Extramedullary Diseases Research Articles

Efficacy and safety of CAR-T therapy targeting CLL1 in patients with extramedullary diseases of acute myeloid leukemia

BackgroundsThe incidence of extramedullary diseases (EMDs) in patients diagnosed with acute myeloid leukemia (AML) is approximately 10–20%. These patients exhibit a significantly distinct etiology, therapeutic response, and prognosis compared to patients without EMDs. CLL1 CAR-T therapy has been demonstrated satisfactory efficacy and safety in the treatment of refractory and relapsed AML patients. However, concerns have been raised regarding the potential impact of extramedullary niduses on the effectiveness of CLL1 CAR-T therapy.MethodsA total of 47 patients were enrolled in this study, including 27 patients with isolated AML tumor bone marrow infiltration and 20 patients with both extramedullary and bone marrow infiltration of AML. CLL1 CAR-T cells were manufactured and subjected to rigorous quality control in the hematology laboratory of Tianjin First Central Hospital. The efficacy and adverse reactions were assessed following CAR-T cell infusion, while expansion of CAR-T cells, levels of cytokines releasing, and other indicators were closely monitored.ResultsAmong the 20 patients with EMDs and the 27 individuals without EMDs, complete remission in bone marrow was achieved by 65.00% and 81.48% of patients, respectively. Meanwhile, among the patients with EMDs, 55.00% achieved complete remission while 10.00% achieved partial remission when assessing the efficacy of CLL1 CAR-T cells against extramedullary niduses. Although the overall survival, progression-free survival, and duration of remission period appeared to be shorter for patients with EMDs compared to those without EMDs, this difference did not reach statistical significance. The incidence rates of complications were comparable between both groups. Meanwhile, there were no significant differences observed in the levels of CAR-T cell expansion and accompanying cytokines release between patients with and without EMDs.ConclusionsOur study findings have demonstrated the efficacy of CLL1 CAR-T therapy in the treatment of AML patients with EMDs, while also indicating manageable occurrence rates of complications within a tolerable range. The CLL1 CAR-T therapy, serving as an ideal strategy for AML patients irrespective of the presence of EMDs, effectively ameliorates the conditions of AML patients and provides them with an opportunity to undergo curative hematopoietic stem cell transplantation while significantly enhancing their prognosis.

Myeloid sarcoma, spectrum of clinical characteristics, prognostic impact, and treatment outcome

BackgroundAcute leukemia may present with extramedullary (EM) tissues. Myeloid sarcoma (MS) and leukemia cutis (LC) are considered extramedullary diseases. This study aims to evaluate the incidence, clinical characteristics, and prognostic factors affecting the outcome of pediatric patients with myeloid sarcoma at the pediatric oncology center from July 2007 to December 2017. Radiological imaging was used to stage the tumor, biopsy was done for pathological diagnosis, and bone marrow aspirate for morphology, flow cytometry, cytogenetics, and molecular analysis. Patients received chemotherapy protocols based on those used by the Children's Cancer Group for acute myeloid leukemia (AML).ResultThe study included 91 (13.2%) of 687 pediatric patients with acute myeloid leukemia, with a male-to-female ratio of 1.9 to 1. Prognostic factors that improved the patient’s 5-year overall survival (OS) were age > 5 years old, molecular and cytogenetic detection of t (8; 21), inv 16, presence of a single and small size lesion < 5 cm, negative CNS lesion, and achieved radiological response in isolated disease or radiological and marrow complete remission in disseminated disease post induction 1 with significant P value. Relapse, particularly early relapse, worsens the OS and EFS by 10% and 7.7%, respectively.ConclusionPatients over five with low-risk disease based on cytogenetics, a small, single, negative CNS lesion, and a complete response post induction 1 had better outcomes, with no significant difference between those with isolated extramedullary disease and those with marrow dissemination. Early relapse harms the outcome. The study group’s 5-year OS and EFS rates are 51.4% and 49.4%, respectively.

Different extramedullary disease shown in chemokine receptor 4 targeted PET/CT with [ 68 Ga]Ga-pentixafor in patients with Waldenström macroglobulinemia and smoldering disease.

It is important to distinguish Waldenström macroglobulinemia from smoldering Waldenström macroglobulinemia (sWM), because only patients with Waldenström macroglobulinemia require treatment, however the distinction can be clinically complex. The aim of this study is to investigate whether [ 68 Ga]Ga-pentixafor PET/CT shows different characteristics in sWM and Waldenström macroglobulinemia patients and therefore can help to differentiate Waldenström macroglobulinemia and sWM. Thirty-seven patients with newly diagnosed Waldenström macroglobulinemia and 11 sWM patients were analyzed [35 men and 13 women; 64.3 ± 10.7 (range, 29-87) years old]. The SUV max of bone marrow disease, lymph nodes, and other extramedullary diseases on [ 68 Ga]Ga-pentixafor were significantly higher than those on 2-[ 18 F]FDG PET/CT ( P < 0.05). On [ 68 Ga]Ga-pentixafor PET/CT, patients with Waldenström macroglobulinemia had more lymph node regions involved, significantly higher incidence of involvement in more than three lymph node regions, larger nodal disease, and higher incidence of other extramedullary disease when compared with sWM patients ( P < 0.05). Waldenström macroglobulinemia patients showed significantly higher total lesions uptake, total lesion volume, and SUV max of extramedullary disease than sWM patients did ( P < 0.05). None of the visual or semiquantitative indexes in 2-[ 18 F]FDG PET/CT showed significant difference between Waldenström macroglobulinemia and sWM patients. [ 68 Ga]Ga-pentixafor PET/CT had better diagnostic performance than 2-[ 18 F]FDG PET/CT in Waldenström macroglobulinemia. Patients with Waldenström macroglobulinemia presented with more extensive extramedullary disease shown in [ 68 Ga]Ga-pentixafor PET/CT than sWM patients did.

Predictive model for CAR-T cell therapy success in patients with relapsed/refractory B-cell acute lymphoblastic leukaemia.

Chimeric antigen receptor T-cell (CAR-T) therapy has demonstrated remarkable efficacy in treating relapsed/refractory acute B-cell lymphoblastic leukaemia (R/R B-ALL). However, a subset of patients does not benefit from CAR-T therapy. Our study aims to identify predictive indicators and establish a model to evaluate the feasibility of CAR-T therapy. Fifty-five R/R B-ALL patients and 22 healthy donors were enrolled. Peripheral blood lymphocyte subsets were analysed using flow cytometry. Sensitivity, specificity, accuracy, positive and negative predictive values and receiver operating characteristic (ROC) areas under the curve (AUC) were determined to evaluate the predictive values of the indicators. We identified B lymphocyte, regulatory T cell (Treg) and peripheral blood minimal residual leukaemia cells (B-MRD) as indicators for predicting the success of CAR-T cell preparation with AUC 0.936, 0.857 and 0.914. Furthermore, a model based on CD3+ T count, CD4+ T/CD8+ T ratio, Treg and extramedullary diseases (EMD) was used to predict the response to CAR-T therapy with AUC of 0.938. Notably, a model based on CD4+ T/CD8+ T ratio, B, Treg and EMD were used in predicting the success of CAR-T therapy with AUC 0.966 [0.908-1.000], with specificity (92.59%) and sensitivity (91.67%). In the validated group, the predictive model predicted the success of CAR-T therapy with specificity (90.91%) and sensitivity (100%). We have identified several predictive indicators for CAR-T cell therapy success and a model has demonstrated robust predictive capacity for the success of CAR-T therapy. These results show great potential for guiding informed clinical decisions in the field of CAR-T cell therapy.

