CN117586344A - Antigenic peptide targeting FLT3-D835 mutation and application thereof in tumor immunotherapy - Google Patents
Antigenic peptide targeting FLT3-D835 mutation and application thereof in tumor immunotherapy Download PDFInfo
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- CN117586344A CN117586344A CN202210968824.2A CN202210968824A CN117586344A CN 117586344 A CN117586344 A CN 117586344A CN 202210968824 A CN202210968824 A CN 202210968824A CN 117586344 A CN117586344 A CN 117586344A
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Abstract
The invention relates to an antigen peptide targeting FLT3-D835 mutation and application thereof in tumor immunotherapy. In particular, the invention provides an antigenic peptide for eliciting an immune response that targets FLT3-D835 mutations. And wild-type polypeptides and HLA-A * 02:01 molecule without affinity, the antigenic peptide of the invention is compared with HLA-A * 02:01 minThe seed has high affinity. The invention also provides application of the antigen peptide. The antigen peptide has good immunogenicity, can activate specific immune response, and has important clinical significance for targeted elimination of leukemia cells, stabilization of the remission state of AML patients and prolongation of disease-free survival time.
Description
Technical Field
The invention belongs to the field of polypeptide medicaments and polypeptide vaccines, and particularly relates to an antigen peptide targeting FLT3-D835 mutation and application thereof in tumor immunotherapy.
Background
Acute Myeloid Leukemia (AML) is a malignant clonal proliferative disease that results from the progressive accumulation of acquired genetic abnormalities in hematopoietic stem/progenitor cells, which arrest the differentiation of the myeloid lineage at different stages. AML is the most common hematological malignancy in adults, accounting for over 70% of all leukemias. At present, the clinical AML treatment is mainly combined with hematopoietic stem cell transplantation by chemical drug treatment, the overall treatment effect is poor, the cure rate of patients under 60 years old is only 35% -40%, the cure rate of patients over 60 years old is lower to 5% -15%, and nearly 50% of patients lose treatment opportunity due to disease recurrence or drug resistance. The continuous accumulation of genetic variation, including somatic mutation, gene insertion/deletion, gene fusion, etc., is a key cause of refractory/recurrent AML. Therefore, the mutant gene is taken as an entry point, abnormal leukemia cells are removed in a targeted manner, and the method has important scientific significance for reducing the recurrence rate of AML and improving the cure rate.
The FMS-like tyrosine kinase 3 (FLT 3) gene is located on chromosome 13q12 and encodes a 993 amino acid-containing protein. Wherein the transmembrane region is located between amino acids 542 to 564 and the kinase domain is located between amino acids 610 to 944, including a kinase insert of about 50 amino acids. FLT3 variation is one of the most common genetic abnormalities in AML, occurring in about 30% of patients. Variation of FLT3 results in an increase in FLT3 kinase activity, thereby promoting leukemia cell proliferation and growth, which is closely related to poor prognosis in AML patients.
About 5-10% of FLT3 mutations were found to involve single nucleotide mutations of the FLT3 tyrosine kinase domain (FLT 3-TKD), with the most common mutation being at amino acid residue 835, aspartic acid (D835), typically located within the activation loop. Aspartic acid is a key regulatory residue of the tyrosine kinase receptor, and is highly conserved in structure. Wild-type D835 residue (D835 wt) is critical to maintaining the inactive conformation of FLT3, and the D835 point mutation results in poor patient prognosis. The therapeutic mode aiming at FLT3-D835 mutation is found, leukemia cells are cleared in a targeted mode, and the therapeutic mode has important clinical significance for stabilizing the relieving state of AML patients and prolonging the disease-free survival time.
In recent years, with the development of immunological, genomic and molecular biological techniques, immunotherapy has become another innovative mode of tumor treatment. Among them, therapeutic tumor vaccines are a research hotspot for immunotherapy. Tumor vaccines are based on tumor-immune circulation theory, and stimulate or enhance the anti-tumor immune response of an organism by promoting the recognition of tumor antigens by immune cells, and clear tumor cells in cooperation with the immune system. However, the conventional tumor vaccine contains a plurality of targets, namely tumor-associated antigens, and the targets are expressed in tumor and normal tissues, so that the vaccine has low immunogenicity and is easy to induce immune tolerance, and the vaccine is frequently frustrated in clinical experiments and has few clinical popularization.
The neoantigen is a protein sequence derived from tumor cells on the basis of genetic variation and comprising mutated amino acids. After the new antigen is presented by antigen presenting cells, the new antigen can be effectively recognized by T cells, and the T cells are activated, so that specific immune response is activated to attack and eliminate tumor cells. The new antigen peptide derived from genetic variation is artificially synthesized to construct therapeutic tumor vaccine, and the therapeutic tumor vaccine is returned to the patient to activate immune cells, so that tumor cells expressing the same new antigen can be targeted and killed. In addition, the new antigen is only expressed in tumor cells, is not expressed in normal cells or tissues, has high immunogenicity and does not induce immune tolerance, and is a dominant target of tumor vaccines. However, current neoantigen vaccine strategies require sequencing of the individual subject genome and HLA typing and calculation of the likely applicable individual neoantigen peptides, a process that is at least 2-3 months and even longer, limiting to some extent the popularization of therapeutic strategies. Furthermore, the accuracy of the calculated new antigen is not high, and the immunogenicity of the genetically mutated polypeptide still needs to be further evaluated.
