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Background: The prognosis of refractory/relapsed aggressive B-cell non-Hodgkin lymphoma (r/r B-NHL) and multiple myeloma (r/r MM) is extremely poor, especially for the patients who failed to CAR-T cells therapy and/or ASCT. Aims: Forr/r B-NHLand r/r MM, a clinical trial using Allo-HSCT with conditioning including donor humanized CAR-T cells from the same donor (allo-CAR-T) has been registered, and the safety and efficacy will be evaluated. Methods: From September 2020 to May 2021, 11 patients were enrolled.The median age was 41 (26-64) years old. The diagnosis included high grade B-cell lymphoma (n=9) and Multiple myeloma (n=2). Seven cases were with TP53 mutations.All patients was progressive disease (PD) who failed to multi-line therapies, including chemotherapy (n=11), ASCT (n=4), autologous CAR-T (n=11).In order to further reduce the tumor burden, all patients were treated with combination therapy before transplantation. Before the trial, the expression of CD19 and/or CD22 or CD20 antigen in tumor tissue of r/r B-NHL and BCMA antigen in r/r MM patients was positive confirmed by immunohistochemistry.There were matched sibling identical donor in 1 case,matched unrelated donor in 1 case and haploidentical donor in 9 cases；Conditioning with busulfan, fludarabine-based regimen combined with allo-CAR-T was applied. Tacrolimus, mycophenolate mofetil, a short-term methotrexate and antithymocyte globulin were used for GVHD prophylaxis. The kinetics and function of CAR-T cells was monitored by quantitative PCR and flow cytometry. The efficacy was evaluated by PET-CT in r/r NHL as well as bone marrow puncture and immunofixation electrophoresis in r/r MM every 2 month after CAR-T infusion. Results: The median allo-CAR-T cells infused were 4 (range,0.78-4.88)×10 6/kg. CRS occurred in all cases with 6 cases in grade I, 1 case in grade II and 4 cases in grade III.The peak of cytokine IFN-γ and IL-6 in grade III CRS were significantly higher than those with grade I-II.No ICANS was noted. Four cases with grade III CRS were relieved with methylprednisolone. G-CSF-mobilized PBSC were infused 7 days after allo-CAR-T with the median CD34 + cells 6 (range,3-8.19)×10 6/kg. The neutrophil and platelets engraftment was achieved in all cases on median days 13 (range,11-24) and 16 (range,14-85) respectively post-transplant .All cases were donor type by STR analysis.Three cases of grade II acute GVHD were seen. CMV viremia occurred in 7 cases.For allo-CAR-T cell expansion,the peak time in vivo was on median 14(range,7-28) days after infusion.The median peak lever was 221 (range,0.191-1502)×10 6/L, which positively correlated with the number of allo-CAR-T infused. The tumor burden before transplantation was not significantly associated with allo-CAR-T expansion.Levels of allo-CAR-T cells were very low after the first 2 months of HSCT which detected persistently in 9/11(81.8%) patients, and the longest lasting time was 239 days post-transplant so far. B-cell aplasia was documented in 8/9 cases of r/r B-NHL during the follow-up. With the median follow-up 171 (range,100-295) days, 7/11(63.6%) patients survived,five cases(5/11,45.5%) achieved CR,one cases(1/11,9.1%) obtained PR, and 1 case(1/11,9.1%) of MM achieved SD and survival with tumor .Three cases(3/11,27.3%) with DLBCL died of PD whose disease status before transplantation were SD or PD, one patient(1/11,9.1%) died of infection.Significantly lower levels of Cumulative CAR T cell levels (AUC) during the first 2 month post transplantation were observed in patients who relapsed compared with those who had durable responses (P=0.0001).aGVHD were not associated directly with in vivo CAR T-cell expansion(P=0.193). Conclusion: Our preliminary results have shown that CRS is manageable and has no influence on hematopoiesis reconstitution. Allo-CAR-T cells still exist persistently post-transplant in majority of patients, which may contribute a long-term anti-lymphoma effect.With current protocol, aGVHD and viral reactivation was mild. Allo-HSCT with conditioning including allo-CAR-T cells is a safe and effective strategy for r/r B-NHL and MM. The Poor clinical efficacy was associated with high tumor burden before transplantation. [Key words] refractory/relapsed B-cell non-Hodgkin lymphoma; refractory/relapsed multiple myeloma;allogeneic CAR-T cell; allogeneic hematopoietic stem cell transplantation Disclosures No relevant conflicts of interest to declare.

