Construction and application of novel T cell receptor fusion construct (TRuC)-T cell containing CD28 or 4-1BB costimulatory domain

Abstract

The invention discloses construction and application of two novel T cell receptor fusion constructs (TRuC)-T cells containing a CD28 or 4-1BB costimulatory structural domain. According to the invention, a novel TRuC28-T cell and a TRuC4-1BB-T cell are constructed by adding a CD28 or 4-1BB costimulatory structural domain to the C terminal of a CD3 epsilon structural domain in a TRuC cell. Compared with original TRuC-T cells, the novel TRuC-T cells can simultaneously integrate a first signal and a second signal to activate T cells, so that the cracking capacity of the novel TRuC-T cells on target cells is remarkably enhanced, the in-vivo durability of the novel TRuC-T cells is remarkably prolonged, disease recurrence is effectively prevented and treated, and the curative effect of the TRuC-T cells is improved.

Description

Technical Field

[0001] This patent belongs to the field of biotechnology engineering, and specifically relates to the construction and application of novel T cell receptor fusion constructs (TRuC)-T cells containing CD28 or 4-1BB costimulatory domains. Background Art

[0002] Chimeric antigen receptor (CAR) T cell therapy is one of the revolutionary therapies for treating cancer and has achieved milestone breakthroughs in hematological malignancies. The basic principle of CAR-T cell technology is mainly to install a positioning and navigation device, CAR (chimeric antigen receptor), on the surface of T cells through genetic engineering technology; CAR-T cells use their CAR to specifically recognize tumor cells in the body and release various effector factors through immune effects, thereby efficiently killing tumor cells. CAR is the core component of CAR-T, endowing T cells with the ability to recognize tumor antigens in an HLA-independent manner. Severe cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS) and other toxic and side effects, easy recurrence after remission, and poor efficacy in solid tumors are major challenges currently faced by CAR-T therapy. The efficacy of CAR-T cell immunotherapy in solid tumors is far less than that in hematological malignancies. The important reasons include tumor heterogeneity, lack of specific tumor antigens, limited chemotaxis and penetration of CAR-T cells to tumor lesions, poor persistence, and tumor immunosuppressive microenvironment.

[0003] TCR is composed of 6 types and a total of 8 subunits, and each subunit interacts with each other in a unique but coordinated manner to precisely regulate the quality, intensity, and duration of intracellular signal transduction in T cells. TRuC-T cells overcome both HLA dependence and have the potential not to induce CRS. TRuC mainly includes two parts: an extracellular antigen-binding region and CD3ε. TRuC-T cells make full use of the natural TCR signaling pathway, can induce a controllable anti-tumor immune response, and have lower toxic and side effects. Therefore, both the efficacy and safety are superior to classical CAR-T cells: 1) They can more effectively recognize tumor cells without being restricted by HLA; 2) Similar to the activation of conventional T cells, using the activation and regulation mechanism of the complete TCR, a large amount of cytokine release can be avoided, resulting in better safety; 3) They can quickly migrate to the tumor site, enhance penetration, and maintain a relatively persistent reactivity, so that tumors (including solid tumors) can be efficiently eliminated.

[0004] T cells require at least two signals to be fully activated: the first signal is through the TCR, and the second signal or co-stimulation is mediated by the ligation of CD80 or CD86 to CD28. T cells are unresponsive to the induction of TCR signal alone. The first-generation CAR is only coupled to the TCR-CD3ζ chain and can provide a stimulatory signal similar to that of the entire CD3 complex. However, due to the lack of the second signal, the persistence and anti-tumor effect of such CAR-T cells in vivo are very limited, and there is no obvious clinical efficacy. To enhance the stimulatory effect, the second-generation CAR adds a co-stimulation module on the basis of the first-generation CAR, enabling the CAR-T cells to expand and improve their persistence, thereby significantly improving the clinical efficacy.

[0005] In view of the necessity of the second signal in the activation of T cells (including CAR-T), the present invention has successfully developed and optimized a novel TRuC-T cell containing the second signal CD28 or 4-1BB. Summary of the Invention

[0006] Aiming at the serious toxic and side effects of CAR-T therapy (such as CRS and ICANS) and its limited efficacy against solid tumors, the present study invented novel TRuC(CD28)-T cells and TRuC(4-1BB)-T cells containing the CD28 or 4-1BB co-stimulation domain (taking TRuC(CD28)-T cells and TRuC(4-1BB)-T cells targeting BCMA as examples).