Ixazomib, Pomalidomide and Dexamethasone (IxaPD) Pan-Oral Regimen in the Treatment of Refractory and Relapsed Multiple Myeloma

Aim To analyze the efficacy and safety of Ixazomib, pomalidomide and dexamethasone (IxaPD) regimen in patients with refractory and relapsed multiple myeloma（RRMM）. Methods Clinical data of RRMM patients were collected from 5 centers in north China from Jan 2021 to Aug 2023, who were treated by IxaPD regimen for at least two courses. Response, time to next treatment and overall survival were analyzed as end points. Results A total of 34 patients were enrolled, including 21 in multicenter real-world study and 13 in investigator initiated IxaPD trial (NCT04989140) who were naïve to ixazomib or pomalidomide with previous treatment of 1 to 3 lines. There were 18 males and 16 females with the median age 65.5 (range 41-81) years old. The median duration from diagnosis to enrollment was 31 months (range 1-117), including 23/34 (67.3%) of patients were in first relapse. Thirteen in 24 (54.2%) patients had high-risk cytogenetic abnormalities. Regarding previous history, 20.6% presented renal insufficiency, 3 cases (8.8%) had extramedullary diseases, and 5 cases (14.7%) received autologous hematopoietic stem cell transplantation. After median 5 courses (range 2-24) of IxaPD chemotherapy, the overall response rate (ORR) was 64.7%, with 47.1% VGPR or better. The median time to achieve the best response was 2 months (range 1-6). After a median follow-up of 13 months (range 2-28), 52.9% of patients progressed and the median time to next treatment was 7 months (range 3-21). The mortality rate was 17.6%, and the median overall survival was not reached. In the multivariate analysis, response to first-line induction therapy significantly affected the time to next treatment(P&amp;lt;0.05), while age, first relapse, R-ISS stage, high-risk cytogenetics abnormalities, and renal insufficiency did not impact significantly. Neither did those factors also have significant impact on overall survival. Most of the adverse effects (AEs) were grade 1-2, mainly skin rash, peripheral neuropathy, constipation, diarrhea, edema, arthralgia, herpes zoster, and shortness of breath. Only one case of grade 3 pulmonary infection occurred. Conclusion The pan-oral IxaPD regimen showed anticipated effect and good tolerance for RRMM patients, especially appropriate for patients achieving deep response in first-line therapy. More patients are being enrolled and update data will be reported.

Phase I Clinical Study of Humanized BCMA-Single-Domain Antibodies-Targeting CAR T (BCMA-CART) in Patients with Relapsed/Refractory Multiple Myeloma

Background:The CAR structure adopted by CART-BCMA is composed of a single-domain antibody targeting BCMA, CD8 hinge region, transmembrane region, 4-1BB co-stimulatory domain, and CD3-ζ T cell activation domain. In the phase I clinical study (NCT05346198) carried out in China, the safety and efficacy of CART-BCMA were initially evaluated through the dose escalation + dose expansion design, and the recommended dose (RD dose) for follow-up studies was determined as well. Method:Enrolled patients with relapsed and/or refractory multiple myeloma (RRMM) had previously received at least 3 lines of therapy including at least one proteasome inhibitor and one immune modulator. After lymphatic preconditioning chemotherapy (program: fludarabine 30mg/m 2/d and cyclophosphamide 300mg/m 2/d, -5, -4, -3, for 3 consecutive days), all patients received a single dose of CART-BCMA at 2.5×10 6, 5×10 6 and 7.5×10 6 CAR-positive T cells/kg (subject body weight), according to the dose level in which they were enrolled. R esults :Up to June 26, 2023, a total of 15 cases of RRMM received 2.5×10 6 CAR-positive T cells/kg (n=3), 5×10 6 CAR-positive T cells/kg (n=11) or 7.5×10 6 CAR-positive T cells/kg (n=1) dose level of CART-BCMA cell infusion therapy on the basis of the dose level in which they were enrolled. The median follow-up time was 14.42 months (range: 10.2-26.6 months). The median age of the subjects was 63.0 years old (range: 36-73 years old) (Table 1), 73.3% of the subjects (n=11) had previously received ≥4 lines of multiple myeloma therapy, and the median line of treatment was 5 lines (range: 3-9 lines), among which, 2 subjects (13.3%) had previously received autologous hematopoietic stem cell transplantation, and 4 subjects (26.7%) had relapsed/refractory diseases after previous treatment with proteasome inhibitors, immune modulators and CD38 monoclonal antibodies (three-drug relapsed/refractory). Nine subjects (60.0%) had high-risk cytogenetic abnormalities according to sMART 3.0 criteria, and 6 subjects (40.0%) had extramedullary plasmacytoma. The most common grade ≥3 adverse events were hematological toxicity, including decreased neutrophil count (n=13, 86.7%), decreased white blood cell count (n=12, 80.0%), decreased lymphocyte count (n=10, 66.7%), anemia (n=8, 53.3%), decreased platelet count (n=4, 26.7%). The first patient in the 7.5×10 6 CAR-positive T cells/kg dose group had a dose-limiting toxicity (DLT) event of grade 4 platelet count decrease, and no DLT event occurred in the other low-dose groups (2.5 and 5×10 6 CAR-positive T cells/kg). All 15 patients (100.0%) developed grade 1-2 cytokine release syndrome (CRS), no grade ≥3 CRS occurred, and the median time to CRS was 2.0 days (range: 1-13 days) , with a median duration of 7.0 days (range: 2 to 17 days). After the occurrence of CRS, all patients recovered without sequelae after supportive treatment, and 6 patients (40%) received tocilizumab (3 subjects received 2 doses, 3 subjects received 1 dose), 4 patients (26.7%) used glucocorticoids. There was no event of immune effector cell-associated neurotoxicity syndrome (ICANS), and no death event occurred. The median time to the first response (TTR) of the patients was 0.953 months (range: 0.92 to 2.23 months). All 15 patients who received CART-BCMA infusion achieved remission, and the overall remission rate (ORR) ( At least achieve PR or better efficacy) was 100% (95%CI, 78.20%-100.00%), as shown in Figure 1, among which, the best response of 9 patients (60%) was strict complete remission ( sCR), 5 patients (33.3%) achieved very good partial response (VGPR), and 1 patient (6.7%) achieved PR. All sCR patients achieved MRD negativity. The ORR of 6 patients with extramedullary plasmacytoma was 100%, of which 2 achieved sCR and 4 achieved VGPR. Notably, gradual deepening of remission was observed in 12 patients (80%), of which 10 patients (66.7%) further deepened the depth of remission on days 60-90 after infusion, and 2 patients (13.3%) were assessed sCR at day 360. Conclusion:Data from this phase 1 clinical study showed that CART-BCMA is well tolerated and highly efficacious in patients with RRMM. Patients with extramedullary plasmacytoma had the comparable response to those without extramedullary diseases. Notably, 12 patients (80%) patients elicted deeper response after 3 months of CART-BCMA infusion

CLAG combined with total body irradiation as intensive conditioning chemotherapy prior to allogeneic hematopoietic stem cell transplantation in patients with refractory or relapsed acute myeloid leukemia.

Refractory or relapsed acute myeloid leukemia (R/R AML) remains the major challenge of AML treatment. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only valid option to achieve cure, but the prognosis is still dismal. We conducted a retrospective analysis for the feasibility of CLAG regimens (cladribine, cytarabine, and granulocyte colony-stimulating factor) combined with total body irradiation (TBI) as new intensive conditioning chemotherapy prior to HSCT in R/R AML. A total of 70 patients, including 21 primary refractory and 49 relapsed AML, were analyzed. Forty-nine (70%) patients had extramedullary diseases, and 54 (77%) patients received haploidentical transplantation. Except for one who died before white blood cell engraftment, all of the 69 evaluable patients achieved measurable residual disease (MRD) negative complete remission. The 3-year overall survival (OS) and relapse-free survival (RFS) rates were 46.0% (95% confidence interval [CI], 33.5-57.7%) and 38.5% (95%CI, 26.8-50.0%). The 1-year cumulative incidences of relapse and non-relapse mortality (NRM) were 38.6% (95%CI, 27.3-49.3%) and 11.6% (95%CI: 5.4-20.3%), respectively. The presence of chronic graft-versus-host disease (cGVHD) showed a trend to be associated with a lower risk of relapse (P = 0.054) and extramedullary diseases with a higher risk of NRM (P = 0.074). Multivariate analyses identified low leukemia burden pre-HSCT (defined as bone marrow blasts ≤ 50%) and cGVHD as independent factors associated with favorable OS and RFS. In conclusion, intensive conditioning with CLAG regimens plus TBI may be an effective and well-tolerated choice for R/R AML patients undergoing allo-HSCT.

