CAR-T cell capable of improving cell viability and targeting B7-H3 and application of CAR-T cell
By introducing endoplasmic reticulum retention protein into CAR-T cells, the problems of mutual killing and exhaustion among B7-H3 CAR-T cells were solved, thereby improving cell viability and tumor killing ability.
Patent Information
- Application Number
- CN202510956339.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-11
- Publication Date
- 2025-11-14
AI Technical Summary
Current CAR-T cell therapies, when targeting B7-H3, face challenges such as difficulty in target selection, tumor microenvironment challenges, and mutual killing and depletion among CAR-T cells, resulting in a weakened tumor-killing ability.
We designed a CAR-T cell that targets B7-H3, carrying a chimeric antigen receptor and an endoplasmic reticulum retention protein. The P2A gene recognizes internal ribosome entry sites or ribosome codon skipping sites, and independently translates the endoplasmic reticulum retention protein to reduce the expression level of B7-H3 on the surface of T cells.
It effectively reduced the mutual killing of CAR-T cells, increased the number and quality of cells, and enhanced the ability to kill tumors.
Smart Images
Figure CN120944826A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and more specifically, to a B7-H3-targeting CAR-T cell with enhanced cell viability and its application. Background Technology
[0002] CAR-T cell therapy is gradually becoming one of the treatment methods for cancer, especially in the treatment of hematological malignancies, where it has achieved remarkable efficacy. However, for solid tumors, which account for 90% of cancers, CAR-T cell therapy still faces challenges related to target selection, invasion, and the tumor microenvironment. Among these challenges, selecting a suitable target for solid tumors remains paramount. Numerous preclinical and clinical studies have tested various targets for CAR-T therapy targeting solid tumors, among which B7-H3, as an effective target that is highly expressed in tumors and lowly expressed in normal tissues, has gained increasing attention.
[0003] B7-H3, along with PD-L1 and PD-L2, belongs to the B7 immunomodulatory factor family. Early literature reported it as a T-cell co-stimulatory molecule that promotes the activation of CD4+ and CD8+ T cells and cytokine secretion. Currently, most studies consider B7-H3 to be an immunosuppressive molecule that can inhibit the activation and killing of T cells and NK cells, as well as graft-versus-host disease and autoimmune diseases. As a transmembrane protein, B7-H3 is expressed at low levels in normal human tissues but is highly expressed in various tumors (such as glioma, ovarian cancer, pancreatic cancer, and liver cancer). It is also highly expressed in tumor stem cells, tumor-associated angiogenesis, and fibroblasts of the stroma, making it a relatively ideal target for CAR-T therapy. Several CAR-T products targeting B7-H3 are currently being tested in clinical trials for different types of solid tumors, and some clinical studies have shown certain therapeutic effects.
[0004] Although B7-H3 expression is low in most human tissues, it can be induced to express after T cell activation. Since CAR-T cell preparation often involves activating T cells first to enhance viral infection efficiency, this activation also induces B7-H3 expression on the CAR-T cell surface, leading to fratricide-like interactions between B7-H3-targeting CAR-T cells. This makes the expansion of B7-H3-targeting CAR-T cells more difficult and also causes CAR-T cell depletion, thus weakening their tumor-killing ability. Therefore, reducing B7-H3 expression levels on T cells using bioengineering methods can effectively reduce fratricide-like interactions and depletion of B7-H3 CAR-T cells, improving their quantity and quality. This invention discovers that using endoplasmic reticulum retention proteins can effectively reduce the level of B7-H3 expressed on the surface of activated T cells, thereby effectively avoiding fratricide-like interactions between B7-H3 CAR-T cells and increasing the number of CAR-T cells and their tumor-killing ability.
[0005] Therefore, a new solution is needed to address this problem. Summary of the Invention
[0006] To address the shortcomings of existing technologies, the present invention aims to provide a CAR-T cell with enhanced cell viability targeting B7-H3 and its application.
[0007] The above-mentioned technical objective of the present invention is achieved through the following technical solution: a CAR-T cell with enhanced cell viability targeting B7-H3, wherein the CAR-T cell can co-express a chimeric antigen receptor targeting B7-H3 and an endoplasmic reticulum retention protein, wherein the endoplasmic reticulum retention protein is one or more.
