Bifunctional car-nrr-t cells and uses thereof

Abstract

The present application relates to a kind of bifunctional CAR-NRR-T cell and its application, the bifunctional CAR-NRR-T cell is obtained by the intracellular signal transduction domain fusion of NRR-scFv and CD19 CAR construction.The present application is verified by experiment, in MEC-1 cell (human CLL cell line) xenograft mouse model, compared with traditional CD19 CAR-T monotherapy, using the present application reinforced combination therapy (NRR-scFv-T cell→bifunctional CAR-NRR-T cell) realizes the fastest, deepest tumor clearance, curative effect is superior to all other treatment groups.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy technology, and specifically relates to a bifunctional CAR-NRR-T cell and its application. Background Technology

[0002] Chronic lymphocytic leukemia (CLL) is the most common adult B-cell malignancy, characterized by the progressive accumulation of CD19-positive clonal B lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. Although targeted therapies such as BTK inhibitors (e.g., ibrutinib) and BCL-2 inhibitors (e.g., veneclade) have significantly improved the prognosis of CLL patients, new treatment strategies are still needed for relapsed / refractory patients, especially those with high-risk genetic abnormalities (e.g., TP53 mutations, 17p deletions).

[0003] Chimeric antigen receptor T-cell (CAR-T) therapy, particularly CD19-targeting CAR-T cells (such as tisagenlecleucel and axicabtagene ciloleucel), has shown some efficacy in relapsed / refractory CLL, but the overall response rate is lower than in acute lymphoblastic leukemia and diffuse large B-cell lymphoma. Major limiting factors include: low and easily downregulated CD19 antigen density on the surface of CLL cells, a highly suppressive tumor microenvironment, and the tendency for CAR-T cells to be depleted in vivo.

[0004] The NOTCH1 signaling pathway plays a crucial role in the development and progression of CLL. Approximately 10% of CLL patients carry gain-of-function mutations in NOTCH1, leading to persistent activation of its downstream signaling pathways, promoting tumor cell survival and drug resistance. Notably, studies have shown that even in CLL patients without NOTCH1 gene mutations, over 50% exhibit activation of this signaling pathway. However, current therapies lack strategies to enhance CAR-T efficacy by modulating the NOTCH1 pathway. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a bifunctional CAR-NRR-T cell and its application, to overcome the problem of poor efficacy of existing CD19 CAR-T therapy for chronic lymphocytic leukemia (CLL) due to factors such as downregulation of CD19 antigen on the surface of CLL cells and inhibition of the tumor microenvironment, and to provide a combined treatment strategy that can enhance the killing effect of CAR-T cells on CLL cells and prolong the duration of response.

[0006] This invention provides a bifunctional CAR-NRR-T cell, which is constructed by fusing the intracellular signal transduction domain of NRR-scFv with that of CD19 CAR.

[0007] The present invention also provides the application of bifunctional CAR-NRR-T cells (CAR-NRR sequence as shown in SEQ NO.2) combined with NRR-scFv-T cells (NRR-scFv sequence as shown in SEQ NO.1) in the preparation of drugs for chronic lymphocytic leukemia.

[0008] Furthermore, the NRR-scFv-T cells are T cells expressing a single-chain antibody fragment against the negative regulatory region of NOTCH1. The NRR-scFv cells can bind to the negative regulatory region of the NOTCH1 receptor on the surface of CLL cells, inhibiting the NOTCH1 signaling pathway, thereby upregulating RAB31 expression in CLL cells and reducing the downregulation of CD19 antigen on the surface of CLL cells during subsequent co-culture with CAR-T cells. They can also be delivered in soluble protein form or via other carriers (such as lentiviruses or nanoparticles), as long as transient inhibition of NOTCH1 signaling in CLL cells can be achieved. However, NRR-scFv-T cells are preferred because they can persist in vivo and exert a pretreatment effect for a certain period.

[0009] The present invention also provides a pharmaceutical composition for treating chronic lymphocytic leukemia, comprising an effective amount of bifunctional CAR-NRR-T cells and NRR-scFv-T cells, and a pharmaceutically acceptable carrier.

[0010] Preferably, the pharmaceutically acceptable carrier includes at least one of a diluent, binder, surfactant, humectant, adsorbent, lubricant, filler, and disintegrant.

[0011] Beneficial effects

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] (1) Significantly enhances anti-CLL efficacy: In the MEC-1 cell (human CLL cell line) xenograft mouse model, compared with traditional CD19 CAR-T monotherapy, the enhanced combination therapy of this invention (NRR-scFv-T cells → bifunctional CAR-NRR-T cells) achieved the fastest and deepest tumor clearance, with efficacy superior to all other treatment groups.

[0014] (2) Prolonging the median survival of CLL-bearing mice: The group receiving enhanced combined treatment achieved the longest median survival.

