Alpha-beta type TCR molecular library as well as construction method and application thereof

Abstract

The invention discloses an alpha-beta type TCR (T cell receptor) molecular library as well as a construction method and application thereof. According to the present invention, with the constructed library containing the vector coding the TCR alpha functional V-J gene combination and the vector coding the TCR beta functional V-J gene combination, the TCR molecule of the target object can be rapidly constructed by replacing the CDR3 sequence based on the structural analysis of the target object, such that the customization and the preparation of the target object TCR molecule can be efficiently and rapidly achieved on a large scale. Therefore, powerful technical support is provided for TCR function research, and a new way is opened up for TCR drug research and development and clinical application.

Description

Technical Field

[0001] The present invention relates to the technical field of library construction, and in particular to an αβ-type TCR molecular library and a construction method and application thereof. Background Art

[0002] Human specific cellular immunity is mainly mediated by T cells, which play an important role in immune responses such as anti-infection and anti-tumor. T cells can be divided into αβ T cells and γδ T cells according to the type of TCR molecules expressed on the cell surface. αβ T cells express αβTCR molecules, while γδ T cells express γδTCR molecules. αβ T cells account for 95%-99% of human T cells. The TCR molecules they express are heterodimers composed of diverse glycosylated TCRα subunits and TCRβ subunits, which can non-covalently bind to non-diversified membrane-bound CD3 proteins to form functional TCR-CD3 complexes on the cell surface. TCR molecules are highly diverse molecules that can recognize a variety of peptides from pathogens or cancer cells bound to MHC. These peptides are derived from peptides produced by proteasome digestion of target antigens. These peptides are transported to the endoplasmic reticulum and then loaded and embedded in the surface grooves of MHC molecules to form epitopes recognized by αβTCR molecules. Among them, the outer surfaces of the α chain and β chain are involved in the interaction of the pMHC (peptide-MHC) complex, while the CD3 protein on the membrane is involved in signal transduction.

[0003] The high diversity of TCR is due to the fact that it is encoded by many gene fragments (such as variable region (Variable, V), D region (diversity, D), joining region (Joining, J) and constant region (Constant, C) gene fragments). These germline gene fragments undergo V(D)J rearrangement in thymocytes to produce functional TCR genes. In this process, the sequence of the VJ joining region is randomly increased or decreased at the same time, making the rearranged VJ sequence more diverse, forming a hypervariable region also known as complementarity determining region 3 (complementarity determining region, CDR3) sequence. These CDR3 sequences serve as the main region for the binding of TCR subunits to MHC-peptide complexes, determining the antigen recognition specificity of TCR.

[0004] The human TCRα gene contains 45 functional V gene segments (AV), 53 functional J gene segments (AJ), and one C gene segment (AC). The human TCRβ gene contains 48 functional V gene segments (BV), 2 functional D gene segments, 13 functional J gene segments (BJ), and 2 functional C gene segments (BC). The differences in TCR molecules affect the antigen recognition specificity of T cells, so they are key molecules for T cell function. Although the number of TCR molecules is huge, each T cell only expresses a pair of functional TCR molecules. Therefore, the TCR sequence of a single T cell consists of limited diversity of V, J and highly diverse CDR3 sequences. Among them, the number of VJ combinations of the TCRα subunit is 2385, and the diversity of VJ combinations of the TCRβ subunit is 624, which covers all possible VJ combinations of αβ-type TCRs in nature.

[0005] The popularity of the current second-generation sequencing technology has greatly promoted the progress of basic scientific research, drug development and clinical research of TCR. For example, bulk sequencing relies on the sequencing of a large number of T cells to generate TCR repertoire sequences, and single cell TCR sequencing of T cells can generate paired TCRab sequences of T cells. These high-throughput sequencing technologies can reveal the specific recognition mechanism of T cells in immune response, and provide unprecedented opportunities for finding antigen-specific TCR gene sequences. However, the existing sequencing technology and gene cloning are inefficient and time-consuming and labor-intensive. For example, when conducting functional studies on candidate TCRs, full synthesis of the DNA sequence of the full-length TCR is generally chosen. If the functional study involves multiple TCR gene combinations, full gene synthesis of each TCR is required, which is extremely time-consuming and economically expensive. Moreover, in the face of the huge amount of TCR data, traditional cloning and expression vector construction methods have been difficult to meet the requirements of high efficiency and speed. Therefore, there is an urgent need for a method that can improve the efficiency of TCR construction and reduce the construction cost, so as to solve the defects of the above methods. Summary of the invention

[0006] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the purpose of the present invention is to provide an αβ-type TCR molecule library and a construction method and application thereof.

[0007] In a first aspect, the present invention provides a library of αβ-type TCR molecules, the library comprising a vector encoding a functional VJ gene combination of TCRα and a vector encoding a functional VJ gene combination of TCRβ.

