Search

Refractory and/or relapsed (r/r) diffuse large B-cell lymphomas after treatment with two lines of systemic chemoimmunotherapy exhibit diversity in genetics, tissue biology, and pathology, as well as poor prognosis. Patient TCRαβ cells engineered with a CD19-specific chimeric antigen receptor (CAR) have shown promising clinical outcomes in r/r diffuse large B-cell lymphoma. The ZUMA-1 study, the JULIET study, and the TRANSCEND NHL 001 study of three prototype 19CAR-T cells have indicated an overall response rate of 52–82%, a complete response rate of 40–58%, and a 12-month progression-free survival of 33.2%–46.6%, with clinically manageable treatment related toxicity. At the 5-year follow-up, relapse was observed in approximately 57% of patients within 1 year. Understanding of the risk factors for non-response remains insufficient. In addition to intrinsic tumor resistance, such as aberrant apoptotic signaling, downregulation or loss of tumor-associated antigens (TAA), an immunosuppressive tumor microenvironment, and CAR-T cell exhaustion in vivo have been suggested to be important risk factors. Mechanisms underlying 19CAR-T cell exhaustion under chronic TAA exposure, and limited 19CAR-T cell trafficking and infiltration into the tumor mass have been reported. Moreover, tumor escape in the presence of low TAA density remains a challenge in 1928ζ CAR-T cell treatment. In this review, we provide an overview of modified modular CAR elements and their synergistic effects in controlling T-cell function. We then briefly discuss novel strategies against tumors with low TAA density, such as bispecific tandem or loop CAR recognition domains, the development of human leukocyte antigen-independent synthetic TCRαβ double-chain receptors integrated into the constant region of the TCRα chain, and armored CAR-T cells targeting the tumor microenvironment.

PURPOSEPatients with relapsed or refractory T-cell acute lymphoblastic leukemia (r/r T-ALL) have few options and poor prognosis. The aim was to assess donor-derived anti-CD7 chimeric antigen receptor (CAR) T-cell safety and efficacy in patients with r/r T-ALL.METHODSIn this single-center, phase I trial, we administered anti-CD7 CAR T cells, manufactured from either previous stem-cell transplantation donors or new donors, to patients with r/r T-ALL, in single infusions at doses of 5 × 105or 1 × 106(±30%) cells per kilogram of body weight. The primary end point was safety with efficacy secondary.RESULTSTwenty participants received infusions. Adverse events including cytokine release syndrome grade 1-2 occurred in 90% (n = 18) and grade 3-4 in 10% (n = 2), cytopenia grade 3-4 in 100% (n = 20), neurotoxicity grade 1-2 in 15% (n = 3), graft-versus-host disease grade 1-2 in 60% (n = 12), and viral activation grade 1-2 in 20% (n = 4). All adverse events were reversible, except in one patient who died through pulmonary hemorrhage related to fungal pneumonia, which occurred at 5.5 months, postinfusion. Ninety percent (n = 18) achieved complete remission with seven patients proceeding to stem-cell transplantation. At a median follow-up of 6.3 months (range, 4.0-9.2), 15 remained in remission. CAR T cells were still detectable in five of five patients assessed in month 6, postinfusion. Although patients' CD7-positive normal T cells were depleted, CD7-negative T cells expanded and likely alleviated treatment-related T-cell immunodeficiency.CONCLUSIONAmong 20 patients with r/r T-ALL enrolled in this trial, donor-derived CD7 CAR T cells exhibited efficient expansion and achieved a high complete remission rate with manageable safety profile. A multicenter, phase II trial of donor-derived CD7 CAR T cells is in progress ( NCT04689659 ).

Although recent clinical trials have demonstrated the efficacy of CD19-directed chimeric antigen receptor (CAR) T-cell therapy for refractory or relapsed B acute lymphoblastic leukemia (r/r B-ALL), most trials exclude patients with high-burden CNS leukemia (CNSL) to avoid the risk of severe neurotoxicity. There were only sparse cases describing the effect of CAR T cells on low-burden CNSL, and the safety and effectiveness of CAR T cells in high-burden CNSL remains unknown. Here, we retrospectively analyzed the results of CD19 CAR T-cell therapy in 12 pediatric patients that had low (Blasts 5/μL blasts in CSF or a solid mass before CAR T-cell expansion, four developed severe (grade 3–4) neurotoxicity featured by persistent cerebral edema and seizure, and they fully recovered after intensive managements. Sustained remission was achieved in 9 of the 12 patients, resulted in a 6-month leukemia-free survival rate of 81.8% (95% CI 59.0–100). Only one patient has CNS relapse again. Our study demonstrates that CAR T cells are effective in clearing both low- and high-burden CNSL, but a high CNSL burden before CAR T-cell expansion may cause severe neurotoxicity requiring intense intervention.

