CD7 protein plays a crucial role in T cell infiltration in tumors.

CD7 protein as a target is being used to treat CD7+ lymphoma; however, the role of CD7 in the hematopoietic system remains largely unknown. Therefore, we evaluated the effects of CD7 KO in mice. The differentiation of the hematopoietic system in the bone marrow and the number of various cell types in the thymus and spleen did not differ between CD7 KO and WT mice. After subcutaneous inoculation of B16-F10 melanoma cells, tumors from CD7 KO mice grew more rapidly, and the proportion of CD8+ T cells in the spleen and tumors decreased. In vitro, the infiltration and adhesion of CD8+ T cells from the spleen of CD7 KO mice were weakened. Blocking CD7 in normal T cells did not alter the migration and infiltration, but in Jurkat, CCRF-CEM, and KG-1a tumor cell lines, migration and invasion were significantly reduced after blocking CD7. Therefore, CD7 does not affect hematopoietic system development but plays a crucial role in T cell infiltration into tumors.

B7-H3 chimeric antigen receptor-modified T cell shows potential for targeted treatment of acute myeloid leukaemia.

BACKGROUND AND AIMS: Chimeric antigen receptor (CAR)-T cell therapy is a novel type of immunotherapy. However, the use of CAR-T cells to treat acute myeloid leukaemia (AML) has limitations. B7-H3 is expressed in several malignancies, including some types of AML cells. However, its expression in normal tissues is low. Therefore, B7-H3 is ideal for targeted AML therapy. MATERIALS AND METHODS: First, we constructed B7-H3 CAR that can target B7-H3, and then constructed B7-H3-CAR-T cells in vitro, which were co-incubated with six AML cell lines expressing different levels of B7-H3, respectively. The toxicity and cytokines were detected by flow cytometry. In vivo, AML model was established in B-NSG mice to study the toxicity of B7-H3-CAR T on AML cells. RESULTS: In vitro functional tests showed that B7-H3-CAR-T cells were cytotoxic to B7-H3-positive AML tumor cells and had good scavenging effect on B7-H3-expressing AML cell lines, and the cytokine results were consistent. In vivo, B7-H3-CAR-T cells significantly inhibited tumor cell growth in a mouse model of AML, prolonging mouse survival compared with controls. CONCLUSION: B7-H3-CAR-T cells may serve as a novel therapeutic method for the targeted treatment of AML.

RPRM deletion preserves hematopoietic regeneration by promoting EGFR-dependent DNA repair and hematopoietic stem cell proliferation post ionizing radiation.

Reprimo (RPRM), a target gene of p53, is a known tumor suppressor. DNA damage induces RPRM, which triggers p53-dependent G2 arrest by inhibiting cyclin B1/Cdc2 complex activation and promotes DNA damage-induced apoptosis. RPRM negatively regulates ataxia-telangiectasia mutated by promoting its nuclear-cytoplasmic translocation and degradation, thus inhibiting DNA damage. Therefore, RPRM plays a crucial role in DNA damage response. Moreover, the loss of RPRM confers radioresistance in mice, which enables longer survival and less severe intestinal injury after radiation exposure. However, the role of RPRM in radiation-induced hematopoietic system injury remains unknown. Herein, utilizing a RPRM-knockout mouse model, we found that RPRM deletion did not affect steady-state hematopoiesis in mice. However, RPRM knockout significantly alleviated radiation-induced hematopoietic system injury and preserved mouse hematopoietic regeneration in hematopoietic stem cells (HSCs) against radiation-induced DNA damage. Further mechanistic studies showed that RPRM loss significantly increased EGFR expression and phosphorylation in HSCs to activate STAT3 and DNA-PKcs, thus promoting HSC DNA repair and proliferation. These findings reveal the critical role of RPRM in radiation-induced hematopoietic system injury, confirming our hypothesis that RPRM may serve as a novel target for radiation protection.

Nanobody-based anti-CD22-chimeric antigen receptor T cell immunotherapy exhibits improved remission against B-cell acute lymphoblastic leukemia.

Chimeric antigen receptor (CAR) T-cell immunotherapies targeting CD19 can achieve impressive clinical remission rates in the treatment of B-cell non-Hodgkin lymphoma and B-cell acute lymphoblastic leukemia. However, relapse after CD19-CAR T treatment remains a major issue, with CD19 antigen-negative relapse being one of the main reasons. CD22, another antigen expressed in a B-cell lineage-specific pattern, is retained following CD19 loss. Accordingly, we hypothesized that CD22 could represent an alternative target to alleviate or compensate for the ineffectiveness of CD19-CAR T therapy. To this end, we generated camelid-derived CD22 nanobodies, whose smaller size, greater stability, and lower immunogenicity offer better quality than classical antibodies, and we used them to construct third-generation CD22-CARs containing 4-1BB and ICOS co-stimulatory domains. The novel CD22-CAR T cells exhibited impressive cytotoxicity both in vitro and in vivo and significantly prolonged the overall survival of tumor-bearing NSG mice. These findings provide the basis for further translational studies employing CD22-CARs.

