CAR-T Cell Persistence Research Articles

Epigenetic reprogramming of CAR T cells for in vivo functional persistence against solid tumors.

Limited CAR T-cell expansion and persistence hinder therapeutic responses in solid cancer patients. To enhance the functional persistence of engineered T-cell therapies, we performed genetic disruption in human CAR T cells of SUV39H1, a histone 3 lysine 9 methyltransferase that promotes heterochromatin formation. This resulted in phenotypic CAR-T reprogramming that elicited optimal and sustained antitumor functionality. Single-cell transcriptomic (scRNA-seq) and chromatin accessibility (scATAC-seq) analyses of tumor-infiltrating CAR T cells showed early reprogramming into self-renewing, stem-like populations with decreased expression of dysfunction genes in all subpopulations. Moreover, we provided evidence that SUV39H1 inactivation elicits potent and durable functional persistence upon multiple tumor rechallenges. This opens a safe path to enhancing adoptive cell therapies for solid tumors.

CAR T-cells for T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) has a dismal prognosis in case of relapsed or refractory disease. Contrary to B-ALL, few immunotherapies are available for T-ALL. Use of autologous CAR T-cells is challenging due to shared antigen between leukemic and normal T-cells responsible for fratricide and T-cell aplasia in case of persistence of CAR T-cells. Moreover, risk of contamination of the apheresis product by lymphoblasts remains an issue. To counteract these challenges several methods are used to edit T-cell such as protein expression blocker, CRISPR/Cas9 and base-editing. Other possibility is to use autologous T-cells naturally selected in vitro or donor-derived T-cells allowing gene edition and reduction of the risk of graft vs host disease. Encouraging results are obtained in preclinical and clinical studies for early response rate but several questions remain. Is the persistence of these cells requiring for maintaining the remission? Is it feasible to recover a target-negative T-cell population without risk of profound immunosuppression? Has an allogeneic stem cell transplantation to be planned for patients after CAR T-cells infusion? What about the risk of engineered T-cells in the long term?

Targeting the activated microenvironment with endosialin (CD248)-directed CAR-T cells ablates perivascular cells to impair tumor growth and metastasis

BackgroundTargeting of solid cancers with chimeric antigen receptor (CAR)-T cells is limited by the lack of suitable tumor-specific antigens and the immunosuppressive, desmoplastic tumor microenvironment that impedes CAR-T cell infiltration,...

Single-cell analysis of refractory anti-SRP necrotizing myopathy treated with anti-BCMA CAR-T cell therapy

Immune-mediated necrotizing myopathy (IMNM) is an autoimmune disorder associated with the presence of autoantibodies, characterized by severe clinical presentation with rapidly progressive muscular weakness and elevated levels of creatine kinase, while traditional pharmacological approaches possess varying and often limited effects. Considering the pathogenic role of autoantibodies, chimeric antigen receptor (CAR)-T cells targeting B cell maturation antigen (BCMA) have emerged as a promising therapeutic strategy. We reported here a patient with anti-signal recognition particle IMNM refractory to multiple available therapies, who was treated with BCMA-targeting CAR-T cells, exhibited favorable safety profiles, sustained reduction in pathogenic autoantibodies, and persistent clinical improvements over 18 mo. Longitudinal single-cell RNA, B cell receptor, T cell receptor sequencing analysis presented the normalization of immune microenvironment after CAR-T cell infusion, including reconstitution of B cell lineages, replacement of T cell subclusters, and suppression of overactivated immune cells. Analysis on characteristics of CAR-T cells in IMNM demonstrated a more active expansion of CD8+ CAR-T cells, with a dynamic phenotype shifting pattern similar in CD4+ and CD8+ CAR-T cells. A comparison of CD8+ CAR-T cells in patients with IMNM and those with malignancies collected at different timepoints revealed a more NK-like phenotype with enhanced tendency of cell death and neuroinflammation and inhibited proliferating ability of CD8+ CAR-T cells in IMNM while neuroinflammation might be the distinct characteristics. Further studies are warranted to define the molecular features of CAR-T cells in autoimmunity and to seek higher efficiency and longer persistence of CAR-T cells in treating autoimmune disorders.

Elevating the potential of CAR-T cell therapy in solid tumors: exploiting biomaterials-based delivery techniques.

Chimeric antigen receptor (CAR) T cells exhibit promising progress in addressing hematologic malignancies. However, CAR-T therapy for solid tumors remains limited, with no FDA-approved CAR-T products available for clinical use at present. Primary reasons include insufficient infiltration, accumulation, tumor immunosuppression of the microenvironment, and related side effects. Single utilization of CAR-T cannot effectively overcome these unfavorable obstacles. A probable effective pathway to achieve a better CAR-T therapy effect would be to combine the benefits of biomaterials-based technology. In this article, comprehensive biomaterials strategies to break through these obstacles of CAR-T cell therapy at the tumor sites are summarized, encompassing the following aspects: 1) generating orthotopic CAR-T cells; 2) facilitating CAR-T cell trafficking; 3) stimulating CAR-T cell expansion and infiltration; 4) improving CAR-T cell activity and persistence; 5) reprogramming the immunosuppressive microenvironments. Additionally, future requirements for the development of this field, with a specific emphasis on promoting innovation and facilitating clinical translation, are thoroughly discussed.

Melatonin as an immunomodulator in CD19-targeting CAR-T cell therapy: managing cytokine release syndrome

BackgroundChimeric antigen receptor CAR-T cell therapies have ushered in a new era of treatment for specific blood cancers, offering unparalleled efficacy in cases of treatment resistance or relapse. However, the emergence of cytokine release syndrome (CRS) as a side effect poses a challenge to the widespread application of CAR-T cell therapies. Melatonin, a natural hormone produced by the pineal gland known for its antioxidant and anti-inflammatory properties, has been explored for its potential immunomodulatory effects. Despite this, its specific role in mitigating CAR-T cell-induced CRS remains poorly understood.MethodsIn this study, our aim was to investigate the potential of melatonin as an immunomodulatory agent in the context of CD19-targeting CAR-T cell therapy and its impact on associated side effects. Using a mouse model, we evaluated the effects of melatonin on CAR-T cell-induced CRS and overall survival. Additionally, we assessed whether melatonin administration had any detrimental effects on the antitumor efficacy and persistence of CD19 CAR-T cells.ResultsOur findings demonstrate that melatonin effectively mitigated the severity of CAR-T cell-induced CRS in the mouse model, leading to improved overall survival outcomes. Remarkably, melatonin administration did not compromise the antitumor effectiveness or persistence of CD19 CAR-T cells, indicating its compatibility with therapeutic goals. These results suggest melatonin's potential as an immunomodulatory compound to alleviate CRS without compromising the therapeutic benefits of CAR-T cell therapy.ConclusionThe study's outcomes shed light on melatonin's promise as a valuable addition to the existing treatment protocols for CAR-T cell therapies. By attenuating CAR-T cell-induced CRS while preserving the therapeutic impact of CAR-T cells, melatonin offers a potential strategy for optimizing and refining the safety and efficacy profile of CAR-T cell therapy. This research contributes to the evolving understanding of how to harness immunomodulatory agents to enhance the clinical application of innovative cancer treatments.

