Chimeric Antigen Receptor T-cell Expansion Research Articles

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.

Cryopreserved leukapheresis material can be transferred from controlled rate freezers to ultracold storage at warmer temperatures without affecting downstream CAR-T cell culture performance and in-vitro functionality

Chimeric antigen receptor (CAR) T-cell therapies are increasingly adopted as a commercially available treatment for hematologic and solid tumor cancers. As CAR-T therapies reach more patients globally, the cryopreservation and banking of patients’ leukapheresis materials is becoming imperative to accommodate intra/inter-national shipping logistical delays and provide greater manufacturing flexibility. This study aims to determine the optimal temperature range for transferring cryopreserved leukapheresis materials from two distinct types of controlled rate freezing systems, Liquid Nitrogen (LN2)-based and LN2-free Conduction Cooling-based, to the ultracold LN2 storage freezer (≤−135 °C), and its impact on CAR T-cell production and functionality. Presented findings demonstrate that there is no significant influence on CAR T-cell expansion, differentiation, or downstream in-vitro function when employing a transfer temperature range spanning from −30 °C to −80 °C for the LN2-based controlled rate freezers as well as for conduction cooling controlled rate freezers. Notably, CAR T-cells generated from cryopreserved leukapheresis materials using the conduction cooling controlled rate freezer exhibited suboptimal performance in certain donors at transfer temperatures lower than −60 °C, possibly due to the reduced cooling rate of lower than 1 °C/min and extended dwelling time needed to reach the final temperatures within these systems. This cohort of data suggests that there is a low risk to transfer cryopreserved leukapheresis materials at higher temperatures (between −30 °C and −60 °C) with good functional recovery using either controlled cooling system, and the cryopreserved materials are suitable to use as the starting material for autologous CAR T-cell therapies.

Multi-centers experience using therapeutic plasma exchange for corticosteroid/tocilizumab-refractory cytokine release syndrome following CAR-T therapy

The chimeric antigen receptor T (CAR-T) cell therapy significantly enhances the prognosis of various hematologic malignancies; however, the systemic expansion of CAR-T cells also gives rise to severe cytokine release syndrome (CRS), and immune effector cell-associated neurotoxicity syndrome (ICANS). Despite the successful application of corticosteroids and tocilizumab in alleviating severe CRS in most patients, there are still individuals who experience life-threatening CRS without responding to the aforementioned therapies. In our retrospective cohort, we conducted an analysis of clinical and laboratory parameters, including inflammatory cytokines, in 17 patients from three centers who underwent therapeutic plasma exchange (TPE) for refractory CRS with or without ICANS following CAR-T products treatment. Our findings demonstrate a significant improvement in both clinical symptoms and laboratory parameters subsequent to TPE treatment. The rapid decrease in temperature and levels of inflammatory indexes indicates the remarkable scavenging efficacy of TPE against cytokine storm following CAR-T therapy. In conclusion, TPE may serve as a valuable and safe adjunct to corticosteroids and tocilizumab in the management of severe CRS resulting from CAR-T cell infusion. We eagerly await further prospective studies to validate this finding.

Lymphodepletion - an essential but undervalued part of the chimeric antigen receptor T-cell therapy cycle.

Lymphodepletion (LD) or conditioning is an essential step in the application of currently used autologous and allogeneic chimeric antigen receptor T-cell (CAR-T) therapies as it maximizes engraftment, efficacy and long-term survival of CAR-T. Its main modes of action are the depletion and modulation of endogenous lymphocytes, conditioning of the microenvironment for improved CAR-T expansion and persistence, and reduction of tumor load. However, most LD regimens provide a broad and fairly unspecific suppression of T-cells as well as other hematopoietic cells, which can also lead to severe side effects, particularly infections. We reviewed 1271 published studies (2011-2023) with regard to current LD strategies for approved anti-CD19 CAR-T products for large B cell lymphoma (LBCL). Fludarabine (Flu) and cyclophosphamide (Cy) (alone or in combination) were the most commonly used agents. A large number of different schemes and combinations have been reported. In the respective schemes, doses of Flu and Cy (range 75-120mg/m2 and 750-1.500mg/m2) and wash out times (range 2-5 days) differed substantially. Furthermore, combinations with other agents such as bendamustine (benda), busulfan or alemtuzumab (for allogeneic CAR-T) were described. This diversity creates a challenge but also an opportunity to investigate the impact of LD on cellular kinetics and clinical outcomes of CAR-T. Only 21 studies explicitly investigated in more detail the influence of LD on safety and efficacy. As Flu and Cy can potentially impact both the in vivo activity and toxicity of CAR-T, a more detailed analysis of LD outcomes will be needed before we are able to fully assess its impact on different T-cell subsets within the CAR-T product. The T2EVOLVE consortium propagates a strategic investigation of LD protocols for the development of optimized conditioning regimens.

Long Term Durable Responses in Relapsed/Refractory (r/r) ALL, DLBCL, and FL Patients Treated with Tisagenlecleucel and Its Association with Persistence of CAR T-Cells

Introduction: Monitoring of chimeric antigen receptor (CAR) transgene levels in peripheral blood post tisagenlecleucel (tisa-cel) infusion provides information on expansion and persistence of CAR T-cells. In adult diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) patients (pts), the expansion of CAR T-cells was similar between responding and non-responding pts, while in pediatric and young adult acute lymphoblastic leukemia (ALL) pts, higher expansion in responding pts was observed. Based on long term follow up data from tisa-cel treated pts, analyses were performed to delineate the impact of CAR persistence and B cell aplasia on duration of remission (DOR). Methods: Transgene levels in blood measured by quantitative polymerase chain reaction were available from pivotal phase II studies in pts with r/r ALL (ELIANA [N=79], ENSIGN [N=64], NCT03123939 [N=69], NCT01626495 [N=60]), r/r DLBCL (JULIET [N=115]), and r/r FL (ELARA [N=97]), along with the long term follow up study (NCT02445222). The time related to loss of persistence of CAR T-cells (T loss) was defined as the time at which transgene levels first dropped below 50 transgene copies/µg DNA (approximates to lower limit of quantification (LLOQ) of the assay) after maximal expansion. Kaplan Meier analysis was performed to investigate the impact of duration of T loss and time corresponding to last quantifiable level (T last) on DOR/relapse. The impact of time to B cell recovery (>1% CD19+ B cells/WBCs or >3% CD19+ B cells/lymphocytes for ALL, and 80-616 cells/µL for DLBCL and FL), on DOR/relapse was also investigated. Results: Long term CAR persistence in tisa-cel treated pts has been observed for up to 9, 6, and 2.5 years for ALL, DLBCL, and FL pts, respectively, reflecting differing length of follow up based on initiation of trials in respective indications. Across indications, pts who lost transgene within 6 months or between 6-12 months had shorter DOR relative to pts with persistent transgene (Fig 1, ALL pts). In ALL, the median T loss was 27.4, 18.2, 9.7, and 18.0 months for ongoing complete response (CR) > 12 months, CR pts between 6-12 months, relapsed pts < 12 months, and relapsed pts > 12 months, respectively. However, of the pts who lost transgene within first 6 months, some pts maintained durable responses for ≥12 months (29%, 15%, and 50% in ALL, DLBCL, and FL, respectively). Among ALL relapsed pts, majority (17/26, 65%) of the pts with T loss < 6 months showed B cell recovery, however 6 pts had T loss at < 6 months as well as B-cell recovery but maintained response for ≥ 12 months. In ALL, Pulsipher et al.,(Blood Cancer Discov. 2022)have shown that NGS MRD within the first 6 months to 1 year post infusion may be a more reliable predictor of potential relapse than B cell recovery. The median time to B-cell recovery was 266 days in pts who relapsed/censored within 12 months but was not reached for pts with ongoing response at 12 months. On the contrary, B cell recovery seems to have no association with relapse in DLBCL or FL pts. Of the ALL pts with CAR persistence, pts with <50% morphologic blasts at any time prior to infusion of tisa-cel had longer DOR compared to pts with ≥50% blasts. This may also reflect a more resistant high risk ALL at the time of study entry or greater potential for stochastic loss of CD19 in pts with the higher disease burden. Conclusions: Long term sustained remission/response was observed in tisa-cel treated pts in the pivotal trials. The analyses demonstrate a positive association between CAR persistence and durable clinical responses across indications, however, some pts maintained durable responses despite early loss of transgene and/or early B cell recovery (< 6 months post infusion; Myers et al., J Clin Oncol. 2021). To note, in DLBCL and FL, the transgene levels in blood may not represent the levels at target sites including lymph nodes, moreover, T last and T loss are dependent on the duration of follow up, LLOQ etc. Of note, these analyses represent data from pivotal tisa-cel clinical studies with a heavily treated pt population post multiple lines of therapy including prior HSCT, and may not represent pts treated with commercial product, at earlier times post diagnosis in current studies with fewer lines of prior treatment, lower pre-infusion disease burden, and/or other CAR product. In pts with early loss of transgene, further research is needed to identify potential factors or patient characteristics resulting in durable responses.

