WO2022179568A1

WO2022179568A1 - Neoantigenic peptide and its use for treating braf gene mutation related diseases

Info

Publication number: WO2022179568A1
Application number: PCT/CN2022/077710
Authority: WO
Inventors: Xiaofei GAO; Yang YUAN; Yun GE; Shao Xu
Original assignee: Westlake Therapeutics (Hangzhou) Co., Limited
Priority date: 2021-02-25
Filing date: 2022-02-24
Publication date: 2022-09-01
Also published as: CN117377683A

Abstract

The present disclosure provides an isolated neoantigenic peptide comprises a tumor-specific neoepitope that is capable of binding to MHC-1 to form a MHC-neoantigen complex, a neoantigen binding peptide, T cells comprising TCR or CAR comprising the neoantigen binding peptide, and their use for the immunotherapy of cancer, especially for cancer having a BRAF G469V mutation.

Description

Neoantigenic peptide and its use for treating BRAF gene mutation related diseases

TECHNICAL FIELD

The present application generally relates to immunotherapeutic peptides, nucleic acids encoding the peptides, peptide binding agents, and their use, for example, in the immunotherapy of cancer, and particularly to neoantigenic peptide and its use for treating BRAF gene mutation related diseases.

BACKGROUND

Lung cancer is by far the leading cause of cancer death among both men and women, making up almost 25%of all cancer deaths. Lung cancers usually are grouped into two main types called small cell and non-small cell. Non-small cell lung cancer (NSCLC) is more common than small cell lung cancer, accounting for 85%of all lung cancers. In recent years, the clinical application of immunotherapy, represented by immunosuppressant (ICIs) anti-PD-1/PD-L1 antibodies, has brought long-term survival hopes to patients with advanced NSCLC. The latest data show that the five-year survival rate of patients with advanced NSCLC using PD-1 antibodies in the first line has reached 23.2%, and the five-year survival rate of patients treated with PD-1 antibodies on the second line has reached 16%, which is 2-3 times higher than traditional treatment.

Unfortunately, studies to date have shown that late-stage NSCLCs carrying gene mutations rarely benefit from ICIs immunotherapy for complex reasons, with fewer new antigens in patients with genetic mutations and the inability to stimulate the body's effective immune response considered to be one of the main reasons. How to enhance the response ability of NSCLC patients with gene mutation to ICIs, so that they can still benefit from follow-up immunotherapy after targeted treatment resistance, is of great significance to improve the five-year survival rate of patients with late NSCLC, and is an important problem that needs to be solved urgently in clinical practice.

Effective anti-tumor immunity in human has been associated with the presence of T cells directed at cancer neoantigens, a class of HLA-bound peptides that arise from tumor-specific mutations and emerging data suggest that recognition of such neoantigens is a major factor in the activity of clinical immunotherapies. Massively parallel whole exome sequencing has been used to detect all mutations within the tumor to predict neoantigens. Vaccination with neoantigens can expand pre-existing neoantigen specific T cell populations and induce new cancer specific T cells. Thus, neoantigens have emerged as potentially ideal targets for anti-tumor immune responses.

BRAF gene is a proto-oncogene found on chromosome 7 with a wide type amino acid as shown in e.g., NCBI Reference Sequence: NP_001365403.1, and becomes an oncogene when mutated. Although BRAF mutation and its connection to cancer was only identified in 2002, research done since then has pinpointed hundreds of different types of mutation that could be associated with cancers. BRAF mutations were categorized into class-1 (kinase-activated, codon 600) , class-2 (kinase-activated, non-codon 600) and class-3 (kinase-impaired) , based on the newly proposed classification scheme (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, mutations do not mean successful identification of neoantigens having immunogenicity, not to mention the capability of inducing antigen-specific cytotoxic T cells (CTLs) that recognize and lyse cancer or tumor cells. As a matter of fact, only a very low proportion of mutations can lead to meaningful neoantigenic peptides.

Accordingly, there is still a need in the art for developing additional cancer therapeutics.

SUMMARY

In an aspect, provided is an isolated neoantigenic peptide, wherein the isolated neoantigenic peptide comprises, consisting essentially of or consisting of an amino acid sequence as shown 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 that is capable of binding to MHC-I to form a MHC-neoantigen complex.

In some embodiments, the functionally equivalent variant has at least 80%sequence identity to SEQ ID NO: 19 or comprises one or two amino acid alterations as compared to SEQ ID NO: 19.

In some embodiments, the amino acid alterations are conservative amino acid substitutions.

In some embodiments, the isolated neoantigenic peptide is linked, optionally via a linker such as a poly-glycine or poly-serine linker, to one or more additional neoantigenic peptide.

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

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

In an aspect, provided is a polynucleotide encoding the neoantigenic peptide as described herein.

In an aspect, provided is a vector comprising the polynucleotide as described herein.

In some embodiments, the vector is selected from the group consisting of a plasmid, a cosmid, a RNA, a 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 alpha virus vector, a Venezuelan equine encephalitis (VEE) virus vector, a sindbis virus vector, a semliki forest virus vector, a simian or human cytomegalovirus vector, a lymphocyte choriomenigitis virus vector, a retroviral vector, a lentiviral vector, an adenovirus vector, or combination thereof.

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

In an aspect, provided is a composition comprising the polynucleotide and/or the vector as described herein, optionally the composition is in the form of in vivo delivery system for example viruses, virus-like particles, plasmids, bacterial plasmids, nanoparticles, or any combination thereof.

