CD19 Chimeric Antigen Receptor T Research Articles

Comprehensive characterization of cytopenia after chimeric antigen receptor-T cell infusion in patients with relapsed or refractory multiple myeloma

BackgroundMany studies have demonstrated the effectiveness of chimeric antigen receptor-T (CAR-T) cell therapy for relapsed or refractory multiple myeloma (RRMM), but the hematologic toxicity has not been well characterized. MethodsA total of 111 adults with RRMM who received BCMA CAR-T cells, BCMA + CD19 CAR-T cells or tandem BCMA/CD19 dual-target (BC19) CAR-T cells infusion were enrolled. We characterized cytopenia and hematologic recovery at different time points after CAR-T-cell therapy, analyzed the effect of cytopenia on prognosis and identified the risk factors. ResultsPatients had a high probability of cytopenia, with anemia, neutropenia and thrombocytopenia occurring in 92%, 95% and 73%, respectively. There were 60 (54%) patients had prolonged hematologic toxicity (PHT) after D28. The median hemoglobin and platelet count were significantly lower at D28 post-CAR-T cell therapy than at baseline. Hemoglobin increased to above baseline at D90. The median absolute neutrophil count was lower than baseline at D0 and D28, and it recovered to baseline at D180. The baseline level of lactate dehydrogenase was associated with thrombocytopenia. Extramedullary involvement was associated with hemoglobin recovery, while the baseline level of albumin and types of CAR-T were related to platelet recovery. Patients with anemia at baseline and at D0, D180 and D360 had shorter progression-free survival (PFS), while anemia at D0, D60, D180 and D360 was associated with shorter overall survival (OS). Neutropenia at D0 was associated with shorter PFS and patients with neutropenia at D90 or D180 had shorter OS. Patients with thrombocytopenia at any time had shorter PFS and OS. Compared to patients without PHT, patients with PHT had shorter PFS and OS. ConclusionsThe majority of RRMM patients treated with CAR-T cells experienced cytopenia. Cytopenia occurred at specific time points was associated with a poorer prognosis.

CD19 chimeric antigen receptor-T cells as bridging therapy to allogeneic hematopoietic cell transplantation improves outcome in patients with refractory/relapsed B-cell acute lymphoblastic leukemia

Chimeric antigen receptor (CAR)-T cell therapy has been confirmed improving remission rates in refractory patients or relapsed B-cell acute lymphoblastic leukemia (R/R B-ALL). However, the added benefits of undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) following CAR-T therapy remain a subject of debate. In this research we investigated the efficiency and long-term outcomes of CD19 CAR-T bridging with allo-HSCT in R/R B-ALL patients. A total of 42 patients were brought into the cohort studies. Our findings revealed that patients who appected CAR-T followed by HSCT had a 1-year overall survival (OS) rate of 70 % and a 1-year leukemia-free survival (LFS) rate of 95 %. Moreover, patients who underwent this combined treatment had higher OS and LFS rates compared to those who received CAR-T therapy alone. In conclusion, the results of this clinical trial provide compelling evidence for the safety and efficacy of using CAR-T therapy as a bridging strategy to allo-HSCT in patients with R/R B-ALL.

Co-stimulation of CD28/CD40 signaling molecule potentiates CAR-T cell efficacy and stemness

CD19 chimeric antigen receptor T (CD19CAR-T) cells have achieved promising outcomes in relapsed/refractory B-cell malignancies. However, recurrences occur due to the loss of CAR-T cell persistence. We developed dual T/B-cell co-stimulatory molecules (CD28 and CD40) in CAR-T cells to enhance intense tumoricidal activity and persistence. CD19.28.40z CAR-T cells promoted pNF-κB and pRelB downstream signaling while diminishing NFAT signaling upon antigen exposure. CD19.28.40z CAR-T cells demonstrated greater proliferation which translated into effective anti-tumor cytotoxicity in long-term co-culture assay. Repetitive weekly antigen stimulation unveiled continuous CAR-T cell expansion while preserving central memory T-cell subset and lower expression of exhaustion phenotypes. The intrinsic genes underlying CD19.28.40z CAR-T cell responses were compared to conventional CARs and demonstrated the up-regulated genes associated with T-cell proliferation and memory as well as down-regulated genes related to apoptosis, exhaustion, and glycolysis pathway. Enrichment of genes toward T-cell stemness, particularly SELL, IL-7r, TCF7, and KLF2, was observed. Effective and continuing anti-tumor cytotoxicity in vivo was exhibited in both B-ALL and B-NHL xenograft models while demonstrating persistent T-cell memory signatures. The functional enhancement of CD37.28.40z CAR-T cell activities against CD37+ tumor cells was further validated. The modification of dual T/B-cell signaling molecules remarkably maximized the efficacy of CAR-T cell therapy.