[0008] In some embodiments of the present invention, the vector encoding the TCRα functional VJ gene combination comprises the following structural units connected in sequence:

[0009] A functional fragment (AV) encoding any one of the TCRα subunit variable regions numbered 001-045 in Table 1, or a variant fragment thereof; and

[0010] A functional fragment (AJ) encoding any one of the TCRα subunit joining regions numbered 046-098 in Table 1, or a variant fragment thereof.

[0011] In some embodiments of the invention, the variant fragment has at least 85% homology to the corresponding functional fragment.

[0012] In the present invention, the term "homology" refers to the percentage of identity between two polynucleotides or polypeptide parts. In the present invention, homology can be measured by existing methods, including but not limited to sequence alignment using tools such as CLUSTAL, BLAST, ClustalW, etc.

[0013] In some embodiments of the invention, the variant fragment comprises:

[0014] Variant fragments that retain the same function and have at least 85% homology to the sequences shown in Nos. 001-045 in Table 1;

[0015] Variant fragments retaining the same function and having at least 85% homology to the sequences shown in Nos. 046-098 in Table 1.

[0016] In some embodiments of the present invention, the library includes all vectors (2385 types) encoding TCRα functional VJ gene combinations, that is, vectors containing structural units of all different arrangements and combinations of AV and AJ.

[0017] In some embodiments of the invention, the variant fragment has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology.

[0018] In some embodiments of the present invention, the variant fragment is obtained by adding amino acids (at both ends, rather than inserting in the middle) to the corresponding fragment.

[0019] In some embodiments of the present invention, the vector encoding the TCRα functional VJ gene combination comprises the following structural units connected in sequence:

[0020] A functional fragment of a TCRα subunit variable region having a sequence as shown in No. 001-045 in Coding Table 1, or a variant fragment thereof; and

[0021] A functional fragment of the TCRα subunit connecting region having the sequence shown in No. 046-098 in Encoding Table 1, or a variant fragment thereof.

[0022] In some embodiments of the invention, the variant fragment comprises:

[0023] A variant fragment having at least 85% homology to the sequence shown in No. 001-045 in Coding Table 1;

[0024] A variant fragment having at least 85% homology to the sequence shown in No. 046-098 in Coding Table 1.

[0025] In some embodiments of the invention, the variant fragment has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology.

[0026] In some embodiments of the present invention, the vector encoding the TCRβ functional VJ gene combination comprises the following structural units connected in sequence:

[0027] A functional fragment (BV) encoding any one of the TCRβ subunit variable regions numbered 100-147 in Table 1, or a variant fragment thereof; and

[0028] A functional fragment (BJ) encoding any one of the TCRβ subunit joining regions No. 148-160 in Table 1, or a variant fragment thereof.

[0029] In some embodiments of the invention, the variant fragment comprises:

[0030] Variant fragments that retain the same function and have at least 85% homology to the sequences shown in Nos. 148-160 in Table 1;

[0031] Variant fragments retaining the same function and having at least 85% homology to the sequences shown in Nos. 148-160 in Table 1.

[0032] In some embodiments of the present invention, the library includes all vectors (624 types) encoding TCRβ functional VJ gene combinations, that is, vectors containing structural units of all different arrangements and combinations of BV and BJ.

[0033] In some embodiments of the invention, the variant fragment has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology.

[0034] In some embodiments of the present invention, the variant fragment is obtained by adding amino acids (at both ends, rather than inserting in the middle) to the corresponding fragment.

[0035] In some embodiments of the present invention, the vector encoding the TCRβ functional VJ gene combination comprises the following structural units connected in sequence:

[0036] A functional fragment of a TCRβ variable region having a sequence as shown in Nos. 100-147 in Coding Table 1, or a variant fragment thereof; and

[0037] A functional fragment of the TCRβ joining region having a sequence as shown in Nos. 148-160 in Coding Table 1, or a variant fragment thereof.

[0038] In some embodiments of the invention, the variant fragment comprises:

[0039] A variant fragment having at least 85% homology to the sequence shown in No. 100-147 in Coding Table 1;

[0040] A variant fragment having at least 85% homology to the sequence shown in No. 148-160 in Coding Table 1.

[0041] In some embodiments of the invention, the variant fragment has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology.

[0042] In some embodiments of the present invention, the two structural units have at least one terminal binding site at opposite ends, and the two structural units are connected to each other through the terminal binding site.

[0043] In some embodiments of the invention, both building blocks have a terminal binding site at opposite ends.

[0044] In some embodiments of the present invention, both ends of the two structural units have a terminal binding site.

[0045] In some embodiments of the present invention, in the two structural units, a linker sequence is inserted between the functional fragment of the variable region and the functional fragment of the connecting region.

[0046] In some embodiments of the present invention, the linker sequence contains two IIS type restriction endonucleases, so that it can be assembled by the Golden Gate method to replace the CDR3 sequence during construction.

