A bicistronic CAR-T cell structure that simultaneously targets CD19 and CD72 and its applications

Abstract

This invention provides a bicistronic CAR-T cell structure that simultaneously targets CD19 and CD72 and its application. The nucleic acid molecule encoding the corresponding chimeric antigen receptors includes at least nucleotide sequences encoding a first chimeric antigen receptor, a second chimeric antigen receptor, and a cleavage peptide. The nucleotide sequence encoding the cleavage peptide is located between the nucleotide sequences encoding the first and second chimeric antigen receptors. The first antigen-binding domain is specific for CD19, and the second antigen-binding domain is specific for CD72. This nucleic acid molecule is a bicistronic structure capable of independently expressing both CD19 and CD72, achieving balanced co-expression of these two independent CAR-T molecules (CD19 CAR and CD72 CAR), thus providing an effective treatment option for relapsed / refractory patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a bicistronic CAR-T cell that simultaneously targets CD19 and CD72 and its application, comprising a nucleic acid molecule encoding the corresponding chimeric antigen receptor, a vector containing the nucleic acid molecule, and a cell. Background Technology

[0002] CAR-T cell therapy is a personalized immunotherapy primarily used to treat certain hematologic malignancies. It involves genetically modifying the patient's own T cells to enable them to precisely identify and attack cancer cells.

[0003] Currently, CAR-T cell therapy is mainly single-target, meaning that T cells are modified to recognize a single target antigen. Due to the antigen escape problem inherent in single-target therapy, dual / multi-target CAR-T therapy needs to be developed to overcome the limitations of single-target therapy in order to improve efficacy and reduce relapse. In particular, the widely used CD19 or CD22 CARs suffer from significantly increased negative relapse rates due to antigen escape issues, necessitating novel CAR-T designs that can address the antigen escape problem and associated risks. Summary of the Invention

[0004] To address the aforementioned issues, this invention proposes the design of a bicistronic CAR-T cell structure that simultaneously targets CD19 and CD72, along with related immune cells and their applications.

[0005] Specifically, this invention provides a nucleic acid molecule comprising at least nucleotide sequences encoding a first chimeric antigen receptor, a second chimeric antigen receptor, and a cleavage peptide. The first chimeric antigen receptor comprises a first antigen-binding domain, the second chimeric antigen receptor comprises a second antigen-binding domain, and the nucleotide sequence encoding the cleavage peptide is located between the nucleotide sequences encoding the first and second chimeric antigen receptors. The first antigen-binding domain is antigen-specific to CD19 and comprises CD19 VL and CD19 VH, wherein the amino acid sequence of CD19 VL is shown in SEQ ID NO.7, and the amino acid sequence of CD19 VH is shown in SEQ ID NO.8. The second antigen-binding domain is antigen-specific to CD72 and comprises CD72 VHH, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] The nucleic acid molecule provided by the present invention may also have the following technical features: the first chimeric antigen receptor further includes a first hinge region, a first transmembrane region, a first co-stimulatory domain, and a first signal domain; the second chimeric antigen receptor further includes a second hinge region, a second transmembrane region, a second co-stimulatory domain, and a second signal domain, wherein the nucleotide sequence encoding the cleavage peptide is located between the nucleotide sequence encoding the first signal domain and the nucleotide sequence encoding the second antigen-binding domain.

[0007] Furthermore, the nucleic acid molecule provided by the present invention may also have the following technical features, wherein the first hinge region and the first transmembrane region are composed of a CD8 molecular hinge region and a CD8 transmembrane region, or are composed of a CD28 molecular hinge region and a CD28 transmembrane region; the second hinge region and the second transmembrane region are composed of a CD8 molecular hinge region and a CD8 transmembrane region, or are composed of a CD28 molecular hinge region and a CD28 transmembrane region; the CD8 molecular hinge region and the CD8 transmembrane region together constitute CD8 Hinge+TM, the amino acid sequence of which is shown in SEQ ID NO.4, and the amino acid sequences of the CD28 molecular hinge region and the CD28 transmembrane region are shown in SEQ ID No.11.

