The Mechanism Of T Cell Dysfunction Related Resistance In CAR T Therapy For Lymphoma

Part I:Clinical Characteristics of Primary Resistance Factor to CD19/CD22 Cocktail CAR T-Cell Immunotherapy in Diffuse Large B-cell LymphomaObjective:Despite impressive progress,a significant portion（27%～48%）of patients still experience primary resistance to chimeric antigen receptor（CAR）T-cell immunotherapy for relapsed/refractory diffuse large B-cell lymphoma（r/r DLBCL）.The primary resistance mechanism closely involves T-cell dysfunction,showing weaker expansion and/or shorter persistence of CAR T cells in vivo.But its influence factors and mechanisms remain unclear.Therefore,this study proposes to establish a method for evaluating T cell function in r/r DLBCL patients receiving CD19/CD22 cocktail CAR T-cell immunotherapy and to identify risk factors for CAR T-cell dysfunction by statistical analysis of clinical characteristics.Methods:In summary,a total of 135 patients of DLBCL treated with murine CD19/CD22cocktail CAR T-therapy between January 2019 and August 2020 in the department of hematology in Tongji hospital were enrolled retrospectively.Based on four criteria(maximal expansion of the transgene/CAR-positive T-cell levels in vivo post-infusion[C_max],initial persistence of the transgene by the CAR transgene level at+3 months[T_last],CD19⁺B-cell levels[B-cell recovery],and the initial response to CAR T-cell therapy),48patients were included in the research and divided into two groups（a T-normal group[n=22]and a T-defect[n=26]group）.Univariate and multivariate regression analyses were performed to identify the risk factors and protective factors for CAR T cellular dynamics.Results:There was no significant difference in the baseline characteristics between the two groups.In the multivariable regression analysis,compared to T-normal group,patients with T-cell dysfunction（T-defect group）were associated with a significantly higher risk of LDH/upper limit of normal prior to leukapheresis（hazard ratio（HR）=1.922,95%confidence interval（95%CI）1.015-3.641,*P=0.045）and decreased risk of CRS grade（HR=0.150,95%CI 0.028-0.795,*P=0.026）but no increased risk in maximal tumor diameter（MTD）（HR=1.346;95%CI=0.737-2.456;P=0.334）.A higher LDH level before leukapheresis is an independent risk factor for T-cell dysfunction.In contrast,patients with T-cell dysfunction tend to present a lower level of CRS grade.Conclusion:Leukapheresis under low LDH levels was beneficial for patients in CAR T-cell immunotherapy.CRS grade evaluation after CAR T-cell infusion could assist doctors in assessing T-cell function and CAR T-cell efficacy in the early stages of treatment.This work may help explain the underlying mechanisms of primary resistance and provide clinical practice guidelines in CAR T-cell immunotherapy.Part Ⅱ:Establish the Methods for T cell Dysfunctional Functional Evaluation in vitroObjective:Therapeutic effects and resistance of CAR T-cell immunotherapy were significantly influenced by T cell dysfunction.The current research was focused on abnormal T cell phenotype,abnormal expression of exhaustion-related RNA and protein on T cells,and disruption of CAR T-cell in patients.Few research paid attention to T-cell defects in the resistance of CAR T-cell immunotherapy.Therefore,this study aims to create the“T cell Functional Evaluation Model,”which will provide new insight into evaluating patients’T cell function.Methods:Next-generation sequencing（NGS）and T cell function evaluation in vitro were performed to evaluate T-cell function.NGS was performed on the 46 patients who received CAR T-cell immunotherapy,including targeted high-throughput sequencing on tumor tissue or circulating tumor DNA（ct DNA）to figure out somatic alterations within 57 genes frequently altered in DLBCL and whole exome sequencing（WES）on peripheral blood mononuclear cells（PBMCs）for germline alterations analysis within 124 T-cell related-genes.Furthermore,T-cell functional