Therefore, there is an urgent need in the art to develop a safe, efficient and economical antigenic peptide that widens the therapeutic range of FLT3 mutant tumor patients and provides a new choice for immunotherapy of patients.
Disclosure of Invention
It is an object of the present invention to provide an antigenic peptide targeting the FLT3-D835 mutation.
It is another object of the invention to provide the use of antigenic peptides targeting FLT3-D835 mutation in tumor immunotherapy.
In a first aspect of the invention there is provided an antigenic peptide for eliciting an immune response targeting FLT3-D835 mutation, said antigenic peptide being capable of forming a complex with an MHC molecule and said antigenic peptide being selected from the group consisting of:
(i) A polypeptide shown in SEQ ID NO. 6:
X 1 IMSDSNYV
wherein X is 1 V, H, I or F;
(ii) Dividing the amino acid sequence of the polypeptide of (i) by X 1 A derivative polypeptide formed by substitution of other amino acids by 1, 2 or 3 amino acids, and/or insertion of 1, 2 or 3 amino acids, and/or deletion of 1 or 2 amino acids, and retaining X 1 。
In another preferred embodiment, the antigenic peptide has the structure of formula I:
X 0 -X 1 -Z 1 -X 10 (I)
wherein,
X 0 is none or R;
X 1 v, H, I or F;
Z 1 is IMSDSNYV;
X 10 is none or V.
In another preferred embodiment, in formula II, X 0 Is none or R, X 10 Is none or V.
In another preferred embodiment, the antigenic peptide has the structure of formula II,
X 1 -Z 1 (II)
wherein,
X 1 v, H, I or F;
Z 1 is IMSDSNYV.
In another preferred embodiment, X 1 V or H.
In another preferred embodiment, the antigenic peptide is a combination of two or more antigenic peptides.
In another preferred embodiment, the antigenic peptide is 1 of the polypeptides of the amino acid sequence shown in any one of SEQ ID NO.1-4, or a combination of 2, 3 or 4 polypeptides.
In another preferred embodiment, the combination of antigenic peptides further comprises additional antigenic peptides directed against other tumor antigens or sites.
In another preferred embodiment, the additional antigenic peptide comprises the polypeptide shown in SEQ ID No. 5.
In a second aspect of the invention there is provided a pMHC complex comprising an antigenic peptide according to the first aspect of the invention.
In another preferred embodiment, the antigenic peptide in the pMHC complex has a polypeptide having the amino acid sequence shown in SEQ ID NO. 6.
In another preferred example, the type of MHC molecule is HLA-A x 02.
In another preferred embodiment, the type of MHC molecule is HLA-A x 02:01.
In a third aspect of the invention there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding an antigenic peptide according to the first aspect of the invention or a complement thereof.
In a fourth aspect of the invention there is provided a vector comprising a nucleic acid molecule according to the third aspect of the invention.
In a fifth aspect of the invention there is provided a host cell comprising a vector according to the fourth aspect of the invention.
In a sixth aspect of the invention, there is provided a method of preparing specific T lymphocytes in vitro comprising the steps of:
a) Providing a PBMC (micro-electro mechanical systems),
b) Contacting and culturing said PBMCs with an antigenic peptide according to the first aspect of the invention in the presence of said antigenic peptide, thereby obtaining specific T lymphocytes activated by the antigenic peptide.
In another preferred embodiment, the concentration of the antigenic peptide is 20. Mu.g/mL.
In another preferred embodiment, the PBMCs are cultured with the antigen peptide for 10 days.
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
In another preferred embodiment, the PBMCs are autologous or allogeneic cells.
In another preferred embodiment, in step (b), further comprising:
(b1) Sorting CD8 from PBMC + Cell and CD8 - The cell is selected from the group consisting of,
(b2) The antigen peptide of the first aspect of the present invention is directed against CD8 as described in (b 1) - The cells are sensitized to obtain sensitized CD8 - The cell is selected from the group consisting of,
(b3) The sensitized CD8 as described in (b 2) - Cell and CD8 + Cells are co-incubated to obtain antigen peptide activated specific T lymphocytes.
In a seventh aspect of the invention there is provided a pharmaceutical composition comprising (ii) a pharmaceutically acceptable carrier and (ii) an antigenic peptide according to the first aspect of the invention, a pMHC complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, or a specific T lymphocyte activated by an antigenic peptide according to the first aspect of the invention.
In another preferred embodiment, the pharmaceutical composition is a vaccine composition.
In another preferred embodiment, the pharmaceutical composition is in a dosage form selected from the group consisting of liquid, solid, and gel.
In another preferred embodiment, the pharmaceutical composition is administered by a means selected from the group consisting of: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, or oral administration.