Bispecific CS1-BCMA CAR-T Cells Are Clinically Active in Relapsed or Refractory Multiple Myeloma: An Updated Clinical Study

Background Bispecific CAR-T cell therapy has been proposed to mitigate some limitations of single-targeted CAR-T cells. CS1 plays a vital role in myeloma pathogenesis, and is also highly expressed on tumor cells in almost all patients (pts) with multiple myeloma (MM). Therefore, we aim to augment BCMA targeting with CS1. Bispecific CS1-BCMA CAR-T cells are effective in targeting MM cells in preclinical studies (Biomedicines 2021). Here we report the outcomes of 16 pts with refractory or relapsed (RR) MM in our phase I clinical trial (NCT04662099). Methods CS1-BCMA bispecific CAR contains a murine anti-CS1 scFv (clone 7A8D5) and a murine anti-BCMA scFv (clone 4C8) in tandem, and a 4-1BB costimulatory domain. The enrolled pts must have received at least 2 prior lines of therapy, and previous BCMA- or CS1-targeted immunotherapies were allowed. Pts were subjected to lymphodepleting regimens with cyclophosphamide (250 mg/m2, d-5 to d-3) and fludarabine (30 mg/m2, d-5 to d-3) daily prior to the CAR-T infusion (d0). Planned dose levels were 0.75x106, 1.5x106, and 3.0x106 CAR+T cells/kg, and repeated infusions were allowed. Primary objectives were incidence of adverse events (AEs). Cytokine release syndrome (CRS) and neurotoxicity were graded using the ASTCT criteria, and other AEs using CTCAE v5.0. Secondary objectives were overall response rate (ORR), overall survival (OS), duration of response (DOR), and progress-free survival (PFS). Response was assessed per the IMWG criteria 2016. Other objectives included in vivo kinetics of CAR-T cells, changes of soluble BCMA, immune assessment of infused CAR-T cells and patients’ lymphoid and immunosuppressive phenotype before and after infusion. Results As of July 31, 2022, 16 patients received the infusion of CS1-BCMA CAR-T cells and were included in the final analysis. Nine pts (56%) carried cytogenetic abnormalities, and 3 pts had extramedullary diseases (EMD) alone without detectable MM cells in bone marrow (BM). BCMA and CS1 were highly expressed on MM cells in BM by flow cytometry (mean BCMA+76.7% and CS1+ 96.5%) and EMD by trichromatic immunofluorescence. Nine pts (56%) were refractory, and 7 pts (44%) were relapsed and refractory. The median prior lines of treatment were 5.4 (range 2-10). All pts (100%) were exposed to bortezomib, 6 (38%) to ixazomib and 1 (6%) to cafilzomib. 13 pts (81%) received lenalidomide, 6 (38%) received thalidomide and 5 (31%) received pomadomide. 7 pts (44%) received autologous transplantation, 6 (38%) were exposed to daratumumab, 2 (13%) received selinexor and 2 (13%) had previous BCMA-targeted CAR-T therapy. Six pts (38%) experienced CRS, with median duration of 4 days (range 4-7). Grade 3 CRS occurred once and lasted for 5 days. Neurotoxicity was not observed. The ORR was 81% (13/16) for all patients, including 6 stringent complete, 3 very good partial, and 4 partial responses (Fig.1A). The ORR was 100% (13/13) for patients with MM cells in BM. Among them, 7 pts (54%) maintained ongoing response, 2 pts progressed after response, 1 withdraw from the study and 3 were lost due to poor compliance. Pt 16 had received prior anti-BCMA CAR-T treatment and achieved a partial response after CS1-BCMA CAR-T infusion. Although CAR-T cells infiltrated into the tumor tissue, response was not observed in the 3 pts with EMD alone (Fig.1B). Soluble BCMA decreased remarkably in peripheral blood (PB) and BM after infusion. At a median follow-up of 290 days, the median OS, PFS and DOR weren't reached, and OS, PFS and DOR at 12-month were 83.9%, 55.2% and 68.8%, respectively. On day 14 after infusion, CAR-T cells peaked at 138245 copies/ug DNA in PB (n=16) and 86654 copies/ug DNA in BM (n=13) by droplet digital PCR; CAR-T cells peaked at 493 CAR+T cells/ul PB (n=16) and 312 CAR+T cells/ul BM (n=13) by flow cytometry. CAR was detectable in vivo at a median of 6 months. Because CS1 is expressed on T and NK cells, we systematically evaluated the immune characteristics (differentiation, Fas, LAG3, PD1, TIM3, CS1) of infused products, absolute quantification and CS1 expression on T and NK cells in PB as well as myeloid derived suppressor cells, regulatory B and T cells in BM and PB. Further data will be released in the meeting. Conclusions Our study demonstrates bispecific CS1-BCMA CAR-T cells are clinical active with a good safety profile in heavily pretreated pts with MM, even after BCMA-targeted CAR-T cell therapy. Further studies are needed for pts with EMD alone. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Final Results of Phase I/II Study of Donor-Derived CAR T Cells Engineered with Sleeping Beauty in Pediatric and Adult Patients with B-Cell Acute Lymphoblastic Leukemia Relapsed Post-HSCT

Introduction Non-viral engineering of allogeneic T cells is a promising alternative option to produce a fit CAR T cell product. We recently proposed the use of Sleeping Beauty (SB) transposon to engineer donor-derived cytokine-induced killer (CIK) cells (CARCIK-CD19) for the treatment of B-cell acute lymphoblastic leukemia (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT) (Magnani C et al.: JCI 2020). Here we report the final results of a phase I/II study evaluating the safety and efficacy of CARCIK-CD19 cells (FT01CARCIK, Clinical trial.gov, NCT03389035). Methods We conducted a multi-center, phase I/II dose-escalation (n=12) and dose expansion (n=9) trial in patients relapsed after HSCT. After approval by the Agenzia Italiana del Farmaco (AIFA), 6 additional patients were treated on a compassionate base use and were considered in the final analysis. CARCIK-CD19 cells were manufactured as described (Magnani C et al.: JCI 2020). CARCIK-CD19 were obtained by HLA-identical sibling (n=7, 26%), matched unrelated (n= 10, 37%), and haploidentical donors (n=10, 37%). After lymphodepletion with Fludarabine (30 mg/m2/day x 4 days) and Cyclophosphamide (500 mg/m2/day x 2 days), patients received a single CARCIK-CD19 infusion. Bridging therapy was allowed before CAR-T cell infusion. The Bayesian Optimal Interval (BOIN) design was used for the dose escalation part of the trial. The primary objectives were to define the Maximum Tolerated Dose (MTD) and the recommended Phase II dose as well as the safety. Secondary objectives included the rate of complete hematologic response (CR), duration of response (DOR), event-free (EFS), and overall survival (OS). DOR and EFS were censored when patient underwent a new anticancer therapy. Results A total of 27 patients (4 children and 23 adults) were infused between February 2018 and August 2021. A successful production of CARCIK-CD19 cells was achieved for all patients. The first 9 patients were treated with increasing numbers of CARCIK cells, in groups of three patients per dose level (1x106 /kg, 3x106/kg and 7.5x106/kg), whereas the remaining 18 patients (67%) received the highest planned dose level of 15x106 cells/Kg, as no dose limiting toxicity (DLT) was observed. The median number of prior therapies was 4 (range, 2-8) with a median time interval from HSCT to relapse of 8 months. We enrolled 25 patients (93%) with >5% bone marrow (BM) blasts with a median of 50% (range, 5-100%) at enrollment and 4% (range, 0-96%) after lymphodepletion. Two patients in the compassionate use group, presented extramedullary diseases without BM involvement. CRS occurred in 9 patients (4 patients with grade I and 5 with grade II) and immune effector cell-associated neurotoxicity-ICAN (grade 3) in 2 patients at the two highest doses. Although 10 out of 27 had experienced GvHD after the previous HSCT, GvHD never occurred after treatment with CARCIK-CD19. Nine patients had infections, of which 6 of grade 3 or higher. CR was achieved by 18 out of 27 patients (66.7%, 95%CI=46.0-83.5%) and by 16 out of 21 patients treated with the two highest doses (76.2%, 95%CI=52.8-91.8%). Fourteen (77.8%) of the overall responders and 13 of the responders at the highest doses (81.3%) were MRD-negative. With a median follow up of 2.8 years (range, 0.05-4.4 years, last update as of July 2022), a significantly better EFS was observed in patients treated with the highest doses (p=0.0265). For the 21 patients treated at the two highest dose levels, the median EFS and OS were 4 and 12 months, respectively (Figure 1). At 6 months, the EFS and OS were 41.5% (SE=11.6) and 71.4% (SE=9.9), respectively. The median DOR of the 16 patients in CR was 9.5 months, with a 6-months DOR of 54.4% (SE=13.8). By univariate analysis, EFS was significantly associated with the % of blasts after lymphodepletion (≥ 5% vs <5%) (p=0.0245). Conclusions In this phase I/II dose-finding study, SB-engineered CAR T cells demonstrated an excellent safety profile with anti-leukemia activity in heavily pretreated patients with B-ALL relapsed after HSCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A New and Cost-Effective Approach to Risk Stratification for Children with Precursor B-Cell Acute Lymphoblastic Leukemia Based on Competing Outcomes

