T cell receptor of acute myeloid leukemia epitope peptide sh2b3-p242s and application thereof

Abstract

The application discloses a T cell receptor of an acute myeloid leukemia epitope peptide SH2B3-P242S and application thereof. The T cell receptor disclosed by the application contains an alpha chain and a beta chain, and the variable region sequences are respectively 20-112 of SEQ ID No. 2 and 20-112 of SEQ ID No. 4. Experiments prove that the T cell receptor of the application can specifically recognize and combine with an SH2B3-P242S epitope peptide / HLA-A11 complex; meanwhile, it is verified in vitro that a TCR transgenic positive human CD8 cell (TCR-T) has the ability of killing human target cells in a SH2B3-P242S epitope peptide dependent manner. The T cell receptor of the application of the HLA-A11 restricted SH2B3-P242S epitope peptide has important application value.

Description

Technical Field

[0001] This invention relates to the biomedical field, specifically to a T-cell receptor for the acute myeloid leukemia epitope peptide SH2B3-P242S and its applications. Background Technology

[0002] Leukemia is a blood disorder caused by the malignant clonal proliferation of hematopoietic stem cells. Clinically, it manifests as an abnormally high number of white blood cells in the blood and bone marrow, severely impairing hematopoietic function and affecting various tissues and organs throughout the body, thus seriously endangering health; hence, it is also known as "blood cancer." Acute leukemia (AL) has the highest incidence among all types of leukemia. Its pathogenic cells are generally hematopoietic cells in the early differentiation stage, and its onset is rapid, making clinical treatment more difficult. Acute leukemia is further divided into acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) depending on the affected cell type.

[0003] AML is a highly heterogeneous clonal disease, and its pathogenesis is closely related to gene mutations in hematopoietic progenitor cells. Studies show that common mutated genes in AML patients mainly involve functional categories such as signal activation, myeloid transcription factors, DNA methylation, chromatin splicing, and nucleolar phosphoprotein localization. Mutations in these genes can lead to dysregulation of normal cellular processes such as transcription, protein kinases, and epigenetic modifications, resulting in abnormal cell proliferation and hematopoietic function. Clinical studies have shown that the occurrence and relapse of AML are often accompanied by a series of gene mutations. Some hotspot mutations occur more frequently, such as NPM1 (25%-35%), FLT3 (20%-27%), DNMT3A (18%-22%), TET2 (7%-22%), and TP53 (8%-15%). The significance of gene mutations in AML is crucial for disease progression, the development of effective therapies, and post-treatment prognosis. In addition, some hotspot mutations may indicate the existence of new targets, which may provide new insights for clinical immunotherapy of AML.

[0004] The SH2B3 (SH2B adapter protein 3) gene encodes a lymphocyte regulatory protein, a growth factor that regulates hematopoietic and vascular cells. Studies have shown that SH2B3 has a negative regulatory function on multiple signaling pathways, and alterations in the SH2B3 gene are closely associated with the development of leukemia, hypertension, diabetes, and kidney disease. In particular, the P242S amino acid point mutation at the SH2B3 Exon2 has a high detection rate in some diseases.

[0005] Antigens generated by gene mutations cannot be expressed on the surface of ALL cells and targeted by CAR-T, but they can be processed by MHC molecules and presented on the cell surface to be targeted by CD8. + T-cell recognition allows specific gene mutations to serve as specific targets for AML TCR-T therapy. Therefore, using TCR-T, rather than CAR-T, to target gene mutation antigens for AML immunotherapy may be a highly effective new approach.

[0006] Human leukocyte antigen (HLA) is the human MHC and an important human genetic marker. The HLA gene complex is the most complex known human genetic complex, exhibiting high polymorphism and playing a crucial role in immune regulation. Currently, there are over 20,000 known HLA alleles, including HLA class I molecules (HLA-A / B / C) and HLA class II molecules (HLA-DR / DQ / DP), which are expressed in a mixed and specific manner on the surface of human cells, resulting in rich HLA polymorphism among individuals. Studies show that the HLA-A3 superfamily (A11, A33, A31, A68) accounts for the largest proportion of all HLA subtypes in the Chinese population, approximately 52.7%, with the HLA-A11 subtype having the highest frequency. Existing research on therapeutic T cells is mainly limited to the HLA-A2 population, while attention to other HLA populations is significantly insufficient.

[0007] Therefore, TCR-T studies targeting HLA-A11-restricted SH2B3-P242S specificity are of great significance for immunotherapy in the Chinese population.