[0008] Furthermore, the endoplasmic reticulum retention proteins include one or more of PEBL1, PEBL2, and PEBL3.
[0009] Furthermore, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:1; the amino acid sequence of PEBL1 is shown in SEQ ID NO:7; the amino acid sequence of PEBL2 is shown in SEQ ID NO:8; and the amino acid sequence of PEBL3 is shown in SEQ ID NO:9.
[0010] The present invention also provides a nucleic acid molecule that encodes, as described above, a chimeric antigen receptor targeting B7-H3 and an endoplasmic reticulum retention protein that can simultaneously express these two proteins.
[0011] Furthermore, the nucleic acid molecule includes a CAR gene targeting B7-H3, a P2A gene, and an endoplasmic reticulum retention protein gene. The P2A gene can recognize internal ribosome entry sites or ribosome codon skipping sites to initiate independent translation of the endoplasmic reticulum retention protein gene.
[0012] Furthermore, the nucleotide sequence of the CAR gene is shown in SEQ ID NO:13; the nucleotide sequence of the P2A gene is shown in SEQ ID NO:14; the nucleotide sequence of the PEBL1 gene is shown in SEQ ID NO:15; the nucleotide sequence of the PEBL2 gene is shown in SEQ ID NO:16; and the nucleotide sequence of the PEBL3 gene is shown in SEQ ID NO:17.
[0013] The present invention also provides a retrovirus, characterized in that: the retrovirus is prepared in 293T cells from a retroviral vector containing the above-mentioned nucleic acid molecules and a packaging vector.
[0014] The present invention also provides a pharmaceutical composition that targets B7-H3 and enhances cell viability, and is applicable to the diagnosis, prevention or treatment of tumor diseases, comprising any of the above-mentioned CAR-T cells, nucleic acid molecules, and retroviruses.
[0015] In summary, the present invention has the following beneficial effects: The CAR-T cells in this invention can effectively enhance the tumor-killing function of B7-H3 CAR-T cells by downregulating the level of B7-H3 molecules on the surface of B7-H3 CAR-T cells. Attached Figure Description
[0016] Figure 1 A graph showing the expression levels of B7-H3 molecules on the surface of activated T cells, untransduced T cells (NT), CD19 CAR-T cells, and B7-H3 CAR-T cells; Figure 2 In the diagram, A represents the cell state of untransduced T cells (NT) and B7-H3 CAR-T cells after T cell transduction. Figure 2 B in the figure represents the survival rate plot. Figure 2 C in the diagram represents the CD4+ / CD8+ T cell ratio. Figure 2 In this context, D represents the expression level of PD-1, a depleted molecule on the surface of T cells. Figure 3 The single vector structure diagram for the three co-expression vectors of CAR and PEBL proteins designed for this invention; Figure 4The graph shows the expression levels of B7-H3 molecules on the surface of blank 293T cells and 293T cells transiently transfected with co-expression of GFP / PEBL protein and B7-H3 CAR vector. Figure 5 A diagram showing the cellular state of B7-H3 CAR-T cells co-expressing control GFP or PEBL proteins during expansion; Figure 6 This is a graph showing the expression levels of B7-H3 molecules on the surface of untransduced T cells (NT) and B7-H3 CAR-T cells co-expressing control GFP or PEBL proteins. Figure 7 This is a diagram showing the number of cells during the expansion of B7-H3 CAR-T cells co-expressing control GFP or PEBL proteins. Figure 8 In the diagram, A represents the expression level of B7-H3 molecules on the surface of LN229 glioma cells; Figure 8 The figure shows the residual status of T cells and tumor cells after co-culturing untransduced T cells (NT), B7-H3 CAR-T cells co-expressing control GFP or PEBL protein, and LN229 cells. Detailed Implementation
[0017] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.
[0018] Example: The 293T cell line used in this invention was cultured in IMDM medium containing 10% fetal bovine serum (FBS), 1% GlutaMAX, and 1% penicillin-streptomycin (P / S). The glioma cell line LN229 was cultured in RPMI-1640 medium containing 10% FBS and 1% P / S, wherein GFP-positive LN229 cells were prepared by transfection with a GFP-encoding retrovirus. Primary T and CAR-T cells were cultured in X-VIVO 15 medium containing 5% FBS, with the addition of interleukin-7 (IL-7) and interleukin-15 (IL-15) cytokines at final concentrations of 10 ng / mL and 5 ng / mL, respectively.