Single-cell analysis of the survival mechanisms of fratricidal CAR-T targeting of T cell malignancies

Chimeric antigen receptor T (CAR-T) cell therapy targeting T-cell tumors still faces many challenges, one of which is its fratricide due to the target gene expressed on CAR-T cells. Despite this, these CAR-T cells can be expanded in vitro via extending culture time and effectively eliminating malignant T cells. However, the mechanisms underlying CAR-T cell survival from cell subpopulations, molecules and regulation are still unknown. We performed single-cell transcriptome profiling to investigate the fratricidal CAR-T products (CD26 CAR-T and CD44v6 CAR-T) targeting T cells, taking CD19 CAR-T targeting B cells from the same donor as a control. Compared with CD19 CAR-T, fratricidal CAR-T cells exhibit no unique cell subpopulation, but have more exhausted T cells, fewer cytotoxic T cells and more TCR clonal amplification. Furthermore, we observed that fratricidal CAR-T cell survival was accompanied by target gene expression. Gene expression results suggests that fratricidal CAR-T cells may downregulate their HLA molecules to evade T-cell recognition. Single-cell regulatory network analysis and suppression experiments revealed that exhaustion mediated by critical regulatory factors may contribute to fratricidal CAR-T cell survival. Together, these data provide valuable and first-time insights into the survival of fratricidal CAR-T cells.

Emerging Innate Immune Cells in Cancer Immunotherapy: Promises and Challenges.

Immune checkpoint inhibitor (ICI)-based therapy has made an unprecedented impact on survival benefit for a subset of cancer patients; however, only a subset of cancer patients is benefiting from ICI therapy if all cancer types are considered. With the advanced understanding of interactions of immune effector cell types and tumors, cell-based therapies are emerging as alternatives to patients who could not benefit from ICI therapy. Pioneering work of chimeric antigen receptor T (CAR-T) therapy for hematological malignancies has brought encouragement to a broad range of development for cellular-based cancer immunotherapy, both innate immune cell-based therapies and T-cell-based therapies. Innate immune cells are important cell types due to their rapid response, versatile function, superior safety profiles being demonstrated in early clinical development, and being able to utilize multiple allogeneic cell sources. Efforts on engineering innate immune cells and exploring their therapeutic potential are rapidly emerging. Some of the therapies, such as CD19 CAR natural killer (CAR-NK) cell-based therapy, have demonstrated comparable early efficacy with CD19 CAR-T cells. These studies underscore the significance of developing innate immune cells for cancer therapy. In this review, we focus on the current development of emerging NK cells, γδ T cells, and macrophages. We also present our views on potential challenges and perspectives to overcome these challenges.

Abstract 4002: Investigating CAR-T cell efficacy and activation in the disseminated NALM6-luc human B-cell acute lymphoblastic leukemia model

Abstract Introduction: Chimeric Antigen Receptor T (CAR-T) cell therapy has emerged as a promising approach for treating hematological malignancies, particularly B-cell acute lymphoblastic leukemia (B-ALL). This study aimed to delve into the activation dynamics and therapeutic efficacy of CD19 CAR-T cells in a disseminated NALM6-luc human B-ALL murine model. Experimental design: NALM6-Luc-mCh-Puro cells were implanted intravenously in female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. Animals were treated with CD19 CAR-T cells alone and in combination with ibrutinib. The anti-tumor activity was assessed, and the persistence and activation of CAR-T cells in the blood were evaluated using flow cytometry. Additionally, ddPCR analysis was employed to quantify CAR copy numbers, providing insights into the T-cell persistence over time. Results: Treatment with CD19 CAR-T cells exhibited notable anti-cancer activity and that activity was enhanced in combination with ibrutinib, in line with other reports. The increased time to progression was noted as 69.2% for the CAR-T group and 138.4% for the combination of CAR-T and ibrutinib compared to control. Flow cytometry analysis showed that CAR-T cells persisted in the blood throughout the study. The mean CAR-T absolute cell count for both CAR-T and CAR-T plus ibrutinib treated groups increased from 2 cell/µL blood on Day 0 to 11 and 51 cells/µL of blood, respectively, by Day 39. The progressive increase of PD-1+ and LAG-3+ percentage cells correlates positively with tumor burden, indicating a dynamic link between biomarker upregulation and the presence of tumor cells in mice. ddPCR analysis revealed an initial CAR copy decrease in all groups, followed by gradual increase in tumor-bearing mice treated with CD19 CAR-T cells, with a dramatic increase observed by Day 39. Results from CAR-T treated and CAR-T plus ibrutinib treated groups were comparable, showing mean copy numbers per ng of DNA increasing from less than 1 on day 4 to 17.9 and 23.3, respectively, on day 39. Importantly, no changes were noted in non-tumor implanted mice, emphasizing the tumor dependency for CAR-T cell expansion. Conclusion: This study emphasizes the marked anti-cancer activity of CD19 CAR-T cells in the NALM6-Luc-mCh-Puro B-ALL murine model. Comprehensive analysis of CAR-T cell persistence and activation, including insights from ddPCR, provides a robust understanding of therapeutic response dynamics. The observed in vivo anti-cancer efficacy aligns with the persistence of CAR-T cells revealed by flow cytometry and the dynamic expansion captured through ddPCR analysis. Our results indicate ddPCR and flow cytometry as complementary and independent methods for assessing the systemic CAR-T cell persistence, contributing to optimizing therapy for B-cell acute lymphoblastic leukemia. Citation Format: Prashant Pandey, David Draper, Sheri Barnes, Yewei Xing, Derrik Germain, Lauren Kucharczyk, Roys Stacey. Investigating CAR-T cell efficacy and activation in the disseminated NALM6-luc human B-cell acute lymphoblastic leukemia model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4002.