Background: Acute lymphoblastic leukemia (ALL) is a malignancy of the white blood cells characterized by its rapid and aggressive progress that needs immediate treatment. ALL could affect both adults and children. Various patient- and disease-related factors may be involved in ALL patients’ prognosis. Therefore, it is critical to identify important risk factors related to the competing outcomes of patients with ALL. Objectives: This study aimed to stratify the risk of outcomes of children with precursor B-cell ALL using demographic characteristics, laboratory characteristics, and extramedullary diseases. To achieve this goal, we used the best competing risks model to make an appropriate decision for children's treatment according to this classification. Methods: In this retrospective cohort study, 393 patients with ALL were included. ALL with B cell origins (CD20, CD19, CD10, and CD22 positive markers) was differentiated using flow cytometry. Complete remission was defined by a lymphoblast count of less than 5% in the bone marrow, presence of no blasts in cerebrospinal fluid (CSF), as well as complete disappearance of clinical symptoms. Patients with ALL were treated based on Berlin-Frankfurt-Münster (BFM). Competing outcomes were first-relapse and non-relapse mortality, respectively. Risk factors affecting competing outcomes were assessed based on a fully specified sub-distribution model. Results: Five-year estimates for overall survival and event free survival were 75% (95% CI: 69 - 79%) and 71% (95% CI: 66 - 75%), respectively. Five-year incidence rates for first-relapse and non-relapse mortality in children were 11.4% (95% CI: 8.32 - 15.16%) and 17.6% (95% CI: 13.98 - 21.67%), respectively. Moreover, according to the results, children with WBC &gt; 50000, hemoglobin 8, and tumor lysis syndrome for the first-relapse outcome, and children with central nervous system (CNS) involvement and tumor lysis syndrome for the non-relapse mortality (NRM) outcome were considered as high-risk groups. Conclusions: We found that extramedullary diseases could have a crucial role in the risk stratification of children with precursor B-cell ALL. Therefore, for a targeted and effective treatment of high-risk children, in addition to chemotherapy, using appropriate PI3K pathway inhibitors, JAK2 pathway inhibitors, and antibody-based immunotherapy is recommended to reduce minimal residual disease and, consequently, mortality rate.

Prospective Comparison Study of Prognostic Value of MRD Detected By 8-Color MFC (EuroFlow-NGF) and NGS in Patients with Multiple Myeloma in ASCT Setting

Background: Novel agents capable of inducing deeper responses dramatically improve the prognosis of patients with multiple myeloma (MM). Innovative technologies such as multiparameter flow cytometry (MFC) and next-generation sequencing (NGS) are utilized to assess minimal residual disease (MRD) for further stratification of patients who achieve a complete response (CR). EuroFlow-next-generation flow (EuroFlow-NGF) is one of the gold standard MFC methods. Recently, both NGF and NGS have been used in many clinical trials to assess MRD levels associated with progression-free survival (PFS) and overall survival (OS). The present study prospectively assessed MRD levels by both NGF and NGS to elucidate the prognostic impact of both methods and clarify their characteristics in MM patients in an autologous stem cell transplantation (ASCT) setting.Methods: We prospectively assessed the response in Japanese patients with newly diagnosed MM who underwent ASCT and lenalidomide-based maintenance therapy at multiple Japanese medical centers between September 2016 and July 2021. The diagnosis of MM and patients' responses to therapy were assessed using the IMWG criteria. Only patients with CR or stringent CR on days 100-365 post-ASCT were included, and bone marrow (BM) samples were obtained to assess MRD. Four milliliters of BM was divided equally. Cells derived from 2 mL BM were analyzed by the NGF method (Flores-Montero et al., Leukemia 2017) at Kanazawa University, and DNA extracted from the remaining 2 mL BM cells was processed by Adaptive Biotechnologies' standardized NGS-MRD assay (Seattle, WA) (Ching et al., BMC Cancer 2020) to assess MRD levels. MRD levels in BM were also monitored at 1-year (± 20 days) and 2-year (± 20 days) post-ASCT. The prognostic value of MRD levels in BM was assessed, and their correlation between NGF and NGS was compared at a cut-off value of 1×10 -5. Sustained MRD negativity was defined as the maintenance of MRD negativity in the BM for more than 6 months. BM cells were analyzed for high-risk cytogenetics (del(17p), t(4;14), and t(14;16)) by FISH.Results: A total of 60 patients (male = 29, female = 31) underwent bortezomib-based induction therapy, ASCT conditioned with high-dose melphalan, and lenalidomide-based maintenance. The median age was 62 years at the ASCT (range 36-71; ISS 1 [n = 13], 2 [n = 24], and 3 [n = 23]). Thirty-three percent of patients showed high-risk chromosomal abnormalities (del17p (n=11), t(4;14) (n=10), t(14;16) (n=2)), 3 patients had double hit diseases, and five patients had extramedullary diseases. With a median follow-up of 3 years, the 3-year progression-free survival (PFS) and 3-year overall survival (OS) rates were 69.2% and 94.2%, respectively. In total, 148 samples were analyzed using NGF and 138 were analyzed using NGS. The rates of MRD negativity at least once using NGF and NGS were 80% and 61%, respectively. The patients who achieved at least one MRD negativity exhibited significantly better 3-year PFS (82.9% by NGF; 84.8% by NGS) than those who did not (P < 0.0001, 0% by NGF; P = 0.005, 49.1% by NGS). Patients who sustained MRD negativity for more than 6 months also showed significantly better 3-year PFS (96.7% by NGF; 92.3% by NGS) compared with those without sustained MRD negativity (Figure; P < 0.0001, 37.1% by NGF; P < 0.01, 50.9% by NGS). The MRD levels between the NGF and NGS methods were significantly correlated with each other (r = 0.9295, P < 0.0001). Among the 17 patients who developed PD after ASCT, seven cases showed discrepancies in the MRD results and two cases in which one case was MRD-positive and the other was MRD-negative by both methods progressed with extramedullary diseases. Five of the seven cases were MRD-positive by NGS and MRD-negative by NGF.Conclusions: In this prospective comparison study of MRD assessment in BM cells using EuroFlow-NGF and NGS approaches, MRD levels highly correlated with each other, and MRD negativity and sustained MRD negativity were significantly associated with prolonged PFS. Multiple MRD assessments by NGF or NGS are essential for predicting durable remission and prolonged clinical outcomes. [Display omitted] DisclosuresTakamatsu: Bristol-Myers Squibb: Honoraria, Research Funding; Adaptive Biotechnologies, Eisai: Honoraria; SRL: Consultancy; Janssen: Consultancy, Honoraria, Research Funding. Yoshihara: Bristol-Myers Squibb: Honoraria; Janssen: Honoraria; Novartis: Honoraria. Matsumoto: Sanofi: Honoraria; Janssen: Honoraria; Ono: Honoraria; Bristol-Myers Squibb: Honoraria. Yamashita: Janssen: Honoraria; Bristol-Myers Squibb: Honoraria; celgene: Honoraria; Takeda: Honoraria. Fuchida: Takeda Pharmaceutical Co., Ltd.: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Sanofi: Honoraria; Bristol-Myers Squibb Co., Ltd.: Honoraria; Celgene Co., Ltd.: Honoraria. Hiragori: BML: Current Employment. Suzuki: Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; ONO: Honoraria; Novartis: Honoraria; Sanofi: Honoraria; Abie: Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Nakao: Symbio: Consultancy; Kyowa Kirin: Honoraria; Novartis Pharma: Honoraria; Alexion Pharma: Research Funding. Durie: Amgen: Other: fees from non-CME/CE services ; Amgen, Celgene/Bristol-Myers Squibb, Janssen, and Takeda: Consultancy.

