WO2024031811A1 - 靶向flt3-d835突变的抗原肽及其在肿瘤免疫治疗中的应用 - Google Patents

靶向flt3-d835突变的抗原肽及其在肿瘤免疫治疗中的应用 Download PDF

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WO2024031811A1
WO2024031811A1 PCT/CN2022/123390 CN2022123390W WO2024031811A1 WO 2024031811 A1 WO2024031811 A1 WO 2024031811A1 CN 2022123390 W CN2022123390 W CN 2022123390W WO 2024031811 A1 WO2024031811 A1 WO 2024031811A1
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cells
antigen peptide
peptide
antigen
nucleic acid
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French (fr)
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周炜均
王侃侃
喻瑾怡
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上海交通大学医学院附属瑞金医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001111—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes

Definitions

  • the invention belongs to the field of polypeptide drugs and polypeptide vaccines, and specifically relates to antigen peptides targeting FLT3-D835 mutation and their application in tumor immunotherapy.
  • AML Acute myeloid leukemia
  • AML is a malignant clonal proliferation disease caused by the continuous accumulation of acquired genetic abnormalities in hematopoietic stem/progenitor cells, which blocks myeloid differentiation at different stages.
  • AML is the most common hematologic malignancy in adults, accounting for more than 70% of all leukemias.
  • clinical treatment of AML is mainly based on chemotherapy combined with hematopoietic stem cell transplantation. The overall therapeutic effect is poor.
  • the cure rate for patients under 60 years old is only 35% to 40%, and the cure rate for patients over 60 years old is as low as 5% to 15%. And nearly 50% of patients lose treatment opportunities due to disease recurrence or drug resistance.
  • 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 amino acids 542 and 564, and the kinase domain is located between amino acids 610 and 944, including a kinase insert of approximately 50 amino acids.
  • FLT3 mutations are one of the most common genetic abnormalities in AML, occurring in approximately 30% of patients. FLT3 mutations lead to enhanced FLT3 kinase activity, thereby promoting the proliferation and growth of leukemia cells, and are closely related to the poor prognosis of AML patients.
  • FLT3-TKD FLT3 tyrosine kinase domain
  • D835 aspartic acid
  • Aspartate is a key regulatory residue in tyrosine kinase receptors and is highly conserved in structure.
  • the wild-type D835 residue (D835wt) is critical for maintaining the inactive conformation of FLT3, and D835 point mutations lead to poor patient prognosis. Finding treatments for FLT3-D835 mutations and targeting the elimination of leukemia cells has important clinical significance for stabilizing the remission status of AML patients and prolonging disease-free survival.
  • tumor vaccines are based on the tumor-immune cycle theory. By promoting the recognition of tumor antigens by immune cells, they stimulate or enhance the body's anti-tumor immune response and cooperate with the immune system to eliminate tumor cells.
  • targets of traditional tumor vaccines are mostly tumor-related antigens, which are expressed in both tumors and normal tissues. As a result, the vaccine has low immunogenicity and is easy to induce immune tolerance. It has been repeatedly frustrated in clinical trials and has been rarely promoted in the clinic. Not much.
  • Neoantigens are protein sequences containing mutated amino acids derived from tumor cells on the basis of genetic mutations. After neoantigens are presented by antigen-presenting cells, they can be effectively recognized by T cells and activate T cells, thereby activating specific immune responses to attack and eliminate tumor cells. Neoantigen peptides derived from genetic mutations are artificially synthesized to construct therapeutic tumor vaccines, which are then infused back into the patient's body to activate immune cells, which can target and kill tumor cells expressing the same neoantigen. In addition, because neoantigens are only expressed in tumor cells and not in normal cells or tissues, they are highly immunogenic and do not induce immune tolerance, making them an advantageous target for tumor vaccines.
  • the current neoantigen vaccine strategy requires sequencing the genome and HLA typing of individual subjects and calculating possible applicable individual neoantigen peptides. This process takes at least 2-3 months or even longer, which is limiting to a certain extent. Promotion of treatment strategies. Moreover, the accuracy of the calculated neoantigens is not high, and the immunogenicity of polypeptides derived from genetic variations still needs further evaluation.
  • the purpose of the present invention is to provide an antigen peptide targeting the FLT3-D835 mutation.
  • Another object of the present invention is to provide the application of antigen peptides targeting FLT3-D835 mutation in tumor immunotherapy.
  • an antigenic peptide for inducing an immune response targeting the FLT3-D835 mutation is provided, the antigenic peptide is capable of forming a complex with an MHC molecule, and the antigenic peptide is selected from the group consisting of:
  • X 1 is V, H, I or F;
  • the antigen peptide has the structure shown in Formula I:
  • X 0 is None or R
  • X 1 is V, H, I or F
  • Z 1 is IMSDSNYV
  • X 10 is None or V.
  • X 0 is none or R
  • X 10 is none or V.
  • the antigen peptide has the structure of formula II,
  • X 1 is V, H, I or F
  • Z 1 is IMSDSNYV
  • X 1 is V or H.
  • the antigen peptide is a combination of two or more antigen peptides.
  • the antigen peptide is one of the polypeptides with the amino acid sequence shown in any one of SEQ ID NO: 1-4, or a combination of 2, 3 or 4 polypeptides.
  • the combination of antigen peptides also contains additional antigen peptides targeting other tumor antigens or sites.
  • the additional antigenic peptide includes the polypeptide shown in SEQ ID No: 5.
  • a pMHC complex comprising the antigen peptide according to the first aspect of the present invention.
  • the antigenic peptide in the pMHC complex has a polypeptide with the amino acid sequence shown in SEQ ID NO: 6.
  • the type of MHC molecule is HLA-A*02.
  • the type of MHC molecule is HLA-A*02:01.
  • nucleic acid molecule comprises a nucleic acid sequence encoding the antigenic peptide of the first aspect of the present invention or its complementary sequence.
  • a vector is provided, the vector containing the nucleic acid molecule according to the third aspect of the present invention.
  • a host cell is provided, the cell containing the vector according to the fourth aspect of the present invention.
  • a method for preparing specific T lymphocytes in vitro including the steps:
  • the concentration of the antigen peptide is 20 ⁇ g/mL.
  • the number of days of culture between the PBMC and the antigen peptide is 10 days.
  • the method is non-diagnostic and non-therapeutic.
  • the PBMC are autologous cells or allogeneic cells.
  • step (b) further includes:
  • composition which composition contains (ii) a pharmaceutically acceptable carrier and (ii) the antigen peptide described in the first aspect of the present invention, the second aspect of the present invention
  • the pharmaceutical composition is a vaccine composition.
  • the dosage form of the pharmaceutical composition is selected from the group consisting of liquid, solid, or gel.
  • the pharmaceutical composition is administered by a method selected from the group consisting of: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, or oral administration.
  • a method for preventing or treating malignant tumor-related diseases includes administering an appropriate amount of the antigen peptide described in the first aspect of the present invention and the pMHC described in the second aspect of the present invention to a subject in need.
  • the antigenic peptide according to the first aspect of the present invention is used to prepare drugs for preventing or treating malignant tumors.
  • the malignant tumor is a hematological malignant tumor.
  • the malignant tumor is acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • the malignant tumor is a malignant tumor with FLT-D835 mutation.
  • Figure 1 shows the stability of the complex formed between the antigenic peptide and the HLA-A*02:01 molecule.
  • Figure 2 shows Tetramer flow cytometry to detect the generation of specific T cells induced by antigenic peptides.
  • Figure 3 shows the ELISPOT experiment to detect the level of IFN- ⁇ secreted by specific T cells activated by antigen peptides.
  • Figure 4 shows that antigenic peptides can induce the generation of CTLs from the peripheral blood of healthy volunteers.
  • Figure 5 shows that the ability of CTLs to secrete IFN- ⁇ in healthy volunteers is increased.
  • the inventor unexpectedly obtained a safe, efficient and economical antigen peptide targeting the FLT3-D835 mutation.
  • the antigen peptide of the present invention (SEQ ID No.1-4) has no affinity with HLA-A*02:01 molecules.
  • 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- ⁇ is increased. On this basis, the present invention was completed.
  • antigenic peptide of the present invention and the "polypeptide of the present invention” or “short peptide of the present invention” can be used interchangeably, and both refer to the antigenic peptide targeting the FLT3-D835 mutation of the present invention.
  • FMS-like tyrosine kinase 3 FLT3-D835
  • 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 amino acids 542 and 564, and the kinase domain is located between amino acids 610 and 944, including a kinase insert of approximately 50 amino acids.
  • FLT3 mutations are one of the most common genetic abnormalities in AML, occurring in approximately 30% of patients. FLT3 mutations lead to enhanced FLT3 kinase activity, thereby promoting the proliferation and growth of leukemia cells, and are closely related to the poor prognosis of AML patients.