[0047] In some embodiments of the present invention, the IIS type restriction endonuclease includes PaqCI (the PaqCI cleavage site is 5'-CACCTGCNNNN-3').

[0048] In some embodiments of the present invention, the linker sequence includes a sequence as shown in SEQ ID NO: 1. Of course, those skilled in the art can also reasonably use other linker sequences to connect the functional fragments of the variable region and the functional fragments of the connecting region to achieve the same effect.

[0049] In some embodiments of the present invention, the linker sequence connects the 3' end of AV and the 5' end of AJ, respectively.

[0050] In some embodiments of the present invention, linker sequences are connected to the 3' end of BV and the 5' end of BJ, respectively.

[0051] In some embodiments of the present invention, in both structural units, the functional fragment of the connecting region is also connected to the constant region fragment.

[0052] In some embodiments of the invention, the constant region fragment and the functional fragment of the joining region encode the same TCR subunit.

[0053] In some embodiments of the invention, the constant region fragment is a murine, cysteine ​​engineered, or wild-type human constant region fragment, or a combination thereof.

[0054] In some embodiments of the present invention, the constant region fragment is a constant region fragment of mouse or human origin.

[0055] In some embodiments of the present invention, the amino acid sequence of the TCRα subunit constant region fragment is shown in SEQ ID NO:2.

[0056] In some embodiments of the present invention, the amino acid sequence of the TCRβ subunit constant region fragment is shown in SEQ ID NO:3.

[0057] In some embodiments of the present invention, the 5' end of AV is further connected to a BstB I restriction enzyme site, the corresponding linker sequence contains an NsiI restriction enzyme site, and the 3' end of the constant region is further connected to a SalI restriction enzyme site.

[0058] In some embodiments of the present invention, the 5' end of BV is also connected to a BstB I restriction enzyme site, the corresponding linker sequence contains an NsiI restriction enzyme site, and the 3' end of the constant region is also connected to MluI and SalI restriction enzyme sites.

[0059] Therefore, when the subsequent co-expression vector is constructed, the TCRα subunit can be connected to the downstream end of the TCRβ subunit through the MluI and SalI sites, thereby realizing the connection of the co-expression fragments.

[0060] In some embodiments of the present invention, the carrier is not limited. In the present invention, any carrier that can be used to store and extract the above-mentioned structural units can be used as a carrier, including but not limited to conventional commercially available carriers.

[0061] In some embodiments of the present invention, the vector encoding the TCRα functional VJ gene combination and / or the vector encoding the TCRβ functional VJ gene combination is further integrated into a framework vector or an expression vector.

[0062] In some embodiments of the present invention, the framework vector or expression vector includes an in vitro transcribed mRNA (ivtRNA) vector, a retroviral vector, and a lentiviral vector. Of course, those skilled in the art may also use other framework vectors or expression vectors for loading, transfection, and storage of products extracted from vectors encoding TCRα functional VJ gene combinations and / or vectors encoding TCRβ functional VJ gene combinations, including but not limited to the above-mentioned in vitro transcribed mRNA (ivtRNA) vectors, retroviral vectors, and lentiviral vectors.

[0063] In some embodiments of the present invention, the ivtRNA vector comprises at least one RNA stabilizing sequence.

[0064] In some embodiments of the present invention, the RNA stabilizing sequence includes but is not limited to a polyadenine tail.

[0065] In some embodiments of the present invention, the vector encoding the TCRα functional VJ gene combination and the vector encoding the TCRβ functional VJ gene combination are further integrated into the same framework vector or expression vector to achieve co-expression of TCRα and TCRβ.

[0066] In some embodiments of the present invention, the integration includes: obtaining the TCRα functional VJ gene combination coding sequence and the TCRβ functional VJ gene combination coding sequence from the vector encoding the TCRα functional VJ gene combination and the vector encoding the TCRβ functional VJ gene combination, connecting the TCRα functional VJ gene combination coding sequence and the TCRβ functional VJ gene combination coding sequence together through a linker, and then inserting them into a framework vector or an expression vector.

[0067] In some embodiments of the invention, the linker comprises an internal ribosome entry site (IRES) or a 2A peptide.

[0068] In some embodiments of the invention, the 2A peptides include P2A, T2A, E2A and F2A.

[0069] In some embodiments of the present invention, the IRES and 2A peptides can be obtained from commercial sources or synthesized according to sequences disclosed in the prior art.

[0070] In some embodiments of the present invention, when constructing the library, the variable region, linker sequence and constant region may be replaced in a single cloning step to obtain a target vector.

[0071] The second aspect of the present invention provides the use of the library described in the above aspect in any one of the following (1)-(4);

[0072] (1) constructing αβ-type TCR molecule expression vectors or transformants;

[0073] (2) Screening of αβ-type TCR molecules;

[0074] (3) Construction of αβ-type TCR protein library;

[0075] (4) Constructing αβ-type T cell receptors or receptor libraries.

