T cell antigen receptor, polymer complex thereof, and preparation method and application of polymer complex

[0169] Example 1: Construction and effect detecting CMV epitope tetramer

[0170] I. Construction of CMV epitope tetramer

[0171] 1) expressed sequence optimized HLA-A * 1101 (which is the amino acid sequence SEQ ID NO: shown as the nucleotide sequence of SEQ ID NO 24: 25 shown) of the α chain and β2m chain (amino acid sequence shown in SEQ ID 26, nucleotide sequence SEQ ID NO:: NO in Fig. 27). Wherein the configuration α chain extracellular domain linked to the corresponding sequence of the HLA α chain Avi-tag sequences, BamHI restriction sites at intervals to provide a biotinylated sites. removed β2m chain signal peptide sequence, the mature peptide sequence is in front of the two amino acids (M and A). Expression vector PET28a +, the expression of bacteria or transetta BL21. IPTG concentration of 0.5mM, induce expression 4h. Extraction α chain and β2m chain protein inclusions.

[0172] 2) CMV epitopes (antigen peptides) selection: HLA-A * 1101-type counterparts epitope ATVQGQNLK (SEQ IDNO: 13).

[0173] 3) pMHC I monomer folding and Purification: Step 2) The antigenic peptide corresponding step 1) of the β2m protein multiplex chain and α chain protein at a molar ratio of 40: 2: 1 ratio was added in order to restore the system , the folding reaction is 72h. The resulting product had Superdex75 10 / 300GL column purification. Purified product was collected avidity biotinylation kit, again to give biotinylated monomers, monomer purity gel electrophoresis.

[0174] 4) The step 3) the biotinylated streptavidin monomer with APC-labeled biotin binding reaction, to give the corresponding tetramer, designated A1101-ATVQGQNLK- tetramer (abbreviated A1101-ATV tetramer ).

[0175] Two, CMV epitope tetramer effect detection

[0176] 1. An isolated human peripheral blood mononuclear cells (PBMC) or preparation TCR-T cells, preparing a cell suspension, a cell density of 1 × 10 6 Cells / mL.

[0177] 2. The cells were centrifuged, 3000rpm, 5min. The supernatant was removed, PBS containing 1% serum with 50μL resuspended.

[0178] 3. Add incubated for 30min 2μL tetramer rt.

[0179] 4. Add 2μL CD8 antibody, incubated on ice for 20min.

[0180] 5. Add 1mL PBS, 3000rpm, 5min.

[0181] 6. The supernatant was removed, added 1mL PBS, 3000rpm, 5min.

[0182] 7. Remove the supernatant with 4% 500μL paraformaldehyde resuspended cells, the cell suspension was filtered through a membrane filter.

[0183] 8. The positive cells detected by flow cytometry.

[0184] Third, experimental results

[0185] SDS PAGE detection result of the monomer See figure 1. like figure 1, The refolding and purified by HPLC to yield monomeric clearly shows the heavy chain (C-terminus is connected Avi-tag sequence α chain extracellular region) and light chain (removal of the signal peptide chain p2M region) corresponding to the size of the protein, and high purity.

[0186] The constructed tetramers are HLA type corresponding fished PBMC cells were incubated with specific T cells. A1101 type corresponding TCR (C44-TCR) can be simultaneously the same epitope independent development and commercialization of tetramers (MBL, TS-M012-1) are identified (see figure 2 ), And the commercialization of tetramer-positive cells accounted for 90.5% of infected cells, independent research and development tetramer-positive cells accounted for 91% of infected cells. C45-TCR see results Figure 4.

[0187] Example 2: Construction pHAGE-TCR-RFP vector

[0188] A, α and β Get CMV pp65 gene fragments specific for an epitope of the TCR

[0189] 1) self-made or purchased from MBL from our HLA-A * 1101-ATVQGQNLK- tetramer, according to the product specification and the peripheral blood staining, tetramer staining positive T cells were single cell sorting flow, reverse transcription to obtain cDNA ( IV Reverse Transcriptase, Invitrogen). The multiplex PCR (Multiplex PCR) principle, variable region fragment was amplified by two TCRβ gene PCR (KOD-Plus-Neo, TOYOBO).