[0007] Plasmid vectors of plenti-anti-BCMA TRuC, plenti-anti-BCMA TRuC(CD28), and plenti-anti-BCMA TRuC(4-1BB) were constructed by genetic engineering techniques, and then lentiviral vectors were packaged by a large-scale production process of high-titer and high-purity lentivirus to transduce T cells. After culturing for five days, the expression rates of anti-BCMA TRuC were detected by flow cytometry respectively, and the in vitro killing effect of anti-BCMA TRuC cells on BCMA-positive cells and the efficacy on the NSG mouse model of human transplanted tumors (such as multiple myeloma) were verified in vitro. Compared with the prior art, the present invention has the following advantages.

[0008] 1. The present invention constructs novel TRuC(CD28)-T cells and TRuC(4-1BB)-T cells containing the co-stimulatory domains of CD28 or 4-1BB, enabling TRuC(CD28)-T cells and TRuC(4-1BB)-T cells to simultaneously possess the first signal and the second signal. Among them, TRuC(CD28) includes an antigen recognition domain, CD3ε, and the intracellular domain of CD28, encoded by the amino acid sequence of SEQ ID NO.1. TRuC(4-1BB)-T cells include an antigen recognition domain, CD3ε, and the 4-1BB domain, encoded by the amino acid sequence of SEQ ID NO.2.

[0009] 2. The present invention directly connects CD3ε through the 4-1BB or CD28 domain, making TRuC(CD28)-T cells and TRuC(4-1BB)-T cells have strong killing ability, long-term tumor suppression in vivo, and good cell persistence.

[0010] 3. The present invention directly connects the internal domain within the CD28 domain to the TRuC structure, making TRuC(CD28)-T cells proliferate rapidly. BRIEF DESCRIPTION OF THE DRAWINGS: Figure 1 It is a schematic diagram of plenti-anti-BCMA TRuC, plenti-anti-BCMA TRuC(CD28), and plenti-anti-BCMA TRuC(4-1BB) plasmids, and the expression of TRuC on the surface of BCMA TRuC-T cells, BCMA TRuC(CD28)-T cells, and BCMA TRuC(4-1BB)-T cells is evaluated by flow cytometry.

[0012] Figure 2 It is the killing activity of BCMA TRuC-T cells, BCMA TRuC(CD28)-T cells, and BCMA TRuC(4-1BB)-T cells against luciferase-expressing target cells MMIS, U266, and BCMA-MDA-MB231 (triple-negative breast cancer) under different effector-to-target ratios measured by the luciferase method, and the cytokine secretion after co-incubation of BCMA TRuC-T cells, BCMA TRuC(CD28)-T cells, and BCMA TRuC(4-1BB)-T cells with target cells is verified by ELISA.

[0013] Figure 3 It is the determination of CD28 promoting the proliferation and activation of BCMA TRuC-T cells and 4-1BB maintaining the low differentiation of BCMA TRuC-T cells by CFSE and flow cytometry.

[0014] Figure 4BCMA TRuC-T cells, BCMA TRuC(CD28)-T cells, and BCMA TRuC(4-1BB)-T cells were used to treat NSG mice bearing multiple myeloma tumors, and the therapeutic effect was evaluated by in vivo imaging technology.

[0015] Figure 5 BCMA TRuC-T cells, BCMA TRuC(CD28)-T cells, and BCMA TRuC(4-1BB)-T cells were used to treat NSG mice bearing BCMA-MDA-231 tumors, and the therapeutic effect was evaluated by in vivo imaging technology. Specific implementation methods

[0016] Main experimental materials: EcoRⅠ-HF and PmeⅠ-HF restriction enzymes (NEB), seamless cloning enzyme (Heyuan Biotechnology), high-fidelity Prime GXL STAR enzyme (TAKARA), TransStbl3 competent cells (TransGen Biotech Co., Ltd.), Plasmid Mini Kit I (OMEGA), EndoFree® Plasmid Maxi Kit (QIAGEN), DMEM, RPMI-1640, Opti-MEM medium, Gibco FBS (Thermo Fisher Scientific), Sanger sequencing (Shanghai Sunny Biotechnology Co., Ltd.), Nacl, yeast powder, peptone, EDTA, NaOH (Shanghai Sangon Biotech Co., Ltd.), primers (Genewiz, Inc.).