[0015] (3) Protecting CD19 antigen on the surface of CLL cells: In vitro co-culture experiments showed that after NRR-scFv-Jurkat cell pretreatment, the downregulation of CD19 antigen on the surface of MEC-1 cells was significantly reduced when co-cultured with CD19 CAR-T cells, thereby maintaining the recognition and killing ability of CAR-T cells. Attached Figure Description

[0016] Figure 1A shows a schematic diagram of the NRR-scFv structure; Figure 1B shows a schematic diagram of the CD19 CAR-NRR-scFv structure.

[0017] CD represents wild-type (WT) cells and corresponding effector cells co-cultured at a 1:1 ratio to a specified time point; where C represents the specific cytotoxicity calculated by analyzing target cell apoptosis using Annexin V staining and flow cytometry, with the calculated value subtracted from the baseline apoptosis value of target cells cultured alone; D represents the expression of CD19 on the surface of surviving target cells after co-culture, as detected by flow cytometry.

[0018] E is a schematic diagram of the control eGFP-scFv structure;

[0019] F represents WT cells co-cultured with NRR-scFv-Jurkat cells or control eGFP-scFv-Jurkat cells for 36 hours. After separating the target cells, the expression of the intracellular NOTCH1 fragment, HEY1, and RAB31 was detected by Western blotting.

[0020] GH represents WT cells co-cultured with NRR-scFv-Jurkat or control eGFP-scFv-Jurkat cells for 36 hours, followed by pre-treated cells as a baseline control, or continued co-culture with Jurkat cells or CD19 CAR-Jurkat cells for 12, 24, and 36 hours; G represents the expression of CD19 on the surface of surviving target cells after co-culture, assessed by flow cytometry; H represents the apoptosis of target cells detected by Annexin V staining flow cytometry, with the calculated specific cytotoxicity (%) subtracting the background apoptosis rate of target cells cultured alone.

[0021] Figure 2 A shows the experimental protocols for the six treatment groups in the mouse model; B shows in vivo imaging monitoring of tumor growth in each group of mice; C shows the radiation flux in each group after tumor cell implantation; and D shows the Kaplan-Meier survival curves for each group of mice.

[0022] In the picture:

[0023] NRR-scFv: Single-chain antibody fragment against the negative regulatory region of NOTCH1;

[0024] CAR: Chimeric antigen receptor;

[0025] Jurkat: Human T-cell leukemia cell line (used to construct CAR-T effector cells).

[0026] MEC-1: Human chronic lymphocytic leukemia (CLL) cell line (CD19 positive);

[0027] RAB31: Ras-associated protein 31;

[0028] eGFP: Enhanced green fluorescent protein (negative control). Detailed Implementation

[0029] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0030] Example 1

[0031] In vitro validation of the functional activity of NRR-scFv in CLL cells:

[0032] (1) Cell construction: Using lentiviral transduction technology, Jurkat cells stably expressing NRR-scFv (NRR-scFv-Jurkat) and control eGFP-scFv-Jurkat cells were constructed respectively.

[0033] (2) CLL cell line: MEC-1 cells are a human chronic lymphocytic leukemia cell line, CD19 positive, and the NOTCH1 signaling pathway is active.

[0034] (3) Functional verification: NRR-scFv-Jurkat cells and MEC-1 cells were co-cultured for 36 hours at an effector cell:target cell ratio of 1:1. MEC-1 cells were collected, and the expression levels of HEY1 and RAB31, downstream signaling molecules of NOTCH1, were detected by Western blot.

[0035] In vitro pretreatment enhances the killing effect of CD19 CAR-T cells on CLL cells:

[0036] (1) Pretreatment: MEC-1 cells were co-cultured with NRR-scFv-Jurkat cells or control eGFP-scFv-Jurkat cells for 36 hours.

[0037] (2) Killing phase: Pretreated MEC-1 cells and conventional CD19 CAR-Jurkat cells were co-cultured at an effector cell:target cell ratio of 1:1 for 12, 24, and 36 hours. The percentage of viable tumor cells was detected by flow cytometry (Annexin V), and the specific killing rate was calculated.

[0038] (3) CD19 expression detection: staining with anti-CD19 antibody and flow cytometry to detect the mean fluorescence intensity (MFI) of CD19 on the surface of live tumor cells.

[0039] Discussion: To establish a NOTCH1 strategy capable of precisely targeting the tumor microenvironment, a single-chain antibody fragment (NRR-scFv) targeting the negative regulatory region of NOTCH1 was designed (Figure 1A). This NRR-scFv was fused with the intracellular signal transduction domain of a CD19 CAR to construct a bifunctional CAR (named CAR-NRR-T), which was stably expressed in Jurkat cells via lentiviral transduction (CAR-NRR-Jurkat) (Figure 1B). Co-culture results showed that, compared with conventional CD19 CAR-Jurkat cells, CAR-NRR-Jurkat cells did not exhibit superior sustained killing activity (Figure 1C); both rapidly downregulated CD19 antigen expression on the tumor cell surface after co-culture (Figure 1D).

