| Part Ⅰ: In vivo expressing of CD19 BiTE for the treatment of B-cell hematological malignancies by liver-targeted adenoassociated virus[Objective]Bispecific T-cell engagers（BiTE）is an “off-the-shelf” tumor immunotherapy drug,which can efficiently activate T cells and kill tumor cells.Currently,it is a research hotspot in the field of cancer therapy.CD19-targeted BiTE（CD19BiTE）has significant efficacy in the treatment of relapsed/refractory B-cell hematologic malignancies,but it has a short half-life that necessitates long-term continuous intravenous infusion,which undoubtedly increases the treatment cost and reduces patient compliance.Meanwhile,unstable plasma concentration compromises the efficacy of CD19 BiTE.Therefore,it is of great clinical significance and broad application value to explore the continuous and stable in vivo expression of CD19 BiTE.In this study,the liver-targeted adeno-associated virus（AAV）expressing CD19 BiTE was applied to treat B-cell hematological malignancies,in order to achieve long-term anti-tumor activity with one AAV injection,improve the efficacy of CD19 BiTE and reduce the treatment cost,which is expected to become a new strategy for the treatment of B-cell hematological malignancies.[Methods]（1）Construction of liver-targeted AAV vector: Firstly,CD19 BiTE sequence was identified by literature investigation and integrated into the AAV8 vector,and then liver-targeted promoter was inserted to construct a liver-targeted AAV vector expressing CD19 BiTE.（2）Acquisition and purification of AAV: The CD19 BiTE plasmid and helper plasmid were co-transfected into HEK293 T cells by lipid transfection,and the supernatant was collected and concentrated after 72 h,and then the concentrated AAV was purified by CsCl gradient centrifugation.Finally,Real-time quantitative polymerase chain reaction（RT-qPCR）was used to detect the AAV titers.（3）Detection of liver-targeted AAV-CD19 BiTE expression: 293 T and two hepatoma cells（PLC/PRF/5 and Hep G2 cells）were transfected with AAV-CD19 BiTE.After 72 h,the expression of CD19 BiTE was analyzed by antibody competition and immunofluorescence assays.（4）Analysis of anti-tumor activity of AAV-CD19 BiTE in vitro: T cells were coincubated with CD19+ NALM-6 and Raji cells,and CD19-K562 cells based on the effector-target ratio of 5:1,and then the culture supernatant of Hep G2 cells transfected with AAV-CD19 BiTE was added.The killing ability of AAV-CD19 BiTE on the above three cells was analyzed by flow cytometry after 48 h.（5）Treatment of B-ALL with AAV-CD19 BiTE: NCG mice were injected with NALM-6 cells via the tail vein to construct the B-ALL model,and then AAV-CD19 BiTE(5×1012 gc/kg)and human peripheral blood mononuclear cells（PBMNC）were injected into the mice.Meanwhile,AAV-GFP was used as the control group to observe the changes of tumor burdens by small living animal imaging machine at regular intervals to evaluate the therapeutic effect of AAV-CD19 BiTE.（6）Treatment of B-cell lymphoma with AAV-CD19 BiTE: NCG mice were subcutaneously injected with Raji lymphoma cells to construct a CDX（cell linederived xenograft）model of B-cell lymphoma,and then AAV-CD19 BiTE(5×1012 gc/kg)and PBMNC were injected into the mice.Meanwhile,AAV-GFP was used as a control group to observe the changes of tumor burdens by small living animal imaging machine at regular intervals.In addition,tumor samples of patients with Bcell lymphoma were collected,and a PDX（patient-derived xenograft）model of Bcell lymphoma was constructed in NCG mice,which was grouped identical to CDX model,and the sizes of the tumors were observed and recorded regularly.（7）Ameliorating effect of AAV-CD19 BiTE on lymphoma microenvironment: NCG mice were subcutaneously injected with Raji lymphoma cells to construct a CDX model of B-cell lymphoma.Then AAV-CD19 BiTE(5×1012 gc/kg)and PBMNC were injected into the mice,and AAV-GFP was used as control group.After 3 weeks,the mice were sacrificed,and the contents of CD3+,CD4+,CD8+ T cells,and the activated CD8+ T cells in lymphoma were analyzed.（8）In vivo safety investigation of AAV-CD19 BiTE therapy: Balb/C mice were injected with an experimental dose of AAV-CD19 BiTE via the tail vein,and the same volume of PBS was used as the control group at the same time.After four weeks,peripheral blood of mice was collected for blood cell counts,biochemical tests,and various cytokines analysis,and the mouse organs were observed by HE staining to evaluate the safety of AAV-CD19 BiTE.