  • FLT3-TKD FLT3 tyrosine kinase domain
  • D835 point mutations that have been identified in AML patients include: alanine (A), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine Acid (I), aspartic acid (N), valine (V), tyrosine (Y), among which D835F, D835H, D835I, D835V and D835Y are the most common. D835 point mutation leads to poor patient prognosis.
  • Neoantigen peptide (neoantigen)
  • Neoantigens are protein sequences containing mutated amino acids derived from tumor cells on the basis of genetic mutations. After neoantigens are presented by antigen-presenting cells, they can be effectively recognized by T cells and activate T cells, thereby activating specific immune responses to attack and eliminate tumor cells. Neoantigen peptides derived from genetic mutations are artificially synthesized to construct therapeutic tumor vaccines, which are then infused back into the patient's body to activate immune cells, which can target and kill tumor cells expressing the same neoantigen. Moreover, because neoantigens are only expressed in tumor cells and not in normal cells or tissues, they are highly immunogenic and do not induce immune tolerance, making them an advantageous target for tumor vaccines.
  • an antigen peptide for inducing an immune response targeting the FLT3-D835 mutation is provided, the antigen peptide is capable of forming a complex with an MHC molecule, and the antigen peptide is selected from Next group:
  • X 1 is V, H, I or F;
  • Amino acid substitution means that an amino acid residue is replaced by another amino acid residue at the same position.
  • the inserted amino acid residues may be inserted at any position, all or part of the inserted amino acid residues may be adjacent to each other, or none of the inserted amino acid residues may be adjacent to each other.
  • the peptides of the present invention may be post-translationally modified at one or more positions between the amino acid sequences. Examples of post-translational modifications can be found in Engelhard et al. Curr Opin Immunol. 2006 Feb;18(1):92-7, and include phosphorylation, acetylation, and deamidation.
  • the peptide of the present invention binds to MHC at the peptide binding site of the MHC molecule.
  • the modified amino acids described above do not disrupt the ability of the peptide to bind MHC.
  • the amino acid modification increases the ability of the peptide to bind to MHC.
  • mutations may occur at the site where the peptide binds to the MHC.
  • the length of the amino acids of the peptide of the present invention may be 8-15, preferably 8-10, preferably 9.
  • polypeptide of the present invention can be composed of any polypeptide in SEQ ID NO. 1-4 in Table 1.
  • the present invention also provides analogs of the proteins or peptides shown in SEQ ID NO: 1-4.
  • the difference between these analogs and natural peptides may be differences in amino acid sequences, differences in modified forms that do not affect the sequences, 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, site-directed mutagenesis or other techniques known in molecular biology.
  • Analogues also include analogs with residues that differ from natural L-amino acids (eg, D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (eg, beta, gamma-amino acids). It should be understood that the peptides of the present invention are not limited to the representative peptides exemplified above.
  • Modified forms include chemically derivatized forms of the peptide such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications of the peptide during its synthesis and processing or during further processing steps. This modification can be accomplished by exposing the peptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are peptides that have been modified to increase their resistance to proteolysis or to optimize solubility properties.
  • glycosylation such as those resulting from glycosylation modifications of the peptide during its synthesis and processing or during further processing steps. This modification can be accomplished by exposing the peptide to an enzyme that performs glycosylation, such as a mammalian glycosylase
  • the protein conservative variant peptide shown in SEQ ID NO: 1-4 means that compared with the amino acid sequence of SEQ ID NO: 1-4, at most 3, and more preferably at most 2 amino acids are substituted. Amino acids with similar or similar properties are replaced to form peptides. These conservative variant peptides are preferably produced by amino acid substitutions according to Table 1.
  • substitutions Ala(A) Val;Leu;Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg gnc Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro;Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile
  • Lys(K) Arg Gln; Asn Arg Met(M) Leu;Phe;Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp;Phe;Thr;Ser Phe Val(V) Ile;Leu;Met;Phe;Ala Leu
  • the peptides of the present invention can be simply synthesized using the Merrifield synthesis method (also known as polypeptide solid-phase synthesis). GMP-grade peptides can be synthesized using solid-phase synthesis technology from Multiple Peptide Systems (San Diego, CA). Alternatively, the peptide can be synthesized recombinantly, if desired, using methods known in the art. Typical such methods involve the use of a vector comprising a nucleic acid sequence encoding a polypeptide for expression of the polypeptide in vivo; for example, expression in bacterial, yeast, insect or mammalian cells. Alternatively, in vitro cell-free systems can also be used for expression. Such systems are known in the art and are commercially available.
  • the peptide may be isolated and/or provided in substantially pure form. For example, they may be provided in a form that is substantially free of other peptides or proteins.
  • a second aspect of the invention provides a pMHC complex comprising the peptide of the first aspect of the invention.
  • the polypeptide binds to the peptide binding groove of the MHC molecule.
  • the MHC molecule may be an MHC class I molecule or an MHC class II molecule.
  • the MHC molecule is an MHC class I molecule.
  • the MHC molecule is HLA-A*02, more preferably, the MHC molecule is HLA-A*0201.
  • the pMHC complex of the present invention may exist in the form of a multimer, for example, a dimer, a tetramer, a pentamer, a hexamer, an octamer, or larger.
  • Appropriate methods for generating pMHC multimers can be found in relevant literature, such as (Greten et al., Clin. Diagnostic Lab. Immunol. 2002: 216-220).
  • pMHC complexes bearing biotin residues can be complexed with fluorescently labeled streptavidin to produce pMHC multimers.
  • the pMHC multimers can also be formed using immunoglobulins as molecular scaffolds. In this system, the extracellular region of the MHC molecule is joined to the constant region of the immunoglobulin heavy chain through a short linker sequence.
  • carrier molecules such as dextran (WO02072631)
  • pMHC multimers help improve detection of the moieties to which they bind, such as T-cell receptors. Alternatively, improve the effect of pMHC complexes in related applications, such as activating T cells.
  • the pMHC complexes of the present invention can be provided in soluble form.
  • the MHC molecule in the pMHC complex does not contain a transmembrane region.
  • the MHC class I molecule can be composed of its light chain and all or part of the extracellular domain of its heavy chain.
  • an MHC molecule is a fragment containing only its functional domains.
  • MHC molecules in the soluble pMHC complexes of the invention can also be produced synthetically and then refolded with the peptides of the invention. By determining whether the peptide and MHC molecules can refold, it can be determined which type of MHC molecules the peptide of the present invention can form a complex with.
  • the soluble pMHC complex of the present invention can be used to screen or detect molecules that bind to it, such as TCR or antibodies.
  • the method includes contacting the pMHC complex with a binding moiety to be tested, and determining whether the binding moiety to be tested binds to the complex.
  • Methods for determining binding of pMHC complexes 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, and microscopy.
  • the binding can be detected by functional assays of the biological response resulting from the binding, such as cytokine release or apoptosis.
  • the soluble pMHC complex of the present invention can also be used to screen TCR or antibody libraries.
  • the use of phage display technology to construct antibody libraries is well known in the art, as described in the reference Aitken, Antibody phage display: Methods and Protocols (2009, Humana, New York).
  • the pMHC complex of the present invention is used to screen a diverse TCR library displayed on the surface of phage particles.
  • the TCRs displayed by the library may contain unnatural mutations.
  • the soluble pMHC complex of the present invention can be immobilized on an appropriate solid phase carrier through a linker.
  • solid supports include, but are not limited to, beads, membranes, agarose gels, magnetic beads, substrates, tubes, and columns.
  • pMHC complexes can be immobilized on ELISA reaction plates, magnetic beads, or surface plasmon resonance biosensor chips.
  • Methods of immobilizing pMHC complexes to solid supports are known to those skilled in the art and include, for example, the use of affinity binding pairs, such as biotin and streptavidin, or antibodies and antigens.
  • the pMHC complex is labeled with biotin and immobilized on a streptavidin-coated surface.
  • the peptides of the invention can be presented to the cell surface together with MHC complexes. Therefore, the invention also provides a cell capable of presenting the pMHC complex of the invention to its surface.
  • Such cells may be mammalian cells, preferably immune system cells, and 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).
  • Cells presenting a peptide or pMHC complex according to the invention may be isolated, preferably in the form of a population of cells, or provided in substantially pure form.
  • the cells may not naturally present the complexes of the invention, or the cells may present the complexes at a higher level than in the natural state.
  • Such cells can be obtained by pulse treatment with the peptides of the invention. Pulse treatment involves incubating cells with the peptide for several hours, preferably at a concentration of 10-5-10-12M. Additionally, the cells can be transduced with HLA-A*02 molecules to further induce peptide presentation.
  • Cells presenting the pMHC complex of the present invention can be used to isolate T cells and T cell receptors. The T cells are activated by the cells and further sorted out, and then the expression in the T cells can also be obtained. Surface T cell receptors.