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

In some embodiments, the modulator of a checkpoint molecule is selected from the group consisting of: (a) an agonist of a tumor necrosis factor receptor superfamily member, preferably of CD27, CD40, 0X40, GITR, or CD137; and (b) an antagonist of PD-1, PD-L1, CD274, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3, VISTA, or an antagonist of a B7-CD28 superfamily member, preferably of CD28 or ICOS or an antagonist of a ligand thereof; 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 an aspect, provided is a T cell receptor (TCR) capable of binding the neoantigenic peptide as described herein or an MHC-I-peptide complex comprising the neoantigenic peptide as described herein.

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

In an aspect, provided is a T cell comprising the T cell receptor or the chimeric antigen receptor as 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 antigen presenting cells such as artificial antigen presenting cells and the neoantigenic peptide as 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 an aspect, provided is a method for activating tumor specific T cells 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 as described herein for a sufficient time to activate the T cells.

In an aspect, provided is a modified CD8+ T cell transfected or transduced with a nucleic acid encoding the TCR as described herein or the chimeric antigen receptor as described herein.

In an aspect, provided is a composition comprising the T cell as described herein, the activated tumor specific T cells produced by the method as described herein, and/or the modified CD8+ T cell as described herein.

In an aspect, provided is a method of treating or preventing a BRAF mutation-related cancer in a subject comprising administering to the subject a therapeutically effective amount of the isolated neoantigenic peptide as described herein, the polynucleotide as described herein, the vector as described herein, the composition as described herein, the T cell as described herein, the activated tumor specific T cells produced by the method as described herein, and/or the modified CD8+ T cell as described herein.

In an aspect, provided is a method of inhibiting growth of a tumor cell having a BRAF mutation, comprising administering to the subject a therapeutically effective amount of the isolated neoantigenic peptide as described herein, the polynucleotide as described herein, the vector as described herein, the composition as described herein, the T cell as described herein, the activated tumor specific T cells produced by the method as described herein, and/or the modified CD8+ T cell as described herein.

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

BRIEF DESCRIPTION OF THE DRAWINGS

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

Fig. 1 shows HLA I ELISpot results of BRAF G469V donor.

Fig 2 shows candidate peptides prediction results of BRAF G469V mutation site. In the prediction model, a high rank score indicates the high binding affinity.

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

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

Fig. 5 shows ELISpot results of a healthy honor.

Fig. 6 shows ELISpot analysis results of 16 healthy donor.

Fig. 7 shows ELISpot analysis results of 4 HLA A 0201 healthy donor.

Fig. 8 shows plasmid profile of MSCV plasmid which codes HLA A 0201 and BRAF-19 peptide.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of this disclosure is thereby intended.

In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings as generally understood by a person skilled in the art. Although any methods and materials similar or equivalent to those described herein find use 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 Specification as a whole.

As used herein, the singular terms “a, ” “an, ” and “the” include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. 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 they are used by those of skills in the art.

As used herein, the term “consisting essentially of” in the context of an amino acid sequence is meant the recited amino acid sequence together with additional one, two, three, four or five amino acids at the N-or C-terminus.

Unless the context requires otherwise, the terms “comprise” , “comprises” and “comprising” , or similar terms are intended to mean a non-exclusive inclusion, such that a recited list of elements or features does not include those stated or listed elements solely, but may include other elements or features that are not listed or stated.

The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent 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 such as a parameter, an amount or the like, are meant to encompass variations of the given value, such as variations of +/-10%or less, +/-5%or less, +/-1%or less, and +/-0.1%or less of and from the given value.

As used herein, the terms “patient” , “individual” and “subject” are used in the context of any mammalian recipient of a treatment or composition disclosed herein. Accordingly, the methods and composition 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 meant to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the 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 “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) .

Neoantigens /Neoantigenic peptides

As used herein, the term “neoantigen” or “neoantigenic peptide” is meant to refer to a class of tumor antigens which are derived from tumor-specific changes in proteins. Neoantigens encompass, but are not limited to, tumor antigens arising from, for example, substitution in the protein sequence, frame shift mutation, in-frame deletion, insertion, and tumor-specific overexpression of polypepitdes. The term “tumor-specific changes” refers to changes that are not present in a normal non-cancerous cell but is found in cancer or tumor cells.

Neoantigens have rarely been used in cancer vaccine or immunogenic compositions due to technical difficulties in identifying them, selecting optimized antigens, and producing neoantigens for use in a vaccine or immunogenic composition.

Neoantigens binding MHC is a dynamic process and no chemical change occurs during its transportation. Since the level of T cells activation is determined by the concentration of successful MHC-neoantigen complex, the task that hunts active neoantigen turns to identifying stable MHC-peptide complex. Neural network based learning approaches with validated binding and non-binding peptides have advanced the accuracy of prediction algorithms for identifying stable MHC-peptide complex.

Sequencing methods are used to identify tumor specific mutations. Any suitable sequencing method can be used, for example, Next Generation Sequencing (NGS) technologies. “NGS” may include all novel high throughput sequencing technologies which read nucleic acid templates randomly in parallel along the entire genome by breaking the entire genome into small pieces. Such NGS technologies (massively parallel sequencing technologies) are able to deliver nucleic acid sequence information of a whole genome, exome, transcriptome (all transcribed sequences of a genome) or methylome (all methylated sequences of a genome) in very short time periods.

