Work

Chimeric antigen receptor T cells targeting CD312 as a therapy for acute myeloid leukemia

Year: 2024

Type: article

Abstract: To the Editor: Chimeric antigen receptor (CAR)-T-cell therapy has made considerable breakthroughs in the treatment of lymphoma and myeloma, but the lack of suitable or specific targets for acute myelocytic leukemia (AML) has limited the progress of CAR-T-cell therapy in the clinical treatment of AML. Cluster of differentiation (CD312), a member of the adhesion G protein-coupled receptor family, is highly expressed in acute myeloid leukemia and absent in normal hematopoietic stem cells (HSCs),[1] making it a potential target antigen for CAR-T-cell therapy. In this study, we assessed the expression of CD312 in AML cell lines and patients and constructed anti-CD312 CAR-T cells, which showed excellent antitumor activity and safety in vitro and in vivo. Notably, CD312 CAR-T cells exhibited no significant off-target effects, suggesting their potential as an effective and safe treatment strategy for AML. The study was reviewed and approved by the Ethics Committee of Tianjin First Center Hospital (No. 2020N212KY). The animal study was approved by the Medical Ethics Committee of Nankai University (No. 2024-SYDWLL-000127). Written informed consent was obtained from all the participants or their legal representatives. To verify whether CD312 can be used as a target for CAR-T-cell treatment of AML, we assessed CD312 expression in a set of AML cell lines and human primary AML bone marrow specimens via flow cytometry and used a negative control cell line, NALM6, which is a human acute lymphoblastic leukemia (ALL) cell line that does not express CD312. The myeloid tumor cell lines (K562, SKM1, THP-1, MOLM13, and U937) exhibited specific expression of CD312 [Figure 1A]. Next, we analyzed the expression of CD312 in AML patients with different French–American–British (FAB) subtypes in the cBioPortal database. The results revealed that CD312 expression was lower in patients with the M6 and M7 subtypes than in patients with the M0–M5 subtypes [Figure 1B]. Furthermore, we detected the expression of CD312 on primary AML cells from 106 fresh bone marrow samples (AML: 80; myelodysplastic syndrome [MDS]: 26). Approximately 3.3% of patients had positive CD312 expression, and the median expression rate of CD312 was 73.38% (range 0.90–99.74%) [Figure 1C].Figure 1: (A) Analysis of CD312 expression in tumor cell lines by flow cytometry. (B) The messenger RNA mRNA expression of CD312 in each type of AML based on FAB typing in the cBioPortal database. (C) The distribution of CD312+ cells in bone marrow samples of primary AML (AML n = 90; MDS n = 26). (D) The illustration of CD312 CAR. (E) CD312 CAR-T cells or untransduced T cells were co-cultured with MOLM13 cells at 1:4, 1:1, and 3:1 ratios and analyzed for cytotoxicity after 24 h. (F) CD312 CAR-T or untransduced T cells were co-cultured with AML primary cells at 5:1 ratio and assayed for cytotoxicity after 24 h. (G) Representative BLI maps for monitoring tumor progression in MOLM13 xenograft mice weekly at fixed intervals. (H) Analysis of colony formation performed with residual live hematopoietic stem and progenitor cells (HSC/Ps) after 6 h of coincubation with CD312 CAR-T cells or untransduced T cells. Diagram shows the absolute number of colonies determined by microscopy on day 14. Three independent repeated experiments were performed, and the data are expressed as mean ± standard deviation. * P <0.05, † P <0.01, ‡ P <0.001 represent significant. AML: Acute myelocytic leukemia; BFU-E: Burst-forming units-erythroid; BLI: Bioluminescence imaging; CAR: Chimeric antigen receptor; CAR-T: Chimeric antigen receptor T; CFU–GM: Colony-forming units-granulocyte-macrophage; CFU–GEMM: Colony-forming units-granulocyte-erythrocyte-macrophage-megakaryocyte; FAB: French–American–British; MDS: Myelodysplastic syndrome.In addition to CD312, we also measured the expression of human C-type lectin-like molecule 1 (CLL1), a classical marker antigen of AML. We found that CD312 and CLL1 appeared to have a “complementary” relationship in 69 AML patients. Most primary AML cells expressed CD312 and/or CLL1 [Supplementary Figure 1A, https://links.lww.com/CM9/C210]. Phenotypic analysis of bone marrow lymphocytes, granulocytes, and monocytes revealed that CD312 was expressed on granulocytes and monocytes but not on lymphocytes, which was consistent with the myeloid-restricted expression profile of CD312 [Supplementary Figure 1B, https://links.lww.com/CM9/C210]. Next, we generated a second-generation CD312-41BB CAR construct [Figure 1D] and evaluated its effects on cell viability [Figure 1E and Supplementary Figure 1C,D, https://links.lww.com/CM9/C210] and cytokine production [Supplementary Figure 1E, https://links.lww.com/CM9/C210] of MOLM13, U937, and THP-1 cells. Overall, in vitro experiments demonstrated that CD312 CAR-T cells exerted cytotoxic effects on these cell lines at different effector-to-target (E:T) ratios (1:4, 1:1, and 3:1), whereas no cytotoxic effects on NALM6 cells [Supplementary Figure 1C, https://links.lww.com/CM9/C210]. So we demonstrated that CD312 CAR-T cells exhibited potent antitumor effects in vitro. We subsequently evaluated the cytotoxicity of CD312 CAR-T cells against primary AML cells from three patients whose tumor burden exceeded 80% [Supplementary Figure 1F, https://links.lww.com/CM9/C210] at an E:T target ratio of 5:1 for 24 h. Compared with uninfected T cells, CD312 CAR-T cells exhibited significant cytotoxic effects on primary AML cells [Figure 1F] and screted higher level of cytokines [Supplementary Figure 1G, https://links.lww.com/CM9/C210]. Overall, our findings demonstrated that CD312 CAR-T cells effectively