[0076] In the present invention, the expression vector refers to a receptor that can insert an exogenous DNA molecule (target fragment) and stably maintain the molecule therein, including but not limited to plasmids, bacteriophages and viruses.

[0077] In the present invention, the transformant refers to a clone obtained by various transformation methods or transduction methods, including but not limited to bacteria, fungi and cells.

[0078] The third aspect of the present invention provides a product obtained based on the library construction described in the above aspects.

[0079] In some embodiments of the present invention, the product includes a cell clone library, a protein library and a TCR molecule expression system.

[0080] In some embodiments of the present invention, the cell clone library contains transformants of at least one vector in the above aspects.

[0081] In some embodiments of the present invention, the protein library is obtained from a transformant containing at least one vector in the above aspects.

[0082] In some embodiments of the present invention, the TCR molecule expression system contains the library described in the above aspects.

[0083] In some embodiments of the present invention, the TCR molecule expression system further comprises a screening module for screening specific TCR molecules from the library.

[0084] In some embodiments of the present invention, the TCR molecule expression system contains: a framework vector or expression vector integrated with a vector encoding a TCRα functional VJ gene combination and / or a vector encoding a TCRβ functional VJ gene combination. Including but not limited to an ivtRNA vector, a retroviral vector, and a lentiviral vector integrated with a vector encoding a TCRα functional VJ gene combination and / or a vector encoding a TCRβ functional VJ gene combination.

[0085] A fourth aspect of the present invention provides a method for constructing an αβ T cell receptor library, comprising:

[0086] (1) designing a primer set containing a CDR3 sequence encoding a target TCR molecule, and annealing to form a double-stranded DNA, wherein the CDR3 sequence includes the CDR3 sequence of the α subunit and the β subunit of the target TCR molecule;

[0087] (2) using the double-stranded DNA obtained in step (1) to replace the linker sequences in the vectors in the above aspects, respectively, to obtain α-subunit and β-subunit vector plasmids;

[0088] (3) Integrate the α-subunit and β-subunit vector plasmids to obtain a co-expression plasmid, and transfer the co-expression plasmid into a cell vector to obtain an αβ-type T cell receptor library.

[0089] In some embodiments of the present invention, in step (3), a TCRα subunit coding sequence and a TCRβ subunit coding sequence integrated into the co-expression plasmid are connected by a linker.

[0090] In some embodiments of the invention, the 2A peptides include P2A, T2A, E2A and F2A.

[0091] A fifth aspect of the present invention provides an αβ T cell receptor library, wherein the αβ T cell receptor library is constructed by the method described in the above aspects.

[0092] The beneficial effects of the present invention are:

[0093] 1. In the present invention, all possible VJ combinations are pre-designed and constructed, and a special restriction site is designed between VJ to construct a library, so as to allow rapid and large-scale insertion of CDR3 sequences, thereby constructing a complete TCR gene. Thus, the target TCR expression sequence can be quickly obtained by analyzing the structural information of the target sequence, thereby achieving the construction of a large number of TCR expression vectors in a short time, greatly improving the experimental efficiency. It not only provides strong technical support for TCR function research, but also opens up new ways for TCR drug development and clinical application.

[0094] 2. By constructing a TCR expression vector based on the library of the present invention, the key regions can be easily replaced, so as to quickly and efficiently construct TCR molecules with highly diverse characteristics. In addition, the constant region can also be transformed and replaced, such as reducing the humanization characteristics, cysteine ​​modification, humanization modification, etc., to meet the needs of different TCR expression strategies. It has high flexibility and versatility. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 The diagram is a schematic diagram of the key structural composition of the TCRαV-J vector and the TCRβV-J vector in the library of the present invention, wherein the TCRαV-J vector contains the mouse TCRα subunit constant region (mCA) and any one of the 2385 human functional AV-AJ permutations and combinations, and the TCRβV-J vector contains the mouse TCRβ subunit constant region (mCB) and any one of the 624 human functional BV-BJ permutations and combinations.

[0096] Figure 2 This is a plasmid map of the TCRα / TCRβ vector system pRVTCR framework vector based on the retroviral vector pMP71 backbone in an embodiment of the present invention.

[0097] Figure 3 Schematic diagram of the process for rapid construction of TCR expression vector, retroviral packaging and T cell transduction.

[0098] Figure 4 This is a flow cytometry antibody staining profile, in which T cells without antibody staining were used as blank controls for flow cytometry detection.

[0099] Figure 5 The results of ELISA detection of the specific secretion of IFN-γ in each group of cells.

[0100] Figure 6 Figure 5 shows the results of tetramer staining analysis, with normal T cells as a control.

[0101] Figure 7The following are the construction results of different methods in the comparative example. DETAILED DESCRIPTION

[0102] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0103] Example 1 Construction of αβ TCR Molecular Library

[0104] In this example, the construction of all vector plasmids was completed by Biosynthetic Company.

