described herein. It will be appreciated by those skilled in the art that, due to redundancy of the genetic code, different nucleic acid sequences may encode the same peptide, and that each nucleic acid falls within the scope of the present invention. The coding nucleic acid may be DNA or RNA, e.g. mRNA, or a combination thereof. In some embodiments, the nucleic acid encoding the peptide is a self-amplifying mRNA. Any suitable polynucleotide encoding a neoantigenic peptide described herein falls within the scope of the invention.

In some embodiments, provided herein are vectors, e.g., expression vectors, useful for the production and administration of the novel antigenic peptides described herein. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Typically, the DNA is inserted into an expression vector, e.g., a plasmid, in the correct orientation and in the correct reading frame for expression. If desired, the DNA may be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host (e.g., bacteria), although such control is typically available in expression vectors. The vector is then introduced into host bacteria for cloning using standard techniques (see, e.g., sambrook et al (1989) Molecular Cloning, A Laboratory Manual, cold Spring Harbor Laboratory, cold Spring Harbor, n.y.).

Expression vectors comprising the polynucleotides described herein and host cells comprising the expression vectors are also contemplated. The neoantigenic peptide may be provided in the form of an RNA or cDNA molecule encoding the desired neoantigenic peptide. One or more of the neoantigenic peptides of the invention may be encoded by a single expression vector.

Provided herein are vector systems comprising an expression vector, wherein the expression vector may be selected from a plasmid, a cosmid, an RNA formulated in a particle, a self-amplifying RNA (SAM), a SAM formulated in a particle, or a viral vector. In some embodiments, the viral vector may be an alphavirus vector, a Venezuelan Equine Encephalitis (VEE) viral vector, a sindbis viral vector, a semliki forest viral vector, a simian or human cytomegalovirus vector, a lymphocytic choriomeningitis viral vector, a retrovirus vector, a lentivirus vector, an adenovirus vector, or a combination thereof.

Suitable host cells for expressing the polypeptide include prokaryotes, yeast, insects, or higher eukaryotic cells under the control of a suitable promoter. Prokaryotes include gram-negative or gram-positive organisms, such as E.coli. Higher eukaryotic cells include established mammalian-derived cell lines. Cell-free translation systems may also be used. Suitable cloning and expression vectors for bacterial, fungal, yeast and mammalian cell hosts are well known in the art.

Novel antigen binding peptides

In one aspect, the disclosure provides binding proteins (e.g., antibodies or antigen binding fragments thereof), or T Cell Receptors (TCRs), or Chimeric Antigen Receptors (CARs) capable of binding with high affinity to a neoantigenic peptide as described herein or an MHC (HLA) -neoantigenic peptide complex as described herein. In some embodiments, the antibody is a monoclonal antibody specific for a neoantigenic peptide described herein.

In some embodiments, provided herein are T Cell Receptors (TCRs) capable of binding to or comprising the neoantigenic peptides described herein or MHC-peptide complexes of the neoantigenic peptides described herein. In some embodiments, the MHC of the MHC-peptide complex is MHC class I. In some embodiments, the TCR may be a bispecific TCR further comprising a domain comprising an antibody or antibody fragment capable of binding an antigen. In some embodiments, the antigen may be a T cell specific antigen, such as CD3. In some embodiments, the antibody or antibody fragment is an anti-CD 3 scFv.

In some embodiments, provided herein is a chimeric antigen receptor comprising: (i) a T cell activating molecule; (ii) a transmembrane region; and (iii) an antigen recognition moiety capable of binding to a neoantigenic peptide described herein or an MHC-peptide complex comprising a neoantigenic peptide described herein. In some embodiments, the T cell activating molecule is CD3- ζ. In some embodiments, the chimeric antigen receptor further comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain may be selected from the group consisting of CD28, 4-1BB, OX40, ITAM, and ICOS. In some embodiments, the antigen recognition moiety is capable of binding to a neoantigenic peptide in the context of class I MHC. In some embodiments, the antigen recognition moiety is an scFv of a monoclonal antibody specific for a neoantigenic peptide described herein. In some embodiments, the MHC of the MHC peptide is MHC class I.

T cell

In one aspect, the present disclosure provides a T cell comprising a T cell receptor or chimeric antigen receptor described herein. Preferably, the T cells are cd8+ T cells or cytotoxic T cells.

In some embodiments, provided herein are T cells comprising a T Cell Receptor (TCR) capable of binding to a neoantigenic peptide described herein or an MHC-peptide complex comprising a neoantigenic peptide described herein, wherein the T cells are T cells isolated from a T cell population from a subject, the T cell population having been incubated with an antigen presenting cell (e.g., an artificial antigen presenting cell) and a neoantigenic peptide described herein for a sufficient time to activate the T cells. In some embodiments, the T cell is a cd8+ T cell or a cytotoxic T cell. In some embodiments, the T cell population from the subject is a cd8+ T cell population from the subject. In some embodiments, activated cd8+ T cells are isolated from antigen presenting cells. In some embodiments, the antigen presenting cells are artificial antigen presenting cells or dendritic cells or CD40L expanded B cells. In some embodiments, the antigen presenting cells are autologous. In some embodiments, the antigen presenting cells have been treated to strip endogenous MHC-related peptides from their surface. In some embodiments, the treatment to strip endogenous MHC-related peptides comprises treating the cells with a weak acid solution. In some embodiments, the antigen presenting cells have been pulsed with a neoantigenic peptide described herein. In some embodiments, pulsing comprises incubating the antigen presenting cells in the presence of at least about 2 μg/ml of the neoantigenic peptides described herein. In some embodiments, the ratio of isolated T cells to antigen presenting cells is between about 0.1:1 to 300:1, e.g., between about 0.5:1 to 200:1, between about 1:1 to 150:1. In some embodiments, in the presence of IL-2 and IL-7 in the incubation of isolated T cell population. In some embodiments, the MHC of the MHC peptide is MHC class I.

