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PURPOSE G protein–coupled receptor, class C group 5 member D (GPRC5D) is considered to be a promising surface target for multiple myeloma (MM) immunotherapy. Here, we report the efficacy and safety of anti-GPRC5D chimeric antigen receptor (CAR) T cells in patients with relapsed or refractory (R/R) MM. METHODS This phase Ⅱ, single-arm study enrolled patients (18-70 years) with R/R MM. Lymphodepletion was performed before patients received 2 × 106/kg anti-GPRC5D CAR T cells. The primary end point was the proportion of patients who achieved an overall response. Safety was also evaluated in eligible patients. RESULTS From September 1, 2021, to March 23, 2022, 33 patients were infused with anti-GPRC5D CAR T cells. At a median follow-up of 5.2 months (range, 3.2‐8.9), the overall response rate was 91% (95% CI, 76 to 98; 30 of 33 patients), including 11 (33%) stringent complete responses, 10 (30%) complete responses, four (12%) very good partial responses, and five (15%) partial responses. Partial responses or better were observed in nine (100%) of nine patients with previous anti–B-cell maturation antigen (BCMA) CAR T-cell therapy, including two patients who had received repeated anti-BCMA CAR T-cell infusions with no responses at the last time. Grade 3 or higher hematologic toxicities were neutropenia (33 [100%]), anemia (17 [52%]), and thrombocytopenia (15 [45%]). Cytokine release syndrome occurred in 25 (76%) of 33 patients (all were grade 1 or 2), and neurotoxicities in three patients (one grade 2 and one grade 3 ICANSs and one grade 3 headache). CONCLUSION Anti-GPRC5D CAR T-cell therapy showed an encouraging clinical efficacy and manageable safety profile in patients with R/R MM. For patients with MM that progressed after anti-BCMA CAR T-cell therapy or that is refractory to anti-BCMA CAR T cell, anti-GPRC5D CAR T-cell therapy might be a potential alternative option.

The considerable efficacy of B-cell maturation antigen-targeted chimeric antigen receptor (CAR)-T-cell therapy has been extensively demonstrated in the treatment of relapsed or refractory multiple myeloma. Nevertheless, in clinical practice, prolonged hematologic toxicity (PHT) extends hospital stay and impairs long-term survival.This retrospective study reviewed 99 patients with relapsed or refractory multiple myeloma who underwent B-cell maturation antigen CAR-T-cell therapy at our institution between April 2018 and September 2021 (ChiCTR1800017404).Among 93 evaluable patients, the incidence of prolonged hematologic toxicities was high after CAR-T-cell infusion, including 38.71% (36/93) of patients with prolonged neutropenia, 22.58% (21/93) with prolonged anemia and 59.14% (55/93) with prolonged thrombocytopenia. In addition, 9.68% (9/93) of patients experienced prolonged pancytopenia. Our multivariate analyses identified that cytokine profiles were independent risk factors for PHTs, whereas a sufficient baseline hematopoietic function and high CD4/CD8 ratio of CAR-T cells were protective factors for PHTs after CAR-T-cell infusion. Subgroup analyses found that the kinetics of post-CAR-T hematologic parameters were primarily determined by the collective effects of cytokine release syndrome and baseline hematopoietic functions, and showed influential weights for the three lineages.Our findings improve the understanding of the impact of cytokines on hematopoietic functions, which could contribute to the mechanism investigation and exploration of potential intervention strategies.

Background aimsB-cell maturation antigen (BCMA)-targeted chimeric antigen receptor-T cell (CAR-T) therapy is used for refractory or relapsed multiple myeloma (r/r MM). However, CAR-T-related tumor lysis syndrome (TLS) has been observed. We aimed to elucidate the incidence, clinical and laboratory characteristics, and prognosis of CAR-T cell-related TLS.MethodsPatients (n=105) with r/r MM treated with BCMA-targeted CAR-T cell therapy were included. Patient characteristics, laboratory parameters, and clinical outcomes were assessed.ResultsEighteen (17.1%) patients developed TLS after BCMA-targeted CAR-T cell therapy. The median time till TLS onset was 8 days. Patients with TLS had steep rise in uric acid (UA), creatinine, and lactate dehydrogenase (LDH) within 6 days following CAR-T cell infusion and presented earlier and persistent escalation of cytokines (C-reactive protein [CRP], interleukin-6 [IL-6], interferon-γ [IFN-γ], and ferritin levels). All 18 patients had cytokine release syndrome (CRS), of which 13 (72.2%) developed grade 3–4 CRS. Three of 18 patients (16.7%) developed immune effector cell-associated neurotoxicity syndrome (ICANS): two patients with grade 1 ICANS and one with grade 2 ICANS. TLS development had a negative effect on the objective response rate (77.8% in the TLS group vs. 95.4% in the non-TLS group, pConclusionTLS is a frequently observed CAR-T therapy complication and negatively influences clinical response and prognosis. Close monitoring for TLS should be implemented during CAR-T cell therapy, especially for those at high TLS risk.

Chimeric antigen receptor-T (CAR-T) therapy remains to be investigated in T-cell malignancies. CD7 is an ideal target for T-cell malignancies but is also expressed on normal T cells, which may cause CAR-T cell fratricide. Donor-derived anti-CD7 CAR-T cells using endoplasmic reticulum retention have shown efficacy in patients with T-cell acute lymphoblastic leukemia (ALL). Here we launched a phase I trial to explore differences between autologous and allogeneic anti-CD7 CAR-T therapies in T-cell ALL and lymphoma. Ten patients were treated and 5 received autologous CAR-T therapies. No dose-limiting toxicity or neurotoxicity was observed. Grade 1–2 cytokine release syndrome occurred in 7 patients, and grade 3 in 1 patient. Grade 1–2 graft-versus-host diseases were observed in 2 patients. Seven patients had bone marrow infiltration, and 100% of them achieved complete remission with negative minimal residual disease within one month. Two-fifths of patients achieved extramedullary or extranodular remission. The median follow-up was 6 (range, 2.7–14) months and bridging transplantation was not administrated. Patients treated with allogeneic CAR-T cells had higher remission rate, less recurrence and more durable CAR-T survival than those receiving autologous products. Allogeneic CAR-T cells appeared to be a better option for patients with T-cell malignancies.

