T cell receptor targeting SLC2A1 mutation, related biological material and preparation method thereof

Abstract

The invention discloses a T cell receptor targeting SLC2A1 mutation as well as a related biological material and a preparation method thereof, and belongs to the field of immunology. The invention aims to solve the technical problem of how to better treat cancers or tumors caused by SLC2A1 gene mutation. Therefore, the invention provides a TCR for targeted recognition of an SLC2A1 mutant peptide fragment. A TCR alpha chain of the TCR comprises a CDR3 sequence of which the amino acid sequence is SEQ ID NO.4; and the TCR beta chain comprises a CDR3 sequence of which the amino acid sequence is SEQ ID NO.7. The TCR sequence targeting the SLC2A1 mutant peptide fragment is obtained through screening, then TCR-T cell drugs or protein drugs and nucleic acid drugs based on the TCR are constructed, target cells expressing the SLC2A1 mutant peptide fragment can be effectively cleared away, the method is beneficial exploration for precise immunotherapy of diseases, and the corresponding TCR sequence is reported for the first time.

Description

Technical Field

[0001] The present invention relates to the field of immunology, and in particular to a T cell receptor targeting SLC2A1 mutation, and related biological materials and preparation methods thereof. Background Art

[0002] The glucose transporter (SLC2A1) on the human cell membrane is an important membrane protein that mediates the transmembrane transport of glucose along the concentration gradient, and plays a vital role in cellular energy supply and maintaining normal life activities. SLC2A1 has been shown to be highly expressed in most human cancers such as brain cancer, breast cancer, head and neck cancer, bladder cancer, kidney cancer, colorectal cancer, lung cancer and ovarian cancer. SLC2A1 (UniProtKB database number: P11166) is encoded by the SLC2A1 gene and is located in the short arm region of chromosome 1. Its protein structure consists of 12 transmembrane helices forming two domains, the N-terminal and C-terminal, and the cavity between the two domains faces the cell. Using the protein crystal structure of SLC2A1, researchers accurately analyzed more than 30 amino acid mutation sites related to the disease and corresponded to the mutation sites of the SLC2A1 gene. Since mutations in the SLC2A1 gene have been found in many tumors, the SLC2A1 peptide expressed on the surface of tumor cells is expected to become a therapeutic target for diseases related to SLC2A1 mutations. Currently, there are techniques such as surgery, radiotherapy and chemotherapy for treating tumors, but all of them will cause damage to the body's normal cells.

[0003] T cell adoptive immunotherapy is to transfer reactive T cells specific to target cell antigens into the patient's body so that they can play a role against target cells. T cell receptor (TCR) is a membrane protein on the surface of T cells, which is the "grip" of T cells to recognize abnormal cells and can recognize antigenic short peptides on the surface of target cells. In the immune system, the binding of TCR specific to antigenic short peptides with short peptide-major histocompatibility complex (pMHC complex) triggers direct physical contact between T cells and antigen presenting cells (APC), and then other cell membrane surface molecules of both T cells and APCs interact, causing a series of subsequent cell signaling and other physiological reactions, so that T cells with different antigen specificities can exert immune effects on their target cells. Therefore, those skilled in the art are committed to isolating TCRs specific to SLC2A1 antigenic short peptides, and transducing the TCRs into T cells to obtain T cells specific to SLC2A1 antigenic short peptides, so that they can play a role in cell immunotherapy. Summary of the invention

[0004] The technical problem to be solved by the present application is: how to construct a recombinant T cell (TCR-T) that can specifically recognize SLC2A1 pathogenic mutations or SLC2A1 pathogenic mutation antigen short peptides, improve the cytotoxicity of tumor cells expressing SLC2A1 pathogenic mutations or SLC2A1 pathogenic mutation antigen short peptides, and treat cancer or tumors caused by SLC2A1 pathogenic mutations (D184Y).

[0005] To solve the above technical problems, the present application provides a TCR (T cell receptor) that targets and recognizes SLC2A1 D184Y, wherein the TCR comprises an α chain and a β chain, wherein both the α chain and the β chain contain a constant region and a variable region, wherein the variable region of the α chain comprises a CDR3 sequence whose amino acid sequence is SEQ ID NO.4; and the variable region of the β chain comprises a CDR3 sequence whose amino acid sequence is SEQ ID NO.7.

[0006] Furthermore, the SLC2A1 D184Y peptide segment is a peptide segment containing the sequence shown in SEQ ID NO.1 and / or the SLC2A1 D184Y peptide segment is a peptide segment shown in SEQ ID NO.1.

[0007] In the present application, the SLCA2A1 D184Y, SLC2A1 (D184Y) and SLC2A1 D184Y The same meaning as , indicating that the 184th position of SLCA2A1 is mutated from aspartic acid (D) to tyrosine (Y). The SLCA2A1D184Y peptide, SLC2A1(D184Y) peptide and SLC2A1 D184Y The peptides all represent peptides containing the sequence shown in SEQ ID NO.1, or the SLCA2A1 D184Y peptide, SLC2A1 (D184Y) peptide and SLC2A1 D184Y The peptide segments all refer to the peptide segments whose amino acid sequence is shown in SEQ ID NO.1.