Chemical genetic control of cytokine signaling in CAR-T cells using lenalidomide-controlled membrane-bound degradable IL-7.

CAR-T cell therapy has emerged as a breakthrough therapy for the treatment of relapsed and refractory hematologic malignancies. However, insufficient CAR-T cell expansion and persistence is a leading cause of treatment failure. Exogenous or transgenic cytokines have great potential to enhance CAR-T cell potency but pose the risk of exacerbating toxicities. Here we present a chemical-genetic system for spatiotemporal control of cytokine function gated by the off-patent anti-cancer molecular glue degrader drug lenalidomide and its analogs. When co-delivered with a CAR, a membrane-bound, lenalidomide-degradable IL-7 fusion protein enforced a clinically favorable T cell phenotype, enhanced antigen-dependent proliferative capacity, and enhanced in vivo tumor control. Furthermore, cyclical pharmacologic combined control of CAR and cytokine abundance enabled the deployment of highly active, IL-7-augmented CAR-T cells in a dual model of antitumor potency and T cell hyperproliferation.

Safety and Efficacy of CD22/ CD19 CAR-T and Auto-HSCT “Sandwich” Strategy As Consolidation Therapy for Ph Negative B Cell Acute Lymphoblastic Leukemia

Introduction: Chimeric antigen receptor T-cell (CAR-T) therapy has a high relapse rate in B cell acute lymphoblastic leukemia (B-ALL). CAR T-cells target multi-antigen or CAR-T combined with other strategy may reduce the relapse post CAR-T. The main purpose of this study is to observe the safety and efficacy of CD22/CD19 CAR-T and Auto-HSCT “Sandwich” strategy in Ph negative (Ph -) B-ALL patients. Methods: A total of 19 newly diagnosed Ph - B-ALL patients were enrolled in this study (NCT05470777). These patients received induction and consolidation chemotherapy according to standard protocols. Autologous lymphocytes were collected at week 4 after induction chemotherapy. Afterwards, autologous CAR T-cells targeting CD22 and CD19 (CAR-T 1, co-stimulatory molecule was 4-1BB and infusion dose was 5×10 6/kg, respectively) were sequential infused after lymphodepletion chemotherapy with fludarabine and cytophosphamide. Autologous stem cells mobilization and collection were performed at 6-8 weeks after CAR T-cells infusion. After conditioned with modified BuCy regimen, autologous stem cells transplantation (auto-HSCT) was conducted. Sequential infusion of CD22 and CD19 CAR-T cells was performed on the second day after transplantation (CAR-T 2, co-stimulatory molecules and dose as CAR-T 1). No maintenance therapy was performed after auto-HSCT. Measurable detectable disease (MRD) was monitored by flow cytometry and IGHV leader-based next generation sequencing. Persistence of CAR T-cells were detected using real time quantitative RT-PCR (qPCR). Results: Three of the enrolled patients were Ph-like B-ALL. 10 (52.6%) patients had poor risk factors according to NCCN guidelines. Grade 1-2 cytokine release syndrome (CRS) occurred in four (21.1%) and seven (36.8%) patients after CAR-T 1 and CAR-T 2, respectively. No grade 3-4 CRS and immune effector cell associated neurotoxicity syndrome (ICANS) occurred. After CAR-T therapy, all patients achieved MRDnegative complete remission (MRD -CR). Until the last follow-up on July 1 st, 2023, the median follow-up was 16 months (range, 7 - 32 months). 84.2% of enrolled patients remain MRD -CR. Duration of continuous MRD -CR was detected in 2 patients for more than 2 years and in 9 patients for more than 1 year. The median overall survival (OS) and leukemia-free survival (LFS) in all 19 patients are not reached. Three (3/19, 15.8%) patients experienced antigen-positive relapse. One Ph-like B-ALL patient relapsed at 10 months after auto-HSCT and underwent allogeneic HSCT. Two patients with Ph - B-ALL relapsed at 5 and 6 months after auto-HSCT and were treated with blinatumomab. All of them achieved MRD -CR again after treatment. All patients are alive and CAR-T cells are detectable in all patients at the last follow-up (range, 5-28 months). Conclusions: Our preliminary study demonstrated that CD22/CD19 CAR-T and Auto-HSCT “Sandwich” strategy as a consolidation strategy showed favorable safety and efficacy in Ph - B-ALL. CD22/CD19 CAR-T resulted in deeper remission before transplantation. The new strategy may benefit patients from LFS and OS.

Efficacy and Safety of a Third Generation CD20 CAR-T (MB-106) for Treatment of Relapsed/Refractory Indolent B-Cell Non-Hodgkin Lymphoma: Phase-1 Results from a Multicenter Trial

Background: Indolent B-cell non-Hodgkin lymphomas (NHL) remain incurable despite multiple therapeutic options, including chemoimmunotherapy, targeted therapies, bispecific antibodies, and CD19-targeted chimeric antigen receptor T (CAR-T) cells. MB-106, a fully human 3 rd-generation CD20-targeted CAR-T product with both 4-1BB and CD28 costimulatory domains, had high overall response rate (100%) and complete response (CR) rate (91%) in follicular lymphoma (FL) patients at doses ≥ 3.3×10 6 cells/kg in a single institution trial (Shadman et al., 17-ICML, 2023). We seek to replicate the favorable efficacy and safety profile of MB-106 in a multicenter trial with centralized manufacturing. Methods: MB-106 is being evaluated in a Phase 1/2, open label, multicenter study utilizing a 3+3 dose-escalation design in R/R B-cell NHL patients with confirmed CD20 expression. Prior treatment with CD19-targeted CAR-T therapy is permitted. Following lymphodepletion (cyclophosphamide 300 mg/m 2/day and fludarabine 30 mg/m 2/day for 3 days), MB-106 is administered to patients with indolent B-cell NHL at one of two dose levels (DL): DL1, 3.3×10 6; and DL2, 1.0×10 7 cells/kg. MB-106 is infused in the outpatient setting if allowed by the institution, except for the first patient in each dose level cohort (overnight observation). Dose-limiting toxicities (DLT) are monitored through day 28. The treatment response is assessed on day 28 and on subsequent visits, and best overall response is reported here. CAR-T persistence is assessed by both flow cytometry and qPCR in peripheral blood mononuclear cells. Results: Three patients with indolent NHL (2 FL, 1 Waldenström macroglobulinemia [WM]) received MB-106 at DL1. In addition, one patient with hairy cell leukemia variant (HCL-v) received non-conforming material (drug product that failed to meet release criteria) at DL1, following FDA authorization. These four patients were heavily pre-treated with a median of 5.5 prior lines of treatment. High-risk features included progression of disease within 24 months of first-line treatment (POD24, n=1), prior autologous stem cell transplant (n=1) and prior CD19-targeted CAR-T therapy (n=1). No DLTs were observed at DL1. Three patients experienced Grade 1 cytokine release syndrome. There have been no occurrences of immune effector cell-associated neurotoxicity syndrome (ICANS). The 2 FL patients achieved CR by both PET-CT and bone marrow biopsy, one of whom had been previously treated with CD19-targeted CAR-T. The WM patient, who had nine prior treatments, including autologous stem cell transplant, and high disease burden, achieved very good partial response, with complete metabolic response by PET-CT, morphologic clearance of lymphoma in bone marrow, and resolution of the IgM paraprotein. The patient with HCL-v, who had been transfusion dependent, continues to have stable disease with decreased disease in the bone marrow, and achieved complete transfusion independence which is ongoing at 6 months. All patients displayed MB-106 expansion with peak levels between 7-14 days post-infusion. Persistence of CAR T-cells is ongoing at 6 months. Conclusion: Treatment with MB-106, a third generation CD20 targeting CAR-T, resulted in responses, including CRs, and CAR-T persistence in patients with R/R indolent NHL and was associated with favorable safety profile with no occurrence of Grade 3 or Grade 4 cytokine release syndrome and no ICANS of any grade. CRs have been observed in patients previously treated with CD19-targeted CAR-T in both this multicenter trial and the original single institution trial. Dosing is ongoing in the final dosing level (1.0×10 7 cells/kg) to establish the recommended Phase 2 dose for a planned pivotal trial in WM. It is anticipated that the complete data set from the Phase 1 indolent NHL arm will be presented.