kill primary AML cells. We inoculated NOD-SCID IL-2 receptor gamma null (NSG) mice with ffLuc + green fluorescent protein (GFP) + MOLM13 or U937 AML cells, and medullar and extramedullary AML manifestations were assessed by bioluminescence imaging (BLI). On day 3, we treated the mice with CD312 CAR-T cells or untransduced T cells. The progression of AML was monitored via BLI from day 7 onward. By day 14, we observed reduction in the systemic leukemia burden via BLI [Supplementary Figure 2A, https://links.lww.com/CM9/C210]. On day 21, the most significant difference in AML tumor burden was observed between the experimental and control groups, and most of the control mice died after day 21, indicating a survival advantage for mice treated with CD312 CAR-T cells [Figure 1G and Supplementary Figure 2B, https://links.lww.com/CM9/C210]. We observed similar results in the MOLM13 and the U937 transplantation models [Supplementary Figure 2C,D, https://links.lww.com/CM9/C210]. To assess the potential impact of CAR-T-cell therapy on liver and kidney function, the biochemical parameters of venous blood from the mice were analyzed. All the liver and kidney indices of the mice were within the normal range, suggesting that CD312 CAR-T cells can be safely administered in vivo without causing toxicity to specific organs [Supplementary Figure 2E,F, https://links.lww.com/CM9/C210]. In conclusion, this study demonstrated that CD312 CAR-T cells have a potent anti-AML effect in vivo while also exhibiting a significant safety profile. To address concerns regarding the potential bone marrow toxicity associated with CAR-T-cell therapy for AML, we investigated the effect of CD312 CAR-T cells on normal hematopoiesis. First, we evaluated the expression of CD312 on progenitor cells (CD34+CD38–) and stem cells (CD34+CD38+) from donor bone marrow samples. The results revealed that CD312 was only expressed in a few normal HSCs [Supplementary Figure 3A, https://links.lww.com/CM9/C210]. We subsequently investigated the effect of CD312 CAR-T cells on the colony formation ability of peripheral blood mononuclear cells (PBMCs) from CD34+ HSC-enriched donors. CAR-T or uninfected T cells were cultured with HSCs at a ratio of 10:1 for 6 h and then transferred to methylcellulose semisolid medium for observation for 14 days. The number of burst-forming units-erythroid (BFU-E), colony-forming units-granulocyte-macrophage (CFU–GM), and colony-forming units-granulocyte-erythrocyte-macrophage-megakaryocyte (CFU–GEMM) were counted on day 14 [Supplementary Figure 3B, https://links.lww.com/CM9/C210]. The results indicated that there was no significant difference in the number of colonies generated between the CAR-T-cell group and uninfected T-cell or untreated group [Figure 1H]. Based on these findings, it can be reasonably concluded that CD312 CAR-T cells do not lead to adverse toxic effects on HSCs. Over the past few years, CAR-T therapy for AML has been limited due to the lack of highly specific antigens espressed in AML cells. Currently, the CLL1 target may be the optimal choice. Our group also reported a phase I study of CLL1 CAR-T cells in adults with relapsed/refractory AML in 2022, and the clinical effect was promising.[2] However, the severe agranulocytosis caused by CLL1 CAR-T cells presents certain challenges for their clinical application. We discovered that CD312 expression in granulocytes is lower than CLL1, which implies that CD312 CAR-T cells may cause a lower degree of agranulocytosis and thus safer for clinical application. Furthermore, we found that CD312 is expressed in monocyte-derived macrophages. An increasing number of studies have indicated that macrophages are important source of toxic cytokines.[3] These toxic cytokines, especially IL-6, play crucial roles in the production, development, and management of cytokine release syndrome (CRS), the most serious complication in CAR-T-cell treatment; thus, we hypothesized that CD312 CAR-T cells can target and destroy macrophages, resulting in a considerably milder CRS response. Additionally, we observed an interesting phenomenon of complementary expression between CLL1 and CD123, which may imply that for AML patients with low expression of single target, dual-target CAR-T cells can be constructed to better eliminate AML cells. We believe that CD312 CAR-T cells have safe and effective anti-AML effects and can also be used in various stages of AML treatment, such as serving as a “bridge to transplant” and eliminating minimal residual disease (MRD). In conclusion, we successfully constructed CAR-T cells targeting CD312 and validated its substantial anti-AML effects both in vitro and in vivo, as well as their favorable safety profile. Importantly, CAR-T cells caused no damage to healthy tissues or typical hematopoiesis. These findings highlight the enormous potential of CD312 CAR-T in the treatment of AML. Funding This work was supported by grants from the General Project of the National Natural Science Foundation of China (No. 81970180), the Science and Technology Project of Tianjin Municipal Health Committee (No. TJWJ2022QN030), the Key Projects of Tianjin Applied Basic Research and Multi-Investment Fund (No. 21JCZDJC01240), the Science and Technology Project of Tianjin Municipal Health Committee (No. TJWJ2022XK018), the Tianjin Key Medical Discipline (Specialty) Construction Project (No. TJYXZDXK-056B), the Tianjin Municipal Natural Science Foundation (No. 22JCQNJC00820), the Tianjin Health Research Project (No. TJWJ2023QN027), the Tianjin Health Bureau Project (No. ZC20074), and the Tianjin Key Medical Discipline (Specialty) Construction Project (No. TJWJ2023XK010). Conflicts of interest None.

Source: Chinese Medical Journal

DOI: 10.1097/cm9.0000000000003362