Chimeric antigen receptor t cells with novel transwell zone and applications thereof
By introducing the CD1a transmembrane region into CAR-T cells, the internalization and recycling of CAR are optimized, overcoming the limitations of existing CAR-T cell therapies, achieving lower activation and cytokine release, and enhancing anti-tumor effects.
Patent Information
- Application Number
- CN202411860141.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-17
- Publication Date
- 2026-01-13
- Estimated Expiration
- 2044-12-17
AI Technical Summary
Existing CAR-T cell therapies have poor efficacy in treating solid tumors and suffer from cytokine release syndrome, immune effector cell-related neurotoxicity syndrome, and T cell dysfunction. There is a lack of research on optimizing the transmembrane region of CAR molecules to regulate internalization and recirculation.
By replacing the traditional CD8α transmembrane region with the CD1a transmembrane region, chimeric antigen receptor T cells were constructed. These cells, combined with the CD8 hinge region, intracellular signaling domain 4-1BB, and CD3ζ region, formed CAR-T cells with novel transmembrane regions.
CAR-T cells in the CD1a transmembrane region have lower CAR levels on their surface, faster internalization and recycling rates, lower activation levels, less cytokine release, and less expression of exhaustion markers, resulting in superior in vivo anti-tumor capabilities.
Smart Images
Figure CN119685260B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of CAR-T cell therapy technology, and specifically relates to a chimeric antigen receptor T cell with a novel transmembrane region and its application. Background Technology
[0002] CAR-T cell therapy targeting CD19 chimeric antigen receptor (CAR-T) has proven effective in treating relapsed or refractory acute B-cell leukemia. However, this therapy still has some limitations, such as poor response to solid tumors, cytokine release syndrome (CRS), immune effector cell-related neurotoxicity syndrome, and T cell dysfunction and exhaustion. To address these limitations, strategies such as CAR molecule structure optimization, co-expression of cytokines, combination therapy with other drugs for CAR-T therapy, and dual-antigen targeted CAR-T have been explored. Among these, CAR structural design has attracted considerable attention. Studies have shown that surface CAR levels affect CAR-T cell cytotoxicity, thus controlling the expression of surface CAR molecules is crucial. CAR molecule optimization strategies, such as altering CAR internalization and recycling by adding the CTLA-4 molecule tail or enhancing CAR recycling by inhibiting 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 modulating CAR internalization and recycling. Considering the structure of CAR, including single-strand variable fragments (scFv), hinges, transmembrane domains (TMD), co-stimulatory domains, and CD3ζ signaling domains, TMD is the motif that is the easiest to optimize. It can regulate the internalization and recycling of CAR without introducing additional domains, unlike introducing the tail of the CTLA-4 molecule to change the internalization and recycling of CAR.
[0003] Hinges and TMDs have been shown to influence CAR-T cell performance. For example, CAR-T cells with CD8α hinges and TMDs produce lower cytokine levels and exhibit less activation-induced cell death compared to hinges and TMDs with CD28 molecules. CD28 TMDs regulate CAR-T cell activity by participating in endogenous CD28, revealing differences between CD8α TMDs and CD28 TMDs. Furthermore, CARs with CD3ζ TMDs form a complex with endogenous CD3ζ, which can enhance CAR-Jurkat cell function. Additionally, CAR-T cells with ICOS hinges and TMDs exhibit superior antitumor activity. Variations in hinge and TMD length also yield different results in CAR-T cells. These studies demonstrate the important role of TMDs in CAR-T cell function. However, the effects of TMDs on internalization and recycling of CAR molecules are currently lacking. 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 modifies the transmembrane region of the CAR molecule without introducing additional structural 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 the 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 the CD8 hinge region, the intracellular signaling domain 4-1BB, and the CD3ζ region.
[0008] Furthermore, the chimeric antigen receptor T cells target CD19 or BCMA.
[0009] The present invention also provides a pharmaceutical composition comprising chimeric antigen receptor T cells as described above, and a pharmaceutically acceptable carrier.
[0010] Furthermore, the pharmaceutically acceptable carrier includes at least one of diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.