[0008] In conclusion, immunotherapy for acute myeloid leukemia (AML), especially TCR-T therapy, has significant advantages in improving the current clinical treatment status of AML, increasing clinical remission rates, and prolonging patient survival, making it a promising future treatment direction. Considering the MHC restriction of TCR-T and the HLA preference in the Chinese population, developing HLA-A11-restricted TCR-T therapy specifically targeting ALL could provide a new treatment option for HLA-A11-restricted AML, which is predominantly present in the Chinese population. Summary of the Invention

[0009] The technical problem to be solved by this invention is how to treat acute myeloid leukemia.

[0010] To address the aforementioned technical problems, this invention first provides a T-cell receptor that recognizes the HLA-A11-restricted SH2B3-P242S epitope peptide, comprising an α chain and a β chain, wherein the α chain contains complementarity-determining regions CDR1α, CDR2α, and CDR3α, and CDR1α is either a1) or a2).

[0011] a1) The polypeptide represented by positions 46-51 of SEQ ID No. 2;

[0012] a2) The T cell receptor α chain complementarity-determining region that has more than 75% identity with a1) obtained by substituting and / or deleting and / or adding one or more amino acid residues (such as 3, 2 or 1 amino acid residues).

[0013] CDR2α is either a3) or a4):

[0014] a3) The polypeptide represented by positions 69-75 of SEQ ID No. 2;

[0015] a4) The T cell receptor α chain complementarity-determining region that has more than 75% identity with a3) obtained by substituting and / or deleting and / or adding one or more amino acid residues (such as 3, 2 or 1 amino acid residues).

[0016] CDR3α is either a5) or a6):

[0017] a5) The polypeptide represented by positions 110-122 of SEQ ID No. 2;

[0018] a6) The T cell receptor α chain complementarity-determining region that has more than 75% identity with a5) obtained by substituting and / or deleting and / or adding one or more amino acid residues (such as 3, 2 or 1 amino acid residues).

[0019] The β chain contains complementarity-determining regions CDR1β, CDR2β, and CDR3β, where CDR1β is either b1) or b2):

[0020] b1) The polypeptide represented by positions 45-49 of SEQ ID No. 4;

[0021] b2) The T cell receptor α chain complementarity-determining region that has more than 75% identity with b1) obtained by substituting and / or deleting and / or adding one or more amino acid residues (such as 3, 2 or 1 amino acid residues).

[0022] CDR2β is either b3) or b4):

[0023] b3) The polypeptide shown at positions 67-72 of SEQ ID No. 4;

[0024] b4) The T cell receptor α chain complementarity-determining region that has more than 75% identity with b3) obtained by substituting and / or deleting and / or adding one or more amino acid residues (such as 3, 2 or 1 amino acid residues).

[0025] CDR3β is either b5) or b6):

[0026] b5) The polypeptide represented by positions 110-122 of SEQ ID No. 4;

[0027] b6) The T-cell receptor α-chain complementarity-determining region with more than 75% identity to b5) obtained by substituting and / or deleting and / or adding one or more amino acid residues (e.g., 3, 2 or 1 amino acid residues).

[0028] In the aforementioned T cell receptor, the variable region of the α chain can be a7) or a8):

[0029] a7) The polypeptide represented by positions 20-112 of SEQ ID No. 2;

[0030] a8) The T cell receptor α chain variable region having more than 75% identity with a7) obtained by substituting and / or deleting and / or adding one or more amino acid residues (such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues).

[0031] The variable region of the β chain can be b7) or b8):

[0032] b7) As shown in bits 20-112 of SEQ ID No. 4;

[0033] b8) The T cell receptor α chain variable region having more than 75% identity with b7) is obtained by substituting and / or deleting and / or adding one or more amino acid residues (such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues).

[0034] In the aforementioned T cell receptors, the α chain and the β chain may contain constant regions of mouse, human, or humanized T cell receptors.

[0035] Specifically, the α chain can be a9) or a10):

[0036] a9) The polypeptide shown in SEQ ID No. 2;

[0037] a10) is a T-cell receptor α chain with more than 75% identity to a9) obtained by substituting and / or deleting and / or adding one or more amino acid residues (such as 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues).

[0038] The β chain is either b9) or b10):

[0039] b9) The polypeptide shown in SEQ ID No. 4;

[0040] b10) is a T-cell receptor β chain with more than 75% identity to b9) obtained by substituting and / or deleting and / or adding one or more amino acid residues (such as 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues).

[0041] Among the aforementioned T cell receptors, 75% or more identity can be 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity.

[0042] The α and β chains of the aforementioned T cell receptors can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0043] The T-cell receptor may be composed of the α-chain and the β-chain, or may be formed by linking the α-chain and the β-chain. In one embodiment of the present invention, the T-cell receptor is formed by linking the α-chain and the β-chain via a linker peptide (such as a P2A self-cleaving polypeptide).