Targeting autophagy overcomes cancer-intrinsic resistance to CAR-T immunotherapy in B-cell malignancies.

Chimeric antigen receptor T (CAR-T) therapy has substantially revolutionized the clinical outcomes of patients with hematologic malignancies, but the cancer-intrinsic mechanisms underlying resistance to CAR-T cells remain yet to be fully understood. This study aims to explore the molecular determinants of cancer cell sensitivity to CAR-T cell-mediated killing and to provide a better understanding of the underlying mechanisms and potential modulation to improve clinical efficacy. The human whole-genome CRISPR/Cas9-based knockout screening was conducted to identify key genes that enable cancer cells to evade CD19 CAR-T-cell-mediated killing. The in vitro cytotoxicity assays and evaluation of tumor tissue and bone marrow specimens were further conducted to confirm the role of the key genes in cancer cell susceptibility to CAR-T cells. In addition, the specific mechanisms influencing CAR-T cell-mediated cancer clearance were elucidated in mouse and cellular models. The CRISPR/Cas9-based knockout screening showed that the enrichment of autophagy-related genes (ATG3, BECN1, and RB1CC1) provided protection of cancer cells from CD19 CAR-T cell-mediated cytotoxicity. These findings were further validated by in vitro cytotoxicity assays in cells with genetic and pharmacological inhibition of autophagy. Notably, higher expression of the three autophagy-related proteins in tumor samples was correlated with poorer responsiveness and worse survival in patients with relapsed/refractory B-cell lymphoma after CD19 CAR-T therapy. Bulk RNA sequencing analysis of bone marrow samples from B-cell leukemia patients also suggested the clinical relevance of autophagy to the therapeutic response and relapse after CD19 CAR-T cell therapy. Pharmacological inhibition of autophagy and knockout of RB1CC1 could dramatically sensitize tumor cells to CD19 CAR-T cell-mediated killing in mouse models of both B-cell leukemia and lymphoma. Moreover, our study revealed that cancer-intrinsic autophagy mediates evasion of CAR-T cells via the TNF-α-TNFR1 axis-mediated apoptosis and STAT1/IRF1-induced chemokine signaling activation. These findings confirm that autophagy signaling in B-cell malignancies is essential for the effective cytotoxic function of CAR-T cells and thereby pave the way for the development of autophagy-targeting strategies to improve the clinical efficacy of CAR-T cell immunotherapy.

Preclinical evaluation of cyclophosphamide and fludarabine combined with CD19 CAR-T in the treatment of B-cell hematologic malignancies in vivo.

Chimeric antigen receptor T (CAR-T) cell therapy has achieved marked therapeutic success in ameliorating hematological malignancies. However, there is an extant void in the clinical guidelines concerning the most effective chemotherapy regimen prior to chimeric antigen receptor T (CAR-T) cell therapy, as well as the optimal timing for CAR-T cell infusion post-chemotherapy. We employed cell-derived tumor xenograft (CDX) murine models to delineate the optimal pre-conditioning chemotherapy regimen and timing for CAR-T cell treatment. Furthermore, transcriptome sequencing was implemented to identify the therapeutic targets and elucidate the underlying mechanisms governing the treatment regimen. Our preclinical in vivo evaluation determined that a combination of cyclophosphamide and fludarabine, followed by the infusion of CD19 CAR-T cells five days subsequent to the chemotherapy, exerts the most efficacious therapeutic effect in B-cell hematological malignancies. Concurrently, RNA-seq data indicated that the therapeutic efficacy predominantly perturbs tumor cell metabolism, primarily through the inhibition of key mitochondrial targets, such as C-Jun Kinase enzyme (C-JUN). In summary, the present study offers critical clinical guidance and serves as an authoritative reference for the deployment of CD19 CAR-T cell therapy in the treatment of B-cell hematological malignancies.

Efficacy and Safety of the Second CAR-T Therapy in Patients with Refractory/Relapsed Acute B-Cell Lymphoblastic Leukemia