In an eighth aspect of the invention there is provided a method of preventing or treating a malignancy-associated disease comprising administering to a subject in need thereof an amount of an antigenic peptide according to the first aspect of the invention, a pMHC complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a specific T lymphocyte activated by an antigenic peptide according to the first aspect of the invention or a pharmaceutical composition according to the seventh aspect of the invention.
Use of an antigenic peptide according to the first aspect of the invention, a pMHC complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a specific T lymphocyte activated by an antigenic peptide according to the first aspect of the invention or a pharmaceutical composition according to the seventh aspect of the invention for the preparation of a medicament for the prevention or treatment of a malignant tumour.
In another preferred embodiment, the malignancy is a hematologic malignancy.
In another preferred embodiment, the malignancy is Acute Myelogenous Leukemia (AML).
In another preferred embodiment, the malignancy is a FLT-D835 mutant malignancy.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows antigenic peptides and HLA-A * Stability of complex 8H formed by 02:01 molecules.
Figure 2 shows the detection of antigen peptide induced generation of specific T cells by tetra mer flow cytometry.
FIG. 3 shows the detection of IFN-gamma secretion by antigen peptide activation-specific T cells by an ELISPOT assay.
Figure 4 shows that antigenic peptides can induce CTLs from peripheral blood of healthy volunteers.
Figure 5 shows that CTLs in healthy volunteers have an increased ability to secrete IFN- γ.
Detailed Description
The inventor of the present invention has conducted extensive and intensive studies and has unexpectedly obtained a safe, efficient and economical antigen peptide targeting the FLT3-D835 mutation through a large number of screening. Experiments show that the polypeptide (SEQ ID No. 7) and the wild type polypeptide (HLA-A) * 02:01 molecule without affinity the antigenic peptide of the invention (SEQID No. 1-4) and HLA-A * The 02:01 molecules have medium or high affinity. Meanwhile, the antigen peptide has good immunogenicity, can activate specific immune response, can induce CTLs from peripheral blood of healthy volunteers, and has high IFN-gamma secretion capacity. The present invention has been completed on the basis of this finding.
It will be appreciated that in the present invention, the term "antigenic peptide" is used interchangeably with "polypeptide of the invention" or "short peptide of the invention" and refers to the antigenic peptide of the invention that targets the FLT3-D835 mutation.
Terminology
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
FMS-like tyrosine kinase 3 (FLT 3) and FLT3-D835
The FMS-like tyrosine kinase 3 (FLT 3) gene is located on chromosome 13q12 and encodes a 993 amino acid-containing protein. Wherein the transmembrane region is located between amino acids 542 to 564 and the kinase domain is located between amino acids 610 to 944, including a kinase insert of about 50 amino acids. FLT3 variation is one of the most common genetic abnormalities in AML, occurring in about 30% of patients. Variation of FLT3 results in an increase in FLT3 kinase activity, thereby promoting leukemia cell proliferation and growth, which is closely related to poor prognosis in AML patients.
About 5-10% of FLT3 mutations were found to involve single nucleotide mutations of the FLT3 tyrosine kinase domain (FLT 3-TKD), with the most common mutation being at amino acid residue 835, aspartic acid (D835), typically located within the activation loop. Aspartic acid is a key regulatory residue of the tyrosine kinase receptor, and is highly conserved in structure. The D835 point mutations have been identified from AML patients at present including: alanine (a), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), aspartic acid (N), valine (V), tyrosine (Y), with D835F, D835H, D835I, D835V and D835Y being particularly common. The D835 point mutation resulted in poor patient prognosis.
New antigenic peptides (neoantigen)
The neoantigen is a protein sequence derived from tumor cells on the basis of genetic variation and comprising mutated amino acids. After the new antigen is presented by antigen presenting cells, the new antigen can be effectively recognized by T cells, and the T cells are activated, so that specific immune response is activated to attack and eliminate tumor cells. The new antigen peptide derived from genetic variation is artificially synthesized to construct therapeutic tumor vaccine, and the therapeutic tumor vaccine is returned to the patient to activate immune cells, so that tumor cells expressing the same new antigen can be targeted and killed. In addition, the new antigen is only expressed in tumor cells, is not expressed in normal cells or tissues, has the characteristic of high immunogenicity and does not induce immune tolerance, and is a dominant target of tumor vaccines.
In particular, in a first aspect of the invention, there is provided an antigenic peptide for eliciting an immune response targeting FLT3-D835 mutation, said antigenic peptide being capable of forming a complex with an MHC molecule and being selected from the group consisting of:
(i) A polypeptide shown in SEQ ID NO. 6:
X 1 IMSDSNYV
wherein X is 1 V, H, I or F;
(ii) Dividing the amino acid sequence of the polypeptide of (i) by X 1 Amino acids other than 1, 2 or 3 ammoniaDerived polypeptides formed by amino acid substitutions, and/or 1, 2 or 3 amino acid insertions, and/or 1 or 2 amino acid deletions, and which retain X 1 。
Amino acid substitutions means that at the same position, one amino acid residue is replaced by another amino acid residue. The inserted amino acid residues may be inserted at any position, and the inserted amino acid residues may be all or partially adjacent to each other, or none of the inserted amino acids may be adjacent to each other. It is known to those skilled in the art that the peptides of the 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 Curr Opin Immunol.2006 by Engelhard et al, 2; 18 (1): 92-7, and includes phosphorylation, acetylation, and deamidation.