Zkscan3 affects erythroblast development by regulating the transcriptional activity of GATA1 and KLF1 in mice.

ZKSCAN3 encodes a zinc-finger transcription factor that regulates the expression of important genes and plays a significant role in tumor development, pathogenesis, and metastasis. However, its biological functions under normal physiological conditions remain largely unknown. In our previous studies, using flow cytometry, we found that the deletion of Zkscan3 may cause abnormal erythropoiesis. In this study, we found that, in a Zkscan3 knockout mice model, the number of splenic early-stage (basophilic-erythroblasts) and late-stage (chromatophilic-erythroblasts to polychromatophilic-erythroblasts through orthochromatophilic-erythroblasts) erythroblasts increased, whereas the number of late erythroblasts in the bone marrow decreased. Moreover, the phenotype was exacerbated after treating mice with phenylhydrazine (PHZ), which causes severe hemolytic anemia. In the knockout mice treated with PHZ, the percentage of reticulocyte in the peripheral blood conspicuously increased, whereas MCHC and red blood cells decreased. Then, we performed RNA-seq and quantitative-polymerase chain reaction assay and found that the expression of GATA1 and Tiam1 in erythroblasts were upregulated, whereas KLF1 was downregulated. Luciferase assays showed that Zkscan3 inhibited the transcription of GATA1 and Tiam1 and promoted the expression of KLF1. Additionally, ChIP and CO-IP results confirmed that Zkscan3 directly interacts with GATA1 and inhibits its transcriptional activity in MEL cells. Our results demonstrate, for the first time, the significant role of Zkscan3 in physiological erythropoiesis through the interaction with GATA1, both at the DNA and protein level, and with Tiam1 and KLF1 at the DNA level.

Chimeric antigen receptor T cells derived from CD7 nanobody exhibit robust antitumor potential against CD7-positive malignancies.

The great success of chimeric antigen receptor T (CAR-T)-cell therapy in B-cell malignancies has significantly promoted its rapid expansion to other targets and indications, including T-cell malignancies and acute myeloid leukemia. However, owing to the life-threatening T-cell hypoplasia caused by CD7-CAR-T cells specific cytotoxic against normal T cells, as well as CAR-T cell-fratricide caused by the shared CD7 antigen on the T-cell surface, the clinical application of CD7 as a potential target for CD7+ malignancies is lagging. Here, we generated CD7ΔT cells using an anti-CD7 nanobody fragment coupled with an endoplasmic reticulum/Golgi retention domain and demonstrated that these cells transduced with CD7-CAR could prevent fratricide and achieve expansion. Additionally, CD7ΔCD7-CAR-T cells exhibited robust antitumor potiential against CD7+ tumors in vitro as well as in cell-line and patient-derived xenograft models of CD7-positive malignancies. Furthermore, we confirmed that the antitumor activity of CD7-CAR-T cells was positively correlated with the antigen density of tumor cells. This strategy adapts well with current clinical-grade CAR-T-cell manufacturing processes and can be rapidly applied for the therapy of patients with CD7+ malignancies.

Research progression on the first-line biological target therapy of advanced / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To develop a new type of CD7 chimeric antigen receptor modified T cell (CD7-CAR-T) for the treatment of CD7 positive acute myeloid leukemia (AML), and to observe its killing effect on CD7 positive AML cells. Methods: The CD7-CAR lentiviral vector was constructed based on the CD7 Nanobody sequence and costimulatory domain sequence of CD28 and 4-1BB. The lentiviral particles were packaged and used to co-transfect human T cells with protein expression blocker (PEBL), so as to prepare CD7- CAR-T cells. Real time cellular analysis (RTCA) was used to monitor the cytotoxicity of CD7-CAR-T cells on CD7 overexpressed 293T cells. Flow cytometry assay was used to detect the effect of CD7-CAR-T cells on proliferation and cytokine secretion of AML cells with high, medium and low CD7 expressions (KG-1, HEL and Kasumi-1 cells, respectively). Results: CD7-CAR-T cell was successfully constructed and its surface expression of CD7 was successfully blocked. Compared with T cells, CD7-CAR-T cells could significantly inhibit the proliferation of CD7-293T cells and promote the release of TNF, Granzyme B and INF-γ; in addition, CD7-CAR-T cells also significantly promoted the apoptosis (t=147.1, P<0.01; t=23.57, P<0.01) and cytokine release (P<0.05 or P<0.01) in CD7 positive KG-1 and HEL cells, but had little effect on Kasumi-1 cells that only expressed minimal CD7 antigen (t=0.7058, P>0.05). Conclusion: CD7-CAR-T cells can specifically kill CD7-positive AML cells in vitro.