Background: Chimeric antigen receptor T (CAR-T) cells therapy has shown significant efficacy in relapsed or refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), but a substantial proportion of patients still relapse after a period of remission. The efficacy of a second CAR-T cell infusion is uncertain. The aim of this study was to explore the efficacy and safety of the second CAR-T therapy in R/R B-ALL patients. Methods: Between August 2018 and October 2022, 35 patients with R/R B-ALL successfully received the second CAR-T treatment. Among them, 18 patients received single CD19 or CD22 CAR-T therapy, 14 patients received tandem CD19/CD22 CAR-T therapy, and 3 patients received sequential CD19 and CD22 CAR-T therapy. Results: The complete remission (CR) rates were 55.6% (10/18) in patients who received CD19 or CD22 CAR-T therapy, 85.7% (12/14) in patients who received tandem CD19/CD22 CAR-T therapy, and 33.3% (1/3) in patients who received sequential CD19 and CD22 CAR-T therapy (tandem CD19/CD22 vs. others, P=0.045). Tandem CD19/CD22 CAR-T therapy remained one of the significant favorable factors in multivariate logistic regression analysis of CR rate in all patients (hazard ratio, 0.128; 95% CI, 0.017-0.974). A total of 5 patients with severe cytokine release syndrome (Grade ≥ 3) were observed, including 2 patients in the single CD19 CAR-T group, 2 patients in the tandem CD19/CD22 CAR-T group, and 1 patient in the sequential CD19 and CD22 CAR-T group. Of the 23 patients who achieved CR, 4 patients bridged to allogeneic hematopoietic stem cell transplantation (allo-HSCT) and 3 patients received decitabine (DAC) consolidation.With the median follow-up of 25.5 months (range, 1.1 to 33.5), the 2-year overall survival, leukemia-free survival (LFS) and cumulative incidence of relapse rates were 23.7%, 30.5% and 69.5%, respectively. Multivariate Cox regression analyses showed that a better LFS related to the absence of high-risk cytogenetics and genetic characteristics, DAC combination with fludarabine and cyclophosphamide lymphodepletion regimen, and bridging allo-HSCT or DAC consolidation. Conclusions: Our study showed that the second infusion of tandem CD19/CD22 CAR-T therapy obtains a better response than other infusion strategies. Bridging allo-HSCT or DAC consolidation had a significant survival benefit in patients with CR after the second CAR-T therapy.

Prophylactic or Preemptive Donor-Derived CD19 CAR-T Cell Infusion for Preventing Relapse in High-Risk B-ALL after Allogeneic Hematopoietic Stem Cell Transplantation

Introduction: Relapse remains a significant challenge in the management of high-risk B-cell acute lymphoblastic leukemia (B-ALL) patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Existing post-transplant relapse prevention strategies for high-risk B-ALL patients after transplantation, particularly in cases of Philadelphia chromosome-negative B-ALL, have limitations and suboptimal efficacy. This study aims to evaluate the efficacy and safety profile of prophylactic or preemptive infusion of donor-derived CD19 chimeric antigen receptor T (CAR-T) cells in high-risk B-ALL patients after allo-HSCT. Methods: Between May 2017 and July 2022, high-risk B-ALL patients with minimal residual disease (MRD) positive or MRD negative status after allo-HSCT were enrolled in clinical trials of donor-derived CD19 CAR-T cell therapy (ChiCTR 2000041025 and ChiCTR-ONN-16009862). The primary outcome was the cumulative incidence of relapse (CIR). Secondary outcomes were progression-free survival (PFS), overall survival (OS) and CAR-T cell treatment-related toxicities. Results: A total of 25 high-risk B-ALL patients who received infusion of donor-derived CD19 CAR-T cells after allo-HSCT were enrolled in the CAR-T cell group, including 8 patients with MRD and 17 patients without MRD. A contemporaneous cohort of 43 high-risk B-ALL patients who did not receive CAR-T cell therapy in MRD-positive or MRD-negative status after transplantation was also included as a control group. The median age of patients enrolled in the CAR-T group was 29 years (range: 9-60 years). The median infused CAR-T cell dose was 2.5×10^6/kg (range: 1.0-5.0×10^6/kg), with a median time from transplantation to CAR-T cell treatment of 122 days (range: 45-315 days). The median follow-up duration was 23.6 months (range: 8.3-60.6 months). Of the 25 patients in CAR-T group, eight MRD-positive patients achieved MRD-negative remission after CAR-T cells treatment (Figure1A). The two-year cumulative incidence of relapse in the CAR-T cell group was significantly lower at 4.8% (95% CI: 0.0%-12.6%) compared to the control group's incidence of 26.6% (95% CI: 11.4%-41.8%, P = 0.03，Figure1B). Additionally, the CAR-T cell group exhibited a higher two-year PFS rate [86.3% (95% CI: 71.8%-100%)] compared to the control group [63.5% (95% CI: 48.0%-79.0%), P = 0.04]. However, the two-year OS rates were comparable between the CAR-T cell group [91.0% (95% CI: 79.0%-100%)] and the control group [79.1% (95% CI: 66.0%-92.2%), P = 0.20]. Adverse events were well tolerated, and no patients experienced grade 3-4 cytokine release syndrome or any grade of immune effector cell-associated neurotoxicity syndrome. Hematological cytopenia was the most severe CAR-T cell related toxicity, but the toxicity was manageable, and only three patients experienced acute graft-versus-host disease following CAR-T cell infusion. Conclusions: Prophylactic or preemptive infusion of CAR-T cells after allo-HSCT demonstrated a reduced incidence of relapse and acceptable adverse events in high-risk B-ALL patients. These findings highlight the potential of CAR-T cell therapy as a promising strategy for relapse prevention in high-risk B-ALL patients after allo-HSCT.

