Peptide vaccines against glioma and uses thereof

Abstract

The present invention provides a peptide vaccine pharmaceutical composition and method of use for stimulating an immune response against glioma H3K27M mutation. The peptide vaccine has a length of at least 12 amino acid residues, is capable of stimulating a CD4T cell response upon administration, optionally also capable of stimulating a CD8T cell response.

Description

Background Art

[0001] Diffuse intrinsic pontine glioma (DIPG) is a rare and devastating brain tumor that primarily affects children, most often diagnosed between the ages of four and seven. Approximately 200-300 children are diagnosed with DIPG in the United States each year. The median survival for DIPG patients is 9-12 months after diagnosis. The tumor's location—the pons—contains nerves essential for basic life functions, making it impossible to surgically remove the tumor. Currently, radiation therapy is the only effective treatment for DIPG. Patient survival has remained stable for over four decades.

[0002] A point mutation in which lysine 27 (K27) in the histone H3 tail is replaced by a methionine residue (M), or H3K27M, has been identified as a driver mutation in DIPG (Schwartzentruber J et al., 2012; Wu G et al., 2012). An estimated 60–70% of DIPG tumors carry heterozygous H3K27M mutations. All H3K27M mutations occur in two histone H3 variants (H3.1 and H3.3), with H3.3 mutations being found more frequently (approximately 70%) (Argersinger DP et al., 2021; Zhang X et al., 2019). Of note, in addition to DIPG, H3K27M mutations have been associated with other pediatric brain tumors (e.g., pediatric high-grade gliomas) and several other adult cancers, including gliomas, acute myeloid leukemia (AML), and melanoma (Lowe BR et al., 2019).

[0003] Previously, a peptide vaccine targeting H3K27M has been developed to treat DIPG (WO2016179326A1). This peptide vaccine essentially contains a 10-amino acid (10-AA) long peptide - (R / A)MSAP(S / A)TGGV (SEQ ID NO: 4), which is an epitope restricted to HLA-A*02. Therefore, this peptide vaccine is designed to specifically trigger CD8 T cell responses or cytotoxic T cell responses. However, another important T cell response, CD4 T cell responses, is intentionally excluded in the vaccine design of the above technology. + T cell responses or T helper cell mediated immune responses (which have profound implications for anti-tumor immunity) because the peptide is as short as 10 amino acids and can only trigger an HLA class I restricted response. It has been shown that CD8 + T cell epitopes range from 8 to 11 residues (Rosa DS et al., 2010; Hemmer B et al., 2000), whereas CD4 +T lymphocytes generally recognize peptides of 12-16 amino acids (Rosa DS et al., 2010; Hemmer B et al., 2000) or 13-17 amino acids (Chicz RM et al., 1992; Sercarz EE et al., 2003). Summary of the Invention

[0004] In a first aspect, the present disclosure provides a pharmaceutical composition, which is essentially a peptide vaccine composition. The pharmaceutical composition comprises a peptide (i.e., a peptide vaccine) having a length of at least 12 amino acid residues and comprising a 4-AA peptide segment RMSA (SEQ ID NO: 26). After being administered to a subject in accordance with its therapeutically effective regimen, the pharmaceutical composition can stimulate a CD4 T cell response to histone 3 (H3) K27M mutation (H3K27M) in the subject. Therefore, compared to the 10-AA peptide vaccine disclosed in WO2016179326A1, which can only stimulate CD8 T cell responses to H3.3K27M mutations, the peptide vaccine provided in the present disclosure can stimulate CD4 T cell responses to H3K27M mutations.

[0005] Here, the 4-AA peptide segment RMSA (SEQ ID NO: 26) in the peptide vaccine substantially corresponds to the "K27M" mutant form of the 4-AA region RKSA (SEQ ID NO: 30) (as cited in the literature), which corresponds to positions 27-30 of human histone 3.1 (ie, H3.1) variant (SEQ ID NO: 22) and human histone 3.3 (ie, H3.3) variant (SEQ ID NO: 24). Therefore, based on the sequence of the peptide vaccine, the peptide vaccine provided herein is capable of stimulating CD4 T cell responses against both the H3.1 K27M mutation and the H3.3 K27M mutation in a subject.

[0006] According to some embodiments, the pharmaceutical composition is further capable of stimulating a CD8 T cell response against the H3K27M mutation in a subject after administration. In other words, some embodiments of the peptide vaccine are capable of stimulating both a CD4 T cell response and a CD8 T cell response after administration.

[0007] According to some embodiments of the pharmaceutical composition, the peptide comprises a 10-AA segment RMSAP(S / A)TGGV (SEQ ID NO: 25). Here, depending on the different mutant H3 genotypes of the target tumor tissue in the subject, the peptide vaccine may comprise a 10-AA segment RMSAPSTGGV (SEQ ID NO: 5), thereby stimulating a CD4 T cell response to the H3.3K27M mutation in the subject, and optionally, a CD8 T cell response, or comprise a 10-AA segment RMSAPATGGV (SEQ ID NO: 29), thereby stimulating a CD4 T cell response to the H3.1K27M mutation in the subject, and optionally, a CD8 T cell response.

[0008] Further according to some embodiments of the pharmaceutical composition, the peptide comprises an 18-AA segment KQLATKAARMSAP(S / A)TGGV (SEQ ID NO: 1). Depending on the different mutant H3 genotypes of the target tumor tissue in the subject, the peptide vaccine may comprise an 18-AA segment KQLATKAARMSAPSTGGV (SEQ ID NO: 2), thereby stimulating a CD4 T cell response to the H3.3K27M mutation in the subject, and optionally, a CD8 T cell response, or comprise an 18-AA segment KQLATKAARMSAPATGGV (SEQ ID NO: 27), thereby stimulating a CD4 T cell response to the H3.1K27M mutation in the subject, and optionally, a CD8 T cell response.

[0009] According to a specific embodiment, the peptide in the pharmaceutical composition consists of 18-AA KQLATKAARMSAPSTGGV (SEQ ID NO: 2). As described in detail in the examples set forth below, this 18-AA peptide vaccine, when formulated into a pharmaceutical composition comprising a polyICLC adjuvant and administered to a patient, is capable of stimulating both CD4 and CD8 T cell responses against the H3.3K27M mutation.

[0010] According to another specific embodiment, the peptide in the pharmaceutical composition consists of 18-AA KQLATKAARMSAPATGGV (SEQ ID NO: 27), which is designed to stimulate a similar T cell response against the H3.1K27M mutation.