[0011] Beneficial effects
[0012] This invention constructed CAR-T cells with a CD1a transmembrane region and demonstrated that, compared with CAR-T cells with a CD8α transmembrane region, CAR-T cells with a CD1a transmembrane region exhibited lower surface CAR levels, faster internalization and recycling rates, lower activation levels, less cytokine release, and less expression of exhaustion markers. Furthermore, CAR-T cells with a CD1a transmembrane region demonstrated superior antitumor activity in vivo. This invention also provides a direction where optimizing the transmembrane region individually may be an effective strategy for alleviating CRS and reducing exhaustion to enhance CAR-T cell function. Attached Figure Description
[0013] Figure 1 This is a schematic diagram of the CAR molecule structure with CD1a or CD8α transmembrane regions.
[0014] Figure 2 CAR-T cells with CD1a transmembrane regions showed lower expression of CAR molecules on their surface.
[0015] Figure 3CAR molecules with the CD1a transmembrane region showed a faster internalization rate.
[0016] Figure 4 CAR molecules with the CD1a transmembrane region exhibit faster recycling rates.
[0017] Figure 5 CAR-T cells with CD1a transmembrane regions showed lower activation levels.
[0018] Figure 6 CAR-T cells with CD1a transmembrane regions showed less cytokine release after activation.
[0019] Figure 7 AC showed that CAR-T cells with CD1a transmembrane regions expressed fewer exhaustion markers after activation.
[0020] Figure 8 AB studies showed that CAR-T cells with CD1a transmembrane regions have superior anti-tumor capabilities in vivo. Detailed Implementation
[0021] 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.
[0022] Example 1
[0023] In this embodiment, the transmembrane sequence of the CD1a molecule (as shown in SEQ NO.1) was integrated into the sequence of a CAR molecule targeting the CD19 antigen, and then a plasmid for lentiviral packaging was constructed. This plasmid was applied to lentiviral packaging to generate infective lentivirus. The lentivirus was then used to infect T cells, forming CAR-T cells with the CD1a transmembrane region. For comparison, commonly used CAR-T cells with the CD8α transmembrane region were also constructed. Schematic diagrams of the two CAR molecule structures are shown below. Figure 1 As shown.
[0024] Subsequently, flow cytometry was used to detect CAR molecules on the surface of two types of CAR-T cells. The results showed that CAR-T cells with the CD1a transmembrane region expressed fewer CAR molecules on their surface, such as... Figure 2 As shown. Based on the detection methods in relevant literature, the internalization and recycling rates of CAR molecules in two types of CAR-T cells were detected. The results showed that CAR molecules with a CD1a transmembrane region had faster internalization and recycling rates, such as... Figure 3 and Figure 4As shown, the CD1a transmembrane region confers distinctly different properties on CAR molecules. Due to the low expression of surface CAR molecules, the activation level of CAR-T cells co-cultured with tumor cells (SEM cell line) was examined. CD69 molecules were used as an indicator of CAR-T cell activation level. The results showed that after 24 hours of co-culture with tumor cells, CAR-T cells with the CD1a transmembrane region expressed even less CD69, indicating a lower activation level. Figure 5 As shown in the image. Subsequently, the cytokine release levels of CAR-T cells and tumor cells were detected using a kit. The results showed that CAR-T cells with CD1a transmembrane regions released fewer cytokines compared to CAR-T cells with CD8α transmembrane regions, such as... Figure 6 As shown, this also indicates that CAR-T cells with CD1a transmembrane regions have low activation levels.
[0025] CAR-T cell activation is related to their exhaustion. This study examined the expression levels of exhaustion markers PD-1, LAG-3, and TIM-3 on the surface of CAR-T cells after co-culturing with tumor cells for 72 hours. The results showed that CAR-T cells with CD1a transmembrane regions expressed fewer exhaustion markers on their surface after co-culture. Figure 7 As shown in AC, CAR-T cells with CD1a transmembrane regions exhibit lower exhaustion levels while killing tumors.
[0026] Finally, this embodiment verified the survival prolongation of tumor-bearing mice by CAR-T cells with CD1a permeabilization regions in mice. A 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 both types of permeabilization regions were injected, respectively. Survival analysis showed that both types of CAR-T cells prolonged the survival time of mice, with CAR-T cells with CD1a permeabilization regions showing significantly better therapeutic effects. 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 on 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 ID NO.
1.
3. The chimeric antigen receptor T cell according to claim 1, characterized in that: The chimeric antigen receptor T cell also includes the CD8 hinge region, the intracellular signaling domain 4-1BB, and the 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 chimeric antigen receptor T cells as described in any one of claims 1-4, 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 the following: diluent, binder, surfactant, humectant, adsorbent, lubricant, filler, and disintegrant.