[0008] In addition, the nucleic acid molecule provided by the present invention may also have the following technical features: the first co-stimulatory domain and the second co-stimulatory domain are independently selected from the 4-1BB co-stimulatory domain, the CD28 co-stimulatory domain, and a combination thereof, respectively, and the amino acid sequence of the 4-1BB co-stimulatory domain is shown in SEQ ID NO.5.

[0009] In addition, the nucleic acid molecule provided by the present invention may also have the following technical features, wherein the first signal domain and the second signal domain are independently selected from the CD3 signal domain and the ICOS third signal domain, respectively, and the amino acid sequence of the CD3 signal domain is shown in SEQ ID NO.6.

[0010] The nucleic acid molecule provided by the present invention may also have the following technical features, wherein the cleavage peptide is any one of P2A, E2A, F2A, T2A or an optimized mutant of any one of them, and the amino acid sequence of the cleavage peptide P2A is shown in SEQ ID NO.10.

[0011] The present invention also provides a vector comprising any of the above-mentioned nucleic acid molecules, wherein the vector is a lentiviral shuttle plasmid, a Sleeping Beauty transposon, or mRNA for electroporation delivery.

[0012] The present invention further provides a cell comprising a nucleic acid molecule as described above, and / or a carrier as described above, wherein the cell is a T cell.

[0013] In addition, the present invention also provides the use of the above-mentioned cells in the preparation of medicaments for treating or improving malignant tumors of the hematopoietic system.

[0014] The design of a bicistronic CAR-T cell structure that simultaneously targets CD19 and CD72, along with related immune cells and their applications, provided by this invention, utilizes a bicistronic structure capable of independently expressing both CD19 and CD72. This is because the corresponding nucleic acid molecule contains nucleotide sequences encoding a first chimeric antigen receptor and a second chimeric antigen receptor. The first antigen-binding domain of the first chimeric antigen receptor is antigen-specific to CD19, and the second antigen-binding domain of the second chimeric antigen receptor is antigen-specific to CD72. Furthermore, the nucleotide sequence encoding the cleavage peptide is located between the nucleotide sequences encoding the first and second chimeric antigen receptors. Therefore, this nucleic acid molecule is a bicistronic structure capable of independently expressing both CD19 and CD72. This avoids the functional limitations caused by the steric hindrance of two scFvs in tandem CAR technology, and also avoids the uneven efficiency problem of dual-vector co-transduction. It achieves balanced co-expression of the two independent CRA molecules, CD19CAR and CD72CAR, thereby providing an effective treatment option for relapsed / refractory patients. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the bicistronic sequence structure according to an embodiment of the present invention.

[0016] Figure 2 This is a schematic diagram of the sequence structure of CD19 CAR according to an embodiment of the present invention.

[0017] Figure 3 This is a schematic diagram of the sequence structure of CD72 CAR according to an embodiment of the present invention.

[0018] Figure 4 This is a graph showing the expression detection results of CAR-T cells in an embodiment of the present invention.

[0019] Figure 5 This is a diagram showing the amplification results of CAR-T cells in an embodiment of the present invention.

[0020] Figure 6 This is a graph showing the changes in the tumor proportion after co-incubating different groups of CAR-T cells with different tumor cell lines in embodiments of the present invention.

[0021] Figure 7 This is a graph showing the detection results of the activation signal expression level of co-incubated T cells according to an embodiment of the present invention.

[0022] Figure 8 This is a graph showing the results of cytokine expression level detection in the co-incubation supernatant of an embodiment of the present invention.

[0023] Figure 9 This is a graph showing the results of CAR-T cell count detection in the peripheral blood of patients after reinfusion, according to an embodiment of the present invention.

[0024] Figure 10 This is a graph showing the CAR copy number detection results in a patient after reinfusion according to an embodiment of the present invention.

[0025] Figure 11 This is a graph showing the detection results of inflammation-related cytokines in patients after reinfusion according to an embodiment of the present invention.

[0026] Figure 12 These are lane diagrams of 33 patients treated according to an embodiment of the present invention.