[0190] Reverse transcription primers: TRBC1-TCAGGCAGTATCTGGAGTCATTG (SEQ ID NO: 136)

[0191] PCR amplification primers:

[0192] The upstream primer 1: T cell receptor variable region β --TRBV_F1 (SEQ ID NO: 56 to 95, wherein SEQ ID NO: 60,59, respectively, may be prepared separately reacting C44, C45 of the variable region of the β gene fragment as a TRBV_F1)

[0193] Upstream primer 2: T cell receptor variable region β --TRBV_F2 (SEQ ID NO: 96 to 135 wherein SEQ ID NO: 102,100 may be separately prepared separately reacting C44, C45 of the variable region of the β gene fragment as a TRBV_F2)

[0194] Downstream primer 1: T cell receptor β constant region --TRBC2-GCACCTCCTTCCCATTCACC (SEQ ID NO: 137)

[0195] Downstream primer 2: T cell receptor β constant region -TRBC3-GCTTCTGATGGCTCAAACACAG (SEQ ID NO: 138)

[0196] Specifically, based on the PCR polymerase KOD-Plus-Neo product specifications, 20 L of a PCR system, the annealing temperature was 60 ℃, 30 cycles. Taking a first round PCR reaction products [mu] L, as a template in two PCR, two PCR system was 30 L, the annealing temperature was 60 ℃, 30 cycles. The second PCR product was then run an agarose gel, the bands corresponding to the size for Gel Extraction (TianGen gel extraction kit), sent to sequencing, the sequencing primer downstream primer 2. Obtained TCRβ gene sequence. β chain nucleotide sequence of C44, C45 as SEQ ID NO: 38,39 shown in FIG.

[0197] 2) as described above, performs tetramer staining positive T cells obtained by reverse transcription of cDNA ( IV ReverseTranscriptase, Invitrogen). According to product brochures gene fragment was amplified by two TCRα PCR (KOD-Plus-Neo, TOYOBO).

[0198] Reverse transcription primer: T-cell receptor α constant region -TRAC1-CGACCAGCTTGACATCACAG (SEQ ID NO: 139)

[0199] PCR amplification primers:

[0200] Upstream primer 3: T cell receptor variable region of the α -TRAV_F1 (SEQ ID NO: 142 to 186 wherein SEQ ID NO: 183,177, respectively, may be used as separately reacting C44, variable region fragment prepared α gene of C45 TRAV_F1 )

[0201] Upstream primer 4: T cell receptor variable region of the α -TRAV_F2 (SEQ ID NO: 42-55,187 to 214, wherein SEQID NO: 52,47, respectively, may be separately prepared α as a variable region gene C44, C45 of fragments TRAV_F2)

[0202] Downstream primer 3: T-cell receptor α constant region -TRAC2-GTTGCTCTTGAAGTCCATAGACCTC (SEQ ID NO: 140)

[0203] Downstream primer 4: T-cell receptor α constant region -TRAC3-CAGGGTCAGGGTTCTGGATA (SEQ ID NO: 141)

[0204] Specifically, based on the PCR polymerase KOD-Plus-Neo product specifications, 20 L of a PCR system, the annealing temperature was 60 ℃, 30 cycles. Taking a first round PCR reaction products [mu] L, as a template in two PCR, two PCR system was 30 L, the annealing temperature was 60 ℃, 30 cycles. The second PCR product was then run an agarose gel, the bands corresponding to the size for Gel Extraction (TianGen gel extraction kit), sent to sequencing, the sequencing primer downstream primer 4. TCRα obtained gene sequence. Specifically, α chain nucleotide sequence of C44, C45 as SEQ ID NO: 40,41 shown in FIG.

[0205] Second, the vector construct pHAGE-TCR

[0206] The (sequence comprising fp2A) overlap-PCR amplification (KOD-Plus-Neo, TOYOBO) TCRβ, fp2A, TCRα obtained over a long primer TCRβ-fp2A-TCRα fragment, were designated as C44 or C45.

[0207] Amplification primers comprises an upstream primer 5 (SEQ ID NO: 14 (C44), SEQ ID NO: 15 (C45)), downstream primer 5 (e.g., SEQ ID NO: 16 shown), an upstream primer 6 (SEQ ID NO: 17 (C44), SEQ ID NO: 18 (C45)), downstream primer 6 (e.g., SEQ ID NO: 19 shown).

