Antigenic peptides targeting FLT3-D835 mutation and their application in tumor immunotherapy

Abstract

The present invention relates to an antigenic peptide targeting FLT3-D835 mutation and its application in tumor immunotherapy. Specifically, the present invention provides an antigenic peptide for eliciting an immune response targeting FLT3-D835 mutation. Compared with the lack of affinity between the wild-type polypeptide and HLA-A * 02:01 molecule, the antigenic peptide of the present invention has high affinity for HLA-A * 02:01 molecule. The present invention also provides the application of the antigenic peptide of the present invention. The antigenic peptide of the present invention has good immunogenicity, can activate specific immune responses, and has important clinical significance for targeting the clearance of leukemia cells, stabilizing the remission state of AML patients, and prolonging the disease-free survival time.

Description

Technical Field

[0001] The present invention belongs to the fields of polypeptide drugs and polypeptide vaccines, and particularly relates to an antigenic peptide targeting FLT3-D835 mutation and its application in tumor immunotherapy. Background Art

[0002] Acute myeloid leukemia (AML) is a malignant clonal proliferative disease caused by the continuous accumulation of acquired genetic abnormalities in hematopoietic stem / progenitor cells, resulting in myeloid differentiation arrest at different stages. AML is the most common hematological malignancy in adults, accounting for more than 70% of all leukemias. Currently, the treatment of AML in clinical practice mainly consists of chemotherapy combined with hematopoietic stem cell transplantation, and the overall treatment effect is poor. The cure rate of patients under 60 years old is only 35% - 40%, and that of patients over 60 years old is even lower, at 5% - 15%. Moreover, nearly 50% of patients lose the opportunity of treatment due to disease recurrence or drug resistance. The continuous accumulation of gene mutations, including somatic mutations, gene insertions / deletions, gene fusions, etc., is the key reason for the refractory / relapsed AML. Therefore, taking the mutated gene as the breakthrough point and targeting the elimination of abnormal leukemia cells have important scientific significance for reducing the recurrence rate of AML and improving the cure rate.

[0003] The FMS-like tyrosine kinase 3 (FLT3) gene is located on chromosome 13q12 and encodes a protein containing 993 amino acids. Among them, the transmembrane region is located between the 542nd and 564th amino acids, and the kinase domain is located between the 610th and 944th amino acids, including a kinase insert fragment of about 50 amino acids. FLT3 variation is one of the most common gene abnormalities in AML, occurring in approximately 30% of patients. FLT3 variation leads to enhanced FLT3 kinase activity, thereby promoting the proliferation and growth of leukemia cells, and is closely related to the poor prognosis of AML patients.

[0004] Studies have found that approximately 5 - 10% of FLT3 variations involve single nucleotide mutations in the FLT3 tyrosine kinase domain (FLT3-TKD), and the most common of which is the mutation of the 835th amino acid residue - aspartic acid (D835), usually located within the activation loop. Aspartic acid is a key regulatory residue of tyrosine kinase receptors and is highly conserved in structure. The wild-type D835 residue (D835wt) is crucial for maintaining the inactive conformation of FLT3, and the D835 point mutation leads to poor prognosis of patients. Finding a treatment method targeting FLT3-D835 mutation and targeting the elimination of leukemia cells has important clinical significance for stabilizing the remission state of AML patients and prolonging the disease-free survival time.

[0005] In recent years, with the development of immunology, genomics, and molecular biology technologies, immunotherapy has become another revolutionary mode of cancer treatment. Among them, therapeutic cancer vaccines are a research hotspot in immunotherapy. Based on the tumor-immune cycle theory, cancer vaccines stimulate or enhance the body's anti-tumor immune response by promoting the recognition of tumor antigens by immune cells, and cooperate with the immune system to eliminate cancer cells. However, traditional cancer vaccines contain mostly tumor-associated antigens that are expressed in both tumors and normal tissues, resulting in low immunogenicity of the vaccines, easy induction of immune tolerance, repeated setbacks in clinical trials, and few clinical promotions.

[0006] Neoantigens are protein sequences containing mutated amino acids derived from cancer cells based on gene mutations. After being presented by antigen-presenting cells, neoantigens can be effectively recognized by T cells, activating T cells, thereby activating specific immune responses to attack and eliminate cancer cells. Synthesizing neoantigen peptides derived from gene mutations to construct therapeutic cancer vaccines and re-infusing them into patients to activate immune cells can target and kill cancer cells expressing the same neoantigens. Moreover, neoantigens, due to their exclusive expression in cancer cells and absence in normal cells or tissues, have the characteristics of high immunogenicity without inducing immune tolerance, making them an advantageous target for cancer vaccines. However, current neoantigen vaccine strategies require sequencing the genomes and HLA typing of individual subjects and calculating possible applicable individual neoantigen peptides, which takes at least 2-3 months or even longer, to some extent limiting the promotion of the treatment strategy. Additionally, the accuracy of the calculated neoantigens is not high, and the immunogenicity of gene mutation-derived polypeptides still needs further evaluation.

[0007] Therefore, there is an urgent need in the art to develop a safe, efficient, and economical antigen peptide to broaden the treatment scope for patients with FLT3-mutated tumors and provide new options for the immunotherapy of patients. Summary of the Invention

[0008] The object of the present invention is to provide an antigen peptide targeting the FLT3-D835 mutation.

[0009] Another object of the present invention is to provide the application of the antigen peptide targeting the FLT3-D835 mutation in tumor immunotherapy.