Background Donor-derived CD7-directed chimeric antigen receptor (CAR) T cells showed feasibility and early efficacy in patients with refractory or relapsed T-cell acute lymphoblastic leukemia (r/r T-ALL), in a previous phase I trial report, at a median follow-up of 6.3 months. Here we report long-term safety and activity of the therapy after a 2-year follow-up. Methods Participants received CD7-directed CAR T cells derived from prior stem cell transplantation (SCT) donors or from HLA-matched new donors after lymphodepletion. The target dose was 1 × 106 (± 30%) CAR T cells per kg of patient weight. The primary endpoint was safety with efficacy secondary. This report focuses on the long-term follow-up and discusses them in the context of previously reported early outcomes. Results Twenty participants were enrolled and received infusion with CD7 CAR T cells. After a median follow-up time of 27.0 (range, 24.0–29.3) months, the overall response rate and complete response rate were 95% (19/20 patients) and 85% (17/20 patients), respectively, and 35% (7/20) of patients proceeded to SCT. Six patients experienced disease relapse with a median time-to-relapse of 6 (range, 4.0–10.9) months, and 4 of these 6 patients were found to have lost CD7 expression on tumor cells. Progression-free survival (PFS) and overall survival (OS) rates 24 months after treatment were respectively 36.8% (95% CI, 13.8–59.8%) and 42.3% (95% CI, 18.8–65.8%), with median PFS and OS of respectively 11.0 (95% CI, 6.7–12.5) months and 18.3 (95% CI, 12.5–20.8) months. Previously reported short-term adverse events ( 30 days after treatment included five infections and one grade 4 intestinal GVHD. Despite good CD7 CAR T-cell persistence, non-CAR T and natural killer cells were predominantly CD7-negative and eventually returned to normal levels in about half of the participants. Conclusions In this 2-year follow-up analysis, donor-derived CD7 CAR T-cell treatment demonstrated durable efficacy in a subset of patients with r/r T-ALL. Disease relapse was the main cause of treatment failure, and severe infection was a noteworthy late-onset adverse event. Trial registration ChiCTR2000034762.

Chimeric antigen receptor (CAR) T-cell therapies that target either CD19 or CD22 alone have potent antilymphoma effects. However, antigen escape–mediated relapse often occurs. CAR T cells targeting both CD19 and CD22 may overcome this limitation. In this study, we developed bispecific CAR T cells simultaneously recognizing CD19- and CD22-expressing targets and assessed their safety and efficacy profiles in patients with relapsed/refractory aggressive B-cell lymphoma. Twenty-four patients were screened, and 16 were found eligible for the study. CAR T-cell–associated toxicities were recorded. Responses, overall survival (OS), and progression-free survival (PFS) were assessed. Of the 16 eligible patients, 14 (87.5%) achieved objective response and 10 (62.5%) achieved complete response (CR). The 2-year OS and PFS rates were 77.3% and 40.2%, respectively. Achieving CR (P = 0.046) and the number of prior chemotherapy lines (n = 2; P = 0.047) were independent prognostic factors associated with favorable PFS. The 2-year OS and PFS among patients who achieved CR were higher than among those who did not (P = 0.015 and P < 0.001, respectively). The 2-year PFS among patients who received two prior lines of chemotherapy was higher than that among patients who received more than two lines of chemotherapy (P = 0.049); OS did not differ between the groups. Severe grade 4 cytokine-release syndrome (CRS) was observed in 1 patient; 4 and 11 patients had grades 1 and 2 CRS, respectively. No patients developed neurotoxicity. CD19/CD22 dual-targeted CAR T cells may be a safe, potent antilymphoma cell-based targeted immunotherapy.

Supplementary materials

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 ).

Purpose: B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T-cell therapy results in high remission rates in patients with relapsed/refractory (R/R) multiple myeloma. However, the factors associated with prognosis following CAR T-cell therapy are unknown. Patients and Methods: Between July 1, 2018 and July 31, 2020, 61 patients with R/R multiple myeloma received anti-BCMA CAR T-cell therapy (Chictr.org number, ChiCTR1800017404). Step-wise multivariate Cox regression and competing risk analyses were conducted to identify poor prognosis–associated risk factors. Results: Sixty patients (98.4%) experienced cytokine release syndrome (CRS), including 33, 23, and 4 cases of CRS grades 1 to 2, 3, and 4, respectively. The objective response rate (ORR) was 98.3%, and the complete remission (CR) rate was 70.3%. With a median follow-up period of 21.1 months, the 1-year overall survival (OS) and progression-free survival (PFS) rates were 78.0% and 50.2%, respectively. The median PFS was 12.7 months. Cox modeling revealed that poor PFS was associated with extramedullary disease [HR = 2.59, 95% confidence interval (95% CI) = 1.29–5.21, P = 0.008], light chain multiple myeloma (HR = 2.53, 95% CI = 1.03–5.97, P = 0.035), high-risk cytogenetics (HR = 2.80, 95% CI = 1.27–6.14, P = 0.01), and prior treatment with more than 3 therapeutic lines (HR = 3.14, 95% CI = 1.34–7.34, P = 0.008). Among the 41 CR cases, competing risk analyses demonstrated higher relapse predispositions in those with extramedullary disease (HR = 4.51, 95% CI = 1.86–10.9, P = 0.001), light chain multiple myeloma (HR = 4.89, 95% CI = 1.52 – 15.7, P = 0.008), or high-risk cytogenetics (HR = 5.09, 95% CI = 1.63–15.9, P = 0.005). Conclusions: Anti-BCMA CAR T-cell therapy is safe and effective for R/R multiple myeloma. For patients with high-risk factors, improvements to extend remission and more specific individualized therapies are needed.