[0017] Construction of recombinant plasmids Construction of plenti-anti-BCMA TRuC, plenti-anti-BCMA TRuC(CD28), and plenti-anti-BCMA TRuC(4-1BB) recombinant plasmids: The sequences of anti-BCMA TRuC, anti-BCMA TRuC(CD28), and anti-BCMA TRuC(4-1BB) were synthesized by Suzhou GeneScript Biotechnology Co., Ltd. The plenti-EF1a-MCS vector was double digested with EcoR I-HF and Pme I-HF. The reaction conditions were 37°C for 2 h and 65°C for 20 min. The enzyme digestion system is shown in Table 5. The target fragment was amplified by PCR (primers are shown in Table 1), and the reaction system is shown in Table 2. The digested product and the PCR product were subjected to 1% agarose gel electrophoresis to obtain the vector fragment, and then the Plenti vector fragment and the PCR product were recovered using the XYGENE Gel Extraction Kit (the operation steps are shown in Table 3 below). The concentration and purity were detected. The vector fragment and the target fragment were ligated by seamless cloning (the system is shown in Table 4), at 37°C for 1 h, and then plasmid transformation was carried out (after the seamless cloning product was placed on ice for 5 min, it was transferred into 50 μl of TransStbl3 competent cells, placed on ice for 30 min, heat shocked at 42°C for 45 s, then placed on ice for 5 min, 500 μl of LB was added, and it was activated in a shaker at 37°C and 225 rpm / min for 1 h. Then, it was centrifuged at 5000 rpm and 20°C for 5 min, the supernatant was discarded, the remaining bacterial solution was mixed evenly and plated, and cultured at 37°C for 12 - 14 hours). Single colonies were picked for bacterial liquid amplification at 37°C and 250 rpm / min for 12 h - 14 h, plasmid extraction was performed, and finally, it was identified by digestion with AFLII-HF restriction endonuclease, and finally Sanger sequencing was carried out. The plasmid schematic diagram is as shown in Figure 1 Table 1 Primer Sequences Table 2 PCR System Reagent Volume GXL buffer 5x 5 μl dNTP 2 μl Prime GXL STAR 1 μl Template 1 μl Primer F (100 μm) 0.1 μl Primer R (100 μm) 0.1 μl <![CDATA[ddH 2 o]]> 15.8 μl Total volume 25 μl Table 3. Gel Extraction 1. Solubilize gel Add 3 times the volume of DE-A (100 mg = 100 μl) of the gel volume to the EP tube containing the gel, and then solubilize the gel at 65 °C for 5 - 10 min. 2. Bind DNA After the gel melts, add DE-B with a volume of 0.5 times that of DE-A, mix well, add the mixture to the nucleic acid binding column, and centrifuge at 12,000 x g for 1 min 3. Wash the membrane <![CDATA[Add 500 μl of W 1 , centrifuge the membrane at 12,000 g for 30 s to wash the membrane]]> 4. Wash the membrane (desalting) <![CDATA[Add 700 μl of W 2 , centrifuge the membrane at 12,000 g for 30 s to wash the membrane]]> 5. Wash the membrane (desalting) Repeat step 4 6. Air dry Centrifuge and air dry at 12,000 g for 1 min 7. Elute Elute DNA with 26 μl of eluent Table 4. Seamless Cloning System Reagent Volume Target fragment 1 Linearly digested vector 2 Seamless cloning enzyme 2 Seamless cloning buffer 4 ddH2O 11 Total volume 20 μl Table 5. Restriction Enzyme Digestion System Reagent Volume EcoR I-HFPme I-HF 1 μl 1 μl Cutsmart buffer 2 μl Plasmid 2 μl <![CDATA[ddH 2 O]]> 14 μl Total volume 20 μl II. Transduce 293T cells with the Plenti vector plasmid and the helper plasmid to package lentivirus, and transfect the packaged lentivirus into Jurkat Cell to calculate the virus titer.