[0019] In this embodiment, the CAR sequence targeting B7-H3 is derived from patent CN112390892B, and the CAR sequence targeting CD19 is derived from the literature "doi: 10.1182 / blood-2010-04-281931". The PEBL protein gene fragment was obtained by PCR of the single-chain antibody (scFv) region of the B7-H3 CAR and the addition of an endoplasmic reticulum retention signal. The CAR-P2A-GFP or CAR-P2A-PEBL gene fragment was obtained by overlapping extension PCR and then cloned into the retroviral vector SFG. The sequence correctness of all vectors was confirmed by sequencing.
[0020] The murine leukemia retrovirus (MMLV) was prepared by transiently transfecting 293T cells using a three-plasmid system consisting of SFG (containing the target gene), Pag-Pam-e, and RDF. The supernatant from the 293T cells was collected 48 and 72 hours after transfection for T cell transfection.
[0021] T cell transduction and expansion were performed using peripheral blood donated from healthy individuals to isolate mononuclear cells. Mononuclear cells were isolated using a lymphocyte density gradient separation method. The isolated mononuclear cells were activated with mouse anti-human CD3 and CD28 antibodies, and then transduced with collected retroviruses. 48-72 hours after viral transduction, successfully transduced CAR-T cells were collected and expanded in T cell culture medium. The T cells were counted and the culture medium was changed every two days. The final CAR-T cells were used for CAR and antigen expression detection and co-culture experiments with tumor cells.
[0022] T cell stimulation and B7-H3 expression in cells were detected. Untransduced T cells (NT) 5-8 days after activation were expressed at a concentration of 1-2 x 10⁻⁶. 6 Cells were seeded per well in 24-well plates, and 50 ng / mL phorbol ester (PMA) and 1000 ng / mL ionomycin were added. After stimulation for 6 hours, NT cells were collected and washed for the detection of B7-H3 expression on their surface. For the detection of B7-H3 expression on the surface of CAR-T, 293T, and LN229 cells, cells were directly collected, washed three times with 1x phosphate-buffered saline (PBS), stained with APC-labeled B7-H3 antibody on ice for 30 minutes, washed with 1x PBS, and then the expression level of B7-H3 on the surface of the various cell types was detected by flow cytometry.
[0023] T cells were co-cultured with tumor cells, and 1-2 x 10⁶ cells were seeded per well in a 24-well plate. 5 GFP-labeled LN229 glioma cells were added 24 hours later with appropriate numbers of T cells at a T cell:tumor cell (E:T) ratio of 1:1 or 2:1. Cells were collected from the wells 3 days later, and the ratio of residual T cells to tumor cells was detected by flow cytometry. T cells were detected using mouse anti-human CD3 antibody, and tumor cells were detected using GFP.
[0024] To determine whether T cell activation induces B7-H3 expression, we activated T cells from healthy individuals using PMA / ionomycin, and then detected the expression level of B7-H3 on the surface of activated T cells using a specific antibody targeting B7-H3 and flow cytometry. We also prepared and tested the expression levels of B7-H3 on the surface of CD19 CAR-T and B7-H3 CAR-T cells. The results are attached. Figure 1 As shown, high concentrations of PMA / ionomycin effectively induced B7-H3 expression on the T cell surface after T cell activation. Compared with activated T cells and CD19 CAR-T (the amino acid sequence of CD19 is shown in SEQ ID NO:2), B7-H3 CAR-T showed a higher proportion of B7-H3 expression on the cell surface. This may be due to the mutual killing among B7-H3 CAR-T cells, which continuously activated T cells and further induced high B7-H3 expression. Subsequently, we prepared B7-H3 CAR-T using T cells from two other healthy individuals and compared its B7-H3 expression level with that of untransduced T cells (NT), confirming that B7-H3 CAR-T had a higher B7-H3 surface expression.