BackgroundNeutropenia and cytokine release syndrome (CRS) are two major toxicities of chimeric antigen receptor (CAR)-T cell therapy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an ideal candidate treatment for neutropenia except for its potential aggravation of CRS. We hypothesized that the optimal timing of supplemental with GM-CSF in a shortage of host immunity and CAR T-cell was chosen as avoidance of CRS. In the study we evaluated the safety and efficacy of GM-CSF intervention post-CAR T-cell therapy while circulating CAR T-cell declined.Materials and methodsNine patients received GM-CSF therapy who displayed moderate neutropenia with absolute neutrophil counts (ANC) < 1,500 cells/mm3 with concomitant declination of circulating CAR T-cell.ResultsThe median duration of GM-CSF intervention was 15 days (4–30). CAR T-cell expansion was observed in peripheral blood (PB) of seven patients (7/9). The median baseline and peak CAR T cells count in PB of the seven patients with CAR T-cell expansion were 0.85 × 106/L (0–50.9) and 6.06 × 106/L (1.43–112.55). And the peaks of CAR T-cell levels in PB appeared in day 7 (2–11) following the initiation of GM-CSF administration with increases of 2.84 × 106/L (0.38–61.65). Also, increased white blood cells in PB were observed in all patients. The median onset and duration time of WBC recovery were 9 (1–14) and 17 (3–53) days. Moreover, the increment of WBC, neutrophil, lymphocyte and CD3-CD16 + CD56 + natural killer cell in PB was observed. In addition, no CRS or fatal infection occurred during GM-CSF treatment.ConclusionThis study provides evidence for the clinical feasibility of combining CAR T-cell therapy with the GM-CSF to treat neutropenia patients with concomitant declination of circulating CAR T-cell.

Chimeric antigen receptor T cell (CAR-T) therapy has revolutionized the clinical treatment of hematological malignancies due to the prominent anti-tumor effects. B cell maturation antigen (BCMA) CAR-T cells have demonstrated promising effects in patients with relapsed/refractory multiple myeloma. However, the dynamics of CAR-T cell proliferation and cytotoxicity in clinical patients remains unexplored. Here, we longitudinally profiled the transcriptomes of 55,488 T cells including CAR-T products, CAR-T cells, and endogenous T cells at the peak and remission phases in a plasma cell leukemia (PCL) patient treated with BCMA CAR-T cells by single-cell transcriptomic analysis. Our results showed distinct CAR-T and endogenous T cell subsets indicating stage-specific expression in proliferation, cytotoxicity, and intercellular signaling pathways. Furthermore, we found that CAR-T cells at peak phase gradually convert to a highly cytotoxic state from a highly proliferative state along a development trajectory. Moreover, re-analysis of a single cell study from CD8(+) CD19 CAR-T confirmed our findings. These commonalities suggest conserved mechanisms for CAR-T treatment across hematological malignancies. Taken together, our current study provides insight into CAR-T cell dynamics during CAR-T therapy and proves that both BCMA CAR-T and CD19 CAR-T have similar transcriptional characteristics, especially at the CAR-T peak phase.

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.

Background: Leukopenia and cytokine release syndrome (CRS) are two major toxicities of CAR-T cell therapy. GM-CSF is an ideal candidate treatment for leukopenia except for its potential aggravation of CRS. We hypothesized that the optimal timing of supplemental with GM-CSF in a shortage of host immunity and CAR T-cell was chosen as avoidance of CRS. In the study we evaluated the safety and efficacy of GM-CSF intervention post-CAR T-cell therapy while circulating CAR T-cell declined. Methods: Nine patients received GM-CSF therapy who displayed moderate leukopenia with white blood cell counts (WBC) 3 and absolute neutrophil counts (ANC) 3 with concomitant declination of circulating CAR T-cell after CAR T-cell therapy. Results: The median duration of GM-CSF intervention was 15 days (4-30). CAR T-cell expansion was observed in eight patients (8/9), including cerebrospinal fluid of one patient and peripheral blood (PB) of other seven patients. And the peaks of CAR T-cell levels in cerebrospinal fluid and PB appeared in day 6 and day 7 (2-11) following the initiation of GM-CSF administration. Also, increased white blood cells in PB was observed in all patients. The median onset and duration time of WBC recovery were 9 (1-14) and 17(3-53) days. Moreover, the increment of WBC, neutrophil, lymphocyte and CD3-CD16+CD56+ natural killer cell in PB was observed. In addition, no CRS or fatal infection occurred during GM-CSF treatment. Conclusion: This study provides evidence for the clinical feasibility of combining CAR T-cell therapy with the GM-CSF to treat leukopenia patients with concomitant declination of circulating CAR T-cell.