[0044] The present invention also provides the T cell receptor-related biomaterial, wherein the biomaterial is any one of the following C1) to C7):

[0045] C1) is the nucleic acid molecule that encodes the T cell receptor;

[0046] C2) An expression cassette containing the nucleic acid molecule described in C1);

[0047] C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0048] C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3);

[0049] C5) A transgenic animal cell line containing the nucleic acid molecule described in C1), or a transgenic animal cell line containing the expression cassette described in C2);

[0050] C6) Transgenic animal tissue containing the nucleic acid molecule described in C1), or transgenic animal tissue containing the expression cassette described in C2);

[0051] C7) A transgenic animal organ containing the nucleic acid molecule described in C1) or a transgenic animal organ containing the expression cassette described in C2).

[0052] In the above-mentioned biological materials, the nucleic acid molecule described in C1) may be a nucleic acid molecule encoding the α chain and the β chain.

[0053] Specifically, in the α chain, the nucleic acid molecule encoding CDR1α can be the DNA molecule shown at positions 136-153 of SEQ ID No. 1 or a DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with it and encodes the same amino acid residue sequence; the nucleic acid molecule encoding CDR2α is the DNA molecule shown at positions 205-225 of SEQ ID No. 1 or a DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with it and encodes the same amino acid residue sequence; and the nucleic acid molecule encoding CDR3α is the DNA molecule shown at positions 328-366 of SEQ ID No. 1 or a DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with it and encodes the same amino acid residue sequence.

[0054] Furthermore, the nucleic acid molecule encoding the variable region of the α chain may be the DNA molecule shown at positions 58-336 of SEQ ID No. 1 or a DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with it and encodes the same amino acid residue sequence.

[0055] Furthermore, the nucleic acid molecule encoding the α chain may be the DNA molecule shown in SEQ ID No. 1 or a DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with it and encodes the same amino acid residue sequence.

[0056] In the β chain, the nucleic acid molecule encoding CDR1β can be the DNA molecule shown at positions 133-147 of SEQ ID No. 3 or a DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with it and encodes the same amino acid residue sequence; the nucleic acid molecule encoding CDR2β is the DNA molecule shown at positions 199-216 of SEQ ID No. 3 or a DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with it and encodes the same amino acid residue sequence; and the nucleic acid molecule encoding CDR3β is the DNA molecule shown at positions 328-366 of SEQ ID No. 3 or a DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with it and encodes the same amino acid residue sequence.

[0057] Furthermore, the nucleic acid molecule encoding the variable region of the β chain may be the DNA molecule shown at positions 58-336 of SEQ ID No. 3 or a DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with it and encodes the same amino acid residue sequence.

[0058] Furthermore, the nucleic acid molecule encoding the β chain may be the DNA molecule shown in SEQ ID No. 3 or a DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with it and encodes the same amino acid residue sequence.

[0059] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequences encoding the α-chain, the β-chain, or functional fragments thereof of the present invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0060] In the aforementioned biological materials, the expression cassette (T-cell receptor gene expression cassette) containing a nucleic acid molecule encoding the T-cell receptor described in B2) refers to DNA capable of expressing the T-cell receptor in host cells. This DNA may include not only a promoter to initiate T-cell receptor gene transcription but also a terminator to terminate T-cell receptor gene transcription. Furthermore, the expression cassette may also include an enhancer sequence.

[0061] Recombinant vectors containing the T cell receptor gene expression cassette can be constructed using existing expression vectors.

[0062] In the above-mentioned biological materials, the vector can be a plasmid, granule, bacteriophage, or viral vector, such as pCDH-MSCV-MCS-2A-GFP (System Biosciences, number: CD731B-1).

[0063] The recombinant vector is a recombinant plasmid obtained by inserting a nucleic acid molecule encoding the α chain of the T cell receptor and a nucleic acid molecule encoding the β chain of the T cell receptor between multiple cloning sites (such as EcoRI and BamHI) of the plasmid pCDH-MSCV-MCS-2A-GFP (System Biosciences, number: CD731B-1).

[0064] In one embodiment of the present invention, the recombinant lentiviral packaging vector may be the recombinant plasmid pCDH-MSCV-TCR-GFP. The recombinant plasmid pCDH-MSCV-TCR-GFP is obtained by replacing the small DNA fragment between the restriction endonucleases EcoRI and BamHI in the lentiviral packaging vector pCDH-MSCV-MCS-2A-GFP with a TCRDNA fragment whose nucleotide sequence is shown in SEQ ID No. 5, while keeping other sequences unchanged. In SEQ ID No. 5, positions 1 to 807 are the complete coding gene for the α chain, positions 808 to 873 are the coding gene for the P2A self-cleaving polypeptide, and positions 874 to 1788 are the complete coding gene for the β chain.

[0065] In the above-mentioned biological materials, the microorganisms may be yeast, bacteria, algae or fungi.