Real-World Retrospective Study of 46 Patients with Macrofocal Multiple Myeloma (MFMM)

Real-world retrospective study of 46 patients with Macrofocal multiple myeloma (MFMM)Introduction:Multiple myeloma is a clonal plasma cell malignant neoplasm characterized by bone disease and marrow involvement. However, we found that some myeloma patients presented multiple lytic lesions, but didn't meet the diagnostic criteria of at least 10% clonal plasma cells in the bone marrow. And these patients were always diagnosed with myeloma by biopsy-proven plasmacytomas. This special myeloma entity has been called macrofocal multiple myeloma (MFMM) [1-2]. Considering that the data of this rare myeloma entity are limited, it is of great significance to investigate the clinical characteristics, genetic abnormality, treatment response and prognosis of MFMM patients.Methods:Based on the definition (BMPCs<20% and multiple lytic lesions/plasmacytomas, without anemia, renal insufficiency or hypercalcemia) [3]，we identified 46 MFMM patients among 791 myeloma patients(5.8%) diagnosed at out hospital between January 2013 and December 2019. In the same period with same therapies, other 92 typical myeloma patients were selected as the control group.Results:Patient characteristics and comparisons between MFMM patients and the control group are depicted in Table 1. Of the 46 MFMM patients, 82.6% were <65 years and 73.9% had at least 4 lytic lesions. And the incidence of plasmacytomas in MFMM patients was significantly higher than the control group (43.5% vs 18.5%, p<0.05). According to the international staging (ISS) and the Revised ISS, advanced stage patients in the MFMM group was less common than controls (p<0.05). Regarding to the cytogenetics, the high-risk features was infrequent in patients with MFMM compared to typical myeloma patients (15.8% vs 32.2%, p=0.058). And t (11;14) could be observed in 32.4% MFMM patients and 9.4% myeloma patients (p<0.05).The treatment patterns of the two groups were similar; about 30% of the patients received ASCT, and 80% patients received proteasome-inhibitors based regimen as the induction therapy. Concerning about the best response to treatment, the CR rate of the MFMM group was significantly higher than the controls (78.2% vs 60.8%, p<0.05). As of June 2021, the median follow-up time was 37.9 months. The median progression-free survival in the study and control groups were 77.5 vs 39.8 months, respectively (p<0.05). The overall survival (OS) of MFMM patients was significantly longer than typical myeloma patients during the same period with similar therapies (not reached vs 68.2m, p<0.05).Conclusion:In conclusion, Macrofocal multiple myeloma is a special entity of MM, which is characterized with multiple lytic lesions, more extramedullary diseases, less bone marrow infiltration, and fewer adverse features. And the MFMM patient could achieve deep remission and prolonged OS in the era of novel agents.

The clinical study of anti-BCMA CAR-T with single-domain antibody as antigen binding domain.

8025 Background: Relapsed/refractory (RR) multiple myeloma (MM), RRMM, remains as an incurable disease and has a 5-year survival rate of nearly 50%. To address the unmet medical need, an autologous CAR-T cell therapy was developed previously with a humanized single-domain antibody (sdAb) targeting BCMA as the antigen binding domain, 4-1BB and CD3ζ as cytoplasmic domain. Methods: An investigator-initiated clinical trial (IIT) was conducted in China to assess the safety and efficacy of the sdAb-based CAR-T. The trail was started in June 2018 and the last patient infused in June 2019. As of 1 February 2021, 34 were treated and followed up. The patients had received multiple lines of prior treatment (including bortezomib, lenalidomide, and others). Following a lymphodepleting regimen of cyclophosphamide (300-600 mg/m2, d-5, -4) and fludarabine (25-30 mg/m2, d-5 to d-3), patients were infused with 2.5-10.0 × 106 CAR+ cells/kg body weight. CAR-T was infused immediately after preparation and quality control performed in all patients except one, who was infused a 10.0 × 106 CAR+ cells/kg dose of frozen cells. Efficacy was assessed based on the IMWG criteria, toxicity was graded by CTCAE 4.02, and CRS grading was based on the grading system by CARTOX working group. Results: All 34 patients had the tumor burden of plasma cells in bone marrow, or M protein or free light chains (FLCs) in serum, 7 patients were accompanied with extramedullary diseases. The efficacy shows the best ORR is 88.2% (30/34), sCR rate is 55.9% (19/34). The mPFS was 12.1 months, several patients shows continuous sCR after 2 years. No obvious correlation between efficacy and dosage were found in three dose groups of 2.5×106 CAR+ cells/kg (6 pts), 5.0×106 CAR+ cells/kg (23 pts) or 10.0×106 CAR+ cells/kg (5 pts). The observed adverse events include thrombocytopenia (≥grade 3, 38.2%), neutropenia (≥grade 3, 44.1%), leukopenia (≥grade 3, 32.4%), lymphopeniPa (≥grade 3, 26.5%), and anemia (≥grade 3, 20.6%). CRS was monitored occurring in 29 patients (any grade, 85.3%, ≥grade 3, 2.9%). Conclusions: Our result demonstrates that the CART employing one humanized sdAb targeting BCMA is safe and efficacious for clinical application. The phase I clinical trial has been initiated in China for searching the RP2D using the cryopreserved CAR-T cells. Clinical trial information: NCT03661554.

Influence of patient characteristics on chimeric antigen receptor T cell therapy in B-cell acute lymphoblastic leukemia

CD19-specific chimeric antigen receptor T cell (CD19 CAR T) therapy has shown high remission rates in patients with refractory/relapsed B-cell acute lymphoblastic leukemia (r/r B-ALL). However, the long-term outcome and the factors that influence the efficacy need further exploration. Here we report the outcome of 51 r/r B-ALL patients from a non-randomized, Phase II clinical trial (ClinicalTrials.gov number: NCT02735291). The primary outcome shows that the overall remission rate (complete remission with or without incomplete hematologic recovery) is 80.9%. The secondary outcome reveals that the overall survival (OS) and relapse-free survival (RFS) rates at 1 year are 53.0 and 45.0%, respectively. The incidence of grade 4 adverse reactions is 6.4%. The trial meets pre-specified endpoints. Further analysis shows that patients with extramedullary diseases (EMDs) other than central nervous system (CNS) involvement have the lowest remission rate (28.6%). The OS and RFS in patients with any subtype of EMDs, higher Tregs, or high-risk genetic factors are all significantly lower than that in their corresponding control cohorts. EMDs and higher Tregs are independent high-risk factors respectively for poor OS and RFS. Thus, these patient characteristics may hinder the efficacy of CAR T therapy.