Preferably, the peptides of the invention bind to MHC at the peptide binding site of an MHC molecule. In general, the modified amino acids described above do not disrupt the binding capacity of the peptide to MHC. In a preferred embodiment, the amino acid modification increases the ability of the peptide to bind to MHC. For example, mutations may occur at binding sites of peptides to MHC. These binding sites and preferred residues on the binding sites are known in the art, especially for which peptides bind HLA-A.02 (see, e.g., parkhurst et al, J.Immunol.157:2539-2548 (1996)).
More specifically, the amino acids of the peptides of the invention may be 8-15, preferably 8-10, preferably 9 in length.
The polypeptide of the present invention may consist of any one of the polypeptides of SEQ ID NO.1-4 in Table 1.
TABLE 1 antigenic peptides of the invention
The invention also provides SEQ ID NO:1-4, or a peptide analog thereof. These analogs may differ from the native peptide by differences in amino acid sequence, by differences in modified forms that do not affect the sequence, or by both. These peptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, by site-directed mutagenesis or other known techniques of molecular biology. Analogs also include analogs having residues other than the natural L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., 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 (typically without altering the primary structure) forms include: chemical derivatization forms of peptides in vivo or in vitro such as acetylation or carboxylation. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the peptide or during further processing steps. Such modification may be accomplished by exposing the peptide to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Peptides modified to improve their proteolytic resistance or to optimize their solubility properties are also included.
In the present invention, "SEQ ID NO:1-4 refers to a protein conserved variant peptide "as set forth in SEQ ID NO:1-4, and more preferably up to 3, more preferably up to 2 amino acids are replaced by amino acids of similar or similar nature to form a peptide. These conservatively mutated peptides are preferably generated by amino acid substitution according to Table 1.
Table A
The peptides of the invention can be simply synthesized using Merrifield synthesis (also known as solid phase synthesis of polypeptides). GMP-grade peptides can be synthesized using solid phase synthesis techniques of the polypeptide system (Multiple Peptide Systems, san Diego, calif.). Alternatively, the peptides may be synthesized recombinantly, and if desired, by methods known in the art. Typically such methods involve the use of vectors 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, expression may also be performed using an in vitro cell-free system. Such systems are known in the art and are commercially available. The peptides 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.
Tumor antigens are processed into polypeptide fragments of 8-16 amino acids in length, i.e., CTL epitopes, by proteolysis within cells, which in turn bind to MHC molecules in the lumen of the endoplasmic reticulum to form polypeptide-MHC complexes (pMHC) that are presented together to the cell surface. Accordingly, in a second aspect the present invention provides a pMHC complex comprising a peptide according to the first aspect of the invention. Preferably, the polypeptide is bound to a peptide binding groove of an MHC molecule. The MHC molecule may 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, more preferably, the MHC molecule is HLA-A * 0201。
The pMHC complexes of the invention may exist in multimeric form, for example, as dimers, or tetramers, or pentamers, or hexamers, or octamers, or greater. Suitable methods for producing pMHC multimers can be found in the literature, for example (Greten et al, clin.diagnostic Lab.immunol.2002:216-220).
In general, pMHC multimers can be generated by complexing a pMHC complex with biotin residues with streptavidin labeled by fluorescence. Alternatively, the pMHC multimer may also be formed by immunoglobulins as a molecular scaffold. In this system, the extracellular region of the MHC molecule is joined to the constant region of the immunoglobulin heavy chain by a short linker sequence (linker). In addition, the formation of pMHC multimers may also utilize carrier molecules, such as dextran (WO 02072631). pMHC multimers help to enhance detection of binding moieties, such as T cell receptors. Alternatively, the effect of the pMHC complex in related applications, such as activating T cells, is increased.
The pMHC complexes of the invention may be provided in soluble form. To obtain a soluble form of the pMHC complex, preferably the MHC molecules in the pMHC complex do not contain a transmembrane region. In particular, in pMHC complexes, MHC class i molecules may consist of the extracellular domain of their light chain and all or part of the heavy chain. Alternatively, an MHC molecule is a fragment comprising only its functional domain.
Methods of producing the soluble pMHC complexes of the invention are known to those skilled in the art and include, but are not limited to, the methods described in the examples of the invention. MHC molecules in the soluble pMHC complexes of the invention may also be produced synthetically and then refolded with the peptides of the invention. By determining whether a peptide is refolded with an MHC molecule, it can be determined with which MHC class the peptide of the invention is capable of forming a complex.