Longitudinal Transcriptomic Analysis of CAR-T Cells at Single-Cell Level Allows the Identification of Molecular Mechanisms Promoting Long-Term CAR-T Cell Persistence in MM

Background: CAR-T therapies targeting BCMA are a promising option for Multiple Myeloma (MM) patients. However, a significant number of patients still relapse and the limited persistence of CAR-T cells after infusion has been associated with lack of long-term responses. To shed light on the potential mechanisms driving CAR-T cell persistence we performed longitudinal transcriptomic analysis, at single cell level, of CAR-T cells from a patient enrolled in CARTBCMA-HCB-01 clinical trial (NCT04309981) (Oliver-Caldes A, et al. Lancet Oncol, 2023), that presented detectable CAR-T cells more than one year after CAR-T cell administration. Methodology: Single-cell transcriptomic and TCR analysis was performed on 42.719 CAR-T cells isolated from infusion product (IP) and paired bone marrow (BM) and peripheral blood (PB) samples at 1, 3, 6, 12 and 18 months after infusion (11 samples), using the Chromium Single-Cell Immune Profiling solution from 10x Genomics. Gene Regulatory Network (GRN) analysis was performed using SimiC, a novel machine learning method developed by our group, that infers regulatory dissimilarities form single cell data (Peng J, et al. Commun Biol., 2022). Results: We observed that only 4 out of the 30 patients included in the first pilot academic clinical trial evaluating ARI0002h presented detectable CAR-T cells 12 months after treatment (Oliver-Caldes A, et al. Lancet Oncol, 2023). We performed a deeper analysis of one patient still presenting high levels of CAR-T cells 18 months after treatment that finally ended up relapsing at month 23. scRNA-seq analysis revealed that post-infusion CAR-T cells were mainly composed by effector/effector-memory CD8 + cells, with a polyclonal TCR repertoire. Interestingly, this patient suffered a respiratory tract infection with CMV reactivation during the first month after infusion that was also reflected on CAR-T cells features. Thus, circulating CAR-T cells at M1 expressed high levels of genes involved in IFN response, with an equivalent anti-viral response in CAR-T cells obtained from BM at M3. However, phenotypes of CAR-T cells from both PB and BM samples converged at M6, with a prevailing CAR-T cell population (termed 'CD8 + GZMK') presenting cytotoxic features and expressing key markers related to T cell memory and survival that could explain the prolonged CAR-T cell persistence. However, at M12 we observed changes on CAR-T cell populations, with increased presence of CAR-T cells with more cytotoxic features, enrichment of effector and activation signatures, and expressing lower levels of memory markers ('CD8 + GZMB' cluster), indicating CAR-T cell reactivation. Finally, applying GRNA analysis with SimiC we identified several regulons (FOS and NR4A2), presenting increased activity at M12 that could represent potential drivers of CAR-T reactivation in response to tumor relapse. Further analysis at month 18 are being performed to elucidate mechanisms of tumoral resistance in the presence of CAR-T cells. Conclusions: Our analysis clearly showed that most CAR-T cells after administration presented expression of key markers related to T cell memory and survival without clonal expansion, suggesting that memory induction is crucial for long-term persistence. Interestingly, prior to tumor relapse, we observed reactivation of CAR-T cell with the enrichment of effector and activation signatures, and GRN analysis identified FOS and NR4A2 regulons as potential drivers involved in CAR-T cell reactivation. Overall, scRNA-seq/scTCR-seq coupled with novel machine learning models provides relevant mechanistic insights that could uncover useful targets to be modulated for the development of long-term persistent CAR-T cells.

BCMA CAR-T-Cell Therapy in Combination with Long-Term Pomalidomide Is a Safe and Effective Treatment for Relapsed/Refractory Multiple Myeloma

Background: B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor T-cell (CAR-T-cell) therapy has exhibited remarkable efficacy in refractory or relapsed multiple myeloma (R/R MM), but recurrence and rapid progression of disease are still observed in some cases within a short time after treatment. Long-term pomalidomide therapy, which potentiates T-cell functionality and has direct antimyeloma effects, might enhance the efficacy of BCMA CAR-T-cell therapy. Here, we present the efficacy of this combination regimen for R/R MM. Methods: We performed a single-center prospective clinical study that was registered with the Chinese Clinical Trial Registration Center (ChiCTR2000036350). Patients with relpased or refractory multiple myeloma receiving BCMA CAR-T cell infusion were randomized to receive long-term pomalidomide treatment (4 mg/day) one month after infusion. The primary endpoint is overall survival (OS) in R/R MM with long-term pomalidomide administration compared to those without pomalidomide therapy after CAR T-cell infusion. The secondary endpoints include time to progression (TTP) and therapeutic response. Also, the copy numbers of CAR were assessed after infusion. Results: A total of 14 R/R MM patients who received BCMA CAT T-cell therapy, were included in this study. The median age of the enrolled R/R MM patients was 59.5 years (44-71). Five patients had high-risk cytogenetic abnormalities (IGH rearrangement, 1q21 amplification, and P53 deletion) at the time of initial diagnosis. In addition, high-risk genetic alterations were newly detected in patient 2 during first-line therapy. All patients had previously received 3 to 6 lines of treatment with proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), or anti-CD38 monoclonal antibodies. As shown in Figure 1, all patients had demonstrated progressive disease prior to CAR T-cell infusion. The objective response rate (ORR) of BCMA-CART was 100%. Three months following CAR-T-cell infusion, of the 6 patients receiving pomalidomide, all patients (6/6) achieved VGPR (very good partial response) or CR (complete response）, while only 5 patients (5/8) who did not receive pomalidomide treatment achieved VGPR or better. At a median follow-up of 19.7 months, for the 8 patients who did not receive pomalidomide administration, the median TTP (time to progression) was 4.5 (1-14) months, while the TTP was not reached in patients who received long-term pomalidomide treatment. Moreover, the dynamic abundance of circulating BCMA CAR-T cells was monitored as shown in Figure 2. In patients who received pomalidomide treatment after CAR-T-cell therapy, the CAR copy numbers of circulating CAR-T cells peaked 8 to 10 days after CAR-T-cell infusion, with a peak copy number of up to 10 6. In addition, BCMA CAR-T cells persisted (>1*10 3 copies/µg DNA) for 3 months in vivo. Without pomalidomide administration, BCMA CAR-T cells persisted for up to 2.5 months in the peripheral circulation (results not shown). Conclusion: Our results confirm the efficacy of the long-term oral pomalidomide administration (4 mg/day) after BCMA CAR T-cell infusion in patients with R/R MM. Notably, this combination regieme reduced the recurrence rate and prolonged progression-free survival for R/R MM patients, providing a promising option for treating R/R MM. In addition, oral administration of pomalidomide did not significantly prolong the persistence of BCMA CAR T-cells in vivo. However, data from studies with larger sample sizes are required to draw valid conclusions.