[0010] In the first aspect of the present invention, there is provided an antigen peptide for eliciting an immune response targeting the FLT3-D835 mutation, the antigen peptide being capable of forming a complex with MHC molecules, and the antigen peptide being selected from the group consisting of:

[0011] (i) the polypeptide shown in SEQ ID NO:6:

[0012] X1IMSDSNYV

[0013] Among them, X1 is V, H, I or F;

[0014] (ii) A derivative polypeptide formed by substituting 1, 2 or 3 amino acids, and / or inserting 1, 2 or 3 amino acids, and / or deleting 1 or 2 amino acids in the amino acid sequence of the polypeptide in (i) except for X1, and the derivative polypeptide retains X1.

[0015] In another preferred example, the antigenic peptide has the structure shown in Formula I:

[0016] X0-X1-Z1-X 10 (I)

[0017] Wherein,

[0018] X0 is none or R;

[0019] X1 is V, H, I or F;

[0020] Z1 is IMSDSNYV;

[0021] X 10 is none or V.

[0022] In another preferred example, in Formula II, X0 is none or R, and X 10 is none or V.

[0023] In another preferred example, the antigenic peptide has the structure of Formula II,

[0024] X1-Z1 (II)

[0025] Wherein,

[0026] X1 is V, H, I or F;

[0027] Z1 is IMSDSNYV.

[0028] In another preferred example, X1 is V or H.

[0029] In another preferred example, the antigenic peptide is a combination of two or more antigenic peptides.

[0030] In another preferred example, the antigenic peptide is 1 kind, or a combination composed of 2, 3 or 4 polypeptides among the polypeptides with the amino acid sequence shown in any one of SEQ ID NO:1-4.

[0031] In another preferred example, the combination of the antigenic peptides further contains additional antigenic peptides directed against other tumor antigens or sites.

[0032] In another preferred example, the additional antigenic peptide includes the polypeptide shown in SEQ ID No:5.

[0033] In a second aspect of the present invention, a pMHC complex is provided, which complex comprises the antigenic peptide described in the first aspect of the present invention.

[0034] In another preferred embodiment, the antigenic peptide in the pMHC complex is a polypeptide having the amino acid sequence shown in SEQ ID NO: 6.

[0035] In another preferred embodiment, the type of the MHC molecule is HLA-A*02.

[0036] In another preferred embodiment, the type of the MHC molecule is HLA-A*02:01.

[0037] In a third aspect of the present invention, a nucleic acid molecule is provided, which nucleic acid molecule comprises a nucleic acid sequence encoding the antigenic peptide described in the first aspect of the present invention or its complementary sequence.

[0038] In a fourth aspect of the present invention, a vector is provided, which vector contains the nucleic acid molecule described in the third aspect of the present invention.

[0039] In a fifth aspect of the present invention, a host cell is provided, which cell contains the vector described in the fourth aspect of the present invention.

[0040] In a sixth aspect of the present invention, a method for preparing specific T lymphocytes in vitro is provided, comprising the steps of:

[0041] a) providing PBMC,

[0042] b) contacting and culturing the PBMC with the antigenic peptide described in the first aspect of the present invention in the presence of the antigenic peptide, thereby obtaining antigenic peptide-activated specific T lymphocytes.

[0043] In another preferred embodiment, the concentration of the antigenic peptide is 20 μg / mL.

[0044] In another preferred embodiment, the number of days for culturing the PBMC with the antigenic peptide is 10 days.

[0045] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0046] In another preferred embodiment, the PBMC is autologous cells or allogeneic cells.

[0047] In another preferred embodiment, step (b) further comprises:

[0048] (b1) sorting out CD8 + cells and CD8 - cells,

[0049] (b2) Sensitize the CD8 cells described in the first aspect of the present invention with the antigenic peptide pair described in (b1), thereby obtaining sensitized CD8 cells. - cells, -

[0050] (b3) Co-incubate the sensitized CD8 cells described in (b2) with CD8 cells, thereby obtaining specifically antigenic peptide-activated T lymphocytes. - cells and CD8 +

[0051] In the seventh aspect of the present invention, there is provided a pharmaceutical composition, which contains (ii) a pharmaceutically acceptable carrier and (ii) the antigenic peptide described in the first aspect of the present invention, the pMHC complex described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, or specifically antigenic peptide-activated T lymphocytes.

[0052] In another preferred embodiment, the pharmaceutical composition is a vaccine composition.

[0053] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from liquid, solid, or gel state.

[0054] In another preferred embodiment, the pharmaceutical composition is administered by a method selected from the following group: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, or oral administration.

[0055] In the eighth aspect of the present invention, there is provided a method for preventing or treating malignant tumor-related diseases, including administering an appropriate amount of the antigenic peptide described in the first aspect of the present invention, the pMHC complex described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, specifically antigenic peptide-activated T lymphocytes, or the pharmaceutical composition described in the seventh aspect of the present invention to a subject in need.

[0056] Use of the antigenic peptide described in the first aspect of the present invention, the pMHC complex described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, specifically antigenic peptide-activated T lymphocytes, or the pharmaceutical composition described in the seventh aspect of the present invention for the preparation of a drug for preventing or treating malignant tumors.

[0057] In another preferred embodiment, the malignant tumor is a hematological malignancy.

[0058] In another preferred embodiment, the malignant tumor is acute myeloid leukemia (AML).

[0059] In another preferred embodiment, the malignant tumor is an FLT-D835 mutant malignant tumor. ​​

[0060] It should be understood that within the scope of the present invention, the above-mentioned various technical features of the present invention and the various technical features specifically described hereinafter (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Displays the stability of the complex formed by the antigen peptide and HLA-A * 02:01 molecule at 8H.