  • the method of obtaining the above-mentioned T cells includes stimulating fresh blood obtained from healthy volunteers using the above-mentioned cells presenting the pMHC complex of the present invention. It can go through several rounds of stimulation, such as 3-4 rounds.
  • Activated T cells can be identified by measuring cytokine release in the presence of T2 cells pulsed with the peptides of the invention (eg, IFN- ⁇ ELISpot assay). Using labeled antibodies, activated cells can be sorted by flow cytometry (FACS), and the sorted cells can be scaled up and further validated, e.g., by ELISpot assay and/or cytotoxicity against target cells and/or pMHC multimerization. Body staining for verification. TCR strands from validated T cell clones can be amplified by rapid amplification of cDNA ends (RACE) and sequenced.
  • RACE rapid amplification of cDNA ends
  • the invention also provides a nucleic acid molecule comprising a nucleic acid sequence encoding a peptide of the invention.
  • the nucleic acid may be cDNA.
  • the nucleic acid molecule may consist essentially of a nucleic acid sequence encoding a peptide according to the invention, or may encode only a peptide according to the invention.
  • Such nucleic acid molecules can be synthesized using methods known in the art. Due to the degeneracy of the genetic code, those skilled in the art will understand that nucleic acid molecules with different nucleic acid sequences can encode the same amino acid sequence.
  • the present invention also provides a vector, which includes the nucleic acid sequence of the present invention.
  • Suitable vectors are known in the art of vector construction, including the selection of promoters and other regulatory elements, such as enhancer elements.
  • the vectors of the present invention include sequences suitable for introduction into cells.
  • 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.
  • vector includes DNA molecules, such as plasmids, phages, viruses or other vectors, which contain one or more heterologous or recombinant nucleic acid sequences.
  • Suitable phages and viral vectors include, but are not limited to: lambda-phage, EMBL phage, simian virus, bovine wart virus, Epstein-Barr virus, adenovirus, herpes virus, mouse sarcoma virus, murine breast cancer virus, lentivirus, etc. .
  • the present invention also provides a binding molecule that can be used as an immunotherapeutic agent or diagnostic reagent.
  • the binding molecule may bind to the peptide alone or to a complex of the peptide and the MHC molecule. In the latter case, the binding molecule may be partially bound to the MHC molecule while it is also bound to the peptide of the invention.
  • the binding moiety of the invention may be isolated and/or soluble, and/or non-naturally occurring, ie having no equivalent in nature, and/or pure, and/or synthetic.
  • the binding molecule is a T cell receptor (TCR).
  • TCR T cell receptor
  • IMGT International Immunogenetics Information System
  • Natural ⁇ heterodimeric TCRs have ⁇ and ⁇ chains. Broadly speaking, each chain contains a variable region, a connecting region and a constant region. The ⁇ chain usually also contains a short variable region between the variable region and the connecting region, but this variable region is often regarded as part of the connecting region.
  • the TCR of the present invention may be in any form known in the art.
  • the TCR may be a heterodimer or exist as a single chain.
  • the TCR may be in a soluble form (ie, without a transmembrane or cytoplasmic region), and in particular, the TCR may comprise all or part of the TCR extracellular domain.
  • the TCR may also be a full-length chain including its transmembrane region.
  • the TCR can be provided to the surface of cells, such as T cells.
  • Soluble TCRs can be obtained by combining existing technologies in the art, for example, introducing an artificial disulfide bond between the constant domains of the ⁇ and ⁇ chains of ⁇ TCR, or between the ⁇ chain variable region and the ⁇ chain constant region of ⁇ TCR. Artificial disulfide bonds are introduced between them.
  • the TCR of the present invention can be used to deliver cytotoxic agents or immunostimulatory agents to target cells, or be transformed into T cells so that T cells expressing the TCR can destroy tumor cells in a treatment process known as adoptive immunotherapy given to the patient.
  • the TCR of the present invention may also contain mutations.
  • the affinity of the mutated TCR for the pMHC complex of the present invention is improved.
  • the TCR of the present invention can be used alone, or can be combined with a conjugate in a covalent or other manner, preferably in a covalent manner.
  • 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 invention), a therapeutic agent, a PK (protein kinase) modifying moiety, or any of the above. Combinatorial binding or coupling.
  • the TCR of the present invention can also be combined with an anti-CD3 antibody, preferably in a covalent manner, to redirect T cells to kill target cells.
  • the binding molecule of the invention is an antibody.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules containing specific binding sites, which may be wholly natural, partially synthetic, or wholly synthetic.
  • antibody includes antibody fragments including an immunoglobulin binding region that is an antibody binding region or that binds to an antibody, its derivatives, functional equivalents, and homologous antibodies, humanized antibodies Homogenous area. It can be completely natural, partially synthetic, or completely synthetic.
  • a humanized antibody can be a modified antibody that contains the variable regions of a non-human antibody (e.g., mouse) and the constant regions of a human antibody.
  • antibodies may be immunoglobulins of the same type (e.g., IgG, IgE, IgM, IgD, and IgA) and subclasses thereof; fragments including antigen-binding regions, such as Fab, scFv, Fv, dAb, Fd; and diabodies.
  • Antibodies can be polyclonal antibodies or monoclonal antibodies, preferably monoclonal antibodies.
  • TCR and antibodies Methods for preparing the above-mentioned TCR and antibodies are known to those skilled in the art, including but not limited to expression and purification from E. coli cells or insect cells.
  • the invention further provides uses of the peptides, pMHC complexes, nucleic acid molecules, vectors, cells and binding molecules of the invention in pharmaceuticals.
  • the peptides, pMHC complexes, nucleic acids, vectors, cells or binding molecules can be used to treat or prevent malignant tumors, preferably acute myeloid leukemia.
  • the present invention also provides a pharmaceutical composition, which contains the antigen peptide of the present invention, the pMHC complex, the nucleic acid molecule of the present invention, the cell of the present invention or the binding molecule of the present invention, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be in any suitable form, depending on the method of administration desired by the patient. They may be provided in unit dosage form, usually in sealed containers, and may be provided as part of a kit. Such kits usually, but do not necessarily, include instructions for use. It may contain a plurality of said unit dosage forms.
  • compositions are suitable for any appropriate route of administration, such as injection (including subcutaneous, intramuscular, intraperitoneal or intravenous injection), inhalation or oral administration, or nasal or anal administration.
  • routes of administration such as injection (including subcutaneous, intramuscular, intraperitoneal or intravenous injection), inhalation or oral administration, or nasal or anal administration.
  • the compositions may be prepared by any method known in the pharmaceutical art, for example by mixing the active ingredient with a carrier or excipient under sterile conditions.
  • the dosage of the formulation of the present invention may vary within a wide range depending on the disease or condition being treated (eg, cancer, viral infection, or autoimmune disease), the individual age and condition of the patient, and the like. The final decision on appropriate dosage will be made by the physician.
  • the disease or condition being treated eg, cancer, viral infection, or autoimmune disease
  • the final decision on appropriate dosage will be made by the physician.
  • 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 function.
  • the peptides, pMHC complexes or pMHC complex-presenting cells of the invention may be provided in the form of a vaccine composition.
  • the vaccine composition can be used to treat or prevent cancer. All such compositions are included in the present invention. It is understood that the vaccine can be in a variety of forms (Schlom J.J Natl Cancer Inst. 2012 104(8):599-613).
  • the peptides of the invention can be used directly in immunized patients (Salgaller ML. Cancer Res. 1996.56 (20): 4749-57 and Marchand M. Int J Cancer. 1999.80 (2): 219-230).
  • the vaccine composition may comprise additional peptides such that the peptide of the invention is one of a mixture of peptides.
  • Adjuvants may be added to the vaccine composition to enhance the immune response.
  • the vaccine composition may be in the form of an antigen-presenting cell presenting a peptide of the invention and an MHC complex.
  • the antigen-presenting cells are immune cells, more preferably dendritic cells.
  • the peptides can also be pulsed to the surface of cells (Thurner BI. et al., J. Exp. Med. 1999.
  • nucleic acids encoding peptides of the invention can be introduced into dendritic cells, e.g., using Electroporation method (Van Tendeloo, VF.etal., Blood 2001.98:49).
  • the present invention aims to overcome the shortcomings of related technologies to a certain extent and provide a new antigen peptide and its application in tumor immunotherapy.
  • the antigen peptide of the present invention has the effect of eliminating tumor cells.
  • the antigen peptide of the present invention can broaden the scope of treatment for patients with FLT3 mutant tumors and provide new options for immunotherapy for patients.
  • %Rank_EL ⁇ 0.500 is considered to have strong binding force
  • 0.500 ⁇ %Rank_EL ⁇ 2.00 is considered to have medium binding force
  • %Rank_EL> 2.000 is regarded as no binding force.
