Combination sequence of intracellular structural domain, CAR plasmid, application of CAR plasmid and CAR-T transformation method

Abstract

The invention provides a combined sequence of intracellular domains, a CAR plasmid and application thereof, and a CAR-T transformation method, and belongs to the field of molecular biology. Firstly, two different intracellular domains are designed, and the two different intracellular domains comprise different costimulatory domains, different signal transduction regions and combination forms of the costimulatory domains and the signal transduction regions; and then carrying out lentivirus packaging on the designed intracellular domain combination sequence to obtain CAR plasmids, carrying out T cell transduction according to a specific proportion, realizing co-expression of two CAR molecules targeting different intracellular domains of the same antigen on the surface of a T cell in the human T cell, and transforming into a single-target heterotype intracellular domain parallel CAR-T cell. Compared with the conventional CAR-T cell, the CAR-T cell has the advantages that the killing ability and the cruising ability are improved, and the depletion condition of the CAR-T cell can be effectively delayed.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and specifically relates to a combination sequence of intracellular domains, and also relates to a group of CAR plasmids comprising the combination sequence of intracellular domains, as well as the application of the combination sequence of intracellular domains or CAR plasmids, and a CAR-T transformation method. Background Art

[0002] Compared with traditional therapies, tumor immunotherapy has fewer side effects and more obvious efficacy. It is gradually becoming the development direction of future tumor treatment and is regarded as the fourth largest tumor treatment technology after surgery, radiotherapy and chemotherapy. Among them, chimeric antigen receptor T cell (CAR-T) therapy has taken the lead in many immunotherapies, such as the technical solution provided by the invention patent with publication number CN105968189A. However, after CAR-T cell immunotherapy was applied in clinical practice, there are still many common problems that have not been well solved, such as how to prolong the retention time of CAR-T cells in the body and how to solve the rapid exhaustion of CAR-T cells.

[0003] The basic structure of CAR-T cells includes three main components, namely ①scFv fragment, which is responsible for recognizing and binding antigens; ②transmembrane domain anchored on the cell membrane; ③intracellular domain responsible for initiating signal cascade reactions. Currently, the recognized structures include one to three generations of CAR-T, and other modifications are attributed to the fourth or fifth generation of CAR-T. The main changes in the first to third generations of CAR-T are in the intracellular domain responsible for initiating signal cascade reactions. The first generation of CAR-T contains only the ζ signal transduction fragment in the CD3 complex; the second generation is a series of scFv, a CD28 or 4-1BB co-stimulatory molecule fragment and downstream CD3ζ; the third generation is a series of scFv, CD28 and 4-1BB two co-stimulatory molecules and downstream CD3ζ. The choice of co-stimulatory molecules in series can also be OX40, ICOS and CD27. There is no recognized structure for the fourth or fifth generation of CAR-T. It is often based on the expression of CAR molecules to overexpress some cytokines or chemokines, or add suicide genes for more precise regulation. Summary of the invention

[0004] Based on the above problems in the prior art, the present invention provides a combination sequence of intracellular domains, which comprises a CD28 intracellular domain, a 41-BB intracellular domain and a CD3ζ intracellular domain; the nucleotide sequence of the CD28 intracellular domain is shown in SEQ ID NO.13, the nucleotide sequence of the 41-BB intracellular domain is shown in SEQ ID NO.12, and the nucleotide sequence of the CD3ζ intracellular domain is shown in SEQ ID NO.14.

[0005] The present invention also provides a group of CAR plasmids, wherein the plasmid sequences thereof comprise the combined sequences of the intracellular domains as described above.

[0006] Preferably, the set of CAR plasmids includes two CAR plasmids, namely CAR plasmid 1 and CAR plasmid 2, wherein the CAR plasmid 1 includes the CD28 intracellular domain, and the CAR plasmid 2 includes the 41-BB intracellular domain and the CD3ζ intracellular domain.

[0007] Preferably, the CAR plasmid 1 comprises the following coding structure:

[0008] CD8 signal peptide (SP), anti-TSHR / HER2 heavy chain variable region (VH), linker, anti-TSHR / HER2 light chain variable region (VL), avidin tag (Strep-Tag II), CD8 hinge region (hinge), CD28 transmembrane region (CD28TM) and CD28 intracellular domain (CD28-ICD).