[0066] Among the aforementioned biological materials, the transgenic animal cell lines, transgenic animal tissues, and transgenic animal organs do not include reproductive materials.

[0067] The cell line may be a T cell line or a Jurkat cell line. Specifically, the T cells may be human T cells or mouse T cells.

[0068] The present invention also provides a pharmaceutical composition comprising the T cell receptor or the biological material.

[0069] The pharmaceutical composition may also contain the SH2B3-P242S polypeptide.

[0070] The active ingredient of the pharmaceutical composition may be the T cell receptor or the biological material, or a composition of the T cell receptor or the biological material and the SH2B3-P242S polypeptide.

[0071] The SH2B3-P242S polypeptide is as shown in SEQ ID No. 7.

[0072] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier.

[0073] The following applications of the T-cell receptor, the biological material, or the pharmaceutical composition also fall within the scope of protection of this invention:

[0074] X1) Treatment and / or prevention of blood disorders;

[0075] X2) Prepare products for the treatment and / or prevention of blood disorders;

[0076] X3) Treatment and / or prevention of acute leukemia;

[0077] X4) Prepare products for the treatment and / or prevention of acute leukemia;

[0078] X5) Treatment and / or prevention of acute myeloid leukemia;

[0079] X6) Prepare products for the treatment and / or prevention of acute myeloid leukemia;

[0080] X7) Treatment and / or prevention of diseases caused by the P242S mutation of SH2B3;

[0081] X8) Prepare products for the treatment and / or prevention of diseases caused by the P242S mutation of SH2B3.

[0082] X9) Treatment and / or prevention of SH2B3-P242S-positive acute myeloid leukemia;

[0083] X10) Prepare products for the treatment and / or prevention of SH2B3-P242S-positive acute myeloid leukemia;

[0084] X11) Kill target cells expressing the SH2B3-P242S epitope peptide in vivo or in vitro;

[0085] X12) to prepare products for killing target cells in vivo or in vitro;

[0086] X13) Clear SH2B3-P242S positive cells;

[0087] X14) Prepare a product for eliminating SH2B3-P242S positive cells;

[0088] X15) to prepare cytotoxic cytokines IFN-γ and / or TNF-α.

[0089] The product may be a drug.

[0090] The application of SH2B3-P242S polypeptide in the preparation of T-cell receptors for acute myeloid leukemia epitope peptides is also within the scope of protection of this invention.

[0091] The T cell receptor in this invention may be an isolated or purified T cell receptor (TCR).

[0092] Through extensive experiments, the inventors of this invention isolated and identified a pair of SH2B3-P242S-specific TCR sequences, successfully constructed the corresponding TCR-T cells, and confirmed that this pair of TCR sequences specifically recognizes and binds to the SH2B3-P242S epitope peptide / HLA-A11 complex. Simultaneously, in vitro testing verified that TCR-transgenic positive human CD8 cells (TCR-T) possess the ability to kill human target cells in a SH2B3-P242S epitope peptide-dependent manner. The HLA-A11-restricted SH2B3-P242S epitope peptide T cell receptor provided by this invention has significant application value.

[0093] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0094] Figure 1 CTL detection was performed on mice immunized with SH2B3-P242S peptide. PMA stimulation served as a control.

[0095] Figure 2 CD8 specific sorting for SH2B3-P242S.

[0096] Figure 3 The staining results of Jurkat cells expressing SH2B3-P242S-specific TCR.

[0097] Figure 4 TCR-T cells were prepared by electroporation of mRNA into human primary T cells.

[0098] Figure 5 Results show that SH2B3-P242S-specific Human TCR-T cells have the ability to kill target cells in vitro. ** indicates significance p < 0.01, **** indicates significance p < 0.0001.

[0099] Figure 6 Results of cytokine release assay for Human TCR-T cells killing target cells in vitro. Detailed Implementation

[0100] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are all commercially available. All quantitative experiments in the following examples were performed in at least three replicates, and the results were averaged. Unless otherwise specified, in the following examples, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA / RNA.

[0101] The HLA-A11 / hTAP-LMP transgenic mouse is described in the following literature: Man Huang, Wei Zhang, Jie Guo, Xundong Wei, Krung Phiwpan, Jianhua Zhang, Xuyu Zhou. Improved Transgenic Mouse Model for Studying HLA Class I Antigen Presentation. Scientific Report. 2016, doi:10.1038 / srep33612.