[0008] In the present application, the SLCA2A1 can also be represented by GTR1 or GLUT1, and belongs to the glucose transporter family. The number in the UniProtKB database is P11166 (URL: https: / / www.uniprot.org / uniprotkb).

[0009] Furthermore, the variable region of the α chain also includes a CDR1 sequence whose amino acid sequence is positions 27-38 of SEQ ID NO.3 and / or the variable region of the α chain also includes a CDR2 sequence whose amino acid sequence is positions 56-65 of SEQ ID NO.3; the variable region of the β chain also includes a CDR1 sequence whose amino acid sequence is positions 27-38 of SEQ ID NO.6 and / or the variable region of the β chain also includes a CDR2 sequence whose amino acid sequence is positions 56-65 of SEQ ID NO.6.

[0010] In the present invention, the sequences of CDR1, CDR2, and CDR3 of the variable regions of the α chain and β chain can be obtained based on the full-length sequences of the variable regions of the above-mentioned α chain and β chain (SEQ ID NO.3, SEQ ID NO.6) according to the Kabat, IMGT, Chothia, AbM, Contact numbering systems or other numbering system definitions (new numbering systems that may be discovered in the future, existing numbering systems). The sequences corresponding to CDR1, CDR2, and CDR3 obtained according to any one or any combination of the Kabat, IMGT, Chothia, AbM, Contact numbering systems or other numbering system definitions (new numbering systems that may be discovered in the future, existing numbering systems) are also included in the protection scope of the present invention.

[0011] In some embodiments of the present application, the sequences of CDR1, CDR2, and CDR3 of the variable regions of the α chain and β chain can be obtained according to the IMGT numbering system based on the full-length sequences of the variable regions of the above-mentioned α chain and β chain (SEQ ID NO.3, SEQ ID NO.6).

[0012] Furthermore, the amino acid sequence of the variable region of the α chain is SEQ ID NO.3, and the amino acid sequence of the variable region of the β chain is SEQ ID NO.6.

[0013] Furthermore, the TCR includes:

[0014] (1) β chain obtained by fusion of the variable region of β chain with the constant region of mTRBC;

[0015] (2) α chain obtained by fusing the variable region of α chain with the constant region of mTRAC.

[0016] The amino acid sequence of the TRBC constant region is SEQ ID NO. 9. The amino acid sequence of the TRAC constant region is SEQ ID NO. 10.

[0017] The present application also provides a biological material related to the TCR, and the biological material may be any of the following:

[0018] B1) a nucleic acid molecule encoding the TCR;

[0019] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0020] B3) a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2);

[0021] B4) A recombinant microorganism containing the nucleic acid molecule described in B1), a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0022] Furthermore, the nucleic acid molecule in B1) may be any of the following:

[0023] g1) the nucleotide sequence or coding sequence is the DNA molecule shown in positions 328-366 of SEQ ID NO.5;

[0024] g2) the nucleotide sequence or coding sequence is the DNA molecule shown in SEQ ID NO.5;

[0025] g3) the nucleotide sequence or coding sequence is the DNA molecule shown in positions 328-378 of SEQ ID NO.8;

[0026] g4) the nucleotide sequence or coding sequence is the DNA molecule shown in SEQ ID NO.8;

[0027] g5) the nucleotide sequence or coding sequence is the DNA molecule shown in positions 2809-3732 of SEQ ID NO.13;

[0028] g6) the nucleotide sequence or coding sequence is the DNA molecule shown in positions 3799-4608 of SEQ ID NO.13;

[0029] g7) the nucleotide sequence or coding sequence is the DNA molecule shown in SEQ ID NO.13, 2809-4608;

[0030] g8) A DNA molecule having 80% or more identity in nucleotide sequence with the DNA molecule defined in any one of g1) to g7) and having the same function.

[0031] In the present application, the 80% or more identity may be 80%, 85%, 90% or more identity.

[0032] The 80% or more identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The 85% or more identity may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The 90% or more identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The greater than 95% identity may be at least 95%, 96%, 97%, 98% or 99% identity.

[0033] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of an amino acid sequence, the value (%) of identity can then be obtained.

[0034] A person skilled in the art can easily mutate the nucleotide sequence encoding the chimeric antigen receptor of the present application by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 780% or higher identity with the nucleotide sequence of the chimeric antigen receptor of the present application, as long as they encode the chimeric antigen receptor and have the function of the chimeric antigen receptor, are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application. The vector described herein refers to a vector capable of carrying exogenous DNA or target gene into host cells for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), viral vector (such as baculovirus vector, retrovirus (including lentivirus), adenovirus, adeno-associated virus or herpes virus (such as herpes simplex virus)). In some embodiments of the present application, the vector can be a lentiviral vector pLVX.