The soluble pMHC complexes of the invention can be used to screen or detect molecules, such as TCRs or antibodies, bound thereto. The method comprises contacting the pMHC complex with a test binding moiety, and determining whether the test binding moiety binds to the complex. Methods for determining the 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 technique, ELISA, flow cytometry, chromatography, microscopy. Alternatively, and in addition, the binding may be detected by performing a functional assay of the biological response produced by the binding, such as cytokine release or apoptosis.
Likewise, the soluble pMHC complexes of the invention may also be used to screen TCR or antibody libraries. The construction of antibody libraries using phage display technology is well known in the art, as described in reference Aitken, antibody phage display: methods and Protocols (2009,Humana,New York). In a preferred embodiment, the pMHC complexes of the invention are used to screen diverse TCR libraries displayed on the surface of phage particles. The library may display TCRs that contain unnatural mutations.
Thus, the soluble pMHC complexes of the invention may be immobilized via a linker to a suitable solid support. 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 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. In a preferred embodiment, the pMHC complex is labeled with biotin and is immobilized on a streptavidin-coated surface.
The peptides of the invention may be presented to the cell surface together with MHC complexes. Thus, the present invention also provides a cell capable of presenting the pMHC complex of the invention to its surface. Such cells may be mammalian cells, preferably cells of the immune system, and preferably are 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 peptide or pMHC complex of 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 pulsing with the peptides of the invention. The pulse treatment involves incubating the cells with the peptide for several hours, preferably at a concentration of 10 -5 -10 -12 M. In addition, the cells may also use HLA-A * The 02 molecule transduces, further inducing peptide presentation. Cells presenting the pMHC complexes of the invention can be used to isolate T cells activated by the cells and further sorted to obtain T cell receptors expressed on the surface of the T cells.
In a preferred embodiment, the method of obtaining the T cells described above comprises stimulating fresh blood obtained from healthy volunteers with the cells presenting pMHC complexes of the invention described above. Several rounds of stimulation, such as 3-4 rounds, may be performed. Identification of activated T cells cytokine release can be determined by the presence of peptide-pulsed T2 cells of the invention (e.g., IFN- γ ELISpot assay). With labeled antibodies, activated cells can be sorted by flow cytometry (FACS), and the sorted cells can be expanded cultured and further validated, for example, by ELISpot detection and/or cytotoxicity against target cells and/or pMHC multimer staining. TCR chains from validated T cell clones can be amplified by Rapid Amplification of CDNA Ends (RACE) and sequenced.
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 the peptide of the invention, or may encode only the peptide of the invention. Such nucleic acid molecules can be synthesized using methods known in the art. Because of the degeneracy of the genetic code, it will be understood by those skilled in the art that nucleic acid molecules of different nucleic acid sequences may encode the same amino acid sequence.
The invention also provides a vector comprising the nucleic acid sequence of the invention. Suitable vectors are known in the art of vector construction and include selection of promoters and other regulatory elements, such as enhancer elements. The vectors of the present invention include sequences suitable for introduction into cells. For example, the vector may be an expression vector in which the coding sequence of the polypeptide is under the control of its own cis-acting regulatory element, the vector being designed to facilitate gene integration or gene replacement in a host cell, etc.
As understood by those of ordinary skill in the art, 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 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 invention also provides a binding molecule that can be used as an immunotherapeutic or diagnostic agent. The binding molecule may bind to the peptide alone or to a complex formed by the peptide and an MHC molecule. In the latter case, the binding molecule may be partially bound to an MHC molecule, while it also binds to the peptide of the invention. The binding moieties of the invention may be isolated and/or soluble and/or non-naturally occurring, i.e., without equivalents in nature, and/or pure, and/or synthetic.
In a preferred embodiment of the invention, the binding molecule is a T Cell Receptor (TCR). TCRs can be described using the international immunogenetic information system (IMGT). The native αβ heterodimeric TCR has an α chain and a β chain. In a broad sense, each chain comprises a variable region, a linking region and a constant region, and the β chain also typically contains a short polytropic region between the variable region and the linking region, but this polytropic region is often considered part of the linking region.
The TCRs of the present invention may be in any form known in the art. For example, the TCR may be heterodimeric, or exist in a single chain form. The TCR may be in a soluble form (i.e. without transmembrane or cytoplasmic domains), in particular the TCR may comprise all or part of the TCR extracellular domain. The TCR may also be a full long chain comprising its transmembrane region. The TCR may be provided to a cell surface, such as a T cell.
Soluble TCRs may be obtained in combination with the prior art in the art, for example, by introducing artificial disulfide bonds between the α and β chain constant domains of an αβ TCR, or by introducing artificial disulfide bonds between the α chain variable region and the β chain constant region of an αβ TCR.
The TCRs of the present invention may be used to deliver a cytotoxic or immunostimulatory agent to a target cell, or be transformed into T cells, enabling T cells expressing the TCR to destroy tumor cells for administration to a patient during a treatment process known as adoptive immunotherapy. In addition, the TCR of the invention may also comprise a mutation, preferably a mutated TCR with increased affinity for the pMHC complex of the invention. The TCRs of the present invention may be used alone or may be covalently or otherwise bound to the conjugate, preferably covalently. The conjugate includes a detectable label (for diagnostic purposes, wherein 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 a combination or coupling of any of the above. The TCRs of the invention may also be conjugated, preferably covalently, with an anti-CD 3 antibody to redirect T cells, thereby killing target cells.