[0027] Figure 13 This is a graph showing the event-free survival (EFS) and overall survival (OS) of 33 patients treated with Bi-72 / 19 CAR-T in an embodiment of the present invention. Detailed Implementation

[0028] The specific embodiments of the present invention are described below with reference to the accompanying drawings and examples. In the following examples, reagents and materials not specified in the description were purchased through conventional commercial channels, and experimental procedures and conditions not specified in the description were performed in accordance with conventional techniques in the art, or by adopting the same experimental procedures and conditions as in other embodiments or test examples in this application specification.

[0029] Example

[0030] This embodiment constructs a chimeric antigen receptor (CAR) that simultaneously targets CD19 and CD72, comprising a CD72 CAR moiety and a CD19 CAR moiety. The CD72 CAR moiety includes a signal peptide, a CD72 antigen-binding domain, a CD8 molecular hinge region, a CD8 transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 signaling domain; the CD19 CAR moiety includes a signal peptide, a CD19 antigen-binding domain, a CD8 molecular hinge region, a CD8 transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 signaling domain.

[0031] Specifically, the CD72 antigen-binding domain contains CD72 VHH, whose amino acid sequence is shown in SEQ ID NO.1.

[0032] In the CD72 CAR region, the signal peptide is composed of CD8α and a MYC tag, whose amino acid sequences are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively. The CD8 molecular hinge region and the CD8 transmembrane region together constitute CD8 Hinge+TM, the amino acid sequence of which is shown in SEQ ID NO.4. In addition, the amino acid sequences of the 4-1BB co-stimulatory domain and the CD3 signal domain are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0033] The CD19 antigen-binding domain comprises CD19 VL and CD19 VH, wherein the amino acid sequence of CD19 VL is shown in SEQ ID NO. 7, and the amino acid sequence of CD19 VH is shown in SEQ ID NO. 8. A linker is also provided between CD19 VL and CD19 VH, and the amino acid sequence of this linker in this embodiment is shown in SEQ ID NO. 9. Furthermore, the signal peptide region, CD8 molecular hinge region, CD8 transmembrane region, 4-1BB co-stimulatory domain, and CD3 signal domain in the CD19 CAR portion are the same as those in the CD72 CAR portion, and will not be described again here.

[0034] In this embodiment, a cleavage peptide P2A is also provided between the CD19 CAR portion and the CD72 CAR portion, and its amino acid sequence is shown in SEQ ID NO.10.

[0035] Figure 1 This is a schematic diagram of the bicistronic sequence structure according to an embodiment of the present invention.

[0036] like Figure 1 As shown, based on the above-mentioned CAR that simultaneously targets CD19 and CD72, this embodiment further designs a corresponding nucleic acid molecule encoding the CAR, which contains a nucleic acid sequence encoding ScFV that is anti-CD72 and anti-CD19. This sequence is connected in parallel with the CD8 molecule hinge region, CD8 transmembrane region, 4-1BB co-stimulatory domain and CD3ζ signal domain through P2A, thereby forming a bicistronic CD72-CD19 CAR sequence.

[0037] Specifically, the bicistronic sequences from 5' to 3' include: 5'-LTR, EF1α (promoter), Anti-CD72 (including the signal peptide SP and VHH), Hinge (i.e., CD8 Hinge+TM), 4-1BB, CD3ζ, P2A, Anti-CD19 (including the signal peptide SP, VH and VL), Hinge, 4-1BB, CD3ζ and 3'-LTR.

[0038] Based on the above-mentioned bicistronic sequence structure design, this embodiment further prepared a vector containing the bicistronic group and corresponding CAR-T cells. The T cells were derived from peripheral blood mononuclear cells (PBMCs) from the patient's own blood or from a healthy donor, obtained through peripheral blood collection and separation processes. The specific separation process employed existing technology and will not be detailed here. The vector used was a third-generation self-inactivated lentivirus.

[0039] The specific preparation process is as follows:

[0040] Step 1: Synthesize a nucleic acid molecule with a bicistronic sequence and clone it into a lentiviral shuttle plasmid to obtain a vector containing the nucleic acid molecule;

[0041] Step 2: The vector and viral packaging plasmid were co-transfected into 293T cells. After culturing, the culture supernatant was collected and the virus was concentrated to obtain a virus with a chimeric antigen receptor gene engineering vector, which is a dual-target lentivirus.

