EBV-associated lymphoproliferative disease post-CAR-T cell therapy.

Epstein-Barr virus (EBV)-associated lymphoproliferative diseases (EBV-LPDs) are common complications that occur after solid organ transplantation or allogeneic hematopoietic stem-cell transplantation (HSCT). However, their occurrence and treatment post-chimeric antigen receptor-modified T (CAR-T) cell therapy has not been reported. Two patients had been diagnosed with EBV-positive aggressive B-cell lymphoma and experienced relapses after multiple lines of treatment. After receiving CAR-T cell therapy in tandem with autologous HSCT, the patients achieved complete remission. However, with a median time of 38.5 months after CAR-T cell therapy, B-cell-derived EBV-LPDs were diagnosed, and they were relieved through the administration of immune checkpoint inhibitor or B-cell-depleting agents. Collectively, our report suggests that EBV-LPDs may represent a long-term adverse event after CAR-T cell therapy, especially in patients who previously had EBV-positive disorders, and they can be resolved by immune normalization strategy or B-cell depleting therapy.

Suppression of cytokine release syndrome during CAR-T-cell therapy via a subcutaneously injected interleukin-6-adsorbing hydrogel.

The infusion of chimaeric antigen receptor (CAR) T cells can trigger the release of life-threatening supraphysiological levels of pro-inflammatory cytokines. However, uncertainty regarding the timing and severity of such cytokine release syndrome (CRS) demands careful monitoring of the conditions required for the administration of neutralizing antibodies. Here we show that a temperature-sensitive hydrogel conjugated with antibodies for the pro-inflammatory cytokine interleukin-6 (IL-6) and subcutaneously injected before the infusion of CAR-T cells substantially reduces the levels of IL-6 during CRS while maintaining the therapy's antitumour efficacy. In immunodeficient mice and in mice with transplanted human haematopoietic stem cells, the subcutaneous IL-6-adsorbing hydrogel largely suppressed CAR-T-cell-induced CRS, substantially improving the animals' survival and alleviating their levels of fever, hypotension and weight loss relative to the administration of free IL-6 antibodies. The implanted hydrogel, which can be easily removed with a syringe following a cooling-induced gel-sol transition, may allow for a shift in the management of CRS, from monitoring to prevention.

Highly distributed amorphous copper catalyst for efficient ammonia electrosynthesis from nitrate.

Electroreduction of nitrate to ammonia, instead of N2, is beneficial toward pollution control and value-added chemical production. Metallic catalysts have been developed for enhancing ammonia evolution efficiency from nitrate based on the crystalline state of the catalyst. However, the development of amorphous metallic catalysts with more active sites is still unexplored. Herein, a highly distributed amorphous Cu catalyst exhibiting an outstanding ammonia yield rate of 1.42 mol h-1 g-1 and Faradaic efficiency of 95.7%, much superior to crystallized Cu, is demonstrated for nitrate-reduction to ammonia. Experimental and computational results reveal that amorphizing Cu increases the number of catalytic sites, enhances the NO3- adsorption strength with flat adsorption configurations, and facilitates the potential determining step of *NO protonation to *NHO. The amorphous Cu catalyst shows good electrochemical stability at - 0.3 V, while crystallization weakens the activity at a more negative potential. This study confirms the crystallinity-activity relationship of amorphous catalysts and unveils their potential-limited electrochemical stability.

Autologous stem cell transplantation in tandem with Anti-CD30 CAR T-cell infusion in relapsed/refractory CD30+ lymphoma.