In some embodiments, provided herein is a method for activating tumor-specific T cells, the method comprising: isolating a population of T cells from a subject; and incubating the isolated population of T cells with antigen presenting cells and the neoantigenic peptides described herein for a time sufficient to activate the T cells. In some embodiments, the T cell is a cd8+ T cell or a cytotoxic T cell. In some embodiments, the T cell population from the subject is a cd8+ T cell population from the subject. In some embodiments, activated cd8+ T cells are isolated from antigen presenting cells. In some embodiments, the antigen presenting cells are artificial antigen presenting cells or dendritic cells or CD40L expanded B cells. In some embodiments, the antigen presenting cells are autologous. In some embodiments, the antigen presenting cells have been treated to strip endogenous MHC-related peptides from their surface. In some embodiments, the treatment to strip endogenous MHC-related peptides comprises treating the cells with a weak acid solution. In some embodiments, the antigen presenting cells have been pulsed with a neoantigenic peptide described herein. In some embodiments, pulsing comprises incubating the antigen presenting cells in the presence of at least about 2 μg/ml of the neoantigenic peptides described herein. In some embodiments, the ratio of isolated T cells to antigen presenting cells is between about 0.1:1 to 300:1, e.g., between about 0.5:1 to 200:1, between about 1:1 to 150:1. In some embodiments, in the presence of IL-2 and IL-7 in the incubation of isolated T cell population. In some embodiments, the MHC of the MHC peptide is MHC class I.

In some embodiments, provided herein are modified T cells transfected or transduced with nucleic acids encoding a TCR or chimeric antigen receptor as described herein. The polynucleotides or nucleic acids described herein are capable of transducing T cells, thereby producing modified T cells having a TCR or CAR described herein. In some embodiments, the T cell is a CTL. In some embodiments, the CTL is injected into the patient.

Composition and method for producing the same

In one aspect, the disclosure provides a composition (e.g., a pharmaceutical composition) comprising an isolated neoantigenic peptide, an antibody or antigen binding fragment thereof, a polynucleotide, a T cell comprising a TCR or CAR, an activated tumor-specific T cell, and/or a modified cd8+ T cell, as described herein.

In some embodiments, the invention relates to immunogenic compositions, e.g., vaccine compositions, that are capable of eliciting a neoantigen-specific response (e.g., a cell-mediated immune response or a humoral immune response). In some embodiments, the immunogenic composition comprises a neoantigen therapeutic (e.g., peptide, polynucleotide, TCR or CAR-containing cell, antibody, etc.) described herein that corresponds to a tumor-specific neoantigen (e.g., BRAF-19) identified herein. In some embodiments, the immunogenic compositions described herein are capable of eliciting a specific cytotoxic T cell response or B cell response. In some embodiments, provided herein are compositions comprising autologous subject T cells that contain a T cell receptor or chimeric antigen receptor described herein.

In some embodiments, the compositions described herein further comprise at least one modulator or immunomodulator of a checkpoint molecule, or a nucleic acid encoding the modulator or immunomodulator, or a vector comprising a nucleic acid encoding the modulator or immunomodulator. In some embodiments, the modulator of the checkpoint molecule is selected from: (a) Agonists of members of the tumor necrosis factor receptor superfamily, preferably agonists of CD27, CD40, 0X40, GITR or CD 137; (b) Antagonists of PD-1, PD-L1, CD274, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3, VISTA, or antagonists of members of the B7-CD28 superfamily, preferably antagonists of CD28 or ICOS, or antagonists of its ligands. In some embodiments, the modulator of the checkpoint molecule is an inhibitor of a checkpoint molecule selected from the group consisting of PD-1, PD-L1, CD274, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3, and VISTA. In some embodiments, the inhibitor of the checkpoint molecule interacts with a ligand of a checkpoint molecule selected from the group consisting of PD-1, PD-L1, CD274, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3, and VISTA. In some embodiments, the modulator is a costimulatory ligand, a TNF ligand, an Ig superfamily ligand, CD28, CD80, CD86, ICOS, CD40L, OX, CD27, GITR, CD30, DR3, CD69, or 4-1BB. In some embodiments, the immunomodulator is a T cell growth factor, preferably IL-2, IL-12 or IL-15.

In some embodiments, the immunogenic compositions described herein may further comprise an adjuvant and/or carrier. An adjuvant is a substance that, when mixed into an immunogenic composition, can increase or otherwise alter the immune response to a therapeutic agent. The carrier is a scaffold structure, such as a polypeptide or polysaccharide, to which the neoantigen polypeptide or polynucleotide is capable of binding. Optionally, the adjuvant is conjugated covalently or non-covalently to a polypeptide or polynucleotide described herein.

In some embodiments, the adjuvant is selected from the group consisting of aluminum salts, amplivax, monophosphoryl lipid A, ruiximad, beta-glucan, acrylic or methacrylic acid polymers, copolymers of maleic anhydride, poly (I: C), poly-ICLC, IC30, IC31, AS15, BCG, CP-870, cpG7909, cyaA, dSLIM, GM-CSF, imiquimod, imuFact IMP321, ISS, ISCOMATRIX, juvlmmune, lipoVac, MF59, montanide ISA 206VG, montanide ISA 50V2, montanide ISA 51VG, OK-432, OM-174, OM-197-MP-EC, ONTAK, moloTAK, molozenges, almond, molozenges, mileages, and the like,SRL172, YF-17D, R848, pam3Cys and Pam3CSK4.