[0009] Preferably, the CAR plasmid 2 comprises the following coding structure:

[0010] CD8 signal peptide (SP), anti-TSHR / HER2 heavy chain variable region (VH), linker, anti-TSHR / HER2 light chain variable region (VL), avidin tag (Strep-Tag II), CD8 hinge region (hinge), CD28 transmembrane region (CD28TM), 41-BB intracellular domain and CD3ζ intracellular domain.

[0011] Preferably, the sequence of the CD8 signal peptide is shown as SEQ ID NO.1, the sequence of the anti-TSHR / HER2 heavy chain variable region is shown as SEQ ID NO.4 or 7, the sequence of the connecting fragment is shown as SEQ ID NO.5, the sequence of the anti-TSHR / HER2 light chain variable region is shown as SEQ ID NO.6 or 8, the sequence of the avidin tag is shown as SEQ ID NO.9, the sequence of the CD8 hinge region is shown as SEQ ID NO.10, and the sequence of the CD28 transmembrane region is shown as SEQ ID NO.11.

[0012] The present invention also discloses the use of the above-mentioned combination sequence of intracellular domains in preparing chimeric antigen receptor T cells targeting differentiated thyroid cancer cell lines or HER2-positive breast cancer cell lines.

[0013] The present invention also discloses the use of a group of CAR plasmids as described above in the preparation of chimeric antigen receptor T cells targeting differentiated thyroid cancer cell lines or HER2-positive breast cancer cell lines.

[0014] The present invention also provides a CAR-T modification method, which includes first designing the combination sequence of the intracellular domains as described above, synthesizing a gene fragment containing the combination sequence of the intracellular domains according to the design and constructing it into a lentiviral vector to form the CAR plasmid as described above, and finally using the CAR plasmid to transduce T cells to prepare CAR-T cells.

[0015] Preferably, when using a CAR plasmid to transduce T cells, CAR plasmid 1 or CAR plasmid 2 is used to transduce the T cells first, and then another CAR plasmid is used to transduce the T cells after the transduction is completed.

[0016] The beneficial effects of the present invention are: designing specific intracellular domain sequences and combinations thereof, realizing the parallel expression of CAR molecules targeting the same antigen on one T cell, utilizing different intracellular domains to achieve functional regulation of T cells, effectively delaying their exhaustion, and enhancing their endurance, so that CAR-T therapy can achieve better therapeutic effects in various solid tumors such as thyroid cancer and breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the structure of six plasmids in the examples.

[0018] Figure 2 This is a representative flow cytometric plot of the expression of a single CAR molecule in differentiated thyroid cancer cells targeting TSHR.

[0019] Figure 3 This is a representative flow cytometric plot of the expression of a single CAR molecule targeting HER2-positive breast cancer cells.

[0020] Figure 4 This is a representative flow cytometric plot of the expression of dual CAR molecules targeting TSHR or HER2.

[0021] Figure 5 It is a curve chart of the killing ability of intracellular heterotypic dual-target CAR-T cells and second-generation CAR-T.

[0022] Figure 6 This is a graph showing the changes in the exhaustion markers PD-1 and TIM3 of different CAR-T cells after two rounds of cancer cell stimulation, with the target cells being K1-TSHR-luc.

[0023] Figure 7 This is a graph showing the results of flow cytometry detection in the target cell K1-TSHR-luc experiment.

[0024] Figure 8This is a diagram of the different CAR-T memory phenotype changes after two rounds of cancer cell stimulation. The target cells are K1-TSHR-luc.

[0025] Fig. 9 This is a graph showing the changes in the exhaustion markers PD-1 and TIM3 of different CAR-T cells after two rounds of cancer cell stimulation, with the target cells being K1-TSHR-luc.

[0026] Fig.10 It is a statistical chart of IFNγ released after intracellular heterotypic dual-target CAR-T cells or second-generation CAR-T and tumor cells are co-cultured.