Cytokine release syndrome (CRS) and immune effector cell‐associated neurotoxicity are two major CAR T related toxicities. With the interventions of Tocilizumab and steroids, many patients can recover from severe CRS. However, some patients are refractory to steroids and develop life‐threatening consequences. Ruxolitinib is an oral JAKs inhibitor and promising drug in inflammatory diseases. In this pilot study, we evaluate the efficacy of Ruxolitinib in CRS. Of 14 r/r B‐ALL children who received CD19 or CD22 CAR T cell therapies, 4 patients developed severe (≥grade 3) CRS with symptoms that were not alleviated with high‐dose steroids and thus received ruxolitinib. Rapid resolution of CRS symptoms was observed in 4 patients after ruxolitinib treatment. Serum cytokines significantly decreased after ruxolitinib intervention. All patients achieved complete remission on day 30 after infusion, and we could still detect CAR T expansion in vivo despite usage of ruxolitinib. There were no obvious adverse events related to ruxolitinib. In vitro assays revealed that ruxolitinib could dampen CAR T expansion and cytotoxicity, suggesting that the timing and dosage of ruxolitinib should be carefully considered to avoid dampening anti‐leukaemia response. Our results suggest that ruxolitinib is active and well tolerated in steroid‐refractory and even life‐threatening CRS.

Objective. The objective is to explore the effectiveness and safety of CAR T-cell therapy in advanced relapsed/refractory central nervous system B-cell lymphoma and compare the impact of autologous stem cell transplantation (ASCT) plus CAR T-cell therapy versus sequential CART therapy on the survival of patients. Methods. The retrospective analysis was based on the data of 17 patients with advanced relapsed/refractory central nervous system B-cell lymphoma. Bridging chemotherapy was applied before CAR T-cell infusion to further reduce the tumor burden. For patients with autologous hematopoietic stem cell successful collection, CD19/20/22CAR T-cell immunotherapy following ASCT was performed with the thiotepa-containing conditioning regimen, while sequential CD19/CD20/CD22CAR T-cell therapy was applied. For lymphodepletion, patients received bendamustine or fludarabine monotherapy or fludarabine combined with cyclophosphamide pre-CART-cell infusion. Results. Out of the 17 patients, 8 completed ASCT plus CART cell therapy, while 9 patients completed CART cell alone therapy. In efficacy assessment at 3 months after infusion, the objective response rate (ORR) was 12/17 (71%) and the complete response rate (CRR) was 11/17 (65%). The CRR of the ASCT group and non-ASCT was 100% and 44.4%, respectively ( P < 0.01 ). The median progression-free survival was 16.3 (2.6–24.5) months, and the median overall survival was 19.3 (6–24.5) months. Patients who underwent ASCT plus CART cell therapy had significantly longer PFS ( P < 0.01 ) and OS ( P < 0.01 ). Grade 3 or higher immune effector cell-associated neurologic toxicity syndrome (≥grade 3 ICANS) and cytokine release syndrome (≥grade 3 CRS) events occurred in 29% and 41% of the patients, respectively. No treatment-related death occurred. Conclusion. The CAR T-cell therapy could augment its efficacy in the treatment of advanced relapsed/refractory CNS B-cell lymphoma, while ASCT in combination with CART can induce durable responses and OS with a manageable side effect.

Pan and colleagues report one of the first prospective evaluations of planned sequential chimeric antigen receptor (CAR) T-cell therapy targeting CD19 and then CD22 in a phase 1 trial, indicating acceptable toxicity and encouraging durable efficacy in pediatric patients with relapsed/refractory (r/r) acute lymphoblastic leukemia (ALL).

Despite worldwide promising clinical outcome of CD19 CAR-T therapy, relapse after this therapy is associated with poor prognosis and has become an urgent problem to be solved. We conducted a CD22 CAR T-cell therapy in 34 relapsed or refractory (r/r) B-ALL pediatric and adult patients who failed from previous CD19 CAR T-cell therapy. Complete remission (CR) or CR with incomplete count recovery (CRi) was achieved in 24 of 30 patients (80%) that could be evaluated on day 30 after infusion, which accounted for 70.5% of all 34 enrolled patients. Most patients only experienced mild cytokine-release syndrome and neurotoxicity. Seven CR patients received no further treatment, and 3 of them remained in remission at 6, 6.6, and 14 months after infusion. Eleven CR patients were promptly bridged to transplantation, and 8 of them remained in remission at 4.6 to 13.3 months after transplantation, resulted in 1-year leukemia-free survival rate of 71.6% (95% CI, 44.2–99.0). CD22 antigen loss or mutation was not observed to be associated with relapsed patients. Our study demonstrated that our CD22 CAR T-cells was highly effective in inducing remission in r/r B-ALL patients, and also provided a precious window for subsequent transplantation to achieve durable remission.