[0105] In this example, an αβ-type TCR molecule library was constructed, which consisted of vectors each encoding 2385 different TCRα functional VJ genes and vectors each encoding 624 different TCRβ functional VJ genes.

[0106] Among them, the vector of the TCRα functional VJ gene is a conventional commercially available blank vector carrying a VJ structural unit. The VJ structural unit is a functional fragment (AV) of the TCRα subunit variable region, a linker sequence (5'-GCTGTGCAGGTGGTCGATGCATCACCTGCTAGA-3' (SEQ ID NO: 1)), a functional fragment (AJ) of the TCRα subunit connecting region, and a TCRα subunit constant region fragment connected in sequence. The amino acid sequence of the functional fragment (AV) of the TCRα subunit variable region or its variant is shown in Table 1, No. 001-045. The amino acid sequence of the functional fragment (AJ) of the TCRα subunit connecting region or its variant is shown in Table 1, No. 046-098. The amino acid sequence of the constant region fragment of the TCRα subunit is: HIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNG AIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKV AGFNLLMTLRLWSS (SEQ ID NO: 2). Each vector of the TCRα functional VJ gene independently contains one of the AVs or its variants, and one of the AJs or its variants, so that different vectors with different AV and AJ arrangements can be produced, thereby obtaining vectors encoding 2385 (45×53) different TCRα functional VJ genes. Of course, variant sequences of AV and AJ can also be used.

[0107] Similarly, the vector of the functional VJ gene of TCRβ is also the same blank vector carrying the VJ structural unit. Its VJ structural unit is a functional fragment (BV) of the variable region of the TCRβ subunit, a linker sequence (as shown in SEQ ID NO: 1), a functional fragment (BJ) of the connecting region of the TCRβ subunit, or a fragment of the constant region of the TCRβ subunit, which are sequentially connected. The amino acid sequence of BV or its variant is shown in Table 1, No. 100-147. The amino acid sequence of BJ or its variant is shown in Table 1, No. 148-160. The amino acid sequence of the TCRβ subunit constant region fragment is: EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQ AYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAW GRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS (SEQ ID NO: 3). It also independently contains one of the BV or its variants, and one of the BJ or its variants, thereby obtaining a vector encoding 624 (48×13) different TCRβ functional VJ genes. Of course, variant sequences of BV and BJ can also be used.

[0108] Table 1 Sequence information of TCRα and β subunits AV and its mutants and AJ and its mutants

[0109]

[0110]

[0111]

[0112] The corresponding position of the IMGT standard protein sequence in Table 1 refers to the position of the sequence corresponding to the human TCR gene standard sequence in the following IMGT database. That is, the sequence composition of the sequence can be determined by selecting the position in Table 1 according to the human TCR gene standard sequence in the following public IMGT database.

[0113] Human TCR gene standard sequence in the IMGT database:

[0114] AV standard sequence:

[0115] https: / / www.imgt.org / IMGTrepertoire / index.php?section=LocusGenes&repertoire=genetable&species=human&group=TRAV.

[0116] AJ standard sequence:

[0117] https: / / www.imgt.org / IMGTrepertoire / index.php?section=LocusGenes&repertoire=genetable&species=human&group=TRAJ.

[0118] BV standard sequence:

[0119] https: / / www.imgt.org / IMGTrepertoire / index.php?section=LocusGenes&repertoire=genetable&species=human&group=TRBV.

[0120] BJ standard sequence:

[0121] https: / / www.imgt.org / IMGTrepertoire / index.php?section=LocusGenes&repertoire=genetable&species=human&group=TRBJ.

[0122] The key structure diagram of the vector in the αβ TCR molecule library is as follows Figure 1 shown.

[0123] Example 2 Cloning and Construction of Melanoma Antigen Family A1 (MAGE-A1) Specific TCR (hereinafter referred to as T1367)

[0124] In this example, the method of the present invention is explained by taking the melanoma antigen family A1 (MAGE-A1) protein as an example. It should be noted that the method of the present invention is not limited to the construction of a specific protein (such as MAGE-A1), and those skilled in the art can reasonably apply it to the construction of a library of other protein TCRs based on the examples in the following examples.

[0125] (1) Screening of αβTCR vector library:

[0126] In this embodiment, according to the published literature (Obenaus M, C,Leisegang M,Chen X,etal.Identification of human T-cell receptors with optimal affinity to cancerantigens using antigen-negative humanized mice.Nat Biotechnol.2015Apr;33(4):402-7.doi:10.1038 / nbt.3147.) obtained the molecular structure information of T1367.