[0011] In any of the embodiments described above, the pharmaceutical composition may further include an adjuvant. According to some embodiments, the adjuvant includes a toll-like receptor (TLR) agonist, which may optionally include a ligand of any one of TLR3, TLR4, TLR7 / TLR8, and TLR9. Optionally, a TLR3 ligand is used as an adjuvant for a pharmaceutical composition comprising a peptide vaccine, such as poly I:C (polyriboinosinic acid: polyribocytidylic acid). Further optionally, certain derivatives of the poly I:C have improved stability, safety, and / or adjuvant properties, such as polyICLC and poly I:C12U. Optionally, other TLR ligands may also be used, and non-limiting examples may include monophosphoryl lipid A (MPL, as a TLR4 ligand) or derivatives thereof, imiquimod and resiquimod (all as TLR7 / 8 ligands), cpG ODN (TLR9 ​​ligand). More examples and related information of such adjuvants can be found in U.S. Patent Application No. US20110038888A1, Toussi DN and Massari P, 2014, the disclosures of which are incorporated by reference in their entirety. According to other embodiments, the adjuvant includes exogenous substances other than TLR agonists, which may include Bacillus Calmette-Guérin (BCG) vaccines, but may also include substances with different properties and material characteristics.

[0012] Optionally, polyICLC is used as an adjuvant, and in the pharmaceutical composition, the weight ratio of polyICLC to the peptide may be approximately 1:0.5 to 1:5 (eg, 1:1, 1:2, 1:3, 1:4).

[0013] Further optionally, the pharmaceutical composition can be formulated to have a dosage form comprising about 0.5 mg of poly-ICLC and about 0.5-2 mg of the peptide.

[0014] Further optionally, the pharmaceutical composition can be formulated into a preparation suitable for subcutaneous injection into a subject.

[0015] In a second aspect, there is further provided a method of stimulating an immune response in a subject in need thereof using the pharmaceutical composition provided in the first aspect.

[0016] As used herein, "subject" refers to a human individual with cancer, and the cancer is characterized by a K27M mutation in histone H3 in the cancer tissue / cell. Non-limiting examples of subjects may include human individuals with glioma, DIPG, acute myeloid leukemia (AML), melanoma (Lowe BR et al., 2019), but other examples of subjects may also include other cancers, as long as the cancer tissue / cell contains H3K27M. It should also be noted that although the K27M mutation has only been identified in two variants of histone H3 (i.e., H3.1 and H3.3), the pharmaceutical compositions disclosed herein and methods of use thereof should also be interpreted as covering other H3 variants (e.g., H3.2, SEQ ID NO: 23) or other H3 mutations, as long as the variant or mutant H3 contains K27M.

[0017] Here, the method essentially comprises the following steps: administering a pharmaceutical composition according to any embodiment of the first aspect above to a subject according to a therapeutically effective regimen, thereby inducing an immune response in the subject. Here, the immune response comprises a CD4 T cell response to the H3K27M mutation, and optionally, the immune response further comprises a CD8 T cell response to the H3K27M mutation.

[0018] According to some embodiments of the method, prior to the administering step, the method further comprises the step of determining the mutation variant of H3 in the subject.

[0019] Here, if the mutation variant of H3 is determined to be H3.3, then in the administering step, the peptide in the pharmaceutical composition comprises RMSAPSTGGV (SEQ ID NO: 5). Further optionally, the peptide in the pharmaceutical composition comprises KQLATKAARMSAP S TGGV (SEQ ID NO: 2). Further optionally, the peptide is 18 AA in length and is composed of KQLATKAARMSAP S The composition of the protein is TGGV (SEQ ID NO: 2).

[0020] Here, if the mutant variant of H3 is determined to be H3.1, then in the administration step, the peptide in the pharmaceutical composition comprises RMSAP A TGGV (SEQ ID NO: 29). Further optionally, the peptide in the pharmaceutical composition comprises KQLATKAARMSAP A TGGV (SEQ ID NO: 27). Further optionally, the peptide is 18 AA in length and is composed of KQLATKAARMSAP A The composition of the protein is TGGV (SEQ ID NO: 27).

[0021] In any embodiment of the above method, the cancer may include at least one of glioma, acute myeloid leukemia (AML) or melanoma. Optionally, the cancer may include glioma, and further optionally, the cancer includes DIPG.

[0022] In any of the embodiments of the above methods, the subject carries the HLA-A*02 allele.

[0023] In any of the embodiments of the above methods, the subject carries at least one of an HLA-DRB1*07:01 allele or an HLA-DRB1*01:01 allele.

[0024] In any of the embodiments of the above methods, the therapeutically effective regimen comprises administering the pharmaceutical composition to the subject by subcutaneous injection.

[0025] In a third aspect, the present disclosure further provides T cells expressing a T cell receptor (TCR) or a functional fragment thereof.

[0026] Here, the TCR or a functional fragment thereof is capable of binding to a peptide / MHC II complex, wherein the peptide in the peptide / MHC II complex has a length of at least 12 amino acid residues and comprises RMSA (SEQ ID NO: 26).

[0027] According to some embodiments of the T cell, the TCR or a functional fragment thereof is further capable of binding to a complex formed between the peptide and MHC I.

[0028] Optionally, the peptide comprises RMSAP(S / A)TGGV (SEQ ID NO: 25). Further optionally, the peptide comprises KQLATKAARMSAP(S / A)TGGV (SEQ ID NO: 1).

[0029] Here, according to some embodiments of the T cell, the peptide comprises KQLATKAARMSAPSTGGV (SEQ ID NO: 2), and further optionally, the peptide is 18 AA in length and consists of KQLATKAARMSAPSTGGV (SEQ ID NO: 2). Optionally, the TCR or fragment thereof cannot bind to a complex formed between a second peptide and an MHC II complex, and the second peptide comprises the amino acid sequence

[0030] Here, according to some other embodiments of the T cell, the peptide comprises KQLATKAARMSAPATGGV (SEQ ID NO: 27), and further optionally, the peptide is 18 AA in length and consists of KQLATKAARMSAPATGGV (SEQ ID NO: 27). Optionally, the TCR or fragment thereof is unable to bind to a complex formed between a second peptide and an MHC II complex, and the second peptide comprises the amino acid sequence KQLATKAARKSAPATGGV (SEQ ID NO: 28).

[0031] According to some embodiments of the T cell, the TCR is heterologous to the T cell. Optionally, the TCR is transduced or transfected into the T cell via a vector such as a retroviral vector.

[0032] All sequences cited here and elsewhere in this application are listed in Table 1. Table 1. Sequences cited in this application definition

[0033] As used herein, the term "about" or "approximately" when used with a number refers to any number that is within 1%, 5%, or 10% of the recited number.