[Results]（1）The purified AAV-CD19 BiTE titer was 1.31×1013 gc/m L,and the antibody competition experiment showed that the hepatocellular carcinoma cells transfected with AAV-CD19 BiTE could secrete CD19 BiTE,while the transfected 293 T cells could not secrete CD19 BiTE,indicating that the AAV-CD19 BiTE produced in this study was hepatocyte-targeted.（2）The results of in vitro experiments indicated that CD19 BiTE secreted by hepatocellular carcinoma cells could specifically kill CD19+ tumor cells,but had no killing ability against CD19-tumor cells.（3）The RT-qPCR results of various organs exhibited that CD19 BiTE could only be expressed in the liver rather than other organs,and the expression time of CD19 BiTE could be maintained for more than half a year.（4）The results of in vivo experiments confirmed that AAV-CD19 BiTE could effectively kill B-ALL cells and significantly reduce tumor burden.Meanwhile,the median survival time of mice in AAV-GFP and AAV-CD19 BiTE groups was 21 days and 27 days,respectively,and AAV-CD19 BiTE could significantly prolong the survival time of mice（P=0.001）.（5）AAV-CD19 BiTE showed significant efficacy in both CDX and PDX models of Bcell lymphoma,which could effectively inhibit the growth of tumor cells and prolong the survival time of mice（P<0.05）.In addition,the results of lymphoma microenvironment analysis indicated that the content of CD3+,CD4+,CD8+ T cells and the ratios of activated CD8+ T cells in the AAV-CD19 BiTE treatment group were significantly higher than those in the AAV-GFP control group（P<0.001）.（6）The in vivo safety analysis of AAV-CD19 BiTE indicated that there were no significant differences in blood cell counts,biochemical indicators of major organs,and various cytokine contents between AAV-CD19 BiTE and PBS groups（P>0.05）,suggesting AAV-CD19 BiTE had a good safety profile in mice.[Conclusion]The AAV-CD19 BiTE produced in this study was liver-targeted,and could achieve sustained expression of CD19 BiTE for more than half a year after a single intravenous injection.AAV-CD19 BiTE exhibited potent anti-tumor activity for the treatment of B-cell hematological malignancies in vitro and in vivo,and it could significantly improve the lymphoma microenvironment.This study provided strong evidences for the treatment of B-cell hematological malignancies by expressing CD19 BiTE in vivo and it could maintain the efficacy of CD19 BiTE while avoiding multiple repeated dosing.AAV-CD19 BiTE was expected to reduce the treatment costs and improve patient accessibility,and provided a new treatment strategy for B-cell hematological malignancies.Part Ⅱ: Construction of CAR-T biomimetic nanosystems and its preclinical study for the treatment of B-cell acute lymphoblastic leukemia[Objective]Chimeric antigen receptor T cells（CAR-T）are highly effective for the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia（B-ALL）,with complete remission（CR）rates of 68%–93%.However,in vitro CAR-T therapy has a long production cycle,which limits its usage for patients with rapidly progressive diseases;meanwhile,it is expensive,greatly reducing the accessibility of patients.In order to avoid the cumbersome production process of CAR-T cells in vitro and reduce the treatment cost,this study aimed to use optimized nanomaterials to encapsulate CD19 CAR mRNA,to realize the production of CAR-T cells in vivo and explore its efficacy in the treatment of B-ALL.[Methods]（1）Construction of CAR-T biomimetic nanosystems: Firstly,classical poly（β β-amino esters）（PβAE）nanomaterials were produced.Then,293 T cells and human Tlymphocytic leukemia Jurkat cells were transfected with PβAE encapsulating GFP mRNA to analyze transfection efficiency.（2）Optimization of CAR-T biomimetic nanosystems: the structure of PβAE was optimized to improve the transfection efficiency of suspension cells Jurkat,and the transfection efficiencies of Jurkat cells were analyzed when the optimized nanomaterial PβAE-447 encapsulating GFP mRNA with different mass ratios.Then,we compared the transfection efficiencies of PβAE-447 and Lipo2000.At the same time,the dynamic changes of GFP mRNA after entering the T cells were tracked.Further,the transfection efficiency of PβAE-447 encapsulating CAR mRNA on T cells and the cytotoxicity of PβAE-447 to T cells were analyzed to determine the optimal encapsulation ratio.Finally,the CD3 antibody was connected to the surface of PβAE-447 to increase the targeting ability of the nanosystems.（3）In vitro functional analyses of CAR-T biomimetic nanoparticles: CAR-T cells produced by nanoparticles were co-incubated with CD19+ NALM-6 and CD19-K562 cells in certain proportions,and the killing abilities of CAR-T cells were measured after 48 h.At the same time,enzyme-linked immunosorbent assay（ELISA）was used to detect the contents of IL-2,TNF-α and IFN-γ in the supernatant of the co-culture systems.