Herein, the inventors predicted neoantigen peptide sequences for patients with BRAF G469V mutation by NGS and machine learning approaches, and confirmed that BRAF-19 peptide as shown in SEQ ID NO: 19 is capable of activating CD8+ cells, suggesting its anti-tumor function in patients with BRAF G469V mutation.

The BRAF G469V mutation arises from a single nucleotide change (c. 1406G>T) and results in an amino acid substitution of the glucine (G) at position 469 by a valine (V) . BRAF G469V is present in 0.07%of AACR GENIE cases, with lung adenocarcinoma, colon adenocarcinoma, bladder urothelial carinoma, cancer of unknown primary, and cancer of unknown primary.

In one aspect, the present disclosure provides an isolated neoantigenic peptide, wherein the isolated neoantigenic peptide comprises an amino acid sequence as shown in SEQ ID NO: 19 (QRIGSGSFVT) or a functionally equivalent variant of SEQ ID NO: 19.

The term “isolated” refers to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Accordingly, isolated neoantigenic peptide described herein does not contain some or all of the materials normally associated with the peptide in their in situ environment. An “isolated” neoantigenic peptide refers to a neoantigenic epitope that does not include the whole sequence of the antigen from which the epitope was derived.

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

In some embodiments, the present disclosure envisages the functionally equivalent variant of the isolated neoantigenic peptide as identified herein. As used herein, the term “functionally equivalent variant” is meant to refer to variant polypeptide species that have one or more amino acid substitutions, insertions, or deletions as compared to the reference peptide, provided that the variant retains or substantially retains its specific binding function. In some embodiments, the functionally equivalent variants have at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%or higher) sequence identity to SEQ ID NO: 19 or comprises one or two amino acid alterations as compared to SEQ ID NO: 19.

In some embodiments, the amino acid alterations are conservative amino acid substitutions. A “conservative amino acid substitution” refers to one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues 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 a phenylalanine for a tyrosine is a conservative substitution. Another example is substitution of glutamine for glutamic acid or asparagine for aspartic acid, which may be considered as a similar substitution in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate peptide function are known to persons skilled in the art.

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

A variety of linkers such as a poly-glycine or poly-serine linker known in the art can be used in the present disclosure. For example, short oligo-or polypeptide linkers, preferably between 2 and 10 amino acids in length, may form the linkage between peptides. A glycine-serine doublet provides a particularly suitable linker.

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

In some embodiments, the isolated neoantigenic peptide binds major histocompatibility complex (MHC) class I. In some embodiments, the isolated neoantigenic peptide binds MHC class I (MHC-I) with a binding affinity of about 500 nM or less. In some embodiments, the isolated neoantigenic peptide binds MHC-I with a binding affinity of about 250 nM or less. In some embodiments, the isolated neoantigenic peptide binds MHC-I with a binding affinity of about 50 nM or less. As used herein, the term “Major Histocompatibility Complex” or “MHC” is a cluster of genes that plays a role in control of the cellular interactions responsible for physiologic immune responses. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. 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 peptide as described herein has a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles.

In some embodiments, the isolated neoantigenic peptide may further comprise a modification which increases in vivo half-life, cellular 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, nanoparticulate encapsulation, conjugation to a ligand, conjugation to an antibody, albumin fusion, Fc fusion, cholesterol fusion, PEGylation, acylation, amidation, glycosylation, phosphorylation, biotinylation, or addition of unnatural amino acids.

In some embodiments, provided herein are polynucleotides encoding the neoantigenic peptides as described herein. Persons skilled in the art would appreciate that various nucleic acid sequences can encode the same peptide due to genetic code redundancy, and each of the nucleic acids falls within the scope of the present invention. The coding nucleic acids can be DNA or RNA, for example, mRNA, or a combination thereof. In some embodiments, a nucleic acid encoding a peptide is a self-amplifying mRNA. Any suitable polynucleotide that encodes the neoantigenic peptide described herein falls within the scope of this invention.

In some embodiments, provided herein is a vector such as an expression vector useful for the production and administration of the neoantigenic peptides described herein. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host (e.g., bacteria) , although such controls are generally available in the expression vector. The vector is then introduced into the 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 as described herein as well as host cells containing the expression vectors are also contemplated. The neoantigenic peptides may be provided in the form of RNA or cDNA molecules encoding the desired neoantigenic peptides. One or more neoantigenic peptides of the invention may be encoded by a single expression vector.

A vector system comprising an expression vector is provided herein, wherein the expression vector may be selected from the group consisting of a plasmid, a cosmid, a RNA, a RNA formulated in a particle, a self-amplifying RNA (SAM) , a SAM formulated in a particle, or a viral vector. In some embodiments, viral vectors may be an alpha virus vector, a Venezuelan equine encephalitis (VEE) virus vector, a sindbis virus vector, a semliki forest virus vector, a simian or human cytomegalovirus vector, a lymphocyte choriomenigitis virus vector, a retroviral vector, a lentiviral vector, an adenovirus vector, or combination thereof.

Suitable host cells for expression of a polypeptide include prokaryotes, yeast, insect or higher eukaryotic cells under the control of suitable promoters. Prokaryotes include gram negative or gram positive organisms, for example E. coli. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems could also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are well known in the art.