[0034] As used herein, the terms "neoplasia," "hyperplasia," and "tumor" are often collectively referred to as "cancer," which is a general term for more than 100 diseases characterized by uncontrolled, abnormal growth of cells. As used herein, "tumor" also includes normal, benign, or malignant tissue masses.

[0035] As used herein, the term "peptide" refers to a polymer of amino acids connected by amide bonds (or peptide bonds) well known to persons skilled in the art. Peptide can be 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acid whose polymers are connected by covalent amide bonds. In some embodiments, peptide is a polymer of 6 to 8, 8 to 10, 10 to 15, 10 to 20, 10 to 25, 10 to 30, 10 to 40, 10 to 50 or 25 to 25 amino acid whose polymers are connected by covalent amide bonds. In certain embodiments, peptide is a polymer of 50 to 65, 50 to 75, 50 to 85, 50 to 95, 50 to 100, 75 to 100 amino acid whose polymers are connected by covalent amide bonds. As used herein, the term can refer to a single peptide chain linked by a covalent amide bond. The term can also refer to multiple peptide chains associated by non-covalent interactions such as ionic contacts, hydrogen bonds, van der Waals contacts, and hydrophobic contacts. Those skilled in the art will recognize that the term includes modified peptides, for example, peptides modified by post-translational processing such as signal peptide cleavage, disulfide bond formation, glycosylation (e.g., N-linked glycosylation), protease cleavage, and lipid modification (e.g., S-palmitoylation).

[0036] As used herein, the term "conservative substitution" refers to replacing an amino acid of one class with another amino acid of the same class. In a specific embodiment, a conservative substitution does not change the structure or function of the peptide, or neither the structure nor the function is changed. The categories of amino acids for conservative substitution purposes include hydrophobic (Met, Ala, Val, Leu, Ile), neutral hydrophilic (Cys, Ser, Thr), acidic (Asp, Glu), basic (Asn, Gln, His, Lys, Arg), conformationally disruptive (Gly, Pro) and aromatic (Trp, Tyr, Phe).

[0037] As used herein, the term "immune response" refers to a reaction that occurs in a human subject that is alleged to be used to fight cancer. The immune response can include both innate and adaptive immune responses; the former includes immune cells such as neutrophils, macrophages, and monocytes, as well as cytokines and complement, while the latter includes immune cells such as dendritic cells, T cells, B cells, and antibodies that stimulate antigen-specific immune responses.

[0038] The term "T cell response" refers to a type of adaptive immune response mediated by T cells, which generally includes peptide-induced proliferation and activation of effector functions against specific antigens in vitro or in vivo. There are two major subtypes of T cells: CD8 +T cells (i.e., killer T cells, cytotoxic T cells, or effector T cells) and CD4 + T cells (i.e., helper T cells), and therefore T cell responses include CD4 T cell responses and CD8 T cell responses.

[0039] The terms “CD8 T cell response”, “CD8 + T cell response" or similar terms refers to a response by CD8 + T cells or CD8 + A type of T cell response mediated by lymphocytes. CD8 + T cells are commonly used to destroy virus-infected cells and tumor cells and typically recognize their targets by binding to short peptides (8-11 AA in length) associated with MHC class I molecules present on the surface of all nucleated cells. + T cells also produce several key cytokines, including TNFα, IL-2, and IFNγ, which can influence the effector functions of other cells, particularly macrophages and NK cells.

[0040] The terms “CD4 T cell response”, “CD4 + T cell response" or similar terms refers to the response of CD4 + T cells or CD4 + Another type of T cell response mediated by lymphocytes. CD4 + T cells usually assist other lymphocytes, including the maturation of B cells and the activation of cytotoxic T cells and macrophages. + When T cells present longer peptide antigens (12-16 AA in length), CD4 + T cells become activated, causing them to divide rapidly and secrete cytokines that regulate or aid the immune response (e.g., IFNγ, IL-2, etc.). As used herein, the term "antigen presenting cell" refers to dendritic cells (DCs), monocytes / macrophages, B lymphocytes, or other cell types that express the necessary MHC / co-stimulatory molecules that effectively allow T cells to recognize presented peptides.

[0041] The term "MHC" stands for "major histocompatibility antigen." In humans, the MHC genes are called HLA ("human leukocyte antigen") genes. Although there is no consistently followed convention, some literature uses HLA to refer to HLA protein molecules and MHC to refer to the genes encoding HLA proteins. Therefore, when used in this article, the terms "MHC" and "HLA" are equivalent. The human HLA system has an equivalent in mice, the H2 system. The most intensively studied HLA genes are the nine so-called classical MHC genes: HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1. In humans, the MHC is divided into three regions: class I, class II, and class III. The A, B, and C genes belong to MHC class I, while the six D genes belong to class II. MHC class I molecules consist of one polymorphic chain containing three domains (α1, 2, and 3) and associate with β2 microglobulin on the cell surface. Class II molecules consist of two polymorphic chains, each containing two chains (α and β).

[0042] Class I MHC molecules are expressed on almost all nucleated cells. Peptide fragments presented in the context of class I MHC molecules are recognized by CD8 T lymphocytes (cytotoxic T lymphocytes or CTLs). + T cells or lymphocytes often mature into cytotoxic effector cells that lyse cells bearing stimulating antigens. Class II MHC molecules are primarily expressed on activated lymphocytes and antigen-presenting cells. CD4 + T lymphocytes (helper T lymphocytes or HTLs) are activated by recognizing unique peptide fragments presented by class II MHC molecules, usually present on antigen-presenting cells such as macrophages or dendritic cells. + T lymphocytes proliferate and secrete cytokines that support antibody-mediated responses through the production of IL-4 and IL-10, or cell-mediated responses through the production of IL-2 and IFNγ.

[0043] Functional HLA is characterized by a deep binding groove to which endogenous and exogenous potential antigenic peptides bind. The groove is further characterized by a clear shape and physicochemical properties. HLA class I binding sites are closed because the peptide ends are constrained within the ends of the groove. They also participate in a hydrogen bond network with conserved HLA residues. In view of these limitations, the length of the bound peptide is limited to 8-10 residues. Unlike HLA class I binding sites, class II sites are open at both ends. This allows the peptide to extend from the actual binding region, thereby "hanging out" at both ends. Therefore, class II HLA can bind peptide ligands of different lengths, usually more than 12 amino acid residues.