Characteristics of Central Nervous System Relapse after CD19 CAR-T Cell Therapy in R/R NHL

Background CD19-targeted chimeric antigen receptor T (CAR-T) cell therapy has shown remarkable antitumor activity against relapsed/refractory (R/R) non-Hodgkin lymphoma (NHL). However, NHL with central nervous system (CNS) relapse carries a poor prognosis and high mortality. We reported an analysis of 80 patients with NHL who underwent CAR-T cell therapy, and described the characteristics including the international prognostic index (IPI) and other clinical variables to evaluate the outcomes of CNS relapse in NHL patients treated with CAR-T cell therapy. Methods A total of 80 patients with NHL treated with CD19 CAR-T cell therapy in our center between April 2017 and November 2022 was retrospectively analyzed. Patient characteristics, laboratory parameters, and clinical outcomes were collected. CNS relapse was diagnosed by cerebrospinal fluid cytology, MRI or PET-CT. Results Baseline characteristics of all 80 patients are summarized in Table 1. The median age was 58.5 (range, 10 to 79) years, and 57.5% of patients were male. Overall, 67 (83.8%) patients achieved complete remission (CR) or partial remission (PR) after CD19 CAR-T therapy. Seven (8.43%) patients developed isolated CNS relapse after CAR-T cell therapy, all of which were heavily pre-treated with multiple lines of chemotherapy prior to CAR-T cell therapy, and one of them had received autologous stem cell transplant before infusion of CAR-T cells. Only 4 of 7 patients with isolated CNS relapse achieved CR (n=4), and the median time to isolated CNS relapse from CAR-T cell infusion was 6.3(range, 1.53 to 25.8) months. Thirty-seven patients (46.2%) developed systemic recurrence without CNS involvement, with the median time from CAR-T to relapse of 3.2 (range, 0.467-30.8) months. The other 36 patients remained in remission at the last follow-up. During the median follow-up of 13.5 months of the whole cohort, the number of recorded deaths in isolated CNS relapse, systemic only recurrence, and no relapse patients was 5 (71.4%), 22 (59.4%), 1(2.78%), respectively. The 1-year OS and 1-year PFS of patients with isolated CNS recurrence was significantly lower compared to the no relapse group (OS, 35.7% [95%CI 12.1-100] vs 97.22% [95%CI 92-100], P<0.001; PFS, 28.6% [95%CI 8.86-92.2] vs 100% [95%CI 100-100], P<0.001), but showed no difference compared to the systemic only recurrence group(OS, 53.3% [95%CI 39.3-72.4], P=0.79; PFS, 13.0% [95%CI 5.53-30.5], P=0.17). Conclusion In conclusion, patients with CNS relapse showed significant difference in 1-year outcomes with no relapse group but not systemic relapse group in our study, which suggested the importance of effective prophylaxis against CNS relapse. Further investigation is necessary to better define the characteristics of patients who will benefit from prophylaxis. Acknowledgements We gratefully thank all the participation from patients enrolled in clinical trials (ChiCTR1800015575; NCT03118180; NCT04532281; NCT04532268; NCT04213469).

Autophagy Inhibition Prevents CAR-T Exhaustion and Terminal Differentiation Via TCF7 Accumulation

Introduction: Chimeric antigen receptor T (CAR-T) cell therapy has achieved remarkable success in hematological malignancies. However, CAR-T cell dysfunction due to exhaustion has been identified as a major roadblock to effective CAR-T cell therapy. Autophagy mediates T cell differentiation, survival, and memory by regulating the degradation of organelles and specific proteins. A recent study reported that inhibition of autophagy significantly enhanced the killing effect of CD19 CAR-T cells. Yet, the impact of autophagy inhibitors on CAR-T cell exhaustion and maintenance is still unclear. The study aims to systematically evaluate the role and mechanism of autophagy inhibitors on CAR-T cells. Methods and Results: To explore whether autophagy inhibitors could prevent CAR-T cell exhaustion triggered by antigen-independent CAR tonic signaling, we applied 3 autophagy inhibitors: SBI0206965, MRT67307, Compound C, respectively, to the culture medium of CD19.4-1BBz CAR-T cells. We found that all the 3 autophagy inhibitors could reduce CAR-T cell exhaustion and terminal differentiation. The results were replicable with GD2.28z CAR-T cells. Among the three inhibitors, SBI0206965 showed the most significant effect. Thus, we chose SBI0206965 for further research. CD19.4-1BBz CAR-T cells pretreated with SBI0206965 demonstrated enhanced killing capacity against Nalm-6 cells in vitro. Using the Nalm-6-bearing leukemia xenograft model, we found that compared to DMSO pretreated CD19.4-1BBz CAR-T cells, SBI0206965 pretreated CD19.4-1BBz CAR-T cells showed superior persistence and antitumor efficacy, exhibited a less exhausted and differentiated state, and further prolonged mice survival (Fig.1). To evaluate whether SBI0206965 could protect CAR-T cell exhaustion triggered by antigen stimulation, we cocultured CD19.4-1BBz CAR-T cells with Nalm-6 cells in the medium with or without SBI0206965. We demonstrated that the addition of SBI0206965 could effectively inhibit CAR-T cell exhaustion and terminal differentiation driven by antigen exposure. By performing proteomics, we identified TCF7 enriched in SBI0206965-treated cells. We validated the differences using Western blot analysis, which showed increased TCF7 levels in CAR-T cells treated with SBI0206965 for 24 hours. Thus we speculated that inhibition of autophagy might contribute to the accumulation of TCF7 and further improve CAR-T maintenance. To further verify whether TCF7 was degraded in an autophagy-dependent manner, we next determined TCF7 turnover rates in the presence of cycloheximide (CHX) in CAR-T cells with or without SBI0206965 treatment. CHX chase assay showed that SBI0206965 treatment could slower the TCF7 degradation rate(Fig.2). Significance: Our research provides a strategy to optimize CAR-T antitumor efficacy and persistence via TCF7 accumulation by autophagy inhibition.

