To do this, we measured the percentage of GFP+ Jurkat cells using circulation cytometry (Figures 5C and S2B). the need for complex T?cell modifications. This approach is usually safer than previous methods and provides a new, simple, and feasible strategy for clinical immunotherapies targeting CD7-positive malignant tumors. Keywords: recombinant anti-CD7 blocking antibody, anti-CD7 CAR-T cell, fratricide, immunotherapy, T-ALL Graphical Abstract Open in a separate windows Ye et?al. statement a way to solve fratricide during the culture of anti-CD7 CAR-T cells by adding blocking MSH6 antibody. It is a new, simple, and feasible strategy to obtain sufficient and effective anti-CD7 CAR-T cells for clinical immunotherapies targeting CD7-positive malignant tumors. Introduction T cell malignancies are a group of heterogeneous diseases that reflect the clonal nature of T?cells with impaired functions.1 In particular, T?cell acute lymphoblastic leukemia (T-ALL) is a highly invasive hematological malignancy caused by the abnormal proliferation of hematopoietic stem cells.2 Despite treatment with multidrug chemotherapy regimens, 30% of patients eventually relapse, and fewer than 15% of relapsed patients have an event-free survival time of 3 years.3,4 In particular, the malignancy of T?cell malignant tumors is greater than that of B cell malignant tumors, which makes T-ALL treatment significantly challenging.5 Chimeric antigen receptor (CAR) T?cell immunotherapy is promising for the treatment of refractory and relapsed B cell acute lymphoblastic leukemia (r/r B-ALL) and the first commercial T?cell therapy approved for malignancy.6,7 This success implies that CAR-T cell therapy is very promising for the treatment of T?cell malignancies. At present, CD1a, CD5, and CD7 are the major selective?target antigens in T-ALL. However, because CD1a expression is characterized in only some subtypes, such as cortical thymocytes,?and CD5 expression is low, targeting CD1a or CD5 is not suitable for long-term treatment.8, 9, 10, 11, 12 CD7 is a 40?kDa single-chain glycoprotein that is expressed by T?cells and natural killer (NK) cells.13 Unlike the CD1a and CD5 antigens, CD7 accounts for more than 95% of the dominant expression in T?cell leukemias and lymphomas and stably maintains high expression.8,14 In addition, clinical data have shown that CD7 is continuously expressed on cancerous T?cells at higher levels than that on T?cells from normal donors and is therefore considered an attractive target for CAR-T cell therapy.15 However, it is almost impossible to produce sufficient anti-CD7 CAR-T cells for clinical therapy because TAS-115 of fratricide.16 At TAS-115 present, two strategies have been reported to avoid fratricide. One strategy is to knock out CD7 using CRISPR-Cas9 gene editing technology,?and the other is to target the CD7 protein with an anti-CD7 single-chain variable fragment (scFv) coupled with an endoplasmic reticulum (ER)/Golgi-retention domain to retain newly synthesized CD7 in the ER or Golgi apparatus. However, both strategies require additional modifications at the DNA level to inhibit the?expression of CD7 on the cell membrane, which may introduce unpredictable risk (such as off-target effects); moreover, these strategies may have an unknown impact on the biological function of CAR-T cells because of the TAS-115 lack of CD7 expression on the cell membrane.10,11,15,17 Therefore, to overcome the shortcomings of the reported methods for preparing anti-CD7 CAR-T cells, we propose a new strategy in which the CD7 antigen on the T?cell surface is blocked with a free anti-CD7 antibody containing the same binding domain as the CAR to avoid fratricide during anti-CD7 CAR-T cell preparation. Our research showed that the anti-CD7 CAR-T cells cultured with the antibody, which was added during the preparation stage, exhibited improved cell viability and proliferation and a relatively ideal subpopulation. Finally, we harvested a sufficient amount of anti-CD7 CAR-T cells with specific and effective cytotoxicity against CD7 antigen-positive target tumor cells for clinical use. This new study provides a simple, safe, and feasible method for preparing anti-CD7 CAR-T cells for the clinical treatment of T-ALL. Results The expression of an anti-CD7 CAR on T?cells leads to fratricide To obtain a CAR that can specifically target the CD7 antigen on the surface of tumor cells, we designed two third-generation anti-CD7 CARs (Figure?1A). Anti-CD7 CAR-T 1 and TAS-115 anti-CD7 CAR-T 2 cells were prepared using lentiviral transduction, and the infection efficiencies measured on day 8 were 51.2% and 25.7%, respectively (Figure?1B). Open in a separate window Figure?1 Expression of anti-CD7 CAR on T?cells results in fratricide (A) Schematics of two third-generation anti-CD7 CAR constructs: anti-CD7 CAR-T 1 (top) and anti-CD7 CAR-T 2 (bottom). (B) Representative histograms of anti-CD7.