[0025] During the preparation of B7-H3 CAR-T cells, we also observed cell status and detected cell viability, CD4+ / CD8+ T cell ratio, and the expression level of PD-1, the T cell exhaustion molecule. The results are attached. Figure 2 As shown, compared with control NT cells, B7-H3 CAR-T cells contained more dead cells and had a significantly lower cell viability than control NT cells (see attached image). Figure 2 A, 2B). Meanwhile, B7-H3 CAR-T cells contain a relatively high proportion of CD4+ T cells and a low proportion of CD8+ T cells (see appendix). Figure 2 (C) This may be because CD8+ T cells, as the primary killer cells, exhibit stronger inter-cell killing than CD4+ T cells, thus resulting in a lower proportion. Furthermore, due to inter-cell killing, B7-H3 CAR-T cells also showed a higher proportion of PD-1 expression, a T cell depletion molecule, compared to control NT cells (see appendix). Figure 2 D).
[0026] To reduce the expression level of B7-H3 on the surface of B7-H3 CAR-T cells, we tested the co-expression of B7-H3-targeting scFvs containing endoplasmic reticulum retention signals in B7-H3 CAR-T cells. (See attached image.) Figure 3 As shown, we designed three different endoplasmic reticulum retention proteins to test their downregulation of B7-H3 on the surface of CAR-T cells. Since T cell transduction expression depends on the virus, validation is time-consuming and complex. Furthermore, 293T cells, which are relatively easy to transfect, also exhibit a high proportion of B7-H3 protein expression on their surface (see attached diagram). Figure 4Therefore, we first tested the downregulation of B7-H3 protein on the surface of 293T cells by transiently transfecting 293T cells with three different endoplasmic reticulum retention proteins (B7-H3 CAR-P2A-PEBL1 protein sequence shown in SEQ ID NO:4; B7-H3 CAR-P2A-PEBL2 protein sequence shown in SEQ ID NO:5; B7-H3 CAR-P2A-PEBL3 protein sequence shown in SEQ ID NO:6). The results are attached. Figure 4 As shown, compared to protein PEBL-1, the other two proteins can more significantly downregulate the B7-H3 level on the surface of 293T cells. Therefore, proteins PEBL-2 and PEBL-3 were selected for subsequent testing. The nucleotide sequence of B7-H3 CAR-P2A-PEBL1 is shown in SEQ ID NO:10; the nucleotide sequence of B7-H3 CAR-P2A-PEBL2 is shown in SEQ ID NO:11; and the nucleotide sequence of B7-H3 CAR-P2A-PEBL3 is shown in SEQ ID NO:12.
[0027] After identifying the candidate molecule, we constructed a retroviral vector co-expressing B7-H3 CAR and the candidate molecule, and simultaneously constructed a vector co-expressing B7-H3 CAR and GFP as a control (the B7-H3 CAR-GS-P2A-GFP protein sequence is shown in SEQ ID NO:3). We then prepared retrovirally transduced T cells, obtaining B7-H3 CAR-T cells that simultaneously expressed B7-H3 CAR and PEBL proteins or the control GFP. We first observed the expansion status of these B7-H3 CAR-T cells under a microscope. (See attached image) Figure 5 As shown in Figure 6, due to the presence of mutual killing, B7-H3 CAR-T cells co-expressing control GFP exhibited more apoptosis under the microscope, while those co-expressing PEBL2 and PEBL3 showed less apoptosis. We then used flow cytometry to confirm the expression level of B7-H3 on the surface of these B7-H3 CAR-T cells. The results are shown in Figure 6. Compared with B7-H3 CAR-T cells co-expressing GFP, B7-H3 CAR-T cells co-expressing PEBL2 and PEBL3 had lower levels of B7-H3 expression on their surface, indicating that endoplasmic reticulum retention proteins can effectively downregulate cell surface B7-H3 protein. We also counted these CAR-T cells at different time points during T cell expansion, and the results are shown in the attached figure. Figure 7As shown, on days 8 and 10 after T cell stimulation, B7-H3 CAR-T cells co-expressing PEBL2 and PEBL3 had significantly more cells compared to B7-H3 CAR-T cells co-expressing control GFP. This indicates that PEBL2 and PEBL3 can reduce the killing effect among B7-H3 CAR-T cells by downregulating B7-H3 protein, thereby obtaining more CAR-T cells during expansion.