[0035] The recombinant vector described in this article refers to a DNA molecule constructed by connecting an exogenous target gene to a vector in vitro. It can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0036] In some embodiments of the present application, the recombinant vector is a recombinant lentiviral vector pLVX-SLC2A1-TCR. The nucleotide sequence of the recombinant lentiviral vector pLVX-SLC2A1-TCR is SEQ ID NO.13.

[0037] The microorganism described herein may be a virus, a bacterium, a yeast, an algae or a fungus. Among them, the bacteria may be from Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., etc., but not limited thereto. The yeast may be from Saccharomyces cerevisiae sp., Pichia sp., Yarrowia sp., Candida sp., Schizosaccharomyces sp., Hansenula sp., Kluyreromyces sp., etc., but not limited thereto. The algae may be from Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., Boekelovia sp., etc., but not limited thereto. The fungi may be from Fusarium sp., Rhizoctonia sp., Verticillium sp., Penicillium sp., Aspergillus sp., Cephalosporium sp., etc., but not limited thereto.

[0038] The recombinant microorganism described in this article refers to a microorganism with changed functions obtained by manipulating and modifying the genes of a target microorganism, such as a recombinant microorganism obtained by introducing an exogenous target gene or a recombinant vector into the target microorganism, or a recombinant microorganism obtained by directly editing the endogenous genes of the target microorganism.

[0039] The present application also provides a recombinant cell, which may be a recombinant cell containing the TCR or the biological material.

[0040] Furthermore, the recombinant cell is achieved by introducing the TCR or the biological material into a recipient cell.

[0041] Furthermore, the receptor cells include T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages and / or mast cells.

[0042] Furthermore, the receptor cell may be a T cell.

[0043] Furthermore, the recipient cell of the recombinant cell may be a peripheral blood mononuclear cell.

[0044] Furthermore, the recipient cell of the recombinant cell may be a lymphocyte.

[0045] Furthermore, the recipient cell of the recombinant cell may be a T cell.

[0046] In some embodiments, the present invention has no particular limitation on the receptor cells, and any receptor cells known to those skilled in the art that can bind to the T cell receptor provided by the present invention to form recombinant cells are within the protection scope of the present invention. In a specific embodiment of the present invention, the receptor cells may be T cells.

[0047] Furthermore, the recombinant cell may be a TCR-T cell.

[0048] In the present application, the TCR-T cell may be a T cell expressing the TCR or a T cell modified by the TCR.

[0049] Specifically, the TCR-T cell described in the present application may be a TCR-T cell targeting SLC2A1 D184Y, and the TCR-T cell expresses the T cell receptor.

[0050] The TCR-T cells described in this article are obtained by genetic engineering in vitro recombination.

[0051] Furthermore, the TCR-T cell described herein may be a recombinant cell obtained by transferring the coding sequence of the TCR (SEQ ID NO. 13, positions 2809-4608) into a lentiviral vector to obtain a recombinant lentiviral vector, wherein the recombinant lentiviral vector is packaged by a packaging cell to obtain a recombinant lentivirus, and then the recombinant lentivirus is introduced into a T cell, wherein the packaging cell may be a HEK293T cell.

[0052] The present application also provides a pharmaceutical composition, which contains the TCR, the biological material and / or the recombinant cell.

[0053] In the present application, the pharmaceutical composition can enhance the tumor cell killing ability of the recombinant cells.

[0054] In the present application, the pharmaceutical composition can be used to prevent or treat SLC2A1 target-related diseases.

[0055] Furthermore, the pharmaceutical composition may further include one or more pharmaceutically acceptable carriers.

[0056] Furthermore, the pharmaceutically acceptable carrier may be an excipient, a stabilizer, a suspending agent or a diluent, etc., which are well known to those skilled in the art.

[0057] Furthermore, the carrier material includes, but is not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acid, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a common preparation, a sustained-release preparation, a controlled-release preparation, and various microparticle delivery systems. In order to make a unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to make the unit dosage form into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc. In order to prepare the unit dosage form into a suppository, various carriers known in the art can be widely used. Examples of carriers include, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. In order to prepare the unit dosage form into an injectable preparation, such as a solution, emulsion, freeze-dried powder injection and suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol may be added to the injection preparation. In addition, conventional solubilizers, buffers, pH adjusters, etc. may also be added.In addition, if necessary, colorants, preservatives, perfumes, flavoring agents, sweeteners or other materials may be added to the pharmaceutical preparations.

[0058] The present application also provides an application, which may be an application of the TCR, the biomaterial and / or the recombinant cell in any of the following:

[0059] D1) Use in the preparation of drugs for preventing or treating tumors;

[0060] D2) Use in the preparation of a drug for preventing or treating a disease associated with the SLCA2A1 D184Y mutation;

[0061] D3) Application in developing drugs for preventing or treating diseases associated with SLCA2A1 D184Y mutation.