[0062] Figure 2 Displays the generation of specific T cells induced by the antigen peptide detected by Tetramer flow cytometry.

[0063] Figure 3 Displays the level of IFN-γ secreted by specific T cells activated by the antigen peptide detected by the ELISPOT assay.

[0064] Figure 4 Displays that the antigen peptide can induce the generation of CTLs from the peripheral blood of healthy volunteers.

[0065] Figure 5 Displays an increase in the ability of CTLs from healthy volunteers to secrete IFN-γ. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0066] Through extensive and in-depth research and a large number of screenings, the present inventors unexpectedly obtained a safe, efficient, and economical antigen peptide targeting the FLT3-D835 mutation. Experiments show that compared with the wild-type polypeptide (SEQ ID No. 7) having no affinity for HLA-A * 02:01 molecule, the antigen peptide of the present invention (SEQ ID No. 1-4) has medium or high affinity for HLA-A * 02:01 molecule. At the same time, the antigen peptide of the present invention has good immunogenicity, can activate specific immune responses, can induce the generation of CTLs from the peripheral blood of healthy volunteers, and the ability of CTLs to secrete IFN-γ increases. Based on this, the present invention was completed.

[0067] It should be understood that in the present invention, the "antigen peptide" of the present invention can be used interchangeably with the "polypeptide of the present invention" or the "short peptide of the present invention", and all refer to the antigen peptide targeting the FLT3-D835 mutation of the present invention.

[0068] TERMS

[0069] To facilitate a better understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the art to which the present invention pertains. As used herein, when referring to a specifically recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0071] FMS-like tyrosine kinase 3 (FLT3) and FLT3-D835

[0072] The FMS-like tyrosine kinase 3 (FLT3) gene is located on chromosome 13q12 and encodes a protein containing 993 amino acids. Among them, the transmembrane region is between the 542nd and 564th amino acids, and the kinase domain is between the 610th and 944th amino acids, including a kinase insert fragment of about 50 amino acids. FLT3 variation is one of the most common genetic abnormalities in AML, occurring in approximately 30% of patients. FLT3 variation leads to enhanced FLT3 kinase activity, thereby promoting the proliferation and growth of leukemia cells and being closely related to poor prognosis of AML patients.

[0073] Studies have found that approximately 5-10% of FLT3 variations involve single nucleotide mutations in the FLT3 tyrosine kinase domain (FLT3-TKD), and the most common of which is the mutation of the 835th amino acid residue - aspartic acid (D835), which is usually located within the activation loop. Aspartic acid is a key regulatory residue of the tyrosine kinase receptor and is highly conserved in structure. Currently, D835 point mutations identified from AML patients include: alanine (A), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), asparagine (N), valine (V), tyrosine (Y), among which D835F, D835H, D835I, D835V, and D835Y are the most common. The D835 point mutation results in poor prognosis of patients.

[0074] Neoantigen

[0075] Neoantigens are protein sequences containing variant amino acids derived from tumor cells based on gene mutations. After being presented by antigen-presenting cells, neoantigens can be effectively recognized by T cells, activating T cells and thus activating specific immune responses to attack and eliminate tumor cells. Synthesizing neoantigen peptides derived from gene mutations to construct therapeutic cancer vaccines and reinfusing them into patients can activate immune cells to target and kill tumor cells expressing the same neoantigens. Moreover, since neoantigens are only expressed in tumor cells and not in normal cells or tissues, they have the characteristics of high immunogenicity without inducing immune tolerance, making them an advantageous target for cancer vaccines.

[0076] Specifically, in the first aspect of the present invention, there is provided an antigenic peptide for eliciting an immune response targeting the FLT3-D835 mutation, which antigenic peptide is capable of forming a complex with an MHC molecule, and the antigenic peptide is selected from the group consisting of:

[0077] (i) the polypeptide shown in SEQ ID NO:6:

[0078] X1IMSDSNYV

[0079] wherein X1 is V, H, I or F;

[0080] (ii) a derivative polypeptide formed by substituting one, two or three amino acids, and / or inserting one, two or three amino acids, and / or deleting one or two amino acids in the amino acid sequence of the polypeptide in (i) except for X1, and the derivative polypeptide retains X1.

[0081] Amino acid substitution means that at the same position, one amino acid residue is replaced by another amino acid residue. The inserted amino acid residues can be inserted at any position, and the inserted amino acid residues can be adjacent to each other in whole or in part, or the inserted amino acids are not adjacent to each other. As is known to those skilled in the art, the peptides of the present invention can be post-translationally modified at one or more positions between amino acid sequences. Examples of post-translational modifications can be found in Engelhard et al. Curr Opin Immunol. February 2006; 18(1): 92-7 and include phosphorylation, acetylation and deamidation.

[0082] Preferably, the peptide of the present invention binds to the peptide-binding site of MHC on the MHC molecule. Generally, the modified amino acids described above do not disrupt the binding ability of the peptide to MHC. In a preferred embodiment, the amino acid modification enhances the binding ability of the peptide to MHC. For example, mutations may occur at the peptide-MHC binding site. These binding sites and the preferred residues at the binding sites are known in the art, especially for peptides that bind HLA-A*02 (see, for example, Parkhurst et al., J. Immunol. 157:2539-2548 (1996)).

[0083] More specifically, the length of the amino acids of the peptide of the present invention can be 8-15, preferably 8-10, and more preferably 9.

[0084] The polypeptide of the present invention may be composed of any one of the polypeptides of SEQ ID NOs. 1-4 in Table 1.