Dendritic Cell Vaccines Extend CD19 CAR-T Cell Persistence and Improve the Outcomes in Refractory/Relapsed Adult B-ALL

Introduction: The long-term efficacy of anti-CD19 chimeric antigen receptor (CAR)-T cell in refractory or relapsed (r/r) adult B-cell acute lymphoblastic (B-ALL) patients is limited, and the recurrence rate is high. CAR-T cell depletion and limited CAR-T cell persistence are some of the most common reasons for relapse. Our previous in vitro studies confirmed that dendritic cell (DC) vaccines targeting tumor antigens could induce CAR-T cell rejuvenation and increase the killing function of CAR-T cells. So, we designed a clinical trial to study CD19 CAR-T cell combined with DC vaccination for adult r/r B-ALL to explore whether this therapy improves LFS. (clinicaltrials.gov, no: NCT03291444). Methods: Adult r/r B-ALL patients who expressed HLA-A1101, A2402, or A0201 and had high expression of EPS8 or WT1 were eligible. An EPS8 peptide-derived DC (EPS8-DCs) vaccine was used in EPS8-high patients, while a WT1 peptide-derived DC (WT1-DC) vaccine was used in EPS8-negative patients with WT1 positivity. Lymphodepleting chemotherapy comprising fludarabine (30 mg/m²) and cyclophosphamide (300 mg/m²) was administered intravenously daily for 3 days before CD19 CAR-T cells infusion. After 4 weeks of CAR-T infusion, if bone marrow morphologic remission had been achieved, DC vaccination was administered intradermally every 2 weeks for 4 doses. Results: Eight adult patients with r/r B-ALL were enrolled and successfully received CAR-T cells and DC cells, of which 4 (50%) relapsed after allogeneic hematopoietic stem cell transplantation. They were successfully administered one dose of CD19 CAR-T with a median dose of 2.26×10 6/kg (range 6.4×10 5/kg to 4.46×10 6/kg) on day 0 and four doses of DC vaccination with a median dose of 5.44×10 6 (range 2.97×10 6/dose to 2.68×10 7/dose) every 2 weeks after 4 weeks of CAR-T infusion. All eight evaluable patients achieved complete response (CR) after receiving CD19 CAR-T. With a median follow-up of 608 days, the median LFS time was 489 days, and the median OS was not reached. Seven of the eight evaluable patients were still alive. Four (50%) were in continuous MRD-negative remission at the cutoff time, and two of them (pt 02 and pt 03) maintained MRD-negative CR for more than 4 years. The median peak of CAR-T cell expansion in the PB was detected on day 7 after infusion of CD19 CAR-T. The median persistence time of CAR-T was 336 days (range 84 to 1549 days). CAR-T cells were reamplified after infusion of the DC vaccine. For patients with an LFS of more than 2 years (pt 02, pt 03, and pt 05), CD19 CAR-T cells were still detectable for more than 1 year, with a maximum of 4.2 years in pt 03. The activity of the CTLs measured by IFN-γ ELIspot showed that IFN-γ-secreting CTLs were significantly increased after DC vaccination. These assays showed that antigen-specific cellular immune activity was enhanced after vaccination. No grade ≥3 cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS) occurred after infusion of 4sCAR19. No grade ≥3 events occurred during the infusion of the DC vaccine. Only 1 of 8 patients experienced local skin reactions after infusion of the DC vaccine. Conclusions: This study reports a novel combination therapy strategy (CAR-T cell combining with individualized DC vaccination) for adult r/r B-ALL. DC vaccination has higher safety, may prolong the persistence of CAR-T cells, and may prolong the survival time and quality of life. CAR-T cell therapy combining with DC vaccination is a potential therapy strategy for adult r/r ALL patients who are not eligible for transplantation or who relapse after transplantation.

Analysis of Metabolic Mechanism of CD19CAR-T with CD79A/CD40 Co-Stimulatory Domain Based on Comparison with Established CAR-Ts