[0062] The present application also provides the use of the TCR, biomaterial, recombinant cell and / or pharmaceutical composition as described below.

[0063] D4) Application in the prevention or treatment of tumors;

[0064] D5) Use in preventing or treating SLCA2A1 D184Y mutation-related diseases.

[0065] Furthermore, in the application, the SLC2A1 target-related disease may be a SLC2A1 D184Y mutation-positive disease.

[0066] Furthermore, in the application, the SLC2A1-positive disease may be SLCA2A1 D184Y mutation-positive cancer.

[0067] Furthermore, the SLC2A1 D184Y mutation positivity may be that the 184th amino acid of SLCA2A1 mutates from aspartic acid (D) to tyrosine (Y).

[0068] In the present application, the tumor and / or the SLCA2A1 D184Y mutation-positive cancer may be, for example, brain cancer, breast cancer, head and neck cancer, bladder cancer, kidney cancer, colorectal cancer, lung cancer, and ovarian cancer.

[0069] The recombinant cell contains and / or expresses the TCR.

[0070] The TCR, biomaterial, recombinant cell and / or pharmaceutical composition described herein have at least any of the following characteristics:

[0071] E1) secretes the specific effector molecule IFN-γ;

[0072] E2) secretes the specific effector molecule IL-2;

[0073] E3) specifically kills tumor cells carrying the SLC2A1 D184Y mutation;

[0074] E4) inhibits the proliferation of tumor cells carrying the SLC2A1 D184Y mutation.

[0075] The present application also provides a method for screening specific T cells targeting SLC2A1, the method comprising the following steps:

[0076] S1, obtaining antigen-presenting cells loaded with the SLC2A1 D184Y peptide, and co-culturing the antigen-presenting cells with T cells;

[0077] S2, sorting CD8-positive and pMHC probe-positive T cells from the T cells in the culture system of step S1, thereby obtaining specific T cells targeting the SLC2A1 D184Y mutation.

[0078] The SLC2A1 D184Y peptide provided in the present application is a SLC2A1 mutant in SLC2A1-positive cancer patients, and the amino acid sequence of the SLC2A1 D184Y peptide is SEQ ID NO. 1. The amino acid sequence of the wild-type SLC2A1 peptide in normal cells is SEQ ID NO. 2.

[0079] In the present application, the antigen presenting cell may be a CD8 negative cell carrying the above-mentioned SLC2A1 D184Y peptide (amino acid sequence is SEQ ID NO.1). In some embodiments of the present application, the D8 negative cell may be a dendritic cell. Further, in step S1 of the present application, the T cell may be a CD8 positive T cell.

[0080] In some embodiments of the present application, the CD8 positive T cells and the CD8 negative cells can be separated by Dynabeads TM It was isolated using CD8 positive isolation kit.

[0081] The present application also provides a method for preparing the recombinant cell, which comprises: transferring the above-mentioned nucleic acid molecule into a recipient cell for stable expression to obtain the recombinant cell.

[0082] Furthermore, the recipient cells may be peripheral blood mononuclear cells.

[0083] Furthermore, the receptor cell may be a lymphocyte.

[0084] Furthermore, the receptor cell may be a T cell.

[0085] Furthermore, the recombinant cell may be a TCR-T cell.

[0086] Furthermore, the recombinant cell may be a SLC2A1 TCR-T cell.

[0087] Furthermore, the method may include the following steps:

[0088] (1) The SLC2A1 TCR gene (SEQ ID NO. 13, positions 2809-4608) was constructed into the lentiviral vector pLVX-IRES-ZsGreen1, and the other nucleotide sequences of the lentiviral vector pLVX-IRES-ZsGreen1 were kept unchanged to obtain a recombinant lentiviral vector pLVX-SLC2A1 TCR;

[0089] (2) introducing the recombinant lentiviral vector pLVX-SLC2A1 TCR into packaging cells for packaging to obtain a recombinant retrovirus;

[0090] (3) Infecting human peripheral blood mononuclear cells (PBMC, such as T cells) with the recombinant retrovirus to obtain recombinant cells, namely SLC2A1 TCR-T cells.

[0091] Furthermore, the packaging cells may be HEK293T cells.

[0092] The present application also provides a method for using the TCR, TCR-T cell or pharmaceutical composition to prevent or treat SLC2A1 target-related diseases, the method comprising administering an effective dose of the TCR, TCR-T cell or pharmaceutical composition to a subject suffering from the SLC2A1 target-related disease.

[0093] The present application also provides a method for preventing or treating a SLC2A1 target-related disease, comprising administering an effective dose of the TCR, TCR-T cell or pharmaceutical composition to a subject suffering from the SLC2A1 target-related disease.