[0044] As used herein, the term "adjuvant" refers to an exogenous substance that, when administered to a subject simultaneously with a specific vaccine (e.g., a peptide vaccine disclosed herein), can enhance the subject's immune response to the vaccine, also referred to as an "immune adjuvant." Adjuvants that can be used simultaneously with the peptide vaccines disclosed herein are not limited and may include exogenous substances of various properties and sources, such as mineral salts, oil-based and water-based emulsions, polymers, microparticles, liposomes, saponins, microbial products, and cytokines. The mechanism of action of the adjuvants in the present disclosure is also not limited and may include nonspecific effects (i.e., antigen reservoirs at the immunization site), as well as specific activation of immune cells, resulting in improved innate and adaptive responses of the host.

[0045] As used herein, the term "poly I:C" refers to polyinosinic:polycytidylic acid, also abbreviated as poly(I:C) or similar terms, which is typically used in the form of a sodium salt to stimulate an immune response. The term "polyICLC" or poly I:CLC or similar terms refers to a mixture of poly I:C with stabilizers carboxymethylcellulose and polylysine.

[0046] As used herein, the term "therapeutically effective regimen" refers to a regimen of dosage, timing, frequency, mode and duration of administration of a pharmaceutical composition comprising a peptide vaccine to a subject in need thereof for the treatment and / or management of a cancer characterized by an H3K27M mutation, and the dosage, timing, frequency and duration of the administration are configured to be sufficient to stimulate a level of immune response (e.g., a CD4 T cell response, and optionally, a CD8 T cell response) against the H3K27M mutation in the subject.

[0047] As used herein, the term "or" should be construed as inclusive unless otherwise stated or obvious from the context.

[0048] As used herein, the terms "a," "an," and "the" are to be construed as referring to either the singular or the plural, unless specifically stated otherwise or clear from the context.

[0049] Unless otherwise specified or obvious from the context, as used herein, the terms "approximately," "about," "around," or similar terms should be understood to mean within the normal tolerance range in the art, for example, within 2 standard deviations of the mean. "About" can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise apparent from the context, all numerical values ​​provided herein are understood to be modified by this term.

[0050] The term "comprising" includes "including" and "consisting of, for example, a composition "comprising" X may consist entirely of X or may include other elements, such as X+Y.

[0051] The term "substantially" does not exclude "completely", for example, a composition that is "substantially free" of Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of the present invention.

[0052] All references, patents, or patent applications mentioned herein are hereby incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 IFN-γ ELISpot results are shown. Briefly, peripheral blood mononuclear cell (PBMC) samples from two DIPG patients, EN-17 and EN-23, were cultured and antigen-stimulated. After culture expansion and antigen stimulation, 20,000 cells were collected into each well for each sample for ELISpot assay to detect cells secreting IFN-γ. PSG1: Peptide stimulation group 1. Cells were stimulated only once with the corresponding peptide and expanded for 2-3 weeks. PSG2: Peptide stimulation group 2. Cells were stimulated with the corresponding peptide at the beginning of culture expansion and then stimulated again with the antigen peptide before ELISpot assay. PHA: Phytohemagglutinin, a positive control antigen for ELISpot. NCG: Negative control group (no peptide stimulation). KQ: long peptide (18aa) carrying a point mutation. RM: short peptide (10aa) carrying a point mutation.

[0054] Figure 2 shows the EN-17C27 TCR validation results. In short, EN-17C27 is a candidate TCR cloned from CD4 T cells. Selected PBMC samples were transduced with a retroviral vector carrying the candidate TCR. The transduced PBMC cells were then stimulated with different peptides and T cell responses were examined using FACS. CD4 T cells and CD8 T cells were gated and studied separately. All FACS results are displayed in the same format, with T cell activation markers shown along the X-axis and SSC (i.e., side scatter, which is related to the granularity of the cells and is therefore an optical measure of each individual cell in the FACS analysis) shown along the Y-axis. (A) CD4 T cell response. (B) CD8 T cell response.

[0055] Figure 3 shows the results of EN-10C01 and C04 TCR validation. In short, EN-10C01 and C04 are two candidate TCRs cloned from CD8 T cells. Selected PBMC samples were transduced with corresponding retroviral vectors carrying candidate TCRs. The transduced PBMC cells were then stimulated with different peptides and T cell responses were examined using FACS. Only CD8 T cell responses are shown. CD4 T cell responses are weaker (data not shown). All FACS results are shown in the same format, with T cell activation markers shown along the X-axis and SSC shown along the Y-axis. (A) EN-10C01. (B) EN-10C04. DETAILED DESCRIPTION

[0056] The present disclosure provides a peptide vaccine that can be used to stimulate CD4 T cell responses, and optionally CD8 T cell responses, against the H3K27M mutation in subjects carrying somatic mutations who have been administered a pharmaceutical composition comprising the peptide vaccine. Because the peptide vaccine can stimulate significant T cell responses against this clinically critical mutation that is closely associated with human gliomas, particularly DIPG in children and adults, the peptide vaccine has the potential to treat the disease.

[0057] In a first aspect, a pharmaceutical composition is provided, comprising a peptide (i.e., a peptide vaccine) having a length of at least 12 amino acid residues (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, etc.) and comprising a 4-AA peptide segment RMSA (SEQ ID NO: 26). The pharmaceutical composition, when administered to a subject according to its therapeutically effective regimen, is capable of stimulating a CD4 T cell response against the H3K27M mutation in the subject. In addition, optionally and preferably, the pharmaceutical composition is further capable of stimulating a CD8 T cell response.

[0058] In other words, according to some embodiments, the pharmaceutical composition comprising the peptide vaccine can only stimulate CD4 T cell responses against the H3K27M mutation, but cannot stimulate CD8 T cell responses against the H3K27M mutation; while according to other embodiments, the pharmaceutical composition comprising the peptide vaccine can stimulate both CD4 T cell responses and CD8 cell responses against the H3K27M mutation.

[0059] Therefore, compared with the 10-AA peptide vaccine disclosed in WO2016179326A1, which can only stimulate CD8 T cell responses against the H3.3K27M mutation, the peptide vaccine in the pharmaceutical composition provided by the present disclosure can differentially stimulate CD4 T cell responses against the H3K27M mutation.

[0060] Herein, H3 may be H3.1 or H3.3, whose wild-type sequences are set forth in SEQ ID NO: 22 and SEQ ID NO: 24, respectively. A subject carrying an H3.1K27M mutation or an H3.3K27M mutation may benefit from the peptide vaccine, and the subject may suffer from at least one of glioma (e.g., DIPG), acute myeloid leukemia (AML), and melanoma.