  • the antigenic peptide (SEQ ID No.1 to SEQ ID No.5) has medium or high affinity with the HLA-A*02:01 molecule, which is significantly higher than the affinity between the wild-type polypeptide (SEQ ID No.7) and HLA-A *02:01 Affinity of molecules (difference of 10 times or more).
  • the wild-type peptide (SEQ ID No. 7) has no or very low affinity to HLA-A*02:01 molecules (Table 3).
  • T2 cells Take T2 cells in the logarithmic growth phase, adjust the concentration of T2 cells to 1 ⁇ 106/mL in serum-free and antibiotic-free IMDM medium (containing 100ng/mL human ⁇ 2m), and incubate them with 100 ⁇ g/mL neoantigen peptide overnight at 37°C. .
  • Collect the cells the next day add serum-free IMDM medium containing 10 ⁇ g/mL Brefeldin A, and incubate for 1 hour; add serum-free IMDM medium containing 0.5 ⁇ g/mL Brefeldin A, and incubate at 37°C at 0, 2, 4, and Collect cells at time points of 6 and 8 hours, resuspend cells in 100 ⁇ L PBS, add FITC-labeled anti-HLA-A2 monoclonal antibody, incubate at room temperature for 30 minutes, detect by flow cytometry, and calculate the average fluorescence of T2 cells at each time point. strength.
  • PBMCs peripheral blood mononuclear cells
  • CD8- cells stimulated by antigen pulses were collected, resuspended in RPMI-1640 medium containing 10% FBS (containing IL-2 50U/mL, IL-7 5ng/mL, IL-15 5ng/mL), and coagulated with CD8+ cells. Incubate, change half of the medium every 2-3 days, and culture for 10-20 days. The cells were collected, stained with PE-labeled antigen peptide-HLA-A*02:01-Tetramer antibody, and detected by flow cytometry.
  • Neoantigen peptide activates peripheral blood-specific T lymphocytes of AML patients
  • PBMCs peripheral blood cells
  • PBMCs Venous blood from healthy volunteers with the same HLA typing was taken, and PBMCs were isolated and purified by Ficoll density gradient centrifugation. Dynabeads magnetic beads separate CD8+ cells and CD14+ cells respectively. Resuspend CD14+ cells in RPMI-1640 medium containing 10% FBS (containing IL-4 1000U/mL, GM-CSF 1000U/mL), place it in an incubator to culture for 5-7 days to induce dendritic cells (DCs), and add TNF - ⁇ (10ng/mL) promotes maturation.
  • FBS containing IL-4 1000U/mL, GM-CSF 1000U/mL
  • DCs stimulated by antigen peptide pulses were collected, resuspended in RPMI-1640 medium containing 10% FBS (containing IL-2 50U/mL, IL-7 5ng/mL, IL-15 5ng/mL), and co-incubated with CD8+ cells , change half of the medium every 2-3 days, and culture for 10-20 days.
  • the cells were collected, stained with PE-labeled antigen peptide-HLA-A*02:01-Tetramer antibody, and detected by flow cytometry.
  • Neoantigen peptide activates specific T lymphocytes in peripheral blood of healthy volunteers
  • the venous blood of healthy volunteers with the same HLA typing was taken to induce the generation of antigen peptide-specific CTLs as in Example 6.
  • remove the cells resuspend them in RPMI-1640 medium containing 10% FBS to a density of 1 ⁇ 106/mL, and add 100 ⁇ L/well of an ELISPOT detection plate pre-coated with Human IFN- ⁇ antibody.
  • DCs loaded with neoantigen peptides were prepared as antigen-presenting cells as in Example 6, and added to corresponding ELIPSOT well plates.
  • DCs not loaded with neoantigen peptides were added to the negative control wells, and PHA (final concentration 4 ⁇ g/mL) was added to the positive control wells, and incubated at 37°C for 18-24 hours. Take out the spot plate, wash the plate, incubate the antibody, develop color according to the instructions, dry and read the plate.

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Abstract

提供一种靶向FLT3-D835 突变的抗原肽，与野生型多肽与HLA-A*02:01分子无亲和力相比，新抗原肽具有高亲和力。该抗原肽能够激活特异性免疫反应，对靶向清除白血病细胞以及稳定AML患者缓解状态、延长无病生存时间。

Description

靶向FLT3-D835突变的抗原肽及其在肿瘤免疫治疗中的应用 技术领域
本发明属于多肽药物和多肽疫苗领域，具体涉及靶向FLT3-D835突变的抗原肽及其在肿瘤免疫治疗中的应用。
背景技术