[0208] Specifically, TCRα and TCRβ respectively first amplification primer and primer 5 out of 6, 50 L of the PCR system, the annealing temperature was 60 ℃, 30 cycles. The PCR product was run on a gel recovered (TianGen gel extraction kit), were taken recovered product 1μL as a template, Coverlap PCR using an upstream primer 5 and downstream primers. 6, PCR system was 50 L, the annealing temperature was 60 ℃, 30 cycles . Agarose gel to obtain a band of about 1800bp performs Gel Extraction.

[0209] The lentivirus vector pHAGE-IRES-RFP double digested with NotI and NheI. 40 L of enzyme systems, which contain NheI and NotI, respectively, 1.5 L, plasmids 2-3μg, 37 ℃ digested 6h, followed by the addition of alkaline phosphatase in the system, there 1μL (NEB), for 1h to reduce self-ligated plasmid, the enzyme after cutting the plasmid recovered gel was run, with nanodrop concentrations tested, as the backbone for the construction of plasmid.

[0210] The Clone Express II One Step Cloning kit product specification, the overlap with the TCR through the digestion of linearized vector pHAGE-IRES-RFP connected (see image 3 ), Transformed into Stbl3 strain cultured 12-16h on ampicillin-containing LB plate, picked monoclonal bacteria were sequenced, the sequencing primer selected primer seq-pHAGE-F and seq-pHAGE-R on pHAGE carrier, and a downstream primer 4. To obtain the corresponding the TCR, abbreviated as C44, respectively (nucleotide sequence as SEQ ID NO: 31, the amino acid sequence as SEQ ID NO: 30 shown), C45 (its nucleotide sequence SEQ ID NO: 33 Suo shows the amino acid sequence as SEQ ID NO: 32 shown).

[0211] Example 3: Expression and affinity membrane pMHC tetramer staining of TCR

[0212] 1 construct cell lines JurkatT knockout endogenous TCR

[0213] Based on sequence features Jurkat cells TCR in the design guide sequence of α-chain and β-chain constant region (TRA_oligo1-CACCGTCTCTCAGCTGGTACACGGC (SEQ ID NO: 20), TRA_oligo2-AAACGCCGTGTACCAGCTGAGAGAC (SEQ ID NO: 21), TRB_oligo1-CACCGGGCTCAAACACAGCGACCTC (SEQ ID NO : 22), TRB_oligo2-AAACGAGGTCGCTGTGTTTGAGCCC (SEQ ID NO: 23)).

[0214] The synthetic sequence of α-chain and β guide chain are constructed to sgRNA-LentiCRISPR-puro sgRNA-LentiCRISPR-BSD and lentivirus vector and packaging plasmids psPAX2, pMD2.G and PEI transfection reagent according to a certain proportion of co-transfection of 293T cells, cells were harvested 48h and 72h of the culture supernatant, after two viruses simultaneously infect humans JurkatT concentrated cell lines. With the appropriate concentration of puromycin and blasticidin medicated killing infected 48h, until the two drugs respective control cells died. 96-well plate and cultured cells viable cell sorting by flow menu. For obtaining the monoclonal cell line, performed with TCRα chain antibody and its expression β chain are identified, both strands of endogenous TCR deficient cell line is the available inter knock Jurkat T cells, designated JC5.

[0215] 2. Construction of stable integration of the CMV-TCR cell line JC5

[0216] Example 2 Construction of the C44 above embodiment, C45, etc. pHAGE-TCR plasmids, pMD2.G and PEI transfection reagent according to certain proportions and packaging plasmids psPAX2, 293T cells were transfected. 48h and 72h to charge the cell culture supernatant, and concentrated JC5 infected cells in logarithmic growth phase (MOI = 0.3). Three days after infection, cells were with anti-human and anti-human CD3 antibody staining TCRαβ flow, will be cultured sorted TCR expression level of the same cell, i.e. JC5-TCR cell line.

[0217] 3, TCR affinity and detection on the membrane where

[0218] Take 1 × 10 6 JC5-TCR cells, with Brilliant Violet 421 TManti-human TCRαβ (Biolegend) and the corresponding tetramer CMVpp65pMHC -APC (Tetramer-APC) staining followed by flow cytometry.

[0219] Depend on Figure 4 , 5 We found for CMV pp65 HLA-A * A1101 ATVQGQNLK specific C44-TCR, C45-TCR expression can be properly prepared and appear outside the cell membrane, and the corresponding tetramer probe having a certain affinity. In summary The results show that the application of the present embodiment obtained in Production Example TCR has a good affinity.