[0102] Example 1: Obtaining and applying TCR-T cells

[0103] I. Using HLA-A11 / hTAP-LMP transgenic mice to screen for HLA-A11-restricted SH2B3-P242S epitope-specific TCR sequences

[0104] 1. Immunoassay of SH2B3-P242S epitope peptide

[0105] (1) Mix and emulsify 100 μL of PBS buffer containing 50 μg of peptide SH2B3-P242S with 100 μL of IFA; then inject subcutaneously at multiple sites into HLA-A11 / hTAP-LMP transgenic mice. HLA-A11 / hTAP-LMP transgenic mice will induce a CTL immune response against the SH2B3-P242S epitope.

[0106] (2) On the 7th day after completing step (1), peripheral blood of immunized mice was collected, red blood cells were lysed and stimulated with peptide SH2B3-P242S for 4 hours, followed by intracellular IFNg staining.

[0107] Meanwhile, peripheral blood was collected from unimmunized HLA-A11 / hTAP-LMP transgenic mice. After lysing the red blood cells, the cells were stimulated with the peptide SH2B3-P242S for 4 hours, followed by intracellular IFNg staining as a control.

[0108] Test results are shown Figure 1 (Control mice were unimmunized HLA-A11 / hTAP-LMP transgenic mice). The results showed that immunization with SH2B3-P242S peptide stimulated specific CD8+ in mice. + T cells produce a CTL response and release IFN-γ.

[0109] 2. Take the IFN-γ-immunized mice obtained in step 1 and administer Tetramer SH2B3-P242S-positive CD8 inhibitors. + T cells are sorted.

[0110] Tetramer SH2B3-P242S positive CD8 + The sorting efficiency and purity results of T cells are shown in the table below. Figure 2 .

[0111] 3. TCR gene fragment amplification and sequencing

[0112] Extraction of Tetramer SH2B3-P242S positive CD8 + RNA from T cells was then reverse transcribed to obtain cDNA from Tetramer SH2B3-P242S-positive CD8+ T cells. Tetramer SH2B3-P242S-positive CD8+ T cells were then used to transcribe cDNA. + Using T cell cDNA as a template, TCRα and TCRβ fragments were amplified using conserved region primers. The amplified TCRα and β fragments were then ligated into a T vector, and the T vector was subsequently sequenced to isolate a pair of TCRs.

[0113] The coding genes for the variable region of the α chain are shown in positions 58-336 of SEQ ID No. 1. In SEQ ID No. 1, positions 136-153, 205-225, and 328-366 are the coding genes for three CDRs, respectively.

[0114] The amino acid sequence of the α-chain variable region is shown at positions 20-112 of SEQ ID No. 2, where positions 46-51, 69-75, and 110-122 are three complementarity-determining regions.

[0115] The coding genes for the β chain variable region are shown in positions 58-336 of SEQ ID No. 3. In SEQ ID No. 3, positions 133-147, 199-216, and 328-366 are the coding genes for three CDRs, respectively.

[0116] The amino acid sequence of the β-chain variable region is shown at positions 20-112 of SEQ ID No. 4, where positions 45-49, 67-72, and 110-122 of SEQ ID No. 4 are three complementarity-determining regions.

[0117] This TCR receptor, consisting of the α-chain sequence shown in SEQ ID No. 2 and the β-chain sequence shown in SEQ ID No. 4, may have a high affinity for the HLA-A11-restricted CTL epitope SH2B3-P242S and originates from the same T cell clone.

[0118] II. Obtaining and Identifying Jurkat Cells Expressing TCR

[0119] 1. Based on the sequences of the α-chain variable region and β-chain variable region obtained in step one, and referring to the α-chain and β-chain constant region sequences of the mouse genome on NCBI, the complete coding genes of the α-chain and β-chain of the HLA-A11-restricted SH2B3-P242S-specific TCR receptor were obtained and artificially synthesized.

[0120] The complete coding gene for the α chain is shown in SEQ ID No. 1, which encodes the α chain shown in SEQ ID No. 2.

[0121] The complete coding gene for the β chain is shown in SEQ ID No. 3, which encodes the β chain shown in SEQ ID No. 4.

[0122] 2. Replace the small DNA fragment between the EcoRI and BamHI recognition sequences of the restriction endonuclease in plasmid pCDH-MSCV-MCS-2A-GFP (System Biosciences, ID: CD731B-1) with the TCR DNA fragment whose nucleotide sequence is shown in SEQ ID No. 5. All other sequences remain unchanged to obtain the recombinant plasmid pCDH-MSCV-TCR-GFP. In SEQ ID No. 5, positions 1 to 807 represent the complete coding gene for the α chain, positions 808 to 873 represent the coding gene for the P2A self-cleaving polypeptide, and positions 874 to 1788 represent the complete coding gene for the β chain.