[0042] Step 3: Transfect T cells with the above dual-target lentivirus to obtain dual-target CAR-T cells (denoted as Bi-72 / 19).

[0043] Figure 2 This is a schematic diagram of the sequence structure of CD19 CAR according to an embodiment of the present invention. Figure 3 This is a schematic diagram of the sequence structure of CD72CAR according to an embodiment of the present invention.

[0044] like Figures 2-3 As shown, nucleic acid sequences for expressing CD19 CAR and CD72 CAR were synthesized in this embodiment, each comprising (from 5' to 3'):

[0045] CD19 CAR sequences: 5'-LTR, EF1α (promoter), Anti-CD19 (including SP, VH, and VL), Hinge, 4-1BB, CD3ζ, and 3'-LTR.

[0046] CD72 CAR sequences: 5'-LTR, EF1α (promoter), Anti-CD72 (including SP and VHH), Hinge, 4-1BB, CD3ζ, and 3'-LTR.

[0047] Based on the above two sequences, this embodiment prepared lentiviruses carrying CD19 CAR, CD72 CAR, and negative control CAR (NC), respectively. T cells of the same origin were transfected using the same process to obtain corresponding CD19 single-target CAR-T cells, CD72 single-target CAR-T cells, and NCCAR-T cells (NC).

[0048] Figure 4 This is a graph showing the expression detection results of CAR-T cells in an embodiment of the present invention.

[0049] like Figure 4 As shown, all four CARs can be stably expressed on cells. The CAR positivity rate of single-target CAR-T cells (CD19CAR PE: 38.8%, CD72CAR APC: 57.2%) is slightly higher than that of dual-target CAR-T cells (CD19: 38.98%, CD72: 40.57%).

[0050] Figure 5This is a diagram showing the amplification results of CAR-T cells in an embodiment of the present invention.

[0051] like Figure 5 As shown, each CAR-T cell was expanded separately, and the cell expansion fold on day 9 and day 2 of culture were compared. It was found that the in vitro expansion capacity of dual-target CAR-T cells was slightly higher than that of the other two groups of single-target CAR-T cells, but the difference was not statistically significant. At the same time, no obvious cell death was observed in any group.

[0052] To explore the cytotoxic activity of different CAR-T cells against CD19-positive or CD19 / CD72 double-positive tumor cells, K562 cells (CD19 / CD72), K562-CD19 cells (CD19+ / CD72-), and Nalm6 cells (CD19+ / CD72+) were used as target cells, and the four types of CAR-T cells in this embodiment were co-incubated as effector cells at an effector-target ratio of 1:4. After 48 hours of co-incubation, the changes in tumor proportion in each group were analyzed by flow cytometry.

[0053] Figure 6 This is a graph showing the changes in the tumor proportion after co-incubating different groups of CAR-T cells with different tumor cell lines in embodiments of the present invention.

[0054] like Figure 6 As shown, compared with single-target CAR-T cells, dual-target CAR-T cells exhibited stronger cytotoxic effects when co-incubated with Nalm6 and K562 CD19 cells; after co-incubation with K562 cells, none of the four types of CAR-T cells showed significant response.

[0055] Figure 7 This is a graph showing the detection results of T cell activation signal expression levels in an embodiment of the present invention. Figure 8 This is a graph showing the results of cytokine expression level detection in the co-incubation supernatant of an embodiment of the present invention.

[0056] like Figure 7 , Figure 8 As shown, after the supernatant of each group was co-incubated, samples were taken and the T cell activation signal CD25, interferon-γ (IFNγ), and IL-2 were detected. The results showed that the expression level of the T cell activation signal CD25 in the dual-target CAR-T cell group was higher than that in the other three groups, but the expression levels of interferon-γ and IL-2 were the highest in CD19 CAR-T cells.