BACKGROUND: Long-term outcome is unfavourable for relapsed/refractory (r/r) lymphoma patients who are resistant to salvage chemotherapy, even after subsequent autologous stem-cell transplantation (ASCT). Although anti-CD30 chimeric antigen receptor (CAR30) T-cell therapy induces high response rates in these patients, the duration of response is relatively limited. METHODS: This open-label, single-center and single-arm pilot study investigated the safety and efficacy of ASCT in tandem with CAR30 T-cell infusion in r/r CD30+ lymphoma. The primary endpoint was safety and key secondary endpoint was overall response rate, overall survival, progression-free survival, and duration of response. RESULTS: Five classical Hodgkin lymphoma (cHL) patients and 1 anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) patient were enrolled. The median age was 24 years. No patient had prior ASCT. Three patients (50.0%) relapsed for ≥ 2 times and 3 patients (50.0%) had primary refractory diseases. All had a Deauville score of 4 or 5, and 5 patients (83.3%) had a stable or progressive disease (SD/PD) at enrollment. All patients received myeloablative chemotherapy and infused CD34-positive hematopoietic stem cells (HSCs) and CAR30 T cells in tandem, with a median dose of 3.9 × 106/kg and 7.6 × 106/kg, respectively. Five paitents presented with cytokine release syndrome (CRS), all of which were grade 1. No neurotoxicity was observed. All patients had successful HSCs engraftment and reached an objective response, including 5 (4 cHL and 1 ALCL, 83.3%) with a complete response (CR) and 1 with a partial response (PR). With a median follow-up of 20.4 (range, 12.1-34.4) months, all remained alive and maintained their responses. CONCLUSION: Our work demonstrates the combined administration of ASCT and CAR30 T-cell therapy is well-tolerate and highly effective in r/r cHL and ALCL, even in PET-positive or chemorefractory patients who are expected to have inferior outcome after ASCT, although further large-scaled validation in prospective clinical trial is warranted. Trial registration The trial was registered with the Chinese Clinical Trial Registry (ChiCTR, number ChiCTR2100053662).

Chimeric Antigen Receptor-Modified T Cell Immunotherapy for Relapsed and Refractory Adult Burkitt Lymphoma.

Patients with Burkitt lymphoma who are refractory to initial therapy or who relapse after undergoing intensive chemotherapy and autologous stem cell transplantation (ASCT) usually have a poor prognosis. While there has been considerable progress in the use of chimeric antigen receptor-modified (CAR) T cell immunotherapy for the treatment of relapsed and refractory (r/r) malignancies, explicit data on adult patients with r/r Burkitt lymphoma are limited. We conducted two single-arm clinical trials to evaluate the clinical efficacy and toxicity of CD19/CD22 CAR T cell immunotherapy both alone (trial A) and in combination with ASCT (trial B) in adult patients with r/r Burkitt lymphoma. In total, 28 adult patients with r/r Burkitt lymphoma were enrolled [trial A (n = 15) and trial B (n = 13)]. The median doses of CD22 and CD19 CAR T cell infusions were 4.1 × 106/kg and 4.0 × 106/kg, respectively. Subsequently, after CAR T cell infusion, overall and complete responses were observed in 19 (67.9%) and 16 (57.1%) patients, respectively. The cumulative incidence rates of grade 2-4 cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome were 39.3% (11/28) and 10.7% (3/28), respectively. After a median follow-up duration of 12.5 months, 16 patients (5 in trial A and 11 in trial B) survived. Both the estimated 1-year progression-free and overall survival rates were 55.6%. Our preliminary results indicated that salvage therapy with CD19/CD22 CAR T cell infusion alone and that in combination with ASCT are effective in treating some adult patients with r/r Burkitt lymphoma.

The third-generation anti-CD30 CAR T-cells specifically homing to the tumor and mediating powerful antitumor activity.

CAR T-cell therapy is well tolerated and effective in patients with Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL). However, even second- generation anti-CD30 CAR T-cells with CD28 (28z) costimulatory domains failed to achieve the desired rate of complete responses. In the present study, we developed second-generation (CD28z) and third-generation (CD28BBz) CAR T-cells targeting CD30 and investigated their efficacy in vitro and in vivo. Both of CD28z and CD28BBz anti-CD30 CAR T cells were similar regarding amplification, T cell subsets distribution, T cell activity, effector/memory and exhaustion. However, we found that the 28BBz anti-CD30 CAR T-cells persist long-term, specifically homing to the tumor and mediating powerful antitumor activity in tumor xenograft models. Subsequently, we also demonstrated that the third generation anti-CD30 CAR T-cells have miner side effects or potential risks of tumorigenesis. Thus, anti-CD30 CAR T-cells represent a safe and effective treatment for Hodgkin lymphoma.