Neoantigen binding peptides

In an aspect, the present disclosure provides a binding protein (e.g., an antibody or antigen-binding fragment thereof) , or a T cell receptor (TCR) , or a chimeric antigen receptor (CAR) capable of binding with a high affinity to the neoantigenic peptide as described herein or a MHC (HLA) -neoantigenic peptide complex as described herein. In some embodiments, the antibody is a monoclonal antibody specific for the neoantigenic peptide described herein.

In some embodiments, provided herein is a T cell receptor (TCR) capable of binding the neoantigenic peptide described herein or an MHC-peptide complex comprising the neoantigenic peptide 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, which further comprises 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 for example CD3. In some embodiments, the antibody or antibody fragment is an anti-CD3 scFv.

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

T cells

In an aspect, the present disclosure provides a T cell comprising the T cell receptor or the chimeric antigen receptor as described herein. Preferably, the T cell is a CD8+T cell or a cytotoxic T cell.

In some embodiments, provided herein is a T cell comprising a T cell receptor (TCR) capable of binding the neoantigenic peptide described herein or an MHC-peptide complex comprising the neoantigenic peptide described herein, wherein the T cell is a T cell isolated from a population of T cells from a subject that has been incubated with antigen presenting cells such as artificial antigen presenting cells and the 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 cytotoxic T cell. In some embodiments, the population of T cells from a subject is a population of CD8+ T cells from the subject. In some embodiments, the activated CD8+ T cells are separated from the 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-associated peptides from their surface. In some embodiments, the treatment to strip the endogenous MHC-associated peptides comprises treating the cells with a mild acid solution. In some embodiments, the antigen presenting cells have been pulsed with the neoantigenic peptide as 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 peptide described herein. In some embodiments, the ratio of isolated T cells to antigen presenting cells is between about 0.1 : 1 and 300 : 1 such as between about 0.5 : 1 and 200 : 1, between about 1 : 1 and 150 : 1. In some embodiments, the incubating of the isolated population of T cells is in the presence of IL-2 and IL-7. 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 peptide described herein for a sufficient time to activate the T cells. In some embodiments, the T cell is a CD8+ T cell or cytotoxic T cell. In some embodiments, the population of T cells from a subject is a population of CD8+ T cells from the subject. In some embodiments, the activated CD8+ T cells are separated from the 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-associated peptides from their surface. In some embodiments, the treatment to strip the endogenous MHC-associated peptides comprises treating the cells with a mild acid solution. In some embodiments, the antigen presenting cells have been pulsed with the neoantigenic peptide as 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 peptide described herein. In some embodiments, the ratio of isolated T cells to antigen presenting cells is between about 0.1 : 1 and 300 : 1 such as between about 0.5 : 1 and 200 : 1, between about 1 : 1 and 150 : 1. In some embodiments, the incubating of the isolated population of T cells is in the presence of IL-2 and IL-7. In some embodiments, the MHC of the MHC-peptide is MHC class I.

In some embodiments, provided herein is a modified T cell transfected or transduced with a nucleic acid encoding the TCR or the chimeric antigen receptor as described herein. The polynucleotide or nucleic acid as described herein is capable of transducing T cell, thus resulting in modified T cells having the TCR or CAR as described herein. In some embodiments, the T cell is a CTL. In some embodiments, CTL is injected into the patient.

Compositions

In an aspect, the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising the isolated neoantigenic peptide, the antibody or antigen-binding fragment thereof, the polynucleotide, the T cells comprising the TCR or CAR, the activated tumor specific T cells, and/or the modified CD8+ T cells, as described herein.

In some embodiments, the present invention is directed to an immunogenic composition such as a vaccine composition that is capable of raising a neoantigen-specific response (e.g., a cell-mediated or humoral immune response) . In some embodiments, the immunogenic composition comprises neoantigen therapeutics (e.g., peptides, polynucleotides, cells containing TCR or CAR, antibody, etc. ) described herein corresponding to the tumor specific neoantigen (e.g., BRAF-19) identified herein. In some embodiments, the immunogenic composition described herein is capable of raising a specific cytotoxic T cells response or a B cell response. In some embodiments, provided herein is a composition comprising autologous subject T cells containing the T cell receptor or chimeric antigen receptor described herein.

In some embodiments, the composition as described herein further comprises at least one modulator of a checkpoint molecule or an immunomodulator, or a nucleic acid encoding the modulator or immunomodulator, or a vector comprising the nucleic acid encoding the modulator or immunomodulator. In some embodiments, the modulator of a checkpoint molecule is selected from the group consisting of: (a) an agonist of a tumor necrosis factor receptor superfamily member, preferably of CD27, CD40, 0X40, GITR, or CD137; and (b) an antagonist of PD-1, PD-L1, CD274, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3, VISTA, or an antagonist of a B7-CD28 superfamily member, preferably of CD28 or ICOS or an antagonist of a ligand thereof. In some embodiments, the modulator of a checkpoint molecule is an inhibitor of checkpoint molecule selecting 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 checkpoint molecule interacts with a ligand of checkpoint molecule selecting 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 co-stimulatory ligand, a TNF ligand, an Ig superfamily ligand, CD28, CD80, CD86, ICOS, CD40L, OX40, 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 composition described herein can further comprise an adjuvant and/or a carrier. Adjuvants are a substance whose admixture into the immunogenic composition can increase or otherwise modify the immune response to the therapeutic agent. Carriers are scaffold structures, for example a polypeptide or a polysaccharide, to which a neoantigenic polypeptide or polynucleotide, is capable of being associated. Optionally, adjuvants are conjugated covalently or non-covalently to the polypeptides or polynucleotides described herein.

