Improved Radiotherapy Segmentation Combined With CAR-T Cell For B Cell Lymphoma

Objective:CD19 chimeric antigen receptor T(CAR-T)cells are an effective method for the treatment of relapsed refractory B-cell lymphoma,but some patients are still ineffective.Analyze the characteristics of ineffective patients and propose an improved CAR-T treatment method.This study shows that a large mass is an undesirable factor for CAR-T therapy.We designed an animal experiment for large mass B-cell lymphoma,adopted radiotherapy combined with CAR-T cell therapy,and further analyzed its mechanism.Further clarify the best combination of improved radiotherapy and CAR-T cell therapy.Methods:1.B-cell lymphoma patients receiving CD19 CAR-T cell therapy,to detect the number of CAR-T expansion in vivo,to evaluate the efficacy and adverse reactions.2.Analysis of disease characteristics of patients with relapsed and refractory B-cell lymphoma receiving CD19 CAR-T cell therapy,including pathological type of disease,disease state before CAR-T infusion,disease stage,B symptoms,extranodal involvement,Massive type(diameter≥7cm),bone marrow involvement,lactate dehydrogenase level,β2 microglobulin level,poor genetics,double shock or double expression,and whether CAR-T treatment is a second-line treatment or more than second-line treatment factors.3.For large tumor type is poor factor of CAR-T cell efficacy,design radiotherapy combined with CAR-T cell therapy Program.(1).Establishing a large-tumor mouse model,the therapeutic effects of CAR-T cells on lymphoma cell lines in vitro and in vivo,respectively.(2).Different radiotherapy segmentation methods,including the conventional 2Gy and large segmentation 4Gy,8Gy mode impact on immunity.(3).The possible mechanism is analyzed from the tumor microenvironment and tumor infiltrating lymphocytes.(4).Observe and detect the side effects of the combined treatment of the two and the management of side effects.Results:Part 1.(1).After infusion of CART cells,the CR rate was 45.5%and the ORR rate was 77.3%;CD19 CAR-T cell proliferation was detected in peripheral blood,and the peak of proliferation was 7 days after treatment(1～19 days);Peripheral blood CAR-T cells accounted for 7.08%(2.23%～28.60%)of the total T lymphocytes;(2).CAR-T cell therapy related adverse reactions:22 patients had different degrees of CRS,9 cases were grade 1 CRS,4 cases were grade 2 CRS,and 1case of grade 3 CRS.CRS was controlled after treatment with glucocorticoid and interleukin 6 antibody.Part 2:After infusion of CD19 CAR-T cells in patients with relapsed and refractory B-cell lymphoma,they are divided into the effective group and the poorly effective group,and the characteristics of these two groups of patients are analyzed.A large mass was an poor factor affecting the efficacy of CD19 CAR-T cells therapy.The difference was statistically significant(P=0.001).Logistic regression multivariate analysis showed that the characteristic of massive tumors was still independent prognostic factors for the poor efficacy of CD19 CAR-T cells in this group of patients(P=0.005,OR=0.039).Part 3:(1).Radiotherapy could kill tumor cells,but the tumor cannot be eradicated.Different treatment groups:Group A:2Gy×5 times,Group B:4Gy×5times and Group C:8Gy×3 times X-ray irradiation in split mode.The tumor volume of group B and C was significantly smaller than that of group A,these three methods of segmentation are not enough to cure a tumor with a volume of 300m~3.2.CAR-T cells were injected 24 hours after radiotherapy.Regardless of the type of radiotherapy,the ratio of CD4/CD8 will increase and return to normal levels after 3～7 days.This may be a mechanism for the body to escape damage caused by radiotherapy.It will also increase significantly in 3～7 days and play an immunosuppressive effect.3.The effect of combined CAR-T with different fractionated radiotherapy.The combination of large-segmentation can rapidly shrink the tumor,the tumor cannot be detected on the 7th day after treatment,and the therapeutic time of CAR-T cells was extended,so that the survival time of mice was prolonged.The survival time of each group was 45days(39～69 days),52 days(39～70 days),103 days(78-182 days)and 100 days(82-193 days).4.Compared with the CAR-T group and the 2Gy group,there was no statistically significant difference in the proportion of infiltrating T lymphocytes P=0.37(P>0.05),indicating that traditional radiotherapy is not sufficient to promote T lymphocyte invasion of tumors;large-segment radiotherapy could promote the infiltration of T lymphocytes into the tumor.The percentage of T lymphocytes in the four groups of different division methods in the tumor tissue are(2.13±0.11%),(2.77±0.21%)(7.63±0.49%)and 8.53±0.62%).CAR-T cells were detected in the peripheral blood of all mice,the peak time was3(2～7)days after treatment,CD19CAR-T cells accounted for 23.17%of T lymphocytes(7.92%～52.16%)and 14.05%(5.90%～28.06%),the proportion of CAR-T cell expansion was higher in large-segment radiotherapy than in traditional radiotherapy group,and there was no statistically significant difference between the two group(P=0.263).5.The mice with adverse reactions above grade 3 accounted for 2 in the 2Gy group,5 in the 4Gy group,and 7 in the 8Gy group.The mice of each group had different degrees of weight loss,of which the 8Gy group was the most obvious,and the difference was not statistically significant(P=0.151).Part 4:For a patient with grade 3 CRS after CAR-T cell therapy,there is no significant improvement after treatment with conventional glucocorticoids and interleukin 6 receptor antagonists.Cytokines of high levels(IL-2R:7500U/ml,IL-6:1000pg/ml,IL-8:103pg/ml,IL-10:615pg/ml,TNF-a:40.6pg/ml,INF-γ:670.8ng/ml,CRP:232.8mg/L)were detected,after three times of plasma exchange,the level of cytokines decreased significantly(IL-2R:7500U/ml,IL-6:43.1pg/ml,IL-8:64.2pg/ml,IL-10:11.7 pg/ml,TNF-a:25.2 pg/ml,INF-γ:31.2 ng/ml,CRP:14.17 mg/L),CRS-related clinical manifestations weree rapidly improved,bone marrow remission.Conclusions:1.CD19 CAR-T is an effective treatment for relapsed and refractory B-cell lymphoma,and the side effects can be controlled.2.A large mass is a poor factor of CAR-T cells for B-cell lymphoma.3.The large-segment radiotherapy program combined with CAR-T cell therapy can improve the efficacy of CAR-T cell therapy in B-cell lymphoma mouse models with large tumor,and prolong survival.It may be related to the tumor microenvironment and infiltration of T lymphocytes.It should be noted that the management of side effects after the two combined treatments.4.Plasma exchange therapy was one of the effective measures to treat severe CRS.