Background: The prognosis of refractory/relapsed aggressive B-cell non-Hodgkin lymphoma (r/r B-NHL) is extremely poor especially for the patients who failed to CD19-Specific chimeric antigen receptor-T (CAR-T) cells therapy.Even sequentially autologous hematopoietic stem cell transplantation(ASCT) could not maintain a durable remission in most patients. Aims: To prolong relapse-free survival, we combined ASCT and another target humanized CAR-T cells to treat r/r B-NHL patients failed to murinized CD19-CAR-T cells therapy with either CD22 or CD20 antigen expression on tumor cell.The safety and efficacy will be evaluated. Methods: From December 2019 to March 2021, 12 patients were enrolled. The median age was 38 (16-68) years old. The diagnosis included DLBCL (n=8) ,BL(n=3) and PMBCL (n=1). The median IPI score was 3 (range,2-4).There were 9 patients(9/12,75%) with extranodal lesions. Six cases(6/12,50%) were with TP53 mutations. The disease status was progressive disease in all patients who failed to multi-line therapies and murinized CD19-CAR-T cells therapy.In order to further reduce the tumor burden, all patients were treated with combined chemotherapy before transplantation. Before the trial, the expression of CD20 and CD22 antigen in tumor tissue was positive confirmed by immunohistochemistry,and the target was selected according to the antigen expression. Conditioning with BEAM-based regimen was applied. The kinetics and function of CAR-T cells was monitored by quantitative PCR and flow cytometry. The efficacy was evaluated by PET-CT every 3 month after transplantation. Results: The autologous peripheral blood stem cells were infused with the median CD34 + cells 3.91(range,0.46-9.46)×10 6/kg.Humanized CAR-T cells with the median 1.85 (range,0.13-3.26)×10 6/kg were infused 2 day after stem cells,including target antigen CD20(7/12,58.3%) and CD22(5/12,41.7%). Cytokine release syndrome (CRS) occurred in 11 cases with 5 cases in grade I, 3 case in grade II and 3 cases in grade III.One case developed immune effector cell-associated neurotoxicity syndrome (ICANS) in grade IV. The peak of cytokine IFN-γ and IL-6 post baseline in patients with grade III CRS were significantly higher than those in patients with grade I-II CRS,especially in ICANS patient.Six cases with grade II and III were relieved with glucocorticoid. The neutrophil and platelets engraftment was achieved in all cases on median days 14 (range,9-22) and 14(range,8-35) respectively post-transplant .Seven cases of bacterial enteritis were seen. Pneumonia occurred in 7 cases.For CAR-T cells expansion,the peak time in vivo was on median 11(range,7-28) days after CAR-T cells infusion.The median peak lever of CAR-T cells was 20.3 (range,0.13-60.4)×10 6/L, which was positively correlated with the number of CART infused. The tumor burden before transplantation was not significantly associated with CAR-T cells expansion.The median duration of CAR-T cells in vivo was 30 days, and the longest lasting time was 139 days post-transplant so far. B-cell aplasia was documented in all cases(7/7,100%) of CD20-CART group and two cases(2/5,40%) of CD22-CART group during the follow-up. With the median follow-up 266 (range,118-565) days, 9/12(75%) patients survived,seven cases(7/12,58.3%) achieved complete remission(CR),2 cases(2/12,16.7%) achieved PD and survival with tumor.Kaplan-Meier survival analysis showed that OS and PFS rates were 71.3% and 66.6% respectively at 9 months after transplantation.Two cases(2/12,16.7%) with BL and one with DLBCL (1/12,8.3%)died of PD.Significantly lower levels of Cumulative CAR T cell levels (AUC) during the first 1 month post transplantation were observed in patients with disease progression compared with those who had durable responses (P<0.0001). Conclusion: CRS is manageable and has no influence on hematopoiesis reconstitution.With current protocol, complication was mild and encouraging disease control was found. ASCT combined with another target humanized CAR-T therapy is a safe and effective salvage strategy for r/r B-NHL after failure of murinized CD19-CAR-T. Long-term follow-up is needed. [Key words] refractory/relapsed B-cell non-Hodgkin lymphoma; failure of CD19-CAR-T; another target CAR-T cell; autologous hematopoietic stem cell transplantation Disclosures No relevant conflicts of interest to declare.