（4）In vivo functional analyses of CAR-T biomimetic nanoparticles: the CDX model of B-ALL was constructed by infusion of NALM-6 leukemia cells via the tail vein of（NOD-Prkdcem26Cd52il2rgem26Cd22/Nju,NCG）mice.Then,the mice were infused with human PBMNC and nanoparticles encapsulating CAR mRNA,and the GFP mRNA was used as control group to observe the changes of tumor burdens in mice by small animal living imager.At the same time,the peripheral blood of mice was collected regularly to monitor the dynamical changes of CAR expressions.In addition,bone marrow samples from patients with B-ALL were collected,and a PDX model was constructed to further analyze the anti-tumor activity of CAR mRNA biomimetic nanoparticles in vivo.（5）In vivo safety analysis of CAR-T biomimetic nanoparticles: the Balb/c mice were injected with PβAE-447 encapsulating 15μg CAR mRNA for 5 consecutive times via tail veins,and the control group was injected with the same volume of PBS.One week after the last injection,the periorbital blood of mice was collected for blood cells counts and biochemical analyses,then,the contents of IL-1α,IL-2,IL-4,IL-5,TNF-α,IFN-γ and other cytokines in plasma was detected by Luminex,and the differences between the nanoparticles and PBS groups were analyzed.At the same time,the liver,heart,spleen,lung,kidney,brain and other organs of mice were collected,formalin fixed,paraffin embedded,and HE stained after section to observe whether there was any difference in organ morphology between the nanoparticles and PBS groups,analyzing the organ toxicity of the CAR mRNA biomimetic nanoparticles.[Results]（1）Encapsulated GFP mRNA with PβAE was a good transfection vehicle for 293 T cells,but it hardly transfected Jurkat cells.The optimized nanoparticles PβAE-447 could encapsulate GFP mRNA well,and the mass ratio of PβAE-447/mRNA was finally determined to be 80/1 by balancing transfection efficiency and cytotoxicity.（2）The size and potential of PβAE-447 encapsulating with GFP mRNA were 273.6 nm ± 4.43 and potential 24.2 m V ± 0.45,respectively.And there was no significant difference in transfection efficiency of Jurkat cells between PβAE-447 and Lipo2000,but the gene expression intensity of PβAE-447 transfection was significantly stronger than that of Lipo2000,indicating that PβAE-447 could better help mRNA achieve endosome escape.In addition,the sustained expression time of GFP mRNA after entering the cell was about 2 weeks.（3）The transfection efficiency of PβAE-447 encapsulating GFP mRNA on activated T cells was 10.05% ± 0.33,and the transfection efficiency was significantly improved by connecting CD3 antibody（P<0.01）,which was 20.27% ± 0.24.（4）PβAE-447 encapsulating CD19 CAR mRNA also could transfect non-activated T cells well,and in vitro functional experiments have shown that CD19 CAR-T cells produced by nanoparticles could specifically kill CD19+ tumor cells and release cytokines.In addition,there was no significant difference in the anti-tumor activity of CD19 CAR-T cells produced by nanoparticles and virus in vitro.（5）In vivo animal experiments confirmed that PβAE-447 connected to CD3 antibody could achieve in vivo T cell editing to produce CAR-T cells after encapsulating CD19 CAR mRNA,and in vivo transfection efficiency was 12.77% ± 2.50.The tumor burdens of mice infused with CAR mRNA nanoparticles were significantly lower than that of the control group,and the survival time was significantly prolonged（P<0.01）.In addition,the PDX model of B-ALL was further constructed to verify the potent anti-tumor activity of CAR-T in vivo.（6）In vivo safety analysis indicated that there were no significant differences between the CAR mRNA nanoparticles and PBS groups in terms of blood cell counts,heart,liver and kidney biochemical indicators and the contents of various cytokines,indicating that CAR mRNA nanosystems had a good safety profile in vivo.