| Background and Objective: Presently,the malignant tumor has become a major public health problem.The most common brain malignant tumors of the central nervous system(CNS)include glioma,medulloblastoma,WHO III grade meningioma,etc.The common strategies for brain malignant tumors therapy are surgical resection,postoperative chemotherapy and radiation,such as the integrated treatment model.However,after the comprehensive treatment,the tumors will inevitably relapse with shorter overall survival time,which remain a challenge and dilemmas in the treatment and clinical management of tumor recurrence.In particular,for glioblastoma(GBM)and medulloblastoma(MB),the treatment options are still limited;the current treatment regimens still do not provide an ideal survival time,and these treatments may lead to serious side effects.Therefore,it is urgent to develop novel and effective therapeutic methods for these relapsed/refractory GBM and MB.In recent years,the development of immunotherapy has brought new hope for the treatment of malignant tumors.Among them,the CAR-T cell immunotherapy is considered to be one of the most promising tumor treatment methods,and this method is effective in the treatment of some hematologic tumors.The principle is that the CAR,which can specifically recognize target tumor cells,is expressed on T cells by gene modification,and these T cells use CAR receptors to specifically recognize and bind to the target cells,this combination triggers the activation of T cells and releases cytokines,perforin,granzyme and other anti-tumor agents to kill tumor cells.The breakthrough of CART cell therapy in hematologic tumors has led to the expectation and exploration of CAR-T cell therapy for solid tumors,such as high-grade gliomas and MB with poor prognosis.The key to this approach is to select an ideal target to construct the CAR molecule,and the ideal target antigen should only be highly expressed in tumor cells,but not expressed or low expressed in normal tissue cells.Recently,based on the literature and our previous studies,it has been found that B7-H3 antigenic molecules are highly expressed in both GBM and MB,but not expressed or low expressed in normal cells,which is specificity and is an ideal target antigen.In addition,B7-H3 is also a negative immunomodulatory molecule,which not only inhibits the activation and proliferation of T cells and NK cells but also inhibits the T cells secretion of the IL-2 and IFN-γ.Therefore,in theory,the B7-H3.CAR-T cells can not only kill tumor cells,but also improve the tumor’s immunosuppressive microenvironment,playing a doubleedged sword role.Based on our previous studies on B7-H3 CAR-T cells for gliomas cells in vitro and in tumor-bearing animal models,we further explored 1.the clinical application of B7-H3.CAR-T cells for recurrent refractory GBM;2.The application of B7-H3.CAR-T cells for relapsed/refractory MB for providing data on the further administrating CAR-T cells for patients.Materials and Methods: Part I1.Patients were recruited and surgically resected for recurrent GBM.Ommaya device was placed in the tumor lumen during operation to provide a channel for subsequent CAR-T cell injection.2.Immunohistochemical staining(ICH)was performed for GBM tissues,and primary cells were isolated and cultured at the same time.Cell immunofluorescence and flow cytometry were performed to evaluate the expression of B7-H3 in primary tumor cells.If the patient can provide the specimen of the previous operation,this analysis procedure can be made firstly to screen whether the patient meets the inclusion criteria.3.B7-H3-target CAR lentivirus vector was constructed,lentivirus packaging,and analyzed whether the related indicators meet the requirements of follow-up studies;4.The peripheral venous blood of the patient was extracted,lymphocytes were isolated and amplified,and the CAR-T cells targeting B7-H3 were manufactured by infecting T cells with the suspension of lentiviral vector virus;5.The phenotype of CAR-T cells was analyzed by flow cytometry,and the specific killing ability of CAR-T cells against primary GBM cells was verified by cell experiments in vitro;6.The CAR-T cells were injected into the tumor lumen of the patient via Ommaya device according to a set time point;7.During the treatment,the patient’s condition and side effects were closely monitored,and the efficacy of treatment was evaluated by head-enhanced MRI and monitoring of CAR-T cells and