Background For CD19-positive relapsed/refractory B-cell acute lymphoblastic leukemia (r/r B-ALL) after treatment with murine CD19 (mCD19) CAR-T, the reinfusion of mCD19 CAR-T cells may be ineffective due to anti-mouse single-chain variable fragment (scFv) antibody caused by mCD19 CAR. To overcome this immunogenicity, we applied humanized CD19 (hCD19) CAR-T cells to treat r/r B-ALL patients with prior mCD19 CAR-T therapy. Methods Nineteen pediatric and adult patients were included, 16 relapsed after and 3 were primarily resistant to mCD19 CAR-T. All patients presented with more than 5% blasts in bone marrow and/or extramedullary disease, and still showed CD19 antigen expression. Humanized CD19-CARs were lentiviral vectors carrying a second generation CAR with 4–1-BB co-stimulatory and CD3ζ signaling domains. Patient-derived cells were collected for producing CAR-T cells, the median dose of infused hCD19 CAR-T cells was 2.4 × 105/kg (range, 1.0–18.0 × 105/kg). Results hCD19 CAR-T resulted in a complete remission (CR) rate of 68% (13/19). Among 13 remission patients, 11 underwent allogeneic hematopoietic cell transplantation (allo-HCT) (3 were second HCT) and 10 remained in CR; the event-free survival rates at 12–18 months were 91% in 11 patients received following allo-HCT and 69% in all CR patients. Six cases had no response to hCD19 CAR-T, 3 died of disease progression; another 3 received salvage second transplantation, of them, 2 relapsed again (one died). Cytokine release syndrome (CRS) occurred in 95% (18/19) of patients, most CRS events were grade 1 and grade 2 (n = 17), there was only one grade 4 CRS. Two cases experienced grade 1 neurotoxicity. Conclusions Humanized CD19 CAR-T cell therapy could be a treatment option for CD19-positive B-ALL patients who relapsed after or resisted prior murine CD19 CAR-T, hCD19 CAR-T followed by allo-HCT provided a longer remission in CR patients. Nevertheless, the prognosis of non-responders to hCD19 CAR-T remained dismal. Trial registration Chinese Clinical Trial Registry/WHO International Clinical Trial Registry (ChiCTR1900024456, URL: www.chictr.org.cn); registered on July 12, 2019.

BackgroundMurine chimeric antigen receptor T (CAR-T) cell therapy has demonstrated clinical benefit in patients with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). However, the potential immunogenicity of the murine single-chain variable fragment domain may limit the persistence of CAR-T cell, leading to relapse.MethodsWe performed a clinical trial to determine the safety and efficacy of autologous and allogeneic humanized CD19-targeted CAR-T cell (hCART19) for R/R B-ALL. Fifty-eight patients (aged 13–74 years) were enrolled and treated between February 2020 and March 2022. The endpoints were complete remission (CR) rate, overall survival (OS), event-free survival (EFS), and safety.ResultsOverall, 93.1% (54/58) of patients achieved CR or CR with incomplete count recovery (CRi) by day 28, with 53 patients having minimal residual disease negativity. With a median follow-up of 13.5 months, the estimated 1-year OS and EFS were 73.6% (95% CI 62.1% to 87.4%) and 46.0% (95% CI 33.7% to 62.8%), with a median OS and EFS of 21.5 months and 9.5 months, respectively. No significant increase in human antimouse antibodies was observed following infusion (p=0.78). Duration of B-cell aplasia in the blood was observed for as long as 616 days, which was longer than that in our prior mCART19 trial. All toxicities were reversible, including severe cytokine release syndrome, which developed in 36% (21/58) of patients and severe neurotoxicity, which developed in 5% (3/58) of patients. Compared with our prior mCART19 trial, patients treated with hCART19 had longer EFS without increased toxicity. Additionally, our data also suggest that patients treated with consolidation therapy, including allogeneic hematopoietic stem cell transplantation or CD22-targeted CAR-T cell, following hCART19 therapy had a longer EFS than those without consolidation therapy.ConclusionhCART19 has good short-term efficacy and manageable toxicity in R/R B-ALL patients.Trial registration numberNCT04532268.

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.

Immunotherapy utilizing chimeric antigen receptor T cell (CAR-T) therapy holds promise for hematologic malignancies, however, response rates and associated immune-related adverse effects widely vary among patients. Here we show, by comparing diversity and composition of the gut microbiome during different CAR-T therapeutic phases in the clinical trial ChiCTR1800017404, that the gut flora characteristically differs among patients and according to treatment stages, and might also reflect patient response to therapy in relapsed/refractory multiple myeloma (MM; n = 43), acute lympholastic leukemia (ALL; n = 23) and non-Hodgkin lymphoma (NHL; n = 12). We observe significant temporal differences in diversity and abundance of Bifidobacterium, Prevotella, Sutterella, and Collinsella between MM patients in complete remission (n = 24) and those in partial remission (n = 11). Furthermore, we find that patients with severe cytokine release syndrome present with higher abundance of Bifidobacterium, Leuconostoc, Stenotrophomonas, and Staphylococcus, which is reproducible in an independent cohort of 38 MM patients. This study has important implications for understanding the biological role of the microbiome in CAR-T treatment responsiveness of hematologic malignancy patients, and may guide therapeutic intervention to increase efficacy. The success rate of CAR-T cell therapy is high in blood cancers, yet individual patient characteristics might reduce therapeutic benefit. Here we show that therapeutic response in MM, ALL and NHL, and occurrence of severe cytokine release syndrome in multiple myeloma are associated with specific gut microbiome alterations.