[0061] According to some embodiments, the peptide comprises a 10-AA segment RMSAP(S / A)TGGV (SEQ ID NO: 25). Here, depending on the different mutant H3 genotypes of the target tumor tissue in the subject, the peptide vaccine may comprise a 10-AA segment RMSAPSTGGV (SEQ ID NO: 5), thereby stimulating a CD4 T cell response to the H3.3K27M mutation in the subject, and optionally, a CD8 T cell response, or may comprise a 10-AA segment RMSAPATGGV (SEQ ID NO: 29), thereby stimulating a CD4 T cell response to the H3.1K27M mutation in the subject, and optionally, a CD8 T cell response, or may comprise the above two peptides, one of which comprises the 10-AA segment of SEQ ID NO: 25 and the other comprises the 10-AA segment of SEQ ID NO: 29.

[0062] Further according to some embodiments of the pharmaceutical composition, the peptide comprises an 18-AA segment KQLATKAARMSAP(S / A)TGGV (SEQ ID NO: 1). Depending on the different mutant H3 genotypes of the target tumor tissue in the subject, the peptide vaccine may comprise an 18-AA segment KQLATKAARMSAPSTGGV (SEQ ID NO: 2), thereby stimulating a CD4 T cell response to the H3.3K27M mutation in the subject, and optionally, a CD8 T cell response, or comprise an 18-AA segment KQLATKAARMSAPATGGV (SEQ ID NO: 27), thereby stimulating a CD4 T cell response to the H3.1K27M mutation in the subject, and optionally, a CD8 T cell response, or comprise both.

[0063] Further according to a specific embodiment, the peptide in the pharmaceutical composition consists of the 18-AA sequence KQLATKAARMSAPSTGGV (SEQ ID NO: 2), which specifically targets H3.3K27M, and its experimental data will be described in more detail below in Example 1. According to another specific embodiment, the peptide in the pharmaceutical composition consists of the 18-AA sequence KQLATKAARMSAPATGGV (SEQ ID NO: 27), which specifically targets H3.1K27M.

[0064] It should be noted that, according to different needs, the pharmaceutical composition may contain one peptide as described above, or may contain a combination of multiple peptides as described above.

[0065] The pharmaceutical composition can be formulated into a dosage form that allows for administration to a subject in need thereof. Thus, in addition to the peptide vaccine, the pharmaceutical composition can further comprise an adjuvant, for example, the adjuvant can comprise polyICLC. The pharmaceutical composition can be formulated to allow for administration by subcutaneous injection, but can also be formulated to allow for other modes of administration known to those skilled in the art.

[0066] In a second aspect, a method of using the pharmaceutical composition as described in the first aspect above is further provided. Such a method may involve using the pharmaceutical composition to stimulate an immune response, particularly a T cell response, in a subject in need thereof, who may have cancer (e.g., DIPG, glioma, AML, or melanoma, etc.) and may carry an H3K27M mutation.

[0067] Here, the method essentially comprises the following steps: administering a pharmaceutical composition according to any embodiment of the first aspect above to a subject according to a therapeutically effective regimen, thereby inducing an immune response in the subject. Here, the immune response comprises a CD4 T cell response against the H3K27M mutation, and optionally, the immune response further comprises a CD8 T cell response against the H3K27M mutation.

[0068] The peptide vaccine in the pharmaceutical composition to be administered to a subject in the methods disclosed herein may comprise 4-AA RMSA (SEQ ID NO: 26), or further optionally may comprise 10-AA RMSAP(S / A)TGGV (SEQ ID NO: 25), or further optionally may comprise 18-AA KQLATKAARMSAP(S / A)TGGV (SEQ ID NO: 1), i.e., 18-AAAKQLATKAARMSAP S TGGV(SEQ ID NO:2) or 18-AA KQLATKAARMSAP A One of TGGV (SEQ ID NO: 27).

[0069] In the methods described, the subject may carry the HLA-A*02 allele, thereby allowing stimulation of a CD8 T cell response in addition to a CD4 T cell response. However, other subjects who may carry HLA alleles other than HLA-A*02 may also be able to stimulate a CD8 T cell response.

[0070] In the methods, the subject may carry at least one of the HLA-DRB1*07:01 allele or the HLA-DRB1*01:01 allele, resulting in a defined CD4 T cell response, but may also carry other alleles, resulting in a CD4 T cell response.

[0071] In the method, the pharmaceutical composition comprising the peptide vaccine can be administered by subcutaneous injection, but can also be administered by other routes of administration.

[0072] In a third aspect, the disclosure further provides a T cell expressing a T cell receptor (TCR) or its functional fragment. Here, the TCR or its functional fragment can be combined with a peptide / MHC II complex, wherein the peptide in the peptide / MHC II complex has a length of at least 12 amino acid residues and comprises a 4-AA segment RMSA (SEQ ID NO: 26), or further optionally comprises a 10-AA segment RMSAP (S / A) TGGV (SEQ ID NO: 25), or further optionally comprises 18-AAKQLATKAARMSAP (S / A) TGGV (SEQ ID NO: 1). According to some embodiments of the T cell, the TCR or its functional fragment can further be combined with the complex formed between the peptide and MHC I.

[0073] Here, according to a specific embodiment of the T cell, the peptide comprises KQLATKAARMSAPSTGGV (SEQ ID NO: 2), and further optionally, the peptide is 18 AA in length and consists of KQLATKAARMSAPSTGGV (SEQ ID NO: 2). Optionally, the TCR or its fragment cannot bind to a complex formed between a second peptide and an MHC II complex, and the second peptide comprises the amino acid sequence KQLATKAARKSAPSTGGV (SEQ ID NO: 6). Thus, the TCR specifically binds to the K27M mutant peptide of the H3.3 variant but not to its wild-type peptide, thereby conferring the ability to specifically stimulate CD4 T cell responses against the H3.3K27M mutation but not against wild-type H3.3.

[0074] According to another specific embodiment of the T cell, the peptide comprises KQLATKAARMSAPATGGV (SEQ ID NO: 27), and further optionally, the peptide is 18 AA in length and consists of KQLATKAARMSAPATGGV (SEQ ID NO: 27). Optionally, the TCR or fragment thereof is unable to bind to a complex formed between a second peptide and an MHC II complex, and the second peptide comprises the amino acid sequence KQLATKAARKSAPATGGV (SEQ ID NO: 28). Thus, the TCR specifically binds to the K27M mutant form of H3.1 but not to the wild-type form, thereby conferring the ability to specifically stimulate CD4 T cell responses against the H3.1 K27M mutation but not against wild-type H3.1.

[0075] According to some embodiments of the T cell, the TCR is heterologous to the T cell. Optionally, the TCR is transduced or transfected into the T cell via a vector such as a retroviral vector.

[0076] A specific example (Example 1) is provided below for testing the ability of a specific embodiment of the peptide vaccine (i.e., 18-AAKQLATKAARMSAPSTGGV (SEQ ID NO: 2)) to induce or stimulate an immune response, particularly a CD4 T cell response and / or a CD8 T cell response against the H3.3K27M mutation, after administration to DIPG patients.