Introduction: Anti-CD19 chimeric antigen receptor T cell (CD19CAR-T) therapy has achieved significant clinical outcomes and changed treatment strategy in B-cell malignancies. However, recurrence after CAR-T therapy remains a major obstacle due to poor peak expansion and short persistence of CAR-T cells. We have reported that CD19CAR-T incorporating a co-stimulatory domain consisting of CD79A and CD40 (79A40CAR) provided superior anti-tumor efficacy and survival compared to CD19CAR-T with CD28 or 4-1BB (CD28CAR/4-1BBCAR) in mice xenograft models. 79A40CAR exhibited sustained NF-κB phosphorylation and superior in vivo efficacy compared to the CD28CAR or 4-1BBCAR. (Molecular Therapy 2021, Julamanee J et al.) In this study, we aimed to clarify the molecular mechanism of 79A40CAR by comparing with CD28/4-1BBCAR in terms of T-cell metabolism. Methods: CD8+T cells were isolated from PBMCs of healthy donors and retrovirally transduced with either CD28IC-, 4-1BBIC-, or 79A40IC-CD19CARs. CD28CAR, 4-1BBCAR, or 79A40CAR were cocultured with 100 Gy irradiated NALM6 at E:T ratio = 1:5, respectively. Repeated antigen stimulations (Ag-stim) were performed, and day 7 after each stimulation was subjected to analysis. RNA-seq, expansion capacity, T-cell phenotype, and metabolic evaluation using a seahorse flux analyzer (Agilent Technologies) were performed. After Ag-stim, CD45RA negative CD62L positive and CD45RA negative CD62L positive CAR-T cell fractions were sorted as central memory (T CM) and effector memory (T EM), respectively. T CM and T EM received Ag-stim. After Ag-stim, we performed evaluation of mitochondrial functionality with MitoTracker based on mitochondrial mass and membrane potential. Results: All CD19CAR genes were successfully transduced into primary CD8+ T-cells. To understand the differences between co-stimulatory domains, we first confirmed bulk RNA-seq, expansion capacity, and T-cell phenotype. After Ag-stim, 79A40CAR showed enhancement of the NF-κB pathway. We detected few DEGs when 79A40CAR compared to 4-1BBCAR, however, we detected 162 upregulated DEGs when 79A40CAR compared to CD28CAR. Principal component analysis of gene expression demonstrated similarity between 4-1BBCAR and 79A40CAR. We confirmed that 79A40CAR had superior expansion capacity than CD28, or 4-1BBCAR after repeated Ag-stim. PD-1 and Tim-3 expression in 79A40CAR was lowest after repeated Ag-stim. In the evaluation of T-cell differentiation, 4-1BB and 79A40CAR showed an earlier transition to the effector phenotype compared to CD28CAR after Ag-stim. To evaluate differences in terms of T-cell metabolism, we performed metabolic analysis using a seahorse flux analyzer. After repeated Ag-stim, 4-1BB and 79A40CAR showed a predominant oxidative phosphorylation metabolic pathway (Fig1A) and higher spare respiratory capacity (SRC), which has been correlated with mitochondrial functionality (Fig1B), than CD28CAR. After repeated Ag-stim, 4-1BB and 79A40CAR demonstrated higher ECAR than CD28CAR. Because we observed differences in the expression of T-cell differentiation phenotype, we performed cell sorting to evaluate T-cell metabolism of each differentiation phenotype. After applying repeated Ag-stim to T CM and T EM-derived CD19CAR-T, mitochondrial functionality was evaluated using Mitotracker. T CM- (Fig1C) and T EM- (Fig1D) derived 4-1BB and 79A40CAR had more functional mitochondria and fewer dysfunctional mitochondria compared to CD28CAR. Conclusion: 79A40CAR seems to preserve the mitochondrial oxidative phosphorylation-dominant metabolic pathway and supply energy demands for a longer period, especially when compared to CD28CAR. 79A40CAR showed functionally very similar to 4-1BBCAR. We showed that CD19CAR-Ts had different metabolic pathway according to the different co-stimulatory domains. To reveal additional metabolic pathway differences between 4-1BBCAR and 79A40CAR, we are now conducting proteomics analysis.

Detection of CAR-T Cell Persistence with Digital Droplet PCR: Correlation of In Vivo Expansion with Clinical Outcomes in a Cohort of B-Cell Lymphoma Patients

Introduction: In vivo CAR-T cell expansion is of central importance to clinical response and toxicity in patients with hematologic malignancies. In clinical trials, the area under the curve (AUC) within the first 28 days of infusion was considered a valid tool to describe the concentration-time profile of CAR-T cells in peripheral blood (PB). However, several longitudinal assessments performed by either flow cytometry or quantitative real-time polymerase chain reaction (qPCR) were required. This study evaluates the clinical utility of assessing CAR-T concentration by digital droplet PCR (ddPCR) in patients with non-Hodgkin lymphoma (B-NHL). Methods: Patients with B-NHL ddPCR test collected on day 28 ± 7 post-commercial CD19 CAR T infusion, and clinical outcome data was extracted retrospectively. In this ddPCR assay, the PCR primers target the mammalian expression vector present in the CAR constructs as well as the ATP2B4 reference gene. The CAR-T and reference gene signals are segregated by differential fluorescence signals of tagged probes. The number of CAR constructs and reference gene signals per microliter (uL) are calculated from sample droplets and data fitting to a Poisson distribution. The assay sensitivity is ~0.003% of CAR construct per reference gene. Results: Between October 2020 and February 2022, 66 patients (62% male) with diffuse large B cell lymphoma (DLBCL, 73%), Burkitt lymphoma (1%), mantle cell lymphoma (MCL, 24%) and follicular lymphoma (FL, 12%) received axicabtagene ciloleucel (83%), brexucabtagene autoleucel (12%), or tisagenlecleucel (5%), with fludarabine/cyclophosphamide (49%) or bendamustine (51%) lymphodepletion and had testing within the timeframe. Bridging therapy was administered in 82%, and elevated serum LDH prior to CAR T was present in 31%. With a median follow-up of 4.5 months (IQR 3.1-6.2 mo), the day 28 overall response rate was 80%, complete remission 57%, and progressive disease 18%. Median CAR copies/microL was 2.3 (range 0-474); of those with a detectable value, the median was 3.57 copies/microL. Excluding the 7 patients who had progression at the same time the sample was drawn, day 28 landmarked 3- and 6-month progression-free survival (PFS) for patients with non-detectable (22%) vs. detectable (78%) ddPCR construct was 31% vs 81% and 31% vs 73%, respectively (p=0.012, Figure 1B). Three- and 6-month PFS for non-detectable (22%), detectable <= 3.57 (41%), and detectable >3.57 (37%) was 31%, 77%, 86% and 31%, 62%, and 86%, respectively (p=0.039, Figure 1A). Univariable analysis of factors predicting detection of CAR T construct by ddPCR at Day 28, including age, sex, gender, elevated LDH, lines of therapy, bridging, CAR T construct, grade 2+ cytokine release syndrome (CRS), and grade 2+ ICANS was significant only for grade 2+ CRS. Patients with grade 0-1 CRS (44%) vs grade 2+ CRS (56%) had a median of 0 (range 0 - 474) vs 4 (0 - 307) CAR copies/microL (p<0.001). Conclusion: Assessment of PB CAR-T concentration performed by ddPCR at a single timepoint reliably predicted CD19 CAR-T cell efficacy in patients with B-NHL, with detectable CAR construct at day 28 predicting better 6-month PFS. Further research is needed to define the best timepoint for testing and the impact of using strategies to mitigate CRS and/or ICANS.

The persistence and antitumor efficacy of CAR-T cells are modulated by tonic signaling within the CDR

Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable clinical efficacy, but challenges related to relapse and CAR-T cell exhaustion persist. One contributing factor to this exhaustion is CAR tonic signaling, where CAR-T cells self-activate without antigen stimulation, leading to reduced persistence and impaired antitumor activity. To address this issue, we conducted a preclinical study evaluating tonic signaling using nanobody-derived CAR-T cells. Our investigation revealed that specific characteristics of the complementary determining regions (CDRs), including low solubility, polarity, positive charge, energy, and area of ionic and positive CDR patches of amino acids, were associated with low antigen-independent tonic signaling. Significantly, we observed that stronger tonic signaling directly impacted CAR-T cell proliferation in vitro, consequently leading to CAR-T cell exhaustion and diminished persistence and effectiveness in vivo. Our findings provide compelling preclinical evidence and lay the foundation for the clinical assessment of CAR-T cells with distinct tonic signaling patterns. Understanding the role of CDRs in modulating tonic signaling holds promise for advancing the development of more efficient and durable CAR-T cell therapies, thereby enhancing the treatment of cancer and addressing the challenges of relapse in CAR-T cell therapy.