急性髓细胞白血病(AML)是由于造血干/祖细胞不断累积获得性遗传学异常，使髓系分化阻滞在不同阶段而产生的恶性克隆增殖性疾病。AML是成人最常见的血液系统恶性肿瘤，占所有白血病的70％以上。目前，临床上AML的治疗以化学药物治疗联合造血干细胞移植为主，治疗效果整体较差，60岁以下患者治愈率仅35％～40％，60岁以上患者更低至5％～15％，并且近50％的患者因疾病复发或耐药丧失治疗机会。基因变异的不断累积，包括体细胞突变、基因插入/缺失、基因融合等，是AML难治/复发的关键原因。因此，以变异基因为切入点，靶向清除异常白血病细胞，对降低AML复发率，提高治愈率具有重要的科学意义。
FMS样酪氨酸激酶3(FLT3)基因位于染色体13q12，编码一个含993个氨基酸的蛋白。其中，跨膜区位于第542位至564位氨基酸之间，激酶结构域位于第610位至第944位氨基酸之间，包括约50个氨基酸的激酶插入片段。FLT3变异是AML最常见的基因异常之一，发生于大约30％的患者。FLT3变异导致FLT3激酶活性增强，从而促进白血病细胞增殖与生长，与AML患者预后差密切相关。
研究发现，约5～10％的FLT3变异涉及FLT3酪氨酸激酶结构域(FLT3-TKD)的单核苷酸突变，其中最常见的是第835位氨基酸残基——天冬氨酸(D835)的突变，通常位于激活环内。天冬氨酸是酪氨酸激酶受体的关键调节残基，在结构上高度保守。野生型D835残基(D835wt)对维持FLT3的非活性构象至关重要，D835点突变导致患者预后不良。寻找针对FLT3-D835突变的治疗方式，靶向清除白血病细胞，对稳定AML患者缓解状态、延长无病生存时间，具有重要的临床意义。
近年来，随着免疫学、基因组学和分子生物学技术的发展，免疫治疗已成为肿瘤治疗的又一革新模式。其中，治疗性肿瘤疫苗是免疫治疗的研究热点。肿瘤疫苗基于肿瘤-免疫循环理论，通过促进免疫细胞对肿瘤抗原的识别，激发或增强机体抗肿瘤免疫应答，协同免疫系统清除肿瘤细胞。然而，传统的肿瘤疫苗包含的靶标多为肿瘤相关抗原，在肿瘤和正常组织均有表达，导致疫苗免疫原性较低，易诱发免疫耐受， 在临床试验中屡次受挫，在临床的推广寥寥无几。
新抗原(neoantigen)是肿瘤细胞在基因变异的基础上衍生的包含变异氨基酸的蛋白序列。新抗原经抗原呈递细胞呈递后，可被T细胞有效识别，活化T细胞，从而激活特异性免疫反应攻击并清除肿瘤细胞。将基因变异衍生的新抗原肽进行人工合成，构建治疗性肿瘤疫苗，回输至患者体内激活免疫细胞，可靶向杀伤表达相同新抗原的肿瘤细胞。并且，新抗原因其仅在肿瘤细胞表达，不在正常细胞或组织表达，具有高免疫原性而不诱导免疫耐受的特性，是肿瘤疫苗的优势靶标。然而，目前的新抗原疫苗策略需要对个体受试者基因组及HLA分型进行测序并计算可能的适用个体户新抗原肽，该过程至少历时2-3个月甚至更久，在一定程度上限制了治疗策略的推广。并且，根据计算获得的新抗原的准确性不高，基因变异衍生多肽的免疫原性仍需要进一步评估。
因此，本领域迫切需要开发出一种安全、高效、经济的抗原肽，扩宽FLT3突变型肿瘤患者的治疗范围，为患者的免疫治疗提供新选择。
发明内容
本发明的目的就是提供一种靶向FLT3-D835突变的抗原肽。
本发明的另一目的是提供靶向FLT3-D835突变的抗原肽在肿瘤免疫治疗中的应用。
在本发明的第一方面，提供了一种用于引发靶向FLT3-D835突变的免疫应答的抗原肽，所述抗原肽能够与MHC分子形成复合物，并且所述抗原肽选自下组：
(i)SEQ ID NO:6所示的多肽：
X 1IMSDSNYV
其中，X 1为V、H、I或F；
(ii)对(i)多肽的氨基酸序列中除X 1以外的氨基酸进行1个、2个或3个氨基酸取代，和/或1个、2个或3个氨基酸插入，和/或1个或2个氨基酸缺失所形成的衍生多肽，并且所述衍生多肽保留X 1。
在另一优选例中，所述抗原肽具有式I所示结构：
X 0-X 1-Z 1-X 10   (I)
其中，
X 0为无或R；
X 1为V、H、I或F；
Z 1为IMSDSNYV；
X 10为无或V。
在另一优选例中，在式II中，X 0为无或R，X 10为无或V。
在另一优选例中，所述的抗原肽具有式II结构，
X 1-Z 1   (II)
其中，
X 1为V、H、I或F；
Z 1为IMSDSNYV；
在另一优选例中，X 1为V或H。
在另一优选例中，所述的抗原肽为两种或两种以上抗原肽的组合。
在另一优选例中，所述抗原肽为SEQ ID NO:1-4中任一所示的氨基酸序列的多肽中1种，或2种、3种或4种多肽构成的组合。
在另一优选例中，所述的抗原肽的组合还含有针对其他肿瘤抗原或位点的额外的抗原肽。
在另一优选例中，所述的额外的抗原肽包括SEQ ID No:5所示的多肽。
在本发明的第二方面，提供了一种pMHC复合物，所述复合物包含本发明第一方面所述的抗原肽。
在另一优选例中，所述pMHC复合物中的抗原肽具有SEQ ID NO:6所示的氨基酸序列的多肽。
在另一优选例中，MHC分子的类型是HLA-A*02。
在另一优选例中，MHC分子的类型是HLA-A*02:01。
在本发明的第三方面，提供了一种核酸分子，所述核酸分子包含编码本发明第一方面所述抗原肽的核酸序列或其互补序列。
在本发明的第四方面，提供了一种载体，所述载体含有本发明第三方面所述的核酸分子。
在本发明的第五方面，提供了一种宿主细胞，所述细胞中含有本发明第四方面所 述的载体。
在本发明的第六方面，提供了一种体外制备特异性T淋巴细胞的方法，包括步骤：
a)提供PBMC，
b)在抗原肽存在下，将所述PBMC与本发明第一方面所述的抗原肽进行接触并培养，从而获得经抗原肽激活的特异性T淋巴细胞。
在另一优选例中，所述抗原肽的浓度为20μg/mL。
在另一优选例中，所述PBMC与抗原肽的培养天数为10天。
在另一优选例中，所述方法是非诊断和非治疗的。
在另一优选例中，所述的PBMC是自体细胞或异体细胞。
在另一优选例中，在步骤(b)中还包括：
(b1)从PBMC中分选出CD8 +细胞和CD8 -细胞，
(b2)将本发明第一方面所述的抗原肽对(b1)中所述的CD8 -细胞进行致敏处理，从而获得经致敏的CD8 -细胞，
(b3)将(b2)中所述的经致敏的CD8 -细胞与CD8 +细胞共孵育，从而获得经抗原肽激活的特异性T淋巴细胞。
在本发明的第七方面，提供了一种药物组合物，所述组合物含有(ii)药学上可接受的载体以及(ii)本发明第一方面所述的抗原肽、本发明第二方面所述的pMHC复合物、本发明第三方面所述的核酸分子、或经本发明第一方面所述的抗原肽激活的特异性T淋巴细胞。
在另一优选例中，所述药物组合物为疫苗组合物。
在另一优选例中，所述的药物组合物的剂型选自液态、固态、或凝胶态。
在另一优选例中，所述的药物组合物用选自下组的方式施用：皮下注射、皮内注射、肌肉注射、静脉注射、腹腔注射、微针注射、或口服。
在本发明的第八方面，提供了一种预防或治疗恶性肿瘤相关疾病的方法，包括给需要的对象施用适量的本发明第一方面所述的抗原肽、本发明第二方面所述的pMHC复合物、本发明第三方面所述的核酸分子、经本发明第一方面所述的抗原肽激活的特异性T淋巴细胞或本发明第七方面所述的药物组合物。
如本发明第一方面所述的抗原肽、本发明第二方面所述的pMHC复合物、本发明第三方面所述的核酸分子、经本发明第一方面所述的抗原肽激活的特异性T淋巴细胞或本发明第七方面所述的药物组合物的用途，用于制备预防或治疗恶性肿瘤的药物。
在另一优选例中，所述的恶性肿瘤为血液系统恶性肿瘤。
在另一优选例中，所述的恶性肿瘤为急性髓细胞白血病(AML)。
在另一优选例中，所述的恶性肿瘤为FLT-D835突变的恶性肿瘤。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了抗原肽与HLA-A*02:01分子形成的复合物的稳定性。
图2显示了Tetramer流式细胞术检测抗原肽诱导生成特异性T细胞。
图3显示了ELISPOT实验检测抗原肽激活特异性T细胞分泌IFN-γ的水平。
图4显示了抗原肽可从健康志愿者外周血诱导生成CTLs。
图5显示了健康志愿者CTLs分泌IFN-γ能力升高。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，意外地获得了一种安全、高效、经济的靶向FLT3-D835突变的抗原肽。实验表明，与野生型多肽(SEQ ID No.7)与HLA-A*02:01分子无亲和力相比，本发明抗原肽(SEQ ID No.1-4)与HLA-A*02:01分子具有中等或高亲和力。同时，本发明抗原肽具有良好的免疫原性，能够激活特异性免疫反应，可从健康志愿者外周血诱导生成CTLs，并且CTLs分泌IFN-γ能力升高。在此基础上完成了本发明。
应理解，在本发明中，本发明的“抗原肽”与“本发明多肽”或“本发明短肽”可互换使用，均指本发明的靶向FLT3-D835突变的抗原肽。
术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
FMS样酪氨酸激酶3(FLT3)和FLT3-D835
FMS样酪氨酸激酶3(FLT3)基因位于染色体13q12，编码一个含993个氨基酸的蛋白。其中，跨膜区位于第542位至564位氨基酸之间，激酶结构域位于第610位至第944位氨基酸之间，包括约50个氨基酸的激酶插入片段。FLT3变异是AML最常见的基因异常之一，发生于大约30％的患者。FLT3变异导致FLT3激酶活性增强，从而促进白血病细胞增殖与生长，与AML患者预后差密切相关。