[0027] Fig.11 This is a graph showing the changes in the exhaustion markers PD-1 and TIM3 of different CAR-T cells after two rounds of cancer cell stimulation, with the target cell being JIMT1.

[0028] Fig.12 This is a graph showing the results of flow cytometry detection in the target cell JIMT1 experiment.

[0029] Fig.13 It shows the different CAR-T memory phenotype changes after two rounds of cancer cell stimulation, and the target cells are JIMT1.

[0030] Fig.14 This is a graph showing the changes in the exhaustion markers PD-1 and TIM3 of different CAR-T cells after two rounds of cancer cell stimulation, with the target cell being JIMT1.

[0031] Fig.15 This is an image of biochemiluminescence detection of mouse tumors.

[0032] Fig.16 This is a graph showing the quantitative results of biochemiluminescence of mouse tumors.

[0033] Fig.17 This is a comparison chart of mouse tumor sizes.

[0034] Fig.18 This is a graph showing the quantitative results of mouse tumor volume.

[0035] in, Figure 2 , 3 The colors in , 4, 7, 15, 16, and 18 are part of the experimental results, and color images are used in preparation for expressing the experimental results. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is described below in conjunction with the accompanying drawings and specific embodiments.

[0037] Example 1: A combination sequence of an intracellular domain.

[0038] A combination sequence of intracellular domains, comprising a CD28 intracellular domain, a 41-BB intracellular domain and a CD3ζ intracellular domain; the nucleotide sequence of the CD28 intracellular domain is shown in SEQ ID NO.13, the nucleotide sequence of the 41-BB intracellular domain is shown in SEQ ID NO.12, and the nucleotide sequence of the CD3ζ intracellular domain is shown in SEQ ID NO.14.

[0039] The combined sequences provided in this example can be used to prepare chimeric antigen receptor T cells targeting differentiated thyroid cancer cell lines or HER2-positive breast cancer cell lines, and to prepare corresponding cancer vaccines, or to treat corresponding cancers.

[0040] Example 2: A group of CAR plasmids.

[0041] A group of CAR plasmids includes two CAR plasmids, namely CAR plasmid 1 and CAR plasmid 2, wherein the CAR plasmid 1 includes the following coding structure: CD8 signal peptide, anti-TSHR / HER2 heavy chain variable region, connecting fragment, anti-TSHR / HER2 light chain variable region, avidin tag, CD8 hinge region, CD28 transmembrane region and CD28 intracellular domain; the CAR plasmid 2 includes the following coding structure: CD8 signal peptide, anti-TSHR / HER2 heavy chain variable region, connecting fragment, anti-TSHR / HER2 light chain variable region, avidin tag, CD8 hinge region, CD28 transmembrane region, 41-BB intracellular domain and CD3ζ intracellular domain.

[0042] The sequence of the CD8 signal peptide is shown in SEQ ID NO.1, the sequence of the anti-TSHR / HER2 heavy chain variable region is shown in SEQ ID NO.4 or 7, the sequence of the connecting fragment is shown in SEQ ID NO.5, the sequence of the anti-TSHR / HER2 light chain variable region is shown in SEQ ID NO.6 or 8, the sequence of the avidin tag is shown in SEQ ID NO.9, the sequence of the CD8 hinge region is shown in SEQ ID NO.10, and the sequence of the CD28 transmembrane region is shown in SEQ ID NO.11.

[0043] The set of CAR plasmids provided in this embodiment can be used to prepare chimeric antigen receptor T cells targeting differentiated thyroid cancer cell lines or HER2-positive breast cancer cell lines, and to prepare corresponding cancer vaccines, or to treat corresponding cancers.

[0044] Example 3: A CAR-T modification method.

[0045] In this example, human T lymphocytes modified with TSHR / HER2-specific chimeric antigen receptors (anti-TSHR / anti-HER2 CAR-T cells) were prepared by transforming primary human T lymphocytes. The specific steps are as follows:

[0046] Step 1. Preparation of CAR gene fragments.