[0077] Example 1

[0078] 1.1. Peptide vaccine design:

[0079] In order to design peptide vaccines, HLA binding and antigen presentation were first computationally predicted based on the Immune Epitope Database (IEDB, www.iedb.org). Specifically, the MHC-II binding prediction module (IEDB recommendation 2.22) was used for prediction. '12-18' was used as the 'selection length' to allow the algorithm to predict all epitopes containing H3K27M mutations with a length ranging from 12AA to 18AA. If there is any corresponding predictor for the HLA allele, 'IEDB recommendation 2.22' uses a consensus method that combines NN-align, SMM-align, CombLib, and Sturniolo, otherwise NetMHCIIpan is used. After prediction, the minimum prediction score (percentile ranking listed in Table 2) is recorded to indicate the specificity of an epitope binding to the corresponding HLA allele. The smaller the score, the stronger the specificity of the epitope binding to the HLA allele. The prediction results are summarized in Table 2. Table 2. Computational predictions of HLA class II binding and antigen presentation Note: The 4-AA core segment common to all sequences is in bold and underlined.

[0080] As shown in the prediction results, various epitopes containing point mutations were predicted to have high affinity for class II HLA molecules, and all epitopes had a core 4-AA segment carrying a K27M mutation (i.e., the second position of the 4-AA segment was an M residue) ( SEQ ID NO: 26). It should be noted that some of the selected HLA class II molecules are high-frequency molecules in the human population, such as HLA-DRB1*01:01 and HLA-DRB1*07:01.

[0081] Based on these prediction results, a series of candidate peptide vaccines were designed. In addition to the high-ranking candidate peptides listed above (i.e., percentile ranking <= 15, including SEQ ID NOs: 8-11 and 15-17), a series of candidate peptide vaccines with 18 amino acids (i.e., A series of experiments were subsequently conducted using this 18-AA peptide vaccine candidate.

[0082] 1.2. Experimental testing of peptide vaccines

[0083] 1.2.1 Patient recruitment:

[0084] Newly diagnosed DIPG patients aged 5 years and older were selected and, after signing informed consent, were given the vaccine if their tumor biopsy showed HLA-A*02 subtype and H3.3K27M genotyping results.

[0085] 1.2.2 Vaccine Administration:

[0086] First, the peptide vaccine was prepared into a suitable pharmaceutical composition. Three dosage forms (Dose #1, #2 and #3) were designed (see Table 3), all of which showed a good safety profile. Finally, the peptide vaccine was tested on patients using the dosage formulation indicated as "Dose #3". Table 3. Vaccine formulation and dose exploration

[0087] In brief, two weeks after conformal radiotherapy was completed, the peptide vaccine was administered by subcutaneous injection (at the dosage formulation dose #3 in Table 2). The date of the first vaccine injection was defined as day 1 (D1). Injections were performed on D3, D15, D29, D57, and D85, and thereafter every 8 weeks until disease progression. Peripheral blood samples were collected at different time points and used for pharmacokinetic (PK) and pharmacodynamic (PD) studies as well as TCR cloning work. Blood samples for TCR cloning were collected from patients after the fifth and sixth vaccine injections.

[0088] All PBMC samples used in TCR cloning were collected from patients who received vaccine dose #3. The PBMC sample used for TCR cloning from patient EN17 was collected after the patient received their sixth vaccine injection. The PBMC sample used for TCR cloning from patient EN10 was collected after the patient received their sixth vaccine injection. The PBMC sample used for TCR cloning from patient EN23 was a combined PBMC sample, obtained from the patient's PBMC samples after the patient received their third and fourth vaccine injections.

[0089] 1.2.3 CD4 and CD8 T cell responses detected in peripheral blood samples of vaccinated patients

[0090] Peripheral blood samples were collected from vaccinated DIPG patients and peripheral blood mononuclear cells (PBMCs) were collected by Ficoll gradient. PBMC samples were stimulated with the corresponding peptide for 2-3 weeks, after which the cells were seeded into IFN-γ detection ELISpot plates and re-stimulated with the corresponding peptide. The increase in IFN-γ signal was considered to be the presence of antigen-specific T cells ( Figure 1 The samples were then subjected to single-cell RNA sequencing by 10× Genomics. Candidate TCRs were selected based on their transcriptional profiles, introduced into primary human T cells, and validated by pulsing the corresponding peptides (Figures 2 and 3). In this technical disclosure, we use these data to demonstrate that our vaccination approach indeed elicits a robust tumor antigen-specific immune response.

[0091] Table 4 summarizes the following terms and / or abbreviations used in this disclosure, including the figures and description. Table 4. Description of terms and abbreviations used in Example 1 Note: Underlined amino acid residues represent key letters in each term / abbreviation.

[0092] More specifically, we culture PBMC samples using specific conditions. These cultures include antigenic peptides and appropriate antigen-presenting cells. This allows us to specifically stimulate the expansion of antigen-specific T cells. After 2-3 weeks, the cells are plated onto IFN-γ detection ELISpot plates and restimulated with the corresponding peptide. Following activation, T cells release IFN-γ in response to antigenic stimulation. IFN-γ secretion can be detected using ELISpot assays. One spot indicates the detection of a single IFN-γ-secreting cell. Figure 1 IFN-γ ELISpot results are shown. The results show that PBMC samples (EN-17 and EN-23) responded to stimulation with the long peptide (KQ) but not the short peptide (RM). Therefore, T cell responses were specifically detected in response to stimulation with the long peptide. These responses were most likely CD4 T cells.

[0093] 1.2.4 TCR cloning and experimental procedures:

[0094] The following is a brief description of the TCR cloning experimental procedure. In short, PB samples are collected from vaccinated patients at different time points. PBMCs are isolated from blood samples. The isolated PBMCs are subjected to culture conditions optimized for T cell expansion and antigen stimulation. Autologous B cells or DC cells that have been cultured and expanded are used as antigen presenting cells (APCs) to present long peptide antigens (MHC class II antigens). At the same time, B cells and DCs are also suitable for presenting short peptides (MHC class I antigens). The patient's T cells are expanded and stimulated by peptide antigens for several weeks. In order to monitor the status of T cells over time, a small number of cultured T cells are collected at different time points. The collected cells are subjected to a second round of antigen stimulation, and T cell responses are measured by ELISpot and FACS. Single-cell transcriptome sequencing is performed on samples that show obvious T cell activation signals. Candidate TCRs are selected from the single-cell transcriptome data analysis and downstream functional validation is performed respectively. In order to functionally validate each candidate TCR, a retroviral vector is used to introduce the TCR into a selected PBMC sample with a suitable HLA subtype by viral transduction. T cells (CD4 + and CD8 + T cells) expressing candidate TCRs as foreign transgenes. Transduced PBMC samples were expanded and subjected to antigen stimulation. T cell responses were examined using FACS analysis. In the FACS analysis, CD4 + T cells and CD8 + T cells were gated to specifically examine each type of immune response. More details are provided below.