Two-Year Follow-up of Investigator-Initiated Phase 1 Trials of the Safety and Efficacy of Fully Human Anti-Bcma CAR T Cells (CT053) in Relapsed/Refractory Multiple Myeloma

Background: B cell maturation antigen (BCMA) is a potential therapeutic target in multiple myeloma. CT053 comprises autologous T cells transduced with a second-generation chimeric antigen receptor (CAR) utilizing a fully human BCMA-specific single-chain fragment variant (25C2) with high binding affinity. CT053 was firstly studied in a single-arm, open-label Phase I, investigator-initiated program (NCT03716856, NCT03302403, and NCT03380039) in eastern China. Here we present the 24-month follow-up results of the study. Methods: The multicenter exploratory Phase 1 studies included adult subjects with relapsed/ refractory multiple myeloma (RRMM) who had received at least 2 prior lines of myeloma treatment. After preconditioning treatment with fludarabine and cyclophosphamide for 2-4 days, 21 subjects received one cycle of 1.5 × 108 T cells CT053 CAR-BCMA T cells. Three subjects respectively received 0.5 × 108, 1 × 108, or 1.8 × 108 cells. The primary objective was subject safety. The secondary objectives were pharmacokinetics and efficacy. Efficacy was assessed according to IMWG 2016 criteria. Results: A total of 24 subjects with a median age of 60.1 years (range, 38.5-69.9) were enrolled from Sept. 10, 2017 to Sept. 22, 2018. The subjects had a median of 4.5 (range, 2-11) prior lines of therapy, and 41.7% (10/24) underwent autologous stem cell transplantation. At baseline, 10 subjects (41.7%) had concomitant extramedullary involvement, 8 subjects (33.3%) had ECOG scores 2-3, and 9 subjects (37.5%) reported ISS Grade III. As of June 30, 2020, 9 subjects completed 24 months of follow-up with responses including 8 stringent complete response (sCR) and 1 complete response (CR). Also, 15 subjects discontinued prior to completing the 24-month follow-up, of whom 13 discontinued due to disease progression (PD), and 2 discontinued for other anticancer therapy. The overall response rate was 87.5% (21/24) including 79.2% (19/24) with complete responses or stringent complete responses (3 CR, 16 sCR). The median duration of response (DOR) was 21.8 months (95%CI: 9.2, not evaluable [NE]). The median progression-free survival (PFS) was 18.8 months (95%CI: 10.1, NE), with 6-month and 12-month PFS rates of 87% and 60.9%, respectively. Thirteen subjects progressed with median PFS of 10.2 months (range, 0.9-23 months): 3 progressed within 6 months, 6 progressed within 6-12 months, and 4 within 12-24 months. Compared to 9 subjects with persistent CR/sCR, the 13 progressed subjects had a higher percentage of ECOG scores 2-3 (46.2% vs 22.2%), ISS Grade III (53.9% vs 11.1%) and high-risk cytogenetics profiles (53.8% vs 33.3%). Rates of concomitant extramedullary diseases were similar, 46.2% and 44.4%, respectively. Hematological toxicities were the most common treatment-related adverse events of grade (G) 3 or higher, including leukopenia (83.3%), neutropenia (85%), lymphocytopenia (79.2%) and thrombocytopenia (20.8%). In general, cytokine release syndrome (CRS) occurred at 1-4 days and resolved in a median 6 days (range, 3-9 days). Low-grade CRS was reported in 15 of 24 (62.5%) subjects. All CRS events (4 G1, 11 G2) resolved within 2-8 days; among them, 9 patients received a low dose of tocilizumab 4-6 mg/kg. One patient experienced G3 neurotoxicity, presenting as epilepsy and accompanied by simultaneous G2 CRS. This patient fully recovered within 3 days after treatment with methylprednisolone, diazepam and sodium valproate. Six patients (25%) experienced 10 cases of treatment-related serious adverse events (SAEs), including lung infection (3), gastroenteritis (1), neutropenic infection (1), fever (1) and hematological toxicities (4). By the cutoff date, one subject died of SAE a (bone morrow failure and neutropenic infection) and PD, and seven subjects died of PD. CAR-BCMA T cell expansion was detectable as early as day 1-7 after infusion and reached peak values on day 7-21 with the highest concentration at 4.5×105 copies/µg genomic DNA. Median T cell persistence was 172 days. The longest persistence of CAR-BCMA copies was measured at 341 days and continues. No immunogenicity was detected. Conclusion: These studies demonstrated that CT053 had excellent efficacy in RRMM, showing early, deep and durable response with 21.8 months DOR. CT053 was well tolerated among the subjects. Figure Disclosures Li: CARsgen Therapeutics Co. LtD: Current Employment, Current equity holder in private company. Wang:CARsgen Therapeutics Corp.: Current Employment. Xiao:CARsgen Therapeutics Corp.: Current Employment. Yuan:CARsgen Therapeutics Corp.: Current Employment. Ma:CARsgen Therapeutics Corp.: Current Employment.

The clinical study of anti-bcma car-t with humanized single-domain antibody

Background & Aim The Chimeric antigen receptor T cells (CART) targeting BCMA has the promised effects to treat the relapsed/refractory (RR) multiple myeloma (MM), RRMM. We developed CART cells (CART-BCMA) using one single-domain antibody as the binding domain for BCMA. The anti-BCMA single-domain antibody was derived from the alpaca with the affinity of 1.14nM, then was humanized, and was used to construct the CAR molecule, which use the 4-1BB and CD3ζ intracellular regions as T cell activation domain. Methods, Results & Conclusion A single-arm clinical study was conducted to assess the safety and efficacy of CART-BCMA. The enrolled RRMM patients had received at least 3 lines of prior treatment. Patients were subjected to a lymphodepletion regimen with Cy (300-600 mg/m2, d-5, -4) and Flu (30 mg/m2, d-5 to d-3) before CART infusion at the dose of 2-10 × 106 CAR+ cells/kg (body weight). The efficacy was assessed based on the IMWG Criteria, and the toxicity was graded by CTCAE 4.02. As of November 30, 2019, 34 patients were infused with autologous CART-BCMA cells and had at least 5 months of follow-up. Many patients have M protein in serum, some patients have extramedullary diseases. The different dose shows a similar efficacy (Table 1), the best ORR is 88.2% (30/34), CR/sCR rate is 52.9% (18/34). Many patients showed no or grade 1/2 CRS, one patient showed the grade 3 CRS. Other ≥grade 3 adverse events (AE) include neutropenia (38.2%), leukopenia (29.4%), thrombocytopenia (38.2%), anemia (23.5%).Our result demonstrates the promising efficacy of anti-BCMA CAR-T using the humanized single domain antibody, and supports further development of this anti-RRMM cellular immunotherapy. Clinical trial information: NCT03661554.