Alternative Lymphodepletion Strategies Lead to Similar Response Rates and Toxicities in Standard-of-Care BCMA-Directed CAR-T Therapy in Relapsed Refractory Multiple Myeloma

Introduction: Lymphodepletion (LD) chemotherapy is critical for the efficacy of CAR-T therapy by eradicating immunosuppressive cells, inducing costimulatory molecules, and promoting expansion and persistence of CAR-T cells. It also can contribute to an increased risk of infections, cytopenia, and other toxicities. We evaluated the utilization of varying LD regimens used in standard-of-care (SOC) autologous B-cell maturation antigen-directed (BCMA) CAR-T therapy with ide-cel or cilta-cel for relapsed/refractory multiple myeloma (RRMM) and the effect on outcomes and toxicity. Methods: Data were retrospectively collected from patients with RRMM who underwent LD and CAR-T cell infusion by December 31, 2022, from 13 US academic institutions with SOC ide-cel or cilta-cel. Response rates and other categorical variables, specifically LD regimen, were tested for association using Wilcoxon rank-sum (or Kruskal-Wallis for three or more groups) or Fisher's exact tests, respectively. Results: A total of 523 patients were infused at the time of data cut-off with 489 patients evaluable for safety and survival analyses; 368 (75%) were infused with ide-cel and 121 (25%) with cilta-cel. The median age was 65 years old (range 30-90) with 206 females (42%) and 283 males (58%), and 13% with ECOG performance status ≥ 2 at time of LD. Fludarabine and cyclophosphamide (Flu/Cy) LD was used in 422 patients while 67 patients received other LD regimens including bendamustine-based (n = 35, 52%), cladribine-based (n = 25, 37%) or cyclophosphamide alone (n = 7, 10%). The primary reason for use of Flu/Cy LD was institutional guidance or SOC in 378 patients (90%), while the primary reason for use of other LD regimens was due to the fludarabine shortage in 65 patients (97%) with 3% citing physician's choice or institutional guidance. No significant difference was identified among response outcomes, with those receiving Flu/Cy LD achieving an ORR of 84% compared to 81% with other LD regimens (p = 0.59) and no difference observed in rates of ≥ VGPR (66% Flu/Cy LD vs 60% other LD; p = 0.48). Of those evaluable for MRD at 1 and 3 months, the MRD negative rate at 10 -6 for those with Flu/Cy LD was 77% (n= 137) and 77% (n=113) and with other LD regimens was 66% (n=35) and 79% (n=28), respectively. At a median follow-up of 9.7 months, no statistically significant difference was noted in median progression free survival (mPFS) with Flu/Cy vs other LD regimens (9.3 mos vs NR) or overall survival (OS) (19.4 mos vs NR). When stratified by type of CAR-T therapy, treatment with bendamustine-based LD was associated with an inferior mPFS compared to those receiving Flu/Cy LD in patients treated with ide-cel (3.8 vs 8.5 mos; p=0.0056), though not by multivariate analysis. Univariate analysis of patients treated with ide-cel between Flu/Cy and bendamustine LD did not reveal any significant differences in age, gender, ECOG, R-ISS stage, extramedullary disease, high-risk cytogenetics or renal dysfunction (Cr < 45 ml/min). LD regimen was not a predictor for PFS on multivariable analyses amongst patients treated with ide-cel, while ECOG ≥ 3 (hazard ratio [HR]= 4.44, 95% confidence interval [CI]= 1.40, 14.08) and t(4,14) (HR= 2.02, 95% CI=1.14, 3.59) were associated with worse PFS. No difference in mPFS by LD regimen was observed in patients receiving cilta-cel, which was a smaller cohort (mPFS: Flu/Cy 11.7 mos, bendamustine-based NR, cladribine-based NR, cyclophosphamide NR). Rates of all grade CRS (81% vs 70%), Grade ≥ 3 CRS (3% vs 3%), all grade ICANS (19% vs 12%, and Grade ≥ 3 ICANS (4% vs 2%) were similar among those receiving Flu/Cy or other LD regimens, respectively. Patients receiving Flu/Cy LD were less likely to have any grade cytopenia at 1 month (33% vs 55%, p < 0.001) and 3 months (52% vs 69%, p < 0.001), with no observed difference in infection rate. Conclusions: The fludarabine shortage experienced in the US in late 2022 necessitated the use of alternative LD regimens with BCMA-directed CAR-T therapy with most centers relying on bendamustine- or cladribine-based regimens. Use of bendamustine-based LD was associated with inferior PFS in patients receiving ide-cel on univariate analysis, but not on multivariate analysis after adjusting for other prognostic factors. In summary, there is no clear difference in efficacy or safety between Flu/Cy and non-Flu/Cy LD in patients receiving BCMA-directed CAR-T therapy. *Authors contributed equally

Evaluating the Impact of Cryopreservation of PBMCs on CAR-T Therapy Efficacy and Safety in DLBCL Patients: An Informative Approach to Optimize Manufacturing Strategies

Background: Chimeric Antigen Receptor (CAR) T-cell therapy has emerged as a pivotal treatment modality for relapsed/refractory (R/R) B-cell malignancies. However, some patients are unable to receive CAR-T therapy due to challenges in obtaining sufficient lymphocytes through leukapheresis, often resulting from intensive prior treatments or impaired lymphocyte function after repeated tumor reductions under high tumor burden. This issue underscores the need to explore strategies such as advance collection and cryopreservation of peripheral blood mononuclear cells (PBMCs), which could provide enhanced flexibility in the pre-CAR-T treatment planning process and potentially widen the accessibility of CAR-T therapy. Aims: This retrospective study compares the safety, efficacy, and adverse reactions of anti-CD19 CAR-T cell therapy for Diffuse Large B-Cell Lymphoma (DLBCL) patients, using cryopreserved versus freshly harvested PBMCs. Methods: A total of 162 R/R DLBCL patients treated with an anti-CD19-CD3zeta-4-1BB CAR-T cell therapy from January 2019 to April 2023 were included in this study, excluding those who received concurrent autologous stem cell transplants. The median age was 52 (15-78) years old, and the majority (87.7%) patients had stage III-IV disease. With a median IPI score of 2 (range, 0-5), 71.0% (115/162) had extranodal lesions and 18.0% (29/162) had bulky disease > 7 cm. Among these, 136 patients received CAR-T cells derived from cryopreserved PBMCs, which were frozen with a controlled rate freezer and CryoSure-DEX40 cryoprotectant and stored in the liquid phase of liquid nitrogen. The remaining 26 patients received CAR-T cells that were directly cultured from freshly collected lymphocytes without any cryopreservation. For all patients, CAR-T cells were freshly infused post-manufacture. The study primarily explores the attainment of CAR-T cell infusion dose (achieving 2x10 6 cells/kg), in vivo expansion of CAR-T cells, and the persistence of CAR-T cells in the body. Secondary aims include assessing overall survival (OS), progression-free survival (PFS), 3-month complete response (CR) rate and objective response rate (ORR). The incidence of cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and levels of cytokines were also recorded. In addition, baseline characteristics of both groups were taken into analysis, including factors such as Eastern Cooperative Oncology Group (ECOG) performance status, International Prognostic Index (IPI) score, age, gender, presence of bulky disease, and extranodal lesions. For statistical analysis, either chi-square test or Fisher's exact test was employed for categorical variables, whereas continuous variables were assessed using the rank-sum test. Survival curves between groups were compared using the log-rank test. All P-values were two-tailed. Results: Baseline characteristics between the cryopreserved and fresh groups showed no significant differences in age, gender, IPI score≥3, ECOG score≥3, and extranodal lesions (P all > .05), while bulky disease prevalence was higher in the cryopreserved group (20.6% vs. 3.85%, P = .049). Metrics of CAR-T cell expansion, including meeting the infusion dosage benchmark of 2x10 6 cells/kg (46.1% vs. 44.9%, P = .90), in vivo CAR-T cell persistence (median duration: 21 days for both groups, P= .48), and the peak proportion of CART19 cells in lymphocytes (median peak: 49.6% vs. 35.2%, P = .41), revealed no significant differences between the fresh and cryopreserved groups. Assessments of CAR-T therapy safety revealed no significant differences in the incidence of grade 3 or higher ICANS and CRS, or peak levels of cytokines including interferon gamma, IL-6, IL-10, soluble CD25, and TNFα (P all > .05) between the two groups. Clinical efficacy measurements (3-month CR and ORR, 1-year OS and PFS) were similar between the two groups, with no statistically significant differences in CR (46.2% vs. 45.5%), ORR (69.2% vs. 61.9%), 1-year OS (75.4% vs. 64.1%, Fig 1), and 1-year PFS (52.1% vs. 44.5%, Fig 2) (P all > .05). Conclusion: Our study affirms that cryopreservation of PBMCs has no significant influence on the safety and efficacy of anti-CD19 CAR-T therapy for patients with R/R DLBCL. These findings widen the therapeutic flexibility and accessibility of anti-CD19 CAR-T therapy for DLBCL patients. Key words: PBMC Cryopreservation, CAR-T, DLBCL