The prognosis of relapsed acute lymphoblastic leukemia (ALL) after allogeneic transplantation is dismal when treated with conventional approaches. While single-target CD19 or CD22 chimeric antigen receptor (CAR) T-cell therapy has achieved high complete remission (CR) rates in refractory/relapsed B-ALL, it could not maintain a durable remission in most patients. To prolong relapse-free survival, we sequentially combined CD19 and CD22 CAR-T cells to treat post-transplant relapsed B-ALL patients with both CD19/CD22 antigen expression on lymphoblasts. Patient-derived donor cells were collected to produce CAR-T cells that were transfected by lentiviral vectors encoding second generation CARs composed of CD3ζ and 4-1BB. The second T-cell infusion was scheduled at least 1 month, and usually within 6 months after the first CAR-T treatment. Twenty-seven adult and pediatric patients, including 11 (41%) with extramedullary diseases (EMD), received the first CD19 CAR-T and 23 (85%) achieved CR. Subsequently, 21 out of 27 patients received the second CD22 CAR-T and were followed-up for a median of 19.7 (range, 5.6-27.3) months; 14 cases remained in CR, seven relapsed and two of them died from disease progression; Kaplan-Meier survival analysis showed overall survival and event-free survival rates of 88.5% and 67.5%, respectively, at both 12 months and 18 months. CAR-T associated graft-versus-host disease (GVHD) occurred in 23% of patients, with 8% new-onset acute GVHD and 15% persistent or worsened pre-existing cGVHD before CAR-T. This combination strategy of sequential CD19 and CD22 CAR-T therapy significantly improved the long-term survival in B-ALL patients who relapsed after transplantation.

7035 Background: Refractory or relapsed (r/r) T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) has a poor prognosis. Our previous report showed that donor-derived CD7 CAR-T cell therapy could induce remission of r/r T-ALL, but graft-versus-host disease (GVHD) occurred in some patients (Pan et al. J Clin Oncol 2021;39:3340-3351). We recently conducted a phase I trial to evaluate autologous CD7 CAR-T therapy in r/r T-ALL/LBL, which may avoid GVHD while showing the efficacy; here we report the early result with the approval of the Data and Safety Monitoring Committee and Institutional Review Board (IRB). Methods: The patients who had CD7+ r/r T-ALL/LBL and no leukemia cells in peripheral blood were eligible for this phase I trial (NCT04840875). The CD7 CAR construct included an endoplasmic reticulum anchor domain fused to a CD7 binding domain to prevent CD7 expression on cell surface, which contributed to minimize CAR T cell fratricide. CAR T product was checked to ensure lack of tumor contamination before infusion. The trial followed a “3+3” dose escalation process. Adverse events and efficacy were primary and secondary endpoints. Results: A total of 5 patients (Pt1-5), with a median age of 3.8 years (range, 1.9-13), were enrolled between Sept 2021 and Jan 2022. At enrollment, Pt1 had mediastinal mass and blasts in pleural fluid; Pt4 was in central nervous system (CNS)-3 status; Pt2, 3 and 5 had marrow disease with a median burden of 1.35% (range, 0.07%-7.31%). Pt1, 2 and 3 received 5×105 (±20%) cells/kg; Pt5 received 1×106 (±20%) cells/kg; Pt4 received cells below the target dose. Three patients had cytokine release syndrome (CRS), including one grade 3; the median onset was on day 5 (range, 1-9) with the median duration of 4 days (range, 3-14). No patient had neurotoxicity, GVHD or infection. Grade 3-4 hematological toxicity occurred in all five patients, which recovered to grade 2 within 30 days. On one month post-infusion, four patients achieved complete remission, and one patient (Pt4) still had leukemia cells in the cerebrospinal fluid. With the median follow-up time of 62 days (range, 35-136), one patient (Pt1) underwent stem cell transplantation (SCT) on 2.9 months post-infusion, and had a CD7- relapse on 1.4 months post-SCT; the other three responders remained in MRD- CR. In the four patients who received target dose, the median peak CAR T cell count in peripheral blood was 4.27×102/uL (range, 2.49-5.61) by flow cytometry, and all five patients had detectable CAR transgene by PCR on their last visits. There was a decrease of CD7-positive normal T cells and an increase of CD7-negative counterparts in all responders. Conclusions: Autologous CD7 CAR T cell therapy was safe and effective in the induction of remission in r/r T-ALL/LBL patients, without signs of GVHD. Longer follow-up time of more cases will be used to further evaluate this therapy. Clinical trial information: NCT04840875.