[0085] Table 1 Antigenic peptides of the present invention

[0086]

[0087] The present invention also provides analogs of the proteins or peptides shown in SEQ ID NO: 1-4. These analogs may differ from the native peptides in terms of amino acid sequence, or in modified forms that do not affect the sequence, or both. These peptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, and also by site-directed mutagenesis or other known molecular biology techniques. Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the peptides of the present invention are not limited to the representative peptides exemplified above.

[0088] Modified (usually without changing the primary structure) forms include: chemically derivatized forms of peptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those peptides that are glycosylated during peptide synthesis and processing or further processing steps. Such modifications can be accomplished by exposing the peptide to glycosylating enzymes (such as mammalian glycosylating enzymes or deglycosylating enzymes). Modified forms also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are peptides that are modified to enhance their proteolytic resistance or optimize their solubility properties.

[0089] In the present invention, the "protein conservative variant peptides shown in SEQ ID NO: 1-4" refer to peptides formed by replacing at most 3, more preferably at most 2 amino acids with amino acids having similar or close properties compared with the amino acid sequences of SEQ ID NO: 1-4. These conservative variant peptides are preferably generated by amino acid substitution according to Table 1.

[0090] Table A

[0091]

[0092]

[0093] The peptides of the present invention can be simply synthesized by the Merrifield synthesis method (also known as solid-phase peptide synthesis). Peptides of GMP grade can be synthesized by solid-phase synthesis technology of Multiple Peptide Systems (San Diego, CA). Alternatively, the peptides can be recombinantly synthesized and, if necessary, can be synthesized by methods known in the art. Typical such methods involve the use of vectors that include nucleic acid sequences encoding the polypeptide and express the polypeptide in vivo; for example, in bacteria, yeast, insect, or mammalian cells. Alternatively, an in vitro cell-free system can also be used for expression. Such systems are known in the art and are commercially available. The peptides can be isolated and / or provided in a substantially pure form. For example, they can be provided in a form that is substantially free of other peptides or proteins.

[0094] Tumor antigens are processed intracellularly by proteolysis into polypeptide fragments 8-16 amino acids in length, i.e., CTL epitopes, which then bind to MHC molecules in the endoplasmic reticulum lumen to form a peptide-MHC complex (pMHC) and are presented together on the cell surface. Therefore, a second aspect of the present invention provides a pMHC complex that contains the peptides described in the first aspect of the present invention. Preferably, the polypeptide binds to the peptide-binding groove of the MHC molecule. The MHC molecule can be an MHC class I molecule or an MHC class II molecule. Preferably, the MHC molecule is an MHC class I molecule. In a preferred embodiment, the MHC molecule is HLA-A * 02, and more preferably, the MHC molecule is HLA-A * 0201.

[0095] The pMHC complex described in the present invention can exist in the form of multimers, for example, dimers, or tetramers, or pentamers, or hexamers, or octamers, or larger. Appropriate methods for generating pMHC multimers can be referred to relevant literature, such as (Greten et al., Clin. Diagnostic Lab. Immunol. 2002: 216-220).

[0096] Generally, pMHC multimers can be generated by complexing a pMHC complex with a biotin residue with fluorescently labeled streptavidin. Alternatively, the pMHC multimers can also be formed using immunoglobulins as molecular scaffolds. In this system, the extracellular region of the MHC molecule is bound to the constant region of the immunoglobulin heavy chain through a short linker sequence. Additionally, carrier molecules such as dextran (WO02072631) can be used to form pMHC multimers. pMHC multimers help improve the detection of their binding partners, such as T cell receptors. Or, enhance the effect of pMHC complexes in related applications, such as activating T cells.

[0097] The pMHC complex described in the present invention can be provided in a soluble form. To obtain a soluble pMHC complex, preferably, the MHC molecule in the pMHC complex does not contain a transmembrane region. Specifically, in the pMHC complex, MHC class I molecules can consist of their light chains and the extracellular domains of all or part of the heavy chains. Alternatively, the MHC molecule is a fragment that only contains its functional domains.

[0098] Methods for generating the soluble pMHC complex of the present invention are known to those skilled in the art, including, but not limited to, the methods described in the examples of the present invention. The MHC molecules in the soluble pMHC complex of the present invention can also be generated using synthetic methods and then refolded with the peptides of the present invention. By determining whether the peptide and the MHC molecule can be refolded, it can be determined which type of MHC molecule the peptide of the present invention can form a complex with.

[0099] The soluble pMHC complex of the present invention can be used to screen or detect molecules that bind to it, such as TCRs or antibodies. The method includes contacting the pMHC complex with the binding moiety to be tested and determining whether the binding moiety to be tested binds to the complex. Methods for determining the binding of the pMHC complex are well known in the art. Preferred methods include, but are not limited to, surface plasmon resonance, or any other biosensing technology, ELISA, flow cytometry, chromatography, microscopy. Alternatively, in addition, the binding can be detected by a functional assay of the biological response generated by the binding, such as cytokine release or apoptosis.

[0100] Similarly, the soluble pMHC complexes of the present invention can also be used to screen TCR or antibody libraries. Constructing antibody libraries using phage display technology is well known in the art, as described in the reference Aitken, Antibody phage display: Methods and Protocols (2009, Humana, New York). In a preferred embodiment, the pMHC complexes of the present invention are used to screen a diverse TCR library displayed on the surface of phage particles. The TCRs displayed by the library may contain unnatural mutations.

