case_images: PMC11772494_01
case_images: First diagnosis of T-ALL and the usage of drugs before CART therapy. (A) Bone marrow pathology. The area of nucleated cells was unevenly distributed, hyperplasia was extremely active in some areas, the original cells are diffuse, amd silver stain was positive. (a, b) hematoxylin and eosin stain. (c) silver stain. (B) Initial diagnosis of bone marrow MRD. 71.9% of nucleated cells expressed CD7, cCD3,CD5, CD38, TdT, some ceclls expressed CD34, CD1a, CD10, CD99, CD33, which did not express CD19,MPO,CD56. (C) The usage of drugs before CAR-T therapy. VDP, Vincristine, Daunorubicin, Dexamethasone; CAM, Cyclophosphamide, Cytarabine, Mercaptopurine; MA, Lipo-MIT, Cytarabine; Lipo-MIT, Mitoxantrone hydrochloride liposome; BUS, Busulfan; ATG, Anti-thymocyte Globulin; CSA, Cyclosporin A.
case_images: fimmu-15-1519055-g001.jpg
case_images: PMC11772494_01_fimmu-15-1519055-g001.jpg
case_images: f1
case_images: Bone marrow biopsy revealed malignant tumor ( Figure 1A ). Peripheral blood smears showed 66% primitive and naive cells. Bone marrow smear cytology showed hyperactive proliferation of primitive cells, and bone marrow flow immunotyping showed: 71.9% cells expressed CD7, CD34part, cCD3, CD5, CD38, CD1a part, CD10part, TDT, CD99 and CD33part ( Figure 1B ). Comprehensive detection of ALL/LBL related genes showed that KRAS variation abundance was 45.20%, NOTCH1 variation abundance was 39.5%, and PHF6 variation abundance was 93.5%. The treatment plan was shown in  Figure 1C . Figure 1 ). Fludarabine and Cyclophosphamide were used for lymphodepletion on March 01, 2024, then 50mL (total 1.80x108 cells) anti-CD7 CAR-T infusion was performed on March 06, 2024 ( Figures 3C,
case_images: Bone marrow analysis. (A) Bone marrow MRD analysis before CAR-T therapy. (B) Bone marrow MRD analysis after CAR-T therapy. On day 26 after infusion, there were no abnormal naive T cells. (C) The fingerprint of the patient after allo-HSCT. (a) Pre-transplant fingerprint. (b) Post-transplant fingerprint (date: 20230929). (c) Fingerprint of relapse after Allo-HSCT treatment (date: 20240116). (d) Fingerprint of CR after CD7 CAR-T infusion (date: 20241107). (D) Changes in chimerism rate. The chimerism rate was 25.3% after relapse.
case_images: fimmu-15-1519055-g002.jpg
case_images: PMC11772494_01_fimmu-15-1519055-g002.jpg
case_images: f2
case_images: The patient was maintained CR for 4 months after allo-HSCT, and then he was diagnosed with a relapse of T-ALL based on peripheral blood smears showing a significant increase in the number of original cells, and bone marrow smears showing that the number of original cells accounted for 76.5%, minimal residual disease (MRD) results showed that 54.9% of the original cells were abnormal naive T cells ( Figure 2A ) and bone marrow chimerism decreased to 26.64% on January 15, 2024. After 2 weeks of treatment with VDP (Vindesine, Daunorubicin, Dexamethasone) +PEG asp, bone marrow smears showed that primitive immature cells accounted for 63.5%, and minimal residual disease (MRD) results showed that 21.9% of original cells were abnormal naive T cells ( Figure 2A ). After one week of treatment, although the flow cytometry results on February 18, 2024 showed an MRD of 0.7% ( Figure 2A ), considering the high risk of disease recurrence and progression, subsequent infusion of allogeneic CAR-T cells aims to improve efficacy. According to the bone marrow MRD analysis of T-ALL, MRD remained negative from the 26th day after CAR-T infusion ( Figure 2B ;  Supplementary Figure 2 ), indicating that the patient's condition was effectively controlled and achieved CR. At the same time, the bone marrow chimerism rate recovered from 26.64% before infusion to 100% ( Figures 2C, D ) and the patient did not experience any symptoms related to GVHD, such as gut microbiota disruption caused by diarrhea ( Supplementary Figure 3 ).
case_images: Construction of CD7-specific CAR and protocol of anti-CD7 CAR-T cell infusions. (A) a schematic diagram of anti-CD7 CAR vector. (B) Flow detection of CD7 and CD7 CAR during cells preparation. The proportion of CD7+T gradually decreased after RV infection. (C) Characterization of the final anti-CD7 CAR-T cell product. VDP, Vindesine, Lipo-MIT, Dexamethasone; FA, Fludarabine; CTX, Cyclophosphamide. (D) a protocol of Treatment. FC for lymphodepletion, FA(30 mg, -D5~-D3) and CTX (300 mg, -D5~-D3). CAR-T cells were infused at a dose of 3x106/kg.
case_images: fimmu-15-1519055-g003.jpg
case_images: PMC11772494_01_fimmu-15-1519055-g003.jpg
case_images: f3
case_images: The structure of the CAR is shown in  Figure 3A . After 16 days culture in vitro, the proportion of CD7+ T cells continued to decrease ( Figure 3B ), indicating that CD7 CAR-T exerted a killing effect on CD7+ T cells. At the same time, the bone marrow chimerism rate recovered from 26.64% before infusion to 100% ( Figures 2C, D ) and the patient did not experience any symptoms related to GVHD, such as gut microbiota disruption caused by diarrhea ( Supplementary Figure 3 ).
case_images: Clinical responses and levels of key indicators to infusions of anti-CD7 CAR-T cells. (A) The changes of patient's WBC, Neut, Lymph, CRP. (B) Levels of IL-6 after CAR-T cell infusion. IL-6 peaked on the 1st day (2801 pg/mL) and returned to normal levels on the 12th day. (C) CAR-T cell expansion levels detected by flow cytometry and CAR DNA copies detected by qPCR. CAR-T peaked on day 14 and CAR DNA copies peaked on day 6 after infusion (CD3+ CAR+T cells: 83 cells/muL, CAR DNA copies:350758 copies/mug). (D) T cell counts analysis. T cell counts (111.8 cells/muL) were lowest on day 6 after infusion.
case_images: fimmu-15-1519055-g004.jpg
case_images: PMC11772494_01_fimmu-15-1519055-g004.jpg
case_images: f4
case_images: At this time, the count of patient's WBC and Neut decreased ( Figure 4A ), the levels of serum CRP ( Figure 4A ) and IL-6 ( Figure 4B ) also increased accordingly, which reached the highest peak within 1 week after infusion. The RV copy numbers of CD7-CARs reached the peak at 350, 758 copies/mug by using qPCR to quantitatively analyse ( Figure 4C ). After one week infusion, a decrease in the proportion of T cells was followed by an increase ( Figure 4D ), indicating that CD7 CAR-T exerted effect for T-ALL.