7028 Background: Despite the manageable safety and encouraging efficacy of donor-derived CD7 chimeric antigen receptor (CAR) T cells in relapsed or refractory T-cell acute lymphoblastic leukemia (r/r T-ALL) (Pan et al. J Clin Oncol 2021;39:3340-3351), a considerable proportion of responding patients eventually relapsed with CD7 antigen loss. CAR T cells targeting another antigen, CD5, which is expressed on blasts of over 80% T-ALL cases, may be capable of treating these patients. Here we present early safety and efficacy results of a phase I trial of donor-derived CD5 CAR T cells in T-ALL. Methods: CD5 CAR T cells that resist fratricide by deletion of CD5 gene (Preclinical data in Dai et al. Mol Ther 2021;29:2707-2722) were manufactured. Patients with prior stem cell transplantation (SCT) (group A) received CAR T cells from prior SCT donors, while patients without SCT history (group B) received CAR T cells from new donors who also provided stem cells for transplantation post CAR T therapy. The trial using bayesian optimal interval phase I/II design to explore optimal biological dose (OBD) from the initial dose of 1×106 (±20%) CAR T cells/kg in each group. If manufactured cells were not sufficient, patients could be treated at a low dose of 5×105 (± 20%)/kg. The primary endpoint was safety with efficacy secondary. Results: Five patients who had CD7-negative relapsed after CD7 CAR therapy were enrolled and received prior SCT donor-derived CD5 CAR T cells between Oct 8th, 2021 and Dec 14th, 2021. Four patients were on initial dose level, while one patient was on low dose level. This early report has approved by the Data and Safety Monitoring Committee, and indicated the OBD in group A was 1×106 (±20%) /kg. No DLT occurred. Adverse events within 30 days included grade 3-4 hematologic toxicity in all (100%) which already existed before enrollment, grade 1-2 cytokine release syndrome in 4 (80%), grade 1 graft-versus-host disease in 1 (20%), grade 2 rash maculo-papular in 3 (60%). One patient developed late grade 5 epstein-barr virus infection accompanied by hemophagocytic lymphohistiocytosis at 2.7 months post-infusion. All five patients achieved complete remission at day 30, and remained MRD-negative with a median follow-up time of 2.7 (range, 1.8-4.1) months. In three patients, low level of CD7 CAR T cells were still detectable for one month post-infusion, but CD5 CAR T cells dominated and persisted well until last visit. Patient CD5-positive healthy T cells were depleted, while CD5-negative T cells increased to a median count of 133 (range, 13-299)/μl at one month, although they were still much lower than normal level. Conclusions: We report the safety and efficacy of donor-derived CD5 CAR T cells in r/r T-ALL. And the depletion of CD5-positive healthy T cells was commonly observed. Longer follow-up assessment is needed to determine the durable remission and the functional immune system reconstitution. Clinical trial information: NCT05032599.

7023 Background: Results of a phase I trial of donor-derived CD7 chimeric antigen receptor (CAR) T cells for relapsed or refractory T-cell acute lymphoblastic leukemia (r/r T-ALL) (Pan et al. J Clin Oncol 2021;39:3340-3351) have been reported previously, and are updated now. Methods: The target dose is 1 × 106 (±30%) CAR T cells per kg of body weight. Patients with prior stem cell transplantation (SCT) received CAR T cells from prior SCT donors, while patients without SCT history received CAR T cells from new donors who also provided stem cells for transplantation post CAR T therapy. The primary endpoint was safety with efficacy secondary. Survival status were continuously followed up while severe adverse events (SAEs) were recorded until receiving other anti-leukemia therapy. Results: Nineteen (95%) of twenty enrolled patients responded and were followed up with a median time of 15.8 months (range 13-18.3) until Feb, 14th, 2022. Short-term adverse events included grade 3 or higher cytokine release syndrome (10%) and grade 1-2 graft-versus-host disease (GVHD, 60%), which were all reversible. Six late-onset ( > 30 days post-infusion) severe adverse events (SAEs) occurred in 5 responders. Two had been reported previously. Four SAEs were newly observed, including a grade 4 intestinal GVHD at month 11 and a grade 5 pneumonia at month 12.3 in one patient, a grade 5 Pseudomonas Aeruginosa pneumonia at month 8.7 in one patient, and a grade 3 cytomegalovirus (CMV) encephalitis at month 11 which recovered at month 13.3 in another patient. All severe infections occurred in patients with no further therapy, and the total T cells in them reached a median count of 300.03/μL (range 121.46-512.83), which were substantially lower than normal levels despite steadily increasing. The objective response and complete remission rate was 95% and 85% at day 30 post-infusion. Of 19 responders that were followed up, two (11%) withdrew for other therapy at day 55 and 271 respectively. Of 10 (53%) patients who received no further therapy, all had continuously detectable CAR T cells until the last visit, three remained in remission, three had a relapse (one CD7+, and two CD7-), and four died of infection; Seven (37%) patients proceeded to SCT and no CAR T cells were detectable after SCT, and among them two remained in remission, four had a relapse (three CD7+, and one CD7-), and one died of transplant-related mortality. Patients relapsed at a median time of 6 (range 4-10.9) months. The one-year progressive-free survival (PFS) and overall survival (OS) rates were 51.6% (95% CI, 24.7-78.4%) and 72.5% (95% CI, 51.9-93.0%). Conclusions: Donor-derived CD7 CAR T cell therapy showed encouraging activity in treating r/r T-ALL. Relapse emerges as major issues impeding long-term outcomes. CD7-negative relapse was commonly observed under CAR T cell surveillance. Late onset GVHD and infections may occur and should be carefully managed. Clinical trial information: ChiCTR2000034762.