[0127] Among them, based on the records in the literature, the structural information of the α subunit of the T1367 molecule is: AV5*01, AJ41*01, and its CDR3 sequence is: CAESIGSNSGYALNF (SEQ ID NO: 4); the structural information of the β subunit is: BV28*01, BJ2-7*01, and its CDR3 sequence is: CASRGLAGYEQYF (SEQ ID NO: 5); the recognition epitope is KVLEYVIKV (SEQ ID NO: 6); the HLA restriction type is HLA-A*02:01. The amino acid sequence is shown in SEQ ID NO: 7.

[0128] MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASRGLAGYEQYFGPGTRLTVTEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNSGSGATNFSLLKQAGDVEENPGPTRMKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAESIGSNSGYALNFGKGTSLLVTPHIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:7)。

[0129] Based on the existing αβTCR vector library framework (see Schambach A, Wodrich H, Hildinger M, et al. Context dependence of different modules for posttranscriptional enhancement of gene expression from retroviral vectors. Mol Ther 2: 435-445, doi: 10.1006 / mthe.2000.0191 and Hildinger M, Abel KL, Ostertag W, et al. Design of 5'Untranslated Sequences in Retroviral Vectors Developed for Medical Use [J]. Journal of Virology, 1999, 73 (5): 4083-4089. DOI: 10.1128 / JVI.73.5.4083-4089.1999.), the framework vector plasmid is as follows Figure 2 As shown, the insertion position of the target fragment or exogenous expression sequence (in this embodiment, it refers to the VJ structural unit) is between BstB I and Sal I) and the αβ type TCR molecular library is constructed according to the method in Example 1. Then, the conventional methods in the art (such as sequencing or detection kits, etc.) are used to screen the vector plasmids corresponding to the T1367 molecule α subunit and β subunit structural information disclosed in the literature from the constructed αβ type TCR molecular library for subsequent construction of the T1367 expression vector. In this embodiment, the screened vector plasmids are: TCRαV5*01-J41*01 and TCRβV28*01-J2-7*01 vectors.

[0130] (2) Construction of expression vectors for the α and β subunits of the T1367 molecule:

[0131] The primer sequence for synthesizing α subunit CDR3 was designed, and the specific primer sequence was:

[0132] 5'-TGTGCCGAGAGCATCGGCAGCAACAGCGGCTACCGCCCTGAAC-3' (SEQ ID NO: 8);

[0133] 5'-CAAAGTTCAGGGCGTAGCCGCTGTTGCTGCCGATGCTCTCGG-3' (SEQ ID NO: 9).

[0134] The primer sequence for synthesizing β subunit CDR3 was designed, and the specific primer sequence was:

[0135] 5'-TGTGCCAGCAGAGGCCTGGCCGGCTACGAGCAGTAT-3' (SEQ ID NO: 10);

[0136] 5'-CAAAATACTGCTCGTAGCCGGCCAGGCCTCTGCTGG-3' (SEQ ID NO: 11).

[0137] Then, according to conventional operations in the art, the two primers of each subunit are annealed to form a double-stranded DNA containing the CDR3 sequence.

[0138] The CDR3 double-stranded DNA synthesized in the above steps is inserted into the TCR subunit vector.

[0139] The specific operations are as follows: Figure 3 As shown in the flowchart, the Goldengate kit (purchased from NEB) was used to replace the VJ linker sequence of the above TCRαV5*01-J41*01 and TCRβV28*01-J2-7*01 vectors with CDR3 double-stranded DNA using PaqCI endonuclease and T4 ligase. After selecting the vector plasmid with correct insertion, it was used to transform DH5α competent bacteria, and then LB agar plates (Amp resistance) were plated and cultured at 37°C overnight.

[0140] Then positive clones were identified: monoclonal colonies were picked after culture, cultured overnight in LB medium, plasmids (T1367 molecule α vector and β vector) were extracted, and then identified after double digestion with NsiI+NheI. Among them, the positive clone of TCRα subunit was about 6.3Kb, and after digestion, it was about 4.7Kb and 1.6Kb. The positive clone of TCRβ subunit was about 6.5Kb, and after digestion, it was about 4.7Kb and 1.8Kb.

[0141] (3) Construction of TCRα and β subunit co-expression vector (pRVTCR-T1367):

[0142] Design the primer sequence for synthesizing the α subunit. The specific primer sequence is:

[0143] 5'-GACGTGGAGGAGAACCCAGGCCCAACGCGTATGAAGACATTTGCTGGATTTTCG TTC-3' (SEQ ID NO: 12);

[0144] 5'-GGAATAAATGGCGGTAAGATGCTCG-3' (SEQ ID NO: 13).

[0145] The above primers were used to perform PCR amplification on the T1367 molecule α vector. The amplified product was 876 bp in length and was purified by gel recovery.