In another preferred embodiment, the binding molecule of the invention is an antibody. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain specific binding sites, that can be all natural, or partially or fully synthetic. The term "antibody" includes antibody fragments, derivatives, functional equivalents, and homologous, humanized antibodies, which comprise immunoglobulin binding regions, which are or are homologous to antibody binding regions. It may be entirely natural, or partially or entirely synthetic. The humanized antibody may be a modified antibody that contains the variable region of a non-human antibody (e.g., mouse) as well as the constant region of a human antibody.
Examples of antibodies may be isotype immunoglobulins (e.g., igG, igE, igM, igD and IgA) and subclasses of their isotypes; fragments include antigen binding regions such as Fab, scFv, fv, dAb, fd; a diabody. The antibody may be a polyclonal or monoclonal antibody, preferably a monoclonal antibody.
Methods for preparing such TCRs and antibodies are known to those skilled in the art and include, but are not limited to, expression from e.coli cells or insect cells and purification.
In a further aspect, the invention further provides the use of the peptides, pMHC complexes, nucleic acid molecules, vectors, cells and binding molecules of the invention in the pharmaceutical field. The peptide, pMHC complex, nucleic acid, vector, cell or binding molecule may be used for the treatment or prevention of malignant tumors, preferably acute myeloid leukemia.
The invention also provides a pharmaceutical composition comprising an antigenic peptide of the invention, a pMHC complex, a nucleic acid molecule of the invention, a cell of the invention or a binding molecule of the invention, and a pharmaceutically acceptable carrier. The pharmaceutical composition may be in any suitable form, (depending on the method of administration required by the patient). It may be provided in unit dosage form, typically in a sealed container, and may be provided as part of a kit. Such kits typically (but not necessarily) comprise instructions for use. Which may comprise a plurality of said unit dosage forms.
The pharmaceutical composition is suitable for any suitable route of administration, such as injection (including subcutaneous, intramuscular, intraperitoneal or intravenous), inhalation or oral, or nasal, or anal. The compositions may be prepared by any method known in the pharmaceutical arts, for example, by mixing the active ingredient with a carrier or excipient under sterile conditions.
The dosage of the formulation of the present invention to be administered may vary within a wide range depending on the disease or disorder to be treated (e.g., cancer, viral infection, or autoimmune disease), the age and condition of the individual patient, etc. The appropriate dosage to be administered will be ultimately determined by the physician.
According to the state of the art, a peptide presented to the cell surface together with an MHC molecule, pMHC complex or a cell presenting pMHC complex can activate T cells or B cells to function.
Thus, the peptides, pMHC complexes or cells presenting pMHC complexes of the invention may be provided in the form of a vaccine composition. The vaccine composition may be used to treat or prevent cancer. All such compositions are included in the present invention. It will be appreciated that the vaccine may take a variety of forms (Schlom J.J Natl Cancer Inst.2012 104 (8): 599-613). For example, the peptides of the invention can be used directly in immunization of patients (Salteller ML. Cancer Res.1996.56 (20): 4749-57and 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. The vaccine composition may be added with an adjuvant to enhance the immune response. Alternatively, the vaccine composition may be in the form of antigen presenting cells presenting the peptide and MHC complex of the invention. Preferably, the antigen presenting cells are immune cells, more preferably dendritic cells. The peptides may also be pulsed onto the surface of the cells (Thurer BI.et al, J. Exp. Med.1999. 190:1669), or the nucleic acids encoding the peptides of the invention may be introduced into dendritic cells, for example, using electroporation (Van Tendelloo, VF. Et al, blood 2001.98:49).
The main advantages of the invention include:
a) The invention aims to overcome the defects of the related technology to a certain extent and provides a novel antigen peptide and application thereof in tumor immunotherapy.
b) The antigen peptide has the function of eliminating tumor cells.
c) The antigen peptide can widen the treatment range of FLT3 mutant tumor patients, and provides a new choice for the immunotherapy of patients.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Example 1 novel antigenic peptides and HLA-A * 02:01 molecular affinity prediction
Predicting neoantigenic peptides and HLA-A using on-line biological software NetMHCpan 4.1 * Affinity of 02:01 molecules,% Rank_EL < 0.500 is considered to have strong binding, 0.500 <% Rank_EL < 2.00 is considered to have medium binding, and% Rank_EL > 2.000 is considered to be unbound.
It was found that the novel antigenic peptides (SEQ ID No.1 to SEQ ID No. 5) and HLA-A were found to be * The 02:01 molecule has moderate or strong binding force, and the wild type polypeptide (SEQ ID No. 7) and HLA-A * The 02:01 molecules were unbound (Table 2).