[0028] Finally, we used a co-culture experiment of CAR-T cells and tumor cells to test the tumor-killing ability of different modified B7-H3 CAR-T cells. Glioma cells LN229 highly express B7-H3 antigen (see attached image). Figure 8 A) This method can be used to test the killing effect of B7-H3 CAR-T cells on tumor cells. After LN229 cells adhered, we added untransduced T cells (NT control) and three types of transduced B7-H3 CAR-T cells at a T cell:tumor cell ratio of 1:1 or 2:1. After co-culturing for 3 days, residual cells were collected, and the ratio of residual T cells to tumor cells was detected by flow cytometry. (See attached image) Figure 8 As shown in Figure B, compared with the NT control, all three B7-H3 CAR-T cells exhibited some tumor cell killing activity. Among them, B7-H3 CAR-T cells co-expressing PEBL2 protein showed significantly better tumor killing than those co-expressing control GFP and PEBL3 proteins. This indicates that PEBL2 molecules can effectively enhance the tumor-killing function of B7-H3 CAR-T cells by downregulating the level of B7-H3 molecules on the surface of B7-H3 CAR-T cells. While PEBL3 protein can also effectively downregulate the level of B7-H3 molecules on the CAR-T cell surface, because PEBL3 protein is also expressed on the cell surface, it interferes with the binding of CAR molecules to tumor antigens, resulting in a lower tumor-killing effect compared to CAR-T cells co-expressing control GFP.
[0029] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.
Claims
1. A CAR-T cell targeting B7-H3 with enhanced cell viability, characterized in that: The CAR-T cells can co-express a chimeric antigen receptor targeting B7-H3 and an endoplasmic reticulum retention protein, wherein the endoplasmic reticulum retention protein is one or more.
2. The CAR-T cell according to claim 1, characterized in that: The endoplasmic reticulum retention protein includes one or more of PEBL1, PEBL2, and PEBL3, wherein the amino acid sequence of PEBL1 is shown in SEQ ID NO:7; the amino acid sequence of PEBL2 is shown in SEQ ID NO:8; and the amino acid sequence of PEBL3 is shown in SEQ ID NO:
9.
3. The CAR-T cell according to claim 1, characterized in that: The chimeric antigen receptor is composed of the amino acid sequence shown in SEQ ID NO:
1.
4. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes, as described in claim 1, a chimeric antigen receptor that simultaneously expresses B7-H3 and an endoplasmic reticulum retention protein.
5. A nucleic acid molecule according to claim 4, characterized in that: The nucleic acid molecules include a CAR gene targeting B7-H3, a P2A gene, and an endoplasmic reticulum retention protein gene. The P2A gene can recognize internal ribosome entry sites or ribosome codon skipping sites and is used to initiate independent translation of the endoplasmic reticulum retention protein gene.
6. A nucleic acid molecule according to claim 5, characterized in that: The nucleotide sequence of the CAR gene is shown in SEQ ID NO:13; the nucleotide sequence of the P2A gene is shown in SEQ ID NO:
14.
7. A nucleic acid molecule according to claim 5, characterized in that: The endoplasmic reticulum retention protein genes include PEBL1, PEBL2, and PEBL3 genes. The nucleotide sequence of the PEBL1 gene is shown in SEQ ID NO:15; the nucleotide sequence of the PEBL2 gene is shown in SEQ ID NO:16; and the nucleotide sequence of the PEBL3 gene is shown in SEQ ID NO:
17.
8. A retrovirus, characterized in that: The retrovirus was prepared in 293T cells using a retroviral vector and packaging vector of the nucleic acid molecule described in claim 4.
9. A pharmaceutical composition targeting B7-H3 with enhanced cell viability, applicable to the treatment of tumor diseases, characterized in that: It includes CAR-T cells as described in any one of claims 1-3, nucleic acid molecules as described in any one of claims 4-7, and retroviruses as described in claim 8.
10. The use of the CAR-T cells according to any one of claims 1-3, the nucleic acid molecules according to any one of claims 4-7, the retrovirus according to claim 8, and the pharmaceutical composition according to claim 9 in the diagnosis, prevention, or treatment of tumors, wherein the tumor is a tumor expressing B7-H3, and the tumor includes one or more of brain cancer, pancreatic cancer, ovarian cancer, kidney cancer, bladder cancer, gastric cancer, intestinal cancer, head and neck cancer, thyroid cancer, prostate cancer, and Kaposi's sarcoma; wherein the brain cancer includes one or more of glioblastoma, astrocytoma, meningioma, oligodendroglioma, and glioma.