[0094] In the present application, "subject" includes a person who is being treated for a disease or preventing a disease as a patient. The methods described herein can be used to treat animal subjects belonging to any classification. Examples of such animals include mammals. Mammals include, but are not limited to, rodents (order Rodentia) mammals, such as mice and hamsters, and lagomorpha (order Logomorpha) mammals, such as rabbits. Mammals can be carnivores (order Carnivora), including felines (cats) and canines (dogs). Mammals can be even-toed ungulates (order Artiodactyla), including bovines (cows) and suids (pigs), or odd-toed ungulates (order Perssodactyla), including equines (horses). Mammals can be primates (order Primate), four-legged monkeys (order Ceboid) or monkeys (order Simoid) (monkeys) or humanoids.

[0095] Order Anthropoid (humans and apes). In some embodiments, the mammal is a human.

[0096] The term "effective" as applied to dosage or amount refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would be effective if administered alone. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the specific drug or drugs employed, the mode of administration, and the like.

[0097] Furthermore, in the method, the SLC2A1 target-related disease may be a SLC2A1-positive disease.

[0098] Furthermore, in the method, the SLC2A1-positive disease may be SLC2A1-positive cancer.

[0099] Furthermore, in the method described above, in the present application, the SLCA2A1-positive cancer may be, for example, brain cancer, breast cancer, head and neck cancer, bladder cancer, kidney cancer, colorectal cancer, lung cancer, and ovarian cancer.

[0100] In the present application, the SLC2A1 positivity may be SLC2A1(D184Y) positivity.

[0101] The TCR targeting SLC2A1 described in the present application is composed of an α chain and a β chain, and recognizes pMHC in the form of a heterodimer. The α chain and the β chain are transmembrane glycoproteins, which are connected by disulfide bonds to form a heterodimer, namely TCR. The extramembrane parts of the α chain and the β chain each contain two Ig-like domains, one is the constant region (C region) at the proximal end of the membrane, and the other is the variable region (V region) at the distal end of the membrane. The V region of the α chain and the β chain together constitute the antigen binding site of the TCR. The V region can be further divided into complementarity-determining regions 1 (CDR1), CDR2 and CDR3. When the TCR recognizes the ligand, the CDR1 and CDR2 with lower diversity contact the α helices on both sides of the pMHC, while the CDR3 contacts the central peptide. CDR3 is the most critical sequence that determines the antigen specificity of TCR recognition. In order to achieve recognition of different pMHC molecules, the CDR3 region has a high diversity. TCR diversity is concentrated in the high variability of the CDR3 region. With the deepening of TCR research, it is now possible to obtain a functional full-length TCR V region sequence through bioinformatics methods by simply identifying the CDR3 sequence, which greatly simplifies the TCR isolation process.

[0102] Compared with the prior art, the beneficial technical effects achieved by this application are as follows:

[0103] Screening TCR sequences targeting SLC2A1 mutant peptides and then constructing TCR-T cell drugs or protein drugs or nucleic acid drugs based on this TCR can effectively eliminate target cells expressing SLC2A1 mutant peptides. This is a beneficial exploration of precise immunotherapy for diseases, and the corresponding TCR sequence is reported for the first time.

[0104] By establishing a screening platform for TCRs targeting SLC2A1 mutant peptides, we can further create conditions for screening TCRs targeting other targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 . Flow cytometry analysis of the proportion of CD8 positive / pMHC probe + double positive T cells.

[0106] Figure 2 is the amino acid sequence of TCR.

[0107] Figure 3 Analyze the gene transduction rate of TCR-T cells by flow cytometry.

[0108] Figure 4 To detect the interferon-γ secretion ability of TCR-T cells.

[0109] Figure 5 To detect the interleukin-2 secretion ability of TCR-T cells.

[0110] Figure 6 To detect the killing ability of TCR-T cells.

[0111] Figure 7 This is the sequencing result of the α chain of TCR in TCR clonotype analysis.

[0112] Figure 8 This is the sequencing result of the β chain of TCR in TCR clonotype analysis. DETAILED DESCRIPTION

[0113] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0114] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0115] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0116] The following examples used GraphPad Prism statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. The t-test was used for the test. P < 0.05 (*) indicated a significant difference, and P < 0.001 (***) indicated a very significant difference.