[0101] Accordingly, the soluble pMHC complexes of the present invention can be immobilized onto a suitable solid support via a linker. Examples of solid supports include, but are not limited to, beads, membranes, agarose gels, magnetic beads, substrates, tubes, columns. The pMHC complexes can be immobilized on ELISA plates, magnetic beads, or surface plasmon resonance biosensor chips. Methods for immobilizing the pMHC complexes onto solid supports are known to those skilled in the art and include, for example, using affinity binding pairs such as biotin and streptavidin, or antibody and antigen. In a preferred embodiment, the pMHC complexes are labeled with biotin and immobilized on a streptavidin-coated surface.

[0102] The peptides of the present invention can be presented together with MHC complexes on the cell surface. Accordingly, the present invention also provides a cell that is capable of presenting the pMHC complexes of the present invention on its surface. Such cells can be mammalian cells, preferably immune system cells, and more preferably specialized antigen-presenting cells such as dendritic cells or B cells. Other preferred cells include T2 cells (Hosken, et al., Science. 1990. 248:367-70). The cells presenting the peptides or pMHC complexes of the present invention can be isolated, preferably provided in the form of a cell population, or in a substantially pure form. The cells may not naturally present the complexes of the present invention, or the level of complex presentation by the cells is higher than in the natural state. Such cells can be obtained by pulsing with the peptides of the present invention. Pulsing involves incubating the cells with the peptides for several hours, preferably at a concentration of 10 -5 -10 -12 M. In addition, the cells can also be transduced with HLA-A * 02 molecules to further induce peptide presentation. The cells presenting the pMHC complexes of the present invention can be used to isolate T cells and T cell receptors, and the T cells activated by the cells can be further sorted out, and thus the T cell receptors expressed on the surface of the T cells can also be obtained.

[0103] In a preferred embodiment, the method for obtaining the above-mentioned T cells includes stimulating fresh blood obtained from healthy volunteers with the cells presenting the pMHC complex of the present invention. It can undergo several rounds of stimulation, such as 3 - 4 rounds. The identification of the activated T cells can be determined by measuring the release of cytokines (such as IFN-γ ELISpot assay) in the presence of the peptide-pulsed T2 cells of the present invention. Using labeled antibodies, the activated cells can be sorted by flow cytometry (FACS), and the sorted cells can be expanded and further verified, for example, by ELISpot assay and / or cytotoxicity against target cells and / or pMHC multimer staining. The TCR chains from the verified T cell clones can be amplified by rapid amplification of cDNA ends (RACE) and sequenced.

[0104] The present invention also provides a nucleic acid molecule, which includes a nucleic acid sequence encoding the peptide of the present invention. The nucleic acid can be cDNA. The nucleic acid molecule can mainly consist of the nucleic acid sequence encoding the peptide of the present invention, or can only encode the peptide of the present invention. Such nucleic acid molecules can be synthesized by methods known in the art. Due to the degeneracy of the genetic code, those skilled in the art should understand that nucleic acid molecules with different nucleic acid sequences can encode the same amino acid sequence.

[0105] The present invention also provides a vector, which includes the nucleic acid sequence of the present invention. Suitable vectors are known in the field of vector construction, including the selection of promoters and other regulatory elements, such as enhancer elements. The vector of the present invention includes sequences suitable for introduction into cells. For example, the vector can be an expression vector, in which the coding sequence of the polypeptide is controlled by its own cis-acting regulatory elements, and the vector is designed to facilitate gene integration or gene replacement in host cells, etc.

[0106] Those of ordinary skill in the art should understand that in the present invention, the term "vector" includes DNA molecules, such as plasmids, phages, viruses or other vectors, which contain one or more heterologous or recombinant nucleic acid sequences. Suitable phage and virus vectors include, but are not limited to: λ-phage, EMBL phage, simian virus, bovine papillomavirus, Epstein-Barr virus, adenovirus, herpes virus, murine sarcoma virus, murine mammary tumor virus, lentivirus, etc.

[0107] The present invention also provides a binding molecule, which can be used as an immunotherapeutic agent or a diagnostic reagent. The binding molecule can bind only to a peptide, or to a complex formed by a peptide and an MHC molecule. In the latter case, the binding molecule can bind partially to the MHC molecule, and at the same time, it also binds to the peptide of the present invention. The binding moiety of the present invention can be isolated and / or soluble, and / or non-naturally occurring, i.e., there is no equivalent in nature, and / or pure, and / or synthetic.

[0108] In a preferred embodiment of the present invention, the binding molecule is a T cell receptor (TCR). The TCR can be described using the International Immunogenetics Information System (IMGT). The native αβ heterodimeric TCR has an α chain and a β chain. Generally speaking, each chain contains a variable region, a joining region, and a constant region. The β chain usually also contains a short hypervariable region between the variable region and the joining region, but this hypervariable region is often regarded as part of the joining region.

[0109] The TCR of the present invention can be in any form known in the art. For example, the TCR can be a heterodimer, or exist in a single-chain form. The TCR can be in a soluble form (i.e., without a transmembrane or cytoplasmic region). Specifically, the TCR can contain all or part of the extracellular domain of the TCR. The TCR can also be a full-length chain containing its transmembrane region. The TCR can be provided on the cell surface, such as on a T cell.

[0110] Soluble TCRs can be obtained by combining with the prior art in the art. For example, an artificial disulfide bond is introduced between the constant domains of the α and β chains of the αβ TCR, or an artificial disulfide bond is introduced between the variable region of the α chain and the constant region of the β chain of the αβ TCR.