[0146] The recovered and purified amplified product (α vector DNA) is seamlessly cloned into the β vector to achieve co-expression of the α subunit and β subunit of the T1367 molecule. The specific steps are: the β vector obtained in step (2) is double-digested with Mlu I + Sal I to recover a 6.5Kb digestion fragment. The PCR fragment of the α vector recovered in the above step and the digestion fragment of the β vector obtained are Gibson assembled (NEB) (based on P2A connection), and the assembled product is used to transform DH5α competent bacteria, plated on LB agar plates (Amp resistance), and cultured at 37°C overnight. Pick a single clone colony, culture it in LB medium overnight, extract the plasmid, and then use BstBI + SalI double digestion for identification. Among them, the digestion products of the pRVTCR-T1367 positive clone are approximately 5.4Kb and 1.8Kb, and the digestion products of the clone that has not been successfully inserted (i.e., TCRβ vector) are approximately 5.5Kb and 1.0Kb. The positive plasmid is sequenced by Sanger sequencing to confirm that the sequence is correct.

[0147] (4) Preparation and functional analysis of T1367 TCR-T cells:

[0148] The retrovirus containing the T1367 expression vector was packaged using a retroviral packaging system. The specific operation was as follows: pRVTCR-T1367 and pRVTCR-EGFP plasmids were transfected into GALV cells using the LipofectamineTM 3000 kit (Invitrogen) (as a control, the target fragment in pRVTCR-T1367 was replaced with the EGFP sequence). The first batch of viral supernatant was collected 48 hours after transfection. The second batch of viral supernatant was collected 4 hours later. The collected viral supernatant was filtered through a 0.45 micron filter (PALL) and concentrated using a 50KD protein concentrator (Merck) to prepare a retroviral solution of pRVTCR-T1367.

[0149] PBMCs were isolated from the peripheral blood of healthy volunteers and T cell stimulation was performed in a 24-well plate coated with OKT3 antibody (Biolegend). The culture medium consisted of 1640 medium (Gibco) containing 500 IU / mL IL-2 (Peprotech), 1 μg / mL αCD28 antibody (Biolegend) and 5% fetal bovine serum (Gibco). After 48 hours of encapsulation culture, the first batch of pRVTCR-T1367 retrovirus solution was added, and the cells were infected by centrifugation at 2500 rpm for 2 hours at 32°C. After repeated infection once the next day, the cells were placed in a 37°C, 5% CO2 cell culture incubator for culture. After the 8th day, the cells were rested and the culture medium was replaced with 1640 medium containing 30 IU / mL IL-2 and 5% fetal bovine serum. After 3 days of continuous culture, the cells were collected for flow cytometry.

[0150] The flow cytometry analysis results are as follows Figure 4 As shown, the T1367 positive rate in the experimental group can reach more than 50%.

[0151] The antigen specificity of TCR-T cells was further analyzed, and the steps were as follows: the prepared T1367 TCR-T cells were co-cultured with T2 cells in a 96U-type well plate, and then the commercially available epitope peptide MAGE-A1 (278-286) (corresponding sequence is SEQ ID NO: 6) was added, and at the same time, the commercially available MART-1 (26-35) (corresponding sequence is ELAGIGILTV (SEQ ID NO: 14)) was used as a control. The culture medium used was 1640 culture medium containing 2% fetal bovine serum. After overnight culture, the supernatant was collected, and the IFN-γ production was detected using Human IFN-γ ELISA (Dayou) as a readout value for T cell activation.

[0152] The results are as follows Figure 5 The experimental group loaded with KVLEYVIKV short peptide can make T1367 TCR-T cells release a large amount of IFN-γ, while the control group has no obvious expression.

[0153] Further tetramer staining analysis was performed, the steps were as follows: the epitope peptide KVLEYVIKV was loaded with the tetramer (Acro) of HLA-A*02:01, thereby obtaining the tetramer of KVLEYVIKV / HLA-A*02:01 / PE, and the tetramer was used for flow cytometry staining of the prepared T1367 TCR-T cells, with ordinary T cells as a control. At the same time, CD3 APC was also used for staining.

[0154] The results are as follows Figure 6 As shown. T1367 TCR-T cells were PE-positive, while control T cells were PE-negative.

[0155] In summary, the above results show that, according to the structural information of the target TCR molecule, the TCR vector library constructed in the above embodiment can be used to quickly construct an expression vector of the TCR molecule, and can be successfully used for T cell function modification, that is, the preparation of TCR-T cells, so that it has the same antigen-specific recognition function as the target TCR molecule.

[0156] Comparative Example 1

[0157] In this comparative example, the α and β subunits of T1367 were constructed respectively using the method in patent document CN111315883A for comparison with the method in the above embodiment.