[0117] Example 1: Capturing SLC2A1 D184Y Neoantigen-specific T cells

[0118] Based on SLC2A1 D184Y HLA-A02:01 restricted epitope (SLC2A1 D184Y The amino acid sequence of the antigen epitope is YLWPLLLSI, and the sequence number is SEQ ID NO.1). The new antigen-specific T cell clone is isolated from the peripheral blood of healthy people and its TCR sequence is analyzed. The specific implementation steps are as follows:

[0119] On the first day, mononuclear lymphocytes were isolated from peripheral blood donated by healthy people using a vacuum venous blood sample collection container (BD Biosciences, catalog number 362761), and CD8 positive T cell subsets and CD8 negative cell subsets were separated using a CD8 positive cell separation kit (Thermo Fisher, catalog number 11333D), wherein the CD8 positive T cell subsets were frozen in liquid nitrogen for standby use, and the CD8 negative cell subsets were used to prepare dendritic cells. The CD8 negative cell subsets containing dendritic cells were inoculated into a six-well plate, 2 ml of CellGro DC culture medium (Cellgenix, catalog number 20801-0500) was added, and 1% human AB serum (Gemini, catalog number 100-512), 500U / mL interleukin 4 (R&D, catalog number 204-IL-500ug) and 1000U / mL granulocyte macrophage colony stimulating factor (Perprotech, catalog number 300-03-250ug) were added. Dendritic cells were cultured at 37°C and 5% CO2 for 3 days. On day 4, all dendritic cells were collected and seeded into 24-well plates at 500,000 per well, and 100 U / mL interferon γ (Perprotech, catalog number 300-02-100ug) and 10 ng / mL lipopolysaccharide (Sigma, catalog number L4391-1MG) were added to induce cell maturation, as well as 10 μg of SLC2A1 D184Y Peptide (amino acid sequence is YLWPLLLSI), and culture was continued for 16 hours. On the 5th day, dendritic cells were treated with 30μg / mL mitomycin C (Sigma, catalog number M4287-2MG) for 30 minutes, and then the revived CD8-positive T cells were mixed with dendritic cells, and 30ng / mL IL-21 (Perprotech, catalog number 200-21-10ug) was added and cultured. Next, 5ng / mL IL-15 (Perprotech, catalog number 200-15-10ug) and 5ng / mL IL-7 (Perprotech, catalog number 200-07-10ug) were supplemented once on the 8th, 10th and 12th days respectively. On the 15th day, the proportion of CD8-positive / pMHC probe + double-positive T cells was analyzed by flow cytometry using CD8 antibody (Proteintech, catalog number FITC-65144) and pMHC probe (custom synthesized by Immundex) (see Figure 1 ) and sorted them out. The sorted cells are used for subsequent TCR clonal type analysis.

[0120] Example 2: TCR clonotype analysis

[0121] The TCR clonal type analysis of the sorted CD8 positive / pMHC probe + double positive T cells was performed by the second generation sequencing method. The specific implementation steps are as follows:

[0122] Total RNA was extracted and sorted using an RNA isolation kit (Life Technologies, catalog number KIT0204). Next, cDNA was prepared using a cDNA library construction kit (Clontech, catalog number 634901), followed by adding adapters with barcode sequences using an indexing kit (Illumina, catalog numbers FC-131-2001, FC-131-2002, FC-131-2003), and finally the prepared library was subjected to 300 bp paired-end sequencing on the Illumina MiSeq platform. The data obtained by sequencing were analyzed with reference to the IMGT / GENE-DB database for Vα (i.e., α chain variable region), Vβ (i.e., β chain variable region) chain sequences and CDR3 sequences, as well as the proportion of each TCR clone (see Figure 7 and Figure 8 ).

[0123] Example 3. Construction of TCR-T cells

[0124] The TCR clones with the highest proportion in the sequencing results were selected for subsequent verification after gene rearrangement. The variable region of the α chain of TCR is composed of the rearrangement of the TRAV12-2 (UniProtKB database number A0A075B6T6) gene and the TRAJ6 (UniProtKB database number P17909) gene. The amino acid sequence of the variable region of the α chain produced by the rearrangement is SEQ ID NO.3, and the amino acid sequence of the CDR3 of the variable region of the α chain is SEQ ID NO.4. The coding sequence of the variable region of the α chain is SEQ ID NO.5. The CDR3 of the variable region of the α chain is encoded by SEQ ID NO.5 from 328 to 366. The variable region of the β chain of TCR is composed of the rearrangement of the TRBV19 (UniProtKB database number A0A075B6N1) gene and the TRBJ2-1 (UniProtKB database number A0A0A0MTA7) gene. The amino acid sequence of the variable region of the β chain of the rearranged TCR is SEQ ID NO.6, and the amino acid sequence of CDR3 of the variable region of the β chain is SEQ ID NO.7. The coding sequence of the variable region of the β chain is SEQ ID NO.8. CDR3 of the variable region of the β chain is encoded by positions 328 to 378 of SEQ ID NO.5. In order to effectively prevent mismatch between exogenous TCR and endogenous TCR, the present invention fuses the variable region of the β chain (amino acid sequence is SEQ ID NO.6) with the mouse mTRBC constant region (amino acid sequence is SEQ ID NO.9) to obtain the β chain of TCR, and fuses the variable region of the α chain (amino acid sequence is SEQ ID NO.3) with the mouse mTRAC constant region (amino acid sequence is SEQ ID NO.10) to obtain the α chain of TCR. The coding gene of the β chain of TCR is SEQ ID NO.11, and the coding gene of the α chain of TCR is SEQ ID NO.12. The sequence structure of TCR is as shown in Figure 2 shown.