In some embodiments, the carrier may, for example, confer stability, increase biological activity, or increase serum half-life. The carrier may be any suitable carrier known to those skilled in the art, such as a protein or antigen presenting cell. Carrier proteins may include, but are not limited to, keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin, or ovalbumin, immunoglobulins, or hormones such as insulin or palmitic acid.

In some embodiments, the pharmaceutical compositions (e.g., immunogenic compositions) described herein for therapeutic treatment are intended for parenteral, topical, nasal, oral, or topical administration. In some embodiments, the pharmaceutical compositions described herein are administered parenterally, for example, intravenously, subcutaneously, intradermally, or intramuscularly. In some embodiments, described herein are compositions for parenteral administration comprising a solution of a neoantigenic peptide, and an immunogenic composition is dissolved or suspended in an acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers may be used, such as water, buffered water, saline, and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered.

In some embodiments, the concentration of the neoantigenic peptides and polynucleotides described herein in the pharmaceutical formulation can vary widely, i.e., from less than about 0.1% up to 50% by weight or more, depending on the particular mode of administration selected.

The neoantigenic peptides and polynucleotides described herein may also be administered via liposomes that target the peptides to specific cellular tissues, such as lymphoid tissues. Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and sterols, such as cholesterol.

In some embodiments, the pharmaceutical composition may further comprise pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The carrier or other material may be selected according to the route of administration. Acceptable carriers, excipients, or stabilizers are those that are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexa-methyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenolic alcohols, butanols or benzyl alcohols, alkyl p-hydroxybenzoates, such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants, e.g. Or polyethylene glycol (PEG).

In some embodiments, vaccines are provided that comprise a neoantigen therapeutic (e.g., peptide, polynucleotide, TCR or CAR-containing cell, antibody, etc.) described herein that corresponds to a tumor-specific neoantigen (e.g., BRAF-19) identified herein. Vaccines can be delivered by a variety of routes. The route of delivery may include, but is not limited to, oral (including buccal and sublingual), parenteral (including intramuscular, intraarterial, intrathecal, intradermal, intraperitoneal, subcutaneous and intravenous), rectal, vaginal, topical, nasal, transdermal patches, pulmonary or suppository administration or in a form suitable for administration by nebulization, inhalation or insufflation. General information about drug delivery systems can be found in Ansel et al, pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins, baltimore Md. (1999): vaccines described herein can be administered intramuscularly or by subcutaneous or transdermal administration, e.g., by iontophoresis.

In some embodiments, the vaccine may be a formulation suitable for nasal administration, and may include a powder having a particle size, for example, in the range of about 10 to about 500 microns, which is administered by nasal inhalation. The formulation may be a nasal spray, nasal drops or aerosol administration by nebulizer.

In some embodiments, the vaccine may be a liquid formulation, such as a suspension, syrup, or elixir. The vaccine may also be a formulation for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injection administration), such as a sterile suspension or emulsion.

In some embodiments, the vaccine may include a substance for a single immunization or for multiple immunizations (e.g., a "multi-dose" kit). Preservatives are preferably included in the multi-dose composition. As an alternative (or in addition) to including a preservative in the multi-dose composition, the composition may be contained in a container with a sterile adapter to remove substances. In some embodiments, the vaccine may be administered in a dose volume of about 0.5mL or higher, but may be administered at half the dose (e.g., about 0.25 mL) depending on the condition and age of the subject. In some embodiments, the vaccine may be administered at a higher dose, e.g., about 1 mL.

In some embodiments, the vaccine may be administered as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more dose regimen. In some embodiments, the vaccine is administered as a 1, 2, 3, or 4 dose course regimen. In some embodiments, the vaccine is administered as a 1 dose course regimen. In some embodiments, the vaccine is administered as a 2 dose course regimen. In some embodiments, the administration of the first and second doses may be separated by about 1 day, 2 days, 5 days, 7 days, 14 days, 21 days, 30 days, 2 months, 4 months, 6 months, 9 months, 1 year, 1.5 years, 2 years, 3 years, 4 years, or more. The examples of dosages are not limiting and are merely intended to illustrate particular dosing regimens for administering the vaccines described herein.

Method

In one aspect, the present disclosure provides for the use of the neoantigen therapeutics described herein (e.g., peptides, polynucleotides, TCR or CAR-containing cells, antibodies, etc.) in a variety of applications, including, but not limited to, therapeutic and/or prophylactic methods, such as the treatment of cancer. In some embodiments, the method of treatment comprises immunotherapy. In certain embodiments, the novel antigenic peptides described herein are useful for activating, promoting, increasing and/or enhancing an immune response, increasing the immunogenicity of a tumor, inhibiting tumor growth, reducing tumor volume, increasing tumor cell apoptosis, and/or reducing the tumorigenicity of a tumor. The method may be an in vitro, ex vivo or in vivo method. In some embodiments, the activation, promotion, increase, and/or enhancement of an immune response comprises increasing T cell activity. In some embodiments, the activation, promotion, increase, and/or enhancement of an immune response comprises increasing CTL activity.