In some embodiments, the adjuvant is selected from the group consisting of aluminium salts, Amplivax, monophosphoryl lipid A, resiquimod, beta-glucan, acrylic or methacrylic 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 206 VG, Montanide ISA 50 V2, Montanide ISA 51 VG, OK-432, OM-174, OM-197-MP-EC, ONTAK,

SRL172, YF-17D, R848, Pam3Cys, and Pam3CSK4.

In some embodiments, the carrier can for example be to confer stability, to increase the biological activity, or to increase serum half-life. The carrier can be any suitable carriers known to the person skilled in the art, for example a protein or an antigen presenting cell. A carrier protein could include but 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 local administration. In some embodiments, the pharmaceutical compositions described herein are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. In some embodiments, described herein are compositions for parenteral administration which comprise a solution of the neoantigenic peptide, and immunogenic compositions are dissolved or suspended in an acceptable carrier, for example, an aqueous carrier. A variety of aqueous carriers can be used, e.g., water, buffered water, saline, and the like. These compositions can be sterilized by conventional, well known sterilization techniques, or can be sterile filtered.

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

The neoantigenic peptides and polynucleotides described herein can also be administered via liposomes, which target the peptides to a particular cells tissue, such as lymphoid tissue. Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, for example, cholesterol.

In some embodiments, the pharmaceutical compositions can further include a pharmaceutically acceptable excipient, carrier, buffer, stabilizer 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 ingredients. The carrier or other material can be selected depending on the route of administration. Acceptable carriers, excipients, or stabilizers are those that are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; 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 counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes) ; and/or non-ionic surfactants such as

or polyethylene glycol (PEG) .

In some embodiments, provided is a vaccine comprising neoantigen therapeutics (e.g., peptides, polynucleotides, cells containing TCR or CAR, antibody, etc. ) described herein corresponding to the tumor specific neoantigen (e.g., BRAF-19) identified herein. Vaccine can be delivered via a variety of routes. Delivery routes can include but not limited to oral (including buccal and sub-lingual) , parenteral (including intramuscular, intraarterial, intrathecal, intradermal, intraperitoneal, subcutaneous and intravenous) , rectal, vaginal, topical, nasal, transdermal patch, pulmonary, or suppository administration or in a form suitable for administration by aerosolization, inhalation or insufflation. General information on drug delivery systems can be found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams &Wilkins, Baltimore Md. (1999) . The vaccine described herein can be administered to muscle, or can be administered via intradermal or subcutaneous injections, or transdermally, such as by iontophoresis. Epidermal administration of the vaccine can be employed.

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

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

In some embodiments, the vaccine can include material for a single immunization, or for multiple immunizations (e.g., a “multi-dose” kit) . The inclusion of a preservative is preferable in multi-dose compositions. As an alternative (or in addition) to including a preservative in multi-dose compositions, the compositions can be contained in a container having an aseptic adaptor for removal of material. In some embodiments, the vaccine can be administered in a dosage volume of about 0.5 mL or higher, although a half dose (e.g., about 0.25 mL) can be administered depending on the condition and age of the subject. In some embodiments, the vaccine can be administered in a higher dose e.g. about 1 mL.

In some embodiments, the vaccine can be administered as a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dose-course 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 dose and second dose can 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 longer. The dosage examples are not limiting and are only used to exemplify particular dosing regiments for administering a vaccine described herein.

Methods

In an aspect, the present disclosure provides the use of the neoantigen therapeutics (e.g., peptides, polynucleotides, cells containing TCR or CAR, antibody, etc. ) described herein in a variety of applications including, but not limited to, treatment and/or prevention methods, such as the treatment of cancer. In some embodiments, the treatment methods comprise immunotherapy. In certain embodiments, the neoantigenic peptide as described herein is 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 methods can be in vitro, ex vivo, or in vivo methods. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing T cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CTL activity.

In some embodiments, the present disclosure provides a method of treating or preventing a cancer in a subject comprising administering to the subject a therapeutically effective amount of the isolated neoantigenic peptide, the polynucleotide, the vector, the composition, the T cell, the activated tumor specific T cells, and/or the modified CD8+ T cell as described herein. In some embodiments, the present disclosure provides a method of inhibiting growth of a tumor cell comprising administering to the subject a therapeutically effective amount of the isolated neoantigenic peptide, the polynucleotide, the vector, the composition, the T cell, the activated tumor specific T cells, and/or the modified CD8+ T cell as described herein. In some embodiments, the present disclosure provides the isolated neoantigenic peptide, the polynucleotide, the vector, the composition, the T cell, the activated tumor specific T cells, and/or the modified CD8+ T cell as described herein for use in treating or preventing a cancer in a subject or for inhibiting growth of a tumor cell. In some embodiments, the present disclosure provides the use of the isolated neoantigenic peptide, the polynucleotide, the vector, the composition, the T cell, the activated tumor specific T cells, and/or the modified CD8+ T cell as described herein in the manufacture of a pharmaceutical composition (e.g., an immunogenic composition or a vaccine) or a medicament for treating or preventing a cancer in a subject or for inhibiting growth of a tumor cell. In some embodiments, the cancer is a BRAF mutation-related cancer. In some embodiments, the tumor cell has a BRAF mutation. In some embodiments, the BRAF mutation is BRAF G469V mutation.