TABLE 2 targeting of FLT3-D835 mutant neoantigenic peptides to HLA-A * 02:01 affinity prediction results
Example 2 neoantigenic peptides with HLA-A * 02:01 molecular affinity validation
Taking logarithmic phase T2 cells, adjusting cell concentration to 1×10 in serum-free and antibiotic-free IMDM culture medium 6 Per mL, neoantigenic peptide (10. Mu.g/mL) and. Beta.2 microglobulin (3. Mu.g/mL) were added, respectively, and co-cultured at 37℃for 4h. And after the culture is finished, taking out the cells, washing with PBS, adding the FITC-labeled anti-HLA-A2 monoclonal antibody, incubating for 30min at room temperature, and detecting by a flow cytometer. The final result is measured by fluorescence coefficient (FI): (sample average fluorescence intensity-background average fluorescence intensity)/background average fluorescence intensity. FI (FI)>1.5 is regarded as a polypeptide having a high affinity for HLA-A2 molecules; 1.0<FI<1.5 is medium affinity, FI<1.0 is low affinity.
The results showed that the antigenic peptides (SEQ ID No.1 to SEQ ID No. 5) and HLA-A * The 02:01 molecule has medium or high affinity, significantly higher than the wild-type polypeptide (SEQ ID No. 7) and HLA-A * 02:01 (10 fold or more difference).
Wild-type polypeptide (SEQ ID No. 7) and HLA-A * The 02:01 molecules have no affinity or are very low (Table 3).
TABLE 3 targeting of FLT3-D835 mutant neoantigenic peptides to HLA-A * 02:01 molecular affinity
EXAMPLE 3 neoantigenic peptide/HLA-A * 02:01 molecular Complex stability validation
Taking logarithmic phase T2 cells, and adjusting the concentration of T2 cells to 1×10 in serum-free and antibiotic-free IMDM culture medium (containing 100ng/mL human β2m) 6 /mL, respectivelyIncubated with 100. Mu.g/mL of the neoantigenic peptide overnight at 37 ℃. The following day, cells were collected and incubated with serum-free IMDM medium containing 10. Mu.g/mL Brefeldin A for 1h; serum-free IMDM medium containing 0.5. Mu.g/mL Brefeldin A was added, incubated at 37℃and cells were collected at time points of 0, 2, 4, 6 and 8h, respectively, 100. Mu.L PBS was used to resuspend the cells, FITC-labeled anti-HLA-A2 monoclonal antibody was added, incubated at room temperature for 30min, and the average fluorescence intensity of T2 cells at each time point was calculated for detection by flow cytometry.
Results
TABLE 4 stability of neoantigenic peptide/HLA-A 02:01 molecular complexes targeting FLT3-D835 mutations (%)
The experimental results are shown in FIG. 1 and Table 4, and the detection shows that the antigen peptide and HLA-A of the invention * The complex formed by the 02:01 molecules is stable, wherein the antigen peptide V and HLA-A * The 02:01 molecule forms the most stable complex.
Example 4 antigenic peptides to induce specific T lymphocytes in AML patients
Peripheral Blood Mononuclear Cells (PBMCs) were isolated and purified by Ficoll density gradient centrifugation from venous blood of AML patients with the same HLA class and the same FLT3-D835 mutation. Dynabeads magnetic beads to select CD8 + Cells, in CD8 - The cells act as antigen presenting cells.
Serum-free RPMI-1640 medium to re-suspend CD8 - Cells were resuspended in PBS after mitomycin (30. Mu.g/mL) and inactivated at 37℃for 30min, serum-free RPMI-1640 medium, antigen peptide V (20. Mu.g/mL) was added, and incubated at 37℃for 2-4h.
Collection of antigen pulse stimulated CD8 - Cells resuspended in RPMI-1640 medium containing 10% FBS (50U/mL containing IL-2, 5ng/mL IL-7, 5ng/mL IL-15), and CD8 + Cells were incubated for 10-20 days with half-cell changes every 2-3 days. Collecting cells, and labeling with PE-labeled antigen peptide-HLA-A * 02:01-Tetramer antibody staining, flow cytometry detection.
Results
The experimental results are shown in fig. 2, and it is found that Tetramer positive cells are increased after the antigen peptide is added for stimulation, and the antigen peptide V can induce specific T lymphocytes (CTL cells) from peripheral blood of AML patients.
EXAMPLE 5 neoantigenic peptide activates Zhou Xiete-specific T lymphocytes in AML patients
The PBMCs are separated and purified by adopting a Ficoll density gradient centrifugation method from venous blood of AML patients with the same HLA type and the same FLT3-D835 mutation, laid on a 96-well plate, added with 10 mug/mL of antigen peptide respectively, and amplified and cultured for 10-20D. After the completion of the culture, the cells were removed, and the cells were resuspended to a density of 1X 10 in RPMI-1640 medium containing 10% FBS 6 100. Mu.L/well of ELISPOT assay plate pre-coated with Human IFN-. Gamma.antibody was added to the corresponding wells, antigen peptide V (final concentration 10. Mu.g/mL) was added to the negative control wells without antigen peptide, PHA (final concentration 4. Mu.g/mL) was added to the positive control wells, and incubated at 37℃for 18-24h. The spot plate is taken out, washed according to instructions, incubated with antibodies, developed, dried and read.