Ferroptosis Inhibition Generates TCF-1 + CAR-T Cells with Enhanced Persistence and Cytotoxicity

[Introduction] Long-term in vivo persistence of chimeric antigen receptor (CAR) T-cells is associated with sustained response after CAR-T cell therapy against hematological malignancies. Ferroptosis is a novel form of programmed cell death that refers to iron-dependent regulated cell death resulting from lipid peroxidation and consequent cytomembrane rupture. The iron, lipid, amino acid, and glutathione metabolisms strictly govern the beginning and execution of ferroptosis. Recent studies demonstrated that ferroptosis limited longevity of virus-specific memory T cells (Wang Y: Nat Immunol 2022). However, the role of ferroptosis in CAR-T cells remains to be clarified. We herein explored the role of ferroptosis in CAR-T cells both in vitro and in vivo. [Methods] For CAR-T cell generation, purified T cells from naïve mice were stimulated with anti-CD3/CD28 antibodies for 48 hrs and then incubated with retroviral vector encoding anti-CD19 CAR (1D3-28Z.1-3; Addgene) for another 48 hrs, in the presence or absence of 32 µM of ferrostatin-1 (Fer-1), an inhibitor of the iron-dependent production of lipid hydroperoxide, or 10 µM of deferoxamine (DFO), an iron chelator. Flow cytometric analyses of phenotype of CAR-T cells, accumulation of reactive oxygen species (ROS), lipid peroxidation, and cytokine production were performed 96 hrs after T-cell stimulation. ROS were quantified using CellROX Green dye, and lipid peroxidation was evaluated using Lipid Peroxidation Assay Kit (abcam). [Results] First, we tested whether CAR-T manufacturing process could induce ferroptosis. We found a substantial accumulation of ROS in CAR-T cells compared to their pre-culture levels, along with a significant increase in lipid peroxidation, indicating that manufacturing process induces ferroptosis of CAR-T cells. Next, we tested whether ferroptosis inhibition by the lipid peroxidation inhibitor Fer-1, could impact the quality of CAR-T cells. When added to the culture, Fer-1 significantly reduced lipid peroxidation in CAR-T cells and significantly increased the proportion of CAR-T cells with a progenitor phenotype, TCF-1 +Tim-3 -, which is known to be associated with longevity of CAR-T cells. Importantly, ferroptosis inhibition significantly enhanced proliferative capacity and cytotoxicity against syngeneic A20 B-cell leukemia in vitro. Next, we tested whether ferroptosis inhibition by the iron chelator DFO could improve CAR-T cell functions. Similar to the effects of Fer-1, DFO mitigated lipid peroxidation of CAR-T cells in association with increased TCF-1 +Tim-3 - population, while it did not affect ROS accumulation. DFO-treated CAR-T cells demonstrated enhanced cytotoxicity against A20 cells compared with control CAR-T cells, confirming that ferroptosis inhibition improves anti-tumor effects of CAR-T cells. Finally, we examined whether ferroptosis inhibition could enhance antitumor effects of CAR-T cells in vivo. Naïve BALB/c mice were intravenously (i.v.) injected with 1.0 x 10 6 luciferase-expressing A20 cells on day 0, followed by i.v. injection of 1.0 x 10 5 Fer-1- or DFO-treated CAR-T cells or control CAR-T cells 7 days later. In mice injected with Fer-1-treated CAR-T cells, the numbers of CAR-T cells in peripheral blood on day 16 was significantly higher than those in mice injected with control CAR-T cells, suggesting that ferroptosis inhibition improved the persistence of CAR-T cells in vivo ( Fig. 1). In vivo bioluminescence imaging showed that both Fer-1-treated and DFO-treated CAR-T cells demonstrated significantly improved anti-tumor effects compared to control CAR-T cells ( Fig. 2). [Conclusions] We for the first time showed that ferroptosis limits the persistence and anti-tumor effects of CAR-T cells, while ferroptosis inhibition induces TCF-1 +Tim-3 - CAR-T cells and improves the therapeutic effect of CAR-T cell therapy. Figure 1. The number of CAR-T cells in the peripheral blood on day 16 after tumor inoculation. Fer-1, ferrostatin-1; *, P < 0.05; P < 0.01. Figure 2. The fold change of the total flux at each timepoint relative to the total flux of the baseline in the mice injected with DFO-treated CAR-T cells, Fer-1-treated CAR-T cells, and control CAR-T cells on day 7 after tumor inoculation. Fer-1, ferrostatin-1; DFO, deferoxamine; *, P < 0.05.