In some embodiments, the cancer is selected from the group consisting of: adrenal, bladder, breast, cervical, colorectal, glioblasoma, head and neck, kidney chromophobe, kidney clear cell, kidney papillary, liver, lung adenocarcinoma, lung squamous, ovarian, pancreatic, melanoma, stomach, uterine corpus endometrial, and uterine carcinosarcoma. In some embodiments, the cancer is selected from the group consisting of: prostate cancer, bladder, lung squamous, NSCLC, breast, head and neck, lung adenocarcinoma, GBM, Glioma, CML, AML, supretentorial ependyomas, acute promyelocytic leukemia, solitary fibrous tumors, and crizotinib resistant cancer. In some embodiments, the cancer is selected from the group consisting of: NSCLC, melanoma and bladder cancer. In some embodiments, the cancer is selected from the group consisting of: lung adenocarcinoma, colon adenocarcinoma, bladder urothelial carinoma, cancer of unknown primary, and cancer of unknown primary. In some embodiments, the cell of the cancer includes BRAF G469V mutation.

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

In some embodiments, the method further comprises administering at least one modulator of a checkpoint molecule or an immunomodulator, or a nucleic acid encoding the modulator or immunomodulator, or a vector comprising the nucleic acid encoding the modulator or immunomodulatory, to the subject. In some embodiments, the modulator of a checkpoint molecule is selected from the group consisting of: (a) an agonist of a tumor necrosis factor receptor superfamily member, preferably of CD27, CD40, 0X40, GITR, or CD137; and (b) an antagonist of PD-1, PD-L1, CD274, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3, VISTA, or an antagonist of a B7-CD28 superfamily member, preferably of CD28 or ICOS or an antagonist of a ligand thereof. In some embodiments, the modulator of a checkpoint molecule is an inhibitor of checkpoint molecule selecting 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 checkpoint molecule interacts with a ligand of checkpoint molecule selecting 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 co-stimulatory ligand, a TNF ligand, an Ig superfamily ligand, CD28, CD80, CD86, ICOS, CD40L, OX40, 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, “treating” , “treat” or “treatment” refers to a therapeutic intervention that at least partially ameliorates, eliminates or reduces a symptom or pathological sign of a disease, disorder or condition such as a cancer after it has begun to develop. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.

As used herein, “preventing” , “prevent” or “prevention” refers to a course of action initiated before the onset of a symptom or pathological sign of the disease, disorder or condition, so as to prevent and/or reduce the symptom or pathological sign. It is to be understood that such preventing need not be absolute to be beneficial to a subject. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of the disease, disorder or condition, or exhibits only early signs for the purpose of decreasing the risk of developing a symptom or pathological sign of the disease, disorder or condition.

As used herein, the terms “effective amount” or “therapeutically effective amount” refer to an amount of a therapeutic effective to treat or prevent a disease or disorder in a subject or mammal. The therapeutically effective amount of a drug has a therapeutic effect and as such can prevent the development of a disease or disorder; slow down the development or progression of a disease or disorder; relieve to some extent one or more of the symptoms associated with a disease or disorder; reduce morbidity and mortality; improve quality of life; or a combination of such effects.

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

The effective amount for use in humans can be determined from animal models. For example, a dose for humans can be formulated to achieve circulating, liver, topical and/or gastrointestinal concentrations that have been found to be effective in animals. Based on animal data and other types of similar data, those skilled in the art can determine the effective amounts of a vaccine composition appropriate for humans.

It will be appreciated by those skilled in the art that other variations of the embodiments described herein may also be practiced without departing from the scope of the invention. Other modifications are therefore possible.

Although the disclosure has been described and illustrated in exemplary forms with a certain degree of particularity, it is noted that the description and illustrations have been made by way of example only. Numerous changes in the details of construction and combination and arrangement of parts and steps may be made. Accordingly, such changes are intended to be included in the invention, the scope of which is defined by the claims.

EXAMPLES

Materials and Methods:

Reagents

Peptides were synthesized by Genscript Inc. The peptides sequences used in this experiment are listed below:

Name	Peptide sequence
CMV HLA A0201	NLVPMVATV
CMV HLA A2402	QYDPVAALF
CMV HLA B0702	TPRVTGGGAM
CMV HLA B18	SDEEEAIVAYTL
CMV HLA B3501	IPSINVHHY
CMV HLA B44	EFFWDANDIY
Neg control (HIV HLA 0201)	LTFGWCFKL

PBMC separation

PBMCs (peripheral blood mononuclear cells) were isolated from human peripheral blood. Whole blood was diluted 1: 1 with phosphate buffer, and PBMCs were isolated by centrifugation at 1200 x g for 15 minutes using a lymphocyte separation solution (Lymphoprep ^TM, STEMCELL Technologies) and a lymphocyte separation tube.

The purpose of this step is to achieve a density gradient centrifugation of the cell components by the lymphocyte separation solution, and to separate the PBMCs from different cells such as red blood cells and platelets.

ELISpot

Human IFN-γ precoated ELISpot kits were purchased from DAKEWEI Inc. ELISpot assays were performed according to protocol. Briefly, 1 x 10 ⁵ PBMC cells were seeded per well, after 48 h incubation with peptides at 37℃ under 5%CO ₂, and pictures were taken by Nikon SMZ18 microscope.