A Bcma and CD19 Bispecific CAR-T for Relapsed and Refractory Multiple Myeloma

Introduction Chimeric Antigen Receptor T cells (CAR-T) therapy, e.g. B Cell Maturation Antigen (BCMA)-directed CAR-T has provided an encouraging modality for relapsed and refractory management of multiple myeloma (MM). However, a significant portion of patients still relapse with progressive disease after monospecific anti-BCMA CAR-T treatment. It has been demonstrated that CD19-directed CAR-T was effective in certain MM patients, likely due to CD19 expression on subsets of MM cells, and/or undetectable level of CD19 on MM cells. In addition, it has been reported that CD19 could express on the myeloma progenitor cells. To further improve the efficacy and to reduce relapse, we have designed a bispecific CAR-T targeting both BCMA and CD19. In addition to the conventionally-manufactured BCMA-CD19 CAR-T, the bispecific CAR-T was also successfully manufactured in our newly developed FasT CAR-T platform, which shortened the production time to one day. Here we report the results from pre-clinical studies and early results from the first-in-human clinical study. Methods The BCMA-CD19 bispecific CAR was constructed by linking BCMA and CD19 scFv, joined by a CD8 hinge, transmembrane domain, co-stimulatory domain and CD3. CAR-T cells were produced using either the conventional process (GC012) or the FasT CAR-T platform (GC012F). Peripheral blood (PB) mononuclear cells were obtained by leukapheresis either from healthy donors for the pre-clinical study or from patients for the clinical trial. T cells were isolated and used for CAR-T manufacturing. A xenograft mouse model was used to determine the efficacy in vivo. From March 2019 to July 2019, 5 adult relapsed/refractory MM patients (Age 50-59), who had previously received multiple lines of therapies, were enrolled (Table). Among them, 2 had extramedullary diseases. One patient did not receive lymphodepletion, and all other 4 patients received i.v. fludarabine and cyclophosphamide for 3 days. All patients received a single infusion of CAR-T cells, either at dose 1x106/Kg (DL1) (2 patients) or at dose 2x106/Kg (DL2) (3 patients), and the dose escalation is still ongoing. The endpoints of the exploratory trial were to evaluate the safety, feasibility, PK, and clinical efficacy of BCMA-CD19 bispecific CAR-T. Results In pre-clinical study, BCMA-CD19 bispecific CAR-T were very effective in killing CD19+ and/or BCMA + target cells including MM cell lines RPMI8226 and MM.1s (Fig 1). Increased IFN production and CD107a up-regulation were also observed. We demonstrated that BCMA-CD19 CAR-T completely eliminated BCMA+ MM cell line RPMI8226, MM.1s, and CD19+ ALL cell line Nalm6 in in vivo xenograft models. Additionally BCMA-CD19 CAR-T cells were shown to be more cytotoxic than single CAR-T both in vitro and in vivo. BCMA-CD19 CAR-T manufactured in the FasT CAR-T platform was more effective in eliminating MM in a xenograft model (Fig. 1). In the clinical study, the median observation time was 68 days (27-144 days up to 2019/7/30). Five patients were evaluated between 15-59 days post CAR-T infusion. Despite the relatively short disease evaluation time, all 5 patients responded to the treatment: 1 patient achieved sCR, 3 achieved VGPR and 1 achieved PR. Notably, although patient KM001 did not receive any pre-conditioning, however, the patient achieved sCR status on Day 15 and has maintained sCR up to now (129 days). CAR-T PK in the PB was monitored by qPCR and flow cytometry. The CAR-T proliferation peak was reached on Day 10 (D7-D14), and the median peak copy number was 34,039 (12,897-128,775) copies /ug DNA (Fig. 2). Remarkably, despite the encouraging clinical response to the CAR-T treatment, no severe adverse events were encountered during the observation period. Three patients experienced only grade 1 cytokine release syndrome (CRS) and no subject suffered from neurotoxicity of any level (Table). Conclusion Pre-clinical data demonstrated BCMA-CD19 CAR-T cells are effective in eliminating MM tumor cells both in vitro and in vivo. The first-in-human clinical trial also showed extraordinary safety profile and efficacy of BCMA-CD19 bispecific CAR-T in treating R/R MM. The long-term benefit and effect on relapse are being further studied. Bispecific CAR-T manufacturing on the FasT CAR-T platform is successful and has been shown to be more potent. A clinical study to evaluate safety and efficacy of FasT BCMA-CD19 CAR-T is ongoing. Disclosures No relevant conflicts of interest to declare.

Corticosteroids Do Not Influence the Efficacy and Kinetics of CAR-T Cells for B-Cell Acute Lymphoblastic Leukemia

Cytokine release syndrome (CRS) is the most prominent and potentially life-threatening toxicity caused by chimeric antigen receptor (CAR) T cell therapy, therefore, effectively controlling severe CRS is critical to ensure patient safety. Tocilizumab, an interleukin-6 receptor antagonist, has been widely used to treat CRS, whereas it is not clear if corticosteroids could be as another optimal choice for managing CRS. We applied corticosteroids instead of tocilizumab as the first-line agent to control CRS in patients with relapsed/refractory B-cell acute lymphoblastic leukemia during CAR-T therapy. The impacts of steroids on treatment efficiency and kinetics of CAR-T cells were assessed by comparing two groups of patients who did (42 cases) or did not (26 cases) receive steroids. Patients followed up less than one month (went to other hospitals for transplantation or died within one month) were excluded. Treatment effects were evaluated on day 30 after T-cell infusion and then monthly in follow-up patients. Minimal residual disease (MRD) was detected by multiparameter flow cytometry (FCM) and quantitative PCR for fusion genes. The dynamic monitoring of CAR-T cells was performed through flow cytometric quantitation of FITC+CD3+ T cells. B-cell aplasia (BCA) was assayed by FCM. Dexamethasone or methylprednisolone or both (alternately) were administrated. Dexamethasone was used in most cases especially for patients with neurologic symptoms; methylprednisolone was preferred for patients with pulmonary or liver dysfunction, and patients accepting high dose steroids. Steroids started with low dose and could be increased if symptoms were not resolved, for severe CRS, steroids would be escalated up to dexamethasone 20mg/m2/d or more higher up to methylprednisolone 10mg/kg/d. Once CRS was improved, steroids were rapidly reduced and stopped. A total of 68 patients (28 adults and 40 children younger than 18 years) were included, 22 (32.4%) presented with extramedullary diseases (EMD), bone marrow blasts in patients without EMD varied between 5%-96.5%, 31 (45.6%) patients had an allogeneic transplantation, 54 (79.4%) cases received CD19-specific and 14 (20.6%) received CD22-specific CAR-T therapy. Forty-two (61.8%) cases, including all (10) of grade III CRS, 68.2% (30/44) of grade II CRS and 2 patients with no CRS but with GVHD (1 case) or neurotoxicity (1 case), were administered steroids, among them, 23/42 (54.8%) received high dose steroids (&amp;gt;10mg/m2/d dexamethasone or equivalent), the duration of steroid use was 1-16 days (78.6% &amp;lt;= 7 days); whereas 26 (38.2%) patients were not given any steroids but the supportive care. We found that there was no difference either in complete remission (CR) rate (95.2% vs 92.3%, p=.344) or in MRD negative CR rate (80.0% vs 79.2%, p=.249) between steroid and non-steroid group, verified that corticosteroids even high dose steroids did not influence the treatment response. Furthermore, we investigated the dynamics of CAR-T cells. Firstly, the expansion of CAR-T cells in peripheral blood (PB) was evaluated, the average CAR-T cell counts in steroid group were significantly higher than those in non-steroid group on D11 (p=.0302), D15 (p=.0053), D20 (p=.0045) and D30 (p=.0028), except for D7 when CAR-T cells began to expand (p=.9815), this demonstrated that steroids did not suppress the proliferation of CAR-T cells in PB. Secondly, the percentages of patients with detectable CAR-T cells in bone marrow (BM) and cerebrospinal fluid (CSF) were compared between steroid and non-steroid group, there were no differences both in BM (85.2% vs 78.6%, p=.923) and in CSF (68.6% vs 57.9%, p=.433), which implied steroids did not influence the trafficking of T-cells to BM and CSF. Thirdly, we monitored B-cell aplasia (BCA) in part of patients followed-up more than 2 months without further treatments, the percentages of patients with BCA in steroid group had no significant differences compared to non-steroid group at 2-month (p=.086) and 3-month (p=.146). Later, although limited cases left, in the steroid group, 100% of patients (4-month, 7/7; 5-month, 7/7; 6-month, 5/5) still maintained BCA and CR, indicating that corticosteroids did not impact the duration of functional CAR-T cells. In conclusion, corticosteroids do not compromise the treatment efficacy and kinetics of CAR-T cells, could be as a feasible and effective approach to manage CAR-T associated CRS. Disclosures No relevant conflicts of interest to declare.