T-Charge™ Manufacturing of the Anti-BCMA CAR-T, Durcabtagene Autoleucel (PHE885), Promotes Expansion and Persistence of CAR-T Cells with High TCR Repertoire Diversity

BCMA targeted CAR-T cells are an effective therapy for patients with relapsed or refractory multiple myeloma (r/r MM). However, autologous CAR-T cell products are highly heterogeneous and the functional roles of various T cell populations within these products have not been established. Stem-like memory T cells (Tscm) are a rare T cell subset which maintain high capacity for self-renewal and multipotency. Previously, we demonstrated that the T-Charge TM platform, a novel rapid manufacturing process that reduces manufacturing time to <2 days, preserves less differentiated CAR-T cells which exhibit potent anti-tumor activity and robust expansion in preclinical models (Dexiu Bu, ASH 2021). We conducted a phase 1 clinical trial in r/r MM (NCT04318327) of durcabtagene autoleucel (PHE885), a fully human product manufactured using the T-Charge platform. Here we present a detailed analysis of CAR-T cell products and subsequent expansion of CAR-T cells in 32 patients enrolled in this clinical trial at the Dana-Farber Cancer Institute. Apheresis products (APH), final products (FP) and post-infusion peripheral blood mononuclear cells (PBMC) were characterized using flow cytometry, mass cytometry (CyTOF) and TCR sequencing. We previously reported a 98% overall response rate (ORR) across all dose levels (2.5-20x10 6 CAR-T cells) and 100% ORR at doses >5x10 6 cell dose (Sperling, ASCO 2023). CAR-T cells expanded rapidly after infusion reaching median peak levels of 3,118 cells/ul (range 373 to 17,865) with a median 87.4% (range, 46.9-97.8) of CD3+ T cells expressing the CAR at a median of 14 days (range 10 to 27) after infusion. CAR-T cells persisted at high levels with transgene detectable by qPCR in 67% of patients at 6 months. Among 28 evaluable patients, 13 (43%) had >20% CAR positive T cells detectable by flow cytometry at 3 months. Phenotypic analysis of APH and FP samples showed that less-differentiated T cell subsets, including Tscm and central memory T cells were maintained in FP. CyTOF evaluation of functional markers revealed high expression of proliferation and activation markers in Tscm in FP and subsequently in CAR-T cells at the time of peak expansion in vivo. Subsequently, more differentiated CAR-T cells increased, accompanied by a decline in activation markers, while no significant changes were observed in inhibitory receptors. The proportion of Tscm in the FP positively correlated with early in vivo CAR-T cell expansion. TCR repertoire diversity and TCR clone tracking were used to characterize product manufacturing and CAR-T cells in peripheral blood after infusion. We sorted naïve/Tscm CD4 T cells, naïve/Tscm CD8 T cells, memory CD4 T cells, and memory CD8 T cells from APH and FP, and isolated CAR-positive CD4 T cells, CAR-positive CD8 T cells, CAR-negative CD4 cells, and CAR-negative CD8 T cells from post-infusion PBMC by fluorescence-activated cell sorting and ran TCR sequence from the extracted gDNA from each T cell subpopulation. Measurement of TCR diversity post-infusion revealed higher TCR repertoire diversity in CAR-T cells than in non-CAR-T cells. Notably, post-infusion CAR-T cells shared significantly more TCR clonotypes with T SCM than with memory T cells in the FP, suggesting that the highly heterogeneous post-infusion CAR-T clones were preferentially derived from T SCM clones in the FP. In three evaluable patients with long-term persistence, a highly diverse TCR repertoire in CAR T cells was maintained 1 year after treatment. Our findings demonstrate that the T-Charge™ manufacturing platform successfully maintains highly heterogeneous transduced Tscm clones with self-renewal potential in durcabtagene autoleucel products. Maintenance of Tscm in manufactured products contributes to robust CAR-T expansion and long-term persistence of CAR-T cells with a highly diverse TCR repertoire after infusion.

CAR-T Cell Therapy in Patients with Refractory Systemic Autoimmune Diseases Exhibits Less Inflammation, Toxicities and Different Cellular Dynamics Compared to Patients with B Cell Lymphoma

Chimeric antigen receptor (CAR) T-cell therapies have shown high efficacy in the treatment of B cell non-Hodgkin lymphoma (NHL) and acute lymphoblastic leukemia (ALL) and are established as a standard treatment for patients with hematological malignancies. Aside from malignant B cells, autoreactive B cells can also cause great harm in patients with autoimmune diseases (AID), resulting in chronic inflammation, organ damage, and increased mortality. We have recently demonstrated that CD19-directed CAR-T cell therapy is feasible and effective in patients with refractory systemic lupus erythematosus (Mougiakakos et al, 2021; Mackensen et al., 2022), antisynthetase syndrome (Müller et al., 2023) and systemic sclerosis (Bergmann et al., 2023). Despite these promising data, there are many uncertainties in the treatment of AID patients with cellular therapies, which were primarily developed for the treatment of cancer patients. Here, we analyzed the occurrence and severity of toxicities, inflammatory markers, and the cellular dynamics of 15 AID patients and 40 NHL patients undergoing CAR-T cell therapy. All patients were treated in our center with either commercial CAR-T cell products (Axicabtagenee ciloleucel, Tisagenlecleucel, Lisocabtagen maraleucel) or with the investigational medicinal product MB-CART19.1 (in-house manufactured 2 nd generation anti-CD19 CAR-T cells with a 4.1BB co-stimulatory domain; Kretschmann et al., 2023). Average peak expansion was reached at day 7 in both patient groups. Despite comparable average peak levels, patients with AID showed lower incidence and severity of cytokine-release syndrome (CRS) and immune-effector cell associated neurotoxicity syndrome (ICANS) compared to the NHL cohort. Consistent with reduced toxicity, AID patients received less tocilizumab and glucocorticoid treatment. Bone marrow toxicity with severe and/or prolonged cytopenia is a frequent long-term CAR-T cell related adverse event. Of interest, AID patients demonstrated a less profound decrease of neutrophils, platelet counts, and hemoglobin after CAR-T cell therapy compared to NHL patients and a faster recovery to normal values. Occurrence and duration of severe neutropenia (<500 neutrophils/µl) also differed between both patient cohorts. There was no late bone marrow toxicity in AID patients, whereas 19 of 40 patients with NHL showed at least one > grade 3 blood count reduction. In line with the Hematotox-Score predicting bone marrow toxicity, baseline inflammation (e.g. C-reactive protein, ferritin) prior to CAR-T cell treatment was higher in lymphoma patients than in AID patients. While AID patients quickly returned to normal ferritin values, elevated ferritin levels often persisted in the NHL cohort after CAR-T cell therapy. Given that AID patients showed fewer late toxicity and lower long-term inflammation, we speculated that persistence of CAR-T cells was lower. In line, longitudinal monitoring of the patient's immune cells including CAR-T cell kinetics demonstrated a typical contraction of CAR-T cell numbers with low-level persistence in both cohorts. We did not observe a difference in absolute CAR-T cell numbers or dynamics within the first three months after treatment. In contrast to NHL patients in remission, AID patients did not develop long-term persistence of CAR-T cells as most patients lost CAR-T cells within 1 year after cell infusion. Consistent with this observation, all AID patients showed B-cell reconstitution after loss of CAR-T cell persistence. Taken together, we observed less toxicities and severe adverse events in AID patients than in NHL patients despite similar CAR-T cell expansion and dynamics. The lack of CAR-T cell persistence and the early recurrence of B cells in AID patients is so far not explained and could provide novel insights in the biological processes controlling long-term CAR-T cell persistence.