BackgroundCD19- and/or CD22-targeted chimeric antigen receptor (CAR) T cells efficiently induced remission in patients with B acute lymphoblastic leukemia (B-ALL), but a considerable proportion of patients relapsed after both CD19- and CD22-CAR therapies associated with the loss or downregulation of target antigen. Re-infusions of the prior used CAR T cells were usually ineffective. In contrast to the frequent loss of CD19, low level of CD22 is usually present on leukemia cells post CAR therapy, suggesting that newly designed CD22-CAR therapies may be effective in these patients.MethodsA yeast full-human single-chain variable fragment (scFv) library and a high-throughput NFAT reporter assay were utilized to screen several full-human CD22-CAR candidates; CD107 assay and in vitro cytotoxicity assay was used to evaluate the effector function of CAR T cells; membrane proteome assay was conducted to determine the specificity of the CAR toward the target antigen; a leukemia animal models was used to test the in vivo efficacy of CAR T cells. A phase I trial (ChiCTR2000028793) was conducted to assess the safety and effectiveness of CD22-CARFH80 therapy in 8 children with B-ALL resistant to or relapsed after prior CD19- and CD22-CAR treatment.ResultsWe identified a full-human CD22-CAR construct termed CD22CARFH80 which could mediate superior anti-leukemia activity in vitro and in a leukemia animal model and had good specificity to the target antigen. Data from the trial showed that with CD22-CARFH80 T-cell therapy, 6/8 (75%) patients including 2 who had CD22low blasts achieved complete remission; 1 patient had a partial response. CAR T cells efficiently expanded in vivo, while the toxic effect is low in most patients. At a median follow-up of 5 months, 4/6 (57%) patients remained in remission.ConclusionsTherapy with a newly invented CD22-CARFH80 overcomes the resistance to prior versions of CD19- and CD22-CAR formats and elicits potent anti-leukemia responses with an acceptable safety profile, representing a promising salvage regimen for B-ALL that fails in prior CD19- and CD22-CAR treatments.Trial registrationClinicalTrials.gov: ChiCTR2000028793; registered 4 January, 2020. http://www.chictr.org.cn/showproj.aspx?proj=47857

Chimeric antigen receptor (CAR)-T cell therapy emerges as a new treatment for refractory or relapsed (r/r) B-cell non-Hodgkin lymphoma (B-NHL); however, the overall response rate (ORR) of which in the B-NHL patients is much lower compared to the patients with r/r B acute lymphoblastic leukemia (B-ALL). We previously confirmed that sequential infusions of CD20 and CD22 CAR-T cells significantly improved the prognosis of the B-NHL patients, while some advanced patients still progressed to death during these CAR-T cell treatments. In this study, we showed that timely sequential administration of the second CAR-T cells could enhance expansion of prior CAR-T cells with stronger tumor-killing capacity in vitro and in vivo. We further conducted compassionate treatments on two advanced B-NHL patients with short-interval sequential infusions of CD19/22/20 CAR-T cells. Disease progression was observed in both patients after primary CAR-T cell infusion but robust re-expansion of prior CAR-T cells and anti-tumor effects was induced by infusion of a secondary CAR-T cells. These results indicate sequential infusions of CAR-T cells with a short interval may improve therapeutic efficacy in the B-NHL patients by promoting expansion of prior CAR-T cells.