[0111] The TCR of the present invention can be used to deliver a cytotoxic agent or an immunostimulatory agent to a target cell, or be transfected into a T cell so that the T cell expressing the TCR can destroy tumor cells, for administration to a patient during a treatment process called adoptive immunotherapy. In addition, the TCR of the present invention can also contain mutations. Preferably, the affinity of the mutated TCR for the pMHC complex of the present invention is increased. The TCR of the present invention can be used alone, or covalently or otherwise combined with a conjugate, preferably covalently. The conjugate includes a detectable label (for diagnostic purposes, where the TCR is used to detect the presence of cells presenting the pMHC complex of the present invention), a therapeutic agent, a PK (protein kinase) modification moiety, or a combination of any of the above substances bound or conjugated. The TCR of the present invention can also be bound to an anti-CD3 antibody, preferably covalently, to redirect T cells to kill target cells.

[0112] In another preferred embodiment, the binding molecule of the present invention is an antibody. As used herein, the term "antibody" refers to an immunoglobulin molecule and the immunologically active portion of an immunoglobulin molecule, i.e., a molecule containing a specific binding site, which can be wholly natural, or partially synthetic, or wholly synthetic. The term "antibody" includes antibody fragments, derivatives thereof, functional equivalents, and homologous antibodies, humanized antibodies, and the antibody fragments include immunoglobulin binding regions, which are antibody binding regions or homologous to antibody binding regions. It can be wholly natural, or partially synthetic, or wholly synthetic. A humanized antibody can be a modified antibody that contains the variable region of a non-human antibody (e.g., murine) and the constant region of a human antibody.

[0113] Examples of antibodies can be isotype immunoglobulins (e.g., IgG, IgE, IgM, IgD, and IgA) and their isotype subclasses; fragments include antigen-binding regions, such as Fab, scFv, Fv, dAb, Fd; and diabodies. The antibody can be polyclonal or monoclonal, preferably monoclonal.

[0114] The preparation methods of the above TCRs and antibodies are known to those skilled in the art, including but not limited to, expression from Escherichia coli cells or insect cells and purification.

[0115] On the other hand, the present invention further provides the use of the peptides, pMHC complexes, nucleic acid molecules, vectors, cells, and binding molecules of the present invention in the pharmaceutical field. The peptides, pMHC complexes, nucleic acids, vectors, cells, or binding molecules can be used for the treatment or prevention of malignant tumors, preferably acute myeloid leukemia.

[0116] The present invention also provides a pharmaceutical composition, which comprises the antigen peptide, pMHC complex, nucleic acid molecule, cell, or binding molecule of the present invention, and a pharmaceutically acceptable carrier. The pharmaceutical composition can be in any suitable form (depending on the administration method required by the patient). It can be provided in unit dosage form, usually placed in a sealed container, and can be provided as part of a kit. Such kits usually (but not necessarily) contain instructions for use. It can contain multiple said unit dosage forms.

[0117] The pharmaceutical composition is suitable for any appropriate administration route, such as injection (including subcutaneous, intramuscular, intraperitoneal, or intravenous injection), inhalation, oral administration, or nasal, or rectal administration, etc. The composition can be prepared by any method known in the pharmaceutical field, for example, by mixing the active ingredient with a carrier or excipient under sterile conditions.

[0118] Depending on the disease or disorder to be treated (such as cancer, viral infection or autoimmune disease), the individual age and condition of the patient, etc., the dosage of the preparation of the present invention can vary within a relatively wide range. The appropriate dosage will be ultimately determined by the physician.

[0119] According to the prior art in the art, peptides, pMHC complexes or cells presenting pMHC complexes that are presented to the cell surface together with MHC molecules can activate T cells or B cells to make them function.

[0120] Therefore, the peptides, pMHC complexes or cells presenting pMHC complexes of the present invention can be provided in the form of a vaccine composition. The vaccine composition can be used for the treatment or prevention of cancer. All such compositions are included in the present invention. It should be understood that the vaccine can be in various forms (Schlom J. J Natl Cancer Inst. 2012 104(8): 599 - 613). For example, the peptides of the present invention can be directly used to immunize patients (Salgaller ML. Cancer Res. 1996. 56(20): 4749 - 57 and Marchand M. Int J Cancer. 1999. 80(2): 219 - 230). The vaccine composition can contain additional peptides such that the peptides of the present invention are one of the peptide mixtures. The vaccine composition can be added with adjuvants to enhance the immune response. Alternatively, the vaccine composition can be in the form of antigen - presenting cells presenting the peptides and MHC complexes of the present invention. Preferably, the antigen - presenting cells are immune cells, more preferably dendritic cells. The peptides can also be pulsed onto the surface of the cells (Thurner BI. et al., J. Exp. Med. 1999. 190: 1669), or the nucleic acid encoding the peptides of the present invention can be introduced into dendritic cells, for example, by electroporation (Van Tendeloo, VF. et al., Blood 2001. 98: 49).

[0121] The main advantages of the present invention include:

[0122] a) The present invention aims to overcome the deficiencies of the related art to a certain extent and provides a novel antigen peptide and its application in tumor immunotherapy.

[0123] b) The antigen peptide of the present invention has the effect of eliminating tumor cells.

[0124] c) The antigen peptide of the present invention can broaden the treatment scope for patients with FLT3 - mutant tumors and provide new options for the immunotherapy of patients.

[0125] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0126] Example 1 Prediction of the Affinity between Neoantigen Peptides and HLA-A * 02:01 Molecule

[0127] Use the online biological software NetMHCpan 4.1 to predict the affinity between neoantigen peptides and HLA-A * 02:01 molecule. %Rank_EL < 0.500 is regarded as having strong binding ability, 0.500 < %Rank_EL < 2.00 is regarded as having medium binding ability, and %Rank_EL > 2.000 is regarded as having no binding ability.