Although chimeric antigen receptor T (CAR-T) cell therapy has proven to be effective in treating relapsed or refractory B-cell hematological malignancies, severe hematological toxicities remain an intractable issue. This retrospective study assessed the characteristics and risk factors of new-onset severe cytopenia following CAR-T cell infusion in 76 patients with r/r acute lymphoblastic leukemia. The rates of new-onset severe cytopenia were high, including severe neutropenia (SN) (39/56, 70%), severe anemia (SA) (35/66, 53%), and severe thrombocytopenia (ST) (31/64, 48%). Comparatively, cohorts with higher cytokine release syndrome (CRS) grades had higher incidence of severe cytopenia with prolonged duration. Multivariable analyses showed that elevated maximum (max) lg D-dimer and delayed peak time of CRS are independent risk factors for SN recovery; increased max lg IL-10 and delayed CRS recovery are risk factors for SA; high max lg ferritin is a risk factor for ST; and longer period to CRS onset or CRS recovery and higher grade of CRS are risk factors for prolonged hematological toxicities. These observations led to the conclusion that profiles of CRS, including its duration, severity and serum markers are correlated to the incidence and recovery of new-onset severe cytopenia, prompting clinical intervention for post-CAR-T severe cytopenia.

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.

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.

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.

Chimeric antigen receptor (CAR) T-cell therapies that target either CD19 or CD22 alone have potent antilymphoma effects. However, antigen escape–mediated relapse often occurs. CAR T cells targeting both CD19 and CD22 may overcome this limitation. In this study, we developed bispecific CAR T cells simultaneously recognizing CD19- and CD22-expressing targets and assessed their safety and efficacy profiles in patients with relapsed/refractory aggressive B-cell lymphoma. Twenty-four patients were screened, and 16 were found eligible for the study. CAR T-cell–associated toxicities were recorded. Responses, overall survival (OS), and progression-free survival (PFS) were assessed. Of the 16 eligible patients, 14 (87.5%) achieved objective response and 10 (62.5%) achieved complete response (CR). The 2-year OS and PFS rates were 77.3% and 40.2%, respectively. Achieving CR (P = 0.046) and the number of prior chemotherapy lines (n = 2; P = 0.047) were independent prognostic factors associated with favorable PFS. The 2-year OS and PFS among patients who achieved CR were higher than among those who did not (P = 0.015 and P < 0.001, respectively). The 2-year PFS among patients who received two prior lines of chemotherapy was higher than that among patients who received more than two lines of chemotherapy (P = 0.049); OS did not differ between the groups. Severe grade 4 cytokine-release syndrome (CRS) was observed in 1 patient; 4 and 11 patients had grades 1 and 2 CRS, respectively. No patients developed neurotoxicity. CD19/CD22 dual-targeted CAR T cells may be a safe, potent antilymphoma cell-based targeted immunotherapy.

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.

Background Consolidative allogeneic hematopoietic stem cell transplantation is a controversial option for patients with relapsed/refractory acute lymphoblastic leukemia after chimeric antigen receptor T cell (CAR-T) therapy. We performed a multicenter retrospective study to assess whether patients can benefit from haploidentical hematopoietic stem cell transplantation after CAR-T therapy. Methods A total of 122 patients after CAR-T therapy were enrolled, including 67 patients without subsequent transplantation (non-transplant group) and 55 patients with subsequent haploidentical hematopoietic stem cell transplantation (transplant group). Long-term outcome was assessed, as was its association with baseline patient characteristics. Results Compared with the non-transplant group, transplantation recipients had a higher 2-year overall survival (OS; 77.0% versus 36.4%; P < 0.001) and leukemia-free survival (LFS; 65.6% versus 32.8%; P < 0.001). Multivariate analysis showed that minimal residual disease (MRD) positivity at transplantation is an independent factor associated with poor LFS (P = 0.005), OS (P = 0.035), and high cumulative incidence rate of relapse (P = 0.045). Pre-transplant MRD-negative recipients (MRD− group) had a lower cumulative incidence of relapse (17.3%) than those in the non-transplant group (67.2%; P < 0.001) and pre-transplant MRD-positive recipients (MRD+ group) (65.8%; P = 0.006). The cumulative incidence of relapse in MRD+ and non-transplant groups did not differ significantly (P = 0.139). The 2-year LFS in the non-transplant, MRD+, and MRD− groups was 32.8%, 27.6%, and 76.1%, respectively. The MRD− group had a higher LFS than the non-transplantation group (P < 0.001) and MRD+ group (P = 0.007), whereas the LFS in the MRD+ and non-transplant groups did not differ significantly (P = 0.305). The 2-year OS of the MRD− group was higher than that of the non-transplant group (83.3% versus 36.4%; P < 0.001) but did not differ from that of the MRD+ group (83.3% versus 62.7%; P = 0.069). The OS in the non-transplant and MRD+ groups did not differ significantly (P = 0.231). Conclusion Haploidentical hematopoietic stem cell transplantation with pre-transplant MRD negativity after CAR-T therapy could greatly improve LFS and OS in patients with relapsed/refractory acute lymphoblastic leukemia. Trial registration The study was registered in the Chinese clinical trial registry (ChiCTR1900023957).

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.

Mantle cell lymphoma (MCL) is a distinct histological type of B-cell lymphoma with a poor prognosis. Several agents, such as proteasome inhibitors, immunomodulatory drugs, and inhibitors of B cell lymphoma-2 and Bruton's tyrosine kinase have shown efficacy for relapsed or refractory (r/r) MCL but often have short-term responses. Chimeric antigen receptor (CAR) T-cell therapy has emerged as a novel treatment modality for r/r non-Hodgkin's lymphoma. However, long-term safety and tolerability associated with CAR T-cell therapy are not defined well, especially in MCL. In this report, we described a 70-year-old patient with r/r MCL with 48-month duration of follow-up who achieved long-term remission after CAR T-cell therapy. CAR T-cell-related toxicities were also mild and tolerated well even in this elderly patient. This report suggested that CAR T-cell therapy is a promising treatment modality for patients with MCL, who are generally elderly and have comorbid conditions.