In Vivo Anti-BCMA CAR T-Cell Expansion Kinetics Correlate with Early IMWG Response in RRMM: A Single Institution Study with Comparative Analysis of Idecabtagene Vicleucel and Ciltacabtagene Autoleucel

Introduction: The most recent breakthrough in the treatment of relapsed/refractory MM (RRMM) is chimeric antigen receptor (CAR) T-cell (CAR T) therapy which led to high overall response rates with acceptable toxicity. Here we present our single-institution experience with MM-specific CAR T performing a comparative analysis of clinical data and expansion kinetics after infusion of two commercially available myeloma CAR-T products. Methods: Out of 76 RRMM patients treated with CAR T, 23 patients provided consent, enrolled, apheresed and treated with commercial CAR-T between 6/20/2022 and 4/24/2023. Majority of patients had relapsed and multi-refractory MM. 11 patients received idecabtagene (idecel) and 12 received ciltacabtagene (ciltacel). Manufacturing slot availability was the primary deterministic factor for CAR T-type assignment. Study was IRB approved and informed consent was obtained to collect blood and clinical data. Blood samples were obtained at apheresis, pre-lymphodepleting (LD) chemotherapy, and on days 0, +7, +14, +21, +28 and +3 months post CAR T-cell infusion. PBMCs were generated from whole blood using density gradient centrifugation and were stained using BCMA or control CAR detection reagents and monoclonal antibodies against T-cell surface antigens. Samples were analyzed using MacsQuant-10 flow cytometer, gating on CD3+ and CAR+ cells. Fisher's Exact Test and Pearson Chi-Square tests were used for assessment of categorical variables whereas Independent-Samples Median Test was employed for non-categorical variables. Mann-Whitney U test was used for CAR T cell expansion analyses at individual timepoints. Results: As of July 10, 2023, 23 commercial patients were enrolled. Baseline clinical characteristics were comparable between the idecel and ciltacel cohorts with regard to age (median 66, p=0.827), prior lines of treatment, ASCT history (Table 1). Two patients in each cohort were penta-refractory. Three patients in each cohort had EMD (27.3%). There was balanced distribution of del 17p and t(4;14) across cohorts (p=1.000, p=0.175). Each cohort had 1 RISS-3 patient (p=0.476). Seven patients (30.4%) were previously treated with belantamab. Pre-apheresis and pre-LD absolute lymphocyte count (ALC) were comparable across cohorts. (Table 1). There were no G3 CRS events whereas G1/2 CRS events were not statistically significant between cohorts (p=0.256). Three G3 ICANS were observed, 2 in idecel and 1 in ciltacel cohorts (18.2% and 8.3%). Distribution of all-grade ICANS across cohorts were not statistically significant (p=0.676). Post-CAR T ferritin max correlated with the baseline b2m (p<0.001) and tocilizumab dose number (p=0.009) but none correlated with peak CAR T expansion (p=0.315). Responses were assessed by IMWG criteria at months 1,3,6,9 for efficacy-evaluable patients at that timepoint (n=19,14,8 and 2 in grand cohort (82.6%, 60.9%, 34.8% and 8.7%)). Global cohort ORR was 78.3% (n=18) and 60.9% (n=14) at days 30 and 90 respectively. +1 month IMWG responses were comparable with 72.7% (8/11) in idecel and 83.3% (10/12) in ciltacel cohort (p=0.625). Similarly, there was no statistically significant ORR difference between cohorts at 3, 6, and 9 months. Two-way ANOVA revealed a trend towards an effect of treatment type on CAR-T expansion (p=0.0595) (Figure 1A). Time-to-peak analysis reached statistical significance (p=0.0127) between two products (Figure 1B). Despite its delayed peak, ciltacel had significantly higher magnitude of expansion (p=0.043) (Figure 1C). Peak CAR-T expansion was associated with clinical responses across cohorts at +1 (p=0.034) and +3 months (p=0.050) but not at +6 months (p=0.449). Day +30 responses were indicative of Day +90 and Day +180 responses (p<0.001, p=0.011). Conclusions: In our single-center study of comparative analysis of commercial BCMA targeting CAR T-cell treatments, peak CAR T-cell expansion showed a positive correlation with +1 and +3 month post-CAR-T clinical responses. Despite the delayed peak expansion with ciltacel, the expansion was more robust with a higher level of circulating CAR T-cells. Baseline ALC or fludarabine dose reduction did not have an impact on overall CAR T expansion in combined cohort. Studies are underway at our institution investigating anti-BCMA CAR T cell responses in more detail and larger numbers of patients and functional studies will be further presented.

Determinants of Response to Anti CD-19 CAR-T Cells for Diffuse Large B-Cell Lymphoma in Pre-Treatment Peripheral Blood Mononuclear Cells Using Single-Cell RNA-Seq