[0128] It was predicted that the neoantigen peptides (SEQ ID No.1 to SEQ ID No.5) had medium or strong binding ability with HLA-A * 02:01 molecule, and the wild-type polypeptide (SEQ ID No.7) had no binding ability with HLA-A * 02:01 molecule (Table 2).

[0129] Table 2 Prediction Results of the Affinity between Neoantigen Peptides Targeting FLT3-D835 Mutation and HLA-A * 02:01

[0130]

[0131] Example 2 Verification of the Affinity between Neoantigen Peptides and HLA-A * 02:01 Molecule

[0132] Take T2 cells in the logarithmic growth phase and adjust the cell concentration to 1×10 in IMDM medium without serum and antibiotics 6 / mL, add the neoantigen peptide (10 μg / mL) and β2-microglobulin (3 μg / mL) respectively, and co-culture at 37 °C for 4 h. After the culture, take out the cells, wash them with PBS, add the FITC-labeled anti-HLA-A2 monoclonal antibody, incubate at room temperature for 30 min, and detect with a flow cytometer. The final result uses the fluorescence index (FI) as the measurement index: (average fluorescence intensity of the sample - average fluorescence intensity of the background) / average fluorescence intensity of the background. FI > 1.5 is considered that the polypeptide has a high affinity with the HLA-A2 molecule; 1.0 < FI < 1.5 is medium affinity, and FI < 1.0 is low affinity.

[0133] The results showed that the antigen peptides (SEQ ID No.1 to SEQ ID No.5) had medium or high affinity with the HLA-A * 02:01 molecule, which was significantly higher than the affinity of the wild-type polypeptide (SEQ ID No.7) with the HLA-A * 02:01 molecule (a difference of 10 times or more).

[0134] The wild-type polypeptide (SEQ ID No.7) had no or extremely low affinity with the HLA-A * 02:01 molecule (Table 3).

[0135] Table 3 Affinity of the neoantigen peptide targeting FLT3-D835 mutation with the HLA-A * 02:01 molecule

[0136]

[0137] Example 3 Verification of the stability of the neoantigen peptide / HLA-A * 02:01 molecular complex

[0138] Take T2 cells in the logarithmic growth phase, adjust the concentration of T2 cells to 1×10 with serum-free and antibiotic-free IMDM medium (containing 100 ng / mL human β2m) 6 / mL, and incubate with 100 μg / mL neoantigen peptide overnight at 37 °C. Collect the cells the next day, add serum-free IMDM medium containing 10 μg / mL Brefeldin A and incubate for 1 h; add serum-free IMDM medium containing 0.5 μg / mL Brefeldin A, incubate at 37 °C, collect the cells at the time points of 0, 2, 4, 6, and 8 h respectively, resuspend the cells with 100 μL PBS, add the FITC-labeled anti-HLA-A2 monoclonal antibody, incubate at room temperature for 30 min, detect with a flow cytometer, and calculate the average fluorescence intensity of T2 cells at each time point.

[0139] Results

[0140] Table 4 Stability (%) of neoantigen peptide / HLA-A*02:01 molecular complex targeting FLT3-D835 mutation

[0141]

[0142]

[0143] The experimental results are as shown in Figure 1 and Table 4. It was detected that the complex formed by the antigen peptide of the present invention and HLA-A * 02:01 molecule was stable, and the complex formed by antigen peptide V and HLA-A * 02:01 molecule was the most stable.

[0144] Example 4 Induction of specific T lymphocytes by antigen peptide in AML patients

[0145] Venous blood was collected from AML patients with the same HLA typing and the same FLT3-D835 mutation. Peripheral blood mononuclear cells (PBMCs) were separated and purified by Ficoll density gradient centrifugation. CD8 + cells were sorted by Dynabeads magnetic beads, and CD8 - cells were used as antigen-presenting cells.

[0146] CD8 - cells were resuspended in serum-free RPMI-1640 medium, added with mitomycin (30 μg / mL), inactivated at 37°C for 30 min, washed with PBS, resuspended in serum-free RPMI-1640 medium, added with antigen peptide V (20 μg / mL), and incubated at 37°C for 2-4 h.

[0147] Antigen-pulsed CD8 - cells were collected, resuspended in RPMI-1640 medium containing 10% FBS (containing IL-2 50 U / mL, IL-7 5 ng / mL, IL-15 5 ng / mL), co-incubated with CD8 + cells, and the medium was changed by half every 2-3 days, and cultured for 10-20 d. The cells were collected, stained with PE-labeled antigen peptide-HLA-A * 02:01-Tetramer antibody, and detected by flow cytometry.

[0148] Results

[0149] The experimental results are as shown in Figure 2 It was detected that after adding antigen peptide stimulation, the number of Tetramer-positive cells increased, and antigen peptide V could induce the generation of specific T lymphocytes (CTL cells) from the peripheral blood of AML patients.