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.

Neovasculature imaging is a promising approach for tumor diagnosis. We constructed tumor neovasculature targeted paramagnetic nanoliposomes with RGD10, F56, and K237 peptides, which can bind to Integrin αvβ3 and VEGFR-1, VEGFR-2, respectively, and compared their potential value as MRI contrast agents for detecting small tumors in animal models.Peptide-Ahx-palmitic acid conjugate was synthesized using Fmoc solid-phase synthesis chemistry. Targeted paramagnetic nanoliposomes were prepared by the thin film dispersion-sonication method. The tumor signal enhancements of liposome particles were evaluated by MRI in a xenograft mice model.The apparent affinity constants of RGD10, K237, and F56 peptides binding to their cell receptors were 9.15 × 10(7), 6.01 × 10(7), and 3.85 × 10(7) mol/L, respectively. RGD10 and K237 targeted paramagnetic nanoliposomes have shown much greater tumor-specific MRI signal enhancement in xenograft of the nude mice compared to F56 targeted paramagnetic nanoliposome. Tumor signal enhancement rate (SER %) increased 2.21 ± 0.09 and 1.82 ± 0.05 fold, respectively, for RGD10 and K237 compared to non-targeted control in T1 weighted MR image.RGD10 and K237 targeted paramagnetic nanoliposomes can be developed as potential tumor-specific MRI contrast agents and are helpful for tumor detection.

Introduction: The source of chimeric antigen receptor (CAR) T cells can be autologous or allogeneic, depending on the history of allogeneic hematopoietic stem cell transplantation (allo-HSCT). For patients with a history of allo-HSCT, CAR T cells are allogeneic, which can be manufactured from donors' peripheral blood mononuclear cells (PBMC) (donor-derived allogeneic CAR T cells, DD-alloCAR) or patients' own PBMC (recipient-derived allogeneic CAR T cells, RD-alloCAR). Unlike autoCAR T cells, which are activated by CAR only, alloCAR T cells receive activation signals from both T-cell receptor and CAR. The difference could affect CAR T-cell biology, resulting in different clinical outcomes. Methods: We retrospectively reviewed 31 consecutive patients who received CAR T-cell therapy in our center. Patients were divided into three groups, as autoCAR group, DD-alloCAR group and RD-alloCAR group. We compared the efficacy and safety profiles among the three groups. We also performed a subgroup analysis of patients received alloCAR. Results: Altogether there were 17 patients in the autoCAR, 11 in the RD-alloCAR and 3 in the DD-alloCAR groups. After a median follow-up of 9 months, the CR rate was 88.2% (95% CI 63.6-98.5%) in the autoCAR group and 100% (95% CI 71.5-100%) in the RD-alloCAR group, which had no significant difference (p=0.73). CR rate in DD-alloCAR was 66.7% (95% CI 9.43-99.1%). The median peak expansion of CAR T cells in the autoCAR was significantly higher than the RD-alloCAR group (p=0.007, Figure A). However, the median OS and EFS did not differ significantly among the groups. As far as adverse effects, RD-alloCAR group had significantly less patients with severe CRS (defined as Grade ≥ 3) than the autoCAR group (p=0.049), with significantly lower peak IL-6 level (p=0.021, Figure B). Neurotoxicity and the degree of cytopenia did not have significant difference among the groups. Acute GVHD occurred in 2 (18.2%) RD-alloCAR patients and 1 (33.3%) DD-alloCAR patients following CAR T-cell infusion. Univariate subgroup analysis of the patients who received alloCAR T-cell therapy showed the presence of cGVHD at the time of PBMC collection was significantly associated with less 6-month relapses (p=0.022); the median RFS in the no-cGVHD group was 2.00 (95% CI 0.72 to 3.28) months, compared to 12.0 (95% CI 2.40 to 21.6) months in the cGVHD group (p=0.084, Figure C); the median OS had no significant difference (Figure D). RD-alloCAR patients with or without cGVHD at PBMC collection did not differ in terms of the peak CAR T-cell expansion, CRS grades or OS. Conclusions: Compared with autoCAR, RD-alloCAR had similar efficacy but less severe CRS. For RD-alloCAR T-cell therapy, the presence of cGVHD at the time of PBMC collection was associated with fewer 6-month relapses. DD-alloCAR and RD-alloCAR T-cell therapies were effective and safe without causing significant GVHD. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Background: CD19-targeting chimeric antigen receptor (CAR) T-cell therapy has shown great efficacy in patients with refractory/relapsed non-Hodgkin lymphoma (NHL), but was associated with serious adverse effects such as cytokine release syndrome (CRS). It has been speculated that NHL baseline disease burden might affect clinical outcome and CRS, but such assumption has not been explored in detail in previous studies. Metabolic tumor volume (MTV) and total lesion glycolysis (TLG), calculated using FDG PET-CT, are quantitative indicators of baseline tumor burden. Methods: Utilizing FDG PET-CT, we calculated MTV and TLG at baseline and post CAR T-cell therapy in 15 patients with NHL. Results: Among all the patients, the median MTV was 72 (range 0.02-3024.9) cm3 and the median TLG was 610.1 (range 0.011-13156.3). After a median follow-up of 6 months, the overall response rate (ORR) was 66.7% (95% CI 38.4-88.2%). The baseline MTV and TLG did not have significant difference in patients with or without response (p=0.271 and 0.95, respectively). Cox-regression analysis did not find lower baseline MTV and TLG significantly associated with better overall survival (p=0.67 and 0.45, respectively). Patients with mild and moderate CRS (defined as Grade 0-2) had significantly lower MTV and TLG than those with severe CRS (Grade 3, 4) (median MTV: 49.3 v.s. 1137.7 cm3, p=0.012; median TLG: 379.1 v.s. 9384, p=0.012, Figure A, B). The median MTV in patients received tocilizumab, an IL-6 antagonist for the treatment of severe CRS, was 963.4 cm3, which was significantly higher than the patients not receiving tocilizumab (58.1 cm3, p=0.037). The median TLG in patients received tocilizumab was 9187.1, compared with 610.1 in patients not receiving tocilizumab (p=0.053). Using FDG PET-CT, we also demonstrated that CAR T-cell therapy in NHL patients could associate with severe local complications such as local compression and local inflammation. Conclusions: Low NHL baseline disease burden is not associated with better response rate or long-term outcome. Patients with higher baseline disease burden have more severe CRS Figure. Figure. Disclosures No relevant conflicts of interest to declare.