研究发现，约5～10％的FLT3变异涉及FLT3酪氨酸激酶结构域(FLT3-TKD)的单核苷酸突变，其中最常见的是第835位氨基酸残基——天冬氨酸(D835)的突变，通常位于激活环内。天冬氨酸是酪氨酸激酶受体的关键调节残基，在结构上高度保守。目前已从AML患者中鉴定出D835点突变包括：丙氨酸(A)、谷氨酸(E)、苯丙氨酸(F)、甘氨酸(G)、组氨酸(H)、异亮氨酸(I)、天冬氨酸(N)、缬氨酸(V)、酪氨酸(Y)，其中，尤以D835F、D835H、D835I、D835V和D835Y最常见。D835点突变导致患者预后不良。
新抗原肽(neoantigen)
新抗原(neoantigen)是肿瘤细胞在基因变异的基础上衍生的包含变异氨基酸的蛋白序列。新抗原经抗原呈递细胞呈递后，可被T细胞有效识别，活化T细胞，从而激活特异性免疫反应攻击并清除肿瘤细胞。将基因变异衍生的新抗原肽进行人工合成，构建治疗性肿瘤疫苗，回输至患者体内激活免疫细胞，可靶向杀伤表达相同新抗原的肿瘤细胞。并且，因新抗原仅在肿瘤细胞表达，不在正常细胞或组织表达，具有高免 疫原性而不诱导免疫耐受的特性，是肿瘤疫苗的优势靶标。
具体地，在本发明的第一方面，提供了一种用于引发靶向FLT3-D835突变的免疫应答的抗原肽，所述抗原肽能够与MHC分子形成复合物，并且所述抗原肽选自下组：
(i)SEQ ID NO:6所示的多肽：
X 1IMSDSNYV
其中，X 1为V、H、I或F；
(ii)对(i)多肽的氨基酸序列中除X 1以外的氨基酸进行1个、2个或3个氨基酸取代，和/或1个、2个或3个氨基酸插入，和/或1个或2个氨基酸缺失所形成的衍生多肽，并且所述衍生多肽保留X 1。
氨基酸取代意味着在相同位置，某个氨基酸残基被其他氨基酸残基替代。插入的氨基酸残基可以在任何位置插入，插入的氨基酸残基也可以全部或部分彼此相邻，或插入的氨基酸之间都不彼此相邻。本领域技术人员已知，本发明所述的肽可以在氨基酸序列之间的一个或多个位置进行翻译后修饰。翻译后修饰的例子可以在Engelhard等Curr Opin Immunol.2006年2月；18(1)：92-7中找到，并且包括磷酸化作用、乙酰化作用和脱酰氨基作用。
较佳地，本发明所述的肽与MHC结合于MHC分子的肽结合位点。通常，上述描述的修饰的氨基酸不会破坏所述肽与MHC的结合能力。在一个优选的实施方式中，所述的氨基酸修饰提高了肽与MHC结合的能力。例如，突变可能发生在肽与MHC的结合位点。这些结合位点和结合位点上优选的残基为本领域已知的，尤其是对哪些结合HLA-A*02的肽来说(参见，比如Parkhurst等，J.Immunol.157:2539-2548(1996))。
更具体地，本发明的肽的氨基酸的长度可以是8-15个，优选8-10个，较佳地，9个。
本发明所述多肽可以由表1中SEQ ID NO.1-4中任一多肽组成。
表1本发明抗原肽
Figure PCTCN2022123390-appb-000001
Figure PCTCN2022123390-appb-000002
本发明还提供SEQ ID NO：1-4所示的蛋白或肽的类似物。这些类似物与天然的肽差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些肽包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分子生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的肽并不限于上述例举的代表性的肽。
修饰(通常不改变一级结构)形式包括：体内或体外的肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的肽。这种修饰可以通过将肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的肽。
在本发明中，“SEQ ID NO：1-4所示的蛋白保守性变异肽”指与SEQ ID NO：1-4的氨基酸序列相比，有至多3个，更佳地至多2个氨基酸被性质相似或相近的氨基酸所替换而形成肽。这些保守性变异肽最好根据表1进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明所述的肽可以用Merrifield合成方法(又被称为多肽固相合成法)简单合成。GMP级别的肽可以用多肽系统(Multiple Peptide Systems，San Diego，CA)的固相合成技术予以合成。或者，所述肽可以重组合成，如果需要，可以用本领域已知的方法予以合成。典型的此类方法涉及载体的使用，所述载体包括编码多肽的核酸序列，在体内表达多肽；例如，在细菌、酵母、昆虫或哺乳动物细胞中表达。或者，还可以使用体外无细胞体系进行表达。此类系统为本领域已知的，并且可以从商业途径获得。所述肽可以是分离的和/或以基本上纯的形式提供。例如，它们可以以一种基本上没有其他肽或蛋白的形式提供。
肿瘤抗原在细胞内通过蛋白水解作用将其加工成为8-16个氨基酸长度的多肽片段，即CTL表位，进而与内质网腔中的MHC分子结合形成多肽-MHC复合物(peptide-MHC complex,pMHC),一起递呈到细胞表面。因此，本发明的第二方面提供了一种pMHC复合物，所述复合物中包含本发明第一方面所述的肽。较佳地，所述多肽结合于MHC分子的肽结合槽上。所述MHC分子可以是MHC I类分子或MHCⅡ类分子，优选地，所述MHC分子是MHC I类分子。在一个优选的实施方式中，所述MHC分子是HLA-A*02，更优选地，所述MHC分子是HLA-A*0201。
本发明所述的pMHC复合物可以以多聚体形式存在，例如，二聚体、或四聚体、或五聚体、或六聚体、或八聚体、或更大。产生pMHC多聚体的适当方法可以参考相关文献，如(Greten et al.，Clin.Diagnostic Lab.Immunol.2002：216-220)。
通常，可以用带有生物素残基的pMHC复合物与通过荧光标记链霉亲和素复合产生pMHC多聚体。或者，所述pMHC多聚体也可以通过免疫球蛋白作为分子支架来形成。在这个系统中，MHC分子的胞外区与免疫球蛋白重链的恒定区通过一个短 的连接序列(linker)结合在一起。另外，形成pMHC多聚体也可以利用载体分子，如右旋糖酐(WO02072631)。pMHC多聚体有助于提高与其结合部分的检测，如T细胞受体。或者，提高pMHC复合物在相关应用中的效应，如激活T细胞。
本发明所述的pMHC复合物可以以可溶形式提供。为获得可溶形式的pMHC复合物，优选地，所述pMHC复合物中MHC分子不含跨膜区。具体地，在pMHC复合物中，MHCⅠ类分子可以由其轻链及全部或部分重链的胞外结构域组成。或者，MHC分子是仅包含其功能结构域的片段。
产生本发明可溶性pMHC复合物的方法是本领域技术人员已知的，包括，但不限于，本发明实施例中所述的方法。本发明可溶性pMHC复合物中的MHC分子也可以利用合成方法产生，然后与本发明的肽重折叠。通过确定肽与MHC分子是否能够重折叠，可以确定本发明肽能够与哪类MHC分子形成复合物。
本发明的可溶性pMHC复合物可以用于筛选或检测与其结合的分子，如TCR或抗体。所述方法包括将所述pMHC复合物与待测结合部分接触，和测定待测结合部分是否与复合物结合。pMHC复合物的结合的测定方法是本领域熟知的。优选的方法包括但不限于，表面等离子体共振，或任何其他的生物传感技术，ELISA、流式细胞术、色谱法、显微镜检查。或者，此外，所述结合可以通过对结合产生的生物响应进行功能测定来检测，如细胞因子释放或细胞凋亡。
同样地，本发明的可溶性pMHC复合物还可以用于筛选TCR或抗体文库。利用噬菌体展示技术来构建抗体文库是本领域熟知的，如参考文献Aitken,Antibody phage display:Methods and Protocols(2009,Humana,New York)中所述。在一个优选的实施方式中，本发明的pMHC复合物被用于筛选展示于噬菌体颗粒表面的多样性TCR文库。所述文库展示的TCR可能含有非天然的突变。
因此，本发明的可溶性pMHC复合物可以通过连接物固定到适当的固相载体上。固相载体的例子包括，但不限于，珠子、膜、琼脂糖凝胶、磁珠、基板、管子、柱。pMHC复合物可以固定在ELISA反应板、磁珠、或表面等离子体共振生物传感器芯片上。将pMHC复合物固定到固相载体的方法为本领域技术人员已知的，并且包括，例如，使用亲和结合对，比如生物素和链霉亲和素，或抗体和抗原。在一个优选的实施方式中，pMHC复合物用生物素标记，并且固定在链霉亲和素包被的表面。
本发明所述的肽可以与MHC复合物一起递呈到细胞表面。因此，本发明还提供了一种细胞，所述细胞能够递呈本发明的pMHC复合物到其表面。此类细胞可以是哺乳动物细胞，较佳地，免疫系统细胞，并且优选为专门的抗原呈递细胞，比如树突 细胞或B细胞。其他优选的细胞包括T2细胞(Hosken,et al.,Science.1990.248:367-70)。呈递本发明所述的肽或pMHC复合物的细胞可以是分离的，较佳地，以细胞群体的形式，或以基本上纯的形式提供。所述细胞可以不是天然递呈本发明所述的复合物的，或所述细胞递呈复合物的水平比天然状态下的高。此类细胞可以用本发明所述的肽进行脉冲处理而获得。脉冲处理涉及用所述肽孵育细胞几小时，优选地，所用肽的浓度为10-5-10-12M。此外，所述细胞还可以用HLA-A*02分子进行转导，进一步诱导肽的递呈。递呈本发明所述pMHC复合物的细胞可以被用于分离T细胞和T细胞受体，所述T细胞由所述细胞激活并进一步被分选出来，进而也能够获得表达在所述T细胞表面的T细胞受体。
