Chimeric antigen receptor T cell with novel transmembrane region and application of chimeric antigen receptor T cell

Abstract

The invention relates to a chimeric antigen receptor T cell with a novel transmembrane region and application of the chimeric antigen receptor T cell. The transmembrane region of the chimeric antigen receptor T cell is a CD1a transmembrane region. According to the chimeric antigen receptor T cell disclosed by the invention, a transmembrane region of a CAR molecule is modified under the condition that an additional structural domain is not introduced, so that internalization and recirculation of the CAR are influenced, and the possibility of generating cytokine release syndrome in CAR-T treatment is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of CAR-T cell therapy, and in particular relates to a chimeric antigen receptor T cell with a novel transmembrane region and an application thereof. Background Art

[0002] Targeted CD19 chimeric antigen receptor T (CAR-T) cell therapy has been shown to be effective in the treatment of relapsed or refractory acute B-cell leukemia. However, this therapy still has some limitations, such as poor efficacy in solid tumors, the presence of cytokine release syndrome (CRS), immune effector cell-related neurotoxicity syndrome, and T cell dysfunction and exhaustion. To address these limitations, strategies such as CAR molecular structure optimization, co-expression of cytokines, drug-combined CAR-T therapy, and dual-antigen targeted CAR-T have been explored. Among them, the structural design of CAR has attracted much attention. Studies have shown that surface CAR levels affect CAR-T cell cytotoxicity, so it is crucial to control the expression of surface CAR molecules. CAR molecule optimization strategies, such as altering CAR internalization and recycling by adding CTLA-4 molecule tails or enhancing CAR recycling by preventing CAR ubiquitination, have been shown to improve CAR-T cell anti-tumor immunity. These strategies highlight the potential to enhance CAR-T cell function by regulating CAR internalization and recycling. Considering the structure of CAR, including single-chain variable fragment (scFv), hinge, transmembrane domain (TMD), co-stimulatory domain and CD3ζ signaling domain, TMD is the easiest motif to optimize and can regulate the internalization and recycling of CAR without introducing additional domains, which is different from introducing the tail of CTLA-4 molecule to change the internalization and recycling of CAR.

[0003] Hinge and TMD have been shown to affect the performance of CAR-T cells. For example, CAR-T cells with CD8α hinge and TMD produced lower cytokine levels and exhibited less activation-induced cell death compared with hinge and TMD with CD28 molecules. CD28 TMD regulates CAR-T cell activity by engaging endogenous CD28, revealing the difference between CD8αTMD and CD28TMD. In addition, CAR with CD3ζTMD forms a complex with endogenous CD3ζ, which can enhance the function of CAR-Jurkat cells. In addition, CAR-T cells with ICOS hinge and TMD exhibit superior anti-tumor effects. Changes in the length of hinge and TMD also show different results on CAR-T cells. These studies indicate the important role of TMD in CAR-T cell function. However, there is a lack of research on the effect of TMD of CAR molecules on internalization and recycling. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a chimeric antigen receptor T cell with a novel transmembrane region and its application. The chimeric antigen receptor T cell transforms the transmembrane region of the CAR molecule without introducing additional domains, thereby affecting the internalization and recycling of CAR and reducing the possibility of cytokine release syndrome in CAR-T therapy.

[0005] The present invention provides a chimeric antigen receptor T cell with a novel transmembrane region, wherein the transmembrane region of the chimeric antigen receptor T cell is a CD1a transmembrane region.

[0006] Furthermore, the sequence of the CD1a transmembrane region is shown in SEQ NO.1.

[0007] Furthermore, the chimeric antigen receptor T cell also includes a CD8 hinge region, an intracellular signaling domain 4-1BB and a CD3ζ region.

[0008] Furthermore, the chimeric antigen receptor T cells target CD19 or BCMA.

[0009] The present invention also provides a pharmaceutical composition, which comprises the chimeric antigen receptor T cell as described above, and a pharmaceutically acceptable carrier.

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

[0011] Beneficial Effects

[0012] The present invention constructs CAR-T cells with CD1a transmembrane regions, and proves that CAR-T cells with CD1a transmembrane regions have lower surface CAR levels, faster internalization and recirculation rates, lower activation levels, less cytokine release, and less exhaustion marker expression compared to CAR-T cells with CD8α transmembrane regions. In addition, CAR-T cells with CD1a transmembrane regions have superior anti-tumor ability in vivo. In addition, the present invention also provides a direction that optimizing the transmembrane region alone may be an effective strategy to alleviate CRS and reduce exhaustion to enhance CAR-T cell function. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the molecular structure of CAR with CD1a or CD8α transmembrane region.

[0014] Figure 2 It shows that CAR-T cells with CD1a transmembrane region express less CAR molecules on their surface.