[0158] The specific steps are as follows: according to the design principle in CN111315883A, the first vector of αV5*01-mCA (Amp resistance), the second vector of αJ41*01 (Kana resistance), and the first vector of βV28*01-mCB (Amp resistance), the second vector of βJ2-7*01 (Kana resistance) were prepared. Then, according to its design principle, the IIS type restriction site of PaqCI was set in each vector and the sequence was matched according to the connection order of the fragments. The first vector of the subunit, the second vector, and the CDR3 double-stranded DNA with a connector were placed in a tube for Goldengate assembly overnight, and the product was transformed into DH5α competent bacteria, coated on LB agar plates (Amp resistance), and cultured at 37°C overnight. 24 clone colonies were picked from each plate, cultured overnight in LB, plasmids were extracted, and then enzyme digestion identification (BstB I+Sal I) was performed, and the correct proportion of enzyme digestion was counted.

[0159] The results are as follows Figure 7 As shown. It can be found that based on the construction method in CN111315883A, the success rate of α subunit construction is only 41.7% (10 / 24), and the success rate of β subunit construction is only 50% (12 / 24). When the method in the above embodiment of the present invention is used for construction simultaneously, under the same conditions (24 bacteria are selected for each construction reaction, and the success rate is identified by enzyme digestion), the success rate of α subunit construction is 79.1% (19 / 24), and the success rate of β subunit construction is 87.5% (21 / 24). The results are also in line with the general rule that the Goldengate assembly success rate decreases with the increase of the number of fragments.

[0160] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A library of αβ-type TCR molecules, characterized in that: The library includes a vector encoding a TCRα functional VJ gene combination and a vector encoding a TCRβ functional VJ gene combination; Wherein, the vector encoding the functional VJ gene combination of TCRα comprises the following structural units connected in sequence: A functional fragment encoding any one of the TCRα subunit variable regions numbered 001-045 in Table 1, or a variant fragment thereof; and A functional fragment encoding any one of the TCRα subunit connecting regions numbered 046-098 in Table 1, or a variant fragment thereof; Wherein, the vector encoding the TCRβ functional VJ gene combination comprises the following structural units connected in sequence: A functional fragment encoding any one of the TCRβ subunit variable regions numbered 100-147 in Table 1, or a variant fragment thereof; and A functional fragment encoding any one of the TCRβ subunit connecting regions numbered 148-160 in Table 1, or a variant fragment thereof; wherein the two structural units have at least one terminal binding site at opposite ends, and the two structural units are connected to each other through the terminal binding site.

2. The library according to claim 1, characterized in that The variant fragment has at least 85% homology with the corresponding functional fragment.

3. The library according to claim 1, characterized in that In the two structural units, a linker sequence is inserted between the functional fragment of the variable region and the functional fragment of the connecting region; preferably, the linker sequence comprises the sequence shown in SEQ ID NO:

1.

4. The library according to any one of claims 1 to 3, characterized in that In both structural units, the functional fragment of the connecting region is also connected to the constant region fragment; preferably, the constant region fragment and the functional fragment of the connecting region encode the same TCR subunit.

5. The library according to claim 4, characterized in that The vector encoding the TCRα functional VJ gene combination and / or the vector encoding the TCRβ functional VJ gene combination is further integrated into a framework vector or an expression vector, and the framework vector or expression vector includes an in vitro transcribed mRNA (ivtRNA) vector, a retroviral vector and a lentiviral vector; preferably, the vector encoding the TCRα functional VJ gene combination and the vector encoding the TCRβ functional VJ gene combination are further integrated into the same framework vector or expression vector.

6. Use of the library according to any one of claims 1 to 5 in any one of the following (1) to (4); (1) constructing αβ-type TCR molecule expression vectors or transformants; (2) Screening of αβ-type TCR molecules; (3) Construction of αβ-type TCR protein library; (4) Constructing αβ-type T cell receptors or receptor libraries.

7. A cell clone library, protein library or TCR molecule expression system constructed based on the library according to any one of claims 1 to 5; in, The cell clone library contains transformants of at least one vector according to any one of claims 1 to 5; The protein library is obtained from a transformant containing at least one vector according to any one of claims 1 to 5; The TCR molecule expression system contains the library according to any one of claims 1 to 5.

8. A method for constructing an αβ T cell receptor library, comprising: (1) designing a primer set containing a CDR3 sequence encoding a target TCR molecule, and annealing to form a double-stranded DNA, wherein the CDR3 sequence includes the CDR3 sequence of the α subunit and the β subunit of the target TCR molecule; (2) using the double-stranded DNA obtained in step (1) to replace the linker sequences in the vectors of claims 3 to 5, respectively, to obtain α-subunit and β-subunit vector plasmids; (3) Integrate the α-subunit and β-subunit vector plasmids to obtain a co-expression plasmid, and transfer the co-expression plasmid into a cell vector to obtain an αβ-type T cell receptor library.

9. The method according to claim 8, characterized in that In step (3), a TCRα subunit coding sequence and a TCRβ subunit coding sequence are integrated into the co-expression plasmid via a linker; preferably, the linker comprises an internal ribosome entry site (IRES) or a 2A peptide.

10. An αβ T cell receptor library, characterized in that: The αβ type T cell receptor library is constructed by the method of claim 8 or 9.

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