[0057] This embodiment also demonstrates a clinical application based on the aforementioned dual-target CAR-T cell protocol, as detailed below:

[0058] 1. A case of CD22-negative B-ALL patient

[0059] One patient presented with relapsed B-cell acute lymphoblastic leukemia (B-ALL) after multiple lines of therapy failure, exhibiting positive ETV6-NTRK3 and PAX5-C200orf112 fusion genes. The patient had previously undergone multiple lines of therapy including the CCCG-ALL-2020 regimen, the CCCG-ALL-2017 relapse regimen, belintumab, and veneclade combined with dasatinib. The bone marrow showed a 74.8% percentage of primitive / immature lymphocytes, an MRD of 7.69%, and tumor cells that were CD22 negative but persistently expressed antigens CD19 and CD72. Therefore, this patient received dual-target CAR-T cell therapy as described in this example. After lymphocyte clearance pretreatment with fludarabine combined with cyclophosphamide (FC), autologous dual-target CAR-T cells were infused, with a supplemental infusion 13 days later.

[0060] Figure 9 This is a graph showing the results of CAR-T cell count detection in the peripheral blood of patients after reinfusion, according to an embodiment of the present invention. Figure 10 This is a graph showing the CAR copy number detection results in a patient after reinfusion according to an embodiment of the present invention.

[0061] like Figure 9 After reinfusion, the CAR-T cells in the patient's body exhibited typical expansion-contraction kinetics: the peripheral blood CAR-T cell count rose to 8.03 cells / μL on day 4, reached a peak of 222.74 cells / μL on day 8, and then gradually declined, dropping to approximately 4.89 cells / μL and 4.38 cells / μL on days 15 and 18, respectively.

[0062] like Figure 10 As shown, ddPCR detection revealed synchronous amplification of CAR copy number, with 19CAR reaching a peak of 2.41 × 10⁻⁶ on day 7. 5 The peak value of 72 CAR copies / μg DNA was 8.71 × 10⁻⁶. 4 The number of copies / μg DNA (day 7) decreased gradually thereafter, reaching 1.27 × 10⁻⁶ on day 15. 4 copies / μg and 2.01×10 3 The number of copies / μg was close to the baseline level on day 17, suggesting that the bicistronic structure can achieve effective co-expression and simultaneous amplification in vivo.

[0063] Figure 11 This is a graph showing the detection results of inflammation-related cytokines in patients after reinfusion according to an embodiment of the present invention.

[0064] like Figure 11As shown, IL-6 levels peaked on day 4 (1948.19 pg / mL), while IFN-γ levels significantly increased to a peak of 4229.04 pg / mL on day 1, before rapidly returning to baseline on day 2, indicating a manageable inflammatory response. Furthermore, grade 3 CRS occurred during treatment, but the symptoms were reversible, and no ICANS, severe infection, or organ failure occurred.

[0065] Efficacy assessment showed that two weeks after CAR-T infusion, bone marrow morphology achieved complete remission (CR), MRD <0.01%, and fusion gene ddPCR became negative; one month later, CR was maintained, MRD <0.01%, and fusion gene remained negative. These results indicate that 72-19 bicistronic CAR-T cells achieved significant in vivo expansion, good durability, and deep molecular remission in this high-tumor-burden relapsed / refractory B-ALL patient, demonstrating excellent anti-leukemic activity and manageable safety.

[0066] 2. Overall prognostic analysis of 33 patients after 72-19 infusion

[0067] Thirty-three children with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) were enrolled after disease screening (29 relapsed, 4 refractory). There were 22 males and 11 females, with a mean age of 9.36 years (range 1.9–18.8 years). Two patients had isolated central nervous system involvement, two had isolated testicular involvement, and the remaining 29 had isolated bone marrow involvement or bone marrow involvement combined with other site involvement (17 isolated bone marrow involvement, 11 with central nervous system leukemia, and 1 with testicular involvement). Some patients relapsed after multiple lines of treatment; nine patients relapsed after receiving antibody therapy such as berintooline, daratum, and bebosa; and two patients relapsed after transplantation.

[0068] Specific patient information is shown in Tables 1-2 below:

[0069] Table 1 Basic Information of Enrolled Patients - 1

[0070]

[0071] Table 2 Basic Information of Enrolled Patients - 2

[0072]

[0073] All 33 patients treated with Bi-72 / 19 CAR-T in this embodiment achieved complete remission of bone marrow and extramedullary lesions, with an overall ORR of 100%. 100% of patients with bone marrow involvement achieved molecular-level remission (MRD<0.01%), and patients with central nervous system involvement achieved negative cerebrospinal fluid after treatment, with imaging gradually improving to complete remission.