The in vivo regulation of T lymphocyte activity by the activation of a suicide mechanism is an essential paradigm for the safety of adoptive cell therapies. In light of reports showing that gamma-retroviral vector-encoded herpes simplex virus thymidine kinase (hsvtk) undergoes recombination, we undertook a thorough investigation of the genomic stability of SFG-based vectors using two variants of the wild-type hsvtk gene. In a large panel of independent clones, we demonstrate that both hsvtk genes undergo recombination with molecular signatures indicative of template switching in GC-rich regions displaying homology at the deletion junctions or RNA splicing. In the absence of ganciclovir selection, the frequency of recombination is 3% per retroviral replication cycle. Our results underscore the importance of the five nucleotide difference between the two hsvtk genes that account for the presence of recombinogenic hot spots in one variant and not the other, indicating that the probability of RNA splicing is influenced by minute nucleotide changes in sequences adjacent to the splice donor and acceptor sites. Furthermore, our mutational analysis in an unbiased panel of human lymphoid cells (that is, without immune or ganciclovir-mediated selective pressure) provides a robust in vitro assay to predict and quantify clinically relevant mutations in hsvtk suicide genes, which can be applied to studying and improving the stability of any transgene expressed in gamma-retroviral or lentiviral vectors.

The ability of specific virally encoded proteins to down-regulate MHC class I molecules may enable infected cells to elude killing by CTL. In the case of HIV-1, Nef appears to be responsible for this effect. Thus, interfering with Nef-induced MHC class I down-regulation would be a strategy for increasing HIV-1-specific CTL activity, particularly towards long-lived T cell populations suchas memory T cells that harbor replication-competent virus. Here, using two Nef-expressing human cell model systems, we show that a dominant-negative mutant derived from the Hck protein-tyrosine kinase, composed of the Hck N-terminal region, as well as the SH3 and SH2 domains, was able to inhibit Nef-induced MHC class I molecule down-regulation. This effect was SH3 domain dependent as it was not evident when the cells were transfected with DN-Hck-W93F, an SH3 domain mutant. The inhibitory effect of dominant-negative-Hck (DN-Hck) on Nef-induced class I down-regulation suggests that this Nef-mediated effect requires an interaction between the Nef polyproline site and an SH3-containing cellular protein that is involved in MHC class I molecule turnover. Interfering with the function of the Nef SH3 binding site in this way represents a strategy for assisting the host CTL response to clear HIV-1-infected cells.

Abstract Introduction: Patients (pts) with relapsed refractory B-ALL are mostly incurable by chemotherapy. The disease free survival (DFS) is low even treatment with allogeneic hematopoietic stem cell transplantation (allo-HSCT). We explored treatment of 47 cases of relapsed refractory B-ALL with low dose CD19 CAR-T cells and assessed the clinical safety and efficacy. Patients and Methods: (6-8)X107 pts' peripheral blood mononuclear cells(PBMCs) were activated with CD3 and CD28 antibodies for 24h, then transduced with the lentivirus encoding anti-CD19-CD3zeta-4-1BB CAR (Image1/Picture1) and cultured for 5-6 days in serum-free media containing IL2,IL7,IL15,IL21. All pts except one who had persistent cytopenia received cyclophosphamide 250 mg/m2/d X 3d, and fludarabine 30 mg/m2/d X 3d, then CAR-T cell infusion. Between Jul.31 2015 and Jul. 15 2016, a total of 47 cases were treated with CAR-T cells (Chart1). 37/47 cases had frank hematologically relapsed refractory B-ALL, who could not achieve complete remission (CR) after more than 1 cycles of chemotherapy. The median prior chemotherapy duration was 18 months. The median pre-treatment bone marrow blast percentage was 67% (6.5-98.5%). The most recently treated 15 cases had their PB blasts Chart 1: Characteristics of Patients Pre-CAR-T Image 1/Picture 1: Results: The pts received a median of 10 (0.5-140) X104cells/kg CAR-T cells, and the most recently treated 15 cases all received 10 X 104cells/kg. The median observation period was 201 days (20-368 days). On day 16-20 after CAR-T infusion, 31/35 (88.6%) relapsed hematological refractory cases achieved CR or incomplete CR(Cri), and 29/35 cases (82.9%) achieved negative MRD by FCM(CMR: complete molecular remission). No extramedullary leukemia was detected in any cases with residual disease. The most recently treated 15 consecutive cases all achieved CR with only mild ( Conclusion: Our anti-CD19 CAR-T cell therapy can result in a high CR/CRi /CMR rate in pts with refractory B-ALL and could overcome pre-existing risk factors for poor outcomes, including complex chromosome abnormalities, poor gene mutations, inherited predisposing gene mutation, extramedullary leukemia etc. Pts could be safely bridged into allo-HSCT for potential cure. The dose of infused CAR-T cells in our patients was far lower than previously reported in the literature and the culture period was only 6-7 days, which could dramatically reduce the cost of the CAR-T therapy. 10X104cells/kg of CAR-T cells was a safe and effective number for treating B-ALL. The major complication was CRS and the severity of CRS was directly correlated with the number of malignant B cells in the PB. The efficacy of CAR-T therapy was correlated to the infused number of CAR-T cells. The most recently treated 15 consecutive cases all achieved CR without severe CRS suggesting that the optimal number of CAR-T cells and patient selection are important for the efficacy and safety. Table. Table. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