[0047] 1.1. Design the fusion gene fragment according to the following coding structure sequence:

[0048] CAR molecule 1: CD8 signal peptide (SP), anti-TSHR / HER2 heavy chain variable region (VH), linker, anti-TSHR / HER2 light chain variable region (VL), avidin tag (Strep-Tag II), CD8 hinge region (hinge), CD28 transmembrane region (CD28TM), CD28 intracellular domain (CD28-ICD);

[0049] CAR molecule 2: CD8 signal peptide (SP), anti-TSHR / HER2 heavy chain variable region (VH), linker, anti-TSHR / HER2 light chain variable region (VL), avidin tag (Strep-Tag II), CD8 hinge region (hinge), CD28 transmembrane region (CD28TM), 41-BB intracellular domain, CD3ζ intracellular domain. The above two CAR gene structures are directly synthesized by whole gene synthesis technology;

[0050] The nucleotide and corresponding amino acid sequences of the above genes are shown in the following table:

[0051]

[0052]

[0053] 1.2. The corresponding gene fragments were synthesized according to the above design and constructed into the PLVX-EF1α-IRES-puro lentiviral vector to form a recombinant plasmid; in this embodiment, six recombinant plasmids were constructed, namely PLV172, PLV201, PLV202, PLV143, PLV204, and PLV205. The schematic diagrams of the six plasmid structures are shown in the attached Figure 1 As shown, specifically:

[0054] The nucleic acid sequences of PLV172 are SEQ ID Nos. 1, 2, 4, 5, 6, 9, 10, 11, 12, 14;

[0055] The PLV201 nucleic acid sequences are SEQ ID Nos. 1, 2, 4, 5, 6, 9, 10, 11, 13, 14;

[0056] The nucleic acid sequences of PLV202 are SEQ ID Nos. 1, 3, 4, 5, 6, 9, 10, 11, and 13;

[0057] The nucleic acid sequences of PLV143 are SEQ ID Nos. 1, 2, 7, 5, 8, 9, 10, 11, 12, 14;

[0058] The nucleic acid sequences of PLV204 are SEQ ID Nos. 1, 2, 7, 5, 8, 9, 10, 11, 13, 14;

[0059] The PLV205 nucleic acid sequence is SEQ ID No. 1, 3, 7, 5, 8, 9, 10, 11, 13.

[0060] Step 2. Anti-TSHR-CAR lentiviral packaging.

[0061] According to the method disclosed in the invention patent with patent number 2020115638326, the above six plasmid gene fragments were respectively subjected to lentiviral packaging, lentiviral titer detection, and separation and activation of human peripheral blood mononuclear cells (PBMCs) to construct six lentiviruses.

[0062] Step 3. Preparation of CAR-T cells.

[0063] 3.1. Count the T cells activated for 24 hours and resuspend them in 500 μl of prepared T culture medium (make sure the final cell density is about 10 7 / ml) were transferred into a 24-well plate;

[0064] 3.2. Single CAR molecule cell preparation: Six CAR-T groups were set up, each corresponding to six lentiviruses. The constructed lentiviruses were added to the CAR-T group at an MOI of 2-8, and an equal volume of T cell culture medium was added to the control group. Polybrene with a final concentration of 4 μg / ml was added to each of the above groups and placed at 37°C, 5% CO 2 Transfection was performed in an incubator for 4-6 hours;

[0065] 3.3. Each group of T cells was placed in a different 6-well plate and supplemented with T cell culture medium until the cell density was 1×10 6 / ml, at 37°C, 5% CO 2 Incubate overnight in an incubator;

[0066] 3.4. Preparation of dual CAR molecule cells: Continue to transfect the second virus within 48 hours after completing step 3.3, and keep the MOI consistent;

[0067] 3.5. Observe the status of T cells every day and count them according to 1×10 6The T cell density was adjusted by adding standard supplemented T cell culture medium and adjusting the T cell density. On the second day after the second virus infection, the positive ratio of CAT-T cells in each group was determined by flow cytometry. The positive rate of CAR-T cells is shown in the attached Figure 2-4 .

[0068] Example 4: Functional performance test of CAR-T cells targeting TSHR / HER2.