Results:

To identify the HLA subtype of BRAF G469V donor, ELISpot assay was performed. The Human IFN-γ ELISpot assay is designed for the detection of human IFN-γsecreting cells at the single cell level and can be used to quantitate the frequency of human IFN-γ secreting cells. This has been frequently used for the quantitation of CD8+ T cell responses. Here, previously discovered CMV peptides for HLA A0201, HLA A2402, HLA B07, HLA B18, HLA B35 and HLA B44 were synthesized, as indicators to testify the HLA subtype of the donor.

1 μg CMV peptides for HLA A0201, HLA A2402, HLA B07, HLA B18, HLA B35 and HLA B44, were incubated 48h with 1 x 10 ⁵ PBMCs separated from the donor. HIV peptide sequence (LTFGWCFKL) was used as negative control, Phytohemagglutinin (PHA) was used as positive control. IFN-γ dots were observed in PHA and HLA A0201 group. The photos were taken with a microscope after the wells were dried out.

The results in Figure1 show that, HLA A0201 and positive control group were positive in ELISpot assay, indicating this donor is harboring HLA A0201 subtype.

Prediction results (peptide sequence)

To predict candidate peptides of BRAF G469V mutation site of HLA A0201 using the model named REDpepper (from Westlake University) . In the prediction model, a high rank score indicates the high binding affinity with human HLA A0201 subtype. The BRAF-19 has a higher binding affinity with MHC-I than other peptides as shown by Table 1 and Figure 2.

Table 1. The prediction results

We then tested which peptide was able to activate CD8+ cell to excrete IFN-γ. 1 μg peptide was incubated 48h with 1 x 10 ⁵ PBMCs separated from the donor. 35 peptides (1 μg) were incubated 48h with 1 x 10 ⁵ PBMCs separated from the donor. HIV peptide sequence (LTFGWCFKL) was used as negative control, Phytohemagglutinin (PHA) was used as positive control. IFN-γ dots were observed in PHA, HLA A0201 and BRAF-19 group. The photos were taken with a microscope after the wells were dried out. Among 38 peptides, BRAF-19 peptide (QRIGSGSFVT) was sufficient to activate CD8+ cell as shown by ELISpot assay, and has a significantly stronger immunogenicity as compared to all of 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 validity of BRAF-19 epitope on BRAF G469V donor, we constructed a MSCV plasmid which codes HLA A 0201 and BRAF-19 peptide as shown in Figure 8. The expressed HLA A 0201 and BRAF-19 protein is anchored on the cell membrane and is able to activate CD8+ cell. We then transfected BRAF-19 MSCV and negative control MSCV plasmid to HEK 293F cells. After 24 h transfection, 1 x 10 ⁴ HEK 293F cells was seeded to ELISpot wells with 1 x 10 ⁵ PBMCs at a ratio of CD8+ cells to HEK 293F cells of about 1 : 1.

Specifically, 4 peptides (BRAF-28, BRAF-31, BRAF-37, BRAF-19; 1 μg) were incubated 48h with 1 x 10 ⁵ PBMCs separated from the donor. BRAF-19 MSCV and Neg MSCV plasmid transfected 293F cells were also seeded with 1 x 10 ⁵ PBMCs. HIV peptide sequence (LTFGWCFKL) was used as negative control, Phytohemagglutinin (PHA) was used as positive control. The photos were taken with a microscope after the wells were dried out. The results in Figure 4 shows that, BRAF-19 MSCV group has significant IFN-γ dots compared to Negative MSCV group.

To verify the specificity of BRAF-19 epitope on non-BRAF-G469V mutation people, we also performed ELISpot assay on 16 healthy donors. 1 μg CMV peptides for HLA A0201, HLA A2402, HLA B MIX (peptide mix by HLA B07, B18, B35 and B44) , were incubated 48h with 1 x 10 ⁵ PBMCs BRAF-19 MSCV and Neg MSCV plasmid transfected 293F cells were also seed with 1 x 10 ⁵ PBMCs. HIV peptide sequence (LTFGWCFKL) was used as negative control, Phytohemagglutinin (PHA) was used as positive control. The photos were taken with a microscope after the wells were dried out. The difference value between BRAF-19 peptide group and negative peptide group and the difference value between BRAF-19 MSCV group and negative MSCV group is calculated by Prism 5. The results in Figures 5 and 6 showed that, the difference value between BRAF-19 peptide group and negative peptide group is 2.5 on average in 16 healthy donors, while in BRAF G469V honor, the difference value is 31. In MSCV plasmid transfection group, the difference value between BRAF-19 MSCV group and negative MSCV group is 3.3 on average in 16 healthy donors, while in BRAF G469V honor, the difference value is 39.

Specifically, in 4 healthy HLA A 0201 donors, the results in Figure 7 showed that, the difference value between BRAF-19 peptide group and negative peptide group is 1.5 on average, and in MSCV plasmid transfection group, the difference value between BRAF-19 MSCV group and negative MSCV group is 1.5 on average. The difference value between BRAF-19 peptide group and negative peptide group and the difference value between BRAF-19 MSCV group and negative MSCV group is calculated by Prism 5.