Introduction: Chimeric antigen receptor T-cell (CART) Therapy has induced high complete remission (CR) in the patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL), but the median event-free survival is only 6.1 months (Park JH. et al. NEJM 2018). To achieve durable leukemia-free survival (LFS), it may helpful to receive allogeneic hematopoietic stem cell transplantation (allo-HSCT) after CR with CART therapy. Objective: In current study, safety and long-term outcomes of allo-HSCT after CART therapy in r/r B-ALL were assessed and risk factors for overall survival (OS) and LFS were analyzed. Methods: Between June 2017 to June 2020, 137 patients with r/r B-ALL who achieved CR with CD19 or CD22 CART then bridge to allo-HSCT in our hospital were evaluated. The median age was 9.7 years (1.8-56). Bone marrow (BM) samples were collected to detect somatic gene mutations of leukemic cells. Blood samples from patients, parents, and potential related donors were collected to analyze germline mutations of hematological and immunological hereditary predisposition genes. The disease status before transplant was minimal residual disease (MRD) negative in 114 (83.2%) patients and MRD positive in 23 (16.8%) patients. The median time from CART infusion to allo-HSCT was 51 days (29-94). The types of transplant were haploidentical (105, 76.6%), unrelated (26, 19.0%), and identical siblings (6, 4.4%). Myeloablative reduced-toxicity conditioning (RTC) with either total body irradiation (TBI)/fludarabine-based (84, 61.3%) or busulfan (BU) /fludarabine-based (53, 38.7%) were applied. Cyclosporine, mycophenolate mofetil and short-term methotrexate were employed for graft-versus-host disease (GVHD) prophylaxis. Results: Full donor engraftment was achieved in all patients. The median time for neutrophil and platelet engraftment was 15 days (9-21) and 13 days (5-33). With a median follow-up of 653 days (481-783), two-year OS was 74.1% (64.4-81.5) and two-year LFS was 64.4% (54.6-72.6). The incidences of grade Ⅱ to Ⅳ and severe acute GVHD (aGVHD) were 27%, 14.5%, respectively. Twenty-five (18.2%) cases developed chronic GVHD (cGVHD) (limited 18, extensive 7). The incidences of CMV and EBV viremia were 20.1%, 12.5%. Hemorrhagic cystitis was found in 32 (22.2%) cases. Thirty-six (26.3%) cases relapsed after transplant, 28/114 (24.6%) in MRD negative cohort and 8/23 (43.5%) in MRD positive cohort. Thirty (21.9%) patients died (relapsed 22, infection 5, multiple organ failure 2, thrombotic microangiopathy 1). Transplant-related mortality was only 5.8%. Total 137 patients with 99 kinds of somatic mutations, the most frequent 15 mutations were TP53, CYP2C19, KRAS, NRAS, PTPN11, CREBBP, KMT2D, KIT, NR3C1, STK11, FLT3, NF1, NUDT15, ABL1, ADAMTS13. LFS was significantly reduced in patients with TP53, KIT, NF1 mutation (P < 0.0001; P < 0.025; P < 0.003). Total 114 evaluable patients with 93 kinds of hematological and immunological hereditary predisposition gene mutations, the most frequent 15 mutations were BLTA, SERPINE1, CYP2C19, F7, TP53, DPYD, MLH1, TNFAIP3, YARS2, HIFIA, ATM, EP300, KIT, ADAMTS13, BRCA2. LFS was significantly reduced in patients with germline EP300 mutation (P < 0.00027). Univariate analysis showed that BU-based regimen (P=0.0194), somatic TP53 mutation (P= 0.002), fungal infection (P= 0.0217), viral infection (P=0.0187) and MRD positive before transplant (P= 0.0227) were adverse factors of OS. The negative factors of LFS were BU-based regimen (P=0.0139), germline EP300 mutation (P<0.001) and fungal infection (P =0.0002). Multivariate analysis showed that TBI-based regimen (P=0.019) and aGVHD (P=0.047) increased LFS remarkably, but fungal infection (P=0.002), somatic TP53 mutation (P<0.001) and germline EP300 mutation (P<0.001) decreased LFS significantly. Conclusions: Our large series and long-term outcomes have demonstrated that LFS and OS have been improved remarkably in r/r B-ALL who achieved CR with CART then quick bridge to RTC allo-HSCT. Our study has shown the somatic and germline mutation profiles in r/r/ B-ALL and found that somatic TP53 mutation and germline EP300 mutation are negative factors for LFS. TBI-based regimen , MRD negative before transplant and aGVHD have positive impact on LFS, and fungal infection, somatic TP53 and germline EP300 have negative impact on LFS. Disclosures No relevant conflicts of interest to declare.