Although recent clinical trials have demonstrated the efficacy of CD19-directed chimeric antigen receptor (CAR) T-cell therapy for refractory or relapsed B acute lymphoblastic leukemia (r/r B-ALL), most trials exclude patients with high-burden CNS leukemia (CNSL) to avoid the risk of severe neurotoxicity. There were only sparse cases describing the effect of CAR T cells on low-burden CNSL, and the safety and effectiveness of CAR T cells in high-burden CNSL remains unknown.Here, we retrospectively analyzed the results of CD19 CAR T-cell therapy in 12 pediatric patients that had low (Blasts 20/μL in CSF) or high-burdens (Blasts or intracranial solid mass) of CNS B-ALL, that are enrolled in three clinical trials and one pilot study at Beijing Boren Hospital RESULTS: Eleven patients (91.7%) achieved complete remission (CR) on day 30, and one patient got CR on day 90 after infusion. Most patient experienced mild cytokine-release syndrome. However, of the five patients who retained 5/μL blasts in CSF or a solid mass before CAR T-cell expansion, four developed severe (grade 3-4) neurotoxicity featured by persistent cerebral edema and seizure, and they fully recovered after intensive managements. Sustained remission was achieved in 9 of the 12 patients, resulted in a 6-month leukemia-free survival rate of 81.8% (95% CI 59.0-100). Only one patient has CNS relapse again.Our study demonstrates that CAR T cells are effective in clearing both low- and high-burden CNSL, but a high CNSL burden before CAR T-cell expansion may cause severe neurotoxicity requiring intense intervention.

INTRODUCTION The phase 1/2 clinical trials of CD19 CAR T-cell that led to US Food and Drug Administration approval excluded patients with central nervous system (CNS) involvement, due to concerns for CAR T-cell related neurotoxicity. The clinical trial (ChiCTR1800014457) examined efficacy and safety of sequential different B cell antigen-targeted CAR T-cell therapy for pediatric refractory/relapsed (r/r) Burkitt Lymphoma with secondary CNS involvement. METHODS Twenty-three patients up to 18 years of age with r/r Burkitt Lymphoma were enrolled between January 17, 2018 and November 8, 2019 including 10 patients with secondary CNS involvement. All enrolled patients received the first mCD19 CAR T-cell infusion. The patients who achieved an ongoing complete response (CR) did not receive further therapy; for patients who retained a partial response (PR) or no response (NR) until mCD19 CAR T cells became undetectable in PB by flow cytometry (FCM), the second hCD22 CAR T-cell infusion was initiated so that it did not prematurely interfere with the antitumor effect of mCD19 CAR T cells, and could prevent disease progression in the setting of continuous mCD19 CAR T-cell contraction; and for patients who had progressive disease (PD) or relapsed disease (RD) after responding to mCD19 CAR T cells, considering that the tumor had already produced immune escape from mCD19 CAR T cells, regardless of whether there were mCD19 CAR T cells detectable in PB by FCM, the second hCD22 CAR T-cell infusion was immediately initiated. After the second hCD22 CAR T-cell infusion, the patients who retained a PR until hCD22 CAR T cells became undetectable in PB by FCM received the third hCD20 CAR T-cell infusion; for patients who suffered relapses after achieving the first CR (CR1) following the first infusion and then attained the second CR (CR2) by the second infusion, the third hCD20 CAR T-cell infusion was given as consolidation therapy after loss of hCD22 CAR T cells detectable in PB by FCM, and the patients who had PD after having obtained a PR immediately received the third hCD20 CAR T-cell infusion even if hCD22 T cells were still detectable in PB by FCM. After the third hCD20 CAR T-cell infusion, the patients who retained a PR until hCD20 CAR T cells became undetectable in PB by FCM were given the fourth hCD19 CAR T-cell infusion. RESULTS The median time from the last infusion to cutoff date was 17 months (range, 15 to 23). At 18 months after the last infusion, all patients had an estimated PFS rate of 78% (95% CI, 55 to 90) and OS rate of 83% (95% CI, 60 to 93). Through mCD19 CAR T-cell expansion in the cerebrospinal fluids (CSF) of ten patients with CNS involvement, 8 patients achieved a CR, and 2 PR. Among the 8 patients who achieved a CR, 4 patients (P5, P15, P16, and P19) maintained an ongoing CR, whereas 4 patients (P7, P11, P17, and P21 ) developed a RD at m6, m2, m3, and m1.5 after infusion. P17 died of rapid intracerebral mass (ICM) progression, which resulted in elevated intracranial pressure (ICP), a contraindication to further CAR T-cell therapy. Two patients (P7 and P21) with CNS RD, P11 with bone marrow RD, and 2 patients (P13 and P20) with PR received the second hCD22 CAR T cells infusion. Among 5 patients experiencing 2 cycles of CAR T-cell infusion, 4 patients (P7, P13, P20, and P21) had hCD22 CAR T-cell expansion in CSF. P13 achieved an ongoing CR1; P20 and P21 died of ICM progression, who did not proceed with the third hCD20 CAR T-cell infusion due to elevated ICP and loss of CD20 in tumor cells, respectively; P7 and P11 achieved a CR2, who underwent the third hCD20 CAR T cells infusion, and then had hCD20 CAR T cells expansion in CSF. P11 achieved an ongoing CR2; P7 had a CNS RD again at m3 after obtaining a CR2 and finally died of rapid disease progression. Six patients achieved ongoing CR after the last infusion. The estimated 18-month PFS rate of 60% (95% CI, 25 to 83) in the patients with CNS involvement was not significantly lower than 92% (95% CI, 56 to 99) in the patients without CNS involvement (p=0.28 ). Grade 3 neurotoxicity occurred in 30% (3/10) of patients with CNS involvement. All adverse events were reversible. Statistically significant difference was not observed in the severity of neurotoxicity between patients with and without CNS involvement (p= 0.62). CONCLUSION Sequential CAR T-cell therapy may result in a durable response and is safe in pediatric r/r Burkitt lymphoma. Patients with secondary CNS involvement may benefit from sequential CAR T-cell therapy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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 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.