experiments were conducted in vitro,including T-cell cytokine release,T-cell activation,degranulation,proliferation,and cytotoxicity.Healthy donors were performed as control samples for average value,and patients who harbored primary immunodeficiencies were enrolled as experimental samples to prove the rationality and effectiveness of the T cell function evaluation experiments.Results:NGS performed on the patients in Part I revealed that compared to the T-normal group,patients in the T-defect group had no difference in somatic mutations while significantly enriched germline variants within 47 T-cell related-genes(96%vs.41%;^****P<0.0001).These genes consisted of CAR vector genes（n=3,e.g.,CD19、CD28）,T-cell signal 1 to signal 3 genes（n=13,e.g.,LAT、TNFRSF18）,T cell immune regulation-and checkpoint-related genes（n=9,e.g.,UNC13D、JAK3）,cytokine-and chemokine-related genes（n=13,e.g.,IFNGR1、CX3CR1）,and T-cell metabolism-related genes（n=9,e.g.,ENTPD2、NFAT5）.Heterozygous germline UNC13D mutations presented the highest intergroup differences（T-defect vs.T-normal:26.9%vs.0%;**P=0.008）.Furthermore,normal values of T-cell functional experiments were 10th to the 90th percentile of values obtained in healthy donors（n=20）.Validation was performed on four patients who carried pathogenic germline alterations（UNC13D,TNFRSF9,MAGT1）,according to the characteristics of the genes and PIDs.Conclusion:Based on the clinical characteristics of patients in the Part I section and revealed by NGS on T-cell related-germline mutations,it indicated that T-cell-related-heterozygous germline genetic nosology might inform resistance mechanisms.Furthermore,the“T-cell Functional Evaluation Model”was built,consequently providing novel insight for T cell dysfunctional evaluation in CAR T-cell immunotherapy.PartⅢ:Clinical Application of T cell Dysfunctional Evaluation MethodsObjective:To verify the effectiveness of the“T-cell Functional Evaluation Model”,the resistance mechanisms in CAR T-cell immunotherapy were explored in patients with rare characteristics and complex genetic backgrounds,both of which were first reported in CAR T-cell immunotherapy.Detailed analysis of these rare cases could provide novel insight into CAR T-cell immunotherapy for lymphoma.Methods:Based on Part I and Part II,comprehensive analyses were performed to explore the inherent resistance mechanism in patients with complex genetic backgrounds.Including T-cell-related clinical characteristics analysis,NGS,and T-cell functional evaluation in vitro.Furthermore,various inspections were applied as auxiliary methods,such as fluorescence in situ hybridization（FISH）,flow cytometry,RNA sequencing,etc.Results:A patient with MYC,BCL2,BCL6 rearrangements,and TP53 deletion was resistant to the CAR19 and CAR22 T cell cocktail therapy.His clinical characteristics met the criterion of the T-defect group.RNA-seq furthering proved T-cell dysfunction on his CD3⁺T cells compared to healthy donors.NGS revealed somatic alterations（TP53,KMT2D,and IGLL5）,and clonal evolution（with new BCL2 mutation and subclones with DDX3X and BCL2 mutations after CAR T-cell infusion）have arisen.And CD19 antigen escape was revealed by flow cytometry.Those factors contributed to resistance in combination.Another male twin developed classical Hodgkin’s lymphoma in succession.A rare MAGT1 germline homozygous mutation was detected by NGS,which affected the degranulation and killing of T cells.Relapse mechanism after chemotherapy and CAR T-cell immunotherapy was associated with primary T cell immunodeficiency.Conclusion:Based on the“T-cell Functional Evaluation Model,”comprehensive analyses were applied in two rare cases with complex genetic backgrounds and resistant or relapsed after CAR T-cell immunotherapy.It is suggested that T-cell defect,T cell-related mutations,and other factors might appear simultaneously and contribute to resistance in combination,further proving the feasibility of the clinical application of this model.