[0018] (1) Culture 293T cells in a 15-cm cell culture dish. When the 293T cells grow to 70% confluence in the entire field of view, resuspend 60 μg of PEI with 1.5 ml of PBS, and resuspend the total mass of 20 μg of the Plenti vector plasmid and the helper plasmid with 1.5 ml of PBS; (2) Let it stand at room temperature for 5 min, add the PBS-PEI mixture to the PBS-DNA mixture, and let it stand at room temperature for 20 min; (3) Prepare OPTI-DMEM complete medium and warm it up in a 37 °C incubator. Aspirate and discard the original DMEM medium in the 293T cells, and add OPTI-DMEM along the wall of the dish to the 293T cells; (4) Add the PEI-DNA-PBS mixture to the culture dish and culture it at 37 °C for 48 h; (5) Collect the lentivirus in the supernatant into a 50 ml centrifuge tube, and then add 20 ml of medium to incubate for 24 h to collect the virus within 72 h; (6) Centrifuge at 1500 rpm for 5 min to remove cell debris, or filter it through a 0.45 µm filter with a syringe, and concentrate the virus at 4 °C; (7) Aspirate and discard the supernatant, and resuspend the virus by adding Vivo complete medium or AIM-V complete medium at a ratio of 1:200 - 1:400 (it is best to add 1% HEPES); (8) Aliquot the virus into 1.5 ml Ep tubes, store it at -80 °C, and avoid repeated freezing and thawing (freezing and thawing reduces the titer by one order of magnitude). Leave a little virus for the next virus titer detection experiment; (9) After centrifuging Jurkat cells at 1500 rpm for 5 min, discard the supernatant, resuspend them in 1 ml of 1640 medium, and count; (10) Add 0.5x10 6 Jurkat cells to a 96-well plate, add the virus at gradient ratios of 1:50, 1:500, 1:1000, 1:2000, etc., and then supplement the medium to a total volume of 200 µl per well; (11) Add 0.1 µl of PolybreneB protein to promote transduction to each well (0.1 µl / 200 µl system); (12) Centrifuge the 96-well plate at 1200 g for 90 min at 32 °C. After centrifugation, incubate it in a 37 °C incubator for 4 h; (13) Pipette and mix the Jurkat cell suspension in each well of the 96-well plate, transfer it to a 1.5 ml Ep tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend it in 1 ml of 1640 complete medium, and then transfer it to a 24-well plate for expansion culture for 48 h at 37 °C. III. Isolate peripheral blood mononuclear cells (PBMCs) from healthy individuals by density gradient centrifugation, transfect T cells with lentivirus, and detect the expression of CAR on the surface of T cells (1) Take 10 ml of peripheral blood from a healthy individual into an EDTA-Na2 anticoagulant tube and mix it with DPBS at a ratio of 1:1; (2) Take four 15-ml sterile centrifuge tubes, add 5 ml of Ficoll separation solution to each tube, and slowly add the mixed solution of peripheral blood and DPBS to the surface of the Ficoll separation solution, taking care not to disrupt the liquid surface. (3) Centrifuge horizontally at 800 g for 20 min at 25 °C, and adjust both the acceleration and deceleration to "0". (4) After centrifugation, use a Pasteur pipette to aspirate the white flocculent layer (i.e., the PBMC layer) in the centrifuge tube and transfer it to a new sterile centrifuge tube. Add PBS and centrifuge the PBMC twice to wash. (5) Centrifuge horizontally at 1500 rpm for 5 min, discard the supernatant, add 1 ml of Buffer1 (DPBS containing 5% FBS), and resuspend and count the PBMC. (6) Use flow cytometry to determine the proportion of CD3-positive cells in PBMC. Add CD3 / CD28 beads to the cell suspension at a ratio of CD3 / CD28 dynabeads: CD3-positive cells = 3:1 (add 30 μl of beads for every 10 6 CD3-positive cells), and rotate and shake at a speed of 1 rpm at 4 °C for 30 min to allow the magnetic beads to fully contact and bind to the cells. (7) After 30 minutes, add enough (more than 1 ml) Buffer1 to the test tube, then place the test tube on a magnetic stand and rotate it left and right for 1 - 2 minutes, and aspirate and discard the supernatant. (8) Prepare Vivo complete medium: Vivo empty medium + 5% FBS + 1% HEPES + 1% sodium pyruvate + 1% non-essential amino acids + 1:30 glutamine + 1:10000 IL-2 + 1:2000 IL-7 + 1:2000 IL-15, and resuspend the cells and magnetic beads with Vivo complete medium and count. (9) Add medium to make the concentration of CD3-positive cells between 0.5 - 1×10 6 / ml. The seeding cell concentration is 0.5 - 1.0×106 / ml, and place it in an incubator at 37 °C for culture. (10) Culture T cells for 24 - 36 h at 5% CO2 and 37 °C. (11) Within 24 - 36 h, transduce the TAC lentiviral vector with an MOI value of 40. MOI (multiplicity of infection) = [virus titer × virus volume (ml)] / number of cells. (12) After centrifugation at 1200 xg for 90 min at 4 °C, incubate in an incubator at 37 °C until the cells in the 96-well plate are confluent, then transfer them to a 24-well plate, and measure the transduction rate of TRuC at 5 - 7 days. IV. Detection of TRuC-T cell activation by flow cytometry (1) Pipette TRuC-T cells and Mock-T cells from the culture plate into the EP tube. The number of cells required for staining each well is approximately 0.1×106 - 0.2×106 cells.