[0069] A thyroid cancer cell line (K1-TSHR-Luc) that stably expresses TSHR and Luciferase reporter genes was constructed by lentiviral overexpression. For detailed method steps and structure, please refer to the invention patent No. 2020115638326. The breast cancer cell line JIMT1 naturally expresses HER2 molecules.

[0070] Three experimental groups were set up on a 96-well plate, corresponding to CAR-T cells containing PLV 172, PLV 201, and PLV 172+PLV 202, respectively. CAR-T cells and target cells K1-TSHR-luc were co-cultured at effector-target ratios of 10:1, 5:1, 2.5:1, 1.25:1, and 0.625:1, with 1×10 per well. 4 The specific groups are shown in Table 2. The 96-well plate was placed in a horizontal low-speed centrifuge and centrifuged at 400 g for 5 minutes, and then placed at 37°C and 5% CO. 2 Incubate in incubator for 6-8 hours.

[0071]

[0072] Table 2. Grouping of Anti-TSHR CAR-T Luc killing experiment.

[0073] After incubation for 6-8 hours, the 96-well plate was removed, 100ul D-luciferin substrate was added to each well, and incubated for another 2 minutes. The biotin luminescence intensity was calculated by a multifunctional microplate reader and converted to killing efficiency. Multiple rounds of killing were performed according to similar steps. Flow cytometry was performed after each round of killing to analyze T cell exhaustion markers and memory phenotype detection. The killing efficiency, exhaustion markers and memory phenotype changes of each group are shown in the attached figure. Figure 5-9 As shown, the killing ability of intracellular heterotypic dual-target CAR-T cells is stronger than that of the complete second-generation CAR-T, that is, intracellular heterotypic dual-target CAR-T cells targeting TSHR (dual CAR molecule expression) show stronger in vitro anti-tumor ability.

[0074] Three experimental groups were set up on a 96-well plate, corresponding to CAR-T cells containing PLV 143, PLV 204, and PLV 143 + PLV 205, respectively. CAR-T cells were co-cultured with target cells JIMT1 at an effector-target ratio of 5:1, with 1×10 4 The specific groups of JIMT1 target cells are shown in Table 3. The 96-well plate was placed in a horizontal low-speed centrifuge and centrifuged at 400 g for 5 minutes, and then placed at 37°C and 5% CO 2 Incubate in an incubator for 6-8 hours;

[0075]

[0076]

[0077] Table 3. Grouping of Anti-HER2 CAR-T in vitro experiments.

[0078] After incubation for 6-8 hours, the 96-well plate was removed and multiple rounds of killing were performed according to similar steps. Flow cytometry was performed after each round of killing to analyze T cell cytokine expression, exhaustion markers, and memory phenotype detection.

[0079] Cytokine secretion, exhaustion markers, and memory phenotype transitions in each group are shown in the attached figure. Figure 10-14 As shown, the IFNγ released after co-culture of intracellular heterotypic dual-target CAR-T cells and tumor cells was higher than that of two complete second-generation CAR-Ts, that is, intracellular heterotypic dual-target CAR-T cells targeting HER2 (dual CAR molecule expression) showed stronger in vitro anti-tumor ability.

[0080] Example 5: Animal model experimental test.

[0081] Twenty 8-week-old NSG (NOD / scid Rag2- / -IL2rg- / -) mice were selected and a mouse thyroid cancer subcutaneous transplant tumor model was established using the K1-TSHR-Luc cell line;

[0082] The mice were observed once a day, and their vital signs (weight, hair, mental state, etc.) were recorded. When the tumor diameter reached 5 mm (about 7 days), they were randomly divided into NT=5 mice, anti-TSHR 4-1BBζ group of 5 mice, anti-TSHR CD28ζ group of 5 mice, and anti-TSHR 4-1BBζ+CD28 group of 5 mice. The above four groups of mice were treated with Mock-T cells or corresponding CAR-T cells by tail vein injection.

