Study On Enrichment Of CAR-T Precursor Cells In Children With Blood Cell Separator

BackgroundChimeric antigen receptor T lymphocyte(CAR-T)treatment is a new tumor immunotherapy method,which has been applied in cancer of children with leukemia,neuroblastoma and rhabdomyosarcoma.Chimeric antigen receptor T lymphocyte treatment has achieved curative effect in recent years.With the advent of CAR-T immunotherapy,obtaining sufficient number of active CAR-T precursor cells with the use of MNC collection function and technique of blood cell separators is one of the key factors for the success of CAR-T cell immunotherapy.However,the application of a safe,feasible,reliable,and effective method for enriching CAR-T precursor cells with a blood cell separator needs to be studied,such as the type of blood cell separator and the selection of apheresis procedures,the setting of MNC function parameters,the prevention and treatment of adverse reactions,the safety assessment and so forth.In young children,unlike adults,the physiological growth of each system is not yet mature,the circulating blood volume is small,the compliance and coordination are low,and no mobilization agent is used before isolation.What is more the absolute number of T cells in peripheral circulating blood is small.Therefore,we designed a protocol for enriching CAR-T precursor cells in children using a continuous flow COM.TEC blood cell separator and evaluated the safety,feasibility,efficacy,and reliability of the protocol.ObjectiveTo evaluate the safety,feasibility and reliability of concentrating the CAR-T precursor cell program in children with blood cell separator.MethodsAccording to the established enrichment program,The CAR-T precursor cells were collected from 16 children by COM.TEC blood cell separator with Auto-MNC program.The obtained peripheral blood mononuclear cells were sorted and counted?Safety was assessed by adverse events,changes of blood cell counts and blood biochemical indexes before and after CAR-T precursor cells collection.Data were analyzed using the SPSS 21.0 software package.The analysis results were expressed as mean � standard deviation('X�S).Before and after CAR-T precursor cells collection,peripheral blood cell counts and blood biochemical indexes changes were expressed by Paired t test.P values less than 0.05 was considered statistically significant.Results1.We performed 22 lymphocyte apheresis on 16 patients using continuous flow cell separators by processing 2.2 times of their total blood volume.A total of 59 ml(30-102ml)of CAR-T precursor cells was collected in(13 5�3 9)min using(340�101)ml ACD by processing the(2271 �294 ml)peripheral blood.2.The main components of the product containing CAR-T precursor cells were lymphocytes.The number of lymphocytes was(32.25 � 22.39)� 109/L,which accounting for about 62.92%of the total amount.The product also has a certain amount of red blood cells,platelets,monocytes and neutrophils.3.The absolute values of WBC,Hb,Hct and mononuclear cells in children before and after CAR-T precursor cells enrichment did not change obviously.There was no significant difference before and after collection(P value>0.05).The PLT decreased from 205 � 109/L to 115 � 109/L after enrichment,with a decrease of 43.9%.However no bleeding occurred.There is no need for transfusion of platelets.After enrichment,the number of lymphocytes was significantly decreased compared with the previous,the difference was significant(P value<0.05).4.Before enriching CAR-T precursor cells;The concentration of peripheral blood potassium,peripheral blood calcium,peripheral blood glucose were(4.10�0.32)mmol/L,(2.27�0.30)mmol/L and(5.74�1.30)mmol/L.Respectively,after enrichment of CAR-T precursor cells,The concentration of peripheral blood potassium,peripheral blood calcium,peripheral blood glucose were(3.58�0.54)mmol/L,(2.52�0.20)mmol/L,and(5.71�1.22)mmol/L.There was no significant difference in electrolyte levels before and after enrichment(P value>0.05).Hypokalemia,hypocalcemia,high glucose or hypoglycemia did not appear during the collection.5.Young patients have a stable vital sign during CAR-T precursor cell enrichment.During the enrichment process,One patient with numbness was observed.One patient had a rash.No citrate poisoning reactions such as numbness and numbness and convulsions were observed.And no adverse reactions such as hypovolemia or circulatory overload were observed.6.The amount of CAR-T cells infused was 6.77 � 3.01 �107cells(1.8 � 107 cells?1.2 � 108cells)on average.All 18 patients were safely transfused with CAR-T cells and no obvious toxic reactions were found.ConclusionOur program of enriching children CAR-T precursor cells is safe,feasible,effective,and reliable.