CD19 CAR-T cell therapy for relapsed or refractory diffuse large B cell lymphoma: Why does it fail?

Chimeric antigen receptor T (CAR-T) cell therapy is an effective treatment for relapsed or refractory diffuse large B cell lymphoma (DLBCL) with 3 CD19 targeting products now FDA-approved for this indication. However, up to 60% of patients ultimately progress or relapse following CAR-T cell therapy. Mechanisms of resistance to CAR-T cell therapy in patients with DLBCL are likely multifactorial and have yet to be fully elucidated. Determining patient, tumor and therapy-related factors that may predict an individual's response to CAR-T cell therapy requires ongoing analysis of data from clinical trials and real-world experience in this population. In this review we will discuss the factors identified to-date that may contribute to failure of CAR-T cell therapy in achieving durable remissions in patients with DLBCL.

Tislelizumab augment the efficacy of CD19/22 dual-targeted chimeric antigen receptor T cell in advanced stage relapsed or refractory B-cell non-Hodgkin lymphoma.

Dual-targeted chimeric antigen receptor T (CAR-T) cell is an important strategy to improve the efficacy of CD19 CAR-T cell against refractory or relapsed B cell non-Hodgkin lymphoma (R/R B-NHL). However, durable responses are not achieved in most patients, in part owing CAR-T cell exhaustion caused by PD-1/PD-L1 pathway.We conducted a prospective, single-arm study of dual-targeted CD19/22 CAR-T cell combined with anti-PD-1 antibody, tislelizumab, in R/R B-NHL (NCT04539444). Tislelizumab was administrated on +1day after patients received infusion of CD19/22 CAR-T cell. Responses, survival and safety were evaluated. From 1 August 2020 to 30 March 2023, 16 patients were enrolled. The median follow-up time is 16.0 (range: 5.0-32.0months) months. Overall response was achieved in 14 of 16 (87.5%) patients, and the complete response (CR) was achieved in 11 of 16 (68.8%) patients. The 1-year progression-free survival and overall survival rates were 68.8% and 81.3%, respectively. Of the 14 patients responded, 9 patients maintained their response until the end of follow-up. Among the 15 out of 16 (93.8%) patients who had extranodal involvement, 14 (93.3%) patients achieved overall response rate with 11 (73.3%) patients achieving CR. Eight (50%) patients experienced cytokine release syndrome. No neurologic adverse events were reported. Gene Ontology-Biological Process enrichment analysis showed that immune response-related signaling pathways were enriched in CR patients. Our results suggest that CD19/22 CAR-T cell combined with tislelizumab elicit a safe and durable response in R/R B-NHL and may improve the prognosis of those patients.

Anti-CD79b/CD3 bispecific antibody combined with CAR19-T cells for B-cell lymphoma treatment.

CD19 CAR-T (chimeric antigen receptor-T) cell immunotherapy achieves a remission rate of approximately 70% in recurrent and refractory lymphoma treatment. However, the loss or reduction of CD19 antigen on the surface of lymphoma cells results in the escape of tumor cells from theimmunekilling of CD19 CAR-T cells (CAR19-T). Therefore, novel therapeutic strategies are urgently required. In this study, an anti-CD79b/CD3 bispecific antibody (BV28-OKT3) was constructed and combined with CAR19-T cells for B-cell lymphoma treatment. When the CD19 antigen was lost or reduced, BV28-OKT3 redirected CAR19-T cells to CD79b+ CD19- lymphoma cells; therefore, BV28-OKT3 overcomes the escape of CD79b+ CD19- lymphoma cells by the killing action of CAR19-T cells in vitro and in vivo. Furthermore, BV28-OKT3 triggered the antitumor function of CAR- T cells in the infusion product and boosted the antitumor immune response of bystander T cells, markedly improving the cytotoxicity of CAR19-T cells to lymphoma cells in vitro and in vivo. In addition, BV28-OKT3 elicited the cytotoxicity of donor-derived T cells toward lymphoma cells in vitro, which depended on the presence of tumor cells. Therefore, our findings provide a new clinical treatment strategy for recurrent and refractory B-cell lymphoma by combining CD79b/CD3 BsAb with CAR19-T cells.

Effectiveness and safety of CD22 and CD19 dual-targeting chimeric antigen receptor T-cell therapy in patients with relapsed or refractory B-cell malignancies: A meta-analysis.