在一个优选的实施方式中，获得上述T细胞的方法包括利用上述递呈本发明pMHC复合物的细胞刺激从健康志愿者处获得的新鲜血液。可以经过几轮的刺激，如3-4轮。激活的T细胞的鉴定可以通过在本发明的肽脉冲的T2细胞的存在下，来测定细胞因子的释放(比如，IFN-γELISpot实验)。利用标记抗体，激活细胞可以通过流式细胞仪(FACS)进行分选，分选的细胞可以扩大培养和进一步验证，例如，通过ELISpot检测和/或针对靶细胞的细胞毒性和/或pMHC多聚体染色进行验证。来自经验证的T细胞克隆的TCR链可以通过cDNA末端快速扩增(RACE)被放大，并进行测序。
本发明还提供了一种核酸分子，所述核酸分子包括编码本发明的肽的核酸序列。所述核酸可以是cDNA。所述核酸分子可以主要由编码本发明所述肽的核酸序列组成，或可以仅编码本发明所述的肽。此类核酸分子可以用本领域已知的方法合成。由于遗传密码的简并性，本领域技术人员应理解，不同核酸序列的核酸分子可以编码相同的氨基酸序列。
本发明还提供了一种载体，所述载体中包括本发明所述的核酸序列。合适的载体是载体构建领域已知的，包括启动子的选择和其他调控元件，比如增强子元件。本发明所述的载体包括适合引入细胞的序列。比如，所述载体可以是表达载体，在该载体中，所述多肽的编码序列受到它自身顺式作用调控元件的控制，载体的设计便于宿主细胞的基因整合或基因替换等。
本领域普通技术人员应理解，在本发明中，术语“载体”包括DNA分子，比如质粒、噬菌体、病毒或其他载体，它含有一个或多个异源的或重组的核酸序列。合适的噬菌体和病毒载体包括，但不限于：λ-噬菌体，EMBL噬菌体、猿猴病毒、牛疣病毒、Epstein-Barr病毒、腺病毒、疱疹病毒、小鼠肉瘤病毒、鼠类乳癌病毒、慢病 毒等。
本发明还提供了一种结合分子，所述分子可以用来作为免疫治疗剂或诊断试剂。该结合分子可以仅和肽结合，或与肽和MHC分子形成的复合物结合。在后一种情况，所述结合分子可以部分结合到MHC分子上，同时，它还与本发明的肽结合。本发明的结合部分可以是分离的和/或可溶的，和/或非天然存在的，即大自然中没有等效物，和/或纯的，和/或人工合成的。
在本发明的一个优选例中，所述结合分子是T细胞受体(TCR)。可以采用国际免疫遗传学信息系统(IMGT)来描述TCR。天然αβ异源二聚TCR具有α链和β链。广义上讲，各链包含可变区、连接区和恒定区，β链通常还在可变区和连接区之间含有短的多变区，但该多变区常视作连接区的一部分。
本发明的TCR可以是本领域已知的任何形式。例如，所述TCR可以是异二聚体，或以单链的形式存在。所述TCR可以是可溶形式(即无跨膜或胞浆区)，具体地，所述TCR可以包含全部或部分TCR胞外结构域。所述TCR也可以是包含其跨膜区的全长链。所述TCR可以被提供到细胞表面，比如T细胞。
可以结合本领域中的现有技术来获得可溶性的TCR，例如，在αβTCR的α与β链的恒定域之间引入人工二硫键，或者在αβTCR的α链可变区与β链恒定区之间引入人工二硫键。
本发明的TCR可用于将细胞毒性剂或免疫刺激剂递送到靶细胞，或被转化入T细胞，使表达该TCR的T细胞能够破坏肿瘤细胞，以便在被称为过继免疫治疗的治疗过程中给予患者。另外，本发明的TCR中也可以含有突变，优选地，突变后的TCR对本发明pMHC复合物的亲和力有所提高。本发明的TCR可以单独使用，也可与偶联物以共价或其他方式结合，优选以共价方式结合。所述偶联物包括可检测标记物(为诊断目的，其中所述TCR用于检测呈递本发明pMHC复合物的细胞的存在)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。本发明的TCR还可以与抗-CD3的抗体结合，优选以共价方式结合，以重定向T细胞，从而杀伤靶细胞。
在另一优选例中，本发明的结合分子是抗体。如本文所用，术语“抗体”指免疫球蛋白分子和免疫球蛋白分子的免疫活性部分，即含有特异性结合位点的分子，其可以全天然、或部分人工合成、或全部人工合成。术语“抗体”包括抗体片段、其衍生物、功能性等效物以及同源抗体、人源化抗体，所述抗体片段包括免疫球蛋白结合区，所述结合区是抗体结合区或与抗体结合区同源。其可以全天然、或部分人工合成、或全部人工合成。人源化抗体可以是修饰的抗体，其含有非人抗体的可变区(例如，小 鼠)以及人抗体的恒定区。
抗体的例子可以是同型免疫球蛋白(例如IgG、IgE、IgM、IgD以及IgA)以及它们同型的亚类；片段包括抗原结合区，比如Fab、scFv、Fv、dAb、Fd；以及双链抗体。抗体可以是多抗或单抗，优选为单克隆抗体。
上述TCR与抗体的制备方法是本领域技术人员已知的，包括但不限于，从大肠杆菌细胞或昆虫细胞中表达，并纯化出来。
在另一方面，本发明进一步提供了本发明的肽、pMHC复合物、核酸分子、载体、细胞以及结合分子在制药方面的用途。所述肽、pMHC复合物、核酸、载体、细胞或结合分子可以被用于治疗或预防恶性肿瘤，优选急性髓细胞白血病。
本发明还提供了一种药物组合物，其包含本发明的抗原肽、pMHC复合物、本发明的核酸分子、本发明的细胞或本发明的结合分子，以及药学上可接受的载体。所述药物组合物可以是任何合适的形式，(取决于患者需要的给药方法)。其可以以单位剂型的形式提供，通常置于密封容器中，并且可以作为试剂盒的一部分提供。此类试剂盒通常(但不是必须)包含使用说明。其可以包含多个所述的单位剂型。
所述药物组合物适用于任何适当的给药途径，如注射(包括皮下，肌肉，腹腔或静脉注射)、吸入或口服、或经鼻、或经肛门等途径。所述组合物可以通过药学领域已知的任何方法制备，例如在无菌条件下，通过将活性成分与载体或赋形剂混合。
根据用于治疗的疾病或病症(例如癌症、病毒性感染或自身免疫疾病)、患者的个体年龄和状况等，本发明制剂的给药剂量可以在较宽的范围内变化。恰当的给药剂量将由医师最终决定。
根据本领域的现有技术，与MHC分子一起被呈递到细胞表面的肽、pMHC复合物或递呈pMHC复合物的细胞，可以激活T细胞或B细胞，使其发挥作用。
因此，本发明的肽、pMHC复合物或递呈pMHC复合物的细胞可以以疫苗组合物的形式提供。所述疫苗组合物可以用于治疗或预防癌症。所有的这类组合物都包括在本发明中。应理解，所述疫苗可以为多种形式(Schlom J.J Natl Cancer Inst.2012 104(8)：599-613)。例如，本发明的肽可以直接用于免疫患者(Salgaller ML.Cancer Res.1996.56(20)：4749-57 and Marchand M.Int J Cancer.1999.80(2)：219-230)。所述疫苗组合物可以包含额外的肽，使得本发明的肽是肽混合物中的一个。所述疫苗组合物可以加入佐剂，以增强免疫反应。或者，所述疫苗组合物可以是呈递本发明肽和MHC复合物的抗原递呈细胞的形式。优选地，所述抗原呈递细胞为免疫细胞，更优选地为树突细胞。所述肽也可以脉冲到细胞的表面(Thurner BI.et al.,J.Exp.Med.1999. 190:1669)，或者可以将本发明肽的编码核酸引入到树突细胞中，例如，利用电穿孔法(Van Tendeloo,VF.etal.,Blood 2001.98:49)。
本发明的主要优点包括：
a)本发明旨在一定程度克服上能够克服相关技术的不足，提供一种新抗原肽及其在肿瘤免疫治疗中的应用。
b)本发明的抗原肽具有清除肿瘤细胞的作用。
c)本发明的抗原肽能够扩宽FLT3突变型肿瘤患者的治疗范围，为患者的免疫治疗提供新选择。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1新抗原肽与HLA-A*02:01分子亲和力预测
利用在线生物学软件NetMHCpan 4.1预测新抗原肽与HLA-A*02:01分子的亲和力，％Rank_EL＜0.500视为具有强结合力，0.500＜％Rank_EL＜2.00视为具有中等结合力，％Rank_EL＞2.000视为无结合力。
经预测发现，新抗原肽(SEQ ID No.1至SEQ ID No.5)与HLA-A*02:01分子具有中等或强结合力，野生型多肽(SEQ ID No.7)与HLA-A*02:01分子无结合力(表2)。
表2靶向FLT3-D835突变的新抗原肽与HLA-A*02:01亲和力预测结果
Figure PCTCN2022123390-appb-000003
实施例2新抗原肽与HLA-A*02:01分子亲和力验证
取对数生长期的T2细胞，无血清无抗生素的IMDM培养基调整细胞浓度至1×106/mL，分别加入新抗原肽(10μg/mL)及β2微球蛋白(3μg/mL)，于37℃共培养4h。培养结束后取出细胞，PBS洗涤，加入FITC标记的anti-HLA-A2单克隆抗体，室温孵育30min，流式细胞仪检测。最终结果以荧光系数(FI)作为衡量指标：(样品平均荧光强度-背景平均荧光强度)/背景平均荧光强度。FI>1.5视为多肽与HLA-A2分子亲和力高；1.0<FI<1.5为中等亲和力，FI<1.0为低亲和力。