[0019] (2) Centrifuge at 1500rpm for 5min (or 2500rpm for 3min), discard the supernatant, and resuspend the cells in 100ul flow cell resuspension buffer (1% serum + PBS).

[0020] (3) Repeat the previous step.

[0021] (4) Prepare flow cytometry antibodies, add to the resuspended cells, and place at 4°C for 20 minutes.

[0022] (5) Add 150ul flow cell resuspension solution (1% serum + PBS) to terminate staining, centrifuge at 1500rpm for 5min (or 1800rpm for 3min), and discard the supernatant.

[0023] (6) Add 100ul of flow cytometry resuspension solution to resuspend the cells, centrifuge at 1500rpm for 5min (or 1800rpm for 3min), and discard the supernatant. Add 200ul of flow cytometry resuspension solution to resuspend the cells and test on the machine. The results are as follows Figure 3 . V. CFSE Cell Proliferation Assay (1) Prepare Buffer I (PBS containing 0.5% FBS), dilute 10mM / ml CFSE to 5uM / ml with PBS; (2) Take MOCK-T cells and TRuC-T cells and place them in a 15 ml centrifuge tube. After demagnetization by a magnetic rack, centrifuge at 1500 rpm for 5 min. (3) Discard the supernatant, resuspend the cells with 1ml Buffer I, take 10ul for counting, and centrifuge at 1500rpm for 5min; (4) Discard the supernatant, resuspend the cells with diluted CFSE (106 cells plus 1 ml CFSE), adjust the cell density to 1×106 cells / ml and transfer to a 1.5 ml EP tube, incubate at 4°C in dark for 10 min, and mix thoroughly during this period; (5) Transfer the cells in the EP tube to a 15ml centrifuge tube, add 8-10ml Buffer I to terminate the reaction, and centrifuge at 1500rpm for 5min; (6) Discard the supernatant, add 10 ml of Buffer I, mix thoroughly by pipetting, and centrifuge at 1500 rpm for 5 min; (7) Discard the supernatant, resuspend the cells in T cell culture medium, and take some cells to detect whether CFSE labeling is successful by flow cytometry; (8) and 0.5×10 6 ​Seed the cells at a density of / ml into 24-well plates and place them in the incubator for further culture; (9) On days 3, 5, and 7, collect some cells and detect the proliferation of T cells by flow cytometry. The results are as Figure 3 . VI. Detect the killing effect of TRuC-T cells on MMIS and BCMA-MDA-MB231 target cells by luciferase method (1) Digest the target cells from the culture flask into a centrifuge tube, centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0024] (2) Resuspend the target cells with 1 ml of complete medium for target cells and count the cells.