[0074] Figure 12 These are lane diagrams of 33 patients treated according to an embodiment of the present invention.

[0075] like Figure 12 As shown in this example, after Bi-72 / 19 CAR-T therapy, one patient developed a post-appendicitis infection one month after CAR-T infusion and died from drug-resistant Pseudomonas aeruginosa infection. The remaining 32 patients survived, of whom 4 underwent bridging hematopoietic stem cell transplantation after CAR-T. Two patients relapsed 3-4 months after CAR-T infusion; both had bone marrow involvement combined with central nervous system involvement, and one of these relapsed after bridging hematopoietic stem cell transplantation.

[0076] Figure 13 This is a graph showing the event-free survival (EFS) and overall survival (OS) of 33 patients treated with Bi-72 / 19 CAR-T in an embodiment of the present invention.

[0077] like Figure 13 As shown, as of the application date of this application, the median enrollment time for the 33 patients was 4 months, with the longest enrollment period approaching six months. Figure 13 As shown, the expected EFS and OS at 6 months were 88.1% and 97.0%, respectively. These excellent short-term efficacy results demonstrate the good safety and efficacy of this Bi-72 / 19 CAR-T cell therapy.

[0078] As described above, in this embodiment, since a CAR targeting CD19 and CD72 is used, and a bicistronic structure capable of independently expressing CD19 and CD72 is employed, the functional limitation caused by the steric hindrance of the two scFvs in tandem CAR technology can be avoided. At the same time, the problem of uneven efficiency in dual-vector co-transduction can be avoided, thus achieving balanced co-expression of the two independent CRA molecules, CD19 CAR and CD72 CAR.

[0079] Furthermore, based on this balanced co-expression, the dual-target CAR-T cells of this embodiment can exert a therapeutic effect on relapsed and refractory children with B-cell acute lymphoblastic leukemia (B-ALL), providing an effective and safe treatment option for such patients.

[0080] Furthermore, the dual-target CAR-T cells of this embodiment can also be extended to CD72 and / or CD19 positive malignant tumors such as ALL, NHL, multiple myeloma, as well as systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, autoimmune encephalitis, and other autoimmune diseases.

[0081] Furthermore, in this embodiment, since a third-generation self-inactivated lentivirus is used as the vector system, it has the advantages of more random insertion sites and lower oncogenic risk compared to gamma retroviruses used in previous technologies. Simultaneously, since both the CD72 CAR and CD19 CAR employ a 4-1BB co-stimulatory domain, forming a CD72.BBζ / CD19.BBζ combination, long-term persistence can be ensured, further reducing the possibility of relapse.

[0082] In this embodiment, since P2A is used as the cleavage peptide, it has the advantage of higher cleavage efficiency compared to other types of cleavage peptides (such as E2A and F2A), which can reduce uncleaved fusion protein and improve expression efficiency.

[0083] Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

[0084] For example, EF1α was used as the promoter in the embodiment, but as an alternative, other types of promoters such as CAG (which can enhance expression), PGK (which can reduce immunogenicity) or inducible promoters (such as the Tet-On system) can also replace EF1α as the promoter in the bicistronic sequence of the present invention.

[0085] In this embodiment, P2A was used as the cleavage peptide, which has the advantage of high cleavage efficiency. However, other cleavage peptides in the art, such as E2A, F2A, T2A, or their optimized mutants, can also be used instead.

[0086] The embodiments use 4-1BB as the co-stimulatory domain, which can also be other co-stimulatory domains used in the art and / or combinations thereof, such as a combination of 4-1BB and CD28ζ (inverse combination). Similarly, the embodiments use the CD8 molecular hinge region and CD8 transmembrane region, but these can also be replaced by the CD28 molecular hinge region and CD28 transmembrane region, the corresponding amino acid sequence of which is shown in SEQ ID No. 11. In addition, the CAR structure can also include an OX40 or ICOS third signal domain, etc.

