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Qing Li,1 Juan Mu,1 Jijun Yuan,2 Zhenxing Yang,2 Jia Wang,1 Qi Deng1 1Department of Hematology, Tianjin First Central Hospital, Tianjin 300192, People’s Republic of China; 2Medical Department, Shanghai Genbase Biotechnology Co., Ltd, Shanghai 201203, People’s Republic of ChinaCorrespondence: Qi Deng Email kachydeng@126.comPurpose: To investigate the donor chimerism changes and curative effects associated with the use of autologous anti-CD19 chimeric antigen receptor (CAR) T cells with B-cell acute lymphoblastic leukemia (B-ALL) presenting with a low donor chimerism level and relapse after allogeneic hematopoietic stem cell transplant (allo-HSCT).Methods: Nine patients with B-ALL showing low donor chimerism level and relapse after allo-HSCT were enrolled. Patients 1– 3 received CD19 CAR-T cell therapy using cells derived from autologous peripheral blood mononuclear cells (PBMCs) (comprising a mixture of patient and original donor cells) as their donors could not provide PBMCs. Samples from the other six patients (Patients A–F) were investigated only in vitro. The changes in the degree of donor chimerism, function of the CD19 CAR-T cells and T cells in all nine patients were analyzed in vitro. The therapeutic effects and adverse events (AEs) were also evaluated in Patients 1– 3.Results: The CAR-T cells and T cells in all nine patients showed complete donor chimerism restoration following a 12-day culture period in vitro. These CD19 CAR-T cells demonstrated strong cytotoxicity towards Nalm 6 cells in vitro except in patients 3 and D. In the latter patients, the absolute numbers of all subsets, especially the CD8 + T-cell absolute numbers in peripheral blood were very low. Patients 3 and D showed relatively short durations from transplant to recurrence and received chemotherapy after relapse. In the patients receiving CD19 CAR-T cell therapy, the most commonly observed AE was grade 1 to 2 cytokine release syndrome. None of the cases showed acute graft-versus-host disease during treatment. Patients 1 and 2 achieved complete response with complete restoration of donor chimerism. Patient 3, who received the same CD19 CAR-T cell therapy, did not respond to this therapy.Conclusion: CD19 CAR-T cells derived from patients relapsed after allo-HSCT with a low level of donor chimerism were effective for salvage therapy and could restore to complete donor chimerism after 12 days’ culture in vitro.Trial Registration: Humanized CD19 CAR-T cell therapy for relapse or refractory B-cell lymphoma or acute B lymphocytic leukemia, ChiCTR1800019622, Registered 24 November 2018, http://www.chictr.org.cn/index.aspx.Keywords: chimeric antigen receptor, acute lymphoblastic leukemia, allogeneic hematopoietic stem cell transplant, relapse, donor chimerism

Purpose: To study the efficacy and side effects of humanized anti-CD19-CAR T-cell combined with multiple combination treatments in relapsed/refractory B-cell non-Hodgkin lymphoma. Methods: Thirty-eight relapsed/refractory B-cell non-Hodgkin lymphoma patients were enrolled in a clinical trial of anti-CD19 chimeric antigen receptor (CAR) modified T-cell expressing humanized anti-CD19 scFv and 4-1BB-CD3ζ costimulatory-activation domains therapy (ChiCTR1800018059) at the Department of Hematology in Tianjin First Center Hospital (Tianjin, China). Except for anti-CD19-CAR T-cell therapy, all the patients received multiple combination treatments before or at the same time of CAR-T cell therapy. The multiple combination treatments included anti-CD22-CAR T-cell, Programmed death 1 (PD-1) inhibitor, Bruton's tyrosine kinase (BTK) inhibitors, Lenalidomide, Advance high-dose chemotherapy and Radiotherapy. Patients were evaluated 2 months after the infusion. The side effects were observed in all the process of the combination treatments. Cytokine release syndrome (CRS) was graded according to the ASBMT Consensus Criteria. Results: The median time from infusion to cutoff day was 7 months (Range: 1 to 16 months). There were 37 patients included in the evaluation of the efficacy and side effects (Except for one patient with high tumor burden died of CRS 7 days after the infusion). The best overall response rate (ORR) was 75.6% (28/37). The complete response (CR) rate was 45.9% (17/37), the partail response (PR) rate was 29.7% (11/37), the stable disease (SD) rate was16.2% (6/37) and the disease progression (PD) rate was 8.1% (3/37). The grade of cytokine release syndrome (CRS) was Grade 1: 25/38, Grade 2: 9/38, Grade 3: 3/38, Grade 4: 0/38 and Grade 5: 1/38. Two patients died from disease progression within 60 days after infusion. One patient with high tumor burden who achieved PR at 14 days died from intestinal perforation after 25 days of infusion. The combination treatments of anti-CD22-CAR T-cell (4/38), treatment dose or 1/2 treatment dose of PD-1 inhibitor (5/38) or BTK inhibitors (3/38) therapy improved the curative effect of humanized anti-CD19-CAR T-cell therapy. Advance high-dose chemotherapy for patients (9/38) with high tumor burden before the anti-CD19-CAR T-cell therapy achived a satisfactory ORR (5/9) and Grade 1-2 CRS. No patients died from the advance and intensive chemotherapy. There were 6 patients with refractory lymphoma received radiotherapy before they enrolled in the anti-CD19-CAR T-cell therapy. There were 5 patients achived CR or PR except for one patient with high tumor burden died of CRS 7 days after the infusion (the IL-6 level was more than 1000 U/mL at day 3). But the side effects were significant in these 6 patients (Grade 1: 0/6, Grade 2: 2/6, Grade 3: 3/6, Grade 4: 0/6, Grade 5: 1/6). There was no efficacy improvement from the combination treatments of lenalidomide (14/38, 3 patients received advance high-dose chemotherapy meanwhile among the 14 patients), and the side effects were not significant. Conclusions: The multiple combination treatments combined with humanized anti-CD19-CAR T-cell therapy could improve the efficacy without increase side effects obviously. Disclosures No relevant conflicts of interest to declare.