Refractory or relapsed B lymphoblastic leukemia (B-ALL) patients have a dismal outcome with current therapy. We treated 42 primary refractory/hematological relapsed (R/R) and 9 refractory minimal residual disease by flow cytometry (FCM-MRD+) B-ALL patients with optimized second generation CD19-directed CAR-T cells. The CAR-T-cell infusion dosages were initially ranged from 0.05 to 14 × 105/kg and were eventually settled at 1 × 105/kg for the most recent 20 cases. 36/40 (90%) evaluated R/R patients achieved complete remission (CR) or CR with incomplete count recovery (CRi), and 9/9 (100%) FCM-MRD+ patients achieved MRD-. All of the most recent 20 patients achieved CR/CRi. Most cases only experienced mild to moderate CRS. 8/51 cases had seizures that were relieved by early intervention. Twenty three of twenty seven CR/CRi patients bridged to allogeneic hematopoietic stem cell transplantation (allo-HCT) remained in MRD- with a median follow-up time of 206 (45-427) days, whereas 9 of 18 CR/CRi patients without allo-HCT relapsed. Our results indicate that a low CAR-T-cell dosage of 1 × 105/kg, is effective and safe for treating refractory or relapsed B-ALL, and subsequent allo-HCT could further reduce the relapse rate.

10001 Background: Despite the success of chimeric antigen receptor T cell therapy in B cell malignances, there is currently no proved CAR T treatment for T cell neoplasms. We provide first evidence support the use of donor derived CAR T cells in T cell leukemia. Methods: In this phase 1 trial, CD7 CAR T cells were manufactured with T cells from prior SCT prior to a single infusion at doses of 5 × 105 or 1 × 106 (± 30%) cells per kilogram of body weight. donors, or from new donors who were HLA-matched or haploidentical, via leukopheresis and transduced with a lentiviral vector which carries a CD7 CAR construct. The primary endpoint was safety. Short-term efficacy was also assessed. Results: Results of 20 enrolled patients who received infusion are reported. Of 20 patients, 12 received previous HSCT-donor derived CAR T cells and 8 received fresh haplo-identical donor derived CAR T cells and plan to received transplantation as consolidation after remission.Adverse events included grade 3-4 hematologic toxicity in all (100%), grade 3-4 and grade 1-2 cytokine release syndrome in 2 (10%) and in 18 (90%), grade 1 neurotoxicity in 3 (15%), grade 1-2 graft-versus-host disease in 12 (60%), and grade 1 viral activation in 3 (15%) patients. Nineteen (95%) patients had a response, including 18 (90%) with complete remission and 1 (5%) with partial remission. Of 19 responders, 7 were bridged to SCT and remained minimal residual disease (MRD)-negative until last visit; 12 were followed up at a medium of 4.4 months, among whom 9 remained MRD-negative, 1 had a relapse, 1 discontinued for other treatment, and 1 died of pulmonary fungal infection at 5.5 months. CAR cells mostly persisted beyond 3 months. Patient CD7-positive healthy T cells were depleted, while CD7-negative T cells increased. Conclusions: We report the initial toxicity profile and anti-leukemia activity of a donor-derived CD7-targeted cellular immunotherapy for patients with relapsed or refractory T-ALL. (Funded by the National Key R&D program; ChiCTR.org number, ChiCTR2000034762). Clinical trial information: ChiCTR2000034762. [Table: see text]