T Cell Defects: New Insights Into the Primary Resistance Factor to CD19/CD22 Cocktail CAR T-Cell Immunotherapy in Diffuse Large B-Cell Lymphoma.

Despite impressive progress, a significant portion of patients still experience primary or secondary resistance to chimeric antigen receptor (CAR) T-cell immunotherapy for relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). The mechanism of primary resistance involves T-cell extrinsic and intrinsic dysfunction. In the present study, a total of 135 patients of DLBCL treated with murine CD19/CD22 cocktail CAR T-therapy were assessed retrospectively. Based on four criteria (maximal expansion of the transgene/CAR-positive T-cell levels post-infusion [Cmax], initial persistence of the transgene by the CAR transgene level at +3 months [Tlast], CD19+ B-cell levels [B-cell recovery], and the initial response to CAR T-cell therapy), 48 patients were included in the research and divided into two groups (a T-normal group [n=22] and a T-defect [n=26] group). According to univariate and multivariate regression analyses, higher lactate dehydrogenase (LDH) levels before leukapheresis (hazard ratio (HR) = 1.922; p = 0.045) and lower cytokine release syndrome (CRS) grade after CAR T-cell infusion (HR = 0.150; p = 0.026) were independent risk factors of T-cell dysfunction. Moreover, using whole-exon sequencing, we found that germline variants in 47 genes were significantly enriched in the T-defect group compared to the T-normal group (96% vs. 41%; p<0.0001), these genes consisted of CAR structure genes (n=3), T-cell signal 1 to signal 3 genes (n=13), T cell immune regulation- and checkpoint-related genes (n=9), cytokine- and chemokine-related genes (n=13), and T-cell metabolism-related genes (n=9). Heterozygous germline UNC13D mutations had the highest intergroup differences (26.9% vs. 0%; p=0.008). Compound heterozygous CX3CR1I249/M280 variants, referred to as pathogenic and risk factors according to the ClinVar database, were enriched in the T-defect group (3 of 26). In summary, the clinical characteristics and T-cell immunodeficiency genetic features may help explain the underlying mechanism of treatment primary resistance and provide novel insights into CAR T-cell immunotherapy.

Outcome of aggressive B-cell lymphoma with TP53 alterations administered with CAR T-cell cocktail alone or in combination with ASCT.

TP53 gene alteration confers inferior prognosis in refractory/relapse aggressive B-cell non-Hodgkin lymphoma (r/r B-NHL). From September 2016 to September 2020, 257 r/r B-NHL patients were assessed for eligibility for two trials in our center, assessing anti-CD19 and anti-CD22 chimeric antigen receptor (CAR19/22) T-cell cocktail treatment alone or in combination with autologous stem cell transplantation (ASCT). TP53 alterations were screened in 123 enrolled patients and confirmed in 60. CAR19/22 T-cell administration resulted in best objective (ORR) and complete (CRR) response rate of 87.1% and 45.2% in patients with TP53 alterations, respectively. Following a median follow-up of 16.7 months, median progression-free survival (PFS) was 14.8 months, and 24-month overall survival (OS) was estimated at 56.3%. Comparable ORR, PFS, and OS were determined in individuals with or without TP53 alterations, and in individuals at different risk levels based on functional stratification of TP53 alterations. CAR19/22 T-cell treatment in combination with ASCT resulted in higher ORR, CRR, PFS, and OS, but reduced occurrence of severe CRS in this patient population, even in individuals showing stable or progressive disease before transplantation. The best ORR and CRR in patients with TP53 alterations were 92.9% and 82.1%, respectively. Following a median follow-up of 21.2 months, 24-month PFS and OS rates in patients with TP53 alterations were estimated at 77.5% and 89.3%, respectively. In multivariable analysis, this combination strategy predicted improved OS. In conclusion, CAR19/22 T-cell therapy is efficacious in r/r aggressive B-NHL with TP53 alterations. Combining CAR-T cell administration with ASCT further improves long-term outcome of these patients.

Lignin-based fluorescence-switchable graphene quantum dots for Fe3+ and ascorbic acid detection.