In some embodiments, the present disclosure provides methods of treating or preventing cancer in a subject, the methods comprising administering to the subject a therapeutically effective amount of an isolated neoantigenic peptide, polynucleotide, vector, composition, T cell, activated tumor-specific T cell, and/or modified cd8+ T cell described herein. In some embodiments, the present disclosure provides methods of inhibiting tumor cell growth comprising administering to a subject a therapeutically effective amount of an isolated neoantigenic peptide, polynucleotide, vector, composition, T cell, activated tumor-specific T cell, and/or modified cd8+ T cell described herein. In some embodiments, the present disclosure provides isolated neoantigenic peptides, polynucleotides, vectors, compositions, T cells, activated tumor-specific T cells, and/or modified cd8+ T cells as described herein for use in treating or preventing cancer in a subject or for inhibiting the growth of tumor cells. In some embodiments, the disclosure provides the use of an isolated neoantigenic peptide, polynucleotide, vector, composition, T cell, activated tumor-specific T cell, and/or modified cd8+ T cell described herein in the manufacture of a pharmaceutical composition (e.g., an immunogenic composition or vaccine) or medicament for treating or preventing cancer or for inhibiting tumor cell growth in a subject. In some embodiments, the cancer is a BRAF mutation-related cancer. In some embodiments, the tumor cells have a BRAF mutation. In some embodiments, the BRAF mutation is a BRAF G469V mutation.

In some embodiments, the cancer is selected from: adrenal cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, glioblastoma, head and neck cancer, renal chromophobe cancer, renal clear cell carcinoma, renal papillary carcinoma, liver cancer, lung adenocarcinoma, lung squamous carcinoma, ovarian cancer, pancreatic cancer, melanoma, gastric cancer, endometrial cancer, and uterine carcinoma sarcoma. In some embodiments, the cancer is selected from: prostate cancer, bladder cancer, lung squamous cell carcinoma, NSCLC, breast cancer, head and neck cancer, lung adenocarcinoma, GBM, glioma, CML, AML, supratentorial ependymoma, acute promyelocytic leukemia, isolated fibroma, and crizotinib resistant cancer. In some embodiments, the cancer is selected from: NSCLC, melanoma, and bladder cancer. In some embodiments, the cancer is selected from: lung adenocarcinoma, colon adenocarcinoma, bladder urothelial carcinoma, primary unknown cancer, and primary unknown cancer. In some embodiments, the cancer cell comprises a BRAF G469V mutation.

In some embodiments, provided herein are methods of treating cancer or eliciting, enhancing or prolonging an anti-tumor response in a subject in need thereof, comprising administering to the subject a peptide, polynucleotide, vector, composition, antibody or cell described herein. In some embodiments, the subject is a human. In some embodiments, the subject has cancer, preferably has a BRAF G469V mutation.

In some embodiments, the method further comprises administering at least one modulator or immunomodulator of a checkpoint molecule, or a nucleic acid encoding the modulator or immunomodulator, or a vector comprising a nucleic acid encoding the modulator or immunomodulator. In some embodiments, the modulator of the checkpoint molecule is selected from: (a) Agonists of members of the tumor necrosis factor receptor superfamily, preferably agonists of CD27, CD40, 0X40, GITR or CD 137; (b) Antagonists of PD-1, PD-L1, CD274, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3, VISTA, or antagonists of members of the B7-CD28 superfamily, preferably antagonists of CD28 or ICOS, or antagonists of its ligands. In some embodiments, the modulator of the checkpoint molecule is an inhibitor of a checkpoint molecule selected from the group consisting of PD-1, PD-L1, CD274, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3, and VISTA. In some embodiments, the inhibitor of the checkpoint molecule interacts with a ligand of a checkpoint molecule selected from the group consisting of PD-1, PD-L1, CD274, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3, and VISTA. In some embodiments, the modulator is a costimulatory ligand, a TNF ligand, an Ig superfamily ligand, CD28, CD80, CD86, ICOS, CD40L, OX, CD27, GITR, CD30, DR3, CD69, or 4-1BB. In some embodiments, the immunomodulator is a T cell growth factor, preferably IL-2, IL-12 or IL-15.

As used herein, "treatment" refers to a therapeutic intervention that at least partially ameliorates, eliminates, or reduces symptoms or pathological signs after the onset of a disease, disorder, or condition (e.g., cancer). Treatment need not be absolutely beneficial to the subject. The beneficial effect can be determined using any method or criteria known to one of ordinary skill.

As used herein, "preventing" or "prevention" refers to a series of actions that begin before the symptoms or pathological signs of a disease, disorder, or condition appear to prevent and/or alleviate the symptoms or pathological signs. It will be appreciated that such prevention need not be absolutely beneficial to the subject. "prophylactic" treatment is a treatment administered to a subject that does not exhibit symptoms of a disease, disorder or condition, or exhibits only early symptoms, with the aim of reducing the risk of developing symptoms or pathological signs of the disease, disorder or condition.

As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic agent effective to treat or prevent a disease or disorder in a subject or mammal. A therapeutically effective amount of the medicament has a therapeutic effect, and thus can prevent the development of a disease or disorder; slowing the progression or progression of the disease or condition; to some extent, alleviate one or more symptoms associated with the disease or condition; reducing morbidity and mortality; improving the quality of life; or a combination of these effects.

In some embodiments, the subject receives a single dose of cells during treatment, or receives multiple doses of cells, e.g., 2 to 5, 10, 20, or more doses. In some embodiments, the course of treatment lasts about 1 week to 12 months or longer, e.g., 1, 2, 3, or 4 weeks or 2, 3, 4, 5, or 6 months. In some embodiments, the subject may be treated about every 2-4 weeks. One of ordinary skill in the art will appreciate that the dosage and/or interval of administration may be selected based on a variety of factors, such as the weight and/or blood volume of the subject, the condition being treated, the response of the subject, and the like. The exact amount required may vary from subject to subject, depending on a variety of factors, such as the species, age, weight, sex and general condition of the subject, the severity of the disease or disorder, the immunogenic therapeutic agent used, the mode of administration, concurrent therapy, and the like.