Background: Chimeric antigen receptor T (CAR-T) cell therapy has become the standard of care in patients with relapsed/refractory diffuse large cell lymphoma (R/R DLBCL), providing durable remission in 40% of patients. Lack of response is likely to be attributed to inter- and intra-patient variability, including PBMC heterogeneity and apheresis material composition, CAR-T infusion product, in-vivo expansion and activity of CAR-T cells and the development of tumor-intrinsic mechanisms. Current methods for evaluating CAR-T cell activation and potency have limited predictive value for clinical outcomes. To address this challenge, we performed single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) obtained from patients with R/R LBCL patients at the time of apheresis for the manufacturing of anti-CD19 CAR-T cell therapy. Methods: Between 10/2020 and 03/2022, 31 patients who underwent lymphopheresis were enrolled. Samples were obtained - 1) At day of lymphopheresis from peripheral blood; 2) On the day of infusion of CAR-T from the product; and 3) On day +7 after infusion from peripheral blood. All patients had day +30 PET-CT. In addition, PBMCs samples from healthy donors were used as control to analyze baseline characteristics compared to the DLBCL patients. PBMCs from patients of healthy donors were purified from frozen blood samples by density gradient separation, stained using human CD45 antibody and loaded onto a 10x Chromium system. libraries were generated using the 10X Genomics Chromium Single Cell 3' Kit. The data was processed and analyzed using the Seurat R package. The study was approved by the local Ethic committee. Results: Patient characteristics: 31 RR DLBCL patients (42% females; median age 72, range 47-81 years), all treated with CAR-T cells therapy as third line of therapy, participated in the study. All patients underwent lymphopheresis, followed by CAR-T production. Disease status at lymphodepletion was complete remission (CR, n=4, 13%), partial remission (n=6, 19%), and progressive disease (n=21, 68%) and patients received either tisagenlecleucel (n=20, 65%) or axicabtagene ciloleucel (n=11, 35%) . PET scan performed on day 30 post treatment revealed that 68% and 32% obtained CR, and PD, respectively. scRNA analysis revealed differences in PBMC composition between healthy donors and LBCL patients (Figure 1A), with lower levels of naïve and memory T cells and elevated levels of activated monocytes in LBCL patients, suggesting a shift towards an inflamed and pre-dysfunctional immune state. Categorization of LBCL patients into responders (PR/CR on day +30 PET-CT) and non-responders (PD on day +30 PET-CT) revealed differences in immune cell subsets between responders and non-responders (Figure 2A), with enriched inflammation-related pathways in non-responders, In contrast, responders exhibited a distinct healthy-like immune profiles, particularly high levels of naive T cells, similar to that obtained in their age matched healthy controls. Of note, non-responders showed alterations in immune cell subsets, including increased levels of T regulatory cells and effector NK cells. Furthermore, non-responders demonstrated a prominent inflammatory immune profile, characterized by gene pathways related to inflammation and activation, such as type I and III IFN-G, and TNFb response (Figure 1B). Notably, an IFN-G score was significantly higher in non-responder's monocytes subpopulations, compared to responders. Conclusions: These findings indicate that the success of CAR-T therapy could be determined by the molecular characteristics and subpopulation distribution of each patient's PBMC. The diverse immune profiles of DLBCL patients might impact the effectiveness of their immune cells in generating successful CAR-T products or providing a conducive environment for CAR T cells to combat cancer. Furthermore, these findings emphasize the potential of scRNA-seq in evaluating PBMC from CAR-T patients prior to treatment, allowing the potential calculation of an “immune potency score” associated with the probability of a positive outcome from CAR-T treatment of DLBCL patients. This score could potentially be used by oncologists in decision-making processes to determine the appropriate candidates for CAR-T therapy.

Axicabtagene Ciloleucel (Axi-Cel) in Patients with Relapsed/Refractory Indolent Non-Hodgkin Lymphoma: 4-Year Follow-up from the Phase 2 ZUMA-5 Trial

Introduction: ZUMA-5 is a multicenter Phase 2 study of axi-cel autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy in patients with relapsed/refractory (R/R) indolent non-Hodgkin lymphoma (iNHL; follicular lymphoma [FL] and marginal zone lymphoma [MZL]). After a median of ≥3 years of follow-up, medians of progression-free survival (PFS) in patients with FL and MZL were 40.2 months and not reached, respectively, and no new safety signals were observed (Neelapu et al. ASH 2022. Abstract 4660). Here, we report updated outcomes from ZUMA-5 after a median follow-up of ≥4 years. Methods: Eligible patients had R/R FL or MZL after ≥2 lines of therapy including an anti-CD20 monoclonal antibody plus an alkylating agent. Patients underwent leukapheresis at enrollment, then received lymphodepletion and axi-cel infusion (2×10 6 CAR T cells/kg). The primary endpoint was overall response rate (ORR; complete response [CR] + partial response [PR]). Time-to-event endpoints were assessed by investigators in all enrolled patients. Exploratory analyses included lymphoma-specific survival, using competing risk assessment, in which deaths unrelated to progression, axi-cel, or lymphodepletion were competing risks. Results: In 159 enrolled patients (FL: 127, MZL: 31) at data cutoff (March 31, 2023), median follow-up was 52.5 months (range, 20.3-69.4; FL: 53.7, MZL: 43.8). The ORR in enrolled patients remained consistent with prior analyses (90% ORR, 75% CR rate). Median duration of response (DOR) was 55.5 months (95% CI, 38.6-not estimable; FL: 55.5, MZL: not reached). Medians for DOR were 60.4 months in those with a best response of CR and 4.9 months in those with a PR. At data cutoff, responses were ongoing in 48% of patients, consistent by disease type. Median PFS was 57.3 months (95% CI, 34.9-not estimable; FL: 57.3, MZL: 46.9); estimated 48-month PFS rate was 52% (FL: 53%, MZL: 47%; Figure 1). After data cutoff of the prior analysis, 1 patient with FL had disease progression. PFS rates at 48 months in patients with FL were consistent regardless of high-risk characteristics, including progression <2 years from initiating first anti-CD20-containing chemoimmunotherapy (POD24). Median time to next therapy was 62.2 months (95% CI, 37.8-not estimable; FL: 62.2, MZL: 46.9). Median overall survival (OS) was not reached (95% CI, 62.2-not estimable); 48-month OS rate was 72% (FL: 72%, MZL: 68%). Among enrolled patients with FL, the 48-month cumulative incidence of lymphoma-specific progression or death was 34%, while the cumulative incidence of competing risks was 13% (Figure 2). Additionally, the cumulative incidence of lymphoma-specific death at 48 months was 14%; the cumulative incidence of other or unknown death was 14%. After the 3-year analysis, among 152 treated patients (124 FL; 28 MZL), 6 experienced serious adverse events, 1 of which was related to axi-cel (FL, Grade 3 myelodysplastic syndrome). No new neurologic events, hypogammaglobulinemia cases, Grade ≥3 cytopenias, or Grade ≥3 infections occurred. Seven additional patients died due to progression (n=2; both patients received subsequent therapy after progression), new malignancy (n=1; not axi-cel related), and other causes (n=4; 2 cardiac arrest, 1 infection, and 1 unknown). Among treated patients with FL, those with ongoing response at 48 months continued to have higher median postinfusion CAR T-cell expansion by peak (52.2 cells/µL) and area under the curve (583.6 cells/µL×days) than those who relapsed (29.6 cells/µL and 337.6 cells/µL×days) or had no response (25.4 cells/µL and 269.9 cells/µL×days). Additionally, those with ongoing response had a higher proportion of naive (CCR7+CD45RA+) T cells in axi-cel product (25%) than relapsed (13%) or nonresponding patients (9%). Similar trends were observed in MZL. Conclusions: With a median ≥4 years of follow-up in ZUMA-5, axi-cel demonstrated continued durable response and long-term survival in patients with R/R FL and R/R MZL. Late progression or lymphoma-specific death was uncommon in FL, suggesting curative potential for those patients. The long-term safety profile of axi-cel was manageable.