Conclusion

Taken together, we successfully predicted 38 BRAF G469V neoantigen sequences, and further confirmed their abilities in activating CD8+ cells by ELISpot assay. We found only BRAF-19 neoantigen peptide is capable to activate CD8+ cells, and proved its specificity by ELISpot assay in 16 healthy donors, suggesting BRAF-19 has anti-tumor function in 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 sequence of MSCV plasmid which codes HLA A 0201 and BRAF-19 peptide is shown in SEQ ID NO: 39 as set forth below:

Claims

An isolated neoantigenic peptide, wherein the isolated neoantigenic peptide comprises an amino acid sequence as shown in SEQ ID NO: 19 (QRIGSGSFVT) or a functionally equivalent variant of SEQ ID NO: 19.
The isolated neoantigenic peptide of claim 1, wherein the isolated neoantigenic peptide comprises a tumor-specific neoepitope that is capable of binding to MHC-I to form a MHC-neoantigen complex.
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 alterations as compared to SEQ ID NO: 19.
The isolated neoantigenic peptide of claim 3, wherein the amino acid alterations are conservative amino acid substitutions.
The isolated neoantigenic peptide of any of claims 1-4, wherein the isolated neoantigenic peptide is linked, optionally via a linker such as a poly-glycine or poly-serine linker, to one or more additional neoantigenic peptide.
The isolated neoantigenic peptide of any of claims 1-5, wherein the isolated neoantigenic peptide is from about 10 to 30 amino acids in length, such as about 10 to 20 amino acids in length e.g., 10, 11, 12, 13, 14 and 15 amino acids in length.
The isolated neoantigenic peptide of any of claims 1-6, wherein the isolated neoantigenic peptide binds MHC-I with a binding affinity of about 500 nM or less, e.g., about 250 nM or less, or about 50 nM or less.
A polynucleotide encoding the neoantigenic peptide of any one of claims 1 to 7.
A vector comprising the polynucleotide of claim 8.
The vector of claim 9, wherein the vector is selected from the group consisting of a plasmid, a cosmid, a RNA, a RNA formulated in a particle, a self-amplifying RNA (SAM) , a SAM formulated in a particle, or a viral vector.
The vector of claim 10, wherein the viral vector is an alpha virus vector, a Venezuelan equine encephalitis (VEE) virus vector, a sindbis virus vector, a semliki forest virus vector, a simian or human cytomegalovirus vector, a lymphocyte choriomenigitis virus vector, a retroviral vector, a lentiviral vector, an adenovirus vector, or combination thereof.
A composition comprising the isolated neoantigenic peptide of any one of claims 1 to 7, optionally the composition is in the form of in vivo delivery system for example nanoparticulate encapsulation, virus like particles, liposomes, or any combination thereof.
A composition comprising the polynucleotide of claim 8 and/or the vector of any of claims 9-11, optionally the composition is in the form of in vivo delivery system for example viruses, virus-like particles, plasmids, bacterial plasmids, nanoparticles, or any combination thereof.
The composition of claims 12 or 13, further comprising at least one modulator of a checkpoint molecule or an immunomodulator, or a nucleic acid encoding the modulator or immunomodulator, or a vector comprising the nucleic acid encoding the modulator or immunomodulator.
The composition of claim 14, wherein the modulator of a checkpoint molecule is selected from the group consisting of: (a) an agonist of a tumor necrosis factor receptor superfamily member, preferably of CD27, CD40, 0X40, GITR, or CD137; and (b) an antagonist of PD-1, PD-L1, CD274, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3, VISTA, or an antagonist of a B7-CD28 superfamily member, preferably of CD28 or ICOS or an antagonist of a ligand thereof; or wherein the immunomodulator is a T cell growth factor, preferably IL-2, IL-12, or IL-15.
The composition of any of claims 12 to 15, further comprising one or more adjuvants.
A T cell receptor (TCR) capable of binding the neoantigenic peptide of any of claims 1-7 or an MHC-I-peptide complex comprising the neoantigenic peptide of any of claims 1-7.
A chimeric antigen receptor comprising: (i) a T cell activation molecule; (ii) a transmembrane region; and (iii) an antigen recognition moiety capable of binding the neoantigenic peptide of any of claims 1-7 or an MHC-peptide complex comprising the neoantigenic peptide of any of claims 1-7.
A T cell comprising the T cell receptor of claim 17 or the chimeric antigen receptor of claim 18.
The T cell of claim 19, wherein the T cell is a T cell isolated from a population of T cells from a subject that has been incubated with antigen presenting cells such as artificial antigen presenting cells and the neoantigenic peptide of any of claims 1-7 for a sufficient time to activate the T cells.
The T cell of claim 19 or 20, wherein the T cell is a CD8+ T cell or a cytotoxic T cell.
A method for activating tumor specific T cells 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 of claims 1-7 for a sufficient time to activate the T cells.
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.
A composition comprising the T cell of any of claims 19-21, activated tumor specific T cells produced by the method of claim 22, and/or the modified CD8+ T cell of claim 23.
A method of treating or preventing a BRAF mutation-related cancer in a subject 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 of claims 9-11, the composition of any of clams 12-16 and 24, the T cell of any of claims 19-21, activated tumor specific T cells produced by the method of claim 22, and/or the modified CD8+ T cell of claim 23.
A method of inhibiting growth of a tumor cell having a BRAF mutation, 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 of claims 9-11, the composition of any of clams 12-16 and 24, the T cell of any of claims 19-21, activated tumor specific T cells produced by the method of claim 22, and/or the modified CD8+ T cell of claim 23.
The method of claim 25 or 26, wherein the BRAF mutation is BRAF G469V mutation.