To reject tumors, T cells must overcome poor tumor immunogenicity and an adverse tumor microenvironment. Providing agonistic costimulatory signals to tumor-infiltrating T cells to augment T cell function remains a challenge for the implementation of safe and effective immunotherapy. We hypothesized that T cells overexpressing selected costimulatory ligands could serve as cellular vehicles mediating powerful, yet constrained, anatomically targeted costimulation. Here, we show that primary human T cells expressing CD80 and 4-1BB ligand (4-1BBL) vigorously respond to tumor cells lacking costimulatory ligands and provoke potent rejection of large, systemic tumors in immunodeficient mice. In addition to showing costimulation of bystander T cells (transcostimulation), we show the effect of CD80 and 4-1BBL binding to their respective receptors in the immunological synapse of isolated single cells (autocostimulation). This new strategy of endowing T cells with constitutively expressed costimulatory ligands could be extended to other ligand-receptor pairs and used to enhance any targeted adoptive transfer therapy.

Recent studies on the integration patterns of different categories of retroviral vectors, the genotoxicity of long-terminal repeats (LTRs) and other genetic elements, the rise of lentiviral technology and the emergence of regulated vector systems providing tissue-restricted transgene expression and RNA interference, are profoundly changing the landscape of stem cell-based therapies. New developments in vector design and an increasing understanding of the mechanisms underlying insertional oncogenesis are ushering in a new phase in hematopoietic stem cell (HSC) engineering, thus bringing the hitherto exclusive reliance on LTR-driven, gamma-retroviral vectors to an end. Based on their ability to transduce non-dividing cells and their genomic stability, lentiviral vectors offer new prospects for the manipulation of HSCs. Tissue-specific vectors, as exemplified by globin vectors, not only provide therapeutic efficacy, but may also enhance safety, insofar that they restrict transgene expression in stem cells, progenitor cells and blood cells in all but the transcriptionally targeted lineage. This review provides a survey of these advances as well as several remaining challenges, focusing in particular on the importance of achieving adequate levels of protein expression from a limited number of vector copies per cell-ideally one to two.

We demonstrate here the capacity of erythroid cells to mediate long-term, systemic and therapeutic protein delivery in vivo. By targeting human factor IX (hFIX) expression to late-stage erythropoiesis, we achieve long-term hFIX secretion at levels significantly higher (>tenfold) than those obtained with an archetypal ubiquitous promoter in a mouse model of hemophilia B. Erythroid cell-derived hFIX is biologically active, resulting in phenotypic correction of the bleeding disorder. In addition to achieving high expression levels and resistance to transcriptional silencing, red cell-mediated protein delivery offers multiple advantages including immune tolerance induction, reduction of the risk of insertional oncogenesis and relative ease of application by either engrafting transduced hematopoietic stem cells or transfusing ex vivo-generated, stem cell-derived erythroid cells.

Top of pageAbstract Various cell types have been targeted for expression of clotting factors in experimental models of hemophilia, with muscle and hepatocyte-directed gene transfer advancing to clinical trials. Hematopoietic stem cells (HSCs) are attractive alternative targets because of their self-renewing properties, their ability to generate blood cells over extended periods of time, and their ease of manipulation ex vivo. However, several hurdles must be overcome to make this approach safe and practical. These include the need to express sufficient amount of therapeutic gene products and minimize the risk of insertional mutagenesis and the toxicity of host conditioning to facilitate HSC engraftment. We have previously developed an erythoid cell-specific platform for long-term and systemic therapeutic protein delivery in vivo using an erythroid- specific lentiviral vector. We demonstrated in an immune competent C57BL/6 hemophilia B mouse model, that therapeutic levels of human factor IX (hFIX) can be expressed at low average vector copy (VC) number (250-350 ng/mL with under 0.5 VC per cell). In this study, we examined the level of erythroid-specific protein delivery obtained after engraftment following minimal conditioning, eventually in the presence of in vivo methylguanine methyltransferase (MGMT)-mediated drug resistance. Low to moderate levels of hFIX expression were achieved in control, lethally irradiated mice (average 100-200 ng/ml), 4-weeks post-transplantation. In non-myeloablated recipients conditioned with 400 cGy or Busulfan (20 mg/kg, given twice at 48 h and 24 h before engraftment), the levels of hFIX expression were ranged from negligible to low (0-20 ng/ml and 0 |[ndash]| 100 ng/ml, respectively). Four weeks after the first round of BG/BCNU (30 mg/kg and 5 mg/kg, respectively), a wide range of hFIX expression levels were achieved, reaching in some mice levels equivalent to 20% of physiological levels in humans. We are currently monitoring long-term hFIX expression, as well as the average VC number in blood cells and CFUs. Further rounds of drug selection are also in progress. In conclusion, our data suggest that it is possible to express high levels of a secreted protein from HSC-derived erythroid cells engrafted under minimal conditioning. The combined features of reduced intensity conditioning, reduced insertional mutagenesis due to low vector copy number requirement and erythroid-specific transgene expression, as well as long-term protein expression at therapeutic levels, increase the potential applicability of adult stem cell-based gene therapy in non-lethal disorders such as hemophilia.