Outsmart™ IL-2/15, a Novel Cytokine Designed to Enhance ROR1-Specific CAR T Anti-Tumor Activity While Minimizing Treg Activation and Systemic Cytokine Exposure

Introduction: Effective treatment with chimeric antigen receptor T (CAR-T) cells against solid and some liquid tumors will require the safe production of immune-modulating cytokines to promote CAR-T cell expansion and to stimulate endogenous anti-tumor immune responses within the tumor microenvironment. OutSmart™ IL-2/15 is a computationally designed, CD8α-targeted, IL-2-based cytokine produced by engineered T cells via a T cell activation-dependent promoter. Here, we demonstrate that inducible production of CD8-targeted IL-2/15 drives proliferation and anti-tumor efficacy of ROR1-specific CAR-T cells, stimulates bystander CD8 + T and NK cells, and minimizes activation of immune suppressive T regulatory cells (Tregs). Methods and Results: Wild-type (wt) IL-2 was modified using Rosetta protein design software package to eliminate IL-2Rα binding while retaining native IL-2Rβγ binding interfaces. Out of 10,355 designs, 38 were prioritized based on in silico analysis and ML-guided structure prediction, and evaluated for IL-2R binding as recombinant proteins. Consistent with the design criteria, none of the designs bound IL-2Rα, and 33 (87%) retained IL-2Rβγ binding and signaling activity. pSTAT5 analysis showed wt IL-2 highly activated CD4 +CD25 +FoxP3 + Treg cells, whereas the novel IL-2 designs showed a >3-log reduction Treg stimulation. These designs were iteratively optimized to generate a version exhibiting improved thermostability and IL-2Rβγ binding affinity equivalent to wt IL-2. To increase the potency against immune effector cells, but not Tregs, an anti-CD8α VHH was tethered to the modified IL-2 molecules (CD8-IL-2/15) to mimic high-affinity IL-2Rα binding and selectively activate CD8 + T and NK cells. This prosthetic binding interaction dramatically increased CD8 + T cell stimulation and CD8α + NK cell activation (>2-logs and 1-log by pSTAT5, respectively), while retaining its reduced capacity to stimulate Tregs. CD8-IL-2/15 gene expression was subsequently placed under the control of a T cell activation-dependent promoter and integrated into a lentiviral construct with a constitutively expressed ROR1-specific CAR. T cells transduced with a lentivirus expressing CD8-IL-2/15 and a ROR1-targeting CAR showed inducible cytokine production following exposure to ROR1 + tumor cells (H1975) leading to enhanced CAR-T cell proliferation and anti-tumor killing during repeat tumor challenge assays in vitro. Using immune-deficient mice (NSG) engrafted with H1975 tumors, inducible CD8-IL-2/15 enhanced ROR1-specific CAR-T cells showed durable tumor control at low doses (1e6 - 4e6 CAR T cells/animal), which was comparable to wt IL-2, whereas mock T cells and ROR1 CAR only T cells failed to control tumor growth ( Figure 1). In addition, CAR-T expansion with CD8-IL-2/15 increased >4-fold over wt IL-2 (p<0.0001), measured by cell counts in peripheral blood. Conclusions: In summary, OutSmart™ IL-2/15 is a genetic module that produces CD8-IL-2/15 in response to T cell activation, promoting robust CAR-T expansion and enhanced anti-tumor efficacy. Furthermore, local production of a CD8-IL-2/15 enhances the effector function of bystander CD8 + T cells and NK cells, but only minimally activates Treg cells due to removing the IL-2Rα binding interface. Inducible production of CD8-IL-2/15 dramatically improves the potency of a ROR1 CAR for treating ROR1 + solid and liquid cancers. More generally, OutSmart™ protein design methods and control technologies can be applied to create other designed cytokines for oncology and beyond.

Efficacy and Safety of CD19-Specific CAR-T Cell Therapy Following Immunochemotherapy in Newly Diagnosed B-Cell Lymphoma Associated Hemophagocytic Lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) represents a grave and potentially fatal systemic inflammatory condition. Lymphoma associated hemophagocytic lymphohistiocytosis (LAHS), as a common type in secondary HLH, suffers the worst outcome among sHLH. B cell Lymphoma associated hemophagocytic lymphohistiocytosis (B-LAHS) typically presents as high-risk lymphoma and is associated with a poorer prognosis compared to B cell lymphoma without HLH with standard first-line chemoimmunotherapy. As a result, there is an urgent need for more effective therapeutic strategies. Recent advancements in chimeric antigen receptor (CAR) T-cell therapy have demonstrated remarkable responses in relapsed or refractory B-cell non-Hodgkin lymphoma (B-NHL), as well as in some high-risk newly diagnosed B-cell lymphoma. However, There have been no clinical reports regarding the application of CAR-T cell treatment as part of first-line therapy in B-LAHS. We assessed the effectiveness and safety of autologous anti-CD19 CAR-T cell therapy following an immunochemotherapeutic regimen based on R-DEP (Rituximab-doxorubicin-etoposide-methylprednisolone) as a first-line treatment option for patients with B-LAHS. As of 1 July 2023, a cohort of 13 patients diagnosed with B-LAHS（with 3 cases enrolled before registration for clinical trials）was recruited for evaluation and met the criteria for assessing treatment efficacy at our institution. The overall response rate (ORR) for HLH was 100% (with 7 cases achieving complete response and 6 cases achieving partial response) prior to infuse CAR-T cells and the ORR for lymphoma also was 100% (with 10 cases achieving complete response and 3 cases achieving partial response) on the time of a month after CAR-T cell infusion. As of the follow-up date on 1 July 2023 (with a median follow-up time of 14 months), all 13 patients were still alive, 12 cases maintained sustained remission, while 1 case experienced relapse at 16 months after CAR-T cell infusion. The median overall survival (OS) has not been reached, and the median progression-free survival (PFS) was 31.5 months. To provide comparison, we included a group of 26 patients diagnosed with B-LAHS who did not receive CAR-T cell therapy during the same period. The non-CAR-T group demonstrated a significantly lower median OS of 5.93 months and a median PFS of 4.43 months, highlighting the superior prognosis of the CAR-T group (p < 0.001). Regarding adverse events, the CAR-T group exhibited a cytokine release syndrome (CRS) incidence of 53.8% (7/13) and an immune effector cell-associated neurotoxicity syndrome (ICANS) incidence of 30.7% (4 /13), all of which were classified as grade 1. None of the patients experienced non-hematologic toxicity of grade ≥3. While all 13 patients developed cytopenias, hematologic toxicity reactions ≥ grade 3 were observed in 76.9% (10 /13) of patients, but blood cell counts recovered within 3 weeks. Robust expansion of CAR T-cells occurred in all patients, with a median time to peak expansion of 8 days. Importantly, no exacerbation of hemophagocytic syndrome occurred following CAR-T cell infusion, and no patients succumbed to adverse events. In conclusion, our findings suggest that CD19 CAR-T cell treatment exhibits high efficacy as part of first-line therapy for B-LAHS, with a manageable safety profile.

A Phase 1 Dose Escalation Trial of Third-Generation CD19-Directed CAR T-Cells Incorporating CD28 and Toll-like Receptor 2 (TLR2) Intracellular Domains for Relapsed or Refractory B-Cell Non-Hodgkin Lymphomas (ENABLE)