[0123] 3. Culture Jurkat cells to a quantity of 2×10⁻⁶. 7 Collect the cells, wash them twice with antibiotic-free 1640 medium, and then resuspend them in antibiotic-free 1640 medium to a density of 5 × 10⁶ cells / mL after the final wash. 7 / mL, divide the above cells into 400μL aliquots and transfer them to an electroporation cuvette (BIO-RAD, catalog number: 165-2088). Simultaneously add 40μg of recombinant plasmid pCDH-MSCV-TCR-GFP, mix well, and place the cuvette into an electroporator (BIO-RAD, Gene Pulser Xcell). TM Electroporation was performed at 250V and 950μF, followed by a 15-minute incubation period. Cells were then filtered through a sterile 200-mesh nylon membrane and transferred to 6-well plates. After 12 hours, 1640 medium containing antibiotics was added for further culture. Expression of the target gene was detected within 12-48 hours. The recombinant plasmid pCDH-MSCV-TCR-GFP was introduced into Jurkat cells to obtain Jurkat cells expressing TCR.

[0124] 4. Jurkat cells expressing TCR obtained in step 3 were stained using Tetramer (HLA-A11 / SH2B3-P242S). The reagents used were Anti-mouse TCR beta (H57-597, Invitrogen), HLA-A*11:01 Monomer UVX (Biolegend), and APC Streptavidin (eBioscience). The negative control was the untreated cell group. Electroporated cells were incubated with wild-type Tetramer (loaded with unmutated peptide) and SH2B3-P242S Tetramer (loaded with mutant peptide), respectively.

[0125] Staining results are shown Figure 3 The results showed that the TCR had a strong affinity for the HLA-A11-restricted SH2B3-P242S epitope.

[0126] III. Human TCR-T cells have the function of killing target cells in vitro.

[0127] To verify the ability of Human TCR-T cells to specifically kill target cells in vitro, an in vitro killing experiment was conducted. The specific steps are as follows:

[0128] 1. Isolation of human peripheral T lymphocytes

[0129] (1) Draw 5 mL of peripheral venous blood from a person.

[0130] (2) After completing step (1), take a centrifuge tube (50 mL), add 5 mL of peripheral blood and 5 mL of PBS buffer, and mix thoroughly.

[0131] (3) After completing step (2), add 5 mL of human peripheral blood lymphocyte separation solution (Tianjin Haoyang Biotechnology Co., Ltd., product number: LTS1077) to the centrifuge tube. Use a disposable sterile dropper to carefully stack the diluted peripheral blood along the tube wall onto the surface of the separation solution, and be careful to keep the interface clear.

[0132] (4) After completing step (3), place the centrifuge tube in the centrifuge, adjust the speed increase and decrease to the lowest, and centrifuge at 800g for 20 minutes.

[0133] (5) After completing step (4), the tube is divided into four layers from top to bottom: the first layer is plasma and PBS, the second layer is a ring-shaped milky white lymphocyte layer, the third layer is a clear separation solution layer, and the fourth layer is a red blood cell and granulocyte layer. Carefully aspirate the second ring-shaped milky white lymphocyte layer into a sterile centrifuge tube (50 mL), add 40 mL of PBS buffer to the centrifuge tube, mix the cells, centrifuge at 800 g for 5 min, discard the supernatant, then resuspend the cells in 1640 medium, count them for later use, and obtain human peripheral T lymphocytes.

[0134] 3. Activation of human peripheral T lymphocytes

[0135] (1) Take a 24-well plate, add 500 μL of anti-CD3 antibody diluent and 500 μL of anti-CD28 antibody diluent to each well, and coat overnight at 4°C.

[0136] anti-CD3 antibody dilution solution: The anti-CD3 antibody (BioXcell, clone number: OKT3) was diluted with PBS buffer to a concentration of 3 μg / mL.

[0137] anti-CD28 antibody dilution solution: dilute anti-CD28 antibody (BioXcell, clone number: CD28.2) with PBS buffer to a concentration of 1 μg / mL.

[0138] (2) After completing step (1), take the 24-well plate, remove the liquid, and wash it once with PBS buffer.

[0139] (3) After completing step (2), take the 24-well plate, add 500 μL of human peripheral T lymphocyte dilution solution, and incubate at 37°C for 48 h; then centrifuge at 400 g for 5 min, collect the precipitate and resuspend it in 1640 medium to obtain activated human peripheral T lymphocytes.

[0140] Human peripheral T lymphocyte dilution medium: Dilute the human peripheral T lymphocytes obtained in step 2 to 4 × 10⁻⁶ using 1640 medium. 6 The number of cells / mL was obtained.

[0141] 4. Prepare mRNA and electroporate it into human peripheral T lymphocytes

[0142] (1) Insert the TCR DNA fragment shown in SEQ ID No. 5 into the vector as a template for transcription reaction, and then purify it with lithium chloride to obtain the corresponding mRNA. The sequence of the obtained mRNA is the sequence obtained by replacing T with U in SEQ ID No. 5. The mRNA can be obtained by common mRNA transcription methods or by artificial synthesis.