An effective amount for use in humans may be determined based on animal models. For example, dosages for humans may be formulated to achieve concentrations of circulation, liver, local and/or gastrointestinal tract found to be effective in animals. Based on animal data and other types of similar data, one skilled in the art can determine an effective amount of vaccine composition suitable for humans.

Those skilled in the art will appreciate that other variations to the embodiments described herein may be practiced without departing from the scope of the invention. Other modifications are therefore possible.

Although the present disclosure has been described and illustrated in an exemplary form with a certain degree of particularity, it should be noted that the description and illustration has been made by way of example only. Many variations in the details of construction and the combination and arrangement of parts and steps are possible. Accordingly, such modifications are intended to be included within the scope of this invention as defined in the claims.

Examples

Materials and methods:

reagent(s)

Peptides were synthesized by Genscript inc. The peptide sequences used in this experiment are shown in the following table:

name of the name	Peptide sequences
		CMV HLA A0201	NLVPMVATV
CMV HLA A2402	QYDPVAALF
		CMV HLA B0702	TPRVTGGGAM
CMV HLA B18	SDEEEAIVAYTL
		CMV HLA B3501	IPSINVHHY
CMV HLA B44	EFFWDANDIY
		Negative control (HIV HLA 0201)	LTFGWCFKL

PBMC isolation

PBMCs (peripheral blood mononuclear cells) were isolated from human peripheral blood. Whole blood was diluted 1:1 with phosphate buffer and lymphocyte separation solution (Lymphoprep was used ^TM STEMCELL Technologies) and lymphocyte separation tubes were centrifuged at 1200Xg for 15 minutes to separate PBMC.

The purpose of this step is to achieve density gradient centrifugation of the cellular components by lymphocyte separation fluid, separating PBMCs from different cells such as erythrocytes and platelets.

ELISpot

Human IFN-gamma pre-coated ELISpot kit was purchased from DAKEWEI inc. ELISpot assay was performed according to the experimental protocol. Briefly, 1X10 ⁵ PBMC cells were seeded into each well at 37℃with 5% CO ₂ After 48 hours incubation with the peptide, and photographs were taken using a nikon SMZ18 microscope.

Results:

to identify the HLA subtype of BRAF G469V donor, ELISpot assay was performed. A human IFN- γ ELISpot assay designed to detect human IFN- γ secreting cells at a single cell level can be used to quantify the frequency of human IFN- γ secreting cells. This is often used for quantification of cd8+ T cell responses. Herein, previously discovered CMV peptides of HLA a0201, HLA a2402, HLA B07, HLA B18, HLA B35, and HLA B44 were synthesized as indicators to demonstrate donor HLA subtypes.

Mu.g of CMV peptides of HLA A0201, HLA A2402, HLA B07, HLA B18, HLA B35 and HLA B44 were isolated from donor 1X10 ⁵ PBMCs were incubated for 48 hours. HIV peptide sequence (LTFGWCFKL) was used as a negative control and Phytohemagglutinin (PHA) was used as a positive control. IFN-. Gamma.points were seen in both PHA and HLA A0201 groups. Photographs were taken with a microscope after the wells were dried.

The results of figure 1 show that both HLA a0201 and positive control group were positive in ELISpot assay, indicating that the donor carried HLA a0201 subtype.

Prediction results (peptide sequence)

Candidate peptides for the BRAF G469V mutation site of HLA a0201 (from Westlake university) were predicted using a model named REDpepper. In the predictive model, high rank scores represent high binding affinities to human HLA a0201 subtype. BRAF-19 binds to MHC-I with higher affinity than other peptides, as shown in Table 1 and FIG. 2.

TABLE 1 prediction results

We then tested which peptides could activate the secretion of IFN- γ by cd8+ cells. 1 μg peptide with 1x 10 isolated from donor ⁵ PBMCs were incubated for 48 hours. 35 peptides (1. Mu.g) were isolated from the donor 1X 10 ⁵ PBMCs were incubated for 48 hours. HIV peptide sequence (LTFGWCFKL) was used as a negative control and Phytohemagglutinin (PHA) was used as a positive control. IFN-gamma points were seen in PHA, HLA A0201, and BRAF-19 groups. Photographs were taken with a microscope after the wells were dried. Of the 38 peptides, BRAF-19 peptide (QRIGSGSFVT) was sufficient to activate cd8+ cells as shown by the ELISpot assay and was significantly more immunogenic than all other BRAF peptides (including BRAF-11, 12, 13, 15, 18, 20, 21, 22, 24, 25, 30 and 31), as shown in figure 3.

To further confirm the effectiveness of the BRAF-19 epitope on BRAF G469V donors, we constructed MSCV plasmids encoding HLA A A0201 and BRAF-19 peptides, as shown in FIG. 8. Expressed HLA A0201 and BRAF-19 proteins are anchored on cell membranes, Can activate CD8+ cells. We then transfected BRAF-19MSCV and negative control MSCV plasmids into HEK 293F cells. 24 hours after transfection, at about 1:1 ratio of CD8+ cells to HEK 293F cells, 1X 10 ⁴ HEK 293F cells and 1X 10 ⁵ Is inoculated into ELISpot wells.