Phase 1 Trial of the Safety and Efficacy of Fully Human Anti-Bcma CAR T Cells in Relapsed/Refractory Multiple Myeloma

Background: B cell maturation antigen (BCMA) is a potential therapeutic target in multiple myeloma. CT053 CAR-BCMA, which is a BCMA-specific chimeric antigen receptor (CAR) T cell, consists of autologous T cells genetically modified with a second-generation CAR incorporating a fully human anti-BCMA single chain fragment variant, a 4-1BB co-stimulatory domain and a CD3-zeta signaling domain. CT053 was studied in a single-arm, open-label phase I program (NCT03716856, NCT03302403, and NCT03380039) in eastern China. Methods: This investigation is a 3-site study in adult subjects with relapsed/refractory multiple myeloma (rrMM) who had received at least 2 prior myeloma regimens. Subjects received one cycle of CT053 CAR-BCMA after fludarabine/cyclophosphamide infusion. The primary objective was the assessment of subject safety. The secondary objectives were pharmacokinetics and efficacy. Efficacy was assessed according to IMWG 2016 criteria. The data cutoff date was June 30, 2019. Results: A total of 24 subjects with median age of 60.1 years (range, 38.5-69.9) were enrolled from Sep 10, 2017 to Sep 22, 2018 (Table 1). The subjects had a median of 4.5 (range, 2-11) prior regimens of therapy, and 41.7% (10/24) underwent autologous stem cell transplantation. At baseline, eleven out of 24 subjects (45.8%) had concomitant extramedullary involvement. Eight subjects (33.3%) had ECOG score 2-3, and 9 subjects (37.5%) reported ISS grade III. All subjects received lymphodepletion preconditioning of fludarabine/cyclophosphamide for 2-4 days, followed by one cycle of CT053 at a dose of 1.5 x 108 T cells except 3 subjects who received 0.5 x 108, 1 x 108, and 1.8 x 108 cells, respectively. The overall response rate was 87.5% (21/24) including 79.2% (19/24) with complete responses or stringent complete response (5 CR, 14 sCR). As shown in Figure 1, P1 who received the lowest dose of 0.5 x 108 experienced partial response (PR) at M1 and very good partial response (VGPR) at M2. P1 then converted to CR at M14 and sCR at M16. Among 20 subjects who underwent the evaluation of minimal residual disease (MRD) status, 17 achieved MRD-negative (≤10−4 nucleated cells) and reported a tumor response (17 CR/sCR). In 13 subjects with ongoing CR/sCR, the median follow-up after CT053 infusion was 383 days (range, 301-467). Nine subjects progressed with median progression-free period of 281 days (range, 57-573); among them, 5 progressed within 6-12 months, 1 at 13 months and 1 at 19 months. Compared to 13 subjects with persistent CR/sCR, the 9 progressed subjects were observed to have a higher percentage of ECOG score 2-3 (66.7% vs 15.4%), ISS Grade III (55.6% vs 15.4%), and concomitant extramedullary diseases (66.7% vs 38.5%) and a decreased hemoglobin (70g/L vs 92g/L) at baseline. Three subjects died of disease progression at the time of analysis. Hematologic toxic effects were the most common treatment-related adverse events of grade (G) 3 or higher, including white blood cell count decreased (87.5%), neutrophil count decreased (66.7%), lymphocyte count decreased (79.2%) and thrombocytopenia (25%). No dose limiting toxicities were observed. Low-grade cytokine release syndrome (CRS) was reported in 15 of 24 (62.5%) subjects. All CRS events (3 G1, 12 G2) recovered within 2-8 days; among them 8 received tocilizumab. Three subjects (12.5%) had neurotoxicity (2 G1, 1 reversible G3). One subject died of bone marrow failure and neutropenic infection. CAR-BCMA T cell expansion was detected as early as Day 1-7 after infusion and reached peak values on Day 7-21 with the highest at 4.5×105 copies/µg genomic DNA. Median T cell persistence was 172 days. The longest persistence of CAR-BCMA copies was measured at 341 days and continues. Conclusion: This study demonstrated that CT053 had an excellent efficacy and a good safety profile in subjects with rrMM. Disclosures Li: CARsgen Therapeutics Co. Ltd: Employment, Equity Ownership. Xiao:CARsgen Therapeutics Co. Ltd: Employment. WANG:CARsgen Therapeutics Co. Ltd: Employment. Yuan:CARsgen Therapeutics Co. Ltd: Employment. Ma:CARsgen Therapeutics Co. Ltd: Employment.

PS1213 SEQUENTIAL CD19‐ AND CD22‐DIRECTED CAR‐T CELL THERAPIES IN 11 REFRACTORY OR RELAPSED B‐ALL CHILDREN PATIENTS

Background:Despite promising efficacy of CD19‐directed chimeric antigen receptors (CD19 CAR) T‐cell therapy in refractory or relapsed B acute lymphoblastic leukemia (r/r B‐ALL), 1‐year relapse rate is still high. The furtherly optimized therapeutic strategies are needed for these patients.Aims:To evaluate the efficacy and safety of sequential CD19‐ and CD22‐directed CAR‐T cell therapies in r/r B‐ALL children who did not received CAR therapy or transplantation previously.Methods:Between August, 2017 and January, 2018, 11 pediatric patients (pts) with r/r B‐ALL were enrolled in Beijing Boren Hospital to receive sequential CD19‐ and CD22‐directed CAR T‐cell therapies. There were 5 males and the median age was 8 (5–13) years old. Seven pts were relapsed diseases (with previous regimens of 22 (7–95) months of treatment) and 4 pts were refractory minimal residual disease by flow cytometry (FCM‐MRD+) (with previous regimens of 18 (3–47) months of treatment)). Extramedullary diseases (EMDs) were commonly observed in 3/7 relapsed pts including 2 with exclusive EMDs. The median marrow blast of 5.58% (rang, 0% to 93.18%) blasts by FCM. Complex chromosome aberration and high‐risk gene mutations were commonly observed including TP53. All pts had CD19 and CD22 expression on blasts. Two Lentiviral vectors were constructed to carry a second‐generation CAR including murine anti‐CD19 or humanized anti‐CD22 ScFv fused to 4–1BB co‐stimulatory and CD3z signaling domains. Manufacture of CAR T cells from PBMCs commenced on day 0 of leukapheresis and was completed in 7–8 days, and both protocols of 19‐CART and 22‐CART manufacture were the same. The doses of CD19 CAR T cells (cycle 1) and CD22 CAR T cells (cycle 2) infused were less than 5 × 106/kg. The duration between 2 cycles were less than 6 months. The CD19 CAR T‐cell and CD22 CAR T‐cell expansion and cytokine releasing syndrome (CRS) were monitored after infusion. The blast in BM was evaluated on day 30 (cycle 1), and EMDs were examined by imaging tests on day 30 (cycle 1). Long term follow‐up was carried out from the date of infusion of CD22 CAR T cells (cycle 2).Results:The median dose of CD19 CAR T cells (cycle 1) infused were 10 (3.3 to 42.8) × 105/kg and CD22 CAR T cells (cycle 2) infused were 5 (0.25 to 47.4) × 105/kg (P = 0.766). The median duration time between 2 cycles was 2.8 (1.3 to 5.2) m. Peak expansion of CD19 CAR T cells (10.63%, range 0.2 to 68.7% CAR T/CD3+) and CD22 CART cells (19.5%, range 0.3 to 47.9% CART/CD3+) was detected at day 10 (cycle 1) and 14 (cycle 2). All pts survived for 30 days or longer and were evaluated for response, 11 (100%) achieved CR/CRi and FCM‐MRD− after cycle 1. EMDs disappeared after cycle 1(Figure 1). CRS occurred in 10/11 (90.9%) pts in cycle 1 and 6/11 (54.5%) pts in cycle 2. Grade 1 neurotoxicity occurred in 1/11 pts in cycle 1 and 2/11 pts in cycle 2. Only 1 pt had grade 3 neurotoxicity in cycle 1 and fully recovered after interventions. With a median follow‐up time of 7 (2.5 to 14) months, 1 pt with TP53 mutation had relapsed on 9.5m, and 1‐year DFS was 50%.Summary/Conclusion:Our study demonstrated that sequential CAR T‐cell therapies are effective and safe for r/r B‐ALL children who can not be bridged to transplantation and achieved improved 1‐year DFS. Future trials based on larger population and longer follow‐up period are needed.image