Despite worldwide promising clinical outcome of CD19 CAR-T therapy, relapse after this therapy is associated with poor prognosis and has become an urgent problem to be solved. We conducted a CD22 CAR T-cell therapy in 34 relapsed or refractory (r/r) B-ALL pediatric and adult patients who failed from previous CD19 CAR T-cell therapy. Complete remission (CR) or CR with incomplete count recovery (CRi) was achieved in 24 of 30 patients (80%) that could be evaluated on day 30 after infusion, which accounted for 70.5% of all 34 enrolled patients. Most patients only experienced mild cytokine-release syndrome and neurotoxicity. Seven CR patients received no further treatment, and 3 of them remained in remission at 6, 6.6, and 14 months after infusion. Eleven CR patients were promptly bridged to transplantation, and 8 of them remained in remission at 4.6 to 13.3 months after transplantation, resulted in 1-year leukemia-free survival rate of 71.6% (95% CI, 44.2-99.0). CD22 antigen loss or mutation was not observed to be associated with relapsed patients. Our study demonstrated that our CD22 CAR T-cells was highly effective in inducing remission in r/r B-ALL patients, and also provided a precious window for subsequent transplantation to achieve durable remission.

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 (>10mg/m2/d dexamethasone or equivalent), the duration of steroid use was 1-16 days (78.6% 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.

Introduction: Recently, chimeric antigen receptor (CAR) T-cell (CART) targeting CD19 has resulted in high complete remission (CR) rate in patients with refractory or relapsed B cell acute lymphoblastic leukemia (r/r B-ALL). Our previous clinical study has achieved more than 90% CR rate with CD19-CART in r/r B-ALL, but approximately 10% of patients have failed even CD19 high expression on their leukemia cells (Pan J, et al. Leukemia 2017; 31: 2587-2593). In current study, we investigate whether leukemia burden in peripheral blood (PB) has any influence in CART preparation ex vivo and its clinical efficacy. Patients and Methods: From June 2017 to June 2019, 143 r/r B-ALL patients received CD19-CART therapy. The median age was 8 years old (10 months to 65 years old). Thirty-six patients had leukemia cells in PB (positive group) and the median leukemia cells proportion after lymphocyte separation was 32.7% (3.23% to 83.2%); 107 patients had no leukemia cells detectable in PB (negative group). After apheresis, peripheral blood mononuclear cells (PBMCs) were isolated using lymphocyte separation liquid and leukemia cells proportion were determined by flow cytometry (FCM). PBMCs were activated with CD3 and CD28 antibodies for 24 hours, then transduced with the lentivirus encoding anti-CD19-CD3zeta-4-1BB CAR and cultured for 4-6 days in serum-free media containing IL2, IL7, IL15, IL21. Mycoplasma, endotoxin, bacteria and fungi were strictly controlled during the culture. Cell viability, expanding fold, percentage of CD3+ T cells, transduction efficiency and B cells were all examined before CART infusion. The patients received cyclophosphamide (250 mg/m2) and fludarabine (30 mg/m2) all for 3 days, and followed by CART infusion with a median number of 4.7 (0.6 to 43) X 105cells/kg. CD19-CART cells' expansion capacity in vivo was analyzed by FCM. Clinical efficacy was evaluated by bone marrow morphology and FCM on day 30. Results: Both normal B cells and leukemia B cells were all undetectable in infused CD19-CART. The median percentage of CD3+ T cells and transduction efficiency in positive and negative groups were 94.1% (80.3% to 98.0%) vs. 96.6% (80.1% to 99.5%) (P=0.056), and 35.6% (4.82% to 56.1%) vs. 52.4% (9.44% to 82%) (P Table Disclosures No relevant conflicts of interest to declare.

Based on the petrological studies of wall rocks, mineralized rocks, ores and veins from the Laowangzhai gold deposit, it is discovered that with the development of silification, carbonation and sulfidation, a kind of black opaque ultracrystalline material runs through the space between grains and amphibole cleavages, which is the product of fast condensing consolidation with magma mantle fluids turning into hydrothermal crustal fluids in the process of mineralization and alteration. It is thought that the water in ore-forming fluids mainly derived from magmatic water through research on H-O isotopes, and C as well as S isotopic compositions, has clear mantle-derived characteristics, and rock (mine) stones contain high 87Sr/86Sr ratios, low 143Nd/144Nd ratios and high 206Pb/204Pb ratios, which also reflects the ore-forming fluids were derived from the metasomatically enriched mantle. In combination with the features of H-O-C-S isotopes and Sr-Nd-Pb isotopes described above, the ore-forming fluids of the Laowangzhai gold deposit in the northern part of the Ailao Mountains were derived mainly from the deep interior of the mantle, and their properties were transformed from magma fluids to hydrothermal fluids in the course of metasomatism and alteration, which initiated crust-mantle contamination simultaneously to be in favor of mineralization.