[0150] Example 5: Neoantigen Peptide Activates Specific T Lymphocytes in the Peripheral Blood of AML Patients

[0151] Collect venous blood from AML patients with the same HLA typing and the same FLT3-D835 mutation. Isolate and purify PBMCs by Ficoll density gradient centrifugation, and plate them in 96-well plates. Add antigen peptide at a concentration of 10 μg / mL and culture for 10 - 20 days. After the culture is completed, take out the cells, resuspend them in RPMI-1640 medium containing 10% FBS to a density of 1×10 6 / mL, and add 100 μL / well to an ELISPOT assay plate pre-coated with Human IFN-γ antibody. Add antigen peptide V (final concentration 10 μg / mL) to the corresponding wells, do not add antigen peptide to the negative control wells, and add PHA (final concentration 4 μg / mL) to the positive control wells. Incubate at 37°C for 18 - 24 h. Take out the spot plate, wash the plate, incubate with antibodies, develop color according to the instructions, and read the plate after drying.

[0152] Results

[0153] The experimental results are as Figure 3 shown. It was detected that after adding the antigen peptide, the ability of PBMCs from AML patients to secrete IFN-γ increased. The antigen peptide has good immunogenicity and can activate specific immune responses.

[0154] Example 6: Neoantigens Induce Specific T Lymphocytes in Healthy Volunteers

[0155] Collect venous blood from healthy volunteers with the same HLA typing. Isolate and purify PBMCs by Ficoll density gradient centrifugation. Use Dynabeads magnetic beads to separately sort out CD8 + cells and CD14 + cells. Resuspend CD14 + cells in RPMI-1640 medium containing 10% FBS (containing IL-4 1000 U / mL, GM-CSF 1000 U / mL), place them in an incubator and culture for 5 - 7 days to induce dendritic cells (DCs), and add TNF-α (10 ng / mL) to promote maturation.

[0156] Recover mature DCs, resuspend the cells in serum-free RPMI-1640 medium, add antigen peptide V (20 μg / mL), and incubate at 37°C for 2 - 4 h. Collect DCs pulsed with the antigen peptide, resuspend them in RPMI-1640 medium containing 10% FBS (containing IL-2 50 U / mL, IL-7 5 ng / mL, IL-15 5 ng / mL), and co-incubate with CD8 + cells. Replace half of the medium every 2 - 3 days and culture for 10 - 20 days. Collect the cells and use antigen peptide-HLA-A labeled with PE *02:01-Tetramer antibody staining and detection by flow cytometry.

[0157] Results

[0158] The experimental results are as Figure 4 shown. It was found through detection that the antigen peptide could induce the generation of CTLs from the peripheral blood of healthy volunteers.

[0159] Example 7 Activation of specific T lymphocytes in the peripheral blood of healthy volunteers by neoantigen peptides

[0160] Collect venous blood from healthy volunteers with the same HLA typing, and induce the generation of antigen peptide-specific CTLs as in Example 6. After the culture, take out the cells, resuspend the cells in RPMI-1640 medium containing 10% FBS to a density of 1×10 6 / mL, and add 100 μL / well to an ELISPOT detection plate pre-coated with Human IFN-γ antibody. Prepare DCs loaded with neoantigen peptides as antigen-presenting cells as in Example 6, and add them to the corresponding ELIPSOT well plates respectively. Add DCs not loaded with neoantigen peptides to the negative control wells, and add PHA (final concentration 4 μg / mL) to the positive control wells, and incubate at 37°C for 18 - 24 h. Take out the spot plate, wash the plate, incubate the antibody, develop color according to the instructions, and read the plate after drying.

[0161] Results

[0162] The experimental results are as Figure 5 shown. It was found through detection that the ability of CTLs in healthy volunteers to secrete IFN-γ increased, and the neoantigen peptides had good immunogenicity and could activate specific immune responses.

[0163] All documents mentioned in the present invention are cited herein as references, as if each document was cited individually as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of an antigen peptide, pMHC complex or nucleic acid molecule, characterized in that, Use for preparing a drug for preventing or treating malignant tumors; The antigenic peptide is a polypeptide shown in SEQ ID NO: 6: X1IMSDSNYV wherein X1 is V or I; The pMHC complex contains the antigenic peptide, and the type of MHC molecule is HLA-A*02; The nucleic acid molecule contains the nucleic acid sequence of the antigenic peptide or its complementary sequence; wherein the malignant tumor is acute myeloid leukemia.

2. The use according to claim 1, characterized in that, The malignant tumor is acute myeloid leukemia with FLT-D835 mutation.

3. The use according to claim 1, characterized in that, The type of MHC molecule is HLA-A*02:

01.

4. The use according to claim 1, wherein, The antigenic peptide is a polypeptide with the amino acid sequence shown in SEQ ID NO:

1.

5. The use according to claim 1, wherein, The drug is a pharmaceutical composition, and the composition contains (i) a pharmaceutically acceptable carrier and (ii) an antigenic peptide, a pMHC complex, a nucleic acid molecule, or specific T lymphocytes activated by the antigenic peptide; The antigenic peptide is a polypeptide shown in SEQ ID NO: 6: X1IMSDSNYV wherein X1 is V; The pMHC complex contains the antigenic peptide, and the type of MHC molecule is HLA-A*02; The nucleic acid molecule contains the nucleic acid sequence of the antigenic peptide or its complementary sequence; The pharmaceutical composition is a vaccine composition.

6. Use of an antigen peptide combination, pMHC complex or nucleic acid molecule, characterized in that, Use for preparing a drug for preventing or treating malignant tumors; The antigenic peptide combination is a combination composed of 1, or 2, or 3 polypeptides selected from SEQ ID NO: 1 and SEQ ID NO: 2-4; The pMHC complex contains the antigenic peptide combination, and the type of MHC molecule is HLA-A*02; The nucleic acid molecule contains the nucleic acid sequence of the antigenic peptide combination or its complementary sequence; wherein the malignant tumor is acute myeloid leukemia.

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