[Conclusion]This study constructed a biomimetic nanosystems to produce CD19 CAR-T in vivo,and good anti-tumor activity against B-ALL was observed in vitro and in vivo experiments.The production of CAR-T cells in vivo avoided the cumbersome preparation process of in vitro CAR-T and was expected to reduce the cost of CAR-T therapy,further improving the accessibility of patients.In addition,unlike the viral production of CAR-T cells,the biomimetic nanoparticles manufacture avoided the risk of potential tumorigenic risk caused by random integration of CAR,and increased the safety and controllability of CAR-T therapy.Part Ⅲ: The study of CD19 CAR-T-associated hematotoxicity and the establishment of early predictive model[Objective]Hematotoxicity is the most common long-term adverse event（AE）after CD19 CART therapy.However,clinical studies often underestimate hematological toxicity after CD19 CAR-T therapy due to the strict patient inclusion and exclusion criteria,especially for some rare but fatal hematological toxicities.Therefore,this study intends to comprehensively and systematically reveal the hematotoxicity after CD19 CAR-T therapy using the US Food and Drug Administration Adverse Event Reporting System（FAERS）,so as to help clinicians early alert those rare but fatal hematological adverse events,reducing the risk of severe hematotoxicity for patients.In addition,we also used the data of our research center to construct an early prediction model for hematological toxicity after CD19 CAR-T therapy,which is helpful for risk stratification of hematotoxicity and early intervention for high-risk patients.[Methods]（1）Firstly,the FAERS database was used to search and collect hematological adverse events after CD19 CAR-T therapy between January 2017 and December 2021,and classified them according to the international Medical Dictionary for Regulatory Activities（Med DRA）.（2）The reporting odds ratio（ROR）and information component（IC）in the disproportionality analysis were used to detect and analyze the signals of potential adverse events after CD19 CAR-T therapy.And the lower limits of the ROR and IC 95% confidence interval（CI）（ROR025 and IC025）exceeding one and zero were considered significant,respectively.（3）The whole hematological adverse events were systematically classified and analyzed based on the preferred term（PT）and high level term（HLT）,mainly focusing on the differences among the top 10 PTs and the 6 HLTs in different CART products,genders and ages.At the same time,the relationship between different PTs,HLTs and cytokine release syndrome（CRS）was explored.Finally,the proportion of deaths between different PTs and HLT was calculated,and the hematological adverse events closely related to death were identified by LASSO regression analysis.（4）Medical information was collected of 71 patients with B-cell hematological malignancies who received CD19 CAR-T therapy in our research center.Then,univariate and multivariate analyses were performed to identify the independent risk factors closely related to hematological toxicity after CD19 CAR-T therapy,further to establish an early prediction model for hematotoxicity.[Results]（1）Among the 105,087,611 reports in FAERS,we identified 5,112 reports of hematotoxicity associated with CD19 CAR-T therapy,of which 23 were statistically significant（ROR025>1）.（2）Among the 23 statistically significant hematological adverse events,hemophagocytic lymphohistiocytosis（HLH;n=136[2.7%],ROR025=21.06）,coagulopathy（n=128[2.5%],ROR025=10.43）,bone marrow failure（n=112[2.2%],ROR025=4.88）,disseminated intravascular coagulation（DIC;n=99[1.9%],ROR025=9.64）and B cell hypoplasia（n=98[1.9%],ROR025=118.16,all IC025>0）were less reported in clinical trials of CD19 CAR-T therapy.（3）Overall,the mortality rate related to hematotoxicity was 41.43%,and the mortality rates of HLH and DIC were 69.9% and 59.6%,respectively.Further,22 adverse events were identified to be closely related to death by LASSO regression analysis.（4）According to the data in our center,the incidence of hematotoxicity after CD19 CAR-T cell therapy in patients with B-ALL and large B-cell lymphoma was 45.5% and 38.5%,respectively.Multivariate analysis showed that the severity of CRS was an independent risk factor for hematotoxicity.（5）We further performed cytokines analyses and found that the peak levels of tumor necrosis factor-α and C-reactive protein within 1 week following CD19 CAR-T therapy were closely related to hematotoxicity.The accuracy of the early prediction model of hematotoxicity based on these two cytokines was 87.7% and 85.0% in the training and validation groups,respectively.[Conclusion]A variety of hematological adverse events related to CD19 CAR-T therapy have been underestimated in clinical trials and associated with high mortality.Meanwhile,these adverse events were closely related to CRS,among which HLH and DIC were largely underestimated but fatal adverse events.In addition,the constructed early prediction model of hematotoxicity after CD19 CAR-T therapy based on our research center data had high accuracy,which could help risk stratification and precise treatment,reducing the risk of severe hematotoxicity for patients. |
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