The efficacy of CD22 or CD19 chimeric antigen receptor T (CAR-T) cells in the management of acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL) was observed. Because antigen loss and lack of CAR-T-cell persistence are the leading causes of progressive disease following single-antigen targeting, we evaluated CD22/CD19 dual-targeting CAR-T-cell therapy efficacy and safety in relapsed/refractory B-cell malignancies. The Web of Science, PubMed, Cochrane, and Embase databases were searched until July 2022. Patients confirmed with any relapsed/refractory B-cell hematological malignancies were included regardless of age, gender, or ethnicity, receiving CD22 and CD19-dual-targeting CAR-T-cell therapy. The studies conducted on patients with coexisting other cancer were excluded. We used random-effect models to explore the outcome, and heterogeneity was investigated by subgroup analysis. Fourteen studies (405 patients) were included. The pooled overall response (OR) and complete remission (CR) were 97% and 93%, respectively, for ALL patients. The 1-year proportions of overall survival (OS) and progression-free survival (PFS) were 70% and 49%, respectively. For NHL, OR occurred in 85% of patients, and 57% experienced CR. The results illustrated that the 1-year OS and 1-year PFS were 77% and 65%, respectively. The subgroup analysis showed that the dual-targeting modality achieved higher CR in the following cases: coadministration of CD22/CD19-CAR-T cells and third-generation CAR-T cells combined with ASCT and BEAM pretreatment. The ALL and NHL groups seemed similar in treatment-related toxicity: all grade cytokine release syndrome (CRS), severe CRS, and neurotoxicity occurred in 86%, 7%, and 12% of patients, respectively. Our meta-analysis demonstrated that the CD22/CD19 dual-targeting CAR-T-cell strategy has high efficiency with tolerable adverse effects in B-cell malignancies.

CD19 chimeric antigen receptor T cell therapy in leukemia xenograft mouse: Anti-leukemic efficacy, kinetics, and 4-week single-dose toxicity

CD19 Chimeric antigen receptor T (CAR-T) cell therapy has shown a promising response rate for relapsed/refractory B-cell malignancies. However, serious side effects such as cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome arose in early case reports. Though several preclinical and clinical studies of CAR-T cell therapy have been reported, there is a lack of toxicological assessments. This study was carried out as a preclinical assessment of CD19 CAR-T cell therapy, including the anti-leukemic efficacy, kinetics in peripheral blood, and 4-week single-dose toxicity evaluation in leukemia xenograft mice. Leukemia xenograft mice model was established by injecting 1.0 × 105 cells/mouse of luciferase-labeled human B cell acute lymphoblastic leukemia (B-ALL) cell line via the tail vein, and after 3 days, 2.0 or 4.0 × 106 cells/mouse of CD19 CAR-T cells were injected intravenously. CD19 CAR-T cells showed significant anti-leukemic efficacy, showing inhibition of tumor progression in the bioluminescence-based in-vivo imaging system. In the kinetics study using qPCR, CAR-T cells peaked in peripheral blood on day 60 in males and day 30 in females. In a 4-week single-dose toxicity study, CD19 CAR-T cell injected groups showed no mortality and toxicological signs, or changes in body weight, food/water consumption, hematology, clinical chemistry, organ weights, and histopathology compared to control groups. These results suggested that 4.0 × 106 cells/mouse of CD19 CAR-T cells were effective in B-ALL xenograft mice without serious side effects, so the no-observed adverse effect level (NOAEL) was estimated to be higher than 4.0 × 106 cells/mouse, under the condition examined in the current study.

Abstract 1847: Engineered cell surface tag-targeted IL-2 and IL-21 selectively and safely enhance CAR-T anti-tumor activity via different mechanisms

Abstract The treatment of some patients with hematological malignancies has been transformed by chimeric antigen receptor T (CAR-T) cells. However, a large clinical need remains for more effective CAR-T therapies and to expand their use to broader patient groups including those with solid tumors. The exogenous administration of IL-2 and IL-21 were shown to enhance the engraftment, persistence, and functionality of CAR-Ts in preclinical models. However, clinical use of such cytokines is limited due to their pleiotropic nature that can result in toxicity and undesired effects on endogenous immune cells. To overcome this, we applied our cis-targeting technology to develop CAR-T specific IL-2 and IL-21 molecules that selectively activate CAR-Ts and exhibit minimal activity on non-CAR cells. Cis-targeted IL-2 and IL-21 molecules are comprised of a targeting antibody fused to an affinity-attenuated cytokine mutein, the activity of which is rescued due to the avidity provided by the targeting arm. This enables the highly selective delivery of cytokine support to human CAR-T cells. We engineered two types of fusions targeting either 1) the CAR directly without blocking CAR antigen recognition (CAR-IL2) or 2) an exogenous cell surface tag (non-signaling truncated epidermal growth factor receptor [EGFRt]) co-expressed with the CAR (EGFRt-IL2 and EGFRt-IL21). We characterized these molecules in vitro and in vivo. Both CAR- and EGFRt-targeted molecules demonstrated >100-fold preferential STAT activation of CAR-T cells over non-CAR cells. Unexpectedly, CAR-targeted fusions induced substantial antigen independent cytokine release, whereas fusions targeting the EGFRt tag did not. We found that antigen independent cytokine release was more suppressed by a LCK inhibitor rather than a JAK inhibitor, suggesting that downstream CAR signaling was the major contributor, compared to cytokine receptor signaling. In vivo studies examined the anti-tumor activity of the tag-targeted cytokine fusions given their desired safety profile. A single dose of either EGFRt-IL2 or EGFRt-IL21 on day 1 or day 7 following a sub-optimal infusion of CD19 CAR-T cells (0.1 x106) induced strong tumor regression and relapse free survival for >70 days in an aggressive NALM-6 leukemia model. EGFRt-IL21 induced a sustained 10-fold expansion of CAR-T numbers over PBS in the blood by day 7. In contrast, EGFRt-IL2 induced a delayed but greater expansion (1000-fold over PBS) by day 14 suggestive of a mechanistic difference of the two cis-targeted molecules. Analysis of EGFRt-IL2 and EGFRt-IL21 in a solid tumor model using CAR-T cells targeting endogenous tumor antigen showed similar anti-tumor effects. Cis-targeted IL-2 and IL-21 fusion molecules directed by anti-tag EGFRt antibodies confer selective enhancement of CAR-T cells with a minimal safety risk, representing a promising approach as an adjuvant CAR-T therapy. Citation Format: Nathan D. Mathewson, Wei Chen, Paul Bessette, Sara Sleiman, Meghana Sukthankar, Kelly D. Moynihan, Chris Kimberlin, Terrence Park, Audrey Hollingsworth, Saar Gill, Andy Yeung, Ivana Djuretic. Engineered cell surface tag-targeted IL-2 and IL-21 selectively and safely enhance CAR-T anti-tumor activity via different mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1847.