[0125] The coding genes of the α chain and β chain of TCR were assembled according to TCRβV-mTRBC-P2A-TCRαV-mTRAC to obtain the TCR coding sequence and constructed into the lentiviral vector pLVX-IRES-ZsGreen1 (Clontech, catalog number 632187). The obtained recombinant lentiviral vector was named pLVX-TCR. The full sequence of pLVX-TCR is SEQ ID NO.13. Among them, positions 2505-2708 of SEQ ID NO.13 are promoter sequences, positions 2809 to 4608 are TCR coding sequences (wherein positions 2809 to 3213 encode the variable region of the β chain, positions 3214 to 3732 encode the constant region of the β chain (mTRBC), positions 3733 to 3798 encode P2A (connecting peptide), positions 3799 to 4194 encode the variable region of the α chain, and positions 4195 to 4605 encode the constant region of the α chain (mTRAC)), and positions 4606 to 4608 are terminators.

[0126] 293T cells (ATCC, catalog number: CRL-3216) were used. TM ) to produce pLVX-TCR lentivirus, the specific implementation steps are as follows: count 3×10 6 293T cells were suspended in 10 mL of serum-free DMEM medium and inoculated on a 10 cm culture dish. Transfection was performed after the cells adhered to the wall and occupied more than 50% of the total area of ​​the culture dish; pLVX-TCR vector and lentiviral packaging plasmid pmd2.G (Addgene product, catalog number #12259) and pSPAX2 (ATCC, catalog number: #12260) were mixed in a ratio of 2:1:1, transfected into 293T cells, and incubated in a 37°C, 5% CO2 cell culture incubator; fresh DMEM medium was replaced once 24 hours after transfection, and the culture supernatant containing the virus was collected 72 hours later and filtered through a 0.45 μm filter to remove cell debris. The supernatant obtained was named viral supernatant, and the virus contained was named pLVX-TCR lentivirus), and was aliquoted and frozen at -80°C for long-term storage.

[0127] Peripheral blood mononuclear cells donated by healthy individuals were transfected with pLVX-TCR lentivirus to prepare TCR-T cells. The specific implementation steps were as follows: peripheral blood mononuclear cells were counted and the cell concentration was adjusted to 1×10 6 / mL, 1mL per well, incubate in a 37°C, 5% CO2 cell culture incubator for 24 hours; add 500μL of viral supernatant, add protamine sulfate (final concentration 8μg / ml, Sigma Aldrich, catalog number 53597-25-4); discard the viral supernatant, add fresh GT-T551 H3 culture medium (Takara product, catalog number: #WK551S), continue to culture for 14 days, collect all cells and name them TCR-T cells, and freeze them in liquid nitrogen for future use.

[0128] The gene transduction rate of TCR-T was analyzed by flow cytometry. The specific implementation steps were as follows: 100,000 TCR-T cells were taken, 1 μL TCRβ flow cytometry antibody (Proteintech, catalog number APC-65106) was added for flow cytometry analysis, and the results were shown in Figure 3 . Figure 3 The results showed that the TCR gene transduction rate reached 50.8%.

[0129] Example 4: Verification of cytokine secretion ability of TCR-T cells

[0130] The cytokine secretion capacity of TCR-T cells is determined by detecting the secretion amount of cytokines. The specific implementation steps are as follows:

[0131] First, prepare the loaded SLC2A1 D184Y The specific implementation steps are as follows: 200,000 C1R cells (ATCC, catalog number CRL-2369) were taken and 10ug of SLC2A1 was added to the serum-free medium. D184Y The polypeptide (amino acid sequence is SEQ ID NO.1) was cultured overnight; at the same time, a portion of C1R cells incubated with wild-type SLC2A1 polypeptide (amino acid sequence is SEQ ID NO.2) was prepared as a control. On the second day, the C1R cells incubated with the polypeptide were washed twice with PBS and set aside.

[0132] 50,000 TCR-T cells were mixed with 20,000 C1R cells incubated with peptides and cultured in a 96-well plate overnight. The next day, the cell culture supernatant was collected and the interferon-γ and interleukin-2 levels in the supernatant were detected using the interferon-γ enzyme-linked immunosorbent assay (BD bioscience, catalog number 555142) and interleukin-2 enzyme-linked immunosorbent assay (BD bioscience, catalog number 555190) according to the instructions. The results are shown in Figure 4 and Figure 5 . Figure 4 and Figure 5 The results showed that TCR-T cells can specifically recognize SLC2A1 D184YThe target cells of the peptide secrete the cytokines interferon-γ and interleukin-2, but are unresponsive to the wild type.