[0015] Figure 3It was shown that the internalization rate of CAR molecules with CD1a transmembrane domain was faster.

[0016] Figure 4 The CAR molecules with CD1a transmembrane domains were shown to recirculate more rapidly.

[0017] Figure 5 It showed that the activation level of CAR-T cells with CD1a transmembrane region was lower.

[0018] Figure 6 It was shown that CAR-T cells with CD1a transmembrane region released less cytokines after activation.

[0019] Figure 7 AC showed that CAR-T cells with CD1a transmembrane region expressed less exhaustion markers after activation.

[0020] Figure 8 AB showed that CAR-T cells with CD1a transmembrane region have superior anti-tumor ability in vivo. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0022] Example 1

[0023] This example uses the transmembrane sequence of the CD1a molecule (as shown in SEQ NO.1) and integrates it into the sequence of the CAR molecule targeting the CD19 antigen, and then constructs a plasmid for lentiviral packaging. This plasmid is used for lentiviral packaging to produce a lentivirus with infectious activity. The lentivirus is then used to infect T cells to form CAR-T cells with a CD1a transmembrane region. For comparison, a commonly used CAR-T cell with a CD8α transmembrane region was also constructed. The schematic diagrams of the two CAR molecular structures are shown in Figure 1. Figure 1 shown.

[0024] Subsequently, this example used flow cytometry to detect the CAR molecules on the surface of the two CAR-T cells. The results showed that the expression of CAR molecules on the surface of CAR-T cells with CD1a transmembrane region was less, such as Figure 2 According to the detection method of the relevant literature, the internalization and recycling rates of CAR molecules on the two CAR-T cells were detected. The results showed that the internalization and recycling rates of CAR molecules with CD1a transmembrane regions were faster, as shown in Figure 2. Figure 3 and Figure 4As shown, the CD1a transmembrane region gives the CAR molecule completely different characteristics. Due to the low expression of surface CAR molecules, the activation level of CAR-T cells after co-culture with tumor cells (SEM cell line) was detected accordingly, and the CD69 molecule was used as an indicator of the activation level of CAR-T cells. The results showed that after 24 hours of co-culture with tumor cells, CAR-T cells with CD1a transmembrane regions expressed less CD69, indicating that they had a lower activation level, as shown in Figure 2. Figure 5 Subsequently, the kit was used to detect the cytokine release level after 72 hours of co-culture of CAR-T cells and tumor cells. The results showed that compared with CAR-T cells with CD8α transmembrane regions, CAR-T cells with CD1a transmembrane regions released less cytokines, as shown in Figure 2. Figure 6 As shown, this also indicates that the activation level of CAR-T cells with CD1a transmembrane region is lower.

[0025] There is a relationship between the activation of CAR-T cells and their exhaustion. This example detected the expression levels of exhaustion indicators PD-1, LAG-3, and TIM-3 after 72 hours of co-culture of CAR-T cells and tumor cells. The results showed that CAR-T cells with CD1a transmembrane regions expressed less exhaustion indicators on their surfaces after co-culture, such as Figure 7 As shown in AC, CAR-T cells with CD1a transmembrane region showed lower exhaustion level while killing tumors.

[0026] Finally, this example verifies the effect of CAR-T cells with CD1a transmembrane domain on the survival of tumor mice in vivo. SEM tumor cell xenograft model was constructed by intravenously injecting SEM-mRuby2-luciferase tumor cells into NOD / SCID mice. Three days after tumor cell injection, T cells and CAR-T cells with two transmembrane domains were injected respectively. Survival analysis showed that both CAR-T cells with CD1a transmembrane domains prolonged the survival time of mice, and the therapeutic effect of CAR-T cells with CD1a transmembrane domain was significantly better, such as Figure 8 As shown in AB.

Claims

1. A chimeric antigen receptor T cell with a novel transmembrane region, characterized in that: The transmembrane region of the chimeric antigen receptor T cell is the CD1a transmembrane region.

2. The chimeric antigen receptor T cell according to claim 1, characterized in that: The sequence of the CD1a transmembrane region is shown in SEQ NO.

1.

3. The chimeric antigen receptor T cell according to claim 1, characterized in that: The chimeric antigen receptor T cell also includes a CD8 hinge region, an intracellular signaling domain 4-1BB and a CD3ζ region.

4. The chimeric antigen receptor T cell according to claim 1, characterized in that: The chimeric antigen receptor T cells target CD19 or BCMA.

5. A pharmaceutical composition, characterized in that It comprises the chimeric antigen receptor T cell according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that: The pharmaceutically acceptable carrier includes at least one of a diluent, a binder, a surfactant, a wetting agent, an adsorption carrier, a lubricant, a filler, and a disintegrant.

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