Specifically, 4 peptides (BRAF-28, BRAF-31, BRAF-37, BRAF-19; 1. Mu.g) were isolated from 1X 10 isolated from the donor ⁵ PBMCs were incubated for 48 hours. 293F cells transfected with BRAF-19MSCV and Neg MSCV plasmids were also transfected with 1X 10 ⁵ PBMC inoculation. HIV peptide sequence (LTFGWCFKL) was used as a negative control and Phytohemagglutinin (PHA) was used as a positive control. Photographs were taken with a microscope after the wells were dried. The results of fig. 4 show that the BRAF-19MSCV group has a significant IFN- γ point compared to the negative MSCV group.

To verify the specificity of the BRAF-19 epitope for the non-BRAF-G469V mutant population, we also performed an ELISPot assay on 16 healthy donors. Mu.g of the CMV peptide of HLA A0201, HLA A2402, HLA B MIX (peptide mixture of HLA B07, B18, B35 and B44) was mixed with 1X 10 ⁵ PBMCs were incubated for 48 hours. 293F cells transfected with BRAF-19MSCV and Neg MSCV plasmids were also transfected with 1X 10 ⁵ PBMC inoculation. HIV peptide sequence (LTFGWCFKL) was used as a negative control and Phytohemagglutinin (PHA) was used as a positive control. Photographs were taken with a microscope after the wells were dried. The difference between the BRAF-19 peptide group and the negative peptide group and the difference between the BRAF-19MSCV group and the negative MSCV group were calculated by Prism 5. The results of fig. 5 and 6 show that the difference between the BRAF-19 peptide group and the negative peptide group was 2.5 on average in 16 healthy donors, while in BRAF G469V donors the difference was 31. In the MSCV plasmid transfected group, the difference between the BRAF-19MSCV group and the negative MSCV group among 16 healthy donors was 3.3 on average, while in the BRAF G469V donor, the difference was 39.

Specifically, the results of fig. 7 show that the difference between the BRAF-19 peptide group and the negative peptide group averages 1.5 in the 4 healthy HLA a 0201 donors, while the difference between the BRAF-19MSCV group and the negative MSCV group averages 1.5 in the MSCV plasmid transfected group. The difference between the BRAF-19 peptide group and the negative peptide group and the difference between the BRAF-19MSCV group and the negative MSCV group were calculated by Prism 5.

Conclusion(s)

Taken together, we successfully predicted 38 BRAF G469V neoantigen sequences and further confirmed their ability to activate cd8+ cells by ELISpot assay. We found that only BRAF-19 neoantigenic peptide was able to activate CD8+ cells and demonstrated its specificity in 16 healthy donors by ELISPot assay, indicating that BRAF-19 has anti-tumor function for patients with BRAF G469V mutation.

While embodiments of the invention have been illustrated and described, it is not intended that these embodiments illustrate and describe all possible forms of the invention. Rather, the words used in the specification are words of description rather than limitation, and it is understood that various changes may be made without departing from the spirit and scope of the invention.

The nucleotide sequences of the MSCV plasmids encoding HLA A0201 and BRAF-19 peptides are shown in SEQ ID NO:39, as shown in:

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sequence listing

<110> Western lake biomedical technology (Hangzhou) Co., ltd

<120> New antigenic peptides and their use in the treatment of diseases related to mutation of BRAF gene

<130> PF 211213PCT

<150> PCT/CN2021/077930

<151> 2021-02-25

<160> 39

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acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60

acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 120

cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 180

attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc 240

tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 300

agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 360

ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 420

cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct 480

tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt 540

gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag 600

aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt 660

aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt 720

agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg 780

atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag 840

gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag 900

taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg 960

acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag 1020

cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag 1080

ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc 1140

tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga 1200

gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc 1260

aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg 1320

gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata 1380

aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440

ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500

acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560

ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620

agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680

tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740

tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggttaacttt 1800

taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc 1860

aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttatcga 1920

tactagagct agagctagca tgtctcgctc cgtggcctta gctgtgctcg cgctactctc 1980

tctttctggc ctggaggctc agaggatcgg cagcggcagc ttcgtgaccg gctgcggcgc 2040

ctccggtggc ggtggctccg gcggtggtgg gtccatccag cgtactccaa agattcaggt 2100

ttactcacgt catccagcag agaatggaaa gtcaaatttc ctgaattgct atgtgtctgg 2160

gtttcatcca tccgacattg aagttgactt actgaagaat ggagagagaa ttgaaaaagt 2220

ggagcattca gacttgtctt tcagcaagga ctggtctttc tatctcttgt actacactga 2280

attcaccccc actgaaaaag atgagtatgc ctgccgtgtg aaccatgtga ctttgtcaca 2340

gcccaagata gttaagtggg atcgagacat gggtggcggt ggctccggcg gtggtgggtc 2400

cggtggcggt ggctccggcg gtggtgggtc cggcagccac agcatgaggt acttcttcac 2460

cagcgtgagc aggcccggca ggggcgagcc caggttcatc gccgtgggct acgtggacga 2520

cacccagttc gtgaggttcg acagcgacgc cgccagccag aggatggagc ccagggcccc 2580

ctggatcgag caggagggcc ccgagtactg ggacggcgag accaggaagg tgaaggccca 2640

cagccagacc cacagggtgg acctgggcac cctgaggggc tactacaacc agagcgaggc 2700

cggcagccac accgtgcaga ggatgtacgg ctgcgacgtg ggcagcgact ggaggttcct 2760

gaggggctac caccagtacg cctacgacgg caaggactac atcgccctga aggaggacct 2820

gaggagctgg accgccgccg acatggccgc ccagaccacc aagcacaagt gggaggccgc 2880

ccacgtggcc gagcagctga gggcctacct ggagggcacc tgcgtggagt ggctgaggag 2940

gtacctggag aacggcaagg agaccctgca gaggaccgac gcccccaaga cccacatgac 3000

ccaccacgcc gtgagcgacc acgaggccac cctgaggtgc tgggccctga gcttctaccc 3060

cgccgagatc accctgacct ggcagaggga cggcgaggac cagaccaccg agctggtgga 3120

gaccaggccc gccggcgacg gcaccttcca gaagtgggcc gccgtggtgg tgcccagcgg 3180

ccaggagcag aggtacacct gccacgtgca gcacgagggc ctgcccaagc ccctgaccct 3240

gaggtgggag atggccgccg ccctggaggt gtccttaagt accactgagg tggcaatgca 3300

cacttcaact tcttcttcag tcacaaagag ttacatctca tcacagacaa atgatacgca 3360

caaacgggac acatatgcag ccactcctag agctcatgaa gtttcagaaa tttctgttag 3420

aactgtttac cctccagaag aggaaaccgg agaaagggta caacttgccc atcatttctc 3480

tgaaccagag ataacactca ttatttttgg ggtgatggct ggtgttattg gaacgatcct 3540

cttaatttct tacggtattc gccgactgat aaagaaaagc ccatctgatg taaaacctct 3600

cccctcacct gacacagacg tgcctttaag ttctgttgaa atagaaaatc cagagacaag 3660

tgatcaagcg gccgctgagg gcagaggaag tcttctaaca tgcggtgacg tggaggagaa 3720

tcccggccct tccggaatgg agagcgacga gagcggcctg cccgccatgg agatcgagtg 3780

ccgcatcacc ggcaccctga acggcgtgga gttcgagctg gtgggcggcg gagagggcac 3840

ccccaagcag ggccgcatga ccaacaagat gaagagcacc aaaggcgccc tgaccttcag 3900

cccctacctg ctgagccacg tgatgggcta cggcttctac cacttcggca cctaccccag 3960

cggctacgag aaccccttcc tgcacgccat caacaacggc ggctacacca acacccgcat 4020

cgagaagtac gaggacggcg gcgtgctgca cgtgagcttc agctaccgct acgaggccgg 4080

ccgcgtgatc ggcgacttca aggtggtggg caccggcttc cccgaggaca gcgtgatctt 4140

caccgacaag atcatccgca gcaacgccac cgtggagcac ctgcacccca tgggcgataa 4200

cgtgctggtg ggcagcttcg cccgcacctt cagcctgcgc gacggcggct actacagctt 4260

cgtggtggac agccacatgc acttcaagag cgccatccac cccagcatcc tgcagaacgg 4320

gggccccatg ttcgccttcc gccgcgtgga ggagctgcac agcaacaccg agctgggcat 4380

cgtggagtac cagcacgcct tcaagacccc catcgccttc gccagatccc gcgctcagtc 4440

gtccaattct gccgtggacg gcaccgccgg acccggctcc accggatctc gctagagctg 4500

aatctaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct 4560

taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc 4620

tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct 4680

ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga 4740

cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc 4800

tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac 4860

aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt 4920

tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt 4980

cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc 5040

tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc 5100

gcctggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa 5160

aagaaaaggg gggactggaa gggctaattc actcccaacg atgtcaagaa ttgtgaaaga 5220

ccccacctgt aggtttggca agttagctta agtaacgcca ttttgcaagg catggaaaat 5280

acataactga gaatagagaa gttcagatca aggttaggaa cagagagaca gcagaatatg 5340

ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg 5400

gtccccagat gcggtcccgc cctcagcagt ttctagcgaa ccatcagatg tttccagggt 5460

gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag tttgcttctt 5520

gcttctgttt gtgtgcttct gctccctgag ctcaataaaa gagcccacaa cccctcactt 5580

ggtgggccag tcctctgata gactgtgtcc cctggatacc cgtatatcga ttctgctttt 5640

tgcttctact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact 5700

agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc 5760

ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa 5820

aatctctagc agtagtagtt catgtcatct tattattcag tatttataac ttgcaaagaa 5880

atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt acaaataaag 5940

caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt 6000

gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc cctaactccg 6060

cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc 6120

gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta 6180

gacttttgca gagacggccc aaattcgtaa tcatggtcat agctgtttcc tgtgtgaaat 6240

tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 6300

ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 6360

tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 6420

ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 6480

ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 6540

gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 6600

gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 6660

cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 6720

ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 6780

tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 6840

gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 6900

tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 6960

ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 7020

ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 7080

ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 7140

accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 7200

tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 7260

cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 7320

taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 7380

caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 7440

gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 7500

gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 7560

ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 7620

attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 7680

gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 7740

tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 7800

agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 7860

gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 7920

actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 7980

tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 8040

attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 8100

tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 8160

tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 8220

aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 8280

tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 8340

cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta 8400

acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt 8460

gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc 8520

gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt 8580

aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg 8640

cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac 8700

tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga 8760

tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa 8820

acgacggcca gtgccaagct g 8841

Claims (27)

1. An isolated neoantigenic peptide, wherein the isolated neoantigenic peptide comprises the amino acid sequence set forth in SEQ ID No. 19 (QRIGSGSFVT), or a functionally equivalent variant of SEQ ID No. 19.

2. The isolated neoantigenic peptide of claim 1, wherein the isolated neoantigenic peptide comprises a tumor-specific neoepitope capable of binding MHC-1 to form an MHC-neoantigenic complex.