[0025] (3) Plate the cells: In a 96-well white plate, add 0.01×106 target cells to each well and fill all wells with medium to 200 μl.

[0026] After placing the target cells for 12 h and waiting for them to adhere, remove and discard half of the medium along the wall.

[0027] (5) Pipette TRuC-T cells and Mock-T cells from the culture flask into a centrifuge tube, centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0028] (6) Resuspend TRuC-T cells and mock T cells with 1 ml of complete medium for T cells and count the cells.

[0029] (7) Calculate the number of TRuC-T cells according to the expression efficiency of TRuC detected by flow cytometry.

[0030] (8) Plate the TRuC-T cells and fill the medium to 200 μl per well.

[0031] The well settings are as follows: (9) After co-incubation for 20 - 24 h, centrifuge at 1800 rpm for 3 min and discard the supernatant.

[0032] (10) Add 200 μl of detection medium (substrate: medium = 1:200) to each well, and place it at room temperature in the dark for 10 min.

[0033] Detect the bioluminescence value using a full-wavelength microplate reader.

[0034] Calculate the killing efficiency: Killing efficiency % = (value of only target cell wells - value of detection wells) × 100 / (value of only target cell wells - value of cell-free wells) The results are as Figure 2 VII. Experiment on NSG mice with multiple myeloma (1)Prepare MMIS-luc-GFP cells expressing luciferase and 4-6-week-old NSG mice in advance.

[0035] (2)Place 4-6-week-old NSG mice in the laboratory for 3-7 days to acclimatize them to the environment, observe their status every day, and avoid stress.

[0036] (3)Wash MMIS-luc-GFP cells twice with pre-cooled PBS, count them, take out an appropriate number of cells, centrifuge at 1500 rpm for 5 min, and carefully discard the supernatant.

[0037] (4)Resuspend the cells with an appropriate amount of PBS, mix the cells well, and place them on ice.

[0038] (5)Inject the mixed cell suspension into the tail vein of the mice using a syringe, and observe the mice every day.

[0039] (6)On the 5th to 7th day after tumor formation, image using the IVIS system to observe the tumor formation in the mice.

[0040] Wait for the tumors in the mice to form, prepare TRuC-T cells and mock-T cells, and inject them into the mice through the tail vein.

[0041] Every 5-7 days after treatment, image using the IVIS system to observe the regression of the tumors in the mice. The results are as Figure 4 .

[0042] VIII. Experiment on BCMA-MDA-MB231 NSG mice (1)Prepare BCMA-MDA-MB231 cells expressing luciferase and 4-6-week-old NSG mice in advance.

[0043] (2)Place 4-6-week-old NSG mice in the laboratory for 3-7 days to acclimatize them to the environment, observe their status every day, and avoid stress.

[0044] (3)Wash BCMA-MDA-MB231 cells twice with pre-cooled PBS, count them, take out an appropriate number of cells, centrifuge at 1500 rpm for 5 min, and carefully discard the supernatant.

[0045] (4)Resuspend the cells with an appropriate amount of PBS and Matrigel, mix the cells well, and place them on ice.

[0046] (5)Inject the mixed cell suspension subcutaneously into the mice using a syringe, and observe the mice every day.

[0047] (6)On the 5th to 7th day after tumor formation, image using the IVIS system to observe the tumor formation in the mice.

[0048] (7) Wait for the mouse tumor to form, prepare TRuC-T cells and mock-T cells, and inject them into the mice via the tail vein.

[0049] Every 5 - 7 days after treatment, use the IVIS system for imaging to observe the regression of the mouse tumors. The results are as Figure 5 .

Claims

1. A novel TRuC-T cell vector comprising a CD28 co-stimulatory domain, characterized in that: The structure includes an antigen binding domain, CD3ε, and an intracellular CD28 domain. The amino acid sequence of the novel TRuC containing the CD28 co-stimulatory domain is SEQ ID NO.

1.

2. A novel TRuC-T cell vector comprising a 4-1BB co-stimulatory domain, characterized in that: The structure includes an antigen binding domain, CD3ε, and an intracellular 4-1BB domain. The amino acid sequence of the novel TRuC containing the 4-1BB intracellular domain is SEQ ID NO.2.

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