[0040] To confirm the effectiveness of NRR-scFv, Jurkat cells expressing only NRR-scFv (NRR-scFv-Jurkat) and control eGFP-scFv-Jurkat cells were constructed (Figure 1E). After co-culturing with wild-type MEC-1 cells for 36 hours, NRR-scFv-Jurkat cells effectively inhibited the NOTCH1 signaling pathway and upregulated RAB31 expression in tumor cells compared to control eGFP-scFv-Jurkat cells, confirming the effectiveness of the constructed NRR-scFv (Figure 1F). Based on this, a "pretreatment" strategy was adopted: tumor cells were first treated with NRR-scFv-Jurkat cells to inhibit NOTCH1 signaling, and then killed with conventional CD19 CAR-Jurkat cells. The results showed that the tumor cell killing effect was more significant after pretreatment. Figure 1G ), the downregulation of surface CD19 antigen is weakened ( Figure 1H The combined results indicate that this sequential strategy of NOTCH1 inhibition combined with CD19 CAR-T cell killing is effective.

[0041] Example 2

[0042] Sequential combination therapy in mouse CLL model:

[0043] (1) Animal model: 4- to 5-week-old female NOD-SCID mice were injected via the tail vein with 1×10 7 A systemic CLL xenograft model was established using luciferase-labeled MEC-1 cells (MEC-1-Luc). Figure 2 A).

[0044] (2) Grouping and treatment regimen: Mice were randomly divided into 6 groups ( Figure 2 B):

[0045] Group 1 (control group);

[0046] Group 2 (NRR-scFv-T monotherapy group): single infusion of 1×10 7 One NRR-scFv-T cell;

[0047] Group 3 (Traditional CAR-T monotherapy group): Single infusion of 1×10 7 A traditional CD19 CAR-T cell (a third-generation CAR targeting CD19, containing CD28 and 4-1BB co-stimulatory domains).

[0048] Group 4 (Bifunctional CAR-NRR-T monotherapy group): Single infusion of 1×10 7 One bifunctional CAR-NRR-T cell (i.e., a bifunctional CAR with the intracellular signaling domain of CD19CAR fused with NRR-scFv).

[0049] Group 5 (Sequential combined therapy group): First, infuse 1×10 7 One NRR-scFv-T cell was infused, followed by an infusion of 1×102 7 One traditional CD19 CAR-T cell.

[0050] Group 6 (Intensive Combined Treatment Group): First, administer 1×10 7 One NRR-scFv-T cell was infused, followed by an infusion of 1×102 7 One bifunctional CAR-NRR-T cell.

[0051] (3) Evaluation of therapeutic effect: In vivo imaging was performed every 4 to 5 days. Figure 2 B, C). Record mouse survival time and plot Kaplan-Meier survival curves (B, C). Figure 2 D). Statistical analysis was performed using the Log-rank test.

[0052] (4) Results:

[0053] The NRR-scFv-T cell monotherapy group (Group 2) showed only a slight tumor-suppressive effect, confirming that it does not possess direct killing function, but it provided a basis for subsequent combination therapy. The sequential combination therapy group (Group 5) showed significantly better efficacy than the traditional CAR-T monotherapy group (Group 3), with more thorough tumor regression and later rebound. This demonstrates the effectiveness of the strategy of "pre-treatment with NRR-scFv-T cells in vivo, followed by CAR-T killing". Encouragingly, the bifunctional CAR-NRR-T monotherapy group (Group 4) showed slightly better efficacy than the traditional CAR-T monotherapy group in the early stages. However, the most powerful anti-tumor effect appeared in the intensified combination therapy group (Group 6). This regimen achieved the fastest and deepest tumor clearance, with significantly better efficacy than all other treatment groups. Survival analysis results were consistent with the tumor growth curve. Figure 2 D). The median survival of the sequential combination therapy group (group 5) was significantly longer than that of the traditional CAR-T monotherapy group. The longest median survival was achieved in the intensified combination therapy group (group 6).

[0054] The above results indicate that the enhanced combined treatment group provided by the present invention can effectively enhance the anti-tumor effect of CD19 CAR-T against chronic lymphocytic leukemia and significantly prolong the survival of mice.

Claims

1. A bifunctional CAR-NRR-T cell, characterized in that, The bifunctional CAR-NRR-T cells were constructed by fusing the intracellular signal transduction domain of NRR-scFv with that of CD19 CAR.

2. Application of a bifunctional CAR-NRR-T cell combined with NRR-scFv-T cells in the preparation of drugs for chronic lymphocytic leukemia.

3. The application according to claim 2, characterized in that, The NRR-scFv-T cells are T cells expressing single-chain antibody fragments against the negative regulatory region of NOTCH1.

4. A pharmaceutical composition for treating chronic lymphocytic leukemia, characterized in that, It contains effective amounts of bifunctional CAR-NRR-T cells and NRR-scFv-T cells, as well as pharmaceutically acceptable vectors.

5. The composition according to claim 1, characterized in that, The pharmaceutically acceptable carrier includes at least one of the following: diluent, binder, surfactant, humectant, adsorbent, lubricant, filler, and disintegrant.

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