Introduction: Chimeric antigen receptor (CAR) T cell therapies directed against CD19 and incorporating either CD28 or 4-1BB intracellular co-stimulatory domains are a standard of care for relapsed or refractory (r/r) B-cell lymphomas. CD19-directed CAR T-cell products employing CD28 co-stimulation yield high response rates, but have been associated with higher rates of immune effector cell-associated neurotoxicity syndrome (ICANS) and severe cytokine release syndrome (CRS) than 4-1BB co-stimulated products. There is a need for CAR T-cell products that combine high efficacy with low toxicity. Toll-like receptor 2 (TLR2) is expressed by T-cells, and its engagement enhances T-cell expansion, modulates cytokine production, and promotes long-lived T-cell memory. In preclinical studies, interposition of an intracellular domain from TLR2 between CD28 and CD3ζ resulted in reduced CAR T-cell production of ICANS-associated cytokines GM-CSF and IFN-γ, while maintaining production of the homeostatic cytokine IL-7. We investigated the safety and efficacy of a novel third generation CD19-directed CAR T-cell product, which combines CD28 and TLR2 co-stimulatory domains ( Figure). Methods: We completed a first-in-human phase 1 dose escalation trial of WZTL-002, comprising autologous 1928T2z CAR T-cells (‘ENABLE’, NCT04049513) for patients with r/r B-cell non-Hodgkin lymphomas (B-NHL). A 3+3 dose escalation design was used, with doses from 5 × 10 4 to 1 × 10 6 viable CAR T-cells/kg body weight. Eligible participants had radiologically assessable disease, satisfactory organ function and no central nervous system (CNS) involvement by lymphoma. Bridging therapy was permitted after leukapheresis and pending CAR T-cell manufacture and release. WZTL-002 CAR T-cells were administered intravenously after 3 days of fludarabine (30mg/m 2/day) and cyclophosphamide (500mg/m 2/day) lymphodepletion. Adverse events (AEs) were graded by CTCAE 5.0 except CRS and ICANS (graded by American Society for Transplantation and Cellular Therapy criteria). Response assessment was by PET/CT at month 3, per Lugano 2014 criteria. Pharmacokinetic analyses were by droplet digital PCR for CAR transgenes in blood mononuclear cells. Results: Of 21 patients treated within the dose escalation trial, median age was 57 years (range 23 - 70); 10 (48%) were female; 4 (19%) Māori. Lymphomas were of large cell histology in 17 (81%, Table). Participants had received a median of 4 prior lines of therapy, including autograft in 11 (52%) and allograft in 1. Product phenotyping showed WZTL-002 CAR T-cells were median 49% CD4 +, 41% CD8 + and 34% CD62L +. As of June 14 2023, all patients had reached the primary follow-up timepoint (3 months after WZTL-002 administration). Grade ≥ 3 AEs occurring in ≥ 10% of recipients were: neutropenia (95%), lymphopenia (57%), hypogammaglobulinemia (57%), anaemia (43%), febrile neutropenia (43%), thrombocytopenia (24%) and tumor pain (19%). Grade 1 - 2 CRS occurred in 13 patients (62%); 6 received tocilizumab and 3 dexamethasone. No CRS of grade ≥ 3, and no ICANS of any grade, occurred. Two grade 4 cytopenia dose limiting toxicities occurred at day 21 (one each at 5 × 10 5 and 1 × 10 6 cells/kg), both resolved to grade 2 by day 90. Maximum tolerated dose was not reached. Responses were seen at all dose levels with complete metabolic response in 11 (52%) at month 3. WZTL-002 CAR T-cells expanded at all dose levels. Among recipients of 0.5 - 1.0 × 10 6 CAR T-cells/kg (n = 12), median peak CAR T-cell level (C max) was 93,950 transgene copies/μg genomic DNA (reached at median day 10) and CAR T-cells persisted in 9 of 11 assessed (82%) at day 90. A recommended phase 2 dose (RP2D) range of 0.5 - 1 × 10 6/kg was selected. Conclusions: In this dose escalation trial of a novel third-generation CD19-directed CAR T-cell product that combines CD28 and TLR2 co-stimulatory domains for r/r B-NHL, we observed no severe CRS, no ICANS of any grade and complete responses at all dose levels. At RP2D, in vivo CAR T-cell expansion was similar to or greater than other CD19-directed products. In conjunction with preclinical findings, this study suggests interposition of a TLR2 domain between CD28 and CD3ζ domains may reduce CAR T-cell-related ICANS risk while retaining efficacy. Enrolment to a dose expansion cohort has commenced and will assess outpatient management and the safety and efficacy of WZTL-002 CAR T-cells manufactured using a closed automated process.

Mitochondrial Isocitrate Dehydrogenase Inhibition Enhances CAR T-Cell Function By Restraining Antioxidant Metabolism and Histone Acetylation

Introduction: The efficacy of chimeric antigen receptor (CAR) T-cell therapy is hampered by relapse in hematologic malignancies and by hyporesponsiveness in solid tumors. Mitochondria are vital for the regulation of memory T-cell formation or exhaustion. A mitochondria-related compound screening was performed and we found that the FDA-approved isocitrate dehydrogenase 2 (IDH2) inhibitor enasidenib enhances long-lived memory CAR T-cell formation, and improves tumor clearance in vivo. IDH2 as the key component of the tricarboxylic acid (TCA) cycle, we hypothesized that IDH2 inhibition might promote CAR T-cell persistence by reprogramming the metabolism. The study aims to systematically evaluate the role and the mechanism of IDH2 inhibition on CAR T cells, and explore the application of enasidenib in CAR T-cell therapy. Methods and Results: To identify mitochondrial components that affect the long-term efficacy of CAR T-cells, we performed an in vitro screening using a mitochondria-related compound library based on the enrichment of the CD62L + CAR T-cell subset, which mainly contains T memory stem cells (T SCM) and central memory T cells (T CM). Among several candidate compounds that promoted memory CAR T-cell formation, enasidenib (ENA), an inhibitor of both wild-type and mutant IDH2 enzymes, was the most effective one. The proportion of CD62L has increased by over 20%. To determine the effect of ENA on exhaustion induced by tonic CAR signaling and tumor antigen stimulation, we measured the expression of inhibitory receptors PD-1, TIM-3, and LAG-3 and the level of apoptosis in freshly expanded CAR T cells and B-ALL encountered CAR T cells. CAR T cells treated with ENA exhibited reduced surface levels of those inhibitory receptors. We measured the production of granzyme B and IFNγ, and tumor rechallenge assay in CAR T cells after ENA treatment to assess the effector function, ENA treated CAR T cells exerted enhanced and sustained cytotoxicity in a tumor rechallenge assay at an extreme E:T ratio of 1:10. These results were observed not only in CD19-41BBζ CAR T cells, but also in CD19-28ζ and GD2-28ζ CAR T cells. CAR T cells expanded in the presence of ENA significantly prolonged survival of recipients after Nalm-6 infusion (p < 0.0001), extended in vivo ENA treatment further enhanced CAR T-cell expansion and tumor suppression. The median survival was extended from 45 to 67.5 days. Consistent with the effect of ENA treatment, IDH2 knockdown increased the proportions of the T scm/T n and T cm subsets, and alleviated CAR T-cell exhaustion. IDH2-knockdown CAR T cells had sustained cytotoxicity and higher CD62L expression after rounds of killing B-ALL cells in vitro. To systematically explore the effects of IDH2 inhibition on the metabolism of CAR T cells, relative metabolite amounts was measured using mass spectrometry (MS). IDH2-inhibited CAR T cells exhibited a substantial reduction of metabolites in the TCA cycle, including succinate, fumarate, and malate, and in the glycolysis pathway, including lactate, phosphoenolpyruvate. Mechanistically, IDH2 inhibition reprogram central carbon metabolism of CAR T cells by redirects glucose carbon utilization from glycolysis to the pentose phosphate pathway. In addition, IDH2 limits cytosolic acetyl-CoA level to prevent histone acetylation that promotes memory cell formation. Significance: Our study indicates that metabolic intervention in CAR T cells with the FDA-approved IDH2 inhibitor enasidenib can advance current treatment, with better tumor eradication and CAR T-cell persistence.