Purpose: To study the efficacy of anti-CD19-CAR T-cell therapy and the changes of donor chimerism in CAR-T cells in vitro culture for patients who relapsed after allo-HSCT with a low level of donor chimerism. Methods: Two patients relapsed after allo-HSCT with a low level of donor chimerism (11.22% and 16.08%). Their autologous PBMCs were selected for anti-CD19-CAR T-cell therapy because their sibling or unrelated donor could not provide PBMC cells. The adverse events (AEs) were observed after CAR T-cell infusion. The cytokine release syndrome (CRS), the expansion of anti-CD19-CAR T-cells, the DNA level of anti-CD19-CAR gene and the changes of the donor chimerism were detected after CAR T-cell infusion. Then we selected other 7 acute leukemia patients relapsed after allo-HSCT with a low level of donor chimerism to detect the changes of donor chimerism in their T cells and anti-CD19-CAR T-cells in vitro culture. Results: Their notable AEs were grade 2 CRS. They had no aGVHD during anti-CD19-CAR T-cell therapy. They achieved CR 14 days after anti-CD19-CAR T-cell therapy with a restoration of complete donor chimerism (98.61% and 98.48%). To date, they had leukemia-free survival (LFS) and complete donor chimerism for 8 and 7 months. It was very interesting and fortunate that the CAR-T cells infused into the two patients were restorated to complete donor chimerism (99.2% and 99.4%). Then we found that complete donor chimerism were restored after 12 days culture in vitro in all the T cells and anti-CD19-CAR T-cells in the other 7 patients relapsed after allo-HSCT with a low level of donor chimerism. Conclusions: Anti-CD19-CAR T-cells derived from patients relapsed after allo-HSCT with a low level of donor chimerism were effective for this salvage therapy and could restorate to complete donor chimerism after 12 days culture in vitro. Disclosures No relevant conflicts of interest to declare.

Purpose: To investigate the immunophenotypic characteristics of CD19 CAR transfected into leukemia cells with CD19 expression during CAR-T cell preparation. Patient and Methods: Here we reported a TP53 positive refractory B-ALL patient relapsed 14 days after Complete response (CR) from the CD19 CAR-T therapy. But her leukemia cells expressed CD19 and expressed aberrantly the anti-CD19 CAR gene at the same time. We transfected CD19 CAR gene into Nalm-6 cells to detect the expression of CD19 CAR Nalm-6 cells and the sensitivity to anti-CD19 CAR T-cell and anti-CD22 CAR T-cell. The binding of CD19 antigen on the surface of CD19 CAR Nalm-6 cells and Nalm-6 cells to CD19 CAR in CD19 CAR Nalm-6 cells was observed by confocal microscopy. The proportion of Nalm-6 cells in the transfection system was increased gradually to observe the change of CD19 expression on CD19 CAR Nalm-6 cells. Results: The patient received a CD22 CAR-T therapy followed by a haploid hematopoietic stem cell transplantation and achieved CR with vanishment of TP53 mutation. To date, she had leukemia-free survival (LFS) and complete donor chimerism for 8 months. We found that there had 1.41% leukemic cells in the peripheral blood mononuclear cells (PBMC) during anti-CD19 CAR T-cell preparation, which may contribute to the relapse of the first CD19 CAR-T therapy. The CD19 expression was only 1.19% in CD19 CAR Nalm-6 cell culture system with a transfection rate of 46.5±3.78%, while the proportion of CD19 expression increased gradually when we increased the proportion of Nalm-6 cells in the transfection system. CD19 CAR of CD19 CAR Nalm-6 cells occupied the CD19 expression site of the cells themselves, meanwhile occupied the CD19 expression site of Nalm-6 cells. We found that the CD19 CAR Nalm-6 cells were sensitive to anti-CD22 CAR T-cell, althought they were resistant to anti-CD19 CAR T-cell. Conclusions: There had a presence that CD19 CAR gene was transferred into leukemia cells during T cell manufacturing. The immunophenotype of leukemia cells of these refractory B-ALL patients who relapsed after CR from anti-CD19 CAR T-cell therapy were CD19+ or CD19-. It was determined by the proportion of CD19 CAR leukemia cells and leukemia cells. Anti-CD22 CAR T-cell therapy could be as a salvage measure to such patients. Figure Disclosures No relevant conflicts of interest to declare.

The autotrophic process for nitrogen removal has attracted worldwide attention in the field of wastewater treatment, and the performance of this process is greatly influenced by the size of granular sludge particles present in the system. In this work, the granular sludge was divided into three groups, i.e. large size (> 1.2 mm), medium size (0.6–1.2 mm) and small size (