[0095] First, suitable samples (EN-17 and EN-23PSG2-KQ) that showed enhanced IFN-γ signaling after restimulation were selected and subjected to downstream single-cell RNA-Seq analysis. More specifically, PBMC samples (post-vaccination) were initially stimulated and expanded for 2-3 weeks. ELISpot analysis was then performed. Samples that showed enhanced IFN-γ after restimulation in this ELISpot experiment, namely the EN-17 and EN-23PSG2-KQ samples, were then subjected to single-cell RNA sequencing.

[0096] After analyzing the single-cell data, candidate TCRs were selected based on the transcriptional profile and subjected to functional validation experiments. More specifically, the candidate TCRs were introduced into primary human T cells via retrovirus (viral backbone pMP71). The corresponding peptide pulse was introduced into the TCR-transduced T cells, and flow cytometry analysis was used to monitor the response. As an example, Figure 2 shows the functional validation results of EN-17C27, a candidate TCR cloned from CD4 T cells of EN-17 PBMC samples. CD4 T cells and CD8 T cells were gated and studied separately. CD134 (OX40) is an activation marker for CD4 T cells. Specifically, CD4 T cells upregulate the cell surface expression of CD134 in response to antigen stimulation. CD107a, CD137, and CD69 are three other T cell activation markers. Both CD4 and CD8 T cells upregulate the expression of CD107a, CD137, and CD69 in response to antigen stimulation.

[0097] Figure 2 shows that EN-17C27 (TCR cloned from CD4 T cells) was validated as an H3K27M antigen-specific TCR. It responds to short peptides (RMS or RKS, Figure 2A ) did not respond to stimulation. It specifically responded to stimulation with a long peptide carrying a point mutation (KQ-RMS), but did not respond to stimulation with a long peptide without a point mutation (KQ-RKS) ( Figure 2A ).

[0098] EN-17C27 is a CD4 TCR introduced as a foreign TCR into PBMC cells. Both CD4 and CD8 T cells can be transduced with retroviral vectors. When EN-17C27 is introduced into CD8 T cells, it can still interact with its cognate antigen (KQ-RMS presented by class II HLA). However, in this case, this interaction cannot be enhanced by the CD4 molecule because the host cell is a CD8 T cell. In addition, the CD8 molecule does not contribute to the interaction between the TCR and the antigen. This is a good test for a candidate TCR. A good candidate TCR with high binding affinity for its antigen can trigger a T cell response independently of CD4 or CD8. The data show that EN-17C27 can trigger a CD8 T cell response independent of CD4 ( Figure 2B ).

[0099] These data demonstrate that EN-17C27 is an H3K27M antigen-specific CD4 TCR with high antigen-binding affinity. To date, we have cloned seven antigen-specific CD4 TCRs from vaccinated patient samples (six from patient EN-17 and one from patient EN-23). ​​Meanwhile, we have only cloned two CD8 TCRs. Compared to CD4 TCRs, the cloned CD8 TCRs were functionally weaker (Figure 3).

[0100] Figure 3 shows the functional validation results of two cloned CD8 TCRs. Both TCRs were cloned from a single patient sample, EN-10. These two TCRs could only trigger weak responses in CD4 T cells (data not shown). Therefore, they had low binding affinity for antigens. EN-10C01 was unable to distinguish between peptide antigens with or without point mutations ( Figure 3A ), but EN-10C04 is specific for the mutant peptide ( Figure 3B ). Interestingly, C01 showed a better response to the long peptide than to the short peptide (Figure 3). Since CD8 TCR can only recognize antigenic peptides of 8-12aa. The originally designed short peptide vaccine (RMS) may not be the optimal antigenic peptide to trigger the C01 response. The subregion of the long peptide (KQ-RMS) that is different from "RMS" may serve as a cognate antigen for EN-10C01. This suggests that compared with previous peptide designs, the long peptide design not only recruits CD4 T cells into the immune response, but also provides a better antigen to trigger the CD8 response.

[0101] 1.2.5 Conclusion:

[0102] Consistent with our vaccine design, both CD8 and CD4 T cell responses were detected in the peripheral blood of vaccinated patients. Importantly, a stronger immune response was detected in CD4 T cells compared with CD8 T cells. These data suggest that the peptide vaccine can stimulate anticancer immunity in DIPG patients. Furthermore, the CD4 T cell-mediated immune response triggered by the long peptide vaccine may play a more important role in cancer treatment than the CD8 T cell response. References Schwartzentruber J et al. Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature 2012 Jan 29;482(7384):226-31. Wu G et al., Somatic histone H3 alterations in pediatric diffuseintrinsic pontine gliomas and non-brainstem glioblastomas. Nat Genet 2012 Jan 29;44(3):251-3. Argersinger DP et al., New Developments in the Pathogenesis, TherapeuticTargeting, and Treatment of H3K27M-Mutant Diffuse Midline Glioma. Cancers 2021, 13(21), 5280. Zhang X et al., Oncohistone Mutations in Diffuse Intrinsic Pontine Glioma. Trends Cancer 2019 Dec;5(12):799-808. Lowe BR et al., Histone H3 Mutations: An Updated View of Their Role in Chromatin Deregulation and Cancer. Cancers. 2019 May;11(5):660. Rosa DS et al., CD4 +T Cell Epitope Discovery and Rational Vaccine Design. Archivum Immunologiae et Therapiae Experimentalis Vol. 58, pp. 121–130 (2010) Hemmer B et al., Minimal peptide length requirements for CD4 + T cell clones—implications for molecular mimicry and T cell survival. International Immunology, March 2000; 12(3):375-383. Chicz RM et al., Predominant naturally-processed peptides bound to HLA-DR1 are derived from MHC-related molecules and are heterogeneous in size. Nature, 1992, vol. 358(764-768). Sercarz EE et al., MHC-guided processing: binding of large antigen fragments. Nat. Rev. Immunol, 2003, vol. 3(621-629) Toussi DN and Massari P, Immune Adjuvant Effect of Molecularly-defined Toll-Like Receptor Ligands. Vaccines June 2014; 2(2):323-353.