Dynamic Study on Tumor Killing Activity of CD19 Chimeric Antigen Receptor-T Lymphocytes

Malignant tumor is a major factor in global disease burden. In recent years, its incidence is still increasing year by year. Because of its complex etiology and the frequent recurrence and metastasis after general surgery, it brings great challenges to global public health. The emergence of new technology, that is, “Chimeric Antigen Receptor-T” immuno-therapy, has created a new era for the treatment of hematological malignant tumors, such as non-Hodgkin’s lymphoma and acute B-lymphocytic leukemia. In order to evaluate the killing effect of CART tumor immuno-therapy on tumor cells more intuitively, CD19-CART cells targeting B-cell lymphoma were constructed in vitro, combined with innovative in vitro real-time cellular analysis technology. Meanwhile, it is compared with common T lymphocytes in the human body to observe their killing activity against targeted tumors. The results showed that in comparison with common T lymphocytes, CD19-CART cells constructed in this paper showed significantly enhanced killing activity for targeting tumors and a dose-dependent effect on the ratio of response to the target. Besides, CD19-CART cells constructed in this paper have a remarkable killing effect on tumor cells and adopt superior technical methods to observe the killing activity more intuitively, which provides a solid theoretical basis for the clinical application of CART cell tumor immuno-therapy technology.

A simple and effective method to purify and activate T cells for successful generation of chimeric antigen receptor T (CAR-T) cells from patients with high monocyte count

BackgroundChimeric antigen receptor T (CAR-T) cells are genetically modified T cells with redirected specificity and potent T-cell-mediated cytotoxicity toward malignant cells. Despite several CAR-T products being approved and commercialized in the USA, Europe, and China, CAR-T products still require additional optimization to ensure reproducible and cost-effective manufacture. Here, we investigated the critical parameters in the CD3+ T-cell isolation process that significantly impacted CAR-T manufacturing's success.MethodsCAR-T cells were prepared from cryopreserved peripheral blood mononuclear cells (PBMC). The thawed PBMC was rested overnight before the CD3+ T cell isolation process using CTS™ Dynabeads™ CD3/CD28. Different isolation media, cell-bead co-incubation time, and cell density were examined in this study. Activated CD3+ T cells were transduced with a gamma retroviral vector carrying the CD19 or BCMA CAR sequence. The CAR-T cells proliferated in a culture medium supplemented with interleukin 2 (IL-2).ResultsCD14+ monocytes hindered T-cell isolation when X-VIVO 15 basic medium was used as the selection buffer. The activation of T cells was blocked because monocytes actively engulfed CD3/28 beads. In contrast, when DPBS was the selection medium, the T-cell isolation and activation were no longer blocked, even in patients whose PBMC contained abnormally high CD14+ monocytes and a low level of CD3+ T cells.ConclusionsIn this study, we discovered that selecting CD3+ T-cell isolation media is critical for improving T-cell activation, transduction, and CAR-T proliferation. Using DPBS as a CD3+ T cell isolation buffer significantly improved the success rate and shortened the duration of CAR-T production. The optimized process has been successfully applied in our ongoing clinical trials.Trial registration NCT03798509: Human CD19 Targeted T Cells Injection Therapy for Relapsed and Refractory CD19-positive Leukemia. Date of registration: January 10, 2019. NCT03720457: Human CD19 Targeted T Cells Injection (CD19 CAR-T) Therapy for Relapsed and Refractory CD19-positive Lymphoma. Date of registration: October 25, 2018. NCT04003168: Human BCMA Targeted T Cells Injection Therapy for BCMA-positive Relapsed/Refractory Multiple Myeloma. Date of registration: July 1, 2019