3-Year Analysis of ZUMA-12: A Phase 2 Study of Axicabtagene Ciloleucel (Axi-Cel) As First-Line Therapy in Patients with High-Risk Large B-Cell Lymphoma (LBCL)

Background: Axi-cel is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for the treatment of relapsed/refractory (R/R) LBCL. ZUMA-12 (NCT03761056) is a Phase 2, multicenter, single-arm study of axi-cel as part of first-line treatment in patients with high-risk LBCL. In the primary efficacy analysis (n=37; median follow-up of 15.9 months), axi-cel demonstrated a high rate of investigator-assessed complete response (CR; 78%; 95% CI, 62-90) and an 89% objective response rate (ORR; 95% CI, 75-97). Axi-cel also had a manageable safety profile, with no new safety signals observed in the first-line setting (Neelapu et al. Nat Med. 2022). Here, we report updated efficacy and safety outcomes from ZUMA-12 in all patients treated with axi-cel after a median follow-up of ≥40 months. Methods: Eligible adults had high-risk LBCL, with MYC and BCL2 and/or BCL6 rearrangements (double- or triple-hit histology) per investigator or an International Prognostic Index (IPI) score ≥3, plus a positive interim PET per Lugano classification (Deauville score 4/5) after 2 cycles of an anti-CD20 monoclonal antibody and anthracycline-containing regimen. Patients underwent leukapheresis, followed by lymphodepleting chemotherapy (cyclophosphamide and fludarabine) on Days -5, -4, and -3, and a single axi-cel infusion of 2×10 6 CAR T cells/kg on Day 0. Per investigator discretion, non-chemotherapy bridging could be administered. The primary endpoint was investigator-assessed CR rate per Lugano classification (Cheson et al. J Clin Oncol. 2014). Secondary endpoints included ORR, duration of response (DOR), event-free survival (EFS), progression-free survival (PFS), overall survival (OS), safety, and levels of CAR T cells in blood and cytokines in serum. Results: As of May 3, 2023, 42 patients were enrolled and 40 were treated with axi-cel, with a median follow-up of 40.9 months (range, 29.5-50.2). In total, 37 patients were evaluable for response (centrally confirmed double- or triple-hit histology or IPI score ≥3). The CR rate was 86% (95% CI, 71-95), and 92% (95% CI, 78-98) of patients had an objective response. Patients with centrally confirmed double- or triple-hit histology (n=10) had a CR rate of 90%. Responses were ongoing in 73% of response-evaluable patients at data cutoff. Medians for DOR, EFS, PFS, and OS were not reached. The 36-month estimates for DOR, EFS (Figure 1), PFS, and OS (Figure 2) were 82%, 73%, 75%, and 81%, respectively. All treated patients (n=40) experienced adverse events (AEs) of any grade and 88% of patients had grade ≥3 AEs. Similar to the primary analysis, the most common any-grade AEs were pyrexia (100%), headache (70%), and neutrophil count decreased (55%). No new cases of cytokine release syndrome (CRS) or neurologic events of any grade occurred since the prior data cut and all cases of CRS and neurologic events reported were resolved by data cutoff. Prolonged cytopenias of any grade (present Day ≥30 post-infusion) occurred in 9 patients (n=7 neutrophil count decreased) and were resolved by data cutoff. There were 8 deaths due to progressive disease (n=5) and other causes not related to axi-cel (1 COVID-19, 1 esophageal adenocarcinoma, 1 septic shock on Days 350, 535, and 287 post axi-cel infusion, respectively). Two of the 8 deaths (1 progressive disease and 1 esophageal adenocarcinoma) occurred after the primary analysis. Among treated patients, post-infusion CAR T-cell expansion by peak and area under the curve was 36.9 cells/µL and 368.0 cells/µL×days, respectively, in those with ongoing responses at data cutoff (n=17), 45.1 cells/µL and 413.4 cells/µL×days in those who relapsed (n=6), and 34.7 cells/µL and 566.8 cells/µL×days in non-responders (n=3). Conclusion: In this updated analysis of ZUMA-12 with a median follow-up of ≥40 months, axi-cel demonstrated a high rate of durable responses and no new safety signals. Axi-cel may benefit patients exposed to fewer prior therapies and those with high-risk LBCL, a population with high unmet need and poor outcomes after standard first-line chemoimmunotherapy.

Assessment of Early Intervention Strategies for Management of Cytokine Release Syndrome and Neurologic Events after Brexucabtagene Autoleucel (Brexu-Cel) Treatment in Patients with Relapsed or Refractory Mantle Cell Lymphoma (R/R MCL) in ZUMA-2

Introduction: Brexu-cel is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved in the United States for the treatment of adults with R/R MCL and in the European Union for adults with R/R MCL after ≥2 prior therapies, including a Bruton tyrosine kinase inhibitor (BTKi). In the Phase 2 ZUMA-2 study, with a median follow-up of 35.6 months, brexu-cel showed an objective response rate of 91%, a median duration of response of 28.2 months, and a median overall survival of 46.6 months with a manageable safety profile in treated patients (pts) with R/R MCL (N=68). Grade (Gr) ≥3 infections, cytokine release syndrome (CRS), and neurologic events (NEs) occurred in 37%, 15%, and 31% of pts, respectively (Wang et al. J Clin Oncol. 2023). In August 2018, CRS and NE management strategies for ZUMA-2 were updated to initiate treatment of these adverse events earlier at the onset of Gr 1 events to improve safety outcomes. Here, we report 4-yr safety outcomes for pts in ZUMA-2 by initial and updated CRS and NE management strategies. Methods: Pts with R/R MCL and ≤5 prior treatments, including a BTKi, underwent leukapheresis and conditioning chemotherapy followed by a single infusion of brexu-cel (2×10 6 CAR T cells/kg). For CRS, the initial toxicity management strategy was to consider tocilizumab ± corticosteroids (methylprednisolone 1 mg/kg bid or dexamethasone 10 mg q6h) for Gr 2 CRS with extensive comorbidities or older age and Gr 3 CRS, and to increase the dose of corticosteroids (methylprednisolone 1 g/day x3, followed by a rapid taper consisting of 250 mg bid x2 days, 125 mg bid x2 days and then 60 mg bid x2 days) for Gr 4 CRS. The updated guidance was to administer tocilizumab for Gr 1/2 CRS that does not improve after 24 hours, and tocilizumab + corticosteroids (only if no resolution after 24 hours) and vasopressors for Gr 2 and 3 CRS. For NEs, the initial guidance was to consider use of tocilizumab for Gr 2 NEs only if other comorbid conditions exist (8 mg/kg IV over 1 hour, not to exceed 800 mg) and to add corticosteroids (dexamethasone 10 mg IV q6h, methylprednisolone 1 mg/kg bid) for Gr ≥3 NEs. In the updated guidance, steroid treatment without concurrent CRS remained the same, but both tocilizumab + dexamethasone 10 mg IV were to be administered for Gr ≥2 NEs concurrent with CRS. Both guidance protocols recommended treatment with antibiotics for management of infections. This exploratory post hoc analysis examined safety outcomes related to CRS, NEs, and infections for pts in each management group, with descriptive statistics reported. Results: As of July 23 2022, the median follow-up in the 68 treated pts was 47.5 months (range, 37.9-68.3). Of these pts, 40 received updated guidance (early intervention group [EIG]) and 28 received initial guidance (later intervention group [LIG]). Gr ≥3 CRS events were experienced at similar rates in both groups while Gr ≥3 NEs and Gr ≥3 infections occurred less often in the EIG vs the LIG (Table). Gr 5 infections occurred in 2 pts in the EIG and 0 pts in the LIG. No Gr 5 CRS or NEs occurred in either group. The median durations of Gr ≥3 CRS and infections were shorter for the EIG than the LIG while the median duration of Gr ≥3 NEs was longer for the EIG. Any Gr CRS and NEs were resolved at similar rates in both groups, but fewer any Gr infections were resolved for the EIG (all unresolved events in both groups were due to pt death before event resolution). Concomitant medications of interest were used in 80% (n=32) and 82% (n=23) of pts in the EIG and LIG, respectively. Tocilizumab was used at similar rates in the EIG and LIG (73% vs 68%), but steroids (55% vs 68%), vasopressors (18% vs 29%), and immunoglobulins (28% vs 50%) were used at lower rates in the EIG. Median CAR T-cell peak levels and area under the curve were lower for the EIG than the LIG while median CAR T-cell levels peaked at similar times in both groups. Conclusions: With an updated safety management protocol emphasizing early intervention with tocilizumab and corticosteroids for CRS and NEs, only 20% of pts had Gr ≥3 NEs and 30% had Gr ≥3 infections. Additionally, a lower percentage of EIG pts required steroid, vasopressor, and immunoglobulin use than pts in the LIG. CAR T-cell expansion levels were numerically lower in the EIG. Although limited by small pt numbers, these findings support the current clinical guidance for safety management during brexu-cel treatment, which is close to the ZUMA-2 updated toxicity management guidance that was examined in this study.