[0087] In addition, lentiviral shuttle plasmids were used as vectors in the embodiments, and CAR-T cells were obtained by transfecting T cells with third-generation self-inactivated lentiviral packaging plasmids. In this invention, the preparation process can also be replaced by the following methods: (1) using Sleeping Beauty transposons as vectors or using electroporation for delivery, which can reduce the preparation cost; (2) using CRISPR to knock bicistronic boxes into the TRAC locus for targeted integration, avoiding random insertion; (3) knocking out TCR and HLA to prepare off-the-shelf bicistronic UCAR-T cells, improving allogeneic universality.

[0088] In clinical applications, in addition to the more typical CAR-T treatment methods described in the examples, the present invention can also be combined with other treatment methods, such as: (1) local administration, intrathecal injection, for CNS leukemia; (2) combined treatment, such as combined treatment with CD22 and / or CD19 CAR-T cells to enhance efficacy; (3) fractionated infusion to reduce the risk of CRS.

Claims

1. A nucleic acid molecule, characterized in that: It contains at least the nucleotide sequences encoding a first chimeric antigen receptor, a second chimeric antigen receptor, and a cleavage peptide. The first chimeric antigen receptor includes a first antigen-binding domain. The second chimeric antigen receptor includes a second antigen-binding domain. The nucleotide sequence encoding the cleaved peptide is located between the nucleotide sequence encoding the first chimeric antigen receptor and the nucleotide sequence encoding the second chimeric antigen receptor. The first antigen-binding domain is specific for CD19 and comprises CD19 VL and CD19 VH, wherein the amino acid sequence of CD19 VL is shown in SEQ ID NO.7 and the amino acid sequence of CD19 VH is shown in SEQ ID NO.

8. The second antigen-binding domain is antigen-specific to CD72 and contains CD72 VHH, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. The nucleic acid molecule according to claim 1, characterized in that: in, The first chimeric antigen receptor further includes a first hinge region, a first transmembrane region, a first co-stimulatory domain, and a first signal domain; The second chimeric antigen receptor further includes a second hinge region, a second transmembrane region, a second co-stimulatory domain, and a second signaling domain. The nucleotide sequence encoding the cleavage peptide is located between the nucleotide sequence encoding the first signal domain and the nucleotide sequence encoding the second antigen-binding domain.

3. The nucleic acid molecule according to claim 2, characterized in that: in, The first hinge region and the first transmembrane region are composed of CD8 molecular hinge regions and CD8 transmembrane regions, or are composed of CD28 molecular hinge regions and CD28 transmembrane regions. The second hinge region and the second transmembrane region are composed of CD8 molecular hinge regions and CD8 transmembrane regions, or are composed of CD28 molecular hinge regions and CD28 transmembrane regions. The CD8 molecular hinge region and the CD8 transmembrane region together constitute CD8 Hinge+TM, and the amino acid sequence of CD8 Hinge+TM is shown in SEQ ID NO.

4. The amino acid sequences of the CD28 molecular hinge region and CD28 transmembrane region are shown in SEQ ID No.

11.

4. The nucleic acid molecule according to claim 2, characterized in that: in, The first and second costimulatory regions are independently selected from the 4-1BB costimulatory region, the CD28 costimulatory region, and combinations thereof. The amino acid sequence of the 4-1BB co-stimulatory domain is shown in SEQ ID NO.

5.

5. The nucleic acid molecule according to claim 2, characterized in that: in, The first signal domain and the second signal domain are independently selected from the CD3 signal domain and the ICOS third signal domain, respectively. The amino acid sequence of the CD3 signal domain is shown in SEQ ID NO.

6.

6. The nucleic acid molecule according to claim 1, characterized in that: in, The cleavage peptide is any one of P2A, E2A, F2A, or T2A, or an optimized mutant of any one of them. The amino acid sequence of the cleavage peptide P2A is shown in SEQ ID NO.

10.

7. A carrier, characterized in that, The vector comprises a nucleic acid molecule as described in any one of claims 1-6, wherein the vector is a lentiviral shuttle plasmid, a Sleeping Beauty transposon, or mRNA for electroporation delivery.

8. A cell, characterized in that, The cell comprises a nucleic acid molecule according to any one of claims 1-6, and / or the vector according to claim 7, wherein the cell is a T cell.

9. The use of the cells as described in claim 8 in the preparation of medicaments for treating or improving hematologic malignancies.

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