3. The isolated neoantigenic peptide of claim 1 or 2, wherein the functionally equivalent variant has at least 80% sequence identity to SEQ ID No. 19 or comprises one or two amino acid changes compared to SEQ ID No. 19.

4. The isolated neoantigenic peptide of claim 3, wherein the amino acid change is a conservative amino acid substitution.

5. The isolated neoantigenic peptide of any one of claims 1-4, wherein the isolated neoantigenic peptide is linked to one or more additional neoantigenic peptides, optionally via a linker, such as a polyglycine or polyserine linker.

6. The isolated neoantigenic peptide of any one of claims 1-5, wherein the isolated neoantigenic peptide is about 10 to 30 amino acids in length, such as about 10 to 20 amino acids in length, such as 10, 11, 12, 13, 14 and 15 amino acids in length.

7. The isolated neoantigenic peptide of any one of claims 1-6, wherein the isolated neoantigenic peptide binds MHC-1 with a binding affinity of about 500nM or less, such as about 250nM or less, or about 50nM or less.

8. A polynucleotide encoding the neoantigenic peptide of any one of claims 1 to 7.

9. A vector comprising the polynucleotide of claim 8.

10. The vector of claim 9, wherein the vector is selected from the group consisting of a plasmid, a cosmid, an RNA formulated in a particle, a self-amplifying RNA (SAM), a SAM formulated in a particle, or a viral vector.

11. The vector of claim 10, wherein the viral vector is an alphavirus vector, a Venezuelan Equine Encephalitis (VEE) viral vector, a sindbis viral vector, a semliki forest viral vector, a simian or human cytomegalovirus vector, a lymphocytic choriomeningitis viral vector, a retrovirus vector, a lentivirus vector, an adenovirus vector, or a combination thereof.

12. A composition comprising the isolated neoantigenic peptide of any one of claims 1 to 7, optionally in the form of an in vivo delivery system, such as nanoparticle encapsulation, virus-like particles, liposomes, or any combination thereof.

13. A composition comprising the polynucleotide of claim 8 and/or the vector of any one of claims 9-11, optionally in the form of an in vivo delivery system, such as a virus, virus-like particle, plasmid, bacterial plasmid, nanoparticle, or any combination thereof.

14. The composition of claim 12 or 13, wherein the composition further comprises at least one modulator or immunomodulator of a checkpoint molecule, or a nucleic acid encoding the modulator or immunomodulator, or a vector comprising a nucleic acid encoding the modulator or immunomodulator.

15. The composition of claim 14, wherein the modulator of the checkpoint molecule is selected from the group consisting of: (a) Agonists of members of the tumor necrosis factor receptor superfamily, preferably agonists of CD27, CD40, 0X40, GITR or CD 137; (b) Antagonists of PD-1, PD-L1, CD274, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3, VISTA, or antagonists of members of the B7-CD28 superfamily, preferably antagonists of CD28 or ICOS, or antagonists of its ligands; or wherein the immunomodulator is a T cell growth factor, preferably IL-2, IL-12 or IL-15.

16. The composition of any one of claims 12 to 15, further comprising one or more adjuvants.

17. A T Cell Receptor (TCR) capable of binding the neoantigenic peptide of any one of claims 1-7 or an MHC-I-peptide complex comprising the neoantigenic peptide of any one of claims 1-7.

18. A chimeric antigen receptor comprising: (i) a T cell activating molecule; (ii) a transmembrane region; and (iii) an antigen recognition moiety capable of binding to the neoantigenic peptide of any one of claims 1 to 7 or to an MHC-peptide complex comprising the neoantigenic peptide of any one of claims 1 to 7.

19. A T cell comprising the T cell receptor of claim 17 or the chimeric antigen receptor of claim 18.

20. The T cell of claim 19, wherein the T cell is a T cell isolated from a population of T cells from a subject, the population of T cells having been incubated with an antigen presenting cell, such as an artificial antigen presenting cell, and the neoantigen peptide of any one of claims 1-7 for a time sufficient to activate the T cell.

21. The T cell of claim 19 or 20, wherein the T cell is a cd8+ T cell or a cytotoxic T cell.

22. A method of activating tumor-specific T cells, the method comprising: (a) isolating a population of T cells from a subject; and (b) incubating the isolated population of T cells with antigen presenting cells, such as artificial antigen presenting cells, and the neoantigenic peptide of any one of claims 1-7 for a time sufficient to activate the T cells.

23. A modified cd8+ T cell transfected or transduced with a nucleic acid encoding the TCR of claim 17 or the chimeric antigen receptor of claim 18.

24. A composition comprising the T cell of any one of claims 19-21, an activated tumor-specific T cell produced by the method of claim 22, and/or the modified cd8+ T cell of claim 23.

25. A method of treating or preventing BRAF mutation related cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of the isolated neoantigenic peptide of any one of claims 1 to 7, the polynucleotide of claim 8, the vector of any one of claims 9-11, the composition of any one of claims 12-16 and 24, the T cell of any one of claims 19-21, an activated tumor-specific T cell produced by the method of claim 22, and/or the modified cd8+ T cell of claim 23.

26. A method of inhibiting the growth of a tumor cell bearing a BRAF mutation, the method comprising administering to the subject a therapeutically effective amount of the isolated neoantigenic peptide of any one of claims 1-7, the polynucleotide of claim 8, the vector of any one of claims 9-11, the composition of any one of claims 12-16 and 24, the T cell of any one of claims 19-21, an activated tumor-specific T cell produced by the method of claim 22, and/or the modified cd8+ T cell of claim 23.

27. The method of claim 25 or 26, wherein the BRAF mutation is a BRAF G469V mutation.