Seven-Day Vein-to-Vein Point-of-Care Manufactured CD19 CAR T Cells (GLPG5201) in Relapsed/Refractory CLL/SLL Including Richter's Transformation: Results from the Phase 1 Euplagia-1 Trial

Background Despite treatment advances, chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) remain incurable. Patients (pts) with concurrent Richter's transformation (RT) have poor prognoses with limited treatment options. Chimeric antigen receptor (CAR) T-cell products targeting CD19 have shown activity in pts with CLL/SLL or RT, yet data are limited and no therapies are approved. Due to limited treatment options and short survival of pts with RT, rapid treatment delivery is essential. A novel decentralized and automated point-of-care (PoC) manufacturing model was developed to administer fresh, autologous CAR-T treatments within 7 days of apheresis. An update on the Phase (Ph) 1 Euplagia-1 trial of PoC manufactured GLPG5201 is presented. Methods Euplagia-1 (CTIS: 2022-501686-47-00) is a Ph1/2 study of PoC-manufactured GLPG5201 in pts with R/R CLL or SLL, including pts with RT. Pts with CD19+ R/R CLL/SLL with ≥2 prior therapy lines are eligible. Pts with RT are eligible regardless of prior therapy. GLPG5201, an anti-CD19/41BB CAR-T therapy, is administered as a fresh product following fludarabine/cyclophosphamide lymphodepleting chemotherapy. Upon screening, pts receive daily ibrutinib until leukapheresis. The primary objectives are safety and determining a recommended Ph2 dose (Ph1), and objective response rate (ORR; Ph2). Secondary objectives include additional efficacy measures, pharmacokinetics and feasibility of PoC manufacturing. Results As of April 26, 2023, 12 pts were enrolled in Ph1 at dose level (DL) 1 (35×10 6 CAR+ T cells, n=6) or DL2 (100×10 6 CAR+ T cells, n=6) at a single institution. All pts were diagnosed with R/R CLL, 7/12 with concurrent RT. Median age was 66 years (range 58-71); 8/12 pts were male. Median prior lines of therapy were 4 (range 2-10); 10/12 pts received a BTK inhibitor, 9/12 pts venetoclax, and 1 pt an allogeneic stem-cell transplant. Five of 7 pts with RT relapsed after, or were refractory to, R-CHOP. Six of 12 pts had a TP53 mutation, 1 pt had a 17p deletion; 11 pts had an unmutated IGHV status and 2 had a complex karyotype reported. GLPG5201 was manufactured for all pts and administered as a fresh infusion. Median vein-to-vein time was 7 days (range 7-14). Due to lower manufactured CAR+ T cell yield, 3 pts received DL1 instead of the intended DL2. A preserved early memory phenotype for CD4+ and CD8+ CAR T cells was observed in the final product, compared with apheresis starting material ( Figure). Most treatment emergent adverse events (TEAEs) were Grade 1-2; most Grade ≥3 events were hematological ( Table). Six (50%) pts had Grade 1-2 cytokine release syndrome (CRS), with no CRS Grade ≥3. No neurotoxicity was reported. A dose limiting toxicity occurred in 1 pt (Grade 4 neutropenia, DL2), which was manageable and within expectations in this pt population. No unexpected GLPG5201-related toxicities were observed; no deaths occurred while pts were on study. Eleven pts responded to treatment (best ORR 92%). Complete response (CR) was observed for 9 pts (CR rate [CRR] 75%). ORR and CRR were 83% and 67% at DL1, and 100% and 83% at DL2, respectively. All but 1 pt with RT responded (ORR 86%); CR was achieved by 5/7 pts with RT (CRR 71%). One pt with RT was refractory with CD19-negative disease. At the time of analysis, 9/11 (82%) pts had ongoing responses; duration was up to 9 months post-infusion (median follow-up 3.2 months; range 1-9). Two pts progressed after an initial response, 1 pt with CD19-negative disease. Robust CAR T-cell expansion was observed in all pts by qPCR, with a median maximum expansion of 4.4×10 5 copies/µg DNA (C max). Median T max was 14 days (range 9-20 days). Median AUC 0-28d was 5.3×10 6 copies/µg DNA × days. GLPG5201 could be detected in peripheral blood up to 9 months post-infusion. Conclusion Data from 12 pts enrolled in the Ph1 Euplagia-1 study demonstrate that PoC CAR-T manufacturing with a short vein-to-vein time is feasible. GLPG5201 is administered as a fresh product; median vein-to-vein time is 7 days. There are no unexpected safety findings, and no CRS Grade ≥3 or any ICANS are reported. Efficacy data are encouraging, with a 92% best ORR and 75% CRR; 6/7 pts with RT responded. With this novel PoC manufacturing model, an early phenotype of less differentiated CAR T cells is preserved in the final product. GLPG5201 demonstrates rapid in vivo expansion and durable persistence post-infusion . Euplagia-1 is ongoing, and updated data will be presented.

Development of a Multiscale Mechanistic Modelling Framework Integrating Differential Cellular Kinetics of CAR-T Cell Subsets and Immunophenotypes in Cancer Patients.

Chimeric antigen receptor (CAR) T-cell subsets and immunophenotypic composition of the pre-infusion product, as well as their longitudinal changes following infusion, are expected to affect CAR-T cell expansion, persistence, and clinical outcomes. Herein, we sequentially evolved our previously described cellular kinetic-pharmacodynamic (CK-PD) model to incorporate CAR-T cell product-associated attributes by utilizing published preclinical and clinical datasets from two affinity variants (FMC63 and CAT19 scFv) anti-CD19 CAR-T cells. In step 1, a unified cell-level PD model was used to simultaneously characterize the in vitro killing datasets of two CAR-Ts against CD19+ cell lines at varying effector: target (E: T) ratios. In step 2, an augmented CK-PD model for anti-CD19 CAR-Ts was developed, by integrating CK dataset(s) from two bioanalytical measurements (qPCR and flow cytometry) in cancer patients. The model described the differential in vivo expansion properties of CAR-T cell subsets. The estimated expansion rate constant was ~1.12-fold higher for CAR+CD8+ cells in comparison to CAR+CD4+ T cells. In step 3, the model was extended to characterize the disposition of four Immunophenotypic populations of CAR-T cells, including stem-cell memory (TSCM ), central memory (TCM ), effector memory (TEM ) and effector (TEFF ) cells. The model adequately characterized the longitudinal changes in immunophenotypes post anti-CD19 CAR-T cell infusion in acute lymphocytic leukemia paediatric patients. Polyclonality in the pre-infusion product was identified as a categorical covariate influencing differentiation of immunophenotypes. In the future, this model could be leveraged a priori towards optimizing the composition of CAR-T cell infusion product, and further understand the CK-PD relationship in patients.