The synthesis of lignin-based graphene quantum dots (GQDs) with excellent fluorescence stability, quantum yield, and biocompatibility for sensitive and selective detection of Fe3+ and ascorbic acid (AA) has remained a challenging endeavor. Using an acidolysis process with 17.5% nitric acid followed by hydrothermal treatment at 200 °C, this study provided an improved synthesis route for the production of high-quality GQDs from alkali lignin. The nitrogen-doped GQDs exhibit remarkable fluorescence stability under a wide range of pH (3-10), duration (1-12 h), and [NaCl] (0-1000 mM) conditions, and have a high quantum yield of 28%. The GQDs or GQDs/Fe3+ sensing systems ([GQDs] at 50 mg L-1, [Fe3+] at 500 µmol L-1, and UV excitation at 370 nm) for fluorescence sensing of Fe3+ or AA have excellent sensitivity, selectivity, and reproducibility. For Fe3+ and AA, the limit of detection is 1.49 and 1.62 µmol L-1, respectively. Mechanism investigation shows that photoluminescence quenching is caused by the formation of GQDs-Fe3+ complexes, whereas fluorescence recovery is due to Fe3+ reduction by AA.

CAR T cells targeting CD99 as an approach to eradicate T-cell acute lymphoblastic leukemia without normal blood cells toxicity.

CAR T cell therapy has shown dramatic clinical success in relapsed or refractory B-ALL and other hematological malignancies. However, the loss of specific antigens, cell fratricide, T cell aplasia, and normal T cell separation are challenges in treating T cell leukemia/lymphoma with CAR T therapy. CD99 is a promising antigen to target T-ALL and AML as it is strongly expressed on the majority of T-ALL and AML. Here, we isolated a low-affinity CD99 (12E7) antibody, which specifically recognizes leukemia cells over normal blood cells. Moreover, T cells transduced with an anti-CD99-specific CAR that contained the 12E7 scFv expanded with minor fratricide and without normal blood cells toxicity. We observed that our anti-CD99 CAR T cells showed robust cytotoxicity specifically against CD99+ T-ALL cell lines and primary tumor cells in vitro and significantly prolonged cell line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) models survival in vivo. Together, our results demonstrate that anti-CD99 CAR T cells could specifically recognize and efficiently eliminate CD99+ leukemia cells.

CD19/CD22 Chimeric Antigen Receptor T Cell Cocktail Therapy following Autologous Transplantation in Patients with Relapsed/Refractory Aggressive B Cell Lymphomas.

High-dose chemotherapy followed by autologous stem cell transplantation (HDT-ASCT) is the standard of care for chemosensitive relapsed or refractory (R/R) aggressive B cell lymphoma. Patients with a positive positron emission tomography (PET) scan before ASCT have a poor prognosis, and those who fail to achieve a therapeutic response better than partial remission after salvage treatment are ineligible candidates for ASCT. We conducted this open-label single-arm prospective clinical study to evaluate the safety and efficacy of sequential infusion of CD19/22 chimeric antigen receptor (CAR) T cells following HDT-ASCT. Eligibility for this study included patients with R/R aggressive B cell non-Hodgkin lymphoma (B-NHL) with 18F-fluorodeoxyglucose-PET positivity and patients with stable or progressive disease after salvage chemotherapy. Between November 14, 2016, and August 15, 2019, 42 patients underwent HDT-ASCT followed by CD19/22 CAR T cell infusion. Grade 3 cytokine release syndrome (CRS) occurred in only 2 patients. Twenty-one percent of patients experienced any grade of neurotoxicity, 5% with severe grade 3. All cases of CRS and neurotoxicity were reversible. The overall response rate was 90.5% (95% confidence interval [CI], 77.4% to 97.3%). At a median follow-up of 24.3 months, the median progression-free survival (PFS) and overall survival were not reached. The 2-year PFS rate was 83.3 % (95% CI, 68.2% to 91.7%). No patients were found to be CD19- and CD22-negative at the time of progression; 97.1% and 68.6% of patients with ongoing complete remission (CR) had consistently detectable levels of CD19 and CD22 CAR transgene, respectively, at 3 months. The median time to onset of sustained B cell recovery was 8.2 months. The high durable CR rates and favorable safety profiles support the strong potential of the HDT-ASCT plus CD19/CD22 CAR T cell cocktail therapy for the suboptimal group of patients with R/R aggressive B-NHL who are less sensitive or fail salvage chemotherapy. These early data are encouraging and informative for future trials to further test the efficacy and safety of HDT-ASCT plus CAR T cell therapy in a larger population. © 2021 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