[0133] Example 5: Verification of the killing ability of TCR-T cells

[0134] The killing ability of TCR-T cells was determined by detecting the amount of lactate dehydrogenase released by antigen presenting cells. The specific implementation steps were as follows: SLC2A1 loaded cells were prepared according to the method of Example 4. D184Y Then, 50,000 TCR-T cells were mixed with 20,000 C1R cells incubated with peptides and cultured overnight in a 96-well plate. The next day, the cell culture supernatant was collected and the lactate dehydrogenase content was determined using a cytotoxicity detection kit (Promega, catalog number G1780) according to the instructions. The results were calculated according to the instructions in the instructions. The results are shown in Figure 6 . Figure 6 The results showed that TCR-T cells can specifically cleave SLC2A1 D184Y The target cells of the peptide were not responsive to the wild type.

Claims

1. A TCR targeting and recognizing the SLC2A1 D184Y peptide segment, the TCR comprising an α chain and a β chain, the α chain and the β chain both comprising a constant region and a variable region, characterized in that: The variable region of the α chain includes a CDR3 sequence whose amino acid sequence is SEQ ID NO.4; the variable region of the β chain includes a CDR3 sequence whose amino acid sequence is SEQ ID NO.

7.

2. The TCR according to claim 1, characterized in that The variable region of the α chain also includes a CDR1 sequence whose amino acid sequence is positions 27-38 of SEQ ID NO.3 and / or the variable region of the α chain also includes a CDR2 sequence whose amino acid sequence is positions 56-65 of SEQ ID NO.3; the variable region of the TCR β chain also includes a CDR1 sequence whose amino acid sequence is positions 27-38 of SEQ ID NO.6 and / or the variable region of the TCR β chain also includes a CDR2 sequence whose amino acid sequence is positions 56-65 of SEQ ID NO.

6.

3. The TCR according to claim 1 or 2, characterized in that The amino acid sequence of the variable region of the α chain is SEQ ID NO.3, and the amino acid sequence of the variable region of the β chain is SEQ ID NO.

6.

4. The biomaterial associated with TCR according to claims 1-3, characterized in that: The biological material is any of the following: B1) a nucleic acid molecule encoding the TCR according to any one of claims 1 to 3; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

5. The biomaterial according to claim 4, characterized in that B1) The nucleic acid molecule may be any of the following: g1) the nucleotide sequence or coding sequence is the DNA molecule shown in positions 328-366 of SEQ ID NO.5; g2) the nucleotide sequence or coding sequence is the DNA molecule shown in SEQ ID NO.5; g3) the nucleotide sequence or coding sequence is the DNA molecule shown in positions 328-378 of SEQ ID NO.8; g4) the nucleotide sequence or coding sequence is the DNA molecule shown in SEQ ID NO.8; g5) the nucleotide sequence or coding sequence is the DNA molecule shown in positions 2809-3732 of SEQ ID NO.13; g6) the nucleotide sequence or coding sequence is the DNA molecule shown in positions 3799-4608 of SEQ ID NO.13; g7) the nucleotide sequence or coding sequence is the DNA molecule shown in positions 2809-4608 of SEQ ID NO.13; g8) A DNA molecule having 80% or more identity in nucleotide sequence with the DNA molecule defined in any one of g1) to g7) and having the same function.

6. A recombinant cell, characterized in that The recombinant cell contains the TCR according to any one of claims 1 to 3 and / or the biological material according to claim 4 or 5.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the TCR according to any one of claims 1 to 3, the biological material according to claim 4 or 5 and / or the recombinant cell according to claim 6.

8. Application, characterized in that, The application is (1) or (2): (1) Use of the TCR according to any one of claims 1 to 3, the biomaterial according to claim 4 or 5, and / or the recombinant cell according to claim 6 in any of the following; D1) Use in the preparation of drugs for preventing or treating tumors; D2) Use in the preparation of a drug for preventing or treating a disease associated with the SLCA2A1 D184Y mutation; D3) Application in the development of drugs for preventing or treating diseases associated with SLCA2A1 D184Y mutation; (2) The use of the SLC2A1 D184Y peptide segment of claim 1, wherein the SLC2A1 D184Y peptide segment is used to induce SLC2A1 D184Y antigen-specific T cells, and the amino acid sequence of the SLC2A1 D184Y peptide segment is SEQ ID NO.

1.

9. A method for screening specific T cells targeting SLC2A1 D184Y mutation, characterized in that: The method comprises the following steps: S1, obtaining antigen-presenting cells loaded with the SLC2A1 D184Y peptide, and co-culturing the antigen-presenting cells with T cells; S2, sorting CD8-positive and pMHC-positive T cells from the T cells in the culture system of step S1, thereby obtaining specific T cells targeting the SLC2A1 D184Y mutation.

10. A method for preparing the recombinant cell according to claim 6, characterized in that: The method comprises: transferring the nucleic acid molecule described in claim 4 or 5 into a recipient cell for stable expression to obtain the recombinant cell.

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