cytokines in cerebrospinal fluid(CSF)and blood.Part IIThe general method and steps of part II are the same as the first part,but there are still some differences.1.Patients with MB were recruited,and the tissue samples of the previous operation were taken for ICH staining to evaluate the expression of B7-H3;2.Medulloblastoma cell lines of Daoy cells and D283 cells were cultured,and the expression of B7-H3 in tumor cells was detected by immunofluorescence assay;3.The construction of B7-H3.CAR vector and the manufacturing of B7-H3.CAR-T cells were the same as those of methods 3 and 4 in part I;4.The phenotype of CAR-T cells was analyzed by flow cytometry,and the specific killing ability of CAR-T cells against Daoy cells and D283 cells was verified in vitro;5.Lumbar puncture was performed at the set time point,and CAR-T cells were injected into the subarachnoid space of the spinal canal through this pathway;6.Treatment monitoring and evaluation are similar to Part I of method 7.Results:Part I1.B7-H3 was highly expressed in GBM tissues,but not in the peri-tumor tissues,and B7-H3 antigen was highly expressed in primary tumor cells by flow cytometry and immunofluorescence staining.2.We prepared B7-H3.CAR-BBζ-T cells.Flow cytometric analysis showed that the CAR-T cells expressed central memory T cell markers(CD45RO and CD62L),but effector T cell markers(CD69 and CD25),PD1 and TIM3 were negative or low.3.B7-H3.CAR-BBζ T cells were co-cultured with tumor cells,and the B7-H3-positive GBM primary cells were specifically killed,but the B7-H3-negative tumor cells were not.In the group of CAR-T cells co-cultured with GBM primary cells,the IL-2 and IFN-γ secreted more in the medium supernatant.4.The brain MRI after infusion of B7-H3.CAR-BBζ-T cells showed a significant reduction in tumor volume compared with pre-infusion.In addition,compared with the brain MRI enhanced signal before injection,the signal intensity of the recurrent tumor was weakened.5.The levels of cytokines IL-1β,IL-Rα,IL-2,IL-5,I-L6,IL-8,IL-17 A,IP-10,IFN-γ,and TNFα in CSF increased significantly before and after the 1st,2nd,and 3rd CAR-T cell therapy,but decreased after the 4th,5th,and 6th CAR-T cell therapy.6.There was no grade 3 or higher adverse events during intra-tumoral injection of B7-H3.CAR-BBζ-T cells.Part II1.B7-H3 molecules were highly expressed in the medulloblastoma tissue,and immunofluorescence staining showed that B7-H3 antigen molecules were expressed on the Daoy cells and D283 cells.2.We made B7-H3.CAR-BBζ-T cells.Flow cytometric analysis showed that the CAR-T cells expressed central memory T cell markers(CD45RO and CD62L),effector T cell marker CD69 was low,PD1 and LAG3 were low.3.B7-H3.CAR-BBζ-T cells are co-cultured with tumor cells,B7-H3 positive Daoy cells and D283 cells are specifically killed,while B7-H3 negative tumor cells will not be killed.B7-H3.CAR-BBζ-T cells co-cultured with Daoy cells and D283 cells secreted more IL-2 and IFN-γ in the medium supernatant.4.After three intrathecal injections of B7-H3.CAR-BBζ-T cells,reexamination of the head MRI showed a significant reduction in tumor volume and a reduced enhancement signal in the recurrent tumor compared to the pre-CAR-T infusion.5.The levels of cytokines IL-1β,IL-Rα,IL-2,IL-6,IL-8,IL-17 A,IP-10,IFN-γ and TNF-α in CSF increased significantly before and after each CAR-T cell injection.6.No grade 3 or higher adverse events occurred during intraocular injection of B7-H3.CAR-BBζ-T cells.Conclusions:1.The CAR-T cells constructed by lentiviral vector with B7-H3 as the target,4-1BB and CD3ζ as the intracellular domain,generated B7-H3.CAR-BBζ-T cells were mainly composed of central memory T cells;2.Vitro experiments showed that B7-H3.CAR-BBζ-T cells could kill B7-H3 positive GBM primary cells,Daoy cells and D283 cells;3.The increasing trend of cytokines in CSF may indirectly reflect the efficacy of B7-H3.CAR-BBζ-T cells for recurrent refractory GBM or MB;4.B7-H3.CAR-BBζ-Tcells are effective against B7-H3-positive recurrent refractory GBM and MB by intramural and intrathecal injection,respectively,and can inhibit tumor growth without serious side effects,providing a new treatment option for these tumors;5.Intrathecal injection of CAR-T cells to treat MB is feasible and safe.It provides a novel administering CAR-T cells that do not rely on the Ommaya pathway,and has the value of clinical application and promotion. |
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