A phase I study of anti-BCMA CAR T cell therapy in relapsed/refractory multiple myeloma and plasma cell leukemia.

BACKGROUND: Relapsed/refractory (R/R) multiple myeloma (MM) patients and primary plasma cell leukemia (PCL) have an unfavorable prognosis and no effective treatment. This study was designed to assess the safety and preliminary efficacy of a novel anti-B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cell in R/R MM and PCL. METHODS: Between February 22, 2017, and June 25, 2018, 28 R/R and two R/R primary PCL patients received a median dose of 11.2 × 106 CAR+ cells/kg. The subjects were refractory to a proteasome inhibitor and/or an immunomodulatory agent. Fludarabine and cyclophosphamide were given as lymphodepletion chemotherapy. RESULTS: Results for these 30 consecutive patients who received an anti-BCMA CAR T cell infusion are reported. The patients had received a median of four prior lines of therapy. A total of 44 different types of adverse events were recorded, and hematologic toxic effects were the most common events of any grade during treatment. Hematologic toxic effects were also the most common events of grade 3 or higher. A total of 29 patients (96.7%) had cytokine release syndrome, which was of grade 1 or 2 in 24 patients (80%) and grade 3 in five patients (16.7%). Neurologic toxic effects only occurred in one patient (3.3%) and were of grade 1. The objective response rate was 90%, and the complete response rate was 43.3%. With a median follow-up of 12.6 months, the median progression-free survival (PFS) and overall survival were 5.2 months and 14.0 months. One of the two primary PCL achieved a complete response with a PFS of 307 days. The other patients achieved a very good partial response with a PFS of 117 days. CONCLUSIONS: Anti-BCMA CAR T cell treatment is safe and highly active in R/R multiple myeloma.

Efficacy and safety of CAR19/22 T-cell cocktail therapy in patients with refractory/relapsed B-cell malignancies.

Antigen-escape relapse has emerged as a major challenge for long-term disease control after CD19-directed therapies, to which dual-targeting of CD19 and CD22 has been proposed as a potential solution. From March 2016 through January 2018, we conducted a pilot study in 89 patients who had refractory/relapsed B-cell malignancies, to evaluate the efficacy and safety of sequential infusion of anti-CD19 and anti-CD22, a cocktail of 2 single-specific, third-generation chimeric antigen receptor-engineered (CAR19/22) T cells. Among the 51 patients with acute lymphoblastic leukemia, the minimal residual disease-negative response rate was 96.0% (95% confidence interval [CI], 86.3-99.5). With a median follow-up of 16.7 months (range, 1.3-33.3), the median progression-free survival (PFS) was 13.6 months (95% CI, 6.5 to not reached [NR]), and the median overall survival (OS) was 31.0 months (95% CI, 10.6-NR). Among the 38 patients with non-Hodgkin lymphoma, the overall response rate was 72.2% (95% CI, 54.8-85.8), with a complete response rate of 50.0% (95% CI, 32.9-67.1). With a median follow-up of 14.4 months (range, 0.4-27.4), the median PFS was 9.9 months (95% CI, 3.3-NR), and the median OS was 18.0 months (95% CI, 6.1-NR). Antigen-loss relapse occurred in 1 patient during follow-up. High-grade cytokine release syndrome and neurotoxicity occurred in 22.4% and 1.12% patients, respectively. In all except 1, these effects were reversible. Our results indicated that sequential infusion of CAR19/22 T cell was safe and efficacious and may have reduced the rate of antigen-escape relapse in B-cell malignancies. This trial was registered at www.chictr.org.cn as #ChiCTR-OPN-16008526.