Background: In our phase I clinical trial, 90% complete remission (CR) in r/r T-ALL/LBL has been achieved with donor-derived CD7-chimeric antigen receptor T-cell (CART) therapy (Pan J. et al. JCO July 29, 2021 online), but hematopoiesis and immune reconstitution is crucial for long-term disease-free survival (DFS) in this setting. Aims: In present study, the safety and efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) after donor-derived CD7-CART therapy for r/r T-ALL/LBL were examined. Methods: Between October 2020 and June 2021, 12 patients with r/r T-ALL/LBL (refractory 2, relapsed 10; T-ALL 10, T-LBL 2) who obtained CR or partial remission (PR) by donor-derived CD7-CART therapy then bridge to allo-HSCT from the same donor were enrolled. The median age was 14 (2-43) years old. The median blasts in bone marrow (BM) were 30 (12-71.5) % before CD7-CART. Eight (66.7%) patients had extramedullary disease (EMD). Two patient relapsed after transplants (allo-HSCT 1, auto-HSCT 1). One case was with MLL-AF6 fusion gene, and 8 cases had somatic gene mutations including NOTCH1 in 8, IL7R in 3, JAK in 2, FBXW7 in 2, WT1 in 2, and NAS in 2. Donor chimerism in BM and peripheral blood (PB) CD3 cells at 4 weeks after CART were 38 (0.14-99.4) %, 72.8 (0.25-100) %, respectively. Before allo-HSCT, minimal residual disease (MRD) in cerebrospinal fluid was all negative, MRD in BM was negative in 10 cases and positive in 2 cases, and EMDs were detectable in 3 patients but with much smaller sizes than before CART. Pancytopenia and transfusion-dependent were seen in 10 cases before allo-HSCT. The median time from CART infusion to allo-HSCT was 34 (30-55) days. The types of HSCT were haploidentical in 8, identical sibling in 3 and unrelated in 1. Conditioning regimens were busulfan/fludarabine-based (n=9) or TBI/fludarabine-based (n=3). ATG was used in haploidentical or unrelated transplants. Cyclosporine, mycophenolate mofetil and short-term methotrexate were employed for GVHD prophylaxis. CART cells were monitored by flow cytometry and PCR. Results: The median time of neutrophil and platelet engraftment was 15 (12-21) days and 38 (6-100) days. With a median follow-up of 234 (14-353) days, the 6-month overall survival (OS) and DFS were 91.67%, 83.33%, respectively. Transplant-related mortality (TRM) was 8.33%. One patient died of multi-organ failure at 14 days post-transplant. All patients achieved full donor chimerism in BM and PB CD3 cells. EMDs were not detected in all patients by the last follow-up.Ten patients achieved MRD- CR at 1 month after transplant. One case was CR with MLL-AF6 positive at 1 month and his fusion gene became negative at 2 months post-transplant after developed aGVHD by immunosuppressant withdrawal. MRD became positive in 2 patients at 3 months, 7.5 months after transplant. The leukemia cells in 1 case was CD7- and MRD turned to negative after cessation of immunosuppressants. The leukemia cells in another case was CD7+ and obtained MRD- by CD7-CART re-infusion. One patient relapsed with 57% blasts in BM at 4.5 months post-transplant and achieved MRD- CR by CD7-CART re-infusion. Three patients developed aGVHD (grade Ⅱ 2, grade Ⅳ 1) and all resolved. Three cases had limited chronic GVHD (cGVHD). Ten patients had intestinal infections (bacteria 9, fungal 1) and 5 cases developed pulmonary infection (fungal 4, bacteria 1). Eight patients had CMV reactivation (CMV viremia 7, CMV retinitis 1). One case had post-transplant lymphoproliferative disease and resolved with rituximab and chemotherapy. Seven cases had hemorrhagic cystitis. After HSCT, CD7-CART cells were detectable in PB in 5 patients at a median time of 1 (0.5-2) month, with the median level of CART cells was 0.069 (0.037-3.61) %. During existence of CD7-CART cells, CD7-CD4+ and CD7-CD8+ cells were emerged which had been demonstrated to be functional for anti-infections (unpublished data). Conclusions: With our protocol, hematopoiesis reconstitution has been achieved in all patients. Donor-derived CD7-CART combined with allo-HSCT has shown powerful anti-T-ALL/T-LBL effects and good safety profile. The existence of CD7-CART early after transplant had no influence on engraftment and may contribute to prolonged anti-tumor effect. Infections are main complication in this setting, therefore, appropriate dosages of immunosuppressants and the timing bridge to allo-HSCT need to be further investigated. Disclosures No relevant conflicts of interest to declare.