Claims

1. A pharmaceutical composition comprising a peptide, wherein the peptide has a length of at least 12 amino acid residues and comprises RMSA (SEQ ID NO: 26), wherein the pharmaceutical composition, after administration to a subject according to a therapeutically effective regimen, is capable of stimulating a CD4 T cell response against histone 3 (H3) K27M mutation (H3K27M) in the subject.

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is further capable of stimulating a CD8 T cell response against the H3K27M mutation in the subject after being administered to the subject according to a therapeutically effective regimen.

3. The pharmaceutical composition of claim 1 or claim 2, wherein the peptide comprises RMSAP(S / A)TGGV (SEQ ID NO: 25).

4. The pharmaceutical composition of claim 3, wherein the peptide comprises the amino acid sequence KQLATKAARMSAP(S / A)TGGV (SEQ ID NO: 1).

5. The pharmaceutical composition of claim 4, wherein the H3 in the subject has the H3.3 variant, wherein the peptide has the amino acid sequence KQLATKAARMSAPSTGGV (SEQ ID NO: 2).

6. The pharmaceutical composition of claim 4, wherein the H3 in the subject has the H3.1 variant, wherein the peptide has the amino acid sequence KQLATKAARMSAPATGGV (SEQ ID NO: 27).

7. The pharmaceutical composition according to any one of claims 1 to 6, further comprising an adjuvant.

8. The pharmaceutical composition of claim 7, wherein the adjuvant comprises a toll-like receptor agonist.

9. The pharmaceutical composition of claim 8, wherein the toll-like receptor agonist is poly I:C.

10. The pharmaceutical composition according to claim 9, wherein the toll-like receptor agonist is polyICLC. 11 . The pharmaceutical composition of claim 10 , wherein the weight ratio of the polyICLC to the peptide is approximately 1:0.5 to 1:

5.

12. The pharmaceutical composition of claim 11, wherein the weight ratio of the polyICLC to the peptide is about 1:

4.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the dosage form of the pharmaceutical composition comprises about 0.5 mg poly-ICLC and about 0.5-2 mg of the peptide.

14. The pharmaceutical composition of claim 13, wherein the dosage form of the pharmaceutical composition comprises about 0.5 mg polyICLC and about 2 mg of the peptide.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the pharmaceutical composition is formulated for subcutaneous injection into a subject.

16. A method of stimulating an immune response in a subject having cancer, wherein the cancer is characterized by a K27M mutation in histone H3, the method comprising the steps of: The pharmaceutical composition according to any one of claims 1 to 10 is administered to the subject in a therapeutically effective regimen, thereby inducing an immune response in the subject.

17. The method of claim 16, wherein the immune response comprises a CD4 T cell response against the H3K27M mutation.

18. The method of claim 17, wherein the immune response further comprises a CD8 T cell response against the H3K27M mutation.

19. The method according to any one of claims 16 to 18, further comprising the step of determining the mutational variant of H3 in the subject prior to the administering step.

20. The method of claim 19, wherein the mutant variant of H3 is identified as H3.3, wherein: In the administering step, the peptide in the pharmaceutical composition comprises RMSAPSTGGV (SEQ ID NO: 5).

21. The method of claim 20, wherein the peptide in the pharmaceutical composition comprises KQLATKAARMSAPSTGGV (SEQ ID NO: 2).

22. The method of claim 21, wherein the peptide in the pharmaceutical composition consists of KQLATKAARMSAPSTGGV (SEQ ID NO: 2).

23. The method of claim 19, wherein the mutant variant of H3 is identified as H3.1, wherein: In the administering step, the peptide in the pharmaceutical composition comprises RMSAPATGGV (SEQ ID NO: 29).

24. The method of claim 23, wherein the peptide in the pharmaceutical composition comprises KQLATKAARMSAPATGGV (SEQ ID NO: 27).

25. The method of claim 24, wherein the peptide in the pharmaceutical composition consists of KQLATKAARMSAPATGGV (SEQ ID NO: 27).

26. The method of any one of claims 16-25, wherein the cancer comprises at least one of glioma, acute myeloid leukemia (AML), or melanoma.

27. The method of claim 26, wherein the cancer comprises glioma.

28. The method of claim 27, wherein the cancer comprises DIPG.

29. The method of any one of claims 16-28, wherein the subject carries the HLA-A*02 allele.

30. The method of any one of claims 16-28, wherein the subject carries at least one of an HLA-DRB1*07:01 allele or an HLA-DRB1*01:01 allele.

31. The method of any one of claims 16-29, wherein the therapeutically effective regimen comprises administering the pharmaceutical composition to the subject by subcutaneous injection.

32. A T cell expressing a T cell receptor (TCR) or a fragment thereof, wherein the TCR is capable of binding to a peptide / MHC II complex, wherein the peptide in the peptide / MHC II complex has a length of at least 12 amino acid residues and comprises RMSA (SEQ ID NO: 26).

33. The T cell of claim 32, wherein the TCR or fragment thereof is further capable of binding to a complex formed between the peptide and MHC I.

34. The T cell of claim 32 or claim 33, wherein the peptide comprises RMSAP(S / A)TGGV (SEQ ID NO: 25).

35. The T cell of claim 34, wherein the peptide comprises the amino acid sequence KQLATKAARMSAP(S / A)TGGV (SEQ ID NO: 1).

36. The T cell of claim 35, wherein the peptide comprises the amino acid sequence KQLATKAARMSAPSTGGV (SEQ ID NO: 2).

37. The T cell of claim 36, wherein the peptide consists of the amino acid sequence KQLATKAARMSAPSTGGV (SEQ ID NO: 2).

38. The T cell of claim 36 or claim 37, wherein the TCR or fragment thereof is incapable of binding to a complex formed between a second peptide and an MHC II complex, wherein the second peptide comprises the amino acid sequence KQLATKAARKSAPSTGGV (SEQ ID NO: 6).

39. The T cell of claim 35, wherein the peptide comprises the amino acid sequence KQLATKAARMSAPATGGV (SEQ ID NO: 27).

40. The T cell of claim 39, wherein the peptide consists of the amino acid sequence KQLATKAARMSAPATGGV (SEQ ID NO: 27).

41. The T cell of claim 39 or claim 40, wherein the TCR or fragment thereof is incapable of binding to a complex formed between a second peptide and an MHC II complex, wherein the second peptide comprises the amino acid sequence KQLATKAARKSAPATGGV (SEQ ID NO: 28).

42. The T cell of any one of claims 32-41, wherein the TCR is heterologous to the T cell.

43. The T cell of claim 42, wherein the TCR is transduced or transfected into the T cell via a vector.

44. The T cell of claim 43, wherein the vector is a retroviral vector.

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