[0083] After CAR-T cell injection, the in vivo anti-tumor effect of CAR-T cells was evaluated by small animal imaging. Imaging was performed once a week, and mice were anesthetized by intraperitoneal injection of D-Lucifer luciferase substrate at a dose of 150 mg / kg and intraperitoneal injection of pentobarbital at a dose of 75 mg / kg. After 12 minutes, the signal was collected using the IVIS small animal imaging system. The in vivo experimental results of mice are shown in the attached figure. Figure 15-18 As shown in the results, after successful tumor modeling, anti-TSHR 4-1BBζ+CD28 with a single target heterotypic intracellular domain had the best in vivo tumor treatment effect, and dual CAR-T cells targeting the intracellular heterotypic domains of TSHR showed a stronger anti-tumor effect in mice.

[0084] The present invention provides the above embodiments only to illustrate rather than limit the technical solutions of the present invention. Although the above embodiments describe the present invention in detail, relevant technical personnel in the field should understand that the present invention can be modified or equivalently replaced, but any modification and partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A combined sequence of intracellular domains, characterized in that: It comprises a CD28 intracellular domain, a 41-BB intracellular domain and a CD3ζ intracellular domain; the nucleotide sequence of the CD28 intracellular domain is shown in SEQ ID NO.13, the nucleotide sequence of the 41-BB intracellular domain is shown in SEQ ID NO.12, and the nucleotide sequence of the CD3ζ intracellular domain is shown in SEQ ID NO.

14.

2. A group of CAR plasmids, characterized in that: The plasmid sequence thereof comprises the combined sequence of the intracellular domain as claimed in claim 1.

3. A set of CAR plasmids according to claim 2, characterized in that: It includes two CAR plasmids, namely CAR plasmid 1 and CAR plasmid 2, wherein CAR plasmid 1 includes the CD28 intracellular domain, and CAR plasmid 2 includes the 41-BB intracellular domain and the CD3ζ intracellular domain.

4. A set of CAR plasmids according to claim 3, characterized in that: The CAR plasmid 1 includes the following coding structure: CD8 signal peptide, anti-TSHR / HER2 heavy chain variable region, connecting fragment, anti-TSHR / HER2 light chain variable region, avidin tag, CD8 hinge region, CD28 transmembrane region and CD28 intracellular domain.

5. A set of CAR plasmids according to claim 4, characterized in that: The CAR plasmid 2 includes the following coding structure: CD8 signal peptide, anti-TSHR / HER2 heavy chain variable region, connecting fragment, anti-TSHR / HER2 light chain variable region, avidin tag, CD8 hinge region, CD28 transmembrane region, 41-BB intracellular domain and CD3ζ intracellular domain.

6. A set of CAR plasmids according to claim 5, characterized in that: The sequence of the CD8 signal peptide is shown in SEQ ID NO.1, the sequence of the anti-TSHR / HER2 heavy chain variable region is shown in SEQ ID NO.4 or 7, the sequence of the connecting fragment is shown in SEQ ID NO.5, the sequence of the anti-TSHR / HER2 light chain variable region is shown in SEQ ID NO.6 or 8, the sequence of the avidin tag is shown in SEQ ID NO.9, the sequence of the CD8 hinge region is shown in SEQ ID NO.10, and the sequence of the CD28 transmembrane region is shown in SEQ ID NO.

11.

7. Use of a combination sequence of an intracellular domain as claimed in claim 1 in preparing chimeric antigen receptor T cells targeting differentiated thyroid cancer cell lines or HER2-positive breast cancer cell lines.

8. Use of a group of CAR plasmids as described in any one of claims 2-6 in the preparation of chimeric antigen receptor T cells targeting differentiated thyroid cancer cell lines or HER2-positive breast cancer cell lines.

9. A CAR-T modification method, characterized in that: First, design the combination sequence of the intracellular domain as described in claim 1, synthesize the gene fragment containing the combination sequence of the intracellular domain according to the design and construct it into the lentiviral vector to constitute the CAR plasmid as described in any one of claims 3-6, and finally use the CAR plasmid to transduce T cells to make CAR-T cells.

10. A CAR-T modification method according to claim 9, characterized in that: When using CAR plasmids to transduce T cells, CAR plasmid 1 or CAR plasmid 2 is used to transduce the T cells first, and then another CAR plasmid is used to transduce the T cells after transduction is completed.

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