CRISPR/Cas9: A Powerful Strategy to Improve CAR-T Cell Persistence.

As an emerging treatment strategy for malignant tumors, chimeric antigen receptor T (CAR-T) cell therapy has been widely used in clinical practice, and its efficacy has been markedly improved in the past decade. However, the clinical effect of CAR-T therapy is not so satisfying, especially in solid tumors. Even in hematologic malignancies, a proportion of patients eventually relapse after receiving CAR-T cell infusions, owing to the poor expansion and persistence of CAR-T cells. Recently, CRISPR/Cas9 technology has provided an effective approach to promoting the proliferation and persistence of CAR-T cells in the body. This technology has been utilized in CAR-T cells to generate a memory phenotype, reduce exhaustion, and screen new targets to improve the anti-tumor potential. In this review, we aim to describe the major causes limiting the persistence of CAR-T cells in patients and discuss the application of CRISPR/Cas9 in promoting CAR-T cell persistence and its anti-tumor function. Finally, we investigate clinical trials for CRISPR/Cas9-engineered CAR-T cells for the treatment of cancer.

Axicabtagene Ciloleucel in Combination with the 4-1BB Agonist Utomilumab in Patients with Relapsed/Refractory Large B-Cell Lymphoma: Phase 1 Results from ZUMA-11.

Chimeric antigen receptor (CAR) T-cell therapies have shown clinical benefit for patients with relapsed/refractory (R/R) large B-cell lymphoma (LBCL), yet approximately 60% of patients do not respond or eventually relapse. We investigated the safety and feasibility of the CD19-directed CAR T-cell therapy axicabtagene ciloleucel (axi-cel) in combination with the 4-1BB agonist antibody utomilumab as an approach to improve efficacy of CAR T-cell therapy. In phase 1 of the single-arm ZUMA-11 trial, patients with R/R LBCL received a single axi-cel infusion (target dose, 2 × 106 cells/kg) plus utomilumab 10 to 200 mg intravenously every 4 weeks for up to 6 months in a dose-escalation design. The primary endpoint was incidence of dose-limiting toxicities (DLT) with utomilumab. Key secondary endpoints were safety, antitumor activity, pharmacokinetics, and pharmacodynamics. No DLTs were observed among patients treated with axi-cel and utomilumab (n = 12). Grade ≥3 adverse events occurred in 10 patients (83%); none were Grade ≥3 cytokine release syndrome or neurologic events. The objective response rate was 75% and seven patients (58%) had a complete response. Peak CAR T-cell levels increased in a utomilumab dose-dependent manner up to 100 mg. Patients who received utomilumab 100 mg had persistently increased CAR T cells on days 57 to 168 compared with other dose levels. Utomilumab was associated with dose-dependent increases in IL2, IFNγ, and IL10. Utomilumab-mediated 4-1BB agonism combined with axi-cel therapy had a manageable safety profile. Dual 4-1BB and CD28 costimulation is a feasible therapeutic approach that may enhance CAR T-cell expansion in patients with LBCL.

Enhancing CAR T-cell Therapy Using Fab-Based Constitutively Heterodimeric Cytokine Receptors.

Adoptive T-cell therapy aims to achieve lasting tumor clearance, requiring enhanced engraftment and survival of the immune cells. Cytokines are paramount modulators of T-cell survival and proliferation. Cytokine receptors signal via ligand-induced dimerization, and this principle has been hijacked utilizing nonnative dimerization domains. A major limitation of current technologies resides in the absence of a module that recapitulates the natural cytokine receptor heterodimeric pairing. To circumvent this, we created a new engineered cytokine receptor able to constitutively recreate receptor-heterodimer utilizing the heterodimerization domain derived from the IgG1 antibody (dFab_CCR). We found that the signal delivered by the dFab_CCR-IL2 proficiently mimicked the cytokine receptor heterodimerization, with transcriptomic signatures like those obtained by activation of the native IL2 receptor. Moreover, we found that this dimerization structure was agnostic, efficiently activating signaling through four cytokine receptor families. Using a combination of in vivo and in vitro screening approaches, we characterized a library of 18 dFab_CCRs coexpressed with a clinically relevant solid tumor-specific GD2-specific chimeric antigen receptor (CAR). Based on this characterization, we suggest that the coexpression of either the common β-chain GMCSF or the IL18 dFab_CCRs is optimal to improve CAR T-cell expansion, engraftment, and efficacy. Our results demonstrate how Fab dimerization is efficient and versatile in recapitulating a cytokine receptor heterodimerization signal. This module could be applied for the enhancement of adoptive T-cell therapies, as well as therapies based on other immune cell types. Furthermore, these results provide a choice of cytokine signal to incorporate with adoptive T-cell therapies.

Impact of fludarabine exposure on the depth and duration of lymphopenia and CAR T-cell outcomes.

10033 Background: Chimeric antigen receptor (CAR) T-cells are highly effective for patients with relapsed or refractory B-ALL. Lymphodepletion has been paramount to CAR T-cell expansion and efficacy. Recent data identified an association of fludarabine exposure with reduced relapse rates, longer leukemia free survival, and more prolonged B-cell aplasia following tisagenlecleucel. However, the relationship of fludarabine exposure to lymphopenia and the mechanism by which dosing impacts outcomes following CAR T-cells remains unknown. Methods: We conducted a retrospective review of children and young adults with B-ALL who received fludarabine for lymphodepletion (LD) prior to infusion of anti-CD19, anti-CD22, or anti-CD19/22 bispecific CAR T-cells. The primary objective of this study was to investigate the impact of fludarabine on the depth and duration of lymphopenia. Secondary objectives included examining the impact of fludarabine exposure on: 1) overall clinical response, and 2) CAR T-cell expansion. All patients in the analysis cohort received fludarabine 25 mg/m2/day x 3 days ( = 75 mg/m2 cumulative dosing). Fludarabine exposure (AUC per dose) for each patient was predicted using a validated population PK model and analyzed with various responses/endpoints using the 3-day cumulative exposure/patient. Results: Of 137 children and young adults, the median cumulative AUC was 8.3 mg*hr/mL (range, 3.00-13.83 mg*hr /mL). The overall complete response (CR) rate was: 67.9% (93/137). The median duration of lymphopenia (defined as an absolute lymphocyte count (ALC) £ 150 K/uL) was 5 days (range, 0-15); and the median ALC on Day 0 was 80 K/uL (range, 0-1250 K/uL). When stratified by the median fludarabine exposure, there was no difference in depth or duration of lymphopenia or CAR T-cell expansion collectively or per trial. The median fludarabine exposure also did not differ between responders and non-responders. However, depth and duration of lymphopenia did differ in the CD22 CAR T-cell trial. Specifically, in those achieving CR, the duration of lymphopenia was longer than in non-responders (7 days versus 4 days, p = 0.030). Similarly, CAR T-cell expansion was higher if lymphopenia duration was greater than 5 days (p = 0.019). These findings were not seen across our less persistent CD19 or CD19/22 CAR T-cell trials. Conclusions: Although lymphodepletion impacts CAR T-cell response, we did not find an association between CR rate, depth of lymphopenia, or duration of lymphopenia with varying fludarabine exposures. This raises additional questions regarding the impact of LD therapy on CAR T-cell outcomes—especially since a more prolonged lymphopenia was associated with high CR rates and greater peak expansion in at least one of our studies (CD22 CAR T-cells). Future analysis will evaluate the impact of fludarabine on Day 0 cytokines and other immune parameters. Clinical trial information: NCT03448393 .