结果表明，抗原肽(SEQ ID No.1至SEQ ID No.5)与HLA-A*02:01分子具有中等或高亲和力，显著高于野生型多肽(SEQ ID No.7)与HLA-A*02:01分子的亲和力(相差10倍或更多)。
野生型多肽(SEQ ID No.7)与HLA-A*02:01分子无亲和力或极低(表3)。
表3靶向FLT3-D835突变的新抗原肽与HLA-A*02:01分子亲和力
Figure PCTCN2022123390-appb-000004
实施例3新抗原肽/HLA-A*02:01分子复合物稳定性验证
取对数生长期的T2细胞，无血清无抗生素的IMDM培养基(含100ng/mL人β2m)调整T2细胞的浓度为1×106/mL，分别与100μg/mL新抗原肽在37℃孵育过夜。第二天收集细胞，加入含有10μg/mL Brefeldin A的无血清IMDM培养基孵育1h；加入含有0.5μg/mL Brefeldin A的无血清IMDM培养基，于37℃孵育，分别在0、2、4、6及8h的时间点收集细胞，100μL PBS重悬细胞，加入FITC标记的anti-HLA-A2单克隆抗体，室温孵育30min，于流式细胞仪检测，计算每个时间点的T2细胞的平均荧光强度。
结果
表4靶向FLT3-D835突变的新抗原肽/HLA-A*02:01分子复合物稳定性(％)
Figure PCTCN2022123390-appb-000005
实验结果如图1和表4所示，经检测发现，本发明的抗原肽与HLA-A*02:01分子形成的复合物稳定，其中抗原肽V与HLA-A*02:01分子形成的复合物最稳定。
实施例4抗原肽在AML患者中诱导特异性T淋巴细胞
取相同HLA分型及相同FLT3-D835突变的AML患者静脉血，Ficoll密度梯度离心法分离和纯化外周血单个核细胞(PBMCs)。Dynabeads磁珠分选出CD8+细胞，以CD8-细胞作为抗原递呈细胞。
无血清RPMI-1640培养基重悬CD8-细胞，加入丝裂霉素(30μg/mL)，在37℃灭活30min后，PBS洗涤细胞，无血清RPMI-1640培养基重悬细胞，加入抗原肽V(20μg/mL)，37℃孵育2-4h。
收集抗原脉冲刺激的CD8-细胞，重悬于含10％FBS的RPMI-1640培养基(含IL-2 50U/mL、IL-7 5ng/mL、IL-15 5ng/mL)，与CD8+细胞共孵育，每2-3天半量换液，培养10-20d。收集细胞，用PE标记的抗原肽-HLA-A*02:01-Tetramer抗体染色，流式细胞仪检测。
结果
实验结果如图2所示，经检测发现，加入抗原肽刺激后，Tetramer阳性细胞增多，抗原肽V可从AML患者外周血诱导生成特异性T淋巴细胞(CTL细胞)。
实施例5新抗原肽激活AML患者外周血特异性T淋巴细胞
取相同HLA分型及相同FLT3-D835突变的AML患者静脉血，Ficoll密度梯度离心法分离和纯化PBMCs，铺于96孔板，分别加入抗原肽10μg/mL，扩增培养10-20d。培养结束后取出细胞，含10％FBS的RPMI-1640培养基重悬细胞至密度1× 106/mL，100μL/孔加入预包被Human IFN-γ抗体的ELISPOT检测板，加入抗原肽V(终浓度10μg/mL)至相应孔，阴性对照孔不加抗原肽，阳性对照孔加入PHA(终浓度4μg/mL)，于37℃孵育18-24h。取出斑点板，按说明书洗板、孵育抗体、显色，干燥后读板。
结果
实验结果如图3所示，经检测发现加入抗原肽后，AML患者PBMCs分泌IFN-γ能力升高，抗原肽具有良好的免疫原性，能够激活特异性免疫反应。
实施例6新抗原在健康志愿者中诱导特异性T淋巴细胞
取相同HLA分型的健康志愿者静脉血，Ficoll密度梯度离心法分离和纯化PBMCs。Dynabeads磁珠分别分选出CD8+细胞及CD14+细胞。含10％FBS的RPMI-1640培养基(含IL-4 1000U/mL、GM-CSF 1000U/mL)重悬CD14+细胞，置于孵箱培养5-7d诱导树突状细胞(DCs)，加入TNF-α(10ng/mL)促成熟。
回收成熟DCs，无血清RPMI-1640培养基重悬细胞，加入抗原肽V(20μg/mL)，37℃孵育2-4h。收集抗原肽脉冲刺激的DCs，重悬于含10％FBS的RPMI-1640培养基(含IL-2 50U/mL、IL-7 5ng/mL、IL-15 5ng/mL)，与CD8+细胞共孵育，每2-3d半量换液，培养10-20d。收集细胞，用PE标记的抗原肽-HLA-A*02:01-Tetramer抗体染色，流式细胞仪检测。
结果
实验结果如图4所示，经检测发现，抗原肽可从健康志愿者外周血诱导生成CTLs。
实施例7新抗原肽激活健康志愿者外周血特异性T淋巴细胞
取相同HLA分型的健康志愿者静脉血，如实施例6诱导生成抗原肽特异性CTLs。培养结束后取出细胞，含10％FBS的RPMI-1640培养基重悬细胞至密度1×106/mL，100μL/孔加入预包被Human IFN-γ抗体的ELISPOT检测板。如实施例6制备负载新抗原肽的DCs作为抗原递呈细胞，分别加入相应的ELIPSOT孔板。阴性对照孔加入不负载新抗原肽的DCs，阳性对照孔加入PHA(终浓度4μg/mL)，于37℃孵育18-24h。取出斑点板，按说明书洗板、孵育抗体、显色，干燥后读板。
结果
实验结果如图5所示，经检测发现健康志愿者CTLs分泌IFN-γ能力升高，新抗原肽具有良好的免疫原性，能够激活特异性免疫反应。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种用于引发靶向FLT3-D835突变的免疫应答的抗原肽，其特征在于，所述抗原肽能够与MHC分子形成复合物，并且所述抗原肽选自下组：
    (i)SEQ ID NO:6所示的多肽：
    X 1IMSDSNYV
    其中，X 1为V、H、I或F；
    (ii)对(i)多肽的氨基酸序列中除X 1以外的氨基酸进行1个、2个或3个氨基酸取代，和/或1个、2个或3个氨基酸插入，和/或1个或2个氨基酸缺失所形成的衍生多肽，并且所述衍生多肽保留X 1。
  2. 如权利要求1所述的抗原肽，特征在于，所述抗原肽具有式I所示结构：
    X 0-X 1-Z 1-X 10  (I)
    其中，
    X 0为无或R；
    X 1为V、H、I或F；
    Z 1为IMSDSNYV；
    X 10为无或V。
  3. 如权利要求1所述的抗原肽，特征在于，所述的抗原肽具有式II结构，
    X 1-Z 1  (II)
    其中，
    X 1为V、H、I或F；
    Z 1为IMSDSNYV。
  4. 如权利要求1所述的抗原肽，特征在于，X 1为V或H。
  5. 如权利要求1所述的抗原肽，特征在于，所述抗原肽为SEQ ID NO:1-4中任一所示的氨基酸序列的多肽中1种，或2种、3种或4种多肽构成的组合。
  6. 一种pMHC复合物，其特征在于，所述复合物包含权利要求1所述的抗原肽。
  7. 一种核酸分子，其特征在于，所述核酸分子包含编码权利要求1所述抗原肽的核酸序列或其互补序列。
  8. 一种载体，其特征在于，所述载体含有权利要求7所述的核酸分子。
  9. 一种宿主细胞，其特征在于，所述细胞中含有权利要求8所述的载体。
  10. 一种体外制备特异性T淋巴细胞的方法，其特征在于，包括步骤：
    a)提供PBMC，
    b)在抗原肽存在下，将所述PBMC与权利要求1所述的抗原肽进行接触并培养，从而获得经抗原肽激活的特异性T淋巴细胞。
  11. 如权利要求10所述的方法，其特征在于，在步骤(b)中还包括：
    (b1)从PBMC中分选出CD8 +细胞和CD8 -细胞，
    (b2)将权利要求1所述的抗原肽对(b1)中所述的CD8 -细胞进行致敏处理，从而获得经致敏的CD8 -细胞，
    (b3)将(b2)中所述的经致敏的CD8 -细胞与CD8 +细胞共孵育，从而获得经抗原肽激活的特异性T淋巴细胞。
  12. 一种药物组合物，其特征在于，所述组合物含有(ii)药学上可接受的载体以及(ii)权利要求1所述的抗原肽、权利要求6所述的pMHC复合物、权利要求7所述的核酸分子、或经权利要求1所述的抗原肽激活的特异性T淋巴细胞。
  13. 如权利要求12所述的药物组合物，其特征在于，所述药物组合物为疫苗组合物。
  14. 一种预防或治疗恶性肿瘤相关疾病的方法，其特征在于，包括给需要的对象施用适量的权利要求1所述的抗原肽、权利要求6所述的pMHC复合物、权利要求7所述的核酸分子、经权利要求1所述的抗原肽激活的特异性T淋巴细胞或权利要求12所述的药物组合物。
  15. 如权利要求1所述的抗原肽、权利要求6所述的pMHC复合物、权利要求7所述的核酸分子、经权利要求1所述的抗原肽激活的特异性T淋巴细胞或权利要求12所述的药物组合物的用途，其特征在于，用于制备预防或治疗恶性肿瘤的药物。
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