[0143] (2) Culture activated human peripheral T lymphocytes until they are in good condition, and use 1×10⁶ cells per sample. 7 To determine the optimal cell ratio, take an appropriate amount of T cell suspension, centrifuge at 1300 rpm for 5 min, discard the supernatant, and then wash the T cells 1-2 times with phenol red-free Opti-MEM medium preheated to 37℃, centrifuging at 1300 rpm for 5 min each time. Resuspend the T cells in 100 μL of Opti-MEM medium for each sample, then mix 20 μg of mRNA from each sample with the T cell suspension, quickly transfer the mixture to an electroporation cuvette, ensuring no air bubbles are generated, and immediately perform electroporation at 200V 800Us 1000ms for 3 cycles. Incubate the cuvette at 37℃ for 20 min. Wash the cuvette 1-2 times with 1640 medium (rhIL2), transfer the cell suspension to 6-well plates, and culture for 48 h to detect the expression of corresponding genes.

[0144] T cells without mRNA were used as a negative control.

[0145] (3) Detection of TCR expression after electroporation, such as Figure 4 The results showed that the T cells transfected with mRNA successfully expressed the target TCR.

[0146] 5. Human TCR-T cells have the function of killing target cells in vitro.

[0147] The substrate, stop solution, and cell lysis buffer were all CytoTox. Components of the Non-Radioactive CytotoxicityAssay kit (Promega, catalog number: G1780).

[0148] (1) Digest and count 293T cells that are stably transfected with HLA-A11. These cells can stably express HLA-A11 and present the acute myeloid leukemia-associated antigen peptide SH2B3-P242S, and can be used as target cells for killing experiments.

[0149] The 293T cells that were stably converted to HLA-A11 were preserved in the laboratory and described in the literature (The splicing isoform Foxp3Δ2 differentially regulates tTreg and pTreg homeostasis. Cell Reports. 2023; 42(8):112877).

[0150] (2) Add the target cells obtained in step (1) to a 96-well plate, 2 × 10⁻⁶ cells per well. 4 Cells / well; then Human TCR-T cells (i.e., the mRNA-transfected T cells obtained in step 4) were added at an effect-to-target ratio of 10:1, 5:1 or 1:1, and 10 mg / ml of SH2B3-P242S polypeptide (the sequence of which is SEQ ID No.7 in the sequence listing) was added to the experimental group and cultured at 37°C and 5% CO2 for 4 h.

[0151] (3) Take the 96-well plate that has completed step (2), centrifuge at 250g for 4 min, then transfer 50 μL of supernatant to a new 96-well plate, add 50 μL of substrate to each well, and incubate at room temperature in the dark for 30 min; then add 50 μL of stop solution to each well, and read the values ​​using an ELISA reader (wavelength 490 nm), which are the values ​​of the experimental group.

[0152] (4) Detection of baseline values ​​in lethality experiments

[0153] ① Following steps (1)-(3) above, replace step (2) with step (2A), keeping all other steps unchanged. The value read by the microplate reader is the T cell self-release value. Step (2A): Take a 96-well plate, add the same number of HumanTCR-T cells as the experimental group, and incubate at 37℃ and 5% CO2 for 4h.

[0154] ② Following steps (1)-(3) above, replace step (2) with step (2B), keeping all other steps unchanged. The value read by the microplate reader is the target cell self-release value. Step (2B): Take a 96-well plate, add the same number of target cells as the experimental group, and incubate at 37℃ and 5% CO2 for 4 hours.

[0155] ③ Following steps (1)-(3) above, replace step (2) with step (2C), keeping all other steps unchanged. The value read by the microplate reader is the maximum release value of the target cells. Step (2C): Take a 96-well plate, add the same number of target cells as the experimental group and 10 μL of cell lysis buffer, and incubate at 37°C and 5% CO2 for 4 h.

[0156] 6. Calculate lethality

[0157] Killing activity = (experimental group value - T cell auto-release value - target cell auto-release value) / (target cell maximum release value - target cell auto-release value) × 100%.

[0158] Replace the Human TCR-T cells in the above method with Human T cells that have not been transfected with TCR, while keeping all other steps unchanged, as a control.

[0159] The results of in vitro detection of target cell killing by human TCR-T cells are shown in the figure. Figure 5 The results showed that Human TCR-T cells can effectively kill target cells in vitro.

[0160] 7. Detect cytokine release

[0161] Adjust the ratio of effector cells to target cells in step 5 to 1:1, with 2 × 10⁶ target cells. 4 Cells were added to 96-well plates, and Monensin was added to the cells to block the transport of proteins from the Golgi apparatus to the extracellular space. At the same time, 10 mg / ml of SH2B3-P242S peptide was added to the experimental group, while no peptide was added to the control group culture system.