Results
The experimental result is shown in figure 3, and after the antigen peptide is detected, the capability of secreting IFN-gamma of PBMCs of AML patients is increased, and the antigen peptide has good immunogenicity and can activate specific immune response.
EXAMPLE 6 New antigen induces specific T lymphocytes in healthy volunteers
PBMCs were isolated and purified by Ficoll density gradient centrifugation from venous blood of healthy volunteers of the same HLA class. Dynabeads magnetic beads are respectively used for separating CD8 + Cells and CD14 + And (3) cells. RPMI-1640 medium (containing IL-4 1000U/mL, GM-CSF 1000U/mL) with 10% FBS resuspended CD14 + Cells were incubated in incubator for 5-7d to induce Dendritic Cells (DCs), and TNF- α (10 ng/mL) was added to promote maturation.
Recovering mature DCs, resuspending cells in serum-free RPMI-1640 medium, adding antigen peptide V (20. Mu.g/mL)Incubation is carried out at 37℃for 2-4h. Antigen peptide pulsed DCs were collected, resuspended in RPMI-1640 medium containing 10% FBS (containing IL-2 50U/mL, IL-7 ng/mL, IL-15 ng/mL), and CD8 + Cells were incubated for 10-20d with half-changes of fluid every 2-3 d. Collecting cells, and labeling with PE-labeled antigen peptide-HLA-A * 02:01-Tetramer antibody staining, flow cytometry detection.
Results
The experimental results are shown in fig. 4, and the antigen peptide can be detected to induce CTLs from peripheral blood of healthy volunteers.
Example 7 activation of T lymphocytes of the Zhou Xiete specificity in healthy volunteers by the neoantigen peptide
Healthy volunteers of the same HLA class were taken for venous blood, and antigen peptide-specific CTLs were induced as in example 6. After the completion of the culture, the cells were removed, and the cells were resuspended to a density of 1X 10 in RPMI-1640 medium containing 10% FBS 6 100. Mu.L/well of ELISPOT assay plate pre-coated with Human IFN-. Gamma.antibody was added per mL. DCs loaded with the neoantigenic peptides were prepared as antigen presenting cells as in example 6 and individually added to corresponding ELIPSOT well plates. DCs not loaded with neoantigenic peptide were added to the negative control wells, PHA (final concentration 4. Mu.g/mL) was added to the positive control wells, and incubated at 37℃for 18-24h. The spot plate is taken out, washed according to instructions, incubated with antibodies, developed, dried and read.
Results
The experimental result is shown in figure 5, and the detection shows that the CTLs of healthy volunteers have high IFN-gamma secretion capacity, and the new antigen peptide has good immunogenicity and can activate specific immune response.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Claims (10)
1. An antigenic peptide for eliciting an immune response targeting FLT3-D835 mutation, characterized in that said antigenic peptide is capable of forming a complex with an MHC molecule and is selected from the group consisting of:
(i) A polypeptide shown in SEQ ID NO. 6:
X 1 IMSDSNYV
wherein X is 1 V, H, I or F;
(ii) Dividing the amino acid sequence of the polypeptide of (i) by X 1 A derivative polypeptide formed by substitution of other amino acids by 1, 2 or 3 amino acids, and/or insertion of 1, 2 or 3 amino acids, and/or deletion of 1 or 2 amino acids, and retaining X 1 。
2. The antigenic peptide of claim 1 which is 1 of the polypeptides of the amino acid sequence shown in any one of SEQ ID NOs 1 to 4, or a combination of 2, 3 or 4 polypeptides.
3. A pMHC complex, characterized in that it comprises the antigenic peptide of claim 1.
4. A nucleic acid molecule comprising a nucleic acid sequence encoding the antigenic peptide of claim 1 or a complement thereof.
5. A vector comprising the nucleic acid molecule of claim 4.
6. A host cell comprising the vector of claim 5.
7. A method for preparing specific T lymphocytes in vitro comprising the steps of:
a) Providing a PBMC (micro-electro mechanical systems),
b) Contacting and culturing said PBMCs with the antigenic peptide of claim 1 in the presence of the antigenic peptide, thereby obtaining specific T lymphocytes activated by the antigenic peptide.
8. A pharmaceutical composition comprising (ii) a pharmaceutically acceptable carrier and (ii) the antigenic peptide of claim 1, the pMHC complex of claim 3, the nucleic acid molecule of claim 4, or specific T lymphocytes activated by the antigenic peptide of claim 1.
9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is a vaccine composition.
10. Use of an antigenic peptide according to claim 1, a pMHC complex according to claim 3, a nucleic acid molecule according to claim 4, a specific T lymphocyte activated by an antigenic peptide according to claim 1 or a pharmaceutical composition according to claim 8 for the preparation of a medicament for the prevention or treatment of a malignancy.
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