[0162] Cells were cultured at 37°C and 5% CO2 for 4 hours, followed by flow cytometry staining to detect the secretion of cytokines IFN-γ and TNF-α.

[0163] See results Figure 6 Human T cells transduced with TCR showed a significant increase in the secretion of cytotoxic cytokines upon stimulation with specific polypeptide antigens.

[0164] In summary, the inventors of this invention isolated and identified a pair of SH2B3-P242S-specific TCR sequences, successfully constructed the corresponding TCR-T cells, and determined that this pair of TCR sequences specifically recognizes and binds to the SH2B3-P242S epitope peptide / HLA-A11 complex. Simultaneously, TCR-transgenic positive human CD8 cells (TCR-T) possess the ability to kill target cells in a SH2B3-P242S epitope peptide-dependent manner and can release effector cytokines. The HLA-A11-restricted SH2B3-P242S epitope peptide T cell receptor provided by this invention has significant application value.

[0165] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A T-cell receptor that recognizes the SH2B3-P242S epitope peptide, comprising an α chain and a β chain, wherein the α chain contains complementarity-determining regions CDR1α, CDR2α and CDR3α, and CDR1α is the polypeptide represented at positions 46-51 of SEQ ID No. 2; CDR2α is the polypeptide represented by positions 69-75 of SEQ ID No. 2; CDR3α is the polypeptide represented by positions 110-122 of SEQ ID No. 2; The β chain contains complementarity-determining regions CDR1β, CDR2β and CDR3β, where CDR1β is the polypeptide represented by positions 45-49 of SEQ ID No. 4; CDR2β is the polypeptide represented by positions 67-72 of SEQ ID No. 4; CDR3β is the polypeptide represented by positions 110-122 of SEQ ID No.

4.

2. The T cell receptor of claim 1, wherein: The α chain and the β chain contain constant regions of mouse, human, or humanized T cell receptors.

3. The T cell receptor of claim 2, wherein: The α chain is either a9 or a10). a9) The polypeptide shown in SEQ ID No. 2; a10) is a T cell receptor α chain with more than 75% identity to a9) obtained by substituting and / or deleting and / or adding one or more amino acid residues. The β chain is either b9 or b10. b9) The polypeptide shown in SEQ ID No. 4; b10) is a T-cell receptor β chain that has more than 75% identity with b9) obtained by substituting and / or deleting and / or adding one or more amino acid residues.

4. The biological material associated with any of the T-cell receptors described in claims 1-3 is any one of the following C1) to C5): C1) A nucleic acid molecule encoding any one of the T cell receptors described in claims 1-3; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3); C5) A cell, tissue, or organ containing the nucleic acid molecule described in C1), or a cell, tissue, or organ containing the expression cassette described in C2).

5. The biomaterial according to claim 4, characterized in that: C1) The nucleic acid molecule is a nucleic acid molecule encoding any of the α chain and the β chain in claims 1-3.

6. The biomaterial according to claim 4 or 5, characterized in that: In the α chain, the nucleic acid molecule encoding CDR1α is the DNA molecule shown in positions 136-153 of SEQ ID No. 1, the nucleic acid molecule encoding CDR2α is the DNA molecule shown in positions 205-225 of SEQ ID No. 1, and the nucleic acid molecule encoding CDR3α is the DNA molecule shown in positions 328-366 of SEQ ID No. 1; In the β chain, the nucleic acid molecule encoding CDR1β is the DNA molecule shown in positions 133-147 of SEQ ID No. 3, the nucleic acid molecule encoding CDR2β is the DNA molecule shown in positions 199-216 of SEQ ID No. 3, and the nucleic acid molecule encoding CDR3β is the DNA molecule shown in positions 328-366 of SEQ ID No.

3.

7. The biomaterial according to claim 6, characterized in that: The nucleic acid molecule encoding the α chain is the DNA molecule shown in SEQ ID No.

1.

8. The biomaterial according to claim 6, characterized in that: The nucleic acid molecule encoding the β chain is the DNA molecule shown in SEQ ID No.

3.

9. A pharmaceutical composition comprising the T-cell receptor of any one of claims 1-3, or the biological material of any one of claims 4-8.

10. The pharmaceutical composition according to claim 9, characterized in that: The pharmaceutical composition also contains the SH2B3-P242S polypeptide.

11. The following applications of the T-cell receptor according to any one of claims 1-3, or the biological material according to any one of claims 4-8, or the pharmaceutical composition according to claim 9 or 10: X1) Prepare products for the treatment of SH2B3-P242S-positive acute lymphoblastic leukemia; X2) Prepare products for the treatment of SH2B3-P242S-positive acute myeloid leukemia.

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